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Parathyroids and Bone Metabolism

Writer and Curator: Larry H. Bernstein, MD, FCAP 

 

 

Parathyroid hormone (PTH), parathormone or parathyrin, is secreted by the chief cells of the parathyroid glands as a polypeptide containing 84 amino acids. It acts to increase the concentration of calcium (Ca2+) in the blood, whereas calcitonin (a hormone produced by the parafollicular cells (C cells) of the thyroid gland) acts to decrease calcium concentration. PTH acts to increase the concentration of calcium in the blood by acting upon the parathyroid hormone 1 receptor (high levels in bone and kidney) and the parathyroid hormone 2 receptor (high levels in the central nervous system, pancreas, testis, and placenta). PTH half-life is approximately 4 minutes.[2] It has a molecular mass of 9.4 kDa.

hPTH-(1-34) crystallizes as a slightly bent, long helical dimer. Analysis reveals that the extended helical conformation of hPTH-(1-34) is the likely bioactive conformation.[4] The N-terminal fragment 1-34 of parathyroid hormone (PTH) has been crystallized and the structure has been refined to 0.9 Å resolution.

The_ribbon_cartoon_structure - hPTH helical dimer

The_ribbon_cartoon_structure – hPTH helical dimer

http://upload.wikimedia.org/wikipedia/commons/1/1e/The_ribbon_cartoon_structure.png

Regulation of serum calcium

PTH was one of the first hormones to be shown to use the G-protein, adenylyl cyclase second messenger system.

Normal total plasma calcium level ranges from 8.5 to 10.2 mg/dL (2.12 mmol/L to 2.55 mmol/L).

Region Effect
bone It enhances the release of calcium from the large reservoir contained in the bones.[7] Bone resorption is the normal destruction of bone by osteoclasts, which are indirectly stimulated by PTH. Stimulation is indirect since osteoclasts do not have a receptor for PTH; rather, PTH binds to osteoblasts, the cells responsible for creating bone. Binding stimulates osteoblasts to increase their expression of RANKL and inhibits their expression of Osteoprotegerin (OPG). OPG binds to RANKL and blocks it from interacting with RANK, a receptor for RANKL. The binding of RANKL to RANK (facilitated by the decreased amount of OPG available for binding the excess RANKL) stimulates these osteoclast precursors to fuse, forming new osteoclasts, which ultimately enhances bone resorption
kidney It enhances active reabsorption of calcium and magnesium from distal tubules and the thick ascending limb. As bone is degraded, both calcium and phosphate are released. It also decreases the reabsorption of phosphate, with a net loss in plasma phosphate concentration. When the calcium:phosphate ratio increases, more calcium is free in the circulation
intestine via kidney It enhances the absorption of calcium in the intestine by increasing the production of activated vitamin D. Vitamin D activation occurs in the kidney. PTH up-regulates25-hydroxyvitamin D3 1-alpha-hydroxylase, the enzyme responsible for 1-alpha hydroxylation of 25-hydroxy vitamin D, converting vitamin D to its active form (1,25-dihydroxy vitamin D). This activated form of vitamin D increases the absorption of calcium (as Ca2+ ions) by the intestine via calbindin.

http://en.wikipedia.org/wiki/Parathyroid_hormone

Development of Present Concepts of the Parathyroid –
The Parathyroids – Progress, problems and practice,
in Current Problems in Surgery, 1971; 8(8): 3-64.
Leon Goldman, Gilbert Gordon, Betty S. Roof
http://dx.doi.org/10.1016/S0011-3840(71)80008-4

The parathyroid gland first achieved clinical significance because of hypoparathyroid tetany. Tetany: a syndrome manifested by painful muscle spasms or rigors; is derived from the Greek:  tetanos, past participle of the verb teinein, meaning “to stretch,” Tetany : stretched, or spastic, in modern terms “up tight.,’ When the word was used by Hippocrates, no differentiation was made between the types of muscular spasms caused by neurotoxins (e.g., lockjaw) and those of metabolic causes. The word ~ went through the Latin, tetanus, and to French. Te’tanie, where the attribute of intermittent muscular spasm was added.

Owea's drawing of parathyroid gland of Indian rhinoceros

Owea’s drawing of parathyroid gland of Indian rhinoceros

Owea’s drawing of parathyroid gland of Indian rhinoceros

According to file Oxford English Dictionary, the relation of tetany to surgical operations was noted in tile year 1805 in The Medical Journal XIV, 304: “tetanie affections very often to|low the great operations. . .” It is not clear from this reference what type of operations were invo]ved.  The relationship of tetany to thyroidectomy was recognized as early as 1878 when WoIfler described convulsions in one of the patients on whom Billroth had performed a total thyroidectomy. The great surgeon WilIiam Stewart Halsted suggested that postoperative hypoparathyroidism had not been reported earlier because before that time total thyroidectomy had always been fatal, leaving insufficient time for tetany to develop. In 1883 Weiss collected 13 cases of tetany, all following total thyroidcctomy. The relation to total thyroidectomy became historically significant later when postoperative tetany was misinterpreted as the acute form of thyroid insufficiency, while myxedema was correctly recognized as the chronic form.
Anatomically, the parathyroid glands had been noted fleetingly by Remak (1855), by Virchow (1863) and probably by others in the course of human dissection. Perhaps better publicized was the description by Sir Richard Owen, published in 1852. As Hunterian Professor and Conservator of the Museum in the Royal College of Surgeons, Owen anatomized animals that died at the London Zoo. In 1849, while performing an autopsy on tile Great Indian rhinoceros, Owen clearly noted, drew and named the parathyroid gland (Fig. 1). However, microscopic examination was not reported, and it was not known at that time whether the parathyroid gland was separate.
The causal relationship of the parathyroid gland to post-thyroidectomy tetany was clarified by the French physiologist Eugdne Gley in 1891. He showed that, in the rabbit, removal of the thyroid gland was not responsible for these seizures but that removal of the parathyroid glands caused fatal convulsions.
Very soon after this, a parallel discovery was made in Berkeley, California, by Jacques Loeb.  Loeb noticed that the rhythmic contractions of a frog muscle in a saline medium were stopped by the addition of calcium. He concluded that calcium has the important function of inhibiting excessive neuromuscular
irritability.  Loeb’s studies led MacCallum, in 1909, to investigate the possibility that a low blood calcium level might be responsible for the increased excitability of the muscles, in hypoparathyroid tetany.  He and Voegtlin removed the parathyroids from dogs and showed that tetany ensued when the serum calcium level fell. They also showed that administration of calcium promptly relieved tetany. Less well known is their publication in the following
year, which entirely recanted the earlier view. Their observations that calcium, magnesium and strontium immediately abolish tetany, and the report of Joseph and Sleltzer that infusion of hypertonic sodium chloride slowly relieves this kind of tetany, led MacCallum to believe that the effect of calcium was nonspecific.
By this time thyroid surgery was being performed widely. The Reverdin brothers in Geneva noted what they considered complex nervous manifestations following total thyroidectomv, Moussu’ s observations in animals were confirmed in patients; post-thyroidectomy convulsions were not necessarily fatal.
Thyroid surgery was now sufficiently improved so that Kocher was able to find symptoms of tetany–and these were transient ….. in only 1 of his 18 cases of total thyroidectomy. How many more would have been identified as victims of hypoparathyroidism by appropriate chemical examination can only be conjectured. By 1907 Halsted had recognized the importance of the parathyroids and how essential the intimate knowledge of their anatomy is to the goiter surgeon. Halsted put a bright young medical student to work on this project as a penalty for delinquent attendance at lectures. The sketch of the beautiful dissection by the student, Herbert McLean Evans, was used by Halsted to illustrate his monograph on The Operative History of Goiter. On the basis of this knowledge, of anatomy, it was established that the parathyroids are usually related to the posterior capsule and that leaving this capsule intact greatly reduces the risk of tetany.
In 1923 the distinguished Norwegian physician-physiologist, Harald Salvesen published beautiful, imaginative and thorough studies in which he showed, that complete parathyroid ablation invariably lowered the blood calcium, that the blood sugar level was not altered and that guanidine accumulation occurred only terminally during agonal convulsions. He further found that parathyroid tetany could be prevented by calcium feeding and confirmed MacCallum’s earlier observation that it could be promptly corrected by calcium infusion. He also noted that one of his dogs with parathyroid tetany developed a cataract. In our opinion, the relation of the parathyroid gland to calcium metabolism was first firmly established by Salvesen in 1923.
Consider the knowledge and use of endocrines in 1923. Desiccated thyroid, which Osler had praised as the miracle of modern metabolic therapy, was the only orally effective endocrine preparation. ]nsulin had just been discovered. Another potent preparation was the hydrochloric acid extract of parathyroid glands made by Adolph Hanson. That it was an effective preparation is perhaps best attested by the fact that it is still used, under the name Parathyroid Extract USP, and that much of the work on the actions of parathyroid hormone has been carried out with this crude extract. In 1925 Collip, who had been of such immeasurable help to Banting, Best and McLeod in preparing a clean, potent insulin extract from normal pancreas, applied his genius to the parathyroid with an equally satisfactory result. His relatively clean parathyroid extract  made it possible for the first time to elucidate the actions of the parathyroid glands in man.
Using this preparation, Albright and Ellsworth in 1929 clarified the two fundamental actions of parathyroid hormone (PTH) identical with those obtained nowadays with the most highly purified preparations. These two actions are:
(1) elevation of serum calcium and
(2) excretion of phosphate by the kidneys, with a consequent lowering of the serum phosphate.
It will later be shown that the action that raises serum calcium levels is, for the most part, an increase in the rate of bone breakdown. It remained for Copp and associates to show in 1961 that another horrnone, calcitonin, with an opposite action, is necessary for maintenance of calcium homeostasis. And still later Chase and Aurbach showed in 1968 that the phosphaturic action of PTH is mediated by the enzyme adenyl cyclase, which stimulates production of cyclic 3’5′-adenosine monophosphate (AMP).
It is now clear that hypophosphatemia predisposes to hyperealcemia and that hyperphosphatemia can actually abolish hypercalcemia. However, numerous experiments, one of them by Albright’s collaborators, Ellsworth and Futeher in 1935 showed that parathyroid extract raised the serum calcium level in the absence of the kidneys.  Clearly, therefore, the calcium-mobilizing effect of PTH is not the result of the phosphate diuretic action only. Conclusive evidence was obtained by Barnicot of Cambridge in 1948. …
The brilliant group at the Massachusetts General Hospital, led by Aub and including two young men destined to make brilliant records in American medicine Fuller Albright and Waiter Bauer soon showed that the kind of hyperparathyroidism described by Recklinghausen, Mandl and Askanazy is, in fact, the end stage of a series of chemical events predictable from the known actions of PTH. Starting with the famous case of Captain Charles Martell, a mariner with severe bone disease, who shrank in stature in 10 years, Albright soon clarified the most significant feature of hyperparathyroidism: the hypercalcemia that is found in at least 99% of patients with proved primary hyperparathyroidism.
It was not until 1953 that Jonas Shota directly demonstrated the other action of excess PTH in hyperparathyroidism: a low rate of tubular reabsorption of phosphate (TRP), as fifteen years later, in 1968, Chase and Aurbach would show that this action is mediated by renal adenyl cyclase and cyclic AMP. Meanwhile, in 1935, Pappenheimer and Wilens had described another form of hyperparathyroidism arising not as a primary tumor, but as a secondary or compensatory response to the metabolic abnormalities of uremia. Goldman independently described this phenomenon. It .is noteworthy that hyperparathyroidism secondary to lack of dietary calcium had already been described by Erdheiqm and that  these 2 causes of secondary hyperparathyroidism, Uremia and intestinal malabsorption, have subsequently been shown, to have in comrnon inadequate intestinal absorption of calcium.
Since the classic studies of Sandstrom, Gley, Loeb, Salvesen, Cotlip, Aub, Bauer and Albright, enormous strides have advanced our knowledge of parathyroid physiology. Isolation, purification, and characterization of  the hormone and development of a highly sensitive  radioimmunoassay for PTH.  Almost slmultaneously in1959, Aurbach, Rasmussen and Craig obtained a purified bovine PTH. These two groups of investigators identified a similar peptide with a molecular weight of about 8,500 and with biological activity of about 3.000 units/mg. This peptide contains 84 amino acid residfies the first 30-45 are necessary for biologic and immunologic activity. A tentative molecular structure reported by Potts, Aurbach and Sherwood in 1965 has subsequently been modified by Brewer and Ronan, with confirmation by Niall et aI. in Potts’s laboratory. The heterogeneous  nature of circulating PTH was first: shown by Berson and Yalow using two antisera prepared from beef PTH but showing quantitative differences in reaction to circulating PTH. They were able  to detect two parathormones, one with a half-life of only 10-20 minutes, and another with a half-life of about 1.5 hours.
The parathyroid hormone-regulated transcriptome in osteocytes: Parallel actions with 1,25-dihydroxyvitamin D3 to oppose gene expression changes during differentiation and to promote mature cell function

Hillary C. St. John, MB Meyer, NA Benkusky, AH Carlson, M Prideaux, et al.
Bone 72 (2015) 81–91
http://dx.doi.org/10.1016/j.bone.2014.11.010

Although localized to the mineralized matrix of bone, osteocytes are able to respond to systemic factors such as the calciotropic hormones 1, 25-(OH)2 D3 and PTH. In the present studies, we examined the transcriptomic response to PTH in an osteocyte cell model and found that this hormone regulated an extensive panel of genes. Surprisingly, PTH uniquely modulated two cohorts of genes, one that was expressed and associated with the osteoblast to osteocyte transition and the other a cohort that was expressed only in the mature osteocyte. Interestingly, PTH’s effects were largely to oppose the expression of differentiation-related genes in the former cohort, while potentiating the expression of osteocyte-specific genes in the latter cohort. A comparison of the transcriptional effects of PTH with those obtained previously with 1, 25-(OH)2 D3 revealed a subset of genes that was strongly overlapping. While 1, 25-(OH)2 D3 potentiated the expression of osteocyte-specific genes similar to that seen with PTH, the overlap between the two hormones was more limited. Additional experiments identified the PKA-activated phospho-CREB (pCREB) cistrome, revealing that while many of the differentiation-related PTH regulated genes were apparent targets of a PKA-mediated signaling pathway, a reduction in pCREB binding at sites associated with osteocyte-specific PTH targets appeared to involve alternative PTH activation pathways. That pCREB binding activities positioned near important hormone-regulated gene cohorts were localized to control regions of genes was reinforced by the presence of epigenetic enhancer signatures exemplified by unique modifications at histones H3 and H4. These studies suggest that both PTH and 1, 25-(OH)2 D3 may play important and perhaps cooperative roles in limiting osteocyte differentiation from its precursors while simultaneously exerting distinct roles in regulating mature osteocyte function. Our results provide new insight into transcription factor-associated mechanisms through which PTH and 1, 25-(OH)2 D3 regulate a plethora of genes important to the osteoblast/osteocyte lineage.

Bone, a dynamic and integrating tissue

The guest editors Bram C.J. van der Eerden, Anna Teti, Willian F. Zambuzzi
Archives of Biochemistry and Biophysics 561 (2014) 1–2
http://dx.doi.org/10.1016/j.abb.2014.08.012

The special issue ‘Bone, a dynamic and integrating tissue’ provides the most recent information regarding the interacting nature of bone cells with their immediate neighboring cells within the skeleton as well as with distant target cells in other organs, using different types of both cellular and non-cellular communication. It should appeal to any scientist or clinician in the field, given the wide variety of topics, covering molecular, experimental cell and animal biology, biomechanics and -physics, genetics and medicine.

This special issue arose from a collaboration between the guest editors within ‘INTERBONE’, a European Union funded Marie Curie Actions – People – International Research Staff Scheme (PIRSESGA-2011-295181) on the interplay among bone cells, matrices and systems.

Over the recent years, many developments have paved new avenues to study signaling pathways and mechanisms in bone in much greater detail. Genetic progress has been made, which has provided us with novel genes behind already known as well as hitherto idiopathic bone diseases. The enormous expansion of specific animal models has enabled us to study new mechanisms and pathways in vivo in great spatial and temporal detail. As a consequence, novel treatment modalities have seen the light, which are predominantly focusing on bone anabolic therapies. These advances will not cease to exist and an exciting biological era lies ahead of us, with many discoveries to be made.

In this special issue of Archives in Biochemistry and Biophysics, experts in the field of bone metabolism have addressed the recent developments in which special attention is paid to the concept that bone is not just a static, isolated organ, but a dynamic and integrating tissue. Over the last decade, discoveries have led to the notion that bone cells are interacting with many other cell types within bone. Besides this intraskeletal communication, bone cells produce factors that are capable of controlling cell types and organs elsewhere in the organism, which are now being recognized as bona fide hormones.

All contributors have explored the recent advances made in their research area. The latest progress in osteoblast/osteocyte and osteoclast biology is revisited with special focus on bone morphogenetic proteins, microRNAs and extracellular vesicles as illustrative examples of different levels of communication between cell types. In separate chapters, the interaction of osteoblasts and osteoclasts, as well as their cross-talk with endothelial cells, fat cells, immune cells, hematopoietic stem cells and different types of cancer cells is discussed extensively, further emphasizing the interactive nature of bone cells in their microenvironment. Beside cell–cell interaction, attention has been paid to the osteointegration of bone cells in a non-cellular context, including extracellular matrix and metal devices, combining main components for bone bioengineering. Finally, the endocrine role of bone is discussed in great detail by several contributors, focusing on the control of bone cell function by the brain as well as the role of bone-produced factors in, amongst others, phosphate homeostasis, energy metabolism and fertility.

The Great Beauty of the osteoclast

Alfredo Cappariello, Antonio Maurizi, Vimal Veeriah, Anna Teti
Archives of Biochemistry and Biophysics 561 (2014) 13–21
http://dx.doi.org/10.1016/j.abb.2014.08.009

Much has been written recently on osteoclast biology, but this cell type still astonishes scientists with its multifaceted functions and unique properties. The last three decades have seen a change in thinking about the osteoclast, from a cell with a single function, which just destroys the tissue it belongs to, to an ‘‘orchestrator’’ implicated in the concerted regulation of bone turnover. Osteoclasts have unique morphological features, organelle distribution and plasma membrane domain organization. They require polarization to cause extracellular bone breakdown and release of the digested bone matrix products into the circulation. Osteoclasts contribute to the control of skeletal growth and renewal. Alongside other organs, including kidney, gut, thyroid and parathyroid glands, they also affect calcemia and phosphatemia. Osteoclasts are very sensitive to pro-inflammatory stimuli, and studies in the ‘00s ascertained their tight link with the immune system, bringing about the question why bone needs a cell regulated by the immune system to remove the extracellular matrix components. Recently, osteoclasts have been demonstrated to contribute to the hematopoietic stem cell niche, controlling local calcium concentration and regulating the turnover of factors essential for hematopoietic stem cell mobilization. Finally, osteoclasts are important regulators of osteoblast activity and angiogenesis, both by releasing factors stored in the bone matrix, and secreting ‘‘clastokines’’ that regulate the activity of neighboring cells. All these facets will be discussed in this review article, with the aim of underscoring The Great Beauty of the osteoclast.

Osteoclasts: more than ‘bone eaters’

Julia F. Charles and Antonios O. Aliprantis
Trends in Molecular Medicine, Aug 2014; 20(8): 449-459
http://dx.doi.org/10.1016/j.molmed.2014.06.001

As the only cells definitively shown to degrade bone, osteoclasts are key mediators of skeletal diseases including osteoporosis. Bone-forming osteoblasts, and hematopoietic and immune system cells, each influence osteoclast formation and function, but the reciprocal impact of osteoclasts on these cells is less well appreciated. We highlight here the functions that osteoclasts perform beyond bone resorption.
First, we consider how osteoclast signals may contribute to bone formation by osteoblasts and to the pathology of bone lesions such as fibrous dysplasia and giant cell tumors.
Second, we review the interaction of osteoclasts with the hematopoietic system, including the stem cell niche and adaptive immune cells. Connections between osteoclasts and other cells in the bone microenvironment are discussed within a clinically relevant framework.

Bone is a composite tissue of protein and mineral which undergoes continual remodeling to grow, heal damage, and regulate calcium and phosphate metabolism. This remodeling process is executed by the concerted and sequential effort of bone-resorbing osteoclasts and bone-forming osteoblasts, acting in what has been termed the basic multicellular unit (BMU) (Figure 1A). Osteocytes, long-lived osteoblast-derived cells that reside within the bone matrix, monitor bone quality and stress, and coordinate remodeling through membrane-bound and secreted factors. Skeletal integrity is maintained throughout the life-span by matching bone formation and resorption, a process referred to as osteoclast:osteoblast  ‘coupling.’ Coupling is thoroughly summarized in recent excellent reviews and in Figure 1.

