VCAM-1 Activates Phosphatidylinositol 3-Kinase and Induces p120 Cbl Phosphorylation in Human Airway Smooth Muscle Cells This information is current as of September 26, 2021. Aili L. Lazaar, Vera P. Krymskaya and Susan K. P. Das J Immunol 2001; 166:155-161; ; doi: 10.4049/jimmunol.166.1.155 http://www.jimmunol.org/content/166/1/155 Downloaded from

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2001 by The American Association of Immunologists All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. VCAM-1 Activates Phosphatidylinositol 3-Kinase and Induces p120Cbl Phosphorylation in Human Airway Smooth Muscle Cells 1

Aili L. Lazaar,2*† Vera P. Krymskaya,* and Susan K. P. Das*

VCAM-1 is a member of the Ig superfamily of receptors the expression of which is up-regulated on human airway smooth muscle (ASM) cells following stimulation with inflammatory mediators. The function of these receptors in adhesion is well known, but there is growing recognition that they also possess “outside-in” signaling functions, such as cytoskeletal reorganization, calcium mobilization, and cytokine release. The present study examined the activation of extracellular signal-regulated kinase and phos- phatidylinositol 3-kinase (PI3K) in ASM cells following VCAM-1 engagement. VCAM-1 ligation activated extracellular signal- regulated kinase 2 and resulted in increased expression of cyclin D1, yet there was neither p27kip1 degradation nor an increase in smooth muscle cell DNA synthesis. VCAM-1 ligation, however, augmented the proliferative response to submitogenic concentra-

tions of epidermal growth factor. VCAM-1 engagement also stimulated a rapid increase in PI3K activity. This was associated with Downloaded from phosphorylation of the adapter protein p120Cbl and an increase in Cbl-associated PI3K activity. These studies suggest that VCAM-1 is linked to multiple signaling pathways in human ASM cells and may function to augment growth factor-induced responses. The Journal of Immunology, 2001, 166: 155–161.

irway or vascular remodeling, in part due to smooth also possess “outside-in” signaling functions (reviewed in Ref. 3). muscle cell hypertrophy and hyperplasia, is a character- Several reports suggest that cross-linking ICAM-1 induces secre- http://www.jimmunol.org/ A istic feature of patients with asthma and atherosclerosis, tion of chemokines and cytokines (4, 5), calcium mobilization (6), although the cellular processes stimulating muscle growth are not and expression of MHC class II (7). Activation of p60src and p53/ known. Smooth muscle cell activation is regulated by cell-cell in- 56lyn, along with transient inhibition of cdc2 kinase activity, has teractions through cell adhesion molecules, adherence to extracel- also been demonstrated following ICAM-1 engagement (7–9). lular matrix proteins, and exposure to soluble cytokines or growth ICAM-3 engagement stimulates a number of cellular functions, factors. Evidence from our laboratory suggests that T lymphocyte ␤ ␤ including increased 1 and 2 integrin function (10), chemokine adhesion via cell adhesion molecules can induce smooth muscle secretion (11), and calcium mobilization as well as phosphoryla- cell DNA synthesis (1). Leukocyte-smooth muscle cell interactions tion of multiple src family kinases and other unidentified proteins therefore may play an important role in triggering smooth muscle (12, 13). In contrast to the many studies of ICAM-1 and ICAM-3 by guest on September 26, 2021 cell growth in inflammatory disease. signaling, little is known about the role of VCAM-1 in cellular VCAM-1 and ICAM-1 are members of the Ig supergene family activation, although two recent studies suggest that VCAM-1 en- (IgSF)3 of receptors. Interactions of these receptors with their re- gagement leads to cytoskeletal rearrangement, myoinositol pro- spective ligands, very late Ag-4 and LFA-1, are critical for the duction, and calcium mobilization (14, 15). recruitment of eosinophils and T lymphocytes into the airway and The lipid kinase phosphatidylinositol 3-kinase (PI3K) is com- for the development of airway hyperresponsiveness in animal posed of an 85-kDa regulatory subunit and a 110-kDa catalytic models of asthma. We have previously demonstrated that subunit. Activation of this enzyme has been shown to be critical VCAM-1 and ICAM-1 are up-regulated on airway smooth muscle (ASM) cells after treatment with inflammatory mediators or after for growth factor-induced proliferation in smooth muscle cells coincubation with activated T cells (1, 2). Although it has long (16–18). There are several pathways by which PI3K becomes ac- been believed that cell adhesion receptors in the IgSF acted only in tivated following association with cell surface receptors, including an adhesive role, there is growing recognition that these receptors recognition of the YXXM motif by Src homology (SH) 2 domains of the p85 subunit (19) and interactions with proline-rich regions via SH3 domains. Recent studies investigating the role of PI3K in *Pulmonary, Allergy and Critical Care Division, Department of Medicine, University CD28-mediated integrin activation have revealed that impaired ty- † of Pennsylvania School of Medicine, Philadelphia, PA 19104; and Wistar Institute, rosine phosphorylation of the adapter protein p120Cbl results in Philadelphia, PA 19104 decreased association of CD28 with PI3K and therefore decreased Received for publication June 16, 2000. Accepted for publication September 29, 2000. integrin activation (20). Cbl is tyrosine phosphorylated in response The costs of publication of this article were defrayed in part by the payment of page to stimulation via numerous receptors, including immune recep- charges. This article must therefore be hereby marked advertisement in accordance tors, growth factors, integrins, and cytokines (reviewed in Refs. 21 with 18 U.S.C. Section 1734 solely to indicate this fact. and 22). Phosphorylated Cbl associates with a number of SH2- and 1 This work was supported by National Institutes of Health Grant HL-03202 and by SH3-containing proteins, including fyn, syk, Crk family proteins, the McCabe Fund of the University of Pennsylvania. Grb2, Shc, and p85 PI3K (reviewed in Refs. 21 and 22). Cbl ex- 2 Address correspondence and reprint requests to Dr. Aili Lazaar, Pulmonary, Allergy and Critical Care Division, University of Pennsylvania School of Medicine, 852 BRB II/III, pression is not restricted to hematopoietic cells, although the func- 421 Curie Boulevard, Philadelphia, PA 19104. E-mail address: [email protected] tion of Cbl in nonlymphoid cells has not been extensively studied, 3 Abbreviations used in this paper: IgSF, Ig supergene family; ASM, airway smooth and there are data to suggest that Cbl plays a role in PI3K activa- muscle; PI3K, phosphatidylinositol 3-kinase; SH, Src homology; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; EGF, epidermal tion following stimulation with growth factors in NIH 3T3 fibro- growth factor; MFI, mean fluorescence intensity. blasts and A549 epithelial cells (23, 24).

