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Results for “Core Binding Factor alpha Subunits”

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PubMed2001

Skeletal malformations caused by overexpression of Cbfa1 or its dominant negative form in chondrocytes.

During skeletogenesis, cartilage develops to either permanent cartilage that persists through life or transient cartilage that is eventually replaced by bone. However, the mechanism by which cartilage phenotype is specified remains unclarified. Core binding factor alpha1 (Cbfa1) is an essential transcription factor for osteoblast differentiation and bone formation and has the ability to stimulate chondrocyte maturation in vitro. To understand the roles of Cbfa1 in chondrocytes during skeletal development, we generated transgenic mice that overexpress Cbfa1 or a dominant negative (DN)-Cbfa1 in chondrocytes under the control of a type II collagen promoter/enhancer. Both types of transgenic mice displayed dwarfism and skeletal malformations, which, however, resulted from opposite cellular phenotypes. Cbfa1 overexpression caused acceleration of endochondral ossification due to precocious chondrocyte maturation, whereas overexpression of DN-Cbfa1 suppressed maturation and delayed endochondral ossification. In addition, Cbfa1 transgenic mice failed to form most of their joints and permanent cartilage entered the endochondral pathway, whereas most chondrocytes in DN-Cbfa1 transgenic mice retained a marker for permanent cartilage. These data show that temporally and spatially regulated expression of Cbfa1 in chondrocytes is required for skeletogenesis, including formation of joints, permanent cartilages, and endochondral bones.

Animals↗

PubMed2001

Overexpression of Cbfa1 in osteoblasts inhibits osteoblast maturation and causes osteopenia with multiple fractures.

Targeted disruption of core binding factor alpha1 (Cbfa1) showed that Cbfa1 is an essential transcription factor in osteoblast differentiation and bone formation. Furthermore, both in vitro and in vivo studies showed that Cbfa1 plays important roles in matrix production and mineralization. However, it remains to be clarified how Cbfa1 controls osteoblast differentiation, bone formation, and bone remodelling. To understand fully the physiological functions of Cbfa1, we generated transgenic mice that overexpressed Cbfa1 in osteoblasts using type I collagen promoter. Unexpectedly, Cbfa1 transgenic mice showed osteopenia with multiple fractures. Cortical bone, which was thin, porous, and enriched with osteopontin, was invaded by osteoclasts, despite the absence of acceleration of osteoclastogenesis. Although the number of neonatal osteoblasts was increased, their function was impaired in matrix production and mineralization. Furthermore, terminally differentiated osteoblasts, which strongly express osteocalcin, and osteocytes were diminished greatly, whereas less mature osteoblasts expressing osteopontin accumulated in adult bone. These data indicate that immature organization of cortical bone, which was caused by the maturational blockage of osteoblasts, led to osteopenia and fragility in transgenic mice, demonstrating that Cbfa1 inhibits osteoblast differentiation at a late stage.

Animals↗

PubMed1999

Mutation analysis of core binding factor A1 in patients with cleidocranial dysplasia.

Cleidocranial dysplasia (CCD) is a dominantly inherited disorder characterized by patent fontanelles, wide cranial sutures, hypoplasia of clavicles, short stature, supernumerary teeth, and other skeletal anomalies. We recently demonstrated that mutations in the transcription factor CBFA1, on chromosome 6p21, are associated with CCD. We have now analyzed the CBFA1 gene in 42 unrelated patients with CCD. In 18 patients, mutations were detected in the coding region of the CBFA1 gene, including 8 frameshift, 2 nonsense, and 9 missense mutations, as well as 2 novel polymorphisms. A cluster of missense mutations at arginine 225 (R225) identifies this residue as crucial for CBFA1 function. In vitro green fluorescent protein fusion studies show that R225 mutations interfere with nuclear accumulation of CBFA1 protein. There is no phenotypic difference between patients with deletions or frameshifts and those with other intragenic mutations, suggesting that CCD is generally caused by haploinsufficiency. However, we were able to extend the CCD phenotypic spectrum. A missense mutation identified in one family with supernumerary teeth and a radiologically normal skeleton indicates that mutations in CBFA1 can be associated exclusively with a dental phenotype. In addition, one patient with severe CCD and a frameshift mutation in codon 402 had osteoporosis leading to recurrent bone fractures and scoliosis, providing first evidence that CBFA1 may help maintain adult bone, in addition to its function in bone development.

