BoneKEy-Osteovision | Meeting Reports
Meeting report from the 23rd annual meeting of the American society for bone and mineral research12th-16th October, 2001 in Phoenix, Arizona, USA
J E Dennis
E T Keller
M A Petit
J C Prior
C F Njeh
G J Strewler
S Zeni
DOI:10.1138/2001043
IntroductionBoneKEy-Osteovision covered this year's Annual Meeting of the ASBMR with a team of seven reporters. In the Meeting Report we have tried to capture recurring themes in oral and poster presentations. To find topics of interest to you, use the Table of Contents below to navigate through the text of the Meeting Report - just click on topics. Through the courtesy of ASBMR, the report is linked to the Abstracts on line, or you may find abstracts in the 2001 ASBMR Annual Meeting Program and Abstracts. We have also included references to some recent papers and to Commentaries published in BoneKEy-Osteovision. They are cited in the text, and links to abstracts or full-text have been included in the References list at the end.
This is an experiment in using web resources to produce a meeting report. We couldn't cover every important theme that ran through the meeting and we apologize if we have slighted an area of interest to you. Please let us know how you like it by sending us your comments. GJS.
Parathyroid hormone, PTHrP and their receptorGordon J Strewler, Susana Zeni
Lactation and PTHrP. One of the highlights of the meeting was the elegant demonstration that PTHrP is indeed, as was expected, the “missing” regulator of bone mass during lactation (ASBMR-Abs.1001). To ensure that calcium can be delivered into the newborn in the face of adversity, homeostatic mechanisms permit the sacrifice of maternal bone mass during lactation. This adaptation protects even against severe maternal calcium deficiency, and occurs despite hypoparathyroidism or vitamin D deficiency - thus, neither of the classic regulators of calcium homeostasis is involved. As a calciotropic agent that is expressed in lactating mammary tissue, PTHrP was a candidate regulator. In this report (ASBMR-Abs.1001), selective removal of the PTHrP gene from breast using cre-lox technology eliminates PTHrP from the maternal circulation and preserves maternal bone mass during lactation. PTHrP is largely a local regulator, although it causes hypercalcemia when it is inappropriately secreted into the systemic circulation by malignant tumors, and this report is the first evidence for a systemic role of PTHrP in the physiology of the normal adult. Deletion of the gene from mammary tissue also also removes PTHrP completely from milk, so that as a dividend of the model, the role of PTHrP in the newborn can also be explored. Newborn mice nursed with PTHrP-deficient milk had increased bone mass, strongly suggesting that PTHrP in milk is a regulator of bone metabolism in the newborn (personal communication, John Wysolmerski). GJS.
Knockout of PTH. Ablation of the PTHrP gene causes abnormalities of endochondral bone formation and hypocalcemia, indicating that PTH cannot replace the loss of PTHrP. Ablation of the shared PTH/PTHrP receptor has a more severe, though similar phenotype. One possible explanation is that PTH as well as PTHrP contributes to normal endochondral bone formation. Two groups have used different methods to ablate PTH expression. One group studied the Hoxa3 knockout, in which deletion of a hox gene expressed in the branchial arches produces a DiGeorge-like syndrome including absence of the parathyroid glands (ASBMR-Abs.1086). The other one directly targeted the PTH gene (ASBMR-Abs.1085). Both concluded that the absence of PTH plus PTHrP produced a more severe phenotype than absence of PTHrP alone: mice were smaller, had shorter limbs, and had a more severe defect in chondrocyte proliferation than did PTHrP(-/-) mice. Both Hoxa(-/-) and PTHrP(-/-) mice were hypocalcemic, and the double knockout mice were severely hypocalcemic. Thus, PTH has effects on growth plate development, albeit smaller than the effects of PTHrP, and PTH is the dominant regulator of systemic fetal calcium homeostasis. GJS.
PTH Receptor Interactions. Aside from well-known relationships with G proteins and β-arrestins, the dancing partners of the PTH/PTHrP receptor (PTH1R) are not known. Using the C-terminal tail of PTH1R as bait in a yeast two-hybrid screen, NHERF2 was identified as a partner of the receptor (ASBMR-Abs.1090). NHERF2 (sodium-hydrogen exchanger regulatory factor-2) is an adapter protein that regulates display of the sodium-proton antiporter (NHE) on the apical surface of the renal tubule cells. Binding of PTH1R to NHERF2 was confirmed, both in vitro and in transfected cells. The most striking finding of the report was that coexpression of NHERF2 with PTH1R “switched” the signaling pathway of the receptor from primarily cAMP to phospholipase C. This accords with previous reports that the apical PTH1R, unlike the basolateral receptor, signals mainly through phospholipase C, and raises the possibility that macromolecular complexes of PTH1R/NHERF2/NHE may exist on apical renal membranes. Similar trimolecular interactions of the PTH1R, NHERF2 and the sodium-phosphate cotransporter are also possible, as Murer has reported evidence that NHERF's regulate trafficking of the phosphate transporter. GJS.
Medical Therapy of Primary Hyperparathyroidism. On the clinical side, a promising treatment of primary hyperparathyroidism was reported (ASBMR-Abs.1106). An interim analysis showed that the calcimimetic AMG 073 was effective in the treatment of mild to moderate primary hyperparathyroidism (baseline serum calcium 10.3-12.5 mg/dl). This was a 24-week interim analysis of a randomized, placebo-controlled multicenter trial of the agent, which acts at the parathyroid calcium receptor to increase the sensitivity to calcium and thereby inhibit the secretion of parathyroid hormone (). Patients were titrated to an effective dose and then followed on that dose for 12 weeks. Serum calcium was normalized in over 85% of patients receiving active drug. There was no difference in the side-effect profile between AMG 073 and placebo. While preliminary, the interim analysis supports the safety and efficacy of a calcimimetic agent as the first specific medical treatment of primary hyperparathyroidism. GJS.
