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Correspondence to: Martin Griffin, Dept. Life Sciences, Nottingham Trent University, Clifton Lane, Nottingham NG11 8NS, UK.
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Summary |
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Using a cytochemical approach, we examined the role of tissue transglutaminase (tTgase, Type II) in the incorporation of latent TGF-ß binding protein-1 (LTBP-1) in the extracellular matrix of Swiss 3T3 fibroblasts in which tTgase expression can be modulated through a tetracycline-controlled promoter. Increased tTgase expression led to an increased rate of LTBP-1 deposition in the matrix, which was accompanied by an increased pool of deoxycholate-insoluble fibronectin. Matrix deposition of LTBP-1 could also be reduced by the competitive amine substrate putrescine. Immunolocalization at the fluorescence and electron microscopic level showed that extracellular tTgase is located at the basal and apical surfaces of cells and at cellcell contacts, and that LTBP-1 is co-distributed with cell surface tTgase, suggesting an early contribution of tTgase to the binding of LTBP-1 to matrix proteins. LTPB-1 was also found to co-localize with both intracellular and extracellular fibronectin, and increased immunoreactivity for LTBP-1 and fibronectin was found in large molecular weight polymers in the deoxycholate-insoluble matrix of fibroblasts overexpressing tTgase. We conclude that regulation of tTgase expression is important for controlling matrix storage of latent TGF-ß1 complexes and that fibronectin may be one extracellular component to which LTBP-1 is crosslinked when LTBP-1 and tTgase interact at the cell surface. (J Histochem Cytochem 47:14171432, 1999)
Key Words: tissue transglutaminase, latent transforming growth factor-ß binding protein-1, extracellular matrix fibronectin
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Introduction |
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In most cell types, transforming growth factor-ß (TGF-ß) is secreted in a biologically inactive form, known as large latent TGF-ß complex, which consists of latent TGF-ß binding protein (LTBP, 120190 kD) disulfide-linked to the latent TGF-ß precursor, also called small latent TGF-ß complex. Latent TGF-ß precursor consists of the mature TGF-ß homodimer (25 kD) noncovalently associated with the amino terminal pro-peptide homodimer (LAP, latency associated peptide; 75 kD) (reviewed in
Four members of the LTBP family have been described thus far (reviewed in
Given the major role of TGF-ß in the regulation of ECM synthesis and degradation and its potent effect in controlling proliferation and differentiation ( (
-glutamyl)lysine bridges. Well-characterized family members include the plasma transglutaminase (Factor XIIIa), which is activated by thrombin during wounding, and the keratinocyte transglutaminase, involved in terminally differentiating keratinocytes (reviewed in
In a recent study using transfected Swiss 3T3 fibroblasts (
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Materials and Methods |
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Cell Culture and tTgase Induction Protocol
Transfected Swiss 3T3 fibroblasts, displaying inducible expression of tTgase (clone TG3 and TG27), were cultured as described by
Indirect Immunofluorescence and Image Analysis
Exponentially growing cells were seeded in 8-well glass chamber slides to obtain 105 cells/well (confluent cultures) at the time of the experiment. The cell seeding density was adjusted considering that the doubling time for clone TG3 and clone TG27 is, respectively, 23 hr and 27 hr, when calculated from cells exponentially grown for 2 days. Cells were stained in culture in serum-free Dulbecco's modified Eagle's medium (DMEM; SigmaAldrich, Poole, Dorset, UK) for 2 hr with the respective antibody or in normal serum-containing medium (deprived of selection reagents). The presence of serum made little difference to the results but prevented cells from dying and releasing tTgase when they were deprived of serum factors. After incubation, the antibody was removed with the medium and cells were fixed in 3.7% (w/v) paraformaldehyde in PBS for 15 min at room temperature (RT). Fixed cells were blocked in 3% (w/v) BSA in PBS for 30 min at RT and incubated with fluorochrome-conjugated secondary