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(Investigative Ophthalmology and Visual Science. 2007;48:27-33.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.06-0826
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Internalization Is Essential for the Antiapoptotic Effects of Exogenous Thymosin ß-4 on Human Corneal Epithelial Cells

Jennifer Hui-Chun Ho,1,2 Chiao-Hui Chuang,3 Chih-Yuan Ho,3 Yu-Ru Vernon Shih,3 Oscar Kuang-Sheng Lee,*,1,3 and Yeu Su*,1

1From the Institute of Biopharmaceutical Science, National Yang-Ming University, Taipei, Taiwan; the 2Department of Ophthalmology, Buddhist Tzu Chi General Hospital-Taipei, Taiwan; and the 3Department of Medical Research and Education, Taipei Veterans General Hospital and School of Medicine, National Yang-Ming University, Taipei, Taiwan.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. Exogenous thymosin ß-4 (Tß4) has been shown to inhibit the apoptosis in nontransformed human corneal epithelial cells that is triggered by ethanol. The purpose of this study is to examine whether exogenous Tß4 protects SV40-immortalized human corneal epithelial T (HCE-T) cells against the toxic effects of Fas ligand (FasL) and hydrogen peroxide (H2O2) and to elucidate its mechanism of action.

METHODS. HCE-T cells were incubated without or with the recombinant histidine-tagged Tß4 produced by Escherichia coli before the addition of FasL or H2O2. Cell viability was determined by MTT or MTS assay, and activation of caspase-8, -9, and -3 was examined by colorimetric and fluorescent substrate cleavage assays. The internalization of exogenous Tß4 in HCE-T cells was analyzed by immunofluorescence staining. Cytochalasin D, an actin depolymerization agent, was added to examine whether the actin cytoskeleton is involved in Tß4 entry and whether the internalization of this peptide is crucial for its cytoprotection.

RESULTS. The death of HCE-T cells induced by both FasL and H2O2 was dramatically reduced by the recombinant Tß4 pretreatment. Moreover, FasL-mediated activation of caspases-8 and -3 as well as H2O2-triggered stimulation of caspases-9 and -3 in these cells was abolished by preincubating them with the exogenous Tß4. Of note, internalization of this G-actin-sequestering peptide into HCE-T cells was found to be essential in cell death prevention, in that disruption of the cellular entry of Tß4 by cytochalasin D abrogated its cytoprotective effects.

CONCLUSIONS. This is the first report to demonstrate that the internalization of exogenous Tß4 is essential for its antiapoptotic activity in human corneal epithelial cells.

Apoptosis is the pathomechanism of many corneal diseases, such as dry eye syndrome,1 2 ultraviolet irradiation,3 infection,4 Fuchs’ dystrophy, and pseudophakic bullous keratopathy.5 Signaling through death receptors and/or mitochondrial activation are the major pathways responsible for apoptosis.6 7 Fas (CD95) is a transmembrane death receptor that could initiate the apoptosis of cells bearing this surface molecule on engaging with Fas ligand (FasL).8 Not only were Fas and FasL expressed but also a Fas-mediated apoptosis was detected in the corneal epithelial cells.9 Moreover, the involvement of FasL/Fas signaling has been proposed in the apoptosis of corneal cells resulting from uniocular anterior chamber (AC) inoculation of HSV-1.10 Significantly higher expression levels of Fas have been found in the pathogenic cornea and conjunctiva in comparison with the normal ones.11 In addition, FasL-induced apoptosis has been shown to play a major role in corneal graft rejection,12 13 Sjögren’s syndrome,2 and nonspecific chronic conjunctivitis. In contrast, apoptosis of corneal epithelial cells triggered by hypoxia14 and low-dose UVB irradiation15 has been demonstrated to be mediated by the mitochondrial pathway due mainly to an increase in intracellular reactive oxygen species (ROS).

