- Email: [email protected]
Effect of Transforming Growth Factor-β1 and -β2 onin vitroRabbit Corneal Epithelial Cell Proliferation Promoted by Epidermal Growth Factor, Keratinocyte Growth Factor, or Hepatocyte Growth Factor
Exp. Eye Res. (1997) 65, 391–396
Effect of Transforming Growth Factor-β1 and -β2 on in vitro Rabbit Corneal Epithelial Cell Proliferation Promoted by Epidermal Growth Factor, Keratinocyte Growth Factor, or Hepatocyte Growth Factor Y O I C H I H O N M A, K O H J I N I S H I D A, C H I E S O T O Z O N O S H I G E R U K I N O S H I T A Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto 602, Japan (Received Lund 3 January 1997 and accepted in revised form 17 April 1997) Corneal epithelial wound healing is intimately controlled by a variety of growth factors, such as epidermal growth factor (EGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and transforming growth factor-βs (TGF-βs). In this study, we investigate the effects of TGF-β1 and TGF-β2 on cultured rabbit corneal epithelial cell proliferation promoted by EGF, KGF, or HGF. Both TGF-β1 and -β2 dose-dependently inhibited corneal epithelial cell proliferation promoted by KGF (40 ng ml−") and HGF (40 ng ml−"), and weakly inhibited cell proliferation promoted by EGF (4 ng ml−"). Furthermore, the inhibitory effect tended to be stronger with TGF-β2 than TGF-β1. These findings suggest that TGF-β1 and -β2 play important roles as negative modulators against the cell proliferation effect of EGF, KGF and HGF. # 1997 Academic Press Limited Key words : transforming growth factor-β1 (TGF-β1) ; transforming growth factor-β2 (TGF-β2) ; epidermal growth factor (EGF) ; keratinocyte growth factor (KGF) ; hepatocyte growth factor (HGF) ; corneal epithelial cell ; cell proliferation.
1. Introduction By regulating proliferation, differentiation, apoptosis and other functions, growth factors play an important role in corneal epithelial wound healing. For example, epidermal growth factor (EGF), keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) have been shown to stimulate the proliferation of corneal epithelial cells (Frati et al., 1972 ; Kitazawa et al., 1990 ; Sotozono et al., 1994 ; Wilson et al., 1994). EGF exists as a constant component of human tear fluid (Ohashi et al., 1989) ; EGF mRNA and EGF receptor mRNA are present in human corneal epithelial cells (Wilson, He and Lloyd, 1992 ; Hongo et al., 1992). KGF and HGF are characterized as paracrine mediators produced by stromal fibroblast cells (Sotozono et al., 1994 ; Wilson et al., 1993). Transforming growth factor-βs (TGF-βs) are a family of approximately 25 kD molecular weight polypeptides that have multifunctional regulatory activities, such as controlling cell growth and differentiation, and stimulating extracellular matrix production (Roberts and Sporn, 1990 ; Lyon and Moses, 1990a ; Sporn and Roberts, 1992). The TGF-β family comprises three closely related isoforms in mammals : TGF-β1, -β2, and -β3 (Roberts and Sporn, 1990). In light of their general actions, TGF-βs may play important roles in corneal epithelial maintenance and wound healing, and may closely correlate with the pathology of corneal diseases. For example, Hayashi et al. (1989), using a vitamin A-deficient rat model, reported that TGF-β may play an important role in 0014–4835}97}09039106 $25.00}0}ey970338
corneal wound healing, as a mediator that draws fibroblasts and macrophages into the inflammatory focus. Moreover, it has been demonstrated that TGF-β antagonizes the actions of EGF on corneal epithelial cells (Mishima et al., 1992). Previous reports have shown that TGF-β2 polypeptide and mRNA are strongly expressed, TGF-β1 and -β3 mRNA also being expressed, in corneal epithelial cells (Nishida et al., 1994, 1995). These findings indicate that TGF-βs may play key roles in corneal wound healing ; however, their function in the cornea is not fully understood. To better understand the coordinated regulatory mechanisms of growth promotion and inhibition, as well as the difference between TGF-β1 and TGF-β2 in effect upon cell proliferation, we investigated the effects of TGF-β1 and TGF-β2 on cultured rabbit corneal epithelial cell proliferation promoted by EGF, KGF, or HGF.
2. Materials and Methods Cell Culture Normal rabbit cell culture was performed as previously described (Sotozono et al., 1994). Briefly, normal rabbit corneal epithelial cells (NRCE) obtained from Kurabo (Osaka, Japan) were cultured in 25 cm# culture flasks (Corning Laboratories, Corning City, NY, U.S.A.) in RCGM (serum-free medium, specific for rabbit corneal epithelial cells, containing 5 µg ml−" of insulin, 0±5 µg ml−" of hydrocortisone, 50 µg ml−" of gentamicin, 0±25 µg ml−" of amphotericin B, 0±03 m # 1997 Academic Press Limited
392
Y. H O N M A E T A L.
Relative number of cells (% of control)
300
200
100
0
0.04 0.4 4 40 0 Concentration of growth factor (ng ml–1)
F. 1. Growth promoting effect of EGF, KGF, and HGF on cultured rabbit corneal epithelial cells. Cells seeded at density of 1±5¬10% cells per well in 96-well microplate were 24 hr later treated with EGF (*), KGF (8), or HGF (+) before further incubation for 72 hr. Data represent mean values³.. (n ¯ 16) of % absorbance at 630 nm compared to control level.
