IOVS Drug Metabolism and Disposition
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lin, L.
Right arrow Articles by Kurpakus-Wheater, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lin, L.
Right arrow Articles by Kurpakus-Wheater, M.
(Investigative Ophthalmology and Visual Science. 2002;43:2615-2621.)
© 2002 by The Association for Research in Vision and Ophthalmology, Inc.

Laminin {alpha}5 Chain Adhesion and Signaling in Conjunctival Epithelial Cells

Lin Lin and Michelle Kurpakus-Wheater

From the Department of Anatomy and Cell Biology, Wayne State University School of Medicine, Detroit, Michigan.


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
PURPOSE. To identify peptides of the LG4 module of the laminin {alpha}5 chain that mediate human conjunctival epithelial cell adhesion to the laminin-10 isoform.

METHODS. A peptide corresponding to a major heparin- and cell-binding domain of the LG4 module of the laminin {alpha}5 chain was analyzed. The attachment of conjunctival epithelial cells to the peptide compared with laminin-10 was determined by colorimetric adhesion assay. The role of glycosaminoglycans in mediating adhesion to the peptide was determined by altering their function at the cell surface and by blocking adhesion with exogenous glycosaminoglycans. The role of syndecan-4 in cell adhesion to the peptide was examined by adhesion assay. The role of the peptide in cell signaling was examined by phosphotyrosine Western blot analysis.

RESULTS. The peptide facilitated the adhesion of conjunctival epithelium, although not as efficiently as laminin-10. Heparinase had no effect on adhesion to the peptide. In contrast, adhesion to the peptide decreased in glycosaminoglycan-deficient cells, heparatinase-treated cells, cells blocked with exogenous heparan sulfate proteoglycan, and cells treated with antibodies to the ectodomain of syndecan-4. Cell adhesion to the peptide for 90 or 120 minutes resulted in a significant increase in focal adhesion kinase (FAK) tyrosine phosphorylation, compared with the nonadherent control.

CONCLUSIONS. Human conjunctival epithelial cells use a heparin- and cell-binding peptide of the LG4 module of laminin {alpha}5 chain in adhesion to laminin-10. Syndecan-4 is one mechanism by which the peptide facilitates adhesion. In addition to adhesion, the peptide may function in cell-signaling events.


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Laminins are heterotrimeric glycoproteins consisting of one {alpha} chain, one ß chain, and one {gamma} chain. Currently, five {alpha} chains ({alpha}1–5), three ß chains (ß1–3), and three {gamma} chains ({gamma}1–3) have been described, forming fifteen isoforms (laminins 1–15). Laminins are localized to epithelial basement membranes, and in general play major roles in cell adhesion, migration, proliferation, differentiation, and survival.1

The widespread distribution of the {alpha}5 chain in developing and adult tissues suggests that it is a major laminin chain in epithelial basement membranes.2 Indeed, it has been suggested that the {alpha}5 chain has the widest expression of all {alpha} chains and thus has a more ubiquitous distribution than the laminin {alpha}1 chain, a component of the classic laminin-1 isoform. Laminin {alpha}5 chain is a component of laminin-10 ({alpha}5ß1{gamma}1 integrin) and laminin-11 ({alpha}5ß2{gamma}1 integrin). Despite its apparent widespread distribution, the exact function of laminin-10 has not been fully elucidated. Using immunofluorescence microscopy, laminin-10 has been localized to both corneal and conjunctival basement membranes.3 4 We have shown that laminin-10 can mediate the rapid attachment of both corneal and conjunctival epithelial cells in vitro.5 Other laminin isoforms are also present in both corneal and conjunctival basement membranes, but the purpose of multiple laminin isoform expression in ocular surface basement membranes is still unknown.

All {alpha} chains contain a tandem of five laminin G domainlike (LG) modules (LG1–5) located within the C terminus of the molecule.6 Analysis of the adhesion characteristics of laminin peptides has demonstrated that those contained within LG modules are responsible for most of the cell-adhesion activity of the laminin {alpha} chains.7 However, it has been suggested by several investigators that the ability of LG module peptides to promote adhesion may be cell-type specific.8 9 In agreement with this Tani et al.10 showed that integrin-mediated mechanisms of adhesion to laminin-10 were dependent on cell type. For example, in JAR cells, adhesion is {alpha}6ß1-dependent; but, in PANC-1 cells, adhesion is mediated by {alpha}3ß1. We showed that in a transformed human corneal epithelial cell line (HCE-T) adhesion to laminin-10 is mediated by the {alpha}3ß1 integrin.5

In addition to integrins, cell surface proteoglycans containing heparan sulfate glycosaminoglycan (GAG) side chains participate in adhesion of cells to extracellular matrix molecules including laminin. In many cases heparan sulfate proteoglycans may function as coreceptors with integrins in regulating cell adhesion activity. The syndecans are a family of four (syndecan 1–4) transmembrane heparan sulfate proteoglycans expressed by nearly all cell types.11 12 Syndecan-4 (amphiglycan or ryudocan) functions as a coreceptor with integrins to regulate epithelial cell spreading and the assembly of focal adhesions.13 In focal adhesions, syndecan-4 and ß1 integrins cooperatively activate Rho and initiate signaling mechanisms.

