Alterations in protein kinase C isoforms in experimentally-induced colitis in the rat

Background. Although protein kinase C (PKC) isoenzymes have been implicated as mediators for multiple physiological processes, PKC also mediates cellular and intestinal mucosal injury. We have investigated the expression of the isoenzymes, PKC alpha, PKC delta PKC epsilon and PKC zeta in colonic mucosal tissue from TNBS-treated and HLA-B27 spontaneous colitis animals. Methods: Colonic mucosal samples were taken at various times (2 h-14 d) after instillation of TNBS (75 mg/kg in 50% ethanol) or from HLA-B27 rats at 16-18 weeks of age. Tissues were homogenized and separated into membrane and cytosolic fractions by centrifugation. PKC activity was measured radioenzymatically. PKC protein for isoforms alpha, delta, epsilon and zeta was assessed by Western blot while corresponding mRNA was analyzed by RT-PCR. Results: PKC activity increased in cytosolic and membrane fractions by 1d after TNBS and returned to normal by d3. PKC alpha protein was translocated from cytosol to membrane by 2 h after TNBS followed by down-regulation until d3. Increases in PKC delta, PKC epsilon and PKC zeta protein occurred initially in membrane fractions as early as 2 h after TNBS. Increases in cytosolic protein occurred at later times after induction of colitis. Protein levels for all isoenzymes remained increased up to 7d after TNBS. RT-PCR revealed that mRNA for PKC alpha decreased while PKC zeta mRNA increased correspondingly with their respective protein levels. In HLA-B27 rats, protein levels for all isoforms were less than was detected in normal colonic tissue. Conclusions: The early increase in gene expression and protein levels for PKC alpha and zeta suggest that these isozymes may play roles in an acute model of colitis induced by TNBS. In contrast, the increase in PKC delta and epsilon protein was not associated with mRNA changes suggesting that these isozymes are not similarly regulated in the inflamed colonic mucosa. In a chronic model of experimental colonic inflammation (HLA-B27), all of these isoforms appeared to be down-regulated.