tration of growth factor, the concentrations of 4 ng ml−" of EGF, 40 ng ml−" of HGF, and 40 ng ml−" of KGF showed equal cell proliferation effect as shown in Fig. 1. These cells were then further incubated for 72 hr. After this incubation, cells were photographed with a phase-contrast microscope and cell proliferation was evaluated by the methylene blue method. Briefly, the cells were fixed with 2±5 % glutaraldehyde for 15 min and stained with 0±5 % methylene blue for 15 min. To extract the methylene blue, 200 µl of 0±33 N HCl was added to each well, and the plate was agitated on a mixer. Absorbance at 630 nm was measured (Yamazaki et al., 1986) by a spectrophotometer (Titertek Multiscan, Flow Laboratories, McLean, VA, U.S.A.). The relative number of cells was expressed as the ratio of the average absorbance of the TGF-βtreated cells to that of controls. Statistical comparisons were performed using one-way ANOVA, including Dunn comparison. A probability level of P ! 0±05 was considered statistically significant. 3. Results
Growth Assays Proliferation assays were performed by plating 1±5¬10% cells per well in 96-well microplates (Corning Laboratories) with 100 µl of RCBM (serum-free medium, specific for rabbit corneal epithelial cells, containing 5 µg ml−" of insulin, 0±5 µg ml−" of hydrocortisone, 50 µg ml−" of gentamicin, 0±25 µg ml−" of amphotericin B, and 0±03 m Ca#+ ; Kurabo Co.) containing no growth factor, serum or other extracts, with incubation in a 5 % CO atmosphere at 37°C. # After 24 hr, 100 µl of growth factors dissolved in RCBM was added to each well to achieve the final concentration of growth factor. TGF-β1 (human recombinant TGF-β1, Boehringer Mannheim Biochemica Co.) or TGF-β2 (human recombinant TGFβ2, Boehringer Mannheim Biochemica Co.) was added to the cultures to final concentrations of 0±1, 1, 10, and 100 ng ml−", using eight wells for each concentration. Combinations of growth factors were also tested. Four nanograms per millilitre of EGF (human recombinant EGF, Otsuka Pharmaceutical Co.), 40 ng ml−" of KGF (human recombinant KGF, Upstate Biotechnology Inc.) or 40 ng ml−" of HGF (human recombinant heterodimeric form HGF, Collaborative Biomedical Products Co.) was added to the cultures with serial dilutions of TGF-β1 or TGF-β2, final concentrations ranging from 0±1 to 10 ng ml−", using eight wells for each concentration. In a preliminary examination to determine the appropriate concen-
Cells were treated with variable concentrations of EGF, KGF, or HGF to determine the appropriate concentration of growth factor. As shown in Fig. 1, the concentrations of 4 ng ml−" of EGF, 40 ng ml−" of HGF, and 40 ng ml−" of KGF showed equal cell proliferation effect, ranging from 250–280 %. The effects of TGF-βs on rabbit corneal epithelial cell proliferation with or without EGF (4 ng ml−"), KGF (40 ng ml−"), or HGF (40 ng ml−") are shown in Figs 2–5. Both TGF-β1 and TGF-β2, each added to the
Relative number of cells (% of control)
Ca#+, 10 ng ml−" of EGF, and 0±4 % of bovine pituitary extract ; Kurabo Co.). Cells were fed every 2 days with RCGM. The initial secondary culture was used for growth assays.
120
*
* *
* *
100 80 60 40 20 0
Control 0.1 1 10 100 Concentration of TGF-β (ng ml–1)
F. 2. Effect of TGF-β1 and TGF-β2 on cultured rabbit corneal epithelial cell proliferation. Cells seeded at density of 1±5¬10% cells per well in 96-well microplate were 24 hr later treated with TGF-β1 (8), TGF-β2 (+), or control treated with RCBM alone (*) before further incubation for 72 hr. Data represent mean values³.. (n ¯ 24) of % absorbance at 630 nm compared to control level. * P ! 0±05.
120
*
100 80 60 40 20 0
Control 0.1 1 10 Concentration of TGF-β (ng ml–1)
Relative number of cells (% of control)
F. 3. Effect of TGF-β1 and TGF-β2 on cultured rabbit corneal epithelial cell proliferation promoted by EGF. Cells seeded at density of 1±5¬10% cells per well in 96-well microplate were 24 hr later treated with EGF (4 ng ml−") and TGF-β1 (8), with EGF (4 ng ml−") and TGF-β2 (+), or control treated with EGF (4 ng ml−") alone (*) before further incubation for 72 hr. Data represent mean values³.. (n ¯ 45) of % absorbance at 630 nm compared to the control level. * P ! 0±05.
120
* *
* *
*
100 80 60 40 20 0
393
Relative number of cells (% of control)
Relative number of cells (% of control)
TGF-β-EFFECT ON CORNEAL EPITHELIAL CELLS
Control 0.1 1 10 Concentration of TGF-β (ng ml–1)
F. 4. Effect of TGF-β1 and TGF-β2 on cultured rabbit corneal epithelial cell proliferation promoted by KGF. Cells seeded at density of 1±5¬10% cells per well in 96-well microplate were 24 hr later treated with KGF (40 ng ml−") and TGF-β1 (8), with KGF (40 ng ml−") and TGF-β2 (+), or control treated with KGF (40 ng ml−") alone (*) before further incubation for 72 hr. Data represent mean values³.. (n ¯ 32) of % absorbance at 630 nm compared to control level. * P ! 0±05.
culture alone, inhibited epithelial cell proliferation in dose-dependent fashion at concentrations ranging from 0±1 to 10 ng ml−" ; at 10 ng ml−", cell proliferation was approximately 40 to 50 % that of control (Fig. 2). Epithelial cell proliferation promotion by KGF
* *
120
*
*
100
* *
* *
80 60 40 20 0
Control 0.1 1 10 Concentration of TGF-β (ng ml–1)
F. 5. Effect of TGF-β1 and TGF-β2 on cultured rabbit corneal epithelial cell proliferation promoted by HGF. Cells seeded at density of 1±5¬10% cells per well in 96-well microplate were 24 hr later treated with HGF (40 ng ml−") and TGF-β1 (8), with HGF (40 ng ml−") and TGF-β2 (+), or control treated with HGF (40 ng ml−") alone (*) before further incubation for 72 hr. Data represent mean values³.. (n ¯ 16) of % absorbance at 630 nm compared to control level. * P ! 0±05.
(40 ng ml−") or HGF (40 ng ml−") was also inhibited in dose-dependent fashion by the addition of TGF-β1 or TGF-β2 ; at 10 ng ml−", cell proliferation was approximately 40 to 65 % that of control (Figs 4 and 5). In the case of EGF, however, cell proliferation was inhibited weakly, and not dose-dependently, by the addition of TGF-β1 or TGF-β2. Cell proliferation was approximately 85 to 95 % that of control at TGF-β concentrations of 0±1 to 10 ng ml−" (Fig. 3). It was noted that the inhibition effect of TGF-β2 was slightly stronger than that of TGF-β1 in all experiments. These results correlated well with the morphologic changes observed under phase contrast microscopy (Fig. 6). In culture combining TGF-β and EGF, many elongated cells were seen, suggesting cell growth or migration [Fig. 6(A), (B) and (C)]. In contrast, in culture combining TGF-β and KGF}HGF, there were few elongated cells [Fig. 6(D)–(I)].
