Expression of Integrin Receptors on Plasma Membranes of Primary Corneal Epithelial Cells is Matrix Specific

Exp. Eye Res. (1997) 64, 323–334

Expression of Integrin Receptors on Plasma Membranes of Primary Corneal Epithelial Cells is Matrix Specific L. S. G R U S H K I N - L E R N E R, R. K E W A L R A M A N I    V. T R I N K A U S - R A N D A L L* Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts 02118, U.S.A. (Received Cleveland 1 March 1996 and accepted in revised form 25 July 1996) Modulation of cell behavior may occur through cell adhesion receptors that bind domains of extracellular matrix molecules and mediate cell-substrate signal transduction. It was hypothesized that while primary corneal epithelial cells seeded onto laminin and fibronectin express and synthesize integrin receptors, they are not detected on the plasma membrane until the appropriate ligand is present. The integrin subunits (α-6, β-4 and β-1) present on the plasma membrane after adherence to laminin and fibronectin were compared with changes that occurred in mRNA expression and protein synthesis. Prior to seeding, the percentage of cells expressing integrin receptors and matrix proteins on their plasma membrane was determined. Negligible laminin and fibronectin (0–7 %) were present on the plasma membrane while the population of epithelial cells expressing β-4 and β-1 on the plasma membrane was low (21–23 %). After 3 hr of adherence the cell population expressing integrin subunits was substrate dependent. The percentage of cells adherent to LM expressing β-4 was four-fold greater than cells adherent to FN. After 24 hr the percentage of cells cultured on fibronectin expressing β-4 increased significantly indicating ligand deposition. The expression and protein synthesis of α-6 and β-4 was evaluated and an increase in the synthesis of α-6 and β-4 was not detected until 18 hr on LM and 21 hr on FN. The present results demonstrate that expression and transport of integrin receptors to the plasma membrane of primary corneal epithelial cells after adhesion is regulated by the presence of specific ligands. # 1997 Academic Press Limited Key words : integrin ; receptors ; epithelium ; adhesion ; matrix.

1. Introduction Cellular interaction with the underlying extracellular matrix (ECM) influences many functions such as cell adhesion, spreading, protein synthesis (Grinnell and Hays, 1978 ; Ben-Ze’ev, Farmer and Penman, 1980), formation of adhesion structures (Kurpakus, Quaranta and Jones, 1991) and maintenance of tissue integrity (Lampugnani et al., 1991). All of these functions depend upon the presence of integrins. Integrins are noncovalently linked α}β heterodimer receptors that mediate communication between the extracellular environment and intracellular cytoskeleton (Hemler, 1990 ; Hynes, 1992). There are at least eight unique β subunits and 16 α subunits that can associate in multiple combinations (Arnout, 1993 ; Clark and Brugge, 1995). Both subunits are characterized by a large extracellular domain, an α-helical transmembrane domain and a cytoplasmic domain (Hemler, 1990 ; Hynes, 1992). Many investigators have demonstrated the presence of integrins at sites of cell-substratum interaction in many cell types (Sonnenberg et al., 1986 ; 1991 ; Grushkin-Lerner and Trinkaus-Randall, 1991 ; Quaranta, 1990 ; Virtanen et al., 1992 ; Carter et al., 1990 ; DeLuca et al., 1991 ; * For correspondence at : Room L903, Department of Biochemistry, Boston University School of Medicine, East Concord Street, Boston, MA 02118, U.S.A.

0014–4835}97}030323­12 $25.00}0}ey960207

Hertle, Adams and Watt, 1991 ; Sonnenberg et al., 1991). The corneal epithelium adheres to its basal lamina through a series of structures termed hemidesmosomes. It has been well demonstrated that the α6β4 integrin localizes to the hemidesmosomes (Stepp et al., 1990). When cells are cultured on living modified stromal substrates multiple cell–matrix attachment complexes (CMAXs) form (Trinkaus-Randall et al., 1988b, Wu, Svoboda and Trinkaus-Randall, 1995). Recently, investigators have demonstrated that the formation of a CMAX may be necessary for epithelial cell survival (Meredith, Fazeli and Schwartz, 1993 ; Frisch and Francis, 1994). When an injury occurs epithelial cells disassemble their hemidesmosomes and migrate along the substrate. As cells migrate, they form adhesion sites along regions of membrane that are closely adherent to the underlying matrix. As this process occurs epithelial cells migrate over an altered matrix that has presumably been remodeled due to either the deposition of ECM proteins or the secretion of ECM components from underlying stromal cells (Fujikawa et al., 1984 ; Zieske, Bukusoglu and Gipson, 1989). The objective of the present study was to evaluate and quantitate the initial expression and synthesis of specific integrin subunits after adherence and to determine whether the response was matrix specific. Laminin and FN were the focus of our study as they # 1997 Academic Press Limited

