Rho Protein Inhibition Blocks Cyclooxygenase-2 Expression by Proinflammatory Mediators in Endothelial Cells

Rho proteins participate in the regulation of inflammatory gene expression in endothelial cells. We made use of Clostridium difficiletoxin B-10643 (TcdB-10463) which inhibites RhoA/Rac1/Cdc42 to analyze their role in expression and regulation of cyclooxygenase-2 (COX-2) in endothelial cells (EC). Pretreatment of EC with TcdB-10643 prevented lipopolysaccharide (LPS)- or tumor necrosis factor-α (TNFα )-related COX-2 expression but had no effect on COX-1 protein levels. TcdB-10463 preincubation suppressed LPS-dependent nuclear factor-κB activation (NF-κ B). Rho inhibition did not affect COX-1 activity. Inactivation of Rho proteins before LPS stimulation blocked arachidonic acid (AA)-, thrombin-, and Escherichia colihemolysin (HlyA)-dependent release of COX-2-related 6-ketoprostaglandin F1α(6k-PGF1α). In contrast, Rho inhibition did not affect COX-2-dependent 6k-PGF1αliberation when TcdB-10643 was added 10 h after LPS or TNFα stimulation of EC. Therefore, RhoA/Rac1/Cdc42 contribute to NF-κ B-dependent LPS- and TNFα-induced expression of PGHS-2 in EC but had no effect on the activity of expressed COX-1 and COX-2.