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Introduction: Osteoarthritis (OA) is a devastating whole-joint disease affecting a large population worldwide with no cure; its mechanism remains poorly defined. Abnormal mechanical stress is the main pathological factor of OA. Objectives: To investigate the effects of Piezo1 activation on OA development and progression and to explore Piezo1-targeting OA treatment. Methods: The expression levels of Piezo1 were determined in human OA cartilage and experimental OA mice. Mice with genetic Piezo1 deletion in chondrocytes or intra-articular injection of the Piezo1 activator Yoda1 were utilized to determine the effects on DMM-induced OA progression. Effects of artemisinin (ART), a potent antimalarial drug, on Piezo1 activation, chondrocyte metabolism and OA lesions were determined. Results: Piezo1 expression was elevated in articular chondrocytes in human OA and DMM-induced mouse OA cartilage. Piezo1 deletion in chondrocytes largely attenuates DMM-induced OA-like phenotypes. In contrast, intra-articular injection of Yoda1 aggravates the knee joint OA lesions in mice. PIEZO1 activation increases, while PIEZO1 siRNA knockdown decreases, expression of RUNX2 and catabolic enzymes MMP13 and ADAMTS5 in primary human articular chondrocytes in a PI3K-AKT dependent manner. We have provided strong evidence supporting that ART is a novel and potent inhibitor of Piezo1 activation in primary OA-HACs and all cell lines examined, including human endothelial HUVEC cells, ATDC5 chondrocyte-like cells and MLO-Y4 osteocytes-like cells. Results from in vitro experiments confirmed that ART decreases the Yoda1-induced increases in the levels of OA-related genes and p-PI3K and p-AKT proteins in OA-HACs and alleviates DMM-induced OA lesions in mice. Conclusions: We establish a critical role of Piezo1 in promoting OA development and progression and define ART as a potential OA treatment.

Cyclin D1 has been recognized as an oncogene due to its abnormal upregulation in different types of cancers. Here, we demonstrated that cyclin D1 is SUMOylated, and we identified Itch as a specific E3 ligase recognizing SUMOylated cyclin D1 and mediating SUMO-induced ubiquitination and proteasome degradation of cyclin D1. We generated cyclin D1 mutant mice with mutations in the SUMOylation site, phosphorylation site, or both sites of cyclin D1, and found that double mutant mice developed a Mantle cell lymphoma (MCL)-like phenotype. We showed that arsenic trioxide (ATO) enhances cyclin D1 SUMOylation-mediated degradation through inhibition of cyclin D1 deSUMOylation enzymes, leading to MCL cell apoptosis. Treatment of severe combined immunodeficiency (SCID) mice grafted with MCL cells with ATO resulted in a significant reduction in tumor growth. In this study, we provide novel insights into the mechanisms of MCL tumor development and cyclin D1 regulation and discover a new strategy for MCL treatment.

Abstract Rheumatoid arthritis (RA) is an autoimmune disease. Early studies hold an opinion that gut microbiota is environmentally acquired and associated with RA susceptibility. However, accumulating evidence demonstrates that genetics also shape the gut microbiota. It is known that some strains of inbred laboratory mice are highly susceptible to collagen-induced arthritis (CIA), while the others are resistant to CIA. Here, we show that transplantation of fecal microbiota of CIA-resistant C57BL/6J mice to CIA-susceptible DBA/1J mice confer CIA resistance in DBA/1J mice. C57BL/6J mice and healthy human individuals have enriched B. fragilis than DBA/1J mice and RA patients. Transplantation of B. fragilis prevents CIA in DBA/1J mice. We identify that B. fragilis mainly produces propionate and C57BL/6J mice and healthy human individuals have higher level of propionate. Fibroblast-like synoviocytes (FLSs) in RA are activated to undergo tumor-like transformation. Propionate disrupts HDAC3-FOXK1 interaction to increase acetylation of FOXK1, resulting in reduced FOXK1 stability, blocked interferon signaling and deactivation of RA-FLSs. We treat CIA mice with propionate and show that propionate attenuates CIA. Moreover, a combination of propionate with anti-TNF etanercept synergistically relieves CIA. These results suggest that B. fragilis or propionate could be an alternative or complementary approach to the current therapies.

Abstract Protein-encoding genes only constitute less than 2% of total human genomic sequences, and 98% of genetic information was previously referred to as “junk DNA”. Meanwhile, non-coding RNAs (ncRNAs) consist of approximately 60% of the transcriptional output of human cells. Thousands of ncRNAs have been identified in recent decades, and their essential roles in the regulation of gene expression in diverse cellular pathways associated with fundamental cell processes, including proliferation, differentiation, apoptosis, and metabolism, have been extensively investigated. Furthermore, the gene regulation networks they form modulate gene expression in normal development and under pathological conditions. In this review, we integrate current information about the classification, biogenesis, and function of ncRNAs and how these ncRNAs support skeletal development through their regulation of critical genes and signaling pathways in vivo. We also summarize the updated knowledge of ncRNAs involved in common skeletal diseases and disorders, including but not limited to osteoporosis, osteoarthritis, rheumatoid arthritis, scoliosis, and intervertebral disc degeneration, by highlighting their roles established from in vivo, in vitro, and ex vivo studies.

Research background: The role of osteocytes in maintaining bone mass has been progressively emphasized. Pip5k1c is the most critical isoform among PIP5KIs, which can regulate cytoskeleton, biomembrane, and Ca2+ release of cells and participate in many processes, such as cell adhesion, differentiation, and apoptosis. However, its expression and function in osteocytes are still unclear. Materials and methods: To determine the function of Pip5k1c in osteocytes, the expression of Pip5k1c in osteocytes was deleted by breeding the 10-kb mouse Dmp1-Cre transgenic mice with the Pip5k1cfl/fl mice. Bone histomorphometry, micro-computerized tomography analysis, immunofluorescence staining and western blotting were used to determine the effects of Pip5k1c loss on bone mass. In vitro, we explored the mechanism by siRNA knockdown of Pip5k1c in MLO-Y4 cells. Results: Pip5k1c expression was decreased in osteocytes in senescent and osteoporotic tissues both in humans and mice. Loss of Pip5k1c in osteocytes led to a low bone mass in long bones and spines and impaired biomechanical properties in femur, without changes in calvariae. The loss of Pip5k1c resulted in the reduction of the protein level of type 1 collagen in tibiae and MLO-Y4 cells. Osteocyte Pip5k1c loss reduced the osteoblast and bone formation rate with high expression of sclerostin, impacting the osteoclast activities at the same time. Moreover, Pip5k1c loss in osteocytes reduced expression of focal adhesion proteins and promoted apoptosis. Conclusion: Our studies demonstrate the critical role and mechanism of Pip5k1c in osteocytes in regulating bone remodeling. The translational potential of this article: Osteocyte has been considered to a key role in regulating bone homeostasis. The present study has demonstrated that the significance of Pip5k1c in bone homeostasis by regulating the expression of collagen, sclerostin and focal adhesion expression, which provided a possible therapeutic target against human metabolic bone disease.

