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(Investigative Ophthalmology and Visual Science. 2004;45:955-963.)
© 2004 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.03-0210
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TGF-ß2–Induced Cell Surface Tissue Transglutaminase Increases Adhesion and Migration of RPE Cells on Fibronectin through the Gelatin-Binding Domain

Siegfried G. Priglinger, Claudia S. Alge, Aljoscha S. Neubauer, Nadine Kristin, Christoph Hirneiss, Kirsten Eibl, Anselm Kampik, and Ulrich Welge-Lussen

From the Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Germany.


   Abstract
Top
 Abstract
Material and Methods
 Results
 Discussion
 References
 
PURPOSE. Migration and adhesion of dislocated retinal pigment epithelial (RPE) cells to a fibronectin-rich extracellular matrix is an initial step in proliferative vitreoretinopathy (PVR). In the present study, the functional role of cell surface tissue transglutaminase (tTG) in adhesion and migration of RPE cells on fibronectin (Fn) and collagen type I (Col I) after stimulation with TGF-ß2 was investigated.

METHODS. Cultured human RPE cells were treated with 1.0 ng/mL TGF-ß2 for 24 hours. Cell surface tTG expression was determined by cell fraction analysis. Attachment on Col I, full-length Fn, and its 45-kDa gelatin-binding and 110-kDa cell-binding fragment was measured with an MTT assay. Migration of RPE cells was measured by a Boyden chamber assay, and cell spreading was determined. Experiments were performed in the presence or absence of anti-tTG antibodies and anti-integrin {alpha}5 and ß1 antibodies.

RESULTS. TGF-ß2 markedly induced expression of cell-surface tTG on RPE cells and increased attachment and migration on Fn and Col I. Blocking cell surface tTG inhibited attachment, migration, and spreading on Fn and its 45-kDa gelatin-binding fragment, whereas no effect was seen on Col I and the 110-kDa cell-binding Fn fragment. In contrast, blocking of integrin {alpha}5 and ß1 suppressed adhesion and migration on full-length Fn and the 110-kDa Fn fragment.

CONCLUSIONS. These data demonstrate that TGF-ß2 increases expression of cell surface tTG, which in turn strengthens adhesion, migration, and spreading of RPE cells on Fn through the 45-kDa gelatin-binding Fn fragment. At the onset of PVR, this mechanism may help RPE cells to attach and migrate on Fn-containing matrices.

Proliferative vitreoretinopathy (PVR) is the most common cause of surgical failure in the treatment of rhegmatogenous retinal detachment. It is characterized by the formation of epi- and subretinal membranes on the neuroretinal surface.1 2 3 4 5 6 There is accumulating evidence that the formation of a PVR membrane is a protracted wound-healing response, a process that is characterized by extracellular matrix (ECM) accumulation and degradation, cell–matrix adhesion, and cell migration. Retinal pigment epithelium (RPE) cells are considered to play an important role in the development and onset of PVR. Detachment of RPE cells from their subretinal monolayer after retinal injury, such as retinal detachment or trauma, appears to be the crucial event in early PVR. These detached RPE cells proliferate, migrate, and attach to a provisional ECM, mainly composed of collagen type I (Col I) and fibronectin (Fn).7 8 Attachment studies have demonstrated that RPE cells have a clear substrate preference for Fn.9 The binding of RPE cells to Fn is mainly mediated by the integrin {alpha}5ß1.10 11 Previous studies have shown that membrane-bound cell surface tissue transglutaminase (tTG) increases the binding of cells to Fn by acting as an integrin-associated adhesion coreceptor for Fn.12

Transglutaminases are a group of protein cross-linking enzymes that polymerize proteins into high-molecular-mass aggregates through intramolecular {epsilon}-({gamma}-glutamyl) lysine bonds. tTG is the most ubiquitous member of this family of enzymes that covalently cross-link proteins in a Ca2+-dependent manner.13 tTG is mainly localized in the cytoplasm. In addition, some amounts of the enzyme are present on the cell surface and in the ECM.14 15 One well-documented property of membrane-bound cell surface tTG is the ability to cross-link ECM proteins, such as Fn, fibrinogen, osteopontin, and laminin–nidogen complexes.16 17 18 19 20 21 This function of cell surface tTG may help to stabilize the ECM and basement membranes.16 17

Recently a novel function of cell surface tTG as an integrin-associated adhesion coreceptor for Fn has been described.12 Surface tTG exerts this function by associating with several ß1 integrins while simultaneously binding to Fn through the gelatin-binding domain (42- or 45-kDa, modules I6II1,2I7-9).12 22 23 24 This Fn-binding site lacks integrin-binding motifs that are located on the cell-binding domain (110-kDa, modules III9III10). Using artificial, immortal, partly transfected human or animal fibroblast and tumor cell lines, investigators in recent studies have demonstrated that surface tTG promotes adhesion, migration, and enhancement of spreading of cells adhering to the isolated gelatin-binding domain of Fn and to whole Fn.12 25 26 27 28 29 The function of cell surface tTG in cell adhesion is distinct from and independent of its enzymatic (cross-linking) activity.

We have recently demonstrated that tTG is highly expressed, both in cultured RPE cells and in PVR membranes.30 After RPE dedifferentiation, tTG accumulates rapidly to very high levels. Treatment with transforming growth factor (TGF)-ß2, a growth factor known to be increased in the vitreous of patients with PVR,31 further increases the expression of tTG.30

Several lines of evidence suggest that cell-surface–associated tTG mediates cell adhesion and migration of different cell types to Fn.12 25 26 27 28 32 It has been shown that TGF-ß affects cell adhesion properties, including stimulation of ECM production and enhancement of cell motility.33 34 Whether TGF-ß2–mediated upregulation of tTG has a functional relevance in the initial process of PVR has not been investigated so far. By studying cell surface tTG as a mediator of RPE adhesion and migration in dedifferentiated and TGF-ß2–treated RPE cells, we addressed the possible functional role of cell surface tTG in vitro.