Osteitis Fibrosa Cystica. Osteitis fibrosa cystica occurs in hyperparathyroidism. To create a model of osteitis fibrosa, PTH(1-34) was infused continuously into adult rats. This produces moderate hypercalcemia, an increase in bone formation and resorption and extensive marrow fibrosis (ASBMR-Abs.1084). Using cDNA microarray techniques, continuous PTH upregulates the expression of platelet-derived growth factor A chain (PDGF-A), a chemotactic factor that increases fibroblast proliferation, suggesting that PDGF-A excess is responsible for PTH-induced marrow fibrosis. To confirm the role of PDGF in osteitis fibrosa, trapidil, an inhibitor of PDGF receptor signaling, was used. Co-treatment with continuous PTH and trapidil markedly reduces marrow fibrosis, at the same time decreasing without normalizing hypercalcemia and maintaining the effect of PTH on bone formation. It appears that signaling through the PDGF receptor may be involved in the pathogenesis of marrow fibrosis in hyperparathyroid states. SZ.
PTH Assays. The new immunoradiometric assay for whole PTH (wPTH) detects only PTH (1-84), while the intact PTH IRMA (iPTH) in common use measures the N-truncated PTH fragment PTH (~7-84) in addition to PTH (1-84). The N-truncated fragment is a weak antagonist at the PTH1 receptor and may be an agonist at the putative C-terminal PTH receptor. There is evidence of intracellular degradation of PTH in primary hyperparathyroidism (PHPT). PTH assays in adenomatous parathyroid glands and in serum of PHPT patients demonstrate the presence of both PTH (1-84) and the N-truncated PTH fragment (ASBMR-Abs.1081). In parathyroid adenoma and in serum, iPTH is higher than wPTH, and the iPTH levels in adenomas correlate with wPTH. The wPTH mean percentage in adenomatous gland and in serum of PHPT patients is similar and no different from the 65% of bioactive hormone present in normal subjects. Thus, the N-truncated PTH fragment is produced by the parathyroid adenomas of PHPT patients, and metabolism in liver and kidney does not alter the proportion of circulating bioactive PTH. SZ.
CartilageGordon J Strewler, Evan T Keller
Hypoxia and Cartilage. Cartilage is famously avascular, and it has been presumed that cartilage, absent capillary blood flow, is poorly oxygenated. An important paper (ASBMR-Abs.1002) reported that the central growth plate is indeed hypoxic, though the periphery is well oxygenated. HIF-1α is an important regulator of genes that must respond to hypoxia. Conditional deletion of the HIF-1α gene in cartilage produced massive cell death in the centers of growth plates, and mice homozygous for the defect were short-limbed and died at birth. The expression of VEGF and glycolytic enzymes was reduced in HIF-1α deleted growth plates. Thus, the threat of hypoxia to the growth plate is real, and active upregulation of compensatory genes is necessary to avoid apoptotic cell death. GJS.
Chondrocyte Proliferation. PTHrP acts in the growth plate to keep chondrocytes in the proliferative pool. Under the control of Indian hedgehog, PTHrP thus regulates the transition from proliferative to hypertrophic states. Absence of the genes for the cell cycle regulators p57kip2 or the rb-related genes p107 and p130 prolongs proliferation and delays the appearance of hypertrophic chondrocytes and mineralization of the bone collar, suggesting that these genes are required for cessation of chondrocyte proliferation. To study the relationship of PTHrP signaling to these cell cycle regulators, double and triple knockout mice were created (ASBMR-Abs.1099). PTHrP/p57 double knockout mice and PTHrP/p107/p130 triple knockout mice have partial reversal of the PTHrP null phenotype. The zone of proliferation is increased and the onset of hypertrophy occurs at about the right time. Thus PTHrP acts partly through downstream effects on these negative cell cycle regulators, not only with regard to proliferation but also to differentiation. GJS.
C-Type Natriuretic Peptide And Ossification. The list of molecules that effect bone remodeling continues to expand. Recently, C-type natriuretic peptide (CNP), an extracellular signaling molecule that increases cGMP has been implicated in increased endochondral ossification (). Messenger RNA for the CNP receptor, GC-B, was identified in the proliferative and prehypertrophic zones of the growth plate (ASBMR-Abs.1013). Deletion of the CNP gene product resulted in dwarfism associated with diminished longitudinal growth of vertebrae, tail and limb bones, and decreased femoral and tibial cortical thickness (ASBMR-Abs.1013,1014). In these animals, the growth plate hypertrophic zone was decreased. In vitro studies revealed that CNP increased alkaline phosphatase activity in osteoblast-like cell lines (ASBMR-Abs.SU217). These studies demonstrate that CNP is an endogenous positive regulator of ossification. ETK.
Regulating osteoblast expressionJames E Dennis
C/EBPβ, a transcription factor linked to adipogenesis, can now to linked to osteogenesis. C/EBPβ was shown to be expressed in calvarial cells, and overexpression of C/EBPβ in 10T1/2 cells induced alkaline phosphatase and osteocalcin expression (ASBMR-Abs.1101). Two independent investigations showed data indicating that C/EBP and Cbfa1 bind to an upstream element of the OC promoter and synergistically upregulate OC expression (ASBMR-Abs.-1154, 1101). These results were supported by another study in which transgenic expression of a truncated, dominant negative C/EBPβ caused not only a reduction in adipose tissue, but also a reduction in bone mass which was not due to effects on resorption (ASBMR-Abs.1102). JED.
