ANGPTL4 binds to the leptin receptor to regulate ectopic bone formation.

Leptin protein was thought to be unique to leptin receptor (LepR), but the phenotypes of mice with mutation in LepR [db/db (diabetes)] and leptin [ob/ob (obese)] are not identical, and the cause remains unclear. Here, we show that db/db, but not ob/ob, mice had defect in tenotomy-induced heterotopic ossification (HO), implicating alternative ligand(s) for LepR might be involved. Ligand screening revealed that ANGPTL4 (angiopoietin-like protein 4), a stress and fasting-induced factor, was elicited from brown adipose tissue after tenotomy, bound to LepR on PRRX1+ mesenchymal cells at the HO site, thus promotes chondrogenesis and HO development. Disruption of LepR in PRRX1+ cells, or lineage ablation of LepR+ cells, or deletion of ANGPTL4 impeded chondrogenesis and HO in mice. Together, these findings identify ANGPTL4 as a ligand for LepR to regulate the formation of acquired HO.

MYL3 protects chondrocytes from senescence by inhibiting clathrin-mediated endocytosis and activating of Notch signaling.

As the unique cell type in articular cartilage, chondrocyte senescence is a crucial cellular event contributing to osteoarthritis development. Here we show that clathrin-mediated endocytosis and activation of Notch signaling promotes chondrocyte senescence and osteoarthritis development, which is negatively regulated by myosin light chain 3. Myosin light chain 3 (MYL3) protein levels decline sharply in senescent chondrocytes of cartilages from model mice and osteoarthritis (OA) patients. Conditional deletion of Myl3 in chondrocytes significantly promoted, whereas intra-articular injection of adeno-associated virus overexpressing MYL3 delayed, OA progression in male mice. MYL3 deficiency led to enhanced clathrin-mediated endocytosis by promoting the interaction between myosin VI and clathrin, further inducing the internalization of Notch and resulting in activation of Notch signaling in chondrocytes. Pharmacologic blockade of clathrin-mediated endocytosis-Notch signaling prevented MYL3 loss-induced chondrocyte senescence and alleviated OA progression in male mice. Our results establish a previously unknown mechanism essential for cellular senescence and provide a potential therapeutic direction for OA.

Mono-phosphorylation at Ser4 of barrier-to-autointegration factor (Banf1) significantly reduces its DNA binding capability by inducing critical changes in its local conformation and DNA binding surface.

Barrier-to-autointegration factor (Banf1) is a small DNA-bridging protein. The binding status of Banf1 to DNA is regulated by its N-terminal phosphorylation and dephosphorylation, which plays a critical role in cell proliferation. Banf1 can be phosphorylated at Ser4 into mono-phosphorylated Banf1, which is further phosphorylated at Thr3 to form di-phosphorylated Banf1. It was observed decades ago that mono-phosphorylated Banf1 cannot bind to DNA. However, the underlying molecular- and atomic-level mechanisms remain unclear. A clear understanding of these mechanisms will aid in interfering with the cell proliferation process for better global health. Herein, we explored the detailed atomic bases of unphosphorylated Banf1-DNA binding and how mono- and di-phosphorylation of Banf1 impair these atomic bases to eliminate its DNA-binding capability, followed by exploring the DNA-binding capability of mono- and di-phosphorylation Banf1, using comprehensive and systematic molecular modelling and molecular dynamics simulations. This work presented in detail the residue-level binding energies, hydrogen bonds and water bridges between Banf1 and DNA, some of which have not been reported. Moreover, we revealed that mono-phosphorylation of Banf1 causes its N-terminal secondary structure changes, which in turn induce significant changes in Banf1's DNA binding surface, thus eliminating its DNA-binding capability. At the atomic level, we also uncovered the alterations in interactions due to the induction of mono-phosphorylation that result in the N-terminal secondary structure changes of Banf1. Additionally, our modelling showed that phosphorylated Banf1 with their dominant N-terminal secondary structures bind to DNA with a significantly lower affinity and the docked binding pose are not stable in MD simulations. These findings help future studies in predicting effect of mutations in Banf1 on its DNA-binding capability and open a novel avenue for the development of therapeutics such as cancer drugs, targeting cell proliferation by inducing conformational changes in Banf1's N-terminal domain.

Tuberous sclerosis complex 1 targeted depletion in macrophages promotes osteogenesis by modulating secretion of Oncostatin M in the inflammatory stage of bone healing.