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are either present in the intact basal lamina (LM) or are detected temporarily after injury (FN). Laminin-1 is not detected under migrating epithelium of keratectomy wounds but is detected after abrasion (Fujikawa et al., 1984). After cellular adherence the expression of mRNA and synthesis of new integrin subunits were compared to the presence of integrin receptors on the plasma membrane. While there was an increase in specific mRNAs with time, the mRNAs for the integrin subunits were detected at all time points in this assay. The greatest increase in β4 protein synthesis occurred between 18–21 hr on LM but not until 21–24 hr on FN. It was also demonstrated that, while the percentage of cells with β4 on their cell membrane increased four-fold 3 hr after adherence on LM, there was no change in those cells adherent on FN. This is supported by data demonstrating that LM and FN are negligible on the plasma membrane prior to seeding of the cells. These results suggest that translocation of integrin receptors to the plasma membrane is highly regulated and is dependent on the presence of the appropriate ligand.

clonal antibodies to laminin (LM) and fibronectin (FN) were purchased from Collaborative Research (Bedford, MA, U.S.A.). The elastin antibody was obtained from the laboratory of Dr C. Franzblau. Antibodies were affinity purified prior to use in the experiments and were made to the concentration of 0±1 mg ml−" for further dilution as described. All of these antibodies have been previously characterized and shown to be specifically reactive with rabbit epithelial integrins (Trinkaus-Randall et al., 1993 ; Wu et al., 1994 ; 1995).

2. Materials and Methods

Cell Adhesion and Spreading Assays

Tissue Culture

To monitor cell spreading, corneal epithelial cells were seeded in serum free medium at a density of 1¬10& cells cm−# on LM or FN coated surfaces for 3, 12 and 24 hr. After each time point, the nonadherent cells were removed with several washes and the remaining attached cells were fixed with 2 % glutaraldehyde and photographed. A minimum of 4–35 mm bacteriologic plates were used for each time point. The cell adhesion assays were conducted by radiolabeling cells prior to seeding as described in TrinkausRandall et al. (1988b). Epithelial cells cultured from explants were washed and radiolabeled for 18 hr in $H-proline serum free medium containing (10 µCi ml−") (Amersham, IL, U.S.A.). The cultured radiolabeled cells were washed, trypsinized and subcultured on LM or FN coated bacteriologic plastic at 1¬10& cells cm−# in the presence of the following antibodies (anti-α6, anti-β4, anti-β1 and anti-α5). The antibodies were titrated by dilution assays to determine the maximum cell adhesion inhibition. The resulting dilutions of antibodies used were anti-α6 (GoH3), 1 : 5 ; anti-α5 (BIE5), 1 : 5 ; anti-β1 (AIIB2), 1 : 5 and anti-β4, 1 : 4. After 3 hr the medium was removed and the cells were washed to remove nonadherent cells. For each substrate and antibody, the adherent radiolabeled cells were removed with trypsin, solubilized and incorporated label was determined. Adhesion was determined as percent of control. These results were confirmed with parallel experiments where adherent cells were trypsinized and counted using a Coulter Counter. The effect of anti-integrin antibodies was shown to be significant when inhibition was equal to or greater than 50 % of control. Immunoblots were conducted to determine whether ECM proteins had

The technique for establishing epithelial cell cultures from corneal explants was previously described (Trinkaus-Randall, Newton and Franzblau, 1988a). Briefly, New Zealand rabbits were killed with 5 ml sodium pentobarbital (325 mg) administered intravenously. The epithelium was cultured from explants for 10 days in Falcon tissue culture plastic dishes containing Dulbecco’s modified Eagles medium 1 : 1 with Ham’s F-12 nutrient medium and supplemented with 3±7 g l−" NaHCO , 1 % nonessential amino acids, $ 1 % penicillin-streptomycin, 4 % dialysed fetal bovine serum (FBS and 0±2 % insulin-transferrin-selenium (ITSG) (Gibco BRL Gaithersburg, MD, U.S.A.). After 10 days, cells were subcultured using trypsin-ethylenediaminetetraacetic acid (EDTA) (1 : 4). All experiments were performed with these first passage cells. Antibodies The following hybridoma supernatant antibodies were used : rat monoclonal anti-α6 (GoH3) was obtained from the laboratory of Dr A. Sonnenberg (Sonnenberg et al., 1987) and was characterized by Hemler, Crouse and Sonnenberg, (1989). It was shown to be a functional blocking antibody by Lee et al., (1992). The rat monoclonal anti-α5 (BIE5) and rat monoclonal anti-β1 (AIIB2) were donations from Dr C. Damsky (Brown et al., 1989). They were characterized for functional blocking by Sonnenberg et al. (1990). In addition the mouse monoclonal antibeta1 (Gibco Lab) was used. The mouse anti-β4 was a donation from Dr V. Quaranta. It was characterized by Kajiji, Tamura and Quaranta, (1989). Mouse mono-

Preparation of Substrates Bacteriologic plastic was coated with either EHS LM type I (Collaborative Biomedical Products-Becton Dickinson Bedford, MA, U.S.A.) or cellular FN (Sigma Chemical Co., St. Louis, MO, U.S.A.) for 2 hr at room temperature. The optimum concentration for the adhesion and spreading experiments was determined to be 30 µg}ml−" for both LM and FN (data not shown).

