Critical role of actin-associated proteins in smooth muscle contraction, cell proliferation, airway hyperresponsiveness and airway remodeling

There is accumulating evidence to suggest that actin filament polymerization transpires in smooth muscle in response to contractile activation, and actin depolymerization occurs during the relaxation of smooth muscle. First, we and others have used an actin fractionation assay and calculated that 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 [11, 16‐18, 27‐30]. By using the fractionation assay, a number of 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 [12, 13, 29‐31]. Furthermore, the F-actin/G-actin ratios are reduced in smooth muscle during relaxation [31‐33]. Second, several groups including our laboratory have also used fluorescent microscopy to evaluate F-actin and G-actin by reacting smooth muscle tissues/cells with phalloidin (for F-actin) and DNase I (for G-actin) conjugated with fluorescent labels [33‐36]. 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 [33, 36, 37]. In other studies, stimulation with vasoconstrictors enhances the F-actin/G-actin ratios in vascular smooth muscle as evaluated by fluorescent microscopy [12, 34, 35]. Third, a decrease of the G-actin pool was observed in contractile airway smooth muscle tissues by using the DNase inhibition assay [11, 14]. In addition, Barany et al. also documented a reduced G-actin content in contractile arterial smooth muscle by measuring the exchange rates of actin-bound nucleotide [11, 38]. Taken together, these studies demonstrate that actin polymerization and depolymerization occur in various smooth muscle cell/tissue types during the contraction-relaxation cycle.

The inhibition of actin polymerization by pharmacological agents such as cytochalasin or latrunculin attenuates the contraction in a variety of smooth muscle tissues including airway smooth muscle [32, 35, 39‐41]. Cytochalasin caps existing actin filaments at the barbed end, preventing F-actin elongation whereas latrunculin binds to G-actin and blocks the assembly of G-actin onto actin filaments [11, 14, 42, 43]. The short-term treatment of smooth muscle with these inhibitors does not impair the organization and ultrastructure of contractile filaments [11, 14]. Furthermore, inhibition of actin polymerization by molecular approaches that interfere with actin-regulatory proteins also attenuates smooth muscle contraction [16‐18, 30, 35, 44‐48]. More importantly, the suppression of actin polymerization inhibits force development in smooth muscle with little or no effects on myosin light chain phosphorylation, a cellular event that is essential for smooth muscle contraction [16‐18, 30, 35, 44‐48]. Therefore, these results suggest that actin filament polymerization and myosin activation are two parallel cellular processes that are fundamental for the regulation of smooth muscle contraction.

How does actin polymerization affect the contraction in smooth muscle? First, 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 extracellular matrix [11, 14, 15, 25, 26, 49, 50]. Thus, the actin-integrin-matrix connection provides the structural basis for the force transmission between the contractile unit and the extracellular matrix [11, 14, 15, 25, 26, 49‐53]. Nascent actin polymerization may occur at the cell cortex of smooth muscle [11, 14, 15, 25, 27, 28, 49, 54]. Cortical actin assembly may strengthen the linkage of actin filaments to integrins and enhance the transmission of contractile force [11, 14, 15, 25, 30, 35, 44, 53, 55].

Second, actin filament assembly may participate in the “latch” formation of contractile filaments, supporting force maintenance under the condition of lower crossbridge phosphorylation [13, 14, 56, 57]. Third, our recent studies suggest that actin polymerization promotes the recruitment of β-catenin to N-cadherin (critical components of intercellular junctions), which may facilitate the cell-to-cell force transmission and contraction [58].

Abl is a non-receptor protein tyrosine kinase that is ubiquitously expressed and has been implicated to function in a variety of cellular processes including the regulation of the actin cytoskeleton that mediates cell migration and adhesion [59‐61]. There is evidence that Abl tyrosine kinase is necessary for airway smooth muscle contraction. Smooth muscle cell–specific knockout of Abl attenuates contractile responses of mouse tracheal rings to acetylcholine (ACh) [5]. Moreover, acute treatment with the Abl pharmacological inhibitors imatinib (Gleevec, STI-571) [5, 47, 62] and GNF-5 [63] 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 [5]. In addition, treatment with imatinib attenuates airway smooth muscle reactivity in vivo [64].

Abl has also been shown to regulate vascular smooth muscle contraction. Knockdown of Abl inhibits force development in arterial smooth muscle tissues during contractile activation [48]. Moreover, treatment with a cell permeable peptide or imatinib attenuates vascular smooth muscle contraction [47]. Interestingly, administration of the Abl inhibitor imatinib lessens the clinical symptoms of patients with pulmonary arterial hypertension [65, 66], suggesting a role for Abl in pulmonary arterial contraction.

