Regulation of human lung fibroblast phenotype and function by vitronectin and vitronectin integrins

Amelia K. Scaffidi1,2, Yuben P. Moodley1,2, Markus Weichselbaum1,2, Philip J. Thompson1,2 and Darryl A. Knight1,2,*

1 Asthma and Allergy Research Institute, Nedlands, Western Australia, 6009
2 Department of Medicine, University of Western Australia, Nedlands, Western Australia, 6009

*Author for correspondence (e-mail: dknight{at}cyllene.uwa.edu.au)

Accepted June 25, 2001


    SUMMARY
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 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Myofibroblasts, characterised by high expression of {alpha}-smooth muscle actin ({alpha}-SMA), are important and transient cells in normal wound healing but are found in increased number in various pathological conditions of the lung including asthma and pulmonary fibrosis. The mechanisms that regulate the myofibroblast phenotype are unknown but are likely to involve signals from the extracellular matrix transmitted via specific integrins. Vitronectin is a glycoprotein released during inflammation and has been shown to regulate the phenotype of vascular smooth muscle cells via {alpha}v and ß1 integrins. In the current study we have examined whether vitronectin influences the phenotype and function of normal human lung fibroblasts (HFL-1). Incubation of HFL-1 cells with vitronectin induced a concentration-dependent reduction in {alpha}-SMA expression. By contrast, function-blocking monoclonal antibodies to the vitronectin integrins {alpha}v, ß1, {alpha}vß3 and {alpha}vß5 induced the expression of {alpha}-SMA and its organization into stress fibers. Expression of {alpha}-SMA induced by all function-blocking monoclonal antibodies was abrogated by inhibition of protein kinase C and phosphatidylinositol-3 kinase, but the effects of inhibition of other signalling pathways was integrin dependent. Exposure to other extracellular matrix proteins such as fibronectin, collagen or their integrins did not influence expression of {alpha}-SMA. The expression and organization of {alpha}-SMA induced by exposure to function-blocking antibodies was translated into an augmented capacity of HFL-1 cells to contract fibroblast populated collagen gels. By contrast, contraction of collagen gels following incubation with vitronectin was not significantly different to control. This study has shown that vitronectin influences the phenotype and behaviour of HFL-1 cells by downregulating the expression of {alpha}-SMA and reducing their contractile ability. By contrast, occupancy of specific integrins by function-blocking antibodies upregulated the expression of {alpha}-SMA and induced the formation of functional stress fibers capable of contracting collagen gels. These results suggest that vitronectin modulates the fibroblast-myofibroblast phenotype, implying an important role in the remodelling process during lung development or response to injury.

Key words: Extracellular matrix, Fibroblast, Asthma, Integrin, Remodeling, Fibroblast


    INTRODUCTION
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
One of the key events in wound repair is the infiltration of fibroblasts from surrounding tissue to an extracellular matrix (ECM) of fibronectin (FN), collagens (CN), vitronectin (VN) and fibrin. Within the ECM fibroblasts proliferate and differentiate into cells called myofibroblasts that share phenotypic and behavioral features resembling those of fibroblasts and of smooth muscle cells (Serini and Gabbiani, 1999). Under normal conditions myofibroblasts play a crucial role in ECM deposition and subsequent wound contraction and then disappear as the fibrotic response diminishes and normal structure and function is achieved (Gailit and Clark, 1994; Welch et al., 1990; Phan, 1996).

In diseases such as asthma, the consequence of repeated inflammation includes structural remodelling of the airway wall, which is in part, characterized by exaggerated ECM deposition and excessive subepithelial collagen deposition (Roche et al., 1989; Brewster et al., 1990). An increased number of myofibroblasts are also observed and several studies have demonstrated a significant correlation between the number of these cells and the thickness of the collagen layer (Brewster et al., 1990; Gizycki et al., 1997), suggesting a causal relationship.

The hallmark of the myofibroblast phenotype is the expression of {alpha}-smooth muscle actin ({alpha}-SMA) (Serini and Gabbiani, 1999). The factors that regulate the expression and fate of myofibroblasts in the lung are unknown, but alterations in the expression of a variety of growth factors and cytokines during the inflammatory and postinflammatory states are likely to contribute. More recent evidence, however, points to these interactions being controlled by the ECM. These cell-ECM interactions are mediated by specific cell-surface receptors or integrins. Integrins are heterodimeric transmembrane glycoproteins consisting of {alpha} and ß chains and they have important roles in many biological processes such as wound healing and cell growth and survival (Hynes, 1992).

Vitronectin is a glycoprotein found in human serum and ECM and is found in increased amounts in inflammatory lung diseases (Teschler et al., 1993) and cryptogenic fibrosing alveolitis (Pohl et al., 1991). Exposure of vascular smooth muscle cells to VN has been shown to result in a loss of contractility as well as changes in morphology, adhesive properties and sensitivity to mitogens. These phenotypic changes induced by VN are mediated via {alpha}v and ß1 integrins (Dahm and Bowers, 1998). Although VN signals through at least two other integrins, {alpha}vß3 and {alpha}vß5, these did not appear to be involved in the phenotypic changes. The resultant noncontractile cell is thought to play an important role in the blood vessel’s response to injury. However, whether VN or other ECM proteins influence the phenotype of other cell types such as fibroblasts is unknown.

