Cleavage of Cdc6 by caspase-3 promotes ATM/ATR kinase–mediated apoptosis of HeLa cells

We show that caspase-3 cleaves Cdc6 at D290/S and D442/G sites, producing p32-tCdc6 (truncated Cdc6) and p49-tCdc6, respectively, during etoposide- or tumor necrosis factor (TNF)-α–induced apoptosis. The expression of these tCdc6 proteins, p32- and p49-tCdc6, promotes etoposide-induced apoptosis. The expression of tCdc6 perturbs the loading of Mcm2 but not Orc2 onto chromatin and activates ataxia telangiectasia mutated (ATM) and ATM and Rad-3 related (ATR) kinase activities with kinetics similar to that of the phosphorylation of Chk1/2. The activation kinetics are consistent with elevated cellular levels of p53 and mitochondrial levels of Bax. The tCdc6-induced effects are all suppressed to control levels by expressing a Cdc6 mutant that cannot be cleaved by caspase-3 (Cdc6-UM). Cdc6-UM expression attenuates the TNF-α–induced activation of ATM and caspase-3 activities. When ATM or ATR is down-expressed by using the small interfering RNA technique, the TNF-α– or tCdc6-induced activation of caspase-3 activities is suppressed in the cells. These results suggest that tCdc6 proteins act as dominant-negative inhibitors of replication initiation and that they disrupt chromatin structure and/or induce DNA damage, leading to the activation of ATM/ATR kinase activation and p53–Bax-mediated apoptosis

High Cleavage Efficiency of a 2A Peptide Derived from Porcine Teschovirus-1 in Human Cell Lines, Zebrafish and Mice

When expression of more than one gene is required in cells, bicistronic or multicistronic expression vectors have been used. Among various strategies employed to construct bicistronic or multicistronic vectors, an internal ribosomal entry site (IRES) has been widely used. Due to the large size and difference in expression levels between genes before and after IRES, however, a new strategy was required to replace IRES. A self-cleaving 2A peptide could be a good candidate to replace IRES because of its small size and high cleavage efficiency between genes upstream and downstream of the 2A peptide. Despite the advantages of the 2A peptides, its use is not widespread because (i) there are no publicly available cloning vectors harboring a 2A peptide gene and (ii) comprehensive comparison of cleavage efficiency among various 2A peptides reported to date has not been performed in different contexts. Here, we generated four expression plasmids each harboring different 2A peptides derived from the foot-and-mouth disease virus, equine rhinitis A virus, Thosea asigna virus and porcine teschovirus-1, respectively, and evaluated their cleavage efficiency in three commonly used human cell lines, zebrafish embryos and adult mice. Western blotting and confocal microscopic analyses revealed that among the four 2As, the one derived from porcine teschovirus-1 (P2A) has the highest cleavage efficiency in all the contexts examined. We anticipate that the 2A-harboring cloning vectors we generated and the highest efficiency of the P2A peptide we demonstrated would help biomedical researchers easily adopt the 2A technology when bicistronic or multicistronic expression is required

AP-1-Targeting Anti-Inflammatory Activity of the Methanolic Extract of Persicaria chinensis

In traditional Chinese medicine, Persicaria chinensis L. has been prescribed to cure numerous inflammatory disorders. We previously analyzed the bioactivity of the methanol extract of this plant (Pc-ME) against LPS-induced NO and PGE2 in RAW264.7 macrophages and found that it prevented HCl/EtOH-induced gastric ulcers in mice. The purpose of the current study was to explore the molecular mechanism by which Pc-ME inhibits activator protein- (AP-) 1 activation pathway and mediates its hepatoprotective activity. To investigate the putative therapeutic properties of Pc-ME against AP-1-mediated inflammation and hepatotoxicity, lipopolysaccharide- (LPS-) stimulated RAW264.7 and U937 cells, a monocyte-like human cell line, and an LPS/D-galactosamine- (D-GalN-) induced acute hepatitis mouse model were employed. The expression of LPS-induced proinflammatory cytokines including interleukin- (IL-) 1β, IL-6, and tumor necrosis factor-α (TNF-α) was significantly diminished by Pc-ME. Moreover, Pc-ME reduced AP-1 activation and mitogen-activated protein kinase (MAPK) phosphorylation in both LPS-stimulated RAW264.7 cells and differentiated U937 cells. Additionally, we highlighted the hepatoprotective and curative effects of Pc-ME pretreated orally in a mouse model of LPS/D-GalN-intoxicated acute liver injury by demonstrating the significant reduction in elevated serum AST and ALT levels and histological damage. Therefore, these results strongly suggest that Pc-ME could function as an antihepatitis remedy suppressing MAPK/AP-1-mediated inflammatory events

