Daozhang Cai

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.

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.

Osteoblasts secrete Cxcl9 to regulate angiogenesis in bone.

Communication between osteoblasts and endothelial cells (ECs) is essential for bone turnover, but the molecular mechanisms of such communication are not well defined. Here we identify Cxcl9 as an angiostatic factor secreted by osteoblasts in the bone marrow microenvironment. We show that Cxcl9 produced by osteoblasts interacts with vascular endothelial growth factor and prevents its binding to ECs and osteoblasts, thus abrogating angiogenesis and osteogenesis both in mouse bone and in vitro. The mechanistic target of rapamycin complex 1 activates Cxcl9 expression by transcriptional upregulation of STAT1 and increases binding of STAT1 to the Cxcl9 promoter in osteoblasts. These findings reveal the essential role of osteoblast-produced Cxcl9 in angiogenesis and osteogenesis in bone, and Cxcl9 can be targeted to elevate bone angiogenesis and prevent bone loss-related diseases.

Activation of mTORC1 in B Lymphocytes Promotes Osteoclast Formation via Regulation of β-Catenin and RANKL/OPG.

The cytokine receptor activator of nuclear factor‐κB ligand (RANKL) induces osteoclast formation from monocyte/macrophage lineage cells. However, the mechanisms by which RANKL expression is controlled in cells that support osteoclast differentiation are still unclear. We show that deletion of TSC1 (tuberous sclerosis complex 1) in murine B cells causes constitutive activation of mechanistic target of rapamycin complex 1 (mTORC1) and stimulates RANKL but represses osteoprotegerin (OPG) expression and subsequently promotes osteoclast formation and causes osteoporosis in mice. Furthermore, the regulation of RANKL/OPG and stimulation of osteoclastogenesis by mTORC1 was confirmed in a variety of RANKL‐expressing cells and in vivo. Mechanistically, mTORC1 controls RANKL/OPG expression through negative feedback inactivation of Akt, destabilization of β‐catenin mRNA, and downregulation of β‐catenin. Our findings demonstrate that mTORC1 activation‐stimulated RANKL expression in B cells is sufficient to induce bone loss and osteoporosis. The study also established a link between mTORC1 and the RANKL/OPG axis via negative regulation of β‐catenin.

mTORC1 Inhibits NF-κB/NFATc1 Signaling and Prevents Osteoclast Precursor Differentiation, In Vitro and In Mice.

The mechanistic target of rapamycin complex 1 (mTORC1) is a critical sensor for bone homeostasis and bone formation; however, the role of mTORC1 in osteoclast development and the underlying mechanisms have not yet been fully established. Here, we found that mTORC1 activity declined during osteoclast precursors differentiation in vitro and in vivo. We further targeted deletion of Raptor (mTORC1 key component) or Tsc1 (mTORC1 negative regulator) to constitutively inhibit or activate mTORC1 in osteoclast precursors (monocytes/macrophages), using LyzM‐cre mice. Osteoclastic formation was drastically increased in cultures of Raptor deficient bone marrow monocytes/macrophages (BMMs), and Raptor‐deficient mice displayed osteopenia with enhanced osteoclastogenesis. Conversely, BMMs lacking Tsc1 exhibited a severe defect in osteoclast‐like differentiation and absorptive function, both of which were restored following rapamycin treatment. Importantly, expression of nuclear factor κ‐light‐chain‐enhancer of activated B cells (NF‐κB) and nuclear factor of activated T cells, cytoplasmic 1 (NFATc1), transcription factors that are essential for osteoclast differentiation was negatively regulated by mTORC1 in osteoclast lineages. These results provide evidence that mTORC1 plays as a critical role as an osteoclastic differentiation‐limiting signal and suggest a potential drawback in treating bone loss–related diseases with mTOR inhibitors clinically.

Loss of Rictor with aging in osteoblasts promotes age-related bone loss

Osteoblast dysfunction is a major cause of age-related bone loss, but the mechanisms underlying changes in osteoblast function with aging are poorly understood. This study demonstrates that osteoblasts in aged mice exhibit markedly impaired adhesion to the bone formation surface and reduced mineralization in vivo and in vitro. Rictor, a specific component of the mechanistic target of rapamycin complex 2 (mTORC2) that controls cytoskeletal organization and cell survival, is downregulated with aging in osteoblasts. Mechanistically, we found that an increased level of reactive oxygen species with aging stimulates the expression of miR-218, which directly targets Rictor and reduces osteoblast bone surface adhesion and survival, resulting in a decreased number of functional osteoblasts and accelerated bone loss in aged mice. Our findings reveal a novel functional pathway important for age-related bone loss and support for miR-218 and Rictor as potential targets for therapeutic intervention for age-related osteoporosis treatment.

