Center for Cell Biology & Cancer Research, Albany Medical College, Albany, New York 12208, USA
*Author for correspondence (e-mail: higginp{at}mail.amc.edu)
Accepted July 16, 2001
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SUMMARY |
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Key words: PAI-1, Gene expression, Signal transduction, TGF-ß1
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INTRODUCTION |
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Injury-induced cell motility is orchestrated by various autocrine/paracrine-acting growth factors (Martin, 1997). Most prominent are members of the transforming growth factor-ß (TGF-ß), fibroblast growth factor (FGF), and epidermal growth factor (EGF) families (Boland et al., 1996; Sato and Rifkin, 1988; Song et al., 2000; Ellis et al., 2001; Goke et al., 2001). TGF-ß1 and activin A, in particular, integrate the complex processes of tissue repair and cell migration (Zambruno et al., 1995; Munz et al., 1999) largely through control of genes that encode matrix components (fibronectin, type I collagen), regulators of ECM homeostasis (e.g., uPA, PAI-1) and the cellular adhesive apparatus (e.g., PAI-1, integrin subunits) (Cajot et al., 1989; Cajot et al., 1990; Wrana et al., 1991; Munz et al., 1999; Lai et al., 2000; Providence et al., 2000). Therefore, growth factor-initiated changes in the expression, focalization and/or relative activity of uPA/PAI-1 may stimulate or inhibit cell migration via ECM barrier proteolysis or by altering cellular adhesive interactions with the ECM (Stefansson and Lawrence, 1996; Mignatti and Rifkin, 2000). Variances in PAI-1 synthesis (Providence et al., 2000) and/or site-localization (Kutz et al., 1997) would specifically impact on cellular migration by affecting uPA activity as well as uPAR/vitronectin- or integrin/vitronectin-dependent cell attachment (Ciambrone and McKeown-Longo, 1990; Deng et al., 1996; Chapman, 1997; Stefansson and Lawrence, 1996; Loskutoff et al., 1999).
Since TGF-ß1 stimulates cell motility (Kutz et al., 2001), PAI-1 induction (Boehm et al., 1999) in response to TGF-ß1 is probably critical to the motile process and the acquisition of epithelial cell plasticity (Akiyoshi et al., 2001; Zavadil et al., 2001). This was confirmed in the present study using the PAI-1-deficient 4HH cell line (Providence et al., 2000) in a quantitative model of induced cell locomotion. Therefore, it was important to define mechanisms involved in TGF-ß1-dependent PAI-1 gene expression. PAI-1 transcription in TGF-ß1-responsive T2 epithelial cells used an immediate-early, tyrosine kinase-mediated, signaling pathway. Moreover, PAI-1 induction and basal as well as TGF-ß1-stimulated T2 cell locomotion was MEK-dependent. The involvement of MEK in TGF-ß1-initiated PAI-1 expression and the adhesion-dependency of MEK activation (Renshaw et al., 1997) suggested that substrate attachment may influence TGF-ß1-induced PAI-1 gene regulation. TGF-ß1, in fact, poorly induced PAI-1 transcription in cells maintained in suspension culture but significantly increased PAI-1 expression during attachment to fibronectin-coated surfaces. Cellular adherence to fibronectin alone (i.e., in the absence of TGF-ß1), and to a lesser extent vitronectin, also stimulated PAI-1 mRNA synthesis indicating that adhesive state can modulate TGF-ß1 signaling to particular target genes (i.e., PAI-1).
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MATERIALS AND METHODS |
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Northern blot analysis
Total cellular RNA was isolated and denatured at 55°C for 15 minutes in 1x MOPS, 6.5% formaldehyde and 50% formamide prior to electrophoresis on agarose/formaldehyde gels (1.2% agarose, 1.1% formaldehyde, 1x MOPS, 50 mM sodium acetate, 1 mM EDTA, pH 8.0). RNA was transferred to Nytran membranes by capillary action in 10x SSC (3M NaCl, 0.3 M Na citrate, pH 7.0), UV crosslinked and incubated for 2 hours at 42°C in 50% formamide, 5x Denhardts solution, 1% SDS, 100 µg/ml sheared/heat-denatured salmon sperm DNA (ssDNA) and 5x SSC. RNA blots were hybridized with 32P-labeled cDNA probes for PAI-1 and A-50 (Ryan and Higgins, 1993) for 24 hours at 42°C in 50% formamide, 2.5x Denhardts solution, 1% SDS, 100 µg/ml ssDNA, 5x SSC and 10% dextran sulfate. Membranes were washed 3 times in 0.1x SSC/0.1% SDS for 15 minutes each at 42°C followed by 3 washes at 55°C prior to exposure to film.
