Hypoxia-increased RAGE expression regulates chemotaxis and pro-inflammatory cytokines release through nuclear translocation of NF-κ B and HIF1α in THP-1 cells

The potential role of hypoxia in mediating the receptor for advanced glycation end products (RAGE) expression deserves to be confirmed. And the role of RAGE in hypoxia-induced chemotaxis and inflammation is still unclear. In present study, THP-1?cells were pretreated with siRNA to block HIF1α, NF-κ B, or RAGE, followed by exposed to hypoxia (combined with H₂O₂ or SNP), and then RAGE expression, nuclear translocation of HIF1α and NF-κ B, release of TNF-α and IL-1β, as well as expression of MCP-1 and CCR2 were measured. The results revealed that RAGE mRNA and protein in THP-1?cells were significantly increased after exposed into hypoxia atmosphere, especially into the solution containing SNP or H₂O₂. Moreover, SNP or H₂O₂ exposure could further amplify hypoxia-induced nuclear translocation of HIF-1α and NF-κ B. Knockdown HIF-1α or NF-κ B by siRNAs could reduce hypoxia- and oxidative stress-induced RAGE hyper-expression. And pretreatment THP-1?cells with RAGE siRNA or NF-κ B siRNA could reduce hypoxia- and oxidative stress-induced expression of MCP-1 and CCR2, and release of TNF-α and IL-1β. Thus, hypoxia not only increases RAGE expression in THP-1?cells by promoting nuclear translocation of NF-κ B and HIF1α, but also regulates chemotaxis and pro-inflammatory cytokines release, which may be partially mediated through upregulation of RAGE expression.     

Redox activation of Ref-1 potentiates cell survival following myocardial ischemia reperfusion injury

A recent study showed that cardiac adaptation could potentiate translocation of thioredoxin-1 (Trx-1) into the nucleus, which then interacted with Ref-1, resulting in a survival signal. Here, we present evidence that such adaptation also causes nuclear translocation of Ref-1, which is almost completely inhibited when the hearts were pretreated with antisense Ref-1 that also abolished the cardioprotective adaptive response. Significant amounts of NFkappaB and Nrf2 were found to be associated with Ref-1 when the nuclear extract obtained from the left ventricle was immunoprecipitated with Ref-1. Such Ref-1-NFkappaB and Ref-1-Nrf2 interactions were significantly inhibited with antisense Ref-1. However, immunoprecipitation of nuclear extract with NFkappaB showed that the association of Trx-1 with NFkappaB is increased in the adapted heart, which was again significantly blocked by antisense Ref-1. Nrf2 was also associated with NFkappaB; however, such association appeared to be independent of Ref-1. In contrast, myocardial adaptation to ischemia inhibited the ischemia reperfusion-induced loss of Nrf2 from the nucleus, which was inhibited by antisense Ref-1. The nuclear translocation and activation of Ref-1 appeared to generate a survival signal as evidenced by the increased phosphorylation of Akt that was inhibited with antisense Ref-1. Finally, confocal microscopy confirmed the results of immunoblotting, clearly showing the nuclear translocation of Ref-1 and nuclear 3D colocalization of Ref-1 with NFkappaB in the adapted heart and its inhibition with antisense Ref-1. Our results show that PC potentiates a survival signal through the phosphorylation of Akt by causing nuclear translocation and activation of Ref-1, where significant interaction among NFkappaB and Ref-1, Trx-1, and Nrf2 appears to regulate Ref-1-induced survival signal.     

低氧诱导因子-1α在低氧促成肌细胞增殖中的作用

目的:探讨低氧对大鼠骨骼肌成肌细胞(SkMs)增殖的影响及低氧诱导因子(HIF-1α)在低氧促成肌细胞增殖中的相关机制。方法:采用流式细胞仪观察了3、10%O2对SkMs细胞数量和增殖指数的影响;用RT-PCR方法检测了HIF-1αmRNA的表达,用Western blot方法检测了SkMs胞浆、胞核及总HIF-1α蛋白的水平。结果:低氧组较常氧组细胞数量和增殖指数增加(P0.05);HIF-1αmRNA、总蛋白水平在常氧组和低氧组中没有明显差异,常氧下胞浆中HIF-1α蛋白水平高于胞核内,低氧下HIF-1α蛋白水平在胞核内高于胞浆。结论:低氧能够促进SkMs增殖,HIF-1α可能是通过氧浓度调控的核转位的方式参与了低氧促SkMs的增殖。     

Thioredoxin-dependent redox regulation of p53-mediated p21 activation

Thioredoxin (TRX) is a dithiol-reducing enzyme that is induced by various oxidative stresses. TRX regulates the activity of DNA-binding proteins, including Jun/Fos and nuclear factor-kappaB. TRX also interacts with an intranuclear reducing molecule redox factor 1 (Ref-1), which enhances the activity of Jun/Fos. Here, we have investigated the role of TRX in the regulation of p53 activity. Electrophoretic mobility shift assay showed that TRX augmented the DNA binding activity of p53 and also further potentiated Ref-1-enhanced p53 activity. Luciferase assay revealed that transfection of TRX enhanced p53-dependent expression of p21 and further intensified Ref-1-mediated p53 activation. Furthermore, Western blot analysis revealed that p53-dependent induction of p21 protein was also facilitated by transfection with TRX. Overexpression of transdominant negative mutant TRX (mTRX) suppressed the effects of TRX or Ref-1, showing a functional interaction between TRX and Ref-1. cis-Diamminedichloroplatinum (II) (CDDP) induced p53 activation and p21 transactivation. The p53-dependent p21 transactivation induced by CDDP was inhibited by mTRX overexpression, suggesting that TRX-dependent redox regulation is physiologically involved in p53 regulation. CDDP also stimulated translocation of TRX from the cytosol into the nucleus. Hence, TRX-dependent redox regulation of p53 activity indicates coupling of the oxidative stress response and p53-dependent repair mechanism.     

Phenethyl isothiocyanate,by virtue of its antioxidant activity,inhibits invasiveness and metastatic potential of breast cancer cells: HIF-1α as a putative target

Hypoxia-inducible factor 1α (HIF-1α) plays a crucial role in facilitating tumor progression and metastasis. Reducing the levels of HIF-1α might therefore be an important anticancer strategy. This could be achieved by understanding the key cellular events involved in HIF-1α activation. Present study explored the effect of phenethyl isothiocyanate (PEITC), a natural isothiocyanate, found in cruciferous vegetables on the expression of HIF-1α and HSP90 in breast adenocarcinoma cell lines (MCF-7 and MDA-MB-231) under both normoxia and hypoxia. This study established the possible role of ROS in the up-regulation of these markers in breast cancer cells. PEITC-induced nuclear accumulation of Nrf2, increased the activities of several antioxidant enzymes, and thus reduced the ROS burden of the tumor cells by acting as an indirect antioxidant. This resulted in the down-regulation of HSP90 and thereby HIF-1α expression. HSP90 was also found to be involved in the regulation of HIF-1α. A probable link between down-regulation of HIF-1α with reduction of ROS by PEITC through induction of Nrf2 was determined. Finally, our study demonstrated that modulation of HIF-1α by PEITC retarded adhesion, aggregation, migration and invasion of the breast cancer cells, thereby showing anti-metastatic effect. Activities of MMPs (2 & 9) and expression of VEGF were also altered by PEITC.