Zee-Won Lee

Hydrogen Peroxide Activates p70S6k Signaling Pathway

We investigated a possible role of reactive oxygen species (ROS) in p70S6k activation, which plays an important role in the progression of cells from G0/G1 to S phase of the cell cycle by translational up-regulation of a family of mRNA transcripts that encode for components of the protein synthetic machinery. Treatment of mouse epidermal cell JB6 with H2O2 generated extracellularly by glucose/glucose oxidase led to the activation of p70S6k and p90Rsk and to phosphorylation of p42MAPK/p44MAPK. The activation of p70S6k and p90Rsk was dose-dependent and transient, maximal activities being in extracts treated for 15 and 30 min, respectively. Further characterization of ROS-induced activation of p70S6kusing specific inhibitors for p70S6k signaling pathway, rapamycin, and wortmannin revealed that ROS acted upstream of the rapamycin-sensitive component FRAP/RAFT and wortmannin-sensitive component phosphatidylinositol 3-kinase, because both inhibitors caused the inhibition of ROS-induced p70S6k activity. In addition, Ca2+ chelation also inhibited ROS-induced activation of p70S6k, indicating that Ca2+ is a mediator of p70S6k activation by ROS. However, down-regulation of 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive protein kinase C (PKC) by chronic pretreatment with TPA or a specific PKC inhibitor Ro-31-8220 did not block the activation of p70S6kby ROS, indicating that the activation of TPA-responsive PKC was not required for stimulation of p70S6k activity by H2O2 in JB6 cells. Exposure of JB6 cells to platelet-derived growth factor or epidermal growth factor led to a rapid increase in H2O2, phosphorylation, and activation of p70S6k, which were antagonized by the pretreatment of catalase. Taken together, the results suggest that ROS act as a messenger in growth factor-induced p70S6k signaling pathway.

Activation of in situ tissue transglutaminase by intracellular reactive oxygen species.

We have investigated the novel function of intracellular reactive oxygen species (ROS) in the activation of in situ tissue transglutaminase (tTGase) by lysophosphatidic acid (LPA) and transforming growth factor-beta (TGF-beta) in Swiss 3T3 fibroblasts. LPA induced a transient increase of intracellular ROS with a maximal increase at 10 min, which was blocked by ROS scavengers, N-acetyl-L-cysteine and catalase. LPA activated tTGase with a maximal increase at 1h, which was inhibited by cystamine and ROS scavengers. Incubation with exogenous H(2)O(2) activated tTGase. TGF-beta also activated tTGase with a maximal activation at 2h and the tTGase activation was inhibited by the ROS scavengers. Scrape-loading of C3 transferase inhibited the ROS production and in situ tTGase activation by LPA and TGF-beta, and the inhibitory effect of C3 transferase was reversed by exogenous H(2)O(2). Microinjection of GTPgammaS inhibited transamidating activity of tTGase stimulated by LPA, TGF-beta, and maitotoxin. These results suggested that intracellular ROS was essential for the activation of in situ tTGase in response to LPA and TGF-beta.

Differential expression of cell surface proteins in human bone marrow mesenchymal stem cells cultured with or without basic fibroblast growth factor containing medium

Mesenchymal stem cells (MSCs) are multipotent cells, which have the capability to differentiate into various mesenchymal tissues such as bone, cartilage, fat, tendon, muscle, and marrow stroma. However, they lose the capability of multi‐lineage differentiation after several passages. It is known that basic fibroblast growth factor (bFGF) increases growth rate, differentiation potential, and morphological changes of MSCs in vitro. In this report, we have used 2‐DE coupled to MS to identify differentially expressed proteins at the cell membrane level in MSCs growing in bFGF containing medium. The cell surface proteins isolated by the biotin–avidin affinity column were separated by 2‐DE in triplicate experiments. A total of 15 differentially expressed proteins were identified by quadrupole‐time of flight tandem MS. Nine of the proteins were upregulated and six proteins were downregulated in the MSCs cultured with bFGF containing medium. The expression level of three actin‐related proteins, F‐actin‐capping protein subunit alpha‐1, actin‐related protein 2/3 complex subunit 2, and myosin regulatory light chain 2, was confirmed by Western blot analysis. The results indicate that the expression levels of F‐actin‐capping protein subunit alpha‐1, actin‐related protein 2/3 complex subunit 2, and myosin regulatory light chain 2 are important in bFGF‐induced morphological change of MSCs.

