Ror2, encoding a receptor-like tyrosine kinase, is required for cartilage and growth plate development.

Receptor tyrosine kinases often have critical roles in particular cell lineages by initiating signalling cascades in those lineages. Examples include the neural-specific TRK receptors, the VEGF and angiopoietin endothelial-specific receptors, and the muscle-specific MUSK receptor. Many lineage-restricted receptor tyrosine kinases were initially identified as 'orphans' homologous to known receptors, and only subsequently used to identify their unknown growth factors. Some receptor-tyrosine-kinase-like orphans still lack identified ligands as well as biological roles. Here we characterize one such orphan, encoded by Ror2 (ref. 12). We report that disruption of mouse Ror2 leads to profound skeletal abnormalities, with essentially all endochondrally derived bones foreshortened or misshapen, albeit to differing degrees. Further, we find that Ror2 is selectively expressed in the chondrocytes of all developing cartilage anlagen, where it essential during initial growth and patterning, as well as subsequently in the proliferating chondrocytes of mature growth plates, where it is required for normal expansion. Thus, Ror2 encodes a receptor-like tyrosine kinase that is selectively expressed in, and particularly important for, the chondrocyte lineage.

Dominant mutations in ROR2, encoding an orphan receptor tyrosine kinase, cause brachydactyly type B.

Inherited limb malformations provide a valuable resource for the identification of genes involved in limb development. Brachydactyly type B (BDB), an autosomal dominant disorder, is the most severe of the brachydactylies and characterized by terminal deficiency of the fingers and toes. In the typical form of BDB, the thumbs and big toes are spared, sometimes with broadening or partial duplication. The BDB1 locus was previously mapped to chromosome 9q22 within an interval of 7.5 cM (refs 9,10). Here we describe mutations in ROR2, which encodes the orphan receptor tyrosine kinase ROR2 (ref. 11), in three unrelated families with BDB1. We identified distinct heterozygous mutations (2 nonsense, 1 frameshift) within a 7-amino-acid segment of the 943-amino-acid protein, all of which predict truncation of the intracellular portion of the protein immediately after the tyrosine kinase domain. The localized nature of these mutations suggests that they confer a specific gain of function. We obtained further evidence for this by demonstrating that two patients heterozygous for 9q22 deletions including ROR2 do not exhibit BDB. Expression of the mouse mouse orthologue, Ror2, early in limb development indicates that BDB arises as a primary defect of skeletal patterning.

Interleukin-1 receptor antagonist and tumor necrosis factor binding protein decrease osteoclast formation and bone resorption in ovariectomized mice.

To investigate the contribution of IL-1, IL-6, and TNF to the increased osteoclastogenesis induced by estrogen deficiency, ovariectomized (ovx) mice were treated with either IL-1 receptor antagonist (IL-1ra), a competitive inhibitor of IL-1, TNF binding protein (TNFbp), an inhibitor of TNF, or the anti-IL-6 antibody (Ab) 20F3 for the first 2 wk after surgery. ovx increased the bone marrow cells secretion of IL-1 and TNF, but not IL-6, and the formation of TRAP-positive osteoclast-like multinucleated cells (MNCs) in bone marrow cultures treated with 1,25(OH)2D3. The increase in MNC formation induced by ovx was prevented by in vivo treatment with either 17 beta estradiol, IL-1ra, TNFbp, or anti-IL-6 Ab. However, the percent change in MNC formation induced by the anti-IL-6 Ab was similar in ovx and sham-operated animals, whereas IL-1ra and TNFbp were effective only in ovx mice. MNC formation was also decreased by in vitro treatment of bone marrow cultures with IL-1ra and TNFbp, but not with anti-IL-6 Ab. Ovx also increased bone resorption in vivo and in vitro, as assessed by the urinary excretion of pyridinoline cross links and the formation of resorption pits, respectively. IL-1ra, TNFbp and estrogen decreased bone resorption in vivo and in vitro whereas the anti-IL-6 Ab inhibited bone resorption in vitro but not in vivo. In conclusion, these data indicate that IL-1 and TNF play a direct role in mediating the effects of ovx on osteoclastogenesis and bone resorption. The data also suggest that IL-6 is not essential for increasing bone resorption in the early postovariectomy period.

Estrogen blocks M-CSF gene expression and osteoclast formation by regulating phosphorylation of Egr-1 and its interaction with Sp-1.