Copyright © 2001 by The American Association of Immunologists 0022-1767/01/$02.00 156 VCAM-1 SIGNALING IN AIRWAY SMOOTH MUSCLE

The interaction between activated T cells and smooth muscle LiCl and 0.2 mM orthovanadate; and twice in 10 mM Tris (pH 7.5), 100 cells via cell adhesion molecules induces critical changes in mM NaCl, 1 mM EDTA, and 0.2 mM orthovanadate. Immunoprecipitates were incubated for 10 min at room temperature in buffer containing 4 mM smooth muscle cell phenotype and synthetic function. We hypoth- ␮ ␮ ␥ 32 MgCl2,50 M ATP, 30 Ci [ - P]ATP, and 0.2 mg/ml sonicated phos- esized that ligation of VCAM-1 on ASM cells would activate sig- phatidylinositol in EGTA. Reactions were stopped by adding 20 ␮lof6N naling pathways that play an important role in smooth muscle cell HCl and extracted with 160 ␮l of chloroform-methanol (1:1). Lipids were responses to inflammation. In this report, we demonstrate activa- separated on oxalate-coated TLC plates using a chloroform-methanol-wa- tion of extracellular signal-regulated kinase (ERK) 2 and PI3K ter-ammonium hydroxide (60:40:11:3.2) solvent system. The lipids were visualized by autoradiography. upon VCAM-1 cross-linking. Our data indicate that VCAM-1 in- duces transient activation of mitogen-activated protein kinase Immunoprecipitation and immunoblotting (MAPK) and a more prolonged activation of PI3K but does not For analysis of cyclin D1 and p27kip1 expression, cells were scraped into induce ASM DNA synthesis. In addition, we demonstrate tyrosine cold PBS; briefly pelleted; and lysed in 300 ␮l of buffer containing 50 mM phosphorylation of p120Cbl following VCAM-1 engagement and HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, ␤ its association with the p85 subunit of PI3K. These data suggest 0.1 mM sodium orthovanadate, 0.5 mM PMSF, 10 mM -glycerophos- phate, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, and 0.1% Triton X-100. that VCAM-1 receptor engagement provides important signals Cells were lysed on ice for 10 min and then sonicated at 2-s intervals for regulating cellular activation. The study of cell adhesion molecule a total of 10 s. Cell lysates were cleared by centrifugation at 13,000 rpm for function on smooth muscle cells, therefore, can serve to emphasize 15 min. Protein concentrations were determined using the bicinchoninic their role not only in cell-cell interactions but also in the perpet- acid kit (Pierce, Rockford, IL). For analysis of Cbl, cells were lysed in uation of inflammation. buffer containing 150 mM NaCl, 50 mM Tris, 1 mM EDTA, 1 mM sodium orthovanadate, 10 mM NaF, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, 1% Triton X-100, and 0.1% deoxycholate. Equal amounts of protein were im- Downloaded from Materials and Methods munoprecipitated with anti-Cbl and protein A-Sepharose-conjugated Abs and reagents beads. Proteins were separated by electrophoresis on an 8% (Cbl) or a 10% (cyclin D1 and p27kip1) gel and transferred to polyvinylidene difluoride. In Murine anti-human VCAM-1 Ab 4B9 (IgG1) (25) was a kind gift from some instances, membranes were blocked with TNB (30 mM Tris (pH 7.6), John Harlan (University of Washington, Seattle, WA) and Timothy Carlos 75 mM NaCl, and 3% BSA) and incubated with polyclonal rabbit Ab for (University of Pittsburgh, Pittsburgh, PA). Rabbit polyclonal Abs specific 1 h, and then washed and incubated with 0.5 ␮Ci of 125I-labeled protein A for ERK-2, cyclin D1, p27kip1, and Cbl were purchased from Santa Cruz (NEN, Boston, MA) in TNB for 30 min at room temperature. The mem- http://www.jimmunol.org/ Biotechnology (Santa Cruz, CA); monoclonal antiphosphotyrosine 4G10 branes were extensively washed and proteins were visualized by and anti-p85 PI3K Abs were purchased from Upstate Biotechnology (Lake autoradiography. Placid, NY); and anti-phospho-MAPK