Up-regulation of COX-2/PGE2 by endothelin-1 via MAPK-dependent NF-κB pathway in mouse brain microvascular endothelial cells

Abstract

Background: Endothelin-1 (ET-1) is a proinflammatory mediator and elevated in the regions of several brain injury and inflammatory diseases. The deleterious effects of ET-1 on endothelial cells may aggravate brain inflammation mediated through the regulation of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) system in various cell types. However, the signaling mechanisms underlying ET-1-induced COX-2 expression in brain microvascular endothelial cells remain unclear. Herein we investigated the effects of ET-1 in COX-2 regulation in mouse brain microvascular endothelial (bEnd.3) cells.

Results: The data obtained with Western blotting, RT-PCR, and immunofluorescent staining analyses showed that ET-1-induced COX-2 expression was mediated through an ETB-dependent transcriptional activation. Engagement of Gi- and Gq-protein-coupled ETB receptors by ET-1 led to phosphorylation of ERK1/2, p38 MAPK, and JNK1/2 and then activated transcription factor NF-κB. Moreover, the data of chromatin immunoprecipitation (ChIP) and promoter reporter assay demonstrated that the activated NF-κB was translocated into nucleus and bound to its corresponding binding sites in COX-2 promoter, thereby turning on COX-2 gene transcription. Finally, up-regulation of COX-2 by ET-1 promoted PGE2 release in these cells.

Conclusions: These results suggested that in mouse bEnd.3 cells, activation of NF-κB by ETB-dependent MAPK cascades is essential for ET-1-induced up-regulation of COX-2/PGE2 system. Understanding the mechanisms of COX-2 expression and PGE2 release regulated by ET-1/ETB system on brain microvascular endothelial cells may provide rationally therapeutic interventions for brain injury or inflammatory diseases.

Figure 1

ET-1 induced COX-2 expression and PGE₂ release. (A) Time and concentration dependence of ET-1-induced COX-2 expression, cells were treated with various concentration ET-1 for the indicated time intervals. (B) Time dependence of ET-1-induced COX-2 mRNA expression, cells were treated with 10 nM ET-1 for the indicated time intervals. COX-2 mRNA was analyzed by RT-PCR. (C) Time dependence of ET-1-induced COX-2 promoter transcription activity, cells were transfected a COX-2 promoter–luciferase reporter gene and then incubated with ET-1 for the indicated times. The promoter reporter assay was performed as described in “Materials and Methods”. (D) PGE₂ release induced by ET-1, the conditioned media were collected to assay PGE₂ level by EIA as described in “Materials and Methods” (n=3 in each group; ^*P<0.05, ^#P<0.01 compared with vehicle).

Figure 2

Involvement of ET_B receptors in ET-1-induced COX-2 expression. (A) RT-PCR analysis of ET receptor expression in bEnd.3 cells. Lane 1: maker. Lane 2: ET_A primers and bEnd.3 RNA. Lane 3: ET_B primers and bEnd.3 RNA. (B, C) Cells were pretreated with BQ-123 or BQ-788 for 1 h and then incubated with ET-1 for (B) 6 h and (C) 1 h. (D) Cells were transfected with siRNA for ET_B receptor for 24 h and then exposed to ET-1 for 6 h. The (B, D) COX-2 protein and (C) mRNA were analyzed by Western blot and RT-PCR, respectively as described in Figure 1. Data are expressed as mean ± SEM of three individual experiments (n=3 in each group, ^#P<0.01 as compared with cells stimulated by ET-1 alone).

Figure 3

ET-1 induces COX-2 expression via a G_i and G_q proteins-coupled ET_B receptor. (A, B) Cells were pretreated with G_i antagonist (GPA2) or G_q antagonist (GPA2A) for 1 h and then exposed to ET-1 for (A) 6 h and (B) 1 h. (C, D) Cells were transfected with siRNA for (C) G_iα or (D) G_qα protein for 24 h and then exposed to ET-1 for 6 h. The (A, C, D) COX-2 protein and (B) mRNA were analyzed by Western blot and RT-PCR, respectively as described in Figure 1. Data are expressed as mean ± SEM of three individual experiments (n=3 in each group, ^#P<0.01 as compared with ET-1 alone).