Central to the pathogenesis of osteoporosis is the ability of estrogen deficiency to increase osteoclast formation by enhancing stromal cell production of the osteoclastogenic cytokine macrophage colony-stimulating factor (M-CSF). We report that stromal cells from ovariectomized mice exhibit increased casein kinase II-dependent phosphorylation of the nuclear protein Egr-1. Phosphorylated Egr-1 binds less avidly to the transcriptional activator Sp-1 and the resulting higher levels of free Sp-1 stimulate transactivation of the M-CSF gene. Estrogen replacement fails to block M-CSF mRNA expression and osteoclast formation in ovariectomized mice lacking Egr-1, confirming the critical role played by this transcription factor in mediating the antiosteoclastogenic effects of estrogen. Thus, by downregulating formation of a novel Egr-1/Sp-1 complex in stromal cells, estrogen deficiency results in enhanced levels of free Sp-1 and increased M-CSF gene expression and osteoclast formation.

Interleukin-1 receptor antagonist decreases bone loss and bone resorption in ovariectomized rats.

Interleukin-1 (IL-1), a cytokine produced by bone marrow cells and bone cells, has been implicated in the pathogenesis of postmenopausal osteoporosis because of its potent stimulatory effects on bone resorption in vitro and in vivo. To investigate whether IL-1 plays a direct causal role in post ovariectomy bone loss, 6-mo-old ovariectomized rats were treated with subcutaneous infusions of IL-1 receptor antagonist (IL-1ra), a specific competitor of IL-1, for 4 wk, beginning either at the time of surgery or 4 wk after ovariectomy. The bone density of the distal femur was measured non invasively by dual-energy X-ray absorptiometry. Bone turnover was assessed by bone histomorphometry and by measuring serum osteocalcin, a marker of bone formation, and the urinary excretion of pyridinoline cross-links, a marker of bone resorption. Ovariectomy caused a rapid increase in bone turnover and a marked decrease in bone density which were blocked by treatment with 17 beta estradiol. Ovariectomy also increased the production of IL-1 from cultured bone marrow cells. Ovariectomy induced-bone loss was significantly decreased by IL-1ra treatment started at the time of ovariectomy and completely blocked by IL-1ra treatment begun 4 wk after ovariectomy. In both studies IL-1ra also decreased bone resorption in a manner similar to estrogen, while it had no effect on bone formation. In contrast, treatment with IL-1ra had no effect on the bone density and the bone turnover of sham-operated rats, indicating that IL-1ra specifically blocked estrogen-dependent bone loss. In conclusion, these data indicate that IL-1, or mediators induced by IL-1, play an important causal role in the mechanism by which ovariectomy induces bone loss in rats, especially following the immediate post ovariectomy period.

The functional block of TNF but not of IL-6 prevents bone loss in ovariectomized mice.

Considerable evidence supports the hypothesis that estrogen prevents bone loss by blocking the bone marrow cell production of pro-osteoclastogenic cytokines. However, controversy remains on the role of candidate factors, such as tumor necrosis factor (TNF) and interleukin-6 (IL-6). To investigate the contribution of these cytokines to the pathogenesis of ovariectomy (OVX)-induced bone loss, OVX mice were treated with either TNF binding protein (TNFbp), an inhibitor of TNF, the anti-(IL-6) antibody (Ab) 20F3, or estrogen for the first 2 weeks after surgery. OVX caused a rapid decrease in trabecular bone volume (TBV) and an increase in in vivo bone resorption, as assessed by bone histomorphometry. Treatment with TNFbp completely prevented bone loss and the increase in both osteoclast formation and bone resorption induced by OVX, but had no effects in sham-operated controls. In contrast, treatment with anti-IL-6 antibody failed to prevent bone loss, and the increase in bone resorption and osteoclastogenesis induced by OVX. These data demonstrate that in nongenetically manipulated mice, the estrogen-regulated cytokine that plays a central role in the mechanism by which estrogen deficiency causes bone loss is not IL-6, but rather TNF.

Simultaneous block of interleukin-1 and tumor necrosis factor is required to completely prevent bone loss in the early postovariectomy period.

