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The Journal of Immunology, 2001, 166: 155-161.
Copyright © 2001 by The American Association of Immunologists

VCAM-1 Activates Phosphatidylinositol 3-Kinase and Induces p120Cbl Phosphorylation in Human Airway Smooth Muscle Cells 1

Aili L. Lazaar2,*,{dagger}, Vera P. Krymskaya* and Susan K. P. Das*

* Pulmonary, Allergy and Critical Care Division, Department of Medicine, University of Pennsylvania School of Medicine, Philadelphia, PA 19104; and {dagger} Wistar Institute, Philadelphia, PA 19104


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
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 phosphatidylinositol 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 concentrations of epidermal growth factor. VCAM-1 engagement also stimulated a rapid increase in PI3K activity. This was associated with 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.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Airway or vascular remodeling, in part due to smooth muscle cell hypertrophy and hyperplasia, is a characteristic feature of patients with asthma and atherosclerosis, although the cellular processes stimulating muscle growth are not known. Smooth muscle cell activation is regulated by cell-cell interactions through cell adhesion molecules, adherence to extracellular matrix proteins, and exposure to soluble cytokines or growth factors. Evidence from our laboratory suggests that T lymphocyte adhesion via cell adhesion molecules can induce smooth muscle cell DNA synthesis (1). Leukocyte-smooth muscle cell interactions therefore may play an important role in triggering smooth muscle cell growth in inflammatory disease.

VCAM-1 and ICAM-1 are members of the Ig supergene family (IgSF)3 of receptors. Interactions of these receptors with their respective ligands, very late Ag-4 and LFA-1, are critical for the recruitment of eosinophils and T lymphocytes into the airway and for the development of airway hyperresponsiveness in animal models of asthma. We have previously demonstrated that VCAM-1 and ICAM-1 are up-regulated on airway smooth muscle (ASM) cells after treatment with inflammatory mediators or after coincubation with activated T cells (1, 2). Although it has long been believed that cell adhesion receptors in the IgSF acted only in an adhesive role, there is growing recognition that these receptors also possess "outside-in" signaling functions (reviewed in Ref. 3). Several reports suggest that cross-linking ICAM-1 induces secretion of chemokines and cytokines (4, 5), calcium mobilization (6), and expression of MHC class II (7). Activation of p60src and p53/56lyn, along with transient inhibition of cdc2 kinase activity, has also been demonstrated following ICAM-1 engagement (7, 8, 9).

ICAM-3 engagement stimulates a number of cellular functions, including increased {beta}1 and {beta}2 integrin function (10), chemokine secretion (11), and calcium mobilization as well as phosphorylation of multiple src family kinases and other unidentified proteins (12, 13). In contrast to the many studies of ICAM-1 and ICAM-3 signaling, little is known about the role of VCAM-1 in cellular activation, although two recent studies suggest that VCAM-1 engagement leads to cytoskeletal rearrangement, myoinositol production, and calcium mobilization (14, 15).

The lipid kinase phosphatidylinositol 3-kinase (PI3K) is composed of an 85-kDa regulatory subunit and a 110-kDa catalytic subunit. Activation of this enzyme has been shown to be critical for growth factor-induced proliferation in smooth muscle cells (16, 17, 18). There are several pathways by which PI3K becomes activated following association with cell surface receptors, including 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 CD28-mediated integrin activation have revealed that impaired tyrosine phosphorylation of the adapter protein p120Cbl results in decreased association of CD28 with PI3K and therefore decreased integrin activation (20). Cbl is tyrosine phosphorylated in response to stimulation via numerous receptors, including immune receptors, growth factors, integrins, and cytokines (reviewed in Refs. 21 and 22). Phosphorylated Cbl associates with a number of SH2- and SH3-containing proteins, including fyn, syk, Crk family proteins, Grb2, Shc, and p85 PI3K (reviewed in Refs. 21 and 22). Cbl expression is not restricted to hematopoietic cells, although the function of Cbl in nonlymphoid cells has not been extensively studied, and there are data to suggest that Cbl plays a role in PI3K activation following stimulation with growth factors in NIH 3T3 fibroblasts and A549 epithelial cells (23, 24).