4. Discussion This study demonstrates that both TGF-β1 and TGFβ2 dose-dependently inhibit in vitro rabbit corneal epithelial cell proliferation promoted by KGF (40 ng ml−") and HGF (40 ng ml−"), but weakly inhibit proliferation promoted by EGF (4 ng ml−"). It is noteworthy that TGF-βs have much less inhibitory effect on EGF than on KGF and HGF, in terms of cell proliferation. EGF exists as a constant component of human tear fluid (Ohashi et al., 1989). On the other hand, KGF and HGF are produced by stromal
394
Y. H O N M A E T A L.
F. 6. Phase-contrast micrographs of rabbit corneal epithelial cell cultures at 72 hr after addition of growth factors. Cells treated with individual growth factor [EGF (4 ng ml−") (A), KGF (40 ng ml−") (D), HGF (40 ng ml−") (G)] formed dense cell layer. Cells treated with combination of growth factors EGF (4 ng ml−") and TGF-β1 (10 ng ml−") (B), or EGF (4 ng ml−") and TGFβ2 (10 ng ml−") (C) were predominantly elongated, with cell-free areas present. Cells treated with KGF (40 ng ml−") and TGFβ1 (10 ng ml−") (E), KGF (40 ng ml−") and TGF-β2 (10 ng ml−") (F), HGF (40 ng ml−") and TGF-β1 (40 ng ml−") (H), or HGF (40 ng ml−") and TGF-β2 (40 ng ml−") (I) remained mostly round ; cell-free areas were frequent. Scale bars ¯ 100 µm.
fibroblasts (Sotozono et al., 1994 ; Wilson et al., 1993) ; HGF is also produced by the lacrimal gland (Li et al., 1996). Li et al. (1996) have reported that HGF expression in keratocytes is upregulated after corneal epithelial wounding, and that HGF concentration in tear fluid is higher in postoperative tears than normal tears. These findings, taken together with the present results, lead us to speculate that the effects of KGF and HGF are regulated by their expression level, and another regulators, like TGF-βs, whereas, the effects of EGF are regulated by the expression of its receptors. In fact, Green et al. (1983) have reported that EGF
activity is regulated through a cell surface receptor that is localized to the proliferative cell layers in stratified squamous epithelium. Recently, Zieske and Wasson (1993) reported that EGF receptors were present in basal cells across the adult cornea, but more numerous in the limbal basal zone, where EGF may maintain stem cells in an undifferentiated state. These findings suggest that, just after corneal epithelial wounding, EGF from human tear fluid may promote limbal basal cell proliferation by binding to receptors without inhibiting TGF-βs present in the anterior segment of the eye (tear fluid, cornea).
TGF-β-EFFECT ON CORNEAL EPITHELIAL CELLS
The present study suggests that the inhibitory effect of TGF-β2 was slightly stronger than that of TGF-β1, although further study may be needed to demonstrate the activity of TGF-β1 and ®β2 using the mink lung assay or other method. This result may be due to the stronger affinity of TGF-β2 for receptors than that of TGF-β1 in the in-vitro rabbit corneal epithelial cells used. In fact, several recent reports have shown that type I, II, and III receptors have equal or higher affinity for TGF-β2 than for TGF-β1 in certain cell types (Woodward et al., 1995 ; Zhou et al., 1995 ; Sankar et al., 1995). Alternatively, it can be speculated that TGF-β1 and -β2 may have different receptors or signal transduction systems, as suggested by a previous report (Zhou et al., 1995). Our result that TGF-β1, -β2 weakly inhibit corneal epithelial cell proliferation promoted by EGF is somewhat different from that of Mishima et al. (1992), whose results showed that TGF-β dose-dependently inhibits corneal epithelial cell proliferation promoted by EGF. One possible explanation for this discrepancy is the different cell conditions and}or different culture media used ; that used in our study, with very low Ca#+ concentration, is specifically designed for rabbit corneal epithelial cell proliferation. The proliferative activity of the corneal epithelial cells in our study might therefore be greater than in that of Mishima et al., leading to different results. A recent study by our laboratory has shown both the protein and mRNA of latent-TGF-β2 to be present in human corneal epithelial cells (Nishida et al., 1995), and that latent TGF-β2 is predominantly present in human tear fluid (Kokawa et al., 1996), which may be of great relevance to our result. It has been reported that plasmin, a serine protease that which is excessively generated at the leading epithelial edge by uPA (Hayashi et al., 1991), can activate latent TGF-β in an in vitro co-culture system of endothelial cells and pericytes (Lyon et al., 1990b ; Sato and Rifkin, 1989 ; Odekon, Blasi and Rifkin, 1994), and may be important for in vivo activation of latent TGFβ in corneal epithelial wound healing. It is possible that latent TGF-β1 or -β2 may be activated at the leading epithelial edge and antagonize the proliferative effect of EGF, KGF, and HGF. In vivo animal studies will be needed to determine the distribution and regulation of TGF-β isoforms during corneal epithelial wound healing, and to elucidate the in vivo activation mechanism of latent TGF-βs on the ocular surface. Acknowledgements This work was supported in part by a research grant (07771557) from the Ministry of Education, Culture and Science of Japan. This work was presented in part at the Annual Meeting of the Association for Research in Vision and Ophthalmology, 1995, in Fort Lauderdale, Florida, U.S.A.
