SUMMARYSOX9 is a transcription factor of the SRY family that regulates sex determination, cartilage development and numerous other developmental events. In the foetal growth plate, Sox9 is highly expressed in chondrocytes of the proliferating and prehypertrophic zone but declines abruptly in the hypertrophic zone, suggesting that Sox9 downregulation in hypertrophic chondrocytes might be a necessary step to initiate cartilage-bone transition in the growth plate. In order to test this hypothesis, we generated transgenic mice misexpressing Sox9 in hypertrophic chondrocytes under the control of a BAC-Col10a1 promoter. The transgenic offspring showed an almost complete lack of bone marrow in newborns, owing to strongly retarded vascular invasion into hypertrophic cartilage and impaired cartilage resorption, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed high levels of Sox9 misexpression in hypertrophic chondrocytes but deficiencies of Vegfa, Mmp13, RANKL and osteopontin expression in the non-resorbed hypertrophic cartilage, indicating that Sox9 misexpression in hypertrophic chondrocytes inhibits their terminal differentiation. Searching for the molecular mechanism of SOX9-induced inhibition of cartilage vascularization, we discovered that SOX9 is able to directly suppress Vegfa expression by binding to SRY sites in the Vegfa gene. Postnatally, bone marrow formation and cartilage resorption in transgenic offspring are resumed by massive invasion of capillaries through the cortical bone shaft, similar to secondary ossification. These findings imply that downregulation of Sox9 in the hypertrophic zone of the normal growth plate is essential for allowing vascular invasion, bone marrow formation and endochondral ossification.

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Previously we have shown that insertion of a LacZ reporter gene into the Col10a1 gene in the context of a bacterial artificial chromosome (BAC) drives strong and specific expression of LacZ in hypertrophic cartilage of transgenic mice [Gebhard S., Hattori T., Bauer E., Bosl M.R., Schlund B., Poschl E., Adam N., de Crombrugghe B., von der Mark K., 2007 Histochem. Cell Biol. 19 127:183–194]. BAC constructs in transgenic reporter mouse lines control efficient and specific LacZ expression in hypertrophic chondrocytes under the complete Col10a1 promoter. Here we report on the generation of Col10a1-specific Cre deleter mice using a BAC recombineering technique based on homologous recombination in E. coli. Sixteen BAC-Col10-Cre transgenic lines were generated containing between 1 and 5 copies of the BAC-Col10-Cre gene. All lines tested so far expressed Cre specifically in hypertrophic chondrocytes of E16.5 and P1 growth plates of long bones, ribs, vertebrae and sternum as examined by crossing with ROSA26 reporter mice. Cre activity was detected as early as E13.5 when hypertrophic cartilage develops in the diaphysis of femur and humerus. The data confirm that expression of Cre under the control of the complete BAC-Col10a1 promoter occurs with high efficiency and specificity in hypertrophic chondrocytes. The BAC-Col10-Cre lines should thus provide a valuable tool to get further insight into the role of genes involved in endochondral ossification by allowing their specific deletion in the hypertrophic zone of the growth plate.

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PTH and PTHrP have been shown to inhibit maturation of growth plate chondrocytes and the expression of type X collagen. In order to examine the regulatory mechanisms involved, fetal bovine growth plate chondrocytes were incubated for 24-48 h under serum-free conditions with PTH and PTHrP and various aminoterminal, midregional, and carboxyterminal fragments of these hormones. Analysis of type X collagen mRNA levels by Northern hybridization showed a significant suppression by PTH (1-84), PTH (1-34), and PTHrP (1-40), but not by PTH (28-48) or PTH (53-84). PTH fragment (3-34) did not reduce alpha1(X) mRNA levels, while bis-indolylmaleimide, an inhibitor of the protein-kinase C pathway, did not affect alpha1(X) mRNA suppression by PTH, supporting the notion that the inhibition of type X collagen expression by PTH involves predominantly the adenylate cyclase pathway of the PTH/PTHrP-receptor. Since PTH and PTHrP have been shown to induce c-fos in osteoblasts and chondrocytes, the possibility was tested that c-fos mediated the suppressive effect of PTH/PTHrP on collagen X expression. In fetal bovine hypertrophic chondrocytes PTH (1-34), but not PTH (3-34) nor the midregional or C-terminal PTH fragments induced c-fos expression. In order to identify cis- and trans-acting elements in the COL10A1 gene involved in c-fos-mediated inhibition of collagen X expression by PTH/PTHrP, reporter gene constructs carrying various fragments of the human COL10A1 promoter coupled to the luciferase gene were transfected into hypertrophic chondrocytes. A tissue-specific, strong enhancer region, which we had previously located in the promoter of the human type X collagen gene COL10A1, was further narrowed down to a 530-bp sequence, located between - 1,870- and - 2,407 bp upstream of the transcription start site. The transcriptional activity of this enhancer element in transfected hypertrophic chondrocytes was significantly reduced after incubation with PTH (1-34) or PTHrP (1-40). Transcription of these reporter genes was also inhibited when chondrocytes were cotransfected with a c-fos expression vector. These results indicate the presence of a PTH/PTHrP responsive element in the human COL10A1 enhancer, which may be represented by multiple putative AP-1 sites located in this region.