Bone and Bones↗

PubMed1994

PEBP2--a modulator of polyoma DNA replication.

Previously, we have shown that integrated copies of polyoma DNA can be induced to replicate in rat fibroblasts (H3 cells) exposed to a DNA-damaging agent. In the current study, we demonstrate that UV-irradiation of mouse fibroblasts (WOP cells), transiently transfected with polyoma DNA, results in repression of polyoma replication. Cotransfection of oligomers representing wild-type but not mutated forms of the PEBP2 target sequence restored levels of viral replication indicating a role of PEBP2 binding proteins in mediating this effect. DNA-binding assays revealed that a different subset of complexes was formed with the PEBP2 target sequence when nuclear proteins from sham and UV-irradiated WOP and H3 cells were compared, suggesting that the activities of PEBP2 binding proteins are differentially regulated upon UV-irradiation in these two cell types. The ability of PEBP2 to modulate polyoma replication following UV-irradiation in WOP cells suggests a potential role of PEBP2 proteins in the cellular response to DNA damage.

Animals↗

PubMed2003

Modulation of cellular differentiation by N-methyl-D-aspartate receptors in osteoblasts.

N-methyl-D-aspartate (NMDA) receptors for the central neurotransmitter l-glutamate (Glu) have been shown to be present in both osteoblasts and osteoclasts. Sustained exposure to the NMDA channel antagonist dizocilpine (MK-801) significantly prevented increases in both alkaline phosphatase activity and Ca2+ accumulation in a concentration-dependent manner in osteoblasts cultured for 7-28 days in vitro (DIV), without significantly affecting cell survivability. Osteocalcin expression was markedly reduced in the presence of MK-801 in osteoblasts cultured for 28 DIV. Both an NMDA domain antagonist and a glycine domain antagonist similarly prevented Ca2+ accumulation in osteoblasts exposed for 28 consecutive DIV. MK-801 was effective in significantly inhibiting Ca2+ accumulation determined at 28 DIV in osteoblasts exposed before 7 DIV but was ineffective in cells exposed after 11-21 DIV. Sustained exposure to MK-801 significantly inhibited DNA binding activity and expression of core binding factor alpha-1 (CBFA1) in osteoblasts exposed after 7 DIV up to 28 DIV, but not in those exposed before 7 DIV. These results suggest that heteromeric NMDA receptor channels may be functionally expressed to regulate mechanisms underlying cellular differentiation rather than proliferation and/or maturation through modulation of expression of CBFA1 in cultured rat calvarial osteoblasts.

Animals↗

PubMed2002

A case of a Japanese patient with cleidocranial dysplasia possessing a mutation of CBFA1 gene.

Cleidocranial dysplasia (CCD) is an autosomal dominant human bone disease characterized by hypoplastic or aplastic clavicles, wide cranial sutures, supernumerary teeth, short stature, and other skeletal disorders. Recently, various mutations of the core binding factor (CBFA1) gene have been detected in CCD patients. The CBFA1 gene is a member of the runt family of transcription factors. We experienced one Japanese case of CCD with open sutures, hypoplasia of clavicles and brachydactyly, combined with atlant-axis dislocation. We performed the sequence analysis of the CBFA1 gene and detected a missense mutation of R225W in exon 3.

Aged↗

PubMed2004

Large fontanelles are a shared feature of haploinsufficiency of RUNX2 and its co-activator CBFB.

CBFB at 16q22 heterodimerizes with either RUNX2 (also known as CBFA1) or RUNX1 (CBFA2) to activate the transcription of downstream molecules. RUNX2 regulates osteoblast differentiation and chondrocyte maturation and its haploinsufficiency leads to cleidocranial dysplasia, characterized large fontanelles, hypoplasia or aplasia of the clavicles, hypoplasia of the distal phalanges, and a wide pubic symphysis. Complete loss of Runx1 or Cbfb in mice is lethal because of the absence of fetal liver hematopoiesis. Fetal rescue in Cbfb(-/-) mice by providing the Cbfb functions in the hematopoietic progenitors leads to wide fontanelle and delayed chondrocyte maturation, presumably resulting from the incomplete function of the transcriptional pathway mediated by the Cbfb-Runx2 heterodimer. The present report describes a patient with a small deletion of chromosome 16q22.1 encompassing CBFB. Skeletal abnormalities included a widely open fontanelle, multiple wormian bones along the sagittal suture, hypoplasia of the distal phalanges, and mildly shortened clavicles. G-banding analysis revealed a shortening of the 16q22.1 band. A fluorescence in situ hybridization analysis, using the BAC probe spanning the CBFB locus at 16q22.1, revealed that the CBFB probe hybridized to only one of the two homologous chromosome 16 regions. Array-comparative genomic hybridization analysis revealed that the deletion spans 1.2 megabases. In reviewing eight previously reported cases of 16q interstitial deletions involving band q22, large cranial sutures were noted in all but one case. Considering the phenotypic similarity of the 16q22 deletion case and Cbfb(-/-) mice rescued for hematopoiesis and the consistency of the phenotype among 16q22 deletion cases, we suggest that the common phenotypic feature of the 16q22 deletion, large fontanelles, can be attributed to a haploinsufficiency of CBFB.

