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PubMed2004

The interferon-inducible p204 protein acts as a transcriptional coactivator of Cbfa1 and enhances osteoblast differentiation.

The differentiation of uncommitted mesenchymal cells into osteoblasts is a fundamental molecular event governing both embryonic development and bone repair. The bone morphogenetic proteins (BMPs) are important regulators of this process; they function by binding to cell surface receptors and signaling by means of Smad proteins. Core binding factor alpha-1 (Cbfa1), a member of the runt family of transcription factors, is an essential transcriptional regulator of osteoblast differentiation and bone formation, and this process is positively or negatively regulated by a variety of coactivators and corepressors. We report that p204, an interferon-inducible protein that was previously shown to inhibit cell proliferation and promote the differentiation of myoblasts to myotubes, is a novel regulator in the course of osteogenesis. p204 is expressed in embryonic osteoblasts and hypertrophic chondrocytes in the growth plate as well as in the calvaria osteoblasts of neonatal mice. Its level is increased in the course of the BMP-2-triggered osteoblast differentiation of pluripotent C2C12 cells. This increase is probably due to the activation of the gene encoding 204 (Ifi204) by Smad transcription factor, including Smad1, -4, and -5. Overexpression of p204 enhances the BMP-2-induced osteoblast differentiation in vitro, as revealed by elevated alkaline phosphatase activity and osteocalcin production. p204 acts as a cofactor of Cbfa1: 1) high levels of p204 augment, whereas the lowering of p204 level decreases, the Cbfa1-dependent transcription, and 2) p204 associates with Cbfa1 both in vitro and in vivo. Two nonoverlapping segments in p204 bind to Cbfa1, and the N-terminal 88-amino acid segment of Cbfa1 is required for binding to p204. p204, which is the first interferon-inducible protein found to associate with Cbfa1, functions as a novel regulator of osteoblast differentiation.

Alkaline Phosphatase↗
PubMed2004

Quantitative transcriptional analysis of fusing and nonfusing cranial suture complexes in mice.

Previous studies have documented the differences in expression of various genes associated with the process of osteogenesis in fusing and nonfusing cranial sutures, including growth factors, growth factor receptors, and extracellular matrix molecules. Most of these studies were performed in rats, and although the biology regulating cranial suture fusion in mice and rats is presumed to be similar, studies are needed to verify these expression patterns as mice become increasingly utilized for scientific inquiry into the molecular biology of suture fusion and patency. The purpose of this study was to determine the differences in expression of several genes known to be critical to osteoblast biology. Posterior frontal and sagittal suture complexes (including the associated dura mater, suture mesenchyme, and osteogenic fronts) were isolated from 5-, 15-, 25-, 35-, and 45-day-old male CD-1 mice (n = 8 per age; n = 40 total). Total cellular RNA was extracted and converted to cDNA. Quantitative real-time reverse transcriptase polymerase chain reaction was then performed for the following genes: transforming growth factor beta1 and beta3, fibroblast growth factor receptor 1, Runx2,Osteopontin, and Osteocalcin. Expression of all genes examined was increased significantly in the posterior frontal suture as compared with the sagittal suture. Peak expression for all genes was observed on day 25. These data demonstrate that the expression of osteogenic growth factors, growth factor receptors, transcription factors, and extracellular matrix molecules is increased in the fusing posterior frontal suture in mice.

Animals↗
PubMed2004

RUNX3 expression in primary and metastatic pancreatic cancer.

AIM: Runx transcription factors are important regulators of lineage specific gene expression, cell proliferation, and differentiation. Runx3 expression is lost in a high proportion of gastric cancers, suggesting a tumour suppressive role in this malignancy. This study investigates the expression and localisation of Runx3 in pancreatic tissues. METHODS: Quantitative polymerase chain reaction was used to measure Runx3 mRNA. Immunohistochemistry was carried out to localise Runx3 in normal pancreatic tissues, and in primary and metastatic pancreatic ductal adenocarcinoma (PDAC). Basal and transforming growth factor beta1 (TGFbeta1) induced Runx3 expression was analysed in cultured pancreatic cancer cell lines. RESULTS: Runx3 expression was low to absent in normal pancreatic tissues, but increased in a third of cancer tissues. Runx3 was present only in islets in normal pancreas, whereas in pancreatic cancers, Runx3 was detected in the cancer cells of seven of 24 samples analysed. In addition, it was expressed by lymphocytes in six of the 16 cases with lymphocyte infiltration. In pancreatic cancer cell lines, Runx3 mRNA was present in Colo-357 and T3M4 cells, but was low to absent in the other cell lines tested. TGFbeta1 repressed Runx3 mRNA expressed in Colo-357 cells, and had no effect on Runx3 expression in the other pancreatic cancer cell lines. CONCLUSION: Runx3 expression is restricted to islets in the normal pancreas. In contrast, a considerable proportion of pancreatic tumours express Runx3, and its expression is localised in the tumour cells and in the infiltrating lymphocytes. Thus, Runx3 might play a role in the pathogenesis of PDAC.

Adenocarcinoma↗
PubMed2001

Bone anabolic effects of sonic/indian hedgehog are mediated by bmp-2/4-dependent pathways in the neonatal rat metatarsal model.

A neonatal rat metatarsal organ culture model has been employed to study the anabolic effects of Sonic/Indian hedgehog and BMP-4. In this culture system, a significant increase in endochondral ossification is measured by an increase in length of mineralized bone, after daily treatment for 7 days with Sonic hedgehog protein (Shh-N). Previous evidence indicated that PTH related protein (PTHrP) is a critical target of hedgehog function in endochondral ossification. Using a PTHrP blocking antibody, it is shown that hedgehog mediates this activity via pathways other than through PTHrP. A dose-related increase in endochondral ossification is observed when metatarsals are treated with 25 ng/ml recombinant human bone morphogenetic protein 4 (BMP-4). However, this activity is not evident at higher doses of BMP-4 (200 ng/ml). High doses of BMP-4 resulted in increased expression of noggin messenger RNA and cotreatment of noggin and Shh-N resulted in reversal of the anabolic action of Shh-N. This observation suggests that BMP-4 signaling can influence the Shh-N mediated increase in endochondral ossification. Finally, we show that the Shh-N and BMP-4 mediated increase in endochondral ossification is reversed by treatment with antisense oligonucleotides targeted against Cbfa1. Thus, this report identifies Shh-N as an inducer of endochondral ossification that mediates its effect via BMP-4 and Cbfa1-dependent pathways.

Animals↗
PubMed1999

Collagenase 3 is a target of Cbfa1, a transcription factor of the runt gene family involved in bone formation.

Collagenase 3 (MMP-13) is a recently identified member of the matrix metalloproteinase (MMP) gene family that is expressed at high levels in diverse human carcinomas and in articular cartilage from arthritic patients. In addition to its expression in pathological conditions, collagenase 3 has been detected in osteoblasts and hypertrophic chondrocytes during fetal ossification. In this work, we have evaluated the possibility that Cbfa1 (core binding factor 1), a transcription factor playing a major role in the expression of osteoblastic specific genes, is involved in the expression of collagenase 3 during bone formation. We have functionally characterized a Cbfa motif present in the promoter region of collagenase 3 gene and demonstrated, by cotransfection experiments and gel mobility shift assays, that this element is involved in the inducibility of the collagenase 3 promoter by Cbfa1 in osteoblastic and chondrocytic cells. Furthermore, overexpression of Cbfa1 in osteoblastic cells unable to produce collagenase 3 leads to the expression of this gene after stimulation with transforming growth factor beta. Finally, we show that mutant mice deficient in Cbfa1, lacking mature osteoblasts but containing hypertrophic chondrocytes which are also a major source of collagenase 3, do not express this protease during fetal development. These results provide in vivo evidence that collagenase 3 is a target of the transcriptional activator Cbfa1 in these cells. On the basis of these transcriptional regulation studies, together with the potent proteolytic activity of collagenase 3 on diverse collagenous and noncollagenous bone and cartilage components, we proposed that this enzyme may play a key role in the process of bone formation and remodeling.

3T3 Cells↗
PubMed2005

A role for Runx2 in normal mammary gland and breast cancer bone metastasis.

The transcription factor Runx2 is essential for the formation of the skeleton. It has therefore primarily been considered as a specific regulator of bone genes. However, mice containing a LacZ insertion at the Runx2 locus also revealed expression in the nascent mammary epithelium. Reports of Runx2 expression in breast cancer cell lines, combined with the fact that breast cancers preferentially metastasise to bone, have also hinted at a potential role for Runx2 in the formation of bone metastasese. These initial observations have prompted further analysis of Runx2 function in mammary epithelial cells and recent findings have demonstrated that Runx2 does indeed contribute to the ability of metastatic breast cancer cell lines to form osteolytic bone lesions. In addition, evidence is accumulating that Runx2 has a role in the regulation of normal mammary gland gene expression and recent data demonstrate that it regulates transcription of the mammary gland-specific gene, beta-casein. In this article I discuss recent advances that link Runx2 with normal mammary epithelial cell function and the development of bone metastasese in breast cancer.

Animals↗
PubMed2002

The large subunit of initiation factor aIF2 is a close structural homologue of elongation factors.

The heterotrimeric factor e/aIF2 plays a central role in eukaryotic/archaeal initiation of translation. By delivering the initiator methionyl-tRNA to the ribosome, e/aIF2 ensures specificity of initiation codon selection. The three subunits of aIF2 from the hyperthermophilic archaeon Pyrococcus abyssi could be overproduced in Escherichia coli. The beta and gamma subunits each contain a tightly bound zinc. The large gamma subunit is shown to form the structural core for trimer assembly. The crystal structures of aIF2gamma, free or complexed to GDP-Mg(2+) or GDPNP-Mg(2+), were resolved at resolutions better than 2 A. aIF2gamma displays marked similarities to elongation factors. A distinctive feature of e/aIF2gamma is a subdomain containing a zinc-binding knuckle. Examination of the nucleotide-complexed aIF2gamma structures suggests mechanisms of action and tRNA binding properties similar to those of an elongation factor. Implications for the mechanism of translation initiation in both eukarya and archaea are discussed. In particular, positioning of the initiator tRNA in the ribosomal A site during the search for the initiation codon is envisaged.

Amino Acid Sequence↗
PubMed1992

Different binding site requirements for binding and activation for the bipartite enhancer factor EF-1A.

The human transcription factor EF-1A binds to the purine-rich E1A core enhancer sequence in the adenovirus E1A and E4 and polyomavirus enhancer regions. The consensus binding site for EF-1A resembles that of members of the ets domain protein family. EF-1A activation of transcription requires a dimeric binding site. Analysis of binding sites containing point mutations revealed that EF-1A binding is determined by the core nucleotides of the binding site, while transcriptional activation is determined both by the core and some peripheral nucleotides that do not affect binding. We have purified EF-1A and analyzed its two constituent subunits, EF-1A alpha and EF-1A beta. EF-1A alpha (MW approximately 60kD) makes the primary DNA contacts. EF-1A beta (MW approximately 50 kD) forms a heteromultimeric complex with EF-1A alpha both in solution and on a dimeric binding site. Binding of both EF-1A subunits is necessary, but not sufficient, for transcriptional activation. We present immunochemical and functional evidence that EF-1A alpha is related to the murine ets-related protein GABP alpha and that EF-1A beta is related to the murine protein GABP beta.

Adenoviridae↗
PubMed2006

Unique human CD133+ leukemia cell line and its modulation towards a mesenchymal phenotype by FGF2 and TGFbeta1.

