Nrf2-Keap1 pathway promotes cell proliferation and diminishes ferroptosis.

Cancer cells are hallmarked by high proliferation and imbalanced redox consumption and signaling. Various oncogenic pathways such as proliferation and evading cell death converge on redox-dependent signaling processes. Nrf2 is a key regulator in these redox-dependent events and operates in cytoprotection, drug metabolism and malignant progression in cancer cells. Here, we show that patients with primary malignant brain tumors (glioblastomas, WHO °IV gliomas, GBM) have a devastating outcome and overall reduced survival when Nrf2 levels are upregulated. Nrf2 overexpression or Keap1 knockdown in glioma cells accelerate proliferation and oncogenic transformation. Further, activation of the Nrf2-Keap1 signaling upregulates xCT (aka SLC7A11 or system Xc-) and amplifies glutamate secretion thereby impacting on the tumor microenvironment. Moreover, both fostered Nrf2 expression and conversely Keap1 inhibition promote resistance to ferroptosis. Altogether, the Nrf2-Keap1 pathway operates as a switch for malignancy in gliomas promoting cell proliferation and resistance to cell death processes such as ferroptosis. Our data demonstrate that the Nrf2-Keap1 pathway is critical for cancer cell growth and operates on xCT. Nrf2 presents the Achilles' heel of cancer cells and thus provides a valid therapeutic target for sensitizing cancer for chemotherapeutics.

Activation of the astrocytic Nrf2/ARE system ameliorates the formation of demyelinating lesions in a multiple sclerosis animal model.

Oxidative stress critically contributes to the pathogenesis of a variety of neurodegenerative diseases such as multiple sclerosis. Astrocytes are the main regulators of oxidative homeostasis in the brain and dysregulation of these cells likely contributes to the accumulation of oxidative damage. The nuclear factor erythroid 2-related factor 2 (Nrf2) is the main transcriptional regulator of the anti-oxidant stress defense. In this study, we elucidate the effects of astrocytic Nrf2-activation on brain-intrinsic inflammation and lesion development. Cells deficient for the Nrf2 repressor kelch-like ECH-associated protein 1 (Keap1) are characterized by hyperactivation of Nrf2-signaling. Therefore, wild type mice and mice with a GFAP-specific Keap1-deletion were fed with 0.25% cuprizone for 1 or 3 weeks. Cuprizone intoxication induced pronounced oligodendrocyte loss, demyelination and reactive gliosis in wild type animals. In contrast, astrocyte-specific Nrf2-activation was sufficient to prevent oligodendrocyte loss and demyelination, to ameliorate brain intrinsic inflammation and to counteract axonal damage. Our results highlight the potential of the Nrf2/ARE system for the treatment of neuroinflammation in general and of multiple sclerosis in particular. © GLIA 2016;64:2219-2230.

[New from old : relevant factors for fracture healing in aging bone]. / Aus alt mach neu : Relevante Faktoren bei der Frakturheilung im Alter.

BACKGROUND: Fracture healing is a complex biological process with specific temporal expression patterns. During this process new bone tissue is formed, which is similar to the original bone in quality and structure. This occurs in four phases: inflammation, formation of a soft tissue callus, formation of a bony callus and remodelling of the bony callus. This needs the precise orchestration of each cell type involved. OBJECTIVES: This article presents details of the fracture healing phases and the relevant factors. During the aging process there is an increase of reactive oxygen species and a change in expression pattern of growth factors that have a negative effect on the fracture healing process. METHODS: A selective review of the literature was carried out in PubMed concerning the influence of aging on fracture healing. CONCLUSION: The healing process is regulated by systemic and local factors. An understanding of these processes and the changes during aging is necessary in order to improve the knowledge of delayed or lack of fracture healing during aging to decide when an intervention is needed.

[Maternal-placental interactions and fetal programming]. / Mütterlich-plazentare Interaktionen und fetale Programmierung.