In the present study, we have used nontransformed normal human lung fibroblasts to demonstrate that VN downregulates {alpha}-SMA expression. By contrast, blocking VN integrin function using specific blocking antibodies induced {alpha}-SMA expression, whereas blocking CN or FN integrin function had no effect. Furthermore, {alpha}-SMA appeared to be involved in functional stress fiber formation, as incubation of HFL-1 cells with blocking antibodies against VN integrins produced a rapid contraction of fibroblast-populated collagen lattices (FPCL). Taken together, our data suggests that VN regulates the phenotype and function of human lung fibroblasts.


    MATERIALS AND METHODS
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Normal diploid human fetal lung fibroblasts (HFL-1) were obtained from American Type Culture Collection (ATCC, Manassas, VA). Cells were cultured at 37°C and 5% CO2 in either DMEM (Life Technologies, Mulgrave, Australia) supplemented with 10% fetal calf serum (Life Technologies), 2 mM L-glutamine, penicillin and gentamicin (Sigma, Botany, Australia), or serum-free DMEM (SF-DMEM) supplemented with insulin, basic fibroblast growth factor (bFGF) (Edward Keller, Hallam, Australia), L-glutamine and antibiotics. For experiments, cells cultured in the presence of serum were washed extensively in PBS and quiesced in DMEM without any additives (basal medium) for 24 hours.

ECM proteins, inhibitors and antibodies
CN I, FN and laminin were purchased from Boehringer Mannheim (Castle Hill, Australia). Wortmannin, actinomycin D, cyclohexamide, VN, poly-lysine, TRITC-conjugated phalloidin, peroxidase-conjugated anti-mouse IgG, monoclonal antibody to {alpha}-SMA (clone 1A4) and streptavidin-HRP were obtained from Sigma. Function-blocking monoclonal antibodies (fb-MoAb) as well as polyclonal antibodies used in the study are outlined in Table 1. Fluorescent Alexa-488 dye was purchased from Molecular Probes (Eugene, OR). PD98059, genestein, herbimycin, calphostin C and cytochalasin D were purchased from Calbiochem (Alexandria, Australia). The Src inhibitor pp2 was a generous gift of Marie Bogoyevitch (Biochemistry Department, University of Western Australia). C3 exoenzyme was purchased from Cayman Chemicals (Ann Arbor, MI). RGD peptides were purchased from Life Technologies.


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Table 1.
 
Measurement of {alpha}-SMA by ELISA
Cells were seeded in a 96-well plate and allowed to adhere for 16 hours, after which the media was replaced with basal DMEM and specific treatments added for a further 24 hours. Cells were washed in PBS/0.5% Tween-20 and then fixed in ice-cold methanol for 20 minutes. Cells were incubated with a monoclonal antibody to {alpha}-SMA for 60 minutes. After washing, cells were incubated with HRP-conjugated anti-mouse IgG (1:4000, 60 minutes) and the reaction was allowed to develop by addition of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) substrate/H2O2 for 60 minutes prior to reading the absorbance at 414 nm using a Spectramax 250 ELISA plate reader (Molecular Devices, Sunnyvale, CA).

Flow cytometry
Cells were cultured in six-well culture plates as described above and harvested using trypsin/EDTA and resuspended in SF-DMEM supplemented with 1% BSA. For cell-surface expression of integrins, cells were incubated with fb-MoAbs to {alpha}vß3, {alpha}vß5 or ß1 for 1 hour at room temperature. Cells were washed in PBS/0.1% Tween-20 before incubation with FITC-conjugated rabbit anti-mouse IgG (1:500) for 30 minutes at 4°C, washed resuspended in SF-DMEM and analyzed on a FACScan flow cytometer (Becton Dickinson, CA).

Confocal microscopy
Cells were dispersed onto poly-lysine-coated coverslips in SF-DMEM and allowed to adhere for 4-6 hours. Cells were then washed in PBS and incubated in basal media for an additional 16 hours after which treatments were added for a further 24 hours. Cells were fixed in 1% paraformaldehyde for 30 minutes and permeabilised for 3 minutes with 0.2% Triton X-100. Coverslips were rinsed in PBS and cells incubated with monoclonal antibody to either {alpha}-SMA or VN for 60 minutes. Following several washes, coverslips were incubated in the dark for 60 minutes in PBS containing rabbit anti-mouse IgGs conjugated to Alexa-488. After washing, coverslips were incubated in DAPI (2 µg/ml) or TRITC-phalloidin (0.5 µg/ml) for 45 minutes, washed and mounted with fade-resistant aqueous mounting medium. Z-series projections of fluorescent images of {alpha}-SMA or F-actin were obtained using a confocal laser-scanning microscope (BioRad MRC 1000) using COMOS software. Image processing was performed using Confocal Assistant software and Adobe Photoshop.

Immunoprecipitation and western blotting
Cells were lysed in RIPA buffer (Boehringer Mannheim) and centrifuged at 11,000 g for 20 minutes at 4°C. The supernatant was pre-cleared by incubation with protein A-agarose (Santa Cruz, San Diego, CA) for 1 hour at 4°C with end-over-end mixing. Following centrifugation, supernatants were incubated with antibodies against {alpha}v integrins for 1 hour at 4°C. Immune complexes were precipitated by addition of protein A-agarose for 2 hours. Agarose beads with immune complexes bound were then washed three times in RIPA buffer, boiled for 5 minutes and fractionated on a 4% stacking/12.5% resolving gel and transferred to PVDF membrane. The membrane was blocked overnight in 5% skim milk powder in Tris-buffered saline/0.25% Tween-20 (TTBS) and then incubated with anti-ß1; 1:500 (P5D2) for 1 hour at room temperature. After washing, HRP-conjugated anti-mouse IgGs were added (1:2000, 1 hour). Protein was visualized using ECL (Amersham).

