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PubMed1995

Transcriptional activity of core binding factor-alpha (AML1) and beta subunits on murine leukemia virus enhancer cores.

Core binding factor (CBF), also known as polyomavirus enhancer-binding protein 2 and SL3 enhancer factor 1, is a mammalian transcription factor that binds to an element termed the core within the enhancers of the murine leukemia virus family of retroviruses. The core elements of the SL3 virus are important genetic determinants of the ability of this virus to induce T-cell lymphomas and the transcriptional activity of the viral long terminal repeat in T lymphocytes. CBF consists of two subunits, a DNA binding subunit, CBF alpha, and a second subunit, CBF beta, that stimulates the DNA binding activity of CBF alpha. One of the genes that encodes a CBF alpha subunit is AML1, also called Cbf alpha 2. This locus is rearranged by chromosomal translocations in human myeloproliferative disorders and leukemias. An exogenously expressed Cbf alpha 2-encoded subunit (CBF alpha 2-451) stimulated transcription from the SL3 enhancer in P19 and HeLa cells. Activity was mediated through the core elements. Three different isoforms of CBF beta were also tested for transcriptional activity on the SL3 enhancer. The longest form, CBF beta-187, increased the transcriptional stimulation by CBF alpha 2-451 twofold in HeLa cells, although it had no effect in P19 cells. Transcriptional activation by CBF beta required binding to the CBF alpha subunit, as a form of CBF beta that lacked binding ability, CBF beta-148, failed to increase activity. These results indicated that at least in certain cell types, the maximum activity of CBF required both subunits. They also provided support for the hypothesis that CBF is a factor in T lymphocytes that is responsible for recognition of the SL3 cores. We also examined whether CBF could distinguish a 1-bp difference between the enhancer core of SL3 and the core of the nonleukemogenic virus, Akv. This difference strongly affects transcription in T cells and leukemogenicity of SL3. However, no combination of CBF alpha and CBF beta subunits that we tested was able to distinguish the 1-bp difference in transcription assays. Thus, a complete understanding of how T cells recognize the SL3 core remains to be elucidated.

Animals↗
PubMed2001

Assessment of gene regulation by bone morphogenetic protein 2 in human marrow stromal cells using gene array technology.

Marrow stromal cells can differentiate into osteoblasts, adipocytes, myoblasts, and chondrocytes. Bone morphogenetic protein 2 (BMP-2) is a potent stimulator of osteoblastic differentiation, and identification of the genes regulated by BMP-2 in these cells should provide insight into the mechanism(s) of osteoblastic differentiation. Thus, we used a conditionally immortalized human marrow stromal cell line (hMS) and a gene expression microarray containing probes for a total of 6800 genes to compare gene expression in control and BMP-2-treated cultures. A total of 51 genes showed a consistent change in messenger RNA (mRNA) frequency between two repeat experiments. Seventeen of these genes showed a change in expression of at least 3-fold in BMP-2-treated cultures over control cultures. These included nuclear binding factors (10 genes), signal transduction pathway genes (2 genes), molecular transport (1 gene), cell surface proteins (2 genes) and growth factors (2 genes). Of particular interest were four of the nuclear binding factor genes ID-1, ID-2, ID-3, and ID-4. These encode dominant negative helix-loop-helix (dnHLH) proteins that lack the nuclear binding domain of the basic HLH proteins and thus have no transcriptional activity. They have been implicated in blocking both myogenesis and adipogenesis. Other transcription factors up-regulated at least 3-fold by BMP-2 included Dlx-2, HES-1, STAT1, and JunB. The changes in these nuclear binding factor mRNA levels were confirmed by real-time reverse-transcriptase-polymerase chain reaction (RT-PCR). A further three transcription factors, core binding factor beta (CBFbeta), AREB6, and SOX4, showed changes in expression of between 2- and 3-fold with BMP-2 treatment. In summary, we have used a gene chip microarray to identify a number of BMP-2 responsive genes in hMS cells. Thus, these studies provide potential candidate genes that may induce osteoblastic differentiation or, in the case of the ID proteins, block differentiation along alternate pathways.

Bone Marrow Cells↗
PubMed2005

The inv(16) cooperates with ARF haploinsufficiency to induce acute myeloid leukemia.

