Membrane adhesion junctions regulate airway smooth muscle phenotype and function

Abstract

The local environment surrounding airway smooth muscle (ASM) cells has profound effects on the physiological and phenotypic properties of ASM tissues. ASM is continually subjected to the mechanical forces generated during breathing and to the constituents of its surrounding extracellular milieu. The smooth muscle cells within the airways continually modulate their properties to adapt to these changing environmental influences. Smooth muscle cells connect to the extracellular cell matrix (ECM) at membrane adhesion junctions that provide mechanical coupling between smooth muscle cells within the tissue. Membrane adhesion junctions also sense local environmental signals and transduce them to cytoplasmic and nuclear signaling pathways in the ASM cell. Adhesion junctions are composed of clusters of transmembrane integrin proteins that bind to ECM proteins outside the cell and to large multiprotein complexes in the submembranous cytoplasm. Physiological conditions and stimuli from the surrounding ECM are sensed by integrin proteins and transduced by submembranous adhesion complexes to signaling pathways to the cytoskeleton and nucleus. The transmission of information between the local environment of the cells and intracellular processes enables ASM cells to rapidly adapt their physiological properties to modulating influences in their extracellular environment: mechanical and physical forces that impinge on the cell, ECM constituents, local mediators, and metabolites. The structure and molecular organization of adhesion junction complexes and the actin cytoskeleton are dynamic and constantly changing in response to environmental influences. The ability of ASM to rapidly accommodate to the ever-changing conditions and fluctuating physical forces within its local environment is essential for its normal physiological function.

CLINICAL HIGHLIGHTS.

The adhesion junctions that bind cells to the extracellular matrix within airway smooth muscle (ASM) tissues have historically been viewed as static structural components of the cell. These junctions are now recognized to play a critical regulatory role in ASM tissues: they detect conditions in the external environment of the cell and transduce that information to intracellular pathways that regulate the physiological properties of the smooth muscle. Pathophysiological conditions alter the extracellular environment of the smooth muscle cell and induce changes in cell structure and function that contribute to its dysfunction. Unraveling the complex molecular events that govern the effects of extracellular influences on the normal physiological function of the ASM as well as their role in inducing the pathological changes that occur with disease may open avenues for the development of new therapeutic approaches.

1. INTRODUCTION

Airway smooth muscle (ASM) serves multiple physiological functions within the lungs. Many of these functions depend on the ability of the muscle to detect and respond to the conditions within the surrounding environment of the tissue. ASM regulates the caliber of airways within the bronchial tree so that airflow can be directed to lung regions with optimal gas exchange, and it constricts airways in response to foreign particles, debris, noxious gases, and other irritating substances to prevent their inhalation (1–5). ASM also dynamically adjusts the compliance and caliber of individual airways to enable the homogeneous inflation and deflation of the lungs and to optimize airflow during inspiration and expiration (6–9). In addition, ASM functions as an immunomodulatory and synthetic organ: it synthesizes and secretes cytokines, chemokines, and autoregulatory molecules into the local extracellular space and makes connective tissue proteins that remodel the local milieu of the tissue (10–14).

ASM is present within the walls of all the airways as well as in the lung parenchyma, where it is found in alveolar ducts and within the tissue surrounding alveoli (1, 2, 15, 16). The structural and histological organization of ASM tissue within the walls of the airways varies among different airway generations in accordance with the differences in the specialized functions of these airways. In the trachea, smooth muscle is localized within the posterior membrane, a thin membranous sheet of tissue that spans the ends of horseshoe-shaped bands of cartilage. In airways within the bronchial tree, individual smooth muscle cells are organized into bundles that are arrayed circumferentially around the airway lumen. In larger bronchi, the circumferential smooth muscle layer is surrounded by plates of cartilage, whereas the noncartilaginous smaller conducting and respiratory airways are embedded tightly within the lung parenchymal tissue.

The smooth muscle within all the airways is subjected to physical forces caused by breathing maneuvers. Inflation and deflation of the lungs stretch and retract the walls of all the airways, resulting in forces on the walls of airways that are transmitted to ASM tissues via the attachments of smooth muscle cells to the surrounding tissues and to each other (6, 17–19). Expansion and retraction of the trachea also result in the circumferential stretch and retraction of tracheal smooth muscle (20). Thus, all ASM must continually respond and adapt to the physical forces imposed on the airways during breathing.

The physiological effects of the mechanical forces exerted on the airways during breathing and the effects of mechanical perturbations on ASM have been recognized for decades and studied extensively in humans, experimental animals, and isolated airway tissues and cells (7–9, 20–32). Studies in experimental animals and in human subjects have demonstrated that inflation of the lungs and deep inspiration during breathing decrease airway resistance and increase expiratory flow (27). The cycles of stretch and retraction imposed on ASM during tidal breathing keep the airways dilated, maintain their patency, and maintain normal low levels of airway reactivity (7, 9, 24, 33–39).

The ability of ASM to rapidly adapt its compliance and contractility to accommodate to externally imposed mechanical forces is well documented (7, 24–26, 36, 40–42). The mechanical oscillation or stretch of isolated ASM tissues reduces their stiffness and decreases their responsiveness to contractile stimuli (7, 20, 21, 24, 38–40). The same contractile stimulus imposed on ASM cells and tissues elicits different responses from the muscle depending on its mechanical history: how it has been stretched or shortened before receiving the stimulus (35, 43, 44). These physiological effects suggest that smooth muscle cell structure and organization are malleable and can be modulated by mechanical forces (3, 33, 37, 45, 46). Thus, the physiological effects of breathing maneuvers on airway responsiveness in vivo appear to result from the intrinsic properties of the ASM cells.

The connections between ASM and other tissues within the airways and lungs enable physiological forces that are imposed on lung tissues to be transmitted to the smooth muscle tissues and mechanical forces generated by the contraction of ASM to be transmitted to adjacent airway and lung tissues (FIGURE 1). Each ASM cell is connected to the extracellular matrix that surrounds it: a network of fibrous proteins and proteoglycans that link individual ASM cells to each other. Components of the extracellular matrix form a hydrated gel in the interstitial space between the individual smooth muscle cells that harbors proteases, mediators, and metabolites that are released into the intercellular space (48, 49). Mechanical forces generated by adjoining airways and lung tissues during breathing are transmitted between ASM tissues and the surrounding lung tissues through the intercellular connections formed by the extracellular matrix.

FIGURE 1.

Structure of airway smooth muscle (ASM) tissue and extracellular matrix connections. A: longitudinal section of smooth muscle tissue [hematoxylin and eosin (H&E)] showing smooth muscle cells connected by extracellular matrix fibers. B and C: electron micrographs of 60-nm-thick longitudinal sections of canine tracheal smooth muscle tissues. B: magnification: ×18,500. C: magnification: ×18,500, ×49,000, and ×95,000. Scale bar, 0.1 or 0.5 µm. D: mechanical force is transmitted between smooth muscle cells within a tissue through the extracellular matrix, which connects to the cells at membrane adhesion junctions. B and C are reprinted from Ref. 47, with permission of the American Thoracic Society.

The properties of the extracellular matrix surrounding ASM cells profoundly influence their physiological responses and phenotypic properties (14, 47, 50–56). Pathophysiological conditions can alter the extracellular milieu by modulating the composition or structure of the extracellular matrix and connective tissues that surround the airways and by causing the release of local humoral mediators (10, 57, 58). Pathophysiological conditions can also cause airway constriction and remodeling that alter the mechanical forces that impinge on ASM tissues during breathing. All these factors can affect the physiological properties and functions of the ASM tissues (59, 60). The ability of ASM to rapidly accommodate ever-changing conditions and fluctuating physical forces within its local extracellular environment is an essential component of its normal physiological function.

