HIGHLIGHTED TOPICS
Signal Transduction in Smooth Muscle
Invited Review: The circle of life: cell cycle regulation in airway smooth muscle

¹ Respiratory Research Group, Faculty of Pharmacy, University of Sydney, New South Wales 2006, Australia; and ² Pulmonary, Allergy and Critical Care Division, Department of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6160

	ABSTRACT

TOP ABSTRACT INTRODUCTION AIRWAY SMOOTH MUSCLE... CELL CYCLE REGULATION REGULATION OF CELL CYCLE... INHIBITION OF AIRWAY SMOOTH... FUTURE DIRECTIONS REFERENCES

Severe asthma is characterized by increased airway smooth muscle (ASM) mass, due predominantly to ASM hyperplasia. Diverse stimuli, which include growth factors, plasma- or inflammatory cell-derived mediators, contractile agonists, cytokines, and extracellular matrix proteins, induce ASM proliferation. Mitogens act via receptor tyrosine kinase, G protein-coupled receptors, or cytokine receptors, to activate p21ras and stimulate two parallel signaling pathways in ASM cells, namely, the extracellular signal-regulated kinase (ERK) or the phosphatidylinositol 3-kinase (PI3K) pathways. ERK and PI3K regulate cell cycle protein expression and thus modulate cell cycle traversal. ERK activation and downstream effectors of PI3K, such as Rac1 and Cdc42, stimulate expression of cyclin D1, a key regulator of G₁ progression in the mammalian cell cycle. In addition, PI3K activates 70-kDa ribosomal S6 kinase, an enzyme that also regulates the translation of many cell cycle proteins, including the elongation factor E2F. The present review examines the mitogens and critical signal transduction pathways that stimulate ASM cell proliferation. Further study in this area may reveal new therapeutic targets to abrogate ASM hyperplasia in diseases such as asthma and chronic obstructive pulmonary disease.

receptor tyrosine kinases; G protein-coupled receptors; p21ras; extracellular signal-related kinase; phosphatidylinositol 3-kinase; cyclin D1

	INTRODUCTION

TOP ABSTRACT INTRODUCTION AIRWAY SMOOTH MUSCLE... CELL CYCLE REGULATION REGULATION OF CELL CYCLE... INHIBITION OF AIRWAY SMOOTH... FUTURE DIRECTIONS REFERENCES

ASTHMA, A CHRONIC DISEASE characterized by airway hyperreactivity, inflammation, and remodeling, occurs in 5-8% of the U.S. population and is an extraordinarily common cause of pulmonary impairment worldwide. Others (14, 19, 21, 57) and our laboratory (51) have recently suggested a prominent role for airway smooth muscle (ASM) in the perpetuation of airway inflammation and induction of the chronic features of airway remodeling that occur in asthma. This assertion is supported by observations that numerous agents that are elevated in the asthmatic airway are mitogenic to ASM in vitro (51) and that ASM expresses adhesion molecules (36) and secretes numerous cytokines (29) after exposure to relevant inflammatory agents. Furthermore, ASM mass is increased in the bronchi of severe chronic asthmatic subjects (17), and this increased mass is due to hyperplasia and/or hypertrophic growth (18, 27). Consequently, much research has centered on the elucidation of the cellular and molecular mechanisms that regulate mitogen-induced ASM proliferation.

Over the past decade, significant advances have been made in identifying the many diverse mitogens and signal transduction pathways that modulate ASM growth (see review, Ref. 26). Because progression of ASM through the cell cycle is a fundamental event in regulating cell proliferation, recent studies have also examined the signal transduction pathways that regulate specific cell cycle protein expression in ASM cells. The present review examines the signaling pathways that stimulate ASM cell proliferation and identify the critical cell cycle events that regulate ASM growth.

	AIRWAY SMOOTH MUSCLE PROLIFERATION

TOP ABSTRACT INTRODUCTION AIRWAY SMOOTH MUSCLE... CELL CYCLE REGULATION REGULATION OF CELL CYCLE... INHIBITION OF AIRWAY SMOOTH... FUTURE DIRECTIONS REFERENCES

Many inflammatory mediators are increased in bronchoalveolar lavage (BAL) from asthmatic airways, and some have been shown to induce ASM mitogenesis in vitro. To date, mitogenic stimuli include: growth factors, such as epidermal growth factor (EGF) (72), insulin-like growth factors (42), platelet-derived growth factor (PDGF) isoforms BB and AB (24), and basic fibroblast growth factor (8); plasma- or inflammatory cell-derived mediators, such as lysomal hydrolases (-hexosaminidases and -glucuronidase) (38), -thrombin (53), tryptase (13), and sphingosine 1-phosphate (SPP) (2); and contractile agonists, such as histamine (55), endothelin-1 (45); substance P (45), phenylephrine (43), serotonin (23), thromboxane A₂ (44), and leukotriene D4 (54).

