Bone undergoes continuous remodeling due to balanced bone formation and resorption mediated by osteoblasts and osteoclasts, respectively. Osteoclasts arise from the macrophage lineage, and their differentiation is dependent on RANKL, a member of the TNF family of cytokines. Here, we have provided evidence that RANKL controls the expression of 3BP2, an adapter protein that is required for activation of SRC tyrosine kinase and simultaneously coordinates the attenuation of β-catenin, both of which are required to execute the osteoclast developmental program. We found that RANKL represses the transcription of the E3 ubiquitin ligase
Yoshinori Matsumoto, Jose Larose, Oliver A. Kent, Melissa Lim, Adele Changoor, Lucia Zhang, Yaryna Storozhuk, Xiaohong Mao, Marc D. Grynpas, Feng Cong, Robert Rottapel
Shohat-type spondyloepimetaphyseal dysplasia (SEMD) is a skeletal dysplasia that affects cartilage development. Similar skeletal disorders, such as spondyloepiphyseal dysplasias, are linked to mutations in type II collagen (COL2A1), but the causative gene in SEMD is not known. Here, we have performed whole-exome sequencing to identify a recurrent homozygous c.408+1G>A donor splice site loss-of-function mutation in DDRGK domain containing 1 (
Adetutu T. Egunsola, Yangjin Bae, Ming-Ming Jiang, David S. Liu, Yuqing Chen-Evenson, Terry Bertin, Shan Chen, James T. Lu, Lisette Nevarez, Nurit Magal, Annick Raas-Rothschild, Eric C. Swindell, Daniel H. Cohn, Richard A. Gibbs, Philippe M. Campeau, Mordechai Shohat, Brendan H. Lee
Cellular identity in metazoan organisms is frequently established through lineage-specifying transcription factors, which control their own expression through transcriptional positive feedback, while antagonizing the developmental networks of competing lineages. Here, we have uncovered a distinct positive feedback loop that arises from the reciprocal stabilization of the tyrosine kinase ABL and the transcriptional coactivator TAZ. Moreover, we determined that this loop is required for osteoblast differentiation and embryonic skeletal formation. ABL potentiated the assembly and activation of the RUNX2-TAZ master transcription factor complex that is required for osteoblastogenesis, while antagonizing PPARγ-mediated adipogenesis. ABL also enhanced TAZ nuclear localization and the formation of the TAZ-TEAD complex that is required for osteoblast expansion. Last, we have provided genetic data showing that regulation of the ABL-TAZ amplification loop lies downstream of the adaptor protein 3BP2, which is mutated in the craniofacial dysmorphia syndrome cherubism. Our study demonstrates an interplay between ABL and TAZ that controls the mesenchymal maturation program toward the osteoblast lineage and is mechanistically distinct from the established model of lineage-specific maturation.
Yoshinori Matsumoto, Jose La Rose, Oliver A. Kent, Melany J. Wagner, Masahiro Narimatsu, Aaron D. Levy, Mitchell H. Omar, Jiefei Tong, Jonathan R. Krieger, Emily Riggs, Yaryna Storozhuk, Julia Pasquale, Manuela Ventura, Behzad Yeganeh, Martin Post, Michael F. Moran, Marc D. Grynpas, Jeffrey L. Wrana, Giulio Superti-Furga, Anthony J. Koleske, Ann Marie Pendergast, Robert Rottapel
Joints that have degenerated as a result of aging or injury contain dead chondrocytes and damaged cartilage. Some studies have suggested that chondrocyte death precedes cartilage damage, but how the loss of chondrocytes affects cartilage integrity is not clear. In this study, we examined whether chondrocyte death undermines cartilage integrity in aging and injury using a rapid 3D confocal cartilage imaging technique coupled with standard histology. We induced autonomous expression of diphtheria toxin to kill articular surface chondrocytes in mice and determined that chondrocyte death did not lead to cartilage damage. Moreover, cartilage damage after surgical destabilization of the medial meniscus of the knee was increased in mice with intact chondrocytes compared with animals whose chondrocytes had been killed, suggesting that chondrocyte death does not drive cartilage damage in response to injury. These data imply that chondrocyte catabolism, not death, contributes to articular cartilage damage following injury. Therefore, therapies targeted at reducing the catabolic phenotype may protect against degenerative joint disease.