In bone healing, earlier bone formation benefits bone repair. The first process of repair following bone injury involves the interaction between macrophage polarization and osteogenic activation of osteoblast linage cells, but the radical difference between the contributions of classically-activated M1 macrophages and alternatively-activated M2 macrophages to osteogenesis remains obscure. To test our hypothesis that M1 macrophages promote bone healing, we generated transgenic mice with myeloid lineage-specific TSC1 deletion (TSC1KO) to investigate the functional roles of M1 macrophages in the process of bone defect healing. We demonstrated that constitutive activation of mammalian target of rapamycin complex 1 (mTORC1) enhances M1 macrophage polarization during bone healing. By creating tibial bone defect as a model of bone repair in TSC1KO mice and their littermates, we surprisingly found osteogenic responses in the defective bone region of TSC1KO mice, where repair occurred by intramembranous ossification (IO) in the mice was promoted due to the enhanced M1-polarized macrophage polarization. We propose that Oncostatin M (OSM) secreted by M1-polarized macrophages but not M2 macrophages likely functions as a paracrine factor in this promoted repair process, as verified by the induction of osteoblastic differentiation and matrix mineralization. Interestingly, the expression level of the OSM receptor (OSMR) was continually upregulated in osteoblast linage cells with M1 medium. Additionally, OSMR activated the signaling transduction system of JAK/STAT/RUNX2 in MSCs, which in turn stimulates the recruitment of osteoblast lineage cells and activates IO. These results indicate that TSC1 targeted depletion in macrophages promotes bone healing by inducing secretion of OSM. This study highlights that regulation of M1 macrophage polarization is a novel basis for the improvement of bone regeneration and that regulation of macrophage polarization can be a potential therapeutic strategy to treat defects in the repair phase of bone healing.

Exosome Release Delays Senescence by Disposing of Obsolete Biomolecules.

Accumulation of obsolete biomolecules can accelerate cell senescence and organism aging. The two efficient intracellular systems, namely the ubiquitin-proteasome system and the autophagy-lysosome system, play important roles in dealing with cellular wastes. However, how multicellular organisms orchestrate the processing of obsolete molecules and delay aging remains unclear. Herein, it is shown that prevention of exosome release by GW4869 or Rab27a-/- accelerated senescence in various cells and mice, while stimulating exosome release by nutrient restriction delays aging. Interestingly, exosomes isolate from serum-deprived cells or diet-restricted human plasma, enriched with garbage biomolecules, including misfolded proteins, oxidized lipids, and proteins. These cellular wastes can be englobed by macrophages, eventually, for disintegration in vivo. Inhibition of nutrient-sensing mTORC1 signaling increases exosome release and delays senescence, while constitutive activation of mTORC1 reduces exosome secretion and exacerbates senescence in vitro and in mice. Notably, inhibition of exosome release attenuates nutrient restriction- or rapamycin-delayed senescence, supporting a key role for exosome secretion in this process. This study reveals a potential mechanism by which stimulated exosome release delays aging in multicellular organisms, by orchestrating the harmful biomolecules disposal via exosomes and macrophages.

Deletion of Rheb1 in Osteocytes Leads to Osteopenia Characterized by Reduced Bone Formation and Enhanced Bone Resorption.

Ras homologue enriched in brain 1 (Rheb1), an upstream activator of the mechanistic target of rapamycin complex 1 (mTORC1), is known to modulate various cellular processes. However, its impact on bone metabolism in vivo remains unknown. The study aimed at understanding the role of Rheb1 on bone homeostasis. We measured the serum parameters and performed histomorphometry, quantitative real-time polymerase chain reaction, and Western blotting, along with the generation of mouse gene knockout (KO) model, and conducted a microcomputed tomography analysis and tartrate-resistant acid phosphatase staining, to delineate the impacts of Rheb1 on bone homeostasis. In the Rheb1 KO mice, the results showed that Rheb1 KO caused significant damage to the bone microarchitecture, indicating that mTORC1 activity was essential for the regulation of bone homeostasis. Specifically, suppressed mineralization activity in primary osteoblasts and a decreased osteoblast number were observed in the Rheb1 KO mice, demonstrating that loss of Rheb1 led to impaired osteoblastic differentiation. Furthermore, the higher apoptotic ratio in Rheb1-null osteocytes could promote Tnfsf11 expression and lead to an increase in osteoclasts, indicating increased bone resorption activity in the KO mice. The findings confirmed that Rheb1 deletion in osteoblasts/osteocytes led to osteopenia due to impaired bone formation and enhanced bone resorption.