For surface biotinylation experiments, cells were labeled with 1 µg/ml NHS-LC-Biotin (Pierce, Rockford, IL) in PBS on ice for 90 minutes, washed in PBS containing 50 mM glycine prior to immunoprecipitation. Biotinylated proteins were probed with peroxidase-conjugated steptavidin and visualized using ECL.

For western blot of {alpha}-SMA, protein was extracted by incubation with lysis-buffer (50 mM Tris, 0.5 mM EGTA, 150 mM NaCl, 1% Triton X-100, pH 7.5) containing a cocktail of protease inhibitors and centrifugation. The protein content of the resultant supernatant was determined using the Bradford method. Equal amounts of protein (40 µg) were added to SDS-PAGE buffer, boiled for 5 minutes and electrophoresed on an 8-16% gradient gel prior to western analysis for {alpha}-SMA. Membranes were then stripped, reblocked and probed with a MoAb against {alpha}-tubulin.

Fibroblast populated collagen lattices
Trypsinized cells (1x105 cells/ml) were mixed with a solution containing rat-tail collagen (final concentration 1.25 mg/ml), DMEM and HEPES. Suspensions were exposed to anti-{alpha}vß5 (P1F6) (2 µg/ml) in the presence or absence of inhibitors as outlined, dispensed into 24-well tissue culture plates and allowed to incubate for 1 hour at 37°C. Basal media containing a similar concentration of P1F6 was then added and gels released from the sides of the well and incubated at 37°C in 5% CO2 for up to 72 hours. Gel contraction was measured at 24 hour intervals, taking the length of both axes to provide a measurement of area and expressed as a percentage of the original gel area.

Transient transfection
Fugene (Roche Diagnostics, Castle Hill, Australia) was used for the transient transfection of HFL-1 cells with the C3-exoenzyme, used in the FPCL experiments. Fugene transfection reagent was purchased from Roche Diagnostics. Transfection was performed according to the manufacturer’s instructions. Transfection efficiency for these experiments was 49%.

Statistical methods
Data are expressed as mean±s.e.m. of at least four experiments performed in duplicate. Statistical comparisons of mean data were performed using one-way ANOVA with Bonferroni correction performed post-hoc to correct for multiple comparisons. A P value <0.05 was considered significant.


    RESULTS
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Detection of VN and VN integrins in HFL-1 cells
To determine whether HFL-1 cells express specific VN integrins, we performed flow cytometry using a panel of antibodies recognizing VN integrins or specific subunits. Fig. 1A shows that HFL-1 cells express {alpha}v- and ß1-containing integrins as well as {alpha}vß5 and {alpha}vß3 on their cell surface. As antibodies to {alpha}vß1 are not available, we performed immunoprecipitation to confirm that the {alpha}v subunit associates with ß1 (Fig. 1B). In some experiments, cells were surface biotinylated prior to immunoprecipitation (lane 1). Cell lysates were immunoprecipitated with L230 and western analysis was performed using peroxidase-conjugated steptavidin (lane 1) or P5D2 (lane 2). HFL-1 cells also demonstrated specific staining for VN as shown by confocal microscopy. Inspection of Z-series projections (Fig. 2a) clearly shows VN immunoreactivity within the cell and in a fibrillar form on the cell surface. High magnification single optical sections (Fig. 2b) show that VN may also be bound to the cell membrane.



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Fig. 1. Integrins recognizing VN are present on HFL-1 cells. (A) HFL-1 cells were stained with no antibody (white peaks) or with monoclonal antibodies LM609 (anti-{alpha}vß3), P1F6 (anti-{alpha}vß5), P5D2 (anti-ß1) or L230 (anti-{alpha}v) (black peaks) followed by FITC-labeled rabbit anti-mouse IgG. Immunostaining was then analyzed by flow cytometry. (B) In lane 1, cells were surface biotinylated, lysed and cell extracts immunoprecipitated with L230 (anti {alpha}v). In lane 2, untreated cells were lysed and extracts were immunoprecipitated with L230. P5D2 (anti ß1) was used in western analysis to establish that the {alpha}vß1 complex was present in HFl-1 cells.

 


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Fig. 2. Detection of VN on HFL-1 cells. HFL-1 cells were stained with a monoclonal antibody recognizing VN followed by FITC-labeled goat anti-mouse IgG. Sections were counterstained with DAPI to visualize cell nuclei (blue) and analyzed using confocal microscopy. (a) A pseudo coloured z-series projection shows specific VN immunoreactivity (green stain) in the intracellular compartment and on the cell membrane (arrows). DAPI staining of nuclei is shown in blue. (b) A higher magnification single optical section shows intense localization to the cell membrane (arrowhead). Bar, 100 µm.