Real-Time Measurement of Intracellular Reactive Oxygen Species Using Mito Tracker Orange (CMH2TMRos)

We have investigated a novel method to monitor real changes of intracellular ROS by the use of CMH2TMRos (a reduced form of MitoTracker orange) in Swiss 3T3 fibroblasts. Arachidonic acid induced a rapid increase of CMTMRos fluorescence with a maximal elevation at 120–150 sec, which was determined by scanning every 10 sec with a confocal microscope. The fluorescence increase by arachidonic acid was completely inhibited by 2-MPG but not by catalase, indicating a major contribution of superoxide to the oxidation of CMH2TMRos. Incubation with glucose oxidase, exogenous H2O2, KO2 and lysophosphatidic acid also increased the CMTMRos fluorescence, which was blocked by 2-MPG. These results suggested that CMH2TMRos is a useful fluorophore for real-time monitoring of intracellular ROS and also indicated that CMH2TMRos detects primarily superoxide in cells even though the fluorophore can be oxidized by both superoxide and H2O2.

Roles of RhoA and Phospholipase A2 in the Elevation of Intracellular H2O2 by Transforming Growth Factor-β in Swiss 3T3 Fibroblasts

We have investigated the mechanisms by which transforming growth factor-beta (TGF-beta) increased intracellular H2O2 in Swiss 3T3 fibroblasts. Increase of intracellular H2O2 by TGF-beta was maximal at 30 min and blocked by catalase from Aspergillus niger. Scrape-loading of C3 transferase, which down-regulated RhoA, inhibited the production of H2O2 in response to TGF-beta. TGF-beta stimulated release of arachidonic acid, which was completely inhibited by mepacrine, a phospholipase A2 inhibitor. Mepacrine also blocked the increase of H2O2 by TGF-beta. In addition, arachidonic acid increased intracellular H2O2. Furthermore, TGF-beta stimulated stress fibre formation, which was blocked by catalase, without membrane ruffling. Catalase also inhibited stimulation of thymidine incorporation by TGF-beta. These results suggested that TGF-beta increased intracellular H2O2 through RhoA and phospholipase A2, and also suggested that intracellular H2O2 was required for the stimulation of stress fibre formation and DNA synthesis in response to TGF-beta.

Proteomic and bioinformatic analysis of membrane proteome in type 2 diabetic mouse liver

Type 2 diabetes mellitus (T2DM) is the most prevalent and serious metabolic disease affecting people worldwide. T2DM results from insulin resistance of the liver, muscle, and adipose tissue. In this study, we used proteomic and bioinformatic methodologies to identify novel hepatic membrane proteins that are related to the development of hepatic insulin resistance, steatosis, and T2DM. Using FT‐ICR MS, we identified 95 significantly differentially expressed proteins in the membrane fraction of normal and T2DM db/db mouse liver. These proteins are primarily involved in energy metabolism pathways, molecular transport, and cellular signaling, and many of them have not previously been reported in diabetic studies. Bioinformatic analysis revealed that 16 proteins may be related to the regulation of insulin signaling in the liver. In addition, six proteins are associated with energy stress‐induced, nine proteins with inflammatory stress‐induced, and 14 proteins with endoplasmic reticulum stress‐induced hepatic insulin resistance. Moreover, we identified 19 proteins that may regulate hepatic insulin resistance in a c‐Jun amino‐terminal kinase‐dependent manner. In addition, three proteins, 14–3‐3 protein beta (YWHAB), Slc2a4 (GLUT4), and Dlg4 (PSD‐95), are discovered by comprehensive bioinformatic analysis, which have correlations with several proteins identified by proteomics approach. The newly identified proteins in T2DM should provide additional insight into the development and pathophysiology of hepatic steatosis and insulin resistance, and they may serve as useful diagnostic markers and/or therapeutic targets for these diseases.

