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Orthopaedic Proceedings
Vol. 102-B, Issue SUPP_7 | Pages 59 - 59
1 Jul 2020
Qiu H Cheng T Chim SM Zhu S Xu H Qin A Wang C Teguh D Zhang G Tickner J Yao F Vrielink A Smithers L Pavlos N Xu J
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Bone is a connective tissue that undergoes constant remodeling. Any disturbances during this process may result in undesired pathological conditions. A single nucleotide substitution (596T-A) in exon eight which leads to a M199K mutation in human RANKL was found to cause osteoclast-poor autosomal recessive osteopetrosis (ARO). Patients with ARO cannot be cured by hematopoietic stem cell transplantation and, without proper treatments, will die in their early age. To date, how this mutation alters RANKL function has not been characterized. We thus hypothesized that hRANKL M199 residue is a structural determinant for normal RANKL-RANK interaction and osteoclast differentiation. By sharing our findings, we aim to achieve an improved clinical outcome in treating bone-related diseases such as osteoporosis, ARO and osteoarthritis.

Site-directed mutagenesis was employed to create three rat RANKL mutants, replacing the methionine 200 (human M199 equivalent residue) with either lysine (M200K), alanine (M200A) or glutamic acid (M200E). Recombinant proteins were subsequently purified through affinity chromatography and visualized by Coomassie blue staining and western blot. MTS was carried out before osteoclastogenesis assay in vitro to measure the cellular toxicity. Bone resorption pit assay, immuno-fluorescent staining, luciferase reporter assay, RT-PCR, western blot and calcium oscillation detection were also conducted to explore the biological effect of rRANKL mutants. Computational modeling, thermal Shift Assay, western blot and protein binding affinity experiments were later carried out for structural analyses.

rRANKL mutants M200K/A/E showed a drastically reduced ability to induce osteoclast formation and did not demonstrate features of competitive inhibition against wild-type rRANKL. These mutants are all incapable of supporting osteoclastic polarization and bone resorption or activating RANKL-induced osteoclast marker gene transcription. Consistently, they were unable to induce calcium flux, and also showed a diminished induction of IκBa degradation and activation of NF-kB and NFATc1 transcriptional activity. Furthermore, the transcriptional activation of the antioxidant response element (ARE) crucial in modulating oxidative stress and providing cytoprotection was also unresponsive to stimulation with rM200s. Structural analyses showed that rM200 is located in a hydrophobic pocket critical for protein folding. Thermal shift and western blot assays suggested that rM200 mutants formed unstructured proteins, with disturbed trimerisation and the loss of affinity to its intrinsic receptors RANK and OPG.

Taken together, we first demonstrates the underlying cause of M199-meidated ARO in a cellular and molecular level by establishing a phenotype in BMMs similar to observed in human samples. Further investigation hints the structural significance of a hydrophobic pocket within the TNF-like region. Combined with pharmaceutical studies on small-molecule drugs, this finding may represent a therapeutic target motif for future development of anti-resorptive treatments.


Orthopaedic Proceedings
Vol. 91-B, Issue SUPP_II | Pages 349 - 350
1 May 2009
Xu J Pavlos N Cheng T Ang E Zheng M
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Osteoclastic bone resorption is a highly dynamic process that requires the tight ordering of intracellular trafficking events in order to maintain the structural and functional polarization of the ruffled border and basolateral domains. Rab3 proteins are a subfamily of GTPases, known to mediate membrane transport in eukaryotic cells and play a role in exocytosis. Our recent data indicates that Rab3D modulates a post-TGN trafficking step that is required for osteoclastic bone resorption (1). Here, to identify down-stream regulatory molecules of Rab3D, we have performed a yeast two-hybrid screen. Amongst several candidate Rab3D-interacting proteins identified, Rab3D was found to associate with calmodulin, an established regulator of osteoclastic bone resorption. As an initial effort to better define the interaction between Rab3D and calmodulin, we generated several mutants of Rab3D which interfere with the GDP/GTP nucleotide exchange (Rab3DQ81L, Rab3DN135I) and/or membrane attachment of Rab3D (Rab3D-CXC). By in vivo bioluminescence resonance energy transfer (BRET) assay, Calmodulin was found to associate equivalently with wild type Rab3D as well as Rab3DN135I and Rab-3DCXC variants. Over expression of constitutively active Rab3D (Rab3DQ81L) enhanced this interaction suggesting that the active form of Rab3D (i.e. GTP-bound) might recruit additional effector molecules which further potentiate it’s binding to calmodulin. In an attempt to address the impact of calmodulin activity on Rab3D-calmodulin interaction and osteoclastic bone resorption, we performed complementary BRET and in vitro bone resorption assays in the presence of the calmodulin inhibitor, calmidazolium chloride. Interestingly, we show that suppression of calmodulin activity via calmidazolium chloride impairs the association of Rab3D with calmodulin, an affect that correlates with a disruption in osteoclastic bone resorption. We propose that the recruitment of calmodulin by Rab3D might be an important requirement for osteoclast-mediated bone resorption.


