Kyeong-Sook Lee

Runx2 is a common target of transforming growth factor beta1 and bone morphogenetic protein 2, and cooperation between Runx2 and Smad5 induces osteoblast-specific gene expression in the pluripotent mesenchymal precursor cell line C2C12.

ABSTRACT When C2C12 pluripotent mesenchymal precursor cells are treated with transforming growth factor β1 (TGF-β1), terminal differentiation into myotubes is blocked. Treatment with bone morphogenetic protein 2 (BMP-2) not only blocks myogenic differentiation of C2C12 cells but also induces osteoblast differentiation. The molecular mechanisms governing the ability of TGF-β1 and BMP-2 to both induce ligand-specific responses and inhibit myogenic differentiation are not known. We identified Runx2/PEBP2αA/Cbfa1, a global regulator of osteogenesis, as a major TGF-β1-responsive element binding protein induced by TGF-β1 and BMP-2 in C2C12 cells. Consistent with the observation that Runx2 can be induced by either TGF-β1 or BMP-2, the exogenous expression of Runx2 mediated some of the effects of TGF-β1 and BMP-2 but not osteoblast-specific gene expression. Runx2 mimicked common effects of TGF-β1 and BMP-2 by inducing expression of matrix gene products (for example, collagen and fibronectin), suppressing MyoD expression, and inhibiting myotube formation of C2C12 cells. For osteoblast differentiation, an additional effector, BMP-specific Smad protein, was required. Our results indicate that Runx2 is a major target gene shared by TGF-β and BMP signaling pathways and that the coordinated action of Runx2 and BMP-activated Smads leads to the induction of osteoblast-specific gene expression in C2C12 cells.

Bone Morphogenetic Protein-2 Stimulates Runx2 Acetylation

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.

Both the Smad and p38 MAPK pathways play a crucial role in Runx2 expression following induction by transforming growth factor-beta and bone morphogenetic protein.

The Runx family of transcription factors plays pivotal roles during normal development and in neoplasias. In mammals, Runx family genes are composed of Runx1 (Pebp2αB/Cbfa2/Aml1), Runx2 (Pebp2αA/Cbfa1/Aml3) and Runx3 (Pebp2αC/Cbfa3/Aml2). Runx1 and Runx3 are known to be involved in leukemogenesis and gastric carcinogenesis, respectively. Runx2, on the other hand, is a common target of transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) and plays an essential role in osteoblast differentiation. Runx2 is induced by the receptor-activated Smad; Runx2 mediates the blockage of myogenic differentiation and induces osteoblast differentiation in C2C12 pluripotent mesenchymal precursor cells. However, Smad does not directly induce Runx2 expression; an additional step of de novo protein synthesis is required. Here we report that Smad-induced junB functions as an upstream activator of Runx2 expression. Furthermore, not only the Smad pathway but also the mitogen-activated protein kinase (MAPK) cascades are involved in the induction of Runx2 by TGF-β1 and BMP-2. Our results demonstrate that following TGF-β and BMP induction, both the Smad and p38 MAPK pathways converge at the Runx2 gene to control mesenchymal precursor cell differentiation.

Inhibitory Effects of 150 Plant Extracts on Elastase Activity, and Their Anti-inflammatory Effects

The inhibitory effects of 150 medicinal plants on elastase activity were investigated. Among the 150 plants, six plant extracts (final concentration 1 mg/ml in methanol) exhibited more than 65% of inhibition of elastase activity. The inhibitory effects of six active plants, including Areca catechu (IC50, 42.4 μg/ml), Cinnamonum cassia (IC50, 208.7 μg/ml), Myristica fragrans (IC50, 284.1 μg/ml), Curcuma longa (IC50, 398.4 μg/ml), Alpinia katsumadai (IC50, 465.7 μg/ml) and Dryopteris cassirrhizoma (IC50, 714.4 μg/ml) on the activity of human leukocyte elastase, hyaluronidase and lipid peroxidation were examined. In the lipid peroxidation assay, using the TBA method, three of the six plants, including Curcuma longa (IC50, 45.5 μg/ml), Areca catechu (IC50, 51.0 μg/ml) and Alpinia katsumadai (IC50, 116.3 μg/ml) exhibited more than 70% inhibition at the concentration of 1 μg/ml, but only one plant, Areca catechu (IC50, 563 μg/ml) showed high inhibitory effect on hyaluronidase activity. The results suggest that medicinal plants showing several biological activities may be potent inhibitors of the anti‐ageing process in skin. This property might be useful for application in cosmetics.

