NPR receptor binding research reagent

Abstract

Drugs that target tumor necrosis factor-α (TNF) are particularly important in the treatment of severe inflammatory progression in rheumatoid arthritis, Crohn's disease and psoriasis. Despite the central role of the TNF/TNF receptor (TNFR) in various disease states, there is a paucity of information concerning TNFR2 signaling. In this study, we have developed a simple and highly sensitive cell-death based assay system for analyzing TNFR2-mediated bioactivity that can be used to screen for TNFR2-selective drugs. Using a lentiviral vector, a chimeric receptor was engineered from the extracellular and transmembrane domain of human TNFR2 and the intracellular domain of mouse Fas and the recombinant protein was then expressed in TNFR1−/−R2−/− mouse preadipocytes. Our results demonstrate that this chimeric receptor is capable of inducing apoptosis by transmembrane- as well as soluble-TNF stimuli. Moreover, we found that our bioassay based on cell death phenotype had an approximately 80-fold higher sensitivity over existing bioassays. We believe our assay system will be an invaluable research tool for studying TNFR2 and for screening TNFR2-targeted drugs.

Introduction

Tumor necrosis factor-α (TNF) is a pleiotropic cytokine that regulates various biological processes such as host defense, inflammation, autoimmunity, apoptosis and tumor cell death through the TNF-receptor 1 (TNFR1) and receptor 2 (TNFR2) (Wajant et al., 2003). TNF/TNFR interaction is considered to be an attractive target for the treatment of refractory diseases, including autoimmune disease and malignant tumors (Aggarwal, 2003, Szlosarek and Balkwill, 2003). In rheumatoid arthritis, for example, biological anti-TNF agents, such as Infliximab and Adalimumab, rapidly reduce signs and symptoms of joint inflammation (Feldmann and Maini, 2003). However, anti-TNF drugs used to treat inflammatory disorders have been reported to increase the risk of infection, in accordance with animal studies (Brown et al., 2002, Nathan et al., 2006).

A thorough understanding of the biology of the TNF/TNFR system is a prerequisite to the safe and effective development of anti-TNF therapeutics. In particular, several factors and mechanisms hypothesized to be involved in the side effects elicited by anti-TNF drugs need to be tested (Curtis et al., 2007, Jacobs et al., 2007, Schneeweiss et al., 2007). These include the differential power of the drugs to neutralize TNF bioavailability and the differential inhibition of TNF signaling events. Despite extensive studies on the molecular biology of TNF/TNFR1 signaling (Micheau and Tschopp, 2003) the functions of TNFR2 are poorly understood. There is an increasing need for a comprehensive understanding of TNF/TNFR2 biology, particularly in terms of the development of TNFR-selective drugs.

In this context, we have used a novel phage-display based screening system (Yamamoto et al., 2003, Shibata et al., 2004, Shibata et al., 2008) to develop structural mutants of TNF to help clarify the biology of TNF/TNFR2 interactions. These TNF variants, which exert TNFR2-mediated agonistic or antagonistic activity, might be extremely valuable for elucidating structure-activity relationships between TNF and TNFR2. So far, in order to evaluate the bioactivity of TNF through TNFR2, many researchers have used the TNFR2 over-expressing cell lines(Heller et al., 1992, Weiss et al., 1998), such as rat/mouse T hybridomas transfected with human TNFR2 (PC60-hR2) (Vandenabeele et al., 1992). The PC60-hR2 assay is based on granulocyte macrophage colony-stimulating factor (GM-CSF) secretion mediated by TNF/TNFR2 stimuli. The GM-CSF secretion level is quantified by proliferation of GM-CSF-dependent cell lines or by ELISA. However, this two-step assay system is complicated and the screening process is highly laborious. Thus, there are increasing demands for the development of a simple, highly sensitive screening system that is TNFR2-selective.

