Bone biology
Abstract
In the brain, the ventral hypothalamus (VHT) regulates energy and bone metabolism. Whether this regulation uses the same or different neuronal circuits is unknown. Alteration of AP1 signaling in the VHT increases energy expenditure, glucose utilization, and bone density, yet the specific neurons responsible for each or all of these phenotypes are not identified. Using neuron-specific genetically targeted AP1 alterations as a tool in adult mice, we found that AgRP- or POMC- expressing neurons, predominantly present in the arcuate nucleus (ARC) within the VHT, stimulate whole body energy expenditure, glucose utilization and bone formation and density, although their effects on bone resorption differed. In contrast, AP1 alterations in Steroidogenic factor 1 (SF1)-expressing neurons, present in the ventromedial hypothalamus (VMH), increase energy, but decrease bone density, suggesting that these effects are independent. Altered AP1 signaling also increased the levels of the neuromediator galanin in the hypothalamus and global galanin deletion, VHT galanin silencing using shRNA, or pharmacological galanin receptor blockade, counteracted the observed effects on energy and bone. Thus, AP1 antagonism reveals that AgRP- and POMC- expressing neurons can stimulate body metabolism and increase bone density, with galanin acting as a central downstream effector. The results obtained with SF1-expressing neurons, however, indicate that bone homeostasis is not always dictated by the global energy status, and vice versa.
Authors
Anna Idelevich, Kazusa Sato, Kenichi Nagano, Glenn Rowe, Francesca Gori, Roland Baron
Abstract
Coupling is the process that links bone resorption to bone formation in a temporally and spatially coordinated manner within the remodeling cycle. Several lines of evidence point to the critical roles of osteoclast-derived coupling factors in the regulation of osteoblast performance. Here, we used a fractionated secretomic approach and identified the axon-guidance molecule SLIT3 as a clastokine that stimulated osteoblast migration and proliferation by activating β-catenin. SLIT3 also inhibited bone resorption by suppressing osteoclast differentiation in an autocrine manner. Mice deficient in Slit3 or its receptor, Robo1, exhibited osteopenic phenotypes due to a decrease in bone formation and increase in bone resorption. Mice lacking Slit3 specifically in osteoclasts had low bone mass, whereas mice with either neuron-specific Slit3 deletion or osteoblast-specific Slit3 deletion had normal bone mass, thereby indicating the importance of SLIT3 as a local determinant of bone metabolism. In postmenopausal women, higher circulating SLIT3 levels were associated with increased bone mass. Notably, injection of a truncated recombinant SLIT3 markedly rescued bone loss after an ovariectomy. Thus, these results indicate that SLIT3 plays an osteoprotective role by synchronously stimulating bone formation and inhibiting bone resorption, making it a potential therapeutic target for metabolic bone diseases.
Authors
Beom-Jun Kim, Young-Sun Lee, Sun-Young Lee, Wook-Young Baek, Young Jin Choi, Sung Ah Moon, Seung Hun Lee, Jung-Eun Kim, Eun-Ju Chang, Eun-Young Kim, Jin Yoon, Seung-Whan Kim, Sung Ho Ryu, Sun-Kyeong Lee, Joseph A. Lorenzo, Seong Hee Ahn, Hyeonmok Kim, Ki-Up Lee, Ghi Su Kim, Jung-Min Koh
Abstract
Genetic forms of vitamin D–dependent rickets (VDDRs) are due to mutations impairing activation of vitamin D or decreasing vitamin D receptor responsiveness. Here we describe two unrelated patients with early-onset rickets, reduced serum levels of the vitamin D metabolites 25-hydroxyvitamin D and 1,25-dihydroxyvitamin D, and deficient responsiveness to parent and activated forms of vitamin D. Neither patient had a mutation in any genes known to cause VDDR, however, using whole exome sequence analysis we identified a recurrent de novo missense mutation c.902T>C (p.I301T) in CYP3A4 in both subjects that alters the conformation of substrate-recognition-site 4 (SRS-4). In vitro, the mutant CYP3A4 oxidized 1,25-dihydroxyvitamin D with 10-fold greater activity than wild-type CYP3A4 and 2-fold greater activity than CYP24A1, the principal inactivator of vitamin D metabolites. As CYP3A4 mutations have not previously been linked to rickets, these findings provide new insight into vitamin D metabolism, and demonstrate that accelerated inactivation of vitamin D metabolites represents a previously undescribed mechanism for vitamin D deficiency.