Thymosin ß-4 (Tß4), a ubiquitous, abundant intracellular peptide consisting of 43 amino acids, regulates the actin monomer pool by sequestering G-actin.16 17 It has pleiotropic effects on different types of cells, such as increasing the rate of attachment and spread of endothelial cells on matrix components and stimulating the migration of human umbilical vein endothelial cells,18 promoting cardiomyocyte migration and cardiac repair through activating integrin-linked kinase19 20 and promoting matrix metalloproteinase expression during dermal wound repair.21 Upregulation of Tß4 increases the invasive ability of human colorectal carcinoma cells by promoting the disruption of cell-cell adhesion and a consequential activation of ß-catenin signaling,22 23 and overexpression of Tß4 renders SW480 colon carcinoma cells more resistant to apoptosis triggered by FasL and two topoisomerase II inhibitors via downregulating Fas and upregulating survivin expression.24 By contrast, the functions of exogenous Tß4 appear to be very different from those of the endogenous peptide. For example, this peptide has been reported to increase hair growth by stimulating the migration of hair follicle stem cells,25 to induce plasminogen activator inhibitor (PAI)-1 secretion by endothelial cells, and to modulate the fibrinolytic potential of endothelial cells.26 27 On the contrary, Tß4 and its N-terminal tetrapeptide, AcSDKP, have been shown to inhibit the proliferation of hematopoietic progenitor cells.28 Anti-inflammatory effects of Tß4 and its sulfoxide adduct have also been demonstrated.29 In corneal epithelial cells, wound healing promotion, inflammation reduction,30 31 and inhibition of ethanol-induced32 or benzalkonium chloride-triggered apoptosis33 by exogenous Tß4 have been reported. However, the precise mechanism of the protective effect of this G-actin-sequestering peptide against various injuries in corneal epithelial cells is not fully understood.

The purpose of this study was to examine the antiapoptotic effect of exogenous Tß4 on immortalized human corneal epithelial T (HCE-T) cells and subsequently to determine its underlying mechanism. In our study, exogenous Tß4 not only prevented the cell death induced by FasL and H2O2 but also inhibited the activation of several key caspases. Furthermore, internalization of exogenous Tß4 in HCE-T cells (demonstrated by immunofluorescence staining), to our surprise, was essential for its protection against the cytotoxicity of both FasL and H2O2.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Cell Culture
The SV-40 immortalized human corneal epithelial HCE-T cells,34 kindly provided by Kaoru Araki-Sasaki (Riken BioResource Center, Ibaraki, Japan), were maintained in DMEM/HamF12 (1:1) supplemented with 5% fetal bovine serum (HyClone, Logan, UT), 5 µg/mL insulin, 0.1 µg/mL cholera toxin (Sigma-Aldrich, St. Louis, MO), 10 ng/mL recombinant human epidermal growth factor (hEGF; BD Biosciences, San Jose, CA), and 0.5% dimethyl sulfoxide (DMSO) in 95% air and 5% CO2 at 37°C.

Preparation of Recombinant Tß4
To generate the histidine-tagged Tß4 fusion protein His6-Tß4, we inserted the mouse Tß4 coding region obtained by PCR amplification into the BamHI/EcoRI sites of the pET6H vector (kindly provided by Jin Jer Lin of the National Yang-Ming University, Taipei, Taiwan). The resultant pET6H-Tß4 plasmid was then transformed into Escherichia coli BL21 (DE3), and His6-Tß4 was prepared as follows. Bacteria were grown at 37°C to an optical density OD600 of 0.5 before 0.25 µM IPTG (isopropyl-ß-D-thiogalactopyranoside) was added to induce protein expression. After incubation at 30°C for 4 hours, harvested bacteria were suspended in 20 mL ice-cold lysis buffer (500 mM NaCl, 20 mM Tris-HCl [pH 80], 5 mM imidazole, and protease inhibitor cocktail [1:200; Calbiochem, Darmstadt, Germany]) and then disrupted by French press three times at 1000 psi, and the debris was removed by centrifugation at 35,000g for 30 minutes. Crude lysate was incubated with 200 µL Ni-NTA agarose (Qiagen, Hilden, Germany) at 4°C overnight with gentle agitation before being washed several times in buffer (500 mM NaCl, 20 mM Tris-HCl [pH 8.0], 10 mM imidazole). Elution was performed in a similar buffer with 30 mM imidazole, and the eluents were dialyzed against PBS at 4°C overnight, to remove the imidazole. The protein solution was sterilized by filtering through a 0.22-µm filter (Millipore, Billerica, MA), and the endotoxin contaminants were removed (Detoxi-Gel Endotoxin Removing Gel; Pierce, Rockford, IL), according to the manufacturer’s protocol. The concentration of His6-Tß4 was determined by bicinchoninic acid (BCA) protein assay (Pierce) and purity was assessed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Preparation of the Anti-Tß4 Polyclonal Antibody
A synthetic peptide spanning the N-terminal 14 amino acids of Tß4 (Kelowna International Scientific Inc., Taipei, Taiwan) was conjugated to KLH (keyhole limpet hemocyanin; Sigma-Aldrich) by EDC (1-ethyl-3-(3-dimethylamino-propyl) carbodiimide; Sigma-Aldrich), by vigorous agitation at room temperature for 2 hours. Two hundred fifty milligrams of peptide-KLH conjugates after being emulsified with the Freund’s complete adjuvant (Sigma-Aldrich) were injected subcutaneously into the back of a New Zealand White rabbit, and 4 weeks later immunization was repeated. Afterward, booster doses were administered by subcutaneous injection of an emulsion made of 125 µg peptide-KLH conjugates and Freund’s incomplete adjuvant (Sigma-Aldrich) every 2 weeks until the anti-Tß4 antibodies were detected in the animals’ sera. The polyclonal anti-Tß4 antibodies were further purified (Hi Trap rProtein A FF; GE Healthcare, Piscataway, NJ), according to the manufacturer’s protocol, with eluents of OD280 greater than 1.5 collected. The animal procedures were performed in accordance with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research.