The interaction between activated T cells and smooth muscle cells via cell adhesion molecules induces critical changes in smooth muscle cell phenotype and synthetic function. We hypothesized that ligation of VCAM-1 on ASM cells would activate signaling pathways that play an important role in smooth muscle cell responses to inflammation. In this report, we demonstrate activation of extracellular signal-regulated kinase (ERK) 2 and PI3K upon VCAM-1 cross-linking. Our data indicate that VCAM-1 induces transient activation of mitogen-activated protein kinase (MAPK) and a more prolonged activation of PI3K but does not induce ASM DNA synthesis. In addition, we demonstrate tyrosine phosphorylation of p120Cbl following VCAM-1 engagement and its association with the p85 subunit of PI3K. These data suggest that VCAM-1 receptor engagement provides important signals regulating cellular activation. The study of cell adhesion molecule function on smooth muscle cells, therefore, can serve to emphasize their role not only in cell-cell interactions but also in the perpetuation of inflammation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Abs and reagents

Murine anti-human VCAM-1 Ab 4B9 (IgG1) (25) was a kind gift from John Harlan (University of Washington, Seattle, WA) and Timothy Carlos (University of Pittsburgh, Pittsburgh, PA). Rabbit polyclonal Abs specific for ERK-2, cyclin D1, p27kip1, and Cbl were purchased from Santa Cruz Biotechnology (Santa Cruz, CA); monoclonal antiphosphotyrosine 4G10 and anti-p85 PI3K Abs were purchased from Upstate Biotechnology (Lake Placid, NY); and anti-phospho-MAPK Abs were purchased from New England Biolabs (Beverly, MA). Wortmannin was purchased from Sigma (St. Louis, MO); LY294002 was purchased from Calbiochem (La Jolla, CA).

Human ASM cell culture

Human ASM cells isolated from the trachealis muscle of transplant donors were maintained in Ham’s F12 medium (Life Technologies, Grand Island, NY) supplemented with 10% FBS (HyClone, Logan, UT) and 2 mM glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (all from Sigma). These cells retain smooth muscle-specific actin staining and responsiveness to contractile agonists, as previously described by Panettieri et al. (26). Confluent ASM cells were growth arrested in serum-free Ham’s F12 containing 0.1% BSA for 48 h before experiments. In these studies, only third- and fourth-passage cells were used.

ERK in vitro kinase assay

Cells were stimulated by the addition of anti-VCAM-1 Ab or epidermal growth factor (EGF) for various times. Cells were lysed in buffer containing 10 mM Tris (pH 7.6), 50 mM NaCl, 1 mM EGTA, 1 mM orthovanadate, 30 mM sodium pyrophosphate, 5 mM benzamidine, 1 mM PMSF, 10 µg/ml aprotinin, 1% Triton X-100, and 0.1% deoxycholate. Cell lysates were clarified by centrifugation at 13,000 rpm for 10 min. Equal amounts of protein were incubated with 5 µl (1 µg) of rabbit anti-ERK-2 Abs for 2 h at 4°C. The immunocomplexes were precipitated with 20 µl of packed protein A-agarose (Life Technologies) for 1 h at 4°C. The cells were 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 orthovanadate. The precipitated proteins were incubated in 30 µl of reaction mix containing 30 mM HEPES (pH 8), 10 mM MgCl2, 1 mM DTT, 5 mM benzamidine, 20 µM ATP, 2 µg myelin basic protein, and 10 µCi [{gamma}-32P]ATP. The reactions were allowed to proceed for 15 min at 30°C and were stopped by adding 6 µl of 6x Laemmli buffer and boiling for 5 min. The proteins were resolved by SDS-PAGE on a 12% gel, which was dried and exposed to film.

