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Transforming growth factor-beta1 inhibits beta2-adrenoceptor gene transcription
J C Mak1, J Rousell, E B Haddad
1Department of Thoracic Medicine, Imperial College School of Medicine at the National Heart and Lung Institute, London, UK. j.mak@ic.ac.uk
Abstract:
Transforming growth factor-beta1 (TGF-beta1) has been shown to modulate beta-adrenoceptor number and function in cultured human tracheal smooth muscle cells and cardiac fibroblasts, but the mechanism is unclear. In this study, we have characterized the beta2-adrenoceptor expression by radioligand binding assay, Northern blot analysis and measurement of intracellular cAMP accumulation in a human embryonic lung fibroblast cell line (HEL299 cells). Treatment with TGF-beta1 caused a time-dependent decrease in beta2-adrenoceptor mRNA, and in receptor number after 24 h. Furthermore, nuclear run-on assays showed a 35% reduction in the transcription rate of the beta2-adrenoceptor gene with no alteration in stability of the beta2-adrenoceptor mRNA. After TGF-beta1 treatment, the basal, procaterol- and forskolin-stimulated cAMP accumulations were also decreased. Cycloheximide inhibited TGF-beta1-mediated reduction of beta2-adrenoceptor mRNA and protein, whilst alone caused induction of beta2-adrenoceptor mRNA without any effect on receptor number. In summary, TGF-beta1 induces beta2-adrenoceptor desensitization through the alteration in adenylyl cyclase activity and down-regulation of beta2-adrenoceptor mRNA and protein through the reduction in the rate of beta2-adrenoceptor gene transcription.
Insights
Transforming growth factor-beta1 (TGF-beta1) reduces beta2-adrenoceptor expression and function in lung fibroblasts. This involves decreased gene transcription and altered adenylyl cyclase activity, leading to desensitization.
Area of Science:
- Cellular and Molecular Biology
- Pharmacology
- Respiratory Medicine
Background:
- Transforming growth factor-beta1 (TGF-beta1) influences beta-adrenoceptor function.
- The precise mechanisms by which TGF-beta1 affects beta-adrenoceptors, particularly in lung cells, remain unclear.
Purpose of the Study:
- To investigate the effects of TGF-beta1 on beta2-adrenoceptor expression and function in human embryonic lung fibroblast HEL299 cells.
- To elucidate the molecular mechanisms underlying TGF-beta1-mediated modulation of beta2-adrenoceptors.
Main Methods:
- Radioligand binding assays to quantify beta2-adrenoceptor number.
- Northern blot analysis to assess beta2-adrenoceptor mRNA levels.
- Measurement of intracellular cAMP accumulation to evaluate receptor function.
- Nuclear run-on assays to determine gene transcription rates.
- Experiments with cycloheximide to assess protein synthesis involvement.
Main Results:
- TGF-beta1 treatment led to a time-dependent decrease in beta2-adrenoceptor mRNA and receptor number.
- Nuclear run-on assays revealed a 35% reduction in beta2-adrenoceptor gene transcription rate without affecting mRNA stability.
- TGF-beta1 decreased basal, procaterol-, and forskolin-stimulated cAMP accumulation.
- Cycloheximide blocked TGF-beta1-induced reduction of beta2-adrenoceptor mRNA and protein.
Conclusions:
- TGF-beta1 induces beta2-adrenoceptor desensitization in lung fibroblasts.
- This desensitization occurs via reduced beta2-adrenoceptor gene transcription and altered adenylyl cyclase activity.
- TGF-beta1 down-regulates beta2-adrenoceptor mRNA and protein levels, impacting cellular signaling pathways.