Cloning, Sequencing, and Functional Characterization of the Rat Homologue of Receptor Activator of NF-κB Ligand

Xu, Jiake; Tan, Jamie; Huang, Lin; Gao, Xiuhui; Laird, Rebecca; Liú, Dan; Wysocki, S.J.; Zheng, Minghao

doi:10.1359/jbmr.2000.15.11.2178

Cited by 156 publications

(162 citation statements)

References 36 publications

(89 reference statements)

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“…Restriction enzymes and reverse transcriptase were purchased from Promega (Sydney, Australia), and DNA polymerase was from GeneWorks (Adelaide, Australia). GST-rRANKL 160 -318 recombinant proteins were expressed and purified in our laboratory as previously described (29). Anti-GFP rabbit polyclonal antibodies were obtained from Abcam (Sapphire Bioscience Pty.…”

Section: Methodsmentioning

confidence: 99%

Cytoplasmic Terminus of Vacuolar Type Proton Pump Accessory Subunit Ac45 Is Required for Proper Interaction with V0 Domain Subunits and Efficient Osteoclastic Bone Resorption

Feng

Cheng

Pavlos

et al. 2008

Journal of Biological Chemistry

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Solubilization of mineralized bone by osteoclasts is largely dependent on the acidification of the extracellular resorption lacuna driven by the vacuolar (H؉)-ATPases (V-ATPases)polarized within the ruffled border membranes. V-ATPases consist of two functionally and structurally distinct domains, V 1 and V 0 . The peripheral cytoplasmically oriented V 1 domain drives ATP hydrolysis, which necessitates the translocation of protons across the integral membrane bound V 0 domain. Here, we demonstrate that an accessory subunit, Ac45, interacts with the V 0 domain and contributes to the vacuolar type proton pump-mediated function in osteoclasts. Consistent with its role in intracellular acidification, Ac45 was found to be localized to the ruffled border region of polarized resorbing osteoclasts and enriched in pH-dependent endosomal compartments that polarized to the ruffled border region of actively resorbing osteoclasts. Interestingly, truncation of the 26-amino acid residue cytoplasmic tail of Ac45, which encodes an autonomous internalization signal, was found to impair bone resorption in vitro. Furthermore, biochemical analysis revealed that although both wild type Ac45 and mutant were capable of associating with subunits a3, c, c؆, and d, deletion of the cytoplasmic tail altered its binding proximity with a3, c؆, and d. In all, our data suggest that the cytoplasmic terminus of Ac45 contains elements necessary for its proper interaction with V 0 domain and efficient osteoclastic bone resorption.Osteoclasts are terminally differentiated multinucleated cells derived from the hematopoietic lineage that specialize in the solubilization of mineralized bone material (1). Upon attachment to the bone surface, the osteoclast undergoes a series of cytoskeletal reorganization and polarization events that culminate in the formation of a unique apical membrane known as the ruffled border. The ruffled border is the "resorptive organelle" of the osteoclast and is formed by the polarized targeting and fusion of acidified intracellular vesicles with the plasma membrane (2-4). These transport vesicles courier acidifying machineries as well as osteolytic enzymes such as, TRACP, cathepsin K, and matrix metalloproteinases (2, 5-7) to the ruffled border membrane domain where each cargo plays a discrete role in the resorptive function. Vacuolar H ϩ -adenosine triphosphatases (V-ATPases) 5 that line the ruffled border have long been established to play a vital role in the resorptive process. V-ATPases function to pump H ϩ into the underlying resorptive lacunae. This elevation in protons results in a highly acidified microenvironment that solubilizes the mineralized component of bone while providing optimal conditions for the degradation of the exposed organic phase by collagenolytic enzymes such cathepsin K (2, 7).The importance of V-ATPase in osteoclastic bone resorption is exemplified by studies employing specific inhibitors and gene knock-out strategies to impair V-ATPase functions (8 -11). Furthermore, mutations in the human TCIRG1 g...

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Section: Methodsmentioning

confidence: 99%

Cytoplasmic Terminus of Vacuolar Type Proton Pump Accessory Subunit Ac45 Is Required for Proper Interaction with V0 Domain Subunits and Efficient Osteoclastic Bone Resorption

Feng

Cheng

Pavlos

et al. 2008

Journal of Biological Chemistry

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“…Recombinant rat GSTrRANKL was expressed and purified as previously described. (10) TPA, LPS, recombinant TNF-␣, interleukin (IL)-1␤, and Goat anti-mouse IgG-conjugated horseradish peroxidase were obtained from Sigma (Sydney, Australia). Polyclonal antibodies to NF-B1 (p50), Rel-A (p65), c-Rel, and B-Rel, and antibodies to phospho-specific p44/42 ERK and p38 were obtained from Santa Cruz Biotechnology (Santa Cruz, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

“…(8) In addition, RANKL was able to induce mature osteoclast activation in vitro (9) and osteoclast polarization and hypercalcemia in vivo. (9,10) RANKLinduced osteoclastogenesis is mediated through its receptor, RANK. The interaction of RANKL with RANK leads to the recruitment of members of the TNF receptor associated factor (TRAF) adapter proteins and the activation of the NF-〉 and c-Jun N-terminal kinase (JNK) signaling pathways.…”

Section: Introductionmentioning

confidence: 99%

12-O-tetradecanoylphorbol-13-acetate (TPA) Inhibits Osteoclastogenesis by Suppressing RANKL-Induced NF-κB Activation