The G domain of the laminin {alpha}4 chain contains both high- and low-affinity heparin-binding sites.14 Binding of heparin and {alpha}-dystroglycan can be localized to the LG4 module of the laminin {alpha}1 chain.15 Recently, a peptide resident in the LG4 module of the laminin {alpha}5 chain, AGQWHRVSVRWG, was identified as a major heparin and cell-binding peptide16 and was termed F4. To extend our observation that laminin-10 is an efficient adhesion protein for human conjunctival epithelial cells, in the present study, we examined the adhesion characteristics of peptide F4 of the laminin {alpha}5 chain.

We hypothesized that peptide F4 mediates conjunctival epithelial adhesion through mechanisms involving heparin or heparan sulfate cellular receptors. In this study, peptide F4 was a cell-binding site for human conjunctival epithelial cells, and heparan sulfate molecules, including syndecan-4, mediated this adhesion. Evidence was also provided that conjunctival epithelial cell adhesion to peptide F4 can initiate cell signaling through the tyrosine phosphorylation of focal adhesion kinase (FAK).


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Synthesis of Peptides
The three peptides used in this study were commercially prepared (United Biochemical Research Inc., Seattle, WA). According to the nomenclature of Nielsen et al.,16 peptide F1, consists of the amino acid sequence RNRLHLSMLVRP, and peptide F4 consists of the amino acid sequence AGQWHRVSVRWG. A scrambled peptide F4 with sequence AVSWHWGAVRGR was prepared for use as a negative control. A preliminary cell adhesion assay showed that attachment to peptide F4 was not significantly different at concentrations of 10 or 100 µg/mL (results not shown). Therefore to conserve peptide, all adhesion assays were performed at a concentration of 10 µg/mL.

Antibodies and Reagents
The monoclonal antibody (mAb) 4C7 was purchased from Chemicon International, Inc. (Temecula, CA). This antibody has been shown to be specific for the laminin {alpha}5 chain.17 mAb specific for the ectodomain of human syndecan-4, mAb against phosphorylated tyrosine residues (PY99), and a rabbit polyclonal antibody directed against FAK were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Unless noted otherwise, all other reagents were purchased from Sigma Chemical Co. (St. Louis, MO).

Cell Culture
The human conjunctival epithelial cell line HC0597 was generously provided by Sherry Ward (The Gillette Company, Gillette Medical Evaluation Laboratories, Boston, MA). Cells were maintained in T-75 flasks in serum-free KGM, which consists of keratinocyte basal medium (KBM) supplemented with 0.1% bovine insulin, 0.1% human epidermal growth factor, 0.4% bovine pituitary extract, and 0.1% hydrocortisone (Clonetics, Inc., San Diego, CA). Medium was replaced every other day, and cells were trypsinized at approximately 80% confluence for use in experiments or for further passage. HC0597 cells were used between passages 12 and 25. The life span of the HC0597 line is approximately 30 passages.

Colorimetric Cell Adhesion Assay
Adhesion assays were performed according to the procedure of Zhang and Kramer18 as described.5 Wells of a 96-well microtiter plate were coated with 100 µL of 10 µg/mL laminin-10 (Gibco-BRL Life Technologies, Gaithersburg, MD), peptide F1, or peptide F4. After a 1-hour incubation at 37°C the wells were washed three times with sterile PBS and blocked with 1% bovine serum albumin (BSA). The wells were washed with PBS again and stored at 4°C until use. Trypsinized HC0597 cells were pelleted by centrifugation in trypsin-neutralizing solution (Cascade Biologics, Portland, OR). Cell pellets were then washed three times in KBM-0.1% BSA and resuspended in the same medium. Cells were plated at 2 x 104 cells/well and incubated for 1 hour at 37°C in 5% CO2. The wells were washed with KBM-0.1% BSA to remove unattached cells, and attached cells were fixed, stained with 2% crystal violet, and lysed in 2% SDS. The absorbance of the lysate was measured at a 570-nm wavelength (A570), and absorbance of cells adherent to BSA only (BSA control) was subtracted. Each adhesion assay was repeated three times, with six wells of each treatment and control(s) assayed each time. The results are reported as the percentage of adhesion compared with the control (set at 100%). Errors are expressed as SEM.

