The authors state that they have no conflicts of interest.
Sp1 Family of Transcription Factors Regulates the Human α2 (XI) Collagen Gene (COL11A2) in Saos-2 Osteoblastic Cells†
Article first published online: 20 FEB 2006
Copyright © 2006 ASBMR
Journal of Bone and Mineral Research
Volume 21, Issue 5, pages 661–673, May 2006
How to Cite
Goto, T., Matsui, Y., Fernandes, R. J., Hanson, D. A., Kubo, T., Yukata, K., Michigami, T., Komori, T., Fujita, T., Yang, L., Eyre, D. R. and Yasui, N. (2006), Sp1 Family of Transcription Factors Regulates the Human α2 (XI) Collagen Gene (COL11A2) in Saos-2 Osteoblastic Cells. J Bone Miner Res, 21: 661–673. doi: 10.1359/jbmr.020605
- Issue published online: 4 DEC 2009
- Article first published online: 20 FEB 2006
- Manuscript Accepted: 13 FEB 2006
- Manuscript Revised: 23 JAN 2006
- Manuscript Received: 24 AUG 2005
- transcription factors;
- type XI collagen
Genes encoding type XI collagen, normally associated with chondrogenesis, are also expressed by osteoblasts. By studying Saos-2 cells, we showed that the transcription factors, Sp1, Sp3, and Sp7 (Osterix), regulate COL11A2 expression through its proximal promoter. The findings indicate both ubiquitous and osteoblast-specific mechanisms of collagen gene regulation.
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 α2(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 α2(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 histone 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.