Animals↗

PubMed1999

Discovery: Osf2/Cbfa1, a master gene of bone formation.

This report reviews the current research that has impacted on our understanding of osteogenesis. Recent studies indicate that the transcription factor Osf2 (osteoblast specific transcription factor 2)/Cbfa1 (core binding factor activity 1) serves as a Master Gene regulating osteoblast-specific gene expression. The gene is expressed in cells of the osteoblast lineage only, and this expression is regulated by calciotropic agents. Moreover, when expressed in non-skeletal cells, the cells assume many of the characteristics of an osteoblast. In knockout experiments designed to assess the importance of the gene in osteogenesis, no evidence of bone formation could be observed in animals that are homozygous for the deletion. Studies of the heterozygote indicate that osteoblast function is compromised: there is a severe reduction in the number of bone cells, the tissue is deficient in bone proteins, and the activity of the enzyme alkaline phosphatase is low. It was noted that the heterozygote displays abnormalities that are remarkably similar to those exhibited by cleidocranial dysplastics. Indeed, Osf2 mapped close to a chromosomal locus on chromosome 6p21, long suspected of being involved with the disease. A search conducted for Osf2 mutations in kindreds with cleidocranial dysplasia revealed deletions, insertions, and missense mutations; these mutations are found to segregate with patients who are defined clinically as cleidocranial dysplastic. Aside from providing a new insight into a disease state that has so far avoided molecular analysis, results of the studies emphasize that the loss of a Master Gene drastically alters the development and maintenance of the appendicular skeleton and the craniofacial complex.

Animals↗

PubMed1995

The t(8;21) fusion protein interferes with AML-1B-dependent transcriptional activation.

The AML-1/CBF beta transcription factor complex is targeted by both the t(8;21) and the inv(16) chromosomal alterations, which are frequently observed in acute myelogenous leukemia. AML-1 is a site-specific DNA-binding protein that recognizes the enhancer core motif TGTGGT. The t(8;21) translocation fuses the first 177 amino acids of AML-1 to MTG8 (also known as ETO), generating a chimeric protein that retains the DNA-binding domain of AML-1. Analysis of endogenous AML-1 DNA-binding complexes suggested the presence of at least two AML-1 isoforms. Accordingly, we screened a human B-cell cDNA library and isolated a larger, potentially alternatively spliced, form of AML1, termed AML1B. AML-1B is a protein of 53 kDa that binds to a consensus AML-1-binding site and complexes with CBF beta. Subcellular fractionation experiments demonstrated that both AML-1 and AML-1/ETO are efficiently extracted from the nucleus under ionic conditions but that AML-1B is localized to a salt-resistant nuclear compartment. Analysis of the transcriptional activities of AML-1, AML-1B, and AML-1/ETO demonstrated that only AML-1B activates transcription from the T-cell receptor beta enhancer. Mixing experiments indicated that AML-1/ETO can efficiently block AML-1B-dependent transcriptional activation, suggesting that the t(8;21) translocation creates a dominant interfering protein.

Alternative Splicing↗

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↗

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↗

PubMed2003

Transcriptional coactivation of bone-specific transcription factor Cbfa1 by TAZ.

