BoneKEy-Osteovision | Meeting Reports

Meeting report from the frontiers of skeletal biology: Ninth workshop on cell biology of bone and cartilage in health and disease



DOI:10.1138/2002034

New genes in bone

LRP-5. LRP-5 emerged last year as a powerful determinant of bone mass (), unique amongst known skeletal genes because different mutations can produce either a marked gain or a marked loss of bone mass in humans. Mark Johnson (Creighton Univ) reviewed a syndrome of high bone mass associated with a mutation in LRP-5. Affected individuals from a single Midwestern U.S. family have a mean Z-score of +5.5, and no member of the family has ever sustained a fracture. They have inherited a dominant missense mutation in the gene for LRP-5 (G171V) that, when expressed as a transgene in mice, causes an increase in trabecular number, cortical thickness, and mechanical strength of bone. On the other side, the osteoporosis-pseudoglioma syndrome, caused by loss-of-function mutations in the gene for LRP-5, is associated with very low bone mass (Z -2.3 in heterozygotes and -4.7 in homozygotes), and deletion of the LRP-5 gene in mice also produces osteoporosis. The possibility that other alleles of LRP-5 contribute to a continuum of bone mass is under investigation.

The LRP-5 gene is expressed in osteoblasts and encodes a membrane protein that can act as a coreceptor for Wnt proteins. As reviewed by Matthew Gillespie, the Wnt family comprises 19 known vertebrate genes and the Wnt receptor, frizzled, has 11 vertebrate homologs, so much work remains to determine how LRP-5 and the Wnt family determine bone mass. It is likely that LRP-5 is a major determinant of the recruitment of osteoblasts during pubertal growth and remodeling, but no data on the age dependence of bone mass in mice or men with LRP-5 mutations have been reported. Johnson showed preliminary data that mice with the HBM allele had increased bone formation in response to skeletal loading, consistent with the possibility that LRP-5 is involved in cell-matrix interactions that determine the response of bone to mechanical loading.

Osterix. The osterix story () was told by Benoit de Crombrugghe (MD Anderson Hospital). His laboratory recently identified a zinc finger transcription factor and gave it the charming name osterix (osx) after they discovered that deletion of the gene gave rise to a normally patterned skeleton composed only of cartilage and without bone — a phenotype strongly reminiscent of the cbfa1/runx2 knockout. Osx is genetically downstream of cbfa1/runx2, because cbfa1 levels are normal in osx(-/-) mice but osx is not expressed in cbfa1(-/-) mice. The functional relationship of the two is probably more complex. Cbfa, though genetically upsteam of osx, binds directly to a downstream target, the osteocalcin promoter: How then do cbfa1/runx2 and osx interact to drive the expression of this and other osteoblast-specific genes?

MMP-13. The metalloprotease MMP-13 is a collagenase that is expressed predominantly in the skeleton. Stephen Krane (Harvard Medical School) reported that MMP-13 (-/-) mice have phenotypes in both cartilage and bone. In cartilage the growth plate is elongated, both because the proliferation of chondrocytes is increased and because the hypertrophic zone is widened. In bone there is a marked decrease in bone resorption, which leads to an increase in bone mass. Is this, as previously suggested, because other cells must secrete collagenase onto the bone surface before osteoblasts can resorb the matrix? Or does MMP-13 release collagen degradation products that activate bone resorption, or release bound ligands by its collagenase activity, or is direct binding of MMP-13 to the osteoclast an integral step in bone resorption?

FGF-23. This newest member of the FGF family has a key role in hypophosphatemic disorders (). Its arrival on the scene was reviewed by Seiji Fukumoto (Tokyo Univ). FGF-23 is produced in large amounts by tumors that cause osteomalacia and itself reproduces the syndrome of hypophosphatemic rickets when expressed or infused in mice. In a second form of hypophosphatemia, autosomal dominant hypophosphatemic rickets, FGF-23 is not normally cleaved because of mutations at the cleavage site; this presumably leads to its accumulation and resultant hypophosphatemia. It is possible that PHEX (the protease that is the locus of mutations causing X-linked hypophosphatemic rickets) is the enzyme responsible for cleaving FGF-23; this would link three hypophosphatemic disorders to one another. But whether FGF-23 is a substrate for PHEX has not been demonstrated definitively.

Sclerostin. Sclerostosis is a recessively inherited osteosclerotic disorder caused by mutations in a protein called sclerostin (). John Latham (Celltech) reported that sclerostin is related in sequence to a family of secreted BMP antagonists which includes Gremlin, Cerberus and Dan. Sclerostin is expressed in osteoblasts and binds BMP5 and BMP6 with high affinity but does not bind other BMPs. Expression in C3H10T1/2 cells blocks acquisition of the osteoblast phenotype, and transgenic expression in osteoblasts under the control of the osteocalcin promoter gives rise to osteoporosis. As a secreted inhibitor of osteoblasts, sclerostin is an attractive target for drug development - small molecule inhibitors would be predicted to increase bone mass.