Bindarit Reduces Bone Loss in Ovariectomized Mice by Inhibiting CCL2 and CCL7 Expression via the NF-κB Signaling Pathway.

OBJECTIVE: To investigate the changes in proinflammatory cytokines and chemokines, namely, C-C motif ligand (CCL) 2 and CCL7, in postmenopausal osteoporosis (PMOP) and to develop a new drug, bindarit (Bnd), for PMOP in an ovariectomized (OVX) mouse model. METHODS: Bone marrow macrophages (BMMs) from the femurs of five women with PMOP and five premenopausal women without osteoporosis were detected by RNA sequencing. BMMs from mice were differentiated into osteoclasts and treated with a synthetic inhibitor of CCL2 and CCL7, Bnd, or 17 beta estradiol (E2 ). Mouse BMMs were differentiated into osteoclasts with or without Bnd for 7 days and analyzed by RNA sequencing. Osteoblasts of mice were induced to undergo osteoblastogenesis and treated with Bnd. OVX mice were treated with E2 or Bnd after surgery. The protein and mRNA expression of CCL2 and CCL7 was detected using immunostaining and qPCR, respectively, in OVX and aged mice and in cells cultured in vitro. Osteoclast formation was detected using a tartrate-resistant acid phosphatase (TRAP) assay in vitro and in vivo. Alkaline phosphatase (ALP), runt-related transcription factor 2 (Runx2) and osteocalcin (OCN) were detected using immunostaining to evaluate osteogenesis. Microcomputed tomography was conducted to analyze trabecular bone parameters, the structure model index, bone mineral density and other variables. Nuclear factor-κB (NF-κB) signaling pathway-related protein phosphorylation of IKKα/ß (p-IKKα/ß) and p-NFκB p65 was examined using western blotting. RESULTS: CCL2, CCL7 and their receptor of C-C chemokine receptor-2 (CCR2), and the NF-κB signaling pathway, were significantly increased in women with PMOP. CCL2 and CCL7 protein and mRNA expression was increased in OVX mice and aged female mice, but the increases were attenuated by E2 and Bnd. E2 and Bnd effectively inhibited osteoclastogenesis and the protein expression of CCL2 and CCL7 both in vitro and in vivo and reduced bone loss in OVX mice. Bnd did not affect the mineralization of osteoblasts directly in vitro but reduced bone turnover in vivo. p-IKKα/ß and p-NFκB p65 levels were increased in BMMs of mice after differentiation into osteoclasts but were significantly decreased by Bnd. CONCLUSION: The proinflammatory cytokines and chemokines CCL2, CCL7 and CCR2 were correlated with PMOP. Bnd attenuated the increases in CCL2 and CCL7 levels to affect osteoporosis in OVX mice via the NFκB signaling pathway. Thus, Bnd may be useful as a new therapeutic for the prevention of PMOP.

DNMT1 is a negative regulator of osteogenesis.

The role and underlying mechanisms of DNA methylation in osteogenesis/chondrogenesis remain poorly understood. We here reveal DNA methyltransferase 1 (DNMT1), which is responsible for copying DNA methylation onto the newly synthesized DNA strand after DNA replication, is overexpressed in sponge bone of people and mice with senile osteoporosis and required for suppression of osteoblast (OB) differentiation of mesenchymal stem cells (MSCs) and osteoprogenitors. Depletion of DNMT1 results in demethylation at the promoters of key osteogenic genes such as RORA and Fgfr2, and consequent upregulation of their transcription in vitro. Mechanistically, DNMT1 binds exactly to the promoters of these genes and are responsible for their 5-mc methylation. Conversely, simultaneous depletion of RORA or Fgfr2 blunts the effects of DNMT1 silencing on OB differentiation, suggesting RORA or Fgfr2 may be crucial for modulating osteogenic differentiation downstream of DNMT1. Collectively, these results reveal DNMT1 as a key repressor of OB differentiation and bone formation while providing us a new rationale for specific inhibition of DNMT1 as a potential therapeutic strategy to treat age-related bone loss.

Mechanical overloading promotes chondrocyte senescence and osteoarthritis development through downregulating FBXW7.