Western Blot Analysis
Aliquots of 1 µg histidine-tagged and synthetic Tß4 peptides were loaded on a 12% SDS-polyacrylamide gel. After electrophoresis, the gel was soaked in 2.5% glutaraldehyde (in PBS) for 15 minutes at room temperature for cross-linking the small-sized Tß4 molecules to facilitate their membrane retention during blotting, and then washed extensively with PBS. The proteins were subsequently electrotransferred to a PVDF membrane and the resultant blot was blocked in 10% skim milk for 1 hour, followed by incubation with a polyclonal anti-Tß4 antibody (1:1000) at 4°C for 16 to 18 hours and then with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:5000) for 1 hour at room temperature. Detection was then conducted by enhanced chemiluminescence (ECL; NEN Life Science, Boston, MA) according to the manufacturer’s protocol.

MTS and MTT Assays
HCE-T cells were seeded in a 96-well plate (4.0 x 103 cells/well) 1 night before being treated without or with various concentrations (100, 200, and 400 µM) of H2O2 for 24 hours. Culture medium in each well was then replaced by serum-free DMEM (100 µL) containing 20 µL MTS reagent (Promega, Madison, WI), and OD490 was measured by a microplate reader (Bio-Rad, Hercules, CA) after cells were incubated in the dark at 37°C for another 1 to 4 hours. MTT assays were performed primarily as previously described35 after HCE-T cells were treated with the exogenous Tß4 (His6-Tß4, 1 µg/mL) for 2 hours, followed by the treatment of anti-Fas IgM (CH-11, 40 ng/mL) or H2O2 (100 µM) for 24 hours with the continuous presence of Tß4.

Flow Cytometry
HCE-T cells were incubated without or with the His6-Tß4 (1 µg/mL) for 24 hours before being harvested and stained with an FITC-conjugated mouse antibody against human CD95 or a mouse IgG as the isotype control (BD Bioscience, San Jose, CA) on ice for 30 minutes. Flow cytometry was then performed (FACS Calibur; BD Biosciences), and the percentages of cells with Fas expression was calculated (Modfit software; Verity Software House, Topsham, ME).

Immunofluorescence Staining
HCE-T cells were incubated with the His6-Tß4, (1 µg/mL) for 1, 2, and 18 hours before being processed immediately, or for 2 hours before the removal of peptides and processing 24 hours later for immunofluorescence staining. After fixation in 4% formaldehyde for 20 minutes and blocking in 5% milk for 1 hour, the cells were incubated with either an anti-histidine antibody (1:1000; Viogen, Sunnyvale, CA) at room temperature for 1 hour, followed by an FITC-conjugated goat anti-rabbit antibody (1:1000; BD Biosciences) at room temperature for 30 minutes, for detecting His-tag, or first with the anti-Tß4 antibody (1:1000) and they with a rhodamine-conjugated mouse anti-rabbit antibody (1:100; Jackson ImmunoResearch, West Grove, PA), for detecting Tß4. After nuclear staining with 4,6-diamidino-2-phenylindole (DAPI), the samples were assessed with a fluorescence microscope (Leitz, Wetzlar, Germany) and imaging was performed (SPOT RT Imaging System; Diagnostic Instruments, Sterling Heights, MI).

