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The Proceedings of the American Thoracic Society 5:80-88 (2008)
© 2008 The American Thoracic Society
doi: 10.1513/pats.200705-057VS
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Phenotype and Functional Plasticity of Airway Smooth Muscle

Role of Caveolae and Caveolins

Andrew J. Halayko1,2, Thai Tran1,2 and Reinoud Gosens1,2,*

1 Departments of Physiology and Internal Medicine, Section of Respiratory Diseases, and National Training Program in Allergy and Asthma, University of Manitoba, Winnipeg, Manitoba, Canada; and 2 Biology of Breathing Theme, Manitoba Institute of Child Health, Winnipeg, Manitoba, Canada

Correspondence and requests for reprints should be addressed to Dr. Andrew J. Halayko, Ph.D., Associate Professor, Canada Research Chair in Airway Cell and Molecular Biology, Section of Respiratory Disease, Health Sciences Centre, RS321–810 Sherbrook Street, Winnipeg, MB, R3A 1R8 Canada. E-mail: ahalayk{at}cc.umanitoba.ca

ABSTRACT

Airway smooth muscle (ASM) cells exhibit phenotype plasticity that is under control of external stimuli such as growth factors and the extracellular matrix, and is regulated by a network of intracellular signaling cascades that control transcription and protein translation of phenotype-specific genes. Phenotype plasticity underpins the ability of airway myocytes to contribute both to acute bronchospasm, and to the features of airway remodeling in chronic asthma. A feature of mature, contractile ASM cells is the presence of abundant caveolae, omega-shaped plasma membrane invaginations that develop from the association of lipid rafts with caveolin-1, a unique protein with structural and functional properties. Caveolae and caveolin-1 modulate signaling from receptors for growth factors and contractile agonists, and thus may modulate functional diversity of myocytes. Caveolin-1 appears to play a suppressive role in ASM cell proliferation, and orchestrates receptor-mediated signal transduction that regulates phenotype expression of ASM cells. Interestingly, in contractile myocytes caveolae are organized in close proximity to intracellular Ca2+-handling organelles, and are partitioned into discrete linear domains aligned with β-dystroglycan, a subunit of the actin-tethered dystrophin glycoprotein complex (DGC). Despite development of transgenic models to investigate caveolin biology, only superficial understanding of the role of these proteins in ASM phenotype expression and modulation of the functional responses of myocytes of a particular phenotype is available. This review summarizes mechanisms regulating ASM cell phenotype plasticity, and the role of caveolae as determinants of the functional diversity of ASM cells of a particular phenotypic state.

Key Words: intracellular Ca2+ • contraction • proliferation • G-protein coupled receptors • dystrophin dystroglycan complex

Airway smooth muscle (ASM) is a primary determinant of airway physiology in health and disease due to its ability to contract and control the diameter of the bronchi and bronchioles that it encircles. Traditional research has focused on factors that determine the contractility of ASM, including mechanical properties, responsiveness to contracting and relaxing factors, and structural association with the lung parenchyma. Contemporary research incorporates understanding gleaned in the last decade that differentiated myocytes retain a capacity for phenotype plasticity. Myocytes undergo reversible switching between a contractile phenotype and a more "immature" phenotype characterized by a tendency to proliferate and/or synthesize extracellular matrix and other biologically active proteins. Thus, in obstructive airways disease myocytes are able to control airway diameter acutely via reversible bronchospasm, and chronically as an effector of key aspects of airway wall remodeling that underpin irreversible reductions in airway conductance (1).

Studies of ASM phenotype have identified molecular markers of the contractile phenotype that include contractile apparatus– and cytoskeleton-associated proteins (2–4). Understanding of signal transduction pathways that regulate gene transcription and protein translation associated with phenotype plasticity is now emerging. Ultrastructural markers of myocyte phenotype have also been identified—for instance, a high cytoplasmic volume fraction of contractile apparatus in mature smooth muscle cells (5, 6). In addition, the formation of abundant caveolae, which can exceed 160,000 in number on a single contractile myocyte, occurs during the differentiation of smooth muscle cells (7, 8). A feature of caveolae is the presence of a unique family of proteins, the caveolins, which serve a scaffolding and signal transduction function and are thought to modulate cell responses to a wide range of external factors, including contractile agonists and mitogens (9). This review provides an overview of existing paradigms for ASM phenotype plasticity, the mechanisms that control phenotype and functional plasticity, and discusses the emerging role of caveolae and caveolins as determinants of the functional responses of proliferative and contractile phenotype ASM cells.

