SOCS3 promotor hypermethylation and STAT3-NF-κB interaction downregulate SOCS3 expression in human coronary artery smooth muscle cells
SOCS3 promotor hypermethylation and STAT3-NF-κB interaction downregulate SOCS3 expression in human coronary artery smooth muscle cells
复制标题
DOI：
10.1152/ajpheart.00570.2012
复制
发表时间：
2013-03-01
影响因子：
4.8
通讯作者：
Agrawal, Devendra K.
中科院分区：
文献类型：
--
作者：
Dhar, Kajari;Rakesh, Kriti;Agrawal, Devendra K.
Dhar K，Rakesh K，Pankajakshan D，Agrawal DK。SOCS3启动子高甲基化和STAT3-NF-kappa B相互作用下调人冠状动脉平滑肌细胞SOCS3的表达。Am J Physiol心脏圈Physiol 304：H776-H785,2013。2013年1月18日首次出版；doi：10.1152/ajpheart.00570.2012。-细胞因子信号转导抑制因子-3(SOCS3)是细胞因子信号转导途径的细胞内负调控因子。我们最近发现SOCS3在动脉粥样硬化猪的冠状动脉平滑肌细胞(CASMCs)和体外培养的细胞中的表达显著降低。在这里，我们研究了胰岛素样生长因子-1和肿瘤坏死因子-α下调人CASMCs(HCASMCs)SOCS3的潜在机制。我们认为SOCS3启动子中CpG岛的高甲基化是导致STAT3和NF-kappa B-p65相互作用的SOCS3表达减少的原因。免疫印迹和定量聚合酶链式反应结果显示，单独应用肿瘤坏死因子-α(100 ng/ml)或胰岛素样生长因子-1(100 ng/ml)时，hCASMC中SOCS3的表达显著上调(6~10倍)。然而，与单独刺激相比，联合应用肿瘤坏死因子-α和胰岛素样生长因子-1的作用显著降低(5倍)。胰岛素样生长因子-1磷酸化STAT3和肿瘤坏死因子-α激活hCASMCs的核因子-kappaB。免疫共沉淀法显示，在经肿瘤坏死因子-α和胰岛素样生长因子-1共同刺激的hCASMCs核提取液中，核转录因子-kappa B-p65与pSTAT3之间存在相互作用。通过小干扰RNA抑制STAT3基因的表达，可抑制胰岛素样生长因子-1对SOCS3的表达。甲基化特异性聚合酶链式反应证实，在肿瘤坏死因子-α和胰岛素样生长因子-1刺激的hCASMCs中，SOCS3启动子高甲基化，这与DNA甲基转移酶-I水平升高(9-10倍)呈正相关。DNMT1基因敲除可增加IGF-1+TNF-α刺激的细胞SOCS3的表达。在同时存在肿瘤坏死因子-α和胰岛素样生长因子-1的情况下，hCASMCs中SOCS3的下调是由于SOCS3启动子的高甲基化涉及STAT3-NF-kappa Bp65的相互作用。由于在冠状动脉介入治疗过程中由于机械损伤而释放了肿瘤坏死因子-α和胰岛素样生长因子-1，因此SOCS3基因高甲基化可能是动脉内膜增生和再狭窄的潜在机制。
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.