antibodies in blocking buffer for 2 hr at RT. To localize LTBP-1, cells were incubated with polyclonal antibody to human platelet LTBP-1 (Ab-39) (Pharmingen; San Diego, CA) diluted 1:100 in culture medium. Secondary antibody was an FITC-conjugated swine anti-rabbit IgG (Dako; High Wycombe, Buckinghamshire, UK). Indirect immunofluorescence of fibronectin and tTgase was performed using mouse monoclonal antibodies against, respectively, the fourth Type III repeat of plasma fibronectin (IST-3) (SigmaAldrich) diluted 1:100 and the active site of tTgase (Cub7402) (Neomarkers; Freemont, CA), 2 µg/ml. Bound antibodies were detected with an FITC-conjugated rabbit anti-mouse IgG (Dako). Double labeling of LTBP-1 and fibronectin and LTBP-1 and tTgase was performed by using Ab-39 and IST-3, and Ab-39 and Cub7402, respectively. Secondary antibodies were FITC-conjugated swine anti-rabbit IgG (Dako) and TRITC-conjugated goat anti-mouse IgG (SigmaAldrich). The concentration of secondary antibodies was approximately 20 µg/ml. Nonimmune rabbit and mouse IgGs (2 µg/ml) were used as controls instead of primary antibodies. Coverslips were mounted with Vectashield (Vector Laboratories; Peterborough, UK) and observed by confocal fluorescent microscopy using a Leica TCSNT confocal laser microscope system (Leica Lasertechnik; Heidelberg, Germany) equipped with an argon/krypton laser adjusted at 488 and 560 nm for fluorescein and rhodamine excitation. For comparison, confocal images of induced and noninduced transfected cells were obtained at constant microscope settings and corresponded to the central section of cells. Fluorescence intensity measurements of LTBP-1 were performed with the Leica TCSNT (version 1.5451) image processing menu on fixed cells. Between 100 and 300 induced and noninduced cells in random fields were compared in each independent experiment and fluorescence values were expressed per cell number after staining of nuclei with propidium iodide. Image analysis of double immunofluorescence staining was undertaken using the software Image Pro Plus (Media Cybernetics; Silver Spring, MD).
Detection of In Situ tTgase Activity
Cells were seeded into 8-well glass chamber slides as described above, allowed to settle for 4 hr and then incubated in the presence of 0.5 mM fluoresceincadaverine (Molecular Probes; Eugene, OR) in normal serum-containing medium for a total of 15 hr. Cells were fixed in methanol at -20C for 10 min and mounted (
Electron Microscopy
Cells were cultured to confluency on 0.5-cm squares of Melinex, previously conditioned by overnight incubation in serum-containing DMEM. For immunostaining of extracellular tTgase, cells were stained live in culture before fixation, using the method previously detailed for immunofluorescence. Cells were fixed in 1% (w/v) paraformaldehyde and 0.05% (w/v) glutaraldehyde in PBS, dehydrated through increasing concentrations of ethanol, and placed in hydrophilic resin [LR Gold resin and glycolmethacrylate (low acid) (6:4), plus 0.1% bezoinethylether (Taab; Berks UK)]. After several changes of resin, the samples were embedded in plastic trays and the resin polymerized using UV light (360 nm) under nitrogen gas for 24 hr at RT. The Melinex was removed and some of the resin-embedded samples were re-embedded, to allow both en face and vertical sectioning of the cells. Ultrathin sections (6090 nm) were collected on collodion [2% (w/v) in amyl acetate]-coated nickel grids. Before immunolabeling, sections were blocked with 0.5% (w/v) BSA in TBS (20 mM Tris, 225 mM NaCl, pH 7.6) and exposed to the primary antibody [mouse anti-fibronectin (IST-3, 1:2000), rabbit anti-LTBP-1 (Ab-39, 1:2500) or, for tTgase intracellular staining, mouse anti-tTgase (Cub7402, 1:500) antibody] in blocking buffer containing 0.1% (v/v) Tween-20. Grids were then incubated with the respective colloidal gold-conjugated secondary antibodies in blocking buffer without Tween-20. Secondary antibodies (BioCell; Cardiff, UK) included goat anti-rabbit antibodies (15-nm and 30-nm gold conjugate, diluted 1:60 and 1:30, respectively) and goat anti-mouse antibodies (5-nm and 15-nm gold conjugate, diluted 1:200 and 1:60, respectively). Some grids were silver-enhanced (BioCell) before counterstaining, with 2% aqueous uranyl acetate and alkaline lead citrate. Samples were viewed on a JEOL transmission electron microscope (100 CX-II).