Coupling: how osteoclasts ‘talk back’ to cells of the osteoblast lineage Coupling of bone formation to resorption is likely achieved through multiple mechanisms, including signals that stimulate the proliferation of pre-osteoblasts, their recruitment to resorption lacunae, and their differentiation into bone-forming cells. Cellular mediators of coupling include osteoclasts, osteoblasts, osteocytes, macrophages, and T cells, which produce a variety of factors including Wnt pathway regulators, such as sclerostin, and cytokines such as oncostatin M

Osteoclasts–osteoblast interactions in the basic multicellular unit (BMU).

Osteoclasts–osteoblast interactions in the basic multicellular unit (BMU).

Osteoclasts–osteoblast interactions in the basic multicellular unit (BMU).
Cell–cell contact mechanisms may also mediate OC-OB communication. Bidirectional signaling from OC ephrins and OB Eph receptors, and reverse signaling through RANKL on OBs, have both been invoked.

Box 1. Usurping local resources: osteoclasts feed bone invaders

Liberation of growth factors embedded in bone matrix by osteoclasts may promote metastatic tumor growth in bone. Reciprocal stimulation of osteoclasts by cancer cell derived parathyroid hormone related protein (PTHrP), and other factors, could potentiate growth factor release in what has been termed the ‘vicious cycle’ ]. Xenograft experiments utilizing breast cancer cells expressing a TGFβ responsive reporter demonstrated osteolytic metastases had high TGFβ activity. Inhibition of osteoclastic bone resorption with pamidronate reduced TGFβ activity and osteolytic lesions, suggesting that matrix resorption is a relevant source of TGFβ for skeletal metastasis in vivo. Although prophylactic pamidronate treatment decreased frequency of bone metastasis, the drug did not decrease disease progression if administered after tumor cell inoculation. Thus, whether inhibiting the release of matrix growth factors by osteoclasts has a substantive effect on tumor growth is unclear. Several bisphosphonates, as well as the anti-RANKL antibody denosumab, reduce skeletal events in metastatic cancer, but data on whether they prevent bone metastasis are inconsistent.

Immunoregulation by osteoclasts. Osteoclast precursors (OCPs) and osteoclasts (OCs) inhibit CD4 and CD8 T cell proliferation via nitric oxide (NO) production in response to T cell derived interferon g (IFNg). IFNg in turn inhibits differentiation of OCPs into mature OCs. OCs also present antigen through major histocompatibility complex class I (MHCI) to skew CD8+ T cells toward an induced Treg phenotype termed OC-iTcreg. OC-iTcreg cells in turn inhibit OCP differentiation to mature OC through IFNg, interleukin 10 (IL10), and IL6.

In mouse models, we suggest that systems for the temporal deletion of conditional alleles in osteoclasts and their precursors be established. Moreover, clinical research in humans with emerging therapeutics which specifically target key regulators of bone remodeling, such as RANKL, cathepsin K, and sclerostin, could include nested translational studies that specifically address their effects on the immune system, HSCs, and tumor growth, where appropriate. In these ways, a clear picture of osteoclast biology beyond their role as ‘bone eaters’ will emerge.

Leukemia inhibitory factor: A paracrine mediator of bone metabolism

Natalie A. Sims & Rachelle W. Johnson
Growth Factors, April 2012; 30(2): 76–87
http://dx.doi.org:/10.3109/08977194.2012.656760

Leukemia inhibitory factor (LIF) is a soluble interleukin-6 family cytokine that regulates a number of physiologic functions, including normal skeletal remodeling. LIF signals through the cytokine co-receptor glycoprotein-130 in complex with its cytokine-specific receptor [LIF receptor (LIFR)] to activate signaling cascades in cells of the skeletal system, including stromal cells, chondrocytes, osteoblasts, osteocytes, adipocytes, and synovial fibroblasts. LIF action on skeletal cells is cell-type specific, and frequently dependent on the state of cell differentiation. This review describes the expression patterns of LIF and LIFR in bone, their regulation by physiological and inflammatory agents, as well as cell-specific influences of LIF on osteoblast, osteoclast, chondrocyte, and adipocyte differentiation. The actions of LIF in normal skeletal growth and maintenance, in pathological states (e.g. autocrine tumor cell signaling and growth in bone) and inflammatory conditions (e.g. arthritis) will be discussed, as well as the signaling pathways activated by LIF and their importance in bone formation and resorption.

In vivo evidence of IGF-I–estrogen crosstalk in mediating the cortical bone response to mechanical strain

Subburaman Mohan, CG Bhat, JE Wergedal and C Kesavan
Bone Research (2014) 2, 14007 http://dx.doi.org:/10.1038/boneres.2014.7

Although insulin-like growth factor-I (IGF-I) and estrogen signaling pathways have been shown to be involved in mediating the bone anabolic response to mechanical loading, it is not known whether these two signaling pathways crosstalk with each other in producing a skeletal response to mechanical loading. To test this, at 5 weeks of age, partial ovariectomy (pOVX) or a sham operation was performed on heterozygous IGF-I conditional knockout (HIGF-I KO) and control mice generated using a Cre-loxP approach. At 10 weeks of age, a 10 N axial load was applied on the right tibia of these mice for a period of 2 weeks and the left tibia was used as an internal non-non-loaded control. At the cortical site, partial estrogen loss reduced total volumetric bone mineral density (BMD) by 5% in control pOVX mice (P50.05, one-way ANOVA), but not in the H IGF-I KO pOVX mice. At the trabecular site, bone volume/total volume (BV/TV) was reduced by 5%–6% in both control pOVX (P,0.05) and H IGF-I KO pOVX (P50.05) mice. Two weeks of mechanical loading caused a 7%–8% and an 11%–13%(P,0.05 vs. non-loaded bones) increase in cortical BMD and cortical thickness (Ct.Th), respectively, in the control sham, control pOVX and H IGF-I KO sham groups. By contrast, the magnitude of cortical BMD (4%, P50.13) and Ct.Th (6%, P,0.05) responses were reduced by 50% in the H IGF-I KO pOVX mice compared to the other three groups. The interaction between genotype and estrogen deficiency on the mechanical loading-induced cortical bone response was significant (P,0.05) by two-way ANOVA. Two weeks of axial loading caused similar increases in trabecular BV/TV (13%–17%) and thickness (17%–23%) in all four groups of mice. In conclusion, partial loss of both estrogen and IGF-I significantly reduced cortical but not the trabecular bone response to mechanical loading, providing in vivo evidence of the above crosstalk in mediating the bone response to loading.

Role of FGF/FGFR signaling in skeletal development and homeostasis: learning from mouse models

Nan Su, Min Jin and Lin Chen
Bone Research (2014) 2, 14003; http://dx.doi.org:/10.1038/boneres.2014.3

Fibroblast growth factor (FGF)/fibroblast growth factor receptor (FGFR) signaling plays essential roles in bone development and diseases. Missense mutations in FGFs and FGFRs in humans can cause various congenital bone diseases, including chondrodysplasia syndromes, craniosynostosis syndromes and syndromes with dysregulated phosphate metabolism. FGF/FGFR signaling is also an important pathway involved in the maintenance of adult bone homeostasis. Multiple kinds of mouse models, mimicking human skeleton diseases caused by missense mutations in FGFs and FGFRs, have been established by knock-in/out and transgenic technologies. These genetically modified mice provide good models for studying the role of FGF/FGFR signaling in skeleton development and homeostasis. In this review, we summarize the mouse models of FGF signaling-related skeleton diseases and recent progresses regarding the molecular mechanisms, underlying the role of FGFs/FGFRs in the regulation of bone development and homeostasis. This review also provides a perspective view on future works to explore the roles of FGF signaling in skeletal development and homeostasis.

Osteoporosis in men: a review

Robert A Adler
Bone Research (2014) 2, 14001; http://dx.doi.org:/10.1038/boneres.2014.1

Osteoporosis and consequent fracture are not limited to postmenopausal women. There is increasing attention being paid to osteoporosis in older men. Men suffer osteoporotic fractures about 10 years later in life than women, but life expectancy is increasing faster in men than women. Thus, men are living long enough to fracture, and when they do the consequences are greater than in women, with men having about twice the 1-year fatality rate after hip fracture, compared to women. Men at high risk for fracture include those men who have already had a fragility fracture, men on oral glucocorticoids or those men being treated for prostate cancer with androgen deprivation therapy. Beyond these high risk men, there are many other risk factors and secondary causes of osteoporosis in men. Evaluation includes careful history and physical examination to reveal potential secondary causes, including many medications, a short list of laboratory tests, and bone mineral density testing by dual energy X-ray absorptiometry (DXA) of spine and hip. Recently, international organizations have advocated a single normative database for interpreting DXA testing in men and women. The consequences of this change need to be determined. There are several choices of therapy for osteoporosis in men, with most fracture reduction estimation based on studies in women.

From skeletal to non skeletal: The intriguing roles of BMP-9: A literature review

  1. Leblanc, G. Drouin, G. Grenier, N. Faucheux, R. Hamdy
    Advances in Bioscience and Biotechnology, 2013; 4: 31-46
    http://dx.doi.org/10.4236/abb.2013.410A4004

In the well-known superfamily of transforming growth factors beta (TGF-), bone morphogenetic proteins (BMPs) are one of the most compelling cytokines for their major role in regulation of cell growth and differentiation in both embryonic and adult tissues. This subfamily was first described for its ability of potentiating bone formation, but nowadays, the power of BMPs is well beyond the bone healing scope. Some of the BMPs have been well studied and described in the literature, but the BMP9 is still worthy of attention. It has been shown by many authors that it is the most potent osteogenic BMP. Moreover, it has been de- scribed as one of the rare circulating BMPs. In this paper, we will review the recent literature on BMP9 and the different avenues for future research in that field. Our primary scope is to review its relation to bone formation and to elaborate on the available literature on other systems.

Fong et al. recently demonstrated in vitro that rhBMP9 can also augment bone resorption. This increase was shown to be functional and not related to osteoclast formation. Furthermore, rhBMP9 could alter the intrinsic apoptosis pathway and increase survival of osteoclasts. The effect of rhBMP9 on osteoclast was explained by the presence of ALK1 and BMPRII co-receptors and their activation of the Smad 1/5/8 and non-smad MAPK/ERK pathways. These results show for the first time that BMP9 can directly affect human osteoclasts, acting on their function and their survival.

Insulin resistance is a systemic multifactorial impairment of glucose uptake. Muscle, a glucose consuming organ, needs Akt2 to be able to activate insulin-induced glucose uptake and this pathway seems to be severely impaired in insulin resistance. Interestingly, a combination of bioinformatic and high- throughput functional analyses have shown BMP9 to be the first hepatic factor to regulate blood glucose concencentration. Moreover, this effect was thought to be mediated by activation of Akt kinase in differentiated myotubes. Then, it has been demonstrated that recombinant BMP9 (1 and 5 mg/kg) improves glucose homeostasis in vivo in diabetic and non-diabetic rodents. The mechanism relied on the upregulation of Smad5 and Akt2 in differentiated rats myotubes. On the opposite side, Smad5 was downregulated in myotubes by de xamethasone, a well known hyperglycemia inducer and Smad5 knockdown in rats decreased Akt2 expression and phosphorylation leading to a decrease in insulin-induced glucose uptake by myotubes. It was then hypothesized that Smad5 regulated glucose uptake in skeletal muscle through Akt2 expression and phosphorylation. These findings also revealed Smad5 as a potential target for the treatment of type 2 diabetes. Hence, BMP9 could be seen as a potential activator of Smad5 for that purpose.

BMP9 is a major member of the TGF- superfamily that is implied in many fundamental developmental and pa- thologic processes. Future research will certainly bring answers to the many questions left open, and those an- swers will unquestionably lead to clinical applications.

Understanding Bone Loss

Max Stanley Chartrand, PhD.
DigiCare® Behavioral Research

During their lifetimes, at least half of those over age 50 will be at risk of developing osteoporosis. When we speak of bone loss we are primarily speaking of three diagnostic stages: Osteoarthritis (1-2% loss per annum), Osteopenia (3% per annum), and Osteoporosis (4-5%+ per annum) that are caused almost entirely by diet, hydration, lifestyle, medications, and environ-mental stressors.

Human bones are highly vascularized and mineralized tissues that are constantly being shaped and developed by cells called osteoblasts and torn down and resorbed by cells called osteoclasts. Recent research confirms that throughout one’s lifespan it is osteoblast activity that controls and dictates osteoclast activity as long as the body receives the nutrients it requires to maintain homeostasis. Growing children, for instance, have a far greater abundance of osteoblasts than of osteoclasts. By the time they reach young adulthood (at about age 26 for men, 22 for women) osteoclasts increase while osteoblasts slow down. Even so, humans of any age can increase osteoblast activity and slow the formation of osteoclasts through weight bearing exercise and other methods.

Long bone

Long bone

Long bone
The problem of bone shrinkage and decline in strength presents most often in health states involving:

  1. Sedentary Lifestyle, making weight bearing exercise a frontline defense against bone loss for everyone.
  2. Acidosis (low pH), from a diet that is nutritionally lacking, genetically modified, degerminated, irradiated, laden with toxins & over-processed.
  3. Chronic dehydration from too much caffeine and high fructose corn syrup (a GMO) and not enough water that is both ionized and alkalized.
  4. Lacking in calcium that is live, ionically charged, as well as phosphorus, magnesium, boron, and other minerals comprised in human bones. On the other hand, commercially available calcium causes atherosclerosis, kidney stones, bone spurs, cataracts, and yet MORE bone loss!
  5. Taking prescription medications, especially acid reflux meds, NSAIs and steroids. These and more interfere with osteoblast activity and weaken immunology. Osteoporosis meds prevent living bone mass!
  6. Unhealed injuries and deterioration of the spine, such as compression fractures (>50% of the US adult population), spinal stenosis, kyphosis, and scoliosis. These cause even more rapid loss of bone mass.
  7. Subclinical infections: tooth and gum sepsis, around artificial joints, keratosis obturans, kidney and bladder infections, neuropathies, and osteomyelitis as a result of injuries and/or shock to the bones.
  8. Heavy metal accumulations: lead, mercury, cadmium, arsenic, formaldehyde, cyanide, etc. found in the drinking water, fresh foods, cosmetics, paints, fuels, and a host of commonly used products.
  9.  Lifestyle Substances– Smoking, alcohol, excess coffee, marijuana, opium (including opiate pain killers), diet sodas, caffeine drinks.

The Kinetics of Skeletal Remodeling

Jan 1, 1966  by Lent C. Johnson
Semin Musculoskelet Radiol. 2000;4(1):1-15.

Bone tumor dynamics: an orthopedic pathology perspective.
Johnson LC1, Vinh TN, Sweet DE.

The diagnosis and classification of primary bone tumors remains as much a challenge today as it has for the last 80 plus years. Although pathology is invariably equated with the image of a diagnostic microscope, the vast majority of diagnoses are made grossly with the unaided eye, as are the tissue specimens selected for microscopic “confirmation.” Radiologic studies, particularly plain radiographs, remain the gold standard in gross pathologic diagnosis of the skeleton. Today, confirmation and final classification continue as the pathologist’s domain, but perhaps not for long, considering the evolving ancillary imaging techniques and progressive sophistication of magnetic resonance (MR) imaging. The bone tumor cases collected and compiled by Ernest Codman, M.D. during the second through fourth decades of this century formed the basis of the first tumor registry. The Codman Bone Sarcoma Registry demonstrated among other things the importance of radiographic/pathologic correlation, underscoring the reliability of a bone tumor’s location, margin (host bone/tumor interface), periosteal reaction, and matrix patterns as an accurate guide to classification and likely future biologic behavior. “A General Theory of Bone Tumors,” written by Lent C. Johnson nearly 50 years ago and published in the Bulletin of The New York Academy of Medicine (February 1953, second series, vol. 29, no. 2, pp. 164-171), provided a conceptual cellular approach to the understanding bone tumor dynamics reinforcing radiologic/pathologic correlation as a reliable diagnostic tool. At the time of Dr. Lent C. Johnson’s death (1910-1998), he was literally working on an updated version of his original article, the latter of which is being reprinted as the core of this illustrated revision. Our continued experience with bone tumors over the past five decades has only served to validate, on a daily basis, the fundamental principles outlined in Johnson’s original article. In like fashion, it is important to keep in mind that terminology and nomenclature has also evolved since 1953, despite a continued inability to achieve complete consensus.
PMID:  11061688    http://www.ncbi.nlm.nih.gov/pubmed/11061688

Interactions between adrenal-regulatory and calcium-regulatory hormones in human health

Brown, J.M., Vaidya, A.

Curr Opinion in Endocr, Diabetes and Obesity 2014; 21 (3), pp. 193-201

Purpose of review: To summarize the evidence characterizing the interactions between adrenal-regulating and calcium-regulating hormones, and the relevance of these interactions to human cardiovascular and skeletal health. Recent findings: Human studies support the regulation of parathyroid hormone (PTH) by the renin-angiotensin-aldosterone system (RAAS): angiotensin II may stimulate PTH secretion via an acute and direct mechanism, whereas aldosterone may exert a chronic stimulation of PTH secretion.
Studies in primary aldosteronism, congestive heart failure, and chronic
kidney disease have identified associations between hyperaldosteronism, hyperparathyroidism, and bone loss, which appear to improve when
inhibiting the RAAS. Conversely, elevated PTH and insufficient vitamin D
status have been associated with adverse cardiovascular outcomes, which
may be mediated by the RAAS. Studies of primary hyperparathyroidism implicate PTH-mediated stimulation of the RAAS, and recent evidence shows that the vitamin D-vitamin D receptor complex may negatively regulate renin expression and RAAS activity. Ongoing human interventional studies are evaluating the influence of RAAS inhibition on PTH and the influence of vitamin D receptor agonists on RAAS activity. Summary: Although previously considered independent endocrine systems, emerging evidence supports a complex web of interactions between adrenal-regulating and calcium-regulating hormones, with implications for human cardiovascular and
skeletal health.

Backbone modification of a polypeptide drug alters duration of action in vivo

Cheloha, R.W., Maeda, A., Dean, T., Gardella, T.J., Gellman, S.H.

Nature Biotechnology 2014; 32 (7), pp. 653-655 http://dx.doi.org/doi:10.1038/nbt.2920

Systematic modification of the backbone of bioactive polypeptides through amino acid residue incorporation could provide a strategy for generating molecules with improved drug properties, but such alterations can result in lower receptor affinity and potency. Using an agonist of parathyroid hormone receptor-1 (PTHR1), a G protein-coupled receptor in the B-family, we present an approach for residue replacement that enables both high activity and improved pharmacokinetic properties in vivo.

Mouse and human BAC transgenes recapitulate tissue-specific expression
of the vitamin D receptor in mice and rescue the VDR-null phenotype

Lee, S.M., Bishop, K.A., Goellner, J.J., O’Brien, C.A., Pike, J.W.
Endocrinology 2014; 155 (6), pp. 2064-2076
http://dx.doi.org/10.1210/en.2014-1107

The biological actions of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) are mediated by the vitamin D receptor (VDR), which is expressed in numerous target tissues in a cell type-selective manner. Recent studies using genomic analyses and recombined bacterial artificial chromosomes (BACs) have defined the specific features of mouse and human VDR gene loci in vitro. In the current study, we introduced recombined mouse and human VDR BACs as transgenes into mice and explored their expression capabilities in vivo. Individual transgenic mouse strains selectively expressed BAC derived mouse or human VDR proteins in appropriate vitamin D target tissues, thereby recapitulating the tissue-specific expression of endogenous mouse VDR. The mouse VDR transgene was also regulated by 1,25(OH)2D3 and dibutyryl-cAMP. When crossed into a VDR-null mouse background, both transgenes restored wild-type basal as well as 1,25(OH)2D3-inducible gene expression patterns in the appropriate tissues. This maneuver resulted in the complete rescue of the aberrant phenotype noted in the VDR-null mouse, including systemic features associated with altered calcium and phosphorus homeostasis and disrupted production of parathyroid hormone and fibroblast growth factor 23, and abnormalities associated with the skeleton, kidney, parathyroid gland, and the skin. This study suggests that both mouse and human VDR transgenes are capable of recapitulating basal and regulated expression of the VDR in the appropriate mouse tissues and restore 1,25(OH)2D 3 function. These results provide a baseline for further dissection of mechanisms integral to mouse and human VDR gene expression and offer the potential to explore the consequence of selective mutations in VDR proteins in vivo.

