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A.H. Kakazu, G. Chandrasekher, H.E. P. Bazan; The survival factor HGF induces changes in members of the Bcl–2 protein family in corneal epithelial cells . Invest. Ophthalmol. Vis. Sci. 2004;45(13):3814.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose:After injury, corneal repair requires that the cells do not enter apoptosis, so that they can migrate and proliferate. Previous studies showed a correlation between a protective effect of hepatocyte growth factor (HGF) against apoptosis of corneal epithelial cells (CEC) and the activation of a PI–3K/Akt signaling pathway (Kakazu et al, ARVO 2002). The protection by HGF and the activation of the signaling system occur with different apoptotic inducers in both rabbit and human CEC (Kakazu et al, ARVO 2003). The purpose of this study was a) to demonstrate the relevance of Akt activation in CEC survival and b) to determine weather Bcl–2 family members were involved in the inhibition of apoptosis by HGF. Methods:Rabbit and human CEC were used. Overexpression of Akt–1 was performed by transfecting the cells with FuGENE 6 and Akt–1 pUSEamp cDNA in the active form. Apoptosis was induced with staurosporine (10–100ng/ml), DNA fragmentation was identified by DNA laddering. Immunofluorescence was performed with a p–Akt–1 antibody. Hoescht was used for nuclear staining. Mitochondrial and cytosolic fractions were isolated and proteins analyzed by Western blot using specific antibodies. Results: Expression of active Akt–1 in CEC prevented laddering induced by staurosporine. Immunofluorescence showed accumulation of p–Akt –1 in the plasma membrane. When non transfected CEC are stimulated with HGF, p–Akt–1 appears as a punctate staining localized in different areas of the cell as soon as 2 min after stimulation. HGF also induced the phosphorylation and inactivation of the Bcl–2 family protein Bad at Ser 136, which was inhibited by the PI–3K inhibitor LY294002. Presence of staurosporine caused de–phosphorylation of Bad in the soluble fraction and translocation of the protein to the mitochondria. Overexpression of active Akt–1 inhibited Bad translocation to the mitochondria by staurosporine. Another pro–apoptotic protein, Bax, increased its expression with staurosporine which was reduced by treatment with HGF. Conclusions: Akt–1 is a central kinase in the survival effect of HGF in CEC. The protective effect is exerted through phosphorylation of Bad and retention of this apoptotic protein in the cytosol and by decreasing the expression of the proapoptotic Bax. The results show that HGF, through PI3K/Akt–1 activation, suppresses mitochondria– initiated apoptosis in CEC. These could represent important points of regulation to restore a healthy epithelium after injury.
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