Motility, Survival, and Proliferation

P1: OTA/XYZ P2: ABCJWBT335-c110018 JWBT335/Comprehensive Physiology September 26, 2011 20:45 Printer Name: Yet to Come

Motility, Survival, and ProliferationWilliam T. Gerthoffer,*1 Dedmer Schaafsma,2 Pawan Sharma,2 Saeid Ghavami,2 and Andrew J. Halayko2

ABSTRACTAirway smooth muscle has classically been of interest for its contractile response linked to bron-choconstriction. However, terminally differentiated smooth muscle cells are phenotypically plasticand have multifunctional capacity for proliferation, cellular hypertrophy, migration, and the syn-thesis of extracellular matrix and inflammatory mediators. These latter properties of airway smoothmuscle are important in airway remodeling which is a structural alteration that compounds theimpact of contractile responses on limiting airway conductance. In this overview, we describethe important signaling components and the functional evidence supporting a view of smoothmuscle cells at the core of fibroproliferative remodeling of hollow organs. Signal transductioncomponents and events are summarized that control the basic cellular processes of proliferation,cell survival, apoptosis, and cellular migration. We delineate known intracellular control mecha-nisms and suggest future areas of interest to pursue to more fully understand factors that regulatenormal myocyte function and airway remodeling in obstructive lung diseases. C© 2012 AmericanPhysiological Society. Compr Physiol 2:255-281, 2012.

IntroductionThe classical role of smooth muscle cells in the surroundingmuscle layer of hollow organs is to regulate dynamic changesin lumen caliber and wall stiffness. Appreciation of the multi-functional behavior of smooth muscle cells in physiology andpathophysiology has steadily increased since initial insightprovided by Wissler’s work on large elastic arteries (334). Inaddition to contraction, terminally differentiated smooth mus-cle cells are also capable of reversibly adopting capacity toexpress and secrete cytokines, chemokines, and extracellularmatrix (ECM) proteins, to proliferate, and to migrate. Thishas led to current paradigms that place smooth muscle cells atthe core of fibroproliferative remodeling of hollow organs indiseases of the vasculature (atherosclerosis and hypertension)and the airways (asthma and chronic obstructive disease).

Remodeling of the airways involves thickening of thebronchial and bronchiolar walls due to multiple events involv-ing multiple cell types. There is epithelial cell denudation, mu-cus gland hyperplasia, increased smooth muscle mass, thick-ening of the lamina reticularis and accumulation of subepithe-lial ECM, increased numbers of submucosal myofibroblasts,increased vascularization, and development of a chronicallyhealing epithelium (172, 185). Evidence points to progres-sive structural change in the airway wall due to rounds ofinflammation-driven wound healing as a fundamental compo-nent for development of fixed airway narrowing (171, 316). Asignificant component of irreversible airway hyperresponsive-ness in long-standing asthma excludes the inflammatory re-sponse, suggesting that fibroproliferative changes associatedwith mesenchymal cell populations in bronchial wall may un-derpin fixed airway dysfunction (144, 185). Local inflamma-tion is complex as it is manifested both by recruited leukocytesand mast cells, but also by the intrinsic capacity of airway

myocytes to express and release cytokines, chemokines, andother proinflammatory molecules (12). Thus airway smoothmuscle (ASM) thickening probably results from a combina-tion of biological signals that induce several trophic myocyteresponses.

Though ASM has classically been of interest for its con-tractile response linked to bronchoconstriction, terminally dif-ferentiated smooth muscle cells are phenotypically plasticand have multifunctional capacity for proliferation, cellularhypertrophy, migration, and the synthesis of ECM and in-flammatory mediators (139, 143, 145, 146). It is this propertyof ASM that positions it as an effector of airway remodel-ing which is a structural alteration that itself compounds theimpact of contractile responses on limiting airway conduc-tance. Understanding ASM-associated cellular mechanismsthat contribute to airway remodeling is of great relevancefor several reasons. First, though remodeling and thickeningconsists of multiple structural changes, the increased massof contractile ASM is the most significant causal featurefor airway hyperreactivity and excessive narrowing that re-duces airflow (1, 204, 332). Second, airway remodeling ischaracterized by increased numbers of myofibroblasts in thesubmucosal compartment. Their accumulation after allergenchallenge is rapid, thus there is growing belief that migra-tion of airway myocytes from the adjacent smooth muscle

*Correspondence to [email protected] of Biochemistry and Molecular Biology, University ofSouth Alabama, Mobile, Alabama2Departments of Physiology and Internal Medicine, University ofManitoba and Biology of Breathing Group, Manitoba Institute ofChild Health, Winnipeg, Manitoba, Canada

Published online, January 2012 (comprehensivephysiology.com)

DOI: 10.1002/cphy.c100018

Copyright C© American Physiological Society

Volume 2, January 2012 255


Motility, Survival, and Proliferation Comprehensive Physiology

Epithelium

Migration

Trophic andsurvival factors

Apoptosis

ProliferationHypertrophy

Trophic and survival factors

Eosinophil Neutrophil Lymphocyte Mast cell

Figure 1 Schematic representation of the role of airway smooth mus-cle (ASM) cell proliferation, cellular hypertrophy, apoptosis, and migra-tion if development of airway remodeling in asthma. A key local drivingforce for airway remodeling are cytokines, chemokines, and growthfactors released by the epithelium that act on the underlying airwaywall (myo)fibroblasts and ASM cells. ASM and fibroblasts also releasetrophic and profibrotic factors that contribute to local inflammation andtissue repair. Central to the initiation and modulation of inflammation,tissue damage and repair is recruitment of active inflammatory cells in-cluding Th-2 and Th-1 polarized lymphocytes, eosinophils, neutrophils,and mast cells.

layer feeds this response (113, 192). Last, in the precedingdecade there has been growing interest in research aimed atdeveloping new therapeutics that target ASM to treat asthma(19, 58, 168).

As an overarching paradigm for this article, Figure 1 pro-vides a schematic model for cellular mechanisms, includingmigration, proliferation, hypertrophy, and apoptosis. Numer-ous studies posit that these processes play affective and ef-fective roles in airway remodeling and hyperresponsivenessin obstructive lung disease. However, it is not entirely clearhow hyperplasia and hypertrophy of smooth muscle in re-modeled airways contributes to hyperreactivity. Nor is it clearyet how smooth muscle hypertrophy alters smooth musclefunction in vivo. This article provides an overview of cur-rent understanding of molecules and cellular processes thatregulate ASM proliferation, hypertrophy, apoptosis and mi-gration. One goal is to stimulate interest in signaling pathwaysand cellular processes that might be targets of antiremodelingtherapy.

Airway Smooth Muscle ProliferationASM cells from asthmatic humans and hyperresponsive ratsproliferate at higher rates than cells from normal humans andrats (187, 349). These observations suggest the ASM hyper-

plasia described in moderate and severe asthmatics may resultfrom modification of cell cycle control and the response tomitogenic stimuli. ASM cells in culture can respond to a vari-ety of mitogenic cues that promote traversing the Gap 1 (G1),S, Gap 2 (G2), and M(itosis) phases of the cell cycle. As anearly response to mitogen stimulation, from a quiescent G0state myocytes enter the G1 phase of the cell cycle coincidentwith increased expression of specific D-cyclins, such as cy-clin D1 (169, 339). Initially, progression through the G1 phasedepends on the binding of one or several D-type cyclins (D1,D2, and/or D3) to existing cyclin-dependent kinases (CDK4and 6), forming active complexes that subsequently activatecyclin E/CDK2. This leads to increased phosphorylation ofretinoblastoma protein (Rb), which in turn dissociates froman elongation factor E2F/Rb complex. E2F/Rb is otherwisebound to E2F responsive genes, effectively halting their tran-scription and creating a cell cycle block; the release of E2Fpermits the transcription of various genes, including DNApolymerase, essential for effective transit of cells through G1and into S phase. G1/S transition represents a restriction point(R) past which DNA will be synthesized (S phase), cells willincrease in size and synthesize microtubules (G2), and even-tually undergo mitosis (288, 289).

This whole process is of course tightly regulated. Theactivity of CDKs and their effects on cell cycle progressioncan be negatively regulated by CDK inhibitors during theG1/S transition. In this regard, two principal families of geneshave been identified based on their structure and specific CDKtargets: (i) the Cip/Kip family (p21Cip1, p27Kip1, and p57Kip2),which interfere with cell cycle in the G1 phase by inactivatingcyclin D-, E-, and A-dependent kinases (288) and (ii) theINK4/ARF family (inhibitor of kinase 4/alternative readingframe; p16INK4a, p15INK4b, p18INK4c, and p19INK4d), whichnegatively affect the catalytic subunits of CDK4 and 6 and assuch prevent interaction with cyclin D1 (288).

During the cell cycle, cells will go through a number of“checkpoints” to ensure that each phase of the cycle has beenaccurately completed before entering the next one; at eachpoint the cell is screened for DNA integrity, and requires acollective of effective temporal mitogen stimulation. The firstcycle checkpoint occurs at the end of the G1 phase, just beforeentering into S phase, where it is typically decided whetherthe cell should proceed, enter a resting/repair stage, or exitthe cycle via apoptosis. At this checkpoint DNA damage ismonitored through a process involving the tumor suppressorprotein, p53, which has capacity to arrest cycling of G1 cellsby activating transcription of p21Cip1, leading to subsequentCDK inhibition (92). Depending on the severity of DNA dam-age, p53 can either activate DNA repair proteins enabling thecell to eventually continue cell cycle or, in cases of irreparableDNA damage, induce apoptosis (92). A second checkpoint islocated at the end of the G2 phase and regulates initiation ofM phase. This checkpoint is subserved by a complex of cyclinB/CDK1 complex (referred to as MPF, maturation promotingfactor), which is responsible for essential phosphorylationevents in a number of proteins required for mitosis (212). A

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Comprehensive Physiology Motility, Survival, and Proliferation

Table 1 Primary Factors Affecting Airway Smooth Muscle Cell Proliferation in Culture

Class Proproliferative Antiproliferative References

Receptor tyrosinekinases

PDGF (A, B, C), IGF-1, bFGF, EGF, NGF, insulin (35, 99, 130, 131,166,167, 199, 234, 297)

G-protein-coupledreceptors

Histamine, thromboxane A2, endothelin-1,α-adrenergic agonists, cysteinyl leukotrienes,thrombin, tryptase, substance P, sphingosinephosphate, lysophosphatidic acid, muscarinic M3receptor agonists, 5-hydroxytryptamine, urotensin II,ATP, UTP, bradykinin

PGE2, β-adrenergicagonistsVIP, sphingosine,atrial natriuretic peptide

(25, 52, 56, 62, 75,97, 124, 129, 131,200, 217, 221, 235,236, 237, 245, 248,249, 262, 310, 348)

Cytokines IL-1β, TNF-α, TGF-β1, IL-6 IL-4, TNF-α, TGF-β1,IFNγ, IFNβ

(9, 10, 35, 55, 61, 70,71, 153, 238, 297,307)

Matrix proteins Fibronectin, collagen I, vitronectin Laminin, chondroitinsulphate

(73, 170, 231)

third checkpoint (the mitotic spindle checkpoint) occurs dur-ing metaphase when chromosomes have aligned at the mitoticplate and are under bipolar tension from the spindle appara-tus. The appropriate tension created by this bipolar attachmentis necessary to initiate progression to anaphase during whichindividual chromosomes are segregated and pulled toward op-posite poles. Thereafter, cytokinesis proceeds and the originalcell spawns two daughter cells that can then continue throughG1 phase and another cell cycle, or be diverted to a quiescentstate in G0 (92).

