Cargando…

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

BACKGROUND: 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 enzym...

Descripción completa

Detalles Bibliográficos
Autores principales: Dusaban, Stephanie S., Chun, Jerold, Rosen, Hugh, Purcell, Nicole H., Brown, Joan Heller
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5455202/
https://www.ncbi.nlm.nih.gov/pubmed/28577576
http://dx.doi.org/10.1186/s12974-017-0882-x
_version_ 1783240996717330432
author Dusaban, Stephanie S.
Chun, Jerold
Rosen, Hugh
Purcell, Nicole H.
Brown, Joan Heller
author_facet Dusaban, Stephanie S.
Chun, Jerold
Rosen, Hugh
Purcell, Nicole H.
Brown, Joan Heller
author_sort Dusaban, Stephanie S.
collection PubMed
description BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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.
format Online
Article
Text
id pubmed-5455202
institution National Center for Biotechnology Information
language English
publishDate 2017
publisher BioMed Central
record_format MEDLINE/PubMed
spelling pubmed-54552022017-06-06 Sphingosine 1-phosphate receptor 3 and RhoA signaling mediate inflammatory gene expression in astrocytes Dusaban, Stephanie S. Chun, Jerold Rosen, Hugh Purcell, Nicole H. Brown, Joan Heller J Neuroinflammation Research BACKGROUND: 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. METHODS: 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. RESULTS: 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. CONCLUSIONS: 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. BioMed Central 2017-06-02 /pmc/articles/PMC5455202/ /pubmed/28577576 http://dx.doi.org/10.1186/s12974-017-0882-x Text en © The Author(s). 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research
Dusaban, Stephanie S.
Chun, Jerold
Rosen, Hugh
Purcell, Nicole H.
Brown, Joan Heller
Sphingosine 1-phosphate receptor 3 and RhoA signaling mediate inflammatory gene expression in astrocytes
title Sphingosine 1-phosphate receptor 3 and RhoA signaling mediate inflammatory gene expression in astrocytes
title_full Sphingosine 1-phosphate receptor 3 and RhoA signaling mediate inflammatory gene expression in astrocytes
title_fullStr Sphingosine 1-phosphate receptor 3 and RhoA signaling mediate inflammatory gene expression in astrocytes
title_full_unstemmed Sphingosine 1-phosphate receptor 3 and RhoA signaling mediate inflammatory gene expression in astrocytes
title_short Sphingosine 1-phosphate receptor 3 and RhoA signaling mediate inflammatory gene expression in astrocytes
title_sort sphingosine 1-phosphate receptor 3 and rhoa signaling mediate inflammatory gene expression in astrocytes
topic Research
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5455202/
https://www.ncbi.nlm.nih.gov/pubmed/28577576
http://dx.doi.org/10.1186/s12974-017-0882-x
work_keys_str_mv AT dusabanstephanies sphingosine1phosphatereceptor3andrhoasignalingmediateinflammatorygeneexpressioninastrocytes
AT chunjerold sphingosine1phosphatereceptor3andrhoasignalingmediateinflammatorygeneexpressioninastrocytes
AT rosenhugh sphingosine1phosphatereceptor3andrhoasignalingmediateinflammatorygeneexpressioninastrocytes
AT purcellnicoleh sphingosine1phosphatereceptor3andrhoasignalingmediateinflammatorygeneexpressioninastrocytes
AT brownjoanheller sphingosine1phosphatereceptor3andrhoasignalingmediateinflammatorygeneexpressioninastrocytes