Phosphorylation of p38 mitogen-activated protein kinase downstream of bax-caspase-3 pathway leads to cell death induced by high D-glucose in human endothelial cells
Phosphorylation of p38 mitogen-activated protein kinase downstream of bax-caspase-3 pathway leads to cell death induced by high D-glucose in human endothelial cells
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DOI：
10.2337/diabetes.50.6.1472
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发表时间：
2001-06-01
期刊：
影响因子：
7.7
通讯作者：
Ogihara, T
中科院分区：
文献类型：
--
作者：
Nakagami, H;Morishita, R;Ogihara, T
由于高糖显著刺激内皮细胞死亡，我们研究了高糖诱导内皮细胞凋亡的分子机制，高糖处理人主动脉内皮细胞与生理浓度相比，甘露醇和L-葡萄糖（25 mmol/l）导致细胞数量显著减少，凋亡细胞显著增加（5 mmol/l）时，高糖处理可显著增加bax蛋白的表达，并伴有bax蛋白从胞浆向富含caspase的重膜组分的移位，而bcl-2蛋白的表达和分布不受高糖的影响。此外，高糖处理后caspase-3蛋白酶活性增加，p38丝裂原活化蛋白激酶（MAPK）被显著磷酸化，并在刺激后显示持续磷酸化。p38 MAPK的特异性抑制剂SB 203580和激酶失活的p38 MAPK的过表达显著减弱了高糖诱导的人主动脉内皮细胞的细胞死亡，而在高D-葡萄糖处理后6小时，SB 203580和激酶失活的p38 MAPK的过表达不减弱高D-葡萄糖诱导的caspase-3活化。重要的是，caspase抑制剂显著减弱高D-葡萄糖诱导的p38 MAPK的持续磷酸化。因此，我们最后集中于MAPK激酶（MEK）激酶1（MEKK 1）以进一步检查p38 MAPK和bax-半胱天冬酶蛋白酶途径之间的相互作用，高D-葡萄糖处理诱导MEKK 1裂解，而半胱天冬酶抑制剂显著减弱了切割。重要的是，激酶失活的MEKK 1也阻断了由高D-葡萄糖诱导的p38 MAPK的磷酸化。在这里，我们证明了高D-葡萄糖通过激活bax-半胱天冬酶蛋白酶途径和通过MEKK 1介导的p38 MAPK磷酸化诱导人内皮细胞凋亡，bax-caspase通路下游p38 MAPK的磷酸化可能在高糖介导的内皮细胞凋亡中起关键作用。
Because high D-glucose significantly stimulates endothelial cell death, we examined the molecular mechanisms of high D-glucose-induced endothelial apoptosis, Treatment of human aortic endothelial cells with high D-glucose (25 mmol/l), but not mannitol and L-glucose, resulted in a significant decrease in cell number and a significant increase in apoptotic cells as compared with a physiological concentration (5 mmol/l), Interestingly, high D-glucose treatment significantly increased bax protein, accompanied by translocation of bax protein from cytosol to mitochondria-enriched heavy membrane fraction, In contrast, the expression and distribution of bcl-2 protein were not altered by high D-glucose, In addition, the activity of caspase-3 proteases was increased after exposure to high glucose, whereas caspase inhibitors prevented endothelial cell death induced by high D-glucose, On the other hand, p38 mitogen-activated protein kinase (MAPK) was markedly phosphorylated and showed sustained phosphorylation after stimulation, A specific inhibitor of p38 MAPK, SB 203580, and the overexpression of kinase-inactive p38 MAPK significantly attenuated cell death induced by high D-glucose in human aortic endothelial cells, whereas at 6 h after high D-glucose treatment, SB 203580 and overexpression of kinase-inactive p38 MAPK did not attenuate caspase-3 activation induced by high D-glucose, Importantly, caspase inhibitors significantly attenuated the sustained phosphorylation of p38 MAPK induced by high D-glucose, Thus, we finally focused the MAPK kinase (MEK) kinase 1 (MEKK1) to further examine the cross-talk between p38 MAPK and the bax-caspase proteases pathway, High D-glucose treatment induced MEKK1 cleavage, whereas caspase inhibitors significantly attenuated the cleavage. Importantly, kinase-inactive MEKK1 also blocked the phosphorylation of p38 MAPK induced by high D-glucose, Here, we demonstrated that high D-glucose induced apoptosis in human endothelial cells through activation of the bax-caspase proteases pathway and through phosphorylation of p38 MAPK mediated by MEKK1, Phosphorylation of p38 MAPK downstream of the bax-caspase pathway may play a pivotal role in endothelial apoptosis mediated by high D-glucose.