Loss of receptor tyrosine kinase-like orphan receptor 2 impairs the osteogenesis of mBMSCs by inhibiting signal transducer and activator of transcription 3.
ABSTRACT: BACKGROUND:Receptor tyrosine kinase-like orphan receptor 2 (Ror2) plays a key role in bone formation, but its signaling pathway is not completely understood. Signal transducer and activator of transcription 3 (Stat3) takes part in maintaining bone homeostasis. The aim of this study is to reveal the role and mechanism of Ror2 in the osteogenic differentiation from mouse bone marrow mesenchymal stem cells (mBMSCs) and to explore the effect of Stat3 on Ror2-mediated osteogenesis. METHODS:Ror2 CKO mice were generated via the Cre-loxp recombination system using Prrx1-Cre transgenic mice. Quantitative real-time PCR and western blot were performed to assess the expression of Stat3 and osteogenic markers in Ror2-knockdown mBMSCs (mBMSC-sh-Ror2). After being incubated in osteogenic induction medium for 3?weeks, Alizarin Red staining and western blot were used to examine the calcium deposit and osteogenic markers in Stat3 overexpression in mBMSC-sh-Ror2. RESULTS:Loss of Ror2 in mesenchymal or osteoblast progenitor cells led to a dwarfism phenotype in vivo. The mRNA expression of osteogenic markers (osteocalcin, osteopontin (OPN), and collagen I) in the ulna proximal epiphysis of Ror2 CKO mice was significantly decreased (P?
Project description:Osteoporosis is caused by an overstimulation of osteoclast activity and the destruction of the bone extracellular matrix. Without the normal architecture, osteoblast cells are unable to rebuild phenotypically normal bone. Hormone replacement therapy with estrogen has been effective in increasing osteoblast activity but also has resulted in the increased incidence of breast and uterine cancer. In this study we designed and synthesized a series of daidzein analogs to investigate their osteogenic induction potentials. Human bone marrow derived mesenchymal stem cells (MSCs) from three different donors were treated with daidzein analogs and demonstrated enhanced osteogenesis when compared to daidzein treatment. The enhanced osteogenic potential of these daidzein analogs resulted in increased osterix (Sp7), alkaline phosphatase (ALP), osteopontin (OPN), and insulin-like growth factor 1 (IGF-1), which are osteogenic transcription factors that regulate the maturation of osteogenic progenitor cells into mature osteoblast cells.
Project description:BACKGROUND:Identifying bone anabolic agents is a superior strategy for the treatment of osteoporosis. Naturally, derived coumarin derivatives have shown osteoanabolic effect in vitro and in vivo. In this study, we investigated the effect of 5'-Hydroxy Auraptene (5'-HA), a coumarin derivative that newly isolated from Lotus lalambensis Schweinf on the differentiation of the mouse bone marrow-derived mesenchymal (skeletal) stem cells (mBMSCs) into osteoblast and adipocyte. METHODS:The effect of 5'-HA on mBMSCs cell proliferation and osteoblast differentiation was assessed by measuring cell viability, quantitative alkaline phosphatase (ALP) activity assay, Alizarin red staining for matrix mineralization and osteogenic gene array expression. Adipogenesis was measured by Oil Red O staining and quantitative real time PCR (qPCR) analysis of adipogenic markers. Regulation of BMPs signaling pathways by 5'-HA was measured by Western blot analysis and qPCR. RESULTS:5'-HA showed to stimulate the differentiation of mBMSCs into osteogenic cell lineage in a dose-dependent manner, without affecting their differentiation into adipocytic cell lineage. Treatment of mBMSCs with 5'-HA showed to promote significantly the BMP2-induced osteogenesis in mBMSCs via activating Smad1/5/8 phosphorylation and increasing Smad4 expression. Blocking of BMP signaling using BMPR1 selective inhibitor LDN-193189 significantly inhibited the stimulatory effect of 5'-HA on osteogenesis. CONCLUSIONS:Our data identified 5'-HA, as a novel coumarin derivative that function to stimulate the differentiation of mBMSCs into osteoblasts in BMP-signaling dependent mechanism.