OBJECTIVES: To investigate the role of mechanical stress in cartilage ageing and identify the mechanistic association during osteoarthritis (OA) progression. METHODS: F-box and WD repeat domain containing 7 (FBXW7) ubiquitin ligase expression and chondrocyte senescence were examined in vitro, in experimental OA mice and in human OA cartilage. Mice with Fbxw7 knockout in chondrocytes were generated and adenovirus-expressing Fbxw7 (AAV-Fbxw7) was injected intra-articularly in mice. Destabilised medial meniscus surgery was performed to induce OA. Cartilage damage was measured using the Osteoarthritis Research Society International score and the changes in chondrocyte senescence were determined. mRNA sequencing was performed in articular cartilage from Fbxw7 knockout and control mice. RESULTS: Mechanical overloading accelerated senescence in cultured chondrocytes and in mice articular cartilage. FBXW7 was downregulated by mechanical overloading in primary chondrocytes and mice cartilage, and decreased in the cartilage of patients with OA, aged mice and OA mice. FBXW7 deletion in chondrocytes induced chondrocyte senescence and accelerated cartilage catabolism in mice, as manifested by an upregulation of p16INK4A, p21 and Colx and downregulation of Col2a1 and ACAN, which resulted in the exacerbation of OA. By contrast, intra-articular injection of adenovirus expressing Fbxw7 alleviated OA in mice. Mechanistically, mechanical overloading decreased Fbxw7 mRNA transcription and FBXW7-mediated MKK7 degradation, which consequently stimulated JNK signalling. In particular, inhibition of JNK activity by DTP3, a MKK7 inhibitor, ameliorated chondrocyte senescence and cartilage degeneration CONCLUSIONS: FBXW7 is a key factor in the association between mechanical overloading and chondrocyte senescence and cartilage ageing in the pathology of OA.

Vangl2 limits chaperone-mediated autophagy to balance osteogenic differentiation in mesenchymal stem cells.

Lysosomes are the recycling center and nutrient signaling hub of the cell. Here, we show that lysosomes also control mesenchymal stem cell (MSC) differentiation by proteomic reprogramming. The chaperone-mediated autophagy (CMA) lysosome subgroup promotes osteogenesis, while suppressing adipogenesis, by selectively removing osteogenesis-deterring factors, especially master transcriptional factors, such as adipogenic TLE3, ZNF423, and chondrogenic SOX9. The activity of the CMA-committed lysosomes in MSCs are controlled by Van-Gogh-like 2 (Vangl2) at lysosomes. Vangl2 directly binds to lysosome-associated membrane protein 2A (LAMP-2A) and targets it for degradation. MSC-specific Vangl2 ablation in mice increases LAMP-2A expression and CMA-lysosome numbers, promoting bone formation while reducing marrow fat. The Vangl2:LAMP-2A ratio in MSCs correlates inversely with the capacity of the cells for osteoblastic differentiation in humans and mice. These findings demonstrate a critical role for lysosomes in MSC lineage acquisition and establish Vangl2-LAMP-2A signaling as a critical control mechanism.

Tsc1 regulates tight junction independent of mTORC1.

Tuberous sclerosis complex 1 (Tsc1) is a tumor suppressor that functions together with Tsc2 to negatively regulate the mechanistic target of rapamycin complex 1 (mTORC1) activity. Here, we show that Tsc1 has a critical role in the tight junction (TJ) formation of epithelium, independent of its role in Tsc2 and mTORC1 regulation. When an epithelial cell establishes contact with neighboring cells, Tsc1, but not Tsc2, migrates from the cytoplasm to junctional membranes, in which it binds myosin 6 to anchor the perijunctional actin cytoskeleton to ß-catenin and ZO-1. In its absence, perijunctional actin cytoskeleton fails to form. In mice, intestine-specific or inducible, whole-body Tsc1 ablation disrupts adherens junction/TJ structures in intestine or skin epithelia, respectively, causing Crohn's disease-like symptoms in the intestine or psoriasis-like phenotypes on the skin. In patients with Crohn's disease or psoriasis, junctional Tsc1 levels in epithelial tissues are markedly reduced, concomitant with the TJ structure impairment, suggesting that Tsc1 deficiency may underlie TJ-related diseases. These findings establish an essential role of Tsc1 in the formation of cell junctions and underpin its association with TJ-related human diseases.