The sclerostin-independent bone anabolic activity of intermittent PTH treatment is mediated by T-cell-produced Wnt10β

Li, J.-Y., Walker, L.D., Tyagi, A.M., (…), Neale Weitzmann, M., Pacifici, R
Journal of Bone and Mineral Research 2014; 29 (1), pp. 43-54
http://onlinelibrary.wiley.com/doi/10.1002/jbmr.2044/pdf

Both blunted osteocytic production of the Wnt inhibitor sclerostin (Scl) and increased T-cell production of the Wnt ligand Wnt10b contribute to the bone anabolic activity of intermittent parathyroid hormone (iPTH) treatment. However, the relative contribution of these mechanisms is unknown. In this study, we modeled the repressive effects of iPTH on Scl production in mice by treatment with a neutralizing anti-Scl antibody (Scl-Ab) to determine the contribution of T-cell-produced Wnt10b to the Scl-independent modalities of action of iPTH. We report that combined treatment with Scl-Ab and iPTH was more potent than either iPTH or Scl-Ab alone in increasing stromal cell production of OPG, osteoblastogenesis, osteoblast life span, bone turnover, bone mineral density, and trabecular bone volume and structure in mice with T cells capable of producing Wnt10b. In T-cell-null mice and mice lacking T-cell production of Wnt10b, combined treatment increased bone turnover significantly more than iPTH or Scl-Ab alone. However, in these mice, combined treatment with Scl-Ab and iPTH was equally effective as Scl-Ab alone in increasing the osteoblastic pool, bone volume, density, and structure. These findings demonstrate that the Scl-independent activity of iPTH on osteoblasts and bone mass is mediated by T-cell-produced Wnt10b. The data provide a proof of concept of a more potent therapeutic effect of combined treatment with iPTH and Scl-Ab than either alone.

N-cadherin restrains PTH activation of Lrp6/β-catenin signaling and osteoanabolic action

Revollo, L., Kading, J., Jeong, S.Y., (…), Mbalaviele, G., Civitelli, R.
Journal of Bone and Mineral Research 2015; 30 (2), pp. 274-28

Interaction between parathyroid hormone/parathyroid hormone-related peptide receptor 1 (PTHR1) and low-density lipoprotein receptor-related protein 6 (Lrp6) is important for parathyroid hormone (PTH) signaling and anabolic action. Because N-cadherin has been shown to negatively regulate canonical Wnt/β-catenin signaling, we asked whether N-cadherin alters PTH signaling and stimulation of bone formation. Ablation of the N-cadherin gene (Cdh2) in primary osteogenic lineage cells resulted in increased Lrp6/PTHR1 interaction in response to PTH1-34, associated with enhanced PTH-induced PKA signaling and PKA-dependent β-catenin C-terminus phosphorylation, which promotes β-catenin transcriptional activity. β-catenin C-terminus phosphorylation was abolished by Lrp6 knockdown. Accordingly, PTH1-34 stimulation of Tcf/Lef target genes, Lef1 and Axin2, was also significantly enhanced in Cdh2-deficient cells. This enhanced responsiveness to PTH extends to the osteo-anabolic effect of PTH, as mice with a conditional Cdh2 deletion in Osx+ cells treated with intermittent doses of PTH1-34 exhibited significantly larger gains in trabecular bone mass relative to control mice, the result of accentuated osteoblast activity. Therefore, N-cadherin modulates Lrp6/PTHR1 interaction, restraining the intensity of PTH-induced β-catenin signaling, and ultimately influencing bone formation in response to intermittent PTH administration.

EphrinB2 signaling in osteoblasts promotes bone mineralization by preventing apoptosis

Tonna, S., Takyar, F.M., Vrahnas, C., (…), Martin, T.J., Sims, N.A.
FASEB Journal 2014; 28 (10), pp. 4482-4496 10.1096/fj.14-254300

Cells that form bone (osteoblasts) express both ephrinB2 and EphB4, and previous work has shown that pharmacological inhibition of the ephrinB2/ EphB4 interaction impairs osteoblast differentiation in vitro and in vivo. The purpose of this study was to determine the role of ephrinB2 signaling in the osteoblast lineage in the process of bone formation. Cultured osteoblasts from mice with osteoblast-specific ablation of ephrinB2 showed delayed expression of osteoblast differentiation markers, a finding that was reproduced by ephrinB2, but not EphB4, RNA interference. Microcomputed tomography, histomorphometry, and mechanical testing of the mice lacking ephrinB2 in osteoblasts revealed a 2-fold delay in bone mineralization, a significant reduction in bone stiffness, and a 50% reduction in osteoblast differentiation induced by anabolic parathyroid hormone (PTH) treatment, compared to littermate sex- and age-matched controls. These defects were associated with significantly lower mRNA levels of late osteoblast differentiation markers and greater levels of osteoblast and osteocyte apoptosis, indicated by TUNEL staining and transmission electron microscopy of bone samples, and a 2-fold increase in annexin V staining and 7-fold increase in caspase 8 activation in cultured ephrinB2 deficient osteoblasts. We conclude that osteoblast differentiation and bone strength are maintained by antiapoptotic actions of ephrinB2 signaling within the osteoblast lineage.-
Bone involvement in primary hyperparathyroidism and changes after parathyroidectomy

Rolighed, L., Rejnmark, L., Christiansen, P.
European Endocrinology 2014; 10 (1), pp. 84-87

Parathyroid hormone (PTH) is produced and secreted by the parathyroid glands and has primary effects on kidney and bone. During the pathological growth of one or more parathyroid glands, the plasma level of PTH increases and causes primary hyperparathyroidism (PHPT). This disease is normally characterized by hyperparathyroid hypercalcemia. In PHPT a continuously elevated PTH stimulates
the kidney and bone causing a condition with high bone turnover, elevated plasma calcium and increased fracture risk. If bone resorption is not followed by a balanced formation of new bone, irreversible bone loss may occur in these patients. Medical treatment can help to minimize the loss of bone but the cure of PHPT is by parathyroidectomy. After operation, bone mineral density increases during the return to normal bone metabolism. Supplementation with calcium and vitamin D after operation may improve the normalization to normal bone metabolism with a secondary reduction in fracture risk.

Primary hyperparathyroidism and the skeleton

Mosekilde, L.
Clinical Endocrinology 2008; 69 (1), pp. 1-19
http://dx.doi.org:/10.1111/j.1365-2265.2007.03162.x

Today, primary hyperparathyroidism (PHPT) in the developed countries is typically a disease with few or no obvious clinical symptoms. However, even in the asymptomatic cases the endogenous excess of PTH increases bone turnover leading to an insidious reversible loss of cortical and trabecular bone because of an expansion of the remodelling space and an irreversible loss of cortical bone due to increased endocortical resorption. In contrast trabecular bone structure and integrity to a large extent is maintained and there may be a slight periosteal expansion. Most studies have reported decreased bone mineral density (BMD) in PHPT mainly located at cortical sites, whereas sites rich in trabecular bone only show a modest reduction or even a slight increase in BMD. The frequent occurrence of vitamin D insufficiency and deficiency in PHPT and increased plasma FGF23 levels may also contribute to the decrease in BMD. The effect of smoking is unsolved. Epidemiological studies have shown that the relative risk of spine and nonspine fractures is increased in untreated PHPT starting up to 10 years before the diagnosis is made. Successful surgery for PHPT normalizes bone turnover, increases BMD and decreases fracture risk based on larger epidemiological studies. However, 10 years after surgery fracture risk appears to increase again due to an increase in forearm fractures. There are no randomized controlled studies (RCTs) demonstrating a protective effect of medical treatment on fracture risk in PHPT. Less conclusive studies suggest that vitamin D supplementation may have a beneficial effect on plasma PTH and BMD in vitamin D deficient PHPT patients. Hormone replacement therapy (HRT) and maybe SERM appear to reduce bone turnover and increase BMD. However, their nonskeletal side-effects preclude their use for this purpose. Bisphosphonates reduce bone turnover and increase BMD in PHPT as in osteoporosis and may be a therapeutical option in selected patients with low BMD. Obviously, there is a need for larger RCTs with fractures as end-points that appraise this possibility. Calcimimetics reduce plasma calcium and PTH in PHPT but has no beneficial effect on bone turnover or BMD. In symptomatic hypercalcemic PHPT with low BMD where curative surgery is impossible or contraindicated a combination of a calcimimetic and a bisphosphonate may be an undocumented therapeutical option that needs further evaluation.

Current Issues in the Presentation of Asymptomatic Primary Hyperparathyroidism: Proceedings of the Fourth International Workshop

Shonni J. Silverberg, Bart L. Clarke, Munro Peacock, Francisco Bandeira, et al. The Journal of Clinical Endocrinology & Metabolism 2014; 99(10) http://dx.doi.org/10.1210/jc.2014-1415

Objective: This report summarizes data on traditional and nontraditional manifestations of primary hyperparathyroidism (PHPT) that have been published since the last International Workshop on PHPT.

Participants: This subgroup was constituted by the Steering Committee to address key questions related to the presentation of PHPT. Consensus was established at a closed meeting of the Expert Panel that followed.

Evidence: Data from the 5-year period between 2008 and 2013 were
presented and discussed to determine whether they support changes in recommendations for surgery or nonsurgical follow-up.

Consensus Process: Questions were developed by the International Task
Force on PHPT. A comprehensive literature search for relevant studies was undertaken. After extensive review and discussion, the subgroup came to agreement on what changes in the recommendations for surgery or nonsurgical follow-up of asymptomatic PHPT should be made to the Expert Panel.

Conclusions:

1) There are limited new data available on the natural history of
asymptomatic PHPT. Although recognition of normocalcemic PHPT
(normal serum calcium with elevated PTH concentrations; no secondary
cause for hyperparathyroidism) is increasing, data on the clinical
presentation and natural history of this phenotype are limited.
2) Although there are geographic differences in the predominant
phenotypes of PHPT (symptomatic, asymptomatic, normocalcemic),
they do not justify geography-specific management guidelines.
3) Recent data using newer, higher resolution imaging and analytic
methods have revealed that in asymptomatic PHPT, both trabecular
bone and cortical bone are affected.
4) Clinically silent nephrolithiasis and nephrocalcinosis can be detected
by renal imaging and should be listed as a new criterion for surgery.
5) Current data do not support a cardiovascular evaluation or surgery
for the purpose of improving cardiovascular markers, anatomical or
functional abnormalities.
6) Some patients with mild PHPT have neuropsychological complaints
and cognitive abnormalities, and some of these patients may benefit
from surgical intervention. However, it is not possible at this time to
predict which patients with neuropsychological complaints or cognitive
issues will improve after successful parathyroid surgery.

Sclerosing Bone Dysplasias: Leads Toward Novel Osteoporosis Treatments

Igor Fijalkowski, Eveline Boudin, Geert Mortier, Wim Van Hul
Current Osteoporosis Reports Sept 2014; 12(3), pp 243-251
http://dx.doi.org:/10.1007/s11914-014-0220-5

Sclerosing bone dysplasias are a group of rare, monogenic disorders characterized by increased bone density resulting from the disturbance in the fragile equilibrium between bone formation and resorption. Over the last decade, major contributions have been made toward better understanding of the pathogenesis of these conditions. These studies provided us with important insights into the bone biology and yielded the identification of numerous drug targets for the prevention and treatment of osteoporosis. Here, we review this heterogeneous group of disorders focusing on their utility in the development of novel osteoporosis therapies.

Clinical development of neridronate: potential for new applications

Gatti D, Rossini M, Viapiana O, Idolazzi L, Adami S
Ther & Clin Risk Manag Apr 2013; 2013(9): Pages 139—147

Neridronate is an aminobisphosphonate, licensed in Italy for the treatment
of osteogenesis imperfecta (OI) and Paget’s disease of bone (PDB).  A characteristic property of neridronate is that it can be administered both intravenously and intramuscularly, providing a useful system for administration in homecare. In this review, we discuss the latest clinical results of neridronate administration in OI and PDB, as well as in osteoporosis and other conditions. We will focus in particular on the latest evidence of the effect of neridronate on treatment of complex regional pain syndrome type I.

Disorders of bone remodeling

Feng, X., McDonald, J.M.
Ann Rev of Pathol: Mechanisms of Disease 2011; 6, pp. 121-145
http://dx.doi.org:/10.1146/annurev-pathol-011110-130203

The skeleton provides mechanical support for stature and locomotion, protects vital organs, and controls mineral homeostasis. A healthy skeleton must be maintained by constant bone modeling to carry out these crucial functions throughout life. Bone remodeling involves the removal of old or damaged bone by osteoclasts (bone resorption) and the subsequent replacement of new bone formed by osteoblasts (bone formation). Normal bone remodeling requires a tight coupling of bone resorption to bone formation to guarantee no alteration in bone mass or quality after each remodeling cycle. However, this important physiological process can be derailed by a variety of factors, including menopause-associated hormonal changes, age-related factors, changes in physical activity, drugs, and secondary diseases, which lead to the development of various bone disorders in both women and men. We review the major diseases of bone remodeling, emphasizing our current understanding of the underlying pathophysiological mechanisms.

Paget’s disease and hypercalcemia: Coincidence or causal relationship?

Green, I., Altman, A.
Harefuah 2009; 148 (10), pp. 708-710

Paget’s disease is a chronic disease in which osteoclast mediated bone resorption precedes imperfect osteoblast mediated bone repair. Symptoms include bone pain, pathological fractures, osteoarthritis and neurological symptoms. There is evidence that genetic and viral component are involved in the etiology. Hypercalcemia is rare and when it is diagnosed, primary hyperparathyroidism should be ruled out. The authors present a patient with Paget’s disease and concomitant hypercalcemia. Evaluation for hypercalcemia revealed an adenoma of the parathyroid. However, despite the removal of the adenoma, the symptoms persisted. Previous studies
showed that hyperparathyroidism causes hypercalcemia in Paget’s disease patients. Removal of the adenoma led to improvement in calcium and alkaline phosphatase (ALP) levels but clinical improvement is seen only in patients with high calcium level prior to the operation. This leads to the assumption that symptoms of Paget’s disease are due to osteoclast hypersensitivity to parathyroid hormone (PTH) and by removing the adenoma the osteoclast activity is also reduced. In summary, the most common cause of hypercalcemia in Paget’s disease patients is hyperparathyroidism and adenectomy may improve the biochemical and sometimes also the clinical symptoms of Paget’s disease.

Signaling networks that control the lineage commitment and differentiation of bone cells

Soltanoff, C.S., Yang, S., Chen, W., Li, Y.-P.
Critical Reviews in Eukaryotic Gene Expression 2009; 19 (1), pp. 1-46

Osteoblasts and osteoclasts are the two major bone cells involved in the bone remodeling process. Osteoblasts are responsible for bone formation while osteoclasts are the bone-resorbing cells. The major event that triggers osteogenesis and bone remodeling is the transition of mesenchymal stem cells into differentiating osteoblast cells and monocyte/macrophage precursors into differentiating osteoclasts. Imbalance in differentiation and function of these two cell types will result in skeletal diseases such as osteoporosis, Paget’s disease, rheumatoid arthritis, osteopetrosis, periodontal disease, and bone cancer metastases.
Osteoblast and osteoclast commitment and differentiation are controlled by complex activities involving signal transduction and transcriptional regulation of gene expression. Recent advances in molecular and genetic studies using gene targeting in mice enable a better understanding of the multiple factors and signaling networks that control the differentiation process at a molecular level.
This review summarizes recent advances in studies of signaling transduction pathways and transcriptional regulation of osteoblast and osteoclast cell lineage commitment and differentiation. Understanding the signaling networks that control the commitment and differentiation of bone cells will not only expand our basic understanding of the molecular mechanisms of skeletal development but will also aid our ability to develop therapeutic means of intervention in skeletal diseases.

Salmon calcitonin: a review of current and future therapeutic indications

  1. H. Chesnut III, M. Azria, S. Silverman, M. Engelhardt, M. Olson, L. Mindeholm Osteoporosis International 2008; 19(4), pp 479-491
    http://dx.doi.org:/10.1007/s00198-007-0490-1

Salmon calcitonin, available as a therapeutic agent for more than 30 years, demonstrates clinical utility in the treatment of such metabolic bone diseases as osteoporosis and Paget’s disease, and potentially in the treatment of osteoarthritis. This review considers the physiology and pharmacology of salmon calcitonin, the evidence based research demonstrating efficacy and safety of this medication in postmenopausal osteoporosis with potentially an effect on bone quality to explain its abilities to reduce the risk of spine fracture, the development of an oral salmon calcitonin preparation, and the therapeutic rationale for this preparation’s chondroprotective effect in osteoarthritis.

Pharmacotherapies to Manage Bone Loss-Associated Diseases:  A Quest for the Perfect Benefit-to-Risk Ratio

Valverde

Current Medicinal Chemistry : 15 (3): Pages 284-304
http://dx.doi.org:/10.2174/092986708783497274

In this review, benefits and side-effects of current and emerging therapies to treat and prevent pathological bone loss are described. Bisphosphonates are the antiresorptive compounds most widely used in the treatment of bone-loss associated diseases. They are generally well-tolerated although have recently been associated with osteonecrosis of the jaw and other complications. Therapies modulating estrogen receptor activation are indicated in the prevention and treatment of either breast cancer or osteoporosis in postmenopausal women. Thus, hormone replacement therapy is effective in prevention of osteoporosis, but its long-term use can increase the risk of breast cancer, stroke and embolism. Tamoxifen benefits all stages of breast cancer, but its use may lead to uterine cancer and thromboembolism. Raloxifene is approved in prevention of breast cancer and treatment of postmenopausal osteoporosis, but its use can increase the risk of fatal stroke. Aromatase inhibitors are superior to tamoxifen at advanced stages of disease and as adjuvants, but their use increase fracture incidence. Fulvestrant is as effective as aromatase inhibitors in the treatment of advanced breast cancer and does not cause bone fractures. Another antiresorptive available for the treatment of postmenopausal osteoporosis, Pagets disease and hypercalcemia is calcitonin, which also exhibits analgesic effects. A promising antiresorptive agent currently in clinical trials is denosumab. Aditional therapies for osteoporosis that decrease fracture risk consist of PTH-like anabolic agents and the dual action bone agent strontium ranelate. Antiseptics and antibiotics are used extensively in periodontal disease intervention to target bacterial biofilm, although hostdirected therapies are also being developed. – See more at: http://www.eurekaselect.com/66301/article#sthash.EGNCH4Eu.dpuf

Parathyroid Hormone An Anabolic Treatment for Osteoporosis

Paul Morley, James F. Whitfield and Gordon E. Willick
Current Pharmaceutical Design Pages 671-687
http://dx.doi.org:/10.2174/1381612013397780

Osteoporosis is a disease characterised by low bone mass, structural deterioration of bone and increased risk of fracture. The prevalence, and cost, of osteoporosis is increasing dramatically with our ageing population and the World Health Organization now considers it to be the second-leading healthcare problem. All currently approved therapies for osteoporosis (eg., estrogen, bisphosphonates, calcitonin and selective estrogen receptor modulators) are anti-resorptive agents that act on osteoclasts to prevent further bone loss. A new class of bone anabolic agent capable of building mechanically strong new bone in patients with established osteoporosis is
in development. While the parathyroid hormone (PTH) is classically considered to be a bone catabolic agent, when delivered intermittently at low doses PTH potently stimulates cortical and trabecular bone growth in animals humans. The native hPTH-(1-84) and its osteogenic fragment, hPTH-(1-34), have already entered Phase III clinical trials. Understanding the mechanism of PTHs osteogenic actions has led to the development of smaller PTH analogues which can also build mechanically normal bone in osteopenic rats. These new PTH analogues are promising candidates for treating osteoporosis in humans as they are as efficacious as hPTH-(1-84) and hPTH-(1-34), but there is evidence that they may have considerably less ability to induce hypercalcemia, the major side effect of PTH therapy. In addition to treating osteoporosis, PTHs may be used to promote fracture healing, to restore bone loss in immobilized patients, or following excessive glucocorticoid or prolonged spaceflight, and to treat psoriasis. http://www.eurekaselect.com/65008/article#sthash.FWa67NrB.dpuf

Effects of Parathyroid Hormone on Cancellous Bone Mass and Structure in Osteoporosis

Naohisa Miyakoshi
Current Pharmaceutical Design  ;10(21): Pages 2615-2627
http://dx.doi.org:/10.2174/1381612043383737

Parathyroid hormone (PTH) is the major hormonal regulator of calcium homeostasis. PTH is a potent stimulator of bone formation and can restore bone to an osteopenic skeleton, when administered intermittently. Osteoblasts are the primary target cells for the anabolic effects of PTH in bone tissue. Anabolic effects of PTH on bone have been demonstrated in animals and humans, by numerous measurement techniques including bone mineral density and bone histomorphometry. Clinically, the most important aspect of treatment for osteoporosis is prevention of fractures. Microstructural alterations, such as loss of trabecular connectivity, have been implicated in increased propensity for fracture. Recent two-dimensional (2D) and three-dimensional (3D) assessments of cancellous bone structure have shown that PTH can re-establish lost trabecular connectivity in animals and humans.
These results provide new insight into the positive clinical effects of PTH in osteoporosis. In recent randomized controlled clinical trials of intermittent
PTH treatment, PTH decreased incidence of vertebral and non-vertebral fractures
in postmenopausal women. Thus, PTH shows strong potential as therapy for osteoporosis. However, 2D and 3D structural analysis of advanced osteopenia in animals has shown that there is a critical limit of trabecular connectivity and bone strength below which PTH cannot completely reverse the condition. Given that PTH treatment fails to completely restore trabecular connectivity and bone strength in animals with advanced osteopenia, early treatment of osteoporosis appears important and efficacious for preventing fractures caused by decreased bone strength resulting from decreased trabecular connectivity. – See more at: http://www.eurekaselect.com/62780/article#sthash.OnoaRPyh.dpuf

Clinical applications of RANK-ligand inhibition

Romas, E.
Internal Medicine Journal 2009; 39 (2), pp. 110-116
http://dx.doi.org:/10.1111/j.1445-5994.2008.01732.x

An enhanced rate of bone remodelling fuelled by osteoclastogenesis mediates diseases such as osteoporosis, arthritic bone destruction, Paget’s disease and malignancy-induced bone loss. Thus, the control of osteoclastogenesis is of major clinical importance. The receptor activator of nuclear factor κB (RANK); its ligand, RANKL and decoy receptor, osteoprotegerin, are critical determinants of osteoclastogenesis, and increased RANK signalling is involved in several bone diseases, providing the rationale for RANKL inhibition. The effects of RANKL inhibition are being witnessed in clinical trials of neutralizing fully human monoclonal antibodies that target RANKL (e.g. denosumab) and which induce profound and sustained inhibition of bone resorption. The relative efficacy, cost-effectiveness and side-effects of targeted RANKL inhibition compared with conventional antiresorptive drugs (i.e. bisphosphonates) should be resolved by clinical trials in coming years.

