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,
*UMR788, INSERM and University Paris-Sud 11, IFR 93, Le Kremlin-Biceˆtre Cedex, France University Paris Descartes, UPR 2228 CNRS, IFR95, Paris Cedex 6, France
àInstitut Cochin CNRS UMR8104, INSERM U 567, Department of Genetic and Development ‘‘Cancer, Apoptosis and Mitochondria’’, University Paris Descartes, Paris, France
§UMR7079, University Paris Pierre et Marie Curie and CNRS, Paris, France
¶UMR 6247, CNRS-INSERM U931 and Clermont Universite´, et Centre de Recherche en Nutrition Humaine d’Auvergne, Aubie`re, France **UMR 7191, CNRS/ULP, University Louis Pasteur Faculte´ de Me´decine, Strasbourg, France
Oligodendrocytes are glial cells that form myelin sheaths in the CNS. In several pathologies like multiple sclerosis (McQualter and Bernard 2007) and Alzheimer’s disease (AD) (Roth et al. 2005), oligodendrocytes are involved in an inflammatory process associated with increased levels of eicosanoids, cytokines, and inflammatory enzymes such as secreted phospholipase A2 (sPLA2). The levels of oxyster-ols, cholesterol oxidation products, are also altered in these diseases. In particular, the concentration of 24(S)-hydroxy-cholesterol (OH), also known as cerebrosterol [for review
Received October 22, 2008; revised manuscript received February 10, 2009; accepted February 11, 2009.
Address correspondence and reprint requests to Charbel Massaad, CNRS UPR 2228, Universite´ Paris Descartes, IFR95, 45 rue des Saints-Pe`res, 75270, Paris Cedex 6, France.
E-mail: charbel.massaad@parisdescartes.fr
Abbreviations used: AD, Alzheimer’s disease; GC/MS, Gas Chroma-tography/Mass Spectrometry; IL, interleukin; LXR, liver-X receptor; OH, hydroxycholesterol; PI, propidium iodide; PPRE, Peroxysome Proliferator Activated Receptor Response Element; PXR, pregnane X receptor; RXR, retinoic X receptor; siLXR, siRNA against LXR; siRNA, short interfering RNA; sPLA2, secreted phospholipase A2; TNF, tumor necrosis factor. Abstract
In several neurodegenerative diseases of the CNS, oligo-dendrocytes are implicated in an inflammatory process associated with altered levels of oxysterols and inflammatory enzymes such as secreted phospholipase A2 (sPLA2). In view of the scarce literature related to this topic, we investi-gated oxysterol effects on these myelinating glial cells. Natural oxysterol 25-hydroxycholesterol (25-OH; 1 and 10 lM) altered oligodendrocyte cell line (158N) morphology and triggered apoptosis (75% of apoptosis after 72 h). These effects were mimicked by 22(S)-OH (1 and 10 lM) which does not activate liver X receptor (LXR) but not by a synthetic LXR ligand (T0901317). Therefore, oxysterol-induced apoptosis appears to be independent of LXR. Interestingly, sPLA2 type IIA (sPLA2-IIA) over-expression partially rescued 158N cells from oxysterol-induced apoptosis. In fact, 25-OH, 24(S)-OH, and T0901317 stimulated sPLA2-IIA promoter and sPLA2 activity
in oligodendrocyte cell line. Accordingly, administration of T0901317 to mice enhanced sPLA2 activity in brain extracts by twofold. Short interfering RNA strategy allowed to establish that stimulation of sPLA2-IIA is mediated by pregnane X receptor (PXR) at high oxysterol concentration (10 lM) and by LXR b at basal oxysterol concentration. Finally, GC coupled to mass spectrometry established that oligodendrocytes contain oxysterols and express their biosynthetic enzymes, suggest-ing that they may act through autocrine/paracrine mechanism. Our results show the diversity of oxysterol signalling in the CNS and highlight the positive effects of the LXR/PXR path-way which may open new perspectives in the treatment of demyelinating and neurodegenerative diseases.
Keywords: apoptosis, liver-X receptor, oligodendrocyte, oxysterol, pregnane X receptor, secreted phospholipase A2, short interfering RNA.
refer to (Bjorkhem 2007)], is increased in plasma and CSF during early stages of AD (Lutjohann et al. 2000; Papasso-tiropoulos et al. 2002), whereas it is reduced in plasma at late stages (Papassotiropoulos et al. 2000). Furthermore, 24(S)-OH concentrations are also modified in multiple sclerosis patients (Leoni et al. 2002; Teunissen et al. 2003).
With regard to inflammation, 24(S)-OH induces the expression of inflammatory genes such as cyclooxygenase-2 and PLA2 in neural cells (Alexandrov et al. 2005). The type IIA sPLA2 is a member of the large family of phospholipases A2 which are involved in the release of arachidonic acid from membrane phospholipids. It is implicated in the inflammatory process in the CNS (Sun et al. 2005), can induce neuronal cell death (Yagami et al. 2002) and also exerts neurotrophic role on cerebellar granule neurons (Arioka et al. 2005). sPLA2-IIA enzyme is up-regulated in AD (Moses et al. 2006) and multiple sclerosis (Cunningham et al. 2004). Its synthesis is activated by pro-inflammatory cytokines, such as interleukin 1b (IL-1b), IL-6, and tumor necrosis factor-1 (TNF-1) and by oxysterols (Antonio et al. 2003).
Oxysterols particularly [22(R)-OH, 24(S)-OH, and 25-OH] are ligands of the two liver X receptor (LXR) isoforms (Janowski et al. 1996), LXRa and LXRb. However, this mechanism of action is not shared by all oxysterols, for example, 22(S)-OH does not activate LXR (Schmidt et al. 1999). While LXRb is ubiquitously expressed, LXRa is prominently expressed in liver, kidney, intestine, adipose tissue, lungs, and cerebellum. Oxysterols can also bind the pregnane X receptor (PXR) (Mitro et al. 2007), a xenobiotic-activated member of the nuclear receptor superfamily. PXR is expressed in liver, kidney, brain, and lung. LXR and PXR receptors form heterodimers with retinoic X receptor (RXR), the nuclear receptor for 9-cis retinoic acid. In the nucleus, they regulate gene expression by binding to specific respon-sive elements.
The implication of LXR receptors in lipid homeostasis (Ulven et al. 2005), bile acid homeostasis (Uppal et al. 2007), inflammation (Zelcer and Tontonoz 2006), and steroid synthesis (Cummins et al. 2006) are very well documented in peripheral tissues, whereas little is known concerning their function in the nervous system. Cholesterol carriers like ABCA1, ABCG2, or ApoE, which enhance cholesterol efflux in the CNS (Fukumoto et al. 2002; Whitney et al. 2002; Abildayeva et al. 2006) and in other tissues (Mouzat et al. 2006) are LXR target genes. LXR double knock-out mice exhibited a lipid homeostasis alteration in the brain, resulting in a loss of neurons, astrocytic proliferation, and disorganized myelin sheaths (Wang et al. 2002). Moreover LXRb)/) mice show motor neuron degeneration evoking disease such as amyotrophic lateral sclerosis (Andersson et al. 2005).
Very few data are available on the roles of oxysterols (particularly 25-OH) in oligodendrocytes. Therefore, the aim of this study is to understand the effects of oxysterols on
oligodendrocyte cell line. We have shown that high concen-trations of oxysterols are able to provoke oligodendrocyte apoptosis. Furthermore, oxysterols [24(S)-OH, 25-OH)] activate the inflammatory sPLA2-IIA via LXRb and PXR receptors at low and high levels of oxysterols, respectively. High levels of sPLA2-IIA were partially relieved from oxysterol-induced apoptosis.
