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
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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DOI：
10.1002/jcb.10260
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发表时间：
2002-01-01
影响因子：
4
通讯作者：
von der Mark, K
中科院分区：
文献类型：
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作者：
Riemer, S;Gebhard, S;von der Mark, K
PTH和PTHrP可抑制生长板软骨细胞的成熟和X型胶原的表达。为探讨其调控机制，将胎牛生长板软骨细胞与甲状旁腺素、甲状旁腺素及这些激素的氨基末端、中段和羧基末端片段在无血清条件下孵育24~48h。Northern杂交结果显示，PTH(1-84)、PTH(1-34)和PTHrP(1-40)对X型胶原的表达有明显的抑制作用，而PTH(28-48)和PTH(53-84)对X型胶原的表达无明显抑制作用。PTH片段(3-34)不降低α1(X)mRNA水平，而蛋白激酶C途径的抑制剂双吲哚马来酰亚胺不影响PTH抑制α1(X)mRNA表达的观点，支持PTH抑制X型胶原表达主要涉及PTH/PTHrP受体的腺苷环化酶途径。由于PTH和PTHrP已被证实可诱导成骨细胞和软骨细胞中c-fos的表达，因此验证了c-fos介导PTH/PTHrP抑制X胶原蛋白表达的可能性。在胎牛肥大软骨细胞中，PTH(1-34)不能诱导c-fos的表达，但PTH(3-34)和微区或C末端的PTH片段不能诱导c-fos的表达。为了确定COL10A1基因中的顺式和反式作用元件参与c-fos介导的PTH/PTHrP抑制X胶原表达，将携带不同片段的人COL10A1启动子片段与荧光素酶基因偶联的报告基因载体导入肥大软骨细胞。我们之前在人X型胶原基因COL10A1的启动子中定位了一个组织特异性的强增强子区域，后来进一步缩小到530个核苷酸的序列，位于转录起始点上游-1,870-2,407个核苷酸之间。与PTH(1-34)或PTHrP(1-40)孵育后，该增强子元件在肥大软骨细胞中的转录活性显著降低。当软骨细胞与c-fos表达载体共转染时，这些报告基因的转录也被抑制。这些结果表明，人COL10A1增强子中存在PTH/PTHrP反应元件，该元件可能由位于该区域的多个推测的AP-1位点表示。(C)2002年Wiley-Liss，Inc.
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 micregional 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. (C) 2002 Wiley-Liss, Inc.