Chuanshu Huang

Vanadate Induces p53 Transactivation through Hydrogen Peroxide and Causes Apoptosis

Vanadium is a metal widely distributed in the environment. Although vanadate-containing compounds exert potent toxic effects on a wide variety of biological systems, the mechanisms controlling vanadate-induced adverse effects remain to be elucidated. The present study investigated the vanadate-induced p53 activation and involvement of reactive oxygen species (ROS) in p53 activation as well as the role of p53 in apoptosis induction by vanadate. Exposure of mouse epidermal JB6 cells to vanadate led to transactivation of p53 activity in a time- and dose-dependent manner. It also caused mitochondrial damage, apoptosis, and generated ROS. Scavenging of vanadate-induced H2O2 byN-acetyl-l-cysteine (a general antioxidant) or catalase (a specific H2O2 inhibitor), or the chelation of vanadate by deferoxamine, resulted in inhibition of p53 activation and cell mitochondrial damage. In contract, an increase in H2O2 generation in response to superoxide dismutase or NADPH enhanced these effects caused by vanadate. Furthermore, vanadate-induced apoptosis occurred in cells expressing wild-type p53 (p53+/+) but was very weak in p53-deficient (p53−/−) cells. These results demonstrate that vanadate induces p53 activation mainly through H2O2 generation, and this activation is required for vanadate-induced apoptosis.

Phosphatidylinositol 3-kinase/Akt positively regulates Fas (CD95)-mediated apoptosis in epidermal Cl41 cells.

Fas (CD95)-mediated apoptosis is an essential mechanism for the maintenance of homeostasis, and disruption of this death pathway contributes to many human diseases. The cell survival protein kinase Akt/protein kinase B (PKB) is a known regulator of apoptosis, but its role in Fas-mediated cell death and its regulatory mechanisms are unclear. In this study, we show that stimulation of the Fas receptor by its ligand (FasL) induces rapid phosphorylation of Akt/PKB and a parallel increase in cell apoptosis in epidermal Cl41 cells. Inhibition of PI3K/Akt by dominant-negative overexpression of PI3K (Δp85) and Akt (Akt-T308A/S473A) protects the cells from apoptosis, indicating an unexpected proapoptotic role of PI3K/Akt in the Fas signaling process. Treatment of the cells with pharmacological inhibitors of PI3K, wortmannin and 2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-1 (LY294002), similarly inhibits FasL-induced apoptosis and Akt/PKB phosphorylation, indicating that PI3K is an upstream mediator of Akt/PKB and is involved in Fas-mediated cell death. Electron spin resonance studies show that FasL treatment induces rapid generation of reactive oxygen species, and inhibition of ROS by antioxidants effectively inhibits Akt/PKB signaling, suggesting that FasL activation of Akt/PKB is redox sensitive. In cells transfected with dominant-negative PI3K/Akt, Fas expression is down-regulated, but FLIP expression is unaffected. Reporter gene and mRNA expression assays show that FasL activates fas transcriptional activity and this effect is inhibited by PI3K/Akt suppression. Together, our results indicate that the PI3K/Akt, in addition to its normal prosurvival role, also plays an apoptotic role in Fas-mediated cell death through a mechanism that involves transcriptional activation of Fas receptor.

Induction of Activator Protein-1 through Reactive Oxygen Species by Crystalline Silica in JB6 Cells

We reported previously that freshly fractured silica (FFSi) induces activator protein-1 (AP-1) activation through extracellular signal-regulated protein kinases (ERKs) and p38 kinase pathways. In the present study, the biologic activities of FFSi and aged silica (ASi) were compared by measuring their effects on the AP-1 activation and phosphorylation of ERKs and p38 kinase. The roles of reactive oxygen species (ROS) in this silica-induced AP-1 activation were also investigated. We found that FFSi-induced AP-1 activation was four times higher than that of ASi in JB6 cells. FFSi also caused greater phosphorylation of ERKs and p38 kinase than ASi. FFSi generated more ROS than ASi when incubated with the cells as measured by electron spin resonance (ESR). Studies using ROS-sensitive dyes and oxygen consumption support the conclusion that ROS are generated by silica-treated cells. N-Acetylcysteine (an antioxidant) and polyvinyl pyridine-N-oxide (an agent that binds to Si-OH groups on silica surfaces) decreased AP-1 activation and phosphorylation of ERKs and p38 kinase. Catalase inhibited phosphorylation of ERKs and p38 kinase, as well as AP-1 activation induced by FFSi, suggesting the involvement of H2O2 in the mechanism of silica-induced AP-1 activation. Sodium formate (an ⋅OH scavenger) had no influence on silica-induced MAPKs or AP-1 activation. Superoxide dismutase enhanced both AP-1 and MAPKs activation, indicating that H2O2, but not O⨪2, may play a critical role in silica-induced AP-1 activation. These studies indicate that freshly ground silica is more biologically active than aged silica and that ROS, in particular H2O2, play a significant role in silica-induced AP-1 activation.

