Inhibition of osteoblast-specific transcription factor Cbfa1 by the cAMP pathway in osteoblastic cells. Ubiquitin/proteasome-dependent regulation.
(9/863)
The cAMP pathway, a major intracellular pathway mediating parathyroid hormone signal, regulates osteoblastic function. Parathyroid hormone (through activation of protein kinase A) has also been shown to stimulate ubiquitin/proteasome activity in osteoblasts. Since the osteoblast-specific transcription factor Osf2/Cbfa1 is important for differentiation of osteoblastic cells, we examined the roles of the cAMP and ubiquitin/proteasome pathways in regulation of Cbfa1. In the osteoblastic cell line, MC3T3-E1, continuous treatment with cAMP elevating agents inhibited both osteoblastic differentiation based on alkaline phosphatase assay and DNA binding ability of Cbfa1 based on a gel retardation assay. Cbfa1 inhibition was paralleled by an inhibitory effect of forskolin on Cbfa1-regulated genes. Northern and Western blot analyses suggested that the inhibition of Cbfa1 by forskolin was mainly at the protein level. Pretreatment with proteasome inhibitors prior to forskolin treatment reversed the effect of forskolin. Furthermore, addition of proteasome inhibitors to forskolin-pretreated samples resulted in recovery of Cbfa1 protein levels and accumulation of polyubiquitinated forms of Cbfa1, indicating a role for the proteasome pathway in the degradation of Cbfa1. These results suggest that suppression of osteoblastic function by the cAMP pathway is through proteolytic degradation of Cbfa1 involving a ubiquitin/proteasome-dependent mechanism. (+info)
Characterization of Osf1, an osteoblast-specific transcription factor binding to a critical cis-acting element in the mouse Osteocalcin promoters.
(10/863)
To elucidate the mechanisms of osteoblast-specific gene expression we are studying the regulation of osteocalcin, the most osteoblast-specific gene. Previous studies of OG2, one of the two mouse osteocalcin genes, identified two osteoblast-specific cis-acting elements, OSE1 and OSE2, the latter being the binding site of Cbfa1, the only osteoblast-specific transcription factor known to date. Here we show that OSE1 is a cis-acting element as important as OSE2 for the osteoblast-specific expression of OG2 in cell culture and transgenic mice. We also show that OSE1 is present in the promoter of several osteoblast-specific genes including Cbfa1 itself. These biological features demonstrate the importance of OSE1 and led us to further characterize this site and the factor binding to it, provisionally termed Osf1. We first defined the core OSE1 sequence, 5'-TTACATCA-3', which is necessary and sufficient for Osf1 binding to DNA. This sequence has no strong homology to any known transcription factor-binding sites. As a first step in identifying Osf1, we performed an analytical purification of this protein using nuclear extracts from two different osteoblastic cell lines. We purified Osf1 to homogeneity through a five-step procedure including a renaturation experiment and found that its apparent molecular mass is 40 kDa. In conclusion, this study indicates the existence of multiple osteoblast-specific cis-acting elements of equal importance in controlling OG2 promoter activity and provides the first biochemical characterization of Osf1, a novel osteoblast-specific transcription factor. (+info)
Mutation analysis of core binding factor A1 in patients with cleidocranial dysplasia.
(11/863)
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. (+info)
AML3/CBFalpha1 is required for androgen-specific activation of the enhancer of the mouse sex-limited protein (Slp) gene.
(12/863)
A complex 120-base pair enhancer, derived from the mouse sex-limited protein (Slp) gene, is activated solely by the androgen receptor (AR) in specific tissues, although it contains a hormone response element recognized by several steroid receptors. The generation of this transcriptional specificity has been ascribed to the interactions of the receptor with tissue-specific nonreceptor factors bound to accessory sites within the enhancer. Protein-DNA interaction assays revealed two factors binding the 5' part of the enhancer that differ widely in abundance between cells showing AR-specific activation of the Slp element compared with those that also permit activation by glucocorticoid receptor (GR). The factor designated B formed a complex centered on the sequence TGTGGT, a core motif recognized by members of the AML/CBFalpha transcription factor family. This complex was competed by a high affinity binding site specific for AML/CBFalpha and was specifically supershifted by an antibody to AML3/CBFalpha1, placing factor B within the AML3/CBFalpha1 subclass. Interestingly, this factor was shown to bind to a second site in the 3' part of the enhancer, positioned between the two critical AR binding sites. Transfection studies revealed that AML1-ETO, a dominant-negative AML/CBFalpha construct, abrogated AR induction of the enhancer, but not of simple hormone response elements. Furthermore, overexpression of AML3/CBFalpha1 could rescue the AML1-ETO repression. Finally, glutathione S-transferase-AML/CBFalpha fusion proteins demonstrated direct interaction between AML/CBFalpha and steroid receptors. Although this interaction was equivalent between AML1/CBFalpha2 and AR or GR, AML3/CBFalpha1 showed stronger interaction with AR than with GR. These data demonstrate that AML3/CBFalpha1 is functionally required for hormonal induction of the Slp enhancer and that direct, preferential protein-protein interactions may contribute to AR-specific activation. These results demonstrate an intriguing role of AML3/CBFalpha1 in steroid- as well as tissue-specific activation of target genes. (+info)
Isolation and characterization of the distal promoter region of mouse Cbfa1.
