Cbfa1, an essential transcription factor for bone formation, is expressed in testis from the same promoter used in bone.
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Cbfa1 is an essential transcription factor for bone formation, but its transcripts have also been detected in thymus and testis. To elucidate the expression of Cbfa1 in testis, we isolated Cbfa1 cDNA from mouse testis. First we examined the length of the transcripts expressed in mouse testis by Northern hybridization. Using a cDNA probe that can detect both Type-I and Type-II Cbfa1 (Type-I: originally reported as Pebp2alphaA by Ogawa et al.; Type-II: originally reported as til-1 by Stewart et al.), 1.8-kb transcripts were detected in testis, and larger transcripts (6.3 kb and 7.4 kb) in T-cell lines, thymus and bone. Using a Type-II specific probe (bone isoform-specific probe), surprisingly, 1.8-kb testis transcript(s) were detected as well as those (6.3 kb) found in bone. In addition, the transcription start site in mouse testis coincides with one of three start sites identified in bone. These results suggest that the testis transcript is generated from the same promoter of Type-II Cbfa1, which is thought to be active only in osteoblasts and some chondrocytes. Next, to define the difference in length of transcripts, we isolated the Cbfa1 cDNA from mouse testis. Sequence analysis of the cDNA showed that alternative splicing around exon 2 and poly-adenylation within exon 8 occurred in the testis isoform, which produce the smaller transcripts. These data revealed that testis Cbfa1 mRNA is transcribed from same promoter used in bones, but the post-transcriptional regulation is different between testis and skeletal tissues. (+info)
A Pro250Arg substitution in mouse Fgfr1 causes increased expression of Cbfa1 and premature fusion of calvarial sutures.
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Pfeiffer syndrome is a classic form of craniosynostosis that is caused by a proline-->arginine substitution at amino acid 252 (Pro252Arg) in fibroblast growth factor receptor 1 (FGFR1). Here we show that mice carrying a Pro250Arg mutation in Fgfr1, which is orthologous to the Pfeiffer syndrome mutation in humans, exhibit anterio-posteriorly shortened, laterally widened and vertically heightened neurocraniums. Analysis of the posterior and anterior frontal, sagittal and coronal sutures of early post-natal mutant mice revealed premature fusion. The sutures of mutant mice had accelerated osteoblast proliferation and increased expression of genes related to osteoblast differentiation, suggesting that bone formation at the sutures is locally increased in Pfeiffer syndrome. Of note, dramatically increased expression of core-binding transcription factor alpha subunit type 1 (Cbfa1) accompanied premature fusion, suggesting that Cbfa1 may be a downstream target of Fgf/Fgfr1 signals. This was confirmed in vitro, where we demonstrate that transfection with wild-type or mutant Fgfr1 induces Cbfa1 expression. The induced expression was also observed using Fgf ligands (Fgf2 and Fgf8). These studies provide direct genetic evidence that the Pro252Arg mutation in FGFR1 causes human Pfeiffer syndrome and uncovers a molecular mechanism in which Fgf/Fgfr1 signals regulate intramembraneous bone formation by modulating Cbfa1 expression. (+info)
A RUNX2/PEBP2alpha A/CBFA1 mutation displaying impaired transactivation and Smad interaction in cleidocranial dysplasia.
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Cleidocranial dysplasia (CCD), an autosomal-dominant human bone disease, is thought to be caused by heterozygous mutations in runt-related gene 2 (RUNX2)/polyomavirus enhancer binding protein 2alphaA (PEBP2alphaA)/core-binding factor A1 (CBFA1). To understand the mechanism underlying the pathogenesis of CCD, we studied a novel mutant of RUNX2, CCDalphaA376, originally identified in a CCD patient. The nonsense mutation, which resulted in a truncated RUNX2 protein, severely impaired RUNX2 transactivation activity. We show that signal transducers of transforming growth factor beta superfamily receptors, Smads, interact with RUNX2 in vivo and in vitro and enhance the transactivation ability of this factor. The truncated RUNX2 protein failed to interact with and respond to Smads and was unable to induce the osteoblast-like phenotype in C2C12 myoblasts on stimulation by bone morphogenetic protein. Therefore, the pathogenesis of CCD may be related to the impaired Smad signaling of transforming growth factor beta/bone morphogenetic protein pathways that target the activity of RUNX2 during bone formation. (+info)
Energetic and functional contribution of residues in the core binding factor beta (CBFbeta ) subunit to heterodimerization with CBFalpha.
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Core-binding factors (CBFs) are a small family of heterodimeric transcription factors that play critical roles in several developmental pathways, including hematopoiesis and bone development. Mutations in CBF genes are found in leukemias and bone disorders. CBFs consist of a DNA-binding CBFalpha subunit (Runx1, Runx2, or Runx3) and a non-DNA-binding CBFbeta subunit. CBFalpha binds DNA in a sequence-specific manner, whereas CBFbeta enhances DNA binding by CBFalpha. Recent structural analyses of the DNA-binding Runt domain of CBFalpha and the CBFbeta subunit identified the heterodimerization surfaces on each subunit. Here we identify amino acids in CBFbeta that mediate binding to CBFalpha. We determine the energy contributed by each of these amino acids to heterodimerization and the importance of these residues for in vivo function. These data refine the structural analyses and further support the hypothesis that CBFbeta enhances DNA binding by inducing a conformational change in the Runt domain. (+info)
Association with the nuclear matrix and interaction with Groucho and RUNX proteins regulate the transcription repression activity of the basic helix loop helix factor Hes1.
