Bone Sialoprotein Gene Transcription Research Articles

Effects of interleukin-11 on the expression of human bone sialoprotein gene

Interleukin-11 (IL-11) is a bone marrow stromal fibroblast-derived cytokine with a wide spectrum of activities in different biological systems. IL-11 and IL-6 are two cytokines known to rely on osteoblast-osteoclast communication for their effects on osteoclast differentiation. Bone sialoprotein (BSP) is a mineralized connective tissue-specific protein expressed in differentiated osteoblasts, odontoblasts, and cementoblasts. To determine the molecular basis of the transcriptional regulation of the human BSP gene by IL-11, we conducted real-time polymerase chain reactions (PCR), transient transfection analyses with chimeric constructs of the human BSP gene promoter linked to a luciferase reporter gene, gel mobility shift assays, and a chromatin immunoprecipitation assay using human osteoblast-like Saos2 cells. IL-11 (20ng/ml) increased BSP, Runx2, and Osterix mRNA levels at 6h and the alkaline phosphatase (ALP) mRNA level at 12h in osteoblast-like Saos2 cells. In a transient transfection assay, IL-11 (20ng/ml, 12h) increased luciferase activities of constructs between -60LUC and -868LUC including the human BSP gene promoter. Transcriptional stimulations by IL-11 were partially inhibited in the constructs that included 2-bp mutations in the cAMP response element 1 (CRE1, -72 to -79) and CRE2 (-667 to -674). When mutations were made in pairs of CRE1 and CRE2 in -868LUC, the effect of IL-11 on luciferase activity was almost totally abrogated. Transcriptional activities induced by IL-11 were inhibited by protein kinase A, tyrosine kinase, ERK1/2, and PI3-kinase inhibitors. Gel mobility shift analyses showed that IL-11 increased nuclear proteins binding to CRE1 and CRE2. CREB1, phospho-CREB1, c-Fos, and c-Jun antibodies disrupted the formation of CRE1 and CRE2 protein complexes. These data demonstrate that IL-11 stimulates BSP gene transcription via CRE1 and CRE2 elements in the human BSP gene promoter.

Unliganded estrogen receptor α stimulates bone sialoprotein gene expression

Estrogen is one of the steroid hormones essential for skeletal development. The estrogen receptor (ER) is a transcription factor and a member of the steroid receptor superfamily. There are two different forms of the ER, usually referred to as α and β, each encoded by a separate gene. Hormone-activated ERs form dimers, since the two forms are coexpressed in many cell types. Bone sialoprotein (BSP) is a tissue-specific acidic glycoprotein that is expressed by differentiated osteoblasts, odontoblasts and cementoblasts during the initial formation of mineralized tissue.To determine the molecular basis of the tissue-specific expression of BSP and its regulation by estrogen and the ER, we have analyzed the effects of β-estradiol and ERα on BSP gene transcription. ERα protein levels were increased after ERα overexpression in ROS17/2.8 cells. While BSP mRNA levels were increased by ERα overexpression, the endogenous and overexpressed BSP mRNA levels were not changed by β-estradiol (10−8M, 24h). Luciferase activities of different sized BSP promoter constructs (pLUC3~6) were increased by ERα overexpression, whereas basal and induced luciferase activities by ERα overexpression were not influenced by β-estradiol. Effects of ERα overexpression were abrogated by 2bp mutations in either the cAMP response element (CRE) or activator protein 1 (AP1)/glucocorticoid response element (GRE). Gel shift analyses showed that ERα overexpression increased binding to the CRE and AP1/GRE elements. Notably, the CRE–protein complexes were disrupted by ERα, CREB and phospho-CREB antibodies. The AP1/GRE–protein complexes were supershifted by the c-Fos antibody. These studies demonstrate that ERα stimulates BSP gene transcription in a ligand-independent manner by targeting the CRE and AP1/GRE elements in the rat BSP gene promoter.

Aggregatibacter actinomycetemcomitans lipopolysaccharide regulates bone sialoprotein gene transcription

Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) is believed to be associated with aggressive periodontitis characterized by a rapid bone loss. A. actinomycetemcomitans lipopolysaccharide (LPS) has a similar structure to Escherichia coli LPS, and they are Toll-like receptor 4 agonists. Bone sialoprotein (BSP) is an early marker of osteoblast differentiation. To investigate the effects of A. actinomycetemcomitans LPS on bone formation, we targeted BSP as a marker for osteogenic differentiation and bone formation. BSP mRNA levels were decreased by 0.1 µg/ml and increased by 0.01 µg/ml A. actinomycetemcomitans LPS at 6 h in osteoblast-like ROS17/2.8 cells. In transient transfection analyses, 0.1 µg/ml decreased and 0.01 µg/ml A. actinomycetemcomitans LPS increased luciferase activities of the construct (-116 to +60). Introduction of 2 bp mutations to the constructs showed that the effects of A. actinomycetemcomitans LPS were mediated by a cAMP response element (CRE), a FGF2 response element (FRE), and a homeodomain protein-binding site (HOX). Tyrosine kinase, ERK1/2, and PI3-kinase/Akt participated in the effects of both 0.1 and 0.01 µg/ml A. actinomycetemcomitans LPS. The results of gel shift showed that 0.1 µg/ml decreased while 0.01 µg/ml A. actinomycetemcomitans LPS increased CRE-, FRE-, and HOX-binding protein complexes formation at 6 h, and revealed that 0.01 µg/ml A. actinomycetemcomitans LPS induced BSP transcription through CREB1, JunD, Fra2, c-Fos, Runx2, Dlx5, and Smad1 targeting those response elements. These studies therefore indicated that 0.1 µg/ml suppressed and 0.01 µg/ml A. actinomycetemcomitans LPS increased BSP gene transcription mediated through CRE, FRE, and HOX elements in the rat BSP gene promoter.

