scholarly journals Inhibition of BMP2-Induced Bone Formation by the p65 Subunit of NF-κB via an Interaction With Smad4

2014 ◽  
Vol 28 (9) ◽  
pp. 1460-1470 ◽  
Author(s):  
Shizu Hirata-Tsuchiya ◽  
Hidefumi Fukushima ◽  
Takenobu Katagiri ◽  
Satoshi Ohte ◽  
Masashi Shin ◽  
...  
Keyword(s):  
Dna Binding ◽  
Bone Formation ◽  
Osteoblast Differentiation ◽  
Bone Diseases ◽  
Bmp Signaling ◽  
Wild Type ◽  
Smad Pathway ◽  
Wild Type Mefs

Bone morphogenic proteins (BMPs) stimulate bone formation in vivo and osteoblast differentiation in vitro via a Smad signaling pathway. Recent findings revealed that the activation of nuclear factor-κB (NF-κB) inhibits BMP-induced osteoblast differentiation. Here, we show that NF-κB inhibits BMP signaling by directly targeting the Smad pathway. A selective inhibitor of the classic NF-κB pathway, BAY11–770682, enhanced BMP2-induced ectopic bone formation in vivo. In mouse embryonic fibroblasts (MEFs) prepared from mice deficient in p65, the main subunit of NF-κB, BMP2, induced osteoblastic differentiation via the Smad complex to a greater extent than that in wild-type MEFs. In p65−/− MEFs, the BMP2-activated Smad complex bound much more stably to the target element than that in wild-type MEFs without affecting the phosphorylation levels of Smad1/5/8. Overexpression of p65 inhibited BMP2 activity by decreasing the DNA binding of the Smad complex. The C-terminal region, including the TA2 domain, of p65 was essential for inhibiting the BMP-Smad pathway. The C-terminal TA2 domain of p65 associated with the MH1 domain of Smad4 but not Smad1. Taken together, our results suggest that p65 inhibits BMP signaling by blocking the DNA binding of the Smad complex via an interaction with Smad4. Our study also suggests that targeting the association between p65 and Smad4 may help to promote bone regeneration in the treatment of bone diseases.

scholarly journals The Type 2 Cannabinoid Receptor Regulates Bone Mass and Ovariectomy-Induced Bone Loss by Affecting Osteoblast Differentiation and Bone Formation

Endocrinology ◽  
2011 ◽  
Vol 152 (6) ◽  
pp. 2141-2149 ◽  
Author(s):  
Antonia Sophocleous ◽  
Euphemie Landao-Bassonga ◽  
Robert J. van‘t Hof ◽  
Aymen I. Idris ◽  
Stuart H. Ralston
Keyword(s):  
Bone Loss ◽  
Bone Formation ◽  
Bone Mass ◽  
Osteoblast Differentiation ◽  
Cannabinoid Receptor ◽  
Nodule Formation ◽  
Wild Type

The type 2 cannabinoid receptor (CB2) has been reported to regulate bone mass and bone turnover but the mechanisms responsible are incompletely understood. In this study we investigated the role that the CB2 pathway plays in bone metabolism using a combination of genetic and pharmacological approaches. Bone mass and turnover were normal in young mice with targeted inactivation of CB2 receptor (CB2−/−), but by 12 months of age, they had developed high-turnover osteoporosis with relative uncoupling of bone resorption from bone formation. Primary osteoblasts from CB2−/− mice had a reduced capacity to form bone nodules in vitro when compared with cells from wild-type littermates and also had impaired PTH-induced alkaline phosphatase (ALP) activity. The CB2-selective agonist HU308 stimulated bone nodule formation in wild-type osteoblasts but had no effect in CB2−/− osteoblasts. Further studies in MC3T3-E1 osteoblast like cells showed that HU308 promoted cell migration and activated ERK phosphorylation, and these effects were blocked by the CB2 selective inverse agonist AM630. Finally, HU308 partially protected against ovariectomy induced bone loss in wild-type mice in vivo, primarily by stimulating bone formation, whereas no protective effects were observed in ovariectomized CB2−/− mice. These studies indicate that the CB2 regulates osteoblast differentiation in vitro and bone formation in vivo.

