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

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.

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.

This article has been cited by other articles:

				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. [Abstract] [Full Text] [PDF]