Anatoly Samoylenko

Transcriptional regulation of heme oxygenase-1 gene expression by MAP kinases of the JNK and p38 pathways in primary cultures of rat hepatocytes.

Heme oxygenase-1 (HO-1) gene expression is induced by various oxidative stress stimuli including sodium arsenite. Since mitogen-activated protein kinases (MAPKs) are involved in stress signaling we investigated the role of arsenite and MAPKs for HO-1 gene regulation in primary rat hepatocytes. The Jun N-terminal kinase (JNK) inhibitor SP600125 decreased sodium arsenite-mediated induction of HO-1 mRNA expression. HO-1 protein and luciferase activity of reporter gene constructs with −754 bp of the HO-1 promoter were induced by overexpression of kinases of the JNK pathway and MKK3. By contrast, overexpression of Raf-1 and ERK2 did not affect expression whereas overexpression of p38α, β, and δ decreased and p38γ increased HO-1 expression. Electrophoretic mobility shift assays (EMSA) revealed that a CRE/AP-1 element (−668/−654) bound c-Jun, a target of the JNK pathway. Deletion or mutation of the CRE/AP-1 obliterated the JNK- and c-Jun-dependent up-regulation of luciferase activity. EMSA also showed that an E-box (−47/−42) was bound by a putative p38 target c-Max. Mutation of the E-box strongly reduced MKK3, p38 isoform-, and c-Max-dependent effects on luciferase activity. Thus, the HO-1 CRE/AP-1 element mediatesHO-1 gene induction via activation of JNK/c-Jun whereas p38 isoforms act through a different mechanism via the E-box.

Upregulation of heme oxygenase-1 gene by turpentine oil-induced localized inflammation: involvement of interleukin-6.

Heme oxygenase-1 (HO-1) is the inducible isoform of an enzyme family responsible for heme degradation and was suggested to be involved in the acute phase response in the liver. However, the mechanisms of the HO-1 regulation under inflammatory conditions are poorly understood. Therefore, the purpose of the current work was to study the expression of HO-1 in the liver and other organs of rats with a localized inflammation after intramuscular injection of turpentine oil (TO). Since interleukin-6 (IL-6) is known to be a principal mediator of inflammation, the levels of this cytokine were also estimated in the animal model used. HO-1 and IL-6 expression was evaluated by Northern blot, in situ hybridization, Western blot, immunohistochemistry and enzyme-linked immunosorbent assay. In the liver and injured muscle, the HO-1 mRNA levels were dramatically increased 4–6 h after TO administration. HO-1 protein levels in the liver were elevated starting from 6–12 h after the treatment. In other internal organs such as the heart, kidney and large intestine, only a slight induction of HO-1 mRNA was observed. IL-6-specific transcripts appeared only in the injured muscle and were in accordance with serum levels of IL-6. In turn, temporal expression of IL-6 in the muscle and circulatory IL-6 levels correlated well with HO-1 expression in the liver and injured muscle. In the liver of control rats HO-1 protein was detected in Kupffer cells, while in TO-injected rats also hepatocytes became strongly HO-1 positive. Conversely, in the injured muscle, HO-1 immunoreactivity was attributed only to macrophages. Our data demonstrate that during localized inflammation HO-1 expression was rapidly and strongly induced in macrophages of injured muscle and in hepatocytes, and IL-6 derived from injured muscle seems to be responsible for the HO-1 induction in the liver.

Oxidative Stress and Hypoxia: Implications for Plasminogen Activator Inhibitor-1 Expression

Plasminogen activator inhibitor-1 (PAI-1) is the major physiological inhibitor of urokinase-type and tissue-type plasminogen activators. It has gained special interest among clinicians because a number of pathological conditions, such as myocardial infarction, atherosclerosis, thrombosis, several types of cancer, and the metabolic syndrome, as well as type 2 diabetes mellitus, are associated with increased PAI-1 levels. Interestingly, a number of these diseases are also accompanied by oxidative stress and the enhanced production of reactive oxygen species or tissue hypoxia. This article tries to summarize some aspects leading to enhanced PAI-1 production under oxidative stress or hypoxia.