Clinical development of neridronate: potential for new applications

Davide Gatti, M Rossini, O Viapiana, L Idolazzi, SAdami
Therapeutics and Clinical Risk Management 2013:9 139–147
http://dx.doi.org/10.2147/TCRM.S35788

Neridronate is an aminobisphosphonate, licensed in Italy for the treatment of osteogenesis imperfecta (OI) and Paget’s disease of bone (PDB). A characteristic property of neridronate is that it can be administered both intravenously and intramuscularly, providing a useful system for administration in homecare. In this review, we discuss the latest clinical results of neridronate administration in OI and PDB, as well as in osteoporosis and other conditions. We will focus in particular on the latest evidence of the effect of neridronate on treatment of complex regional pain syndrome type I.

The Sclerostin‐Independent Bone Anabolic Activity of Intermittent PTH Treatment Is Mediated by T‐Cell–Produced Wnt10β

Jau‐Yi Li, Lindsey D Walker, Abdul Malik Tyagi, Jonathan Adams, et al.
Journal of Bone and Mineral Research, Jan 2014; 29(1): pp 43–54
http://dx.doi.org:/10.1002/jbmr.2044

Both blunted osteocytic production of the Wnt inhibitor sclerostin (Scl) and increased T‐cell production of the Wnt ligand Wnt10β contribute to the bone anabolic activity of intermittent parathyroid hormone (iPTH) treatment. However, the relative contribution of these mechanisms is unknown. In this study, we modeled the repressive effects of iPTH on Scl production in mice by treatment with a neutralizing anti‐Scl antibody (Scl‐Ab) to determine the contribution of T‐cell–produced Wnt10β to the Scl‐independent modalities of action of iPTH. We report that combined treatment with Scl‐Ab and iPTH was more potent than either iPTH or Scl‐Ab alone in increasing stromal cell production of OPG, osteoblastogenesis, osteoblast life span, bone turnover, bone mineral density, and trabecular bone volume and structure in mice with T cells capable of producing Wnt10β. In T‐cell–null mice and mice lacking T‐cell production of Wnt10β, combined treatment increased bone turnover significantly more than iPTH or Scl‐Ab alone. However, in these mice, combined treatment with Scl‐Ab and iPTH was equally effective as Scl‐Ab alone in increasing the osteoblastic pool, bone volume, density, and structure. These findings demonstrate that the Scl‐independent activity of iPTH on osteoblasts and bone mass is mediated by T‐cell–produced Wnt10β. The data provide a proof of concept of a more potent therapeutic effect of combined treatment with iPTH and Scl‐Ab than either alone.

Treatment of Paget’s disease with hypercalcemia

Donald H. Gutteridge – Letter to the Editor
Bone 12 Jan 2006; 39(668)
http://dx.doi.org:/10.1016/j.bone.2006.01.165

Selby et al. [7] “Guidelines on the management of Paget’s disease of bone” produced a very helpful review, with 139 references. I take issue however with their approach to the clinical problem of concurrent Paget’s and hypercalcemia.
Firstly, the combination is not rare. Of 1836 literature and personally reported unselected patients with Paget’s disease, 90 had concurrent hypercalcemia due to primary hyperparathyroidism [PHPT], i.e., 4.9% [4]. The number with unspecified hypercalcemia would have exceeded 5%.                                     Secondly, the authors give similar weight to immobilization and PHPT as causes. Immobilization as a cause of hypercalcemia in Paget’s disease is rare [4,3]. The former paper studied 184 consecutive new referrals with Paget’s disease over 15 years. Hypercalcemia was present in 21: two had malignancy (multiple myeloma, secondary cancer); the remaining 19 had biochemical PHPT with most confirmed by neck exploration; none had hypercalcemia of immobilization. Gillespie [3] reported two patients who died following pagetic fractures with immobilization. One was diagnosed and treated as immobilization hypercalcemia; both had large parathyroid adenomas at autopsy.
Thirdly, they have recommended that “patients with Paget’s disease and hypercalcemia should be treated with bisphosphonate”. Since most patients with this combination have PHPT, since bisphosphonate treatment of Paget’s disease is associated with parathyroid hormone (PTH) stimulation [5] and since activation of Paget’s disease occurs with increased PTH [2], it seems reasonable to exclude PHPT (and other causes— e.g., milk alkali syndrome and vitamin D toxicity) and consider neck exploration before bisphosphonate treatment. The response to parathyroidectomy can be profound—and is predictable. In those with PHPT there is a significant linear relationship between preoperative severity (plasma calcium corrected for plasma albumin) and postoperative improvement in bone turnover (%fall in plasma alkaline phosphatase) [4]. In those 7 patients with a preoperative calcium >3.0 mmol/l, the postoperative mean fall in plasma alkaline phosphatase was 68%. Bisphosphonate treatment may be an option in those with PHPT and mild asymptomatic hypercalcemia; likewise following a reasonable interval (say 6 months) after successful neck exploration, should increased bone turnover and pagetic symptoms persist.

In those rare cases with the combination of Paget’s disease, hypercalcemia and immobilized pagetic fracture, where other causes of hypercalcemia have been excluded [1,6], bisphosphonate treatment is eminently reasonable.

[1] Bannister P, Roberts M, Sheridan P. Recurrent hypercalcaemia in a young man with mono-ostotic Paget’s disease. Postgrad Med J 1986;62:481–3.
[2] Genuth SM, Klein L. Hypoparathyroidism and Paget’s disease: the effect of parathyroid hormone administration. J Clin Endocrinol Metab 1972;35: 693–9.
[3] Gillespie WJ. Hypercalcaemia in Paget’s disease of bone. Aust N Z J Surg 1979;49:84–6.
[4] Gutteridge DH, Gruber HE, Kermode DG, Worth GK. Thirty cases of concurrent Paget’s disease and primary hyperparathyroidism: sex distribution, histomorphometry, and prediction of the skeletal response to parathyroidectomy. Calcif Tissue Int 1999;65:427–35.
[5] Harinck HIJ, Bijvoet OLM, Blanksma HJ, Dahlinghaus-Nienhuys PJ. Efficacious management with aminobisphosphonate (APD) in Paget’s disease of bone. Clin Orthop Relat Res 1987;217:79–98.
[6] Nathan AW, Ludlam HA, Wilson DW, Dandona P. Hypercalcaemia due to immobilization of a patient with Paget’s disease of bone. Postgrad Med J 1982;58:714–5.
[7] Selby PL, Davie MWJ, Ralston SH, Stone MD. Guidelines on the management of Paget’s disease of bone. Bone 2002;31:10–9.

The authors of the article entitled “Guidelines on the management of Paget’s disease of bone” published in BONE 2002:31:10–9, have elected not to respond to the above letter to the Editor.

Safety of Bisphosphonates in the Treatment of Osteoporosis

Robert R. Recker, E. Michael Lewiecki, Paul D. Miller, James Reiffel
The American Journal of Medicine (2009) 122, S22–S32
http://dx.doi.org:/10.1016/j.amjmed.2008.12.004

In this review 4 experts consider the major safety concerns relating to bisphosphonate therapy for osteoporosis. Specific topics covered are skeletal safety (particularly with respect to atypical fractures and delayed healing), gastrointestinal intolerance, hypocalcemia, acute-phase (i.e., postdose) reactions, chronic musculoskeletal pain, renal safety, and cardiovascular safety (specifically, atrial fibrillation).

The bone-remodeling cycle

The bone-remodeling cycle

The bone-remodeling cycle.
Remodeling of bone in a multicellular bone unit starts with osteoblastic activation of osteoclast differentiation, fusion, and activation (A and B).
When resorption lacunae are formed, the osteoclasts leave the area and mononucleated cells of uncertain origin appear and “clean up” the organic matrix remnants left by the osteoclast, also possibly forming the cement line (dotted line) at the bottom of the lacunae
(C). During the resorption process, coupling factors, including insulin-like growth factor–I and transforming growth factor–β, are released from the bone-extracellular matrix, and these growth factors contribute to the recruitment of osteoblasts to the resorption lacunae and their activation.
(D). The osteoblasts will then fill the lacunae with new bone; when the same amount of bone is formed as is being resorbed, the remodeling process is finished, and the mineralized extracellular matrix will be covered by osteoid and a single-cell layer of osteoblasts
(E). (Reprinted with permission from J Dent Res.6)

SUMMARY

Persistent, long-term antifracture efficacy has been demonstrated for bisphosphonates, and there is no evidence that the antifracture efficacy declines during treatment periods lasting as long as 10 years. Bisphosphonate-induced oversuppression of remodeling and return of fracturing remains a theoretical possibility.
It is likely that a few patients who are potential candidates for bisphosphonate treatment have preexisting oversuppression of bone remodeling. Treatment with a bisphosphonate in these cases would not be helpful and might even be harmful. The problem when encountering a patient with fractures and deciding whether to recommend treatment with a bisphosphonate is that no reliable diagnostic method exists that allows detection of the rare instance of preexisting oversuppression of remodeling.  When pretreatment BMD is not particularly low, that is, not lower than normal or mildly osteopenic, the persistence of fracturing during treatment may mean that oversuppression of remodeling was already present and a change in medication would be appropriate. There is no evidence that bisphosphonate treatment impairs fracture healing. Indeed, there are a substantial number of reports involving animal models, as well as a few human case reports, to suggest that bisphosphonate treatment actually improves fracture healing. In general, it is important to bear in mind the positive benefit-to-risk ratio for this therapeutic class when making treatment recommendations for patients with osteoporosis.

Bisphosphonate Safety:

1.               Gastrointestinal Intolerance,2.               Hypocalcemia,

3.               Acute-Phase Reaction, and

4.               Chronic Bone and Muscle Pain

PTH: Potential role in management of heart failure

  1. Gruson, A. Buglioni, J.C. Burnett Jr.
    Clinica Chimica Acta 433 (2014) 290–296
    http://dx.doi.org/10.1016/j.cca.2014.03.029

Biomarkers play an important role for the diagnosis and prognosis of heart failure (HF), a disease with high morbidity and mortality as well as a huge impact on healthcare budgets. Parathyroid hormone (PTH) is a major systemic calcium-regulating hormone and an important regulator of bone and mineral homeostasis. PTH testing is important for differential diagnosis of calcemia related disorders and for the management of patients with chronic kidney disease. As secondary hyperparathyroidism has been evidenced in HF patients, PTH testing might be relevant in HF patients for risk stratification and more personalized selection of treatment.

Heart failure and neurohormonal activation

Heart failure is a syndrome characterized by increasing prevalence, high morbidity, elevated hospital readmission rate and high mortality. The continuing improvement of diagnosis, prognosis, treatment and management of HF requires a better understanding of the different sub-phenotypes and heterogeneity of this syndrome at the cellular, organ, and systemic level. Neurohormonal activation, one of the hallmarks of HF, plays a significant role in the myocardial and multi-organ adaptation. The comprehensive understanding of neurohormonal activation has allowed the identification of several biomarkers, such as natriuretic peptides, which are now playing an important role in HF management. Beside their contribution to the diagnosis of HF, natriuretic peptides are also relevant for follow-up and prognosis of HF patients.  Nevertheless, natriuretic peptides are more related to ventricular stretch, and biomarkers from other biological pathways like cardiac remodeling might provide additional value for the risk stratification of HF patients. The integration of biomarkers from several pathophysiological pathways along with imaging and genetic testing, might therefore be used to define HF subtypes, responding differently to specific therapeutic actions and contributing to more tailored based approaches.
Abnormalities of bone and mineral metabolism are also found in HF.  Secondary hyperparathyroidism has been evidenced in this context and several recent reports have documented the potential use of parathyroid hormone (PTH) testing for a more personalized management of HF patients. The aim of this article is therefore to review some of the cardiac effects of PTH and the potential role of PTH testing in HF.

Parathyroid hormone: biology and cardiac effects
PTH is one of the major regulators of the bone and mineral metabolism and its secretion is modulated by changes in concentration of calcium in the blood; decreased calcium concentrations stimulating PTH secretion via calcium-sensing receptors in the parathyroid gland. In response to hypocalcemia,
PTH has different targets to increase circulating calcium concentration. A fundamental target is the renal tubule where PTH will increase phosphorus excretion in the proximal tract and will enhance calcium reabsorption from the ascending limb of the loop of Henle to the collecting duct. The proximal renal tubule is also a target where PTH will stimulate the 1-α hydroxylation of 25-hydroxyvitamin D to 1,25-dihydroxyvitamin D: this biologically active form of vitamin D acts on the gut to increase absorption of both dietary calcium and phosphorus. Another target of PTH is the osteoclasts, leading to increased bone resorption with release of phosphorus and calcium in the blood.
PTH is a polypeptide containing 84 amino acids secreted by the parathyroid glands after cleavage from preproparathyroid hormone to proparathyroid hormone to the mature hormone. However, it displays several circulating forms and related fragments. PTH is secreted predominantly as an intact molecule, but it is rapidly cleaved in peripheral tissues to amino terminus and carboxy terminus fragments. The amino terminus fragment is biologically active and has a relatively short circulating half-life. The carboxy-terminal species include a 7-84 peptide and a variety of shorter fragments. These fragments can have a longer half-life and accumulate in renal failure. PTH assays measure not only the full-length form of PTH but also isoforms as well as fragments and differences can be observed between assays depending on the antibody specificities.

Cardiac effects of PTH
Primary hyperparathyroidism has been associated with heart diseases, underlying the potential cardiac consequences of increased circulating levels of PTH. Furthermore, as the heart is one of the target organs of PTH, the involvement of this hormone in the pathogenesis of cardiovascular diseases was previously suggested. PTH has different effects on the heart and can stimulate hypertrophy, arrhythmias and a pro-oxidative state. PTH has a direct hypertrophic action on cardiomyocytes. PTH is able, through a direct effect mediated through its receptors, to activate protein kinase C which further stimulates hypertrophic growth and reexpression of fetal type proteins in cardiomyocytes. PTH was also reported as a potent activator of protein kinase A (PKA) and several other downstream effectors related to cardiomyocyte hypertrophy. The hypertrophic effect of PTH on cardiac cells is also reinforced by its ability to stimulate an anti-hypertrophic response, including the natriuretic peptide gene transcription and by the increased of plasma concentrations of N-terminal pro-B-type natriuretic peptide (NT-proBNP) observed in patients with primary hyperparathyroidism. The hypertrophic effect of PTH on the heart was also evidence by a close relation between PTH levels and left ventricular mass.
In addition to its hypertrophic action, PTH can stimulate cardiac arrhythmias. PTH was documented as a chronotropic agent able to cause early death ofmyocytes in rat. Importantly, Bogin et al. showed in cultured heart cells of rat, that both amino-terminal PTH 1–34 and intact PTH 1–84 produced an immediate, sustained and significant rise in beats per minute and that the cells died earlier than control cardiomyocytes. Similar bservations were obtained by Shimoyama et al. In human, recent data showed that chronic renal failure and hyperparathyroidism are associated with a sympathetic over-activity. In that case, PTH 1–34 was able to facilitate norepinephrine release in human heart atria by activating the PTH-receptors. Therefore, highly increased PTH levels that can be observed in HF patients can participate to the elevated sympathetic nerve activity and the associated cardiovascular mortality.
The cardiac impact of PTH is also related to calcium overloading in myocardial cells. This cytoplasmic calcium overloading induced by PTH in myocardial cells appears as a paradox for hyperparathyroidism states. The mechanisms behind the increase of intracellular calcium involve a receptor-mediated rise in L-type calcium channel at the plasma membrane level leading to a secondary entry of calcium into cardiomyocyte and mobilization of calcium from sarcoplasmic reticulum. Both PTH 1–34 and PTH 1–84 produced such a dose dependent increase of intracellular calcium in cardiomyocytes. This increase of cytosolic calcium can be prevented by the calcium channel blocker verapamil.
Hyperparathyroidism has also been documented to trigger oxidative stress. When PTH levels are increased, a higher H2O2 production is observed in peripheral blood mononuclear cells. The increase in intracellular calcium induced by PTH might impair the mitochondrial function and ATP production, inducing the production of reactive oxygen species and leading to oxidative stress as well as inflammation and, at the end, to cardiomyocyte necrosis.
Lastly, increased circulating concentrations of PTH might stimulate adrenal aldosterone synthesis, initiating a vicious circle between hyperparathyroidism and hyperaldosteronism and leading to more proinflammatory, pro-oxidant and pro-fibrotic actions.

The rise of PTH in HF
Through its cardiac effects PTH can participate to the pathophysiology of cardiovascular diseases and a chronic excess to high circulating levels of PTH can have some deleterious consequences for the HF patients. Several factors might explain the increase of circulating PTH levels in HF patients.
First of all, impaired cation homeostasis and calcium loss should be considered.   Alteration in electrolyte equilibrium is frequent in HF patients as a consequence of hormonal changes in this pathological condition (hyperadrenergic state and secondary hyperaldosteronism). Calcium wasting is also triggered by diuretics, used to treat HF patients.
A second important factor can be a deficiency of vitamin D. Low vitamin D levels are frequently observed in HF patients and can lead to a rise in PTH levels.
Another documented factor is the interrelationship between hemodynamic state and serum intact PTH levels in patients with HF. Indeed, in a cross-sectional study including 105 patients with chronic HF, log-transformed intact PTH levels were positively and significantly correlated with pulmonary capillary wedge pressure and inversely correlated with stroke volume index after adjusting for variables associated with PTH.

The cross talk between PTH and aldosterone
The cross talk between PTH and FGF-23
Circulating levels of PTH and heart failure
PTH levels in HF patients
PTH testing and heart failure: conclusions and perspectives
PTH testing: assay matters

secondary hyperparathyroidism

secondary hyperparathyroidism

Potential involvement of secondary hyperparathyroidism in the worsening course of heart failure significant correlations were observed, through generation assays, between PTH and natriuretic peptides aswell as galectin-3. Importantly, the different immunoreactivities might impact on the value of PTH testing in treatment and prognosis of HF.

The measurement of PTH concentrations in HF can, like in patients with chronic kidney disease, help to monitor the efficiency of the treatment (drugs as well as medical devices). The use of PTH testing in HF patients might also allow the selection of more personalized and tailored therapies. HF patients with higher PTH levels could be relevant candidates for vitamin D supplementation or other pharmacological treatment. Based on the positive relationship between aldosterone and PTH, higher PTH levels can be an additional reason to use aldosterone blockers in HF patients.

Parathyroid hormone and cardiovascular disease events: A systematic review and meta-analysis of prospective studies

Adriana J. van Ballegooijen, I Reinders, M Visser, and IA Brouwer
Am Heart J 2013;165:655-664.e5
http://dx.doi.org/10.1016/j.ahj.2013.02.014

The parathyroid hormone (PTH) is a key hormone for the maintenance of calcium homeostasis. Low serum calcium triggers the secretion of PTH from the parathyroid glands.1 This results in a raise in serum calcium by promoting the release of calcium from bone, reduces calcium excretion by the kidneys, and increases the calcium absorption by the small intestine. In turn, the increase in calcium inhibits PTH secretion from the parathyroid glands.
In addition to traditionally known target organs, PTH is of interest for its potential impact on cardiovascular disease (CVD) risk. Observational studies have demonstrated that chronic PTH elevation is linked to hypertension, cardiac hypertrophy, and myocardial dysfunction. Furthermore, PTH receptors are present in the myocardium and exert hypertrophic effects on cardiomyocytes. Taken together, these associations suggest plausible mechanisms whereby elevated PTH concentrations may be involved in pathological processes that lead to CVD.