For adhesion assays with the 4C7 antibody, aliquots of HC0597 cells were incubated with different dilutions of antibody (1:50, 1:100, or 1:1000) at 37°C for 30 minutes. Another aliquot of cells was incubated in nonimmune IgG as control. Cells were then plated at 2 x 104 cells/well on to laminin-10–coated microtiter plates. After a 1-hour incubation the assay was completed as described in the previous paragraph. For adhesion assays with the syndecan-4 antibody, HC0597 cells were incubated with 20 µg/mL antibody at 37°C for 30 minutes or with nonimmune IgG. Cells were then plated at 2 x 104 cells/well on to peptide F4–coated microtiter plates. After a 1-hour incubation the assay was completed as described.

For adhesion assays using the peptides as blocking agents, two aliquots of HC0597 cells were incubated with peptide F4 (10 µg/mL) or scrambled peptide F4 (10 µg/mL) at 37°C for 30 minutes. The cells were then plated at 2 x 104 cells/well onto laminin-10–coated microtiter plates. After a 1-hour incubation, the assay was completed as described.

Disruption of Cell-Surface GAGs
To disrupt the sulfation of cell surface GAGs, HC0597 cells at approximately 70% confluence were cultured in KGM containing 50 mM sodium chlorate for 24 or 48 hours.19 The cells were then trypsinized and used for cell adhesion assays with laminin-10 or peptide F4. Control experiments for this assay included the BSA control plus HC0597 cells that were not treated with sodium chlorate. In a second study, HC0597 cells were trypsinized, and suspended cells were incubated with 1 U/mL heparinase or heparatinase for 30 minutes at 37°C.16 20 Cells (2 x 104) were then plated on laminin-10– or peptide F4–coated microtiter plates for the adhesion assay. In addition to the BSA control, HC0597 cells were incubated with heat-treated, inactivated enzymes (100°C for 10 minutes and the addition of 1 mM ZnCl2) for the same length of time. In some experiments, HC0597 cells were treated with 2 µg/mL cycloheximide for 2 hours to inhibit protein synthesis20 before treatment with the enzymes. However, we did not detect any differences in adhesion results with cycloheximide-treated cells compared with nontreated cells.

Exogenous GAGs
Microtiter plates were coated with laminin-10 or peptide F4, as just described. The wells were then incubated with 100 µL of 25 or 50 µg/mL heparin (from porcine intestinal mucosa; Sigma), heparan sulfate (from bovine kidney; Sigma), or heparan sulfate proteoglycan (from basement membrane of Engelbreth-Holm-Swarm mouse sarcoma; Sigma). The wells were incubated with the GAGs for 60 minutes at 37°C and washed three times with PBS to remove unbound GAG. HC0597 cells were then plated at 2 x 104 cells/well for use in the colorimetric adhesion assay.

Immunofluorescence Microscopy
Sterile glass coverslips (22 x 22 mm) were coated with 10 µg/mL peptide F4 and blocked with BSA. HC0597 cells were seeded at 3 x 105 cells and cultured in KGM for 4 or 24 hours. The cells were fixed in 3.7% formalin, permeabilized in 1% Triton X-100, and incubated with antibodies to syndecan-4 (diluted 1:20 in PBS). After washing in PBS, the cells were incubated with rhodamine-conjugated goat anti-rabbit secondary antibodies. Images were captured with a fluorescence microscope (Axiophot; Carl Zeiss, Oberkochen, Germany) and image-management software (Spot Diagnostics; Sterling Heights, MI).

Phosphotyrosine Western Blot Analysis and Densitometry
HC0597 cells were trypsinized, pelleted, and washed three times in 1:1 DMEM-0.1% BSA, Ham’s F12 containing 20 µM sodium orthovanadate, and 5 mM sodium fluoride. Cells were resuspended in the same medium and plated onto 60-mm tissue culture plates coated with 10 µg/mL peptide F4. After 5, 15, 30, 60, 90, or 120 minutes’ incubation one plate was rinsed with PBS, and the attached cells were lysed in sample buffer containing 10 mM Tris-HCl (pH 6.8), 150 mM NaCl, 10% glycerol, 1% ß-mercaptoethanol, 2% SDS, 200 µM sodium orthovanadate, and 5 mM sodium fluoride.21 Suspended cells at time 0 were used as the control and were pelleted and lysed in the same buffer.

Cell lysates were analyzed for total protein, by the method of Henkel and Beiger.22 Samples containing 15 µg total protein were separated by 7.5% SDS-PAGE and transferred to nitrocellulose membrane for Western blot analysis. Molecular weight standards were run with the gels. The blots were blocked in Tris-buffered saline (TBS) containing 5% milk and incubated in PY99 diluted 1:500 in TBS containing 0.1% Tween-20 and 0.2% milk. Proteins containing phosphorylated tyrosine residues were detected as exposed bands on x-ray film, with alkaline phosphatase–conjugated goat anti-mouse secondary antibodies coupled with a chemiluminescence detection system (Immune-Star; Bio-Rad Laboratories, Hercules, CA). The intensity of individual immunoreactive protein bands was determined by scanning the developed x-ray films and measuring the optical density and area of the bands using image-analysis software (ImageQuant; Molecular Dynamics, Sunnyvale, CA).

