RESUMEN
The mechanisms by which the sensitivity of naive CD4+ T cells to stimulation by the cognate antigen via the T cell antigen receptor (TCR) determines their differentiation into distinct helper T cell subsets remain elusive. Here we demonstrate functional collaboration of the ubiquitin E3 ligases Itch and WWP2 in regulating the strength of the TCR signal. Mice lacking both Itch and WWP2 in T cells showed spontaneous autoimmunity and lung inflammation. CD4+ T cells deficient in Itch and WWP2 exhibited hypo-responsiveness to TCR stimulation and a bias toward differentiation into the TH2 subset of helper T cells. Itch and WWP2 formed a complex and cooperated to enhance TCR-proximal signaling by catalyzing the conjugation of atypical ubiquitin chains to the phosphatase SHP-1 and reducing the association of SHP-1 with the tyrosine kinase Lck. These findings indicate that targeted ubiquitination regulates the strength of the TCR signal and differentiation toward the TH2 lineage.
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Receptores de Antígenos de Linfocitos T/metabolismo , Células Th2/inmunología , Ubiquitina-Proteína Ligasas/fisiología , Animales , Autoinmunidad , Diferenciación Celular , Humanos , Inflamación/genética , Células Jurkat , Proteína Tirosina Quinasa p56(lck) Específica de Linfocito/metabolismo , Ratones , Ratones Noqueados , Proteína Tirosina Fosfatasa no Receptora Tipo 6/metabolismo , Proteínas Represoras/metabolismo , Transducción de Señal , Células Th2/enzimología , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Vertebral bone is subject to a distinct set of disease processes from long bones, including a much higher rate of solid tumour metastases1-4. The basis for this distinct biology of vertebral bone has so far remained unknown. Here we identify a vertebral skeletal stem cell (vSSC) that co-expresses ZIC1 and PAX1 together with additional cell surface markers. vSSCs display formal evidence of stemness, including self-renewal, label retention and sitting at the apex of their differentiation hierarchy. vSSCs are physiologic mediators of vertebral bone formation, as genetic blockade of the ability of vSSCs to generate osteoblasts results in defects in the vertebral neural arch and body. Human counterparts of vSSCs can be identified in vertebral endplate specimens and display a conserved differentiation hierarchy and stemness features. Multiple lines of evidence indicate that vSSCs contribute to the high rates of vertebral metastatic tropism observed in breast cancer, owing in part to increased secretion of the novel metastatic trophic factor MFGE8. Together, our results indicate that vSSCs are distinct from other skeletal stem cells and mediate the unique physiology and pathology of vertebrae, including contributing to the high rate of vertebral metastasis.
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Neoplasias de la Mama , Linaje de la Célula , Metástasis de la Neoplasia , Columna Vertebral , Células Madre , Humanos , Neoplasias de la Mama/patología , Diferenciación Celular , Autorrenovación de las Células , Metástasis de la Neoplasia/patología , Osteoblastos/citología , Osteoblastos/patología , Columna Vertebral/citología , Columna Vertebral/patología , Células Madre/citología , Células Madre/metabolismo , Células Madre/patología , BiomarcadoresRESUMEN
Mutagenic screening is powerful for identifying key genes involved in developmental processes. However, such screens are successful only in lower organisms. Here, we develop a targeted genetic screening approach in mice through combining androgenetic haploid embryonic stem cells (AG-haESCs) and clustered regularly interspaced palindromic repeats/CRISPR-associated protein 9 (CRISPR-Cas9) technology. We produced a mutant semi-cloned (SC) mice pool by oocyte injection of AG-haESCs carrying constitutively expressed Cas9 and an single guide RNA (sgRNA) library targeting 72 preselected genes in one step and screened for bone-development-related genes through skeletal analysis at birth. This yielded 4 genes: Zic1 and Clec11a, which are required for bone development, and Rln1 and Irx5, which had not been previously considered. Whereas Rln1-/- mice exhibited small skeletal size only at birth, Irx5-/- mice showed skeletal abnormalities both in postnatal and adult phases due to decreased bone mass and increased bone marrow adipogenesis. Mechanistically, iroquois homeobox 5 (IRX5) promotes osteoblastogenesis and inhibits adipogenesis by suppressing peroxisome proliferator activated receptor γ (PPARγ) activation. Thus, AG-haESC-mediated functional mutagenic screening opens new avenues for genetic interrogation of developmental processes in mice.
