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Jennifer Hui-Chun Ho, Chiao-Hui Chuang, Chih-Yuan Ho, Yu-Ru Vernon Shih, Oscar Kuang-Sheng Lee, Yeu Su; Internalization Is Essential for the Antiapoptotic Effects of Exogenous Thymosin β-4 on Human Corneal Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2007;48(1):27-33. doi: 10.1167/iovs.06-0826.
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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.
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