Journal of Cellular Biochemistry 100:1301–1312 (2007) TLR3 Signaling in a Hepatoma Cell Line is Skewed Towards Apoptosis Elina Khvalevsky, Ludmila Rivkin, Jacob Rachmilewitz, Eithan Galun, and Hilla Giladi* The Goldyne Savad Institute of Gene Therapy, Hadassah University Hospital, Jerusalem, Israel Abstract Toll-like receptors (TLRs) recognize pathogen-associated molecular patterns (PAMPS) leading to the activation of the innate immune response and subsequently to the shaping of the adaptive immune response. Of the known human TLRs, TLR3, 7, 8, and 9 were shown to recognize nucleic acid ligands. TLR3 signaling is induced by double- stranded (ds)RNA, a molecular signature of viruses, and is mediated by the TRIF (TIR domain-containing adaptor-inducing IFNb) adaptor molecule. Thus, TLR3 plays an important role in the host response to viral infections. The liver is constantly exposed to a large variety of foreign substances, including pathogens such as HBV (hepatitis B virus) and HCV (hepatitis C virus), which frequently establish persistent liver infections. In this work, we investigated the expression and signaling pathway of TLR3 in different hepatoma cell lines. We show that hepatocyte lineage cells express relatively low levels of TLR3 mRNA. TLR3 signaling in HEK293 cells (human embryonic kidney cells) activated NF-kB and IRF3 (interferon regulatory factor 3) and induced IFNb (interferon b) promoter expression, which are known to lead to pro-inflammatory cytokine secretion. In Huh7 cells, there was only a short-term IRF3 activation, and a very low level of IFNb expression. In HepG2 cells on the other hand, while no induction of pro-inflammatory factors was observed, signaling by TLR3 was skewed towards the induction of apoptosis. These results indicate preferential induction of the apoptotic pathway over the cytokine induction pathway by TLR3 signaling in hepatocellular carcinoma cells with potential implications for therapeutic strategies. J. Cell. Biochem. 100: 1301 – 1312, 2007. ß 2007 Wiley-Liss, Inc. Key words: TRIF; NF-kB; IRF3; Rip1 Mammalian Toll-like receptors (TLRs) serve as an early surveillance against infection by pathogens. To date, 10 TLRs have been identi- fied in humans that recognize conserved patho- gen-associated molecular patterns (PAMPs) of microorganisms and viruses. Engagement of TLRs with pathogen components, initiates a signaling cascade in the cell, leading to activa- tion of the innate immune response and shaping of the subsequent adaptive immune response. TLRs are characterized by a ligand-recognizing ectodomain composed of multiple repeats of a leucine-rich motif, and a cytoplasmic signaling domain referred to as a TIR (Toll/IL-I receptor) domain; the TIR domain serves as a platform for assembling multiple protein kinases and adap- tor proteins that initiates the signaling process, reviewed in Akira and Takeda [2004]. Among the TLRs, TLR3, 7, 8, and 9, recognize nucleic acids; TLR7 and TLR8 recognize single stranded (ss) RNA [Diebold et al., 2004; Heil et al., 2004] or siRNA (short-interfering RNA) [Hornung et al., 2005]; TLR9 recognizes DNA containing unmethylated CpG motifs [Ahmad- Nejad et al., 2002], and TLR3 recognizes dsRNA derived from viruses or host RNA [Alexopoulou et al., 2001; Kariko et al., 2004]. All four nucleic acid-recognizing TLRs are expressed mainly in the intracellular compartments, whereas all other TLR members are expressed solely on the cell surface [Nishiya and DeFranco, 2004; Nishiya et al., 2005; Kajita et al., 2006]. With the exception of TLR3, all TLR members recruit the adaptor molecule MyD88 (TIR ß 2007 Wiley-Liss, Inc. Grant sponsor: Salzberg Foundation; Grant sponsor: Israeli Science Ministry grant (Gene Therapy Strategic Center); Grant sponsor: Blum Foundation; Grant sponsor: Green- spoon Foundation; Grant sponsor: Horwitz Foundation (E.G.). *Correspondence to: Hilla Giladi, Goldyne Savad Institute of Gene Therapy, Hadassah University Hospital, P.O. Box 12000, Jerusalem, 91120 Israel. E-mail: giladi@hadassah.org.il Received 5 July 2006; Accepted 2 August 2006 DOI 10.1002/jcb.21119