Minjie Zhang, Sriniwasan B. Mani, Yao He, Amber M. Hall, Lin Xu, Yefu Li, David Zurakowski, Gregory D. Jay, Matthew L. Warman
Achondroplasia (ACH) is the most frequent form of dwarfism and is caused by gain-of-function mutations in the fibroblast growth factor receptor 3–encoding (FGFR3-encoding) gene. Although potential therapeutic strategies for ACH, which aim to reduce excessive FGFR3 activation, have emerged over many years, the use of tyrosine kinase inhibitor (TKI) to counteract FGFR3 hyperactivity has yet to be evaluated. Here, we have reported that the pan-FGFR TKI, NVP-BGJ398, reduces FGFR3 phosphorylation and corrects the abnormal femoral growth plate and calvaria in organ cultures from embryos of the
Davide Komla-Ebri, Emilie Dambroise, Ina Kramer, Catherine Benoist-Lasselin, Nabil Kaci, Cindy Le Gall, Ludovic Martin, Patricia Busca, Florent Barbault, Diana Graus-Porta, Arnold Munnich, Michaela Kneissel, Federico Di Rocco, Martin Biosse-Duplan, Laurence Legeai-Mallet
Both the WNT/β-catenin and hedgehog signaling pathways are important in the regulation of limb development, chondrocyte differentiation, and degeneration of articular cartilage in osteoarthritis (OA). It is not clear how these signaling pathways interact in interzone cell differentiation and synovial joint morphogenesis. Here, we determined that constitutive activation of hedgehog signaling specifically within interzone cells induces joint morphological changes by selectively inhibiting β-catenin–induced
Jason S. Rockel, Chunying Yu, Heather Whetstone, April M. Craft, Katherine Reilly, Henry Ma, Hidetoshi Tsushima, Vijitha Puviindran, Mushriq Al-Jazrawe, Gordon M. Keller, Benjamin A. Alman
Fracture nonunions develop in 10%–20% of patients with fractures, resulting in prolonged disability. Current data suggest that bone union during fracture repair is achieved via proliferation and differentiation of skeletal progenitors within periosteal and soft tissues surrounding bone, while bone marrow stromal/stem cells (BMSCs) and other skeletal progenitors may also contribute. The NOTCH signaling pathway is a critical maintenance factor for BMSCs during skeletal development, although the precise role for NOTCH and the requisite nature of BMSCs following fracture is unknown. Here, we evaluated whether NOTCH and/or BMSCs are required for fracture repair by performing nonstabilized and stabilized fractures on NOTCH-deficient mice with targeted deletion of
Cuicui Wang, Jason A. Inzana, Anthony J. Mirando, Yinshi Ren, Zhaoyang Liu, Jie Shen, Regis J. O’Keefe, Hani A. Awad, Matthew J. Hilton
BACKGROUND. Severe gonadal steroid deficiency induces bone loss in adult men; however, the specific roles of androgen and estrogen deficiency in hypogonadal bone loss are unclear. Additionally, the threshold levels of testosterone and estradiol that initiate bone loss are uncertain.
METHODS. One hundred ninety-eight healthy men, ages 20–50, received goserelin acetate, which suppresses endogenous gonadal steroid production, and were randomized to treatment with 0, 1.25, 2.5, 5, or 10 grams of testosterone gel daily for 16 weeks. An additional cohort of 202 men was randomized to receive these treatments plus anastrozole, which suppresses conversion of androgens to estrogens. Thirty-seven men served as controls and received placebos for goserelin and testosterone. Changes in bone turnover markers, bone mineral density (BMD) by dual-energy x-ray absorptiometry (DXA), and BMD by quantitative computed tomography (QCT) were assessed in all men. Bone microarchitecture was assessed in 100 men.
RESULTS. As testosterone dosage decreased, the percent change in C-telopeptide increased. These increases were considerably greater when aromatization of testosterone to estradiol was also suppressed, suggesting effects of both testosterone and estradiol deficiency. Decreases in DXA BMD were observed when aromatization was suppressed but were modest in most groups. QCT spine BMD fell substantially in all testosterone-dose groups in which aromatization was also suppressed, and this decline was independent of testosterone dose. Estradiol deficiency disrupted cortical microarchitecture at peripheral sites. Estradiol levels above 10 pg/ml and testosterone levels above 200 ng/dl were generally sufficient to prevent increases in bone resorption and decreases in BMD in men.
CONCLUSIONS. Estrogens primarily regulate bone homeostasis in adult men, and testosterone and estradiol levels must decline substantially to impact the skeleton.
TRIAL REGISTRATION. ClinicalTrials.gov, NCT00114114.
FUNDING. AbbVie Inc., AstraZeneca Pharmaceuticals LP, NIH.
