Abstract
Abstract: :
Purpose: 12(R)–HETrE is an arachidonic acid metabolite synthesized by and released from the corneal epithelium in response to injury. 12(R)–HETrE stimulates corneal neovascularization both in vitro and in vivo. We have previously shown that VEGF expression is increased in microvessel endothelial cells (MEC) treated with 12(R)–HETrE and this effect is mediated in part by ERK1/2 activation. In the present study, we further evaluate the role of Akt and PKC in 12–HETrE signal transduction and use microarrays to identify genes that may contribute to its inflammatory and angiogenic activity. Methods: MEC where grown to 70% confluent and starved for 48hr in absence of serum before treatment with 12(R)–HETrE (0.1, 1 and 10nM). In some experiments cells were incubated with the AKT specific inhibitor (LY294002 20µM) or PKC inhibitors (Calphostin C, 500 nM; Bisindolylmeleimide I, 10 nM) prior to the addition of 12–HETrE. Akt phosphorylation and PKC activation was measured by Western Blot analysis. Gene expression profile was performed on mRNA from cells treated with 12(R)–HETrE (1nM) using a Superarray gene profiling system. Results: 12(R)–HETrE induced ERK1/2 phosphorylation in a time– and concentration–dependent manner as previously described. AKT was not phosphorylated by 12(R)–HETrE treatment at any of the concentrations tested. PKC α, ßI, ßII and Δ were not activated in response to 12(R)–HETrE (5 min) since cellular protein extract did not immunoreact with a pan antibody which recognizes α, ßI, ßII and Δ PKC isoforms. DNA microarray analysis of 12(R)–HETrE–treated cells showed a significant increase in IL–8 gene expression compared to control cells. The IL–8 protein increase in cells in response to 12(R)–HETrE was confirmed by ELISA. Conclusions: 12(R)–HETrE–induced ERK1/2 activation in RLME is not mediated by AKT or PKC activation. PKC isoforms other than α, ßI, ßII and Δ may be involved. Interestingly, 12(R)–HETrE stimulated both IL–8 gene and protein expression. These results suggest that IL–8 may play an important role in the angiogenic response of limbal vasculature to mitogenic and inflammatory 12(R)–HETrE.
Keywords: eicosanoids • inflammation • signal transduction: pharmacology/physiology