Negative Regulator Of Osteoblast Differentiation Research Articles

The ATF3-OPG Axis Contributes to Bone Formation by Regulating the Differentiation of Osteoclasts, Osteoblasts, and Adipocytes.

Activating transcription factor 3 (ATF3) has been identified as a negative regulator of osteoblast differentiation in in vitro study. However, it was not associated with osteoblast differentiation in in vivo study. To provide an understanding of the discrepancy between the in vivo and in vitro findings regarding the function of ATF3 in osteoblasts, we investigated the unidentified roles of ATF3 in osteoblast biology. ATF3 enhanced osteoprotegerin (OPG) production, not only in osteoblast precursor cells, but also during osteoblast differentiation and osteoblastic adipocyte differentiation. In addition, ATF3 increased nodule formation in immature osteoblasts and decreased osteoblast-dependent osteoclast formation, as well as the transdifferentiation of osteoblasts to adipocytes. However, all these effects were reversed by the OPG neutralizing antibody. Taken together, these results suggest that ATF3 contributes to bone homeostasis by regulating the differentiation of various cell types in the bone microenvironment, including osteoblasts, osteoclasts, and adipocytes via inducing OPG production.

8-Nitro-cGMP suppresses mineralization by mouse osteoblasts.

Nitric oxide and reactive oxygen species regulate bone remodeling, which occurs via bone formation and resorption by osteoblasts and osteoclasts, respectively. Recently, we found that 8-nitro-cGMP, a second messenger of nitric oxide and reactive oxygen species, promotes osteoclastogenesis. Here, we investigated the formation and function of 8-nitro-cGMP in osteoblasts. Mouse calvarial osteoblasts were found to produce 8-nitro-cGMP, which was augmented by tumor necrosis factor-α (10 ng/ml) and interleukin-1β (1 ng/ml). These cytokines suppressed osteoblastic differentiation in a NO synthase activity-dependent manner. Exogenous 8-nitro-cGMP (30 μmol/L) suppressed expression of osteoblastic phenotypes, including mineralization, in clear contrast to the enhancement of mineralization by osteoblasts induced by 8-bromo-cGMP, a cell membrane-permeable analog of cGMP. It is known that reactive sulfur species denitrates and degrades 8-nitro-cGMP. Mitochondrial cysteinyl-tRNA synthetase plays a crucial role in the endogenous production of RSS. The expression of osteoblastic phenotypes was suppressed by not only exogenous 8-nitro-cGMP but also by silencing of the Cars2 gene, indicating a role of endogenous 8-nitro-cGMP in suppressing the expression of osteoblastic phenotypes. These results suggest that 8-nitro-cGMP is a negative regulator of osteoblastic differentiation.

Osterix Marks a Distinct Population of Circulatory Bone Marrow Mesenchymal Stem Cells That Is Increased upon Oncogenic Stress