PI3K assay

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), 137 mM NaCl, 1 mM MgCl2, 1 mM CaCl2, 0.2 mM orthovanadate, 10% glycerol, 1 mM PMSF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1% Nonidet P-40. Clarified cell lysates were incubated with anti-p85 (5 µg/ml) or anti-Cbl (1:200) Ab overnight at 4°C. Immunocomplexes were added to 60 µl of protein A-Sepharose and incubated for 2 h at 4°C. The immunoprecipitates were washed three times in PBS, 1% Nonidet P-40, and 0.2 mM orthovanadate; three times in 0.1 M Tris (pH 7.5) containing 0.5 M LiCl and 0.2 mM orthovanadate; and twice in 10 mM Tris (pH 7.5), 100 mM NaCl, 1 mM EDTA, and 0.2 mM orthovanadate. Immunoprecipitates were incubated for 10 min at room temperature in buffer containing 4 mM MgCl2, 50 µM ATP, 30 µCi [{gamma}-32P]ATP, and 0.2 mg/ml sonicated phosphatidylinositol in EGTA. Reactions were stopped by adding 20 µl of 6N HCl and extracted with 160 µl of chloroform-methanol (1:1). Lipids were separated on oxalate-coated TLC plates using a chloroform-methanol-water-ammonium hydroxide (60:40:11:3.2) solvent system. The lipids were visualized by autoradiography.

Immunoprecipitation and immunoblotting

For analysis of cyclin D1 and p27kip1 expression, cells were scraped into cold PBS; briefly pelleted; and lysed in 300 µl of buffer containing 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 0.1 mM sodium orthovanadate, 0.5 mM PMSF, 10 mM {beta}-glycerophosphate, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 0.1% Triton X-100. Cells were lysed on ice for 10 min and then sonicated at 2-s intervals for a total of 10 s. Cell lysates were cleared by centrifugation at 13,000 rpm for 15 min. Protein concentrations were determined using the bicinchoninic acid kit (Pierce, Rockford, IL). For analysis of Cbl, cells were lysed in buffer containing 150 mM NaCl, 50 mM Tris, 1 mM EDTA, 1 mM sodium orthovanadate, 10 mM NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 1% Triton X-100, and 0.1% deoxycholate. Equal amounts of protein were immunoprecipitated with anti-Cbl and protein A-Sepharose-conjugated beads. Proteins were separated by electrophoresis on an 8% (Cbl) or a 10% (cyclin D1 and p27kip1) gel and transferred to polyvinylidene difluoride. In some instances, membranes were blocked with TNB (30 mM Tris (pH 7.6), 75 mM NaCl, and 3% BSA) and incubated with polyclonal rabbit Ab for 1 h, and then washed and incubated with 0.5 µCi of 125I-labeled protein A (NEN, Boston, MA) in TNB for 30 min at room temperature. The membranes were extensively washed and proteins were visualized by autoradiography.

Proliferation assays

Proliferation of growth-arrested human ASM cells in response to growth factors or Ab stimulation was assessed as previously described (26).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
TNF-{alpha} and IL-4 synergize to increase ASM expression of VCAM-1

We have previously demonstrated that VCAM-1 is expressed on ASM and is up-regulated by TNF-{alpha} (1). Reports in vascular smooth muscle suggest that IL-4 can synergize with TNF-{alpha} to further increase VCAM-1 expression (27). Human ASM cells were stimulated with TNF-{alpha}, IL-4, or TNF-{alpha} + IL-4 for 24 h. The cells were stained with an isotype-matched control Ab or with an Ab specific for VCAM-1 and analyzed by flow cytometry. TNF-{alpha} induced a 3-fold increase in VCAM-1 expression (mean fluorescence intensity (MFI), 25.9), whereas the combination of TNF-{alpha} and IL-4 induced a 16.5-fold increase (MFI, 125) compared with the expression of VCAM-1 on cells incubated in media alone (MFI, 7.6) (Fig. 1Go). IL-4 alone had no effect on expression of VCAM-1 (data not shown).



