Molecular mechanisms of tumor necrosis factor α gene expression in monocytic cells via hyperglycemia-induced oxidant stress-dependent and -independent pathways
Molecular mechanisms of tumor necrosis factor α gene expression in monocytic cells via hyperglycemia-induced oxidant stress-dependent and -independent pathways
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DOI：
10.1074/jbc.275.23.17728
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发表时间：
2000-06-09
影响因子：
4.8
通讯作者：
Natarajan, R
中科院分区：
文献类型：
--
作者：
Guha, M;Bai, W;Natarajan, R
据报道，在体内糖尿病状态下，氧化应激通过活性氧（ROS）的产生而增加，这种应激势必对循环单核细胞的激活起关键作用，导致糖尿病患者动脉粥样硬化的加速。然而，高糖活化单核细胞的确切分子机制目前尚不清楚。在这里，我们证明慢性高糖（CHG）引起炎症细胞因子肿瘤坏死因子α (TNF α)释放的急剧增加，至少部分是通过ROS通过激活转录因子核因子κ B （nf - κ B）和激活蛋白-1 （AP-1）介导的TNF α mRNA转录的增强，CHG在条件培养基中的TNF α积累增加了10倍，mRNA水平增加了11.5倍。以下观察结果支持NF-kappa B和AP-1通过CHG介导TNF - α转录增强：1)在荧光素酶报告试验中，含有NF-kappa B和AP-1位点的近端TNF - α启动子的295个碱基对片段再现了CHG对TNF - α转录的影响，2)对NF-kappa B和AP-1位点的突变分析消除了90%的荧光素酶活性，3)使用结合位点的凝胶转移分析显示，在CHG核提取物中NF-kappa B和AP-1被激活。4) Western blot分析显示，CHG处理的单核细胞核中p65和p50水平升高，胞浆中I κ B α水平降低。以下证据支持ROS在CHG通路中起关键作用：1)与PMA或TNF α相似的超氧化物水平升高，2)应激反应性丝裂原活化蛋白激酶p38和JNK-1磷酸化升高，3)CHG对TNF α产生、295TNFluc报告蛋白活性、NF κ B激活以及抗氧化剂和p38丝裂原活化蛋白激酶抑制剂抑制I κ B α的作用的抵消。该研究表明，ROS在从葡萄糖升高到单核细胞活化的调节途径中起关键作用。
Increased oxidative stress has been reported in vivo in the diabetic state via the production of reactive oxygen species (ROS), Such stress is bound to play a key role on activation of circulating monocytes, leading to the accelerated atherosclerosis observed in diabetics. However the exact molecular mechanisms of monocyte activation by high glucose is currently unclear. Here, we demonstrate that chronic high glucose (CHG) causes a dramatic increase in the release of the inflammatory cytokine tumor necrosis factor alpha (TNF alpha), at least in part through enhanced TNF alpha mRNA transcription, mediated by ROS via activation of transcription factors nuclear factor kappa B (NF-kappa B) and activating protein-1 (AP-1), TNF alpha accumulation in the conditioned media was increased 10-fold and mRNA levels were increased 11.5-fold by CHG, The following observations supported that both NF-kappa B and AP-1 mediated enhanced TNF alpha transcription by CHG: 1) A 295-base pair fragment of the proximal TNF alpha promoter containing NF-kappa B and AP-1 sites reproduced the effects of CHG on TNF alpha transcription in a luciferase reporter assay, 2) mutational analyses of both NF-kappa B and the AP-1 sites abrogated 90% of the luciferase activity, 3) gel-shift analysis using the binding sites showed activation of NF-kappa B and AP-1 in CHG nuclear extracts, and 4) Western blot analyses demonstrated elevated nuclear levels of p65 and p50 and decreased cytosolic levels of I kappa B alpha in CHG-treated monocytes, That ROS acted as a key intermediate in the CHG pathway was supported by the following evidence: 1) increased superoxide levels similar to those observed with PMA or TNF alpha, 2) increased phosphorylation of stress-responsive mitogen-activated protein kinases p38 and JNK-1, 3) counteraction of the effects of CHG on TNF alpha production, the 295TNFluc reporter activity, activation of NF kappa B, and repression of I kappa B alpha by antioxidants and p38 mitogen-activated protein kinase inhibitors. The study suggests that ROS function as key components in the regulatory pathway progressing from elevated glucose to monocyte activation.