Sp1 and Sp3 regulate the basal transcription of receptor activator of nuclear factor kappa B ligand gene in osteoblasts and bone marrow stromal cells.
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.
Liu
Jianzhong
J
Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA.
Yang
Hongmei
H
Liu
Wei
W
Cao
Xuemei
X
Feng
Xu
X
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D001854:Bone Marrow Cells / Q000378:metabolism*; D002352:Carrier Proteins / Q000235:genetics*; D002478:Cells, Cultured; D016384:Consensus Sequence; D024202:Electrophoretic Mobility Shift Assay; D005786:Gene Expression Regulation; D008562:Membrane Glycoproteins / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009154:Mutation / Q000235:genetics; D009687:Nuclear Proteins / Q000378:metabolism; D009841:Oligonucleotides; D010006:Osteoblasts / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000378:metabolism*; D051705:Sp3 Transcription Factor / Q000378:metabolism*; D017154:Stromal Cells / Q000378:metabolism*; D014158:Transcription, Genetic
D002352:Carrier Proteins; D008562:Membrane Glycoproteins; D009687:Nuclear Proteins; D009841:Oligonucleotides; D053245:RANK Ligand; D053246:Receptor Activator of Nuclear Factor-kappa B; D016329:Sp1 Transcription Factor; C505687:Tnfrsf11a protein, mouse; C505689:Tnfsf11 protein, mouse; D051705:Sp3 Transcription Factor