mTORC1 hyperactivation arrests bone growth in lysosomal storage disorders by suppressing autophagy

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Abstract

The mammalian target of rapamycin complex 1 (mTORC1) kinase promotes cell growth by activating biosynthetic pathways and suppressing catabolic pathways, particularly that of macroautophagy. A prerequisite for mTORC1 activation is its translocation to the lysosomal surface. Deregulation of mTORC1 has been associated with the pathogenesis of several diseases, but its role in skeletal disorders is largely unknown. Here, we show that enhanced mTORC1 signaling arrests bone growth in lysosomal storage disorders (LSDs). We found that lysosomal dysfunction induces a constitutive lysosomal association and consequent activation of mTORC1 in chondrocytes, the cells devoted to bone elongation. mTORC1 hyperphosphorylates the protein UV radiation resistance–associated gene (UVRAG), reducing the activity of the associated Beclin 1–Vps34 complex and thereby inhibiting phosphoinositide production. Limiting phosphoinositide production leads to a blockage of the autophagy flux in LSD chondrocytes. As a consequence, LSD chondrocytes fail to properly secrete collagens, the main components of the cartilage extracellular matrix. In mouse models of LSD, normalization of mTORC1 signaling or stimulation of the Beclin 1–Vps34–UVRAG complex rescued the autophagy flux, restored collagen levels in cartilage, and ameliorated the bone phenotype. Taken together, these data unveil a role for mTORC1 and autophagy in the pathogenesis of skeletal disorders and suggest potential therapeutic approaches for the treatment of LSDs.

Authors

Rosa Bartolomeo, Laura Cinque, Chiara De Leonibus, Alison Forrester, Anna Chiara Salzano, Jlenia Monfregola, Emanuela De Gennaro, Edoardo Nusco, Isabella Azario, Carmela Lanzara, Marta Serafini, Beth Levine, Andrea Ballabio, Carmine Settembre

Parathyroid hormone regulates fates of murine osteoblast precursors in vivo

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Abstract

Teriparatide, a recombinant form of parathyroid hormone (PTH), is the only approved treatment for osteoporosis that increases the rate of bone formation. Teriparatide increases osteoblast numbers by suppressing osteoblast apoptosis and activating bone-lining cells. No direct evidence for teriparatide’s actions on early cells of the osteoblast lineage has been demonstrated. Here, we have employed a lineage-tracing strategy that uses a tamoxifen-dependent, promoter-driven cre to mark early cells of the osteoblast lineage in adult mice. We show that teriparatide increases the numbers of osteoblast precursors and drives their differentiation into mature osteoblasts. Unexpectedly, following withdrawal of teriparatide therapy, bone marrow adipocytes increased dramatically in number. Some of these adipocytes derived from cells marked by Sox9-cre expression weeks earlier. Continued therapy with teriparatide prevented the appearance of adipocytes. Selective, inducible deletion of the PTH receptor in Sox9-cre cells demonstrated that PTH receptor expression is required for teriparatide-mediated increases in early osteoblast precursors. The increase in early precursors after teriparatide administration was associated with robust suppression of precursor apoptosis without affecting their rate of proliferation. Thus, teriparatide increases the numbers of early cells of the osteoblast lineage, hastens their differentiation into osteoblasts, and suppresses their differentiation into adipocytes in vivo.

Authors

Deepak H. Balani, Noriaki Ono, Henry M. Kronenberg

Osteocyte-specific WNT1 regulates osteoblast function during bone homeostasis

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Abstract

Mutations in WNT1 cause osteogenesis imperfecta (OI) and early-onset osteoporosis, identifying it as a key Wnt ligand in human bone homeostasis. However, how and where WNT1 acts in bone are unclear. To address this mechanism, we generated late-osteoblast-specific and osteocyte-specific WNT1 loss- and gain-of-function mouse models. Deletion of Wnt1 in osteocytes resulted in low bone mass with spontaneous fractures similar to that observed in OI patients. Conversely, Wnt1 overexpression from osteocytes stimulated bone formation by increasing osteoblast number and activity, which was due in part to activation of mTORC1 signaling. While antiresorptive therapy is the mainstay of OI treatment, it has limited efficacy in WNT1-related OI. In this study, anti-sclerostin antibody (Scl-Ab) treatment effectively improved bone mass and dramatically decreased fracture rate in swaying mice, a model of global Wnt1 loss. Collectively, our data suggest that WNT1-related OI and osteoporosis are caused in part by decreased mTORC1-dependent osteoblast function resulting from loss of WNT1 signaling in osteocytes. As such, this work identifies an anabolic function of osteocytes as a source of Wnt in bone development and homoeostasis, complementing their known function as targets of Wnt signaling in regulating osteoclastogenesis. Finally, this study suggests that Scl-Ab is an effective genotype-specific treatment option for WNT1-related OI and osteoporosis.

