The Dynamic Actin Cytoskeleton in Smooth Muscle

CHAPTER ONE

The Dynamic Actin Cytoskeleton in Smooth Muscle Dale D. Tang1 Albany Medical College, Albany, NY, United States 1 Corresponding author: e-mail address: [email protected]

Contents 1. Introduction 2. Actin Dynamics and Smooth Muscle Contraction 2.1 Critical Role of Actin Dynamics in Smooth Muscle Contraction 2.2 Actin-Regulatory Signaling Networks in Smooth Muscle Contraction 2.3 Targeting Actin Remodeling May Be a New Avenue to Treat Hypertension and Asthma 3. Actin Dynamics and Smooth Muscle Cell Proliferation 3.1 Regulation of Growth Factor Signaling by the Dynamic Actin Cytoskeleton 3.2 Role of c-Abl-Regulated Contractile Ring in Cytokinesis of Smooth Muscle Cells 4. Actin Dynamics and Smooth Muscle Cell Migration 4.1 Local Actin Dynamics Promotes Lamellipodial Formation 4.2 Localized Actin Dynamics Facilitates Focal Adhesion Assembly 4.3 Actin Dynamics Facilitates Stress Fiber Formation 5. Actin-Regulatory Proteins and Vascular/Pulmonary Diseases 5.1 c-Abl Tyrosine Kinase 5.2 Rho and Rho Kinase 5.3 Others 6. Conclusion Conflict of Interest Acknowledgments References

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Abstract Smooth muscle contraction requires both myosin activation and actin cytoskeletal remodeling. Actin cytoskeletal reorganization facilitates smooth muscle contraction by promoting force transmission between the contractile unit and the extracellular matrix (ECM), and by enhancing intercellular mechanical transduction. Myosin may be viewed to serve as an “engine” for smooth muscle contraction whereas the actin cytoskeleton may function as a “transmission system” in smooth muscle. The actin

Advances in Pharmacology, Volume 81 ISSN 1054-3589 https://doi.org/10.1016/bs.apha.2017.06.001

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cytoskeleton in smooth muscle also undergoes restructuring upon activation with growth factors or the ECM, which controls smooth muscle cell proliferation and migration. Abnormal smooth muscle contraction, cell proliferation, and motility contribute to the development of vascular and pulmonary diseases. A number of actin-regulatory proteins including protein kinases have been discovered to orchestrate actin dynamics in smooth muscle. In particular, Abelson tyrosine kinase (c-Abl) is an important molecule that controls actin dynamics, contraction, growth, and motility in smooth muscle. Moreover, c-Abl coordinates the regulation of blood pressure and contributes to the pathogenesis of airway hyperresponsiveness and vascular/ airway remodeling in vivo. Thus, c-Abl may be a novel pharmacological target for the development of new therapy to treat smooth muscle diseases such as hypertension and asthma.

ABBREVIATIONS Abi1 Abl ACh ADF AHR CAS ERK1/2 EVH1 FAK GBD GEF GMF-γ GPCR HDACs HSP IL ILK MAPK MK2 Nck N-WASP PAK PDGF PI3K PIP2 Pfn-1 VASP VCA WIP WAVE

Abl interactor 1 Abelson tyrosine kinase acetylcholine actin-depolymerization factor airway hyperresponsiveness Crk-associated substrates extracellular signal-regulated kinase 1/2 enabled/VASP homology focal adhesion kinase GTP-binding domain guanine nucleotide exchange factor glia maturation factor-γ G protein-coupled receptor histone deacetylases heat shock protein interleukin integrin-linked kinase mitogen-activated protein kinase MAP kinase-activated protein (MAPKAP) kinase 2 noncatalytic region of tyrosine kinase adaptor protein 1 neuronal Wiskott–Aldrich syndrome protein p21-activated kinase platelet-derived growth factor phosphatidylinositol-4,5-bisphosphate 3-kinase phosphatidylinositol 4,5-bisphosphate profilin-1 vasodilator-stimulated phosphoprotein verprolin, central, and acidic WASP-interacting protein WASP-family verprolin homologous protein

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1. INTRODUCTION Smooth muscle is present in the walls of passageways such as blood vessels and the airways, and in the walls of hollow organs such as the urinary bladder, uterus, stomach, and intestines. Smooth muscle is able to contract in response to external stimulation, which plays a critical role in regulating the functions of the cardiovascular and respiratory systems including blood pressure and airway tone. Smooth muscle cells also proliferate and migrate upon the activation of chemical and mechanical factors, which modulates tissue homeostasis and development. Unfortunately, uncontrolled vascular smooth muscle contractility results in the development of hypertension. Aberrant airway smooth muscle reactivity leads to the progression of airway hyperresponsiveness (AHR), a key feature of asthma. Anomalous smooth muscle growth and migration causes the development of vascular/airway remodeling, a critical pathological process of vascular diseases and asthma. Research on the mechanism of smooth muscle contraction has long been focused on the regulation of myosin activation. Upon contractile stimulation, 20-kDa myosin light chain gets phosphorylated at Ser-19 by myosin light chain kinase, which activates myosin ATPase, and initiates sliding of contractile filaments and smooth muscle contraction (Kamm & Stull, 1989; Tang, Stull, Kubota, & Kamm, 1992). However, there is a wealth of evidence to suggest that actin cytoskeletal remodeling is also critical for smooth muscle contraction. A pool of globular actin (G-actin) is added onto existing filamentous actin (F-actin) in smooth muscle during contractile stimulation. Inhibition of the G-actin to F-actin transition by pharmacological agents (e.g., cytochalasin and latrunculin) and molecular approach attenuates smooth muscle contraction without reducing myosin light chain phosphorylation (Gunst & Zhang, 2008; Kim, Gallant, Leavis, Gunst, & Morgan, 2008; Rembold, Tejani, Ripley, & Han, 2007; Tang, 2015; Tang & Anfinogenova, 2008). Actin filament polymerization may promote smooth muscle force development by enhancing force transmission between the contractile unit and the extracellular matrix (ECM) (Gunst & Zhang, 2008; Kim et al., 2008; Rembold et al., 2007; Tang, 2009; Tang & Anfinogenova, 2008; Wang, Cleary, Wang, & Tang, 2014), and by increasing intercellular connections (Tang, 2015; Wang, Wang, Cleary, Gannon, & Tang, 2015). Thus, myosin can be viewed to serve as an “engine” for smooth muscle contraction

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whereas the actin cytoskeleton may function as a “transmission system” in smooth muscle (Tang, 2015; Wang, Cleary, Wang, Li, & Tang, 2014; Wang, Cleary, Wang, & Tang, 2013). In recent years, accumulating evidence suggests that the actin cytoskeleton of smooth muscle undergoes restructuring amid proliferation and migration. Disruption of actin cytoskeletal reorganization impairs the cellular processes that orchestrate smooth muscle cell growth and migration. This review will recapitulate our current understanding of physiological and cellular properties of the dynamic actin cytoskeleton in smooth muscle. Additionally, the role of actin-regulatory proteins in vascular and pulmonary diseases will be summarized. In particular, there is evidence to suggest that Abelson tyrosine kinase (c-Abl, Abl) is an important molecule that controls actin dynamics in vascular and airway smooth muscle and regulates smooth muscle contraction and cell proliferation/locomotion in vitro, as well as blood pressure, AHR, and vascular/airway remodeling in vivo. These studies indicate that c-Abl may be a novel pharmacological target for the development of new therapy to treat smooth muscle diseases such as hypertension and asthma.

