Several lines of evidence have demonstrated that high levels of PGs, synthesized by inducible COX-2, are involved in inflammatory responses. The up-regulation of COX-2 has been shown to display a wide range of biological activities in different tissues, including development, proliferation, cancers, and inflammation [14, 15]. Moreover, ET-1 is elevated in the regions of vascular injuries and inflammation [7, 8]. Circumstantial evidence has further demonstrated that overexpression of ET-1 on endothelial cells has deleterious effects on ischemic brain [1, 5, 6]. Reid et al. (1995) suggest that the ET-1 model provides new insights into the mechanisms of cerebral ischemia and reperfusion injury, and evaluates the usefulness of novel strategies of neuroprotection . ET-1 has been shown to up-regulate the expression of COX-2 through MAPKs in various cell types [26, 27, 34]. However, little is known about the effect of ET-1 on COX-2 expression in brain vascular endothelial cells. Here, we applied cultured models of mouse bEnd.3 cells coupled with Western blot analysis, selective pharmacological inhibitors, transfection with siRNAs, immunofluorescenct staining, and promoter assay to investigate the molecular mechanisms underlying ET-1-induced COX-2 expression and PGE2 release. Our results demonstrate that in bEnd.3 cells, activation of ETB receptor-dependent MAPKs (ERK1/2, p38, and JNK1/2) and NF-κB signaling cascade is essential for ET-1-induced COX-2 gene expression and PGE2 release.
ET-1 activates ET receptor subtypes (ETA and ETB) which are coupled to various G proteins such as Gq and Gi and then lead to multiple signaling pathways and regulate diverse cellular functions [7, 20–22]. Thus, we first demonstrated a significant expression of ETB receptor in mouse bEnd.3 cells (Figure 2A). The involvement of ETB receptors in these responses is confirmed by that pretreatment with BQ-788 (an ETB receptor antagonist) reduced the ET-1-induced COX-2 protein and mRNA expression (Figure 2B and 2C), promoter activity (Figure 6D), and PGE2 release (Figure 6F), but not by an ETA receptor antagonist BQ-123. Subsequently, we confirmed these results by transfection with ETB siRNA (Figure 2D), suggesting that ETB receptor predominantly mediates ET-1-induced COX-2 expression and PGE2 release in bEnd.3 cells. Next, several subtypes of G proteins are potentially implicated in ET-1-induced COX-2 expression. We use GPA2 (a Gi protein antagonist) and GPA2A (a Gq protein antagonist) to interrupt G protein signaling and consequent COX-2 expression (Figure 3A). Moreover, the inhibitory effects of GPA2 and GPA2A on COX-2 induction by ET-1 were also observed in its mRNA (Figure 3B), promoter activity (Figure 6D), and PGE2 release (Figure 6F), indicating that ET-1-induced COX-2 expression and PGE2 release is mediated through a GPCR (i.e. ETB) coupling to either Gi or Gq protein in bEnd.3 cells, consistent with previous studies from esophageal smooth muscle cells  and rat brain astrocytes . In contrast, previous reports have shown that ET-1 induces COX-2 expression via ETA receptors in peripheral lung microvascular smooth muscle cells  and ET-1 (ETA) receptors linked to phospholipase C and phospholipase A2 activation and prostanoid secretion (e.g. PGE2) in cultured human brain microvascular endothelial cells [35, 36]. However, in respiratory and cardiovascular systems, both ET receptor subtypes, ETA in particular, are involved in progression of several diseases [37, 38]. There differences may be due to cell type specific or different experimental conditions.
