Bone biology
Abstract
Activating mutations in the fibroblast growth factor receptor 3 (FGFR3) or inactivating mutations in guanylyl cyclase B (GC-B), also known as NPR-B or Npr2, cause short-limbed dwarfism. FGFR3 activation causes dephosphorylation and inactivation of GC-B, but the contribution of GC-B dephosphorylation to achondroplasia (ACH) is unknown. GC-B7E/7E mice that express a glutamate-substituted version of GC-B that cannot be inactivated by dephosphorylation were bred with mice expressing FGFR3-G380R, the most common human ACH mutation, to determine if GC-B dephosphorylation is required for ACH. Crossing GC-B7E/7E mice with FGFR3G380R/G380R mice increased naso-anal and long (tibia and femur), but not cranial, bone length twice as much as crossing GC-B7E/7E mice with FGFR3WT/WT mice from 4 to 16 weeks of age. Consistent with increased GC-B activity rescuing ACH, long bones from the GC-B7E/7E/FGFR3G380R/G380R mice were not shorter than those from GC-BWT/WT/FGFR3WT/WT mice. At two weeks of age, male but not female FGFR3G380R/G380R mice had shorter long bones and smaller growth plate hypertrophic zones, whereas female but not male GC-B7E/7E mice had longer bones and larger hypertrophic zones. In two-week old males, crossing FGFR3G380R/G380R mice with GC-B7E/7E mice increased long bone length and hypertrophic zone area to levels observed in mice expressing wild type versions of both receptors. We conclude that preventing GC-B dephosphorylation rescues reduced axial and appendicular skeleton growth in a mouse model of achondroplasia.
Authors
Brandon M. Wagner, Jerid W. Robinson, Yun-Wen Lin, Yi-Ching Lee, Nabil Kaci, Laurence Legeai-Mallet, Lincoln R. Potter
Abstract
Currently, no effective therapies exist for fibrodysplasia ossificans progressiva (FOP), a rare congenital syndrome in which heterotopic bone is formed in soft tissues due to dysregulated activity of the bone morphogenetic protein (BMP) receptor kinase ALK2/ACVR1. From a screen of known biologically active compounds, we identified saracatinib as a potent ALK2 kinase inhibitor. In enzymatic and cell-based assays, saracatinib preferentially inhibited ALK2 compared with other receptors of the BMP/TGFb signaling pathway, and induced dorsalization in zebrafish embryos consistent with BMP antagonism. We further tested the efficacy of saracatinib using an inducible ACVR1Q207D transgenic mouse line, which provides a model of heterotopic ossification, as well as an inducible ACVR1R206H knock-in, which serves as a genetically and physiologically faithful model of FOP. In both models, saracatinib was well tolerated and potently inhibited the development of heterotopic ossification even when administered transiently following soft tissue injury. Together, these data suggest that saracatinib is an efficacious clinical candidate for repositioning in the treatment of FOP, offering an accelerated path to clinical proof of efficacy studies and potentially significant benefits to individuals with this devastating condition.
Authors
Eleanor P. Williams, Jana Bagarova, Georgina Kerr, Dong-Dong Xia, Elsie S. Place, Devaveena Dey, Yue Shen, Geoffrey A. Bocobo, Agustin H. Mohedas, Xiuli Huang, Philip E. Sanderson, Arthur Lee, Wei Zheng, Aris N. Economides, James C. Smith, Paul B. Yu, Alex N. Bullock
Abstract
Osteoclasts are specialized cells of the hematopoietic lineage that are responsible for bone resorption and play a critical role in musculoskeletal disease. JAK2 is a key mediator of cytokine and growth factor signaling; however, its role in osteoclasts in vivo has yet to be investigated. To elucidate the role of JAK2 in osteoclasts, we generated an osteoclast-specific JAK2–KO (Oc-JAK2–KO) mouse using the Cre/Lox-P system. Oc-JAK2–KO mice demonstrated marked postnatal growth restriction; however, this was not associated with significant changes in bone density, microarchitecture, or strength, indicating that the observed phenotype was not due to alterations in canonical osteoclast function. Interestingly, Oc-JAK2–KO mice had reduced osteoclast-specific expression of IGF1, suggesting a role for osteoclast-derived IGF1 in determination of body size. To directly assess the role of osteoclast-derived IGF1, we generated an osteoclast-specific IGF1–KO mouse, which showed a similar growth-restricted phenotype. Lastly, overexpression of circulating IGF1 by human transgene rescued the growth defects in Oc-JAK2–KO mice, in keeping with a causal role of IGF1 in these models. Together, our data show a potentially novel role for Oc-JAK2 and IGF1 in the determination of body size, which is independent of osteoclast resorptive function.
Authors
David W. Dodington, Jenalyn L. Yumol, Jiaqi Yang, Evan Pollock-Tahiri, Tharini Sivasubramaniyam, Sandra M. Sacco, Stephanie A. Schroer, Yujin E. Li, Helen Le, Wendy E. Ward, Minna Woo
Abstract
Staphylococcus aureus is prevalent in surgical site infections (SSI) and leads to death in approximately 1% of patients. Phase IIB/III clinical trial results have demonstrated that vaccination against the iron-regulated surface determinant protein B (IsdB) is associated with an increased mortality rate in patients with SSI. Thus, we hypothesized that S. aureus induces nonneutralizing anti-IsdB antibodies, which facilitate bacterial entry into leukocytes to generate “Trojan horse” leukocytes that disseminate the pathogen. Since hemoglobin (Hb) is the primary target of IsdB, and abundant Hb-haptoglobin (Hb-Hp) complexes in bleeding surgical wounds are normally cleared via CD163-mediated endocytosis by macrophages, we investigated this mechanism in vitro and in vivo. Our results demonstrate that active and passive IsdB immunization of mice renders them susceptible to sepsis following SSI. We also found that a multimolecular complex containing S. aureus protein A–anti-IsdB–IsdB–Hb-Hp mediates CD163-dependent bacterial internalization of macrophages in vitro. Moreover, IsdB-immunized CD163–/– mice are resistant to sepsis following S. aureus SSI, as are normal healthy mice given anti-CD163–neutralizing antibodies. These genetic and biologic CD163 deficiencies did not exacerbate local infection. Thus, anti-IsdB antibodies are a risk factor for S. aureus sepsis following SSI, and disruption of the multimolecular complex and/or CD163 blockade may intervene.
Authors
Kohei Nishitani, Masahiro Ishikawa, Yugo Morita, Noriaki Yokogawa, Chao Xie, Karen L. de Mesy Bentley, Hiromu Ito, Stephen L. Kates, John L. Daiss, Edward M. Schwarz
Abstract
In aging mice, osteoclast number increases in cortical bone but declines in trabecular bone, suggesting that different mechanisms underlie age-associated bone loss in these two compartments. Osteocytes produce the osteoclastogenic cytokine RANKL, encoded by Tnfsf11. Tnfsf11 mRNA increases in cortical bone of aged mice, suggesting a mechanism underlying the bone loss. To address this possibility, we aged mice lacking RANKL in osteocytes. Whereas 24-month-old control mice had lower cortical bone compared to 8-month-old controls, mice lacking RANKL in osteocytes gained cortical bone from 8 to 24 months of age. Mice of both genotypes lost trabecular bone with age. Osteoclasts increased with age in cortical bone of control mice, but not in RANKL conditional knockout mice. Induction of cellular senescence increased RANKL production in murine and human cell culture models, suggesting an explanation for elevated RANKL levels with age. Over-expression of the senescence-associated transcription factor Gata4 stimulated Tnfsf11 expression in cultured murine osteoblastic cells. Lastly, elimination of senescent cells from aged mice using senolytic compounds reduced Tnfsf11 mRNA in cortical bone. Our results demonstrate the requirement of osteocyte-derived RANKL for age-associated cortical bone loss and suggest that increased Tnfsf11 expression with age results from accumulation of senescent cells in cortical bone.
Authors
Ha-Neui Kim, Jinhu Xiong, Ryan S. MacLeod, Srividhya Iyer, Yuko Fujiwara, Keisha M. Cawley, Li Han, Yonghan He, Jeff D. Thostenson, Elisabeth Ferreira, Robert L. Jilka, Daohong Zhou, Maria Almeida, Charles A. O'Brien
Abstract
Myelodysplastic syndromes (MDS) are clonal malignant hematopoietic disorders in the elderly characterized by ineffective hematopoiesis. This is accompanied by an altered bone microenvironment, which contributes to MDS progression and higher bone fragility. The underlying mechanisms remain largely unexplored. Here, we show that myelodysplastic NUP98‑HOXD13 (NHD13) transgenic mice display an abnormally high number of osteoblasts, yet a higher fraction of nonmineralized bone, indicating delayed bone mineralization. This was accompanied by high fibroblast growth factor-23 (FGF-23) serum levels, a phosphaturic hormone that inhibits bone mineralization and erythropoiesis. While Fgf23 mRNA expression was low in bone, brain, and kidney of NHD13 mice, its expression was increased in erythroid precursors. Coculturing these precursors with WT osteoblasts induced osteoblast marker gene expression, which was inhibited by blocking FGF-23. Finally, antibody-based neutralization of FGF-23 in myelodysplastic NHD13 mice improved bone mineralization and bone microarchitecture, and it ameliorated anemia. Importantly, higher serum levels of FGF‑23 and an elevated amount of nonmineralized bone in patients with MDS validated the findings. C‑terminal FGF‑23 correlated negatively with hemoglobin levels and positively with the amount of nonmineralized bone. Thus, our study identifies FGF-23 as a link between altered bone structure and ineffective erythropoiesis in MDS with the prospects of a targeted therapeutic intervention.
Authors
Heike Weidner, Ulrike Baschant, Franziska Lademann, Maria G. Ledesma Colunga, Ekaterina Balaian, Christine Hofbauer, Barbara M. Misof, Paul Roschger, Stéphane Blouin, William G. Richards, Uwe Platzbecker, Lorenz C. Hofbauer, Martina Rauner
Abstract
Musculoskeletal disorders represent the 3rd greatest burden on health in the developed world. Osteoarthritis is the single greatest cause of chronic pain, has no cure, and affects 8.5 and 27 million in the UK and US respectively. Osteoarthritis commonly occurs after joint injury, particularly affecting younger patients. Painful joints are often treated with injections of steroid or hyaluronic acid (HA), but treatments to prevent subsequent joint degeneration remain elusive. In animals, joint injury increases glutamate release into the joint, acting on nerves to cause pain, and joint tissues to cause inflammation and degeneration. This study investigated synovial fluid glutamate concentrations and glutamate receptor (GluR) expression in injured human joints and compared efficacy of GluR antagonists with current treatments in a mouse model of injury-induced osteoarthritis (ACL rupture). GluRs were expressed in ligament and meniscus after knee injury and synovial fluid glutamate concentrations ranged from 19–129 µM. Intra-articular injection of NBQX (GluR antagonist), administered at the time of injury, substantially reduced swelling and degeneration in the mouse ACL rupture model. HA had no effect and depo-medrone reduced swelling for 1 day, but increased degeneration by 50%. Intra-articular administration of NBQX was both symptom and disease modifying to a greater extent than current treatments. There is an opportunity for repurposing related drugs, developed for CNS disorders, with proven safety in man, to prevent injury-induced osteoarthritis. This could quickly reduce the substantial burden associated with osteoarthritis.
Authors
Cleo S. Bonnet, Sophie J. Gilbert, Emma J. Blain, Anwen S. Williams, Deborah J. Mason
Abstract
Heterotopic ossification (HO) is defined as abnormal differentiation of local stromal cells of mesenchymal origin resulting in pathologic cartilage and bone matrix deposition. CCN family members are matricellular proteins that have diverse regulatory functions on cell proliferation and differentiation, including the regulation of chondrogenesis. However, little is known regarding CCN family member expression or function in HO. Here, a combination of bulk and single cell RNA sequencing defined the dynamic temporospatial pattern of CCN family member induction within a mouse model of trauma-induced HO. Among CCN family proteins, Wisp1(also known as Ccn4) was most upregulated during the evolution of HO, and Wisp1 expression corresponded with chondrogenic gene profile. Immunohistochemistry confirmed WISP1/CCN4 expression across traumatic and genetic HO mouse models, as well as in human HO samples. Transgenic Wisp1LacZ/LacZ knockin animals showed an increase in endochondral ossification in HO after trauma. Finally, the transcriptome of Wisp1 null tenocytes revealed enrichment in signaling pathways such as STAT3 and PCP signaling that may explain increased HO in the context of Wisp1 deficiency. In sum, CCN family members, and in particular Wisp1, are spatiotemporally associated with and negatively regulate trauma-induced HO formation.