Microscopy
Cells were washed twice with Ca2+/Mg2+ free phosphate-buffered saline (PBS-CMF; 2.7 mM KC1, 1.2 mM KH2PO4, 0.14 M NaC1, 8.1 mM Na2HPO4-7H2O) and fixed in 10% formalin/PBS-CMF for 10 minutes. Following permeabilization with cold 0.5% Triton X-100/PBS-CMF (for PAI-1 immunolocalization) or 1% NP-40/PBS-CMF (for phalloidin-actin binding) for 10 minutes at 4°C, cells were washed 3 times (5 minutes each) with PBS-CMF then overlayed with rabbit antibodies to PAI-1 (Kutz et al., 1997) in BSA (3 mg/ml)/PBS-CMF. After three PBS-CMF washes, cells were incubated with fluorescein-conjugated goat anti-rabbit IgG (1:20 in BSA/PBS-CMF) for 30 minutes at 37°C, washed, and coverslips mounted with 100 mM n-propylgalate in 50% glycerol/PBS-CMF. Rhodamine-phalloidin was used to visualize actin microfilament structures (Ryan and Higgins, 1993).
MAP kinase assays
Cells were extracted for 30 minutes in cold lysis buffer (0.5% deoxycholate, 50 mM Hepes [pH 7.5], 1% Triton X-100, 1% NP-40, 150 mM NaCl, 50 mM NaF, 1 mM Na-orthovanadate, 0.1% aprotinin, 4 µg/ml pepstatin A, 10 µg/ml leupeptin, 1 mM PMSF) and lysates clarified by centrifugation at 14,000 g for 15 minutes at 4°C. For immunoprecipitation, aliquots containing 500 µg protein were incubated with 2 µg ERK1/2 antibody for 2 hours with gentle rocking. Protein A/G Plus-agarose (30 µl) was added for 2 hours, immune complexes collected by centrifugation, washed twice with lysis buffer and twice with 100 mM NaCl in 50 mM Hepes (pH 8.0) and then incubated at 37°C for 15 minutes in kinase reaction buffer (10 µCi 32P-ATP, 50 µM ATP, 20 mM Hepes (pH 8.0), 10 mM MgCl2, 1 mM DTT, 1 mM benzamidine, 0.3 mg/ml myelin basic protein (MBP)). Electrophoresis buffer (50 mM Tris (pH 6.0), 10% glycerol, 1% SDS, 1% ß-mercaptoethanol) was added, samples boiled for 10 minutes and 15 µl aliquots separated on SDS/15% polyacrylamide gels. Proteins were transferred to nitrocellulose in 25 mM Tris, 190 mM glycine, 20% methanol and membranes exposed to film for visualization of phosphorylated MBP. Western blotting for ERK2 and total MBP detection by Ponceau S staining were used to confirm equivalent loading per lane. For detection of phosphorylated ERK1/2, membranes were washed for 10 minutes in 0.05% Triton X-100/PBS-CMF followed by 2 hours in wash solution containing 3% milk. Phospho-ERK monoclonal antibody (1:1000) was added for an overnight incubation in blocking solution at room temperature. Following 3 washes for 20 minutes, horseradish peroxidase (HRP)-labeled anti-mouse secondary antibody (1:3000 in blocking solution) was added and incubated for an additional 1 hour. Membranes were washed 5 times for 10 minutes each in wash solution, incubated with ECL substrate solution (Amersham, Piscataway, NJ) for 2 minutes with gentle rocking and exposed to film. Membranes were stripped for 90 minutes at room temperature using the Western Stripper Kit (Bioworld, Dublin, Ohio), neutralized then incubated in a ERK1/2 primary antibody mixture (each diluted 1:3000 in blocking solution) followed by HRP-anti-rabbit secondary antibody and ECL reagent as described above.