Determination of the Maximum Tolerated Dose of Cytochalasin D
HCE-T cells were treated in various conditions (10 or 20 µM for 30, 45, or 60 minutes) with cytochalasin D (Sigma-Aldrich), and the maximum tolerated dose was defined as the highest compound concentration that did not cause cell detachment (from dishes), cell shrinkage, and nuclear condensation.

Detection of the Activation of the Various Caspases
After being incubated without or with cytochalasin D (20 µM) for 30 minutes, HCE-T cells were gently washed with PBS and then treated with the His6-Tß4, (1 µg/mL) for 2 hours before FasL (CH11, 40 ng/mL) or H2O2 (200 µM) was added and incubation continued for another 20 hours. For examining the in situ activation of caspase-3, a fluorogenic substrate for this protease (OncoImmunin, College Park, MD) was added directly to the cells for 1 hour, and propidium iodide (BD Biosciences) staining was performed during the last 15 minutes of the 1-hour incubation. After being fixed in 4% paraformaldehyde, the cells were examined by fluorescence microscope (Leitz). For analyzing caspase activity by substrate cleavage assays, each 1 x 106 cells (determined by a hemocytometer) were resuspended in 25 µL cold lysis buffer and were then incubated with substrates of caspase-3, -8 and -9 (R&D Systems, Minneapolis, MN), respectively, at 37°C for 1 to 2 hours before the OD405 was measured by a microplate reader (Bio-Rad, Hercules, CA), according to the manufacturer’s protocol.

Statistics
Statistical analysis was performed (Statistical Package for Social Science (SPSS for Windows package release 10.5; SPSS Inc., Chicago, IL) with one-way ANOVA used to analyze statistical differences. P < 0.05 was considered to be statistically significant.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Preparation of the Recombinant Tß4 and a Polyclonal Anti-Tß4 Antibody
To determine the purity of the recombinant histidine-tagged Tß4 (His6-Tß4), We performed SDS-PAGE with the synthetic Tß4 serving as the positive control. As shown in Figure 1A , the His6-Tß4 exhibited a single band with molecular weight similar to that of the synthetic peptide. Western blot analysis was then conducted to assess the usefulness of the polyclonal anti-Tß4 antibody. This antibody indeed recognized both the recombinant and the synthetic peptides (Fig. 1B , left). By contrast, only the His6-Tß4 was detected by the anti-His antibody (Fig. 1B , right).


Figure 1
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  		FIGURE 1. Preparation of the recombinant His-tagged Tß4 peptides and a polyclonal anti-Tß4 antibody. (A) Coomassie blue staining of a synthetic Tß4 (Syn) and a histidine-tagged Tß4 (His) purified from E. coli after they were separated by SDS-PAGE. (B) Western blot analysis of the synthetic and the His-tagged Tß4 peptides using a polyclonal anti-Tß4 antibody (left) or an anti-His antibody (right).

 
Effects of Tß4 against FasL- and H2O2-Induced Injury in HCE-T Cells
To examine the antiapoptotic effect of exogenous Tß4 on HCE-T cells, we first assessed whether these cells could be killed by FasL and H2O2 Surface expression of Fas (CD95) on HCE-T cells was not altered by Tß4 treatment (Fig. 2A versus 2B), and after incubation with FasL (anti-Fas IgM, clone 11), the changes of cell morphology, including cellular shrinkage, vacuolated cytoplasm, and nuclear condensation, suggested that these cells may be apoptotic (Fig. 2E) . In contrast, most of the HCE-T cells looked unaffected when the His6-Tß4 (1 µg/mL) was added before FasL treatment (Fig. 2F) . Both the MTT (for viability) and substrate cleavage (for caspases-3 and -8 activities) assays were then used to analyze the protective effects of exogenous Tß4 on HCE-T cells against FasL. A marked increase of survival (Fig. 2G) and a diminished activation of both caspase-8 (Fig. 2H) and caspase-3 (Fig. 2I) were indeed observed when cells were pretreated with the exogenous Tß4. A dose-dependent killing of HCE-T cells by H2O2 was also found and the protection of exogenous Tß4 against H2O2-triggered death in these cells was demonstrated by both the viability and caspase activity assays as well (data not shown).


