Dataset Information

Notch signaling maintains bone marrow mesenchymal progenitors by suppressing osteoblast differentiation.

ABSTRACT: Postnatal bone marrow houses mesenchymal progenitor cells that are osteoblast precursors. These cells have established therapeutic potential, but they are difficult to maintain and expand in vitro, presumably because little is known about the mechanisms controlling their fate decisions. To investigate the potential role of Notch signaling in osteoblastogenesis, we used conditional alleles to genetically remove components of the Notch signaling system during skeletal development. We found that disruption of Notch signaling in the limb skeletogenic mesenchyme markedly increased trabecular bone mass in adolescent mice. Notably, mesenchymal progenitors were undetectable in the bone marrow of mice with high bone mass. As a result, these mice developed severe osteopenia as they aged. Moreover, Notch signaling seemed to inhibit osteoblast differentiation through Hes or Hey proteins, which diminished Runx2 transcriptional activity via physical interaction. These results support a model wherein Notch signaling in bone marrow normally acts to maintain a pool of mesenchymal progenitors by suppressing osteoblast differentiation. Thus, mesenchymal progenitors may be expanded in vitro by activating the Notch pathway, whereas bone formation in vivo may be enhanced by transiently suppressing this pathway.

SUBMITTER: Hilton MJ

PROVIDER: S-EPMC2740725 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Notch signaling maintains bone marrow mesenchymal progenitors by suppressing osteoblast differentiation.

Hilton Matthew J MJ Tu Xiaolin X Wu Ximei X Bai Shuting S Zhao Haibo H Kobayashi Tatsuya T Kronenberg Henry M HM Teitelbaum Steven L SL Ross F Patrick FP Kopan Raphael R Long Fanxin F

Nature medicine 20080224 3

Postnatal bone marrow houses mesenchymal progenitor cells that are osteoblast precursors. These cells have established therapeutic potential, but they are difficult to maintain and expand in vitro, presumably because little is known about the mechanisms controlling their fate decisions. To investigate the potential role of Notch signaling in osteoblastogenesis, we used conditional alleles to genetically remove components of the Notch signaling system during skeletal development. We found that di ...[more]

PMID: 18297083

Similar Datasets

Project description:BackgroundChemical biology approaches using small molecule inhibitors targeting specific signaling pathways are useful tools to dissect the molecular mechanisms governing stem cell differentiation and for their possible use in therapeutic interventions.MethodsStem cell signaling small molecule library functional screen was performed employing human bone marrow skeletal (mesenchymal) stem cells (hBMSCs). Alkaline phosphatase (ALP) activity and formation of mineralized matrix visualized by Alizarin red staining were employed as markers for osteoblastic differentiation. Global gene expression profiling was conducted using the Agilent microarray platform, and data normalization and bioinformatics were performed using GeneSpring software. Pathway analyses were conducted using the Ingenuity Pathway Analysis (IPA) tool. In vivo ectopic bone formation was performed using hBMSC mixed with hydroxyapatite-tricalcium phosphate granules that were implanted subcutaneously in 8-week-old female nude mice. Hematoxylin and eosin staining and Sirius red staining were performed to identify bone formation in vivo.ResultsAmong the tested molecules, LY411575, a potent ?-secretase and Notch signaling inhibitor, exhibited significant inhibitory effects on osteoblastic differentiation of hBMSCs manifested by reduced ALP activity, mineralized matrix formation, and decreased osteoblast-specific gene expression as well as in vivo ectopic bone formation. Global gene expression profiling of LY411575-treated cells revealed changes in multiple signaling pathways, including focal adhesion, insulin, TGF?, IL6, and Notch signaling, and decreased the expression of genes associated with functional categories of tissue development. Among the affected signaling networks were TGF?1, SPP1, and ERK regulatory networks.ConclusionsWe identified ?-secretase inhibitor (LY411575) as a potent regulator of osteoblastic differentiation of hBMSC that may be useful as a therapeutic option for treating conditions associated with ectopic bone formation.