Joel S. Finkelstein, Hang Lee, Benjamin Z. Leder, Sherri-Ann M. Burnett-Bowie, David W. Goldstein, Christopher W. Hahn, Sarah C. Hirsch, Alex Linker, Nicholas Perros, Andrew B. Servais, Alexander P. Taylor, Matthew L. Webb, Jonathan M. Youngner, Elaine W. Yu
Paget’s disease (PD) is characterized by focal and dramatic bone resorption and formation. Treatments that target osteoclasts (OCLs) block both pagetic bone resorption and formation; therefore, PD offers key insights into mechanisms that couple bone resorption and formation. Here, we evaluated OCLs from 3 patients with PD and determined that measles virus nucleocapsid protein (MVNP) was expressed in 70% of these OCLs. Moreover, transgenic mice with OCL-specific expression of MVNP (MVNP mice) developed PD-like bone lesions that required MVNP-dependent induction of high IL-6 expression levels in OCLs. In contrast, mice harboring a knockin of p62P394L (p62-KI mice), which is the most frequent PD-associated mutation, exhibited increased bone resorption, but not formation. Evaluation of OCLs from MVNP, p62-KI, and WT mice revealed increased IGF1 expression in MVNP-expressing OCLs that resulted from the high IL-6 expression levels in these cells. IL-6, in turn, increased the expression of coupling factors, specifically ephrinB2 on OCLs and EphB4 on osteoblasts (OBs). IGF1 enhanced ephrinB2 expression on OCLs and OB differentiation. Importantly, ephrinB2 and IGF1 levels were increased in MVNP-expressing OCLs from patients with PD and MVNP-transduced human OCLs compared with levels detected in controls. Further, anti-IGF1 or anti-IGF1R blocked Runx2 and osteocalcin upregulation in OBs cocultured with MVNP-expressing OCLs. These results suggest that in PD, MVNP upregulates IL-6 and IGF1 in OCLs to increase ephrinB2-EphB4 coupling and bone formation.
Jumpei Teramachi, Yuki Nagata, Khalid Mohammad, Yuji Inagaki, Yasuhisa Ohata, Theresa Guise, Laëtitia Michou, Jacques P. Brown, Jolene J. Windle, Noriyoshi Kurihara, G. David Roodman
Osteoblast-derived VEGF is important for bone development and postnatal bone homeostasis. Previous studies have demonstrated that VEGF affects bone repair and regeneration; however, the cellular mechanisms by which it works are not fully understood. In this study, we investigated the functions of osteoblast-derived VEGF in healing of a bone defect. The results indicate that osteoblast-derived VEGF plays critical roles at several stages in the repair process. Using transgenic mice with osteoblast-specific deletion of
Kai Hu, Bjorn R. Olsen
Osteoarthritis (OA) is the most prevalent and debilitating joint disease, and there are currently no effective disease-modifying treatments available. Multiple risk factors for OA, such as aging, result in progressive damage and loss of articular cartilage. Autonomous circadian clocks have been identified in mouse cartilage, and environmental disruption of circadian rhythms in mice predisposes animals to OA-like damage. However, the contribution of the cartilage clock mechanisms to the maintenance of tissue homeostasis is still unclear. Here, we have shown that expression of the core clock transcription factor BMAL1 is disrupted in human OA cartilage and in aged mouse cartilage. Furthermore, targeted
Michal Dudek, Nicole Gossan, Nan Yang, Hee-Jeong Im, Jayalath P.D. Ruckshanthi, Hikari Yoshitane, Xin Li, Ding Jin, Ping Wang, Maya Boudiffa, Ilaria Bellantuono, Yoshitaka Fukada, Ray P. Boot-Handford, Qing-Jun Meng
Bone formation during fracture repair inevitably initiates within or around extravascular deposits of a fibrin-rich matrix. In addition to a central role in hemostasis, fibrin is thought to enhance bone repair by supporting inflammatory and mesenchymal progenitor egress into the zone of injury. However, given that a failure of efficient fibrin clearance can impede normal wound repair, the precise contribution of fibrin to bone fracture repair, whether supportive or detrimental, is unknown. Here, we employed mice with genetically and pharmacologically imposed deficits in the fibrin precursor fibrinogen and fibrin-degrading plasminogen to explore the hypothesis that fibrin is vital to the initiation of fracture repair, but impaired fibrin clearance results in derangements in bone fracture repair. In contrast to our hypothesis, fibrin was entirely dispensable for long-bone fracture repair, as healing fractures in fibrinogen-deficient mice were indistinguishable from those in control animals. However, failure to clear fibrin from the fracture site in plasminogen-deficient mice severely impaired fracture vascularization, precluded bone union, and resulted in robust heterotopic ossification. Pharmacological fibrinogen depletion in plasminogen-deficient animals restored a normal pattern of fracture repair and substantially limited heterotopic ossification. Fibrin is therefore not essential for fracture repair, but inefficient fibrinolysis decreases endochondral angiogenesis and ossification, thereby inhibiting fracture repair.