 
Expression of {alpha}-SMA is regulated by VN and integrins recognizing VN
Vitronectin induced a concentration-dependent reduction in {alpha}-SMA content in HFL-1 cells over a 24 hour period as determined by western blot. However, incubation with FN, CN and laminin had no effect (Fig. 3). The use of fb-MoAb against VN integrins {alpha}v (L230), ß1 (P5D2), {alpha}vß3 (LM609) and {alpha}vß5 (P1F6) augmented the intracellular content of {alpha}-SMA over a 24 hour period (Fig. 4A). The data obtained by western blot was confirmed and extended using ELISA (Fig. 4B). By contrast, exposure of HFL-1 cells to antibodies recognizing {alpha}2, {alpha}5 or ß6 subunits did not increase {alpha}-SMA expression above control levels. Furthermore, exposure to the RGD peptide GRGDdSP or the preferential peptide inhibitor of {alpha}vß3, the cyclic RGD peptide GPenGRGDSPCA did not influence {alpha}-SMA expression above control levels (Fig. 4B). The {alpha}v, ß1 and ß3 subunits mediate binding to a variety of ECM proteins including FN, CN and VN. However, the lack of effect of other integrin subunits involved in binding FN ({alpha}5, ß6) or CN ({alpha}2) suggests that this effect on {alpha}-SMA expression may be more specific for VN integrins.



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Fig. 3. Expression of {alpha}-SMA in HFL-1 cells induced by VN. Equal amounts of protein from HFL-1 cells was exposed to VN (0.01, 0.1, 1 µg/ml), FN (10 µg/ml), CN I (10 µg/ml) or laminin (10 µg/ml), subjected to SDS-PAGE and probed by western blotting using a monoclonal antibody to {alpha}-SMA. Membranes were stripped and reprobed with a monoclonal antibody for tubulin, as a protein loading control. Blots are representative of at least three separate experiments.

 


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Fig. 4. Expression of {alpha}-SMA is induced in HFL-1 cells by function-blocking antibodies to VN integrins. (A) Protein from HFL-1 cells exposed to P1F6 (anti-{alpha}vß5), L230 (anti-{alpha}v), LM609 (anti-{alpha}vß3) or P5D2 (anti-ß1) was subjected to SDS-PAGE and probed by western blotting using a monoclonal antibody to {alpha}-SMA. Membranes were stripped and reprobed with a monoclonal antibody for tubulin, as a protein loading control. Blots are representative of at least three separate experiments. (B) HFL-1 cells were incubated with monoclonal antibodies recognizing specific {alpha}-subunits: L230 ({alpha}v), P1E6 ({alpha}2), P3D10H5 ({alpha}5); specific ß-subunits: 10D5.8 (ß6), P5D2 (ß1), 25E11 (ß3); integrin heterodimers: LM609 ({alpha}vß3) and P1F6 ({alpha}vß5) or RGD peptides GRGDdSP and GPenGRGDSPCA for 24 hours. Cells were permeabilised, fixed in methanol and incubated with a monoclonal antibody to {alpha}-SMA followed by HRP-conjugated rabbit anti-mouse IgGs. Expression of {alpha}-SMA was then analyzed by ELISA. Results are calculated from triplicate wells of at least four different experiments and are expressed as mean±s.e.m. *Significantly greater {alpha}-SMA expression compared to control cells, P<0.05.

 
VN integrins use different intracellular pathways to regulate {alpha}-SMA expression
The effects of inhibition of signal transduction pathways involved in mediating the upregulation of {alpha}-SMA expression are shown in Fig. 5A-D. Expression of {alpha}-SMA induced by P1F6, LM609 and P5D2 was effectively blocked by inhibiting phosphatidylinositol-3 kinase (PI-3 kinase) and PKC activity using wortmannin and calphostin c, respectively. By contrast, inhibition of mitogen-activated protein kinase (MAPK) using PD98059 did not influence the effect of either P1F6 or P5D2, but significantly inhibited {alpha}-SMA expression induced by LM609. Perhaps most striking was the observation that {alpha}-SMA expression induced by P5D2 did not appear to be dependent on either MAPK, tyrosine kinase (using the tyrosine kinase inhibitors genestein and herbimycin) or src activity (using the src inhibitor pp2) (Fig. 5C). Similar findings were obtained using a different fb-MoAb to ß1 integrins, clone P4C10 (data not shown). Additional immunoprecipitation experiments demonstrated that tyrosine phosphorylation of multiple proteins resulted from cell exposure to P5D2.



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Fig. 5. Expression of {alpha}-SMA in HFL-1 cells is mediated by PI-3 kinase and PKC. HFL-1 cells were incubated with either the MAPK inhibitor PD 98059 (50 µM), the tyrosine kinase inhibitors genestein (10-100 µM) and herbimycin (2 µM), the src inhibitor pp2 (10 µM), the PI-3 kinase inhibitor wortmannin (100 nM) or the PKC inhibitor calphostin c (1 µM) for 60 minutes prior to incubation with P1F6 (A) LM609 (B) or P5D2 (C) for a further 24 hours. HFL-1 cells were incubated with the Rho inhibitor C3-exoenzyme (2.5 µg/ml) for 60 minutes prior to the addition of anti-ß1 (P4C10), LM609 or P1F6 for 24 hours (D). Expression of {alpha}-SMA was analyzed using ELISA. Results are calculated from triplicate wells of at least four different experiments and are expressed as mean±s.e.m. *Significantly less {alpha}-SMA expression compared to cells exposed to function-blocking antibody alone, P<0.05.

