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

Abstract

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 in order 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 (hASMC), 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 hASMC, acute treatment with BK triggered sub-cellular 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 hASMC, an ARHGEF1 siRNA 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 ASMC of asthmatic patients and in lungs of OVA-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.

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