Vanadium-induced apoptosis and pulmonary inflammation in mice: Role of reactive oxygen species†

Pulmonary exposure to metals and metal‐containing compounds is associated with pulmonary inflammation, cell death, and tissue injury. The present study uses a mouse model to investigate vanadium‐induced apoptosis and lung inflammation, and the role of reactive oxygen species (ROS) in this process. Aspiration of the pentavalent form of vanadium, V (V), caused a rapid influx of polymorphonuclear leukocytes into the pulmonary airspace with a peak inflammatory response at 6 h post‐exposure and resolution by 72 h. During this period, the number of apoptotic lung cells which were predominantly neutrophils increased considerably with a peak response at 24 h accompanied by no or minimum necrosis. After 24 h when the V (V)‐induced inflammation was in the resolution phase, an increased influx of macrophages and engulfment of apoptotic bodies by these phagocytes was observed, supporting the role of macrophages in apoptotic cell clearance and resolution of V (V)‐induced lung inflammation. Electron spin resonance (ESR) studies using lavaged alveolar macrophages showed the formation of ROS, including O2·−, H2O2, and ·OH radicals which were confirmed by inhibition with free radical scavengers. The mechanism of ROS generation induced by V (V) involved the activation of an NADPH oxidase complex and the mitochondrial electron transport chain. The ROS scavenger, catalase (H2O2 scavenger), effectively inhibited both lung cell apoptosis and the inflammatory response, whereas superoxide dismutase (SOD) (O2·− scavenger) and the metal chelator, deferoxamine (inhibitor of ·OH generation by Fenton‐like reactions) had lesser effects. These results indicate that multiple oxidative species are involved in V (V)‐induced lung inflammation and apoptosis, and that H2O2 plays a major role in this process. Published 2003 Wiley‐Liss, Inc.

Differential role of hydrogen peroxide in UV-induced signal transduction

The present study investigated the differential requirement of ROS in UV-induced activation of these pathways. Exposure of the mouse epidermal Cl41 cells to UV radiation led to generation of ROS as measured by electron spin resonance (ESR) and by H2O2 and O2•−; fluorescence staining assay. Treatment of cells with UV radiation or H2O2 also markedly activated Erks, JNKs, p38 kinase and led to increases in phosphorylation of Akt and p70S6k in mouse epidermal JB6 cells. The scavenging of UV-generated H2O2 by N-acety-L-cyteine (NAC, a general antioxidant) or catalase (a specific H2O2 inhibitor) inhibited UV-induced activation of JNKs, p38 kinase, Akt and p70S6k, while it did not show any inhibitory effects on Erks activation. Further, pretreatment of cells with sodium formate (an •OH radical scavenger) or superoxide dismutase (O2•− radical scavenger) did not inhibit any of these pathways. These results demonstrate that H2O2 generation is required for UV-induced phosphorylation of Akt and p70S6k, and involved in activation of JNKs and p38 kinase, but not Erks.

INVOLVEMENT OF ERKS ACTIVATION IN CADMIUM-INDUCED AP-1 TRANSACTIVATION IN VITRO AND IN VIVO

Cadmium is a potent and effective carcinogen in rodents and has recently been accepted by IARC (International Agency for Research on Cancer) as a category 1 carcinogen. Cadmium-induced up‐regulation of intracellular signaling pathways leading to increased mitogenesis is thought to be a major mechanism for the carcinogenic activity following chronic cadmium exposure. In the present study, we found that exposure of cells to cadmium induced significant activation of AP‐1 and all three members of the MAP kinase family in mouse epidermal JB6 cells. The induction of AP‐1 activity by cadmium appears to involve activation of Erks, since the induction of AP‐1 activity by cadmium was blocked by pretreatment of cells with PD98058. Interestingly, the induction of AP‐1 by cadmium was greatly enhanced by the chemical tumor promoter, TPA and the growth factor EGF, but not by ultraviolet C radiation. In vivo studies demonstrated that cadmium could also induce transactivation of AP‐1 in AP‐1‐luciferase report transgenic mice. Considering the role of AP‐1 activation in tumor promotion, the results presented in this study provide a possible molecular mechanism for cadmium‐induced carcinogenesis.

Vanadate-induced cell growth arrest is p53-dependent through activation of p21 in C141 cells.