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Desarrollo Óseo/genética , Regulación del Desarrollo de la Expresión Génica , Marcación de Gen/métodos , Pruebas Genéticas/métodos , Células Madre Embrionarias de Ratones/metabolismo , Animales , Sistemas CRISPR-Cas , Células Cultivadas , Haploidia , Factores de Crecimiento de Célula Hematopoyética/genética , Factores de Crecimiento de Célula Hematopoyética/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Ratones , Ratones Noqueados , Relaxina/genética , Relaxina/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
During the aging process, bone marrow mesenchymal stem cells (BMSCs) exhibit declined osteogenesis accompanied by excess adipogenesis, which will lead to osteoporosis. Here, we report that the H3 lysine 36 trimethylation (H3K36me3), catalyzed by histone methyltransferase SET-domain-containing 2 (SETD2), regulates lineage commitment of BMSCs. Deletion of Setd2 in mouse bone marrow mesenchymal stem cells (mBMSCs), through conditional Cre expression driven by Prx1 promoter, resulted in bone loss and marrow adiposity. Loss of Setd2 in BMSCs in vitro facilitated differentiation propensity to adipocytes rather than to osteoblasts. Through conjoint analysis of RNA sequencing (RNA-seq) and chromatin immunoprecipitation sequencing (ChIP-seq) data, we identified a SETD2 functional target gene, Lbp, on which H3K36me3 was enriched, and its expression was affected by Setd2 deficiency. Furthermore, overexpression of lipopolysaccharide-binding protein (LBP) could partially rescue the lack of osteogenesis and enhanced adipogenesis resulting from the absence of Setd2 in BMSCs. Further mechanistic studies demonstrated that the trimethylation level of H3K36 could regulate Lbp transcriptional initiation and elongation. These findings suggest that H3K36me3 mediated by SETD2 could regulate the cell fate of mesenchymal stem cells (MSCs) in vitro and in vivo, indicating that the regulation of H3K36me3 level by targeting SETD2 and/or the administration of downstream LBP may represent a potential therapeutic way for new treatment in metabolic bone diseases, such as osteoporosis.
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Células de la Médula Ósea/fisiología , N-Metiltransferasa de Histona-Lisina/fisiología , Histonas/genética , Proteínas de Fase Aguda/genética , Proteínas de Fase Aguda/fisiología , Adipocitos/citología , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/fisiología , Diferenciación Celular/genética , Metilación de ADN/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Lisina/metabolismo , Masculino , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/fisiología , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Endogámicos C57BL , Regiones Promotoras Genéticas/genéticaRESUMEN
The transcription factor signal transducer and activator of transcription 3 (STAT3) plays a central role in cell survival and function. STAT3 has been demonstrated to participate in the maintenance of bone homeostasis in osteoblasts, but its role in osteoclasts in vivo remains poorly defined. Here, we generated a conditional knockout mouse model in which Stat3 was deleted in osteoclasts using a cathepsin K-Cre (Ctsk-Cre) driver. We observed that osteoclast-specific Stat3 deficiency caused increased bone mass in mice, which we attributed to impaired bone catabolism by osteoclasts. Stat3-deficient bone marrow macrophages (BMMs) showed decreased expression of nuclear factor of activated T cells, cytoplasm 1 (NFATc1), and reduced osteoclast differentiation determined by decreases in osteoclast number, tartrate-resistant acid phosphatase activity, and expression of osteoclast marker genes. Enforced expression of NFATc1 in Stat3-deficient BMMs rescued the impaired osteoclast differentiation. Mechanistically, we revealed that STAT3 could drive the transcription of NFATc1 by binding to its promoter. Furthermore, preventing STAT3 activation by using an inhibitor of upstream phosphorylases, AG490, also impaired osteoclast differentiation and formation in a similar way as gene deletion of Stat3 In summary, our data provide the first evidence that STAT3 is significant in osteoclast differentiation and bone homeostasis in vivo, and it may be identified as a potential pharmacological target for the treatment of bone metabolic diseases through regulation of osteoclast activity.
