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Transcriptional Mechanisms that Mediate Downregulation of TSP‐1 by Chromium in Vascular Smooth Muscle Cells in Response to High Glucose
Author(s) -
Ganguly Rituparna,
Sahu Soumyadip,
Raman Priya
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.974.14
Subject(s) - downregulation and upregulation , chemistry , transcription factor , glucose uptake , vascular smooth muscle , microbiology and biotechnology , transcription (linguistics) , biochemistry , biology , gene , endocrinology , linguistics , philosophy , smooth muscle , insulin
Trivalent chromium (Cr 3+ ) is a beneficial mineral nutrient reported to lower the atherogenic index and reduce risks of vascular inflammation in diabetes. We recently reported that Cr 3+ inhibits expression of a proatherogenic matricellular protein, thrombospondin‐1 (TSP‐1), in glucose‐stimulated human aortic smooth muscle cells (HASMC) in vitro . Using luciferase reporter assays, we further showed that Cr 3+ significantly decreases TSP‐1 gene (THBS1) promoter activity in glucose‐stimulated cells. The goal of this study was to determine the molecular mechanisms by which Cr 3+ inhibits high glucose‐induced TSP‐1 transcription in HASMC. Using protein DNA array (Panomics) that allows detection of activated transcription factors (TF) in nuclear extracts, we demonstrate that Cr 3+ reduces activation of glucose‐induced TFs (IRF1, PPARγ), with predicted binding sites in the glucose‐responsive region of the THBS1 promoter. In contrast, Cr 3+ did not have any effect on other glucose‐activated TFs (AhR, CEBP, Egr‐1) under similar experimental conditions. These results were further confirmed in EMSA using relevant THBS1 promoter‐specific oligonucleotide probes; specifically we show that Cr 3+ attenuates THBS1 promoter‐specific IRF1 binding activity in glucose‐treated nuclear extracts. Additionally, immunoprecipitation of cell lysates with anti‐IRF1 followed by immunoblotting with RL2, an antibody that recognizes O‐linked N‐acetylglucosamine (O‐GlcNAc) moieties on proteins, shows a dramatic reduction in O‐GlcNAcylation of IRF1 by Cr 3+ in glucose‐stimulated cells. Together, our data suggest that modulation of IRF1 via O‐GlcNAc modification plays a key role in downregulation of TSP‐1 transcription by Cr 3+ .