Cross-Talk between Transforming Growth Factor-beta(1) and Muscarinic M-2 Receptors Augments Airway Smooth Muscle Proliferation

Transforming growth factor-beta(1) (TGF-beta(1)) 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-beta(1) exerts only weak mitogenic effects on ASM cells. In this study, we hypothesized that the mitogenic effect of TGF-beta(1) 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-beta(1), alone and in combination with the muscarinic receptor agonist methacholine, on human ASM cell proliferation. Acutely, TGF-beta(1) exerted no mitogenic effect. However, prolonged treatment (for 7 d) with TGF-beta(1) 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-beta(1). Interestingly, the integrin-blocking peptide Arg-Gly-Asp-Ser, as well as integrin alpha 5 beta(1) function-blocking antibodies, inhibited the effects of TGF-beta(1) and its combination with methacholine on cell proliferation. Accordingly, prolonged treatment with TGF-beta(1) increased fibronectin expression, which was also synergistically enhanced by methacholine. The synergistic effects of methacholine on TGF-beta(1)-induced proliferation were reduced by the long-acting muscarinic receptor antagonist tiotropium and the M-2 receptor subtype-selective antagonist gallamine, but not the M-3-selective antagonist DAU5884. In line with these findings, the irreversible G(i) protein inhibitor pertussis toxin also prevented the potentiation of TGF-beta(1)-induced proliferation by methacholine. We conclude that prolonged exposure to TGF-beta(1) enhances ASM cell proliferation, which is mediated by extracellular matrix-integrin interactions, and which can be enhanced by muscarinic M-2 receptor stimulation.