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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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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.
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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.

Mentions: Next we investigated whether the anti-inflammatory effects of L-norvaline are dependent on arginase activity. For this purpose, the endothelial cells were either pre-treated with another arginase inhibitor BEC (Fig. 3) or transduced with recombinant adenovirus expressing shRNA targeting arginase-II (Fig. 4), the principle arginase isoform in HUVECs [18]. Our experiments showed that BEC at the concentration of 200 μmol/L exerted inhibitory effects on arginase activity (51.9 ± 7.0% inhibition) which was comparable to that of 20 mmol/L L-norvaline (52.4 ± 3.9% inhibition, n = 4). However, in contrast to L-norvaline, BEC was unable to affect TNFα-induced expression of any of the inflammatory molecules (Fig. 3, n = 5). Similarly, knocking down arginase-II by the two different shRNAs i.e. shRNA-ARG IIa and c (shRNA-ARG IIb had no effect and served as a 2nd control in addition to shRNA-LacZ) also had no effects (Fig. 4, lanes 6 and 8 vs. lanes 5 and 7, n = 5).


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)

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.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2664787&req=5

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.
Mentions: Next we investigated whether the anti-inflammatory effects of L-norvaline are dependent on arginase activity. For this purpose, the endothelial cells were either pre-treated with another arginase inhibitor BEC (Fig. 3) or transduced with recombinant adenovirus expressing shRNA targeting arginase-II (Fig. 4), the principle arginase isoform in HUVECs [18]. Our experiments showed that BEC at the concentration of 200 μmol/L exerted inhibitory effects on arginase activity (51.9 ± 7.0% inhibition) which was comparable to that of 20 mmol/L L-norvaline (52.4 ± 3.9% inhibition, n = 4). However, in contrast to L-norvaline, BEC was unable to affect TNFα-induced expression of any of the inflammatory molecules (Fig. 3, n = 5). Similarly, knocking down arginase-II by the two different shRNAs i.e. shRNA-ARG IIa and c (shRNA-ARG IIb had no effect and served as a 2nd control in addition to shRNA-LacZ) also had no effects (Fig. 4, lanes 6 and 8 vs. lanes 5 and 7, n = 5).

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