Figure 1.
CO treatment enhances SUMOylation of PPARγ.
(A) 293FT cells were transfected in 10 cm dishes with mixtures of expression plasmids, consisting of 2.5 µg HA-PPARγ, 5.5 µg myc-SUMO-1, and 2.0 µg of PIAS1 as indicated. 24 hours after transfection, cells were incubated for 12–18 hours with Rosiglitazone and/or CO (250 ppm) followed by immunoprecipitation of PPARγ (left panel) or SUMO-1 (right panel) from cell lysates and immunoblotting for HA-tag. The western blots are representative of three independent experiments. (B) 293FT cells transfected with the expression plasmids for HA-PPARγ, myc-SUMO-1 and PIAS1 were incubated for 12–18 hours with or without CO (250 ppm) in the presence or absence of N-Acetylcysteine (10 mM). SUMOylated PPARγ was calculated as the ratio of SUMOylated PPARγ versus total PPARγ form pull-down assays. Shown are mean +/− SEM densitometry values of three independent experiments.
Figure 2.
CO requires SUMOylated PPARγ to suppress proinflammatory gene expression.
(A) RAW 264.7 cells were transiently transfected with pcDNA3.1 along with a firefly luciferase reporter construct under the control of the murine Egr-1 promoter (pGL3-Egr1) and pRL-TK as internal control followed by treatment with LPS (10 ng/ml) in the absence or presence of CO (250 ppm). Note that LPS provoked a strong and robust Egr-1 luciferase signal which was inhibited in the presence of CO. (B) RAW 264.7 cells were transiently transfected with a SUMOylation-defective PPARγ mutants (PPARγ-K365R or PPARγ-K77R) or pCDNA3.1 control vector followed by treatment with LPS in the absence or presence of CO (250 ppm). Total proteins were harvested at 6 hr post induction and dual luciferase activities determined. The value of firefly luciferase was normalized by the rennila luciferase to generate the relative luciferase activity. (C) RAW 264.7 cells were transiently transfected with a SUMOylation-defective PPARγ mutant (PPARγ-K365R) or pCDNA3.1 control vector ( = ctrl) followed by treatment with LPS in the absence or presence of CO (250 ppm). RNA was extracted and iNOS mRNA levels (normalized to the Rplp0 gene) measured by real-time PCR. Bars represent mean values ± SEM of three independent experiments.
Figure 3.
CO-mediated PPARγ-SUMOylation and p38MAPK activity cooperate for full suppression of LPS-induced Egr-1 promoter activity.
(A) RAW 264.7 cells were pretreated with 250 ppm CO or Air for 3 hours, followed by a further 30 min incubation with MAPK inhibitors for p38 (SB203580) or ERK1/2 (U0126) prior to stimulation with LPS (10 ng/ml) for 1 hour. Egr-1 protein expression was analyzed by western blotting and β-actin was used as a loading control. The western blot is representative of three independent experiments. (B) RAW 264.7 cells were transiently transfected with PPARγ-K365R or pcDNA3.1 control vector along with pGL3-Egr1 and phRL-Tk. Thereafter, RAW 264.7 cells were pretreated with 250 ppm CO or Air for 3 hours, followed by a further 30 min incubation with SB203580 or vehicle (DMSO) prior to stimulation with LPS (10 ng/ml). Total proteins were harvested at 6 hr postinduction and dual luciferase activities determined. The value of firefly luciferase was normalized by the rennila luciferase to generate the relative luciferase activity. Bars represent mean values ± SEM of three independent experiments (*P<0.05).
Figure 4.
CO increases ROS levels via UCP2 to inhibit Egr-1 expression.
(A) Bone-marrow derived macrophages from wild-type (open bars) or Ucp2−/− (filled bars) mice were treated with CO (250 ppm) and ROS formation was measured with MitoSOX at indicated times by FACS analysis. MitoSOX is specific for mitochondria-derived ROS. Left, the basal effect of UCP2 loss was compared to control cells. Right, we show the effect of CO on ROS formation in Ucp2−/− and wild-type macrophages. (C) Bone-marrow derived macrophages from wild-type or Ucp2−/− mice were pretreated with 250 ppm CO or Air for 3 hours, prior to incubation with LPS (10 ng/ml) for indicated times. Egr-1 protein expression was analyzed by western blotting and β-actin was used as a loading control. (D) The bar graph represents densitometry analysis of western blots after 1 hr LPS stimulation in the presence or absence of CO. Depicted western blot is one of three representative experiments. Bar graphs indicate mean fold change ± SEM of three independent experiments (*P<0.05 and **P<0.001).
Figure 5.
Schematic diagram describing the effects of CO on LPS-activated macrophages.
Exposure of macrophages to CO leads to an increase in mitochondria-derived reactive oxygen species (ROS), which in turn drives SUMOylation of PPARγ and activation of p38 mitogen-activated protein kinase. The activation of PPARγ and p38 both contribute in part to modulation of the inflammatory response.