Materials and methods
Cell cultureThe mouse oligodendrocytes cell line (158N) was kindly provided by Dr. S. M. Ghandour (Strasbourg, France) (Feutz et al. 2001). It originated from FVB mouse strain which is sPLA2-IIA positive. Primary culture of oligodendrocytes was prepared as described in Garcia-Ladona et al. (1997). The mouse Schwann cell line (MSC80) was maintained in Dulbecco’s minimal essential medium supplemented with 10% fetal calf serum (Gibco, Rockville, MD, USA), 100 U/mL penicillin, 100 lL/mL streptomycin, and 0.5 lg/ mL fungizone (Gibco).
Animals
Two-month-old FVB mice were gavaged with 45 mg/kg T0901317 (Cayman Chemicals, Montigny le Bretonneux, France) or vehicle (methyl cellulose) once a day during 4 days as previously described (Volle and Lobaccaro 2007) (see Supporting Information).
Phospholipase A2 activity
Secreted phospholipase A2 activity was measured using a selective fluorimetric assay as described in Couturier et al. (2000). Details of the experimental procedure are in Supporting Information. The sPLA2-enriched medium was prepared from mouse fibroblast lines C127 stably transfected with sPLA2-IIA expression vector as described in Pernas et al. (1991).
Transient transfections
158N cells were transiently transfected using Effecten reagent (Qiagen, Valencia, CA, USA). One day after the transfection, the medium was replaced by Dulbecco’s modified Eagle’s medium containing 5% charcoal-treated fetal calf serum with or without 25-OH or 24(S)-OH (10)5or 10)6M). 25-OH and 24(S)-OH were purchased from Steraloids Inc (Newport, RI, USA). The purity of 24(S)-OH, 25-OH, and 27-OH was assessed by GC/MS (purity > 99%).
Luciferase activity was assayed as described in Massaad et al. (2000). The colorimetric b-galactosidase activity was assayed with UV visible spectrometer by measuring absorbance of the solution at 420 nm. It was used to normalize the transfection efficiency. A value of 100% attributed to the basal activity (control conditions) was the result of the ratio of luciferase activity (arbitrary units, relative light units) over b-galactosidase activity.
RT-PCR
RT-PCR experiments were performed as described in Grenier et al. (2006). Primers are listed in Supporting Information.
Oxysterols quantification by GC/MS
158N cells (27–36· 106) and their corresponding media were collected separately and oxysterols were extracted with 10 volumes MeOH/CHCl3(1 : 1 v/v). Thirty ng of2H10-24(S)-OH cholesterol, as internal standard, and 50 lg butylated hydroxytoluene were added into extracts for oxysterols quantification and for avoiding cholesterol autoxidation processes, respectively.
Cholesterol was separated from oxysterols by solid-phase extraction. Samples were applied in CH3CN/isopropanol (1 : 1 v/v) to a 500 mg C18 cartridge (International Sorbent Technology, Mid Glamorgan, UK) and oxysterols were eluted with 12 mL CH3CN : isopropanol : H2O (55 : 25 : 25 v/v/v). This fraction was further purified by means of a second solid-phase extraction with a recycling procedure (Liere et al. 2004). Details of oxysterol extraction and derivatization are given in Supporting Information.
Atomic force microscopy
Atomic force microscopy measurements were performed on AFM Bioscope (Nanoscope IIIa; Digital Instruments, Veeco Metrology Group, Santa Barbara, CA, USA) in contact mode. Using a fluid cell, AFM contact mode images of living cells were recorded in culture medium using a long V-shape silicon nitride cantilever (320 lm in length), with a nominal spring constant of 0.01 mN/m (MLCT-AU Microlevers, Veeco Probes, Camarillo, CA, USA) to minimize the force applied to the cells. The image fields (60 lm· 60 lm) were obtained at 0.5 Hz; thus 6–10 min were required to scan the entire sample. 158N cells cultured on collagen I-coated Petri dishes (rat tail, BD Biosciences, Le Pont de Claix, France) were mounted directly on the stage of the inverted microscope (Olympus IX 70, Olympus America Inc., Melville, NY, USA) of the Bioscope to choose viable cells at relative low density. The samples were equilibrated in the cell medium for 10–15 min period to avoid problem with drift of the equipment. Morphometric parameters (maximum height, cell sur-face, and cell volume) were determined from the AFM topography images using the Nanoscope software (v5.12, Digital Instruments, Veeco Instruments Inc., Santa Barbara, CA, USA) (see Supporting Information for more details).
Study of apoptosis by flow cytometry
Changes in mitochondrial membrane potential difference (DYm) were evaluated by incubating cells (158N cells, 5· 105
/mL) for 15 min at 37C with 3,3-dihexyl-oxacarbocyanine. Cells were then analyzed by using the FACScalibur cytometer (Becton-Dickinson, Franklin Lakes, NJ, USA). The fluorescence was quantified after suitable compensa-tion in FL-1 channel for 3,3-dihexyl-oxacarbocyanine.
Late apoptotic events and/or secondary necrosis were estimated by staining cells with annexin V-FITC. The annexin V-FITC absorption on cell surface was monitored by flow cytometry in FL-1 channel together with the membrane integrity assay using propidium iodide (PI, 1 lg/mL) which emitted in FL-3 channel. Caspase 3 like activity was assayed by flow cytometry in intact cells incubated with caspase substrate PhiPhilux G1D2 (OncoImmunin Inc., Kensington, MD, USA) which contained the GDEVDG sequence (details are in Supporting Information).
Statistical analysis
Unless otherwise specified, means of treatment groups were compared withANOVA. When the ANOVA showed that there were
significant differences between the groups, Bonferroni’s test was used to identify the sources of these differences. Alternatively, Student’s t-test was used. A p-value of £ 0.05 was considered statistically significant.
Results
Oxysterols alter oligodendrocyte cell line morphology and trigger apoptosis
In plasma and in CSF oxysterol concentrations were high and could reach micromolar range (Bretillon et al. 2000). Furthermore, in demyelinating and neurodegenerative dis-eases as well as in brain trauma, oxysterol levels were altered (Bretillon et al. 2000; Kolsch et al. 2003). Yet, few data were available on the amounts of oxysterol levels in brain. Therefore, we assayed by GC/MS, the concentrations of three oxysterols [24(S)-OH, 25-OH, and 27-OH)] in rat brains. The concentration of 24(S)-OH was high (10350 ± 361 ng/g), while 25-OH and 27-OH were less prominent (65.7 ± 1.1 ng/g and 206.1 ± 5.4 ng/g, respectively). Values for 24(S)-OH were comparable with those obtained by Lu¨tjohann et al. (8600–15100 ng/g) in cerebrum (Lutjohann et al. 1996). These results showed that oxysterol levels were high in rat brain: 24(S)-OH and 25-OH concentrations were about 25 and 0.16 lM, respectively. As little is known about the role of oxysterols, especially 25-OH, in oligodendrocyte homeostasis, this prompted us to examine their impact on these myelinating glial cells.
First, the effects of 25-OH on oligodendrocyte cell line shape were monitored by atomic force microscopy, a technique that provides topographic images as well as morphological parameters (height, surface, and absolute cell volume) of individual living cells (McNally and Ben Borgens 2004; Klembt Andersen et al. 2005). Figure 1a shows typical deflection images of living 158N cells obtained in contact mode, cultured in the absence or in the presence of 25-OH (10 lM) during 24 h. Cytoskeleton elements were clearly visible underneath the plasma membrane of control cells. The exposure of 158N cells to 10 lM 25-OH during 24 h resulted in striking alterations of the cell morphology and important variations both in height and in width (Fig. 1b). As shown in Fig. 1c, treatment with 25-OH elicited a significant increase in maximum height of the cell from 2.11 ± 0.21 to 3.98 ± 0.40 lm, a retracted edge of approximately 10 lm and an enhancement of the spherical shape. The surface of the 25-OH-treated cells was reduced by 59% compared with the control, whereas the total volume was not significantly modified. We have also tested the effects of 25-OH at a lower dose (1 lM), we obtained similar modifications in oligo-dendrocyte cell line morphology (not shown).
