Abstract
Abstract: :
Purpose: The objective of this study was to evaluate the role of protein kinase C (PKC) isoforms in the signaling pathways that modulate PGF2α–induced MMP secretion from human ciliary muscle (HCM) cells. Methods: Primary cultures of human ciliary muscle (HCM) cells were used in this study. Serum–starved cells were treated with agonist (prostaglandin F2α) for 2–24 hr and concentrated media were analyzed for MMP using selective anti–MMP antibodies. PKC isoforms were detected by Western blotting using PKC–isoform specific antibodies. Transfection of the siRNA targeting endogenous gene for PKC isoform was carried out in 70–80% confluent HCM cells using 1–100 nmol/L siRNA duplexes for each PKC isoform. Results: PKCα, Δ, ε, , and isoforms were detected in HCM cells. PGF2α treatment resulted in the translocation of PKCε to the particulate fraction by 3 to 4 fold. PKCα was marginally translocated to the particulate fraction whereas other PKC isoforms were not affected by this treatment. PGF2α increased the secretion of MMP–2 in a dose–dependent manner and this secretion was significantly (P <.05) inhibited in the presence of PKC inhibitors (chelerythrine chloride) in HCM cells. To selectively knockdown endogenous PKC isoforms, siRNA was used to target individual PKC isoform genes. In HCM cells, transfection of human PKCε siRNA resulted in over 80% reduction of endogenous PKCε expression, which subsequently resulted in the suppression of PGF2α–induced MMP–2 secretion from HCM cells. Conclusions: These data provide evidence that intraocular pressure lowering agents such as PGF2α selectively activated and translocated PKCε isoform in ciliary muscle cells. Moreover, pharmacological inhibitors of PKC and biochemical tools, gene silencing by siRNA, blocked the agonist–induced functional response in these cells. We conclude that selective PKC isoforms modulate the function of uveoscleral outflow pathways, and their activity can be altered by ocular hypotensive drugs.
Keywords: outflow: ciliary muscle • ciliary muscle • signal transduction: pharmacology/physiology