Dependence of induction of osteocalcin gene expression on the presence of wild‐type p53 in a murine osteosarcoma cell line

Chandar, Nalini; Campbell, Phil G.; Novak, Josef F.; Smith, Martin L.

doi:10.1002/mc.2940080413

Cited by 17 publications

(29 citation statements)

References 30 publications

(20 reference statements)

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“…In previous studies carried out with a murine osteosarcoma cell line showing characteristics of mature osteoblasts, we found that loss of p53 affects the ability of the cells to secrete osteocalcin, and that this property is regained after stable transfection of the cells with wild type p53 [7]. In other studies with osteoblast cell lines capable of displaying features of bone differentiation, we also observed p53 to have a distinct transcription-activating role, unrelated to growth arrest [33].…”

Section: Introductionsupporting

confidence: 56%

“…the process of bone differentiation, osteoblasts proliferate, secrete matrix proteins and mineralize. We have found modulation of several bone specific genes to be related to the p53 status of the cell ( [7] and preliminary data).…”

Section: Introductionmentioning

confidence: 76%

“…These results contrast with our observations of osteocalcin gene expression. Osteocalcin gene expression requires wild type p53 function and was not expressed when p53 was mutated [7,8,33]. BMP-2 expression did not require wild type p53 function, since both cell types (p53 mutant and wild type) express BMP-2, but its upregulation during differentiation was only detected in cells expressing wild type p53.…”

Section: Transient Transfection Of Wild Type P53 Is Not Sufficient Tomentioning

confidence: 93%

“…In our initial studies, we stably transfected wild type p53 into null osteosarcoma cells and generated a line expressing low levels of p53, which appeared to be sufficient for down regulation of tumor growth when injected into syngeniec hosts [7]. Using a stronger promoter we were able to generate several cell lines stably expressing wild type, temperature sensitive and other mutant expressing cell lines.…”

Section: Wild Type P53 Expression In Osteosarcomas Can Directly Affecmentioning

confidence: 99%

“…These cells express alkaline phosphatase, alpha 1 collagen and osteocalcin during differentiation [7]. To determine changes in BMP gene expression, we exposed these cells to DP media for about 3 weeks and performed gene expression analyses at different time intervals.…”

Section: Several Bmps Show Modulation During Differentiation In Ros 1mentioning

confidence: 99%

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Relationship of bone morphogenetic protein expression during osteoblast differentiation to wild type p53

et al. 2005

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We have previously shown p53 to have a specific role in osteoblast differentiation by its ability to regulate expression of certain bone specific proteins. In this study, we show mineralized matrix formation in vivo to be directly related to the presence of wild type p53 in osteoblastic osteosarcoma cells. In order to further understand the importance of p53 in differentiation, we investigated the relationship between p53 and Bone Morphogenetic Proteins (BMPs) (BMP 1, 2, 3A, 3B (GDF-lo), 4, 5, 6, 7, 8A and 8B) during osteoblast differentiation. The expression of several BMPs were tested using RNase Protection Assay in differentiating ROS17/ 2.8 osteoblastic osteosarcoma cells. The expression of BMPs 1, 2, 3a, 3b and 7 showed time dependent modulation during ih vitro differentiation. In order to determine if p53 has a role in this process, we used a murine osteosarcoma cell line stably expressing a temperature sensitive p53. Cells were exposed to ascorbic acid and glycerophosphates to hasten in vitro osteoblast differentiation and maintained either at 32 or 37 "C for expression of the wild type or mutant p53 phenotype. The expression of BMP-2, BMP-4 and BMP-7 were modulated in a p53 dependent fashion. We were able to confirm the p53 dependency of BMP-2 independently by RT-PCR. While BMP-2 expression was evident in the presence of both wild type and mutant p53, regulated expression was seen only in cells expressing wild type p53. Transient over expression of wild type p53 did not result in the same BMP-2 response as stable expression showing that the presence of p53 may be important for an orderly development of osteoblast differentiation rather than a direct effect on gene expression. The functional relationship between p53 and these bone specific markers is discussed.

