Mitogenic signaling pathways in airway smooth muscle

Respiratory Physiology & Neurobiology 137 (2003) 295 /308 www.elsevier.com/locate/resphysiol

Mitogenic signaling pathways in airway smooth muscle Limei Zhou, Marc B. Hershenson * Department of Pediatrics, University of Chicago, Chicago, IL, USA Accepted 4 December 2002

Abstract Increased airway smooth muscle mass has been demonstrated in patients with asthma, bronchopulmonary dysplasia and most recently, cystic fibrosis. These observations emphasize the need for further knowledge of the events involved in airway smooth muscle mitogenesis and hypertrophy. Workers in the field have developed cell culture systems involving tracheal and bronchial myocytes from different species. An emergent body of literature indicates that mutual signal transduction pathways control airway smooth muscle cell cycle entry across species lines. This article reviews what is known about mitogen-activated signal transduction in airway myocytes. The extracellular signal regulated kinase (ERK) and phosphatidylinositol 3-kinase (PI 3-kinase) pathways appear to be key positive regulators of airway smooth muscle mitogenesis; recent studies have also demonstrated specific roles for reactive oxygen and the JAK/STAT pathway. It is also possible that growth factor stimulation of airway smooth muscle concurrently elicits signaling through negative regulatory intermediates such as p38 mitogen-activated protein (MAP) kinase and protein kinase C (PKC) delta, conceivably as a defense against extreme growth. # 2003 Elsevier B.V. All rights reserved. Keywords: Airways, smooth muscle; Disease, asthma, cystic fibrosis; Enzyme, mitogen-activated protein kinase; Mammals, humans; Muscle, smooth, airways; Oxygen, reactive

1. Introduction Developing bronchi are enclosed by a wellformed layer of smooth muscle cells by the conclusion of the embryonic period of fetal lung development (Sparrow et al., 1999). Fetal airway smooth muscle is spontaneously contractile throughout gestation and this phasic activity is * Corresponding author: Present address: Department of Pediatrics, University of Michigan, 1150 West Medical Center Drive, 3570 MSRB II, Box 0688, Ann Arbor, MI 48109-0688, USA. Tel.:
/1-734-764-4123; fax:
/1-734-936-7635. E-mail address: [email protected] (M.B. Hershenson).

associated with the maintenance of a positive intraluminal pressure (Schittny et al., 2000). Positive intraluminal pressure, in turn, has been implicated in the process of lung growth and development (Nakamura and McCray, 2000). Conversely, mechanical stress promotes bronchial myogenesis via the alternative splicing of serum response factor, which is required for contractile protein expression (Yang et al., 2000). With airway circumferential and axial growth, this layer enlarges, a consequence of both cellular hypertrophy and hyperplasia. It has been shown that the quantity of smooth muscle is abnormally increased in the airways of premature infants with bronch-

1569-9048/03/$ - see front matter # 2003 Elsevier B.V. All rights reserved. doi:10.1016/S1569-9048(03)00154-X

