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.
Highlights
Stimulation of HEL 299 cells with tumor necrosis factor ␣ (TNF-␣) or interleukin 1 (IL-1) had no effect on M2 muscarinic receptor expression
We first sought to determine whether TNF-␣ and IL-1 are capable of affecting the expression of M2 muscarinic receptors in cultured human embryonic lung fibroblasts, a cell line that expresses the muscarinic M2 receptor subtype with no evidence of M1, M3, and M4 receptors
We provide evidence that TNF-␣ and IL-1 synergize to induce down-regulation of muscarinic M2 receptors in HEL 299 cells by a mechanism that is dependent on de novo protein synthesis and mediated by decreased transcription of the m2 receptor gene
Summary
Cycloheximide abolished the TNF-␣ and IL-1 effect, suggesting that de novo protein synthesis is required for receptor down-regulation These results suggest that the TNF-␣ and IL-1 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. These results suggest that M2 receptor expression is under the control of a cytokine network. Prejunctional M2 receptors regulating acetylcholine release from cholinergic nerves have been demonstrated in human airways [9], and in vivo studies suggest that they may be dysfunctional in patients with asthma [10].
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