Smooth muscle cells connect to extracellular cell matrix (ECM) fibers at specialized areas along the plasma membrane that appear as electron-dense patches along the sarcolemma (FIGURE 1). These regions of attachment to the ECM have traditionally been referred to as “membrane-associated dense plaques,” “adhesion plaques,” or “focal adhesions” (61–63). Membrane adhesion junctions provide for the mechanical coupling of smooth muscle cells within a tissue and enable the intercellular transmission of mechanical forces that are generated within the cells or that originate outside the cells from physiological processes and events. Adhesion complexes on the plasma membrane of ASM cells play a fundamental role in detecting stimuli from the extracellular environment of the smooth muscle cells and transducing signals to intracellular pathways that regulate cytoskeletal organization and synthetic processes within the ASM cell (26, 34, 45, 46). The rapid and continuous transmission of information between the extracellular environment and intracellular processes enables ASM cells to rapidly adapt their physiological behavior and phenotypic properties to physical forces, local mediators, and extracellular matrix constituents from their surrounding extracellular milieu. The plasticity and dynamism of the molecular organization of membrane adhesion complexes and the smooth muscle cytoskeleton are fundamental to the ability of ASM cells to rapidly adapt their physiological properties to accommodate to changes in their local tissue environment.

This article describes the molecular processes that underlie the ability of ASM tissues to sense changes in their local extracellular conditions and transduce signals from the extracellular local environment to intracellular pathways that regulate the phenotypic properties and physiological functions of the muscle.

2. STRUCTURE AND CYTOSKELETAL ORGANIZATION OF AIRWAY SMOOTH MUSCLE CELLS

The plasma membrane dense plaques of smooth muscle cells contain integral membrane proteins (integrins) that span the membrane and connect extracellular matrix proteins outside the cell to large submembranous multiprotein complexes, “adhesomes,” on the cytoplasmic side of the cell membrane (FIGURE 2). Membrane adhesion complexes alternate with flask-shaped invaginations of the plasma membrane known as caveolae that are specialized lipid rafts that contain dystrophin, dystroglycans, and the integral proteins caveolin and cavin (64–66).

FIGURE 2.

Smooth muscle cell cytoskeletal structure and organization. A: the extracellular matrix connects cells within the airway smooth muscle (ASM) tissue by binding to the extracellular domains of transmembrane integrin proteins at the cell membrane. Transmembrane integrin proteins also bind to protein complexes (adhesomes) that associate with their cytoplasmic tails, thus linking the extracellular matrix to the actin cytoskeleton. Actin and myosin filaments that traverse the cytoplasm constitute the contractile apparatus. Tension generated by the contractile apparatus can be transmitted between the cells of the ASM tissue through their connections to adhesion complexes. B: molecular organization of membrane adhesion junctions in ASM. Actin filaments are linked to integrin proteins via the actin cross-linking proteins talin and α-actinin, which bind to the cytoplasmic tails of integrin proteins. Scaffolding proteins vinculin (Vin) and paxillin (Pax) regulate the assembly of signaling proteins at adhesion junctions. FAK, focal adhesion kinase; NM myosin, nonmuscle myosin; SM myosin, smooth muscle myosin. Adapted from Ref. 46, with permission from the American Physiological Society.

The cytoplasm of ASM cells is filled with contractile filaments: thick and thin filaments containing polymers of actin and smooth muscle myosin II that traverse the cell and anchor at membrane adhesion plaques. The thin filaments surround thick filaments to form rosette patterns of actin and myosin (67–72). The thin filaments are composed of intertwined polymers of actin and tropomyosin that can associate with caldesmon, calponin, and other actin filament binding proteins (73, 74). The thick filaments consist primarily of polymers of smooth muscle myosin II and associated regulatory proteins (75–79).

Actin filaments bind to proteins within membrane adhesion junctions on the cytoplasmic side of adhesion sites, thus enabling the transmission of mechanical tension generated by the contractile apparatus to the extracellular matrix (63, 67, 72, 75, 80, 81) (FIGURES 1 AND 2). Actin filaments also insert into cytoplasmic dense bodies, electron-dense areas composed predominantly of the actin cross-linking protein α-actinin (80, 82–86). Dense bodies act as anchors for actin filaments during tension generation, thus playing a role analogous to the Z lines of skeletal muscle sarcomeres. In studies using high-resolution three-dimensional (3-D) images, dense bodies appear as cablelike structures that lie parallel to the contractile filaments, which has led to a hypothesis that dense bodies can act as passive tension-bearing elements (72, 87, 88).

Actomyosin cross-bridge cycling is the fundamental mechanism for tension development and shortening in smooth muscle. Contractile stimulation initiates the crawling of the thick filaments along the thin filaments powered by cross-bridge cycling: the repetitive attachment and detachment of head domains of myosin II with actin filaments. The ATPase activity of the head domain of the myosin molecule generates energy that is converted to mechanical force that propels the myosin filament along the actin filament, resulting in shortening and tension generation (71, 72, 89, 90).

Actin and myosin filaments are also found in the submembranous cytoplasm at the cortex of ASM cells (46, 91, 92). The organization and composition of cortical actin and myosin filaments differ from those of the contractile filaments with respect to isoform, filament organization, and binding proteins (45, 91, 92). Cortical actin filaments bind to proteins within membrane adhesion sites, thus linking the smooth muscle cytoskeleton to the extracellular matrix. Filaments of nonmuscle myosin II interact with cortical actin in the cortex of ASM cells during contractile activation of the cell. These cortical actin and myosin filaments have a physiological function that is distinct from that of the thick and thin filaments that traverse the cell cytoplasm and comprise the contractile apparatus.

Historically, cytoskeletal and contractile filaments have been viewed as stable structural elements, with the actin filaments anchored at membrane adhesion sites and at cytosolic dense bodies constituting a fixed and stable network on which the myosin filaments move during shortening and tension development (86, 93). However, the structure and organization of cytoskeletal filaments and membrane adhesion complexes in ASM tissues are now recognized to be highly dynamic (37, 45, 46). Actin and myosin filaments and membrane adhesion complexes undergo constant remodeling in response to external stimuli and changes in the surrounding environment of the cell (37, 46, 47, 91, 94–96). The rapid dynamic reorganization and remodeling of the cytoskeletal apparatus in response to external conditions enable the cell to continually adapt its shape and mechanical properties to its local environmental conditions. Appreciation of the structural dynamics of the cytoskeleton and its role in responding to the external conditions surrounding ASM cells has provided a conceptual basis for understanding the molecular mechanisms underlying the ability of ASM to modulate its functional behavior in response to changes in its local extracellular environment.

3. MOLECULAR ORGANIZATION OF MEMBRANE ADHESION JUNCTIONS IN ASM TISSUES

Membrane adhesion junctions play an essential role in the transduction of signals from virtually all extracellular stimuli that impinge on the cells within the ASM tissue. Signal transduction pathways from neurohumoral and hormonal mediators, mechanical stimuli, cytokines, and local metabolites are modulated by adhesion complexes and transduced to cytoplasmic effector pathways that regulate the functional properties of the cells. Extracellular matrix properties are also important regulators of signaling pathways mediated by adhesion junctions. Cytoskeletal properties within the cell are also sensed and transmitted to the outside of the cell via adhesion junction complexes.

3.1. Integrins Link the ECM to Submembranous Signaling Complexes

The structure and molecular organization of the sites of attachment between cells and their substrates have been studied extensively for decades (62, 97). Whereas the fundamental molecular structure of these junctions is common across many cell types, the specific constituents and molecular processes that occur at these junctions depend on the function of the cell and the nature of its connections to its surrounding cells and tissue components. The molecular organization of adhesion junctions in ASM tissues has much in common with that of other cells; however, the signaling and transduction mechanisms that occur at these sites are likely to be specialized for the unique characteristics and functions of ASM (FIGURE 2B and TABLE 1).

Table 1.

