Andrea Wintersperger

Vitamin E (alpha-tocopherol) attenuates cyclo-oxygenase 2 transcription and synthesis in immortalized murine BV-2 microglia.

One of the immediate early microglial genes that are up-regulated in response to proinflammatory stimuli is cyclo-oxygenase 2 (COX-2). In the present study, we have investigated the effects of alpha-tocopherol (alpha TocH), an essential constituent of the nervous system, on the activation of COX-2 in lipopolysaccharide (LPS)-stimulated mouse BV-2 microglia. In unstimulated BV-2 cells, COX-2 mRNA and protein were almost undetectable but were strongly up-regulated in response to LPS. Activation of COX-2 protein synthesis in LPS-stimulated BV-2 cells involved activation of the extracellular-signal-regulated kinase 1/2 (ERK1/2) and p38 mitogen-activated protein kinase (MAPK) pathway and was sensitive to the protein kinase C (PKC) inhibitors staurosporine and chelerythrine, and the MAP kinase/ERK kinase 1/2 inhibitors PD98059 and U0126. Supplementation of BV-2 cells with alpha TocH before LPS stimulation resulted in pronounced up-regulation of protein phosphatase 2A (PP2A) activity, down-regulation of PKC activity, ERK1/2 phosphorylation and nuclear factor kappa B (NF kappa B) activation. As a result, COX-2 protein levels and prostaglandin E(2) production were significantly lower in alpha TocH-supplemented cells. The effects of alpha TocH on PKC activity could be reverted by calyculin A and okadaic acid, two PP inhibitors. In summary, our results suggest that alpha TocH activates microglial PP2A activity and thereby silences an LPS-activated PKC/ERK/NF kappa B signalling cascade resulting in significantly attenuated COX-2 protein synthesis. These in vitro results imply that alpha TocH could induce quiescence to pathways that are associated with acute or chronic inflammatory conditions in the central nervous system.

Lipoprotein-associated α-tocopheryl-succinate inhibits cell growth and induces apoptosis in human MCF-7 and HBL-100 breast cancer cells

K-Tocopheryl succinate (K-TS) is a potent inhibitor of tumor cell proliferation. The goal of the present study was to investigate whether and to what extent K-TS associates with plasma lipoproteins and if K-TS-enriched lipoproteins inhibit breast cancer cell growth in a manner comparable to the free drug. In vitro enrichment of human plasma revealed that K-TS readily associated with the main lipoprotein classes, findings confirmed in vivo in mice. At the highest K-TS concentrations, lipoproteins carrying 50 000 (VLDL), 5000 (LDL) and 700 (HDL) K-TS molecules per lipoprotein particle were generated. KTS enrichment generated lipoprotein particles with slightly decreased density and increased particle radius. To study whether the level of LDL-receptor (LDL-R) expression affects K-TS uptake from apoB/E containing lipoprotein particles human breast cancer cells with low (MCF-7) and normal (HBL-100) LDL-R expression were used. The uptake of free, VLDL- and (apoE-free) HDL3-associated K-TS was nearly identical for both cell lines. In contrast, uptake of LDL-associated K-TS by HBL-100 cells (normal LDL-R expression) was about twice as high as compared to MCF-7 cells (low LDL-R expression). VLDL and LDL-associated K-TS inhibited proliferation most effectively at the highest concentration of K-TS used (100% inhibition of MCF-7 growth with 20 Wg/ml of lipoprotein-associated K-TS). However, also K-TS-free VLDL and LDL inhibited HBL-100 cell proliferation up to 55%. In both cell lines, K-TS-enriched HDL3 inhibited cell growth by 40^60%. Incubation of both cell lines in the presence of free or lipoprotein-associated K-TS resulted in DNA fragmentation indicative of apoptosis. Collectively, the present findings demonstrate that: (1) K-TS readily associates with lipoproteins in vitro and in vivo; (2) the lipoprotein-enrichment efficacy was dependent on the particle size and/or the triglyceride content of the lipoprotein; (3) uptake of LDL-associated K-TS was apparently dependent on the level of LDL-R expression ; and (4) lipoproteins were efficient K-TS carriers inducing reduced cell proliferation rates and apoptosis in human breast cancer cells as observed for the free drug. fl 2000 Elsevier Science B.V. All rights reserved.

Modulation of microglial superoxide production by α-tocopherol in vitro: attenuation of p67phox translocation by a protein phosphatase-dependent pathway

As in other phagocytic cells, the NADPH‐oxidase system in microglia is thought to be primarily responsible for the production of superoxide anion radicals (O2−·), a potentially cytotoxic reactive oxygen species. The assembly of a functional NADPH‐oxidase complex at the plasma membrane depends on the phosphorylation and subsequent translocation of several cytosolic subunits. Immunocytochemical and subcellular fractionation experiments performed during the present study revealed that the NADPH‐oxidase subunit p67phox translocates from the cytosol to the plasma membrane upon stimulation. Pre‐incubation of microglia in α‐tocopherol (αTocH) containing medium decreased O2−· production in a time‐ and concentration‐dependent manner, findings attributed to attenuated p67phox translocation to the plasma membrane. Moreover, αTocH‐supplementation of the culture medium resulted in decreased microglial protein kinase C (PKC) activities, an effect that could be partially or completely reversed by the addition of protein phosphatase inhibitors (okadaic acid and calyculin A). The addition of the PKC‐inhibitor staurosporine inhibited the microglial respiratory burst in a manner comparable to αTocH. The addition of okadaic acid or calyculin A completely restored O2−· production in αTocH‐supplemented cells. The present findings suggest that αTocH inactivates PKC via a PP1 or PP2A‐mediated pathway and, as a consequence, blocks the phosphorylation‐dependent translocation of p67phox to the plasma membrane. As a result, O2−· production by the microglial NADPH‐oxidase system is substantially inhibited.

Lysophosphatidic acid receptor activation affects the C13NJ microglia cell line proteome leading to alterations in glycolysis, motility, and cytoskeletal architecture

Microglia, the immunocompetent cells of the CNS, are rapidly activated in response to injury and microglia migration towards and homing at damaged tissue plays a key role in CNS regeneration. Lysophosphatidic acid (LPA) is involved in signaling events evoking microglia responses through cognate G protein‐coupled receptors. Here we show that human immortalized C13NJ microglia express LPA receptor subtypes LPA1, LPA2, and LPA3 on mRNA and protein level. LPA activation of C13NJ cells induced Rho and extracellular signal‐regulated kinase activation and enhanced cellular ATP production. In addition, LPA induced process retraction, cell spreading, led to pronounced changes of the actin cytoskeleton and reduced cell motility, which could be reversed by inhibition of Rho activity. To get an indication about LPA‐induced global alterations in protein expression patterns a 2‐D DIGE/LC‐ESI‐MS proteomic approach was applied. On the proteome level the most prominent changes in response to LPA were observed for glycolytic enzymes and proteins regulating cell motility and/or cytoskeletal dynamics. The present findings suggest that naturally occurring LPA is a potent regulator of microglia biology. This might be of particular relevance in the pathophysiological context of neurodegenerative disorders where LPA concentrations can be significantly elevated in the CNS.

Inhibition of lung carcinoma cell growth by high density lipoprotein-associated α-tocopheryl-succinate

Abstractα-Tocopheryl-succinate (αTS) is a synthetic, anti-neoplastic derivative of α-tocopherol. Here we studied the effects of free and high-density lipoprotein subclass 3 (HDL3)-associated αTS on the growth of human (A549) and mouse Lewis (LL2) lung carcinoma cells. Both free and HDL3-associated αTS inhibited A549 growth in a time- and concentration-dependent manner. Treatment of A549 cells with αTS-enriched HDL3 led to DNA fragmentation and a time-dependent decrease in immunoreactivity of poly(ADP-ribose)polymerase. Uptake experiments revealed a high capacity for selective αTS uptake in excess of holoparticle endocytosis. Overexpression of scavenger receptor class B, type I (SR-BI), the prime receptor mediating selective lipid uptake, in A549 cells resulted in significantly increased selective αTS uptake, a finding associated with complete cellular growth arrest. The present in vitro findings were verified in an in vivo model: tumor inoculation in C57BL6 was performed with either wild-type, β-galactosidase- or SR-BI-overexpressing LL2 cells. After tumor inoculation, the animals received six consecutive intravenous injections of αTS. This experimental setup resulted in significantly reduced tumor burden in animals that were inoculated with SR-BI-overexpressing LL2 cells but not in animals inoculated with wild-type or β-galactocidase-transfected cells. Based on our in vitro and in vivo findings, we propose that SR-BI could provide a novel route for HDL3-mediated drug delivery of anti-neoplastic drugs.