Detection of Fibronectin and tTgase in the ECM of Cell Cultures Using a Modified ELISA
Appropriate cell numbers as defined earlier were seeded in 96-well plates and left to grow for the time required, either 48, 24, or 6 hr. For analysis of deoxycholate (DOC)-insoluble fibronectin, cells were washed twice with PBS, pH 7.4, and solubilized in 0.1% (w/v) sodium deoxycholate containing 2 mM EDTA for 10 min at RT (
Methods were first optimized for antibody concentration shown to give linear responses when 102105 cells per well were used. Nonspecific binding of primary antibody was obtained using a monoclonal antibody raised to wheat gliadins. Values (typically in the range of A450nm = 0.010.05) were subtracted from sample data before inclusion (Table 1).
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Isolation of DOC-insoluble ECMs and Immunoblotting for Fibronectin and LTBP-1
Transfected Swiss 3T3 cells, previously induced or repressed for overexpression of tTgase, were reseeded in 60-mm Petri dishes (106 cells) and cultured for 24 hr in serum-containing medium, at which time they reached confluency. Cells were extracted in PBS containing 1% (w/v) DOC and 2 mM EDTA using a modification of the method of
Statistics
Differences between datasets (shown as mean ± SD) were determined by the Student's t-test or MannWhitney test (analysis of modified ELISA) at a significance level of p< 0.05.
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Results |
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Correlation of tTgase Expression with LTBP-1 Deposition in the ECM of Swiss 3T3 Fibroblasts
After modulation of tTgase expression in Swiss 3T3 fibroblasts by tetracycline, LTBP-1 was localized in the cell lines TG3 and TG27 ( (
-glutamyl)lysine crosslinks.
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To follow the time-dependent binding of LTBP-1 to the matrix, cells were plated and stained for LTBP-1 at 6, 24, and 48 hr of culture. The number of cells seeded was adjusted so that the same number of confluent cells was present at each time interval. Consistency in cell density of both induced and noninduced clones was monitored by transmitted light microscopy (not shown) before acquisition of each image. After 6 hr of culture, hardly any LTBP-1 fibrils were detectable in the ECM of the culture exhibiting low expression of tTgase (Figure 2Ad), whereas very thin initial LTBP-1 fibrils were detected in the corresponding culture induced to express high levels of tTgase (Figure 2Aa). Within 24 hr, cells overexpressing tTgase formed a well-defined network of LTBP-1 fibers (Figure 2Ab). Similar fibers were also observed in the ECM of cells expressing background levels of tTgase, but these were shorter and appeared to be at an earlier stage of maturation, as exemplified in Figure 2Ae. At 2-day culture, LTBP-1 was localized in thicker and longer extracellular fibers in both cultures (Figure 2Ac and 2Af), although confocal microscope depth profiles indicated a more organized distribution of LTBP-1 throughout the layers of induced cells (not shown). These findings strongly suggest that an increased expression of tTgase contributes to a more rapid accumulation of LTBP-1 in the ECM of fibroblasts, leading to a more complex arrangement of LTBP-1 fibrillar structures throughout the culture. Quantification by image analysis of the time-dependent deposition of LTBP-1 confirmed these observations that an increased tTgase expression affects the rate of LTBP-1 deposition at 6 and 24 hr rather than the final level accumulated in a 2-day-old matrix (Figure 2b). In parallel to LTBP-1 staining, fibronectin deposition was also followed by immunostaining for fibronectin. At 6 hr from cell plating, fibronectin fibrils were easily detectable in both the induced and the noninduced cells (Figure 2Ag and 2Al), suggesting that fibronectin fibers appear before LTBP-1. At 24 hr, fibronectin fibrils became progressively coarser and thoroughly distributed in the ECM of both cultures (Figure 2Ah and 2Am). The immunolabeling also appeared to suggest that the appearance of