Pregnancy-related complications not only represent a risk for maternal and fetal morbidity and mortality, but are also a risk for several diseases later in life. Many epidemiological studies have shown clear associations between an adverse intrauterine environment and an increased risk of diabetes, hypertension, cardiovascular disease, depression, obesity, and other chronic diseases in the adult. Some of these syndromes could be prevented by avoiding adverse stimuli or insults including psychological stress during pregnancy, intake of drugs, insufficient diet and substandard working conditions. Hence, all of these stimuli have the potential to alter health later in life. The placenta plays a key role in regulating the nutrient supply to the fetus and producing hormones that control the fetal as well as the maternal metabolism. Thus, any factor or stimulus that alters the function of the hormone producing placental trophoblast will provoke critical alterations of placental function and hence could induce programming of the fetus. The factors that change placental development may interfere with nutrient and oxygen supply to the fetus. This may be achieved by a direct disturbance of the placental barrier or more indirectly by, e. g., disturbing trophoblast invasion. For both path-ways, the respective pathologies are known: while preeclampsia is caused by alterations of the villous trophoblast, intra-uterine growth restriction is caused by insufficient invasion of the extravillous trophoblast. In both cases the effect can be undernutrition and/or fetal hypoxia, both of which adversely affect organ development, especially of brain and heart. However, the mechanisms responsible for disturbances of trophoblast differentiation and function remain elusive.

New Insights into the Pathogenesis of Preeclampsia - The Role of Nrf2 Activators and their Potential Therapeutic Impact.

Preeclampsia (PE), characterized by proteinuric hypertension and occurring in 2-3 % of all pregnancies, is one of the leading causes of maternal, fetal and neonatal morbidity and mortality. The etiology of PE still remains unclear and current treatments for this devastating disorder are still limited to symptomatic therapies. Placental oxidative stress may be a key intermediate step in the pathogenesis of PE; it has been related to excessive secretion of multiple antiangiogenic factors, mainly soluble fms-like tyrosine kinase-1 (sFlt-1) and soluble endoglin (sEng). The nuclear factor-erythroid 2-like 2 (Nrf2) pathway is one of the most important systems that enhance cellular protection against oxidative stress. Nrf2 serves as a master transcriptional regulator of the basal and inducible expression of a multitude of genes encoding detoxification enzymes and antioxidative proteins. Evidence for a link between Nrf2 and restoring the balance between pro- and antiangiogenic factors mainly through its downstream target protein heme oxygenase-1 (HO-1) has lately been discussed. HO-1 metabolizes heme to biliverdin, iron and carbon monoxide (CO). CO enhances vascular endothelial growth factor (VEGF) synthesis in vascular smooth muscle and promotes its relaxation and hence vasodilatation. In addition, HO-1 has been shown in vitro to inhibit the production of sFlt-1. A recent animal study demonstrated that the induction of HO-1 in a mouse model of PE attenuates the induced hypertension in pregnant mice. This provides compelling evidence for the protective role of Nrf2/HO-1 in pregnancy and identifies this pathway as a target to treat women with PE. We summarize the recent findings on the involvement of Nrf2 in the pathogenesis of PE, and provide an overview of the possible beneficial effects of Nrf2 inducers in PE.

PP015. Differential expression of Nrf2 and VEGF in human placental beds from normal and pregnancies complicated with preeclampsia and IUGR.

INTRODUCTION: Impaired trophoblast invasion into the uteroplacental arteries is accompanied with an evidence of oxidative stress in the extravillous trophoblast in preeclampsia complicated with IUGR. OBJECTIVES: Preeclampsia is characterised by increased lipid oxidation and diminished antioxidant capacity; recently, we have shown that PE is associated with an increased expression of the nuclear factor erythroid 2-related factor 2 (Nrf2) in villous cytotrophoblast. A possible relationship between the vascular endothelial growth factor (VEGF) and Nrf2 was established in vitro and the activation of Nrf2 pathway could lead to upregulation of VEGF synthesis through the induction of Nrf2-dependent Heme oxygenase-1 (HO-1). In this study the expression of Nrf2 and VEGF was determined in the interstitial and intramural extravillous trophoblast in normal pregnancies and those complicated by preeclampsia and intra-uterine growth restriction (IUGR). METHODS: Full-thickness uterine tissues were obtained from caesarean hysterectomies performed in 5 healthy normotensive women delivering term infants and from 5 women with severe early-onset preeclampsia and IUGR (29-34 week's gestation). The interstitial and intramural trophoblasts were studied by immunohistochemical analysis of paraffin sections stained with anti VEGF and anti Nrf2. RESULTS: Cases suffering from preeclampsia with IUGR were characterised by reduced invasion of extravillous trophoblast into uteroplacental arteries in the endometrial and myometrial segments. In addition, these cells showed an increased expression of Nrf2 in the pathological sections. The overexpression of Nrf2 in cases with preeclampsia was associated with restricted expression of VEGF in these cells compared to controls. CONCLUSION: Our data suggest that besides villous cytotrophoblast, also the extravillous trophoblast is a source of Nrf2-dependent genes. VEGF deficiency may cause higher oxidative stress in extravillous trophoblast in cases with preeclampsia with IUGR. The resulting reduced basal defence against oxidative stress and the higher vulnerability to oxidative damage may play a role in the limited trophoblast invasion into uteroplacental arteries in cases suffering from early onset preeclampsia and IUGR.

