title: C-peptide increases Na,K-ATPase expression via PKC- and MAP kinase-dependent activation of transcription factor ZEB in human renal tubular cells.
abstract: 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.
  
  
  RESULTS
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Section of Integrative Physiology, Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden.
authors.forename: Dana
authors.initials: D
authors.lastname: Galuska
authors.forename: Sergej
authors.initials: S
authors.lastname: Pirkmajer
authors.forename: Romain
authors.initials: R
authors.lastname: Barrès
authors.forename: Karin
authors.initials: K
authors.lastname: Ekberg
authors.forename: John
authors.initials: J
authors.lastname: Wahren
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Chibalin
mesh_terms: D002096:C-Peptide / Q000737:chemistry*; D002467:Cell Nucleus / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D015971:Gene Expression Regulation, Enzymologic; D020868:Gene Silencing; D018398:Homeodomain Proteins / Q000096:biosynthesis*; D006801:Humans; D006943:Hyperglycemia / Q000378:metabolism; D007684:Kidney Tubules / Q000378:metabolism*; D020935:MAP Kinase Signaling System; D008954:Models, Biological; D010042:Ouabain / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D020033:Protein Isoforms; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha / Q000378:metabolism; D051745:Protein Kinase C-delta / Q000378:metabolism; D051744:Protein Kinase C-epsilon / Q000378:metabolism; D015398:Signal Transduction; D012964:Sodium / Q000737:chemistry; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D014157:Transcription Factors / Q000096:biosynthesis*; D000071799:Zinc Finger E-box-Binding Homeobox 1
chemical_list: D002096:C-Peptide; D018398:Homeodomain Proteins; D010455:Peptides; D020033:Protein Isoforms; D014157:Transcription Factors; C486307:ZEB1 protein, human; D000071799:Zinc Finger E-box-Binding Homeobox 1; D010042:Ouabain; D012964:Sodium; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D051745:Protein Kinase C-delta; D051744:Protein Kinase C-epsilon; D048049:Extracellular Signal-Regulated MAP Kinases; D000254:Sodium-Potassium-Exchanging ATPase