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opulmonary dysplasia (Hislop and Haworth, 1989; Sward-Comunelli et al., 1997), due in part to excess cell proliferation (Johnson et al., 1991). Information regarding airway smooth muscle growth during typical postnatal development is not available, however. Increased airway smooth muscle mass has also been shown in non-fatal (Carroll et al., 1993) and fatal asthma (Dunhill et al., 1971; Takizawa and Thurlbeck, 1971; Heard and Hossain, 1973; Sobonya, 1984; James et al., 1989; Ebina et al., 1990; Saetta et al., 1991; Ebina et al., 1993). In the most compelling report to date, Ebina et al. (1993) studied the airway thickness and smooth muscle cell number of patients with fatal asthma with state-of-the-art stereological methods. Two asthmatic subtypes were found, one in which the smooth muscle thickness was increased only in the central bronchi (Type I) and another in which the quantity of smooth muscle was increased throughout the airway tree (Type II). In Type I, an increased number of smooth muscle cell nuclei was present in the central airways, whereas in Type II, cellular hypertrophy was present. However, it should be noted that earlier studies examining lowmagnification circumferential profiles of airway tissue may not have taken into account the fact that airway smooth muscle bundles contain significant amounts of collagen which do not contribute to muscle shortening (Thomson et al., 1996). Subepithelial myofibroblasts have been noted in the airways of patients with chronic severe asthma (Brewster et al., 1990), as well as those undergoing allergen challenge (Gizycki et al., 1997). Recent studies suggest that these cells may be the source of subepithelial fibrosis found in patients with chronic severe asthma (Morishima et al., 2001; Hastie et al., 2002). However, as far as we are aware there are no data suggesting that myofibroblasts contribute to increased airway smooth muscle mass in asthma. Increased protein abundance of epidermal growth factor (EGF), a mitogen for human airway smooth muscle, has been found in asthmatic airways (Vignola et al., 1997; Amishima et al., 1998). EGF receptor expression is also increased (Amishima et al., 1998). Bronchoalveolar lavage

fluid basic fibroblast growth factor concentrations are significantly higher in subjects with atopic asthma than in control subjects without asthma (Redington et al., 2001). Also, bronchoalveolar lavage fluid from asthmatic airways has been shown to increase the extracellular signal regulated kinase (ERK) activation, cyclin D1 protein abundance, [3H]-thymidine incorporation and cell number of cultured human airway smooth muscle cells (Naureckas et al., 1999). Abnormally increased airway smooth muscle DNA synthesis has been shown in two animal models of airways disease, hyperoxic exposure and allergen sensitization (Hershenson et al., 1994; Wang et al., 1995; Panettieri et al., 1998). Finally, increased smooth muscle mass has recently been noted in the airways of patients with cystic fibrosis (Hays et al., 2001). Together, these reports demonstrate that abnormal smooth muscle mitogenesis is present in the airways of patients with chronic airways disease, and emphasize the need for further study of airway smooth muscle mitogenesis. To that end, workers in the field have developed cell culture systems involving tracheal and bronchial myocytes from different species. A review of mitogen-activated signal transduction in airway smooth muscle follows below.

2. Growth factor stimulation of airway smooth muscle cells Multiple reports have examined airway smooth muscle cell mitogenesis in response to growth factors. Airway myocytes proliferate in response to peptide growth factors ligating receptor tyrosine kinases (Hirst et al., 1992; Kelleher et al., 1995; Hirst et al., 1996; Krymskaya et al., 1999a), as well as to bronchoconstrictor substances associated with G protein-coupled seven transmembrane receptors. The latter include histmamine, thrombin, endothelin and tryptase (Panettieri et al., 1990; Stewart et al., 1994; Malarkey et al., 1995; Panettieri et al., 1995; Shapiro et al., 1996; Whelchel et al., 1997; Walker et al., 1998; Vichi et al., 1999; Brown et al., 2002). The response to growth factor activation may be species-specific. For instance, histamine has been reported to

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induce human airway smooth muscle proliferation (Panettieri et al., 1990; Maruno et al., 1995), but does not induce proliferation in bovine cells (Kelleher et al., 1995). Nonetheless, the signaling pathways and downstream transcription factor targets elicited by growth factor stimulation may be surprisingly stable across species lines (see below). For the last 7 years, we have studied the signaling pathways underlying platelet-derived growth factor (PDGF)-induced cyclin D1 expression and DNA synthesis in bovine tracheal myocytes (Fig. 1). Cyclin D1, a G1 cyclin, is expressed in response to growth factor stimulation (Winston and Pledger, 1993). Using microinjection techniques, we have shown that cyclin D1 is required for serum-induced DNA synthesis (Xiong et al., 1997). Accordingly, cyclin D1 expression may be used as a surrogate for cell proliferation when monitoring upstream signaling pathways. The PDGF receptor consists of two individual chains (A and B) that are dimerized by a disulfide

