Cannabinoid Inhibition of Adenylate Cyclase-mediated Signal Transduction and Interleukin 2 (IL-2) Expression in the Murine T-cell Line, EL4.IL-2

Robin Condie,Amy Herring,Woo S Koh,Michael Lee,Norbert E Kaminski

doi:10.1074/jbc.271.22.13175

Robin Condie, Amy Herring + Show 3 more

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https://doi.org/10.1074/jbc.271.22.13175

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Abstract

Cannabinoid receptors negatively regulate adenylate cyclase through a pertussis toxin-sensitive GTP-binding protein. In the present studies, signaling via the adenylate cyclase/cAMP pathway was investigated in the murine thymoma-derived T-cell line, EL4.IL-2. Northern analysis of EL4.IL-2 cells identified the presence of 4-kilobase CB2 but not CB1 receptor-subtype mRNA transcripts. Southern analysis of genomic DNA digests for the CB2 receptor demonstrated identical banding patterns for EL4.IL-2 cells and mouse-derived DNA, both of which were dissimilar to DNA isolated from rat. Treatment of EL4.IL-2 cells with either cannabinol or Delta9-THC disrupted the adenylate cyclase signaling cascade by inhibiting forskolin-stimulated cAMP accumulation which consequently led to a decrease in protein kinase A activity and the binding of transcription factors to a CRE consensus sequence. Likewise, an inhibition of phorbol 12-myristate 13-acetate (PMA)/ionomycin-induced interleukin 2 (IL-2) protein secretion, which correlated to decreased IL-2 gene transcription, was induced by both cannabinol and Delta9-THC. Further, cannabinoid treatment also decreased PMA/ionomycin-induced nuclear factor binding to the AP-1 proximal site of the IL-2 promoter. Conversely, forskolin enhanced PMA/ionomycin-induced AP-1 binding. These findings suggest that inhibition of signal transduction via the adenylate cyclase/cAMP pathway induces T-cell dysfunction which leads to a diminution in IL-2 gene transcription.

Highlights

Cannabinoid compounds mediate many if not most of their actions on the immune and central nervous system through interactions with cannabinoid receptors
These findings strongly suggest that the T-cell is a sensitive target for tein; PMA, phorbol 12-myristate 13-acetate; IL-2, interleukin 2; bp, base pair(s); kb, kilobase(s); rc, recombinant; RT-PCR, reverse transcription-polymerase chain reaction; IS, internal standard; DTT, dithiothreitol; PKA, protein kinase A; cAMP regulatory element (CRE), cAMP response element; ELISA, enzyme-linked immunosorbent assay; CREB, cAMP response elementbinding protein; ATF, activating transcription factor; PKI, protein kinase A inhibitor
We demonstrate in the mouse thymoma line EL4.IL-2 that cannabinoid treatment significantly inhibits modulation of the cAMP signal transduction pathway leading to a disruption in IL-2 transcription and production

Summary

Introduction

Cannabinoid compounds mediate many if not most of their actions on the immune and central nervous system through interactions with cannabinoid receptors. One of the most sensitive immune responses to inhibition by cannabinoids that we have far identified is the IgM antibody-forming cell response to the T-cell dependent antigen, sheep erythrocytes [2, 17]. The lack of an effect by cannabinoids on humoral immune responses to T-cell independent antigens suggests that the adenylate cyclase/cAMP signaling pathway is not critical for immunoglobulin production by B-cells. The involvement of adenylate cyclase inhibition in immune dysfunction is supported by the observation that membrane-permeable cAMP analogs (dibutyryl-cAMP and 8-bromo-cAMP) reverse ⌬9-THC-mediated suppression of both the sRBC antibody-forming cell response and lymphoproliferation following PMA plus ionomycin stimulation [16]. Treatment of splenocytes with pertussis toxin abrogates ⌬9-THC inhibition of the sRBC antibody-forming cell response and the inhibition of adenylate cyclase activity [16]

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