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. 2017 Mar;91(3):197–207. doi: 10.1124/mol.116.106963

Regulator of G Protein Signaling 10 (Rgs10) Expression Is Transcriptionally Silenced in Activated Microglia by Histone Deacetylase Activity

Mohammed Alqinyah 1, Nagini Maganti 1, Mourad W Ali 1, Ruchi Yadav 1, Mei Gao 1, Ercan Cacan 1, Han-Rong Weng 1, Susanna F Greer 1, Shelley B Hooks 1,✉
PMCID: PMC5325084  PMID: 28031332

Abstract

RGS10 has emerged as a key regulator of proinflammatory cytokine production in microglia, functioning as an important neuroprotective factor. Although RGS10 is normally expressed in microglia at high levels, expression is silenced in vitro following activation of TLR4 receptor. Given the ability of RGS10 to regulate inflammatory signaling, dynamic regulation of RGS10 levels in microglia may be an important mechanism to tune inflammatory responses. The goals of the current study were to confirm that RGS10 is suppressed in an in vivo inflammatory model of microglial activation and to determine the mechanism for activation-dependent silencing of Rgs10 expression in microglia. We demonstrate that endogenous RGS10 is present in spinal cord microglia, and RGS10 protein levels are suppressed in the spinal cord in a nerve injury–induced neuropathic pain mouse model. We show that the histone deacetylase (HDAC) enzyme inhibitor trichostatin A blocks the ability of lipopolysaccharide (LPS) to suppress Rgs10 transcription in BV-2 and primary microglia, demonstrating that HDAC enzymes are required for LPS silencing of Rgs10. Furthermore, we used chromatin immunoprecipitation to demonstrate that H3 histones at the Rgs10 proximal promoter are deacetylated in BV-2 microglia following LPS activation, and HDAC1 association at the Rgs10 promoter is enhanced following LPS stimulation. Finally, we have shown that sphingosine 1-phosphate, an endogenous microglial signaling mediator that inhibits HDAC activity, enhances basal Rgs10 expression in BV-2 microglia, suggesting that Rgs10 expression is dynamically regulated in microglia in response to multiple signals.

Introduction

Microglia are central nervous system (CNS)–resident macrophages that serve protective functions to combat infection and clear cellular debris, as well as developmental functions, including synaptic pruning (Gehrmann et al., 1995; Stevens et al., 2007; Trang et al., 2011). In addition to these normal physiologic functions, dysregulated microglial activation has been implicated in the initiation and progression of neurodegenerative disorders such as multiple sclerosis, Alzheimer’s disease, and Parkinson’s disease (Fu et al., 2014), and in neuropathic pain (Trang et al., 2011). Identifying signaling pathways regulating microglial functions bears significance in the development of strategies for the treatment of such neurologic disorders.

Regulator of G protein signaling (RGS) 10 has emerged as an important anti-inflammatory regulator in microglia. RGS10 is a member of the RGS superfamily of proteins that deactivate heterotrimeric G proteins, with profound effects on G protein–coupled receptor (GPCR) signaling in neural diseases (Zachariou et al., 2003; Nishiguchi et al., 2004; Hurst and Hooks, 2009; Okahisa et al., 2011; Vellano et al., 2011). RGS proteins are a highly diverse group of proteins that regulate signaling pathways downstream of GPCRs. The classic role of RGS proteins is to regulate the duration and amplitude of G protein signaling through their ability to function as GTPase-activating proteins to accelerate the deactivation of G proteins by increasing the rate of GTP hydrolysis (Posner et al., 1999). RGS10 selectively deactivates Gi family G proteins (Hunt et al., 1996), and it is expressed at high levels in the brain (Gold et al., 1997) and immune tissues (Haller et al., 2002), with specific enrichment in microglia (Waugh et al., 2005).

Recent studies suggest that RGS10 protein in microglia serves to suppress microglial activation, proliferation, and nuclear factor-κB (NF-κB) activity downstream of Toll-like receptor 4 (TLR4) receptors, and loss of RGS10 enhances microglial-mediated neuroinflammation and neurotoxicity. RGS10 knockout mice display significantly more activated microglia in brain tissue (Lee et al., 2008). Furthermore, in a mouse model of Parkinson’s disease, RGS10 knockout animals display exacerbated dopaminergic neuron cell death compared with wild-type animals. The anti-inflammatory role of microglial RGS10 is also observed and consistently modeled in the mouse microglial cell line BV-2 (Henn et al., 2009). Knockdown of RGS10 in BV-2 cells enhances activity of the transcription factor NF-κB and increases expression of inflammatory cytokines, including tumor necrosis factor (TNF)-α and interleukin (IL)-1β in response to the classic TLR4 activator lipopolysaccharide (LPS) (Henn et al., 2009). In complementary experiments, RGS10 overexpression suppressed microglial activation, proinflammatory cytokine release, and inflammatory neurotoxicity, and inhibited activation of microglial NF-κB (Lee et al., 2008, 2011). Therefore, RGS10 is an important regulator of inflammatory signaling in microglia in vivo and in vitro, and changes in RGS10 levels have significant effects on microglial inflammatory signaling.

RGS10 is normally found in microglia at high levels, but Lee et al. (2008) have reported that RGS10 protein levels are markedly reduced following microglial activation by LPS or TNF-α in microglia. Given the ability of RGS10 to regulate inflammatory signaling, understanding the mechanisms that control RGS10 levels in microglia may reveal new therapeutic strategies to address neuroinflammatory disease. The most commonly described mechanism for regulation of RGS protein abundance involves critical post-translational mechanisms that control protein stability (Sjögren et al., 2012; Raveh et al., 2014). However, our studies in ovarian cancer cells suggest that Rgs10 is transcriptionally regulated by DNA and histone-targeted epigenetic mechanisms (Ali et al., 2013; Cacan et al., 2014). It is unclear whether and how expression of Rgs10 in microglia is regulated via epigenetic mechanisms. Furthermore, it is unknown whether Rgs10 expression can be suppressed by pathologic microglial activation and neuroinflammation in vivo.

The goals of the current study were to determine whether RGS10 is suppressed in an in vivo neuroinflammatory model of microglial activation and to determine the mechanism for activation-dependent silencing of Rgs10 expression in microglia. We observed significant loss of RGS10 in the spinal dorsal horn following nerve injury–induced inflammation, and we define a role for histone deacetylase (HDAC) regulation of histone acetylation at the Rgs10 promoter following activation with LPS.

Materials and Methods

Cells and Reagents.

