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 ethanols 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 ethanols 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 nuclear translocation of TORC3 may play a more critical role than CREB phosphorylation in the observed changes in the cAMP driven reporter gene activity. Furthermore, the ethanol effect on cAMP regulated reporter gene expression is due to a change in the amount of cAMP, which most likely results from the enhancement of AC7 activity by ethanol.
| Identifer | oai:union.ndltd.org:LSU/oai:etd.lsu.edu:etd-06282016-221816 |
| Date | 08 July 2016 |
| Creators | Hill, Rebecca Ann |
| Contributors | McFarland, Alison, Li, Shisheng, Yoshimura, Masami, Strain, George, Xu, Wu |
| Publisher | LSU |
| Source Sets | Louisiana State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.lsu.edu/docs/available/etd-06282016-221816/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached herein a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to LSU or its agents the non-exclusive license to archive and make accessible, under the conditions specified below and in appropriate University policies, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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