23 research outputs found

    Osteoblast-Specific Transcription Factor Osterix Increases Vitamin D Receptor Gene Expression in Osteoblasts

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    Osterix (Osx) is an osteoblast-specific transcription factor required for osteoblast differentiation from mesenchymal stem cells. In Osx knock-out mice, no bone formation occurs. The vitamin D receptor (VDR) is a member of the nuclear hormone receptor superfamily that regulates target gene transcription to ensure appropriate control of calcium homeostasis and bone development. Here, we provide several lines of evidence that show that the VDR gene is a target for transcriptional regulation by Osx in osteoblasts. For example, calvaria obtained from Osx-null embryos displayed dramatic reductions in VDR expression compared to wild-type calvaria. Stable overexpression of Osx stimulated VDR expression in C2C12 mesenchymal cells. Inhibition of Osx expression by siRNA led to downregulation of VDR. In contrast, Osx levels remained unchanged in osteoblasts in VDR-null mice. Mechanistic approaches using transient transfection assays showed that Osx directly activated a 1 kb fragment of the VDR promoter in a dose-dependent manner. To define the region of the VDR promoter that was responsive to Osx, a series of VDR promoter deletion mutants were examined and the minimal Osx-responsive region was refined to the proximal 120 bp of the VDR promoter. Additional point mutants were used to identify two GC-rich regions that were responsible for VDR promoter activation by Osx. Chromatin immunoprecipitation assays demonstrated that endogenous Osx was associated with the native VDR promoter in primary osteoblasts in vivo. Cumulatively, these data strongly support a direct regulatory role for Osx in VDR gene expression. They further provide new insight into potential mechanisms and pathways that Osx controls in osteoblasts and during the process of osteoblastic cell differentiation

    Semaphorin 3B Is a 1,25-Dihydroxyvitamin D 3 -Induced Gene in Osteoblasts that Promotes Osteoclastogenesis and Induces Osteopenia in Mice

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    The vitamin D endocrine system is important for skeletal homeostasis. ] is the bioactive metabolite of vitamin D. This hormone functions through the vitamin D receptor (VDR), a member of the nuclear hormone receptor superfamily, to regulate the transcription of target genes in a number of tissues including the intestine, bone, parathyroid gland, skin, and a variety of other systems (1, 2). The 1,25-(OH) 2 D 3 /VDR endocrine system functions in diverse biological processes, such as hair follicle cycling, mammary gland development, and immune cell function (2). One of the most profound actions of 1,25-(OH) 2 D 3 is to protect skeletal integrity because deficiencies in either the hormone or the receptor result in undermineralized bones Acting in concert with PTH, 1,25-(OH) 2 D 3 preserves bone mineralization primarily by maintaining calcium and phosphate homeostasis. 1,25-(OH) 2 D 3 controls serum levels of these minerals by stimulating calcium and phosphate absorption by the intestine, by increasing reabsorption of calcium and phosphate in the kidney, and by liberating calcium and phosphate from skeletal stores (3). When dietary sources of calcium are inadequate, 1,25-(OH) 2 D 3 promotes osteoclastogenesis and bone resorption, in part, by stimulating osteoblasts to express receptor activator of nuclear factor-B ligand (RANKL) (5), a molecule essential for osteoclast formation and function (6, 7). Under conditions of normocalcemia, the 1,25-(OH) 2 D 3 /VDR endocrine system also modulates osteoblast differentiation and mineralization (8-11). Thus, 1,25-(OH) 2 D 3 functions both systemically to regulate serum concentrations of calcium and phosphate and locally to fine-tune the balance between bone formation and bone resorption. However, with the exception of RANKL and a few bone matrix protein

    Nuclear coactivator-62 kDa/Ski-interacting protein is a nuclear matrix-associated coactivator that may couple vitamin D receptor-mediated transcription and RNA splicing

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    Nuclear coactivator-62 kDa/Ski-interacting protein (NCoA62/SKIP) is a putative vitamin D receptor (VDR) and nuclear receptor coactivator protein that is unrelated to other VDR coactivators such as those in the steroid receptor coactivator (SRC) family. The mechanism through which NCoA62/SKIP functions in VDR-activated transcription is unknown. In the present study, we identified a nuclear localization sequence in the COOH terminus of NCoA62/SKIP and showed that NCoA62/SKIP was targeted to nuclear matrix subdomains. Chromatin immunoprecipitation studies revealed that endogenous NCoA62/SKIP associated in a 1,25-dihydroxyvitamin D3-dependent manner with VDR target genes in ROS17/2.8 osteosarcoma cells. A cyclic pattern of promoter occupancy by VDR, SRC-1, and NCoA62/SKIP was observed, with NCoA62/SKIP entering these promoter complexes after SRC-1. These studies provide strong support for the proposed role of NCoA62/SKIP as a VDR transcriptional coactivator, and they indicate that key mechanistic differences probably exist between NCoA62/SKIP and SRC coactivators. To explore potential mechanisms, NCoA62/SKIP-interacting proteins were purified from HeLa cell nuclear extracts and identified by mass spectrometry. The identified proteins represent components of the spliceosome as well as other nuclear matrix-associated proteins. Here, we show that a dominant negative inhibitor of NCoA62/SKIP (dnNCoA62/SKIP) interfered with appropriate splicing of transcripts derived from 1,25-dihydroxyvitamin D3-induced expression of a growth hormone minigene cassette. Taken together, these data show that NCoA62/SKIP has properties that are consistent with those of nuclear receptor coactivators and with RNA spliceosome components, thus suggesting a potential role for NCoA62/SKIP in coupling VDR-mediated transcription to RNA splicing