The murine microglial BV-2 cell line is a well-established model widely used to study microglial functions (Henn et al., 2009) and was a generous gift from G. Hasko at University of Medicine and Dentistry of New Jersey (Newark, NJ). The BV-2 cell line was generated by infecting primary microglial cell cultures with a v-raf/v-myc oncogene-carrying retrovirus (J2) (Blasi et al., 1990). BV-2 cells were maintained in Dulbecco's modified Eagle's medium (American Type Culture Collection, Manassas, VA) supplemented with 10% fetal bovine serum (GE Healthcare, Pittsburgh, PA). LPS, H89 dihydrochloride, trichostatin A (TSA), and 5-Aza-2′-deoxycytidine (5-Aza) were obtained from Sigma-Aldrich (St. Louis, MO). Sphingosine 1-phosphate was obtained from Avanti Polar Lipids (Alabaster, AL). TNF-α antagonist III, R-7050, was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). For Western blot analysis, the following antibodies were used: phosphor-p44/p42 extracellular signal-regulated kinase (ERK; Cell Signaling Technologies, Danvers, MA), total ERK (Cell Signaling Technologies), and anti-RGS10 antibody (C10; Santa Cruz Biotechnology). For chromatin immunoprecipitation (ChIP) experiments, we used anti-histone H3 and anti-acetylated histone H3 (Millipore, Lake Placid, NY) and anti-HDAC1 (Santa Cruz Biotechnology).

Quantitative Real-Time Polymerase Chain Reaction.

mRNA was isolated using TRIzol reagent (Invitrogen/Life Technologies, Carlsbad, CA), and cDNA was synthesized from 2 μg total RNA using the High Capacity Reverse Transcriptase cDNA kit (Life Technologies/Thermo Fisher Scientific, Carlsbad, CA). Quantitative real-time polymerase chain reaction (PCR) was performed using Superscript III kit for reverse-transcription PCR (RT-PCR; Invitrogen/Life Technologies) and Power SYBR Green reagent (Life Technologies/Thermo Fisher Scientific). Reactions were normalized using the housekeeping gene [actin and/or glyceraldehyde-3-phosphate dehydrogenase (GAPDH), as indicated], and calculations were performed according to the 2−ΔΔCT method. Fold change in expression was determined in triplicate in three independent experiments. Primers used were based on algorithm-generated sequences from Primer Bank (http://pga.mgh.harvard.edu/primerbank/). RGS10 forward, 5′-CCT GGA GAA TCT TCT GGA AGA CC-3′; RGS10 reverse, 5′-CTG CTT CCT GTC CTC CGT TTT C-3′; TNF-α forward, 5′-CCT GTA GCC CAC GTC GTA G-3′; TNF-α reverse, 5′-GGG AGT AGA CAA GGT ACA ACC C-3′; IL-1β forward, 5′-GAA ATG CCA CCT TTT GAC AGT G-3′; IL-1β reverse, 5′-TGG ATG CTC TCA TCA GGA CAG-3′; actin forward, 5′-GGC TGT ATT CCC CTC CAT CG-3′; actin reverse, 5′-CCA GTT GGT AAC AAT GCC ATG T-3′; GAPDH forward, 5′-TGG CCT TCC GTG TTC CTA C-3′; and GAPDH reverse, 5′-GAG TTG CTG TTG AAG TCG CA-3′. All primers were purchased from Integrated DNA Technologies, IDT (Coralville, IA).

Western Blot Analysis.

To evaluate protein expression in cell lines, 105 cells were lysed in SDS-PAGE sample buffer. The lysates were boiled for 5 minutes and analyzed using SDS-PAGE. Membranes were incubated with primary antibodies and appropriate horseradish peroxidase–conjugated rabbit secondary antibodies (Thermo Fisher Scientific; Pierce, Rockford, IL) and visualized using enhanced chemiluminescence reagents (Thermo Fisher Scientific; Pierce). Membranes were subsequently blotted with GAPDH antibodies (Millipore Technologies) as a loading control and quantified using ImageJ 1.46 software (NIH).

To analyze RGS10 protein expression in the spinal dorsal horn, mice were deeply anesthetized with urethane (1.3–1.5 g/kg, i.p.). The L4 to L5 spinal segment was exposed by surgery and removed from the mice, and the animals were then euthanized by cervical dislocation. The dorsal quadrant of the spinal segment ipsilateral to the nerve injury site or sham operation site was isolated. The isolated tissues were quickly frozen in liquid nitrogen and stored at −80°C for later use. The frozen tissues were homogenized with a hand-held pellet in lysis buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1 mM EDTA, 0.1% SDS, 1% deoxycholic acid, 2 mM orthovanadate, 100 mM NaF, 1% Triton X-100, 0.5 mM phenylmethylsulfonyl fluoride, 20 μM leupeptin, 100 IU ml−1 aprotinin) for 0.5 hour at 37°C. The samples were then centrifuged for 20 minutes at 12,000g at 4°C, and the supernatants containing proteins were collected. The quantification of protein contents was made by the bicinchoninic acid method. Protein samples (40 μg) were electrophoresed in 8% SDS polyacrylamide gels and transferred to a polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membranes were blocked with 5% milk and incubated overnight at 4°C with antibodies against RGS10 (1:1000, C10; Santa Cruz Biotechnology) or β-actin (1:2000; Sigma-Aldrich) as a loading control. The blots were then incubated for 1 hour at room temperature with the corresponding horseradish peroxidase–conjugated secondary antibody (1:5000; Santa Cruz Biotechnology), visualized in enhanced chemiluminescence solution (SuperSignal West Pico Chemiluminescent Substrate; Pierce) for 1 minute, and exposed onto FluorChem HD2 System. The intensity of immunoreactive bands was quantified using ImageJ 1.46 software (NIH). Results were expressed as the ratio of each marker over β-actin control.

ChIP Assay.