Masato Yuasa, Nicholas A. Mignemi, Jeffry S. Nyman, Craig L. Duvall, Herbert S. Schwartz, Atsushi Okawa, Toshitaka Yoshii, Gourab Bhattacharjee, Chenguang Zhao, Jesse E. Bible, William T. Obremskey, Matthew J. Flick, Jay L. Degen, Joey V. Barnett, Justin M.M. Cates, Jonathan G. Schoenecker
The unfolded protein response (UPR) is a cellular adaptive mechanism that is activated in response to the accumulation of unfolded proteins in the endoplasmic reticulum. The inositol-requiring protein-1α/X-box–binding protein–mediated (IRE1α/XBP1-mediated) branch of the UPR is highly conserved and has also been shown to regulate various cell-fate decisions. Herein, we have demonstrated a crucial role for the IREα/XBP1-mediated arm of the UPR in osteoclast differentiation. Using murine models, we found that the conditional abrogation of IRE1α in bone marrow cells increases bone mass as the result of defective osteoclastic bone resorption. In osteoclast precursors, IRE1α was transiently activated during osteoclastogenesis, and suppression of the IRE1α/XBP1 pathway in these cells substantially inhibited the formation of multinucleated osteoclasts in vitro. We determined that XBP1 directly binds the promoter and induces transcription of the gene encoding the master regulator of osteoclastogenesis nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Moreover, activation of IRE1α was partially dependent on Ca2+ oscillation mediated by inositol 1,4,5-trisphosphate receptors 2 and 3 (ITPR2 and ITPR3) in the endoplasmic reticulum, as pharmacological inhibition or deletion of these receptors markedly decreased
Takahide Tohmonda, Masaki Yoda, Takao Iwawaki, Morio Matsumoto, Masaya Nakamura, Katsuhiko Mikoshiba, Yoshiaki Toyama, Keisuke Horiuchi
Bone marrow mesenchymal stem cells (BMSCs) exhibit an age-dependent reduction in osteogenesis that is accompanied by an increased propensity toward adipocyte differentiation. This switch increases adipocyte numbers and decreases the number of osteoblasts, contributing to age-related bone loss. Here, we found that the level of microRNA-188 (miR-188) is markedly higher in BMSCs from aged compared with young mice and humans. Compared with control mice, animals lacking miR-188 showed a substantial reduction of age-associated bone loss and fat accumulation in bone marrow. Conversely, mice with transgenic overexpression of miR-188 in osterix+ osteoprogenitors had greater age-associated bone loss and fat accumulation in bone marrow relative to WT mice. Moreover, using an aptamer delivery system, we found that BMSC-specific overexpression of miR-188 in mice reduced bone formation and increased bone marrow fat accumulation. We identified histone deacetylase 9 (HDAC9) and RPTOR-independent companion of MTOR complex 2 (RICTOR) as the direct targets of miR-188. Notably, BMSC-specific inhibition of miR-188 by intra–bone marrow injection of aptamer-antagomiR-188 increased bone formation and decreased bone marrow fat accumulation in aged mice. Together, our results indicate that miR-188 is a key regulator of the age-related switch between osteogenesis and adipogenesis of BMSCs and may represent a potential therapeutic target for age-related bone loss.
