Biomedical subjects

R H Kim

Publications and source records attributed to R H Kim.

At least 19 recordsLinked to original sources

PubMed2001

SNIP1 inhibits NF-kappa B signaling by competing for its binding to the C/H1 domain of CBP/p300 transcriptional co-activators.

SNIP1 is a 396-amino acid nuclear protein shown to be an inhibitor of the TGF-beta signal transduction pathway and to be important in suppressing transcriptional activation dependent on the co-activators CBP and p300. In this report we show that SNIP1 potently inhibits the activity of NF-kappa B, which binds the C/H1 domain of CBP/p300, but does not interfere with the activity of transcription factors such as p53, which bind to other domains of p300, or factors such as VP16, which are independent of these co-activators. Inhibition of NF-kappa B activity is a function of the N-terminal domain of SNIP1 and involves competition of SNIP1 and the NF-kappa B subunit, RelA/p65, for binding to p300, similar to the mechanism of inhibition of Smad signaling by SNIP1. Immunohistochemical staining shows that expression of SNIP1 is strictly regulated in development and that it colocalizes, in certain tissues, with nuclear staining for RelA/p65 and for p300, suggesting that they may regulate NF-kappa B activity in vivo in a spatially and temporally controlled manner. These data led us to suggest that SNIP1 may be an inhibitor of multiple transcriptional pathways that require the C/H1 domain of CBP/p300.

Animals↗

PubMed2001

Ceruloplasmin enhances DNA damage induced by cysteine/iron in vitro.

Ceruloplasmin (Cp) was found to promote the oxidative damage to DNA in vitro, as evidenced by the formation of 8-hydroxy-2'-deoxyguanosine and strand breaks, when incubated with a cysteine metal-catalyzed oxidation system (Cys-MCO) comprised of Fe(3+), O(2), and cysteine as an electron donor. The capacity of Cp to enhance oxidative damage to DNA was inhibited by hydroxyl radical scavengers such as sodium azide and mannitol, a metal chelator, diethylenetriaminepentaacetic acid, a spin-trapping agent, 5,5-dimethyl-1-pyrroline N-oxide (DMPO) and catalase. Ceruloplasmin also caused the two-fold enhancement of a mutation in the pUC18 lacZ' gene in the presence of Cys-MCO when measured as a loss of alpha-complementation. Incubation of Cp with Cys-MCO resulted in an increase in the content of carbonyl groups and the significant alteration of the ferroxidase activity, as well as the proteolytic susceptibility. The deoxyribose assay and the salicylate hydroxylation assay showed that hydroxyl free radicals were generated in the reaction of Cp with Cys-MCO. The release of a portion of Cu from Cp was observed, and conformational alterations were indicated by the changes in fluorescence spectra. Based on these results, we interpret the enhancing effect of Cp on DNA damage and mutagenicity induced by Cys-MCO as due to reactive oxygen species, probably hydroxyl free radicals, formed by the reaction of free Cu(2+), released from oxidatively damaged Cp, and H(2)O(2) produced by Cys-MCO. The release of Cu from Cp during oxidative stress could enhance the formation of reactive oxygen species and could also potentiate cellular damage.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2001

The molecular basis of O2-sensing and hypoxia tolerance in pheochromocytoma cells.

Hypoxia is a common environmental stimulus. However, very little is known about the mechanisms by which cells sense and respond to changes in oxygen. Our laboratory has utilized the PC12 cell line in order to study the biophysical and molecular response to hypoxia. The current review summarizes our results. We demonstrate that the O2-sensitive K(+) channel, Kv1.2, is present in PC12 cells and plays a critical role in the hypoxia-induced depolarization of PC12 cells. Previous studies have shown that PC12 cells secrete a variety of autocrine/paracrine factors, including dopamine, norepinephrine, and adenosine during hypoxia. We investigated the mechanisms by which adenosine modulates cell function and the effect of chronic hypoxia on this modulation. Finally, we present results identifying the mitogen- and stress-activated protein kinases (MAPKs and SAPKs) as hypoxia-regulated protein kinases. Specifically, we show that p38 and an isoform, p38gamma, are activated by hypoxia. In addition, our results demonstrate that the p42/p44 MAPK protein kinases are activated by hypoxia. We further show that p42/p44 MAPK is critical for the hypoxia-induced transactivation of endothelial PAS-domain protein 1 (EPAS1), a hypoxia-inducible transcription factor. Together, these results provide greater insight into the mechanisms by which cells sense and adapt to hypoxia.