Abnormal MAPK regulation might be implicated in several models of CNS injury and inflammation . Several lines of evidence demonstrate that MAPKs could be activated by GPCR agonists through different signaling pathways . MAPKs activation by ET-1 has been shown to modulate various cellular responses in several cell types [22, 25]. Activation of ERK1/2 (p44/p42 MAPK) might be implicated in the expression of inflammatory genes in several models of vascular injury and inflammation [17, 28]. In this study, we demonstrated that ET-1 stimulated an ETB receptor-dependent cascade of sequential ERK1/2 phosphorylation (Figure 4E), which contributes to induction of COX-2 protein and mRNA levels (Figure 4A and 4B), promoter activity (Figure 6D), and PGE2 release (Figure 6F). The involvement of ERK1/2 in COX-2 expression and PGE2 release was furthe confirmed by transfection of cells with p42 siRNA (Figure 4D). These results are consistent with those of obtained with COX-2 expression induced by BK, thrombin, or ET-1 in various cell types [17, 26, 28]. Additionally, we found that expression of COX-2 and release of PGE2 induced by ET-1 were also attenuated by the inhibitor of p38 MAPK or JNK1/2. Pretreatment with SB202190 or SP600125 both markedly reduced ET-1-induced expression of COX-2 protein and mRNA (Figure 4A and 4B), promoter activity (Figure 6D), and PGE2 release (Figure 6F). Moreover, we also demonstrated that ET-1 stimulates phosphorylation of p38 MAPK and JNK via an ETB-dependent manner (Figure 4C and 4E). Similarly, we further confirmed these results by transfection with siRNA for p38 MAPK or JNK1 that attenuated ET-1-induced COX-2 expression (Figure 4D). These data clearly indicated that in bEnd.3 cells, three MAPK cascades (i.e. ERK1/2, p38 MAPK, and JNK1/2) are required for ET-1-induced COX-2 expression and PGE2 release. These results are consistent with those of obtained with up-regulation of COX-2 by ET-1 via p38 MAPK in glomerular mesangial cells or esophageal smooth muscle cells [27, 34]. For the role of JNK1/2, we are the first presented that JNK1/2 plays a critical role in induction of COX-2 by ET-1 in endothelial (bEnd.3) cells.
It has been well established that inflammatory responses following exposure to extracellular stimuli are highly dependent on activation of NF-κB transcription factor, which plays an important role in regulation of several gene expression . The 5’-flanking region of the COX-2 promoter has been shown to contain several binding sequences for various transcription factors including NF-κB . Therefore, the regulation of COX-2 transcription may be mediated by aberrant activation of several distinct transcription factors dependent on agonists [29, 42]. These reports suggest that NF-κB plays a critical role in the regulation of COX-2 expression in the development of the inflammatory responses. Our data showed that ET-1-induced COX-2 gene expression and PGE2 release was significantly abolished by a selective NF-κB inhibitor Bay11-7082 (Figures 5 and 6) or NF-κB p65 siRNA (Figures 5E and 6F), suggesting that NF-κB (p65) is involved in ET-1-induced COX-2 expression in bEnd.3 cells. Moreover, ET-1-stimulated NF-κB p65 translocation (Figure 5C), binding to COX-2 promoter region (Figure 6C and 6E), and NF-κB transcriptional activity (Figure 6A) was significantly inhibited by Bay11-7082 and the MAPK inhibitor U0126 (MEK1/2), SB202190 (p38 MAPK), or SP600125 (JNK1/2) (Figures 5 and 6). Our data further showed that ET-1-stimulated NF-κB transcriptional activity was significantly attenuated by blocking Gi and Gq protein-coupled ETB receptor-dependent pathways (Figure 6B), indicating that ET-1-induced activation of NF-κB is mediated through ETB receptor-dependent activation of three MAPKs cascades. These findings are consistent with recent studies indicating that COX-2 expression and prostacyclin release induced by thrombin were mediated through MAPKs and NF-κB activation in endothelial cells  and vascular smooth muscle cells  and COX-2 expression and PGE2 release induced by BK via ERK1/2 linking to NF-κB activation in astrocytes . The involvement of NF-κB in ET-1-induced COX-2 expression is also consistent with previous reports indicating that ET-1-stimulated activation of NF-κB regulates expression of target genes involved in various CNS inflammatory processes . Moreover, our recent data have also demonstrated that in bEnd.3 cells, c-Src-dependent transactivation of EGFR/PI3K/Akt and MAPKs linking to c-Jun/AP-1 cascade is essential for ET-1-induced COX-2/PGE2 upregulation . We suggest that the findings of these two studies might have a crosstalk in MAPKs and lead to COX-2 expression induced by ET-1 in these cells. The interplay between these two pathways in the induction of COX-2 will be investigated in the future.