DEPTOR Prevents Osteoarthritis Development Via Interplay With TRC8 to Reduce Endoplasmic Reticulum Stress in Chondrocytes.

Endoplasmic reticulum (ER) stress has been shown to promote chondrocyte apoptosis and osteoarthritis (OA) progression, but the precise mechanisms via which ER stress is modulated in OA remain unclear. Here we report that DEP domain-containing mTOR-interacting protein (DEPTOR) negatively regulated ER stress and OA development independent of mTOR signaling. DEPTOR is ubiquitinated in articular chondrocytes and its expression is markedly reduced along with OA progression. Deletion of DEPTOR in chondrocytes significantly promoted destabilized medial meniscus (DMM) surgery-induced OA development, whereas intra-articular injection of lentivirus-expressing DEPTOR delayed OA progression in mice. Proteomics analysis revealed that DEPTOR interplayed with TRC8, which promoted TRC8 auto-ubiquitination and degraded by the ubiquitin-proteasome system (UPS) in chondrocytes. Loss of DEPTOR led to TRC8 accumulation and excessive ER stress, with subsequent chondrocyte apoptosis and OA progression. Importantly, an inhibitor of ER stress eliminated chondrocyte DEPTOR deletion-exacerbated OA in mice. Together, these findings establish a novel mechanism essential for OA pathogenesis, where decreasing DEPTOR in chondrocytes during OA progression relieves the auto-ubiquitination of TRC8, resulting in TRC8 accumulation, excessive ER stress, and OA progression. Targeting this pathway has promising therapeutic potential for OA treatment. © 2020 American Society for Bone and Mineral Research (ASBMR).

Mesenchymal Stem Cell-Specific and Preosteoblast-Specific Ablation of TSC1 in Mice Lead to Severe and Slight Spinal Dysplasia, Respectively.

BACKGROUND: TSC1-related signaling plays a pivotal role in intramembranous and endochondral ossification processes during skeletogenesis. This study was aimed at determining the significance of the TSC1 gene at different stages of spinal development. Materials and Methods. TSC1-floxed mice (TSC1flox/flox) were crossed with Prrx1-Cre or BGLAP-Cre transgenic mice or mesenchymal stem cell- and osteoblast-specific TSC1-deficient mice, respectively. Somatic and vertebral differences between WT and Prrx1-TSC1 null mice were examined at 4 weeks after birth. RESULTS: No apparent body size abnormalities were apparent in newborn and 4-week- to 2-month-old mice with BGLAP-Cre driver-depleted TSC1. Vertebral and intervertebral discs displayed strong dysplasia in Prrx1-TSC1 null mice. In contrast, vertebrae were only slightly affected, and intervertebral discs from skeletal preparations displayed no apparent changes in BGLAP-TSC1 null mice. CONCLUSION: Our data suggest that the TSC1 gene is crucial for endochondral ossification during postnatal spine development but plays discriminative roles at different stages. Mesenchymal stem cell-specific ablation of TSC1 led to severe spinal dysplasia at early stages of endochondral ossification while osteoblast-specific deletion of TSC1 affected vertebrae slightly and had no detectable effects on intervertebral discs.

Characterization of a Novel Calcific Achilles Tendinopathy Model in Mice: Contralateral Tendinopathy Induced by Unilateral Tenotomy.

Achilles tendinopathy is a significant clinical disease characterized by activity-related pain, focal movement limitation, and intratendinous imaging changes. However, treatment of Achilles tendinopathy has been based mainly on theoretical rationale and clinical experience because of its unclear underlying pathogenesis and mechanism. The purpose of the study was to develop a simple but reproducible overuse-induced animal model of Achilles tendinopathy in mice to better understand the underlying mechanism and prevent calcific Achilles tendinopathy. A total of 80 C57/B6 mice (8 or 9 weeks old) were employed and randomly divided into control and experimental groups. Unilateral Achilles tenotomy was performed on the right hind limbs in the experiment group. 12 weeks after unilateral Achilles tenotomy, the onset of Achilles tendinopathy in the contralateral Achilles tendon was determined by radiological assessment, histologic analysis, electron microscopy observation, and biomechanical test. The onset of calcific Achilles tendinopathy in contralateral Achilles tendon was confirmed after 12 weeks of unilateral tenotomy. The contralateral Achilles tendon in the experimental group was characterized as hypercellularity, neovascularization, and fused collagen fiber disarrangement, compared with the control group. Importantly, intra-tendon endochondral ossification and calcaneus deformity were featured in contralateral Achilles tendon. In addition, poor biomechanical properties in the contralateral Achilles tendon revealed the incidence of Achilles tendinopathy. We hereby introduce a novel, simple, but reproducible spontaneous contralateral calcific Achilles tendinopathy model in mice, which represents overuse conditions during tendinopathy development in humans. It should be a useful tool to further study the underlying pathogenesis of calcific Achilles tendinopathy.