Animals↗

PubMed2000

A novel smad nuclear interacting protein, SNIP1, suppresses p300-dependent TGF-beta signal transduction.

Members of the transforming growth factor-beta superfamily play critical roles in controlling cell growth and differentiation. Effects of TGF-beta family ligands are mediated by Smad proteins. To understand the mechanism of Smad function, we sought to identify novel interactors of Smads by use of a yeast two-hybrid system. A 396-amino acid nuclear protein termed SNIP1 was cloned and shown to harbor a nuclear localization signal (NLS) and a Forkhead-associated (FHA) domain. The carboxyl terminus of SNIP1 interacts with Smad1 and Smad2 in yeast two-hybrid as well as in mammalian overexpression systems. However, the amino terminus of SNIP1 harbors binding sites for both Smad4 and the coactivator CBP/p300. Interaction between endogenous levels of SNIP1 and Smad4 or CBP/p300 is detected in NMuMg cells as well as in vitro. Overexpression of full-length SNIP1 or its amino terminus is sufficient to inhibit multiple gene responses to TGF-beta and CBP/p300, as well as the formation of a Smad4/p300 complex. Studies in Xenopus laevis further suggest that SNIP1 plays a role in regulating dorsomedial mesoderm formation by the TGF-beta family member nodal. Thus, SNIP1 is a nuclear inhibitor of CBP/p300 and its level of expression in specific cell types has important physiological consequences by setting a threshold for TGF-beta-induced transcriptional activation involving CBP/p300.

Amino Acid Sequence↗

PubMed2000

Parathyroid hormone regulation of bone sialoprotein (BSP) gene transcription is mediated through a pituitary-specific transcription factor-1 (Pit-1) motif in the rat BSP gene promoter.

Bone sialoprotein (BSP) is a mineralized tissue-specific protein expressed by differentiated osteoblasts that appears to function in the initial mineralization of bone. Parathyroid hormone (PTH), which regulates serum calcium through its actions on bone cells, increases the expression of BSP in the rat osteosarcoma cell line (ROS 17/2.8). At 10(-8) M PTH (human 1-34 PTH), stimulation of BSP mRNA was first evident at 3 h ( approximately 3.8-fold), reached maximal levels at 6 h ( approximately 4.7-fold), and declined slowly thereafter. The effects of PTH, which were abrogated by cycloheximide (28 microg/ml), did not alter the stability of the BSP mRNA. The increased transcription was mimicked by both forskolin (10(-6) M) and isoproterenol (10(-7) M), and was also increased by 3-isobutyl-1-methylxanthine (IBMX; 10(-5) M), while the transcriptional activity induced by PTH was inhibited by the protein kinase A inhibitor, H89 (5x10(-6) M). From transient transfection assays using various BSP promoter-luciferase constructs, a pituitary-specific transcription factor-1 (Pit-1) regulatory element (nts -111 to -105) was identified as the target of transcriptional activation by PTH. Thus, transcriptional activity of constructs including the Pit-1 was enhanced approximately 4.7-fold by 10(-8) M PTH while 5'-ligation of the Pit-1 element conferred PTH regulation in an SV40 promoter construct. Binding of a nuclear protein, recognized by anti-Pit-1 antibodies, to a radiolabelled Pit-1-BSP probe was decreased in nuclear extracts prepared from PTH, forskolin and isoproterenol-stimulated ROS 17/2.8 cells. Moreover, co-transfection of ROS cells with a double-stranded Pit-1 oligonucleotide also increased luciferase activity. Collectively, these results indicate that PTH acts through a protein kinase A pathway involving cAMP to stimulate BSP transcription by blocking the action of a Pit-1-related nuclear protein that suppresses BSP transcription by binding a cognate element in the BSP promoter. Thus, we have identified a novel Pit-1 suppressor element in the rat BSP gene promoter that is the target of PTH-stimulated transcription of the BSP gene.

Amino Acid Motifs↗

PubMed2000

Ceruloplasmin enhances DNA damage induced by hydrogen peroxide in vitro.

Ceruloplasmin (Cp) was found to promote the oxidative damage to DNA, as evidenced by the formation of 8-hydroxy-2'-deoxyguanosine and strand breaks, when incubated with H2O2 in vitro. The capacity of Cp to enhance oxidative damage to DNA was inhibited by hydroxyl radical scavengers such as sodium azide and mannitol, a metal chelator, diethylenetriaminepentaacetic acid, and catalase. Although the oxidized protein resulted in an increase in the content of carbonyl groups, the ferroxidase activity and the proteolytic susceptibility were not significantly altered. The release of a portion of Cu from Cp was observed, and conformational alterations were indicated by the changes in fluorescence spectra. Based on these results, we suggest that damage to DNA is mediated in the H2O2/Cp system via the generation of *OH by released Cu2+ and/or loosely bound Cu exposed from oxidatively damaged Cp through the conformational change. The release of Cu from Cp during oxidative stress could enhance the formation of reactive oxygen species and could also potentiate cellular damage.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1999

Transcription of the bone sialoprotein gene is stimulated by v-Src acting through an inverted CCAAT box.

Bone sialoprotein (BSP) is an early marker of differentiated osteoblasts that has been implicated in the nucleation of hydroxyapatite crystal formation during de novo bone formation. Although essentially specific to mineralizing connective tissues, BSP is also expressed ectopically by carcinomas that exhibit microcalcification and which metastasize to bone with high frequency. However, it is not known how BSP is regulated in transformed cells. Because the v-src oncogene induces expression of a number of genes that are involved in tumor growth and metastasis, including osteopontin, we have studied the effects of v-Src on transcription of the BSP gene. Transfection of mouse src-/- cells with a v-src expression vector increased the transcriptional activity of rat BSP promoter/luciferase chimeric constructs approximately 5-fold. Deletion analysis revealed that the v-Src activity was targeted to an inverted CCAAT box located immediately upstream from an inverted TATA box in the BSP promoter. Although mutation of the CCAAT box diminished the basal transcription activity of the BSP promoter, the Src-induced stimulation was completely abolished. Gel mobility shift analysis identified four nuclear factors that bound to this region of the BSP promoter, two of which required an intact CCAAT sequence. Monoclonal antibodies identified nuclear factor-Y (NF-Y) as the principal nuclear factor that bound to the CCAAT box; the second factor (beta) showing strong binding only in short constructs containing the CCAAT sequence. Transcription analyses with a dominant negative NF-Y expression vector confirmed that NF-Y mediated the action of v-Src. These studies indicate that BSP gene expression in transformed cells can be up-regulated by Src kinase activity through a mechanism mediated by the NF-Y transcription factor, which targets an inverted CCAAT box in the BSP gene promoter.

3T3 Cells↗

PubMed1999

Bone sialoprotein.

The search for a protein nucleator of hydroxyapatite crystal formation has been a focus for the isolation and characterization of the major non-collagenous proteins in bone. Of the proteins characterized to date, bone sialoprotein (BSP) has emerged as the only bona fide candidate for nucleation. BSP is a highly glycosylated and sulphated phosphoprotein that is found almost exclusively in mineralized connective tissues. Characteristically, polyglutamic acid and arginine-glycine-aspartate (RGD) motifs with the ability to bind hydroxyapatite and cell-surface integrins, respectively, have been conserved in the protein sequence. Expression of the BSP gene, which is induced in newly formed osteoblasts, is up-regulated by hormones and cytokines that promote bone formation and down-regulated by factors that suppress bone formation. Thus, BSP has the biophysical and chemical properties of a nucleator, and its temporo-spatial expression coincides with de novo mineralization in bone and cementum. Moreover, BSP has been associated with mineral crystal formation in several pathologies, including breast carcinomas. However, the ability of BSP to mediate cell attachment and to signal through the RGD motif points to alternate functions for BSP which need further investigation. In combination, the hydroxyapatite-binding polyglutamic acid sequences and the RGD provide bi-functional entities through which BSP may mediate the targeting and attachment of normal and metastasizing cells to the bone surface.