Abstract Given afferent functions, sensory nerves have recently been found to exert efferent effects and directly alter organ physiology. Additionally, several studies have highlighted the indirect but crucial role of sensory nerves in the regulation of the physiological function of osteoclasts. Nonetheless, evidence regarding the direct sensory nerve efferent influence on osteoclasts is lacking. In the current study, we found that high levels of efferent signals were transported directly from the sensory nerves into osteoclasts. Furthermore, sensory hypersensitivity significantly increased osteoclastic bone resorption, and sensory neurons (SNs) directly promoted osteoclastogenesis in an in vitro coculture system. Moreover, we screened a novel neuropeptide, Cyp40, using an isobaric tag for relative and absolute quantitation (iTRAQ). We observed that Cyp40 is the efferent signal from sensory nerves, and it plays a critical role in osteoclastogenesis via the aryl hydrocarbon receptor (AhR)-Ras/Raf-p-Erk-NFATc1 pathway. These findings revealed a novel mechanism regarding the influence of sensory nerves on bone regulation, i.e., a direct promoting effect on osteoclastogenesis by the secretion of Cyp40. Therefore, inhibiting Cyp40 could serve as a strategy to improve bone quality in osteoporosis and promote bone repair after bone injury.

Abstract Objective While joint immobilization is a useful repair method for intra-articular ligament injury and periarticular fracture, prolonged joint immobilization can cause multiple complications. A better understanding how joint immobilization and remobilization impact joint function and homeostasis will help clinicians develop novel strategies to reduce complications. Design We first determined the effects of long-term immobilization on joint pain and osteophyte formation in patients after an extraarticular fracture or ligament injury. We then developed a mouse model of joint immobilization and harvested the knee joint samples at 2, 4, and 8 weeks. We further determined the effects of remobilization on recovery of the osteoarthritis (OA) lesions induced by immobilization in mice. Results We found that the long-term (6 weeks) joint immobilization caused significant joint pain and osteophytes in patients. In mice, 2-week immobilization already induced moderate sensory innervation and increased pain sensitivity and infiltration in synovium without inducing marked osteophyte formation and cartilage loss. Long-term immobilization (4 and 8 weeks) induced more severe sensory innervation and inflammatory infiltration in synovium, massive osteophyte formation on both sides of the femoral condyle, and the edge of the tibial plateau and significant loss of the articular cartilage in mice. Remobilization, which ameliorates normal joint load and activity, restored to certain extent some of the OA lesions and joint function in mice. Conclusions Joint immobilization caused multiple OA-like lesions in both mice and humans. Joint immobilization induced progressive sensory innervation, synovitis, osteophyte formation, and cartilage loss in mice, which can be partially ameliorated by remobilization.

Osteoarthritis (OA) is a painful degenerative joint disease and is the leading cause of chronic disability among elderly individuals. To improve the quality of life for patients with OA, the primary goal for OA treatment is to relieve the pain. During OA progression, nerve ingrowth was observed in synovial tissue and articular cartilage. These abnormal neonatal nerves act as nociceptors to detect OA pain signals. The molecular mechanisms for transmitting OA pain in the joint tissues to the central nerve system (CNS) is currently unknown. MicroRNA miR-204 has been demonstrated to maintain the homeostasis of joint tissues and have chondro-protective effect on OA pathogenesis. However, the role of miR-204 in OA pain has not been determined. In this study, we investigated interactions between chondrocytes and neural cells and evaluated the effect and mechanism of miR-204 delivered by exosome in the treatment of OA pain in an experimental OA mouse model. Our findings demonstrated that miR-204 could protect OA pain by inhibition of SP1- LDL Receptor Related Protein 1 (LRP1) signaling and blocking neuro-cartilage interaction in the joint. Our studies defined novel molecular targets for the treatment of OA pain.

Background: Genetically modified mice are the most useful tools for investigating the gene functions in articular cartilage biology and the pathogenesis of osteoarthritis. The AggrecanCreERT2 mice are one of the most reported mouse lines used for this purpose. The Prg4 (proteoglycan 4) gene encodes the lubricin protein and is expressed selectively in chondrocytes located at the superficial layer of the articular cartilage. While the Prg4GFPCreERT2 knock-in inducible-Cre transgenic mice were generated a while ago, so far, few studies have used this mouse line to perform gene functional studies in cartilage biology. Methods: We have recently reported that deleting the Fermt2 gene, which encodes the key focal adhesion protein Kindlin-2, in articular chondrocytes by using the AggrecanCreERT2 transgenic mice, results in spontaneous osteoarthritis (OA) lesions, which highly mimics the human OA pathologies. In this study, we have compared the Kindlin-2 deficiency-caused OA phenotypes induced by Prg4GFPCreERT2 with those caused by AggrecanCreERT2 using imaging and histological analyses. Results: We find that Kindlin-2 protein is deleted in about 75% of the superficial articular chondrocytes in the tamoxifen (TAM)-treated Prg4GFPCreERt2/+; Fermt2fl/fl mice compared to controls. At 6 months after TAM injections, the OARSI scores of AggrecanCreERT2/+; Fermt2fl/fl and Prg4GFPCreERt2/+; Fermt2fl/fl mice were 5 and 3, respectively. The knee joints histological osteophyte and synovitis scores were also significantly decreased in Prg4GFPCreERT2/+; Fermt2fl/fl mice compared to those in AggrecanCreERT2/+; Fermt2fl/fl mice. Furthermore, magnitudes of upregulation of the extracellular matrix-degrading enzymes Mmp13 and hypertrophic chondrocyte markers Col10a1 and Runx2 were decreased in Prg4GFPCreERT2/+; Fermt2fl/fl versus AggrecanCreERT2/+; Fermt2fl/fl mice. We finally examined the susceptibility of Prg4GFPCreERT2/+; Fermt2fl/fl mouse model to surgically induce OA lesions. The pathological features of OA in the TAM-DMM model exhibited significant enhancement in cartilage erosion, proteoglycan loss, osteophyte, and synovitis and an increase in OARSI score in articular cartilage compared with those in corn-oil DMM mice. Conclusion: Kindlin-2 loss causes milder OA-like lesions in Prg4GFPCreERT2/+; Fermt2fl/fl than in AggrecanCreERT2/+; Fermt2fl/fl mice. In contrast, Kindlin-2 loss similarly accelerates the destabilization of the medial meniscus-induced OA lesions in both mice.Translational Potential of this Article: Our study demonstrates that Prg4GFPCreERT2 is a useful tool for gene functional study in OA research. This study provides useful information for investigators to choose appropriate Cre mouse lines for their research in cartilage biology.