Constituents of adhesion complexes in airway smooth muscle

Constituent	Cell Function	References
Transmembrane integrins
α1, α3, β1, and β3	ASM tissues in situ	(60, 95, 98, 99)
α1–7, α9, αv, β1, β3, and β5	Cultured ASM cells	(47, 98, 100–103)
Linker proteins
Talin		(104, 105)
α-Actinin		(95)
Scaffolding proteins
Vinculin	Vinculin-paxillin complex	(94, 104, 105)
Paxillin	Vinculin-paxillin complex	(106, 107)
ILK	ILK-PINCH-Parvin complex	(108–111)
α-Parvin	ILK-PINCH-Parvin complex	(108, 111)
β-Parvin	ILK-PINCH-Parvin complex	(108)
PINCH	ILK-PINCH-Parvin complex	(108, 110, 111)
Zyxin		(112)
Adapter proteins
CrkII		(113)
Abl1		(114)
CAS		(263, 264)
Small GTPase, GEF, and GAP
Cdc42	Small GTPase	(115, 116)
RhoA	Small GTPase	(117, 118)
βPIX	GEF, activates cdc42, GIT-PAK-PIX	(111, 119)
GIT1	GAP, activates cdc42	(111, 119)
Actin polymerization regulator proteins
N-WASp	Actin filament branching	(92, 111)
Arp2/3	Actin filament nucleation	(92)
VASP	Actin filament elongation	(120)
Profilin	G-actin chaperone	(120–122)
ADF/cofilin	Actin filament severing	(123)
Cortactin	Adapter protein and actin branching	(121)
GMF-γ	Actin severing, ADF/cofilin family	(124, 125)
Kinases
FAK	Tyrosine kinase	(56, 107, 126)
PAK1	Serine/threonine kinase	(119, 127)
Abl	Tyrosine kinase	(128)

ASM, airway smooth muscle; FAK, focal adhesion kinase; GAP, GTPase activating protein; GEF, guanine nucleotide exchange factor; GIT1‐βPIX‐Pak, G protein receptor kinase interacting tyrosine phosphorylated (GIT), Pak‐interacting exchange factor (PIX), p21‐activated kinase (Pak); ILK, integrin‐linked kinase; N‐WASp, neuronal Wiskott–Aldrich syndrome protein; VASP, Vasodilator‐stimulated Phosphoprotein.

All cell-ECM adhesion junctions consist of clusters of transmembrane integrin proteins that form an anchor for extracellular ligands and for large submembranous cytoplasmic adhesion protein complexes, adhesomes (129). Integrin proteins constitute the primary cellular sensors for local extracellular conditions and are capable of transducing complex information about the extracellular environmental milieu to the cell cytoplasm and nucleus via the adhesome complexes that associate with integrin cytoplasmic tails (130–136). Integrins bind to extracellular matrix ligands, cell-surface ligands, soluble ligands, and humoral mediators and are sensitive to physical stimuli such as mechanical tension, substrate rigidity, and fluid flow. Integrins were initially characterized as ligands for extracellular matrix proteins and were thus conceived to be a form of inert cellular “glue” that binds the individual cells within tissues together. However, it is now recognized that integrins also directly bind to many other protein constituents within the extracellular matrix, including proteases, growth factors, and immunoglobulins. Integrins can also sometimes form cell-to-cell connections.

Transmembrane integrin proteins are large membrane-spanning proteins consisting of α- and β-subunits that form heterodimers. There are at least 18 α- and 8 β-subunits that form 24 different αβ-heterodimer combinations in humans (136–138). A somewhat smaller subset of these integrin heterodimers has been documented in ASM cells and tissues (TABLE 1). The expressions of α1-, α3-, β1-, and β3-integrins have been reported in ASM tissue in situ, whereas ASM cells in culture are known to express α1–7-, α9-, αv-, β1-, β3-, and β5-integrins (47, 60, 95, 98–103).

Integrin heterodimers act as specific receptors for extracellular matrix proteins and other ligands. The specificity of engagement of integrin receptors with extracellular ligands is determined by the combination of α- and β-subunits in each heterodimer. Each protein subunit within the integrin heterodimer has a large extracellular multidomain region, a single-pass transmembrane region, and a short cytoplasmic tail that mediates adherence to actin filaments and adhesome proteins (139, 140). The binding of integrins to extracellular ligands requires a stable noncovalent association between the extracellular portions of the integrin subunits.

The extracellular domains of integrins can assume different conformations that differ in their affinity for ligand binding; these include a low-affinity “bent” conformation, an intermediate-affinity “extended-closed” conformation, and a high affinity “extended-open” conformation (138, 139) (FIGURE 3). Upon binding to an extracellular ligand, integrins undergo conformational changes within their transmembrane and cytoplasmic domains that regulate the binding and activation state of cytoplasmic adhesion proteins to the integrin cytoplasmic domain (131, 141). This is referred to as “outside-in” signaling. Changes in the conformation of integrin proteins that affect their binding to extracellular ligands can also be induced by the binding of cytoplasmic ligands such as talin, integrin-linked kinase (ILK), and focal adhesion kinase (FAK) to the cytoplasmic domains of integrin proteins, termed “inside-out” signaling. The coupling between the transmembrane domains of α- and β-integrin subunits is necessary for propagating conformational changes from the cytoplasmic tails of integrins to their extracellular domain. Signals transmitted by integrin complexes regulate cellular processes that determine ASM cell contractility, differentiation, proliferation, and secretory status (58–60, 99).

FIGURE 3.

A: the extracellular domains of integrins assume conformations that differ in their affinity for ligand binding to the extracellular matrix. The cleavage of talin by calpain separates the globular head domain from the tail domain and prevents talin from cross-linking and activating β-integrins. Talin cleavage and integrin inactivation prevent the activation of vinculin (Vin) and its binding to adhesome ligands and to actin filaments. Stimulation with the inflammatory mediator IL-4 causes integrins to assume an inactive low-affinity “bent” conformation, resulting in the cleavage of talin. Stimulation with ACh causes talin to bind to integrins, triggering them to assume a high-affinity “extended-open” conformation. B: talin cleavage is mediated by calpain, resulting in the separation of talin’s globular head domain from its rod domain. This prevents talin from cross-linking actin filaments and activating integrins. C: autoinhibited vinculin in the cytoplasm is maintained in an inactive “closed conformation” in which the binding sites for actin and talin are unavailable. Inactive vinculin remains in a complex with paxillin (Pax). Contractile stimulation of the airway smooth muscle (ASM) cell induces recruitment of the vinculin-paxillin protein complex to adhesion junctions, where vinculin interacts with talin and assumes an open ligand-binding conformation. Activated vinculin can bind to F-actin, which strengthens the adhesion junction and supports the transmission of tension generated by actomyosin cross-bridge cycling. FAK, focal adhesion kinase; NM myosin, nonmuscle myosin.

3.2. Talin and α-Actinin Act as Linker Proteins

The small cytoplasmic domains of transmembrane integrins bind to proteins within submembranous adhesomes (FIGURE 3). Adhesome proteins that bind directly to both integrins and actin filaments provide a continuous mechanical link between the actin cytoskeleton and the extracellular matrix (131). In ASM tissues, talin and α-actinin link β-integrins directly to actin filaments and provide a foundation for the assembly of layers of scaffolding and signaling proteins within adhesome complexes (95, 131, 142). Vinculin, integrin-linked kinase (ILK), and paxillin act as intermediary scaffolding proteins that connect linker proteins to actin filaments (104, 143–148). Scaffolding and adaptor proteins also serve as a platform for the assembly of signaling complexes that transduce information about extracellular conditions transmitted by integrin proteins.

Both talin and α-actinin form dimers that enable them to cross-link actin filaments (131, 141, 149). The talin molecule (270 kDa) consists of an NH₂-terminal 50-kDa head domain and an elongated helical rod of ∼220 kDa connected by a linker region (FIGURE 3B) (131). The NH₂-terminal head domain of talin binds to the β1-integrin cytoplasmic tail; the talin rod domain mediates the formation of talin homodimers that cross-link actin filaments via a COOH-terminal F-actin binding site. The rod domain also contains binding sites for the scaffolding protein vinculin. Additional integrin binding proteins that bind to actin filaments such as filamin and kindlin have been documented in other cell types (133, 134, 150); however, their expression and function in ASM have not been demonstrated to date.