Immunological features of GM-490 cells, a new blood cell line from a patient with acute lymphoblastic leukemia, included lack of CD34, CD38, CD45, CD14, HLA-DR, and lymphoid and myeloid markers and expression of CD29, CD36, CD44, CD54, CD71, CD105, and CD133. Molecular analysis indicated CD45 gene expression was absent but CD34 mRNA was present. GM-490 cells constitutively produced fibronectin (FN), type III and traces of type I collagen, collagenases, glycosaminoglycans (GAG) and biglycan and betaglycan proteoglycans (PG) as well as FGF2 and TGFbeta1. When FGF2 and/or TGFbeta1 were added to cells in vitro, they stimulated cell proliferation and differently modulated matrix production and growth factor receptor expression. Reverse transcription-polymerase chain reaction (RT-PCR) detection of transcripts encoding for osteocalcin and RUNX2 suggests GM-490 cells differentiate towards the osteoblast pathway. GM-490 cells expressed the low affinity nerve growth factor receptor (p75LNGFR), a somatic stem cell marker that is not detected in hematopoietic cells, leading to the hypothesis that GM-490 has mesenchymal stem cell properties. The reciprocal modulating effects of FGF2 and TGFbeta1 on each other's receptors make the GM-490 cell line a new model for investigating the relationship between these growth factors and their receptors in autocrine loops which are believed to sustain the malignant clone in hematological diseases.

AC133 Antigen↗
PubMed2001

Dimerization with PEBP2beta protects RUNX1/AML1 from ubiquitin-proteasome-mediated degradation.

The RUNX family genes are the mammalian homologs of the Drosophila genes runt and lozenge, and members of this family function as master regulators of definitive hematopoiesis and osteogenesis. The RUNX genes encode the alpha subunit of the transcription factor PEBP2/CBF. The beta subunit consists of the non-RUNX protein PEBP2beta. We found that RUNX1/AML1, which is essential for hematopoiesis, is continuously subjected to proteolytic degradation mediated by the ubiquitin-proteasome pathway. When PEBP2beta is present, however, the ubiquitylation of RUNX1 is abrogated and this causes a dramatic inhibition of RUNX1 proteolysis. Heterodimerization between PEBP2beta and RUNX1 thus appears to be an essential step in the generation of transcriptionally competent RUNX1. Consistent with this notion, RUNX1 was barely detected in PEBP2beta(-/-) mouse. CBF(PEBP2)beta- SMMHC, the chimeric protein associated with inv(16) acute myeloid leukemia, was found to protect RUNX1 from proteolytic degradation more efficiently than PEBP2beta. These results reveal a hitherto unknown and major role of PEBP2beta, namely that it regulates RUNX1 by controlling its turnover. This has allowed us to gain new insights into the mechanism of leukemogenesis by CBFbeta-SMMHC.

Amino Acid Sequence↗
PubMed2004

The C-terminal domain of Escherichia coli Hfq increases the stability of the hexamer.

The Hfq (Host factor 1) polypeptide is a nucleic acid binding protein involved in the synthesis of many polypeptides. Hfq particularly affects the translation and the stability of several RNAs. In an earlier study, the use of fold recognition methods allowed us to detect a relationship between Escherichia coli Hfq and the Sm topology. This topology was further validated by a series of biophysical studies and the Hfq structure was modelled on an Sm protein. Hfq forms a beta-sheet ring-shaped hexamer. As our previous study predicted a large number of alternative conformations for the C-terminal region, we have determined whether the last 19 C-terminal residues are necessary for protein function. We find that the C-terminal truncated protein is fully capable of binding a polyadenylated RNA (K(d) of 120 pm vs. 50 pm for full-length Hfq). This result shows that the functional core of E. coli Hfq resides in residues 1-70 and confirms previous genetic studies. Using equilibrium unfolding studies, however, we find that full-length Hfq is 1.8 kcal x mol(-1) more stable than its truncated variant. Electron microscopy analysis of both truncated and full-length proteins indicates a structural rearrangement between the subunits upon truncation. This conformational change is coupled to a reduction in beta-strand content, as determined by Fourier transform infra-red. On the basis of these results, we propose that the C-terminal domain could protect the interface between the subunits and stabilize the hexameric Hfq structure. The origin of this C-terminal domain is also discussed.

