Regulation of Lens Fiber Membrane Gene Expression

晶状体纤维膜基因表达的调控

• 批准号: 6826532
• 负责人: ANA B CHEPELINSKY
• 金额: --
• 依托单位: NATIONAL EYE INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6826532
• 关键词: cataract; cell differentiation; cell line; cell membrane; confocal scanning microscopy; crystallins; fibroblast growth factor; fluorescence microscopy; gene expression; genetic regulation; genetic regulatory element; genetically modified animals; immunoprecipitation; laboratory mouse; lens; lens proteins; macrophage inflammatory proteins; protein protein interaction; protein signal sequence; protein structure function; transcription factor; water channel

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.

该项目研究了编码MIP/Aquaporin 0的基因的表达，这是透镜纤维膜的主要固有蛋白，该基因在眼镜镜片中特别表达，对于透镜的透明度和正确的折射率至关重要。 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.我们的结果表明，MIP基因5'-频序序列包含对响应FGF2的晶状体细胞中MIP基因表达所需的调节元素。我们目前正在表征与晶状体细胞中FGF2激活MIP基因表达的信号通路。我们发现MAPK（ERK1/2）信号通路参与透镜细胞分化过程中MIP基因的激活。我们还发现，PKC信号通路不需要FGF2诱导MIP表达，但是在透镜细胞质膜中MIP积分是必需的。 MIP/Aquaporin 0充当水通道。但是，它可能在镜头中具有其他功能来维持透镜透明度。我们的目标是识别和表征与MIP相互作用的蛋白质，以阐明这些相互作用在MIP函数中的作用。我们已经确定γe-晶体蛋白是一种在晶状体纤维中特异性表达的水溶性蛋白，是与MIP C末端肽的结合蛋白。共免疫沉淀分析表明，γe-Crystallin在哺乳动物细胞中与全长MIP特别相互作用。有趣的是，MIP与高度保守的γ-晶状体基因家族的另一个成员Gamma D-Crystallin不相互作用。共聚焦荧光显微镜表明，MIP与哺乳动物细胞中的γe-晶体蛋白相互作用，并且这种相互作用导致γe-Crystallin从细胞质募集到质膜。 MIP和γ-晶状蛋白都在晶状体纤维中特别表达。伽马电子结晶蛋白在小鼠镜头的透明度中起作用。在鼠γe-Crystallin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。我们的结果证明了MIP和伽马晶链蛋白之间的首次具体相互作用，提供了MIP和γ-晶状体之间功能联系的证据。我们的数据还表明，MIP和Gamma e -Crystallin之间的相互作用可能对MIP和Gamma -Crystallins如何参与透镜白内障发生有重要意义。