FEATURES OF PHENOTYPE PLASTICITY

Phenotypic plasticity occurs in differentiated smooth muscle cells and is manifest as the reversible modulation and maturation of individual myocytes both in vitro and in vivo (3, 4). Phenotype switching requires changes in expression of phenotype-specific genes and the subsequent accumulation of the proteins that they encode. Primary culture models of smooth muscle cells, including those from the airways, reveal that contractile myocytes undergo spontaneous phenotype modulation when seeded at subconfluence in the presence of mitogens (2, 3, 5). Phenotype modulation results in the acquisition of a synthetic phenotype characterized by the presence of abundant organelles for protein and lipid synthesis, and numerous mitochondria (Figure 1). The cells also exhibit increased proliferative capacity, but diminished abundance of contractile apparatus and associated proteins, with a concomitant suppression of responsiveness to contractile agonists (5, 10). The reacquisition of a contractile phenotype is called maturation, and in cell culture it occurs as cells grow to confluence and achieve contact inhibition (Figure 1). Maturation is marked by accumulation of contractile apparatus and associated proteins, reacquisition of responsiveness to physiologic contractile agonists, and decreased abundance of synthetic organelles (10, 11). Protein markers for the contractile phenotype include smooth muscle (sm)-{alpha}-actin, sm-{gamma}-actin, sm-myosin heavy chain (smMHC), calponin, h-caldesmon, SM22, desmin, and smoothelin (for review see References 3 and 4). Concomitant with the expression of molecular markers for the contractile phenotype, recent studies have revealed that the number of caveolae, and their associated marker proteins caveolin-1 and cavelolin-2, are markedly increased in ASM and vascular smooth muscle cells during myocyte maturation (12–14). As discussed in later sections, caveolae appear to play a central role in modulating receptor-mediated cell responses, and thus are key determinants of the functional diversity of individual smooth muscle cells.


Figure 1
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  		Figure 1. Phenotypic plasticity of airway smooth muscle. Modulation of myocytes to a functional "synthetic/proliferative" state is induced by placing cells in low-density primary culture, exposure to mitogens, and adherence to extracellular matrix (ECM) proteins such as fibronectin and collagen-1. Maturation to a functional "contractile" state occurs in cell culture at high cell density, in response to mitogen withdrawal, exposure to a number hypertrophic stimuli, and adherence to laminin-rich ECM. "Synthetic/proliferative" myocytes predominate in the developing lung and primary cell culture, and their numbers are thought to be increased in adult airways in association with fibroproliferative pathologies. These cells exhibit a high volume fraction of synthetic organelles (e.g., Golgi apparatus), but have relatively few caveolae and low levels of contractile apparatus–associated proteins. "Contractile" myocytes predominate in normal adult tissues, and exhibit variable degrees of maturation based on the level of molecular and functional markers. These cells exhibit a high volume fraction of contractile apparatus, many caveolae in ordered linear arrays, and abundant levels of contractile apparatus–associated proteins.

 
Phenotype maturation of myocytes in culture is accelerated by mitogen withdrawal and is promoted by supplementation with insulin or transforming growth factor β (11, 15, 16). A distinct subset of ASM cells mature into a functionally contractile phenotype with elongated morphology, fully reconstituted contractile apparatus, abundant contractile protein content, and cell surface re-coupling of muscarinic M3 receptors for acetylcholine (10, 11, 17). Maturation appears to require endogenously expressed laminin-2, and is reliant on individual cells expressing a unique repertoire of laminin-binding {alpha}-integrin subunits (18, 19). Notably, maturation of the large contractile cells appears to parallel the process of cellular hypertrophy, as it requires signaling pathways that control protein translation (15, 20, 21). As increased smooth muscle mass is a feature of airways remodeling in asthma, further evaluation of mechanisms controlling myocyte maturation is likely to contribute better understanding to the remodeling process in asthma.

CAVEOLAE AND AIRWAY SMOOTH MUSCLE CELL PHENOTYPE

Since caveolae were first identified by electron microscopy as 50- to 100-nm invaginations of the plasma membrane, there has been considerable progress in our understanding of their role in cell biology (9). It is now clear that they comprise specialized plasma membrane regions to which numerous receptors, signaling proteins, and ion channels are sequestered, thus creating foci for the regulation of cellular activity (9, 22). Caveolae are formed from cholesterol and sphingolipid-rich, "liquid-ordered" lipid rafts that bind to and are stabilized by oligomeric complexes of distinct caveolae proteins, the caveolins. Three caveolins are expressed by separate genes, and of these caveolin-1 and caveolin-3 independently induce caveolae formation (9). Overexpression of caveolin-1 in cells that are caveolae deficient is sufficient to induce formation of caveolae (23, 24). Caveolin-1, which can form oligomeric complexes alone or with caveolin-2, is the most abundant isoform in vascular and ASM, whereas caveolin-3 is more or less skeletal muscle specific (25, 26).

