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

Dhar K, Rakesh K, Pankajakshan D, Agrawal DK. SOCS3 promotor hypermethylation and STAT3-NF-kappa B interaction down-regulate SOCS3 expression in human coronary artery smooth muscle cells. Am J Physiol Heart Circ Physiol 304: H776-H785, 2013. First published January 18, 2013; doi:10.1152/ajpheart.00570.2012.-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-alpha 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 NF kappa B-p65 interaction. Western blot and qPCR data revealed significant upregulation of SOCS3 (6- to 10-fold) in hCASMC when treated individually with TNF-alpha (100 ng/ml) or IGF-1 (100 ng/ml). However, a significant decrease (5-fold) was observed by the combined treatment with TNF-alpha and IGF-1 compared with individual stimulation. IGF-1 phosphorylated STAT3 and TNF-alpha-activated NF-kappa B in hCASMCs. In the nuclear extract of hCASMCs stimulated with both TNF-alpha and IGF-1, there was an interaction between NF-kappa B-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-alpha 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-alpha-stimulated cells. Downregulation of SOCS3 in the presence of both TNF-alpha and IGF-1 in hCASMCs is due to SOCS3 promoter hypermethylation involving STAT3-NF kappa Bp65 interaction. Because TNF-alpha 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.