Internalization Is Essential for the Antiapoptotic Effects of Exogenous Thymosin β-4 on Human Corneal Epithelial Cells

Abstract

purpose. Exogenous thymosin β-4 (Tβ₄) 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 Tβ₄ 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 Tβ₄ 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 Tβ₄ 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 Tβ₄ 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 Tβ₄ 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 Tβ₄. 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 Tβ₄ by cytochalasin D abrogated its cytoprotective effects.

conclusions. This is the first report to demonstrate that the internalization of exogenous Tβ₄ 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 hypoxia ¹⁴ and low-dose UVB irradiation ¹⁵ has been demonstrated to be mediated by the mitochondrial pathway due mainly to an increase in intracellular reactive oxygen species (ROS). 

Thymosin β-4 (Tβ₄), 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 kinase ^{19 20} and promoting matrix metalloproteinase expression during dermal wound repair. ²¹ Upregulation of Tβ₄ increases the invasive ability of human colorectal carcinoma cells by promoting the disruption of cell-cell adhesion and a consequential activation of β-catenin signaling, ^{22 23} and overexpression of Tβ₄ 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 Tβ₄ 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, Tβ₄ and its N-terminal tetrapeptide, AcSDKP, have been shown to inhibit the proliferation of hematopoietic progenitor cells. ²⁸ Anti-inflammatory effects of Tβ₄ and its sulfoxide adduct have also been demonstrated. ²⁹ In corneal epithelial cells, wound healing promotion, inflammation reduction, ^{30 31} and inhibition of ethanol-induced ³² or benzalkonium chloride-triggered apoptosis ³³ by exogenous Tβ₄ 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 Tβ₄ on immortalized human corneal epithelial T (HCE-T) cells and subsequently to determine its underlying mechanism. In our study, exogenous Tβ₄ not only prevented the cell death induced by FasL and H₂O₂ but also inhibited the activation of several key caspases. Furthermore, internalization of exogenous Tβ₄ 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

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 Tβ₄

To generate the histidine-tagged Tβ₄ fusion protein His₆-Tβ₄, we inserted the mouse Tβ₄ 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-Tβ₄ plasmid was then transformed into Escherichia coli BL21 (DE3), and His₆-Tβ₄ was prepared as follows. Bacteria were grown at 37°C to an optical density OD₆₀₀ of 0.5 before 0.25 μM IPTG (isopropyl-β-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-Tβ₄ was determined by bicinchoninic acid (BCA) protein assay (Pierce) and purity was assessed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). 

Preparation of the Anti-Tβ₄ Polyclonal Antibody

A synthetic peptide spanning the N-terminal 14 amino acids of Tβ₄ (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-Tβ₄ antibodies were detected in the animals’ sera. The polyclonal anti-Tβ₄ 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 Tβ₄ 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 Tβ₄ 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-Tβ₄ 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 × 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 described ³⁵ after HCE-T cells were treated with the exogenous Tβ₄ (His₆-Tβ₄, 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 Tβ₄. 

Flow Cytometry

HCE-T cells were incubated without or with the His₆-Tβ₄ (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-Tβ₄, (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-Tβ₄ antibody (1:1000) and they with a rhodamine-conjugated mouse anti-rabbit antibody (1:100; Jackson ImmunoResearch, West Grove, PA), for detecting Tβ₄. 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-Tβ₄, (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 × 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

Preparation of the Recombinant Tβ₄ and a Polyclonal Anti-Tβ₄ Antibody

To determine the purity of the recombinant histidine-tagged Tβ₄ (His₆-Tβ₄), We performed SDS-PAGE with the synthetic Tβ₄ serving as the positive control. As shown in Figure 1A , the His₆-Tβ₄ 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-Tβ₄ antibody. This antibody indeed recognized both the recombinant and the synthetic peptides (Fig. 1B , left). By contrast, only the His₆-Tβ₄ was detected by the anti-His antibody (Fig. 1B , right). 

Effects of Tβ₄ against FasL- and H₂O₂-Induced Injury in HCE-T Cells

To examine the antiapoptotic effect of exogenous Tβ₄ 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 Tβ₄ treatment (Fig. 2Aversus 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-Tβ₄ (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 Tβ₄ 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 Tβ₄. A dose-dependent killing of HCE-T cells by H₂O₂ was also found and the protection of exogenous Tβ₄ against H₂O₂-triggered death in these cells was demonstrated by both the viability and caspase activity assays as well (data not shown). 