These morphological changes, particularly the decrease in volume and the loss of focal adhesion, could be early
prerequisites for apoptosis (Hessler et al. 2005). Therefore, we studied the effects of oxysterols on oligodendrocyte viability. 158N cells were incubated with 25-OH (1 and 10 lM) during 24, 48, and 72 h and then imaged by optical microscopy. 25-OH (1 and 10 lM) seemed to induce a time-dependent decrease in the number of viable cells (Fig. 2a). This incited us to study the effects of 25-OH on oligoden-drocyte apoptosis by flow cytometry. As depicted in Fig. 2b, the number of cells with high mitochondrial membrane potential was decreased in a time- and concentration-dependent manner after a treatment with 25-OH. After 24 h of treatment with 25-OH the percentage of cells with high mitochondrial membrane potential was 74% for 1 lM and 40% for 10 lM versus 92% in the control conditions. After 48 h of treatment with 25-OH, the percentage of cells with high membrane potential was 48% for 1 lM and 17% for 10 lM. Finally, after 3 days of treatment with 25-OH, this percentage has fallen to 11% at 1 lM and 3% at 10 lM. This mitochondrial membrane potential drop appeared to be associated with caspase 3 enzyme activation (Fig. 2c), as a matter of fact, at 10 lM 25-OH dramatically enhanced caspase 3 cleavage activity (41% at 24 h, 76% at 48 h, and
87% at 72 h). The effect of 25-OH on caspase 3 is not transcriptional because the promoter activity of caspase 3 is not enhanced by 25-OH (not shown). Accordingly, annexin V and PI staining indicated that the cells underwent either apoptosis or necrosis. In fact, 14.2% of 158N cells underwent apoptosis (vs. 3.8% in control cells) after 24 h of 25-OH treatment and this rate increased to 57.7% after 48 h and to 75.8% after 72 h (Fig. 2d). Interestingly, a time-dependent effect of 25-OH was not observed on the number of cells undergoing necrosis (PI+/A+ cells). In conclusion, 25-OH enhanced oligodendrocyte cell line apoptosis (PI)/A+cells) which was aggravated by a extended exposure and increased concentration of this product.
We then wondered whether 25-OH-induced apoptosis was mediated by a nuclear receptor of oxysterols, namely, LXRs. Therefore, we tested the effect of a LXR synthetic ligand, T0901317, on the viability of oligodendrocytes. 158N cell viability was assessed by light microscopy after a 24, 48, and 72 h treatment with T0901317 (1 and 10 lM). T0901317 did not elicit any toxic effect on oligodendrocyte cell line (not shown). This observation was confirmed by flow cytometry as the number of cells with Control (a) (c) (b) Control Maxim u m height (µm) Cell surface (µm 2) Cell v olume (µm 3) Height (µm)
25-OH Control 25-OH Control
Distance (µm) 25-OH 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ** ** 0.0 0 100 200 300 400 500 600 0 100 200 300 400 500 600 0.0 5 0 20 µm 15 10 20 25 30 35 25-OH Control 0.5 1.0 1.5 2.0 2.5 3.0 4.0 3.5 700 800 25-OH
Fig. 1 Alteration of oligodendrocyte cell line morphology by oxysterols. (a) Representative AFM deflection images of living oligodendrocytes. 158N cells were cultured on a matrix of collagen I during 24 h and then cells were incubated or not with 25-OH (10)5M) during 24 h. The cells were then imaged with AFM in contact mode. This experiment was repeated three times, and typical images are presented here. Image
scan size: 60· 60 lm. (b) Height profiles corresponding to the cross-sections of the same cells as in (a). (c) Histogram of the morphometric parameters: maximum cell height, cell surface, and total cell volume of 158N cells incubated or not with 25-OH (10)5M). All means are cal-culated from five cells. *p < 0.05 and **p < 0.01 when compared with control by Student’s t-test. AFM, atomic force microscopy.
high mitochondrial membrane potential remained un-changed (Supporting Information). These results demon-strated that a specific ligand of LXR did not alter oligodendrocyte viability.
Finally, we assessed the effects on cell viability of an oxysterol, 22(S)-OH, that do not activate to LXRs. 158N cells were incubated with 22(S)-OH (1 and 10 lM) during 24, 48, and 72 h. Interestingly, this compound altered 158N cell viability (Fig. 3a) and provoked a significant decrease in the number of cells with high mitochondrial potential (Fig. 3b). The effect of 22(S)-OH was more potent at 10 lM than at 1 lM (29% vs. 68% of high mitochondrial potential cells). Then, percentage of apoptotic and necrotic cells were evaluated after a treatment with 22(S)-OH (Fig. 3c). As expected the total amount of dead cells
increased in a time-dependent manner. We detected not only an increase in apoptotic death (23% at 24 h, 27% at 48 h, and 41% at 72 h) but also an increase in necrotic death (16% at 24 h, 24% at 48 h, and 26% at 72 h), yet to a lesser extent. In conclusion, 25-OH triggered oligodendrocyte cell line apoptosis. This effect was not LXR-dependent as a synthetic ligand of LXR did not alter cell viability, while an oxysterol (22S) unable to activate LXR provoked oligodendrocyte death.
sPLA2-IIA expression attenuates oxysterol toxicity Cytosolic as well as secreted PLA2 were known to stimulate proliferation of smooth muscle (Jaulmes et al. 2005), uterine (Specty et al. 2001), and endothelial cells. Therefore, we thought that sPLA2 might be able to protect 158N from
(a) (b)
(c) (d)
Fig. 2 Role of 25-OH in oligodendrocyte cell line death. 158N cells were treated with vehicle (EtOH) or with 25-OH (1 or 10 lM) during 24, 48, or 72 h. (a) Cells were viewed using phase-contrast microscope. This experiment was repeated three times; images represent a typical experiment. (b) The mitochondrial membrane potential was deter-mined by 3,3-dihexyl-oxacarbocyanine and the percentage of cells with high mitochondrial membrane potential was calculated and spotted. (c) Caspase 3 activity was determined as described in Materials and methods. These results represent the mean ± SD of three independent experiments. (d) Histograms of the 158N cells
treated with vehicle or 25-OH (10 lM) during 24, 48, or 72 h. Apoptotic cells were identified as Annexin V-FITC-positive/PI-negative whereas necrotic cells or cells exhibiting secondary necrosis are considered as Annexin V-FITC-positive and PI-positive. The addition of the two populations set the total cell death in the present conditions. The different categories of cell populations have been simultaneously plotted. In all cases 10000 cells have been counted. The SDs have been extracted from five different experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 when compared with control by usingANOVAwith Bonferroni’s post hoc test.
oxysterol-triggered apoptosis. Culture medium was enriched with sPLA2-IIA, a sPLA2 isoform isolated from C127 mouse fibroblast cells, over-expressing this enzyme (Pernas et al. 1991). As shown in Fig. 4a, sPLA2 activity was 50-fold higher in the sPLA2-IIA-enriched medium compared with the control medium, and comparable with that reached after stimulation by inflammatory components, such as IL-1b, IL-6, TNFa, and ecosanoids. Thus, 158N cells were incubated with 10 lM of 25-OH in plain medium or sPLA2-IIA-enriched medium during 24, 48, and 72 h. As expected, 25-OH produced a dramatic alteration of cell morphology and viability which was exacerbated after 48 and 72 h of treatment. sPLA2-IIA-enriched medium did not alter oligo-dendrocyte morphology (Fig. 4b) or viability (Fig. 4c), but interestingly, partially rescued oligodendrocyte cell line from
oxysterol-triggered death. Interestingly, sPLA2-IIA medium counteracted 25-OH effects, increasing the cell viability by 20%.
