Quantitative Bio-Science 33(1), 41 45(2014) Attenuation of Translocator Protein 18 kDa (TSPO) Up-Regulation by Peroxisome Proliferator-Activated Receptor γ Ligand in Activated Microglia Hyojin Cho, Hyun-Jung Shim, Seong-Woon Yu* Department of Brain Science, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu 711-873, Korea (Received May 13, 2014; Revised May 23, 2014; Accepted May 24, 2014) Translocator protein (18 kDa) (TSPO) is a five transmembrane domain protein localized primarily in the outer mitochon- drial membrane. Recently, we reported that TSPO is a negative regulator of neuroinflammation in microglia. Peroxisomeproliferator-activated receptor γ (PPARγ) is a ligand-specific transcriptional factor belonging to the nuclear receptor super-family and predicted as a putative TSPO transcriptional factor. A number of studies suggest that the activation of PPARγhas anti-inflammatory effects. In this study, we observed that treatment of rosiglitazone, a PPARγ ligand significantlydecreased the NO production in lipopolysaccharide-stimulated BV2 microglia cell, indicating inhibition of microglialactivation. The inhibitory effect of rosiglitazone extended to attenuated protein level of TSPO. TSPO up-regulation seemsan adaptive anti-inflammatory response to overcome microglia activation, according to our previous report. Taken together,these results indicate that PPARγ activation by rosiglitazone attenuates neuroinflammation and leads to reduced expressionof TSPO in the BV2 microglial cells.
Key words : Microglia, Neuroinflammation, PPARγ, Translocator protein 18 kDa (TSPO)
inflammatory responses upon lipopolysaccharide (LPS) treat- ment while TSPO knockdown led to the opposite events [5].
A transcription factor (a sequence-specific DNA-binding Microglia are resident macrophages of the brain and spinal factor) is a protein that controls the activity of a gene by bind- cord sensitive to brain injury and disease. Under normal condi- ing to specific DNA sequences. Transcription factors exert its tions, microglia cells contribute to brain development and main- activity by promoting or blocking the recruitment of RNA poly- tenance of tissue homeostasis. However, microglia also have a merase to specific genes through complex with other proteins potential to cause neuronal damage when activated inflamma- or alone [6]. Using the bioinformatics tools peroxisome proli- tory responses process chronically [1-3].
ferator-activated receptor γ (PPARγ) was predicted as a putative Translocator protein (18 kDa) (TSPO) is a five transmembrane TSPO transcriptional factor. PPARs are a group of nuclear domain protein localized primarily in the outer mitochondrial receptor proteins that function as transcription factors regulat- membrane. TSPO is expressed in various tissue types including ing the expression of genes [7,8]. Recently, there are reports the central nervous system, especially in microglia and reactive showing inhibitory effects of PPARγ ligands on the production astrocytes [4]. In the previous study, we demonstrated that TSPO of microglia-derived proinflammatory molecules. It is known is a negative regulator of neuroinflammation in microglia. Over- that thiadiazolidinones (TDZDs) and thiazolidinediones (TZDs), expression of TSPO diminished microglia activation and pro- such as rosiglitazone, pioglitazone, and troglitazone, are ago- * Correspondence should be addressed to Dr. Seong-Woon Yu, Department of Brain Science, Daegu Gyeongbuk Institute of Science and Technology (DGIST),Daegu 711-873, Korea. Tel: +82-53-785-6113, Fax: +82-53-785-6109, E-mail: yusw@dgist.ac.kr Quantitative Bio-Science Vol. 33, No. 1, 2014 nists of PPARγ. It has recently been shown that TDZDs inhi- te assay. This assay is to measure the accumulated level of the bit inflammatory activation of cultured brain astrocytes and NO metabolite nitrite (NO - 2 ) in the supernatant as a surrogate microglia [9-12]. These studies suggest the possibility that index of NO using a colorimetric reaction with Griess reagent PPARγ is related to the regulation of TSPO gene expression, (0.1% naphthylethylenediamine, 1% sulfanylamide and 2.5% exhibiting anti-inflammatory activities in common. Here we H3PO4; Promega) 24 h after LPS treatment. Absorbance was report that administration of rosiglitazone, a specific ligand measured at 540 nm with a microplate reader.
for PPARγ, attenuated neuroinflammation in BV2 microgliacells. The improved neuroinflammation upon rosiglitazone 5. RNA isolation and real-time reverse transcription- administration was associated with the attenuated expression polymerase chain reaction (RT-PCR) level of TSPO.
