Reduced expression of adipose triglyceride lipase enhances tumor necrosis factor alpha-induced intercellular adhesion molecule-1 expression in human aortic endothelial cells via protein kinase C-dependent activation of nuclear factor-kappaB

Abstract

We examined the effects of adipose triglyceride lipase (ATGL) on the initiation of atherosclerosis. ATGL was recently identified as a rate-limiting triglyceride (TG) lipase. Mutations in the human ATGL gene are associated with neutral lipid storage disease with myopathy, a rare genetic disease characterized by excessive accumulation of TG in multiple tissues. The cardiac phenotype, known as triglyceride deposit cardiomyovasculopathy, shows massive TG accumulation in both coronary atherosclerotic lesions and the myocardium. Recent reports show that myocardial triglyceride content is significantly higher in patients with prediabetes or diabetes and that ATGL expression is decreased in the obese insulin-resistant state. Therefore, we investigated the effect of decreased ATGL activity on the development of atherosclerosis using human aortic endothelial cells. We found that ATGL knockdown enhanced monocyte adhesion via increased expression of TNFα-induced intercellular adhesion molecule-1 (ICAM-1). Next, we determined the pathways (MAPK, PKC, or NFκB) involved in ICAM-1 up-regulation induced by ATGL knockdown. Both phosphorylation of PKC and degradation of IκBα were increased in ATGL knockdown human aortic endothelial cells. In addition, intracellular diacylglycerol levels and free fatty acid uptake via CD36 were significantly increased in these cells. Inhibition of the PKC pathway using calphostin C and GF109203X suppressed TNFα-induced ICAM-1 expression. In conclusion, we showed that ATGL knockdown increased monocyte adhesion to the endothelium through enhanced TNFα-induced ICAM-1 expression via activation of NFκB and PKC. These results suggest that reduced ATGL expression may influence the atherogenic process in neutral lipid storage diseases and in the insulin-resistant state.

FIGURE 1.

RNAi-induced ablation of ATGL in HAECs. HAECs were transfected with double-stranded ATGL-specific siRNA at a final concentration of 50 nm and cultured for 24–72 h at 37 °C. Control HAECs were incubated with the siRNA-negative universal control. A, ATGL and β-actin mRNA were evaluated using real-time RT-PCR. Bars, mean ± S.E. (error bars) (n = 10); *, p < 0.0001 versus control. B, Western blots (after SDS-PAGE separation of 30 μg of HAEC cell protein) were developed using anti-ATGL or anti-β-actin antibodies. Bars, mean ± S.E. (error bars) (n = 5). *, p < 0.001 versus control. Open bars, control siRNA; closed bars, ATGL siRNA.

FIGURE 2.

Knockdown of ATGL increased TNFα-induced ICAM-1 expression in HAECs. HAECs were transfected with either control or ATGL siRNA for 6 h and then treated with or without 1 ng/ml TNFα for 18 h. A, the levels of ICAM-1 and β-actin mRNA were evaluated using real-time RT-PCR. Bars, mean ± S.E. (error bars) (n = 10); *, p < 0.05 versus control. B and C, Western blots (after SDS-PAGE separation of 30 μg of HAEC cell protein) were developed using anti-ICAM-1 or anti-β-actin antibodies. Bars, mean ± S.E. (error bars) (n = 5). *, p < 0.05 versus control. Open bars, control siRNA; closed bars, ATGL siRNA.

FIGURE 3.

Effects of ATGL knockdown on U937 cell adhesion to TNFα-stimulated HAECs. HAECs were transfected with control or ATGL siRNA for 6 h and then incubated with or without TNFα (1 ng/ml) for 18 h. U937 cells were incubated on each monolayer for 15 min. The number of adherent U937 cells within a high power field (0.01 mm²/field) was counted. Photographs represent randomly chosen fields from three separate experiments. Data represent the mean ± S.E. of five different fields.

FIGURE 4.

Effects of ATGL knockdown on TNFα signaling molecules in HAECs. HAECs were transfected with either control or ATGL siRNA and then treated with TNFα (1 ng/ml) for the indicated times. Western blots (after SDS-PAGE separation of 30 μg of HAEC cell protein) were developed using anti-IκBα (A), anti-NFκB p65 (B), or anti-β-actin antibodies. Bars, mean ± S.E. (error bars) (n = 3). The bar graphs represent the percentage of the maximum from three independent experiments. *, p < 0.05; **, p < 0.005 versus control. Open bars, control siRNA; closed bars, ATGL siRNA.