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RESULTS |
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DISCUSSION |
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TGF-ß1 exerts concentration-dependent effects on cellular locomotion in 3D culture systems (Gajdusek et al., 1993) as well as in the more spatially restricted planar model of denudation injury (this study) (Gajdusek et al., 1993; Zicha et al., 1999). Wound repair analysis of the PAI-1-deficient 4HH cell line, in which PAI-1 synthesis is specifically ablated by antisense targeting (Higgins et al., 1997; Providence et al., 2000), supports the contention that PAI-1 is an important component in the motile program in this model. Since TGF-ß1 stimulates PAI-1 synthesis and PAI-1 impacts directly on cell motility (this study) (Deng et al., 1999; Providence et al., 2000), it was important to assess TGF-ß1-dependent controls on PAI-1 expression as well as on cellular migration. TGF-ß1 initiates PAI-1 transcription in quiescent T2 cells via an immediate-early response, tyrosine kinase-dependent pathway that involves MEK, an upstream activator of ERK1/2. However, unlike the typical rapid ERK phosphorylation associated with serum-stimulation (i.e., within 15 minutes), TGF-ß1-mediated ERK activation (as assessed by phosphorylation of the target substrate MBP) was delayed by 30-60 minutes. The MEK dependency for PAI-1 expression and TGF-ß1-stimulated as well as basal migration in T2 cells suggests that these events are related. Although MEK blockade probably interferes with cell movement at several levels (Klemke et al., 1997; Rikitake et al., 2000), genetic targeting approaches confirmed that both basal and TGF-ß1-stimulated cell migration (over a 24-36 hour period) requires PAI-1 expression (Providence et al., 2000; Kutz et al., 2001). Clearly, MEK inhibition may not affect basal locomotion in all cell types (Nguyen et al., 1998) but results may depend on the specific system studied. For example, in the monolayer denudation model (unlike random motility assays), basal migration is most probably growth factor-mediated. Indeed, monolayer wounding in various cell types, in and of itself, is a sufficient stimulus to initiate autocrine growth factor expression (e.g., TGF-ß1, basic FGF, heparin-binding EGF) (Sato and Rifkin, 1988) and activate MAP kinases (Dieckgraefe et al., 1997). Moreover, growth factor synthesis and ERK phosphorylation/nuclear translocation occurs specifically in cells adjacent to the injury site (Dieckgraefe et al., 1997; Song et al., 2000; Ellis et al., 2001) similar to the distribution of locomoting PAI-1-expressing cells (Providence et al., 2000).
Matrix attachment, perhaps as part of the motile response, also stimulates PAI-1 expression. This has particular physiologic relevance. Although the present data suggest that not all matrices have equivalent inductive capability, during the process of wound healing cells switch their integrin complement to accommodate the composition of the provisional ECM (Yamada et al., 1996). In certain instances, TGF-ß1 directly mediates changes in integrin availability and, therefore, cellular adhesive traits (Collo and Pepper, 1999; Dalton et al., 1999; Lai et al., 2000). Engagement of particular integrins (i.e. vß3,
3ß1) by immobilized antibodies or ligands has been implicated in PAI-1 gene control (Ghosh et al., 2000; Khatib et al., 2001). The
3ß1 ligands laminin-5 and collagen I, when presented immobilized on beads, were also effective inducers of uPA synthesis (Ghosh et al., 2000). Similar to data reported in this study with regard to adhesive controls on PAI-1 expression, ß1 integrin aggregation-induced uPA synthesis was also MEK-dependent as PD98059 inhibited ERK activation and uPA expression. Perhaps not coincidentally, both uPA and PAI-1 are induced by pharmacologic disorganization of the actin-based microfilament system as is ERK activation (Higgins et al., 1992; Irigoyen et al., 1997). Since integrin ligation/clustering and cell adhesion result in various levels of cytoskeletal reorganization and recruitment of signaling intermediates (Zhu and Assoian, 1995; Lin et al., 1997; Miyamoto et al., 1998; Renshaw et al., 1997), the control of specific protease/protease inhibitors may be a common event in outside-in signaling initiated by adhesive state and integrin engagement. Several matrices (i.e., fibronectin vs vitronectin) clearly differ in relative capacity to induce PAI-1 expression in T2 cells allowed to adhere under growth factor-free conditions. Most novel, however, is the observation that only certain matrix attachments synergize with TGF-ß1 to achieve maximal PAI-1 expression. Whether matrix-type variations in the amplitude and duration of ERK signaling underlies this differential response in T2 epithelial cells is currently under study. Nevertheless, the present findings indicate that adhesive influences also modulate TGF-ß1 signaling to target genes (i.e. PAI-1).
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ACKNOWLEDGMENTS |
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