ChIP assays were performed as described in Cacan et al. (2014). Briefly, BV-2 cells were plated at a density of 2.50 × 106 in 10-cm tissue culture plates, and the next day the cells were treated with vehicle or LPS (100 ng/mL) for 24 hours. Cells were harvested and cross-linked with 1% formaldehyde at room temperature. The cross-linking reaction was stopped by the addition of 0.125 M glycine, and cell nuclei were isolated and concentrated by lysing in fresh SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris, pH 8.0, dH2O), including protease inhibitors, followed by flash freezing in liquid nitrogen. Nuclei were sonicated using a Bioruptor water bath sonicator to generate an average of 500–600 bp of sheared DNA. Sonication efficiency was confirmed by subjecting lysates to 1% agarose gel electrophoresis. Sonicated lysates were then precleared with salmon-sperm/agarose beads (Upstate/Millipore, Lake Placid, NY), and 5% of the total lysate was stored as input for normalization. Half of the remaining lysate was immunoprecipitated with 5 μg indicated antibody overnight at 4°C, and the other half was immunoprecipitated with control antibody. Following an additional 2-hour immunoprecipitation with 60 μl salmon-sperm–coated agarose beads, all samples were washed with each of the following buffers: low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris, pH 8.0, 150 mM NaCl, dH2O), high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris, pH 8.0, 500 mM NaCl, dH2O), LiCl (0.25 M LiCl, 1% NP40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris, pH 8.0, dH2O), and 1× Tris-EDTA. DNA was eluted with SDS elution buffer (1% SDS, 0.1 M NaHCO3, dH2O). Following elution, cross-links were reversed overnight with 5 M NaCl at 65°C, and immunoprecipitated DNA was isolated using phenol:chloroform:isopropanol mix (Invitrogen/Life Technologies) as per the manufacturer’s instructions. Isolated DNA was quantified by real-time PCR on an ABI prism 7900 (Applied Biosystems) using the following primers and probe for RGS10: forward, 5′-ACC CAA GTG TCG TCC AAG TTA-3′, reverse, 5′-TGG AGC CTC TGC GGT TTC-3′, and probe, 5′-TGC TGG CGC GCT CAG ATC CA-3′; and for GAPDH: forward, 5′- CCA TCC GGG TTC CTA TAA ATA CG-3′, reverse, 5′-CGG CCG TCT CTG GAA CA-3′, and probe, 5′-CTG CAG CCC TCC CTG GTG CTC TCT-3′. Values generated from real-time PCRs were calculated based on standard curves generated, were run in triplicate reactions, and were analyzed using the SDS 2.0 program. RT-PCR experiments were analyzed with both actin and GAPDH internal controls, with similar results.

Mice.

C57BL/6J mice were obtained from Harlan Laboratories (Envigo, Indianapolis, IN). Male mice of 6–8 weeks old were used for partial sciatic nerve ligation and sham operation. Primary microglia cultures were prepared from cerebral cortexes of 2-day-old neonatal mice pups. Protocols were approved by the Institutional Animal Care and Use Committee at the University of Georgia and were fully compliant with the National Institutes of Health Guidelines for the Use and Care of Laboratory Animals.

Primary Microglia Isolation.

Total glial cultures were isolated from mouse pups at postnatal day 2 by dissecting total forebrain, removing meninges, dissociating with trypsin, and isolating individual cells by passage over a 40-µm filter. Total cell isolates were plated in T-75 flasks (∼three brains per flask), and then the mixed cultures were grown for 14 days in Eagle’s minimum essential medium (obtained from American Type Culture Collection) supplemented with 10% fetal bovine serum, L-glutamine (glutamax), 4.5 mg/mL glucose, and penicillin/streptomycin. Microglia were isolated by shaking the confluent mixed glial cultures at 250 rpm for 1.5 hours. The supernatant containing microglia was removed, passed through a 35-µm screen, and plated on poly-D-lysine–coated six-well plates (Suzumura et al., 1987; Levison and McCarthy, 1991).

Immunofluorescence.

Mice were deeply anesthetized with urethane (1.3–1.5 g/kg, i.p.) and transcardially perfused with heparinized phosphate-buffered saline (PBS) solution (0.1 M PBS), pH 7.35, followed by a solution of 4% formaldehyde in (0.1 M PBS), pH 7.35. The L4 and L5 spinal cord was removed and fixed for next 24 hours at 4°C in fresh 4% formaldehyde. L4–L5 spinal lumbar region was dehydrated with gradient ethanol and embedded in paraffin. The transverse sections of the spinal cord were sliced at a 10-µm thickness and mounted on microscope slides. After paraffin-embedded sections were deparaffinized using xylene and ethanol washes, and antigen retrieval treatment was performed by incubating slides in 50 mM sodium citrate (pH 8.0) at 80°C for 30 minutes. Slides were then allowed to cool to room temperature and washed in water and PBS. Tissue was blocked and permeabilized in 0.1× PBS containing 0.1% Triton, 0.5% bovine serum albumin, and 5% horse serum for 2 hours at room temperature. Primary antibodies (RGS10-1:50; Iba1-1:250) were incubated with the tissue in 0.1× PBS containing 0.5% bovine serum albumin and 5% horse serum overnight at 4°C. Following washes in PBS, secondary antibodies (anti-goat 594-fluorophore and anti-rabbit 488-flurophore, both at 1:1000) were incubated with the tissue in PBS for 2 hours at room temperature. Following final washes in PBS, slides were mounted using ProLong Gold with 4′,6′-diamidino-2-phenylindole (ThermoScientific). Slides were imaged using an Olympus U-CMAD3 camera and the Olympus-cell Sens Dimensions software. Exposure and contrast settings were held constant for all images. No manipulation of images was performed, except that the 10× image in Fig. 3A represents a composite of two images of the same section (one left, one right) to display the entire dorsal surface of the spinal cord.

Fig. 3.

Fig. 3.

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Suppression of spinal RGS10 levels in pSNL model of inflammatory neuropathic pain. (A) Tissue slices (10 µm) obtained the L4–L5 region of mouse spinal cord, and expression of RGS10 (red) and Iba1 (microglial marker, green) was visualized using immunofluorescence, as described in Materials and Methods. Yellow indicates overlap of RGS10 and Iba1 expression. Center panel: original magnification, 10×. Left inset: original magnification, 20×. Right inset: original magnification, 40×. (B) Spinal dorsal horn (L4–L5) tissue from SHAM (n = 3) or pSNL (n = 3) mice was isolated 3 days after surgery, subjected to SDS-PAGE separation (one animal per lane), and analyzed using Western blotting with RGS10 and β-actin antibody. Data shown are average RGS10 levels normalized to β-actin, ±S.E.M. Data were normalized by dividing band intensity of RGS10 immunoblot by band intensity of β-actin immunoblot, and analyzed for statistical significance using an unpaired t test. *P < 0.05.

Partial Sciatic Nerve Ligation Animal Model and Behavioral Tests.

Eight-week-old male wild-type C57BL/6J mice were subjected to either partial sciatic nerve ligation (pSNL) or SHAM surgery groups, as described (Seltzer et al., 1990). Briefly, the left sciatic nerve at the upper thigh was exposed and tightly ligated with 5-0 silk sutures to approximately one-third to one-half its original thickness, and the wound was closed with muscle sutures and skin staples. The sham surgery group was subjected to surgical exposure of the sciatic nerve, but without ligation. Behavioral tests were used to determine the development of neuropathic pain in animals with pSNL. Briefly, behavioral tests were conducted in a quiet room with the room temperature at 22°C. To test possible changes in mechanical sensitivity, animals were placed on a wire mesh, loosely restrained under a plexiglass cage (12 × 20 × 15 cm3), and allowed to acclimate for at least 30 minutes for rats and 1.5 hours for mice. A series of von Frey monofilaments (bending force from 0.07 to 2.00 g) were tested in ascending order to generate response-frequency functions for each animal. Each von Frey filament was applied five times to the mid-plantar area of each hind paw from beneath for about 1 second. The response-frequency [(number of withdrawal responses of both hind paws/10) × 100%] for each von Frey filament was determined. Withdrawal response mechanical threshold was defined as the lowest force filament that evoked a 50% or greater response-frequency. This value was later averaged across all animals in each group to yield the group response threshold.

Statistical Analysis.

All quantitative data shown are compiled from three independent experimental repeats each performed in duplicate, unless otherwise noted. Data were analyzed for statistical differences using an analysis of variance, followed by Bonferroni’s multiple comparison test or Tukey’s test between groups. *P < 0.05; **P < 0.01; and ***P < 0.001 indicate the levels of significance.

Results

Suppression of RGS10 Expression Enhances GPCR Signaling in BV-2 Microglia.

Previous studies have demonstrated the effect of RGS10 suppression on multiple inflammatory signaling pathways in microglia. To demonstrate the effect of RGS10 suppression on a classic GPCR signaling pathway, we treated BV-2 microglial cells with control or RGS10-targeted small interfering RNA, and determined the effect of CXCL12/Sdf-1–stimulated ERK mitogen-activated protein (MAP) kinase phosphorylation. Knockdown of RGS10 by 70% had no effect on basal MAP kinase phosphorylation, but significantly (P = 0.045) increased CXCL12-stimulated MAP kinase phosphorylation (Fig. 1). This suggests that endogenous mechanisms that control the level of RGS10 expression may significantly impact the strength of GPCR signaling pathways.

Fig. 1.

Fig. 1.

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Suppression of RGS10 enhances CXCL2 signaling in microglia. BV-2 cells were treated with either scrambled small interfering RNA as negative control or RGS10 small interfering RNA for 48 hours. Cells were starved overnight prior to treatment with CXCL12 (100 ng/ml) for 20 minutes. Cells were lysed, and Western blotting was performed using specific antibodies against phospho-ERK (P-ERK), total ERK, RGS10, and GAPDH. Phospho-ERK and intensity were quantified and normalized to total ERK intensity, and statistical comparison was determined using an unpaired t test of compiled data from three independent experiments, each with duplicate samples (*P = 0.45).

LPS Silencing of RGS10 Transcript and Protein Expression in BV-2 Microglia.

RGS10 protein levels are reportedly suppressed in microglia following activation by LPS or TNF-α, but early studies suggested that the effect may not follow a typical dose response (Lee et al., 2008), and the effect on transcription of Rgs10 was not defined. To further characterize RGS10 suppression, we analyzed Rgs10 transcript and RGS10 protein levels after treating mouse microglial BV-2 cells with increasing doses of LPS using quantitative RT-PCR and Western blotting, respectively. LPS treatment resulted in suppression of both Rgs10 transcript (measured at 4 hours; Fig. 2A) and RGS10 protein (measured at 24 hours; Fig. 2B) in a dose-dependent fashion, with maximal effects observed at 1 µg/mL. We simultaneously measured LPS-induced TNF-α inflammatory cytokine transcript production to confirm robust microglial activation (Fig. 2A, lower panel). The fold change and dose dependency of suppression of Rgs10 transcript expression were very similar to protein expression changes, suggesting a primarily transcriptional mechanism of silencing. A time course of LPS treatment (100 ng/mL) showed Rgs10 transcript was maximally inhibited at approximately 6 hours and remained suppressed up to 72 hours (Fig. 2C). These results demonstrate activation of microglia by LPS triggers transcriptional suppression of Rgs10 while enhancing proinflammatory cytokine expression.

Fig. 2.

Fig. 2.

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Rgs10 transcript and RGS protein suppressed in BV-2 mouse microglial cells following LPS treatment in a dose-dependent fashion. (A) BV2 cells were treated with vehicle (serum-free media), 10 ng/ml, 100 ng/ml, 1 µg/ml, and 10 µg/ml LPS for 4 hours. Cells were harvested in TRIzol, and RNA was isolated. Rgs10 (top panel) and Tnf-α (bottom panel) transcripts were quantified using SYBR Green RT-PCR reagents and normalized to the housekeeping gene actin (2−ΔΔCT). (B) BV-2 cells were treated with vehicle, 10 ng/ml, 100 ng/ml, 1 µg/ml, and 10 µg/ml LPS for 24 hours, and total cell lysates were collected in SDS sample buffer and assessed using Western blotting with RGS10 and GAPDH antibodies. (C) BV-2 cells were treated with 100 ng/mL LPS for the times indicated, and Rgs10 transcript expression was determined using SYBR Green RT-PCR reagents and normalized to the housekeeping gene actin (2−ΔΔCT). Data are compiled from three independent experimental repeats, each performed in duplicate. Data were analyzed for statistical differences using an analysis of variance, followed by Tukey’s test between groups. *P < 0.05; and ***P < 0.001 indicate the levels of significance. Similar RT-PCR results were obtained when normalized to GAPDH (data not shown).

Silencing of Endogenous RGS10 in an In Vivo Inflammatory Model of Microglial Activation.

Microglial activation and, specifically, endogenous activation of microglial TLR4 receptors are directly implicated in the development of pain sensitization in the mouse pSNL model of neuropathic pain (Raghavendra et al., 2003; Bettoni et al., 2008). This suggests that the TLR4-mediated suppression of RGS10 we have observed in vitro may occur endogenously in the spinal cord following pSNL injury. To test this prediction, we first confirmed RGS10 levels in resident microglia of the spinal dorsal horn. Immunofluorescence staining with RGS10 and Iba1 microglial marker show robust RGS10 abundance in the spinal cord and clear colocalization in Iba-1–positive cells with distinct microglial morphology (Fig. 3A). We next performed SHAM or pSNL surgeries on three mice each and assessed RGS10 protein levels in the spinal dorsal horn 3 days postsurgery, at which time robust microglial activation occurs (Weng et al., 2014). Baseline pain sensitivity was determined immediately prior to surgeries, and again 3 days after surgery immediately before sacrifice to confirm the development of pain sensitization in the pSNL animals (data not shown). The L4–L5 region of the spinal cord was isolated, and total lysates were subjected to Western blotting with RGS10 and β-actin control antibodies. Our results show an approximately 50% reduction in total RGS10 level in the spinal cord of pSNL animals, suggesting that RGS10 silencing occurs in vivo under conditions that trigger endogenous microglial activation (Fig. 3B).

The Role of DNMT, DNA Methyl Transferase, and HDAC Enzymes in LPS-Mediated RGS10 Silencing in Microglia.