Abs were purchased from New En- gland Biolabs (Beverly, MA). Wortmannin was purchased from Sigma (St. Proliferation assays Louis, MO); LY294002 was purchased from Calbiochem (La Jolla, CA). Proliferation of growth-arrested human ASM cells in response to growth Human ASM cell culture factors or Ab stimulation was assessed as previously described (26). Human ASM cells isolated from the trachealis muscle of transplant donors Results were maintained in Ham’s F12 medium (Life Technologies, Grand Island, TNF-␣ and IL-4 synergize to increase ASM expression of NY) supplemented with 10% FBS (HyClone, Logan, UT) and 2 mM glu- tamine, 100 U/ml penicillin, and 100 ␮g/ml streptomycin (all from Sigma). VCAM-1 by guest on September 26, 2021 These cells retain smooth muscle-specific actin staining and responsiveness We have previously demonstrated that VCAM-1 is expressed on to contractile agonists, as previously described by Panettieri et al. (26). ␣ Confluent ASM cells were growth arrested in serum-free Ham’s F12 con- ASM and is up-regulated by TNF- (1). Reports in vascular taining 0.1% BSA for 48 h before experiments. In these studies, only third- smooth muscle suggest that IL-4 can synergize with TNF-␣ to and fourth-passage cells were used. further increase VCAM-1 expression (27). Human ASM cells were ␣ ␣ ϩ ERK in vitro kinase assay stimulated with TNF- , IL-4, or TNF- IL-4 for 24 h. The cells were stained with an isotype-matched control Ab or with an Ab Cells were stimulated by the addition of anti-VCAM-1 Ab or epidermal specific for VCAM-1 and analyzed by flow cytometry. TNF-␣ growth factor (EGF) for various times. Cells were lysed in buffer contain- ing 10 mM Tris (pH 7.6), 50 mM NaCl, 1 mM EGTA, 1 mM orthovana- induced a 3-fold increase in VCAM-1 expression (mean fluores- date, 30 mM sodium pyrophosphate, 5 mM benzamidine, 1 mM PMSF, 10 cence intensity (MFI), 25.9), whereas the combination of TNF-␣ ␮g/ml aprotinin, 1% Triton X-100, and 0.1% deoxycholate. Cell lysates and IL-4 induced a 16.5-fold increase (MFI, 125) compared with were clarified by centrifugation at 13,000 rpm for 10 min. Equal amounts the expression of VCAM-1 on cells incubated in media alone ␮ ␮ of protein were incubated with 5 l(1 g) of rabbit anti-ERK-2 Abs for 2 h (MFI, 7.6) (Fig. 1). IL-4 alone had no effect on expression of at 4°C. The immunocomplexes were precipitated with 20 ␮l of packed protein A-agarose (Life Technologies) for1hat4°C. The cells were VCAM-1 (data not shown). washed three times in lysis buffer and once in kinase buffer containing 10 mM Tris (pH 7.2), 100 mM NaCl, 5 mM benzamidine, and 1 mM or- VCAM-1 induces activation of ERK-2 in ASM cells thovanadate. The precipitated proteins were incubated in 30 ␮l of reaction The interaction between activated T cells and smooth muscle cells mix containing 30 mM HEPES (pH 8), 10 mM MgCl ,1mMDTT,5mM 2 via cell adhesion molecules induces critical changes in smooth benzamidine, 20 ␮M ATP, 2 ␮g myelin basic protein, and 10 ␮Ci [␥-32P]ATP. The reactions were allowed to proceed for 15 min at 30°C and muscle cell phenotype and synthetic function. To investigate the were stopped by adding 6 ␮lof6ϫ Laemmli buffer and boiling for 5 min. signaling events that may be mediated through adhesion receptors The proteins were resolved by SDS-PAGE on a 12% gel, which was dried on ASM, we first examined changes in tyrosine phosphorylation of and exposed to film. p42/p44 MAPK. The MAPK family of proteins is activated fol- PI3K assay lowing a wide variety of stimuli, and in ASM cells from some species, activation of the MAPK family appears to be necessary to Cells were stimulated by the addition of anti-VCAM-1 Ab or EGF for various times. Cells were lysed in buffer containing 20 mM Tris (pH 7.5), stimulate growth (28, 29). Growth-arrested ASM cells were treated