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  		FIGURE 1. ASM cells were cultured in serum-free media or stimulated with TNF (500 U/ml) or TNF + IL-4 (10 ng/ml) for 24 h. Cells were stained with an isotype control Ab or mouse anti-human VCAM-1 Ab (10 µg/ml), followed by FITC-conjugated goat anti-mouse IgG. Flow cytometric analysis was performed and analyzed using CellQuest software (Becton Dickinson, San Jose, CA). The data are representative of three separate experiments.

 
VCAM-1 induces activation of ERK-2 in ASM cells

The interaction between activated T cells and smooth muscle cells via cell adhesion molecules induces critical changes in smooth muscle cell phenotype and synthetic function. To investigate the signaling events that may be mediated through adhesion receptors on ASM, we first examined changes in tyrosine phosphorylation of p42/p44 MAPK. The MAPK family of proteins is activated following a wide variety of stimuli, and in ASM cells from some species, activation of the MAPK family appears to be necessary to stimulate growth (28, 29). Growth-arrested ASM cells were treated with TNF/IL-4 for 24 h and then stimulated for various times with anti-VCAM-1 or isotype-matched control Abs. We performed immunoblot analysis of VCAM-1-stimulated ASM cells using an Ab specific for phospho-MAPK, which revealed a time-dependent increase in phosphorylated MAPK. Phosphorylation was maximal at 10–15 min and diminished by 30 min (Fig. 2GoA). This effect was not seen in cells treated with control Ab (data not shown). The degree of MAPK phosphorylation was significantly less than that observed following EGF stimulation (Fig. 2GoA). Previous studies from our laboratory have demonstrated a rapid and prolonged activation of MAPK in ASM cells stimulated with EGF (30, 31).



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  		FIGURE 2. Growth-arrested ASM cells were incubated in serum-free media and treated with TNF/IL-4 for 24 h. Cells were stimulated with anti-VCAM-1 Abs for the indicated times or with EGF (10 ng/ml) for 5 min. A, Equal amounts of protein were separated by SDS-PAGE on an 8% gel and immunoblotted with an Ab recognizing the phosphorylated form of MAPK (n = 2). B, Cell lysates were immunoprecipitated with polyclonal Abs specific for ERK-2, and an in vitro kinase assay was performed as described (n = 3).

 
To correlate protein phosphorylation with enzymatic activity, cell lysates were immunoprecipitated with Abs to ERK-2, and an 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. 2GoB). 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 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. Tyrosine-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. 3GoA). 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 engagement are specific (data not shown). Treatment with the specific PI3K inhibitor wortmannin or LY294002 markedly inhibited VCAM-1-induced activation of PI3K (Fig. 3GoB). 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 follows a similar time course (33).



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  		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 µg/ml) for the indicated times, or with an isotype-matched control Ab (iso, 10 µg/ml) for 5 min (n = 3). B, Growth-arrested ASM cells were incubated in serum-free media (C) and treated with TNF/IL-4 (cyto) for 24 h. Cells were stimulated with anti-VCAM-1 Abs for 5 min in the absence (5') or presence of LY294004 (LY, 10 µM) or wortmannin (WM, 100 nM) (n = 2). Cell lysates were immunoprecipitated with anti-p85 Abs, and an in vitro assay of PI3K activity was performed as described. Lipids were separated on TLC plates and visualized by autoradiography. Or, Origin; PIP, [32P]phosphatidylinositol monophosphate.

 
VCAM-1 engagement induces tyrosine phosphorylation of Cbl

It has been reported that integrin engagement stimulates tyrosine phosphorylation of the adapter protein p120Cbl and its subsequent association with the p85 subunit of PI3K (20, 34, 35). To investigate the possibility that Cbl was phosphorylated following VCAM-1 engagement, lysates of ASM cells were immunoprecipitated with Abs specific for Cbl and analyzed by immunoblotting for phosphotyrosine. In resting cells and cells treated with cytokine alone, there was no evidence of Cbl tyrosine phosphorylation (Fig. 4GoA). A gradual increase in phosphotyrosine was seen following engagement of VCAM-1 with Ab. No increase was seen following stimulation with control Ab (data not shown). In contrast to the gradual increase in tyrosine phosphorylation of Cbl following VCAM-1 engagement, EGF induced a rapid and sustained increase (Fig. 4GoB). These data suggest that different mechanisms regulate Cbl phosphorylation following stimulation via growth factors or adhesion receptors.



