SYNERGY BETWEEN TUMOR-NECROSIS-FACTOR-ALPHA AND INTERLEUKIN-1-BETA ININDUCING 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-alp ha) 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 expr ession and uncoupled M(2) receptors from adenylyl cyclase, There was n o 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 kina se inhibitor H8 provided a significant protection against receptor dow n-regulation, the protein kinase C inhibitor GF109203X had no effect. The ceramide analog C-2-ceramide (N-acetylsphingosine) was without eff ect on m2 receptor expression, However, a strong synergistic effect wa s demonstrated when cells were treated with the combination of C-2-cer amide and TNF-alpha or IL-1 beta. TNF-alpha and/or IL-1 beta combinati on also activated the 46- and 55-kDa c-Jun NH2-terminal protein kinase s 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 dow n-regulation, These results suggest that the TNF-alpha and IL-1 beta s ynergize to induce transcriptional down regulation of the M(2) muscari nic receptor, which seems to be mediated through activation of both ce ramide and cAMP-dependent protein kinase pathways, Furthermore, these results suggest that M(2) receptor expression is under the control of a cytokine network.