2. ACTIN DYNAMICS AND SMOOTH MUSCLE CONTRACTION 2.1 Critical Role of Actin Dynamics in Smooth Muscle Contraction Cortical actin filaments of smooth muscle cells attach to the plasma membrane at dense plaques. At dense plaques, the cytoplasmic tails of transmembrane integrins link to actin filaments via linker proteins such as vinculin and talin whereas the extracellular domains of integrins interact with the ECM. Another end of cortical actin filaments connect with dense bodies in the cytoplasm. For “deeper” actin filaments (actin filaments between the nucleus and cortical actin), their two ends anchor to adjacent dense bodies in the myoplasm. The majority of actin (thin) filaments localize around myosin (thick) filaments in a rosette array, forming the contractile apparatus. This pool of actin filaments is referred to as “contractile actin.” In addition, a portion of actin filaments does not structurally interact with myosin filaments, which is called “cytoskeletal actin” and plays a role in maintaining the structural integrity of smooth muscle cells (Gunst & Tang, 2000; Small & Gimona, 1998; Tang & Anfinogenova, 2008). Thus, the actin– integrin–ECM connection forms structural basis for the force transmission

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Fig. 1 Schematic illustration of the actin cytoskeleton, contractile units, and the integrin-associated complex in smooth muscle. (A and B) Contractile actin (ConA) localize around myosin filaments (My) in a rosette array, forming the contractile apparatus. Cytoskeletal actin (CskA) does not interact with myosin filaments during the contraction–relaxation cycle, but is important for structural integrity. IC, integrinassociated complex; M, membrane; DB, dense bodies. (C) Interactions of signaling proteins and structural proteins with the integrin-associated complex and actin filaments (see detailed protein interactions at adhesomes in the review by Tang, 2015). Abi1, Abl interactor 1; c-Abl, Abelson tyrosine kinase; Arp2/3, actin-related protein 2/3; CAS, Crkassociated substrates; CTTN, cortactin; GMF-γ, glia maturation factor-γ; HDAC8, histone deacetylase 8; HSP, heat shock protein; N-WASP, neuronal Wiskott–Aldrich syndrome protein; Pfn-1, profilin-1.

between the contractile units and the ECM (Fig. 1). In addition, actin filaments connect with N-cadherin-associated complex (an important intercellular junction) via catenins. Therefore, contractile force can be transmitted among smooth muscle cells (Tang, 2015; Wang et al., 2015).

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2.1.1 Actin Cytoskeleton Is Dynamic in Smooth Muscle Upon Contractile and Mechanical Stimulation Actin filament polymerization transpires in vascular and airway smooth muscle in response to contractile activation, and actin depolymerization occurs during the relaxation of smooth muscle. Several technologies have been used to analyze actin dynamics in cells and tissues. First, the actin fractionation assay has been used to quantify the F-actin/G-actin ratio in cells/ tissues. Studies on vascular smooth muscle have shown that the ratio of F-actin to G-actin is approximately 1.8 in unstimulated arterial smooth muscle tissues, suggesting that approximately 25%–30% of total actin exists in the form of actin monomers. The F-actin/G-actin ratio increases to 5.8 in response to contractile stimulation, indicating that approximately 10%–15% of total actin is G-actin (Kim et al., 2008, 2010; Tang & Tan, 2003a, 2003b). By using a similar assay, a number of other research teams have found that contractile stimulation increases the amount of F-actin and/or decreases the G-actin level in arterial smooth muscle tissues (Chen, Pavlish, Zhang, & Benoit, 2006; Kim et al., 2008; Rembold et al., 2007; Zhang, Huang, & Gunst, 2012). The dynamic state of the actin cytoskeleton has also been verified in airway smooth muscle. In unstimulated airway smooth muscle cells/tissues, 70%–80% of total actin exists in insoluble actin (F-actin) whereas 20%–30% of total actin is found in soluble actin (G-actin). In response to contractile activation, 85%–90% of total actin is F-actin in airway smooth muscle cells/tissues (Gunst & Zhang, 2008; Tang & Gunst, 2004; Tang, Zhang, & Gunst, 2005; Wang, Cleary, Wang, Li, et al., 2014; Wang, Cleary, et al., 2013; Wang, Cleary, Wang, & Tang, 2014; Zhang et al., 2012). Furthermore, the F-actin/ G-actin ratios are reduced in smooth muscle during relaxation (Chen et al., 2006; Hirshman, Zhu, Pertel, Panettieri, & Emala, 2005; Tang & Tan, 2003a). Second, several research groups have also used fluorescent microscopy to evaluate F-actin and G-actin by staining smooth muscle tissues/cells with phalloidin (for F-actin) and DNase I (for G-actin) conjugated with fluorescent labels (Chen, Wang, Li, & Tang, 2009; Hirshman, Zhu, Panettieri, & Emala, 2001; Hirshman et al., 2005; Kim et al., 2010). Treatment of human airway smooth muscle cells with carbachol and endothelin-1 leads to the increase in the ratio of F-actin to G-actin as estimated by fluorescence microscopy, whereas treatment with smooth muscle relaxants (isoproteronol and forskolin) decreases the F-actin/G-actin ratios (Hirshman & Emala, 1999; Hirshman et al., 2001, 2005). In other studies, stimulation with vasoconstrictors enhances the F-actin/G-actin ratios in

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vascular smooth muscle as evaluated by phalloidin and DNase I staining (Chen et al., 2009; Kim et al., 2008, 2010). Third, a decrease of the G-actin pool was observed in contractile airway smooth muscle tissues by using the DNase inhibition assay (Gunst & Zhang, 2008; Tang & Anfinogenova, 2008). Fourth, bioenergetics evidence suggests that actin dynamics was necessary for smooth muscle contraction ( Jones et al., 1999). In addition, Barany and colleagues (Barany, Barron, Gu, & Barany, 2001) have reported a high concentration of G-actin in vascular smooth muscle tissues by assessing the exchange rates of actin-bound nucleotide and observed a decrease of the G-actin pool in response to contractile stimulation. Taken together, these studies demonstrate that actin polymerization and depolymerization occur in various smooth muscle cell/tissue types during the contraction–relaxation cycle. Alterations in mechanical environments also affect the reorganization of the actin cytoskeleton. There is evidence that mechanical stretch induces fluidization and resolidification of smooth muscle cells, suggesting actin cytoskeletal remodeling (actin depolymerization and polymerization) in the cells upon transient stretch (Chen et al., 2010). Passive tension also induces the tyrosine phosphorylation of the cytoskeletal protein paxillin, an index of the actin cytoskeleton reorganization (Tang, 2015; Tang & Gunst, 2001b; Tang, Mehta, & Gunst, 1999). Furthermore, mechanical signals locally imposed on cells triggers structural reorganization of the actin architecture in smooth muscle cells (Deng, Fairbank, Fabry, Smith, & Maksym, 2004; Tang, 2015; Tang & Anfinogenova, 2008). The dynamic feature of the actin architecture in differentiated smooth muscle is evidently distinctive from striated muscle. Skeletal muscle tissues do not have a significant G-actin pool and have lower exchange rate of actinbound nucleotides (Barany et al., 2001). Smooth muscle tissues also contain a much higher ratio of F-actin to myosin than skeletal muscle, and they maintain a significant pool of F-actin that does not interact with myosin (Small, Rottner, & Kaverina, 1999; Tang, 2015; Tang & Anfinogenova, 2008). 2.1.2 Actin Polymerization Pharmacological Agents Regulate Smooth Muscle Contractile State To assess the functional role of actin polymerization in smooth muscle contraction, various pharmacological agents have been used. Cytochalasins are able to cap existing actin filaments at the barbed end, preventing F-actin elongation (Adler, Krill, Alberghini, & Evans, 1983; Cooper, 1987; Tang, 2015; Tang & Anfinogenova, 2008). Latrunculin A binds to G-actin and

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blocks the assembly of G-actin onto actin filaments (Coue, Brenner, Spector, & Korn, 1987; Gunst & Zhang, 2008; Tang & Anfinogenova, 2008). Treatment with cytochalasins or latrunculin A attenuates the contraction in a variety of smooth muscle tissues including vascular and airway smooth muscle (Adler et al., 1983; Kim et al., 2010; Obara & Yabu, 1994; Tang & Tan, 2003a, 2003b). The acute treatment of smooth muscle with these inhibitors does not impair the organization and ultrastructure of contractile filaments (Gunst & Zhang, 2008; Tang & Anfinogenova, 2008). In addition, inhibition of actin polymerization by molecular approaches that interfere with actin-regulatory proteins also attenuates smooth muscle contraction (Anfinogenova, Wang, Li, Spinelli, & Tang, 2007; Jia & Tang, 2010; Kim et al., 2010; Wang, Cleary, Wang, Li, et al., 2014; Wang, Cleary, et al., 2013; Wang, Cleary, Wang, & Tang, 2014; Wu & Gunst, 2015; Zhang et al., 2012; Zhao, Du, Huang, Wu, & Gunst, 2008). Furthermore, actin polymerization by jasplakinolide or F-actin stabilization by phalloidin enhances smooth muscle contraction (Cipolla, Gokina, & Osol, 2002; Jones et al., 1999). More importantly, the suppression of actin polymerization inhibits force development in smooth muscle without reducing myosin light chain phosphorylation, a cellular event that is essential for smooth muscle contraction (Anfinogenova et al., 2007; Jia & Tang, 2010; Kim et al., 2010; Wang, Cleary, Wang, Li, et al., 2014; Wang, Cleary, et al., 2013; Wang, Cleary, Wang, & Tang, 2014; Wu & Gunst, 2015; Zhang et al., 2012; Zhao et al., 2008). Therefore, these results suggest that both actin filament polymerization and myosin activation are necessary for smooth muscle contraction, but they are two parallel cellular processes for the regulation of smooth muscle contraction. Actin polymerization may facilitate smooth muscle contraction by several mechanisms. The actin filaments of smooth muscle cells connect to the cytoplasmic domain of β-integrins via linker proteins such as vinculin, talin, and α-actinin whereas the extracellular portion of β-integrins engages with the ECM (Burridge & Chrzanowska-Wodnicka, 1996; Gunst & Tang, 2000; Gunst, Tang, & Opazo, 2003; Gunst & Zhang, 2008; Small & Gimona, 1998; Tang, 2009; Tang & Anfinogenova, 2008) (Fig. 1C). Thus, the β-integrin-associated complex is able to transmit mechanical force between the contractile unit and the ECM (Burridge & ChrzanowskaWodnicka, 1996; Gerthoffer & Gunst, 2001; Gunst & Tang, 2000; Gunst et al., 2003; Gunst & Zhang, 2008; Small & Gimona, 1998; Tang, 2009; Tang & Anfinogenova, 2008). Expression of an α-actinin mutant hinders the binding of actin filaments to the cytoplasmic tail of β-integrins as well

