Cell Biology Group, Department of Surgery, University of Maryland School of Medicine, Baltimore, MD, USA.
Baltimore Veterans Affairs Medical Center, Baltimore, MD, USA.
Physiol Rep. 2021 May;9(9):e14864. doi: 10.14814/phy2.14864.
Gut epithelial restitution after superficial wounding is an important repair modality regulated by numerous factors including Ca signaling and cellular polyamines. Transient receptor potential canonical-1 (TRPC1) functions as a store-operated Ca channel in intestinal epithelial cells (IECs) and its activation increases epithelial restitution by inducing Ca influx after acute injury. α4 is a multiple functional protein and implicated in many aspects of cell functions by modulating protein phosphatase 2A (PP2A) stability and activity. Here we show that the clonal populations of IECs stably expressing TRPC1 (IEC-TRPC1) exhibited increased levels of α4 and PP2A catalytic subunit (PP2Ac) and that TRPC1 promoted intestinal epithelial restitution by increasing α4/PP2Ac association. The levels of α4 and PP2Ac proteins increased significantly in stable IEC-TRPC1 cells and this induction in α4/PP2Ac complexes was accompanied by an increase in IEC migration after wounding. α4 silencing by transfection with siRNA targeting α4 (siα4) or PP2Ac silencing destabilized α4/PP2Ac complexes in stable IEC-TRPC1 cells and repressed cell migration over the wounded area. Increasing the levels of cellular polyamines by stable transfection with the Odc gene stimulated α4 and PP2Ac expression and enhanced their association, thus also promoting epithelial restitution after wounding. In contrast, depletion of cellular polyamines by treatment with α-difluoromethylornithine reduced α4/PP2Ac complexes and repressed cell migration. Ectopic overexpression of α4 partially rescued rapid epithelial repair in polyamine-deficient cells. These results indicate that activation of TRPC1-mediated Ca signaling enhances cell migration primarily by increasing α4/PP2Ac associations after wounding and this pathway is tightly regulated by cellular polyamines.
肠上皮细胞在受到轻度损伤后的修复是一种重要的修复方式，其受到许多因素的调节，包括钙信号和细胞多胺。瞬时受体电位经典型 1（TRPC1）在肠上皮细胞（IECs）中作为一种储存操纵钙通道发挥作用，其激活后通过急性损伤后诱导钙内流，增加上皮修复。α4 是一种多功能蛋白，通过调节蛋白磷酸酶 2A（PP2A）的稳定性和活性，参与细胞功能的许多方面。在这里，我们发现稳定表达 TRPC1 的 IEC 克隆群体（IEC-TRPC1）表现出 α4 和 PP2A 催化亚基（PP2Ac）水平升高，并且 TRPC1 通过增加α4/PP2Ac 结合来促进肠上皮修复。稳定的 IEC-TRPC1 细胞中α4 和 PP2Ac 蛋白水平显著增加，α4/PP2Ac 复合物的这种诱导伴随着损伤后 IEC 迁移的增加。用靶向α4 的 siRNA（siα4）转染或 PP2Ac 沉默转染进行α4 沉默，破坏了稳定的 IEC-TRPC1 细胞中α4/PP2Ac 复合物的稳定性，并抑制了细胞在创伤区域的迁移。通过稳定转染 Odc 基因增加细胞多胺水平可刺激α4 和 PP2Ac 的表达，并增强它们的结合，从而促进损伤后的上皮修复。相比之下，用α-二氟甲基鸟氨酸处理以耗尽细胞多胺，减少了α4/PP2Ac 复合物，并抑制了细胞迁移。细胞内多胺缺乏时，过表达α4 可部分挽救上皮修复的快速损伤。这些结果表明，TRPC1 介导的钙信号的激活通过在损伤后主要增加α4/PP2Ac 结合来增强细胞迁移，并且该途径受细胞多胺的紧密调节。
