Lipopolysaccharide induces mouse translocator protein (18 kDa) expression via the AP-1 complex in the microglial cell line, BV-2

Shuji Shimoyama; Tomonori Furukawa; Yoshiki Ogata; Yoshikazu Nikaido; Kohei Koga; Yui Sakamoto; Shinya Ueno; Kazuhiko Nakamura

doi:10.1371/journal.pone.0222861

. 2019 Sep 19;14(9):e0222861. doi: 10.1371/journal.pone.0222861

Lipopolysaccharide induces mouse translocator protein (18 kDa) expression via the AP-1 complex in the microglial cell line, BV-2

Shuji Shimoyama ^1,², Tomonori Furukawa ², Yoshiki Ogata ², Yoshikazu Nikaido ^2,^¤a, Kohei Koga ^2,^¤b, Yui Sakamoto ³, Shinya Ueno ^1,², Kazuhiko Nakamura ^1,^3,^*

Editor: Leo TO Lee⁴

PMCID: PMC6752844 PMID: 31536603

Abstract

It has been reported that neuroinflammation occurs in the central nervous system (CNS) in patients with neuropathic pain, Alzheimer’s disease and autism spectrum disorder. The 18-kDa translocator protein TSPO is used as an imaging target in positron emission tomography to detect neuroinflammation, and its expression is correlated with microglial activation. However, the mechanism underlying the transcriptional regulation of Tspo induced by inflammation is not clear. Here, we revealed that lipopolysaccharide (LPS) -induced Tspo expression was activated by the AP-1 complex in a mouse microglial cell line, BV-2. Knockdown of c-Fos and c-Jun, the components of AP-1, reduced LPS-induced Tspo expression. Furthermore, the enrichment of Sp1 in the proximal promoter region of Tspo was increased in the presence of LPS. In addition, the binding of histone deacetylase 1 (HDAC1) to the enhancer region, which contains the AP-1 site, was decreased by LPS treatment, but there were no significant differences in HDAC1 binding to the proximal promoter region with or without LPS. These results indicated that HDAC1 is involved not in the proximal promoter region but in the enhancer region. Our study revealed that inflammatory signals induce the recruitment of AP-1 to the enhancer region and Sp1 to the proximal promoter region of the Tspo gene and that Sp1 may regulate the basal expression of Tspo.

Introduction

Microglia are resident immune cells of the central nervous system (CNS) and play a pivotal role in maintaining the neuronal environment. Resting (ramified) microglia monitor neurons by “palpating” synapses [1]. Individual microglia have their own territory in the CNS and are distributed throughout the brain so that they do not overlap. In the case of homeostatic disruption in the CNS, microglia transform into an amoeboid (activated) morphology and migrate toward the damaged regions to release neurotoxic or neuroprotective molecules. M1 phenotype microglia produce various types of proinflammatory cytokines, such as IL-1β and TNF-α [2,3], and M2 phenotype microglia also produce neuroprotective and neurotrophic factors, such as BDNF and IGF-1 according to the conditions [4,5]. In addition, if possible, damaged cells are repaired; excessively damaged cells are phagocytosed by activated microglia [6].

Recent studies showed that neuronal inflammation occurs in patients with neuropathic pain, Alzheimer’s disease and autism spectrum disorder (ASD) [7–9]. To visualize inflammation in the CNS, positron emission tomography with a radiotracer, such as [¹¹C] (R) PK-11195 or [¹¹C] PBR28, is widely used in animal models or human subjects [10,11]. These radiotracers are specific ligands for the 18-kDa translocator protein TSPO, alternatively called peripheral-type benzodiazepine receptor (PBR) [12]. These tracers are helpful for evaluating the level of neuronal inflammation in vivo, thereby determining the treatment plan for CNS disease.

TSPO is expressed in various organs, including glial cells in the CNS, and contributes to the regulation of steroid hormone production by transporting cholesterol, the precursor of pregnenolone, from the cytosol to the mitochondrial intermembrane space [13,14]. Additionally, TSPO is distinct from the central benzodiazepine receptor [γ-aminobutyric acid type A (GABA_A) receptor] and plays different roles, for example, in the regulation of cell growth [15] and ATP production [16].

The expression of TSPO is correlated with M1 phenotype microglial activation; however, the mechanism underlying the upregulation of TSPO expression during neuroinflammation has not been elucidated. Previous studies have suggested two possible mechanisms: an increase in the number of microglia in the CNS or of monocytes and macrophages that infiltrate the CNS in response to inflammation [17] and transcriptional activation of Tspo in microglia. Expression profiling data (GEO NCBI, #GSE37611) revealed that LPS, a classical inducer of inflammation, increased Tspo expression in mouse microglial cell lines [18]; therefore, upregulation of TSPO was the result of transcriptional activation. Accordingly, we focused on transactivation of Tspo gene expression in this study, and LPS was used as an inducer of neuroinflammation to elucidate the transcriptional regulation of TSPO.

In the present study, we showed that recruitment of both c-Fos and c-Jun, which are components of the AP-1 complex, to the enhancer region of the Tspo gene was increased upon LPS treatment in the microglial cell line BV-2. This evidence was supported by the observation that Tspo expression was reduced by knockdown of c-Fos and c-Jun. In addition, we found that the concentration of histone deacetylase 1 (HDAC1) at the AP-1 binding site in the Tspo enhancer region was decreased by LPS treatment. These data suggest that LPS-induced Tspo gene expression in BV-2 was upregulated by AP-1 activation and that the release of HDAC1 from the AP-1 site was increased by LPS treatment.

Materials and methods

Cell culture and chemical treatment

The mouse microglial cell line BV-2 (RRID: CVCL_0182) was purchased from Banca Biologica e Cell Factory (San Martino, Italy). Cells were maintained in DMEM containing 10% FBS and antibiotics and grown at 37°C in an atmosphere containing 5% CO₂. Cells were treated with LPS from E. coli (O111, Wako, Osaka, Japan) at 1, 10, 100 or 500 ng/ml for various duration. Recombinant murine IFN-γ and IL-1β were purchased from PeproTech (Rocky Hill, NJ, USA).

