Abstract
BackgroundReactive 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 (S1P3) signaling and SphK1 in activated rat astrocytes has not been defined.Methodology/Principal FindingsUsing immunohistochemistry we observed the upregulation of S1P3 and SphK1 expression on reactive astrocytes and SphK1 on macrophages in MS lesions. Increased mRNA and protein expression of S1P3 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 S1P3 agonist led to increased phosphorylation of extracellular signal-regulated kinase (ERK)-1/2), which was further elevated with a LPS pre-challenge, suggesting that S1P3 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 S1P3 signaling compared to S1P1 signaling was demonstrated by the use of selective S1P3 or S1P1 agonists.Conclusion/SignificanceIn summary, our data demonstrate that the SphK1/S1P3 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.
Highlights
Astrocytes are the most abundant glial cells in the mammalian central nervous system (CNS)
Coexpression of S1P3 and glial fibrillary acidic protein (GFAP) on all reactive astrocytes in multiple sclerosis (MS) lesions and surrounding tissues was confirmed by double-immunostaining (Fig. 2A), but only small numbers of S1P3-expressing CD68-positive macrophages could be detected
Co-expression of sphingosine kinase 1 (SphK1) and GFAP on reactive astrocytes in MS lesions and their borders was confirmed by double immunostaining (Fig. 2B), and SphK1 expression on macrophages in MS lesions, lesion borders and around blood vessels was confirmed by double immunostaining for SphK1 and CD68 (Fig. 2C, D)
Summary
Astrocytes are the most abundant glial cells in the mammalian central nervous system (CNS) They have important functions in maintenance of homeostasis and are involved in synaptic function and physical structuring of the CNS during development [1], and become reactive in response to pathological insults [2,3]. They can upregulate genes involved in amplification of inflammation by attracting inflammatory cells to specific sites and limit immune cell invasion of adjacent healthy parenchyma [4,5]. 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 (S1P3) signaling and SphK1 in activated rat astrocytes has not been defined
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