After chemiluminescence analysis, the blots were stripped by incubation for 30 minutes at 37°C in 62.5 mM Tris-HCl (pH 6.7), 2% SDS, and 0.1 mM ß-mercaptoethanol. The blot was then reprobed with a rabbit polyclonal antibody to FAK. This confirmed the identification of a 125-kDa tyrosine phosphorylated protein as FAK and allowed for normalization of the loading of FAK protein in each lane of the gel.

Statistical Analysis
For all statistical analyses of significant differences in adhesion, the two-tailed unpaired Student’s t-test was used, with significance set at P <= 0.05.


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Adhesion of Conjunctival Epithelial Cells to Peptide F4 Compared with Laminin-10
Our previous studies have shown that laminin-10 is a major adhesive laminin in human conjunctival epithelial cells.21 To determine whether G-domains of the {alpha}5 chain of laminin-10 play a role in the adhesion of this cell type, rapid attachment of HC0597 cells to immobilized laminin-10 was assayed in the presence of monoclonal antibody 4C7. ELISA has been used to demonstrate that the 4C7 epitope is present in the commercial preparation of laminin used in our adhesion assay.23 The 4C7 antibody binds at or near the G domain of the laminin {alpha}5 chain.7 Compared with control IgG, the presence of 4C7 antibody in the assay served to inhibit rapid HC0597 adhesion to laminin-10 in a dose-dependent manner (Fig. 1) . The maximal amount of antibody tested inhibited cell binding by approximately 50% (P < 0.0001 compared with the control), suggesting that the G domains play at least a partial role in the adhesion of conjunctival epithelial cells to laminin-10.



View larger version (15K):
[in this window]
[in a new window]
  		Figure 1. Effect of laminin {alpha}5 chain antibody on human conjunctival epithelial cell adhesion to laminin-10. The results of the assay are expressed as the percentage of cell adhesion in the presence of 4C7 antibody compared with nonimmune IgG control. Six values were averaged for each treatment, and data are the mean ± SEM of results in three experiments. At all antibody dilutions, adhesion was significantly inhibited (P < 0.0001) compared with the IgG control.

 
To identify amino acid sequences within the G domains that participate in conjunctival epithelial cell adhesion to laminin-10, the peptide AGQWHRVSVRWG, termed F4 by Nielsen et al.,16 was examined. In addition, the peptide RNRLHLSMLVRP, termed F1 by Nielsen et al.,16 which is the laminin {alpha}5 chain peptide homologous to AG73 of laminin {alpha}1, was assayed.20 Compared with the BSA control both F1 and F4 peptides facilitated the rapid adhesion of HC0597 cells (Fig. 2 ; P < 0.0001 for both F1 and F4 adhesion compared with BSA). However, the cells adhered less readily to either peptide, compared with laminin-10, with both peptides exhibiting approximately 70% of the adhesion capability of laminin-10. Throughout this study, we noted that the adhesion characteristics of peptides F4 and F1 were consistently the same; however, the F1 peptide could not promote the tyrosine phosphorylation of FAK (data not shown, see Fig. 9 ). Therefore, we report only the results using peptide F4.



View larger version (13K):
[in this window]
[in a new window]
  		Figure 2. Comparison of human conjunctival epithelial cell adhesion to laminin-10 and laminin {alpha}5 chain peptides. Six values were averaged for each treatment, and data are the mean ± SEM of results in three experiments.

 


View larger version (29K):
[in this window]
[in a new window]
  		Figure 9. Relative levels of tyrosine phosphorylation of FAK in human conjunctival epithelial cells adherent to peptide F4. FAK protein content and tyrosine phosphorylation was normalized to control, which is a lysate prepared from suspended cells at time 0. The resultant relative multiple of the difference in FAK tyrosine phosphorylation is indicated for the 60-, 90-, and 120-minute adhesion times.

 
To determine how significant peptide F4 is in laminin-10–mediated cell adhesion, it was used as a blocking peptide in adhesion assays. Peptide F4 partially inhibited (by 16%) adhesion of HC0597 cells to laminin-10 (Fig. 3) ; however, this inhibition was not significant (P = 0.0779 compared with control). A peptide synthesized from a scrambled F4 amino acid sequence (sF4), AVSWHWGAVRGR, however, did not inhibit adhesion at all (P = 0.8494 compared with control). Our observation that peptide F4 affords only a partial inhibition of HC0597 adhesion to laminin-10 suggests that other modules of the intact molecule mediate conjunctival epithelial cell adhesion in addition to those in the LG4 module of the {alpha}5 chain.