the fibronectin fibers was slightly more dense in cells overexpressing tTgase (Figure 2Ag and 2Ah) at these early time periods. Fibronectin staining eventually assumed an intense meshwork pattern after 48 hr in both cultures (Figure 2Ai and 2An). Immunolocalization of tTgase at the same time intervals revealed that in cells induced to overexpress the enzyme, extracellular tTgase was detectable starting from 6-hr culture (Figure 2Ao2Aq). However, unlike fibronectin and LTBP-1, tTgase was not found uniformly distributed in the monolayer of 2-day cultured cells but was often present in dense patches localized between cells in areas of high cell confluency (Figure 2Aq). The endogenous level of tTgase expressed by the noninduced cells was hardly detectable at each time interval using immunofluorescence (Figure 2Ar2At), although we have previously confirmed the presence of extracellular tTgase in noninduced Swiss 3T3 fibroblasts by developing cell surface-specific tTgase activity assays (
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Detection of DOC-insoluble Fibronectin and Extracellular tTgase by Modified ELISA
Changes in matrix fibronectin on induction of tTgase expression, which were apparent from immunofluorescent stainings, were examined in more depth using a procedure designed to detect more specifically ECM-associated fibronectin. DOC-insoluble fibronectin was quantified using a modified immunoassay based on ELISA. Cells were first removed from the matrix by extraction with 0.1% DOC containing 2 mM EDTA (to inhibit tTgase-mediated crosslinking of fibronectin) (
Because there is a general degree of difficulty in detecting externalized tTgase by immunocytochemical staining, we sought other methods to quantify the amount of tTgase deposited in the extracellular space over the time periods the cells were cultured. To this end, we developed a modified ELISA optimized for detection of extracellular tTgase. In keeping with the method used for immunofluorescence studies, primary antibody was added to cells in live culture, thus preventing measurement of intracellular enzyme that might bind to the ECM during cell lysis, given its high affinity for fibronectin (
LTBP-1 Co-localizes with In Situ tTgase Activity
In previous reports, tTgase activity was visualized in situ by incorporation of the competitive amine substrate fluoresceincadaverine into endogenous substrates (-glutamyl residues that are used by the enzyme during protein crosslinking. In cells induced to overexpress tTgase, the fibrillar nature of LTBP-1 staining, which was revealed by immunofluorescence in a 15-hr culture (Figure 3B), was found to co-localize with in situ tTgase activity (Figure 3A). Fluoresceincadaverine was incorporated into a network of fibrils that appeared to cover the surface of adherent cells, indicating that under normal conditions tTgase is predominantly active extracellularly. However, the labeling was more prominent in certain fibers likely to correspond to areas of higher cell density, where LTBP-1 was also found to be co-localized (see arrows in Figure 3A and Figure 3B). Interestingly, the fluorescence was almost completely abolished on treatment of monolayer with plasmin (0.1 U/ml) for 1 hr at 37C (not shown), indicating that fluoresceincadaverine was incorporated into proteins that are plasmin-sensitive, such as LTBP-1 (see also Figure 1B1H). When measured by image analysis, 63.6 ± 20.8% (n = 4) of the total LTBP-1 was found to co-localize with the fluoresceincadaverine labeling (the high standard deviation was caused by occasional spots of insoluble fluorescent substrates that remained on the specimen after methanol washing). The highly reduced level of immunoreactive LTBP-1 in the confluent monolayer of noninduced cells expressing low endogenous levels of tTgase (Figure 3D) suggests that fluoresceincadaverine led to some inhibition of LTBP-1 deposition in the matrix (compare the LTBP-1 immunostaining in Figure 3D and Figure 1C) in a manner comparable to that of high concentrations of putrescine in the high tTgase-expressing cells (Figure 1I1L).
