Woodchuck lymphotoxin-α, -β and tumor necrosis factor genes: structure, characterization and biological activity

Abstract

We cloned and characterized the woodchuck tumor necrosis factor (TNF) and lymphotoxin-α, -β (LT-α, -β) cDNAs, genes and proteins to facilitate study of the functions of these cytokines during the course of woodchuck hepatitis virus (WHV) infection. Woodchuck cDNA and genomic DNA libraries were screened with woodchuck-specific DNA probes to isolate the cDNA and gene clones for TNF, LT-α and LT-β. The cDNAs for woodchuck TNF, LT-α and LT-β code for proteins of 233, 205 and 310 amino acids respectively. The polypeptide encoded by each gene among woodchucks, humans and mice can differ: the human TNF, LT-α and LT-β genes encode polypeptides of 233, 205 and 244 amino acids respectively, whereas the mouse TNF, LT-α and LT-β genes encode polypeptides of 235, 202 and 306 amino acids respectively. In the woodchuck, there are four exons for TNF, four exons for LT-α and three exons for LT-β. The RNA splicing patterns for TNF, LT-α and LT-β genes are identical among woodchucks, humans and mice, except that the human LT-β gene contains four exons. The woodchuck TNF gene promoter contains consensus sequences for binding of AP-1, AP-2, C/EBPβ, CRE, Egr-1, Ets, NF-AT, NF-κB and SP-1 transcription factors. LT-α has AP-2, Ets, NF-κB, SP-1 and STAT binding sites, and LT-β has Egr-1/SP-1, Ets and NF-κB binding sites. The bacterially expressed woodchuck TNF and LT-α proteins exhibited cytotoxic activities on both mouse L929B and woodchuck A₂ cells in the presence of actinomycin D. The specific activities of TNF and LT-α were 2.62×10⁸ units/mg and 2.22×10³ units/mg respectively for L929B cells, and 1.05×10⁹ units/mg and 3.56×10⁴ units/mg respectively for A₂ cells. However, only woodchuck TNF showed cytotoxic activity on human HepG₂ cells, with a specific activity of 6.55×10⁷ units/mg in the presence of actinomycin D. The data obtained from this study will be useful to future investigations of the TNF and LT anti-tumor and anti-viral activities, and their therapeutic potential in the woodchuck model for human hepatitis B virus (HBV).

Introduction

Tumor necrosis factor (TNF) and lymphotoxin-α, -β (LT-α, -β) are well known as pluripotent mediators of inflammatory and immune defense mechanisms, and regulators of lymphoid organ development. TNF and LT-α, -β are members of the TNF ligand family that also includes 4-1BBL, CD27L, CD30L, CD40L, FasL, Neurotrophins (NGF), OX40L, TRAIL, TWEAK [reviewed by Ware et al. (1998)]. TNF is expressed in both secreted and membrane-bound forms and is mainly produced by activated macrophages and T lymphocytes. LT-α only exists as a secreted protein, whereas LT-β is expressed solely as a transmembrane protein. LT-α, -β are mainly produced by activated T and B lymphocytes, and natural killer cells [reviewed by Millet and Ruddle (1997) and Zhang and Tracey (1998)]. Secreted TNF or LT-α monomers can form homotrimers (Eck et al., 1992, Jones et al., 1989). Additionally, the LT-α monomer can also form heterotrimers with the LT-β monomer (Browning et al., 1993).

The receptors for TNF and LT-α homotrimers and LT-α2β1 heterotrimers are TNFR-60 and TNFR-80. The main LT-αβ complex is the LT-α1β2 heterotrimer and its receptor is the LT-β receptor (LTβR). Upon the binding of TNF or LT-α homotrimers to TNFR-60 on the cell surface, TNFR-60 trimerizes and associates with the cytosolic adapter protein TRADD (TNFR-associated death domain). The death domain was originally defined as a domain within the cytoplasmic portion of the receptor that signals programmed cell death and is present only in TNFR-60, but not in TNFR-80 and LTβR. TRADD then recruits several cytosolic signaling proteins including FADD (Fas-associating protein with death domain), RIP (receptor interacting protein) and TRAF2 (TNFR-associated factor 2) to the activated receptor. This leads either to activation of nuclear factor kappa B (NF-κB) and of c-Jun N-terminal kinase/activating protein-1 (JNK/AP-1), or to programmed cell death (apoptosis) [reviewed by Ashkenazi and Dixit (1998)]. TNFR-80 and LTβR use TRAF2 and other members of the set of adapter proteins termed TRAFs (TNFR-associated factors) to signal the pathways leading either to the activation of NF-κB and of AP-1/JNK, or to apoptosis. The family of TRAFs currently has six members including TRAF1, 2, 3, 4, 5 and 6; TRAF1 and 2 bind TNFR-80, whereas TRAF2, 3 and 5 bind LTβR [reviewed by Arch et al. (1998)].

The two major functions of TNF that are related to human hepatitis B virus (HBV) and that have been previously demonstrated are the inhibition of viral replication (Cavanaugh et al., 1998, Guidotti et al., 1996) and the induction of apoptosis in viral DNA transfected cells (Guilhot et al., 1996, Su and Schneider, 1997). The function of LT-α in the course of HBV infection remains to be elucidated, although the amounts of LT-α are elevated in HBV-infected patients (Jung et al., 1991).

The woodchuck (Marmota monax) has been used as a model for study of HBV infection since it is a natural host of woodchuck hepatitis virus (WHV), which is closely related to HBV. In woodchucks, WHV causes acute infection, chronic hepatitis and hepatocellular carcinoma. These conditions are identical to those occurring in HBV-infected patients. For this reason, the woodchuck has been used extensively for studying the molecular mechanisms of the viral life cycle (Seeger and Maragos, 1990, Yang et al., 1996), the immunomodulation of infection (Menne et al., 1998), the mechanisms of carcinogenesis (Wang et al., 1998, Wei et al., 1998), and the development of therapeutic vaccination (Lu et al., 1999) and antiviral agents (Cullen et al., 1997).

To study the functions of TNF and LT affecting WHV infection in the woodchuck model, it is essential to understand the gene structure of TNF and LT, their transcriptional and translational regulatory elements, and their biological activities. For the above reason, attempts were made to clone both the cDNAs and the genes for woodchuck TNF, LT-α and LT-β.