Background: Cell culture studies demonstrate the importance of β3 integrin in osteocyte mechanotransduction. However, the in vivo roles of osteocyte β3 integrin in the regulation of bone homeostasis and mechanotransduction are poorly defined. Materials and methods: To study the in vivo role of osteocyte β3 integrin in bone, we utilized the 10-kb Dmp1 (dentin matrix acidic phosphoprotein 1)-Cre to delete β3 integrin expression in osteocyte in mice. Micro-computerized tomography (μCT), bone histomorphometry and in vitro cell culture experiments were performed to determine the effects of osteocyte β3 integrin loss on bone mass accrual and biomechanical properties. In addition, in vivo tibial loading model was applied to study the possible involvement of osteocyte β3 integrin in the mediation of bone mechanotransduction. Results: Deletion of β3 integrin in osteocytes resulted in a low bone mass and impaired biomechanical properties in load-bearing long bones in adult mice. The loss of β3 integrin led to abnormal cell morphology with reduced number and length of dentritic processes in osteocytes. Furthermore, osteocyte β3 integrin loss did not impact the osteoclast formation, but significantly reduced the osteoblast-mediated bone formation rate and reduced the osteogenic differentiation of the bone marrow stromal cells in the bone microenvironment. In addition, mechanical loading failed to accelerate the anabolic bone formation in mutant mice. Conclusions: Our studies demonstrate the essential roles of osteocyte β3 integrin in regulating bone mass and mechanotransduction.

Cyclin D1 is a key regulator of cell cycle progression, which forms a complex with CDK4/6 to regulate G1/S transition during cell cycle progression. Cyclin D1 has been recognized as an oncogene since it was upregulated in several different types of cancers. It is known that the post-translational regulation of cyclin D1 is controlled by ubiquitination/proteasome degradation system in a phosphorylation-dependent manner. Several cullin-associated F-box E3 ligases have been shown to regulate cyclin D1 degradation; however, it is not known if additional cullin-associated E3 ligases participate in the regulation of cyclin D1 protein stability. In this study, we have screened an siRNA library containing siRNAs specific for 154 ligase subunits, including F-box, SOCS, BTB-containing proteins, and DDB proteins. We found that multiple cullin-associated E3 ligases regulate cyclin D1 activity, including Keap1, DDB2, and WSB2. We found that these E3 ligases interact with cyclin D1, regulate cyclin D1 ubiquitination and proteasome degradation in a phosphorylation-dependent manner. These E3 ligases also control cell cycle progression and cell proliferation through regulation of cyclin D1 protein stability. Our study provides novel insights into the regulatory mechanisms of cyclin D1 protein stability and function.

Abstract Spine degeneration is an aging-related disease, but its molecular mechanisms remain unknown, although elevated β-catenin signaling has been reported to be involved in intervertebral disc degeneration. Here, we determined the role of β-catenin signaling in spinal degeneration and in the homeostasis of the functional spinal unit (FSU), which includes the intervertebral disc, vertebra and facet joint and is the smallest physiological motion unit of the spine. We showed that pain sensitivity in patients with spinal degeneration is highly correlated with β-catenin protein levels. We then generated a mouse model of spinal degeneration by transgenic expression of constitutively active β-catenin in Col2 + cells. We found that β-catenin-TCF7 activated the transcription of CCL2, a known critical factor in osteoarthritic pain. Using a lumbar spine instability model, we showed that a β-catenin inhibitor relieved low back pain. Our study indicates that β-catenin plays a critical role in maintaining spine tissue homeostasis, its abnormal upregulation leads to severe spinal degeneration, and its targeting could be an avenue to treat this condition.

The skeletal system, which contains bones, joints, tendons, ligaments and other elements, plays a wide variety of roles in body shaping, support and movement, protection of internal organs, production of blood cells and regulation of calcium and phosphate metabolism. The prevalence of skeletal diseases and disorders, such as osteoporosis and bone fracture, osteoarthritis, rheumatoid arthritis, and intervertebral disc degeneration, increases with age, causing pain and loss of mobility and creating a huge social and economic burden globally. Focal adhesions (FAs) are macromolecular assemblies that are composed of the extracellular matrix (ECM), integrins, intracellular cytoskeleton and other proteins, including kindlin, talin, vinculin, paxillin, pinch, Src, focal adhesion kinase (FAK) and integrin-linked protein kinase (ILK) and other proteins. FA acts as a mechanical linkage connecting the ECM and cytoskeleton and plays a key role in mediating cell–environment communications and modulates important processes, such as cell attachment, spreading, migration, differentiation and mechanotransduction, in different cells in skeletal system by impacting distinct outside-in and inside-out signaling pathways. This review aims to integrate the up-to-date knowledge of the roles of FA proteins in the health and disease of skeletal system and focuses on the specific molecular mechanisms and underlying therapeutic targets for skeletal diseases.

Abstract Osteoarthritis (OA) is a chronic degenerative joint disorder that leads to disability and affects more than 500 million population worldwide. OA was believed to be caused by the wearing and tearing of articular cartilage, but it is now more commonly referred to as a chronic whole-joint disorder that is initiated with biochemical and cellular alterations in the synovial joint tissues, which leads to the histological and structural changes of the joint and ends up with the whole tissue dysfunction. Currently, there is no cure for OA, partly due to a lack of comprehensive understanding of the pathological mechanism of the initiation and progression of the disease. Therefore, a better understanding of pathological signaling pathways and key molecules involved in OA pathogenesis is crucial for therapeutic target design and drug development. In this review, we first summarize the epidemiology of OA, including its prevalence, incidence and burdens, and OA risk factors. We then focus on the roles and regulation of the pathological signaling pathways, such as Wnt/β-catenin, NF-κB, focal adhesion, HIFs, TGFβ/ΒΜP and FGF signaling pathways, and key regulators AMPK, mTOR, and RUNX2 in the onset and development of OA. In addition, the roles of factors associated with OA, including MMPs, ADAMTS/ADAMs, and PRG4, are discussed in detail. Finally, we provide updates on the current clinical therapies and clinical trials of biological treatments and drugs for OA. Research advances in basic knowledge of articular cartilage biology and OA pathogenesis will have a significant impact and translational value in developing OA therapeutic strategies.

Background: Osteoarthritis (OA) is a common degenerative joint disease with significant negative impact on the quality of life. It has been reported that abnormal upregulation of β-catenin signaling could lead to OA development; however, the upstream regulatory mechanisms of β-catenin signaling have not been determined. Methods: Primary rat chondrocytes and ATDC5 chondrocyte cell line were stimulated with AKT2 and treated with or without metformin, an adenosine 5′-monophosphate-activated protein kinase (AMPK) activator. Westerrn blot analysis, luciferase reporter assay and immunofluorescent (IF) staining were performed to examine changes in β-cateninS552 phosphorylation and β-catenin nuclear translocation in ATDC5 cells and in primary chondrocytes. Results: We found that metformin inhibited β-cateninS552 phosphorylation in ATDC5 cells and in primary chondrocytes in a time-dependent manner. Metformin inhibited β-catenin nuclear translocation and β-catenin reporter activity. In addition, metformin also attenuated the expression of β-catenin downstream target genes. We also demonstrated that metformin inhibited β-cateninS552 phosphorylation in articular cartilage in mice. Conclusion: These findings suggest that metformin may exert its chondro-protective effect at least in part through the inhibition of β-catenin signaling in chondrocytes. The translational potential of this article: This study demonstrated the interaction between AMPK and β-catenin signaling in chondrocytes and defined novel molecular targets for the treatment of OA disease.

Abstract Osteoarthritis (OA) is the most common degenerative joint disease that causes painful swelling and permanent damage to the joints in the body. The molecular mechanisms of OA are currently unknown. OA is a heterogeneous disease that affects the entire joint, and multiple tissues are altered during OA development. To better understand the pathological mechanisms of OA, new approaches, methods, and techniques need to be used to understand OA pathogenesis. In this review, we first focus on the epigenetic regulation of OA, with a particular focus on DNA methylation, histone modification, and microRNA regulation, followed by a summary of several key mediators in OA-associated pain. We then introduce several innovative techniques that have been and will continue to be used in the fields of OA and OA-associated pain, such as CRISPR, scRNA sequencing, and lineage tracing. Next, we discuss the timely updates concerning cell death regulation in OA pathology, including pyroptosis, ferroptosis, and autophagy, as well as their individual roles in OA and potential molecular targets in treating OA. Finally, our review highlights new directions on the role of the synovial lymphatic system in OA. An improved understanding of OA pathogenesis will aid in the development of more specific and effective therapeutic interventions for OA.

Abstract The progressive destruction of condylar cartilage is a hallmark of the temporomandibular joint (TMJ) osteoarthritis (OA); however, its mechanism is incompletely understood. Here, we show that Kindlin-2, a key focal adhesion protein, is strongly detected in cells of mandibular condylar cartilage in mice. We find that genetic ablation of Kindlin-2 in aggrecan-expressing condylar chondrocytes induces multiple spontaneous osteoarthritic lesions, including progressive cartilage loss and deformation, surface fissures, and ectopic cartilage and bone formation in TMJ. Kindlin-2 loss significantly downregulates the expression of aggrecan, Col2a1 and Proteoglycan 4 (Prg4), all anabolic extracellular matrix proteins, and promotes catabolic metabolism in TMJ cartilage by inducing expression of Runx2 and Mmp13 in condylar chondrocytes. Kindlin-2 loss decreases TMJ chondrocyte proliferation in condylar cartilages. Furthermore, Kindlin-2 loss promotes the release of cytochrome c as well as caspase 3 activation, and accelerates chondrocyte apoptosis in vitro and TMJ. Collectively, these findings reveal a crucial role of Kindlin-2 in condylar chondrocytes to maintain TMJ homeostasis.

Background: The key focal adhesion protein β1 integrin plays an essential role in early skeletal development. However, roles of β1 integrin expression in osteocytes during the regulation of bone homeostasis and mechanotransduction are incompletely understood. Materials and methods: To study the in vivo function of osteocyte β1 integrin in bone, we utilized the 10-kb Dmp1 (Dentin matrix acidic phosphoprotein 1)-Cre to generate mice with β1 integrin deletion in this cell type. Micro-computerized tomography, bone histomorphometry and immunohistochemistry were performed to determine the effects of osteocyte β1 integrin loss on bone mass accrual and biomechanical properties. In vivo tibial loading model was applied to study the possible involvement of osteocyte β1 integrin in bone mechanotransduction. Results: Loss of β1 integrin expression in osteocytes resulted in a severe low bone mass and impaired biomechanical properties in load-bearing long bones and spines, but not in non-weight-bearing calvariae, in mice. The loss of β1 integrin led to enlarged size of lacunar-canalicular system, abnormal cell morphology, and disorientated nuclei in osteocytes. Furthermore, β1 integrin loss caused shortening and disorientated collagen I fibers in long bones. Osteocyte β1 integrin loss did not impact the osteoclast activities, but significantly reduced the osteoblast bone formation rate and, in the meantime, enhanced the adipogenic differentiation of the bone marrow stromal cells in the bone microenvironment. In addition, tibial loading failed to accelerate the anabolic bone formation and improve collagen I fiber integrity in mutant mice. Conclusions: Our studies demonstrate an essential role of osteocyte β1 integrin in regulating bone homeostasis and mechanotransduction.The transnational potential of this article: This study reveals the regulatory roles of osteocyte β1 integrin in vivo for the maintenance of bone mass accrual, biomechanical properties, extracellular matrix integrity as well as bone mechanobiology, which defines β1 integrin a potential therapeutic target for skeletal diseases, such as osteoporosis.