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as smooth muscle contraction (Gunst & Zhang, 2008). The results suggest that the linkage of actin filaments to integrins is critical for smooth muscle reactivity. Nascent actin polymerization transpires at the near membrane region of smooth muscle (Gunst & Tang, 2000; Gunst et al., 2003; Gunst & Zhang, 2008; Tang, 2009; Tang & Anfinogenova, 2008; Tang & Gunst, 2004; Tang et al., 2005), which may strengthen the engagement of actin filaments to β-integrins and enhance the transmission of contractile force (Gerthoffer & Gunst, 2001; Gunst & Tang, 2000; Gunst & Zhang, 2008; Kim et al., 2010; Tang, 2009; Tang & Anfinogenova, 2008; Wu & Gunst, 2015; Zhang et al., 2012; Zhang, Wu, Wu, & Gunst, 2007). In addition, actin filament assembly may participate in the “latch bridge” formation of contractile filaments, supporting force maintenance under the condition of lower crossbridge phosphorylation (Meeks, Ripley, Jin, & Rembold, 2005; Murphy & Rembold, 2005; Rembold et al., 2007; Tang & Anfinogenova, 2008). In the initial phase of contractile response (<5–10 min after stimulation), relatively fast rates of contraction are associated with transiently high levels of myosin light chain phosphorylation. In sustained contraction, the levels of myosin light chain phosphorylation are relatively low, a phenomenon termed “latch bridge.” Because F-actin levels are high in sustained contraction, it is likely that cytoskeletal actin may tether unphosphorylated myosin head to contractile actin filaments (Rembold et al., 2007; Tang, 2009, 2015; Tang & Anfinogenova, 2008). Furthermore, our recent studies suggest that actin polymerization promotes the recruitment of β-catenin to N-cadherin, which may facilitate the cell-to-cell force transmission and contraction (Wang et al., 2015). Adherens junctions are protein complexes that exist at cell–cell junctions in various cell types including epithelial cells, endothelial cells, and muscle cells, which plays an essential role in intercellular connection and mechanotransduction (Belkin et al., 1994; Jamora & Fuchs, 2002; Pokutta & Weis, 2007; Wang et al., 2015). β-Catenin, a member of the armadillo family of proteins, is a key component of the cadherin–catenin complex in the plasma membrane (Pokutta & Weis, 2007). β-Catenin is a necessary component of the cellular process that regulates smooth muscle contraction ( Jansen, Van Ziel, Baarsma, & Gosens, 2010; Wang et al., 2015). However, β-catenin is not involved in the regulation of actin polymerization, myosin activation, or contractile protein expression ( Jansen et al., 2010; Wang et al., 2015). Contractile stimulation promotes the recruitment of β-catenin to N-cadherin in smooth muscle cells/tissues. This recruitment of β-catenin to N-cadherin is critical for smooth muscle

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Fig. 2 Actin dynamics promotes recruitment of β-catenin to N-cadherin in smooth muscle. Contractile activation of smooth muscle induces actin polymerization, which promotes the coupling of β-catenin with N-cadherin. The increase in the protein– protein interaction may enhance the linkage of actin filaments to the adherens junctions and enhance the intercellular force transmission and smooth muscle contraction. Linkers, linker proteins such as α-catenin, vinculin, and VASP.

contraction (Wang et al., 2015). More importantly, actin polymerization has a positive role in regulating the recruitment of β-catenin to N-cadherin. Together, these findings reveal a novel of adherens junctions in smooth muscle contraction: Contractile stimulation promotes actin polymerization, which may increase the coupling of β-catenin with N-cadherin and facilitate intercellular mechanotransduction (Fig. 2).

2.2 Actin-Regulatory Signaling Networks in Smooth Muscle Contraction In the last 10–15 years, a number of laboratories have investigated the mechanisms that regulate actin dynamics in smooth muscle. In general, the actinregulatory proteins are effector molecules that orchestrate actin dynamics in smooth muscle. Protein tyrosine kinases, serine/threonine kinases, integrins, and small GTPases are upstream regulators for the dynamic actin cytoskeleton. 2.2.1 Regulation of Actin Dynamics by c-Abl Tyrosine Kinase c-Abl is a nonreceptor protein tyrosine kinase that has been implicated to modulate actin cytoskeletal reorganization that mediates cell migration

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(Cleary, Wang, Waqar, Singer, & Tang, 2014; Hu, Bliss, Wang, & Colicelli, 2005; Wang, 2004). c-Abl has been shown to regulate vascular smooth muscle contraction. Knockdown of c-Abl by RNAi inhibits force development in arterial smooth muscle tissues during contractile activation (Anfinogenova et al., 2007). This is the first evidence to demonstrate the role of c-Abl in smooth muscle contraction. Moreover, treatment with a cell permeable peptide or imatinib attenuates vascular smooth muscle contraction ( Jia & Tang, 2010). Administration of the c-Abl inhibitor imatinib relieves the symptoms of patients with pulmonary arterial hypertension (Ghofrani, Seeger, & Grimminger, 2005; ten Freyhaus et al., 2012). The results suggest a pivotal role for c-Abl in pulmonary arterial contraction in humans. However, it should be noted that imatinib may not be specific to c-Abl. c-Abl tyrosine kinase is also necessary for airway smooth muscle contraction. Contractile responses of mouse tracheal rings to acetylcholine (ACh) are reduced in smooth muscle cell-specific knockout mice (Cleary, Wang, Wang, & Tang, 2013). Moreover, short-term treatment with the c-Abl pharmacological inhibitors imatinib (Gleevec, STI-571) (Cleary et al., 2013; Jia & Tang, 2010) and GNF-5 (Zhang et al., 2010) significantly inhibits force development in mouse tracheal rings induced by ACh. Furthermore, treatment with imatinib or GNF-5 induces the relaxation of tracheal rings precontracted by ACh (Cleary et al., 2013). In addition, treatment with imatinib attenuates airway smooth muscle reactivity in vivo (Berlin & Lukacs, 2005). Because smooth muscle contraction is dependent upon myosin light chain phosphorylation and actin polymerization, the effects of c-Abl knockdown or inhibition on myosin phosphorylation and actin dynamics were evaluated. Silencing or inhibition of c-Abl does not affect myosin light chain phosphorylation in smooth muscle (Anfinogenova et al., 2007; Jia & Tang, 2010). However, knockdown of c-Abl attenuates actin polymerization in smooth muscle cells/tissues during contractile stimulation (Anfinogenova et al., 2007; Tang & Anfinogenova, 2008; Wang, Cleary, et al., 2013). Inhibition of c-Abl activation by a cell permeable peptide also diminishes actin dynamics in smooth muscle stimulated by contractile agonists ( Jia & Tang, 2010). These results indicate that c-Abl regulates smooth muscle contraction by controlling actin polymerization during contractile stimulation (Gunst & Zhang, 2008; Kim et al., 2008, 2010; Wang, Cleary, Wang, Li, et al., 2014; Wang, Cleary, et al., 2013; Wang, Cleary, Wang, & Tang, 2014; Zhao et al., 2008).

