Leptin augments recruitment of IRF-1 and CREB to thrombospondin-1 gene promoter in vascular smooth muscle cells in vitro.
We previously reported that high pathophysiological concentrations of leptin, the adipocyte-secreted peptide, upregulate the expression of a potent proatherogenic matricellular protein, thrombospondin-1 (TSP-1), in vascular smooth muscle cells. Moreover, this regulation was found to occur at the level of transcription; however, the underlying molecular mechanisms remain unknown. The goal of the present study was to investigate the specific transcriptional mechanisms that mediate upregulation of TSP-1 expression by leptin. Primary human aortic smooth muscle cell cultures were transiently transfected with different TSP-1 gene (THBS1) promoter-linked luciferase reporter constructs, and luciferase activity in response to leptin (100 ng/ml) was assessed. We identified a long THBS1 promoter (-1270/+750) fragment with specific leptin response elements that are required for increased TSP-1 transcription by leptin. Promoter analyses, protein/DNA array and gel shift assays demonstrated activation and association of transcription factors, interferon regulatory factor-1 (IRF-1) and cAMP response element-binding protein (CREB), to the distal fragment of the THBS1 promoter in response to leptin. Supershift, chromatin immunoprecipitation, and coimmunoprecipitation assays revealed formation of a single complex between IRF-1 and CREB in response to leptin; importantly, recruitment of this complex to the THBS1 promoter mediated leptin-induced TSP-1 transcription. Finally, binding sequence decoy oligomer and site-directed mutagenesis revealed that regulatory elements for both IRF-1 (-1019 to -1016) and CREB (-1198 to -1195), specific to the distal THBS1 promoter, were required for leptin-induced TSP-1 transcription. Taken together, these findings demonstrate that leptin promotes a cooperative association between IRF-1 and CREB on the THBS1 promoter driving TSP-1 transcription in vascular smooth muscle cells.
Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, Ohio; and School of Biomedical Sciences, Kent State University, Kent, Ohio.
Soumyadip
S
Sahu
Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, Ohio; and School of Biomedical Sciences, Kent State University, Kent, Ohio.
Rituparna
R
Ganguly
Department of Integrative Medical Sciences, Northeast Ohio Medical University, Rootstown, Ohio; and School of Biomedical Sciences, Kent State University, Kent, Ohio praman@neomed.edu.
Priya
P
Raman
D001665:Binding Sites / Q000235:genetics; D002478:Cells, Cultured; D047369:Chromatin Immunoprecipitation / Q000379:methods; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000235:genetics; D006801:Humans; D050856:Interferon Regulatory Factor-1 / Q000378:metabolism*; D020738:Leptin / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed / Q000379:methods; D032389:Myocytes, Smooth Muscle / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics; D020218:Response Elements / Q000235:genetics; D019700:Thrombospondin 1 / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic / Q000235:genetics; D015533:Transcriptional Activation / Q000235:genetics; D014162:Transfection / Q000379:methods; D015854:Up-Regulation / Q000235:genetics
C115163:CREB1 protein, human; D017362:Cyclic AMP Response Element-Binding Protein; C494257:IRF1 protein, human; D050856:Interferon Regulatory Factor-1; D020738:Leptin; D019700:Thrombospondin 1; C000611726:thrombospondin-1, human
cAMP response element-binding protein; interferon regulatory factor-1; leptin; thrombospondin-1; transcription; vascular smooth muscle cells