Abstract Postmenopausal osteoporosis is a common bone metabolic disorder characterized by deterioration of the bone microarchitecture, leading to an increased risk of fractures. Recently, circular RNAs (circRNAs) have been demonstrated to play pivotal roles in regulating bone metabolism. However, the underlying functions of circRNAs in bone metabolism in postmenopausal osteoporosis remain obscure. Here, we report that circStag1 is a critical osteoporosis-related circRNA that shows significantly downregulated expression in osteoporotic bone marrow mesenchymal stem cells (BMSCs) and clinical bone tissue samples from patients with osteoporosis. Overexpression of circStag1 significantly promoted the osteogenic capability of BMSCs. Mechanistically, we found that circStag1 interacts with human antigen R (HuR), an RNA-binding protein, and promotes the translocation of HuR into the cytoplasm. A high cytoplasmic level of HuR led to the activation of the Wnt signaling pathway by stabilizing and enhancing low-density lipoprotein receptor-related protein 5/6 (Lrp5/6) and β-catenin expression, thereby stimulating the osteogenic differentiation of BMSCs. Furthermore, overexpression of circStag1 in vivo by circStag1-loaded adeno-associated virus (circStag1-AAV) promoted new bone formation, thereby preventing bone loss in ovariectomized rats. Collectively, we show that circStag1 plays a pivotal role in promoting the regeneration of bone tissue via HuR/Wnt signaling, which may provide new strategies to prevent bone metabolic disorders such as postmenopausal osteoporosis.

Here, the authors show that expression of kindlin-2 is increased in patients with nonalcoholic fatty liver disease (NAFLD). In mouse models, specific deletion of kindlin-2 in liver ameliorates, while its overexpression exacerbates, NAFLD by modulating Foxo1 in hepatocytes.

Abstract Intervertebral disc (IVD) degeneration (IVDD) is the main cause of low back pain with major social and economic burdens; however, its underlying molecular mechanisms remain poorly defined. Here we show that the focal adhesion protein Kindlin-2 is highly expressed in the nucleus pulposus (NP), but not in the anulus fibrosus and the cartilaginous endplates, in the IVD tissues. Expression of Kindlin-2 is drastically decreased in NP cells in aged mice and severe IVDD patients. Inducible deletion of Kindlin-2 in NP cells in adult mice causes spontaneous and striking IVDD-like phenotypes in lumbar IVDs and largely accelerates progression of coccygeal IVDD in the presence of abnormal mechanical stress. Kindlin-2 loss activates Nlrp3 inflammasome and stimulates expression of IL-1β in NP cells, which in turn downregulates Kindlin-2. This vicious cycle promotes extracellular matrix (ECM) catabolism and NP cell apoptosis. Furthermore, abnormal mechanical stress reduces expression of Kindlin-2, which exacerbates Nlrp3 inflammasome activation, cell apoptosis, and ECM catabolism in NP cells caused by Kindlin-2 deficiency. In vivo blocking Nlrp3 inflammasome activation prevents IVDD progression induced by Kindlin-2 loss and abnormal mechanical stress. Of translational significance, adeno-associated virus-mediated overexpression of Kindlin-2 inhibits ECM catabolism and cell apoptosis in primary human NP cells in vitro and alleviates coccygeal IVDD progression caused by mechanical stress in rat. Collectively, we establish critical roles of Kindlin-2 in inhibiting Nlrp3 inflammasome activation and maintaining integrity of the IVD homeostasis and define a novel target for the prevention and treatment of IVDD.

Background: To comprehensively analyze the global level and trends of prevalence, incidence and years lived with disability (YLDs) for low back pain (LBP) from 1990 to 2019 by age, sex and sociodemographic index (SDI). Methods: Publicly available modelled data and methods were obtained from the Global Burden of Diseases (GBD) study 2019, and used to evaluate the global burden of LBP through a systematic analysis. Results: Globally, the age-standardized prevalence, incidence and YLDs rate of LBP were slightly decreased from 1990 to 2019, but the number of the prevalent cases, incident cases and YLDs had substantially increased, and LBP remains the leading cause of YLDs in 2019 worldwide. The number of prevalent cases was increased with age and peaked at the age of 45–54 years for both sexes, and the global prevalence rate was higher in females than in males and increased with age, peaking at the 80–84 age group in both sexes in 2019. Overall, a positive association between the age-standardized YLD rate and SDI was observed over the past thirty years. At the national revel, the United States, Denmark and Switzerland had the three highest levels of age-standardized prevalence, while Zambia, Zimbabwe and Canada showed the highest increase in the age-standardized prevalence during 1990–2019. Conclusions: LBP is a major public health issue globally, and its burden remains high. Increasing population awareness about its risk factors and preventive measures for LBP are needed to reduce the future burden of this condition. The translational potential of this article: Due to the high prevalence and heavy burden of LBP globally, it is important to update its epidemiological data. This systematic analysis provides researchers and healthcare policy makers with up-to-date, comprehensive and comparable information on global LBP burden, which is of clinical translational significance.

Background: Our recent studies demonstrate that the focal adhesion protein Kindlin-2 exerts crucial functions in the mesenchymal stem cells, mature osteoblasts and osteocytes in control of early skeletal development and bone homeostasis in mice. However, whether Kindlin-2 plays a role in osteoprogenitors remains unclear. Materials and methods: Mice lacking Kindlin-2 expression in osterix (Osx)-expressing cells (i.e., osteoprogenitors) were generated. Micro-computerized tomography (μCT) analyses, histology, bone histomorphometry and immunohistochemistry were performed to determine the effects of Kindlin-2 deletion on skeletal development and bone mass accrual and homeostasis. Bone marrow stromal cells (BMSCs) from mutant mice (Kindlin-2fl/fl; OsxCre) and control littermates were isolated and determined for their osteoblastic differentiation capacity. Results: Kindlin-2 was highly expressed in osteoprogenitors during endochondral ossification. Deleting Kindlin-2 expression in osteoprogenitors impaired both intramembranous and endochondral ossifications. Mutant mice displayed multiple severe skeletal abnormalities, including unmineralized fontanel, limb shortening and growth retardation. Deletion of Kindlin-2 in osteoprogenitors impaired the growth plate development and largely delayed formation of the secondary ossification center in the long bones. Furthermore, adult mutant mice displayed a severe low-turnover osteopenia with a dramatic decrease in bone formation which exceeded that in bone resorption. Primary BMSCs isolated from mutant mice exhibited decreased osteoblastic differentiation capacity. Conclusions: Our study demonstrates an essential role of Kinlind-2 expression in osteoprogenitors in regulating skeletogenesis and bone mass accrual and homeostasis in mice. The translational potential of this article: This study reveals that Kindlin-2 through its expression in osteoprogenitor cells controls chondrogenesis and bone mass. We may define a novel therapeutic target for treatment of skeletal diseases, such as chondrodysplasia and osteoporosis.