Authors

Kyu Sang Joeng, Yi-Chien Lee, Joohyun Lim, Yuqing Chen, Ming-Ming Jiang, Elda Munivez, Catherine Ambrose, Brendan H. Lee

Ubiquitin ligase RNF146 coordinates bone dynamics and energy metabolism

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Abstract

Cleidocranial dysplasia (CCD) is an autosomal dominant human disorder characterized by abnormal bone development that is mainly due to defective intramembranous bone formation by osteoblasts. Here, we describe a mouse strain lacking the E3 ubiquitin ligase RNF146 that shows phenotypic similarities to CCD. Loss of RNF146 stabilized its substrate AXIN1, leading to impairment of WNT3a-induced β-catenin activation and reduced Fgf18 expression in osteoblasts. We show that FGF18 induces transcriptional coactivator with PDZ-binding motif (TAZ) expression, which is required for osteoblast proliferation and differentiation through transcriptional enhancer associate domain (TEAD) and runt-related transcription factor 2 (RUNX2) transcription factors, respectively. Finally, we demonstrate that adipogenesis is enhanced in Rnf146^–/– mouse embryonic fibroblasts. Moreover, mice with loss of RNF146 within the osteoblast lineage had increased fat stores and were glucose intolerant with severe osteopenia because of defective osteoblastogenesis and subsequent impaired osteocalcin production. These findings indicate that RNF146 is required to coordinate β-catenin signaling within the osteoblast lineage during embryonic and postnatal bone development.

Authors

Yoshinori Matsumoto, Jose La Rose, Melissa Lim, Hibret A. Adissu, Napoleon Law, Xiaohong Mao, Feng Cong, Paula Mera, Gerard Karsenty, David Goltzman, Adele Changoor, Lucia Zhang, Megan Stajkowski, Marc D. Grynpas, Carsten Bergmann, Robert Rottapel

MYC-dependent oxidative metabolism regulates osteoclastogenesis via nuclear receptor ERRα

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Abstract

Osteoporosis is a metabolic bone disorder associated with compromised bone strength and an increased risk of fracture. Inhibition of the differentiation of bone-resorbing osteoclasts is an effective strategy for the treatment of osteoporosis. Prior work by our laboratory and others has shown that MYC promotes osteoclastogenesis in vitro, but the underlying mechanisms are not well understood. In addition, the in vivo importance of osteoclast-expressed MYC in physiological and pathological bone loss is not known. Here, we have demonstrated that deletion of Myc in osteoclasts increases bone mass and protects mice from ovariectomy-induced (OVX-induced) osteoporosis. Transcriptomic analysis revealed that MYC drives metabolic reprogramming during osteoclast differentiation and functions as a metabolic switch to an oxidative state. We identified a role for MYC action in the transcriptional induction of estrogen receptor–related receptor α (ERRα), a nuclear receptor that cooperates with the transcription factor nuclear factor of activated T cells, c1 (NFATc1) to drive osteoclastogenesis. Accordingly, pharmacological inhibition of ERRα attenuated OVX-induced bone loss in mice. Our findings highlight a MYC/ERRα pathway that contributes to physiological and pathological bone loss by integrating the MYC/ERRα axis to drive metabolic reprogramming during osteoclast differentiation.

Authors

Seyeon Bae, Min Joon Lee, Se Hwan Mun, Eugenia G. Giannopoulou, Vladimir Yong-Gonzalez, Justin R. Cross, Koichi Murata, Vincent Giguère, Marjolein van der Meulen, Kyung-Hyun Park-Min

Efficacy of anti-sclerostin monoclonal antibody BPS804 in adult patients with hypophosphatasia

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Abstract

BACKGROUND. Hypophosphatasia (HPP) is a rare genetic disorder resulting in variable alterations of bone formation and mineralization that are caused by mutations in the ALPL gene, encoding the tissue-nonspecific alkaline phosphatase (ALP) enzyme.