137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 0.2 mM orthovanadate, 10% with TNF/IL-4 for 24 h and then stimulated for various times with glycerol, 1 mM PMSF, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, and 1% anti-VCAM-1 or isotype-matched control Abs. We performed im- Nonidet P-40. Clarified cell lysates were incubated with anti-p85 (5 ␮g/ml) munoblot analysis of VCAM-1-stimulated ASM cells using an Ab or anti-Cbl (1:200) Ab overnight at 4°C. Immunocomplexes were added to 60 ␮l of protein A-Sepharose and incubated for2hat4°C. The immuno- specific for phospho-MAPK, which revealed a time-dependent in- precipitates were washed three times in PBS, 1% Nonidet P-40, and 0.2 crease in phosphorylated MAPK. Phosphorylation was maximal at mM orthovanadate; three times in 0.1 M Tris (pH 7.5) containing 0.5 M 10–15 min and diminished by 30 min (Fig. 2A). This effect was not The Journal of Immunology 157

VCAM-1 engagement leads to activation of PI3K activity. Growth-arrested cells were pretreated with TNF/IL-4 and then stimulated with anti-VCAM-1 Abs for the indicated times. Ty- rosine-phosphorylated proteins were immunoprecipitated with Abs specific for the p85 subunit of PI3K, and lipid kinase activity was measured. In a time-dependent manner, VCAM-1 Abs induced a rapid increase in kinase activity as early as 1 min, which was sustained over 60 min (Fig. 3A). This was significantly increased from the PI3K activity induced by cytokine treatment alone and was not seen using an isotype-matched control Ab. Stimulation of the cells with an anti-ICAM-1 Ab also did not result in activation of PI3K activity, suggesting that the effects of VCAM-1 engage- ment are specific (data not shown). Treatment with the specific PI3K inhibitor wortmannin or LY294002 markedly inhibited VCAM-1-induced activation of PI3K (Fig. 3B). These data suggest that VCAM-1 can induce a significant and specific increase in PI3K enzymatic activity. This increase in PI3K activity is less robust than that seen in ASM cells stimulated with EGF but fol-