Leptin, fat, and bone

Ian Reid (Univ of Auckland) reviewed a mass of data that suggest a direct relationship between fat mass and bone mass that is independent of load-bearing and other mechanical factors. It has previously been proposed that the adipocyte hormone leptin is the mediator of these effects, and Gerard Karsenty (Baylor Univ) presented considerable new data on a central action of leptin to control bone mass. The site of leptin action is on antiosteogenic neurons in the ventromedial nuclei (VMN). Neurons of the arcuate nucleus and VMN have leptin receptors. Chemical lesions of the arcuate nucleus with monosodium glutamate have no effect on bone mass, however; lesioning the VMN with gold thioglucose produces the high bone mass phenotype of leptin deficiency, and leptin infusion does not lower bone mass after the VMN is lesioned. These antiosteogenic neurons are distinct from anorexogenic neurons in the VMN that respond to α-MSH. The agouti mouse (Ay/a), which is resistant to the anorexogenic effect of leptin, has normal bone mass; mice from whom the α-MSH receptor MC4R was removed have normal bone mass; and treatment with the MSH receptor agonist MT2 produces weight loss but no change in bone mass. In addition, leptin infused into the 3rd ventricle at low doses (1-4 ng/hr) causes bone volume to decrease without affecting body weight. What is the pathway by which central effects of leptin are transmitted to bone? In parabiosis experiments intraventricular infusion of leptin reduces bone mass only in the recipient ob/ob mouse, suggesting that no humoral messenger is produced and raising the possibility of a neural mechanism.

Does leptin act solely centrally or does it also have peripheral effects on bone mass? Reid reviewed the abundant evidence that osteoblasts and chondrocytes have leptin receptors and respond biochemically to leptin. Evidence of increased trabecular bone volume and bone strength in response to leptin has also been reported. Yet Karsenty cannot identify leptin receptors or signal transduction in mouse osteoblasts and reports no effects of osteoblast-specific expression of leptin using the COLIA1 promoter. Although these contrasting views of the peripheral actions of leptin on bone were not reconciled, the interpretation that leptin is part of a hypothalamic system for the central control of bone mass is compatible with its also having direct effects on bone. The physiological significance of the peripheral effects of leptin remains to be determined, however, while the reversal of high bone mass in the ob/ob mouse by leptin infusion into the 3rd ventricle provides evidence that central control of bone mass is physiologically important. Though the case for central regulation of bone mass is strengthened by the new data, leptin itself is unlikely to be the main messenger from the fat cell to the bone cell — raising peripheral leptin levels does not induce bone loss.

The RANK/RANKL system

William Dougall (Immunex) reviewed the RANK/RANKL system in osteoclast development. He described a number of studies, most of them published, that establish the efficacy of recombinant soluble RANK-Fc as a decoy receptor to block bone resorption. sRANK-Fc is effective in models of solid tumors and multiple myeloma and also blocks periarticular bone loss in a variety of arthritis models. Its potential advantages over the endogenous decoy receptor, OPG, are at least two - it has a very long half-life in vivo, and it does not block the TNF family member TRAIL, a proapoptotic signal that could be beneficial to the host by killing cancer cells.

RANKL is justly celebrated as the principal regulator of the osteoclast, but is it also a key to development of the osteoblast? Steven Teitelbaum (Washington Univ, St Louis) told a surprising tale. It began with the unexpected finding that subcutaneous injection of a GST-RANKL fusion protein into mice produces a massive increase in trabecular bone, with increased numbers of osteoblasts and increased osteoblast activity. Followup experiments demonstrated that GST-RANKL is mitogenic in primary cultures of mouse calvarial bone cells and induces large increases in osteoblast colonies in CFU-OB assays. Yet soluble RANKL itself does not share these remarkable properties. What accounts for the startling osteoblast tropism of GST-RANKL? Primary mouse osteoblast cultures signal in response to both RANKL and GST-RANKL, but the response to GST-RANKL is markedly prolonged. Confocal microscopy suggests that GST-RANKL is not internalized, though native RANKL is, and gel filtration discloses that the GST-RANKL preparation consists of oligomers much larger than the native RANKL triplet, likely concatemers of triplets attached via dimerization of the GST domain. Teitelbaum suggested that osteoblasts have low levels of the RANK receptor and have a greatly exaggerated response to GST-RANKL because it is not internalized. Much remains to explain these effects, to account for the lack of a significant increase in osteoclast number or bone resorption (intermittent versus continuous RANKL, a la PTH?), and rigorously to exclude a contribution of the GST moiety to the effect of GST-RANKL.

