Functions of receptor activator of NF-κB ligand (RANKL) and its receptors, RANK and OPG, are evolutionarily conserved

The tumour necrosis factor (TNF) superfamily is a group of cytokines that orchestrate a variety of functions, both in the development of the architecture of immune organs and of the immune response. The mammalian TNF superfamily consists of 19 ligands and 29 receptors, whereas in the chicken only 10 ligands and 15 receptors are present. Chickens do not develop lymph nodes, possibly due to the absence of the lymphotoxin genes (TNF superfamily members) in their genome. New members of the chicken TNF superfamily have recently been identified in the genome, namely chicken receptor activator of NF-κB ligand (chRANKL), its signalling receptor, chRANK, and its decoy receptor, osteoprotegerin (chOPG). In mammals, RANKL and RANK are transmembrane proteins expressed on the surface of Th1 cells and mature dendritic cells (DC), respectively. OPG is expressed as a soluble protein from osteoblasts and DC, regulating the interaction between RANKL and RANK. To investigate the bioactivity of this triad of molecules, the extracellular soluble domains of chRANKL and chRANK and full-length chOPG were identified and cDNAs cloned. ChRANKL, chRANK and chOPG mRNA are ubiquitously expressed across non-lymphoid and lymphoid tissues and immune cells in the chicken. Similar to mammals, chRANK and chOPG mRNA expression levels are upregulated in mature bone marrow-derived DC (BMDC). ChRANKL transcription is regulated by Ca2+-mobilisation and is further enhanced by the activation of the protein kinase C pathway, as seen in mammals. The biological activities of chRANKL, chRANK and chOPG were investigated by the production of recombinant soluble fusion proteins. The extracellular, TNF-homology, domain of chRANKL (schRANKL) was sub-cloned into a modified pCI-neo vector expressing an in-frame isoleucine zipper to encourage trimer formation. FLAG-tagged schRANKL produced in COS-7 cells predominantly forms homotrimers and chOPG is expressed as homodimers, both signatures of their mammalian TNF superfamily orthologues. SchRANKL enhances the mRNA expression levels of pro-inflammatory cytokines in mature BMDC and BM-derived macrophages (BMDM). Pre-incubation with soluble chRANK-Fc or chOPG-Fc blocked the schRANKL-mediated increase in pro-inflammatory cytokine mRNA expression levels in BMDC. Expression of surface markers on BMDC and BMDM were not affected by schRANKL treatment. SchRANKL enhances the survival rates of BMDC and BMDM and can drive osteoclast differentiation from monocyte/macrophage progenitor cells. The chRANKL signalling receptor, chRANK, does not contain an intracellular catalytic domain but requires the binding of intracellular TNF receptor-associated factors (TRAF) to initiate signalling. TRAFs are a family of seven proteins (TRAF1-7) grouped due to their highly conserved RING domains, zinc finger domains, TRAF-N and TRAF-C domains. ChRANK possesses four of the five TRAF peptide-binding motifs found in mammalian RANK. The “missing” chRANK TRAF peptide-binding motif is TRAF6-specific, a vital protein for RANKL-mediated osteoclastogenesis. All seven members of the mammalian TRAF family are present in the chicken genome. To investigate the conservation of RANK-specific TRAF signalling proteins, chicken TRAF2 (chTRAF2), chTRAF5, chTRAF6 and a newly found member, chTRAF7, were identified and their cDNAs cloned. ChTRAF5, chTRAF6 and chTRAF7 had mRNA expression patterns, in non-lymphoid and lymphoid tissues and in a number of immune cells, similar to their orthologues in mammals. Interestingly, chTRAF2 has two variants, the full-length chTRAF2 and a novel isoform (chTRAF2S) lacking exon 4. ChTRAF2S lacks a portion of zinger finger one, all of zinc finger two and a portion of zinc finger three, producing a protein with a hybrid of zinc fingers 1 and 3 and intact zinc fingers 4 and 5. RT-PCR analyses indicated differential expression of both of the chTRAF2 isoforms in a number of non-lymphoid and lymphoid tissues, splenocyte subsets and in a kinetic study of ConA-stimulated splenocytes. ChTRAF2S is biologically active compared to chTRAF2, inducing higher levels of NF-κB activation. Co-transfections indicate that chTRAF2 may regulate chTRAF2S bioactivity as no synergistic effect was identified when cells were transfected with both isoforms. Knowledge gained from this study will help work to further dissect the interactions between chRANKL-expressing T cells and chRANK-expressing DC to drive Th1 immune responses and to understand how the chicken mounts an effective immune response while expressing a minimal essential repertoire of the TNF superfamily.