Abstract
Basic fibroblast growth factor (FGF2) stimulates photoreceptor survival in vivo and in vitro, but the molecular signaling mechanism(s) involved are unknown. Immunohistochemical and immunoblotting analyses of pure photoreceptors, inner retinal neurons, and Müller glial cells (MGC) in vitro revealed differential expression of the high affinity FGF receptors (FGFR1-4), as well as many cytoplasmic signaling intermediates known to mediate the extracellular signal-regulated kinase (ERK1/2) pathway. FGF2-induced tyrosine phosphorylation in vitro exhibited distinct profiles for each culture type, and FGF2-induced ERK1/2 activation was observed for all three preparations. Whereas U0126, a specific inhibitor of ERK kinase (MEK), completely abolished FGF2-induced ERK1/2 tyrosine phosphorylation and survival in cultured photoreceptors, persistent ERK1/2 phosphorylation was observed in cultured inner retinal cells and MGC. Furthermore U0126 treatment entirely blocked nerve growth factor-induced ERK1/2 activation in MGC, as well as FGF2-induced ERK1/2 activation in cerebral glial cells. Taken together, these data indicate that FGF2-induced ERK1/2 activation is entirely mediated by MEK within photoreceptors, which is responsible for FGF2-stimulated photoreceptor survival. In contrast, inner retina/glia possess alternative, cell type, and growth factor-specific MEK-independent ERK1/2 activation pathways. Hence signaling and biological effects elicited by FGF2 within retina are mediated by cell type-specific pathways.
Highlights
Basic fibroblast growth factor (FGF2) stimulates photoreceptor survival in vivo and in vitro, but the molecular signaling mechanism(s) involved are unknown
FGF signal transduction has been intensively studied, mostly in non-neuronal tissue, using immortalized cell lines overexpressing one or more FGFR (10 –12), and a variety of molecules involved in the FGF signal transduction cascade have been described: phospholipase C␥1 (PLC␥1), a regulator of phosphatidyl inositol mechanism [13]; the adaptor protein Shc; son of sevenless (SOS), a guanine nucleotide exchange factor for ras and growth factor receptor-binding protein 2 (Grb2) [12]; a phosphotyrosine phosphatase designated as SHPTP2 [14]; and protein kinase B (Akt) [15]
Immunostaining of cultured retinal cells with antisera directed against second messenger molecules (PLC␥1, SOS1, SOS2, extracellular signal-regulated kinases (ERK), SHPTP2, SHC) showed uniform PR, neuronal, and glial labeling
Summary
Basic fibroblast growth factor (FGF2) stimulates photoreceptor survival in vivo and in vitro, but the molecular signaling mechanism(s) involved are unknown. FGF signal transduction has been intensively studied, mostly in non-neuronal tissue, using immortalized cell lines overexpressing one or more FGFR (10 –12), and a variety of molecules involved in the FGF signal transduction cascade have been described: phospholipase C␥1 (PLC␥1), a regulator of phosphatidyl inositol mechanism [13]; the adaptor protein Shc; son of sevenless (SOS), a guanine nucleotide exchange factor for ras and growth factor receptor-binding protein 2 (Grb2) [12]; a phosphotyrosine phosphatase designated as SHPTP2 [14]; and protein kinase B (Akt) [15] These molecules activate downstream signaling pathways, including those of the mitogen-activated protein kinases [16], known as extracellular signal-regulated kinases (ERK), a family of serine/ threonine protein kinases. We have exploited primary cultures of different retinal cells (purified PR, inner retina (IR, without PR), and purified Muller glial cells (MGC)) to determine whether FGF2-related signaling molecules have distinct expression patterns between the different cell populations and whether FGF2 possesses multiple pathways for signal transduction in the retina This is the case, PR survival depending uniquely upon ERK activation via MEK. Whereas in FGF2-treated PR, MEK seems to be the only upstream ERK activator, additional MEK-independent pathways exist in IR and MGC
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