The ability of caveolae to sequester signaling proteins lies in the unique structural properties of the caveolin proteins (Figure 2). Caveolin-1 is a 22-kD protein that integrates with the sarcolemma via two membrane association domains (MADS) such that both the C- and N-termini remain intracellular (9). The intracellular N-terminal region harbors a 20–amino acid (residues 82–101) caveolin-scaffolding domain (CSD) that has affinity for signaling molecules with a conserved caveolin-binding domain (CBD) (27–29). The CSD provides the means for caveolin to co-segregate signaling proteins and regulate cross-talk between different pathways (30, 31). Caveolins appear to associate preferentially with inactive signaling proteins that harbor a CBD, and may modulate basal activity; however, upon activation of the signaling effectors by internal or external stimuli caveolin-mediated inhibition is removed (9, 30). A large number of caveolae-associated proteins that possess a CBD have been identified (Figure 2); many of these have well-defined roles in control of smooth muscle contraction and proliferation: for example, G-protein {alpha}-subunits, members of the Rho small GTPase family and downstream effectors such as Rho kinases, protein kinase C (PKC), adenylate cyclase isoforms, G protein receptor kinases (GRKs), receptor tyrosine kinases such as receptors for platelet-derived growth factor (PDGF) and epidermal growth factor (EGF), members of the Src tyrosine kinase family, and a number of glycosyl phosphatidylinositol (GPI)-linked proteins (for a review see Reference 9). Thus, caveolins interact with signaling proteins but do not transduce intracellular signals per se, however they do appear to tweak the activity of signal transduction cascades that control the full range of smooth muscle phenotype and function.


Figure 2
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  		Figure 2. Schematic representation of structural and functional domains in caveolin-1. The 22-kD caveolin-1 protein is composed of 178 amino acids (a.a.). It is anchored to the cytosolic leaflet of the plasmalemma bilayer by two membrane association domains, N-MAD (black, a.a. 70-101) and C-MAD (green, a.a. 134-156). N- and C-termini are cytoplasmic. C-MADS has three palmitoylation sites, cysteines 133, 143, and 156 (C133, C143, C156). The highly conserved caveolin scaffolding domain (CSD) (shown in red) includes a.a. 82 to 101 of the N-MAD, and is essential for interactions with signaling molecules such as G{alpha}-subunits, PKC isozymes, and RhoA that carry a caveolin-binding domain (CBD) sequence; the CBD sequence shown corresponds to G{alpha}ia1. Caveolin-1 also contains a WW domain (WW) (shown in blue) from a.a. 99 to 132 that is marked by conserved aromatic and proline residues (important residues are boxed). The WW domain has selective binding affinity for proteins with a conserved PPXY motif; the WW binding domain shown corresponds to the C-terminal tail of β-dystroglycan.

 
As described in subsequent sections, recent in vitro and in vivo evidence suggests caveolae and caveolin-1 may modulate contractile and proliferative activity of smooth muscle cells. Also, a number of signaling pathways that control transcription and translation of phenotype marker proteins are known to be associated with caveolae; this implies that these membrane structures, which themselves are significantly increased in number in contractile phenotype myocytes, may be involved with the integration of mechanisms that control myocyte differentiation. Caveolin-1 protein abundance increases greatly as vascular and ASM cells acquire a contractile phenotype, and this results in the appearance of significant numbers of caveolae that become organized into longitudinally oriented rib-like plasma membrane arrays (8, 32, 33). Individual caveolae appear as flask-like invaginations of the cell membrane, and in smooth muscle can form tube-like cavities or grape-like clusters just beneath the plasma membrane in close proximity to sarcoplasmic reticulum, which can also be associated with mitochondria (7, 34, 35). Caveolin-1 exhibits a partial shift in distribution to the Golgi apparatus in proliferating myocytes compared with quiescent, contractile phenotype cells (36). Phenotype-associated differences in caveolae number and distribution are likely important in the functional diversity of myocytes, as caveolins provide a scaffold for signaling proteins, receptors, and ion channels that are known to control muscle cell development, differentiation, and proliferation.

MECHANISMS FOR PHENOTYPIC PLASTICITY

ASM phenotype is regulated by growth factors, contractile agonists acting on G protein–coupled receptors (GPCRs), and extracellular matrix proteins. In a stimulus-dependent manner, smooth muscle cells can be induced to undergo modulation to a proliferative/synthetic phenotype or to follow a maturation process leading to the expression of a functionally contractile state. For example, whereas TGF-β, insulin, and laminin support the induction of a contractile ASM phenotype, PDGF, fetal bovine serum, and fibronectin promote the proliferative function of the smooth muscle cells (37–40). The differential capacity for these factors to induce modulation or maturation of smooth muscle phenotype appears to be related to the induction of unique cassettes of intracellular signaling pathways.

Maturation of ASM cells requires the marked accumulation of contractile proteins and those that regulate the contractile apparatus, for instance SM22, sm-{alpha}-actin, smMHC, calponin, and desmin. Expression of these proteins requires coordinated control of transcriptional and translational processes (1, 4). The Rho/Rho kinase pathway and PI3K (phosphatidyl inositide 3-kinase)–dependent pathways have been associated with transcription and translational control of smooth muscle–specific proteins (20, 21, 41–43). These pathways are summarized schematically in Figure 3.