Project description:Long noncoding RNAs (lncRNAs) have been demonstrated to be important regulators during the osteogenic differentiation of mesenchymal stem cells (MSCs). We analyzed the lncRNA expression profile during osteogenic differentiation of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) and identified a significantly downregulated lncRNA RP11-527N22.2, named osteogenic differentiation inhibitory lncRNA 1, ODIR1. In hUC-MSCs, ODIR1 knockdown significantly promoted osteogenic differentiation, whereas overexpression inhibited osteogenic differentiation in vitro and in vivo. Mechanistically, ODIR1 interacts with F-box protein 25 (FBXO25) and facilitates the proteasome-dependent degradation of FBXO25 by recruiting Cullin 3 (CUL3). FBXO25 increases the mono-ubiquitination of H2BK120 (H2BK120ub) which subsequently promotes the trimethylation of H3K4 (H3K4me3). Both H2BK120ub and H3K4me3 form a loose chromatin structure, inducing the transcription of the key transcription factor osterix (OSX) and increasing the expression of the downstream osteoblast markers, osteocalcin (OCN), osteopontin (OPN), and alkaline phosphatase (ALP). In summary, ODIR1 acts as a key negative regulator during the osteogenic differentiation of hUC-MSCs through the FBXO25/H2BK120ub/H3K4me3/OSX axis, which may provide a novel understanding of lncRNAs that regulate the osteogenesis of MSCs and a potential therapeutic strategy for the regeneration of bone defects.
Project description:Transcription factors (TFs) regulate the expression of target genes, inducing changes in cell morphology or activities needed for cell fate determination and differentiation. The BMP signaling pathway is widely regarded as one of the most important pathways in vertebrate skeletal biology, of which BMP2 is a potent inducer, governing the osteoblast differentiation of bone marrow stromal cells (BMSCs). However, the mechanism by which BMP2 initiates its downstream transcription factor cascade and determines the direction of differentiation remains largely unknown. In this study, we used RNA-seq, ATAC-seq, and animal models to characterize the BMP2-dependent gene regulatory network governing osteoblast lineage commitment. Sp7-Cre; Bmp2<sup>fx/fx</sup> mice (BMP2-cKO) were generated and exhibited decreased bone density and lower osteoblast number (n?>?6). In vitro experiments showed that BMP2-cKO mouse bone marrow stromal cells (mBMSCs) had an impact on osteoblast differentiation and deficient cell proliferation. Osteogenic medium induced mBMSCs from BMP2-cKO mice and control were subjected to RNA-seq and ATAC-seq analysis to reveal differentially expressed TFs, along with their target open chromatin regions. Combined with H3K27Ac CUT&Tag during osteoblast differentiation, we identified 2338 BMP2-dependent osteoblast-specific active enhancers. Motif enrichment assay revealed that over 80% of these elements were directly targeted by RUNX2, DLX5, MEF2C, OASIS, and KLF4. We deactivated Klf4 in the Sp7?+?lineage to validate the role of KLF4 in osteoblast differentiation of mBMSCs. Compared to the wild-type, Sp7-Cre; Klf4<sup>fx/+</sup> mice (KLF4-Het) were smaller in size and had abnormal incisors resembling BMP2-cKO mice. Additionally, KLF4-Het mice had fewer osteoblasts and decreased osteogenic ability. RNA-seq and ATAC-seq revealed that KLF4 mainly "co-bound" with RUNX2 to regulate downstream genes. Given the significant overlap between KLF4- and BMP2-dependent NFRs and enriched motifs, our findings outline a comprehensive BMP2-dependent gene regulatory network specifically governing osteoblast differentiation of the Sp7?+?lineage, in which Klf4 is a novel transcription factor.
