Activation of protein kinase C by tumor necrosis factor-alpha in human non-pigmented ciliary epithelium.
Previously, we have shown that tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine, increases the synthesis and release of endothelin-1 (ET-1), a potent vasoactive peptide from human non-pigmented ciliary epithelial (HNPE) cells, in a protein kinase C (PKC)-dependent manner. Diacylglycerol (DAG) and intracellular calcium ([Ca2+]i) are well known activators of PKC. Some cytokines induce PKC activation by stimulating phospholipase C that hydrolyzes phosphatidylinositol bisphosphate (PIP2) into IP3 (intracellular calcium mobilizer) and DAG. In this study, the existence of a similar pathway was evaluated in HNPE cells treated with TNF-alpha, using intracellular calcium ([Ca2+]i) measurements, PKC translocation assays and thin-layer chromatography (TLC) for quantification of DAG. Incubation times for agonists and inhibitors ranged from 1-30 minutes. The increase in DAG levels with TNF-alpha treatment was consistent with the observed translocation of the calcium-dependent PKC alpha isoform from the cytosol to the plasma membrane. However, these observations were not accompanied by a concomitant increase in [Ca2+]i. Similar translocation responses were observed with phorbol ester (phorbol 12-myristate 13-acetate) treatment. Our results indicate that TNF-alpha-induced PKC activation in HNPE cells occurs as a result of elevated DAG levels and is not due to an increase in intracellular calcium. Activated PKC, could enhance the pro-inflammatory responses of TNF-alpha in part by increasing the production of endothelins in the eye.
Prasanna
G
G
Department of Pharmacology and North Texas Eye Research Institute, University of North Texas Health Science Center at Fort Worth, 76107, USA.
Dibas
A
A
Brown
K
K
Yorio
T
T
D002118:Calcium / Q000378:metabolism; D002460:Cell Line; D002924:Ciliary Body / Q000187:drug effects* / Q000201:enzymology; D004075:Diglycerides / Q000378:metabolism; D019332:Endothelin-1 / Q000494:pharmacology; D004789:Enzyme Activation; D006801:Humans; D007527:Isoenzymes / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism*; D051571:Protein Kinase C-alpha; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D004075:Diglycerides; D019332:Endothelin-1; D007527:Isoenzymes; D014409:Tumor Necrosis Factor-alpha; C496628:PRKCA protein, human; D011493:Protein Kinase C; D051571:Protein Kinase C-alpha; D002118:Calcium