Orthopaedic Proceedings
Vol. 91-B, Issue SUPP_II | Pages 348 - 348
1 May 2009
Cheng T Feng H Leong CL Pavlos N Zheng M Xu J
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Vacuolar adenosine triphosphatase (V-ATPase) proton pumps play an essential role in the acidification of the bone matrix during osteoclast-mediated bone resorption. Recently, mice lacking the V-ATPase d2 subunit have been shown to be osteopetrotic due to defective osteoclasts (Lee et al., Nature Med, 2006). Here, to investigate the transcriptional regulation of the d2 gene during RANKL-induced osteoclastogenesis, we have cloned and characterized its promoter region. By semi-quantitative RT-PCR, expression of d2 and NFATc1 was found to be strongly up-regulated by RANKL but not by other pro-osteoclastic factors including TNF, LPS and M-CSF. Bioinformatic analysis of the cloned 3 kb d2 promoter region revealed several candidate transcription factor binding sites including NFATc1, a key transcription factor for osteoclastogenesis.

To explore the influence of RANKL on d2 transcription, we generated a series of d2 promoter constructs using the pGL-3 reporter plasmid. Using luciferase assays, the d2 promoter was found to be induced by RANKL stimulation. Chromatin immunoprecipitation (ChIP) assays demonstrate that NFATc1 forms a complex with the d2 promoter. Using EMSA assays, we have defined a specific NFATc1 biding site between nucleotide − 555 to −561 upstream from the translation start site of d2 gene. Furthermore, targeted mutagenesis of the putative NFAT transcription binding site was found to significantly reduce the luciferase activity as induced by NFATc1 over expression. Addition of the NFAT inhibitor cyclosporin A was found to blunt the mRNA expression of d2 induced by RANKL in RAW264.7 cells.

We propose that NFATc1 is an important regulator of d2 transcription during RANKL-induced osteoclastogenesis.


Orthopaedic Proceedings
Vol. 91-B, Issue SUPP_II | Pages 349 - 349
1 May 2009
Lin Z Pavlos N Cake M Wood D Xu J Zheng M
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Calcitonin has been recently shown to have a direct protective effect on articular cartilage against joint degenerative disease. It has been proposed that calcitonin might act through the calcitonin receptor (CTR) to activate the cyclic AMP pathway and protect type II collagen degradation. In this study, we examined the presence of the CTR in human articular cartilage and chondrocytes and investigated the potential pharmacological effects and transduction pathway of salmon calcitonin in human chondrocytes.

Five human articular cartilage samples were examined for the expression of the CTR by polymerase chain reaction (PCR), immunostaining and Western blotting. Cyclic AMP levels in human chondrocyte stimulated with salmon calcitonin were measured by ELISA. The effect of salmon calcitonin on the gene expression profiles, including aggrecan, type II collagen, matrix metalloproteinase (MMP)-1, MMP-3 and MMP-13, of human chondrocytes was also examined by Real-time PCR.

It was shown that CTR was not detectable in human cartilage and chondrocytes. The cAMP level in human chondrocytes in vitro was significantly increased by forskolin (100μM) by > 10 fold (P< 0.001), but was not induced by salmon calcitonin (10^-7M, 10^-8M, 10^-9M). Real-time PCR demonstrated that salmon calcitonin tended to reduce the gene expression of MMPs, yet without statistical significance. In contrast to previous reports, our data showed that human cartilage and chondrocytes do not express calcitonin receptors. There was no direct effect of salmon calcitonin on human chondrocytes.

The result suggests that the chondroprotective effect of calcitonin observed in vivo may be indirect via its effect on subchondral bone resorptive activity.