The inv(16) is one of the most frequent chromosomal translocations associated with acute myeloid leukemia (AML) and creates a chimeric fusion protein consisting of most of the runt-related X1 co-factor, core binding factor beta fused to the smooth muscle myosin heavy chain MYH11. Expression of the ARF tumor suppressor is regulated by runt-related X1, suggesting that the inv(16) fusion protein (IFP) may repress ARF expression. We established a murine bone marrow transplant model of the inv(16) in which wild type, Arf+/-, and Arf-/- bone marrow were engineered to express the IFP. IFP expression was sufficient to induce a myelomonocytic AML even when expressed in wild type bone marrow, yet removal of only a single allele of Arf greatly accelerated the disease, indicating that Arf is haploinsufficient for the induction of AML in the presence of the inv(16).

Alleles↗
PubMed2006

Vascular calcification: pathobiological mechanisms and clinical implications.

Once thought to result from passive precipitation of calcium and phosphate, it now appears that vascular calcification is a consequence of tightly regulated processes that culminate in organized extracellular matrix deposition by osteoblast-like cells. These cells may be derived from stem cells (circulating or within the vessel wall) or differentiation of existing cells, such as smooth muscle cells (SMCs) or pericytes. Several factors induce this transition, including bone morphogenetic proteins, oxidant stress, high phosphate levels, parathyroid hormone fragments, and vitamin D. Once the osteogenic phenotype is induced, cells gain a distinctive molecular fingerprint, marked by the transcription factor core binding factor alpha1. Alternatively, loss of inhibitors of mineralization, such as matrix gamma-carboxyglutamic acid Gla protein, fetuin, and osteopontin, also contribute to vascular calcification. The normal balance between promotion and inhibition of calcification becomes dysregulated in chronic kidney disease, diabetes mellitus, atherosclerosis, and as a consequence of aging. Once the physiological determinants of calcification are perturbed, calcification may occur at several sites in the cardiovascular system, including the intima and media of vessels and cardiac valves. Here, calcification may occur through overlapping yet distinct molecular mechanisms, each with different clinical ramifications. A variety of imaging techniques are available to visualize vascular calcification, including fluoroscopy, echocardiography, intravascular ultrasound, and electron beam computed tomography. These imaging modalities vary in sensitivity and specificity, as well as clinical application. Through greater understanding of both the mechanism and clinical consequences of vascular calcification, future therapeutic strategies may be more effectively designed and applied.

Animals↗
PubMed1996

The biological significance of the multidrug resistance gene MRP in inversion 16 leukemias.

Multidrug resistance represents an important mechanism by which leukaemic and solid tumour cells escape cell death after exposure to anthracyclines and other natural products. Acute myeloid leukaemia (AML) associated with the inversion chromosome 16: inv(16)(p13q22) has a favourable prognosis and is known to be chemosensitive. The inversion chromosome is seen in a number of FAB subclasses but is most commonly associated with acute myelomonocytic leukaemia with abnormal eosinophils, M4Eo. It results in the creation of a fusion between the myosin heavy chain gene (MYH11) on the short arm and the gene for a transcription factor, core binding factor beta (CBFB) on the long arm. In a subset of these inv(16) AML patients, inversion also results in loss of the gene for the multidrug resistance protein (MRP) at the short arm breakpoint. This gene maps to 16p13.13, centromeric to the primary short arm breakpoint, separated from MYH11 by a distance of approximately 150kb. Deletion of the MRP gene has been demonstrated by in situ hybridisation, gene dosage studies and by loss of heterozygosity of a flanking microsatellite marker (D16S405). Twenty two patients with inv(16) leukaemia were analysed for deletion of the MRP gene. Deletion of the gene was detected in seven patients, fourteen patients showed retention of the gene and in one case the findings were indeterminate. Clinical data from 13 of these patients were analysed revealing deletion of the MRP gene to be significantly associated with longer time from diagnosis until failure (death or relapse from complete remission) in these patients (p = 0.007). From this work and the growing literature concerning MRP, it appears likely that the deletion of an MRP allele, may favourably affect the biology of inv(16) AML and may have important prognostic implications.