lows a similar time course (33). Downloaded from FIGURE 1. ASM cells were cultured in serum-free media or stimulated with TNF (500 U/ml) or TNF ϩ IL-4 (10 ng/ml) for 24 h. Cells were VCAM-1 engagement induces tyrosine phosphorylation of Cbl stained with an isotype control Ab or mouse anti-human VCAM-1 Ab (10 ␮g/ml), followed by FITC-conjugated goat anti-mouse IgG. Flow cyto- It has been reported that integrin engagement stimulates tyrosine Cbl metric analysis was performed and analyzed using CellQuest software phosphorylation of the adapter protein p120 and its subsequent (Becton Dickinson, San Jose, CA). The data are representative of three association with the p85 subunit of PI3K (20, 34, 35). To inves- separate experiments. tigate the possibility that Cbl was phosphorylated following http://www.jimmunol.org/ VCAM-1 engagement, lysates of ASM cells were immunoprecipi- tated with Abs specific for Cbl and analyzed by immunoblotting seen in cells treated with control Ab (data not shown). The degree for phosphotyrosine. In resting cells and cells treated with cytokine of MAPK phosphorylation was significantly less than that ob- alone, there was no evidence of Cbl tyrosine phosphorylation (Fig. served following EGF stimulation (Fig. 2A). Previous studies from 4A). A gradual increase in phosphotyrosine was seen following our laboratory have demonstrated a rapid and prolonged activation engagement of VCAM-1 with Ab. No increase was seen following of MAPK in ASM cells stimulated with EGF (30, 31). stimulation with control Ab (data not shown). In contrast to the To correlate protein phosphorylation with enzymatic activity, cell lysates were immunoprecipitated with Abs to ERK-2, and an by guest on September 26, 2021 in vitro kinase assay was performed using myelin basic protein as a substrate. Compared with cells treated with cytokine alone, cells treated with anti-VCAM-1 displayed a rapid increase in ERK-2 kinase activity that corresponded to the time course of MAPK tyrosine phosphorylation (Fig. 2B). We repeated the kinase assay in the presence of PI3K inhibitors, LY294002, or wortmannin, and found no inhibition of VCAM-1-induced ERK-2 activity (data not shown). This suggests that ERK-2 activation is not dependent on the activation of PI3K in this system.

VCAM-1 induces activation of PI3K in ASM cells Previous studies have shown that activation of PI3K is important in modulating smooth muscle cell proliferation in response to growth factors (16, 18, 32). We tested the hypothesis that

FIGURE 3. A, Growth-arrested ASM cells were incubated in serum- free media and treated with TNF/IL-4 for 24 h. Cells were stimulated with vehicle (C), with anti-VCAM-1 Abs (10 ␮g/ml) for the indicated times, or with an isotype-matched control Ab (iso, 10 ␮g/ml) for 5 min (n ϭ 3). B, FIGURE 2. Growth-arrested ASM cells were incubated in serum-free Growth-arrested ASM cells were incubated in serum-free media (C) and media and treated with TNF/IL-4 for 24 h. Cells were stimulated with treated with TNF/IL-4 (cyto) for 24 h. Cells were stimulated with anti- anti-VCAM-1 Abs for the indicated times or with EGF (10 ng/ml) for 5 VCAM-1 Abs for 5 min in the absence (5Ј) or presence of LY294004 (LY, min. A, Equal amounts of protein were separated by SDS-PAGE on an 8% 10 ␮M) or wortmannin (WM, 100 nM) (n ϭ 2). Cell lysates were immu- gel and immunoblotted with an Ab recognizing the phosphorylated form of noprecipitated with anti-p85 Abs, and an in vitro assay of PI3K activity MAPK (n ϭ 2). B, Cell lysates were immunoprecipitated with polyclonal was performed as described. Lipids were separated on TLC plates and Abs specific for ERK-2, and an in vitro kinase assay was performed as visualized by autoradiography. Or, Origin; PIP, [32P]phosphatidylinositol described (n ϭ 3). monophosphate. 158 VCAM-1 SIGNALING IN AIRWAY SMOOTH MUSCLE

with Cbl. Lysates of ASM cells treated with cytokine alone or in combination with either anti-VCAM-1 or an isotype-matched con- trol Ab were prepared and immunoprecipitated with anti-Cbl Abs. PI3K activity was measured in the precipitates using an in vitro kinase assay. There was a low level of kinase activity associated with resting cells (Fig. 5A). Stimulation of the cells with anti- VCAM-1 resulted in an increase in Cbl-associated lipid kinase activity that compared with the coassociation of Cbl and PI3K FIGURE 4. Left, Growth-arrested ASM cells were incubated in serum- following EGF stimulation (Fig. 5A). No detectable increase in free media (C) and treated with TNF/IL-4 (cyto) for 24 h. Cells were kinase activity was seen in cells treated with the control Ab. stimulated with anti-VCAM-1 Abs for the indicated times. Cell lysates We then performed a time course of Cbl-associated PI3K ac- containing equal amounts of protein were immunoprecipitated with Abs tivity. Lysates of Ab-treated ASM cells were immunoprecipitated specific for Cbl. Proteins were separated by SDS-PAGE on an 8% gel and with anti-Cbl, and PI3K activity was determined. Lipid kinase ac- ϭ immunoblotted with 4G10 (PY, n 3). Blots were stripped and reprobed tivity was maximal at 2 min and decreased over 15 min (Fig. 5B). with anti-Cbl. Right, Growth-arrested ASM cells were incubated in serum- Again, no increase in kinase activity was seen in cells stimulated free media and stimulated with EGF (10 ng/ml) for the indicated times (n ϭ 2). Cbl immunoprecipitation and immunoblotting for phosphoty- with control Ab. The rapid increase in Cbl-associated PI3K activ- rosine were performed as described. ity paralleled the increase seen in total cytosolic lipid kinase ac- tivity (Fig. 3A), although it did not appear to be as sustained.

