Signal transduction pathways regulating cyclooxygenase‐2 in lipopolysaccharide‐activated primary rat microglia

Abstract

Microglia are the major cell type involved in neuroinflammatory events in brain diseases such as encephalitis, stroke, and neurodegenerative disorders, and contribute significantly to the release of prostaglandins (PGs) during neuronal insults. In this report, we studied the immediate-early intracellular signalling pathways in microglia, following bacterial lipopolysaccharide (LPS) stimulation, leading to the synthesis and release of PGE2. Here we show that LPS induces cyclooxygenase (COX) 2 by activating sphingomyelinases leading to the release of ceramides, which in turn, activate the p38 mitogen-activated protein kinases (MAPK), but not the p42/44 MAPK. We further show that exogenously added ceramide analogue (C2-ceramide) also induce PGE2 synthesis through a p38 MAPK-dependent pathway. This potential nature of ceramides in activating microglia suggests that endogenously produced ceramides during neuronal apoptosis in ischemia or neurodegenerative diseases could also contribute to the amplification of neuroinflammatory events. In contrast to protein kinase C (PKC) and phosphocholine-specific phospholipase C (PC-PLC), which transcriptionally regulate LPS-induced COX-2 synthesis, inhibition of phospholipase A2 (PLA2) has no effect on COX-2 transcription, although it inhibits the release of PGE2. Transcriptional regulation of LPS-induced COX-2 by PKC is further proved by the ability of the PKC inhibitor, Gö 6976, to inhibit LPS-induced 8-isoprostane synthesis, but not affecting LPS-induced COX-2 activity. Our data with 8-isoprostane also indicates that COX-2 plays a major role in ROS production in LPS-activated microglia. This detailed view of the intracellular signaling pathway in microglial activation and COX-2 expression opens a new therapeutic window in the search for new and more effective central anti-inflammatory agents.

Z-ligustilide attenuates lipopolysaccharide-induced proinflammatory response via inhibiting NF-κB pathway in primary rat microglia

“…Upon stimulation with LPS, IκB is phosphorylated and degraded through proteosome-mediated pathways. The dissociated NF-κB p65 subunit then translocates into the nucleus and triggers the transcription of multiple genes, which leads to the production and release of proinflammatory cytokines (eg, TNF-α, IL-1β, and MCP-1), the induction of COX-2 and iNOS expression resulting in the biosynthesis and release of prostaglandins (PGs) and NO, and activation of NADPH oxidase-generating superoxide anions that combine with NO from iNOS to form the more damaging peroxynitrite (ONOO-) free radical [10,[22][23][24] . The collective insult by microglia-released cytokines, reactive oxygen species, and lipid metabolites eventually leads to neurotoxicity.…”

“…Primary microglial culture and treatment Primary mixed glial cultures were prepared from postnatal day 1 Sprague-Dawley rat pups as previously described [10] . Briefly, their cerebral cortices were aseptically excised and carefully stripped of their blood vessels and meninges in cold D-Hank's solution.…”

Iptakalim Alleviates Rotenone-Induced Degeneration of Dopaminergic Neurons through Inhibiting Microglia-Mediated Neuroinflammation

“…Mitogen-activated protein kinases (MAPKs), the predominant signaling transduction pathway responsible for synthesis and production of proinflammatory factors in microglia, can be regulated by mitochondrial function (Akundi et al, 2005;Ciallella et al, 2005;Lund et al, 2005;Waetzig et al, 2005). In the present study, microglia were treated with rotenone (10 nM) for different intervals of time to determine the involvement of MAPK after rotenone stimulation.…”

“…However, the exact mechanism underlying the inhibition of microglial activation by opening mitoK ATP channels remains unknown. Mitochondrial membrane potential and the downstream MAPKs have been demonstrated to regulate microglial activation and the production of proinflammatory factors from microglia (Akundi et al, 2005;Ciallella et al, 2005;Lund et al, 2005;Waetzig et al, 2005). The inhibitory effects of IPT on rotenone-induced DC m loss and p38/JNK MAPK activation were abolished by addition of 5-HD, indicating IPT might regulate mitochondrial function and MAPKs pathways through opening mitoK ATP channels.…”

Studying neurodegenerative diseases in culture models

“…For instance, primary microglial cells can be isolated from cerebral cortices of 1-day-old Wistar rats. [47][48][49][50][51] It is important to take extreme care to avoid lipopolysaccharide contamination, thus to keep microglia in a resting or ''surveying'' state instead of an activated state. Floating microglia can be harvested from 10-to 14-day-old mixed astroglial and microglial primary cultures.…”