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bond and can occur in three isoforms, AA, AB, or BB. Classically, binding of a growth factor to its receptor tyrosine kinase stimulates the receptor’s intracellular kinase domain, inducing phosphorylation of particular tyrosine residues within the kinase domain. Ligand binding of PDGF brings about creation of a stable receptor dimer, which in turn induces one receptor molecule to phosphorylate the other in the dimer (Ullrich and Schlessinger, 1990). EGF, a monomeric molecule, binds concurrently to two receptor molecules (Lemmon and Schlessinger, 1994). Phosphorylation of tyrosine residues inside the kinase domain augments kinase activity further, provoking phosphorylation of additional receptor residues outside the kinase domain (White et al., 1988). These phosphotyrosine residues, in turn, function as docking positions for downstream signal transduction molecules containing Src-homology 2 (SH2) domains. The PDGF receptor possesses nine phosphotyrosine domains, one of which is critical for receptor tyrosine kinase activity and eight that

Fig. 1. Signaling pathways regulating PDGF-induced transcription from the cyclin D1 promoter in bovine tracheal myocytes. The ERK and phosphatidylinositol (PI) 3-kinase/Rac1 pathways positively regulate transcription, whereas PKC-d negatively regulates promoter activity. The PI 3-kinase/Rac1 signal is transduced by the generation of reactive oxygen. Growth factor stimulation and activation of PI 3-kinase/Rac1 induce cAMP response element binding protein (CREB)-1 DNA binding and CRE transactivation, respectively, whereas PKCd attenuates CREB1 activity. Finally, it should be noted that other receptors, for example, G-protein coupled receptors, may also initiate mitogenic signaling pathways.

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interact with various signal transduction intermediates. These include the growth factor receptor binding protein Grb2, phosphatidylinositol 3-kinase (PI 3-kinase), phospholipase C-gamma, the GTPase-activating protein GAP, the Src tyrosine kinase, and protein tyrosine phosphatase 1D (Claesson-Welsh, 1994). We have described signaling pathways arising from at least three of these PDGF receptor-binding proteins in bovine tracheal myocytes (Karpova et al., 1997; Page et al., 2000, 2002).

3. Role of the ERK signaling pathway in cell cycle progression The mitogen-activated protein (MAP) kinases are a superfamily of cytoplasmic serine/threonine kinases that participate in the transfer of growth and differentiation-promoting signals to the cell nucleus (Fig. 2). They share a common activation mechanism which involves the phosphorylation of tyrosine and threonine residues in a Thr-X-Tyr (TXY) motif positioned in their activation loop. Based on the identity of the residue between the threonine and tyrosine, the MAP kinase superfamily can be divided into three main groups: ERKs (Thr-Glu-Tyr); Jun amino terminal kinases

Fig. 2. MAP kinase superfamily of serine/threonine kinases. Each MAP kinase family member */ERK, JNK and p38 */is activated by successive activation of a MAP kinase kinase kinase and MAP kinase kinase.

