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Preosteoblasts are precursor cells that are committed to the osteoblast lineage. Differentiation of these cells to mature osteoblasts is regulated by the extracellular factors and environmental cues. Recent studies have implicated mTOR signaling in the regulation of osteoblast differentiation. However, mTOR exists in two distinct protein complexes (mTORC1 and mTORC2), and the specific role of mTORC1 in regulating the progression of preosteoblasts to mature osteoblastis still unclear. In this study, we first deleted Raptor, a unique and essential component of mTORC1, in primary calvarial cells. Deletion of Raptor resulted in loss of mTORC1 but an increase in mTORC2 signaling without overtly affecting autophagy. Under the osteogenic culture condition, Raptor-deficient cells exhibited a decrease in matrix synthesis and mineralization. qPCR analyses revealed that deletion of Raptor reduced the expression of late-stage markers for osteoblast differentiation (Bglap, Ibsp, and Col1a), while slightly increasing early osteoblast markers (Runx2, Sp7, and Alpl). Consistent with the findings in vitro, genetic ablation of Raptor in osterix-expressing cells led to osteopenia in mice. Together, our findings have identified a specific role for mTORC1 in the transition from preosteoblasts to mature osteoblasts. 相似文献
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Xin Zhou Klaus von der Mark Stephen Henry William Norton Henry Adams Benoit de Crombrugghe 《PLoS genetics》2014,10(12)
One of the crucial steps in endochondral bone formation is the replacement of a cartilage matrix produced by chondrocytes with bone trabeculae made by osteoblasts. However, the precise sources of osteoblasts responsible for trabecular bone formation have not been fully defined. To investigate whether cells derived from hypertrophic chondrocytes contribute to the osteoblast pool in trabecular bones, we genetically labeled either hypertrophic chondrocytes by Col10a1-Cre or chondrocytes by tamoxifen-induced Agc1-CreERT2 using EGFP, LacZ or Tomato expression. Both Cre drivers were specifically active in chondrocytic cells and not in perichondrium, in periosteum or in any of the osteoblast lineage cells. These in vivo experiments allowed us to follow the fate of cells labeled in Col10a1-Cre or Agc1-CreERT2 -expressing chondrocytes. After the labeling of chondrocytes, both during prenatal development and after birth, abundant labeled non-chondrocytic cells were present in the primary spongiosa. These cells were distributed throughout trabeculae surfaces and later were present in the endosteum, and embedded within the bone matrix. Co-expression studies using osteoblast markers indicated that a proportion of the non-chondrocytic cells derived from chondrocytes labeled by Col10a1-Cre or by Agc1-CreERT2 were functional osteoblasts. Hence, our results show that both chondrocytes prior to initial ossification and growth plate chondrocytes before or after birth have the capacity to undergo transdifferentiation to become osteoblasts. The osteoblasts derived from Col10a1-expressing hypertrophic chondrocytes represent about sixty percent of all mature osteoblasts in endochondral bones of one month old mice. A similar process of chondrocyte to osteoblast transdifferentiation was involved during bone fracture healing in adult mice. Thus, in addition to cells in the periosteum chondrocytes represent a major source of osteoblasts contributing to endochondral bone formation in vivo. 相似文献
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Ichiro Kadouchi Kei Sakamoto Liu Tangjiao Takashi Murakami Eiji Kobayashi Yuichi Hoshino Akira Yamaguchi 《Biochemical and biophysical research communications》2009,378(3):600-604
Latexin is the only known carboxypeptidase A inhibitor in mammals. We previously demonstrated that BMP-2 significantly induced latexin expression in Runx2-deficient mesenchymal cells (RD-C6 cells), during chondrocyte and osteoblast differentiation. In this study, we investigated latexin expression in the skeleton and its role in chondrocyte differentiation. Immunohistochemical studies revealed that proliferating and prehypertrophic chondrocytes expressed latexin during skeletogenesis and bone fracture repair. In the early phase of bone fracture, latexin mRNA expression was dramatically upregulated. BMP-2 upregulated the expression of the mRNAs of latexin, Col2a1, and the gene encoding aggrecan (Agc1) in a micromass culture of C3H10T1/2 cells. Overexpression of latexin additively stimulated the BMP-2-induced expression of the mRNAs of Col2a, Agc1, and Col10a1. BMP-2 treatment upregulated Sox9 expression, and Sox9 stimulated the promoter activity of latexin. These results indicate that latexin is involved in BMP-2-induced chondrocyte differentiation and plays an important role in skeletogenesis and skeletal regeneration. 相似文献
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Pharmacological inhibition of S6K1 impairs self‐renewal and osteogenic differentiation of bone marrow stromal cells
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Xiaohui Gu Xuejie Fu Jihang Lu Saijilafu Bin Li Zong‐ping Luo Jianquan Chen 《Journal of cellular biochemistry》2018,119(1):1041-1049
mTORC1 signaling not only plays important physiological roles in the regulation of proliferation and osteogenic differentiation of BMSCs, but also mediates exogenous Wnt‐induced protein anabolism and osteoblast differentiation. However, the downstream effectors of the mTORC1 signaling in the above processes are still poorly understood. In this study, we explored the specific role of S6K1, one of the major targets of the mTORC1 pathway, in BMSCs self ‐ renewal and osteogenic differentiation. We first found that S6K1 was active in primary mouse bone marrow stromal cells, and further activated upon osteogenic induction. We then determined the effects of S6K1 inhibition by LY2584702 Tosylate, a selective inhibitor of S6K1 (hereafter S6KI), using both primary mouse bone marrow stromal cells and ST2 cells. Colony‐Forming Unit‐Fibroblast (CFU‐F) assays showed that S6KI dramatically reduced the total number of colonies formed in primary BMSCs cultures. Under the basal osteogenic culture condition, S6KI significantly inhibited mRNA expression of osteoblast marker genes (Sp7, Bglap, Ibsp, and Col1a1), ALP activity and matrix mineralization. Upon Wnt3a treatments, S6KI inhibited Wnt3a‐induced osteoblast differentiation and expression of protein anabolism genes in ST2 cells, but to a much lesser degree than rapamycin (a specific inhibitor of mTORC1 signaling). Collectively, our findings have demonstrated that pharmacological inhibition of S6K1 impaired self ‐ renewal and osteogenic differentiation of BMSCs, but only partially suppressed exogenous Wnt3a‐induced osteoblast differentiation and protein anabolism. 相似文献
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Katsuyuki Imamura Shingo Maeda Ichiro Kawamura Kanehiro Matsuyama Naohiro Shinohara Yuhei Yahiro Satoshi Nagano Takao Setoguchi Masahiro Yokouchi Yasuhiro Ishidou Setsuro Komiya 《The Journal of biological chemistry》2014,289(14):9865-9879
Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis. 相似文献
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Objectives: Dental follicle cells (DFCs) provide the origin of periodontal tissues, and Runx2 is essential for bone formation and tooth development. In this study, pluripotency of DFCs was evaluated and effects of Runx2 on them were investigated. Materials and methods: The DFCs were induced to differentiate towards osteoblasts, adipocytes or chondrocytes, and alizarin red staining, oil red O staining or alcian blue staining was performed to reveal the differentiated states. Bone marrow stromal cells (BMSCs) and primary mouse fibroblasts served as controls. DFCs were also infected with recombinant retroviruses encoding either full‐length Runx2 or mutant Runx2 without the VWRPY motif. Western blot analysis, real‐time real time RT‐PCR and in vitro mineralization assay were performed to evaluate the effects of full‐length Runx2 or mutant Runx2 on osteogenic/cementogenic differentiation of the cells. Results: The above‐mentioned staining methods demonstrated that DFCs were successfully induced to differentiate towards osteoblasts, adipocytes or chondrocytes respectively, confirming the existence of pluripotent mesenchymal stem cells in dental follicle tissues. However, staining intensity in DFC cultures was weaker than in BMSC cultures. Real‐time PCR analysis indicated that mutant Runx2 induced a more pronounced increase in expression levels of OC, OPN, Col I and CP23 than full‐length Runx2. Mineralization assay also showed that mutant Runx2 increased mineralization nodule formation more prominently than full‐length Runx2. Conclusions: Multipotent DFCs can be induced to differentiate towards osteoblasts, adipocytes or chondrocytes in vitro. Runx2 over‐expression up‐regulated expression levels of osteoblast/cementoblast‐related genes and in vitro enhanced osteogenic differentiation of DFCs. In addition, mutant Runx2‐induced changes in DFCs were more prominent than those induced by full‐length Runx2. 相似文献
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With the establishment of methods that provide evidence for the generation of chondrocyte and osteoblast cell types from ESCs, there is a need for reagents that will enable their further characterization. Here we report on the derivation of chondrocyte and osteoblast reporter ESCs from previously generated and characterized transgenic mouse lines, Collagen type 2 alpha 1(Col2a1)‐ECFP, Bone Sialoprotein (BSP)‐Topaz, and BSP‐Topaz/Dentin Matrix Protein 1 (DMP1)‐Cherry dual reporter mice. Col2a1‐ECFP is highly expressed in chondrocytes, while BSP‐Topaz and DMP1‐Cherry are highly expressed in osteoblasts and osteocytes, respectively. These new skeletal reporter mouse ESC lines will serve as valuable reagents to investigate the functionality of ESC derived chondrocyte and osteoblast cell types. genesis 53:294–298, 2015. © 2015 Wiley Periodicals, Inc. 相似文献
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Insulin‐like growth factor‐1 promotes osteogenic differentiation and collagen I alpha 2 synthesis via induction of mRNA‐binding protein LARP6 expression
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Yue Guo Chen‐Yi Tang Xiao‐Fei Man Hao‐Neng Tang Jun Tang Ci‐La Zhou Shu‐Wen Tan Min Wang Yun‐Zhi Feng Hou‐De Zhou 《Development, growth & differentiation》2017,59(2):94-103
