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

TY - JOUR

T1 - C-peptide increases Na,K-ATPase expression via PKC- and MAP kinase-dependent activation of transcription factor ZEB in human renal tubular cells

AU - Galuska, Dana

AU - Pirkmajer, Sergej

AU - Barres, Romain

AU - Ekberg, Karin

AU - Wahren, John

AU - Chibalin, Alexander V

PY - 2011/12/5

Y1 - 2011/12/5

N2 - 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 (86Rb+) 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 α1-subunit protein expression, accompanied with increase in 86Rb+ uptake, both in normal- and hyperglycemic conditions. Na,K-ATPase α1-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 α1-subunit mRNA expression. Effects of 1 nM C-peptide on Na,K-ATPase α1-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.

AB - 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 (86Rb+) 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 α1-subunit protein expression, accompanied with increase in 86Rb+ uptake, both in normal- and hyperglycemic conditions. Na,K-ATPase α1-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 α1-subunit mRNA expression. Effects of 1 nM C-peptide on Na,K-ATPase α1-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.

KW - C-Peptide

KW - Cell Nucleus

KW - Extracellular Signal-Regulated MAP Kinases

KW - Gene Expression Regulation, Enzymologic

KW - Gene Silencing

KW - Homeodomain Proteins

KW - Humans

KW - Hyperglycemia

KW - Kidney Tubules

KW - MAP Kinase Signaling System

KW - Models, Biological

KW - Ouabain

KW - Peptides

KW - Phosphorylation

KW - Protein Isoforms

KW - Protein Kinase C

KW - Protein Kinase C-alpha

KW - Protein Kinase C-delta

KW - Protein Kinase C-epsilon

KW - Signal Transduction

KW - Sodium

KW - Sodium-Potassium-Exchanging ATPase

KW - Transcription Factors

U2 - 10.1371/journal.pone.0028294

DO - 10.1371/journal.pone.0028294

M3 - Journal article

C2 - 22162761

SN - 1932-6203

VL - 6

SP - e28294

JO - P L o S One

JF - P L o S One

IS - 12

ER -