Elitsa Y. Dimova

Reactive oxygen species, nutrition, hypoxia and diseases: Problems solved?

Within the last twenty years the view on reactive oxygen species (ROS) has changed; they are no longer only considered to be harmful but also necessary for cellular communication and homeostasis in different organisms ranging from bacteria to mammals. In the latter, ROS were shown to modulate diverse physiological processes including the regulation of growth factor signaling, the hypoxic response, inflammation and the immune response. During the last 60–100 years the life style, at least in the Western world, has changed enormously. This became obvious with an increase in caloric intake, decreased energy expenditure as well as the appearance of alcoholism and smoking; These changes were shown to contribute to generation of ROS which are, at least in part, associated with the occurrence of several chronic diseases like adiposity, atherosclerosis, type II diabetes, and cancer. In this review we discuss aspects and problems on the role of intracellular ROS formation and nutrition with the link to diseases and their problematic therapeutical issues.

Metabolic, hormonal and environmental regulation of plasminogen activator inhibitor-1 (PAI-1) expression: Lessons from the liver

Plasminogen activator inhibitor-1 (PAI-1) controls the regulation of the fibrinolytic system in blood by inhibiting both urokinase-type and tissue-type plasminogen activators. Enhanced levels of PAI-1 are found in patients with type 2 diabetes mellitus which is associated with a dysbalance in glucose and lipid homeostasis. Especially a defective insulin response in the liver contributes to the development of hyperglycemia, dyslipidemia and peripheral insulin resistance and may contribute to hepatic over-expression of PAI-1 in diabetes type 2. Furthermore, a substantial upregulation of PAI-1 expression has also been shown in a variety of liver injury models. Thus, the liver appears to be not only a major site of PAI-1 synthesis in response to hormonal changes, but also in response to a variety of other pathological events. PAI-1 expression in liver largely depends on activation of signalling pathways and transcriptional regulators which may be the basis for a new level of cross-talk between different signalling pathways and thus may represent attractive therapeutic candidates. This article will primarily focus on the regulation of PAI-1 expression in liver cells and discuss potential cross-talks between metabolic, hormonal and environmental signals.

FoxO1 and HNF-4 Are Involved in Regulation of Hepatic Glucokinase Gene Expression by Resveratrol

Resveratrol, a polyphenol derived from grapes, exerts important effects on glucose and lipid metabolism, yet detailed mechanisms mediating these effects remain unknown. The liver plays a central role in energy homeostasis, and glucokinase (GK) is a key enzyme involved in glucose utilization. Resveratrol activates SIRT1 (sirtuin 1), which promotes deacetylation of the forkhead transcription factor FoxO1. Previously, we reported that FoxO1 can suppress and that HNF-4 can stimulate GK expression in the liver. Here, we examined the role of FoxO1 and HNF-4 in mediating resveratrol effects on liver GK expression. Resveratrol suppressed hepatic GK expression in vivo and in isolated hepatocytes, and knocking down FoxO1 with shRNAs disrupted this effect. Reporter gene, gel shift, supershift assay, and chromatin immunoprecipitation studies show that FoxO1 binds to the GK promoter and that the interplay between FoxO1 and HNF-4 within the GK promoter is essential for mediating the effects of resveratrol. Resveratrol promotes deacetylation of FoxO1 and enhances its recruitment to the FoxO-binding element. Conversely, resveratrol suppresses recruitment of HNF-4 to its binding site, and knockdown of FoxO1 blocks this effect of resveratrol. Coprecipitation and chromatin immunoprecipitation studies show that resveratrol enhances interaction between FoxO1 and HNF-4, reduces binding of HNF-4 to its own site, and promotes its recruitment to the FoxO site in a FoxO1-dependent manner. These results provide the first evidence that resveratrol represses GK expression via FoxO1 and that the interaction between FoxO1 and HNF-4 contributes to these effects of resveratrol.

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.

Cell type-dependent regulation of the hypoxia-responsive plasminogen activator inhibitor-1 gene by upstream stimulatory factor-2.

Transcriptional regulation of the plasminogen activator inhibitor type-1 (PAI-1) gene is an important issue since PAI-1 plays a crucial role in various pathological conditions. The transcription factor USF-2 was shown to be a negative regulator for rat PAI-1 expression, and therefore it was the aim of this study to evaluate the role of USF-2 for human PAI-1 expression. We found in human hepatoma cells (HepG2) that USF-2 induced human PAI-1 expression via two classical E-boxes and the hypoxia-responsive element (HRE) within the promoter. Gel-shift analyses showed that E-box 4 and E-box 5 bound USFs, and although the HRE contributed to the USF-dependent effects, it did not bind them. By contrast, USF-2 inhibited PAI-1 promoter activity in primary rat hepatocytes suggesting that PAI-1 expression depends on either the promoter context or USF activity which might be cell type-specific. Cotransfection of human or rat PAI-1 promoter luciferase constructs with expression vectors for wild-type USF-2 or USF-2 mutants in human HepG2 and rat H4IIE cells as well as in primary rat hepatocytes revealed that the effects of USF on PAI-1 expression depend on the cell type rather than the promoter context and that the USF-specific region domain of USF accounts for the observed cell type-specific effects.