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Wdr5 is developmentally expressed in osteoblasts and is required for osteoblast differentiation. Mice overexpressing Wdr5 under the control of the mouse α(1)I collagen promoter (Col I-Wdr5) display accelerated osteoblast differentiation as well as accelerated chondrocyte differentiation, suggesting that overexpression of Wdr5 in osteoblasts affects chondrocyte differentiation. To elucidate the molecular mechanism by which overexpression of Wdr5 in the perichondrium regulates chondrocyte differentiation, studies were undertaken using skeletal elements and cultured metatarsals isolated from wild-type and Col I-Wdr5 embryos. FGF18 mRNA levels were decreased in Col I-Wdr5 humeri. Furthermore, local delivery of FGF18 to the bone collar of ex vivo cultures of metatarsals attenuated the chondrocyte phenotype of the Col I-Wdr5 metatarsals. Impairing local FGF action in wild-type metatarsals resulted in a chondrocyte phenotype analogous to that of Col I-Wdr5 metatarsals implicating impaired FGF action as the cause of the phenotype observed. The expression of Twist-1, which regulates chondrocyte differentiation, was increased in Col I-Wdr5 humeri. Chromatin immunoprecipitation analyses demonstrated that Wdr5 is recruited to the Twist-1 promoter. These findings support a model in which overexpression of Wdr5 in the perichondrium promotes chondrocyte differentiation by modulating the expression of Twist-1 and FGF18. 相似文献
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Wu‐Xia Qiu Xiao‐Li Ma Xiao Lin Fan Zhao Di‐Jie Li Zhi‐Hao Chen Ke‐Wen Zhang Ru Zhang Pai Wang Yun‐Yun Xiao Zhi‐Ping Miao Kai Dang Xiao‐Yang Wu Ai‐Rong Qian 《Journal of cellular and molecular medicine》2020,24(1):317-327
Microtubule actin cross‐linking factor 1 (Macf1) is a spectraplakin family member known to regulate cytoskeletal dynamics, cell migration, neuronal growth and cell signal transduction. We previously demonstrated that knockdown of Macf1 inhibited the differentiation of MC3T3‐E1 cell line. However, whether Macf1 could regulate bone formation in vivo is unclear. To study the function and mechanism of Macf1 in bone formation and osteogenic differentiation, we established osteoblast‐specific Osterix (Osx) promoter‐driven Macf1 conditional knockout mice (Macf1f/fOsx‐Cre). The Macf1f/fOsx‐Cre mice displayed delayed ossification and decreased bone mass. Morphological and mechanical studies showed deteriorated trabecular microarchitecture and impaired biomechanical strength of femur in Macf1f/fOsx‐Cre mice. In addition, the differentiation of primary osteoblasts isolated from calvaria was inhibited in Macf1f/fOsx‐Cre mice. Deficiency of Macf1 in primary osteoblasts inhibited the expression of osteogenic marker genes (Col1, Runx2 and Alp) and the number of mineralized nodules. Furthermore, deficiency of Macf1 attenuated Bmp2/Smad/Runx2 signalling in primary osteoblasts of Macf1f/fOsx‐Cre mice. Together, these results indicated that Macf1 plays a significant role in bone formation and osteoblast differentiation by regulating Bmp2/Smad/Runx2 pathway, suggesting that Macf1 might be a therapeutic target for bone disease. 相似文献
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Akifumi Yamada Takashi Yokoo Shinya Yokote Shuichiro Yamanaka Luna Izuhara Yuichi Katsuoka Yohta Shimada Akinori Shukuya Hirotaka James Okano Toya Ohashi Hiroyuki Ida 《Human cell》2014,27(2):59-67
We previously showed that mesenchymal stem cells (MSCs) can differentiate into a functional miniature kidney, suggesting that MSCs may be a cell source for kidney regeneration. However, MSCs from long-term dialysis patients, which have been exposed to uremic toxin, can exhibit reduced viability. Therefore, the aim of this study was to examine the gene expression profiles and differentiation capabilities of bone marrow- and adipose-derived MSCs from chronic kidney disease (CKD) model rats. CKD was induced in rats by adenine feeding, and then MSCs were isolated from bone marrow (BMSCs) and adipose tissue (ASCs). After confirming MSC surface marker expression, comprehensive gene expression profiles were obtained by RT-PCR array. MSCs were differentiated into adipocytes, osteoblasts, and chondrocytes, and histological and/or functional assays were performed. Tgfb3 expression was up-regulated, while Bmp6, Gdf15, Mmp2, and Vegfa were down-regulated in CKD-ASCs compared with Control-ASCs. There were no significant differences in the gene expression of stemness markers, and the morphology of cells that underwent adipogenesis, osteogenesis, and chondrogenesis, or GPDH activity between CKD and control groups. Comparing BMSCs with ASCs, gene expression of Bglap, Bmp4, Igf1, Itgax, Pparg, Ptprc, and Tnf were up-regulated, while Col1a1, Mmp2, Sox9, and Vegfa were down-regulated in both CKD and control groups. Uremic toxin in CKD rats had a small effect on the gene expression and differentiation of MSCs. However, long-term exposure to uremic toxin and the differences in gene expression of MSCs derived from bone marrow or adipose tissue may affect renal regeneration. 相似文献