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T I Percentage of cells expressing integrin subunits on plasma membrane (Relative Fluorescence) Time (after seeding) t¯0 (cells in suspension) 3 hr laminin fibronectin 24 hr laminin fibronectin

α '

β "

β %

61±14³3±34

22±89³1±94

21±25³2±94

79±79³0±59 69±47³3±95

21±72³0±66 20±71³0±3

82±47³2±44 16±77³4±16

86±7³0±47 66±46³5±88

32±96³0±99 9±53³0±37

62±64³2±61 56±69³4±15

Control values for α6 (rat IgG) ; 7±63³8±59 and for the β subunits (mouse IgG) ; 1±25³0±014. n ¯ 3.

been secreted or deposited after 3 hr of incubation using the chemiluminescence-ECL system (Amersham, IL, U.S.A.). To determine the role of the integrin subunits on spreading, experiments were conducted in the presence or absence of antibodies. Epithelial cells were seeded on bacteriologic plastic coated with FN or LM in serum free medium at a density of 1¬10& cells cm−# in the presence of specific antibodies to integrin subunits for 6 hr at 37°C. Negative controls either lacked the primary antibody or used a nonspecific monoclonal antibody (anti-elastin 1 : 10). After 6 hr, the nonadherent cells were removed with several washes and the attached cells were fixed with 2 % glutaraldehyde and photographed. A minimum of 4 wells were used for each time point and antibody. Four random areas of each well were photographed and the amount of spreading was averaged from 16 measurements. Using previously established criteria, cells possessing lamellipodial extensions were considered spread (Trinkaus-Randall et al., 1988b). The difference in surface area between spread and round cells was determined. Cell tracings were imaged with a Hamamatsu C2400 newvicon video camera for analog enhancement. The video signal was captured to a Macintosh Quadra 950 computer equipped with a Data Translations video capture board. The cell areas were computed using NIH image (v 1±45. NIH Research Services Branch) and analysis of variance determined. The Student’s t test was used to determine whether any of the antibodies significantly inhibited cell spreading in comparison to the controls where P ! 0±05 (t test) was significant. Immunoprecipitation To compare the synthesis of α6 and β4, epithelial cells cultured from explants were trypsinized and plated on P-100 bacteriologic dishes coated with LM or FN at a density of 5±1¬10' cells cm−# in medium. This medium differed from the other experiments in that it contained cysteine and methionine free

Dulbecco’s modified Eagles medium and Ham’s F-12 (1 : 1). All other components remained the same. Cells were radiolabeled for 3 hr periods (0–3, 3–6, 9–12, 18–21, 21–24) with 100 mCi ml−" of 35S-cysteinemethionine (specific activity 1,100 CimMol−") (NEN Boston, MA, U.S.A.). For each time point the medium was removed and the cells were harvested in PBS containing 0±3  p-hydroxymercuribenzoic acid (PCMB), 10 m N-ethylmaleimide (NEM), 50 m phenylmethylsulfonylfluoride (PMSF) and 0±3 mg ml−" leupeptin. The cells were centrifuged at 800 rpm and the pellet was lysed in 0±05  Tris–HCl}0±15  NaCl pH 7±5 containing 1±0 m EDTA, 0±5 % Nonidet P-40, 0±2 m PMSF and leupeptin. The lysate was precleared with Protein A-Sepharose (Pharmacia, Uppsala, Sweden). The majority of the β1 integrin was removed by incubating the lysate with a mouse antibeta1 complex overnight at 4°C. To precipitate α6, the remaining unbound cell lysate (following depletion of β1) was then added to a Protein-A Sepharose, rabbit anti-rat, rat anti-α6 complex and incubated overnight at 4°C. Rat IgG was used as a control (Jackson ImmunoResearch, Jackson Harbor, Maine). The immunoprecipitates were analysed by 5 % SDS-PAGE (Laemmli, 1970) and autoradiography. Immunohistochemistry Four well glass chamberslides were coated with LM and FN and cells were cultured for 24 hr in serum free medium. The procedure for staining corneal epithelial cell populations was described previously (Wu et al., 1994 ; Grushkin-Lerner and Trinkaus-Randall, 1991). Cells were fixed with 3±7 % formaldehyde at room temperature, rinsed three times with PBS and permeabilized with 0±1 % Triton-X 100. They were rinsed three times with PBS containing 1 % bovine serum albumin (BSA). Briefly, the cells were incubated with anti-α6 (GoH3), anti-β1 or anti-β4 overnight at 4°C, rinsed with PBS and washed for 10 min in 3 % BSA}PBS (dilution of antibodies previously described in Wu et al., 1994). Cells were then incubated with