Figure 2
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  		FIGURE 2. Exogenous Tß4 protected HCE-T cells against FasL-induced damage. Surface expression of Fas (CD95) in HCE-T cells before (A) and after (B) Tß4 (1 µg/mL) treatment for 24 hours was examined by flow cytometry. HCE-T cells were incubated without (C, E) or with (D, F) exogenous Tß4 (1 µg/mL) for 2 hours before being treated with an anti-Fas IgM (40 ng/mL) for another 18 hours (E, F) Images were obtained by light microscope. Bar, 30 µm. (G) HCE-T cells were treated without or with exogenous Tß4 for 2 hours before FasL injury. Twenty-four hours later, cell viability was determined by MTT assay (G), and intracellular caspase-8 (H) and caspase-3 (I) activities were analyzed by colorimetric assays. Data were analyzed by ANOVA with the Tukey’s post hoc test, and different characters represent different levels of signifigance (P < 0.05) (n = 3).

 
Entry of Exogenous Tß4 into HCE-T Cells
We next asked whether exogenous Tß4 could enter the HCE-T cells, because internalization of this G-actin-binding peptide in other cell types has been reported.19 36 Immunofluorescence staining was performed after cells were incubated with the His6-Tß4 (1 µg/mL) for various lengths of time. As can be seen in Figure 3A , the background FITC signals representing the proteins cross-reacted with anti-His antibody were extremely low, whereas the background rhodamine signals, which represent the endogenous Tß4, were detected mainly in the cytosol (Figs 3B 3D) . Of note, dramatic increases in the intensity of both the FITC (Fig. 3E) and rhodamine (Fig. 3F) signals were detected after the cells were incubated with exogenous Tß4 for 2 hours. Colocalization of the histidine tag and Tß4 signals was also found in the nucleus (Figs. 3G 3H) . Moreover, even 24 hours after the removal of the extracellular peptides, intracellular—in fact, nuclear—retention of Tß4 was observed (Figs. 3K 3L) . Besides HCE-T cells, internalization of Tß4 was also found in bovine corneal endothelial cells and human bone marrow-derived mesenchymal stem cells (data not shown).


Figure 3
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  		FIGURE 3. Exogenous Tß4 entered the HCE-T cells. HCE-T cells were incubated without (A–D) or with (E–H) the exogenous Tß4 (1 µg/mL) for 2 hours before being processed for immunofluorescence staining with anti-His, anti-Tß4, and DAPI. Similar cells were incubated without (I, J) or with (K, L) the exogenous Tß4 for 2 hours and washed with serum-free medium several times before being cultured in a regular medium for another 24 hours. Immunofluorescence staining was then performed Bar, 15 µm.

 
Cytochalasin D and the Internalization of Tß4 in HCE-T Cells
To elucidate the mechanisms of Tß4 internalization in HCE-T cells, we used immunofluorescence staining to examine whether the entry of this peptide was affected by cytochalasin D, a fungal toxin that depolymerizes actin filaments. After being treated with cytochalasin D (20 µM) for 30 minutes, the cells remained attached, but their boundaries became more distinct (Fig. 4B) . In contrast, even though the background FITC signals were increased by cytochalasin D (Figs. 4E 4F) , pretreatment with this agent abolished the internalization of exogenous Tß4 (Figs. 4G 4H 4I 4J) .


Figure 4
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  		FIGURE 4. Cytochalasin D blocked the internalization of Tß4 in HCE-T cells. The morphology of HCE-T cells (and their local enlargements) treated without (A) or with (B) 20 µM cytochalasin D for 30 minutes. HCE-T cells were stained for His-tag, Tß4, and were stained with DAPI after they were incubated without (C, D) or with (E, F) 20 µM of cytochalasin D for 30 minutes. The colocalization of the His and Tß4 signals (G, H) disappeared when the cells were treated with cytochalasin D before the addition of Tß4 (I, J). Bar, 15 µm.

 
Internalization and the Antiapoptotic Effect of Exogenous Tß4
To examine whether the internalization of exogenous Tß4 is necessary for its antiapoptotic effects, in situ caspase-3 activation was analyzed in HCE-T cells in various conditions. Although no caspse-3 activity was detected in the control groups (Figs. 5A 5F) , more than 80% of the FasL- or H2O2-treated cells were caspase-3 positive (Figs. 5B 5G) . As expected, preincubating HCE-T cells with the His6-Tß4 significantly diminished the activation of caspase-3 triggered by both FasL (Fig. 5C) and H2O2 (Fig. 5H) . In contrast, even though no significant elevation of caspase-3 activity was detected in HCE-T cells 20 hours after they were pulsed with cytochalasin D for 30 minutes (Figs. 5D 5I) , such treatment interfered with the protective effects of the exogenous Tß4 (Figs. 5E 5J) . In addition, suppression of the activation of caspases induced by FasL (Figs. 6A 6B) and H2O2 (Fig. 6C) in HCE-T cells by this G-actin-sequestering peptide was also abrogated by cytochalasin D pretreatment.


