August 2003
Volume 44, Issue 8
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Cornea  |   August 2003
Production of Fibronectin and Tenascin Isoforms and Their Role in the Adhesion of Human Immortalized Corneal Epithelial Cells
Author Affiliations
  • Sissi Filenius
    From the Institute of Biomedicine and Anatomy, University of Helsinki, Helsinki, Finland; and the
    Department of Ophthalmology, Helsinki University Central Hospital, Helsinki, Finland.
  • Timo Tervo
    Department of Ophthalmology, Helsinki University Central Hospital, Helsinki, Finland.
  • Ismo Virtanen
    From the Institute of Biomedicine and Anatomy, University of Helsinki, Helsinki, Finland; and the
Investigative Ophthalmology & Visual Science August 2003, Vol.44, 3317-3325. doi:10.1167/iovs.02-1146
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      Sissi Filenius, Timo Tervo, Ismo Virtanen; Production of Fibronectin and Tenascin Isoforms and Their Role in the Adhesion of Human Immortalized Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2003;44(8):3317-3325. doi: 10.1167/iovs.02-1146.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. To study the production and deposition of fibronectin (Fn) isoforms and tenascin-C (Tn-C) by immortalized human corneal epithelial (HCE) cells and their integrin-dependent adhesion characteristics.

methods. Indirect immunofluorescence with isoform-specific monoclonal antibodies (mAbs) was used to study extracellular matrix (ECM) protein composition and their integrin receptors in HCE cells. The synthesis of proteins was studied by Western blot analysis and adhesion by quantitative adhesion assay.

results. HCE cells deposited fibrillar matrix containing extradomain EDA-Fn and sparser deposits of Onc-Fn, whereas Tn-C was deposited diffusely. EDA-Fn was present both in ECM and in culture medium, whereas Tn-C was present only in ECM. Fn-binding integrin (Int) α5 subunit was present in subconfluent cells in focal adhesions (FAs) and matrix adhesions, whereas Int αvβ5 was present in FAs in sparse cultures and as ringlike structures in denser cultures. Int αvβ6 was colocalized with Int α5 in FAs, only in cells adhering to growth substratum coated with Fn or Fn/Tn-C. Ints α5β1 and αvβ6 mediated adhesion to Fn and Int αvβ5 mediated adhesion to Vn, and both were inhibited by RGD peptide. The cells failed to adhere to Tn-C but adhered to Fn/Tn-C and were then more efficiently inhibited by the function-blocking integrin mAbs and RGD peptide.

conclusions. The results suggest corneal epithelial cells as the possible source for Fn isoforms and Tn-C in wound healing and pathologic conditions. The presence of Tn-C only in ECM suggest a vectorial deposition and adhesion experiments also indicate a role for Tn-C in Fn functioning.