395
References Frati, L., Daniele, S., Delogu, A. and Covelli, I. (1972). Selective binding of the epidermal growth factor and its specific effects on the epithelial cells of the cornea. Exp. Eye Res. 14, 135–41. Green, M. R., Basketter, D. A., Couchman, J. R. and Rees, D. A. (1983). Distribution and number of epidermal growth factor receptors in skin is related to epithelial cell growth. Dev. Biol. 100, 506–12. Hayashi, K., Frangieh, G., Wolf, G. and Kenyon, K. (1989). Expression of transforming growth factor-β in wound healing of vitamin A-deficient rat corneas. Invest. Ophthalmol. Vis. Sci. 30, 239–47. Hayashi, K., Berman, M., Smith, D., El-Ghatit, A., Pease, S. and Kenyon, K. R. (1991). Pathogenesis of corneal epithelial defects : role of plasminogen activator. Curr. Eye Res. 10, 381–98. Hongo, M., Itoi, M., Yamaguchi, N. and Imanishi, J. (1992). Distribution of epidermal growth factor (EGF) receptors in rabbit corneal epithelial cells, keratocytes and endothelial cells, and the changes induced by transforming growth factor-β1. Exp. Eye Res. 54, 9–16. Kitazawa, T., Kinoshita, S., Fujita, K., Araki, K., Watanabe, H., Ohashi, Y. and Manabe, R. (1990). The mechanism of accelerated corneal epithelial healing by human epidermal growth factor. Invest. Ophthalmol. Vis. Sci. 31, 1773–8. Kokawa, N., Sotozono, C., Nishida, K. and Kinoshita, S. (1996). High total TGF-β2 levels in normal human tears. Curr. Eye Res. 15, 341–3. Li, Q., Weng, J., Mohan, R. R., Bennett, G. L., Schwall, R., Wang, Z-F., Tabor, K., Kim, J., Hargrave, S., Cuevas, K. H. and Wilson, S. E. (1996). Hepatocyte growth factor and hepatocyte growth factor receptor in the lacrimal gland, tears, and cornea. Invest. Ophthalmol. Vis. Sci. 37, 727–39. Lyon, R. M. and Moses, H. L. (1990a). Transforming growth factor and regulation of cell proliferation. Eur. J. Biochem. 187, 467–73. Lyon, R. M., Gentry, L. E., Purchio, A. F. and Moses, H. L. (1990b). Mechanism of activation of latent recombinant transforming growth factor β1 by plasmin. J. Cell Biol. 110, 1361–7. Mishima, H., Nakamura, M., Murakami, J., Nishida, T. and Otori, T. (1992). Transforming growth factor-β modulates effect of epidermal growth factor on corneal epithelial cells. Curr. Eye Res. 11, 691–6. Nishida, K., Kinoshita, S., Yokoi, N., Kaneda, M., Hashimoto, K. and Yamamoto, S. (1994). Immunohistochemical localization of transforming growth factor-β1, -β2, and -β3 latency-associated peptide in human cornea. Invest. Ophthalmol. Vis. Sci. 35, 3289–94. Nishida, K., Sotozono, C., Adachi, W., Yamamoto, S., Yokoi, N. and Kinoshita, S. (1995). Transforming growth factor-β1, -β2 and -β3 mRNA expression in human cornea. Curr. Eye Res. 14, 235–41. Odekon, L. E., Blasi, F. and Rifkin, D. B. (1994). Requirement for receptor-bound urokinase in plasmin-dependent cellular conversion of latent TGF-beta to TGF-beta. J. Cell. Physiol. 158, 398–407. Ohashi, Y., Motokura, M., Kinoshita, Y., Mano, T., Watanabe, H., Kinoshita, S., Manabe, R., Oshiden, K. and Yanaihara, C. (1989). Presence of epidermal growth factor in human tears. Invest. Ophthalmol. Vis. Sci. 30, 1879–82. Roberts, A. B. and Sporn, M. B. (1990). The transforming growth factor-betas. In Peptide growth factors and their receptors, handbook of experimentary pharmacology (Eds
396
Sporn, M. B. and Roberts, A. B.). Pp. 419–72. SpringerVerlag : Heidelberg. Sankar, S., Mahooti-Brooks, N., Centrella, M., McCarthy, T. L. and Madri, J. A. (1995). Expression of transforming growth factor type III receptor in vascular endothelial cells increases their responsiveness to transforming growth factor β2. J. Biol. Chem. 270, 13567–72. Sato, Y. and Rifkin, D. B. (1989). Inhibition of endothelial cell movement by pericytes and smooth muscle cells : activation of a latent transforming growth factor-β1like molecule by plasmin during co-culture. J. Cell Biol. 109, 309–15. Sotozono, C., Kinoshita, S., Kita, M. and Imanishi, J. (1994). Paracrine role of keratinocyte growth factor in rabbit corneal epithelial cell growth. Exp. Eye Res. 59, 385–92. Sporn, M. B. and Roberts, A. B. (1992). Transforming growth factor-β : recent progress and new challenges. J. Cell. Biol. 119, 1017–21. Wilson, S. E., He, Y-G. and Lloyd, S. A. (1992). EGF, EGF receptor, basic FGF, TGF beta-1, and IL-1 alpha mRNA in human corneal epithelial cells and stromal fibroblasts. Invest. Ophthalmol. Vis. Sci. 33, 1756–65. Wilson, S. E., Walker, J. W., Chwang, E. L. and He, Y-G. (1993). Hepatocyte growth factor, keratinocyte growth factor, their receptors, fibroblast growth factor receptor-
Y. H O N M A E T A L.
2, and the cells of the cornea. Invest. Ophthalmol. Vis. Sci. 34, 2544–61. Wilson, S. E., He, Y-G., Weng, J., Zieske, J. D., Jester, J. V. and Schultz, G. S. (1994). Effect of epidermal growth factor, hepatocyte growth factor, and keratinocyte growth factor, on proliferation, motility and differentiation of human corneal epithelial cells. Exp. Eye Res. 59, 665–78. Woodward, T. L., Dumont, N., O’Connor-McCort, M., Turner, J. D. and Philip, A. (1995). Characterization of transforming growth factor-β growth regulatory effects and receptors on bovine mammary cells. J. Cell. Physiol. 165, 339–48. Yamasaki, S., Onishi, E., Enami, K., Natori, K., Kohase, M., Sakamoto, H., Tanouchi, M. and Hayashi, H. (1986). Proposal of standardized methods and reference for assaying recombinant human tumor necrosis factor. Japan., J. Med. Sci. Biol. 39, 105–18. Zhou, G-H. K., Sechrist, G. L., Periyasamy, S., Brattain, M. G. and Mulder, K. M. (1995). Transforming growth factor β isoform-specific differences in interactions with type I and II transforming growth factor β receptors. Cancer Res. 55, 2056–62. Zieske, J. D. and Wasson, M. (1993). Regional variation in distribution of EGF receptor in developing and adult corneal epithelium. J. Cell. Sci. 106, 145–52.
