Mitogenic and Antiapoptotic Effects of Various Growth Factors on Human Corneal Fibroblasts

Abstract

purpose. To investigate both the effects of various growth factors on the proliferation of human corneal fibroblasts and the abilities of these factors to protect the cells from apoptosis.

methods. Cultured human corneal fibroblasts were incubated separately with 11 different growth factors whose receptors are expressed by these cells. Cell proliferation was evaluated by measurement of [³H]thymidine incorporation. The activation of the protein kinase Akt, which plays an important role in antiapoptotic signaling, was assessed by immunoblot analysis with antibodies specific for a phosphorylated form of the enzyme. Apoptosis was quantitated by the TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay.

results. Of the 11 growth factors examined, platelet-derived growth factor, insulin, insulin-like growth factors-1 and -2, and epidermal growth factor, each stimulated the proliferation of corneal fibroblasts, induced the activation of Akt in these cells, and protected them from apoptosis induced by sodium nitroprusside (SNP). Basic fibroblast growth factor, keratinocyte growth factor, nerve growth factor, and hepatocyte growth factor stimulated cell proliferation but did not induce Akt activation or protect the cells from SNP-induced apoptosis. Transforming growth factor-β1 and -β2 did not stimulate proliferation and had no effect on Akt activity or on SNP-induced apoptosis.

conclusions. In terms of their modulatory effects on the proliferation and apoptosis of human corneal fibroblasts, the 11 growth factors examined can be classified into three groups. These growth factors may both contribute to maintenance of the cornea and coordinate the proliferative and apoptotic responses of corneal fibroblasts during corneal wound healing.

The balance between cell proliferation and cell death is important for maintenance of the integrity of the cornea. An early event in the response to injury of the corneal epithelium, including that resulting from refractive surgery or corneal incision, is programmed cell death, or apoptosis, of corneal fibroblasts. ^{1 2 3 4 5 6 7} The subsequent wound-healing process includes the proliferation and migration of corneal fibroblasts and the infiltration of inflammatory cells, ^{8 9} events that are modulated by various cytokines and growth factors. Characterization of the mechanisms by which growth factors regulate the functions of corneal fibroblasts is thus important for an understanding of both the physiology and pathophysiology of the cornea. 

Corneal fibroblasts produce various growth factors and express the receptors for such factors. ¹⁰ These cells have thus been shown to express, at the mRNA or protein level or both, the receptors for platelet-derived growth factor (PDGF), ^{10 11} transforming growth factor (TGF), ^{10 12} basic fibroblast growth factor (bFGF), ^{10 13} keratinocyte growth factor (KGF), ^{10 13} nerve growth factor (NGF), ¹⁴ hepatocyte growth factor (HGF), ^{10 13} insulin, ¹⁵ insulin-like growth factor (IGF)-1, ¹⁰ IGF-2, ¹⁰ and epidermal growth factor (EGF). ^{10 16} Both KGF ^{10 13 17} and HGF ^{10 13} are produced by corneal fibroblasts; PDGF is produced by corneal epithelial cells ^{10 11} ; and TGF-β1, ^{10 12} TGF-β2, ^{10 12} bFGF, ¹⁰ NGF, ¹⁴ and IGF-1 ¹⁰ are produced by both corneal fibroblasts and epithelial cells. In addition, the expression of KGF, HGF, and EGF increases in the lacrimal gland after corneal epithelial injury. ^{18 19} Thus, these various endogenous growth factors may regulate the functions of corneal fibroblasts, including proliferation and apoptosis, by autocrine or paracrine mechanisms. 

Certain growth factors inhibit apoptosis and promote cell survival by activating a signaling pathway mediated by phosphatidylinositol (PI) 3-kinase and Akt. ^{20 21} The activation of PI 3-kinase results in the generation of 3′-phosphorylated phosphoinositides, which, in turn, results in the activation of several protein kinases, including Akt. ²² The activation of Akt requires its phosphorylation on residues threonine-308 and serine-473. Activated Akt probably inhibits apoptosis by phosphorylating components of the apoptotic machinery. To clarify the mechanisms by which growth factors modulate the functions of corneal fibroblasts, we examined both the effects of various such factors on the proliferation of cultured human corneal fibroblasts and the abilities of these factors to trigger antiapoptotic signaling. 