Factors controlling airway smooth muscle cellproliferationASM proliferation can be affected by at least three groupsof mitogens: polypeptide growth factors, G-protein-coupledreceptor (GPCR) agonists, and proinflammatory cytokines(169) (Table 1). In addition, ECM proteins are importantregulators of mitogen-induced proliferation (73, 170). Inasthma, excessive accumulation of (contractile) smooth mus-cle has frequently been described in central and small air-ways (22, 88, 335), and is typically associated with my-ocyte hyperplasia and hypertrophy. Thus, increased ASMmass may, in part, to be due to cellular proliferation driven bygrowth factors, inflammatory mediators and neurotransmitters(129, 230).

Polypeptide growth factors

Polypeptide growth factors induce proliferation by activat-ing receptors with intrinsic protein tyrosine kinase (RTK)activity and are among the most effective inducers of ASMproliferation. This group of mitogens includes for instancebasic fibroblast growth factor (bFGF), epidermal growth fac-tor (EGF), platelet-derived growth factor (PDGF), insulin-like growth factor-1 (IGF-1), and insulin, which all havebeen shown to induce ASM cell proliferation (Table 1). Sev-eral RTK growth factors, including EGF, PDGF, and IGF-1,have been implicated in asthma pathogenesis based on eitherincreased immunoreactivity of the growth factor, bioavail-

ability, and/or receptor expression (5, 14, 263, 276). Im-portantly, some combinations of these growth factors (e.g.,EGF + insulin + PDGF) can produce synergistic prolifera-tive responses in airway myocytes (90, 130, 167, 234, 297).A number of RTKs, for example the PDGF and EGF re-ceptors, are located in caveolae in the plasma membrane,where they associate with caveolin-1 (134). This may repre-sent a mechanism for additive or synergistic effects of mito-gens. For instance PDGF and EGF receptors uncouple fromcaveolin-1 in response to mitogen stimulation and thus ac-tivated, traffic to peripheral caveolae-free membrane sites,where p42/44 mitogen-activated protein kinases (MAPK) ac-tivation can take place (125, 134).

G-protein coupled receptor agonists

Contractile agonists, such as acetylcholine and cysteinylleukotrienes, acting via GPCRs have been associated withincreased ASM thickening in asthma (Table 1) and in ani-mal models of asthma (34, 123, 158, 321). However, stim-ulation of muscarinic receptors or cysteinyl leukotriene re-ceptors alone is not sufficient induce ASM cell proliferation.Rather, these GPCR agonists exert profound promitogenic ef-fects in the presence of a peptide growth factor, manifest as asynergistic increase in the proliferative response induced bythe growth factor in isolation (129, 200, 248). In addition tomuscarinic M3 and CysLT1 receptor agonists, it has becomeapparent that these effects are also observed for a number ofother contractile agonists, including histamine, bradykinin,and thrombin (28, 35, 124, 196, 200). The synergistic effectsof contractile agonists on growth factor-induced proliferationare principally mediated through receptors that are coupled totrimeric G-proteins of the Gq subfamily (129, 196, 200). In ad-dition to Gq-protein coupled receptors, several agonists [e.g.,thromboxane, thrombin, and lysophosphatidic acid (LPA)]that mediate effects via Gi-coupled GPCRs also have syner-gistic effects on growth factor-induced ASM proliferation (28,48, 52). Notably, the intracellular mechanisms for this effectdiffers from that of Gq-coupled receptors, as these agonists




do not necessarily require interaction with RTKs. Specificdetails on this issue are discussed below in a section describ-ing molecular mechanisms of proliferation.

In contrast to Gq- and Gi-coupled receptor agonists,various Gs-protein-coupled receptor agonists, includingprostaglandin E2 (PGE2) and β2-receptor agonists, inhibitASM cell proliferation (97, 310, 348). These effects appearto rely on the potency of these agonists to induce prolongedcAMP production and subsequent protein kinase A (PKA)activation (138, 311).

Proinflammatory cytokines

The involvement of proinflammatory cytokines, such as TNF,IL-6, and IL1β in ASM cell proliferation is controversial.Several reports suggest modest proliferative effects (70, 71),whereas others demonstrate no effects or even growth in-hibition (Table 1) (219, 242). It has become apparent thatfor IL-6, IL-1β, and TNF that these paradoxical findingsmight be explained by cytokine-induced production of an-tiproliferative mediators such as the cyclooxygenase-2 prod-uct prostaglandin E2 or IFNβ, which exert an autocrine effecton the ASM cells (224, 297, 307). Most of the cytokinesof interest exert their effects on gene regulation through cellsurface glycoprotein complexes, comprising 2 to 4 receptorchains that couple to several non-RTKs, such as Src fam-ily proteins and components of the MAPK and Janus kinase(JAK) and signal transducer and activator of transcription(STAT) cascades (169) (Fig. 2). The balance between paral-lel and functionally opposing signaling pathways and uniquephenotype of the cell population are ultimately the determi-nants of the effects of cytokines on ASM proliferation.

Extracellular matrix proteins

Several ECM proteins have emerged as regulators of growthfactor-induced ASM cell proliferation (Table 1). Cells cul-tured on monomeric collagen I or fibronectin matricesprogress toward a more proliferative phenotype, as evidencedby an augmented basal proliferative response (32, 73) and anaugmented mitogenic response toward either RTK or GPCRligands (32, 73, 170, 231). Conversely, when cultured on alaminin or laminin-rich Matrigel substrate, growth factor-induced proliferation is markedly suppressed (73, 74, 170).These observations could be of significant relevance to airwaywall remodeling and asthma pathogenesis, as both the quan-tity and the composition of the ECM is altered in the airwaysof chronic asthmatics. Deposition of collagen IV and elastinis decreased in the airway wall of asthmatic patients, whereascollagen I, III, V, fibronectin, tenascin, hyaluran, versican, andlaminin α2/β2 chains are increased (4, 202, 203, 271). Im-portantly, changes in matrix composition directly surroundingASM cells have also been reported: collagen I, hyaluronan,and versican increased in patients with asthma (270, 333).Human ASM cells also secrete ECM proteins in response toasthmatic sera (188) suggesting a cellular source for ECMdeposition in airways and implicating a novel mechanismin which ASM cells may modulate autocrine proliferativeresponses.

ECM proteins interact with smooth muscle cells throughintegrins, which are heterodimeric glycoproteins consistingof membrane-spanning, noncovalently associated, α and β

subunits (111). Enhancement of growth factor-induced pro-liferation of ASM cells on a collagen I or fibronectin matrix isdependent on activation of α2β1, α4β1, and α5β1 integrins,

Cytokinereceptor

Src

JAK

Ras

Raf

MEK

ERK

STAT

Shc

Grb

Grb

SOS

SOS

α

αβ γ Shc

Src

PDKPKC

PI3 kinasePI3-K

Akt

mTOR

p7056KGSK3

PIP3

RTK

ASM proliferation

RTK

GPCR

Gq

Giβγ

GPCR

Figure 2 Schematic representation of key signaling mechanisms associated with control of airwaysmooth muscle cell proliferation. See text for details and Table 1 for list of factors that control activationof these pathways.




of which α5β1 has emerged as a crucial signaling integrin forproliferation both in healthy and asthmatic ASM cells (231).Laminin most likely exerts its antiproliferative effects throughthe α7β1 integrin (328).

Molecular signaling pathways in airway smoothmuscle cell proliferationMajor pathways described below are shown schematically inFigure 2.

Citogen-activated protein kinases

The mitogen-activated protein (MAP) kinases are a super-family of serine/threonine-directed protein kinases involvedin transcriptional regulation in response to a variety of ex-tracellular stimuli, including growth factors (354), therebybeing responsible for intracellular transmission of extracellu-lar trophic signals. MAP kinases share a common activationmechanism which involves the phosphorylation of tyrosineand threonine residues in a Thr-X-Tyr (TXY) motif posi-tioned in their activation loop. Based on the identity of theresidue between the threonine and tyrosine, the MAP kinasesuperfamily can be divided into three main groups: ERKs(Thr-Glu-Tyr); Jun amino terminal kinases (JNKs) (Thr-Pro-Tyr); and p38s (Thr-Gly-Tyr). Each MAP kinase is activatedby successive activation of a MAP kinase kinase kinase anda MAP kinase kinase. Activation of the ERK pathway consti-tutes an important regulator of cell cycle entry and G1 pro-gression, and is required for DNA synthesis and proliferationin an extensive variety of mammalian cell systems, includ-ing bovine, rat, and human ASM (189, 207, 242, 331). Thetraditional path to ERK activation is comprised of the growthfactor receptor binding protein Grb2, the nucleotide exchangefactor Son of sevenless (Sos), the monomeric 21 kDa GTPaseRas, the 74 kDa cytosolic serine/threonine kinase Raf-1, andthe 45 kDa dual function kinase MAP kinase/ERK kinase ki-nase (MEK)-1. Grb2 is found in a stable complex with thenucleotide exchange factor Sos. Docking of Grb2 to a RTKcauses Sos to bind to and activate Ras. Ras then escorts Raf-1 to the cell membrane, resulting in Raf-1 activation (298).Raf-1 phosphorylates MEK1 on two serine residues, Ser218

and Ser222 (344) MEK1 phosphorylates tyrosine and threo-nine residues in the ERK activation loop. Induction of theRas/Raf1/MEK/ERK1/2 pathway has emerged to be a keypathway in the transcriptional activation of the cyclin D1promoter, cyclin D1 activity, and protein expression (8, 169,243).

It has been suggested that p21Ras can act as a point of con-vergence for mitogenic signals induced by different receptor-operated mechanisms (6, 169). Activation of p21Ras resultsnot only in its binding to Raf-1 but also phosphoinositide 3-kinase (PI3-kinase) (the latter effect is described in the nextsection) (Fig. 2). Notably, the mechanistic difference betweenthe proproliferative effects of Gi- and Gq-coupled receptorsmay be explained by the differential involvement of the p42/44

MAPK cascade. Thus, Gi, but not Gq, activates p21Ras inASM cells (93). For example, Ras/Raf/MEK/p42/p44MAPKsignaling is involved in the mitogenic effects of the Gi-protein-coupled receptor agonists thromboxane A2, thrombin, andLPA Citro, 2005 (59, 89, 209, 210). Gi mediates p42/p44MAPK activation via its βγ-receptor subunits, which havebeen shown to increase p21Ras activation through an aug-mented tyrosine phosphorylation of Shc leading to an in-creased functional association between Shc, Grb2, and SOS(299, 315) (Fig. 2). Along with p42/44 MAPK, p38 MAPKhas emerged as a regulator of ASM cell proliferation (95,229). However, the involvement of p38MAPK appears to bestimulus dependent, as it is not involved in TGFβ1-inducedproliferation of human ASM cells (338).

Ras-dependent PI3-kinase pathways

Activation of RTKs results in the intracellular phosphoryla-tion of receptor tyrosine residues (receptor autophosphoryla-tion), which serve as docking sites for other kinases, includ-ing Src and phosphatidyl inositol 3 kinase (PI3-kinase), andmediates p21Ras activation through the guanine nucleotideexchange factor Sos (356). PI3-kinase has emerged as a keysignaling molecule of proliferation and cellular hypertrophyof ASM (141, 201, 320) (Fig. 2). Three distinct classes ofPI3-kinase, specifically IA, II, and III, have been identi-fied in ASM, of which class IA is primarily involved incell proliferation, being required for both RTK and GPCRmitogen effects (197). PI3-kinase regulates cell function byphosphorylating phosphoinositides (PIP) at the 3 position ofthe inositol ring. This results in PI3P, PIP2, and PIP3 forma-tion, of which the latter appears to be the most important ofthese second messengers (308, 337). Subsequent recruitmentof phosphoinositide-dependent kinase 1 (PDK1) to the cellmembrane results in Akt1 activation, which acts as an in-hibitor of the constitutively active glycogen synthase kinase3 (GSK-3) and an activator mammalian target of rapamycin(mTOR) and p70 S6 kinase (44, 65, 201, 283). These activ-ities are important for transcriptional activation and proteintranslation leading to ASM cell proliferation and hypertrophy(126, 283).

