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Mutations in the coactivator CREB-binding protein (CBP) are a major cause of the human skeletal dysplasia Rubinstein-Taybi syndrome (RTS); however, the mechanism by which these mutations affect skeletal mineralization and patterning is unknown. Here, we report the identification of 3-phosphoinositide-dependent kinase 1 (PDK1) as a key regulator of CBP activity and demonstrate that its functions map to both osteoprogenitor cells and mature osteoblasts. In osteoblasts, PDK1 activated the CREB/CBP complex, which in turn controlled runt-related transcription factor 2 (RUNX2) activation and expression of bone morphogenetic protein 2 (BMP2). These pathways also operated in vivo, as evidenced by recapitulation of RTS spectrum phenotypes with osteoblast-specific Pdk1 deletion in mice (Pdk1osx mice) and by the genetic interactions observed in mice heterozygous for both osteoblast-specific Pdk1 deletion and either Runx2 or Creb deletion. Finally, treatment of Pdk1osx and Cbp+/- embryos with BMPs in utero partially reversed their skeletal anomalies at birth. These findings illustrate the in vivo function of the PDK1-AKT-CREB/CBP pathway in bone formation and provide proof of principle for in utero growth factor supplementation as a potential therapy for skeletal dysplasias. [ABSTRACT FROM AUTHOR]

The periosteum contains skeletal stem/progenitor cells that contribute to bone fracture healing. However, the in vivo identity of periosteal skeletal stem cells (P-SSCs) remains unclear, and membrane protein markers of P-SSCs that facilitate tissue engineering are needed. Here, we identified integral membrane protein 2A (Itm2a) enriched in SSCs using single-cell transcriptomics. Itm2a+ P-SSCs displayed clonal multipotency and self-renewal and sat at the apex of their differentiation hierarchy. Lineage-tracing experiments showed that Itm2a selectively labeled the periosteum and that Itm2a+ cells were preferentially located in the outer fibrous layer of the periosteum. The Itm2a+ cells rarely expressed CD34 or Osx, but expressed periosteal markers such as Ctsk, CD51, PDGFRA, Sca1, and Gli1. Itm2a+ P-SSCs contributed to osteoblasts, chondrocytes, and marrow stromal cells upon injury. Genetic lineage tracing using dual recombinases showed that Itm2a and Prrx1 lineage cells generated spatially separated subsets of chondrocytes and osteoblasts during fracture healing. Bone morphogenetic protein 2 (Bmp2) deficiency or ablation of Itm2a+ P-SSCs resulted in defects in fracture healing. ITM2A+ P-SSCs were also present in the human periosteum. Thus, our study identified a membrane protein marker that labels P-SSCs, providing an attractive target for drug and cellular therapy for skeletal disorders.

Osteogenesis imperfecta (OI) is a disorder of low bone mass and increased fracture risk due to a range of genetic variants that prominently include mutations in genes encoding type I collagen. While it is well known that OI reflects defects in the activity of bone-forming osteoblasts, it is currently unclear whether OI also reflects defects in the many other cell types comprising bone, including defects in skeletal vascular endothelium or the skeletal stem cell populations that give rise to osteoblasts and whether correcting these broader defects could have therapeutic utility. Here, we find that numbers of skeletal stem cells (SSCs) and skeletal arterial endothelial cells (AECs) are augmented in Col1a2 oim/oim mice, a well-studied animal model of moderate to severe OI, suggesting that disruption of a vascular SSC niche is a feature of OI pathogenesis. Moreover, crossing Col1a2 oim/oim mice to mice lacking a negative regulator of skeletal angiogenesis and bone formation, Schnurri 3 (SHN3), not only corrected the SSC and AEC phenotypes but moreover robustly corrected the bone mass and spontaneous fracture phenotypes. As this finding suggested a strong therapeutic utility of SHN3 inhibition for the treatment of OI, a bone-targeting AAV was used to mediate Shn3 knockdown, rescuing the Col1a2 oim/oim phenotype and providing therapeutic proof-of-concept for targeting SHN3 for the treatment of OI. Overall, this work both provides proof-of-concept for inhibition of the SHN3 pathway and more broadly addressing defects in the stem/osteoprogenitor niche as is a strategy to treat OI., (© 2024. The Author(s).)

The skeleton has been suggested to function as an endocrine organ controlling whole organism energy balance, however the mediators of this effect and their molecular links remain unclear. Here, utilizing Schnurri-3 -/- (Shn3 -/- ) mice with augmented osteoblast activity, we show Shn3 -/- mice display resistance against diet-induced obesity and enhanced white adipose tissue (WAT) browning. Conditional deletion of Shn3 in osteoblasts but not adipocytes recapitulates lean phenotype of Shn3 -/- mice, indicating this phenotype is driven by skeleton. We further demonstrate osteoblasts lacking Shn3 can secrete cytokines to promote WAT browning. Among them, we identify a C-terminal fragment of SLIT2 (SLIT2-C), primarily secreted by osteoblasts, as a Shn3-regulated osteokine that mediates WAT browning. Lastly, AAV-mediated Shn3 silencing phenocopies the lean phenotype and augmented glucose metabolism. Altogether, our findings establish a novel bone-fat signaling axis via SHN3 regulated SLIT2-C production in osteoblasts, offering a potential therapeutic target to address both osteoporosis and metabolic syndrome., (© 2024. The Author(s).)

Tissue-resident stem cells are essential for development and repair, and in the skeleton, this function is fulfilled by recently identified skeletal stem cells (SSCs). However, recent work has identified that SSCs are not monolithic, with long bones, craniofacial sites, and the spine being formed by distinct stem cells. Recent studies have utilized techniques such as fluorescence-activated cell sorting, lineage tracing, and single-cell sequencing to investigate the involvement of SSCs in bone development, homeostasis, and disease. These investigations have allowed researchers to map the lineage commitment trajectory of SSCs in different parts of the body and at different time points. Furthermore, recent studies have shed light on the characteristics of SSCs in both physiological and pathological conditions. This review focuses on discussing the spatiotemporal distribution of SSCs and enhancing our understanding of the diversity and plasticity of SSCs by summarizing recent discoveries., (© The Author(s) 2024. Published by Oxford University Press on behalf of Higher Education Press.)

Transforming growth factor beta (TGF-β) is ubiquitously found in bone and plays a key role in bone turnover. Mice expressing constitutively active TGF-β receptor type I ( Mx1;TβRI CA mice) are osteopenic. Here, we identified the candidate genes involved in bone turnover in Mx1;TβRI CA mice using RNA sequencing analysis. A total of 285 genes, including 87 upregulated and 198 downregulated genes, were differentially expressed. According to the KEGG analysis, some genes were involved in osteoclast differentiation ( Fcgr4 , Lilrb4a ), B cell receptor signaling ( Cd72 , Lilrb4a ), and neutrophil extracellular trap formation ( Hdac7 , Padi4 ). Lilrb4 is related to osteoclast inhibition protein, whereas Hdac7 is a Runx2 corepressor that regulates osteoblast differentiation. Silencing Lilrb4 increased the number of osteoclasts and osteoclast marker genes. The knocking down of Hdac7 increased alkaline phosphatase activity, mineralization, and osteoblast marker genes. Therefore, our present study may provide an innovative idea for potential therapeutic targets and pathways in TβRI -associated bone loss.

