Cytokine-induced F-actin reorganization in endothelial cells involves RhoA activation

Campos SB, Ashworth SL, Wean S, Hosford M, Sandoval RM, Hallett MA, Atkinson SJ, Molitoris BA. Cytokine-induced F-actin reorganization in endothelial cells involves RhoA activation. Am J Physiol Renal Physiol 298: F487-F495, 2009. First published January 14, 2009; doi:10.1152/ajprenal.00112.2008.-Acute ischemic kidney injury results in marked increases in local and systemic cytokine levels. IL-1 alpha, IL-6, and TNF-alpha orchestrate various inflammatory reactions influencing endothelial permeability by altering cell-to-cell and cell-to-extracellular matrix attachments. To explore the role of actin and the regulatory proteins RhoA and cofilin in this process, microvascular endothelial cells (MS1) were exposed to individual cytokines or a cytokine cocktail. Within minutes, a marked, time-dependent redistribution of the actin cytoskeleton occurred with the formation of long, dense F-actin basal stress fibers. The concentration of F-actin, normalized to nuclear staining, significantly increased compared with untreated cells (up 20%, P <= 0.05). Western blot analysis of MS1 lysates incubated with the cytokine cocktail for 4 h showed an increase in phosphorylated/inactive cofilin (up 25 +/- 15%, P <= 0.05) and RhoA activation (up to 227 +/- 26% increase, P <= 0.05) compared with untreated cells. Decreasing RhoA levels using small interfering RNA blocked the effect of cytokines on stress fiber organization. Treatment with Y-27632, an inhibitor of the RhoA effector p160-ROCK, decreased levels of phosphorylated cofilin and reduced stress fiber fluorescence by 22%. In cells treated with Y-27632 followed by treatment with the cytokine cocktail, stress fiber levels were similar to control cells and cofilin phosphorylation was 55% of control levels. Taken together, these studies demonstrate cytokine stimulation of RhoA, which in turn leads to cofilin phosphorylation and formation of numerous basal actin stress fibers. These results suggest cytokines signal through the Rho-ROCK pathway, but also through another pathway to affect actin dynamics.