Cross-talk between transforming growth factor-ß1 and muscarinic M2 receptors augments airway smooth muscle proliferation.

Transforming growth factor-ß1 (TGF-ß1) 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-ß1 exerts only weak mitogenic effects on ASM cells. In this study, we hypothesized that the mitogenic effect of TGF-ß1 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-ß1, alone and in combination with the muscarinic receptor agonist methacholine, on human ASM cell proliferation. Acutely, TGF-ß1 exerted no mitogenic effect. However, prolonged treatment (for 7 d) with TGF-ß1 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-ß1. Interestingly, the integrin-blocking peptide Arg-Gly-Asp-Ser, as well as integrin α5ß1 function-blocking antibodies, inhibited the effects of TGF-ß1 and its combination with methacholine on cell proliferation. Accordingly, prolonged treatment with TGF-ß1 increased fibronectin expression, which was also synergistically enhanced by methacholine. The synergistic effects of methacholine on TGF-ß1-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-ß1-induced proliferation by methacholine. We conclude that prolonged exposure to TGF-ß1 enhances ASM cell proliferation, which is mediated by extracellular matrix-integrin interactions, and which can be enhanced by muscarinic M2 receptor stimulation.

Muscarinic receptor stimulation augments TGF-ß1-induced contractile protein expression by airway smooth muscle cells.

Acetylcholine (ACh) is the primary parasympathetic neurotransmitter in the airways. Recently, it was established that ACh, via muscarinic receptors, regulates airway remodeling in animal models of asthma and chronic obstructive pulmonary disease (COPD). The mechanisms involved are not well understood. Here, we investigated the functional interaction between muscarinic receptor stimulation and transforming growth factor (TGF)-ß(1) on the expression of contractile proteins in human airway smooth muscle (ASM) cells. ASM cells expressing functional muscarinic M(2) and M(3) receptors were stimulated with methacholine (MCh), TGF-ß(1), or their combination for up to 7 days. Western blot analysis revealed a strong induction of sm-α-actin and calponin by TGF-ß(1), which was increased by MCh in ASM cells. Immunocytochemistry confirmed these results and revealed that the presence of MCh augmented the formation of sm-α-actin stress fibers by TGF-ß(1). MCh did not augment TGF-ß(1)-induced gene transcription of contractile phenotype markers. Rather, translational processes were involved in the augmentation of TGF-ß(1)-induced contractile protein expression by muscarinic receptor stimulation, including phosphorylation of glycogen synthase kinase-3ß and 4E-binding protein 1, which was enhanced by MCh. In conclusion, muscarinic receptor stimulation augments functional effects of TGF-ß(1) in human ASM cells on cellular processes that underpin ASM remodeling in asthma and COPD.