Corneal wound healing has been the subject of intensive research during the past years. Understanding of the healing response of the cornea to refractive surgery, injuries, chronic ulcers, and recurrent erosion syndrome is of utmost importance in developing therapeutic measures or attempting to enhance wound healing. This process requires complex epithelial and stromal interactions mediated by growth factors and extracellular matrix components. 1 2 3  
Fibronectin (Fn) and tenascin (Tn), both multifunctional extracellular matrix glycoproteins, play a major role in corneal wound healing. Fn is present both in plasma and the extracellular matrix. It also plays a role in embryogenesis, hemostasis, and thrombosis and functions in cell adhesion, migration, and maintenance of normal cell morphology. 4 The primary transcript of Fn is alternatively spliced and therefore different isoforms, such as ED-A, ED-B, and CS-1 and a further isoform, Onc-Fn, are expressed by alternative glycosylation. 4 5 6 7 Fn is widely expressed in human and rabbit cornea and EDA-Fn emerges during wound healing. 8 9 ED-A and Onc-Fn have been shown to be present in the basement membrane (BM) zone of the cornea, limbus, and conjunctiva. 10 Recent studies suggest that Staphylococcus aureus, one of the major causes of bacterial ulcerative keratitis, becomes internalized by corneal epithelial cells by using their integrin (Int) α5β1 and Fn. 11 12  
The ability to promote cell adhesion to Fn requires specific Fn domains and surface integrin receptors of adhering cells. 13 The classic cell adhesion domain of Fn contains an RGD tripeptide sequence and is recognized by Ints α5β1 and αvβ3. 14 15 Fn also serves as a ligand for several other integrins, such as α4β1, α8β1, α9β1, αvβ1 and αvβ6 which bind to different regions of the Fn molecule. 16 The production of Fn has been attributed to both mesenchymal and epithelial cells. 17  
Among the Tn family of proteins, Tn-C and -X participate in various epithelial-mesenchymal interactions during development. Tn-C has been found in stromal areas of carcinomas, wound healing, and inflammation. 18 Although Tn-C has been widely studied, little is still known about its precise functions. Unlike Fn, Tn-C has been associated with both adhesive and antiadhesive properties. 19 20 Because it is widely expressed in the preterm cornea but is restricted to the limbal area of child and adult corneas, it has been presumed that Tn-C is involved in corneal development, differentiation, or proliferation of stem cells. 21 In keratoconus and bullous keratopathy, the level of Tn-C is increased beneath the epithelium. 22 23 Tn-C is highly increased during healing of corneal wounds, implying a role for Tn-C in the healing process. 9 24 Several studies in Tn-C knockout mice have suggested, however, that their epidermal, corneal, and limbal wounds heal normally. 25 26 27 Forsberg et al. 25 and Matsuda et al. 27 discovered, in contrast, that in Tn-C knockout mice, both in skin and cornea, the wounds appear to have reduced Fn expression and that corneal healing defects occur after suture injuries. 24 25 27 Among the integrins, at least Ints α2β1, α8β1, α9β1, αvβ3, and αvβ6 have been reported to bind epithelial cells to Tn-C. 6 28 29 Furthermore, in bullous keratopathy the increase in Tn-C content parallels an increase of Ints α8β1, α9β1, and αvβ6. 30 Both mesenchymal cells and epithelial cells have been shown to synthesize Tn-C. 17 31 32 33 34 35  
Masur et al. 36 have shown that corneal myofibroblasts adhere to Fn by using Int α5β1. Less is known about corneal epithelial cells, and the purpose of our study was therefore to investigate the production and deposition of Fn and Tn isoforms by human corneal epithelial (HCE) cells as well as to explore the cells’ tendency to adhere to those proteins. The results suggest that understanding the cooperation between Tn-C and Fns is essential when adhesion characteristics of corneal epithelial cells are considered. 
Materials and Methods
Culture of Immortalized Human Corneal Epithelial Cells
Simian virus (SV)40 immortalized HCE cells were cultured in DMEM/F12, supplemented with 15% fetal bovine serum (FBS), 5 μg/mL insulin, 0.1 μg/mL cholera toxin, 10 ng/mL human epidermal growth factor, and 40 μg/mL gentamicin (all from GibcoBRL, Grand Island, NY), as described by Araki-Sasaki et al. 37 The cells were kindly provided by Kaoru Araki-Sasaki (Osaka University, School of Medicine, Osaka, Japan). Human embryonic skin fibroblasts were obtained from a local source and were cultured in RPMI 1640 supplemented with 10% PBS and antibiotics. 
Indirect Immunofluorescence Microscopy
For indirect immunofluorescence microscopy, HCE cells were cultured on glass coverslips and fixed in −20°C methanol for 10 minutes. After cells were washed in phosphate-buffered saline (PBS), they were incubated with mAbs for 30 minutes. After they were washed three times with PBS, the cells were exposed to fluorescein isothiocyanate (FITC)-coupled goat anti-mouse or anti-rat IgG (both from Jackson ImmunoResearch, West Grove, PA) for 30 minutes. Finally, the cells were washed in PBS and embedded in veronal-glycerol buffer (1:1; pH 8.4) and examined under a microscope (Axiophot; Leica, Heidelberg, Germany) equipped with appropriate filters. 
Experiments to determine adhesion of HCE cells to purified (p)Fn, Tn-C (Sigma-Aldrich, St. Louis, MO) or vitronectin (Vn) were also performed on glass coverslips. pFn was isolated from outdated human plasma (Finnish Red Cross Blood Transfusion Service, Helsinki, Finland) by using gelatin-Sepharose (Amersham Pharmacia Biotech, Uppsala, Sweden), according to Engvall and Ruoslahti, 38 and Vn by using heparin-Sepharose (Amersham Pharmacia Biotech), according to Yatohgo et al. 39 The wells were coated with pFn, Tn-C, or both (4 μg/mL) at 37°C for 4 hours. PBS was then used to wash the glass coverslips twice, and 3% bovine serum albumin (BSA) in PBS was used for postcoating. After incubation at 37°C for 1 hour, the glass coverslips were washed once with PBS. The cells were incubated with cycloheximide (10 μg/mL; Sigma-Aldrich) to inhibit protein synthesis at 37°C for 1 hour. The cells were trypsinized, and soybean trypsin inhibitor (STI; Sigma-Aldrich) was added, according to the instructions of the manufacturer. The cells were then suspended in serum-free culture medium containing cycloheximide (10 μg/mL) and centrifuged for 5 minutes. The supernatant was removed, and the cells were suspended in serum-free culture medium in the presence of cycloheximide. The cell suspension was added to the glass coverslips, allowed to spread at 37°C for 1 hour, and fixed as described earlier. 
The following mAbs were used for immunostaining: 100EB2 against Tn-C, BC-2 against the large subunit of Tn-C, 52DH1 against EDA-Fn, FDC-6 against Onc-Fn, BC-1 detecting EDB-Fn, 102DF5 against the Int β1 subunit, 90BB10 against Int β3, 1A9 against Int β5, LM 142.69 against Int αv, E7P6 against Int αvβ6, Y9A2 against Int α9, and BIE5 against the Int α5 subunit. 7 40 41 42 43 44 45 46 47 48 49 The mAb against talin was from Serotec (Oxford, UK) and tetramethylrhodamine isothiocyanate (TRITC)-coupled phalloidin was from Molecular Probes (Eugene, OR). The polyclonal antiserum against Tn-X has been described. 50 For double immunostaining experiments HCE cells were first exposed to polyclonal antibody against Fn (Sigma-Aldrich) or rat mAb, followed by incubation with the TRITC-conjugate and finally with the mouse mAb and FITC conjugate. 
Quantitative Cell Adhesion Assays
Adhesion experiments with HCE cells were performed in 96-well plates with a method based on intracellular acid phosphatase. 51 52 The wells were coated with pFn, Tn-C, or Vn (4 μg/mL) at room temperature for 1 hour, washed twice in PBS and were postcoated with 3% BSA. After incubation at room temperature for 1 hour, the wells were washed twice with PBS. Subsequently, the cells were incubated with cycloheximide (10 μg/mL) at 37°C for 1 hour. They were then trypsinized, and STI was added. The cells were washed once with serum-free culture medium and suspended in the same medium, and cycloheximide was added. The antibody-cell suspension was added to the wells and incubated at 37°C for 90 minutes. Cells were washed with PBS and 100 μL of the substrate solution (5 mg/mL, Sigma-Aldrich 104 phosphatase in 50 mM acetate buffer [pH 5]; containing 1% Triton X-100) was added to each well. The wells were incubated at 37°C for 1 hour, and the reaction was stopped by adding 50 μL of 1 M NaOH. The air bubbles were carefully removed, and the absorbancy measured at 405 nm. The following function-blocking mAbs were used to inhibit cell adhesion: PIB5 against Int α3 (Chemicon, Temecula, CA), BIE5 against Int α5, 13 against Int β1, PIF6 against Int αvβ5, and E7P6 against Int αvβ6 (Chemicon). 48 53 54 GRGDSP peptide (Sigma-Aldrich) was used at concentrations of 100 to 500 μg/mL. The data were analyzed with paired Student’s t-tests with two-tailed hypothesis and significance set at P ≤ 0.05. 
