Cannabinol-mediated inhibition of nuclear factor-kappa B, cAMP response element-binding protein, and interleukin-2 secretion by activated thymocytes

Cannabinol (CBN), an immunosuppressive cannabinoid and ligand for the perip heral cannabinoid receptor CB2, inhibits the cAMP signaling cascade in fors kolin-stimulated thymocytes. The objective of the present studies was to fu rther characterize the mechanism of CBN immune modulation by investigating its effects on interleukin-2 (IL-2) secretion, cAMP response element (CRE), and kappa B DNA binding activity in phorbol ester (phorbol-12-myristate-13 -acetate, PMA) plus calcium ionophore (PMA/Io)-activated thymocytes. PMA/Io treatment induced CRE and kB DNA binding activity that was attenuated in t he presence of CBN. A concomitant and concentration-related inhibition of I L-2 also was produced by CBN in PMA/Io-activated thymocytes. PMA/Io induced two CRE DNA binding complexes, a major complex consisting of a cAMP respon se element-binding protein (CREB)-1 homodimer, and a minor CREB-1/activatin g transcription factor (ATF)-2 complex. Both CRE complexes were inhibited b y CBN. Conversely, two kappa B DNA binding complexes were observed, but onl y one was PMA/Io-inducible. However, the DNA binding activity of both compl exes was diminished in the presence of CBN. The PMA/Io-inducible kappa B co mplex was a p65/c-Rel heterodimer. Analysis of up-stream regulation reveale d a decrease in phosphorylated CREB/ATF nuclear proteins in PMA/Io-activate d thymocytes after CBN treatment. Similarly, CBN prevented the phosphorylat ion-dependent degradation of the nuclear factor-kappa B inhibitory protein I kappa B-alpha. These results provide a potential link between the CBN-med iated inhibition of thymocyte function, including IL-2 production, and the inhibition of two critical transcription factor families, CREB/ATF and NF-k appa B/Rel.