Internalization is essential for the antiapoptotic effects of exogenous thymosin β-4 on human corneal epithelial cells

Jennifer Hui Chun Ho, Chiao Hui Chuang, Chih Yuan Ho, Yu Ru Vernon Shih, Oscar Kuang Sheng Lee, Yeu Su

Research output: Contribution to journalArticle

32 Citations (Scopus)

Abstract

PURPOSE. Exogenous thymosin β-4 (Tβ 4) 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 Tβ 4 protects SV40-immortalized human corneal epithelial T (HCE-T) cells against the toxic effects of Fas ligand (FasL) and hydrogen peroxide (H 2O 2) and to elucidate its mechanism of action. METHODS. HCE-T cells were incubated without or with the recombinant histidine-tagged Tβ 4 produced by Escherichia coli before the addition of FasL or H 2O 2. 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 Tβ 4 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 Tβ 4 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 H 2O 2 was dramatically reduced by the recombinant Tβ 4 pretreatment. Moreover, FasL-mediated activation of caspases-8 and -3 as well as H 2O 2-triggered stimulation of caspases-9 and -3 in these cells was abolished by preincubating them with the exogenous Tβ 4. 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 Tβ 4 by cytochalasin D abrogated its cytoprotective effects. CONCLUSIONS. This is the first report to demonstrate that the internalization of exogenous Tβ 4 is essential for its antiapoptotic activity in human corneal epithelial cells.

Original languageEnglish
Pages (from-to)27-33
Number of pages7
JournalInvestigative Ophthalmology and Visual Science
Volume48
Issue number1
DOIs
Publication statusPublished - Jan 2007
Externally publishedYes

Fingerprint

Thymosin
Epithelial Cells
Fas Ligand Protein
T-Lymphocytes
Cytochalasin D
Caspase 9
Caspase 8
Caspase 3
Actins
Peptides
Cytoprotection
Poisons
Actin Cytoskeleton
Histidine
Human Activities
Hydrogen Peroxide
Fluorescent Antibody Technique
Cell Survival
Cell Death
Ethanol

ASJC Scopus subject areas

  • Ophthalmology

Cite this

Internalization is essential for the antiapoptotic effects of exogenous thymosin β-4 on human corneal epithelial cells. / Ho, Jennifer Hui Chun; Chuang, Chiao Hui; Ho, Chih Yuan; Shih, Yu Ru Vernon; Lee, Oscar Kuang Sheng; Su, Yeu.

In: Investigative Ophthalmology and Visual Science, Vol. 48, No. 1, 01.2007, p. 27-33.

Research output: Contribution to journalArticle

Ho, Jennifer Hui Chun ; Chuang, Chiao Hui ; Ho, Chih Yuan ; Shih, Yu Ru Vernon ; Lee, Oscar Kuang Sheng ; Su, Yeu. / Internalization is essential for the antiapoptotic effects of exogenous thymosin β-4 on human corneal epithelial cells. In: Investigative Ophthalmology and Visual Science. 2007 ; Vol. 48, No. 1. pp. 27-33.
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abstract = "PURPOSE. Exogenous thymosin β-4 (Tβ 4) 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 Tβ 4 protects SV40-immortalized human corneal epithelial T (HCE-T) cells against the toxic effects of Fas ligand (FasL) and hydrogen peroxide (H 2O 2) and to elucidate its mechanism of action. METHODS. HCE-T cells were incubated without or with the recombinant histidine-tagged Tβ 4 produced by Escherichia coli before the addition of FasL or H 2O 2. 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 Tβ 4 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 Tβ 4 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 H 2O 2 was dramatically reduced by the recombinant Tβ 4 pretreatment. Moreover, FasL-mediated activation of caspases-8 and -3 as well as H 2O 2-triggered stimulation of caspases-9 and -3 in these cells was abolished by preincubating them with the exogenous Tβ 4. 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 Tβ 4 by cytochalasin D abrogated its cytoprotective effects. CONCLUSIONS. This is the first report to demonstrate that the internalization of exogenous Tβ 4 is essential for its antiapoptotic activity in human corneal epithelial cells.",
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T1 - Internalization is essential for the antiapoptotic effects of exogenous thymosin β-4 on human corneal epithelial cells

AU - Ho, Jennifer Hui Chun

AU - Chuang, Chiao Hui

AU - Ho, Chih Yuan

AU - Shih, Yu Ru Vernon

AU - Lee, Oscar Kuang Sheng

AU - Su, Yeu

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N2 - PURPOSE. Exogenous thymosin β-4 (Tβ 4) 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 Tβ 4 protects SV40-immortalized human corneal epithelial T (HCE-T) cells against the toxic effects of Fas ligand (FasL) and hydrogen peroxide (H 2O 2) and to elucidate its mechanism of action. METHODS. HCE-T cells were incubated without or with the recombinant histidine-tagged Tβ 4 produced by Escherichia coli before the addition of FasL or H 2O 2. 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 Tβ 4 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 Tβ 4 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 H 2O 2 was dramatically reduced by the recombinant Tβ 4 pretreatment. Moreover, FasL-mediated activation of caspases-8 and -3 as well as H 2O 2-triggered stimulation of caspases-9 and -3 in these cells was abolished by preincubating them with the exogenous Tβ 4. 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 Tβ 4 by cytochalasin D abrogated its cytoprotective effects. CONCLUSIONS. This is the first report to demonstrate that the internalization of exogenous Tβ 4 is essential for its antiapoptotic activity in human corneal epithelial cells.

AB - PURPOSE. Exogenous thymosin β-4 (Tβ 4) 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 Tβ 4 protects SV40-immortalized human corneal epithelial T (HCE-T) cells against the toxic effects of Fas ligand (FasL) and hydrogen peroxide (H 2O 2) and to elucidate its mechanism of action. METHODS. HCE-T cells were incubated without or with the recombinant histidine-tagged Tβ 4 produced by Escherichia coli before the addition of FasL or H 2O 2. 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 Tβ 4 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 Tβ 4 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 H 2O 2 was dramatically reduced by the recombinant Tβ 4 pretreatment. Moreover, FasL-mediated activation of caspases-8 and -3 as well as H 2O 2-triggered stimulation of caspases-9 and -3 in these cells was abolished by preincubating them with the exogenous Tβ 4. 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 Tβ 4 by cytochalasin D abrogated its cytoprotective effects. CONCLUSIONS. This is the first report to demonstrate that the internalization of exogenous Tβ 4 is essential for its antiapoptotic activity in human corneal epithelial cells.

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