Biochemical and genetic characterization of a yeast TFIID mutant that alters transcription in vivo and DNA binding in vitro

1992 ◽  
Vol 12 (5) ◽  
pp. 2372-2382
Author(s):  
K M Arndt ◽  
S L Ricupero ◽  
D M Eisenmann ◽  
F Winston
Keyword(s):  
Amino Acid ◽  
Dna Binding ◽  
Direct Repeat ◽  
Single Amino Acid ◽  
Wild Type ◽  
Dna Binding Specificity ◽  
Selection For

A mutation in the gene that encodes Saccharomyces cerevisiae TFIID (SPT15), which was isolated in a selection for mutations that alter transcription in vivo, changes a single amino acid in a highly conserved region of the second direct repeat in TFIID. Among eight independent spt15 mutations, seven cause this same amino acid change, Leu-205 to Phe. The mutant TFIID protein (L205F) binds with greater affinity than that of wild-type TFIID to at least two nonconsensus TATA sites in vitro, showing that the mutant protein has altered DNA binding specificity. Site-directed mutations that change Leu-205 to five different amino acids cause five different phenotypes, demonstrating the importance of this amino acid in vivo. Virtually identical phenotypes were observed when the same amino acid changes were made at the analogous position, Leu-114, in the first repeat of TFIID. Analysis of these mutations and additional mutations in the most conserved regions of the repeats, in conjunction with our DNA binding results, suggests that these regions of the repeats play equivalent roles in TFIID function, possibly in TATA box recognition.

scholarly journals Hedgehog Signaling Inhibition by Smoothened Antagonist BMS-833923 Reduces Osteoblast Differentiation and Ectopic Bone Formation of Human Skeletal (Mesenchymal) Stem Cells

2019 ◽  
Vol 2019 ◽  
pp. 1-12 ◽  
Author(s):  
Nihal AlMuraikhi ◽  
Nuha Almasoud ◽  
Sarah Binhamdan ◽  
Ghaydaa Younis ◽  
Dalia Ali ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bone Formation ◽  
Osteoblast Differentiation ◽  
Global Gene Expression ◽  
Ectopic Bone Formation ◽  
Ectopic Bone ◽  
In Vitro Mineralization ◽  
Global Gene Expression Profiling

Background. Hedgehog (Hh) signaling is essential for osteoblast differentiation of mesenchymal progenitors during endochondral bone formation. However, the critical role of Hh signaling during adult bone remodeling remains to be elucidated. Methods. A Smoothened (SMO) antagonist/Hedgehog inhibitor, BMS-833923, identified during a functional screening of a stem cell signaling small molecule library, was investigated for its effects on the osteoblast differentiation of human skeletal (mesenchymal) stem cells (hMSC). Alkaline phosphatase (ALP) activity and Alizarin red staining were employed as markers for osteoblast differentiation and in vitro mineralization capacity, respectively. Global gene expression profiling was performed using the Agilent® microarray platform. Effects on in vivo ectopic bone formation were assessed by implanting hMSC mixed with hydroxyapatite-tricalcium phosphate granules subcutaneously in 8-week-old female nude mice, and the amount of bone formed was assessed using quantitative histology. Results. BMS-833923, a SMO antagonist/Hedgehog inhibitor, exhibited significant inhibitory effects on osteoblast differentiation of hMSCs reflected by decreased ALP activity, in vitro mineralization, and downregulation of osteoblast-related gene expression. Similarly, we observed decreased in vivo ectopic bone formation. Global gene expression profiling of BMS-833923-treated compared to vehicle-treated control cells, identified 348 upregulated and 540 downregulated genes with significant effects on multiple signaling pathways, including GPCR, endochondral ossification, RANK-RANKL, insulin, TNF alpha, IL6, and inflammatory response. Further bioinformatic analysis employing Ingenuity Pathway Analysis revealed significant enrichment in BMS-833923-treated cells for a number of functional categories and networks involved in connective and skeletal tissue development and disorders, e.g., NFκB and STAT signaling. Conclusions. We identified SMO/Hedgehog antagonist (BMS-833923) as a powerful inhibitor of osteoblastic differentiation of hMSC that may be useful as a therapeutic option for treating conditions associated with high heterotopic bone formation and mineralization.

scholarly journals CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

2004 ◽  
Vol 279 (44) ◽  
pp. 45887-45896 ◽  
Author(s):  
Mark J. Demma ◽  
Serena Wong ◽  
Eugene Maxwell ◽  
Bimalendu Dasmahapatra
Keyword(s):  
Dna Binding ◽  
Mammalian Cells ◽  
P53 Protein ◽  
Binding Activity ◽  
Mutant P53 ◽  
Dependent Manner ◽  
Wild Type ◽  
Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

scholarly journals Fibrillin-1 and -2 differentially modulate endogenous TGF-β and BMP bioavailability during bone formation

