Synergy between tumor necrosis factor alpha and interleukin 1 beta in inducing transcriptional down-regulation of muscarinic M(2) receptor gene expression - Involvement of protein kinase A and ceramide pathways

Stimulation of HEL 299 cells with tumor necrosis factor alpha (TNF-alpha) or interleukin 1 beta (IL-1 beta) had no effect on M(2) muscarinic receptor expression, However, the combination of Obese two cytokines markedly down-regulated muscarinic M(2) receptor protein and mRNA expression and uncoupled M(2) receptors from adenylyl cyclase, There was no effect of TNF-alpha and IL-1 beta 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 C-2-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 C-2-ceramide and TNF-alpha or IL-1 beta. TNF-alpha and/or IL-1 beta 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-alpha and IL-1 beta effect, suggesting that de novo protein synthesis is required for receptor down-regulation, These results suggest that the TNF-alpha and IL-1 beta synergize to induce transcriptional down regulation of the M(2) muscarinic receptor, which seems to be mediated through activation of both ceramide and cAMP-dependent protein kinase pathways, Furthermore, these results suggest that M(2) receptor expression is under the control of a cytokine network.