title: Internalization is essential for the antiapoptotic effects of exogenous thymosin beta-4 on human corneal epithelial cells.
abstract: 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.
authors.affiliation: Institute of Biopharmaceutical Science, National Yang-Ming University, Taipei, Taiwan.
authors.forename: Jennifer Hui-Chun
authors.initials: JH
authors.lastname: Ho
authors.forename: Chiao-Hui
authors.initials: CH
authors.lastname: Chuang
authors.forename: Chih-Yuan
authors.initials: CY
authors.lastname: Ho
authors.forename: Yu-Ru Vernon
authors.initials: YR
authors.lastname: Shih
authors.forename: Oscar Kuang-Sheng
authors.initials: OK
authors.lastname: Lee
authors.forename: Yeu
authors.initials: Y
authors.lastname: Su
mesh_terms: 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)
chemical_list: 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