The process of adhesome complex assembly and organization is highly dynamic in ASM tissues and is constantly modulated in response to changing extracellular stimuli and intracellular signals (3, 94, 95, 115, 145, 151–153). The assembly and activation of adhesion complexes at the membrane of differentiated ASM cells and tissues can be triggered by humoral and mechanical stimuli (46, 105, 115, 153, 154). Both α-actinin and talin associate with integrin adhesion sites in ASM, but inactive pools of both proteins are also maintained sequestered in the cytoplasm.

Contractile or other physiological stimuli such as mechanical force trigger the recruitment of talin and α-actinin to the cytoplasmic tails of β-integrins at adhesion sites. This provides the mechanical strength required to support force transmission between the contractile apparatus and the extracellular matrix of the smooth muscle cell (94, 95, 104, 105). These proteins also form a scaffold for the assembly of additional structural and signaling molecules within the adhesome complex. When the connections between α-actinin and the cytoplasmic tails of β-integrins are disrupted by the use of molecular interventions to block the binding of α-actinin to β-integrins, force development by the ASM tissue in response to a contractile stimulus is impaired (95).

The binding of talin to integrin heterodimers at the plasma membrane regulates integrin activation and adhesome assembly (141) (FIGURE 3B). Talin can undergo calpain-mediated cleavage in its linker region in response to external stimuli that results in separation of the head and rod domains (155, 156). In ASM tissues, stimulation with the contractile agonist acetylcholine triggers the recruitment of talin to adhesion junctions and the activation of β1-integrin proteins and β-integrin-linked adhesome proteins (47, 94, 157). Treatment of ASM tissues with the proprotein convertase furin, a protease that can act on ECM proteins, also activates β1-integrins and associated adhesome proteins (157). Conversely, treatment of ASM tissues with the Th2 inflammatory mediators IL-4 and IL-13 or the protease neutrophil elastase induces cleavage of the head and tail domains of talin and prevents the activation of β1-integrins and associated adhesome proteins (54, 157). Talin cleavage disrupts its ability to bind to vinculin, which results in the disruption of linkages between the actin cytoskeleton and integrin complexes. This reduces the support for force transmission and prevents the activation of vinculin-binding adhesome proteins such as paxillin (54, 157).

3.3. Vinculin and Paxillin Serve as Scaffolds for Signaling Modules

Vinculin and its closely related muscle-specific isoform metavinculin (158) are abundant constituents of adhesion complexes in ASM (94, 104, 151). They act as scaffolding proteins and provide mechanical support for connections between the actin cytoskeleton and integrin proteins. The vinculin molecule consists of a globular head domain with four helical bundle domains connected by a flexible linker to a short tail domain. The conformational state of the vinculin molecule can be reversibly regulated between an activated “open” state and an inactive “autoinhibited” state (104, 105, 159–161) (FIGURE 3C).

Vinculin binds to α-actinin and to the rod domain of talin, both of which induce its conversion to an “open” activated conformation in which the binding sites for actin filaments are exposed (144, 160). When vinculin is in an open state, the exposure of binding sites for talin and α-actinin on its head domain and for F-actin on its tail domain enables it to form connections between the actin cytoskeleton and integrin adhesion complexes (FIGURE 3C). When vinculin is in a closed conformation, the head and tail domains bind to each other and mask the binding sites for actin, talin, and α-actinin. The conformation and scaffolding activity of the vinculin molecule is regulated by its phosphorylation on tyrosine 1065 on its tail domain (105). The activation of vinculin and its binding to talin, α-actinin, and actin strengthen linkages between the actin cytoskeleton and integrin proteins and provide a scaffold for the assembly of signaling modules at adhesion sites (162). The recruitment of vinculin to adhesion sites is an essential step in the development of contractile tension in ASM (104, 105).

In ASM, vinculin remains in a stable complex with its binding partner paxillin (94). Paxillin provides a scaffold for the assembly of a number of signaling modules that mediate both cytoskeletal and nuclear signaling pathways at integrin adhesion sites (94, 104, 105, 146, 163). A pool of vinculin-paxillin complexes is maintained in the cytoplasm of ASM cells in an inactive state, and the complexes are recruited to adhesion sites in response to physiological stimulation (94, 104, 105). Once localized to the membrane, vinculin undergoes a conformational shift that enables it to bind to talin, α-actinin, and actin filaments. The activation of vinculin exposes docking sites for recruitment of additional adaptor and signaling proteins to adhesomes (46, 94, 104, 105). The conversion of vinculin to an activated open conformation promotes the phosphorylation of paxillin at multiple sites. Paxillin phosphorylation facilitates its binding to signaling modules that mediate both cytoskeletal and nuclear signaling pathways (104, 105, 108, 113, 119, 146, 164). The signaling modules that are recruited to bind to paxillin and vinculin depend on the specific stimulus and the local environmental conditions affecting the ASM cell. These signaling modules can mediate pathways to diverse effectors within both the cytoplasm and the nucleus of the smooth muscle cell.

Historically, membrane adhesion junctions in intact smooth muscle tissues were viewed as a stable “cyto-skeletal” structure, with vinculin, talin, and α-actinin considered to be inactive structural components (80, 84). However, these proteins are now recognized to be highly dynamic adhesome constituents that are actively regulated by external stimulation and by changes in extracellular conditions. Adhesion complex assembly in intact ASM tissues involves processes that are analogous in many respects to those at the focal adhesion sites of migrating cells (165, 166).

4. ACTIN POLYMERIZATION IS CATALYZED AT ADHESION COMPLEXES IN AIRWAY SMOOTH MUSCLE TISSUES

4.1. Physiological Functions of Stimulus-Induced Actin Polymerization During ASM Contraction

In ASM tissues, actin polymerization occurs in response to contractile stimulation concurrently with the activation of actomyosin cross‐bridge cycling (45, 46, 115). Both actin polymerization and cross-bridge cycling are necessary for the development of contractile tension; however, they are regulated by distinct and independent molecular processes and serve different physiological functions during the tension development (46, 167, 168) (FIGURE 4). The polymerization of actin in the submembranous region of the smooth muscle cell strengthens molecular connections between the contractile apparatus and membrane adhesion junctions, thus supporting the transmission of force across the cell membrane (40, 46, 92, 95, 168). Force generated by the contractile apparatus is transmitted out of the cell, and external tension exerted on the ASM tissue is transmitted into the cell to the actin cytoskeleton and contractile apparatus. This actin polymerization is mediated by proteins that are recruited to membrane adhesion complexes, where they undergo activation and catalyze the formation of filamentous actin within the submembranous space (47, 92, 168, 169). Thus, these molecular processes for regulating actin polymerization occur within the submembranous cell cortex.

FIGURE 4.

The contraction of airway smooth muscle (ASM) in response to stimulation requires adhesome complex assembly and actin polymerization as well as the activation of smooth muscle (SM) myosin and cross-bridge cycling. These processes are regulated by independent signaling pathways. Contractile stimulation catalyzes the assembly and activation of adhesome proteins at integrin adhesion sites and the polymerization of cortical actin, which is activated through adhesome signaling pathways. These processes strengthen integrin-mediated connections between the contractile apparatus and the extracellular matrix (ECM), providing a pathway for the transmission of tension generated by the contractile apparatus to the outside of the cell. Concurrently, the rise in intracellular Ca²⁺ triggers the activation of myosin light chain kinase (MLCK), which phosphorylates the regulatory light chain of myosin. Myosin light chain phosphorylation activates the myosin ATPase, which stimulates cross-bridge cycling and tension development. Activation of the contractile apparatus generates tension that is transmitted out of the cell at fortified adhesion sites. The increase in mechanical tension at adhesion sites provides additional stimulation for the assembly and fortification of adhesion complexes and actin polymerization (solid blue line). The increase in mechanical tension also increases the load on the contractile apparatus, which results in a decrease in the rate of ATP hydrolysis and cross-bridge cycling (broken blue line).

