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...

Runx2 is a critical transcription factor for osteoblast differentiation. Regulation of Runx2 expression levels and transcriptional activity is important for bone morphogenetic protein (BMP)-induced osteoblast differentiation. Previous studies have shown that extracellular signal-regulated kinase (Erk) activation enhances the transcriptional activity of Runx2 and that BMP-induced Runx2 acetylation increases Runx2 stability and transcriptional activity. Because BMP signaling induces Erk activation in osteoblasts, we sought to investigate whether BMP-induced Erk signaling regulates Runx2 acetylation and stability. Erk activation by overexpression of constitutively active MEK1 increased Runx2 transcriptional activity, whereas U0126, an inhibitor of MEK1/2, suppressed basal Runx2 transcriptional activity and BMP-induced Runx2 acetylation and stabilization. Overexpression of constitutively active MEK1 stabilized Runx2 protein via up-regulation of acetylation and down-regulation of ubiquitination. Erk activation increased p300 protein levels and histone acetyltransferase activity. Knockdown of p300 using siRNA diminished Erk-induced Runx2 stabilization. Overexpression of Smad5 increased Runx2 acetylation and stabilization. Erk activation further increased Smad-induced Runx2 acetylation and stabilization, whereas U0126 suppressed these functions. On the other hand, knockdown of Smad1 and Smad5 by siRNA suppressed both basal and Erk-induced Runx2 protein levels. Erk activation enhanced the association of Runx2 with p300 and Smad1. Taken together these results indicate that Erk signaling increases Runx2 stability and transcriptional activity, partly via increasing p300 protein levels and histone acetyltransferase activity and subsequently increasing Runx2 acetylation by p300. In addition to the canonical Smad pathway, a BMP-induced non-Smad Erk signaling pathway cooperatively regulates osteoblast differentiation partly via increasing the stability and transcriptional activity of Runx2.The bone morphogenetic proteins (BMPs) 3 are members of the transforming growth factor-␤ superfamily and are primary growth factors that induce formation of both cartilage and bone. Receptors for BMP are serine/threonine kinase receptors and consist of type I (BMPR-I) and II (BMPR-II) receptors. After ligand binding, BMPR-I kinases are activated by BMPR-II kinase-induced phosphorylation. R-Smad proteins are then recruited to activated receptors and play a role in transmitting the BMP signal from the receptor to target genes such as alkaline phosphatase (ALP), bone sialoprotein, osteocalcin (OC), Runx2, and Dlx5 (1, 2). In addition to the Smad pathway, diverse intracellular signaling molecules also participate in BMP-induced osteoblast differentiation. These are collectively called the non-Smad pathway of BMP signaling and include extracellular signal-regulated protein kinase (Erk), p38 mitogen-activated protein kinase, c-Jun N-terminal kinase, phosphatidylinositol 3-kinase, and protein kinases C and D (3-7). These cooperate with and/or reg...

Runx2 is a key transcription factor in osteoblast differentiation, and its activity is regulated by fibroblast growth factors (FGFs). Craniosynostosis, characterized by premature suture closure, results from mutations that generate constitutively active FGF receptors (FGFRs). We previously showed that FGF/FGFR-activated protein kinase C (PKC) is involved in the expression and activity of Runx2. Activated PKCdelta physically interacts with Runx2 in FGF2-stimulated MC3T3-E1 preosteoblastic cells. Immunopurified Runx2 protein reacted with PKCdelta kinase, and a phosphorylated 1460-Da peptide fragment (amino acids 241-252, 1380-Da) derived from Runx2 was also detected in MS analysis. Computer analysis predicted that Ser247 in this Runx2 can be a possible phosphorylation site by PKCdelta. We also showed that Runx2 activity after FGF stimulation correlates with the presence of the Runx2 Ser247 residue. The S247A (Ser --> Ala) mutation confers decreased transcriptional activity on a Runx2-responsive promoter after FGF treatment.

Background: Genetic interaction between Runx2 and Pin1 is critical for embryonic bone formation. Results: Pin1 is a critical modifying enzyme promoting both subnuclear accumulation and protein acetylation of Runx2. Conclusion: Pin1 determines the fate of Runx2 protein in osteoblast differentiation. Significance: The modulation of Pin1 activity may be a clinical target for the regulation of bone formation.

Background:The cell type-specific induction of Bmp2 expression by Wnt3a indicates that Bmp2 is controlled by epigenetic mechanisms. Results: Epigenetic modification activates Bmp2 and Alp expression by Wnt3a in nonosteogenic cells. Conclusion: Epigenetic induction of Bmp2 production by Wnt3a reveals a new mechanistic dimension in morphogen-mediated control of osteogenesis. Significance: Epigenetic modifications/canonical Wnt3a signaling may provide a new method for trans-differentiation of nonosteogenic cells to osteoblasts.