   Material and Methods
Top
 Abstract
 Material and Methods
Results
 Discussion
 References
 
Human RPE Cell Culture
Eighteen human donor eyes were obtained from the Munich University Hospital Eye Bank and processed within 5 to 13 hours after death. The donors ranged in age between 15 and 71 years. None of the donors had a known history of eye disease. Methods for securing human tissue were humane, included proper consent and approval and complied with the Declaration of Helsinki. Human retinal pigment epithelial (RPE) cells were harvested according to a procedure described previously.30 35 In brief, whole eyes were thoroughly cleansed. After removal of the anterior segment from each donor eye, the neural retinas were carefully peeled away from the RPE-choroid-sclera. The eyecup was rinsed with Ca2+ and Mg2+-free Hanks’ balanced salt solution and treated with 0.25% trypsin (Invitrogen-Gibco) for 1 hour at 37°C. The trypsin was aspirated and the eyecup filled with Dulbecco’s modified Eagle’s medium (DMEM; Biochrom, Berlin, Germany) supplemented with 20% fetal calf serum (FCS; Biochrom).

The RPE cell suspension was transferred to a 50-mL flask (Falcon, Wiesbaden, Germany) containing 20 mL of DMEM supplemented with 20% FCS and maintained at 37°C and 5% carbon dioxide. Epithelial origin and the absence of contaminating macrophages (anti-CD11; Sigma-Aldrich, Diesenhofen, Germany) and endothelial cells (anti-von Willebrand factor; Sigma-Aldrich) was confirmed as described (data not shown).30 34

For growth factor experiments, dedifferentiated RPE cells were grown to confluence and incubated overnight in serum-free DMEM. The next day, the medium was replaced with serum-free DMEM supplemented with 1.0 ng/mL TGF-ß2 (R&D Systems, Wiesbaden, Germany) for 24 hours. Control cells were incubated under identical conditions without growth factors in the medium.

Western Blot Analysis
Cytosolic and membrane protein fractions were isolated as described previously by Kim et al.37 RPE cells grown on 35-mm2 tissue culture dishes were washed twice with ice-cold PBS, collected, and lysed with 0.1 M Tris acetate (pH 7.5), 1 mM EDTA, containing protease inhibitors (5 µg/mL leupeptin, 5 µg/mL aprotinin, 50 µg/mL calpain inhibitor I, 100 µg/mL bestatin, and 1 mM phenylmethylsulfonyl fluoride; Complete Mini; Roche, Mannheim, Germany). The homogenates then were centrifuged at 10,000g for 30 minutes at 4°C in a microfuge (5810R; Eppendorf, Hamburg, Germany). The resultant supernatant, which contained the cytosolic protein fraction, was removed and stored at -80°C for future use, whereas the pellet containing the membrane fraction was extracted with the same buffer, supplemented with 1% Triton X-100, incubated for 10 minutes at room temperature, and pelleted again. This procedure released the membrane-bound tTG into the supernatant. Both fractions were used for Western blot analysis. The protein content was measured using the bicinchoninic acid (BCA) protein assay reagent (Pierce Biotechnology, Rockford, IL). Denatured proteins (2 µg) were separated under reducing conditions by electrophoresis, using a 5% SDS-polyacrylamide stacking gel and a 8% SDS-polyacrylamide separating gel, transferred with semidry blotting onto a polyvinyl difluoride membrane (Roche) and probed with a mouse-anti-tTG antibody, as described before.30 Visualization of the alkaline phosphatase was achieved using chemiluminescence. Luminescent agent (CDP-star; Roche) was diluted 1:100 in detection buffer and the filter was incubated for 5 minutes at room temperature. After it had air dried, the semidry membrane was sealed in a plastic bag. Chemiluminescence was detected with an imager (LAS-1000; RayTest, Pforzheim, Germany). To show that Western blot conditions during final development were linear, showing stable relations of protein fractions, different charge-coupled device (CCD) exposure times (1–30 minutes) of each gel were routinely compared. Quantification of chemiluminescence was performed with the accompanying software package (AIDA; RayTest).

Cell Adhesion Assay
Ninety-six-well plates (Nunc, Wiesbaden, Germany) were coated with Col I, full-length Fn, and its 45-kDa gelatin-binding (Sigma-Aldrich) and 110-kDa cell-binding fragments (Upstate Biotechnology, Hamburg, Germany)24 for 16 hours at 4°C. Each coating protein was solubilized in 1x PBS (pH: 7.4) to yield a final concentration of 50 µg/mL, and a volume of 70 µL was added to individual wells. The plates were then blocked with 2 mg/mL ovalbumin (Sigma-Aldrich) in PBS for 1 hour at 37°C.

RPE cells, pretreated or not with TGF-ß2, were isolated by trypsinization, were washed once in DMEM with 10% FCS to stop trypsin activity and twice with serum-free DMEM to remove serum components. For function-blocking experiments, RPE cell suspensions were incubated with 10 µg/mL blocking polyclonal anti-tTG (Upstate Biotechnology) or mouse antibody CUB 7402 against tTG (Quartett, Berlin, Germany), function-blocking mouse antibodies against {alpha}5 and ß1 integrins (Chemicon, Hofheim, Germany), or control nonimmune mouse IgG for 1 hours at 4°C. All antibodies were used as purified IgG.

