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Abstract #22060 Published in IGR 10-4
Role of PKCepsilon in PGF2alpha-stimulated MMP-2 secretion from human ciliary muscle cells
Husain S; Crosson CEJournal of Ocular Pharmacology and Therapeutics 2008; 24: 268-277
Studies were designed to examine the roles of individual protein kinase C (PKC) isoforms in the prostaglandin F2α (PGF2α)-induced matrix metalloproteinase-2 (MMP-2) secretion from human ciliary muscle cells. Studies utilized primary cultures of human ciliary muscle cells. Individual PKC isoforms were detected by Western blotting, using PKC-isoform-specific antibodies. To evaluate MMP-2 secretion, cells were serum-starved overnight, treated with PGF2α (1 µmol/L) for 4 h and the media analyzed for MMP-2 by Western blotting. To assess ERK1/2 activation, cells were serum-starved overnight, treated with PGF2α (1 µmol/L) for 5 min and cell lysates analyzed for ERK1/2 phosphorylation by Western blot analysis. To evaluate the roles of individual PKC isoforms, cells were pretreated with PKC inhibitors or siRNAs prior to the addition of PGF2α. In cultured human ciliary muscle cells, the PKC isoforms exhibiting the highest level of expression were PKCα, ε, ι and λ. The δ and η isoforms exhibited moderate levels of expression and β, γ, and &phi: were not detected. The administration of PGF2α (1 µmol/L) primarily induced the translocation of PKCε from cytosol to the membrane fraction, as well as increased MMP-2 secretion and ERK1/2 phosphorylation. The secretion of MMP-2 was inhibited by pretreatment with the broad-range PKC inhibitor, chelerythrine chloride; however, this response was not blocked by Go-6976, an inhibitor of conventional PKC isoforms. The PGF2α-induced secretion of MMP-2 was also blocked by pretreatment with the PKCε-selective peptide translocation inhibitor, EAVSLKPT, or the transfection of siRNA-targeting PKCε. The activation of ERK1/2 was inhibited by chelerythrine and the PKCε translocation inhibitor. Human ciliary muscle cells express the α, ε, ι and λ PKC isoforms. Stimulation of FP receptors in these cells activates PKCε, resulting in ERK1/2 activation and an eventual increase in MMP-2 secretion. These data support the idea that the activation of FP receptors in vivo modulate uveoscleral outflow through the PKCε-dependent secretion of MMPs.
Dr. S. Husain, Department of Ophthalmology, Hewitt Laboratory of the Ola B. Williams Glaucoma Center, Medical University of South Carolina, Charleston, SC 29425, USA. Husain@musc.edu
