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Am J Physiol Cell Physiol 290: C972-C980, 2006. First published November 9, 2005; doi:10.1152/ajpcell.00136.2005
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VASCULAR BIOLOGY

Modulation of {alpha}7-integrin-mediated adhesion and expression by platelet-derived growth factor in vascular smooth muscle cells

Jun-Tzu Chao,1 Luis A. Martinez-Lemus,1 Stephen J. Kaufman,2 Gerald A. Meininger,1 Kenneth S. Ramos,3 and Emily Wilson1

1Division of Vascular Biology, Cardiovascular Research Institute, The Texas A&M University System Health Science Center, College Station, Texas; 2Department of Cell and Structural Biology, University of Illinois, Urbana, Illinois; and 3Department of Biochemistry and Molecular Biology, University of Louisville, Louisville, Kentucky

Submitted 23 March 2005 ; accepted in final form 2 November 2005


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
We showed previously that the expression of {alpha}7-integrin in aortic vascular smooth muscle cells (VSMC) is enhanced in a rat model of atherosclerosis. In the present study, we investigated the effects of platelet-derived growth factor (PDGF) on {alpha}7-integrin expression and VSMC adhesion and migration. Expression of the {alpha}7-integrin gene was determined by real-time RT-PCR, whereas protein levels were determined by fluorescence-activated cell sorting analysis. PDGF increased {alpha}7 cell surface protein expression (12 and 24 h: 3.3 ± 0.8- and 3.6 ± 0.4-fold, P < 0.05 vs. control) and mRNA levels (24 h: 3.1-fold, P < 0.05 vs. control) in a time-dependent manner. Actinomycin D and cycloheximide attenuated PDGF-induced increases in {alpha}7-integrin, indicating the involvement of de novo mRNA and protein synthesis. Treatment with the MAPK inhibitors PD-98059, SP-600125, and SB-203580 attenuated PDGF-induced increases in mRNA. In contrast, PD-98059 and SP-600125, but not SB-203580, attenuated PDGF-induced increases in cell surface protein levels. PDGF-treated VSMC adhered to laminin more efficiently (42 ± 6% increase, P < 0.01), and this increase was partially inhibited by anti-{alpha}7-integrin function-blocking antibody. However, PDGF did not alter migration on laminin, and there was no effect of the anti-{alpha}7-integrin function-blocking antibody on basal or PDGF-stimulated migration. Immunofluorescence imaging revealed an increase in {alpha}7-integrin distribution along the stress fibers. Together, these observations indicate that PDGF enhances {alpha}7-integrin expression in VSMC and promotes {alpha}7-integrin-mediated adhesion to laminin.

vascular injury; laminin; mitogen-activated protein kinase

INTEGRINS PLAY ESSENTIAL ROLES in cellular functions such as migration, adhesion, differentiation, and proliferation. Upon binding to the extracellular matrix (ECM), integrins undergo conformational changes in the cytoplasmic domain that initiate recruitment of adaptor and cytoskeletal proteins and signaling molecules. Along with the activation of the signaling molecules, integrins are able to transduce signals from extracellular stimuli into the nucleus that may alter gene expression. Consequently, integrins modulate cellular phenotypic changes (29). In this regard, integrins and growth factors share many common elements in their respective signal transduction pathways (14, 34), and significant cross talk and interaction between growth factor and integrin signaling have been shown previously (14, 35, 36, 41). For example, {alpha}vbeta3- and {alpha}vbeta5-integrins mediate a mechanically induced mitogenic response in vascular smooth muscle cells (VSMC) by initiating the secretion of platelet-derived growth factor (PDGF) (40). Whereas integrins are known to be involved extensively in the modulation of growth factor signaling, the role of growth factor in modulating integrin expression is not well defined.

PDGF, one of the major growth factors present in blood vessels, plays a critical role in several vascular pathologies. In response to arterial injury such as balloon angioplasty, PDGF stimulates proliferation and migration of VSMC (25). PDGF modulates these cellular functions by activating signaling cascades, such as phosphatidylinositol 3-kinase, Ras, Raf, and mitogen-activated protein kinases (MAPK), including ERK, JNK, and p38 (17, 26). Through these signaling pathways, PDGF modulates the expression of a number of genes that contribute to vascular remodeling. Among the genes regulated are ECM proteins such as fibronectin, laminin-5, and osteopontin in VSMC (22, 26, 39). In addition, PDGF modulates the expression of integrins, such as {alpha}vbeta3-integrin complex and {alpha}5-integrin subunit in VSMC (19). However, little is known regarding the effect of PDGF on {alpha}7-integrin subunit expression.