How does Abl regulate force development in smooth muscle? To answer the question, the effects of Abl knockdown or inhibition on actin dynamics and myosin phosphorylation was evaluated. Knockdown of Abl attenuates actin polymerization in smooth muscle cells/tissues during contractile stimulation [14, 17, 48]. Inhibition of Abl activation by a cell permeable peptide also diminishes actin dynamics in smooth muscle stimulated by contractile agonists [47]. However, silencing or inhibition of Abl does not affect myosin light chain phosphorylation in smooth muscle [47, 48]. These observations suggest that Abl regulate smooth muscle contraction by controlling actin polymerization, but not myosin activation during contractile stimulation [11, 12, 16‐18, 35, 45].

Contractile stimulation induces phosphorylation of Abl tyrosine kinase at Tyr-412 in smooth muscle [47, 48]. Tyr-412 is located at the activation loop of Abl kinase domain. When unstimulated, the activation loop of the Abl kinase domain folds into the active site, thereby preventing binding of both the substrate and ATP. Phosphorylation at Tyr-412 induces conformation changes; the activation loop no longer blocks the active site, which leads the increase in kinase activity [14, 47, 48, 60]. Abl tyrosine phosphorylation is regulated by Src in smooth muscle cells/tissues in response to contractile activation [15, 48, 67]. Similarly, Abl phosphorylation is also mediated by Src in fibroblasts [68] and cancer cells [69].

Abl regulates actin dynamics in smooth muscle by controlling several downstream effectors. Abi1 is an adapter protein that has been implicated in the regulation of actin dynamics in vitro [70], cell adhesion and migration [71, 72]. In human airway smooth muscle cells/tissues, contractile stimulation induces an increase in the association of Abi1 with N-WASP (an actin nucleation activator) [17]. N-WASP is known to regulate the Arp2/3-mediated actin polymerization and branching [55, 73, 74]. Furthermore, contractile stimulation activates N-WASP in live smooth muscle cells as evidenced by changes in fluorescence resonance energy transfer efficiency of an N-WASP sensor [17]. Abi1 is necessary for N-WASP activation, actin polymerization and the contraction in smooth muscle. However, Abi1 does not affect myosin light chain phosphorylation [17]. More importantly, contractile activation induces the formation of a multiprotein complex including Abl, Crk-associated substrate (CAS) and Abi1. CAS is an adapter protein that participates in the regulation of smooth muscle tension development [15, 32, 39, 67]. Knockdown of Abl and CAS attenuates the activation of Abi1 during contractile activation. Collectively, the results indicate that Abl tyrosine kinase is able to regulate the formation of the CAS/Abi1/N-WASP complex, which subsequently activates N-WASP and actin polymerization [15, 17, 32, 39] (Fig. 1).

Cortactin is an actin-regulatory protein that is able to regulate actin filament assembly in in vitro studies as well as adhesion, migration, endocytosis of nonmuscle cells [75, 76]. In human airway smooth muscle cells/tissues, cortactin controls actin polymerization and force development without affecting myosin activation [18]. 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 [18, 32, 39]. The cortactin/Pfn-1 complex is also rapidly recruited to the membrane upon contractile activation. The disruption of the protein-protein interaction by a cell permeable peptide attenuates actin polymerization and smooth muscle contraction. More importantly, Abl has a critical role in regulating the agonist-induced cortactin phosphorylation and the interaction of cortactin with Pfn-1 [18]. These results suggest that cortactin phosphorylation and cortactin/Pfn-1 coupling play a pivotal role in regulating actin dynamics and smooth muscle contraction. Abl regulates the association of cortactin with Pfn-1 and actin polymerization by catalyzing cortactin phosphorylation (Fig. 1).

GMF-γ is a member of actin-depolymerizing factor (ADF)/cofilin family that is widely expressed in eukaryotes and plays a central role in reorganizing the actin cytoskeleton by inducing actin network debranching [77, 78]. GMF-γ is able to inhibit actin nucleation in vitro [78]. Moreover, in vitro biochemical studies show that GMF-γ may induce debranching of the actin filament networks [77, 78]. Knockdown of GMF-γ enhances actin polymerization and the contraction in human airway smooth muscle cells/tissues with little or no effect on myosin phosphorylation [16]. 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 Abl tyrosine kinase. Furthermore, expression of mutant Y104F GMF-γ attenuates actin polymerization and contraction in smooth muscle [16]. Thus, Abl tyrosine kinase is able to phosphorylate GMF-γ at Tyr-104, which inhibits the ability of GMF-γ to induce actin network disassembly (Fig. 1).