Western Blot Analysis
For Western blot analysis, HCE cells were cultured in serum-free HCE medium overnight. Purified U251Mg glioma cell Tn-C was purchased from Sigma-Aldrich. Samples, which were taken from the spent culture medium and ECM preparations, were analyzed by SDS-PAGE on 6.5% slab gels under reducing conditions, and fibroblasts were analyzed similarly in RPMI-1640 medium. Finally, the proteins were transferred onto nitrocellulose sheets and exposed to mAb 52DHI to EDA −Fn, mAb FDC6 to Onc-Fn, mAb BCI to EDB-Fn, mAb 100EB2 to Tn-C, and mAb BC-2 to the large subunit of TN-C. Bound antibodies were detected by using the avidin-biotin bridge method according to the instructions of the manufacturer (Vectastain; Vector Laboratories, Burlingame, CA). 
Results
Fn-Isoforms and Tn-C in HCE Cell Cultures
First, we wanted to clarify by indirect immunofluorescence microscopy the deposition of Tn-C and Fn isoforms in HCE cultures. A bright immunoreactivity was detected with mAb against the EDA-Fn isoform (Fig. 1A) , mostly located to focal adhesions (FAs) and the margins of HCE cell islands. Reaction with mAb to EDB-Fn remained negative (not shown), whereas a less intense immunoreactivity was observed for Onc-Fn (Fig. 1B) . A distinct nonfibrillar immunoreactivity was noted for Tn-C as detected with mAbs against both Tn-C subunits (Fig. 1C) or against large subunit only (Fig. 1D) . With both mAbs, immunoreactivity was especially associated with cell substratum beneath the cells. After treatment with monensin, HCE cells showed Fn in cytoplasmic granules (Fig. 1E) , whereas double immunostaining revealed that in the same cells Tn-C was in different granules (Fig. 1F) . No immunoreactivity for Tn-X was present in any of the HCE cultures (not shown). 
Production of Fn Isoforms and Tn-C by HCE Cells
Results of the immunofluorescence studies prompted us to study the secretion of Fn and Tn-C isoforms into the culture medium and deposition to cell-free ECM. Production of Fn isoforms and Tn-C was studied with mAbs, using direct Western blot analysis of the culture medium and cell-free ECM. In the culture medium a prominent Mr 240,000 band was detected for EDA-Fn (Fig. 2 ; lane 1), and a weak reaction was obtained for Onc-Fn (Fig. 2 ; lane 2). mAb against the large subunit of Tn-C did not detect any polypeptides in the culture medium of HCE cells (Fig. 2 ; lane 3), and a similar result was obtained with mAb detecting both subunits of Tn-C (Fig. 2 ; lane 4; 100EB2). In cell-free ECM material, produced by Na-deoxycholate treatment, a prominent band of Mr 240,000 was detected for EDA-Fn (Fig. 3 ; lane 1) and a similar but weaker band for Onc-Fn (Fig. 3 ; lane 2). ECM from human fibroblasts, used as a positive control, showed a prominent Mr 240,000 band (Fig. 3 ; lane 3). Prominent Mr 290,000 bands appeared with mAbs against both subunits of Tn-C in both HCE ECM (Fig. 3 ; lane 4) and in U251Mg Tn-C (Fig. 3 , lane 6) and a weak band of Mr 190,000 in HCE ECM and a distinct band in U251Mg Tn-C. mAb BC-2 showed only the large subunit of Tn-C in both specimens (Fig. 3 ; lanes 5, 7). 
Distribution of Fn-, Tn-, and Vn-Binding Integrins on HCE Cells
We studied the expression of Fn-, Tn-C–, and Vn-binding integrins in HCE cells to obtain data for cell adhesion studies. In indirect immunofluorescence, Int α5 subunit–specific reaction in HCE cultures was found in streaklike structures, corresponding to FAs (Fig. 4A , arrows) as well as to matrix adhesions on the cell surface (Fig. 4B) . A bright immunoreaction was observed for the Int β1 subunit in FAs (Fig. 4C) . mAbs against the Int β3 (not shown) and the Int β6 (Fig. 4D) subunits showed no detectable immunoreaction. 
Immunoreactions for both the Int αv and β5 subunits (not shown) were rather similar, although those for Int αv showed a wider immunostaining. In addition to point- and ringlike structures elsewhere (Fig. 4E , arrowhead), streaklike FAs were observed in the marginal cells of cell islands in subconfluent cultures (Int αv, Fig. 4E ; arrows). In dense cultures, both mAbs showed only such peculiar ringlike structures on the substratum-adherent side of the cells (Int αv, Fig. 4F ). 
Adhesion of HCE Cells to pFn, Tn-C, and Vn
The aforementioned results prompted us to study, first in immunofluorescence, the expression of the HCE integrins during adhesion to these ECM proteins. We then studied how the adhesion process could be modulated by inhibition of integrin functioning by function-blocking mAbs. We first studied the adhesion of HCE cells to purified pFn. The function-blocking mAb against the Int α3 subunit inhibited adhesion more effectively than mAbs against Ints α5 and β1 (Fig. 5A) . Our previous observation that laminin-5 is deposited early during adhesion beneath HCE cells, prompted us to investigate the effect of cycloheximide, an inhibitor of protein synthesis, on the adhesion process. 55 This adhesion experiment showed that both the mAb against Int β1 and the mAb against Int α5 effectively inhibited the adhesion of the cells to pFn. When the early synthesis, secretion, and deposition of laminin-5 was inhibited, it did not bind to Int α3β1. The differences between the adhesion of the untreated and cycloheximide-treated HCE cells were statistically significant (P < 0.05). When cycloheximide was present, no inhibition occurred with mAb against Int α3 (Fig. 5A) . mAb against Int αvβ6 did not alone affect adhesion to pFn (not shown). However, when mAbs against Ints α5 and αvβ6 were applied together, the adhesion of cells to pFn was reduced significantly more than with mAbs against Int α5 or β1 alone (Fig. 5B) . The cells did not adhere at all to purified Tn-C (Fig. 5C) . When Fn and Tn-C (pFn/Tn-C) were both absorbed into the growth substratum, HCE cells adhered to pFn/Tn-C as they did to pFn (Fig. 5C) . However, now the mAbs against both the Int β1 and α5 subunits appeared to inhibit the adhesion process more effectively (Fig. 5C ; see also Fig. 5D ). This inhibition was statistically significant with Ints β1 and α5 (P < 0.05). The experiments with GRGDSP peptide showed that it inhibited the adhesion of HCE cells to pFn/Tn-C clearly more effectively than to pFn alone. This increase in inhibition was statistically significant with each mAb (Fig. 5D)
HCE cells also adhered to purified Vn. This adhesion process was decreased with mAb against Int β1 subunit and more effectively with mAb against Int αvβ5 complex, as well as with GRGDSP peptide (Fig. 6) . The presence of cycloheximide increased the effects of the function-blocking mAbs and GRGDSP peptide significantly (P < 0.05 in each mAb). 
Microscopic cell adhesion experiments in HCE cells showed that the cells spread both on pFn- and pFn/Tn-C–coated growth substrata within 1 hour. There was less spreading on the Vn coating, whereas no spreading occurred on the Tn-C coating. HCE cells that were plated on uncoated glass coverslips and without cycloheximide spread much slower. 
On pFn-coated glass coverslips, the cells showed talin-reactive FAs as tiny streaklike peripheral structures (Fig. 7A) . mAbs against the Int α5 subunit (Fig. 7B) and the Int β6 subunit (not shown) showed similar immunoreactions located in the FAs in the cells on pFn-coated glass coverslips. No immunoreaction was seen for the Int β6 subunit in HCE cells on Vn-coated growth substratum. Further immunostaining against Int α5 on Vn-coated coverslips remained negative (not shown). When spreading on pFn/Tn-C–coated substratum, immunoreactivities for both Ints α5 (Fig. 6C) and β6 (Fig. 6D) showed a similar location to FAs. 
