Bone Sialoprotein Promoter Research Articles

Thymosin β4 is associated with bone sialoprotein expression via ERK and Smad3 signaling pathways in MDPC-23 odontoblastic cells.

Thymosin β4 (Tβ4) regulates the expression of molecules associated with dentinogenesis, including bone sialoprotein (BSP). BSP regulates the initiation of mineralization and the direction of dentin growth. However, the association between Tβ4 signaling and BSP expression in odontoblasts remains unclear. Therefore, the aim of the present study was to investigate Tβ4 mRNA expression in odontoblasts during dentinogenesis and the association between the Tβ4 signaling pathway and BSP expression in MDPC‑23 odontoblastic cells. Expression and localization of Tβ4 mRNA was determined by insitu hybridization during mouse tooth development. The effect of Tβ4 signaling on BSP expression was investigated by reverse transcription polymerase chain reaction, western blot analysis, immunofluorescence and a luciferase reporter assay in the presence or absence of specific inhibitors of mitogen activated protein kinase kinase (PD98059) and mothers against decapentaplegic homolog 3 (Smad3; SIS3) in MDPC‑23 cells. The expression of Tβ4 mRNA in the odontoblast layer was highest at postnatal day 5, known as the advanced bell stage, when odontoblasts actively secrete dentin matrix proteins. Tβ4 increased BSP mRNA and protein levels in MDPC‑23 cells, but this was inhibited by PD98059 or SIS3 treatment. Tβ4 increased levels of phosphorylated (p) extracellular signal‑regulated kinase (ERK)1/2, pSmad3, pβ‑catenin, and runt‑related transcription factor 2 (Runx2) protein, but these effects were inhibited by PD98059 or SIS3. Tβ4 induced the nuclear translocation of Runx2 and pSmad3, while nuclear translocation of β‑catenin was decreased. Tβ4 significantly increased BSP promoter activity, which was decreased by PD98059 or SIS3 treatment. Tβ4 induced BSP expression in MDPC‑23 cells via ERK and Smad3 signaling pathways, suggesting its role as a signaling molecule in odontoblasts for regulating BSP secretion during dentinogenesis.

Inhibition of Runx2 signaling by TNF-α in ST2 murine bone marrow stromal cells undergoing osteogenic differentiation.

Tumor necrosis factor-alpha (TNF-α) inhibits osteogenic differentiation of murine bone marrow stromal cells, and transcription factor Runx2 serves as an essential regulation target in the process. The underlying mechanism may involve the regulation of Runx2 expression and the Runx2 activity in downstream gene transcription, which has not been fully elucidated. In this study, ST2 murine bone marrow-derived stromal cells were treated with bone morphogenetic protein-2 (BMP-2) and/or TNF-α in osteogenic medium, and the expression of Runx2 was estimated. Cells were transfected with Runx2, p65, inhibitor of κBα (IκBα), 9.0kb bone sialoprotein (BSP) promoter-luciferase or osteoblast-specific cis-acting element 2 (OSE2)-luciferase reporter vectors, and then real time-PCR and dual luciferase analysis were used to investigate the effect of TNF-α on Runx2-activated osteogenic gene transcription and the molecular mechanism. We found that TNF-α inhibited BMP-2-induced osteogenic marker expression and both the spontaneous and BMP-2-induced Runx2 expression. TNF-α stimulation or overexpression of nuclear factor-kappa B (NF-κB) p65 subunit repressed the Runx2-activated BSP and osteocalcin (OC) transcriptions. The Runx2-induced 9.0kb BSP promoter activity was attenuated by TNF-α or p65, while the OSE2 activity was not affected. Besides, blockage of NF-κB by IκBα overexpression eliminated these inhibitory effects of TNF-α on Runx2 signaling. These results suggest that in murine bone marrow stromal cells undergoing osteogenic differentiation, TNF-α and it activated NF-κB pathway inhibit the expression of Runx2 gene, and suppress the Runx2-mediated osteogenic gene transcription via the 9.0kb BSP promoter.

