Deletion of beta catenin in hypertrophic growth plate chondrocytes impairs trabecular bone formation.

In order to elucidate the role of ß-catenin in hypertrophic cartilage zone of the growth plate, we deleted the ß-catenin gene ctnnb1specifically from hypertrophic chondrocytes by mating ctnnb1(fl/fl) mice with BAC-Col10a1-Cre-deleter mice. Surprisingly, this resulted in a significant reduction of subchondral trabecular bone formation in BACCol10Cre; ctnnb1(Δ/Δ) (referred to as Cat-ko) mice, although Cre expression was restricted to hypertrophic chondrocytes. The size of the Col10a1 positive hypertrophic zone was normal, but qRT-PCR revealed reduced expression of Mmp13, and Vegfa in Cat-ko hypertrophic chondrocytes, indicating impaired terminal differentiation. Immunohistological and in situ hybridization analysis revealed the substantial deficiency of collagen I positive mature osteoblasts, but equal levels of osterix-positive cells in the subchondral bone marrow space of Cat-ko mice, indicating that the supply of osteoblast precursor cells was not reduced. The fact that in Cat-ko mice subchondral trabeculae were lacking including their calcified cartilage core indicated a strongly enhanced osteoclast activity. In fact, TRAP staining as well as in situ hybridization analysis of Mmp9 expression revealed denser occupation of the cartilage erosion zone with enlarged osteoclasts as compared to the control growth plate, suggesting increased RANKL or reduced osteoprotegerin (Opg) activity in this zone. This notion was confirmed by qRT-PCR analysis of mRNA extracted from cultured hypertrophic chondrocytes or from whole epiphyses, showing increased Rankl mRNA levels in Cat-ko as compared to control chondrocytes, whereas changes in OPG levels were not significant. These results indicate that ß-catenin levels in hypertrophic chondrocytes play a key role in regulating osteoclast activity and trabecular bone formation at the cartilage-bone interface by controlling RANKL expression in hypertrophic chondrocytes.

SOX9 is a major negative regulator of cartilage vascularization, bone marrow formation and endochondral ossification.

SOX9 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.

Specific expression of Cre recombinase in hypertrophic cartilage under the control of a BAC-Col10a1 promoter.

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.

BAC constructs in transgenic reporter mouse lines control efficient and specific LacZ expression in hypertrophic chondrocytes under the complete Col10a1 promoter.

During endochondral ossification hypertrophic chondrocytes in the growth plate of fetal long bones, ribs and vertebrae play a key role in preparing growth plate cartilage for replacement by bone. In order to establish a reporter gene mouse to facilitate functional analysis of genes expressed in hypertrophic chondrocytes in this process, Col10a1- BAC reporter gene mouse lines were established expressing LacZ specifically in hypertrophic cartilage under the control of the complete Col10a1 gene. For this purpose, a bacterial artificial chromosome (BAC RP23-192A7) containing the entire murine Col10a1 gene together with 200 kb flanking sequences was modified by inserting a LacZ-Neo cassette into the second exon of Col10a1 by homologous recombination in E. coli. Transgenic mice containing between one and seven transgene copies were generated by injection of the purified BAC-Col10a1- lLacZ DNA. X-gal staining of newborns and embryos revealed strong and robust LacZ activity exclusively in hypertrophic cartilage of the fetal and neonatal skeleton of the transgenic offspring. This indicates that expression of the reporter gene in its proper genomic context in the BAC Col10a1 environment is independent of the integration site and reflects authentic Col10a1 expression in vivo. The Col10a1 specific BAC recombination vector described here will enable the specific analysis of effector gene functions in hypertrophic cartilage during skeletal development, endochondral ossification, and fracture callus healing.

A highly conserved enhancer in mammalian type X collagen genes drives high levels of tissue-specific expression in hypertrophic cartilage in vitro and in vivo.

Previously we have identified a cis-acting regulatory domain in the human type X collagen gene upstream of the transcription start site which acts as a strong enhancer in hypertrophic, but not in resting chondrocytes. Here we show that this enhancer is highly conserved also in the murine and bovine Col10a1 genes, but not found in the known promoter sequences of chicken Col10a1. It contains a functionally active AP-1 site (TPA Responsive Element, TRE) which is essential for the high transcriptional activity of the COL10A1 enhancer in transiently transfected hypertrophic chondrocytes. Gel-shift experiments with nuclear extracts of hypertrophic chondrocytes revealed FosB and Fra-1 as candidates regulating AP-1 factors binding to the TRE site. In fact, coexpression of FosB and Fra-1 in reporter gene assays greatly stimulated transcriptional activity of enhancer bearing reporter genes. Quantitative analysis of AP-1 factor mRNA levels in distinct fractions of fetal bovine epiphyseal chondrocytes by real-time PCR confirmed significant levels of FosB and Fra-1 mRNA besides other AP-1 factors in hypertrophic chondrocytes. A key role of the enhancer element in regulating tissue-specific expression of the Col10a1 gene was shown by establishing transgenic mouse lines with a reporter gene containing a 4.6 kb murine Col10a1 promoter fragment which included the enhancer, exon 1, part of exon 2 and the first intron. Reporter gene expression was seen exclusively in hypertrophic cartilages in the growth plates of long bones, ribs, vertebrae, sternum and mandibles of 17.5-18.5 dpc embryos, confirming that the 4.6 kb promoter is able to drive specific expression of Col10a1 in hypertrophic cartilage. These established transgenic lines should facilitate the genetic analysis of regulatory pathways of chondrocyte maturation and Col10a1 gene expression in the future.

Role of c-fos in the regulation of type X collagen gene expression by PTH and PTHrP: localization of a PTH/PTHrP-responsive region in the human COL10A1 enhancer.

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.