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  		FIGURE 4. Left, Growth-arrested ASM cells were incubated in serum-free media (C) and treated with TNF/IL-4 (cyto) for 24 h. Cells were stimulated with anti-VCAM-1 Abs for the indicated times. Cell lysates containing equal amounts of protein were immunoprecipitated with Abs specific for Cbl. Proteins were separated by SDS-PAGE on an 8% gel and immunoblotted with 4G10 (PY, n = 3). Blots were stripped and reprobed with anti-Cbl. Right, Growth-arrested ASM cells were incubated in serum-free media and stimulated with EGF (10 ng/ml) for the indicated times (n = 2). Cbl immunoprecipitation and immunoblotting for phosphotyrosine were performed as described.

 
PI3K activity associates with Cbl following VCAM-1 cross-linking

We next performed coimmunoprecipitations to determine whether VCAM-1 engagement resulted in the association of PI3K activity with Cbl. Lysates of ASM cells treated with cytokine alone or in combination with either anti-VCAM-1 or an isotype-matched control 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. 5GoA). 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 following EGF stimulation (Fig. 5GoA). No detectable increase in kinase activity was seen in cells treated with the control Ab.



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  		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 stimulated with anti-VCAM-1 or isotype control Abs (10 µg/ml) for 20 min or with EGF (10 ng/ml) for 10 min. B, Cells were stimulated with isotype control Ab for 10 min or with anti-VCAM-1 for the indicated times. Cell lysates containing equal amounts of protein were immunoprecipitated with Abs specific for Cbl, and a lipid kinase assay was performed as described. Or, Origin; PIP, [32P]phosphatidylinositol monophosphate. C, Following stimulation with anti-VCAM-1 or an isotype-matched Ab, equal amounts of protein from cell lysates were immunoprecipitated (ip) with anti-Cbl Abs and immunoblotted (ib) with anti-p85.

 
We then performed a time course of Cbl-associated PI3K activity. Lysates of Ab-treated ASM cells were immunoprecipitated with anti-Cbl, and PI3K activity was determined. Lipid kinase activity was maximal at 2 min and decreased over 15 min (Fig. 5GoB). Again, no increase in kinase activity was seen in cells stimulated with control Ab. The rapid increase in Cbl-associated PI3K activity paralleled the increase seen in total cytosolic lipid kinase activity (Fig. 3GoA), although it did not appear to be as sustained.

To determine whether Cbl and PI3K were physically associated, we immunoprecipitated Cbl and immunoblotted for p85. We found that Cbl and the p85 subunit of PI3K were constitutively associated in ASM cells (Fig. 5GoC). No increase in the degree of associated Cbl and p85 was detected following stimulation with anti-VCAM-1 Abs.

Effects of VCAM-1 ligation on smooth muscle cell proliferation

In some cell types, expression of G1 phase cell cycle proteins requires activation of PI3K. We tested whether VCAM-1 engagement 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-regulated by 4 h following VCAM-1 stimulation (Fig. 6GoA). Expression 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 following VCAM-1 engagement but were dramatically decreased after stimulation with EGF (Fig. 6GoB).



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  		FIGURE 6. A, Growth-arrested ASM cells were treated with TNF/IL-4 for 24 h (cyto) and then stimulated with anti-VCAM-1 Abs (10 µg/ml) for 1–22 h or with EGF alone (10 ng/ml) for 22 h. Equal amounts of protein were separated by SDS-PAGE on a 10% gel and immunoblotted with an Ab recognizing cyclin D1 (n = 3). B, Growth-arrested, cytokine-treated cells were stimulated with anti-VCAM-1 Abs for 6, 12, and 24 h. Parallel cultures were treated with EGF alone for the same time periods. Equal amounts of protein were separated by SDS-PAGE on a 10% gel and immunoblotted with an Ab recognizing p27kip1 (n = 2).

