Osteoblast-specific Cis-acting Element Research Articles

Cyclophilin A Promotes Osteoblast Differentiation by Regulating Runx2.

Cyclophilin A (CypA) is a ubiquitously expressed and highly conserved protein with peptidyl-prolyl cis-trans isomerase activity that is involved in various biological activities by regulating protein folding and trafficking. Although CypA has been reported to positively regulate osteoblast differentiation, the mechanistic details remain largely unknown. In this study, we aimed to elucidate the mechanism of CypA-mediated regulation of osteoblast differentiation. Overexpression of CypA promoted osteoblast differentiation in bone morphogenic protein 4 (BMP4)-treated C2C12 cells, while knockdown of CypA inhibited osteoblast differentiation in BMP4-treated C2C12. CypA and Runx2 were shown to interact based on immunoprecipitation experiments and CypA increased Runx2 transcriptional activity in a dose-dependent manner. Our results indicate that this may be because CypA can increase the DNA binding affinity of Runx2 to Runx2 binding sites such as osteoblast-specific cis-acting element 2. Furthermore, to identify factors upstream of CypA in the regulation of osteoblast differentiation, various kinase inhibitors known to affect osteoblast differentiation were applied during osteogenesis. Akt inhibition resulted in the most significant suppression of osteogenesis in BMP4-induced C2C12 cells overexpressing CypA. Taken together, our results show that CypA positively regulates osteoblast differentiation by increasing the DNA binding affinity of Runx2, and Akt signaling is upstream of CypA.

Inhibition of Runx2 signaling by TNF-α in ST2 murine bone marrow stromal cells undergoing osteogenic differentiation.

Tumor necrosis factor-alpha (TNF-α) inhibits osteogenic differentiation of murine bone marrow stromal cells, and transcription factor Runx2 serves as an essential regulation target in the process. The underlying mechanism may involve the regulation of Runx2 expression and the Runx2 activity in downstream gene transcription, which has not been fully elucidated. In this study, ST2 murine bone marrow-derived stromal cells were treated with bone morphogenetic protein-2 (BMP-2) and/or TNF-α in osteogenic medium, and the expression of Runx2 was estimated. Cells were transfected with Runx2, p65, inhibitor of κBα (IκBα), 9.0kb bone sialoprotein (BSP) promoter-luciferase or osteoblast-specific cis-acting element 2 (OSE2)-luciferase reporter vectors, and then real time-PCR and dual luciferase analysis were used to investigate the effect of TNF-α on Runx2-activated osteogenic gene transcription and the molecular mechanism. We found that TNF-α inhibited BMP-2-induced osteogenic marker expression and both the spontaneous and BMP-2-induced Runx2 expression. TNF-α stimulation or overexpression of nuclear factor-kappa B (NF-κB) p65 subunit repressed the Runx2-activated BSP and osteocalcin (OC) transcriptions. The Runx2-induced 9.0kb BSP promoter activity was attenuated by TNF-α or p65, while the OSE2 activity was not affected. Besides, blockage of NF-κB by IκBα overexpression eliminated these inhibitory effects of TNF-α on Runx2 signaling. These results suggest that in murine bone marrow stromal cells undergoing osteogenic differentiation, TNF-α and it activated NF-κB pathway inhibit the expression of Runx2 gene, and suppress the Runx2-mediated osteogenic gene transcription via the 9.0kb BSP promoter.

BMP2 Protein Regulates Osteocalcin Expression via Runx2-mediated Atf6 Gene Transcription