Figure 3
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  		Figure 3. Mechanisms of airway smooth muscle phenotype maturation. Maturation of airway smooth muscle cells is under coordinated control of transcriptional and translational processes. Smooth muscle–specific gene transcription is chiefly regulated by the transcription factor SRF and its co-activators myocardin (Mcdn) and MAL. Nuclear localization of SRF is regulated by Rho kinase-mediated actin polymerization. Protein kinase C activation can counter effects of Rho kinase by inducing actin depolymerization. Myocardin binding to SRF can be prevented competitively by phosphorylated Elk-1, which is targeted by p42/p44 MAP kinase (not shown). Smad signaling, activated by TGF-β receptors, can enhance smooth muscle specific gene transcription by the interaction of SRF with regulatory Smads -2, -3, and -4; inhibitory Smad, Smad-7, suppresses effects of regulatory Smads on SRF. Translation of smooth muscle–specific gene transcripts into proteins requires intracellular signaling by PI3K dependent pathways. PI3K signaling also disrupts the interaction of the inhibitory transcription factor, Foxo4, with nuclear SRF (not shown). The pathways involved with gene transcription and protein translation are regulated by extracellular stimuli such as acetylcholine, TGF-β, insulin, and growth factors (e.g., platelet-derived growthnfactor). Abbreviations used: InsR, insulin receptor; M3R, muscarinic M3 receptor; Mcdn, myocardin; mTOR, mammalian target of rapamycin; PI3K, phosphatidyl inositide 3-kinase; PKC, protein kinase C; SRF, serum response factor.

 
RhoA, Rho Kinase, and PKC in ASM Phenotype
Transcription of smooth muscle genes such smMHC, SM22, calponin, and {alpha}-actin that encode markers of the contractile phenotype is regulated by combinatorial control involving a number of key transcription factors (reviewed in Reference 44). The 5' promoters of smooth muscle–specific genes have at least two CArG box elements [CC(A/T)6GG] that each bind dimers of serum response factor (SRF); these sites are essential for smooth muscle–specific gene expression as their mutation renders their promoters inactive (1, 45, 46). In the nucleus, SRF associates with transcriptional coactivators such as myocardin and MAL/MKL1 (megakaryocytic acute leukemia/megakaryoblastic leukemia), which direct SRF to smooth muscle–specific promoters (47, 48). Activation of smooth muscle gene transcription by SRF is further regulated by the Rho/Rho kinase pathway, which promotes SRF nuclear localization in association with increasing filamentous actin in the myoplasm (43, 49, 50) (Figure 3). The ability of the Rho/Rho kinase pathway to promote actin polymerization leads to a loss of globular actin (g-actin), which results in the release of the SRF co-activator MAL, a g-actin–binding protein (47). Thus, SRF, a central regulator of smooth muscle–specific gene transcription, is under tight control by the Rho/Rho kinase pathway.

Activation of Rho/Rho kinase signal transduction by receptor tyrosine kinases and GPCRs occurs through the action of Rho-specific guanine exchange factors (RhoGEFs). For example, muscarinic M3 receptors coupled to G{alpha}q can induce RhoA via p63RhoGEF and promote Rho kinase-dependent actin polymerization that leads to nuclear translocation of SRF and the induction of smooth muscle–specific gene expression (42, 51). Interestingly, the concomitant induction of conventional protein kinase C isoforms by G{alpha}q-coupled receptors, phorbol esters, or the application of cyclic bi-axial mechanical strain likely balances the actin-dependent, pro-transcriptional effects of RhoA, as PKC induction leads to filamentous actin de-polymerization, which inhibits SRF activation (52, 53) (Figure 3). Collectively, these observations indicate that mechanisms that induce or modulate RhoA–Rho kinase and/or PKC are likely to be an important determinant of ASM phenotype expression through actions on SRF-mediated gene transcription. Notably the activation of PKC, RhoA, and Rho kinase isoforms is regulated by interactions with the caveolin-1 CSD (54). Moreover, as caveolae and caveolin-1 increase during ASM maturation, it is plausible that they play an active role in regulating smooth muscle phenotype expression. To date, systematic assessment of the regulatory role of caveolins in ASM phenotype plasticity has not been reported, though based on existing evidence this may be an important area for future research.

