Static magnetic fields-induced bone sialoprotein (BSP) expression is mediated through FGF2 response element and pituitary-specific transcription factor-1 motif

Emi Shimizu, Yuko Matsuda-Honjyo, Hiroshi Samoto, Ryoichiro Saito, Yu Nakajima, Youhei Nakayama, Naoko Kato, Muneyoshi Yamazaki, Yorimasa Ogata

Research output: Contribution to journalArticle

Abstract

Bone sialoprotein (BSP) is a sulfated and phosphorylated glycoprotein found almost exclusively in mineralized connective tissues. Recent studies on the developmental expression of BSP mRNA and temporo-spatial appearance of the protein during bone formation in vivo and in vitro have demonstrated that BSP is expressed by differentiated osteoblasts, and that it may function in the initial nucleation of hydroxyapatite crystals in de novo bone formation. Physical forces may play a fundamental role in the regulation of cell function in bone, but little is known about how cells are able to sense mechanical loads and signal transduction. Magnetic fields of sufficient magnitude have been shown to affect various biologic systems at organ, tissue, cellular, and subcellular levels. In the present study, rat osteosarcoma-derived osteoblast-like cells, UMR106, were used to assess the effect of static magnetic fields (SMF) on gene transcription of BSP. In our culture system, application of 300 and 800 Gauss SMF increased BSP mRNA levels after 24 h stimulation. To determine the molecular basis of the transcriptional regulation of BSP gene transcription by SMF, we conducted transient transfection analyses with chimeric constructs of the rat BSP gene promoter linked to a luciferase (LUC) reporter gene. SMF (300 and 800 Gauss) increased expression of the construct (pLUC3; -116 to +60) after 24 h treatment. Further deletion analysis of the BSP promoter showed that a region within nt -116 to -84 was targeted by SMF, the effect of which was inhibited by the tyrosine kinase inhibitor herbimycin A (HA). Mutations (2 bp) were made in an inverted CCAAT box between nt -50 and -46, a cyclicAMP response element (CRE; between nt -75 and -68), a fibroblast growth factor-2 response element (FRE; -92 to -85), and a pituitary-specific transcription factor-1 motif (Pit-1; nt -111 to -105) within the pLUC3 construct. Transcriptional stimulation by SMF was almost completely abrogated in constructs that included 2-bp mutations in the FRE and Pit-1. Binding of nuclear proteins to a radiolabeled FRE was increased and that to a Pit-1 was decreased in nuclear extracts prepared from SMF-stimulated UMR 106 cells. Further, the stimulatory and inhibitory effects of SMF on FRE and Pit-1 DNA-protein complexes were completely abolished by HA treatment. These studies, therefore, show that SMF increases BSP transcription through a tyrosine kinase-dependent pathway and that the SMF effects are mediated through juxtaposed FRE and Pit-1 elements in the proximal promoter of the BSP gene.

Original languageEnglish (US)
Pages (from-to)1183-1196
Number of pages14
JournalJournal of Cellular Biochemistry
Volume91
Issue number6
DOIs
StatePublished - 2004

Fingerprint

Integrin-Binding Sialoprotein
Response Elements
Fibroblast Growth Factor 2
Magnetic Fields
Transcription Factors
Magnetic fields
Genes
Transcription
Bone
Osteoblasts
Protein-Tyrosine Kinases
Osteogenesis
Rats
Transcription Factor Pit-1
Cellular Mechanotransduction
Tissue
Magnetic field effects
Signal transduction
Messenger RNA
Mutation

Keywords

  • Bone sialoprotein
  • Ferritic magnet
  • Gene regulation
  • Mineralized tissues
  • Static magnetic fields
  • Transcription

ASJC Scopus subject areas

  • Biochemistry
  • Cell Biology

Cite this

Static magnetic fields-induced bone sialoprotein (BSP) expression is mediated through FGF2 response element and pituitary-specific transcription factor-1 motif. / Shimizu, Emi; Matsuda-Honjyo, Yuko; Samoto, Hiroshi; Saito, Ryoichiro; Nakajima, Yu; Nakayama, Youhei; Kato, Naoko; Yamazaki, Muneyoshi; Ogata, Yorimasa.

In: Journal of Cellular Biochemistry, Vol. 91, No. 6, 2004, p. 1183-1196.

Research output: Contribution to journalArticle

Shimizu, E, Matsuda-Honjyo, Y, Samoto, H, Saito, R, Nakajima, Y, Nakayama, Y, Kato, N, Yamazaki, M & Ogata, Y 2004, 'Static magnetic fields-induced bone sialoprotein (BSP) expression is mediated through FGF2 response element and pituitary-specific transcription factor-1 motif', Journal of Cellular Biochemistry, vol. 91, no. 6, pp. 1183-1196. https://doi.org/10.1002/jcb.20002
Shimizu, Emi ; Matsuda-Honjyo, Yuko ; Samoto, Hiroshi ; Saito, Ryoichiro ; Nakajima, Yu ; Nakayama, Youhei ; Kato, Naoko ; Yamazaki, Muneyoshi ; Ogata, Yorimasa. / Static magnetic fields-induced bone sialoprotein (BSP) expression is mediated through FGF2 response element and pituitary-specific transcription factor-1 motif. In: Journal of Cellular Biochemistry. 2004 ; Vol. 91, No. 6. pp. 1183-1196.
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AU - Shimizu, Emi