Runt-Related Transcription Factor RUNX3 Is a Target of MDM2-Mediated Ubiquitination

The p14(ARF)-MDM2-p53 pathway constitutes an effective mechanism for protecting cells from oncogenic stimuli such as activated Ras and Myc. Importantly, Ras activation induces p14(ARF) and often occurs earlier than p53 inactivation during cancer development. Here, we show that RUNX3, a tumor suppressor in various tumors including stomach, bladder, colon, and lung, is stabilized by Ras activation through the p14(ARF)-MDM2 signaling pathway. RUNX3 directly binds MDM2 through its Runt-related DNA-binding domain. MDM2 blocks RUNX3 transcriptional activity by interacting with RUNX3 through an acidic domain adjacent to the p53-binding domain of MDM2 and ubiquitinates RUNX3 on key lysine residues to mediate nuclear export and proteasomal degradation. Our data indicate that the lineage-specific tumor suppressor RUNX3 and the ubiquitous p53 protein are both principal responders of the p14(ARF)-MDM2 cell surveillance pathway that prevents pathologic consequences of abnormal oncogene activation.

Jab1/CSN5 induces the cytoplasmic localization and degradation of RUNX3

Runt‐related (RUNX) transcription factors play pivotal roles in neoplastic development and have tissue‐specific developmental roles in hematopoiesis (RUNX1), osteogenesis (RUNX2), as well as neurogenesis and thymopoiesis (RUNX3). RUNX3 is a tumor suppressor in gastric carcinoma, and its expression is frequently inactivated by DNA methylation or its protein mislocalized in many cancer types, including gastric and breast cancer. Jun‐activation domain‐binding protein 1 (Jab1/CSN5), a component of the COP9 signalosome (CSN), is critical for nuclear export and the degradation of several tumor suppressor proteins, including p53, p27Kip1, and Smad4. Here, we find that Jab1 facilitates nuclear export of RUNX3 that is controlled by CSN‐associated kinases. RUNX3 sequestered in the cytoplasm is rapidly degraded through a proteasome‐mediated pathway. Our results identify a novel mechanism of regulating nuclear export and protein stability of RUNX3 by the CSN complex. J. Cell. Biochem. 107: 557–565, 2009.

Anti-elastase and anti-hyaluronidase of phenolic substance from Areca catechu as a new anti-ageing agent.

Synopsis We have previously screened 150 medicinal plants for the inhibition of elastase and found significant inhibitory effects of the extracts of Areca catechu L. on the ageing and inflammation of skin tissues. To isolate and identify the compounds having biological activity, they were further purified by each fraction of solvents, silica gel column chromatography, preparative TLC and reversed‐phase HPLC. The peak in HPLC, which coincided with the inhibitory activity against elastase, was identified as a phenolic substance by using various colorimetric methods, UV and IR. IC50 values of this phenolic substance were 26.9 μg mL−1 for porcine pancreatic elastase (PPE) and 60.8 μg mL−1 for human neutrophil elastase (HNE). This phenolic substance showed more potent activity than that of reference compounds, oleanolic acid (76.5 μg mL−1 for PPE, 219.2 μg mL−1 for HNE) and ursolic acid (31.0 μg mL−1 for PPE, 118.6 μg mL−1 for HNE). According to the Lineweaver–Burk plots, the inhibition against both PPE and HNE by this phenolic substance was competitive inhibition with the substrate. The phenolic substance from A. catechu effectively inhibited hyaluronidase activity (IC50 : 210 μg mL−1 ).

E1A physically interacts with RUNX3 and inhibits its transactivation activity

The adenoviral gene, termed early region 1A (E1A), is crucial for transformation and has been used very effectively as a tool to determine the molecular mechanisms that underlie the basis of cellular transformation. pRb, p107, p130, p300/CBP, p400, TRRAP, and CtBP were identified to be E1A‐binding proteins and their roles in cellular transformation have been established. Although the major function of E1A is considered to be the regulation of gene expression that is critical for differentiation and cell cycle exit, one of the most significant questions relating to E1A transformation is how E1A mediates this regulation. RUNX3 is a transcription factor that was first described as a gastric cancer tumor suppressor but is now known to be involved in many different cancers. Exogenous expression of RUNX3 strongly inhibits the growth of cells. Here, we show that the adenovirus oncoprotein E1A interacts with RUNX3 in vitro and in vivo. RUNX3 interacts with the N‐terminus (amino acids 2‐29) of E1A, which is known to interact with p300/CBP, p400, and TRRAP. E1A interacts directly with the Runt domain of RUNX3 but does not interfere with CBFβ‐RUNX3 interactions. In addition, E1A inhibits the transactivation activity of RUNX3 on the p21WAF1/CIP1 promoter. Consistent with these observations, the growth inhibition induced by RUNX3 is reduced by E1A. These results demonstrate that E1A specifically binds to RUNX3 and inactivates its transactivation activity. We propose that one of the mechanisms for the oncogenic activity of E1A is the inhibition of RUNX3, similar to that of RB and p300/CBP. J. Cell. Biochem. 105: 236–244, 2008.