Evidence for functional selectivity at CB1 cannabinoid receptor constitutively expressed in neuroblastoma cells

CB1 cannabinoid receptor activation is frequently associated with acute alterations of neurotransmitter release as well as more profound modifications of gene expression through regulation of complex intracellular signalling pathways. In addition to the well described Gi/o-mediated reduction of cAMP accumulation, cannabinoids have been shown to activate different MAPK family members, to regulate Ca2+ and K+ currents. Putative coupling with Gs and Gq type G-proteins has also been suggested. We recently reported a CB1-mediated regulation of tyrosine hydroxylase expression in N1E-115 neuroblastoma. Noteworthy, different classes of full CB1 agonists were shown to induce opposite effects1, raising the question of the activation of distinct cell signallings by unrelated CB1 ligands. In order to investigate the CB1-mediated signalling pathways involved in the regulation of gene transcription, we performed luciferase reporter gene assay in N1E-115 neuroblastoma. Cells were transiently tranfected with luciferase reporter plasmids containing repeats of either CRE or AP1 cis-enhancer elements, both involved in the control of tyrosine hydroxylase expression2,3,4. In this system, the properties of HU 210 and CP 55,940, two synthetic analogues of 9-THC, the major psychoactive component of the plant Cannabis sativa were evaluated. Consistent with their full agonist properties, both ligands strongly decreased cAMP accumulation and consecutively reduced CRE-driven luciferase expression (pEC50 HU 210 : 7.8 0.2 and CP 55,940 : 8.3 0.4). In contrast, in cells engineered to examine the AP1-controlled luciferase activity, HU 210 exhibited a dose-dependent reduction of luciferase activity (pEC50 : 7,5 0,2) whereas no significant response was obtained with CP 55,940. In the presence of SR 141716A, a CB1 selective antagonist, both responses were prevented, confirming the involvement of the CB1 receptor. Different kinase inhibitors were used to discriminate between the signalling pathways involved in the paradoxical results obtained using the AP1 construct. The use of PKC inhibitors was effective in blocking HU 210-mediated decreased transcription in cells expressing the AP1 construct, suggesting an agonist selective regulation of PKC-depending responses. In addition, these responses were sensitive to pertussis toxin, thus suggesting that they are Gi/o-mediated. Taken together, our results demonstrate that different agonists acting at CB1 receptor may induce differential effects regarding AP1 cis-enhancer element activity. This could be due to their ability to selectively regulate distinct intracellular signalling pathways, supporting the concept of functional selectivity