A portion of the synergizing effects of GPCRs on growthfactor-induced proliferation can be explained by augmentedPI3-kinase activity. Together with a peptide growth factor theβγ-subunit derived from a Gq-coupled receptor can synergis-tically stimulate PI3-kinase, Akt, and p70 S6kinase (28, 126,196, 200), resulting in increased proliferation.

RTK-induced PI3 kinase activity also results in phospho-rylation of the non-RTK Src; activation is required for ASMcell proliferation (198), and therefore represents an impor-tant pathway by which PI-kinase modulates mitogenesis. An-other route through which PI3-kinase affects ASM cell cycleprogression is through Rho family GTPases (281). Indeed,PI3-kinase-dependent activation of Rac1 and Cdc42, but notRhoA, and subsequent induction of cyclin D1 promoter ac-tivity has been demonstrated in ASM; importantly, this effect




appeared to be independent of ERK1/2, suggesting parallelpathways in the induction of cyclin D1 (21, 244).

Protein kinase C

In addition to synergistic activation of p42/44 MAPK and PI3-kinase pathways, there are also synergistic effects of GPCRson RTK-stimulated ASM proliferation mediated by proteinkinase C (PKC) (126, 127, 343) (Fig. 2). PKC is a super-family that includes three classes of isoenzymes. So-called,conventional isoforms (α, β1, β2, and γ) are activated by cal-cium, phorbol esters, and phosphatidylserine; novel isoforms(δ, ε, ι, θ, and μ) are calcium insensitive and activated byphorbol esters and phosphatidylserine; and, atypical isoforms(ζ and τ/λ) are calcium and phorbol ester insensitive and ac-tivated by phosphatidylserine. PKC α, β1, β2, δ, and ζ, butnot γ or ι, are expressed in bovine tracheal myocytes (327),whereas PKC α, β1, β2, δ, ε, θ, ι, ζ, τ, and μ have each beenidentified in human tracheal myocytes (49).

It has been postulated that the synergistic effects of PKCactivation are mediated through inhibition of glycogen syn-thase kinase-3β (GSK-3β). In its unphosphorylated form,GSK-3β is constitutively active and negatively regulates sev-eral promitogenic transcription factors and cell cycle regula-tory proteins in quiescent cells (83). Thus far, the involve-ment of this pathway has been elucidated for muscarinicreceptor-mediated synergism only (126), however, PKC de-pendency has also been demonstrated for other Gq-coupledreceptor agonists, including bradykinin and endothelin (ET)(124, 343). This indicates that PKC activity, and likely sub-sequent GSK-3β inhibition, could represent a general path-way in GPCR-mediated synergism of RTK-induced ASMproliferation.

Reactive oxygen species

In parallel with the activation of MAPKs and PI3-kinase,RTKs can activate a signaling cascade involving the smallG-protein Rac1, which constitutes part of the nicotinamideadenine dinucleotide phosphate (NADPH) oxidase complexthat produces reactive oxygen species (such as H2O2 andO2

−). Induction of this pathway is linked to cyclin D1 pro-moter activity and ASM cell proliferation, likely via the in-volvement of NF-κB (37, 38, 244). In addition, a role forJanus kinase 2 (JAK2) and signal transducer and activatorof transcription-3 (STAT3) in response to reactive oxygenspecies that are generated by PDGF stimulation appears to bean important regulatory pathway in the expression of c-mycand cyclin D1, and subsequent DNA-synthesis (291). In linewith these findings, inhibition of p22-and p67phox, subunitsof NADPH oxidase, prevents mitogen-induced cyclin D1 pro-moter activity (281) and DNA synthesis (37) in ASM. More-over, a role for the nonphagocyte NADPH oxidase catalytichomolog Nox4 in the regulation of TGFβ1-induced mitosisis evident, as silencing of this molecule prevents TGFβ1-induced phosphorylation of Rb and 4E-BP-1 that is essential

for ASM cell proliferation (300). ROS is also implicated asan intermediate signal during transactivation of EGF recep-tor activation in ASM by leukotriene D4 (265). Collectively,these findings implicate an important role for reactive oxygenspecies in the promotion of growth factor-induced ASM cellproliferation.

Rho-Rho kinase signaling

In ASM, the Rho-Rho kinase signaling pathway has emergedas an important regulator of many cellular functions (281).The role of Rho-Rho kinase signaling in ASM cell prolifera-tion is unclear, with some studies suggesting a rather limitedrole for the pathway in PDGF- and EGF-induced proliferation(89, 132). In contrast, other studies with human ASM cellssuggest a key role for RhoA and Rho kinase, as preventionof RhoA activation and/or pharmacological inhibition of Rhokinase prevent proliferation induced by fetal bovine serum(FBS) (302). Furthermore, the proliferative response of hu-man ASM cells to the GPCR agonist LPA alone and its strongsynergism with EGF can be markedly diminished by Rho in-hibition (89). Parallel effects of Rho kinase inhibition on LPA,LPA/EGF, and FBS-induced proliferation likely relates to thefact that LPA is a major component of FBS. The differencein Rho-Rho kinase dependency between FBS and individualRTK mitogens may also be explained by the observation thatPDGF-induced proliferation relies more on Rac- and Cdc42-mediated pathways (21), whereas FBS-induced proliferationof human ASM cells appears independent of Rac- and Cdc42-mediated signaling Takeda, 2006 (302). Thus, Rho-Rho ki-nase signaling may regulate proliferation of ASM cells; how-ever, the level of activation and relative contribution of thispathway is stimulus dependent.

Integrin-mediated signaling in airway smoothmuscle cell proliferation

Integrins mediate signals in response to ECM protein stimula-tion through (auto)phosphorylation of a number of signalingmolecules, including the nonreceptor cytoplasmic tyrosinekinases, focal adhesion kinase (FAK), and c-Src. These ki-nases subsequently activate other effector proteins, like PI3-kinase, p38 MAPK, and ERK 1/2, which are, as describedpreviously, associated with growth factor-induced prolifera-tion (106, 336). However, the exact mechanisms by whichECM proteins modulate ASM cell proliferation distinct fromgrowth factor-induced signaling are still elusive and mightvery well be species and stimulus dependent. For instance,in human lung carcinoma cells fibronectin has been shown toaffect proliferation by reducing expression of the cell cycleinhibitory protein p21Cip1 in an ERK 1/2- and Rho-kinase-dependent fashion (148). In contrast, in bovine ASM cellsproliferation induced by PDGF has been shown to be de-pendent on ERK 1/2, p38 MAPK, and PI3-kinase, but notRho-kinase (132).




NF-κB signaling

The NF-κB pathway probably contributes to distal signalingevents in ASM remodeling in asthma because it is activatedby many mediators that elicit airways inflammation, ASMproliferation, and cell migration. Prostanoids, IL-β, TNFα,peptide growth factors, and Toll-like receptor ligands all actin part by activating NF-κB signaling in ASM (15, 135, 186,250, 286, 301, 352). Elements of the canonical NF-κB signal-ing pathway have been described in ASM and they appear tobe conserved at both the molecular and functional levels whencompared to cells that participate in immunity. Inhibitors ofNF-κB signaling can reduce synthesis of peptide and proteinmediators in ASM as they do in cells of the immune system(180, 284). NF-κB signaling is profoundly important for cellsignaling and is a highly conserved pathway suggesting itmight not be an ideal target of drug therapy due to potentialoff-target effects. Nevertheless, there is evidence it may be aviable target in treating lung inflammation and asthma. Theactive component of an herbal medicine from Andrographispaniculata, andrographolide, inhibits NF-κB signaling, andis effective after parenteral dosing in reducing markers of in-flammation in ovalbumin-sensitized mice (18). Further studyof this novel anti-inflammatory agent using lung-restricteddrug delivery methods seems to be warranted.

Regulation of airway smooth musclehypertrophyIn asthma, excessive accumulation of contractile smooth mus-cle in central and small airways is associated not only withmyocyte hyperplasia, but also smooth muscle cell hypertro-phy (22, 88, 335). With respect to myocyte hypertrophy itis clear that the process requires coordinated and selectiveprotein synthesis that supports accumulation of contractileproteins. Therefore it is important to understand the signalingpathways that regulate hypertrophic ASM cell growth.

In cell culture, the levels of contractile protein markersvary depending upon cell confluence and the exogenous stim-uli provided by the media. Plating ASM cell at low density inthe presence of FBS represses expression of contractile pro-teins such as sm-α-actin, myosin light chain kinase (MLCK),and smooth muscle myosin heavy chain (smMHC) (30, 142,225, 247). Conversely, long-term serum deprivation of con-fluent myocyte cultures promotes accumulation of contractileproteins and induces the formation of large contractile my-ocytes (47, 140, 141). Interestingly the transcriptional activityof contractile protein genes actually peaks whilst myocytesare undergoing proliferation and nearing confluence; this be-comes dramatically reduced at confluence when mRNA lev-els of contractile markers such as SM22 and smMHC reach amaximum and is sustained thereafter during prolonged serumdeprivation (47, 141). During this period of reduced tran-scription, contractile proteins do, however, accumulate greatlyas cells acquire an enlarged, contractile phenotype morphol-ogy. Collectively this suggests that accumulation of smooth

muscle proteins associated with myocyte enlargement is reg-ulated by critical posttranscriptional mechanisms. Posttran-scriptional regulation of ASM contractile protein expressionis consistent with studies of hypertrophy in other systems in-cluding cardiac and skeletal muscles, and vascular smoothmuscle (VSM) (104, 114, 162, 182, 226, 232, 278).

The study of smooth muscle cell hypertrophy led to thedevelopment of novel cell lines and interventions that enablerepression of cell cycle transit and promote myocyte growth.As described in a previous section, the proliferation of eu-karyotic cells is tightly regulated through a balance of pos-itive and negative regulatory proteins that exert their effectsduring the first gap phase (G1) of the cell cycle (176, 289).Transit through the cell cycle requires accumulation of G1cyclins that leads to activation of CDKs and phosphorylationof downstream targets that ultimately allows entry into the Sphase. The activity of G1 cyclin kinases is modulated by sev-eral key proteins, including p21CIP1, p16INK4, and p27Kip1 (91,151, 285, 340). Based on this paradigm, adenovirus-mediatedoverexpression of cell cycle inhibitors p27Kip1 and p21Cip1

has been used as an experimental means of inducing cellularhypertrophy (305). For cultured human ASM cells, transfor-mation using temperature-sensitive simian virus 40 large tu-mor antigen to induce p21Cip/Waf p57Kip2 expression has beenshown to evoke cell cycle arrest in mid-G1 with concomitantaccumulation of contractile proteins and an increase in cellsize (22, 355). With cell cycle is blockade, serum-inducedcell division is prevented, however, hypertrophic growth ap-pears to continue as contractile protein abundance increases(without affecting mRNA levels). These observations furthersupport the concept that hypertrophic protein accumulationin ASM is regulated in a posttranscriptional manner, likelybeing under control of effectors that modulate protein transla-tion (96). This paradigm is consistent with Woodruff and col-leagues (335) who reported increased smooth muscle α-actinprotein (without any change in mRNA) in airway biopsiesfrom mild asthmatics.

