Transforming growth factor-beta 1 induces transcriptional down-regulation of m2 muscarinic receptor gene expression.
In human embryonic lung fibroblasts, transforming growth factor-beta 1 (TGF-beta 1) induced a time-dependent down-regulation of M2 muscarinic receptor binding sites as measured with the nonselective hydrophilic ligand [3H]N-methylscopolamine (NMS). This down-regulation was slow, with 58% loss of all receptors after 24 hr of treatment. The affinity of [3H]NMS for the remaining sites was unaltered by TGF-beta 1. The loss in [3H]NMS binding was accompanied by reduced adenylyl cyclase activity and functional desensitization of M2 muscarinic receptors. Northern blot analyses showed a 72% decrease in the steady state levels of m2 muscarinic receptor mRNA after 24-hr TGF-beta 1 treatment. Recovery of m2 muscarinic receptor mRNA after TGF-beta 1 treatment was slow, with a half-life of approximately 8 hr. There was no effect of TGF-beta 1 on the m2 muscarinic receptor mRNA half-life measured in the presence of actinomycin D, but the rate of m2 muscarinic receptor gene transcription measured with nuclear run-on assay was reduced by 50%, indicating reduced gene transcription. Cycloheximide (10 micrograms/ml) pretreatment abolished the TGF-beta 1 effect, indicating that de novo protein synthesis was required for receptor downregulation. In summary, we have shown that TGF-beta 1 induced desensitization and down-regulation of M2 muscarinic receptor protein and gene that was mediated through reduction in the rate of m2 receptor gene transcription.
Haddad
E B
EB
Department of Thoracic Medicine, National Heart and Lung Institute, London, United Kingdom.
Rousell
J
J
Mak
J C
JC
Barnes
P J
PJ
D000262:Adenylyl Cyclases / Q000378:metabolism; D002460:Cell Line; D005576:Colforsin / Q000494:pharmacology; D003513:Cycloheximide / Q000494:pharmacology; D015232:Dinoprostone / Q000494:pharmacology; D015536:Down-Regulation / Q000187:drug effects*; D006801:Humans; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D012333:RNA, Messenger / Q000235:genetics; D011976:Receptors, Muscarinic / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D011500:Protein Synthesis Inhibitors; D012333:RNA, Messenger; D011976:Receptors, Muscarinic; D016212:Transforming Growth Factor beta; D005576:Colforsin; D003513:Cycloheximide; D000262:Adenylyl Cyclases; D015232:Dinoprostone