To determine whether Cbl and PI3K were physically associated, Downloaded from gradual increase in tyrosine phosphorylation of Cbl following we immunoprecipitated Cbl and immunoblotted for p85. We found VCAM-1 engagement, EGF induced a rapid and sustained increase that Cbl and the p85 subunit of PI3K were constitutively associ- (Fig. 4B). These data suggest that different mechanisms regulate ated in ASM cells (Fig. 5C). No increase in the degree of associ- Cbl phosphorylation following stimulation via growth factors or ated Cbl and p85 was detected following stimulation with adhesion receptors. anti-VCAM-1 Abs. http://www.jimmunol.org/ PI3K activity associates with Cbl following VCAM-1 cross- Effects of VCAM-1 ligation on smooth muscle cell proliferation linking In some cell types, expression of G1 phase cell cycle proteins We next performed coimmunoprecipitations to determine whether requires activation of PI3K. We tested whether VCAM-1 engage- VCAM-1 engagement resulted in the association of PI3K activity ment would result in up-regulated expression of cyclin D1, which may be important for growth factor-induced DNA synthesis (36). Growth-arrested, cytokine-treated ASM cells were stimulated with anti-VCAM-1 Abs for 4–24 h. Control cells were treated with EGF for 24 h. There was a low constitutive expression of cyclin D1 in serum-deprived and cytokine-treated cells that was up-reg- by guest on September 26, 2021 ulated by 4 h following VCAM-1 stimulation (Fig. 6A). Expres- sion remained elevated for up to 24 h. The increase in cyclin D1 expression following VCAM-1 ligation was less robust, however, than that induced by EGF. Levels of p27kip1 were unchanged fol- lowing VCAM-1 engagement but were dramatically decreased af- ter stimulation with EGF (Fig. 6B). Our data demonstrate some similarities in EGF- and VCAM-1- mediated signaling pathways known to be important for smooth

FIGURE 5. Growth-arrested ASM cells were incubated in serum-free media (C) and treated with TNF/IL-4 (cyto) for 24 h. A, Cells were stim- ulated with anti-VCAM-1 or isotype control Abs (10 ␮g/ml) for 20 min or FIGURE 6. A, Growth-arrested ASM cells were treated with TNF/IL-4 with EGF (10 ng/ml) for 10 min. B, Cells were stimulated with isotype for 24 h (cyto) and then stimulated with anti-VCAM-1 Abs (10 ␮g/ml) for control Ab for 10 min or with anti-VCAM-1 for the indicated times. Cell 1–22 h or with EGF alone (10 ng/ml) for 22 h. Equal amounts of protein lysates containing equal amounts of protein were immunoprecipitated with were separated by SDS-PAGE on a 10% gel and immunoblotted with an Abs specific for Cbl, and a lipid kinase assay was performed as described. Ab recognizing cyclin D1 (n ϭ 3). B, Growth-arrested, cytokine-treated Or, Origin; PIP, [32P]phosphatidylinositol monophosphate. C, Following cells were stimulated with anti-VCAM-1 Abs for 6, 12, and 24 h. Parallel stimulation with anti-VCAM-1 or an isotype-matched Ab, equal amounts cultures were treated with EGF alone for the same time periods. Equal of protein from cell lysates were immunoprecipitated (ip) with anti-Cbl amounts of protein were separated by SDS-PAGE on a 10% gel and im- Abs and immunoblotted (ib) with anti-p85. munoblotted with an Ab recognizing p27kip1 (n ϭ 2). The Journal of Immunology 159