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Huesos/metabolismo , Factores de Transcripción NFATC/metabolismo , Osteoclastos/metabolismo , Osteogénesis , Factor de Transcripción STAT3/metabolismo , Animales , Femenino , Regulación de la Expresión Génica , Homeostasis , Humanos , Macrófagos/citología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados , Factores de Transcripción NFATC/genética , Osteoclastos/citología , Factor de Transcripción STAT3/genética , Transducción de SeñalRESUMEN
The tumor suppressor phosphatase and tensin homolog (PTEN) plays a central role in regulating phosphatidylinositol 3-kinase (PI3K) signaling, and its gene is very frequently mutated in various human cancers. Numerous studies have revealed that PTEN levels are tightly regulated by both transcriptional and posttranslational modifications, with especially ubiquitylation significantly regulating PTEN protein levels. Although several ubiquitin ligases have been reported to mediate PTEN ubiquitylation in vitro, the ubiquitin ligase that promotes PTEN degradation in vivo has not been reported. Here we took advantage of specific knockout mouse models to demonstrate that WW domain-containing E3 ubiquitin protein ligase 2 (WWP2) promotes PTEN degradation under physiological conditions, whereas another ubiquitin ligase, carboxyl terminus of Hsp70-interacting protein (CHIP), had no such effect. WWP2 knockout mice exhibited reduced body size, elevated PTEN protein levels, and reduced phosphorylation levels of the serine/threonine kinase and PTEN target AKT. In contrast, we observed no elevation of PTEN protein levels in CHIP knockout tissues and mouse embryonic fibroblasts. Furthermore, PTEN protein levels in CHIP/WWP2 double knockout mice were very similar to those in WWP2 single knockout mice and significantly higher than in WT and CHIP knockout mice. Our results demonstrate that WWP2, rather than CHIP, is an ubiquitin ligase that promotes PTEN degradation in vivo Considering PTEN's significant role in tumor development, we propose that WWP2 may be a potential target for fine-tuning PTEN levels in anticancer therapies.
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Fosfohidrolasa PTEN/metabolismo , Proteolisis , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Fosfohidrolasa PTEN/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
The APC/Cdh1 E3 ubiquitin ligase plays an essential role in both mitotic exit and G1/S transition by targeting key cell-cycle regulators for destruction. There is mounting evidence indicating that Cdh1 has other functions in addition to cell-cycle regulation. However, it remains unclear whether these additional functions depend on its E3 ligase activity. Here, we report that Cdh1, but not Cdc20, promotes the E3 ligase activity of Smurf1. This is mediated by disruption of an autoinhibitory Smurf1 homodimer and is independent of APC/Cdh1 E3 ligase activity. As a result, depletion of Cdh1 leads to reduced Smurf1 activity and subsequent activation of multiple downstream targets, including the MEKK2 signaling pathway, inducing osteoblast differentiation. Our studies uncover a cell-cycle-independent function of Cdh1, establishing Cdh1 as an upstream component that governs Smurf1 activity. They further suggest that modulation of Cdh1 is a potential therapeutic option for treatment of osteoporosis.