Suspensions of 2 x 104 viable RPE cells were then added to each well and allowed to attach for 1 hour at 37°C and 5% CO2. To determine RPE cell adhesion, plates were then carefully washed three times with PBS using an automated plate washer (Molecular Devices, Garching, Germany). The cell viability was evaluated using trypan blue staining (data not shown).

For determination of the attachment strength, a modified assay was used.38 39 Coating, treatment, and plating of RPE cells was performed as described earlier. After incubation for 1 hour at 37°C and 5% CO2, the plates were inversely centrifuged in the swinging-bucket microplate holder of a centrifuge (Mikrofuge; Eppendorf) at 500, 1000, and then 2000g for 2 minutes.

The adherent cells, both after washing and centrifugation experiments, were detected by the tetrazolium dye-reduction assay (MTT; 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide). The test was performed as described by Mosmann40 with some modifications. In brief, after complete removal of remaining medium, 200 µL MTT working solution per well (1.5 mL MTT stock [2 mg/mL in PBS] plus 28.5 mL DMEM) was added. RPE cells were then incubated at 37°C for 1 hour and carefully monitored so that the Formazan crystals did not form outside the cells. Formazan crystals were then dissolved by the addition of dimethyl sulfoxide (DMSO; 200 µL/well) and allowed to dissolve with careful rocking on a plate shaker for 10 minutes. Absorption was measured by a scanning multiwell spectrophotometer at 550 nm (Molecular Devices). The number of attached living cells was proportional to the absorbance of MTT at 550 nm.

Cell Migration Assay
Migration was assayed by a modification of the Boyden chamber method, using microchemotaxis chambers (Neuro Probe, Gaithersburg, MD) and polycarbonate filters (Nucleopore, Karlsruhe, Germany) with a pore size of 8.0 µm.41 42 The filters were coated with 50 µg/mL of Col I, full-length Fn or its 45-kDa gelatin-binding or 110-kDa cell-binding fragment, or ovalbumin and placed between the chambers. RPE cells with or without TGF-ß2 treatment were trypsinized and suspended at a concentration of 5 x 105 cells/mL in DMEM supplemented with 0.5% FCS. For function-blocking experiments RPE cells were pretreated as described earlier. The RPE suspension (500 µL) was placed in the upper chamber, and 125 µL of DMEM containing 20 ng/mL human recombinant platelet-derived growth factor (PDGF-BB; PeproTech, London, UK) was placed in the lower chamber.43 The chamber was incubated at 37°C and 5% CO2 for 5 hours. The filter then was removed, and the RPE cells on the upper side of the filter were scraped off with a cotton tip. The RPE cells that migrated to the lower side of the filter were fixed in methanol and stained with hematoxylin. Five randomly chosen fields were counted at 200x magnification with a phase-contrast microscope (Leica, Wetzlar, Germany). Experiments were performed in triplicate and were repeated at least three times.

Cell-Spreading Assay
Cell spreading was assayed on 24-well tissue culture plates coated with 50 µg/mL of full-length Fn or its 45-kDa gelatin-binding or 110-kDa cell-binding fragment or on uncoated plates. RPE cells were treated as described earlier, isolated by trypsinization (0.25% trypsin in 5 mM EDTA), washed once in DMEM with 10% FCS and twice with medium without serum, plated at a concentration of 5 x 104 cells per well, and allowed to spread for 4 hours at 37°C in 5% CO2.25 Cells were washed three times with PBS, fixed with 4% paraformaldehyde in PBS, stained with Coomassie brilliant blue for 3 to 4 minutes, and rinsed again with PBS. To quantify cell spreading, four separate fields were photographed by phase contrast microscope (Leica) and spread cells, which had a clearly defined halo of cytoplasm around the nucleus, were counted. Each experiment was performed in triplicate wells and repeated at least three times.


   Results
Top
 Abstract
 Material and Methods
 Results
Discussion
 References
 
Cell Surface TTG Induction by TGF-ß2
To elucidate whether TGF-ß2 influences the expression of the membrane-bound fraction of tTG in RPE cells, protein lysates containing different cellular compartments were prepared. Western blot analysis of untreated RPE cells revealed that approximately 30% of the total tTG protein resided in the membrane fraction (Fig. 1) . After treatment with TGF-ß2, the amount of the membrane-bound surface tTG increased sixfold. In contrast, the cytosolic fraction showed only a 2.5-fold increase after TGF-ß2 treatment. Different CCD exposure times of each gel revealed stable relations of protein fractions, confirming linear response range of Western blot conditions during final development (data not shown).



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  		FIGURE 1. Western blot analysis of the cellular distribution of tTG from cultured RPE. Cells were treated with 1.0 ng/mL TGF-ß2 for 24 hours. Lysates from approximately equal amounts of membrane rich and cytosolic fractions (2 µg) were separated by SDS-polyacrylamide gel electrophoresis and blotted for immunochemical detection of tTG content. To prove that Western blot conditions during final development were linear, different CCD exposure times of each gel were compared. The number below each band shows the chemiluminescence measurement.

 
Cell Surface tTG and RPE Cell Adhesion
For an analysis of whether the TGF-ß2–mediated increase of surface tTG has an influence on RPE cell attachment and adhesion on the ECM components Fn and Col I, quantitative adhesion assays were performed with untreated and TGF-ß2–treated RPE cells. To determine the influence of surface tTG on RPE cell adhesion and attachment strength, control and TGF-ß2–treated RPE cells were incubated with function-blocking antibodies against tTG before attachment was assayed.