Further immunofluorescence studies with mAb against the Int β5 subunit showed bright spots oriented to cell surface corresponding to point adhesions on Vn-coated HCE cells (Fig. 7E) , whereas no reactivity was found in HCE cells for this integrin subunit on pFn or pFn/Tn-C coating (not shown). mAb against the Int αv subunit induced a bright reaction on both Vn- and pFn-coated (Fig. 7F) glass coverslips, with some cells appearing to show a brighter point adhesion-like reaction in the cells. A similar immunoreactivity was present in cells growing on the pFn/Tn-C substratum. Unlike Int αv, Int β1 showed a diffuse reaction on HCE cells adhering to the Vn coating. No immunoreaction was visible with mAb against Int β3 on HCE cells cultured on Vn-, pFn-, or pFn/Tn-C–coated glass coverslips (not shown). Also, immunoreaction for Int α9 remained negative on the Vn coating (not shown). 
Discussion
Understanding corneal wound healing fully requires the consideration of stromal and epithelial cell interactions. 3 According to current knowledge, the expression of Fn isoforms and Tn-C is increased during wound healing. 56 57 Although several reports have concluded that keratinocytes are the major source of Tn-C during the healing process of epidermal wounds, 33 56 58 there are still no data showing the origin of Tn-C during corneal wound healing. In the healing corneas the synthesis of Fn isoforms has been attributed to both stromal and epithelial cells. 59 60 61 Our results show, based on both immunoreactivity and Western blot analysis, that human corneal epithelial cells could be the source of the Tn-C and Fn isoforms. Although Tn-X has been found in the BM zone of rat and human corneas, 62 our negative results did not support the idea that it is produced by HCE cells. 
Fn isoforms and Tn-C showed a distinctly different distribution in HCE cells. Fn isoforms were present in distinct fibrillary deposits, whereas Tn-C appeared as diffuse patches underneath the cells. Also, the secretory patterns of these two ECM proteins appeared to differ: EDA- and Onc-Fn were present both in the culture medium and the detergent-resistant ECM, whereas Tn-C was present only in the ECM and was not detectable in the cell culture medium. This suggests that the secretory mechanism for these proteins are different. Some evidence for this was obtained by using the monovalent ionophore monensin as a secretory blocking agent. 63 After treatment with monensin and subsequent accumulation of the cytoplasmic secretory granules immunoreactivities for Fn and Tn-C were clearly located to different granules. 
We then studied the localization of Fn-binding integrins in subconfluent HCE cell cultures and during cell adhesion to different ECM protein-coated substrata. Indirect immunofluorescence studies showed Int α5 subunit in tiny FAs as well as in ECM-adhesions. Int β1 subunit was also localized to the FAs in HCE cell cultures. The adhesion experiments on Fn, which were performed under conditions in which HCE cells did not synthesize endogenous matrix proteins, showed that Int β1 and α5 subunits as well as Int αvβ6 were localized in the FAs within 1 hour. Our results on HCE cells conform with the previous studies showing that Int α5β1 serves as an Fn adhesion receptor in epidermal keratinocytes, but show for the first time that during adhesion to pFn, Int α5β1 acts in concert with Int αvβ6. 64 65 Studies concerning the integrin composition of corneal epithelium have shown the expression of the Int β1 subunit, whereas the expression of the Int α5 subunit has been detected in rat but not in human cornea. 66 67  
Various cells have been shown to use a dozen integrins as receptors binding to the same or different sites in the Fn molecule. 16 Previous studies have suggested that Int α3β1 would be a predominant integrin, being a receptor for laminins, collagens, and Fn, and a recent review has suggested that α3β1 is a receptor for laminin and Fn. 16 54 We started our quantitative cell adhesion assays without cycloheximide treatment, which showed that, indeed, Int α3β1 appeared to mediate the adhesion of HCE cells to pFn. This observation was supported by several earlier studies. 54 68 69 However, Zhang and Kramer 70 have suggested that in human keratinocytes, the endogenous deposition of laminin-5 could mediate such an adhesion through Int α3β1, which is supported by our previous study, which also showed that Int α3β1 mediates the adhesion of HCE cells to laminin-5. 55  
When we performed adhesion experiments with cycloheximide, which inhibited endogenous protein synthesis and secretion, the results showed that Int α5β1 mediated adhesion of HCE cells to Fn. However, a combination of antibodies to Ints α5β1 and αvβ6 inhibited the adhesion of the cells to pFn even more completely. Our results showed that Int α3β1 is not a receptor for Fn, conforming well with the results of Eble et al., 71 showing that soluble human Int α3β1 does not bind to Fn. 
These results therefore suggest that when using epithelial cells capable of producing laminin-5 in experiments, caution must be used, because neither pretreatment with native BSA nor with heat-denatured BSA blocked rapid deposition of laminin-5 during the assay. Therefore, many experiments involving epithelial cells or epithelial-like cells, such as HT1080 cells, should be reinterpreted with respect to the results of cell adhesion to ECM proteins. Similar findings have been reported in endothelial cells and their endogenous production of Fn. 72  
Tn-C belongs to a group of proteins referred to as the matricellular proteins. 73 74 The members of this heterogeneous group of proteins strengthen so-called intermediate adhesion and weaken the stationary stage of cells, promoting strong adhesion. The focus of our study was also to examine the adhesion characteristics of HCE cells to Tn-C and to the growth substratum containing both Tn-C and pFn. Quantitative cell adhesion assays with Tn-C, which has been associated with both adhesive and anti-adhesive properties, 19 20 74 showed that HCE cells did not adhere to it at all. HCE cells adhered to growth substratum containing both Tn-C and pFn as avidly as to pFn. These studies were confirmed by indirect immunofluorescence analysis, which showed that the cells were able to make FAs on both substrata, containing Int α5, αv, β1, and β6. However, when Tn-C was also present, the inhibition of HCE cell adhesion by mAbs against Int α5β1 and synthetic RGD-peptide was clearly increased, suggesting altered adhesion characteristics. This result suggests that the cooperation between Tn-C and pFn may be essential in the adhesion and migration of epithelial cells, because during wound healing, both proteins are produced underneath the cells in the provisional matrix. These results with HCE cells also show that, unlike in fibroblasts, Tn-C does not necessarily affect Fn-induced FA formation and is not excluded at the sites of FAs. 75  
Finally, we studied the adhesion characteristics of HCE cells to substrata coated with Vn—also referred to as serum-spreading factor and therefore present in the provisional matrix during wound healing and affecting keratinocyte motility. 76 77 Vitronectin has been reported to be expressed only variably in human cornea. 30 Although cornea is avascular, tear fluid contains Vn, and therefore corneal wounds are exposed to it. 78 Many of the integrins binding Tn have also been reported to bind Vn. 16 Int αvβ5 and the Int β1 subunit seemed to mediate this adhesion, which raises the question of an additional Int α subunit participating in this process. Because Int α9β1 could be excluded by its absence in immunostaining, there remains the possibility that Int α8β1 could be the additional receptor for Vn. However, because function-blocking mAbs are not available, this could not be tested. The Int αv subunit assembled into naillike FAs and more punctate structures in sparse cultures and only into ringlike structures in dense cultures. Such a differential distribution of Int αvβ5 has been described in various human cells. 79 In addition, we found that Int αvβ6 and Int αvβ5, which is a Vn receptor, localized to FAs when HCE cells adhered to Vn. 
In summary, the present results show that HCE cells produce two Fn isoforms and the large subunit of Tn-C and adhere to Fn through distinct integrins in a process that can be modulated by Tn-C. 
 