Factors affecting airway smoothmuscle hypertrophyCellular hypertrophy is largely mediated by signaling throughpeptide growth factors: insulin-like growth factor (IGF)-1and growth hormone (GH), the latter acting predominantlyvia increased production of IGF-1 (214). Although levels ofinterleukin (IL)-1β, IL-6, and tumor necrosis factor (TNF)are increased in the bronchoalveolar lavage fluid derivedfrom asthmatics (42), whether these cytokines stimulate ASMgrowth in vitro remains controversial. IL-1β and IL-6 stimu-late hyperplasia and hypertrophy of cultured guinea pig ASMcells (71). ET-1, which is secreted by the epithelium andis elevated in lung lavage fluid from asthmatics (266, 275,294), is also a potent inducer of hypertrophy human ASMcells that is marked by accumulation of contractile pheno-type marker proteins such as smMHC, calponin, and α-SMA(220). When IGF-1, insulin, and other growth factors bind




Integrins

P110α PDK1 P110Y

PKC

elF2B

elF2elF4E

Ribosomesynthesis

5' TOPmRNAs

S6 Kinase

miRNAs

4E-BP1

mTOR GSK3ββ

Translation

Akt

TSC-2?

ILK

Growth factorsGPCRs

ASM hypertrophy

GqGs

ααβ

βγγ

Figure 3 Schematic representation of key signaling mechanisms associated with control of hypertrophiccell growth. See text for details.

to their membrane tyrosine kinase receptors, a 110-kDa lipidkinase, phosphatidylinositol-3 kinase class IA (also referredto as p110α) is activated (272). Accumulated data suggestthat PI3-kinase signaling is a critical underpinning for hyper-trophy. Gene knockout of p110α is lethal at E9.5-E10.5 inmice (showing a severe proliferative defect) (27). Indeed, acentral role of the p110α pathway in IGF-1-induced growthand hypertrophy has been demonstrated in different cell sys-tems (2, 31, 141, 154, 155, 272). The details of this signalingcascade are provided in a subsequent section and are outlinedin Figure 3.

The ECM appears to affect the full functional repertoire ofsmooth muscle cells. Asthmatic airways smooth muscle cellsin culture produce increased amounts and an altered composi-tion of ECM proteins (188). Airway remodeling is character-ized by the deposition of ECM proteins in the airways (268).ECM proteins (collagen I, III, and V; fibronectin; tenascin;hyaluronan; versican; and laminin 2/β2) are increased inprofusion in asthmatic airways (269). Seeding ASM ontofibronectin or collagen type-1 promotes a proliferative phe-notype, whereas laminin-rich matrices promote retention ormaturation of a contractile phenotype (170, 314). Moreover,endogenously expressed laminin-2, which is required for my-ocyte maturation and hypertrophy (314), is increased in theasthmatic airway. Notably, the ability of laminin-2 to pro-mote maturation and support hypertrophy of human airwayssmooth muscle cells is mediated selectively via a α7bβ1 in-tegrin heterodimer (313). Thus an intrinsic autocrine mecha-nism appears to exist wherein myocytes can express both an

ECM element (laminin-2) and requisite receptor (α7β1) tosupport accumulation of contractile proteins and hypertrophicgrowth. Given the association of laminin accumulation withairway remodeling, the expression of this glycoprotein andit receptors may be a central intrinsic mechanism regulatingASM hypertrophy in the adult airway.

Signal transduction pathways that regulateASM hypertrophyPathways discussed in detail below are shown schematicallyin Figure 3.

PI3-kinase

Insulin or IGF-I have been proposed to regulate develop-mental and physiological growth of the cells. Ligand bind-ing to the IGF-I receptor activates PI3-kinase of the Iα IAsubgroup; p110α, which phosphorylates the membrane phos-pholipid phosphatidylinositol 4,5 bisphosphate at the 3′ posi-tion of the inositol ring (272). Through this mechanism PI3-kinases thus recruit effector proteins containing PI(3,4,5)P3-binding pleckstrin homology (PH) domains to the plasmamembrane (206). These include Akt (also called PKB), a57-kD serine/threonine kinase encoded by three genes, and3-phosphoinositide-dependent protein kinase-1 (PDK1) (40).This enforced colocalization of Akt and PDK1 causes the lat-ter to phosphorylate the former at its Thr308 residue, a nec-essary step in for Akt activation (296). Full Akt1 activation




requires membrane localization and phosphorylation at itsThr308 and Ser473 residues. Phosphorylation of Ser473 onAkt is proposed to be mediated by PDK-2, which appearsto be identical to the so-called mTOR complex 2 appears(described below) (183, 345).

Downstream targets of PI(3) kinase and Akt that are as-sociated with promoting protein synthesis and accumulationinclude GSK-3β, p70S6 kinase (p70S6K), and PHAS-1/4E-BP (39, 272) (Fig. 3). Akt1 phosphorylates and inhibits GSK-3β resulting in downstream deinhibition of the translationinitiator eIF2 (65, 329, 330). Akt1 can also phosphorylate,and in part activate, the rapamycin-sensitive threonine/serinekinase, mTOR, a 290-kD protein similar in structure to phos-phoinositide kinases, that can be effectively inhibited by theimmunosuppressor compound rapamycin when the latter isbound to intracellular FK506-binding protein. Of relevanceto a role in cellular hypertrophy, mTOR has downstream tar-gets that include the mitogen- and amino acid-sensitive ser-ine/threonine kinase, p70S6K, and the translation repressorPHAS-1/4E-BP1 (43, 45, 319). mTOR-mediated phosphory-lation of PHAS-1/4E-BP1 releases the latter from binding tothe protein translation mediator, eukaryotic initiation factor4E (eIF4E), thereby increasing the availability of eIF4E toform an active complex with eIF4F and promote translationof specific sets of mRNA transcripts (43).

Activation of p70S6K activation regulates efficiency ofprotein translation by phosphorylating of the 40S ribosomalprotein S6 (43, 86), and is required for PI3-kinase-mediateddifferentiation and hypertrophy of skeletal myotubes (31,272), angiotensin II-induced VSM hypertrophy (112), andautocrine loop-mediated ASM cell maturation and hypertro-phy (140, 141). Phosphorylation of ribosomal S6 protein in-creases translation of mRNAs with 5′ TOP tracts, many ofwhich are involved in mRNA-translation-like elongation fac-tors and ribosomal proteins. Though the principal site requiredfor mTOR-dependent activation of p70S6K is Thr389, whichresides in a region between catalytic and autoinhibitory do-mains (256), full activation of p70S6K is achieved throughhierarchical phosphorylation of seven Ser/Thr sites targetedby mTOR, PDK1, and other PI(3) kinase-dependent kinases(267). As phosphorylation of p70S6K is sensitive to inhibi-tion by both rapamycin and chemical inhibitors of PI3-kinase,researchers often place PI3-kinase, mTOR, and S6 kinase intoa linear signaling pathway. Such a linear scheme is too sim-plistic, however, as a rapamycin-resistant mutant of S6 ki-nase is still sensitive to inhibition by the PI3-kinase inhibitorwortmanin (54), indicating that mTOR and PI3-kinase sig-nals to p70S6K can be dissociated. Indeed, S6 kinase mayalso be phosphorylated by PDK-1 (309, 317), thus providinga mechanism for mTOR-independent, PI3-kinase-dependentactivation. Similarly, mTOR-independent mechanisms of 4E-BP1 phosphorylation may exist. Recent evidence shows thatclass IA PI3-kinases may function as 4E-BP1 kinases (98),and ERK can reportedly also phosphorylate 4E-BP1 (160).

Recent studies have elucidated the role of two signalingmolecules that link Akt and mTOR in the regulation of cell

size. PI3-kinase may positively regulate cell size via activa-tion of Akt, inactivation of TSC2, activation of Rheb, andactivation of mTOR (102, 119, 178, 260). It is now knownthat mTOR exists in two distinct multiprotein complexes, onerapamycin-sensitive (mTOR complex 1) and one rapamycin-insensitive (mTOR complex 2) (184). mTOR complex 1 in-cludes mTOR and Raptor; mTOR complex 2 is comprisedof mTOR-Rictor and mammalian stress-activated protein ki-nase interacting protein. Furthermore, as noted above, mTORcomplex 2 appears to be identical to the proposed Akt kinase,PDK-2, which phosphorylates serine 473 on Akt (183, 345).Thus, Akt acts as both an upstream activator of mTOR com-plex 1, and is a target for activation by mTOR via mTORcomplex 2 to permit high-level PIK/Akt signaling (163).

Clycogen synthase kinase-3β

GSK-3β is a constitutively active serine/threonine kinasethat phosphorylates multiple substrates including eIF2Bε, cy-clin D1, and p21 (3, 81, 273, 329). Phosphorylation by Aktinactivates GSK-3β, leading to dephosphorylation and theactivation of eIF2B, as well as a general enhancement ofribosomal 43S preinitiation complex formation (329). GSK-3β also negatively regulates transcription factors involved inmuscle-specific gene expression, including nuclear factors ofactivated T cells (NFAT), GATA4, and β-catenin (13, 122,149, 150, 227) suggesting a critical role in ASM growth. Thephosphorylation of GSK-3β by Akt indicates that PI3-kinasemay regulate mRNA translation via three distinct mechanisms(see Fig. 3): (i) regulation of cap-dependent mRNAs via ac-tivation of the Akt/TSC2/Rheb/mTOR/4E-BP1 pathway, (ii)regulation of 5′ TOP tract-containing mRNAs via activationof p70S6K (through either mTOR or PDK-1), and (iii) a gen-eral enhancement of translation initiation via activation of theAkt/GSK-3β/eIF2B pathway. Recent studies also implicateregulation of GSK-3β as a key downstream mechanism forthe effects of integrin-mediated effects of ECM proteins oncell growth; this involving the signaling intermediate, integrinlinked kinase (ILK) (63, 76, 218).

Despite human studies indicating the presence of ASMhypertrophy and increased contractile protein expression inasthma, little information is available concerning the signal-ing intermediates and translation initiation factors involved. Inconfluent serum-deprived canine tracheal myocyte cultures,PI3-kinase and p70S6K activities are increased five and twodays after serum deprivation, respectively, and immunohisto-chemical studies show selective phosphorylation of Akt andp70S6K in elongated cells expressing smMHC five to sevendays after serum deprivation (141). LY294002 and rapamycinblocked S6 kinase phosphorylation and phenotypic change,implying that PI3-kinase, mTOR, and p70S6K are responsi-ble for contractile protein accumulation and myocyte hyper-trophy. Recently it has been shown by Deng et al. (78) thatinhibition of GSK-3β (which activates eIF2B) contributesto ASM hypertrophy in vitro and in vivo. More strongly ina mouse model of allergic asthma it has been shown that




phosphorylation and inactivation of GSK-3β is associatedwith ASM hypertrophy (23) while p70S6K alone is responsi-ble for the myocyte enlargement, without changing the con-tractile protein expression in vitro (79).

Rho GTPases

Rho kinase signaling plays an important role in regulationof smooth muscle gene transcription, which promotes serumresponse factor (SRF) nuclear localization and increased cy-toplasmic actin filaments (213, 215, 322). The ability of theRho-Rho kinase pathway to promote actin polymerizationleads to a reduction of globular actin (G-actin) concentration,which results in the release of the SRF coactivator MAL, aG-actin binding protein (223). Thus, SRF, a central regulatorof smooth muscle-restricted gene transcription, is under tightcontrol by the Rho-Rho kinase pathway (133, 146). Rho-Rhokinase activation is regulated by RTKs and GPCRs through theaction of Rho-specific guanine exchange factors (RhoGEFs).Ligand binding to muscarinic M3 receptors coupled to Gαqcan induce RhoA activation, likely via p63RhoGEF, and pro-motes Rho-kinase-dependent actin polymerization leading toSRF translocation and the induction of smooth muscle spe-cific gene expression. Insulin-induced expression of contrac-tile phenotype markers and the induction of a functionallyhypercontractile phenotype also requires the Rho-Rho-kinasepathway, though the GEFs involved have not yet been identi-fied (130, 280). It indicates that Rho-Rho kinase signalingplays an important role in the transcription of genes thatencode mRNA required for synthesis and accumulation ofcontractile proteins in hypertrophic ASM.

