Osteoporosis results from an imbalance in skeletal remodeling that favors bone resorption over bone formation. Bone matrix is degraded by osteoclasts, which differentiate from myeloid precursors in response to the cytokine RANKL. To gain insight into the transcriptional regulation of bone resorption during growth and disease, we generated a conditional knockout of the transcription factor nuclear factor of activated T cells c1 (Nfatc1). Deletion of Nfatc1 in young mice resulted in osteopetrosis and inhibition of osteoclastogenesis in vivo and in vitro. Transcriptional profiling revealed NFATc1 as a master regulator of the osteoclast transcriptome, promoting the expression of numerous genes needed for bone resorption. In addition, NFATc1 directly repressed osteoclast progenitor expression of osteoprotegerin, a decoy receptor for RANKL previously thought to be an osteoblast-derived inhibitor of bone resorption. “Cherubism mice”, which carry a gain-of-function mutation in SH3-domain binding protein 2 (Sh3bp2), develop osteoporosis and widespread inflammation dependent on the proinflammatory cytokine, TNF-α. Interestingly, deletion of Nfatc1 protected cherubism mice from systemic bone loss but did not inhibit inflammation. Taken together, our study demonstrates that NFATc1 is required for remodeling of the growing and adult skeleton and suggests that NFATc1 may be an effective therapeutic target for osteoporosis associated with inflammatory states.
Antonios O. Aliprantis, Yasuyoshi Ueki, Rosalyn Sulyanto, Arnold Park, Kirsten S. Sigrist, Sudarshana M. Sharma, Michael C. Ostrowski, Bjorn R. Olsen, Laurie H. Glimcher
The Sox9 transcription factor plays an essential role in promoting chondrogenesis and regulating expression of chondrocyte extracellular-matrix genes. To identify genes that interact with Sox9 in promoting chondrocyte differentiation, we screened a cDNA library generated from the murine chondrogenic ATDC5 cell line to identify activators of the collagen, type II, α 1 (Col2a1) promoter. Here we have shown that paraspeckle regulatory protein 54-kDa nuclear RNA-binding protein (p54nrb) is an essential link between Sox9-regulated transcription and maturation of Sox9-target gene mRNA. We found that p54nrb physically interacted with Sox9 and enhanced Sox9-dependent transcriptional activation of the Col2a1 promoter. In ATDC5 cells, p54nrb colocalized with Sox9 protein in nuclear paraspeckle bodies, and knockdown of p54nrb suppressed Sox9-dependent Col2a1 expression and promoter activity. We generated a p54nrb mutant construct lacking RNA recognition motifs, and overexpression of mutant p54nrb in ATDC5 cells markedly altered the appearance of paraspeckle bodies and inhibited the maturation of Col2a1 mRNA. The mutant p54nrb inhibited chondrocyte differentiation of mesenchymal cells and mouse metatarsal explants. Furthermore, transgenic mice expressing the mutant p54nrb in the chondrocyte lineage exhibited dwarfism associated with impairment of chondrogenesis. These data suggest that p54nrb plays an important role in the regulation of Sox9 function and the formation of paraspeckle bodies during chondrogenesis.
Kenji Hata, Riko Nishimura, Shuji Muramatsu, Akio Matsuda, Takuma Matsubara, Katsuhiko Amano, Fumiyo Ikeda, Vincent R. Harley, Toshiyuki Yoneda
cGMP-dependent protein kinase II (cGKII; encoded by PRKG2) is a serine/threonine kinase that is critical for skeletal growth in mammals; in mice, cGKII deficiency results in dwarfism. Using radiographic analysis, we determined that this growth defect was a consequence of an elongated growth plate and impaired chondrocyte hypertrophy. To investigate the mechanism of cGKII-mediated chondrocyte hypertrophy, we performed a kinase substrate array and identified glycogen synthase kinase–3β (GSK-3β; encoded by Gsk3b) as a principal phosphorylation target of cGKII. In cultured mouse chondrocytes, phosphorylation-mediated inhibition of GSK-3β was associated with enhanced hypertrophic differentiation. Furthermore, cGKII induction of chondrocyte hypertrophy was suppressed by cotransfection with a phosphorylation-deficient mutant of GSK-3β. Analyses of mice with compound deficiencies in both protein kinases (Prkg2–/–Gsk3b+/–) demonstrated that the growth retardation and elongated growth plate associated with cGKII deficiency were partially rescued by haploinsufficiency of Gsk3b. We found that β-catenin levels decreased in Prkg2–/– mice, while overexpression of cGKII increased the accumulation and transactivation function of β-catenin in mouse chondroprogenitor ATDC5 cells. This effect was blocked by coexpression of phosphorylation-deficient GSK-3β. These data indicate that hypertrophic differentiation of growth plate chondrocytes during skeletal growth is promoted by phosphorylation and inactivation of GSK-3β by cGKII.
Yosuke Kawasaki, Fumitaka Kugimiya, Hirotaka Chikuda, Satoru Kamekura, Toshiyuki Ikeda, Naohiro Kawamura, Taku Saito, Yusuke Shinoda, Akiro Higashikawa, Fumiko Yano, Toru Ogasawara, Naoshi Ogata, Kazuto Hoshi, Franz Hofmann, James R. Woodgett, Kozo Nakamura, Ung-il Chung, Hiroshi Kawaguchi
Osteoclasts (OCs) function to reabsorb bone and are responsible for the bone loss associated with inflammatory arthritis and osteoporosis. OC numbers are elevated in most disorders of accelerated bone destruction, reflecting altered rates of precursor differentiation and apoptosis. Both of these processes are regulated by the JNK family of MAP kinases. In this study, we have demonstrated that the NF-κB subunit RelA/p65 inhibits JNK-mediated apoptosis during a critical period of commitment to the OC phenotype in response to the cytokine RANKL. This RelA/p65-mediated arrest of cell death led to enhanced OC differentiation. Hence, Rela–/– OC precursors displayed prolonged JNK activation in response to RANKL, and this was accompanied by an increase in cell death that prevented efficient differentiation. Although complete blockade of JNK activity inhibits osteoclastogenesis, both short-term blockade in RelA-deficient cultures and suppression of the downstream mediator, Bid rescued apoptosis and differentiation. These antiapoptotic effects were RelA specific, as overexpression of RelA, but not RelB, blocked apoptosis and rescued differentiation in Rela–/– precursors. Thus, RelA blocks a RANKL-induced, apoptotic JNK-Bid pathway, thereby promoting OC differentiation. Consistent with this, mice lacking RelA/p65 in the hematopoietic compartment were shown to have a deficient osteoclastogenic response to RANKL and were protected from arthritis-induced osteolysis.
Sergio Vaira, Muhammad Alhawagri, Imani Anwisye, Hideki Kitaura, Roberta Faccio, Deborah Veis Novack
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