View larger version (14K):
[in this window]
[in a new window]
  		Figure 3. Effect of peptide F4 competitive blocking on human conjunctival epithelial cell adhesion to immobilized laminin-10. HC0597 cells were incubated on immobilized laminin-10 alone, on laminin-10 in the presence of peptide F4, or on laminin-10 in the presence of a scrambled amino acid version of peptide F4 (sF4) for 1 hour before the colorimetric adhesion assay. The results of the assay are expressed as the percentage of cell adhesion compared with laminin-10. Six values were averaged for each treatment, and data are the mean ± SEM of results in three experiments.

 
Role of Cell Surface GAGs in Conjunctival Epithelial Cell Adhesion to Laminin-10 and Peptide F4
Cell surface GAGs, components of proteoglycans, participate in attachment of cells to extracellular matrix molecules. We hypothesized that GAGs participate in adhesion of conjunctival epithelial cells to peptide F4. To test this hypothesis GAG function was inhibited in HC0597 cells before adhesion assays were performed. Altered sulfation of GAGs synthesized by conjunctival epithelial cells was achieved by treating the cultures with sodium chlorate, an inhibitor of 3-phosphoadenosine 5'-phosphosulfate synthesis.19 Adhesion of sulfated GAG-deficient HC0597 cells to laminin-10 was inhibited by 20%, which was not a significant inhibition (Fig. 4) . In contrast, a 41% inhibition of adhesion to peptide F4 was noted in sulfated GAG-deficient HC0597 cells. This inhibition was statistically significant compared with the control (P < 0.0001). Similar results were noted in cells treated with sodium chlorate for 24 (Fig. 4) or 48 hours (results not shown).



View larger version (21K):
[in this window]
[in a new window]
  		Figure 4. Role of cell surface GAG integrity in adhesion of human conjunctival epithelial cells to laminin-10. For both laminin-10 and peptide F4 the results of the assay are expressed as the percentage of cell adhesion compared with control cells, which were not treated with sodium chlorate. Six values were averaged for each treatment, and data are the mean ± SEM of results in three experiments.

 
Adhesion assays performed in the presence of sodium chlorate suggest that cell surface sulfated GAGs may play a role in mediating adhesion of conjunctival epithelial cells to peptide F4. To examine this further, cell surface GAG structure was enzymatically disrupted by treatment with heparinase or heparatinase. Treatment with heparinase had a minimal effect on the adhesion of HC0597 cells to laminin-10 and no effect on adhesion to peptide F4 (Fig. 5) . In contrast, treatment with heparatinase resulted in a statistically significant inhibition of conjunctival epithelial cell adhesion to laminin-10 (35%; P < 0.0001 compared with control) as well as to peptide F4 (23%; P = 0.0065 compared with control).



View larger version (29K):
[in this window]
[in a new window]
  		Figure 5. Effect of heparinase and heparatinase on human conjunctival epithelial cell adhesion to laminin-10. For both laminin-10 and peptide F4 the results of the assay are expressed as the percentage of cell adhesion compared with control cells incubated with inactive enzyme. Six values were averaged for each treatment, and data are the mean ± SEM of results in three experiments.

 
We examined the potential role of cell surface GAGs in mediating cell adhesion by using two methods to disrupt GAG function. To confirm these observations, we next used exogenous GAGs as blocking agents in cell adhesion assays. We examined the ability of heparin, heparan sulfate, or heparan sulfate proteoglycan (HSPG; Fig. 6 ) to block HC0597 adhesion to immobilized laminin-10 or peptide F4. Only heparan sulfate proteoglycan significantly inhibited adhesion of conjunctival epithelial cells to laminin-10 (by 47%; P < 0.0001 compared with control). Although all GAGs used as blocking agents significantly inhibited the adhesion of conjunctival epithelial cells to peptide F4 (P < 0.0001 for all treatments compared with control), heparan sulfate proteoglycan was the most effective (53% inhibition). Figure 6 demonstrates the effect of exogenous GAGs on cell adhesion at a concentration of 50 µg/mL, but similar inhibitory effects were also observed at concentrations of 25 µg/mL (results not shown).



View larger version (33K):
[in this window]
[in a new window]
  		Figure 6. Effect of exogenous GAGs on human conjunctival epithelial cell adhesion to laminin-10. For both laminin-10 and peptide F4 the results of the assay are expressed as the percentage of cell adhesion compared with the control. Six values were averaged for each treatment, and data are the mean ± SEM of results in three experiments.

 
Mediation of Conjunctival Epithelial Cell Adhesion to Peptide F4 by Syndecan-4
Our adhesion assays using enzymes that cleave heparan sulfate moieties coupled with those that used heparan sulfate proteoglycan as a competitive blocking substrate suggest that heparan sulfate proteoglycan can mediate adhesion of conjunctival epithelial cells to peptide F4. Syndecan-4 is an integral membrane proteoglycan of epithelial cells that contains heparan sulfate GAGs. We therefore investigated whether syndecan-4 plays a role in the adhesion of conjunctival epithelial cells to peptide F4.