Authors
Jeffrey D. Roizen, Dong Li, Lauren O'Lear, Muhammad K. Javaid, Nicholas J. Shaw, Peter R. Ebeling, Hanh H. Nguyen, Christine P. Rodda, Kenneth E. Thummel, Tom D Thacher, Hakon Hakonarson, Michael A. Levine
Abstract
The skeleton has emerged as an important regulator of systemic glucose homeostasis, with osteocalcin and insulin representing prime mediators of the interplay between bone and energy metabolism. However, genetic evidence indicates that osteoblasts can influence global energy metabolism through additional, as yet unknown, mechanisms. Here, we report that constitutive or postnatally induced deletion of the hypoxia signaling pathway component von Hippel–Lindau (VHL) in skeletal osteolineage cells of mice led to high bone mass as well as hypoglycemia and increased glucose tolerance, not accounted for by osteocalcin or insulin. In vitro and in vivo data indicated that Vhl-deficient osteoblasts displayed massively increased glucose uptake and glycolysis associated with upregulated HIF-target gene expression, resembling the Warburg effect that typifies cancer cells. Overall, the glucose consumption by the skeleton was increased in the mutant mice, as revealed by 18F-FDG radioactive tracer experiments. Moreover, the glycemia levels correlated inversely with the level of skeletal glucose uptake, and pharmacological treatment with the glycolysis inhibitor dichloroacetate (DCA), which restored glucose metabolism in Vhl-deficient osteogenic cells in vitro, prevented the development of the systemic metabolic phenotype in the mutant mice. Altogether, these findings reveal a novel link between cellular glucose metabolism in osteoblasts and whole-body glucose homeostasis, controlled by local hypoxia signaling in the skeleton.
Authors
Naomi Dirckx, Robert J. Tower, Evi M. Mercken, Roman Vangoitsenhoven, Caroline Moreau-Triby, Tom Breugelmans, Elena Nefyodova, Ruben Cardoen, Chantal Mathieu, Bart Van der Schueren, Cyrille B. Confavreux, Thomas L. Clemens, Christa Maes
Abstract
Enthesopathy is a disorder of bone, tendon, or ligament insertion. It represents one-fourth of all tendon-ligament diseases and is one of the most difficult tendon-ligament disorders to treat. Despite its high prevalence, the exact pathogenesis of this condition remains unknown. Here, we show that TGF-β was activated in both a semi-Achilles tendon transection (SMTS) mouse model and in a dorsiflexion immobilization (DI) mouse model of enthesopathy. High concentrations of active TGF-β recruited mesenchymal stromal stem cells (MSCs) and led to excessive vessel formation, bone deterioration, and fibrocartilage calcification. Transgenic expression of active TGF-β1 in bone also induced enthesopathy with a phenotype similar to that observed in SMTS and DI mice. Systemic inhibition of TGF-β activity by injection of 1D11, a TGF-β–neutralizing antibody, but not a vehicle antibody, attenuated the excessive vessel formation and restored uncoupled bone remodeling in SMTS mice. 1D11-treated SMTS fibrocartilage had increased proteoglycan and decreased collagen X and matrix metalloproteinase 13 expression relative to control antibody treatment. Notably, inducible knockout of the TGF-β type II receptor in mouse MSCs preserved the bone microarchitecture and fibrocartilage composition after SMTS relative to the WT littermate controls. Thus, elevated levels of active TGF-β in the enthesis bone marrow induce the initial pathological changes of enthesopathy, indicating that TGF-β inhibition could be a potential therapeutic strategy.
Authors
Xiao Wang, Liang Xie, Janet Crane, Gehua Zhen, Fengfeng Li, Ping Yang, Manman Gao, Ruoxian Deng, Yiguo Wang, Xiaohua Jia, Cunyi Fan, Mei Wan, Xu Cao
Abstract
The molecular mechanisms that transduce the osteoblast response to physical forces in the bone microenvironment are poorly understood. Here, we used genetic and pharmacological experiments to determine whether the polycystins PC1 and PC2 (encoded by Pkd1 and Pkd2) and the transcriptional coactivator TAZ form a mechanosensing complex in osteoblasts. Compound-heterozygous mice lacking 1 copy of Pkd1 and Taz exhibited additive decrements in bone mass, impaired osteoblast-mediated bone formation, and enhanced bone marrow fat accumulation. Bone marrow stromal cells and osteoblasts derived from these mice showed impaired osteoblastogenesis and enhanced adipogenesis. Increased extracellular matrix stiffness and application of mechanical stretch to multipotent mesenchymal cells stimulated the nuclear translocation of the PC1 C-terminal tail/TAZ (PC1-CTT/TAZ) complex, leading to increased runt-related transcription factor 2–mediated (Runx2-mediated) osteogenic and decreased PPARγ-dependent adipogenic gene expression. Using structure-based virtual screening, we identified a compound predicted to bind to PC2 in the PC1:PC2 C-terminal tail region with helix:helix interaction. This molecule stimulated polycystin- and TAZ-dependent osteoblastogenesis and inhibited adipogenesis. Thus, we show that polycystins and TAZ integrate at the molecular level to reciprocally regulate osteoblast and adipocyte differentiation, indicating that the polycystins/TAZ complex may be a potential therapeutic target to increase bone mass.
