Common Regulation of Growth Arrest and Differentiation of Osteoblasts by Helix-Loop-Helix Factors

ABSTRACT Cellular differentiation entails the coordination of cell cycle arrest and tissue-specific gene expression. We investigated the involvement of basic helix-loop-helix (bHLH) factors in differentiation of osteoblasts using the human osteoblastic cell line MG63. Serum starvation induced growth arrest at G1 phase, accompanied by expression of cyclin-dependent kinase inhibitor p21WAF1/Cip1. Reporter assays with the p21 gene promoter demonstrated that the combination of E2A (E12 or E47) and coactivator CBP was responsible for p21 induction independent of p53. Twist inhibited E2A-CBP-dependent activation of the exogenous and endogenous p21 promoters. Ids similarly inhibited the exogenously transfected p21 promoter; however less antagonistic effect on the endogenous p21 promoter was observed. Twist was predominantly present in nuclei in MG63 cells growing in complete medium, while it localized mainly in the cytoplasm after serum starvation. The fibroblast growth factor receptor 3 gene (FGFR3), which generates signals leading to differentiation of osteoblasts, was found to be controlled by the same transcriptional regulation as the p21 gene. E2A and Twist influenced alkaline phosphatase expression, a consensus marker of osteoblast differentiation. Expression of E2A and FGFR3 was seen at the location of osteoblast differentiation in the calvaria of mouse embryos, implicating bHLH molecules in physiological osteoblast differentiation. These results demonstrate that a common regulatory system is involved in at least two distinct steps in osteoblastic differentiation. Our results also provide the molecular basis of Saethre-Chotzen syndrome, caused by mutations of the TWISTand FGFR3 genes.

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Probiotic Propionibacterium freudenreichii MJ2 Enhances Osteoblast Differentiation and Mineralization by Increasing the OPG/RANKL Ratio

Microorganisms ◽

10.3390/microorganisms9040673 ◽

2021 ◽

Vol 9 (4) ◽

pp. 673

Author(s):

Jiah Yeom ◽

Seongho Ma ◽

Young-Hee Lim

Keyword(s):

Signaling Pathway ◽

Osteoblast Differentiation ◽

Surface Proteins ◽

Bone Morphogenetic Protein 2 ◽

Ovariectomized Rats ◽

Osteoblastic Cell ◽

Enhancement Effect ◽

Propionibacterium Freudenreichii ◽

Alizarin Red ◽

Osteoblastic Cell Line

Osteoblast differentiation is important for the development of bone and the maintenance of bone density. Propionibacterium freudenreichii is a probiotic with an anti-inflammatory property. The aim of this study was to investigate the enhancement effect of P. freudenreichii MJ2 (MJ2) isolated from raw milk on osteoblast differentiation, mineralization, and its signaling pathway. For in vitro and in vivo experiments, human fetal osteoblastic cell line hFOB 1.19 and an ovariectomized rat model were used, respectively. Expression levels of genes and proteins related to osteoblast differentiation and mineralization were measured by real-time polymerase chain reaction (qPCR) and Western blotting, respectively. Alizarin red S staining was performed to measure osteoblast mineralization. Heat-killed MJ2 (hkMJ2)-treated cells showed significantly increased osteoblast differentiation via an increase in the osteoprotegerin (OPG)/receptor activator of nuclear factor-κB ligand (RANKL) ratio and significantly increased osteoblast mineralization by stimulating the expression of bone morphogenetic protein 2 and runt-related transcription factor 2. Additionally, oral administration of live or heat-killed MJ2 to ovariectomized rats inhibited osteoporosis-induced bone loss. Specifically, surface proteins isolated from MJ2 promoted osteoblast differentiation and mineralization. In conclusion, MJ2 enhanced osteoblast differentiation and mineralization through the OPG/RANKL signaling pathway and the effective component of MJ2 might be its surface proteins.