2010 ◽  
Vol 190 (6) ◽  
pp. 1107-1121 ◽  
Author(s):  
Harikiran Nistala ◽  
Sui Lee-Arteaga ◽  
Silvia Smaldone ◽  
Gabriella Siciliano ◽  
Luca Carta ◽  
...  
Keyword(s):  
Bone Formation ◽  
Transforming Growth Factor ◽  
Bone Mineralization ◽  
Bmp Signaling ◽  
Direct Role ◽  
Morphogenetic Protein ◽  
Fibrillin 1 ◽  
Osteoblast Maturation

Extracellular regulation of signaling by transforming growth factor (TGF)–β family members is emerging as a key aspect of organ formation and tissue remodeling. In this study, we demonstrate that fibrillin-1 and -2, the structural components of extracellular microfibrils, differentially regulate TGF-β and bone morphogenetic protein (BMP) bioavailability in bone. Fibrillin-2–null (Fbn2−/−) mice display a low bone mass phenotype that is associated with reduced bone formation in vivo and impaired osteoblast maturation in vitro. This Fbn2−/− phenotype is accounted for by improper activation of latent TGF-β that selectively blunts expression of osterix, the transcriptional regulator of osteoblast maturation, and collagen I, the structural template for bone mineralization. Cultured osteoblasts from Fbn1−/− mice exhibit improper latent TGF-β activation as well, but mature faster because of increased availability of otherwise matrix-bound BMPs. Additional in vitro evidence excludes a direct role of microfibrils in supporting mineral deposition. Together, these findings identify the extracellular microfibrils as critical regulators of bone formation through the modulation of endogenous TGF-β and BMP signaling.

ONO-1301 Enhances in vitro Osteoblast Differentiation and in vivo Bone Formation Induced by Bone Morphogenetic Protein

Spine ◽  
2018 ◽  
Vol 43 (11) ◽  
pp. E616-E624 ◽  
Author(s):  
Sadaaki Kanayama ◽  
Takashi Kaito ◽  
Kazuma Kitaguchi ◽  
Hiroyuki Ishiguro ◽  
Kunihiko Hashimoto ◽  
...  
Keyword(s):  
Bone Formation ◽  
Bone Morphogenetic Protein ◽  
Osteoblast Differentiation ◽  
Morphogenetic Protein ◽  
In Vivo Bone Formation

scholarly journals Regulation of cell proliferation by ERK and signal-dependent nuclear translocation of ERK is dependent on Tm5NM1-containing actin filaments

2015 ◽  
Vol 26 (13) ◽  
pp. 2475-2490 ◽  
Author(s):  
Galina Schevzov ◽  
Anthony J. Kee ◽  
Bin Wang ◽  
Vanessa B. Sequeira ◽  
Jeff Hook ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Actin Filaments ◽  
Nuclear Translocation ◽  
Genetic Manipulation ◽  
Wild Type ◽  
Proximity Ligation ◽  
Mouse Embryo Fibroblasts ◽  
Wild Type Mefs

ERK-regulated cell proliferation requires multiple phosphorylation events catalyzed first by MEK and then by casein kinase 2 (CK2), followed by interaction with importin7 and subsequent nuclear translocation of pERK. We report that genetic manipulation of a core component of the actin filaments of cancer cells, the tropomyosin Tm5NM1, regulates the proliferation of normal cells both in vitro and in vivo. Mouse embryo fibroblasts (MEFs) lacking Tm5NM1, which have reduced proliferative capacity, are insensitive to inhibition of ERK by peptide and small-molecule inhibitors, indicating that ERK is unable to regulate proliferation of these knockout (KO) cells. Treatment of wild-type MEFs with a CK2 inhibitor to block phosphorylation of the nuclear translocation signal in pERK resulted in greatly decreased cell proliferation and a significant reduction in the nuclear translocation of pERK. In contrast, Tm5NM1 KO MEFs, which show reduced nuclear translocation of pERK, were unaffected by inhibition of CK2. This suggested that it is nuclear translocation of CK2-phosphorylated pERK that regulates cell proliferation and this capacity is absent in Tm5NM1 KO cells. Proximity ligation assays confirmed a growth factor–stimulated interaction of pERK with Tm5NM1 and that the interaction of pERK with importin7 is greatly reduced in the Tm5NM1 KO cells.

scholarly journals Zfp521 antagonizes Runx2, delays osteoblast differentiation in vitro, and promotes bone formation in vivo

Bone ◽  
2009 ◽  
Vol 44 (4) ◽  
pp. 528-536 ◽  
Author(s):  
Meilin Wu ◽  
Eric Hesse ◽  
Frederic Morvan ◽  
Jian-Ping Zhang ◽  
Diego Correa ◽  
...  
Keyword(s):  
Bone Formation ◽  
Osteoblast Differentiation

scholarly journals Porcine Collagen–Bone Composite Induced Osteoblast Differentiation and Bone Regeneration In Vitro and In Vivo

