Osteoclasts are multinucleated cells found exclusively in bone and are derived from the haematopoietic cells of monocytes/macrophage lineage. The cell-to-cell interaction between osteoblastic/stromal cells and osteoclast precursor cells is necessary for osteoclastogenesis. Receptor Activator of NF-κB ligand (RANKL) was identified as a membrane-bound TNF ligand family member that is the ‘master’ cytokine expressed on osteoblastic/stromal cells, which stimulate osteoclastogenesis through cell-to-cell contact with osteoclast precursors. RANKL is considered to be a factor that is necessary and sufficient for the induction of osteoclastogenesis (Lacey, et al., 1998). RANKL is a type II transmembrane cytokine of the TNF ligand superfamily and has an active TNF-like core domain at the extracellular domain. This active TNF-like core domain is thought to be the region through which it binds to it’s active receptor, RANK, for the activation of signal transduction pathways for the initiation of processes leading to osteoclastogenesis (Lacey, et al., 1998; Li, et al., 1999). It was hypothesized that any change in the active TNF-like core domain might affect the ability of RANKL binding to RANK and consequently affect the activation of signal transduction pathways and osteoclastogenesis. Hence, this thesis sought to investigate the effects of changes in the active TNF-like core domain by truncation mutation on the ability of RANKL binding to RANK and consequently affect the activation of signal transduction pathways and osteoclastogenesis. A cDNA fragment encoding the full-length TNF-like core domain of rat RANKL (rRANKL) (aa160-318) was cloned into the bacterial expression pGEX vectors and stably expressed in Eschechia coli as a fusion protein with the C-terminus of glutathione S-transferase (GST). Four mutants (aa160-302, aa160-268, aa239-318 and aa246-318) were also generated by truncation mutation in the TNF-like core domain, and cloned into the pGEX vector to produce GST-rRANKL mutants. The proteins were over-expressed and affinity purified to 95% in purity. GST-rRANKL (160-318) containing the full length TNF-like core domain was able to induced osteoclastogenesis in spleen cells in the presence of M-CSF and in RAW264.7 cells in the absence of M-CSF. It was also found to activate mature osteoclast activity in vitro, ex vivo and in vivo. It has the highest binding affinity to RANK and the greatest potency for NF-κB activation as well as the induction of osteoclastogenesis compared to the truncated mutants. Mutants generated by truncation of the TNF-like core domain revealed that the TNF-like core domain is important for the interaction with the RANK, for high binding affinity, NF-κB activation and induction of osteoclastogenesis. In general, the truncated mutants not only displayed a reduction in the binding affinity to RANK, but also a reduction in NF-κB activation, and significantly reduced potency in the induction of osteoclastogenesis. Interestingly, mutant GST-rRANKL (160-268) showed a higher affectivity than the other mutants did, in that it had greater binding affinity to RANK, and in NF-κB activation than the rest of the truncated mutants. Mutants GST-rRANKL (239-318) and GST-rRANKL (246-318) on the other hand, showed little potency in the induction of osteoclast formation, however, might have an inhibitory effect through competition with full length GST-rRANKL (160-318) as well as inducing a response in vivo resulting in an increase in the serum calcium level. In conclusion, this thesis demonstrated that the TNF-like core domain of RANKL is active, and imperative in the binding to RANK, activating signal transduction pathways and induction of osteoclastogenesis. Changes in the active TNF-like core domain affected the ability, affinity and efficiency of RANKL binding to the receptor, RANK and consequently affected the activation of signal transduction pathways and osteoclastogenesis.
| Identifer | oai:union.ndltd.org:ADTP/221008 |
| Date | January 2003 |
| Creators | Tan, Jamie We-Yin |
| Publisher | University of Western Australia. School of Surgery and Pathology |
| Source Sets | Australiasian Digital Theses Program |
| Language | English |
| Detected Language | English |
| Rights | Copyright Jamie We-Yin Tan, http://www.itpo.uwa.edu.au/UWA-Computer-And-Software-Use-Regulations.html |
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