Transcriptional regulation of bone sialoprotein gene by CO2 laser irradiation

Bone sialoprotein (BSP), an early marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during de novo bone formation. Low-power laser irradiation has a stimulating effect on cells and tissues. Although the carbon dioxide (CO(2)) laser is a hard surgical laser, we have attempted to use it at low energy density to achieve biological alterations. To investigate the effects of CO(2) laser irradiation on BSP gene transcription, we used rat osteoblast-like ROS17/2.8 cells. BSP mRNA levels were increased at 12 h after irradiation with the CO(2) laser (2 W, 20 s). Transient transfection assays using various sizes of the rat BSP gene promoter linked to the luciferase reporter gene showed that CO(2) laser irradiation induced luciferase activity of a -116 to +60 BSP promoter construct (pLUC3) at 12 h in the cells. Transcriptional stimulation by CO(2) laser irradiation was abrogated in the pLUC3 construct containing a 2-bp mutation in the fibroblast growth factor 2 response element (FRE). Gel shift analyses showed that CO(2) laser irradiation increased the binding of nuclear protein to FRE. These studies demonstrate that CO(2) laser irradiation increases BSP transcription via FRE in the rat BSP gene promoter.

Kaempferol stimulates bone sialoprotein gene transcription and new bone formation

Kaempferol is a typical flavonol-type flavonoid that is present in a variety of vegetables and fruits, and has a protective effect on postmenopausal bone loss. Bone sialoprotein (BSP) is thought to function in the initial mineralization of bone and could be crucial for osteoblast differentiation, bone matrix mineralization and tumor metastasis. In the present study we investigated the regulation of BSP transcription by kaempferol in rat osteoblast-like UMR106 cells, and the effect of kaempferol on new bone formation. Kaempferol (5 microM) increased BSP and Osterix mRNA levels at 12 h and up-regulated Runx2 mRNA expression at 6 h. Kaempferol increased luciferase activity of the construct pLUC3, which including the promoter sequence between nucleotides -116 to +60. Transcriptional stimulation by kaempferol abrogated in constructs included 2 bp mutations in the inverted CCAAT, CRE, and FRE elements. Gel shift analyses showed that kaempferol increased nuclear protein binding to CRE and FRE elements, whereas the CCAAT-protein complex did not change after kaempferol stimulation. Twelve daily injections of 5 microM kaempferol directly into the periosteum of parietal bones of newborn rats increased new bone formation. These data suggest that kaempferol increased BSP gene transcription mediated through inverted CCAAT, CRE, and FRE elements in the rat BSP gene promoter, and could induce osteoblast activities in the early stage of bone formation.

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 microg/ml and increased by 0.01 microg/ml P. gingivalis LPS at 12 h. Results of luciferase assays showed that 0.1 microg/ml decreased and 0.01 microg/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 microg/ml P. gingivalis LPS. Furthermore, 0.1 microg/ml LPS decreased the CRE- and FRE-protein complexes formation, whereas 0.01 microg/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 microg/ml P. gingivalis LPS. These studies therefore indicated that 0.1 microg/ml suppressed, and 0.01 microg/ml P. gingivalis LPS increased BSP gene transcription mediated through CRE and FRE elements in the rat BSP gene promoter.

骨シアロタンパク質（BSP）の転写に対するステロイドレセプターの効果

骨シアロタンパク質（BSP）の転写に対するステロイドレセプターの効果

Fibroblast growth factor 2 regulates bone sialoprotein gene transcription in human breast cancer cells

Bone sialoprotein (BSP) is a major non-collagenous, extracellular matrix glycoprotein associated with mineralized tissues. Fibroblast growth factor 2 (FGF2) is recognized as a potent mitogen for a variety of mesenchymal cells. FGF2 produced by osteoblasts accumulates in the bone matrix and acts as an autocrine/paracrine regulator of osteoblasts. We previously reported that FGF2 regulates BSP gene transcription through the FGF2 response element (FRE) and activator protein 1 (AP1) binding site overlapping with the glucocorticoid response element in the rat BSP gene promoter. In the present study, FGF2 (10 ng/ml) increased BSP and Runx2 mRNA levels at 6 h in MCF7 human breast cancer cells. Transient transfection analyses were performed using chimeric constructs of the human BSP gene promoter linked to a luciferase reporter gene. Treatment of MCF7 cells with FGF2 (10 ng/ml) increased the luciferase activity of the constructs between -84LUC and -927LUC. Gel mobility shift analyses showed that FGF2 increased the binding of AP1 and CRE2. The CRE2- and AP1-protein complexes were disrupted by antibodies against CREB1, c-Fos, c-Jun, Fra2, p300 and Runx2. These studies demonstrate that FGF2 stimulates BSP transcription in MCF7 human breast cancer cells by targeting the AP1 and CRE2 elements in the human BSP gene promoter.