Although the cytokines interleukin (IL) 1 (IL-1), IL-6, and tumor necrosis factor- (TNF-) are also increased in BAL of asthmatic subjects (12), whether these cytokines stimulate ASM proliferation in vitro remains controversial. In 1995, De et al. (15) reported that IL-1 and IL-6 cause hyperplasia and hypertrophy of cultured guinea pig ASM cells; however, other studies have shown that IL-1 (6) and IL-6 (39) are not mitogenic for human ASM cells. McKay et al. (39) also reported that TNF- (~30 pM) had no immediate mitogenic effect on human ASM cells. These results were in contrast to those of Stewart et al. (69), who reported that the proliferative effect of TNF- on human ASM cells appeared to be biphasic such that low concentrations of TNF- (0.3-30 pM) were promitogenic, whereas at higher concentrations (300 pM) the mitogenic effect was abolished. Such conflicting reports may be due to cytokine-induced cyclo-oxygenase 2-dependent prostanoid production (6). Cyclooxygenase products, such as prostaglandin E₂, inhibit DNA synthesis (6). Therefore, cytokine-induced proliferative responses in ASM may be greater under conditions of cyclo-oxygenase inhibition, in which the expression of growth inhibitory prostanoids, such as prostaglandin E₂, is limited (6, 15, 69).

Airway remodeling, a key feature of persistent asthma, is also characterized by the deposition of extracellular matrix (ECM) proteins in the airways (35, 61). ECM proteins collagen I, III, V, fibronectin, tenascin, hyaluronan, versican, and laminin 2/2 are increased in asthmatic airways (1, 9, 35, 62). Components of the ECM also modulate mitogen-induced ASM growth. Fibronectin and collagen I increase human ASM cell mitogenesis in response to PDGF-BB or -thrombin, whereas laminin inhibits proliferation (25). Recently, human ASM cells were shown to secrete ECM proteins in response to asthmatic sera (28), suggesting a cellular source for ECM deposition in airways and implicating a novel mechanism in which ASM cells may modulate autocrine proliferative responses.

	CELL CYCLE REGULATION

TOP ABSTRACT INTRODUCTION AIRWAY SMOOTH MUSCLE... CELL CYCLE REGULATION REGULATION OF CELL CYCLE... INHIBITION OF AIRWAY SMOOTH... FUTURE DIRECTIONS REFERENCES

Extracellular stimuli transduce proliferative responses that move the cell through the cell cycle, which comprises distinct phases termed G₁, S (DNA synthesis), G₂, and M (mitosis). ASM growth appears to occur by activating cell cycle events similar to those described in other cell types. Hence, the following section provides an overview of the mammalian cell cycle (reviewed in Refs. 65, 66) with particular emphasis on the G₁-to-S transition, the most widely studied cell cycle phase in ASM biology, shown schematically in Fig. 1.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Schematic representation of the G1-to-S transition phase in the cell cycle. In response to mitogens, cells enter the cell cycle from the G0/G1A phase. D-type cyclins (D1 shown here) are expressed, whereas the levels of the cyclin-dependent kinase (CDK) inhibitor p27Kip1, usually high in quiescent cells, fall in response to mitogenic stimulation. Progression through G1 phase initially depends on holoenzymes composed of D-type cyclins in association with CDK4 or CDK6. Most p27Kip1 becomes complexed with cyclin D-CDK, allowing activation of the cyclin E-CDK2 complex. Together, cyclin E and CDK2 act in a cascade to hyperphosphorylate retinoblastoma protein (pRb), which then releases the elongation factor E2F that activates DNA polymerase. Cell commitment to traverse completely through to mitosis is achieved on or near this point, termed the restriction point (R), in the cell cycle. Subsequently, cells initiate DNA synthesis (S phase).