Effect of Transforming Growth Factor-β1 and -β2 on in vitro Rabbit Corneal Epithelial Cell Proliferation Promoted by Epidermal Growth Factor, Keratinocyte Growth Factor, or Hepatocyte Growth Factor Y O I C H I H O N M A, K O H J I N I S H I D A, C H I E S O T O Z O N O S H I G E R U K I N O S H I T A Department of Ophthalmology, Kyoto Prefectural University of Medicine, Kawaramachi Hirokoji, Kamigyo-ku, Kyoto 602, Japan (Received Lund 3 January 1997 and accepted in revised form 17 April 1997) Corneal epithelial wound healing is intimately controlled by a variety of growth factors, such as epidermal growth factor (EGF), keratinocyte growth factor (KGF), hepatocyte growth factor (HGF), and transforming growth factor-βs (TGF-βs). In this study, we investigate the effects of TGF-β1 and TGF-β2 on cultured rabbit corneal epithelial cell proliferation promoted by EGF, KGF, or HGF. Both TGF-β1 and -β2 dose-dependently inhibited corneal epithelial cell proliferation promoted by KGF (40 ng ml−") and HGF (40 ng ml−"), and weakly inhibited cell proliferation promoted by EGF (4 ng ml−"). Furthermore, the inhibitory effect tended to be stronger with TGF-β2 than TGF-β1. These findings suggest that TGF-β1 and -β2 play important roles as negative modulators against the cell proliferation effect of EGF, KGF and HGF. # 1997 Academic Press Limited Key words : transforming growth factor-β1 (TGF-β1) ; transforming growth factor-β2 (TGF-β2) ; epidermal growth factor (EGF) ; keratinocyte growth factor (KGF) ; hepatocyte growth factor (HGF) ; corneal epithelial cell ; cell proliferation.
1. Introduction By regulating proliferation, differentiation, apoptosis and other functions, growth factors play an important role in corneal epithelial wound healing. For example, epidermal growth factor (EGF), keratinocyte growth factor (KGF) and hepatocyte growth factor (HGF) have been shown to stimulate the proliferation of corneal epithelial cells (Frati et al., 1972 ; Kitazawa et al., 1990 ; Sotozono et al., 1994 ; Wilson et al., 1994). EGF exists as a constant component of human tear fluid (Ohashi et al., 1989) ; EGF mRNA and EGF receptor mRNA are present in human corneal epithelial cells (Wilson, He and Lloyd, 1992 ; Hongo et al., 1992). KGF and HGF are characterized as paracrine mediators produced by stromal fibroblast cells (Sotozono et al., 1994 ; Wilson et al., 1993). Transforming growth factor-βs (TGF-βs) are a family of approximately 25 kD molecular weight polypeptides that have multifunctional regulatory activities, such as controlling cell growth and differentiation, and stimulating extracellular matrix production (Roberts and Sporn, 1990 ; Lyon and Moses, 1990a ; Sporn and Roberts, 1992). The TGF-β family comprises three closely related isoforms in mammals : TGF-β1, -β2, and -β3 (Roberts and Sporn, 1990). In light of their general actions, TGF-βs may play important roles in corneal epithelial maintenance and wound healing, and may closely correlate with the pathology of corneal diseases. For example, Hayashi et al. (1989), using a vitamin A-deficient rat model, reported that TGF-β may play an important role in 0014–4835}97}09039106 $25.00}0}ey970338
corneal wound healing, as a mediator that draws fibroblasts and macrophages into the inflammatory focus. Moreover, it has been demonstrated that TGF-β antagonizes the actions of EGF on corneal epithelial cells (Mishima et al., 1992). Previous reports have shown that TGF-β2 polypeptide and mRNA are strongly expressed, TGF-β1 and -β3 mRNA also being expressed, in corneal epithelial cells (Nishida et al., 1994, 1995). These findings indicate that TGF-βs may play key roles in corneal wound healing ; however, their function in the cornea is not fully understood. To better understand the coordinated regulatory mechanisms of growth promotion and inhibition, as well as the difference between TGF-β1 and TGF-β2 in effect upon cell proliferation, we investigated the effects of TGF-β1 and TGF-β2 on cultured rabbit corneal epithelial cell proliferation promoted by EGF, KGF, or HGF.
2. Materials and Methods Cell Culture Normal rabbit cell culture was performed as previously described (Sotozono et al., 1994). Briefly, normal rabbit corneal epithelial cells (NRCE) obtained from Kurabo (Osaka, Japan) were cultured in 25 cm# culture flasks (Corning Laboratories, Corning City, NY, U.S.A.) in RCGM (serum-free medium, specific for rabbit corneal epithelial cells, containing 5 µg ml−" of insulin, 0±5 µg ml−" of hydrocortisone, 50 µg ml−" of gentamicin, 0±25 µg ml−" of amphotericin B, 0±03 m # 1997 Academic Press Limited
392
Y. H O N M A E T A L.
Relative number of cells (% of control)
300
200
100
0
0.04 0.4 4 40 0 Concentration of growth factor (ng ml–1)
F. 1. Growth promoting effect of EGF, KGF, and HGF on cultured rabbit corneal epithelial cells. Cells seeded at density of 1±5¬10% cells per well in 96-well microplate were 24 hr later treated with EGF (*), KGF (8), or HGF (+) before further incubation for 72 hr. Data represent mean values³.. (n ¯ 16) of % absorbance at 630 nm compared to control level.
