Exp.EyeRes.(1992) 54, 9-16

Distribution of Epidermal Growth Factor Rabbit Cornea1 Epithelial Cells, Keratocytes and the Changes Induced by Transforming MIKI

HONGO,

Departments

(Received

MOTOKAZU

ITOI,

NOZOMI

(EGF) Receptors in and Endothelial Cells, Growth Factor-@I

YAMAGUCHPAND

JIRO

of Ophthalmology and aMicrobiology, Kyoto Prefectural University Kawaramachi- Hirokoji, Kamikyo-ku, Kyoto 602, Japan Chicago

17 September

IMANISHIa* of Medicine,

1990 and accepted in revised form 30 January

7991)

Three kinds of rabbit cornea1cells (epithelial cells, keratocytes and endothelial cells) were cultured, and the growth promoting effectsof epidermal growth factor (EGF)were examined in these cells. It was found that the sensitivity of the epithelial cells to EGFwas the highest, that of the endothelial cells was a little lower, and that of the keratocytes was the lowest. Transforming growth factor-p1 (TGF-Pl) did not influence growth of the three kinds of cornea1cells. It was found that TGF-/31enhanced the growth promoting effect of EGFin the keratocytes. but not in the epithelial and endothelial cells. EGF receptors in the cornea1 cells were labelled with [1251]EGFand analysed by Scatchard plot. In the epithelial and endothelial cells, both high and low affinity receptors were found, while the keratocytes had only low affiity receptor. In the epithelial cells, the number and the association constant of the high afEn@ receptors were, respectively, 2.79 x lo4 per cell and 0.034 nM, and those of the low affinity receptors were, respectively, 13.4 x lo4 per cell and 0.700 nM. In the endothelial cells, the number and the association constant of the high affinity receptors were 1.27 x lo4 per cell and 0.086 nM. respectively, and those of the low affinity receptors were 8.91 x lo4 per cell and 1.536 nM. In the keratocytes, the number of receptors and the association constant were 9.49 x lo4 per cell and 1.535 nM, respectively. The cornea1cells were treated with TGF-Pl for 24 hr and its influence on EGFreceptors was examined. The results showed that TGF-Pl induced the high affiity receptors in the keratocytes, although TGF-/31 did not influence EGF receptors in the epithelial and endothelial cells. The number of high affinity receptors in keratocytes treated with TGF-Pl was 1.02 x lo4 per cell and the association constant was 0.171 nM (this was approximately tenfold higher than that of the receptor of keratocytes not treated with TGF-Pl). Key words: epidermal growth factor (EGF):transforming growth factor-,& (TGF-/?l); cornea1epithelial cell ; cornea1keratocyte ; cornea1endothelial cell : EGFreceptor ; Scatchard plot ; cell growth.

1. Introduction

Growth factors have an important role in cornea1 wound healing. Epidermal growth factor (EGF),which exists as a constant component of human tears (Ohashi et al., 1989; Van Setten et al., 1989), accelerateseyelid opening and incisory teeth eruption in newborn mice (Cohen, 1962) and, in addition, stimulates the proliferation of cornea1 epithelial (Frati et al., 1972 ; Savage and Cohen, 1973 ; Watanabe, Nakagawa and Nishida, 1987) and endothelial

cells

(Gospodarowicz, Mescher and Birdwell, 19 77 ; Gospodarowicz and Greenburg, 19 79). The distribution of EGF receptors in various rat tissues was examined with 1311-labelledEGF administered by intraperitoneal injection. The 1311-labelled EGF accumulated in cornea1 epithelial cells (Covelli et al., 19 72) and, furthermore, EGFreceptors have also been found in cat cornea1 endothelial cells (Fabricant et al., 198 1). Thus, it is conjectured that EGFis closely related to the regulation of cornea1 cell proliferation

Conversely, transforming growth factors (TGFs)are autocrine polypeptide growth factors produced in murine sarcoma virus-transformed mouse fibroblasts (Todaro, De Larco and Cohen, 1976; Todaro et al., 1977). TGFs stimulate normal cells to divide in monolayer culture and also to form colonies which grow progressively in soft agar (De Larco and Todaro, 19 78). Type-P TGF (TGF-P),isolated from TGFs,causes the anchorage-independent growth of non-neoplastic indicator cells (Roberts et al., 1981). TGF-P does not compete with EGFfor receptor binding, but can induce the phenotypic transformation of fibroblasts when potentiated by EGF (Roberts et al., 1983). However, the role of TGF-P in cornea1wound healing is not yet known. In order to clarify the effects of EGF and TGF-,&on cornea1wound healing, the sensitivities of three kinds of cultured cornea1 cells (keratocytes, epithelial and endothelial cells) to EGF and TGF-Pl were examined. In addition, the distribution of EGF receptors in three

kinds

of cultured

cornea1 cells was also

determined.

and wound healing. * For correspondence. 00144835/92/010009+08

$03.00/O

0 1992 Academic Press Limited

M. HONG0

10

2. Materials and Methods Growth Factors Purified EGF from human urine was a kind gift from JCR Pharmaceutical Co. (Kobe, Japan). The EGF was dissolved in distilled water at 100 ,ug ml-’ and stored at - 8O’C. For experiments, it was diluted in TC-19 9 medium (Nissui Pharmaceutical Co., Tokyo, Japan) containing 15 mM N-2-hydroxyethyl-piperazine-N’2-ethanesulfonic acid (Hepes, Nacalai Tesque Inc., Kyoto, Japan) and 1.4 g I-’ NaHCO, at pH 6.6 (TCM). Human recombinant TGF-p1 (Nacalai Tesque Inc.) was diluted in TCM containing 0.1 y0 bovine serum albumin (BSA, fraction V, Sigma Chemical Co., St. Louis, MO, U.S.A.) at pH 6.75.

ET AL.

based on their morphological characteristics. Namely, the epithelial cells possessed a needle-like axopodium in one direction and flattened lamellipodia with ruffles in the other directions [Fig. l(A)]. The keratocytes in each piece of stromal mass migrated onto the flask surface and showed a fibroblast-like spindle-shaped pattern [Fig. l(B)]. The endothelial cells on each piece of Descemet’s membrane migrated onto the flask surface and showed a mosaic arrangement pattern [Fig. l(C)]. The three kinds of cornea1 cells showed the morphological uniformity in each culture. Endothelial cells and keratocytes were cultured for 2 weeks then used in our experiments, while the epithelial cells were cultured for 4 days prior to use. Assay Methods for Cell Growth

Cornea1 Cell Cultures Three kinds of cornea1 cells were obtained from 4month-old female albino rabbits (New Zealand White, body weight 2.0 kg) by the modified method described by Nakagawa et al. (1990). Rabbits were killed with sodium pentobarbital (60 mg kg-’ body weight, Pitman-Moore, Inc., Washington, U.S.A.). The eyeballs were enucleated and washed twice with sterile Cat+- Mg2+-free phosphate buffered saline [PBS(-)]. After removal of conjunctivae, the cornea-scleral bottoms were extracted, and lenses and irises were removed. Under stereomicroscope, the sclerae were excised, and the endothelium with Descemet’s membrane was peeled off with jeweller’s forceps. The epithelial and stromal layers were incubated in serumfree TCM containing 2.4 mg ml-l dispase (Godo Shusei Co., Tokyo, Japan) at 37’C for 60 min. Thereafter, the epithelial layer was scraped from the stromal layer by spatula, and cut into small pieces with a sterile razor blade. Epithelial cells were treated with 0.02% ethylene diamine tetraacetic acid (EDTA, Wako Pure Chemical Industries, Osaka, Japan) and 100 U ml-’ trypsin (Mochida Pharmaceutical Co., Tokyo, Japan) in PBS(-) for 15 min at 37’C and dissociated by repeated passage through a 23-gauge hypodermic needle. The endothelial layer was also cut into small pieces together with Descemet’s membrane with a sterile razor blade. The stromal layers including keratocytes were cut into small blocks with sterile scissors. All cells were cultured in TCM containing 20% fetal bovine serum (FBS, Cell Culture Laboratories, Cleveland, OH, U.S.A.) in 25cm2 culture flasks (Corning Laboratories, NY, U.S.A.) in a 5% CO? atmosphere at 37°C. After 1 week, the cultured cells were treated with 0.02% EDTA and 100 U ml-’ trypsin in PBS(-) and dispersed. After washing, they were transferred into 75-cm2 culture flasks (Corning Laboratories) and cultured in TCM containing 10% FBS at 3 7°C in a 5 % CO, atmosphere. Identification of all three of cornea1 cell types was

Cornea1 cells were adjusted to a density of 5 x IO4 cells ml-’ in TCM containing 1 “/o FBS, and 100 ,ul of this suspension (5 x lo3 cells) were added to 9 6-well microplates (Corning Laboratories). Onehundred microlitres of EGF dissolved in serum-free TCM at 0.001, 0.01, 0.1, 1, 10 and 10Ongml~’ or 100 ~1 TGF-/31 dissolved in TCM containing 0.02 % BSA at 0.1, 1 and 10 ng ml-’ were added (,final concentration of FBS was 0.5%: Gospodarowicz and Moran, 19 74, 19 75). These cells were incubated at 37°C at 5 % CO, atmosphere for 2. 3 and 4 days. To examine the combined effect of EGF and TGF-,!?l, 100 ,ul of EGF and TGF-/?l mixtures were added to the cells in the microplates, and incubated at 3 7°C in a 5 “/o CO, atmosphere for 3 days. After incubation, the cells were fixed with 2.5% glutaraldehyde for 15 min and stained with 0.05% methylene blue for 15 min. To extract the methylene blue, 200 ~1 of O-33 N HCI was added to each well, and the plate was agitated with a mixer. The absorbance at 665 nm was measured (Yamazaki et al.. 1986) by spectrophotometer (Titertek Multiskan PLUS MK II, Flow Laboratories Inc., McLean, VA, U.S.A.). The growth promoting effects of EGF and TGF-/j’l were expressed as the ratio of the average absorbance in cells treated with these growth factors to that in the untreated controls. Preparation of 1z51-labelledEGF Highly purified human EGF (JCR Pharmaceutical Co.) was iodinated with [1251]NaI (Du Pont Inc., Boston, MA, U.S.A.) by chloramine-T (Greenwood and Hunter, 19 63). In brief, 37000 KBq of [‘251]NaI were added to 1 ,ug EGF in 10 ,ul distilled water, and maintained at 5°C on ice. Chloramine-T was added and mixed at 5°C for 90 sec. Sodium metabisulphite and potassium iodide solutions were added and the mixture was transferred onto a Bio Gel P-6 (Bio-Rad Laboratories, Richmond, CA, U.S.A.) column and eluted with equilibrated buffer [ 50 mM PBS(-), pH 7.4, containing O-l y’ BSA]. The void volume fractions