Entry of Exogenous Tβ₄ into HCE-T Cells

We next asked whether exogenous Tβ₄ 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-Tβ₄ (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 Tβ₄, 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 Tβ₄ for 2 hours. Colocalization of the histidine tag and Tβ₄ 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 Tβ₄ was observed (Figs. 3K 3L) . Besides HCE-T cells, internalization of Tβ₄ was also found in bovine corneal endothelial cells and human bone marrow-derived mesenchymal stem cells (data not shown). 

Cytochalasin D and the Internalization of Tβ₄ in HCE-T Cells

To elucidate the mechanisms of Tβ₄ 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 Tβ₄ (Figs. 4G 4H 4I 4J) . 

Internalization and the Antiapoptotic Effect of Exogenous Tβ₄

To examine whether the internalization of exogenous Tβ₄ 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-Tβ₄ 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 Tβ₄ (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. 

Discussion

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} Tβ₄, 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 Tβ₄ on corneal epithelial cells are not fully elucidated. In the present study, we generated a recombinant Tβ₄ (His₆-Tβ₄) as well as a polyclonal antibody against this G-actin-sequestering peptide, examined the protection by exogenous Tβ₄ of immortalized HCE-T cells against different insults, and attempted to elucidate its underlying mechanism. 

The size of the recombinant Tβ₄ generated by us was similar to that of the synthetic one (Fig. 1A) . Both of these peptides were recognized by the polyclonal anti-Tβ₄ 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 Tβ₄, 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 Tβ₄ 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 Tβ₄ These results are in good agreement with an earlier observation that Tβ₄ 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 Tβ₄ 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 Tβ₄ interferes with both the intrinsic and extrinsic death-signaling pathways. 

To elucidate the antiapoptotic mechanism of exogenous Tβ₄ 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 Tβ₄ ^{19 26 36} Of note, a rapid internalization of exogenous Tβ₄ (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 Tβ₄ entry was abolished completely by a transient treatment of cytochalasin D, ^{45 46} an actin filament disrupting agent (Fig. 4Gversus 4I). In the meantime, pretreating cells with this drug also abrogated the protective effects of exogenous Tβ₄ against both FasL- and H₂O₂-triggered apoptosis (Figs. 5 6) , suggesting that the internalization of Tβ₄ is critical for its cytoprotection. An explanation for observations may be either that the Tβ₄ surface receptor is absent in HCE-T cells or the interaction between such a receptor and Tβ₄ is insufficient (even failing) to trigger an antiapoptotic response. More work is needed to distinguish between these possibilities. 

Although Tβ₄ 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 Tβ₄ 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 Tβ₄ 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, Tβ₄ 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 Tβ₄ 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 Tβ₄ into human corneal epithelial cells as well as its detailed inhibitory effects on caspase activation are currently under way in our laboratory. 

 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.

 

Figure 1.

 

Preparation of the recombinant His-tagged Tβ₄ peptides and a polyclonal anti-Tβ₄ antibody. (A) Coomassie blue staining of a synthetic Tβ₄ (Syn) and a histidine-tagged Tβ₄ (His) purified from E. coli after they were separated by SDS-PAGE. (B) Western blot analysis of the synthetic and the His-tagged Tβ₄ peptides using a polyclonal anti-Tβ₄ antibody (left) or an anti-His antibody (right).

Figure 2.

 

Exogenous Tβ₄ protected HCE-T cells against FasL-induced damage. Surface expression of Fas (CD95) in HCE-T cells before (A) and after (B) Tβ₄ (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 Tβ₄ (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 Tβ₄ 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).

Figure 3.

 

Exogenous Tβ₄ entered the HCE-T cells. HCE-T cells were incubated without (A–D) or with (E–H) the exogenous Tβ₄ (1 μg/mL) for 2 hours before being processed for immunofluorescence staining with anti-His, anti-Tβ₄, and DAPI. Similar cells were incubated without (I, J) or with (K, L) the exogenous Tβ₄ 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.

Figure 4.

 

Cytochalasin D blocked the internalization of Tβ₄ 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, Tβ₄, 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 Tβ₄ signals (G, H) disappeared when the cells were treated with cytochalasin D before the addition of Tβ₄ (I, J). Bar, 15 μm.

Figure 5.

 

Cytochalasin D blocked the antiapoptotic effect of exogenous Tβ₄. 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 Tβ₄ (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 Tβ₄ on the activation of caspase-3 by FasL (E) or H₂O₂ (J).

Figure 6.

 

Cytochalasin D reduces the suppressive effect of exogenous Tβ₄ on caspase activation. HCE-T cells were incubated with cytochalasin D (20 μM, 30 minutes), followed by the exogenous Tβ₄ (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 Tβ₄ 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).

The authors thank Li-Jen Lin and Hui-Ju Lee for preparing the polyclonal anti-Tβ₄ antibody and the recombinant Tβ₄, respectively.