Animals and primary microglial cell culture

Male C57BL/6 mice approximately 12 weeks old were used in this study. Mice were housed as described previously [19]. This study was carried out in accordance with the recommendations the guidelines for animal research issued by the Physiological Society of Japan. The experimental procedures were approved by the Animal Research Committee of Hirosaki University (Approval Number: M12007), and all efforts were made to minimize the number of animals used and their suffering. Isolation of microglia from adult mice and primary microglial cell culture were performed as described before [20]. Primary microglial cells were seeded in 35-mm dishes at a density of 1.0×10⁴ cells/dish. Twenty-four hours after seeding, cells were stimulated with 100 ng/ml LPS for 8 hours.

RNA isolation, cDNA synthesis and quantitative real-time reverse transcription PCR (qRT-PCR)

BV-2 cells were seeded in 35-mm dishes at a density of 1.0×10⁵ cells/dish. After chemical treatment, cells were harvested by centrifugation. Total RNA was extracted from cells using ISOGEN II (NIPPON GENE, Tokyo, Japan) according to the manufacturer’s instructions. cDNA synthesis and qRT-PCR were performed as described previously [19]. The primer sets used in this study are shown in S1 Table. The expression level of each transcript was measured using the standard curve method, Gapdh as an internal control.

Preparation of whole-cell extracts and cell fractionation, Western blotting and densitometric analysis

Whole-cell extracts were prepared as described previously [21]. BV-2 cells were seeded at a density of 1.0×10⁵ cells/35-mm dish (for whole-cell extracts) or at a density of 1.0×10⁶ cells/100-mm dish (for cell fractionation). The mitochondrial, membrane, cytosolic and nuclear fractions were prepared according to the following method. After washing the cells with cold PBS (-) containing 1 mM PMSF, cells were harvested via centrifugation. Cells were suspended in hypotonic buffer (10 mM HEPES [pH7.9], 10 mM KCl, 1 mM MgCl₂, 0.5 mM DTT and Complete^™ Protease Inhibitor Cocktail: Roche, Mannheim, Germany) and incubated on ice for 15 min. Cells were homogenized using a Dounce Tissue Grinder with a tight pestle (WHEATON, Millville, NJ, USA). Nuclei and unhomogenized cells were precipitated via centrifugation at 3,300×g for 5 min at 4°C, and then, the supernatants were then centrifuged at 8,000×g for 15 min at 4°C to obtain the mitochondrial fraction. In addition, the supernatants were centrifuged at 100,000×g for 60 min at 4°C, and the pellets were recovered as the membrane fraction. The resulting supernatants were used as the cytosolic fraction. The nuclear, mitochondrial and membrane fractions were washed 3 times with isotonic buffer (20 mM HEPES [pH7.9], 150 mM KCl, 3 mM MgCl₂, 0.5 mM DTT and Complete^™ Protease Inhibitor Cocktail) and were then suspended in isotonic buffer containing 1% SDS. The protein concentrations were determined by Bradford protein assay (Bio-Rad, Hercules, CA, USA). Western blotting and densitometric analysis were performed as described previously [19]. The antibodies used in this study are shown in S2 Table.

Immunocytochemistry

BV-2 cells were seeded on cover glasses at a density of 2.0×10⁴ cells/well (Nunc^™ Cell-Culture Treated Multidishes, Thermo Fisher Scientific, Waltham, MA, USA). After chemical treatment, cells were fixed with 4% paraformaldehyde/PBS (-) for 15 min at room temperature and were then permeabilized with 0.1% Triton X-100/PBS (-) for 15 min at room temperature. After blocking with 1% BSA/PBS (-) for 60 min at room temperature, cells were incubated with the primary antibody at 4°C overnight. Cells were incubated with the secondary antibody for 2 hours at room temperature, and nuclei were then stained with DAPI for 15 min at room temperature. Cells on the cover glasses were mounted with VECTASHIELD® Mounting Medium (VECTOR LABORATORIES, Burlingame, CA, USA). All steps were carried out in the dark and cells were washed 3 times with PBS (-) or 0.1% Tween 20/PBS (-) after incubation. The antibodies and dilution ratios used in this study are shown in S2 Table. Images were acquired using a confocal laser scanning microscope and analyzed with NIS-Elements AR Analysis software (Nikon, Tokyo, Japan). To label mitochondria, cells were stained with MitoTracker Orange® CMTMRos (Thermo Fisher Scientific) in accordance with the manufacturer’s instructions.

Reporter gene assay

The regulatory region of the mouse Tspo gene was amplified using forward primers and a common reverse primer with the addition of the Spe I (forward) or Bam HI (reverse) sites as described in S1 Table. The products were ligated to the pMCS-Gaussia Luc Vector (Thermo Fisher Scientific), and the sequences were confirmed via sequencing (S1 Table) using a BigDye® Terminator v3.1 Cycle Sequencing Kit (Thermo Fisher Scientific); the reaction products were then purified using a Gel Filtration Cartridge (Edge Bio, San Jose, CA, USA). The sample sequences were analyzed using an ABI PRISM® 3100 Genetic Analyzer (Applied Biosystems, Waltham, MA, USA).

Various constructs were transfected into BV-2 cells using Attractene (QIAGEN, Hilden, Germany) in accordance with the manufacturer’s instructions. A reporter gene assay was performed using a Gaussia Luciferase Glow Assay Kit (Thermo Fischer Scientific) according to the manufacturer’s instructions.

Chromatin immunoprecipitation (ChIP) assay and real-time PCR

A ChIP assay was performed as described previously [21]. The antibodies used in this study are shown in S2 Table. Purified DNA was subjected to real-time PCR with primer pairs specific for the Tspo enhancer region containing the AP-1 binding site or the proximal promoter region containing the Sp1 binding site, as described in S1 Table. All quantified values were normalized to that of the corresponding input for each sample using the standard curve method.