Actomyosin cross-bridge cycling is initiated by the activation of the Ca²⁺/calmodulin-dependent enzyme myosin light chain kinase (MLCK), which is triggered by an increase in cytoplasmic Ca²⁺. MLCK phosphorylates the myosin regulatory light chain (RLC) subunit of filamentous smooth muscle myosin II, which initiates actin-activated myosin ATPase activity and cross-bridge cycling (170–173). The independence of the molecular processes that regulate actin polymerization from those that regulate cross-bridge cycling has been well documented in ASM (46, 92, 106, 117, 168, 174). The inhibition of actin polymerization during contractile activation does not prevent myosin light chain phosphorylation or cross-bridge cycling; conversely, the inhibition of MLCK or the prevention of myosin RLC phosphorylation during contractile stimulation does not inhibit actin polymerization.

During contractile stimulation, the velocity of smooth muscle shortening is determined by the cross-bridge cycling rate, which is driven by the rate of ATP hydrolysis by the myosin ATPase (71, 72, 170, 172, 173, 175–180). The velocity of muscle shortening and the cross-bridge cycling rate are inversely related to the mechanical load on the contractile apparatus: the rate of ATP hydrolysis and the cross-bridge cycling rate decline as the load on the muscle increases, yielding a hyperbolic relationship between mechanical load and velocity of shortening during isotonic contraction (89, 170, 181).

In ASM, the rates of ATP hydrolysis and cross-bridge cycling increase rapidly during the initial phase of force development and decline after force reaches a plateau, which results in a decline in the energy cost of tension maintenance (177, 182). ASM stiffness also increases rapidly during the force development phase of contraction; however, muscle stiffness also continues to increase slowly during the plateau phase of contraction while the rate of ATP hydrolysis and cross-bridge cycling are declining (21, 177, 182, 183). This increase in muscle stiffness during the plateau phase of contraction has been attributed to the polymerization of new actin filaments and the strengthening of the linkages between contractile filaments and adhesion complexes (21, 174, 182, 183). These cytoskeletal processes increase the internal load on the contractile apparatus, resulting in a decline in the rates of cross-bridge cycling and ATP hydrolysis while the stiffness of the muscle increases (46, 94, 95, 177, 182) (FIGURE 4). The inhibition of actin polymerization during the contractile activation of ASM suppresses force development; however, it also causes an increase in the rate of ATP hydrolysis (177, 182). Disruption of the formation of actin cytoskeletal connections to membrane adhesion sites decreases the internal load on the contractile filaments, resulting in an increase in the rate of ATP hydrolysis and cross-bridge cycling. However, the transmission of force generated by the contractile apparatus to the outside of the cell is impaired by inhibition of the formation of actin filament connections to the membrane; thus, force development is suppressed.

The mechanical force generated by the contractile apparatus increases the tension on transmembrane integrin proteins and their connections to the extracellular matrix. This triggers the recruitment of adhesome proteins such as talin, FAK, and ILK to the cytoplasmic domains of integrin proteins, which leads to conformational changes in integrin proteins that enhance their affinity for extracellular matrix proteins in addition to activating cytoskeletal signaling pathways (81, 134, 184, 185).

The plasticity and dynamism of cytoskeletal structure and organization enable ASM cells to continuously modulate their shape and structural arrangement in response to changes in their local physical environment and to adjust to changes in mechanical forces that are imposed on the ASM tissue during breathing (33, 34, 71, 183). This structural malleability is an important physiological property of ASM that enables it to reorganize its cytoskeleton to reduce its stiffness as it is stretched during inflation of the airways, thus promoting opening of the airways and a lower airway resistance (25). The motion of continuous stretch and retraction that occurs during tidal breathing reduces the stiffness and contractility of ASM both in vivo and in vitro: ASM that is subjected to static mechanical conditions slowly increases its stiffness and increases its force generation in response to contractile stimulation (7–9, 21–32, 40). The property of structural plasticity is likely to underlie the heightened airway responsiveness and decreased airway resistance observed in human subjects and experimental animals subjected to prolonged periods of tidal breathing at low volume without intermittent deep breaths (8, 32, 186).

4.2. Molecular Mechanisms for Actin Polymerization at Membrane Adhesion Junctions

The proportion of actin that undergoes polymerization in ASM tissues in response to contractile stimulation is relatively small. According to biochemical analyses, filamentous (F)-actin constitutes 70–80% of the total actin in the cells of unstimulated ASM tissues, whereas 20–30% of the cellular actin exists as soluble monomeric actin (G-actin) (45, 92, 168). Contractile stimulation triggers a 10–15% increase in the amount of F-actin and a 30–40% decrease in G-actin. These estimates of the proportion of actin that undergoes polymerization in ASM tissues are consistent with assessments based on alternative methods such as electron microscopic and fluorescence imaging (167, 168, 187–191). These methods also indicate that most of the actin is in filamentous form, with a relatively small proportion undergoing polymerization. The relative sizes of the pools of F- and G-actin in ASM tissues are comparable to estimates from arterial, uterus, and urinary bladder smooth muscle tissues (187, 188, 192, 193). Electron microscopic data also suggest that contractile stimulation can also induce an increase in the density of the cytoplasmic actin filaments spanning ASM tissues; however, it is unclear whether the same molecular mechanisms regulate the polymerization of this pool of actin filaments (188, 189).

Actin polymerization can occur through the elongation of existing actin filaments at the “barbed” (fast growing) end of the filament or through the formation of side branches on existing actin filaments (194, 195) (FIGURE 5). There is evidence for both processes in ASM tissues (92, 120, 196, 197). Wiskott–Aldrich syndrome proteins (WASps) catalyze the formation of new actin filaments as branches on a “mother” filament by the Actin-related protein complex (Arp2/3 complex). Actin filament lengthening can be catalyzed by Vasodilator-stimulated Phosphoprotein (VASP), which binds to the barbed ends of existing actin filaments. The actin filament elongation mechanism requires VASP oligomerization and its binding to profilin, a G-actin chaperone (120, 198, 199).

FIGURE 5.

Mechanisms for actin polymerization in airway smooth muscle. A: actin polymerization mediated by neuronal Wiskott–Aldrich syndrome protein (N-WASp) results in the formation of side branches on existing actin filaments. N-WASp and the Arp 2/3 complex are recruited to the adhesome complex, where N-WASp is coupled to tyrosine phosphorylated paxillin (Pax) by Crk II. N-WASp binds to GTP-cdc42 and undergoes activation and a conformational change that enables it to activate the Arp2/3 complex. The Arp2/3 complex forms a template for the polymerization of a new actin filament that branches from the side of an existing actin filament. B: actin polymerization by Vasodilator-stimulated Phosphoprotein (VASP) induces the elongation of existing actin filaments. VASP binds to vinculin (Vin) and assembles into tetrameric oligomers. Actin filament elongation then occurs via the recruitment of profilin-G-actin complexes to the VASP tetramers. This facilitates the transfer and assembly of G-actin monomers into the barbed ends of the actin filaments. FAK, focal adhesion kinase; PIP₂, phosphatidylinositol 4,5-bisphosphate.

N-WASp (neuronal Wiskott–Aldrich syndrome protein) is essential for the process of stimulus-induced actin polymerization in ASM (92). WASp family proteins bind to the Arp2/3 complex and induce its activation (194, 200–202). The Arp2/3 complex consists of seven strongly associated protein subunits that form a stable complex in cells. It includes the actin-related proteins Arp2 and Arp3, which are structurally similar to monomeric actin (G-actin) and provide a template for the formation of new actin filaments. The Arp2/3 complex binds to the sides of existing actin filaments and nucleates new filaments that branch from the mother filament. The actin depolymerizing factor ADF/cofilin regulates the supply of G-actin monomers available for catalyzing actin polymerization by the Arp2/3 complex (123).