Adeno-associated viral (AAV) vectors have emerged as crucial tools in advancing gene therapy for skeletal diseases, offering the potential for sustained expression with low postinfection immunogenicity and pathogenicity. Preclinical studies support both the therapeutic efficacy and safety of these vectors, illustrating the promise of AAV-mediated gene therapy. Emerging technologies and innovations in AAV-mediated gene therapy strategies, such as gene addition, gene replacement, gene silencing, and gene editing, offer new approaches to clinical application. Recently, the increasing preclinical applications of AAV to rare skeletal diseases, such as fibrodysplasia ossificans progressiva (FOP) and osteogenesis imperfecta (OI), and prevalent bone diseases, such as osteoporosis, bone fracture, critical-sized bone defects, and osteoarthritis, have been reported. Despite existing limitations in clinical use, such as high cost and safety, the AAV-mediated gene transfer platform is a promising approach to deliver therapeutic gene(s) to the skeleton to treat skeletal disorders, including those otherwise intractable by other therapeutic approaches. This review provides a comprehensive overview of the therapeutic advancements, challenges, limitations, and solutions within AAV-based gene therapy for prevalent and rare skeletal diseases.

Loss of refreshment in nucleus pulposus (NP) cellularity leads to intervertebral disc (IVD) degeneration. Nevertheless, the cellular sequence of NP cell differentiation remains unclear, although an increasing body of literature has identified markers of NP progenitor cells (NPPCs). Notably, due to their fragility, the physical enrichment of NP-derived cells has limited conventional transcriptomic approaches in multiple studies. To overcome this limitation, a spatially resolved transcriptional atlas of the mouse IVD is generated via the 10x Genomics Visium platform dividing NP spots into two clusters. Based on this, most reported NPPC-markers, including Cathepsin K (Ctsk), are rare and predominantly located within the NP-outer subset. Cell lineage tracing further evidence that a small number of Ctsk-expressing cells generate the entire adult NP tissue. In contrast, Tie2, which has long suggested labeling NPPCs, is actually neither expressed in NP subsets nor labels NPPCs and their descendants in mouse models; consistent with this, an in situ sequencing (ISS) analysis validated the absence of Tie2 in NP tissue. Similarly, no Tie2-cre-mediated labeling of NPPCs is observed in an IVD degenerative mouse model. Altogether, in this study, the first spatial transcriptomic map of the IVD is established, thereby providing a public resource for bone biology., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

The development of bone-targeting drug delivery systems holds immense promise for improving the treatment of skeletal diseases. By precisely delivering therapeutic agents to the affected areas of bone, these strategies can enhance drug efficacy, minimize off-target effects, and promote patient adherence, ultimately leading to improved treatment outcomes and an enhanced quality of life for patients. This review aims to provide an overview of the current state of affinity-based bone-targeting agents and recent breakthroughs in innovative bone-targeting adeno-associated virus (AAV) strategies to treat skeletal diseases in mice. In particular, this review will delve into advanced AAV engineering, including AAV serotype selection for bone targeting and capsid modifications for bone-specific tropism. Additionally, we will highlight recent advancements in AAV-mediated gene therapy for skeletal diseases and discuss challenges and future directions of this promising therapeutic approach.

In the complex tumor microenvironment (TME), mesenchymal cells are key players, yet their specific roles in prostate cancer (PCa) progression remain to be fully deciphered. This study employs single-cell RNA sequencing to delineate molecular changes in tumor stroma that influence PCa progression and metastasis. Analyzing mesenchymal cells from four genetically engineered mouse models (GEMMs) and correlating these findings with human tumors, we identify eight stromal cell populations with distinct transcriptional identities consistent across both species. Notably, stromal signatures in advanced mouse disease reflect those in human bone metastases, highlighting periostin's role in invasion and differentiation. From these insights, we derive a gene signature that predicts metastatic progression in localized disease beyond traditional Gleason scores. Our results illuminate the critical influence of stromal dynamics on PCa progression, suggesting new prognostic tools and therapeutic targets., (© 2024. The Author(s).)

Chronic inflammation contributes to the development of skeletal disorders in patients with systemic lupus erythematosus (SLE). Activation of the host immune response stimulates osteoclast activity, which in turn leads to bone loss. Regenerating bone in the inflammatory microenvironments of SLE patients with critical bone defects remains a great challenge. In this study, we utilized lipopolysaccharide (LPS) to imitate locally and systemically pathogenic bacterial infection and examined the bone regeneration performance of LPS-associated mandibular and tibial bone regeneration impairment in FcγRIIB -/- mice. Our results indicated that a loss of FcγRIIB alleviates bone regeneration in both mandibles and tibiae. After LPS induction, FcγRIIB -/- mice were susceptible to impaired fracture healing in tibial and mandibular bones. LPS decreased the mineralization to collagen ratio in FcγRIIB -/- mice, indicating a mineralization defect during bone repair. An osteoblast-associated gene ( Col1a1 ) was attenuated in FcγRIIB -deficient mice, whereas Bglap, Hhip, and Creb5 were further downregulated with LPS treatment in FcγRIIB -/- mice compared to FcγRIIB -/- mice. Alpl and Bglap expression was dcreased in osteoblasts derived from bone chips. An osteoclast-associated gene, Tnfsf11/Tnfrsf11 ratio, ewas increased in LPS-induced FcγRIIB -/- mice and in vitro. Furthermore, systemic LPS was relatively potent in stimulating production of pro-inflammatory cytokines including TNF-α, IL-6, and MCP-1 in FcγRIIB -/- mice compared to FcγRIIB -/- mice. The levels of TNF-α, IFN-β, IL-1α, and IL-17A were increased, whereas IL-10 and IL-23 were decreased in Fcγ RIIB -/- mice treated locally with LPS. These findings suggest that both local and systemic LPS burden can exacerbate bone regeneration impairment, delay mineralization and skeletal repair, and induce inflammation in SLE patients., Competing Interests: The authors declare no conflict of interest.

We have previously reported that the cortical bone thinning seen in mice lacking the Wnt signaling antagonist Sfrp4 is due in part to impaired periosteal apposition. The periosteum contains cells which function as a reservoir of stem cells and contribute to cortical bone expansion, homeostasis, and repair. However, the local or paracrine factors that govern stem cells within the periosteal niche remain elusive. Cathepsin K (Ctsk), together with additional stem cell surface markers, marks a subset of periosteal stem cells (PSCs) which possess self-renewal ability and inducible multipotency. Sfrp4 is expressed in periosteal Ctsk-lineage cells, and Sfrp4 global deletion decreases the pool of PSCs, impairs their clonal multipotency for differentiation into osteoblasts and chondrocytes and formation of bone organoids. Bulk RNA sequencing analysis of Ctsk-lineage PSCs demonstrated that Sfrp4 deletion down-regulates signaling pathways associated with skeletal development, positive regulation of bone mineralization, and wound healing. Supporting these findings, Sfrp4 deletion hampers the periosteal response to bone injury and impairs Ctsk-lineage periosteal cell recruitment. Ctsk-lineage PSCs express the PTH receptor and PTH treatment increases the % of PSCs, a response not seen in the absence of Sfrp4 . Importantly, in the absence of Sfrp4 , PTH-dependent increase in cortical thickness and periosteal bone formation is markedly impaired. Thus, this study provides insights into the regulation of a specific population of periosteal cells by a secreted local factor, and shows a central role for Sfrp4 in the regulation of Ctsk-lineage periosteal stem cell differentiation and function.

Background: Artificial intelligence (AI) conversational agents, or chatbots, are computer programs designed to simulate human conversations using natural language processing. They offer diverse functions and applications across an expanding range of healthcare domains. However, their roles in laboratory medicine remain unclear, as their accuracy, repeatability, and ability to interpret complex laboratory data have yet to be rigorously evaluated., Content: This review provides an overview of the history of chatbots, two major chatbot development approaches, and their respective advantages and limitations. We discuss the capabilities and potential applications of chatbots in healthcare, focusing on the laboratory medicine field. Recent evaluations of chatbot performance are presented, with a special emphasis on large language models such as the Chat Generative Pre-trained Transformer in response to laboratory medicine questions across different categories, such as medical knowledge, laboratory operations, regulations, and interpretation of laboratory results as related to clinical context. We analyze the causes of chatbots' limitations and suggest research directions for developing more accurate, reliable, and manageable chatbots for applications in laboratory medicine., Summary: Chatbots, which are rapidly evolving AI applications, hold tremendous potential to improve medical education, provide timely responses to clinical inquiries concerning laboratory tests, assist in interpreting laboratory results, and facilitate communication among patients, physicians, and laboratorians. Nevertheless, users should be vigilant of existing chatbots' limitations, such as misinformation, inconsistencies, and lack of human-like reasoning abilities. To be effectively used in laboratory medicine, chatbots must undergo extensive training on rigorously validated medical knowledge and be thoroughly evaluated against standard clinical practice., (© Association for Diagnostics & Laboratory Medicine 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Bone morphogenetic protein (BMP) gene delivery to Lewis rat lumbar intervertebral discs (IVDs) drives bone formation anterior and external to the IVD, suggesting the IVD is inhospitable to osteogenesis. This study was designed to determine if IVD destruction with a proteoglycanase, and/or generating an IVD blood supply by gene delivery of an angiogenic growth factor, could render the IVD permissive to intra-discal BMP-driven osteogenesis and fusion. Surgical intra-discal delivery of naïve or gene-programmed cells (BMP2/BMP7 co-expressing or VEGF 165 expressing) +/- purified chondroitinase-ABC (chABC) in all permutations was performed between lumbar 4/5 and L5/6 vertebrae, and radiographic, histology, and biomechanics endpoints were collected. Follow-up anti-sFlt Western blotting was performed. BMP and VEGF/BMP treatments had the highest stiffness, bone production and fusion. Bone was induced anterior to the IVD, and was not intra-discal from any treatment. chABC impaired BMP-driven osteogenesis, decreased histological staining for IVD proteoglycans, and made the IVD permissive to angiogenesis. A soluble fragment of VEGF Receptor-1 (sFlt) was liberated from the IVD matrix by incubation with chABC, suggesting dysregulation of the sFlt matrix attachment is a possible mechanism for the chABC-mediated IVD angiogenesis we observed. Based on these results, the IVD can be manipulated to foster vascular invasion, and by extension, possibly osteogenesis.

Vertebral bone is subject to a distinct set of disease processes from long bones, including a much higher rate of solid tumour metastases 1-4 . The basis for this distinct biology of vertebral bone has so far remained unknown. Here we identify a vertebral skeletal stem cell (vSSC) that co-expresses ZIC1 and PAX1 together with additional cell surface markers. vSSCs display formal evidence of stemness, including self-renewal, label retention and sitting at the apex of their differentiation hierarchy. vSSCs are physiologic mediators of vertebral bone formation, as genetic blockade of the ability of vSSCs to generate osteoblasts results in defects in the vertebral neural arch and body. Human counterparts of vSSCs can be identified in vertebral endplate specimens and display a conserved differentiation hierarchy and stemness features. Multiple lines of evidence indicate that vSSCs contribute to the high rates of vertebral metastatic tropism observed in breast cancer, owing in part to increased secretion of the novel metastatic trophic factor MFGE8. Together, our results indicate that vSSCs are distinct from other skeletal stem cells and mediate the unique physiology and pathology of vertebrae, including contributing to the high rate of vertebral metastasis., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Craniosynostosis is a group of disorders of premature calvarial suture fusion. The identity of the calvarial stem cells (CSCs) that produce fusion-driving osteoblasts in craniosynostosis remains poorly understood. Here we show that both physiologic calvarial mineralization and pathologic calvarial fusion in craniosynostosis reflect the interaction of two separate stem cell lineages; a previously identified cathepsin K (CTSK) lineage CSC 1 (CTSK + CSC) and a separate discoidin domain-containing receptor 2 (DDR2) lineage stem cell (DDR2 + CSC) that we identified in this study. Deletion of Twist1, a gene associated with craniosynostosis in humans 2,3 , solely in CTSK + CSCs is sufficient to drive craniosynostosis in mice, but the sites that are destined to fuse exhibit an unexpected depletion of CTSK + CSCs and a corresponding expansion of DDR2 + CSCs, with DDR2 + CSC expansion being a direct maladaptive response to CTSK + CSC depletion. DDR2 + CSCs display full stemness features, and our results establish the presence of two distinct stem cell lineages in the sutures, with both populations contributing to physiologic calvarial mineralization. DDR2 + CSCs mediate a distinct form of endochondral ossification without the typical haematopoietic marrow formation. Implantation of DDR2 + CSCs into suture sites is sufficient to induce fusion, and this phenotype was prevented by co-transplantation of CTSK + CSCs. Finally, the human counterparts of DDR2 + CSCs and CTSK + CSCs display conserved functional properties in xenograft assays. The interaction between these two stem cell populations provides a new biologic interface for the modulation of calvarial mineralization and suture patency., (© 2023. The Author(s), under exclusive licence to Springer Nature Limited.)

Transforming growth factor beta (TGF-β) is a key factor mediating the intercellular crosstalk between the hematopoietic stem cells and their microenvironment. Here, we investigated the skeletal phenotype of transgenic mice expressing constitutively active TGF-β receptor type I under the control of Mx1-Cre ( Mx1;TβRI CA mice). μCT analysis showed decreased cortical thickness, and cancellous bone volume in both femurs and mandibles. Histomorphometric analysis confirmed a decrease in cancellous bone volume due to increased osteoclast number and decreased osteoblast number. Primary osteoblasts showed decreased ALP and mineralization. Constitutive TβRI activation increased osteoclast differentiation. qPCR analysis showed that Tnfsf11/Tnfrsf11b ratio, Ctsk , Sufu , and Csf1 were increased whereas Runx2 , Ptch1, and Ptch2 were decreased in Mx1;TβRI CA femurs. Interestingly, Gli1, Wnt3a , Sp7, Alpl, Ptch1, Ptch2 , and Shh mRNA expression were reduced whereas Tnfsf11/Tnfrsf11b ratio was increased in Mx1;TβRI CA mandibles. Similarly, osteoclast-related genes were increased in Mx1;TβRI CA osteoclasts whereas osteoblast-related genes were reduced in Mx1;TβRI CA osteoblasts. Western blot analysis indicated that SMAD2 and SMAD3 phosphorylation was increased in Mx1;TβRI CA osteoblasts, and SMAD3 phosphorylation was increased in Mx1;TβRI CA osteoclasts. CTSK was increased while RUNX2 and PTCH1 was decreased in Mx1;TβRI CA mice. Microindentation analysis indicated decreased hardness in Mx1;TβRI CA mice. Our study indicated that Mx1;TβRI CA mice were osteopenic by increasing osteoclast number and decreasing osteoblast number, possibly by suppressing Hedgehog signaling pathways.

Aldehyde dehydrogenase 2 (ALDH2) deficiency affects 35% to 45% of East Asians and 8% of the world population. ALDH2 is the second enzyme in the ethanol metabolism pathway. The common genetic variant ALDH2*2 allele has a glutamic acid-to-lysine substitution at position 487 (E487K) that reduces the enzyme activity, resulting in an accumulation of acetaldehyde after ethanol consumption. The ALDH2*2 allele is associated with increased risk of osteoporosis and hip fracture. Our prior study showed that administration of an adeno-associated virus (AAV) serotype rh.10 gene transfer vector expressing the human ALDH2 cDNA (AAVrh.10hALDH2) before initiation of ethanol consumption prevented bone loss in ALDH2-deficient homozygous knockin mice carrying the E487K mutation ( Aldh2 E487K+/+ ). We hypothesized that AAVrh.10hALDH2 administration after establishment of osteopenia would be able to reverse bone loss due to ALDH2 deficiency and chronic ethanol consumption. To test this hypothesis, male and female Aldh2 E487K+/+ mice ( n  = 6) were given ethanol in the drinking water for 6 weeks to establish osteopenia and then administered AAVrh.10hALDH2 (10 11 genome copies). Mice were evaluated for an additional 12 weeks. AAVrh.10hALDH2 administration after osteopenia was established corrected weight loss and locomotion phenotypes and, importantly, increased midshaft femur cortical bone thickness, the most important component of bone in the resistance to fractures, and showed a trend toward increased trabecular bone volume. AAVrh.10hALDH2 is a promising therapeutic for osteoporosis in ALDH2-deficient individuals. © 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research., Competing Interests: RGC has equity in and is a consultant to LEXEO Therapeutics, and RGC and OEP are participants in a patent disclosure regarding gene therapy for ALDH2 deficiency. All other authors state that they have no conflicts of interest., (© 2023 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.)

M-CSF is a critical growth factor for myeloid lineage cells, including monocytes, macrophages, and osteoclasts. Tissue-resident macrophages in most organs rely on local M-CSF. However, it is unclear what specific cells in the bone marrow produce M-CSF to maintain myeloid homeostasis. Here, we found that Adipoq-lineage progenitors but not mature adipocytes in bone marrow or in peripheral adipose tissue, are a major cellular source of M-CSF, with these Adipoq-lineage progenitors producing M-CSF at levels much higher than those produced by osteoblast lineage cells. The Adipoq-lineage progenitors with high CSF1 expression also exist in human bone marrow. Deficiency of M-CSF in bone marrow Adipoq-lineage progenitors drastically reduces the generation of bone marrow macrophages and osteoclasts, leading to severe osteopetrosis in mice. Furthermore, the osteoporosis in ovariectomized mice can be significantly alleviated by the absence of M-CSF in bone marrow Adipoq-lineage progenitors. Our findings identify bone marrow Adipoq-lineage progenitors as a major cellular source of M-CSF in bone marrow and reveal their crucial contribution to bone marrow macrophage development, osteoclastogenesis, bone homeostasis, and pathological bone loss., Competing Interests: KI, YQ, YX, JH, RY, JS, RX, MG No competing interests declared, JJ, BZ Reviewing editor, eLife, (© 2023, Inoue, Qin, Xia et al.)

Treating osteoporosis and associated bone fractures remains challenging for drug development in part due to potential off-target side effects and the requirement for long-term treatment. Here, we identify recombinant adeno-associated virus (rAAV)-mediated gene therapy as a complementary approach to existing osteoporosis therapies, offering long-lasting targeting of multiple targets and/or previously undruggable intracellular non-enzymatic targets. Treatment with a bone-targeted rAAV carrying artificial microRNAs (miRNAs) silenced the expression of WNT antagonists, schnurri-3 (SHN3), and sclerostin (SOST), and enhanced WNT/β-catenin signaling, osteoblast function, and bone formation. A single systemic administration of rAAVs effectively reversed bone loss in both postmenopausal and senile osteoporosis. Moreover, the healing of bone fracture and critical-sized bone defects was also markedly improved by systemic injection or transplantation of AAV-bound allograft bone to the osteotomy sites. Collectively, our data demonstrate the clinical potential of bone-specific gene silencers to treat skeletal disorders of low bone mass and impaired fracture repair., Competing Interests: Declaration of interests J.-H.S. is a scientific co-founder of AAVAA Therapeutics and holds equity in this company. G.G. is a scientific co-founder of AAVAA Therapeutics, Voyager Therapeutics, and Aspa Therapeutics and holds equity in these companies. G.G. is an inventor on patents with potential royalties licensed to Voyager Therapeutics, Aspa Therapeutics, and other biopharmaceutical companies. D.S.O. is a chief scientific officer of Osteogene Tech. These pose no conflicts for this study. The other authors declare no competing interests., (Copyright © 2022 The Author(s). Published by Elsevier Inc. All rights reserved.)

Skeletal stem cells (SSCs) are tissue-specific stem cells that can self-renew and sit at the apex of their differentiation hierarchy, giving rise to mature skeletal cell types required for bone growth, maintenance, and repair. Dysfunction in SSCs is caused by stress conditions like ageing and inflammation and is emerging as a contributor to skeletal pathology, such as the pathogenesis of fracture nonunion. Recent lineage tracing experiments have shown that SSCs exist in the bone marrow, periosteum, and resting zone of the growth plate. Unraveling their regulatory networks is crucial for understanding skeletal diseases and developing therapeutic strategies. In this review, we systematically introduce the definition, location, stem cell niches, regulatory signaling pathways, and clinical applications of SSCs., (© The Author(s) 2022. Published by Oxford University Press on behalf of Higher Education Press.)

Although skeletal progenitors provide a reservoir for bone-forming osteoblasts, the major energy source for their osteogenesis remains unclear. Here, we demonstrate a requirement for mitochondrial oxidative phosphorylation in the osteogenic commitment and differentiation of skeletal progenitors. Deletion of Evolutionarily Conserved Signaling Intermediate in Toll pathways (ECSIT) in skeletal progenitors hinders bone formation and regeneration, resulting in skeletal deformity, defects in the bone marrow niche and spontaneous fractures followed by persistent nonunion. Upon skeletal fracture, Ecsit-deficient skeletal progenitors migrate to adjacent skeletal muscle causing muscle atrophy. These phenotypes are intrinsic to ECSIT function in skeletal progenitors, as little skeletal abnormalities were observed in mice lacking Ecsit in committed osteoprogenitors or mature osteoblasts. Mechanistically, Ecsit deletion in skeletal progenitors impairs mitochondrial complex assembly and mitochondrial oxidative phosphorylation and elevates glycolysis. ECSIT-associated skeletal phenotypes were reversed by in vivo reconstitution with wild-type ECSIT expression, but not a mutant displaying defective mitochondrial localization. Collectively, these findings identify mitochondrial oxidative phosphorylation as the prominent energy-driving force for osteogenesis of skeletal progenitors, governing musculoskeletal integrity., (© 2022. The Author(s).)

Osteoclasts are bone-resorbing cells that undergo extensive changes in morphology throughout their differentiation. Altered osteoclast differentiation and activity lead to changes in pathological bone resorption. The mammalian target of rapamycin (mTOR) is a kinase, and aberrant mTOR complex 1 (mTORC1) signaling is associated with altered bone homeostasis. The activation of mTORC1 is biphasically regulated during osteoclastogenesis; however, the mechanism behind mTORC1-mediated regulation of osteoclastogenesis and bone resorption is incompletely understood. Here, we found that MYC coordinates the dynamic regulation of mTORC1 activation during osteoclastogenesis. MYC-deficiency blocked the early activation of mTORC1 and also reversed the decreased activity of mTORC1 at the late stage of osteoclastogenesis. The suppression of mTORC1 activity by rapamycin in mature osteoclasts enhances bone resorption activity despite the indispensable role of high mTORC1 activation in osteoclast formation in both mouse and human cells. Mechanistically, MYC induces Growth arrest and DNA damage-inducible protein (GADD34) expression and suppresses mTORC1 activity at the late phase of osteoclastogenesis. Taken together, our findings identify a MYC-GADD34 axis as an upstream regulator of dynamic mTORC1 activation in osteoclastogenesis and highlight the interplay between MYC and mTORC1 pathways in determining osteoclast activity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Bae, Oh, Tsai, Park, Greenblatt, Giannopoulou and Park-Min.)

Emerging evidence supports that osteogenic differentiation of skeletal progenitors is a key determinant of overall bone formation and bone mass. Despite extensive studies showing the function of mitogen-activated protein kinases (MAPKs) in osteoblast differentiation, none of these studies show in vivo evidence of a role for MAPKs in osteoblast maturation subsequent to lineage commitment. Here, we describe how the extracellular signal-regulated kinase (ERK) pathway in osteoblasts controls bone formation by suppressing the mechanistic target of rapamycin (mTOR) pathway. We also show that, while ERK inhibition blocks the differentiation of osteogenic precursors when initiated at an early stage, ERK inhibition surprisingly promotes the later stages of osteoblast differentiation. Accordingly, inhibition of the ERK pathway using a small compound inhibitor or conditional deletion of the MAP2Ks Map2k1 (MEK1) and Map2k2 (MEK2), in mature osteoblasts and osteocytes, markedly increased bone formation due to augmented osteoblast differentiation. Mice with inducible deletion of the ERK pathway in mature osteoblasts also displayed similar phenotypes, demonstrating that this phenotype reflects continuous postnatal inhibition of late-stage osteoblast maturation. Mechanistically, ERK inhibition increases mitochondrial function and SGK1 phosphorylation via mTOR2 activation, which leads to osteoblast differentiation and production of angiogenic and osteogenic factors to promote bone formation. This phenotype was partially reversed by inhibiting mTOR. Our study uncovers a surprising dichotomy of ERK pathway functions in osteoblasts, whereby ERK activation promotes the early differentiation of osteoblast precursors, but inhibits the subsequent differentiation of committed osteoblasts via mTOR-mediated regulation of mitochondrial function and SGK1., Competing Interests: JK, YY, JH, SC, HC, Mv, RX, MG No competing interests declared, JS is a scientific co-founder of the AAVAA Therapeutics and holds equity in this company. These pose no conflicts for this study, (© 2022, Kim et al.)

Osteocytes play a critical role in bone remodeling through the secretion of paracrine factors regulating the differentiation and activity of osteoblasts and osteoclasts. Sclerostin is a key osteocyte-derived factor that suppresses bone formation and promotes bone resorption, therefore regulators of sclerostin secretion are a likely source of new therapeutic strategies for treatment of skeletal disorders. Here, we demonstrate that protein kinase CK2 (casein kinase 2) controls sclerostin expression in osteocytes via the deubiquitinase ubiquitin-specific peptidase 4 (USP4)-mediated stabilization of Sirtuin1 (SIRT1). Deletion of CK2 regulatory subunit, Csnk2b, in osteocytes (Csnk2b Dmp1 ) results in low bone mass due to elevated levels of sclerostin. This phenotype in Csnk2b Dmp1 mice was partly reversed when sclerostin expression was downregulated by a single intravenous injection with bone-targeting adeno-associated virus 9 (AAV9) carrying an artificial-microRNA that targets Sost. Mechanistically, CK2-induced phosphorylation of USP4 is important for stabilization of SIRT1 by suppressing ubiquitin-dependent proteasomal degradation. Upregulated expression of SIRT1 inhibits sclerostin transcription in osteocytes. Collectively, the CK2-USP4-SIRT1 pathway is crucial for the regulation of sclerostin expression in osteocytes to maintain bone homeostasis., (© 2022. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

It is well-established that receptor activator of NF-κB ligand (RANKL) is the inducer of physiological osteoclast differentiation. However, the specific drivers and mechanisms driving inflammatory osteoclast differentiation under pathological conditions remain obscure. This is especially true given that inflammatory cytokines such as tumor necrosis factor (TNF) demonstrate little to no ability to directly drive osteoclast differentiation. Here, we found that transforming growth factor β (TGFβ) priming enables TNF to effectively induce osteoclastogenesis, independently of the canonical RANKL pathway. Lack of TGFβ signaling in macrophages suppresses inflammatory, but not basal, osteoclastogenesis and bone resorption in vivo. Mechanistically, TGFβ priming reprograms the macrophage response to TNF by remodeling chromatin accessibility and histone modifications, and enables TNF to induce a previously unrecognized non-canonical osteoclastogenic program, which includes suppression of the TNF-induced IRF1-IFNβ-IFN-stimulated-gene axis, IRF8 degradation and B-Myb induction. These mechanisms are active in rheumatoid arthritis, in which TGFβ level is elevated and correlates with osteoclast activity. Our findings identify a TGFβ/TNF-driven inflammatory osteoclastogenic program, and may lead to development of selective treatments for inflammatory osteolysis., (© 2022. The Author(s).)

