Abstract
BackgroundEndothelin-1 (ET-1) is elevated and participates in the regulation of several brain inflammatory disorders. The deleterious effects of ET-1 on endothelial cells may aggravate brain inflammation mediated through the upregulation of cyclooxygenase-2 (COX-2) gene expression. However, the signaling mechanisms underlying ET-1-induced COX-2 expression in brain microvascular endothelial cells remain unclear.ObjectiveThe goal of this study was to examine whether ET-1-induced COX-2 expression and prostaglandin E2 (PGE2) release were mediated through a c-Src-dependent transactivation of epidermal growth factor receptor (EGFR) pathway in brain microvascular endothelial cells (bEnd.3 cells).MethodsThe expression of COX-2 induced by ET-1 was evaluated by Western blotting and RT-PCR analysis. The COX-2 regulatory signaling pathways were investigated by pretreatment with pharmacological inhibitors, short hairpin RNA (shRNA) or small interfering RNA (siRNA) transfection, chromatin immunoprecipitation (ChIP), and promoter activity reporter assays. Finally, we determined the PGE2 level as a marker of functional activity of COX-2 expression.ResultsFirst, the data showed that ET-1-induced COX-2 expression was mediated through a c-Src-dependent transactivation of EGFR/PI3K/Akt cascade. Next, we demonstrated that ET-1 stimulated activation (phosphorylation) of c-Src/EGFR/Akt/MAPKs (ERK1/2, p38 MAPK, and JNK1/2) and then activated the c-Jun/activator protein 1 (AP-1) via Gq/i protein-coupled ETB receptors. The activated c-Jun/AP-1 bound to its corresponding binding sites within COX-2 promoter, thereby turning on COX-2 gene transcription. Ultimately, upregulation of COX-2 by ET-1 promoted PGE2 biosynthesis and release in bEnd.3 cells.ConclusionsThese results demonstrate 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 upregulation. Understanding the mechanisms of COX-2 expression and PGE2 release regulated by ET-1/ETB system on brain microvascular endothelial cells may provide rational therapeutic interventions for brain injury and inflammatory diseases.
Highlights
Endothelin-1 (ET-1) is elevated and participates in the regulation of several brain inflammatory disorders
Upregulation of COX-2 by ET-1 promoted prostaglandin E2 (PGE2) biosynthesis and release in bEnd.3 cells. These results demonstrate that in bEnd.3 cells, c-Src-dependent transactivation of epidermal growth factor receptor (EGFR)/PI3K/Akt and mitogen-activated protein kinases (MAPKs) linking to c-Jun/activator protein 1 (AP-1) cascade is essential for ET-1-induced COX-2 upregulation
These findings suggested that ET-1 induces COX-2 expression at the transcriptional and translational levels, which is mediated through the ETB receptor-mediated c-Src-dependent transactivation of EGFR and activation of PI3K/Akt, extracellular signal-regulated protein kinase 1/2 (ERK1/2), p38 MAPK, JNK1/2, and c-Jun/AP-1 pathways, leading to PGE2 biosynthesis in mouse bEnd.3 cells
Summary
Endothelin-1 (ET-1) is elevated and participates in the regulation of several brain inflammatory disorders. The deleterious effects of ET-1 on endothelial cells may aggravate brain inflammation mediated through the upregulation of cyclooxygenase-2 (COX-2) gene expression. Cyclooxygenase (COX) is a rate-limiting key enzyme in the synthesis of prostaglandins (PGs) and thromboxane. In this process, phospholipase A2 catalyzes the release of arachidonic acid (AA) from membrane phospholipids, while COX catalyzes the conversion of AA into PGH2, various inflammatory tissues, including synovial macrophage and vascular cells of patients with arthritis and atherosclerosis, respectively. Several lines of evidence have further confirmed COX-2 as a major therapeutic target for the treatment of inflammatory disorders such as arthritis [1]. Upregulation of COX-2 leads to increased production of PGs, which are potent inflammatory mediators associated with neurodegenerative disorders [6]. COX2 and its metabolites PGs may act as a major pathological factor in brain inflammatory diseases
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