Figure 4

ET-1-induced COX-2 expression is mediated through MAPKs phosphorylation. (A, B) Cells were treated with 10 nM ET-1 for (A) 6 h and (B) 1 h in the absence or presence of U0126, SB202190, or SP600125. The COX-2 protein and mRNA expression were determined by Western blot and RT-PCR. (C) Time dependence of ET-1-stimulated ERK1/2, p38 MAPK, and JNK1/2 phosphorylation, cells were incubated with 10 nM ET-1 for the indicated times in the absence or presence of U0126 (1 μM), SB202190 (300 nM), or SP600125 (300 nM). (D) Cells were transfected with siRNA of ERK2, p38 MAPK, or JNK1 and then exposed to ET-1 for 6 h. (E) Cells were pretreated with BQ-788 (1 μM), GPA2 (1 μM), or GPA2A (1 μM) for 1 h and then incubated with ET-1 (10 nM) for the indicated times. The cell lysates were collected and analyzed by Western blotting using an anti-COX-2, anti-phospho-ERK1/2, anti-phospho-p38 MAPK, anti-phospho-JNK1/2, anti-ERK2, anti-p38 MAPK, anti-JNK1, or anti-GAPDH (as an internal control) antibody. Data are expressed as mean ± SEM of at least three individual experiments (n=3 in each group; ^*P<0.05, ^#P<0.01 as compared with ET-1 alone).

Figure 5

NF-κB (p65) is essential for ET1-1-induced COX-2 expression. (A, B) Cells were treated with 10 nM ET-1 for 6 h in the absence or presence of Bay11-7082. The COX-2 protein and mRNA expression were determined by Western blot and RT-PCR as described in Figure 1. (C) Time dependence of ET-1-stimulated p65 NF-κB translocation by subcellular isolation, Western blot, and immunofluorescent stain. (D) Cells were pretreated with U0126 (1 μM), SB202190 (300 nM), SP600125 (300 nM), BQ-788 (1 μM), or Bay11-7082 (10 nM) for 1 h and then incubated with ET-1 (10 nM) for 90 min. The nuclear fraction was analyzed by Western blot. (E) Cells were transfected with p65 siRNA and then exposed to ET-1 for 6 h. The cell lysates were collected and analyzed by Western blotting using an anti-COX-2, anti-p65, anti-Lamin A, or anti-GAPDH (as an internal control) antibody. Data are expressed as mean ± SEM of at least three individual experiments (n=3 in each group; ^#P<0.01 as compared with ET-1 alone).

Figure 6

ET-1-stimulated COX-2 promoter activity is mediated through NF-κB-dependent pathway. (A) Time dependence of ET-1-enhanced NF-κB transcription activity, cells were transfected with a NF-κB-luciferase reporter gene and then exposed to ET-1 for the indicated times. (B) After transfection, the cells were pretreated with BQ-788 (1 μM), GPA2 (1 μM), GPA2A (1 μM), U0126 (1 μM), SB202190 (300 nM), SP600125 (300 nM), or (A) Bay11-7082 (10 nM) for 1 h and then incubated with ET-1 (10 nM) for 60 min. (C) Cells were pretreated with BQ-788, U0126, SB202190, or SP600125 for 1 h and then incubated with ET-1. The p65 NF-κB binding activity was analyzed by ChIP-PCR as described in Methods. (D) For COX-2 promoter activity, cells were transfected with a COX-2-promoter-luciferase reporter gene and then exposed to ET-1. After transfection, the cells were pretreated with BQ-788, GPA2, GPA2A, U0126, SB202190, SP600125, or Bay11-7082 for 1 h and then incubated with ET-1 for 6 h. (E) Schematic representation of a 5′-promoter regions of the mouse different COX-2 promoter constructs, either wild-type (WT) or mutation of the κB-binding site (mt-κB) fused to the pGL-luciferase reporter gene, the translational start site (+1) of the luciferase reporter gene was indicated by an arrow. Cells were transfected with WT COX-2 promoter reporter gene (WT-COX-2) or NF-κB mutated COX-2 promoter reporter gene (mt-κB-COX-2) and then incubated with or without ET-1 for 6 h. The promoter reporter assay was performed as described in Methods. (F) Cells were pretreated with BQ-123, BQ-788, GPA2, GPA2A, U0126, SB202190, SP600125, Bay11-7082, or transfected with p65 siRNA and then incubated with ET-1 for 6 h. The PGE₂ levels were analyzed by EIA. Data are expressed as mean ± SEM of at least three individual experiments (n=3 in each group; ^#P<0.01 as compared with ET-1 alone).