Protein kinase C

Although there are several studies suggesting the role ofPKC’s in ASM proliferation (127, 128, 211, 341, 342), theirrole in hypertrophy is not entirely clear. Data from other tissueand cell types indicate that overexpression of these select PKCisoforms can induce cardiac hypertrophy in transgenic mice(84). Moreover, activation of PKC isoenzymes via GPCRshas been linked to GSK-3β phosphorylation, suggesting thisclass of enzymes could play a permissive role in protein trans-lation via eIF2B (126). On this basis, future focus on the roleof PKCs and PKC inhibitors in airway myocyte hypertrophyappears to be warranted.

Regulation of Airway SmoothMuscle ApoptosisTissue development and homeostasis is subject to rounds ofcell division and differentiation, but of equal importance is theduration of cell survival and the capacity to orchestrate self-termination to cull infected, damaged, and unwanted cells.Such programmed cell death, dubbed apoptosis, follows spe-cific patterns and includes shrinkage of the cell, margination

of chromatin, and nuclear fragmentation (108, 193). Apop-tosis occurs in response to environmental or developmentalsignals, cellular stresses, and specific cell death signals. Thisself-inflicted death involves a number of evolutionarily con-served biochemical pathways that have been intensively stud-ied for over two decades (72).

In mammals, programmed cell death can be initiated bytwo major pathways: (i) the extrinsic pathway, which canbe triggered by ligation of death receptors and subsequentcaspase 8 activation and (ii) the intrinsic pathway, which isinitiated by cellular stress followed by activation of caspase9 (Fig. 4). Each of these pathways converges to a commonexecution phase that requires the activation of caspases-3 or-7 from their inactive zymogen form to their processed, activeform (107, 108, 277, 306). The proximal activators (caspase-8 and -9) have a primary specificity for cleavage at Asp297located in a region that delineates the large and small subunitsof active caspases-3 and -7. Apoptotic cell death is centrallycontrolled by both caspase activation cascades and/or mito-chondrial membrane permeabilization (MMP), processes thatare inextricably linked (110, 137, 323). Indeed, MMP itselfstimulates caspase activation through the release of severalcaspase-activating proteins, in particular cytochrome c (107,323), and caspase activation of proteins such as truncated Bid,Bad, and Bcl-XL triggers MMP (41, 194, 208). MMP man-ifests at the level of the outer membrane, which allows forthe release of cytochrome c, as well as at the level of the in-ner membrane as a loss of the mitochondrial transmembranepotential (�ψm) (109, 137, 323).

Airway smooth muscle apoptosis and asthmaAsthma, particularly if severe and/or of long duration, is ac-companied by increased ASM mass due to myocyte hyper-plasia and hypertrophy (17, 22, 88, 335). The potential formyocyte proliferation to contribute to ASM remodeling wasdiscussed above, but it needs to be pointed out that thereis not compelling data from animal models or from humanspecimens that confirm a place for proliferation as the pri-mary underpinning of remodeling. Indeed more recent worksuggests that apoptosis may be of equal importance to pro-liferation in determining the extent of airway remodeling inanimal models of asthma (161, 205, 264). In another studyusing rats, reduced ASM apoptosis was shown to contributeto the airway remodeling process (82). Furthermore, dexam-ethasone was shown to induce myocyte apoptosis possiblyby increasing proapoptotic Bax expression and the decreas-ing antiapoptotic Bcl-2 expression (82). Using a rat modelfor emphysema, it has also been confirmed that Fas and FasLparticipate in apoptosis of myocytes in small airways (233).Interestingly, injection of the Chinese herbal remedy, shen-mai, modulated Fas and FasL protein expression and reducedASM cell apoptosis, likely associated with inhibitory effectson TNF and inflammation (233).

Fas (CD95, the receptor for FasL) is expressed by ASMtissue in vivo and on the surface of cultured human airway




Death receptors(extrinsic pathway)

(e.g., Fas, TNFR1, DR5)

TYPE IIpathway

Caspase 8

Bid

Caspase 3

Caspase 9

TYPE Ipathway

Death

Apaf-1“apoptosome”

Cytochrome C

Mitochondrialfragmentation

DNA damage, stress(intrinsic pathway)

Figure 4 Simplified schematic representation of essential pathways for caspase-dependent apoptoticcell death. Apoptosis is triggered by internal cellular stress (intrinsic pathway) or extracellular signals (ex-trinsic pathway) that mediate effects via the binding of ligands (e.g., Fas, TNFR1, and DR5) to cell surfacedeath receptors. Extrinsic pathways directly activate executioner caspases (caspase-3) through initiatorcaspases (e.g., caspase-8 and -9) ultimately leading to cell death. In intrinsic pathways, death signalsare conducted through mitochondria, increasing permeability that leads to the release of cytochrome c.Cytosolic cytochrome c binds Apaf-1 to activate the apoptosome and caspase-9 which ultimately leadsto downstream activation of executioner caspase-3.

myocytes in vitro (147). Moreover, cross linking of surfaceFas induces apoptosis in a significant number of cultured my-ocytes, an effect that is: (i) potentiated by stimulation withTNF-α, which upregulates surface Fas expression and (ii) re-duced by prolonged serum deprivation, which, in the absenceof TNF-α treatment, reduces surface Fas expression. This ef-fect could be very important considering that even a smallsustained level of apoptosis might have a significant impacton smooth muscle accumulation within intact asthmatic air-ways because the proliferative index of ASM appears to below even in the presence of substantial airway inflammation(246).

ECM protein alterations are a characteristic feature ofasthmatic airway remodeling (66, 185). These changes in-clude modification such as collagen I, III, and V increase,changes in glycoproteins (fibronectin and tenascin), and alter-ations in deposition of various proteoglycans (PG) [versican,biglycan, and decorin (66, 68, 69, 175, 185, 257)]. It has beenreported that culturing cells on different ECM matrices canvariably affect ASM number, with laminin in particular im-parting a prosurvival response (66, 100). Culture on decorinresulted in a persistent decrease in cell number via its effectson both proliferation and apoptosis (66), therefore the antipro-liferative and/or proapoptotic effect of decorin could serve tolimit the growth of ASM beyond its usual compartment.

The endothelins (ET) are a family of three isopeptides,acting through two G-protein-coupled receptors, ETA and

ETB. ET-1, in particular, elevates smooth muscle tone (53)and causes a marked potentiation of cholinergic nerve-evokedcontraction of ASM (115). ET-1 expression is increased inasthma and is primarily released from the bronchial epithe-lium (266, 275, 294). Bronchial smooth muscle cells highlyexpress the ETB receptor which represents about 82%-88% ofthe total ET receptor population (116). ET-1 is a potent inducerof hypertrophy of human ASM cells and at the same time in-creases the contractile potential of these cells by increasingexpression of sm-MHC, calponin, and α-smooth muscle actin(220). ET-1-induced-ASM survival has been causally linkedwith apoptosis inhibition (220), and is a concomitant mecha-nism leading to increased size and synthetic activity of thesecells in primary cell culture.

Cigarette smoke has long been considered as a majorcausative factor for chronic obstructive pulmonary disease(COPD) (20, 174). A number of mechanisms have been sug-gested for the pathogenesis of COPD, including dispropor-tionate activities of proteases and antiproteases (77), influx ofinflammatory cells into the lung, and oxidative stress (195).In addition to these mechanisms, gathering evidence suggeststhat apoptosis may play a significant role in clinical and ex-perimental COPD pathogenesis (174, 177, 347). It has beenreported that cigarette smoke extract (CSE) could induce ox-idative stress and apoptosis in ASM cells through activation ofboth the mitochondrial pathway and death receptor pathway(174). Neutrophilia is a common feature of smoking-induced




inflammation and of severe asthma and these cells are a richsource of elastases in the human lung (290). The degradationof ECM by neutrophil elastases is believed to contribute todecreased airway stability (36). Neutrophils can also induceapoptosis in ASM, for example, detachment-induced apopto-sis (defined as anoikis) with characteristic caspase-3 cleavage(239). Neutrophil-induced myocyte apoptosis appears to re-sult from the proteolytic activity of proteins released by neu-trophils as concomitant fibronectin degradation occurs, andthe serine protease inhibitor, α1-antitrypsin, has a protectiveeffect (239).

Most recently it has been reported that simvastatin, aninhibitor of HMG-CoA reductase which is the proximal rate-limiting enzyme in cholesterol biosynthesis, can induce apop-tosis in primary cultured human airways smooth muscle cells(110). This effect involves a novel p53-dependent pathwaywith selective release of mitochondrial protein, Smac andOmi, which inactivate inhibitor of apoptosis protein (XIAP),allowing for cytochrome c-independent activation of caspase-9. The proapoptosis effects of simvastatin is mainly initi-ated by depletion of the intracellular pool of cholesterol in-termediates called isoprenoids [farnesylpyrophosphate (FPP)and geranylgeranyl pyrophosphate (GGPP)], which are es-sential for membrane anchoring and activation of small RhoGTPase proteins. This finding suggests there may be meansfor development of future new asthma therapy to target ASMhyperplasia in asthma.

Airway Smooth Muscle Cell MigrationAirway smooth muscle elongation and smoothmuscle differentiation in lung developmentThickening of the ASM layer in diseased airways could resultfrom migration of ASM cells or smooth muscle precursorsthat recapitulates events of embryonic development. Duringembryogenesis formation of smooth-muscle containing hol-low organs is thought to include migration and reorienta-tion of smooth muscle cells. Cell migration is a commonprocess in formation of blood vessels, the airways, and thegastrointestinal system. During lung development migrationand differentiation of ASM precursor cells is orchestrated byautocrine and paracrine factors as well as cell-matrix interac-tions that promote maturation of the airway wall (16, 287).The molecular and cellular remodeling that occurs duringsmooth muscle migration may contribute to lung develop-ment by mediating elongation of mesenchymal progenitorcells. In the developing airways mesenchymal progenitorsdifferentiate into elongated cells that express smooth muscle-restricted contractile proteins. Elongation is required for dif-ferentiation and both depend on activation of the Rho A-Rhokinase pathway (24), which is also known to mediate ASMcell migration (164, 251). Cell elongation and expression ofdifferentiation marker proteins during development appearsto be a mechanical signaling phenomenon because soluble

signals that drive smooth muscle differentiation in culture(TGFβ1 and retinoic acid) have no effect on upregulation ofsmooth muscle marker protein expression (16). The role ofsmooth muscle migration in airway development appears tobe to more of an effect on cell elongation and orientationrather than a long range chemotactic migration of progenitorcells that occurs in the developing vasculature. In the mouse,the shape change of mesenchymal smooth muscle progen-itor cells and ultimately smooth muscle cell differentiationdepends critically on expression of laminin 1 and laminin 2(24, 353). Both laminins 1 and 2 can ligate integrin α7, andintegrin α7 is a protein known to promote vascular and ASMdifferentiation (57, 328, 346). Disrupting critical changes incell shape by knocking down laminin 1 and laminin 2 resultsin bronchial smooth muscle hypoplasia (24, 353). Badri andcolleagues suggest mechanical forces in the developing lungare transmitted through integrin-laminin interactions leadingto upregulation of serum-response factor expression and ex-pression of smooth muscle-restricted genes in differentiatedASM Badri, 2008 (16). The hypothesized mechanical signalsare integrated with epithelium-derived soluble signals includ-ing FGF10, BMP4, and components of the Wnt/catenin andhedgehog signaling families. The combined effect of mechan-ical signals and biochemical signals is to drive mesenchy-mal precursor cells to an elongated, differentiated smoothmuscle phenotype. Because cellular processes underlyingtube formation are highly conserved from Drosophila to hu-mans (87), it seems reasonable to infer an important role ofsmooth muscle migration in airway development. However,there are no definitive lineage marker studies of the sourceof new ASM cells in vivo during lung development. Thekey question is what percentage of new muscle originatesfrom existing smooth muscle versus progenitor cells migrat-ing from the surrounding mesenchyme that deposit in theairway wall?