Natural oxysterols and T0901317 enhance secreted phospholipase A2 activity in oligodendrocyte cell line and in brain
We wondered if oxysterols were able to stimulate sPLA2 activity in oligodendrocytes and in the brain. 158N cells were incubated with natural oxysterols 24(S)-OH or 25-OH at 1 and 10 lM during 24 h, and the total sPLA2 activity was measured. As shown in Fig. 5a, 24(S)-OH enhanced by twofold the sPLA2 activity at 1 lM, and by threefold at 10 lM (p < 0.01), while 25-OH treatment resulted in a weak stimu-latory effect on sPLA2 activity at 1 lM, and in a threefold
(a)
(c)
(b)
Fig. 3 Role of 22(S)-OH in oligodendrocyte cell death. 158N cells were treated with vehicle (EtOH) or with 22(S)-OH (1 or 10 lM) during 24, 48, or 72 h. (a) Cells were viewed using phase-contrast microscope. This experiment was repeated three times; images represent a typical experiment. (b) The mitochondrial membrane potential was determined by 3,3-dihexyl-oxacarbocyanine and the percentage of cells with high mitochondrial membrane potential was calculated and spotted. (c) Histograms of the 158N cells treated with vehicle or 25-OH (10 lM) during 24, 48, or 72 h. Apoptotic cells were
identified as Annexin V-FITC-positive/PI-negative whereas necrotic cells or cells exhibiting secondary necrosis are considered as Ann-exin V-FITC-positive and PI-positive. The addition of the two popu-lations set the total cell death in the present conditions. The different categories of cell populations have been simultaneously plotted. In all cases 10000 cells have been counted. The SDs have been extracted from three different experiments. *p < 0.05, **p < 0.01, and ***p < 0.001 when compared with control by using ANOVA with Bonferroni’s post hoc test.
stimulation at 10 lM (p < 0.05). Similar results were obtained with the synthetic LXR agonist T0901317 (data not shown).
Stimulation of sPLA2 activity was not restricted to oligodendrocyte cell line. We examined the effect of T0901317 on total sPLA2 activity in brain in vivo. Adult mice were fed with either T0901317 (45 mg/kg/day) or vehicle methylcellulose during 4 days, and sPLA2 activity was assayed in brain extracts. T0901317 significantly increased sPLA2 activity (2.5-fold) compared with vehicle treatment (p < 0.01) (Fig. 5b) as observed in the oligoden-drocyte cell line.
sPLA2-IIA promoter activity is regulated by oxysterols in oligodendrocytes
Secretory phospholipase A2 type IIA gene is a target of oxysterols in smooth muscle cells (Antonio et al. 2003). Therefore we evaluated the effect of oxysterols on sPLA2-IIA promoter activity. 158N cells were transfected with the sPLA2-IIA promoter [)1160; +46]-Luc (Fig. 5c) and treated with either 24S-OH or 25-OH at 1 and 10 lM. As shown in Fig. 5d, treatment of the cells with 1 lM 24S did not significantly affect the promoter activity while a 10 lM treatment enhanced by twofold the promoter activity
(a)
(b)
(c)
Fig. 4 Role of sPLA2-IIA in oligodendrocyte cell line apoptosis triggered by 25-OH. (a) sPLA2-IIA enriched medium was prepared from C127 cells stably transfected with sPLA2-IIA expression vec-tor. sPLA2 activity was tested on either a control medium or an aliquot of the medium of C127 transfected with sPLA2-IIA expres-sion vector (sPLA2 medium). Results are expressed as arbitrary units over the amount of proteins in mg. ***p < 0.001 when com-pared with control by Student’s t-test. (b) 158N cells were cultured either in control medium or sPLA2-IIA-enriched medium (sPLA2
medium) and treated with EtOH or 25-OH (10 lM) during 24, 48, or 72 h. Photographies of living cells were then taken, and panel b shows a typical experiment. (c) Cell viability was analyzed by flow cytometry using PI staining. PI-negative cells are viable cells. Ten thousand cells have been counted. The SDs have been extracted from two different experiments performed in duplicate. *p < 0.05 and **p < 0.01 when compared between two conditions ‘25-OH’ and ‘25-OH + sPLA2 medium’ by usingANOVAwith Bonferroni’s post hoc
(p < 0.01). Treatment of 158N cells with 1 lM 25-OH modestly activated the sPLA2-IIA promoter (50%-activation, p < 0.05), while 10 lM 25-OH stimulated the promoter by 2.5-fold (p < 0.01). The synthetic T0901317 elicited a
twofold enhancement of the promoter transactivation at 1 lM and 2.5-fold stimulation at 10 lM (data not shown). Interestingly, neither 24S-OH nor 25-OH stimulated the sPLA2-IIA promoter activity in MSC80 cells (Fig. 5d)
(a)
(c)
(d) (e)
(b)
Fig. 5 Regulation of sPLA2 enzymatic activity and promoter by ox-ysterols. (a) sPLA2 activity in 158N cells: cells were incubated during 24 h with ethanol (EtOH), 24S. or 25-OH at the concentrations indi-cated and then sPLA2 activity was measured. Results are expressed as arbitrary units per mg of proteins; they represent the mean ± SEM of at least three independent experiments performed in duplicate. *p < 0.05 and **p < 0.01 when compared with control by Student’s t-test. (b) sPLA2 activity in mouse brains: Mice were gavaged with vehicle or T0901317 (45 mg/kg/day) during 4 days (five mice per group). Brains were removed and extracts were prepared to assay sPLA2 activity. Results are expressed as arbitrary units per mg of protein. They represent the mean ± SEM of two independent experi-ments. **p < 0.01 when compared with control by Student’s t-test. (c) Schematic representation of sPLA2-IIA promoter and its deletion or mutation fragments. (d) Oxysterols effects on sPLA2-IIA promoter activity: 158N cells were transiently transfected with 0.2 lg of
sPLA2-IIA [)1160, +46] and 0.1 lg of pRSV-bGal plasmids. Eighteen hours after transfection, cells were incubated with ethanol (EtOH), 24S or 25-OH (1 or 10 lM) for 24 h, and then luciferase and b-galactosidase activities were measured. Results are expressed as percentage of the basal activity; they represent the mean ± SEM of five independent experiments performed in duplicate. (e) Localization of oxysterol ef-fects on the level of sPLA2-IIA promoter: 158N cells were transiently transfected with 0.2 lg either of IIA [)1160; +46] or sPLA2-DPPRE; sPLA2-IIA [)488; +46] or sPLA2-IIA [)398; +46]; and 0.1 lg of pRSV-bGal plasmids. Eighteen hours after transfection, cells were incubated with EtOH or 25-OH (10 lM) for 24 h, and then luciferase and b-galactosidase activities were analyzed. Results are expressed as percentage of the basal activity; they represent the mean ± SEM of five independent experiments performed in duplicate. In all experi-ments, *p < 0.05, **p < 0.01 when compared with control by using
indicating that oxysterol effects on sPLA2-IIA promoter activity are cell-line specific.