(JNKs) (Thr-Pro-Tyr); and p38s (Thr-Gly-Tyr). Each MAP kinase is activated by successive activation of a MAP kinase kinase kinase and a MAP kinase kinase. Activation of ERK is required for DNA synthesis in an extensive variety of mammalian cell systems, including bovine, rat and human airway smooth muscle (Karpova et al., 1997; Whelchel et al., 1997; Lew et al., 1999; Orsini et al., 1999). Perhaps this is to be expected, as many aspects of MAP kinase cascades, GTPase signaling pathways and cell cycle regulation are highly conserved in eukaryotic species, including mammals, Drosophila , nematodes and yeast (Herskowitz, 1995; Waskiewicz and Cooper, 1995; Edgar and Lehner, 1996; Elledge, 1996; Treisman, 1996; Watanabe et al., 1996b, 1997). The traditional path to ERK activation is comprised of the growth factor receptor binding protein Grb2, the nucleotide exchange factor Son of sevenless (Sos), the 21 kDa GTPase Ras, the 74 kDa cytosolic serine/threonine kinase Raf-1, and the 45 kDa dual function kinase MAP kinase/ ERK kinase kinase (MEK)-1. Grb2 is found in a stable complex with the nucleotide exchange factor Sos. Docking of Grb2 to a receptor tyrosine kinase causes Sos to bind to and activate Ras. Ras then escorts Raf-1 to the cell membrane, resulting in Raf-1 activation (Stokoe et al., 1994). Raf-1 phosphorylates MEK1 on two serine residues, Ser218 and Ser222 (Yan and Templeton, 1994). As noted above, MEK1 phosphorylates tyrosine and threonine residues in the ERK activation loop. Several studies verify that these proteins are required for ERK activation in airway myocytes. In human cells, microinjection of the anti-pan Ras neutralizing antibody inhibits DNA synthesis (Ammit et al., 1999), and overexpression of a dominant-negative form of H-Ras inhibits PDGFinduced ERK activation in bovine cells (Page et al., 1999a) (Ras did not appear to be necessary for phorbol ester-induced ERK activation, however). Overexpression of a kinase-dead mutant of Raf-1 in rat tracheal myocytes inhibits endothelinmediated ERK activation (Vichi et al., 1999). Chemical or dominant-negative inhibition of MEK-1 attenuates ERK activation and DNA synthesis in bovine, rat and human airway smooth

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muscle cells (Karpova et al., 1997; Whelchel et al., 1997; Lew et al., 1999; Orsini et al., 1999). While activated ERK has been shown to stimulate phosphorylation or interact with various nuclear transcription factors, the precise downstream targets of ERK in airway smooth muscle cells are not known. However, as in other cell types (Albanese et al., 1995; Lavoie et al., 1996; Watanabe et al., 1996a), we have shown in bovine tracheal myocytes that ERK is an upstream activator of transcription from the cyclin D1 promoter (Ramakrishnan et al., 1998). Thus, the ERK pathway appears to constitute an important regulator of entry into the cell cycle and G1 progression in airway smooth muscle. Activation of MEK-1 in NIH 3T3 cells, though sufficient for both activation of ERK and expression of cyclin D1, is inadequate for three additional G1 cell cycle events, namely maximal phosphorylation of the retinoblastoma protein, degradation of the cyclin dependent kinase inhibitor p27, and expression of cyclin A (Cheng et al., 1998). Moreover, Ras, but not ERK is necessary for growth factor-mediated break down of p27 in IIC9 fibroblasts (Weber et al., 1997). Lastly, ectopic overexpression of cyclin D1 is inadequate for DNA synthesis (Quelle et al., 1993; Resnitzky et al., 1994). Jointly, these reports imply that the ERK/cyclin D1 pathway is insufficient for S phase traversal, and that Ras coordinates cell cycle progression by regulating output through both ERK-dependent and ERK-independent pathways. Further, these data demonstrate that measurements of cyclin D1 expression may reflect activity through some but not all mitogenic signaling pathways.

4. Role of PI 3-kinase As noted above, the PDGF receptor possesses nine phosphotyrosine domains, one of which is critical for receptor tyrosine kinase activity and eight that interact with various signal transduction intermediates (Claesson-Welsh, 1994). One such phosphotyrosine residue interacts with PI 3-kinase, a heterodimeric lipid kinase comprised of an 85 kDa regulatory subunit and a 110 kDa catalytic

