Department of Molecular Pharmacology, University of Groningen, Groningen, The Netherlands.
Am J Respir Cell Mol Biol. 2013 Jul;49(1):18-27. doi: 10.1165/rcmb.2012-0261OC.
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.
转化生长因子-β₁（TGF-β₁）是组织重塑过程中的中心介质，包括纤维化和气道平滑肌（ASM）增生，如哮喘中所见。然而，这种反应的机制尚不清楚，因为 TGF-β₁对 ASM 细胞仅具有微弱的有丝分裂作用。在这项研究中，我们假设 TGF-β₁对 ASM 的有丝分裂作用是间接的，需要长时间暴露才能允许细胞外基质（ECM）沉积。为了验证这一假设，我们研究了单独和与毒蕈碱受体激动剂乙酰甲胆碱联合使用时，TGF-β₁对人 ASM 细胞增殖的急性和长期作用。急性时，TGF-β₁没有有丝分裂作用。然而，长期（7 天）处理 TGF-β₁可增加 ASM 细胞增殖，并增强血小板衍生生长因子诱导的有丝分裂反应。毒蕈碱受体刺激用乙酰甲胆碱协同增强了 TGF-β₁的作用。有趣的是，整合素阻断肽 Arg-Gly-Asp-Ser 以及整合素α5β1功能阻断抗体抑制了 TGF-β₁及其与乙酰甲胆碱的协同作用对细胞增殖的影响。因此，长期 TGF-β₁处理增加了纤连蛋白的表达，这也被乙酰甲胆碱协同增强。毒蕈碱对 TGF-β₁诱导的增殖的协同作用被长效毒蕈碱受体拮抗剂噻托溴铵和 M₂受体亚型选择性拮抗剂加兰他敏减弱，但 M₃选择性拮抗剂 DAU5884 则没有。与这些发现一致，不可逆的 Gi 蛋白抑制剂百日咳毒素也阻止了乙酰甲胆碱对 TGF-β₁诱导的增殖的增强作用。我们得出结论，长时间暴露于 TGF-β₁可增强 ASM 细胞增殖，这是由细胞外基质-整合素相互作用介导的，并且可以通过毒蕈碱 M₂受体刺激增强。