Nutritional Countermeasures Targeting Reactive Oxygen Species in Cancer: From Mechanisms to Biomarkers and Clinical Evidence

Reactive oxygen species (ROS) exert various biological effects and contribute to signaling events during physiological and pathological processes. Enhanced levels of ROS are highly associated with different tumors, a Western lifestyle, and a nutritional regime. The supplementation of food with traditional antioxidants was shown to be protective against cancer in a number of studies both in vitro and in vivo. However, recent large-scale human trials in well-nourished populations did not confirm the beneficial role of antioxidants in cancer, whereas there is a well-established connection between longevity of several human populations and increased amount of antioxidants in their diets. Although our knowledge about ROS generators, ROS scavengers, and ROS signaling has improved, the knowledge about the direct link between nutrition, ROS levels, and cancer is limited. These limitations are partly due to lack of standardized reliable ROS measurement methods, easily usable biomarkers, knowledge of ROS action in cellular compartments, and individual genetic predispositions. The current review summarizes ROS formation due to nutrition with respect to macronutrients and antioxidant micronutrients in the context of cancer and discusses signaling mechanisms, used biomarkers, and its limitations along with large-scale human trials.

Opposite expression of the antioxidant heme oxygenase-1 in primary cells and tumor cells: regulation by interaction of USF-2 and Fra-1.

Heme oxygenase-1 is the rate-limiting enzyme for the degradation of the prooxidant heme. Previously, we showed that an E-box within the HO-1 promoter is crucial for the regulation of HO-1 expression in primary hepatocytes. Further to investigate the importance of this E-box, we determined the regulatory capacity of the E-box-binding factor USF-2 in primary cells in comparison with transformed cell lines. We found that HO-1 expression was inhibited by USF-2 in primary cells, whereas it was induced in tumor cell lines. Mutation of either the E-box or the AP-1 site within the HO-1 promoter only partially affected the USF-dependent regulation. However, this regulation was dramatically reduced in tumor cells and completely abolished in primary cells transfected with an HO-1 promoter construct containing mutations in both the E-box and the AP-1 site, suggesting that AP-1 factors and USF-2 may act in a cooperative manner. Indeed, protein-protein interaction studies revealed that USF proteins interacted with Fra-1. Further, the USF-dependent HO-1 promoter activity was not detectable with an USF-2 mutant lacking residues of the USF-specific region (USR) or the transactivation domain encoded by exon 4. Together, these data suggest that USF-2 has opposite regulatory roles for HO-1 gene expression in primary cells and tumor cell lines.

FOXO4 Induces Human Plasminogen Activator Inhibitor-1 Gene Expression via an Indirect Mechanism by Modulating HIF-1α and CREB Levels

The plasminogen activator inhibitor-1 (PAI-1) expression can be enhanced by hypoxia and various stimuli associated with oxidative stress. Among the FOXO transcription factors, FOXO4 appears to be crucial in the response against oxidative stress. Therefore, it was the aim of this study to investigate the role of peroxide-induced oxidative stress and FOXO4 on PAI-1 expression under normoxia and hypoxia. Treatment of cells with hydrogen peroxide increased PAI-1 mRNA, protein, and promoter activity, and knocking down FOXO4 abolished the peroxide-dependent PAI-1 induction. PAI-1 promoter reporter gene assays revealed that the peroxide and FOXO4-dependent induction was mediated through the HIF-1 and CREB-binding HRE within the PAI-1 promoter. Western blot analyses then indicated that peroxide and FOXO4 downregulated HIF-1alpha levels, whereas CREB levels were increased. Chromatin immunoprecipitations showed that FOXO4 did not bind the PAI-1 promoter, whereas CREB binding was enhanced on FOXO4 overexpression. In addition, knockdown of CREB abolished the FOXO4-mediated PAI-1 induction. Together, these findings provide the first evidence that oxidative stress and FOXO4 induce PAI-1 expression through an indirect mechanism involving modulation of HIF-1alpha and CREB protein levels and that enhanced CREB binding to the PAI-1 promoter is critical for the PAI-1 induction under oxidative stress.