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either FITC rabbit anti-rat IgG (1 : 100) or FITC horse anti-mouse IgG (1 : 00) (Vector Labs Burlingame, CA, U.S.A.) for 1 hr at 4°C. The controls were incubated with either pure rat IgG or pure mouse IgG. Images were recorded using confocal laser scanning microscopy (CLSM). Fluorescence-Activated Cell-Sorted Analysis The procedure for staining corneal epithelial cell populations for integrin receptors has been described previously (Wu et al., 1994). Briefly, epithelial cells were seeded in serum free medium on LM or FN and evaluated at t ¯ 0 (prior to seeding), 3 and 24 hr. Cells were trypsinized and 10' cells were incubated with the appropriate primary antibody or with control serum (mouse or rat IgG) at 4°C. The cells were washed, incubated with appropriate secondary antibody for 30 min at 4°C, washed and fixed in 2±5 % paraformaldehyde. The monoclonal antibodies (MAb) used were against the following proteins : β1, β4, α6, laminin and fibronectin. Analysis was performed on a Becton Dickinson FASCAN flow cytometer 9. Data are presented in the form of histograms and in Table I. The x axis represents mean channel fluorescence and the y axis represents cell number. A shift to the right indicates an increase in fluorescence. The percent CV or standard deviation was low in all cases. In all conditions 10' cells were examined per treatment group and averages of three runs were taken at three separate times. Confocal Laser Scanning Microscopy Fluorescently labeled samples were analysed on a Leica upright confocal laser scanning microscope (CLSM) equipped with an argon ion laser with an output power of 2–50 mV, two photomultiplier tubes and a narrow band filter for double labeling experiments. All control cells were analysed under the same conditions as the experimental cells. The same procedures were used as for Wu et al. (1995). The images were stored on an optical disc and were photographed with T-MAX 100 film (Kodak, Rochester, NY, U.S.A.) using a 35 mm camera.

L. S. G R U S H K I N -L E R N E R E T A L.

(Stratagene La Jolla, CA, U.S.A.). Probes were labeled with a-32P-dCTP using T7 DNA polymerase and random primers (Pharmacia, Piscataway, NJ, U.S.A.). Specific activities ranged from 10)–10* cpm µg−" cDNA). Hybridizations were carried out at 67°C for 2 hr. Following low (2¬SSC 0±1 % SDS, 25°C) and high stringency (0±2¬SSC 0±1 % SDS, 50°C) washes, membranes were exposed to Amersham Hyperfilm for 24 to 48 hr. Autoradiograms were quantitated by densitometry and integration of peaks was determined using a Molecular Dynamics scanning densitometer. Probes The following clones were used : glyceraldehyde 3phosphate dehydrogenase (GAPDH) : a 545 bp XbaI} Hind3 restriction fragment of human cDNA (Tso et al., 1985), α6 : a6±1 a 1 kb human restriction fragment (Tamura et al., 1990) and β4 : pGEM1-b4ES1, a 2 kb human restriction fragment (Suzuki and Naitoh, 1990). In situ Hybridization To determine the percentage of cells that expressed the integrin subunits on LM or FN, non-isotopic in situ hybridization was conducted following the procedures of Svoboda (1991), Singer et al. (1986) and Lawrence and Singer (1986). The cDNA restriction fragments (α6 ; 300 bp, β4 ; 600 bp and GAPDH ; 545 bp) were prepared prior to labeling. The cDNA probes were labeled using the Nick Translation Kit (Boehringer Mannheim) using Digoxigenin-11-dUTP (Boehringer Mannheim). Following hybridization the cells were incubated in anti-digoxigenin-FITC. The cells were washed, coverslipped and the images were recorded using CLSM. Simultaneous transmitted light images were recorded to calculate the number of cells expressing the integrin subunits as a percentage of the total cell population. A minimum of 50 cells was counted for each treatment group and time point. 3. Results Characterization of the in vitro System

RNA Analysis Epithelial cells were harvested after 3, 6, 9, 12 and 24 hr of culture on LM or FN. Total cellular RNA was extracted with guanidinium isothiocyanate using the procedure of Chomczynski and Sacchi (1987). For Northern blot analysis, equal samples (15 µg) of total cellular RNA were denatured and separated by electrophoresis on 1 % agarose-formaldehyde gels. Equal loading and integrity of the 18S and 28S RNA was verified on gels stained with ethidium bromide. RNA was transferred to Duralose using the PosiBlot pressure blotter (Stratagene ; La Jolla, CA, U.S.A.) and UV crosslinked to the membrane with a Stratalinker

To examine the ability of corneal epithelial cells to synthesize and express specific integrin subunits on their plasma membrane it was necessary to characterize the cellular response to LM and FN. It was demonstrated that plating efficiency and cell doubling of cells was substrate independent, while cell spreading in the first three hours was substrate dependent. The total percentage of spread cells and the extent of membrane ruffling was significantly greater on FN than on LM (7³1 %, 31³3 %) (P ! 0±005) (Students one-tailed t test). After 24 hr, spreading was extensive on both surfaces and differences in cellular morphology could not be detected (data not shown). These

INTEGRINS ON PLASMA MEMBRANE AFTER ADHESION 140

120

% spread of control

100

80

60

40

20

0

α5

α5β1 β1 β4 α6 α6β1 α6β4 Anti-integrin antibodies

F. 1. Role of integrin subunits in cell spreading on LM and FN. Epithelial cells were incubated on LM or FN for 6 hr in the presence or absence (control -ctrl) of anti-integrin antibodies. The percentage of cell spreading was calculated in the presence or absence of antibody. The data are presented as percent of control. , LM ; , FN.

results suggest that the initial cellular responses are matrix specific. To characterize the role of integrin subunits in cell adhesion and spreading on FN and LM, monoclonal antibodies to α5, α6, β1, and β4 were used. Preliminary studies were conducted by performing dose response experiments to assure that the antibodies were not used at subsaturating concentrations. Adhesion to LM was inhibited by anti-α6 by more than