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c-Abl undergoes phosphorylation at Tyr-412 in smooth muscle upon contractile activation (Anfinogenova et al., 2007; Jia & Tang, 2010). Tyr-412 is located at the activation loop of c-Abl kinase domain. When unstimulated, the activation loop of the kinase domain folds into the active site, thereby preventing binding of both the substrate and the ATP. Phosphorylation at Tyr-412 induces conformation changes; the activation loop no longer blocks the active site, which leads the increase in kinase activity (Anfinogenova et al., 2007; Jia & Tang, 2010; Tang & Anfinogenova, 2008; Wang, 2004). Src appears to be a kinase that mediates c-Abl tyrosine phosphorylation in smooth muscle cells/tissues (Anfinogenova et al., 2007; Ogden et al., 2006; Tang, 2009) as well as in fibroblasts (Plattner, Kadlec, DeMali, Kazlauskas, & Pendergast, 1999) and cancer cells (Khusial et al., 2010). How exactly does c-Abl regulate actin dynamics in smooth muscle? There is accumulating evidence to suggest that c-Abl regulates several effectors which subsequently affect actin dynamics. Abl interactor 1 (Abi1) is an adapter protein that has been implicated in the regulation of actin dynamics in vitro (Innocenti et al., 2005), cell adhesion, and migration (Ryu, Echarri, Li, & Pendergast, 2009; Stradal et al., 2001). N-WASP (an actin nucleation activator) is known to regulate the Arp2/3-mediated actin polymerization and branching (Higgs & Pollard, 2000; Pollard, 2007). Contractile stimulation promotes the association of Abi1 with N-WASP in human smooth muscle cells/tissues (Wang, Cleary, et al., 2013). Furthermore, contractile stimulation activates N-WASP in live smooth muscle cells as evidenced by changes in FRET (fluorescence resonance energy transfer) efficiency of an N-WASP sensor (Wang, Cleary, et al., 2013). Abi1 is necessary for N-WASP activation, actin polymerization, and the contraction in smooth muscle. However, Abi1 does not affect myosin light chain phosphorylation (Wang, Cleary, et al., 2013). More importantly, contractile activation induces the formation of a multiprotein complex including c-Abl, Crk-associated substrates (CAS), and Abi1. CAS is an adapter protein that participates in the regulation of smooth muscle tension development and actin cytoskeletal remodeling (Ishida, Ishida, Suero, Takahashi, & Berk, 1999; Ogden et al., 2006; Tang, 2009; Tang & Tan, 2003a, 2003b). Knockdown of c-Abl and CAS attenuates the activation of Abi1 during contractile activation. Collectively, the results indicate that c-Abl tyrosine kinase regulates the formation of the CAS/Abi1/N-WASP complex, which subsequently activates N-WASP and actin polymerization (Tang, 2009; Tang & Tan, 2003a, 2003b; Wang, Cleary, et al., 2013) (Fig. 3). Moreover, c-Abl tyrosine kinase regulates phosphorylation of CAS, the coupling of CAS with CrkII and

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Fig. 3 Signaling networks for the regulation of actin dynamics in smooth muscle. Contractile agonists and/or mechanical signals activate proteins kinases (c-Abl, FAK, Src, etc.), small GTPases (Rho, Cdc42, and Rac), vimentin, and HDAC8 that regulate the downstream mediators and effectors (Abi1, cortactin, GMF-γ, CAS/CrkII, N-WASP, the Arp2/3 complex, profilin, paxillin, HSP27, mDia, vinculin, etc.) and actin polymerization.

N-WASP activation in arterial smooth muscle (Anfinogenova et al., 2007; Chen & Tang, 2014; Cleary et al., 2014; Ogden et al., 2006; Tang et al., 2005). Cortactin is an actin-regulatory protein that regulates actin filament assembly in in vitro studies as well as adhesion, migration, and endocytosis of nonmuscle cells (Ammer & Weed, 2008; Cosen-Binker & Kapus, 2006). In human smooth muscle cells/tissues, cortactin controls actin polymerization and force development without affecting myosin activation (Wang, Cleary, Wang, Li, et al., 2014). Furthermore, contractile stimulation induces cortactin phosphorylation at Tyr-421 and the association of cortactin with profilin-1 (Pfn-1) that is able to transport actin monomers onto actin filaments (Tang & Tan, 2003a, 2003b; Wang, Cleary, Wang, Li, et al., 2014). The cortactin/Pfn-1 complex is also rapidly recruited to the membrane upon contractile activation. The disruption of the protein–protein

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interaction by a cell permeable peptide attenuates actin polymerization and smooth muscle contraction. More importantly, c-Abl is required for the agonist-induced cortactin phosphorylation and the interaction of cortactin with Pfn-1 (Wang, Cleary, Wang, Li, et al., 2014). These results suggest that c-Abl regulates cortactin phosphorylation, which enhances cortactin/Pfn-1 coupling and smooth muscle contraction (Tang, 2015; Wang, Cleary, Wang, Li, et al., 2014) (Fig. 3). Glia maturation factor-γ (GMF-γ) is a member of actin-depolymerization factor (ADF)/cofilin family that induces actin depolymerization and debranching (Gandhi et al., 2010; Ydenberg et al., 2013). GMF-γ is able to inhibit actin nucleation in vitro (Ydenberg et al., 2013). Moreover, in vitro biochemical studies show that GMF-γ may induce debranching of the actin filament networks (Gandhi et al., 2010; Ydenberg et al., 2013). Knockdown of GMF-γ regulates actin polymerization and the contraction in smooth muscle cells/tissues with little or no effect on myosin phosphorylation (Wang, Cleary, Wang, & Tang, 2014). Contractile activation induces GMF-γ phosphorylation at Tyr-104 and dissociation of GMF-γ from Arp2 of the Arp2/3 complex, which is regulated by c-Abl tyrosine kinase. Furthermore, expression of mutant Y104F GMF-γ attenuates actin polymerization and contraction in smooth muscle (Wang, Cleary, Wang, & Tang, 2014). Thus, c-Abl tyrosine kinase phosphorylates GMF-γ at Tyr-104, which inhibits the ability of GMF-γ to induce actin network disassembly (Fig. 3). 2.2.2 Role of Intermediate Filament-Associated Proteins in Actin Dynamics Vimentin is a major intermediate filament protein in smooth muscle cells/ tissues (Tang, 2008; Wang, Li, Anfinogenova, & Tang, 2007; Wang, Li, & Tang, 2006). The vimentin network is able to regulate the actin cytoskeleton in smooth muscle. Vimentin phosphorylation at Ser-56 by PAK1 and Plk1 leads to its disassembly in smooth muscle, which results in the release of CAS from cytoskeletal vimentin. CAS translocates to the cell cortex and promotes the Arp2/3 complex-mediated actin polymerization (Helfand et al., 2011; Li et al., 2006; Li, Wang, Gannon, et al., 2016; Tang, 2008, 2009, 2015; Tang & Anfinogenova, 2008; Wang et al., 2007). Vimentin dephosphorylation at Ser-56 is mediated by type 1 protein phosphatase in smooth muscle (Li, Wang, & Tang, 2016). In addition, caldesmon is a component of thin filaments in smooth muscle cells. Caldesmon is able to interact with intermediate filaments and F-actin,

