title: Synergy between tumor necrosis factor alpha and interleukin 1beta in inducing transcriptional down-regulation of muscarinic M2 receptor gene expression. Involvement of protein kinase A and ceramide pathways.
abstract: Stimulation of HEL 299 cells with tumor necrosis factor alpha (TNF-alpha) or interleukin 1beta (IL-1beta) 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-alpha and IL-1beta 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 C2-ceramide and TNF-alpha or IL-1beta. TNF-alpha and/or IL-1beta 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-1beta effect, suggesting that de novo protein synthesis is required for receptor down-regulation. These results suggest that the TNF-alpha and IL-1beta 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.
authors.affiliation: Department of Thoracic Medicine, National Heart and Lung Institute, Imperial College of Science, Technology, and Medicine, Dovehouse Street, London SW3 6LY, United Kingdom. p.j.barnes@ic.ac.uk
authors.forename: E B
authors.initials: EB
authors.lastname: Haddad
authors.forename: J
authors.initials: J
authors.lastname: Rousell
authors.forename: M A
authors.initials: MA
authors.lastname: Lindsay
authors.forename: P J
authors.initials: PJ
authors.lastname: Barnes
mesh_terms: D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002478:Cells, Cultured; D002518:Ceramides / Q000502:physiology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000502:physiology*; D003513:Cycloheximide / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects; D005786:Gene Expression Regulation; D006801:Humans; D007375:Interleukin-1 / Q000502:physiology*; D011493:Protein Kinase C / Q000502:physiology; D012333:RNA, Messenger / Q000235:genetics; D011976:Receptors, Muscarinic / Q000502:physiology*; D015398:Signal Transduction; D014158:Transcription, Genetic; D014409:Tumor Necrosis Factor-alpha / Q000502:physiology*
chemical_list: D002518:Ceramides; D007375:Interleukin-1; D012333:RNA, Messenger; D011976:Receptors, Muscarinic; D014409:Tumor Necrosis Factor-alpha; D003513:Cycloheximide; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases