Fibroblast growth factor 23 (FGF23) is a phosphate (Pi)-regulating hormone produced by bone. Hereditary hypophosphatemic disorders are associated with FGF23 excess, impaired skeletal growth and osteomalacia. Blocking FGF23 became an effective therapeutic strategy in X-linked hypophosphatemia, but testing remains limited in autosomal recessive hypophosphatemic rickets (ARHR). This study investigates the effects of Pi repletion and bone specific deletion of Fgf23 on bone and mineral metabolism in the Dmp1 knockout (Dmp1KO) mouse model of ARHR.At 12 weeks, Dmp1KO mice showed increased serum FGF23 and PTH levels, hypophosphatemia, impaired growth, rickets and osteomalacia. Six weeks of dietary Pi supplementation exacerbated FGF23 production, hyperparathyroidism, renal Pi excretion and osteomalacia. In contrast, osteocyte-specific deletion of Fgf23 resulted in a partial correction of FGF23 excess, which was sufficient to fully restore serum Pi levels, but only partially corrected the bone phenotype. In vitro, we show that FGF23 directly impairs osteoprogenitors differentiation and that DMP1 deficiency contributes to impaired mineralization independently of FGF23 or Pi levels. In conclusion, FGF23-induced hypophosphatemia is only partially responsible for the bone defects observed in Dmp1KO mice. Our data suggest that combined DMP1 repletion and FGF23 blockade could effectively correct ARHR-associated mineral and bone disorders.
Guillaume Courbon, Dominik Kentrup, Jane Joy Thomas, Xueyan Wang, Hao-Hsuan Tsai, Jadeah J. Spindler, John Von Drasek, Laura Mazudie Ndjonko, Marta Martinez-Calle, Sana Lynch, Lauriane Hivert, Xiaofang Wang, Wenhan Chang, Jian Q. Feng, Valentin David, Aline Martin
Osteoclasts specialize in bone resorption and are critical for bone remodeling. Previous studies have shown that osteoclasts possess abundant mitochondria and derive most energy through oxidative phosphorylation (OXPHOS). However, the energy substrates fueling OXPHOS in osteoclasts remain to be fully defined. Here, we showed that osteoclast differentiation was coupled with increased oxidation of glucose, glutamine and oleate. Transcriptomic analyses with RNA sequencing revealed marked upregulation of genes participating in OXPHOS and mitochondrial fatty acids oxidation, during osteoclast differentiation. Increased mitochondrial oxidation of long-chain fatty acids was required for osteoclast differentiation in vitro. However, blocking fatty acid oxidation in vivo, by deletion of Cpt1a in osteoclast progenitors, impaired osteoclast formation only in the female mice. The Cpt1a-deficient females were further protected from osteoclast activation by a high fat diet. The males, on the contrary, exhibited normal bone resorption despite Cpt1a deletion, regardless of the dietary fat content. Moreover, concurrent deletion of Mpc1 and Cpt1a, blocking mitochondrial oxidation of both glucose and fatty acids in the osteoclast lineage, failed to impede bone resorption in the males. The study therefore uncovers a female-specific dependence on mitochondrial oxidation of fatty acids and glucose in osteoclasts in vivo.
Chao Song, Arianna Valeri, Fangfang Song, Xing Ji, Xueyang Liao, Tyler Marmo, Rebecca A. Seeley, Jared Rutter, Fanxin Long
Osteocytes express parathyroid hormone (PTH)/PTH-related protein (PTHrP) receptors and respond to the PTHrP analog abaloparatide (ABL) and to the PTH 1-34 fragment teriparatide (TPTD), which are used to treat osteoporosis. Several studies indicate overlapping but distinct skeletal responses to ABL or TPTD, but their effects on cortical bone may differ. Little is known about their differential effects on osteocytes. We compared cortical osteocyte and skeletal responses to ABL and TPTD in sham-operated and ovariectomized mice. Administered 7 weeks after ovariectomy for 4 weeks at a dose of 40 μg/kg/d, TPTD and ABL had similar effects on trabecular bone, but ABL showed stronger effects in cortical bone. In cortical osteocytes, both treatments decreased lacunar area, reflecting altered peri-lacunar remodeling favoring matrix accumulation. Osteocyte RNA-Seq revealed that several genes and pathways were altered by ovariectomy and affected similarly by TPTD and ABL. Notwithstanding, several signaling pathways were uniquely regulated by ABL. Thus, in mice, TPTD and ABL induced a positive osteocyte peri-lacunar remodeling balance, but ABL induced stronger cortical responses and affected the osteocyte transcriptome differently. We concluded that ABL affected the cortical osteocyte transcriptome in a manner subtly different from TPTD, resulting in more beneficial remodeling/modeling changes and homeostasis of the cortex.
Zhengtao Lv, Jiaming Zhang, Shuang Liang, Chenhe Zhou, Dorothy Hu, Daniel J. Brooks, Mary L. Bouxsein, Beate Lanske, Paul Kostenuik, Francesca Gori, Roland Baron
Overactive fibroblast growth factor receptor 3 (FGFR3) signaling drives pathogenesis in a variety of cancers and a spectrum of short-limbed bone dysplasias, including the most common form of human dwarfism, achondroplasia (ACH). Targeting FGFR3 activity holds great promise as a therapeutic approach for treatment of these diseases. Here, we established a receptor/adaptor translocation assay system that can specifically monitor FGFR3 activation, and we applied it to identify FGFR3 modulators from complex natural mixtures. An FGFR3-suppressing plant extract of Amaranthus viridis was identified from the screen, and two bioactive porphyrins, pheophorbide a (Pa) and pyropheophorbide a (PyroPa) were sequentially isolated from the extract and functionally characterized. Further analysis showed that Pa reduced excessive FGFR3 signaling by decreasing its half-life in FGFR3-overactivated multiple myeloma (MM) cells and chondrocytes. In an ex vivo culture system, Pa alleviated defective long bone growth in humanized ACH mice (FGFR3ACH mice). Overall, our study presents a novel approach to discovery and validation of plant extracts or drug candidates that target FGFR3 activation. The compounds identified by this approach may have potential applications as therapeutics for FGFR3-associated cancers and skeletal dysplasias.
Yun-Wen Lin, Hsiao-Jung Kao, Wei-Ting Chen, Cheng-Fu Kao, Jer-Yuarn Wu, Yuan-Tsong Chen, Yi-Ching Lee
Osteogenesis imperfecta (OI), brittle bone disease, is a disorder characterized by bone fragility and increased fracture incidence. All forms of OI also feature short stature, implying an effect on endochondral ossification. Using the Aga2+/- mouse, which has a mutation in type I collagen, we show an affected growth plate primarily due to a shortened proliferative zone. We used scRNAseq analysis of tibial and femoral growth plate tissues to understand transcriptional consequences on growth plate cell types. We show that perichondrial cells, which express abundant type I procollagen, and growth plate chondrocytes, which were found to express low amounts of type I procollagen, had ER stress and dysregulation of the same UPR pathway as previously demonstrated in osteoblasts. Aga2+/- proliferating chondrocytes showed increased FGF and MAPK signaling, findings consistent with accelerated differentiation. There was also increased Sox9 expression throughout the growth plate, which is expected to accelerate early chondrocyte differentiation but reduce late hypertrophic differentiation. These data reveal that mutant type I collagen expression in OI has a previously unappreciated impact on the cartilage growth plate. These effects on endochondral ossification indicate that OI is a biologically complex phenotype going beyond its known impacts on bone to negatively affect linear growth.
Jennifer Zieba, Lisette Nevarez, Davis Wachtell, Jorge H. Martin, Alexander Kot, Sereen Wong, Daniel H. Cohn, Deborah Krakow
Mucopolysaccharidosis VI (MPS VI) is a rare lysosomal disease arising from impaired function of the enzyme Arylsulfatase B (ARSB). This causes aberrant accumulation of dermatan sulfate, a glycosaminoglycan (GAG) abundant in cartilage. While clinical severity varies along with age at first symptom manifestation, MPS VI usually presents early and strongly affects the skeleton. Current enzyme replacement therapy (ERT) does not provide effective treatment for the skeletal manifestations of MPS VI. This lack of efficacy may be due to inability of ERT to reach affected cells, or irreversibility of disease. To address the question of reversibility of skeletal phenotypes, we generated a conditional by inversion (COIN) mouse model of MPS VI, ArsbCOIN/COIN, wherein Arsb is initially null and can be restored to wild type using Cre. We restored Arsb at different times during postnatal development, using a tamoxifen-dependent global Cre driver. By restoring Arsb at postnatal days 7, 21, and 56-70 (P7, P21, and P56-P70), we determined that skeletal phenotypes can be fully rescued if Arsb restoration occurs at P7, while only achieving partial rescue at P21, and no significant rescue at P56-70. This work has highlighted the importance of early intervention in MPS VI patients to maximize therapeutic impact.
Elizabeth Hwang-Wong, Gabrielle Amar, Nanditha Das, Xiaoli Zhang, Nina A. Aaron, Kirsten Gale, Nyanza J. Rothman, Massimo Fante, Andrew D. Baik, Ajay Bhargava, Arun D. Fricker, Michelle McAlister, Jeremy S. Rabinowitz, John Lees-Shepard, Kalyan Nannuru, Aris N. Economides, Katherine D. Cygnar
Osteoarthritis (OA) is the most common joint disorder, and disease-modifying OA drugs (DMOADs) represent a major need in OA management. Krüppel-like factor 4 (KLF4) is a central transcription factor upregulating regenerative and protective functions in joint tissues. This study was aimed to identify small molecules activating KLF4 expression and to determine functions and mechanisms of the hit compounds. High-throughput screening (HTS) with 11,948 clinical-stage compounds was performed using a reporter cell line detecting endogenous KLF4 activation. Eighteen compounds were identified through the HTS and confirmed in a secondary screen. After testing in SW1353 chondrosarcoma cells and human chondrocytes, mocetinostat — a class I selective histone deacetylase (HDAC) inhibitor — had the best profile of biological activities. Mocetinostat upregulated cartilage signature genes in human chondrocytes, meniscal cells, and BM-derived mesenchymal stem cells, and it downregulated hypertrophic, inflammatory, and catabolic genes in those cells and synoviocytes. I.p. administration of mocetinostat into mice reduced severity of OA-associated changes and improved pain behaviors. Global gene expression and proteomics analyses revealed that regenerative and protective effects of mocetinostat were dependent on peroxisome proliferator-activated receptor γ coactivator 1-α. These findings show therapeutic and protective activities of mocetinostat against OA, qualifying it as a candidate to be used as a DMOAD.
Manabu Kawata, Daniel B. McClatchy, Jolene K. Diedrich, Merissa Olmer, Kristen A. Johnson, John R. Yates, Martin K. Lotz
Denosumab is an anti-RANKL antibody that potently suppresses bone resorption, increases bone mass, and reduces fracture risk. Discontinuation of denosumab causes rapid rebound bone resorption and bone loss but the molecular mechanisms are unclear. We generated humanized RANKL mice and treated them with denosumab to examine the cellular and molecular conditions associated with rebound resorption. Denosumab potently suppressed both osteoclast and osteoblast number in cancellous bone in humanized RANKL mice. The decrease in osteoclast number was not associated with changes in osteoclast progenitors in bone marrow. Long-term but not short-term denosumab administration reduced OPG mRNA in bone. Localization of OPG expression revealed that OPG mRNA is produced by a subpopulation of osteocytes. Long-term denosumab administration reduced osteocyte OPG mRNA suggesting that OPG expression declines as osteocytes age. Consistent with this, osteocyte expression of OPG was more prevalent near the surface of cortical bone in humans and mice. These results suggest that new osteocytes are an important source of OPG in remodeling bone and that suppression of remodeling reduces OPG abundance by reducing new osteocyte formation. The lack of new osteocytes and the OPG they produce may contribute to rebound resorption after denosumab discontinuation.
Qiang Fu, Nancy C. Bustamante-Gomez, Humberto Reyes-Pardo, Igor Gubrij, Diana Escalona-Vargas, Jeff D. Thostenson, Michela Palmieri, Joseph J. Goellner, Intawat Nookaew, C. Lowry Barnes, Jeffrey B. Stambough, Elena Ambrogini, Charles A. O'Brien
Reactive oxygen species (ROS) are natural products of mitochondrial oxidative metabolism and oxidative protein folding. ROS levels must be well controlled as elevated ROS has been shown to have deleterious effects on osteoblasts. Moreover, excessive ROS is thought to underly many of the skeletal phenotypes associated with aging and sex steroid deficiency in mice and humans. The mechanisms by which osteoblasts regulate ROS and how ROS inhibits osteoblasts are not well understood. Here, we demonstrate that de novo glutathione (GSH) biosynthesis is essential to neutralize ROS and establish a pro-osteogenic REDOX environment. Using a multifaceted approach, we demonstrate that reducing GSH biosynthesis leads to acute degradation of RUNX2, impaired osteoblast differentiation and reduced bone formation. Conversely, reducing ROS using Catalase enhances RUNX2 stability and promotes osteoblast differentiation and bone formation when GSH biosynthesis is limited. Highlighting the therapeutic implications of these findings, in utero antioxidant therapy stabilizes RUNX2 and improves bone development in the Runx2+/- haploinsufficient mouse model of human Cleidocranial Dysplasia. Thus, our data establish RUNX2 as a molecular sensor of the osteoblast REDOX environment and mechanistically clarifies how ROS negatively impacts osteoblast differentiation and bone formation.
Guoli Hu, Yilin Yu, Deepika Sharma, Shondra M. Pruett-Miller, Yinshi Ren, Guo-Fang Zhang, Courtney M. Karner
Hypochondroplasia (HCH) is a mild dwarfism caused by missense mutations in fibroblast growth factor receptor 3 (FGFR3), with the majority of cases resulting from a heterozygous p.Asn540Lys gain-of-function mutation. Here, we report the generation and characterization of the first mouse model (Fgfr3Asn534Lys/+) of HCH to our knowledge. Fgfr3Asn534Lys/+ mice exhibited progressive dwarfism and impairment of the synchondroses of the cranial base, resulting in defective formation of the foramen magnum. The appendicular and axial skeletons were both severely affected and we demonstrated an important role of FGFR3 in regulation of cortical and trabecular bone structure. Trabecular bone mineral density (BMD) of long bones and vertebral bodies was decreased, but cortical BMD increased with age in both tibiae and femurs. These results demonstrate that bones in Fgfr3Asn534Lys/+ mice, due to FGFR3 activation, exhibit some characteristics of osteoporosis. The present findings emphasize the detrimental effect of gain-of-function mutations in the Fgfr3 gene on long bone modeling during both developmental and aging processes, with potential implications for the management of elderly patients with hypochondroplasia and osteoporosis.
Léa Loisay, Davide Komla-Ebri, Anne Morice, Yann Heuzé, Camille Viaut, Amélie de La Seiglière, Nabil Kaci, Danny Chan, Audrey Lamouroux, Geneviève Baujat, J.H. Duncan Bassett, Graham R. Williams, Laurence Legeai-Mallet
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