Genetic Susceptibility on CagA-Interacting Molecules and Gene-Environment Interaction with Phytoestrogens: A Putative Risk Factor for Gastric Cancer

OBJECTIVES: To evaluate whether genes that encode CagA-interacting molecules (SRC, PTPN11, CRK, CRKL, CSK, c-MET and GRB2) are associated with gastric cancer risk and whether an interaction between these genes and phytoestrogens modify gastric cancer risk. METHODS: In the discovery phase, 137 candidate SNPs in seven genes were analyzed in 76 incident gastric cancer cases and 322 matched controls from the Korean Multi-Center Cancer Cohort. Five significant SNPs in three genes (SRC, c-MET and CRK) were re-evaluated in 386 cases and 348 controls in the extension phase. Odds ratios (ORs) for gastric cancer risk were estimated adjusted for age, smoking, H. pylori seropositivity and CagA strain positivity. Summarized ORs in the total study population (462 cases and 670 controls) were presented using pooled- and meta-analysis. Plasma concentrations of phytoestrogens (genistein, daidzein, equol and enterolactone) were measured using the time-resolved fluoroimmunoassay. RESULTS: SRC rs6122566, rs6124914, c-MET rs41739, and CRK rs7208768 showed significant genetic effects for gastric cancer in both the pooled and meta-analysis without heterogeneity (pooled OR = 3.96 [95% CI 2.05-7.65], 1.24 [95% CI = 1.01-1.53], 1.19 [95% CI = 1.01-1.41], and 1.37 [95% CI = 1.15-1.62], respectively; meta OR = 4.59 [95% CI 2.74-7.70], 1.36 [95% CI = 1.09-1.70], 1.20 [95% CI = 1.00-1.44], and 1.32 [95% CI = 1.10-1.57], respectively). Risk allele of CRK rs7208768 had a significantly increased risk for gastric cancer at low phytoestrogen levels (p interaction<0.05). CONCLUSIONS: Our findings suggest that SRC, c-MET and CRK play a key role in gastric carcinogenesis by modulating CagA signal transductions and interaction between CRK gene and phytoestrogens modify gastric cancer risk

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

Metallothionein 3 Promotes Osteoblast Differentiation in C2C12 Cells via Reduction of Oxidative Stress

Metallothioneins (MTs) are intracellular cysteine-rich proteins, and their expressions are enhanced under stress conditions. MTs are recognized as having the ability to regulate redox balance in living organisms; however, their role in regulating osteoblast differentiation is still unclear. In this research, we found that the expression of MT3, one member of the MT protein family, was specifically upregulated in the differentiation process of C2C12 myoblasts treated with bone morphogenetic protein 4 (BMP4). Transfection with MT3-overexpressing plasmids in C2C12 cells enhanced their differentiation to osteoblasts, together with upregulating the protein expression of bone specific transcription factors runt-related gene 2 (Runx2), Osterix, and distal-less homeobox 5 (Dlx5). Additionally, MT3 knockdown performed the opposite. Further studies revealed that overexpression of MT3 decreased reactive oxygen species (ROS) production in C2C12 cells treated with BMP4, and MT3 silencing enhanced ROS production. Treating C2C12 cells with antioxidant N-acetylcysteine also promoted osteoblast differentiation, and upregulated Runx2/Osterix/Dlx5, while ROS generator antimycin A treatment performed the opposite. Finally, antimycin A treatment inhibited osteoblast differentiation and Runx2/Osterix/Dlx5 expression in MT3-overexpressing C2C12 cells. These findings identify the role of MT3 in osteoblast differentiation and indicate that MT3 may have interesting potential in the field of osteogenesis research