Naoko Kato

Insulin-like growth factor-I increases bone sialoprotein (BSP) expression through fibroblast growth factor-2 response element and homeodomain protein-binding site in the proximal promoter of the BSP gene.

Insulin‐like growth factor‐I (IGF‐I) promotes bone formation by stimulating proliferation and differentiation of osteoblasts. Bone sialoprotein (BSP), is thought to function in the initial mineralization of bone, is selectively expressed by differentiated osteoblast. To determine the molecular mechanism of IGF‐I regulation of osteogenesis, we analyzed the effects of IGF‐I on the expression of BSP in osteoblast‐like Saos2 and in rat stromal bone marrow (RBMC‐D8) cells. IGF‐I (50 ng/ml) increased BSP mRNA levels at 12 h in Saos2 cells. In RBMC‐D8 cells, IGF‐I increased BSP mRNA levels at 3 h. From transient transfection assays, a twofold increase in transcription by IGF‐I was observed at 12 h in pLUC3 construct that included the promoter sequence from −116 to +60. Effect of IGF‐I was abrogated by 2‐bp mutations in either the FGF2 response element (FRE) or homeodomain protein‐binding site (HOX). Gel shift analyses showed that IGF‐I increased binding of nuclear proteins to the FRE and HOX elements. Notably, the HOX‐protein complex was supershifted by Smad1 antibody, while the FRE‐protein complex was shifted by Smad1 and Cbfa1 antibodies. Dlx2 and Dlx5 antibodies disrupted the formation of the FRE‐ and HOX‐protein complexes. The IGF‐I effects on the formation of FRE‐protein complexes were abolished by tyrosine kinase inhibitor herbimycin A (HA), PI3‐kinase/Akt inhibitor LY249002, and MAP kinase kinase inhibitor U0126, while IGF‐I effects on HOX‐protein complexes were abolished by HA and LY249002. These studies demonstrate that IGF‐I stimulates BSP transcription by targeting the FRE and HOX elements in the proximal promoter of BSP gene. J. Cell. Physiol. 208: 326–335, 2006.

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

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, UMR 106, 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‐1elements in the proximal promoter of the BSP gene.

Androgen receptor stimulates bone sialoprotein (BSP) gene transcription via cAMP response element and activator protein 1/glucocorticoid response elements.

Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. Androgens are steroid hormones that are essential for skeletal development. The androgen receptor (AR) is a transcription factor and a member of the steroid receptor superfamily that plays an important role in male sexual differentiation and prostate cell proliferation. To determine the molecular mechanism involved in the stimulation of bone formation, we have analyzed the effects of androgens and AR effects on BSP gene transcription. AR protein levels were increased after AR overexpression in ROS17/2.8 cells. BSP mRNA levels were increased by AR overexpression. However, the endogenous and overexpressed BSP mRNA levels were not changed by DHT (10−8 M, 24 h). Whereas luciferase (LUC) activities in all constructs, including a short construct (nts −116 to +60), were increased by AR overexpression, the basal and LUC activities enhanced by AR overexpression were not induced by DHT (10−8M, 24 h). The effect of AR overexpression was abrogated by 2 bp mutations in either the cAMP response element (CRE) or activator protein 1 (AP1)/glucocorticoid response element (GRE). Gel shift analyses showed that AR overexpression increased binding to the CRE and AP1/GRE elements. Notably, the CRE‐protein complexes were supershifted by phospho‐CREB antibody, and CREB, c‐Fos, c‐Jun, and AR antibodies disrupted the complexes formation. The AP1/GRE‐protein complexes were supershifted by c‐Fos antibody and c‐Jun, and AR antibodies disrupted the complexes formation. These studies demonstrate that AR stimulates BSP gene transcription by targeting the CRE and AP1/GRE elements in the promoter of the rat BSP gene. J. Cell. Biochem. 102: 240–251, 2007.

Effect of TNF-α on human osteosarcoma cell line Saos2-TNF-α regulation of bone sialoprotein gene expression in Saos2 osteoblast-like cells

Tumor necrosis factor‐alpha (TNF‐α) is a major mediator of inflammatory response in many diseases. It inhibits bone formation and stimulates bone resorption. To determine the molecular mechanisms involved in the regulation of gene expression of osteoblast‐like cells, we analyzed the effects of TNF‐α on the human osteosarcoma cell line Saos2. We used RT‐PCR to examine the effects of TNF‐α on bone sialoprotein (BSP), core binding factor a1 (Cbfa1), osterix, α1 (I) collagen, cyclooxygenase‐2 (COX‐2), interleukin‐6 (IL‐6), cathepsin B, cathepsin L and tissue inhibitors of metalloproteinase‐1 (TIMP‐1). TNF‐α (10 ng/ml) increased BSP, IL‐6 and COX‐2 mRNA levels after 3 h, reaching maximal levels at 12 h. Cbfa1 mRNA levels increased after 3 h, but decreased by 24 h. Osterix, cathepsin B, cathepsin L and TIMP‐1 mRNA levels did not change after stimulation with TNF‐α. On the other hand, α1 (I) collagen mRNA expression was suppressed by TNF‐α at 24 h.

Regulation of bone sialoprotein (BSP) gene transcription by lipopolysaccharide.