Polymers ◽  
2020 ◽  
Vol 12 (1) ◽  
pp. 93 ◽  
Author(s):  
Eisner Salamanca ◽  
Chia Chen Hsu ◽  
Wan Ling Yao ◽  
Cheuk Sing Choy ◽  
Yu Hwa Pan ◽  
...  
Keyword(s):  
Bone Formation ◽  
Bone Regeneration ◽  
Osteoblast Differentiation ◽  
Critical Size ◽  
Guided Bone Regeneration ◽  
Autogenous Bone ◽  
Critical Size Defects ◽  
Bone Formation Markers

Due to autogenous bone limitations, some substitute bone grafts were developed. Collagenated porcine graft (CPG) is able to regenerate new bone, although the number of studies is insufficient, highlighting the need for future studies to better understand the biomaterial. In order to understand better CPG′s possible dental guided bone regeneration indications, the aim of this work was to determine CPG′s biological capacity to induce osteoblast differentiation in vitro and guided bone regeneration in vivo, whilst being compared with commercial hydroxyapatite and beta tricalcium phosphate (HA/β-TCP) and porcine graft alone. Cell cytotoxicity (WST-1), alkaline phosphatase activity (ALP), and real-time polymerase chain reaction (qPCR) were assessed in vitro. Critical size defects of New Zealand white rabbits were used for the in vivo part, with critical size defect closures and histological analyses. WST-1 and ALP indicated that CPG directly stimulated a greater proliferation and confluency of cells with osteoblastic differentiation in vitro. Gene sequencing indicated stable bone formation markers, decreased resorption makers, and bone remodeling coupling factors, making the transition from osteoclast to osteoblast expression at the end of seven days. CPG resulted in the highest new bone regeneration by osteoconduction in critical size defects of rabbit calvaria at eight weeks. Nonetheless, all biomaterials achieved nearly complete calvaria defect closure. CPG was found to be osteoconductive, like porcine graft and HA/β-TCP, but with higher new bone formation in critical size defects of rabbit calvaria at eight weeks. CPG can be used for different dental guided bone regeneration procedures; however, further studies are necessary.

scholarly journals ER stress arm XBP1s plays a pivotal role in proteasome inhibition-induced bone formation

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Dan Zhang ◽  
Kim De Veirman ◽  
Rong Fan ◽  
Qiang Jian ◽  
Yuchen Zhang ◽  
...  
Keyword(s):  
Bone Formation ◽  
Er Stress ◽  
Osteogenic Differentiation ◽  
Osteoblast Differentiation ◽  
Bone Destruction ◽  
Proteasome Inhibition ◽  
Stress Signaling ◽  
Alizarin Red

Abstract Background Bone destruction is a hallmark of multiple myeloma (MM). It has been reported that proteasome inhibitors (PIs) can reduce bone resorption and increase bone formation in MM patients, but the underlying mechanisms remain unclear. Methods Mesenchymal stem cells (MSCs) were treated with various doses of PIs, and the effects of bortezomib or carfilzomib on endoplasmic reticulum (ER) stress signaling pathways were analyzed by western blotting and real-time PCR. Alizarin red S (ARS) and alkaline phosphatase (ALP) staining were used to determine the osteogenic differentiation in vitro. Specific inhibitors targeting different ER stress signaling and a Tet-on inducible overexpressing system were used to validate the roles of key ER stress components in regulating osteogenic differentiation of MSCs. Chromatin immunoprecipitation (ChIP) assay was used to evaluate transcription factor-promoter interaction. MicroCT was applied to measure the microarchitecture of bone in model mice in vivo. Results We found that both PERK-ATF4 and IRE1α-XBP1s ER stress branches are activated during PI-induced osteogenic differentiation. Inhibition of ATF4 or XBP1s signaling can significantly impair PI-induced osteogenic differentiation. Furthermore, we demonstrated that XBP1s can transcriptionally upregulate ATF4 expression and overexpressing XBP1s can induce the expression of ATF4 and other osteogenic differentiation-related genes and therefore drive osteoblast differentiation. MicroCT analysis further demonstrated that inhibition of XBP1s can strikingly abolish bortezomib-induced bone formation in mouse. Conclusions These results demonstrated that XBP1s is a master regulator of PI-induced osteoblast differentiation. Activation of IRE1α-XBP1s ER stress signaling can promote osteogenesis, thus providing a novel strategy for the treatment of myeloma bone disease.

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