Positive-Feedback Regulation of Subchondral H-Type Vessel Formation by Chondrocyte Promotes Osteoarthritis Development in Mice.

Vascular-invasion-mediated interactions between activated articular chondrocytes and subchondral bone are essential for osteoarthritis (OA) development. Here, we determined the role of nutrient sensing mechanistic target of rapamycin complex 1 (mTORC1) signaling in the crosstalk across the bone cartilage interface and its regulatory mechanisms. Then mice with chondrocyte-specific mTORC1 activation (Tsc1 CKO and Tsc1 CKOER ) or inhibition (Raptor CKOER ) and their littermate controls were subjected to OA induced by destabilization of the medial meniscus (DMM) or not. DMM or Tsc1 CKO mice were treated with bevacizumab, a vascular endothelial growth factor (VEGF)-A antibody that blocks angiogenesis. Articular cartilage degeneration was evaluated using the Osteoarthritis Research Society International score. Immunostaining and Western blotting were conducted to detect H-type vessels and protein levels in mice. Primary chondrocytes from mutant mice and ADTC5 cells were treated with interleukin-1ß to investigate the role of chondrocyte mTORC1 in VEGF-A secretion and in vitro vascular formation. Clearly, H-type vessels were increased in subchondral bone in DMM-induced OA and aged mice. Cartilage mTORC1 activation stimulated VEGF-A production in articular chondrocyte and H-type vessel formation in subchondral bone. Chondrocyte mTORC1 promoted OA partially through formation of VEGF-A-stimulated subchondral H-type vessels. In particular, vascular-derived nutrients activated chondrocyte mTORC1, and stimulated chondrocyte activation and production of VEGF, resulting in further angiogenesis in subchondral bone. Thus a positive-feedback regulation of H-type vessel formation in subchondral bone by articular chondrocyte nutrient-sensing mTORC1 signaling is essential for the pathogenesis and progression of OA. © 2018 American Society for Bone and Mineral Research.

Chondrocyte-Specific Knockout of TSC-1 Leads to Congenital Spinal Deformity in Mice.

Congenital spinal deformity is the most severe clinical orthopedic issue worldwide. Among all the pathological processes of congenital spinal deformity, the imbalance of endochondral ossification is considered to be the most important developmental cause of spinal dysplasia. We established chondrocyte-specific TSC-1 knockout (KO) mice to overactivate the energy metabolic component, mammalian target of rapamycin complex 1 (mTORC1), and measured the spinal development by general, imaging, histological, and Western-blot assessments. In addition to skeletal dysplasia, the KO mice displayed severe congenital spinal deformity and significant intervertebral disc changes. This study suggests that, in the process of endochondral ossification, excessive activation of mTORC1 signaling in chondrocytes induces obvious spinal deformity, and the chondrocytes may be the cell type responsible for congenital spinal deformity.

Osteoblasts support megakaryopoiesis through production of interleukin-9.

Severe thrombocytopenia is a significant challenge in patients undergoing myelosuppressive chemotherapy for malignancies. Understanding the biology of platelet-producing megakaryocytes development in the bone marrow microenvironment may facilitate the development of novel therapies to stimulate platelet production and prevent thrombocytopenia. We report here that osteoblasts supported megakaryopoiesis by secreting interleukin-9 (IL-9), which stimulated IL-9 receptor (IL-9R)/Stat3 signaling in promoting megakaryopoiesis. IL-9 production in osteoblasts was negatively regulated by the mechanistic target of rapamycin complex 1 (mTORC1) signaling in a NF-κB-dependent manner. Constitutive activation of mTORC1 inhibited IL-9 production in osteoblasts and suppressed megakaryocytic cells expansion, whereas mTORC1 inactivation increased IL-9 production and enhanced megakaryocyte and platelet numbers in mice. In mouse models, we showed that IL-9 administration stimulated megakaryopoiesis, whereas neutralizing endogenous IL-9 or IL-9R depletion inhibited the process. Importantly, we found that low doses of IL-9 efficiently prevented chemotherapy-induced thrombocytopenia (CIT) and accelerated platelet recovery after CIT. These data indicate that IL-9 is an essential regulator of megakaryopoiesis and a promising therapeutic agent for treatment of thrombocytopenia such as CIT.

mTORC1 regulates PTHrP to coordinate chondrocyte growth, proliferation and differentiation.