Authors
Zhousheng Xiao, Jerome Baudry, Li Cao, Jinsong Huang, Hao Chen, Charles R. Yates, Wei Li, Brittany Dong, Christopher M. Waters, Jeremy C. Smith, L. Darryl Quarles
Abstract
Although peptides are safe and useful as therapeutics, they are often easily degraded or metabolized. Dampening the clearance system for peptide ligands is a promising strategy for increasing the efficacy of peptide therapies. Natriuretic peptide receptor B (NPR-B) and its naturally occurring ligand, C-type natriuretic peptide (CNP), are potent stimulators of endochondral bone growth, and activating the CNP/NPR-B system is expected to be a powerful strategy for treating impaired skeletal growth. CNP is cleared by natriuretic peptide clearance receptor (NPR-C); therefore, we investigated the effect of reducing the rate of CNP clearance on skeletal growth by limiting the interaction between CNP and NPR-C. Specifically, we generated transgenic mice with increased circulating levels of osteocrin (OSTN) protein, a natural NPR-C ligand without natriuretic activity, and observed a dose-dependent skeletal overgrowth phenotype in these animals. Skeletal overgrowth in OSTN-transgenic mice was diminished in either CNP- or NPR-C–depleted backgrounds, confirming that CNP and NPR-C are indispensable for the bone growth–stimulating effect of OSTN. Interestingly, double-transgenic mice of CNP and OSTN had even higher levels of circulating CNP and additional increases in bone length, as compared with mice with elevated CNP alone. Together, these results support OSTN administration as an adjuvant agent for CNP therapy and provide a potential therapeutic approach for diseases with impaired skeletal growth.
Authors
Yugo Kanai, Akihiro Yasoda, Keita P. Mori, Haruko Watanabe-Takano, Chiaki Nagai-Okatani, Yui Yamashita, Keisho Hirota, Yohei Ueda, Ichiro Yamauchi, Eri Kondo, Shigeki Yamanaka, Yoriko Sakane, Kazumasa Nakao, Toshihito Fujii, Hideki Yokoi, Naoto Minamino, Masashi Mukoyama, Naoki Mochizuki, Nobuya Inagaki
Abstract
Osteocalcin (OCN) is an osteoblast-derived hormone that increases energy expenditure, insulin sensitivity, insulin secretion, and glucose tolerance. The cDNA sequence of OCN predicts that, like many other peptide hormones, OCN is first synthesized as a prohormone (pro-OCN). The importance of pro-OCN maturation in regulating OCN and the identity of the endopeptidase responsible for pro-OCN cleavage in osteoblasts are still unknown. Here, we show that the proprotein convertase furin is responsible for pro-OCN maturation in vitro and in vivo. Using pharmacological and genetic experiments, we also determined that furin-mediated pro-OCN cleavage occurred independently of its γ-carboxylation, a posttranslational modification that is known to hamper OCN endocrine action. However, because pro-OCN is not efficiently decarboxylated and activated during bone resorption, inactivation of furin in osteoblasts in mice resulted in decreased circulating levels of undercarboxylated OCN, impaired glucose tolerance, and reduced energy expenditure. Furthermore, we show that Furin deletion in osteoblasts reduced appetite, a function not modulated by OCN, thus suggesting that osteoblasts may secrete additional hormones that regulate different aspects of energy metabolism. Accordingly, the metabolic defects of the mice lacking furin in osteoblasts became more apparent under pair-feeding conditions. These findings identify furin as an important regulator of bone endocrine function.
Authors
Omar El-Rifai, Jacqueline Chow, Julie Lacombe, Catherine Julien, Denis Faubert, Delia Susan-Resiga, Rachid Essalmani, John W.M. Creemers, Nabil G. Seidah, Mathieu Ferron
Abstract
The short-limbed dwarfism metaphyseal chondrodysplasia type Schmid (MCDS) is linked to mutations in type X collagen, which increase ER stress by inducing misfolding of the mutant protein and subsequently disrupting hypertrophic chondrocyte differentiation. Here, we show that carbamazepine (CBZ), an autophagy-stimulating drug that is clinically approved for the treatment of seizures and bipolar disease, reduced the ER stress induced by 4 different MCDS-causing mutant forms of collagen X in human cell culture. Depending on the nature of the mutation, CBZ application stimulated proteolysis of misfolded collagen X by either autophagy or proteasomal degradation, thereby reducing intracellular accumulation of mutant collagen. In MCDS mice expressing the Col10a1.pN617K mutation, CBZ reduced the MCDS-associated expansion of the growth plate hypertrophic zone, attenuated enhanced expression of ER stress markers such as Bip and Atf4, increased bone growth, and reduced skeletal dysplasia. CBZ produced these beneficial effects by reducing the MCDS-associated abnormalities in hypertrophic chondrocyte differentiation. Stimulation of intracellular proteolysis using CBZ treatment may therefore be a clinically viable way of treating the ER stress–associated dwarfism MCDS.
Authors
Lorna A. Mullan, Ewa J. Mularczyk, Louise H. Kung, Mitra Forouhan, Jordan M.A. Wragg, Royston Goodacre, John F. Bateman, Eileithyia Swanton, Michael D. Briggs, Raymond P. Boot-Handford
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
Copyright © 2018 American Society for Clinical Investigation
ISSN: 0021-9738 (print), 1558-8238 (online)