Total RNA was purified using the QIAzol reagent (Qiagen, Valencia, CA, USA) and cDNA was prepared from total RNAusing Reverse Transcription kit (Qiagen) according to the manu- Materials and Methods
facturer's instructions. cDNA generation and RT-PCR reactionwere performed according to the previous report with slight 1. Chemicals and reagents modifications [5] using QuantiTechSYBR Green PCR kit (Qia- Hyclone DME/F-12 was purchased from Thermo scientific.
gen) and the Rotor gene Q real-time amplification instrument Penicillin-streptomycin, and fetal bovine serum (FBS) were (Qiagen). The PCR conditions and the sequences of the primers purchased from Invitrogen (Carlsbad, CA, USA). LPS was pur- for TSPO and glyceraldehyde-3-phosphate dehydrogenase chased from Calbiochem (La Jolla, CA). Rosiglitazone was (GAPDH) were as reported [5]. Each PCR reaction was perform- purchased from Sigma-Aldrich.
ed in triplicates and for each sample, the levels of TSPO mRNAexpression were normalized to GAPDH levels using the com- parative CT (2-ΔΔCT) method and were expressed as fold induc-tion.
BV2 microglial cells were maintained in DME/F-12 supple- mented with 5% FBS and 1% penicillin-streptomycin. Cells 6. Western blot analysis were grown at 37� C in an atmosphere containing 5% CO2. For experiments the cells were plated at a density of 1.0×105 cells BV2 cells were harvested at the indicated time point and per cm2. For stock solutions, rosiglitazone was prepared in lysed in cell lysis buffer (250 mM sucrose, 50 mM NaCl, 1% dimethyl sulfoxide (DMSO) and LPS was prepared in phos- Triton X-100, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl phate buffered saline (PBS), respectively.
fluoride (PMSF) in 20 mM Tris-HCL, pH 7.2) containing 1×protease cocktail inhibitors (Roche Diagnostics, Indianapolis, 3. Cell viability IN, USA) and 1× phosphatase cocktail inhibitors (Pierce, Rock-ford, IL, USA) for 30 min on ice. The cell lysates were cleared Viability of BV2 cells was determined using The CellTiter- by centrifugation at 14,000 rpm for 15 min. The protein con- Blue® Cell Viability Assay (Promega, Madison, WI, USA).
centration of cell lysates was measured using a BCA protein This assay provides a homogeneous, fluorescent method for assay kit (Thermo Scientific, Rockford, IL, USA). Typically, monitoring cell viability. It is based on the ability of living 10-20 μg of proteins per well were loaded for Western blotting cells to convert a redox dye (resazurin) into a fluorescent end analysis. The proteins were electrotransferred to polyvinylidene product (resorufin). The 10 μL of reagent was directly added fluoride (PVDF) membrane (Millipore, Bedford, MA, USA) to the assay plate. The plate was incubated at 37� using a Trans-Blot SD Semi-Dry Electrophoretic Transfer an incubation step, data were recorded at 490 nm with a Spectra- Cell (Bio-Rad, Hercules, CA, USA), and the membranes were MAX 190 microplate reader (Molecular Devices, Sunnyvale, blocked for 1 h at room temperature in a blocking solution of 5% nonfat dry milk in PBS-0.1% Tween 20 (PBST). Themembranes were incubated overnight with primary antibody.
After rinsing with blocking solution, the membranes were incu- The production of nitric oxide (NO) was determined by nitri- bated for 1 h at room temperature in blocking solution contain- Cho H et al. : TSPO and PPARγ ing horseradish peroxidase conjugated secondary antibodies.
After washing, the membranes were processed for analysisusing an enhanced chemiluminescence kit (Thermo Scientific)as described by the manufacturer.
7. Statistical methods The difference between two groups was analyzed by the t- test. Multiple comparisons among groups were performed byone-way ANOVA. All statistical analyses were performed usingGraphPad Prism (Graphpad software, Inc).