tration of growth factor, the concentrations of 4 ng ml−" of EGF, 40 ng ml−" of HGF, and 40 ng ml−" of KGF showed equal cell proliferation effect as shown in Fig. 1. These cells were then further incubated for 72 hr. After this incubation, cells were photographed with a phase-contrast microscope and cell proliferation was evaluated by the methylene blue method. Briefly, the cells were fixed with 2±5 % glutaraldehyde for 15 min and stained with 0±5 % methylene blue for 15 min. To extract the methylene blue, 200 µl of 0±33 N HCl was added to each well, and the plate was agitated on a mixer. Absorbance at 630 nm was measured (Yamazaki et al., 1986) by a spectrophotometer (Titertek Multiscan, Flow Laboratories, McLean, VA, U.S.A.). The relative number of cells was expressed as the ratio of the average absorbance of the TGF-βtreated cells to that of controls. Statistical comparisons were performed using one-way ANOVA, including Dunn comparison. A probability level of P ! 0±05 was considered statistically significant. 3. Results
Growth Assays Proliferation assays were performed by plating 1±5¬10% cells per well in 96-well microplates (Corning Laboratories) with 100 µl of RCBM (serum-free medium, specific for rabbit corneal epithelial cells, containing 5 µg ml−" of insulin, 0±5 µg ml−" of hydrocortisone, 50 µg ml−" of gentamicin, 0±25 µg ml−" of amphotericin B, and 0±03 m Ca#+ ; Kurabo Co.) containing no growth factor, serum or other extracts, with incubation in a 5 % CO atmosphere at 37°C. # After 24 hr, 100 µl of growth factors dissolved in RCBM was added to each well to achieve the final concentration of growth factor. TGF-β1 (human recombinant TGF-β1, Boehringer Mannheim Biochemica Co.) or TGF-β2 (human recombinant TGFβ2, Boehringer Mannheim Biochemica Co.) was added to the cultures to final concentrations of 0±1, 1, 10, and 100 ng ml−", using eight wells for each concentration. Combinations of growth factors were also tested. Four nanograms per millilitre of EGF (human recombinant EGF, Otsuka Pharmaceutical Co.), 40 ng ml−" of KGF (human recombinant KGF, Upstate Biotechnology Inc.) or 40 ng ml−" of HGF (human recombinant heterodimeric form HGF, Collaborative Biomedical Products Co.) was added to the cultures with serial dilutions of TGF-β1 or TGF-β2, final concentrations ranging from 0±1 to 10 ng ml−", using eight wells for each concentration. In a preliminary examination to determine the appropriate concen-
Cells were treated with variable concentrations of EGF, KGF, or HGF to determine the appropriate concentration of growth factor. As shown in Fig. 1, the concentrations of 4 ng ml−" of EGF, 40 ng ml−" of HGF, and 40 ng ml−" of KGF showed equal cell proliferation effect, ranging from 250–280 %. The effects of TGF-βs on rabbit corneal epithelial cell proliferation with or without EGF (4 ng ml−"), KGF (40 ng ml−"), or HGF (40 ng ml−") are shown in Figs 2–5. Both TGF-β1 and TGF-β2, each added to the
Relative number of cells (% of control)
Ca#+, 10 ng ml−" of EGF, and 0±4 % of bovine pituitary extract ; Kurabo Co.). Cells were fed every 2 days with RCGM. The initial secondary culture was used for growth assays.
120
*
* *
* *
100 80 60 40 20 0
Control 0.1 1 10 100 Concentration of TGF-β (ng ml–1)
F. 2. Effect of TGF-β1 and TGF-β2 on cultured rabbit corneal epithelial cell proliferation. Cells seeded at density of 1±5¬10% cells per well in 96-well microplate were 24 hr later treated with TGF-β1 (8), TGF-β2 (+), or control treated with RCBM alone (*) before further incubation for 72 hr. Data represent mean values³.. (n ¯ 24) of % absorbance at 630 nm compared to control level. * P ! 0±05.
120
*
100 80 60 40 20 0
Control 0.1 1 10 Concentration of TGF-β (ng ml–1)
Relative number of cells (% of control)
F. 3. Effect of TGF-β1 and TGF-β2 on cultured rabbit corneal epithelial cell proliferation promoted by EGF. Cells seeded at density of 1±5¬10% cells per well in 96-well microplate were 24 hr later treated with EGF (4 ng ml−") and TGF-β1 (8), with EGF (4 ng ml−") and TGF-β2 (+), or control treated with EGF (4 ng ml−") alone (*) before further incubation for 72 hr. Data represent mean values³.. (n ¯ 45) of % absorbance at 630 nm compared to the control level. * P ! 0±05.
120
* *
* *
*
100 80 60 40 20 0
393
Relative number of cells (% of control)
Relative number of cells (% of control)
TGF-β-EFFECT ON CORNEAL EPITHELIAL CELLS
Control 0.1 1 10 Concentration of TGF-β (ng ml–1)
F. 4. Effect of TGF-β1 and TGF-β2 on cultured rabbit corneal epithelial cell proliferation promoted by KGF. Cells seeded at density of 1±5¬10% cells per well in 96-well microplate were 24 hr later treated with KGF (40 ng ml−") and TGF-β1 (8), with KGF (40 ng ml−") and TGF-β2 (+), or control treated with KGF (40 ng ml−") alone (*) before further incubation for 72 hr. Data represent mean values³.. (n ¯ 32) of % absorbance at 630 nm compared to control level. * P ! 0±05.
culture alone, inhibited epithelial cell proliferation in dose-dependent fashion at concentrations ranging from 0±1 to 10 ng ml−" ; at 10 ng ml−", cell proliferation was approximately 40 to 50 % that of control (Fig. 2). Epithelial cell proliferation promotion by KGF
* *
120
*
*
100
* *
* *
80 60 40 20 0
Control 0.1 1 10 Concentration of TGF-β (ng ml–1)
F. 5. Effect of TGF-β1 and TGF-β2 on cultured rabbit corneal epithelial cell proliferation promoted by HGF. Cells seeded at density of 1±5¬10% cells per well in 96-well microplate were 24 hr later treated with HGF (40 ng ml−") and TGF-β1 (8), with HGF (40 ng ml−") and TGF-β2 (+), or control treated with HGF (40 ng ml−") alone (*) before further incubation for 72 hr. Data represent mean values³.. (n ¯ 16) of % absorbance at 630 nm compared to control level. * P ! 0±05.
(40 ng ml−") or HGF (40 ng ml−") was also inhibited in dose-dependent fashion by the addition of TGF-β1 or TGF-β2 ; at 10 ng ml−", cell proliferation was approximately 40 to 65 % that of control (Figs 4 and 5). In the case of EGF, however, cell proliferation was inhibited weakly, and not dose-dependently, by the addition of TGF-β1 or TGF-β2. Cell proliferation was approximately 85 to 95 % that of control at TGF-β concentrations of 0±1 to 10 ng ml−" (Fig. 3). It was noted that the inhibition effect of TGF-β2 was slightly stronger than that of TGF-β1 in all experiments. These results correlated well with the morphologic changes observed under phase contrast microscopy (Fig. 6). In culture combining TGF-β and EGF, many elongated cells were seen, suggesting cell growth or migration [Fig. 6(A), (B) and (C)]. In contrast, in culture combining TGF-β and KGF}HGF, there were few elongated cells [Fig. 6(D)–(I)].
