Inducible overexpression of cingulin in stably transfected MDCK cells does not affect tight junction organization and gene expression
Inducible overexpression of cingulin in stably transfected MDCK cells does not affect tight junction organization and gene expression
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DOI：
10.1080/09687680701474009
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发表时间：
2008-01-01
影响因子：
--
通讯作者：
Citi, Sandra
中科院分区：
文献类型：
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作者：
Paschoud, Serge;Citi, Sandra
Cingulin是脊椎动物紧密连接（TJ）细胞质结构域的一个组成部分。在培养细胞中，环谷蛋白突变或下调导致基因表达的改变。其中一些变化依赖于RhoA，其活性由GEF-H1调节，GEF-H1通过在连接处与粘接蛋白结合而失活。为了进一步了解cingulin通过显性负作用的功能，我们克隆并测序了犬cingulin，并建立了稳定的MDCK细胞系，其中全长cingulin或头或杆+尾结构域被诱导过表达。令人惊讶的是，通过免疫印迹、微阵列、免疫荧光、经上皮耐药和细胞密度分析这些克隆表明，过表达全长cingulin或其结构域对TJ蛋白水平、基因表达、RhoA活性、细胞密度、倍增时间以及TJ的组织和功能没有显著影响。这些结果表明，补偿机制在该模型系统中阻止了显性负效应，并且环带蛋白对细胞功能的调节发生在生理蛋白水平内。
Cingulin is a component of the cytoplasmic domain of vertebrate tight junctions (TJ). Mutation or down-regulation of cingulin in cultured cells results in changes in gene expression. Some of these changes are dependent on RhoA, whose activity is regulated by GEF-H1, which is inactivated by binding to cingulin at junctions. To gain further insights on the function of cingulin through dominant-negative effects, we cloned and sequenced canine cingulin, and developed stable MDCK cell lines where either full-length cingulin, or head or rod+tail domains were inducibly overexpressed. Surprisingly, analysis of these clones by immunoblotting, microarray, immunofluorescence, measurement of transepithelial resistance, and cell density showed that the overexpression of either full-length cingulin or its domains does not significantly affect TJ protein levels, gene expression, RhoA activity, cell density, doubling time, and the organization and function of TJ. These results suggest that compensatory mechanisms prevent dominant-negative effects in this model system, and that modulation of cellular functions by cingulin occurs within physiological protein levels.