title: Cross-talk between transforming growth factor-β₁ and muscarinic M₂ receptors augments airway smooth muscle proliferation.
abstract: Transforming growth factor-β₁ (TGF-β₁) is a central mediator in tissue remodeling processes, including fibrosis and airway smooth muscle (ASM) hyperplasia, as observed in asthma. The mechanisms underlying this response, however, remain unclear because TGF-β₁ exerts only weak mitogenic effects on ASM cells. In this study, we hypothesized that the mitogenic effect of TGF-β₁ on ASM is indirect and requires prolonged exposure to allow for extracellular matrix (ECM) deposition. To address this hypothesis, we investigated the effects of acute and prolonged treatment with TGF-β₁, alone and in combination with the muscarinic receptor agonist methacholine, on human ASM cell proliferation. Acutely, TGF-β₁ exerted no mitogenic effect. However, prolonged treatment (for 7 d) with TGF-β₁ increased ASM cell proliferation and potentiated the platelet-derived growth factor-induced mitogenic response. Muscarinic receptor stimulation with methacholine synergistically enhanced the effect of TGF-β₁. Interestingly, the integrin-blocking peptide Arg-Gly-Asp-Ser, as well as integrin α5β1 function-blocking antibodies, inhibited the effects of TGF-β₁ and its combination with methacholine on cell proliferation. Accordingly, prolonged treatment with TGF-β₁ increased fibronectin expression, which was also synergistically enhanced by methacholine. The synergistic effects of methacholine on TGF-β₁-induced proliferation were reduced by the long-acting muscarinic receptor antagonist tiotropium and the M₂ receptor subtype-selective antagonist gallamine, but not the M₃-selective antagonist DAU5884. In line with these findings, the irreversible Gi protein inhibitor pertussis toxin also prevented the potentiation of TGF-β₁-induced proliferation by methacholine. We conclude that prolonged exposure to TGF-β₁ enhances ASM cell proliferation, which is mediated by extracellular matrix-integrin interactions, and which can be enhanced by muscarinic M₂ receptor stimulation.
authors: Department of Molecular Pharmacology, University of Groningen, Groningen, The Netherlands. t.a.oenema@rug.nl
authors: Tjitske A
authors: TA
authors: Oenema
authors: Gerrianne
authors: G
authors: Mensink
authors: Lyanne
authors: L
authors: Smedinga
authors: Andrew J
authors: AJ
authors: Halayko
authors: Johan
authors: J
authors: Zaagsma
authors: Herman
authors: H
authors: Meurs
authors: Reinoud
authors: R
authors: Gosens
authors: Bart G J
authors: BG
authors: Dekkers
mesh_terms: D002461:Cell Line, Transformed; D049109:Cell Proliferation; D016895:Culture Media, Serum-Free; D004261:DNA Replication; D004357:Drug Synergism; D005109:Extracellular Matrix / Q000378:metabolism; D005353:Fibronectins / Q000378:metabolism; D005703:Gallamine Triethiodide / Q000494:pharmacology; D006801:Humans; D039081:Integrin alpha5beta1 / Q000378:metabolism; D016210:Methacholine Chloride / Q000494:pharmacology; D008934:Mitogens / Q000494:pharmacology; D032389:Myocytes, Smooth Muscle / Q000187:drug effects* / Q000378:metabolism; D009842:Oligopeptides / Q000494:pharmacology; D037342:Pertussis Toxin / Q000494:pharmacology; D020239:Receptor Cross-Talk; D043585:Receptor, Muscarinic M2 / Q000819:agonists / Q000378:metabolism*; D012137:Respiratory System / Q000166:cytology; D013997:Time Factors; D053773:Transforming Growth Factor beta1 / Q000494:pharmacology*
chemical_list: D016895:Culture Media, Serum-Free; D005353:Fibronectins; D039081:Integrin alpha5beta1; D008934:Mitogens; D009842:Oligopeptides; D043585:Receptor, Muscarinic M2; D053773:Transforming Growth Factor beta1; D016210:Methacholine Chloride; C046617:arginyl-glycyl-aspartyl-serine; D037342:Pertussis Toxin; D005703:Gallamine Triethiodide