Potent activation of multiple signalling pathways by C-peptide in opossum kidney proximal tubular cells.
OBJECTIVE
Proinsulin C-peptide is generally believed to be inert without any appreciable biological functions. However, it has been shown to modulate a variety of cellular processes important in the pathophysiology of diabetic complications. We therefore investigated the ability of C-peptide to stimulate intracellular signalling pathways in kidney proximal tubular cells, the altered activation of which may possibly be related to the development of diabetic nephropathy.


METHODS
Extracellular signal-regulated kinase (ERK) and Akt phosphorylation were evaluated by western blotting. ERK activity was measured by in vitro kinase assay. Intracellular Ca(2+) was evaluated by confocal imaging. The membrane and cytosol-associated fractions of protein kinase C (PKC) isoforms were evaluated by western blotting. Proliferation was assessed by thymidine incorporation assay.


RESULTS
Using the opossum proximal tubular kidney cell line as a model, we demonstrated that at high picomolar to low nanomolar concentrations, C-peptide stimulates extracellular signal-regulated mitogen-activated kinase (3.3+/-0.1-fold over basal at 3 minutes) and phosphatidylinositol 3-kinase (4.1+/-0.05-fold over basal at 5 minutes). ERK activation was attenuated by pre-treatment with a PKC inhibitor and abolished by pertussis toxin. Elevations of intracellular [Ca(2+)] are seen in response to 5 nmol/l C-peptide with consequent activation of PKC-alpha. Pre-treatment with pertussis toxin abolished PKC-alpha. C-peptide is also a functional mitogen in this cell type, stimulating significantly increased cell proliferation. Proliferation was attenuated by wortmannin and pertussis toxin pre-treatments. None of these effects is reproduced by scrambled C-peptide.


CONCLUSIONS
This study provides evidence that C-peptide, within physiological concentration ranges, stimulates many signalling pathways in opossum kidney cells.
Al-Rasheed
N M
NM
Department of Cell Physiology and Pharmacology, Faculty of Medicine and Biological Sciences, University of Leicester, Medical Sciences Building, University Road, Leicester LE1 9HN, United Kingdom.
Meakin
F
F
Royal
E L
EL
Lewington
A J
AJ
Brown
J
J
Willars
G B
GB
Brunskill
N J
NJ
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D015153:Blotting, Western / Q000379:methods; D002096:C-Peptide / Q000737:chemistry / Q000494:pharmacology / Q000502:physiology*; D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D049109:Cell Proliferation / Q000187:drug effects; D048049:Extracellular Signal-Regulated MAP Kinases / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000502:physiology*; D006801:Humans; D007211:Indoles / Q000494:pharmacology; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism; D018613:Microscopy, Confocal / Q000379:methods; D009893:Opossums; D037342:Pertussis Toxin / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000187:drug effects / Q000378:metabolism; D051571:Protein Kinase C-alpha; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000187:drug effects / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D013936:Thymidine / Q000378:metabolism; D014316:Tritium / Q000378:metabolism; D000077191:Wortmannin
D000730:Androstadienes; D002096:C-Peptide; D007211:Indoles; D011518:Proto-Oncogene Proteins; D014316:Tritium; D037342:Pertussis Toxin; C494918:AKT1 protein, human; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D048049:Extracellular Signal-Regulated MAP Kinases; D002118:Calcium; D013936:Thymidine; C064758:Ro 31-8220; D000077191:Wortmannin