Previous results from our laboratory indicate that immune suppression by cannabinoids is mediated through cannabinoid receptors expressed on immunocompetent cells, most notably T-cells which we have found to exhibits a marked sensitivity. This is indicated by an inhibition of T-cell mediated responses and Il-2 expression in the presence of cannabinoids. We have also shown that ligand binding to cannabinoid receptors inhibits cAMP signal transduction in T-cells as evidenced by decreased; (i) cAMP formation; (ii) protein kinase A activity; and (iii) binding by CREB/ATF transcription factors to their cognate DNA binding site, cAMP responsive element (CRE). CREB/ATF regulatory proteins also dimerize with Fos and Jun family members to help regulate gene transcription at AP-1 sites (e.g., IL- 2). Our studies suggest that the inhibition of cAMP signaling is responsible for immune inhibition by cannabinoids based on three critical observations: 91) membrane permeable cAMP analogs reversed the immunoinhibitory effects produced by cannabinoids; (ii) pertussis toxin pretreatment of splenocytes abrogated cannbinoid-induced immune suppression and inhibition of adenylate cyclase; and (iii) glucagon, which stimulates adenylate cyclase activity through interaction with its own G-protein coupled receptor, reversed the inhibitory effects produced by cannabinoids. Based on the observations described above our present investigation will have two objectives; (1) to identify which specific cAMP-regulated transcription factors are inhibited by cannabinoids during T-cell activation; and (2) to identify specific cAMP regulated genes that exhibit altered expression during T-cell activation in athe presence of cannabinoids. In SA#1, we will identify the DNA binding proteins within the CREB/ATF family of transcription regulators that are involved in T-cell activation and which concomitantly exhibit decreased DNA binding in the presence of cannabinoids. In SA#2, we will characterize the specific time course and magnitude of transcriptional dysregulation at CRE and AP-1 DNA regulatory sites by cannabinoids following T-cell activation using CRE and AP-1 luciferase reporter constructs. In SA#3, we will characterize cannabinoid-mediated inhibition of IL-2 gene expression using I-2 luciferase reporter constructs. In SA#4, we will identify cAMP-regulated genes involved in T-cell activation whose expression is altered by cannabinoid-treatment. Lastly, in SA#5 we will, characterize genes involved in T-cell activation that exhibit sensitivity to dysregulation by cannabinoids.
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