Bone marrow mesenchymal stem cells (BM-MSCs) are multipotent stromal cells that can differentiate into a variety of lineages and play a critical role in tissue homeostasis upon injury and repair. Previous studies using Osx-Cre transgenic mice have demonstrated that the expression of Sp7 (Osterix) marks a population of mesenchymal progenitor cells that can differentiate to osteoblasts as well as bone marrow stromal cells (Mizoguchi et al., 2014). Using a lineage-tracing system, we show that in addition to marking mesenchymal progenitor cells in the bone marrow, Osx-Cre also marks a population of BM-MSCs that circulate throughout the body and home in different tissues such as lung, spleen, intestine and muscle. Osx-Cre mice crossed with R26-mTmG reporter mice were analyzed at E14.5 and adulthood by fluorescence microscopy and flow cytometry. At E14.5, GFP+ cells were exclusively located in bone tissues. At two months of age, GFP+ cells were detectable in blood and many other tissues such as lung, liver, spleen and intestine. Flow cytometric profiling indicated that the GFP+ cells were positive for BM-MSC markers CD105, CD73 and CD140a. In order to exclude the possibility of non-specific recombination of the reporter in the non-osteoblast-lineage, as previously reported, we performed a pulse chase experiment utilizing Osx-CreER;mTmG mice. Fluorescence microscopy of the bone marrow upon Tamoxifen injection revealed that Cre activity was primarily limited to the osteoblast-lineage and bone tissue, whereas GFP+ cells were undetectable in lung and spleen, indicating that the GFP+ cells in the lung migrated from the bone marrow. Given that previous reports identified p53 as a negative regulator of osteoblast differentiation (Lengner et al., 2006), we further sought to determine the effect of p53 on circulatory BM-MSCs. Flow cytometric analysis of Osx-Cre;p53Fx/Fx;mTmG peripheral blood cells revealed a significant reduction of circulatory GFP+ cells as compared to p53 wild type mice (p<0.0001) suggesting a role for p53 in expansion of circulatory BM-MSCs. To further characterize the population of circulatory BM-MSCs in a cancer model, we analyzed the population of GFP+ cells in a syngeneic leukemia using fluorescence microscopy and flow cytometry. We transplanted p53 wildtype (Osx-Cre;mTmG) and p53 mutant (Osx-Cre;p53Fx/R172H;mTmG) reporter mice with AML-ETO-Turquoise leukemia cells and the population of GFP+ cells were analyzed three weeks after transplant. The population of GFP+MSCs were significantly increased in bone marrow and spleen, indicating the recruitment of circulatory BM-MSCs. Conclusion: We present the Osx-Cre;mTmG mouse as a faithful model to study circulatory BM-MSCs in vivo and identified a role for p53 in the regulation of circulatory BM-MSCs. We previously reported that BM-derived MSCs home to solid tumors and their metastases and can be successfully used as gene-delivery vehicles, both in murine models and in patients (Studeny et al. JCI 2001, Andreeff et al. AACR 2018). This model is the first to conduct studies of circulating MSCs and to further analyze their role in tumor biology and therapy. Lengner, C.J., Steinman, H.A., Gagnon, J., Smith, T.W., Henderson, J.E., Kream, B.E., Stein, G.S., Lian, J.B., and Jones, S.N. (2006). Osteoblast differentiation and skeletal development are regulated by Mdm2-p53 signaling. J Cell Biol 172, 909-921. Mizoguchi, T., Pinho, S., Ahmed, J., Kunisaki, Y., Hanoun, M., Mendelson, A., Ono, N., Kronenberg, H.M., and Frenette, P.S. (2014). Osterix marks distinct waves of primitive and definitive stromal progenitors during bone marrow development. Dev Cell 29, 340-349. Figure Disclosures Andreeff: Daiichi-Sankyo; Breast Cancer Research Foundation; CPRIT; NIH/NCI; Amgen; AstraZeneca: Research Funding; Centre for Drug Research & Development; Cancer UK; NCI-CTEP; German Research Council; Leukemia Lymphoma Foundation (LLS); NCI-RDCRN (Rare Disease Clin Network); CLL Founcdation; BioLineRx; SentiBio; Aptose Biosciences, Inc: Membership on an entity's Board of Directors or advisory committees; Daiichi-Sankyo; Jazz Pharmaceuticals; Celgene; Amgen; AstraZeneca; 6 Dimensions Capital: Consultancy; Amgen: Research Funding.

Trabecular Bone is Increased in a Rat Model of Polycystic Ovary Syndrome.

Polycystic ovary syndrome (PCOS) in an intricate disorder characterized by reproductive and metabolic abnormalities that may affect bone quality and strength along with the lifespan. The present study analysed the impact of postnatal androgenization (of a single dose of testosterone propionate 1.25 mg subcutaneously at day 5 of life) on bone development and markers of bone metabolism in adult female Wistar rats. Compared with healthy controls, the results of measurements of micro-computed tomography (microCT) of the distal femur of androgenized rats indicated an increased cortical bone volume voxel bone volume to total volume (VOX BV/TV) and higher trabecular number (Tb.n) with reduced trabecular separation (Tb.sp). A large magnitude effect size was observed in the levels of circulating bone formation Procollagen I N-terminal propeptide (P1NP) at day 60 of life; reabsorption cross-linked C-telopeptide of type I collagen (CTX) markers were similar between the androgenized and control rats at days 60 and 110 of life. The analysis of gene expression in bone indicated elements for an increased bone mass such as the reduction of the Dickkopf-1 factor (Dkk1) a negative regulator of osteoblast differentiation (bone formation) and the reduction of Interleukin 1-b (Il1b), an activator of osteoclast differentiation (bone reabsorption). Results from this study highlight the possible role of the developmental programming on bone microarchitecture with reference to young women with PCOS.

