Weiming Ouyang

Nickel Compounds Render Anti-apoptotic Effect to Human Bronchial Epithelial Beas-2B Cells by Induction of Cyclooxygenase-2 through an IKKβ/p65-dependent and IKKα- and p50-independent Pathway

The carcinogenicity of nickel compounds has been well documented both in vitro and in vivo; however, the molecular mechanisms by which nickel compounds cause cancers are far from understood. Because suppression of apoptosis is thought to contribute to carcinogenesis, we investigated the mechanisms implicated in nickel-induced anti-apoptotic effect in human bronchial epithelial (Beas-2B) cells. We found that exposure of Beas-2B cells to nickel compounds resulted in increased cyclooxygenase-2 (COX-2) expression and that small interfering RNA (siCOX-2) knockdown of COX-2 expression resulted in increased cell sensitivity to nickel-triggered cell apoptosis, demonstrating that COX-2 induction has an anti-apoptotic effect on Beas-2B cells. Overexpression of IKKβ-KM, a kinase inactive mutant of IKKβ, blocked NF-κB activation and COX-2 induction by nickel compounds, indicating that activated NF-κB may be a mediator for COX-2 induction. To further explore the contribution of the NF-κB pathway in COX-2 induction and in protection from nickel exposure, mouse embryonic fibroblasts deficient in IKKβ, IKKα, p65, and p50 were analyzed. Loss of IKKβ impaired COX-2 induction by nickel exposure, whereas knockout of IKKα had a marginal effect. Moreover, the NF-κB p65, and not the p50 subunit, was critical for nickel-induced COX-2 expression. In addition, a deficiency of IKKβ or p65 rendered cells more sensitive to nickel-induced apoptosis as compared with those in wild type cells. Finally, it was shown that reactive oxygen species H2O2 were involved in both NF-κB activation and COX-2 expression. Collectively, our results demonstrate that COX-2 induction by nickel compounds occurs via an IKKβ/p65 NF-κB-dependent but IKKα- and p50-independent pathway and plays a crucial role in antagonizing nickel-induced cell apoptosis in Beas-2B cells.

Cyclin D1 Induction through IκB Kinase β/Nuclear Factor-κB Pathway Is Responsible for Arsenite-Induced Increased Cell Cycle G1-S Phase Transition in Human Keratinocytes

Environmental and occupational exposure to arsenite is associated with an increased risk of human cancers, including skin, urinary bladder, and respiratory tract cancers. Although much evidence suggests that alterations in cell cycle machinery are implicated in the carcinogenic effect of arsenite, the molecular mechanisms underlying the cell cycle alterations are largely unknown. In the present study, we observed that exposure of human keratinocyte HaCat cells to arsenite resulted in the promotion of cell cycle progression, especially G1-S transition. Further studies found that arsenite exposure was able to induce cyclin D1 expression. The induction of cyclin D1 by arsenite required nuclear factor-κB (NF-κB) activation, because the inhibition of IκB phosphorylation by overexpression of the dominant-negative mutant, IKKβ-KM, impaired arsenite-induced cyclin D1 expression and G1-S transition. The requirement of IκB kinase β (IKKβ) for cyclin D1 induction was further confirmed by the findings that arsenite-induced cyclin D1 expression was totally blocked in IKKβ knockout (IKKβ−/−) mouse embryo fibroblasts. In addition, knockdown of cyclin D1 expression using cyclin D1–specific small interference RNA significantly blocked arsenite-induced cell cycle progression in HaCat cells. Taken together, our results show that arsenite-induced cell cycle from G1 to S phase transition is through IKKβ/NF-κB/cyclin D1–dependent pathway.

Loss of Tumor Suppressor p53 Decreases PTEN Expression and Enhances Signaling Pathways Leading to Activation of Activator Protein 1 and Nuclear Factor κB Induced by UV Radiation

