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-lra), 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 TRAPpositive 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 17/8 estradiol, IL-lra, 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 shamoperated animals, whereas IL-lra and TNFbp were effective only in ovx mice. MNC formation was also decreased by in vitro treatment of bone marrow cultures with IL-lra 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-lra, 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. (J. Clin. Invest. 1994.

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.

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 shamoperated 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 (M-CSF). 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.It is now recognized that one of the main mechanisms by which estrogen blocks bone loss is inhibition of proliferation and differentiation of osteoclast (OC) 1 precursors (1). However, the mechanism of these effects and the cellular targets of estrogen in bone are still controversial (2).Osteoclastogenesis is a complex phenomenon that is facilitated by the interaction of bone marrow stromal cells (SC) with hematopoietic OC precursors. SC contribute to osteoclastogenesis by providing a physical support for nascent OCs and by producing soluble and membrane-associated factors that stimulate the proliferation and/or the differentiation of hematopoietic OC precursors (3). Among these factors are M-CSF (3-5), interleukin (IL)-6 (6), and IL-11 (7).M-CSF is essential for the proliferation and differentiation of OC precursors (5) and appears to play a critical role in murine osteoclastogenesis because antibodies against this cytokine completely block OC formation in bone marrow cultures (5). The relevance of M-CSF is further demonstrated by the ability of M-CSF replacement to cure osteopetrosis in op/op mice, a strain characterized by the production of defective M-CSF (3). SC production of M-CSF is induced by IL-1 and tumor necrosis factor (TNF) ␣ and ␤ (8, 9), cytokines produced mainly by bone marrow mononuclear cells (10, 11) and recognized for their ability to promote OC formation and bone resorption (12, 13)....

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.