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Hairy/Enhancer of split 1 (Hes1) is a mammalian transcriptional repressor that plays crucial roles in the regulation of several developmental processes, including neuronal differentiation. The aim of this study was to elucidate the molecular mechanisms that regulate the transcription repression activity of Hes1. It is shown here that Hes1 associates with the nuclear matrix, the ribonucleoprotein network of the nucleus that plays important roles in transcriptional regulation. Nuclear matrix binding is mediated by the same Hes1 C-terminal domain that is also required for transcriptional repression. This domain contains the WRPW motif that acts as a binding site for the transcriptional corepressor Groucho, which also localizes to the nuclear matrix. Both the nuclear matrix association and transcription repression activity of Hes1 are inhibited by deletion of the WRPW motif, indicating that Groucho acts as a transcriptional corepressor for Hes1. This corepressor role is not modulated by the Groucho-related gene product Grg5. In contrast, the Runt-related protein RUNX2, which localizes to the nuclear matrix and interacts with Groucho and Hes1, can inhibit both the Groucho.Hes1 interaction and the transcription repression ability of Hes1. Together, these observations suggest that transcriptional repression by Hes1 requires interactions with Groucho at the nuclear matrix and that RUNX proteins act as negative regulators of the repressive activity of Groucho.Hes1 complexes. (+info)
Decreased c-Src expression enhances osteoblast differentiation and bone formation.
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c-src deletion in mice leads to osteopetrosis as a result of reduced bone resorption due to an alteration of the osteoclast. We report that deletion/reduction of Src expression enhances osteoblast differentiation and bone formation, contributing to the increase in bone mass. Bone histomorphometry showed that bone formation was increased in Src null compared with wild-type mice. In vitro, alkaline phosphatase (ALP) activity and nodule mineralization were increased in primary calvarial cells and in SV40-immortalized osteoblasts from Src(-/-) relative to Src(+/+) mice. Src-antisense oligodeoxynucleotides (AS-src) reduced Src levels by approximately 60% and caused a similar increase in ALP activity and nodule mineralization in primary osteoblasts in vitro. Reduction in cell proliferation was observed in primary and immortalized Src(-/-) osteoblasts and in normal osteoblasts incubated with the AS-src. Semiquantitative reverse transcriptase-PCR revealed upregulation of ALP, Osf2/Cbfa1 transcription factor, PTH/PTHrP receptor, osteocalcin, and pro-alpha 2(I) collagen in Src-deficient osteoblasts. The expression of the bone matrix protein osteopontin remained unchanged. Based on these results, we conclude that the reduction of Src expression not only inhibits bone resorption, but also stimulates osteoblast differentiation and bone formation, suggesting that the osteogenic cells may contribute to the development of the osteopetrotic phenotype in Src-deficient mice. (+info)
Runx2 is a common target of transforming growth factor beta1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12.
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When C2C12 pluripotent mesenchymal precursor cells are treated with transforming growth factor beta1 (TGF-beta1), terminal differentiation into myotubes is blocked. Treatment with bone morphogenetic protein 2 (BMP-2) not only blocks myogenic differentiation of C2C12 cells but also induces osteoblast differentiation. The molecular mechanisms governing the ability of TGF-beta1 and BMP-2 to both induce ligand-specific responses and inhibit myogenic differentiation are not known. We identified Runx2/PEBP2alphaA/Cbfa1, a global regulator of osteogenesis, as a major TGF-beta1-responsive element binding protein induced by TGF-beta1 and BMP-2 in C2C12 cells. Consistent with the observation that Runx2 can be induced by either TGF-beta1 or BMP-2, the exogenous expression of Runx2 mediated some of the effects of TGF-beta1 and BMP-2 but not osteoblast-specific gene expression. Runx2 mimicked common effects of TGF-beta1 and BMP-2 by inducing expression of matrix gene products (for example, collagen and fibronectin), suppressing MyoD expression, and inhibiting myotube formation of C2C12 cells. For osteoblast differentiation, an additional effector, BMP-specific Smad protein, was required. Our results indicate that Runx2 is a major target gene shared by TGF-beta and BMP signaling pathways and that the coordinated action of Runx2 and BMP-activated Smads leads to the induction of osteoblast-specific gene expression in C2C12 cells. (+info)
Cbfa1: a molecular switch in osteoblast biology.
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During the past 4 years, our molecular understanding of osteoblast biology has made rapid progress due to the characterization of the function of one molecule, Cbfa1. This member of the runt/Cbfa family of transcription factors was first identified as the nuclear protein binding to an osteoblast-specific cis-acting element activating the expression of Osteocalcin, the most osteoblast-specific gene. Cbfa1 was then shown to regulate the expression of all the major genes expressed by osteoblasts. Consistent with this ability, genetic experiments identified Cbfa1 as a key regulator of osteoblast differentiation in vivo. Indeed, analysis of Cbfa1-deficient mice revealed that osteoblast differentiation is arrested in absence of Cbfa1, demonstrating both that it is required for this process and that no parallel pathway can overcome its absence. The importance of Cbfa1 in controlling osteoblast differentiation was further emphasized by the identification of Cbfa1 haploinsufficiency as the cause of cleidocranial dysplasia in humans and mice, a syndrome characterized by generalized bone defects. Lastly, Cbfa1 was shown to have a role beyond development and differentiation, regulating the rate of bone matrix deposition by differentiated osteoblasts. Thus, Cbfa1 is a critical gene not only for osteoblast differentiation but also for osteoblast function. These aspects, as well as the more recent progresses in understanding Cbfa1 biology, are the focuses of this review. (+info)