proliferation in smooth muscle cells (30, 31, 33, 40). In addition, PI3K activation and increased expression of cyclin D1 appear to be both necessary and sufficient for growth factor-induced smooth muscle cell proliferation (33, 36); however, we were not able to demonstrate an increase in smooth muscle cell DNA synthesis fol- lowing VCAM-1 engagement alone. This may be due to the tran- sient nature of VCAM-1-induced MAPK activation or the less ro- bust activation of PI3K when compared with EGF. Sustained activation of MAPK is required for smooth muscle cell mitogen- esis in response to growth factors (31). One could postulate that MAPK and PI3K activation transduced through cell adhesion molecules such as VCAM-1 might serve to “prime” the cell for response to mitogenic signals, such as growth FIGURE 7. Growth-arrested ASM cells (C) were treated with TNF/IL-4 factors. For example, sustained activation of ERK and expression for 24 h (cyto) and then stimulated with anti-VCAM-1 Abs (VCAM, 10 of cyclin D1 in fibroblasts following stimulation with EGF are ␮g/ml). Where indicated, growth-arrested cells were stimulated with EGF dependent on integrin-mediated adhesion (41). Our data suggest at 1 (EGF1) or 10 (EGF10) ng/ml in the presence or absence of LY294002 that VCAM-1 engagement can augment smooth muscle cell pro- (LY, in ␮M) or wortmannin (WM, in nM). Incorporation of [3H]thymidine liferation in the presence of submitogenic concentrations of growth was assessed as described. The data are representative of six similar ex- factors, an effect that was completely abrogated by pharmacologic Downloaded from periments, two of which were performed using LY294002 and wortmam- inhibition of PI3K activity. Alternatively, VCAM-1 or other IgSF- Ͻ ء Ϯ min. Data are expressed as mean cpm SEM. , p 0.001 compared with mediated signals may be important for cellular processes other .p Ͻ 0.001 compared with EGF10 ,ءء ;EGF1 ϩ VCAM than proliferation. Our preliminary data suggest that VCAM-1 ligation induces activation of p70 ribosomal S6 kinase (our un- published results), an enzyme that is downstream of PI3K and is

muscle cell proliferation. We therefore examined the effects of critical for regulation of protein translation. As such, VCAM-1- http://www.jimmunol.org/ VCAM-1 cross-linking on ASM proliferation in comparison with mediated signals may be linked to the process of cellular hyper- EGF, a known smooth muscle cell mitogen. Growth-arrested, cy- trophy, rather than to proliferation. For example, agents that induce tokine-treated cells were stimulated with anti-VCAM-1 Abs or vascular smooth muscle hypertrophy, such as angiotensin II, have EGF, and incorporation of [3H]thymidine was measured. EGF in- also been shown to increase cyclin D1 expression in the absence of duced a dose-dependent increase in thymidine incorporation with p27kip1 degradation (42–44). 10 ng/ml causing an ϳ14-fold increase in thymidine incorporation We have demonstrated tyrosine phosphorylation of the protoon- (Fig. 7), which is consistent with our previous experience (30). In cogene p120Cbl following VCAM-1 cross-linking. Although Cbl contrast, VCAM-1 engagement had no effect on thymidine incor- tyrosine phosphorylation occurs in response to diverse stimuli, poration by ASM cells. However, combined stimulation with low such as binding of the B and T cell Ag receptors, growth factors, by guest on September 26, 2021 dose EGF (1 ng/ml) and anti-VCAM-1 Abs augmented the prolif- and cytokines, and integrin ligation (21, 22), this is the first dem- erative response observed with low dose EGF alone. This response onstration of Cbl phosphorylation in response to IgSF cross-link- was completely inhibited by the addition of either LY294002 or ing. The gradual onset of Cbl phosphorylation following VCAM-1 wortmannin (Fig. 7), suggesting that the synergy between the EGF engagement is consistent with other studies in macrophages fol- and VCAM-1 is mediated entirely through activation of PI3K. lowing integrin-mediated stimulation (34, 45), but it contrasts with the rapid and sustained tyrosine phosphorylation observed follow- Discussion ing EGF stimulation. These data suggest that while both IgSF- and In the airway, a variety of cell types reside in or infiltrate the EGF-mediated signaling stimulate tyrosine phosphorylation of Cbl inflamed submucosa that can potentially interact with smooth mus- in ASM cells, the mechanisms are likely to be different. cle cells and alter myocyte function. Our studies have shown that Interestingly, despite a time-dependent increase in Cbl-associ- adhesion of activated T lymphocytes can induce DNA synthesis in ated PI3K activity, we found a constitutive association between quiescent ASM cells (1). We have also recently demonstrated that Cbl and the p85 subunit of PI3K. This suggests that other proteins engagement of CD40, a member of the TNF receptor superfamily, might be recruited into the signaling complex, which would then results in increases in cytosolic calcium, activation of NF-␬B, and act directly to activate PI3K. In turn, Cbl phosphorylation, which augmentation of IL-6 secretion (37). Others have shown that ad- occurs more slowly than PI3K activation, may occur as part of a hesion of activated T lymphocytes induced expression of matrix feedback mechanism, possibly allowing for retention of PI3K metalloproteinases in vascular smooth muscle, via CD40-CD40L within the signaling complex. Further studies will be necessary to interactions (38). These studies highlight the fact that direct inter- elucidate these pathways. The physiologic significance of the Cbl- actions between T cells and smooth muscle cells via immune re- p85 complexes in smooth muscle cells, as in other cells, has yet to ceptors such as CD40 or adhesion receptors such as ICAM-1 or be fully determined. As postulated by Hartley and Corvera (46) VCAM-1 contribute to the modulation of the local milieu, result- and Ojaniemi et al. (34), these complexes may be important, not ing in smooth muscle cell activation. for mitogenic signaling, but rather for other responses such as In this study, we examined VCAM-1-associated signaling path- adhesion. ways in human ASM cells. We found that VCAM-1 ligation re- Another question that remains to be answered is how VCAM-1 sulted in a transient increase in p42/44 MAPK activity as well as engagement initiates activation of intracellular kinases, as the rel- a more sustained activation of PI3K activity. Activation of PI3K atively short cytoplasmic domain has no known intrinsic enzy- ␤ was not required for ERK2 enzymatic activity, in contrast to 1- matic activity, nor does it contain motifs such as SH2 or SH3 integrin-mediated activation of MAPK (39). In addition, we found domains that might facilitate recruitment of intracellular kinases that VCAM-1 induced expression of cyclin D1. Sustained activa- (47). However, since VCAM-1 engagement has been shown to tion of MAPK and PI3K are critical for growth factor-induced result in cytoskeletal rearrangement (15), it is possible that 160 VCAM-1 SIGNALING IN AIRWAY SMOOTH MUSCLE

