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

Abstract

Acute ischemic kidney injury results in marked increases in local and systemic cytokine levels. IL-1alpha, 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 < or = 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 < or = 0.05) and RhoA activation (up to 227 +/- 26% increase, P < or = 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.

Probing the mechanical properties of TNF-&alpha; stimulated endothelial cell with atomic force microscopy

“…The actin filaments are known to form a network under the cell membrane providing mechanical strength and to undergo extensive remodeling and reorganization upon TNF-α stimulation. [34][35][36] The observed increase in filament density could explain the higher stiffness of the stimulated cells as compared with the controls. Also, the level of expression of ICAM-1 was measured through ELISA, demonstrating a statistically significant increase in the surface density for these adhesive molecules (Figure 4), normally overexpressed on the inflamed endothelium.…”

Interleukin-1&beta; and tumor necrosis factor-&alpha; increase stiffness and impair contractile function of articular chondrocytes

“…Indeed, both IL-1β [43][44][45] and TNF-α [46][47][48] have been shown to induce F-actin reorganization in a variety of cell types, but further changes of cell mechanical properties remain controversial. Qi et al [49] reported a decrease of tenocyte stiffness (cellular level) but an increase of tendon stiffness (tissue level) [50] in response to IL-1β; Bakker et al [30] showed that both IL-1β and TNF-α decreased the stiffness of osteocytes; and Lee et al [51] found that TNF-α increased the stiffness of all three endothelial cell lines tested.…”

The biology of interleukin-1: emerging concepts in the regulation of the actin cytoskeleton and cell junction dynamics

“…For instance, IL-1 is known to induce actin stress fiber formation in different cell types [60, 61], and to increase tight junction (TJ) permeability in the intestinal epithelium [62] and in the blood–brain barrier [63]. IL-1 is also involved in the non-inflammatory-related restructuring of cell junctions, which occurs during normal tissue homeostasis.…”