Elsevier

Experimental Cell Research

Volume 313, Issue 17, 15 October 2007, Pages 3779-3788
Experimental Cell Research

Research Article
RhoE participates in the stimulation of the inflammatory response induced by ethanol in astrocytes

https://doi.org/10.1016/j.yexcr.2007.07.018Get rights and content

Abstract

Astroglial cells are involved in the neuropathogenesis of several inflammatory diseases of the brain, where the activation of inflammatory mediators and cytokines plays an important role. We have previously demonstrated that ethanol up-regulates inflammatory mediators in both brain and astroglial cells. Since Rho GTPases are involved in inflammatory responses of astrocytes where loss of stress fibers takes place and RhoE/Rnd3 disorganizes the actin cytoskeleton, the aim of the present study was to investigate the implication of this protein in the stimulation of inflammatory signaling induced by ethanol. Our findings show that RhoE expression induces a decrease in both RhoA and Rac. In addition, RhoE not only induces actin cytoskeleton disorganization but it also stimulates both the IRAK/ERK/NF-κB pathway and the COX-2 expression associated with the inflammatory response in these cells. Our results also show that ethanol exposure induces RhoE signaling in astrocytes. Preincubation of astrocytes with GF109203X, an inhibitor of PKCs, reduces the RhoE levels and abolishes the ethanol-induced activation of IRAK, NF-κB and the COX-2 expression. Furthermore, RhoE overexpression restores ethanol responses in astrocytes treated with the PKCs inhibitor. Altogether, our findings suggest that this small GTPase is involved in the stimulation of the inflammatory signaling induced by ethanol in astrocytes. These findings provide new insights into the molecular mechanism involved in the inflammatory responses in astrocytes.

Introduction

Astrocytes represent the largest glial population in the mammalian brain and evidence from several studies suggests that these cells are crucial regulators of the inflammatory response in the CNS [1]. Astroglial cells respond vigorously to brain injury and it has been postulated that they play an important role in the fine tuning of brain inflammation [2]. Stimulation of glial cells leads to the secretion of cytokines and to the activation of inflammatory mediators, which might induce brain damage and neurodegeneration [3], [4].

Alcohol consumption can cause brain injury and neurodegeneration [5], [6]. Our recent findings demonstrate that ethanol treatment up-regulates cytokines and inflammatory mediators in both brain and astroglial cells, activating signaling events associated with inflammation [7]. We have further shown that ethanol activates TLR4/IL-1RI receptors in astrocytes, stimulating similar signaling pathways to those observed by LPS and IL-1β [8]. However, the mechanisms by which alcohol activates these receptors triggering the inflammatory response in the brain and in astroglial cells are still poorly understood.

Toll-like receptors (TLRs) are type 1 transmembrane proteins which play important roles in the innate immune system as a first line of defense against pathogens [9]. The TLRs belong to a large superfamily that includes the interleukin-1 receptors (IL-1Rs) [10]. TLRs and IL-1Rs have a conserved region of ∼ 200 amino acids in their cytoplasmic tails, which is known as Toll/IL-1R (TIR) domain [10]. After ligand binding, TLRs/IL-1Rs dimerize and undergo the conformational change required for the recruitment of downstream signaling molecules, including IL-1R-associated kinase (IRAK), TNFR-associated factor 6 (TRAF6) and NF-κB-inducing kinase (NIK) [10]. The recruitment of these molecules triggers the stimulation of downstream kinases, including MAPKs and SAPK/JNK, as well as the activation of the transcriptional factors NF-κB and AP-1 [10]. This activation contributes to the induction of genes encoding cytokines, chemokines and reactive mediators including nitric oxide, cyclooxygenase-2 (COX-2) and reactive oxygen species.

Emerging evidence suggests the participation of Rho GTPases as key regulatory components of TLR signaling cascades [9]. Rho GTPases act as molecular switches regulating many basic cellular functions including actin cytoskeletal organization, cell growth, apoptosis and recently, in innate immune response [30]. Indeed, Rho proteins are involved in cytokine signaling [11], [12] participating in inflammatory responses in astrocytes, where an important actin cytoskeleton reorganization takes place, with loss of stress fibers and disruption of focal adhesions [1]. Among Rho GTPases, RhoE/Rnd3 is a member of the Rnd subfamily that acts antagonistically to RhoA [13] and induces stress fiber disassembly [14], [15]. RhoE binds and inhibits ROCK-I from phosphorylating its downstream target [16]. RhoE itself is regulated by phosphorylation by ROCK-I on multiple sites [17].

Considering the role of GTPases as regulators of TLRs response, and the involvement of RhoE in actin cytoskeleton disorganization [14], in the current study we set out to investigate the molecular events underlying the activation of inflammatory mediators induced by ethanol and the potential involvement of RhoE in this process. Here we report that RhoE expression induces signaling associated with inflammation as IRAK, the NF-κB transcription factor and COX-2 expression. Furthermore, ethanol exposure not only stimulates RhoE signaling but this GTPase is also involved in the stimulation of the inflammatory response induced by ethanol in astrocytes.

Section snippets

Astrocyte culture

Primary cultures of astrocytes from 21-day-old rat fetuses were prepared from brain hemispheres as described in detail previously [18]. The purity of astrocytes was assessed by immunofluorescence using: anti-glial fibrillary acidic protein (anti-GFAP, astrocyte marker, Sigma-Aldrich), anti-CD-68 (microglial marker, Serotec), anti-myelin basic protein (MBP, oligodendroglial marker, Sigma-Aldrich) and anti-microtubule associated protein 2 (MAP-2, neuronal marker, Sigma-Aldrich). The astrocyte

RhoE disrupts the actin cytoskeleton organization of primary astrocytes

Since Rho GTPases are involved in inflammatory responses in astrocytes [1], we analyzed whether RhoE was involved in the ethanol-induced stimulation of inflammatory mediators in these cells [7]. However, as the role of RhoE in astroglial cells is unknown, we first investigated the effect of RhoE in the actin cytoskeleton organization of primary astrocytes. Cells were transfected with a plasmid that expresses the RhoE protein, together with a plasmid expressing the green fluorescent protein

Discussion

Rho family of proteins has been implicated in inflammatory responses of astrocytes, where a dramatic reorganization of actin cytoskeleton with loss of stress fibers and disruption of focal adhesions was found [1]. We and others have previously reported that RhoE/Rnd3 protein induces those actin cytoskeleton alterations [14], [15]. In addition, our recent findings demonstrated that ethanol caused a disorganization of actin cytoskeleton in astrocytes [20] and also that alcohol triggered

Acknowledgments

We would like to thank Dr. Anne J Ridley for the generous gift of pCMV5-FLAG-RhoE plasmid and M. March for her excellent technical help. This work was supported by grants from MCYT (SAF 2003-06217; SAF 2006-02178), Instituto de Salud Carlos III (CP03/00105 and PI051207) and Generalitat Valenciana (GV05/084). A.P.A. was supported by the Fundación Valenciana de Investigaciones Biomédicas.

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    Present address: Parque Tecnológico Valencia. C/Benjamín Franklin 5-11. Paterna 46980, Valencia, Spain.

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