Runx2/Cbfa1/Pebp2aA is a global regulator of osteogenesis and is crucial for regulating the expression of bone-specific genes. Runx2 is a major target of the bone morphogenetic protein (BMP) pathway. Genetic analysis has revealed that Runx2 is degraded through a Smurf-mediated ubiquitination pathway, and its activity is inhibited by HDAC4. Here, we demonstrate the molecular link between Smurf, HDACs and Runx2, in BMP signaling. BMP-2 signaling stimulates p300-mediated Runx2 acetylation, increasing transactivation activity and inhibiting Smurf1-mediated degradation of Runx2. HDAC4 and HDAC5 dea-cetylate Runx2, allowing the protein to undergo Smurf-mediated degradation. Inhibition of HDAC increases Runx2 acetylation, and potentiates BMP-2-stimulated osteoblast differentiation and increases bone formation. These results demonstrate that the level of Runx2 is controlled by a dynamic equilibrium of acetylation, deacetylation, and ubiquitination. These findings have important medical implications because BMPs and Runx2 are of tremendous interest with regard to the development of therapeutic agents against bone diseases.

RUNX3 has been suggested to be a tumor suppressor of gastric cancer. The gastric mucosa of the Runx3-null mouse develops hyperplasia due to enhanced proliferation and suppressed apoptosis accompanied by a decreased sensitivity to transforming growth factor ␤1 (TGF-␤1). It is known that TGF-␤1 induces cell growth arrest by activating CDKN1A (p21 WAF1/Cip1 ), which encodes a cyclin-dependent kinase inhibitor, and this signaling cascade is considered to be a tumor suppressor pathway. However, the lineage-specific transcription factor that cooperates with SMADs to induce p21 expression is not known. Here we show that RUNX3 is required for the TGF-␤-dependent induction of p21 expression in stomach epithelial cells. Overexpression of RUNX3 potentiates TGF-␤-dependent endogenous p21 induction. In cooperation with SMADs, RUNX3 synergistically activates the p21 promoter. In contrast, RUNX3-R122C, a mutation identified in a gastric cancer patient, abolished the ability to activate the p21 promoter or cooperate with SMADs. Furthermore, areas in mouse and human gastric epithelium where RUNX3 is expressed coincided with those where p21 is expressed. Our results suggest that at least part of the tumor suppressor activity of RUNX3 is associated with its ability to induce p21 expression.The RUNX family of transcription factors plays pivotal roles in normal development and neoplasias. In mammals, the RUNX family genes consist of RUNX1/AML1, RUNX2, and RUNX3 (see reference 37 for the nomenclature). All RUNX family members share the central Runt domain, which is well conserved and recognizes a specific DNA sequence, but each has relatively divergent N-and C-terminal regions. RUNX1 and RUNX2 are required for hematopoiesis and osteogenesis, respectively, and are genetically altered in leukemia and bone disease (15,20,26,27,29). In contrast, RUNX3 is involved in neurogenesis (10, 17) and thymopoiesis (36, 41) and functions as a tumor suppressor of gastric cancer (7,18). About 60% of primary gastric cancer specimens do not express RUNX3 due to a combination of hemizygous deletion and DNA hypermethylation of the RUNX3 promoter region (7,13,18,28).

NFATc1 (nuclear factor of activated T-cells c1), a key transcription factor, plays a role in regulating expression of osteoclast-specific downstream target genes such as TRAP (tartrate-resistant acid phosphatase) and OSCAR (osteoclast-associated receptor). It has been shown that RANKL [receptor activator of NF-κB (nuclear factor κB) ligand] induces NFATc1 expression during osteoclastogenesis at a transcriptional level. In the present study, we demonstrate that RANKL increases NFATc1 protein levels by post-translational modification. RANKL stimulates NFATc1 acetylation via HATs (histone acetyltransferases), such as p300 and PCAF [p300/CREB (cAMP-response-element-binding protein)-binding protein-associated factor], thereby stabilizing NFATc1 proteins. PCAF physically interacts with NFATc1 and directly induces NFATc1 acetylation and stability, subsequently increasing the transcriptional activity of NFATc1. In addition, RANKL-mediated NFATc1 acetylation is increased by the HDAC (histone deacetylase) inhibitors sodium butyrate and scriptaid. Overexpression of HDAC5 reduces RANKL- or PCAF-mediated NFATc1 acetylation, stability and transactivation activity, suggesting that the balance between HAT and HDAC activities might play a role in the regulation of NFATc1 levels. Furthermore, RANKL and p300 induce PCAF acetylation and stability, thereby enhancing the transcriptional activity of NFATc1. Down-regulation of PCAF by siRNA (small interfering RNA) decreases NFATc1 acetylation and stability, as well as RANKL-induced osteoclastogenesis. Taken together, the results of the present study demonstrate that RANKL induces HAT-mediated NFATc1 acetylation and stability, and subsequently increases the transcriptional activity of NFATc1 during osteoclast differentiation.

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*IL-3 plays important roles in the growth and survival of hematopoietic progenitor cells, processes modeled in studies of the IL-3-dependent cell line Ba/F3. To gain insights into molecular mechanisms governing cell fate, we examined the patterns of proteins up-regulated following stimulation of Ba/F3 cells with IL-3. Through two-dimensional electrophoresis and proteomicsbased approaches, we identified 11 proteins. Of these, expression of 14-3-3␥ was significantly increased by IL-3 stimulation at both the transcriptional and translational levels. 14-3-3␥ overexpression in Ba/F3 cells abrogated dependence on IL-3 and was associated with activation of PI3K and MAPK signaling cascades, suggesting that the functions of 14-3-3␥ in normal hematopoietic progenitors are to promote survival and growth through the activation of distinct signaling pathways. Additionally, the upregulation of Bax and Bad was seen with the ablation of 14-3-3␥, resulting in cell death. These results indicate that deregulated expression of 14-3-3␥ may contribute to malignant transformation, possibly providing a new target for therapeutic intervention in hematopoietic neoplasms.