Indian Hedgehog and Osteoblasts. The essential role of Indian hedgehog (Ihh) in bone collar and spongiosa formation was investigated in mouse chimeras containing wild-type cells and cells derived from Smoothened (Smo) K/Os which were marked with β-gal (ASBMR-Abs.1031). (Smoothened encodes a membrane bound protein that mediates Hedgehog signaling.) The results demonstrated that Smo-deficient cells were incapable of contributing to bone formation either in the bone collar or the primary spongiosa, thus implicating Ihh in formation of the primary spongiosa. Interestingly, Smo-knockout mice were able to form craniofacial bones, indicating that neural crest-derived cells are regulated differently than paraxial or lateral plate mesoderm-derived osteogenic cells. JED.
Bone morphogenetic proteinsEvan T Keller, James E Dennis
eNOS and BMP's. Bone morphogenetic proteins (BMPs) are a large class of molecules in the transforming growth factor-beta family. Statins, drugs that are used to lower cholesterol, were previously identified to induce bone formation, in part, through induction of BMP-2. An inhibitor of endothelial nitric oxide synthase (eNOS), L-NAME, diminished statin-mediated bone formation by 86% in murine calvarial cultures (ASBMR-Abs.1018). Mice deficient for eNOS (eNOS -/-) demonstrate osteopenia (ASBMR-Abs.M056). Furthermore, statin-induced bone formation in calvaria from eNOS -/- mice was reduced compared to those from eNOS +/+ mice. Statins inhibit the HMG-CoA reductase pathway, which is required for production of mevalonate. It was found that mevalonate reduced BMP-2 expression. Taken together, these data suggest that statins inhibit eNOS production, resulting in decreased BMP-2 expression, and that mevalonate is a natural inhibitor of bone production. ETK.
Sclerostin. Another inhibitor of bone production, sclerostin, was shown to mediate its inhibitory effects through blocking BMP-induced bone formation (ASBMR-Abs.-1109,SU033). Sclerostin, a product of the SOST gene, contains a cysteine-knot similar to other BMP-antagonist family molecules (). SOST mRNA increased in bone marrow cultures after the onset of mineralization and addition of recombinant sclerostin blocked BMP-6 and BMP-4-induced alkaline phosphatase (ALP) activity in SF-9 cells (ASBMR-Abs.1109). Sclerostin binds to several BMPs (ASBMR-Abs.SU034). In situ hybridization revealed SOST mRNA expression in mineralized bones of mice and humans, although it was not clear what cells expressed the SOST mRNA (ASBMR-Abs.1109). These studies suggest that sclerostin operates as a negative-feedback regulator of BMP-induced mineralization. ETK.
Smads. In addition to blocking BMPs through direct binding, such as noggin and sclerostin, other regulatory mechanisms on the signaling pathways downstream of BMPs exist. For example, upon binding to their receptors, BMPs induce Smad activity, which results in gene transcription. Smad 4 is a common mediator of TGF-beta, activin and BMP signaling. In a yeast two-hybrid assay, it was demonstrated that JAB-1, a protein initially identified as a co-activator of c-Jun, bound Smad 4, but neither Smad 1 nor Smad 3 (ASBMR-Abs.1110). Inhibitors of the 26S proteasome blocked JAB-1-induced degradation of Smad 4. Furthermore, JAB-1 induced ubiquitination of Smad 4 (ASBMR-Abs.1110). These data demonstrate that JAB-1 regulates Smad-4 degradation through the ubiquitin-proteasome pathway, suggesting that JAB-1 may modulate BMP-induced activation and bone production. Evidence was also presented that osteogenic expression could be regulated through Smad1 via the action of proteasomes. Proteosome inhibitors increase bone formation and were shown to dramatically increased the amounts of Smad1, and in transfectants containing a dominant negative mutant of Smurf1 (a ubiquitin E3 ligase) there was an increase in osteogenic markers in response to BMP-2 (ASBMR-Abs.1033).ETK., JED.
…And more Smads. An isoform of FosB was shown to play a key role in regulating osteoblast expression. Δ2ΔFosB, a truncated spliciform of FosB, was compared to full length FosB in BMP-2 induced osteogenesis of C2C12 and ST2 cells (ASBMR-Abs.1035). Only Δ2ΔFosB transfected cells showed an increase in osteoblast markers in response to BMP-2. Δ2ΔFosB increased mRNA expression of Smad1 and reduced the expression of the Smad6, an inhibitory Smad. Therefore, the Δ2ΔFosB isoform is a regulator of both inductive and inhibitory Smads in the BMP-2 signaling pathway. Evidence was presented that osteogenic expression could also be regulated through Smad1 via the action of proteasomes. JED.
Novel Signaling Pathways for BMP-2. Evidence was presented showing that the PI 3 kinase/Akt pathway is part of the signaling cascade for BMP-2 in osteogenesis (ASBMR-Abs.1032). Dominant negative PI-3 kinase and dominant negative Akt blocked BMP-2-induced alkaline phosphatase and inhibited BMP-2 induced BMP expression and SMAD5/SMAD1 dependant BMP expression. In addition, a novel BMP-2 induced gene was identified by differential display which could accelerate osteoblast differentiation. Transfection into MC3T3 cells of the gene, termed BIG-3, was shown to accelerate expression of alkaline phosphatase expression and nodule formation, and to increase total calcium accumulation (ASBMR-Abs.1034). JED.