Background Parathyroid hormone (PTH) excess might play a role in cardiovascular health. We therefore conducted a systematic review and meta-analysis to evaluate the association between PTH and cardiovascular disease (CVD) events, and intermediate outcomes.
Methods We conducted a systematic and comprehensive database search using MEDLINE and Embase between 1947 and October 2012. We included English-language prospective studies that reported risk estimates for PTH and CVD events, and intermediate outcomes. The characteristics of study populations, exposure, and outcomes of total CVD events, fatal and non-fatal CVD events were reported, and a quality assessment was conducted. Results were extracted for the highest versus lowest PTH concentrations, and meta-analyses were carried out using random effects models.
Results The systematic literature search yielded 5770 articles, and 15 studies were included. Study duration ranged between 2 and 14 years. All studies were performed primarily in whites with a mean age between 55 and 75 years. The metaanalyses included 12 studies, of which 10 investigated total CVD events; 7, fatal CVD events; and 3, non-fatal CVD events. PTH excess indicated an increased risk for total CVD events: pooled HR (95% CI), 1.45 (1.24-1.71). The results for fatal CVD events and non-fatal CVD events were: HR 1.50 (1.18-1.91) and HR 1.48 (1.14-1.92). Heterogeneity was moderately present; however, sensitivity analyses for follow-up duration, prior CVD, or PTH as dichotomous values showed similar results.
Conclusions The meta-analysis indicates that higher PTH concentrations are associated with increased risk of CVD events.

Impact of estrogen on mechanically stimulated cells in vitro

Jörg Neunzehn, Ulrich Meyer and Hans-Peter Wiesman
Int.J.Curr.Microbiol.App.Sci (2014) 3(5) 898-906
Estrogen deficiency and decreased exercise known to be major causes for osteoporosis in elderly patients are assumed on important role in implant failure. Hormone replacement therapy and exercise are established methods to prevent the accompanying bone loss, thereby improving the conditions for implant osseointegration. Whereas the clinical effects of estrogen on bone are well documented, less is known about estrogen effects on loaded and unloaded osteoblasts on a cellular level. This study was aimed at investigating the effects of estrogen on mechanically stimulated osteoblast like cells in culture. Mechanically unstimulated cultures served as controls. Our investigations revealed that estrogen had a suppressive effect on the proliferative response of osteoblasts towards mechanical strain. Estrogen increased the synthesis of bone specific proteins in mechanically stimulated cultures whereas estrogen had no effect on unstimulated cells. The differentiation effects significant altered at estrogen doses of 10nmol and 10 μmol. Our data suggest a positive effect of hormone substitution on the composition of the extracellular matrix in loaded bones. In the context of implant dentistry, hormone repaints therapy should be regarded as a medical tool to improve the conditions for an undisturbed implant healing.

Normal bone physiology, remodelling and its hormonal regulation

Jennifer S Walsh
Surgery 2014; 33:1

The skeleton has structural and locomotor functions, and is a mineral reservoir. Bone turnover by osteoclasts and osteoblasts is a lifelong process, incorporating growth, modelling and remodeling to repair microdamage and access the mineral reservoir.
Bone formation and resorption are the basis of growth, modeling and remodeling. The bone remodeling cycle is an ongoing process that renews bone to repair microdamage and maintain strength. It also maintains serum calcium in the normal physiological range by release of mineral from the bone matrix as required. About 5-10% of the adult skeleton is replaced by remodeling each year.
On trabecular bone and at the endocortical surface, remodeling takes place on the surface of bone, but within cortical bone the osteoclasts form a cutting cone through the bone matrix. The signal to initiate remodeling may be endocrine (such as increased parathyroid hormone (PTH) in response to hypocalcaemia), which leads to generalized increases in osteoclast activation. Localized remodeling is initiated in response to microdamage, probably by signals from osteocytes. During a remodeling cycle, osteoclasts on the bone surface become activated and resorb bone matrix, creating a defect which is filled in by osteoblasts. The cycle usually takes about 200 days to complete. The bone remodeling cycle is highly regulated, and resorption and formation are closely coupled.
Signaling between bone cells is essential for the coordination of these processes. Osteoblasts regulate osteoclast activity through the receptor activator of nuclear factor-kB (RANK)/RANK ligand/osteoprotegerin system, and osteocytes regulate osteoblast activity through sclerostin secretion. If resorption and formation are balanced there is no net change in bone mass after each cycle, but with ageing and some disease states resorption exceeds formation leading to remodeling imbalance, decreased bone mass and loss of microstructural integrity. The rate of remodeling is determined by loading and endocrine influences. The most important endocrine regulator of bone turnover is probably estrogen, but other hormones regulating bone metabolism include insulin-like growth factor-1, parathyroid hormone and gut and adipocyte hormones.

Differential Diagnosis, Causes, and Management of Hypercalcemia

Fredriech K. W. Chan, et al.
Current Problems In Surgery June 1997; 34(6)

Hypercalcemia is a challenging clinical syndrome, both in diagnosis and therapy. The two most common causes of hypercalcemia, primary hyperparathyroidism and malignancy, account for approximately 90% of all patients with an elevated calcium level. In the general population, primary hyperparathyroidism is more common than malignancy. In a hospitalized population, malignancy is by far the more common. The differential diagnosis of hypercalcemia should be focused initially on the distinction between primary hyperparathyroidism and malignancy.

Primary hyperparathyroidism is caused by excessive, abnormally regulated secretion of parathyroid hormone from one or more adenomatous or hyperplastic parathyroid glands. In 80% of cases, primary hyperparathyroidism is due to a single adenoma. In 15% to 20% of patients, all four glands are enlarged as a result of hyperplasia. Parathyroid hyperplasia is also encountered in patients with Multiple Endocrine Neoplasia, Type I or II. Rarely, in fewer than 0.5% of patients, primary hyperparathyroidism is due to parathyroid carcinoma. The clinical features of primary hyperparathyroidism result from the hypercalcemia and the excessive output of parathyroid hormone (PTH).
The major target organs are the bones and the kidneys. The classic but rare bone disease of primary hyperparathyroidism is osteitis fibrosa cystica. Since the advent of the multichannel autoanalyzer in the early 1970s, an era marked by a great increase in incidence of primary hyperparathyroidism, the prevalence of radiologically apparent bone disease in patients with primary hyperparathyroidism has declined from 10% to 15% to a vanishingly small 1% to 2%. Sensitive technologies such as bone densitometry and bone histomorphometry, however, have revealed skeletal involvement with preferential reduction of cortical bone mass and relative preservation of cancellous bone mass. Although the incidence of nephrolithiasis in primary hyperparathyroidism has also decreased markedly, from approximately 60% in the 1940s and 1950s to 15% to 20% now, nephrolithiasis is still the most frequent complication of primary hyperparathyroidism.
Primary hyperparathyroidism also can be associated with neuropsychiatric, gastrointestinal, and cardiovascular manifestations. However, evidence that these features are pathophysiologically linked to the hyperparathyroid process or are reversible after successful parathyroidectomy is not compelling.

Management of Skeletal Health in Patients With Asymptomatic Primary Hyperparathyroidism

  1. Michael Lewiecki
    J Clin Densitometry: Assessment of Skeletal Health, 2010; 13(4), 324e334.
    http://dx.doi.org:/10.1016/j.jocd.2010.06.004

Asymptomatic primary hyperparathyroidism (PHPT) may cause adverse skeletal effects that include high bone remodeling, reduced bone mineral density (BMD), and increased fracture risk. Parathyroid surgery, the definitive treatment for PHPT, has been shown to increase BMD and appears to reduce fracture risk. Current guidelines recommend parathyroid surgery for patients with symptomatic PHPT or asymptomatic PHPT with serum calcium > 1 mg/dL above the upper limit of normal, calculated creatinine clearance < 60 mL/min, osteoporosis, previous fracture, or age > 50 yr. The type of operation performed (parathyroid exploration or minimally invasive procedure) and localizing studies to identify the abnormal parathyroid glands preoperatively should be individualized according to the skills of the surgeon and the resources of the institution. In patients who choose not to be treated surgically or who have contraindications for surgery, medical therapy should include a daily calcium intake of at least 1200 mg and maintenance of serum 25-hydroxyvitamin D levels of at least 20 ng/mL (50 nmol/L). Bisphosphonates and estrogens have been shown to provide skeletal benefits that appear to be similar to parathyroid surgery. Cinacalcet reduces serum calcium in PHPT patients with intractable hypercalcemia but has not been shown to improve BMD. It is not known whether any medical intervention reduces fracture risk in patients with PHPT. There are insufficient data on the natural history and treatment of normocalcemic PHPT to make recommendations for management of this disorder.

Hyperparathyroidism

William D Fraser
thelancet July 11, 2009; 374: 145-158 – Seminar

Hyperparathyroidism is due to increased activity of the parathyroid glands, either from an intrinsic abnormal change altering excretion of parathyroid hormone (primary or tertiary hyperparathyroidism) or from an extrinsic abnormal change affecting calcium homoeostasis stimulating production of parathyroid hormone (secondary hyperparathyroidism). Primary hyperparathyroidism is the third most common endocrine disorder, with the highest incidence in postmenopausal women. Asymptomatic disease is common, and severe disease with renal stones and metabolic bone disease arises less frequently now than it did 20–30 years ago. Primary hyperparathyroidism can be cured by surgical removal of an adenoma, increasingly by minimally invasive parathyroidectomy. Medical management of mild disease is possible with bisphosphonates, hormone replacement therapy, and calcimimetics. Vitamin D deficiency is a common cause of secondary hyperparathyroidism, particularly in elderly people. However, the biochemical definition of vitamin D deficiency and its treatment are subject to much debate. Secondary hyperparathyroidism as the result of chronic kidney disease is important in the genesis of renal bone disease, and several new treatments could help achieve the guidelines set out by the kidney disease outcomes quality initiative.

Table 1: Changing clinical presentation of primary hyperparathyroidism
1930–1970 1970–2000
Nephrolithiasis 51–57% 17–37%
Hypercalciuria 36% 40%
Overt skeletal disease 10–23% 4–14%
Asymptomatic 6–18% 22–80%
Modified from reference 12
Panel 1: Recommendations for surgery from the National Institutes of Health
consensus conference on primary hyperparathyroidism in 1990 and 2002• Serum albumin-adjusted calcium greater than 0·25 mmol/L
above the upper limit of local laboratory reference range

• Urine calcium greater than 10 mmol per 24 h

• Creatinine clearance reduced by 30% or more

• Bone mineral density T score less than –2·5 (at any site)

• Age younger than 50 years

• Patient request; adequate follow-up unlikely

Aldosterone and parathyroid hormone interactions as mediators of metabolic and cardiovascular disease

Andreas Tomaschitz, Eberhard Ritz, Burkert Pieske, Jutta Rus-Machan
Metabolism Clinical and  Experimental 2014; 63: 20 31
http://dx.doi.org/10.1016/j.metabol.2013.08.016

Several studies demonstrated a strong link between dysregulation of the aldosterone and parathyroid hormone (PTH) axes on the one hand and CV pathology on the other hand. Such evidence documents clinically relevant interactions between aldosterone and PTH and a resulting impact on CV health. This review provides an up to date overview discussing the mechanisms and the clinical relevance underlying the interactions between aldosterone and PTH.

Inappropriate aldosterone and parathyroid hormone (PTH) secretion is strongly linked with development and progression of cardiovascular (CV) disease. Accumulating evidence suggests a bidirectional interplay between parathyroid hormone and aldosterone. This interaction may lead to a disproportionally increased risk of CV damage, metabolic and bone diseases.

This review focuses on mechanisms underlying the mutual interplay between aldosterone and PTH as well as their potential impact on CV, metabolic and bone health. PTH stimulates aldosterone secretion by increasing the calcium concentration in the cells of the adrenal zona glomerulosa as a result of binding to the PTH/PTH-rP receptor and indirectly by potentiating angiotensin 2 induced effects. This may explain why after parathyroidectomy lower aldosterone levels are seen in parallel with improved cardiovascular outcomes.

Aldosterone mediated effects are inappropriately pronounced in conditions such as chronic heart failure, excess dietary salt intake (relative aldosterone excess) and primary aldosteronism.

PTH is increased as a result of
(1) the MR (mineralocorticoid receptor)mediated calciuretic and magnesiuretic effects with a trend of hypocalcemia and hypomagnesemia; the resulting secondary hyperparathyroidism causes myocardial fibrosis and disturbed bone metabolism; and

(2) direct effects of aldosterone on parathyroid cells via binding to the MR. This adverse sequence is interrupted by mineralocorticoid receptor blockade and adrenalectomy.

Hyperaldosteronism due to klotho deficiency results in vascular calcification, which can be mitigated by spironolactone treatment. In view of the documented reciprocal interaction between aldosterone and PTH as well as the potentially ensuing target organ damage, studies are needed to evaluate diagnostic and therapeutic strategies to address this increasingly recognized pathophysiological phenomenon.

The classical view that aldosterone acts exclusively on the electrolyte transport in epithelial cells has been broadened after the mineralocorticoid receptor (MR) has been identified in non-epithelial cells as well, e.g. vascular smooth muscle cells and cardiomyocytes. Apart from classical genomic effects, non-genomic aldosterone mediated effects have been identified in various tissues and organs outside of the kidneys and colon, e.g. inner ear, choroid plexus, endothelial cells and cardiomyocytes.

In the past it had been documented that primary aldosteronism (PA; absolute aldosterone excess) contributed to the development of CVD. Several studies suggested, however, that “absolute aldosterone excess” is only the tip of the iceberg leading to the concept of “relative aldosterone excess” . Several large cross-sectional and prospective studies demonstrated a consistent relationship between circulating aldosterone levels, CV risk factors and mortality risk.

Such recent studies also document that even circulating aldosterone concentrations in the “normal” range may result in inappropriate aldosterone–MR interaction which may be reversed by MR blockade.
The identification of PTH receptors within the CV system e.g. in cardiomyocytes, vascular smooth muscle, and endothelial cells, indicates that inappropriate PTH secretion may impact on the CV health beyond the dysregulation of calcium and phosphate homeostasis.

Application of PTH after myocardial infarction attenuates ischaemic cardiomyopathy by increasing migration of bone marrow-derived stem cells to the ischaemic myocardium. On the other hand the PTH excess in primary hyperparathyroidism (pHPT) is linked in the long-term to a spectrum of adverse effects e.g. bone loss and increased fracture risk, coronary microvascular dysfunction, derangement of lipid and glucose metabolism, subclinical aortic valve calcification, increased aortic stiffness, endothelial dysfunction and arterial hypertension.

Interactions between vitamin D, klotho and aldosterone
Increased activity of systemic or local renin–angiotensin systems (RAS) is linked to increased target organ damage. The organ and tissue protective effects of vitamin D have in part been explained by vitamin D induced modulation of RAS activity.

In landmark experiments Li et al. documented markedly elevated renin mRNA expression in the juxtaglomerular apparatus of vitamin D receptor (VDR) knock-out mice compared to wild type mice. Furthermore, 1,25-dihydroxy vitamin D (1,25(OH2)D3) modulated renin gene transcription and renin synthesis and this was independent of serum calcium, PTH and angiotensin 2. Angiotensin 2 in turn reduces renal klotho expression resulting in modulations of FGF-23-signaling and of 1-α hydroxylase activity. Klotho is a membrane (and circulating) protein which is highly expressed in the kidney and modulates the inhibitory effects of FGF-23 on calcitriol formation; klotho contributes to the regulation of renal tubular calcium and phosphate reabsorption. The modulatory effects of vitamin D on the RAS might result in a lower risk of development and progression of CV morbidity and mortality.

Evidence for stimulating effects of PTH on adrenal aldosterone secretion Aldosterone synthesis is mainly initiated by angiotensin 2 and potassium via activating the Ca2+-messenger system in zona glomerulosa (ZG) cells to stimulate the steroidogenic cascade within the mitochondria. The Ca2+-messenger system further participates in the initiation of steroidogenesis by facilitating the cholesterol transfer into the mitochondria. Findings from experimental, mechanistic, observational and interventional studies suggest that PTH contributes to the regulation of aldosterone secretion in the ZG of the adrenal glands.

The interaction between aldosterone and Klotho and its relationship to vascular osteoinduction

The interaction between aldosterone and Klotho and its relationship to vascular osteoinduction

The interaction between aldosterone and Klotho and its relationship to vascular osteoinduction

Estradiol determines the effects of PTH on ERa-dependent transcription in MC3T3-E1 cells

Monika H.E. Christensen, IS Fenne, MH Flågeng, B Almås, et al.
Biochemical and Biophysical Research Communications 450 (2014) 360–365
http://dx.doi.org/10.1016/j.bbrc.2014.05.109

Bone remodeling is a continuous process regulated by several hormones such as estrogens and parathyroid hormone (PTH). Here we investigated the influence of PTH on estrogen receptor alpha (ERa)-dependent transcriptional activity in MC3T3-E1 osteoblasts. Cells that were transfected with an ER-responsive reporter plasmid and treated with PTH showed increased luciferase activity. However, in the presence of 17b-estradiol, we observed that PTH inhibited ERa-mediated transcription. cAMP mimicked the effects by PTH, and the findings were confirmed in COS-1 cells transfected with expression vector encoding the catalytic subunit of cAMP-dependent protein kinase (PKA). Furthermore, PTH exhibited specific effects on the mRNA expression of the decoy receptor osteoprotegerin (OPG) and the receptor activator of NF kappa-B ligand (RANKL) in MC3T3-E1 osteoblasts. In the absence of 17b-estradiol, PTH and cAMP enhanced the OPG/RANKL ratio, whereas, OPG/RANKL was suppressed when estradiol was present. In conclusion, our results indicate that the presence of estradiol determines whether PTH and cAMP stimulates or inhibits ERa-dependent activity and the OPG/RANKL mRNA expression in an osteoblastic cell line.

Ginsenoside-Rb2 displays anti-osteoporosis effects through reducing oxidative damage and bone-resorbing cytokines during osteogenesis

Qiang Huang, Bo Gao, Qiang Jie, Bo-Yuan Wei, et al.
Bone 66 (2014) 306–314
http://dx.doi.org/10.1016/j.bone.2014.06.010

Reactive oxygen species (ROS) are a significant pathogenic factor of osteoporosis. Ginsenoside-Rb2 (Rb2), a 20(S)-protopanaxadiol glycoside extracted from ginseng, is a potent antioxidant that generates interest regarding the bone metabolism area. We tested the potential anti-osteoporosis effects of Rb2 and its underlying mechanism in this study. We produced an oxidative damage model induced by hydrogen peroxide (H2O2) in osteoblastic MC3T3-E1 cells to test the essential anti-osteoporosis effects of Rb2 in vitro. The results indicated that treatment of 0.1 to 10 μMRb2 promoted the proliferation of MC3T3-E1 cells, improved alkaline phosphatase (ALP) expression, elevated calcium mineralization and mRNA expressions of Alp, Col1a1, osteocalcin (Ocn) and osteopontin (Opn) against oxidative damage induced by H2O2. Importantly, Rb2 reduced the expression levels of receptor activator of nuclear factor kappa-B ligand (RANKL) and IL-6 and inhibited the H2O2-induced production of ROS. The in vivo study indicated that the Rb2 administered for 12 weeks partially decreased blood malondialdehyde (MDA) activity and elevated the activity of reduced glutathione (GSH) in ovariectomized (OVX)mice. Moreover, Rb2 improved the micro-architecture of trabecular bones and increased bone mineral density (BMD) of the 4th lumbar vertebrae (L4) and the distal femur. Altogether, these results demonstrated that the potential anti-osteoporosis effects of Rb2 were linked to a reduction of oxidative damage and bone-resorbing cytokines, which suggests that Rb2 might be effective in preventing and alleviating osteoporosis.

Inflammatory cytokines in Paget’s disease of bone

GRW de Castro, Z Buss, JS Da Rosa, TS Fröde
International Immunopharmacology 18 (2014) 277–281
http://dx.doi.org/10.1016/j.intimp.2013.12.003

This study was undertaken to evaluate the expression of inflammatory cytokines in patients with Paget’s disease of bone (PDB). Serum levels of tumoral necrosis factor-α, interleukin 1β, interleukin-6 and interleukin-17
were measured in 51 patients with PDB and in 24 controls with primary osteoarthritis. Compared to controls, patients with Paget’s disease of bone presented higher levels of interleukin 6 and reduced interleukin 17, but levels of tumoral necrosis factor α and interleukin 1 β did not differ significantly. We found no significant differences when patients were compared according to disease activity or current treatment. There were no correlations between cytokine levels and bone-specific alkaline phosphatase or extension of Paget’s disease of bone on bone scintigraphs. In conclusion, patients with PDB present significant differences on levels of certain cytokines in comparison to primary osteoarthritis patients, but these alterations did not appear to have a clear correlation with parameters of disease activity or severity.