MicroRNA-204 Deficiency in Human Aortic Valves Elevates Valvular Osteogenic Activity.

Aortic valve interstitial cells (AVICs) play a major role in valvular calcification associated with calcific aortic valve disease (CAVD). Although AVICs from diseased valves display a pro-osteogenic phenotype, the underlying mechanism causing this remains unclear. MicroRNA-204 (miR-204) is a negative regulator of osteoblast differentiation. We sought to analyze miR-204 expression in diseased human aortic valves and determine the role of this miR in AVIC osteogenic activity associated with CAVD pathobiology. In situ hybridization and PCR analysis revealed miR-204 deficiency in diseased valves and in AVICs from diseased valves. MiR-204 mimic suppressed alkaline phosphatase (ALP) expression and calcium deposition in AVICs from diseased valves. MiR-204 antagomir enhanced ALP expression in AVICs from normal valves through induction of Runx2 and Osx, and expression of miR-204 antagomir in mouse aortic valves promoted calcium deposition through up-regulation of Runx2 and Osx. Further, miR-204 mimic suppressed the osteogenic responses to TGF-β1 in AVICs of normal valves. In conclusion, miR-204 deficiency contributes to the mechanism underlying elevated osteogenic activity in diseased aortic valves, and miR-204 is capable of reversing the pro-osteogenic phenotype of AVICs of diseased valves and suppressing AVIC osteogenic response to stimulation. Exogenous miR-204 may have therapeutic potential for inhibiting valvular calcification associated with CAVD progression.

Monocarboxylate transporter-1 promotes osteoblast differentiation via suppression of p53, a negative regulator of osteoblast differentiation

Monocarboxylate transporter-1 (MCT-1) is a transmembrane transporter for monocarboxylates including lactate and pyruvate. Silencing Mct1 by its small interfering RNA (siRNA) suppressed the expression of marker genes for osteoblast differentiation, namely, Tnap, Runx2, and Sp7, induced by BMP-2 in mouse myoblastic C2C12 cells. Mct1 siRNA also suppressed alkaline phosphatase activity, as well as expressions of Tnap and Bglap mRNAs in mouse primary osteoblasts. On the other hand, Mct1 siRNA did not have effects on the Smad1/5 or ERK/JNK pathways in BMP-2-stimulated C2C12 cells, while it up-regulated the mRNA expression of p53 (Trp53) as well as nuclear accumulation of p53 in C2C12 cells in a BMP-2-independent manner. Suppression of osteoblastic differentiation by Mct1 siRNA in C2C12 cells was abolished by co-transfection of Trp53 siRNA. Together, these results suggest that MCT-1 functions as a positive regulator of osteoblast differentiation via suppression of p53.

MiR-98-TXLNG1 (FIAT)/Sp7 function loop mediates osteoblast mineralization.

To investigate the role of microRNAs (miRNAs) and its mechanism in osteoblast mineralization. Real-time polymerase chain reaction (PCR), Northern Blot, and Western Blot were used to identify the expression mode of regulators. Overexpression and down-regulation experiments were carried out to study the role of miR-98 and interactions between regulators. Bioinformatics calculation and luciferase reporter assay were used to prove the target gene. Electrophoretic mobility shift assay (EMSA), chromatin immunoprecipitation (CHIP), and promoter luciferase reporter assay confirmed the relationship between the regulator and the promoter of miR-98. MiR-98 was up-regulated during osteoblast mineralization. Overexpression of miR-98 promoted osteoblast mineralization. Factor inhibiting activating transcription factor 4 (ATF4)-mediated transcription (FIAT), a negative regulator of osteoblast differentiation, was confirmed to be a target of miR-98. As a motivator in osteoblast mineralization, Sp7 transcription factor 7 (Sp7) promoted miR-98 transcription by a combination on the promoter region. Our study showed that miR-98 was an important regulator in osteoblast mineralization and miR-98 carried out its function through a novel miR-98-FIAT/Sp7 regulatory loop. It provides new insights into the roles of miRNAs in osteoblast mineralization.