Osteoblasts: Programmed death and induced immortalityJames E Dennis
Osteoblast Apoptosis. Considerable attention was paid to the issue of osteoblast apoptosis and its possible role in bone turnover. The anti-fracture efficacy of bisphosphonate treatment may be partly a function of anti-apoptotic effects on osteoblasts or osteocytes. Data were presented showing that the anti-apoptotic effects of bisphosphonates was absent in Cx43 deficient cells and that ERK activation via Src and MEK is required for this effect (ASBMR-Abs.1130). The anti-apoptotic effects of estrogen were also linked to the ERK pathway as well as to the PI3K pathway. The next downstream target of these kinases is likely to be BAD, which upon phosphorylation is inactivated and prevented from initiating apoptosis (ASBMR-Abs.1131). Apoptosis can also be induced by exposure of osteoblastic cells to TNFα produced by osteoclasts. OPG was shown to bind to TNFα by immunoprecipitation and carbodiimide cross-linking experiments and to impart protection from TNFα apoptosis in MG63 cells. (ASBMR-Abs.1132). TNFα induced apoptosis was shown to be inhibited in osteoblasts subjected to shear forces. Shear forces promote release of PGE2, which was also shown to offer protection from apoptosis (ASBMR-Abs.F157). These data showing the production of PGE2 in response to shear forces and the resultant protection from apoptosis seem to contradict other results showing the production of SFRP-1 (secreted frizzled-related protein-1) in response to PGE2, which, in turn, was shown to increase the rate of cell death (ASBMR-Abs.1129). An assay for the production of SFRP-1 in TNFα-induced apoptosis would shed light on this issue. JED.
Contrasting the studies on apoptosis, osteoblastic cells transduced with hTERT, a protein permissive to telomerase function, are able to increase telomerase activity and mean telomerase length (ASBMR-Abs.1127). These hTERT transduced cells showed increased in vivo bone formation, no chromosome abnormalities, and have an extended in vitro lifespan of over 130 population doublings. JED.
Nongenomic Effects of Estrogens and Androgens on the Osteoblast. Evidence was presented indicating a link between nongenotropic and genotropic regulation via estrogen (ER) and androgen receptors (AR) (ASBMR-Abs.1092, ) Using a set of reporter constructs, it was shown that cells transfected with ER, AR, or with the ligand binding domain of the ER fused to a membrane localization signal upregulated Elk-1 when incubated with E2 or dihydrotestosterone. Activation was dependent on downstream activation of Src, Shc or ERK. Using a similar approach, both steroids were shown to downregulate AP-1 transcription. On the other hand, the ERα activated by estradiol stimulates AP-1 activity directly. Therefore, the response of the cell to androgen or estrogen is a balance between direct nuclear transcriptional (genotropic) effects and nongenotropic effects via ERK kinase. Along the same lines, an estrogen receptor antagonist, ICI 182,780, and an androgen receptor antagonist, flutamide, were equally effective at abrogating the pro-apoptotic effects of an estrogen (17β-estradiol) or an androgen (dihydrotestosterone) on male or female cells, indicating that these effects occur independently of gender (ASBMR-Abs.1093). JED.
Osteoblasts and Hematopoiesis. While it is accepted that osteoblasts modulate lineage progression in cells of the hematopoietic lineage, osteoblasts may actually be essential for marrow hematopoiesis, based on results in a transgenic mouse containing the gene for thymidine kinase (TK) under the control of the collagen 2.3 promotor (ASBMR-Abs.F049). In TK transgenics treated with Gancyclovir, the osteoblasts become depleted and the mice show significant bone loss after treatment. But these mice also lose hematopoietic cellularity in the bone marrow and hematopoiesis migrates to embryonic sites in liver and spleen. Hematopoietic stem cells are still present in marrow and can differentiate in vitro, but are incapable of lineage progression without osteoblasts. This will be a useful model for dissecting the contribution of early and late osteoblastic cells to the hematopoietic microenvironment and can be informative about how embryonic hematopoietic microenvironments are induced and how hematopoietic stem cells migrate to these new sites. JED.
Integrins and bone resorptionEvan T Keller
Integrins play an important role in bone biology through their ability to model cell structure, influence cell adhesion, and induce cell signaling. Several reports highlighted the role of integrins in bone pathophysiology. The β3 integrin is required for induction of c-src and c-abl. Transfection of a β3-/- preosteoclast with a β3 construct that contained the extracellular and transmembrane domains, but lacked the cytoplasmic domain did not rescue c-src nor c-abl activation, demonstrating that the cytoplasmic domain is required for this event (ASBMR-Abs.1164). A point mutation (S752P) of β3 blocked adhesion-induced c-src and c-abl activation, resulting in failure of localization of αvβ3 integrin to the ruffled membrane in mature osteoclasts (ASBMR-Abs.1164). However, the absence of β3 did not diminish phosphorylation of the FAK-like kinase Pyk2, which is considered an important mediator of bone resorption. Patients with β3 null type Glanzmann thrombasthenia, in which complete absence of the β3 chain occurs, demonstrate upregulated α2β1 integrin, offsetting the lack of β3. In vitro resorption assays revealed that osteoclast precursors from the patients were able to induce bone resorption in vitro, albeit at a lesser extent than those from normal patients (ASBMR-Abs.1070) demonstrating that compensatory mechanisms exist for integrin activity. ETK.
Genetics of osteoporosisGordon J Strewler
The Mouse, the Fly, the Eye, the Frog, and the Bone. This sounds like a Halloween incantation, but it is a new recipe for the genetics of osteoporosis. Reported at the ASBMR meeting was the finding that bone formation and bone mass are low in mice in which low density lipoprotein receptor-related protein-5 (LRP5) is deleted (ASBMR-Abs.1061). In the simultaneous meeting of the American Society for Human Genetics, loss-of-function mutations at the LRP5 locus were identified in the osteoporosis-pseudoglioma syndrome (OMIM 259770), a recessive bone fragility disorder with visual impairment (ASHG-Abs.67). Remarkably, scientists at Creighton University and Genome Therapeutics of Waltham, MA reported in an ASBMR satellite symposium and simultaneously at the American Society for Human Genetics meeting that a point mutation in a family with high bone mass maps to the same region on human chromosome 11q12-13 (ASHG-Abs.2423). It appears that both gain-of-function and loss-of-function mutations in LRP5 have a profound effect on bone mass, and the biology of LRP5 gene is consistent with its having an important regulatory function in osteoblasts. The LRP5 gene encodes a single-pass transmembrane protein that is a member of a small family (LRP5/LRP6 and Drosophila Arrow) - required for Wnt signaling. Deletion of Arrow produces a Wingless phenotype in the fly, and deletion of LRP6 interferes with signaling of several Wnt proteins in the mouse (). LRP5 as well as LRP6 interacts with Wnt signaling in Xenopus (). Finally, Arrow and LRP6 are receptors for secreted inhibitors of Wnt signaling in the fly and vertebrate, the dickkopf family (), three members of which are expressed in developing bones. We will be hearing much more about LRP5 and dickkopf in years to come. GJS.
