Cytoskeletal architecture regulates cyclooxygenase-2 in human endothelial cells : autocrine modulation by prostacyclin

Endothelium is a highly dynamic tissue that controls vascular homeostasis. This requires constant rearrangements of the shape or function of endothelial cells that cannot set aside the role of the cytoskeleton. The aim of this study was to determine the mechanisms by means of which cytoskeletal alterations induce cyclooxygenase-2 expression in human endothelial cells using compounds that interfere with microtubule or actin architecture. Microtubule disruption by nocodazole markedly increased cyclooxygenase-2 expression and activity, and provoked paracellular gap formation, a cardinal feature of endothelial barrier dysfunction. The cyclooxygenase-2 metabolite prostacyclin down-regulated cyclooxygenase-2 through an autocrine receptor-mediated mechanism, and partially prevented the disassembly of endothelial monolayers. There was also an interaction between microtubules and actin filaments in nocodazole-induced cyclooxygenase-2 expression. Nocodazole provoked the dissolution of the F-actin cortical ring and stress fibre formation, increased actin glutathionylation and concomitantly lowered intracellular levels of reduced glutathione. The restoration of glutathione levels by N-acetylcysteine opposed cyclooxygenase-2 expression and preserved the integrity of endothelial monolayers. Among the signalling pathways connecting microtubule disruption with cyclooxygenase-2 up-regulation, crucial roles are played by Src family kinase activation, serine/threonine phosphatase 2A inhibition, and the phosphorylation of mitogen activated protein kinase p38. Our findings provide a mechanistic insight into the observation that cyclooxygenase-2 is induced in endothelial cells under cytoskeleton-perturbing conditions such as those occurring in the presence of atherogenic/inflammatory stimuli and oxidative stress. In this scenario, cyclooxygenase-2 up-regulation by endothelia exposed to noxious conditions can be considered protective of the vasodilatory and anti-thrombotic properties of the vessel wall.