Figure 1.
 
Distribution of Fn isoforms and Tn-C in subconfluent HCE cells. Fibrillar cell surface–associated immunofluorescence was present after incubation with antibodies against EDA-Fn (A) and Onc-Fn (B). mAbs against both subunits of Tn-C (C) and against the large subunit of Tn-C (D) showed a diffuse cell substratum–associated patchlike immunoreaction. After monensin treatment, most cells showed double immunostained cytoplasmic granules positive for polyclonal antibodies against Fn (E), whereas immunoreactivity to Tn-C was observed in only a subpopulation of cells, in which it was located in distinctly different granules (F). Magnification, ×800.
Figure 1.
 
Distribution of Fn isoforms and Tn-C in subconfluent HCE cells. Fibrillar cell surface–associated immunofluorescence was present after incubation with antibodies against EDA-Fn (A) and Onc-Fn (B). mAbs against both subunits of Tn-C (C) and against the large subunit of Tn-C (D) showed a diffuse cell substratum–associated patchlike immunoreaction. After monensin treatment, most cells showed double immunostained cytoplasmic granules positive for polyclonal antibodies against Fn (E), whereas immunoreactivity to Tn-C was observed in only a subpopulation of cells, in which it was located in distinctly different granules (F). Magnification, ×800.
Figure 2.
 
Western blot analysis of culture medium of HCE cells. mAbs against ED-A Fn showed a prominent Mr 240,000 band (lane 1), whereas that to Onc-Fn showed a weak band of similar Mr (lane 2). mAbs against the large subunit of Tn-C (lane 3) and that to both subunits of Tn-C (lane 4) did not show any reactivity. Left: molecular weight standards (103).
Figure 2.
 
Western blot analysis of culture medium of HCE cells. mAbs against ED-A Fn showed a prominent Mr 240,000 band (lane 1), whereas that to Onc-Fn showed a weak band of similar Mr (lane 2). mAbs against the large subunit of Tn-C (lane 3) and that to both subunits of Tn-C (lane 4) did not show any reactivity. Left: molecular weight standards (103).
Figure 3.
 
