The AML/CBFA family of runt homology domain (rhd) transcription factors regulates expression of mammalian genes of the hematopoietic lineage. AML1, AML2 and AML3 are the three AML genes identified to date which influence myeloid cell growth and differentiation. Recently AML-related proteins were identified in an osteoblast-specific promoter binding complex that functionally modulates bone-restricted transcription of the osteocalcin gene. In the present study we demonstrate that in primary rat osteoblasts AML-3 is the AML family member present in the osteoblast-specific complex. Antibody specific for AML-3 completely supershifts this complex, in contrast to antibodies with specificity for AML-1 or AML-2, AML-3 is present as a single 5.4 kb transcript in bone tissues. To establish the functional involvement of AML factors in osteoblast differentiation, we pursued antisense strategies to alter expression of rhd genes. Treatment of osteoblast cultures with rhd antisense oligonucleotides significantly decreased three parameters which are linked to differentiation of normal diploid osteoblasts: the representation of alkaline phosphatase-positive cells, osteocalcin production, and the formation of mineralized nodules. Our findings indicate that AML-3 is a key transcription factor in bone cells and that the activity of rhd proteins is required for completion of osteoblast differentiation.

Two major isoforms of theHere, Runx2-II expression was found to be specifically stimulated by BMP-2 treatment or by Dlx5 overexpression. In addition, BMP-2, Dlx5, and Runx2-II were found to be expressed in osteogenic fronts and parietal bones of the developing cranial vault and Runx2-I and Msx2 in the sutural mesenchyme. Furthermore, Runx2 P1 promoter activity was strongly stimulated by Dlx5 overexpression, whereas Runx2 P2 promoter activity was not. Runx2 P1 promoter deletion analysis indicated that the Dlx5-specific response is due to sequences between ؊756 and ؊342 bp of the P1 promoter, where three Dlx5-response elements are located. Dlx5 responsiveness to these elements was confirmed by gel mobility shift assay and site-directed mutagenesis. Moreover, Msx2 specifically suppressed the Runx2 P1 promoter, and the responsible region overlaps with that recognized by Dlx5. In summary, Dlx5 specifically transactivates the Runx2 P1 promoter, and its action on the P1 promoter is antagonized by Msx2.The Runt-related transcription factor Runx2 plays an essential role in osteoblast differentiation and bone mineralization (1, 2). Two major isoforms are expressed from the mouse Runx2 locus, and these isoforms are generated by different promoter usage. Runx2 type I (Runx2-I), 2 referred to as the Cbfa1/p56 isoform or PEBP2␣A, is a 513-amino acid protein that starts with the amino acid sequence MRIPV (3) and is derived from the proximal P2 promoter of the gene (4). More recently, upstream exons of the Runx2 gene that potentially encode the N termini of Runx2 isoforms expressed in osteoblasts have been identified (5, 6). These upstream exons contain a 5Ј-untranslated region and encode the N-terminal 19 amino acids of Runx2 type II (Runx2-II; also referred to as Cbfa1/p57 and OSF2), which starts with the sequence MASNSL (7). This isoform is expressed from the P1 or "bone-related" upstream promoter (8), and its expression is predominant in osteoblasts (9). The alternative promoter usage strongly implies that the expression pattern of each isoform differs temporally and/or spatially. Indeed, they exhibit distinct expression patterns during bone development (10, 11). Thus, it is natural to assume that these two promoters differently respond to different extracellular signals or their downstream transcription factors because these promoters have distinct transcription factor-binding sites.Runx2 plays a central role in the BMP-2-induced trans-differentiation of C2C12 cells at an early restriction point by diverting them from the myogenic pathway to the osteogenic pathway (12, 13). We found that the homeobox gene Dlx5 is an upstream target of BMP-2 signaling and that it plays a pivotal role in stimulating the downstream osteogenic master transcription factor Runx2. In turn, Runx2 acts simultaneously or sequentially to induce the expression of bone-specific genes that represent BMP-2-induced osteogenic trans-differentiation. In addition, it has also been suggested that Dlx5 is a critical target of the inhibitory action of transform...

The runt related transcription factor CBFA1 (AML3/PEBP2alphaA/RUNX2) regulates expression of several bone- and cartilage-related genes and is required for bone formation in vivo. The gene regulatory mechanisms that control activation and repression of CBFA1 gene transcription during osteoblast differentiation and skeletal development are essential for proper execution of the osteogenic program. We have therefore defined functional contributions of 5' regulatory sequences conserved in rat, mouse and human CBFA1 genes to transcription. Deletion analysis reveals that 0.6 kB of the bone-related rat or mouse CBFA1 promoter (P1, MASNS protein isoform) is sufficient to confer transcriptional activation, and that there are multiple promoter domains which positively and negatively regulate transcription. Progressive deletion of promoter segments between nt -351 and -92 causes a striking 30- to 100-fold combined decrease in promoter activity. Additionally, 5' UTR sequences repress reporter gene transcription 2- to 3-fold. Our data demonstrate that CBFA1 is a principal DNA binding protein interacting with the 5' region of the CBFA1 gene in osseous cells, that there are at least three CBFA1 recognition motifs in the rat CBFA1 promoter, and that there are three tandemly repeated CBFA1 sites within the 5' UTR. We find that forced expression of CBFA1 protein downregulates CBFA1 promoter activity and that a single CBFA1 site is sufficient for transcriptional autosuppression. Thus, our data indicate that the CBFA1 gene is autoregulated in part by negative feedback on its own promoter to stringently control CBFA1 gene expression and function during bone formation.

Bone formation involves several tightly regulated gene expression patterns of bone-related proteins. To determine the expression patterns of bone-related proteins during the MC3T3-E1 osteoblast-like cell differentiation, we used Northern blotting, enzymatic assay, and histochemistry. We found that the expression patterns of bone-related proteins were regulated in a temporal manner during the successive developmental stages including proliferation (days 4-10), bone matrix formation/maturation (days 10-16), and mineralization stages (days 16-30). During the proliferation period (days 4-10), the expression of cell-cycle related genes such as histone H3 and H4, and ribosomal protein S6 was high. During the bone matrix formation/maturation period (days 10-16), type I collagen expression and biosynthesis, fibronectin, TGF-beta 1 and osteonectin expressions were high and maximal around day 16. During this maturation period, we found that the expression patterns of bone matrix proteins were two types: one is the expression pattern of type I collagen and TGF-beta 1, which was higher in the maturation period than that in both the proliferation and mineralization periods. The other is the expression pattern of fibronectin and osteonectin, which was higher in the maturation and mineralization periods than in the proliferation period. Alkaline phosphatase activity was high during the early matrix formation/maturation period (day 10) and was followed by a decrease to a level still significantly above the baseline level seen at day 4. During the mineralization period (days 16-30), the number of nodules and the expression of osteocalcin were high. Osteocalcin gene expression was increased up to 28 days. Our results show that the expression patterns of bone-related proteins are temporally regulated during the MC3T3-E1 cell differentiation and their regulations are unique compared with other systems. Thus, this cell line provides a useful in vitro system to study the developmental regulation of bone-related proteins in relation to the different stages during the osteoblast differentiation.

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