Theranostic Niosomes for Efficient siRNA/MicroRNA Delivery and Activatable Near-Infrared Fluorescent Tracking of Stem Cells.

RNA interference-mediated gene regulation in stem cells offers great potential in regenerative medicine. In this study, we developed a theranostic platform for efficient delivery of small RNAs [small interfering RNA (siRNA)/microRNA (miRNA)] to human mesenchymal stem cells (hMSCs) to promote differentiation, and meanwhile, to specifically label the transfected cells for the in vivo tracking purpose. We encapsulated indocyanine green (ICG) in a nonionic surfactant vesicle, termed "niosome", that is mainly composed of a nonionic surfactant sorbitan monooleate (Span 80) and a cationic lipid 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP). This novel ICG-containing niosome system (iSPN) demonstrated highly efficient siRNA and miRNA delivery in hMSCs. Specific inhibition of miR-138, a negative regulator of osteoblast differentiation, was achieved by iSPN/miR-138, which significantly promoted osteogenesis of hMSCs. Furthermore, iSPN exhibited OFF/ON activatable fluorescence upon cellular internalization, resulting in efficient near-infrared labeling and the capability to dynamically monitor stem cells in mice. In addition, iSPN/siRNA achieved simultaneous long-term cell tracking and in vivo gene silencing after implantation in mice. These results indicate that our theranostic niosomes could represent a promising platform for future development of stem cell-based therapy.

CRMP4 Inhibits Bone Formation by Negatively Regulating BMP and RhoA Signaling.

We identified the neuroprotein collapsing response mediator protein-4 (CRMP4) as a noncanonical osteogenic factor that regulates the differentiation of mouse bone marrow skeletal stem cells (bone marrow stromal stem cells [mBMSCs]) into osteoblastic cells. CRMP4 is the only member of the CRMP1-CRMP5 family to be expressed by mBMSCs and in osteoprogenitors of both adult mouse and human bones. In vitro gain-of-function and loss-of-function of CRMP4 in murine stromal cells revealed its inhibitory effect on osteoblast differentiation. In addition, Crmp4-deficient mice (Crmp4-/- ) displayed a 40% increase in bone mass, increased mineral apposition rate, and bone formation rate, compared to wild-type controls. Increased bone mass in Crmp4-/- mice was associated with enhanced BMP2 signaling and BMP2-induced osteoblast differentiation in Crmp4-/- osteoblasts (OBs). Furthermore, Crmp4-/- OBs exhibited enhanced activation of RhoA/focal adhesion kinase (FAK) signaling that led to cytoskeletal changes with increased cell spreading. In addition, Crmp4-/- OBs exhibited increased cell proliferation that was mediated via inhibiting cyclin-dependent kinase inhibitor 1B, p27Kip1 and upregulating cyclin D1 expression which are targets of RhoA signaling pathway. Our findings identify CRMP4 as a novel negative regulator of osteoblast differentiation. © 2016 American Society for Bone and Mineral Research.

Cyclic AMP Response Element-binding Protein H (CREBH) Mediates the Inhibitory Actions of Tumor Necrosis Factor α in Osteoblast Differentiation by Stimulating Smad1 Degradation

Endoplasmic reticulum (ER) stress transducers, such as old astrocyte specifically induced substance (OASIS) and activating transcription factor 6 (ATF6), which are induced by bone morphogenetic protein 2 (BMP2), regulate bone formation and osteoblast differentiation. Here, we examined the role of cAMP response element-binding protein H (CREBH), a member of the same family of ER membrane-bound basic leucine zipper (bZIP) transcription factors as OASIS and ATF6, in osteoblast differentiation and bone formation. Proinflammatory cytokine TNFα increased CREBH expression by up-regulating the nuclear factor-κB (NF-κB) signaling pathway in osteoblasts, increased the level of N-terminal fragment of CREBH in the nucleus, and inhibited BMP2 induction of osteoblast specific gene expression. Overexpression of CREBH suppressed BMP2-induced up-regulation of the osteogenic markers runt-related transcription factor 2 (Runx2), alkaline phosphatase (ALP), and osteocalcin (OC) in MC3T3-E1 cells and primary osteoblasts, as well as BMP2-induced ALP activity and OC protein production. In contrast, knockdown of CREBH attenuated the inhibitory effect of TNFα on BMP2-induced osteoblast differentiation. Mechanistic studies revealed that CREBH increased the expression of Smad ubiquitination regulatory factor 1 (Smurf1), leading to ubiquitin-dependent degradation of Smad1, whereas knockdown of CREBH inhibited TNFα-mediated degradation of Smad1 by Smurf1. Consistent with these in vitro findings, administration of Ad-CREBH inhibited BMP2-induced ectopic and orthotopic bone formation in vivo. Taken together, these results suggest that CREBH is a novel negative regulator of osteoblast differentiation and bone formation.