The overall attachment rate of RPE cells was higher on Fn than on Col I–coated wells (Fig. 2) . Pretreatment with TGF-ß2 significantly (P < 0.01) increased the attachment on Fn and on Col I (Fig. 2) . Preincubation with antibodies against cell surface tTG markedly reduced adhesion of both untreated and TGF-ß2–treated RPE cells on Fn (Fig. 2) . In contrast, anti-tTG antibodies did not reduce the attachment of RPE cells on Col I (Fig. 2) .



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  		FIGURE 2. Attachment of human RPE cells on Fn and Col I. RPE cells were treated for 24 hours with 1 ng/mL TGF-ß2 and then plated on wells coated with Fn or Col I. RPE cell suspensions were preincubated for 1 hour with control nonimmune IgG, function-blocking anti-tTG antibody. The antibodies were kept in the medium during the assay. The means of results in three separate experiments performed in duplicate are shown. Co, control; Ab, anti-tTG antibody.

 
Using a centrifugal force–based adhesion assay, the strength of RPE cell attachment on Fn and Col I was measured (Fig. 3) . With increasing centrifugal forces a decrease in attachment of both untreated and TGF-ß2–treated RPE cells was observed. The centrifugal force of 500g yielded no significant difference between control and TGF-ß2–treated cells. However, at 1000 and 2000g, RPE cells pretreated with TGF-ß2 showed a significant (P < 0.01) increase in the strength of binding to Fn and Col I (Fig. 3) . Pretreatment of RPE cells with function–blocking antibodies against cell surface tTG significantly reduced attachment of TGF-ß2–treated and nontreated RPE cells on Fn (Fig. 3A) , whereas attachment strength to Col I was not influenced (Fig. 3B) . These results suggest a substrate specificity of cell surface tTG for Fn.



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  		FIGURE 3. Attachment strength of RPE cells on Fn (A) and Col I (B). The attachment strength was measured by a centrifugal force-based adhesion assay. The number of cells attached at each centrifugal force point was compared with the number of similarly treated cells attached at zero gravity, and the result was expressed as a percentage. TGF-ß2 (1 ng/mL) pretreated RPE cells showed significantly increased strength of attachment to Fn and Col I at 1000g centrifugal force compared with control RPE cells (P < 0.01). Pretreatment with blocking antibody against cell surface tTG markedly reduced adhesion on Fn (A). Such an effect was not seen on Col I (B). Data are expressed as the mean results ± SEM of nine experiments with three different RPE cell cultures. Co, control.

 
Fn-Binding Site of Cell Surface tTG
RPE Cell Adhesion.
For further identification of the Fn-binding site of RPE cell surface tTG, attachment assays were performed using full-length Fn and the 110-kDa cell-binding fragment and the 45-kDa gelatin-binding fragment of Fn as a substrate. Again, to evaluate the surface tTG-specific effect, tTG activity was blocked by incubation with function-blocking antibodies before the experiment. Blocking surface tTG reduced the attachment of both untreated and TGF-ß2–treated RPE cells to full-length Fn (Fig. 4A) . An even more marked reduction was seen, when untreated and TGF-ß2–treated RPE cell binding to the 45 kDa gelatin-binding fragment of Fn (Fig. 4B) was assayed. In contrast, blocking with anti-tTG antibodies did not reduce adhesion of untreated and TGF-ß2–treated RPE cells on the 110-kDa cell-binding Fn fragment (Fig. 4C) . These observations indicate that surface tTG-mediated binding of RPE cells to Fn may be mediated through the 45-kDa gelatin-binding domain of Fn.



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  		FIGURE 4. Blocking cell surface tTG decreased attachment of RPE cells on Fn and the 45-kDa gelatin-binding Fn fragment. RPE cells were treated for 24 hours with 1 ng/mL TGF-ß2 and then plated (2 x 104) on wells coated with Fn (A), the 45-kDa Fn fragment (B), or the 110-kDa cell-binding Fn fragment (C). RPE cell suspensions were preincubated for 1 hour with control nonimmune IgG, function-blocking polyclonal anti-tTG antibody, mAb against tTG, or blocking mAbs against {alpha}5 or ß1 integrins. The antibodies were kept in the medium during the assay. The mean results of three separate experiments performed in duplicate are shown.

 
To gain further insight into this mechanism we studied whether the Fn-binding integrins {alpha}5 and ß1 are involved in cell surface tTG-mediated adhesion of RPE cells to Fn. For this purpose the respective integrins were blocked by specific antibodies before the attachment assay. Blocking of integrin {alpha}5 and ß1 decreased adhesion of untreated and TGF-ß2–treated RPE cells on full-length Fn, although the effect was not as strong as when cell surface tTG was blocked (Fig. 4A) . The interference with integrins had the strongest effect on RPE adhesion to the 110-kDa cell-binding fragment of Fn (Fig. 4C) . Both untreated and TGF-ß2–treated RPE cells showed decreased adhesion to the 110 kDa cell-binding fragment of Fn after treatment with integrin antibodies (Fig. 4C) . In contrast to the results obtained from perturbation of cell surface tTG, anti-integrin antibodies had no effect on the adhesion of untreated and TGF-ß2–treated RPE cells to the 45-kDa gelatin-binding Fn fragment (Fig. 4B) , suggesting that RPE adhesion on the fragment may be independent of integrin {alpha}5 and ß1.

RPE Cell Migration.
To analyze the involvement of surface tTG in migration of untreated and TGF-ß2–treated RPE cells, the undersurfaces of Boyden chamber membranes were coated with ECM proteins and Fn fragments. To ensure an efficient directional migration, platelet-derived growth factor (PDGF)-BB was added to the lower chambers as a chemotactic agent.9

Untreated RPE cells showed only slight migratory activity, with most cells migrating on full-length Fn followed by Col I, and the 45-kDa gelatin-binding and the 110-kDa cell-binding Fn fragments (Fig. 5) . Pretreatment with TGF-ß2 markedly increased the number of migrating cells on full-length Fn and its 45-kDa gelatin-binding fragment (Fig. 5) . TGF-ß2 only slightly enhanced migration on Col I and on the 110-kDa cell-binding fragment of Fn (Fig. 5) .



