Functions of receptor activator of NF-κB ligand (RANKL) and its receptors, RANK and OPG, are evolutionarily conserved
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Sutton2014.docx (11.72Mb)
Date
28/11/2014Author
Sutton, Kate Maurice
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Abstract
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.