We then searched for the response element targeted by oxysterols in the sPLA2-IIA promoter. Antonio et al. (2003) previously showed that in smooth muscle cells oxysterols exerted their action by way of LXR–RXR binding to a DR4 response element of the sPLA2-IIA promoter (Fig. 5c). Yet, we identified by means of MatInspector software (Genomatix Software, Munich, Germany) another putative binding site for LXR–RXR that was also bound by Peroxysome Proliferator Activated Receptor (PPAR) (Ravaux et al. 2007). The segment [)1160; +46] of the sPLA2 promoter allowed 25-OH to enhance transcriptional activity in 158N by twofold. Truncated forms of the promoter lacking the PPAR Response Element (PPRE) (sPLA2-IIA DPPRE) as well as segment [)488; +46] were activated to the same extent by 25-OH (Fig. 5e). Finally, a short form of the promoter [)398; +46] lacking the segment [)1160; )398] that contains the potential oxysterol response element (DR4) did not respond to 25-OH. Hence, deletion of the DR4 response element, contrary to the deletion of PPRE, abolished the transactivation of the sPLA2-IIA promoter by 25-OH.
LXRb and PXR are involved in the regulation of sPLA2-IIA by oxysterols
Oxysterols can bind and activate LXR or PXR but with different affinities. We wanted to identify which nuclear receptor was responsible for the observed stimulation of sPLA2-IIA. LXRa and LXRb are both present in whole brain but their specific expression in myelinating cells is not known. Therefore, we analyzed their expression in oligo-dendrocyte (158N) and in MSC80 cell lines by RT-PCR (Fig. 6a) and real time PCR (not shown). LXRa transcript was undetectable in 158N and was only weakly expressed in MSC80 cells, whereas LXRb mRNA was present in both cell lines. To determine if LXRb isoform mediated the effects observed on sPLA2-IIA, specific short interfering RNA (siRNA) was used to block its expression. LXRb knock-down was ascertained by RT-PCR (Fig. 6b) and real time PCR (not shown). However, the oxysterol-induced stimula-tion of the sPLA2-IIA promoter was unaffected; 25-OH stimulated by 2.4-fold sPLA2-IIA promoter with a non-targeting siRNA and by 2.6-fold when transfected with a siRNA against LXR (siLXR). Surprisingly, siLXR inhibited the basal activity of sPLA2 promoter (p < 0.05).
To determine why the basal promoter activity of sPLA2-IIA was decreased in absence of LXRb, levels of 24S-OH, 25-OH, and 27-OH were measured by GC/MS in 158N cells and in culture medium (72 h in culture plates). As shown in Fig. 6c, 24S-OH, 25-OH, and 27-OH were detected in oligodendrocyte cell line and culture medium. 24(S)-OH prevailed over 25-OH in the cells (1.27 ± 0.18 ng/106cells vs. 0.71 ± 0.06 ng/106 cells) while both oxysterols were
present at equivalent amounts in the medium (1.11 ± 0.11 ng/106cells vs. 1.07 ± 0.11 ng/106cells, respectively), which corresponded to a 2 nM concentration. 27-OH was present at a lower amount than 24(S)-OH and 25-OH in the cells and the culture medium. In cell-free medium, 24(S)-OH and 27-OH were present at very low concentrations (0.095 ± 0.095 ng/106 cells and 0.08 ± 0.02 ng/106 cells, respectively) while 25-OH was undetectable. These results showed that 24(S)-OH, 25-OH, and 27-OH were present in oligodendrocytes and suggest that they may exert their signalling effects in situ. In brain, essentially in neurons, cholesterol can be metabolised into various oxysterols, mainly 24(S)-OH, 25-OH, or 27-OH, by means of cholesterol hydroxylases enzymes, Cyp46A1, 25-hydroxylase, and 27-hydroxylase, respectively (Russell 2000). Transcripts of each of these enzymes were detected by RT-PCR in mouse brain extracts in primary culture of oligodendrocyte and in 158N (Fig. 6d). Therefore, we believe that there is a de novo production of oxysterols in oligodendrocytes.
As LXR did not seem to support sPLA2-IIA stimulation by exogenous oxysterols, we raised the question of PXR contribution to this stimulation. First, we demonstrated by RT-PCR (Fig. 6e), real time PCR (not shown), and western blot that PXR transcript and protein were expressed in brain and 158N (Fig. 6f) and that PXR expression was knocked-down by siRNA (Fig. 6g). PXR knock-knocked-down did not affect the basal activity of sPLA2-IIA promoter, but dampened down the stimulation by 25-OH (p < 0.05): 1.7-fold (siLXR) versus 2.9-fold (non-targeting siRNA) (Fig. 6g). Together these results suggest that oxysterols are able to activate sPLA2-IIA through LXR at physiological concentration and through PXR at higher concentration (10 lM).
Discussion
Oxysterol exerted essential effects on brain homeostasis and their levels were often altered in neurodegenerative and demyelinating diseases. These variations could either reflect dysfunction or degeneration of neuronal or glial cells, or be a response to the disease, a local synthesis modulation in order to exert signalling effects. In this study, we analyzed the effects of some oxysterols on oligodendrocytes, myelinating glial cells. In addition, we have established a relationship between oxysterols levels and activation of sPLA2 enzyme in the brain. Apoptosis was not provoked by a massive cholesterol efflux because of activation to cholesterol transporters for two reasons: (i) treatment of the cells with a hydrosoluble form of cholesterol did not relieve from 25-OH triggered apoptosis (not shown), (ii) T091317 that was able to stimulate cholesterol efflux did not provoke apoptosis.
When incubated with high doses of 25-OH (1 and 10 lM), oligodendrocyte cell line displayed a dramatic shape mod-ification depicted by atomic force microscopy, accompanied
with initiation of apoptosis after a 24 h 25-OH treatment. These events were concomitant with an increase in sPLA2-IIA expression and activity. Therefore, one could speculate that sPLA2 could mediate oxysterol apoptotic effects on oligodendrocytes as suggested in Krabbe disease (globoid cell leukodystrophy) (Giri et al. 2006). Interestingly, sPLA2-IIA over-expression did not alter oligodendrocyte viability invalidating the latter assumption. To the contrary, sPLA2 partially protected against the deleterious effects of oxyster-ols by increasing cell viability. Moreover, 158N treatment with sPLA2 inhibitors, 7,7-dimethyl eicosadienoic acid and LY311727, did not block oxysterol-induced apoptosis ruling out any implication of sPLA2 enzymatic activity in oligo-dendrocyte death (data not shown). sPLA2 was not just implicated in apoptosis but it had proliferative effect towards smooth muscle cells (Jaulmes et al. 2005), uterine cell line (Specty et al. 2001), and monocytes (Saegusa et al. 2008) and was protective towards neurons (Arioka et al. 2005). As a matter of fact, ammodytoxin, a sPLA2, inhibited G2
cell-cycle arrest suggesting that sPLA2 are able to cause the opposing effects: proliferation and apoptosis in cells (Petro-vic et al. 2005).
Secreted PLA2-IIA activity was stimulated after cerebral ischemia and cerebral cortex injuries and could have harmful effects in brain lesions (Muralikrishna Adibhatla and Hatcher 2006). The activity of this inflammatory enzyme was enhanced in several diseases affecting the nervous system like AD (Moses et al. 2006), Krabbe (Giri et al. 2006), and Parkinson’s diseases as well as in multiple sclerosis (Kalyvas and David 2004). Yet sPLA2 cannot only be considered as a ‘harmful’ enzyme as it is involved in phospholipid turnover, membrane remodeling, exocytosis, phospholipid peroxide detoxification, and neurotransmitter release (Farooqui et al. 1997).
We have dissected the mechanism of action of oxysterols on sPLA2 and found that 25-OH and 24(S)-OH enhanced sPLA2-IIA promoter activity in oligodendrocytes. Interest-ingly, this stimulation does not implicate the classical oxysterols receptor LXR only, as the knock-down of its
(a) (b)
(c)
(e) (f) (g)
expression did not significantly affect the stimulatory effects of oxysterols, although it altered the basal activity of the promoter. The involvement of LXR in the regulation of the basal transactivation of the sPLA2-IIA promoter could rely on the local production of oxysterols in oligodendrocytes. As a matter of fact, we evidenced by GC/MS that these glial cells contained 24(S)-OH, 25-OH, and 27-OH, and by functional knock-down experiments that these oxysterols could enhance the basal activity of sPLA2-IIA promoter by means of the LXRb. Moreover, we revealed by RT-PCR that the mRNAs of the enzymes synthesizing these oxysterols (Cyp46A1, Cyp25, and Cyp27) were expressed in oligoden-drocytes suggesting autocrine/paracrine actions of oxysterols in oligodendrocytes.