Cell number (linear scale)

100

(A)

100

327

50 % while adhesion to FN was not significantly inhibited (32 %). Cell adhesion did not decrease further when anti-α6 was combined with either anti-β1 or anti-β4. The results indicate that only anti-α6 antibodies significantly inhibited adhesion to LM (P ! 0±05, one tailed t test) (data not shown), suggesting that α6 is an important receptor subunit in corneal epithelial cell adhesion. The extent of cellular spreading on FM and LM in the presence of anti-integrin antibodies was evaluated using image analysis and the surface area of spread and non-spread cells was determined. The percent of spread cells cultured on LM or FN in the absence of antibody were used as controls (ctrl) and results were analysed as percent of cell spreading. Forty-five percent of the cells spread on either matrix protein in the absence of antibodies (Fig. 1 ctrl). Cellular spreading on LM in the presence of anti-α6 was 21 % of control (P ! 0±05, one tailed t test) (Fig. 1). In the presence of the MAb to β4, cells spread 39 % of control indicating that β4 also had a significant role in regulating spreading on LM (P ! 0±05, one-tailed t test) (Fig. 1). No further change in percent cell spreading occurred when anti-α6 was combined with antibodies to either β1 or β4 (Fig. 1). The antibody to α5 did not inhibit cellular spreading on LM (Fig. 1). The effect of anti-α6 on cellular spreading was similar on both FN and LM (Fig. 1). However, on FN neither anti-β1 nor anti-β4 inhibited epithelial spreading. In addition, the percent of spread cells did not decrease further when the medium contained anti-α6 in combination with antibodies to β1 or β4 subunits (Fig. 1). These observations suggest that α6 functions as one of the primary receptors regulating epithelial 100

(B)

50

50

50

0

0

0

100

(D)

50

0 100

100

(C)

(E)

50

101

0 100 101 102 103 104 102 Mean fluorescence intensity (log scale)

103

104

F. 2. Fluorescence-activated cell-sorted analysis of α6, β4 and β1 was performed to quantitate the percentage of cells possessing integrin subunits on the plasma membrane 3 hr after culture on LM and FN. (A) α6, (B) β1, (C) β4, (D) and (E) Rat and mouse IgG controls respectively. Nonspecific fluorescence of epithelial cell bound FITC-labeled secondary and control IgG antibodies were negative. The y-axis indicates the relative cell number and the x-axis indicates mean fluorescence intensity. (—— FN ; ----LM).

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F. 3. Localization of α6, β1 and β4 in cells cultured on LM (A, C, E) and on FN (B, D, F). Confocal laser scanning micrographs represent images of cells from the basal surface. (A), (B) : Cells on LM and FN labeled with anti-α6. Arrows indicate localization of α6 along the lateral cell membrane between adjacent cells. (C), (D) Cells on LM and FN labeled with anti-β1. Arrows indicate localization of β1 along the cell membrane. Arrowheads indicate localization at lamellipodia. (E), (F) Cells on LM and FN labeled with anti-β4. Distribution is diffuse and β4 is minimal in cells cultured on FN. (G) F-actin filaments of epithelial cells cultured on LM and stained with rhodamine conjugated to phalloidin. Arrowheads show F-actin filaments that terminate just short of the focal contacts. (H) A control image where the primary antibody was omitted. 115¬.

cell spreading on LM and FN. In contrast, the β4 subunit plays an important role in regulating cell spreading only on LM. Cell Surface Expression of Integrin Subunits Since the time course of spreading and the role of integrin subunits differed between cells cultured on

LM and FN, we investigated the relative cell surface expression of integrin protein subunits. The percentage of cells expressing the integrin subunits prior to seeding (t ¯ 0) was determined for each integrin subunit and used as the initial point of reference (Table I). Rat IgG, used as the control for anti-α6 had a value of 7±63 % and mouse IgG, used as the control for anti-β1 and β4 had a value of 1±25 %. To ensure

