Evidence of cannabinoid modulation in nuclear signaling

Abstract

The endocannabinoid system (ECS) is a signaling mechanism involved in many pathophysiological processes, especially in the nervous system. Former studies from our lab demonstrated the presence of the different components of the ECS in nuclei from rat cerebellum and cerebral cortex (CC). We detected diacylglycerol lipase and monoacylglycerol lipase activities which are involved in maintaining the levels of the endocannabinoid 2-arachidonoyl-glycerol. In addition, we demonstrated a nuclear CB1 protein expression by Western Blot and immunocytochemistry. CB1 is a GPCR receptor that triggers different signaling cascades, modulating intracellular Ca²+ levels, ERK1/2 phosphorylation, and other second messengers at the plasma membrane. Nevertheless, we also observed an increase in ERK phosphorylation in isolated nuclei from rat cerebral cortex (CCN) incubated with a CB1 synthetic agonist (WIN 55-212-2). Phosphorylated ERK could be involved in activating MSK1/2, a nuclear kinase that phosphorylates histone 3 (H3). The aim of this work was therefore to study how a cannabinoid agonist modulates ERK1/2 and H3 phosphorylation in CCN. To this end, CC from Wistar rats were dissected and homogenized, and highly purified nuclei (CCN) were isolated on a sucrose-density ultracentrifugation. CCN were subsequently incubated at 37 °C with WIN and ERK1/2 and H3 signaling cascades were studied by Western Blot. Interestingly, it was observed that ERK1/2 and H3 phosphorylation increased in nuclei treated with WIN 5 µM for 30 min with respect to controls (p<0.05). As expected, this was reversed by pre-incubating for 10 min with a MEK inhibitor (U0126, 10 µM), however, no changes were observed when a CB1 antagonist (SR141716, 1 µM) was used (p<0.05). Taken together, these results demonstrate that cannabinoids at nuclear level could modulate H3 phosphorylation by ERK1/2 signaling. This indicates a potential role of these lipids in chromatin regulation and gene expression in cerebral cortex