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Co-distribution of LTBP-1 with Extracellular tTgase and Fibronectin
To further evaluate the role of tTgase and fibronectin in the matrix deposition of LTBP-1, studies were undertaken to localize LTBP-1 with respect to extracellular tTgase and fibronectin by double immunolabeling analysis at the fluorescence and electron microscopic levels. Immunolocalizations were undertaken on confluent Swiss 3T3 fibroblasts after 15-hr culture. To facilitate the detection of extracellular tTgase, cells induced for increased expression of the transfected enzyme were used. Double immunofluorescent labeling of LTBP-1 and tTgase revealed that the fibrillar nature of LTBP-1 staining (Figure 4A) partly overlapped with the staining of extracellular tTgase (Figure 4B). Unlike LTBP-1, tTgase appeared to be preferentially concentrated at the cell surface at areas of cellcell contact (see arrows in Figure 4B) where LTBP-1 and tTgase occurred together, as clearly shown by the yellow-orange fluorescence observed by superimposition of the two fluorochromes (see arrows in Figure 4C). The use of image analysis to quantify the percentage of total tTgase staining co-localized with that of LTBP-1 indicated that 67.3 ± 9.8% (n = 10) of the extracellular tTgase was associated with LTBP-1. At the ultrastructural level, immunogold labeling of tTgase revealed very dense labeling of cells on their apical and basal surfaces and at cellcell contacts (Figure 5A), thus showing agreement with the immunofluorescence data shown in Figure 4B. The co-localization of LTBP-1 and tTgase was further confirmed by double immunogold labeling of induced Swiss 3T3 cells using antibodies against the two proteins, which were revealed by secondary antibodies combined with different-sized gold particles. A close association between LTBP-1 and tTgase (represented by 30-nm and 15-nm gold particles, respectively) was found in proximity of the cell surface (Figure 5B) and at cell junctions (Figure 5C, arrows), but tTgase and LTBP-1 were found to be less co-distributed in the extracellular matrix (see arrow in Figure 5D). This finding was consistent with the immunofluorescence data (Figure 4A4C) and suggests that the initial association of LTBP-1 with tTgase takes place close to the cell surface and at cellcell contacts, where the enzyme is often found in dense clusters (see Figure 5A). When cells were stained for LTBP-1 and fibronectin by immunofluorescence, the fibrillar staining pattern of LTBP-1 (Figure 4D) and fibronectin (Figure 4E) also demonstrated co-localization (Figure 4F). By using image analysis, we found that 66.1 ± 4.9% (n = 3) of the total fibronectin staining co-localized with that of LTBP-1. When double immunogold labeling experiments were performed to demonstrate this association at the electron microscopic level, LTBP-1 and fibronectin, represented by the 15-nm and 5-nm gold particle, respectively, showed extensive co-localization (Figure 6), once again agreeing with immunofluorescence data. Interestingly, LTBP-1 and fibronectin, both of which use the ER/Golgi secretory pathway, were also detected in close proximity to one another in the intracellular environment (see arrowheads in Figure 6), suggesting that they may be present in the same secretory vesicle during their passage to the outside of the cell.
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Analysis of DOC-insoluble ECM Proteins by SDS-PAGE
DOC-insoluble ECM proteins were prepared from previously induced and noninduced Swiss 3T3 cells (clone TG3), which were cultured for a further 24 hr to reach confluency under the same conditions shown in Figure 2 and then analyzed by immunoblotting. Proteins were fractionated by nonreducing SDS-PAGE, Western-blotted, and immunoprobed with antibodies to fibronectin and LTBP-1 consecutively, with stripping of the blot between application of each antibody. By probing with anti-fibronectin antibody, increased amounts of fibronectin polymers, consisting of both disulfide-linked and (
-glutamyl)lysine-linked proteins, were found at the top of both the resolving and stacking gel of the induced fibroblasts (Figure 7B) compared to noninduced cells (Figure 7A). Given that polymer formation is related to fibronectin fibril assembly, this result is in agreement with a significantly increased level of fibronectin measured by ELISA in the DOC-insoluble ECM of induced fibroblasts compared to noninduced fibroblasts (Table 1, FN). Similarly, the immunoprobing of the same blot for LTBP-1, which is reactive in all its forms with Ab-39 only in a nonreduced state (
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Discussion |