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subunit. The catalytic subunit phosphorylates phosphatidylinositol at the D-3 hydroxyl of the inositol ring, forming the phosphatidylinositides phosphatidylinositol 3-phosphate, phosphatidylinositol 3,4-diphosphate and phosphatidylinositol 3,4,5-triphosphate. D-3 phosphorylated phosphoinositide products of PI 3-kinase may induce the translocation of additional intermediates to the cell membrane via their pleckstrin homology domains, thereby activating a diverse group of signaling pathways. Pleckstrin-containing signaling intermediates include protein kinase B (c-Akt), phosphoinositide-dependent kinase-1 and guanine nucleotide exchange factors, the upstream activators of GTPases (Lemmon et al., 1995). Phosphoinositide-dependent kinase-1, in turn, activates protein kinase B and the 70 kDa ribosomal S6 kinase (Alessi et al., 1998; Pullen et al., 1998). Activation of S6 kinase also appears to require FRAP, the mammalian homolog of the yeast TOR proteins (Brown et al., 1994). S6 kinase, through the phosphorylation of the 40S ribosomal protein, upregulates the translation of mRNAs containing an oligopyrimidine tract at their transcriptional start site, including ribosomal proteins and elongation factors (Jefferies et al., 1997). S6 kinase has been shown to increase the translation and protein abundance of cyclin D1 (Hashemolhosseini et al., 1998). The activation of PI 3-kinase and its requirement for airway smooth muscle proliferation has been well-studied. Growth factors stimulate PI 3kinase in human (Krymskaya et al., 1997) and bovine airway smooth muscle cells (Page et al., 2000). Wortmannin and LY294002, chemical inhibitors of PI 3-kinase, inhibit airway smooth muscle transcription from the cyclin D1 promoter, as well as cyclin D1 protein abundance (Page et al., 2000) and DNA synthesis (Scott et al., 1996; Krymskaya et al., 1999b; Page et al., 2000). Overexpression of the catalytic subdomain of PI 3-kinase in bovine tracheal myocytes is sufficient for transcription from the cyclin D1 promoter but does not induce ERK activation (Page et al., 2000), implying that PI 3-kinase signaling occurs independently of ERK. Likewise, in human airway smooth muscle cells, wortmannin and LY-294002 attenuated EGF-induced activation of PI 3-kinase

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but had no effect on ERK activation (Krymskaya et al., 1999b). An important downstream target of PI 3-kinase appears to be the 21 kDa Rho GTPase Rac1. The Rho family GTPases (Rho A-C, Rac1 and 2, and Cdc42), via their regulation of the actin cytoskeleton and interactions with multiple target proteins, influence cell cycle progression. In Swiss 3T3 fibroblasts, Rac1 is necessary for cell cycle progression (Olson et al., 1995; Lamarche et al., 1996). Ample evidence exists that Rac1 signaling pathway is regulated by PI 3-kinase. Generation of phosphatidylinositol triphosphate by PI 3-kinase is essential for receptor-mediated activation of Rac in mammalian cells (Hall, 1998). PI 3-kinase is necessary for PDGF activation of Rac- and Rhomediated rearrangements of the actin cytoskeleton (Wennstrom et al., 1994) and GTP-loading of Rac (Hawkins et al., 1995). Expression of an active PI 3-kinase induces membrane ruffling and focal complex formation which are dependent on endogenous Rac and Rho function (Reif et al., 1996). Finally, guanine nucleotide exchange factors, the upstream activators of GTPases, contain D-3 phosphorylated phosphoinositide-responsive pleckstrin homology domains (Lemmon et al., 1995). We have shown that Rac1 (Page et al., 1999b) and Cdc42, but not RhoA (Bauerfeld et al., 2001) are required for cyclin D1 expression in bovine tracheal myocytes. Overexpression of active Rac1 does not activate ERK in bovine tracheal myocytes, and Rac1-induced transcription from the cyclin D1 promoter is insensitive to the chemical MEK inhibitor PD98059 (Page et al., 1999b), suggesting that Rac1-mediated cell cycle progression, like that following activation of PI 3kinase, is independent of ERK activity. Further, active PI 3-kinase, Rac1 and Cdc42 each activate the cyclin D1 promoter via the cAMP response element binding protein (CREB)/activating transcription factor (ATF)-2 binding site, suggesting that these intermediates lie on the same signaling pathway. Another important downstream target of PI 3kinase appears to be phosphoinositide-dependent kinase-1, which in turn activates protein kinase B and 70 kDa ribosomal S6 kinase (Alessi et al., 1998; Pullen et al., 1998). Inhibition of S6 kinase

by rapamycin attenuates growth factor-induced DNA synthesis in both bovine (Scott et al., 1996) and human airway smooth muscle (Krymskaya et al., 1999b).