Proliferative responses in ASM cells are studied by using cell culture models. ASM cells are grown to confluence, then growth-arrested in a low serum media or serum-free conditions for 24-48 h (52, 55). This experimental design synchronizes ASM cells in the G₀ or early G₁ phase (G_1A) of the cell cycle, in which ASM minimally incorporates [³⁵S]methionine and [³H]thymidine (52, 55). As cells enter the cycle from G₀/G_1A, one or more D-type cyclins (D1, D2, and D3) are expressed as part of the delayed early response to mitogen stimulation, as shown in Fig. 1. Progression through the G₁ phase initially depends on holoenzymes composed of one or more of the D-type cyclins (D1, D2, and/or D3) in association with cyclin-dependent kinases (CDKs), CDK4 or CDK6. This is followed by activation of cyclin E-CDK2 complex as cells approach the G₁/S transition. Together, cyclin E and CDK2 act to hyperphosphorylate retinoblastoma protein (pRb), which then releases the elongation factor E2F that activates DNA polymerase. This step, termed the restriction point, represents the point of no return; cell commitment to undergo DNA synthesis (S phase) and mitosis is inevitable. In ASM cells, S phase is commonly detected by using incorporation of radiolabeled thymidine (52, 55) or by immunofluorescent detection of the thymidine analog 5-bromo-2'-deoxyuridine (3). At each phase of G₁-to-S transition, CDK activities can also be constrained by CDK inhibitors (CKIs). CKIs are assigned to two families on the basis of their structures and CDK targets: 1) INK4 family (p16^INK4a, p15^INK4b, p18^INK4c, and p19^INK4d) specifically inhibit the catalytic subunits of CDK4 and CDK6; and 2) Cip/Kip family (p21^Cip1, p27^Kip1, and p57^Kip2) inhibit the activities of cyclin D-, E-, and A-dependent kinases (67).

	REGULATION OF CELL CYCLE IN AIRWAY SMOOTH MUSCLE CELL PROLIFERATION

TOP ABSTRACT INTRODUCTION AIRWAY SMOOTH MUSCLE... CELL CYCLE REGULATION REGULATION OF CELL CYCLE... INHIBITION OF AIRWAY SMOOTH... FUTURE DIRECTIONS REFERENCES

ASM mitogens may act via different receptor-operated mechanisms (reviewed in Ref. 26), as shown in Fig. 2. Whereas growth factors induce ASM cell mitogenesis by activating receptors with intrinsic protein tyrosine kinase (RTK) activity, contractile agonists released from inflammatory cells mediate their effects via activation of seven transmembrane G protein-coupled receptors (GPCRs). Cytokines signal through cell surface glycoprotein receptors that function as oligomeric complexes consisting of typically two to four receptor chains (4) coupled to Src family nonreceptor tyrosine kinases, such as lyn (7).

View larger version (28K): [in this window] [in a new window]   Fig. 2.   Schematic representation of signal transduction mechanisms that regulate airway smooth muscle (ASM) cell proliferation. ASM mitogens act via intrinsic protein tyrosine kinases (RTKs), cytokine receptors, or G protein-coupled receptors (GPCRs) to activate the small GTPase p21ras. p21ras proteins then interact with downstream effectors Raf-1 and phosphatidylinositol 3-kinase (PI3K). Raf-1 activates mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinase (MEK1), which then phosphorylates ERK. PI3K activates the downstream effectors p70S6k or members of the Rho family GTPases, Rac1 and Cdc42 (although whether Cdc42 acts upstream of Rac1 or cross-talk exists is unknown at present; indicated by dashed lines). ERK, PI3K, and the downstream effectors of PI3K regulate cell cycle proteins, and thus the ERK and PI3K pathways are considered to be two major independent signaling pathways regulating ASM cell growth.

Despite disparate receptor-operated mechanisms, recent evidence suggests that the small GTPase p21ras acts as a point of convergence for diverse extracellular signal-stimulated pathways in ASM cells as shown in Fig. 2 (3). Interestingly, synergy can occur between RTK and GPCRs that promotes human ASM mitogenesis and p21ras activation (33). In their GTP-bound active state, p21ras proteins interact with downstream effectors, namely, Raf-1 and PI3K. By recruiting Raf-1, a 74-kDa cytoplasmic serine/threonine kinase, to the plasma membrane, GTP-bound p21ras activates the ERK pathway, although Raf-1-independent signaling to ERK also has been shown (31). p21ras also binds and activates PI3K by using specific regions termed switch I (Asp³⁰-Asp³⁸) and switch II (Gly⁶⁰-Glu⁷⁶) (47, 63). Although alternative pathways do exist (e.g., protein kinase C-dependent pathways, reviewed in Ref. 26) or reactive oxygen-dependent pathways (10), ERK and PI3K activation appears to be the dominant signal transduction pathway for RTK-, GPCR-, or cytokine-stimulated growth of ASM cells.

