This project studies the regulation of expression of the gene encoding MIP/Aquaporin 0, the major intrinsic protein of the lens fiber membrane, specifically expressed in the ocular lens and is essential for transparency and correct refractive index of the lens. We study the regulatory elements in the MIP gene locus and the signaling pathways responsible for the lens specific expression of the MIP gene and activation of this gene in FGF2-induced differentiation of explanted lens epithelia into fibers. Our results indicate that the MIP gene 5'-flanking sequence contains regulatory elements required for MIP gene expression in differentiating lens cells that are responsive to FGF2. We are currently characterizing the signaling pathways involved in the activation of MIP gene expression by FGF2 in differentiating lens cells. We found that MAPK (ERK1/2) signaling pathway is involved in activation of the MIP gene during lens cell differentiation. We also found that the PKC signaling pathway is not required to induce MIP expression by FGF2 but is required for MIP integration in the lens cell plasma membrane. MIP/Aquaporin 0 functions as a water channel. However, it may have additional functions in the lens to maintain lens transparency. Our goal is to identify and characterize proteins that interact with MIP to elucidate the role of these interactions in MIP functions. We have identified gamma E-crystallin, a water soluble protein specifically expressed in lens fibers, as a binding protein to the MIP C-terminal peptide. Co-immunoprecipitation assays demonstrated that gamma E-crystallin interacts specifically with full-length MIP in mammalian cells. Interestingly, MIP does not interact with gamma D-crystallin, another member of the highly conserved gamma-crystallin gene family. Confocal fluorescence microscopy demonstrated that MIP interacts with gamma E-crystallin in mammalian cells and that this interaction results in the recruitment of gamma E-crystallin from the cytoplasm to the plasma membrane. Both MIP and gamma-crystallins are specifically expressed in the lens fibers. Gamma E-crystallin plays a role in transparency of the mouse lens; mutations resulting in genetic cataracts with a dominant phenotype have been identified in the murine gamma E-crystallin and MIP genes. Our results demonstrate for the first time specific interaction between MIP and gamma E-crystallin, providing evidence for a functional link between MIP and gamma-crystallins. Our data also suggest that interaction between MIP and gamma E-crystallin may have important implications for how MIP and gamma -crystallins are involved in lens cataractogenesis.
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