Using siRNA knock-down system, we demonstrated that PXR was able to mediate the stimulatory effects of oxysterols on the sPLA2-IIA promoter. This receptor has already been shown to intervene in oxysterol signalling pathway in hepatocytes (Shenoy et al. 2004) where PXR but not LXR mediated oxysterol action. We hypothesize that oxysterols could bind and activate LXR at physiological doses, while these compounds would act through PXR at high pharmaco-logical concentrations. It is important to keep in mind that in the brain the relative amount of PXR is lower than that of
LXRb while in liver they are present in equivalent amount (Nishimura et al. 2004). Therefore, we suggest that the predominant LXRb was active under low concentrations of oxysterols, while the less abundant PXR was only activated with high amounts of oxysterols. Such a dual mechanism has already been described for glucocorticoids (Meijer 2002). Indeed, cortisol at low doses binds with high affinity to the mineralocorticoid receptor, resulting in beneficial effects on the nervous system; while cortisol at high levels binds also with low affinity to the glucocorticoid receptor.
Liver X receptor effects on inflammation are subject to controversy. LXR agonists were shown to antagonize the expression of a battery of inflammatory genes in activated macrophages (Joseph et al. 2003). Consistent with these in vitro effects, LXR null mice exhibited enhanced responses to inflammatory stimuli and LXR ligands reduced inflam-mation in rodent models. These observations suggest that LXR agonists may exert their antiatherogenic effects not only by promoting cholesterol efflux but also by limiting the production of inflammatory mediators in the artery wall (Tontonoz and Mangelsdorf 2003). To the opposite, LXRs have been shown to play a role in the production of the pro-inflammatory cytokine TNFa. Human monocytes co-treat-ment with 22(R)-OH and of the RXR-specific ligand 9-cis Fig. 6 LXRs and PXR expression and implication in sPLA2-IIA
pro-moter regulation. (a) Expression of LXRa and LXRb: Total RNA from MSC80 and 158N cells was prepared. RT-PCR experiments were performed by using primers recognizing specifically LXRa and LXRb. PCR products were analyzed on agarose gel (2%) and visualized under UV. (b) Implication of LXRb in sPLA2-IIA promoter expression. The efficacy of the siRNA was analyzed by RT-PCR. 158N cells were transiently transfected with a non-targeting siRNA or siRNA specifi-cally directed against LXRb. Total RNA was prepared 48 h after transfection. RT-PCR experiments were performed by using primers recognizing specifically LXRb. PCR products were analyzed on aga-rose gel (2%) and visualized under UV. 18S RNA was detected by specific primers and used to normalize LXR expression levels. 158N cells were transiently transfected with 0.2 lg of sPLA2-IIA [)1160; +46], 0.1 lg of pRSV-bGal plasmids and non-targeting siRNA or siRNA specifically directed against LXRb as indicated. Eighteen hours after transfection, cells were incubated with 25-OH (10)5M) for 24 h, and then luciferase and b-galactosidase activities were analyzed. Results are expressed as percentage of the basal activity, they rep-resent the mean ± SEM of at least three independent experiments performed in duplicate. *p < 0.05 when compared with control by usingANOVAwith Bonferroni’s post hoc. Beside the arrows are indi-cated the fold induction of sPLA2-IIA promoter activity triggered by 25-OH over the control. (c) Detection of 24(S)-hydroxycholesterol (25-OH), 25-OH, and 27-OH in 158N oligodendrocytes cell line by GC/MS. 158N cells were cultured in normal conditions during 72 h. Then, medium and cells were separated to measure oxysterols by GC/MS analysis. Results are expressed in ng per million of cells and represent the mean ± SEM of three independent experiments performed in duplicate. *p < 0.05, **p < 0.01, and ***p < 0.001 when compared with ‘cell-free medium’ condition by usingANOVAwith Bonferroni’s post hoc
test. (d) Detection of Cyp46A1, 25-hydroxylase, and 27-hydroxylase mRNAs in brain, oligodendrocytes, and 158N cells. Primary culture of oligodendrocytes and 158N cells were cultured in normal conditions and were harvested and total RNA was prepared. Total RNA was also extracted from mouse brains. RT-PCR was performed by using spe-cific primers recognizing Cyp46A1, 25-hydroxylase, and 27-hydroxy-lase. PCR products were analyzed on agarose gel (2%) and visualized under UV. (e) Expression of PXR: total RNAs from mouse brains and 158N were prepared. RT-PCR experiments were performed by using primers recognizing specifically PXR. PCR products were analyzed on agarose gel (2%) and visualized under UV. (f) Western blot analysis of PXR expression in total brain extract and in 158N cells. (g) Implication of PXR in sPLA2-IIA promoter activity. 158N cells were transiently transfected with 0.2 lg of sPLA2-IIA [)1160; +46], 0.1 lg of pRSV-bGal plasmids, and with non-targeting siRNA or a siRNA specifically directed against PXR as indicated. Eighteen hours after transfection, cells were incubated with EtOH or 25-OH (10)5M) for 24 h, and then luciferase and b-galactosidase activities were analyzed. Results are expressed as percentage of the basal activity; they represent the mean ± SEM of at least three independent experiments performed in duplicate. *p < 0.05 when compared between cells transfected or not with siRNA against PXR by usingANOVAwith Bonferroni’s post hoc. Besides, the arrows are indicated the fold induction of sPLA2-IIA pro-moter activity triggered by 25-OH over the control. The efficacy of the siRNA was analyzed by RT-PCR. 158N cells were transiently trans-fected with non-targeting siRNA or a siRNA specifically directed against PXR. Total RNA was prepared 48 h after transfection. RT-PCR experiments were performed by using primers recognizing specifically PXR. PCR products were analyzed on agarose gel (2%) and visualized under UV. 18S RNA was detected by specific primers and used to normalize PXR expression levels. NT, Non Targeting siRNA.
retinoic acid resulted in a dramatic increase in TNF-a production (Landis et al. 2002; Millatt et al. 2003).
LXR agonists were shown to attenuate the inflammation in AD. For example, cyclooxygenase 2, IL-6, and RANTES gene stimulation by lipopolysaccharide were hampered by synthetic LXR agonist GW3695 (Zelcer and Tontonoz 2006). Furthermore, in APP23 transgenic mice, several inflamma-tory genes were down-regulated by T0901317 (Zelcer and Tontonoz 2006). These observations are not in opposition with our description of oxysterol stimulation of sPLA2 gene because they were made in astrocytes and microglia in AD context, while ours were made in non-pathological oligo-dendrocytes. Furthermore, as we have mentioned previously, we must keep in mind that sPLA2 was not only involved in inflammation but it was also implicated in various cell functions like neurotransmission, membrane remodelling, apoptosis, and cell cycle.
In conclusion, we have shown that some oxysterols provoke damaging effects on oligodendrocyte cell line shape and viability. They are able to stimulate sPLA2-IIA expres-sion and activity (via LXR and PXR) which has a protective outcome. Altogether, these observations allow a better understanding of the roles of oxysterols in the brain and highlight the apparent complexity of their toxic/beneficial actions in the CNS. Therefore, a better targeting and modulation of the action of oxysterols could allow the development of new therapies for neurodegenerative dis-eases, where oxysterol levels are altered, based on safer and selective oxysterol receptors modulation.