Development and validation of a novel cell-based assay for potency determination of human parathyroid hormone (PTH)
Axel Hohenstein, Meike Hebell, Heidi Zikry, Maria El Ghazaly, et al.
Journal of Pharmaceutical and Biomedical Analysis 98 (2014) 345–350
http://dx.doi.org/10.1016/j.jpba.2014.06.004

Disorders of bone metabolism
Orthopaedics I: General Principles

Nicola Peel
Surgery 33:1

Bone remodeling is critical to bone health. Alterations in the normal processes and regulation of remodeling may impact on bone mass and bone strength. Changes may be generalized or focal and underlie many of the common disorders of bone metabolism. This article focuses on the changes in bone remodeling which underlie both the development and treatment of osteoporosis. Osteomalacia, as an example of a mineralization disorder and Paget’s disease as an example of a focal disorder of bone remodeling, are also briefly reviewed.

There are many causes of increased bone turnover with the most common being the loss of estrogen at menopause. Increased bone turnover is initiated by increased activation frequency of osteoclasts. The consequent increase in remodeling space leads to bone loss which is, at least in part, reversible. Increased bone turnover is also associated with an increased risk of trabecular perforation with the increased number of remodeling sites acting as stress risers within the trabecular architecture. Bone loss within the trabecular compartment occurs preferentially from the horizontal, non-weight bearing plates resulting in disproportionate loss of bone strength for the reduction in bone mass.
Alterations in bone turnover also have potential to affect bone.

strength by changing the degree of mineralization. Primary mineral apposition occurs early after production of bone matrix by osteoblasts. After completion of the cycle, secondary mineral apposition occurs over many months. Increased bone turnover leads to reduced mineralization as the time between remodeling cycles reduces. Conversely, decreased bone turnover rates reduce the average time between remodeling at any site and hence lead to a greater degree of mineralization. Biomechanical principles indicate that the yield strength (stiffness) of highly mineralized bone increases but that it will withstand less deformation before fracture and therefore becomes brittle. A reduced degree of mineralization results in greater pliability but a reduction in bone strength.
Alterations in bone remodeling underpin changes in bone mass and bone strength. The impact of these changes is manifest in the development and clinical presentation of osteoporosis.

Paget’s disease

Paget’s disease

Paget’s disease: (a) increased uptake on nuclear medicine scanning in the right hemipelvis, sacrum and left femur and (b) left femur showing radiological changes of Paget’s including a fissure fracture in the proximal lateral cortex

Paget’s disease is an example of a localised disorder of bone turnover. Its aetiology remains unclear. Paget’s disease is not uncommon but is often asymptomatic and diagnosed coincidentally. It is estimated to affect approximately 2% of adults over the age of 55 in the UK but the prevalence varies markedly between populations. It is increasingly prevalent with increasing age and affects men more frequently than women. In 80% of cases more than one bone is involved, characteristically in an asymmetric distribution.
Pagetic bone is characterized by the presence of giant multinucleated osteoclasts resulting in dramatic increases in bone resorption in the affected bones. These regions undergo a lytic phase followed by a compensatory increase in bone formation. Rapid bone formation results in an accumulation of woven bone, which is mechanically abnormal resulting in loss of bone strength.
The typical clinical manifestation is of bone pain, which may be associated with bone expansion and deformity. Complications of Paget’s disease include the development of secondary osteoarthritis, fissure fractures and very rarely, osteosarcomatous change (<1% of cases).

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Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?


Introduction – The Evolution of Cancer Therapy and Cancer Research: How We Got Here?

Author and Curator: Larry H Bernstein, MD, FCAP

The evolution of progress we have achieved in cancer research, diagnosis, and therapeutics has  originated from an emergence of scientific disciplines and the focus on cancer has been recent. We can imagine this from a historical perspective with respect to two observations. The first is that the oldest concepts of medicine lie with the anatomic dissection of animals and the repeated recurrence of war, pestilence, and plague throughout the middle ages, and including the renaissance.  In the awakening, architecture, arts, music, math, architecture and science that accompanied the invention of printing blossomed, a unique collaboration of individuals working in disparate disciplines occurred, and those who were privileged received an education, which led to exploration, and with it, colonialism.  This also led to the need to increasingly, if not without reprisal, questioning long-held church doctrines.

It was in Vienna that Rokitansky developed the discipline of pathology, and his student Semelweis identified an association between then unknown infection and childbirth fever. The extraordinary accomplishments of John Hunter in anatomy and surgery came during the twelve years war, and his student, Edward Jenner, observed the association between cowpox and smallpox resistance. The development of a nursing profession is associated with the work of Florence Nightengale during the Crimean War (at the same time as Leo Tolstoy). These events preceded the work of Pasteur, Metchnikoff, and Koch in developing a germ theory, although Semelweis had committed suicide by infecting himself with syphilis. The first decade of the Nobel Prize was dominated by discoveries in infectious disease and public health (Ronald Ross, Walter Reed) and we know that the Civil War in America saw an epidemic of Yellow Fever, and the Armed Services Medical Museum was endowed with a large repository of osteomyelitis specimens. We also recall that the Russian physician and playwriter, Anton Checkov, wrote about the conditions in prison camps.

But the pharmacopeia was about to open with the discoveries of insulin, antibiotics, vitamins, thyroid action (Mayo brothers pioneered thyroid surgery in the thyroid iodine-deficient midwest), and pitutitary and sex hormones (isolatation, crystal structure, and synthesis years later), and Karl Landsteiner’s discovery of red cell antigenic groups (but he also pioneered in discoveries in meningitis and poliomyelitis, and conceived of the term hapten) with the introduction of transfusion therapy that would lead to transplantation medicine.  The next phase would be heralded by the discovery of cancer, which was highlighted by the identification of leukemia by Rudolph Virchow, who cautioned about the limitations of microscopy. This period is highlighted by the classic work – “Microbe Hunters”.

John Hunter

John Hunter

Walter Reed

Walter Reed

Robert Koch

Robert Koch

goldberger 1916 Pellagra

goldberger 1916 Pellagra

Louis Pasteur

Louis Pasteur

A multidisciplinary approach has led us to a unique multidisciplinary or systems view of cancer, with different fields of study offering their unique expertise, contributions, and viewpoints on the etiology of cancer.  Diverse fields in immunology, biology, biochemistry, toxicology, molecular biology, virology, mathematics, social activism and policy, and engineering have made such important contributions to our understanding of cancer, that without cooperation among these diverse fields our knowledge of cancer would never had evolved as it has. In a series of posts “Heroes in Medical Research:” the work of researchers are highlighted as examples of how disparate scientific disciplines converged to produce seminal discoveries which propelled the cancer field, although, at the time, they seemed like serendipitous findings.  In the post Heroes in Medical Research: Barnett Rosenberg and the Discovery of Cisplatin (Translating Basic Research to the Clinic) discusses the seminal yet serendipitous discoveries by bacteriologist Dr. Barnett Rosenberg, which eventually led to the development of cisplatin, a staple of many chemotherapeutic regimens. Molecular biologist Dr. Robert Ting, working with soon-to-be Nobel Laureate virologist Dr. James Gallo on AIDS research and the associated Karposi’s sarcoma identified one of the first retroviral oncogenes, revolutionizing previous held misconceptions of the origins of cancer (described in Heroes in Medical Research: Dr. Robert Ting, Ph.D. and Retrovirus in AIDS and Cancer).   Located here will be a MONTAGE of PHOTOS of PEOPLE who made seminal discoveries and contributions in every field to cancer   Each of these paths of discovery in cancer research have led to the unique strategies of cancer therapeutics and detection for the purpose of reducing the burden of human cancer.  However, we must recall that this work has come at great cost, while it is indeed cause for celebration. The current failure rate of clinical trials at over 70 percent, has been a cause for disappointment, and has led to serious reconsideration of how we can proceed with greater success. The result of the evolution of the cancer field is evident in the many parts and chapters of this ebook.  Volume 4 contains chapters that are in a predetermined order:

  1. The concepts of neoplasm, malignancy, carcinogenesis,  and metastatic potential, which encompass:

(a)     How cancer cells bathed in an oxygen rich environment rely on anaerobic glycolysis for energy, and the secondary consequences of cachexia and sarcopenia associated with progression

invasion

invasion

ARTS protein and cancer

ARTS protein and cancer

Glycolysis

Glycolysis

Krebs cycle

Krebs cycle

Metabolic control analysis of respiration in human cancer tissue

Metabolic control analysis of respiration in human cancer tissue

akip1-expression-modulates-mitochondrial-function

akip1-expression-modulates-mitochondrial-function

(b)     How advances in genetic analysis, molecular and cellular biology, metabolomics have expanded our basic knowledge of the mechanisms which are involved in cellular transformation to the cancerous state.

nucleotides

nucleotides

Methylation of adenine

Methylation of adenine

ampk-and-ampk-related-kinase-ark-family-

ampk-and-ampk-related-kinase-ark-family-

ubiquitylation

ubiquitylation

(c)  How molecular techniques continue to advance our understanding  of how genetics, epigenetics, and alterations in cellular metabolism contribute to cancer and afford new pathways for therapeutic intervention.

 genomic effects

genomic effects

LKB1AMPK pathway

LKB1AMPK pathway

mutation-frequencies-across-12-cancer-types

mutation-frequencies-across-12-cancer-types

AMPK-activating drugs metformin or phenformin might provide protection against cancer

AMPK-activating drugs metformin or phenformin might provide protection against cancer

pim2-phosphorylates-pkm2-and-promotes-glycolysis-in-cancer-cells

pim2-phosphorylates-pkm2-and-promotes-glycolysis-in-cancer-cells

pim2-phosphorylates-pkm2-and-promotes-glycolysis-in-cancer-cells

pim2-phosphorylates-pkm2-and-promotes-glycolysis-in-cancer-cells

2. The distinct features of cancers of specific tissue sites of origin

3.  The diagnosis of cancer by

(a)     Clinical presentation

(b)     Age of onset and stage of life

(c)     Biomarker features

hairy cell leukemia

hairy cell leukemia

lymphoma leukemia

lymphoma leukemia

(d)     Radiological and ultrasound imaging

  1. Treatments
  2. Prognostic differences within and between cancer types

We have introduced the emergence of a disease of great complexity that has been clouded in more questions than answers until the emergence of molecular biology in the mid 20th century, and then had to await further discoveries going into the 21st century.  What gave the research impetus was the revelation of

1     the mechanism of transcription of the DNA into amino acid sequences

Proteins in Disease

Proteins in Disease

2     the identification of stresses imposed on cellular function

NO beneficial effects

NO beneficial effects

3     the elucidation of the substructure of the cell – cell membrane, mitochondria, ribosomes, lysosomes – and their functions, respectively

pone.0080815.g006  AKIP1 Expression Modulates Mitochondrial Function

AKIP1 Expression Modulates Mitochondrial Function

4     the elucidation of oligonucleotide sequences

nucleotides

nucleotides

dna-replication-unwinding

dna-replication-unwinding

dna-replication-ligation

dna-replication-ligation

dna-replication-primer-removal

dna-replication-primer-removal

dna-replication-leading-strand

dna-replication-leading-strand

dna-replication-lagging-strand

dna-replication-lagging-strand

dna-replication-primer-synthesis

dna-replication-primer-synthesis

dna-replication-termination

dna-replication-termination

5     the further elucidation of functionally relevant noncoding lncDNA

lncRNA-s   A summary of the various functions described for lncRNA

6     the technology to synthesis mRNA and siRNA sequences

RNAi_Q4 Primary research objectives

Figure. RNAi and gene silencing

7     the repeated discovery of isoforms of critical enzymes and their pleiotropic properties

8.     the regulatory pathways involved in signaling

signaling pathjways map

Figure. Signaling Pathways Map

This is a brief outline of the modern progression of advances in our understanding of cancer.  Let us go back to the beginning and check out a sequence of  Nobel Prizes awarded and related discoveries that have a historical relationship to what we know.  The first discovery was the finding by Louis Pasteur that fungi that grew in an oxygen poor environment did not put down filaments.  They did not utilize oxygen and they produced used energy by fermentation.  This was the basis for Otto Warburg sixty years later to make the comparison to cancer cells that grew in the presence of oxygen, but relied on anaerobic glycolysis. He used a manometer to measure respiration in tissue one cell layer thick to measure CO2 production in an adiabatic system.

video width=”1280″ height=”720″ caption=”1741-7007-11-65-s1 Macromolecular juggling by ubiquitylation enzymes.” mp4=”https://pharmaceuticalintelligence.files.wordpress.com/2014/04/1741-7007-11-65-s1-macromolecular-juggling-by-ubiquitylation-enzymes.mp4“][/video]

An Introduction to the Warburg Apparatus

http://www.youtube.com/watch?v=M-HYbZwN43o

Lavoisier Antoine-Laurent and Laplace Pierre-Simon (1783) Memoir on heat. Mémoirs de l’Académie des sciences. Translated by Guerlac H, Neale Watson Academic Publications, New York, 1982.

Instrumental background 200 years later:   Gnaiger E (1983) The twin-flow microrespirometer and simultaneous calorimetry. In Gnaiger E, Forstner H, eds. Polarographic Oxygen Sensors. Springer, Heidelberg, Berlin, New York: 134-166.

otto_heinrich_warburg

otto_heinrich_warburg

Warburg apparatus

The Warburg apparatus is a manometric respirometer which was used for decades in biochemistry for measuring oxygen consumption of tissue homogenates or tissue slices.

The Warburg apparatus has its name from the German biochemist Otto Heinrich Warburg (1883-1970) who was awarded the Nobel Prize in physiology or medicine in 1931 for his “discovery of the nature and mode of action of the respiratory enzyme” [1].

The aqueous phase is vigorously shaken to equilibrate with a gas phase, from which oxygen is consumed while the evolved carbon dioxide is trapped, such that the pressure in the constant-volume gas phase drops proportional to oxygen consumption. The Warburg apparatus was introduced to study cell respiration, i.e. the uptake of molecular oxygen and the production of carbon dioxide by cells or tissues. Its applications were extended to the study of fermentation, when gas exchange takes place in the absence of oxygen. Thus the Warburg apparatus became established as an instrument for both aerobic and anaerobic biochemical studies [2, 3].

The respiration chamber was a detachable glass flask (F) equipped with one or more sidearms (S) for additions of chemicals and an open connection to a manometer (M; pressure gauge). A constant temperature was provided by immersion of the Warburg chamber in a constant temperature water bath. At thermal mass transfer equilibrium, an initial reading is obtained on the manometer, and the volume of gas produced or absorbed is determined at specific time intervals. A limited number of ‘titrations’ can be performed by adding the liquid contained in a side arm into the main reaction chamber. A Warburg apparatus may be equipped with more than 10 respiration chambers shaking in a common water bath.   Since temperature has to be controlled very precisely in a manometric approach, the early studies on mammalian tissue respiration were generally carried out at a physiological temperature of 37 °C.

The Warburg apparatus has been replaced by polarographic instruments introduced by Britton Chance in the 1950s. Since Chance and Williams (1955) measured respiration of isolated mitochondria simultaneously with the spectrophotometric determination of cytochrome redox states, a water chacket could not be used, and measurements were carried out at room temperature (or 25 °C). Thus most later studies on isolated mitochondria were shifted to the artifical temperature of 25 °C.

Today, the importance of investigating mitochondrial performance at in vivo temperatures is recognized again in mitochondrial physiology.  Incubation times of 1 hour were typical in experiments with the Warburg apparatus, but were reduced to a few or up to 20 min, following Chance and Williams, due to rapid oxygen depletion in closed, aqueous phase oxygraphs with high sample concentrations.  Today, incubation times of 1 hour are typical again in high-resolution respirometry, with low sample concentrations and the option of reoxygenations.

“The Nobel Prize in Physiology or Medicine 1931”. Nobelprize.org. 27 Dec 2011 www.nobelprize.org/nobel_prizes/medicine/laureates/1931/

  1. Oesper P (1964) The history of the Warburg apparatus: Some reminiscences on its use. J Chem Educ 41: 294.
  2. Koppenol WH, Bounds PL, Dang CV (2011) Otto Warburg’s contributions to current concepts of cancer metabolism. Nature Reviews Cancer 11: 325-337.
  3. Gnaiger E, Kemp RB (1990) Anaerobic metabolism in aerobic mammalian cells: information from the ratio of calorimetric heat flux and respirometric oxygen flux. Biochim Biophys Acta 1016: 328-332. – “At high fructose concen­trations, respiration is inhibited while glycolytic end products accumulate, a phenomenon known as the Crabtree effect. It is commonly believed that this effect is restric­ted to microbial and tumour cells with uniquely high glycolytic capaci­ties (Sussman et al, 1980). How­ever, inhibition of respiration and increase of lactate production are observed under aerobic condi­tions in beating rat heart cell cultures (Frelin et al, 1974) and in isolated rat lung cells (Ayuso-Parrilla et al, 1978). Thus, the same general mechanisms respon­sible for the integra­tion of respiration and glycolysis in tumour cells (Sussman et al, 1980) appear to be operating to some extent in several isolated mammalian cells.”

Mitochondria are sometimes described as “cellular power plants” because they generate most of the cell’s supply of adenosine triphosphate (ATP), used as a source of chemical energy.[2] In addition to supplying cellular energy, mitochondria are involved in other tasks such as signalingcellular differentiationcell death, as well as the control of the cell cycle and cell growth.[3]   The organelle is composed of compartments that carry out specialized functions. These compartments or regions include the outer membrane, the intermembrane space, the inner membrane, and the cristae and matrix. Mitochondrial proteins vary depending on the tissue and the species. In humans, 615 distinct types of proteins have been identified from cardiac mitochondria,[9   Leonor Michaelis discovered that Janus green can be used as a supravital stain for mitochondria in 1900.  Benjamin F. Kingsbury, in 1912, first related them with cell respiration, but almost exclusively based on morphological observations.[13] In 1913 particles from extracts of guinea-pig liver were linked to respiration by Otto Heinrich Warburg, which he called “grana”. Warburg and Heinrich Otto Wieland, who had also postulated a similar particle mechanism, disagreed on the chemical nature of the respiration. It was not until 1925 when David Keilin discovered cytochromes that the respiratory chain was described.[13]    

The Clark Oxygen Sensor

Dr. Leland Clark – inventor of the “Clark Oxygen Sensor” (1954); the Clark type polarographic oxygen sensor remains the gold standard for measuring dissolved oxygen in biomedical, environmental and industrial applications .   ‘The convenience and simplicity of the polarographic ‘oxygen electrode’ technique for measuring rapid changes in the rate of oxygen utilization by cellular and subcellular systems is now leading to its more general application in many laboratories. The types and design of oxygen electrodes vary, depending on the investigator’s ingenuity and specific requirements of the system under investigation.’Estabrook R (1967) Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios. Methods Enzymol. 10: 41-47.   “one approach that is underutilized in whole-cell bioenergetics, and that is accessible as long as cells can be obtained in suspension, is the oxygen electrode, which can obtain more precise information on the bioenergetic status of the in situ mitochondria than more ‘high-tech’ approaches such as fluorescent monitoring of Δψm.” Nicholls DG, Ferguson S (2002) Bioenergetics 3. Academic Press, London.

Great Figures in Cancer

Dr. Elizabeth Blackburn,

Dr. Elizabeth Blackburn,

j_michael_bishop onogene

j_michael_bishop onogene

Harold Varmus

Harold Varmus

Potts and Habener (PTH mRNA, Harvard MIT)  JCI

Potts and Habener (PTH mRNA, Harvard MIT) JCI

JCI Fuller Albright and hPTH AA sequence

JCI Fuller Albright and hPTH AA sequence

Dr. E. Donnall Thomas  Bone Marrow Transplants

Dr. E. Donnall Thomas Bone Marrow Transplants

Dr Haraldzur Hausen  EBV HPV

Dr Haraldzur Hausen EBV HPV

Dr. Craig Mello

Dr. Craig Mello

Dorothy Hodgkin  protein crystallography

Lee Hartwell - Hutchinson Cancer Res Center

Lee Hartwell – Hutchinson Cancer Res Center

Judah Folkman, MD

Judah Folkman, MD

Gertrude B. Elien (1918-1999)

Gertrude B. Elien (1918-1999)

The Nobel Prize in Physiology or Medicine 1922   

Archibald V. Hill, Otto Meyerhof

AV Hill –

“the production of heat in the muscle” Hill started his research work in 1909. It was due to J.N. Langley, Head of the Department of Physiology at that time that Hill took up the study on the nature of muscular contraction. Langley drew his attention to the important (later to become classic) work carried out by Fletcher and Hopkins on the problem of lactic acid in muscle, particularly in relation to the effect of oxygen upon its removal in recovery. In 1919 he took up again his study of the physiology of muscle, and came into close contact with Meyerhof of Kiel who, approaching the problem differently, arrived at results closely analogous to his study. In 1919 Hill’s friend W. Hartree, mathematician and engineer, joined in the myothermic investigations – a cooperation which had rewarding results.

