Synergy between tumor necrosis factor α and interleukin 1β in inducing transcriptional down-regulation of muscarinic M₂ receptor gene expression: Involvement of protein kinase A and ceramide pathways

Stimulation of HEL 299 cells with tumor necrosis factor α (TNF-α) or interleukin 1β (IL-1β) had no effect on M₂ muscarinic receptor expression. However, the combination of these two cytokines markedly down-regulated muscarinic M₂ receptor protein and mRNA expression and uncoupled M₂ receptors from adenylyl cyclase. There was no effect of TNF-α and IL-1β on the m2 muscarinic receptor mRNA stability, and nuclear run-on assays showed reduced m2 receptor gene transcription. Sequential cytokine addition suggests that the synergy involves postreceptor events. Although the cAMP-dependent protein kinase inhibitor H8 provided a significant protection against receptor down-regulation, the protein kinase C inhibitor GF109203X had no effect. The ceramide analog C₂-ceramide (N-acetylsphingosine) was without effect on m2 receptor expression. However, a strong synergistic effect was demonstrated when cells were treated with the combination of C₂2-ceramide and TNF-α or IL-1β. TNF-α and/or IL-1β combination also activated the 46- and 55-kDa c-Jun NH₂-terminal protein kinases and to a lesser extent p42 and p44 mitogen-activated protein kinase isoforms. Cycloheximide abolished the TNF-α and IL-1β effect, suggesting that de novo protein synthesis is required for receptor down-regulation. These results suggest that the TNF-α and IL-1β synergize to induce transcriptional down-regulation of the M₂ muscarinic receptor, which seems to be mediated through activation of both ceramide and cAMP-dependent protein kinase pathways. Furthermore, these results suggest that M₂ receptor expression is under the control of a cytokine network.