Abstract
BackgroundSmall GTPases (guanosine triphosphate, GTP) are involved in many critical cellular processes, including inflammation, proliferation, and migration. GTP loading and isoprenylation are two important post-translational modifications of small GTPases, and are critical for their normal function. In this study, we investigated the role of post-translational modifications of small GTPases in regulating endothelial cell inflammatory responses and junctional integrity.Methods and ResultsConfluent human umbilical vein endothelial cell (HUVECs ) treated with atorvastatin demonstrated significantly decreased lipopolysaccharide (LPS)-mediated IL-6 and IL-8 generation. The inhibitory effect of atorvastatin (Atorva) was attenuated by co-treatment with 100 µM mevalonate (MVA) or 10 µM geranylgeranyl pyrophosphate (GGPP), but not by 10 µM farnesyl pyrophosphate (FPP). Atorvastatin treatment of HUVECs produced a time-dependent increase in GTP loading of all Rho GTPases, and induced the translocation of small Rho GTPases from the cellular membrane to the cytosol, which was reversed by 100 µM MVA and 10 µM GGPP, but not by 10 µM FPP. Atorvastatin significantly attenuated thrombin-induced HUVECs permeability, increased VE-cadherin targeting to cell junctions, and preserved junction integrity. These effects were partially reversed by GGPP but not by FPP, indicating that geranylgeranylation of small GTPases plays a major role in regulating endothelial junction integrity. Silencing of small GTPases showed that Rho and Rac, but not Cdc42, play central role in HUVECs junction integrity.ConclusionsIn conclusion, our studies show that post-translational modification of small GTPases plays a vital role in regulating endothelial inflammatory response and endothelial junction integrity. Atorvastatin increased GTP loading and inhibited isoprenylation of small GTPases, accompanied by reduced inflammatory response and preserved cellular junction integrity.
Highlights
Statins, a family of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) inhibitors, have been used extensively to block cholesterol biosynthesis and reduce serum cholesterol
In conclusion, our studies show that post-translational modification of small GTPases plays a vital role in regulating endothelial inflammatory response and endothelial junction integrity
We showed that atorvastatin inhibited endothelial cellular inflammation and cytoskeletal rearrangement by inhibiting small GTPase geranylgeranylation, which was reversed by exogenous geranylgeranyl pyrophosphate
Summary
A family of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMG-CoA reductase) inhibitors, have been used extensively to block cholesterol biosynthesis and reduce serum cholesterol. Key to our current study, statins treatment produced an outstanding enhancement of endothelial barrier function, including inhibition of stress fiber formation induced by various factors [3,4]. These beneficial effects clearly show that statins have a fundamental mechanistic effect on the endothelium, independent from inhibiting cholesterol synthesis [5]. Small GTPases can activate myosin light chain kinase (MLCK), and the phosphorylation of myosin light chain by activated MLCK leads to cytoskeletal rearrangement, including cellular constriction or relaxation [9] This pathway accompanies changes in cellular junction proteins and endothelial barrier function, which are critical for many processes, including neutrophil and macrophage migration, lamellipodia formation, and regulation of endothelial barrier integrity. We investigated the role of posttranslational modifications of small GTPases in regulating endothelial cell inflammatory responses and junctional integrity
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