TGFβ in ASM Phenotype: Transcription and Protein Translation
TGF-β is a potent inducer of a hypertrophic and hypercontractile ASM phenotype that can promote SRF-dependent gene transcription through activation of Smad signaling (15, 41). TGF-β, via TGF-β receptor-1 and TGF-β receptor-2, induces phosphorylation and nuclear translocation of "regulatory" Smad-2, -3, and -4. Regulatory Smads can bind with nuclear SRF to promote smooth muscle–specific gene transcription, whereas Smad-7 counteracts this effect (41) (Figure 3). Of note, caveolin-1 appears to suppress TGF-β–induced regulatory-Smad activation, further suggesting a role for caveolae in myocyte phenotype expression. Notably, recent evidence related to lung fibroblast proliferation and pro-fibrotic function, which is induced by TGF-β1, in idiopathic pulmonary fibrosis is linked to a marked reduction in caveolin-1 expression (55). Due to the common origin of lung mesenchymal cells, these results are germane to the assessment of the role of caveolae in ASM phenotype and function, as they suggest that caveolin-1 likely prevents phenotype modulation and that its expression during maturation may be related with a change in myocyte responses to TGF-β.

The transcripts from smooth muscle–specific genes need to be translated into proteins to impact on myocyte phenotype and function. Protein translation is under control of several pathways that converge at the level of the ribosome, and a number of these are required for smooth muscle maturation. Mature smooth muscle cells express elevated levels of active, phosphorylated kinases with known effects on protein translation, including PI3K, Akt1, mTOR (mammalian target of rapamycin), and p70 ribosomal S6 kinase (20). Pharmacologic inhibition of PI3K and mTOR are sufficient to prevent p70 ribosomal S6 kinase activation and accumulation of smooth muscle–specific proteins (20). Furthermore, active mTOR can phosphorylate and activate 4E-BP1, a protein that binds to and activates the eukaryotic initiation factor, eIF4, which initiates protein translation and contractile protein accumulation (21). Activation of these PI3K-dependent signaling pathways is required for TGF-β and insulin-induced airway smooth muscle maturation (15, 16, 39), indicating that in addition to SRF-dependent gene transcription, the translation of smooth muscle–specific proteins is tightly regulated by specific intracellular signaling pathways (Figure 3). A recent study has also revealed that PI3K-Akt1 signaling may also control transcription of smooth muscle–specific genes through the phosphorylation of Foxo4 forkhead transcription factor (56). In its unphosphorylated state Foxo4 binds to nuclear myocardin and inhibits its interaction with SRF, thereby damping transcription of smooth muscle–specific genes. Targeting of Foxo4 by Akt1 releases a suppressive mechanism by allowing for myocardin–SRF binding, which activates transcription of smooth muscle–specific genes. Importantly, ongoing studies in our laboratory have revealed that PI3 kinase is closely associated with caveolin-1 in cultured human ASM cells, thus providing another potential link for a role of caveolins in protein translation and transcriptional mechanisms that regulate phenotype expression.

Mitogen-Activated Protein Kinases and ASM Phenotype
In comparison to phenotype maturation, less is known about mechanisms that trigger modulation of smooth muscle cells to a proliferative/synthetic phenotype. Highly mitogenic growth factors such as PDGF or FBS induce a proliferative ASM phenotype that is accompanied by a loss in contractile function due to loss of contractile and contraction-regulatory proteins (4, 57). Paradoxically, activation of the early response gene c-fos, which regulates cell proliferation, requires SRF to bind to CArG elements embedded in serum response elements that are present in the c-fos promoter. The apparent duality of SRF to induce pro-differentiation and pro-proliferative genes can be explained by the existence of multiple transcriptional co-activators that compete for binding to SRF, and thereby direct selective induction of gene transcription. When bound to myocardin, SRF induces smooth muscle–specific genes; in contrast, when bound to ternary complex factors (TCFs) such as phospho-Elk-1, SRF induces proliferative genes such as c-fos (48). As Elk-1 is phosphorylated by p42/p44 mitogen-activated protein (MAP) kinase, the regulation of p42/p44 MAP kinase is a key determinant of smooth muscle cell phenotype; for example, mitogen-induced phenotype modulation is prevented by inhibitors of the p42/p44 MAP kinase pathway (48, 57, 58). Thus, there appears to be a crucial role for growth factor–induced p42/p44 MAP kinase signaling in determining SRF-dependent gene transcription targets, and in controlling pro-contractile to pro-mitogenic functional responses of smooth muscle cells. Furthermore, as described in the next section, recent data from our group indicate that caveolin-1 plays a central role in modulating p42/p44 MAP kinase activation in human ASM cells (59); thus, this could represent an additional arm by which caveolins are involved in the dynamics of myocyte phenotype plasticity.

CAVEOLAE AND AIRWAY SMOOTH MUSCLE PROLIFERATION

There is considerable evidence that caveolins regulate proliferation of many cell types. Loss of caveolin expression is associated with a hyper-proliferative state; this includes ASM and smooth muscle cells of other visceral origins (33, 60, 61). In smooth muscle cells from the airways and other organs, caveolin-1 levels are diminished in myocytes that modulate to an immature, proliferative phenotype (8, 33, 36). Peterson and coworkers (14) recently reported that exposure of cultured vascular smooth muscle cells to PDGF resulted in a rapid 60% reduction in the abundance of caveolin-1 protein. This is consistent with our recent studies using ASM cells in which reduction or ablation in caveolin-1 expression by siRNA silencing induces spontaneous myocyte proliferation (33, 59). Collectively, these data indicate a potent anti-mitogenic role for caveolin-1 in ASM cells. Notably, though caveolin expression in the airways of individuals with asthma has not been assessed, fibroblastic foci in the lungs of patients suffering from idiopathic pulmonary fibrosis exhibit greatly reduced caveolin-1 expression (55).

