Bone sialoprotein (BSP), an early marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during de novo bone formation. Prostaglandin E 2 (PGE 2 ) has anabolic effects on proliferation and differentiation of osteoblasts via diverse signal transduction systems. Because PGE 2 increases the proportion of functional osteoblasts in fetal rat calvarial cell cultures, we investigated the regulation of BSP, as an osteoblastic marker, by PGE 2 . Treatment of rat osteosarcoma UMR 106 cells with 3 M, 300 nM, and 30 nM PGE 2 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 M and 30 nM PGE 2 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 pituitaryspecific transcription factor-1 motif (between nt ؊111 and ؊105) within pLUC3 and pLUC7 constructs. Transcriptional stimulation by PGE 2 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 PGE 2 . These studies show that PGE 2 induces BSP transcription in UMR 106 cells through juxtaposed CRE and FRE elements in the proximal promoter of the BSP gene.Prostaglandins are considered important local factors that modulate bone metabolism through their effects on osteoblastic cells and osteoclasts (1, 2). Prostaglandin E 2 (PGE 2 ), 1 a major eicosanoid produced by osteoblasts, is a potent stimulator of bone resorption (3) that can stimulate the formation of osteoclast-like multinuclear cells in mouse bone marrow cultures (4, 5). The effects of PGE 2 on osteoclastogenesis are, at least in part, mediated by osteogenic cells, which express macrophage colony-stimulating factor (6) and receptor activator of nuclear factor B ligand (RANKL) (7) that promote, and osteoprotegerin, a decoy receptor for RANKL (8), that suppresses osteoclast formation. PGE 2 has been shown to stimulate RANKL and inhibit osteoprotegerin production (7, 9) and also increases production of interleukin-6, which can further enhance osteoclastogenesis (10 -12). In contrast, studies have revealed that PGE 2 also has bone-forming activity (2, 13). Treatment of male, female, and overiectomized mice with PGE 2 increases bone mass in vivo (14), whereas PGE 2 stimulates collagen and DNA synthesis and induces bone growth in calvarial organ (15) and cell cultures in vitro (16, 17). However, PGE 2 can either stimulate or inhibit cellular growth and differentiation of osteoblastic cells depending on PGE 2 concentration (15,18,19).To explain the diverse effects of PGE 2 , th...

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.

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.

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.

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.