5. Role of reactive oxygen intermediates in airway smooth muscle mitogenesis Rac1 constitutes part of the NADPH oxidase complex that generates reactive oxygen species such as H2O2 (Abo et al., 1991, 1992). This enzyme, by donating an electron, catalyzes the reaction 2O2
/NADPH 0/2O2
/NADP
/H
. The superoxide produced is subsequently converted to H2O2. The human NADPH oxidase consists of at least seven components: two membrane spanning polypeptides, p22phox and gp91phox (which comprise cytochrome b 558); three cytoplasmic polypeptides, p47phox, p67phox and p40phox; rap1A; and Rac1, the last of which is required for oxidase activation. Intracellular reactive oxygen intermediates are increased following growth factor treatment of rat tracheal myocytes (Brar et al., 1999), bovine tracheal myocytes (Page et al., 1999b) and human bronchial smooth muscle cells (Brar et al., 2002). Accordingly, treatment with chemical antioxidants attenuates both growth factor induced cyclin D1 expression and DNA synthesis in these cells (Brar et al., 1999; Page et al., 1999b; Brar et al., 2002). Further, selective inhibition of p67phox attenuates PDGF-induced transcription from the cyclin D1 promoter in bovine airway smooth muscle (Page et al., 1999a,b), and inhibition of p22phox blocks cell proliferation in human airway smooth muscle cells (Brar et al., 2002). p22phox and p47phox were recently detected in human airway smooth muscle by immunoblotting (Thabut et al., 2002). Together, these data strongly suggest that an NADPH oxidase regulates growth factor-induced airway smooth muscle proliferation via the production of reactive oxygen intermediates. While it seems clear that reactive oxygen plays a role in airway smooth muscle mitogenesis, the relevant downstream effector(s) are not precisely known. In bovine cells, Rac1 induces transactivation of the cyclin D1 promoter CREB/ATF2

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binding site, and transactivation is attenuated by antioxidants (Page et al., 2000), suggesting that CREB family transcription factors are involved. In human cells, antioxidants block activation of the transcription factor nuclear factor (NF)-kB, suggesting that NF-kB is a downstream target of NADPH oxidase-generated reactive oxygen (Brar et al., 2002). Finally, a role for Janus kinase (JAK)/signal transducer and activator of transcription (STAT) signaling has recently been demonstrated in airway smooth muscle mitogenesis. PDGF treatment of human bronchial epithelial cells induces phosphorylation of JAK2 and STAT3, and chemical inhibition of JAK by AG490 attenuates PDGF-induced cyclin D1 protein expression and DNA synthesis (Simon et al., 2002). Further, expression of catalase blocks STAT3 phosphorylation, suggesting that JAK/ STAT activation is redox-dependent. It is, therefore, conceivable that JAK/STAT constitutes an alternative downstream target of reactive oxygen.

6. Inhibition of airway smooth muscle cell proliferation Persistent elevations of intracellular cyclic AMP (cAMP) concentration have long been known to inhibit airway smooth muscle growth (Panettieri et al., 1990; Lew et al., 1992; Tomlinson et al., 1995; Stewart et al., 1997; Musa et al., 1999). In bovine tracheal myocytes, pre-treatment with forskolin decreases cyclin D1 protein abundance and promoter activity while inducing the phosphorylation and DNA binding of CREB-1. Taken together, these data suggest that cAMP suppresses cyclin D1 gene expression via phosphorylation and transactivation of CREB. However, growth factor treatment also induces CREB1 activation in this system (Page et al., 2002), suggesting that other factors, for example, CREB phosphorylation, the presence of other CREB family transcription factors and co-activators, and/or alternative signaling pathways, may also play a critical role. cAMP fails to attenuate ERK activation in bovine (Hershenson et al., 1995) or rat (Whelchel et al., 1997) airway smooth muscle, suggesting that the effect of cAMP on growth is ERK-independent. Glucocorticoids