{alpha}7beta1-Integrin is a laminin receptor present in skeletal and cardiac muscle (37). In skeletal muscle, {alpha}7beta1-integrin stabilizes the structure of myofibers by providing linkages between laminin in the ECM and the actin cytoskeleton. Increased expression of {alpha}7-integrin subunit is observed in patients with muscular dystrophy caused by dystrophin deficiency (18) and in mice with sarcoglycan or dystroglycan deficiency (3). Elevation of {alpha}7-integrin subunit levels also is seen in regenerating myofibers in rats with transection-induced muscle injury (20). The above-described findings suggest that {alpha}7-integrin subunit plays an essential role in the structural stability of myofibers and in the skeletal muscle response to injury. Furthermore, studies by Babbitt et al. (4) demonstrated increased expression of {alpha}7-integrin subunit in a rat model of pressure overload left ventricular hypertrophy. These studies suggest a role of {alpha}7-integrin subunit in the adaptive response of myocardium to pressure overload (4).

The role of {alpha}7-integrin subunit in VSMC is less well characterized. The presence of {alpha}7-integrin subunit in VSMC was reported by Yao et al. (42). Our group demonstrated previously (10) the pathological significance of {alpha}7-integrin subunit in VSMC by showing increased {alpha}7-integrin subunit expression and adhesion to laminin in VSMC in a chemical model of atherosclerosis. Nonetheless, the physiological regulation and function of {alpha}7-integrin subunit is not clear. Thus the objective of this study was to determine the effect of PDGF on {alpha}7-integrin subunit expression and whether changes in gene expression induced by PDGF correspond to the functional alterations of VSMC. Using PDGF-treated VSMC as an in vitro model to study vascular injury, we investigated the potential modulation of {alpha}7-integrin subunit expression and the regulatory mechanisms responsible for these changes. The role of {alpha}7-integrin subunit in VSMC adhesion and migration also was investigated.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and treatment. VSMC were isolated by enzymatic digestion of aortas from Sprague-Dawley rats as previously described (11). Cells were grown in medium 199 (M199; Invitrogen, Carlsbad, CA) supplemented with 10% FBS (Hyclone, Logan, UT), 2 mmol/l glutamine, 100 units of penicillin, and 100 µg of streptomycin (Invitrogen) in 5% CO2-95% air at 37°C. Recombinant human PDGF-BB, epidermal growth factor (EGF), fibroblast growth factor (FGF), and transforming growth factor (TGF)-beta were purchased from PeproTech (Rocky Hill, NJ). Cells were used between passages 4 and 10 to verify that passage number is not a primary factor in {alpha}7-integrin subunit expression. VSMC were quiesced by serum starvation (0.5% FBS M199) for 48 h before PDGF treatment (10 ng/ml for 0, 4, 12, and 24 h). At the end of treatment, cells were processed for either RNA isolation or determination of {alpha}7-integrin subunit protein by fluorescence-activated cell sorting (FACS) analysis. Actinomycin D, cycloheximide, laminin-1, and all other reagents were purchased from Sigma (St. Louis, MO). PD-98059 (30 µmol/l), SP-600125 (5 µmol/l), and SB-230580 (5 µmol/l) were used to block p42/44, JNK, and p38, respectively, as described by others (2, 5, 13). For the inhibitor studies, cells were pretreated with the various agents for 1 h before coincubation with PDGF and inhibitors for an additional 23 h. PD-98059, SP-600125, and SB-23058 were purchased from Biomol (Plymouth Meeting, PA).

RNA quantification. Cellular RNA was isolated using TRIzol (Invitrogen) according to the manufacturer's protocol. RNase-free DNase (Ambion, Austin, TX) was used to remove genomic DNA contamination. Reverse transcription was performed with total cellular RNA (500 ng) using Superscript reverse transcriptase (Invitrogen) according to the manufacturer's protocol. As a negative control, equal amounts of total RNA were processed as described above without reverse transcriptase. Transcribed cDNA (3 µl) was amplified using standard real-time PCR performed with an ABI 7700 sequence detection system (Applied Biosystems, Foster City, CA). The quantification of {alpha}7-integrin subunit mRNA levels and conditions of real-time RT-PCR were performed as described previously (10). Results are presented relative to the expression level of 18S rRNA.

FACS analysis. The effect of PDGF and MAPK inhibitors on {alpha}7-integrin subunit surface expression in VSMC was determined by flow cytometry using a FACSCalibur apparatus as described previously (10). FITC-conjugated mouse IgG isotype (BD Pharmingen, San Diego, CA) in the absence of primary antibody was used as a negative control. Cells (10,000 cells per sample) were counted for determination of the fluorescence intensity. Values of <2% total positive fluorescence were set as negative expression.

Immunoblot analysis. VSMC were quiesced for 48 h as described previously and then treated with 10 ng/ml PDGF-BB, EGF, FGF, or TGF-beta for 24 h, followed by total protein isolation, as described by Chao et al. (10). For specific protein detection, rabbit anti-{alpha}7 cytoplasmic domain (347) antiserum was used at 1:1,000 dilution as the primary antibody, and donkey anti-rabbit IgG conjugated with horseradish peroxidase was used at 1:20,000 dilution as the secondary antibody (Jackson Laboratory, Bar Harbor, ME). The blot also was reacted with anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) at a 1:1,000 dilution (Advanced Immunochemical, Long Beach, CA) to serve as a loading control. Expression of {alpha}-actin was detected using anti-{alpha}-actin antibody (Sigma) at 1:1,000 dilution. The chemiluminescent signal was detected using SuperSignal West Pico chemiluminescent substrate (Pierce Biotechnology, Rockford, IL). Band intensity of {alpha}7-integrin was determined using densitometry as described previously (10).