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The storage and subsequent release of matrix-bound latent TGF-ß is now recognized as a key mechanism in matrix remodeling that has important consequences for tissue repair, wound healing, and development (-glutamyl residues, in these cells. In a time-dependent study in which induced and noninduced cells were grown to confluency for 6, 24, and 48 hr, we demonstrate that cells overexpressing tTgase accumulated LTBP-1 in the matrix more rapidly than those carrying endogenous levels of tTgase. Moreover, the distribution pattern, particularly at 24-hr culture, appeared more fibrillar and complex, thus confirming the importance of extracellular tTgase in LTBP-1 deposition. However, the small differences in LTBP-1 immunostaining shown between the 48-hr cultures of induced and noninduced cells indicate that the total amount of LTBP-1 bound by tTgase in a long-term culture is likely to be the same in fibroblasts with endogenous and increased level of tTgase. We do not know the reason for this, but our findings (not shown) rule out a saturation of the immunolocalization assay at the antibody concentration employed. A possible explanation is that the matrix becomes saturated with LTBP-1 at 48 hr, which is in keeping with any enzyme reaction given that there is only one variable, the enzyme concentration. Parallel staining of fibronectin at similar time intervals indicated the appearance of a fibronectin network before the deposition of LTBP-1, suggesting that fibronectin may be essential for LTBP-1 deposition to take place. The density of the fibronectin network appeared slightly greater in cells overexpressing tTgase. This microscopic observation was confirmed by quantifying fibronectin with a modified ELISA technique. This procedure, which was specifically designed to detect DOC-insoluble, ECM-associated fibronectin, showed that cells displaying increased tTgase accumulated significantly more fibronectin fibrils at each time of culture. Interestingly, differences measured in the pool of ECM fibronectin were greater than predicted from immunofluorescence cell staining of the total pool of extracellular fibronectin, thus showing that modulation of tTgase affects the insoluble fibronectin fraction. In the high tTgase-expressing cells, tTgase accumulation paralleled that of LTBP-1, whereas in the cells expressing endogenous tTgase, deposition of extracellular tTgase was clearly detectable by immunofluorescence cell staining only after 2-day culture. However, as previously reported (
Our results strongly suggest that tTgase and LTBP-1 interact close to the cell surface. Superimposition of the different staining patterns for tTgase and LTBP-1 obtained by double immunofluorescent labeling suggests that co-localization is always at its greatest around the immediate vicinity of the cell. This observation was confirmed at the electron microscopic level by use of double immunogold labeling of tTgase and LTBP-1, which showed a close co-distribution of the two proteins, particularly at the cell surface and at areas between cells. This finding was also supported by immunogold staining of tTgase, which showed a preferential localization of the enzyme in dense clusters close to the cell surface and at areas of cellcell contact. It is noteworthy that these data provide the first evidence at the electron microscopic level for the extracellular location of tTgase antigen obtained in cells in culture (Figure 5A). The degree of partial co-localization of tTgase with LTBP-1 that was measured at the fluorescent level is consistent with the association of tTgase with other proteins at the cell surface.
When the distribution of LTBP-1 and fibronectin was analyzed by double immunogold labeling, LTBP-1 was found to be associated with fibronectin in the extracellular and intracellular environment, suggesting that these two proteins share a common secretory vesicle. This is the first evidence to indicate that LTBP-1 and fibronectin may share a common secretory pathway. However, it should be noted that other workers have shown co-localization of LTBP-1 with fibrillin (
It is known that purified tTgase has high affinity for fibronectin ( (
-glutamyl)lysine crosslinks than their noninduced counterparts and also display nonreducible high molecular weight polymers of cellular fibronectin (
The binding of large latent TGF-ß1 complex to the matrix has been suggested as another level of control for TGF-ß, whereby this important cytokine can exert its effects on cells at a later date after release from the matrix and subsequent activation (vß6 integrin during injury (
In conclusion, this study is the first to demonstrate that the specific regulation of tTgase expression leads to changes in stored LTBP-1 in the matrix when detected directly by immunofluorescence. Using a novel modified ELISA, we also demonstrate for the first time that increased expression of tTgase leads to an increased deposition of fibronectin in the matrix. By modulating tTgase expression in cells, we also provide novel data on the co-localization of tTgase and LTBP-1 and provide evidence strongly suggesting that the crosslinking of LTBP-1 by tTgase might occur when both proteins are in close association with each other at the cell surface. This study has also demonstrated the close association of LTBP-1 with fibronectin, both intracellularly and in the extracellular matrix, and given that both these proteins are found at greater levels in high molecular weight protein polymers in cells overexpressing tTgase, our data strongly suggest that fibronectin is one of the proteins to which LTBP-1 is linked via tTgase.
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Acknowledgments |
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Supported by a grant from the EPSRC (Ref GR/L43688).
We wish to thank P.J.A. Davies (University of Texas Health Center, Houston) for interesting discussions on TGF-ß/tTgase induction in fetal development, M. Armitage and J. Adams (Smith & Nephew; York, UK) for providing assistance in image analysis, and R. Jones, A. Mezzogiorno, and A. Hargreaves for helpful advice in this study.
Received for publication April 23, 1999; accepted June 4, 1999.
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