Methods

Materials

Human recombinant IGF-1 and -2 were obtained from Becton Dickinson (Franklin Lakes, NJ); TGF-β1, TGF-β2, KGF, NGF, HGF, and EGF from Genzyme (Cambridge, MA); PDGF-BB from Peprotech (Rocky Hill, NJ); and bFGF from R&D Systems (Minneapolis, MN). Rabbit polyclonal antibodies to Akt and phospho-Akt (phosphoserine-473) as well as LY294002 were obtained from Cell Signaling (Beverly, MA), horseradish peroxidase-conjugated donkey antibodies to rabbit immunoglobulin G from Promega (Madison, WI), human recombinant insulin and sodium nitroprusside (SNP) from Sigma (St. Louis, MO), fetal bovine serum (FBS) from GibcoBRL (Grand Island, NY), and [methyl-³H]thymidine (specific radioactivity, 3.2 TBq/mmol) from Amersham Pharmacia Biotech (Little Chalfont, UK). 

Cell Culture

Human corneas were obtained from Mid-America Transplant Service (St. Louis, MO), Northwest Lions Eye Bank (Seattle, WA), and the Eye Bank of Wisconsin (Madison, WI). The use of human tissue was in accordance with the Declaration of Helsinki. The donors were white males and females ranging in age from 4 to 65 years. Corneal fibroblasts were prepared from the tissue remaining after corneal transplantation surgery and were cultured as described previously. ²³ The cells prepared from each cornea were maintained separately in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated FBS until they had achieved ∼90% confluence in 90-mm culture dishes. Cells in the third to seventh passages were used for the experiments described in the present study. The purity of the cell cultures was assessed on the basis of both the distinctive morphology of human corneal fibroblasts and their reactivity with antibodies to vimentin in immunofluorescence analysis. All cells were positive for vimentin and negative for cytokeratin, suggesting the absence of contamination by epithelial cells. All data presented were obtained with the fibroblasts derived from one cornea, but similar results were obtained with cells derived from two additional corneas. 

Measurement of [³H]Thymidine Incorporation

Corneal fibroblasts (1 × 10⁵ cells per well) were cultured in 24-well plates for 24 hours in DMEM containing 0.1% bovine serum albumin (BSA), after which the culture medium was further supplemented with growth factors. The concentrations of growth factors tested were (in nanomolar) 243 PDGF-BB, 8.25 TGF-β1, 8.5 TGF-β2, 80 bFGF, 76 KGF, 224 NGF, 560 HGF, 203 insulin, 305 IGF-1, 53.8 IGF-2, and 242 EGF. Control cultures were incubated in the absence of growth factor. After incubation of the cells for 20 hours, [³H]thymidine (1 kBq per well) was added to the medium, and the cells were cultured for an additional 1 hour. The culture medium was then removed, 0.5 mL of 5% trichloroacetic acid was added to the cells, and the plate was incubated for 20 minutes at 4°C. The cells were washed with 0.5 mL H₂O and then incubated with 0.2 mL of 1 M NaOH for 30 minutes at 37°C. The contents of each well were neutralized by the addition of 0.22 mL of 1 M HCl, and the associated radioactivity was measured with a scintillation counter (Aloka, Tokyo, Japan). 

