Proinsulin C-peptide : activation of intracellular signalling pathways and modulation of transcription factors in opossum kidney proximal tubular cells

In recent years an increasingly substantial body of data, supports a role for C-peptide in several biological activities. However, the precise molecular mechanisms of C-peptide action are not fully understood. The aim of this thesis was to study the intracellular signalling pathways and the transcription factors that C-peptide activates in proximal tubular cells using opossum kidney cells (OK) as a model. Using specific inhibitors and phospho-specific antibodies, intracellular signalling pathways activated by C-peptide were examined by kinase assay and Western blotting. The results show that C-peptide is able to activate extracellular signal regulated kinase (ERK), phosphatidylinositol 3-kinase (PI 3-kinase) and PKC-a. ERK activation was attenuated by PKC inhibitor pre-treatment and activation of ERK and PKC-a were abolished in the absence of extracellular Ca2+. Elevations of [Ca2+]i were examined using confocal microscopy. C-peptide induced transient increase in [Ca2+]i but the response of cells was variable. Thymidine incorporation assay was used to assess proliferation. C-peptide was found to be a functional mitogen in this cell type stimulating significantly increased cell proliferation. Proliferator-activated receptor (PPAR) transcriptional activity was measured using a luciferase reporter assay in OK cells. C-peptide induced concentration-dependent stimulation of PPARy activity. C-peptide also substantially augmented ciglitazone-stimulated PPARy activity. GW9662, an irreversible PPARy antagonist, blocked PPARy activation by ciglitazone, but had no effect on C-peptide-stimulated PPARy activity. C-peptide stimulation of PPARy was attenuated by wortmannin pre-treatment, and by expression of a dominant negative PI 3-kinase p85 regulatory subunit (Ap85). C-peptide had no effect on protein expression levels of PPARy. PPARy phosphorylation was examined by [32P]-orthophosphate labelling of OK cells and immunoprecipitation of phospho-PPARy. C-peptide-induced PI 3-kinase dependent phosphorylation of PPARy. C-peptide is able to protect against tumor necrosis factor-alpha- (TNF-a) induced proximal tubular cells toxicity. Stimulation with 300ng/ml TNF-a for 24 hours resulted in significant reduction of cell viability which was reversed by pretreatment with C-peptide. TNF-a induced apoptosis was detected by measuring histone associated DNA fragments and DNA nick end-labelling of OK cells. Incubation of cells with 300ng/ml TNF-a for 24 hours induced apoptosis, but C-peptide pr-etreatment protected against TNF-a induced apoptosis. The protective effects of C-peptide were associated with activation of nuclear factor kB (NFkB) and increased expression of TNF receptor-associated factor 2, the product of an NFkB-dependent survival gene. This was dependent upon activation of PI 3-kinase, but not ERK. All C-peptide effects were abolished by pretreatment with PTX implicating a G-protein coupled receptor (GPCR), to either Goii or GOo, in the transduction of these events. C-peptide increased [35S]-GTPyS binding to Ga* in OK cell membranes. This study has now for the first time demonstrated specifically that Ga* proteins are activated by C-peptide binding to a GPCR. Despite being ignored for many years it is now clear that C-peptide possesses important biological properties and may potentially protect against diabetic complications