ERK pathway. Raf-1 activation induces phosphorylation and activation of mitogen-activated protein (MAP) kinase/ERK kinase (MEK1). Activated MEK1 then directly phosphorylates (on both tyrosine and threonine residues) and activates the 42-kDa ERK2 and 44-kDa ERK1, also collectively referred to as p42/p44 MAP kinases, as shown in Fig. 2. In bovine ASM, inhibition of MEK1 and ERK activity attenuates PDGF-induced DNA synthesis, suggesting that activation of MEK1 and ERKs is required for proliferation (30). In human ASM (46), mitogens, including EGF, PDGF-BB, and thrombin, produced a robust and sustained activation of ERK1 and ERK2 that was correlated with ASM growth responses and was inhibited by MEK1 inhibition. Studies such as these suggest that the ERK pathway is a key signaling event mediating mitogen-induced ASM proliferation.

D-type cyclins (cyclins D1, D2, and D3) are key regulators of G1 progression in mammalian cells, and, consequently, cyclin D1 has been the most widely studied cyclin in ASM biology. In bovine ASM, mitogenic stimulation with PDGF induced cyclin D1 transcriptional activation and protein synthesis, with consequent hyperphosphorylation of pRb, whereas microinjection with a neutralizing antibody against cyclin D1 inhibited serum-induced S-phase traversal (75). These studies suggested that cyclin D1 is a key downstream target of ERKs and that downstream transcription factor targets of ERKs regulate cyclin D1 promoter transcriptional activity and cell cycle progression. This was also suggested in studies in which a MEK1 inhibitor and a dominant negative mutant of MEK1 or ERK abolished PDGF-induced cyclin D1 promoter activity or cyclin D1 expression (58). Expression of a constitutively active p21ras induced ERK activation and transcriptional activation of the cyclin D1 promoter, suggesting a role of p21ras in regulating the ERK pathway (48).

Evidence now suggests that ERK activation induces expression of cyclin D1 in ASM cells. Hence, recent studies have focused on the transcriptional regulation of ERK-induced cyclin D1 accumulation. The promoter region of the cyclin D1 (22) contains multiple cis-elements potentially important for transcriptional activation, including binding sites for simian virus 40 protein 1 (Sp1); activator protein-1 (AP-1); signal transducers and activators of transcription; nuclear factor B (NF-B); and cAMP response element binding protein (CREB)/activating transcription factor-2 (ATF-2) (41). In 1999, Orsini et al. (46) showed that mitogen-induced ERK activation, thymidine incorporation, and Elk-1 and AP-1 reporter activity were similarly abrogated by MEK1 inhibition. These studies suggest a linkage between ERK activation, transcription factor activation, cyclin D1 expression, and ASM proliferation. Similarly, MEK1 inhibition also attenuated expression of c-Fos (37), suggesting that c-Fos may be one or both of the dimer pairs in the AP-1 transcription factor complex responsible for cyclin D1 expression in ASM cells. Whether ERK-dependent transcriptional regulation of cyclin D1 gene expression is via direct cis-activation with AP-1 dimers (composed of c-Fos) or via Elk-1-mediated trans-activation still requires further investigation. In addition, cyclin D1 protein, but not mRNA levels, was affected by MEK1 inhibition (60), suggesting that posttranscriptional control of cyclin D1 protein levels may also occur independently of the MEK1/ERK signaling pathways.

Another critical cell cycle protein is p27^Kip1 (66), as shown in Fig. 1. In quiescent cells, the cytosolic protein levels of p27^Kip1 remain high. A coordinated increase of cyclin D1 expression promotes complexing of unbound p27^Kip1 molecules with cyclin D-dependent kinases, relieving cyclin E-CDK2 from CKI constraint and thereby facilitating cyclin E-CDK2 activation later in the G₁ phase (66). In human ASM cells (2), SPP, an agonist that activates multiple GPCRs, was shown to increase cyclin D1 levels and decrease p27^Kip1, possibly via an ERK-mediated pathway (56). SPP also appeared to augment EGF- and thrombin-induced DNA proliferation by increasing G₁/S progression (2). This was due to an enhancement of the stimulatory and inhibitory effect of EGF and thrombin on cyclin D1/p27^Kip1 expression by SPP (2).

