Sphingosine Kinase 1 and Sphingosine 1-Phosphate Receptor 3 Are Functionally Upregulated on Astrocytes under Pro-Inflammatory Conditions

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.