A number of signaling proteins involved in proliferation of ASM cells can associate with caveolin-1, and this association appears to suppress pro-mitogenic mechanisms. Binding of caveolin-1 with receptor tyrosine kinases (RTKs), including epidermal growth factor receptor (EGFR) and platelet derived growth factor receptor (PDGFR), inhibits tyrosine kinase activity in human ASM cells (33, 59). Indeed, in quiescent ASM cells the vast majority of unphosphorylated (inactive) EGFR and PDGFR are sequestered to caveolae microdomains. Overexpression of caveolin-1 abrogates PDGF-induced DNA synthesis, and inhibits activation of the MEK-ERK1/2 axis in fibroblasts and vascular smooth muscle cells (60, 62). In human ASM cells, knockdown of caveolin-1 with siRNA or disruption of caveolae with the cholesterol-depleting compound, methyl-β-cyclodextrin, leads to the activation of p42/p44 MAP kinase to an extent that mimics the maximum induction achieved with PDGF (33). Furthermore, the induction of p42/p44 MAP kinase by caveolin-1 knockdown is sufficient to underpin ASM cell replication in vitro (33). These observations support a paradigm in which caveolin-1 suppresses proliferative activity of ASM cells, and is consistent with the accumulation of the protein and caveolae that occurs with phenotype maturation.

Caveolin-1 plays a dynamic role in the regulation of pro-mitogenic signaling pathways. The tyrosine 14 residue of caveolin-1 is phosphorylated by Src upon growth factor exposure in a temporally similar pattern as is seen for p42/p44 MAP kinase activation in ASM cells (59). Phosphorylation of Try-14 in caveolin-1 is thought to be a positive regulator for cell proliferation either by generating docking sites for SH2-containing proteins and/or by releasing receptor tyrosine kinases (63). Indeed, in human ASM cells, exposure to a mitogen such as EGF evokes the rapid trafficking of EGFR to caveolae-free plasma membrane domains that are enriched in p42/p44 MAP kinase (59). Likewise, although in quiescent cells immunoprecipitates of caveolin-1 include an abundance of the PDGFRβ subunit, the association of the receptor with caveolin-1 is rapidly lost after PDGF exposure (34). Interestingly, in human ASM cells p42/p44 MAP kinase is not sequestered to caveolae microdomains, indicating that membrane trafficking of RTKs away from caveolae is a critical early event leading to induction of pro-mitogenic signaling cassettes (59). Collectively, these studies suggest that growth factor–induced airway myocyte proliferation involves, consecutively, the release of the activated RTK from caveolin-1, followed by RTK trafficking from caveolae to caveolae-free membrane, where p42/p44 MAP kinase is activated.

An even broader organizing role for caveolae in cellular signaling associated with proliferation has been suggested as, in addition to RTKs, several GPCRs, and monomeric and heterotrimeric G proteins (including Gi, Gq and Ras) that have been associated with ASM proliferation, also localize to caveolae (9). Importantly, some GPCRs, such as the muscarinic M3 receptor for acetylcholine and the CysLT1 receptor for cysteinyl leukotrienes, significantly potentiate EGF- and PDGF-mediated smooth muscle proliferation (64–66). It is unclear how the diverse signal pathways of different receptor subtypes might be integrated to potentiate proliferation; however, there is evidence that caveolae could be involved. For example, angiotensin II or endothelin-1 induce transactivation of the EGF receptor in vascular smooth muscle cells and glomerular mesangial cells (67, 68). Notably, in vascular smooth muscle cells, EGFR and ERK1/2 transactivation by angiotensin II is reduced by depletion of cholesterol-rich plasma membrane lipid rafts and caveolae (67). Future studies to more precisely dissect the role of caveolin-1 in coordinating proliferative activity of ASM cells are needed to clarify these mechanisms.