Transmembrane integrins are localized in membrane-associated dense plaques (similar to focal adhesion sites of cultured cells) and connect the extracellular matrix with the actin cytoskeleton in smooth muscle [11, 14, 15]. In addition, a number of structural and signaling proteins are found in the integrin-associated structure (Fig. 2). During cell migration, the engagement of integrins with the extracellular matrix initiates the recruitment of actin-associated proteins to the membrane, which eventually promotes actin polymerization [11, 61, 79]. 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 [30, 35, 44, 45, 47, 55, 80, 81].

In canine tracheal smooth muscle, 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 the processes of actin cytoskeletal remodeling and mechanotransduction [11, 55].

Paxillin, a scaffolding and signaling protein, is required for tracheal smooth muscle contraction [82, 83] and is also recruited to membrane adhesion sites in response to contractile stimulation, where it associates with the ILK-associated complex [55]. Paxillin has been shown to undergo tyrosine phosphorylation in response to contractile stimulation in many smooth muscle cell and tissue types [13, 84‐86]. The tyrosine phosphorylation of paxillin at Tyr-31 and −118 promotes its interaction with the SH2/SH3 adaptor protein CrkII, which binds to N-WASP and contributes to N-WASP and Arp2/3 activation [27]. Paxillin phosphorylation is mediated by FAK in smooth muscle [87, 88].

Integrin activation may also stimulate FAK and paxillin in smooth muscle; the tyrosine phosphorylation of FAK and paxillin is mechanosensitive (integrin is a known mechanosensor) in smooth muscle [86, 88].

in smooth muscle [86, 88]. Cyclic strain promotes Pyk2 and FAK phosphorylation at focal adhesion sites in cells. The integrin-mediated activation of tyrosine kinases in turn modulates the functional status of downstream molecules such as paxillin and Hic-5 that are related to remodeling of the actin architecture [14].

Contractile stimulation also induces the recruitment of structural proteins that participate in the linkage of actin filaments to β-integrin in adhesion sites, which may strengthen the connection of actin filaments with the membrane. The cytoskeletal protein vinculin is recruited to the ILK-associated complex at adhesion sites [55]. Vinculin binds to talin via its head domain and to actin filaments via its tail domain. Contractile activation induces vinculin phosphorylation, which promotes the binding of vinculin to talin and actin filaments and tension development [29, 51].

α-Actinin is an actin cross-linking protein that also binds to integrin proteins. Stimulation with a contractile agonist induces rapidly the recruitment of α-actinin to the integrin-associated complexes of canine tracheal smooth muscle cells/tissues [52]. In the tracheal tissues, the recruitment of α-actinin is required for smooth muscle contraction but not for actin polymerization or myosin activation [52]. These observations demonstrate that disruption of the linkage of actin filaments to integrin-associated adhesions is sufficient to inhibit smooth muscle contraction [11, 14, 35] (Fig. 2).

Rho, Cdc42, and Rac are the members of the small GTPase Rho family. There is ample evidence that Cdc42 regulates actin polymerization in various cell types including smooth muscle cells [27, 28, 30, 89‐92]. Activated Cdc42 binds to the GTP binding domain of N-WASP, inducing a conformational change and activating N-WASP, and triggering the nucleation of actin polymerization and actin filament branching [11, 27, 28, 73, 74]. In canine tracheal smooth muscle tissues, introduction of a dominant Cdc42 mutant depresses the activity of N-WASP concurrently with the decrease in actin polymerization and force development [11, 27, 28], suggesting that Cdc42-mediated actin assembly is essential for smooth muscle contraction.

Cdc42 and Rac are known to activate PAKs in mammalian cells [80, 93‐96]. Although 6 isoforms have been found thus far, PAK1 is a major isoform in smooth muscle cells [80, 91‐93, 95‐98]. PAK has been shown to participate in the regulation of p38 MAP kinase in smooth muscle upon external activation [53, 93]. The activation of p38 MAP kinase may modulate the actin cytoskeleton and cell migration via heat shock protein 27 (HSP27) [14, 15, 53, 93]. 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 [53, 94, 99, 100]. p38 MAP kinase may phosphorylate MAP kinase-activated protein (MAPKAP) kinase 2 (MK2), which subsequently catalyzes HSP27 phosphorylation and promotes actin polymerization and contraction [14, 53, 101] (Fig. 2).