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Expression of ID, a negative regulator of helix-loop-helix DNA binding proteins, is down-regulated at confluence and enhanced by dexamethasone in a mouse osteoblastic cell line, MC3T3E1

Biochemical and Biophysical Research Communications ◽

10.1016/s0006-291x(05)81322-1 ◽

1991 ◽

Vol 180 (3) ◽

pp. 1194-1199 ◽

Cited By ~ 26

Author(s):

Toshiko Ogata ◽

Masaki Noda

Keyword(s):

Dna Binding ◽

Cell Line ◽

Binding Proteins ◽

Dna Binding Proteins ◽

Negative Regulator ◽

Osteoblastic Cell ◽

Osteoblastic Cell Line ◽

Helix Loop Helix

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Gas2, a growth arrest-specific protein, is a component of the microfilament network system

The Journal of Cell Biology ◽

10.1083/jcb.117.6.1251 ◽

1992 ◽

Vol 117 (6) ◽

pp. 1251-1261 ◽

Cited By ~ 73

Author(s):

C Brancolini ◽

S Bottega ◽

C Schneider

Keyword(s):

Growth Arrest ◽

Specific Protein ◽

Protein Product ◽

Serum Starvation ◽

Specific Gene ◽

Relative Level ◽

Actin Fiber ◽

Phosphorylation Mechanism ◽

Microfilament System ◽

Nih 3T3

In this report we analyze the protein product of a growth arrest-specific gene, gas2, by means of an affinity-purified antibody raised against the protein produced in bacteria. The regulation of Gas2 biosynthesis reflects the pattern of mRNA expression (Schneider, C., R. King, and L. Philipson. 1988. Cell. 54:787-793): its relative level is tightly associated with growth arrest. Gas2 seems to be regulated also at the posttranslational level via a phosphorylation mechanism. Gas2 is well conserved during the evolution with the same apparent molecular mass (36 kD) between mouse and human. We also demonstrate that Gas2 is a component of the microfilament system. It colocalizes with actin fiber, at the cell border and also along the stress fiber, in growth-arrested NIH 3T3 cells. The pattern of distribution, detected in arrested cells, can also be observed in growing cells when they are microinjected with the purified GST-Gas2 protein. In none of the analyzed oncogene-transformed NIH 3T3 cell lines was Gas2 expression induced under serum starvation.

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Two distinct osteoblast-specific cis-acting elements control expression of a mouse osteocalcin gene.

Molecular and Cellular Biology ◽

10.1128/mcb.15.4.1858 ◽

1995 ◽

Vol 15 (4) ◽

pp. 1858-1869 ◽

Cited By ~ 441

Author(s):

P Ducy ◽

G Karsenty

Keyword(s):

Cell Line ◽

Cell Lines ◽

Osteoblastic Cell ◽

Specific Gene ◽

Osteoblastic Cell Line ◽

Specific Element ◽

Cis Acting ◽

Nuclear Extracts ◽

Osteocalcin Gene ◽

Primary Osteoblasts

Osteoblasts are cells of mesodermal origin that play a pivotal role during bone growth and mineralization. The mechanisms governing osteoblast-specific gene expression are still unknown. To understand these mechanisms, we analyzed the cis-acting elements of mouse osteocalcin gene 2 (mOG2), the best-characterized osteoblast-specific gene, by DNA transfection experiments in osteoblastic and nonosteoblastic cell lines and by DNA-binding assays. 5' deletion analysis of an mOG2 promoter-luciferase chimeric gene showed that a region located between -147 and -34 contained most if not all of the regulatory elements required for osteoblast-specific expression. Three different binding sites, called A, B, and C, for factors present in nuclear extracts of osteoblasts were identified in this short promoter by DNase I footprint assays. In gel retardation assays, the A element, located between bp -64 and -47, bound a factor present only in nuclear extracts of osteoblastic cell lines and nonmineralizing primary osteoblasts. The B element, located between bp -110 and -83, bound a ubiquitously expressed factor. The C element, located between bp -146 and -132, bound a factor present only in nuclear extracts of osteoblastic cell lines and nonmineralizing and mineralizing primary osteoblasts. When cloned upstream of a minimum osteocalcin promoter or a heterologous promoter, multimers of the A element strongly increased the activities of these promoters in osteoblastic cell lines at two different stages of differentiation but in no other cell line; we named this element osteocalcin-specific element 1 (OSE1). Multimers of the C element increased the activities of these promoters predominantly in a differentiated osteoblastic cell line; we named this element OSE2. This study demonstrates that two distinct cis-acting elements are responsible for osteoblast expression of mOG2 and provides for the first time a functional characterization of osteoblast-specific cis-acting elements. We speculate that these two elements may be important at several stages of osteoblast differentiation.

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Transcriptional stimulation of the retina-specific QR1 gene upon growth arrest involves a Maf-related protein.

