BBRC Biochemical and Biophysical Research Communications 307 (2003) 956–961 www.elsevier.com/locate/ybbrc Characterization of chicken TNFR superfamily decoy receptors, DcR3 and osteoprotegerinq Jamie T. Bridgham and Alan L. Johnson* Department of Biological Sciences and the Walther Cancer Research Center, The University of Notre Dame, P.O. Box369, Notre Dame, IN 46556, USA Received 31 May 2003 Abstract Tumor necrosis factor (TNF) family ligands bind to death domain-containing TNF receptors (death receptors), which can subsequently activate intracellular signaling pathways to initiate caspase activity and apoptotic cell death. Decoy receptors, without intracellular death domains, have been reported to prevent cytotoxic effects by binding to and sequestering such ligands, or by interfering with death receptor trimerization. The chicken death receptors, Fas, TNFR1, DR6, and TVB, are constitutively ex- pressed in a relatively wide variety of hen tissues. In this study, two chicken receptors with sequence homology to the mammalian decoys, DcR3 and osteoprotegerin, were identified and their pattern of expression was characterized. Unlike the previously iden- tified chicken death receptors, the newly characterized decoy receptors show comparatively limited expression among tissues, suggesting a tissue-specific function. Finally, characterization of these chicken receptors further contributes to understanding the evolutionary divergence of TNFR superfamily members among vertebrate species. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Tumor necrosis factor receptor superfamily; TNF ligands; Death receptor; Decoy receptor; Ovary; Chicken; Apoptosis Death receptors (DRs) are characterized by the pres- Alternatively, many of the death receptors are capa- ence of a variable number of extracellular cysteine-rich ble of signaling through intracellular pathways that ac- motifs that comprise the ligand binding domain, a single- tually promote cell survival. For example, TNFR1 is pass (type I) transmembrane domain and, in particular, a known to activate nuclear factor jB(NFjB) resulting in cytoplasmic death-domain. When bound to their cog- increased transcription of cell survival signals such as X- nate ligands, DRs typically form homotrimers, a process linked inhibitor of apoptosis protein (XIAP) [2], Bcl- that modifies the conformation of the cytoplasmic do- XLong [3], and FLICE-like inhibitory protein (FLIP) [4]. main and initiates cell signaling. DRs are capable of The ability of TNF family ligands to activate DR initiating apoptotic pathways in a cell type-dependent signaling is proposed to be modulated by the presence of fashion by signaling through binding of their death-do- decoy receptors (DcRs) that can bind to and sequester mains with death domains of cytoplasmic adaptor pro- ligands from the death inducing receptors and/or inter- teins. The adaptor proteins (e.g., Fas associated via fere with receptor trimerization. Similar to DRs, DcRs death domain, FADD), in turn, interact with initiator contain cysteine-rich ligand binding domains, but do not caspases (e.g., caspase-8) via a death effector domain possess intracellular death domains and may, or may (DED). These interactions ultimately promote a cascade not, contain a transmembrane domain. Mammalian of caspase enzyme activation leading to cell death [1]. DcRs are known to exist for TNF-related apoptosis inducing ligand (TRAIL) and Fas ligand (FasL). Ad- ditionally, splice variants encoding soluble forms of Fas, q Supported by USDA 99-35203-7736, NIH HD36095, and DOD DAMD17-03-1-0206 to A.L.J. which are predicted to function as decoys, have been * Corresponding author. Fax: 1-574-631-7413. identified in the human [5] and chicken [6], and soluble E-mail address: [email protected] (A.L. Johnson). Fas variants are capable of inhibiting apoptosis induced 0006-291X/03/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/S0006-291X(03)01304-4 J.T. Bridgham, A.L. Johnson / Biochemical and Biophysical Research Communications 307 (2003) 956–961 957 by the FasL in human T (HUT) lymphoma cells [5]. It Two chicken EST sequences with similarity to human OPG were has been proposed that the relative ratio of DR to DcR identified in the University of Delaware Chicken EST database (pitu- may determine susceptibility of cells to DR-mediated itary gland, hypothalamus, and pineal gland library, clone No. BI393190, and breast muscle, leg muscle, and epiphyseal growth plate cell signaling [7]. library, clone No. BM488261; http://www.chickest.udel.edu/). These More specifically, in the mammalian ovary there is sequences encompass a partial open reading frame with homology to limited immunohistochemical evidence to suggest that the cysteine-rich domain of mammalian OPGs, but lacking