Immunofluorescence imaging. After PDGF treatment, VSMC were fixed with 2% paraformaldehyde in PBS for 15 min at room temperature, followed by two washes with PBS to remove the excess paraformaldehyde. Cells were then incubated with H36 anti-{alpha}7-integrin antibody overnight at 4°C (1:350 dilution) and washed six times with a solution containing 15 mmol/l sodium citrate, 150 mmol/l sodium chloride, and 0.05% Triton X-100. After being washed, cells were incubated with the secondary antibody, Alexa 488-conjugated goat anti-mouse IgG (5 µg/ ml), for 30 min at room temperature (Molecular Probes, Corvallis, OR). Alexa 647-conjugated phalloidin (66 nmol/l; Molecular Probes) was used to stain actin after the incubation with the secondary antibody. Cells also were incubated with mouse IgG isotype as the primary antibody and with the secondary antibody Alexa 488-conjugated goat anti-mouse IgG as a negative control. An additional negative control consisted of incubation with secondary antibody only. Immunofluorescence imaging was performed using a laser scanning confocal system (Ultima-Z312; Meridian Instruments, Okemos, MI) connected to an inverted microscope (Zeiss 135). Images were collected with a x63 oil objective (numerical aperture 1.4). Excitation of the fluorophores was achieved using the 488- and 647-nm lines of an argon-krypton laser combination.

Adhesion assay. VSMC adhesion to laminin-1 was determined using CytoMatrix cell adhesion strips from Chemicon (Temecula, CA) following the manufacturer's protocol. Cells were quiesced for 48 h before PDGF treatment. Control and PDGF-treated VSMC were detached with Versene (1:5,000 dilution) at 37°C for 5 min to prepare the cell suspension. Cells (106 cells/ml) were added to laminin-1-coated wells and incubated at 37°C for 1 h. Nonadherent cells were removed by washing (twice) gently with Dulbecco's PBS (DPBS) in the presence of Ca2+ and Mg2+, followed by staining with 0.1% crystal violet for 5 min. Excess stain was removed by washing with DPBS in the absence of Ca2+ and Mg2+. The adhered cells were solubilized with acetate buffer (pH 4.5) and quantified by determining the absorbance at 550 nm. The effect of blocking {alpha}7-integrin subunit was tested by preincubating the cells with mouse anti-rat {alpha}7-integrin antibody (37) (O26, 1:350 dilution) at room temperature for 30 min before the assay. Cells attached to the wells coated with BSA were used to adjust for nonspecific binding (negative control), which was <5% of maximal binding.

Alternative adhesion assays also were performed by using wells coated with anti-{alpha}7- (provided by S. J. Kaufman), anti-{alpha}5-, and anti-beta1-integrin antibodies (BD Pharmingen, San Diego, CA). Wells were coated with anti-{alpha}7-integrin antiserum at a 1:500 dilution; anti-{alpha}5- and anti-beta1-integrin antibodies were used at 1 µg/ml overnight at 4°C. The excess antibody was removed by washing with DPBS before use. Control and PDGF-treated VSMC were detached as described previously. Cells (106 cells/ml) were added to antibody-coated wells and incubated at 37°C for 1 h. Nonadherent cells were removed by washing (twice) gently with DPBS with Ca2+ and Mg2+, followed by staining with 0.1% crystal violet for 5 min. Excess stain was removed by washing with DPBS in the absence of Ca2+ and Mg2+. The adhered cells were solubilized and quantified as described previously.

Migration assay. Cell migration on laminin-1 was determined using a 48-well chemotaxis chamber and polyvinylpyrrolidone-free polycarbonate filters with pores of 8 µm (Neuro Probe, Gaithersburg, MD) modified from those described by Partridge et al. (30). Each filter was precoated with 20 µg/ml laminin-1 (Sigma) and blocked with 1 mg/ml BSA before the assay was performed. Briefly, control and PDGF-treated VSMC were prepared and detached as described for the adhesion assay. A 28-µl volume of 10% FBS M199 was used as the chemoattractant placed in each well of the lower chamber. A 40-µl volume of cell suspension containing 30,000 cells was placed in the upper chamber and incubated for 4 h at 37°C in a 5% CO2 incubator. The filter was removed, fixed in methanol, and stained (0.1% amido black, 30% methanol, and 10% acetic acid). Staining intensity of migrated cells was determined by densitometry using Multi-Analyst software version 1.0 (Bio-Rad, Hercules, CA).