Silencing by RNAi

The c-Fos and c-Jun knockdown experiments were performed using siRNA purchased from Thermo Fisher Scientific (Silencer® Select siRNAs for Fos/mouse: #s66198, #s66199; Jun/mouse: #s68564; and for Negative Control No.1 [siCont]). siRNAs (10 nM) were transfected into BV-2 cells using Lipofectamine RNAiMAX (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions.

Statistical analyses

All numerical data are presented as the means ± S. Ds, with n = 3 (qRT-PCR, Western blotting and ChIP assay) or n = 4 (reporter gene assay). Comparisons were assessed using Student’s t-test or ANOVA. A value of p<0.05 was considered statistically significant. Statistical analyses were performed using StatPlus:mac Pro software (AnalystSoft, Walnut, CA, USA).

Results

LPS treatment induced Tspo expression in BV-2 cells

To determine the optimum assay conditions, BV-2 cells were treated with LPS for various durations and at various concentrations. The expression of Tspo mRNA was induced by a high concentration of LPS (100 or 500 ng/ml), peaked at 8 hours and gradually returned to normal. By contrast, the expression of Tspo mRNA did not increase at low concentrations of LPS (1 or 10 ng/ml). Under the same conditions, the expression of pro-inflammatory mediators such as Tnf-α, Il-1β and Il-6 was also upregulated by LPS stimulation (Fig 1A). Furthermore, the transcriptional activation of Tspo was induced by LPS stimulation in primary microglial cells from adult mice (Fig 1B).

Fig 1 — **(A)** BV-2 cells were treated for the indicated time and with the indicated dose of LPS, and RNA was then extracted and subjected to qRT-PCR for *Tspo*, *Tnf-α*, *IL-1β*, *IL-6* and *Gapdh*. The expression level of each mRNA was measured and normalized to that of *Gapdh* mRNA. The asterisks indicate statistically significant differences compared to 1 ng/ml LPS (n = 3, *p<0.05, **p<0.01). (B) Primary microglial cells from adult mice were stimulated with 100 ng/ml LPS for 8 hours and were then subjected to qRT-PCR. **(C)** BV-2 cells were treated as described above. The protein expression level of TSPO was measured using Western blot analysis. **(D)** Densitometric analysis of TSPO expression using ImageJ. The expression value was measured and normalized to that of GAPDH as the internal control. The asterisks indicate statistically significant differences (n = 3, *p<0.05, **p<0.01). **(E)** Microscopy images showing TSPO expression. BV-2 cells were stimulated with 1, 10, 100 or 500 ng/ml LPS for 8 hours and immunocytochemistry was then carried out. Images of nuclear (DAPI, blue) and TSPO (green) staining were acquired at 40× magnification. Scale bar: 100 μm.

Subsequent to analysis of the Tspo mRNA expression levels, Western blotting and densitometric analysis were also performed to the measure protein levels (Fig 1C and 1D). The protein expression of TSPO was correlated with its mRNA expression. In addition, the results of Western blotting and immunocytochemistry revealed that TSPO expression was increased at high concentrations of LPS (Fig 1E and S1 Fig). These results indicated that LPS was a potent inducer of Tspo in mouse microglial cells, as reported previously [22]. In addition, we examined whether proinflammatory mediators such as IFN-γ or IL-1β induce the expression of Tspo. BV-2 cells were stimulated with 100 ng/ml IFN-γ and/or IL-1β for 8 hours. The expression of Tspo mRNA was also induced by administration of IFN-γ, IL-1β, or both (S2 Fig).

LPS treatment increased TSPO expression in the mitochondrial and membrane fractions

The TSPO protein is mainly localized in mitochondria in the adrenal gland homogenate [23]. To investigate whether TSPO in microglial cells is localized in mitochondria and whether the intracellular localization of TSPO is affected by LPS treatment, subcellular fractionation analysis was performed. BV-2 cells were collected, and cell fractions were prepared for Western blotting after stimulation with 100 ng/ml LPS for 8 hours. First, the purity of the fractions was confirmed by fraction markers, such as COXIV, the alpha1 subunit of the Na⁺/K⁺ ATPase, GAPDH and Lamin-B1, and there was no contamination or variation in expression between the control and LPS-treated groups in the same fractions. Similar to the fractionation analysis results, TSPO expression was significantly increased in the mitochondrial fraction by LPS treatment (Fig 2A and 2B, p = 0.03). Interestingly, TSPO was also localized in the membrane fraction in the control group and was 1.94-fold higher in the LPS-treated group than in the control group (p = 0.005). To verify whether TSPO is localized in the plasma membrane, TSPO, the Na⁺/K⁺ ATPase and mitochondria were stained by immunocytochemistry and analyzed by laser confocal microscopy. As previously reported, almost all TSPO protein was localized in the mitochondrial outer membrane. However, some fluorescence signals corresponding to TSPO protein were detected in non-mitochondrial regions in both the absence and presence of LPS (Fig 2C, upper, arrowhead). In addition, some TSPO signals were colocalized with or very close to Na⁺/K⁺ ATPase signals (Fig 2C, lower, arrowhead). These results indicated that a small amount of TSPO was localized in the plasma membrane.

Fig 2 — **(A)** Western blot analysis of the TSPO protein and fraction markers with or without 100 ng/ml LPS treatment for 8 hours. COXIV, ATPase, GAPDH and Lamin-B1 were used as the internal markers of mitochondria, membranes, the cytosol and nuclei, respectively. **(B)** The relative expression of the TSPO protein in each fraction was measured and normalized using each fraction marker. The asterisks indicate statistically significant differences from the control in the same fraction (n = 3, p<0.05). **(C)** Images showing TSPO localization in BV-2 cells. BV-2 cells were stimulated with 100 ng/ml LPS for 8 hours, and immunocytochemistry was then carried out. Images of mitochondrial (red, upper), Na⁺/K⁺ ATPase (red, lower), TSPO (green) and nuclear (DAPI, blue) staining were acquired at 100× magnification with a confocal microscope. Arrowhead: TSPO protein signals in non-mitochondrial regions. Scale bar: 10 μm.