PP119. The role of the nuclear factor erythroid 2-related factor 2 (Nrf2) in preeclampsia.

INTRODUCTION: Preeclampsia is a multi-organ syndrome characterized by maternal endothelial damage, is an independent long-term risk factor for hypertension and cardiovascular disease. OBJECTIVES: In animal models the administration of the Vascular Endothelial Growth Factor (VEGF) could reverse the hypertensive signs accompanying this disease. In addition VEGF is implicated in placental oxidative stress during preeclampsia. One of the major cellular defence mechanisms against oxidative stress is the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2). Therefore, the activation of Nrf2 up regulates the HO-1/CO system. The principal aim of this work is to investigate whether the activation of Nrf2 raises VEGF levels by up regulation of CO release. METHODS: This study took place in vitro, the choriocarcinoma cell line BeWo cells and the primary human umbilical vein endothelial cells (HUVECs) were used to study the relationship between VEGF and an Nrf2 inducer Sulforaphane, a naturally occurring compound derived from broccoli. ELISA, Western blot assay and the Dual Luciferase Assay were both mainly applied for protein and VEGF activity analysis. RESULTS: It was found that activation of HO-1 expression via Nrf2/ARE pathway augmented the production of CO, which in turn up-regulated the gene expression of VEGF, and down regulated the production of the antiangiogenic protein, the VEGF antagonist sFlt-1. CONCLUSION: Nrf2 driven HO-1 expression elevates the levels of VEGF via CO production. In particular, activating of Nrf2 via sulforaphane, may have therapeutic potential in preeclampsia.

Platelet-released growth factors can accelerate tenocyte proliferation and activate the anti-oxidant response element.

Little is know about the pathophysiology of acute and degenerative tendon injuries. Although most lesions are uncomplicated, treatment is long and unsatisfactory in a considerable number of cases. Besides the common growth factors that were shown to be relevant for tendon integrity more recently protection against oxidative stress was shown to promote tendon healing. To improve tendon regeneration, many have advocated the use of platelet-rich plasma (PRP), a thrombocyte concentrate that can serve as an autologous source of growth factors. In this study, we investigated the effect of platelet-released growth factors (PRGF) on tenocytes. Tenocytes were isolated from the Achilles tendon of postnatal rats. Tenocyte cell cultures were stimulated with PRGF. We used a CyQuant assay and WST assay to analyse tendon cell growth and viability in different concentrations of PRGF. Migration and proliferation of cells grown in PRGF were assessed by a scratch test. A dual-luciferase assay was used to demonstrate the activation of the anti-oxidant response element (ARE) in tenocytes. A positive effect of PRGF could be shown on tendon cell growth and migratory capacity. PRGF activated the Nrf2-ARE pathway in a dose-dependent manner. Here, we provide evidence of a biological effect of PRGF on tenocytes by the promotion of tenocyte growth and activation of the Nrf2-ARE pathway. This is a novel aspect of the action of platelet concentrates on tendon growth.

Complex patterns of ADAM12 mRNA and protein splice variants in the human placenta.

AIMS: Trophoblast fusion in the placenta is prerequisite to successful pregnancy and the pathological conditions related to it. The presence of syncytin-1, is not sufficient to explain the complete event and ADAM12 is a major co-player candidate. Via differential splicing, the ADAM12 gene produces a short and a long form, being the ADAM12-S and the ADAM12-L respectively. METHODS AND RESULTS: We investigated the localisation of both variants in the human placenta using whole mount in situ hybridisation, immunohistochemistry and Northern blotting in 1st (n=8) and 3rd (n=8) trimester placentae and in the case of NB in several cell lines. In Northern blotting, 1st and 3rd trimester placentae were positive for the ADAM12-S and Bewo, 293HEK, JAR, leucocytes, macrophages, 1st and 3rd trimester placentae were positive for ADAM12-L. In whole mount in situ hybridisation, the 1st and 3rd trimester placental syncytium was positive for both variants. In immunohistochemistry, ADAM12-L localised in the cytotrophoblast of both 1st and 3rd trimester placentae, while ADAM12-S localised in the complete syncytium, often including the cytotrophoblast. CONCLUSION: The different localisation of ADAM12-S and ADAM12-L indicates a possible different role making ADAM12-L a candidate for the fusion event, while the syncytial localisation of the ADAM12-S makes it a candidate for cell-cell and cell-matrix interactions between the placental syncytium and the maternal interface.