Both N-WASp and the Arp2/3 complex are sequestered in inactive form in the cytoplasm in unstimulated ASM tissues. They are recruited to membrane adhesion complexes in response to stimulation, where they undergo activation and catalyze the polymerization of new actin filaments (92, 116). The activation of N-WASp is regulated in part by the phosphorylation of a conserved tyrosine residue, Tyr256, by focal adhesion kinase (FAK) (203). FAK is also recruited to focal adhesion complexes in response to the contractile stimulation of ASM (204).

VASP also plays an essential role in stimulus-induced actin polymerization in ASM (120). VASP assembles into tetrameric oligomers; filament elongation then occurs via the recruitment of profilin-G-actin complexes to the VASP tetramers. This promotes the transfer and assembly of G-actin monomers into the barbed ends of the actin filaments that are also bound to VASP (205–207). In ASM tissues, stimulation with ACh induces the phosphorylation of VASP on Ser157 by protein kinase C, the membrane localization of VASP, VASP oligomerization, and its binding to profilin (120). The prevention of VASP activation using molecular interventions inhibits actin polymerization and contractile tension development in ASM.

Both VASP and N-WASp are recruited to adhesion complexes in response to the stimulation of ASM, where they bind to scaffolding proteins and undergo activation (FIGURE 5). In airway muscle, the coupling of VASP to activated vinculin is prerequisite to its function as a catalyst for actin polymerization (120). The stimulation of ASM with agonists that activate adenyl cyclase, such as forskolin, induces VASP phosphorylation on Ser157 as well as the recruitment of VASP to the membrane. However, they do not induce vinculin activation, the binding of VASP to vinculin, or actin polymerization (120). Thus, the phosphorylation of VASP alone is not sufficient to cause its activation and catalyze actin polymerization.

The coupling of N-WASp to scaffolding proteins at adhesion sites is required for its activation in ASM. N-WASp couples to paxillin via Crk II, an SH2-SH3 adaptor protein (113, 119, 204, 208). After paxillin localizes to adhesion sites, it undergoes tyrosine phosphorylation on residues 31 and 118 by focal adhesion kinase (FAK) (126, 209). Tyrosine phosphorylated paxillin is recognized by the SH2 domains on CrkII, which also binds to N-WASp via its SH3 domains. In ASM, the coupling of N-WASp to paxillin by Crk II is prerequisite to its activation, association with the Arp2/3 complex, and tension development in response to a contractile stimulus (113). N-WASp can also bind to vinculin at adhesion junctions in other cell types (210); thus vinculin might also act as a scaffold for the localization and activation of N-WASp at adhesion sites in ASM.

The coupling of N-WASp to paxillin positions N-WASp for activation by the small GTPase cdc42. N-WASp binds to cdc42 in GTP-bound form; this triggers the conversion of N-WASp to an open conformation and enables it to activate the Arp2/3 complex (200, 202). In ASM, N-WASp cannot undergo activation if cdc42 activation is inhibited or if the coupling of N-WASp to cdc42 is prevented (116, 119). Crk II facilitates the activation of cdc42 and its coupling to N-WASp by guanine nucleotide exchange factors (GEFs) that regulate the activation of cdc42 (119, 204, 211).

The contractile stimulation of ASM also triggers the phosphorylation of paxillin on Ser273 by the serine-threonine kinase p21-activated kinase (Pak), which regulates paxillin’s function as a scaffold that positions cdc42 and N‐WASp in proximity to GEFs for cdc42 (119). The serine phosphorylation of paxillin promotes its coupling to the GIT1-βPIX-Pak [G protein receptor kinase interacting tyrosine phosphorylated (GIT), Pak-interacting exchange factor (PIX), Pak] signaling module at adhesomes (212, 213). GIT and PIX proteins both have GEF activity toward cdc42 in ASM (213, 214). The binding of GIT molecules to the LD4 domain of paxillin regulates the localization of the GIT1-βPIX-Pak signaling module at adhesomes (119, 204, 211, 215). GIT proteins are ArfGAPs that also regulate cdc42, whereas PIX proteins have GEF activity toward cdc42 and bind to Pak as well as to GIT proteins (213, 214). These signaling events and catalytic processes at adhesion complexes are all activated by the contractile stimulation of ASM. The disruption or inhibition of any single step in the molecular events involved in stimulus-induced actin dynamics significantly impairs force development (FIGURE 6).

FIGURE 6.

Paxillin acts as a scaffold for the formation of a protein complex at adhesion junctions that activates cdc42 and neuronal Wiskott–Aldrich syndrome protein (N-WASp), catalyzing actin polymerization. ACh stimulation induces the recruitment of the paxillin/vinculin protein complex and the GIT1-βPIX-Pak [G protein receptor kinase interacting tyrosine phosphorylated (GIT), Pak-interacting exchange factor (PIX), p21-activated kinase (Pak)] complex to the adhesion junction, where focal adhesion kinase (FAK) phosphorylates paxillin on Tyr31 and Tyr118. Paxillin binds to the SH2/SH3 adaptor protein CrkII at its tyrosine phosphorylated sites, and CrkII couples paxillin to N‐WASp. Pak phosphorylates paxillin on Ser273, enabling the binding of GIT1 to Ser273 phosphorylated paxillin. This couples the GIT1-βPIX-Pak complex to paxillin, generating a signaling complex that activates cdc42 via the guanine nucleotide exchange factor (GEF) activity of βPIX. cdc42-GTP binds to N-WASp and catalyzes N‐WASp activation, which activates the Arp2/3 complex and initiates actin polymerization. GPCR, G protein-coupled receptor.

Bronchodilators and stimuli that induce ASM relaxation elicit actin depolymerization in ASM tissues and cells (216–219). Stimulation of ASM tissues and cells with isoproterenol or forskolin causes the depolymerization of actin in actively contracted tissues and can also stimulate the depolymerization of actin stress fibers in cultured ASM cells. This can occur through both PKA-dependent and PKA-independent pathways (218, 219). Forskolin does not stimulate vinculin phosphorylation and activation, which is required for its binding to actin and talin at adhesion sites, which suggests that it may prevent adhesome assembly (120). However, there is little information on the effects of agents that induce ASM relaxation on the assembly of adhesome complexes or on the regulation of adhesion complex proteins. The elucidation of the molecular mechanisms for the effects of bronchodilating agents on actin polymerization and the assembly of membrane adhesome complexes in ASM could provide novel targets for the development of bronchodilators.

Actin cytoskeletal dynamics serves an important function in strengthening the links between the cytoskeleton and the ECM for the transmission of force developed by the contractile apparatus. However, the lattice of actin filament “tracks” at the cortex of the cell also serves a broader function: it is necessary for the assembly of adhesion complexes and signaling modules at adhesion junctions that govern the physiological and phenotypic responses of the cell to all external stimuli.

5. ASSEMBLY OF ADHESION JUNCTION COMPLEXES IN RESPONSE TO STIMULATION OF AIRWAY SMOOTH MUSCLE

The stimulation of ASM induces the trafficking of inactive cytoplasmic adhesion junction and signaling molecules from the cytoplasm of ASM tissues to membrane adhesion sites, where they are assembled into signaling complexes and activated (45, 46, 94, 104, 105, 119, 204). The recruitment of these proteins is extremely rapid and is an upstream step in the development of contractile tension. The assembly of signaling modules at adhesion complexes also occurs in response to noncontractile stimuli and mechanical stimuli (54, 108, 154).