The transcription factor AP-1 is the candidate transcriptional activator of Tspo after LPS treatment

Prior to the construction of vectors for the reporter gene assay, the Tspo regulatory sequence was analyzed using ChIP-seq data in the UCSC Genome Browser (http://genome.ucsc.edu/ENCODE/) and prediction tools for transcription binding sites, such as STAMP (http://www.benoslab.pitt.edu/stamp/), JASPAR (http://jaspardev.genereg.net/) and DBCLS Galaxy (http://galaxy.dbcls.jp/). The presumed transcription factors and their binding sites were identified via a comprehensive assessment using several tools and are shown in Fig 3A. To determine which transcription factor and cis element regulates Tspo expression during LPS treatment, serial deletion constructs were designed for the Tspo reporter gene assay according to the schematic in Fig 3A. Forty-eight hours after transfection of each vector, BV-2 cells were treated with vehicle or 100 ng/ml LPS for an additional 8 hours, and a reporter gene assay was then performed. As shown in Fig 3B, deletion of the AP-1 site (-517) significantly reduced luciferase activity; however, basal expression was not altered. These results indicated that AP-1 was the candidate transcriptional activator of Tspo during LPS stimulation.

Fig 3 — **(A)** Schematic of the predicted and established transcription factor binding sites upstream of the mouse *Tspo* gene. The blue letters indicate established transcription factors that regulate the mouse *Tspo* genes based on a previous study. TSS: Transcriptional start site, UTR: Untranslated region **(B)** Forty-eight hours after transfection of the indicated vectors, BV-2 cells were treated with 100 ng/ml LPS for an additional 8 hours. Luciferase activity was measured as described in the Materials and methods section. The asterisks indicate statistically significant differences (n = 4, **p<0.01).

AP-1, Sp1 and HDAC1 regulated Tspo expression

Based on the results of the reporter gene assay and previous studies [24,25], we examined via a ChIP assay whether the enrichment of AP-1 at the binding site in the Tspo regulatory region was promoted by LPS treatment. A previous study showed that TSPO expression is regulated by the binding of Sp1, Sp3 and/or Sp4 to the proximal promoter region in human breast cancer cell lines, and a similar sequence was observed in the mouse Tspo gene [26]. In addition, there is the possibility that HDAC1 acts as an inhibitory factor of Tspo expression by binding to Sp1 in a transcriptionally repressed state [21,27]. Furthermore, since few studies have examined the interaction between HDAC1 and Sp3 or Sp4, we excluded Sp3 and Sp4 from this study and focused on the interaction between Sp1 and HDAC1 in the proximal promoter region of the Tspo gene. Based on these observations, the primer sets used for the ChIP assay were designed as shown in the schematic in Fig 4A. The ChIP experiment revealed that the enrichment of the AP-1 components c-Fos and c-Jun at the AP-1 binding site was increased by approximately 3.83-and 2.88-fold, respectively, in the presence of LPS (Fig 4B). In the proximal promoter region, the enrichment of RNA polymerase II (RNAP II) and Sp1 was increased at the time of transcriptional activation compared to that in control cells (Fig 4C). These results clearly showed that the binding of AP-1 and Sp1 the upstream region of the Tspo gene was increased by LPS treatment. Interestingly, after LPS treatment, enrichment of HDAC1 was decreased in the enhancer region, which contains the AP-1 site, but enrichment of HDAC1 was not altered in the proximal promoter region.

Fig 4 — **(A)** Schematic of the upstream region of the mouse *Tspo* gene and the positions of the primers used for the ChIP assay. **(B)** Enrichment of c-Fos, c-Jun and HDAC1 at the AP-1 binding site. BV-2 cells were treated with 100 ng/ml LPS for 2 hours. Then, ChIP analyses were performed as described in the Materials and methods section. **(C)** Enrichment of RNAP II, Sp1 and HDAC1 in the proximal promoter region. The Y-axis indicates the quantity of PCR products normalized to that of the input for each sample. The asterisks indicate statistically significant differences from the control (n = 3, p<0.01).

Silencing of c-Fos and c-Jun decreased Tspo expression

To confirm the involvement of AP-1 in Tspo expression, silencing of c-Fos and c-Jun was performed via RNAi. First, Western blot analyses were performed to evaluate the knockdown efficiency of c-Fos and c-Jun. Forty-eight hours after transfection of c-Fos siRNA, c-Jun siRNA or siCont, cells were harvested and subjected to Western blot analysis of c-Fos and c-Jun. As shown in Fig 5A and 5B, the expression of c-Fos and c-Jun was clearly decreased by siRNA compared to siCont. Under these conditions, cells were stimulated with 100 ng/ml LPS for an additional 8 hours and analyzed to measure the expression level of Tspo mRNA. The results of qRT-PCR analysis revealed that knockdown of c-Fos or c-Jun repressed the LPS-promoted expression of Tspo (Fig 5C). Similar results were obtained at the protein level for TSPO (Fig 5D and 5E). Therefore, c-Fos and c-Jun are essential transcriptional activators of LPS-induced Tspo regulation in mice.

Fig 5 — **(A)** Efficiency of c-Fos and c-Jun knockdown by siRNAs. Forty-eight hours after transfection of siRNAs against c-Fos or c-Jun or the control siRNA, BV-2 cells were harvested and used to measure the expression of c-Fos and c-Jun by Western blotting. **(B)** Densitometric analysis of the bands corresponding to the c-Fos (left) and c-Jun (right) proteins. The asterisks indicate statistically significant differences compared with siCont (n = 3, p<0.05). **(C)** Relative expression of *Tspo* mRNA with or without 100 ng/ml LPS treatment and siRNA against c-Fos or c-Jun. Forty-eight hours after transfection of c-Fos siRNA, c-Jun siRNA or siCont, BV-2 cells were treated with 100 ng/ml LPS for an additional 8 hours, and total RNA was then isolated and subjected to qRT-PCR. The asterisks indicate statistically significant differences compared with the control (n = 3, *p<0.05, **p<0.01). **(D)** BV-2 cells were treated as described above. The protein expression level of TSPO was measured via Western blot analysis. **(E)** Microscopy images showing TSPO expression. Images of nuclear (DAPI, blue) and TSPO (green) staining were acquired at 100× magnification with a confocal microscope. Scale bar: 10 μm.