We previously reported that the Rgs10 gene is epigenetically suppressed in chemoresistant ovarian cancer cells via both DNA methyl transferase (DNMT) and histone deacetylase (HDAC) activities (Hooks et al., 2010). However, the development of chemoresistance and the accompanying suppression of RGS10 reflect chronic culturing conditions that cause global gene expression changes, and it is therefore unclear whether the acute silencing of Rgs10 observed in microglia in response to LPS may involve distinct mechanisms. To test whether Rgs10 expression is regulated by DNMT enzymes in BV-2 cells following acute LPS treatment, we analyzed Rgs10 transcript and RGS10 protein expression following pharmacological inhibition of DNMT enzymes. BV-2 cells were treated with vehicle or LPS (10 ng/mL) with or without cotreatment with 5-Aza (10 µM). In experiments measuring transcript, cells were treated with LPS for 6 hours; in experiments measuring protein expression, cells were treated with LPS for 48 hours. In both experiments, either 5-Aza or vehicle control was added to cells 1 hour before LPS and then maintained in media during the LPS treatment. Under these conditions, 5-Aza treatment did not alter basal or LPS-treated Rgs10 transcript or RGS10 protein expression levels (Fig. 4), suggesting that DNMT enzymes do not play a major role in regulating Rgs10 expression in BV-2 microglia under these conditions.

Fig. 4.

Fig. 4.

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The 5-Aza does not affect LPS-induced suppression of Rgs10 transcription. (A) BV-2 cells were plated in 12-well plate and allowed to adhere overnight. Cells were treated with vehicle (serum-free media) or LPS (10 ng/ml, for 6 hours) with or without 5-Aza (AZA, 10 µM). Cells were harvested in TRIzol, and RNA was isolated. mRNA transcript levels were measured by quantitative real-time PCR. Transcripts were normalized to the housekeeping gene actin. (B) BV-2 cells were plated in 24-well plate and allowed to adhere overnight. Cells were treated with either vehicle (serum-free media) or LPS (10 ng/ml) for 48 hours or with 5-Aza (AZA, 10 µM). Cells were harvested, and protein levels were assessed by Western blot analysis with RGS10 and GAPDH control antibodies. Data are compiled from three independent experimental repeats, each performed in duplicate. Data were analyzed for statistical differences using an analysis of variance, followed by Tukey’s test between groups. **P < 0.01 indicates the levels of significance.

To test whether Rgs10 expression is regulated by HDAC enzymes in BV-2 cells, we analyzed Rgs10 transcript expression following pharmacological inhibition of HDAC enzymes using the HDAC inhibitor, TSA. Basal Rgs10 transcript expression was increased in BV-2 cells in a dose-dependent fashion following 24-hour TSA treatment, with maximal effect observed at 250 nM, suggesting that basal Rgs10 gene expression is epigenetically regulated through HDAC activity (Fig. 5A). Next, we tested whether TSA could block LPS-induced Rgs10 suppression in BV-2 cells. To that end, we treated BV-2 cells with vehicle or LPS (10 ng/mL) for 6 hours following pretreatment with TSA (100 nM) for 1 hour. TSA was also maintained in the cell media during the LPS treatment. We found that 1-hour pretreatment increased basal activity and significantly blocked LPS-stimulated suppression of Rgs10 transcript expression. To explore the ability of TSA to block the effect of higher doses of LPS, we determined that a 20-hour pretreatment with 250 nM TSA fully blocked the effect of 1 µg/mL LPS on Rgs10 expression (Fig. 5C). Finally, we also confirmed that the effect of TSA on Rgs10 expression was not limited to transcript, but resulted in restored RGS10 protein levels, as shown in Fig. 5E. These results suggest that LPS-induced Rgs10 suppression in BV-2 cells is epigenetically mediated via histone acetylation of the RGS10 promoter.

Fig. 5.

Fig. 5.

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HDAC inhibitor TSA enhances basal Rgs10 expression and blocks LPS-stimulated suppression in microglia. (A) BV-2 cells were plated in 12-well plate and allowed to adhere overnight. Cells were treated with vehicle, 100, 250, and 500 nM TSA for 24 hours. Cells were harvested in TRIzol, and RNA was isolated. Rgs10 transcript was quantified using quantitative RT-PCR relative to actin. (B) BV-2 cells were treated with vehicle or 10 ng/mL LPS for 6 hours with or without 100 nM TSA, and Rgs10 transcript was quantified relative to the actin. (C) BV-2 cells were treated with vehicle or 1 µg/mL LPS for 4 hours with or without 250 nM TSA, and Rgs10 transcript was quantified relative to the actin. (D) Primary microglia were isolated from 2- to 4-day-old mouse pups, as described in Materials and Methods. Cells were treated with vehicle or LPS (10 ng/mL) for 6 hours with or without TSA (100 nM). Rgs10 transcript expression was determined using quantitative RT-PCR relative to actin. (E) BV-2 cells were plated in six-well plate and allowed to adhere overnight. Cells were treated with either vehicle or LPS (10 ng/ml) for 48 hours with or without TSA (100 nM). Cells were harvested, and protein levels were assessed by Western blot analysis. Blot presented is a representative of three independent experiments. Densitometry values were normalized to GAPDH loading control and then to vehicle-treated condition (right panel). Data in each graph are compiled from three independent experimental repeats, each performed in duplicate. Data were analyzed for statistical differences using an analysis of variance, followed by Tukey’s test between groups. **P < 0.01; and ***P < 0.001 indicate the levels of significance.

BV-2 cells were selected as a model system to study the mechanism of silencing because they recapitulate RGS10 silencing and they are a widely used reliable model of microglial signaling (Henn et al., 2009). However, it is possible that gene regulation in an immortalized cell culture model does not accurately represent regulation in primary microglia. To address this concern, we isolated primary microglia from neonatal mice brain tissue to assess regulation of Rgs10 expression by LPS and TSA. Using immunostaining with Iba1 (microglia), glial fibrillary acidic protein (astrocytes), and NeuN (neurons), we confirmed our microglia-enriched cultures contain approximately 90–95% microglia, with a few astrocytes and no neurons detectable (data not shown). In these primary cultures, 1-hour pretreatment with 100 nM TSA fully blocked silencing of Rgs10 expression stimulated by LPS (10 ng/mL, 6 hours), consistent with the effects observed in BV-2 cells (Fig. 5D).

Role of TNF-α in LPS-Stimulated RGS10 Silencing.

