Bone Morphogenetic Protein-2 Restores Mineralization in Glucocorticoid-Inhibited MC3T3-E1 Osteoblast Cultures

Luppen, Cynthia A.; Smith, Elisheva; Spevak, Lyudmila; Boskey, Adele L.; Frenkel, Baruch

doi:10.1359/jbmr.2003.18.7.1186

Cited by 105 publications

(123 citation statements)

References 71 publications

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“…Interestingly, the glucocorticoidmediated inhibition of terminal differentiation in this osteoblast culture model is not accompanied by either inhibition of Runx2 (4,31) or stimulation of apoptosis (12,30). Using standard (short-term) transient transfection assays, we were initially unable to recapitulate the strong glucocorticoid-mediated repression seen with the endogenous OC gene in these cells (4,11).…”

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confidence: 86%

“…The present study used MC3T3-E1 osteoblast cells with robust mineralization potential that is strongly inhibited when glucocorticoids are administered at a defined commitment stage around the time of confluency (4,6,7,30). Interestingly, the glucocorticoidmediated inhibition of terminal differentiation in this osteoblast culture model is not accompanied by either inhibition of Runx2 (4,31) or stimulation of apoptosis (12,30).…”

mentioning

confidence: 96%

“…However, repression by competition at basal cis-acting regulatory elements is unlikely shared by the as-yet-unidentified glucocorticoidinhibited osteoblastic genes for which OC serves as a model. In addition, glucocorticoid-mediated repression of transiently transfected OC promoter constructs in the aforementioned studies was typically on the order of ϳ20%, suggesting misrepresentation of molecular mechanisms that confer several-fold repression on the endogenous OC gene (4,11,28).…”

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confidence: 99%

“…2 and 3). Impaired osteoblast function is multifaceted and includes inhibition of the action of growth factors, such as insulin-like growth factor 1 (IGF-1) and IGF binding proteins, hepatocyte growth factor (2,3), and bone morphogenetic protein 2 (4); inhibition of osteoblastic cell proliferation (5), in particular a cell cycle that persists during commitment to the osteoblast phenotype (6,7); a decrease in the messenger RNA (mRNA) and protein levels of bone phenotypic markers, including Runx2 (8) and type I collagen (4,(9)(10)(11); and acceleration of apoptosis (5,12).…”

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confidence: 99%

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Glucocorticoids inhibit osteocalcin transcription in osteoblasts by suppressing Egr2/Krox20‐binding enhancer

Leclerc¹,

Noh²,

Khokhar³

et al. 2005

Arthritis & Rheumatism

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Objective. Glucocorticoids are widely used for the management of rheumatoid arthritis. Osteoporosis is a major side effect of glucocorticoid therapy and is attributable to inhibition of bone formation. We developed an osteoblast culture system in which glucocorticoids strongly inhibit development of the osteoblast phenotype, including expression of the bone-specific osteocalcin (OC) gene. Using this gene as a model, the goal of this study was to discover glucocorticoid-sensitive transcriptional mechanisms in osteoblasts.Methods. Dexamethasone (DEX; 1 M) was administered to murine MC3T3-E1 osteoblastic cultures under conditions that inhibit mineralized extracellular matrix formation and OC messenger RNA levels by >10-fold. Because standard (short-term) transient transfection assays with OC promoter-reporter constructs did not recapitulate the strong DEX-mediated repression, mapping of OC negative glucocorticoid response elements (GREs) was performed initially by stable transfection and then with long-term transient transfection assays. Transcription factor binding to the OC negative GRE was studied by electrophoretic mobility shift assays.Results. Several-fold repression of OC-luciferase constructs was recapitulated in stable and long-term transient transfection assays, in which the transfected cells were allowed to progress to a sufficiently advanced developmental stage. Analysis of a 5 promoter deletion series mapped an OC negative GRE to a 15-bp G/C-rich motif (؊161/؊147) located just upstream of the binding site for the osteoblast master transcription factor Runx2. Oligonucleotides encompassing this element and MC3T3-E1 cell extracts formed a protein-DNA complex that contained an Egr/Krox family member(s). Complex formation was competed by either an oligonucleotide containing 2 consensus Egr motifs or by anti-Egr2/Krox20 antibodies. Three copies of this Kroxbinding element conferred 20-fold transcriptional activation on the 147-bp basal OC promoter in osteoblasts, and the enhancer activity was inhibited by DEX. Enhancer activity was not observed in 10T1/2 fibroblasts unless these cells were cotransfected with Runx2.Conclusion. An Egr2/Krox20-binding site located immediately upstream of the Runx2 site of the mouse OC promoter was identified as an enhancer in osteoblasts, whose activity is repressed by glucocorticoids. Sequence similarity suggests that such a mechanism is likely operative in both murine and human cells. Because glucocorticoids inhibit Egr2/Krox20 expression in osteoblasts, and because trabecular bone formation is arrested in Egr2/Krox20-knockout mice, the inhibition of Egr2/Krox20 activity likely contributes to glucocorticoid-induced osteoporosis.Since the 1950 Nobel laureates E. Kendall, T. Reichstein, and P. Hench introduced glucocorticoids for the treatment of rheumatoid arthritis, these drugs have been widely used for the treatment of autoimmune and inflammatory diseases. The enthusiasm with which glucocorticoids were initially accepted was quickly dampened upon realization of their deleter...

