Role of an Adenylyl Cyclase Isoform in Alcohol's Effect on Cyclic AMP Regulated Gene Expression in Mammalian Cells

Abstract

Research suggests that the cyclic AMP (cAMP) signaling pathway including CREB-CRE regulated expression of various genes is implicated in the predisposition to and development of alcoholism in humans. Alcohol also induces changes in inflammatory and immune responses; these changes increase the incidence of pneumonias and other infections, which can negatively affect recovery from infections. Cyclic AMP (cAMP) is known for its immunosuppressive effects and is also required for proper development of the immune system. Previous work in our laboratory has demonstrated that ethanol enhances the activity of adenylyl cyclase (AC) in an isoform-specific manner; type 7 AC (AC7) is most enhanced by ethanol. Therefore, we hypothesize that the AC isoform expressed in the cells will play a role in ethanol’s effects on cAMP regulated gene expression. We further hypothesize that alcohol modulates cAMP signaling in immune cells by enhancing the activity of AC7; thus, AC7 may play a role in ethanol’s effects on immune function. Our objectives include: 1) evaluate the AC isoform specific effects of ethanol on cAMP regulated gene in NIH 3T3 cells by overexpressing two AC isoforms: AC3 and AC7; 2) employ immune cell lines endogenously expressing AC7, RAW 264.7 and BV-2, to further elucidate the role of AC7 in the effect of ethanol on cAMP regulated gene expression. To examine these objectives, time-lapse fluorescent resonance energy transfer (FRET) and cAMP accumulation assays were used to monitor cAMP levels within the cells. A reporter gene (luciferase) driven by an artificial promoter inducible with cAMP was utilized to evaluate the effect of ethanol on cAMP regulated gene expression. CREB phosphorylation and nuclear translocation of transducers of regulated CREB (TORCs) were examined by western blotting. Stimulation of AC activity by the addition of dopamine caused an increase in the reporter gene activity. Ethanol potentiated the increase of reporter gene activity in NIH 3T3 cells expressing AC7, while cells expressing AC3 did not respond to ethanol. Cyclic AMP pathway activation via stimulation with prostaglandin E1 (PGE1) showed an increase in cAMP and reporter gene expression in RAW 264.7 and BV-2 cells. The effect observed was potentiated in the presence of ethanol. Cyclic AMP analog, 8-Bromo-cAMP, induced luciferase activity was not significantly affected by ethanol. The level of CREB phosphorylation did not change by cAMP stimulation or in the presence of ethanol. However, there were significant changes in the TORC3 amount in nuclei depending on stimulation conditions. The results suggest that