Bone morphogenetic protein 2 (BMP2) activates unfolded protein response (UPR) transducers, such as PERK and OASIS, in osteoblast cells. ATF6, a bZIP transcription factor, is also a UPR transducer. However, the involvement of ATF6 in BMP2-induced osteoblast differentiation has not yet been elucidated. In the present study, BMP2 treatment was shown to markedly induce the expression and activation of ATF6 with an increase in alkaline phosphatase (ALP) and OC expression in MC3T3E1 cells. In contrast, ATF6 activation by BMP2 was not observed in the Runx2(-/-) primary calvarial osteoblasts, and Runx2 overexpression recovered BMP2 action. BMP2 stimulated ATF6 transcription by enhancing the direct binding of Runx2 to the osteoblast-specific cis-acting element 2 (OSE2, ACCACA, -205 to -200 bp) motif of the Atf6 promoter region. In addition, the overexpression of ATF6 increased the Oc promoter activity by enhancing the direct binding to a putative ATF6 binding motif (TGACGT, -1126 to -1121 bp). The inhibition of ATF6 function with the dominant negative form of ATF6 (DN-ATF6) blocked BMP2- or Runx2-induced OC expression. Interestingly, OASIS, which is structurally similar to ATF6, did not induce Oc expression. ALP and Alizarin red staining results confirmed that BMP2-induced matrix mineralization was also dependent on ATF6 in vitro. Overall, these results suggest that BMP2 induces osteoblast differentiation through Runx2-dependent ATF6 expression, which directly regulates Oc transcription.

C/EBPβ and RUNX2 cooperate to degrade cartilage with MMP-13 as the target and HIF-2α as the inducer in chondrocytes

To elucidate the molecular mechanism underlying the endochondral ossification process during the skeletal growth and osteoarthritis (OA) development, we examined the signal network around CCAAT/enhancer-binding protein-β (C/EBPβ, encoded by CEBPB), a potent regulator of this process. Computational predictions and a C/EBP motif-reporter assay identified RUNX2 as the most potent transcriptional partner of C/EBPβ in chondrocytes. C/EBPβ and RUNX2 were induced and co-localized in highly differentiated chondrocytes during the skeletal growth and OA development of mice and humans. The compound knockout of Cebpb and Runx2 in mice caused growth retardation and resistance to OA with decreases in cartilage degradation and matrix metalloproteinase-13 (Mmp-13) expression. C/EBPβ and RUNX2 cooperatively enhanced promoter activity of MMP13 through specific binding to a C/EBP-binding motif and an osteoblast-specific cis-acting element 2 motif as a protein complex. Human genetic studies failed to show the association of human CEBPB gene polymorphisms with knee OA, nor was there a genetic variation around the identified responsive region in the human MMP13 promoter. However, hypoxia-inducible factor-2α (HIF-2α), a functional and genetic regulator of knee OA through promoting endochondral ossification, was identified as a potent and functional inducer of C/EBPβ expression in chondrocytes by the CEBPB promoter assay. Hence, C/EBPβ and RUNX2, with MMP-13 as the target and HIF-2α as the inducer, control cartilage degradation. This molecular network in chondrocytes may represent a therapeutic target for OA.

FGF8 regulates myogenesis and induces Runx2 expression and osteoblast differentiation in cultured cells

In the current study, treatment of the rat osteogenic cell line ROB-C26 cells with fibroblast growth factor 8 (FGF8) stimulated alkaline phosphatase (ALP) activity, and also induced the expression of the Runx2 transcription factor, and increased the activity of a luciferase reporter gene containing the osteocalcin (OCN) promoter and six copies of the osteoblast specific cis-acting element 2 (OSE2) response element. In contrast, FGF8 treatment of the mouse myoblast cell line C2C12 inhibited the expression of desmin and the synthesis of myotubes. The expression of MyoD, Myogenin, Foxc2, and Hand1 was also decreased by FGF8. Transient expression of Foxc2 in C2C12 cells induced the expression of Hand1, and chromatin immunoprecipitation (ChIP) analysis indicated that Foxc2 binds to the promoter region of the Hand1 gene. These results indicated that Foxc2 is directly involved in the regulation of Hand1 expression. The results of the current study indicate that FGF8 regulates myoblast differentiation through the regulation of MyoD expression, and that this regulation is independent of Hand1 in cultured cells. Conversely, FGF8 supports bone development and cell differentiation though the induction of Runx2 expression.