Authors
Ginny Ching-Yun Hsu, Simone Marini, Stefano Negri, Yiyun Wang, Jiajia Xu, Chase A. Pagani, Charles Hwang, David M. Stepien, Carolyn A. Meyers, Sarah Miller, Edward McCarthy, Karen M. Lyons, Benjamin Levi, Aaron W. James
Abstract
Bone fractures are a major cause of morbidity and mortality, particularly in diabetic patients, who have a high incidence of fractures and exhibit poor fracture healing. Coordinated expression of osteoblast-derived vascular endothelial growth factor (VEGF) and bone morphogenic proteins (BMPs) is essential for fracture repair. The NO/cGMP/protein kinase G (PKG) signaling pathway mediates osteoblast responses to estrogens and mechanical stimulation, but the pathway’s role in bone regeneration is unknown. Here, we used a mouse cortical defect model to simulate bone fractures and studied osteoblast-specific PKG1 knockout and diabetic mice. The knock-out mice had normal bone micro-architecture, but after injury exhibited poor bone regeneration, with decreased osteoblasts, collagen deposition, and microvessels in the bone defect area. Primary osteoblasts and tibiae from the knock-out mice expressed low amounts of Vegfa and Bmp2/4 mRNAs, and PKG1 was required for cGMP-stimulated expression of these genes. Diabetic mice also demonstrated low Vegfa and Bmp2/4 expression in bone and impaired bone regeneration after injury; notably, the cGMP-elevating agent cinaciguat restored Vegfa and BMP2/4 expression, and full bone healing. We conclude that PKG1 is a key orchestrator of VEGF and BMP signaling during bone regeneration and propose pharmacological PKG activation as a novel therapeutic approach to enhance fracture healing.
Authors
Nadine Schall, Julian J. Garcia, Hema Kalyanaraman, Shyamsundar Pal China, Jenna J. Lee, Robert L. Sah, Alexander Pfeifer, Renate B. Pilz
Abstract
The worldwide prevalence of type 2 diabetes (T2D) is increasing. Despite normal to higher bone density, patients with T2D paradoxically have elevated fracture risk resulting, in part, from poor bone quality. Advanced glycation endproducts (AGEs) and inflammation as a consequence of enhanced receptor for AGE (RAGE) signaling are hypothesized culprits, although the exact mechanisms underlying skeletal dysfunction in T2D are unclear. Lack of inducible models that permit environmental (in obesity) and temporal (after skeletal maturity) control of T2D onset has hampered progress. Here, we show in C57BL/6 mice that a one-time pharmacological intervention (streptozotocin [STZ]) initiated in adulthood combined with high-fat diet (HFD)-induced obesity caused hallmark features of human adult-onset T2D, including prolonged hyperglycemia, insulin resistance, and pancreatic β-cell dysfunction, but not complete destruction. In addition, HFD/STZ (i.e., T2D) resulted in several changes in bone quality that closely mirror those observed in humans, including compromised bone microarchitecture, reduced biomechanical strength, impaired bone material properties, altered bone turnover, and elevated levels of the AGE, CML, in bone and blood. Furthermore, T2D led to the premature accumulation of senescent osteocytes with a unique pro-inflammatory signature. These findings highlight the RAGE pathway and senescent cells as potential targets to treat diabetic skeletal fragility.
Authors
Brittany A. Eckhardt, Jennifer L. Rowsey, Brianne S. Thicke, Daniel G. Fraser, Katherine L. O’Grady, Olga P. Bondar, Jolaine M. Hines, Ravinder J. Singh, Andrew R. Thoreson, Kuntol Rakshit, Anthony B. Lagnado, João F. Passos, Adrian Vella, Aleksey V. Matveyenko, Sundeep Khosla, David G. Monroe, Joshua N. Farr
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