Immunoblot Analysis of Akt and Phospho-Akt

Corneal fibroblasts (2 × 10⁶ cells) were cultured in 35-mm dishes first for 48 hours in DMEM supplemented with 0.5% FBS and then for an additional 2 hours in serum-free medium. They were then incubated for 5 minutes at 37°C with fresh medium containing growth factors at the concentrations indicated earlier. The cells were washed with ice-cold phosphate-buffered saline (0.8% NaCl, 0.29% Na₂HPO₄ · 12H₂O, 0.02% KCl, 0.02% KH₂PO₄ [pH 7.4]) and then lysed in 100 μL of a solution containing 2% SDS, 150 mM NaCl, 2 mM NaF, 2 mM Na₃VO₄, 5 mM EDTA, and 20 mM Tris-HCl (pH 7.5). The protein concentration of the lysate was determined by the method of Lowry et al., ²⁴ and portions (30 μg protein per lane) were fractionated by SDS-polyacrylamide gel electrophoresis. The separated proteins were transferred to a nitrocellulose membrane, nonspecific binding sites on which were then blocked by incubation with 5% nonfat dried milk in Tris-buffered saline (50 mM Tris-HCl [pH 7.5]) and 150 mM NaCl) containing 0.1% Tween 20. The membrane was then incubated with antibodies to Akt or to phospho-Akt (dilution, 1:1000) in Tris-buffered saline containing 5% BSA and 0.1% Tween 20, after which immune complexes were detected with horseradish peroxidase-conjugated secondary antibodies and enhanced chemiluminescence. The amounts of Akt and phospho-Akt were quantitated by densitometric scanning (Lane and Spot Analyzer; ATTO, Tokyo, Japan). The extent of Akt phosphorylation was determined as the amount of phospho-Akt divided by that of Akt, and was expressed as a percentage of the value obtained for EGF-treated cells. 

TUNEL Analysis of SNP-Induced Apoptosis

Corneal fibroblasts (1.25 × 10⁴ cells per well) were cultured in eight-well chamber slides for 24 hours with 0.4 mL DMEM supplemented with 0.5% FBS. After the addition of various growth factors, the cells were cultured for an additional 48 hours and then exposed to 3 mM SNP for 24 hours. The cells were fixed with 4% formaldehyde in phosphate-buffered saline for 25 minutes, washed with phosphate-buffered saline, permeabilized with 0.2% Triton X-100, and then stained with both TdT-mediated dUTP-biotin nick-end labeling (TUNEL; Promega) and propidium iodide (Sigma). The cells were examined by fluorescence microscopy, and the percentage of nonapoptotic cells was calculated as the average of values from duplicate cultures, in each of which cells were counted in three microscope fields. 

Statistical Analysis

Data are presented as the mean ± SE and were analyzed by the Dunnett multiple comparison test with the use of statistical analysis software (StatView for Windows, ver. 5.0; SAS Institute, Cary, NC). P < 0.05 was considered statistically significant. 

Results

Effects of Growth Factors on Proliferation of Corneal Fibroblasts

To investigate the effects of various growth factors on the proliferation of human corneal fibroblasts, we measured the incorporation of [³H]thymidine into cells incubated in the absence or presence of PDGF-BB, TGF-β1, TGF-β2, bFGF, KGF, NGF, HGF, insulin, IGF-1, IGF-2, or EGF. The concentrations of the growth factors chosen were 10 times the reported dissociation constant (K _d) values for the interaction with their receptors. These concentrations were similar to or higher than those used in previous studies with corneal fibroblasts or other cells. ^{25 26 27 28 29 30 31 32 33 34} Incubation of the cells with PDGF-BB, bFGF, NGF, HGF, insulin, IGF-1, IGF-2, or EGF resulted in significant increases in the extent of [³H]thymidine incorporation, compared with that apparent in control cells (Fig. 1) . Although KGF also induced a small increase in [³H]thymidine incorporation, this effect was not statistically significant; this growth factor did, however, induce a significant increase in [³H]thymidine incorporation at a concentration of 760 nM (data not shown), which is 100 times the corresponding K _d value. TGF-β1 and TGF-β2 each had no significant effect on the extent of [³H]thymidine incorporation (Fig. 1) . 

Effects of Growth Factors on Akt Activation in Corneal Fibroblasts

We next examined whether the various growth factors induce the activation of Akt, which inhibits apoptosis and promotes cell survival. Immunoblot analysis revealed that the abundance of Akt was not affected by exposure of corneal fibroblasts to the growth factors (Fig. 2A) . The activation of Akt was assessed by immunoblot analysis with antibodies specific for Akt phosphorylated on serine-473 (Fig. 2A) . Quantitative analysis revealed that PDGF-BB, insulin, IGF-1, IGF-2, and EGF each induced significant phosphorylation of Akt, whereas TGF-β1, TGF-β2, bFGF, KGF, NGF, or HGF had no effect on Akt phosphorylation (Fig. 2B) . 

To determine whether the growth factor-induced activation of Akt in corneal fibroblasts was mediated by PI 3-kinase, we examined the effects of the PI 3-kinase inhibitor LY294002. LY294002 inhibited almost completely the phosphorylation of Akt induced by PDGF-BB, insulin, IGF-1, IGF-2, or EGF (Fig. 3) , indicating that the activation of Akt by these growth factors is mediated by PI 3-kinase. 

Effects of Growth Factors on Apoptosis in Corneal Fibroblasts

To determine the relation between the growth factor-induced activation of Akt in corneal fibroblasts and protection of these cells from apoptosis, we examined the effects of growth factors on nitric oxide (NO)-induced apoptosis. Exposure of corneal fibroblasts to the NO donor SNP for 24 hours resulted in the apoptotic death of most cells, as revealed by their TUNEL positivity (Figs. 4A 4B) . Propidium iodide staining also revealed that the TUNEL-positive cells contained condensed chromatin (data not shown), a characteristic of apoptotic cells. Pretreatment of the cells with PDGF-BB, insulin, IGF-1, or IGF-2 resulted in the survival of more than 90% of the cells after their exposure to SNP (Figs. 4C 4D) ; pretreatment with EGF resulted in the survival of more than 60% of the cells. Neither TGF-β1, TGF-β2, bFGF, KGF, NGF, nor HGF protected corneal fibroblasts from SNP-induced apoptosis. 

Discussion

Apoptosis in corneal fibroblasts is the first detectable response of the corneal stroma to injury. We have now shown that PDGF-BB, insulin, IGF-1, IGF-2, and EGF induce the activation of Akt in human corneal fibroblasts in a manner dependent on PI 3-kinase, an effect that is thought to play a pivotal role in the inhibition of apoptosis and the promotion of cell survival. Indeed, these growth factors significantly inhibited SNP-induced apoptosis in corneal fibroblasts. The antiapoptotic action of these growth factors may thus contribute to wound healing in the cornea. 

Our observations indicate that the 11 growth factors tested, for all of which the receptors are expressed by corneal fibroblasts, can be classified into three groups according to their effects on proliferation and apoptosis in these cells: PDGF-BB, insulin, IGF-1, IGF-2, and EGF stimulate cell proliferation and inhibit apoptosis; bFGF, KGF, NGF, and HGF stimulate proliferation but do not inhibit apoptosis; and TGF-β1 and TGF-β2 do not stimulate proliferation and have no protective effect against apoptosis. During corneal wound healing, the proliferation of corneal fibroblasts occurs after the initial apoptotic response. ^{6 7 8 9} Our data therefore suggest that combinations of these growth factors may orchestrate the apoptotic and proliferative responses of corneal fibroblasts during corneal wound healing. 

The activation of PI 3-kinase and Akt by growth factors has not been demonstrated in the cornea. We have now shown that PDGF-BB, insulin, IGF-1, IGF-2, and EGF activate Akt through PI 3-kinase in corneal fibroblasts, resulting in inhibition of apoptosis. PDGF, bFGF, insulin, and EGF have previously been shown to activate Akt in Rat-1 fibroblasts and in NIH 3T3 cells that overexpress the insulin receptor. ²⁰ Similarly, NGF and HGF induce the activation of Akt in neuronal cells and NIH 3T3 cells, respectively. ^{35 36 37} Thus, although bFGF, NGF, and HGF do not activate Akt in human corneal fibroblasts, they do so in other cell types. The absence of effect of the latter growth factors on the activity of Akt in corneal fibroblasts is not due to the absence of functional receptors, given that all three growth factors stimulate the proliferation of these cells. A dissociation of the stimulation of proliferation from the promotion of cell survival by a particular growth factor has been observed in other cell types. In Rat-1 fibroblasts, for example, IGF-1 and PDGF each markedly promote cell survival but exhibit only a small mitogenic effect. ³⁸  

Consistent with our present results, PDGF and NGF have been shown to stimulate the proliferation of corneal fibroblasts. ¹⁴ In addition, Wilson et al. ¹³ showed that the proliferation of corneal fibroblasts was stimulated by bFGF and EGF but not by HGF and KGF; in our study, all four of these growth factors stimulated cell proliferation, although high concentrations of KGF were required for this effect. Although the reason for this discrepancy is unclear, the inclusion by Wilson et al. of insulin in their proliferation assays may have masked the effects of HGF and KGF. 

