High glucose alters the response of mesangial cell protein kinase C isoforms to endothelin-1
High glucose alters the response of mesangial cell protein kinase C isoforms to endothelin-1
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DOI：
10.1046/j.1523-1755.1999.00284.x
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发表时间：
1999-02-01
影响因子：
19.6
通讯作者：
Whiteside, C
中科院分区：
文献类型：
--
作者：
Glogowski, EA;Tsiani, E;Whiteside, C
背景高糖引起肾小球系膜生长和基质合成增加，从而导致糖尿病性肾小球病。我们的目的是确定高糖是否改变内皮素-1（ET-1）或血小板源性生长因子-B对系膜细胞二酰基甘油敏感蛋白激酶C（PKC）亚型的激活以及随后对丝裂原活化蛋白激酶（MAPK; p42，p44）的刺激。原代培养的大鼠肾小球系膜细胞在葡萄糖5.6 mM（NG）或30 mM（HG）中生长停滞48小时。使用同种型特异性单克隆抗体通过免疫印迹法测量PKC-α、PKC-δ和PKC-β从胞质至膜和胞质至颗粒（细胞骨架、细胞核）细胞组分的易位。还通过共聚焦免疫荧光显微镜观察PKC亚型。通过磷酸化MAPK抗体的免疫印迹和磷酸化MAPK免疫沉淀后Elk-1融合蛋白磷酸化的检测来测量MAPK活化。在NG中，ET-1刺激PKC-δ和PKC-β的胞质-膜易位，但不刺激PKC-α。在HG中，ET-1刺激的PKC-δ和PKC-β的模式改变为细胞溶胶到颗粒的分布，这通过共聚焦免疫荧光成像证实。血小板源性生长因子-B在NG或HG中均不引起PKC-α、PKC-δ或PKC-β易位。高糖组MAPK基础活性和ET-1刺激活性均显著升高。在高糖状态下，佛波酯下调PKC亚型的表达，可抑制ET-1对MAPK的激活作用。在HG中，ET-1增强的系膜细胞MAPK活化是PKC依赖性的，并且与PKC-δ和PKC-β的改变的易位有关。高糖状态下肾小球系膜细胞对血管活性肽的反应性增强可能是糖尿病肾病发生的重要机制。
Background. High glucose causes glomerular mesangial growth and increased matrix synthesis contributing to diabetic glomerulopathy. Our purpose was to determine if high glucose alters endothelin-1 (ET-1) or platelet-derived growth factor-B activation of mesangial cell diacylglycerol-sensitive protein kinase C (PKC) isoforms and subsequent stimulation of mitogen-activated protein kinase (MAPK; p42, p44).Methods. Rat mesangial cells in primary culture were growth arrested for 48 hours in glucose 5.6 mM (NG) or 30 mM (HG). PKC-alpha, PKC-delta, and PKC-epsilon translocation from the cytosol-to-membrane and cytosol-to-particulate (cytoskeleton, nucleus) cellular fractions were measured by immunoblot using isoform-specific monoclonal antibodies. PKC isoforms were visualized also by confocal immunofluorescence microscopy. MAPK activation was measured by immunoblot using phospho-MAPK antibody and by detection of Elk-1 fusion protein phosphorylation following phospho-MAPK immunoprecipitation.Results. In NG, ET-1 stimulated cytosol-to-membrane translocation of PKC-delta and PKC-epsilon but not PKC-alpha. In HG, the pattern of ET-1-stimulated PKC-delta and PKC-epsilon changed to a cytosol-to-particulate distribution, which was confirmed by confocal immunofluorescence imaging. Platelet-derived growth factor-B did not cause translocation of PKC-alpha, PKC-delta, or PKC-epsilon in either NG or HG. In HG, both basal and ET-1-stimulated MAPK activities were increased significantly. In HG, down-regulation of PKC isoforms with phorbol ester prevented the increased stimulation of MAPK by ET-1.Conclusion. In HG, the enhanced activation of mesangial cell MAPK by ET-1 is PKC dependent and associated with altered translocation of PKC-delta and PKC-epsilon. Enhanced mesangial cell signaling responsiveness to vasoactive peptides in HG may constitute an important mechanism contributing to diabetic nephropathy.