 
Transfection of cells with C3-exoenzyme to inhibit Rho-GTPases did not significantly influence {alpha}-SMA expression induced by any of the fb-MoAb used in the study (Fig. 5D).

Cycloheximide (CHX) and actinomycin D (AD) inhibit {alpha}-SMA expression
Pretreatment of HFL-1 cells with the protein synthesis inhibitor CHX inhibited the effects of P1F6 by approximately 50%, suggesting that de novo protein synthesis only partly accounts for {alpha}vß5 integrin-induced {alpha}-SMA expression (Fig. 6A). By contrast, LM609-induced {alpha}-SMA expression was almost totally inhibited by both CHX and AD (Fig. 6B). For P5D2, {alpha}-SMA expression showed a similar pattern of responsiveness to that seen with P1F6 (Fig. 6C).



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Fig. 6. Cells were treated with the translation inhibitor cyclohexamide (10 µM) or the transcription inhibitor actinomycin D (1 µM) for 45 minutes prior to incubation with P1F6 (A) LM609 (B) or P5D2 (C) for a further 24 hours. Expression of {alpha}-SMA was analyzed using ELISA. Results are calculated from triplicate wells of at least four different experiments and are expressed as mean±s.e.m. *Significantly less {alpha}-SMA expression compared to cells exposed to function-blocking antibody alone, P<0.05.

 
{alpha}-SMA expression – confocal microscopy
To determine whether increased {alpha}-SMA expression was organized into stress fiber assembly (F-actin), cells were subjected to double staining immunofluoresence analysis for {alpha}-SMA and total filamentous actin expression. Untreated cells rarely expressed {alpha}-SMA organized as stress-fibers (Fig. 7a). Expression of F-actin, however, appeared to be uniform throughout the cell population. Incubation with fb-MoAb against {alpha}vß3 (LM609, Fig. 7b), ß1 (P5D2, Fig. 7c) or {alpha}vß5 (P1F6) all appeared to increase the amount and organization of {alpha}-SMA stress fiber assembly. By contrast, incubation with fb-MoAb against ß6, (Fig. 7e) was without effect. Incubation with VN (1 µg/ml, Fig. 7f) also failed to induce {alpha}-SMA expression or stress fiber assembly.



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Fig. 7. Assembly of {alpha}-SMA and F-actin in HFL-1 cells is induced by function-blocking antibodies to VN integrins. HFL-1 cells were incubated with no antibody (a) or LM609 (anti-{alpha}vß3, b), P5D2 (anti-ß1, c), P1F6 (anti-{alpha}vß5, d), 10D5.8 (anti-ß6, e) or with VN itself (f). Dual-label images of HFL-1 cells were obtained by confocal immunofluoresence microscopy of fixed cells following staining with {alpha}-SMA (green) and TRITC-phalloidin to label filamentous actin (red). Yellow/orange staining represents intense areas of colocalization of green and red fluorescence. Bar, 50 µm.

 
Ligation of {alpha}vß5 induces contraction of FPCL
Control FPCL consisting of type I collagen and fibroblasts in basal medium contracted slowly over a 48 hour period, reaching 78% of their original area and further decreased in area over the following 24 hours reaching a maximum of 69% of their initial area (Fig. 8A). By contrast, FPCL incubated with P1F6 contracted rapidly, reaching 27% of their initial area within the first 48 hours and by 72 hours, gels were 23% of their initial area. Contraction of FPCL induced by P1F6 was blocked by inhibition of Rho-GTPases (C3-exoenzyme), F-actin assembly (cytochalasin-D), tyrosine kinase activity (genestein), PKC (calphostin c) and PI-3 kinase (wortmannin). By contrast, inhibition of MAPK was only partially effective. Addition of two concentrations of VN resulted in contraction of FPCL, although the rate and magnitude of contraction was not different to control FPCL (Fig. 8B). Using confocal microscopy, all inhibitors were shown to disrupt {alpha}-SMA cytoskeletal formation and colocalization with F-actin (Fig. 9a-f).



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Fig. 8. Contraction of FPCL is induced following exposure of HFL-1 to function-blocking antibodies against VN integrins. (A) Fibroblast populated collagen gels were exposed to culture medium alone ({blacksquare}), P1F6 ({blacktriangleup}), MAPK inhibitor PD98059 ({blacktriangledown}) or P1F6 in the presence of PD98059 ({blacklozenge}), PKC inhibitor calphostin c, PI-3 kinase inhibitor wortmannin, Rho-GTPases inhibitor C3-exoenzyme, F-actin inhibitor cytochalasin D or tyrosine kinase inhibitor genestein (all ). Gel dimensions were determined after 24, 48 and 72 hours. (B) Fibroblast populated collagen gels were exposed to culture medium alone ({blacksquare}) or VN 1 µg/ml ({blacktriangleup}) or 10 µg/ml ({blacktriangledown}). Gel dimensions were determined after 24, 48 and 72 hours. Results are calculated from duplicate wells of at least four different experiments and are expressed as mean±s.e.m. *Significantly less gel area compared to cells exposed to medium alone, P<0.05.