INTEGRINS ON PLASMA MEMBRANE AFTER ADHESION

that matrix proteins were not prominent on the plasma membrane after trypsinization, we used antibodies to FN and LM to quantitate their presence. Prior to seeding we found that the cells had either minimal or no surface associated LM (7 %) or FN (0 %). Only values of adherent cells were determined as cells in suspension downregulate the population of cells expressing integrin receptors (ie. the average fluorescence intensity of cells labelled for alpha6 receptors decreased from a mean of 190 at t ¯ 0 to a mean of 157 after 45 min in suspension). The population of cells expressing α6 on their surface prior to seeding was 61±1 %. Three hours after seeding, the percentage of cells expressing α6 was significantly greater on LM (79±8³0±6 %) than on FN (69±5³3±9 %) (P ! 0±05 one tailed t test) (Fig. 2, Table I). Therefore, out of the population of cells that did not already express α6 (38±9 %), almost 50 % of these cells expressed α6 after attachment to LM while only 20 % expressed α6 after attachment to FN. These experiments also demonstrated that the percentage of cells on LM expressing α6 increased between 3 and 24 hr (Table I). These results suggest that while α6 is a prominent receptor maintained on the surface of cells cultured on both LM and FN, only those cells adherent to LM showed a significant increase in expression. There was no change in the percentage of cells that expressed β1 after 3 hr of adherence on either LM or FN (Fig. 2, Table I). However, after 24 hr there was a 50 % increase in the population of cells on LM expressing β1. In contrast, the population of cells on FN expressing β1 on their plasma membrane decreased from 20±7 % at 3 hr to 9±5 % after 24 hr. These results indicate that β1 was not a principle component of integrin receptor complexes of cells cultured on FN. The matrix specific response to substrate was greatest in the population expressing β4 on their cell membrane after 3 hr. Eighty-two percent of the population cultured on LM expressed β4 while only 16±8 % of the population cultured on FN expressed β4. The latter value did not differ significantly from values recorded prior to adherence (t ¯ 0 : 21±2 %). After 24 hr and matrix deposition (Trinkaus-Randall et al., 1988a), the population of cells expressing β4 was similar on both LM and FN suggesting that β4 is not detected on the plasma membrane until the matrix has been remodeled (Fig. 2 Table I). Our data demonstrated that the initial adherence of cells to LM upregulated the cell surface expression of α6 and β4 relative to FN. Intracellular Localization of Integrin Subunits Having demonstrated that the presence of specific extracellular matrix proteins altered the cell surface expression of integrin subunits, we investigated the distribution of the integrin subunit proteins in corneal

329

F. 4. Epithelial cells were radiolabeled with 35S-cysteinemethionine for 3 hr periods to evaluate the synthesis of α6 and β4 integrin subunits. Cell lysates were sequentially immunoprecipitated and immunoprecipitates were run under nonreducing conditions on a 5 % SDS-PAGE. Cells incubated on LM (A) and on FN (B). Lane 1, control ; Lane 2 : 0–3 hr ; Lane 3 : 3–6 hr ; Lane 4 : 6–9 hr ; Lane 5 : 18–21 hr ; Lane 6 : 21–24 hr.

epithelial cells cultured on LM and FN. In general α6 was diffuse ; however, it was detected along lateral cell membranes of cells on both LM and FN (arrows) [Figs 3 (A), (B)]. The β1 integrin subunit was localized in the basal region of cells cultured on either LM or FN [Fig. 3 (C), (D)]. It was present also at lamellipodia of cells on LM (arrowheads) and was present along lateral cell membranes on FN [Fig. 3 (C), (D) ; arrows]. The β4 subunit was present in the basal optical plane of cells on both LM and FN and was diffuse on LM [Figs 3 (E), (F)]. β4 was negligible in optical sections above the nucleus in cells cultured on both surfaces (data not shown). The cytoarchitecture of the cell is shown after cells were stained with rhodamine conjugated to phalloidin [Fig. 3 (G)]. Fluorescence was not detected when the primary antibody was omitted [Fig. 3 (H)]. Synthesis of α6 and β4 Subunits To evaluate integrin synthesis, cells were radiolabeled with 35-S methionine and the synthesis of α6 and β4 was examined after the following 3 hr pulses : 0–3, 3–6, 9–12, 18–21 and 21–24 hr. To compare the synthesis of α6 and β4 after adherence to LM and FN, the complex was immunoprecipitated with GoH3 from equivalent TCA precipitable counts at the specific times given. These experiments were performed in triplicate with duplicates done in each run. The same trend was seen in all experiments. β1 was not detected as it was removed from the lysate prior to immunoprecipitation of the α6β4 complex with GoH3 (see methodology). Nonspecific protein (anti-laminin) was not precipitated with controls [Fig. 4 (A), (B), lane 1].

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F. 5. Steady state mRNA of α6, β4, β1 and GAPDH from cells harvested 3, 6, 9, 12 and 24 hr after culture on (A). LM and (B) FN. 15 µg of total RNA was loaded per lane.

The remainder of the lanes represent α6 and β4 immunoprecipitated from cells radiolabeled for 3 hr periods. In the first pulse period after seeding (0–3 hr) more α6 was synthesized than β4 on both surfaces. Cells cultured on LM have more precipitable counts than on FN [Fig. 4 (A), (B) lane 2]. No other significant differences were observed until 18 hr when the synthesis of both α6 and β4 increased on LM [Fig. 4 (A) lane 5]. From 21 to 24 hr synthesis remained comparatively high on LM while the increase on FN was not detected until the 21–24 hr pulse [Figure 4 (B) lane 6]. These results demonstrated that the increase in synthesis of α6 and β4 was delayed on FN. Expression of Integrin Subunits in Response to LM and FN To determine if the level of integrin mRNA was induced by matrix proteins or was expressed continuously, steady state levels of α6, β1 and β4 mRNA were evaluated over a 24 hr period on LM and FN. The mRNA of the glycolytic enzyme GAPDH was used as a

F. 6. Localization of α6 mRNA within cells cultured on LM or FN for 3 hr. (A) localization of α6 mRNA in cells adherent to FN. (B) Localization of α6 mRNA in cells adherent to LM. (C) RNAse control. 120¬.