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Cadherinas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Osteoblastos/metabolismo , Complejos de Ubiquitina-Proteína Ligasa/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Ciclosoma-Complejo Promotor de la Anafase , Animales , Antígenos CD , Proteínas Cdh1 , Diferenciación Celular , Humanos , MAP Quinasa Quinasa Quinasa 2/metabolismo , Sistema de Señalización de MAP Quinasas , Ratones , Osteoblastos/citología , Unión Proteica , Multimerización de Proteína , Ubiquitina-Proteína Ligasas/antagonistas & inhibidores , Ubiquitina-Proteína Ligasas/química , UbiquitinaciónRESUMEN
The functional changes of nucleus pulposus (NP) cells are considered to be the initiating factors of intervertebral disc degeneration (IDD), and the differentially expressed circRNAs in NP cells may play an important role in the process of IDD. To identify circular RNAs (circRNAs) associated with human IDD, we isolated the NP cells from human degenerated and non-degenerated intervertebral disc and identified NP cells by microscopy and cell proliferation. CircRNA microarray expression profiles were obtained from NP cells of degenerated and non-degenerated intervertebral disc and further validated by quantitative reverse transcription PCR (qRT-PCR). The expression data were analyzed by bioinformatics. Microarray analysis identified 7294 circRNAs differentially expressed in degenerated human IDD NP cells. Among them, 3724 circRNAs were up-regulated and 3570 circRNAs were down-regulated by more than 2 folds. After validating by qRT-PCR, we predicted the possible miRNAs of the top dysregulated circRNAs using TargetScan, and miRanda. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis showed that the most modulated circRNAs regulate the viability, degradation, apoptosis and oxidative stress in NP cells, and the possible mechanism underlying IDD was discussed. These results revealed that circRNAs may play a role in IDD and might be a promising candidate molecular target for gene therapy.
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Biología Computacional/métodos , Perfilación de la Expresión Génica/métodos , Degeneración del Disco Intervertebral/genética , Núcleo Pulposo/metabolismo , ARN Circular/genética , Adulto , Apoptosis/genética , Supervivencia Celular/genética , Células Cultivadas , Femenino , Ontología de Genes , Humanos , Disco Intervertebral/metabolismo , Disco Intervertebral/patología , Degeneración del Disco Intervertebral/patología , Masculino , MicroARNs/genética , Núcleo Pulposo/patologíaRESUMEN
Mammalian target of rapamycin complex 1 (mTORC1) is involved in anabolic metabolism in both osteoblasts and chondrocytes, but the role of mTORC1 in osteoclast biology in vivo remains to be elucidated. In this study, we showed that deletion of regulatory-associated protein of mTOR (Raptor) in osteoclasts led to an increase in bone mass with decreased bone resorption. Raptor-deficient bone marrow-derived macrophages exhibited lower mTORC1-S6K1 signaling and retarded osteoclast differentiation, as determined by the number of osteoclasts, tartrate-resistant acid phosphatase activity, and expression of osteoclast-specific genes. Enforced expression of constitutively active S6K1 rescued the impaired osteoclast differentiation in Raptor-deficient bone marrow-derived macrophages. Furthermore, pharmacological inhibition of mTORC1 signaling by rapamycin could also inhibit osteoclast differentiation and osteoclast-specific gene expression. Taken together, our findings demonstrate that mTORC1 plays a key role in the network of catabolic bone resorption in osteoclasts and may serve as a potential pharmacological target for the regulation of osteoclast activity in bone metabolic disorders.
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Proteínas Adaptadoras Transductoras de Señales/antagonistas & inhibidores , Huesos/patología , Diferenciación Celular , Complejos Multiproteicos/antagonistas & inhibidores , Osteoclastos/patología , Osteogénesis/fisiología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Proteínas Adaptadoras Transductoras de Señales/fisiología , Animales , Densidad Ósea , Huesos/metabolismo , Células Cultivadas , Macrófagos/metabolismo , Macrófagos/patología , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Ratones Noqueados , Complejos Multiproteicos/genética , Complejos Multiproteicos/metabolismo , Osteoclastos/metabolismo , Fosforilación , Proteína Reguladora Asociada a mTOR , Serina-Treonina Quinasas TOR/genética , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Small molecule inhibitors for bromodomain and extra-terminal (BET) proteins have recently emerged as potential therapeutic agents in clinical trials for various cancers. However, to date, it is unknown whether these inhibitors have side effects on bone structures. Here, we report that inhibition of BET bromodomain proteins may suppress chondrocyte differentiation and restrain bone growth. We generated a luciferase reporter system using the chondrogenic cell line ATDC5 in which the luciferase gene was driven by the promoter of Col2a1, an elementary collagen of the chondrocyte. The Col2a1-luciferase ATDC5 system was used for rapidly screening both activators and repressors of human collagen Col2a1 gene expression, and we found that BET bromodomain inhibitors reduce the Col2a1-luciferase. Consistent with the luciferase assay, BET inhibitors decrease the expression of Col2a1 Furthermore, we constructed a zebrafish line in which the enhanced green fluorescent protein (EGFP) expression was driven by col2a1 promoter. The transgenic (col2a1-EGFP) zebrafish line demonstrated that BET inhibitors I-BET151 and (+)-JQ1 may affect EGFP expression in zebrafish. Furthermore, we found that I-BET151 and (+)-JQ1 may affect chondrocyte differentiation in vitro and inhibit zebrafish growth in vivo Mechanistic analysis revealed that BET inhibitors influenced the depletion of RNA polymerase II from the Col2a1 promoter. Collectively, these results suggest that BET bromodomain inhibition may have side effects on skeletal bone structures.
