1 TGF-β 1 induced-TIAF1 self-association leads to apoptosis M.H. Lee, S.R. Lin, N.S. Chang* Institute of Molecular Medicine, National Cheng Kung University Medical College, Tainan, Taiwan, ROC, and Department of Neuroscience and Physiology, SUNY Upstate Medical University, Syracuse, NY, USA *Address correspondence to NS Chang at nschang13827@gmail.com Summary TIAF1 is a small TGF-β1-induced factor, which supports fibroblast growth, T helper cell activation and probably regulatory T (TREG) cell differentiation. TIAF1 is involved in allograft organ rejection and Hirschsprung's disease. Here, we demonstrate that TIAF1 self-association leads to apoptosis of many types of cells. When cells were overexpressed with CFP-TIAF1 and YFP-TIAF1, TIAF1 underwent self-association and the cells died. Also, TGF-β1 enhanced the self-association for causing apoptosis in TGF- β-sensitive normal epithelial cells. Most interestingly, significant upregulation of amyloid β occurred rapidly following TIAF1 self-association, suggesting a novel mechanism regarding TGF-β1-induced aggregation of TIAF1, subsequent generation of amyloid β, and then cell death. Introduction Neurodegenerative diseases are frequently encountered in the aging populations in the human societies. Effective treatment for Alzheimer’s disease (AD), for example, has not been successful (1). The neuropathology of AD is characterized by extracellular accumulation of fibrillar β-amyloid (Aβ) peptide and intracellular deposition of neurofibrillary tangles (NFT) made of cytoskeletal protein tau (2,3). These protein aggregates invoke neuronal death and block neurogenesis and learning and memory capabilities in AD patients. TGF-β is involved in the pathogenesis of AD (4-7). The type II receptor for TGF-β (TβRII) is central to TGF-β signaling and is needed to maintain normal brain cell physiology, and that reduced levels of TβRII appear to correlate with increased symptom in AD (4). TGF-β1 is upregulated in a transgenic AD mouse model and causes neuronal apoptosis (5). Also, increased levels of TGF-β in AD are associated with perivascular deposition of extracellular matrix, which may hinder clearance of Aβ and allow the development of cerebral amyloid angiopathy (6,7). To investigate the underlying mechanism, here we show that that TGF-β-induced self- association of TIAF1 is essential for superinduction of Aβ that leads to subsequent cell death. TIAF1, known as TGF-β-induced antiapoptotic factor (8-10), is involved in the TNF signaling pathway (8-10). Also, TIAF1 participates in the TGF-β/Smad signaling and controls p53 activation (9,10). TIAF1 is significantly upregulated in TH2 helper lymphocytes in patients with chronic kidney and liver allograft rejection (11), and plays a role in regulatory T cell differentiation (12). Most recently, TIAF1 is shown to be associated with Hirschsprung's disease (13). Materials and Methods Cell lines and cDNA expression constructs. Mink lung epithelial Mv1Lu, human lung cancer H1299 cells, and neuroblastoma SH-SY5Y cells were used in this study. An EGFP-TIAF1 construct was made by tagging the TIAF1 cDNA with enhanced green fluorescence protein (EGFP) to the N-terminus (in pEGFP-C1, Clontech) (8). Also, TIAF1 was tagged to its N-terminus with enhanced cyan or yellow fluorescence protein (ECFP or EYFP). Where indicated, cells were electroporated with the above constructs