the C- DcR-1 (a TRAIL DcR) is expressed in porcine granu- terminus. Homologous primers based on the two EST sequences were 0 losa cells and that decreased levels of DcR-1 are asso- used to amplify a 454 bp fragment (forward primer 5 -TTC TTC CAC CAA CCA CAA GA AC-30 and reverse primer 50-GGG CAA CTT ciated with follicle death via atresia [8]. This finding is of TTA CAA ACA GTG TCA C-30) using an ovarian stromal tissue potential significance, given the importance of granulosa template with the identical amplification conditions described above. cell survival to the viability of differentiating ovarian Each PCR product was cloned into TA cloning vector (Invitrogen, follicles [9]. Brook trout (Salvelinus fontinalis) granulosa Carlsbad, CA), transformed into INVaF0cells (Invitrogen), and sub- cells also express a decoy receptor (TDcR) that becomes sequently sequenced using the dideoxychain-termination method with Sequenase version 2.0 (USB Corporation, Cleveland, OH). Final se- upregulated just after the completion of ovulation [10], quences for full-length clones were obtained through Canadian Mo- presumably at the time of ovarian regression. lecular Research Service (Ottawa, Ont., Canada). Sequence analysis In ongoing studies, we utilize the hen ovary as a and alignments were performed using MacVector 6.5 software (Oxford model system to study DR activation and function in Molecular, Genetics Computer Group, Madison, WI). relation to ovarian follicle viability and development Animals and tissue collection. Single-comb white Leghorn hens (H&H Poultry, Portland IN), 25–35 weeks of age and laying regular (e.g., [6,11,12]). The potential importance of signaling sequences of at least 5–6 eggs, were used in all studies described. Birds through death receptors as one pathway to initiate were housed individually in laying batteries, provided with free access ovarian follicle atresia has been proposed for several to feed (Purina Layeena Mash, Purina Mills, St. Louis, MO) and vertebrate species (bovine, [13]; rat, [14]; and chicken, water, and were exposed to a photoperiod of 15 h light, 9 h darkness, [6]). Significantly, four recently identified chicken death with lights on at midnight. Individual laying cycles were monitored by the daily timing of oviposition. Hens were killed approximately 16– receptors (Fas, TNFR1, DR6, and TVB) are expressed 18 h prior to a midsequence ovulation by cervical dislocation. All at higher levels in atretic follicles as compared to healthy procedures described herein were reviewed and approved by the Uni- follicles [6,11,12]. These studies also demonstrate that versity of Notre Dame Institutional Animal Care and Use Committee, levels of receptor expression are regulated by growth and were performed in accordance with the Guiding Principles for the factors and gonadotropins that promote granulosa cell Care and Use of Laboratory Animals. Tissues were collected from the chicken ovarian stroma, post- survival. The presence of multiple death receptors in this ovulatory follicle, brain, spleen, bone marrow, oviduct, kidney, and single cell type, each controlled by a unique set of in- heart for isolation of total RNA. Ovarian follicle tissues included tracellular signaling pathways, suggests a precise and granulosa and theca from 3 to 5 and 6 to 8 mm diameter prehierarchal intricate regulation of DR activation and signaling, in- (undifferentiated) follicles, and the largest (F1) and second largest (F2) cluding the potential for modulation by DcRs. Ac- preovulatory follicles. Morphologically normal and atretic follicles (3– 8 mm) were processed without separating the granulosa and theca cordingly, in the present report, we provide evidence for layers, as previously described [16]. Samples were collected and pro- two new chicken DcRs, DcR3 and osteoprotegerin cessed immediately or frozen at )70 °C. (OPG), and characterize their selective expression in Northern blot analysis. EcoRI restriction digests were performed to ovarian and extra-ovarian tissues. isolate the chDcR3 558 bp and chOPG 454 bp inserts from the clones described above for use as cDNA probe templates. Templates were random-prime labeled with the Megaprime DNA Labeling System (Amersham, Piscataway, NJ) and [32P]dCTP (3000 Ci/mmol; Amer- Materials and methods sham). Prehybridization was carried out for at least 30 min at 60 °C, at which time probes were added and hybridization was allowed to Isolation and characterization of chicken (ch) decoy receptors. The proceed overnight. Stringent washes were conducted by rinsing with chDcR3 sequence was initially identified in the University of Man- 2Â SSC (single strength SSC ¼ 150 mM sodium chloride and 15 mM chester Institute of Science and Technology (UMIST) expressed se- sodium citrate,