Preameloblast-Derived Factors Mediate Osteoblast Differentiation of Human Bone Marrow Mesenchymal Stem Cells by Runx2-Osterix-BSP Signaling.

Epithelial-mesenchymal interaction occurs during development of various tissues, including teeth and bone. Recently, a preameloblast-conditioned medium (PA-CM) from mouse apical bud cells (ABCs), a type of dental epithelial cell, was found to induce odontogenic differentiation of dental pulp stem cells and promote dentin formation. The aims of the present study were to investigate the effects of PA-CM on human bone marrow mesenchymal stem cells (hBMSCs) in vitro, and to investigate the bone regenerative capacity in vivo through epithelial-mesenchymal interactions of developmental osteogenesis. Coculturing with ABCs and PA-CM treatment upregulated osteoblast differentiation markers of hBMSCs compared to cells cultured alone. PA-CM accelerated mineralized nodule formation and also increased bone sialoprotein promoter activity in hBMSCs. PA-CM facilitated the migration of hBMSCs, but did not significantly influence proliferation. PA-CM promoted bone formation of hBMSCs in vivo. Radiographic and histologic findings showed that PA-CM induced the bony regeneration at calvarial defects in rat. Taken together, these data show that PA-CM enhances the migration and osteogenic differentiation of hBMSCs in vitro and induces bone formation in vivo.

Different Methylation Patterns of RUNX2, OSX, DLX5 and BSP in Osteoblastic Differentiation of Mesenchymal Stem Cells.

Runt-related transcription factor 2 (RUNX2) and osterix (OSX) as two specific osteoblast transcription factors and distal-less homeobox 5 (DLX5) as a non-specific one are of paramount importance in regulating osteoblast related genes including osteocalcin, bone sialoprotein (BSP), osteopontin and collagen type Iα1. The present study sets out to investigate whether epigenetic regulation of these genes is important in osteoblastic differentiation of mesenchymal stem cells (MSCs). In this experimental study, MSCs were differentiated to osteoblasts under the influence of the osteogenic differentiation medium. DNA and RNA were extracted at days 0, 7, 14 and 21 from MSCs differentiating to osteoblasts. Promoter regions of RUNX2, OSX, DLX5 and BSP were analyzed by methylation-specific PCR (MSP). Gene expression was analyzed during osteoblastic differentiation by quantitative real-time polymerase chain reaction (PCR). MSP analysis revealed that promoter methylation status did not change in RUNX2, DLX5 and BSP during MSC osteoblastic differentiation. In contrast, OSX promoter showed a dynamic change in methylation pattern. Moreover, RUNX2, OSX, DLX5 and BSP promoter regions showed three different methylation patterns during MSC differentiation. Gene expression analyses confirmed these results. The results show that in differentiation of MSCs to osteoblasts, epigenetic regulation of OSX may play a leading role.

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.

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.

ERRα regulates osteoblastic and adipogenic differentiation of mouse bone marrow mesenchymal stem cells

The orphan nuclear receptor estrogen-related receptor-α (ERRα) has been reported to have both a positive and a negative regulatory role in osteoblastic and adipocytic differentiation. We have studied the role of ERRα in osteoblastic and adipogenic differentiation of mesenchymal stem cells. Bone marrow mesenchymal stem cells were isolated from ERRα deficient mice and their differentiation capacities were compared to that of the wild-type cells. ERRα deficient cultures displayed reduced cellular proliferation, osteoblastic differentiation, and mineralization. In the complementary experiment, overexpression of ERRα in MC3T3-E1 cells increased the expression of osteoblastic markers and mineralization. Alterations in the expression of bone sialoprotein (BSP) may at least partially explain the effects on mineralization as BSP expression was reduced in ERRα deficient MSCs and enhanced upon ERRα overexpression in MC3T3-E1 cells. Furthermore, a luciferase reporter construct driven by the BSP promoter was efficiently transactivated by ERRα. Under adipogenic conditions, ERRα deficient cultures displayed reduced adipocytic differentiation. Our data thus propose a positive role for ERRα in osteoblastic and adipocytic differentiation. The variability in the results yielded in the different studies implies that ERRα may play different roles in bone under different physiological conditions.

Endothelin-1 regulates rat bone sialoprotein gene transcription

Endothelin-1 (ET-1) was originally discovered as a vasoconstrictor protein excreted by vascular endothelial cells. Recently, tumor-produced ET-1 has been considered to stimulate osteoblasts to form new bone, and to be an important mediator of osteoblastic bone metastasis. ET-1 has high affinity for two different membrane receptors, ET(A)R and ET(B)R, which are expressed by many types of cells including osteoblasts. Bone sialoprotein (BSP) is a phosphorylated and sulfated glycoprotein associated with mineralized connective tissues. To investigate the effects of ET-1 on BSP transcription, we used rat osteoblast-like ROS17/2.8 cells. Levels of BSP and osteopontin mRNA were increased at 12 h after treatment with ET-1 (10 ng/ml), and ET-1 at the same concentration induced luciferase activity of a -116 to +60 BSP promoter construct at 6 h. Transcriptional activity of -84BSPLUC, which contains the cAMP response element (CRE), was increased by ET-1. Furthermore, at 6 h, ET-1 (10 ng/ml) increased the binding of nuclear protein to CRE, the FGF2 response element (FRE) and the homeodomain protein-binding site (HOX). Antibodies against CREB1, JunD and Fra2 disrupted the formation of CRE-protein complexes, while antibodies against Runx2 and Dlx5 reduced the formation of FRE- and HOX-protein complexes. These findings indicate that ET-1 increases BSP transcription via the CRE, FRE and HOX sites in the rat BSP gene promoter.

Conditionally replicating adenovirus therapy utilizing bone sialoprotein promoter (Ad-BSP-E1a) in an in vivo study of treating androgen-independent intraosseous prostate cancer

Background Adenoviral based gene therapy has been used in clinical trials in control of advanced prostate cancer. In this study, a promising conditionally replicating adenovirus (CRAd) driven by a tissue specific bone sialoprotein promoter in controlling prostate cancer both in vitro and in vivo is demonstrated. Methods C4-2B, an androgen-independent prostate cancer cell line, was treated with PBS, Ad-BSP-TK, or the Ad-BSP-E1a in vitro, and in subcutaneous and intraosseous xenographs. Cell proliferation, PSA level in condition medium, tumor volume, and/or serum PSA were followed. Results The growth of C4-2B and the PSA production was dramatically suppressed by Ad-BSP-E1a at very low dosage (0.3 MOI) compared with PBS and Ad-BSP-TK treatment in vitro. In the subcutaneous model, the tumor volume was significantly lower statistically in the Ad-BSP-E1a treated group than the Ad-BSP-TK control group ( P = 0.02). In the intraosseous model, the mice treated in the Ad-BSP-E1a treatment group demonstrated a significant lower PSA compared to that in the control group ( P < 0.01) at week 8 and week 16 post-treatment. Conclusions The CRAd Ad-BSP-E1a revealed potential in treating prostate cancer in this model system. Using viral or none-viral mediated gene therapy to treat prostate carcinoma continues to be a potential avenue to treat afflicted men with prostate cancer.

Dlx5, a Positive Regulator of Osteoblastogenesis, is Essential for Osteoblast-Osteoclast Coupling

The homeodomain protein Dlx5 is an activator of Runx2 (a key regulator of osteogenesis) and is thought to be an important regulator of bone formation. At present, however, the perinatal lethality of Dlx5-null mice has hampered the elucidation of its function in osteogenesis. Here we provide the first analysis of the effects of Dlx5 inactivation on bone development. Femurs of Dlx5-null mouse embryos at the end of gestation exhibit a reduction in both total and trabecular bone volume associated with increased trabecular separation and reduced trabecular number. These parameters are often associated with pathological conditions characterized by reduced osteoblast activity and increased bone resorption. Dlx5(-/-) osteoblasts in culture display reduced proliferation and differentiation rate and reduction of Runx2, Osx, Osteocalcin and Bone Sialoprotein expression. In addition to impaired osteoblast function, Dlx5(-/-) femurs exhibit significant increases in osteoclast number. As Dlx5 is not expressed by osteoclasts, we suggest that its osteoblastic expression might control osteoblast/osteoclast coupling. Cultured Dlx5(-/-) osteoblasts displayed a higher RANKL/OPG ratio. Furthermore, Dlx5(-/-) osteoblasts induced a higher number of TRAP-positive multinucleated cells in normal spleen cultures with a globally increased resorption activity. These findings suggest that Dlx5 is a central regulator of bone turnover as it activates bone formation directly and bone resorption indirectly.

Haploinsufficiency of Runx2 results in bone formation decrease and different BSP expression pattern changes in two transgenic mouse models

Runx2 has been identified as "a master gene" for the differentiation of osteoblasts and Runx2-deficient mice has demonstrated a complete absence of mature osteoblast and ossification. To further characterize the Runx2 responsive elements within the bone sialoprotein (BSP) promoter and further investigate into the role of Runx2 haploinsufficiency in osteoblast differentiation, mBSP9.0Luc mice and mBSP4.8Luc mice were crossed with Runx2-deficient mice respectively. Luciferase assay, micro CT scan, and histological analysis were performed using tissues isolated from mBSP9.0luc/Runx2+/- mice, mBSP4.8luc/Runx2+/- mice and their corresponding Runx2+/+ littermates. Alkaline phosphatase activity, mineralization assays and RT-PCR analysis using calvarial osteoblasts isolated from these transgenic mice were also performed. Luciferase assay demonstrated an early increase in luciferase expression in mBSP9.0luc/Runx2+/- mice before the expression level of luciferase dramatically decreased and turned lower than that in their control littermates in later stages. In contrast, luciferase expression in mBSP4.8luc/Runx2+/- failed to show such an early increase. Micro CT scan and histological analysis showed that BMD and trabecular bone volume were decreased and bone formation was delayed in Runx2+/- mice. Furthermore, mineralization assay and semi-quantitative RT-PCR assay demonstrated a gene-dose-dependent decrease in bone nodule formation and bone marker genes expression levels in cultured calvarial osteoblasts derived from Runx2 knockout mice. Reconstitution of Runx2-null cells with Runx2 vector partially rescued the osteoblast function defects. In conclusion, the 9.0 kb BSP promoter demonstrated a higher tissue-specific regulation of the BSP gene by Runx2 in vivo and full Runx2 gene dose is essential for osteoblast differentiation and normal bone formation.

Runx2- and Histone Deacetylase 3-mediated Repression Is Relieved in Differentiating Human Osteoblast Cells to Allow High Bone Sialoprotein Expression

Bone sialoprotein (BSP) is a bone matrix glycoprotein whose expression coincides with terminal osteoblastic differentiation and the onset of mineralization. In this study we show that BSP expression is considerably increased in confluent Saos-2 human osteosarcoma cells and in differentiating normal human osteoblasts, concomitantly with the decrease of Runx2, a key transcription factor controlling bone formation. Therefore, we investigated the role of Runx2 in the regulation of BSP expression in Saos-2 cells. Using a mobility shift assay, we demonstrated that Runx2 binds to the BSP promoter only in preconfluent cells. Histone deacetylase 3 (HDAC3) has been recently shown to act as a Runx2 co-repressor. Chromatin immunoprecipitation assays demonstrated that both Runx2 and HDAC3 are detectable at the BSP promoter in preconfluent Saos-2 cells but not when they are confluent and overexpress BSP. Consistently, nuclear Runx2 protein level is down-regulated, whereas Saos-2 cells became increasingly confluent. Finally, the suppression of HDAC3, Runx2, or both by RNA interference induced the expression of BSP at both mRNA and protein levels in Saos-2 cells. Our data demonstrate that Runx2 and HDAC3 repress BSP gene expression and that this repression is suspended upon osteoblastic cell differentiation. Both the nuclear disappearance of Runx2 and the non-recruitment of HDAC3 represent new means to relieve Runx2-mediated suppression of BSP expression, thus allowing the acquisition of a fully differentiated and mineralization-competent phenotype by osteoblast cells.

CREB-1 and AP-1 transcription factors JunD and Fra-2 regulate bone sialoprotein gene expression in human breast cancer cells

Bone sialoprotein (BSP) expression is detected in a variety of human osteotropic cancers. High expression of BSP in breast and prostate primary carcinomas is associated with progression and bone metastases development. In this study, we examined the transcriptional regulation of BSP gene expression in MDA-MB-231 and MCF-7 human breast cancer cells compared with Saos-2 human osteoblast-like cells. BSP human promoter deletion analyses delineated a − 56/− 84 region, which comprises a cAMP response element (CRE) that was sufficient for maximal promoter activity in breast cancer cell lines. We found that the basic fibroblast growth factor response element (FRE) also located in the proximal promoter was a crucial regulator of human BSP promoter activity in Saos-2 but not in breast cancer cells. Promoter activity experiments in combination with DNA mobility shift assays demonstrated that BSP promoter activity is under the control of the CRE element, through CREB-1, JunD and Fra-2 binding, in MDA-MB-231, MCF-7 and in Saos-2 cells. Forskolin, a protein kinase A pathway activator, failed to enhance BSP transcriptional activity suggesting that CRE site behaves as a constitutive rather than an inducible element in these cell lines. Over-expression of JunD and Fra-2 increased BSP promoter activity and upregulated endogenous BSP protein expression in MCF-7 and Saos-2 cells while siRNA-mediated inhibition of both factors expression significantly reduced BSP protein level in MDA-MB-231. Collectively, these data provide with new transcriptional mechanisms, implicating CREB and AP-1 factors, that control BSP gene expression in breast cancer cells.

Fibroblast growth factor 2 and cyclic AMP synergistically regulate bone sialoprotein gene expression

Bone sialoprotein (BSP) is a noncollagenous protein of the mineralized bone extracellular matrix. We here report that FGF2 and cAMP act synergistically to stimulate BSP gene expression. Treatment of ROS 17/2.8 cells with either 10 ng/ml FGF2 or 1 μM FSK for 6 h resulted in 5.4- and 8.2-fold increases, respectively, in the levels of BSP mRNA. However, in the presence of both FGF2 and forskolin (FGF/FSK), BSP mRNA levels were increased synergistically by 20.4-fold. Using a luciferase reporter construct, encompassing BSP promoter nucleotides −116 to +60, transcription was also increased synergistically by 15.0-fold with FGF/FSK, compared to stimulations of 2.6- and 5.3-fold, respectively, for FGF2 and FSK alone. Transcriptional stimulation by FGF/FSK abrogated in constructs included 2 bp mutations in the inverted CCAAT, CRE, FRE and Pit-1 elements. Whereas the FRE–protein complex was increased by FGF2 and FGF/FSK, the Pit-1–protein complex was decreased by FSK and FGF/FSK. Notably, transcriptional activity induced by FGF/FSK was blocked by protein kinase A, tyrosine kinase and MEK inhibitors. These studies indicate that the combinatorial effects of FGF and FSK act through PKA, tyrosine kinase and MAP-kinase-dependent pathways, which target the inverted CCAAT, CRE, FRE and Pit-1 elements in the BSP gene to synergistically increase BSP expression.

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.

An In Vivo Model to Study Osteogenic Gene Regulation: Targeting an Avian Retroviral Receptor (TVA) to Bone With the Bone Sialoprotein (BSP) Promoter

To study bone development in vivo, a transgenic mouse model was established in which an avian retroviral receptor (TVA) gene driven by the BSP promoter was selectively expressed in skeletal tissues. The model was validated by showing suppressed BSP expression and delayed bone and tooth formation after infection with a virus expressing a mutated Cbfa1/Runx2 gene. Tissue-specific expression of the avian retroviral (TVA) receptor can be used to efficiently target ectopic expression of genes in vivo. To determine the use of this approach for studies of osteogenic differentiation and bone formation at specific developmental stages, transgenic mice expressing the TVA receptor under the control of a 5-kb bone sialoprotein (BSP) promoter were generated. The mice were first analyzed for tissue-specific expression of the TVA gene and then, after infection with a viral construct, for the effects of a dominant-negative form of the Cbfa1/Runx2 transcription factor on bone formation. We first generated transgenic mice (BSP/TVA) in which the TVA gene was expressed under the control of a 4.9-kb mouse BSP promoter. The tissue-specific expression of the TVA gene was analyzed by RT-PCR, in situ hybridization, and immunohistochemistry and compared with the expression of the endogenous BSP gene. A 396-bp fragment of mutated Cbfa1/Runx2 (Cbfa1mu) encoding the DNA-binding domain was cloned into a RCASBP (A) viral vector, which was used to infect neonatal BSP/TVA mice. Expression of the TVA receptor mRNA and protein in the transgenic mice was consistent with the expression of endogenous BSP. Four days after systemic infection with the Cbfa1mu-RCASBP (A) vector, RT-PCR analyses revealed that the expression of BSP mRNA in tibia and mandibles was virtually abolished, whereas a 30% reduction was seen in calvarial bone. After 9 days, BSP expression in the tibia and mandible was reduced by 45% in comparison with control animals infected with an empty RCASBP vector, whereas BSP expression in the membranous bone of calvariae was decreased approximately 15%. However, after 4 and 8 weeks, there was almost no change in BSP expression in any of the bone tissues. In comparison, a reduction in osteopontin expression was only observed 9 days after viral transfection in the three bones. Histomorphological examination revealed that bone formation and tooth development were delayed in some of the mice infected with mutated Cbfa1. These studies show that BSP/TVA transgenic mice can be used to target genes to sites of osteogenesis, providing a unique system for studying molecular events associated with bone formation in vivo.

Tissue- and bone cell-specific expression of bone sialoprotein is directed by a 9.0 kb promoter in transgenic mice

Bone sialoprotein (BSP) is a phosphorylated glycoprotein that is expressed almost exclusively in mineralizing connective tissues. In bone, expression of BSP correlates with the differentiation of osteoblasts and the onset of mineralization. To determine how the tissue- and differentiation-specific transcription of BSP is regulated, various lengths of promoter sequence were ligated to a luciferase reporter and stably transfected into a rat stromal bone marrow cell line, RBMC-D8 and undifferentiated C3H10T1/2 cells. Luciferase transcription of reporter constructs including 5.4 kb (mBSP5.4Luc) and 9.0 kb (mBSP9.0Luc) of the BSP promoter was strongly up-regulated in parallel with endogenous BSP mRNA in differentiating SBMCs, but not in C3H10T1/2 cells. In contrast, 0.1 kb and 1.4 kb BSP promoter constructs did not show selective expression. To determine tissue-specific expression in vivo, transgenic mice expressing reporter constructs for the 9.0 kb promoter and a 4.8 kb promoter lacking two upstream Cbfa1/Runx2 elements (mBSP9.0Luc and mBSP4.8Luc, respectively) were generated. Analysis of various tissues collected from 1-, 4-, 7-, 14-, and 42-day-old mice revealed extremely high levels of luciferase activity in calvaria, mandible, and tibia of the mBSP9.0Luc mice. In contrast, soft tissues showed negligible luciferase expression. Mice harboring the 4.8 kb transgene also showed selective luciferase expression but displayed a significantly lower activity in mineralized tissues. Northern hybridization of endogenous BSP mRNA and immunostaining of BSP in mBSP9.0Luc mice showed a temporo-spatial expression pattern consistent with the luciferase activity. These results indicate that regulatory elements within the 9.0 kb region of the promoter are required for strong, tissue- and differentiation-specific expression of BSP.