Afamin is synthesized by cerebrovascular endothelial cells and mediates α-tocopherol transport across an in vitro model of the blood-brain barrier.

α‐Tocopherol (αTocH), a member of the vitamin E family, is essential for normal neurological function. Despite the importance of αTocH transport into the CNS, transfer mechanisms across the blood–brain barrier (BBB) are not entirely clear. We here investigate whether afamin, a known αTocH‐binding protein, contributes to αTocH transport across an in vitro model of the BBB consisting of primary porcine brain capillary endothelial cells (BCEC) and basolaterally cultured astrocytoma cells. Exogenously added afamin had no adverse effects on BCEC viability or barrier function and was transported across BCEC Transwell cultures. Furthermore, αTocH transport across polarized BCEC cultures to astrocytoma cells is facilitated by afamin, though to a lesser extent than by high‐density lipoprotein‐mediated transport, an essential and in vivo operating αTocH import pathway at the cerebrovasculature. We also demonstrate that porcine BCEC endogenously synthesize afamin. In line with these in vitro findings, afamin was detected by immunohistochemistry in porcine, human postmortem, and mouse brain, where prominent staining was observed almost exclusively in the cerebrovasculature. The demonstration of afamin mRNA expression in isolated brain capillaries suggests that afamin might be a new family member of binding/transport proteins contributing to αTocH homeostasis at the BBB in vivo.

On the role of 25-hydroxycholesterol synthesis by glioblastoma cell lines. Implications for chemotactic monocyte recruitment

Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor and is invariably fatal to affected patients. Oxysterols belong to a class of bioactive lipids that are implicated in neurological disease and are associated with various types of cancer. Here, we investigated expression and transcriptional regulation of cholesterol 25-hydroxylase (CH25H) in human U87MG and GM133 glioblastoma cell lines. We demonstrate that in both cell lines transcription and translation of CH25H are increased in response to TNFα and IL1β. In parallel, both cell lines upregulate 25-hydroxycholesterol (25-OHC) synthesis and secretion to levels comparable to bone marrow-derived mouse macrophages under inflammatory conditions. To determine whether 25-OHC acts as chemoattractant for tumor-associated macrophages, the human THP-1 monoblastic leukemia cell line was treated with varying amounts of the oxysterol. Experiments revealed that 25-OHC and lipid extracts isolated from GM133-conditioned medium (containing 7-fold higher 25-OHC concentrations than U87MG medium) induce chemotactic migration of THP-1 cells. Of note, 25-OHC also induced the migration of primary human peripheral blood monocytes. In response to exogenously added 25-OHC, THP-1 cells reorganized intermediate filament-associated vimentin to more cortical and polarized structures. Chemotactic migration of monocytes in response to 25-OHC was pertussis toxin-sensitive, indicating the involvement of G protein-coupled receptors. Using RNA interference we demonstrated that G protein-coupled receptor 183 (EBI2) contributes to 25-OHC-mediated chemotactic migration of THP-1 cells. These in vitro data indicate that GBM-derived and secreted 25-OHC may be involved in the recruitment of immune-competent cells to a tumor via EBI2.

Protein kinase D2 regulates migration and invasion of U87MG glioblastoma cells in vitro.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor, which, despite combined modality treatment, reoccurs and is invariably fatal for affected patients. Recently, a member of the serine/threonine protein kinase D (PRKD) family, PRKD2, was shown to be a potent mediator of glioblastoma growth. Here we studied the role of PRKD2 in U87MG glioblastoma cell migration and invasion in response to sphingosine-1-phosphate (S1P), an activator of PRKD2 and a GBM mitogen. Time-lapse microscopy demonstrated that random cell migration was significantly diminished in response to PRKD2 silencing. The pharmacological PRKD family inhibitor CRT0066101 decreased chemotactic migration and invasion across uncoated or matrigel-coated Transwell inserts. Silencing of PRKD2 attenuated migration and invasion of U87MG cells even more effectively. In terms of downstream signaling, CRT0066101 prevented PRKD2 autophosphorylation and inhibited p44/42 MAPK and to a smaller extent p54/46 JNK and p38 MAPK activation. PRKD2 silencing impaired activation of p44/42 MAPK and p54/46 JNK, downregulated nuclear c-Jun protein levels and decreased c-JunS73 phosphorylation without affecting the NFκB pathway. Finally, qPCR array analyses revealed that silencing of PRKD2 downregulates mRNA levels of integrin alpha-2 and -4 (ITGA2 and -4), plasminogen activator urokinase (PLAU), plasminogen activator urokinase receptor (PLAUR), and matrix metallopeptidase 1 (MMP1). Findings of the present study identify PRKD2 as a potential target to interfere with glioblastoma cell migration and invasion, two major determinants contributing to recurrence of glioblastoma after multimodality treatment.