Acknowledgments
This article has been prepared with financial supports from INSERM, CNRS, University Paris-Sud, University Paris Descartes, and the Association Franc¸aise contre les Myopathies (AFM). JMAL is supported by grants from the CNRS, the Universite´ Blaise Pascal, the Fondation pour la Recherche Me´dicale (FRM-INE2000-407031/1), and the Fondation BNP-Paribas. We thank Dr. Sylvie Soues for her critical reading of the manuscript.
Supporting Information
Additional Supporting Information may be found in the online version of this article:
Figure S1 Role of LXR agonist, T0901317, in oligodendrocyte cell death.
Please note: Wiley-Blackwell are not responsible for the content or functionality of any supporting materials supplied by the authors. Any queries (other than missing material) should be directed to the corresponding author for the article.
References
Abildayeva K., Jansen P. J., Hirsch-Reinshagen V. et al. (2006) 24(S)-hydroxycholesterol participates in a liver X
receptor-con-trolled pathway in astrocytes that regulates apolipoprotein E-mediated cholesterol efflux. J. Biol. Chem. 281, 12799–12808. Alexandrov P., Cui J. G., Zhao Y. and Lukiw W. J. (2005)
24S-hy-droxycholesterol induces inflammatory gene expression in primary human neural cells. Neuroreport 16, 909–913.
Andersson S., Gustafsson N., Warner M. and Gustafsson J. A. (2005) Inactivation of liver X receptor beta leads to adult-onset motor neuron degeneration in male mice. Proc. Natl Acad. Sci. USA 102, 3857–3862.
Antonio V., Janvier B., Brouillet A., Andreani M. and Raymondjean M. (2003) Oxysterol and 9-cis-retinoic acid stimulate the group IIA secretory phospholipase A2 gene in rat smooth-muscle cells. Biochem. J. 376, 351–360.
Arioka M., Cheon S. H., Ikeno Y., Nakashima S. and Kitamoto K. (2005) A novel neurotrophic role of secretory phospholipases A2 for cerebellar granule neurons. FEBS Lett. 579, 2693–2701. Bjorkhem I. (2007) Rediscovery of cerebrosterol. Lipids 42, 5–14. Bretillon L., Siden A., Wahlund L. O. et al. (2000) Plasma levels of
24S-hydroxycholesterol in patients with neurological diseases. Neuro-sci. Lett. 293, 87–90.
Couturier C., Antonio V., Brouillet A., Bereziat G., Raymondjean M. and Andreani M. (2000) Protein kinase A-dependent stimulation of rat type II secreted phospholipase A(2) gene transcription involves C/EBP-beta and -delta in vascular smooth muscle cells. Arterios-cler. Thromb. Vasc. Biol. 20, 2559–2565.
Cummins C. L., Volle D. H., Zhang Y. et al. (2006) Liver X receptors regulate adrenal cholesterol balance. J. Clin. Invest. 116, 1902– 1912.
Cunningham T. J., Souayah N., Jameson B., Mitchell J. and Yao L. (2004) Systemic treatment of cerebral cortex lesions in rats with a new secreted phospholipase A2 inhibitor. J. Neurotrauma 21, 1683–1691.
Farooqui A. A., Yang H. C., Rosenberger T. A. and Horrocks L. A. (1997) Phospholipase A2 and its role in brain tissue. J. Neurochem. 69, 889–901.
Feutz A. C., Pham-Dinh D., Allinquant B., Miehe M. and Ghandour M. S. (2001) An immortalized jimpy oligodendrocyte cell line: defects in cell cycle and cAMP pathway. Glia 34, 241–252. Fukumoto H., Deng A., Irizarry M. C., Fitzgerald M. L. and Rebeck
G. W. (2002) Induction of the cholesterol transporter ABCA1 in central nervous system cells by liver X receptor agonists increases secreted Abeta levels. J. Biol. Chem. 277, 48508– 48513.
Garcia-Ladona F. J., Huss Y., Frey P. and Ghandour M. S. (1997) Oli-godendrocytes express different isoforms of beta-amyloid precur-sor protein in chemically defined cell culture conditions: in situ hybridization and immunocytochemical detection. J. Neurosci. Res. 50, 50–61.
Giri S., Khan M., Rattan R., Singh I. and Singh A. K. (2006) Krabbe disease: psychosine-mediated activation of phospholipase A2 in oligodendrocyte cell death. J. Lipid Res. 47, 1478–1492. Grenier J., Trousson A., Chauchereau A., Cartaud J., Schumacher M.
and Massaad C. (2006) Differential recruitment of p160 coactiva-tors by glucocorticoid receptor between Schwann cells and astro-cytes. Mol. Endocrinol. 20, 254–267.
Hessler J. A., Budor A., Putchakayala K., Mecke A., Rieger D., Banaszak D. and Orr B. G. (2005) Atomic force microscopy study of early morphological changes during apoptosis. Langmuir 21, 9280–9286.
Janowski B. A., Willy P. J., Devi T. R., Falck J. R. and Mangelsdorf D. J. (1996) An oxysterol signalling pathway mediated by the nuclear receptor LXR alpha. Nature 383, 728–731.
Jaulmes A., Janvier B., Andreani M. and Raymondjean M. (2005) Autocrine and paracrine transcriptional regulation of type IIA
secretory phospholipase A2 gene in vascular smooth muscle cells. Arterioscler. Thromb. Vasc. Biol. 25, 1161–1167.
Joseph S. B., Castrillo A., Laffitte B. A., Mangelsdorf D. J. and Ton-tonoz P. (2003) Reciprocal regulation of inflammation and lipid metabolism by liver X receptors. Nat. Med., 9, 213–219. Kalyvas A. and David S. (2004) Cytosolic phospholipase A2 plays a key
role in the pathogenesis of multiple sclerosis-like disease. Neuron 41, 323–335.
Klembt Andersen L., Antoranz Contera S., Justesen J., Duch M., Hansen O., Chevallier J., Foss M., Pedersen F. S. and Besenbache F. (2005) Cell volume increase in murine MC3T3-E1 pre-osteoblasts attaching onto biocompatible tantalum observed by atomic force microscopy. Eur. Cells Mat. 10, 61–69.
Kolsch H., Lutjohann D., von Bergmann K. and Heun R. (2003) The role of 24S-hydroxycholesterol in Alzheimer’s disease. J. Nutr. Health Aging 7, 37–41.
Landis M. S., Patel H. V. and Capone J. P. (2002) Oxysterol activators of liver X receptor and 9-cis-retinoic acid promote sequential steps in the synthesis and secretion of tumor necrosis factor-alpha from human monocytes. J. Biol. Chem. 277, 4713–4721.
Leoni V., Masterman T., Diczfalusy U., De Luca G., Hillert J. and Bjorkhem I. (2002) Changes in human plasma levels of the brain specific oxysterol 24S-hydroxycholesterol during progression of multiple sclerosis. Neurosci. Lett. 331, 163–166.
Liere P., Pianos A., Eychenne B., Cambourg A., Liu S., Griffiths W., Schumacher M., Sjovall J. and Baulieu E. E. (2004) Novel lipoidal derivatives of pregnenolone and dehydroepiandrosterone and ab-sence of their sulfated counterparts in rodent brain. J. Lipid Res. 45, 2287–2302.
Lutjohann D., Breuer O., Ahlborg G., Nennesmo I., Siden A., Diczfalusy U. and Bjorkhem I. (1996) Cholesterol homeostasis in human brain: evidence for an age-dependent flux of 24S-hydroxycholes-terol from the brain into the circulation. Proc. Natl Acad. Sci. USA 93, 9799–9804.