Amino Acid Sequence↗

PubMed1998

Rheolytic thrombectomy of chronic coronary occlusion.

Percutaneous intervention in patients with intracoronary thrombus continues to pose a significant clinical challenge. In this report, we describe the successful treatment of a 44-year-old patient with an extensive chronic thrombotic occlusion of the right coronary artery using a rheolytic thrombectomy catheter. Despite angiographic documentation of coronary thrombosis 104 days prior to treatment and a voluminous thrombus burden (60 mm in length x 3 mm in diameter), rapid recanalization was accomplished with this device without embolic complications. At 1 year clinical follow-up, the patient has remained symptom free. The design of this novel device and its mechanism of action are described.

Adult↗

PubMed1998

Enhancement by copper, zinc superoxide dismutase of DNA damage and mutagenicity with hydrogen peroxide.

Oxidative DNA damage caused by hydrogen peroxide was enhanced by copper, zinc superoxide dismutase (CuZnSOD) in a concentration-dependent manner, as reflected by the formation of 8-hydroxy-2'-deoxyguanosine (8-OH-dG) and strand breaks. Hydroxyl radical scavengers such as sodium azide, mannitol and 5,5-dimethyl-1-pyrroline N-oxide (DMPO), a metal chelator, diethylenetriamine-pentaacetic acid, and catalase decreased strand breaks and 8-OH-dG formation in DNA. The deoxyribose assay showed that hydroxyl free radicals were generated in the reaction of CuZnSOD with H2O2. CuZnSOD also caused enhancement of mutation in the pUC18 lacZ' gene in the presence of H2O2 when measured as a loss of alpha-complementation. Based on these results, we interpret the effects of CuZnSOD on hydrogen peroxide induced DNA damage and mutation as due to reactive oxygen species, probably hydroxyl free radicals, formed predominantly by the reaction of hydrogen peroxide and free Cu2+ released from oxidatively damaged CuZnSOD.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed1998

Characteristics of vitamin D3 receptor (VDR) binding to the vitamin D response element (VDRE) in rat bone sialoprotein gene promoter.

Bone sialoprotein (BSP) is a mineralised tissue-specific protein that is highly expressed during the initial formation of bone and cementum. Expression of BSP is suppressed by the osteotropic hormone, 1,25-dihydroxyvitamin D3 (vitamin D3), which regulates bone remodelling. In previous studies, we have identified a vitamin D response element (VDRE) that is integrated with a novel inverted TATA box in the rat BSP promoter which mediates the suppression of BSP transcription (1). Although the nucleotide sequences of VDREs in different genes conform to a direct (hexamer) repeat, spaced by three nucleotides, the precise sequences are unique for each VDRE. To determine whether the nucleotide differences in the VDRE influence VDR binding, we have compared interactions of VDR proteins with various VDREs using gel mobility shift analysis. Both natural and recombinant VDRs bound to rat BSP and both mouse and porcine osteopontin (OPN) VDRE oligonucleotides in a concentration-dependent manner with a strong preference for dimer formation, whereas equal amounts of dimer and monomer were bound to the human osteocalcin VDRE. However, whereas a truncated VDR comprising the DNA binding domain alone bound the mouse osteopontin VDRE, it failed to interact with the porcine OPN and rat BSP VDREs. VDR binding to the BSP was sequence specific, as shown by mutagenesis analysis, and could be abolished by heat and VDR antibody. These studies demonstrate that subtle differences in the nucleotide sequence of VDREs affect VDR binding, which mediates the vitamin D3 response.

Animals↗

PubMed1997

Transforming growth factor-beta 1 regulation of bone sialoprotein gene transcription: identification of a TGF-beta activation element in the rat BSP gene promoter.

