Bioregulatory Aspect of Retinal Pigment Epithelial Cell

该项目研究MIP/Aquaporin 0编码基因的表达调控，MIP/Aquaporin 0是晶状体纤维膜的主要内在蛋白，在晶状体中特异表达，对晶状体的透明度和正确的折射率至关重要。 我们研究了在FGF2诱导晶状体上皮细胞向纤维分化过程中，MIP基因位点上的调控元件和导致晶状体特异性表达MIP基因和激活该基因的信号通路。我们的结果表明，MIP基因5‘侧翼序列包含对FGF2有反应的分化晶状体细胞中MIP基因表达所需的调控元件。我们目前正在研究在晶状体细胞分化过程中，FGF2激活MIP基因表达的信号通路。我们发现MAPK(ERK1/2)和JNK信号通路参与了晶状体细胞分化过程中MIP基因的激活。我们还发现，PKC信号通路不是FGF2诱导MIP表达所必需的，而是MIP整合到晶状体细胞膜上所必需的。 MIP/Aquaporin 0起到水通道的作用。然而，它可能在镜头中有额外的功能来保持镜头的透明度。我们的目标是识别和鉴定与MIP相互作用的蛋白质，以阐明这些相互作用在MIP功能中的作用。我们已经发现，γE-晶体蛋白是一种在晶状体纤维中特异表达的水溶性蛋白，是一种与MIP C端肽结合的蛋白。免疫共沉淀实验表明，在哺乳动物细胞中，γE-晶状体蛋白与全长MIP发生特异性的相互作用。有趣的是，MIP不与伽马D-晶体蛋白相互作用，后者是高度保守的伽马-晶体蛋白基因家族的另一个成员。共聚焦荧光显微镜显示，在哺乳动物细胞中，MIP与γE-晶体蛋白相互作用，这种相互作用导致γE-晶体蛋白从细胞质募集到质膜。MIP和γ-晶状体蛋白都在晶状体纤维中特异表达。Gamma E-晶状体蛋白在小鼠晶状体的透明性中起作用；已在小鼠Gamma E-晶状体蛋白和MIP基因中发现了导致显性表型遗传性白内障的突变。我们的结果首次证明了MIP和伽马E-晶体蛋白之间的特异性相互作用，为MIP和伽马-晶体蛋白之间的功能联系提供了证据。我们的数据还表明，MIP和伽马E-晶体蛋白之间的相互作用可能对MIP和伽马-晶体蛋白如何参与晶状体白内障的发生具有重要的意义。

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) 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 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.