Statistical analysis. Results are presented as means ± SE of at least three independent experiments. Statistical differences were analyzed using either ANOVA or unpaired t-test with P < 0.05 considered significant. All analyses were performed using GraphPad Prism version 4.02 software for Macintosh (GraphPad Software, San Diego, CA). ANOVA with Tukey's multiple comparison posttest was performed.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
PDGF increases {alpha}7-integrin subunit expression in a time-dependent manner. We first examined the time-related effect of PDGF on {alpha}7-integrin subunit mRNA and protein expression in VSMC. Cells incubated at the time points indicated without PDGF were used as control. As shown in Fig. 1A, a time-dependent increase of {alpha}7 mRNA levels was observed. A significant increase of {alpha}7-integrin subunit mRNA was noted at 24 and 48 h after PDGF treatment (3.1- and 4.4-fold, respectively; P < 0.05 vs. control). The surface expression of {alpha}7 protein levels also was increased significantly with PDGF treatment compared with control (12 h, 3.30-fold; 24 h, 3.6-fold; and 48 h, 6.7-fold) (Fig. 1B). Although a more significant increase in {alpha}7-integrin subunit levels was observed at 48 h, more cell loss also was noted (data not shown), suggesting that long-term maintenance of the cells in low serum was detrimental. Thus, for the following studies, cells were treated with PDGF for 24 h.


Figure 1
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  		Fig. 1. Time-dependent modulation of {alpha}7-integrin subunit mRNA (A) and protein expression (B) in vascular smooth muscle cells (VSMC). mRNA expression of {alpha}7-integrin subunit is presented as relative (i.e., -fold) changes compared with control cells. Data are normalized to 18S rRNA expression levels. {alpha}7-Integrin subunit surface protein levels were determined using fluorescence-activated cell sorting (FACS) analysis. Protein levels of {alpha}7-integrin are presented as relative changes compared with control cells. Values represent means ± SE based on at least 3 independent experiments performed in duplicate. The SE at 48 h of treatment in B is too small to be shown at the current scale. *P < 0.05 vs. control.

 
Effect of growth factors on {alpha}7-integrin subunit expression. To extend the results of FACS and real-time RT-PCR analyses and to determine whether increases in {alpha}7-integrin were specific for PDGF, we quiesced VSMC for 48 h and then treated cells with 10 ng/ml PDGF, EGF, FGF, or TGF-beta for 24 h. Total protein was isolated, and immunoblot analyses were performed as described. Figure 2, A and B, shows that only PDGF treatment significantly increased {alpha}7-integrin protein level (42 ± 2.7%; P < 0.05 vs. control), whereas no statistically significant difference in {alpha}7 protein level was seen in TGF-beta-, EGF-, or FGF-treated cells. When comparing the expression of {alpha}-actin or GAPDH between control and growth factor-treated cells (Fig. 2A, b and c), we observed no difference.


Figure 2
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  		Fig. 2. Effect of platelet-derived growth factor (PDGF), transforming growth factor (TGF)-beta, epidermal growth factor (EGF), and fibroblast growth factor (FGF) on {alpha}7-integrin protein level. Each growth factor (10 ng/ml) was used to treat VSMC after 48 h of starvation. Total cellular protein was isolated at the end of 24 h of treatment and was processed for immunoblot analysis. A: a representative image of immunoblot analyses of {alpha}7 protein level: lanes 1 and 2, control; lanes 3 and 4, PDGF-treated VSMC; lane 5, TGF-beta-treated VSMC; lanes 6 and 7, EGF-treated VSMC; lane 8, FGF-treated cells. a, expression level of {alpha}7-integrin; b, level of GAPDH as loading control; c, level of {alpha}-actin. B: bar graph representing the average band intensity as determined by densitometry. {alpha}7 Protein levels are normalized to control. Data are presented as means ± SE. Experiments were done in duplicate, and 3 individual blots were performed. *P < 0.05 vs. control.

 
PDGF modulates {alpha}7-integrin subunit expression at transcriptional level. To determine whether the observed changes induced by PDGF were mediated at the transcriptional or translational level, we treated cells with PDGF for 24 h in the presence of 2 µg/ml of either actinomycin D or cycloheximide. mRNA levels of {alpha}7-integrin subunit were determined using real-time RT-PCR. As shown in Fig. 3, both actinomycin D and cycloheximide treatments prevented the increase of {alpha}7-integrin subunit mRNA level by PDGF (0.9- and 0.8-fold, respectively; P < 0.05 vs. PDGF). These results suggest that increased {alpha}7-integrin mRNA is regulated at the transcriptional level and that new protein synthesis is required in this process.


Figure 3
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  		Fig. 3. Influence of actinomycin D (ActD; 2 µg/ml) and cycloheximide (Cyclohex; 2 µg/ml) on PDGF (10 ng/ml)-mediated expression of {alpha}7-integrin subunit mRNA levels after 24 h of exposure to agonist. mRNA expression of {alpha}7-integrin subunit is presented as relative changes in expression compared with control cells. Values represent means ± SE of 3 independent experiments performed in duplicate. *P < 0.05 vs. control.