PI3K pathway. PI3K isoforms are divided into three classes on the basis of their structure and substrate specificity (59). Class IA PI3Ks are cytoplasmic heterodimers composed of a 110-kDa (p110, -, or -) catalytic subunit and an 85-kDa (p85, p55, or p50) adaptor protein. Class IA isoforms can be activated by RTKs and nonreceptor tyrosine kinases, whereas class IB p110 is activated by G subunits of GPCRs. Class II isoforms are mainly associated with the phospholipid membranes, are concentrated in the trans-Golgi network, and are present in clathrin-coated vesicles (16). Class III isoforms are structurally related to the yeast vesicular sorting protein Vps34p (73). Recent data (32) show that human ASM cells express class IA, II, and III PI3K but not the class IB p110 isoform.

PI3K phosphorylates membrane phosphoinositides on the D3 hydroxyl of the inositol ring to form phosphoinositide 3-phosphate, phosphoinositide 3,4-diphosphate, and phosphoinositide 3,4,5-triphosphate. These D3 phosphoinositides function as second messengers and activate downstream effector molecules, such as 70-kDa ribosomal S6 kinase (p70^S6k) (34, 64) or members of the Rho family GTPases [Rac1 (49) and Cdc42 (5), but not RhoA (5)] to regulate cell cycle protein expression and thus modulate cell cycle traversal in ASM cells (Table 1).

View this table: [in this window] [in a new window]   Table 1.   Effect of mitogens and signaling events on cell cycle protein expression in ASM cells

The use of PI3K inhibitors has shown that activation of PI3K is critical for ASM cell cycle progression in human (34) and bovine ASM (64, 74). Transfection or microinjection of cells with constitutively active class IA PI3K alone markedly increased DNA synthesis (32). This is the first study to show that a constitutively active signaling molecule is capable of inducing DNA synthesis in human ASM cells. The extent of DNA synthesis stimulated in cells microinjected with constitutively active PI3K, however, was substantially less than that induced by receptor-mediated pathways. These data suggest that, although active PI3K is sufficient to stimulate ASM DNA synthesis, other signaling events are also necessary to promote maximal ASM growth responses. Interestingly, PI3K inhibition, however, does not alter ERK activation (34), confirming that PI3K regulates DNA synthesis in an ERK-independent or possibly parallel manner.

In bovine (64) and human ASM (34), rapamycin, an inhibitor of p70^S6k, attenuates growth factor-induced DNA synthesis, showing that p70^S6k is an essential step in the pathway toward ASM cellular proliferation, as shown in Fig. 2. Through the phosphorylation of the 40S ribosomal protein, p70^S6k upregulates the translation of mRNA that contain an oligopyrimidine tract at their transcriptional start site. Such mRNA moieties encode proteins required for cell cycle progression in the G₁ phase, such as the elongation factor E2F (11).

Recent studies have examined the involvement of the Rho GTPases, Rac1 (49), Cdc42 (5), and RhoA (5) in cyclin D1 upregulation and bovine ASM proliferation. Rac1 overexpression induced transcription of a cyclin D1 promoter construct, whereas a dominant-negative allele of Rac1 inhibited PDGF-induced cyclin D1 transcription (49). Rac1-induced cyclin D1 promoter activation was also independent of ERK because inhibition of MEK1 had little effect (49). In other studies, Page et al. (50) demonstrated that overexpression of the catalytically active subunit of PI3K (p110^PI3KCAAX) was sufficient to activate the cyclin D1 promoter and that cyclin D1 promoter activation could be attenuated by inhibitors of Rac1 signaling. These results suggest that Rac1 may be downstream of PI3K; however, further study is necessary to confirm this observation. Other studies using overexpression constructs of Cdc42 and RhoA also showed that overexpression of Cdc42, but not of RhoA, induced transcription from the cyclin D1 promoter in an ERK-independent manner (5). In addition, p110^PI3KCAAX (50), Rac1 (50), and Cdc42 (5) were shown to activate the cyclin D1 promoter via the CREB/ATF-2 binding site. These results led the investigators to speculate that Cdc42 acts upstream of Rac1 (5). Whether this implicates PI3K in a linked signaling cascade remains unknown. A summary of the signal transduction pathways that modulate cell cycle events in ASM is described in Table 1.