Pinch1 and Pinch2 are LIM domain-containing proteins with crucial functions in mediating focal adhesion formation. Our previous studies have demonstrated that Pinch1/2 expression is essential for cartilage and bone formation during skeletal development in mice. Loss of Pinch expression (Prx1Cre; Pinch1flox/flox; Pinch2−/−) inhibits chondrocyte proliferation and promotes chondrocyte apoptosis, resulting in severe chondrodysplasia and limb shortening. Based on these observations, we wonder if Pinch proteins have a role in adult cartilage and whether Pinch deficiency will compromise cartilage homeostasis and promote osteoarthritis (OA)-related defects in adult mice. To this end, we generated the AggrecanCreERT2; Pinch1flox/flox; Pinch2−/− mice, in which the Pinch1 gene can be inducibly deleted in aggrecan-expressing chondrocytes by tamoxifen and the Pinch2 gene is globally inactivated. Immunofluorescent staining confirmed that the expression of Pinch proteins was significantly decreased in articular cartilage in tamoxifen-treated adult AggrecanCreERT2; Pinch1flox/flox; Pinch2−/− mice. Unexpectedly, our results showed that Pinch loss did not induce marked abnormalities in articular cartilage and other joint tissues in the knee joints of either adult (10-month-old) mice or aged (17-month-old) mice. In a destabilization of the medial meniscus (DMM)-induced OA model, the surgically-induced OA lesions were comparable between Pinch-deficient mice and control mice. Given the fact that Pinch proteins are essential for chondrogenesis and cartilage formation during skeletal development, these findings suggest that Pinch expression is seemingly not indispensable for adult cartilage homeostasis in mice.

Inflammatory liver diseases are a major cause of morbidity and mortality worldwide; however, underlying mechanisms are incompletely understood. Here we show that deleting the focal adhesion protein Kindlin-2 expression in hepatocytes using the Alb-Cre transgenic mice causes a severe inflammation, resulting in premature death. Kindlin-2 loss accelerates hepatocyte apoptosis with subsequent compensatory cell proliferation and accumulation of the collagenous extracellular matrix, leading to massive liver fibrosis and dysfunction. Mechanistically, Kindlin-2 loss abnormally activates the tumor necrosis factor (TNF) pathway. Blocking activation of the TNF signaling pathway by deleting TNF receptor or deletion of Caspase 8 expression in hepatocytes essentially restores liver function and prevents premature death caused by Kindlin-2 loss. Finally, of translational significance, adeno-associated virus mediated overexpression of Kindlin-2 in hepatocytes attenuates the D-galactosamine and lipopolysaccharide-induced liver injury and death in mice. Collectively, we establish that Kindlin-2 acts as a novel intrinsic inhibitor of the TNF pathway to maintain liver homeostasis and may define a useful therapeutic target for liver diseases.

Axin1 is a key regulator of canonical Wnt signaling pathway. Roles of Axin1 in skeletal development and in disease occurrence have not been fully defined. Here, we report that Axin1 is essential for lower limb development. Specific deletion of Axin1 in limb mesenchymal cells leads to fibular hemimelia (FH)-like phenotype, associated with tarsal coalition. Further studies demonstrate that FH disease is associated with additional defects in Axin1 knockout (KO) mice, including decreased osteoclast formation and defects in angiogenesis. We then provide in vivo evidence showing that Axin1 controls limb development through both canonical β-catenin and BMP signaling pathways. We demonstrate that inhibition of β-catenin or BMP signaling could significantly reverse the FH phenotype in mice. Together, our findings reveal that integration of β-catenin and BMP signaling by Axin1 is required for lower limb development. Defect in Axin1 signaling could lead to the development of FH disease.

Abstract Mechanotransduction is a fundamental ability that allows living organisms to receive and respond to physical signals from both the external and internal environments. The mechanotransduction process requires a range of special proteins termed mechanotransducers to convert mechanical forces into biochemical signals in cells. The Piezo proteins are mechanically activated nonselective cation channels and the largest plasma membrane ion channels reported thus far. The regulation of two family members, Piezo1 and Piezo2, has been reported to have essential functions in mechanosensation and transduction in different organs and tissues. Recently, the predominant contributions of the Piezo family were reported to occur in the skeletal system, especially in bone development and mechano-stimulated bone homeostasis. Here we review current studies focused on the tissue-specific functions of Piezo1 and Piezo2 in various backgrounds with special highlights on their importance in regulating skeletal cell mechanotransduction. In this review, we emphasize the diverse functions of Piezo1 and Piezo2 and related signaling pathways in osteoblast lineage cells and chondrocytes. We also summarize our current understanding of Piezo channel structures and the key findings about PIEZO gene mutations in human diseases.

Qin et al. show the involvement of Kindlin-2 in osteocyte mechanotransduction. They show Kindlin-2 mediates skeletal responses to mechanical stimulation through Smad 2/3 mediated inhibition of Sclerostin.

Abstract In vertebrates, the type 1 parathyroid hormone receptor (PTH1R) is a critical regulator of skeletal development and homeostasis; however, how it is modulated is incompletely understood. Here we report that deleting Kindlin-2 in osteoblastic cells using the mouse 10-kb Dmp1-Cre largely neutralizes the intermittent PTH-stimulated increasing of bone volume fraction and bone mineral density by impairing both osteoblast and osteoclast formation in murine adult bone. Single-cell profiling reveals that Kindlin-2 loss increases the proportion of osteoblasts, but not mesenchymal stem cells, chondrocytes and fibroblasts, in non-hematopoietic bone marrow cells, with concomitant depletion of osteoblasts on the bone surfaces, especially those stimulated by PTH. Furthermore, haploinsufficiency of Kindlin-2 and Pth1r genes, but not that of either gene, in mice significantly decreases basal and, to a larger extent, PTH-stimulated bone mass, supporting the notion that both factors function in the same genetic pathway. Mechanistically, Kindlin-2 interacts with the C-terminal cytoplasmic domain of PTH1R via aa 474–475 and Gsα. Kindlin-2 loss suppresses PTH induction of cAMP production and CREB phosphorylation in cultured osteoblasts and in bone. Interestingly, PTH promotes Kindlin-2 expression in vitro and in vivo, thus creating a positive feedback regulatory loop. Finally, estrogen deficiency induced by ovariectomy drastically decreases expression of Kindlin-2 protein in osteocytes embedded in the bone matrix and Kindlin-2 loss essentially abolishes the PTH anabolic activity in bone in ovariectomized mice. Thus, we demonstrate that Kindlin-2 functions as an intrinsic component of the PTH1R signaling pathway in osteoblastic cells to regulate bone mass accrual and homeostasis.