Although patients with either β-thalassemia or chronic kidney disease (CKD) clinically correlate with severe osteoporosis, the mechanism by which CKD exposed to high phosphate affects bone turnover has not been characterized in β-thalassemia. We aimed to determine the effects of renal insufficiency on high phosphate intake induced changes in bone metabolism after 5/6th nephrectomy in hemizygous β-globin knockout (BKO) mice. Male BKO mice manifested severe anemia and osteopenia. Nephrectomy induced renal fibrosis and reduced renal function as assessed by increased serum urea nitrogen levels. Moreover, nephrectomy increased bone turnover leading to bone loss in wild type (WT) but not BKO mice. In nephrectomized BKO, PBS in drinking water induced hyperphosphatemia, and hypercalcemia along with osteopenia in both cancellous and cortical bone. Histomorphometric analysis confirmed reduced cancellous bone volume due to decreased bone formation rate, osteoblast number and osteoclast number. The mRNA levels for Alpl, Sp7, Kl, Tnfsf11, and Tnfsf11/Tnfrsf11b were decreased in nephrectomized BKO mice drinking PBS. Interestingly, Fgf23, a bone-derived hormone produced by osteocytes and osteoblasts in response to hyperphosphatemia, were remarkably increased in nephrectomized BKO mice following PBS intake. Serum FGF23 and erythropoietin levels were markedly elevated in BKO mice. Nephrectomy decreased serum erythropoietin but not FGF23 levels. Hyperphosphatemia in BKO mice increased serum erythropoietin, FGF23, and PTH levels, nominating these factors as candidate mediators of bone loss in thalassemic mice with CKD during phosphate retention., Competing Interests: The authors have declared that no competing interests exist.

Aims: To develop an early implant instability murine model and explore the use of intermittent parathyroid hormone (iPTH) treatment for initially unstable implants., Methods: 3D-printed titanium implants were inserted into an oversized drill-hole in the tibiae of C57Bl/6 mice (n = 54). After implantation, the mice were randomly divided into three treatment groups (phosphate buffered saline (PBS)-control, iPTH, and delayed iPTH). Radiological analysis, micro-CT (µCT), and biomechanical pull-out testing were performed to assess implant loosening, bone formation, and osseointegration. Peri-implant tissue formation and cellular composition were evaluated by histology., Results: iPTH reduced radiological signs of loosening and led to an increase in peri-implant bone formation over the course of four weeks (timepoints: one week, two weeks, and four weeks). Observational histological analysis shows that iPTH prohibits the progression of fibrosis. Delaying iPTH treatment until after onset of peri-implant fibrosis still resulted in enhanced osseointegration and implant stability. Despite initial instability, iPTH increased the mean pull-out strength of the implant from 8.41 N (SD 8.15) in the PBS-control group to 21.49 N (SD 10.45) and 23.68 N (SD 8.99) in the immediate and delayed iPTH groups, respectively. Immediate and delayed iPTH increased mean peri-implant bone volume fraction (BV/TV) to 0.46 (SD 0.07) and 0.34 (SD 0.10), respectively, compared to PBS-control mean BV/TV of 0.23 (SD 0.03) (PBS-control vs immediate iPTH, p < 0.001; PBS-control vs delayed iPTH, p = 0.048; immediate iPTH vs delayed iPTH, p = 0.111)., Conclusion: iPTH treatment mediated successful osseointegration and increased bone mechanical strength, despite initial implant instability. Clinically, this suggests that initially unstable implants may be osseointegrated with iPTH treatment. Cite this article: Bone Joint Res  2022;11(5):260-269.

Leptin receptor-positive skeletal progenitors constitute an essential cell population in the bone, yet their heterogeneity remains incompletely understood. In this issue, Mo et al (2021) report a single-cell RNA sequencing resource that deconvolutes the pool of LEPR + skeletal cells under homeostatic and various pathologic conditions, uncovering context-dependent contributions to diverse cell types and functions., (© 2022 The Authors.)

Extracellular signal-regulated kinases (ERKs) are evolutionarily ancient signal transducers of the mitogen-activated protein kinase (MAPK) family that have long been linked to the regulation of osteoblast differentiation and bone formation. Here, we review the physiological functions, biochemistry, upstream activators, and downstream substrates of the ERK pathway. ERK is activated in skeletal progenitors and regulates osteoblast differentiation and skeletal mineralization, with ERK serving as a key regulator of Runt-related transcription factor 2, a critical transcription factor for osteoblast differentiation. However, new evidence highlights context-dependent changes in ERK MAPK pathway wiring and function, indicating a broader set of physiological roles associated with changes in ERK pathway components or substrates. Consistent with this importance, several human skeletal dysplasias are associated with dysregulation of the ERK MAPK pathway, including neurofibromatosis type 1 and Noonan syndrome. The continually broadening array of drugs targeting the ERK pathway for the treatment of cancer and other disorders makes it increasingly important to understand how interference with this pathway impacts bone metabolism, highlighting the importance of mouse studies to model the role of the ERK MAPK pathway in bone formation.

Risk genes for Mendelian (single-gene) disorders (SGDs) are consistent across populations, but pathogenic risk variants that cause SGDs are typically population-private. The goal was to develop "QChip1," an inexpensive genotyping microarray to comprehensively screen newborns, couples, and patients for SGD risk variants in Qatar, a small nation on the Arabian Peninsula with a high degree of consanguinity. Over 10 8 variants in 8445 Qatari were identified for inclusion in a genotyping array containing 165,695 probes for 83,542 known and potentially pathogenic variants in 3438 SGDs. QChip1 had a concordance with whole-genome sequencing of 99.1%. Testing of QChip1 with 2707 Qatari genomes identified 32,674 risk variants, an average of 134 pathogenic alleles per Qatari genome. The most common pathogenic variants were those causing homocystinuria (1.12% risk allele frequency), and Stargardt disease (2.07%). The majority (85%) of Qatari SGD pathogenic variants were not present in Western populations such as European American, South Asian American, and African American in New York City and European and Afro-Caribbean in Puerto Rico; and only 50% were observed in a broad collection of data across the Greater Middle East including Kuwait, Iran, and United Arab Emirates. This study demonstrates the feasibility of developing accurate screening tools to identify SGD risk variants in understudied populations, and the need for ancestry-specific SGD screening tools., (© 2022. The Author(s).)

Currently, the cell of origin for osteosarcoma or other primary skeletal tumors is largely unknown. Recent reports identifying specific cell types comprising bone now newly enable investigation of this topic. Specifically, CXC motif chemokine 12 (CXCL12)-abundant reticular (CAR) cells are a specific skeletal stromal cell type that orchestrate the bone marrow microenvironment through cross-talk with hematopoietic and endothelial cells and a likely candidate cell of origin for at least a subset of primary skeletal tumors. Here, we analyze osteosarcomas via immunohistochemistry for known markers of CAR cells such as leptin receptor (LEPR), B-cell factor 3 (EBF3), CXCL12, and platelet-derived growth factor receptor alpha (PDGFRA). A large proportion of high-grade tumors expressed LEPR, PDGFRA, and EBF3 but not CXCL12. These data raise the hypothesis that CAR cells are the cell of origin of this osteoblastic osteosarcoma subset, a finding with implications for the cellular oncogenesis of primary osteosarcoma and the development of effective targeted therapies. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research., (© 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.)

