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Results for “Core Binding Factor alpha Subunits”

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PubMed2002

Core-binding factor alpha 1 (Cbfa1) induces osteoblastic differentiation of C2C12 cells without interactions with Smad1 and Smad5.

Core-binding factor alpha(1) (Cbfa1) is an essential transcription factor for osteoblastic differentiation and osteogenesis. Bone morphogenetic protein (BMP) is also a powerful inducer of differentiation of pluripotent mesenchymal cells to osteoblast lineage and bone formation. Recent studies suggest that Cbfa1 plays a critical role during BMP-induced osteoblastic differentiation through association with cytoplasmic BMP signaling molecules, Smads. However, other studies have suggested that Cbfa1 may exhibit its osteogenic function without interaction with Smads. Therefore, it remains unclear whether association with Smad is essential for Cbfa1 function. In this study we examine the effects of Cbfa1 on osteoblastic differentiation in the presence or absence of interactions with Smad1 or Smad5 using C2C12 undifferentiated mesenchymal cells. Cbfa1 expression was induced upon stimulation with BMP-2 in C2C12 cells. Introduction of Cbfa1 into C2C12 cells induced osteoblastic differentiation and promoted transactivation of osteocalcin gene promoter without forming the complex with Smad1 or Smad5. Furthermore, in C2C12 cells in which the association of Cbfa1 with Smad1/Smad5 was prevented by the overexpression of the natural antagonist, Smad6, Cbfa1 still induced osteoblastic differentiation and transactivated osteocalcin gene promoter, regardless of BMP-2 stimulation. These results suggest that the interactions with Smad1 or Smad5 are not essential for Cbfa1 to demonstrate its osteogenic actions. However, interactions with Smad1/Smad5 enhance these osteogenic actions of Cbfa1. Of note, BMP-2-induced or Smad-induced osteoblastic differentiation was inhibited by dominant-negative Cbfa1, suggesting that the function of Cbfa1 is critical for BMP-2-induced osteoblastic differentiation. Our results suggest that Cbfa1 is essential and also sufficient to induce osteoblastic differentiation in undifferentiated mesenchymal cells, and establishment of an association with Smad1/Smad5 enhances the osteogenic actions of Cbfa1. On the other hand, Cbfa1 expression requires the activation of Smad1/Smad5 by BMP-2.

Animals↗

PubMed1999

Morphological characterization of skeletal cells in Cbfa1-deficient mice.

To clarify the mechanisms by which core-binding factor-alpha1 (Cbfa1), an essential transcription factor in osteogenesis, functions in osteoblast matrix formation, as well as in chondrocyte differentiation and osteoclastic bone resorption, Cbfa1-deficient embryonic mice were investigated ultrastructurally and histocytochemically at 18.5 days postcoitum. In homozygotic mice, both endochondral and intramembranous ossification were arrested, although bone tissue had already formed at this stage in the wild type. The tibiae of homozygotic mice were characterized by calcified cartilage and alkaline phosphatase (ALP)-positive perichondrium, whereas membranous structures indicating the presence of ALP activity in the lateral portion were observed in the calvariae, rather than the bone tissue. Most of the ALP-positive perichondrial cells in homozygotic tibiae possessed a spindle-shaped cell contour and small cytoplasm, the extracellular matrix of which contained neither type I collagen nor calcifying matrix vesicles. In contrast, some perichondrial cells at the very middle part of tibiae became flattened. In the vicinity of these cells, a thin layer of type I collagen-based calcified matrix, containing osteopontin, bone sialoprotein, or osteocalcin, was observed. In the cartilage of mutant mice, we observed a hypoplasic zone of proliferative chondrocytes, the flattening of hypertrophic chondrocyte-like cells, and calcified chondrocytes which, while not degraded, did display a high level of cell function. Mononuclear osteoclastic cells were found in the perichondrium, near calcified chondrocytes, in mutant mice. Multinuclear osteoclasts possessing H+-ATPase and ruffled borders were also present, although only in limited numbers. Neither the development of ruffled borders nor intracellular polarization was complete. Because the majority of osteogenic cells in Cbfa1-deficient mice can neither form nor calcify the bone matrix, Cbfa1 principally plays essential roles in osteoblastic differentiation and bone matrix formation. Cbfa1 also affects both the proliferation and the differentiation of chondrocytes, whereas its absence prevents normal osteoclast formation and related functions.