Transcription factor p53 and phosphatase PTEN are two tumor suppressors that play essential roles in suppression of carcinogenesis. However, the mechanisms by which p53 mediates anticancer activity and the relationship between p53 and PTEN are not well understood. In the present study, we found that pretreatment of mouse epidermal Cl41 cells with pifithrin-alpha, an inhibitor for p53-dependent transcriptional activation, resulted in a marked increase in UV-induced activation of activator protein 1 (AP-1) and nuclear factor kappaB (NF-kappaB). Consistent with activation of AP-1 and NF-kappaB, pifithrin-alpha was also able to enhance the UV-induced phosphorylation of c-Jun-NH2-kinases (JNK) and p38 kinase, whereas it did not show any effect on phosphorylation of extracellular signal-regulated kinases. Furthermore, the UV-induced signal activation, including phosphorylation of JNK, p38 kinase, Akt, and p70S6K, was significantly enhanced in p53-deficient cells (p53-/-), which can be reversed by p53 reconstitution. In addition, knockdown of p53 expression by its small interfering RNA also caused the elevation of AP-1 activation and Akt phosphorylation induced by UV radiation. These results show that p53 has a suppressive activity on the cell signaling pathways leading to activation of AP-1 and NF-kappaB in cell response to UV radiation. More importantly, deficiency of p53 expression resulted in a decrease in PTEN protein expression, suggesting that p53 plays a critical role in the regulation of PTEN expression. In addition, overexpression of wild-type PTEN resulted in inhibition of UV-induced AP-1 activity. Because PTEN is a well-known phosphatase involved in the regulation of phosphatidylinositol 3-kinase (PI-3K)/Akt signaling pathway, taken together with the evidence that PI-3K/Akt plays an important role in the activation of AP-1 and NF-kappaB during tumor development, we anticipate that inhibition of AP-1 and NF-kappaB by tumor suppressor p53 seems to be mediated via PTEN, which may be a novel mechanism involved in anticancer activity of p53 protein.

PI-3K/Akt pathway-dependent cyclin D1 expression is responsible for arsenite-induced human keratinocyte transformation.

Background Long-term exposure of arsenite leads to human skin cancer. However, the exact mechanisms of arsenite-induced human skin carcinogenesis remain to be defined. Objectives In this study, we investigated the potential role of PI-3K/Akt/cyclin D1in the transformation of human keratinocytic cells upon arsenite exposure. Methods We used the soft agar assay to evaluate the cell transformation activity of arsenite exposure and the nude mice xenograft model to determine the tumorigenesis of arsenite-induced transformed cells. We used the dominant negative mutant and gene knockdown approaches to elucidate the signaling pathway involved in this process. Results Our results showed that repeated long-term exposure of HaCat cells to arsenite caused cell transformation, as indicated by anchorage-independent growth in soft agar. The tumorigenicity of these transformed cells was confirmed in nude mice. Treatment of cells with arsenite also induced significant activation of PI-3K and Akt, which was responsible for the anchorage-independent cell growth induced by arsenite exposure. Furthermore, our data also indicated that cyclin D1 is an important downstream molecule involved in PI-3K/Akt–mediated cell transformation upon arsenite exposure based on the facts that inhibition of cyclin D1 expression by dominant negative mutants of PI-3K, and Akt, or the knockdown of the cyclin D1 expression by its specific siRNA in the HaCat cells resulted in impairing of anchorage-independent growth of HaCat cells induced by arsenite. Conclusion Our results demonstrate that PI-3K/Akt–mediated cyclin D1 expression is at least one key event implicated in the arsenite human skin carcinogenic effect.

Cyclooxygenase-2 Induction by Arsenite Is through a Nuclear Factor of Activated T-cell-dependent Pathway and Plays an Antiapoptotic Role in Beas-2B Cells

Arsenite is a well known metalloid human carcinogen, and epidemiological evidence has demonstrated its association with the increased incidence of lung cancer. However, the mechanism involved in its lung carcinogenic effect remains obscure. The current study demonstrated that exposure of human bronchial epithelial cells (Beas-2B) to arsenite resulted in a marked induction of cyclooxygenase (COX)-2, an important mediator for inflammation and tumor promotion. Exposure of the Beas-2B cells to arsenite also led to significant transactivation of nuclear factor of activated T-cells (NFAT), but not activator protein-1 (AP-1) and NFκB, suggesting that NFAT, rather than AP-1 or NFκB, is implicated in the responses of Beas-2B cells to arsenite exposure. Furthermore, we found that inhibition of the NFAT pathway by either chemical inhibitors, dominant negative mutants of NFAT, or NFAT3 small interference RNA resulted in the impairment of COX-2 induction and caused cell apoptosis in Beas-2B cells exposed to arsenite. Site-directed mutation of two putative NFAT binding sites between–111 to +65 in the COX-2 promoter region eliminated the COX-2 transcriptional activity induced by arsenite, confirming that those two NFAT binding sites in the COX-2 promoter region are critical for COX-2 induction by arsenite. Moreover, knockdown of COX-2 expression by COX-2-specific small interference RNA also led to an increased cell apoptosis in Beas-2B cells upon arsenite exposure. Together, our results demonstrate that COX-2 induction by arsenite is through NFAT3-dependent and AP-1- or NFκB-independent pathways and plays a crucial role in antagonizing arsenite-induced cell apoptosis in human bronchial epithelial Beas-2B cells.

