Bioregulatory Aspect of Retinal Pigment Epithelial Cell

本项目研究编码MIP/Aquaporin 0的基因的表达调控，MIP/Aquaporin 0是透镜纤维膜的主要内在蛋白，特异性地在眼透镜中表达，并且对于透镜的透明度和正确的折射率是必需的。 我们研究了MIP基因位点的调控元件和负责MIP基因的透镜特异性表达的信号通路，以及该基因在FGF 2诱导的透镜上皮细胞分化为纤维中的激活。我们的研究结果表明，MIP基因5 '侧翼序列包含MIP基因表达所需的调节元件在分化透镜细胞，是响应于FGF 2。目前，我们正在表征分化中的透镜细胞中由FGF 2激活MIP基因表达所涉及的信号通路。我们发现MAPK（ERK 1/2）和JNK信号通路参与了透镜细胞分化过程中MIP基因的激活。我们还发现，PKC信号通路是不需要诱导MIP表达的FGF 2，但需要MIP整合在透镜细胞质膜。 MIP/Aquaporin 0作为水通道发挥作用。然而，它可以在透镜中具有额外的功能以保持透镜的透明度。我们的目标是确定和表征与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。我们已经确定γ E-晶状体蛋白，一种在透镜纤维中特异性表达的水溶性蛋白，作为MIP C-末端肽的结合蛋白。免疫共沉淀试验表明，γ E-晶状体蛋白相互作用，特别是与全长MIP在哺乳动物细胞。有趣的是，MIP不与高度保守的γ-晶体蛋白基因家族的另一个成员γ D-晶体蛋白相互作用。共聚焦荧光显微镜表明，MIP与γ E-晶状体蛋白在哺乳动物细胞中相互作用，这种相互作用的结果在招聘γ E-晶状体蛋白从细胞质到质膜。MIP和γ-晶体蛋白都在透镜纤维中特异性表达。γ-E-晶状体蛋白在小鼠透镜的透明度中起作用;在鼠γ-E-晶状体蛋白和MIP基因中已经鉴定出导致具有显性表型的遗传性白内障的突变。我们的研究结果首次证明了MIP和γ-晶状体蛋白之间的特异性相互作用，为MIP和γ-晶状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γ-晶状体蛋白之间的相互作用可能对MIP和γ-晶状体蛋白如何参与透镜白内障的发生具有重要意义。

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.