Lipopolysaccharide (LPS) is a major mediator of inflammatory responses in periodontal disease that inhibits bone formation and stimulates bone resorption. To determine the molecular mechanisms involved in the suppression of bone formation, we have analyzed the effects of LPS on BSP gene expression. Bone sialoprotein (BSP) is a mineralized tissue‐specific protein that appears to function in the initial mineralization of bone. Treatment of osteoblast‐like ROS 17/2.8 cells with LPS (1 µg/ml) for 12 h caused a marked reduction in BSP mRNA levels. The addition of antioxidant N‐acetylcysteine (NAC; 20 mM) 30 min prior to stimulation with LPS 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 LPS (1 µg/ml) suppressed expression of luciferase construct, encompassing BSP promoter nucleotides −108 to +60, transfected into ROS17/2.8 cells. The effects of LPS were inhibited by protein kinase A (PKA) inhibitor, H89 and the tyrosine kinase inhibitor, herbimycin A (HA). Introduction of 2 bp mutations in the inverted CCAAT box (ATTGG; nts −50 and −46), a cAMP response element (CRE; nts −75 to −68), a FGF response element (FRE; nts −92 to −85), and a pituitary specific transcription factor binding element (Pit‐1; nts −111 to −105) showed that the LPS effects were mediated by the CRE and FRE. Whereas the FRE and 3′‐FRE DNA‐protein complexes were decreased by LPS, CRE DNA‐protein complex did not change after LPS treatment. These studies, therefore, show that LPS suppresses BSP gene transcription through PKA and tyrosine kinase‐dependent pathways and that the LPS effects are mediated through CRE and FRE elements in the proximal BSP gene promoter.

Transcriptional regulation of bone sialoprotein gene by Porphyromonas gingivalis lipopolysaccharide

Lipopolysaccharide (LPS) is a major mediator of inflammatory response. Periodontopathic bacterium Porphyromonas gingivalis LPS has quite different character from Escherichia coli LPS. E. coli LPS is agonist for Toll‐like receptor 4 (TLR4), whereas P. gingivalis LPS worked as antagonist for TLR4. Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. To investigate the effects of P. gingivalis LPS on BSP transcription, we used rat osteoblast‐like ROS17/2.8 cells. BSP mRNA levels were decreased by 0.1 µg/ml and increased by 0.01 µg/ml P. gingivalis LPS at 12 h. Results of luciferase assays showed that 0.1 µg/ml decreased and 0.01 µg/ml P. gingivalis LPS increased BSP transcription in −116 to +60 BSP construct. The effects of P. gingivalis LPS were abrogated by double mutations in cAMP response element (CRE) and FGF2 response element (FRE). Tyrosine kinase inhibitor herbimycin A, ERK1/2 inhibitor and antioxidant N‐acetylcystein inhibited effects of P. gingivalis LPS. Protein kinase A inhibitor and PI3‐kinase/Akt inhibitor only abolished the effect of 0.01 µg/ml P. gingivalis LPS. Furthermore, 0.1 µg/ml LPS decreased the CRE– and FRE–protein complexes formation, whereas 0.01 µg/ml P. gingivalis LPS increased the nuclear protein binding to CRE and FRE. ChIP assays revealed increased binding of CREB1, JunD, Fra2, Runx2, Dlx5, and Smad1 to a chromatin fragment containing the CRE and FRE by 0.01 µg/ml P. gingivalis LPS. These studies therefore indicated that 0.1 µg/ml suppressed, and 0.01 µg/ml P. gingivalis LPS increased BSP gene transcription mediated through CRE and FRE elements in the rat BSP gene promoter. J. Cell. Biochem. 110: 823–833, 2010.

Effect of chlorpromazine on bone sialoprotein (BSP) gene transcription

Bone sialoprotein (BSP), an early marker of osteoblast differentiation. Whereas physical forces may play an important role in the regulation of bone cell function, little is known about how cells are able to sense mechanical loads. Chlorpromazine, a tranquilizing agent for treatments of psychiatric disorders, mimics hypotonic stress and causes membrane deformation. Application of 10 µg/ml of chlorpromazine suppressed BSP mRNA levels after 12 and 24 h in osteoblast‐like ROS17/2.8 cells and rat stromal bone marrow cells (SBMC‐D8). Chlorpromazine (10 µg/ml) decreased luciferase activity of the construct (pLUC3; −116 to +60 of the rat BSP gene promoter) after 12 h, the effect was inhibited by the tyrosine kinase inhibitor herbimycin A (HA) and MAP kinase kinase inhibitor U0126. Introduction of 2‐bp mutation in the pLUC3 construct showed that the chlorpromazine effects were mediated by cAMP response element (CRE) and FGF2 response element (FRE). In gel shift assays, using radiolabeled double‐stranded CRE and FRE oligonucleotides, which revealed decreased binding of nuclear proteins from chlorpromazine‐stimulated cells. These studies, therefore, show that chlorpromazine suppresses BSP gene transcription through tyrosine and MAP kinases‐dependent pathways and that the chlorpromazine effects are mediated by CRE and FRE elements in the proximal promoter of the BSP gene. J. Cell. Biochem. 97: 1198–1206, 2006.