NFAT3 is specifically required for TNF-α-induced cyclooxygenase-2 (COX-2) expression and transformation of Cl41 cells

NFAT family is recognized as a transcription factor for inflammation regulation by inducing the expression of proinflammatory cytokines, such as tumor necrosis factor-α (TNF-α), the key mediator of inflammation, which was reported to induce cell transformation in mouse epidermal Cl41 cells. In this study, we demonstrated that TNF-α was able to induce NFAT activation, as well as the expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). The induction of COX-2 by TNF-α was abolished by knockdown of NFAT3 with its siRNA, while the induction of iNOS was not effected. Moreover, TNF-α-induced anchorage-independent cell growth was significantly inhibited by NFAT3 siRNA and cyclosporine A, a chemical inhibitor for the calcineurin/NFAT pathway, which suggests the importance of NFAT3 in regulating TNF-α-induced anchorage-independent cell growth. Consequently, impairment of COX-2 by its siRNA or selective inhibitor also inhibited TNF-α-induced anchorage-independent cell growth. Taken together, our results indicate that NFAT3 plays an important role in the regulation of TNF-α-induced anchorage-independent cell growth, at least partially, by inducing COX-2 expression in Cl41 cells. These findings suggest that NFAT3/cyclooxygenase-2 act as a link between inflammation and carcinogenesis by being involved in the tumor promotion stage.

p85α Acts as a Novel Signal Transducer for Mediation of Cellular Apoptotic Response to UV Radiation

ABSTRACT Apoptosis is an important cellular response to UV radiation (UVR), but the corresponding mechanisms remain largely unknown. Here we report that the p85α regulatory subunit of phosphatidylinositol 3-kinase (PI-3K) exerted a proapoptotic role in response to UVR through the induction of tumor necrosis factor alpha (TNF-α) gene expression. This special effect of p85α was unrelated to the PI-3K-dependent signaling pathway. Further evidence demonstrated that the inducible transcription factor NFAT3 was the major downstream target of p85α for the mediation of UVR-induced apoptosis and TNF-α gene transcription. p85α regulated UVR-induced NFAT3 activation by modulation of its nuclear translocation and DNA binding and the relevant transcriptional activities. Gel shift assays and site-directed mutagenesis allowed the identification of two regions in the TNF-α gene promoter that served as the NFAT3 recognition sequences. Chromatin immunoprecipitation assays further confirmed that the recruitment of NFAT3 to the endogenous TNF-α promoter was regulated by p85α upon UVR exposure. Finally, the knockdown of the NFAT3 level by its specific small interfering RNA decreased UVR-induced TNF-α gene transcription and cell apoptosis. The knockdown of endogenous p85α blocked NFAT activity and TNF-α gene transcription, as well as cell apoptosis. Thus, we demonstrated p85α-associated but PI-3K-independent cell death in response to UVR and identified a novel p85α/NFAT3/TNF-α signaling pathway for the mediation of cellular apoptotic responses under certain stress conditions such as UVR.

Benzo[a]pyrene diol-epoxide (B[a]PDE) upregulates COX-2 expression through MAPKs/AP-1 and IKKβ/NF-κB in mouse epidermal Cl41 cells†

Benzo[α]pyrene‐7,8‐diol‐9,10‐epoxide (B[a]PDE), the major metabolite of benzo[a]pyrene (B[a]P), shows an ultimate complete carcinogen in various animals and is a causative agent for human cancers. However, its effects on the activation of signal pathways and the expression of genes involved in its carcinogenic effect remain largely unknown. In this study, the effects of B[a]PDE on induction of cyclooxygenase (COX)‐2 and the signal pathways leading to the induction were investigated. Treatment of mouse epidermal Cl41 cells with B[a]PDE caused an increase in the expression of COX‐2 at both transcription and protein levels, while its parental compound B[a]P did not show significant inductive effect. The COX‐2 induction by B[a]PDE was dependent on the activation of mitogen‐activated protein kinases (MAPK)s/activation protein (AP)‐1 pathway, because inhibition of AP‐1 by either overexpression of TAM67 (dominant negative mutant of c‐jun), or pretreatment of cells with PD98059 (MEK1/2‐ERKs pathway inhibitor) or SB202190 (p38K inhibitor), markedly inhibited B[a]PDE‐induced COX‐2 expression. In addition, impairment of NF‐κB pathway by either NEMO‐BDBP (an NF‐κB specific inhibitor) or IκB kinase (IKK)β‐KM (dominant negative mutant of IKKβ) also caused marked reduction of COX‐2 induction by B[a]PDE. In contrast, inhibition of nuclear factor of activated T cells (NFAT) with FK506, did not show any effect on B[a]PDE‐induced COX‐2 expression. Collectively, these data indicate that exposure of Cl41 cells to B[a]PDE can induce COX‐2 expression by increasing its transcription, which requires the activation of MAPKs/AP‐1 and IKKβ/NF‐κB pathways, but not NFAT pathway. In view of the importance of COX‐2 in carcinogenesis, we anticipate that the induction of COX‐2 by B[a]PDE may coordinate its mutagenic effects to facilitate the development of skin cancer.

