title: Proinsulin C-peptide antagonizes the profibrotic effects of TGF-beta1 via up-regulation of retinoic acid and HGF-related signaling pathways.
abstract: Novel signaling roles for C-peptide have recently been discovered with evidence that it can ameliorate complications of type 1 diabetes. Here we sought to identify new pathways regulated by C-peptide of relevance to the pathophysiology of diabetic nephropathy. Microarray analysis was performed to identify genes regulated by either C-peptide and/or TGF-beta1 in a human proximal tubular cell line, HK-2. Expression of retinoic acid receptor beta (RARbeta), hepatocyte growth factor (HGF), cellular retinoic acid-binding protein II (CRABPII), vimentin, E-cadherin, Snail, and beta-catenin was assessed by immunoblotting. The cellular localization of vimentin and beta-catenin was determined by immunocytochemistry. Changes in cell morphology were assessed by phase contrast microscopy. Gene expression profiling demonstrated differential expression of 953 and 1458 genes after C-peptide exposure for 18 h or 48 h, respectively. From these, members of the antifibrotic retinoic acid (RA)- and HGF-signaling pathways were selected. Immunoblotting demonstrated that C-peptide increased RARbeta, CRABPII, and HGF. We confirmed a role for RA in reversal of TGF-beta1-induced changes associated with epithelial-mesenchymal transition, including expression changes in Snail, E-cadherin, vimetin, and redistribution of beta-catenin. Importantly, these TGF-beta1-induced changes were inhibited by C-peptide. Further, effects of TGF-beta1 on Snail and E-cadherin expression were blocked by HGF, and inhibitory effects of C-peptide were removed by blockade of HGF activity. This study identifies a novel role for HGF as an effector of C-peptide, possibly via an RA-signaling pathway, highlighting C-peptide as a potential therapy for diabetic nephropathy.
authors.affiliation: Department of Infection, Immunity and Inflammation, University of Leicester, College of Medicine Biological Sciences and Psychology, Leicester, United Kingdom.
authors.forename: Claire E
authors.initials: CE
authors.lastname: Hills
authors.forename: Gary B
authors.initials: GB
authors.lastname: Willars
authors.forename: Nigel J
authors.initials: NJ
authors.lastname: Brunskill
mesh_terms: D002096:C-Peptide / Q000494:pharmacology*; D002460:Cell Line; D017228:Hepatocyte Growth Factor / Q000378:metabolism*; D006801:Humans; D015151:Immunoblotting; D007150:Immunohistochemistry; D008858:Microscopy, Phase-Contrast; D020411:Oligonucleotide Array Sequence Analysis; D018168:Receptors, Retinoic Acid / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects*; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*; D014212:Tretinoin / Q000378:metabolism*; D014746:Vimentin / Q000378:metabolism; D051176:beta Catenin / Q000378:metabolism
chemical_list: D002096:C-Peptide; D018168:Receptors, Retinoic Acid; D053773:Transforming Growth Factor beta1; D014746:Vimentin; D051176:beta Catenin; C082928:retinoic acid binding protein II, cellular; D014212:Tretinoin; D017228:Hepatocyte Growth Factor