Discussion

In this study, LPS-induced gene expression of Tspo in a microglial cell line was examined. Our experiments clearly demonstrated that AP-1, Sp1 and HDAC1 are involved in the transactivation of Tspo expression. Specifically, the release of HDAC1 from the enhancer region of the Tspo gene in the presence of LPS was a novel finding.

The transcription factors c-Jun and STAT3 bind to the enhancer region of the Tspo gene in the mouse cell lines MA-10 and NIH-3T3 via PKCε activation through phorbol 12-myristate 13-acetate (PMA) stimulation [25]. PKCε activation via PMA activates the Raf1, MEK1/2 and ERK1/2 pathways. This signal transduction promotes Tspo gene expression following the activation and DNA binding of STAT3 and c-Jun. Our results indicating that LPS stimulation led to the binding of c-Fos and c-Jun to the enhancer region were partially consistent with those of previous studies. In general, LPS binds directly to Toll-like receptor 4 (TLR4) [28] and activates downstream factors such as mitogen-activated protein kinases (MAPKs) [29,30]. The MKK3/6 and p38 MAPK pathways induce phosphorylation of c-Fos and enhance transcriptional activity [31]. Additionally, c-Jun phosphorylation is induced by TLR4 signaling via the MKK4/7 and JNK pathways, and dimerization of c-Jun and c-Fos is promoted [32,33]. Collectively, these results indicate that LPS treatment is likely to activate the AP-1 complex in a manner mediated by several transduction pathways, such as the MAPK pathway, and that Tspo expression is consequently promoted by the binding of the AP-1 complex to DNA. In contrast to the facilitation of AP-1 binding, the enrichment of HDAC1 at the AP-1 binding site was decreased by LPS treatment. The reduction in HDAC1 enrichment in the enhancer region by LPS treatment possibly led to hyperinduction of Tspo expression via recruitment of AP-1 or another transcriptional activator. A recent study revealed that deletion of sequences between -593 and -520 bp from the TSS, which contains the AP-1, Stat3 and Ets.2 cis elements, abolished the transactivation of Tspo gene expression in BV-2 and Raw-264.7 macrophages [34]. Since our results were consistent with those of that recent report, it is convincingly concluded that the AP-1 and Sp1 are involved in the LPS-induced transcriptional regulation of Tspo. In addition to the involvement of AP-1 and Sp1, Rashid et al reported that the Pu.1 and its binding motif, Ets, play an important role in the transactivation of Tspo gene expression. Prediction tools for transcription binding sites were used in this study; however, Pu.1/Ets was not identified as a candidate transcription factor for the regulation of the Tspo gene. Contrary to the prediction results, the Ets sequence (5’-GGAA-3’) is located 3 bp from the AP-1 binding site and acts as a transcriptional activator of Tspo in response to LPS stimulation.

In addition to LPS stimulation, treatment with IFN-γ and IL-1β also induced Tspo transcription. Generally, IFN-γ activates the JAK-STAT pathway, and STAT acts as a transcriptional activator of inflammatory genes. The consensus sequence of STAT3 is located the upstream region of the Tspo gene, which overlaps with the AP-1 and Ets sites [25]. The IL-1β signaling pathway activates several transcription factors, including AP-1, via MAPKs such as p38, JNK and ERK1/2 [35]. These lines of evidences indicate that the general inflammatory response induces the expression of Tspo in microglial cells via the activation of transcription factors such as AP-1 or STATs.

Sp1, which belongs to the C₂H₂-type zinc-finger protein family, binds to GC-rich motifs and regulates numerous genes [36]. A previous study showed that there are several GC-rich sequences in the proximal promoter region of the mouse Tspo gene [26]. In this study, LPS treatment of BV-2 cells increased the enrichment of Sp1 in the proximal promoter region of Tspo. This evidence suggests that Sp1 binds to GC-rich sequences to regulate various genes by interacting with a transcriptional activator or repressor such as CBP/p300 or HDAC1 [37,38]. In the proximal promoter region of the Tspo gene, Sp1 enrichment was increased by LPS treatment, but HDAC1 enrichment was not affected. This result indicated that HDAC1 localized in the proximal promoter region was not involved in the pathway downstream of TLR4 activation by LPS. Furthermore, the finding that Tspo was expressed to some extent even under the control condition suggested that HDAC1 might minimally bind to the proximal promoter region under physiological conditions.

The expression of TSPO can be upregulated in noninflammatory conditions. According to the Human Brain Transcriptome database (http://hbatlas.org/) [39], human TSPO expression increases during the embryonic to the prenatal period and decreases during the neonatal period, and the TSPO expression level in the neonatal period is maintained throughout life. These data suggest that the functions of TSPO, such as increasing cholesterol transport from the cytosol to mitochondria and promoting pregnenolone production, might be important for fetal development. In addition, there is a possibility that the increase in TSPO expression during the neural developmental stage might be reflective of activated microglia. During neural development, microglia are activated and maintain CNS homeostasis by removing apoptotic cells, myelin debris and other endogenous redundant debris [40]. It is unknown which transcription factor or factors regulate the transcription of the TSPO gene in the fetal period; however, the unknown pathway might activate AP-1 and Sp1 or a different transcription factor.