VCAM-1 interacts with cytoskeletal proteins to assemble a func- 16. Krymskaya, V. P., R. Hoffman, A. Eszterhas, S. Kane, V. Ciocca, and tional signaling complex. For example, ICAM-1 ligation with Ab R. A. Panettieri, Jr. 1999. EGF activates ErbB-2 and stimulates phosphatidylino- sitol 3-kinase in human airway smooth muscle cells. Am J. Physiol. 276:L246. leads to phosphorylation of cortactin (8, 48), as well association 17. Scott, P. H., C. M. Belham, J. Al-Hafidh, E. R. Chilvers, A. J. Peacock, with ␣-actinin and ezrin (49, 50), while monocyte adhesion to G. W. Goulds, and R. Plevin. 1996. A regulatory role for cAMP in phosphati- dylinositol 3-kinase/p70 ribosomal S6 kinase-mediated DNA synthesis in plate- endothelial cells induces receptor clustering of ICAM-1 and let-derived-growth-factor-stimulated bovine airway smooth-muscle cells. Bio- VCAM-1 along with their colocalization with ezrin (51). chem. J. 318:965. Although our data are consistent with a role for Cbl in the ac- 18. Walker, T. R., S. M. Moore, M. F. Lawson, R. A. Panettieri, Jr., and E. R. Chilvers. 1998. Platelet-derived growth factor-BB and thrombin activate tivation of PI3K, the fact that only a small percentage of PI3K phosphoinositide 3-kinase and protein kinase B: role in mediating airway smooth enzymatic activity coassociates with Cbl suggests that there are muscle proliferation. Mol. Pharmacol. 54:1007. other phosphoproteins involved. Potential candidates include src 19. Songyang, Z., S. E. Shoelson, M. Chaudhuri, G. Gish, T. Pawson, W. G. Haser, lyn F. King, T. Roberts, S. Ratnofsky, R. J. Lechleider, et al. 1993. SH2 domains family kinases and Rho, a GTP binding protein. Rho and p53 recognize specific phosphopeptide sequences. Cell 72:767. activation have been noted following ICAM-1 cross-linking (7, 48, 20. Zell, T., C. S. Warden, A. S. H. Chan, M. E. Cook, C. L. Dell, S. W. Hunt III, fyn lck ␤ 52), while activation of p59 and p56 result from ICAM-3 and Y. Shimizu. 1998. Regulation of 1-integrin-mediated cell adhesion by the cross-linking in T cells (12). In addition, ligation of ICAM-1 in- Cbl adaptor protein. Curr. Biol. 8:814. 21. Smit, L., and J. Borst. 1997. The Cbl family of signal transduction molecules. duces activation of Rho (48), while inhibition of Rho inhibits IgSF Crit. Rev. Oncol. 8:359. receptor clustering and cytoskeletal reorganization (48, 51). Future 22. Lupher, M. L., Jr., N. Rao, M. J. Eck, and H. Band. 1999. The Cbl protoonco- studies will be necessary to elucidate the role of other kinases in protein: a negative regulator of immune receptor signal transduction. Immunol. Today 20:375. linking PI3K to a VCAM-1-mediated signaling pathway. 23. Meisner, H., and M. P. Czech. 1995. Coupling of the proto-oncogene product

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