TNF-α suppresses bone sialoprotein (BSP) expression in ROS17/2.8 Cells

Hiroshi Samoto, Emi Shimizu, Yuko Matsuda-Honjo, Ryoichiro Saito, Muneyoshi Yamazaki, Kazutaka Kasai, Shunsuke Furuyama, Hiroshi Sugiya, Jaro Sodek, Yorimasa Ogata

Research output: Contribution to journalArticle

Abstract

Tumor necrosis factor-alpha (TNF-α) is a major mediator of inflammatory responses in many diseases that inhibits bone formation and stimulates bone resorption. To determine molecular mechanisms involved in the suppression of bone formation we have analyzed the effects of TNF-α on BSP gene expression. Bone sialoprotein (BSP) is a mineralized tissue-specific protein that appears to function in the initial mineralization of bone. Previous studies have demonstrated that BSP mRNA expression is essentially restricted to fully-differentiated cells of mineralized connective tissues and that the expression of BSP is developmentally regulated. Treatment of rat osteosarcoma ROS 17/2.8 cells with TNF-α (10ng/ml) for 24 h caused a marked reduction in BSP mRNA levels. The addition of antioxidant N-acetylcysteine (NAC; 20 mM) 30 min prior to stimulation with TNF-α attenuated the inhibition of BSP mRNA levels. Transient transfection analyses, using chimeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene, revealed that TNF-α (10 ng/ml) suppressed expression in all constructs, including a short construct (pLUC3; nts-116 to +60), transfected into ROS17/2.8 cells. Further deletion analysis of the BSP promoter showed that a region within nts -84 to -60 was targeted by TNF-α, the effects which were inhibited by NAC and the tyrosine kinase inhibitor, herbimycin A (HA). Introduction of 2bp mutations in the inverted CCAAT box (ATTGG; nts -50 and -46), a putative cAMP response element (CRE; nts -75 to -68), and a FGF response element (FRE; nts -92 to -85) showed that the TNF-α effects were mediated by the CRE. These results were supported by gel mobility shift assays, using a radiolabeled double-stranded CRE oligonucleotide, which revealed decreased binding of a nuclear protein from TNF-α-stimulated ROS 17/2.8 cells. Further, the inhibitory effect of TNF-α on CRE DNA-protein complex was completely abolished by NAC or HA treatment. These studies, therefore, show that TNF-α suppresses BSP gene transcription through a tyrosine kinase-dependent pathway that generates reactive oxygen species and that the TNF-α effects are mediated by a CRE element in the proximal BSP gene promoter.

Original languageEnglish (US)
Pages (from-to)313-323
Number of pages11
JournalJournal of Cellular Biochemistry
Volume87
Issue number3
DOIs
StatePublished - 2002

Fingerprint

Integrin-Binding Sialoprotein
Tumor Necrosis Factor-alpha
Bone
Genes
Response Elements
Osteogenesis
Protein-Tyrosine Kinases
Messenger RNA
Rats
Tissue
Connective Tissue Cells
Physiologic Calcification
Acetylcysteine
Electrophoretic Mobility Shift Assay
Osteosarcoma
Transcription
Bone Resorption
Nuclear Proteins
Luciferases
Reporter Genes

Keywords

  • Bone sialoprotein
  • Gene regulation
  • Mineralized tissues
  • TNF-α
  • Transcription

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

Cite this

Samoto, H., Shimizu, E., Matsuda-Honjo, Y., Saito, R., Yamazaki, M., Kasai, K., ... Ogata, Y. (2002). TNF-α suppresses bone sialoprotein (BSP) expression in ROS17/2.8 Cells. Journal of Cellular Biochemistry, 87(3), 313-323. https://doi.org/10.1002/jcb.10301

TNF-α suppresses bone sialoprotein (BSP) expression in ROS17/2.8 Cells. / Samoto, Hiroshi; Shimizu, Emi; Matsuda-Honjo, Yuko; Saito, Ryoichiro; Yamazaki, Muneyoshi; Kasai, Kazutaka; Furuyama, Shunsuke; Sugiya, Hiroshi; Sodek, Jaro; Ogata, Yorimasa.

In: Journal of Cellular Biochemistry, Vol. 87, No. 3, 2002, p. 313-323.