 
Signaling pathways contributing to PDGF-induced {alpha}7-integrin subunit expression. To elucidate the signaling pathways that contribute to PDGF-induced increases in {alpha}7-integrin subunit expression, we determined the effects of the MAPK inhibitors PD-98059 (30 µmol/l), SP-600125 (5 µmol/l), and SB-203580 (5 µmol/l) using real-time RT-PCR. As shown in Fig. 4A, all three MAPK inhibitors attenuated PDGF-induced increases in {alpha}7-integrin subunit mRNA levels, albeit to different degrees (PD-98059, 55.8 ± 11.3%; SB-203580, 66.7 ± 14.5%; and SP-600125, 81.2 ± 10.3%; n = 4). In comparison, PD-98059 and SP-600125, but not SB-203580, attenuated increased {alpha}7-integrin subunit protein levels by PDGF (PD-98059, 62 ± 6.7%; SP-600125, 72.1 ± 11%; n = 5) (Fig. 4B).


Figure 4
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  		Fig. 4. Influence of MAPK inhibitors PD-98059 (30 µmol/l), SB-203580 (5 µmol/l), and SP-600125 (5 µmol/l) on PDGF (10 ng/ml)-mediated increases in {alpha}7-integrin subunit expression after 24 h of exposure to agonist. A: effect of MAPK inhibitors on mRNA levels of {alpha}7-integrin subunit. mRNA expression of {alpha}7-integrin subunit is presented as %response to PDGF stimulation. Values represent means ± SE based on at least 3 independent experiments performed in duplicate. ***P < 0.001 vs. untreated control VSMC. **P < 0.001; *P < 0.05, #P < 0.01 vs. PDGF. B: effect of PD-98059, SB-203580, and SP-600125 on cell surface expression of {alpha}7-integrin subunit. Surface protein levels of {alpha}7-integrin subunit are presented as %response to PDGF stimulation. Values represent means ± SE based on at least 3 independent experiments performed in duplicate. ***P < 0.0001 vs. untreated control VSMC. **P < 0.001; *P < 0.05 vs. PDGF.

 
PDGF increases VSMC adhesion but not migration on laminin-1. To determine the extent to which PDGF-mediated increases in {alpha}7-integrin subunit alter cellular functions, we determined cell adhesion to and migration on laminin-1. As shown in Fig. 5A, PDGF treatment significantly increased the adhesion of VSMC to laminin-1 (42.4 ± 6.3%; P = 0.0037 vs. control). When PDGF-treated VSMC were incubated with a function-blocking anti-{alpha}7-integrin antibody before the adhesion assay, adhesion to laminin-1 was partially but significantly decreased (23 ± 7%; P = 0.0219 vs. PDGF). These results indicate that PDGF treatment (24 h) increases the adhesion to laminin and that this increase is partially mediated by {alpha}7-integrin subunit. Mouse IgG isotype was used as a negative control, and no significant difference was observed compared with PDGF-treated VSMC (data not shown).


Figure 5
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  		Fig. 5. Influence of anti-{alpha}7 antibody on PDGF (10 ng/ml)-treated VSMC cell function. A: effect of anti-{alpha}7- and anti-{alpha}1-integrin antibodies on PDGF-treated VSMC adhesion to laminin-1. Data are normalized to %response to PDGF stimulation. Ab, antibody. *P < 0.01 vs. control. #P < 0.05 vs. control; **P < 0.05 vs. PDGF-treated VSMC. B: effect of PDGF on VSMC adhesion to anti-{alpha}7-, anti-{alpha}5-, and anti-beta1-integrin antibody-coated wells. Data are normalized to %response to PDGF stimulation. *P < 0.01 vs. control; #P < 0.05 vs. {alpha}5 and beta1. C: effect of anti-{alpha}7- and anti-{alpha}1-integrin antibodies on PDGF-treated VSMC migration on laminin. Data are normalized to %response of control. Values represent means ± SE based on at least 3 independent experiments performed at least in triplicate. *P < 0.05 vs. control. #P < 0.05 vs. PDGF-treated VSMC.

 
To exclude the possibility that PDGF modulates the expression of other integrins leading to increased adhesion, we performed the adhesion assay using control and PDGF-treated cells on wells coated with anti-{alpha}7-, anti-{alpha}5-, or anti-beta1-integrin antibodies. Adhesion of PDGF-treated VSMC to anti-{alpha}7-integrin antibody-coated wells increased by ~53% (P < 0.01 vs. control; Fig. 5B). Minimal adhesion to anti-{alpha}5-integrin antibody-coated wells was observed in either control or PDGF-treated cells. Among the three integrins examined, anti-{alpha}7-integrin antibody appeared to mediate adhesion more effectively in both control and PDGF-treated cells (P < 0.05 vs. {alpha}5 and beta1). Minimal adhesion to wells coated with IgG or uncoated wells was observed.

We also determined the effect of PDGF on VSMC migration on laminin-1. As shown in Fig. 5C, PDGF-treated cells displayed no migratory advantage on laminin-1 compared with control. Also, no effect of incubation with anti-{alpha}7-integrin antibody was observed for either PDGF-treated or control cells. In contrast, incubation with anti-{alpha}1-integrin antibody with both control and PDGF-treated VSMC reduced the migration on laminin-1 significantly.