Wang

Steer

Joyce

et al. 2003

Journal of Bone and Mineral Research

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The mechanism by which TPA-induced PKC activity modulates osteoclastogenesis is not clear. Using a RAW 264.7 cell culture system and assays for NF-B nuclear translocation, NF-B reporter gene activity, and MAPK assays, we demonstrated that TPA inhibits osteoclastogenesis through the suppression of RANKLinduced NF-〉 activation. Introduction:The protein kinase C (PKC) pathway has been suggested to be an important regulator of osteoclastic bone resorption. The role of PKC in RANKL-induced osteoclastogenesis, however, is not clear. In this study, we examined the effects of 12-O-tetradecanoylphorbol-13-acetate (TPA), a PKC activator, on osteoclastogenesis and studied its role in RANKL-induced signaling. Materials and Methods: RANKL-induced RAW 264.7 cell differentiation into osteoclast-like cells was used to assess the effect of TPA on osteoclastogenesis. Assays for NF-B nuclear translocation, NF-B reporter gene activity, protein kinase activity, and Western blotting were used to examine the effects of TPA on RANKL-induced NF-〉, c-Jun N-terminal kinase (JNK), and MEK/ERK and p38 signal transduction pathways. Results: We found that TPA inhibited RANKL-induced RAW 264.7 cell differentiation into osteoclasts in a dosedependent manner. Time course analysis showed that the inhibitory effect of TPA on RANKL-induced osteoclastogenesis occurs predominantly at an early stage of osteoclast differentiation. TPA alone had little effect on NF-〉 activation in RAW 264.7 cells, but it suppresses the RANKL-induced NF-〉 activation in a dose-dependent fashion. Interestingly, the suppressive effect of TPA on RANKL-induced NF-〉 activation was prevented by a conventional PKC inhibitor, Go6976. Supershift studies revealed that the RANKL-induced DNA binding of NF-〉 complexes consisted of C-Rel, NF-B1 (p50), and RelA (p65). In addition, TPA induced the activation of JNK in RAW 264.7 cells but had little effect on RANKL-induced activation of JNK. TPA also inhibited RANKL-induced activation of ERK but had little effect on p38 activation. Conclusion: Given that NF-B activation is obligatory for osteoclast differentiation, our studies imply that inhibition of osteoclastogenesis by TPA is, at least in part, caused by the suppression of RANKL-induced activation of NF-〉 during an early stage of osteoclastogenesis. Selective modulation of RANKL signaling pathways by PKC activators may have important therapeutic implications for the treatment of bone diseases associated with enhanced bone resorption.

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“…Many researchers have shown that RANKL is directly involved in the differentiation of macrophages into osteoclasts (Wang et al, 2003;Kukita and Kukita, 2013). RANKL is able to activate mature osteoclasts (Burgess et al, 1999), and induce osteoclast polarization and hypercalcemia (Xu et al, 2000). During stimulation with RANKL, the receptor RANK binds to signaling adaptor molecules such as tumor necrosis factor receptorassociated factor adapter proteins (Keating et al, 2007), which leads to the activation of the NFkappaB and c-Jun N-terminal kinase signaling pathways (Darnay et al, 1998).…”

Section: Introductionmentioning

confidence: 99%

Inhibition of andrographolide in RAW 264.7 murine macrophage osteoclastogenesis by downregulating the nuclear factor-kappaB signaling pathway

Ren

Yuan

2015

Genet. Mol. Res.

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ABSTRACT. This study aims to investigate the effects of andrographolide (AGP) on osteoclast formation in RAW 264.7 murine macrophage cells. The effects of AGP on cell viability were determined in RAW 264.7 cells using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The effects of AGP on osteoclast formation were tested by osteoclast staining with tartrate-resistant acid phosphatase (TRAP). The effects of AGP on receptor activator of nuclear factor-kappaB (NFkappaB) ligand (RANKL)-induced, NF-kappaB-dependent transcription in RAW 264.7 cells were assessed using luciferase reporter assays. The results demonstrated that the viability of osteoclast precursor RAW 264.7 cells was not affected by AGP treatment at a concentration of 0.4 to 10 μM. Additionally, the number of TRAP-positive osteoclasts was significantly reduced by the same concentrations of AGP treatment. AGP also inhibited RANKL-induced NF-kappaB activation in a dose-dependent 15955-15961 (2015) fashion as evidenced by luciferase reporter assays. In summary, this study demonstrates that AGP inhibits osteoclastogenesis in RAW 264.7 murine macrophage cells through downregulation of the NF-kappaB signaling pathway.

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Cloning, Sequencing, and Functional Characterization of the Rat Homologue of Receptor Activator of NF-κB Ligand

Cited by 156 publications

References 36 publications

Cytoplasmic Terminus of Vacuolar Type Proton Pump Accessory Subunit Ac45 Is Required for Proper Interaction with V0 Domain Subunits and Efficient Osteoclastic Bone Resorption

Cytoplasmic Terminus of Vacuolar Type Proton Pump Accessory Subunit Ac45 Is Required for Proper Interaction with V0 Domain Subunits and Efficient Osteoclastic Bone Resorption

12-O-tetradecanoylphorbol-13-acetate (TPA) Inhibits Osteoclastogenesis by Suppressing RANKL-Induced NF-κB Activation

Inhibition of andrographolide in RAW 264.7 murine macrophage osteoclastogenesis by downregulating the nuclear factor-kappaB signaling pathway

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