Considerable evidence supports the hypothesis that estrogen prevents bone loss by blocking the production of cytokines in bone or bone marrow. However, controversy remains on the role of candidate factors, such as interleukin-1 (IL-1), IL-6, and tumor necrosis factor (TNF). As IL-1 and TNF have many additive and/or synergistic effects in bone, we tested the hypothesis that the simultaneous block of IL-1 and TNF is required to prevent the initial phase of rapid bone loss that follows ovariectomy (ovx). To this aim, rats were ovariectomized and treated for 2 weeks with either IL-1 receptor antagonist (IL-1ra), an inhibitor of IL-1, or TNF-binding protein (TNFbp), an inhibitor of TNF. Ovx increased bone marrow cell secretion of IL-1 and TNF and decreased the bone density of the distal femur, as measured by dual energy x-ray absorptiometry. Ovx-induced bone loss was decreased by both IL-1ra and TNFbp and completely prevented by simultaneous treatment with IL-1ra and TNFbp. Combined treatment with IL-1ra and TNFbp decreased urinary pyridinoline cross-links, a marker of bone resorption that reflects osteoclast number and osteoclast activity, whereas treatment with either inhibitor alone was less effective. Both IL-1ra and TNFbp decreased the number of osteoclasts on the endocortical surfaces and stimulated bone formation, but the two inhibitors had no additive effects on these indexes, suggesting that inhibition of osteoclastogenesis and stimulation of bone formation do not account for the additive bone-sparing effects of IL-1ra and TNFbp. These inhibitors had no effect in sham-operated rats, indicating that they specifically blocked estrogen-dependent events. In conclusion, these data indicate that in the early post-ovx period, IL-1 and TNF play a critical causal role in inducing bone loss and do so by stimulating bone resorption and inhibiting bone formation.

Skeletal overexpression of noggin results in osteopenia and reduced bone formation.

Skeletal cells synthesize bone morphogenetic proteins (BMPs) and BMP antagonists. Noggin is a glycoprotein that binds BMPs selectively and antagonizes BMP actions. Noggin expression in osteoblasts is induced by BMPs and noggin opposes the effects of BMPs on osteoblastic differentiation and function in vitro. However, its effects in vivo are not known. We investigated the direct in vivo effects of noggin on bone remodeling in transgenic mice overexpressing noggin under the control of the osteocalcin promoter. Noggin transgenics suffered long bone fractures in the first month of life. Total, vertebral, and femoral bone mineral densities were reduced by 23-29%. Static and dynamic histomorphometry of the femur revealed that noggin transgenic mice had decreased trabecular bone volume, number of trabeculae, and bone formation rate. Osteoblast surface and number of osteoblasts/trabecular area were not significantly decreased, indicating impaired osteoblastic function. Osteoclast surface and number were normal/decreased, there was no increase in bone resorption, and the tissue had the appearance of woven bone. Vertebral microcomputed tomography scanning confirmed decreased trabecular bone volume and trabecular number. In conclusion, transgenic mice overexpressing noggin in the bone microenvironment have decreased trabecular bone volume and impaired osteoblastic function, leading to osteopenia and fractures.

Monocytic secretion of interleukin-1 receptor antagonist in normal and osteoporotic women: effects of menopause and estrogen/progesterone therapy.

Interleukin-1 (IL-1), a potent stimulant of bone resorption, has been implicated in the pathogenesis of postmenopausal osteoporosis, because monocyte IL-1 bioactivity increases after the menopause and is decreased by estrogen and progesterone (EP) replacement. As IL-1 bioactivity reflect the production of both IL-1 and the IL-1 inhibitor, IL-1 receptor antagonist (IL-1ra), EP treatment could decrease IL-1 bioactivity by regulating the secretion of either IL-1 or IL-1ra. We now report that EP treatment in vivo decreased the secretion into the medium of cultured monocytes of IL-1ra and IL-1 beta as well as the IL-1 beta/IL-1ra ratio. We also found that in normal women the production of IL-1ra was within premenopausal levels in the first 7 yr after the menopause and increased linearly thereafter. In these women, monocyte IL-1 beta, IL-1 beta/IL-1ra ratio, and IL-1 bioactivity were all increased in the first 7 yr after the menopause and within the premenopausal range thereafter. In osteoporotic women, IL-1 beta, IL-1 beta/IL-1ra ratio, and IL-1 bioactivity increased after the menopause and returned to premenopausal levels after 15 yr from the menopause. In these women monocyte IL-1ra secretion was above the premenopausal range at all times after the menopause, but did not change with the passage of time since menopause. We conclude that hormone replacement decreases the in vitro secretion of both IL-1ra and IL-1 beta. The data also suggest that in normal women a progressive increase in the secretion of IL-1ra contributes to restore a normal IL-1/IL-1ra ratio after the menopause, a phenomenon which, in turn, may play a role in limiting postmenopausal bone loss.