AU - Matsuda-Honjyo, Yuko

AU - Samoto, Hiroshi

AU - Saito, Ryoichiro

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AU - Kato, Naoko

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N2 - Bone sialoprotein (BSP) is a sulfated and phosphorylated glycoprotein found almost exclusively in mineralized connective tissues. Recent studies on the developmental expression of BSP mRNA and temporo-spatial appearance of the protein during bone formation in vivo and in vitro have demonstrated that BSP is expressed by differentiated osteoblasts, and that it may function in the initial nucleation of hydroxyapatite crystals in de novo bone formation. Physical forces may play a fundamental role in the regulation of cell function in bone, but little is known about how cells are able to sense mechanical loads and signal transduction. Magnetic fields of sufficient magnitude have been shown to affect various biologic systems at organ, tissue, cellular, and subcellular levels. In the present study, rat osteosarcoma-derived osteoblast-like cells, UMR106, were used to assess the effect of static magnetic fields (SMF) on gene transcription of BSP. In our culture system, application of 300 and 800 Gauss SMF increased BSP mRNA levels after 24 h stimulation. To determine the molecular basis of the transcriptional regulation of BSP gene transcription by SMF, we conducted transient transfection analyses with chimeric constructs of the rat BSP gene promoter linked to a luciferase (LUC) reporter gene. SMF (300 and 800 Gauss) increased expression of the construct (pLUC3; -116 to +60) after 24 h treatment. Further deletion analysis of the BSP promoter showed that a region within nt -116 to -84 was targeted by SMF, the effect of which was inhibited by the tyrosine kinase inhibitor herbimycin A (HA). Mutations (2 bp) were made in an inverted CCAAT box between nt -50 and -46, a cyclicAMP response element (CRE; between nt -75 and -68), a fibroblast growth factor-2 response element (FRE; -92 to -85), and a pituitary-specific transcription factor-1 motif (Pit-1; nt -111 to -105) within the pLUC3 construct. Transcriptional stimulation by SMF was almost completely abrogated in constructs that included 2-bp mutations in the FRE and Pit-1. Binding of nuclear proteins to a radiolabeled FRE was increased and that to a Pit-1 was decreased in nuclear extracts prepared from SMF-stimulated UMR 106 cells. Further, the stimulatory and inhibitory effects of SMF on FRE and Pit-1 DNA-protein complexes were completely abolished by HA treatment. These studies, therefore, show that SMF increases BSP transcription through a tyrosine kinase-dependent pathway and that the SMF effects are mediated through juxtaposed FRE and Pit-1 elements in the proximal promoter of the BSP gene.

AB - Bone sialoprotein (BSP) is a sulfated and phosphorylated glycoprotein found almost exclusively in mineralized connective tissues. Recent studies on the developmental expression of BSP mRNA and temporo-spatial appearance of the protein during bone formation in vivo and in vitro have demonstrated that BSP is expressed by differentiated osteoblasts, and that it may function in the initial nucleation of hydroxyapatite crystals in de novo bone formation. Physical forces may play a fundamental role in the regulation of cell function in bone, but little is known about how cells are able to sense mechanical loads and signal transduction. Magnetic fields of sufficient magnitude have been shown to affect various biologic systems at organ, tissue, cellular, and subcellular levels. In the present study, rat osteosarcoma-derived osteoblast-like cells, UMR106, were used to assess the effect of static magnetic fields (SMF) on gene transcription of BSP. In our culture system, application of 300 and 800 Gauss SMF increased BSP mRNA levels after 24 h stimulation. To determine the molecular basis of the transcriptional regulation of BSP gene transcription by SMF, we conducted transient transfection analyses with chimeric constructs of the rat BSP gene promoter linked to a luciferase (LUC) reporter gene. SMF (300 and 800 Gauss) increased expression of the construct (pLUC3; -116 to +60) after 24 h treatment. Further deletion analysis of the BSP promoter showed that a region within nt -116 to -84 was targeted by SMF, the effect of which was inhibited by the tyrosine kinase inhibitor herbimycin A (HA). Mutations (2 bp) were made in an inverted CCAAT box between nt -50 and -46, a cyclicAMP response element (CRE; between nt -75 and -68), a fibroblast growth factor-2 response element (FRE; -92 to -85), and a pituitary-specific transcription factor-1 motif (Pit-1; nt -111 to -105) within the pLUC3 construct. Transcriptional stimulation by SMF was almost completely abrogated in constructs that included 2-bp mutations in the FRE and Pit-1. Binding of nuclear proteins to a radiolabeled FRE was increased and that to a Pit-1 was decreased in nuclear extracts prepared from SMF-stimulated UMR 106 cells. Further, the stimulatory and inhibitory effects of SMF on FRE and Pit-1 DNA-protein complexes were completely abolished by HA treatment. These studies, therefore, show that SMF increases BSP transcription through a tyrosine kinase-dependent pathway and that the SMF effects are mediated through juxtaposed FRE and Pit-1 elements in the proximal promoter of the BSP gene.

KW - Bone sialoprotein

KW - Ferritic magnet

KW - Gene regulation

KW - Mineralized tissues

KW - Static magnetic fields

KW - Transcription

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