ATP-Binding Cassette Transporters↗
PubMed2000

Developmental regulation of collagenase-3 mRNA in normal, differentiating osteoblasts through the activator protein-1 and the runt domain binding sites.

Collagenase-3 mRNA is initially detectable when osteoblasts cease proliferation, increasing during differentiation and mineralization. We showed that this developmental expression is due to an increase in collagenase-3 gene transcription. Mutation of either the activator protein-1 or the runt domain binding site decreased collagenase-3 promoter activity, demonstrating that these sites are responsible for collagenase-3 gene transcription. The activator protein-1 and runt domain binding sites bind members of the activator protein-1 and core-binding factor family of transcription factors, respectively. We identified core-binding factor a1 binding to the runt domain binding site and JunD in addition to a Fos-related antigen binding to the activator protein-1 site. Overexpression of both c-Fos and c-Jun in osteoblasts or core-binding factor a1 increased collagenase-3 promoter activity. Furthermore, overexpression of c-Fos, c-Jun, and core-binding factor a1 synergistically increased collagenase-3 promoter activity. Mutation of either the activator protein-1 or the runt domain binding site resulted in the inability of c-Fos and c-Jun or core-binding factor a1 to increase collagenase-3 promoter activity, suggesting that there is cooperative interaction between the sites and the proteins. Overexpression of Fra-2 and JunD repressed core-binding factor a1-induced collagenase-3 promoter activity. Our results suggest that members of the activator protein-1 and core-binding factor families, binding to the activator protein-1 and runt domain binding sites are responsible for the developmental regulation of collagenase-3 gene expression in osteoblasts.

Animals↗
PubMed2000

Regulation of osteoblast differentiation mediated by bone morphogenetic proteins, hedgehogs, and Cbfa1.

Osteoblasts arise from common progenitors with chondrocytes, muscle and adipocytes, and various hormones and local factors regulate their differentiation. We review here regulation of osteoblast differentiation mediated by the local factors such as bone morphogenetic proteins (BMPs) and hedgehogs and the transcription factor, core-binding factor alpha-1 (Cbfa1). BMPs are the most potent regulators of osteoblast differentiation among the local factors. Sonic and Indian hedgehogs are involved in osteoblast differentiation by interacting with BMPs. Cbfa1, a member of the runt domain gene family, plays a major role in the processes of a determination of osteoblast cell lineage and maturation of osteoblasts. Cbfa1 is an essential transcription factor for osteoblast differentiation and bone formation, because Cbfa1-deficient mice completely lacked bone formation due to maturation arrest ofosteoblasts. Although the regulatory mechanism of Cbfa1 expression has not been fully clarified, BMPs are an important local factor that up-regulates Cbfa1 expression. Thus, the intimate interaction between local factors such as BMPs and hedgehogs and the transcription factor, Cbfa1, is important to osteoblast differentiation and bone formation.

Animals↗
PubMed2001

Differentiation of embryonic mesenchymal cells to odontoblast-like cells by overexpression of dentin matrix protein 1.

Cells of the craniofacial skeleton are derived from a common mesenchymal progenitor. The regulatory factors that control their differentiation into various cell lineages are unknown. To investigate the biological function of dentin matrix protein 1 (DMP1), an extracellular matrix gene involved in calcified tissue formation, stable transgenic cell lines and adenovirally infected cells overexpressing DMP1 were generated. The findings in this paper demonstrate that overexpression of DMP1 in pluripotent and mesenchyme-derived cells such as C3H10T1/2, MC3T3-E1, and RPC-C2A can induce these cells to differentiate and form functional odontoblast-like cells. Functional differentiation of odontoblasts requires unique sets of genes being turned on and off in a growth- and differentiation-specific manner. The genes studied include transcription factors like core binding factor 1 (Cbfa1), bone morphogenetic protein 2 (BMP2), and BMP4; early markers for extracellular matrix deposition like alkaline phosphatase (ALP), osteopontin, osteonectin, and osteocalcin; and late markers like DMP2 and dentin sialoprotein (DSP) that are expressed by terminally differentiated odontoblasts and are responsible for the formation of tissue-specific dentin matrix. However, this differentiation pathway was limited to mesenchyme-derived cells only. Other cell lines tested by the adenoviral expression system failed to express odontoblast-phenotypic specific genes. An in vitro mineralized nodule formation assay demonstrated that overexpressed cells could differentiate and form a mineralized matrix. Furthermore, we also demonstrate that phosphorylation of Cbfa1 (osteoblast-specific transcription factor) was not required for the expression of odontoblast-specific genes, indicating the involvement of other unidentified odontoblast-specific transcription factors or coactivators. Cell lines that differentiate into odontoblast-like cells are useful tools for studying the mechanism involved in the terminal differentiation process of these postmitotic cells.