Effects of Polycyclic Aromatic Hydrocarbons (PAHs) on Vascular Endothelial Growth Factor Induction through Phosphatidylinositol 3-Kinase/AP-1-dependent, HIF-1α-independent Pathway

Previous studies have demonstrated that exposure to polycyclic aromatic hydrocarbons (PAHs) and its derivatives is associated with an increased risk of skin cancers, and the carcinogenic effect of PAHs is thought to involve both tumor initiation and promotion. Whereas PAH tumor initiation is well characterized, the mechanisms involved in the tumor promotion of PAHs remain elusive. In the present study, we investigated the effects of PAHs on vascular endothelial growth factor (VEGF) expression by comparison of its induction between the active metabolite and its parent compound (B[a]PDE versus B[a]P) or between active compound and its relatively inactive analog (5-MCDE versus CDE). We found that exposure of cells to (±)-anti-benzo-[a]pyrene-7,8-diol-9,10-epoxide (B[a]PDE) or (±)-anti-5-methylchrysene-1,2-diol-3,4-epoxide (5-MCDE) led to marked induction of VEGF in Cl41 cells, whereas benzo[a]pyrene (B[a]P) or chrysene-1,2-diol-3,4-epoxide (CDE) did not exhibit significant inductive effects. Exposure of cells to B[a]PDE and 5-MCDE did not induce HIF-1α activation, whereas AP-1 was significantly activated. Moreover, overexpression of TAM67 (a dominant-negative mutant c-Jun) dramatically blocked that VEGF induction. Electrophoretic mobility shift assay showed that AP-1 was only able to specifically recognize and bind to its AP-1 potential binding site within –1136 and –1115 of the VEGF promoter region. Site-directed mutation of this AP-1 binding site eliminated the VEGF transcriptional activity induced by B[a]PDE, suggesting that the AP-1 binding site between –1136 and –1115 in the VEGF promoter region is critical for VEGF induction by B[a]PDE. In addition, overexpression of Δp85 (a dominant-negative mutant PI-3K) impaired B[a]PDE- and 5-MCDE-induced VEGF induction. Considering our previous findings that PI-3K is an upstream mediator for c-Jun/AP-1 activation, we conclude that the VEGF induction by B[a]PDE and 5-MCDE is through PI-3K/AP-1-dependent and HIF-1α-independent pathways. These findings may help us to understand the mechanisms involved in PAH carcinogenic effects.

PI-3K/Akt signal pathway plays a crucial role in arsenite-induced cell proliferation of human keratinocytes through induction of cyclin D1.

Exposure of arsenite can induce hyperproliferation of skin cells, which is believed to play important roles in arsenite‐induced carcinogenesis by affecting both promotion and progression stages. However, the signal pathways and target genes activated by arsenite exposure responsible for the proliferation remain to be defined. In the present study, we found that: (1) exposure of human keratinocytic HaCat cells to arsenite caused an increase in cell proliferation, which was significantly inhibited by pretreatment of wortmannin, a specific chemical inhibitor of PI‐3K/Akt signal pathway; (2) arsenite exposure was also able to activate PI‐3K/Akt signal pathway, which thereby induced the elevation of cyclin D1 expression level in both HaCat cells and human primary keratinocytes based on that inhibition of PI‐3K/Akt pathway by either pretreatment of wortmannin or the transfection of their dominant mutants, significantly inhibited cyclin D1 expression upon arsenite exposure; (3) PI‐3K/Akt pathway is implicated in arsenite‐induced proliferation of HaCat cells through the induction of cyclin D1 because either knockdown of cyclin D1 by its siRNA or inhibition of PI‐3K/Akt signal pathway by their dominant mutants markedly impaired the proliferation of HaCat cells induced by arsenite exposure. Taken together, we provide the direct evidence that PI‐3K/Akt pathway plays a role in the regulation of cell proliferation through the induction of cyclin D1 in human keratinocytes upon arsenite treatment. Given the importance of aberrant cell proliferation in cell transformation, we propose that the activation of PI‐3K/Akt pathway and cyclin D1 induction may be the important mediators of human skin carcinogenic effect of arsenite. J. Cell. Biochem. 101: 969–978, 2007.