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Desarrollo Óseo/efectos de los fármacos , Diferenciación Celular/efectos de los fármacos , Condrogénesis/efectos de los fármacos , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas de Unión al ARN/antagonistas & inhibidores , Factores de Transcripción/antagonistas & inhibidores , Animales , Proteínas de Ciclo Celular , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Condrocitos/citología , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Colágeno Tipo II/metabolismo , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Humanos , Ratones , Familia de Multigenes , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Bibliotecas de Moléculas Pequeñas/farmacología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Pez Cebra/crecimiento & desarrollo , Pez Cebra/metabolismoRESUMEN
Bone is regarded as one of few tissues that heals without fibrous scar. The outer layer of the periosteum is covered with fibrous tissue, whose function in bone formation is unknown. We herein developed a system to distinguish the fate of fibrous-layer periosteal cells (FL-PCs) from the skeletal stem/progenitor cells (SSPCs) in the cambium-layer periosteum and bone marrow in mice. We showed that FL-PCs did not participate in steady-state osteogenesis, but formed the main body of fibrocartilaginous callus during fracture healing. Moreover, FL-PCs invaded the cambium-layer periosteum and bone marrow after fracture, forming neo-SSPCs that continued to maintain the healed bones throughout adulthood. The FL-PC-derived neo-SSPCs expressed lower levels of osteogenic signature genes and displayed lower osteogenic differentiation activity than the preexisting SSPCs. Consistent with this, healed bones were thinner and formed more slowly than normal bones. Thus, the fibrous periosteum becomes the cellular origin of bones after fracture and alters bone properties permanently.
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Diferenciación Celular , Curación de Fractura , Fracturas Óseas , Osteogénesis , Periostio , Animales , Periostio/metabolismo , Ratones , Osteogénesis/fisiología , Curación de Fractura/fisiología , Fracturas Óseas/patología , Fracturas Óseas/metabolismo , Células Madre/metabolismo , Células Madre/citología , Ratones Endogámicos C57BL , Callo Óseo/metabolismo , Callo Óseo/patología , MasculinoRESUMEN
Ferroptosis is a necrotic form of iron-dependent regulatory cell death. Estrogen withdrawal can interfere with iron metabolism, which is responsible for the pathogenesis of postmenopausal osteoporosis (PMOP). Here, it is demonstrated that estrogen withdrawal induces iron accumulation in the skeleton and the ferroptosis of osteocytes, leading to reduced bone mineral density. Furthermore, the facilitatory effect of ferroptosis of osteocytes is verified in the occurrence and development of postmenopausal osteoporosis is associated with over activated osteoclastogenesis using a direct osteocyte/osteoclast coculture system and glutathione peroxidase 4 (GPX4) knockout ovariectomized mice. In addition, the nuclear factor erythroid derived 2-related factor-2 (Nrf2) signaling pathway is confirmed to be a crucial factor in the ferroptosis of osteocytic cells. Nrf2 regulates the expression of nuclear factor kappa-B ligand (RANKL) by regulating the DNA methylation level of the RANKL promoter mediated by DNA methyltransferase 3a (Dnmt3a), which is as an important mechanism in osteocytic ferroptosis-mediated osteoclastogenesis. Taken together, this data suggests that osteocytic ferroptosis is involved in PMOP and can be targeted to tune bone homeostasis.