Immunofluorescence microscopy confirmed that syndecan-4 was polarized to cell–substrate attachment sites in response to culture on exogenous F4 peptide. As HC0597 cells adhered and spread on exogenous F4 peptide, syndecan-4 localization initially appeared at the cell edges (Fig. 7A) . After 24 hours of interaction with F4 peptide, syndecan-4 was localized to discreet focal structures distributed throughout the cell (Fig. 7B) . Cell adhesion assays were then performed in the presence of an antibody to the ectodomain of syndecan-4. Compared with irrelevant IgG control, antibodies to syndecan-4 significantly inhibited the adhesion of HC0597 cells to the F4 peptide, demonstrating an 82% reduction in adhesion (Fig. 8 ; P < 0.0001 compared with control).



View larger version (114K):
[in this window]
[in a new window]
  		Figure 7. Localization of syndecan-4 in cultured human conjunctival epithelial cells. HC0597 cells were cultured on peptide F4 for 4 (A) or 24 (B) hours before processing for immunofluorescence microscopy with an antibody to syndecan-4. After 4 hours in culture, syndecan-4 was localized in small deposits within the cytoplasm of the spread edges of adherent cells (A, arrow). After 24 hours in culture, syndecan-4 was localized in focal adhesion-like plaque structures distributed throughout the adherent cells (B, arrows).

 


View larger version (16K):
[in this window]
[in a new window]
  		Figure 8. Effect of syndecan-4 antibody on adhesion of human conjunctival epithelial cell to peptide F4. HC0597 cells were incubated with 20 µg/mL antibody to the ectodomain of syndecan-4 for 60 minutes. The cells were then incubated on peptide F4 for 1 hour before the colorimetric adhesion assay. The results of the assay are expressed as the percentage of cell adhesion compared with the nonimmune IgG control. Six values were averaged for each treatment, and data are the mean ± SEM of results in three experiments.

 
Effect of Peptide F4 Interaction with Conjunctival Epithelial Cells on Tyrosine Phosphorylation
Syndecan-4 is a component of focal adhesions. In addition to mediating cell–extracellular matrix adhesion, focal adhesions contain many protein kinases involved in cell signaling pathways. The ability of peptide F4 to localize syndecan-4 to focal adhesion-like structures implies that the peptide may play a role in cellular laminin-10 signaling events. To provide evidence of this, we assayed for the ability of exogenous peptide F4 to promote the tyrosine phosphorylation of FAK. A densitometric comparison of tyrosine phosphorylation levels of FAK demonstrated that compared with control, phosphorylation levels were increased 2.8-fold in conjunctival epithelial cells after 90 minutes and 4.3-fold after 120 minutes of adhesion to peptide F4 (Fig. 9) .


   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
The basement membrane underlying conjunctival epithelium contains laminin isoforms that incorporate the {alpha}5 chain, including laminin-10 and laminin-11.3 4 We were interested in determining why the conjunctival basement membrane incorporates several laminin isoforms and the functions of each. In an effort to elucidate whether each isoform performs unique biological functions in conjunctival epithelial cell metabolism, we initially concentrated on the ability of the isoforms to mediate cell adhesion. Our previous studies have shown that a commercially available placental laminin preparation, consisting mainly of laminin-10 and some amounts of laminin-11, is a very efficient adhesive substrate for human conjunctival epithelial cells in vitro.21 Indeed, in agreement with several other studies, laminin-10 appears to be a stronger adhesive protein than either laminin-1 or -2. Although many reports by other investigators suggest that laminin-10 is crucial to cell adhesion to extracellular matrix, the mechanisms by which this occurs have not been fully described.

Nielsen et al.16 identified several peptides of the LG4 module of the laminin {alpha}5 chain and stated that the amino acid sequence termed F4 was a major heparin- and cell-binding peptide of this domain. We subsequently determined the role of F4 peptide in human conjunctival epithelial cell adhesion and cell signaling. In agreement with these investigators, our findings showed that F4 peptide has cell-binding capability; however, the peptide alone does not facilitate adhesion to the same degree as the intact laminin-10 molecule. They used human submandibular gland epithelial cell culture in their study. We extended their observation by using a human ocular surface epithelial cell type. Several investigators have raised the possibility that the ability of LG module peptides to promote adhesion may be cell-type specific.8 9 Therefore, further investigation is needed to determine whether peptide F4 is a universal heparin- and cell-binding peptide within the LG4 module.