control to normalize for RNA loading. The following description takes into account the relative ratio of specific probe to GAPDH. The steady state expression of α6 : GAPDH did not change significantly when cells were cultured on LM [Fig. 5 (A)], but increased several fold on FN from 3 to 6 hr and then increased gradually at this elevated level for the rest of the time course [Fig. 5 (B)]. The steady state level of β4 : GAPDH did not change significantly with time on LM ; however, on FN the ratio increased at the later time points. Increases in steady state level of β1 : GAPDH were detected on both surfaces at the later time points [Fig. 5 (A), (B)]. These results demonstrated that the mRNAs for all three integrin subunits were expressed for 24 hr after

INTEGRINS ON PLASMA MEMBRANE AFTER ADHESION

T II Percentage of cells expressing α and β mRNA after ' % 3 hr of adherence

Laminin Fibronectin

α '

β %

22³4 50³5±5

30³5 35³4±5

Expression was determined by viewing the negative controls on the CLSM and setting the voltage such that the image could not be detected. All experimentals were then evaluated at the set voltage. A minimum of 50 cells were counted per group.

adherence indicating that these messages were expressed constitutively under the experimental culture conditions. The intracellular localization of α6 and β4 mRNA was examined in cells using confocal laser scanning microscopy and the percentage of cells expressing specific mRNA was calculated. α6 mRNA was detected in a reticular pattern around the nucleus and did not extend to the lamellipodia of cells on either FN [Fig. 6 (A)] or LM [Fig. 6 (B)] at 3 hr. Three controls were included to assess the specificity of the in situ hybridization. The localization of α6 mRNA was compared to that of the cytoplasmic GAPDH mRNA which was localized throughout the cytoplasm. In addition the anti-DIG-FITC antibody did not bind nonspecifically when incubated in the absence of the probe nor was fluorescence detected when cells were pretreated with RNAse prior to hybridization (Fig. 6 (C)]. The localization of β4 was also examined and did not differ from that of α6 (data not shown). The nature of the substrate did not alter the localization of integrin mRNAs examined. In addition, the percentage of cells expressing α6 and β4 mRNA was determined 3 and 24 hr after adherence using confocal laser scanning microscopy. A matrix specific response in the percentage of cells expressing α6 or β4 mRNA was detected (Table II). In the initial response to the matrix proteins the percentage of cells expressing α6 mRNA on FN was two-fold that of LM. A small difference was detected in the percentage of cells expressing β4 mRNA. By 24 hr 50 % of the cells expressed the integrin mRNAs on both surfaces. In addition preliminary results from in vitro translation experiments have demonstrated that the mRNA for both β4 and α6 have equivalent translatability from cells cultured on either LM or FN. Taken together the results suggest that the regulation of expression occurred at the level of translation. 4. Discussion The regulation of integrin receptor expression on the plasma membrane is a complex phenomenon and may be important in modulating outside-inside signaling. This is confirmed by our results demonstrating that in response to a foreign matrix cells

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will continue to express integrin mRNA subunits constitutively but will delay the synthesis and appearance of the protein on the plasma membrane until the proper matrix has been deposited. Here signals are hypothesized to have been transduced via integrin receptors from the extracellular environment to the cell. In these experiments we evaluated the response of primary corneal epithelial cells to LM and FN. We observed that primary epithelial cells spread more rapidly on FN than LM in the first 3 hr after adhesion but that the population of cells expressing α6 and β4 integrin receptors on the plasma membrane was significantly greater on LM. Our results suggest that the initial cellular response to existing matrix proteins is to differentially modulate cell surface expression of specific integrin subunits. There is a large body of research that has examined whether certain proteins are up or down regulated in corneal epithelial cells in response to injury. Zieske et al. (1989) showed that vinculin was up regulated when the cells began to migrate over a wound bed. Our previous results demonstrated that the localization of α6 is modified in vivo after corneal injury (GrushkinLerner and Trinkaus-Randall, 1990) and with age (Trinkaus-Randall et al., 1993). In addition we showed that α5 is upregulated 24 hr after injury in vivo (Grushkin-Lerner and Trinkaus-Randall, 1990). Specific matrix molecules such as LM, FN and collagen also have been shown to alter the synthetic profile of epithelial cells in vitro (Trinkaus-Randall et al., 1988a). More recently, Wu et al., (1994) has shown that synthetic polymers containing different surface charges alter the integrin receptors (α6, α2, α3, β1 and β4) that are present on the plasma membrane. These experimental results led us to evaluate the presence of integrin receptors on their membrane in response to the substrate prior to matrix deposition and to correlate the appearance of these receptors with changes in their synthesis and expression. To quantitate the percentages of the cell populations that expressed specific integrins subunits on their plasma membrane before and after seeding (3 and 24 hr), cell surface labeling (FACS) experiments were conducted. Prior to seeding, a small percentage of cells expressed both β1 and β4 subunits on their plasma membrane. A larger percentage of cells expressed α6, however, this value decreased by 18 % when cells were kept in suspension for as little as 45 min. In response to adhesion on LM, the percentage of cells expressing β1 did not change while the percentage of cells expressing β4 increased four-fold. No significant change was detected on FN. The early detection of β4 on cells adherent to LM supports our current results demonstrating that anti-β4 inhibited cellular spreading on LM and our previous results demonstrating that adhesion site formation was inhibited in culture (Grushkin-Lerner and Trinkaus-Randall, 1990). The percentage of cells expressing α6 also increased on LM but only by 1±3 fold. Taken together these results