Localization of {alpha}7-integrin in PDGF-treated VSMC. We next examined the distribution pattern of {alpha}7-integrin subunit in PDGF-treated or control cells. In Fig. 6, the immunofluorescence of {alpha}7-integrin subunit is shown in green (A, C, D, and F), F-actin is shown in red (B and E), and the overlap of {alpha}7-integrin subunit and F-actin is shown in yellow (C and F). Punctate staining of {alpha}7-integrin subunit in the cells and on the plasma membrane was found in both control and PDGF-treated cells (Fig. 6, A and D). PDGF treatment increased the intensity of {alpha}7-integrin subunit staining, which indicates the increased levels of {alpha}7-integrin compared with control. This result is consistent with the results of FACS and immunoblot analyses. Spatial association of {alpha}7-integrin subunit with F-actin was observed mainly at the periphery of the control cells (Fig. 6C). In PDGF-treated cells, {alpha}7-integrin subunit staining localized along with the stress fibers (Fig. 6, E and F). Cells also were incubated with mouse IgG isotype as the primary antibody, with the secondary antibody Alexa 488-conjugated goat anti-mouse IgG as a negative control. An additional negative control consisted of incubation with secondary antibody only. Both negative controls showed no fluorescence (data not shown).


Figure 6
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  		Fig. 6. Representative immunofluorescence images of {alpha}7-integrin subunit protein expression and localization in control and PDGF (10 ng/ml)-treated VSMC. A–C: control VSMC. D–F: PDGF-treated VSMC. A and D show staining of {alpha}7-integrin subunit only. B and E show phalloidin-F-actin staining. C and F show merged images of {alpha}7-integrin subunit and phalloidin-F-actin staining. Cells incubated with mouse IgG isotype as the primary antibody and with Alexa 488-conjugated anti-mouse IgG as the secondary antibody displayed no fluorescence (data not shown). Original magnification, x63.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
{alpha}7beta1-Integrin is one of the laminin receptors present in cardiac, skeletal, and vascular smooth muscle. The significance of {alpha}7beta1-integrin expression is demonstrated by increased expression in patients with muscular dystrophy and murine models of muscular dystrophy caused by dystrophin deficiency. Previous findings by others suggest that in muscular dystrophy, enhanced expression of {alpha}7-integrin subunit compensates for the loss of the dystrophin glycoprotein complex, another laminin receptor present in skeletal muscle that stabilizes myofibers (3, 18). The compensatory role of {alpha}7-integrin subunit is underscored by the observation that overexpression of {alpha}7-integrin subunit rescues the lethal muscular dystrophy phenotype in mice (7). Whereas the function of {alpha}7-integrin subunit has been characterized in skeletal muscle, the role of {alpha}7-integrin subunit in VSMC has not been explored.

Our group (10) previously demonstrated increased expression of {alpha}7-integrin subunit in a chemical model of atherosclerosis. In our present study, a significant increase of {alpha}7-integrin subunit expression was observed in VSMC with prolonged treatment (24 and 48 h) of PDGF. We also investigated the regulatory mechanisms by which PDGF modulates the expression of {alpha}7-integrin subunit.

To determine whether increased {alpha}7-integrin expression is a universal response to growth factor stimulation after 48 h of starvation, we treated VSMC with EGF, FGF, and TGF-beta, in addition to PDGF, and performed immunoblot analyses to examine the expression of {alpha}7-integrin. {alpha}7-Integrin protein levels in control VSMC were more variable than the higher levels observed consistently in PDGF-treated cells. Among the growth factors examined, only VSMC treated with PDGF displayed increased expression of {alpha}7-integrin consistently. Cells treated with TGF-beta displayed a trend of decreasing {alpha}7-integrin levels, but this trend was not statistically significant. Notably, increased {alpha}7-integrin expression by PDGF is not due to the alteration in the differentiation status of VSMC, because no significant difference was observed in the expression of {alpha}-actin among the various growth factors used. Overall, these results support the observation that increased {alpha}7-integrin expression is specific to PDGF.

In addition to our findings with PDGF, others have demonstrated altered expression of {alpha}7-integrin subunit by growth factors. Rosbottom et al. (33) demonstrated induction of {alpha}7-integrin subunit by TGF-beta in differentiated mucosal mast cells. Also, in a porcine coronary artery occlusion model, threefold less expression of {alpha}7-integrin subunit was observed in control animals compared with animals transfected with a secretory form of FGF, as determined by microarray analysis (Forough R, unpublished observation). The differences in these various models may be attributed to differences in cell type responses to the growth factors.

Treatment with either actinomycin D or cycloheximide attenuated PDGF-induced increases of {alpha}7-integrin subunit mRNA levels. This observation suggests that PDGF modulates {alpha}7-integrin subunit expression at the transcriptional level and may depend on de novo protein synthesis. Notably, cell surface levels of {alpha}7-integrin subunit determined by FACS analysis showed significant increases at an earlier time point (12 h) than was seen with the mRNA levels. This observation is consistent with the findings that PDGF modulates the membrane mobility and trafficking of integrins (1, 32). Thus the steady-state cell surface levels of integrins may be modulated independently of increases in new mRNA and protein synthesis.