Western blot analysis of ECM produced by HCE cells. Western blot analysis of the ECM, produced by deoxycholate treatment, showed a distinct band of Mr 240,000 with mAb to EDA-Fn (lane 1) and a similar weak band with mAb to Onc-Fn (lane 2). Control Western blot analysis of ECM produced by human embryonic fibroblasts showed a similar Mr 240,000 band with mAb to Onc-Fn (lane 3). mAb to both subunits of Tn-C showed a distinct reaction with Tn-CH (lane 4) and a very weak reaction with Tn-CL (lane 4), whereas mAb against the large subunit of Tn-C showed only the high molecular weight band (lane 5). Control immunoblots with the two mAbs with Tn-C purified from the culture supernatant of U251Mg glioma cells showed, respectively, both subunits of Tn-C (lane 6) and the high molecular weight Tn-C band (lane 7). Left: molecular weight standards of the control protein (103).
Figure 3.
 
Western blot analysis of ECM produced by HCE cells. Western blot analysis of the ECM, produced by deoxycholate treatment, showed a distinct band of Mr 240,000 with mAb to EDA-Fn (lane 1) and a similar weak band with mAb to Onc-Fn (lane 2). Control Western blot analysis of ECM produced by human embryonic fibroblasts showed a similar Mr 240,000 band with mAb to Onc-Fn (lane 3). mAb to both subunits of Tn-C showed a distinct reaction with Tn-CH (lane 4) and a very weak reaction with Tn-CL (lane 4), whereas mAb against the large subunit of Tn-C showed only the high molecular weight band (lane 5). Control immunoblots with the two mAbs with Tn-C purified from the culture supernatant of U251Mg glioma cells showed, respectively, both subunits of Tn-C (lane 6) and the high molecular weight Tn-C band (lane 7). Left: molecular weight standards of the control protein (103).
Figure 4.
 
Distribution of Fn-, Tn- and Vn-binding integrins in HCE cells. Immunoreactivity for Int a5 was detectable in cell membranes and in small streak-like FAs (A, arrows). When focused on the dorsal aspect of the cells Int a5 immunoreactivity was found in cell surface–associated streaks representing ECM adhesions (B, arrows). Int β1 immunoreactivity was found in ventral FAs (C, arrows). Int β6 remained negative in HCE cells (D). Int αv immunoreactivity was present in sparse cultures in peripheral FAs (E, arrows) as well as in spot and ringlike structure in the central part of the cells (E, arrowheads). In confluent HCE cells Int αv immunoreactivity was present only in the ventral variable ringlike structures (F). Magnifications, ×800
Figure 4.
 
Distribution of Fn-, Tn- and Vn-binding integrins in HCE cells. Immunoreactivity for Int a5 was detectable in cell membranes and in small streak-like FAs (A, arrows). When focused on the dorsal aspect of the cells Int a5 immunoreactivity was found in cell surface–associated streaks representing ECM adhesions (B, arrows). Int β1 immunoreactivity was found in ventral FAs (C, arrows). Int β6 remained negative in HCE cells (D). Int αv immunoreactivity was present in sparse cultures in peripheral FAs (E, arrows) as well as in spot and ringlike structure in the central part of the cells (E, arrowheads). In confluent HCE cells Int αv immunoreactivity was present only in the ventral variable ringlike structures (F). Magnifications, ×800
Figure 5.
 
Quantitative cell adhesion assay of HCE cells adhering to Fn (AD) and to Tn-C and Fn/Tn-C (C, D). Without cycloheximide treatment, cell adhesion to Fn was most avidly inhibited by the mAb against the Int α3 chain, whereas, after treatment with cycloheximide, mAbs against the β1 and α5 chain showed distinct inhibition, which was not observed with the mAb against the α3 chain (A). (B) The adhesion to Fn was clearly diminished with mAbs against the Int β1 and α5 chains, but the inhibition was nearly complete only when the mAbs against αvβ6 and α5 were applied together. (C) HCE cells did not adhere at all to purified Tn-C and, when compared with adhesion to Fn, the adhesion of the cells to Fn/Tn was more avidly inhibited by mAbs against the Int β1 and α5 chains. (D) Cell adhesion to Fn was slightly inhibited with RGD peptide at the concentration of 100 μg/mL, but the inhibition was much higher with an RGD peptide concentration of 500 μg/mL, and the inhibition of adhesion with both concentrations of the RGD peptide was much higher when the cells adhered to Fn/Tn-coated substratum. Similarly, the mAb against Int α5 inhibited the adhesion to the Tn/Fn coating more than that to the Fn-only coating.
Figure 5.
 
Quantitative cell adhesion assay of HCE cells adhering to Fn (AD) and to Tn-C and Fn/Tn-C (C, D). Without cycloheximide treatment, cell adhesion to Fn was most avidly inhibited by the mAb against the Int α3 chain, whereas, after treatment with cycloheximide, mAbs against the β1 and α5 chain showed distinct inhibition, which was not observed with the mAb against the α3 chain (A). (B) The adhesion to Fn was clearly diminished with mAbs against the Int β1 and α5 chains, but the inhibition was nearly complete only when the mAbs against αvβ6 and α5 were applied together. (C) HCE cells did not adhere at all to purified Tn-C and, when compared with adhesion to Fn, the adhesion of the cells to Fn/Tn was more avidly inhibited by mAbs against the Int β1 and α5 chains. (D) Cell adhesion to Fn was slightly inhibited with RGD peptide at the concentration of 100 μg/mL, but the inhibition was much higher with an RGD peptide concentration of 500 μg/mL, and the inhibition of adhesion with both concentrations of the RGD peptide was much higher when the cells adhered to Fn/Tn-coated substratum. Similarly, the mAb against Int α5 inhibited the adhesion to the Tn/Fn coating more than that to the Fn-only coating.
Figure 6.
 
