Banan A, Zhang L J, Shaikh M, Fields J Z, Farhadi A, Keshavarzian A
Department of Internal Medicine, Rush University Medical Center, Chicago, IL 60612, USA.
Am J Physiol Cell Physiol. 2004 Jul;287(1):C218-34. doi: 10.1152/ajpcell.00575.2003. Epub 2004 Feb 25.
Using intestinal Caco-2 cells, we previously showed that assembly of cytoskeleton is required for monolayer barrier function, but the underlying mechanisms remain poorly understood. Because the theta-isoform of PKC is present in wild-type (WT) intestinal cells, we hypothesized that PKC-theta is crucial for changes in cytoskeletal and barrier dynamics. We have created the first multiple sets of gastrointestinal cell clones transfected with varying levels of cDNA to stably inhibit native PKC-theta (antisense, AS; dominant negative, DN) or to express its activity (sense). We studied transfected and WT Caco-2 cells. First, relative to WT cells, AS clones underexpressing PKC-theta showed monolayer injury as indicated by decreased native PKC-theta activity, reduced tubulin phosphorylation, increased tubulin disassembly (decreased polymerized and increased monomeric pools), reduced architectural integrity of microtubules, reduced stability of occludin, and increased barrier hyperpermeability. In these AS clones, PKC-theta was substantially reduced in the particulate fractions, indicating its inactivation. In WT cells, 82-kDa PKC-theta was constitutively active and coassociated with 50-kDa tubulin, forming an endogenous PKC-theta/tubulin complex. Second, DN transfection to inhibit the endogenous PKC-theta led to similar destabilizing effects on monolayers, including cytoskeletal hypophosphorylation, depolymerization, and instability as well as barrier disruption. Third, stable overexpression of PKC-theta led to a mostly cytosolic distribution of theta-isoform (<10% in particulate fractions), indicating its inactivation. In these sense clones, we also found disruption of occludin and microtubule assembly and increased barrier dysfunction. In conclusion, 1). PKC-theta isoform is required for changes in the cytoskeletal assembly and barrier permeability in intestinal monolayers, and 2). the molecular event underlying this novel biological effect of PKC-theta involves changes in phosphorylation and/or assembly of the subunit components of the cytoskeleton. The ability to alter the cytoskeletal and barrier dynamics is a unique function not previously attributed to PKC-theta.
我们之前利用肠道Caco-2细胞表明，细胞骨架的组装是单层屏障功能所必需的，但其潜在机制仍知之甚少。由于野生型（WT）肠道细胞中存在蛋白激酶C（PKC）的θ亚型，我们推测PKC-θ对于细胞骨架和屏障动力学的变化至关重要。我们构建了多组胃肠道细胞克隆，用不同水平的互补DNA（cDNA）进行转染，以稳定抑制内源性PKC-θ（反义，AS；显性负性，DN）或表达其活性（正义）。我们研究了转染的和WT Caco-2细胞。首先，相对于WT细胞，低表达PKC-θ的AS克隆表现出单层损伤，表现为内源性PKC-θ活性降低、微管蛋白磷酸化减少、微管蛋白解聚增加（聚合池减少，单体池增加）、微管的结构完整性降低、闭合蛋白稳定性降低以及屏障高通透性增加。在这些AS克隆中，PKC-θ在颗粒组分中显著减少，表明其失活。在WT细胞中，82-kDa的PKC-θ持续激活，并与50-kDa的微管蛋白共缔合，形成内源性PKC-θ/微管蛋白复合物。其次，通过DN转染抑制内源性PKC-θ对单层细胞产生了类似的破坏作用，包括细胞骨架低磷酸化、解聚和不稳定以及屏障破坏。第三，PKC-θ的稳定过表达导致θ亚型主要分布在细胞质中（颗粒组分中<10%），表明其失活。在这些正义克隆中，我们还发现闭合蛋白和微管组装受到破坏，屏障功能障碍增加。总之，1）. PKC-θ亚型是肠道单层细胞中细胞骨架组装和屏障通透性变化所必需的，2）. PKC-θ这种新生物学效应的潜在分子事件涉及细胞骨架亚基成分的磷酸化和/或组装变化。改变细胞骨架和屏障动力学的能力是PKC-θ以前未被归因的独特功能。