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.

Enhancement of osteogenesis post‐splenectomy does not attenuate bone loss in ovariectomized rats

The roles of different immune cell populations and cytokines in bone metabolism have been extensively investigated. However, the influence of whole immune organ removal on osteopathology remains unknown. In the current study, we investigated the effects of splenectomy on bone metabolism and microarchitecture in rats with or without concurrent ovariectomy. Ovariectomized (OVX) rats were used as osteoporosis model. Sixty 12‐week‐old female rats were randomized into 4 groups (n = 15): sham, splenectomized (SP), ovariectomized, as well as ovariectomized and splenectomized (OVX + SP). Bone microarchitecture was assessed by micro CT analysis at 4 week and 12 week post‐operation, respectively. Bone pathology and metabolism were evaluated via immunohistochemical staining. The serum levels of alkaline phosphatase (ALP), tumor necrosis factor‐alpha (TNF‐α), tartrate‐resistant acid phosphatase 5b (Tracp5b), and C‐terminal telopeptide (CTx) were analyzed at 4 and 12 weeks post‐operation. Removal of the spleen led to alterations in the homeostasis of bone metabolism and increased bone formation in rats. In this study, our findings indicate that the spleen is involved in skeletal metabolism.

Tyrosine kinase Fyn promotes osteoarthritis by activating the β-catenin pathway

Objectives To investigate the role of tyrosine kinase Fyn in the development of osteoarthritis (OA) and the underlying mechanisms, and to define whether targeting Fyn could prevent OA in mice. Methods Cartilage samples from normal and aged mice were analysed with proteome-wide screening. Fyn expression was examined with immunofluorescence in human and age-dependent or experimental mouse OA cartilage samples. Experimental OA in Fyn-knockout mice was induced by destabilisation of the medial meniscus. Primary cultured mouse chondrocytes were treated with proinflammatory cytokine interleukin-1β. The inhibitor of Src kinase family, AZD0530 (saracatinib), and inhibitor of Fyn, PP1, were used to treat experimental OA in mice. Results Fyn expression was markedly upregulated in human OA cartilage and in cartilage from aged mice and those with post-traumatic OA. Fyn accumulates in articular chondrocytes and interacts directly with and phosphorylates β-catenin at Tyr142, which stabilises β-catenin and promotes its nuclear translocation. The deletion of Fyn effectively delayed the development of post-traumatic and age-dependent OA in mice. Fyn inhibitors AZD0530 and PP1 significantly attenuated OA progression by blocking the β-catenin pathway and reducing the levels of extracellular matrix catabolic enzymes in the articular cartilage. Conclusions Fyn accumulates and activates β-catenin signalling in chondrocytes, accelerating the degradation of the articular cartilage and OA development. Targeting Fyn is a novel and potentially therapeutic approach to the treatment of OA.

Inactivation of mTORC1 Signaling in Osterix-Expressing Cells Impairs B-cell Differentiation: mTORC1 IN OSX-EXPRESSING CELLS REGULATES B-CELL DEVELOPMENT

Osteoblasts provide a microenvironmental niche for B‐cell commitment and maturation in the bone marrow (BM). Any abnormity of osteoblasts function may result in the defect of B lymphopoiesis. Signaling from mechanistic target of rapamycin complex 1 (mTORC1) has been implicated in regulating the expansion and differentiation of osteoblasts. Thus, we raise a hypothesis that mTORC1 signaling in osteoblasts plays a vital role in B‐cell development. Inactivation of mTORC1 in osterix‐expressing cells (mainly osteoblast lineage) through Osx‐Cre‐directed deletion of Raptor (an mTORC1‐specific component) resulted in a reduction in the total B‐cell population in the BM, which was due to a block in early B‐cell development from the pro‐B to pre‐B cell stage. Further mechanistic studies revealed that this defect was the result of reduction of interleukin‐7 (IL‐7) expression in osterix‐expressing immature osteoblasts, which caused the abnormality of IL‐7/Stat5 signaling in early B lymphocytes, leading to an increased apoptosis of pre‐B plus immature B cells. In vitro and in vivo studies demonstrated that the addition of exogenous IL‐7 partially restored B lymphopoiesis in the BM of Raptor mutant mice. Furthermore, total BM cells cultured in conditioned media from Raptor null immature osteoblasts or media with anti‐IL‐7 neutralizing antibody failed to differentiate into pre‐B and immature B cells, indicating that inactivation of mTORC1 in immature osteoblast cannot fully support normal B‐cell development. Taken together, these findings demonstrate a novel role for mTORC1 in the regulation of bone marrow environments that support B‐cell differentiation via regulating IL‐7 expression.