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indicate that the presence of the integrin subunits is not constant on the plasma membrane, that they become internalized if not in contact with an appropriate ligand and only increase on the plasma membrane within the first 3 hr in response to the ligand suggesting an adhesion dependent regulatory mechanism. This conclusion is supported by Dalton et al., (1995) who demonstrated in fibroblast cell lines that integrin receptor expression is downregulated in the absence of an appropriate ligand and that the internalization and degradation of α}β1 complexes is blocked upon adhesion to the appropriate ligand. The cellular response 24 hr after culture on LM and FN and subsequent matrix deposition was determined. Increases in the percentage of cells cultured on LM after 24 hr expressing α6 and β1 suggest an upregulation in the α6β1 complex and a downregulation in the α6β4 complex. The response on LM may be due to an over expression from the initial cellsubstrate recognition followed by receptor downregulation. In contrast the number of cells adherent to FN that expressed β1 decreased while the expression of β4 increased significantly between 3 and 24 hr (P ! 0±05, t test). One possible explanation for the decrease in β1 is the expression of other FN adhesion receptors. The delay in the expression of β4 on the plasma membrane of cells adherent to FN may be due to the initial lack of appropriate ligand(s) on the surface. Taken together these results suggest that the synthesis and translocation of specific subunits change with time in response to the matrix and reflect the level of outside-to-inside signaling. We evaluated the percentage of cells expressing integrin receptors on their cell membrane and asked whether there was a relationship to the specific integrin subunits that participated in cellular adhesion to and spreading on LM and FN. We demonstrated that only the antibody to α6 significantly inhibited adhesion to LM whereas antibodies to the beta subunits had minimal effects on the adhesion of cells to either protein. Therefore the α6 subunit plays a critical role in cellular adhesion to LM suggesting that the subunit confers a ligand binding specificity to the receptor complex. In contrast, none of the other antibodies evaluated to date significantly inhibited cellular adhesion to FN suggesting that adhesion to FN is mediated through other integrin receptors. This is supported by the observation that only a small percentage of the population expressed β1 or β4 after the 3 hr incubation. While the beta subunits did not participate in epithelial adhesion, antibodies to β1 and β4 did inhibit cellular spreading on LM although they were not as effective as anti-α6. These results suggest that both α6β1 and α6β4 participated in cellular spreading on LM. While anti-α6 did not significantly inhibit the adhesion of cells to FN it did inhibit cell spreading. These results suggest that the antibody to α6 impedes the signal for spreading. Therefore α6 is an important

L. S. G R U S H K I N -L E R N E R E T A L.

integrin subunit in both adhesion and spreading and that β4 plays a role in spreading on LM. To determine whether changes in the expression of integrin receptors at the plasma membrane were a reflection of synthesis and expression, steady state integrin subunit mRNA levels were evaluated and protein synthesis was followed nearly every 3 hr for the first 24 hr. While the increase in steady state mRNA was minimal on LM, the synthesis of α6 and β4 protein increased between 18 and 21 hr. An increase in α6 mRNA was detected on FN. However, the increase in synthesis of α6 and β4 protein did not occur on FN until 21–24 hr. These results correlate well with the cell surface labeling experiments indicating an immediate response when the appropriate ligand is present. In addition, preliminary in vitro translation plasmas have shown that the mRNA extracted from cells cultured on either FN or LM has equivalent translatability. Therefore, our results suggest that matrix-specific regulation of α6 and β4 expression occurs at the level of translation and that this mechanism is sensitive to the extracellular matrix. These hypotheses are supported by the work of Delcommenne and Streuli (1995) who compared the response of mammary epithelial cells cultured on EHS to that on plastic. They demonstrated that the ECM controls integrin subunit expression at either transcriptional or posttranslational levels depending on the specific subunit. Collectively, these findings suggest that cells respond to components of their native matrix in a predicted pattern. However, when they interact with an abnormal matrix, protein or surface charge the cells compensate and deposit a more normal matrix. The presence of selective integrin subunits on the plasma membrane and the interaction of these subunits with their ligands plays a role in the formation of CMAX’s (cell matrix adhesion complexes) (Wu et al., 1994). This response seems to be sensitive to changes in the extracellular environment in vitro and in vivo. The results from this study suggest that integrin subunits may be translocated to the plasma membrane within 3 hr after adherence when the appropriate ligand is detected in the matrix. In response to a foreign matrix cells will continue to express the integrin mRNA subunits constitutively but synthesis and appearance of the protein on the plasma membrane will be delayed until the proper matrix has been deposited. The results predict that under different remodeling conditions such as injury or development specific subunits are recruited to the plasma membrane. Acknowledgements Supported by EY-06000 from NEI and in part by departmental grants from Research to Prevent Blindness Inc and the Massachusetts Lions Eye Research Fund, Inc. We thank Drs Kathy Svoboda, Matthew Nugent and Robert Moreland for critical discussions. Special thanks are due to

INTEGRINS ON PLASMA MEMBRANE AFTER ADHESION

Christopher Brown and Dan Orlow for photographic and computer work.

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