Otto Meyerhof

otto-fritz-meyerhof

otto-fritz-meyerhof

lactic acid production in muscle contraction Under the influence of Otto Warburg, then at Heidelberg, Meyerhof became more and more interested in cell physiology.  In 1923 he was offered a Professorship of Biochemistry in the United States, but Germany was unwilling to lose him.  In 1929 he was he was placed in charge of the newly founded Kaiser Wilhelm Institute for Medical Research at Heidelberg.  From 1938 to 1940 he was Director of Research at the Institut de Biologie physico-chimique at Paris, but in 1940 he moved to the United States, where the post of Research Professor of Physiological Chemistry had been created for him by the University of Pennsylvania and the Rockefeller Foundation.  Meyerhof’s own account states that he was occupied chiefly with oxidation mechanisms in cells and with extending methods of gas analysis through the calorimetric measurement of heat production, and especially the respiratory processes of nitrifying bacteria. The physico-chemical analogy between oxygen respiration and alcoholic fermentation caused him to study both these processes in the same subject, namely, yeast extract. By this work he discovered a co-enzyme of respiration, which could be found in all the cells and tissues up till then investigated. At the same time he also found a co-enzyme of alcoholic fermentation. He also discovered the capacity of the SH-group to transfer oxygen; after Hopkins had isolated from cells the SH bodies concerned, Meyerhof showed that the unsaturated fatty acids in the cell are oxidized with the help of the sulfhydryl group. After studying closer the respiration of muscle, Meyerhof investigated the energy changes in muscle. Considerable progress had been achieved by the English scientists Fletcher and Hopkins by their recognition of the fact that lactic acid formation in the muscle is closely connected with the contraction process. These investigations were the first to throw light upon the highly paradoxical fact, already established by the physiologist Hermann, that the muscle can perform a considerable part of its external function in the complete absence of oxygen.

But it was indisputable that in the last resort the energy for muscle activity comes from oxidation, so the connection between activity and combustion must be an indirect one, and observed that in the absence of oxygen in the muscle, lactic acid appears, slowly in the relaxed state and rapidly in the active state, disappearing in the presence of oxygen. Obviously, then, oxygen is involved when muscle is in the relaxed state. http://upload.wikimedia.org/wikipedia/commons/e/e1/Glycolysis.jpg

The Nobel Prize committee had been receiving nominations intermittently for the previous 14 years (for Eijkman, Funk, Goldberger, Grijns, Hopkins and Suzuki but, strangely, not for McCollum in this period). Tthe Committee for the 1929 awards apparently agreed that it was high time to honor the discoverer(s) of vitamins; but who were they? There was a clear case for Grijns, but he had not been re-nominated for that particular year, and it could be said that he was just taking the relatively obvious next steps along the new trail that had been laid down by Eijkman, who was also now an old man in poor health, but there was no doubt that he had taken the first steps in the use of an animal model to investigate the nutritional basis of a clinical disorder affecting millions. Goldberger had been another important contributor, but his recent death put him out of consideration. The clearest evidence for lack of an unknown “something” in a mammalian diet was presented by Gowland Hopkins in 1912. This Cambridge biochemist was already well known for having isolated the amino acid tryptophan from a protein and demonstrated its essential nature. He fed young rats on an experimental diet, half of them receiving a daily milk supplement, and only those receiving milk grew well, Hopkins suggested that this was analogous to human diseases related to diet, as he had suggested already in a lecture published in 1906. Hopkins, the leader of the “dynamic biochemistry” school in Britain and an influential advocate for the importance of vitamins, was awarded the prize jointly with Eijkman. A door was opened. Recognition of work on the fat-soluble vitamins begun by McCollum. The next award related to vitamins was given in 1934 to George WhippleGeorge Minot and William Murphy “for their discoveries concerning liver therapy in cases of [then incurable pernicious] anemia,” The essential liver factor (cobalamin, or vitamin B12) was isolated in 1948, and Vitamin B12  was absent from plant foods. But William Castle in 1928 had demonstrated that the stomachs of pernicious anemia patients were abnormal in failing to secrete an “intrinsic factor”.

1937   Albert von Szent-Györgyi Nagyrápolt

” the biological combustion processes, with special reference to vitamin C and the catalysis of fumaric acid”

http://www.biocheminfo.org/klotho/html/fumarate.html

structure of fumarate

Szent-Györgyi was a Hungarian biochemist who had worked with Otto Warburg and had a special interest in oxidation-reduction mechanisms. He was invited to Cambridge in England in 1927 after detecting an antioxidant compound in the adrenal cortex, and there, he isolated a compound that he named hexuronic acid. Charles Glen King of the University of Pittsburgh reported success In isolating the anti-scorbutic factor in 1932, and added that his crystals had all the properties reported by Szent-Györgyi for hexuronic acid. But his work on oxidation reactions was also important. Fumarate is an intermediate in the citric acid cycle used by cells to produce energy in the form of adenosine triphosphate (ATP) from food. It is formed by the oxidation of succinate by the enzyme succinate dehydrogenase. Fumarate is then converted by the enzyme fumarase to malate. An enzyme adds water to the fumarate molecule to form malate. The malate is created by adding one hydrogen atom to a carbon atom and then adding a hydroxyl group to a carbon next to a terminal carbonyl group.

In the same year, Norman Haworth from the University of Birmingham in England received a Nobel prize from the Chemistry Committee for having advanced carbohydrate chemistry and, specifically, for having worked out the structure of Szent-Györgyi’s crystals, and then been able to synthesize the vitamin. This was a considerable achievement. The Nobel Prize in Chemistry was shared with the Swiss organic chemist Paul Karrer, cited for his work on the structures of riboflavin and vitamins A and E as well as other biologically interesting compounds. This was followed in 1938 by a further Chemistry award to the German biochemist Richard Kuhn, who had also worked on carotenoids and B-vitamins, including riboflavin and pyridoxine. But Karrer was not permitted to leave Germany at that time by the Nazi regime. However, the American work with radioisotopes at Lawrence Livermore Laboratory, UC Berkeley, was already ushering in a new era of biochemistry that would enrich our studies of metabolic pathways. The importance of work involving vitamins was acknowledged in at least ten awards in the 20th century.

1.   Carpenter, K.J., Beriberi, White Rice and Vitamin B, University of California Press, Berkeley (2000).

2.  Weatherall, M.W. and Kamminga, H., The making of a biochemist: the construction of Frederick Gowland Hopkins’ reputation. Medical History vol.40, pp. 415-436 (1996).

3.  Becker, S.L., Will milk make them grow? An episode in the discovery of the vitamins. In Chemistry and Modern Society (J. Parascandela, editor) pp. 61-83, American Chemical Society,

Washington, D.C. (1983).

4.  Carpenter, K.J., The History of Scurvy and Vitamin C, Cambridge University Press, New York (1986).

Transport and metabolism of exogenous fumarate and 3-phosphoglycerate in vascular smooth muscle.

D R FinderC D Hardin

Molecular and Cellular Biochemistry (Impact Factor: 2.33). 05/1999; 195(1-2):113-21.  http://dx.doi.org/10.1023/A:1006976432578

The keto (linear) form of exogenous fructose 1,6-bisphosphate, a highly charged glycolytic intermediate, may utilize a dicarboxylate transporter to cross the cell membrane, support glycolysis, and produce ATP anaerobically. We tested the hypothesis that fumarate, a dicarboxylate, and 3-phosphoglycerate (3-PG), an intermediate structurally similar to a dicarboxylate, can support contraction in vascular smooth muscle during hypoxia. 3-PG improved maintenance of force (p < 0.05) during the 30-80 min period of hypoxia. Fumarate decreased peak isometric force development by 9.5% (p = 0.008) but modestly improved maintenance of force (p < 0.05) throughout the first 80 min of hypoxia. 13C-NMR on tissue extracts and superfusates revealed 1,2,3,4-(13)C-fumarate (5 mM) metabolism to 1,2,3,4-(13)C-malate under oxygenated and hypoxic conditions suggesting uptake and metabolism of fumarate. In conclusion, exogenous fumarate and 3-PG readily enter vascular smooth muscle cells, presumably by a dicarboxylate transporter, and support energetically important pathways.

Comparison of endogenous and exogenous sources of ATP in fueling Ca2+ uptake in smooth muscle plasma membrane vesicles.

C D HardinL RaeymaekersR J Paul

The Journal of General Physiology (Impact Factor: 4.73). 12/1991; 99(1):21-40.   http://dx.doi.org:/10.1085/jgp.99.1.21

A smooth muscle plasma membrane vesicular fraction (PMV) purified for the (Ca2+/Mg2+)-ATPase has endogenous glycolytic enzyme activity. In the presence of glycolytic substrate (fructose 1,6-diphosphate) and cofactors, PMV produced ATP and lactate and supported calcium uptake. The endogenous glycolytic cascade supports calcium uptake independent of bath [ATP]. A 10-fold dilution of PMV, with the resultant 10-fold dilution of glycolytically produced bath [ATP] did not change glycolytically fueled calcium uptake (nanomoles per milligram protein). Furthermore, the calcium uptake fueled by the endogenous glycolytic cascade persisted in the presence of a hexokinase-based ATP trap which eliminated calcium uptake fueled by exogenously added ATP. Thus, it appears that the endogenous glycolytic cascade fuels calcium uptake in PMV via a membrane-associated pool of ATP and not via an exchange of ATP with the bulk solution. To determine whether ATP produced endogenously was utilized preferentially by the calcium pump, the ATP production rates of the endogenous creatine kinase and pyruvate kinase were matched to that of glycolysis and the calcium uptake fueled by the endogenous sources was compared with that fueled by exogenous ATP added at the same rate. The rate of calcium uptake fueled by endogenous sources of ATP was approximately twice that supported by exogenously added ATP, indicating that the calcium pump preferentially utilizes ATP produced by membrane-bound enzymes.

Evidence for succinate production by reduction of fumarate during hypoxia in isolated adult rat heart cells.

C HohlR OestreichP RösenR WiesnerM Grieshaber

Archives of Biochemistry and Biophysics (Impact Factor: 3.37). 01/1988; 259(2):527-35. http://dx.doi.org:/10.1016/0003-9861(87)90519-4   It has been demonstrated that perfusion of myocardium with glutamic acid or tricarboxylic acid cycle intermediates during hypoxia or ischemia, improves cardiac function, increases ATP levels, and stimulates succinate production. In this study isolated adult rat heart cells were used to investigate the mechanism of anaerobic succinate formation and examine beneficial effects attributed to ATP generated by this pathway. Myocytes incubated for 60 min under hypoxic conditions showed a slight loss of ATP from an initial value of 21 +/- 1 nmol/mg protein, a decline of CP from 42 to 17 nmol/mg protein and a fourfold increase in lactic acid production to 1.8 +/- 0.2 mumol/mg protein/h. These metabolite contents were not altered by the addition of malate and 2-oxoglutarate to the incubation medium nor were differences in cell viability observed; however, succinate release was substantially accelerated to 241 +/- 53 nmol/mg protein. Incubation of cells with [U-14C]malate or [2-U-14C]oxoglutarate indicates that succinate is formed directly from malate but not from 2-oxoglutarate. Moreover, anaerobic succinate formation was rotenone sensitive.

We conclude that malate reduction to succinate occurs via the reverse action of succinate dehydrogenase in a coupled reaction where NADH is oxidized (and FAD reduced) and ADP is phosphorylated. Furthermore, by transaminating with aspartate to produce oxaloacetate, 2-oxoglutarate stimulates cytosolic malic dehydrogenase activity, whereby malate is formed and NADH is oxidized.

In the form of malate, reducing equivalents and substrate are transported into the mitochondria where they are utilized for succinate synthesis.

1953 Hans Adolf Krebs –

 ” discovery of the citric acid cycle” and In the course of the 1920’s and 1930’s great progress was made in the study of the intermediary reactions by which sugar is anaerobically fermented to lactic acid or to ethanol and carbon dioxide. The success was mainly due to the joint efforts of the schools of Meyerhof, Embden, Parnas, von Euler, Warburg and the Coris, who built on the pioneer work of Harden and of Neuberg. This work brought to light the main intermediary steps of anaerobic fermentations.

In contrast, very little was known in the earlier 1930’s about the intermediary stages through which sugar is oxidized in living cells. When, in 1930, I left the laboratory of Otto Warburg (under whose guidance I had worked since 1926 and from whom I have learnt more than from any other single teacher), I was confronted with the question of selecting a major field of study and I felt greatly attracted by the problem of the intermediary pathway of oxidations.

These reactions represent the main energy source in higher organisms, and in view of the importance of energy production to living organisms (whose activities all depend on a continuous supply of energy) the problem seemed well worthwhile studying.   http://www.johnkyrk.com/krebs.html

Interactive Krebs cycle

There are different points where metabolites enter the Krebs’ cycle. Most of the products of protein, carbohydrates and fat metabolism are reduced to the molecule acetyl coenzyme A that enters the Krebs’ cycle. Glucose, the primary fuel in the body, is first metabolized into pyruvic acid and then into acetyl coenzyme A. The breakdown of the glucose molecule forms two molecules of ATP for energy in the Embden Meyerhof pathway process of glycolysis.

On the other hand, amino acids and some chained fatty acids can be metabolized into Krebs intermediates and enter the cycle at several points. When oxygen is unavailable or the Krebs’ cycle is inhibited, the body shifts its energy production from the Krebs’ cycle to the Embden Meyerhof pathway of glycolysis, a very inefficient way of making energy.  

Fritz Albert Lipmann –

 “discovery of co-enzyme A and its importance for intermediary metabolism”.

In my development, the recognition of facts and the rationalization of these facts into a unified picture, have interplayed continuously. After my apprenticeship with Otto Meyerhof, a first interest on my own became the phenomenon we call the Pasteur effect, this peculiar depression of the wasteful fermentation in the respiring cell. By looking for a chemical explanation of this economy measure on the cellular level, I was prompted into a study of the mechanism of pyruvic acid oxidation, since it is at the pyruvic stage where respiration branches off from fermentation.

For this study I chose as a promising system a relatively simple looking pyruvic acid oxidation enzyme in a certain strain of Lactobacillus delbrueckii1.   In 1939, experiments using minced muscle cells demonstrated that one oxygen atom can form two adenosine triphosphate molecules, and, in 1941, the concept of phosphate bonds being a form of energy in cellular metabolism was developed by Fritz Albert Lipmann.

In the following years, the mechanism behind cellular respiration was further elaborated, although its link to the mitochondria was not known.[13]The introduction of tissue fractionation by Albert Claude allowed mitochondria to be isolated from other cell fractions and biochemical analysis to be conducted on them alone. In 1946, he concluded that cytochrome oxidase and other enzymes responsible for the respiratory chain were isolated to the mitchondria. Over time, the fractionation method was tweaked, improving the quality of the mitochondria isolated, and other elements of cell respiration were determined to occur in the mitochondria.[13]

The most important event during this whole period, I now feel, was the accidental observation that in the L. delbrueckii system, pyruvic acid oxidation was completely dependent on the presence of inorganic phosphate. This observation was made in the course of attempts to replace oxygen by methylene blue. To measure the methylene blue reduction manometrically, I had to switch to a bicarbonate buffer instead of the otherwise routinely used phosphate. In bicarbonate, pyruvate oxidation was very slow, but the addition of a little phosphate caused a remarkable increase in rate. The phosphate effect was removed by washing with a phosphate free acetate buffer. Then it appeared that the reaction was really fully dependent on phosphate.

A coupling of this pyruvate oxidation with adenylic acid phosphorylation was attempted. Addition of adenylic acid to the pyruvic oxidation system brought out a net disappearance of inorganic phosphate, accounted for as adenosine triphosphate.   The acetic acid subunit of acetyl CoA is combined with oxaloacetate to form a molecule of citrate. Acetyl coenzyme A acts only as a transporter of acetic acid from one enzyme to another. After Step 1, the coenzyme is released by hydrolysis to combine with another acetic acid molecule and begin the Krebs’ Cycle again.

Hugo Theorell

the nature and effects of oxidation enzymes”

From 1933 until 1935 Theorell held a Rockefeller Fellowship and worked with Otto Warburg at Berlin-Dahlem, and here he became interested in oxidation enzymes. At Berlin-Dahlem he produced, for the first time, the oxidation enzyme called «the yellow ferment» and he succeeded in splitting it reversibly into a coenzyme part, which was found to be flavin mononucleotide, and a colourless protein part. On return to Sweden, he was appointed Head of the newly established Biochemical Department of the Nobel Medical Institute, which was opened in 1937.

Succinate is oxidized by a molecule of FAD (Flavin Adenine Dinucleotide). The FAD removes two hydrogen atoms from the succinate and forms a double bond between the two carbon atoms to create fumarate.

1953

double-stranded-dna

double-stranded-dna

crick-watson-with-their-dna-model.

crick-watson-with-their-dna-model.

Watson & Crick double helix model 

A landmark in this journey

They followed the path that became clear from intense collaborative work in California by George Beadle, by Avery and McCarthy, Max Delbruck, TH Morgan, Max Delbruck and by Chargaff that indicated that genetics would be important.

1965

François Jacob, André Lwoff and Jacques Monod  –

” genetic control of enzyme and virus synthesis”.

In 1958 the remarkable analogy revealed by genetic analysis of lysogeny and that of the induced biosynthesis of ß-galactosidase led François Jacob, with Jacques Monod, to study the mechanisms responsible for the transfer of genetic information as well as the regulatory pathways which, in the bacterial cell, adjust the activity and synthesis of macromolecules. Following this analysis, Jacob and Monod proposed a series of new concepts, those of messenger RNA, regulator genes, operons and allosteric proteins.

Francois Jacob

Having determined the constants of growth in the presence of different carbohydrates, it occurred to me that it would be interesting to determine the same constants in paired mixtures of carbohydrates. From the first experiment on, I noticed that, whereas the growth was kinetically normal in the presence of certain mixtures (that is, it exhibited a single exponential phase), two complete growth cycles could be observed in other carbohydrate mixtures, these cycles consisting of two exponential phases separated by a-complete cessation of growth.

Lwoff, after considering this strange result for a moment, said to me, “That could have something to do with enzyme adaptation.”

“Enzyme adaptation? Never heard of it!” I said.

Lwoff’s only reply was to give me a copy of the then recent work of Marjorie Stephenson, in which a chapter summarized with great insight the still few studies concerning this phenomenon, which had been discovered by Duclaux at the end of the last century.  Studied by Dienert and by Went as early as 1901 and then by Euler and Josephson, it was more or less rediscovered by Karström, who should be credited with giving it a name and attracting attention to its existence.

Lwoff’s intuition was correct. The phenomenon of “diauxy” that I had discovered was indeed closely related to enzyme adaptation, as my experiments, included in the second part of my doctoral dissertation, soon convinced me. It was actually a case of the “glucose effect” discovered by Dienert as early as 1900.   That agents that uncouple oxidative phosphorylation, such as 2,4-dinitrophenol, completely inhibit adaptation to lactose or other carbohydrates suggested that “adaptation” implied an expenditure of chemical potential and therefore probably involved the true synthesis of an enzyme.

With Alice Audureau, I sought to discover the still quite obscure relations between this phenomenon and the one Massini, Lewis, and others had discovered: the appearance and selection of “spontaneous” mutants.   We showed that an apparently spontaneous mutation was allowing these originally “lactose-negative” bacteria to become “lactose-positive”. However, we proved that the original strain (Lac-) and the mutant strain (Lac+) did not differ from each other by the presence of a specific enzyme system, but rather by the ability to produce this system in the presence of lactose.  This mutation involved the selective control of an enzyme by a gene, and the conditions necessary for its expression seemed directly linked to the chemical activity of the system.

1974

Albert Claude, Christian de Duve and George E. Palade –

” the structural and functional organization of the cell”.

I returned to Louvain in March 1947 after a period of working with Theorell in Sweden, the Cori’s, and E Southerland in St. Louis, fortunate in the choice of my mentors, all sticklers for technical excellence and intellectual rigor, those prerequisites of good scientific work. Insulin, together with glucagon which I had helped rediscover, was still my main focus of interest, and our first investigations were accordingly directed on certain enzymatic aspects of carbohydrate metabolism in liver, which were expected to throw light on the broader problem of insulin action. But I became distracted by an accidental finding related to acid phosphatase, drawing most of my collaborators along with me. The studies led to the discovery of the lysosome, and later of the peroxisome.