Figure 5
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  		FIGURE 5. Cytochalasin D blocked the antiapoptotic effect of exogenous Tß4. Immunofluorescence staining for in situ caspase-3 activity of HCE-T cells was performed after the cells were treated without (A–C, F–H) or with (D, E, I, J) cytochalasin D. Positive caspase-3 activity was detected in cells after they were incubated with either FasL (B) or H2O2 (G) for 20 hours. The activity was dramatically reduced by pretreatment with exogenous Tß4 (C, H). Even though no significant elevation of caspase-3 activity (D, I) was found in the cells after a transient (30-minute) incubation with cytochalasin D (20 µM), this treatment diminished the suppressive effects of exogenous Tß4 on the activation of caspase-3 by FasL (E) or H2O2 (J).

 

Figure 6
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  		FIGURE 6. Cytochalasin D reduces the suppressive effect of exogenous Tß4 on caspase activation. HCE-T cells were incubated with cytochalasin D (20 µM, 30 minutes), followed by the exogenous Tß4 (1 µg/mL, 2 hours), and then with FasL or H2O2 (24 hours) The transient cytochalasin D treatment did not affect the baseline caspase-3 and -9 activities (B, C) but slightly increased caspase-8 activity (A). By contrast, the suppressive effects of exogenous Tß4 on caspase activation was diminished by cytochalasin D. Data were analyzed by ANOVA with the Tukey’s post hoc test, and different characters represent different levels of significance (P < 0.05) (n = 3).

 

   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
The corneal epithelial covering on the cornea surface, which serves as the first defense of the eye against exogenous pathogens,37 38 daily UV damage,39 and chemical injury,40 is sensitive to tear film insufficiency,1 2 topical eyedrops,41 42 and contact lens-induced hypoxia.43 Because of their finite lifespan, corneal epithelial cells shed and undergo apoptosis regularly,44 and the latter could be accelerated by various diseases or stress.1 2 3 4 5 10 11 12 13 14 15 Tß4, a G-actin-sequestering peptide, has been shown to promote wound healing, decrease inflammation, and inhibit ethanol- and benzalkonium chloride-induced apoptosis of corneal epithelial cells when administered exogenously,30 31 32 33 suggesting its potential for treating various ocular diseases. However, the molecular mechanisms underlying the protective (especially antiapoptotic) effects of exogenous Tß4 on corneal epithelial cells are not fully elucidated. In the present study, we generated a recombinant Tß4 (His6-Tß4) as well as a polyclonal antibody against this G-actin-sequestering peptide, examined the protection by exogenous Tß4 of immortalized HCE-T cells against different insults, and attempted to elucidate its underlying mechanism.

The size of the recombinant Tß4 generated by us was similar to that of the synthetic one (Fig. 1A) . Both of these peptides were recognized by the polyclonal anti-Tß4 antibody prepared herein and the former could also be detected by the anti-His antibody (Fig. 1B) . To simulate the FasL-mediated T-cell attack during corneal inflammation, Fas-expressing HCE-T cells (Fig. 2A) were treated with an agonistic Fas antibody (CH11) and significant death, which was effectively suppressed by the pretreatment of exogenous Tß4, was detected (Fig. 2G) . A severe decrease of viability in HCE-T cells was also found after they were incubated with H2O2, which likewise was dramatically reduced by Tß4 pretreatment (data not shown). Accordingly, activation of caspase-8 by FasL (Fig. 2H) , stimulation of caspase-9 by H2O2 (data not shown), and induction of casapse-3 by both insults (Figs. 2I 5C 5H) in these cells were all diminished by preincubation with exogenous Tß4 These results are in good agreement with an earlier observation that Tß4 inhibits the activation of caspase-2, -3, -8, and -9 induced by ethanol in nontransformed human corneal epithelial cells (HCECs).32 Even though the decreased deleterious mitochondrial alterations and cytochrome c release from mitochondria in HCECs by Tß4 may account for its suppression of caspase-9 activity,32 the reason for its inhibition of FasL-triggered caspase-8 activation in HCE-T cells remains to be determined. Nonetheless, these data suggest that Tß4 interferes with both the intrinsic and extrinsic death-signaling pathways.