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Ferroptosis , Osteoporosis Posmenopáusica , Ratones , Humanos , Animales , Femenino , Osteocitos/metabolismo , Osteoporosis Posmenopáusica/metabolismo , Factor 2 Relacionado con NF-E2/metabolismo , Estrógenos/metabolismo , Hierro/metabolismoRESUMEN
Osteoarthritis (OA) is one of the most common joint diseases, there are no effective disease-modifying drugs, and the pathological mechanisms of OA need further investigation. Here, we show that H3K36 methylations were decreased in senescent chondrocytes and age-related osteoarthritic cartilage. Prrx1-Cre inducible H3.3K36M transgenic mice showed articular cartilage destruction and osteophyte formation. Conditional knockout Nsd1Prrx1-Cre mice, but not Nsd2Prrx1-Cre or Setd2Prrx1-Cre mice, replicated the phenotype of K36M/+; Prrx1-Cre mice. Immunostaining results showed decreased anabolic and increased catabolic activities in Nsd1Prrx1-Cre mice, along with decreased chondrogenic differentiation. Transcriptome and ChIP-seq data revealed that Osr2 was a key factor affected by Nsd1. Intra-articular delivery of Osr2 adenovirus effectively improved the homeostasis of articular cartilage in Nsd1Prrx1-Cre mice. In human osteoarthritic cartilages, both mRNA and protein levels of NSD1 and OSR2 were decreased. Our results indicate that NSD1-induced H3K36 methylations and OSR2 expression play important roles in articular cartilage homeostasis and OA. Targeting H3K36 methylation and OSR2 would be a novel strategy for OA treatment.
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Cartílago Articular , Osteoartritis , Ratones , Humanos , Animales , Condrocitos/metabolismo , Metiltransferasas/metabolismo , Osteoartritis/patología , Cartílago Articular/metabolismo , Cartílago Articular/patología , Ratones Transgénicos , Homeostasis , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismoRESUMEN
Bone secretory proteins, termed osteokines, regulate bone metabolism and whole-body homeostasis. However, fundamental questions as to what the bona fide osteokines and their cellular sources are and how they are regulated remain unclear. In this study, we analyzed bone and extraskeletal tissues, osteoblast (OB) conditioned media, bone marrow supernatant (BMS), and serum, for basal osteokines and those responsive to aging and mechanical loading/unloading. We identified 375 candidate osteokines and their changes in response to aging and mechanical dynamics by integrating data from RNA-seq, scRNA-seq, and proteomic approaches. Furthermore, we analyzed their cellular sources in the bone and inter-organ communication facilitated by them (bone-brain, liver, and aorta). Notably, we discovered that senescent OBs secrete fatty-acid-binding protein 3 to propagate senescence toward vascular smooth muscle cells (VSMCs). Taken together, we identified previously unknown candidate osteokines and established a dynamic regulatory network among them, thus providing valuable resources to further investigate their systemic roles.
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Osteoblastos , Animales , Osteoblastos/metabolismo , Osteoblastos/citología , Ratones , Huesos/metabolismo , Proteómica , Ratones Endogámicos C57BL , Masculino , Envejecimiento/metabolismo , Humanos , Senescencia Celular , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/citología , MultiómicaRESUMEN
The periosteum contains skeletal stem/progenitor cells that contribute to bone fracture healing. However, the in vivo identity of periosteal skeletal stem cells (P-SSCs) remains unclear, and membrane protein markers of P-SSCs that facilitate tissue engineering are needed. Here, we identified integral membrane protein 2A (Itm2a) enriched in SSCs using single-cell transcriptomics. Itm2a+ P-SSCs displayed clonal multipotency and self-renewal and sat at the apex of their differentiation hierarchy. Lineage-tracing experiments showed that Itm2a selectively labeled the periosteum and that Itm2a+ cells were preferentially located in the outer fibrous layer of the periosteum. The Itm2a+ cells rarely expressed CD34 or Osx, but expressed periosteal markers such as Ctsk, CD51, PDGFRA, Sca1, and Gli1. Itm2a+ P-SSCs contributed to osteoblasts, chondrocytes, and marrow stromal cells upon injury. Genetic lineage tracing using dual recombinases showed that Itm2a and Prrx1 lineage cells generated spatially separated subsets of chondrocytes and osteoblasts during fracture healing. Bone morphogenetic protein 2 (Bmp2) deficiency or ablation of Itm2a+ P-SSCs resulted in defects in fracture healing. ITM2A+ P-SSCs were also present in the human periosteum. Thus, our study identified a membrane protein marker that labels P-SSCs, providing an attractive target for drug and cellular therapy for skeletal disorders.
