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Keywords:

  • inflammation;
  • peroxisome proliferator-activated receptors;
  • superinduction;
  • toll-like receptors

Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References
Thumbnail image of graphical abstract

Peroxisome proliferator-activated receptor β/δ (PPARβ/δ) is a potential regulator of neuroinflammation. Toll-like receptors (TLR) are innate immunity-related receptors of inflammatory stimuli. In the present report, we evaluate the molecular mechanisms of regulation of mRNA, protein, and transcriptional activity levels of PPARβ/δ by agonists of TLR4, TLR1/2, and TLR5, using lipopolysaccharide (LPS), peptidoglycan, and flagellin, respectively. We found that these stimuli increase the PPARβ/δ levels in astrocytes. Expression and activity of PPARβ/δ are separately regulated by inhibitors of p38, MEK1/2, extracellular signal-regulated kinases 1/2, and c-Jun N-terminal Kinase mitogen-activated protein kinases. The LPS-induced kinetics of PPARβ/δ expression is similar to that of the proinflammatory gene cyclooxygenase 2. Moreover, for both genes the expression depends on nuclear factor kappa-light-chain-enhancer of activated B cells and p38, and is induced after inhibition of protein synthesis. The up-regulation of the expression after inhibition of protein synthesis signifies the participation of a labile protein in regulation of PPARβ/δ expression. In contrast to cyclooxygenase 2, the cycloheximide-sensitive PPARβ/δ expression was not responsive to nuclear factor kappa-light-chain-enhancer of activated B cells inhibition. Measurements of PPARβ/δ mRNA stability showed that the PPARβ/δ mRNA levels are regulated post-transcriptionally. We found that in LPS-stimulated astrocytes, the half-life of PPARβ/δ mRNA was 50 min. Thus, we demonstrate that PPARβ/δ expression and activity are regulated in TLR agonist-stimulated astrocytes by mechanisms that are widely used for regulation of proinflammatory genes.

Protein expression level of nuclear receptor PPARβ/δ is important for functions of this transcription factor. We investigate the regulatory mechanisms of PPARβ/δ in rat primary astrocytes stimulated by agonists of toll-like receptors (TLR): TLR4, TLR1/2, and TLR5. Expression, activity, mRNA stability, and superinduction of PPARβ/δ were up-regulated after TLR stimulation. These processes are sensitive to MAPKs and NF-kB inhibitors. Superinduction is up-regulation of mRNA expression after inhibition of protein synthesis.

Abbreviations used
AP-1

activator protein 1

COX-2

cyclooxygenase 2

DMEM

Dulbecco's modified Eagle's medium

ERK

extracellular signal-regulated kinases

FGL

flagellin

GAPDH

glyceraldehyde 3-phosphate dehydrogenase

JNK

c-Jun N-terminal Kinase

LPS

lipopolysaccharide

MAPK

mitogen-activated protein kinases

MEK

MAP kinase/ERK kinase

NF-κB

nuclear factor kappa-light-chain-enhancer of activated B cells

p38

p38 mitogen-activated protein kinases

PGE2

prostaglandin E2

PGN

peptidoglycan

PPAR

peroxisome proliferator-activated receptors

TLR

toll-like receptors

TNFα

tumor necrosis factor α

Peroxisome proliferator-activated receptors (PPAR) are ligand-activated transcription factors, which are implicated in the regulation of lipid and glucose homeostasis (Bensinger and Tontonoz 2008). These nuclear receptors are also involved in the regulation of inflammatory and immune responses in the brain (Heneka and Landreth 2007; Bensinger and Tontonoz 2008). Since neuroinflammation is a hallmark of a variety of brain disorders, PPAR-dependent pathways are considered to be targets for treatment of various brain pathologies (Heneka and Landreth 2007; Bernardo and Minghetti 2008). The exploration of PPAR mechanisms in the brain cells is important for understanding the biochemical basis of these diseases.

The three known PPAR subtypes are PPARα, PPARβ/δ, and PPARγ. Although all three are expressed in the brain, so far PPARα and PPARγ subtypes have been the main focus of studies concentrated on the role of PPARs in neuroinflammation (Heneka and Landreth 2007; Hall et al. 2008). The protective efficiency of synthetic PPARβ/δ agonists in animal models of neurodegenerative diseases has been proven (Hall et al. 2008; Aleshin et al. 2013). Modulation of neuroinflammation and oxidative stress are important PPARβ/δ-dependent mechanisms of brain pathology (Schnegg and Robbins 2011; Aleshin and Reiser 2013). This concept has raised interest in the elucidation of the molecular mechanisms of PPARβ/δ regulation in various models of brain disorders.