We also quantified TNF-α transcript expression following LPS and/or TSA treatment using quantitative RT-PCR, and found that the trend in TNF-α transcript expression was opposite that of RGS10 expression; LPS significantly enhanced TNF-α expression, and HDAC inhibition blocked LPS-induced TNF-α production (Fig. 6A). Previous studies by Lee et al. (2008) show that TNF-α as well as LPS can trigger suppression of RGS10 expression in microglia, suggesting that LPS-mediated suppression of Rgs10 may, in part, be secondary to LPS-stimulated Tnf-α production and TNF-α–mediated suppression of RGS10. Furthermore, given that HDAC inhibition by TSA suppressed Tnf-α as well as enhanced Rgs10 expression, it is possible that the effect of TSA on Rgs10 expression could also be secondary to effects on Tnf-α expression. To clarify the role of TNF-α in LPS-mediated suppression of Rgs10, we determined the effect of inhibition of TNF-α receptor activation on LPS-stimulated Rgs10 suppression. Cells were treated with LPS as above with or without a 1-hour pretreatment with 10 µM R-7050, a TNF-α receptor inhibitor, and Rgs10 transcript was quantified, as above. TNF-α receptor inhibition had no effect on LPS-stimulated Rgs10 transcript or protein suppression, indicating that TNF-α receptor activity is not required for LPS effects on Rgs10 (Fig. 6, B and D). The same treatment with R-7050 had a significant effect on LPS-stimulated IL-1β production, confirming that the compound is active in the conditions tested (Fig. 6C).

Fig. 6.

Fig. 6.

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LPS effects on TNF-α do not mediate RGS10 silencing. (A) BV-2 cells were treated with LPS and/or TSA (as in Fig. 5C), and Tnfα transcript was quantified using SYBR Green RT-PCR reagents and normalized to actin control. (B and C) BV-2 cells were treated with vehicle or LPS (10 ng/ml, for 6 hours) with or without the TNF-α receptor antagonist, R-7050 (10 µM). Rgs10 (B) and IL-1β (C) transcript levels were measured by quantitative real-time PCR and normalized to the housekeeping gene actin. R-7050 treatment had no effect on Rgs10 suppression, but robust effects on IL-1β confirm effective inhibition. (D) BV-2 cells were treated with either vehicle or LPS (10 ng/ml) for 48 hours with or without R7050 (10 µM). Cells were harvested, and protein levels were assessed by Western blot analysis with RGS10 and GAPDH antibodies. Quantitative data in (A–C) are compiled from three independent experimental repeats, each performed in duplicate. Data were analyzed for statistical differences using an analysis of variance, followed by Tukey’s test between groups. *P < 0.05; **P < 0.01; and ***P < 0.001 indicate the levels of significance. Blot presented in (D) is representative of two independent experiments.

LPS Effects on Histone Acetylation and HDAC1 Recruitment at the RGS10 Promoter in Microglia.

The ability of TSA to enhance Rgs10 expression and block LPS-mediated silencing suggests a role for HDAC enzymes in controlling Rgs10 expression, but TSA is not completely selective for HDAC enzymes and may have off-target effects. To directly test the role of histone acetylation in the regulation of Rgs10 expression in BV-2 cells, we performed ChIP assays to quantify the amount of acetylated histones and HDAC1 enzyme bound to the proximal Rgs10-1 promoter with and without LPS treatment (100 ng/ml, 24 hours). LPS treatment resulted in a nearly fivefold increase in the association of HDAC1 at the Rgs10-1 promoter (Fig. 7A). The change in HDAC1 binding was accompanied by a marked decrease in acetylated histone H3 levels at the RGS10 promoter, whereas total histone H3 levels remained similar (Fig. 7, B and C). These results suggest the Rgs10 gene is epigenetically silenced in microglia following LPS stimulation via HDAC1 recruitment and decreased histone H3 acetylation at the Rgs10-1 promoter.

Fig. 7.

Fig. 7.

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LPS increases HDAC1 binding and decreases histone H3 acetylation at the Rgs10 promoter in BV2 cells. ChIP assays were carried out in LPS (100 ng/ml)-treated and vehicle-treated BV-2 cells. Lysates were immunoprecipitated with control, anti-HDAC1 (A), anti-acetyl histone H3 (B), or anti-histone H3 (C) antibodies. Associated DNA was isolated and analyzed via RT-PCR using primers and probes (see Materials and Methods) spanning the Rgs10 promoter and GAPDH promoters. Real-time PCR values were normalized to the total amount of DNA added (input). Input values represent 5% of the total cell lysate. Values were normalized to GAPDH promoter and represent mean ± S.E.M. of two independent experiments, and RT-PCRs were run in triplicate reactions. Data were analyzed using the SDS 2.0 program, and significance was tested using a t test. *P < 0.05.

Sphingosine 1-Phosphate Enhances RGS10 Expression and Alters Histone Acetylation at Rgs10 Promoter.

Our results suggest that activation of TLR4 receptors by LPS triggers a signaling cascade that culminates in recruitment of HDAC to Rgs10 promoters, resulting in transcriptional silencing. However, the ability of TSA to dramatically enhance basal Rgs10 expression (Fig. 5A) suggests that even in the absence of an activation signal, HDAC activity suppresses Rgs10 expression. This suggests that signaling pathways that inhibit HDAC activity in microglia may increase RGS10 expression in resting microglia. Sphingosine 1-phosphate (S1P) is an endogenous bioactive lipid mediator in neurons and glia, and S1P activates G protein–coupled receptors in microglia to regulate inflammatory signaling (Kimura et al., 2007; Nayak et al., 2010; Durafourt et al., 2011; Noda et al., 2013). S1P has also been shown to inhibit HDAC1 enzymes via receptor-independent nuclear mechanisms (Hait et al., 2009). Therefore, we tested the effect of S1P on Rgs10 expression and histone modifications at the Rgs10 promoter. BV-2 cells treated with S1P showed a fivefold increase in Rgs10 transcript expression following 48 hours of S1P treatment (Fig. 8A). To confirm that the ability of S1P to regulate Rgs10 expression reflected a change in HDAC activity, we determined histone acetylation levels at the proximal Rgs10-1 promoter following S1P treatment, and observed a significant increase in H3 histone acetylation, with no change in total H3 histone levels (Fig. 8B). Together, our data demonstrate that histone acetylation of the Rgs10 promotor is dynamically regulated in microglia by endogenous signals to increase or decrease expression of this critical regulator of inflammatory signaling.

Fig. 8.

Fig. 8.