Alcohol, cytokines, and estrogen in the control of bone remodeling.

Alcohol has been identified as a risk factor for the development of osteoporosis. Chronic alcohol abuse has been shown to decrease bone mass and increase the incidence of fractures. Although the exact mechanism by which alcohol influences bone metabolism is not clear, it is likely a combination of both direct and indirect effects on bone cells. Alcohol has the potential to alter bone metabolism indirectly through its effects on gonadal hormones and through the secretion of cytokines shown to be critical factors in postmenopausal osteoporosis. Data are summarized here demonstrating that interleukin-1 and tumor necrosis factor play a direct causal role in the bone loss due to estrogen deficiency. In addition, studies are cited showing that these same cytokines are produced in alcohol-induced liver disease with the potential to enhance bone loss in post-menopausal women and in male patients. Although there are not yet data directly supporting the role of cytokines in bone loss due to alcohol consumption, the studies presented herein are intended to stimulate further research on the role of alcohol, estrogen, and cytokines in osteoporotic bone loss.

Estrogen deficiency increases the ability of stromal cells to support murine osteoclastogenesis via an interleukin-1and tumor necrosis factor-mediated stimulation of macrophage colony-stimulating factor production.

To analyze how estrogen blocks osteoclastogenesis, we investigated the effects of ovariectomy on osteoclast (OC) formation in co-cultures of purified OC precursors and purified stromal cells (SC). OC formation was higher in co-cultures containing SC from ovariectomized mice than in those containing SC from sham-operated mice, thus suggesting that estrogen regulates osteoclastogenesis by targeting SC. Ovariectomy also increased the mononuclear cell secretion of interleukin (IL)-1) and tumor necrosis factor (TNF) and the SC production of macrophage colony-stimulating factor (MCSF). Osteoclastogenesis and SC production of M-CSF were not blocked by in vitro estrogen treatment but were decreased by in vivo treatment of donor mice with either estrogen or a combination of the IL-1 inhibitor, IL-1 receptor antagonist, and the TNF inhibitor, TNF binding protein. IL-1 and TNF production were also blocked by in vivo estrogen treatment, demonstrating that the increased bone marrow levels of IL-1 and TNF characteristic of ovariectomized mice induce the formation of a SC population characterized by a high production of M-CSF and increased pro-osteoclastogenic activity. Since in co-cultures of SC and OC precursors M-CSF levels correlated with OC production (r = 0.7, p < 0.0001), the data also indicate that the pro-osteoclastogenic activity of SC is proportional to their secretion of M-CSF. The ability of estrogen to decrease SC production of M-CSF and the pro-osteoclastogenic activity of these cells by regulating IL-1 and TNF production is a previously undescribed mechanism by which estrogen down-regulates osteoclastogenesis.

Persistent bone-sparing effect of interleukin-1 receptor antagonist: a hypothesis on the role of IL-1 in ovariectomy-induced bone loss.

The recent finding that treatment with the interleukin-1 (IL-1) inhibitor, interleukin-1 receptor antagonist (IL-1ra) decreases bone loss and bone resorption in ovariectomized rats, strongly suggested that IL-1 mediates, at least in part, the effects of estrogen deficiency on bone resorption. Although in vitro studies have shown that IL-1 activates mature osteoclasts and stimulates osteoclastogenesis, the two main mechanisms by which estrogen deficiency stimulates bone resorption, it is still unclear whether IL-1 mediates both effects of estrogen deficiency in vivo. To investigate this matter, we have examined the changes in bone mineral density (BMD) which occur in ovariectomized rats after completion of 1 month of estrogen or IL-1ra treatment begun at the time of ovariectomy. Ovariectomy caused a marked decreased in BMD which was blocked by 17 beta estradiol and decreased by IL-1ra. Cessation of estrogen therapy was followed by a rapid induction of bone loss, indicating that estrogen blocks the activation and utilization of mature osteoclasts without depleting the bone microenvironment of osteoclast precursors and mature, inactive osteoclasts. In contrast, ovariectomized rats treated with IL-1ra maintained a stable bone density for the first 4 weeks after completion of the treatment. In these rats, bone loss resumed not earlier than 6 weeks after discontinuation of the IL-1ra treatment. Estrogen deficiency was necessary to unveil the bone-sparing effect of IL-1ra because in a control experiment in which rats were treated with IL-1ra for the 4 weeks before ovariectomy, BMD began to decrease immediately after ovariectomy.(ABSTRACT TRUNCATED AT 250 WORDS)