Osteoclastogenesis is a tightly regulated biological process, and deregulation can lead to severe bone disorders such as osteoporosis. The regulation of osteoclastic signaling is incompletely understood, but ubiquitination of TNF receptor–associated factor 6 (TRAF6) has recently been shown to be important in mediating this process. We therefore investigated the role of the recently identified deubiquitinating enzyme CYLD in osteoclastogenesis and found that mice with a genetic deficiency of CYLD had aberrant osteoclast differentiation and developed severe osteoporosis. Cultured osteoclast precursors derived from CYLD-deficient mice were hyperresponsive to RANKL-induced differentiation and produced more and larger osteoclasts than did controls upon stimulation. We assessed the expression pattern of CYLD and found that it was drastically upregulated during RANKL-induced differentiation of preosteoclasts. Furthermore, CYLD negatively regulated RANK signaling by inhibiting TRAF6 ubiquitination and activation of downstream signaling events. Interestingly, we found that CYLD interacted physically with the signaling adaptor p62 and thereby was recruited to TRAF6. These findings establish CYLD as a crucial negative regulator of osteoclastogenesis and suggest its involvement in the p62/TRAF6 signaling axis.
Wei Jin, Mikyoung Chang, Emmanuel M. Paul, Geetha Babu, Andrew J. Lee, William Reiley, Ato Wright, Minying Zhang, Jun You, Shao-Cong Sun
Patients with X-linked hypophosphatemia (XLH) and the hyp-mouse, a model of XLH characterized by a deletion in the Phex gene, manifest hypophosphatemia, renal phosphate wasting, and rickets/osteomalacia. Cloning of the PHEX/Phex gene and mutations in affected patients and hyp-mice established that alterations in PHEX/Phex expression underlie XLH. Although PHEX/Phex expression occurs primarily in osteoblast lineage cells, transgenic Phex expression in hyp-mouse osteoblasts fails to rescue the phenotype, suggesting that Phex expression at other sites underlies XLH. To establish whether abnormal Phex in osteoblasts and/or osteocytes alone generates the HYP phenotype, we created mice with a global Phex knockout (Cre-PhexΔflox/y mice) and conditional osteocalcin-promoted (OC-promoted) Phex inactivation in osteoblasts and osteocytes (OC-Cre-PhexΔflox/y). Serum phosphorus levels in Cre-PhexΔflox/y, OC-Cre-PhexΔflox/y, and hyp-mice were lower than those in normal mice. Kidney cell membrane phosphate transport in Cre-PhexΔflox/y, OC-Cre-PhexΔflox/y, and hyp-mice was likewise reduced compared with that in normal mice. Abnormal renal phosphate transport in Cre-PhexΔflox/y and OC-Cre-PhexΔflox/y mice was associated with increased bone production and serum FGF-23 levels and decreased kidney membrane type IIa sodium phosphate cotransporter protein, as was the case in hyp-mice. In addition, Cre-PhexΔflox/y, OC-Cre-PhexΔflox/y, and hyp-mice manifested comparable osteomalacia. These data provide evidence that aberrant Phex function in osteoblasts and/or osteocytes alone is sufficient to underlie the hyp-mouse phenotype.
Baozhi Yuan, Masanori Takaiwa, Thomas L. Clemens, Jian Q. Feng, Rajiv Kumar, Peter S. Rowe, Yixia Xie, Marc K. Drezner
Aggrecan loss from cartilage in arthritis is mediated by aggrecanases. Aggrecanases cleave aggrecan preferentially in the chondroitin sulfate–2 (CS-2) domain and secondarily at the E373↓374A bond in the interglobular domain (IGD). However, IGD cleavage may be more deleterious for cartilage biomechanics because it releases the entire CS-containing portion of aggrecan. Recent studies identifying aggrecanase-2 (ADAMTS-5) as the predominant aggrecanase in mouse cartilage have not distinguished aggrecanolysis in the IGD from aggrecanolysis in the CS-2 domain. We generated aggrecan knockin mice with a mutation that rendered only the IGD resistant to aggrecanases in order to assess the contribution of this specific cleavage to cartilage pathology. The knockin mice were viable and fertile. Aggrecanase cleavage in the aggrecan IGD was not detected in knockin mouse cartilage in situ nor following digestion with ADAMTS-5 or treatment of cartilage explant cultures with IL-1α. Blocking cleavage in the IGD not only diminished aggrecan loss and cartilage erosion in surgically induced osteoarthritis and a model of inflammatory arthritis, but appeared to stimulate cartilage repair following acute inflammation. We conclude that blocking aggrecanolysis in the aggrecan IGD alone protects against cartilage erosion and may potentiate cartilage repair.
