Internalization is essential for the antiapoptotic effects of exogenous thymosin beta-4 on human corneal epithelial cells.
OBJECTIVE
Exogenous thymosin beta-4 (Tbeta(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 Tbeta(4) protects SV40-immortalized human corneal epithelial T (HCE-T) cells against the toxic effects of Fas ligand (FasL) and hydrogen peroxide (H(2)O(2)) and to elucidate its mechanism of action.


METHODS
HCE-T cells were incubated without or with the recombinant histidine-tagged Tbeta(4) produced by Escherichia coli before the addition of FasL or H(2)O(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 Tbeta(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 Tbeta(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(2)O(2) was dramatically reduced by the recombinant Tbeta(4) pretreatment. Moreover, FasL-mediated activation of caspases-8 and -3 as well as H(2)O(2)-triggered stimulation of caspases-9 and -3 in these cells was abolished by preincubating them with the exogenous Tbeta(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 Tbeta(4) by cytochalasin D abrogated its cytoprotective effects.


CONCLUSIONS
This is the first report to demonstrate that the internalization of exogenous Tbeta(4) is essential for its antiapoptotic activity in human corneal epithelial cells.
Ho
Jennifer Hui-Chun
JH
Institute of Biopharmaceutical Science, National Yang-Ming University, Taipei, Taiwan.
Chuang
Chiao-Hui
CH
Ho
Chih-Yuan
CY
Shih
Yu-Ru Vernon
YR
Lee
Oscar Kuang-Sheng
OK
Su
Yeu
Y
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D020169:Caspases / Q000378:metabolism; D018929:Cell Culture Techniques; D002461:Cell Line, Transformed; D015638:Cytochalasin D / Q000494:pharmacology; D004705:Endocytosis / Q000502:physiology*; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D053222:Fas Ligand Protein / Q000633:toxicity; D005434:Flow Cytometry; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D006861:Hydrogen Peroxide / Q000633:toxicity; D020714:Maximum Tolerated Dose; D011817:Rabbits; D011994:Recombinant Proteins / Q000494:pharmacology; D013539:Simian virus 40; D013778:Tetrazolium Salts; D013844:Thiazoles; D013947:Thymosin / Q000494:pharmacology*; D000099201:Thymosin beta(4)
D020169:Caspases; D015638:Cytochalasin D; D053222:Fas Ligand Protein; D006861:Hydrogen Peroxide; D011994:Recombinant Proteins; D013778:Tetrazolium Salts; D013844:Thiazoles; D013947:Thymosin; D000099201:Thymosin beta(4); C022616:thiazolyl blue