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and is required for maintaining the intermediate filament network and actin filaments in smooth muscle cells (Deng, Mohanan, Polyak, & Chacko, 2007). Caldesmon phosphorylation by the serine/threonine protein kinase PFTAIRE1 promotes its binding to F-actin and stress fiber formation (Leung, Ching, & Wong, 2011). Furthermore, Caldesmon phosphorylation by MAP kinase and protein kinase C affects its interaction with myosin and smooth muscle contraction (Adam, Haeberle, & Hathaway, 1989; Gerthoffer et al., 1996; Riseman, Lynch, Nefsky, & Bretscher, 1989; Sutherland & Walsh, 1989). 2.2.3 Regulation of Actin Polymerization by Histone Deacetylase 8 Histone deacetylases (HDACs) are a family of enzymes that regulate nucleosomal histone deacetylation, nucleosome stability, and gene transcription. In general, HDACs induce histone deacetylation and repress gene transcription (de Ruijter, van Gennip, Caron, Kemp, & van Kuilenburg, 2003). HDACs have long been thought to regulate histone deacetylation solely. However, recent studies suggest that some members of HDACs are present in the cytoplasm of mammalian cells, which has been implicated in regulating nonhistone protein deacetylation (Zhang, Yuan, et al., 2007; Zhang et al., 2009). HDAC8 is localized both in the cytoplasm and the nucleus of smooth muscle cells. HDAC8 is required for the contraction of smooth muscle tissues (Li et al., 2014) and cultured smooth muscle cells (Waltregny et al., 2005). Cortactin is an actin-regulatory protein that can be regulated by acetylation/deacetylation (Zhang, Yuan, et al., 2007). When cortactin gets acetylated, its binding to F-actin decreases, and inhibits actin filament assembly. In contrast, deacetylated cortactin interacts with F-actin and increases actin polymerization (Zhang, Yuan, et al., 2007; Zhang et al., 2009). Contractile stimulation induces cortactin deacetylation in smooth muscle tissues. Knockdown or pharmacological inhibition of HDAC8 attenuates cortactin deacetylation, actin polymerization, and contraction without affecting myosin activation. Furthermore, expression of a charge-neutralizing cortactin mutant inhibits contraction and actin dynamics during agonist activation (Li et al., 2014). Taken together, these results suggest a novel paradigm for the regulation of actin dynamics in smooth muscle: Upon contractile activation, HDAC8 induces cortactin deacetylation, which subsequently promotes actin polymerization and the contraction in smooth muscle (Fig. 3).

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2.2.4 The Integrin-Associated Complex Formation in Smooth Muscle As described earlier, transmembrane integrins are localized in membraneassociated dense plaques and connect the ECM with actin filaments in smooth muscle (Gunst & Zhang, 2008; Tang, 2009; Tang & Anfinogenova, 2008). In addition, a number of structural and signaling proteins are present in the integrin-associated structure (Tang, 2015). There is evidence to suggest that contractile stimulation induces the assembly of the multiprotein complex at the membrane, which is critical for the regulation of actin polymerization in smooth muscle ( Jia & Tang, 2010; Kim et al., 2010; Tang, 2015; Tang & Anfinogenova, 2008; Wang et al., 2007; Wu & Gunst, 2015; Zhang et al., 2012; Zhang, Wu, et al., 2007; Zhao et al., 2008). A stable protein complex containing ILK (a β-integrin binding scaffolding protein and protein kinase), PINCH (an ILK binding partners), and α-parvin (an actin-binding protein) is recruited to integrin adhesion sites in response to contractile stimulation, where it interacts with β-integrins and forms a platform for the recruitment of other structural and signaling proteins that are required for processes of actin cytoskeletal remodeling and mechanotransduction (Gunst & Zhang, 2008; Zhang, Wu, et al., 2007). The integrin-associated protein complex may recruit and activate signaling proteins (FAK, paxillin, etc.) (Tang, Turner, & Gunst, 2003; Tang, Wu, Opazo Saez, & Gunst, 2002; Zhang, Wu, et al., 2007) and structural proteins (vinculin, talin, and α-actinin) (Gunst & Zhang, 2008), which eventually promotes actin filament polymerization and strengthens the connection of the actin filaments to the membrane (Tang et al., 2005). 2.2.5 Regulation by Small GTPases in Smooth Muscle Rho, Cdc42, and Rac are the major members of the small GTPase Rho family that functions in a variety of cellular processes including actin polymerization (Gunst & Zhang, 2008; Tang, 2015). Contractile stimulation activates Cdc42 in smooth muscle tissues, and introduction of a dominant Cdc42 mutant attenuates the activity of N-WASP concurrently with the decrease in actin polymerization and force development with little or no effect on myosin light chain phosphorylation (Gunst & Zhang, 2008; Tang & Gunst, 2004; Tang et al., 2005). The results suggest that Cdc42-mediated actin assembly is essential for smooth muscle contraction. Activated Cdc42 binds to the GTP binding domain of N-WASP, inducing a conformational change and activating N-WASP, and triggering actin nucleation and polymerization (Gunst & Zhang, 2008; Higgs & Pollard, 2000; Pollard, 2007; Tang & Gunst, 2004; Tang et al., 2005). Cdc42 activation

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in smooth muscle is regulated by Cdc42GAP, which is reduced upon contractile activation (Cau & Hall, 2005; Li, Spinelli, & Tang, 2009; Li & Tang, 2009; Tang & Gunst, 2004; Tang et al., 2005; Zhang et al., 2012; Zigmond et al., 1998). Cdc42 and Rac are able to activate PAKs in various cell types including smooth muscle (Dechert, Holder, & Gerthoffer, 2001; Jaffer & Chernoff, 2002; Li, Spinelli, & Tang, 2007; Li et al., 2009, 2006; Tang, 2008; Wang et al., 2007). Expression of an inactivate PAK1 (a major isoform in smooth muscle) (Li et al., 2009; Li & Tang, 2009; Tang, 2008; Wang et al., 2007) inhibits the phosphorylation of p38 MAP kinase in smooth muscle cells (Dechert et al., 2001; Gerthoffer & Gunst, 2001). In vitro biochemical studies have shown that unphosphorylated HSP25 (mouse and chicken homologs of HSP27) inhibits actin polymerization whereas phosphorylated HSP27 loses the ability to inhibit actin filament assembly (Gerthoffer & Gunst, 2001; Tang, 2008). There is evidence to suggest that PAK1regulated p38 MAP kinase may phosphorylate MAP kinase-activated protein (MAPKAP) kinase 2 (MK2), which subsequently catalyzes heat shock protein 27 (HSP27) phosphorylation and promotes actin polymerization and contraction (Dechert et al., 2001; Gerthoffer, 2005; Gerthoffer & Gunst, 2001; Tang, 2009; Tang & Anfinogenova, 2008) (Fig. 3). Rho activation promotes actin polymerization in smooth muscle tissues during contractile stimulation (Zhang et al., 2012), and actin stress fiber formation in cultured smooth muscle cells (Hirshman, Togashi, Shao, & Emala, 1998). In smooth muscle tissues, contractile stimulation activates RhoA, which induces the independent recruitment of paxillin–vinculin complexes and FAK to adhesomes on or near the plasma membrane. Activated FAK induces the phosphorylation of paxillin, which facilitates the formation of a complex containing paxillin and Crk II with DOCK180 and PIX GEFs. This complex induces the activation of Cdc42, which in turn promotes the activation of N-WASP, which interacts with the Arp2/3 complex to induce actin polymerization in the cortical region of the smooth muscle cell (Tang, 2015; Zhang et al., 2012). 2.2.6 Regulation by Vasodilator-Stimulated Phosphoprotein (VASP) and Cofilin/ADF Members of the Ena/VASP protein family can promote actin filament elongation. Contractile activation induces VASP Ser-157 phosphorylation in smooth muscle (Wu & Gunst, 2015). Treatment with ACh triggers the formation of VASP–VASP complexes as well as VASP–vinculin and

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VASP–profilin complexes at membrane sites, which may promote cortical actin polymerization (Wu & Gunst, 2015) (Fig. 3). Cofilin/ADF are members of a family of actin-depolymerizing proteins, which bind to aging F-actin, sever actin filaments and provide more free barbed ends of actin filaments for nascent actin polymerization. Cofilin activity is regulated by its phosphorylation at Ser-3, which abolishes the ability of cofilin to bind to F-actin and thus inhibits its severing function (Gunst & Zhang, 2008; Zhao et al., 2008).