Precise coordination of cell growth, proliferation and differentiation is essential for the development of multicellular organisms. Here, we report that although the mechanistic target of rapamycin complex 1 (mTORC1) activity is required for chondrocyte growth and proliferation, its inactivation is essential for chondrocyte differentiation. Hyperactivation of mTORC1 via TSC1 gene deletion in chondrocytes causes uncoupling of the normal proliferation and differentiation programme within the growth plate, resulting in uncontrolled cell proliferation, and blockage of differentiation and chondrodysplasia in mice. Rapamycin promotes chondrocyte differentiation and restores these defects in mutant mice. Mechanistically, mTORC1 downstream kinase S6K1 interacts with and phosphorylates Gli2, and releases Gli2 from SuFu binding, resulting in nuclear translocation of Gli2 and transcription of parathyroid hormone-related peptide (PTHrP), a key regulator of bone development. Our findings demonstrate that dynamically controlled mTORC1 activity is crucial to coordinate chondrocyte proliferation and differentiation partially through regulating Gli2/PTHrP during endochondral bone development.

Chondrocyte-Specific Ablation of AMPKα1 Does Not Affect Bone Development or Pathogenesis of Osteoarthritis in Mice.

AMP-activated protein kinase (AMPK) acts as an intracellular sensor that modulates the energy balance within the cell. AMPKα1 is the dominant catalytic isoform expressed in the bone, but the significance of AMPKα1 in articular cartilage has not been well studied. In this study, we aimed to assess the in vivo function of AMPKα1 in chondrocytes. We created chondrocyte-specific AMPKα1 conditional knockout (KO) mice using Col2α1-Cre and analyzed and compared growth characteristics, HE staining, and AMPKα gene expression between wild-type (WT) mice and AMPKα1 conditional KO mice under normal physiological conditions or following activation of AMPK by metformin intake or treadmill exercise. Microcomputed tomography and safranin O-fast green staining were compared between WT and KO mice after induction of experimental osteoarthritis (OA). Our data showed that there was no somatic difference between WT mice and KO mice of the same age. Metformin intake and treadmill exercise did not alter the phenotype of KO mice, and no difference in cartilage degradation was observed in WT mice or in KO mice after induction of traumatic arthritis. We thought that chondrocyte-specific ablation of AMPKα1 had no effect on bone growth or on pathogenesis of OA in mice, probably because the feedback overexpression of AMPKα2 compensated for loss of AMPKα1 and maintained the combination of AMPKα subunits.

mTORC1 Prevents Preosteoblast Differentiation through the Notch Signaling Pathway.

The mechanistic target of rapamycin (mTOR) integrates both intracellular and extracellular signals to regulate cell growth and metabolism. However, the role of mTOR signaling in osteoblast differentiation and bone formation is undefined, and the underlying mechanisms have not been elucidated. Here, we report that activation of mTOR complex 1 (mTORC1) is required for preosteoblast proliferation; however, inactivation of mTORC1 is essential for their differentiation and maturation. Inhibition of mTORC1 prevented preosteoblast proliferation, but enhanced their differentiation in vitro and in mice. Activation of mTORC1 by deletion of tuberous sclerosis 1 (Tsc1) in preosteoblasts produced immature woven bone in mice due to excess proliferation but impaired differentiation and maturation of the cells. The mTORC1-specific inhibitor, rapamycin, restored these in vitro and in vivo phenotypic changes. Mechanistically, mTORC1 prevented osteoblast maturation through activation of the STAT3/p63/Jagged/Notch pathway and downregulation of Runx2. Preosteoblasts with hyperactive mTORC1 reacquired the capacity to fully differentiate and maturate when subjected to inhibition of the Notch pathway. Together, these findings identified the role of mTORC1 in osteoblast formation and established that mTORC1 prevents preosteoblast differentiation and maturation through activation of the Notch pathway.