	INHIBITION OF AIRWAY SMOOTH MUSCLE PROLIFERATION BY ANTI-ASTHMA THERAPIES

TOP ABSTRACT INTRODUCTION AIRWAY SMOOTH MUSCLE... CELL CYCLE REGULATION REGULATION OF CELL CYCLE... INHIBITION OF AIRWAY SMOOTH... FUTURE DIRECTIONS REFERENCES

The most widely used therapies for the control of asthma symptoms are the corticosteroids and the ₂-agonists. Inhaled corticosteroids inhibit inflammatory cell activation, whereas ₂-agonists are effective bronchodilators. In addition, these anti-asthma therapies are potent inhibitors of ASM cell proliferation.

In human ASM (20), corticosteroids dexamethasone and fluticasone propionate were shown to arrest ASM cells in the G₁ phase of the cell cycle. In this study (20), corticosteroids reduced thrombin-stimulated increases in cyclin D1 protein and mRNA levels and attenuated pRb phosphorylation via a pathway either downstream or parallel to the ERK cascade.

₂-Agonists activate the ₂-adrenergic receptor G_s-adenylyl cyclase pathway to elevate cAMP in ASM cells. Because of their cAMP-elevating ability (71), albuterol (72) and fenoterol (70) have been shown to inhibit mitogen-induced proliferation of human ASM cells. ₂-adrenergic receptor agonists, and other cAMP-elevating agents, are thought to induce G₁ arrest by posttranscriptionally inhibiting cyclin D1 protein levels via action on a proteasome-dependent degradation pathway (68). Musa et al. (40) examined the effects of forskolin, an activator of adenylate cyclase, on DNA synthesis, cyclin D1 expression, and CREB phosphorylation and DNA binding in bovine ASM. By increasing cAMP in ASM cells, this study (40) showed that forskolin suppressed cyclin D₁ gene expression via phosphorylation and transactivation of CREB, suggesting that the effect of cAMP on cyclin D1 gene expression is via cis-repression of cyclin D1 promoter.

Further elucidation of the signaling and transcriptional targets for the inhibition of cell cycle progression by corticosteroids and ₂-agonists may indicate how these anti-asthma therapies could be used optimally, and possibly in combination, to modulate airway wall remodeling in asthma.

	FUTURE DIRECTIONS

TOP ABSTRACT INTRODUCTION AIRWAY SMOOTH MUSCLE... CELL CYCLE REGULATION REGULATION OF CELL CYCLE... INHIBITION OF AIRWAY SMOOTH... FUTURE DIRECTIONS REFERENCES

Although significant advances have been made in identifying the many diverse mitogens and signal transduction pathways that modulate proliferation of ASM cells, only recently have investigators examined the signal transduction pathways that regulate specific cell cycle protein expression in ASM cells (Table 1). Such studies have focused on the G₁-to-S transition, in particular the role of cyclin D1 in modulating G₁ progression to S-phase traversal in ASM cells. Further studies examining the critical signaling events that integrate multiple upstream pathways will provide new therapeutic targets to abrogate ASM cell growth and possibly ASM hyperplasia. Cell cycle protein expression may be an ideal target for such therapy especially if aerosolized therapy can offer organ-specific drug delivery. A final short section detailing actions of anti-asthma therapies on cell cycle traversal and biochemistry might provide additional emphasis for looking to the cell cycle for possible future therapeutic targets. In addition, new investigations into animal models of ASM hyperplasia are also needed to address the relevance of current in vitro studies to complex diseases such as asthma and COPD.

	ACKNOWLEDGEMENTS

This work was funded by the National Health and Medical Research Council of Australia C. J. Martin Fellowship 977301 to A. J. Ammit and National Heart, Lung, and Blood Institute Grants HL-55301 and HL-64063 and National Institute of Allergy and Infectious Diseases Grant AI-40203 to R. A. Panettieri, Jr.

	FOOTNOTES

Address for reprint requests and other correspondence: R. A. Panettieri, Jr., Pulmonary, Allergy and Critical Care Division, Dept. of Medicine, Univ. of Pennsylvania, 805 BRB II/III, 421 Curie Boulevard, Philadelphia PA 19104-6160 (E-mail: rap{at}mail.med.upenn.edu).

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TOP ABSTRACT INTRODUCTION AIRWAY SMOOTH MUSCLE... CELL CYCLE REGULATION REGULATION OF CELL CYCLE... INHIBITION OF AIRWAY SMOOTH... FUTURE DIRECTIONS REFERENCES

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