LIMK2 is required for membrane cytoskeleton reorganization of contracting airway smooth muscle

Abstract

Airway smooth muscle (ASM) has developed a mechanical adaption mechanism by which it transduces force and responds to environmental forces, which is essential for periodic breathing. Cytoskeletal reorganization has been implicated in this process, but the regulatory mechanism remains to be determined. We here observe that ASM abundantly expresses cytoskeleton regulators Limk1 and Limk2, and their expression levels are further upregulated in chronic obstructive pulmonary disease (COPD) animals. By establishing mouse lines with deletions of Limk1 or Limk2, we analyse the length-sensitive contraction, F/G-actin dynamics, and F-actin pool of mutant ASM cells. As LIMK1 phosphorylation does not respond to the contractile stimulation, LIMK1-deficient ASM develops normal maximal force, while LIMK2 or LIMK1/LIMK2 deficient ASMs show approximately 30% inhibition. LIMK2 deletion causes a significant decrease in cofilin phosphorylation along with a reduced F/G-actin ratio. As LIMK2 functions independently of cross-bridge movement, this observation indicates that LIMK2 is necessary for F-actin dynamics and hence force transduction. Moreover, LIMK2-deficient ASMs display abolishes stretching-induced suppression of 5-hydroxytryptamine (5-HT) but not acetylcholine-evoks force, which is due to the differential contraction mechanisms adopted by the agonists. We propose that LIMK2-mediated cofilin phosphorylation is required for membrane cytoskeleton reorganization that is necessary for ASM mechanical adaption including the 5-HT-evoked length-sensitive effect.

Introduction

During physiological breathing, the airway is primarily maintained by tonic force from airway smooth muscle (ASM). The production of maximal force depends on cross-bridge movement of myofilaments and transduction by cytoskeleton reorganization (Horowitz et al., 1996; Gunst and Zhang, 2008). The ASM is exposed to mechanical oscillation which profoundly affects airway tone and responsiveness. To adapt to this mechanical condition, ASM has developed unique contractile abilities including hyperresponsiveness, length-sensitive contraction and extracellular force transduction (Gunst et al., 1990; Shen et al., 1997; Gunst and Tang, 2000). It has been demonstrated that through these abilities, ASM also participates in diverse pathological processes including asthma and chronic obstructive pulmonary disease (COPD) (Postma and Kerstjens, 1998; Prakash, 2016). Current knowledge suggests that cytoskeletal reorganization is fundamental for these abilities (Gunst et al., 1995, 2003; Zhang and Gunst, 2019). However, the underlying regulatory mechanism remains unclear.

Similar to other types of smooth muscle, ASM produces force through cross-bridge movement triggered by myosin regulatory light chain (RLC) phosphorylation (Kamm and Stull, 1985; Saito et al., 1996; Thirstrup, 2000; Zhang et al., 2010), and the resultant force is then transduced outside the muscle (Small, 1995; Zhang and Gunst, 2008). It has been demonstrated that the latter process is primarily mediated by cytoskeletal reorganization which is activated by actin polymerization (Hirshman et al., 1998; Mehta and Gunst, 1999). In light of the fact that smooth muscle maintains a highly dynamic turnover of the G/F-actin pool in an RLC phosphorylation-independent manner (Obara and Yabu, 1994; Saito et al., 1996; Hoover et al., 2012), actin polymerization is usually considered a critical process for force transduction (Gunst and Zhang, 2008). Because inhibition of actin dynamics markedly inhibits length-induced force suppression (Mehta and Gunst, 1999), actin polymerization may also regulate the length-sensitive effect. It has been shown that focal adhesion kinase (FAK) signalling regulates actin polymerization and cytoskeleton reorganization at dense plaques, which are similar to focal adhesion sites in cultured cells (Gabella, 1984; Draeger et al., 1990; Schaller, 2010). Since the plasma membrane of ASM cells makes more contact with the extracellular matrix than dense plaques, we hypothesize that cytoskeletal reorganization and actin polymerization within the plasma membrane may also underlie the mechanical properties of ASM.

Generally, actin dynamics are regulated by LIM-kinases (LIMKs, LIMK1 and LIMK2) through phosphorylation of cofilin at Ser-3, and phosphorylated cofilin inhibits F-actin depolymerization (Zhao et al., 2008; Mizuno, 2013; Ohashi, 2015). Cofilin phosphorylation may also be regulated by other kinases including testicular protein kinases (TESKs) and phosphatases such as Slingshot family protein phosphatases (SSHs), chronophin (CIN), protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A) (Mizuno, 2013). The functional importance of LIMKs for cofilin phosphorylation in ASM contraction remains to be determined. As Limk1 and Limk2 are abundantly expressed in airway smooth muscle cells and their expression levels are further upregulated in COPD animals, we speculate that LIMKs serve as central factors in cytoskeletal reorganization in ASM. Here, we establish two mouse lines with deletion of LIMK1 or LIMK2, and found that LIMK2, but not LIMK1, regulates contractile responses through membrane F-actin polymerization. Deletion of LIMK2 abolishes the force suppression in response to stretching, suggesting that LIMK2-mediated contraction contributes to the length-sensitive effect of ASM.