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also inhibit human airway smooth muscle cyclin D1 expression and DNA synthesis in an ERKindependent manner (Fernandes et al., 1999). On the other hand, PI 3-kinase activation has been shown to be cAMP-sensitive (Scott et al., 1996). Mitogen treatment of airway smooth muscle cells also induces activation of the two stressactivated MAP kinases, p38 and JNK (Shapiro et al., 1996; Pyne and Pyne, 1997; Page et al., 1999a; Conway et al., 2000), consistent with the idea that these MAP kinases, like ERKs, play a role in the growth regulation. However, based on the other types of signals that activate p38 and JNK (cellular stress and proinflammatory cytokines), it is conceivable that these kinases are involved in growth inhibition, rather than proliferation. The p38 MAP kinase family now consists of four isoforms. p38a was originally identified in lipopolysaccharide-stimulated mouse macrophages and was found to have substantial homology to the yeast high osmolarity glycerol kinase (Han et al., 1994; Derijard et al., 1995; Lin et al., 1995). Since then, three additional isoforms, b, g and d, have been cloned (Jiang et al., 1996, 1997; Stein et al., 1997; Wang et al., 1997). p38a, b and d are somewhat ubiquitously expressed, whereas g is primarily restricted to skeletal muscle (Wang et al., 1997). All isoforms are activated primarily by cellular stress and proinflammatory cytokines. p38a and b are inhibitable by pyridinyl imidazole compounds such as SB202190 or SB203580, whereas p38g and d are not (Jiang et al., 1997). The p38 MAP kinases are phosphorylated and activated by MAP kinase kinase (MKK)-3, MKK4, and MKK-6. MKK-6 appears to strongly activate all p38 isoforms, whereas MKK-3 preferentially activates p38a and d (Jiang et al., 1996; Raingeaud et al., 1996; Holland et al., 1997; Lu et al., 1997; Tournier et al., 1997; Enslen et al., 1998). MKK-4 appears to phosphorylate and activate both JNK1 and p38a (Derijard et al., 1995; Lin et al., 1995). A number of distinct MAP kinase kinase kinases have been found to activate MKK-3 and MKK-6, including MAP kinase/ ERK kinase kinase (MEKK)-1 (Xu et al., 1996; Cuenda and Dorow, 1998), mixed lineage kinase (MLK)-2 (Cuenda and Dorow, 1998) and MLK-3 (Tibbles et al., 1996), MAP three kinase (MTK)-1

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(Takekawa et al., 1997), apoptosis signal-regulating kinase (ASH) (Ichijo et al., 1997) and TAK-1, a potential mediator of TGF-b signaling (Yamaguchi et al., 1995). We have recently obtained data suggesting that selective activation of p38 inhibits airway smooth muscle cell cycle progression (Page et al., 2001), as it does in CCL39 hamster lung fibroblasts (Lavoie et al., 1996). Selective inhibition of p38 by pyridinyl imidazole compounds or dominant-negative forms of MKK3 or MKK6 each increased transcription from the cyclin D1 promoter and cyclin D1 protein abundance. Further, overexpression of constitutively active mutants of MKK3 or MKK6 each attenuated both basal and PDGFmediated cyclin D1 promoter activity. Paradoxically, p38 may be activated by Ras and Rac1 (Minden et al., 1995; Bagrodia et al., 1995; Zhang et al., 1995; Page et al., 1999a), implying that GTPases, like growth factors, may concurrently stimulate positive and negative growth regulatory pathways, conceivably as protection against inordinate cell proliferation.