Both O-glycosides and nucleosides are essential biomolecules with important roles in a variety of biological processes. Chemical synthesis of both O-glycosides and nucleosides is a scalable and reliable method to develop new therapeutic agents and decipher their functions. However, the efficient synthesis of both O-glycosides and nucleosides remains one of long-standing challenges in chemical synthesis. In particular, ether-type glycosyl donors are rarely developed to achieve the efficient synthesis of both O-glycosides and nucleosides due to the stronger conditions required for breaking of the glycosidic C–O bond. Here we report that ortho-(1-phenylvinyl)benzyl glycosides are new ether-type donors for efficient synthesis of both O-glycosides and nucleosides under mild reaction conditions. This glycosylation method enables glycosylation with both alcoholic acceptors and nucleobases with a variety of reactive functionalities. Furthermore, the latent-active synthesis of glycans and the efficient synthesis of nucleosides antibiotics have also been successfully demonstrated in the current glycosylation protocol, thereby representing an important step toward streamlining the chemical synthesis of both O-glycosides and nucleosides.

O-glycosides and nucleosides are important biomolecules. Here, the authors developed a protocol for the synthesis of such molecules with a broad substrate scope using glycosyl ortho-(1-phenylvinyl)benzoates as glycosyl donors that allow for mild reaction conditions and high yields.

Beta cell dysfunction and reduction in beta cell mass are hallmark events in the pathogenesis of diabetes mellitus. We identify focal adhesion protein Kindlin-2 as a key factor that controls insulin synthesis and secretion and beta cell mass by modulating MafA and beta-catenin proteins in pancreatic beta cells.

Osteoarthritis involves whole-joint tissue degeneration. Here, the authors show that miR-204 and miR-211 in mesenchymal joint cells regulate their proliferation, catabolic and osteogenic responses, and that disease progression is ameliorated by intra-articular miR-204 delivery in mice.

The mechano-properties of the Extracellular Matrix (ECM) are important for tumorigenesis. Here, the authors show that the stiffening of the ECM promotes translocation of the focal adhesion protein—Kindlin-2—to the mitochondria, where it interacts with the proline synthesis enzyme PYCR1, stimulating proline synthesis and cell proliferation.

In vertebrate, the nucleus pulposus (NP), which is an essential component of the intervertebral disk, is constantly impacted by fluid shear stress (FSS); however, molecular mechanism(s) through which FSS modulates the NP homeostasis is poorly understood. Here we show that FSS regulates the extracellular matrix (ECM) homeostasis in NP cells. A moderate dose of FSS (i.e., 12 dyne/cm2) increases the sulfated glycosaminoglycan (sGAG) content and protein levels of Col2a1 and Aggrecan and decreases those of matrix metalloproteinase 13 (MMP13) and a disintegrin and metalloproteinase with thrombospondin motif 5 (ADMATS5) in rat NP cells, while a higher dose of FSS (i.e., 24 dyne/cm2) displays opposite effects. Results from RNA sequencing analysis, quantitative real-time RT-PCR analysis and western blotting establish that the heme oxygenase-1 (HO-1) is a key downstream mediator of the FSS actions in NP cells. HO-1 knockdown abolishes FSS-induced alterations in ECM protein production and sGAG content in NP cells, which is reversed by HO-1 induction. Furthermore, FSS activates the autophagic pathway by increasing the LC3-II/LC3-I ratio, Beclin-1 protein level, and formation of autophagosome and autolysosome and thereby regulates ECM protein and sGAG production in a HO-1 dependent manner. Finally, we demonstrate that the intraflagellar transport (IFT) 88, a core trafficking protein of primary cilia, is critically involved in the HO-1-mediated autophagy activation and ECM protein and sGAG production in FSS-treated NP cells. Thus, we for the first time demonstrate that FSS plays an important role in maintaining ECM homeostasis through HO-1-dependent activation of autophagy in NP cells.

Summary: T cells are strongly regulated by oxidizing environments and amino acid restriction. How T cells reprogram metabolism to adapt to these extracellular stress situations is not well understood. Here, we show that oxidizing environments and amino acid starvation induce ATF4 in CD4+ T cells. We also demonstrate that Atf4-deficient CD4+ T cells have defects in redox homeostasis, proliferation, differentiation, and cytokine production. We further reveal that ATF4 regulates a coordinated gene network that drives amino acid intake, mTORC1 activation, protein translation, and an anabolic program for de novo synthesis of amino acids and glutathione. ATF4 also promotes catabolic glycolysis and glutaminolysis and oxidative phosphorylation and thereby provides precursors and energy for anabolic pathways. ATF4-deficient mice mount reduced Th1 but elevated Th17 immune responses and develop more severe experimental allergic encephalomyelitis (EAE). Our study demonstrates that ATF4 is critical for CD4+ T cell-mediated immune responses through driving metabolic adaptation. : Oxidizing environments and availability of extracellular amino acids are major mechanisms that regulate T cell proliferation and function. Yang et al. demonstrate that ATF4 drives metabolic reprogramming, which allows CD4+ T cells to adapt to these stresses. Keywords: T cell, metabolism, EAE, Th17, Th1, ATF4, amino acid, glycolysis, TCA cycle, autoimmune

p204, a murine member of an interferon-inducible p200 family, was reported to recognize intracellular viral and bacterial DNAs, however, its role in the innate immunity in vivo remains unknown due to the lack of p204-deficient animal models. In this study we first generated the p204−/− mice. Unexpectedly, p204 deficiency led to significant defect in extracellular LPS signaling in macrophages, as demonstrated by dramatic reductions of LPS-mediated IFN-β and pro-inflammatory cytokines. The serum levels of IFN-β and pro-inflammatory cytokines were also significantly reduced in p204−/− mice following LPS challenge. In addition, p204−/− mice were resistant to LPS-induced shock. LPS-activated NF-ĸB and IRF-3 pathways were all defective in p204-deficient macrophages. p204 binds to TLR4 through its Pyrin domain, and it is required for the dimerization of TLR4 following LPS-challenge. Collectively, p204 is a critical component of canonical LPS-TLR4 signaling pathway, and these studies also suggest that p204 could be a potential target to prevent and treat inflammatory and infectious diseases. Keywords: p204, LPS, TLR4, IFN-β, Inflammatory responses, Macrophages