To begin to understand the signal transduction pathways by which PDGF modulates the expression of {alpha}7-integrin subunit, we investigated the contribution of various MAP kinases in {alpha}7-integrin subunit expression by using MAPK inhibitors. Our results indicate that all three MAP kinases (ERK, p38, and JNK) contribute to the regulation of {alpha}7-integrin subunit mRNA levels, because PDGF-increased {alpha}7 mRNA levels were attenuated by all three MAPK inhibitors (PD-98059, SB-203580, and SP-600125). However, whereas PD-98059 and SP-600125 significantly reduced the PDGF-increased {alpha}7-integrin subunit surface protein levels, SB-203580, a p38 kinase inhibitor, displayed no inhibitory effect. Thus our data imply that all three MAP kinases play some role in PDGF-induced changes in {alpha}7-integrin subunit mRNA levels; however, they may differentially alter cellular functions by regulating cell surface levels such as endocytic recycling of membrane proteins. This concept is supported by the observation that p38, but neither ERK nor JNK, is involved in the endocytic recycling of membrane proteins. p38 has been shown to modulate endocytic trafficking via the formation of GDI-Rab5 complex, a process that can be inhibited by SB-203580 (9). In addition, PDGF has been shown to promote Rab4-dependent recycling of {alpha}vbeta3-integrin from the early endosome back to the cell surface (32). Although it is beyond the scope of the present study, the effect of PDGF on {alpha}7-integrin subunit recycling and endocytosis cannot be excluded at this time.

The effect of PDGF on VSMC adherence and migration on ECM proteins, such as fibronectin and collagen, has been well studied (6, 21), whereas the effect of PDGF on VSMC adhesion to and migration on laminin has received limited attention. Because our results demonstrate increased expression of {alpha}7-integrin subunit, a laminin receptor in VSMC, we further identified potential functional alterations of VSMC on laminin. Thus, in the current studies, we examined the effect of PDGF on VSMC adhesion to and migration on laminin-1. We found that PDGF augments VSMC adhesion to laminin-1 and that this increase is mediated at least in part by {alpha}7-integrin subunit, because a function-blocking anti-{alpha}7-integrin antibody partially prevents the adhesion to laminin-1. Incubation with mouse IgG isotype control displayed no difference in adhesion to laminin-1, indicating that the blockade by {alpha}7 antibody is specific to the {alpha}7-integrin subunit. The incomplete inhibition of adhesion to laminin-1 by anti-{alpha}7-integrin antibody suggests the presence and potential involvement of other laminin receptors, such as {alpha}1-integrin subunit or the dystrophin glycoprotein complex (8, 27). Incubation with anti-{alpha}1-integrin antibody alone had no effect on adhesion to laminin-1. When PDGF-treated VSMC were incubated with the combination of anti-{alpha}7- and anti-{alpha}1-integrin antibodies, increased VSMC adhesion to laminin-1 was repressed to the basal level. This additive effect of antibodies suggests that collaboration between integrins may play a role in VSMC adhesion to laminin-1. In our studies, we used laminin-1 as the substrate, because {alpha}7beta1 has been shown previously to mediate VSMC adhesion and migration on laminin-1 (42, 43). Others have shown that {alpha}7beta1-integrin modulates adhesion of trophoblasts (23), MCF-7 carcinoma cells (38), and skeletal myoblasts (12) in a laminin isoform-specific manner.

The choice to compare the effect of {alpha}7-integrin with that of {alpha}1-integrin rather than with other laminin-binding integrins was based on our previous studies showing that {alpha}3-integrin, another laminin-binding integrin, is not present in VSMC under the culture conditions used (Wilson E, unpublished observation), and that {alpha}6-integrin, another laminin-binding integrin, is not present in VSMC (31). We also examined the effect of PDGF on {alpha}1-integrin expression by performing FACS analysis, and no significant difference was observed (data not shown).

Previously, PDGF was shown to increase the expression of other integrins such as {alpha}5-integrin (16, 19). Thus, to examine the possibility that increased adhesion of PDGF-treated VSMC to laminin-1 results from increased {alpha}5-integrin expression, we performed alternative adhesion assays with wells coated with anti-{alpha}5-, anti-{alpha}7-, and anti-beta1-integrin antibodies. Both control and PDGF-treated cells adhered to anti-{alpha}7-integrin antibody-coated wells more effectively; no significant cell adherence was observed with the wells coated with anti-{alpha}5-integrin antibody. We also determined the expression of other integrins in control and PDGF-treated cells. Of the integrins tested ({alpha}1, {alpha}5, {alpha}v, {alpha}7, beta1, and beta3), only {alpha}7 and beta3 showed increased expression. Minimal expression of {alpha}5-integrin level was detected under basal conditions, and PDGF has no effect on its expression (data not shown).