Parathyroid hormone therapyJerilynn C Prior
Clinical Trials of PTH. Treatment of osteoporosis with parathyroid hormone (PTH) was a recurring and important theme in this ASBMR meeting. The bone world changed since PTH showed significant fracture prevention and massive increases in BMD in a randomized, double blind placebo controlled trial (). That study documented that bone formation agents, not just drugs decreasing bone resorption, can prevent fracture. PTH (high dose at 40 ug/d) was also superior to alendronate (10 mg/d) in a randomized, double-blind 14-month trial in 146 menopausal women with osteoporosis (ASBMR-Abs.1172). PTH gave a greater increase in spine and hip BMD and in total body BMC, and nonvertebral fractures were significantly less on PTH than alendronate. However, 27% of those on PTH had adverse-effect related dose reductions to the 20 ug/d dose (ASBMR-Abs.1172). In a randomized, double blind placebo controlled trial in men with low bone mineral density, treatment with PTH for 11 months, followed by an 18 month observation period, was associated with a reduction in the incidence of vertebral fractures (ASBMR-Abs.1104). Interpretation of the study is complicated by dropout at the conclusion of the treatment period and the concomitant use of other treatments of osteoporosis by up to 29% of men during the observation period. JCP.
Mechanisms of PTH Effects. It has long puzzled clinicians that hyperparathyroidism stimulates net bone loss but PTH therapy increases net bone gain. Intermittent but not continuous PTH dosing decreases osteoblast apoptosis which, in turn, correlates with an increased bone formation rate in vivo in mice (ASBMR-Abs.1169). This provides additional evidence that the antiapoptotic effect of PTH is important in the induction of bone formation, though evidence that the antiapoptotic effect is quantitatively sufficient to account for the increase in bone formation is still lacking. Bone histomorphometry on paired biopsies from those on PTH therapy showed an approximate 20% increase in cortical and trabecular thickness (ASBMR-Abs.1170). Back scattering assessments, however, suggest a lower calcium density in bone from men who had been on high dose PTH for 22 months and women who had been on PTH with ovarian hormone therapy for 32 months (ASBMR-Abs.1170), consistent with undermineralization of the new bone formed during treatment with PTH. In another study of paired biopsies, the second obtained only one month after initiation of therapy, PTH causes bone formation by increased remodeling (formation preceded by resorption) on the endocortical bone surface but increased modeling (without preceding resorption) on both cancellous and periosteal bone surfaces as well as by a novel mechanism, the resumption of previously quiescent bone formation (ASBMR-Abs.1171). These data offer the most comprehensive picture to date of the effects of PTH at the level of the BMU. JCP.
Sex steroids in osteoporosis pathophysiology and therapyJerilynn C Prior, Susana Zeni
Which Sex Steroid in Men? Although decreased menopausal estrogen levels have long been understood to explain lower bone mineral density (BMD) and increased fracture risks in older women, the role of gonadal steroids in osteoporosis risks for men is less clear. Cross-sectional studies suggest that estrogen correlates more highly than testosterone with BMD in men. A recent complex experiment proposes that testosterone decreases but estrogen increases osteoprotegerin (OPG), the protective receptor that inactivates RANK ligand, a potent mediator of osteoclastogenesis (ASBMR-Abs.1037). The study rendered older men hypogonadal (GnRH agonist), prevented aromatisation of testosterone to estrogen (letrazole) and initially replaced both estradiol and testosterone. Subsequent randomized withdrawal of both, neither or only estrogen or testosterone showed that the serum level of OPG was significantly decreased during testosterone treatment, but increased during treatment with estrogen (ASBMR-Abs.1037). JCP.
Which Estrogen Receptor in Women? The effects of estrogen in bone and in uterus are mediated by the estrogen receptor (ER) - but is it ERα or ERβ? A number of abstracts dealt with the receptor specificity of estrogen effects. Female ERβ null (BERKO) mice have longer, thicker bones than WT when young, and these disparities increase with age (ASBMR-Abs.1095). ERβ thus inhibits the acquisition of bone length and circumferential growth, and stimulates bone loss during the aging process. OVX induces cancellous bone loss. Replacement therapy with 17β-estradiol in OVX C57BI/6J mice prevents distal femur bone loss at low levels and increases bone formation at high levels. OVX decreases uterus weight and induces distal femur bone loss in wild type (wt), in ERβ null (BERKO), ERα null (ERKO) (ASBMR-Abs.SA331, SU515) and in double ER (α and β) knockout mice (DRKO). The same doses of estrogen protects BERKO and wt mice with regard to uterus weight and distal femur bone loss, suggesting that bone loss prevention by estrogen is not maintained through ERβ in mice (ASBMR-Abs.SA331). In female ERKO mice estrogen prevents bone loss but only partially; higher doses of estrogen are more effective. Thus ERβ is less effective than ERα and estrogen action is exerted mainly via ERα (ASBMR-Abs.SU515). DRKO needs 3-fold higher doses of estrogen than wt mice to be protected against uterine and bone loss (ASBMR-Abs.1040). The demonstration of estrogen effects in DRKO is of interest and suggests: 1) the existence of a third receptor (not present in humans), 2) Estrogen may act via an androgen receptor 3) a non-genomic effect of estrogen or 4) mediation by ERα splice variants expressed in these DRKO mice. SZ.