    Overexpression of Osx activates VDR gene expression in C2C12 mesenchymal cells.

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    <p>(A) Western immunoblot analysis of the Dox-regulated Osx-expressing C2C12 cells. Osx expression is turned on in the absence of Dox. Beta-actin was used as a loading control. (B) VDR mRNA levels in a stable, Tet-off C2C12 mesenchymal cell line. RNA was obtained from cultures treated with or without Doxycycline. Osx expression is induced in the absence of Doxycycline in this line. VDR mRNA levels were quantitated by real-time RT-PCR. The VDR RNA level obtained from the cells cultured with Dox was normalized to a value of 1. Values are presented as the mean ±S.D. (C) RNA expression level for the osteoblastic marker gene alkaline phosphatase (ALP). (D) RNA expression level for osteoblastic gene osteocalcin (OC). Conditions are identical to those described in panel B. A paired <i>t</i>-test was performed comparing Dox (−) and Dox (+) groups. *: A star indicates statistical significance compared to Dox (+) group.</p

    Osx ablation reduces VDR gene expression <i>in vivo</i>.

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    <p>Calvaria RNAs were isolated from E18.5 <i>Osx</i> wild-type and <i>Osx</i>-null embryos. RNA expression levels for Osx, osteocalcin (OC), Runx2 and VDR were analyzed by real-time RT-PCR. The level of each RNA from <i>Osx</i>-null calvaria was normalized to a value of 1. *: A star indicates statistical significance compared to Osx wild type group.</p

    Osx-regulated expression of VDR is important in Osx-induced osteoblast differentiation.

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    <p>(A) SiRNA directed against VDR knocks down VDR expression. C2C12 mesenchymal cells stably expressing Osx were transfected with VDR siRNA at different concentrations. RNA was isolated and specific RNA levels were measured by real-time RT-PCR. (B,C) Cells were transfected with 300 nM of siRNA targeting VDR, and Osx expression was induced by removing Dox. Osteoblast marker gene expression was examined including (B) ALP and (C)osteocalcin (OC). The RNA level from control siRNA group (Si-C) was normalized to a value of 1. Values were presented as the mean ±S.D. *: A star indicates statistical significance compared to Dox (+) group (p<0.05). **: Two stars indicate statistical significance compared to si-control (Si-C) (p<0.05). (D) Western analysis of the VDR knockdown. C2C12 stable cells without Dox were transfected with 300 nM of siRNA targeting VDR. Protein was isolated from whole cell lysates of control siRNA group (si-C) and VDR siRNA group (si-VDR) and then analyzed by western blot using rabbit anti-VDR, anti-ALP or anti-OC polyclonal antibodies. Beta-actin was used as a loading control.</p

    SiRNA-directed knockdown of Osx impairs VDR gene expression in MC3T3 osteoblasts.

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    <p>(A) RNA expression levels as determined by quantitative real-time RT-PCR. MC3T3 osteoblasts were transfected with siRNA targeting mouse Osx. RNA was isolated 24 h post-transfection and quantitated by real-time RT-PCR. The RNA level from the control siRNA group was normalized to a value of 1. Values were presented as the mean ±S.D. A paired <i>t</i>-test was performed comparing si-control group and si-Osx group. (B) Western analysis of the Osx knockdown. Protein was isolated by acetone precipitation of whole cell lysates and then analyzed by western blot using rabbit anti-VDR or anti-Osx polyclonal antibodies. Beta-actin was used as a loading control.</p

    Both Osx and VDR are upregulated during osteoblast differentiation.

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    <p>MC3T3 osteoblast differentiation experiments were performed in which osteogenic factors were added into the medium, including BMP2, ascorbic acid and β–glycerophosphate. MC3T3 osteoblastic cells were harvested after 0 hr, 8 hr, 24 hr, 72 hr and 104 hr after incubating with differentiation medium. RNA was isolated from cell lysates. RNA levels for VDR and Osx were analyzed by real-time RT-PCR. The level of RNA from 0 hr was normalized to a value of 1. Values were presented as the mean ±S.D.</p
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