Adiponectin inhibits tumor necrosis factor-α-induced vascular inflammatory response via caveolin-mediated ceramidase recruitment and activation - PubMed (original) (raw)

Adiponectin inhibits tumor necrosis factor-α-induced vascular inflammatory response via caveolin-mediated ceramidase recruitment and activation

Yajing Wang et al. Circ Res. 2014.

Abstract

Rationale: Anti-inflammatory and vascular protective actions of adiponectin are well recognized. However, many fundamental questions remain unanswered.

Objective: The current study attempted to identify the adiponectin receptor subtype responsible for adiponectin's vascular protective action and investigate the role of ceramidase activation in adiponectin anti-inflammatory signaling.

Methods and results: Adiponectin significantly reduced tumor necrosis factor (TNF)α-induced intercellular adhesion molecule-1 expression and attenuated TNFα-induced oxidative/nitrative stress in human umbilical vein endothelial cells. These anti-inflammatory actions were virtually abolished by adiponectin receptor 1 (AdipoR1-), but not AdipoR2-, knockdown (KD). Treatment with adiponectin significantly increased neutral ceramidase (nCDase) activity (3.7-fold; P<0.01). AdipoR1-KD markedly reduced globular adiponectin-induced nCDase activation, whereas AdipoR2-KD only slightly reduced. More importantly, small interfering RNA-mediated nCDase-KD markedly blocked the effect of adiponectin on TNFα-induced intercellular adhesion molecule-1 expression. AMP-activated protein kinase-KD failed to block adiponectin-induced nCDase activation and modestly inhibited adiponectin anti-inflammatory effect. In contrast, in caveolin-1 KD (Cav1-KD) cells, >87% of adiponectin-induced nCDase activation was lost. Whereas adiponectin treatment failed to inhibit TNFα-induced intercellular adhesion molecule-1 expression, treatment with sphingosine-1-phosphate or SEW (sphingosine-1-phosphate receptor agonist) remained effective in Cav1-KD cells. AdipoR1 and Cav1 colocalized and coprecipitated in human umbilical vein endothelial cells. Adiponectin treatment did not affect this interaction. There is weak basal Cav1/nCDase interaction, which significantly increased after adiponectin treatment. Knockout of AdipoR1 or Cav1 abolished the inhibitory effect of adiponectin on leukocyte rolling and adhesion in vivo.

Conclusions: These results demonstrate for the first time that adiponectin inhibits TNFα-induced inflammatory response via Cav1-mediated ceramidase recruitment and activation in an AdipoR1-dependent fashion.

Keywords: adipokines; endothelial cells; inflammation; sphingolipids; vascular system injuries.

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Figures

Figure 1

Figure 1. Effect of AdipoR1/AdipoR2 knoc`kdown upon APN’s anti-ICAM-1 effect

HUVEC were transfected with scramble or siRNA against AdipoR1/AdipoR2 to knockdown AdipoR expression (A). 48 hours after transfection, cells were pretreated with vehicle, gAPN or fAPN followed by TNFα treatment. Effect of APN upon TNFα-induced ICAM-1 expression (12 hours post-TNFα treatment) was determined in scramble (B), AdipoR1 siRNA (C) and AdipoR2 siRNA (D) transfected cells. *P<0.05, **P<0.01 vs. TNFα-treated animals without APN treatment.

Figure 2

Figure 2. Effect of AdipoR1/AdipoR2 knockdown upon APN’s anti-oxidative and anti-nitrative effect

Cells were treated as described in Figure 1. Effect of APN upon TNFα-induced superoxide production and nitrotyrosine formation was determined in scramble (A/D), AdipoR1 (B/E) and AdipoR2 (C/F) transfected cells. *<0.05, **P<0.01 vs. TNFα-treated animals without APN treatment.