CAVEOLAE AND AIRWAY SMOOTH MUSCLE CONTRACTION

In contractile phenotype ASM cells, caveolae are highly organized into discrete longitudinal arrays in parallel with domains of adherens junctions and dense plaques on contractile smooth muscle cells (32, 69, 70). Due to their proximity to organelles associated with intracellular Ca2+ flux, such as the peripheral sarcoplasmatic reticulum and mitochondria, caveolae have been postulated to be involved with Ca2+ release that is required for receptor-mediated activation of the contractile apparatus (34, 71). Notably, the plasma membrane Ca2+ ATPase, L-type Ca2+ channels, Na2+/Ca2+ exchanger, large conductance Ca2+-activated K+ channels, transient receptor potential protein C1 (TRPC1), and Ca2+-binding proteins calsequestrin and calreticulin localize to myocyte caveolae (25, 72–77). Our laboratory's recent work indicates that cholesterol depletion, siRNA knockdown of caveolin-1, or incubation of human ASM cells with cell-permeable CSD peptide decreases sensitivity of muscarinic M3 receptor–induced mobilization of intracellular Ca2+ and contraction of ASM strips ex vivo (78, 79). Emerging evidence is revealing a paradigm for a complex and dynamic association of receptors and signaling proteins with caveolae and caveolin-1 (Figure 4).


Figure 4
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  		Figure 4. Relationship of caveolin-1 with the dystrophin glycoprotein complex (DGC) and association with G protein–coupled receptor–mediated intracellular signaling. For simplicity a single caveolin homodimer is shown. Caveolin-1 is anchored to the cytosolic leaflet of the plasmalemma by N-MAD (red) and C-MAD (green) regions. The WW domain (blue) of both caveolin-1 and dystrophin has affinity for the WW binding domain with consensus PPXY motif of β-dystroglycan (β-DG). Dystrophin acts as a linker between the DGC and the filamentous (F)-actin cytoskeleton. The DGC is stabilized by other transmembrane subunits, including a tetrameric sarcoglycan complex (SGC) that includes, in vascular smooth muscle, β-, {delta}-, {varepsilon}-, and {gamma}- or {zeta}-sarcoglycans, and sarcospan (SP). The DGC is also linked to the surrounding basal lamina via binding of its {alpha}-dystroglycan ({alpha}-DG) subunit to laminin-1 and -2. The caveolin scaffolding domain (CSD, shown in red) of caveolin-1 overlaps the the N-MAD region, and serves as a anchor point for signaling effector proteins such as G{alpha}-subunits, PKC isozymes, and RhoA that carry a caveolin binding domain (CBD). Upon exchanging GDP for GTP, G{alpha}q-subunits associate with and activate phospholipase C-β1 (PLCβ1), which catalyzes hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to produce the intracellular messengers diacylglyceraol (DAG) and inositol 1,4,5-trisphosphate (IP3). IP3 binds to its receptor on proximal sarcolasmic reticulum (SR) membranes, and mediates a rapid release of SR Ca2+ into the cytosol. Mobilization of cytosolic Ca2+ is required to initiate contraction of smooth muscle cells.

 
Notably, contractile smooth muscle cells are unique in that they exhibit highly ordered distribution of plasma membrane caveolae, which appear as rib-like longitudinal arrays (32, 69, 70). Caveolae membrane domains run parallel to regions that bind fibronectin and contain dense plaques and adherens junctions (69). Caveolae arrays appear to be linked indirectly with the intracellular actin network through the binding of a WW domain in caveolin-1 to a WW-binding domain (WBD) in the intracellular tail of β-dystroglycan, the core transmembrane subunit of the dystrophin glycoprotein complex (DGC) (32, 80, 81) (Figures 3 and 4). Through the WBD of β-dystroglycan the DGC is linked to intracellular actin by dystrophin, whereas an extracellular {alpha}-dystroglycan subunit binds laminin (82) (Figure 4). Dystrophin—a large, flexible protein with an actin-binding domain at the amino-terminus—is anchored to cortical actin composed of β- and {gamma}-actin (83). In smooth muscle, the DGC also includes a transmembrane sarcoglycan complex (SGC) consisting β-, {delta}-, {varepsilon}-, and {zeta}- or {gamma}-sarcoglycan, and sarcospan (84, 85) (Figure 4). One of the key roles for the DGC in skeletal muscle is in stabilizing the plasma membrane to protect against damage from mechanical force. Interaction between β-dystroglycan and caveolin-1, which is critically important for the maintenance of the caveolae invaginations, has also been has been suggested to be the primary determinant of the unique caveolae distribution in ASM cells, and as such is essential for the spatial coordination of Ca2+-handling channels and organelles (32) (Figure 4).

A central role has been attributed to caveolins in vascular smooth muscle in relation to Ca2+ seensitization, a phenomenon by which contractile responses are regulated independent of intracellular Ca2+ flux (86). In this regard, caveolin-1 is required for plasma membrane targeting of some effectors that underpin Ca2+ sensitization during agonist-induced smooth muscle contraction. For example, competing peptides for the caveolin-1 CSD inhibit {alpha}-adrenergic receptor-mediated membrane translocation of protein kinase C{alpha} (PKC{alpha}) and the monomeric GTPase, RhoA, in aortic smooth muscle (54, 87); this concomitantly blocks receptor-mediated PKC and RhoA activation, and effectively inhibits contractile responses of aortic rings and cells. PKC and RhoA contribute to contraction through pathways involving the phospho-protein CPI-17, and the serine threonine kinase ROCK-1, respectively; these pathways cause Ca2+ sensitization of the contractile apparatus by inhibiting myosin light chain phosphatase through different mechanisms (for review see Reference 86).