To investigate the extent to which increased {alpha}7-integrin subunit levels cause migratory changes of VSMC on laminin-1, we performed the migration assays using the Boyden chamber. Prolonged incubation (24 h) with PDGF did not alter serum-induced migration of VSMC on laminin-1, and preincubation with anti-{alpha}7-integrin antibody did not alter the migration of control or PDGF-treated VSMC in our study. In this respect, our results appear to contradict the observations of Yao et al. (42). Such a discrepancy may be explained on the basis of the following difference in the experimental procedures. 1) Cells were treated differently before the migration assay was performed. In our studies, VSMC were quiesced with 0.5% FBS M199 for 48 h and then treated with PDGF for 24 h before the migration assay; whereas in the study by Yao et al., cells were serum starved overnight just before the assay. 2) The choice of chemoattractant was different. We used 10% FBS M199 instead of PDGF as the chemoattractant in the lower chamber in our studies. In addition, a different source of VSMC was used in the study by Yao et al. However, our current study is consistent with the study by Ziober et al. (44), in which overexpression of {alpha}7-integrin subunit in the highly metastatic M2 cells caused the cells to be less migratory.

Studies from other groups have shown that PDGF stimulation increases expression of laminin-5 in VSMC and increases migration of VSMC on laminin-5 (22). There are several differences between these studies and our current study that may explain the discrepancy in PDGF-stimulated migration on laminin. For example, we treated the cells with PDGF 24 h before the start of the migration assay, and we used laminin-1 as a substrate, not laminin-5. Thus there may be differences in the time course of activation of migration for PDGF and differences in the specific isoform of laminin on which the cells migrate. Similarly, Crawley et al. (12) showed that anti-{alpha}7 antibody blocked migration of skeletal myoblasts on laminin-1 but suggested that the interaction with laminin-2/4 was stronger. In addition, Yao et al. (43) previously showed that {alpha}7-integrin subunit does not interact with laminin-5. Overall, these studies suggest that cellular context and specific treatments are critical in determining the migration on laminin.

Localization of {alpha}7-integrin subunit in PDGF-treated and control VSMC was assessed using immunofluorescence imaging techniques. {alpha}7-Integrin subunit displays a punctate staining pattern in control cells, whereas in PDGF-treated VSMC, a more intense staining of {alpha}7-integrin subunit is shown and displays a stress fiber-like pattern. This result further confirms our findings with FACS analysis that PDGF augments the expression of {alpha}7-integrin subunit. The stress fiber-like staining pattern of {alpha}7-integrin subunit is consistent with the staining patterns observed in skeletal myoblast (24) and suggests that the increased expression of {alpha}7-integrin subunit is spatially associated with F-actin distribution. PDGF treatment also altered the staining of F-actin, which is consistent with previous observations showing that PDGF induces the reorganization of actin (28).

The current understanding of {alpha}7-integrin subunit in cardiovascular biology is limited. A previous study by our group (10) demonstrated that {alpha}7-integrin subunit was the only integrin with substantial increases in a chemical model of atherosclerosis. A recent study by Flintoff-Dye et al. (15) demonstrated cerebrovascular defects in embryonic mice lacking {alpha}7-integrin, which implicated compromised vascular integrity. In this study, PDGF enhanced the expression of {alpha}7-integrin and consequently increased VSMC adhesion mediated by {alpha}7-integrin.

In summary, our studies demonstrate for the first time that PDGF modulates {alpha}7-integrin subunit expression in VSMC, as well as the contribution of MAP kinases (p38, ERK, and JNK) in regulating {alpha}7-integrin subunit expression. In addition, PDGF enhances {alpha}7-integrin subunit-mediated VSMC adhesion to laminin. The results of these studies and others suggest that the interaction between PDGF and {alpha}7-integrin may be important in vasculature development and during the remodeling process.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by National Institutes of Health Grants HL-62863 (to G. A. Meininger, K. S. Ramos, and E. Wilson), HL-62539 (to K. S. Ramos and E. Wilson), HL-58690 (to G. A. Meininger), and AG-14632 (to S. J. Kaufman); the Muscular Dystrophy Association (to S. J. Kaufman and E. Wilson); and Center for Environmental and Rural Health, Texas A&M University Grant ES09106. E. Wilson is a fellow of the Michael E. DeBakey Institute, Texas A&M University.


   ACKNOWLEDGMENTS
 
We thank Jane Miller for technical support with flow cytometry, Nandina Paria for technical support with the MAPK inhibitor study, Jeffrey Bray for excellent technical assistance with immunoblot analysis, and Stephanie Hilliard for editorial assistance. We thank Jan Patterson and Lisa Auckland for general help with this study.

Present addresses: J.-T. Chao, Department of Medical Pharmacology and Physiology, University of Missouri, Columbia, Columbia, MO 65212; G. A. Meininger, Dalton Cardiovascular Research Center, University of Missouri, Columbia, Columbia, MO 65212.


   FOOTNOTES
 

Address for reprint requests and other correspondence: E. Wilson, Cardiovascular Research Institute, The Texas A&M Univ. System Health Science Center, 336 Joe Reynolds Medical Bldg., College Station, TX 77843 (e-mail: emilyw{at}tamu.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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