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H.–C.J. Ho, O.K. Lee, Y. Su; Cellular Uptake Is Essential for the Anti–Apoptotic Effect of Exogenous Thymosin Beta–4 on Human Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):4845.
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© ARVO (1962-2015); The Authors (2016-present)
To define the mechanism of anti–apoptotic effect of exogenous thymosin ß–4 (Tß4) on human corneal epithelial (HCE–T) cells .
The recombinant histidine–tagged Tß4 produced by Eschericha coli (E. coli) was added to HCE–T cells. Membrane tracking was inhibited by cytochalasin D. Immunofluorescence stained for histidine and Tß4 to colocalize exogenous Tß4. Apoptotic HCE–T cells was induced by Fas ligand (FasL) or hydrogen peroxide (H2O2). Using immunofluorescence stain for caspase–3 activity and colorimetric assays for caspase–3, –8, –9 activties to measure the protective effect of exogenous Tß4.
In the present study, we found that pretreatment of the HCE–T cells with Tß4 reduced their sensitivity to FasL and H2O2. Moreover, activation of caspases–8 and –3 by FasL as well as that of caspases–9 and –3 by H2O2 in these cells was also abolished by Tß4 pretreatment. Interestingly, internalization of this G–actin sequestering peptide into HCE–T cells was demonstrated by immunofluorescence staining. Not only was the internalization of Tß4 but also its anti–apoptotic effect abrogated when HCE–T cells were incubated with cytochalasin D, an inhibitor of endocytosis, prior to the addition of exogenous Tß4.
This is the first study to demostrate that internalization of exogenous Tß4 is essential for its anti–apoptotic effect on human corneal epithelial cells.
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