Osteoblasts participate in the innate immunity of the bone by producing human beta defensin-3.

Gram-positive bacterial bone infections are an important cause of morbidity particularly in immunocompromised patients. Antimicrobial peptides (AP) are effectors of the innate immune system and directly kill microorganisms in the first hours after microbial infection. The aim of the present investigation was to study the expression and regulation of gram-positive specialized human beta-defensin-3 (HBD-3) in bone. Samples of healthy and osteomyelitic human bone were assessed for the expression of HBD-3. Using primary and immortalized osteoblasts (SAOS-2 cells), release and regulation of HBD-3 was evaluated after exposure to Staphylococcus aureus supernatant and/or corticosteroids using PCR, immunohistochemistry, Western blot and ELISA. To determine the role of toll-like-receptors-2 and -4 (TLR-2/-4), shRNA was used to downregulate TLRs. An osteomyelitis mouse model was created performed to investigate the release of murine beta-defensins using immunohistochemistry and RT-PCR. Cultured osteoblasts and human bone produce HBD-3 under standard conditions. The release increases within hours of bacterial supernatant exposure in cultured osteoblasts. This observation was not made in chronically infected bone samples. The shRNA-technology revealed the necessity of TLR-2 and -4 in HBD-3 induction in osteoblasts. Blocking protein synthesis with cycloheximide showed that the rapid release of HBD-3 is not dependent on a translational de novo synthesis and is not affected by glucocorticoids. The murine osteomyelitis model confirmed the in vivo release uptake of mouse beta-defensins-4 (MBD-4) in bone. This report shows the bacterial induction of HBD-3 via TLR-2 and -4 in osteoblasts and suggests a central role of antimicrobial peptides in the prevention of bacterial bone infection. The rapid and effective induction of HBD-3 in osteoblasts incubated with conditioned media from bacteria is more likely a result of a rapid secretion of preformed HBD-3 by osteoblasts rather than a result of enhanced biosynthesis. The increased incidence of gram-positive bacterial bone infection in patients with regular intake of glucocorticoids does not seem to be caused by a deranged HBD-3 release in osteoblasts.

The role of human beta-defensin-2 in bone.

Osteomyelitis often causes functional impairment due to tissue destruction. This report demonstrates a novel previously unappreciated role of osteoblasts. Samples of osteomyelitic bone and bacterially challenged osteoblasts produce increased amounts of antimicrobial peptides in order to combat bacterial bone infection. An osteomyelitis mouse model confirmed the osseous induction of the murine homologue of human beta-defensin-2, suggesting a central role in the prevention of bacterial bone infection. Antimicrobial peptides are effectors of the innate defence system and play a key role in host protection at cellular surfaces. Some of them are produced constitutively, whereas others are induced during infection. Human beta-defensins represent a major subclass of antimicrobial peptides and act as a first line of defence through their broad spectrum of potent antimicrobial activity. The aim of the present in-vitro and in-vivo investigations was to study the expression and regulation of human beta-defensin-2 in the case of bacterial bone infection and to analyse the effects of immunosuppressive drugs on bone-derived antimicrobial peptide expression. Samples of healthy human bone, osteomyelitic bone and cultured osteoblasts (hFOB cells) were assessed for the expression of human beta-defensin-2. Regulation of human beta-defensin-2 was studied in hFOB cells after exposure to bacterial supernatants, proinflammatory cytokines and immunosuppressive drugs (glucocorticoids and methotrexate) and was assayed by enzyme-linked immunosorbent assay. An osteomyelitis mouse model was performed to demonstrate the regulation of the murine homologue of human beta-defensin-2, named murine beta-defensin-3, by real-time reverse transcription-polymerase chain reaction and immunohistochemistry. Healthy human bone and cultured osteoblasts are able to produce human beta-defensin-2 under standard conditions. Samples of infected bone produce higher levels of endogenous antibiotics, such as human beta-defensin-2, when compared with samples of healthy bone. A clear induction of human beta-defensin-2 was observed after exposure of cultured osteoblasts to gram-positive bacteria or proinflammatory cytokines. Additional treatment with glucocorticoids or methotrexate prevented bacteria-mediated antimicrobial peptide induction in cultured osteoblasts. The osteomyelitis mouse model demonstrated transcriptional upregulation of the murine homologue of human beta-defensin-2, namely murine beta-defensin-3, in bone after intraosseous contamination of the tibia. Human and murine bone have the ability to produce broad-spectrum endogenous antibiotics when challenged by micro-organisms in vitro and in vivo. Immunosuppressive drugs, such as glucocorticoids or methotrexate, may increase the susceptibility to bone infection by decreasing antimicrobial peptide expression levels in case of microbial challenge. The induction of human beta-defensin-2 following bacterial contact suggests a central role of antimicrobial peptides in the prevention of bacterial bone infection.