1-Oleyl-lysophosphatidic acid (LPA) promotes polarization of BV-2 and primary murine microglia towards an M1-like phenotype

BackgroundMicroglia, the immunocompetent cells of the CNS, rapidly respond to brain injury and disease by altering their morphology and phenotype to adopt an activated state. Microglia can exist broadly between two different states, namely the classical (M1) and the alternative (M2) phenotype. The first is characterized by the production of pro-inflammatory cytokines/chemokines and reactive oxygen and/or nitrogen species. In contrast, alternatively activated microglia are typified by an anti-inflammatory phenotype supporting wound healing and debris clearance. The objective of the present study was to determine the outcome of lysophosphatidic acid (LPA)-mediated signaling events on microglia polarization.MethodsLPA receptor expression and cyto-/chemokine mRNA levels in BV-2 and primary murine microglia (PMM) were determined by qPCR. M1/M2 marker expression was analyzed by Western blotting, immunofluorescence microscopy, or flow cytometry. Cyto-/chemokine secretion was quantitated by ELISA.ResultsBV-2 cells express LPA receptor 2 (LPA2), 3, 5, and 6, whereas PMM express LPA1, 2, 4, 5, and 6. We show that LPA treatment of BV-2 and PMM leads to a shift towards a pro-inflammatory M1-like phenotype. LPA treatment increased CD40 and CD86 (M1 markers) and reduced CD206 (M2 marker) expression. LPA increased inducible nitric oxide synthase (iNOS) and COX-2 levels (both M1), while the M2 marker Arginase-1 was suppressed in BV-2 cells. Immunofluorescence studies (iNOS, COX-2, Arginase-1, and RELMα) extended these findings to PMM. Upregulation of M1 markers in BV-2 and PMM was accompanied by increased cyto-/chemokine transcription and secretion (IL-1β, TNFα, IL-6, CCL5, and CXCL2). The pharmacological LPA5 antagonist TCLPA5 blunted most of these pro-inflammatory responses.ConclusionsLPA drives BV-2 and PMM towards a pro-inflammatory M1-like phenotype. Suppression by TCLPA5 indicates that the LPA/LPA5 signaling axis could represent a potential pharmacological target to interfere with microglia polarization in disease.

Silencing of protein kinase D2 induces glioma cell senescence via p53-dependent and -independent pathways

Background Glioblastoma multiforme (GBM) is a highly aggressive tumor of the central nervous system with a dismal prognosis for affected patients. Aberrant protein kinase C (PKC) signaling has been implicated in gliomagenesis, and a member of the PKC-activated protein kinase D (PRKD) family, PRKD2, was identified as mediator of GBM growth in vitro and in vivo. Methods The outcome of PRKD2 silencing and pharmacological inhibition on glioma cell proliferation was established with different glioma cell lines. Western blotting, senescence assays, co-immunoprecipitation, fluorescence activated cell sorting, quantitative PCR, and immunofluorescence microscopy were utilized to analyze downstream signaling. Results RNA-interference (21-mer siRNA) and pharmacological inhibition (CRT0066101) of PRKD2 profoundly inhibited proliferation of p53wt (U87MG, A172, and primary GBM2), and p53mut (GM133, T98G, U251, and primary Gli25) glioma cells. In a xenograft experiment, PRKD2 silencing significantly delayed tumor growth of U87MG cells. PRKD2 silencing in p53wt and p53mut cells was associated with typical hallmarks of senescence and cell cycle arrest in G1. Attenuated AKT/PKB phosphorylation in response to PRKD2 silencing was a common observation made in p53wt and p53mut GBM cells. PRKD2 knockdown in p53wt cells induced upregulation of p53, p21, and p27 expression, decreased phosphorylation of CDK2 and/or CDK4, hypophosphorylation of retinoblastoma protein (pRb), and reduced transcription of E2F1. In p53mut GM133 and primary Gli25 cells, PRKD2 silencing increased p27 and p15 and reduced E2F1 transcription but did not affect pRb phosphorylation. Conclusions PRKD2 silencing induces glioma cell senescence via p53-dependent and -independent pathways.