Overexpression of TLR2 and TLR4 susceptibility to serum deprivation-induced apoptosis in CHO cells Wei Fan a , Tuanzhu Ha a , Yan Li a , Tammy Ozment-Skelton a , David L. Williams a , Jim Kelley b , I. William Browder a , Chuanfu Li a, * a Department of Surgery, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA b Department of Internal Medicine, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN 37614, USA Received 16 September 2005 Available online 29 September 2005 Abstract We examined the effect of overexpression of TLR2 and TLR4 on apoptosis. TLR2 and TLR4 transfected CHO cells were subjected to serum deprivation for 0, 24, and 48 h. CHO cells served as control. The survival was 80.4% and 66.8% in CHO cells, 73.8% and 47.6% in TLR2/CHO, and 70.5% and 53.0% in TLR4/CHO, respectively. Flow cytometry examination suggested that apoptotic cells were 7.17% and 32.91% in control CHO cells, 29.0% and 64.6% in TLR2/CHO, and 41.4% and 64.6% in TLR4/CHO, respectively. The levels of FasL and caspase-8 activity in TLR2/CHO and TLR4/CHO cells were significantly higher than that of CHO cells. Transfection of dom- inant negative FADD into TLR2/CHO and TLR4/CHO cells significantly reduced apoptosis. Our results suggest that overexpression of TLR2 and TLR4 in CHO cells sensitizes the cells to serum deprivation-induced apoptosis and that the mechanisms are involved in the death receptor-mediated signaling pathway. Ó 2005 Elsevier Inc. All rights reserved. Keywords: Toll-like receptors; Serum deprivation; Apoptosis; Signaling transduction Toll-like receptors (TLRs) are pattern recognition recep- tors that play an important role in induction of innate immunity by recognition of exogenous pathogen associated molecular patterns (PAMPS) and endogenous ligands [1]. TLR-mediated signaling mainly activates nuclear factor jB (NFjB) which plays a critical role in regulation of the expression of groups of genes involved in immune and inflammatory responses, cell death and survival, and cell growth and cell cycle [2]. TLR2 mainly recognizes compo- nents from Gram-positive bacteria while TLR4 has been characterized to specifically recognize Gram-negative bac- terial lipopolysaccharide (LPS) [1] and endogenous ligands such as heat shock protein 60 (Hsp60) and Hsp70 [3]. The main pathway of TLR-mediated signaling that leads to NFjB activation involves the adaptor molecule named myeloid differentiation factor 88 (MyD88), kinases of the IL-1 receptor associated kinase (IRAK) family, tumor necrosis factor receptor associated factor 6 (TRAF6), NFjB-inducing kinase (NIK), transforming growth factor (TGF)-b-activated kinase 1 (TAK1), and IjB kinase (IKK) complexes [4,5]. Activated IKKs will phosphorylate IjBa, an inhibitory protein for NFjB, resulting in IjBa degrada- tion. The NFjB will then translocate into the nucleus and bind at specific DNA binding sites to stimulate gene expression [6], including various cytokines, adhesion mole- cules, and cell survival and cell death. MyD88 is an impor- tant adaptor protein which plays a critical role in the TLR/ interleukin (IL)-receptor (TIR) mediated NFjB activation pathway [1]. Recent studies have suggested that stimulation of TLRs can result in apoptosis by triggering pro-apoptotic signal- ing [7–9]. For example, stimulation of TLR2 and TLR6 with bacterial-lipoproteins induces apoptotic cell death while transfection of dominant negative MyD88 or domi- nant negative FADD (dnFADD) reduced cell death [9]. 0006-291X/$ - see front matter Ó 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.09.123 * Corresponding author. Fax: +1 423 439 6259. E-mail address: Li@MAIL.ETSU.EDU (C. Li). www.elsevier.com/locate/ybbrc Biochemical and Biophysical Research Communications 337 (2005) 840–848 BBRC