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, 6 (8), e23905

Sphingosine Kinase 1 and Sphingosine 1-phosphate Receptor 3 Are Functionally Upregulated on Astrocytes Under Pro-Inflammatory Conditions

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Sphingosine Kinase 1 and Sphingosine 1-phosphate Receptor 3 Are Functionally Upregulated on Astrocytes Under Pro-Inflammatory Conditions

Iris Fischer et al. PLoS One.

Abstract

Background: Reactive astrocytes are implicated in the development and maintenance of neuroinflammation in the demyelinating disease multiple sclerosis (MS). The sphingosine kinase 1 (SphK1)/sphingosine1-phosphate (S1P) receptor signaling pathway is involved in modulation of the inflammatory response in many cell types, but the role of S1P receptor subtype 3 (S1P(3)) signaling and SphK1 in activated rat astrocytes has not been defined.

Methodology/principal findings: Using immunohistochemistry we observed the upregulation of S1P(3) and SphK1 expression on reactive astrocytes and SphK1 on macrophages in MS lesions. Increased mRNA and protein expression of S1P(3) and SphK1, as measured by qPCR and Western blotting respectively, was observed after treatment of rat primary astrocyte cultures with the pro-inflammatory stimulus lipopolysaccharide (LPS). Activation of SphK by LPS stimulation was confirmed by SphK activity assay and was blocked by the use of the SphK inhibitor SKI (2-(p-hydroxyanilino)-4-(p-chlorphenyl) thiazole. Treatment of astrocytes with a selective S1P(3) agonist led to increased phosphorylation of extracellular signal-regulated kinase (ERK)-1/2), which was further elevated with a LPS pre-challenge, suggesting that S1P(3) upregulation can lead to increased functionality. Moreover, astrocyte migration in a scratch assay was induced by S1P and LPS and this LPS-induced migration was sensitive to inhibition of SphK1, and independent of cell proliferation. In addition, S1P induced secretion of the potentially neuroprotective chemokine CXCL1, which was increased when astrocytes were pre-challenged with LPS. A more prominent role of S1P(3) signaling compared to S1P(1) signaling was demonstrated by the use of selective S1P(3) or S1P(1) agonists.

Conclusion/significance: In summary, our data demonstrate that the SphK1/S1P(3) signaling axis is upregulated when astrocytes are activated by LPS. This signaling pathway appears to play a role in the establishment and maintenance of astrocyte activation. Upregulation of the pathway in MS may be detrimental, e.g. through enhancing astrogliosis, or beneficial through increased remyelination via CXCL1.

Conflict of interest statement

Competing Interests: The study is part of a PhD project and stands apart from any internal project. The first author of the study, Iris Fischer, is a PhD student employed by and working at Merck Serono. Chantal Alliod, Nicolase Martinier and Corinne Brana are also employed by Merck Serono and helped in the study design, experiments and/or analyses. Sandrine Pouly, also from Merck Serono, is the manager of Iris Fischer and has supervised the study. Jia Newcombe who is working for the NeuroResource, UCL Institute of Neurology in London, provided the human tissues and gave expert inputs on the stainings. The authors confirm that employment by Merck Serono does not alter their adherence to any of the PLoS ONE policies on sharing data and materials.

