A genome-wide association study of third molar agenesis in Japanese and Korean populations.

Tooth agenesis is the most common developmental anomaly of human dentition, occurring most often in the third molar (wisdom tooth). It is affected by genetic variation, so this study aimed to identify susceptibility genes associated with third molar agenesis. Examination of panoramic radiographs and medical history about third molar extraction were used to diagnose third molar agenesis. We then conducted a genome-wide association study of 149 cases with at least one-third molar agenesis and 338 controls from Japan and Korea using the Illumina HumanOmniExpress BeadChip. After rigorous quality-control filtering, approximately 550 000 single-nucleotide polymorphisms (SNPs) were analyzed in association tests with the status. We identified three SNPs showing evidence of association at P<1 × 10(-5) and 69 SNPs showing evidence of association at P<1 × 10(-4). SNP rs1469622, which maps to an intron of THSD7B (thrombospondin, type I, domain containing 7B) on chromosome 2, showed the strongest association (combined odds ratio=1.88, 95% confidence interval=1.43-2.47, P=7.5 × 10(-6)). The identified SNPs may be considered candidates for future replication studies in independent samples.

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 microg/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 microg/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 microg/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 microg/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.

Regulation of rat bone sialoprotein gene transcription by enamel matrix derivative.

BACKGROUND: Enamel matrix derivative (EMD) has recently been developed for use as a periodontal regenerative treatment. While EMD is believed to induce regeneration of periodontal tissue, the precise mechanism is not known. Bone sialoprotein (BSP), an early phenotypic marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during bone formation. In this study, we examined the ability of EMD to regulate BSP gene transcription in osteoblast-like cells. METHODS: To determine the molecular basis of the transcriptional regulation of BSP gene transcription by EMD, we conducted Northern hybridization, transient transfection analyses with chimeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene, and gel mobility shift assays. RESULTS: Using the osteoblastic cell line ROS 17/2.8, we determined that BSP mRNA levels increased approximately 2.8-fold by EMD. In transient transfection analyses, EMD (50 microg/ml, 12 hours) increased luciferase activities of pLUC4 (nt -425 to +60) and pLUC5 (nt -801 to +60), transfected into ROS 17/2.8 cells. Within the pLUC4 and 5, a homeodomain binding element (HOX) and a transforming growth factor (TGF)-beta activation element (TAE) are present. Gel mobility shift assays with radiolabeled HOX and TAE ds-oligonucleotides revealed increased binding of nuclear proteins from EMD stimulated ROS 17/2.8 cells. CONCLUSION: These studies have, therefore, identified EMD response elements in the rat BSP gene promoter that may mediates the effects of EMD on BSP gene transcription.

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.

Prostaglandin E2 stimulates bone sialoprotein (BSP) expression through cAMP and fibroblast growth factor 2 response elements in the proximal promoter of the rat BSP gene.

Bone sialoprotein (BSP), an early marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during de novo bone formation. Prostaglandin E2 (PGE2) has anabolic effects on proliferation and differentiation of osteoblasts via diverse signal transduction systems. Because PGE2 increases the proportion of functional osteoblasts in fetal rat calvarial cell cultures, we investigated the regulation of BSP, as an osteoblastic marker, by PGE2. Treatment of rat osteosarcoma UMR 106 cells with 3 microm, 300 nm, and 30 nm PGE2 increased the steady state levels of BSP mRNA about 2.7-, 2.5-, and 2.4-fold after 12 h. From transient transfection assays, the constructs including the promoter sequence of nucleotides (nt) -116 to +60 (pLUC3) were found to enhance transcriptional activity 3.8- and 2.2-fold treated with 3 microm and 30 nm PGE2 for 12 h. 2-bp mutations were made in an inverted CCAAT box (between nt -50 and -46), a cAMP response element (CRE; between nt -75 and -68), a fibroblast growth factor 2 response element (FRE; nt -92 to -85), and a pituitary-specific transcription factor-1 motif (between nt -111 and -105) within pLUC3 and pLUC7 constructs. Transcriptional stimulation by PGE2 was almost completed abrogated in constructs that included 2-bp mutations in either the CRE and FRE. In gel shift analyses an increased binding of nuclear extract components to double-stranded oligonucleotide probes containing CRE and FRE was observed following treatment with PGE2. These studies show that PGE2 induces BSP transcription in UMR 106 cells through juxtaposed CRE and FRE elements in the proximal promoter of the BSP gene.

TNF-alpha suppresses bone sialoprotein (BSP) expression in ROS17/2.8 cells.

Tumor necrosis factor-alpha (TNF-alpha) 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-alpha 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-alpha (10 ng/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-alpha 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-alpha (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-alpha, 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-alpha 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-alpha-stimulated ROS 17/2.8 cells. Further, the inhibitory effect of TNF-alpha on CRE DNA-protein complex was completely abolished by NAC or HA treatment. These studies, therefore, show that TNF-alpha suppresses BSP gene transcription through a tyrosine kinase-dependent pathway that generates reactive oxygen species and that the TNF-alpha effects are mediated by a CRE element in the proximal BSP gene promoter.