 


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Fig. 9. Assembly of {alpha}-SMA induced by P1F6 in HFL-1 cells is disrupted by agents that inhibit collagen gel contraction. HFL-1 cells were incubated with or P1F6 in the presence of PKC inhibitor calphostin c (a), tyrosine kinase inhibitor genestein (b), src inhibitor pp2 (c), MAPK inhibitor PD98059 (d), PI-3 kinase inhibitor wortmannin (e) or Rho-GTPases inhibitor C3-exoenzyme (f). Dual-label images of HFL-1 cells were obtained by confocal immunofluoresence microscopy of fixed cells following staining with {alpha}-SMA (green) and TRITC-phalloidin to label F-actin (red). Bar, 50 µm.

 

    DISCUSSION
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
It is increasingly recognized that fibroblasts have a plastic phenotype and are capable of fulfilling distinct functions in normal and pathological situations (Schmitt-Graff et al., 1994). The transient appearance of myofibroblasts, distinguishable by the expression {alpha}-SMA, is crucial for normal wound repair. However, increased numbers of these cells are seen in fibrotic diseases of several organs, including the lung and in chronic asthma (Phan, 1996). However, the mechanisms involved in modulating {alpha}-SMA expression are not well understood. By using specific ELISA, western analysis and FACS we have demonstrated that human lung fibroblasts express the VN integrins {alpha}vß3, {alpha}vß5 and {alpha}vß1 and that fb-MoAbs against these integrins induce the expression and organization of {alpha}-SMA in human lung fibroblasts. This process appeared to be specific to VN integrins, as fb-MoAbs to {alpha}5, {alpha}2, {alpha}9 or ß6 integrins were without effect. Furthermore, incubation with VN itself induced a dose-dependent decrease in {alpha}-SMA expression, whereas incubation with FN and CN were without effect. Increases in {alpha}-SMA expression were associated with an increase in actin stress fiber assembly and enhanced contraction of FPCL. Taken together, this data strongly suggests that VN can regulate the phenotype and function of human lung fibroblasts.

Using confocal microscopy, we have shown that HFL-1 cells stain positive for VN. Previous studies have demonstrated that VN binds in a fibrillar pattern to the ECM immediately adjacent to fibroblast monolayers (Panetti and McKeown-Longo, 1993; Memmo and McKeown-Longo, 1998). Our data showing distinct ‘cables’ of VN immunoreactivity support this finding. In addition, we have also demonstrated that VN is present on the cell membrane and in intracellular pools. Whether the intracellular VN was endocytosed (Pijuan-Thompson and Gladson, 1997; Memmo and McKeown-Longo, 1998) from the ECM or, alternatively, was synthesized endogenously (Chakravortty and Kumar, 1999) is unknown. Nevertheless, the presence of VN on the cell membrane suggests that VN integrins may be ligated and active.

The clustering of integrins into focal adhesions on the cell surface caused either by antibodies or by binding to ECM leads to the activation of a variety of intracellular events that promote re-organization of the actin cytoskeleton (Aplin et al., 1998). In the current study, we have shown that exposure of HFL-1 cells to VN dose-dependently reduced the expression of {alpha}-SMA, whereas exposure to other ECM proteins FN, CN or LN had no effect. By contrast, exposure of these cells to fb-MoAb against {alpha}v, ß1, {alpha}vß3 and {alpha}vß5 integrins over a 24 hour period significantly increased {alpha}-SMA expression, whereas exposure to fb-MoAb recognizing FN or CN integrins was ineffective. Furthermore, the use of cyclic RGD peptides specific for VN integrins failed to influence {alpha}-SMA expression, suggesting that rather than acting as passive integrin blocking agents, fb-MoAbs are capable of initiating an intracellular response (Miyamoto et al., 1995). In a more recent study, Cruz et al. (Cruz et al., 1997) demonstrated that activating antibodies to ß1 integrins will bind a distinct population of integrins that is different from that seen when a function-blocking antibody is used. These authors suggested that the activating and function-blocking antibodies are able to distinguish between two functional states of the integrin.

The majority of signaling molecules implicated in integrin/ECM interactions, including Rho-GTPases, focal adhesion kinase (FAK) and MAPK, are ubiquitous mediators of signal transduction. However, in the present study we have shown that signal transduction pathways involved in the induction of {alpha}-SMA expression are integrin dependent. Expression of {alpha}-SMA induced by fb-MoAb against VN integrins was abrogated by inhibiting PI-3 kinase and PKC pathways. Expression of {alpha}-SMA induced by P1F6 and LM609 was also reduced by the tyrosine kinase inhibitors genestein and herbimycin, as well as the src inhibitor pp2. Our results suggest that FAK may be involved given that autophosphorylation of FAK facilitates binding to the SH2 domain of src and FAK (Howe et al., 1998), which, in turn, is linked to a number of intracellular pathways including the activation of PI3-K. Only LM609-mediated {alpha}-SMA expression was affected by inhibition of MAPK. In the absence of soluble factors, autophosphorylation of FAK can lead to a Src- and Ras-dependent activation of MAPK (Howe et al., 1998). However, in normal fibroblasts the activation of MAPK may occur independently of FAK (Wary et al., 1996). Our results using P1F6 support this latter finding and suggest that there is a divergence in integrin-mediated signaling. Surprisingly, expression of {alpha}-SMA induced by P5D2 was also independent of tyrosine kinase, src and MAPK activity. Immunoprecipitation demonstrated that P5D2 induced tyrosine phosphorylation of FAK. Thus, it does not appear that FAK is involved in P5D2-mediated {alpha}-SMA expression in HFL-1 cells.