Glial cells (astrocytes and microglia) play an active role in the pathological processes of many neurological diseases that are accompanied by inflammation (Farina et al. 2007). The innate immune system is the major component of inflammation, and its role in glial cells is under intensive investigation. Important targets of the glial innate immune system activation are the toll-like receptors (TLR). Therefore, TLR agonists are widely used for investigation of glial inflammatory responses (Akira and Takeda 2004). Astrocytes are equipped with several types of TLR. Among them, TLR1/2 is sensitive to peptidoglycan (PGN), TLR4 is sensitive to lipopolysaccharide (LPS) (van Noort and Bsibsi 2009), and TLR5 is sensitive to flagellin (FGL) (Akira and Takeda 2004; Pedras-Vasconcelos et al. 2009; Rivest 2009; van Noort and Bsibsi 2009). We have previously shown that LPS increases the PPARβ/δ expression levels in rat primary astrocytes (Aleshin et al. 2009). The involvement of other TLR agonists in the regulation of PPARβ/δ expression still has to be elucidated to clarify the role of TLR-PPAR signaling pathways in astrocytes.

The dual role of inflammatory genes, up-regulated after the onset of inflammatory responses was shown for cyclooxygenase 2 (COX-2) (Gilroy et al. 2004). After proinflammatory stimulation, COX-2 produces proinflammatory mediators, the prostaglandins. Nevertheless, prostaglandins are later converted into cyclopentenone prostaglandins, which have anti-inflammatory and proresolution properties. We found that PPARβ/δ expression levels are up-regulated after proinflammatory stimulation similar to COX-2 (Aleshin et al. 2009). This observation and the fact that synthetic PPARβ/δ agonists have anti-inflammatory properties (Hall et al. 2008; Aleshin et al. 2013) raise the question of whether PPARβ/δ has a dual role in inflammation, like COX-2, in astrocytes. The comparison of COX-2 and PPARβ/δ expression features in the course of cellular responses to TLR agonists is relevant to this question.

The role of PPARβ/δ in the resolution process can be clarified with the help of MAPK inhibitors. The MAPK family has three branches: extracellular signal-regulated kinases (ERK/MEK), p38, and c-Jun N-terminal kinase (JNK). All three branches play critical roles in neuroinflammation via interactions with receptors, enzymes, scaffolding proteins of signaling cascades, and transcription factors (Chico et al. 2009; Gaestel et al. 2009). For two reasons, MAPK inhibitors are interesting substances. Firstly, activation of TLRs in various cell types, including astrocytes, leads to activation of the MAPK signaling pathways (Gaestel and Kracht 2009; Kaminska et al. 2009). Therefore, MAPK inhibitors have a significant potential as modulators of brain inflammation and gliosis in neurological disorders (Borders et al. 2008; Chico et al. 2009). Secondly, ERK/MEK p38 and JNK activation influence the DNA-binding activity of PPARα and PPARγ via phosphorylation. Consequently, these transcription factors regulate gene expression in astrocytes and other cell types (Burns and Vanden Heuvel 2007; Burgermeister and Seger 2008; Kaplan et al. 2010). Although PPARβ/δ has computer-predicted phosphorylation sites for protein kinase C and other serine/threonine protein kinases (Burns and Vanden Heuvel 2007; Burgermeister and Seger 2008), the role of the MAPK in the regulation of PPARβ/δ expression and DNA-binding activity has not yet been tested before.