Smooth muscle cell migration and airwaysremodelingAs discussed above, hyperplasia can result from increasedproliferation and diminished apoptosis. In addition, increasedcell number could be a result of migration of new cells into theairway wall. There is evidence for two sources of migratingcells in the airways; the lung parenchyma and the blood. Evi-dence for parenchymal cells as a source of new smooth muscleis from a structural study of lung biopsies from asthmatics inwhich lung myofibroblasts were found to migrate in responseto allergen challenge (113). An important question is whetherthe myofibroblasts are resident cells or are derived from cir-culating fibrocytes, which are CD34+, collagen I+, and α

smooth muscle actin+ progenitor cells. Fibrocytes are hy-pothesized to differentiate to myofibroblasts, to contribute tosubepithelial fibrosis and possibly to become contractile cellsin ASM bundles (14, 279, 282). A central question for stud-ies of ASM cell migration is whether tube formation duringdevelopment, wall thickening, and epithelial-mesenchymal




transformation all require smooth muscle cell migration. Anargument can be made for cell migration during tube for-mation based on analogous events in vascular development.Some interesting questions that need to be tested critically arewhether differentiated smooth muscle cells originating in themuscularis migrate in response to cues such as inflammationor lung injury, and does this recapitulate events that occurredduring development (324). Recent evidence for cell migra-tion in remodeling of asthmatic airways is more consistentwith immigration of blood-borne fibrocytes (14, 279). Fibro-cytes are present in increased numbers in the lamina propriain patients with asthma (279), the number of fibrocytes inthe muscularis increases after allergen challenge (113), andmigration of fibrocytes is enhanced by coculture with differ-entiated ASM cells. ASM cells in culture can secrete a varietyof promigratory substances (Table 2), with PDGF and TGF-β1 being particularly noteworthy. While the recent data arequite provocative it remains to be proven by lineage markingapproaches that the migrating cells contribute to subepithelialfibrosis, differentiate to contractile ASM cells or both. An-other important question is whether some fibrocytes remainin the muscle layer as a population of progenitor cells that canbe activated to proliferate then differentiate to smooth mus-cle cells. These are important questions because increasedASM mass and myofibroblast numbers are thought to be im-portant determinants of fixed airway obstruction that is un-responsive to corticosteroid and bronchodilator therapy (22).

Table 2 Summary of Agents that Modulate Airway Smooth Muscle(ASM) Cell Migration

Promigratory agents Antimigratory agents

Growth factors and cytokinesbFGF (179) CXCL10/CXCR3(279) CC Chemokine ligand 19(CCL19) (190) IL-1β (156) IL-8(136) Leukotriene B4 (325)Leukotriene E4 (251) PDGF(156) TGFβ1 (156)

β-adrenergic agonists and thePKA pathway Dibutyryl cAMP(164) Formeterol (51) Forskolin(118) Cilomilast (118)Salmeterol (118) Theophylline(164)

Extracellular matrix Collagens I,III, V (251) Fibronectin (251)Integrins α5, αV (251) Laminin(251) MMP-3 (181)

Immunomodulating drugsFluticasone (118) Pyrimidinesynthesis inhibitor, FK778 (80)Sirolimus (80)

Other promigratory agentsCyclodextrin (50)Lysophosphatidic acid (164)Thrombin (157) Urokinaseplasminogen activator (121)

Protease inhibitors4-(2-Aminoethyl)benzenesulfonylfluoride HCl(AEBSF) (157) Ilomastat (157)Prinomastat (152) TIMPs 1-4(157)

Protein kinase and phosphataseinhibitors LY294002 (67, 251)PP1 (252) PD98059 (156)SB203580 (156) U-0126 (51)(67) Vanadate (51) Y27632(164, 251)

Other antimigratory agentsPertussis toxin (51)Prostaglandin E2 (251) Retinoicacid (67) SB649146 (SP-1inverse agonist) (326)

Another unresolved issue is what initiates influx of fibrocytesand differentiation of myofibroblasts to smooth muscle cells.As described below many growth factors (e.g., PDGF) andproinflammatory signaling proteins (e.g., interleukins) stim-ulate ASM cell migration. Many recent studies of ASM cellmigration have focused on the molecular mechanisms thattransduce progrowth and proinflammatory signals to cell mo-tion. The following sections summarize conserved featuresof migration of motile cells, the known promigratory and an-timigratory signals affecting ASM migration and some of thekey signal transduction pathways that underlie cell migrationin ASM and other cell types.

Cellular processes and molecular structuresnecessary for migrationCell migration begins with stimulation of receptors that trig-ger cytoskeletal remodeling and repositioning of organellesas illustrated in Figure 5. There are many receptor systemsthat sense promigratory stimuli, but we will limit the discus-sion of these events to the three major classes of receptorsinvolved in cell migration: GPCR, RTKs, and matrix adhe-sive proteins (integrins). One of the earliest events followingreceptor ligation and signal transduction is polymerization ofactin at the leading edge of a motile cell. This is a funda-mentally important process that extends the edge of the cellin the direction of the stimulus during chemotaxis (Fig. 5A).For the leading edge of the cell to stick to the substrate andaffect forward motion focal contacts must assemble just be-hind the leading edge (Fig. 5B). Myosin II motors bind actinfilaments in the body of the cell to generate traction forcethat moves the cell forward. Myosin I motors at the leadingedge are thought to control cortical stiffness and membranetension. Simultaneously the actin and microtubule cytoskele-tal systems remodel, and focal contacts at the rear of the celldetach to allow the body of the cell to follow the leadingedge toward the stimulus. The nucleus, mitochondria, golgi,and endoplasmic reticulum are tethered by adaptor proteinsand motors to the cytoskeleton. One role of myosin II mo-tors is to move cellular organelles along with the remodelingcytoskeleton. Depending on the experimental approach cellsin vitro will move about randomly in the absence of a chem-ical gradient (chemokinesis), or move directionally as theyfollow concentration gradients of soluble attractants (chemo-taxis). Migrating cells can also follow paths of varying matrixadhesiveness and stiffness (durotaxis) and varying concentra-tions of bound chemical attractants (haptotaxis). Durotaxisand haptotaxis are critically important phenomena for properorgan formation during embryonic development. A commongoal of cell migration studies is to establish the sufficiencyand necessity of particular extracellular chemicals and intra-cellular signaling pathways in migration. Another commongoal is to define the cellular machinery necessary for cellmovement, and to determine if the function of the machineryis compromised in disease. Studies over the past ten yearsin ASM have illustrated several important characteristics of




Actinpolymerization

module

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CaM

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TensinTalin

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A. Actin polymerizationat the leading edge

B. Nascent focal contact

Vinculin

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Figure 5 Schematic model illustrating the prominent features of a migrating cell. The leading edge of the cell is representedby the cross hatched region on the right. (A) The actin polymerization module located at the leading edge is a site of rapidactin polymerization, depolymerization, and filament branching. Actin nucleating proteins (mDia1, mDia2, and VASP) promotefilament formation at the plus (barbed) end. G-actin monomers are added by the action of profilin. Actin filaments are severedby gelsolin and depolymerized by cofilin. Actin branching is regulated by small G-proteins acting on Wiskott-Aldrich syndromeprotein (WASP)-family verprolin-homologous protein, WASP, and proteins of the ARP2/3 complex. The stiffness of the actingel and traction forces on the matrix are controlled in part myosin II motor proteins that are regulated by activation ofmultiple kinases [myosin light chain kinase (MLCK), p21-activated protein kinases (PAK), and Rho-activated protein kinases(ROCK)] and myosin light chain phosphatase (MLCP). (B) Signaling and actin attachment modules in the leading edge promoteformation of nascent focal contacts (red bars) that rapidly assemble to transiently attach the cell to the matrix. Actin attachmentcomponents include integrins, adaptor proteins (talin, vinculin, tensin, and paxillin). Signaling module components controlassembly and maturation of the focal contact. These include regulatory proteins [Src, CAS, and focal adhesion kinase (FAK)]and proteins controlling actomyosin assembly and myosin II activation and (MLCK, PAK, MLCP, and ROCK). As the cellmigrates, nascent focal contacts mature and move toward the rear of cell. Focal contacts at the rear of the cell (red bars onthe left) are disassembled as the cell advances. Disassembly requires the action of multiprotein complexes that depend onmicrotubules (gray filaments) emanating from the microtubule organizing center. Reprinted from (105) with permission fromthe American Thoracic Society.

migration relevant to airway development and airway remod-eling in asthma. The remainder of the article summarizesextrinsic molecules that modify ASM migration and the sig-naling pathways involved in controlling migration. For a moregeneral overview of cell migration and protocols for assayingwound healing and chemotactic migration there are severalelegant reviews published by members of the Cell MigrationConsortium (www.cellmigration.org). The reader is also re-ferred to previous reviews of smooth muscle cell migrationthat provide references to methods used in studies of ASMcell migration (105, 120, 216).

Conserved biochemical processes known to occur in mi-grating cells are illustrated in Figure 5. The figure summarizesliterature from both nonmuscle and muscle cell motility stud-ies (318, 350). We will summarize the consensus for how

migration occurs in many cell types and then highlight theknown and unknown features of ASM migration. In all mi-grating cells actin polymerization and depolymerization isrequired. There are numerous actin-associated proteins thatcoordinate polymerization and depolymerization with someof the best defined proteins being illustrated in the inset Figure5A. Some of the earliest events in chemotactic cell migrationare receptor activation, changes in cell Ca2+ signaling, pro-duction of phosphatidyl inositol bis phosphate (PIP2), andactivation of monomeric and trimeric G-proteins (Fig. 6).Each of these proximal signal transduction events can acti-vate multiple signaling cascades. It is impossible to representall the known signaling mechanisms in a simple schematic,so Figure 6 was designed to make the point that signal-ing occurs at multiple levels via parallel signaling pathways




RTKsGPCRs

αβ γSrc P13-K

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PLC

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Raf

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HSP27 mDIA1 Cofilin Myosin II

LIMK

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Figure 6 Signaling pathways that regulate actin polymerization and myosin II motors in smooth mus-cle cell migration. Activation of G-protein-coupled receptors (GPCR) and receptor tyrosine kinases (RTK)initiates activation of parallel signaling cascades that culminate in actin filament remodeling, changesmatrix adhesiveness, and regulation of myosin II motors that generate traction force. Immediate postre-ceptor events include activation of trimeric G proteins, Src family tyrosine kinases, phospholipase C(PLC) and phosphatidyl inositol bis phosphate (PIP2), PI3-kinases (PI3-K), and increased Ca2+. Multiplesmall G-proteins (RhoA, Rac, and Cdc42) and calmodulin (CaM) then activate downstream targets thatare shown here in darker shades of red. Some targets are effector proteins that regulate actin poly-merization including the formins (mDIA1 and mDIA2), Wiskott-Aldrich syndrome protein (WASP)-familyverprolin-homologous protein and WASP, and the ARP2/3 complex. Other targets include members ofthe mitogen-activated protein (MAP) kinase family (p38 MAPK and ERK), Rho kinases (ROCK), and p21-activated protein kinases (PAK). The signaling kinases phosphorylate other protein kinases (MAPKAPKand LIMK) or myosin light chain phosphatases (MLCP) to regulate effector proteins (dark blue ovals)that control actin polymerization and traction forces generated by myosin II. Most of the schematic isorganized as sets of parallel linear signaling cascades, which is an oversimplification for the sake ofclarity. Pathway convergence and crosstalk are known to occur between the pathways shown. Regulationof myosin light chain kinase (MLCK) is a good example where both positive and negative inputs areintegrated to determine the level of myosin II regulatory light chain phosphorylation and traction force.Reprinted from (105) with permission from the American Thoracic Society.

converging on actin polymerization and myosin II motors,both of which are necessary for traction forces required forcell migration. We will focus on signaling events triggered byPDGF in this article because it plays a critical role in smoothmuscle cell migration. However, the reader should be awarethat numerous promigratory stimuli have been described forASM (Table 2), and that each stimulus acts via some of thesame signaling pathways as well as stimulus-specific path-ways not shown in Figure 6. With these limitations in mindwe focus on PDGF family members to illustrate the principlesof smooth muscle cell migration.