The recruitment of adhesion proteins is mediated by nonmuscle (NM) myosin II, which acts as a motor for the transport of proteins along the actin filament lattice at the cortex of the cell (91, 115). NM myosin II constitutes ∼20% of the total amount of myosin II in ASM (91). Smooth muscle myosin provides the molecular motor for cell shortening and tension generation in all smooth muscle tissues; however, NM myosin II serves a distinct function by mediating the assembly and fortification of adhesion complexes at the sites where actin filaments connect to the extracellular matrix. The activation of NM myosin is essential for tension generation in response to contractile stimulation in ASM tissues (91). The selective inactivation of NM myosin II by molecular or pharmacological interventions prevents the recruitment of the adhesion proteins to the membrane in response to a contractile stimulus and inhibits tension generation (91). NM myosin plays a somewhat analogous role in nonmuscle cells, in which it mediates the assembly and disassembly of adhesion complexes at the leading edge of the lamellipodium during cell migration (165).

Nonmuscle (NM) and smooth muscle (SM) isoforms of myosin II have an analogous structure, and both myosin isoforms are regulated by phosphorylation of the 20-kDa regulatory light chain (RLC), which activates actomyosin cross‐bridge cycling (78, 220–222). SM and NM myosin II both exist in monomeric as well as filamentous form in cells (165, 222–226). The contractile stimulation of ASM stimulates the assembly of NM myosin II monomers into filamentous myosin at the cortex of the cell (91) (FIGURE 7). The assembly of NM myosin into filaments is prerequisite to the recruitment of adhesion proteins to the membrane: if NM myosin polymerization is prevented by molecular interventions or pharmacological inhibitors during contractile stimulation, tension generation by ASM is inhibited (91).

FIGURE 7.

Nonmuscle (NM) myosin mediates the recruitment of adhesome components to the cell membrane in response to contractile stimulation. Stimulation of the muscle with ACh activates the small GTPase RhoA, which regulates phosphorylation of the regulatory light chain (RLC) of NM myosin and catalyzes the conversion of inactive folded NM myosin II monomers to an assembly‐competent conformation. ACh also stimulates the phosphorylation of NM myosin heavy chain (HC) on Ser1943, which induces NM myosin filament assembly. NM myosin interacts with cortical actin filaments to transport inactive adhesome proteins to the membrane, where they bind to adhesion complexes to catalyze additional cortical actin polymerization and strengthen adhesion junctions to support force transmission. GPCR, G protein-coupled receptor; Pax, paxillin; Vin, vinculin.

Myosin II monomers can assume a folded inactive conformation that is unable to assemble into filaments and an open “assembly‐competent” conformation (224, 226–229) (FIGURE 7). Both NM and SM myosin isoforms contain a short isoform‐specific nonhelical COOH‐terminal tailpiece that regulates filament assembly. In ASM, the small GTPase RhoA regulates phosphorylation of the regulatory light chain (RLC) of NM myosin and its polymerization (115). NM myosin RLC phosphorylation promotes the conversion of inactive folded NM myosin II monomers to an assembly‐competent conformation and activates cross-bridge cycling. In contrast, RhoA has little effect on SM myosin II activation or polymerization in ASM (91, 117, 230). The low-molecular-weight Ca²⁺-binding protein S100A4 is also regulated by RhoA GTPase in ASM and binds to the nonhelical tail domain on NM myosin II to promote myosin filament assembly (230). Contractile stimulation catalyzes the phosphorylation of the heavy chain NM myosin on Ser1943 in its nonhelical tail domain, which also promotes NM myosin polymerization (91).

In ASM, the activation of RhoA GTPase by a contractile stimulus is a critical upstream event in the development of tension generation that regulates the dynamic reorganization and assembly of the actin cytoskeleton through its effects on NM myosin II polymerization. RhoA activation is necessary for the activation of NM myosin filament assembly, the recruitment and assembly of membrane adhesome complexes, cortical actin polymerization, and tension generation (115, 117, 230). The selective regulation of nonmuscle myosin by RhoA provides a mechanism for its regulation that is distinct from that of smooth muscle myosin II, which serves an entirely separate physiological function in ASM.

6. SIGNAL TRANSDUCTION BY MEMBRANE ADHESION COMPLEXES IN THE REGULATION OF PHENOTYPIC EXPRESSION IN AIRWAY SMOOTH MUSCLE

Adhesion complexes play a central role in the regulation of a wide array of cellular functions that are important to normal physiological processes and to the pathophysiological processes that occur during lung disease (11, 50, 51, 53, 231–236) (FIGURE 8). Cytokines, proteases, and other local mediators activate signaling pathways in ASM cells and other resident lung cells that regulate phenotypic expression. Pathophysiological or physiological conditions can also alter the local surrounding environment of the cells by causing edema, changes in ECM composition or ECM degradation, or changes in the mechanical forces that impinge on ASM and other resident airway cells. Integrin complexes play a critical role in the transduction of signals from inflammatory mediators and other extracellular stimuli to intracellular signaling pathways. Alterations in the extracellular milieu are sensed by integrin receptors and are transduced to downstream pathways that regulate the differentiation state of ASM, its proliferative status, the synthesis and secretion of extracellular matrix proteins, cytokines, and hormones, and the expression of contractile proteins.

FIGURE 8.

Airway inflammation alters the extracellular environment of airway smooth muscle (ASM) cells to induce changes in ASM phenotypic expression that contribute to airway dysfunction. Pathophysiological changes in the extracellular milieu of ASM tissues can then stimulate further phenotypic alterations in airway cells and promote airway remodeling. ECM, extracellular matrix.

The role of transmembrane integrin receptors as regulators of cell differentiation and phenotype expression is well documented (99, 135). In ASM, the deletion of specific integrin subunits, blocking of integrin heterodimers, or modifications in integrin expression can induce changes in airway muscle contractility, proliferation, and phenotypic expression (55, 60, 98–100, 103). Changes in the composition, stiffness, or mechanical tension on the extracellular matrix are sensed by integrins and transduced by adhesomes to nuclear signaling pathways that regulate phenotypic expression, cell cycling, and physiological properties in ASM (51, 52, 54–56, 100, 157, 231). Transitions in ASM phenotype can be characterized experimentally by evaluating the expression of genes encoding the smooth muscle phenotype-specific proteins smooth muscle myosin heavy chain (SmMHC), SM22α, α- and γ-actin, and calponin-h1. ASM phenotype can also be evaluated by measuring changes in the expression and secretion of cytokines, chemokines, or ECM proteins or the proliferation status of the cells (11, 12, 237).

The downstream signaling pathways that regulate ASM phenotype expression, proliferation, and physiological properties have been extensively investigated in ASM (10, 236, 238–240); however, there is more limited information regarding the molecular mechanisms by which integrin-mediated signals are transduced to these pathways. A full understanding of the molecular events that mediate phenotype transitions in ASM tissues under physiological conditions requires the use of experimental models in which the natural extracellular matrix environment of the ASM cell is preserved, as the characteristics of the cell matrix and the mechanical stresses imposed on the ASM cell are important determinants of phenotype expression.

7. MOLECULAR MECHANISMS FOR THE PHENOTYPE MODULATION BY INTEGRIN ADHESOMES IN AIRWAY SMOOTH MUSCLE

Integrin adhesion complexes transduce signals from integrin receptors to cytosolic signaling pathways that regulate phenotypic expression: the synthetic kinases Akt/PKB and ERK MAP kinase are key intermediaries in these signaling pathways (56, 108–110, 154, 240, 241). The activation of Akt promotes an inflammatory phenotype in ASM characterized by the synthesis and secretion of cytokines, whereas Akt inhibition facilitates a differentiated phenotype characterized by the expression of smooth muscle-specific proteins (10, 11, 54, 108–110). Akt can be activated by inflammatory cytokines such as IL-13 and IL-4 and is sensitive to mechanical tension: high levels of mechanical tension imposed on ASM suppress Akt activation, whereas low levels of mechanical tension increase Akt activation (54, 109, 110, 154, 235). Akt activation suppresses the expression of smooth muscle differentiation marker proteins by inhibiting the nuclear translocation of serum response factor (SRF) (109, 242), a transcription factor that regulates the expression of genes encoding smooth muscle differentiation marker proteins (243, 244). In ASM tissues, the adhesome proteins focal adhesion kinase (FAK) and paxillin play a critical role in the transduction of signals from integrin receptors to pathways that regulate phenotype expression by regulating the activation of both Akt and MAPK (54, 56, 108, 110, 154, 157) (FIGURE 9).