Animals↗

PubMed2004

Identification of differentially expressed genes in a renal cell carcinoma tumor model after endostatin-treatment.

Endostatin is a cleavage product of collagen XVIII that has shown to inhibit tumor-angiogenesis in experimental tumor models. At present, the exact molecular mechanism of action of endostatin is not completely elucidated. In this study, we wanted to identify specific target genes of endostatin. For this purpose, the human renal cell carcinoma RC-9 was subcutaneously implanted in nude mice and treated with endostatin. Tumor growth was inhibited by endostatin after 4 days of treatment. Using immunohistochemistry and the hypoxia marker pimonidazole, we demonstrate disintegration of blood vessels and hypoxia and anoxia as a result of the treatment. Hereafter, we applied the polymerase chain reaction (PCR)-based subtractive suppression hybridization (SSH) method, together with the mirror orientation selection (MOS) technique to identify specifically induced and suppressed genes after endostatin-treatment. We found eight genes to be specifically induced and 11 to be suppressed by the endostatin-treatment. Among other genes, core binding factor a-1/osteoblast-specific factor-2 (cbfa1/osf2) was found to be specifically suppressed by endostatin. Unexpectedly, cbfa1/osf2 was found to be specifically expressed in granulocytes in the tumor, not only in the experimental RC-9 tumor model, but in sections of human breast cancer as well. Since an effect of antiangiogenic therapy on granulocytes has been reported before, this might lead to new insights in the role of granulocytes in antiangiogenic therapy in general. In conclusion, the SSH-PCR implemented with the MOS-technique is a powerful tool to identify differentially expressed genes. Using these techniques, we have identified several target genes of endostatin, of which cbfa1/osf2 was found to be specifically expressed in granulocytes in the tumor.

Angiogenesis Inhibitors↗

PubMed2002

Core-binding factor beta interacts with Runx2 and is required for skeletal development.

Core-binding factor beta (CBFbeta, also called polyomavirus enhancer binding protein 2beta (PEBP2B)) is associated with an inversion of chromosome 16 and is associated with acute myeloid leukemia in humans. CBFbeta forms a heterodimer with RUNX1 (runt-related transcription factor 1), which has a DNA binding domain homologous to the pair-rule protein runt in Drosophila melanogaster. Both RUNX1 and CBFbeta are essential for hematopoiesis. Haploinsufficiency of another runt-related protein, RUNX2 (also called CBFA1), causes cleidocranial dysplasia in humans and is essential in skeletal development by regulating osteoblast differentiation and chondrocyte maturation. Mice deficient in Cbfb (Cbfb(-/-)) die at midgestation, so the function of Cbfbeta in skeletal development has yet to be ascertained. To investigate this issue, we rescued hematopoiesis of Cbfb(-/-) mice by introducing Cbfb using the Gata1 promoter. The rescued Cbfb(-/-) mice recapitulated fetal liver hematopoiesis in erythroid and megakaryocytic lineages and survived until birth, but showed severely delayed bone formation. Although mesenchymal cells differentiated into immature osteoblasts, intramembranous bones were poorly formed. The maturation of chondrocytes into hypertrophic cells was markedly delayed, and no endochondral bones were formed. Electrophoretic mobility shift assays and reporter assays showed that Cbfbeta was necessary for the efficient DNA binding of Runx2 and for Runx2-dependent transcriptional activation. These findings indicate that Cbfbeta is required for the function of Runx2 in skeletal development.

Animals↗

PubMed1999

A full-length Cbfa1 gene product perturbs T-cell development and promotes lymphomagenesis in synergy with myc.

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.

Animals↗

PubMed2000

Exogenous cdk4 overcomes reduced cdk4 RNA and inhibition of G1 progression in hematopoietic cells expressing a dominant-negative CBF - a model for overcoming inhibition of proliferation by CBF oncoproteins.