Chang-Jun Li, Peng Cheng, Meng-Ke Liang, Yu-Si Chen, Qiong Lu, Jin-Yu Wang, Zhu-Ying Xia, Hou-De Zhou, Xu Cao, Hui Xie, Er-Yuan Liao, Xiang-Hang Luo
Osteoclasts are bone-resorbing cells that are important for maintenance of bone remodeling and mineral homeostasis. Regulation of osteoclast differentiation and activity is important for the pathogenesis and treatment of diseases associated with bone loss. Here, we demonstrate that retinoid X receptors (RXRs) are key elements of the transcriptional program of differentiating osteoclasts. Loss of RXR function in hematopoietic cells resulted in formation of giant, nonresorbing osteoclasts and increased bone mass in male mice and protected female mice from bone loss following ovariectomy, which induces osteoporosis in WT females. The increase in bone mass associated with RXR deficiency was due to lack of expression of the RXR-dependent transcription factor v-maf musculoaponeurotic fibrosarcoma oncogene family, protein B (MAFB) in osteoclast progenitors. Evaluation of osteoclast progenitor cells revealed that RXR homodimers directly target and bind to the
María P. Menéndez-Gutiérrez, Tamás Rőszer, Lucía Fuentes, Vanessa Núñez, Amelia Escolano, Juan Miguel Redondo, Nora De Clerck, Daniel Metzger, Annabel F. Valledor, Mercedes Ricote
WNT signaling stimulates bone formation by increasing both the number of osteoblasts and their protein-synthesis activity. It is not clear how WNT augments the capacity of osteoblast progenitors to meet the increased energetic and synthetic needs associated with mature osteoblasts. Here, in cultured osteoblast progenitors, we determined that WNT stimulates glutamine catabolism through the tricarboxylic acid (TCA) cycle and consequently lowers intracellular glutamine levels. The WNT-induced reduction of glutamine concentration triggered a general control nonderepressible 2–mediated (GCN2-mediated) integrated stress response (ISR) that stimulated expression of genes responsible for amino acid supply, transfer RNA (tRNA) aminoacylation, and protein folding. WNT-induced glutamine catabolism and ISR were β-catenin independent, but required mammalian target of rapamycin complex 1 (mTORC1) activation. In a hyperactive WNT signaling mouse model of human osteosclerosis, inhibition of glutamine catabolism or
Courtney M. Karner, Emel Esen, Adewole L. Okunade, Bruce W. Patterson, Fanxin Long
Osteoclastogenesis requires activation of RANK signaling as well as costimulatory signals from immunoreceptor tyrosine-based activation motif-containing (ITAM-containing) receptors/adaptors, predominantly tyrosine kinase–binding proteins DAP12 and FcRγ, in osteoclast precursors. It is not well understood how costimulatory signals are regulated and integrated with RANK signaling. Here, we found that osteopetrotic bone phenotypes in mice lacking DAP12 or DAP12 and FcRγ are mediated by the transcription factor RBP-J, as deletion of
Susan Li, Christine H. Miller, Eugenia Giannopoulou, Xiaoyu Hu, Lionel B. Ivashkiv, Baohong Zhao
Both maternal and offspring-derived factors contribute to lifelong growth and bone mass accrual, although the specific role of maternal deficiencies in the growth and bone mass of offspring is poorly understood. In the present study, we have shown that vitamin B12 (B12) deficiency in a murine genetic model results in severe postweaning growth retardation and osteoporosis, and the severity and time of onset of this phenotype in the offspring depends on the maternal genotype. Using integrated physiological and metabolomic analysis, we determined that B12 deficiency in the offspring decreases liver taurine production and associates with abrogation of a growth hormone/insulin-like growth factor 1 (GH/IGF1) axis. Taurine increased GH-dependent IGF1 synthesis in the liver, which subsequently enhanced osteoblast function, and in B12-deficient offspring, oral administration of taurine rescued their growth retardation and osteoporosis phenotypes. These results identify B12 as an essential vitamin that positively regulates postweaning growth and bone formation through taurine synthesis and suggests potential therapies to increase bone mass.
Pablo Roman-Garcia, Isabel Quiros-Gonzalez, Lynda Mottram, Liesbet Lieben, Kunal Sharan, Arporn Wangwiwatsin, Jose Tubio, Kirsty Lewis, Debbie Wilkinson, Balaji Santhanam, Nazan Sarper, Simon Clare, George S. Vassiliou, Vidya R. Velagapudi, Gordon Dougan, Vijay K. Yadav
NOTCH-dependent signaling pathways are critical for normal bone remodeling; however, it is unclear if dysfunctional NOTCH activation contributes to inflammation-mediated bone loss, as observed in rheumatoid arthritis (RA) patients. We performed RNA sequencing and pathway analyses in mesenchymal stem cells (MSCs) isolated from transgenic
Hengwei Zhang, Matthew J. Hilton, Jennifer H. Anolik, Stephen L. Welle, Chen Zhao, Zhenqiang Yao, Xing Li, Zhiyu Wang, Brendan F. Boyce, Lianping Xing
Patient bone mineral density (BMD) predicts the likelihood of osteoporotic fracture. While substantial progress has been made toward elucidating the genetic determinants of BMD, our understanding of the factors involved remains incomplete. Here, using a systems genetics approach in the mouse, we predicted that bicaudal C homolog 1 (
Larry D. Mesner, Brianne Ray, Yi-Hsiang Hsu, Ani Manichaikul, Eric Lum, Elizabeth C. Bryda, Stephen S. Rich, Clifford J. Rosen, Michael H. Criqui, Matthew Allison, Matthew J. Budoff, Thomas L. Clemens, Charles R. Farber