Doris Schneider

Selenium supplementation restores the antioxidative capacity and prevents cell damage in bone marrow stromal cells in vitro

Bone marrow stromal cells (BMSCs) and other cell populations derived from mesenchymal precursors are developed for cell‐based therapeutic strategies and undergo cellular stress during ex vivo procedures. Reactive oxygen species (ROS) of cellular and environmental origin are involved in redox signaling, cumulative cell damage, senescence, and tumor development. Selenium‐dependent (glutathione peroxidases [GPxs] and thioredoxin reductases [TrxRs]) and selenium‐independent (superoxide dismutases [SODs] and catalase [CAT]) enzyme systems regulate cellular ROS steady state levels. SODs process superoxide anion to hydrogen peroxide, which is subsequently neutralized by GPx and CAT; TrxR neutralizes other ROS, such as peroxinitrite. Primary BMSCs and telomerase‐immortalized human mesenchymal stem cells (hMSC‐TERT) express GPx1–3, TrxR1, TrxR2, SOD1, SOD2, and CAT. We show here that in standard cell cultures (5%–10% fetal calf serum, 5–10 nM selenite), the activity of antioxidative selenoenzymes is impaired in hMSC‐TERT and BMSCs. Under these conditions, the superoxide anion processing enzyme SOD1 is not sufficiently stimulated by an ROS load. Resulting oxidative stress favors generation of micronuclei in BMSCs. Supplementation of selenite (100 nM) restores basal GPx and TrxR activity, rescues basal and ROS‐stimulated SOD1 mRNA expression and activity, and reduces ROS accumulation in hMSC‐TERT and micronuclei generation in BMSCs. In conclusion, BMSCs in routine cell culture have low antioxi‐dative capacity and are subjected to oxidative stress, as indicated by the generation of micronuclei. Selenite supplementation of BMSC cultures appears to be an important countermeasure to restore their antioxidative capacity and to reduce cell damage in the context of tissue engineering and transplantation procedures.

Pulse treatment with zoledronic acid causes sustained commitment of bone marrow derived mesenchymal stem cells for osteogenic differentiation

The aminobisphosphonate zoledronic acid (ZA) is a bone seeking specific inhibitor of protein farnesylation and geranylgeranylation, which causes inhibition of osteoclast function and apoptosis. It is widely used as an osteoclast targeted antiresorptive treatment of metastatic bone disease, Pagets disease and osteoporosis. Mesenchymal stem cells (MSC) and osteoblast precursors can also be targets of bisphosphonates, but the clinical relevance of these effects is under debate. We show here that ZA in vitro causes inhibition of proliferation and induction of apoptosis in hMSC, when applied in concentrations of 20 and 50 microM for more than 24 h which can be rescued by treatment with 10 microM geranylgeranyl pyrophosphate (GGPP). However, pulse stimulation for 3 and 6 h with these concentrations and subsequent culture for up to 2 weeks under osteogenic conditions exerts sustained regulation of osteogenic marker genes in hMSC. The effect on gene regulation translates into marked enhancement of mineralization, as shown by alizarin red and alkaline phosphatase staining after 4 weeks of osteogenic culture. ZA, when applied as a pulse stimulus, might therefore also stimulate osteogenic differentiation in vivo, since muM plasma concentrations can be achieved by intravenous application of 5 mg in patients. These data set the stage for the future dissection of the effects of ZA and other aminobisphosphonates on cells beyond osteoclasts, with respect to cell differentiation in benign metabolic and to antitumor efficacy in metastatic bone diseases, as well as adverse events due to putative substance accumulation in bone during long-term treatment.