Research output: Contribution to journalArticle

Samoto, H, Shimizu, E, Matsuda-Honjo, Y, Saito, R, Yamazaki, M, Kasai, K, Furuyama, S, Sugiya, H, Sodek, J & Ogata, Y 2002, 'TNF-α suppresses bone sialoprotein (BSP) expression in ROS17/2.8 Cells', Journal of Cellular Biochemistry, vol. 87, no. 3, pp. 313-323. https://doi.org/10.1002/jcb.10301
Samoto H, Shimizu E, Matsuda-Honjo Y, Saito R, Yamazaki M, Kasai K et al. TNF-α suppresses bone sialoprotein (BSP) expression in ROS17/2.8 Cells. Journal of Cellular Biochemistry. 2002;87(3):313-323. https://doi.org/10.1002/jcb.10301
Samoto, Hiroshi ; Shimizu, Emi ; Matsuda-Honjo, Yuko ; Saito, Ryoichiro ; Yamazaki, Muneyoshi ; Kasai, Kazutaka ; Furuyama, Shunsuke ; Sugiya, Hiroshi ; Sodek, Jaro ; Ogata, Yorimasa. / TNF-α suppresses bone sialoprotein (BSP) expression in ROS17/2.8 Cells. In: Journal of Cellular Biochemistry. 2002 ; Vol. 87, No. 3. pp. 313-323.
@article{f3ccacc4a03a48ee8517cb6a65323d43,
title = "TNF-α suppresses bone sialoprotein (BSP) expression in ROS17/2.8 Cells",
abstract = "Tumor necrosis factor-alpha (TNF-α) is a major mediator of inflammatory responses in many diseases that inhibits bone formation and stimulates bone resorption. To determine molecular mechanisms involved in the suppression of bone formation we have analyzed the effects of TNF-α on BSP gene expression. Bone sialoprotein (BSP) is a mineralized tissue-specific protein that appears to function in the initial mineralization of bone. Previous studies have demonstrated that BSP mRNA expression is essentially restricted to fully-differentiated cells of mineralized connective tissues and that the expression of BSP is developmentally regulated. Treatment of rat osteosarcoma ROS 17/2.8 cells with TNF-α (10ng/ml) for 24 h caused a marked reduction in BSP mRNA levels. The addition of antioxidant N-acetylcysteine (NAC; 20 mM) 30 min prior to stimulation with TNF-α attenuated the inhibition of BSP mRNA levels. Transient transfection analyses, using chimeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene, revealed that TNF-α (10 ng/ml) suppressed expression in all constructs, including a short construct (pLUC3; nts-116 to +60), transfected into ROS17/2.8 cells. Further deletion analysis of the BSP promoter showed that a region within nts -84 to -60 was targeted by TNF-α, the effects which were inhibited by NAC and the tyrosine kinase inhibitor, herbimycin A (HA). Introduction of 2bp mutations in the inverted CCAAT box (ATTGG; nts -50 and -46), a putative cAMP response element (CRE; nts -75 to -68), and a FGF response element (FRE; nts -92 to -85) showed that the TNF-α effects were mediated by the CRE. These results were supported by gel mobility shift assays, using a radiolabeled double-stranded CRE oligonucleotide, which revealed decreased binding of a nuclear protein from TNF-α-stimulated ROS 17/2.8 cells. Further, the inhibitory effect of TNF-α on CRE DNA-protein complex was completely abolished by NAC or HA treatment. These studies, therefore, show that TNF-α suppresses BSP gene transcription through a tyrosine kinase-dependent pathway that generates reactive oxygen species and that the TNF-α effects are mediated by a CRE element in the proximal BSP gene promoter.",
keywords = "Bone sialoprotein, Gene regulation, Mineralized tissues, TNF-α, Transcription",
author = "Hiroshi Samoto and Emi Shimizu and Yuko Matsuda-Honjo and Ryoichiro Saito and Muneyoshi Yamazaki and Kazutaka Kasai and Shunsuke Furuyama and Hiroshi Sugiya and Jaro Sodek and Yorimasa Ogata",
year = "2002",
doi = "10.1002/jcb.10301",
language = "English (US)",
volume = "87",
pages = "313--323",
journal = "Journal of Cellular Biochemistry",
issn = "0730-2312",
publisher = "Wiley-Liss Inc.",
number = "3",