2.3 Targeting Actin Remodeling May Be a New Avenue to Treat Hypertension and Asthma As described earlier, smooth muscle hypercontractility contributes to the pathogenesis of hypertension and asthma. The actin-regulatory protein c-Abl regulates smooth muscle contraction by controlling actin remodeling (Anfinogenova et al., 2007; Berlin & Lukacs, 2005; Tang, 2015; ten Freyhaus et al., 2012; Wang, Cleary, Wang, Li, et al., 2014; Wang, Cleary, et al., 2013). Preclinical and clinical studies suggest that the c-Abl pharmacological inhibitors may be used to treat asthma and pulmonary hypertension (Berlin & Lukacs, 2005; Cleary et al., 2013; Tang, 2015; Tang & Gerlach, 2017; ten Freyhaus et al., 2012). This raises a possibility that targeting actin reorganization in smooth muscle may be a new strategy to develop pharmacological therapy to treat hypertension and asthma. Actin-associated proteins including Abi1, cortactin, Pfn-1, GMF-γ, ILK, parvins, and VASP have been documented to regulate smooth muscle force development ( Jia & Tang, 2010; Kim et al., 2008; Tang, 2015; Wang, Cleary, Wang, Li, et al., 2014; Wang, Cleary, et al., 2013; Wang, Cleary, Wang, & Tang, 2014; Wu & Gunst, 2015; Zhang, Wu, et al., 2007). Thus, these proteins may be used as targets to inhibit actin remodeling and develop new treatment for hypertension and asthma. However, it is challenging to develop small molecules that specifically target these proteins. β-Adrenergic receptor agonists are the first-line drugs to treat asthma worldwide. β-Agonists are able to activate the cAMP/PKA pathway to relax airway smooth muscle (Morgan et al., 2014). It has been suggested that the β-agonist isoproteronol is able to decrease the F-actin/G-actin ratios (Hirshman & Emala, 1999; Hirshman et al., 2001, 2005). However, it is currently unknown how β-agonists affect actin remodeling in smooth muscle. Do β-agonists antagonize cytoskeletal signaling induced by contractile agonists? Or do they actually turn on additional mechanisms to affect actin remodeling? Answer to these questions may open new doors to understand

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how β-agonists induce smooth muscle relaxation, and to help develop new class of bronchodilators that can inhibit actin remodeling only or can inhibit both contractile activation and cytoskeletal remodeling.

3. ACTIN DYNAMICS AND SMOOTH MUSCLE CELL PROLIFERATION Cell proliferation is affected by adhesion, spreading, and migration that are controlled by actin cytoskeletal reorganization. Disruption of actin dynamics diminishes cell adhesion, spreading, and migration, which eventually attenuates cell proliferation (Chen & Tang, 2014; Cleary et al., 2014; Dechert et al., 2001; Ding, Lambrechts, Parepally, & Roy, 2006; Gerthoffer, 2008; Jia, Wang, & Tang, 2012; Panetti, 2002; Tang, 2015; Wang, Mercaitis, Jia, Panettieri, & Tang, 2013; Woodring et al., 2004). In this section of the review, I will focus on the role of actin dynamics and c-Abl in growth factor-mediated signaling and cytokinesis.

3.1 Regulation of Growth Factor Signaling by the Dynamic Actin Cytoskeleton Upon the binding of ligands to growth factor receptors, Raf-1 kinase translocates to the plasma membrane, which subsequently activates Raf-1. Activated Raf-1 phosphorylates MEK1/2 (MAPK kinase), which in turn phosphorylates and activates ERK1/2 and promotes cell proliferation (Chang & Karin, 2001; Widmann, Gibson, Jarpe, & Johnson, 1999). ERK1/2 has been shown to control functional state of transcription factors (Elk-1, Ets-2), ribosomal S6 kinase, MNK kinase, and cPLA2, which regulate gene transcription, protein translation, and other processes that facilitate cell proliferation (Billington et al., 2005; Jiang & Tang, 2015; Roberts & Der, 2007). The actin cytoskeleton is able to regulate Raf-1 kinase activation in smooth muscle cells. Platelet-derived growth factor (PDGF) activation of smooth muscle cells induces an increase in the association of Raf-1 with cytoskeletal actin as well as Raf-1 recruitment to the plasma membrane. Inhibition of actin polymerization by latrunculin A attenuates the interaction of Raf-1 with F-actin and Raf-1 redistribution during PDGF stimulation ( Jia et al., 2012; Wang, Mercaitis, et al., 2013). Moreover, inhibition of actin dynamics by latrunculin A diminishes the PDGF-induced MEK1/2 and ERK1/2 phosphorylation, and the proliferation in smooth muscle cells ( Jia et al., 2012; Tang, 2015; Wang, Mercaitis, et al., 2013).

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To rule out the potential nonspecific effects of latrunculin A, RNAi is utilized to inhibit the expression of c-Abl in smooth muscle cells. As described earlier, c-Abl is a nonreceptor tyrosine kinase that is able to specifically regulate actin polymerization in smooth muscle cells (Anfinogenova et al., 2007; Chen et al., 2009; Jia et al., 2012; Tang, 2009, 2015; Tang & Anfinogenova, 2008; Wang, 2004). Knockdown of c-Abl attenuates the PDGF-induced coupling of Raf-1 with F-actin, which is rescued by the reexpression of c-Abl. These results strongly suggest that actin polymerization regulates the interaction of Raf-1 with actin in smooth muscle cells during proliferative response ( Jia et al., 2012; Tang, 2015; Wang, Mercaitis, et al., 2013) (Fig. 4A). Furthermore, Abl knockdown inhibits the PDGF-induced MEK and ERK1/2 phosphorylation and the proliferation in smooth muscle cells ( Jia et al., 2012; Wang, Mercaitis, et al., 2013). However, c-Abl does not affect AKT activation in smooth muscle cells upon growth factor stimulation ( Jia et al., 2012; Wang, Mercaitis, et al., 2013). c-Abl regulation of ERK1/2

Fig. 4 c-Abl regulates the MAPK pathway and cytokinesis in smooth muscle cells. (A) The binding of ligands to growth factor receptors induces c-Abl tyrosine phosphorylation, which promotes Raf-1 activation by controlling actin dynamics. Activated Raf-1 subsequently modulates the activation of MEK and ERK. (B) c-Abl is recruited to the midzone during cytokinesis, which mediates cortactin phosphorylation. Phosphorylated cortactin promotes F-actin assembly, which facilitates contractile ring formation and cytokinesis. F-actin recruits c-Abl to the midzone, providing a positive feedback.

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phosphorylation has also been reported in nonmuscle cells (e.g., fibroblasts) (Mitra, Beach, Feng, & Plattner, 2008). Stimulation with PDGR and endothelin-1 activates c-Abl tyrosine kinase in smooth muscle cells ( Jia et al., 2012; Wang, Mercaitis, et al., 2013). Endothilin-1 receptor is a member of G protein-coupled receptor (GPCR) family whereas PDGF receptor belongs to the family of tyrosine kinase-containing receptors. Both GPCR and tyrosine kinase receptors play an important role in the regulation of smooth muscle cell functions (Goncharova et al., 2012; Widmann et al., 1999). In rat vascular smooth muscle cells, activation of GPCR by agonists induces phosphorylation of Src at Tyr-416, which in turn triggers the activation of c-Abl ( Jia & Tang, 2010; Jia et al., 2012; Ogden et al., 2006). Similarly, cellular challenge with PDGF is able to activate Src, which subsequently mediates phosphorylation of c-Abl in mammalian cells (Hantschel & Superti-Furga, 2004; Jia et al., 2012). Actin polymerization has also been implicated in smooth muscle differentiation. Intraluminal blood pressure stretches the vascular wall and stimulates protein synthesis and contributes to the maintenance of the smooth muscle contractile phenotype. Mechanical stretch of mouse portal veins increased actin polymerization and contractile protein expression, which may be mediated by Rho kinase and cofilin (Albinsson, Nordstrom, & Hellstrand, 2004).

3.2 Role of c-Abl-Regulated Contractile Ring in Cytokinesis of Smooth Muscle Cells During cytokinesis, a contractile ring consisting of actin filaments and myosin II assembles equatorially at the cell cortex. Activated myosin promotes constriction of the contractile ring to induce cleavage furrow ingression and cytoplastic division of a mother cell (Glotzer, 2005). Recent studies suggest that c-Abl also regulates cytokinesis of smooth muscle cells (Chen & Tang, 2014). c-Abl is localized in the contractile ring of smooth muscle cells during cell division. Knockdown or inhibition of c-Abl attenuates cytokinesis in smooth muscle cells (Chen & Tang, 2014). c-Abl regulates cytokinesis by controlling cortactin, a tyrosinephosphorylated protein that has been implicated in the regulation of actin polymerization (Ammer & Weed, 2008; Cosen-Binker & Kapus, 2006). Phosphorylated cortactin is also found in the midzone of dividing cells. c-Abl knockdown or inhibition attenuates cortactin phosphorylation in the midzone and contractile ring formation. Furthermore, the expression

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of a nonphosphorylatable cortactin mutant diminishes cytokinesis. Interestingly, inhibition of actin dynamics diminishes the recruitment of c-Abl in the midzone. Thus, these results uncover a novel mechanism: c-Abl is recruited to the midzone during cytokinesis, which mediates cortactin phosphorylation. Phosphorylated cortactin promotes actin filament assembly, which facilitates contractile ring formation and cytokinesis. F-actin conversely promotes the recruitment of c-Abl in the contractile ring (Fig. 4B) (Chen & Tang, 2014; Tang, 2015).