In this study, we found that TSPO was not only localized in mitochondria but also in the plasma membrane fraction, as evidenced by the cell fractionation, Western blotting and immunocytochemical analysis results. The results of Western blot analysis indicated that not only the mitochondrial fraction but also in the membrane fraction contained TSPO. Consistent with this finding, the immunocytochemical results revealed that TSPO was mainly colocalized with mitochondria and that small amounts of TSPO signals were not distant from but were closely localized with Na⁺/K⁺ ATPase signals regardless of LPS stimulation. Generally, the Na⁺/K⁺ ATPase localizes to the transmembrane region and is used as a marker of the plasma membrane. However, the plasma membrane is not solely constituted by the Na⁺/K⁺ ATPase. Consequently, the TSPO signals were not completely colocalized with the plasma membrane signals. In addition to mitochondria and the plasma membrane, other organelles appeared to feature TSPO immunostaining signals. Thus, as it is reported that not all of the TSPO protein colocalizes with mitochondria, it is possible that TSPO localizes in organelles other than mitochondria [22]. Further research is needed to assess the localization and physiological function of TSPO induced by LPS stimulation.

In patients with ASD, the binding potential of [¹¹C] (R) PK-11195 to TSPO is increased in multiple brain regions, such as the cerebellum, brainstem and anterior cingulate cortex (ACC) [9]. These results indicate that the activation of microglia, i.e., neuroinflammation, and the expression of TSPO are increased. However, the conditions in our ex vivo study using a microglial cell line and LPS differed from the those of the CNS in patients with ASD; therefore, further investigation is needed to clarify the transcriptional regulation of Tspo using an animal model of ASD. It is necessary to elucidate whether this alteration in TSPO is due to transcriptional activation via AP-1 or a different transcriptional factor or factors in patients with ASD or animal model of ASD.

It is also important to clarify whether expressed TSPO leads to biological protection against or promotion of inflammation in the CNS. Because TSPO expression is increased under inflammatory conditions, it is thought that TSPO may act as a mediator of inflammation [41]. However, a recent study reported that TSPO acts as a negative regulator of inflammation [22]. Furthermore, TSPO overexpression rescues LPS-induced cognitive impairment. TSPO reduced inflammatory cytokines production and microglial activation, and neurogenesis in the hippocampus was promoted. Consequently, LPS-induced cognitive dysfunction was ameliorated [42]. TSPO may play a neuro-protective role in inflammation, and thus, it could be expected that elucidating the mechanism of TSPO induction under conditions other than inflammation could lead to the development of molecular targeted drugs.

Supporting information

S1 Table. Oligonucleotide sequences used in this study.

Oligonucleotide sequences used for real-time PCR and vector construction; sequencing primers used for the vectors and ChIP assay.

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S2 Table. List of antibodies used in this study.

Antibodies used in this study are shown with their supplier, product number and experimental procedure. WB: Western blotting, ICC: Immunocytochemistry, ChIP: Chromatin immunoprecipitation

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S1 Fig. Microscopy images showing TSPO expression.

BV-2 cells were stimulated with 1, 10, 100 or 500 ng/ml LPS for 4, 8, 12 or 24 hours and immunocytochemistry was then carried out. Images of nuclear (DAPI, blue) and TSPO (green) staining were acquired at 40× magnification. Scale bar: 100 μm.

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S2 Fig. IFN-γ and IL-1β induced Tspo expression in the microglial cell line, BV-2.

BV-2 cells were treated with 100 ng/ml IFN-γ and/or IL-1β for 8 hours and RNA was then extracted and subjected to qRT-PCR. The mRNA levels were measured and normalized to those of Gapdh mRNA. The asterisks indicate statistically significant differences compared to the control (n = 3, *p<0.05, **p<0.01).

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Click here for additional data file.^{(7MB, tiff)}

Acknowledgments

We are thankful to Sachiko Kamikawa and Noriaki Kawai for their technical assistance. We would like to thank Springer Nature Author Services (https://authorservices.springernature.com/) for English language editing. This work was performed in part at the Institute for Animal Experimentation, Hirosaki University.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

This work was supported by Hirosaki University Grant for Exploratory Research by Young Scientists and Newly appointed Scientists (to SS), Hirosaki University Institutional Research Grant (to SU and KN) and Grants-in-Aid for Scientific Research (C) #18K08846 (to SU). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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PLoS One. 2019 Sep 19;14(9):e0222861. doi: 10.1371/journal.pone.0222861.r001

Author response to previous submission

5 Jun 2019

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PLoS One. doi: 10.1371/journal.pone.0222861.r002

Decision Letter 0

Leo TO Lee

17 Jul 2019

PONE-D-19-15932

AP-1 complex induces mouse translocator protein (18 kDa) expression by lipopolysaccharide in microglial cell line, BV-2

PLOS ONE

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Reviewer #1: The manuscript could be improved by considering the following points

1. I think it would be important to include something about PU.1 in the discussion (The binding motif of PU.1 is in enhancer region (-593-520) and lies 3 bp apart from the AP1 core binding motif). The expression pattern of TSPO in microglia is very specific, and therefore Lineage dependent transcription factors (LDTFs) such as PU.1 have to play a role (Especially since we have already provided evidence of strong Pu.1 enrichment in this region). Infact, the initial steps of enhancer selection in closed chromatin regions containing regularly positioned nucleosomes involves the binding of LDTFs. This binding results in the depletion/sliding of nucleosomes to expose enhancer DNA sequences. Indeed, in microglia/macrophages, enhancers controlling endotoxin-stimulated gene expression are almost invariably bound by the lineage dependent Ets transcription factor Pu.1 (Ghisletti et al., 2010; Holtman et al., 2017). These LDTFs bound to enhancers induce histone modifications (such as H3K4me1) associated with a primed state of activity (Smale and Natoli, 2014). Histone mark H3K4me1 is a core chromatin signature of primed enhancers and serves as a beacon for signal-dependent effectors of signaling pathways such as nuclear factor-κB (NFκB), interferon responsive factors (IRFs) and activator protein 1 (AP-1) (Ghisletti et al., 2010; Heinz et al., 2010; Smale and Natoli, 2014).

Ghisletti, S., Barozzi, I., Mietton, F., Polletti, S., De Santa, F., Venturini, E., Gregory, L., Lonie, L., Chew, A., Wei, C.-L., et al. (2010). Identification and Characterization of Enhancers Controlling the Inflammatory Gene Expression Program in Macrophages. Immunity 32, 317–328.

