SOCS3 promotor hypermethylation and STAT3-NF-κB interaction downregulate SOCS3 expression in human coronary artery smooth muscle cells

Kajari Dhar, Kriti Rakesh, Divya Pankajakshan, Devendra K. Agrawal

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24 Citations (Scopus)

Abstract

Suppressor of cytokine signaling-3 (SOCS3) is an intracellular negative regulator of cytokine signaling pathway. We recently found significant reduction in SOCS3 expression in coronary artery smooth muscle cells (CASMCs) of atherosclerotic swine and also in vitro cultured cells. Here, we investigated the underlying mechanisms of SOCS3 downregulation by IGF-1 and TNF-α in human CASMCs(hCASMCs). We propose that hypermethylation of CpG islands in the SOCS3 promoter is responsible for decrease in SOCS3 expression involving STAT3 and NFkB-p65 interaction. Western blot and qPCR data revealed significant upregulation of SOCS3 (6- to 10-fold) in hCASMC when treated individually with TNF-α (100 ng/ml) or IGF-1 (100 ng/ml). However, a significant decrease (5-fold) was observed by the combined treatment with TNF-α and IGF-1 compared with individual stimulation. IGF-1 phosphorylated STAT3 and TNF-α-activated NF-κB in hCASMCs. In the nuclear extract of hCASMCs stimulated with both TNF-α and IGF-1, there was an interaction between NF-κB-p65 and pSTAT3, as determined by co-immunoprecipitation. Knockdown of STAT3 by small interfering RNA abolished SOCS3 expression in response to IGF-1. Methylation-specific PCR confirmed hypermethylation of SOCS3 promoter in hCASMCs stimulated with both TNF-α and IGF-1, and this was positively associated with elevated levels of DNA methyltransferase-I (9- to 10-fold). Knockdown of DNMT1 increased SOCS3 expression in IGF-1+TNF-α- stimulated cells. Downregulation of SOCS3 in the presence of both TNF-α and IGF-1 in hCASMCs is due to SOCS3 promoter hypermethylation involving STAT3-NFkBp65 interaction. Because TNF-α and IGF-1 are released due to mechanical injury during coronary intervention, hypermethylation of SOCS3 gene could be an underlying mechanism of intimal hyperplasia and restenosis.

Original languageEnglish
JournalAmerican Journal of Physiology - Heart and Circulatory Physiology
Volume304
Issue number6
DOIs
StatePublished - 2013

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Smooth Muscle Myocytes
Coronary Vessels
Down-Regulation
Insulin-Like Growth Factor I
Cytokines
Tunica Intima
CpG Islands
Methyltransferases
Immunoprecipitation
Methylation
Small Interfering RNA
Hyperplasia
Cultured Cells
Up-Regulation
Swine
Western Blotting
Polymerase Chain Reaction

All Science Journal Classification (ASJC) codes

  • Physiology
  • Physiology (medical)
  • Cardiology and Cardiovascular Medicine

Cite this

@article{162d0f58c06048f8a1e6b71cf6c04b44,
title = "SOCS3 promotor hypermethylation and STAT3-NF-κB interaction downregulate SOCS3 expression in human coronary artery smooth muscle cells",
abstract = "Suppressor of cytokine signaling-3 (SOCS3) is an intracellular negative regulator of cytokine signaling pathway. We recently found significant reduction in SOCS3 expression in coronary artery smooth muscle cells (CASMCs) of atherosclerotic swine and also in vitro cultured cells. Here, we investigated the underlying mechanisms of SOCS3 downregulation by IGF-1 and TNF-α in human CASMCs(hCASMCs). We propose that hypermethylation of CpG islands in the SOCS3 promoter is responsible for decrease in SOCS3 expression involving STAT3 and NFkB-p65 interaction. Western blot and qPCR data revealed significant upregulation of SOCS3 (6- to 10-fold) in hCASMC when treated individually with TNF-α (100 ng/ml) or IGF-1 (100 ng/ml). However, a significant decrease (5-fold) was observed by the combined treatment with TNF-α and IGF-1 compared with individual stimulation. IGF-1 phosphorylated STAT3 and TNF-α-activated NF-κB in hCASMCs. In the nuclear extract of hCASMCs stimulated with both TNF-α and IGF-1, there was an interaction between NF-κB-p65 and pSTAT3, as determined by co-immunoprecipitation. Knockdown of STAT3 by small interfering RNA abolished SOCS3 expression in response to IGF-1. Methylation-specific PCR confirmed hypermethylation of SOCS3 promoter in hCASMCs stimulated with both TNF-α and IGF-1, and this was positively associated with elevated levels of DNA methyltransferase-I (9- to 10-fold). Knockdown of DNMT1 increased SOCS3 expression in IGF-1+TNF-α- stimulated cells. Downregulation of SOCS3 in the presence of both TNF-α and IGF-1 in hCASMCs is due to SOCS3 promoter hypermethylation involving STAT3-NFkBp65 interaction. Because TNF-α and IGF-1 are released due to mechanical injury during coronary intervention, hypermethylation of SOCS3 gene could be an underlying mechanism of intimal hyperplasia and restenosis.",
author = "Kajari Dhar and Kriti Rakesh and Divya Pankajakshan and Agrawal, {Devendra K.}",
year = "2013",
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language = "English",
volume = "304",
journal = "American Journal of Physiology - Heart and Circulatory Physiology",
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TY - JOUR