Osteocytes secrete the glycoprotein sclerostin to inhibit bone formation by osteoblasts, but how sclerostin production is regulated in osteocytes remains unclear. Here, we show that tuberous sclerosis complex 1 (TSC1) in osteocytes promotes sclerostin secretion through inhibition of mechanistic target of rapamycin complex 1 (mTORC1) and downregulation of Sirt1. We generated mice with DMP1-Cre-directed Tsc1 gene deletion (Tsc1 CKO) to constitutively activate mTORC1 in osteocytes. Although osteocyte TSC1 disruption increased RANKL expression and osteoclast formation, it markedly reduced sclerostin production in bone, resulting in severe osteosclerosis with enhanced bone formation in mice. Knockdown of TSC1 activated mTORC1 and decreased sclerostin, while rapamycin inhibited mTORC1 and increased sclerostin mRNA and protein expression levels in MLO-Y4 osteocyte-like cells. Furthermore, mechanical loading activated mTORC1 and prevented sclerostin expression in osteocytes. Mechanistically, TSC1 promotes sclerostin production and prevents osteogenesis through inhibition of mTORC1 and downregulation of Sirt1, a repressor of the sclerostin gene Sost. Our findings reveal a role of TSC1/mTORC1 signalling in the regulation of osteocyte sclerostin secretion and bone formation in response to mechanical loading in vitro. Targeting TSC1 represents a potential strategy to increase osteogenesis and prevent bone loss-related diseases.

ABSTRACT Carbapenem-resistant (CR) sequence type 258 (ST258) Klebsiella pneumoniae has become an urgent health care threat, causing an increasing number of high-mortality infections. Its resistance to numerous antibiotics and threat to immunocompromised patients necessitate finding new therapies to combat these infections. Previous successes in the laboratory, as well as the conservation of capsular polysaccharide (CPS) among the members of the ST258 clone, suggest that monoclonal antibody (MAb) therapy targeting the outer polysaccharide capsule of K. pneumoniae could serve as a valuable treatment alternative for afflicted patients. Here, we isolated several IgG antibodies from mice inoculated with a mixture of CR K. pneumoniae CPS conjugated to anthrax protective antigen. Two of these MAbs, 17H12 and 8F12, bind whole and oligosaccharide epitopes of the CPS of clade 2 ST258 CR K. pneumoniae, which is responsible for the most virulent CR K. pneumoniae infections in the United States. These antibodies were shown to agglutinate all clade 2 strains and were also shown to promote extracellular processes killing these bacteria, including biofilm inhibition, complement deposition, and deployment of neutrophil extracellular traps. Additionally, they promoted opsonophagocytosis and intracellular killing of CR K. pneumoniae by human-derived neutrophils and cultured murine macrophages. Finally, when mice were intratracheally infected with preopsonized clade 2 CR K. pneumoniae, these MAbs reduced bacterial dissemination to organs. Our data suggest that broadly reactive anticapsular antibodies and vaccines against clade 2 ST258 CR K. pneumoniae are possible. Such MAbs and vaccines would benefit those susceptible populations at risk of infection with this group of multidrug-resistant bacteria. IMPORTANCE Carbapenem-resistant Klebsiella pneumoniae is an enteric bacterium that has been responsible for an increasing number of deadly outbreaks and hospital-acquired infections. The pathogen’s resistance to numerous antibiotics, including new drugs, leaves few therapeutic options available for infected patients, who often are too sick to fight the infection themselves. Immunotherapy utilizing monoclonal antibodies has been successful in other medical fields, and antibodies targeting the outer polysaccharide capsule of these bacteria could be a valuable treatment alternative. This study presents two anticapsular antibodies, 17H12 and 8F12, that were found to be protective against the most virulent carbapenem-resistant K. pneumoniae clinical strains. These antibodies are shown to promote the killing of these strains through several extracellular and intracellular processes and prevent the spread of infection in mice from the lungs to distal organs. Thus, they could ultimately treat or protect patients infected or at risk of infection by this multidrug-resistant bacterium.

Lung cancer is the leading cause of cancer-related deaths. The morbidity and mortality of lung cancer have markedly increased in the past decade with at least 75% of patients with lung cancer having evidence of metastases at the time of diagnosis. It frequently metastasizes to bone resulting in osteolytic lesions with unknown mechanisms. The aim of this study was to identify factors that mediate lung cancer–induced osteoclast activity in vivo. Using a human cytokine antibody array, we first determined cytokine levels in a conditioned medium collected from non– small cell lung cancer A549 and H1299 cells and the non-neoplastic human bronchial epithelial BEAS2B cells. Both A549 and H1229 cells produced significantly higher amount of several cytokines including monocyte chemotactic protein 1 (MCP-1) and interleukin 8 (IL-8) compared with BEAS2B cells. These findings were confirmed by ELISA. From clinical serum specimens, we also observed that MCP-1 and IL-8 levels were increased in lung cancer patients with bone metastases compared with the patients with localized tumor. Next, we investigated the effects of MCP-1 on osteoclast formation in vitro using murine bone marrow–derived monocytes. A549 conditioned medium induced osteoclast formation that was inhibited by neutralizing antibodies against MCP-1. Finally, A549 cells were stably transfected with MCP-1 short hairpin RNA. The MCP-1 knockdown A549 cells were implanted into the tibia of severe combined immunodeficient mice for 4 weeks. The MCP-1 knockdown significantly diminished A549 cell growth. We conclude that MCP-1 promotes lung cancer–induced osteoclast activity and thus bone resorptive lesions in vivo.

Parathyroid hormone (PTH) is a potent anabolic agent for the treatment of osteoporosis. However, its mechanism of action in osteoblast and bone is not well understood. In this study, we show that the anabolic actions of PTH in bone are severely impaired in both growing and adult ovariectomized mice lacking bone-related activating transcription factor 4 (ATF4). Our study demonstrates that ATF4 deficiency suppresses PTH-stimulated osteoblast proliferation and survival and abolishes PTH-induced osteoblast differentiation, which, together, compromise the anabolic response. We further demonstrate that the PTH-dependent increase in osteoblast differentiation is correlated with ATF4-dependent up-regulation of Osterix. This regulation involves interactions of ATF4 with a specific enhancer sequence in the Osterix promoter. Furthermore, actions of PTH on Osterix require this same element and are associated with increased binding of ATF4 to chromatin. Taken together these experiments establish a fundamental role for ATF4 in the anabolic actions of PTH on the skeleton.