Rosiglitazone (μM) 1. Rosiglitazone reduces NO production We first confirmed that a representative PPARγ ligand, rosi- glitazone suppresses microglia activation and neuroinflamma- tion. BV2 cells are an immortalized murine microglia cell line[13]. LPS was used as a microglia activator since LPS is the major component of the outer membrane of gram-negative bacteria and is commonly used as an inducer in immune cells[14]. To observe neuroinflammatory response of BV2 micro- glial cells, we carried out NO production assay that can detect Rosiglitazone (μM) 2 ) as a surrogate marker of NO. Rosiglitazone was pretreated 1 h prior to LPS treatment in BV2 cell cultures. The Fig. 1. (A) Rosiglitazone was pretreated 1 h prior to LPS treatment in
BV2 cell cultures and then the level of NO - was measured as markers as a marker of BV2 cell activation were mea- on BV2 cell activation. The NO - levels were determined at 24 h after sured at 24 h after LPS treatment. The results showed that 100 LPS treatment. (B) The viability of BV2 cells was not affected by the ng/mL LPS treatments induced about 5 fold increase in NO pro- doses of rosiglitazone used in this study, when measured by CellTiter duction, compared with untreated control at 24 h after LPS treat- Blue assay. Rosiglitazone was pretreated 1h prior to LPS treatment inBV2 cell cultures. The data are presented as mean±SD (n=3). **p⁄ ment. However, pretreatment with rosiglitazone attenuated the 0.01, compared with control. NS, not significant.
in a dose dependent manner (Fig. 1A). The doses of LPS and rosiglitazone (Fig. 1B) used in this study showed ed that TSPO level in activated microglia increases in response no toxicity on BV2 cells, as confirmed by CellTiter Blue Assay.
to inflammatory stimuli as an adaptive response to resolve sus-tained microglia activation. Therefore, if rosiglitazone suppress- 2. Rosigiltazone attenuated TSPO expression level es inflammation, the increase of TSPO level is expected to be BV2 cells were treated with 100 ng/mL LPS and then the prevented. The current data are in accordance with this notion levels of TSPO expression were measured by RT-PCR and that rosiglitazone administration reduces neuroinflammation Western blot analyses. RT-PCR revealed a moderate reduction and thus, up-regulation of TSPO expression is suppressed.
in TSPO mRNA level 24 h after rosiglitazone administration,but it did not reach a statistically significant level (Fig. 2). How-ever, the levels of TSPO protein were significantly decreased by rosiglitazone treatment, as shown by Western blot analysiswhen measured 24 h after LPS treatment (a representative image Microglia are a resident macrophage population in the cen- of three experiments in Fig. 3A and quantification of protein tral nervous system (CNS). Activated microglia are a source band intensities in Fig. 3B). In our previous report, we suggest- of proinflammatory cytokines and chemokines, which act to

Quantitative Bio-Science Vol. 33, No. 1, 2014 mation of murine microglial BV2 cell line [5].
The peroxisome proliferator-activated receptors (PPARs) are a member of the nuclear receptor superfamily of ligand-dependent transcription factors. It is known that PPARγ is especially detected in adipose tissue, intestine and macrophages[16]. A growing body of evidence suggests that PPARγ agonists exert the anti-inflammatory activities [17]. These studies sug- Fold induction of TSPO/actin gest the possibility that PPARγ may be related to the regulation of TSPO gene expression, both of which have anti-inflamma- tory activities in common.
Rosiglitazone (μM) The present study demonstrates that treatment of rosiglita- Fig. 2. mRNA expression level of TSPO following LPS (100 ng/mL)
zone, a prototype PPARγ ligand, significantly decreased the treatment was measured by RT-PCR in BV2 cells. mRNA expressionlevels were normalized to GAPDH.
levels of NO production in a dose dependent manner, indicat-ing inhibition of microglial activation (Fig. 1A). The reductionof neuroinflammation induced the attenuated expression level of TSPO on rosiglitazone administration (Figs. 2, 3). Takentogether with these data, it is suggested that PPARγ activationby rosiglitazone mitigated neuroinflammation and reduced levels of TSPO.
Rosiglitazone (μM) This work was supported by the DGIST Convergence Science Center Program of the Ministry of Science, ICT and FuturePlanning (14-BD-04).
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