Pharmacological Inhibition of Protein Kinase G1 Enhances Bone Formation by Human Skeletal Stem Cells Through Activation of RhoA-Akt Signaling.

Development of novel approaches to enhance bone regeneration is needed for efficient treatment of bone defects. Protein kinases play a key role in regulation of intracellular signal transduction pathways, and pharmacological targeting of protein kinases has led to development of novel treatments for several malignant and nonmalignant conditions. We screened a library of kinase inhibitors to identify small molecules that enhance bone formation by human skeletal (stromal or mesenchymal) stem cells (hMSC). We identified H-8 (known to inhibit protein kinases A, C, and G) as a potent enhancer of ex vivo osteoblast (OB) differentiation of hMSC, in a stage- and cell type-specific manner, without affecting adipogenesis or osteoclastogenesis. Furthermore, we showed that systemic administration of H-8 enhances in vivo bone formation by hMSC, using a preclinical ectopic bone formation model in mice. Using functional screening of known H-8 targets, we demonstrated that inhibition of protein kinase G1 (PRKG1) and consequent activation of RhoA-Akt signaling is the main mechanism through which H-8 enhances osteogenesis. Our studies revealed PRKG1 as a novel negative regulator of OB differentiation and suggest that pharmacological inhibition of PRKG1 in hMSC implanted at the site of bone defect can enhance bone regeneration. Stem Cells 2015;33:2219-2231.

CXXC5 is a negative-feedback regulator of the Wnt/β-catenin pathway involved in osteoblast differentiation.

The positive roles of the Wnt/β-catenin pathway in osteoblast differentiation and bone mineral density (BMD) maintenance have been clearly demonstrated in both animal experiments and clinical investigations. CXXC finger protein 5 (CXXC5), a recently identified negative regulator of the Wnt/β-catenin pathway, showed altered cellular localization and function, which were dependent on the cell type in previous studies. However, the in vivo function of CXXC5 has not been clearly investigated yet. Here, we characterized CXXC5 as a negative regulator of osteoblast differentiation and bone formation. Deficiency of CXXC5 resulted in elevated BMD in mice without any severe gross developmental abnormalities. CXXC5 exerted a negative-feedback effect on the Wnt/β-catenin pathway via Wnt-dependent binding to Dishevelled (Dvl) during osteoblast differentiation. Suppression of the Dvl-CXXC5 interaction using a competitor peptide resulted in the activation of the Wnt/β-catenin pathway and osteoblast differentiation, and accelerated thickness growth of ex vivo-cultured calvariae. Overall, CXXC5 is a negative-feedback regulator induced by Wnt/β-catenin signaling that inhibits osteoblast differentiation and bone formation via interaction with Dvl.

AP2 suppresses osteoblast differentiation and mineralization through down-regulation of Frizzled-1.

Transcription factor activating protein 2 (AP2) plays an important role in cellular differentiation. Although profound craniofacial and long bone developmental abnormalities have been observed in AP2-knockout mice, the molecular effects of AP2 on osteoblasts are poorly defined. We demonstrated that AP2 regulates the expression of human Frizzled 1 (FZD1), a co-receptor for the Wnt signalling pathway, in human osteoblast cell lines and primary bone marrow stromal cells (BMSCs). We also identified a putative AP2-binding site in the FZD1 proximal promoter in silico and characterized this binding element further in Saos2 invitro by ChIP, electrophoretic mobility shift and promoter reporter assays. The transcriptional repression of the FZD1 promoter by AP2 was confirmed in normal human fetal osteoblasts (hFOB). Furthermore, overexpression of AP2 resulted in a significant reduction in both differentiation and mineralization of Saos2 cells. Knockdown of FZD1 expression before AP2 up-regulation diminished the AP2-dependent inhibition of Saos2 cell differentiation and mineralization. Similarly, overexpressing FZD1 before AP2 treatment in both Saos2 and BMSCs diminished the inhibitory effect of AP2 on osteoblast differentiation and mineralization. Taken together, these results demonstrate that AP2 is a negative regulator of osteoblast differentiation and mineralization, and its inhibitory effect may be mediated in part through down-regulation of FZD1 expression.

AMP-activated protein kinase (AMPK) activity negatively regulates chondrogenic differentiation

Chondrocytes are derived from mesenchymal stem cells, and play an important role in cartilage formation. Sex determining region Y box (Sox) family transcription factors are essential for chondrogenic differentiation, whereas the intracellular signal pathways of Sox activation have not been clearly elucidated. AMP-activated protein kinase (AMPK) is a serine–threonine kinase generally regarded as a key regulator of cellular energy homeostasis. It is known that the catalytic alpha subunit of AMPK is activated by upstream AMPK kinases (AMPKKs) including liver kinase B1 (LKB1). We have previously reported that AMPK is a negative regulator of osteoblastic differentiation. Here, we have explored the role of AMPK in chondrogenic differentiation using in vitro culture models. The phosphorylation level of the catalytic AMPK alpha subunit significantly decreased during chondrogenic differentiation of primary chondrocyte precursors as well as ATDC-5, a well-characterized chondrogenic cell line. Treatment with metformin, an activator of AMPK, significantly reduced cartilage matrix formation and inhibited gene expression of sox6, sox9, col2a1 and aggrecan core protein (acp). Thus, chondrocyte differentiation is functionally associated with decreased AMPK activity.

Twist-related protein 1 negatively regulated osteoblastic transdifferentiation of human aortic valve interstitial cells by directly inhibiting runt-related transcription factor 2

Valve calcification involves transdifferentiation of valve interstitial cells (VICs) into osteoblasts. Twist-related protein 1 (TWIST1) has been established as a negative regulator of osteoblast differentiation in both mouse and human mesenchymal stem cells, but its function in human aortic VICs is unknown. In our study, we determined the mechanism of TWIST1 action in regulating osteoblastic transdifferentiation of human aortic VICs. Human calcified and noncalcified aortic valves were examined for TWIST1 expression. Human aortic VICs were isolated and cultured. The data showed that calcified aortic valves express lower levels of TWIST1. In vitro experiments showed that TWIST1 overexpression inhibited the transdifferentiation of VICs into osteoblasts by decreasing the expression of runt-related transcription factor 2 (RUNX2) and its downstream osteoblastic markers. Through chromatin immunoprecipitation and dual luciferase assays, we found that TWIST1 repressed the expression of RUNX2 by directly binding to an E-box located at -820 bp of the RUNX2 P2 promoter region and inhibiting its activity. Our study results suggest that TWIST1 could play an important role in preventing human aortic valve calcification by negatively regulating osteoblastic transdifferentiation of human aortic VICs through direct inhibition of RUNX2.

ER stress-inducible ATF3 suppresses BMP2-induced ALP expression and activation in MC3T3-E1 cells

Endoplasmic reticulum (ER) stress suppresses osteoblast differentiation. Activating transcription factor (ATF) 3, a member of the ATF/cAMP response element-binding protein family of transcription factors, is induced by various stimuli including cytokines, hormones, DNA damage, and ER stress. However, the role of ATF3 in osteoblast differentiation has not been elucidated. Treatment with tunicamycin (TM), an ER stress inducer, increased ATF3 expression in the preosteoblast cell line, MC3T3-E1. Overexpression of ATF3 inhibited bone morphogenetic protein 2-stimulated expression and activation of alkaline phosphatase (ALP), an osteogenic marker. In addition, suppression of ALP expression by TM treatment was rescued by silencing of ATF3 using shRNA. Taken together, these data indicate that ATF3 is a novel negative regulator of osteoblast differentiation by specifically suppressing ALP gene expression in preosteoblasts.