Heinz, S., Benner, C., Spann, N., Bertolino, E., Lin, Y.C., Laslo, P., Cheng, J.X., Murre, C., Singh, H., and Glass, C.K. (2010). Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities. Mol. Cell 38, 576–589.

Holtman, I.R., Skola, D., and Glass, C.K. (2017). Transcriptional control of microglia phenotypes in health and disease. J. Clin. Invest. 127, 3220–3229.

Smale, S.T., and Natoli, G. (2014). Transcriptional control of inflammatory responses. Cold Spring Harb. Perspect. Biol. 6, a016261.

2. It would be important to comment on differences observed in TSPO expression following knockdown of cFos and cJun. In a recent paper (Raashid et al.Biochim Biophys Acta Gene Regul Mech. 2018 Dec;1861(12):1119-1133.), decreased TSPO expression was only observed following a combined knockdown of cJun and cFos. In the current paper, the authors report a decrease in TSPO expression when either cJun or cFos is knockdown. Is this due to cell-line differences? (I noticed they used a different BV-2 cell-line which was maintained in DMEM medium).

3. In paragraph 4 of the discussion, Sp1 transcription factor is discussed. There is no mention of either Sp3 or Sp4, despite Rashid et al. showing that these factors bind the GC-boxes in the proximal promoter (Sp1, Sp3 and Sp4 share common binding site motifs) and that knockdown of either Sp3 or Sp4 significantly reduces TSPO promoter activity in BV-2 microglia.

4. In paragraph 5 of the discussion, the authors state “ human TSPO expression increases during the embryo to prenatal period and decreases during the neonatal period, and the TSPO expression level in the neonatal period is maintained throughout life. These data suggest that the TSPO functions, such as increasing cholesterol transport from cytosol to mitochondria and promoting pregnenolone production, might be important for fetal development.” While this statement might be true, I think it also important to acknowledge that the increase in TSPO expression during development might simply be a reflection of an activated microglia state. During development, microglia are predominantly in the amoeboid state as they play a major role in removing excessive produced apoptotic neurons and large amounts of extracellular debris from the developing CNS.

Reviewer #2: The authors adequately addressed the issues from the original review. The paper describes a data-supported mechanism for transcriptional regulation of TSPO in LPS-treated microglia. A few minor points:

1) Regarding M1/M2 classification - it should be noted that the range of microglial activation phenotypes is complex and that the M1/M2 classification in the paper is being used for the sake of simplicity.

2) The antibodies and oligonucleotide sequences used can be placed in supplemental information.

3) There were a few grammatical errors throughout the manuscript.

Reviewer #3: In this manuscript the authors described a potential mechanism of the increased TSPO expression after LPS treatment which were regulated by AP-1 complex and HDAC1 in BV-2 microglia cell at the transcriptional level. I have some specific issues that need clarification.

1. The title should be replaced as “Lipopolysaccharide induces mouse translocator protein (18 kDa) expression by AP-1 complex in microglial cell line, BV-2”.

2. As the authors stated in the manuscript, the concentration of HDAC1 (a transcriptional repressor) was decreased in the enhancer region of TSPO at the AP-1 binding site after LPS treatment. I have concerns about the potential effects of HDAC1 intervention on TSPO expression or LPS induced inflammatory reaction.

3. Writing of the paper might be improved to meet sufficient quality.

**********

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Reviewer #2: No

Reviewer #3: No

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PLoS One. 2019 Sep 19;14(9):e0222861. doi: 10.1371/journal.pone.0222861.r003

Author response to Decision Letter 0

18 Aug 2019

Reviewer 1

1. Thank you for the constructive comments to improve our study. We should have mentioned about Pu.1/Ets.2 in the discussion. We added the sentences as below (P17, line 15-21).

“... transcriptional regulation of Tspo. In addition to the involvement of AP-1 and Sp1, Rashid et al reported that the Pu.1 and its binding motif, Ets, play an important role in the transactivation of Tspo gene expression. Prediction tools for transcription binding sites were used in this study; however, Pu.1/Ets was not identified as a candidate transcription factor for the regulation of the Tspo gene. Contrary to the prediction results, the Ets sequence (5’-GGAA-3’) is located 3 bp from the AP-1 binding site and acts as a transcriptional activator of Tspo in response to LPS stimulation.”

2. Thank you for the constructive comments to improve our study. We also wondered the differences between our results and a recent study (Rashid et al, Biochim Biophys Acta. Gene Regul Mech, 2018). Batarseh et al reported that PMA-induced Tspo expression was reduced by the solely knockdown of c-Jun in MA-10 cells and NIH-3T3 cells (ref#.25, Figure 4 and 6), hence, it is thought to be that the expression of Tspo was inhibited by the knockdown of either of c-Fos or c-Jun. In case of our study, the protocols such as cell and its culture condition, the dose or time of LPS stimulation and the measurement of gene expression by qRT-PCR or Western blotting were diffed from previous studies, the results were not completely consistent with those studies.

3. Thank you for your comments. Rashid et al (Ref. #34) and Batarseh et al (Ref. #26) reported that the enrichment of Sp1, Sp3 and Sp4 were increased at the GC-boxes in the proximal and distal promoter region of Tspo gene and the knockdown experiments of Sp3 or Sp4 significantly reduced the transactivation of Tspo, hence, it is considered to be a certain phenomenon that the involvement of Sp family in the Tspo gene expression.

Previous studies showed that Sp1 and HDAC1 forms a transcriptional repressor complex at the transcriptional inactive state. Upon the stimulation, the enrichment of Sp1 did not affect, however, HDAC1 was released from Sp1 and replaced to CBP/p300 (Ref. #21 and 27).

We hypothesized that the same molecular mechanism was occurred in the transcriptional regulation of Tspo gene. Furthermore, since there were few studies that the interaction between HDAC1 and Sp3 or Sp4, we excluded Sp3 and Sp4 from this study. The additional information is shown in the results section as described below. (P14, line1-4)

“... a, transcriptionally repressed state [21,27]. Furthermore, since few studies have examined the interaction between HDAC1 and Sp3 or Sp4, we excluded Sp3 and Sp4 from this study and focused on the interaction between Sp1 and HDAC1 in the proximal promoter region of the Tspo gene.”