Molecular and Cellular Biology ◽

10.1128/mcb.15.10.5563 ◽

1995 ◽

Vol 15 (10) ◽

pp. 5563-5575 ◽

Cited By ~ 21

Author(s):

C Pouponnot ◽

M Nishizawa ◽

G Calothy ◽

A Pierani

Keyword(s):

Leucine Zipper ◽

Ectopic Expression ◽

Growth Arrest ◽

Protein S ◽

Serum Starvation ◽

Specific Gene ◽

Related Protein ◽

Proliferating Cells ◽

Stimulation Of ◽

Transcriptional Stimulation

The avian neural retina (NR) is derived from proliferating neuroectodermal precursors which differentiate after terminal mitosis and become organized in cell strata. Proliferation of postmitotic NR cells can be induced by infection with Rous sarcoma virus (RSV) and requires the expression of a functional v-Src protein. QR1 is a retina-specific gene expressed exclusively at the stage of growth arrest and differentiation during retinal development. In NR cells infected with tsPA101, an RSV mutant conditionally defective in pp60v-src mitogenic capacity, QR1 expression is downregulated in proliferating cells at 37 degrees C and is fully restored when the cells become quiescent as a result of pp60v-src inactivation at 41 degrees C. We were able to arrest proliferation of tsPA101-infected quail NR cells expressing an active v-Src protein by serum starvation at 37 degrees C. This allowed us to investigate the role of cell growth in regulating QR1 transcription. We report that QR1 transcription is stimulated in growth-arrested cells at 37 degrees C compared with that in proliferating cells maintained at the same temperature. Growth arrest-dependent stimulation of QR1 transcription requires the integrity of the A box, a previously characterized cis-acting element responsible for QR1 transcriptional stimulation upon v-Src inactivation and during retinal differentiation. We also show that formation of the C1 complex on the A box is increased upon growth arrest by serum starvation in the presence of an active v-Src oncoprotein. Thus, the C1 complex represents an important link between cell cycle and developmental control of QR1 gene transcription during NR differentiation and RSV infection. By using antibodies directed against different Maf proteins of the leucine zipper family and competition with Maf consensus site-containing oligonucleotides in a gel shift assay, we show that the C1 complex is likely to contain a Maf-related protein. We also show that a purified bacterially expressed v-Maf protein is able to bind the A box and that the level of a 43-kDa Maf-related protein is increased upon growth arrest in infected retinal cells. Moreover, ectopic expression of c-mafI, c-mafII, and mafB cDNAs in quiescent tsPA101-infected quail NR cells is able to stimulate transcription of a QR1 reporter gene through the A box. Therefore, QR1 appears to be the first target gene for a Maf-related protein(s) in the NR.

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Inhibitory Effects of a Reengineered Anthrax Toxin on Canine and Human Osteosarcoma Cells

Toxins ◽

10.3390/toxins12100614 ◽

2020 ◽

Vol 12 (10) ◽

pp. 614

Author(s):

Jonathan Mackowiak da Fonseca ◽

Ivone Izabel Mackowiak da Fonseca ◽

Marcia Kazumi Nagamine ◽

Cristina de Oliveira Massoco ◽

Adriana Tomoko Nishiya ◽

...

Keyword(s):

Cell Cycle ◽

Wound Healing ◽

Anthrax Toxin ◽

Osteoblastic Cell ◽

Wound Healing Assay ◽

Inhibitory Effects ◽

Osteoblastic Cell Line ◽

Human Osteosarcoma Cells ◽

Mg63 Cells ◽

Migration Capacity

Canine and human osteosarcomas (OSA) share similarities. Novel therapies are necessary for these tumours. The Bacillus anthracis toxin was reengineered to target and kill cells with high expressions of matrix metalloproteinases (MMPs) and urokinase plasminogen activator (uPA). Since canine OSA express MMPs and uPA, we assessed whether the reengineered toxin could show efficacy against these tumours. Two OSA cell lines (canine D17 and human MG63) and a non-neoplastic canine osteoblastic cell line (COBS) were used. Cells were treated with different concentrations of the reengineered anthrax toxin and cell viability was quantified using MTT assay. The cell cycle, apoptosis, and necrosis were analysed by flow cytometry. The wound-healing assay was performed to quantify the migration capacity of treated cells. D17 and MG63 cells had significantly decreased viability after 24 h of treatment. Cell cycle analysis revealed that OSA cells underwent apoptosis when treated with the toxin, whereas COBS cells arrested in the G1 phase. The wound-healing assay showed that D17 and MG63 cells had a significantly reduced migration capacity after treatment. These results point for the first time towards the in vitro inhibitory effects of the reengineered anthrax toxin on OSA cells; this reengineered toxin could be further tested as a new therapy for OSA.

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