Patients with systemic lupus erythematosus (SLE) have increased inflammatory cytokines, leading to periodontitis and alveolar bone loss. However, the mechanisms driving this phenomenon are still unknown. Here, we have identified novel therapeutic targets for and mediators of lupus-mediated bone loss using RNA-sequencing (RNA-seq) in a FcγRIIB -/- mouse model of lupus associated osteopenia. A total of 2,710 upregulated and 3,252 downregulated DEGs were identified. The GO and KEGG annotations revealed that osteoclast differentiation, bone mineralization, ossification, and myeloid cell development were downregulated. WikiPathways indicated that Hedgehog, TNFα NF-κB and Notch signaling pathway were also decreased. We identified downregulated targets, Sufu and Serpina12, that have important roles in bone homeostasis. Sufu and Serpina12 were related to Hedgehog signaling proteins, including Gli1, Gli2, Gli3, Ptch1, and Ptch2. Gene knockdown analysis demonstrated that Sufu, and Serpina12 contributed to osteoclastogenesis and osteoblastogenesis, respectively. Osteoclast and osteoblast marker genes were significantly decreased in Sufu-deficient and Serpina12-deficient cells, respectively. Our results suggest that alterations in Hedgehog signaling play an important role in the pathogenesis of osteopenia in FcγRIIB -/- mice. The novel DEGs and pathways identified in this study provide new insight into the underlying mechanisms of mandibular bone loss during lupus development., (© 2021. The Author(s).)

The number of total joint replacements (TJRs) in the United States is increasing annually. Cementless implants are intended to improve upon traditional cemented implants by allowing bone growth directly on the surface to improve implant longevity. One major complication of TJR is implant loosening, which is related to deficient osseointegration in cementless TJRs. Although poor osseointegration in aged patients is typically attributed to decreased basal bone mass, little is known about the molecular pathways that compromise the growth of bone onto porous titanium implants. To identify the pathways important for osseointegration that are compromised by aging, we developed an approach for transcriptomic profiling of peri-implant tissue in young and aged mice using our murine model of osseointegration. Based on previous findings of changes of bone quality associated with aging, we hypothesized that aged mice have impaired activation of bone anabolic pathways at the bone-implant interface. We found that pathways most significantly downregulated in aged mice relative to young mice are related to angiogenic, Notch, and Wnt signaling. Downregulation of these pathways is associated with markedly increased expression of inflammatory and immune genes at the bone-implant interface in aged mice. These results identify osseointegration pathways affected by aging and suggest that an increased inflammatory response in aged mice may compromise peri-implant bone healing. Targeting the Notch and Wnt pathways, promoting angiogenesis, or modulating the immune response at the peri-implant site may enhance osseointegration and improve the outcome of joint replacement in older patients. © 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research., (© 2021 The Authors. JBMR Plus published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research.)

Hedgehog signaling is essential for bone formation, including functioning as a means for the growth plate to drive skeletal mineralization. However, the mechanisms regulating hedgehog signaling specifically in bone-forming osteoblasts are largely unknown. Here, we identified SLIT and NTRK-like protein-5(Slitrk5), a transmembrane protein with few identified functions, as a negative regulator of hedgehog signaling in osteoblasts. Slitrk5 is selectively expressed in osteoblasts and loss of Slitrk5 enhanced osteoblast differentiation in vitro and in vivo. Loss of SLITRK5 in vitro leads to increased hedgehog signaling and overexpression of SLITRK5 in osteoblasts inhibits the induction of targets downstream of hedgehog signaling. Mechanistically, SLITRK5 binds to hedgehog ligands via its extracellular domain and interacts with PTCH1 via its intracellular domain. SLITRK5 is present in the primary cilium, and loss of SLITRK5 enhances SMO ciliary enrichment upon SHH stimulation. Thus, SLITRK5 is a negative regulator of hedgehog signaling in osteoblasts that may be attractive as a therapeutic target to enhance bone formation., (© 2021. The Author(s).)

Patients with systemic lupus erythematosus are at increased risk for alveolar bone loss due to periodontitis possibly as a result of a pathogenic immune response to oral bacteria and inflammation. The aim of the present study was to investigate whether an anti-TNF-α antagonist could prevent mandibular bone loss in the FcγRIIb-/- mouse model of lupus. Mice lacking FcγRIIb had decreased cancellous and cortical bone volume at 6 months of age. Etanercept increased cancellous but not cortical bone volume in WT and increased both cancellous bone volume and cortical thickness in FcγRIIb-deficient mice. FcγRIIb deficiency decreased mRNA levels for osteoblast marker genes, Osx, Col1a1 and Alp without any change in osteoclast marker genes. Etanercept increased Osx, Alp, and Ocn in both WT and FcγRIIb-/- mice. Osteoclast marker genes including TNF-α, Trap and RANKL/OPG ratio was decreased in WT. Serum markers of proinflammatory cytokines, TNF-α, IFNγ, IL-6, and IL-17A, were increased in FcγRIIb-/- mice and etanercept antagonized these effects in FcγRIIb-/- mice. Etanercept increased serum PTH levels in the FcγRIIb-/- mouse model of lupus. Our results suggest that deletion of FcγRIIb induces osteopenia by increasing the level of proinflammatory cytokines. Etanercept is effective in preventing mandibular bone loss in FcγRIIb-/- mice, suggesting that anti-TNF-α therapy may be able to ameliorate mandibular bone loss in SLE patients with periodontitis., Competing Interests: The authors have declare that no competing interests exist.

Neurofibromatosis type I (NF1) is characterized by prominent skeletal manifestations caused by NF1 loss. While inhibitors of the ERK activating kinases MEK1/2 are promising as a means to treat NF1, the broad blockade of the ERK pathway produced by this strategy is potentially associated with therapy limiting toxicities. Here, we have sought targets offering a more narrow inhibition of ERK activation downstream of NF1 loss in the skeleton, finding that MEKK2 is a novel component of a noncanonical ERK pathway in osteoblasts that mediates aberrant ERK activation after NF1 loss. Accordingly, despite mice with conditional deletion of Nf1 in mature osteoblasts (Nf1 fl/fl ;Dmp1-Cre) and Mekk2 -/- each displaying skeletal defects, Nf1 fl/fl ;Mekk2 -/- ;Dmp1-Cre mice show an amelioration of NF1-associated phenotypes. We also provide proof-of-principle that FDA-approved inhibitors with activity against MEKK2 can ameliorate NF1 skeletal pathology. Thus, MEKK2 functions as a MAP3K in the ERK pathway in osteoblasts, offering a potential new therapeutic strategy for the treatment of NF1.