3T3 Cells↗
PubMed2002

High bone resorption in adult aging transgenic mice overexpressing cbfa1/runx2 in cells of the osteoblastic lineage.

The runt family transcription factor core-binding factor alpha1 (Cbfa1) is essential for bone formation during development. Surprisingly, transgenic mice overexpressing Cbfa1 under the control of the 2.3-kb collagen type I promoter developed severe osteopenia that increased progressively with age and presented multiple fractures. Analysis of skeletally mature transgenic mice showed that osteoblast maturation was affected and that specifically in cortical bone, bone resorption as well as bone formation was increased, inducing high bone turnover rates and a decreased degree of mineralization. To understand the origin of the increased bone resorption, we developed bone marrow stromal cell cultures and reciprocal coculture of primary osteoblasts and spleen cells from wild-type or transgenic mice. We showed that transgenic cells of the osteoblastic lineage induced an increased number of tartrate-resistant acid phosphatase-positive multinucleated cells, suggesting that primary osteoblasts as well as bone marrow stromal cells from transgenic mice have stronger osteoclastogenic properties than cells derived from wild-type animals. We investigated the candidate genes whose altered expression could trigger this increase in bone resorption, and we found that the expression of receptor activator of NF-kappaB ligand (RANKL) and collagenase 3, two factors involved in bone formation-resorption coupling, was markedly increased in transgenic cells. Our data thus suggest that overexpression of Cbfa1 in cells of the osteoblastic lineage does not necessarily induce a substantial increase in bone formation in the adult skeleton but has a positive effect on osteoclast differentiation in vitro and can also dramatically enhance bone resorption in vivo, possibly through increased RANKL expression.

Aging↗
PubMed1992

A phorbol ester response element within the human T-cell receptor beta-chain enhancer.

The activity of the T-cell receptor beta-chain gene enhancer is increased by activators of the protein kinase C pathway during T-cell activation. Analysis of mutant enhancer constructs identified two elements, beta E2 and beta E3, conferring phorbol ester inducibility. Multimerized beta E2 acted in isolation as a phorbol ester-responsive element. Both beta E2 and beta E3, which contain a consensus Ets-binding site, were shown to bind directly to the product of the c-ets-1 protooncogene. Both regions also bound a second factor, core-binding factor. Mutation of the beta E2 Ets site abolished the inducibility of the beta E2 multimer. beta E2 and beta E3 Ets site mutations also profoundly affected activity and inducibility of the enhancer. In contrast, enhancer activity but not its inducibility was affected by mutation of the beta E2 core-binding factor site. Cotransfection studies showed that Ets-1 specifically repressed activity of the multimerized beta E2 element and the complete T-cell receptor beta-chain enhancer. These data show that the T-cell receptor beta-chain enhancer responds to protein kinase C-mediated activation signals via a functional domain, composed of two elements, which contains binding sites for Ets transcription factors and which is negatively regulated by Ets-1.

Base Sequence↗
PubMed2003

Butyrates, as a single drug, induce histone acetylation and granulocytic maturation: possible selectivity on core binding factor-acute myeloid leukemia blasts.

