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Sp1 and Sp3 regulate the basal transcription of receptor activator of nuclear factor kappa B ligand gene in osteoblasts and bone marrow stromal cells
Author(s) -
Liu Jianzhong,
Yang Hongmei,
Liu Wei,
Cao Xuemei,
Feng Xu
Publication year - 2005
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/jcb.20569
Subject(s) - rankl , stromal cell , microbiology and biotechnology , transcription factor , osteoblast , osteoclast , activator (genetics) , biology , electrophoretic mobility shift assay , chemistry , transcription (linguistics) , promoter , cancer research , receptor , gene expression , gene , biochemistry , in vitro , linguistics , philosophy
Receptor activator of nuclear factor kappa B ligand (RANKL), a potent regulator of osteoclast formation and function, is expressed by osteoblasts and bone marrow stromal cells. However, the molecular mechanism underlying RANKL expression in osteoblast/stromal cells remains largely unclear. Here, we characterized the molecular mechanism controlling RANKL basal transcription in osteoblast/stromal cells. We cloned a 1,103‐bp murine RANKL promoter (from −953 to +150, relative to the transcription start site). Using a series of deletion mutants of the 1,103‐bp promoter, we identified a 100‐bp region (−154 to −54) mediating RANKL basal transcription in both osteoblasts and bone marrow stromal cells. Electrophoretic mobility shift assay (EMSA) using five overlapping oligonucleotides (Probes 1–5) spanning the 100‐bp region showed that Probes 1 and 2 specifically bound nuclear proteins with high affinity from both cell types. Computer analysis revealed that Probes 1 and 2 contain a putative Sp1‐binding site. Supershift assays with Sp1 and Sp3 antibodies confirmed that the nuclear proteins binding to Probes 1 and 2 are Sp1 and Sp3. Functionally, the mutation of the Sp1/Sp3 site in Probe 1 profoundly reduced the basal promoter activity while the mutation of the one in Probe 2 resulted in moderate reduction in the basal promoter activity. Moreover, the mutation of both sites abrogated the RANKL basal promoter activity, indicating that Sp1 and Sp3 play a key role in the RANKL basal transcription in osteoblasts and bone marrow stromal cells. © 2005 Wiley‐Liss, Inc.