4. Discussion This study demonstrates that both TGF-β1 and TGFβ2 dose-dependently inhibit in vitro rabbit corneal epithelial cell proliferation promoted by KGF (40 ng ml−") and HGF (40 ng ml−"), but weakly inhibit proliferation promoted by EGF (4 ng ml−"). It is noteworthy that TGF-βs have much less inhibitory effect on EGF than on KGF and HGF, in terms of cell proliferation. EGF exists as a constant component of human tear fluid (Ohashi et al., 1989). On the other hand, KGF and HGF are produced by stromal
394
Y. H O N M A E T A L.
F. 6. Phase-contrast micrographs of rabbit corneal epithelial cell cultures at 72 hr after addition of growth factors. Cells treated with individual growth factor [EGF (4 ng ml−") (A), KGF (40 ng ml−") (D), HGF (40 ng ml−") (G)] formed dense cell layer. Cells treated with combination of growth factors EGF (4 ng ml−") and TGF-β1 (10 ng ml−") (B), or EGF (4 ng ml−") and TGFβ2 (10 ng ml−") (C) were predominantly elongated, with cell-free areas present. Cells treated with KGF (40 ng ml−") and TGFβ1 (10 ng ml−") (E), KGF (40 ng ml−") and TGF-β2 (10 ng ml−") (F), HGF (40 ng ml−") and TGF-β1 (40 ng ml−") (H), or HGF (40 ng ml−") and TGF-β2 (40 ng ml−") (I) remained mostly round ; cell-free areas were frequent. Scale bars ¯ 100 µm.
fibroblasts (Sotozono et al., 1994 ; Wilson et al., 1993) ; HGF is also produced by the lacrimal gland (Li et al., 1996). Li et al. (1996) have reported that HGF expression in keratocytes is upregulated after corneal epithelial wounding, and that HGF concentration in tear fluid is higher in postoperative tears than normal tears. These findings, taken together with the present results, lead us to speculate that the effects of KGF and HGF are regulated by their expression level, and another regulators, like TGF-βs, whereas, the effects of EGF are regulated by the expression of its receptors. In fact, Green et al. (1983) have reported that EGF
activity is regulated through a cell surface receptor that is localized to the proliferative cell layers in stratified squamous epithelium. Recently, Zieske and Wasson (1993) reported that EGF receptors were present in basal cells across the adult cornea, but more numerous in the limbal basal zone, where EGF may maintain stem cells in an undifferentiated state. These findings suggest that, just after corneal epithelial wounding, EGF from human tear fluid may promote limbal basal cell proliferation by binding to receptors without inhibiting TGF-βs present in the anterior segment of the eye (tear fluid, cornea).
TGF-β-EFFECT ON CORNEAL EPITHELIAL CELLS
The present study suggests that the inhibitory effect of TGF-β2 was slightly stronger than that of TGF-β1, although further study may be needed to demonstrate the activity of TGF-β1 and ®β2 using the mink lung assay or other method. This result may be due to the stronger affinity of TGF-β2 for receptors than that of TGF-β1 in the in-vitro rabbit corneal epithelial cells used. In fact, several recent reports have shown that type I, II, and III receptors have equal or higher affinity for TGF-β2 than for TGF-β1 in certain cell types (Woodward et al., 1995 ; Zhou et al., 1995 ; Sankar et al., 1995). Alternatively, it can be speculated that TGF-β1 and -β2 may have different receptors or signal transduction systems, as suggested by a previous report (Zhou et al., 1995). Our result that TGF-β1, -β2 weakly inhibit corneal epithelial cell proliferation promoted by EGF is somewhat different from that of Mishima et al. (1992), whose results showed that TGF-β dose-dependently inhibits corneal epithelial cell proliferation promoted by EGF. One possible explanation for this discrepancy is the different cell conditions and}or different culture media used ; that used in our study, with very low Ca#+ concentration, is specifically designed for rabbit corneal epithelial cell proliferation. The proliferative activity of the corneal epithelial cells in our study might therefore be greater than in that of Mishima et al., leading to different results. A recent study by our laboratory has shown both the protein and mRNA of latent-TGF-β2 to be present in human corneal epithelial cells (Nishida et al., 1995), and that latent TGF-β2 is predominantly present in human tear fluid (Kokawa et al., 1996), which may be of great relevance to our result. It has been reported that plasmin, a serine protease that which is excessively generated at the leading epithelial edge by uPA (Hayashi et al., 1991), can activate latent TGF-β in an in vitro co-culture system of endothelial cells and pericytes (Lyon et al., 1990b ; Sato and Rifkin, 1989 ; Odekon, Blasi and Rifkin, 1994), and may be important for in vivo activation of latent TGFβ in corneal epithelial wound healing. It is possible that latent TGF-β1 or -β2 may be activated at the leading epithelial edge and antagonize the proliferative effect of EGF, KGF, and HGF. In vivo animal studies will be needed to determine the distribution and regulation of TGF-β isoforms during corneal epithelial wound healing, and to elucidate the in vivo activation mechanism of latent TGF-βs on the ocular surface. Acknowledgements This work was supported in part by a research grant (07771557) from the Ministry of Education, Culture and Science of Japan. This work was presented in part at the Annual Meeting of the Association for Research in Vision and Ophthalmology, 1995, in Fort Lauderdale, Florida, U.S.A.