Acute myeloid leukemia (AML) is a disease characterized by a block of maturation. Genes coding for core binding factors are rearranged in a considerable subset of AML cases and result in an altered interaction of core binding factor (CBF) subunits with transcriptional coregulators (NCoR/SMRT). Recruitment of histone deacetylase is also altered in AML, and a subsequent transcriptional repression of target genes involved in myeloid maturation is determined. We determined here the effects of two histone deacetylase inhibitors, sodium butyrate and the stable prodrug xylitol butyrate derivative (D1), on a t(8;21)-positive cell line (Kasumi-1) as well as primary AML blasts. Exposure (24-96 h) to butyrates (1 mM) of Kasumi-1 cells induced histone H4 acetylation, whereas H3 acetylation was unchanged. Induction of morphological and immunophenotypic granulocytic maturation (96 h), also confirmed by an increased expression of CAAT/enhancer binding protein alpha, was observed. Inhibition of proliferation and apoptosis via activation of caspase-9 was also observed. In primary AML blasts, butyrates (0.5 mM) increased histone H4 acetylation of 18 of 19 cases tested. Terminal granulocytic maturation was observed in all cases (5 of 5) characterized by chromosomal translocations involving CBF, whereas in non-CBF cases, maturation was incomplete (4 of 8) or absent (4 of 8). Our data indicate the possibility to effectively remove, in CBF AML cases, the maturation block generated by histone deacetylase stable recruitment, contributing to a possible development of molecularly targeted therapies of AML.

Acetylation↗
PubMed2003

Vitamin D and dexamethasone inversely regulate parathyroid hormone-induced regulator of G protein signaling-2 expression in osteoblast-like cells.

The PTH/PTHrP receptor stimulates both adenylate cyclase- and phospholipase C-dependent signaling pathways via different G proteins. The biological actions of PTH on bone are modified by steroid hormones. PTH induces expression of regulator of G protein signaling (RGS)-2, a putative preferential inhibitor of G(q)-mediated phospholipase C activation. We investigated whether steroid hormones interfere with PTH signaling by modulating PTH-induced RGS-2 expression in osteoblast-like UMR 106-01 cells. PTH (1-34) rapidly and transiently induced expression of RGS-2 mRNA and protein via the cAMP/protein kinase A pathway within 30 min, with maximal protein abundance after 2 h. PTH-induced RGS-2 preferentially bound to Galpha(q), compared with Galpha(s) protein. 1,25-(OH)(2)D(3) pretreatment enhanced PTH-induced RGS-2 mRNA and protein accumulation, whereas dexamethasone preincubation had an attenuating effect. These effects were due to modulation of the RGS-2 gene transcription rate, which increased by 35% with 1,25-(OH)(2)D(3) and decreased by 63% with dexamethasone pretreatment. RGS-2 mRNA half-life was not affected by either steroid. The transcriptional effects of dexamethasone and 1,25-(OH)(2)D(3) were independent of PTH/PTHrP receptor activation and were not explained by effects on cAMP accumulation, cAMP response element-binding protein expression or phosphorylation, or the abundance of the osteoblast-specific transcription factor core-binding factor alpha (CBFa1/Runx2), a known activator of RGS-2 expression. In conclusion, glucocorticoids and 1,25-(OH)(2)D(3) inversely modulate PTH-induced RGS-2 gene transcription. Regulation of RGS-2 may constitute a novel mechanism by which steroids modulate signaling via the PTH/PTHrP receptor and other G protein-coupled receptors in bone.

Animals↗
PubMed1998

CBF alpha3 (AML2) is induced by TGF-beta1 to bind and activate the mouse germline Ig alpha promoter.

TGF-beta1 directs class switching to IgA by splenic B cells and by the surface IgM+ B cell line, I.29mu, by inducing germline (GL) Ig alpha transcripts. The promoter segment between -130 and +46, relative to the first initiation site for mouse GL alpha transcripts, is sufficient for expression and TGF-beta1 inducibility of a reporter gene in B cell lines. Within this segment resides a TGF-beta1-responsive element (TbetaRE) that is required for induction of the promoter by TGF-beta1 and, when multimerized, is sufficient to transfer TGF-beta1 inducibility to another promoter. In this report we show that a TGF-beta1-inducible complex binds the TbetaRE and contains the transcription factor core-binding factor (CBF; also known as acute myeloid leukemia, AML). Although all three CBF alpha family members activate the GL alpha promoter, only CBF alpha3 (AML-2) is induced by TGF-beta1 in splenic B and I.29mu cells. The TbetaRE contains two CBF binding sites. Mutation of both sites reduces but does not eliminate induction of the GL alpha promoter by TGF-beta1 or by overexpression of CBF, possibly due to the presence of an additional CBF site in the promoter. In addition, the TbetaRE contains two copies of another sequence motif. Mutation of these motifs eliminates TGF-beta1 induction of the GL alpha promoter. Together the data indicate that TGF-beta1 induction of the alpha promoter involves induction of CBF alpha3, which binds to the TbetaRE of the promoter along with one or more proteins.