OstemiR: A Novel Panel of MicroRNA Biomarkers in Osteoblastic and Osteocytic Differentiation from Mesencymal Stem Cells

MicroRNAs (miRNAs) are small RNA molecules of 21–25 nucleotides that regulate cell behavior through inhibition of translation from mRNA to protein, promotion of mRNA degradation and control of gene transcription. In this study, we investigated the miRNA expression signatures of cell cultures undergoing osteoblastic and osteocytic differentiation from mesenchymal stem cells (MSC) using mouse MSC line KUSA-A1 and human MSCs. Ninety types of miRNA were quantified during osteoblastic/osteocytic differentiation in KUSA-A1 cells utilizing miRNA PCR arrays. Coincidently with mRNA induction of the osteoblastic and osteocytic markers, the expression levels of several dozen miRNAs including miR-30 family, let-7 family, miR-21, miR-16, miR-155, miR-322 and Snord85 were changed during the differentiation process. These miRNAs were predicted to recognize osteogenic differentiation-, stemness-, epinegetics-, and cell cycle-related mRNAs, and were thus designated OstemiR. Among those OstemiR, the miR-30 family was classified into miR-30b/c and miR-30a/d/e groups on the basis of expression patterns during osteogenesis as well as mature miRNA structures. In silico prediction and subsequent qRT-PCR in stable miR-30d transfectants clarified that context-dependent targeting of miR-30d on known regulators of bone formation including osteopontin/spp1, lifr, ccn2/ctgf, ccn1/cyr61, runx2, sox9 as well as novel key factors including lin28a, hnrnpa3, hspa5/grp78, eed and pcgf5. In addition, knockdown of human OstemiR miR-541 increased Osteopontin/SPP1 expression and calcification in hMSC osteoblastic differentiation, indicating that miR-541 is a negative regulator of osteoblastic differentiation. These observations indicate stage-specific roles of OstemiR especially miR-541 and the miR-30 family on novel targets in osteogenesis.

1α,25-Dihydroxyvitamin D3 inhibits osteoblastic differentiation of mouse periodontal fibroblasts

ObjectivePeriodontal ligament (PDL) is a non-mineralized tissue connecting cementum and alveolar bone. Recent studies have suggested that periodontal fibroblasts can differentiate into mineralized tissue-forming cells such as osteoblasts and cementoblasts. We elucidated the immunolocalization of vitamin D receptor (VDR) and the effects of vitamin D3 (VD3) on mouse periodontal fibroblasts to clarify the role of VDR and VD3 in the differentiation of periodontal fibroblasts. DesignImmunohistochemical analysis using anti-VDR antibody was performed in paraffin sections of mouse mandibles at E13, E14, E17, P10, and P35. The roles of VD3 in osteoblastic differentiation and mineralization were estimated by alkaline phosphatase (ALP) and alizarin red (AR) staining. In addition, the mRNA expression of osteoblast markers and mineralization inhibitors was examined by real-time PCR. ResultsAt the bud, cap and early bell stages, immunoreactivity for VDR was scarcely seen in the cells of dental follicles. Labelling was detected in the nuclei of cemetoblasts, periodontal fibroblasts and osteoblasts during root formation. VD3 inhibited ALP activity and AR-positive mineralized nodule formation of periodontal fibroblasts in osteogenic culture medium. Real-time PCR revealed that VD3 down-regulated the levels of osteoblast markers. In contrast, VD3 significantly up-regulated the level of periodontal ligament associated protein (PLAP)-1, a negative regulator of osteoblastic differentiation. ConclusionThese results suggest that VD3 negatively regulates the osteoblastic differentiation of mouse periodontal fibroblasts and prevents the periodontal ligament from mineralization via increase of PLAP-1.

Deletion of Amp-activated Protein Kinase (AMPK)-Alpha 1 catalytic subunit in mice impairs the in vivo response of bones to hormonal challenges

Deletion of Amp-activated Protein Kinase (AMPK)-Alpha 1 catalytic subunit in mice impairs the in vivo response of bones to hormonal challenges

Oxytocin receptor nuclear translocation mediates upregulation of osteoblast specific genes

Oxytocin receptor nuclear translocation mediates upregulation of osteoblast specific genes