Pharmacokinetics of Glutathione and Its Metabolites in Normal Subjects

To determine the loading and maintenance dosage of glutathione (GSH) for patients suffering from reactive oxygen species (ROS) injury such as acute paraquat intoxication, a kinetic study of reduced GSH was performed in synchrony with that of cysteine (Cys), cystine (Cys2), and methionine (Met). Human subjects porticipitation was voluntary. The effective dose of Cys, Cys2, and Met against ROS in fibroblast cells generated by paraquat was assessed using laser scanning confocal microscopy. Both Cys and Met suppressed ROS in a dose-dependent manner at concentrations of 1-1,000 µM; the concentration required to suppress ROS by 50% was 10 µM for Cys and 50 µM for Met. Using metabolite kinetics with the assumption that Cys and Met are the metabolites of GSH, expected concentrations of Cys and Met of above 20 and 50 µM were estimated when GSH was administered at 50 mg/kg body weights every 205.4 min for Cys and 427.4 min for Met.

Direct Monitoring of the Inhibition of Protein–Protein Interactions in Cells by Translocation of PKCδ Fusion Proteins†

In the field of drug discovery and development, the increasing use of cell-based assays has resulted in an increased demand for novel cellular bioassays. Such bioassays are expected to detect a wide variety of functional molecules in live cells. Fluorescence-based imaging techniques such as fluorescence resonance energy transfer (FRET) and biomolecular fluorescence complementation (BiFC) have been developed to analyze protein–protein interactions (PPIs) and inhibition of PPIs (iPPIs) in live mammalian cells. Although these techniques have been useful, they require a variety of fusion constructs to determine the relative locations of fluorophores and binding pairs for optimal performance as well as appropriate linker domains. Alternatively, translocation-based cellular assays (redistribution approaches), which are cell-based assay techniques utilizing protein translocation as the primary readout, have been used to study the PPIs between specific proteins and other intracellular events. These methods use a bait (target) molecule fused to a protein that changes its localization within the cell following a stimulus. Such assays can be formatted as agonist or antagonist assays, in which compounds are tested for their ability to promote or inhibit, respectively, protein translocation caused by a known agonist. Translocation-based cellular assays do not require much construct optimization and boast a high signal-to-noise ratio. These assays are robust, fast, and flexible; thus, these systems have been considered as an ideal assay for high-contentscreening approaches to drug discovery. Despite these advantages, few experimental applications of translocationbased cellular assays have been reported. Most of these have been based on regulated transport between the cell nucleus and the cytoplasm using a combination of nuclear localization signals and/or nuclear export signals. Several technologies are already commercially available. Recently, the groups of Schultz and Heo independently reported that PPIs can be visualized by cotranslocation of a target protein from the cytoplasm to the plasma membrane and to the endosome, respectively. Schultz et al. demonstrated the direct cotranslocation of a protein complex through the Ca-induced translocation of a bait protein fused to Annexin A4, a phospholipidand Ca-binding protein. Heo and colleagues showed that Rab5, an endosome-localized protein, recruited an interacting protein to the endosome through an FKBP–rapamycin–FRB complex intermediate. These studies were focused on the visual detection of PPIs so that new conceptual and novel applications of redistribution approaches have vastly expanded what can be explored in live cells. Herein we demonstrate that the inhibition of protein– protein interactions (iPPI) using a small molecular inhibitor can be monitored directly by a redistribution approach. Protein kinase C (PKC) is known to translocate from the cytoplasm to the plasma membrane in response to physiological stimuli, as well as exogenous ligands such as phorbol esters. In a study using PKC tagged with green fluorescent protein (GFP) the dynamics of PKC translocation in response to different stimuli was monitored in real time in live cells. PKCd has a C1 domain that binds diacylglycerol, but an impaired C2 domain that does not bind Ca ions. Thus, PKCd responds to an increase in phorbol esters in the cell but not Ca ions. Therefore we hypothesized that a PKCd-fused bait protein would guide cotranslocation with the target protein, and a chemical inhibitor would interrupts PPI, making it possible to monitor iPPI (Scheme 1). To verify our approach, we examined iPPI using the p53 (tumor suppressor)/MDM2 (negative regulator of the p53) protein pair and Nutlin-3 (see the Supporting Information for experimental details). The small molecular inhibitor Nutlin-3 is a cis-imidazoline analogue commonly used in anticancer studies that inhibits the interaction between p53 and MDM2; this inhibitor resulted from the optimization of a lead structure identified by the screening of a chemical library. We prepared the C-terminal fusion constructs PKCd/monomeric red fluorescent protein (mRFP)/p53 (bait) and enhanced GFP (eGFP)/MDM2 (target). Both the pmRFP plasmid encoding PKCd–mRFP–p53 and the peGFP plasmid encoding eGFP–MDM2 were transiently cotransfected into HEK-293T cells. When the exogenous ligand phorbol 12-myristate 13-acetate (PMA) was added, both p53 [*] Dr. K.-B. Lee, J. M. Hwang, Dr. J.-S. Choi, Dr. G.-H. Kim, Dr. S. I. Kim, Dr. S. Kim, Dr. Z.-W. Lee Division of Life Science, Korea Basic Science Institute (KBSI) Daejeon 305-333 (Korea) Fax: (+82)42-865-3419 E-mail: shkim@kbsi.re.kr ezone@kbsi.re.kr J. M. Hwang, Prof. Dr. J. Rho Department of Bioscience and Biotechnology Chungnam National University, Daejeon 305-764 (Korea)