Christopher B. Little, Clare T. Meeker, Suzanne B. Golub, Kate E. Lawlor, Pamela J. Farmer, Susan M. Smith, Amanda J. Fosang
This study illustrates that Plekhm1 is an essential protein for bone resorption, as loss-of-function mutations were found to underlie the osteopetrotic phenotype of the incisors absent rat as well as an intermediate type of human osteopetrosis. Electron and confocal microscopic analysis demonstrated that monocytes from a patient homozygous for the mutation differentiated into osteoclasts normally, but when cultured on dentine discs, the osteoclasts failed to form ruffled borders and showed little evidence of bone resorption. The presence of both RUN and pleckstrin homology domains suggests that Plekhm1 may be linked to small GTPase signaling. We found that Plekhm1 colocalized with Rab7 to late endosomal/lysosomal vesicles in HEK293 and osteoclast-like cells, an effect that was dependent on the prenylation of Rab7. In conclusion, we believe PLEKHM1 to be a novel gene implicated in the development of osteopetrosis, with a putative critical function in vesicular transport in the osteoclast.
Liesbeth Van Wesenbeeck, Paul R. Odgren, Fraser P. Coxon, Annalisa Frattini, Pierre Moens, Bram Perdu, Carole A. MacKay, Els Van Hul, Jean-Pierre Timmermans, Filip Vanhoenacker, Ruben Jacobs, Barbara Peruzzi, Anna Teti, Miep H. Helfrich, Michael J. Rogers, Anna Villa, Wim Van Hul
Paget disease is the most exaggerated example of abnormal bone remodeling, with the primary cellular abnormality in the osteoclast. Mutations in the p62 (sequestosome 1) gene occur in one-third of patients with familial Paget disease and in a minority of patients with sporadic Paget disease, with the P392L amino acid substitution being the most commonly observed mutation. However, it is unknown how p62P392L mutation contributes to the development of this disease. To determine the effects of p62P392L expression on osteoclasts in vitro and in vivo, we introduced either the p62P392L or WT p62 gene into normal osteoclast precursors and targeted p62P392L expression to the osteoclast lineage in transgenic mice. p62P392L-transduced osteoclast precursors were hyperresponsive to receptor activator of NF-κB ligand (RANKL) and TNF-α and showed increased NF-κB signaling but did not demonstrate increased 1,25-(OH)2D3 responsivity, TAFII-17 expression, or nuclear number per osteoclast. Mice expressing p62P392L developed increased osteoclast numbers and progressive bone loss, but osteoblast numbers were not coordinately increased, as is seen in Paget disease. These results indicate that p62P392L expression on osteoclasts is not sufficient to induce the full pagetic phenotype but suggest that p62 mutations cause a predisposition to the development of Paget disease by increasing the sensitivity of osteoclast precursors to osteoclastogenic cytokines.
Noriyoshi Kurihara, Yuko Hiruma, Hua Zhou, Mark A. Subler, David W. Dempster, Frederick R. Singer, Sakamuri V. Reddy, Helen E. Gruber, Jolene J. Windle, G. David Roodman
T cell–produced cytokines play a pivotal role in the bone loss caused by inflammation, infection, and estrogen deficiency. IFN-γ is a major product of activated T helper cells that can function as a pro- or antiresorptive cytokine, but the reason why IFN-γ has variable effects in bone is unknown. Here we show that IFN-γ blunts osteoclast formation through direct targeting of osteoclast precursors but indirectly stimulates osteoclast formation and promotes bone resorption by stimulating antigen-dependent T cell activation and T cell secretion of the osteoclastogenic factors RANKL and TNF-α. Analysis of the in vivo effects of IFN-γ in 3 mouse models of bone loss — ovariectomy, LPS injection, and inflammation via silencing of TGF-β signaling in T cells — reveals that the net effect of IFN-γ in these conditions is that of stimulating bone resorption and bone loss. In summary, IFN-γ has both direct anti-osteoclastogenic and indirect pro-osteoclastogenic properties in vivo. Under conditions of estrogen deficiency, infection, and inflammation, the net balance of these 2 opposing forces is biased toward bone resorption. Inhibition of IFN-γ signaling may thus represent a novel strategy to simultaneously reduce inflammation and bone loss in common forms of osteoporosis.
Yuhao Gao, Francesco Grassi, Michaela Robbie Ryan, Masakazu Terauchi, Karen Page, Xiaoying Yang, M. Neale Weitzmann, Roberto Pacifici
Genomic actions induced by 1α25-dihydroxyvitamin D3 [1,25(OH)2D3] are crucial for normal bone metabolism, mainly because they regulate active intestinal calcium transport. To evaluate whether the vitamin D receptor (VDR) has a specific role in growth-plate development and endochondral bone formation, we investigated mice with conditional inactivation of VDR in chondrocytes. Growth-plate chondrocyte development was not affected by the lack of VDR. Yet vascular invasion was impaired, and osteoclast number was reduced in juvenile mice, resulting in increased trabecular bone mass. In vitro experiments confirmed that VDR signaling in chondrocytes directly regulated osteoclastogenesis by inducing receptor activator of NF-κB ligand (RANKL) expression. Remarkably, mineral homeostasis was also affected in chondrocyte-specific VDR-null mice, as serum phosphate and 1,25(OH)2D levels were increased in young mice, in whom growth-plate activity is important. Both in vivo and in vitro analysis indicated that VDR inactivation in chondrocytes reduced the expression of FGF23 by osteoblasts and consequently led to increased renal expression of 1α-hydroxylase and of sodium phosphate cotransporter type IIa. Taken together, our findings provide evidence that VDR signaling in chondrocytes is required for timely osteoclast formation during bone development and for the endocrine action of bone in phosphate homeostasis.
Ritsuko Masuyama, Ingrid Stockmans, Sophie Torrekens, Riet Van Looveren, Christa Maes, Peter Carmeliet, Roger Bouillon, Geert Carmeliet
Excessive bone loss in arthritic diseases is mostly due to abnormal activation of the immune system leading to stimulation of osteoclasts. While phospholipase Cγ (PLCγ) isoforms are known modulators of T and B lymphocyte–mediated immune responses, we found that blockade of PLCγ enzymatic activity also blocks early osteoclast development and function. Importantly, targeted deletion of Plcg2 in mice led to an osteopetrotic phenotype. PLCγ2, independent of PLCγ1, was required for receptor activator of NF-κB ligand–induced (RANKL-induced) osteoclastogenesis by differentially regulating nuclear factor of activated T cells c1 (NFATc1), activator protein–1 (AP1), and NF-κB. Specifically, we show that NFATc1 upregulation is dependent on RANKL-mediated phosphorylation of PLCγ2 downstream of Dap12/Fc receptor γ (Dap12/FcRγ) receptors and is blocked by the PLCγ inhibitor U73122. In contrast, activation of JNK and NF-κB was not affected by U73122 or Dap12/FcRγ deletion. Interestingly, we found that in osteoclasts, PLCγ2 formed a complex with the regulatory adapter molecule GAB2, was required for GAB2 phosphorylation, and modulated GAB2 recruitment to RANK. Thus, PLCγ2 mediates RANKL-induced osteoclastogenesis and is a potential candidate for antiresorptive therapy.
Dailing Mao, Holly Epple, Brian Uthgenannt, Deborah V. Novack, Roberta Faccio
Individuals with neurofibromatosis type 1 (NF1) have a high incidence of osteoporosis and osteopenia. However, understanding of the cellular and molecular basis of these sequelae is incomplete. Osteoclasts are specialized myeloid cells that are the principal bone-resorbing cells of the skeleton. We found that Nf1+/– mice contain elevated numbers of multinucleated osteoclasts. Both osteoclasts and osteoclast progenitors from Nf1+/– mice were hyperresponsive to limiting concentrations of M-CSF and receptor activator of NF-κB ligand (RANKL) levels. M-CSF–stimulated p21ras-GTP and Akt phosphorylation was elevated in Nf1+/– osteoclasts associated with gains of function in survival, proliferation, migration, adhesion, and lytic activity. These gains of function are associated with more severe bone loss following ovariectomy as compared with that in syngeneic WT mice. Intercrossing Nf1+/– mice and mice deficient in class 1A PI3K (p85α) restored elevated PI3K activity and Nf1+/– osteoclast functions to WT levels. Furthermore, in vitro–differentiated osteoclasts from NF1 patients also displayed elevated Ras/PI3K activity and increased lytic activity analogous to those in murine Nf1+/– osteoclasts. Collectively, our results identify a what we believe to be a novel cellular and biochemical NF1-haploinsufficient phenotype in osteoclasts that has potential implications for the pathogenesis of NF1 bone disease.
Feng-Chun Yang, Shi Chen, Alexander G. Robling, Xijie Yu, Todd D. Nebesio, Jincheng Yan, Trent Morgan, Xiaohong Li, Jin Yuan, Janet Hock, David A. Ingram, D. Wade Clapp
The use of estrogens and androgens to prevent bone loss is limited by their unwanted side effects, especially in reproductive organs and breast. Selective estrogen receptor modulators (SERMs) partially avoid such unwanted effects, but their efficacy on bone is only moderate compared with that of estradiol or androgens. Estrens have been suggested to not only prevent bone loss but also exert anabolic effects on bone while avoiding unwanted effects on reproductive organs. In this study, we compared the effects of a SERM (PSK3471) and 2 estrens (estren-α and estren-β) on bone and reproductive organs to determine whether estrens are safe and act via the estrogen receptors and/or the androgen receptor (AR). Estrens and PSK3471 prevented gonadectomy-induced bone loss in male and female mice, but none showed true anabolic effects. Unlike SERMs, the estrens induced reproductive organ hypertrophy in both male and female mice and enhanced MCF-7 cell proliferation in vitro. Estrens directly activated transcription in several cell lines, albeit at much higher concentrations than estradiol or the SERM, and acted for the most part through the AR. We conclude that the estrens act mostly through the AR and, in mice, do not fulfill the preclinical efficacy or safety criteria required for the treatment or prevention of osteoporosis.
Sara H. Windahl, René Galien, Riccardo Chiusaroli, Philippe Clément-Lacroix, Frederic Morvan, Liên Lepescheux, François Nique, William C. Horne, Michèle Resche-Rigon, Roland Baron