Here, we observed a similarity in the effects of LPS, PGN, and FGL on the expression and activity levels of PPARβ/δ. This suggests an involvement of TLR/PPARβ/δ pathways in the regulation of glial cell responses to various TLR agonists. We observed a similar dynamics of LPS-induced PPARβ/δ and COX-2 expression. Increase of expression levels of both genes is sensitive to the inhibition of the transcription factor nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). Moreover, expression levels of both genes are up-regulated in the presence of cycloheximide, an inhibitor of protein synthesis. Measurements of PPARβ/δ mRNA stability by actinomycin D treatment showed that LPS induced rapid mRNA degradation. This phenomenon is known for several inflammation-related genes (Anderson 2010). The data shown here reveal new pathways involved in the post-transcriptional regulation of PPARβ/δ in the course of inflammatory responses.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Reagents

SP600125 Dulbecco's modified Eagle's medium (DMEM, cat.no FG 0435); fetal calf serum, penicillin, and streptomycin were obtained from Biochrom (Berlin, Germany). LPS, purified by phenol extraction from Escherichia coli strain 0111:B4, Actinomycin D, Bay 11-7085, PPARα, PPARβ/δ, PPARγ, and β-tubulin antibodies were purchased from Sigma Chemicals (Germany, Taufkirchen). PD 98059, SB 203580, U0126, SP600125 were ordered from ALEXIS Biochemicals (Grünberg, Germany). FGL (Bacillus subtilis) and peptidoglycan (Bacillus subtilis) originated from Invivogen (San Diego, CA, USA). Cycloheximide and polymyxin B sulfate were purchased from Calbiochem, (Darmstadt, Germany).

Primary cell culture

Cultures of primary rat astrocytes were established and maintained as described before (Strokin et al. 2003; Aleshin et al. 2009). All experiments using brain tissue from animals were done in agreement with guidelines from Sachsen-Anhalt (Germany) on the ethical use of animals, and all efforts were made to minimize the number of animals used. Briefly, brains from newborn rats were triturated by the use of nylon meshes of 250 and 136 μm pore width, in consecutive order. Dissociated cells were plated into 75 cm2 culture flasks at a density 6 × 105 cells per mL. The cells were cultured in DMEM containing 10% fetal calf serum and 100 μg/mL penicillin and streptomycin (37°C, 10% CO2). After 5 days of cultivation in DMEM, culture medium was changed on to a fresh one. After 2 days, the monolayer of astrocytes was trypsinized and plated into six-well plates and maintained 2 days in DMEM. After this, the medium was changed, and the cells were used for experiments.

Measurement of the relative RNA expression level

Real-time PCR was performed as described previously (Aleshin et al. 2009) In brief, total RNA was isolated using total RNA isolation kit RNeasy (Qiagen, Hilden, Germany). cDNA was generated from 1 μg of total RNA with iScript cDNA synthesis kit (Bio-Rad, Munich, Germany) according to the manufacturer's protocol. Real-time PCR was performed using SYBR green PCR Master Mix (Bio-Rad), as described by the manufacturer. Amplification specificity of PCR products was confirmed by melting curve analysis and Tris-borate-EDTA agarose (2%) gel electrophoresis with ethidium bromide.

The sequences of PCR primers used in this study were as follows: glyceraldehyde 3-phosphate dehydrogenase (GAPDH): sense, 5′-CCTGGAGAAACCTGCCAAGTAT-3′; antisense, 5′-AGCCCAGGATGCCCTTTAGT-3′; PPARβ/δ: sense, 5′-CTCCTGCTCACTGACAGATG-3′; antisense, 5′-TCTCCTCCTGTGGCTGTTC-3′; COX-2: sense, 5′-TGTACAAGCAGTGGCAAAGG-3′; antisense, 5′-TAGCATCTGGACGAGGCTTT-3′. The target cDNA copy number was normalized to the cDNA copy number of GAPDH or actin amplified from the same cDNA sample. The relative RNA expression level of a gene was normalized for GAPDH mRNA and expressed relative to that in control cells treated with vehicle.

