Bone biology
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
Abstract
Increased subchondral bone angiogenesis with blood vessels breaching the tidemark into the avascular cartilage is a diagnostic feature of human osteoarthritis. However, the mechanisms that initiate subchondral bone angiogenesis remain unclear. We show that abnormally increased platelet-derived growth factor-BB (PDGF-BB) secretion by mononuclear preosteoclasts induces subchondral bone angiogenesis, contributing to osteoarthritis development. In mice after destabilization of the medial meniscus (DMM), aberrant joint subchondral bone angiogenesis developed during an early stage of osteoarthritis, before articular cartilage damage occurred. Mononuclear preosteoclasts in subchondral bone secrete excessive amounts of PDGF-BB, which activates platelet-derived growth factor receptor β (PDGFRβ) signaling in pericytes for neo-vessel formation. Selective knockout of PDGF-BB in preosteoclasts attenuates subchondral bone angiogenesis and abrogates joint degeneration and subchondral innervation induced by DMM. Transgenic mice that express PDGF-BB in preosteoclasts recapitulate pathological subchondral bone angiogenesis and develop joint degeneration and subchondral innervation spontaneously. Our study provides the first evidence that PDGF-BB derived from preosteoclasts is a key driver of pathological subchondral bone angiogenesis during osteoarthritis development and offers a new avenue for developing early treatments for this disease.
Authors
Weiping Su, Guanqiao Liu, Xiaonan Liu, Yangying Zhou, Qi Sun, Gehua Zhen, Xiao Wang, Yihe Hu, Peisong Gao, Shadpour Demehri, Xu Cao, Mei Wan
Abstract
We reported that transgenic mice expressing measles virus nucleocapsid protein (MVNP) in OCLs (MVNP mice) are a Paget’s disease (PD) model, and that osteoclasts (OCLs) from PD patients and MVNP mice express high levels of OCL-derived IGF1 (OCL-IGF1). To determine OCL-IGF1’s role in PD and normal bone remodeling, we generated WT and MVNP mice with targeted deletion of Igf1 in OCLs (Igf1-cKO) and MVNP/Igf1-cKO mice and assessed OCL-IGF1’s effects on bone mass, bone formation rate, ephrinB2/EphB4 expression on OCLs and osteoblasts (OBs) and pagetic bone lesions (PDLs). Forty percent of MVNP mice but no MVNP/Igf1-cKO mice had PDLs. BV/TV was decreased 60% in lumbar vertebrae and femurs of MVNP/Igf1-cKO vs. MVNP mice with PDLs and by 45% vs. all MVNP mice tested. Bone formation rates were decreased 50% in Igf1-cKO and MVNP/Igf1-cKO mice vs. WT and MVNP mice. MVNP mice had increased ephrinB2 and EphB4 levels in OCLs/OBs vs. WT and MVNP/Igf1-cKO, with none detectable in OCLs/OBs of Igf1-cKO mice. Mechanistically, IL-6 induced the increased OCL-IGF1 in MVNP mice. These results suggest that high OCL-IGF1 levels increase bone formation and PDLs in PD by enhancing ephrinB2/EphB4 expression in vivo, and that OCL-IGF1 may possibly contribute to normal bone remodeling.
Authors
Kazuaki Miyagawa, Yasuhisa Ohata, Jesus Delgado-Calle, Jumpei Teramachi, Hua Zhou, David W. Dempster, Mark A. Subler, Jolene J. Windle, John Chirgwin, G. David Roodman, Noriyoshi Kurihara
Abstract
We previously established that DNA methyltransferase 3b (Dnmt3b) is the sole Dnmt responsive to fracture repair and that Dnmt3b expression is induced in progenitor cells during fracture repair. In the current study, we confirmed that Dnmt3b ablation in mesenchymal progenitor cells (MPCs) resulted in impaired endochondral ossification, delayed fracture repair, and reduced mechanical strength of the newly formed bone in Prx1-Cre;Dnmt3bf/f (Dnmt3bPrx1) mice. Mechanistically, deletion of Dnmt3b in MPCs led to reduced chondrogenic and osteogenic differentiation in vitro. We further identified Rbpjκ as a downstream target of Dnmt3b in MPCs. In fact, we located 2 Dnmt3b binding sites in the murine proximal Rbpjκ promoter and gene body and confirmed Dnmt3b interaction with the 2 binding sites by ChIP assays. Luciferase assays showed functional utilization of the Dnmt3b binding sites in murine C3H10T1/2 cells. Importantly, we showed that the MPC differentiation defect observed in Dnmt3b deficiency cells was due to the upregulation of Rbpjκ, evident by restored MPC differentiation upon Rbpjκ inhibition. Consistent with in vitro findings, Rbpjκ blockage via dual antiplatelet therapy reversed the differentiation defect and accelerated fracture repair in Dnmt3bPrx1 mice. Collectively, our data suggest that Dnmt3b suppresses Notch signaling during MPC differentiation and is necessary for normal fracture repair.