4. ACTIN DYNAMICS AND SMOOTH MUSCLE CELL MIGRATION Smooth muscle cell migration plays a critical role in the development of vascular and pulmonary systems. Smooth muscle cell motility has also been implicated in the pathogenesis of vascular and pulmonary diseases such as injury-induced plaque formation, hypertension, and asthma (Cleary et al., 2014; Tang & Gerlach, 2017). Almost universally, cell migration includes the cycles of the following four steps: protrusion (lamellipodia) formation at the front, new focal adhesion formation in the front, retraction of the rear, and detachment at the tail (Cleary et al., 2014; Gerthoffer, 2008; Pollard & Cooper, 2009). The actin cytoskeleton undergoes dynamic reorganization during cell crawling, which regulates cell protrusion, focal adhesion assembly/disassembly, and stress fiber formation (Cleary et al., 2014; Gerthoffer, 2008; Pollard & Cooper, 2009). The dynamic actin architecture is regulated by a range of actin-regulatory proteins and signaling networks. In this section, I will summarize our current understanding of physiological properties of actin dynamics and actin-regulatory proteins in cell migration in general and in smooth muscle cell migration in particular.

4.1 Local Actin Dynamics Promotes Lamellipodial Formation The lamellipodia are thin, sheet-like membrane protrusions of motile cells. During directional migration, cells undergo cyclic extension and retraction of lamellipodia to explore their environment. The extent of protrusion at the front is greater than retraction. Thus, the overall consequence of cyclic extension and retraction of the lamellipodium is to promote cell movement forward (Cleary et al., 2014; Gerthoffer, 2008; Krause & Gautreau, 2014; Pollard & Cooper, 2009). The dynamic formation of lamellipodia is regulated

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by local actin network rearrangement, which is characterized by actin branching, elongation, debranching, and depolymerization. As described earlier, actin filament branching is largely mediated by the Arp2/3 complex that consists of seven subunits (Arp2, Arp3, p16, p20, p21, p34, and p44) and is coordinated by upstream regulators (Pollard, 2016; Pollard & Borisy, 2003; Pollard & Cooper, 2009; Tang, 2015). Upon activation of extracellular cues (e.g., the ECM, growth factors, cytokines, and chemoattractant), the small GTPases Cdc42 and Rac1 are able to activate nucleation promoting factors such as neuronal Wiskott–Aldrich syndrome protein (N-WASP) and WASP-family verprolin homologous protein (WAVE) (Pollard, 2016; Pollard & Borisy, 2003; Pollard & Cooper, 2009; Tang et al., 2005). The binding of Cdc42 and Rac1 to the GTPbinding domain of N-WASP induces structural changes of N-WASP from a “closed” conformation to an “open” conformation. The activated N-WASP promotes the interaction of Arp2/3 to the CA (central and acidic) domain and the binding of G-actin to VC (verprolin and acidic) domain, which induces actin filament branching (Fig. 5) (Pollard & Cooper, 2009; Tang et al., 2005). Recent studies suggest that the pleckstrin homology and RhoGEF domain containing G3 (PLEKHG3) protein is a GEF for both Rac1 and Cdc42. PLEKHG3 is recruited and

Fig. 5 N-WASP activation by multiple molecules. In inactive state, N-WASP is autoinhibited through the interaction between the GBD and the VCA region. GTP-loaded Cdc42 binds to GBD, resulting in activation of N-WASP. Binding of SH3 domains to N-WASP can independently compete with the autoinhibitory interaction, and thus can activate N-WASP. SH3-domain containing proteins that activate N-WASP include cortactin, Abi1, CrkII, and Nck. PIP2 and WIP are able to bind to the B domain and EVH1 domain, respectively, and activate N-WASP. Abi1, Abl interactor 1; EVH1, enabled/VASP homology; GBD, GTP-binding domain; Nck, noncatalytic region of tyrosine kinase adaptor protein 1; PIP2, phosphatidylinositol 4,5-bisphosphate; VCA, verprolin, central, and acidic; WIP, WASP-interacting protein.

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selectively binds to new F-actin at the leading edge of migrating fibroblasts. Moreover, PLEKHG3 is regulated by phosphatidylinositol-4,5bisphosphate 3-kinase (PI3K) (Nguyen et al., 2016). N-WASP activity may also be regulated by other proteins such as cortactin, Abi1, Nck1, CrkII, and PIP2 (Anfinogenova et al., 2007; Cleary et al., 2014; Pollard, 2007; Wang, Cleary, et al., 2013) (Fig. 5). In addition, c-Abl has been shown to regulate smooth muscle cell migration (Cleary et al., 2014; Tang, 2015). During smooth muscle cell migration, integrin β1 is localized in the leading cell edge, which recruits and activates c-Abl (Cleary et al., 2014). c-Abl regulates the phosphorylation of the actin-regulatory protein cortactin, which may control the activation of N-WASP, and promote actin filament branching in lamellipodia (Cleary et al., 2014; Lapetina, Mader, Machida, Mayer, & Koleske, 2009; Tang, 2015). Moreover, activated cortactin may recruit Pfn-1 to the leading edge, which promotes actin elongation by transporting G-actin to the barbed end of F-actin (Cleary et al., 2014; Tang, 2015) (Fig. 6).

Fig. 6 Role of c-Abl in smooth muscle cell migration. c-Abl is recruited to the leading cell edge via β1-integrin during spreading and migration. c-Abl induces cortactin tyrosine phosphorylation, which may recruit Pfn-1 to the cell edge and promote local actin filament elongation (1). In addition, phosphorylated cortactin activates N-WASP and Arp2/3-mediated branching (2). Furthermore, c-Abl catalyzes GMF-γ phosphorylation, which may inhibit the Arp2/3-mediated debranching and facilitate more actin dynamics (3). The c-Abl-mediated elongation, branching, and inhibition of debranching promote local actin remodeling and cell migration.

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Vasodilator-stimulated phosphoprotein (VASP) undergoes phosphorylation at Ser-239 during cell adhesion and invasion, which promotes filament polymerization and smooth muscle cell motility (Defawe et al., 2010; Kim et al., 2010). Additionally, the Rho effector formin mDia can nucleate and polymerize actin filaments at barbed end and enhance cell migration (Gerthoffer, 2008). Actin filament elongation and branching in the lamellipodia is balanced by actin filament capping, severing, debranching, and depolymerization. The capping protein CapZ binds to the barbed end, preventing actin filament extension. The actin severing protein gelsolin interacts with aging filaments and severs them to short actin filaments. The actin-depolymerization protein ADF/cofilin has higher affinity for ADP-actin fragments, which eventually leads to actin filament depolymerization (Pollard & Borisy, 2003). Moreover, GMF-γ is necessary for human smooth muscle cell movement (Gerlach, Cleary, Gannon, & Tang, 2015). c-Abl mediated GMF-γ phosphorylation at Tyr-104 regulates lamellipodial formation of motile smooth muscle cells. GMF-γ phosphorylation at Tyr-104 may inhibit the Arp2/3-mediated debranching and stabilize the actin network, which may promote cell migration (Gerlach et al., 2015; Tang, 2015; Wang, Cleary, Wang, & Tang, 2014) (Fig. 6).

4.2 Localized Actin Dynamics Facilitates Focal Adhesion Assembly Focal adhesions are large macromolecular assemblies that form mechanical links between intracellular actin bundles and the ECM. Thus, cell adhesion to the ECM at focal adhesions allows cells to crawl during migration. Nascent adhesions form at the leading edge and grow into focal complexes in lamellipodia. Some focal complexes undergo a rapid turnover at the rear of the lamellipodia whereas others become mature focal adhesions that will ultimately disassemble at the cell rear (Tang & Gerlach, 2017). Engagement of integrins with the specific motif of the ECM (e.g., RGD sequence) triggers focal adhesion formation by inducing integrin aggregation and recruiting the structural proteins such as talin, vinculin, tensin, α-actinin, integrin-linked kinase (ILK), and parvins as well as signaling proteins including FAK, paxillin, Cdc42, and Rac1 (Tang, 2015). The recruitment of structural proteins and signaling proteins leads to focal adhesion maturation characterized by increases in the sizes of focal complexes (μm ranges) and enhanced linkage of the actin cytoskeleton to the ECM. Focal adhesion maturation is driven in part by myosin II-dependent stress fiber

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formation and enhanced tension at adhesion sites (Oakes, Beckham, Stricker, & Gardel, 2012). Signaling proteins in focal adhesions are also able to activate cascades to promote actin polymerization (Tang, 2009, 2015; Tang & Anfinogenova, 2008; Tang & Gunst, 2001a; Tang et al., 2002).