Dexamethasone promotes DMP1 mRNA expression by inhibiting negative regulation of Runx2 in multipotential mesenchymal progenitor, ROB‐C26

Dentin matrix protein 1 (DMP1) is an acidic phosphorylated extracellular protein and essential for mineralization of dentin and bone; however, the precise mechanism regulating DMP1 expression is not fully understood. A synthetic glucocorticoid (GC), dexamethasone (Dex), promotes an early osteoblast differentiation of a mesenchymal progenitor, ROB-C26 (C26), in parallel with inductive expression of an osteoblast-specific transcription factor, Runx2, and other extracellular matrix proteins such as osteocalcin and bone sialoprotein (BSP). We have examined the effect of Dex on DMP1 expression via induction of Runx2 in C26 cells. Real time RT-PCR showed that Dex increases DMP1 mRNA expression levels at time- and dose-dependent manners and a GC antagonist, RU486, drastically inhibited DMP1 mRNA expression levels. Furthermore, Dex increased the luciferase activity of six-repeated osteoblast-specific cis-acting element 2 (6 x OSE2), which is the binding sequence of Runx2, suggesting that Dex stimulates DMP1 expression via activation of Runx2. However, unexpected results showed that overexpression of exogenous Runx2 depressed DMP1 mRNA expression level, even after cells had been treated with Dex, while downregulated expression of endogenous Runx2 enhanced Dex-induced DMP1 mRNA expression level. These results imply that large amounts of exogenous Runx2 inhibit DMP1 expression, whereas small amounts are more effective for Dex-induced DMP1 expression in C26 cells. Therefore, Dex may activate some factors that inhibit negative action of Runx2 on DMP1 expression. Since mitogen-activating protein kinase (MAPK) phosphatase-1 (MKP-1) has been reported to affect the Dex-induced osteoblast differentiation via decrease of Runx2-phosphorylation, we focus on the relationship between MKP-1 and DMP1 expression. Dex increases MKP-1 expression, and overexpression of exogenous MKP-1 showed significant increase of luciferase activity of 6 x OSE up to the level detected in Dex-treated C26 cells. However, no inductive DMP1 mRNA expression level was found in C26 cells unlike BSP and OPN. These results suggest that although MKP-1 increases DNA-binding activity of Runx2, DMP1 expression may require the collaboration of MKP-1 and additive factors to stimulate Runx2-mediated DMP1 expression in the post-transcriptional event of Dex-treated C26 cells.

Runx2: of bone and stretch

Runx2 is a key transcriptional modulator of osteoblast differentiation that plays a fundamental role in osteoblast maturation and homeostasis. Runx2-null mice despite normal skeletal patterning have no osteoblasts and consequently bone tissue. Mutations of the runx2 gene in humans cause cleidocranial dysplasia. As a member of the Runx family of transcription factors, Runx2 operates by binding to the osteoblast-specific cis-acting element 2 (OSE2), which is found in the regulatory region of all main osteoblast-related genes controlling their expression. Its expression and/or activity are dictated by a number of different external cues while multiple signalling pathways that affect osteoblast function merge to and are integrated by Runx2. Among the various stimuli that modulate Runx2 activity, mechanical loading (strain/stretching) has been revealed to be one of the most critical signals that connect Runx2 with osteoblast function and bone remodelling through mechanotransduction.

CCAAT/enhancer-binding protein δ activates the Runx2-mediated transcription of mouse osteocalcin II promoter

CCAAT/enhancer-binding proteins (C/EBPs) are involved in the regulation of cell proliferation, differentiation, and control of metabolic function. Although the roles of C/EBPs in osteoblasts are largely unknown, both C/EBPbeta and -delta have been shown to enhance rat osteocalcin promoter activity through the synergistic activation of Runx2 at the C/EBP element. Here we show that in the mouse, C/EBPdelta increases the expression of osteocalcin whereas C/EBPbeta does not. This increased expression was found to occur at the transcriptional level, as demonstrated by the increased transcriptional activity from mouse osteocalcin II (OG2) promoter by C/EBPdelta. Although we found three putative C/EBP sites in the -637/+/-34 region of the OG2 promoter, none of these sites showed binding activity with in vitro translated C/EBP proteins. Notably, we show that C/EBPdelta physically interacts with Runx2 and that C/EBPdelta overexpression increases binding between the Runx2-C/EBPdelta complex and the OSE2 element, a critical osteoblast-specific cis-acting element in the OG2 promoter. Consistent with these DNA binding data, a mutation in OSE2 abrogated the stimulatory effect of C/EBPdelta on this promoter activity. Finally, chromatin immunoprecipitation analysis in MC3T3-E1 cells showed in vivo occupancy of the OG2 promoter by Runx2 and C/EBPdelta. In conclusion, C/EBPdelta was found to regulate mouse osteocalcin OG2 promoter activity indirectly by interacting with Runx2 in the context of the OSE2 element and this subsequently resulted in the cooperative activation of the OG2 promoter.