We investigated potential mechanisms by which peptide F4 might facilitate conjunctival epithelial cell adhesion to laminin-10. One method that we used was to determine the role of GAG in cell-laminin adhesion. Heparinase digests the relatively more sulfated, heparin-like regions of GAGs.20 Although heparinase treatment of cell-surface HC0597 GAGs did not inhibit adhesion to F4 peptide, exogenous heparin used as a blocking agent served to partially inhibit adhesion. Heparin affinity indicates a potential for a given molecule to bind heparan sulfate proteoglycan through clusters of basic amino acids.24 Nielsen et al.16 confirmed that the arginine residues of the F4 peptide are critical for both heparin- and cell-binding activity. Several lines of evidence strongly suggest that heparan sulfate proteoglycans indeed mediate adhesion of human conjunctival epithelial cells to peptide F4. First, the treatment of cells with sodium chlorate, which results in the altered sulfation of cell surface GAGs, significantly inhibited adhesion to peptide F4. Second, the use of heparitinase, which digests relatively less sulfated, or the more heparan sulfatelike, regions of cell surface GAGs also significantly inhibited adhesion to peptide F4. Last, the use of exogenous heparan sulfate as a competitive molecule in adhesion assays significantly inhibited cell attachment to peptide F4.

Within basement membranes, laminins function in the assembly of macromolecular protein complexes, in the adhesion of cells to extracellular matrix, and in the initiation of cell signaling. We have demonstrated that a peptide of the LG4 domain of laminin {alpha}5 chain mediates cell–extracellular matrix adhesion in human conjunctival epithelium. Because maximal disruption of adhesion was observed when an antibody to syndecan-4 was used, we hypothesize that at least one mechanism of LG4 domain adhesion is through syndecans. Syndecan-4 is a known component of focal adhesions, specialized sites of cell–extracellular matrix adhesion. Therefore peptide F4 is implicated in not only cell adhesion but in cell signaling as well. In support of this, we have results suggesting that cell adhesion to peptide F4 results in increased tyrosine phosphorylation of FAK.

In addition to syndecan-4, the heparan sulfate proteoglycan perlecan also inhibited conjunctival epithelial cell adhesion to F4 peptide (results not shown). Perlecan is not a cell membrane receptor like syndecan-4, but is a resident protein of basement membranes. Perlecan may bind to F4 peptide through its heparan sulfate side chains at the same sites that would be occupied by cell surface syndecan-4 heparan sulfate side chains in vivo. This would certainly account for our observation that perlecan inhibits cell adhesion in solid phase assays and provides additional evidence that cell binding to F4 peptide is dependent on the GAGs, not the core protein, of cell surface proteoglycans. However, whether our observations regarding perlecan’s blocking of adhesion also suggests that this proteoglycan may interact with laminin-10 to participate in the macromolecular assembly of the basement membrane is currently unknown.

Laminin-10 is localized to basement membranes underlying many epithelia and is a component of both the corneal and conjunctival structures. We have provided evidence that peptides of laminin-10 are essential for both cell–basement membrane adhesion and tyrosine kinase-mediated cell signaling. Because laminin-10 is such an efficient substrate for adhesion, it may be crucial to maintaining conjunctival–corneal environments by ensuring epithelial cell adhesion to the proper region of the ocular surface. Our studies, however, indicate that laminin-10 may also be an extracellular signaling molecule. The role of laminin-10 in regulating ocular surface epithelial cell differentiation is a focus of studies currently in progress in our laboratory.


   Acknowledgements
 
The authors thank Mark Ireland, PhD, for helpful discussions during the preparation of the manuscript.


   Footnotes
 
Supported by National Eye Institute Grant EY12282 (MK-W).

Submitted for publication February 4, 2002; revised March 22, 2002; accepted April 1, 2002.

Commercial relationships policy: F.

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: Michelle Kurpakus-Wheater, Department of Anatomy and Cell Biology, Wayne State University School of Medicine, 540 E. Canfield, Detroit, MI 48201; mkurpaku{at}med.wayne.edu.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

