Youhei Nakayama

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.

Regulation of human bone sialoprotein gene transcription by platelet-derived growth factor-BB

Platelet-derived growth factor (PDGF) is produced by mesenchymal cells and released by platelets following aggregation and is synthesized by osteoblasts. In bone, PDGF stimulates proliferation and differentiation of osteoblasts. PDGF also increases bone resorption, most likely by increasing the number of osteoclasts. Bone sialoprotein (BSP) is thought to function in the initial mineralization of bone, selectively expressed by differentiated osteoblast. To determine the molecular mechanisms PDGF regulation of human BSP gene transcription, we have analyzed the effects of PDGF-BB on osteoblast-like Saos2 and ROS17/2.8 cells. PDGF-BB (5 ng/ml) increased BSP mRNA and protein levels at 12 h in Saos2 cells, and induced BSP mRNA expression at 3 h, reached maximal at 12 h in ROS17/2.8 cells. Transient transfection analyses were performed using chimeric constructs of the human BSP gene promoter linked to a luciferase reporter gene. Treatment of Saos2 cells with PDGF-BB (5 ng/ml, 12 h) increased luciferase activities of all constructs between -184LUC to -2672LUC including the human BSP gene promoter. Effects of PDGF-BB abrogated in constructs included 2 bp mutations in the two cAMP response elements (CRE1 and CRE2), activator protein 1(3) (AP1(3)) and shear stress response element 1 (SSRE1). Luciferase activities induced by PDGF-BB were blocked by protein kinase A inhibitor H89 and tyrosine kinase inhibitor herbimycin A. Gel mobility shift analyses showed that PDGF-BB increased binding of CRE1, CRE2, AP1(3) and SSRE1 elements. CRE1- and CRE2-protein complexes were supershifted by CREB1 and phospho-CREB1 antibodies. Notably, AP1(3)-protein complexes were supershifted by c-Fos and JunD, and disrupted by CREB1, phospho-CREB1, c-Jun and Fra2 antibodies. These studies, therefore, demonstrate that PDGF-BB stimulates human BSP transcription by targeting the CRE1, CRE2, AP1(3) and SSRE1 elements in the human BSP gene promoter.

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.

AP1 binding site is another target of FGF2 regulation of bone sialoprotein gene transcription

Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. We previously reported that fibroblast growth factor 2 (FGF2) regulates BSP gene transcription via FGF2 response element (FRE) in the proximal promoter of rat BSP gene. We here report that activator protein 1 (AP1) binding site overlapping with glucocorticoid response element (GRE) AP1/GRE in the rat BSP gene promoter is another target of FGF2. Using the osteoblastic cell line ROS17/2.8, we determined that BSP mRNA levels increased by 10 ng/ml FGF2 at 6 and 12 h. Runx2 protein levels increased by FGF2 (10 ng/ml) at 3 h. Treatment of ROS17/2.8 cells with FGF2 (10 ng/ml, 12 h) increased luciferase activities of constructs including -116 to +60 and -938 to +60 of the rat BSP gene promoter. Effects of FGF2 abrogated in constructs included 2 bp mutations in the FRE and AP1/GRE elements. Luciferase activities induced by FGF2 were blocked by tyrosine kinase inhibitor herbimycin A, src-tyrosine kinase inhibitor PP1 and MAP kinase kinase inhibitor U0126. Gel shift analyses showed that FGF2 increased binding of FRE and AP1/GRE elements. Notably, the AP1/GRE-protein complexes were supershifted by Smad1 and c-Fos antibodies, c-Jun and Dlx5 antibodies disrupted the complexes formation, on the other hand AP1/GRE-protein complexes did not change by Runx2 antibody. These studies demonstrate that FGF2 stimulates BSP gene transcription by targeting the FRE and AP1/GRE elements in the rat BSP gene promoter.

Parathyroid hormone regulation of the human bone sialoprotein gene transcription is mediated through two cAMP response elements

Parathyroid hormone (PTH) regulates serum calcium and inorganic phosphate levels through its actions on kidney and bone. Bone sialoprotein (BSP) is an early marker of osteoblast differentiation and bone metabolism. We here report that two cAMP response elements (CRE) in the human BSP gene promoter are target of PTH. In human osteoblast‐like Saos2 cells, PTH (human 1−34 PTH, 10 nM) increased BSP mRNA and protein levels at 3 h. From transient transfection assays, 2‐ to 2.5‐fold increase in transcription by PTH was observed at 3 and 6 h in −184, −211, −428, −868, and −927 luciferase constructs that included the human BSP gene promoter. Effect of PTH was abrogated by 2 bp mutations in either the CRE1 (−79 to −72) or CRE2 (−674 to −667). Luciferase activities induced by PTH were blocked by protein kinase A inhibitor H89 and tyrosine kinase inhibitor herbimycin A. Gel shift analyses showed that PTH increased binding of nuclear proteins to the CRE1 and CRE2 elements. The CRE1–protein and CRE2–protein complexes were disrupted by CRE binding protein 1 (CREB1) antibodies and supershifted by phospho‐CREB1 antibody. ChIP assays detected binding of CREB1 and phospho‐CREB1 to a chromatin fragment containing CRE1 and CRE2, and increased binding of phospho‐CREB1 to the both sites. These studies demonstrate that PTH stimulates human BSP gene transcription by targeting the two CREs in the promoter of the human BSP gene. J. Cell. Biochem. 106: 618–625, 2009.

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.

Collection and culture of alveolar bone marrow multipotent mesenchymal stromal cells from older individuals

In this work, we examined the culture condition of alveolar bone marrow multipotent mesenchymal stromal cells (ABMMSCs), aiming to apply regenerative therapy to older periodontitis patients. To better understand the character of cultured cells from alveolar bone marrow, the expression profiles of well‐known genes and their responses to the induction of osteogenic, chondrogenic, or adipogenic differentiation were examined. Using αMEM‐based culture, ABMMSCs could be obtained from older individuals than in previous reports. Interestingly, ABMMSCs expressing Klf4 were able to differentiate into osteoblasts. The prediction of differentiation potential by Klf4 could be a useful guide for further improvement of the culture conditions required to culture ABMMSCs derived from older individuals. J. Cell. Biochem. 107: 1198–1204, 2009.

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.

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.