Changes in Runx2/Cbfa1 expression and activity during osteoblastic differentiation of human bone marrow stromal cells.

Runx2/Cbfa1 has been identified as a "master gene" controlling osteoblast differentiation. However, its role in inducing the osteoblast phenotype has been characterized primarily in rodent systems. Thus, we examined Runx2/Cbfa1 messenger RNA, protein, and activity levels during osteoblastic differentiation of human bone marrow stromal (BMSC) cells. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) analysis demonstrated that the expression of alkaline phosphatase and osteocalcin mRNAs increased in a time-dependent manner with the development of the osteoblast phenotype by these cells (hMS2-15). Type II Runx2/Cbfa1 messenger RNA was found to be constitutively expressed in hMS2-15 cells and not altered during differentiation; there was no detectable expression of the type I Runx2/Cbfa1 transcript. Interestingly, despite the absence of any change in Runx2/Cbfa1 messenger RNA levels during osteoblastic differentiation of these cells, the activity of Runx2/Cbfa1, as assessed by binding to the osteoblast-specific cis-acting element 2 (OSE2), increased markedly at all time-points examined, with the highest activity level seen at day 7. Similar results were observed in primary cultures of less differentiated human marrow-derived mesenchymal stem cells. Immunoprecipitation and Western blot analysis revealed that whereas there was no increase in Runx2/Cbfa1 protein levels with differentiation in hMS2-15 cells, there was an increase in Runx2/Cbfa1 phosphorylation. Thus, in contrast to rodent systems where osteoblast differentiation is associated with increased synthesis of Runx2/Cbfa1, we find that in human BMSC, osteoblastic differentiation is associated primarily with increases in Runx2/Cbfa1 activity, without a change in messenger RNA or protein levels. Our findings also show that the increase in Runx2/Cbfa1 activity occurs through a posttranslational mechanism involving phosphorylation of key residues.

Regulation of the activity of the transcription factor Runx2 by two homeobox proteins, Msx2 and Dlx5.

Runx2, formerly called PEBP2alphaA or Cbfa1, is a transcription factor whose deletion causes a complete lack of ossification. It directly regulates the expression of osteoblast-specific genes through the osteoblast-specific cis-acting element found in the promoter region of these genes. In this study, we have found conditions in which induction of the expression of Runx2 is not accompanied by expression of an osteoblast-specific gene, osteocalcin in C2C12 cells. This finding suggests the existence of a repressor of the activity of Runx2. We have then found that the homeobox protein Msx2 is able to repress the transcription activity of Runx2 by interacting with it. Furthermore, our results have shown that the other homeobox protein Dlx5 has an activity which interferes with both abilities of Msx2 to interact with Runx2 and repress its transcription activity. It has previously been shown that a missense mutation of Msx2 (P148H) causes Boston-type craniosynostosis in humans. Interestingly, while this mutant form of Msx2 was able to bind to Runx2 and repress its activity, these abilities of Msx2 (P148H) were not subject to regulation by Dlx5. These results suggest that regulation of the activity of Runx2 by Msx2 and Dlx5 plays an important role in the mammalian skull development.