FIGURE 9.

Mechanism for phenotype modulation in airway smooth muscle (ASM) tissues. A: paxillin tyrosine phosphorylation is not catalyzed by inflammatory stimuli or when the level of mechanical tension on the muscle tissue is low. In the absence of paxillin phosphorylation, α-parvin integrin-linked kinase/PINCH/parvin (IPP) complexes bind to paxillin at integrin adhesion junctions. Akt is recruited to adhesion junctions where it binds to integrin-linked kinase (ILK) within the α-parvin IPP complex. The interaction of Akt with ILK facilitates Akt activation by phosphatidylinositol 3-kinase (PI3-kinase) and results in an inflammatory phenotype in the ASM that is characterized by the synthesis of inflammatory mediators such as eotaxin. Akt activation suppresses the nuclear localization of serum response factor (SRF), which inhibits expression of the differentiated phenotype. B: stimulation of smooth muscle cells with ACh or high mechanical tension on the muscle tissue causes the tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin; promoting the binding of paxillin to β-parvin IPP complexes at membrane adhesomes. β-Parvin prevents the binding of Akt to ILK, which inhibits the activation of Akt. The inhibition of Akt activation promotes the nuclear localization of SRF and expression of the differentiated smooth muscle phenotype. ECM, extracellular matrix; SmMHC, smooth muscle myosin heavy chain. Adapted from Ref. 108, with permission from the American Physiological Society.

Focal adhesion kinase (FAK) and paxillin are mechanosensitive and are involved in the transduction of inte-grin-mediated mechanical signals in ASM (108, 126, 153, 154). The level of tyrosine phosphorylation of both FAK and paxillin is sensitive to the amount of mechanical strain or load on the muscle (126, 153, 154). FAK binds to talin at adhesomes and catalyzes the phosphorylation of paxillin, which acts as a scaffold for signaling modules that activate pathways to the nucleus. The inactivation or inhibition of FAK in ASM promotes an inflammatory phenotype characterized by the synthesis of inflammatory cytokines and decreases in the expression of phenotype-specific contractile proteins. Molecular interventions that prevent or inhibit paxillin phosphorylation have a similar effect: the phosphorylation of paxillin promotes the differentiation of ASM, whereas the inhibition of paxillin phosphorylation promotes an inflammatory phenotype in the muscle (108, 154). FAK and paxillin also regulate phenotype transitions that are induced by ECM components in ASM (54, 56).

The molecular mechanisms for the transduction of mechanical and humoral stimuli by FAK and paxillin to regulate nuclear signaling pathways mediated by Akt in ASM involve the recruitment and binding of “IPP” complexes to integrin adhesomes (54, 108, 110, 111, 157) (FIGURE 9). The IPP complex is a heterotrimeric complex consisting of integrin-linked kinase (ILK), PINCH (Particularly interesting new cysteine-histidine rich protein, an adaptor protein that consists of 5 LIM domains), and parvin (148, 245–249). Parvin is expressed in both α- and β-isoforms that can both bind to ILK (148, 245). ILK can interact exclusively with each of the parvin isoforms; thus, an IPP complex can contain either α-parvin or β-parvin but not both (248, 250, 251). Pools of both α- and β-parvin IPP complexes are maintained in the cytoplasm of ASM in stable inactive form and are recruited to the adhesion complexes in response to extracellular stimuli (108, 251). IPP complexes containing either α-parvin or β-parvin localize to integrin adhesomes by binding directly to paxillin (252–254); however, their recruitment and activation are differentially regulated by paxillin (108). The tyrosine phosphorylation of paxillin promotes its interaction with β-parvin IPP complexes at adhesomes, whereas α-parvin IPP complexes interact with unphosphorylated paxillin. The binding of Akt to the α-parvin IPP complex facilitates the activation of Akt by ILK (108, 255). Although the β-parvin IPP complex can also bind to Akt, β-parvin inhibits Akt activation by blocking the interaction of Akt with ILK (108, 256, 257). Thus, phenotype modulation in ASM tissues can be regulated by the differential coupling of α- and β-parvin IPP complexes to paxillin at integrin adhesomes; this determines whether ILK is positioned to activate Akt (108, 154). When paxillin is unphosphorylated, α-parvin IPP complexes are recruited to adhesomes and induce the activation of Akt by ILK, which promotes an inflammatory phenotype. When paxillin is phosphorylated, β-parvin IPP complexes are recruited to adhesomes, and the activation of Akt is inhibited.

Integrin adhesome complexes regulate the activation of other critical signaling intermediaries that mediate ASM phenotypic properties; however, the molecular processes that regulate the coupling of signals from integrin receptors to many of these pathways remain to be elucidated.

8. IMPLICATIONS FOR PATHOPHYSIOLOGY

Pathophysiological conditions of the lungs such as airway inflammation and asthma alter the extracellular milieu of the cells and induce changes in ASM phenotypic expression that can contribute to ASM dysfunction (FIGURE 8). With chronic disease, this can ultimately lead to tissue remodeling and structural changes in the airways and lungs that significantly impact lung function (27, 57, 258–260). Such structural changes can be self-perpetuating, as pathologically induced changes in the structure of lung and airway tissues can themselves stimulate phenotypic alterations in airway cells that promote continued airway remodeling. Lung tissue remodeling is one of the most intractable problems in chronic pulmonary diseases and can result in the irreversible loss of lung function. The elucidation of integrin-mediated molecular pathways that regulate the transduction of extracellular signals to intracellular pathways that regulate the expression of phenotypic properties of ASM could provide new targets for therapeutic interventions to prevent or reverse remodeling processes that contribute to lung pathology (55, 58, 60, 98, 261, 262).

9. CONCLUSIONS

The membrane adhesion junctions on ASM bind individual cells within muscle tissues to components of the extracellular matrix. These junctions enable the transmission of mechanical tension generated by the smooth muscle cells throughout the tissue, and they also provide for the transmission of external forces imposed on the muscle to the actin cytoskeleton. Whereas adhesion junctions have historically been viewed as static structural components of the cell, there is now a wealth of evidence that these junctions play regulatory roles that are critical to the normal physiological functions of smooth muscle tissue. Adhesion junction complexes are highly dynamic structures that transduce information regarding the external cellular environment to intracellular pathways that enable the cell to rapidly adapt its physiological properties to external mechanical forces or alterations in the extracellular matrix environment. Membrane adhesion complexes orchestrate the reorganization of the cortical actin cytoskeleton, which enables the ASM cells to modulate their mechanical behavior and contractility in response to the external forces imposed on the muscle tissues during breathing. Signals from adhesion complexes to the cell nucleus mediate transitions in phenotypic expression induced by changes in the extracellular environment. Pathophysiological conditions alter the local extracellular environment of the smooth muscle cell and induce changes in cell structure and function that contribute to its dysfunction. There have been many advances in our understanding of the complexity of the influences of the extracellular environment on the functional properties of ASM; however, much more work will be needed to unravel the complex molecular events that determine the physiological responses of the cell to changing environmental conditions and to establish the role of extracellular influences on the changes in ASM cell structure and function that contribute to its dysfunction under disease conditions.

GRANTS

These studies were supported by National Heart, Lung, and Blood Institute Grant HL029289.

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

W.Z., Y.W. and S.G. prepared figures; drafted manuscript; edited and revised manuscript; and approved final version of manuscript.