EXPRESSION VON SELENOPROTEINEN IN MONOZYTEN UND MAKROPHAGEN : IMPLIKATIONEN FUR DAS IMMUNSYSTEM

Zusammenfassung□ Monozyten differenzieren unter dem Einfluß von 1,25(OH)2 Vitamin D3 und anderen Faktoren aus myeloischen Vorläuferzellen. Koloniestimulierende Faktoren wie (Granulozyten-)Makrophagen-stimulierender Faktor (GMCSF und MCSF) propagieren die weitere Differenzierung zu Makrophagen. Die Aktivierung von Makrophagen und die Phagozytose von fremden Partikeln sind regelmä\ig begleitet von einem sogenannten „respiratory burst“, einer erhöhten Produktion von reaktiven Sauerstoffspezies (ROS), die durch den Enzymkomplex NADPH-Oxidase bewerkstelligt wird. Gleichzeitig wird eine Anzahl antioxidativer Enzyme exprimiert, um die Zelle vor den zytotoxischen Effekten der ROS zu schützen, die gegen die eingeschlossenen Mikroorganismen gerichtet sind und möglicherweise auch Genregulationen bewirken. Gut charakterisierte Selenoproteine, die in die antioxidative „Defense“-Reaktion der Zelle involviert sind, sind die selenabhängigen Glutathionperoxidasen (zytosolische [cGPx] und plasmatische [pGPx]) und die Thioredoxinreduktasen α und β (TrxRα/β). Das zytosolische Isoenzym der GPx (cGPx) und die TrxRα werden beide im Rahmen der Differenzierung durch 1,25 (OH)2 Vitamin D3 stimuliert. GPx-Isoenzyme neutralisieren H2O2. TrxR sind entweder direkt oder indirekt über ihren Kofaktor Thioredoxin in die Proteinfaltung involviert. Sie reduzieren Sulfhydrylgruppen und beeinflussen so zum Beispiel kritische Protein/Protein-Interaktionen und Protein/DNA-Interktionen; sie modulieren somit die Dimerisation und/oder die DNA-Bindung von Transkriptionsfaktoren (Glukokortikoidrezeptor und andere Steroidhormonrezeptoren, NFκB). Darüber hinaus wurde gezeigt, daß das antibiotische und zytotoxische Peptid NK-Lysin ein Substrat für die TrxRα ist (mit der Folge der Inaktivierung des Peptids), was nahelegt, daß TrxR ein protektiver Faktor für die Zelle selbst ist. Selen wird kontrolliert und spezifisch in Selenoproteine in Form von Selenozystein (Secys) eingebaut, welches in Anwesenheit einer dredimensionalen Haarnadelstruktur der 3′UTR (Secis-Element) am Codon UGA abgelesen wird, das ohne ein geeignetes Secis-Element und im Selenmangel als opales Stop-Codon fungiert. Die oben diskutierten Prozesse können also im Selenmangel alteriert sein und andererseits durch Selensupplementation moduliert werden.□ Wir haben die TrxRα als 1,25(OH)2 Vitamin D3-responsives Protein in Monozyten charakterisiert und gezeigt, daß die Aktivität der GPx und der TrxR durch Selensupplementation in vitro und ex vivo stimuliert wird. Neuere Arbeiten zeigen, daß Thioredoxin, ein wichtiges Substrat der TrxR, nach Behandlung von Zellen mit H2O2 schnell in den Zellkern wandert. Darüber hinaus ist aber wenig bekannt über die Kompartimentalisierung des „respiratory burst“ in der Zelle und die intrazelluläre Lokalisation der antioxidativen Enzyme während dieses Vorgangs. Die Makrophagenfunktion ist alteriert, wenn der „respiratory burst“ insuffizient ist, wie zum Beispiel bei der hereditären chronischen granulomatösen Erkrankung. Andererseits ist diese aber auch gestört, wenn ein Defizit an antioxidativen Enzymen besteht. Thioredoxin wurde als Wachstumsfaktor für Lymphozyten identifiziert und ist in den „Crosstalk“ zwischen Makrophagen und Lymphozyten involviert. Die Relevanz der beschriebenen und anderer noch nicht charakterisierter Selenoproteine von Monozyten bleibt näher zu charakterisieren, wie auch die der Supplementation von Selen generell in der Nahrungskette und speziell in Situationen von kritischen Infektionen und bei der Entwicklung der Autoimmunität.Abstract□ Monocytes differentiate from myeloid precursors towards the macrophage state of differentiation under the influence of 1,25-dihydroxy vitamin D3 (1,25 [OH]2 vitamin D3) and other factors and this is further propagated by colony stimulating factors (MCSF and GMCSF). Macrophage activation and phagocytosis of foreign particles are regularly accompanied by a so called “respiratory burst”, an increase in the production of reactive oxygen species (ROS), exerted by the enzyme complex NADPH oxidase. A number of antioxidant enzymes is expressed at the same time to protect the cells from the cytotoxic effects of ROS directed against engulfed microorganisms. The selenium-dependent glutathione peroxidases and thioredoxin reductases are important examples. The cytosolic GPx isoenzyme (cGPx) and thioredoxin reductase α (TrxRα) are upregulated during the process of differentiation and under the influence of 1,25 (OH)2 vitamin D3. GPx isoenzymes neutralize H2O2. TrxR reduce sulfhydryl-groups like in cysteins either directly or via their cofactor thioredoxin and thus are involved in protein folding and critical protein-protein and protein-DNA interactions, e. g. modulation of dimerization and/or DNA-binding and ligand binding of transcription factors (glucocorticoid receptor and other steroid receptors, NFκB). In addition, the antibiotic peptide NK-lysin was shown to be a substrate for TrxRα, suggesting that TrxR protects the cell itself from the cytotoxic effects of NK-lysin. Selenium is incorporated into selenocysteine (Secys) in a regulated fashion in the presence of a hairpin structure (Secis element) in the 3′UTR of selenoprotein genes. Secis elements direct the insertion of Secys at UGA codons, which function as opal stop codons in the absence of a suitable Secis element and in selenium deficiency. The above mentioned processes might therefore be altered in relative selenium deficiency or vice versa be upregulated through selenium supplementation.□ We have shown that TrxRα is a 1,25 (OH)2 vitamin D3-responsive early gene in monocytic cells and that TrxR activity as well as GPx activity in these cells can be upregulated by the addition of selenium in vitro and ex vivo. Recent work demonstrates that thioredoxin rapidly enters the cell nucleus upon treatment of cells with H2O2, but little is known about the compartimentalization of the respiratory burst and the intracellular localization of antioxidant enzymes during that process. Macrophage function is insufficient if the generation of a respiratory burst is altered like in hereditary chronic granulomatous disease on one hand, but on the other hand is as well disturbed, if there is a lack in antioxidant enzyme activity. Thioredoxin has been identified as a lymphocyte growth factor and might therefore be involved in the crosstalk between macrophages and lymphocytes. The relevance of the above mentioned and other yet undefined monocytic selenoproteins remains to be elucidated in detail as well as the relevance of selenium supplementation in nutrition in general and in situations of critical infectious disease and autoimmunity.

