PERSPECTIVE
Mitogen-Activated Signaling in Airway Smooth Muscle
A Central Role for Ras

Marc B. Hershenson and Mark K. Abe

Department of Pediatrics, University of Chicago, Chicago, Illinois

The first detailed report of airway structural changes in asthma was published over 75 years ago in the Archives of Internal Medicine by Huber and Koessler (1). In this report, the authors demonstrated that patients with fatal asthma had substantial thickening of the airway smooth-muscle layer. Half a century later, this early observation was confirmed by a number of investigators (2). Carroll and colleagues (10) found that in both fatal and nonfatal cases of asthma, the airway smooth-muscle area of the larger membranous bronchioles was significantly greater than in control cases, suggesting that increases in airway smooth-muscle mass are not exclusive to fatal asthma.

Few studies have examined the local mechanism of muscular thickening (i.e., hyperplasia versus hypertrophy). Heard and Hossain (4) found a threefold increase in cell number in the bronchi of asthmatic patients, suggesting that smooth-muscle hyperplasia is present in the airways of patients with fatal asthma. Although the realization that objects must be counted directly in three-dimensional space to obtain unbiased estimates cast doubt on the validity of this early work (11), Ebina and colleagues (9) examined the airways of patients with fatal asthma using a combination of the dissector method with a serial sectioning technique. Two subgroups of asthmatic airways were found: in Type I, smooth-muscle hyperplasia was responsible for central airway smooth-muscle thickening, whereas in Type II, cellular hypertrophy was evident over the entire length of the airway. Finally, excess airway smooth-muscle DNA synthesis has been demonstrated in two animal models of airways disease, hyperoxic exposure, and antigen challenge (12).

The above data, which strongly suggest that excessive smooth-muscle proliferation is present in the airways of patients with asthma, highlight the need for a precise understanding of the events involved in airway smooth-muscle mitogenesis. To that end, numerous investigators have developed cell culture systems adopting tracheal and bronchial myocytes from different species. A large number of smooth-muscle mitogens have been identified, some of which are species specific in their effect. For example, histamine is mitogenic for human airway smooth muscle (15, 16), but does not induce proliferation in bovine cells (17). Nevertheless, a growing body of literature suggests that common signal transduction pathways regulate airway smooth-muscle cell cycle entry across species lines. Indeed, the signaling pathways regulating airway smooth-muscle proliferation may not be substantially different from those regulating the growth of other mesenchymal cells such as fibroblasts. Perhaps this is to be expected, as many aspects of mitogen-activated protein kinase (MAPK) cascades, guanine triphosphatase (GTPase) signaling pathways, and cell-cycle regulation are highly conserved in eukaryotic species, including mammals, Drosophila, nematodes, and yeast (18).

A major signal transduction pathway activated by growth factors is the extracellular signal-regulated kinase (ERK) pathway. ERKs (p44^ERK1 and p42^ERK2) are cytosolic serine/threonine kinases of the MAPK superfamily. ERKs participate in the transduction of growth and differentiation-promoting signals to the nucleus. Studies in airway smooth-muscle cells using selective overexpression of either dominant-negative or constitutively active forms of Ras, Raf-1, and MAPK/ERK kinase-1 (MEK1) suggest that, as in other mesenchymal cells, these signaling intermediates constitute the major route toward ERK activation (25). These data, combined with additional studies using selective chemical inhibitors of MEK1 (27), suggest that signaling through the ERK pathway is required for the airway smooth-muscle cell cycle progression.

However, activation of ERK and expression of cyclin D₁, a downstream affector of ERK signaling (31), may not be sufficient for cell-cycle entry. In NIH3T3 cells, constitutive activation of MEK1, although sufficient to induce cyclin D₁ protein accumulation, is insufficient for maximal phosphorylation of retinoblastoma protein, degradation of the cyclin-dependent kinase inhibitor p27, and cyclin A expressionadditional key events required for the G₁-to- S-phase transition (32). Moreover, in IIC9 fibroblasts, Ras, but not ERK, is required for growth-factor-induced degradation of p27 (33). Together, these data suggest that Ras coordinates cell-cycle progression by regulating signaling through both ERK-dependent and -independent signaling pathways.

In this month's issue of the Journal, Ammit and colleagues examined the requirement of Ras isoforms for human airway smooth-muscle DNA synthesis (34). Microinjection of the anti-pan Ras neutralizing antibody, Y13-259, attenuated fractional labeling with bromodeoxyuridine, demonstrating that in human airway smooth muscle Ras isoforms are required for cell-cycle traversal. This study firmly establishes the 21 kD GTPase Ras as a key regulator of growth in airway smooth-muscle cells. Yet the question remains: What are the additional downstream targets of Ras (other than ERK) which regulate airway smooth-muscle proliferation?

Ras has been noted to interact with a number of candidate effectors besides Raf-1, including phosphotidylinositol (PI) 3-kinase (35). In airway smooth-muscle cells, chemical inhibitors of PI 3-kinase inhibit DNA synthesis (36, 37), consistent with the notion that Ras regulates cell growth by the stimulation of both ERK-dependent and -independent pathways.

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. Translocation of protein kinase B (c-Akt) and phosphoinositide-dependent kinase-1 activates ribosomal S6 kinase (38, 39), a modulator of translation that also appears as a requirement for DNA synthesis in bovine tracheal myocytes (36).