Cbfa1 Contributes to the Osteoblast-specific Expression of type I collagen Genes

Type I collagen is composed of two chains, alpha1(I) and alpha2(I), encoded by two distinct genes, the alpha1(I) and alpha2(I) collagen genes, that are highly expressed in osteoblasts. In most physiological situations, alpha1(I) and alpha2(I) collagen expression is coregulated, suggesting that identical transcription factors control their expression. Here, we studied the role of Cbfa1, an osteoblast-specific transcription factor, in the control of alpha1(I) and alpha2(I) collagen expression in osteoblasts. A consensus Cbfa1-binding site, termed OSE2, is present at the same location in the alpha1(I) collagen promoter at approximately -1347 base pairs (bp) of the rat, mouse, and human genes. Cbfa1 can bind to this site, as demonstrated by electrophoretic mobility shift assay (EMSA) and supershift experiments using an anti-Cbfa1 antibody. Mutagenesis of the alpha1(I) collagen OSE2 at -1347 bp reduced the activity of a alpha1(I) collagen promoter fragment 2- to 3-fold. Moreover, multimers of this OSE2 at -1347bp confer osteoblast-specific activity to a minimum alpha1(I) collagen promoter fragment in DNA transfection experiments as well as in transgenic mice. An additional Cbfa1-binding element is present in the alpha1(I) collagen promoter of mouse, rat, and human at approximately position -372. This site binds Cbfa1 only weakly and does not act as a cis-acting activator of transcription when tested in DNA transfection experiments. Similar to alpha1(I) collagen, the mouse alpha2(I) collagen gene contains multiple OSE2 sites, of which one is conserved across multiple species. In EMSA, Cbfa1 binds to this site and multimers of this alpha2(I) OSE2 element confer osteoblast-specific activity to the minimum alpha1(I) collagen promoter in DNA transfection experiments. Thus, our results suggest that Cbfa1 is one of the positive regulators of the osteoblast-specific expression of both type I collagen genes.

Cbfa1: a molecular switch in osteoblast biology.

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.

Functional Hierarchy between Two OSE2 Elements in the Control of Osteocalcin Gene Expression in Vivo

Osteocalcin gene expression is initiated perinatally and is restricted to mature osteoblasts and odontoblasts. Because their pattern of expression is highly restricted, the osteocalcin genes are excellent tools to study osteoblast-specific gene expression. To define the mechanisms of osteocalcin cell-specific gene expression in vivo, we generated transgenic mice harboring deletion mutants of the promoter region of OG2, one of the mouse osteocalcin genes. We show here that only 647 base pairs of this promoter are sufficient to confer cell-specific and time-specific expression to a reporter gene in vivo. This promoter fragment contains two copies of OSE2. This osteoblast-specific cis-acting element binds Osf2, a recently characterized osteoblast-specific transcription factor (Ducy, P., Zhang, R., Geoffroy, V., Ridall, A. L., and Karsenty, G. (1997) Cell 89, 747-754). We also demonstrate that the proximal OSE2 element is critical to confer an osteoblast-specific, developmentally regulated pattern of expression to a reporter gene. The other OSE2 element, located more upstream and presenting a lower affinity for Osf2, affects only weakly OG2 promoter activity. These data demonstrate the crucial role of Osf2 in controlling osteocalcin gene expression. Since osteocalcin synthesis is a hallmark of the differentiated osteoblast phenotype, these results suggest that, beyond its developmental function, Osf2 is also required for the maintenance of the osteoblast phenotype postnatally.

Transcriptional regulation of osteoblast differentiation during development.

The osteoblast is the bone-forming cell. The molecular basis of osteoblast-specific gene expression and differentiation begin to be understood. Following the characterization of OSE2, an osteoblast-specific cis-acting element present in the Osteocalcin promoter Osf2/Cbfa1, the protein that binds to OSE2, was identified. Osf2/Cbfa1 is a member of the runt family of transcription factors. Its expression is initiated in the mesenchymal condensations of the developing skeleton and is strictly restricted to cells of the osteoblast lineage thereafter. Osf2/Cbfa1 binds to and regulates the expression of multiple genes expressed in osteoblasts, and forced expression of Osf2/Cbfa1 in nonosteoblastic cells induces the expression of the principal osteoblast-specific genes. Osf2/Cbfa1 gene inactivation in mice leads to failure of mesenchymal progenitor cells to differentiate into osteoblasts. Lastly, heterozygous mutations in the Osf2/Cbfa1 gene cause Cleidocranial dysplasia in human and mice, a condition marked by generalized bone defects. These result demonstrate that Osf2/Cbfa1 is an osteoblast-specific transcriptional activator of osteoblast differentiation whose function is non redundant with the function of other gene products during development.