Lutjohann D., Papassotiropoulos A., Bjorkhem I. et al. (2000) Plasma 24S-hydroxycholesterol (cerebrosterol) is increased in Alzheimer and vascular demented patients. J. Lipid Res. 41, 195–198. Massaad C., Garlatti M., Wilson E. M., Cadepond F. and Barouki R.
(2000) A natural sequence consisting of overlapping glucocor-ticoid-responsive elements mediates glucocorticoid, but not androgen, regulation of gene expression. Biochem. J. 350, 123– 129.
McNally H. and Ben Borgens R. (2004) Three-dimensional imaging of living and dying neurons with atomic force microscopy. J. Neur-ocyt. 33, 251–258.
McQualter J. L. and Bernard C. C. (2007) Multiple sclerosis: a battle between destruction and repair. J. Neurochem. 100, 295–306. Meijer O. (2002) Coregulator proteins and corticosteroid action in the
brain. J. Neuroendocrinol. 14, 499–505.
Millatt L. J., Bocher V., Fruchart J. C. and Staels B. (2003) Liver X receptors and the control of cholesterol homeostasis: potential therapeutic targets for the treatment of atherosclerosis. Biochim. Biophys. Acta 1631, 107–118.
Mitro N., Vargas L., Romeo R., Koder A. and Saez E. (2007) T0901317 is a potent PXR ligand: implications for the biology ascribed to LXR. FEBS Lett. 581, 1721–1726.
Moses G. S., Jensen M. D., Lue L. F., Walker D. G., Sun A. Y., Simonyi A. and Sun G. Y. (2006) Secretory PLA2-IIA: a new inflammatory factor for Alzheimer’s disease. J. Neuroinflammation 3, 28. Mouzat K., Prod’homme M., Volle D. H., Sion B., Dechelotte P.,
Gauthier K., Vanacker J. M. and Lobaccaro J. M. (2006) Oxysterol nuclear receptor LXRbeta regulates cholesterol homeostasis and contractile function in mouse uterus. J. Biol. Chem. 13, 13.
Muralikrishna Adibhatla R. and Hatcher J. F. (2006) Phospholipase A2, reactive oxygen species, and lipid peroxidation in cerebral ische-mia. Free Radic. Biol. Med. 40, 376–387.
Nishimura M., Naito S. and Yokoi T. (2004) Tissue-specific mRNA expression profiles of human nuclear receptor subfamilies. Drug Metab. Pharmacokinet. 19, 135–149.
Papassotiropoulos A., Lutjohann D., Bagli M., et al. (2000) Plasma 24S-hydroxycholesterol: a peripheral indicator of neuronal degeneration and potential state marker for Alzheimer’s disease. Neuroreport 11, 1959–1962.
Papassotiropoulos A., Lutjohann D., Bagli M., et al. (2002) 24S-hy-droxycholesterol in cerebrospinal fluid is elevated in early stages of dementia. J. Psychiatr. Res. 36, 27–32.
Pernas P., Masliah J., Olivier J. L., Salvat C., Rybkine T. and Bereziat G. (1991) Type II phospholipase A2 recombinant overexpression enhances stimulated arachidonic acid release. Biochem. Biophys. Res. Commun. 178, 1298–1305.
Petrovic U., Sribar J., Matis M., Anderluh G., Peter-Katalinic J., Krizaj I. and Gubensek F. (2005) Ammodytoxin, a secretory phospholipase A2, inhibits G2 cell-cycle arrest in the yeast Saccharomyces ce-revisiae. Biochem. J. 391, 383–388.
Ravaux L., Denoyelle C., Monne C., Limon I., Raymondjean M. and El Hadri K. (2007) Inhibition of interleukin-1beta-induced group IIA secretory phospholipase A2 expression by peroxisome proliferator-activated receptors (PPARs) in rat vascular smooth muscle cells: cooperation between PPARbeta and the proto-oncogene BCL-6. Mol. Cell. Biol. 27, 8374–8387.
Roth A. D., Ramirez G., Alarcon R. and Von Bernhardi R. (2005) Oli-godendrocytes damage in Alzheimer’s disease: beta amyloid tox-icity and inflammation. Biol. Res. 38, 381–387.
Russell D. W. (2000) Oxysterol biosynthetic enzymes. Biochim. Bio-phys. Acta 1529, 126–135.
Saegusa J., Akakura N., Wu C. Y., Hoogland C., Ma Z., Lam K. S., Liu F. T., Takada Y. K. and Takada Y. (2008) Pro-inflammatory secretory phospholipase A2 type IIA binds to integrins alphavbeta3 and alpha4beta1 and induces proliferation of monocytic cells in an integrin-dependent manner. J. Biol. Chem. 283, 26107–26115. Schmidt A., Vogel R., Holloway M. K., Rutledge S. J., Friedman O.,
Yang Z., Rodan G. A. and Friedman E. (1999) Transcription control and neuronal differentiation by agents that activate the LXR nuclear receptor family. Mol. Cell. Endocrinol. 155, 51– 60.
Shenoy S. D., Spencer T. A., Mercer-Haines N. A., Alipour M., Gargano M. D., Runge-Morris M. and Kocarek T. A. (2004) CYP3A induction by liver x receptor ligands in primary cultured rat and mouse hepatocytes is mediated by the pregnane X receptor. Drug Metab. Dispos. 32, 66–71.
Specty O., Pageaux J. F., Dauca M., Lagarde M., Laugier C. and Fayard J. M. (2001) Control of cell proliferation via transduction of sPLA(2)-I activity and possible PPAR activation at the nuclear level. FEBS Lett. 490, 88–92.
Sun G. Y., Xu J., Jensen M. D., Yu S., Wood W. G., Gonzalez F. A., Simonyi A., Sun A. Y. and Weisman G. A. (2005) Phospholi-pase A2 in astrocytes: responses to oxidative stress, inflamma-tion, and G protein-coupled receptor agonists. Mol. Neurobiol. 31, 27–41.
Teunissen C. E., Dijkstra C. D., Polman C. H., Hoogervorst E. L., von Bergmann K. and Lutjohann D. (2003) Decreased levels of the brain specific 24S-hydroxycholesterol and cholesterol pre-cursors in serum of multiple sclerosis patients. Neurosci. Lett. 347, 159–162.
Tontonoz P. and Mangelsdorf D. J. (2003) Liver X receptor signal-ing pathways in cardiovascular disease. Mol. Endocrinol. 17, 985– 993.
Ulven S. M., Dalen K. T., Gustafsson J. A. and Nebb H. I. (2005) LXR is crucial in lipid metabolism. Prostaglandins Leukot. Essent. Fatty Acids 73, 59–63.
Uppal H., Saini S. P., Moschetta A., et al. (2007) Activation of LXRs prevents bile acid toxicity and cholestasis in female mice. Hepa-tology 45, 422–432.
Volle D. H. and Lobaccaro J. M. (2007) Role of the nuclear receptors for oxysterols LXRs in steroidogenic tissues: beyond the ‘‘foie gras’’, the steroids and sex? Mol. Cell. Endocrinol. 265-266, 183–189.
Wang L., Schuster G. U., Hultenby K., Zhang Q., Andersson S. and Gustafsson J. A. (2002) Liver X receptors in the central nervous
system: from lipid homeostasis to neuronal degeneration. Proc. Natl Acad. Sci. USA 99, 13878–13883.
Whitney K. D., Watson M. A., Collins J. L., et al. (2002) Regulation of cholesterol homeostasis by the liver X receptors in the central nervous system. Mol. Endocrinol. 16, 1378–1385.
Yagami T., Ueda K., Asakura K., et al. (2002) Human group IIA secretory phospholipase A2 induces neuronal cell death via apoptosis. Mol. Pharmacol. 61, 114–126.
Zelcer N. and Tontonoz P. (2006) Liver X receptors as integrators of metabolic and inflammatory signaling. J. Clin. Invest. 116, 607– 614.
