Transforming growth factor‐β enhances Rho‐kinase activity and contraction in airway smooth muscle via the nucleotide exchange factor ARHGEF1

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. AbstractTransforming 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.

Arhgef12 drives IL17A-induced airway contractility and airway hyperresponsiveness in mice

“…Recent studies have implicated GEFs in both airway inflammation and contractility (25,26). This study adds to this growing literature by revealing Arhgef12 to be dominantly expressed among GEFs in the smooth muscle compartment and by showing its relevance to the IL17A pathway, a driver of severe asthma.…”

Ablation of RhoA impairs Th17 cell differentiation and alleviates house dust mite-triggered allergic airway inflammation

“…Y16 is a specific RhoA inhibitor developed in our laboratory, which fits into a surface groove of the DH‐PH domain of leukemia‐associated Rho GEF (LARG), a G‐protein‐regulated Rho GEF involved in RhoA activation . Y16 has been used for RhoA functional studies of a wide range, from endothelial barrier and macrophage phenotypes to contraction of intrapulmonary artery and airway smooth muscle . The main purpose for using Y16 in this study is to complement genetic deletion of RhoA to demonstrate the role of RhoA in Th17‐cell differentiation in vitro and Th17‐involved allergic airway inflammation in vivo.…”

“…36 Y16 has been used for RhoA functional studies of a wide range, from endothelial barrier and macrophage phenotypes to contraction of intrapulmonary artery and airway smooth muscle. [47][48][49][50] The main purpose for using Y16 in this study is to complement genetic deletion of RhoA to demonstrate the role of RhoA in Th17-cell differentiation in vitro and Th17-involved allergic airway inflammation in vivo. Nonetheless, our finding that Y16 alleviated HDM-induced murine allergic airway inflammation might demonstrate a proof-of-principle for pharmacological targeting of RhoA in prevention and therapy of asthma.…”

RhoA/Rho‐kinases in asthma: from pathogenesis to therapeutic targets

“…The ARHGEF12‐deficient mice showed significantly decreased AHR relative to wild‐type mice 59 . ARHGEF1, another RhoGEF, is an activator of RhoA increased in the airway smooth muscles from patients with asthma and in the lung tissues from OVA‐challenged mice 61 . A small interfering RNA (siRNA) against ARHGEF1 attenuated the TGF‐β1‐enhanced RhoA expression, Rho‐kinase activation and airway smooth muscle contraction, respectively 60 .…”

“…ARHGEF1, another RhoGEF, is an activator of RhoA increased in the airway smooth muscles from patients with asthma and in the lung tissues from OVA‐challenged mice 61 . A small interfering RNA (siRNA) against ARHGEF1 attenuated the TGF‐β1‐enhanced RhoA expression, Rho‐kinase activation and airway smooth muscle contraction, respectively 60 . In addition, TGF‐β1 enhanced basal and methacholine‐induced cytoskeletal stiffness in isolated human airway smooth muscle in a time‐ and dose‐dependent manner 61 .…”