Endogenous FGF1-induced Activation and Synthesis of Extracellular Signal-regulated Kinase 2 Reduce Cell Apoptosis in Retinal-pigmented Epithelial Cells

Abstract

Retinal-pigmented epithelial (RPE) cell survival is critical to the maintenance of the function of the neural retinal and in the development of various retina degenerations. We investigated molecular mechanisms involved in this function by assessing apoptosis in RPE cells following serum deprivation. Apoptosis induced by serum withdrawal is lower in aged RPE cells because of higher endogenous acidic fibroblast growth factor (FGF1) synthesis and secretion. These experiments examined several aspects of FGF signaling and the contribution of endogenous FGF1 to activation of the extracellular signal-regulated kinase 2 (ERK2). In aged RPE cells, FGFR1 was rapidly activated, and its autophosphorylation followed the kinetics of endogenous FGF1 secretion, before the onset of apoptosis. ERK2 phosphorylation, activity, and de novo synthesis increased at the same time. In marked contrast, no de novo JNK1 synthesis was observed. MEK1 inhibition resulted in lower levels of ERK2 activation and synthesis and higher levels of apoptosis. Treatment with neutralizing anti-FGF1 or blocking anti-FGFR1 antibodies mimics these effects. Thus, this study strongly suggests that the survival-increasing effect of FGF1 in aged RPE cells is because of an autocrine/paracrine loop in which the ERK2 cascade plays a pivotal role. Fibroblast growth factors (FGFs)1 are a family of at least 15 polypeptides that stimulate growth and differentiation in cells of various mesenchymal and ectodermal origins. (for reviews, see Refs. 1-5). Acidic FGF (FGF1) and basic FGF (FGF2) are the prototype members of this family. FGF1 and 2 lack a classic signal peptide (6, 7), implying that they are not secreted by the classical secretion pathway. There is evidence that FGF1 and 2 are exported from the cell and subsequently act as autocrine or paracrine factors (8, 9). FGF1 and 2 exert their effects via high affinity tyrosine kinase receptors (FGFR1-FGFR4) (for reviews, see Refs. 10 -12) and via lower affinity heparan sulfate proteoglycan binding sites (13-15). FGFR activation causes tyrosine phosphorylation of the receptor itself, intracellular proteins including phospholipase C␥ (PLC␥) (16), extracellular signal-regulated kinases (ERKs) (17), and of several uncharacterized proteins of [80][81][82][83][84][85][86][87][88][89][90]19). All cell layers in normal adult retina produce FGF1, as cells no longer differentiate or proliferate. The pattern of FGF1 production suggests that FGF1 may be involved in the regulation of specific spatiotemporal events, including proliferation, migration, differentiation and survival in the retina. In vivo, despite the presence of FGFs in the retinal interphotoreceptor matrix, RPE cells have a limited proliferation capacity consistent with the normal increase in retinal space associated with growth and age. In adult, RPE cells cannot divide in vivo, but they may still require survival factors to inhibit their apoptosis. Cell survival and proliferation may be determined by the amount of factor available and the amount of receptor produc...

Both FGF1 and Bcl-x synthesis are necessary for the reduction of apoptosis in retinal pigmented epithelial cells by FGF2: role of the extracellular signal-regulated kinase 2

“…The importance of the long-term activation of ERK2 for cell survival is consistent with studies showing that acute activation of the ERK cascade by growth factors potentiates proliferation whereas a chronic increase in ERK activity is cytoprotective (Carter et al, 1998). In addition, it was shown that overproduction of ERKs in human breast cancer was required for cell survival (Sivaraman et al, 1997) and that cell survival is correlated with the overproduction of ERK2 during aging of RPE cells (Guillonneau et al, 1998b). The importance of both activation and production of proteins of the ERK pathway for cell survival was recently con®rmed (Mishima et al, 1998;Yazlovitskaya et al, 1999;Sahl et al, 1999).…”

“…The MEK inhibitors, PD098059 (Calbiochem, Meudon, France) and UO126 (Promega, Charbonniere, France), were added on days 1 and 3 to ®nal concentrations of 10-and 4 mM respectively. There were no non-speci®c cytotoxic e ects of the MEK inhibitors, as previously reported (Guillonneau et al, 1998b).…”

“…Inhibition of MEK by PD098059 and UO126 also leads to ERK2 inactivation, inhibition of BcL-x production and RPE cell apoptosis Stimulation of Bcl-x by FGF2 is mediated by ERK2 M Bryckaert et al controlled by ERK1/2 activation. This seems to be con®rmed by previous studies showing that cell survival is correlated with the overexpression of ERK2 protein during the aging of RPE cells (Guillonneau et al, 1998b).…”

“…In some experiments, anti-FGF1 neutralizing polyclonal antibody (R & D Systems) and blocking anti-FGFR monoclonal antibody (clone VBS1, Chemicon International) were added on days 1 and 3 to ®nal concentrations of 200 mg/ml (anti-FGF1) and 20 mg/ml (anti-FGFR1) of culture medium. The speci®city of the anti-FGF1 and anti-FGFR1 has been previously veri®ed (Guillonneau et al, 1998b). The MEK inhibitors, PD098059 (Calbiochem, Meudon, France) and UO126 (Promega, Charbonniere, France), were added on days 1 and 3 to ®nal concentrations of 10-and 4 mM respectively.…”

“…In growing RPE cells, ERKs are rapidly and transiently activated in response to exogenous FGFs (Malecaze et al, 1993). We have shown that FGF2 stimulated the production of endogenous FGF1 (Guillonneau et al, 1997) and that high-level expression of endogenous FGF1 is correlated with a reduction in apoptosis during RPE cell aging (Guillonneau et al, 1998b). This suggests that there is an FGF paracrine pathway supporting RPE cell survival in vitro, though no loop of FGF1 activation involving endogenous FGF2 was demonstrated in vivo.…”