Quantitative adhesion analysis of HCE cells adhering to Vn-coated substratum. Cell adhesion to Vn was without cycloheximide clearly inhibited with mAbs against Int β1, Int αvβ5, and RGD-peptide. Inhibition was more pronounced with mAbs against Int αvβ5 and with the RGD peptide when cycloheximide was present.
Figure 6.
 
Quantitative adhesion analysis of HCE cells adhering to Vn-coated substratum. Cell adhesion to Vn was without cycloheximide clearly inhibited with mAbs against Int β1, Int αvβ5, and RGD-peptide. Inhibition was more pronounced with mAbs against Int αvβ5 and with the RGD peptide when cycloheximide was present.
Figure 7.
 
Distribution of integrins in pFn, Tn-C, and Vn-adherent HCE cells. Bright streaklike FA-associated immunofluorescence was present with antibodies against talin (A) and Int α5 (B) on the Fn coating. Similar immunoreactions occurred with both Ints α5 (C) and β6 (D) on the pFn/Tn-C coating. Int β5 immunoreactivity was present on the cell surface as bright spots corresponding to point adhesions on Vn-coated growth substratum (E). On the Fn coating Int αv immunoreactivity was also present in the FAs (F, arrows).
Figure 7.
 
Distribution of integrins in pFn, Tn-C, and Vn-adherent HCE cells. Bright streaklike FA-associated immunofluorescence was present with antibodies against talin (A) and Int α5 (B) on the Fn coating. Similar immunoreactions occurred with both Ints α5 (C) and β6 (D) on the pFn/Tn-C coating. Int β5 immunoreactivity was present on the cell surface as bright spots corresponding to point adhesions on Vn-coated growth substratum (E). On the Fn coating Int αv immunoreactivity was also present in the FAs (F, arrows).
The authors thank Pipsa Kaipainen, Marja-Leena Piironen, and Reijo Karppinen for skillful technical assistance and Kenneth M. Yamada, Zena Werb, Martin E. Hemler, Dean Sheppard, Luciano Zardi, and David E. Cheresh for providing mAbs for the study. 
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Figure 1.
 
Distribution of Fn isoforms and Tn-C in subconfluent HCE cells. Fibrillar cell surface–associated immunofluorescence was present after incubation with antibodies against EDA-Fn (A) and Onc-Fn (B). mAbs against both subunits of Tn-C (C) and against the large subunit of Tn-C (D) showed a diffuse cell substratum–associated patchlike immunoreaction. After monensin treatment, most cells showed double immunostained cytoplasmic granules positive for polyclonal antibodies against Fn (E), whereas immunoreactivity to Tn-C was observed in only a subpopulation of cells, in which it was located in distinctly different granules (F). Magnification, ×800.
Figure 1.
 
Distribution of Fn isoforms and Tn-C in subconfluent HCE cells. Fibrillar cell surface–associated immunofluorescence was present after incubation with antibodies against EDA-Fn (A) and Onc-Fn (B). mAbs against both subunits of Tn-C (C) and against the large subunit of Tn-C (D) showed a diffuse cell substratum–associated patchlike immunoreaction. After monensin treatment, most cells showed double immunostained cytoplasmic granules positive for polyclonal antibodies against Fn (E), whereas immunoreactivity to Tn-C was observed in only a subpopulation of cells, in which it was located in distinctly different granules (F). Magnification, ×800.
Figure 2.
 
Western blot analysis of culture medium of HCE cells. mAbs against ED-A Fn showed a prominent Mr 240,000 band (lane 1), whereas that to Onc-Fn showed a weak band of similar Mr (lane 2). mAbs against the large subunit of Tn-C (lane 3) and that to both subunits of Tn-C (lane 4) did not show any reactivity. Left: molecular weight standards (103).
Figure 2.
 
Western blot analysis of culture medium of HCE cells. mAbs against ED-A Fn showed a prominent Mr 240,000 band (lane 1), whereas that to Onc-Fn showed a weak band of similar Mr (lane 2). mAbs against the large subunit of Tn-C (lane 3) and that to both subunits of Tn-C (lane 4) did not show any reactivity. Left: molecular weight standards (103).
Figure 3.
 
Western blot analysis of ECM produced by HCE cells. Western blot analysis of the ECM, produced by deoxycholate treatment, showed a distinct band of Mr 240,000 with mAb to EDA-Fn (lane 1) and a similar weak band with mAb to Onc-Fn (lane 2). Control Western blot analysis of ECM produced by human embryonic fibroblasts showed a similar Mr 240,000 band with mAb to Onc-Fn (lane 3). mAb to both subunits of Tn-C showed a distinct reaction with Tn-CH (lane 4) and a very weak reaction with Tn-CL (lane 4), whereas mAb against the large subunit of Tn-C showed only the high molecular weight band (lane 5). Control immunoblots with the two mAbs with Tn-C purified from the culture supernatant of U251Mg glioma cells showed, respectively, both subunits of Tn-C (lane 6) and the high molecular weight Tn-C band (lane 7). Left: molecular weight standards of the control protein (103).
Figure 3.
 
Western blot analysis of ECM produced by HCE cells. Western blot analysis of the ECM, produced by deoxycholate treatment, showed a distinct band of Mr 240,000 with mAb to EDA-Fn (lane 1) and a similar weak band with mAb to Onc-Fn (lane 2). Control Western blot analysis of ECM produced by human embryonic fibroblasts showed a similar Mr 240,000 band with mAb to Onc-Fn (lane 3). mAb to both subunits of Tn-C showed a distinct reaction with Tn-CH (lane 4) and a very weak reaction with Tn-CL (lane 4), whereas mAb against the large subunit of Tn-C showed only the high molecular weight band (lane 5). Control immunoblots with the two mAbs with Tn-C purified from the culture supernatant of U251Mg glioma cells showed, respectively, both subunits of Tn-C (lane 6) and the high molecular weight Tn-C band (lane 7). Left: molecular weight standards of the control protein (103).
Figure 4.
 