Amino Acid Sequence↗
PubMed1997

Structural and functional analysis of hypoxia-inducible factor 1.

Hypoxia-inducible factor 1 (HIF-1) is a basic helix-loop-helix protein that activates transcription of hypoxia-inducible genes, including those encoding: erythropoietin, vascular endothelial growth factor, heme oxygenase-1, inducible nitric oxide synthase, and the glycolytic enzymes aldolase A, enolase 1, lactate dehydrogenase A, phosphofructokinase I, and phosphoglycerate kinase 1. Hypoxia response elements from these genes consist of a HIF-1 binding site (that contains the core sequence 5'-CGTG-3') as well as additional DNA sequences that are required for function, which in some elements include a second HIF-1 binding site. HIF-1 is a heterodimer. The HIF-1 alpha subunit is unique to HIF-1, whereas HIF-1 beta (ARNT) can dimerize with other bHLH-PAS proteins. Structural analysis of HIF-1 alpha revealed that dimerization with HIF-1 beta (ARNT) requires the HLH and PAS domains, DNA binding is mediated by the basic domain, and that HIF-1 alpha contains a carboxyl-terminal transactivation domain. Co-transfection of HIF-1 alpha and HIF-1 beta (ARNT) expression vectors and a reporter gene containing a wild-type hypoxia response element resulted in increased transcription in non-hypoxic cells and a superinduction of transcription in hypoxic cells, whereas HIF-1 expression vectors had no effect on the transcription of reporter genes containing a mutation in the HIF-1 binding site. HIF-1 alpha and HIF-1 beta (ARNT) protein levels were induced by hypoxia in all primary and transformed cell lines examined. In HeLa cells, the levels of HIF-1 alpha and HIF-1 beta protein and HIF-1 DNA-binding activity increased exponentially as cellular oxygen tension decreased, with maximum values at 0.5% oxygen and half-maximal values at 1.5 to 2% oxygen. HIF-1 alpha and HIF-1 beta (ARNT) mRNAs were detected in all human, mouse, and rat organs assayed and mRNA expression was modestly induced in rodents subjected to hypoxia. HIF-1 alpha protein levels were induced in vivo when animals were subjected to anemia or hypoxia. The HIF1A gene was mapped to human chromosome 14q21-q24 and mouse chromosome 12.

Animals↗
PubMed2002

RUNX: a trilogy of cancer genes.

The RUNX family of transcription factors plays pivotal roles during normal development and in neoplasias. Recent data involve RUNX3 as an important tumor suppressor in gastric cancers and pose interesting questions about how perturbed levels and interspecific competition among RUNX family members may contribute to tumorigenesis.

Animals↗
PubMed2006

Genetic regulation of bone mass and susceptibility to osteoporosis.

Osteoporosis is a common disease with a strong genetic component characterized by reduced bone mass and increased risk of fragility fractures. Twin and family studies have shown that the heritability of bone mineral density (BMD) and other determinants of fracture risk-such as ultrasound properties of bone, skeletal geometry, and bone turnover-is high, although heritability of fracture is modest. Many different genetic variants of modest effect size are likely to contribute to the regulation of these phenotypes by interacting with environmental factors such as diet and exercise. Linkage studies in rare Mendelian bone diseases have identified several previously unknown genes that play key roles in regulating bone mass and bone turnover. In many instances, subtle polymorphisms in these genes have also been found to regulate BMD in the general population. Although there has been extensive progress in identifying the genetic variants that regulate susceptibility to osteoporosis, most of the genes and genetic variants that regulate bone mass and susceptibility to osteoporosis remain to be discovered.

Animals↗
PubMed1993

Crystal structure of a flavoprotein related to the subunits of bacterial luciferase.