Osf2/Cbfa1: A Transcriptional Activator of Osteoblast Differentiation

The osteoblast is the bone-forming cell. The molecular basis of osteoblast-specific gene expression and differentiation is unknown. We previously identified an osteoblast-specific cis-acting element, termed OSE2, in the Osteocalcin promoter. We have now cloned the cDNA encoding Osf2/Cbfa1, the protein that binds to OSE2. Osf2/Cbfa1 expression is initiated in the mesenchymal condensations of the developing skeleton, is strictly restricted to cells of the osteoblast lineage thereafter, and is regulated by BMP7 and vitamin D 3. Osf2/Cbfa1 binds to and regulates the expression of multiple genes expressed in osteoblasts. Finally, forced expression of Osf2/Cbfa1 in nonosteoblastic cells induces the expression of the principal osteoblast-specific genes. This study identifies Osf2/Cbfa1 as an osteoblast-specific transcription factor and as a regulator of osteoblast differentiation.

1,25-Dihydroxyvitamin D3 Inhibits Osteocalcin Expression in Mouse through an Indirect Mechanism

1,25-Dihydroxyvitamin D3 (1,25-(OH)2D3), a key regulator of mineral metabolism, regulates the expression of several genes that are expressed in osteoblasts. In particular, in rat and human osteoblasts, 1,25-(OH)2D3 increases the expression of Osteocalcin by interacting, through a hormone-receptor complex, with a vitamin D-responsive element present in the promoter of the genes. Here we show that in mouse, 1,25-(OH)2D3 inhibits the expression of both osteocalcin genes, OG1 and OG2. This inhibition was observed in primary osteoblast cultures and in the whole animal. From sequence inspection, DNA transfection experiments, and DNA binding assays, we could not identify a functional vitamin D-responsive element in the promoter of OG2 or in the first 3.3 kilobases of the OG1 promoter. However, we show that 1,25-(OH)2D3 treatment of primary osteoblasts abolishes the binding of OSF2, an osteoblast-specific activator of transcription that binds to OSE2, a critical osteoblast-specific cis-acting element present in OG1 and OG2 promoters. Consistent with these DNA binding data, a mutation in OSE2 in the OG2 promoter abrogated the inhibitory effect of 1,25-(OH)2D3 treatment on this promoter activity. This study illustrates that 1,25-(OH)2D3 can play different roles in the expression of the same gene in various species and indicates that this regulation in mouse occurs through an indirect mechanism, 1,25-(OH)2D3 acting on a gene genetically located upstream of Osteocalcin.

A PEBP2α/AML-1-related Factor Increases Osteocalcin Promoter Activity through Its Binding to an Osteoblast-specific cis-Acting Element

To identify osteoblast-specific cis-acting elements and trans-acting factors, we initiated an analysis of the promoter of a mouse osteocalcin gene, an osteoblast-specific gene. In this promoter, we identified two osteoblast-specific cis-acting elements (Ducy, P. and Karsenty, G. (1995) Mol. Cell. Biol. 15, 1858-1869). The sequence of one of these elements, OSE2, is identical to the DNA-binding site of the PEBP2 alpha/AML-1 transcription factors, the mammalian homologues of the Drosophila Runt protein. Here we show, using nuclear extracts, recombinant protein, and a specific antiserum against AML-1 proteins in DNA-binding assays, that one member of this family, AML-1B, binds specifically to OSE2 and is immunologically related to OSF2, the factor present in osteoblast nuclear extracts that binds to OSE2. By DNA cotransfection experiments, we also demonstrate that AML-1B can increase the activity of a short osteocalcin promoter through its binding to OSE2. Lastly, the different mobilities of osteoblast nuclear extract-DNA complexes compared with T-cell nuclear extract-DNA complexes, along with the inability of OSF2 to be upregulated by retinoic acid, unlike the other PEBP2 alpha factors, suggest that OSF2 is a new member of this family of transcription factors. Thus, this study demonstrates that AML-1B can increase gene expression of an osteoblast-specific gene through its binding to an osteoblast-specific cis-acting element and presents evidence that OSF2 is a member of the PEBP2 alpha/AML-1 family of transcription factors.