Stefania Merighi1, Stefania Gessi1, Katia Varani1, Carolina Simioni1, Debora Fazzi1, Prisco Mirandola1, Pier Andrea Borea1. 1. Department of Clinical and Experimental Medicine, Pharmacology Section and Interdisciplinary Center for the Study of Inflammation, University of Ferrara, Via Fossato di Mortara, Ferrara, ItalyDepartment of Human Anatomy, Pharmacology and Forensic Medicine, Institute of Normal Human Anatomy, Ospedale Maggiore, University of Parma, Parma, Italy.
Abstract
BACKGROUND AND PURPOSE: Cannabinoid (CB) receptor agonists have potential utility as anti-inflammatory drugs in chronic immune inflammatory diseases. In the present study, we characterized the signal transduction pathways affected by CB(2) receptors in quiescent and lipopolysaccharide (LPS)-stimulated murine microglia. EXPERIMENTAL APPROACH: We examined the effects of the synthetic CB(2) receptor ligand, JWH-015, on phosphorylation of MAPKs and NO production. KEY RESULTS: Stimulation of CB(2) receptors by JWH-015 activated JNK-1/2 and ERK-1/2 in quiescent murine microglial cells. Furthermore, CB(2) receptor activation increased p-ERK-1/2 at 15 min in LPS-stimulated microglia. Surprisingly, this was reduced after 30 min in the presence of both LPS and JWH-015. The NOS inhibitor L-NAME blocked the ability of JWH-015 to down-regulate the LPS-induced p-ERK increase, indicating that activation of CB(2) receptors reduced effects of LPS on ERK-1/2 phosphorylation through NO. JWH-015 increased LPS-induced NO release at 30 min, while at 4 h CB(2) receptor stimulation had an inhibitory effect. All the effects of JWH-015 were significantly blocked by the CB(2) receptor antagonist AM 630 and, as the inhibition of CB(2) receptor expression by siRNA abolished the effects of JWH-015, were shown to be mediated specifically by activation of CB(2) receptors. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate that CB(2) receptor stimulation activated the MAPK pathway, but the presence of a second stimulus blocked MAPK signal transduction, inhibiting pro-inflammatory LPS-induced production of NO. Therefore, CB(2) receptor agonists may promote anti-inflammatory therapeutic responses in activated microglia.
BACKGROUND AND PURPOSE: Cannabinoid (CB) receptor agonists have potential utility as anti-inflammatory drugs in chronic immune inflammatory diseases. In the present study, we characterized the signal transduction pathways affected by CB(2) receptors in quiescent and lipopolysaccharide (LPS)-stimulated murine microglia. EXPERIMENTAL APPROACH: We examined the effects of the synthetic CB(2) receptor ligand, JWH-015, on phosphorylation of MAPKs and NO production. KEY RESULTS: Stimulation of CB(2) receptors by JWH-015 activated JNK-1/2 and ERK-1/2 in quiescent murine microglial cells. Furthermore, CB(2) receptor activation increased p-ERK-1/2 at 15 min in LPS-stimulated microglia. Surprisingly, this was reduced after 30 min in the presence of both LPS and JWH-015. The NOS inhibitor L-NAME blocked the ability of JWH-015 to down-regulate the LPS-induced p-ERK increase, indicating that activation of CB(2) receptors reduced effects of LPS on ERK-1/2 phosphorylation through NO. JWH-015 increased LPS-induced NO release at 30 min, while at 4 h CB(2) receptor stimulation had an inhibitory effect. All the effects of JWH-015 were significantly blocked by the CB(2) receptor antagonist AM 630 and, as the inhibition of CB(2) receptor expression by siRNA abolished the effects of JWH-015, were shown to be mediated specifically by activation of CB(2) receptors. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate that CB(2) receptor stimulation activated the MAPK pathway, but the presence of a second stimulus blocked MAPK signal transduction, inhibiting pro-inflammatory LPS-induced production of NO. Therefore, CB(2) receptor agonists may promote anti-inflammatory therapeutic responses in activated microglia.