We have shown that the activation of Akt through PI 3-kinase in corneal fibroblasts parallels the inhibition of SNP-induced apoptosis by growth factors. The mechanism by which Akt protects corneal fibroblasts from apoptosis remains to be determined. In other cell types, several proteins have been shown to be targeted by Akt to achieve its antiapoptotic function, including the proapoptotic Bcl-2 homologue BAD, ³⁹ the proapoptotic cysteine protease caspase-9, ⁴⁰ and forkhead transcription factors such as FKHR1. ^{41 42}  

In conclusion, we have shown that growth factors can be categorized into three groups according to their effect on apoptosis and the proliferation and human corneal fibroblasts. Further studies are necessary to determine the mechanisms by which each growth factor contributes to maintenance of the cornea and by which together they coordinate the apoptotic and proliferative responses of corneal fibroblasts during corneal wound healing. 

 Supported by grants-in-aid from the Ministry of Education, Science, Sports, and Culture of Japan.

 Submitted for publication October 9, 2001; revised January 8, 2002; accepted March 12, 2002.

 Commercial relationships policy: N.

 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked “advertisement” in accordance with 18 U.S.C. §1734 solely to indicate this fact.

 

Figure 1.

 

Effects of growth factors on [³H]thymidine incorporation into human corneal fibroblasts. The cells were cultured for 20 hours in DMEM containing 0.1% BSA in the absence or presence of the indicated growth factors at the concentrations provided in the Methods section. They were then incubated further for 1 hours after the addition of [³H]thymidine. The incorporation of [³H]thymidine into cellular DNA was then determined. Data are means ± SE of values from four experiments. *P < 0.05, **P < 0.01, ***P < 0.001 compared with the value for cells incubated in the absence of growth factor.

Figure 2.

 

Effects of growth factors on the activation of Akt in human corneal fibroblasts. (A) Cells were incubated for 5 minutes with the indicated growth factors at the concentrations provided in the Methods section. Cell lysates were then prepared and subjected to immunoblot analysis with antibodies to Akt or to phospho-Akt (P-Akt), as indicated. (B) The extent of Akt phosphorylation was determined by densitometric scanning of immunoblots similar to that shown in (A). Data are expressed as a percentage of the level in EGF-treated cells and are means ± SE of values from four experiments. ***P < 0.001 compared with the level in cells incubated in the absence of growth factor.

Figure 3.

 

Effects of the PI 3-kinase inhibitor LY294002 on growth factor-induced activation of Akt in human corneal fibroblasts. Cells were incubated first for 60 minutes in the absence or presence of 50 μM LY294002 and then for 5 minutes in the additional presence of the indicated growth factors. Cell lysates were then subjected to immunoblot analysis with antibodies to Akt or to phospho-Akt.

Figure 4.

 

Effects of growth factors on SNP-induced apoptosis in human corneal fibroblasts. (A–C) Cells were incubated first for 48 hours in the absence (A, B) or presence (C) of IGF-1 and then for 24 hours in the additional absence (A) or presence (B, C) of 3 mM SNP. They were then subjected to staining both by the TUNEL technique (green) and with propidium iodide (red). Scale bar, 100 μm. (D) Cells were incubated first for 48 hours in the absence or presence of the indicated growth factors and then for 24 hours in the additional presence of 3 mM SNP. The number of TUNEL-negative (nonapoptotic) cells was then determined as a percentage of the total cell number. Data are means ± SE of data from six experiments. ***P < 0.001 compared with the level in cells incubated in the absence of growth factor.