Bone fracture is repaired predominantly through endochondral ossification. However, the regulation of endochondral ossification by key factors during fracture healing remains largely enigmatic. Here, we identify histone modification enzyme LSD1 as a critical factor regulating endochondral ossification during bone regeneration. Loss of LSD1 in Prx1 lineage cells severely impaired bone fracture healing. Mechanistically, LSD1 tightly controls retinoic acid signaling through regulation of Aldh1a2 expression level. The increased retinoic acid signaling in LSD1-deficient mice suppressed SOX9 expression and impeded the cartilaginous callus formation during fracture repair. The discovery that LSD1 can regulate endochondral ossification during fracture healing will benefit the understanding of bone regeneration and have implications for regenerative medicine., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Bone remodeling is tightly regulated by a cross-talk between bone-forming osteoblasts and bone-resorbing osteoclasts. Osteoblasts and osteoclasts communicate with each other to regulate cellular behavior, survival and differentiation through direct cell-to-cell contact or through secretory proteins. A direct interaction between osteoblasts and osteoclasts allows bidirectional transduction of activation signals through EFNB2-EPHB4, FASL-FAS or SEMA3A-NRP1, regulating differentiation and survival of osteoblasts or osteoclasts. Alternatively, osteoblasts produce a range of different secretory molecules, including M-CSF, RANKL/OPG, WNT5A, and WNT16, that promote or suppress osteoclast differentiation and development. Osteoclasts also influence osteoblast formation and differentiation through secretion of soluble factors, including S1P, SEMA4D, CTHRC1 and C3. Here we review the current knowledge regarding membrane bound- and soluble factors governing cross-talk between osteoblasts and osteoclasts.

The osteoblast differentiation capacity of skeletal stem cells (SSCs) must be tightly regulated, as inadequate bone formation results in low bone mass and skeletal fragility, and over-exuberant osteogenesis results in heterotopic ossification (HO) of soft tissues. RUNX2 is essential for tuning this balance, but the mechanisms of posttranslational control of RUNX2 remain to be fully elucidated. Here, we identify that a CK2/HAUSP pathway is a key regulator of RUNX2 stability, as Casein kinase 2 (CK2) phosphorylates RUNX2, recruiting the deubiquitinase herpesvirus-associated ubiquitin-specific protease (HAUSP), which stabilizes RUNX2 by diverting it away from ubiquitin-dependent proteasomal degradation. This pathway is important for both the commitment of SSCs to osteoprogenitors and their subsequent maturation. This CK2/HAUSP/RUNX2 pathway is also necessary for HO, as its inhibition blocked HO in multiple models. Collectively, active deubiquitination of RUNX2 is required for bone formation and this CK2/HAUSP deubiquitination pathway offers therapeutic opportunities for disorders of inappropriate mineralization.

The axon guidance cue SLIT3 was identified as an osteoanabolic agent in two recent reports. However, these reports conflict in their nomination of osteoblasts versus osteoclasts as the key producers of skeletal SLIT3 and additionally offer conflicting data on the effects of SLIT3 on osteoclastogenesis. Here, aiming to address this discrepancy, we found no observable SLIT3 expression during human or mouse osteoclastogenesis and the only modest SLIT3-mediated effects on osteoclast differentiation. Conditional deletion of SLIT3 in cathepsin K (CTSK)-positive cells, including osteoclasts, had no effect on the number of osteoclast progenitors, in vitro osteoclast differentiation, overall bone mass, or bone resorption/formation parameters. Similar results were observed with the deletion of SLIT3 in LysM-positive cells, including osteoclast lineage cells. Consistent with this finding, bone marrow chimeras made from Slit3 -/- donors that lacked SLIT3 expression at all stages of osteoclast development displayed normal bone mass relative to controls. Taken in context, multiple lines of evidence were unable to identify the physiologic function of osteoclast-derived SLIT3, indicating that osteoblasts are the major source of skeletal SLIT3., Competing Interests: Competing interestsThe authors declare no competing interests., (© The Author(s) 2020.)

Inflammatory signals arising from the microenvironment have emerged as critical regulators of hematopoietic stem cell (HSC) function during diverse processes including embryonic development, infectious diseases, and myelosuppressive injuries caused by irradiation and chemotherapy. However, the contributions of cellular subsets within the microenvironment that elicit niche-driven inflammation remain poorly understood. Here, we identify endothelial cells as a crucial component in driving bone marrow (BM) inflammation and HSC dysfunction observed following myelosuppression. We demonstrate that sustained activation of endothelial MAPK causes NF-κB-dependent inflammatory stress response within the BM, leading to significant HSC dysfunction including loss of engraftment ability and a myeloid-biased output. These phenotypes are resolved upon inhibition of endothelial NF-κB signaling. We identify SCGF as a niche-derived factor that suppresses BM inflammation and enhances hematopoietic recovery following myelosuppression. Our findings demonstrate that chronic endothelial inflammation adversely impacts niche activity and HSC function which is reversible upon suppression of inflammation.

Aldehyde dehydrogenase type 2 (ALDH2), a key enzyme in ethanol metabolism, processes toxic acetaldehyde to nontoxic acetate. ALDH2 deficiency affects 8% of the world population and 35-45% of East Asians. The ALDH2*2 allele common genetic variant has a glutamic acid-to-lysine substitution at position 487 (E487K) that reduces the oxidizing ability of the enzyme resulting in systemic accumulation of acetaldehyde with ethanol ingestion. With chronic ethanol ingestion, mutations in ALDH2 are associated with a variety of hematological, neurological, and dermatological abnormalities, and an increased risk for esophageal cancer and osteoporosis. Based on our prior studies demonstrating that a one-time administration of an adeno-associated virus (AAV) serotype rh.10 gene transfer vector expressing the human ALDH2 cDNA (AAVrh.10hALDH2) prevents the acute effects of ethanol administration (the "Asian flush syndrome"), we hypothesized that AAVrh.10hALDH2 would also prevent the chronic disorders associated with ALDH2 deficiency and chronic ethanol ingestion. To assess this hypothesis, AAVrh.10hALDH2 (10 11 genome copies) was administered intravenously to two models of ALDH2 deficiency, Aldh2 knockout homozygous ( Aldh2 -/- ) and knockin homozygous ( Aldh2 E487K+/+ ) mice ( n  = 10 per group). Four weeks after vector administration, mice were given drinking water with 10-15% ethanol for 12 weeks. Strikingly, compared with nonethanol drinking littermates, AAVrh.10hALDH2 administration prevented chronic ethanol-induced serum acetaldehyde accumulation and elevated liver malondialdehyde levels, loss of body weight, reduced hemoglobin levels, reduced performance in locomotor activity tests, accumulation of esophageal DNA damage and DNA adducts, and development of osteopenia. AAVrh.10hALDH2 should be considered as a preventative therapy for the increased risk of chronic disorders associated with ALDH2 deficiency and chronic alcohol exposure.

Bone is composed of a complex mixture of many dynamic cell types. Flow cytometry and in vivo lineage tracing have offered early progress toward deconvoluting this heterogeneous mixture of cells into functionally well-defined populations suitable for further studies. Single-cell sequencing is poised as a key complementary technique to better understand the cellular basis of bone metabolism and development. However, single-cell sequencing approaches still have important limitations, including transcriptional effects of cell isolation and sparse sampling of the transcriptome, that must be considered during experimental design and analysis to harness the power of this approach. Accounting for these limitations requires a deep knowledge of the tissue under study. Therefore, with the emergence of accessible tools for conducting and analyzing single-cell RNA sequencing (scRNA-seq) experiments, bone biologists will be ideal leaders in the application of scRNA-seq to the skeleton. Here we provide an overview of the steps involved with a single-cell sequencing analysis of bone, focusing on practical considerations needed for a successful study. © 2019 American Society for Bone and Mineral Research., (© 2019 American Society for Bone and Mineral Research.)