Phosphoinositide products of PI 3-kinase may also induce the translocation of guanine nucleotide exchange factors, the upstream activators of GTPases (40). The Rho family GTPases (Rho A-C, Rac1 and Rac2, and Cdc42), through their regulation of the actin cytoskeleton and interactions with multiple-target proteins, may influence such diverse cellular activities as morphologic change, motility, adhesion, cell-cycle progression, transformation, and apoptosis. Rac1 plays an essential role for cell-cycle progression through G₁ in Swiss 3T3 fibroblasts (41, 42) and has recently been shown to function as an upstream activator of cyclin D₁ expression in tracheal myocytes (43). Rac1 forms part of the nicotinamide adenine dinucleotide phospate (NADPH) oxidase complex that generates reactive oxygen species such as H₂O₂. In both bovine (43) and rat (44) smooth-muscle cells, growth factor treatment stimulates a substantial increase in intracellular reactive oxygen species, and antioxidant treatment attenuates growth-factor-induced DNA synthesis. Finally, overexpression of an N-terminal fragment of p67^phox, a component of NADPH oxidase that interacts with Rac1, attenuates platelet-derived growth factor (PDGF)-induced cyclin D₁ promoter activity in bovine cells (43). Together, these data suggest that the generation of reactive oxygen species by Rac1/NADPH oxidase is required for cell-cycle progression in airway smooth-muscle cells. Further, they imply a model by which two Ras-dependent signaling pathways, the ERK and PI 3-kinase pathways, positively regulate cyclin D₁ and smooth-muscle growth.

It is also conceivable that additional signaling pathways negatively regulate airway smooth-muscle cell growth. Growth factor treatment of airway smooth-muscle cells induces modest activation of the two stress-activated MAPK families, the Jun amino-terminal kinases (JNKs) and p38 (25, 45). This is consistent with the notion that these intermediates, like ERKs, play a role in the growth control. However, based on the types of signals that activate JNK and p38, it is likely that these pathways are involved in growth inhibition rather than mitogenesis. Indeed, p38 attenuates transcription from the cyclin D₁ promoter in both CCL39 hamster lung fibroblasts (46) and bovine airway smooth-muscle cells (47). Paradoxically, these pathways may be stimulated by Ras and Rac1 (25, 48), suggesting that GTPases may simultaneously activate positive and negative growth regulatory pathways, perhaps as a safeguard against excessive growth.

If, as noted above, Ras activates multiple signaling pathways leading to both airway smooth-muscle cell-cycle progression and arrest, what factors determine the ultimate outcome of GTPase activation? An easy answer might be that the outcome is dependent on the relative amplitude or duration of the two signals. However, it has become increasingly evident that signaling intermediates may be used in various combinations to achieve distinct biologic responses, and that additional strategies must be employed to obtain specific signaling outcomes. One such strategy is the use of scaffolding proteins. Scaffolding proteins may bind several signaling molecules to create multienzyme complexes, or conversely, may sequester signaling proteins so they do not interact with other proteins. Scaffolding can also control subcellular localization. One example of a mammalian scaffolding protein is JNK-interacting protein-1 (JIP1). JIP1 selectively interacts with JNK and its upstream activators, MAPK kinase-7 and the mixed lineage protein kinase (51). A putative scaffold protein for the ERK MAPK module has also been identified (52). Two recently described MAPKs, ERK5 and ERK7, contain C-terminal tails, which may serve some sort of scaffolding function (53, 54). Other scaffolding proteins include the RACKs (receptors for activated protein kinase C) (55) and AKAPs (A kinase anchoring proteins) (56). Finally, caveolae, vesicular invaginations in the plasma membrane, which are believed to concentrate many signaling intermediates (including ERKs) (57), also serve as scaffolds for signaling complexes.

Adding to the confusion are the potential effects of "phenotype switching" on airway smooth-muscle signaling and cell-cycle traversal. Recent studies suggest that prolonged serum starvation at cell confluence can dramatically increase contractile protein expression in a subset of cultured canine airway smooth-muscle cells (58). However, although "contractile myocytes" do not lose the ability to proliferate upon re-exposure to serum, as suggested by their incorporation of bromodeoxyuridine, fractional labeling is decreased (A. Halayko, M. Hershenson, and J. Solway, unpublished data). The mechanisms underlying this apparent reduction in growth potential are unclear. Studies of cultured vascular smooth-muscle cells grown on Matrigel, which manifest a contractile phenotype, suggest that reduced levels of both ERK activation and expression may play a role (59). Preliminary studies also suggest that PI 3-kinase and S6 kinase are required for the translation of contractile protein messenger RNAs (mRNAs), whereas activation of the ERK pathway attenuates transcription from contractile protein gene promoters (A. Halayko, M. Hershenson, and J. Solway, unpublished data). These studies are consistent with the notion that signaling molecules may participate in the induction of diverse signaling outcomes.

The work of Ammit and colleagues (34) highlights the importance of Ras for airway smooth-muscle growth. Insight gained from studies such as this may shed light on parallel mechanisms that may operate in asthma and eventually lead to therapeutic interventions against airway remodeling. However, until we understand how Ras and other signaling molecules are linked to specific cellular events, we cannot hope to treat asthma by the modulation of airway smooth-muscle growth.

	Footnotes

Abbreviations: extracellular signal-regulated kinase, ERK; guanine triphosphatase, GTPase; Jun amino-terminal kinases, JNKs; mitogen-activated protein kinase, MAPK; nicotinamide adenine dinucleotide phosphate, NADPH; phosphotidylinositol, PI.

(Received in original form August 12, 1999).

Acknowledgments: Supported by National Institutes of Health Grants HL03867 (M.K.A.), HL54685, HL56399, HL63314 and the Blowitz-Ridgeway Foundation (M.B.H.).

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