Animals↗
PubMed2003

Bone development in neurofibromatosis 1.

Bony abnormalities are common findings in cases of neurofibromatosis 1. We might hypothesize that neurofibromin, the protein encoded by the neurofibromatosis 1 gene, plays important roles in bone development. Loss of function of oligodendrocyte-myelin glycoprotein gene and increased activity of ras p21 might increase the level of c-fos proto-oncogene in bones with formation of fibrous dysplasia-like tissue. Also, increased ras p21 might disturb collagen I synthesis by osteoblasts. Moreover, increased ras activity might increase the mitogenic signals to the nucleus through mitogen-activated protein kinase (MAPK) and disturb the level of the transcription factor core-binding factor alpha(1) (Cbfa1). Abnormal fibrous tissue and neurofibromas formed at the site of pseudarthrosis might represent abnormal response of periosteal fibroblasts for injury, an effect simulating the response of skin fibroblasts in neurofibromatosis 1 to injury.

Animals↗
PubMed2005

Characterisation of cytosolic FK506 binding protein 12 and its role in modulating expression of Cbfa1 and osterix in ROS 17/2.8 cells.

FK506 is a commonly used immunosuppressant that mediates its action by exclusively interacting with the cytosolic immunophilin, FK506 binding protein 12 (FKBP12). Although FK506-induced acute osteoporosis is now well recognised, its precise mode of action in osteoblasts remains unclear. Therefore, in the present study we characterised FKBP12 in osteoblasts and investigated the role of FK506 in modulating osteoblast-specific transcription factors, core-binding factor alpha1 (Cbfa1) and osterix gene expression in ROS 17/2.8 cells. RT-PCR, immunolocalisation and Western blotting studies were employed to identify and characterise FKBP12 in rat primary osteoblasts and osteoblast-like osteosarcoma ROS 17/2.8 cells. Western blotting extracts of these cells revealed the 12 kDa and hitherto unreported 10 kDa FKBP isoform that were immunolocalised predominantly to the cytosol. The transient exposure of ROS 17/2.8 cells to H2O2 (100 microM) was found to elevate FKBP12 mRNA after 10 min and protein expression after 24 h. Both PTH (10(-9) M) and 1,25 (OH)2D3 (Vitamin D3) (10(-7) M) suppressed FKBP12 protein expression. FK506 in the therapeutic range (25 nmol/L) suppressed expression of Cbfa1 and osterix mRNA. The inhibition of Cbfa1 isoforms II/III expression was evident at 30 min and the extent of inhibition was sustained at 6 h. Osterix inhibition was also seen after 30 min, however, it became maximal after 6 h. The dose-dependant inhibition of osterix in these cells, carried out using 1.25, 12.5 and 125 nmol/L of FK506 was maximal at 1.25 nmol/L. Cbfa1 isoforms II/III were also maximally inhibited at 1.25 nmol/L; interestingly, the inhibition became less marked at higher concentrations of FK506. Similar dose of FK506 was found to inhibit ROS 17/2.8 cell proliferation; the inhibitory effect however was greater in insulin-stimulated cells. The results of this study suggest that immunosuppressant-induced osteoporosis, which is known to involve accelerated bone resorption by increase in osteoclastogenesis, may in fact also be accentuated by the inhibition of osteoblast differentiation and function.

Alkaline Phosphatase↗
PubMed1995

E1BF/Ku interacts physically and functionally with the core promoter binding factor CPBF and promotes the basal transcription of rat and human ribosomal RNA genes.

