Cannabinoid-receptor-independent cell signalling by N-acylethanolamines

Anandamide and other polyunsaturated N-acylethanolamines (NAEs) exert biolo gical activity by binding to cannabinoid receptors. These receptors are lin ked to G(1/0) proteins and their activation leads to extracellular-signal-r egulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) mitogen-act ivated protein kinase (MAP kinase) activation, inhibition of cAMP-dependent signalling and complex changes in the expression of various genes. Saturat ed and monounsaturated NAEs cannot bind to cannabinoid receptors and may th us mediate cell signalling through other targets. Here we report that both saturated/monounsaturated NAEs and anandamide (20:(4n-6) NAE) stimulate can nabinoid-receptor-independent ERK phosphorylation and activator protein-1 ( AP-1)-dependent transcriptional activity in mouse epidermal JB6 cells. Usin g a clone of JB6 P+ cells with an AP-1 collagen-luciferase reporter constru ct, we found that 16:0, 18:1(n-9), 18:1(n-7), 18:2(n-6) and 20:4(n- 6) NAEs stimulated AP-1-dependent transcriptional activity up to 2-fold, with maxi mal stimulation at approx. 10-15 muM. Higher NAE concentrations had toxic e ffects mediated by alterations in mitochondrial energy metabolism. The AP-1 stimulation appeared to be mediated by ERK but not JNK or p38 signalling p athways, because all NAEs stimulated ERK1/ERK2 phosphorylation without havi ng any effect on JNK or p38 kinases. Also, overexpression of dominant negat ive ERK1/ERK2 kinases completely abolished NAE-induced AP-I activation. In contrast with 18:1(n-9) NAE and anandamide, the cannabinoid receptor agonis t WIN 55,212-2 did not stimulate AP-1 activity and inhibited ERK phosphoryl ation. The NAE-mediated effects were not attenuated by pertussis toxin and appeared to be NAE-specific, as a close structural analogue, oleyl alcohol, failed to induce ERK phosphorylation. The data support our hypothesis that the major saturated and monounsaturated NAEs are signalling molecules acti ng through intracellular targets without participation of cannabinoid recep tors.