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S1P upregulates Rgs10 expression and histone acetylation at RGS10 promoters in BV-2. (A) BV-2 cells were serum starved overnight and treated with vehicle or S1P (10 µM) for 48 hours, and then Rgs10 mRNA was isolated and assessed with RT-PCR relative to actin controls, as described. (B) Acetylated and total H3 Histone ChIP assays were carried out as described in BV-2 cells treated with vehicle or S1P (10 μM). Lysates were immunoprecipitated with control, anti-histone H3, or anti-acetyl histone H3 antibodies. Associated DNA was isolated and analyzed via RT-PCR using primers and probes spanning the Rgs10 promoter and GAPDH promoter. Real-time PCR values obtained were normalized to the total amount of DNA added (input). Input values represent 5% of the total cell lysate. Rgs10 promoter values were normalized to GAPDH promoter values and represent mean ± S.E.M. of two independent experiments, and RT-PCRs were run in triplicate reactions. Data were analyzed using the SDS 2.0 program, and significance was tested using a t test. *P < 0.05; **P < 0.01; and ***P < 0.001.

Discussion

Microglia, CNS-resident macrophages, have been implicated in the initiation and progression of multiple neurologic disorders, including neurodegenerative disease and neuropathic pain (Schwartz et al., 2013). Microglia are activated by inflammatory agents such as LPS, resulting in activation of NF-κB transcriptional pathways and production of proinflammatory cytokines. RGS10 has emerged as an important regulator of proinflammatory cytokine production in microglia. Microglia from RGS10 knockout mice show enhanced NF-κB reporter activity and higher expression of inflammatory cytokines, including TNF-α and IL-1β following LPS stimulation, and restoration of RGS10 normalizes responses to LPS (Lee et al., 2008, 2011). Furthermore, RGS10 loss enhances LPS-induced dopaminergic neuronal death (Lee et al., 2008), whereas restoration of RGS10 rescues these neurons (Lee et al., 2011). Importantly, small interfering RNA-mediated reduction of microglial RGS10 expression by 50–75% was sufficient to significantly enhance cytokine levels (Lee et al., 2008). Our data also show that suppression of RGS10 by 70% significantly enhances chemokine signaling, demonstrating that even partial loss of RGS10 has an important impact on inflammatory signaling in microglia. Although RGS10 is normally present in microglia at high levels, expression is silenced by activation of TLR4 or TNF-α receptors. Following activation by the TLR4 activator LPS, microglial Rgs10 transcript expression levels were reduced by up to 75%, comparable to the fold suppression sufficient to amplify NF-κB signaling and inflammatory cytokine production (Lee et al., 2008). This suggests that silencing of RGS10 is sufficient to amplify microglial inflammatory signaling in a feed-forward mechanism that may contribute to the dysregulation of inflammatory signaling in chronic neuroinflammation and disease. Understanding the molecular mechanisms of RGS10 suppression in microglia may reveal novel targets for therapeutic intervention in diseases with underlying chronic neuroinflammation. With this in mind, we sought to define the molecular mechanism of RGS10 suppression following TLR4 activation in vitro.

In this study, we find that activation of microglia by LPS triggers a rapid and robust transcriptional silencing of Rgs10. Lee et al. (2008) previously reported that a low dose of LPS (10 ng/ml) decreased RGS10 protein in BV-2 cells, whereas a higher dose of LPS (1 μg/ml) did not change RGS10 protein level. In this study, we report that Rgs10 transcript and protein levels were suppressed in BV-2 cells in a typical dose-dependent fashion. LPS-induced suppression of Rgs10 transcript reflected and preceded the reduction in protein levels, which suggests that the reduction in RGS10 protein level observed following LPS treatment is mainly due to inhibition of transcription.

Previously, we reported an epigenetic regulation of Rgs10 expression in chemoresistant ovarian cancer cells mediated by the combined activities of HDAC and DNMT enzymes. In this work, we investigated whether Rgs10 expression in microglia is regulated in a similar mechanism after acute LPS treatment. We found that the HDAC inhibitor TSA significantly enhances basal Rgs10 transcript expression and blocks LPS-induced Rgs10 suppression in microglia, whereas the DNMT inhibitor had no effect under the conditions tested. We further show that LPS increases HDAC1 association and decreases histone H3 acetylation at the Rgs10 promoter, observations that strengthen the notion that Rgs10 genes are epigenetically silenced via histone deacetylation in LPS-activated microglia. We also show that HDAC inhibition caused a decrease in LPS-mediated proinflammatory cytokine TNF-α (and IL-1β; data not shown) production in microglia. Consistent with our observations, HDAC inhibitors have been reported to possess anti-inflammatory effects by lowering proinflammatory cytokine production in microglia (Suh et al., 2010). The HDAC inhibitor suberoylanilide hydroxamic acid has been shown to reduce the production of proinflammatory cytokine production in activated microglia (Faraco et al., 2009), whereas TSA reduces proinflammatory cytokine production in microglia and reduces brain injury in a model of LPS-sensitized neonatal hypoxic-ischemia (Fleiss et al., 2012).

The mouse Rgs10 gene contains two transcriptional start sites, yielding two transcript isoforms with distinct first exons, and four common exons (Haller et al., 2002; Lee and Tansey, 2015). Mouse transcripts mRgs10-1 and mRgs10-2 correspond to human transcripts with conserved exon structure and encoding highly conserved human RGS10 protein isoforms RGS10a (181 amino acids) and RGS10b (167 amino acids). A third human transcript (173 amino acids) has been proposed, resulting from an alternate transcriptional start site upstream of the first shared exon (Hunt et al., 1996), but the relevance of this transcript in mouse cells is unclear (Haller et al., 2002). We have shown that Rgs10-1 is the predominant form expressed in human cancer cells, Rgs10-1 is selectively silenced in chemoresistant cells, and HDAC1 is recruited to the unique Rgs10-1 promoter in these cells (Hooks et al., 2010; Ali et al., 2013; Cacan et al., 2014). Our studies and others have indicated that the long isoform (Rgs10-1→RGS10a) is also the predominantly expressed form in microglia and other immune cells (Lee and Tansey, 2015). For these reasons, our studies in microglia focused on regulation of the mouse Rgs10-1 transcript, and ChIP experiments were targeted to the mouse Rgs10-1 proximal promoter.

Microglial activation in the spinal dorsal horn enhances nociception and is a causative factor in central sensitization that contributes to neuropathic pain (Raghavendra et al., 2003; Zhuo et al., 2011). The pSNL model of injury-induced neuropathic pain results in robust microglial activation and spinal sensitization. During nerve injury–induced sensitization, endogenous mediators activate microglial TLR4 receptors, which trigger production of proinflammatory cytokines (Tanga et al., 2005; Bettoni et al., 2008; Wu et al., 2010). Blocking either microglial activation or endogenous TLR4 receptors has been shown to attenuate neuropathic pain (Raghavendra et al., 2003; Saito et al., 2010; Sorge et al., 2011; Yoon et al., 2012). Furthermore, enhanced HDAC activity is observed in the spinal cord following nerve injury (Lv et al., 2011; Lu et al., 2013), and HDAC inhibitors reduce inflammatory cytokine release and neuroinflammation (Blanchard and Chipoy, 2005; Lv et al., 2011). Thus, the pSNL model of neuropathic pain is suitable for the in vivo study of molecular signaling pathways in microglia activated by TLR4.