PAI-1 modulates cell migration in a LRP1-dependent manner via β-catenin and ERK1/2

Plasminogen activator inhibitor-1 (PAI-1) is the major and most specific acting urokinase (uPA) and tissue plasminogen activator (tPA) inhibitor. Apart from its function in the fibrinolytic system, PAI-1 was also found to contribute to processes like tissue remodelling, angiogenesis, and tumour progression. However, the role of PAI-1 in those processes remains largely controversial with respect to the influence of PAI-1 on cell signalling pathways. Although PAI-1 does not possess its own cellular receptor, it can be bound to low-density lipoprotein receptor-related protein 1 (LRP1) which was proposed to modulate the β-catenin pathway. Therefore, we used wild-type mouse embryonic fibroblasts (MEFs), and MEFs deficient of LRP1 to study PAI-1 as modulator of the β-catenin pathway. We found that PAI-1 influences MEF proliferation and motility in a LRP1-dependent manner and that β-catenin is important for that response. In addition, expression of β-catenin and β-catenin-dependent transcriptional activity were induced by PAI-1 in wild type MEFs, but not in LRP1-deficient cells. Moreover, PAI-1-induced ERK1/2 activation was more prominent in the LRP1-deficient cells and interestingly knockdown of β-catenin abolished this effect. Together, the data of the current study show that PAI-1 can promote cell migration via LRP1-dependent activation of the β-catenin and ERK1/2 MAPK pathway which may be important in stage-specific treatment of human diseases associated with high PAI-1 levels.

Urokinase is a negative modulator of Egf‐dependent proliferation and motility in the two breast cancer cell lines MCF‐7 and MDA‐MB‐231

The epidermal growth factor receptor (EGFR) is involved in the regulation of various cellular processes and dysregulation of its signalling plays a critical role in the etiology of a variety of malignancies like breast cancer. At the same time, elevated levels of urokinase (uPA), its receptor uPAR, and other components of the plasminogen activation system are found to be correlated with a poor prognosis in breast cancer. Interestingly, EGFR appears to participate in transducing the signal generated upon binding of uPA to uPAR. However, whether uPA signalling would thereby interfere with ligand‐driven EGFR signalling was not described before. Therefore, it was the aim of the present study to investigate the combined effects of uPA and EGF in the low invasive and high invasive breast adenocarcinoma cell lines MCF‐7 and MDA‐MB‐231, respectively. Simultaneous exposure of cells to both signals negatively affected ERK1/2 and AKT activation whereas positive effects on p38 and Src kinase phosphorylation were noted in both cell lines. Furthermore, uPA attenuated the mitogenic effect of EGF on cellular proliferation, invasion and motility in both MCF‐7 and MDA‐MB‐231 cells. Experiments with the uPA amino terminal fragment (ATF) revealed that the negative effects of uPA were independent from its protease activity. Together, these data suggest that enhanced levels of uPA in breast cancer modulate the mitogenic effects of EGF and thus, this knowledge may help to better understand breast cancer pathogenesis as well as to develop new therapeutic options.

The adaptor protein Ruk/CIN85 activates plasminogen activator inhibitor-1 (PAI-1) expression via hypoxia-inducible factor-1α

Increased levels of plasminogen activator inhibitor-1 (PAI-1) indicate an enhanced risk of ischaemic/hypoxic cardiovascular events and a poor prognosis. The expression of PAI-1 can be induced by various stimuli including hypoxia, insulin and insulin-like growth factor 1 (IGF-1). The hypoxia-inducible factor-1 (HIF-1) is critical for hypoxia or insulin/IGF-1 mediated PAI-1 induction, but the components involved in merging the signals are not known so far. The adaptor/scaffold protein Ruk/CIN85 may be a candidate since it plays important roles in the regulation of processes associated with cardiovascular and oncological diseases such as downregulation of receptor tyrosine kinases, apoptosis, adhesion and invasion. Therefore, it was the aim of this study to investigate the involvement of Ruk/CIN85 in the regulation of PAI-1 expression. It was found that Ruk/CIN85 induced PAI-1 mRNA and protein expression both under normoxia and hypoxia. The induction of PAI-1 expression by Ruk/CIN85 occurred at the transcriptional level since the half-life of PAI-1 mRNA was not affected in cells overexpressing Ruk/CIN85 and reporter gene assays using wild-type and mutant human PAI-1 promoter luciferase constructs showed that the hypoxia responsive element was responsible for Ruk/CIN85 effects. Further, knocking down HIF-1alpha abolished not only the hypoxia-dependent but also the Ruk/CIN85-dependent PAI-1 induction. In addition, transient or stable overexpression of Ruk/CIN85 also induced HIF-1alpha protein levels and HIF-1 activity and knocking down Ruk/CIN85 reversed these effects. Thereby, Ruk/CIN85 interfered with the proline hydroxylation-dependent HIF-1alpha protein destabilisation. Together, these results provide the first evidence that Ruk/CIN85 induces PAI-1 expression via modulation of HIF-1alpha stability.