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
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SUMMARY |
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Key words: Extracellular matrix, Fibroblast, Asthma, Integrin, Remodeling, Fibroblast
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INTRODUCTION |
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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 -smooth muscle actin (
-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
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 v and ß1 integrins (Dahm and Bowers, 1998). Although VN signals through at least two other integrins,
vß3 and
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 vessels 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 -SMA expression. By contrast, blocking VN integrin function using specific blocking antibodies induced
-SMA expression, whereas blocking CN or FN integrin function had no effect. Furthermore,
-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.
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MATERIALS AND METHODS |
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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 -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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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 vß3,
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 -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
-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 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 -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
-SMA. Membranes were then stripped, reblocked and probed with a MoAb against
-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-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 manufacturers 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.
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RESULTS |
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Cycloheximide (CHX) and actinomycin D (AD) inhibit -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 vß5 integrin-induced
-SMA expression (Fig. 6A). By contrast, LM609-induced
-SMA expression was almost totally inhibited by both CHX and AD (Fig. 6B). For P5D2,
-SMA expression showed a similar pattern of responsiveness to that seen with P1F6 (Fig. 6C).
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DISCUSSION |
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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 -SMA, whereas exposure to other ECM proteins FN, CN or LN had no effect. By contrast, exposure of these cells to fb-MoAb against
v, ß1,
vß3 and
vß5 integrins over a 24 hour period significantly increased
-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
-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 -SMA expression are integrin dependent. Expression of
-SMA induced by fb-MoAb against VN integrins was abrogated by inhibiting PI-3 kinase and PKC pathways. Expression of
-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
-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
-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
-SMA expression in HFL-1 cells.
Confocal microscopy was used to visualize the expression and assembly of -SMA in response to VN or antibodies to VN integrins. Immunofluorescent staining of untreated fibroblasts revealed a low level of expression of
-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,
-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
-SMA or F-actin. Similarly, addition of VN itself did not influence the organization of
-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 -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
-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 -SMA expression. By contrast, function-blocking antibodies against VN integrins upregulated
-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).
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ACKNOWLEDGMENTS |
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