Contact of myeloma cells induces a characteristic transcriptome signature in skeletal precursor cells –Implications for myeloma bone disease

Physical interaction of skeletal precursors with multiple myeloma cells has been shown to suppress their osteogenic potential while favoring their tumor-promoting features. Although several transcriptome analyses of myeloma patient-derived mesenchymal stem cells have displayed differences compared to their healthy counterparts, these analyses insufficiently reflect the signatures mediated by tumor cell contact, vary due to different methodologies, and lack results in lineage-committed precursors. To determine tumor cell contact-mediated changes on skeletal precursors, we performed transcriptome analyses of mesenchymal stem cells and osteogenic precursor cells cultured in contact with the myeloma cell line INA-6. Comparative analyses confirmed dysregulation of genes which code for known disease-relevant factors and additionally revealed upregulation of genes that are associated with plasma cell homing, adhesion, osteoclastogenesis, and angiogenesis. Osteoclast-derived coupling factors, a dysregulated adipogenic potential, and an imbalance in favor of anti-anabolic factors may play a role in the hampered osteoblast differentiation potential of mesenchymal stem cells. Angiopoietin-Like 4 (ANGPTL4) was selected from a list of differentially expressed genes as a myeloma cell contact-dependent target in skeletal precursor cells which warranted further functional analyses. Adhesion assays with full-length ANGPTL4-coated plates revealed a potential role of this protein in INA-6 cell attachment. This study expands knowledge of the myeloma cell contact-induced signature in the stromal compartment of myelomatous bones and thus offers potential targets that may allow detection and treatment of myeloma bone disease at an early stage.

Overexpression of tissue-nonspecific alkaline phosphatase increases the expression of neurogenic differentiation markers in the human SH-SY5Y neuroblastoma cell line.

Patients suffering from the rare hereditary disease hypophosphatasia (HPP), which is based on mutations in the ALPL gene, tend to develop central nervous system (CNS) related issues like epileptic seizures and neuropsychiatric illnesses such as anxiety and depression, in addition to well-known problems with the mineralization of bones and teeth. Analyses of the molecular role of tissue-nonspecific alkaline phosphatase (TNAP) in transgenic SH-SY5Y(TNAPhigh) neuroblastoma cells compared to SH-SY5Y(TNAPlow) cells indicate that the enzyme influences the expression levels of neuronal marker genes like RNA-binding protein, fox-1 homolog 3 (NEUN) and enolase 2, gamma neuronal (NSE) as well as microtubule-binding proteins like microtubule-associated protein 2 (MAP2) and microtubule-associated protein tau (TAU) during neurogenic differentiation. Fluorescence staining of SH-SY5Y(TNAPhigh) cells reveals TNAP localization throughout the whole length of the developed projection network and even synapsin Ι co-localization with strong TNAP signals at some spots at least at the early time points of differentiation. Additional immunocytochemical staining shows higher MAP2 expression in SH-SY5Y(TNAPhigh) cells and further a distinct up-regulation of tau and MAP2 in the course of neurogenic differentiation. Interestingly, transgenic SH-SY5Y(TNAPhigh) cells are able to develop longer cellular processes compared to control cells after stimulation with all-trans retinoic acid (RA). Current therapies for HPP prioritize improvement of the bone phenotype. Unraveling the molecular role of TNAP in extraosseous tissues, like in the CNS, will help to improve treatment strategies for HPP patients. Taking this rare disease as a model may also help to dissect TNAPs role in neurodegenerative diseases and even improve future treatment of common pathologies.