Figures

Figure 1
Figure 1. S1P3 and SphK1 are upregulated in MS lesions.
(A, B): Immunohistochemical peroxidise staining shows that S1P3 receptor and SphK1 enzyme expressions are increased in a chronic-active MS lesion which was located in parietal subventricular white matter. S1P3 expression is strong on reactive astrocytes in this lesion border (C), in the lesion (D) and on perivascular cells (G), but is weak in normal control brain white matter (H). SphK1 expression is increased in reactive astrocytes (E) and in macrophages in this MS lesion (F). A particularly high expression of SphK1 is seen in perivascular inflammatory cells (I), whereas its expression is very low in normal control brain white matter (J). This Figure shows representative stainings. Scale bars are 200 µm for A and B, and 20 µm for C–J. The sections were counterstained with haematoxylin.
Figure 2
Figure 2. S1P3 and SphK1 are expressed by reactive astrocytes and macrophages in MS lesions.
Immunofluorescence co-localization studies confirmed that S1P3 is predominately expressed by reactive astrocytes (A). SphK1 is also expressed on reactive astrocytes (B), but the major cell type expressing SphK1 is macrophage in lesions and perivascular cuffs (C–D). Scale bars are 10 µm.
Figure 3
Figure 3. S1P3 and SphK1 mRNAs and protein are upregulated in rat primary astrocytes by LPS stimulation.
Primary astrocytes were incubated in culture medium for 5 or 24 h with LPS (100 ng/ml). The mRNA levels of S1P1 (A), S1P3 (B), SphK1 (C) and SphK2 (D) were assessed after 5 h and 24 h incubation. Quantitative PCR results are shown as the percentage expression of HKG (GAPDH) and represent mean ± SEM of three independent experiments. LPS mediated sustained upregulation of S1P3 (E–F) and SphK1 (G–H) as shown by Western blots of plasma membrane fractions. Primary rat astrocytes were incubated with LPS (100 ng/ml) in serum-free medium containing 0.25% BSA for 12 and 48 h. Representative immunoblots are shown. Graphs represent the mean ± SEM of three independent experiments and are reported as protein expression normalized to actin, expressed as fold change over basal level. One-Way ANOVA followed by Bonferroni's multiple comparison test: *p<0.05, **p<0.01 vs. respective control.
Figure 4
Figure 4. SphK1 is activated in response to LPS.
Serum-deprived astrocytes were incubated for 30 min with 100 ng/ml LPS in the presence or absence of SKI (10 µg/ml). 100 µg of cell extracts were used to determine SphK1 activity by thin layer chromatography. Data represents the mean ± SEM of three independent experiments. One-Way ANOVA followed by Bonferroni's multiple comparison test: *p<0.05, **p<0.01.
Figure 5
Figure 5. Increased S1P3-mediated ERK-1/2, but not Akt signaling by LPS.
Astrocytes were serum-deprived during the 12 h treatment with LPS and were then stimulated for 20 min with 10 µM S1P3 agonist (Compound 20) or 1 µM AUY954. The results show the relative ERK-1/2 phosphorylation (A, C ) and Akt phosphorylation (B, D) ± SEM normalized against actin for three (A, B) and two (C, D) independent experiments. One-Way ANOVA followed by Bonferroni's multiple comparison test: *p<0.05, **p<0.01, ***p<0.001.
Figure 6
Figure 6. S1P and LPS induce astrocyte migration in a scratch assay.
Astrocytes were stimulated with S1P (500 nM), LPS (100 ng/ml) or combined treatments of S1P+LPS. Cells were allowed to migrate into the scratch for 48 h. The images show the representative migration of astrocytes into a scratch in response to the different stimuli 48 h after treatment (A–D). The surface of area covered by GFAP immunoreactivity was plotted and each scratch was evaluated with an average of four photographs, and each treatment group represented five to six replicates (bar graph). Data are representative of at least three independent experiments ± SEM. One-Way ANOVA followed by Dunnett's post-test: *p<0.05, **p<0.01, ***p<0.001.
Figure 7
Figure 7. LPS-induced astrocyte migration is SphK1-dependent, but proliferation-independent.
Astrocyte were treated with increasing concentrations of S1P (10 nM, 100 nM, 1000 nM) (A) or LPS (1 ng/ml, 10 ng/ml, 100 ng/ml) (B), and cell proliferation was measured using a [3H]- thymidine uptake assay. Data are representative of three independent experiments ± SEM. One-Way ANOVA followed by Dunnett's post-test: (A) *p<0.05 vs. control, ***p<0.001 vs. control. Astrocytes were pre-treated or not with SKI (10 µg/ml, 1 h) and then stimulated with LPS (100 ng/ml) (C) or stimulated with S1P (500 nM) and LPS (100 ng/ml), respectively, in the presence or absence of 10 µM antimitotic treatment (D). The graphs show the SKI-mediated inhibition of LPS-induced migration (C) and the influence of antimitiotic treatment on S1P- or LPS-induced migration after 48 h incubation (D). The surface of area covered by GFAP immunoreactivity is plotted. Each scratch was evaluated with an average of four photographs, and each treatment group represented five to six replicates. Data are representative of three independent experiments ± SEM. One-Way ANOVA followed by Dunnett's post-test: (C) *p<0.05, **p<0.01; (D) *p<0.05 **p<0.01 vs. respective control.
Figure 8
Figure 8. S1P3 contributes to the S1P-induced CXCL1 release by primary astrocytes.
Astrocytes were pre-treated with or without LPS (100 ng/ml) for 12 h in serum-free medium and then stimulated or not with S1P (1 µM), a S1P3 agonist (Compound 20, 10 µM) or a S1P1 agonist (AUY954, 10 µM) for 5 h. The graph shows the effect of LPS pre-treatment on the release of CXCL1 by S1P, S1P3 or S1P1 agonists. Data are a pool of two independent experiments each performed in six replicates ± SEM. One-Way ANOVA followed by Bonferroni's multiple comparison test: **p<0.01, ***p<0.001.

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