Figure 3

Figure 3. AdipoR1-dependent nCDase activation mediates APN anti-inflammatory action

(A): Transfection of HUVEC reduced nCDase expression (left panel) and blocked nCDase activation by APN (right panel); (B): Effect of AdipoR1 and AdipoR2 knockdown upon APN-induced nCDase activation; (C): Effect of nCDase knockdown upon the inhibitory action of APN upon TNFα-induced ICAM-1 expression. **P<0.01 vs. Con (A and B). *P<0.05, **P<0.01 vs. TNFα-treated animals without APN treatment (C).

Figure 4

Figure 4. Role of AMPK in APN-induced nCDase activation

Transfection of HUVEC with AMPKα siRNA reduced AMPKα expression (A) and blocked gAPN-induced ACC phosphorylation (B). In contrast, APN-induced nCDase activation was not affected (C) and APN’s anti-TNFα (ICAM-1 expression, D) was only partially inhibited when AMPK expression was genetically inhibited. *P<0.05, **P<0.01 vs. respective control (Con).

Figure 5

Figure 5. Role of Cav1 in APN-induced nCDase activation and anti-inflammatory action

HUVEC Cav1 expression was genetically inhibited by Cav1 siRNA (A, left panel). Cav1 knockdown blocked APN-induced nCDase activation (A, right panel) and anti-inflammatory effects of APN (ICAM-1 expression, B). However, Cav1-KD had no significant effect upon anti-inflammatory effects of S1P and SEW2187 (C). **P<0.01 vs. respective control (A) or vs. TNFα-treated animals without APN treatment (B/C).

Figure 6

Figure 6. Effect of AdipoR1/AdipoR2 or Cav1 knockdown upon cellular levels of ceramide

(A) and S1P (B) after TNFα treatment in the presence and absence of gAPN. Representative photos from at least 5 repeated experiments. C: Cav1/AdipoR1 co-localization determined by immunofluorescent staining. Representative photos from at least 5 repeated experiments; D: Cav1/AdipoR1 interaction determined by co-immunoprecipitation in HUVEC. Cav1 was immunoprecipitated with antibody against Cav1 and samples were immunoblotted with antibody against AdipoR1 (upper panels). To immunoprecipitate AdipoR1, HUVEC were transfected with Myc-tagged AdipoR1 expressing vector and immunoprecipitated with antibody against Myc. Samples were then immunoblotted with antibody against Cav1 (lower panel). E: Cav1/AdipoR1 interaction determined by co-immunoprecipitation in RAEC. MUT=Re-expression in Cav1-KD cells of a mutated Cav1, in which 5 aromatic residues within the scaffolding domain responsible for Cav1 interaction with partner proteins were converted to alanine. F: Cav1/nCDase interaction determined by co-immunoprecipitation in aortic segment from mice treated with vehicle or gAPN. Representative blots from at least 5 repeated experiments; G: APN/Cav1 complex forming (upper panel) and lack of AdipoR2/Cav1 interaction determined by immunoprecipitation (lower panel). Representative blots from at least 5 repeated experiments.

Figure 7

Figure 7. Cellular distribution of nCDase without APN treatment

(A: top panel) and effect of APN treatment upon Cav1 and nCDase interaction in WT, Cav1-KD or AdipoR1KD HUVEC (A: confocal microscopy; B: immunoprecipitation). Note that APN-enhanced Cav1/nCDase interaction is blocked by AdipoR1, but not AdipoR2 knockdown. Cav1/AdipoR1/nCDase complex formation determined by immunoprecipitation (C/D). Representative images from at least 5 repeated experiments/experimental conditions.

Figure 8

Figure 8. Effect of AdipoR1/AdipoR2 knockout upon APN inhibition of TNFα-induced leukocyte rolling and adhesion

WT, AdipoR1KO and AdipoR2KO mice were pre-treated with vehicle or gAPN and leukocyte rolling and adhesion was observed using intravital microscopy following TNFα injection. The inhibitory effect of APN upon TNFα-induced leukocyte rolling (A) and adhesion (B) observed in WT mice was virtually abolished in AdipoR1 but not in AdipoR2 knockout mice. **P<0.01 vs. TNFα-treated animals without APN treatment; #P<0.05, ##P<0.01 vs. WT with the same treatment.

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