Interestingly, a dynamic and variable relationship exists between GPCRs, downstream signaling effectors, and caveolae. Upon binding ligand, receptors may exit (e.g., β2 adrenergic receptor) (88), remain localized (e.g., endothelin ET1A receptor) (89), or translocate into caveolae (e.g., B1 & B2 bradykinin receptors) (90). In caveolae enriched membrane fractions from human ASM, we have identified muscarinic M3 and histamine H1 receptors and their associated trimeric Gq{alpha} protein subunits (91). Loss of contractile response to endothelin and serotonin has been demonstrated in blood vessels treated with the cholesterol-depleting agent, β-methyl-cyclodextrin, whereas contractile responses to {alpha}1-adrenergic agonists are unaffected in blood vessels from caveolin-l knockout mice (76, 92); this disparity of effects on receptor-mediated responses may reveal the significance of the differential association of receptors and signaling proteins in caveolae. This is further illustrated in a report showing that though both B1 and B2 bradykinin receptors traffic to caveolae and induce phospholipase Cβ1 signaling, B2 receptors are rapidly internalized after ligand binding, whereas B1 receptors remain in membrane caveolae, where they can subsequently also activate phospholipase A2 via a Ras-dependent pathway (90). Ostrom and Insel (22) reported that in ASM only a select set of adenylate cyclase (AC) isoforms (AC3, AC5, and AC6) associate with caveolae and lipid rafts, whereas the most abundantly expressed isoforms, AC2 and AC4, are not concentrated in caveolae-rich membrane fractions. AC isoforms are activated by Gs heterotrimeric G protein–linked GPCRs such as the β2 adrenergic receptor (β2AR) and the prostanoid EP2 receptor. In ASM cells the β2AR associates with caveolae-rich membrane fractions, whereas the EP2 does not, suggesting that due to unique compartmentalization each receptor could preferentially activate select sets of AC isoforms.

Collectively it appears that the expression of caveolin-1, the spatial distribution of the caveolae that are formed, and the precise static and dynamic association of receptors and signaling proteins contributes to contractile responses of ASM cells (Figure 4). Therefore, future studies using novel molecular and cellular tools, and transgenic mouse models, are needed to more fully understand the impact of these issues in airway biology and physiology in health and disease. Indeed, very recent studies by our group (93) and by Prakash and colleagues (94) have confirmed that caveolin-1 plays a central facilitator role in contractile agonist-induced intracellular Ca2+ mobilization and contraction of human ASM cells.

CONCLUSIONS

The dynamic functional behavior of ASM is a unique feature that may be at the root of biological mechanisms that lead to changes in airway function and structure in obstructive airways disease. Increased contractile responses and chronic changes in airway wall structure that manifest as airways hyperresponsiveness likely results from the plastic and multifunctional behavior of ASM cells. Alterations in myocyte phenotype, involving altered expression of key regulators of contractility, or changes in pharmacologic responsiveness of airway myocytes, can occur in response to asthma-associated mediators, leading to increased sensitivity to inhaled allergic and nonallergic contractile agonists. Airway remodeling in asthma includes an excessive accumulation of extracellular matrix, ASM, and submucosal myofibroblasts; each of these features can be linked to dynamic changes in airway myocyte phenotype and function.

Recent evidence indicates that caveolae and caveolin-1 are central processors in the signaling and functional responses of ASM cells to extracellular signals. An important role for caveolins in the role of fibroblasts in idiopathic pulmonary fibrosis has recently been demonstrated; however, to date, whether these proteins play a parallel role in fibroproliferative aspects of asthma pathogenesis involving ASM cells has yet to be explored, but research in this direction would appear to hold significant promise in identifying new targets for treatment.

ACKNOWLEDGMENTS

The authors thank Mr. Gerald L. Stelmack for the preparation of Figure 4.

FOOTNOTES

Supported by Sick Kids Foundation/Institute of Human Development, Child and Youth Health (#XG05-011), Canadian Institutes of Health Research (CIHR), CIHR-National Training Program in Allergy and Asthma, Manitoba Institute of Child Health, and Canada Research Chairs Program. R.G. holds a Marie Curie Outgoing International Fellowship from the European Community (MOIF-2005-008823). A.J.H. holds a Canada Research Chair in Airway Cell and Molecular Biology.

* Current address: Department of Molecular Pharmacology, University of Groningen, The Netherlands. Back

Conflict of Interest Statement: A.J.H. has received an operating research grant ($65,000) from a pharmaceutical company, Merck Frosst Inc. T.T. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. R.G. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

(Received in original form May 7, 2007; accepted in final form May 11, 2007)

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