7. Potential role of protein kinase C (PKC) isoforms Protein kinase C (PKC) is a superfamily including three types of isoenzymes. The conventional isoforms (a, b1, b2 and g) are activated by calcium, phorbol esters and phosphatidylserine, whereas the novel isoforms (d, o, i, u and m) are calciuminsensitive and activated by phorbol esters and phosphatidylserine. The atypical isoforms (z, t/l) are calcium and phorbol ester-insensitive and activated by phosphatidylserine. PKC a, b1, b2, d, o, and z, but not g or i, are expressed in bovine tracheal myocytes (Webb et al., 1997), whereas PKC a, b1, b2, d, o, u, i, z, t and m have each been identified in human tracheal myocytes (Carlin et al., 1999). Conventional and novel PKCs may be activated in vivo by diacylglycerol that is formed from phospholipids upon receptor-mediated activation of phospholipases. As noted above, ligand binding of the PDGF receptor leads to the phosphorylation of tyrosine residues that serve as

docking sites for SH2-containing signal transduction molecules, including phospholipase C-g. Different PKC isoforms may have distinct roles in the regulation of cell proliferation. PKCz activity increases in proliferating human airway smooth muscle (Carlin et al., 1999). Enhanced activation of PKCb1 and decreased activation of PKCd have been noted in hyperproliferative airway smooth muscle cells derived from hyperresponsive Fischer strain rats, compared with those isolated from control Lewis rats (Zacour and Martin, 2000). Consistent with the association of decreased PKCd activation and excess airway smooth muscle proliferation, we have found in bovine tracheal myocytes that overexpression of a dominant-negative PKCd and pre-treatment with a PKCd-specific pseudosubstrate peptide increase cyclin D1 promoter activity and cyclin D1 protein abundance, respectively (Page et al., 2002). Conversely, overexpression of the active catalytic subunit of PKCd attenuated PDGF-mediated transcription from the cyclin D1 promoter. We examined the transcriptional regulation of cyclin D1, focusing on the effects of PKCd (Page et al., 2002). We found that the /57 to /52 CREB/ ATF2 site functions as a basal level and PDGF enhancer, whereas the /39 to /30 NF-kB site functions as a basal level suppressor. Further, PDGF and PKCd responsiveness of the cyclin D1 promoter was maintained following 5? deletion to the Ets-containing /22 minimal promoter. Finally, using electrophoretic mobility gel shift and reporter assays, we determined that PKCd inhibits CRE/ATF2 binding and transactivation, activates NF-kB binding and transactivation, and attenuates Ets transactivation. These data suggest that PKCd attenuates cyclin D1 promoter activity via the regulation of three distinct cis -acting regulatory elements. The observation that the cyclin D1 promoter NF-kB site functions as a basal level suppressor in bovine tracheal myocytes may seem inconsistent with the aforementioned finding that IkB activation is required for human airway smooth muscle mitogenesis (Brar et al., 2002). However, NF-kB functions as a negative regulator of cyclin D1 in other systems (Amanatullah et al., 2000; Nakamura et al., 2002).

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8. Summary In recent years, the signaling pathways regulating airway smooth muscle growth have been elucidated. Although the substances mitogenic for airway smooth muscle may vary across species lines, the signal transduction mechanisms linking receptor ligation with DNA synthesis appear to be highly conserved. For example, the ERK and PI 3kinase signaling pathways appear to constitute the major paths required for cell proliferation in both human (Krymskaya et al., 1997; Orsini et al., 1999) and bovine airway smooth muscle cells (Scott et al., 1996; Karpova et al., 1997; Page et al., 2000). The generation of reactive oxygen also appears to be necessary for mitogenesis in rat, bovine and human cells (Brar et al., 1999; Page et al., 1999b; Brar et al., 2002; Simon et al., 2002). Elucidation of the signal transduction and cell cycle mechanisms regulating airway smooth muscle growth may provide insight into similar mechanisms that occur in the airways of patients with the chronic airways diseases asthma, bronchopulmonary dysplasia and cystic fibrosis, and lead to therapeutic interventions.

Acknowledgements These studies were supported by National Institutes of Health Grants HL54685, HL56399, HL63314 and grants from the Cystic Fibrosis Foundation.

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