Anti-proliferative effects of ginsenosides extracted from mountain ginseng on lung cancer

ObjectiveTo investigate the effect of three major ginsenosides from mountain ginseng as anticancer substance and explore the underlying mechanism involved in lung cancer.MethodsThe inhibitory proliferation of lung cancer by major five ginsenosides (Rb1, Rb2, Rg1, Rc, and Re) was examined using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide assay. Calculated 50% inhibition (IC50) values of five ginsenosides were determined and compared each other. Apoptosis by the treatment of single ginsenoside was performed by fluorescence-assisted cytometric spectroscopy. The alterations of apoptosis-related proteins were evaluated by Western blot analysis.ResultsThe abundance of ginsenosides in butanol extract of mountain ginseng (BX-MG) was revealed in the order of Rb1, Rg1, Re, Rc and Rb2. Among them, Rb1 was the most effective to lung cancer cell, followed by Rb2 and Rg1 on the basis of relative IC50 values of IMR90 versus A549 cell. The alterations of apoptotic proteins were confirmed in lung cancer A549 cells according to the administration of Rb1, Rb2 and Rg1. The expression levels of caspase-3 and caspase-8 were increased upon the treatment of three ginsenosides, however, the levels of caspase-9 and anti-apoptotic protein Bax were not changed.ConclusionMajor ginsenosides such as Rb1, Rb2 and Rg1 comprising BX-MG induced apoptosis in lung cancer cells via extrinsic apoptotic pathway rather than intrinsic mitochondrial pathway.

Regulation of reactive oxygen species and stress fiber formation by calpeptin in Swiss 3T3 fibroblasts

We have investigated a novel function of calpeptin, a commonly used inhibitor of calpain, in the production of intracellular reactive oxygen species (ROS) in Swiss 3T3 fibroblasts. Calpeptin induced a rapid increase of intracellular ROS by a dose-dependent manner, with a maximal increase at 10 min, which was inhibited by ROS scavengers, catalase and 2-MPG. However, other calpain inhibitors, E64d and N-acetyl-Leu-Leu-Nle-CHO (ALLN), had no effect on the level of intracellular ROS, indicating that calpain was not involved in the ROS production by calpeptin. The role of Rho in the ROS production by calpain was studied by scrape-loading of C3 transferase. C3 transferase, which inhibited stress fiber formation by calpeptin, had no effect on the ROS production in response to calpeptin, suggesting that Rho was not involved in the ROS production by calpeptin. But the elevation of intracellular ROS was inhibited by mepacrine, a phospholipase A2 inhibitor. In addition, scavenging intracellular ROS by the incubation with catalase and 2-MPG had no effect on the stress fiber formation by calpeptin. These results suggested that calpeptin stimulated the production of intracellular ROS and stress fiber formation by independent mechanisms.