4.3 Actin Dynamics Facilitates Stress Fiber Formation During migration, external signals induce stress fiber assembly and activate actomyosin ATPase, which generate traction force to propel the cell forward. Stress fibers are contractile bundles containing actin filaments and myosin II filaments. The engagement of integrins with the ECM activates the small GTPase RhoA, which is able to promote actin nucleation and stress fiber assembly by activating mDia. In addition, Rac1 activates p21-activated kinase (PAK), which phosphorylates and activates Lim kinase (LIMK). Activated LIMK mediates cofilin phosphorylation and inhibits actin filament depolymerization, thus limiting the amount of actin turnover and increasing stress fiber formation (Murali & Rajalingam, 2014; Schoenwaelder & Burridge, 1999).

5. ACTIN-REGULATORY PROTEINS AND VASCULAR/ PULMONARY DISEASES As described earlier, actin-regulatory proteins have been shown to regulate actin dynamics, which orchestrates smooth muscle contraction, cell proliferation, and migration. In the last decade, scientists in this field have identified a number of actin-regulatory proteins that contribute to the pathogenesis of vascular and pulmonary diseases.

5.1 c-Abl Tyrosine Kinase (1) Pulmonary arterial hypertension: Pulmonary arterial hypertension is characterized by arterial contraction and remodeling in the lung. c-Abl plays a critical role in regulating arterial constriction and smooth muscle cell proliferation (Anfinogenova et al., 2007; Chen et al., 2009; Jia & Tang, 2010; Vallieres, Petitclerc, & Laroche, 2009). The potential therapeutic role of imatinib in pulmonary arterial hypertension has been tested in patients. Treatment with imatinib relieves the symptoms of a patient with pulmonary arterial hypertension (Ghofrani et al., 2005). Results from Phase II and III clinical trials suggest that imatinib has potent and prolonged efficacy in patients with severe pulmonary arterial hypertension (ten Freyhaus et al., 2012). (2) Regulation of blood pressure: Compared to wild-type mice, c-Abl knockout mice display lower blood pressure (Chen et al., 2009). Moreover,

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smooth muscle specific knockout of c-Abl (Cleary et al., 2013) reduces Angiotensin II-induced hypertension (Our unpublished data). (3) Asthma pathogenesis: Asthma is characterized by AHR and airway remodeling, which are largely due to increased airway smooth muscle contractility (Amrani et al., 2004; Cleary et al., 2013; Tang, 2015) and cell proliferation (Ammit & Panettieri, 2003; Chen & Tang, 2014; Dekkers et al., 2010; Wang, Mercaitis, et al., 2013). The expression of c-Abl is upregulated in airway smooth muscle tissues of an animal model of asthma and in asthmatic human airway smooth muscle cells (Cleary et al., 2013; Liao, Panettieri, & Tang, 2015). Conditional knockout of c-Abl in smooth muscle attenuates airway resistance in an animal model of asthma. Intranasal instillation with the c-Abl inhibitors imatinib and GNF-5 also inhibits airway resistance in the animals of asthma. These results are supported by a previous study by others (Berlin & Lukacs, 2005). The allergen-induced smooth muscle mass and cell proliferation are also reduced in the airways of c-Abl knockout mice or the inhibitor-treated mice exposed to the allergen (Cleary et al., 2013). (4) Atherosclerosis: c-Abl has been implicated in the pathogenesis of atherosclerosis; inhibition of c-Abl by imatinib attenuates the progression of diabetesassociated atherosclerosis (Lassila et al., 2004).

5.2 Rho and Rho Kinase The roles of RhoA and Rho kinase in smooth muscle contraction and cell proliferation/locomotion are well described (Bond, Wu, Sala-Newby, & Newby, 2008; Murali & Rajalingam, 2014; Sakurada et al., 2003; Schoenwaelder & Burridge, 1999; Zhang et al., 2012). Increased RhoA and Rho kinase activities play a causative role in the pathogenesis of hypertension in various animal models through combined effects in arteries, kidney, and central nervous system. Rho kinase inhibitors (Y27632, fasudil) decreases blood pressure in experimental hypertension (Loirand & Pacaud, 2014). In addition, Th2 cytokines could increase the expression of RhoA in airway smooth muscle (Gour & Wills-Karp, 2015). Inhibition of the RhoA/Rho kinase hinders the development of airway remodeling in experimental asthma (Kume, 2008).

5.3 Others There is evidence that p38 inhibition reduced smooth muscle cell migration. Treatment with an inactive PAK1 attenuated p38 activation and smooth muscle migration (Gerthoffer, 2008). Moreover, p38 activity is associated with aortic smooth muscle contraction (Lee et al., 2007). Interestingly,

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inhibition of p38 lowered blood pressure in rats (Komers et al., 2007), and suppressed airway remodeling in an animal model of asthma (Liang et al., 2013). β1-integrin is associated with asthma pathogenesis; treatment with RGD peptide blocks integrin activation and reduces airway remodeling in asthmatic animals (Dekkers et al., 2010). In addition, the expression of α1and α5-integrin was upregulated in experimental pulmonary hypertension (Umesh, Paudel, Cao, Myers, & Sham, 2011). Furthermore, neointima formation is a major pathological process after percutaneous coronary intervention, bypass operation, or graft vasculopathy. It has been widely accepted that intimal smooth muscle cells in proliferative vascular diseases are derived largely from resident medial smooth muscle cells (Daniel et al., 2010). Inhibition of β1-integrin expression is associated with reduced neointima formation in vivo (Daniel et al., 2010; Karki, Kim, & Kim, 2013). Vascular remodeling is a key feature of systemic hypertension. Pfn-1 has been shown to mediate vascular remodeling in animal models. Pfn-1 knockdown inhibits arterial remodeling in hypertensive rats whereas overexpression of Pfn-1 promotes vascular remodeling (Wang, Zhang, et al., 2014). A common cortactin gene variation has been found to confer susceptibility of severe asthma (Ma et al., 2008). Since cortactin regulates smooth muscle contraction (Wang, Cleary, Wang, Li, et al., 2014) and cell protrusion formation (Cleary et al., 2014), it is likely that cortactin-associated contraction and migration may contribute to asthma pathogenesis. FAK is able to regulate smooth muscle contraction (Tang, 2015; Tang & Gunst, 2001a) and cell migration by controlling dynamics of focal adhesions and the actin cytoskeleton (Serrels et al., 2007; Tang, 2009, 2015; Tang & Anfinogenova, 2008; Tang & Gunst, 2001a; Tang et al., 2002). Inhibition of FAK phosphorylation and activation are associated with reduced neointima formation in vivo (Daniel et al., 2010; Karki et al., 2013). In addition, formin mDia1 has been shown to mediate neointima expansion in an animal model (Toure et al., 2012). Furthermore, β-catenin (Kumawat, Koopmans, & Gosens, 2014) and PI3K-γ (Lim et al., 2009) have been implicated in asthma pathogenesis.

6. CONCLUSION Actin cytoskeletal remodeling plays a critical role in smooth muscle contraction, cell proliferation, and migration. Understanding how actin

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dynamics is regulated in smooth muscle is fundamental to our knowledge regarding smooth muscle biology and disease. A variety of proteins have been discovered to orchestrate actin dynamics in smooth muscle. c-Abl is an important molecule that controls actin dynamics, contraction, growth, and motility in smooth muscle in vitro; and contributes to the pathogenesis of vascular/airway remodeling and AHR in vivo. Although the roles of actin-associated proteins (e.g., Abi1, cortactin, Pfn-1, ILK, and parvins) in smooth muscle functions have been documented in in vitro studies, their roles in blood pressure, airway tone, vascular/airway wall homeostasis, and vascular/pulmonary diseases in vivo are yet to be elucidated. Furthermore, targeting actin remodeling via these proteins or other molecules may be a new avenue to develop pharmacological treatment of hypertension and asthma.

CONFLICT OF INTEREST The author declares no competing interests.

ACKNOWLEDGMENTS This work was supported by NHLBI Grants HL-110951, HL-113208, and HL-130304 from the National Institutes of Health (to D.D.T.).

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