FIGURE 5.

Phosphorylated forms of Akt, JNK kinase, p38 kinase, or PKC induced by TNFα (1 ng/ml) in ATGL knockdown HAECs. HAECs were transfected with either control or ATGL siRNA and then treated with TNFα (1 ng/ml) for the indicated times. Western blots (after SDS-PAGE separation of 20 μg of HAEC cell protein) were developed using anti-phospho-Akt (A), anti-phospho-JNK (B), anti-phospho-p38 (C), anti-phospho-PKC (D), or anti-β-actin antibodies. Bars, mean ± S.E. (error bars) (n = 4). The bar graphs represent the percentage of the maximum from four independent experiments. *, p < 0.05 versus control. Open bars, control siRNA; closed bars, ATGL siRNA.

FIGURE 6.

Inhibition of PKC prevented TNFα-induced ICAM-1 up-regulation in ATGL knockdown HAECs. HAECs were transfected with either control or ATGL siRNA for 6 h and then treated with or without the indicated inhibitors (0.10 μm calphostin C, 10 μm GF109203X) for 30 min followed by incubation with 1 ng/ml TNFα for 17.5 h. Western blots (after SDS-PAGE separation of 30 μg of HAEC cell protein) were developed using anti-ICAM-1 or anti-β-actin antibodies. Bars, mean ± S.E. (n = 6). *, p < 0.05 versus control. Open bars, control siRNA; closed bars, ATGL siRNA; hatched bars, PKC inhibitors.

FIGURE 7.

Knockdown of ATGL increased the diglyceride content of HAECs. HAECs were transfected with either control or ATGL siRNA. Total DAG levels and those of the individual classes of DAG were determined using high performance liquid chromatography-tandem mass spectrometry. Bars, mean ± S.E. (error bars) (n = 5). *, p < 0.01; **, p < 0.005 versus control. Open bars, control siRNA; closed bars, ATGL siRNA.

FIGURE 8.

Knockdown of ATGL increased [³H]palmitate incorporation into both cells and intracellular DAG. HAECs were transfected with either control or ATGL siRNA for 6 h and then incubated for 18 h with medium containing 2% FBS and [³H]palmitate (20 μCi/ml). Total lipids (A) were extracted, DAGs (B) were separated by thin layer chromatography, and the radioactivity was counted. Bars, mean ± S.E. (n = 5). *, p < 0.05 versus control. Open bars, control siRNA; closed bars, ATGL siRNA.

FIGURE 9.

Knockdown of ATGL increased CD36 expression in HAECs. HAECs were transfected with either control or ATGL siRNA. A, CD36 and β-actin mRNA levels were evaluated using real-time RT-PCR. Bars, mean ± S.E. (n = 8); *, p < 0.0001 versus control. B, Western blots (after SDS-PAGE separation of 30 μg of HAEC cell protein) were developed using anti-CD36 or anti-β-actin antibodies. Bars, mean ± S.E. (n = 5). *, p < 0.0001 versus control. Open bars, control siRNA; closed bars, ATGL siRNA.

FIGURE 10.

Knockdown of ATGL increased PPARγ mRNA expression in HAECs. HAECs were transfected with either control or ATGL siRNA. PPARγ and β-actin mRNA levels were evaluated using real-time RT-PCR. Bars, mean ± S.E. (error bars) (n = 6). *, p < 0.0001 versus control. Open bars, control siRNA; closed bars, ATGL siRNA.

FIGURE 11.

Knockdown of ATGL did not affect HSL mRNA expression and phosphoprotein expression in HAECs. HAECs were transfected with either control or ATGL siRNA. A, HSL and β-actin mRNA levels were evaluated using real-time RT-PCR. Bars, mean ± S.E. (n = 6). B, Western blots (after SDS-PAGE separation of 30 μg of HAEC cell protein) were developed using anti-phospho-HSL or anti-β-actin antibodies. Bars, mean ± S.E. (n = 5). Open bars, control siRNA; closed bars, ATGL siRNA.