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<a href="http://doi.org/10.1152/ajpcell.00068.2016" target="_blank" rel="noreferrer noopener">http://doi.org/10.1152/ajpcell.00068.2016</a>
Pages
C212–224
Issue
2
Volume
311
Dublin Core
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Title
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Leptin augments recruitment of IRF-1 and CREB to thrombospondin-1 gene promoter in vascular smooth muscle cells in vitro.
Publisher
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American journal of physiology. Cell physiology
Date
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2016
2016-08
Subject
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*cAMP response element-binding protein; *interferon regulatory factor-1; *leptin; *thrombospondin-1; *transcription; *vascular smooth muscle cells; Binding Sites/genetics; Cells; Chromatin Immunoprecipitation/methods; Cultured; Cyclic AMP Response Element-Binding Protein/*metabolism; Gene Expression Regulation/genetics; Genetic/genetics; Humans; Interferon Regulatory Factor-1/*metabolism; Leptin/*metabolism; Muscle; Mutagenesis; Myocytes; Promoter Regions; Response Elements/genetics; Site-Directed/methods; Smooth; Smooth Muscle/*metabolism; Thrombospondin 1/*genetics/*metabolism; Transcription; Transcriptional Activation/genetics; Transfection/methods; Up-Regulation/genetics; Vascular/*metabolism
Creator
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Sahu Soumyadip; Ganguly Rituparna; Raman Priya
Description
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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.
Identifier
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<a href="http://doi.org/10.1152/ajpcell.00068.2016" target="_blank" rel="noreferrer noopener">10.1152/ajpcell.00068.2016</a>
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Article information provided for research and reference use only. All rights are retained by the journal listed under publisher and/or the creator(s).
*cAMP response element-binding protein
*interferon regulatory factor-1
*leptin
*thrombospondin-1
*Transcription
*vascular smooth muscle cells
2016
American journal of physiology. Cell physiology
Binding Sites/genetics
Cells
Chromatin Immunoprecipitation/methods
Cultured
Cyclic AMP Response Element-Binding Protein/*metabolism
Department of Integrative Medical Sciences
Ganguly Rituparna
Gene Expression Regulation/genetics
Genetic/genetics
Humans
Interferon Regulatory Factor-1/*metabolism
Leptin/*metabolism
Muscle
Mutagenesis
Myocytes
NEOMED College of Medicine
Promoter Regions
Raman Priya
Response Elements/genetics
Sahu Soumyadip
Site-Directed/methods
Smooth
Smooth Muscle/*metabolism
Thrombospondin 1/*genetics/*metabolism
Transcription
Transcriptional Activation/genetics
Transfection/methods
Up-Regulation/genetics
Vascular/*metabolism