Sp1 family of transcription factors regulates the human α2 (XI) collagen gene (COL11A2) in Saos-2 osteoblastic cells
Sp1 family of transcription factors regulates the human α2 (XI) collagen gene (COL11A2) in Saos-2 osteoblastic cells
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DOI：
10.1359/jbmr.020605
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发表时间：
2006-05-01
影响因子：
6.2
通讯作者：
Yasui, N
中科院分区：
文献类型：
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作者：
Goto, T;Matsui, Y;Yasui, N
XI型胶原蛋白是骨骼形态发生所必需的。胶原XI基因调控已在软骨细胞中研究，但在成骨细胞中尚未研究。材料和方法：我们培养了人骨肉瘤来源的成骨细胞Saos-2细胞。分析α 2（XI）蛋白和COL11A2的调控机制。结果与结论：虽然Saos-2细胞沉积的胶原以I型和V型为主，但它们表达COL11A2 mRNA，细胞外基质中存在α 2（XI）链。COL11A2启动子区域（从-149到-40）包含三个Sp1结合位点，在瞬时转染试验中启动子活性是必需的。所有三个Sp1位点都是电泳迁移迁移试验中核蛋白结合的关键位点。进一步的分析使用一致的寡核苷酸和特异性抗体以及染色质免疫沉淀试验表明Sp1和Sp3与该启动子区域结合。过表达Sp1或Sp3显著增加COL11A2启动子活性和内源性COL11A2基因表达，这一作用被Sp1结合抑制剂米霉素A抑制。进一步的实验表明，Sp1、Sp3、creb结合蛋白（CBP）、p300和历史性去乙酰化酶（HDAC）存在物理关联，HDAC抑制剂（曲古斯汀A或NaB）上调COL11A2启动子活性和内源性基因表达。Sp1家族的另一个成员Sp7 （Osterix）在Saos-2细胞中表达，但在软骨细胞中不表达，并通过染色质免疫沉淀显示占据COL11A2启动子。过表达Sp7增加COL11A2启动子活性和内源基因表达，这一作用也被米霉素a阻断。使用siRNA敲低Sp1、Sp3或Sp7，结果表明，抑制其中任何一个都能降低COL11A2启动子活性和内源基因表达。最后，原代培养的成骨细胞表达COL11A2和Sp7，当Sp1、Sp3或Sp7过表达时，COL11A2启动子活性和内源基因表达上调，当Sp1、Sp3或Sp7被选择性抑制时，COL11A2启动子活性和内源基因表达下调。结果表明Sp1蛋白通过结合COL11A2的近端启动子并直接与CBP、p300和HDAC相互作用来调节COL11A2的转录。
Introduction: Type XI collagen is essential for skeletal morphogenesis. Collagen XI gene regulation has been studied in chondrocytes but not in osteoblasts.Materials and Methods: We cultured Saos-2 cells, a human osteosarcoma-derived line of osteoblasts. and analyzed them for alpha 2(XI) protein and COL11A2 regulatory mechanisms.Results and Conclusions: Although types I and V were the dominant collagens deposited by Saos-2 cells, they expressed COL11A2 mRNA, and alpha 2(XI) chains were present in the extracellular matrix. The COL11A2 promoter region (from -149 to -40) containing three Sp1 binding sites was required for promoter activity in transient transfection assays. All three Sp1 sites were critical for binding by nuclear proteins in electrophoretic mobility shift assays. Further analysis using consensus oligonucleotides and specific antibodies as well as chromatin immunoprecipitation assay implicated Sp1 and Sp3 in binding to this promoter region. Overexpressing Sp1 or Sp3 significantly increased COL11A2 promoter activity and endogenous COL11A2 gene expression, an effect that was suppressed by the Sp1-binding inhibitor mithramycin A. Further experiments showed that Sp1, Sp3, CREB-binding protein (CBP), p300, and historic deacetylase (HDAC) were physically associated and HDAC inhibitors (trichostatin A or NaB) upregulated COL11A2 promoter activity and endogenous gene expression. Another Sp1 family member, Sp7 (Osterix), was expressed in Saos-2 cells, but not in chondrocytes, and was shown by chromatin immunoprecipitation to occupy the COL11A2 promoter. Overexpressing Sp7 increased COL11A2 promoter activity and endogenous gene expression, an effect also blocked by mithramycin A. Using siRNA to knockdown Sp1, Sp3, or Sp7, it was shown that depression of any of them decreased COL11A2 promoter activity and endogenous gene expression. Finally, primary cultures of osteoblasts expressed COL11A2 and Sp7, upregulated COL11A2 promoter activity and endogenous gene expression when Sp1, Sp3, or Sp7 were overexpressed, and downregulated them when Sp1, Sp3, or Sp7 were selectively depressed. The results establish that Sp1 proteins regulate COL11A2 transcription by binding to its proximal promoter and directly interacting with CBP, p300, and HDAC.