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confidence: 86%

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confidence: 96%

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confidence: 99%

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confidence: 99%

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Glucocorticoids inhibit osteocalcin transcription in osteoblasts by suppressing Egr2/Krox20‐binding enhancer

Leclerc¹,

Noh²,

Khokhar³

et al. 2005

Arthritis & Rheumatism

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“…[1][2][3][4][5][6] In addition to their direct effects on osteoblasts, they have also been demonstrated to modify the synthesis of growth factors produced by osteoblastic cells. [7][8][9] These effects lead to the suppression of bone formation, a central feature in the pathogenesis of glucocorticoid induced osteoporosis. Hepatocyte growth factor (HGF), which was initially thought to be of mesodermal origin, and its receptor are expressed in osteoblasts and osteoclasts.…”

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confidence: 99%

Hydrocortisone Inhibits Cellular Proliferation by Downregulating Hepatocyte Growth Factor Synthesis in Human Osteoblasts

Tsunashima

Kondo

Matsuda

et al. 2011

Biological & Pharmaceutical Bulletin

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Glucocorticoids impair the proliferation, differentiation, and function of osteoblasts and induce apoptosis in mature osteoblasts. [1][2][3][4][5][6] In addition to their direct effects on osteoblasts, they have also been demonstrated to modify the synthesis of growth factors produced by osteoblastic cells. [7][8][9] These effects lead to the suppression of bone formation, a central feature in the pathogenesis of glucocorticoid induced osteoporosis. Hepatocyte growth factor (HGF), which was initially thought to be of mesodermal origin, and its receptor are expressed in osteoblasts and osteoclasts. [10][11][12] Previous studies have shown the inhibitory effects of glucocorticoids on HGF mRNA expression in rat calvarial osteoblasts 13) and human osteoblast-like cells, 14) and the stimulatory effects of HGF on thymidine incorporation in osteoblasts.11) These findings led us to investigate whether the effects of glucocorticoids on cellular proliferation are mediated by HGF in human osteoblastic cells. In this study, using osteoblastic cells (SaM-1) and osteogenic sarcoma cells (SaOS-2, HOS, and MG-63), we demonstrate that HGF functions in an autocrine and/or paracrine manner, resulting in mitogenesis. Furthermore, we show that the inhibitory effects of glucocorticoid on the proliferation of human osteoblastic cells can probably be explained by the concomitant glucocorticoidinduced inhibition of HGF production. MATERIALS AND METHODS MaterialsHydrocortisone was obtained from Sigma (St. Louis, MO, U.S.A.), and recombinant human HGF protein and a human HGF enzyme-linked immunosorbent assay (ELISA) system were obtained from R&D systems (Minneapolis, MN, U.S.A.). The bromodeoxyuridine (BrdU) cell proliferation ELISA was from Roche (Penzberg, Germany). QIAzol lysis reagent was obtained from Qiagen (Hilden,was obtained from CalBiochem (San Diego, CA, U.S.A.). Normal human osteoblasts (SaM-1 cells) were kindly provided by Dr. Koshihara (Tokyo Metropolitan Institute of Gerontology), who prepared them from an explant of ulnar periosteum obtained from a 20-year-old male patient undergoing curative surgery who gave his informed consent for its experimental use. SaM-1 cells have a mitotic life span of 34 population doubling levels (PDL), 15) and we used them at 23-24 PDL in our experiments. HOS and SaOS-2 human osteogenic sarcoma cells were obtained from the RIKEN Cell Bank (Tsukuba, Japan). MG-63 human osteogenic sarcoma cells were from the American Type Culture Collection (Rockville, MD, U.S.A.). Alpha-modified minimum essential medium (a-MEM) was purchased from Gibco BRL (Grand Island, NY, U.S.A.), and fetal calf serum (FCS) was obtained from Cell Culture Laboratories (Cleveland, OH, U.S.A.) and Irvine Scientific (Santa Ana, California, U.S.A.). All other chemicals used were of reagent grade.Cell Culture The medium used for all cell lines was a-MEM containing 10% fetal calf serum (FCS), and the cells were incubated in a humidified incubator at 37°C in 95% air and 5% CO 2 . In an analysis of proliferation, SaM-1 cells were seed...

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Infrared Microscopy and Imaging of Hard and Soft Tissues

Menedelsohn¹,

Boskey²,

Camacho³

2005

Spectrochemical Analysis Using Infrared Multichannel Detectors

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Bone Morphogenetic Protein-2 Restores Mineralization in Glucocorticoid-Inhibited MC3T3-E1 Osteoblast Cultures

Cited by 105 publications

References 71 publications

Glucocorticoids inhibit osteocalcin transcription in osteoblasts by suppressing Egr2/Krox20‐binding enhancer

Glucocorticoids inhibit osteocalcin transcription in osteoblasts by suppressing Egr2/Krox20‐binding enhancer

Hydrocortisone Inhibits Cellular Proliferation by Downregulating Hepatocyte Growth Factor Synthesis in Human Osteoblasts

Infrared Microscopy and Imaging of Hard and Soft Tissues

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