Distribution of Fn-, Tn- and Vn-binding integrins in HCE cells. Immunoreactivity for Int a5 was detectable in cell membranes and in small streak-like FAs (A, arrows). When focused on the dorsal aspect of the cells Int a5 immunoreactivity was found in cell surface–associated streaks representing ECM adhesions (B, arrows). Int β1 immunoreactivity was found in ventral FAs (C, arrows). Int β6 remained negative in HCE cells (D). Int αv immunoreactivity was present in sparse cultures in peripheral FAs (E, arrows) as well as in spot and ringlike structure in the central part of the cells (E, arrowheads). In confluent HCE cells Int αv immunoreactivity was present only in the ventral variable ringlike structures (F). Magnifications, ×800
Figure 4.
 
Distribution of Fn-, Tn- and Vn-binding integrins in HCE cells. Immunoreactivity for Int a5 was detectable in cell membranes and in small streak-like FAs (A, arrows). When focused on the dorsal aspect of the cells Int a5 immunoreactivity was found in cell surface–associated streaks representing ECM adhesions (B, arrows). Int β1 immunoreactivity was found in ventral FAs (C, arrows). Int β6 remained negative in HCE cells (D). Int αv immunoreactivity was present in sparse cultures in peripheral FAs (E, arrows) as well as in spot and ringlike structure in the central part of the cells (E, arrowheads). In confluent HCE cells Int αv immunoreactivity was present only in the ventral variable ringlike structures (F). Magnifications, ×800
Figure 5.
 
Quantitative cell adhesion assay of HCE cells adhering to Fn (AD) and to Tn-C and Fn/Tn-C (C, D). Without cycloheximide treatment, cell adhesion to Fn was most avidly inhibited by the mAb against the Int α3 chain, whereas, after treatment with cycloheximide, mAbs against the β1 and α5 chain showed distinct inhibition, which was not observed with the mAb against the α3 chain (A). (B) The adhesion to Fn was clearly diminished with mAbs against the Int β1 and α5 chains, but the inhibition was nearly complete only when the mAbs against αvβ6 and α5 were applied together. (C) HCE cells did not adhere at all to purified Tn-C and, when compared with adhesion to Fn, the adhesion of the cells to Fn/Tn was more avidly inhibited by mAbs against the Int β1 and α5 chains. (D) Cell adhesion to Fn was slightly inhibited with RGD peptide at the concentration of 100 μg/mL, but the inhibition was much higher with an RGD peptide concentration of 500 μg/mL, and the inhibition of adhesion with both concentrations of the RGD peptide was much higher when the cells adhered to Fn/Tn-coated substratum. Similarly, the mAb against Int α5 inhibited the adhesion to the Tn/Fn coating more than that to the Fn-only coating.
Figure 5.
 
Quantitative cell adhesion assay of HCE cells adhering to Fn (AD) and to Tn-C and Fn/Tn-C (C, D). Without cycloheximide treatment, cell adhesion to Fn was most avidly inhibited by the mAb against the Int α3 chain, whereas, after treatment with cycloheximide, mAbs against the β1 and α5 chain showed distinct inhibition, which was not observed with the mAb against the α3 chain (A). (B) The adhesion to Fn was clearly diminished with mAbs against the Int β1 and α5 chains, but the inhibition was nearly complete only when the mAbs against αvβ6 and α5 were applied together. (C) HCE cells did not adhere at all to purified Tn-C and, when compared with adhesion to Fn, the adhesion of the cells to Fn/Tn was more avidly inhibited by mAbs against the Int β1 and α5 chains. (D) Cell adhesion to Fn was slightly inhibited with RGD peptide at the concentration of 100 μg/mL, but the inhibition was much higher with an RGD peptide concentration of 500 μg/mL, and the inhibition of adhesion with both concentrations of the RGD peptide was much higher when the cells adhered to Fn/Tn-coated substratum. Similarly, the mAb against Int α5 inhibited the adhesion to the Tn/Fn coating more than that to the Fn-only coating.
Figure 6.
 
Quantitative adhesion analysis of HCE cells adhering to Vn-coated substratum. Cell adhesion to Vn was without cycloheximide clearly inhibited with mAbs against Int β1, Int αvβ5, and RGD-peptide. Inhibition was more pronounced with mAbs against Int αvβ5 and with the RGD peptide when cycloheximide was present.
Figure 6.
 
Quantitative adhesion analysis of HCE cells adhering to Vn-coated substratum. Cell adhesion to Vn was without cycloheximide clearly inhibited with mAbs against Int β1, Int αvβ5, and RGD-peptide. Inhibition was more pronounced with mAbs against Int αvβ5 and with the RGD peptide when cycloheximide was present.
Figure 7.
 
Distribution of integrins in pFn, Tn-C, and Vn-adherent HCE cells. Bright streaklike FA-associated immunofluorescence was present with antibodies against talin (A) and Int α5 (B) on the Fn coating. Similar immunoreactions occurred with both Ints α5 (C) and β6 (D) on the pFn/Tn-C coating. Int β5 immunoreactivity was present on the cell surface as bright spots corresponding to point adhesions on Vn-coated growth substratum (E). On the Fn coating Int αv immunoreactivity was also present in the FAs (F, arrows).
Figure 7.
 
Distribution of integrins in pFn, Tn-C, and Vn-adherent HCE cells. Bright streaklike FA-associated immunofluorescence was present with antibodies against talin (A) and Int α5 (B) on the Fn coating. Similar immunoreactions occurred with both Ints α5 (C) and β6 (D) on the pFn/Tn-C coating. Int β5 immunoreactivity was present on the cell surface as bright spots corresponding to point adhesions on Vn-coated growth substratum (E). On the Fn coating Int αv immunoreactivity was also present in the FAs (F, arrows).
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