A role for endogenous TGFalpha and associated signaling pathways in the differentiation of lens fiber cells.

Abstract

TGFalpha is hypothesized to be an endogenous regulator of lens fiber terminal differentiation. With immunofluorescence, TGFalpha was localized to differentiating cells in the lens epithelium and superficial fiber cell mass of the adult chicken. A similar pattern of localization was also noted when differentiating epithelial cells were cultured. Immunoneutralization of endogenous TGFalpha inhibited the accumulation of filensin, a unique intermediate filament protein subunit restricted to developing vertebrate lens fibers. ELISA assays quantified the effects of TGFalpha on filensin expression. Surprisingly, inhibition of the TGFalpha receptors' tyrosine kinase activity with nanomolar concentrations of PD153035 increased the accumulation of differentiated characteristics in the presence or absence of ligand. Morphologically, PD153035-treated cells grew as aggregated masses and spread less well onto the substrate. Accompanying these morphologic changes was a complete inhibition of cell division. Post-receptor signaling events were examined with cAMP assays and Western blotting. TGFalpha did not affect cAMP levels while isoproterenol, an additional mediator of lens cell differentiation, caused significant increases in cAMP levels. Activation of ERK2 via dual phosphorylation was noted in response to TGFalpha but not isoproterenol. PD153035 reduced, but did not eliminate, ERK2 phosphorylation in response to TGFalpha. Phosphorylation of the CREB transcription factor was also observed in response to TGFalpha or isoproterenol. These data indicate that endogenous ligands can influence the expression of differentiated characteristics in cultured chick lens cells. A focus of multiple signaling pathways affecting filensin expression is the CREB transcription factor. While increased ERK2 activation may be involved in stimulating cell division, lower levels of persistent ERK2 activation promotes differentiation thus indicating some form of signaling pathway compartmentalization. This could also mean that additional receptors for TGFalpha not inhibited by PD153035 are responsible for promoting differentiation in chick lens cells.