Luteolin protects rat PC12 and C6 cells against MPP+ induced toxicity via an ERK dependent Keap1-Nrf2-ARE pathway.

Oxidative stress is central to neuronal damage in neurodegenerative diseases such as Parkinson's disease and Alzheimer's disease. In consequence, activation of the cerebral oxidative stress defence is considered as a promising strategy of therapeutic intervention. Here we demonstrate that the flavone luteolin confers neuroprotection against oxidative stress via activation of the nuclear factor erythroid-2-related factor 2 (Nrf2), a transcription factor central to the maintenance of the cellular redox homeostasis. Luteolin protects rat neural PC12 and glial C6 cells from N-methyl-4-phenyl-pyridinium (MPP+) induced toxicity in vitro and effectively activates Nrf2 as shown by ARE-reporter gene assays. This protection critically depends on the activation of Nrf2 since downregulation of Nrf2 by shRNA completely abrogates the protection of luteolin in vitro. Furthermore, the neuroprotective effect of luteolin is abolished by the inhibition of the luteolin-induced ERK1/2-activation. Our results highlight the relevance of Nrf2 for neural cell survival conferred by flavones. In particular, we identified luteolin as a promising lead for the search of orally available, blood brain barrier permeable compounds to support the therapy of neurodegenerative disorders.

Influence of polymorphisms of ABCB1 and ABCC2 on mRNA and protein expression in normal and cancerous kidney cortex.

There is increasing evidence that polymorphisms of the adenosine 5' triphosphate membrane transporters ABCB1 (P-glycoprotein, MDR1) may affect expression and function, whereas less information is available about the impact of ABCC2 (multidrug resistance-associated protein (MRP2)) single-nucleotide polymorphisms . Particularly, their role in human kidney for drug elimination and in the etiology of renal cell carcinoma is poorly understood. ABCB1 and ABCC2 mRNA and protein expression levels were determined by real-time polymerase chain reaction or immunohistochemistry in kidney cancer and adjacent unaffected cortex tissue of 82 nephrectomized renal cell cancer (RCC) patients (63 clear-cell RCC (CCRCC), 19 non-CCRCC). The DNA of all patients was genotyped for ABCB1 -2352G>A, -692T>C, 2677G>T/A (Ala893Ser/Thr), and 3435C>T, and ABCC2 -24C>T, 1249G>A (Val417Ile) and 3972C>T. ABCB1 and ABCC2 were less expressed in CCRCC than in normal cortex on mRNA as well as on protein level. Although the overall genotype frequency distribution did not differ between the patients and a matched control group, ABCB1 2677T/A and 3435T genotypes were associated with higher (P=0.02 and P=0.04) and ABCC2 -24 T with lower mRNA levels in normal tissues (0.03). The expression of ABCB1 and ABCC2 was not related to genetic variants in RCC tissue. In a reporter gene assay in HepG2 cells, the ABCC2 -24T construct showed an 18.7% reduced activity (P=0.003). In conclusion, ABCB1 and ABCC2 genotypes modulate the expression in the unaffected renal cortex of RCC patients, possibly contributing to inter-individual differences in drug and xenobiotics elimination. Their role in RCC cancer susceptibility or chemotherapy resistance needs further elucidation.