Overexpression of pkc-ç in mesangial cells increases expression of cox-d and alters cell morphology

Inducible cyclooxygenase (COX-II) is critical for renal development and involved in many pathological processes of the adult kidney by catalyzing the reaction producing prostaglandin Ej (PGE-2) from arachidonic acid. COX II expression in mesangial cells under basal conditions is minimal but dramatically increases with a variety of stimuli including IL-1. The downstream signals controlling this pathway are not known. Previous experiments with protein kinase inhibitors staurosporine and calphostin C suggest a role for the zeta isoform of protein kinase C (PKC-ζ) in the regulation of mis pathway. To investigate this a full length cDNA of rat PKCC-ζ was ligated in forward and reverse orientation into the retroviral expression vector pLXSN. After calcium-phosphate transformation of a retroviral packaging cell line, replication defective virus was harvested and used to infect primary rat mesangial cells. Cells were selected in G418 and immunoblotted for PKCÇ. Expression of cDNA in the forward orientation produced a 40 fold increase in PKCζ. Western analysis also revealed constitutive COX II expression under basal conditions. PGE2 production increased 6-10 fold over control as measured by gas chromatography-mass spectrometry. At confluence the mesangial cells overexpressing PKCÇ had an exaggerated tendency towards hillock formation as compared to control. Neither altered morphology nor significant increases in COX n or PGE2 were seen in control mesangial cells or cells expressing PKCζ cDNA in the reverse orientation. These results suggest PKCζ is involved in the regulation of COX II in mesangial cells.