(13/863)
Cbfa1 is an essential transcription factor for bone formation, and as such little is known about the region responsible for the transcriptional regulation of this gene. Here we report the determination of the transcription start sites, isolation and partial characterization of distal promoter region of this gene. Three transcription start sites were identified by the 5'-Cap site method, recently invented for rapid examination of the 5'-end of genes of interest. A reporter construct containing 1.8 kb of 5' of transcription start sites had approximately 25-fold more luciferase activity than the promoter-less vector in osteoblastic cell lines. Deletion analysis of the reporter construct demonstrated that the minimal region to express promoter activity lies between bp -168 and -99, taking the most downstream transcription start site as +1. By Northern blot analysis, mRNA expression from the distal promoter was detected in the differentiated osteoblastic cell lines, UMR-106, ROS17/2.8 and MC3T3-E1, but not in cell lines of immature phenotype or originated from other organs. Luciferase activity was strongest in UMR-106 and ROS17/2.8, and weakest in COS-1 and HepG2, which are cell lines originating from other organs, corresponding to the level of mRNA expression. These results demonstrated that the distal promoter region examined here is important for tissue- and cell-type-specific gene expression of Cbfa1. (+info)
CBFA1 mutation analysis and functional correlation with phenotypic variability in cleidocranial dysplasia.
(14/863)
Cleidocranial dysplasia (CCD) is a dominantly inherited skeletal dysplasia caused by mutations in the osteoblast-specific transcription factor CBFA1. To correlate CBFA1 mutations in different functional domains with the CCD clinical spectrum, we studied 26 independent cases of CCD and a total of 16 new mutations were identified in 17 families. The majority of mutations were de novo missense mutations that affected conserved residues in the runt domain and completely abolished both DNA binding and transactivation of a reporter gene. These, and mutations which result in premature termination in the runt domain, produced a classic CCD phenotype by abolishing transactivation of the mutant protein with consequent haploinsufficiency. We further identified three putative hypomorphic mutations (R391X, T200A and 90insC) which result in a clinical spectrum including classic and mild CCD, as well as an isolated dental phenotype characterized by delayed eruption of permanent teeth. Functional studies show that two of the three mutations were hypomorphic in nature and two were associated with significant intrafamilial variable expressivity, including isolated dental anomalies without the skeletal features of CCD. Together these data show that variable loss of function due to alterations in the runt and PST domains of CBFA1 may give rise to clinical variability, including classic CCD, mild CCD and isolated primary dental anomalies. (+info)
The murine Bapx1 homeobox gene plays a critical role in embryonic development of the axial skeleton and spleen.
(15/863)
Our previous studies in both mouse and human identified the Bapx1 homeobox gene, a member of the NK gene family, as one of the earliest markers for prechondrogenic cells that will subsequently undergo mesenchymal condensation, cartilage production and, finally, endochondral bone formation. In addition, Bapx1 is an early developmental marker for splanchnic mesoderm, consistent with a role in visceral mesoderm specification, a function performed by its homologue bagpipe, in Drosophila. The human homologue of Bapx1 has been identified and mapped to 4p16.1, a region containing loci for several skeletal diseases. Bapx1 null mice are affected by a perinatal lethal skeletal dysplasia and asplenia, with severe malformation or absence of specific bones of the vertebral column and cranial bones of mesodermal origin, with the most severely affected skeletal elements corresponding to ventral structures associated with the notochord. We provide evidence that the failure of the formation of skeletal elements in Bapx1 null embryos is a consequence of a failure of cartilage development, as demonstrated by downregulation of several molecular markers required for normal chondroblast differentiation (&agr; 1(II) collagen, Fgfr3, Osf2, Indian hedgehog, Sox9), as well as a chondrocyte-specific alpha1 (II) collagen-lacZ transgene. The cartilage defects are correlated with failed differentiation of the sclerotome at the time when these cells are normally initiating chondrogenesis. Loss of Bapx1 is accompanied by an increase in apoptotic cell death in affected tissues, although cell cycling rates are unaltered. (+info)
A full-length Cbfa1 gene product perturbs T-cell development and promotes lymphomagenesis in synergy with myc.
(16/863)
The Cbfa1/PEBP2 alpha A/AML3 gene plays an essential role in osteogenesis but is also expressed in the T-cell lineage where it has been implicated in lymphoma development as a target for retroviral insertional mutagenesis. As lymphoma cells with til-1 insertion express at least five distinct Cbfa1 isoforms, it is important to establish which, if any, have intrinsic oncogenic potential. We have generated transgenic mice in which the most abundant lymphoma isoform (G1/p57) is expressed under the control of the CD2 locus control region. Co-precipitation analysis of transgenic thymus revealed high levels of Cbfa1 protein in an abundant complex containing the binding cofactor Cbfb. CD2-Cbfa1-G1 mice displayed abnormal T-cell development, with a pronounced skew towards CD8 SP cells in the thymus and developed a low incidence of spontaneous lymphomas (6% at 12 months) with cells of similar phenotype. Strongly synergistic tumour development was seen when CD2-Cbfa1-G1 mice were crossed with lines carrying myc transgenes (CD2-myc or tamoxifen-regulatable CD2-mycER) and Cbfa1 was found to rescue expression of the CD2-myc transgene in pre-leukaemic mice. However, synergy did not appear to be due to a dominant block of myc-induced apoptosis by Cbfa1 as explanted primary tumours and cell lines from CD2-Cbfa1-G1/CD2-mycER mice showed accelerated death on induction with tamoxifen at similar rates to CD2-mycER controls. Moreover, thymocytes from preleukaemic CD2-Cbfa1-G1 mice showed reduced survival in vitro and increased sensitivity to the inhibitory effects of TGF-beta. This study demonstrates that a full-length Cbf alpha-chain gene can act as an oncogene without fusion to a heterologous protein. (+info)