T1 - SOCS3 promotor hypermethylation and STAT3-NF-κB interaction downregulate SOCS3 expression in human coronary artery smooth muscle cells

AU - Dhar, Kajari

AU - Rakesh, Kriti

AU - Pankajakshan, Divya

AU - Agrawal, Devendra K.

PY - 2013

Y1 - 2013

N2 - Suppressor of cytokine signaling-3 (SOCS3) is an intracellular negative regulator of cytokine signaling pathway. We recently found significant reduction in SOCS3 expression in coronary artery smooth muscle cells (CASMCs) of atherosclerotic swine and also in vitro cultured cells. Here, we investigated the underlying mechanisms of SOCS3 downregulation by IGF-1 and TNF-α in human CASMCs(hCASMCs). We propose that hypermethylation of CpG islands in the SOCS3 promoter is responsible for decrease in SOCS3 expression involving STAT3 and NFkB-p65 interaction. Western blot and qPCR data revealed significant upregulation of SOCS3 (6- to 10-fold) in hCASMC when treated individually with TNF-α (100 ng/ml) or IGF-1 (100 ng/ml). However, a significant decrease (5-fold) was observed by the combined treatment with TNF-α and IGF-1 compared with individual stimulation. IGF-1 phosphorylated STAT3 and TNF-α-activated NF-κB in hCASMCs. In the nuclear extract of hCASMCs stimulated with both TNF-α and IGF-1, there was an interaction between NF-κB-p65 and pSTAT3, as determined by co-immunoprecipitation. Knockdown of STAT3 by small interfering RNA abolished SOCS3 expression in response to IGF-1. Methylation-specific PCR confirmed hypermethylation of SOCS3 promoter in hCASMCs stimulated with both TNF-α and IGF-1, and this was positively associated with elevated levels of DNA methyltransferase-I (9- to 10-fold). Knockdown of DNMT1 increased SOCS3 expression in IGF-1+TNF-α- stimulated cells. Downregulation of SOCS3 in the presence of both TNF-α and IGF-1 in hCASMCs is due to SOCS3 promoter hypermethylation involving STAT3-NFkBp65 interaction. Because TNF-α and IGF-1 are released due to mechanical injury during coronary intervention, hypermethylation of SOCS3 gene could be an underlying mechanism of intimal hyperplasia and restenosis.

AB - Suppressor of cytokine signaling-3 (SOCS3) is an intracellular negative regulator of cytokine signaling pathway. We recently found significant reduction in SOCS3 expression in coronary artery smooth muscle cells (CASMCs) of atherosclerotic swine and also in vitro cultured cells. Here, we investigated the underlying mechanisms of SOCS3 downregulation by IGF-1 and TNF-α in human CASMCs(hCASMCs). We propose that hypermethylation of CpG islands in the SOCS3 promoter is responsible for decrease in SOCS3 expression involving STAT3 and NFkB-p65 interaction. Western blot and qPCR data revealed significant upregulation of SOCS3 (6- to 10-fold) in hCASMC when treated individually with TNF-α (100 ng/ml) or IGF-1 (100 ng/ml). However, a significant decrease (5-fold) was observed by the combined treatment with TNF-α and IGF-1 compared with individual stimulation. IGF-1 phosphorylated STAT3 and TNF-α-activated NF-κB in hCASMCs. In the nuclear extract of hCASMCs stimulated with both TNF-α and IGF-1, there was an interaction between NF-κB-p65 and pSTAT3, as determined by co-immunoprecipitation. Knockdown of STAT3 by small interfering RNA abolished SOCS3 expression in response to IGF-1. Methylation-specific PCR confirmed hypermethylation of SOCS3 promoter in hCASMCs stimulated with both TNF-α and IGF-1, and this was positively associated with elevated levels of DNA methyltransferase-I (9- to 10-fold). Knockdown of DNMT1 increased SOCS3 expression in IGF-1+TNF-α- stimulated cells. Downregulation of SOCS3 in the presence of both TNF-α and IGF-1 in hCASMCs is due to SOCS3 promoter hypermethylation involving STAT3-NFkBp65 interaction. Because TNF-α and IGF-1 are released due to mechanical injury during coronary intervention, hypermethylation of SOCS3 gene could be an underlying mechanism of intimal hyperplasia and restenosis.

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