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

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.