title: Role of PKCepsilon in PGF2alpha-stimulated MMP-2 secretion from human ciliary muscle cells.
abstract: Studies were designed to examine the roles of individual protein kinase C (PKC) isoforms in the prostaglandin F(2alpha) (PGF(2alpha))-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 PGF(2alpha) (1 micromol/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 PGF(2alpha) (1 micromol/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 PGF(2alpha). In cultured human ciliary muscle cells, the PKC isoforms exhibiting the highest level of expression were PKCalpha, epsilon, iota and lambda. The delta and eta isoforms exhibited moderate levels of expression and beta, gamma, and phi were not detected. The administration of PGF(2alpha) (1 micromol/L) primarily induced the translocation of PKCepsilon 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 PGF(2alpha)-induced secretion of MMP-2 was also blocked by pretreatment with the PKCepsilon-selective peptide translocation inhibitor, EAVSLKPT, or the transfection of siRNA-targeting PKCepsilon. The activation of ERK1/2 was inhibited by chelerythrine and the PKCepsilon translocation inhibitor. Human ciliary muscle cells express the alpha, epsilon, iota and lambda PKC isoforms. Stimulation of FP receptors in these cells activates PKCepsilon, 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 PKCepsilon-dependent secretion of MMPs.
authors.affiliation: Department of Ophthalmology, Hewitt Laboratory of the Ola B. Williams Glaucoma Center, Medical University of South Carolina, Charleston, SC 29425, USA. Husain@musc.edu
authors.forename: Shahid
authors.initials: S
authors.lastname: Husain
authors.forename: Craig E
authors.initials: CE
authors.lastname: Crosson
mesh_terms: D000328:Adult; D000368:Aged; D053119:Benzophenanthridines / Q000494:pharmacology; D002924:Ciliary Body / Q000166:cytology / Q000201:enzymology*; D015237:Dinoprost / Q000494:pharmacology*; D004789:Enzyme Activation; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D005260:Female; D006801:Humans; D066298:In Vitro Techniques; D007202:Indicators and Reagents; D007527:Isoenzymes / Q000378:metabolism; D008297:Male; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism*; D008875:Middle Aged; D009130:Muscle, Smooth / Q000166:cytology / Q000201:enzymology*; D051744:Protein Kinase C-epsilon / Q000037:antagonists & inhibitors / Q000502:physiology*; D021381:Protein Transport / Q000187:drug effects; D034741:RNA, Small Interfering / Q000494:pharmacology; D014162:Transfection
chemical_list: D053119:Benzophenanthridines; D007202:Indicators and Reagents; D007527:Isoenzymes; D034741:RNA, Small Interfering; D015237:Dinoprost; C016299:chelerythrine; D051744:Protein Kinase C-epsilon; D048049:Extracellular Signal-Regulated MAP Kinases; D020778:Matrix Metalloproteinase 2