Glutathione peroxidase-1 modulates lipopolysaccharide-induced adhesion molecule expression in endothelial cells by altering CD14 expression

Abstract

CD14 contributes to LPS signaling in leukocytes through formation of toll-like receptor 4/CD14 receptor complexes; however, a specific role for endogenous cell-surface CD14 in endothelial cells is unclear. We have found that suppression of glutathione peroxidase-1 (GPx-1) in human microvascular endothelial cells increases CD14 gene expression compared to untreated or siControl (siCtrl)-treated conditions. Following LPS treatment, GPx-1 deficiency augmented LPS-induced intracellular reactive oxygen species accumulation, CD14 expression, and intercellular adhesion molecule-1 (ICAM-1) mRNA and protein expression compared to LPS-treated control cells. GPx-1 deficiency also transiently augmented LPS-induced vascular cell adhesion molecule-1 (VCAM-1) expression. Adenoviral overexpression of GPx-1 significantly diminished LPS-mediated responses in adhesion molecule expression. Consistent with these findings, LPS responses were also greater in endothelial cells derived from GPx-1-knockout mice, whereas adhesion molecule expression was decreased in cells from GPx-1-overexpressing transgenic mice. Knockdown of CD14 attenuated LPS-mediated up-regulation of ICAM-1 and VCAM-1 mRNA and protein, and it mitigated the effects of GPx-1 deficiency on LPS-induced adhesion molecule expression. Taken together, these data suggest that GPx-1 modulates the endothelial cell response to LPS, in part, by altering CD14-mediated effects.

Figure 1.

CD14 gene expression in endothelial cells. HMVECs were transfected with siGPx-1 or siRNA control (siCtrl) for 48 h, and CD14 mRNA was measured by qRT-PCR (n=4). Data are presented as means ± se. Means were significantly different by ANOVA (P<0.0001). *P < 0.0001, #P < 0.03 vs. other groups; post hoc analysis using Fisher’s protected least-square difference (PLSD). UT, untransfected.

Figure 2.

LPS and ICAM-1 and VCAM-1 expression. A) HMVECs were incubated with LPS (1 μg/ml) for 3–24 h, and ICAM-1 and VCAM-1 transcripts were measured by qRT-PCR (n=3). B, C) Proteins were separated on 4–15% SDS-PAGE gels and transferred to HyBond membrane. Antibodies against GPx-1 (MBL), ICAM-1 and VCAM-1 (Santa Cruz), and actin (Sigma) were used to detect the effect of LPS in a time-dependent (n=4) (B), and dose-dependent (n=2) fashion (C). Representative blots are shown. D) After LPS treatment for 3–24 h, CD14 mRNA was measured by qRT-PCR (n=3). Data are presented as means ± se. *P < 0.0001, #P < 0.001 vs. no treatment; Fisher’s PLSD pairwise comparison.

Figure 3.

Knockdown of GPx-1 and LPS-induced ROS accumulation and adhesion molecule expression. A) ROS accumulation was measured by DCF fluorescence 30 min after 1 μg/ml LPS treatment (n=3). ANOVA, P < 0.0001. *P < 0.05; pairwise comparison. B, C) HMVECs were transfected with siGPx-1 or siRNA control (siCtrl) for 24 h and treated with 1 μg/ml LPS for 24 h. CD14 (B) and ICAM-1 and VCAM-1 (C) mRNA was measured by qRT-PCR (n=4). ANOVA, P < 0.05. *P < 0.05; post hoc pairwise analysis. D) Effect of 1 μg/ml LPS on GPx-1, ICAM-1, and VCAM-1 protein expression in GPx-1-deficient cells compared to siControl cells was determined by immunoblotting after 24 h of treatment. Densitometry was performed on 5 blots. Data are presented as means ± se; ANOVA, P < 0.0001. *P < 0.05; post hoc tests. E) Dose-dependent effects of LPS on ICAM-1 and VCAM-1 expression were analyzed by Western blot following 24 h of treatment in GPx-1-deficient and control cells (n=3). F) Time course of LPS-stimulated ICAM-1 and VCAM-1 expression was monitored by Western blot using 1000 and 100 ng/ml LPS (n=2–3). Representative blots are shown.

Figure 4.

Overexpression of GPx-1 and LPS-induced ICAM-1 and VCAM-1 expression. HMVECs were infected with AdGPx-1 or empty vector control (Ad5Bgl II) for 24 h, followed by treatment with 1 μg/ml LPS for 24 h. A) GPx-1 transcript levels were measured by qRT-PCR; GPx-1 protein was assessed by Western blotting. B) ICAM-1 and VCAM-1 transcripts were measured by qRT-PCR (n=3). Data are presented as means ± se; ANOVA, P < 0.0001. *P < 0.0001, #P < 0.05; pairwise comparisons.

Figure 5.

Knockdown of CD14 and LPS-induced adhesion molecule expression. A–C) GPx-1 (A), CD14 (B), and ICAM-1 and VCAM-1 (C) transcripts were measured by qRT-PCR after transfection with siRNA (siCtrl, siGPx-1, siCD14, siGPx-1/siCD14) for 24 h followed by 1 μg/ml LPS treatment for 24 h (n=4). D, E) Western blots were performed to show the effect of 1000 ng/ml LPS (D) and 100 ng/ml LPS (E) on GPx-1, ICAM-1, and VCAM-1 protein expression in GPx-1-deficient and CD14-deficient cells compared to siControl cells. Representative blots are shown. F) HMVECs were transfected with siGPx-1 or siRNA control (siCtrl) for 48 h, and transcripts for TLR-4, MyD88, and MD-2 were measured by qRT-PCR (n=3). Data are presented as means ± se; ANOVA, P < 0.001. *P < 0.0001, #P < 0.01; Fisher’s PLSD test.

Figure 6.

Pulmonary endothelial cells and lung tissue from GPx-1 KO and tgGPx-1 mice and LPS-induced ICAM-1 and VCAM-1 expression. A, B) Pulmonary endothelial cells (passage 4) from WT, GPx-1 KO, and tgGPx-1 mice were incubated with LPS (1 μg/ml) for 24 h. GPx-1 mRNA was measured by qRT-PCR (A); GPx-1, ICAM-1, and VCAM-1 protein were measured by immunoblotting (B) (n=3). C) GPx-1 activity was measured in lung tissue from WT, GPx-1 KO, and tgGPx-1 mice. *P < 0.0001 vs. WT, KO vs. tg.

Figure 7.

GPx-1 modulates LPS response in endothelial cells by CD14 mechanisms. LPS forms a complex with soluble CD14 (sCD14) or membrane-bound CD14 (mCD14). CD14-LPS complex formation facilitates TLR4-mediated activation of endothelial cells, which results in ROS production and adhesion molecule expression. Indirectly, oxidative stress, caused by GPx-1 deficiency, increases CD14 gene expression, augmenting LPS-mediated responses. GPx-1 deficiency also increases adhesion molecule expression, contributing to endothelial cell activation. Overexpression of GPx-1 reduces intracellular ROS, decreasing adhesion molecule expression, CD14 expression, and the cellular response to LPS.