Project description:The authors' previous study demonstrated that miR?128 may exert an inhibitory effect on the osteogenic differentiation of bone marrow?derived mesenchymal stem cells (BM?MSCs), but its downstream mechanisms remain to be elucidated. The aim of the present study was to investigate the microRNA (miRNA/miR) and mRNA profiles of differentiated and undifferentiated BM?MSCs and explore new downstream targets for miR?128. The sequencing datasets of GSE107279 (miRNA) and GSE112318 (mRNA) were downloaded from the Gene Expression Omnibus database. The differentially expressed miRNAs (DEMs) and genes (DEGs) were identified using the DESeq2 method. The target genes of DEMs were predicted by the miRwalk 2.0 database. The hub target genes of miR?128 were screened by constructing the protein?protein interaction (PPI) network and module analysis. The expression levels of miR?128 and crucial target genes were validated by reverse transcription?quantitative (RT?q) PCR before or after transfection of miR?128 mimics to BM?MSCs. The miRNA expression profile analysis identified miR?128 as one of the significantly downregulated DEMs (total 338) in differentiated BM?MSCs compared with the undifferentiated control. A total of 103 predicted target genes of miR?128?3p were overlapped with upregulated DEGs. By calculating the topological properties of each protein in the PPI network, 6 upregulated genes (KIT, NTRK2, YWHAB, GAB1, AXIN1 and RUNX1; fold change was the highest for NTRK2) were considered to be hub genes. Of these, 4 were enriched in module 4 (RUNX1, KIT, GAB1 and AXIN1; RUNX1 was particularly crucial as it can interact with the others), while one was enriched in module 7 (YWHAB). The expression levels of miR?128 and these 6 target genes during the osteogenic differentiation were experimentally confirmed by RT?qPCR. In addition, the expression levels of these 6 genes were significantly reversed after transfection of miR?128?3p mimics into rat BM?MSCs compared with the miR?control group. These findings indicated that miR?128?3p may inhibit the osteoblast differentiation of BM?MSCs by downregulation of these 6 genes, particularly RUNX1, YWHAB and NTRK2.

Project description:Osteoblasts are the main functional cells in bone formation, which are responsible for the synthesis, secretion and mineralization of bone matrix. The PI3K/AKT signaling pathway is strongly associated with the differentiation and survival of osteoblasts. The 3‑phosphoinositide‑dependent protein kinase‑1 (PDK‑1) protein is considered the master upstream lipid kinase of the PI3K/AKT cascade. The present study aimed to investigate the role of PDK‑1 in the process of mouse osteoblast differentiation in vitro. In the BX‑912 group, BX‑912, a specific inhibitor of PDK‑1, was added to osteoblast induction medium (OBM) to treat bone marrow mesenchymal stem cells (BMSCs), whereas the control group was treated with OBM alone. Homozygote PDK1flox/flox mice were designed and generated, and were used to obtain BMSCsPDK1flox/flox. Subsequently, an adenovirus containing Cre recombinase enzyme (pHBAd‑cre‑EGFP) was used to disrupt the PDK‑1 gene in BMSCsPDK1flox/flox; this served as the pHBAd‑cre‑EGFP group and the efficiency of the disruption was verified. Western blot analysis demonstrated that the protein expression levels of phosphorylated (p)‑PDK1 and p‑AKT were gradually increased during the osteoblast differentiation process. Notably, BX‑912 treatment and disruption of the PDK‑1 gene with pHBAd‑cre‑EGFP effectively reduced the number of alkaline phosphatase (ALP)‑positive cells and the optical density value of ALP activity, as well as the formation of cell mineralization. The mRNA expression levels of PDK‑1 in the pHBAd‑cre‑EGFP group were significantly downregulated compared with those in the empty vector virus group on days 3‑7. The mRNA expression levels of the osteoblast‑related genes RUNX2, osteocalcin and collagen I were significantly decreased in the BX‑912 and pHBAd‑cre‑EGFP groups on days 7 and 21 compared with those in the control and empty vector virus groups. Overall, the results indicated that BX‑912 and disruption of the PDK‑1 gene in vitro significantly inhibited the differentiation and maturation of osteoblasts. These experimental results provided an experimental and theoretical basis for the role of PDK‑1 in osteoblasts.

Dataset Information

Notch signaling maintains bone marrow mesenchymal progenitors by suppressing osteoblast differentiation.

Publications

Notch signaling maintains bone marrow mesenchymal progenitors by suppressing osteoblast differentiation.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets