Gene Expression of CNTF, CT1, OSM and their receptor subunits were analyzed on human RPE by quantitative RT-PCR. Membrane Localization of the receptor subunits were determined by immunoblot and immunofluorescence analysis using primary culture of human fetal RPE (hfRPE). Phosphorylation assays were used to check the signal transduction pathway activated by CNTF, CT1 and OsM in RPE. BrdU incorporation assay was used to test the cell proliferation and cell survival effects of these neurotrophic factors. Capacitance probe technique was used to test the effect of CNTF on fluid transport (JV) across RPE from the subretinal space to choroid. The mRNA of LIF and CT1 are highly expressed in hfRPE compared to CNTF and OsM. The receptor subunits of these neurotrophic factors (CNTFR, LIFR, gp130 and OsMR) were all detected in primary cultures of hfRPE, although the protein level of CNTFR is under the detection level of immunoblots. Binding of CNTF, CT1 and OsM to their receptors activate the JAK/STAT3 signaling pathway in primary culture of hfRPE and adult RPE (ARPE-19). While OsM significantly activated P44/P42 (ERK) MAP kinase pathway, both CNTF and CT1 has no apparent effects on the phosphorylation of ERK. CNTF has small but significant stimulatory effect on hfRPE proliferation (P <0.05). CT1 show dose response stimulatory effect on RPE proliferation and the maximum stimulatory effect (25%) was observed at 100 ng/ml;OsM show dose-dependent inhibitory effect on hfRPE proliferation from 10-80 ng/ml. Furthermore, CNTF significantly increased fluid transport (JV) across RPE from 8.7 b 0.7 to 20.7 b 3.3 lcm-2 hr-1 (n= 3;P <0.05).