395
References Frati, L., Daniele, S., Delogu, A. and Covelli, I. (1972). Selective binding of the epidermal growth factor and its specific effects on the epithelial cells of the cornea. Exp. Eye Res. 14, 135–41. Green, M. R., Basketter, D. A., Couchman, J. R. and Rees, D. A. (1983). Distribution and number of epidermal growth factor receptors in skin is related to epithelial cell growth. Dev. Biol. 100, 506–12. Hayashi, K., Frangieh, G., Wolf, G. and Kenyon, K. (1989). Expression of transforming growth factor-β in wound healing of vitamin A-deficient rat corneas. Invest. Ophthalmol. Vis. Sci. 30, 239–47. Hayashi, K., Berman, M., Smith, D., El-Ghatit, A., Pease, S. and Kenyon, K. R. (1991). Pathogenesis of corneal epithelial defects : role of plasminogen activator. Curr. Eye Res. 10, 381–98. Hongo, M., Itoi, M., Yamaguchi, N. and Imanishi, J. (1992). Distribution of epidermal growth factor (EGF) receptors in rabbit corneal epithelial cells, keratocytes and endothelial cells, and the changes induced by transforming growth factor-β1. Exp. Eye Res. 54, 9–16. Kitazawa, T., Kinoshita, S., Fujita, K., Araki, K., Watanabe, H., Ohashi, Y. and Manabe, R. (1990). The mechanism of accelerated corneal epithelial healing by human epidermal growth factor. Invest. Ophthalmol. Vis. Sci. 31, 1773–8. Kokawa, N., Sotozono, C., Nishida, K. and Kinoshita, S. (1996). High total TGF-β2 levels in normal human tears. Curr. Eye Res. 15, 341–3. Li, Q., Weng, J., Mohan, R. R., Bennett, G. L., Schwall, R., Wang, Z-F., Tabor, K., Kim, J., Hargrave, S., Cuevas, K. H. and Wilson, S. E. (1996). Hepatocyte growth factor and hepatocyte growth factor receptor in the lacrimal gland, tears, and cornea. Invest. Ophthalmol. Vis. Sci. 37, 727–39. Lyon, R. M. and Moses, H. L. (1990a). Transforming growth factor and regulation of cell proliferation. Eur. J. Biochem. 187, 467–73. Lyon, R. M., Gentry, L. E., Purchio, A. F. and Moses, H. L. (1990b). Mechanism of activation of latent recombinant transforming growth factor β1 by plasmin. J. Cell Biol. 110, 1361–7. Mishima, H., Nakamura, M., Murakami, J., Nishida, T. and Otori, T. (1992). Transforming growth factor-β modulates effect of epidermal growth factor on corneal epithelial cells. Curr. Eye Res. 11, 691–6. Nishida, K., Kinoshita, S., Yokoi, N., Kaneda, M., Hashimoto, K. and Yamamoto, S. (1994). Immunohistochemical localization of transforming growth factor-β1, -β2, and -β3 latency-associated peptide in human cornea. Invest. Ophthalmol. Vis. Sci. 35, 3289–94. Nishida, K., Sotozono, C., Adachi, W., Yamamoto, S., Yokoi, N. and Kinoshita, S. (1995). Transforming growth factor-β1, -β2 and -β3 mRNA expression in human cornea. Curr. Eye Res. 14, 235–41. Odekon, L. E., Blasi, F. and Rifkin, D. B. (1994). Requirement for receptor-bound urokinase in plasmin-dependent cellular conversion of latent TGF-beta to TGF-beta. J. Cell. Physiol. 158, 398–407. Ohashi, Y., Motokura, M., Kinoshita, Y., Mano, T., Watanabe, H., Kinoshita, S., Manabe, R., Oshiden, K. and Yanaihara, C. (1989). Presence of epidermal growth factor in human tears. Invest. Ophthalmol. Vis. Sci. 30, 1879–82. Roberts, A. B. and Sporn, M. B. (1990). The transforming growth factor-betas. In Peptide growth factors and their receptors, handbook of experimentary pharmacology (Eds
396
Sporn, M. B. and Roberts, A. B.). Pp. 419–72. SpringerVerlag : Heidelberg. Sankar, S., Mahooti-Brooks, N., Centrella, M., McCarthy, T. L. and Madri, J. A. (1995). Expression of transforming growth factor type III receptor in vascular endothelial cells increases their responsiveness to transforming growth factor β2. J. Biol. Chem. 270, 13567–72. Sato, Y. and Rifkin, D. B. (1989). Inhibition of endothelial cell movement by pericytes and smooth muscle cells : activation of a latent transforming growth factor-β1like molecule by plasmin during co-culture. J. Cell Biol. 109, 309–15. Sotozono, C., Kinoshita, S., Kita, M. and Imanishi, J. (1994). Paracrine role of keratinocyte growth factor in rabbit corneal epithelial cell growth. Exp. Eye Res. 59, 385–92. Sporn, M. B. and Roberts, A. B. (1992). Transforming growth factor-β : recent progress and new challenges. J. Cell. Biol. 119, 1017–21. Wilson, S. E., He, Y-G. and Lloyd, S. A. (1992). EGF, EGF receptor, basic FGF, TGF beta-1, and IL-1 alpha mRNA in human corneal epithelial cells and stromal fibroblasts. Invest. Ophthalmol. Vis. Sci. 33, 1756–65. Wilson, S. E., Walker, J. W., Chwang, E. L. and He, Y-G. (1993). Hepatocyte growth factor, keratinocyte growth factor, their receptors, fibroblast growth factor receptor-
Y. H O N M A E T A L.
2, and the cells of the cornea. Invest. Ophthalmol. Vis. Sci. 34, 2544–61. Wilson, S. E., He, Y-G., Weng, J., Zieske, J. D., Jester, J. V. and Schultz, G. S. (1994). Effect of epidermal growth factor, hepatocyte growth factor, and keratinocyte growth factor, on proliferation, motility and differentiation of human corneal epithelial cells. Exp. Eye Res. 59, 665–78. Woodward, T. L., Dumont, N., O’Connor-McCort, M., Turner, J. D. and Philip, A. (1995). Characterization of transforming growth factor-β growth regulatory effects and receptors on bovine mammary cells. J. Cell. Physiol. 165, 339–48. Yamasaki, S., Onishi, E., Enami, K., Natori, K., Kohase, M., Sakamoto, H., Tanouchi, M. and Hayashi, H. (1986). Proposal of standardized methods and reference for assaying recombinant human tumor necrosis factor. Japan., J. Med. Sci. Biol. 39, 105–18. Zhou, G-H. K., Sechrist, G. L., Periyasamy, S., Brattain, M. G. and Mulder, K. M. (1995). Transforming growth factor β isoform-specific differences in interactions with type I and II transforming growth factor β receptors. Cancer Res. 55, 2056–62. Zieske, J. D. and Wasson, M. (1993). Regional variation in distribution of EGF receptor in developing and adult corneal epithelium. J. Cell. Sci. 106, 145–52.