Asunto(s)
Curación de Fractura , Proteínas de la Membrana , Periostio , Animales , Periostio/metabolismo , Periostio/citología , Ratones , Curación de Fractura/genética , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Humanos , Células Madre/metabolismo , Células Madre/citología , Proteína Morfogenética Ósea 2/metabolismo , Proteína Morfogenética Ósea 2/genética , Fracturas Óseas/patología , Fracturas Óseas/metabolismo , Fracturas Óseas/terapia , Fracturas Óseas/genética , Osteoblastos/metabolismo , Osteoblastos/citología , Diferenciación Celular , Condrocitos/metabolismo , Condrocitos/citología , Masculino , Linaje de la CélulaRESUMEN
BACKGROUND: Megakaryocytes (MKs) are polyploid cells responsible for producing â¼1011 platelets daily in humans. Unraveling the mechanisms regulating megakaryopoiesis holds the promise for the production of clinical-grade platelets from stem cells, overcoming significant current limitations in platelet transfusion medicine. Previous work identified that loss of the epigenetic regulator SET domain containing 2 (SETD2) was associated with an increased platelet count in mice. However, the role of SETD2 in megakaryopoiesis remains unknown. OBJECTIVES: Here, we examined how SETD2 regulated MK development and platelet production using complementary murine and human systems. METHODS: We manipulated the expression of SETD2 in multiple in vitro and ex vivo models to assess the ploidy of MKs and the function of platelets. RESULTS: The genetic ablation of Setd2 increased the number of high-ploidy bone marrow MKs. Peripheral platelet counts in Setd2 knockout mice were significantly increased â¼2-fold, and platelets exhibited normal size, morphology, and function. By knocking down and overexpressing SETD2 in ex vivo human cell systems, we demonstrated that SETD2 negatively regulated MK polyploidization by controlling methylation of α-tubulin, microtubule polymerization, and MK nuclear division. Small-molecule inactivation of SETD2 significantly increased the production of high-ploidy MKs and platelets from human-induced pluripotent stem cells and cord blood CD34+ cells. CONCLUSION: These findings identify a previously unrecognized role for SETD2 in regulating megakaryopoiesis and highlight the potential of targeting SETD2 to increase platelet production from human cells for transfusion practices.
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Plaquetas , N-Metiltransferasa de Histona-Lisina , Megacariocitos , Ratones Noqueados , Poliploidía , Trombopoyesis , Tubulina (Proteína) , Megacariocitos/metabolismo , Megacariocitos/citología , Animales , Plaquetas/metabolismo , Humanos , Trombopoyesis/genética , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/genética , Metilación , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Ratones Endogámicos C57BL , Ratones , Recuento de PlaquetasRESUMEN
Heterotopic ossification (HO) comprises the abnormal formation of ectopic bone in extraskeletal soft tissue. The factors that initiate HO remain elusive. Herein, we found that calcified apoptotic vesicles (apoVs) led to increased calcification and stiffness of tendon extracellular matrix (ECM), which initiated M2 macrophage polarization and HO progression. Specifically, single-cell transcriptome analyses of different stages of HO revealed that calcified apoVs were primarily secreted by a PROCR+ fibroblast population. In addition, calcified apoVs enriched calcium by annexin channels, absorbed to collagen I via electrostatic interaction, and aggregated to produce calcifying nodules in the ECM, leading to tendon calcification and stiffening. More importantly, apoV-releasing inhibition or macrophage deletion both successfully reversed HO development. Thus, we are the first to identify calcified apoVs from PROCR+ fibroblasts as the initiating factor of HO, and might serve as the therapeutic target for inhibiting pathological calcification.