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.

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.

Effects of Inorganic Polyphosphates on Bone Sialoprotein Gene Transcription

Effects of Inorganic Polyphosphates on Bone Sialoprotein Gene Transcription

ヒト骨シアロタンパク質の転写に対する血小板由来成長因子(PDGF)の影響

ヒト骨シアロタンパク質の転写に対する血小板由来成長因子(PDGF)の影響

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.

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(-8)M, 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.

Effects of quercetin and quercetin 3‐glucuronide on the expression of bone sialoprotein gene

Quercetin is a typical flavonol-type flavonoid and is present in a variety of vegetables, and their antioxidant effect implies their possible role in the prevention of oxidative stress related chronic diseases. Bone sialoprotein (BSP) is a noncollagenous protein of the extracellular matrix in the mineralized connective tissues that has been implicated in the nucleation of hydroxyapatite crystals. Previously, we reported that isoflavone (genistein) activated BSP gene transcription is mediated through an inverted CCAAT box in the proximal BSP gene promoter. The present study investigates the regulation of BSP transcription in a rat osteoblast-like cell line, ROS 17/2.8 cells, by quercetin and its conjugated metabolite quercetin 3-glucuronide. Quercetin and quercetin 3-glucuronide (5 microM) increased the BSP mRNA levels at 12 h and quercetin upregulated the Cbfa1/Runx2 mRNA expression at 12 h. From transient transfection assays using various sized BSP promoter-luciferase constructs, quercetin increased the luciferase activity of the construct (pLUC3), including the promoter sequence nucleotides -116 to -43. Transcriptional stimulations by quercetin were almost completely abrogated in the constructs that included 2 bp mutations in the inverted CCAAT and FRE elements whereas the CCAAT-protein complex did not change after stimulation by quercetin according to gel shift assays. Quercetin increased the nuclear protein binding to the FRE and 3'-FRE. These data suggest that quercetin and quercetin 3-glucuronide increased the BSP mRNA expression, and that the inverted CCAAT and FRE elements in the promoter of the BSP gene are required for quercetin induced BSP transcription.

血小板由来成長因子(PDGF)による骨シアロタンパク質の転写の調節

血小板由来成長因子(PDGF)による骨シアロタンパク質の転写の調節

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 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.

Amelogenin Stimulates Bone Sialoprotein (BSP) Expression Through Fibroblast Growth Factor 2 Response Element and Transforming Growth Factor‐β1 Activation Element in the Promoter of the BSP Gene

Amelogenins are a complex mixture of hydrophobic proteins that are the major organic component of developing enamel. The principal function of the amelogenins and their degradation products has been assigned to structural roles in creating the space and milieu for promoting enamel mineralization. Enamel matrix derivative (EMD) has been used clinically for periodontal regeneration and its therapeutic effectiveness has been attributed to amelogenin, non-amelogenin enamel matrix proteins, and growth factors. While EMD is believed to induce periodontal regeneration, the precise mechanism is not known. Bone sialoprotein (BSP), an early phenotypic marker of osteoblast and cementoblast differentiation, has been implicated in the nucleation of hydroxyapatite during bone formation. In this study, we examined the ability of amelogenin to regulate BSP gene transcription in osteoblast like cells. We conducted Northern hybridization, transient transfection analyses, and gel mobility shift assays using full-length recombinant amelogenin to determine the molecular basis of the transcriptional regulation of BSP gene by amelogenin. Recombinant amelogenin (1 microg/ml, 12 hours) increased BSP mRNA levels approximately 2.4-fold. In transient transfection analyses, amelogenin (1 microg/ml, 12 hours) increased luciferase activity approximately 1.5-fold in pLUC3 (nucleotides -116 to +60) and further increased pLUC5 (nucleotides -801 to +60) activity approximately 2.3-fold transfected into ROS 17/2.8 cells. Amelogenin also increased luciferase activities in rat stromal bone marrow cells. The effect of amelogenin was inhibited by the tyrosine kinase inhibitor herbimycin A. Transcriptional stimulation by amelogenin was almost completely abrogated in cells expressing a BSP promoter construct with a mutation in the fibroblast growth factor 2 (FGF2) response element (FRE). Gel mobility shift assays with radiolabeled FRE and transforming growth factor-beta1 (TGF-beta1) activation element (TAE) ds-oligonucleotides revealed increased binding of nuclear proteins from amelogenin-stimulated ROS 17/2.8 cells. Amelogenin stimulation alters BSP gene transcription by inducing nuclear proteins that bind to the FRE and TAE in the rat 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.