4. Thank you for the constructive comments to improve our study. As you pointed out, we should have mentioned about the activation of microglia during CNS development. We added the sentences in the discussion section with reference (Hanisch UK, Kettenmann H. Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nat Neurosci. 2007 Nov;10(11):1387-94. Review. PubMed PMID: 17965659) as below. (P19, line2-5)

“... for fetal development. In addition, there is a possibility that the increase in TSPO expression during the neural developmental stage might be reflective of activated microglia. During neural development, microglia are activated and maintain CNS homeostasis by removing apoptotic cells, myelin debris and other endogenous redundant debris [40].”

Reviewer 2

1. Thank you for your advice. As you pointed out, the classification of microglia is complicated according to various studies. We described only classical classification of microglia in introduction section and did not mention detailed classification such as M (LPS) and M (IL-4 and IL-13) (previously named as M2a) that reported recently (Michell-Robinson MA et al, Brain. 2015 May;138(Pt 5):1138-59). The objective of this study is clarification of transcriptional mechanism of Tspo gene in mice, hence, we did not intend to mention the classification of microglial cell. LPS and IFN-γ were used for induction of activated microglia and Tspo gene expression as pro-inflammatory inducers.

2. Thank you for your advice. The lists of oligonucleotide sequences and antibodies were transferred to S1 and S2 Table.

3. Thank you for your advice. We revised the manuscript and was proofread by the Springer Nature Author Services.

Reviewer 3

1. Thank you for your advice. The title was replaced as follows. “Lipopolysaccharide induces mouse translocator protein (18 kDa) expression via the AP-1 complex in the microglial cell line, BV-2.”

2. Thank you for your comments. Upon LPS or pro-inflammatory stimulation, various pathways were activated and HDAC1 was released from AP-1 binding site, transcriptional activators were then recruited to the Tspo promoter. Of course, since it is thought that not all HDAC1 molecules were released from AP-1 binding site, some HDAC1 molecules remained at the AP-1 binding site. Furthermore, the enrichment of HDAC1 might gradually returns to normal condition in time-dependent manner after once upon release from AP-1 binding site to inhibit activated gene expression.

3. Thank you for your advice. We revised the manuscript and was proofread by the Springer Nature Author Services.

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PLoS One. doi: 10.1371/journal.pone.0222861.r004

Decision Letter 1

Leo TO Lee

10 Sep 2019

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Lipopolysaccharide induces mouse translocator protein (18 kDa) expression via the AP-1 complex in the microglial cell line, BV-2

PONE-D-19-15932R1

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PLoS One. doi: 10.1371/journal.pone.0222861.r005

Acceptance letter

Leo TO Lee

12 Sep 2019

PONE-D-19-15932R1

Lipopolysaccharide induces mouse translocator protein (18 kDa) expression via the AP-1 complex in the microglial cell line, BV-2

Dear Dr. Nakamura:

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

S1 Table. Oligonucleotide sequences used in this study.

Oligonucleotide sequences used for real-time PCR and vector construction; sequencing primers used for the vectors and ChIP assay.

(XLSX)

Click here for additional data file.^{(10.3KB, xlsx)}

S2 Table. List of antibodies used in this study.

Antibodies used in this study are shown with their supplier, product number and experimental procedure. WB: Western blotting, ICC: Immunocytochemistry, ChIP: Chromatin immunoprecipitation

(XLSX)

Click here for additional data file.^{(10.3KB, xlsx)}

S1 Fig. Microscopy images showing TSPO expression.

(TIFF)

Click here for additional data file.^{(7MB, tiff)}

S2 Fig. IFN-γ and IL-1β induced Tspo expression in the microglial cell line, BV-2.

(TIFF)

Click here for additional data file.^{(7MB, tiff)}

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Data Availability Statement

All relevant data are within the paper and its Supporting Information files.

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PERMALINK

Lipopolysaccharide induces mouse translocator protein (18 kDa) expression via the AP-1 complex in the microglial cell line, BV-2

Shuji Shimoyama

Tomonori Furukawa

Yoshiki Ogata

Yoshikazu Nikaido

Kohei Koga

Yui Sakamoto

Shinya Ueno

Kazuhiko Nakamura

Roles

Abstract

Introduction

Materials and methods

Cell culture and chemical treatment

Animals and primary microglial cell culture

RNA isolation, cDNA synthesis and quantitative real-time reverse transcription PCR (qRT-PCR)

Preparation of whole-cell extracts and cell fractionation, Western blotting and densitometric analysis

Immunocytochemistry

Reporter gene assay

Chromatin immunoprecipitation (ChIP) assay and real-time PCR

Silencing by RNAi

Statistical analyses

Results

LPS treatment induced Tspo expression in BV-2 cells

Fig 1. LPS induced Tspo expression in the microglial cell line BV-2.

LPS treatment increased TSPO expression in the mitochondrial and membrane fractions

Fig 2. TSPO protein expression was increased in the mitochondrial and membrane fractions by LPS treatment.

The transcription factor AP-1 is the candidate transcriptional activator of Tspo after LPS treatment

Fig 3. Involvement of AP-1 in the regulation of Tspo expression by LPS stimulation.

AP-1, Sp1 and HDAC1 regulated Tspo expression

Fig 4. Enrichment of AP-1 and Sp1 was increased and enrichment of HDAC1 was decreased in the Tspo gene regulatory region by LPS treatment.

Silencing of c-Fos and c-Jun decreased Tspo expression

Fig 5. Knockdown of c-Fos and c-Jun reduced Tspo expression.

Discussion

Supporting information

Acknowledgments

Data Availability

Funding Statement

References

Author response to previous submission

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Leo TO Lee

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Author response to Decision Letter 0

Decision Letter 1

Leo TO Lee

Roles

Acceptance letter

Leo TO Lee

Roles

Associated Data

Supplementary Materials

Data Availability Statement

ACTIONS

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