In 1962, I was appointed a professor at the Rockefeller Institute in New York, now the Rockefeller University, the institution where Albert Claude had made his pioneering studies between 1929 and 1949, and where George Palade had been working since 1946.  In New York, I was able to develop a second flourishing group, which follows the same general lines of research as the Belgian group, but with a program of its own.

1968

Robert W. Holley, Har Gobind Khorana and Marshall W. Nirenberg –

“interpretation of the genetic code and its function in protein synthesis”.

1969

Max Delbrück, Alfred D. Hershey and Salvador E. Luria –

” the replication mechanism and the genetic structure of viruses”.

1975 David Baltimore, Renato Dulbecco and Howard Martin Temin –

” the interaction between tumor viruses and the genetic material of the cell”.

1976

Baruch S. Blumberg and D. Carleton Gajdusek –

” new mechanisms for the origin and dissemination of infectious diseases” The editors of the Nobelprize.org website of the Nobel Foundation have asked me to provide a supplement to the autobiography that I wrote in 1976 and to recount the events that happened after the award. Much of what I will have to say relates to the scientific developments since the last essay. These are described in greater detail in a scientific memoir first published in 2002 (Blumberg, B. S., Hepatitis B. The Hunt for a Killer Virus, Princeton University Press, 2002, 2004).

1980

Baruj Benacerraf, Jean Dausset and George D. Snell 

” genetically determined structures on the cell surface that regulate immunological reactions”.

1992

Edmond H. Fischer and Edwin G. Krebs 

“for their discoveries concerning reversible protein phosphorylation as a biological regulatory mechanism”

1994

Alfred G. Gilman and Martin Rodbell –

“G-proteins and the role of these proteins in signal transduction in cells”

2011

Bruce A. Beutler and Jules A. Hoffmann –

the activation of innate immunity and the other half to Ralph M. Steinman – “the dendritic cell and its role in adaptive immunity”.

Renato L. Baserga, M.D.

Kimmel Cancer Center and Keck School of Medicine

Dr. Baserga’s research focuses on the multiple roles of the type 1 insulin-like growth factor receptor (IGF-IR) in the proliferation of mammalian cells. The IGF-IR activated by its ligands is mitogenic, is required for the establishment and the maintenance of the transformed phenotype, and protects tumor cells from apoptosis. It, therefore, serves as an excellent target for therapeutic interventions aimed at inhibiting abnormal growth. In basic investigations, this group is presently studying the effects that the number of IGF-IRs and specific mutations in the receptor itself have on its ability to protect cells from apoptosis.

This investigation is strictly correlated with IGF-IR signaling, and part of this work tries to elucidate the pathways originating from the IGF-IR that are important for its functional effects. Baserga’s group has recently discovered a new signaling pathway used by the IGF-IR to protect cells from apoptosis, a unique pathway that is not used by other growth factor receptors. This pathway depends on the integrity of serines 1280-1283 in the C-terminus of the receptor, which bind 14.3.3 and cause the mitochondrial translocation of Raf-1.

Another recent discovery of this group has been the identification of a mechanism by which the IGF-IR can actually induce differentiation in certain types of cells. When cells have IRS-1 (a major substrate of the IGF-IR), the IGF-IR sends a proliferative signal; in the absence of IRS-1, the receptor induces cell differentiation. The extinction of IRS-1 expression is usually achieved by DNA methylation.

Janardan Reddy, MD

Northwestern University

The central effort of our research has been on a detailed analysis at the cellular and molecular levels of the pleiotropic responses in liver induced by structurally diverse classes of chemicals that include fibrate class of hypolipidemic drugs, and phthalate ester plasticizers, which we designated hepatic peroxisome proliferators. Our work has been central to the establishment of several principles, namely that hepatic peroxisome proliferation is associated with increases in fatty acid oxidation systems in liver, and that peroxisome proliferators, as a class, are novel nongenotoxic hepatocarcinogens.

We introduced the concept that sustained generation of reactive oxygen species leads to oxidative stress and serves as the basis for peroxisome proliferator-induced liver cancer development. Furthermore, based on the tissue/cell specificity of pleiotropic responses and the coordinated transcriptional regulation of fatty acid oxidation system genes, we postulated that peroxisome proliferators exert their action by a receptor-mediated mechanism. This receptor concept laid the foundation for the discovery of

  • a three member peroxisome proliferator-activated receptor (PPARalpha-, ß-, and gamma) subfamily of nuclear receptors.
  •  PPARalpha is responsible for peroxisome proliferator-induced pleiotropic responses, including
    • hepatocarcinogenesis and energy combustion as it serves as a fatty acid sensor and regulates fatty acid oxidation.

Our current work focuses on the molecular mechanisms responsible for PPAR action and generation of fatty acid oxidation deficient mouse knockout models. Transcription of specific genes by nuclear receptors is a complex process involving the participation of multiprotein complexes composed of transcription coactivators.  

Jose Delgado de Salles Roselino, Ph.D.

Leloir Institute, Brazil

Warburg effect, in reality “Pasteur-effect” was the first example of metabolic regulation described. A decrease in the carbon flux originated at the sugar molecule towards the end metabolic products, ethanol and carbon dioxide that was observed when yeast cells were transferred from anaerobic environmental condition to an aerobic one. In Pasteur´s works, sugar metabolism was measured mainly by the decrease of sugar concentration in the yeast growth media observed after a measured period of time. The decrease of the sugar concentration in the media occurs at great speed in yeast grown in anaerobiosis condition and its speed was greatly reduced by the transfer of the yeast culture to an aerobic condition. This finding was very important for the wine industry of France in Pasteur time, since most of the undesirable outcomes in the industrial use of yeast were perceived when yeasts cells took very long time to create a rather selective anaerobic condition. This selective culture media was led by the carbon dioxide higher levels produced by fast growing yeast cells and by a great alcohol content in the yeast culture media. This finding was required to understand Lavoisier’s results indicating that chemical and biological oxidation of sugars produced the same calorimetric results. This observation requires a control mechanism (metabolic regulation) to avoid burning living cells by fast heat released by the sugar biological oxidative processes (metabolism). In addition, Lavoisier´s results were the first indications that both processes happened inside similar thermodynamics limits.

In much resumed form, these observations indicates the major reasons that led Warburg to test failure in control mechanisms in cancer cells in comparison with the ones observed in normal cells. Biology inside classical thermo dynamics poses some challenges to scientists. For instance, all classical thermodynamics must be measured in reversible thermodynamic conditions. In an isolated system, increase in P (pressure) leads to decrease in V (volume) all this in a condition in which infinitesimal changes in one affects in the same way the other, a continuum response. Not even a quantic amount of energy will stand beyond those parameters. In a reversible system, a decrease in V, under same condition, will led to an increase in P.

In biochemistry, reversible usually indicates a reaction that easily goes from A to B or B to A. This observation confirms the important contribution of E Schrodinger in his What´s Life: “This little book arose from a course of public lectures, delivered by a theoretical physicist to an audience of about four hundred which did not substantially dwindle, though warned at the outset that the subject-matter was a difficult one and that the lectures could not be termed popular, even though the physicist’s most dreaded weapon, mathematical deduction, would hardly be utilized. The reason for this was not that the subject was simple enough to be explained without mathematics, but rather that it was much too involved to be fully accessible to mathematics.”

Hans Krebs describes the cyclic nature of the citrate metabolism. Two major research lines search to understand the mechanism of energy transfer that explains how ADP is converted into ATP. One followed the organic chemistry line of reasoning and therefore, searched how the breakdown of carbon-carbon link could have its energy transferred to ATP synthesis. A major leader of this research line was B. Chance who tried to account for two carbon atoms of acetyl released as carbon dioxide in the series of Krebs cycle reactions. The intermediary could store in a phosphorylated amino acid the energy of carbon-carbon bond breakdown. This activated amino acid could transfer its phosphate group to ADP producing ATP. Alternatively, under the possible influence of the excellent results of Hodgkin and Huxley a second line of research appears.

The work of Hodgkin & Huxley indicated the storage of electrical potential energy in transmembrane ionic asymmetries and presented the explanation for the change from resting to action potential in excitable cells. This second line of research, under the leadership of P Mitchell postulated a mechanism for the transfer of oxide/reductive power of organic molecules oxidation through electron transfer as the key for energetic transfer mechanism required for ATP synthesis. Paul Boyer could present how the energy was transduced by a molecular machine that changes in conformation in a series of 3 steps while rotating in one direction in order to produce ATP and in opposite direction in order to produce ADP plus Pi from ATP (reversibility). Nonetheless, a victorious Peter Mitchell obtained the correct result in the conceptual dispute, over the B. Chance point of view, after he used E. Coli mutants to show H gradients in membrane and its use as energy source.

However, this should not detract from the important work of Chance. B. Chance got the simple and rapid polarographic assay method of oxidative phosphorylation and the idea of control of energy metabolism that bring us back to Pasteur. This second result seems to have been neglected in searching for a single molecular mechanism required for the understanding of the buildup of chemical reserve in our body. In respiring mitochondria the rate of electron transport, and thus the rate of ATP production, is determined primarily by the relative concentrations of ADP, ATP and phosphate in the external media (cytosol) and not by the concentration of respiratory substrate as pyruvate. Therefore, when the yield of ATP is high as is in aerobiosis and the cellular use of ATP is not changed, the oxidation of pyruvate and therefore of glycolysis is quickly (without change in gene expression), throttled down to the resting state. The dependence of respiratory rate on ADP concentration is also seen in intact cells. A muscle at rest and using no ATP has very low respiratory rate.

I have had an ongoing discussion with Jose Eduardo de Salles Roselino, inBrazil. He has made important points that need to be noted.

  1. The constancy of composition which animals maintain in the environment surrounding their cells is one of the dominant features of their physiology. Although this phenomenon, homeostasis, has held the interest of biologists over a long period of time, the elucidation of the molecular basis for complex processes such as temperature control and the maintenance of various substances at constant levels in the blood has not yet been achieved. By comparison, metabolic regulation in microorganisms is much better understood, in part because the microbial physiologist has focused his attention on enzyme-catalyzed reactions and their control, as these are among the few activities of microorganisms amenable to quantitative study. Furthermore, bacteria are characterized by their ability to make rapid and efficient adjustments to extensive variations in most parameters of their environment; therefore, they exhibit a surprising lack of rigid requirements for their environment, and appears to influence it only as an incidental result of their metabolism. Animal cells on the other hand have only a limited capacity for adjustment and therefore require a constant milieu. Maintenance of such constancy appears to be a major goal in their physiology (Regulation of Biosynthetic Pathways H.S. Moyed and H EUmbarger Phys Rev,42 444 (1962)).
  2. A living cell consists in a large part of a concentrated mixture of hundreds of different enzymes, each a highly effective catalyst for one or more chemical reactions involving other components of the cell. The paradox of intense and highly diverse chemical activity on the one hand and strongly poised chemical stability (biological homeostasis) on the other is one of the most challenging problems of biology (Biological feedback Control at the molecular Level D.E. Atkinson Science vol. 150: 851, 1965). Almost nothing is known concerning the actual molecular basis for modulation of an enzyme`s kinetic behavior by interaction with a small molecule. (Biological feedback Control at the molecular Level D.E. Atkinson Science vol. 150: 851, 1965). In the same article, since the core of Atkinson´s thinking seems to be strongly linked with Adenylates as regulatory effectors, the previous phrases seems to indicate a first step towards the conversion of homeostasis to an intracellular phenomenon and therefore, one that contrary to Umbarger´s consideration could be also studied in microorganisms.
  3.  Most biochemical studies using bacteria, were made before the end of the third upper part of log growth phase. Therefore, they could be considered as time-independent as S Luria presented biochemistry in Life an Unfinished Experiment. The sole ingredient on the missing side of the events that led us into the molecular biology construction was to consider that proteins, a macromolecule, would never be affected by small molecules translational kinetic energy. This, despite the fact that in a catalytic environment and its biological implications S Grisolia incorporated A K Balls observation indicating that the word proteins could be related to Proteus an old sea god that changed its form whenever he was subjected to inquiry (Phys Rev v 4,657 (1964).
  1. In D.E. Atkinson´s work (Science vol 150 p 851, 1965), changes in protein synthesis acting together with factors that interfere with enzyme activity will lead to “fine-tuned” regulation better than enzymatic activity regulation alone. Comparison of glycemic regulation in granivorous and carnivorous birds indicate that when no important nutritional source of glucose is available, glycemic levels can be kept constant in fasted and fed birds. The same was found in rats and cats fed on high protein diets. Gluconeogenesis is controlled by pyruvate kinase inhibition. Therefore, the fact that it can discriminate between fasting alone and fasting plus exercise (carbachol) requirement of gluconeogenic activity (correspondent level of pyruvate kinase inhibition) the control of enzyme activity can be made fast and efficient without need for changes in genetic expression (20 minute after stimulus) ( Migliorini,R.H. et al Am J. Physiol.257 (Endocrinol. Met. 20): E486, 1989). Regrettably, this was not discussed in the quoted work. So, when the control is not affected by the absorption of nutritional glucose it can be very fast, less energy intensive and very sensitive mechanism of control despite its action being made in the extracellular medium (homeostasis).

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Recently, a lot of attention has been given to developing better cancer diagnostic methods. Finding and validating cancer biomarkers has become an important tool for scientists and physicians in the frontline battle against this chronic epidemic. Various methods (e.g. microarray analysis) have been used to glean which specific proteins whose perturbations (upregulation or downregulation) are an indication of cancerous (or pre-cancerous) activity. One such molecule that is often mentioned is stanniocalcin (Chang et al., 2003).
It is a small family with two members, STC1 and STC2, that are thought to be secreted glycosylated proteins. And, both are found in a wide variety of cancers. Originally found in bony fish as a calcium/phosphate-regulating hormone, it is a homodimeric phosphoglycoprotein structurally. And, the proteins are thought to function in an autocrine/paracrine (rather than the classic endocrine) loop regulating intracellular calcium and/or phosphate levels (Yoskiko and Aubin, 2004).

Originally, STC1 showed up in a screen for mRNA differential display for genes that were related to cellular immortalization (Chang et al., 1995). While STC1 and STC2 are expressed in different tissues, they seem to have a special relationship to the reproductive tissues, hinting at a role in reproduction: STC1 expression is highest in the ovaries and STC2 is induced by the estrogen receptor. And, both are involved in breast cancer pathology. Other tissues where they are highly expressed include the kidney, bones, muscle, neurons (Worthington et al., 1999).
Fig2Physiologically, the proteins play a role in calcium and Pi homeostasis as demonstrated by studies on mouse transgenic models. In addition to cancer, the protein has been linked to atherosclerosis, hypoxia response and in wound repair (Lal et al., 2001; Iyer et al., 1999). Pharmacologically, an STC1 receptor has been deduced from studies and thought to be localized to the mitochondria where it has been shown to have a relationship with the mitochondrial electron transfer (McCudden et al., 2002).  Recent studies show that STC1 activates the mitochondrial antioxidant pathway through its regulation of intracellular calcium (Sheikh-Hamad, 2010).  Overall, STC1 and STC2 are thought to be secreted as phosphoproteins as demonstrated by coimmunoprecipitations of cellular lysates. And, it’s thought the proteins play a role in mineralizing tissues (e.g. bone) to control the levels of calcium and Pi via their influence on calcium channels and sodium/Pi co-transporters.  A schematic diagram showing how stannniocalcin might be have pro-apoptic functions is shown in Figure 1 (Yeung et al., 2012).

Table1However, stanniocalcin’s more prominent role is arguably as a cancer biomarker. Its expression has been shown to be affected in a number of different cancer pathologies. Table 1 shows a representative list of cancers where stanniocalcin levels are differentially expressed depending on the cancer. Thus, it appears that stanniocalcin is a good candidate cancer biomarker.  It is hypothesized that this is due in part to its role in tumor vasculature (Chang et al., 2003).  It should be noted that the list is but a brief compilation while stanniocalcin has been linked to other cancers as well.

At Vanderbilt University, studies were being done to evaluate the expression levels of YAP1 (Hippo pathway) during CNS development. Surprisingly, it was restricted to the choroid plexus (CP), a layer of epithelial cells lining the ventricles of the brain which are thought to act as a filtration system removing metabolic wastes. As such, primary cultures from mice (P=4) were cultured and evaluated. And, it was reported previously that stanniocalcin is expressed highly in CP. The expression of STC1 in choroid plexus epithelium would be consistent with the notion that stanniocalcin may have a role in regulation of calcium and Pi levels in cerebrospinal fluid (Franzén  et al., 2000). To verify that the primary culture were indeed CP cells, an immunofluorescent (IF) assay was done with CP markers including STC1 and STC2.  The following IF micrograph shows a generally a nuclear localization of STC2. In addition, since an extra channel was available for immunofluorescence, an acetlyated tubulin antibody was used to evaluate the cytoskelton.  Surprisingly, there was colocalization of this protein to the primary cilia/centriole (Fig. 2: Blue = DAPI (Nucleus); Red = Acetylated tubulin (primary cilia/centriole); Green = STC2.  The boxed regions show representative colocalizations of STC2 to the primary cilium/centriole).

Fig1

If the colocalization of the STC2 antibody is correct, this will be the first time that stanniocalcin has been localized to the primary cilium. Since the primary cilium has already been linked to different cancer pathologies due to its role as the gatekeeper of the cell cycle (Veland et al., 2009), it seems interesting that another cancer biomarker may now also be linked to the primary cilium.  Studies have shown that STC1 affects the cell cycle by regulating cyclin D1 and ERK 1/2 (Wang et al., 2012).  Thus, it raises more questions:

Is there cross-talk between the mitochondria and the primary cilium via stanniocalcin which might then have further repercussions on cell cycle fate?

Is the the primary cilia helping to coordinate calcium/Pi signal systems?

It almost seems logical that there would be a link between the primary cilium and this important class of protein due to their respective roles in cancer.  But, further research (including validation) is needed to further delineate whether this relationship exists.

REFERENCES

Chang AC, Janosi J, Hulsbeek M, de Jong D, Jeffrey KJ, Noble JR, Reddel RR 1995 A novel human cDNA highly homologous to the fish hormone stanniocalcin. Mol Cell Endocrinol. 112:241-247.

Chang AC, Jellinek DA, Reddel RR. 2003 Mammalian stanniocalcins and cancer. Endocr Relat Cancer 10:359-373.

Franzén AM, Zhang KZ, Westberg JA, Zhang WM, Arola J, Olsen HS, Andersson LC 2000 Expression of stanniocalcin in the epithelium of human choroid plexus. Brain Res 887:440-443.

Iyer VR, Eisen MB, Ross DT, Schuler G, Moore T, Lee JCF, Trent JM, Staudt LM, Hudson J Jr, Boguski MS, Lashkari D, Shalon D, Botstein D & Brown PO 1999 The transcriptional program in the response of human fibroblasts to serum. Science 283 83–87.

Lal A, Peters H, St Croix B, Haroon ZA, Dewhirst MW, Strausberg RL, Kaanders JHAM, van der Kogel AJ & Riggins GJ 2001 Transcriptional response to hypoxia in human tumors. J National Cancer Institute 93 1337–1343.

McCudden CR, James KA, Hasilo C & Wagner GF 2002 Characterization of mammalian stanniocalcin receptors: mitochondrial targeting of ligand and receptor for regulation of cellular metabolism. J Biol Chem 277: 45249–45258.

Sheikh-Hamad D. 2010  Mammalian stanniocalcin-1 activates mitochondrial antioxidant pathways: new paradigms for regulation of macrophages and endothelium. Am J Physiol Renal Physiol. 298:F248-F254.

Veland IR, Awan A, Pedersen LB, Yoder BK, Christensen ST 2009 Primary cilia and signaling pathways in mammalian development, health and disease. Nephron Physiol 111:39-53.

Wang H, Wu K, Sun Y, Li Y, Wu M, Qiao Q, Wei Y, Han ZG, Cai B. 2012 STC2 is upregulated in hepatocellular carcinoma and promotes cell proliferation and migration in vitro. BMB Rep. 45:629-634.

Worthington RA, Brown L, Jellinek D, Chang AC, Reddel RR, Hambly BD, Barden JA. 1999 Expression and localisation of stanniocalcin 1 in rat bladder, kidney and ovary. Electrophoresis 20:2071-2076.

Yeung BH, Law AY, Wong CK 2012 Evolution and roles of stanniocalcin. Mol Cell Endocrinol 349:272-280.

Yoshiko Y and Aubin JE 2004 Stanniocalcin 1 as a pleiotropic factor in mammals. Peptides 25:1663-1669.

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