We have demonstrated that RGS10 is abundant in the spinal cord, with confirmed localization in microglia. Nonmicroglial cell types also stain positively for RGS10, consistent with reports of predominantly microglial and lesser neuronal expression in brain (Waugh et al., 2005). Following pSNL injury, we observe downregulation of RGS10 protein expression by about 50% in spinal dorsal horn as measured by Western blotting in total L4–L5 spinal dorsal horn tissue. Our observation that RGS10 is suppressed in the spinal cord in the pSNL model provides supporting evidence that the suppression observed in cultured BV-2 and primary microglia also occurs in vivo. However, our approach most likely underestimates the cellular suppression of RGS10; given that RGS10 is predominantly expressed in microglia, the expansion of microglia during inflammation following pSNL most likely masks the degree of RGS10 suppression in individual microglial cells. Furthermore, the expression of RGS10 in neurons, although less than microglia, also limits quantitative interpretation of the degree of suppression in microglia. Thus, future studies should evaluate RGS10 levels in individual microglia isolated from in vivo neuroinflammation models. It will also be important for future studies to determine whether HDAC inhibitors, which are known to inhibit inflammatory signaling, also restore Rgs10 expression in the spinal cord, and whether this effect contributes to their ability to impact inflammation. Lee and colleagues have shown that reintroduction of RGS10 protein expression in microglia from knockout animals normalizes inflammatory signaling and dopaminergic neuron cell death in vivo (Lee et al., 2011). Therefore, strategies that restore RGS10 levels in microglia may blunt neuroinflammation and associated diseases.

In recent years, RGS proteins have emerged as promising novel drug targets in diverse disease states, but progress has lagged due to the lack of traditional small-molecule ligand or substrate interaction sites. Strategies to target regulation of RGS protein expression have focused on post-translational mechanisms that control protein stability (Sjögren et al., 2012; Raveh et al., 2014). In contrast, our data suggest predominantly transcriptional regulation of Rgs10 expression. Our results demonstrate that Rgs10 silencing is completely reversible with small-molecule HDAC inhibitors and provide proof of concept for targeting RGS10 suppression in neuroinflammatory disease models. Multiple HDAC inhibitors are being assessed in clinical trials, but specificity, pharmacokinetic features, and toxicity remain significant barriers to the use of broad-spectrum HDAC inhibitors in CNS diseases. Thus, more selective pharmacological approaches are needed to target Rgs10 silencing.

A potential alternative to targeting HDAC enzymes directly to regulate Rgs10 expression is to target more selective pathways that may indirectly modulate HDAC activity. S1P activates a family of GPCRs, including several expressed in microglial cells, and S1P is a key regulator of microglial activity (Tham et al., 2003; Nayak et al., 2010; Noda et al., 2013; Blaho and Hla, 2014). Our results in this work show that S1P may enhance Rgs10 expression through regulation of HDAC. Notably, the time course of S1P effects on RGS10 expression (48 hours) was much longer than that of LPS effects on Rgs10 expression (6 hours), and it remains to be determined whether S1P receptor activation is sufficient to reverse LPS-mediated silencing of RGS10. However, the involvement of GPCRs in regulating Rgs10 expression offers another opportunity for therapeutic intervention, as GPCRs are relatively easy to target with reasonable selectivity compared with HDACs. Strikingly, the S1P receptor functional antagonist FTY-720 (Fingolimod, Gilenya) has been approved for use in relapsing-remitting multiple sclerosis (MS), and a recent study demonstrated that RGS10 knockout animals exhibit blunted symptoms in the experimental autoimmune encephalomyelitis model of MS (Lee et al., 2016). Given the role of microglial activation in MS pathology, it is tempting to postulate a connection between our observations connecting S1P receptors and RGS10 in microglia with these findings in models of MS. However, the predicted mechanisms of action of both FTY-720 and RGS10 in MS models are based on their effects on peripheral immune cells, not microglia, so the relevance of the observed regulation in microglia to MS is unclear at this time.

Summary

In this study, we demonstrate dose-dependent suppression of Rgs10 in response to LPS activation in microglia, demonstrate that the HDAC inhibitor TSA blocks the ability of LPS to suppress Rgs10 expression, and demonstrate HDAC1 recruitment and H3 histone deacetylation at the Rgs10-1 proximal promoter following LPS stimulation. These observations suggest that LPS-stimulated silencing of Rgs10 expression is rapid, robust, and reversible in BV-2 microglial cells, and this model system reflects the mechanism observed in primary microglia. We further show that activation of microglial S1P receptors increases Rgs10 expression, demonstrating that Rgs10 expression may be precisely dialed up or down in microglia in response to multiple cues. Finally, we demonstrate that RGS10 is suppressed in an in vivo mouse model of injury-induced inflammation. Together, our results suggest that the ability of RGS10 to regulate microglial inflammatory pathways can be modulated by various pathways through HDAC regulation of Rgs10 expression with significant implications on neuroinflammatory disease states.

Abbreviations

5-Aza

5-Aza-2′-deoxycytidine

ChIP

chromatin immunoprecipitation

CNS

central nervous system

DNMT

DNA methyl transferase

ERK

extracellular signal-regulated kinase

GAPDH

glyceraldehyde-3-phosphate dehydrogenase

GPCR

G protein–coupled receptor

HDAC

histone deacetylase

IL

interleukin

LPS

lipopolysaccharide

MAP

mitogen-activated protein

MS

multiple sclerosis

NF-κB

nuclear factor-κB

PBS

phosphate-buffered saline

PCR

polymerase chain reaction

pSNL

partial sciatic nerve ligation

RGS

regulator of G protein signaling

RT-PCR

reverse-transcription PCR

S1P

sphingosine 1-phosphate

TLR

Toll-like receptor

TNF

tumor necrosis factor

TSA

trichostatin A

Authorship Contributions

Participated in research design: Alqinyah, Maganti, Weng, Greer, Hooks.

Conducted experiments: Alqinyah, Maganti, Ali, Yadav, Cacan, Gao.

Performed data analysis: Alqinyah, Maganti, Greer, Hooks.

Wrote or contributed to the writing of the manuscript: Alqinyah, Hooks.

Footnotes

This work was supported by grants from the National Multiple Sclerosis Society (to S.B.H.) and National Institutes of Health National Institutes of Neurologic Disorders and Stroke [Grant RO1NS064289] (to H.-R.W.).

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