Confocal microscopy was used to visualize the expression and assembly of {alpha}-SMA in response to VN or antibodies to VN integrins. Immunofluorescent staining of untreated fibroblasts revealed a low level of expression of {alpha}-SMA that did not appear to be polymerized. Expression of F-actin was readily detectable and present in the form of stress fibers in both treated and untreated cells, with no apparent differences. Upon exposure to P1F6, P5D2 and LM609, {alpha}-SMA expression was markedly increased and appeared to become orientated into stress fibers, whereas exposure to an antibody recognizing the ß6 integrin did not influence the expression of either {alpha}-SMA or F-actin. Similarly, addition of VN itself did not influence the organization of {alpha}-SMA.

The reorganization of collagen fibrils by fibroblasts has been proposed as the mechanism by which these cells contract collagen gels. Indeed, fibroblasts embedded in three-dimensional collagen gels demonstrate contractile properties similar to wound fibroblasts in vivo and hence provide a useful model to study particular events of connective tissue re-organization and wound healing (Stephens et al., 1997; Defilippi et al., 1999; Ehrlich et al., 1998). The current study demonstrated that P1F6 markedly enhanced the contraction of FPCL over a 72 hour period. By contrast, VN did not influence the contraction of FPCL when compared to control gels. Analysis of signaling pathways used in the contraction of FPCL showed that MAPK was not significantly involved. By contrast, tyrosine kinases and PI-3 kinase, as well as PKC, appear to be required for P1F6-mediated contraction of FPCL. Transfection of fibroblasts with C3-exoenzyme also prevented contraction of FPCL, implicating the Rho family of GTPases. This family of small peptides has been previously shown to regulate the actin cytoskeleton and formation of stress fibers within several cell types. Disruption of F-actin stress fiber assembly with cytochalasin-D also completely prevented FPCL contraction. These functional results were supported by confocal microscopy, which demonstrated the disruption of {alpha}-SMA assembly and colocalization with F-actin. The contractility of fibroblasts in a three dimensional collagen gel is dependent on a variety of factors including cell number, collagen concentration and the presence of serum or other soluble factors (Defilippi et al., 1999). However, in this study, a constant number of cells were added to the same concentration of collagen and all experiments were performed in serum-free conditions. This suggests that the increased {alpha}-SMA expression induced by P1F6 is organized so as to facilitate contractile responses.

In conclusion, this study has shown that exposure of human lung fibroblasts to the ECM protein VN dose-dependently downregulated {alpha}-SMA expression. By contrast, function-blocking antibodies against VN integrins upregulated {alpha}-SMA expression, organization and function in these cells. Other ECM proteins such as FN, CN and LN or their ligands were without effect. These results suggest that exposure to VN may act to retain human lung fibroblasts in a noncontractile and possibly pluripotential state, the behaviour of which would be influenced by the local ECM environment. The phenotypic modulation of fibroblasts into myofibroblasts during wound healing represents an example of cellular adaptation, resulting in the appearance of cells with specialized functions. Indeed, due to their number, function(s) and location, myofibroblasts are potentially important effector cells in a number of lung diseases including idiopathic pulmonary fibrosis and asthma (Phan, 1996; Zhang et al., 1994; Zhang et al., 1997).


    ACKNOWLEDGMENTS
 
The authors thank David Cheresh, Scripps Research Institute, for the provision of the LM609 antibody and Marie Bogoyevitch, Department of Biochemistry, University of Western Australia for provision of the pp2. We also acknowledge Dean Sheppard, University of California at San Francisco for provision of Y9A2, P3D10H5, L230, P5D2 and 10D5.8 antibodies and for his helpful comments during the preparation of this manuscript. We also thank Paul Rigby, Biomedical Confocal Microscopy Research Centre (BCMRC), University of Western Australia for assistance with the confocal microscopy as well as Neil Misso and Steven Mutsaers for their critical appraisal of this manuscript. This work is funded by the National Health and Medical Research Council (NHMRC) of Australia.


    REFERENCES
 Top
 SUMMARY
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

Aplin, A. E., Howe, A., Alahari, S. K. and Juliani, R. L. (1998). Signal transduction and signal modulation by cell adhesion receptors – the role of integrins, cadherins, immunoglobulin-cell adhesion molecules and selectins. Pharmacol. Rev. 50, 197-263.[Abstract/Free Full Text]

Brewster, C. E., Howarth, P. H., Djukanovic, R., Wilson, J., Holgate, S. T. and Roche, W. R. (1990). Myofibroblasts and subepithelial fibrosis in bronchial asthma. Am. J. Respir. Cell Mol. Biol. 3, 507-511.[Medline]

Chakravortty, D. and Kumar, K. S. (1999). Interaction of lipopolysaccharide with human small intestinal lamina propria fibroblasts favors neutrophil migration and peripheral blood mononuclear cell adhesion by the production of proinflammatory mediators and adhesion molecules. Biochim. Biophys. Acta. 1453, 261-272.[Medline]

Cruz, M. T., Dalgard, C. L. and Ignatius, M. J. (1997). Functional partitioning of ß1 integrins revealed by activating and inhibitory mAbs. J. Cell Sci. 110, 2647-2659.[Abstract/Free Full Text]

Dahm, L. M. and Bowers, C. W. (1998). Vitronectin regulates smooth muscle contractility via {alpha}v and ß1 integrin. J. Cell Sci. 111, 1175-1183.[Abstract/Free Full Text]