}

TY - JOUR

T1 - TNF-α suppresses bone sialoprotein (BSP) expression in ROS17/2.8 Cells

AU - Samoto, Hiroshi

AU - Shimizu, Emi

AU - Matsuda-Honjo, Yuko

AU - Saito, Ryoichiro

AU - Yamazaki, Muneyoshi

AU - Kasai, Kazutaka

AU - Furuyama, Shunsuke

AU - Sugiya, Hiroshi

AU - Sodek, Jaro

AU - Ogata, Yorimasa

PY - 2002

Y1 - 2002

N2 - Tumor necrosis factor-alpha (TNF-α) is a major mediator of inflammatory responses in many diseases that inhibits bone formation and stimulates bone resorption. To determine molecular mechanisms involved in the suppression of bone formation we have analyzed the effects of TNF-α on BSP gene expression. Bone sialoprotein (BSP) is a mineralized tissue-specific protein that appears to function in the initial mineralization of bone. Previous studies have demonstrated that BSP mRNA expression is essentially restricted to fully-differentiated cells of mineralized connective tissues and that the expression of BSP is developmentally regulated. Treatment of rat osteosarcoma ROS 17/2.8 cells with TNF-α (10ng/ml) for 24 h caused a marked reduction in BSP mRNA levels. The addition of antioxidant N-acetylcysteine (NAC; 20 mM) 30 min prior to stimulation with TNF-α attenuated the inhibition of BSP mRNA levels. Transient transfection analyses, using chimeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene, revealed that TNF-α (10 ng/ml) suppressed expression in all constructs, including a short construct (pLUC3; nts-116 to +60), transfected into ROS17/2.8 cells. Further deletion analysis of the BSP promoter showed that a region within nts -84 to -60 was targeted by TNF-α, the effects which were inhibited by NAC and the tyrosine kinase inhibitor, herbimycin A (HA). Introduction of 2bp mutations in the inverted CCAAT box (ATTGG; nts -50 and -46), a putative cAMP response element (CRE; nts -75 to -68), and a FGF response element (FRE; nts -92 to -85) showed that the TNF-α effects were mediated by the CRE. These results were supported by gel mobility shift assays, using a radiolabeled double-stranded CRE oligonucleotide, which revealed decreased binding of a nuclear protein from TNF-α-stimulated ROS 17/2.8 cells. Further, the inhibitory effect of TNF-α on CRE DNA-protein complex was completely abolished by NAC or HA treatment. These studies, therefore, show that TNF-α suppresses BSP gene transcription through a tyrosine kinase-dependent pathway that generates reactive oxygen species and that the TNF-α effects are mediated by a CRE element in the proximal BSP gene promoter.

AB - Tumor necrosis factor-alpha (TNF-α) is a major mediator of inflammatory responses in many diseases that inhibits bone formation and stimulates bone resorption. To determine molecular mechanisms involved in the suppression of bone formation we have analyzed the effects of TNF-α on BSP gene expression. Bone sialoprotein (BSP) is a mineralized tissue-specific protein that appears to function in the initial mineralization of bone. Previous studies have demonstrated that BSP mRNA expression is essentially restricted to fully-differentiated cells of mineralized connective tissues and that the expression of BSP is developmentally regulated. Treatment of rat osteosarcoma ROS 17/2.8 cells with TNF-α (10ng/ml) for 24 h caused a marked reduction in BSP mRNA levels. The addition of antioxidant N-acetylcysteine (NAC; 20 mM) 30 min prior to stimulation with TNF-α attenuated the inhibition of BSP mRNA levels. Transient transfection analyses, using chimeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene, revealed that TNF-α (10 ng/ml) suppressed expression in all constructs, including a short construct (pLUC3; nts-116 to +60), transfected into ROS17/2.8 cells. Further deletion analysis of the BSP promoter showed that a region within nts -84 to -60 was targeted by TNF-α, the effects which were inhibited by NAC and the tyrosine kinase inhibitor, herbimycin A (HA). Introduction of 2bp mutations in the inverted CCAAT box (ATTGG; nts -50 and -46), a putative cAMP response element (CRE; nts -75 to -68), and a FGF response element (FRE; nts -92 to -85) showed that the TNF-α effects were mediated by the CRE. These results were supported by gel mobility shift assays, using a radiolabeled double-stranded CRE oligonucleotide, which revealed decreased binding of a nuclear protein from TNF-α-stimulated ROS 17/2.8 cells. Further, the inhibitory effect of TNF-α on CRE DNA-protein complex was completely abolished by NAC or HA treatment. These studies, therefore, show that TNF-α suppresses BSP gene transcription through a tyrosine kinase-dependent pathway that generates reactive oxygen species and that the TNF-α effects are mediated by a CRE element in the proximal BSP gene promoter.

KW - Bone sialoprotein

KW - Gene regulation

KW - Mineralized tissues

KW - TNF-α

KW - Transcription

UR - http://www.scopus.com/inward/record.url?scp=6444244431&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=6444244431&partnerID=8YFLogxK

U2 - 10.1002/jcb.10301

DO - 10.1002/jcb.10301

M3 - Article

VL - 87

SP - 313

EP - 323

JO - Journal of Cellular Biochemistry

JF - Journal of Cellular Biochemistry

SN - 0730-2312

IS - 3

ER -