Analysis of promoter regions regulating basal and interleukin-4-inducible expression of the human CB1 receptor gene in T lymphocytes.
The majority of effects of cannabinoids are mediated by the two receptors CB1 and CB2. In addition to neuronal cells, CB1 receptors are expressed in T lymphocytes, in which they are involved in cannabinoid-induced T helper cell biasing. Although basally expressed only weakly in T cells, CB1 receptors are up-regulated in these cells by stimuli such as cannabinoids themselves. This effect is mediated by interleukin-4. In this study, we investigated basal and interleukin-4-inducible expression of the CB1 gene in T lymphocytes. In a promoter analysis, two regions [nucleotides (nts) -3086 to -2490 and nts -1950 to -1653] were identified, which suppress basal transcription of the gene in Jurkat T cells, whereas the region between nts -648 and -559 enhanced basal CB1 transcription. Interleukin-4 markedly induced transcription of CB1 in Jurkat cells and primary human T cells. Experiments using transcription factor decoy oligonucleotides demonstrated that STAT6 mediates regulation of the gene by interleukin-4. Using reporter gene assays and the transcription factor decoy oligonucleotide approach, a binding site for STAT6 was identified at nt -2769 on the human CB1 gene promoter. Interleukin-4 also caused up-regulation of functional CB1 receptor proteins. In interleukin-4 pretreated, but not in naive Jurkat cells, the CB1 agonist R(+)-methanandamide caused a significant inhibition of forskolin-induced cAMP formation. This effect was blocked by the CB1-selective antagonists N-(piperidin-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251) and 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl-N-4-mo rpholinyl-1H-pyrazole-3-carboxamide (AM281). Taken together, these data show that CB1 receptors are expressed and up-regulated by interleukin-4 in T lymphocytes, which enables CB1-mediated communication to cells of other systems, such as neuronal cells.
D000107:Acetylation; D002478:Cells, Cultured; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D000242:Cyclic AMP / Q000032:analysis; D018274:Electroporation; D004797:Enzyme-Linked Immunosorbent Assay; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D006801:Humans; D015847:Interleukin-4 / Q000235:genetics* / Q000494:pharmacology; D019169:Jurkat Cells; D007700:Kinetics; D010880:Piperidines / Q000037:antagonists & inhibitors; D010957:Plasmids; D011401:Promoter Regions, Genetic; D011720:Pyrazoles / Q000037:antagonists & inhibitors; D043884:Receptor, Cannabinoid, CB1 / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D050800:STAT6 Transcription Factor / Q000378:metabolism; D013223:Statistics as Topic; D013601:T-Lymphocytes / Q000378:metabolism*; D014158:Transcription, Genetic; D014162:Transfection
D010880:Piperidines; D011720:Pyrazoles; D043884:Receptor, Cannabinoid, CB1; D050800:STAT6 Transcription Factor; C494101:STAT6 protein, human; D015847:Interleukin-4; C103505:AM 251; D000242:Cyclic AMP; D015500:Chloramphenicol O-Acetyltransferase