Bisphosphates and statins (round one of a scheduled six round bout)

Mike Rogers (Univ of Aberdeen) recounted the mechanism of action of the nitrogen-containing bisphosphonates, which we now know to act in the mevalonate pathway of cholesterol biosynthesis by blocking farnesyl diphosphate synthase, an enzyme that is essential to prenylate proteins and thereby target them to their appropriate membrane location. Rogers described an elegant experiment to confirm what has been suspected - that bisphosphates target osteoclasts selectively. Neonatal rabbits injected with a bisphosphonate have a selective accumulation of unprenylated protein in vitronectin (VN) receptor positive osteoclasts, whereas rabbits treated with cerevestatin, which acts in the same pathway, have impaired prenylation in both VN-receptor positive and VN receptor negative bone cells. The prenylation step most important to the mechanism of action of bisphosphonates is geranylgeranylation of small GTP-binding proteins (Rho, Rac, Cdc42, which regulate the cytoskeleton; and Rab, which regulates vesicle and membrane trafficking). Osteoclasts can be rescued by geranylgeraniol.. Inhibition of geranylgeranyl transfer mimicks bisphosphonate action. And NE10790, a specific inhibitor of Rab prenylation, blocks bone resorption by osteoclasts, probably by a selective effect on membrane trafficking, because the actin ring is not disrupted as it is by bisphosphonates. This begins to assign specific cellular events induced by bisphosphonates to particular GTPases.

What about cell death itself? Al Reszka (Merck) reported in a short talk that geranylgeranyltransferase inhibitors inhibit protein phosphorylation by the p70 S6 protein kinase, an antiapoptic signaling molecule. Rapamycin, an inhibitor of the p70 S6 kinase activator mTOR, also blocked S6 phosphorylation and induced osteoclast apoptosis (). Conversely, several antiapoptic signals, such as sRANKL, activate the S6 kinase pathway. The S6 kinase thus emerges as a prime candidate for proapoptotic effects of the bisphosphonates. The direct target of bisphosphonates in this pathway has not yet been identified.

Greg Mundy (Univ Texas, San Antonio) then updated the previously told story of statins and bone (). His group previously reported that statins increase osteoblastic bone formation. The statins in clinical use to lower cholesterol, however, are strongly directed to the liver, the most important site of cholesterol synthesis. Higher and more sustained blood levels are achieved with topical than with oral administration of lovastatin. Treatment for five days with topical lovastatin produced a marked increase in trabecular bone volume at the end of 35 days.

The Mundy group now has data to suggest that statins target the osteoblast by a mechanism that involves the endothelial form of nitric oxide synthase (eNOS). eNOS null mice have a marked abrogation of the effect of statins on bone formation. In endothelial cells the effect of statins on eNOS activity appears to be mediated by blockade of geranylgeranylation of the GTPase Rho, but it is not known how the mevalonate pathway is linked to osteoblast differentiation. In a short talk, however, Agi Grigoriades reported that the Pasteurella multocida toxin activates the GTPase Rho and inhibits bone formation, but specific Rho kinase inhibitors increase osteoblast differentiation (). It remains to be established how early effects of statins on Rho or eNOS might be linked to downstream events in osteoblast formation, such as the expression of BMP-2.

Drug discovery

Roland Baron (Yale Univ and Aventis) reported on the structure of an industrial program to identify targets for drug discovery by screening for genes in the pathway of osteoblast differentiation. This involves high-throughput screening using gene chips, novel bioinformatic approaches to deal with large masses of information (60 samples in one experiment yielded 20,386,000 data points!), and strategies to validate candidate genes as targets for drug discovery.

Mark Nuttall (Smith-Kline Glaxo) gave a report on progress in four areas. A potent and specific inhibitor of cathepsin K, the principal cathepsin in osteoclastic bone resorption, (10 nM affinity) has been shown to inhibit bone turnover markedly in a macaque monkey model of lupron-induced menopause. Antagonists of the parathyroid calcium receptor are being used to mimic the effect of intermittent injections of PTH by stimulating intermittent secretion of endogenous PTH, and have now been shown to have an anabolic effect on bone. Small molecule agonists of the PTH receptor have also been developed by high-throughput screening of about seven million compounds, using cells transfected with N-terminal modified PTH receptor and CRE-luciferase as a reporter. An antagonist of the PPAR receptor, which regulates reciprocally the entry of marrow stromal cells into adipocyte and osteoblast pathways, has been shown to protect against bone loss in an OVX rat model. Interesting targets all.


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