This project studied 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 essential for transparency and correct refractive index of the lens. ? ? We studied the signaling pathways responsible for the lens specific expression of the MIP gene and its activation in FGF2-induced differentiation of explanted lens epithelia into fibers. Our results indicated 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 characterized the signaling pathways involved in the activation of MIP gene expression by FGF2 in differentiating lens cells. We found that MAPK (ERK1/2) and JNK signaling pathways are 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, besides functioning as a water channel, may have additional functions in the lens to maintain lens transparency. 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. 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. MIP does not interact with the Elo mutant of gamma E-crystallin, which has been linked to a dominant cataract phenotype in mice. Confocal fluorescence microscopy demonstrated that gamma E- and F- crystallins co-localize specifically with full-length MIP in mammalian cells while other gamma-crystallins including gamma A-, B-, C-, D- and S- do not. As a result of this interaction, only gamma E- and F-crystallins were recruited to the plasma membrane from the cytoplasm. 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 demonstrating specific interaction between MIP and gamma E- or F-crystallins, provides evidence for a functional link between MIP and gamma-crystallins. The interaction between MIP and two members of the gamma-crystallin family may have important implications for MIP and gamma -crystallins involvement in lens cataractogenesis.
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