C-peptide increases Na,K-ATPase expression via PKC- and MAP kinase-dependent activation of transcription factor ZEB in human renal tubular cells

• Published in: PloS one Vol. 6; no. 12; p. e28294
• Main Authors: Galuska, Dana, Pirkmajer, Sergej, Barrès, Romain, Ekberg, Karin, Wahren, John, Chibalin, Alexander V
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 05.12.2011; Public Library of Science (PLoS)
• Subjects: Abundance; Adenosine triphosphatase; ATPases; Binding; Binding sites; Biology; C-Peptide - chemistry; Cell Nucleus - metabolism; Coronary vessels; Deoxyribonucleic acid; Diabetes; Diabetes mellitus; DNA; DNA binding; Electrophoretic mobility; Exposure; Extracellular signal-regulated kinase; Extracellular Signal-Regulated MAP Kinases - metabolism; Gene expression; Gene Expression Regulation, Enzymologic; Gene Silencing; Genetic research; Glucose; Homeodomain Proteins - biosynthesis; Humans; Hyperglycemia; Hyperglycemia - metabolism; Incubation; Insulin; Kidney diseases; Kidney transplantation; Kidney Tubules - metabolism; Kinases; MAP kinase; MAP Kinase Signaling System; Medicin och hälsovetenskap; Medicine; Membranes; Models, Biological; Musculoskeletal system; Na+/K+-exchanging ATPase; Nephrectomy; Ouabain; Ouabain - pharmacology; Peptides; Peptides - chemistry; Phosphorylation; Physiological aspects; Physiological effects; Physiology; Protein expression; Protein Isoforms; Protein kinase C; Protein Kinase C - metabolism; Protein Kinase C-alpha - metabolism; Protein Kinase C-delta - metabolism; Protein Kinase C-epsilon - metabolism; Proteins; RNA; Rodents; Rubidium; Signal Transduction; Signaling; siRNA; Sodium; Sodium - chemistry; Sodium-Potassium-Exchanging ATPase - metabolism; Surgery; Transcription activation; Transcription Factors - biosynthesis; Type 1 diabetes; Western blotting; Zinc Finger E-box-Binding Homeobox 1
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0028294

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. HRTC were cultured from the outer cortex obtained from patients undergoing elective nephrectomy. Ouabain-sensitive rubidium ((86)Rb(+)) 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 α(1)-subunit protein expression, accompanied with increase in (86)Rb(+) uptake, both in normal- and hyperglycemic conditions. Na,K-ATPase α(1)-subunit expression and Na,K-ATPase activity were reduced in BLM isolated from cells cultured in presence of high glucose. Exposure to1 nM, but not 10 nM of C-peptide increased PKCε 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 α(1)-subunit mRNA expression. Effects of 1 nM C-peptide on Na,K-ATPase α(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. 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.