Asunto(s)
Vesículas Extracelulares , Osificación Heterotópica , Humanos , Receptor de Proteína C Endotelial , Vesículas Extracelulares/patología , Osificación Heterotópica/patología , Osificación Heterotópica/terapia , Matriz Extracelular , FibroblastosRESUMEN
Tibial cortex transverse distraction is a surgical method for treating severe diabetic foot ulcers (DFUs), but the underlying mechanism is unclear. We show that antioxidant proteins and small extracellular vesicles (sEVs) with multiple-tissue regenerative potential are released during bone transport (BT) in humans and rats. These vesicles accumulate in diabetic wounds and are enriched with microRNAs (miRNAs) (e.g., miR-494-3p) that have high regenerative activities that improve the circulation of ischemic lower limbs while also promoting neovascularization, fibroblast migration, and nerve fiber regeneration. Deletion of miR-494-3p in rats reduces the beneficial effects of BT on diabetic wounds, while hydrogels containing miR-494-3p and reduced glutathione (GSH) effectively repair them. Importantly, the ginsenoside Rg1 can upregulate miR-494-3p, and a randomized controlled trial verifies that the regimen of oral Rg1 and GSH accelerates wound healing in refractory DFU patients. These findings identify potential functional factors for tissue regeneration and suggest a potential therapy for DFUs.
Asunto(s)
Cicatrización de Heridas , Animales , Cicatrización de Heridas/efectos de los fármacos , Humanos , Ratas , Masculino , MicroARNs/metabolismo , MicroARNs/genética , Vesículas Extracelulares/metabolismo , Ratas Sprague-Dawley , Pie Diabético/metabolismo , Pie Diabético/patología , Diabetes Mellitus Experimental/metabolismo , Glutatión/metabolismo , Persona de Mediana Edad , Regeneración/efectos de los fármacos , Femenino , Huesos/metabolismoRESUMEN
Transcortical vessels (TCVs) provide effective communication between bone marrow vascular system and external circulation. Although osteocytes are in close contact with them, it is not clear whether osteocytes regulate the homeostasis of TCVs. Here, we show that osteocytes maintain the normal network of TCVs by transferring mitochondria to the endothelial cells of TCV. Partial ablation of osteocytes causes TCV regression. Inhibition of mitochondrial transfer by conditional knockout of Rhot1 in osteocytes also leads to regression of the TCV network. By contrast, acquisition of osteocyte mitochondria by endothelial cells efficiently restores endothelial dysfunction. Administration of osteocyte mitochondria resultes in acceleration of the angiogenesis and healing of the cortical bone defect. Our results provide new insights into osteocyte-TCV interactions and inspire the potential application of mitochondrial therapy for bone-related diseases.
Asunto(s)
Angiogénesis , Osteocitos , Osteocitos/metabolismo , Células Endoteliales , Huesos , MitocondriasRESUMEN
TGFbeta activated kinase 1 (TAK1), a member of the MAPKKK family, controls diverse functions ranging from innate and adaptive immune system activation to vascular development and apoptosis. To analyse the in vivo function of TAK1 in cartilage, we generated mice with a conditional deletion of Tak1 driven by the collagen 2 promoter. Tak1(col2) mice displayed severe chondrodysplasia with runting, impaired formation of secondary centres of ossification, and joint abnormalities including elbow dislocation and tarsal fusion. This phenotype resembled that of bone morphogenetic protein receptor (BMPR)1 and Gdf5-deficient mice. BMPR signalling was markedly impaired in TAK1-deficient chondrocytes as evidenced by reduced expression of known BMP target genes as well as reduced phosphorylation of Smad1/5/8 and p38/Jnk/Erk MAP kinases. TAK1 mediates Smad1 phosphorylation at C-terminal serine residues. These findings provide the first in vivo evidence in a mammalian system that TAK1 is required for BMP signalling and functions as an upstream activating kinase for Smad1/5/8 in addition to its known role in regulating MAP kinase pathways. Our experiments reveal an essential role for TAK1 in the morphogenesis, growth, and maintenance of cartilage.