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. 2009 Mar 13;9:12.
doi: 10.1186/1471-2261-9-12.

Inhibition of S6K1 Accounts Partially for the Anti-Inflammatory Effects of the Arginase Inhibitor L-norvaline

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Free PMC article

Inhibition of S6K1 Accounts Partially for the Anti-Inflammatory Effects of the Arginase Inhibitor L-norvaline

Xiu-Fen Ming et al. BMC Cardiovasc Disord. .
Free PMC article

Abstract

Background: Pharmacological inhibition of endothelial arginase-II has been shown to improve endothelial nitric oxide synthase (eNOS) function and reduce atherogenesis in animal models. We investigated whether the endothelial arginase II is involved in inflammatory responses in endothelial cells.

Methods: Human endothelial cells were isolated from umbilical veins and stimulated with TNFalpha (10 ng/ml) for 4 hours. Endothelial expression of the inflammatory molecules i.e. vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), and E-selectin were assessed by immunoblotting.

Results: The induction of the expression of endothelial VCAM-1, ICAM-1 and E-selectin by TNFalpha was concentration-dependently reduced by incubation of the endothelial cells with the arginase inhibitor L-norvaline. However, inhibition of arginase by another arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC) had no effects. To confirm the role of arginase-II (the prominent isoform expressed in HUVECs) in the inflammatory responses, adenoviral mediated siRNA silencing of arginase-II knocked down the arginase II protein level, but did not inhibit the up-regulation of the adhesion molecules. Moreover, the inhibitory effect of L-norvaline was not reversed by the NOS inhibitor L-NAME and L-norvaline did not interfere with TNFalpha-induced activation of NF-kappaB, JNK, p38mapk, while it inhibited p70s6k (S6K1) activity. Silencing S6K1 prevented up-regulation of E-selectin, but not that of VCAM-1 or ICAM-1 induced by TNFalpha.

Conclusion: The arginase inhibitor L-norvaline exhibits anti-inflammatory effects independently of inhibition of arginase in human endothelial cells. The anti-inflammatory properties of L-norvaline are partially attributable to its ability to inhibit S6K1.

Figures

Figure 1
Figure 1
Effect of L-norvaline on TNFα-induced expression of VCAM-1, ICAM-1, and E-selectin. (A) Stimulation of HUVECs with TNFα (10 ng/ml, 4 hours) increased expression of VCAM-1, ICAM-1, and E-selectin, which was significantly reduced by L-norvaline in a concentration-dependent manner. Tubulin in bottom panel served as loading control. (B) Quantification of the signals shown in panel A. Representative data from five independent experiments are reported as mean ± SEM. Values are given as percentage relative to stimulation with TNFα. *p < 0.05, **p < 0.01, ***p < 0.001 vs. stimulation with TNFα. o/n = overnight.
Figure 2
Figure 2
L-norvaline-mediated inhibition of VCAM-1, ICAM-1, and E-selectin: independence of NOS activity. (A) The effects of L-norvaline on TNFα-induced expression of VCAM-1, ICAM-1, and E-selectin could not be reversed by the NOS inhibitor L-NAME (100 μmol/L). Tubulin in bottom panel served as loading control. Shown are representative blots from five independent experiments. (B) Quantification of the signals shown in panel A. Values are given as percentage relative to stimulation with TNFα in the absence of L-NAME. **p < 0.01, ***p < 0.001 vs. stimulation with TNFα in the absence of L-NAME.
Figure 3
Figure 3
Role of the arginase inhibitor BEC on TNFα-induced expression of VCAM-1, ICAM-1, and E-selectin. Stimulation of the cells with TNFα (10 ng/ml, 4 hours) increased inflammatory response which was not affected by the arginase inhibitor BEC (200 μmol/L, o/n). Shown are representative blots from five independent experiments.
Figure 4
Figure 4
Role of arginase II silencing on TNFα-induced expression of VCAM-1, ICAM-1, and E-selectin. HUVECs were transduced with recombinant adenovirus expressing shRNA against LacZ as control (lanes 1 and 5) or arginase II (lanes 2–4 and 6 – 8). On day 4 of post transduction, cells were serum-starved for 20 h followed by stimulation with TNFα (10 ng/ml, 4 hours) and extracted. The knocking down effects of various shRNA targeting sequences were assessed by immunoblotting as shown in panel A. Panel B shows the effects on expression of VCAM-1, ICAM-1, and E-selectin. Shown are representative blots from five independent experiments.
Figure 5
Figure 5
Effects of L-norvaline on TNFα-induced activation of various signaling pathways. After pre-treatment of the cells with L-norvaline (Nor. 20 mmol/L), the cells were stimulated with TNFα (10 ng/ml, 15 min) as indicated. The extracts were subjected to immunoblotting to examine the activation of NF-κB by monitoring degradation and phosphorylation of IκB. The activation of JNK, p38mapk and S6K1 pathways were assessed by monitoring phosphorylation of their substrate c-jun, CREB and S6, respectively. Shown are representative blots from four independent experiments.
Figure 6
Figure 6
Effects of silencing S6K1 on expression of VCAM-1, ICAM-1, and E-selectin. HUVECs were transduced with recombinant adenovirus expressing shRNA against LacZ as control (lanes 1 and 2) or S6K1 (lanes 3 and 4). On day 4 of post transduction, cells were serum-starved for 20 h followed by stimulation with TNFα (10 ng/ml) for 4 hours and extracted. (A) The knocking-down effects of shRNA targeting S6K1 were assessed by immunoblotting for the expression of S6K1 (panel a) and for phosphorylation of its substrate S6 (panel b). Panels c to e reveal the effects on expression of VCAM-1, ICAM-1, and E-selectin. Shown are representative blots from five independent experiments. (B) Quantification of the signals in (A) (panels c – e). All blots were normalized to tubulin expression. Representative data from five independent experiments are reported as mean ± SEM. Values are given as percentage relative to stimulation with TNFα in the control LacZ-shRNA-transduced cells. ***p < 0.001 vs. stimulation with TNFα in the control LacZ-shRNA-transduced cells.

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