Upregulation of COX-2/PGE2 by ET-1 mediated through Ca2+-dependent signals in mouse brain microvascular endothelial...

Endothelin-1 (ET-1), a proinflammatory mediator, is elevated in the regions of several brain inflammatory disorders, implying that ET-1 may contribute to inflammatory responses. The deleterious effects of ET-1 on brain endothelial cells may aggravate brain inflammation mediated through the upregulation of cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) system. However, the signaling mechanisms underlying ET-1-induced COX-2 expression in mouse brain microvascular endothelial cells (bEnd.3 cells) remain unclear. Herein, we investigated the effects of Ca2+-dependent protein kinases on ET-1-induced COX-2 expression and PGE2 release in bEnd.3 cells. The data obtained with Western blotting, reverse transcription PCR, and intracellular Ca2+ analyses showed that ET-1-induced COX-2 expression was mediated through phosphatidylinositol-phospholipase C (PI-PLC) and phosphatidylcholine-phospholipase C (PC-PLC)/Ca2+-dependent activation of protein kinase C-alpha (PKC-α) and calmodulin kinase II (CaMKII) cascades. Next, we demonstrated that ET-1 stimulated intracellular Ca2+ increase, phoshorylation of PKC-α, CaMKII, and mitogen-activated protein kinases (MAPKs) (ERK1/2, p38 MAPK, and JNK1/2) and then activated the activating transcription factor 2 (ATF2)/activator protein 1 (AP-1) via Gq/i protein-coupled ETB receptors. Moreover, the data of chromatin immunoprecipitation and promoter reporter assay demonstrated that the activated ATF2/AP-1 and p300 bound to its corresponding binding sites within COX-2 promoter, thereby turning on COX-2 gene transcription. Finally, upregulation of COX-2 by ET-1 promoted PGE2 biosynthesis and release in these cells. Taken together, these results demonstrate that in bEnd.3 cells, Ca2+-dependent PKC-α and CaMKII linking to MAPKs, ATF2/AP-1, and p300 cascade is essential for ET-1-induced COX-2 upregulation. Understanding the mechanisms of COX-2/PGE2 system upregulated by ET-1 on brain microvascular endothelial cells may provide rational therapeutic interventions for brain injury and inflammatory diseases.