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P. C. Kothary, S. K. Gupta, M. A. Del Monte; Cross-Talk Between the ERK Kinase and Adenyl Cyclase Signalling Pathways in Vascular Endothelial Growth Factor Synthesis. Invest. Ophthalmol. Vis. Sci. 2009;50(13):3426.
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© ARVO (1962-2015); The Authors (2016-present)
Human retinal pigment epithelium (hRPE) cells have been shown to play a role in choroidal neovascularization. Since vascular endothelial growth factor (VEGF) also plays an important role in choroidal neovascularization, we investigated the role of various signaling pathways in regulating VEGF synthesis in cultured hRPE cells.
hRPE cultures were established from four human eyes. hRPE cell proliferation was determined by 3H-thymidine incorporation (3H-thy). Confluent hRPE cells were then washed with Minimum Essential Media prior to their exposure to experimental reagents for 24 to 48 hours, depending on the experiment. Anti-VEGF was used for immuno-precipitation of 14C-methionine-VEGF and immuno-histochemical analysis. Data were analyzed by Student 't' test. hRPE cells were treated with varying concentrations of fetal bovine serum (FBS), PD98059 (a selective inhibitor of MAP kinase), H89, (a selective cAMP-dependent protein kinase inhibitor) and AG490 (a specific JAK-2 protein tyrosine kinase inhibitor).
Increasing concentrations of FBS stimulated hRPE proliferation as determined by 3H-thy. FBS (0-10%) also stimulated 14C-VEGF synthesis in a dose-dependent manner. FBS (10%) stimulated 14C-VEGF synthesis was inhibited by PD98059 (50 µM) (846.2±245.1 vs.1234.6±194.2, CPM±SEM, p<0.05, n=4) and H89 (10 µM) (522.6±60.0 vs. 759.9±95.2, CPM±SEM, p<0.05, n=4). However, AG490 did not inhibit, but instead possibly stimulated 14C-VEGF synthesis (6055.6±125.9 vs. 4450±742.8, CPM±SEM, p<0.05, n=4). In addition, H89 had no effect on 14C-ERK1-2 synthesis (1119.9±306.1 vs.1148.6±189.2, CPM±SEM, p≥0.05, n=4). Immuno-histochemical studies showed an increase in VEGF immuno-reactive cells in the presence of FBS (10%) when compared to controls and hRPE cells exposed to FBS (10%) supplemented with PD98059 (50 µM).
We have identified a cross-talk between ERK kinase and adenylyl cyclase signaling that may be involved in regulating VEGF synthesis in hRPE cells. Understanding the complex regulation of VEGF synthesis should provide critical information that will contribute to the development of therapeutic pharmaco-therapy for choroidal neovascularization.
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