Prahalad Priya, Calvo Ignacio, Waechter Holly, Matthews Jeffrey B, Zuk Anna, Matlin Karl S
Department of Biology, Massachusetts Institute of Technology, Cambridge 02142, USA.
Am J Physiol Cell Physiol. 2004 Mar;286(3):C693-707. doi: 10.1152/ajpcell.00124.2003. Epub 2003 Nov 26.
The attachment of epithelial cells to the extracellular matrix substratum is essential for their differentiation and polarization. Despite this, the precise adhesion mechanism and its regulation are poorly understood. In the kidney, an ischemic insult causes renal tubular epithelial cells to detach from the basement membrane, even though they remain viable. To understand this phenomenon, and to probe the regulation of epithelial cell attachment, we used a model system consisting of newly adherent Madin-Darby canine kidney (MDCK) cells subjected to ATP depletion to mimic ischemic injury. We found that MDCK cells detach from collagen I after 60 min of ATP depletion but reattach when resupplied with glucose. Detachment is not caused by degradation or endocytosis of beta(1)-integrins, which mediate attachment to collagen I. Basal actin filaments and paxillin-containing adhesion complexes are disrupted by ATP depletion and quickly reform on glucose repletion. However, partial preservation of basal actin by overexpression of constitutively active RhoA does not significantly affect cell detachment. Furthermore, Y-27632, an inhibitor of the RhoA effector Rho-kinase, does not prevent reattachment of cells on glucose addition, even though reformation of central stress fibers and large adhesion complexes is blocked. In contrast, reattachment of ATP-depleted cells and detachment of cells not previously subjected to ATP depletion are prevented by ML-7, an inhibitor of myosin light chain kinase (MLCK). We conclude that initial adherence of MDCK cells to a collagen I substratum is mediated by peripheral actin filaments and adhesion complexes regulated by MLCK but not by stress fibers and adhesion complexes controlled by RhoA.
上皮细胞与细胞外基质底层的附着对于其分化和极化至关重要。尽管如此，精确的黏附机制及其调控仍知之甚少。在肾脏中，缺血性损伤会导致肾小管上皮细胞从基底膜脱离，尽管它们仍保持存活。为了理解这一现象并探究上皮细胞附着的调控机制，我们使用了一个模型系统，该系统由新贴壁的Madin-Darby犬肾（MDCK）细胞组成，通过消耗ATP来模拟缺血性损伤。我们发现，MDCK细胞在ATP耗尽60分钟后从I型胶原上脱离，但在重新供应葡萄糖时会重新附着。脱离并非由介导与I型胶原附着的β(1)整合素的降解或内吞作用引起。基础肌动蛋白丝和含桩蛋白的黏附复合物在ATP耗尽时被破坏，并在葡萄糖补充后迅速重新形成。然而，通过组成型活性RhoA的过表达部分保留基础肌动蛋白，对细胞脱离没有显著影响。此外，RhoA效应物Rho激酶的抑制剂Y-27632并不能阻止细胞在添加葡萄糖时的重新附着，尽管中央应力纤维和大型黏附复合物的重新形成被阻断。相反，肌球蛋白轻链激酶（MLCK）的抑制剂ML-7可阻止ATP耗尽细胞的重新附着以及未预先经历ATP耗尽的细胞的脱离。我们得出结论，MDCK细胞对I型胶原底层的初始黏附是由外周肌动蛋白丝和受MLCK调控的黏附复合物介导的，而非由RhoA控制的应力纤维和黏附复合物介导。