  1. Lin, CQ, Bissell, MJ. (1993) Multi-faceted regulation by extracellular matrix FASEB J 7,737-743[Abstract]
  2. Miner, JH, Lewis, RM, Sanes, JR. (1995) Molecular cloning of a novel laminin chain, alpha5, and widespread expression in adult mouse tissues J Biol Chem 270,28523-28526[Abstract/Free Full Text]
  3. Ljubimov, AV, Burgeson, RE, Butkowski, RJ, Michael, AF, Sun, TT, Kenney, MC. (1995) Human corneal basement membrane heterogeneity: topographical differences in the expression of type IV collagen and laminin isoforms Lab Invest 72,461-473[Medline][Order article via Infotrieve]
  4. Touri, A, Uusitalo, H, Burgeson, RE, Terttunen, J, Virtanen, I. (1996) The immunohistochemical composition of the human corneal basement membrane Cornea 15,286-294[Medline][Order article via Infotrieve]
  5. Kurpakus, MA, Daneshvar, C, Davenport, J, Kim, A. (1999) Human corneal epithelial cell adhesion to laminins Curr Eye Res 19,106-114[Medline][Order article via Infotrieve]
  6. Timpl, R, Tisi, D, Talts, JF, Andac, Z, Sasaki, T, Hohenester, E. (2000) Structure and function of laminin LG modules Matrix Biol 19,309-317[Medline][Order article via Infotrieve]
  7. Engvall, E, Davis, GE, Dickerson, K, Ruoslahti, E, Varon, S, Manthorpe, M. (1986) Mapping of domains in human laminin using monoclonal antibodies: localization of the neurite-promoting site J Cell Biol 103,2457-2458[Abstract/Free Full Text]
  8. Richard, BL, Nomizu, M, Yamada, Y, Kleinman, HK. (1996) Identification of synthetic peptides derived from laminin alpha1 and alpha2 chains with cell type specificity for neurite outgrowth Exp Cell Res 228,98-105[Medline][Order article via Infotrieve]
  9. Nomizu, M, Kuratomi, Y, Malinda, KM, et al (1998) Cell binding sequences in mouse laminin {alpha}1 chain J Biol Chem 273,32491-32499[Abstract/Free Full Text]
  10. Tani, T, Lehto, VP, Virtanen, I. (1999) Expression of laminins 1 and 10 in carcinoma cells and comparison of their roles in cell adhesion Exp Cell Res 248,115-121[Medline][Order article via Infotrieve]
  11. Bernfield, M, Kokenyesi, R, Kato, M, et al (1992) Biology of the syndecans: a family of transmembrane heparan sulfate proteoglycans Ann Rev Cell Biol 8,365-393
  12. Kim, CW, Goldberger, OA, Gallo, RL, Bernfield, M. (1994) Members of the syndecan family of heparan sulfate proteoglycans are expressed in distinct cell-, tissue-, and development-specific patterns Mol Biol Cell 5,797-805[Abstract]
  13. Saoncella, S, Echtermeyer, F, Denhez, F, et al (1999) Syndecan-4 signals cooperatively with integrins in a Rho-dependent manner in the assembly of focal adhesions and actin stress fibers Proc Natl Acad Sci USA 96,2805-2810[Abstract/Free Full Text]
  14. Yamaguchi, H, Yamashita, H, Mori, H, et al (2000) High and low affinity heparin-binding sites in the G domain of the mouse laminin {alpha}4 chain J Biol Chem 275,29458-29465[Abstract/Free Full Text]
  15. Andac, Z, Sasaki, T, Mann, K, Brancaccio, A, Deutzmann, R, Timpl, R. (1999) Analysis of heparin, {alpha}-dystroglycan and sulfatide binding to the G domain of the laminin {alpha}1 chain by site-directed mutagenesis J Mol Biol 287,253-264[Medline][Order article via Infotrieve]
  16. Nielsen, PK, Gho, YS, Hoffman, MP, et al (2000) Identification of a major heparin and cell binding site in the LG4 module of the laminin {alpha}5 chain J Biol Chem 275,14517-14523[Abstract/Free Full Text]
  17. Tiger, C-F, Champliaud, M-F, Pedrosa-Domellof, F, Thornell, L-E, Ekblom, P, Gullberg, D. (1997) Presence of laminin {alpha}5 chain during human muscle development and in muscular dystrophies J Biol Chem 272,28590-28599[Abstract/Free Full Text]
  18. Zhang, K, Kramer, RH. (1996) Laminin 5 deposition promotes keratinocyte motility Exp Cell Res 227,309-322[Medline][Order article via Infotrieve]
  19. Chen, N, Chen, CC, Lau, LF. (2000) Adhesion of human skin fibroblasts to Cyr61 is mediated through integrin {alpha}6ß1 and cell surface heparan sulfate proteoglycans J Biol Chem 275,24953-24961[Abstract/Free Full Text]
  20. Hoffman, MP, Nomizu, M, Roque, E, et al (1998) Laminin-1 and laminin-2 G-domain synthetic peptides bind syndecan-1 and are involved in acinar formation of a human submandibular gland cell line J Biol Chem 273,28633-28641[Abstract/Free Full Text]
  21. Lin, L, Daneshvar, C, Kurpakus-Wheater, M. (2000) Evidence for differential signaling in human conjunctival epithelial cells adherent to laminin isoforms Exp Eye Res 70,537-546[Medline][Order article via Infotrieve]
  22. Henkel, AW, Beiger, SC. (1994) Quantification of proteins dissolved in an electrophoresis sample buffer Anal Biochem 223,329-331[Medline][Order article via Infotrieve]
  23. Lee, SP, Cunningham, ML, Hines, PC, Joneckis, CC, Orringer, EP, Parise, LV. (1998) Sickle cell adhesion to laminin: potential role for the {alpha}5 chain Blood 92,2951-2958[Abstract/Free Full Text]
  24. Lander, AD. (1994) Targeting the glycosaminoglycan-binding sites on proteins Chem Biol 1,73-78[Medline][Order article via Infotrieve]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lin, L.
Right arrow Articles by Kurpakus-Wheater, M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lin, L.
Right arrow Articles by Kurpakus-Wheater, M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS