Increased activity of epithelial Cdc42 Rho GTPase and tight junction permeability in the Cftr knockout intestine.

IF 3.9 3区 医学 Q1 GASTROENTEROLOGY & HEPATOLOGY American journal of physiology. Gastrointestinal and liver physiology Pub Date : 2024-10-01 Epub Date: 2024-08-06 DOI:10.1152/ajpgi.00211.2022
Rowena A Woode, Ashlee M Strubberg, Jinghua Liu, Nancy M Walker, Lane L Clarke
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Abstract

Increased intestinal permeability is a manifestation of cystic fibrosis (CF) in people with CF (pwCF) and in CF mouse models. CF transmembrane conductance regulator knockout (Cftr KO) mouse intestine exhibits increased proliferation and Wnt/β-catenin signaling relative to wild-type mice (WT). Since the Rho GTPase Cdc42 plays a central role in intestinal epithelial proliferation and tight junction remodeling, we hypothesized that Cdc42 may be altered in the Cftr KO crypts. Immunofluorescence showed distinct tight junction localization of Cdc42 in Cftr KO fresh crypts and enteroids, the latter indicating an epithelial-autonomous feature. Quantitative PCR and immunoblots revealed similar expression of Cdc42 in the Cftr KO crypts/enteroids relative to WT, whereas pulldown assays showed increased GTP-bound (active) Cdc42 in proportion to total Cdc42 in Cftr KO enteroids. Cdc42 activity in the Cftr KO and WT enteroids could be reduced by inhibition of the Wnt transducer Disheveled. With the use of a dye permeability assay, Cftr KO enteroids exhibited increased paracellular permeability to 3 kDa dextran relative to WT. Leak permeability and Cdc42 tight junction localization were reduced to a greater extent by inhibition of Wnt/β-catenin signaling with endo-IWR1 in Cftr KO relative to WT enteroids. Increased proliferation or inhibition of Cdc42 activity with ML141 in WT enteroids had no effect on permeability. In contrast, inhibition of Cdc42 with ML141 increased permeability to both 3 kDa dextran and tight junction impermeant 500 kDa dextran in Cftr KO enteroids. These data suggest that increased constitutive Cdc42 activity may alter the stability of paracellular permeability in Cftr KO crypt epithelium.NEW & NOTEWORTHY Increased tight junction localization and GTP-bound activity of the Rho GTPase Cdc42 was identified in small intestinal crypts and enteroids of cystic fibrosis (CF) transmembrane conductance regulator knockout (Cftr KO) mice. The increase in epithelial Cdc42 activity was associated with increased Wnt signaling. Paracellular flux of an uncharged solute (3 kDa dextran) in Cftr KO enteroids indicated a moderate leak permeability under basal conditions that was strongly exacerbated by Cdc42 inhibition. These findings suggest increased activity of Cdc42 in the Cftr KO intestine underlies alterations in intestinal permeability.

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上皮细胞 Cdc42 Rho GTPase 活性增强和 Cftr 基因敲除肠的紧密连接通透性增加
在囊性纤维化患者(pwCF)和囊性纤维化小鼠模型中,肠道通透性增加是囊性纤维化(CF)的一种表现形式。与野生型小鼠(WT)相比,CF 跨膜传导调节因子基因敲除(Cftr KO)小鼠肠道的增殖和 Wnt/β-catenin 信号传导增加。由于 Rho GTPase Cdc42 在肠上皮细胞增殖和紧密连接重塑中起着核心作用,我们推测 Cdc42 可能在 Cftr KO 小鼠隐窝中发生了改变。免疫荧光显示,在 Cftr KO 新鲜隐窝和肠管中,Cdc42 的紧密连接定位不同,后者显示了上皮自主的特征。定量 PCR 和免疫印迹显示,相对于 WT,Cftr KO 隐窝/肠固有体中 Cdc42 的表达量相似,而牵引试验显示,Cftr KO 肠固有体中 GTP 结合(活性)Cdc42 与总 Cdc42 的比例增加。抑制 Wnt 转导因子 Disheveled 2 可以降低 Cftr KO 和 WT 肠道中 Cdc42 的活性。使用染料渗透性试验,Cftr KO 肠道相对于 WT 表现出对 3kD 右旋糖酐的旁细胞渗透性增加。通过 Endo-IWR1 抑制 Wnt/β-catenin 信号传导,Cftr KO 肠道相对于 WT 肠道的渗漏通透性和 Cdc42 紧密连接定位的降低程度更大。用 ML141 增加增殖或抑制 Cdc42 活性对 WT 肠道通透性没有影响。相反,用 ML141 抑制 Cdc42 会增加 Cftr KO 肠道对 3kD 右旋糖酐和紧密连接不通透的 500 kD 右旋糖酐的通透性。这些数据表明,组成型 Cdc42 活性的增加可能会改变 Cftr KO 隐窝上皮细胞旁通透性的稳定性。
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来源期刊
CiteScore
9.40
自引率
2.20%
发文量
104
审稿时长
1 months
期刊介绍: The American Journal of Physiology-Gastrointestinal and Liver Physiology publishes original articles pertaining to all aspects of research involving normal or abnormal function of the gastrointestinal tract, hepatobiliary system, and pancreas. Authors are encouraged to submit manuscripts dealing with growth and development, digestion, secretion, absorption, metabolism, and motility relative to these organs, as well as research reports dealing with immune and inflammatory processes and with neural, endocrine, and circulatory control mechanisms that affect these organs.
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