We have previously characterized an RNA polymerase (pol) I transcription factor, E1BF, from rat cells. This protein is immunologically related to Ku autoantigen and is required in pol-I directed transcription of rodent ribosomal RNA gene (rDNA). Glycerol density gradient fractionation and in situ UV cross-linking analysis of the purified factor showed directly that it consists of a heterodimer of 85 and 72 kDa polypeptides. E1BF also interacted with the human core promoter and augmented transcription of human rDNA as much as fivefold in HeLa nuclear extract, whereas transcription from adenovirus major late promoter, CMV or SV40 early promoters by pol II and of U6 and 5S RNA genes by pol III were either unaffected or minimally inhibited by the antibodies. Purified rat E1BF partially restored the suppression of human rDNA transcription by anti-Ku antibodies. Immunoprecipitation of rat cell extract with the anti-Ku antibodies followed by SDS-PAGE of the precipitated proteins and Southwestern analysis showed that E1BF interacts with CPBF, a core promoter binding factor. When the majority of CPBF and E1BF was removed from the reaction mixture by preincubation with a core promoter oligo nucleotide fragment, rDNA transcription was severely impaired. Addition of exogenous CPBF or E1BF to such a reaction resulted in significant restoration of the transcription, whereas inclusion of both factors caused further enhancement of rDNA transcription. These data demonstrate that E1BF is a basal pol I transcription factor that interacts with a core promoter binding factor both physically and functionally, and that is not a general pol II or pol III transcription factor.

Adenoviridae↗
PubMed2003

The teleost fish medaka (Oryzias latipes) as genetic model to study gravity dependent bone homeostasis in vivo.

Long-term space flight and microgravity result in bone loss that can be explained by reduced activity of bone-forming cells (osteoblasts) and/or an increase in activity of bone resorbing cells (osteoclasts). Osteoprotegerin (OPG) has been shown to regulate the balance between osteoblast and osteoclast cell numbers and is involved in maintaining constant bone mass under normal gravitational conditions. The small bony fish medaka (Oryzias latipes) has attracted increasing attention as a genetic model system to study normal embryonic developmental and pathological processes. To analyze the molecular mechanisms of bone formation in this small vertebrate, we have isolated two opg genes, opgl and opg2, from medaka. Our phylogenetic analysis reveals that both genes originated from a common ancestor by fish-specific gene duplication and represent the orthologs of the mammalian opg gene. Both opg genes are differentially expressed during embryonic and larval development, in adult tissues and in cultured primary osteoblast-like cells. Furthermore, we have characterized the opg2 promoter region and identified consensus binding sites for the transcription factor core-binding-factor-1A (CBFA1). In mammals, CBFA1 has been shown to be a regulator of opg expression and to be essential for several steps during osteoblast differentiation. Here we show that sequence and expression domains of opg, cbfal and a member of the dlx gene family are highly conserved between medaka and higher vertebrates. This suggests that not only single genes but entire genetic networks for bone formation are conserved between teleosts and mammals. These findings will open medaka fish as a genetic model to monitor bone formation under different gravity conditions in a living whole animal allowing the identification of novel factors involved in bone homeostasis.

Animals↗
PubMed2003

Diabetes interferes with the bone formation by affecting the expression of transcription factors that regulate osteoblast differentiation.

Type 1 diabetes in humans has as one of its complications inadequate bone formation, resulting in osteopenia and delayed fracture healing. To investigate the mechanisms by which diabetes affects bone formation, experiments were performed in a marrow ablation model. Mice were made diabetic by multiple low-dose streptozotocin treatment, and controls were treated with vehicle alone. Killing occurred 0, 2, 4, 6, 10, and 16 d following marrow ablation. Histologic analysis demonstrated that the amount of immature mesenchymal tissue was equivalent in both the experimental and control groups on d 4. On d 6 a burst of bone formation occurred in the control group that was significantly reduced in the diabetic group. This deficit was evident at the molecular level as shown by diminished expression of osteocalcin, collagen types I. When transcription factors were examined, core-binding factor alpha1 (Cbfa1)/runt domain factor-2 (Runx-2) and human homolog of the drosophila distal-less gene (Dlx5) expression were substantially reduced in the diabetic, compared with control, groups on d 4 and 6. C-fos but not c-jun expression was also suppressed in the diabetic group but not closely linked to bone formation. Insulin treatment substantially reversed the effect of diabetes on the expression of bone matrix osteocalcin and collagen type I and transcription factors Cbfa1/Runx2 and Dlx5. These results indicate that diabetic animals produce sufficient amounts of immature mesenchymal tissue but fail to adequately express genes that regulate osteoblast differentiation, Cbfa1/Runx-2 and Dlx5, which in turn, leads to decreased bone formation.

Animals↗