REGULATION OF THE ACTIN CYTOSKELETON BY THROMBIN IN HUMAN ENDOTHELIAL-CELLS - ROLE OF RHO PROTEINS IN ENDOTHELIAL BARRIER FUNCTION

Endothelial barrier function is regulated at the cellular level by cyt oskeletal- dependent anchoring and retracting forces. Ln the present s tudy we have examined the signal transduction pathways underlying agon ist-stimulated reorganization of the actin cytoskeleton in human umbil ical vein endothelial cells. Receptor activation by thrombin, or the t hrombin receptor (proteinase-activated receptor 1) agonist peptide, le ads to an early increase in stress fiber formation followed by cortica l actin accumulation and cell rounding. Selective inhibition of thromb in-stimulated signaling systems, including Gi/o (pertussis toxin sensi tive), p42/p44, and p38 MAP kinase cascades, Src family kinases, PI-3 kinase, or S6 kinase pathways had no effect on the thrombin response. In contrast, staurosporine and KT5926, an inhibitor of myosin light ch ain kinase, effectively blocked thrombin-induced cell rounding and ret raction. The contribution of Rho to these effects was analyzed by usin g bacterial toxins that either activate or inhibit the GTPase. Escheri chia call cytotoxic necrotizing factor 1, an activator of Rho, induced the appearance of dense actin cables across cells without perturbing monolayer integrity. Accordingly, lysophosphatidic acid, an activator of Rho-dependent stress fiber formation in fibroblasts, led to reorgan ization of polymerized actin into stress fibers but failed to induce c ell rounding. Inhibition of Rho with Clostridium botulinum exoenzyme C 3 fused to the B fragment of diphtheria toxin caused loss of stress fi bers with only partial attenuation of thrombin-induced cell rounding. The implication of Rac and Cdc42 was analyzed in transient transfectio n experiments using either constitutively active (V12) or dominant-int erfering (N17) mutants. Expression of RacV12 mimicked the effect of th rombin on cell rounding, and RacNI7 blocked the response to thrombin, whereas Cdc42 mutants were without effect. These observations suggest that Rho is involved in the maintenance of endothelial barrier functio n and Rac participates in cytoskeletal remodeling by thrombin in human umbilical vein endothelial cells.