FGF–induced ERK and JNK signaling are required for MIP/Aquaporin 0 expression in Rat Lens Explants

Abstract

Abstract: : Purpose: Lens Major Intrinsic Protein (MIP)/Aquaporin 0 is specifically expressed in the ocular lens fibers and plays an important role in lens transparency. We study the FGF–induced signaling pathways involved in MIP expression in rat lens epithelia explants. Methods: MIP expression was analyzed by Real Time PCR, immunohistochemistry, Western blot and luciferase reporter assay in rat lens epithelia explants cultured with FGF2 for 3 days. The role of MAPK pathways in the activation of MIP gene expression was studied using specific Erk and JNK phosphorylation inhibitors, UO126 and SP600125, respectively. Results: Real Time PCR revealed that endogenous MIP gene expression in the lens epithelia explants is FGF2 dose dependent. FGF2 also induces MIP promoter (–1648/+44) activity in the rat lens explants as observed by luciferase assay. Western blot analysis of the lens explants showed that FGF2 induces ERK1/2 activation in a concentration–dependent manner. This activation is totally abolished by the inhibitor UO126. Real Time PCR revealed a 5–fold decrease in MIP transcript level in the presence of UO126 in FGF–treated explants. Luciferase assay showed partial inhibition of MIP –1648/+44 promoter activity in the presence of the ERK1/2 phosphorylation inhibitor UO126. Western blot analysis revealed that FGF treatment led to a slight increase in phosphorylation of JNK1 (p46) but not of JNK2 (p54). Real Time PCR analysis revealed that MIP mRNA level was decreased 3–fold in the presence of specific inhibitor of JNK (SP600125). Immunofluorescent analysis by confocal microscopy showed abrogation of MIP protein expression in SP600125–treated explants. Conclusions: Our results indicate that MIP gene transcriptional activation involves factors downstream of ERK and JNK pathways. It is possible that these two pathways, in spite of their ability to activate some common transcription factors, distinctly regulate specific transcription factors that activate the MIP promoter in the lens explants. We also demonstrate that ERK activation is not sufficient to induce MIP expression and that JNK signaling synergizes with the ERK pathway to induce efficient expression of MIP upon FGF–2 stimulation. Further studies are required to precisely delineate the factors in the FGF/ERK and FGF/JNK signaling pathways that are responsible for MIP expression and to identify their response elements in the MIP promoter.

Keywords: gene/expression • signal transduction • phosphorylation