EGF

ECEPTORS

IN CORNEA1

11

CELLS

were collected and SO-y1 aliquots maintained at - 80°C for the 1251-labelledEGF ([lz51]EGF)binding assay. The specific activity of [1251]EGF was 24 70025 000 Bq ng-‘. EGF Receptor Assay

The EGF binding assay was performed by the method describedby Carpenter and Cohen (19 76) and Sarup, Rao and Fox (1988) with some modifications. After cultured cells were dissociated with 0.02 % EDTA and 100 U ml-’ trypsin in PBS(-), they were washed and collected twice by centrifugation at 800 g for 5 min in TCM containing 10% FBS. The cell density was adjusted to l-2 x lo5 cells ml-‘. One-hundred microlitres of celi suspension was placed into 24-weli plates (Corning Laboratories) and 900 ,ul TCM containing 10% FBS was added to each well. After incubation at 3 7°C in a 5 y0CO, atmosphere for 24 hr, the cultured cells were washed twice in serum-free TCM and 300 ,.ulTCM containing 0.2 % FBSat pH 6.75 was added. After incubation at 37°C in a 5 % CO, atmosphere for 24 hr, the supernatants were removed, and the monolayer cells were washed twice with binding medium consisting of TC-199 medium containing 0.1% BSA, 20 mM Hepes and 1.4 g 1-l NaHCO,, pH 6-75. Binding medium ( 300 141)containing 240 Bq of [lz51]EGFwas added to the wells, and immediately after that, 6.3, 63, 250, 500. 1000, 2000 and 4000 pg EGF was added in triplicate, respectively. For the determination of non-specific binding, a lOOO-fold excess of unlabelled EGF (about 5 pg per well) was added in triplicate to wells containing [‘“SIIEGF. After incubation at 5°C for 24 hr with gentle rocking, the medium was removed and the monolayer cells were washed four times with binding medium. These cells were dissolved in 300 ~1 of 1 N NaOH, and the radioactivity counted in a liquid scintillation counter (Packard Auto-Gamma 500 C type, Packard Japan Co., Tokyo, Japan). The binding association constants were determined by a Scatchard plot (Scatchard, 1949 : Rosenthal, 1967).

At the same time as the above procedure, cells in a duplicate plate cultured under the same conditions as that used for the receptor assay, were dispersed with

FIG. 1. Phase-contrast micrographs of cornea1 epithelial cells, keratocytes and endothelial cells. A, Epithelial cells after 4 days in culture. These cells possessedaxopodium (large arrows) and lamellipodia (small arrows). B, Keratocytes after 7 days in culture. Keratocytes migrated onto the culture flask from a piece of stromal mass and were spindie-shaped. C. Endothelial cells after 7 days in culture. Endothelial cells migrated onto the culture flask from a piece of Descemet’smembrane and showed mosaic arrangement. x 200.

M. HONG0

12

ET AL.

TABLE I Growth promoting

EGF concentration (ng ml-‘) 100 10 1 0.1 0.01 0.001 Control Absorbance

eflectsof EGF in cornea1epithelial cells Ratio of promoting effects of EGF at different incubation times (days) 4

3

2

116.20+8.57 121.80+34.16 133.80f 5.32* 136.60~15~13~

157.00+13.43$

178.00 + 5.73$ 199.00f 14.13$ 146.40 + 14.588 103.00_+6.17

100~90_+4~04 106.90f10.34

98.301fr9.43

100.00 + 5.56

100~00 * 16.88 0~077f0*013

0~072+0~004

200.40+13.18$ 210.90+23.72$

191.90+26.05$ 160.20 + 10*09$ 110.60+9.38 102.7Ok13.27 100.00 + 16.92 0.065f0.011

Cells were cultured in TCM containing 0.5% FBS, 1.4 g I-’ NaHCO, and 15 mM Hepes in each experiment. The incubation times for cells were 2. 3 and 4 days. Concentrations of EGFwere from O-001 to 100 ng ml-‘. Controt cells were incubated without EGF.Values are mean+s.o. (n = 4) of percentage absorbance at 665 nm to the control. ‘P