Project description:Epigenetic mechanisms control skeletal development and osteoblast differentiation. Pharmacological inhibition of the histone 3 Lys-27 (H3K27) methyltransferase enhancer of zeste homolog 2 (EZH2) in WT mice enhances osteogenesis and stimulates bone formation. However, conditional genetic loss of Ezh2 early in the mesenchymal lineage (i.e. through excision via Prrx1 promoter-driven Cre) causes skeletal abnormalities due to patterning defects. Here, we addressed the key question of whether Ezh2 controls osteoblastogenesis at later developmental stages beyond patterning. We show that Ezh2 loss in committed pre-osteoblasts by Cre expression via the osterix/Sp7 promoter yields phenotypically normal mice. These Ezh2 conditional knock-out mice (Ezh2 cKO) have normal skull bones, clavicles, and long bones but exhibit increased bone marrow adiposity and reduced male body weight. Remarkably, in vivo Ezh2 loss results in a low trabecular bone phenotype in young mice as measured by micro-computed tomography and histomorphometry. Thus, Ezh2 affects bone formation stage-dependently. We further show that Ezh2 loss in bone marrow-derived mesenchymal cells suppresses osteogenic differentiation and impedes cell cycle progression as reflected by decreased metabolic activity, reduced cell numbers, and changes in cell cycle distribution and in expression of cell cycle markers. RNA-Seq analysis of Ezh2 cKO calvaria revealed that the cyclin-dependent kinase inhibitor Cdkn2a is the most prominent cell cycle target of Ezh2 Hence, genetic loss of Ezh2 in mouse pre-osteoblasts inhibits osteogenesis in part by inducing cell cycle changes. Our results suggest that Ezh2 serves a bifunctional role during bone formation by suppressing osteogenic lineage commitment while simultaneously facilitating proliferative expansion of osteoprogenitor cells.
Project description:Multiple myeloma (MM) is a B lymphocyte malignancy that remains incurable despite extensive research efforts. This is due, in part, to frequent disease recurrences associated with the persistence of myeloma cancer stem cells (mCSCs). Bone marrow mesenchymal stromal cells (BMSCs) play critical roles in supporting mCSCs through genetic or biochemical alterations. Previously, we identified mechanical distinctions between BMSCs isolated from MM patients (mBMSCs) and those present in the BM of healthy individuals (nBMSCs). These properties of mBMSC contributed to their ability to preferentially support mCSCs. To further illustrate mechanisms underlying the differences between mBMSCs and nBMSCs, here we report that (i) mBMSCs express an abnormal, constitutively high level of phosphorylated Myosin II, which leads to stiffer membrane mechanics, (ii) mBMSCs are more sensitive to SDF-1?-induced activation of MYL2 through the G(i./o)-PI3K-RhoA-ROCK-Myosin II signaling pathway, affecting Young's modulus in BMSCs and (iii) activated Myosin II confers increased cell contractile potential, leading to enhanced collagen matrix remodeling and promoting the cell-cell interaction between mCSCs and mBMSCs. Together, our findings suggest that interfering with SDF-1? signaling may serve as a new therapeutic approach for eliminating mCSCs by disrupting their interaction with mBMSCs.
Project description:Hesperetin (3',5,7-trihydroxy-4-methoxyflavanone) is a metabolite of hesperidin (hesperetin-7-O-rutinoside), which belongs to the flavanone subgroup and is found mainly in citrus fruits. Hesperetin has been reported to be an effective osteoinductive compound in various in vivo and in vitro models. However, how hesperetin effects osteogenic differentiation is not fully understood. In this study, we investigated the capacity of hesperetin to stimulate the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) and to relieve the anti-osteogenic effect of high glucose. Osteogenesis of PDLSCs was assessed by measurement of alkaline phosphatase (ALP) activity, and evaluation of the mRNA expression of ALP, runt-related gene 2 (Runx2), osterix (OSX), and FRA1 as osteogenic transcription factors, as well as assessment of protein expression of osteopontin (OPN) and collagen type IA (COLIA). When PDLSCs were exposed to a high concentration (30 mM) of glucose, osteogenic activity decreased compared to control cells. Hesperetin significantly increased ALP activity at doses of 1, 10, and 100 µM. Pretreatment of cells with hesperetin alleviated the high-glucose-induced suppression of the osteogenic activity of PDLSCs. Hesperetin scavenged intracellular reactive oxygen species (ROS) produced under high glucose condition. Furthermore, hesperetin increased the activity of the PI3K/Akt and ?-catenin pathways. Consistent with this, blockage of Akt or ?-catenin diminished the protective effect of hesperetin against high glucose-inhibited osteogenic differentiation. Collectively, our results suggest that hesperetin alleviates the high glucose-mediated suppression of osteogenic differentiation in PDLSCs by regulating ROS levels and the PI3K/Akt and ?-catenin signaling pathways.
Project description:Osterix (Osx), a transcriptional factor essential for osteogenesis, is also critical for in vivo cellular cementum formation. However, the molecular mechanism by which Osx regulates cementoblasts is largely unknown. In this study, we initially demonstrated that overexpression of Osx in a cementoblast cell line upregulated the expression of markers vital to cementogenesis such as osteopontin (OPN), osteocalcin (OCN), and bone sialoprotein (BSP) at both mRNA and protein levels, and enhanced alkaline phosphatase (ALP) activity. Unexpectedly, we demonstrated a sharp increase in the expression of DKK1 (a potent canonical Wnt antagonist), and a great reduction in protein levels of ?-catenin and its nuclear translocation by overexpression of Osx. Further, transient transfection of Osx reduced protein levels of TCF1 (a target transcription factor of ?-catenin), which were partially reversed by an addition of DKK1. We also demonstrated that activation of canonical Wnt signaling by LiCl or Wnt3a significantly enhanced levels of TCF1 and suppressed the expression of OPN, OCN, and BSP, as well as ALP activity and formation of extracellular mineralized nodules. Importantly, we confirmed that there were a sharp reduction in DKK1 and a concurrent increase in ?-catenin in Osx cKO mice (crossing between the Osx loxP and 2.3 Col 1-Cre lines), in agreement with the in vitro data. Thus, we conclude that the key role of Osx in control of cementoblast proliferation and differentiation is to maintain a low level of Wnt-?-catenin via direct up-regulation of DKK1.
Project description:Although the role of glycogen synthase kinase 3? (GSK3?) in osteogenic differentiation of bone marrow-derived mesenchymal stromal cells (BMSCs) is well-characterized as a negative regulator of ?-catenin, its effect on osteogenesis of adipose-derived stromal cells (ADSCs) is poorly understood. Here, we show that GSK3? positively regulates osteogenic differentiation of murine ADSCs. Gain-of-function studies showed that GSK3? promotes in vitro osteogenesis of ADSCs. Regulation of GSK3? activity in ADSCs, either by small interfering RNA (siRNA)-mediated GSK3? silencing or by pharmacological inhibitors, blunted osteogenesis and the expression of osteogenic markers. Importantly, we demonstrated that transgenic mice, engineered to overexpress the constitutively active GSK3? (GSK3?-S9A) mutant, exhibited a marked increase in osteogenesis, whereas expression of the catalytically inactive GSK3? (GSK3?-K85A) in mice inhibits osteogenic differentiation. Molecular analyses showed that the enhanced osteoblast differentiation induced by GSK3? was mediated by downregulation of ?-catenin. Remarkably, ?-catenin silencing enhances osteogenesis and osteoblast marker gene expression such as alkaline phosphatase (ALP) and osterix. Taken together, these findings demonstrate a novel role for GSK3? in the regulation of osteogenic differentiation in ADSCs.
Project description:The lineage specification of mesenchymal stem/stromal cells (MSCs) is tightly regulated by a wide range of factors. Recently, the versatile functions of ZBP1 (also known as DAI or DLM-1) have been reported in the blood circulation and immune systems. However, the biological function of ZBP1 during the lineage specification of MSCs is still unknown. In the present study, we found that ZBP1 was upregulated during osteogenesis but downregulated during adipogenesis in mouse bone marrow-derived MSCs (mBMSCs). ZBP1 was highly expressed in osteoblasts but expressed at a relatively low level in marrow adipocytes. Knockdown of ZBP1 inhibited alkaline phosphataseactivity, extracellular matrix mineralization, and osteogenesis-related gene expression in vitro and reduced ectopic bone formation in vivo. Knockdown of ZBP1 also promoted adipogenesis in MSCs in vitro. Conversely, the overexpression of ZBP1 increased the osteogenesis but suppressed the adipogenesis of MSCs. When the expression of ZBP1 was rescued, the osteogenic capacity of ZBP1-depleted mBMSCs was restored at both the molecular and phenotypic levels. Furthermore, we demonstrated that ZBP1, a newly identified target of Wnt/?-catenin signaling, was required for ?-catenin translocation into nuclei. Collectively, our results indicate that ZBP1 is a novel regulator of bone and fat transdifferentiation via Wnt/?-catenin signaling.