Transforming growth factor-beta (TGF-beta) increases steady-state mRNA levels of several extracellular matrix proteins in mineralized connective tissues. Bone sialoprotein (BSP) is a major constituent of the bone matrix, thought to initiate and regulate the formation of mineral crystals. To determine the molecular pathways of TGF-beta 1 regulation of bone proteins, we have analyzed the effects of the TGF-beta 1 on the expression of the BSP in the rat osteosarcoma cell line (ROS 17/2.8). TGF-beta 1 at 1 ng/ml, increased BSP mRNA levels in ROS 17/2.8 cells approximately 8-fold: the stimulation was first evident at 3 hr, reached maximal levels at 12 hr and slowly declined thereafter. Since the stability of the BSP mRNA was not significantly affected by TGF-beta 1, and nuclear "run-on" transcription analyses revealed only a approximately 2-fold increase in the transcription of the BSP gene, most of the increase in BSP mRNA appeared to involve a nuclear post-transcriptional mechanism. Moreover, the effects of TGF-beta 1 were indirect, since the increase in BSP mRNA was abrogated by cycloheximide (28 micrograms/ml). To identify the site of transcriptional regulation by TGF-beta 1, transient transfection analyses were performed using BSP gene promoter constructs linked to a luciferase reporter gene. Constructs that included nt -801 to -426 of the promoter sequence were found to enhance transcriptional activity approximately 1.8-fold in cells treated with TGF-beta 1. Within this sequence, approximately 500 nt upstream of the transcription start site, a putative TGF-beta activation element (TAE) was identified that contained the 5'-portion of the nuclear factor-1 (NF-1) canonical sequence (TTGGC) overlapping a consensus sequence for activator protein-2 (AP-2). The functionality of the TAE was shown by an increased binding of a nuclear protein from TGF-beta 1 stimulated cells in gel mobility shift assays and from the attenuation of TGF-beta 1-induced luciferase activity when cells were co-transfected with a double-stranded TAE oligonucleotide. Competition gel mobility shift analyses revealed that the nuclear protein that binds to the TAE has similar properties to, but is distinct from, NF-1 nuclear protein. These studies have therefore identified a TGF-beta activation element (TAE) in the rat BSP gene promoter that mediates the stimulatory effects of TGF-beta 1 on BSP gene transcription.

Animals↗

PubMed1996

Identification of a vitamin D3-response element that overlaps a unique inverted TATA box in the rat bone sialoprotein gene.

Bone sialoprotein (BSP), an early marker of osteoblast differentiation, has been implicated in the nucleation of hydroxyapatite during bone formation de novo. Our studies, using the osteoblastic cell line ROS 17/2.8, have revealed that rat BSP gene expression is suppressed by 1,25-dihydroxyvitamin D3[1,25(OH)2D3], which is a powerful regulator of bone formation and resorption. To determine the molecular basis of the transcriptional suppression of BSP gene transcription by 1,25(OH)2D3, we have conducted transient transfection analyses with chimaeric constructs of the rat BSP gene promoter linked to a luciferase reporter gene. 1,25(OH)2D3 suppressed expression in all constructs, including a short construct (pLUC 3; nt -116 to +60) that contained a putative vitamin D3-response element (VDRE; AGGGTTTATAGGTCA; nt -28 to -14) that overlaps a unique inverted TATA (TTTATA) box. Mobility shift assays demonstrated strong binding of recombinant human vitamin D3 receptor protein (hVDR) to the VDRE. Point mutations introduced into each half-site and analysed for 1,25(OH)2D3-mediated suppression of transcription and for hVDR binding either decreased or increased both transcriptional suppression and binding. In comparison with activating VDREs, the rat BSP VDRE bound VDR-VDR homodimers more avidly than VDR-RXR alpha heterodimers (where RXR is retinoid X receptor). These studies have therefore identified a novel 1,25(OH)2D3 suppressor element that overlaps the inverted TATA box in the rat BSP gene and indicate that transcriptional suppression of the rat BSP gene by 1,25(OH)2D3 might involve competition between the VDR and the TATA binding protein (TBP).

Animals↗

PubMed1996

Nonoperative removal of sialoliths and sialodochoplasty of salivary duct strictures.

OBJECTIVE: To describe the nonsurgical removal of sialoliths and treatment of salivary duct strictures. DESIGN: Case series. SETTING: Two 200-bed general community hospitals. PATIENTS: Twelve consecutive patients from April 1985 to November 1994-8 with calculi, 3 with salivary duct strictures, and 1 with calculi and stricture. RESULTS: Successful nonoperative removal of calculi in 7 of 9 patients. All 4 sialodochoplasties were successful. All 10 patients with successful procedures had no recurrent symptoms. Seven patients have been symptom-free for 10 months to 10 years. Communication with 3 patients has been impossible recently, although these patients were symptom-free for at least 3 years. CONCLUSIONS: These methods of nonsurgical sialolith removal and sialodocoplasty were highly successful and should be used as the initial therapies for patients with these conditions.

Catheterization↗

PubMed1996

AP-1 regulation of the rat bone sialoprotein gene transcription is mediated through a TPA response element within a glucocorticoid response unit in the gene promoter.

Bone sialoprotein (BSP), a protein which has been implicated in the initial mineralization of newly-formed bone, provides an early phenotypic marker for differentiated osteoblasts. BSP expression is induced by glucocorticoids in association with osteoblast differentiation, and a glucocorticoid response element (GRE) overlapping a putative TRE (TPA, 12-O-tetradecanoyl-phorbol 13-acetate, response element) site has been identified in the rat BSP promoter (Ogata et al., 1995). Since AP-1 and the glucocorticoid receptor have a central role in regulating cell proliferation and differentiation, we have studied AP-1 activity, stimulated by 100 ng/ml TPA in normal fetal rat calvarial cells and in transformed rat osteosarcoma cells (ROS 17/2.8). A transient induction of both c-fos and c-jun mRNAs by TPA was observed in both cell populations, together with an associated suppression of BSP mRNA in the fetal rat calvarial cells. Rat BSP promoter constructs, transiently transfected into ROS 17/2.8 cells, were used to show that TPA suppressed transcription of a luciferase construct (-938/+60; pLUC6) that included the GRE/TRE, but not transcription of shorter contructs lacking this element. Notably, suppression of pLUC6 transcription by TPA was abrogated in the presence of the synthetic glucocorticoid, dexamethasone. Gel mobility shift analyses were performed using two double-stranded synthetic oligonucleotides. These encompassed the TRE and either the distal pair of GRE half-sites (-936/ -910; GRE3) or the proximal pair of GRE half-sites (-925/-899; GRE 4) that comprise the GRE/AP-1 element. The assay showed binding of both AP-1 complexes and recombinant c-Jun homodimers. Additionally, either the c-Jun or glucocorticoid receptor could displace its counterpart from the GRE/TRE but not from consensus GRE and TRE oligonucleotides, indicating that the abrogation of AP-1-mediated gene suppression by glucocorticoids could involve competitive binding. These studies, therefore, have identified a glucocorticoid response unit through which c-Fos and c-Jun can suppress the expression of BSP in proliferating pre-osteoblastic cells and through which glucocorticoids can ameliorate the effects of AP-1 and promote osteoblast differentiation and the associated expression of BSP.

Animals↗

PubMed1996

Characterization of the bone sialoprotein (BSP) gene promoter.

Bone sialoprotein is a 34 kDa phosphorylated and sulphated glycoprotein that is essentially unique to mineralizing connective tissues. Recent studies on the developmental expression of BSP mRNA and the 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. To study the cell-specific regulation of BSP we have isolated genomic clones that encompass the BSP promoter regions of both the human and rat genes. These promoters are characterized by a highly conserved region (BSP Box) that extends upstream from the transcription start site to nt -370. Within this region the immediate promoter is further characterized by a unique inverted TATA box and an inverted CCAAT box, both of which are required for basal transcriptional activity. The TATA box is overlapped by a vitamin D3 response element (VDRE) which appears to mediate vitamin D suppression of BSP gene transcription by competing with the TATA-binding protein (TBP) for occupancy of the site of the pre-initiation complex formation. Mutation of the inverted TATA box into a normal TATA sequence increases transcription slightly but does not affect the functionality of the VDRE indicating that the orientation of the TATA box is not critical for these functions. Further upstream an AP-1 site, overlapped by a steroid hormone response-like sequence, mediates down-regulation of BSP transcription induced by TPA that is abrogated by a complex interaction between Jun and the glucocorticoid receptor protein induced by dexamethasone. Thus, the characterization of approximately 3 kb of the BSP promoter and approximately 2 kb of the first intron has revealed several sites of transcriptional regulation that are important in regulating BSP expression and, consequently, bone formation.

