IFN-γ and TNF-α regulate human NHE3 gene expression by modulating the Sp family transcription factors in human intestinal epithelial cell line C2BBe1
IFN-γ and TNF-α regulate human NHE3 gene expression by modulating the Sp family transcription factors in human intestinal epithelial cell line C2BBe1
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DOI：
10.1152/ajpcell.00630.2005
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发表时间：
2006-11-01
影响因子：
5.5
通讯作者：
Ramaswamy, Krishnamurthy
中科院分区：
文献类型：
--
作者：
Amin, Md. Ruhul;Malakooti, Jaleh;Ramaswamy, Krishnamurthy
与炎症性肠病相关的腹泻归因于促炎细胞因子（如 IFN-γ 和 TNF-α）的刺激分泌，这些细胞因子已被证明可以下调钠氢交换器 3 (NHE3) 基因的表达。在本研究中，我们研究了 C2BBe1 细胞中 IFN-γ 和 TNF-α 对 NHE3 基因调控的机制。为了响应 IFN-γ (30 ng/ml) 和 TNF-α (20 ng/ml)，含有人 NHE3 启动子的 bp -95 至 +5 区域（包含许多顺式元件，包括四个潜在 Sp1 结合位点）的构建体显示出 60% 的最大抑制。使用小干扰 RNA 敲低 Sp1 和 Sp3 表达可显着抑制 NHE3 启动子活性并抵抗细胞因子效应。分别通过 RT-PCR 和蛋白质印迹分析评估，这些细胞因子对 Sp1 和 Sp3 mRNA 的表达和蛋白质水平没有影响。凝胶迁移率变化分析和染色质免疫沉淀分析表明，用细胞因子处理后，Sp1 和 Sp3 蛋白与 NHE3 启动子的结合显着降低。两种细胞因子对 NHE3 启动子的抑制作用均被广谱激酶抑制剂十字孢菌素和选择性蛋白激酶 A (PKA) 抑制剂 8-溴腺苷-3', 5'-环单硫代磷酸酯 Rp 异构体完全阻断。 PKA α 催化亚基体外磷酸化核蛋白后，Sp1 和 Sp3 蛋白与 NHE3 Sp1 探针的结合亲和力显着降低。我们的数据表明，IFN-γ和TNF-α可能通过PKA介导的Sp1和Sp3转录因子磷酸化来抑制C2BBe1细胞中NHE3启动子活性。
Diarrhea associated with inflammatory bowel disease has been attributed to stimulated secretion of proinflammatory cytokines like IFN-gamma and TNF-alpha, which have been shown to downregulate the expression of the sodium-hydrogen exchanger-3 (NHE3) gene. In this study, we have investigated the mechanism of NHE3 gene regulation by IFN-gamma and TNF-alpha in C2BBe1 cells. In response to both IFN-gamma (30 ng/ml) and TNF-alpha (20 ng/ml), the construct containing the bp -95 to +5 region of the human NHE3 promoter, which harbors a number of cis-elements including four potential Sp1 binding sites, showed a maximum repression of 60%. Knockdown of Sp1 and Sp3 expression using small interfering RNA resulted in a significant inhibition of the NHE3 promoter activity and resistance to cytokines effects. These cytokines showed no effects on the expression of Sp1 and Sp3 mRNA and protein levels as assessed by RT-PCR and Western blot analyses, respectively. After treatment with cytokines, the binding of Sp1 and Sp3 proteins to NHE3 promoter decreased significantly, as seen by gel mobility shift assays and chromatin immunoprecipitation assays. The inhibitory effects of both cytokines on the NHE3 promoter were completely blocked by the broad-range kinase inhibitor staurosporine and the selective protein kinase A (PKA) inhibitor 8-bromoadenosine-3' , 5'-cyclic monophosphorothioate, Rp-isomer. The binding affinity of Sp1 and Sp3 proteins for NHE3 Sp1 probe was significantly decreased after in vitro phosphorylation of nuclear proteins by the alpha-catalytic subunit of PKA. Our data indicate that IFN-gamma and TNF-alpha may repress the NHE3 promoter activity in C2BBe1 cells by PKA-mediated phosphorylation of Sp1 and Sp3 transcription factors.