Sphingosine 1-phosphate receptor 3 and RhoA signaling mediate inflammatory gene expression in astrocytes

Abstract

Sphingosine 1-phosphate (S1P) signals through G protein-coupled receptors to elicit a wide range of cellular responses. In CNS injury and disease, the blood-brain barrier is compromised, causing leakage of S1P from blood into the brain. S1P can also be locally generated through the enzyme sphingosine kinase-1 (Sphk1). Our previous studies demonstrated that S1P activates inflammation in murine astrocytes. The S1P 1 receptor subtype has been most associated with CNS disease, particularly multiple sclerosis. S1P 3 is most highly expressed and upregulated on astrocytes, however, thus we explored the involvement of this receptor in inflammatory astrocytic responses. Astrocytes isolated from wild-type (WT) or S1P 3 knockout (KO) mice were treated with S1P 3 selective drugs or transfected with short interfering RNA to determine which receptor subtypes mediate S1P-stimulated inflammatory responses. Interleukin-6 (IL-6), and vascular endothelial growth factor A (VEGFa) messenger RNA (mRNA) and cyclooxygenase-2 (COX-2) mRNA and protein were assessed by q-PCR and Western blotting. Activation of RhoA was measured using SRE.L luciferase and RhoA implicated in S1P signaling by knockdown of Gα 12/13 proteins or by inhibiting RhoA activation with C3 exoenzyme . Inflammation was simulated by in vitro scratch injury of cultured astrocytes. S1P 3 was highly expressed in astrocytes and further upregulated in response to simulated inflammation. Studies using S1P 3 knockdown and S1P 3 KO astrocytes demonstrated that S1P 3 mediates activation of RhoA and induction of COX-2, IL-6, and VEGFa mRNA, with some contribution from S1P 2 . S1P induces expression of all of these genes through coupling to the Gα 12/13 proteins which activate RhoA. Studies using S1P 3 selective agonists/antagonists as well as Fingolimod (FTY720) confirmed that stimulation of S1P 3 induces COX-2 expression in astrocytes. Simulated inflammation increased expression of Sphk1 and consequently activated S1P 3 , demonstrating an autocrine pathway through which S1P is formed and released from astrocytes to regulate COX-2 expression. S1P 3 , through its ability to activate RhoA and its upregulation in astrocytes, plays a unique role in inducing inflammatory responses and should be considered as a potentially important therapeutic target for CNS disease progression.