Core-binding factor 1 (Cbfa1; also called Runx2) is a transcription factor belonging to the Runt family of transcription factors that binds to an osteoblast-specific cis-acting element (OSE2) activating the expression of osteocalcin, an osteoblast-specific gene. Using the yeast two-hybrid system, we identified a transcriptional coactivator, TAZ (transcriptional coactivator with PDZ-binding motif), that binds to Cbfa1. A functional relationship between Cbfa1 and TAZ is demonstrated by the coimmunoprecipitation of TAZ by Cbfa1 and by the fact that TAZ induces a dose-dependent increase in the activity of osteocalcin promoter-luciferase constructs by Cbfa1. A dominant-negative construct of TAZ in which the coactivation domains have been deleted reduces osteocalcin gene expression down to basal levels. NIH 3T3, MC 3T3, and ROS 17/2.8 cells showed the expected nuclear localization of Cbfa1, whereas TAZ was distributed throughout the cytoplasm with some nuclear localization when transfected with either Cbfa1 or TAZ. Upon cotransfection by both Cbfa1 and TAZ, the transfected TAZ shows predominant nuclear localization. The dominant-negative construct of TAZ shows minimal nuclear localization upon cotransfection with Cbfa1. These data indicate that TAZ is a transcription coactivator for Cbfa1 and may be involved in the regulation of osteoblast differentiation.

3T3 Cells↗

PubMed2001

Smooth muscle cell phenotypic transition associated with calcification: upregulation of Cbfa1 and downregulation of smooth muscle lineage markers.

Bovine aortic smooth muscle cell (BASMC) cultures undergo mineralization on addition of the organic phosphate donor, beta-glycerophosphate (betaGP). Mineralization is characterized by apatite deposition on collagen fibrils and the presence of matrix vesicles, as has been described in calcified vascular lesions in vivo as well as in bone and teeth. In the present study, we used this model to investigate the molecular mechanisms driving vascular calcification. We found that BASMCs lost their lineage markers, SM22alpha and smooth muscle alpha-actin, within 10 days of being placed under calcifying conditions. Conversely, the cells gained an osteogenic phenotype as indicated by an increase in expression and DNA-binding activity of the transcription factor, core binding factor alpha1 (Cbfa1). Moreover, genes containing the Cbfa1 binding site, OSE2, including osteopontin, osteocalcin, and alkaline phosphatase were elevated. The relevance of these in vitro findings to vascular calcification in vivo was further studied in matrix GLA protein null (MGP(-/-)) mice whose arteries spontaneously calcify. We found that arterial calcification was associated with a similar loss in smooth muscle markers and a gain of osteopontin and Cbfa1 expression. These data demonstrate a novel association of vascular calcification with smooth muscle cell phenotypic transition, in which several osteogenic proteins including osteopontin, osteocalcin, and the bone determining factor Cbfa1 are gained. The findings suggest a positive role for SMCs in promoting vascular calcification.

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

Transactivation of core binding factor alpha1 as a basic mechanism to trigger parathyroid hormone-induced osteogenesis.

During 28-day culture of bone marrow- and calvaria-derived osteoblasts, the constant presence of parathyroid hormone (PTH)(1-34) retarded differentiation and nodule formation (NF) in a dose-dependent fashion (C-phase). In contrast, addition of PTH(1-34) in late stage cultures (from day 10 to 21) accelerated NF (A-phase). The stable production of such an A-phase allowed us to study the mechanism of bone anabolic action of PTH(1-34). Subcellular localization studies of core binding factor alpha1 (Cbfa1) and reporter assays provided the results indicating that in the A-phase, PTH(1-34) triggers its bone anabolic action via enhancement of Cbfa1 transactivation. RT-PCR and Northern blot analyses revealed that alkaline phosphatase, osteocalcin and bone sialoprotein expression decreased in the C-phase and increased in the A-phase; however, expression of other bone proteins (Cbfa1, PTH/PTH-related peptide-receptor, osteopontin, collagen I alpha1, collagen I alpha2, vitamin K-dependent gamma-glutamyl carboxylase) did not change in a phase transition-related manner. Ovariectomized osteopenic mice, treated with PTH(1-34) (4 and 40 microg/kg, s.c., every other day, 4 or 6 weeks), recovered lost bone, displayed elevated nuclear localization of Cbfal in tibiae without alteration of its cytosolic level and exhibited upregulation of expressions of the same set of proteins (alkaline phosphatase, osteocalcin and bone sialoprotein) in femora. These results obtained by a concerted study in vitro and in vivo suggest that PTH triggers its osteogenic action via promotion of the transactivation of Cbfa1.

Alkaline Phosphatase↗

PubMed2001

Lipophilic statins can be osteogenic by promoting osteoblastic calcification in a Cbfa1- and BMP-2-independent manner.