The Return of Progesterone for Osteoporosis. PTH trials have shifted attention to bone formation, thus reviving the idea that progesterone is anabolic. Two randomized, double blind placebo controlled trials showed positive changes in BMD with 2.5 mg/d of medroxyprogesterone (MP) (ASBMR-Abs.1175) or 1 mg/d norethindrone (NE) (ASBMR-Abs.F384) in estrogen-treated menopausal women. Two previous controlled trials of progestin with estrogen versus estrogen alone have shown positive BMD results () but one trial was negative (). These observations fit with progesterone's osteoblast receptor-mediated actions and that progesterone positively related to cancellous bone change in premenopausal women (). Cyclic MP also significantly increased spine BMD in a randomized, double blind placebo controlled trial in amenorrheic premenopausal women (). Fracture outcomes using progestin/progesterone are needed. JCP.
Osteoporosis epidemiology and hip fractureJerilynn C Prior
A 16-yr prospective epidemiological study showed that lower initial femoral neck BMD is related to incident fracture (289 fractures, Relative Hazard [RH]1.58). However, osteoporosis by BMD (T ≤ −2.5) was initially present in only 42% and 53% of those with hip and non-vertebral fractures respectively in a 10-yr study accumulating 469 low trauma hip and 2055 non-vertebral fractures (ASBMR-Abs.1077). Recurrent hip fractures occurred with visual impairment (decreased depth perception) or weight loss in older women — calcium supplements, estrogen therapy or reported walking were independently protective (ASBMR-Abs.1133). A case control study reported associations with hip fracture in the Veterans’ population. Both African-American and Caucasian male veterans with hip fracture were more likely to be alcoholics. Cigarette and anticonvulsant use increased and thiazide diuretic or statin use were protective for hip fracture (ASBMR-Abs.1135). Bone loss occurred rapidly following hip fracture in a case control study that matched for age and total hip BMD (ASBMR-Abs.1075)-these results may explain high recurrent hip fracture rates (10.3% within 3 years) (ASBMR-Abs.F291). Even in women who were heavy, 5% weight loss was importantly related to the risk for hip fracture in older women and this risk was as important in those with voluntary weight loss (ASBMR-Abs.1076). Weight variability has been related to increased hip fracture in middle-aged men and women () and lifetime weight cycling (l0 lb. loss/regain) to past fragility fractures in men (ASBMR-Abs.SU359). Weight loss and change probably alter bone strength. JCP.
Epidemiology of vertebral fractureJerilynn C Prior
African American women had a higher incidence of vertebral fracture if they had lower total hip BMD or had previously fractured but a lower incidence if they were stronger and walked faster - similar risk factors overall to white women (ASBMR-Abs.1136). Chinese men and women over 60 have nearly identical vertebral fracture prevalence (ASBMR-Abs.1137). Vertebral fractures in men were inversely associated with hip BMD and related to lower weight and physical performance measures- vertebral fractures tended to increase with heavy labor (ASBMR-Abs.1137). The overall 11% vertebral fracture prevalence in Chinese men may be lower than the 22% prevalence reported in men in a population-based study that also showed equal vertebral fracture prevalence in men and women (). Kyphosis increased over 17 years by 10o in 50% of women and 25% of men, however, it did not relate to vertebral deformities (ASBMR-Abs.1074). JCP.
Vitamin D physiology and therapyJerilynn C Prior
Vitamin D loomed large in this ASBMR (109 presentations). High levels of Vitamin D are required (3,800 IU/d!) in men to maintain serum 25-OHD3 levels over the winter (in latitudes ≥ 41° ) (ASBMR-Abs.1038). A 49% within-person decrease in falls over 3 months in a randomized, controlled trial of 800 IU/d of Vitamin D with calcium (1200 mg/d) versus calcium alone suggested that increased leg strength mediated the effect (ASBMR-Abs.1108). Serum 25OH D3 levels also negatively related to falls in elders in intermediate or nursing home care (ASBMR-Abs.1134). Incident hip fracture was importantly decreased by monthly Vitamin D therapy (3 drops = 294,000 IU Vitamin D2) in a controlled nursing home study (ASBMR-Abs.1174). The decrease was significant only during the first 12 months (RR 0.66 [0.46-0.93]) of a 3-yr trial, however it also prevented recurrent hip fractures (RR 0.28 [0.14, 0.59]). This dose of Vitamin D2 cost only 50 cents/participant/yr! JCP.
Diagnostic assessment of osteoporosisChristopher F Njeh
Diagnostic Thresholds. The problem with the WHO thresholds approach to the diagnosis of osteoporosis is now apparent. Different measurement techniques will identify different subjects as osteoporotic when using T-scores to diagnose osteoporosis. At the conference possible solutions were presented (ASBMR-Abs.-F084, 1115, 1116, SU122). It was suggested (ASBMR-Abs.1115) that using age, prevalent fractures and BMD expressed as Z-score improves risk prediction. The general opinion is that criteria based on fracture risk will be better than the current T-score approach (ASBMR-Abs.1116). It was made clear, however, that even with a fracture-risk based approach, the quantitative estimates of fracture risk derived from densitometry depend on the relative risk value of the densitometric technique as well as the skeletal status of the patient, and should not be equated between techniques (ASBMR-Abs.SU103). Because of the general uneasiness over the level of discordance from measurement made at different sites and due to differences in techniques, it has always been advocated that the best predictor of fracture at a specific site is a measurement carried out at that site. This was further validated by an in vitro study carried out using human samples from the femur, spine and radius (ASBMR-Abs.1119). CFN.