PDGF signaling is necessary for tube formation duringvascular development as well as wound healing in responseto injury and inflammation. The β isoform of PDGF recep-tor (PDGFR-β) is coupled via PI3-K and phospholipase Cγ

which elicits changes in myoplasmic calcium, hydrolysis ofPIP2, and activation of MAP kinases (33, 156). These signal-ing intermediates act together with the small G proteins (Racand Cdc42) to initiate nucleation of F-actin. Nucleation is pro-

moted in several ways: de novo at the minus (pointed) end,uncapping of plus (barbed) ends by dissociation of actin cap-ping proteins, or by forming new branches (Fig. 5A). Nucle-ation and branching are promoted by proteins of the ARP2/3complex, profilin and the formins (mDia 1 and 2). The net ef-fect of these proteins is to increase polymerization at the plusends of existing actin filaments. Profilin is bound to mem-brane phospholipids in the absence of promigratory stimuli.In the presence of stimuli that activate phospholipases plasmamembrane PIP2 levels decrease which releases sequesteredprofilin. Profilin then enhances adenine nucleotide exchangeon G-actin and drives actin polymerization. The formins areactivated by binding monomeric G-proteins-–mDia1 is acti-vated by RhoA, and mDia2 is activated by Cdc42 and Rac.Small G-proteins also promote filament branching by ac-tivating Wiskott-Aldrich syndrome protein (WASP)-familyverprolin-homologous protein (WAVE) complex and WASP,respectively. WAVE and WASP proteins activate componentsof the ARP2/3 complex to increase the number of nucleation




sites and the number of sites for branching. Increased F-actinnucleation, polymerization, and branching are necessary forformation of filopodia and the lamellipodium leading to ex-tension of leading edge of a migrating cell (cross-hatched areaof the cell in Fig. 5). In addition to nucleation and branching,actin filaments must be severed and depolymerized to produceeffective migration. Actin severing is mediated by several pro-teins including gelsolin and cofilin. Gelsolin is activated byboth increased Ca2+ concentrations and by PIP2 (Fig. 5A).The number of actin nucleation sites increases when gelsolinis released from the plus end of actin filaments. Migrationdepends critically on filament growth at the plus ends and fil-ament shrinkage at the minus end. The dynamic behavior ofactin filaments is greatly enhanced by cofilin, which promotesdepolymerization at the minus end and severs actin filamentsthus increasing nucleation sites (Fig. 5A). The net effect of allthe processes just described is to generate propulsive force atthe leading edge of the cell extending filopodia and the lamel-lipodium toward the stimulus (261). During the initial stagesof lamellipodial extension focal contacts must form betweenthe cell membrane and the ECM for cells to move (Fig. 5B).Focal contacts are critically important adhesive structures thatare dynamic in a motile cell, forming rapidly, maturing, andeventually disassembling at the rear of the cell thus releasingtail of the cell from the matrix.

Focal contacts in airway smooth muscle cellsand tissuesThe protein composition of the focal contact “adhesome” andthe function of focal contacts to sense the biochemical andphysical environment surrounding a motile cell has been re-viewed recently (103, 351). Geiger and colleagues divided thecomponents and functions of focal contacts into a signalingmodule, an actin-linking module and an actin-polymerizingmodule (see Fig. 5A and B). In this section, we will focuson the components of focal contacts that have been describedin cultured ASM cells and intact ASM tissue. Several com-ponents of the actin linking module have been described in-cluding paxillin (292, 304), vinculin (240), and talin (292).Elements of the signaling module have also been describedin ASM including FAK (292, 304), Src (165, 198, 252), PI3-kinase (7), Ca2+ and phospholipase C (136), and several MAPkinases (see below). Signaling module proteins catalyze a va-riety of reactions, including phospholipid metabolism, proteinphosphorylation and dephosphorylation, and oscillations incell [Ca2+], all of which contribute to dynamic formation anddegradation of focal contacts during migration. Phosphoryla-tion of focal contact components including FAK, paxillin andtalin has been shown in ASM tissue during contraction (255,304) and following strain of cultured cells (292). In migrat-ing ASM cells, FAK is phosphorylated and degraded duringurokinase-stimulated migration (50). Carlin et al. (50) alsofound Src trafficked to the cell membrane during urokinase-induced migration consistent with Src being phosphorylatedand activated during ASM cell migration (198, 252). Com-

ponents of the signaling module are critical for catalyzingphosphorylation and dephosphorylation events that promoteboth formation and turnover of the nascent focal contacts atthe leading edge.

In addition to an important role for protein phosphory-lation there is also a requirement for proteolysis of focalcontact proteins by metalloproteinases. Turnover of maturefocal contracts is due in part to proteolysis occurring at thetrailing edge. In migrating ASM cells, as in many other celltypes, upregulation of MMPs 1, 2, and 3 increases duringmigration. The necessity for MMP activity was demonstratedclearly by the fact that both tissue inhibitors of metallopro-teinases (TIMPs) and chemical protease inhibitors reduced orcompletely blocked ASM cell migration (152, 157). Proteaseinhibitors block migration because stable focal contacts at therear of the cell must eventually disassemble for the cell tomove forward.

There are some interesting unaddressed questions aboutthe spatial and temporal features of proteins in nascent andmature focal contacts in ASM. It is not clear which compo-nents are most sensitive to inflammation, which are alteredby mechanical strain during tidal breathing or how the focaladhesion composition and spatial distribution changes as afunction of the differentiation state of ASM cells. We assumethat many components of focal contacts are similar to those ofmigrating nonmuscle cells, and evidence to date has largelyconfirmed this assumption. However, identifying unique pro-tein components of the ASM adhesome and its constituentmodules (signaling, actin binding, and polymerization) is im-portant for identifying novel targets for inhibiting or reversingairway remodeling.

Mechanics of cell migrationThe primary sources of force generation during for cell mi-gration are actin polymerization promoting protrusion of theleading edge and force generated by myosin motors. Myosin IImotors produce traction force that is transmitted to the matrixthrough the focal contacts (Fig. 5B). Smooth muscle myosin IIis a phosphoprotein that is activated by Ca2+-calmodulin acti-vation of MLCK. Activated myosin II binds to actin filamentsthat contain tropomyosins and caldesmon (predominantly l-caldesmon in cultured ASM cells). Phosphorylation of myosinregulatory light chains increases actomyosin ATPase activityand crossbridge cycling, thus generating traction force. Inaddition to the canonical Ca2+-calmodulin-MLCK activationpathway, there may also be an important Ca2+-independentactivation of myosin II mediated by RhoA activation of Rhokinases. Rho A and Rho kinases are well-known inhibitorsof myosin light chain phosphatase via phosphorylation of themyosin binding subunit of the phosphatase (293). Activationof Rho A and Rho kinases inhibits myosin phosphatase ac-tivity thus increasing phosphorylation of myosin II in ASMtissues. This is a conserved pathway that has been describedin cultured smooth muscle cells, fibroblasts, and cancer celllines [reviewed by (60, 228)]. There is also evidence for




direct phosphorylation of myosin regulatory light chains byRho kinases in 3T3 fibroblasts (312), but there is no directevidence yet for this reaction in ASM. In addition to the cen-tral role of phosphorylation in activation of myosin II motorsthere is evidence in cultured ASM that assembly of myosinfilaments and therefore the number of motors available to gen-erate traction force is also dynamic. The assembly of myosinII filaments depends on the transition of myosin II from afolded (10S) configuration to an extended (6S) configuration(222). It seems likely that promigratory stimuli could increasemyosin phosphorylation and actomyosin ATPase activity aswell as increase the number of myosin motors available togenerate force. This notion needs to be critically tested by as-sessing myosin II filament assembly and distribution duringASM migration.

Another interesting question that remains unexplored ishow myosin motors respond to the physical nature of thematrix. This is potentially important given that there are dy-namic changes in matrix composition and stiffness of thelung parenchyma during development and disease. Ingber andcoworkers showed that decreasing stiffness of an artificial fi-bronectin matrix reduced myosin light chain phosphorylationin cultured pulmonary artery smooth muscle cells (258). Inaddition, inhibition of myosin ATPase with 2,3-butanedione2-monoxime (BDM)-reduced myosin light chain phosphory-lation, suggesting that traction forces are necessary for properfunction of the Ca2+-calmodulin-MLCK signaling pathway.In the same study, disrupting microtubules with nocodazole-increased myosin phosphorylation. The authors suggestedthat decreased adhesiveness, decreased matrix stiffness, andreduced force from myosin II motors all reduced the prestresson the cytoskeleton. Reduced prestress then inhibited myosinphosphorylation, possibly by altering proper assembly of theenzymes and other proteins regulating myosin phosphoryla-tion. Ingber et al. have proposed a model where myosin IIgenerates traction force on the matrix, the matrix modifiesmyosin phosphorylation rate and level, and therefore the ac-tivity of actomyosin as a function of matrix stiffness. If this istrue, several interesting questions arise related to airway re-modeling in inflammatory lung diseases. Does inflammationenhance migration of fibrocytes, myofibroblasts or existingsmooth muscle cells through increased contractile tone andthus increased prestress? Do anti-inflammatory drugs reverseor inhibit such an effect? Does a decrease in elastic modulus ofthe lung parenchyma influence migration of these cells in asth-matic airways? Would reversing the changes in parenchymalmechanics prevent immigration of fibrocytes and their sub-sequent differentiation to myofibroblasts and smooth musclecells? While there are no direct studies of both matrix compo-sition and migration in asthma, the promigratory influencesof collagen, elastin, and laminin on ASM cell migration invitro is consistent with the hypothesis that matrix composi-tion and matrix mechanics could be a key regulator of ASMcell migration in vivo (252, 253). Further studies of migrationof fibrocytes and myofibroblasts on matrices that mimic theremodeled asthmatic airway are warranted.