 
Our data demonstrate some similarities in EGF- and VCAM-1-mediated signaling pathways known to be important for smooth muscle cell proliferation. We therefore examined the effects of VCAM-1 cross-linking on ASM proliferation in comparison with EGF, a known smooth muscle cell mitogen. Growth-arrested, cytokine-treated cells were stimulated with anti-VCAM-1 Abs or EGF, and incorporation of [3H]thymidine was measured. EGF induced a dose-dependent increase in thymidine incorporation with 10 ng/ml causing an ~14-fold increase in thymidine incorporation (Fig. 7Go), which is consistent with our previous experience (30). In contrast, VCAM-1 engagement had no effect on thymidine incorporation by ASM cells. However, combined stimulation with low dose EGF (1 ng/ml) and anti-VCAM-1 Abs augmented the proliferative response observed with low dose EGF alone. This response was completely inhibited by the addition of either LY294002 or wortmannin (Fig. 7Go), suggesting that the synergy between the EGF and VCAM-1 is mediated entirely through activation of PI3K.



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  		FIGURE 7. Growth-arrested ASM cells (C) were treated with TNF/IL-4 for 24 h (cyto) and then stimulated with anti-VCAM-1 Abs (VCAM, 10 µg/ml). Where indicated, growth-arrested cells were stimulated with EGF at 1 (EGF1) or 10 (EGF10) ng/ml in the presence or absence of LY294002 (LY, in µM) or wortmannin (WM, in nM). Incorporation of [3H]thymidine was assessed as described. The data are representative of six similar experiments, two of which were performed using LY294002 and wortmammin. Data are expressed as mean cpm ± SEM. *, p < 0.001 compared with EGF1 + VCAM; **, p < 0.001 compared with EGF10.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the airway, a variety of cell types reside in or infiltrate the inflamed submucosa that can potentially interact with smooth muscle cells and alter myocyte function. Our studies have shown that adhesion of activated T lymphocytes can induce DNA synthesis in quiescent ASM cells (1). We have also recently demonstrated that engagement of CD40, a member of the TNF receptor superfamily, results in increases in cytosolic calcium, activation of NF-{kappa}B, and augmentation of IL-6 secretion (37). Others have shown that adhesion of activated T lymphocytes induced expression of matrix metalloproteinases in vascular smooth muscle, via CD40-CD40L interactions (38). These studies highlight the fact that direct interactions between T cells and smooth muscle cells via immune receptors such as CD40 or adhesion receptors such as ICAM-1 or VCAM-1 contribute to the modulation of the local milieu, resulting in smooth muscle cell activation.

In this study, we examined VCAM-1-associated signaling pathways in human ASM cells. We found that VCAM-1 ligation resulted in a transient increase in p42/44 MAPK activity as well as a more sustained activation of PI3K activity. Activation of PI3K was not required for ERK2 enzymatic activity, in contrast to {beta}1-integrin-mediated activation of MAPK (39). In addition, we found that VCAM-1 induced expression of cyclin D1. Sustained activation of MAPK and PI3K are critical for growth factor-induced 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 following VCAM-1 engagement alone. This may be due to the transient nature of VCAM-1-induced MAPK activation or the less robust activation of PI3K when compared with EGF. Sustained activation of MAPK is required for smooth muscle cell mitogenesis 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 factors. For example, sustained activation of ERK and expression of cyclin D1 in fibroblasts following stimulation with EGF are dependent on integrin-mediated adhesion (41). Our data suggest that VCAM-1 engagement can augment smooth muscle cell proliferation in the presence of submitogenic concentrations of growth factors, an effect that was completely abrogated by pharmacologic inhibition of PI3K activity. Alternatively, VCAM-1 or other IgSF-mediated signals may be important for cellular processes other than proliferation. Our preliminary data suggest that VCAM-1ligation induces activation of p70 ribosomal S6 kinase (our unpublished results), an enzyme that is downstream of PI3K and is critical for regulation of protein translation. As such, VCAM-1-mediated signals may be linked to the process of cellular hypertrophy, rather than to proliferation. For example, agents that induce vascular smooth muscle hypertrophy, such as angiotensin II, have also been shown to increase cyclin D1 expression in the absence of p27kip1 degradation (42, 43, 44).