Vanadium is widely used in industry. It is a potent toxic agent and carcinogen. The mechanisms involved in its toxicity and carcinogenesis are still unclear. Improper cell growth is believed to be involved in cancer development. The present study investigated the regulation of p53 on vanadate-induced cell growth arrest using both p53 wild type C141 cells and p53 deficient embryo fibroblasts (p53 -/-). On vanadate stimulation, C141 cells exhibited a dose- and time-dependent S phase arrest as determined by DNA content analysis. In contrast, vanadate was unable to increase the percentage of S phase in p53 -/- cells. Luciferase assay showed that vanadate induced p53 activation in a dose- and time-dependent manner in p53 wild type C141 cells. Addition of pifithrin-alpha (PFT), a specific inhibitor of p53, reduced the activation of p53 with a concomitant decrease in growth arrest at S phase. Western blotting analysis demonstrated that vanadate caused a dose- and time-dependent increase of p21 level in C141 cells. Pretreatment of C141 cells with PFT decreased p21 expression induced by vanadate while the p21 expression did not vary in vanadate stimulated p53 -/- cells. The results obtained from the present study suggest that vanadate is able to induce S phase arrest through p53- and p21-dependent pathway.

Involvement of hydrogen peroxide in asbestos- induced NFAT activation

The present study investigated the role of reactive oxygen species (ROS) in activation of nuclear factor of activated T cells (NFAT), a pivotal transcription factor responsible for regulation of cytokines, by asbestos in mouse embryo fibroblast PW cells. Exposure of cells to asbestos led to the transactivation of NFAT in a time- and dose-dependent manner. Scavenging of asbestos-induced H2O2 with N-acety-L-cyteine (NAC, a general antioxidant) or catalase (a specific H2O2 inhibitor) resulted in inhibition of NFAT activation. In contrast, an increase in H2O2 generation by the addition of superoxide dismutase (SOD) slightly enhanced asbestos-induced NFAT activation. In addition, pretreatment of cells with sodium formate did not exhibit any inhibition of NFAT activity induced by asbestos. These results demonstrated that H2O2 appeared to play an important role in asbestos-induced NFAT transactivation. Furthermore, it was observed that incubation of cells with 12-O-tetradecanoylphorbol-13-acetate (TPA) not only resulted in NFAT activation by itself, but also enhanced asbestos-induced NFAT induction. Pretreatment of cells with cyclosporin A (CSA), a pharmacological inhibitor of the phosphatase calcineurin, blocked both asbestos- and TPA plus asbestos-induced NFAT activation. These data suggest that asbestos is able to induce NFAT activation through H2O2-dependent and CSA-sensitive pathways, which may be involved in asbestos-induced carcinogenesis.

Activation of aPKC is required for vanadate-induced phosphorylation of protein kinase B (Akt), but not p70S6k in mouse epidermal JB6 cells.

Vanadium is a metal widely distributed in the environment. Although vanadate-containing compounds exert potent toxic effects on a wide variety of biological systems, the mechanisms by which vanadate mediates adverse effects are not well understood. The present study investigated the vanadate-induced phosphorylation of Akt and p70S6K, two kinases known to be vital for cell survival, growth, transformation, and transition of the cell cycle in mammals. Exposure of mouse epidermal JB6 cells to vanadium led to phosphorylation of Akt and p70S6K in a time- and dose-dependent manner. Vanadium exposure also caused translocation of atypical isoforms of PKC (λ, ζ) from the cytosol to the membrane, but had no effect on PKCα translocation, suggesting that the atypical PKCs (aPKC) were specifically involved in vanadium-induced cellular response. Importantly, overexpression of a dominant negative mutant PKCλ blocked Akt phosphorylation at Ser473 and Thr308, whereas it did not inhibit p70S6k phosphorylation at Thr389 and Thr421/Ser424, suggesting that aPKC activation is specifically involved in vanadium-induced activation of Akt, but not in activation of p70S6k. Furthermore, vanadium-induced p70S6k phosphorylation at Thr389 and Thr421/Ser424 and Akt phosphorylation at Thr308 occurred through a PI-3K-dependent pathway because a PI-3K dominant negative mutant inhibited induction as compared with vector control cells. These results indicate that there was a differential role of aPKC in vanadate-induced phosphorylation of Akt and p70S6k, suggesting that signal transduction pathways leading to the activation of Akt and p70S6k were different.

The role of phosphatidylinositol-3 kinase in vanadate-promoted S phase entry.

Phosphatidylinositil-3 kinase (PI3K) is a heterodimer of catalytic and regulatory subunits. It is involved in various signaling pathways and key functions of the cells. The present study investigated the role of PI3K in vanadate-induced alteration in cell cycle regulation in C141 mouse epidermal cells. Vanadate caused a time- and dose-dependent increase in PI3K activity and phosphorylation of p70 S6 kinase (p70S6K) at Thr421/Ser424 and Thr389 sites. The phosphorylation at these sites was inhibited by PI3K inhibitor, LY294002, and p70S6K mutation. Vanadate promoted S phase entry and this promotion was inhibited by LY294002 and rapmycin, a p70S6K inhibitor. Vanadate-induced enhancement in S phase entry was also inhibited in transfection with dominant negative p70S6K mutant cells. The results obtained show that vanadate is able to increase PI3K activity through phosphorylation. PI3K activated p70S6K, which phosphated protein S6, and promoted S phase entry.