Microtubules and cell migrationActin polymerization/depolymerization and focal contact re-modeling have justifiably been the focus of many studies ofcell migration. However, it is clear that microtubules mustalso remodel during migration, and that this critical processis not as well defined as remodeling of the actin cytoskeleton.In stationary cells such as ASM embedded in ECM of theairway walls the microtubule organizing center (MTOC) andthe nucleus are centered in the cell. However, during migra-tion the nucleus is relocated toward the trailing edge of thecell. One important mediator of this relocation is Cdc42 reg-ulation of myotonic dystrophy kinase-related Cdc42 bindingkinase (MRCK) (117). Gomes et al. (117) found that nuclearrelocation required phosphorylation of myosin II by MRCK.Whether a similar event occurs in ASM cells is unknown,but there is some evidence that dynamic instability of mi-crotubules is required for migration of VSM cells. Paclitaxel(Taxol), which stabilizes microtubules by binding the sides ofthe tubulin polymer, blocks VSM cell migration (254). Mi-crotubules clearly affect the degree of prestress in the ASMcytoskeleton and influence traction forces in cultured cells(11, 295), but it is not known to what extent dynamic instabil-ity is necessary during ASM migration. In nonmuscle cells,dynamic instability of microtubules is important for disassem-bly of stable focal contacts at the rear of migrating cells. Focalcontact disassembly is required for disengagement of the trail-ing edge from the matrix (191). At this time there is only indi-rect evidence to infer a signaling pathway that would promotedynamic instability in ASM. A study of urokinase-stimulatedASM cell motility showed that urokinase induces ASM mi-gration via a pathway including PI3-kinase (51). Studies inVSM cell migration indicate urokinase also activates Akt andGSK-3β (101). GSK-3β interacts with adenomatous polypo-sis coli (APC), which is known to regulate cell polarity byinteracting with the plus end of microtubules (94). Whetherthis signaling model functions in ASM migration is unclear,but it is likely that a functionally analogous system is requiredfor microtubule remodeling during detachment and transloca-tion of the rear of a migrating ASM cell. Blocking detachmentof the trailing edge of the cell might in theory be beneficialfor blocking remodeling events in the asthmatic airway. Inhi-bition of Akt and GSK-3β signaling might have the appealingfeature of reducing hypertrophy as well as reducing immigra-tion of new cells to the muscle layers of diseased airways (seediscussion of Akt and GSK-3β signaling above).

Soluble and solid state signals that modulatemigrationThere are many chemically and structurally diverse moleculesthat enhance or inhibit ASM cell migration (Table 2). Manyare soluble signaling molecules, but some are components ofthe ECM that are presented to ASM cells as solid-state sig-nals. The first soluble promigratory molecule used to stimulateASM migration in vitro was PDGF (156). Subsequent studies




identified biogenic amines, lipids, growth factors, cytokines,and chemokines as soluble modulators of ASM migration.Many of the soluble promigratory compounds are autocrineor paracrine signaling molecules that are secreted at elevatedlevels in diseased airways. The earliest studies of solid-statesignals that promote migration were by Schuger and col-leagues who showed that laminin β1 chain as necessary formigration of smooth muscle cells from mouse lung explants(353). Later in vitro studies described the promigratory effectsof collagens, fibronectin, laminins and matrix metallopro-teinases, and antimigratory effects of tissue inhibitors of met-aloproteinases, and chemical protease inhibitors (152, 157,252). Although it is clear that matrix composition changes inthe asthmatic airway and that matrix expression by culturedASM cells changes upon exposure to proinflammatory agents(253, 270), it is not known whether matrix composition altersmigration in vivo in the lungs of asthmatic humans or in exper-imental asthma in mice. Whether a given signaling moleculeor pathway is necessary for ASM migration could be tested inknockout and transgenic mouse models using lineage mark-ing strategies. Lineage marking has been used successfully todefine the source of VSM cells in atherosclerotic plaques [re-viewed by (241)], and to demonstrate the necessity for PDGFsignaling in pericyte migration during blood vessel develop-ment (26).

Signaling cascadesMultiple highly conserved signal transduction cascades areactivated during cell migration. The pathways studied mostfrequently in both nonmuscle and smooth muscle cell mi-gration are illustrated in Figure 6. In this simplified scheme,signal transduction events are shown as cascades beginningwith receptor activation. We focus the illustration on three fun-damentally important types of receptors: RTKs, GPCR, andintegrins, which are each known to promote cell migration.Coupling of early activation to G-proteins is common to manypromigratory stimuli. Both small G-proteins (RhoA, Rac, andCdc42) and trimeric G-proteins are known to participate inpromigratory signaling depending of the stimulant used inthe experiment. Activated G-proteins, Ca2+, and changes inphospholipids including PIP2 and IP3 activate protein kinasecascades that include PI3-kinase, Ca2+-dependent protein ki-nases, Rho-activated protein kinases (ROCK), and MAPK(Fig. 6). The substrates for the various protein kinases includeother protein kinases (MAPKAP kinase and LIM kinase) aswell as proteins that interact with or regulate actin filamentformation (HSP27, cofilin, and myosin II). The monomeric G-proteins (RhoA, rac, and cdc42) also frequently regulate pro-teins that influence F-actin formation (mDia1, WAVE, WASP,and ARP2/3). The more distal effector proteins in this scheme(blue ellipses in Fig. 6) regulate two critical cellular processes:actin polymerization and activation of myosin II. The proteinsrequired for actin polymerization and coupling of F-actin tothe cell membrane integrins are illustrated in Figure 5A and B.Stimuli that increase myoplasmic Ca2+ oscillations or mean

Ca2+ concentration in a cell activate MLCK which phospho-rylates the regulatory light chains of myosin II, which is theprotein that generates traction forces necessary to move thecell. Some of these pathways have been described in somedetail in ASM (Src, ERK, p38 MAPK, and PI3-kinase), butother aspects of signaling are less well defined or undefinedin ASM migration (Rac, RhoA, ROCK, LIM kinase, cofilin,and ARP2/3). The reader is referred to a previous reviewfor more details of signal transduction pathways known toparticipate in ASM migration (105). A more complete defi-nition of signal transduction processes in ASM migration isimportant because migration is a fundamental process in lungdevelopment, and migration is presumed to be altered by lungdiseases possibly contributing to airway wall thickening inasthmatics.

Modulation of ASM cell migration by drugsOne of the exciting aspects of studies of ASM cell migrationis that biochemical processes mediating migration might benovel therapeutic targets for preventing or reversing airwayremodeling in asthma. This is frequently cited as a rationalefor exploring novel aspects of ASM migration. However, thereis no evidence of ASM-restricted target proteins or processesunique to ASM migration that would serve as selective targetsof antimigratory drugs. All promigratory and antimigratoryagents described thus far (Table 2) and all the proteins andprocesses proven to mediate migration of ASM (Fig. 5) arehighly conserved among motile cells. Identifying lung- orASM-restricted features of cell migration is a problem inneed of some attention.

Identifying novel drug targets has also been a drivingforce in studies of VSM cell migration. In fact a numberof cardiovascular drugs have beneficial effects in reducingatherogenesis and promoting recovery from vascular injuryin part by reducing VSM proliferation and cell migration[reviewed by (105)]. A clear proof of principle comes fromthe effects of statins, rapamycin, and taxol, which all reduceproliferation, inhibit cell migration, and reduce vascular wallremodeling (64, 254, 259). In the case of statins, the thera-peutic goal is to inhibit cholesterol synthesis to reduce serumLDL levels. There may also be a secondary benefit result-ing from inhibiting mevalonate synthesis and isoprenylationof small G-proteins. As discussed above, statins reduce air-way hyperreactivity in a mouse asthma model (57), reducecell proliferation (302), and increase apoptosis (110). Thepleiotropic effects are very likely due to disrupting signalingvia small G-proteins. Small G-proteins participate in multiplebiochemical processes including cell migration (see Figs. 5and 6). Extended, low dose therapy with statins may well in-hibit or reverse pathological airway remodeling by multiplemechanisms including reduced migration of ASM cells, fi-brocytes, or myofibroblasts. Whether such an effect occurs inthe airways is an interesting question that has been raised inrecent reviews of ASM as a target for novel asthma therapies(46, 274).




Several established drugs used to treat asthma also havesignificant antimigratory effects (Table 2). Corticosteriodsand β-adrenergic agonists are mainstays of combination ther-apy for long-term asthma control. Both classes of drugs as wellas other drugs acting via cAMP have antimigratory effects.This suggests the hypothesis that combination of corticos-teroids and long acting β-agonists might act by a combinationof reducing ASM proliferation, preventing matrix remodel-ing, and reducing ASM cell migration. Although this is aprovocative hypothesis, there is no in vivo animal or humanclinical data that critically tests this notion.

Other potential drug targets that should inhibit ASM cellmigration include MAP kinases and Rho kinases. P38 MAPkinases have been targeted for development of drugs to treatinflammatory diseases since the mid-1990s, but developmenthas been limited by hepatotoxicity of first generation in-hibitors. The advent of second generation inhibitors increasesthe possibility that expression of numerous contractile, proin-flammatory, and promigratory signaling proteins might bereduced by blocking p38 MAP kinase signaling (29, 85, 173).In theory, inhibiting p38 MAP kinases could reduce expres-sion of the extracellular signals for cell migration (e.g., PDGF,IL1β, and IL8) as well as block migration directly (156). Ev-idence from asthma models is consistent with this hypothesis(85), but preclinical studies in animal models of asthma andstudies in humans using less toxic p38 MAPK inhibitors areneeded to critically test this strategy (29).

Rho kinases are also potential antimigratory target pro-teins. It is clear that blocking Rho kinases inhibits ASM cellmigration (164), and a Rho kinase inhibitor (Fasudil) hasbeen tested in humans to reduce cerebral vasospasm and treatangina pectoris. The latter effects are due to vasodilation. Rhokinase inhibitors are also effective bronchodilators in mousemodels of asthma (159, 303), but there is no published evi-dence of clinical benefit to humans with asthma. In addition,there are no data demonstrating a significant effect of Rhokinase inhibition on cell migration in vivo and airway wall re-modeling. Nevertheless, Rho kinase inhibitors and p38 MAPkinase inhibitors are mechanistically appealing for modify-ing multiple aspects of the cell biology of airway dysfunc-tion including smooth muscle contraction, proliferation, andcell migration. Off-target effects are the major limitationsof protein kinase inhibitors. However, new generation drugswith enhanced selectivity combined with local delivery to thelungs might address this problem and thereby expand the toolsavailable to the pulmonary physician for long-term therapy ofasthma.

ConclusionRapid progress has been made in the past decade in studies ofkey processes underlying airway remodeling. Based on clin-ical studies and animal models of asthma it is clear that bothASM hyperplasia and hypertrophy occurs. To increase cellnumber and size in the airway wall cells it is assumed that

cells proliferate, survive for longer time periods and increaseexpression of proteins. In addition, it is possible that some im-migration of progenitor cells from beyond the muscularis oc-curs in diseased airways as well as shape changes in residentcells that differentiate to contractile smooth muscle. Thesenotions stimulated a host of studies of biochemical pathwaysthat control the fundamental processes of proliferation, apop-tosis, and cell migration. Many of the key stimuli, receptorsand transduction pathways are conserved molecules known toparticipate in remodeling of the vasculature and in tumorige-nesis. Developing novel drugs or novel uses of existing drugsto modify organ remodeling is one of the compelling reasonsfor studying many of pathways described. While a compre-hensive view of some pathways and processes is emergingthere are still basic and applied science questions remaining.Some outstanding basic science questions include the degreeto which ASM cells proliferate in vivo in diseased lungs, thesource of migrating cells, and the potential for novel featuresof translational control of protein expression and cell survivalto be discovered. The latter point is particularly important fordeveloping organ-selective drugs targeting pathways uniquein airway remodeling. In addition, even if organ or cell speci-ficity is not possible it is possible that known drugs beingtested or used currently in cancer chemotherapy and cardio-vascular medicine can be delivered in a lung-restricted mannerto alter airway remodeling. Broad-based, multidiscliplinaryapproaches employing cell, animal and human studies will berequired to integrate the basic molecular and cell signalingstudies into an effective translational strategy for developingnovel therapy of obstructive lung diseases.

AcknowledgementsSupported by NIH grant HL077726 (WTG) and The CanadianInstitutes of Health Research (CIHR), GlaxoSmithKline Col-laborative Innovation Research Fund, Manitoba Institute ofChild Health (MICH), and Canada Foundation for Innovation(AJH). S. Ghavami is supported by a Parker B. Francis Fel-lowship in Pulmonary Research. D. Schaafsma is supportedby a CIHR Postdoctoral Fellowship. P. Sharma is supportedby the Manitoba Health Research Council, MICH, and CIHR.A.J. Halayko holds a Canada Research Chair in Airway Celland Molecular Biology.

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