To elucidate the antiapoptotic mechanism of exogenous Tß4 in more detail, we first tried to determine the locations (i.e., extra- or intracellular) in which this peptide exerts its effect, since both sites have been shown to play a role in mediating the signal of Tß419 26 36 Of note, a rapid internalization of exogenous Tß4 (Figs. 3E 3F 3G 3H) which could be retained in cells for at least 24 hours (Figs. 3K 3L) , was detected, and the mobilization of this peptide appeared to be dependent on actin polymerization, because Tß4 entry was abolished completely by a transient treatment of cytochalasin D,45 46 an actin filament disrupting agent (Fig. 4G versus 4I). In the meantime, pretreating cells with this drug also abrogated the protective effects of exogenous Tß4 against both FasL- and H2O2-triggered apoptosis (Figs. 5 6) , suggesting that the internalization of Tß4 is critical for its cytoprotection. An explanation for observations may be either that the Tß4 surface receptor is absent in HCE-T cells or the interaction between such a receptor and Tß4 is insufficient (even failing) to trigger an antiapoptotic response. More work is needed to distinguish between these possibilities.

Although Tß4 has been reported to reduce the release of cytochrome c from mitochondria; increase Bcl-2 expression; and suppress the activation of caspase-2, -3, -8, and -9 in ethanol-treated HCECs,32 the detailed antiapoptotic mechanisms of this peptide have not yet been determined. In contrast, internalization of the exogenous Tß4 and a consequential activation of integrin-linked kinase (ILK) and Akt by this peptide seem to be the major mechanisms that promote the survival of cardiomyocytes.19 20 In this regard, even though the precise mechanism of Tß4 cellular entry remains to be determined, our results support the idea that its intracellular action(s) is essential for the protective effect of this G-actin-binding protein on HCE-T cells. In addition to the aforementioned antiapoptotic activities, Tß4 may use its sole methionine residue to react with intracellular oxygen to generate a sulfoxide adduct, thereby not only reduces the oxidative stress but also produces a novel anti-inflammatory mediator.29

In summary, exogenous Tß4 is able to increase the resistance of corneal epithelial cells against apoptosis induced by both extrinsic and intrinsic pathways and, to that effect, intracellular localization is essential. Studies aimed at dissecting the internalization mechanism of Tß4 into human corneal epithelial cells as well as its detailed inhibitory effects on caspase activation are currently under way in our laboratory.


   Acknowledgements
 
The authors thank Li-Jen Lin and Hui-Ju Lee for preparing the polyclonal anti-Tß4 antibody and the recombinant Tß4, respectively.


   Footnotes
 
Supported by Grant VGHUST95-P7-26 from the Veteran General Hospital-University System, Taiwan, and the Ministry of Education; Grant 95A-C-D01-PPG-0 from Aim for the Top University Plan (YS); and Intramural Grant VGH-94-365-13 from the Taipei Veterans General Hospital (OKL).

Submitted for publication July 19, 2006; revised August 15, 2006; accepted October 16, 2006.

Disclosure: J.H.-C. Ho, None; C.-H. Chuang, None; C.-Y. Ho, None; Y.-R.V. Shih, None; O.K.-S. Lee, None; Y. Su, None

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.

* Each of the following is a corresponding author: Yeu Su, National Yang-Ming University, 155, Li-Nong Street, Sec. 2, Peitou, Taipei 11221, Taiwan; yeusu{at}ym.edu.tw.; Oscar Kuang-Sheng Lee, Taipei Veterans General Hospital, No. 201, Sec. 2, Shih-Pai Road, Taipei 11217, Taiwan; kslee{at}vghtpe.gov.tw. Back


   References
Top
 Abstract
 Materials and Methods
 Results
 Discussion
 References
 

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J H-C Ho, K-C Tseng, W-H Ma, K-H Chen, O K-S Lee, and Y Su
Thymosin beta-4 upregulates anti-oxidative enzymes and protects human cornea epithelial cells against oxidative damage
Br. J. Ophthalmol., July 1, 2008; 92(7): 992 - 997.
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