METHODS. In this phase IIA open-label, single-center, intra-patient, dose-escalating study, adult patients with HPP received 3 ascending intravenous doses of 5, 10, and 20 mg/kg BPS804, a fully human anti-sclerostin monoclonal antibody, on days 1, 15, and 29, respectively. Patients were followed for 16 weeks after the last dose. We assessed the pharmacodynamics, pharmacokinetics, preliminary efficacy, and safety of BPS804 administrations at specified intervals during treatment and follow-up.

RESULTS. Eight patients (mean age 47.8 years) were enrolled in the study (6 females, 2 males). BPS804 treatment increased mean ALP and bone-specific ALP enzymatic activity between days 2 and 29. Transient increases in the bone formation markers procollagen type-I N-terminal propeptide (PINP), osteocalcin, and parathyroid hormone as well as a transient decrease in the bone resorption marker C-telopeptide of type I collagen (CTX-1) were observed. Lumbar spine bone mineral density showed a mean increase by day 85 and at end of study. Treatment-associated adverse events were mild and transient.

CONCLUSION. BPS804 treatment was well tolerated and resulted in increases in bone formation biomarkers and bone mineral density, suggesting that sclerostin inhibition could be applied to enhance bone mineral density, stability, and regeneration in non-life-threatening clinical situations in adults with HPP.

TRIAL REGISTRATION. Clinicaltrials.gov NCT01406977.

FUNDING. Novartis Institutes for BioMedical Research, Basel, Switzerland.

Authors

Lothar Seefried, Jasmin Baumann, Sarah Hemsley, Christine Hofmann, Erdmute Kunstmann, Beate Kiese, Yue Huang, Simon Chivers, Marie-Anne Valentin, Babul Borah, Ronenn Roubenoff, Uwe Junker, Franz Jakob

RANKL coordinates multiple osteoclastogenic pathways by regulating expression of ubiquitin ligase RNF146

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Abstract

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 RNF146 through an NF-κB–related inhibitory element in the RNF146 promoter. RANKL-mediated suppression of RNF146 results in the stabilization of its substrates, 3BP2 and AXIN1, which consequently triggers the activation of SRC and attenuates the expression of β-catenin, respectively. Depletion of RNF146 caused hypersensitivity to LPS-induced TNF-α production in vivo. RNF146 thus acts as an inhibitory switch to control osteoclastogenesis and cytokine production and may be a control point underlying the pathogenesis of chronic inflammatory diseases.

Authors

Yoshinori Matsumoto, Jose Larose, Oliver A. Kent, Melissa Lim, Adele Changoor, Lucia Zhang, Yaryna Storozhuk, Xiaohong Mao, Marc D. Grynpas, Feng Cong, Robert Rottapel

Loss of DDRGK1 modulates SOX9 ubiquitination in spondyloepimetaphyseal dysplasia

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Abstract

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 (DDRGK1) in 4 families affected by SEMD. In zebrafish, ddrgk1 deficiency disrupted craniofacial cartilage development and led to decreased levels of the chondrogenic master transcription factor sox9 and its downstream target, col2a1. Overexpression of sox9 rescued the zebrafish chondrogenic and craniofacial phenotype generated by ddrgk1 knockdown, thus identifying DDRGK1 as a regulator of SOX9. Consistent with these results, Ddrgk1^–/– mice displayed delayed limb bud chondrogenic condensation, decreased SOX9 protein expression and Col2a1 transcript levels, and increased apoptosis. Furthermore, we determined that DDRGK1 can directly bind to SOX9 to inhibit its ubiquitination and proteasomal degradation. Taken together, these data indicate that loss of DDRGK1 decreases SOX9 expression and causes a human skeletal dysplasia, identifying a mechanism that regulates chondrogenesis via modulation of SOX9 ubiquitination.

Authors

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

Reciprocal stabilization of ABL and TAZ regulates osteoblastogenesis through transcription factor RUNX2

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Abstract

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.

Authors

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

Induced superficial chondrocyte death reduces catabolic cartilage damage in murine posttraumatic osteoarthritis

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Abstract

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.

Authors

Minjie Zhang, Sriniwasan B. Mani, Yao He, Amber M. Hall, Lin Xu, Yefu Li, David Zurakowski, Gregory D. Jay, Matthew L. Warman