C-peptide stimulates ERK1/2 and JNK MAP kinases via activation of protein kinase C in human renal tubular cells
C-peptide stimulates ERK1/2 and JNK MAP kinases via activation of protein kinase C in human renal tubular cells
复制标题
DOI：
10.1007/s00125-004-1602-5
复制
发表时间：
2005-01-01
期刊：
影响因子：
8.2
通讯作者：
Chibalin, AV
中科院分区：
文献类型：
--
作者：
Zhong, Z;Davidescu, A;Chibalin, AV
目的/假设：越来越多的证据表明，1型糖尿病患者服用C肽可改善神经和肾功能障碍，但其分子机制尚不完全清楚。C-肽与G-蛋白偶联的膜结合部位有特异性结合，导致钙离子内流，激活丝裂原激活的蛋白激酶信号通路，刺激Na+，K+-ATPase和内皮型一氧化氮合酶。这项研究检测了C-肽激活的人肾小管上皮细胞内信号通路。方法：体外培养人肾小管上皮细胞，取自择期肾切除患者的肾组织外皮质。用磷酸化特异性抗体检测细胞外信号调节激酶1/2(ERK1/2)、c-jun氨基末端激酶(JNK)和Akt/蛋白激酶B(PKB)的活性。蛋白激酶C(PKC)和RhoA的活性是通过使用异构体特异性抗体测量它们到细胞膜部分的移位来确定的。结果：人C肽以浓度和时间依赖的方式促进肾小管上皮细胞ERK1/2和Akt/PKB的磷酸化。C-肽的C-末端五肽与全长C-肽等效，而杂乱的C-肽不起作用。C-肽刺激也会导致JNK的磷酸化，但不会导致p38丝裂原活化蛋白激酶的磷酸化。MEK1/2抑制剂PD98059阻断C肽对ERK1/2磷酸化的影响。C-肽导致肾小管上皮细胞内PKC异构体Delta和epsilon的特异性转位。百日咳毒素可完全消除C-肽的刺激作用。异构体特异性PKC-Delta抑制剂rotlerin和广谱PKC抑制剂GF109203X均可消除C-肽对ERK1/2磷酸化的影响。C-肽的刺激也会导致小的GTP酶RhoA从胞浆移位到细胞膜。抑制磷脂酶C可阻断C肽对ERK1/2、JNK和PKC-Delta磷酸化的刺激作用。结论/解释：人肾小管上皮细胞的C-肽信号转导涉及磷脂酶C、PKC-Delta、PKC-epsilon和RhoA的激活，然后是ERK1/2和JNK的磷酸化，Akt的平行激活。
Aims/hypothesis: Accumulating evidence indicates that replacement of C-peptide in type 1 diabetes ameliorates nerve and kidney dysfunction, but the molecular mechanisms involved are incompletely understood. C-peptide shows specific binding to a G-protein-coupled membrane binding site, resulting in Ca2+ influx, activation of mitogen-activated protein kinase signalling pathways, and stimulation of Na+, K+-ATPase and endothelial nitric oxide synthase. This study examines the intracellular signalling pathways activated by C-peptide in human renal tubular cells. Methods: Human renal tubular cells were cultured from the outer cortex of renal tissue obtained from patients undergoing elective nephrectomy. Extracellular-signal- regulated kinase 1/2 (ERK1/2), c-Jun N-terminal kinase (JNK) and Akt/protein kinase B (PKB) activation was determined using phospho-specific antibodies. Protein kinase C (PKC) and RhoA activation was determined by measuring their translocation to the cell membrane fraction using isoform-specific antibodies. Results: Human C-peptide increases phosphorylation of ERK1/2 and Akt/PKB in a concentration- and time-dependent manner in renal tubular cells. The C-terminal pentapeptide of C-peptide is equipotent with the full-length C-peptide, whereas scrambled C-peptide has no effect. C-peptide stimulation also results in phosphorylation of JNK, but not of p38 mitogen-activated protein kinase. MEK1/2 inhibitor PD98059 blocks the C-peptide effect on ERK1/2 phosphorylation. C-peptide causes specific translocation of PKC isoforms delta and epsilon to the membrane fraction in tubular cells. All stimulatory effects of C-peptide were abolished by pertussis toxin. The isoform-specific PKC-delta inhibitor rottlerin and the broad-spectrum PKC inhibitor GF109203X both abolish the C-peptide effect on ERK1/2 phosphorylation. C-peptide stimulation also causes translocation of the small GTPase RhoA from the cytosol to the cell membrane. Inhibition of phospholipase C abolished the stimulatory effect of C-peptide on phosphorylation of ERK1/2, JNK and PKC-delta. Conclusions/interpretation: C-peptide signal transduction in human renal tubular cells involves the activation of phospholipase C and PKC-delta and PKC-epsilon, as well as RhoA, followed by phosphorylation of ERK1/2 and JNK, and a parallel activation of Akt.