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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

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Abstract

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 α1(X) mRNA levels, while bis-indolylmaleimide, an inhibitor of the protein-kinase C pathway, did not affect α1(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. J. Cell. Biochem. 86: 688–699, 2002. © 2002 Wiley-Liss, Inc.

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