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Rho protein inhibition blocks cyclooxygenase-2 expression by proinflammatory mediators in endothelial cells
Bernd Schmeck1, Marco Brunsch, Joachim Seybold
1Charité, Department of Internal Medicine, Humboldt-University, Berlin, Germany.
Abstract:
Rho proteins participate in the regulation of inflammatory gene expression in endothelial cells. We made use of Clostridium difficile toxin 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-alpha (TNFalpha)-related COX-2 expression but had no effect on COX-1 protein levels. TcdB-10463 preincubation suppressed LPS-dependent nuclear factor-kappaB activation (NF-kappaB). Rho inhibition did not affect COX-1 activity. Inactivation of Rho proteins before LPS stimulation blocked arachidonic acid (AA)-, thrombin-, and Escherichia coli hemolysin (HlyA)-dependent release of COX-2-related 6-ketoprostaglandin F(1alpha), (6k-PGF(1alpha)). In contrast, Rho inhibition did not affect COX-2-dependent 6k-PGF(1alpha) liberation when TcdB-10643 was added 10 h after LPS or TNFalpha stimulation of EC. Therefore, RhoA/Rac1/Cdc42 contribute to NF-kappaB-dependent LPS- and TNFalpha-induced expression of PGHS-2 in EC but had no effect on the activity of expressed COX-1 and COX-2.
Insights
Rho proteins regulate inflammatory gene expression. Inhibiting RhoA, Rac1, and Cdc42 prevented lipopolysaccharide-induced cyclooxygenase-2 (COX-2) expression in endothelial cells by blocking nuclear factor-kappaB activation.
Area of Science:
- Cell Biology
- Molecular Biology
- Immunology
Background:
- Rho proteins are key regulators of inflammatory gene expression in endothelial cells.
- Cyclooxygenase-2 (COX-2) plays a critical role in inflammatory processes.
- Understanding the upstream regulators of COX-2 is crucial for developing anti-inflammatory strategies.
Purpose of the Study:
- To investigate the role of RhoA, Rac1, and Cdc42 in the regulation of cyclooxygenase-2 (COX-2) expression in endothelial cells.
- To determine the impact of Rho protein inhibition on lipopolysaccharide (LPS)- and tumor necrosis factor-alpha (TNFα)-induced COX-2 expression.
- To elucidate the signaling pathways, specifically nuclear factor-kappaB (NF-κB), involved in Rho-mediated COX-2 regulation.
Main Methods:
- Endothelial cells (EC) were treated with Clostridium difficile toxin B-10643 (TcdB-10643) to inhibit RhoA, Rac1, and Cdc42.
- COX-2 and COX-1 protein levels were assessed following stimulation with LPS or TNFα.
- NF-κB activation was measured to evaluate its dependence on Rho proteins.
- The release of 6-ketoprostaglandin F1α (6k-PGF1α), a product of COX-2 activity, was quantified under various experimental conditions.
Main Results:
- TcdB-10643 pretreatment prevented LPS- or TNFα-induced COX-2 expression but did not affect COX-1 levels.
- Inhibition of Rho proteins suppressed LPS-dependent NF-κB activation.
- Rho inactivation blocked the release of 6k-PGF1α induced by arachidonic acid, thrombin, and E. coli hemolysin (HlyA) when performed before LPS stimulation.
- Rho inhibition did not affect COX-2-dependent 6k-PGF1α release when TcdB-10643 was added after LPS or TNFα stimulation.
Conclusions:
- RhoA, Rac1, and Cdc42 are essential for NF-κB-dependent induction of COX-2 (encoded by PGHS-2) in endothelial cells stimulated by LPS and TNFα.
- Rho proteins do not influence the enzymatic activity of expressed COX-1 or COX-2.
- These findings highlight Rho proteins as critical upstream regulators of inflammatory mediator production in endothelial cells.