Assessment of Bone Structure. It has always been thought that measuring bone structure in addition to bone mineral density will improve fracture risk prediction. Measurement of structure in vivo has been difficult, however, Magnetic Resonance (MR) provides interesting possibilities (ASBMR-Abs.- M112, F092). It was reported that applying digital topological analysis (ASBMR-Abs.F092) to in vivo MR images provided better discrimination of vertebral fracture subjects. Another exciting arena for bone structure analysis is micro-Computed Tomography (µCT), although at the moment it is limited to in vitro studies. Using µCT to analyze bone biopsies acquired before and after menopause in a group of women, the 3-dimensional changes in trabecular network occurring after menopause at the iliac crest were demonstrated. They found that 3D analysis provided more significant evidence of structural changes due to loss of estrogen than 2D analysis, with the structure shifting from “plate-like to rod like” after menopause (ASBMR-Abs.1055). Such changes have a significant impact on the mechanical competence of bone. CFN.
Quantitative Ultrasound. QUS was initially hailed as the measurement technique that would provide more information about fracture risk. This was because of the possibility that QUS could provide information about the bone structure. Prospective studies have shown QUS to be similar to central DXA for fracture risk prediction. This conference also provided further evidence of the clinical utility of QUS especially from large European prospective studies (ASBMR-Abs.-F101, F103). There were indications that not all QUS devices perform similarly in vertebral prediction (ASBMR-Abs.F103). Contrary to previous reports (even from the same group), a large American prospective study failed to show a significant association between BUA or SOS (measured using a gel-coupled device) with hip fracture in women, but significant association with hip fracture in men (ASBMR-Abs.1118). This re-emphasizes the fact that all QUS devices are not the same and each device must be evaluated for its clinical efficacy. CFN.
Bone acquisition and pediatric boneMoira A Petit
Noninvasive Techniques to Assess Bone in Children and Adolescents. A highlight of the meeting was increased use and development of non-invasive techniques for assessing bone strength and structural parameters, rather than BMC or BMD alone. Potential technologies for use in children and adolescents include the Hip Structural Analysis (HSA) program (ASBMR-Abs.-1007, 1145, 1147, SA021), Magnetic Resonance Imaging (MRI) (ASBMR-Abs.1150), and peripheral QCT (ASBMR-Abs.1151) - all of which can be used to assess bone strength (section modulus), cortical thickness, periosteal diameter, and bone cross-sectional area. pQCT can also separate cortical and trabecular bone components and estimate muscle mass. Although pQCT has great potential for assessment of bone and muscle strength in healthy and clinical pediatric groups, there was much discussion about the appropriate voxel size, location of scan, etc. Standardized parameters have yet to be established. MAP.
Growth and Exercise. HSA was used to show changes in bone strength and structure during growth (ASBMR-Abs.-1007, 1145) and with exercise intervention (ASBMR-Abs.1147). Girls and boys who were the most active during adolescence had greater increases in femoral neck bone strength compared with low active children (ASBMR-Abs.1147). A school-based exercise intervention increased femoral neck bone strength and cortical thickness in early-pubertal (Tanner stage 2 & 3) girls but had no effect in pre-pubertal (Tanner stage 1) girls (ASBMR-Abs.1147). In boys, the same exercise intervention was effective in increasing BMC and BMD only in those who had low or normal body mass. The intervention had no benefit in boys who were in the highest quartile of body mass for height (ASBMR-Abs.1146). Together these data suggest a complex response to exercise intervention which is dependent on stage of maturation, body weight, gender and is region specific. MAP.
Calcium Supplementation in Adolescents. The long-term benefit of calcium supplementation in children and adolescents has been a contentious issue. Although calcium supplementation with milk, calcium foods, or elemental calcium (i.e. calcium citrate malate, CCM) in early adolescence is associated with increased bone accrual, no benefit persists after withdrawal of supplementation. A nicely controlled study presented at the conference showed that 11-12y old girls had increased bone accrual after 18 months of supplementation with CCM (ASBMR-Abs.1149). After two-years of follow-up with no supplementation, there was no difference in BMD between supplemented and placebo groups (ASBMR-Abs.1149). The authors propose that the effect of CCM is due to suppression of bone remodeling that is reversed when supplementation withdrawn (ASBMR-Abs.1149). It was noted that different types of supplementation may influence the bone modeling response. In contrast to the lost benefit with CCM, a recently published study showed positive effects of milk-extracted calcium phosphate were retained 3.5 years after discontinuation in young girls (). Studies comparing these two forms of supplementation are underway and will provide important information about the optimal form of supplementation and the long-term benefits. MAP.
Exercise-Calcium Interactions. Interestingly, weight-bearing exercise (ASBMR-Abs.1008) or calcium supplementation from foods alone (J. Lappe, personal communication) did not appear to increase proximal femur bone accrual over one year in 9-10 year old prepubertal girls (J. Lappe, personal communication). In contrast, the combination of exercise and increased calcium (to 1500 mg/d) from foods had a positive effect on hip BMC (ASBMR-Abs.1008). These data are from the first year of a three-year randomized intervention. The long-term follow-up will provide important information about whether these benefits persist. MAP.
These data (ASBMR-Abs.-1008,1149,1145,1146,1147) further highlight the complexity of the bone response to intervention during growth. In each of the pediatric intervention and follow-up studies presented at the meeting, researchers acknowledged the difficulty in controlling for the substantial individual and group differences in body size and rates of maturation which dramatically confound intervention effects. Follow-up studies when adult bone mass is reached are necessary to distinguish maturational differences between groups and ultimate differences in fracture risk. As acknowledged by presenters, it is unclear from studies using BMC or BMD as the primary outcome if differences in bone accrual were due to changes in bone size, length, or the amount of mineral within the bone. Several of the new technologies (i.e. HSA, pQCT, MRI) discussed at the meeting will assist in answering these important questions about changes in bone geometry and strength with intervention. MAP.
