Abstract

Key points Transforming growth‐factor‐β (TGF‐β) and RhoA/Rho‐kinase are independently implicated in the airway hyper‐responsiveness associated with asthma, but how these proteins interact is not fully understood.We examined the effects of pre‐treatment with TGF‐β on expression and activity of RhoA, Rho‐kinase and ARHGEF1, an activator of RhoA, as well as on bradykinin‐induced contraction, in airway smooth muscle.TGF‐β enhanced bradykinin‐induced RhoA translocation, Rho‐kinase‐dependent phosphorylation and contraction, but partially suppressed bradykinin‐induced RhoA activity (RhoA‐GTP content).TGF‐β enhanced the expression of ARHGEF1, while a small interfering RNA against ARHGEF1 and a RhoGEF inhibitor prevented the effects of TGF‐β on RhoA and Rho‐kinase activity and contraction, respectively.ARHGEF1 expression was also enhanced in airway smooth muscle from asthmatic patients and ovalbumin‐sensitized mice.ARHGEF1 is a key TGF‐β target gene, an important regulator of Rho‐kinase activity and therefore a potential therapeutic target for the treatment of asthmatic airway hyper‐responsiveness. Transforming growth factor‐β (TGF‐β), RhoA/Rho‐kinase and Src‐family kinases (SrcFK) have independently been implicated in airway hyper‐responsiveness, but how they interact to regulate airway smooth muscle contractility is not fully understood. We found that TGF‐β pre‐treatment enhanced acute contractile responses to bradykinin (BK) in isolated rat bronchioles, and inhibitors of RhoGEFs (Y16) and Rho‐kinase (Y27632), but not the SrcFK inhibitor PP2, prevented this enhancement. In cultured human airway smooth muscle cells (hASMCs), TGF‐β pre‐treatment enhanced the protein expression of the Rho guanine nucleotide exchange factor ARHGEF1, MLC20, MYPT‐1 and the actin‐severing protein cofilin, but not of RhoA, ROCK2 or c‐Src. In hASMCs, acute treatment with BK triggered subcellular translocation of ARHGEF1 and RhoA and enhanced auto‐phosphorylation of SrcFK and phosphorylation of MYPT1 and MLC20, but induced de‐phosphorylation of cofilin. TGF‐β pre‐treatment amplified the effects of BK on RhoA translocation and MYPT1/MLC20 phosphorylation, but suppressed the effects of BK on RhoA‐GTP content, SrcFK auto‐phosphorylation and cofilin de‐phosphorylation. In hASMCs, an ARHGEF1 small interfering RNA suppressed the effects of BK and TGF‐β on RhoA‐GTP content, RhoA translocation and MYPT1 and MLC20 phosphorylation, but minimally influenced the effects of TGF‐β on cofilin expression and phosphorylation. ARHGEF1 expression was also enhanced in ASMCs of asthmatic patients and in lungs of ovalbumin‐sensitized mice. Our data indicate that TGF‐β enhances BK‐induced contraction, RhoA translocation and Rho‐kinase activity in airway smooth muscle largely via ARHGEF1, but independently of SrcFK and total RhoA‐GTP content. A role for smooth muscle ARHGEF1 in asthmatic airway hyper‐responsiveness is worthy of further investigation.

Highlights

  • In healthy subjects, airway smooth muscle (ASM) tone and airflow resistance are low, while in subjects with allergic asthma, exposure to allergens sensitizes airways to constrictor stimuli, resulting in inappropriate episodic ASM constriction and long-term remodelling (Doeing & Solway, 2013)

  • Considering that the Transforming growth factor-β (TGF-β)-induced enhancement of contraction is dependent on Rho-specific guanine nucleotide exchange factors (RhoGEFs) and Rho-kinase, we examined the effects of TGF-β on expression, activity and translocation of RhoA, expression and activity of Rho-kinase and the influence of the RhoGEF ARHGEF1 on those effects, in human airway smooth muscle cells (hASMCs)

  • A, representative blots showing effects of acute BK treatment (1 μM, 30 s) with or without TGF-β pre-treatment (10 ng ml−1, 24 h) in hASMCs transfected with either ARHGEF1 small interfering RNA (siRNA), or a scrambled siRNA control, on phospho-MYPT-1 (Thr-696), total myosin phosphatase targeting subunit-1 (MYPT1) or GAPDH as a loading control

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Summary

Introduction

Airway smooth muscle (ASM) tone and airflow resistance are low, while in subjects with allergic asthma, exposure to allergens sensitizes airways to constrictor stimuli, resulting in inappropriate episodic ASM constriction (airway hyper-responsiveness, AHR) and long-term remodelling (Doeing & Solway, 2013) These smooth muscle phenotypic changes are triggered by inflammatory mediators, produced in the airways in response to allergen sensitization. GEF-H1 and Net, are up-regulated by TGF-β in epithelia, enhancing RhoA-dependent F-actin expression/cell migration and epithelial-to-mesenchymal transition, respectively (Tsapara et al 2010; Papadimitriou et al.2012) Another RhoGEF, ARHGEF1 (p115-RhoGEF, Lsc), not previously linked with TGF-β, has been implicated in the T-cell dysfunction associated with allergic asthma, and in the contractile effects of angiotensin II on hypertensive vascular smooth muscle (Brown et al 2007; Guilluy et al 2010). The role of ARHGEF1 in ASM contraction remains to be determined

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