Authors
Jun Ying, Taotao Xu, Cuicui Wang, Hongting Jin, Peijian Tong, Jianjun Guan, Yousef Abu-Amer, Regis O’Keefe, Jie Shen
Abstract
Extracellular matrix and osmolarity influence the development and homeostasis of skeletal tissues through Rho GTPase-mediated alteration of the actin cytoskeleton. This study investigated whether the actin-branching Arp2/3 complex, a downstream effector of the Rho GTPases Cdc42 and Rac1, plays a critical role in maintaining the health of matrix-rich and osmotically loaded intervertebral discs and cartilage. Mice with constitutive intervertebral disc and cartilage-specific deletion of the critical Arp2/3 subunit Arpc2 (Col2-Cre; Arpc2f/f) developed chondrodysplasia and spinal defects. Since these mice did not survive to adulthood, we generated mice with inducible Arpc2 deletion in disc and cartilage (Acan-CreERT2; Arpc2f/f). Inactivation of Arp2/3 at skeletal maturity resulted in growth plate closure, loss of proteoglycan content in articular cartilage, and degenerative changes in the intervertebral disc at 1 year of age. Chondrocytes with Arpc2 deletion showed compromised cell spreading on both collagen and fibronectin. Pharmacological inhibition of Cdc42 and Arp2/3 prevented the osmoadaptive transcription factor TonEBP/NFAT5 from recruiting co-factors in response to a hyperosmolarity challenge. Together, these findings suggest that Arp2/3 plays a critical role in cartilaginous tissues through the regulation of cell-extracellular matrix interactions and modulation of TonEBP-mediated osmoadaptation.
Authors
Steven Tessier, Alexandra C Doolittle, Kimheak Sao, Jeremy D. Rotty, James E. Bear, Veronica Ulici, Richard F. Loeser, Irving M. Shapiro, Brian O. Diekman, Makarand V. Risbud
Abstract
Background: Inflammation is implicated in many aging-related disorders. In animal models, menopause leads to increased gut permeability and inflammation. Our primary objective was to determine if gut permeability increases during the menopause transition (MT) in women. Our exploratory objectives were to examine whether greater gut permeability is associated with more inflammation and lower bone mineral density (BMD).Methods: We included 65 women from the Study of Women’s Health Across the Nation. Key measures were markers of gut permeability (gut barrier dysfunction [fatty acid binding protein 2 [FABP2]) and immune activation secondary to gut microbial translocation (lipopolysaccharide binding protein [LBP], soluble CD14 [sCD14]); inflammation (high-sensitivity CRP); and lumbar spine (LS) or total hip (TH) BMD. Results: In our primary analysis, FABP2, LBP, and sCD14 increased by 22.8% (P = 0.001), 3.7% (P = 0.05), and 8.9% (P = 0.0002), respectively from pre- to postmenopause. In exploratory, repeated measures, mixed-effects linear regression (adjusted for age at the premenopausal visit, body mass index, race/ethnicity, and study site), greater gut permeability was associated with greater inflammation, and lower LS and TH BMD. Conclusions: Gut permeability increases during the MT. Greater gut permeability is associated with more inflammation and lower BMD. Future studies should examine the longitudinal associations of gut permeability, inflammation, and BMD.Funding: NIH, Department of Health and Human Services, through the National Institute on Aging, National Institute of Nursing Research, and NIH Office of Research on Women’s Health (U01NR004061, U01AG012505, U01AG012535, U01AG012531, U01AG012539, U01AG012546, U01AG012553, U01AG012554, U01AG012495).
Authors
Albert Shieh, Marta Epeldegui, Arun S Karlamangla, Gail A. Greendale
Abstract
Dietary salt consumption leads to cutaneous Na+ storage and is associated with various disorders, including osteopenia. Here, we explore the impact of Na+ and the osmoprotective transcription factor nuclear factor of activated T cell 5 (NFAT5) on bone density and osteoclastogenesis. Compared with treatment of mice with high-salt diet, low-salt diet (LSD) increased bone density, decreased osteoclast numbers, and elevated Na+ content and Nfat5 levels in the BM. This response to LSD was dependent on NFAT5 expressed in myeloid cells. Simulating in vivo findings, we exposed osteoclast precursors and osteoblasts to elevated Na+ content (high-salt conditions; HS¢), resulting in increased NFAT5 binding to the promotor region of RANKL decoy receptor osteoprotegerin (OPG). These data not only demonstrate that NFAT5 in myeloid cells determines the Na+ content in BM, but that NFAT5 is able to govern the expression of the osteoprotective gene OPG. This provides insights into mechanisms of Na+-induced cessation of osteoclastogenesis and offers potentially new targets for treating salt-induced osteopenia.
Authors
Agnes Schröder, Patrick Neubert, Jens Titze, Aline Bozec, Wolfgang Neuhofer, Peter Proff, Christian Kirschneck, Jonathan Jantsch
Abstract
Observations in transgenic α-Klotho (Kl) mice (KlTg) defined the antiaging role of soluble Klotho (sKL130). A genetic translocation that elevates sKL levels in humans is paradoxically associated with increased circulating fibroblast growth factor 23 (FGF23) levels and the potential of both membrane KL (mKL135) and sKL130 to act as coreceptors for FGF23 activation of fibroblast growth factor receptors (FGFRs). Neither FGF23 expression nor the contributions of FGF23, mKL135, and sKL130 codependent and independent functions have been investigated in KlTg mice. In the current study, we examined the effects of Kl overexpression on FGF23 levels and functions in KlTg mice. We found that mKL135 but not sKL130 stimulated FGF23 expression in osteoblasts, leading to elevated Fgf23 bone expression and circulating levels in KlTg mice. Elevated FGF23 suppressed 1,25(OH)2D and parathyroid hormone levels but did not cause hypophosphatemic rickets in KlTg mice. KlTg mice developed low aldosterone–associated hypertension but not left ventricular hypertrophy. Mechanistically, we found that mKL135 and sKL130 are essential cofactors for FGF23-mediated ERK activation but that they inhibited FGF23 stimulation of PLC-γ and PI3K/AKT signaling. Thus, increased longevity in KlTg mice occurs in the presence of excess FGF23 that interacts with mKL and sKL to bias FGFR pathways.
Authors
Zhousheng Xiao, Gwendalyn King, Salvatore Mancarella, Undral Munkhsaikhan, Li Cao, Chun Cai, Leigh Darryl Quarles
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