The molecular structure of the luxF protein from the bioluminescent bacterium Photobacterium leiognathi has been determined by X-ray diffraction techniques and refined to a conventional R-factor of 17.8% at 2.3 A resolution. The 228 amino acid polypeptide exists as a symmetrical homodimer and 33% of the monomer's solvent-accessible surface area is buried upon dimerization. The monomer displays a novel fold that contains a central seven-stranded beta-barrel. The solvent-exposed surface of the monomer is covered by seven alpha-helices, whereas the dimer interface is primarily a flat surface composed of beta-strands. The protein monomer binds two molecules of flavin mononucleotide, each of which has C6 of the flavin isoalloxazine moiety covalently attached to the C3' carbon atom of myristic acid. Both myristyl groups of these adducts are buried within the hydrophobic core of the protein. One of the cofactors contributes to interactions at the dimer interface. The luxF protein displays considerable amino acid sequence homology with both alpha- and beta-subunits of bacterial luciferase, especially the beta-subunit. Conserved amino acid residues shared between luxF and the luciferase subunits cluster predominantly in two distinct regions of the luxF protein molecule. These homologous regions in the luciferase subunits probably share a three-dimensional fold similar to that of the luxF protein.

Amino Acid Sequence↗
PubMed1995

Indirect and direct disruption of transcriptional regulation in cancer: E2F and AML-1.

The disruption of transcriptional regulatory circuits through the elimination of negative regulatory factors (tumor suppressors), the activation of positive acting factors (oncogenes), or when chimeric proteins result from chromosomal translocations, is likely a key event in multistep tumorigenesis. Here, using the transcription factors E2F and AML-1 as model systems, we discuss the disruption of coordinate transcriptional regulation in oncogenesis. E2F oncogenic signals are released when the pRb tumor suppressor is inactivated, and E2F activation may necessitate the coordinate inactivation of a second tumor suppressor, p53. AML-1 is the target of the (8;21) translocation, found in approximately 15% of acute myeloid leukemia (AML) cases, and the t(12;21), found in up to 30% of childhood B-cell acute lymphoblastic leukemias. The t(8;21) creates a fusion protein between AML-1 and a gene of unknown function, mtg8 (ETO), whereas the t(12;21) fuses the TEL (translocation-ets-leukemia) transcription factor to the N-terminus of AML-1. The inv(16), which is the most frequent anomaly found in AML, also targets AML-1, by fusing the gene that encodes AML-1's heterodimeric partner CBF beta to the smooth muscle myosin heavy chain gene MYHll. Thus, E2F and AML-1 provide excellent models for the disruption of transcriptional regulation in cancer.

Carrier Proteins↗
PubMed1997

A novel transcript encoding an N-terminally truncated AML1/PEBP2 alphaB protein interferes with transactivation and blocks granulocytic differentiation of 32Dcl3 myeloid cells.

The gene AML1/PEBP2 alphaB encodes the alpha subunit of transcription factor PEBP2/CBF and is essential for the establishment of fetal liver hematopoiesis. Rearrangements of AML1 are frequently associated with several types of human leukemia. Three types of AML1 cDNA isoforms have been described to date; they have been designated AML1a, AML1b, and AML1c. All of these isoforms encode the conserved-Runt domain, which harbors the DNA binding and heterodimerization activities. We have identified a new isoform of the AML1 transcript, termed AML1 deltaN, in which exon 1 is directly connected to exon 4 by alternative splicing. The AML1 deltaN transcript was detected in various hematopoietic cell lines of lymphoid to myeloid cell origin, as revealed by RNase protection and reverse transcriptase PCR analyses. The protein product of AML1 deltaN lacks the N-terminal region of AML1, including half of the Runt domain, and neither binds to DNA nor heterodimerizes with the beta subunit. However, AML1 deltaN was found to interfere with the transactivation activity of PEBP2, and the molecular region responsible for this activity was identified. Stable expression of AML1 deltaN in 32Dcl3 myeloid cells blocked granulocytic differentiation in response to granulocyte colony-stimulating factor. These results suggest that AML1 deltaN acts as a modulator of AML1 function and serves as a useful tool to dissect the functional domains in the C-terminal region of AML1.

Amino Acid Sequence↗