Human Osteocalcin and Bone Sialoprotein Mediating Osteomimicry of Prostate Cancer Cells: Role of cAMP-Dependent Protein Kinase A Signaling Pathway

Osteocalcin and bone sialoprotein are the most abundant noncollagenous bone matrix proteins expressed by osteoblasts. Surprisingly, osteocalcin and bone sialoprotein are also expressed by malignant but not normal prostate epithelial cells. The purpose of this study is to investigate how osteocalcin and bone sialoprotein expression is regulated in prostate cancer cells. Our investigation revealed that (a) human osteocalcin and bone sialoprotein promoter activities in an androgen-independent prostate cancer cell line of LNCaP lineage, C4-2B, were markedly enhanced 7- to 12-fold in a concentration-dependent manner by conditioned medium collected from prostate cancer and bone stromal cells. (b) Deletion analysis of human osteocalcin and bone sialoprotein promoter regions identified cyclic AMP (cAMP)-responsive elements (CRE) as the critical determinants for conditioned medium-mediated osteocalcin and bone sialoprotein gene expression in prostate cancer cells. Consistent with these results, the protein kinase A (PKA) pathway activators forskolin and dibutyryl cAMP and the PKA pathway inhibitor H-89, respectively, increased or repressed human osteocalcin and bone sialoprotein promoter activities. (c) Electrophoretic mobility shift assay showed that conditioned medium-mediated stimulation of human osteocalcin and bone sialoprotein promoter activities occurs through increased interaction between CRE and CRE-binding protein. (d) Conditioned medium was found to induce human osteocalcin and bone sialoprotein promoter activities via increased CRE/CRE-binding protein interaction in a cell background-dependent manner, with marked stimulation in selected prostate cancer but not bone stromal cells. Collectively, these results suggest that osteocalcin and bone sialoprotein expression is coordinated and regulated through cAMP-dependent PKA signaling, which may define the molecular basis of the osteomimicry exhibited by prostate cancer cells.

Limited specificity of promoter constructs for gene therapy in osteosarcoma.

Osteosarcoma (OS), a malignant bone neoplasia in childhood, has poor prognosis if metastases appear in the lung. A novel therapeutic approach could consist in a gene therapeutic treatment of OS metastases. However, if promiscuous viral vectors are applied for the delivery of potentially toxic transgenes, their misdelivery into normal tissues could cause severe complications. This problem could be circumvented by application of OS-specific promoters for transgene expression control. We analysed the function of promoters described to be tumour-, osteosarcoma- or osteoblast-specific. Expression rates driven by osteoblast- specific fragments from the collagen1A1-promoter, the human Osteocalcin-promoter, the bone-sialoprotein promoter and the beta-catenin promoter depending on vitamin supplementation were analysed in five OS cell lines, in normal lung fibroblasts and in a non-osteoblastic prostate cancer cell line (LNCaP) by dual luciferase assays. In addition, an unspecific but doxycyclin-repressible promoter construct (pAd.3r-luc) was examined. We found that all constructs were active in OS cell lines to varying extents. The complete human Osteocalcin promoter and the bone-sialoprotein promoter were partially induced by vitamin D3 or C respectively while the pAd.3r-luc activity could be shut down by doxycyclin. In contrast, the human Osteocalcin-promoter was not activated by vitamin D3 in LNCaP cells; its action remained relatively low. Interestingly, excepting the beta-catenin promoter, we measured strong activities of all promoters in lung fibroblast cells. Our study demonstrates that promoter activity should be evaluated not only for the target cells of the gene therapeutic approaches, but also for neighbouring normal tissues. Unspecific but repressible promoters could represent an alternative.