Defilippi, P., Olivo, C., Venturino, M., Dolce, L., Silengo, L. and Tarone, G. (1999). Actin cytoskeleton organization in response to integrin-mediated adhesion. Microsc. Res. Tech. 47, 67-78.[Medline]

Ehrlich, H. P., Cremona, O. and Gabbiani, G. (1998). The expression of {alpha}2 ß1 integrin and alpha smooth muscle actin in fibroblasts grown on collagen. Cell. Biochem. Funct. 16, 129-137.[Medline]

Gailit, J. and Clark, R. A. (1994). Wound repair in the context of extracellular matrix. Curr. Opin. Cell Biol. 6, 717-725.[Medline]

Gizycki, M. J., Adelroth, E., Rogers, A. V., O’Byrne, P. M. and Jeffery, P. K. (1997). Myofibroblast involvement in the allergen-induced late response in mild atopic asthma. Am. J. Respir. Cell Mol. Biol. 16, 664-673.[Abstract]

Howe, A., Aplin, A. E., Alahari, S. K. and Juliano, R. L. (1998). Integrin signaling and cell growth control. Curr. Opin. Cell Biol. 10, 220-231.[Medline]

Hynes, R. O. (1992). Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69, 11-25.[Medline]

Liaw, L., Skinner, M. P., Raines, E. W., Ross, R., Cheresh, D. A., Schwartz, S. M. and Giachelli, C. M. (1995). The adhesive and migratory effects of osteopontin are mediated via distinct cell surface integrins. J. Clin. Invest. 95, 713-724.[Medline]

Memmo, L. M. and McKeown-Longo, P. (1998). The alpha v beta 5 integrin functions as an endocytic receptor for vitronectin. J. Cell Sci. 111, 425-433.[Abstract/Free Full Text]

Miyamoto, S., Akiyama, S. K. and Yamada, K. M. (1995). Synergistic roles for receptor occupancy and aggregation in integrin transmembrane function. Science 267, 883-885.[Medline]

Panetti, T. S. and McKeown-Longo, P. J. (1993). The alpha v beta 5 integrin receptor regulates receptor-mediated endocytosis of vitronectin. J. Biol. Chem. 268, 11492-11495.[Abstract/Free Full Text]

Phan, S. H. (1996). Role of the myofibroblast in pulmonary fibrosis. Kidney Int. Suppl. 54, S46-48.[Medline]

Pijuan-Thompson, V. and Gladson, C. L. (1997). Ligation of integrin alpha5beta1 is required for internalization of vitronectin by integrin alphavbeta3. J. Biol. Chem. 272, 2736-2743.[Abstract/Free Full Text]

Pohl, W. R., Conlan, M. G., Thompson, A. B., Ertl, R. F., Romberger, D. J., Mosher, D. F. and Rennard, S. I. (1991). Vitronectin in broncholveolar lavage fluid is increased in patients with interstitial lung disease. Am. Rev. Respir. Dis. 143, 1369-1375.[Medline]

Roche, W. R., Beasley, R., Williams, J. H. and Holgate, S. T. (1989). Subepithelial fibrosis in the bronchi of asthmatics. Lancet i, 520-524.

Schmitt-Graff, A., Desmouliere, A. and Gabbiani, G. (1994). Heterogeneity of myofibroblast phenotypic features: an example of fibroblastic cell plasticity. Virchows Arch. 425, 3-24.[Medline]

Schoenwaelder, S. M. and Burridge, K. (1999). Bidirectional signaling between the cytoskeleton and integrins. Curr. Opin. Cell Biol. 11, 274-286.[Medline]

Serini, G. and Gabbiani, G. (1999). Mechanisms of myofibroblast activity and phenotypic modulation. Exp. Cell Res. 250, 273-283.[Medline]

Stephens, P., Genever, P. G., Wood, E. J. and Raxworthy, M. J. (1997). Integrin receptor involvement in actin cable formation in an in vitro model of events associated with wound contraction. Int. J. Biochem. Cell Biol. 29, 121-128.[Medline]

Teschler, H., Pohl, W. R., Thompson, A. B., Konietzko, N., Mosher, D. F., Costabel, U. and Rennard, S. I. (1993). Elevated levels of bronchoalveolar lavage vitronectin in hypersensitivity pneumonitis. Am. Rev. Respir. Dis. 147, 332-337.[Medline]

Wary, K. K., Maineiro, F., Isoda, H. and Guan, J. L. (1996). The adapter protein shc couples a class of integrins to the control of cell cycle progression. Cell 87, 733-743.[Medline]

Welch, M. P., Odland, G. F. and Clark, R. A. (1990). Temporal relationships of F-actin bundle formation, collagen and fibronectin matrix assembly, and fibronectin receptor expression to wound contraction. J. Cell Biol. 110, 133-145.[Abstract]

Zhang, H. Y., Gharaee-Kermani, M. and Phan, S. H. (1997). Regulation of lung fibroblast alpha-smooth muscle actin expression, contractile phenotype, and apoptosis by IL-1beta. J. Immunol. 158, 1392-1399.[Abstract]

Zhang, K., Rekhter, M. D., Gordon, D. and Phan, S. H. (1994). Myofibroblasts and their role in lung collagen gene expression during pulmonary fibrosis. A combined immunohistochemical and in situ hybridization study. Am. J. Pathol. 145, 114-125.[Abstract]