Activation of the Lens MIP/Aquaporin 0 Gene Promoter during Induced Differentiation of Lens Epithelia Explants by FGF2

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 MIP gene regulatory elements and the signaling pathways responsible for the activation of the MIP promoter in FGF2-induced differentiation of explanted lens epithelia. Methods: Various murine MIP gene 5'-flanking sequences were inserted upstream of the luciferase and GFP reporter genes. These constructs were transfected into rat lens epithelia explants cultured with FGF2 and assayed for promoter activity. The possible roles of ERK 1/2 phosphorylation and PLC in the activation of promoter activity by FGF2 were studied using specific inhibitors UO126 (for ERK 1/2) and U-73122 (for PLC). The effect of these inhibitors was studied by real time PCR, scanning electron microscopy (SEM) and luciferase transient assays. Results: Lens epithelia explants cultured with FGF2 express MIP, whereas no MIP transcripts are observed in its absence. Lens explants cultured with 100ng/ml FGF2 show significant morphological changes in the apical surface of epithelial cells by SEM, not observed at low concentration of FGF2. MIP gene 5'-flanking sequence -1648/+44 activates the expression of the luciferase gene more efficiently than -342/+44 in differentiating lens explants. MIP -1648/+44 promoter activation by FGF2 is dose dependent, with a 10-fold increase in reporter activity observed between 0.05 and 100ng/ml FGF2. UO126 partially inhibits the MIP -1648/+44 promoter activity induced by FGF2. Conclusions: Our results indicate that MIP gene 5'-flanking sequence -1648/+44 contains regulatory elements required for MIP gene expression in differentiating lens cells. These promoter elements are not functional in rabbit kidney and mouse lens epithelial cell lines, suggesting their involvement in regulating lens-fiber specific expression of the MIP gene. Partial inhibition of the FGF2-induced MIP promoter activation by UO126 suggests the involvement of not only MAPK (ERK1/2) signaling pathway but possibly also other pathways such as PLC and PKC in this activation. Future experiments will allow us to further elucidate the FGF2 signaling pathways involved in the activation of the MIP promoter in lens fibers.

Keywords: gene/expression • molecular biology • transcription