C-Peptide Increases Na,K-ATPase Expression via PKC- and MAP Kinase-Dependent Activation of Transcription Factor ZEB in Human Renal Tubular Cells
C-Peptide Increases Na,K-ATPase Expression via PKC- and MAP Kinase-Dependent Activation of Transcription Factor ZEB in Human Renal Tubular Cells
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DOI：
10.1371/journal.pone.0028294
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发表时间：
2011-12-05
期刊：
影响因子：
3.7
通讯作者：
Chibalin, Alexander V.
中科院分区：
文献类型：
--
作者：
Galuska, Dana;Pirkmajer, Sergej;Chibalin, Alexander V.
背景：1型糖尿病患者应用胰岛素原C肽替代治疗可改善神经和肾功能障碍，而这些功能障碍与Na，K-ATPase活性降低有关。我们研究了在对照组和高血糖状态下，长期暴露于C-肽刺激原代人肾小管细胞(HRTC)Na，K-ATPase表达和活性的分子机制。方法/主要研究结果：HRTC取自择期肾切除患者的外皮质。测定哇巴因敏感型Rb-86(+)摄取和Na，K-ATPase活性。用Western blotting检测完整细胞或分离的基底膜(BLM)中Na，K-ATPase的丰度。电迁移率改变分析(EMSA)检测DNA结合活性。在正常和高血糖条件下，用1 NM而不是10 NM的人C肽培养HRTCs 5天，可导致Na，K-ATPaseα(1)亚单位蛋白表达增加，并伴有Rb-86(+)摄取增加。高糖培养的BLM细胞Na，K-ATPaseα(1)亚基表达和Na，K-ATPase活性降低。无论葡萄糖浓度如何，暴露于1 nM而不是10 nM的C-肽可增加PKC epsilon的磷酸化、磷酸化和核ERK1/2的丰度。1 nM的C-肽可增加转录因子ZeB(AREB6)的DNA结合活性，并伴随Na，K-ATPase A1亚单位mRNA的表达。PKC或MEK1/2抑制剂和Zeb siRNA沉默可阻断1 nM C肽对HRTC Na，K-ATPaseα(1)-亚基表达和/或Zeb DNA结合活性的影响。结论：尽管高血糖激活了ERK1/2和PKC，但C肽对Na，K-ATPase表达和活性的调节涉及不同的PKC和ERK1/2池。最有可能的是C-肽通过激活ZeB来刺激钠泵的表达，ZeB是一种转录因子，以前还没有涉及到C-肽介导的信号转导。重要的是，只有生理浓度的C-肽才能引起这种效应。
Background: Replacement of proinsulin C-peptide in type 1 diabetes ameliorates nerve and kidney dysfunction, conditions which are associated with a decrease in Na,K-ATPase activity. We determined the molecular mechanism by which long term exposure to C-peptide stimulates Na,K-ATPase expression and activity in primary human renal tubular cells (HRTC) in control and hyperglycemic conditions.Methodology/Principal Findings: HRTC were cultured from the outer cortex obtained from patients undergoing elective nephrectomy. Ouabain-sensitive rubidium (Rb-86(+)) uptake and Na,K-ATPase activity were determined. Abundance of Na,K-ATPase was determined by Western blotting in intact cells or isolated basolateral membranes (BLM). DNA binding activity was determined by electrical mobility shift assay (EMSA). Culturing of HRTCs for 5 days with 1 nM, but not 10 nM of human C-peptide leads to increase in Na,K-ATPase alpha(1)-subunit protein expression, accompanied with increase in Rb-86(+) uptake, both in normal- and hyperglycemic conditions. Na,K-ATPase alpha(1)-subunit expression and Na,K-ATPase activity were reduced in BLM isolated from cells cultured in presence of high glucose. Exposure to 1 nM, but not 10 nM of C-peptide increased PKC epsilon phosphorylation as well as phosphorylation and abundance of nuclear ERK1/2 regardless of glucose concentration. Exposure to 1 nM of C-peptide increased DNA binding activity of transcription factor ZEB (AREB6), concomitant with Na,K-ATPase a1-subunit mRNA expression. Effects of 1 nM C-peptide on Na,K-ATPase alpha(1)-subunit expression and/or ZEB DNA binding activity in HRTC were abolished by incubation with PKC or MEK1/2 inhibitors and ZEB siRNA silencing.Conclusions/Significance: Despite activation of ERK1/2 and PKC by hyperglycemia, a distinct pool of PKCs and ERK1/2 is involved in regulation of Na,K-ATPase expression and activity by C-peptide. Most likely C-peptide stimulates sodium pump expression via activation of ZEB, a transcription factor that has not been previously implicated in C-peptide-mediated signaling. Importantly, only physiological concentrations of C-peptide elicit this effect.