Mevastatin (3-10 microM) and fluvastatin (0.1-10 microM), but not pravastatin, were found to promote calcification of MC3T3-E1 cells and their subclone MC4, in either the presence or absence of 3 mM inorganic phosphate stimulus. The mechanism of action was examined. Gel retardation assay and immunocytochemical analysis of core binding factor (Cbfa1) revealed that mevastatin and fluvastatin completed the nuclear export of Cbfa1, possibly thereby reducing the induction of the stably transfected p6OSE2-luc gene, and then promoted Cbfa1-independent calcification, which invariably occurred in both wild type and dominant negative Cbfa1-expressing cells. The induction of the bone morphogenetic protein-2 (BMP-2) gene promoter failed to respond to the statins. All the effects of the cell-permeable statins were negated by mevalonate pathway metabolites (geranylgeranylpyrophosphate > farnesylpyrophosphate > mevalonate) and reproduced by toxin B (a Rho-specific inhibitor), but not totally by Y27632 (a ROCK-specific inhibitor). The results suggest that lipophilic statins can be osteogenic by promoting Cbfa1- and BMP-2-independent calcification processes.

Animals↗

PubMed1999

Medium perfusion enhances osteogenesis by murine osteosarcoma cells in three-dimensional collagen sponges.

In this study, we examined in vitro histogenesis by murine K8 osteosarcoma cells maintained in three-dimensional (3D) collagen sponges. We tested the hypothesis that perfusion of medium enhances cell viability and their biosynthetic activity as assessed by expression of the osteoblastic phenotype and mineral deposition. At intervals, samples were harvested and analyzed histologically, biochemically, and by Northern hybridization for type I collagen, osteopontin (OPN), osteocalcin (OC), and core binding factor alpha 1 (Cbfa1). Histologic evaluation showed greater viability, more alkaline phosphatase (ALP)-positive cells, and more mineralized tissue in the perfused sponges after 21 days. Immunohistological assessment of proliferating cell nuclear antigen revealed 5-fold more proliferating cells in the perfused sponges compared with the controls (p = 0.0201). There was 3-fold more ALP activity in the perfused sponges than the controls at 6 days and 14 days (p = 0.0053). The perfused sponges contained twice the DNA and eight times more calcium than the nonperfused controls after 21 days (p < 0.0001 for both). Northern hybridization analysis revealed more mRNA for collagen type I (2-fold) and 50% more for OC at 14 days and 21 days, whereas OPN and Cbfa1 mRNA expression remained unaffected by the medium perfusion. These results show that medium perfusion had beneficial effects on the proliferation and biosynthetic activity of this osteosarcoma cell line. This system mimics the 3D geometry of bone tissue and has the potential for revealing mechanisms of regulation of osteogenesis.

Alkaline Phosphatase↗

PubMed1999

Cloning of a 2.5 kb murine bone sialoprotein promoter fragment and functional analysis of putative Osf2 binding sites.

Bone sialoprotein (BSP) is an extracellular matrix protein that is intimately associated with the process of biomineralization. Osf2, a member of the Cbf/runt family of transcription factors, is required for the development of osteoblasts in vivo and has been reported to stimulate the transcription of BSP when overexpressed in mesenchymal cell lines. To investigate the role of Osf2 in BSP expression, we cloned a 2.5 kb fragment of a 5' untranscribed sequence from the murine BSP gene and evaluated it for putative Osf2 binding sites. This promoter, which was able to direct 5- to 10-fold higher levels of luciferase reporter expression in osteoblastic cells than in nonbone cell lines, contains two consensus core binding sites for members of the Cbf/runt family. One, at -61 relative to the start of transcription, is within a region having 75% overall sequence identity with the rat and human BSP promoters. The other is located at -1335, outside this highly conserved region. Neither site is completely conserved in the rat or human sequences. Only the -1335 site was able to bind a protein in nuclear extracts of osteoblastic cells, and this protein was identified as Osf2. Despite this in vitro binding ability, we detected no significant enhancer activity in the -1335 element when placed in front of a minimal osteocalcin promoter driving a luciferase reporter gene in osteoblastic cells nor any loss in transcriptional activity of a 5' promoter deletion which eliminated this element as compared with the full-length 2.5 kb promoter. These results suggest that Osf2 binding to the BSP promoter is not essential for its osteoblast-selective expression.

3T3 Cells↗