Keap1 regulates the constitutive expression of GST A1 during differentiation of Caco-2 cells
Keap1 regulates the constitutive expression of GST A1 during differentiation of Caco-2 cells
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DOI：
10.1021/bi800199z
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发表时间：
2008-06-10
期刊：
影响因子：
2.9
通讯作者：
Uchida, Koji
中科院分区：
文献类型：
--
作者：
Kusano, Yuri;Horie, Shunsuke;Uchida, Koji
kelch样ech相关蛋白1 （Keap1）是一种btbkelch底物衔接蛋白，用于cul3依赖性泛素连接酶复合体，通过抑制转录因子Nrf2调节谷胱甘肽s -转移酶（GST）等2期酶的诱导。众所周知，在人类胃肠道中，GST A1和P1都是组成性表达的主要GST同工酶。本研究通过稳定转染野生型Keap1，利用人结肠癌细胞系Caco-2细胞的过表达Keap1衍生物，研究了GST同工酶在分化过程中组成性表达的分子机制。结果表明，过表达Keap1完全抑制GST A1的组成表达，而不抑制GST P1的组成表达。在过表达keap1的细胞中，圆顶的形成消失，细胞与细胞接触部位完整的肌动蛋白细胞骨架组织的形成以及E-cadherin和β -catenin在粘附连接处的募集受到抑制。Caco-2细胞中组成型GST A1的表达受到e -cadherin介导的细胞-细胞粘附破坏的抑制，提示在Caco-2分化过程中上皮细胞极化与诱导基础型GST A1表达之间存在相关性。Keap1过表达确实抑制了小鸟苷三磷酸酶Rac1对e -cadherin介导的细胞-细胞粘附形成的激活。将组成型活性的Rac1突变体V12Rac1转染到过表达的Keap1细胞中，促进了GST A1的基础表达，提示Keap1通过激活Rac1介导的e -cadherin介导的细胞-细胞粘附形成，调控Caco-2分化过程中GST A1的基础表达。本研究的结果表明，在上皮细胞分化过程中，一种新的依赖keap1的信号通路参与了诱导构成性GST A1表达。
Kelch-like ECH-associated protein 1 (Keap1), a BTB-Kelch substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex, regulates the induction of the phase 2 enzymes, such as glutathione S-transferase (GST), by repressing the transcription factor Nrf2. It is known that, in the human gastrointestinal tract, both GST A1 and P1 are constitutively expressed as the major GST isozymes. In the present study, using the Keap1-overexpressing derivatives of Caco-2 cells, human carcinoma cell line of colonic origin, by stable transfection of wild type Keap1, we investigated the molecular mechanism underlying the constitutive expression of these GST isozymes during differentiation. It was revealed that the overexpression of Keap1 completely repressed the constitutive expression of GST A1, but not GST P1. In Keap1-overexpressed cells, dome formation disappeared, and the formation of the intact actin cytoskeletal organization at cell-cell contact sites and the recruitment of E-cadherin and beta-catenin to adherens junctions were inhibited. The constitutive GST A1 expression in Caco-2 cells was repressed by disruption of E-cadherin-mediated cell-cell adhesion, suggesting the correlation between epithelial cell polarization and induction of the basal GST A1 expressions during Caco-2 differentiation. Keap1 overexpression indeed inhibited the activation of the small guanosine triphosphatase Rac1 on the formation of E-cadherin-mediated cell-cell adhesion. The transfection of V12Rac1, the constitutively active Rac1 mutant, into Keap1-overexpressed cells promoted the basal GST A1 expression, suggesting that Keap1 regulated the basal GST A1 expression during Caco-2 differentiation via Rac1 activation on the formation of E-cadherin-mediated cell-cell adhesion. The results of this study suggest the involvement of a novel Keap1-dependent signaling pathway for the induction of the constitutive GST A1 expression during epithelial cell differentiation.