We have demonstrated tyrosine phosphorylation of the protooncogene p120Cbl following VCAM-1 cross-linking. Although Cbl tyrosine phosphorylation occurs in response to diverse stimuli, such as binding of the B and T cell Ag receptors, growth factors, and cytokines, and integrin ligation (21, 22), this is the first demonstration of Cbl phosphorylation in response to IgSF cross-linking. The gradual onset of Cbl phosphorylation following VCAM-1 engagement is consistent with other studies in macrophages following integrin-mediated stimulation (34, 45), but it contrasts with the rapid and sustained tyrosine phosphorylation observed following EGF stimulation. These data suggest that while both IgSF- and EGF-mediated signaling stimulate tyrosine phosphorylation of Cbl in ASM cells, the mechanisms are likely to be different.

Interestingly, despite a time-dependent increase in Cbl-associated PI3K activity, we found a constitutive association between Cbl and the p85 subunit of PI3K. This suggests that other proteins might be recruited into the signaling complex, which would then act directly to activate PI3K. In turn, Cbl phosphorylation, which occurs more slowly than PI3K activation, may occur as part of a feedback mechanism, possibly allowing for retention of PI3K within the signaling complex. Further studies will be necessary to elucidate these pathways. The physiologic significance of the Cbl-p85 complexes in smooth muscle cells, as in other cells, has yet to be fully determined. As postulated by Hartley and Corvera (46) and Ojaniemi et al. (34), these complexes may be important, not for mitogenic signaling, but rather for other responses such as adhesion.

Another question that remains to be answered is how VCAM-1 engagement initiates activation of intracellular kinases, as the relatively short cytoplasmic domain has no known intrinsic enzymatic activity, nor does it contain motifs such as SH2 or SH3 domains that might facilitate recruitment of intracellular kinases (47). However, since VCAM-1 engagement has been shown to result in cytoskeletal rearrangement (15), it is possible that VCAM-1 interacts with cytoskeletal proteins to assemble a functional signaling complex. For example, ICAM-1 ligation with Ab leads to phosphorylation of cortactin (8, 48), as well association with {alpha}-actinin and ezrin (49, 50), while monocyte adhesion to endothelial cells induces receptor clustering of ICAM-1 and VCAM-1 along with their colocalization with ezrin (51).

Although our data are consistent with a role for Cbl in the activation of PI3K, the fact that only a small percentage of PI3K enzymatic activity coassociates with Cbl suggests that there are other phosphoproteins involved. Potential candidates include src family kinases and Rho, a GTP binding protein. Rho and p53lyn activation have been noted following ICAM-1 cross-linking (7, 48, 52), while activation of p59fyn and p56lck result from ICAM-3 cross-linking in T cells (12). In addition, ligation of ICAM-1 induces activation of Rho (48), while inhibition of Rho inhibits IgSF receptor clustering and cytoskeletal reorganization (48, 51). Future studies will be necessary to elucidate the role of other kinases in linking PI3K to a VCAM-1-mediated signaling pathway.


   Acknowledgments
 
We thank Drs. Timothy Carlos and John Harlan for their generous gift of 4B9 Ab and Dr. Reynold Panettieri, Jr., for helpful discussion.


   Footnotes
 
1 This work was supported by National Institutes of Health Grant HL-03202 and by the McCabe Fund of the University of Pennsylvania. Back

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, 421 Curie Boulevard, Philadelphia, PA 19104. Back

3 Abbreviations used in this paper: IgSF, Ig supergene family; ASM, airway smooth muscle; PI3K, phosphatidylinositol 3-kinase; SH, Src homology; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; EGF, epidermal growth factor; MFI, mean fluorescence intensity. Back

Received for publication June 16, 2000. Accepted for publication September 29, 2000.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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