Hydrogen peroxide-induced cellular apoptosis is mediated by TGF-beta2 signaling pathway in cultured human lens epithelial cells.
The objective of this study was to investigate the signaling characteristics of transforming growth factor-beta2 (TGF-beta2) and the Smads (Caenorhabditis elegans, Sma; Drosophila mothers against dpp, Mad) signal pathway of cellular apoptosis induced by hydrogen peroxide with human lens epithelial cells (HLECs). HLECs were starved for 24 h before exposure to 0.1 mumol/ml of hydrogen peroxide in the presence and in the absence of 0.01 mug/ml of AF-302-NA, a monoclonal anti-TGF-beta2 neutralization antibody. Non-stimulated cells served as controls. Cell apoptosis was examined by in situ immunocytochemistry using terminal deoxynucleotidyl transferase dUTP-mediated biotin nick end labeling (TUNEL) and by flow cytometry (FCM) using Annexin V-FITC apoptosis detection. Gene expression was assessed using the reverse transcription-polymerase chain reaction (RT-PCR). Smad-4 localization was observed by immunocytochemistry. Hydrogen peroxide induced the accumulation of Smad-4 in the nucleus of HLECs, and upregulated the expression of TGF-beta receptors (TbetaRs) mRNA in HLECs, as well as upregulated the expression of the apoptotic gene bax, which leads HLECs to apoptosis. AF-302-NA decreased cellular apoptosis induced by hydrogen peroxide in HLECs and inhibited the translocation of Smad-4 from the cytoplasm to the cell nucleus. Moreover, AF-302-NA upregulated the expression of TbetaRs mRNA and downregulated the expression of bax mRNA in HLECs incubated with hydrogen peroxide. Our study demonstrated that the TGF-beta2 signal pathway participated in the apoptotic signal transfer and might be an initiator of cellular apoptosis of HLECs after incubation with hydrogen peroxide. Interruption of the TGF-beta2 signal pathway could partially protect HLECs from apoptosis induced by incubation with hydrogen peroxide.
Cao
Xiaoguang
X
Department of Ophthalmology, People's Hospital of Peking University, Xizhimen Nan Street No.11, XiCheng District, Beijing, 100044, People's Republic of China. cxgcat@hotmail.com
Li
Xiaoxin
X
Hu
Jianxin
J
Bao
Yongzhen
Y
D017209:Apoptosis / Q000187:drug effects; D002467:Cell Nucleus / Q000378:metabolism; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism; D017931:DNA Primers; D004847:Epithelial Cells / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D007908:Lens, Crystalline / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D012333:RNA, Messenger / Q000235:genetics; D018125:Receptors, Transforming Growth Factor beta / Q000235:genetics; D015398:Signal Transduction / Q000502:physiology; D051901:Smad4 Protein / Q000378:metabolism; D053781:Transforming Growth Factor beta2 / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation
D017931:DNA Primers; D012333:RNA, Messenger; D018125:Receptors, Transforming Growth Factor beta; D051901:Smad4 Protein; D053781:Transforming Growth Factor beta2; D012313:RNA; D006861:Hydrogen Peroxide