Core Binding Factor (CBF) is required for the development of definitive hematopoiesis, and the CBF oncoproteins AML1-ETO, TEL-AML1, and CBFbeta-SMMHC are commonly expressed in subsets of acute leukemia. CBFbeta-SMMHC slows the G1 to S cell cycle transition in hematopoietic cells, but the mechanism of this effect is uncertain. We have sought to determine whether inhibition of CBF-mediated trans-activation is sufficient to slow proliferation. We demonstrate that activation of KRAB-AML1-ER, a protein containing the AML1 DNA-binding domain, the KRAB repression domain, and the Estrogen receptor ligand binding domain, also slows G1, if its DNA-binding domain is intact. Also, exogenous AML1 overcame CBFbeta-SMMHC-induced inhibition of proliferation. Representational difference analysis (RDA) identified cdk4 RNA expression as an early target of KRAB-AML1 activation. Inhibition of CBF activities by KRAB-AML1-ER or CBFbeta-SMMHC rapidly reduced endogenous cdk4 mRNA levels, even in cells proliferating at or near control rates as a result of exogenous cdk4 expression. Over-expression of cdk4, especially a variant which cannot bind p16INK4a, overcame cell cycle inhibition resulting from activation of KRAB-AML1-ER, although cdk4 did not accelerate proliferation when expressed alone. These findings indicate that mutations which alter the expression of G1 regulatory proteins can overcome inhibition of proliferation by CBF oncoproteins. Oncogene (2000).

Animals↗

PubMed2006

Decreased intranuclear mobility of acute myeloid leukemia 1-containing fusion proteins is accompanied by reduced mobility and compartmentalization of core binding factor beta.

Acute myeloid leukemia 1 (AML1) gene on chromosome 21 is involved in several chromosomal translocations, including t(8;21) and t(16;21), that produce chimeric fusion proteins AML1-eight twenty-one (ETO) and AML-myeloid transforming gene chromosome 16 (MTG16), which contribute to leukemogenesis. The molecular basis for the leukemogenic effects of these fusion proteins is incompletely understood. Using gel-shift assay, we showed that AML1-ETO and AML1-MTG16 bound to a series of AML1 consensus DNA-binding sites with different affinities. Using fluorescence recovery after photobleaching (FRAP), we demonstrated that a fusion of AML1 with ETO or MTG16 exhibits reduced intranuclear mobility compared with wild-type AML1 or either fusion partner. The dimerization domain (nervy homology region 2) of ETO is responsible for the reduced mobility of AML1-ETO. Dual FRAP studies revealed that CBFbeta colocalized with AML1-ETO within the nucleus, resulting in reduced mobility of CBFbeta. Therefore, AML1 fusion proteins may interfere with normal AML1 function due to aberrant nuclear dynamics, which leads to spatial and temporal sequestration of CBFbeta and perhaps other coregulators critical for myeloid differentiation.

Animals↗

PubMed2004

Identification of genes that synergize with Cbfb-MYH11 in the pathogenesis of acute myeloid leukemia.

Acute myeloid leukemia subtype M4 with eosinophilia is associated with a chromosome 16 inversion that creates a fusion gene CBFB-MYH11. We have previously shown that CBFB-MYH11 is necessary but not sufficient for leukemogenesis. Here, we report the identification of genes that specifically cooperate with CBFB-MYH11 in leukemogenesis. Neonatal injection of Cbfb-MYH11 knock-in chimeric mice with retrovirus 4070A led to the development of acute myeloid leukemia in 2-5 months. Each leukemia sample contained one or a few viral insertions, suggesting that alteration of one gene could be sufficient to synergize with Cbfb-MYH11. The chromosomal position of 67 independent retroviral insertion sites (RISs) was determined, and 90% of the RISs mapped within 10 kb of a flanking gene. In total, 54 candidate genes were identified; six of them were common insertion sites (CISs). CIS genes included members of a zinc finger transcription factors family, Plag1 and Plagl2, with eight and two independent insertions, respectively. CIS genes also included Runx2, Myb, H2T24, and D6Mm5e. Comparison of the remaining 48 genes with single insertion sites with known leukemia-associated RISs indicated that 18 coincide with known RISs. To our knowledge, this retroviral genetic screen is the first to identify genes that cooperate with a fusion gene important for human myeloid leukemia.

Acute Disease↗

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↗

PubMed1997

Proviral insertions induce the expression of bone-specific isoforms of PEBP2alphaA (CBFA1): evidence for a new myc collaborating oncogene.

The til-1 locus was identified as a common retroviral integration site in virus-accelerated lymphomas of CD2-myc transgenic mice. We now show that viral insertions at til-1 lead to transcriptional activation of PEBP2alphaA (CBFA1), a transcription factor related to the Drosophila segmentation gene product, Runt. Insertions are upstream and in the opposite orientation to the gene and appear to activate a variant promoter that is normally silent in T cells. Activity of this promoter was detected in rodent osteogenic sarcoma cells and primary osteoblasts, implicating bone as the normal site of promoter activity. The isoforms encoded by the activated gene all encompass the conserved runt DNA-binding domain and share a novel N terminus different from the previously reported PEBP2alphaA products. Minor products include isoforms with internal deletions due to exon skipping and a novel C-terminal domain unrelated to known runt domain factors. The major isoform expressed from the activated til-1 locus (G1) was found to account for virtually all of the core binding factor activity in nuclear extracts from its corresponding lymphoma cell line. Another member of this gene family, AML1(CBFA2), is well known for its involvement in human hemopoietic tumors. These results provide evidence of a direct oncogenic role for PEBP2alphaA and indicate that the Myc and Runt family genes can cooperate in oncogenesis.

Amino Acid Sequence↗

PubMed1998

Smad2 overexpression enhances Smad4 gene expression and suppresses CBFA1 gene expression in osteoblastic osteosarcoma ROS17/2.8 cells and primary rat calvaria cells.

Mothers against decapentaplegic-related proteins (Smads) are essential intracellular components for the signal transduction of transforming growth factor-beta (TGF-beta) family members. Smad1 mediates bone morphogenetic protein (BMP) signals, whereas Smad2 functions downstream of TGF-beta. TGF-beta is expressed in osteoblastic cells and acts as an autocrine and/or paracrine factor in regulation of osteoblastic functions. In this study, we examined the levels and functions of Smad2 in osteoblastic cells. Smad2 mRNA expression was hardly detectable by Northern blot analysis in an osteoblast-like cell line, ROS17/2.8, as well as in primary rat calvaria (PRC) cells. Overexpression of Smad2 gene enhanced endogenous Smad4 gene expression in both ROS17/2.8 and PRC cells, while Smad3 levels were not altered. Smad2 overexpression suppressed osteocalcin mRNA expression in ROS17/2.8 cells. Furthermore, Smad2 overexpression also suppressed transcriptional activity of the 1-kilobase pair osteocalcin gene promoter, which was linked to chloramphenicol acetyltransferase reporter gene in both ROS and PRC cells. Since core binding factor A1 (CBFA1) is involved in osteocalcin gene expression, we further examined CBFA1 expression in the Smad2-overexpressing ROS17/2.8 and PRC cells. The levels of CBFA1 mRNA were suppressed by the overexpression of Smad2 by about 50% in both ROS17/2.8 and PRC cells. TGF-beta treatment enhanced Smad4 expression in PRC cells, and this TGF-beta effect was blocked by the cotreatment with BMP, indicating that TGF-beta signaling pathway is interfered by BMP. These data indicate that Smad2 regulates Smad4 specifically and that CBFA1 gene is one of the downstream targets of Smad2.

Animals↗

PubMed2000

Parathyroid hormone regulation of the rat collagenase-3 promoter by protein kinase A-dependent transactivation of core binding factor alpha1.

Previously we showed that the activator protein-1 site and the runt domain binding site in the collagenase-3 promoter act cooperatively in response to parathyroid hormone (PTH) in the rat osteoblastic osteosarcoma cell line, UMR 106-01. Our results of the expression pattern of core binding factor alpha1 (Cbfa1), which binds to the runt domain site, indicated that there is no change in the levels of Cbfa1 protein or RNA under either control conditions or after PTH treatment. The importance of posttranslational modification of Cbfa1 in the signaling pathway for PTH-induced collagenase-3 promoter activity was analyzed. PTH stimulation of collagenase-3 promoter activity was completely abrogated by protein kinase A (PKA) inhibition. To determine the role of PKA activity with respect to Cbfa1 activation (in addition to its known activity of phosphorylating cAMP-response element-binding protein to enhance c-fos promoter activity), we utilized the heterologous Gal4 transcription system. PTH stimulated the transactivation of activation domain-3 in Cbfa1 through the PKA site. In vitro phosphorylation studies indicated that the PKA site in the wild type activation domain-3 is a substrate for phosphorylation by PKA. Thus, we demonstrate that PTH induces a PKA-dependent transactivation of Cbfa1, and this transactivation is required for collagenase-3 promoter activity in UMR cells.

Animals↗

PubMed2002

Superoxide mediates shock wave induction of ERK-dependent osteogenic transcription factor (CBFA1) and mesenchymal cell differentiation toward osteoprogenitors.

Extracorporeal shock wave (ESW) is an alternative non-invasive method for the promotion of bone growth and tendon repair. In an animal model, we have reported that ESW promoted bone marrow osteoprogenitor growth through transforming growth factor-beta1 induction. We have further explored the mechanism for the ESW promotion of osteogenesis. Results showed that an optimal ESW treatment at 0.16 mJ/mm(2) for 500 impulses rapidly induced a higher O(2)(-) and ONOO(-) production associated with a decrease of nitric oxide level in 1 h, and induced a higher transforming growth factor-beta1 production in 24 h, and a higher colony-forming units-osteoprogenitor formation in 12 days. The colony-forming units-osteoprogenitor colonies revealed positive staining of bone alkaline phosphatase and turned into bone nodules in 21 days. Early scavenging of O(2)(-) but not Ca(2+), H(2)O(2), or prostaglandin E(2) suppressed osteoprogenitor cell growth and maturation. Scavenging of O(2)(-) by superoxide dismutase raised the nitric oxide level back to the basal level and suppressed ESW-promoted osteoprogenitor cell growth, whereas inhibition of ONOO(-) by urate or NO by N-nitro-l-arginine methyl ester did not affect ESW promotion of osteogenesis, indicating that O(2)(-) acted as an early signal for ESW-induced cell growth. Further studies demonstrated that ESW induced ERK activation, and blockage of O(2)(-) production or inhibition of tyrosine kinase, but not protein kinase A and C inhibitors, suppressed ESW-induced ERK activation. In support that O(2)(-) mediated the ESW-induced ERK activation and osteogenic differentiation, we further demonstrated that scavenging of O(2)(-) by superoxide dismutase and inhibition of ERK activation by PD98059 decreased specific osteogenic transcription factor, core binding factor A1 activation, and decreased osteocalcin expression. Taken together, we showed that ESW-induced O(2)(-) production followed by tyrosine kinase-mediated ERK activation and core binding factor A1 activation resulted in osteogenic cell growth and maturation. Thus, an appropriate modulation of redox reaction by ESW may have some positive effect on the bone regeneration.

Animals↗

PubMed2002

The bone-specific transcriptional regulator Cbfa1 is a target of mechanical signals in osteoblastic cells.

A primary goal of bone research is to understand the mechanism(s) by which mechanical forces dictate the cellular and metabolic activities of osteoblasts, the bone-forming cells. Several studies indicate that osteblastic cells respond to physical loading by transducing signals that alter gene expression patterns. Accumulated data have documented the fundamental role of the osteoblast-specific transcription factor Cbfa1 (core-binding factor) in osteoblast differentiation and function. Here, we demonstrate that low level mechanical deformation (stretching) of human osteoblastic cells directly up-regulates the expression and DNA binding activity of Cbfa1. This effect seems to be fine tuned by stretch-triggered induction of distinct mitogen-activated protein kinase cascades. Our novel finding that activated extracellular signal-regulated kinase mitogen-activated protein kinase physically interacts and phosphorylates endogenous Cbfa1 in vivo (ultimately potentiating this transcription factor) provides a molecular link between mechanostressing and stimulation of osteoblast differentiation. Elucidation of the specific modifiers and cofactors that operate in this mechanotranscription circuitry will contribute to a better understanding of mechanical load-induced bone formation which may set the basis for nonpharmacological intervention in bone loss pathologies.

Cells, Cultured↗

PubMed2003

E3 ubiquitin ligase Smurf1 mediates core-binding factor alpha1/Runx2 degradation and plays a specific role in osteoblast differentiation.

Osteoblast differentiation and bone formation is stimulated by bone morphogenetic protein (BMP)-2 and its downstream signaling molecules Smad1 and -5 and the osteoblast-specific transcription factor core-binding factor alpha1 (Cbfa1). Proteolytic degradation of Smad1 and Cbfa1 is proteasome-dependent, and intracellular concentrations of Smad1 and Cbfa1 are enhanced by inhibition of the 26 S proteasome. Smad1 degradation is mediated by the E3 ubiquitin ligase Smurf1 (Smad ubiquitin regulatory factor 1), but the specific E3 ligase responsible for Cbfa1 degradation has not been identified. Because Cbfa1 interacts with Smad1, whose degradation is mediated by Smurf1, we examined the effect of Smurf1 on Cbfa1 degradation in osteoblast precursor cells. Smurf1 interacts directly with Cbfa1 and mediates Cbfa1 degradation in a ubiquitin- and proteasome-dependent manner. Because Smurf1 controls the intracellular concentrations of several key molecules in the bone formation cascade, we examined the effect of a mutant form of Smurf1 in osteoblasts and found that expression of mutant Smurf1 markedly enhanced osteoblast differentiation. Smurf1 therefore appears to be an important regulatory factor in osteoblast differentiation and a potential molecular target for identification of bone anabolic agents.

Animals↗

PubMed2004

Strong and rapid induction of osteoblast differentiation by Cbfa1/Til-1 overexpression for bone regeneration.

Core binding factor alpha-1 (Cbfa1), known as an essential transcription factor for osteogenic lineage, has two major N-terminal isoforms: Pebp2alphaA and Til-1. To study the roles of these isoforms in bone regeneration, we applied an adenoviral vector carrying their genes to transduce primary osteoprogenitor cells in vitro and in vivo. Overexpression of the two isoforms induced rapid and marked osteoblast differentiation, with Til-1 being more effective in vitro, by examination of the alkaline phosphatase activity, calcium content, and Alizarin red staining. Til-1 overexpressing cells/porous ceramic composites were transplanted into subcutaneous and bone defect sites in Fischer rats (cultured bone transplantation model) and markedly affected in vivo bone formation and osteoblast markers. The results demonstrated that the reconstitution of bone tissues, such as cortical bone and trabecular bone was accelerated by implantation of Til-1 overexpressing cells/porous ceramic composites. Moreover, the new bone formation by Til-1 overexpression appeared to reflect replacement of new bone within the implant boundaries. To ascertain whether implanted Cbfa1 overexpressing cells could differentiate into osteogenic cells to create bone or whether it stimulated the surrounding recipient tissue to regenerate bone, implanted male donor cells were visualized by fluorescent in situ hybridization analysis. The proportion of implanted cells in the presumptive bone forming region was over 80% and did not change throughout from 3 days to 8 weeks after implantation. These findings suggested that the newly formed bone in the porous area of the scaffold is mostly produced by the implanted donor cells or their derived cells, effectively by Til-1 overexpression.

Adenoviridae↗

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

A rapid RT-PCR screening assay incorporating multiplexed validated control genes for CBF rearrangements at diagnosis in AML.

AIMS: Our objective was to establish a multiplexed assay using the Biomed 1 primers to detect AML1-ETO transcripts and 10 different CBFB-MYH11 transcripts, using BCR and ABL transcripts as controls. METHODS: Control genes were systematically tested for characteristics of optimal controls. The final assay was validated on 50 AML patient samples. RESULTS: Testing confirmed that the designated control gene criteria were fulfilled. Of 50 patient samples tested, four RT-PCR results were discordant with the cytogenetic result. In three cytogenetically negative cases, RT-PCR detected cryptic CBF rearrangements (one AML1-ETO and two CBFB-MYH11). The fourth case was inv(16) positive but negative by RT-PCR; however, the control gene result revealed suboptimal RNA quality. CONCLUSIONS: We have described a robust multiplex RT-PCR assay that incorporates experimentally validated control genes that are important for accurate interpretation. The assay is more sensitive than cytogenetics in the detection of CBF AML. Application to large patient cohorts will determine the prognostic significance of cryptic CBF rearrangements compared with their cytogenetic counterparts.

Acute Disease↗