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Section: Introductionsupporting

confidence: 56%

Section: Introductionmentioning

confidence: 76%

Section: Transient Transfection Of Wild Type P53 Is Not Sufficient Tomentioning

confidence: 93%

Section: Wild Type P53 Expression In Osteosarcomas Can Directly Affecmentioning

confidence: 99%

Section: Several Bmps Show Modulation During Differentiation In Ros 1mentioning

confidence: 99%

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Relationship of bone morphogenetic protein expression during osteoblast differentiation to wild type p53

et al. 2005

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p53 transactivity during in vitro osteoblast differentiation in a rat osteosarcoma cell line

et al. 1999

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We previously demonstrated a correlation between wild-type p53 expression and appearance of osteoblastic-specific differentiation characteristics, as evidenced by basal osteocalcin gene expression in a mouse osteosarcoma tumor. The study reported here further explored the possibility of p53's having a distinct transcription-activating role in bone differentiation, in addition to its proposed role in G1 arrest and apoptosis. ROS17/2.3 osteoblastic osteosarcoma cells were stably transfected with a plasmid containing wild-type p53 binding sequences fused to the chloramphenicol acetyltransferase reporter gene. These cells were used to determine the transactivating role of p53 in regulation of osteocalcin gene expression. We chose two conditions under which osteocalcin expression is known to be upregulated: exposure of osteoblastic cells to differentiation-promoting medium and to vitamin D3. Exposure of the transfected cells to differentiation-promoting medium produced an increase in p53 transactivating activity correlating with the appearance of osteocalcin expression after about 1 wk. Vitamin D3 treatment resulted in upregulation of osteocalcin activity without a corresponding change in p53 transactivation activity or expression. In separate experiments, we tested whether changes in osteocalcin expression accompanied changes in p53 activity under conditions of downregulation of cell proliferation mediated by inhibition of DNA synthesis. Hydroxyurea treatment was used to inhibit DNA synthesis and produce growth arrest in osteoblastic cells. Inhibition of osteoblast cell proliferation was associated with a fourfold increase in p53 transactivating activity and a transient increase in osteocalcin steady-state expression. These results demonstrated a close relationship between p53 and osteocalcin and suggested a regulatory role for wild-type p53 in the control of basal osteocalcin gene expression in osteoblasts.

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Expression of osteocalcin and its transcriptional regulators core‐binding factor alpha 1 and MSX2 in osteoid‐forming tumours

Hopyan

Gökgöz

Bell

et al. 1999

Journal Orthopaedic Research

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Osteosarcoma, fibrous dysplasia, and myositis ossificans contain osteoid-producing cells that are not necessarily morphologically typical osteoblasts. Nevertheless, these pathologic cells may share differentiation steps with osteoblasts at the molecular level. Osteocalcin, a bone-specific extracellular matrix protein, is a marker of mature osteoblasts. Osteocalcin is upregulated by the transcription factor core-binding factor alpha 1, which is responsible for commitment to the osteoblastic lineage, and is downregulated by MSX2, a homeobox-containing transcription factor expressed during the early proliferative phase of osteoblast differentiation. Semiquantitative reverse transcription-polymerase chain reaction was used to compare expression levels of osteocalcin, core-binding factor alpha 1, and MSX2 in 34 osteosarcoma, five fibrous dysplasia, and five myositis ossificans specimens, as well as in seven normal cortical bone samples. Despite normal or elevated levels of core-binding factor alpha-1 expression in most specimens, osteocalcin expression was low or undetectable in most cases of osteosarcoma (25 of 34) and myositis ossificans (4 of 5). Single-strand conformation polymorphism and sequencing did not identify any mutations in the DNA-binding domain of core-binding factor alpha 1. However, a high level of MSX2 expression was demonstrated in these lesions, which may inhibit osteocalcin transcription. The presence of moderate levels of osteocalcin in fibrous dysplasia may contribute to the characteristic disconnected appearance of trabeculae in that entity because osteocalcin is a negative regulator of bone formation.

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Dependence of induction of osteocalcin gene expression on the presence of wild‐type p53 in a murine osteosarcoma cell line

Cited by 17 publications

References 30 publications

Relationship of bone morphogenetic protein expression during osteoblast differentiation to wild type p53

Relationship of bone morphogenetic protein expression during osteoblast differentiation to wild type p53

p53 transactivity during in vitro osteoblast differentiation in a rat osteosarcoma cell line

Expression of osteocalcin and its transcriptional regulators core‐binding factor alpha 1 and MSX2 in osteoid‐forming tumours

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