Transcriptional regulation of plasminogen activator inhibitor-1 expression by insulin-like growth factor-1 via MAP kinases and hypoxia-inducible factor-1 in HepG2 cells

Insulin-like growth factor 1 (IGF-1) and plasminogen activator inhibitor-1 (PAI-1) appear to play a crucial role in a number of processes associated with growth and tissue remodelling. IGF-1 was shown to enhance PAI-1 expression in primary hepatocytes and HepG2 hepatoma cells, but the molecular mechanisms underlying this effect have not been fully elucidated. In this study, we investigated the transcriptional mechanism and the signaling pathway by which IGF-1 mediates induction of PAI-1 expression in HepG2 cells. By using human PAI-1 promoter reporter gene assays we found that mutation of the hypoxia responsive element (HRE), which could bind hypoxia-inducible factor-1 (HIF-1), nearly abolished the induction by IGF-1. We found that IGF-1-induced up-regulation of PAI-1 expression was associated with activation of HIF-1 alpha. Furthermore,IGF-1 enhanced HIF-1alpha protein levels and HIF-1 DNA-binding to each HRE,E4 and E5 as shown by EMSA. Mutation of the E-boxes, E4 and E5, did not affect the IGF-1-dependent induction of PAI-1 promoter constructs under normoxia but abolished the effect of IGF-1 under hypoxia. Inhibition of either the PI3K by LY294002 or ERK1/2 by U0126 reduced HIF-1alpha protein levels while both inhibitors together completely abolished the IGF-1 effect on HIF-1alpha. Remarkably, transfection of HepG2 cells with vectors expressing a dominant-negative PDK1 or the PKB inhibitor, TRB3, did not influence while dominant-negative Raf inhibited the IGF-1 effect on HIF-1alpha. Thus, IGF-1 activates human PAI-1 gene expression through activation of the PI3-kinase and ERK1/2 via HIF-1alpha.

Hypoxia-Inducible Factors (HIFs) and Phosphorylation: Impact on Stability, Localization, and Transactivity

The hypoxia-inducible factor α-subunits (HIFα) are key transcription factors in the mammalian response to oxygen deficiency. The HIFα regulation in response to hypoxia occurs primarily on the level of protein stability due to posttranslational hydroxylation and proteasomal degradation. However, HIF α-subunits also respond to various growth factors, hormones, or cytokines under normoxia indicating involvement of different kinase pathways in their regulation. Because these proteins participate in angiogenesis, glycolysis, programmed cell death, cancer, and ischemia, HIFα regulating kinases are attractive therapeutic targets. Although numerous kinases were reported to regulate HIFα indirectly, direct phosphorylation of HIFα affects HIFα stability, nuclear localization, and transactivity. Herein, we review the role of phosphorylation-dependent HIFα regulation with emphasis on protein stability, subcellular localization, and transactivation.

Kinases as Upstream Regulators of the HIF System: Their Emerging Potential as Anti-Cancer Drug Targets

The hypoxia-inducible factor-1 (HIF-1) is a key regulator in the mammalian response to oxygen deficiency under both physiological and pathological conditions such as cancer. A number of studies indicated an association between tumor hypoxia, increased hypoxia-inducible factor (HIF-1alpha) levels and a poor prognosis. The HIF-1alpha regulation in response to hypoxia occurs primarily on the level of protein stability due to posttranslational hydroxylation. However, HIFalpha-subunits also respond to various growth factors, hormones, or cytokines under non-hypoxic conditions implicating the involvement of different kinase pathways in their regulation thereby increasing the interest in HIF-1alpha as a new drug target. Herein, we review current knowledge about phosphorylation-dependent HIF-1alpha regulation, HIF-1alpha protein-protein interactions and subcellular localization with emphasis on new therapeutic strategies targeting the HIF pathway.

Obesity and inflammation: reduced cytokine expression due to resveratrol in a human in vitro model of inflamed adipose tissue.

Obesity is associated with an inflammatory status and linked with a number of pathophysiological complications among them cardiovascular disease, type 2 diabetes mellitus, or the metabolic syndrome. Resveratrol was proposed to improve obesity-related inflammatory problems, but the effect of resveratrol on cytokine expression in obesity is not completely understood. In this study, we used an in vitro model of human adipose tissue inflammation to examine the effects of resveratrol on the production of the inflammatory cytokines interleukin 6 (IL-6), IL-8, and monocyte chemoattractant protein 1 (MCP-1). We found that resveratrol reduced IL-6, IL-8, and MCP-1 levels in a concentration-dependent manner in adipocytes under inflammatory conditions. Further experiments showed that the action of resveratrol was mainly due to its NFκB inhibitory potential. Thus, our data support the concept that resveratrol can alleviate obesity-induced up-regulation of inflammatory cytokines providing a new insight toward novel treatment options in obesity.

The MAPK Pathway and HIF‐1 Are Involved in the Induction of the Human PAI‐1 Gene Expression by Insulin in the Human Hepatoma Cell Line HepG2

Abstract:  Enhanced levels of plasminogen activator inhibitor‐1 (PAI‐1) are considered to be a risk factor for pathological conditions associated with hypoxia or hyperinsulinemia. The expression of the PAI‐1 gene is increased by insulin in different cells, although, the molecular mechanisms behind insulin‐induced PAI‐1 expression are not fully known yet. Here, we show that insulin upregulates human PAI‐1 gene expression and promoter activity in HepG2 cells and that mutation of the hypoxia‐responsive element (HRE)–binding hypoxia‐inducible factor‐1 (HIF‐1) abolished the insulin effects. Mutation of E‐boxes E4 and E5 abolished the insulin‐dependent activation of the PAI‐1 promoter only under normoxia, but did not affect it under hypoxia. Furthermore, the insulin effect was associated with activation of HIF‐1α via mitogen‐activated protein kinases (MAPKs) but not PDK1 and PKB in HepG2 cells. Furthermore, mutation of a putative FoxO1 binding site which was supposed to be involved in insulin‐dependent PAI‐1 gene expression influenced the insulin‐dependent activation only under normoxia. Thus, insulin‐dependent PAI‐1 gene expression might be regulated by the action of both HIF‐1 and FoxO1 transcription factors.