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  		FIGURE 5. TGF-ß2 increased the migration of RPE cells on Fn and its 45-kDa gelatin-binding fragment. Migration was observed, using filters coated on one side with Col I, Fn, or the 45-kDa gelatin-binding or 110-kDa cell-binding fragments of Fn. Cultured RPE cells were treated for 24 hours with 1.0 ng/mL TGF-ß2 before they were added to the chamber. After 5 hours at 37°C, cells that migrated into the lower surface of the filter were fixed, stained, and quantitated. All results are expressed as the average number of cells ± SD per x200 microscope field from three independent experiments performed in triplicate. Co, control.

 
Treatment with blocking antibodies against cell surface tTG markedly reduced migration of TGF-ß2 pretreated RPE cells on full-length Fn and its 45-kDa gelatin-binding fragment, whereas no changes were observed regarding the 110-kDa cell-binding fragment of Fn (Fig. 6) . Function-blocking antibodies to either {alpha}5 or ß1 integrin inhibited migration of RPE cells on full-length Fn, although they had a more modest effect than anti-tTG antibodies. Migration of RPE cells on the 110-kDa Fn fragment was efficiently suppressed by the anti-{alpha}5 and -ß1 integrin, whereas anti-tTG antibodies had essentially no effect on this substrate (Fig. 6) . This is in accordance with the observations of RPE cell adhesion.



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  		FIGURE 6. Blocking cell surface tTG inhibited migration of RPE cells on Fn and the 45-kDa gelatin-binding Fn fragment. Migration was performed using filters coated on one side with Fn, the 45-kDa gelatin-binding Fn fragment, or the 110-kDa cell-binding Fn fragment. Cultured RPE cells were treated for 24 hours with 1.0 ng/mL TGF-ß2 before they were added to the chamber and incubated with control nonimmune IgG, function-blocking polyclonal anti-tTG antibody, anti-tTG mAb, or blocking mAbs against {alpha}5 or ß1 integrins. The antibodies were kept in the medium during the assay. Antibodies were used at 10 µg/mL. After 5 hours at 37°C, cells that migrated into the lower surface of the filter were fixed, stained, and quantitated. Data shown are the mean ± SD from at least three experiments performed in triplicate, and the results are expressed as the percentage of migration in the presence of control nonimmune IgG.

 
RPE Cell Spreading.
In a last step, spreading of RPE cells was measured on tissue culture plates coated with full-length Fn, the 45-kDa gelatin-binding Fn fragment, and the 110-kDa cell-binding Fn fragment and on noncoated tissue culture plastic surfaces (Figs. 7 8) . After 4 hours of incubation, cell spreading of both untreated and TGF-ß2–stimulated RPE cells was found to be increased on all coated plates when compared with the noncoated control. The overall increase was most pronounced on full-length Fn. TGF-ß2 treatment resulted in even more marked spreading of RPE cells grown on full-length Fn (Figs. 7 8) , on the 45-kDa Fn fragment, and on the 110-kDa Fn fragment when compared with the uncoated control (Fig. 7) .



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  		FIGURE 7. Blocking cell surface tTG inhibited spreading of TGF-ß2–stimulated RPE cells. Quantitative assessment of RPE cell spreading on tissue culture plastic surfaces and on 24-well tissue culture plates coated with 50 µg/mL Fn, the 45-kDa gelatin-binding Fn fragment, or the 110-kDa cell-binding Fn fragment. Untreated and TGF-ß2–treated RPE cells (5 x 104 cells/mL) were plated in the wells and incubated with a function-blocking polyclonal anti-tTG antibody. Cells were incubated in DMEM for 4 hours at 37°C before examination. The data represent the mean ± SEM of results obtained from four separate fields by examining at least 100 cells per field.

 


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  		FIGURE 8. Morphology of cells used for collection of data in Figure 7 . Representative fields are shown. (A) Untreated RPE cells grown on tissue culture plastic surface; (B) untreated RPE cells on Fn; (C) after preincubation of RPE cells with TGF-ß2 on Fn; and (D) after preincubation of RPE cells with TGF-ß2 and anti-tTG on Fn. S, spreading cells; NS, nonspreading cells. Magnification, x100.

 
Anti-tTG antibodies markedly inhibited TGF-ß2–induced spreading of RPE cells on full-length Fn (Figs. 7 8) and the 45-kDa gelatin-binding fragment of Fn (Fig. 7) , whereas only a slight effect was found in RPE cells grown on the 110-kDa cell-binding Fn fragment (Fig. 7) . Control RPE cells without TGF-ß2 pretreatment showed similar results in reduction of cell spreading compared with TGF-ß2–pretreated RPE cells (Fig. 7) .


   Discussion
Top
 Abstract
 Material and Methods
 Results
 Discussion
References
 
The present study provides evidence that TGF-ß2 induces expression of cell surface tTG, which enhances adhesion, migration, and spreading of human RPE cells on (Fn) through the 45-kDa gelatin-binding domain of Fn. To our knowledge, this is the first study to describe these mechanisms of surface tTG in RPE cells. In previous studies, artificial, immortal, partly transfected human or animal fibroblast and tumor cell lines where used to describe these tTG-related mechanisms.12 25 26 27 28 29 44 45 A major difference of most cell lines used in previous studies compared with human RPE cells is the absence of basal tTG expression and the need for transfection to produce active tTG to perform experiments. In RPE cells basal tTG expression and increase in tTG activity during dedifferentiation or stimulation of growth factors are natural phenomena.

