S100A4 is activated by RhoA and catalyzes the polymerization of non-muscle myosin, adhesion complex assembly and contraction in airway smooth muscle.

KEY POINTS: S100A4 is expressed in many tissues including smooth muscle, but its physiologic function is unknown. S100A4 regulates the motility of metastatic cancer cells by binding to non-muscle myosin II. Contractile stimulation causes the polymerization of NM myosin in airway smooth muscle, which is necessary for tension development. NM myosin regulates the assembly of adhesion junction signalling complexes (adhesomes) that catalyze actin polymerization. In airway smooth muscle, ACh stimulated the binding of S100A4 to the NM myosin heavy chain, which was catalyzed by RhoA GTPase via the RhoA-binding protein, rhotekin. The binding of S100A4 to NM myosin was required for NM myosin polymerization, adhesome assembly and actin polymerization. S100A4 plays a critical function in regulating airway smooth muscle contraction by catalyzing NM myosin filament assembly. The interaction of S100A4 with NM myosin is likely to play an important role in the physiologic function of other tissues.

ABSTRACT: S100A4 binds to the heavy chain of non-muscle (NM) myosin II and can regulate the motility of crawling cells. S100A4 is widely expressed in many tissues including smooth muscle (SM), but its role in regulating their physiologic function is not known. We hypothesized that S100A4 contributes to the regulation of contraction in airway SM by regulating a pool of NM myosin II at the cell cortex. NM myosin II undergoes polymerization in airway SM and regulates contraction by catalyzing the assembly of integrin-associated adhesome complexes that activate pathways that catalyze actin polymerization. Acetylcholine (ACh) stimulated the interaction of S100A4 with NM myosin II in airway SM at the cell cortex and catalyzed NM myosin filament assembly. RhoA GTPase regulated the activation of S100A4 via rhotekin, which facilitated the formation of a complex between RhoA, S100A4 and NM myosin II. The depletion of S100A4, RhoA or rhotekin from airway SM tissues using shRNA or siRNA prevented NM myosin II polymerization, the recruitment of vinculin and paxillin to adhesome signalling complexes in response to ACh, and inhibited actin polymerization and tension development. S100A4 depletion did not affect ACh-stimulated SM myosin RLC phosphorylation. Results show that S100A4 plays a critical role in tension development in airway SM tissue by catalyzing NM myosin filament assembly, and that the interaction of S100A4 with NM myosin in response to contractile stimulation is activated by RhoA GTPase. These results may be broadly relevant to the physiologic function of S100A4 in other cell and tissue types. PLA performed on freshly dissociated tracheal smooth muscle cells and on sections on airway smooth muscle tissues to evaluate the effect of ACh stimulation on the molecular interactions between talin and vinculin. The effect of contractile stimulation with ACh on the interaction between vinculin and talin was assessed using PLA in both isolated freshly dissociated airway smooth muscle cells and in airway smooth muscle tissue sections. Very thin smooth muscle tissue sections or freshly dissociated cells are fixed, permeabilized and labelled with primary antibodies against target epitopes for vinculin and talin using Abs from different species. Secondary antibodies conjugated to oligonucleotide-labelled + or - PLA probes are then targeted to the primary antibodies. When the target proteins (vinculin and talin) are within 40 nm of each other, the oligonucleotide probes form circular DNA strands that undergo amplification to generate a fluorescent signal. Qualitatively similar results are observed for both isolated airway smooth muscle cells (left) and airway smooth muscle tissues (right): ACh stimulates interactions between vinculin and talin that result in fluorescent spots. Unstimulated (US) cells and tissues have few fluorescent spots. This article is protected by copyright. All rights reserved.