Pediatric Bone Disease. On the clinical side, there is much concern about increased fracture risk in pediatric groups with debilitating conditions and in those with long-term use of corticosteroids. A nicely done case-control study showed asthma itself, rather than use of inhaled corticosteroids, was associated with increased fracture risk (ASBMR-Abs.1152). Mechanisms underlying these risks are yet to be determined. Exciting data showed a positive increase in bone strength and mass (measured by QCT) in children with cerebral palsy and cystic fibrosis using high frequency, low magnitude platform (ASBMR-Abs.1148). In this nicely designed randomized clinical trials, children were randomized to either placebo or active platforms. Although compliance to the intervention was low, children randomized to intervention had positive increase in spine trabecular BMD after 6 months (ASBMR-Abs.1148). MAP.
Other disordersGordon J Strewler, Susana Zeni
Hypophosphatemic Disorders And FGF23. A new member of the fibroblast growth factor family, FGF23, has recently been implicated in the pathogenesis of autosomal dominant hypophosphatemic rickets (ADHR). The same molecule has been identified in tumors that produce acquired osteomalacia (oncogenic osteomalacia), strongly suggesting that it is critical in the pathogenesis of both disorders. In this meeting, it was reported that recombinant FGF23 inhibits phosphate transport by a renal epithelial cell line, OK (ASBMR-Abs.1058). Others had previously been unable to show a direct effect of FGF23 on phosphate transport. The new result strongly suggests that FGF23 has a direct phosphaturic effect on the renal tubule. Transgenic mice overexpressing FGF23 develop severe hypophosphatemia and osteomalacia (ASBMR-Abs.1059), in agreement with a previous report that FGF23 was phosphaturic when injected or when implanted in CHO cells. In ADHR, point mutations found in several families were previously found to be clustered at two closely spaced arginine residues in FGF23. Introduction of these mutations into recombinant FGF23 rendered the molecule resistant to proteolysis (ASBMR-Abs.1064), consistent with the possibility that the mutant molecule accumulates in ADHR patients because it is resistant to degradation. It is possible that the protease that degrades FGF23 is PHEX, the membrane-associated metalloprotease whose gene is inactivated in X-linked hypophosphatemic rickets (XLH). If FGF23 is indeed a phosphaturic factor, then phosphaturia in XLH (inactivation of FGF23 degradation) would be neatly tied together with ADHR (inability of FGF23 to be degraded) and oncogenic osteomalacia (overproduction of FGF23). Two recent reports disagree on the enzymatic specificity of PHEX, however, one reporting degradation of FGF23 () and the other reporting a different enzymatic specificity of PHEX (). GJS.
Fibrodysplasia Ossificans Progressiva. Fibrodysplasia ossificans progressiva (FOP) is a genetic disorder of ectopic bone formation, with an incidence of 1/2000000 but no gender, geographical or racial bias. In normal conditions appropriate bone formation is the result of a balanced activity between osteogenic factors and inhibitors that prevent them from interacting with their receptors. In this regard, osteoblastic cells increase the expression of two antagonists of bone morphogenetic protein-4 (BMP-4), noggin and gremlin, in response to a BMP stimulus. Conversely, cells from FOP patients fail to upregulate the expression of these inhibitors (ASBMR-Abs.1083). FOP patients present an increase in BMP-4 mRNA and protein expression (ASBMR-Abs.SU026) and in lymphoblastoid cell lines from FOP patients there is a 10-fold increment in the levels of BMP receptor type I (ASBMR-Abs.SU026). The increment in ligand and receptor, along with the reduction in antagonist activity, could increase the activity of BMP-4, thereby inducing ectopic bone formation. Hence, BMP antagonists could be a successful therapy in FOP. The matrix metalloproteinases (MMPs) MMP-2 and MMP-9 play a role in the angiogenesis of pre-osseous lesions in FOP (ASBMR-Abs.M072). They can be measured in urine and, expressed as a creatinine ratio, are increased in FOP patients compared to normal subjects. MMP-2 and MMP-9 could serve as markers for anti-angiogenesis treatment (ASBMR-Abs.M072). SZ.
Paget's Disease. Osteoclasts from marrow cultures of patients with Paget's disease are characterized by an increase in number, size, and nuclei per cell as well as increased resorptive activity. Osteoclast precursors from pagetic patients and normal osteoclast precursors infected with measles virus nuclecapsid gene (MVNP) are hypersensitive to 1,25(OH)2D3 (ASBMR-Abs.1182). This response may be mediated by the induction of a coactivator of the vitamin D receptor (VDR). This cofactor is a 17-kDa peptide, identified as TAFII-17/TAFII20 (a component of the TFIID transcription complex) that binds to VDR. TAFII-17 mRNA is overexpressed in both pagetic preosteoclasts and MVNP-infected normal preosteoclasts, and NIH3T3 cells transfected with this protein are hypersensitive to 1,25(OH)2D3. The data support a pathophysiological role of measles virus in Paget's disease. SZ.
Vascular Calcification. Atherogenic oxidized lipids increase atherosclerotic vascular calcification by increasing the osteoblastic activity and mineralization in calcifying vascular cells, but the same oxidized lipid products inhibit the osteogenic effect of osteoblast cells, suggesting a common mechanism for vascular calcification and osteoporosis. Low density lipoprotein receptor (LDLr) null mice present atherosclerotic lesions which are important for calcification. When these mice are fed a high fat diet these lesions are increased and body weight is lower than animals fed the control diet (ASBMR-Abs.1144). In addition, they present an increase in cholesterol, in unesterificated-cholesterol and hydroxyperoxides (LOOH) while high density lipoprotein (HDL) cholesterol is not changed. Quantitative CT scanning shows a decreased BMD without changes in BMC, suggesting that atherosclerosis on the high fat diet decreases bone mineralization in mice (). This effect is associated with increased levels of LOOH (markers of lipoxidation). Indeed, analysis of the bone marrow lipids of C57BL/6 mice fed a high fat diet show an increased level of LOOH and cholesterol as well as an accumulation of oxidized lipid products similar to those observed in atherosclerotic arteries. SZ.
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