Animals↗

PubMed1995

An inverted TATA box directs downstream transcription of the bone sialoprotein gene.

The orientation of the TATA box is thought to direct downstream transcription of eukaryotic genes by RNA polymerase II. However, the putative TATA box in the promoter of the bone sialoprotein (BSP) gene, which codes for a tissue-specific and developmentally regulated bone matrix protein, is inverted (5'-TTTATA-3') relative to the consensus TATA box sequence (5'-TATAAA-3') and is overlapped by a vitamin D3-response element. Here we show that the inverted TATA sequence in the rat BSP gene binds to recombinant TATA-box-binding protein (TBP) with an affinity similar to that observed with the consensus TATA box, and site-directed point mutations in the inverted TATA sequence (mutating TTTATA into TCTCTA) abrogate both TBP binding and BSP promoter activity. However, when the inverted TATA sequence is changed to a canonical TATAAA, the TBP- and vitamin D3 receptor-binding properties together with the BSP promoter activity are retained. In addition, we found that the TBP is required to reconstitute in vitro transcription driven by the BSP promoter. These studies, which have revealed a naturally occurring inverted TATA box that can bind TBP and direct downstream transcription, demonstrate that the orientation of the TATA box does not determine the direction of transcription in higher eukaryotic genes. Consequently, the inverted TATA box that is conserved in the human, rat and mouse BSP gene promoters will provide an excellent in vivo model to investigate the polarity of the transcription factor IID-DNA complex and its relation to downstream transcription.

Animals↗

PubMed1995

Glucocorticoid regulation of bone sialoprotein (BSP) gene expression. Identification of a glucocorticoid response element in the bone sialoprotein gene promoter.

Glucocorticoids modulate the development and growth of many organs through interactions with a specific intracellular receptor (glucocorticoid receptor) that regulates gene transcription through a cognate element, the glucocorticoid response element (GRE), in the promoter of target genes. In bone formation glucocorticoids stimulate osteoblast differentiation and the formation of bone matrix. Recent studies have demonstrated that the induction of the bone sialoprotein (BSP) gene is associated with osteoblast differentiation and de novo bone formation. To determine the molecular pathways of glucocorticoid regulation of BSP expression, we have analyzed the effects of the synthetic glucocorticoid, dexamethasone, on the expression of the BSP by bone cells in vitro. At 10 nM, dexamethasone induced BSP expression in association with bone tissue formation by confluent fetal rat calvarial cells and adult rat marrow cells and also stimulated BSP expression up to sixfold in osteoblastic cells (UMR 106-6 and ROS 17/2.8 cells). Most of the stimulation was blocked by cycloheximide, indicating direct and indirect mechanisms of BSP gene regulation. Nuclear 'run-on' transcription analysis revealed an up to twofold increase in transcription corresponding to the increase in mRNA that was unaffected by cycloheximide. Analysis of BSP mRNA in the presence of a transcription inhibitor (5,6-dichloro-1-beta-D-ribofuanosyl benzimidazole) by Northern hybridization revealed that the stability of the BSP mRNA was not significantly altered by dexamethasone, indicating that the major, indirect, stimulation of BSP expression involves a nuclear post-transcriptional mechanism. To study the direct effects of dexamethasone, nucleotide sequence analysis of the rat BSP promoter was extended upstream to position -2992 and downstream to +2282 in the first intron. Transient transfection analyses, using various rat BSP promoter constructs linked to a luciferase reporter gene, and gel mobility shift assays were used to identify a putative glucocorticoid response unit comprising three GRE half-sites and a putative AP-1 site, located within positions -906 to -931 upstream from the translation start site of the BSP gene promoter. BSP transcription was stimulated approximately 1.5-fold by dexamethasone through this GRE, indicating that its direct effects are mediated by glucocorticoid receptor binding to this site. These studies, therefore, have identified both indirect and direct pathways of glucocorticoid regulation of BSP gene expression, the direct effects being mediated by a GRE in the rat BSP promoter through which the effects of glucocorticoids on BSP gene transcription appear to be regulated.

Animals↗