Abstract
Abstract: :
Purpose: Prostaglandin (PG) F2α and its analog latanoprost lower intraocular pressure (IOP) and increase uveosclereal outflow, in part through receptor-mediated mechanisms in the ciliary muscle. The increased uveoscleral outflow is primarily attributed to remodeling of extracellular matrix (ECM) between the muscle bundles. Matrix metalloproteinases (MMPs) have a specialized function of degrading ECM. The purpose of the present study was to investigate the effects of prostaglandins on mitogen activated protein kinases (ERK1/ERK2) activation and their role in the regulation of MMPs in HCM cells. Methods: The HCM cells were treated with PGF2α or latanoprost for 5 min and activity of ERK1/ERK2 was measured using an in-gel-kinase assay. Phosphorylation of ERK1/ERK2 was determined by Western blot analysis using anti-phospho-ERK1/ERK2 antibodies. The secretion of MMP-1 and MMP-2 was determined in conditioned medium by Western blot analysis using anti-MMP-1 and MMP-2 antibodies after 6-48 h of PGF2α treatment. The activity of MMPs was measured by zymography. Results: Both PGF2α and latanoprost activate ERK1/ERK2 in a dose dependent manner with EC50 values of 5 x 10-11 M and 8 x 10-12 M, respectively. Furthermore, 1 µM PGF2α or 1 µM latanoprost induced a sustained activation of ERK1/ERK2 for up to 24 h. In contrast, 1 µM PGE2 had inhibitory effects on ERK1/ERK2 activation. PGF2α or latanoprost induced activation of ERK1/ERK2 was inhibited 75 ± 3 and 70 ± 5 %, respectively when cells were pre-incubated with 1 µM PGE2 for 2 h. PGF2α induced activity was inhibited 64 ± 3 and 75 ± 5 % in the presence of protein kinase C (PKC) inhibitors, chelerythrine chloride (1µM) and calphostin (1µM), respectively. Whereas latanoprost induced ERK1/ERK2 activity was completely inhibited in the presence of these inhibitors. PGF2α also increased pro-MMP-1 and pro-MMP-2 secretion in a dose-dependent manner with EC50 values of 3.5 x 10-9 M and 4.1 x 10-9 M, respectively. PGF2α induced pro-MMP-2 activation and secretion was completely inhibited by 1µM PD-98059, an ERK1/ERK2 inhibitor. Furthermore, PGF2α induced pro-MMP-1 expression was completely inhibited in the presence of PGE2. Conclusion: These data suggest that (a) different signaling mechanisms are coupled to FP and EP receptor-subtypes, (b) PGF2α (FP-agonist) and PGE2 (EP-agonist) have different responses on ERK1/ERK2 activation and MMPs secretion, (c) PKC is an upstream regulator of ERK1/ERK2, and (d) ERK1/ERK2 may act as upstream regulators of MMPs in HCM cells.
Keywords: signal transduction: pharmacology/physiology • eicosanoids • second messengers: pharmacology/physiology