Hydrogen peroxide-induced cellular apoptosis is mediated by TGF-β2 signaling pathway in cultured human lens epithelial cells

Abstract

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.

The role of TGF-β2 in cartilage development and diseases

“…Smad proteins are canonical intracellular mediators of TGF-β2 signalling pathways, and their name is based on the names of the caenorhabditis elegans (Sma) and drosophila (Mad). 58 Based on the structural and functional characteristics, these proteins are divided into three groups: regulated Smads (Smads1/2/3/5/8), Co-Smad (Smad4), and inhibitory Smads (Smads6/7). 53 , 59 In addition, there is a unique structure of Smad proteins that contains highly conserved N-terminal (MH1) and CTGF-terminal (MH2) domains and a proline-rich linker region that is of divergent length and sequence.…”