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Inhibition of S6K1 accounts partially for the anti-inflammatory effects of the arginase inhibitor L-norvaline.

Ming XF, Rajapakse AG, Carvas JM, Ruffieux J, Yang Z - BMC Cardiovasc Disord (2009)

Bottom Line: However, inhibition of arginase by another arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC) had no effects.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.The arginase inhibitor L-norvaline exhibits anti-inflammatory effects independently of inhibition of arginase in human endothelial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Vascular Biology, Department of Medicine, Division of Physiology, University of Fribourg, Switzerland. xiu-fen.ming@unifr.ch

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.

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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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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.

Mentions: The role of S6K1 in up-regulation of VCAM-1, ICAM-1, and E-selectin was then further investigated by recombinant adenovirus-mediated RNA interference (RNAi). In parallel to the abrogated expression of S6K1 (Fig. 6A, panel a), cells transduced with the rAd-shRNA against S6K1 exhibited significantly decreased kinase activity upon stimulation with TNFα (10 ng/ml, 4 hours), as measured by phosphorylation of its substrate S6 at serine235/serine236 (Fig. 6A, panel b). In the cells transduced with control viral vector expressing LacZ-shRNA, TNFα (10 ng/ml, 4 hours) induced up-regulation of VCAM-1, ICAM-1, and E-selectin (Fig. 6A, panels c to e, lane 2; n = 5, p < 0.001 vs. LacZ alone lane 1), silencing S6K1 significantly reduced TNFα-induced up-regulation of E-selectin, but not that of VCAM-1 and ICAM-1 (Fig. 6B, lane 4; n = 5, p < 0.001 vs. LacZ + TNFα, lane 2).


Inhibition of S6K1 accounts partially for the anti-inflammatory effects of the arginase inhibitor L-norvaline.

Ming XF, Rajapakse AG, Carvas JM, Ruffieux J, Yang Z - BMC Cardiovasc Disord (2009)

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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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.
Mentions: The role of S6K1 in up-regulation of VCAM-1, ICAM-1, and E-selectin was then further investigated by recombinant adenovirus-mediated RNA interference (RNAi). In parallel to the abrogated expression of S6K1 (Fig. 6A, panel a), cells transduced with the rAd-shRNA against S6K1 exhibited significantly decreased kinase activity upon stimulation with TNFα (10 ng/ml, 4 hours), as measured by phosphorylation of its substrate S6 at serine235/serine236 (Fig. 6A, panel b). In the cells transduced with control viral vector expressing LacZ-shRNA, TNFα (10 ng/ml, 4 hours) induced up-regulation of VCAM-1, ICAM-1, and E-selectin (Fig. 6A, panels c to e, lane 2; n = 5, p < 0.001 vs. LacZ alone lane 1), silencing S6K1 significantly reduced TNFα-induced up-regulation of E-selectin, but not that of VCAM-1 and ICAM-1 (Fig. 6B, lane 4; n = 5, p < 0.001 vs. LacZ + TNFα, lane 2).

Bottom Line: However, inhibition of arginase by another arginase inhibitor S-(2-boronoethyl)-L-cysteine (BEC) had no effects.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.The arginase inhibitor L-norvaline exhibits anti-inflammatory effects independently of inhibition of arginase in human endothelial cells.

View Article: PubMed Central - HTML - PubMed

Affiliation: Vascular Biology, Department of Medicine, Division of Physiology, University of Fribourg, Switzerland. xiu-fen.ming@unifr.ch

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.

Show MeSH
Related in: MedlinePlus