TGF-ß2 is known to be secreted in the initial phase of retinal detachment46 and has been found elevated in the vitreous body of patients with PVR.31 We have recently demonstrated that TGF-ß2 increases total cellular tTG expression in cultured RPE.30 Using subcellular fraction analysis we found a cell-surface associated expression of tTG on RPE cells, which was markedly upregulated by TGF-ß2. Similar observations have been made after TGF-ß treatment of fibroblasts.27

Functionally, TGF-ß2 treatment increased adhesion, migration, and spreading of RPE cells on full-length Fn. These cellular processes in part appeared to be mediated by surface tTG, because preincubation with anti-tTG significantly reduced adhesion, migration, and spreading of untreated and TGF-ß2–treated RPE cells on full-length Fn. This is in accordance with studies that have indicated that downregulation of cell surface tTG activity by inhibition of the tTG-Fn interaction completely inhibits cell attachment and migration on Fn.26 27 28 Furthermore, TGF-ß2 increased adhesion and migration of RPE cells on Col I, but this effect was not influenced by function-blocking antibodies to tTG. These findings corroborate those in a previous study describing no influence of blocking antibodies against surface tTG or downregulation of tTG by antisense strategies on adhesion and migration on Col I.28

It has repeatedly been shown that TGF-ß2 has an influence on the expression of integrins (mainly the {alpha}1, {alpha}2, {alpha}3, {alpha}5, and ß1 chains) in numerous cell types, including chondrocytes, fibroblasts, and carcinoma cells,47 48 which in turn support cell adhesion on Fn. Accordingly, we found the binding of untreated and TGF-ß2–treated RPE cells to full-length Fn to be reduced when the Fn-binding integrins {alpha}5 and ß1 were blocked. However, this binding appears to be mediated through the 110-kDa cell-binding fragment of Fn: integrin antibodies reduced binding to the 110-kDa cell-binding Fn fragment, but had no effect on the 45-kDa gelatin-binding Fn fragment. This appears reasonable, because the 110-kDa fragment is known to contain integrin-binding motifs, whereas the 45-kDa fragment has been shown not to contain them.18 19 In clear contrast, attachment, migration, and spreading of RPE cells on the 45-kDa gelatin-binding Fn fragment was reduced after blocking tTG function, whereas no effect was seen on the 110-kDa Fn fragment. These observations indicate that cell surface tTG-mediated binding of RPE cells to Fn is mainly exerted through the 45-kDa gelatin-binding Fn fragment, suggesting the presence of an integrin independent interaction.

The present study demonstrated that TGF-ß2 enhances adhesion, migration, and spreading of human RPE cells on Fn. Adhesion of RPE cells to Fn can be mediated by integrins and surface tTG, although the binding effects appear to be exerted through different Fn domains. In addition to that, Akimov et al.12 suggested a direct interaction of integrins and surface tTG in cell adhesion, which may finally lead to a localized strengthening of ECM, especially when cells require enhanced adhesion and anchoring to the underlying substrate. We believe that these mechanisms may have relevance in the pathogenesis of early PVR. In the onset of PVR, RPE-dedifferentiation, together with a TGF-ß2–mediated increase of cell surface tTG expression may help RPE cells to attach to and migrate on Fn-containing matrices, which may ultimately contribute to the formation of tractional fibrocellular sub- and epiretinal membranes. Although fully speculative at this point, the inhibition of cell surface tTG may offer a future therapeutic approach to prevent adhesion and migration of detached RPE cells. Further studies will help to elucidate the physiological significance of this interaction and its regulation at the molecular level.


   Acknowledgements
 
The authors thank Katja Obholzer for expert technical assistance.


   Footnotes
 
Supported by Deutsche Forschungsgemeinschaft WE 2577/2-1, and the Vera und Volker Doppelfeld Stiftung.

Submitted for publication March 3, 2003; revised July 15, 2003; accepted July 31, 2003.

Disclosure: S.G. Priglinger, None; C.S. Alge, None; A.S. Neubauer, None; N. Kristin, None; C. Hirneiss, None; K. Eibl, None; A. Kampik, None; U. Welge-Lussen, None

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

Corresponding author: Ulrich Welge-Lussen, Department of Ophthalmology, Ludwig-Maximilians-University, Munich, Mathildenstrasse 8, 80336 Munich, Germany; ulrich.welge-luessen{at}ak-i.med.uni-muenchen.de.


   References
Top
 Abstract
 Material and Methods
 Results
 Discussion
 References
 

  1. Kampik A, Kenyon KR, Michels RG, Green WR, de la Cruz ZC. Epiretinal and vitreous membranes: comparative study of 56 cases. Arch Ophthalmol. 1981;99:1445–1454.[ISI][Medline][Order article via Infotrieve]
  2. Machemer R, Laqua H. Pigment epithelial proliferation in retinal detachment (massive periretinal proliferation). Am J Ophthalmol. 1975;80:1–23.[ISI][Medline][Order article via Infotrieve]
  3. Machemer R, van Horn D, Aaberg TM. Pigment epithelial proliferation in human retinal detachment with massive periretinal proliferation. Am J Ophthalmol. 1978;85:181–191.[ISI][Medline][Order article via Infotrieve]
  4. Hiscott PS, Grierson I, McLeod D. Retinal pigment epithelial cells in epiretinal membranes: an immunohistochemical study. Br J Ophthalmol. 1984;68:708–715.[Abstract/Free Full Text]
  5. Grierson I, Hiscott PS, Hitchins C, McKechnie NM, White VA, McLeod D. Which cells are involved in the formation of epiretinal membranes?. Semin Ophthalmol. 1987;2:99–109.
  6. Hiscott P, Sheridan C, Magee RM, Grierson I. Matrix and the retinal pigment epithelium in proliferative retinal disease. Prog Retin Eye Res. 1999;18:167–190.[CrossRef][ISI][Medline][Order article via Infotrieve]
  7. Charteris DG. Proliferative vitreoretinopathy: pathobiology, surgical management, and adjunctive treatment. Br J Ophthalmol. 1994;79:953–960.
  8. Casaroli Marano RP, Vilaro S. The role of fibronectin, laminin, vitronectin and their receptors on cellular adhesion in proliferative vitreoretinopathy. Invest Ophthalmol Vis Sci. 1994;35:2791–2803.[Abstract/Free Full Text]
  9. Hinton DR, He S, Graf K, et al. Mitogen-activated protein kinase activation mediates PDGF-directed migration of RPE cells. Exp Cell Res. 1998;239:11–15.[CrossRef][ISI][Medline][Order article via Infotrieve]
  10. Elner SG, Elner VM. The integrin superfamily and the eye. Invest Ophthalmol Vis Sci. 1996;37:696–701.[Abstract/Free Full Text]
  11. Anderson DH, Johnson LV, Hageman GS. Vitronectin receptor expression and distribution at the photoreceptor-retinal pigment epithelial interface. J Comp Neurol. 1995;360:1–16.[CrossRef][ISI][Medline][Order article via Infotrieve]
  12. Akimov SS, Krylov D, Fleischman LF, Belkin AM. Tissue transglutaminase is an integrin-binding adhesion coreceptor for fibronectin. J Cell Biol. 2000;148:825–838.[Abstract/Free Full Text]
  13. Folk JE. Transglutaminases. Ann Rev Biochem. 1980;49:517–531.[CrossRef][ISI][Medline][Order article via Infotrieve]
  14. Upchurch HF, Conway E, Maxwell MD. Localization of cellular transglutaminase on the extracellular matrix after wounding: characteristics of the matrix-bound enzyme. J Cell Physiol. 1991;149:375–382.[CrossRef][ISI][Medline][Order article via Infotrieve]
  15. Aeschlimann D, Kaupp O, Paulsson M. Transglutaminase-catalyzed matrix cross-linking in differentiating cartilage: identification of osteonectin as a major glutaminyl substrate. J Cell Biol. 1995;129:881–892.[Abstract/Free Full Text]
  16. Aeschlimann D, Paulsson M. Cross-linking of laminin-nidogen complexes by tissue transglutaminase: a novel mechanism for basement membrane stabilization. J Biol Chem. 1991;266:15308–15317.[Abstract/Free Full Text]
  17. Martinez J, Chalupowicz DG, Roush RK, Sheth A, Barsigian C. Transglutaminase-mediated processing of fibronectin by endothelial cell monolayers. Biochemistry. 1994;33:2538–2545.[CrossRef][Medline][Order article via Infotrieve]
  18. Kleman JP, Aeschlimann D, Paulsson M, van der Rest M. Transglutaminase-catalyzed cross-linking of fibrils of collagen V/XI in A204rhabdomyosarcoma cells. Biochemistry. 1995;34:13768–13775.[CrossRef][Medline][Order article via Infotrieve]
  19. Kaartinen MT, Pirhonen A, Linnala-Kankunen A, Maenpaa PH. Transglutaminase-catalyzed crosslinking of osteopontin is inhibited by osteocalcin. J Biol Chem. 1997;272:22736–22741.[Abstract/Free Full Text]
  20. Bowness JM, Folk JE, Timpl R. Identification of a substrate site for liver transglutaminase on the aminopropeptide of type III collagen. J Biol Chem. 1987;262:1022–1024.[Abstract/Free Full Text]
  21. Achyuthan KE, Mary A, Greenberg CS. The binding sites on fibrin(ogen) for guinea pig liver transglutaminase are similar to those of blood coagulation factor XIII: characterization of the binding of liver transglutaminase to fibrin. J Biol Chem. 1988;263:14296–14301.[Abstract/Free Full Text]
  22. Turner PM, Lorand L. Complexation of fibronectin with tissue transglutaminase. Biochemistry. 1989;28:628–635.[CrossRef][Medline][Order article via Infotrieve]
  23. Radek JT, Jeong JM, Murthy SN, Ingham KC, Lorand L. Affinity of human erythrocyte transglutaminase for a 42-kDa gelatin-binding fragment of human plasma fibronectin. Proc Natl Acad Sci USA. 1993;90:3152–3156.[Abstract/Free Full Text]
  24. Skorstengaard K, Thogersen HC, Vibe-Pedersen K, Petersen TE, Magnusson S. Purification of twelve cyanogen bromide fragments from bovine plasma fibronectin and the amino acid sequence of eight of them: overlap evidence aligning two plasmic fragments, internal homology in gelatin-binding region and phosphorylation site near C terminus. Eur J Biochem. 1982;128:605–623.[ISI][Medline][Order article via Infotrieve]
  25. Jones RA, Nicholas B, Mian S, Davies PJA, Griffin M. Reduced expression of tissue transglutaminase in a human endothelial cell line leads to changes in cell spreading, cell adhesion and reduced polymerisation of fibronectin. J Cell Sci. 1997;110:2461–2472.[Abstract]
  26. Verderio E, Nicholas B, Gross S, Griffin M. Regulated expression of tissue transglutaminase in swiss 3T3 fibroblasts: effects on the processing of fibronectin, cell attachment, and cell death. Exp Cell Res. 1998;239:119–138.[CrossRef][ISI][Medline][Order article via Infotrieve]
  27. Akimov SS, Belkin AM. Cell-surface transglutaminase promotes fibronectin assembly via interaction with the gelatin-binding domain of fibronectin: a role in TGFbeta-dependent matrix deposition. J Cell Sci. 2001;114:2989–3000.
  28. Akimov SS, Belkin AM. Cell surface tissue transglutaminase is involved in adhesion and migration of monocytic cells on fibronectin. Blood. 2001;98:1567–1576.[Abstract/Free Full Text]
  29. Balklava Z, Verderio E, Collighan R, Gross S, Adams J, Griffin M. Analysis of tissue transglutaminase function in the migration of Swiss 3T3 fibroblasts: the active-state conformation of the enzyme does not affect cell motility but is important for its secretion. J Biol Chem. 2002;277:16567–16575.[Abstract/Free Full Text]
  30. Priglinger SG, May CA, Neubauer AS, et al. Tissue transglutaminase as a modifying enzyme of the extracellular matrix in PVR-membranes. Invest Ophthalmol Vis Sci. 2003;44:355–364.[Abstract/Free Full Text]
  31. Kon CH, Occleston NL, Aylward GW, Khaw PT. Expression of vitreous cytokines in proliferative vitreoretinopathy: a prospective study. Invest Ophthalmol Vis Sci. 1999;40:705–712.[Abstract]
  32. Aeschlimann D, Paulsson M. Transglutaminases: protein cross-linking enzymes in tissues and body fluids. Thromb Haemost. 1994;71:402–415.[ISI][Medline][Order article via Infotrieve]
  33. Heldin CH, Miyazono K, ten Dijke P. TGF-beta signalling from cell membrane to nucleus through SMAD proteins. Nature. 1997;390:465–471.[CrossRef][Medline][Order article via Infotrieve]
  34. Derynck R, Feng XH. TGF-beta receptor signaling. Biochim Biophys Acta. 1997;1333:F105–F150.[Medline][Order article via Infotrieve]
  35. Campochiaro PA, Jerdon JA, Glaser BM. The extracellular matrix of human retinal pigment epithelial cells in vivo and its synthesis in vitro. Invest Ophthalmol Vis Sci. 1986;27:1615–1621.[Abstract/Free Full Text]
  36. Leschey KH, Hackett SF, Singer JH, Campochiaro PA. Growth factor responsiveness of human retinal pigment epithelial cells. Invest Ophthalmol Vis Sci. 1990;31:839–846.[Abstract/Free Full Text]
  37. Kim SY, Grant P, Lee JH, Pant HC, Steinert PM. Differential expression of multiple transglutaminases in human brain: increased expression and cross-linking by transglutaminases 1 and 2 in Alzheimer’s disease. J Biol Chem. 1999;274:30715–30721.[Abstract/Free Full Text]
  38. McClay DR, Wessel GM, Marchase RB. Intercellular recognition: quantitation of initial binding events. Proc Natl Acad Sci USA. 1981;78:4975–4979.[Abstract/Free Full Text]
  39. Lotz MM, Burdsal CA, Erickson HP, McClay DR. Cell adhesion to fibronectin and tenascin: quantitative measurements of initial binding and subsequent strengthening response. J Cell Biol. 1989;109:1795–1805.[Abstract/Free Full Text]
  40. Mosmann T. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Imunol Methods. 1983;65:55–63.[CrossRef][ISI][Medline][Order article via Infotrieve]
  41. Dai Y, Dean TP, Church MK, Warner JO, Shute JK. Desensitisation of neutrophil responses by systemic interleukin 8 in cystic fibrosis. Thorax. 1994;49:867–871.[Abstract/Free Full Text]
  42. Figari IS, Mori NA, Palladino MA, Jr. Regulation of neutrophil migration and superoxide production by recombinant tumor necrosis factors-alpha and -beta: comparison to recombinant interferon-gamma and interleukin-1 alpha. Blood. 1987;70:979–984.[Abstract/Free Full Text]
  43. Jin M, He S, Worpel V, Ryan SJ, Hinton DR. Promotion of adhesion and migration of RPE cells to provisional extracellular matrices by TNF-alpha. Invest Ophthalmol Vis Sci. 2000;41:4324–4332.[Abstract/Free Full Text]
  44. Cai D, Ben T, De Luca LM. Retinoids induce tissue transglutaminase in NIH-3T3 cells. Biochem Biophys Res Commun. 1991;175:1119–1124.[CrossRef][ISI][Medline][Order article via Infotrieve]
  45. Ball DJ, Mayhew S, Vernon DI, Griffin M, Brown SB. Decreased efficiency of trypsinization of cells following photodynamic therapy: evaluation of a role for tissue transglutaminase. Photochem Photobiol. 2001;73:47–53.[CrossRef][ISI][Medline][Order article via Infotrieve]
  46. Guerin CJ, Hu L, Scicli G, Scicli AG. Transforming growth factor beta in experimentally detached retina and periretinal membranes. Exp Eye Res. 2001;73:753–764.[CrossRef][ISI][Medline][Order article via Infotrieve]
  47. Heino J, Ignotz RA, Hemler ME, Crouse C, Massague J. Regulation of cell adhesion receptors by transforming growth factor-beta: concomitant regulation of integrins that share a common beta 1 subunit. J Biol Chem. 1989;264:380–388.[Abstract/Free Full Text]
  48. Kim LT, Yamada KM. The regulation of expression of integrin receptors. Proc Soc Exp Biol Med. 1997;214:123–131.[Abstract]



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