PAK1 also regulates smooth muscle contraction by controlling the vimentin intermediate filament network [80, 91, 92, 94, 95, 97, 102]. Vimentin network undergoes phosphorylation at Ser-56 and spatial reorganization in smooth muscle cells/tissues in response to contractile stimulation, which is critical for smooth muscle force development [80, 95, 97, 102]. PAK1 deficiency reduces vimentin phosphorylation, reorientation of the vimentin network and smooth muscle contraction. Moreover, PAK1 is able to catalyze vimentin phosphorylation in vitro [80, 91, 92, 94, 95, 97, 102, 103]. Thus, PAK1 regulates smooth muscle force development in part by modulating vimentin phosphorylation and reorientation of the vimentin network.

Rho activation promotes actin stress fiber formation in cultured smooth muscle cells [104, 105], and actin polymerization in smooth muscle tissues during contractile stimulation [30]. In canine tracheal smooth muscle, contractile stimulation activates RhoA, which induces the independent recruitment of paxillin-vinculin complexes and FAK to cell adhesomes. Activated FAK induces the phosphorylation of paxillin, which remains bound to activated vinculin. Paxillin phosphorylation also 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 [30] (Fig. 2).

Histone deacetylases (HDACs) are a family of enzymes that are originally identified as key regulators of histone deacetylation, nucleosome stability and gene transcription [106, 107]. The HDAC family members have long been thought to regulate nucleosomal histone acetylation solely. HDACs typically induce histone deacetylation and repress gene transcription [108]. However, recent studies suggest that some HDACs are present in the cytoplasm of nonmuscle cells, which has been implicated in regulating cell migration and microtubule dynamics [109, 110].

HDAC8 is localized both in the cytoplasm and the nucleus of mouse and human airway smooth muscle cells. HDAC8 is required for the contraction of smooth muscle tissues [111] and cultured smooth muscle cells [112]. In addition, cortactin is an actin-regulatory protein that undergoes deacetylation during migration of NIH 3 T3 cells [110]. Contractile stimulation induces cortactin deacetylation in mouse and human 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 [111]. 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 in turn promotes actin polymerization and the contraction in airway smooth muscle (Fig. 2).

Cofilin/ADF are members of a family of “actin-dynamizing proteins,” which are able to regulate the availability of actin monomers for actin filament assembly [11]. Cofilin binds to F-actin, severs actin filaments and provides 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 [11, 45, 113].

In canine tracheal smooth muscle, contractile activation decreases cofilin phosphorylation at Ser-3, which is associated with force development [45]. Expression of an inactive phospho-cofilin mimetic (cofilin S3E) in the smooth muscle tissues inhibits endogenous ADF/cofilin dephosphorylation and actin polymerization. Expression of cofilin S3E in the tissues depresses tension development in response to ACh, but it does not influence myosin light chain phosphorylation. These observations verify the role of cofilin in regulating the availability of actin monomers for actin polymerization in smooth muscle [11, 45]. In addition, cofilin phosphorylation at Ser-3 is regulated by protein phosphatase 2B [11, 45].

Members of the Ena/VASP protein family can promote actin polymerization by elongating actin filaments. The elongation mechanism involves VASP oligomerization and its binding to profilin and vinculin. In tracheal smooth muscle, treatment with ACh or forskolin (an adenylyl cyclase activator) increases VASP Ser-157 phosphorylation [44]. VASP undergoes phosphorylation at Ser-157 in a biphasic manner in aortic smooth muscle during stimulation with phenylephrine [35]. Treatment with ACh but not forskolin triggers the formation of VASP-VASP complexes as well as VASP-vinculin and VASP-profilin complexes at membrane sites. VASP-VASP complex formation and the interaction of VASP with vinculin and profilin are inhibited by an inactive vinculin mutant, but VASP phosphorylation and membrane localization are unaffected [44]. Gunst et al. conclude that VASP phosphorylation at Ser-157 mediates its localization at the membrane, but Ser-157 phosphorylation and membrane localization are not sufficient to activate its actin polymerization activity (as in the forskolin case). The interaction of VASP with activated vinculin at membrane sites is essential for VASP-mediated actin polymerization (Fig. 2) [44].