In the present study, we developed a simple but highly sensitive cell death-based assay system for evaluating TNFR2-mediated activity. We constructed a lentiviral vector expressing a chimeric receptor derived from the extracellular (EC) and transmembrane (TM) domain of human TNFR2 (hTNFR2) and the intracellular (IC) domain of mouse Fas (mFas). Additionally, to eliminate the influence of the endogenous TNFR1, the chimeric receptor was expressed on TNFR1−/−R2−/− preadipocytes (Xu et al., 1999). We found that hTNFR2/mFas-expressing preadipocyte (hTNFR2/mFas-PA) showed about 80-times higher sensitivity after treatment with soluble-TNF and over the conventional method. Furthermore, hTNFR2/mFas-PA could detect not only transmembrane TNF- (tmTNF) but also soluble TNF-activity. The technology described herein will be highly useful both as an assay system for various TNF variants via TNFR2 and also as a cell-based drug discovery system for TNFR2 agonists/antagonists.

Section snippets

Cell culture

TNFR1−/−R2−/−, TNFR1−/−, and wild-type (wt) preadipocytes established from day 16–17 mouse embryos were generously provided by Dr. Hotamisligil (Harvard School of Public Health, Boston MA). Preadipocytes, 293T cells and HeLaP4 cells were cultured in Dulbecco's modified Eagle's medium (DMEM; Sigma-Aldrich, Inc., Tokyo, Japan) with 10% bovine fetal serum (FBS) and 1% antibiotic cocktail (penicillin 10,000 u/ml, streptomycin 10 mg/ml, and amphotericin B 25 μg/ml; Nacalai Tesque, Kyoto, Japan). The

Fas- but not TNFR-mediates induction of cell-death in TNFR1−/−R2−/− preadipocytes

Initially, we established a cell line that could be used to evaluate TNFR2-specific bioactivity by means of the chimeric receptor (hTNFR2/mFas) strategy. The parental cell line must possess both Fas-sensitivity and TNF-resistance. Thus, we selected TNFR1−/−R2−/− preadipocytes as the parental cell line and then examined the susceptibility of this cell line against TNFR1- and Fas-induced cell death. TNFR1−/−R2−/− preadipocytes were resistant to TNF-induced cell death, while WT preadipocytes,

Discussion

Here, we developed a hTNFR2/mFas-PA cell-based assay system in order to investigate hTNF activity through hTNFR2. The assay is simple to perform and can detect hTNF-mediated hTNFR2 activity with high sensitivity. Because the hTNFR2/mFas-PA system was engineered in TNFR1−/−R2−/− preadipocytes, we were able to analyze hTNF-mediated hTNFR2 activity without affecting TNFR1-related apoptosis. Moreover, not only tmTNF- but also soluble TNF-mediated hTNFR2 activity was detectable using the

Acknowledgements

This study was supported in part by Grants-in-Aid for Scientific Research (No. 18015055, 17689008) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, in part by Health Labor Sciences Research Grant from the Ministry of Health, Labor and Welfare of Japan, in part by Health Sciences Research Grants for Research on Health Sciences focusing on Drug Innovation from the Japan Health Sciences Foundation, and in part by JSPS Research Fellowships for Young Scientists from

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• Fine tuning of receptor-selectivity for tumor necrosis factor-α using a phage display system with one-step competitive panning 2011, Biomaterials HEp-2 cells, a human fibroblast cell line, were provided by Cell Resource Center for Biomedical Research (Tohoku University, Sendai, Japan) and were maintained in RPMI 1640 (Sigma–Aldrich Japan, Tokyo, Japan) supplemented with 10% bovine fetal serum (FBS) 1 mm sodium pyruvate, 50 mm 2-mercaptoethanol, and antibiotics. hTNFR2/mFas-PA cells are preadipocytes derived from TNFR1-/-R2-/- mice expressing a chimeric receptor, the extracellular and transmembrane domain of human TNFR2, and intracellular domain of mouse Fas; these cells were cultured in RPMI 1640 supplemented with 10% FBS, 5 μg/ml Blasticidin S HCl (Invitrogen, Carlsbad, CA), and antibiotics [26]. Protocol for the construction of phage-display library displaying structural mutants of human TNF has been described previously [23].
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