Western blot analysis

For protein isolation, astrocytes were lysed in modified radio immunoprecipitation assay buffer (50 mM Tris, pH 7.4, 1% NP-40 (Sigma Chemicals), 0.25% Na-deoxycholate, 150 mM NaCl, 1 mM EDTA, 1 mM Na3VO4, 1 mM NaF, and one tablet of protease inhibitor cocktail from Roche Molecular Biochemicals, Mannheim, Germany) per 50 mL. The protein concentration was determined by the Bradford method. Protein of 20 μg was loaded on each lane of a 10% sodium dodecyl sulphate–polyacrylamide gel. After electrophoresis, proteins were transferred onto nitrocellulose membrane. Membranes were blocked in 10% Rotiblock (Roth, Nurnberg, Germany) solution, after 1-h the membrane was incubated 1 h with PPARβ/δ antibodies (1 : 700), washed with phosphate-buffered saline with Tween 20 0.05%, and incubated for 1 h at 23°C again with a species-specific polyclonal antibodies labeled with horseradish peroxidase. For β-tubulin analysis, the blot was stripped at 23°C for 20 min with stripping buffer Restore Western Blot Stripping Buffer (Pierce, Bonn, Germany). Membranes were reprobed with antibody against β-tubulin (1 : 10 000) from Sigma Chemicals and secondary anti-mouse IgG (Dianova, Hamburg, Germany) to control for protein loading. Protein bands were visualized by enhanced chemiluminescence (Super-Signal West Pico; Pierce). Band intensity was measured using a GS-800 calibrated densitometer and Quantity One software (Bio-Rad), and normalized to the intensity of the respective bands obtained for β-tubulin.

Determination of TNFα and PGE2 by enzyme-linked immunoassay

For studies of prostaglandin E2 (PGE2) and tumor necrosis factor α (TNFα) production, astrocytes were grown in six-well plates. After the experiment, the supernatant was collected and stored at −80°C. PGE2 concentration was detected using an enzyme-linked immunoassay (Cayman Chemical, Steinheim, Germany) and TNFα concentration was detected using an enzyme-linked immunoassay (Pierce) according to the manufacturer's instructions. TNFα and PGE2 concentrations were determined by using a Thermo Multiskan EX plate reader (Thermo Scientific, Bonn, Germany) according to the instruction of the manufacturer. The results are presented as picograms per milliliter of cell culture medium.

Transcription activity of PPARβ/δ

Transcription activity and DNA-binding activity of PPARβ/δ were assayed using an enzyme-linked immunosorbent assay-based PPARs Complete Transcription Factor Assay Kit according to the manufacturer's instructions (Cayman Chemical). Nuclear proteins were extracted from astrocytes using a nuclear extraction kit (Cayman Chemical) according to the manufacturer's instructions. This assay is a non-radioactive method to trace DNA-binding activity of transcription factors in nuclear extracts. Specific double-stranded DNA sequences which contain the PPAR-responsive element are immobilized onto the bottom of wells of a 96-well plate. PPARβ/δ binds specifically to this sequence and is detected by a selective primary antibody. Clarified cell lysates are supplied as positive controls for PPARβ/δ DNA binding. Specificity of binding is demonstrated using wells with no nuclear protein added. In these wells, no binding was detected (data not shown). A secondary antibody conjugated with horseradish peroxidase is applied to provide a sensitive colorimetric signal at 450 nm. 50 μg of nuclear extract protein per sample was used for determination of PPARβ/δ activity. Binding activity was calculated as activity ratio, using the lysates from untreated astrocytes as reference value.

Experimental data analysis and statistics

All data and figures presented in text and figures as mean and SEM are from at least three independent experiments, each characterized in groups of at least three independent replicas. Data were subjected to a one-way anova with Dunnett's post hoc comparison. Statistical significance was established at p < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

Regulation of PPARβ/δ by agonists of different types of TLR

It is known that astrocytes express plasma membrane TLR, type 1, 2, 4, and 5 (Borysiewicz et al. 2009; van Noort and Bsibsi 2009). Therefore, we tested PGN (5 μg/mL), LPS (100 ng/mL), and flagellin (FGL, 5 μg/mL). These substances are agonists of TLR1/2, TLR4, and TLR5, respectively. The concentrations of agonists were chosen from previously published data, where these concentrations were shown to activate the synthesis of various proinflammatory molecules in astrocytes (Farina et al. 2005; Gurley et al. 2008; Aleshin et al. 2009). Treatment of astrocytes with each of these agonists for 4 h increased the mRNA levels of PPARβ/δ (Fig. 1a). FGL, at the tested concentration, induced lower but still significant increase of PPARβ/δ levels.

image

Figure 1. Comparison of influence of toll-like receptor (TLR) agonists on peroxisome proliferator-activated receptor β/δ (PPARβ/δ) mRNA expression (a), protein level (b, c) and activity (d). Astrocytes were stimulated for 4 h with the TLR agonists: peptidoglycan (PGN; 5 μg/mL, TLR1/2), lipopolysaccharide (LPS; 100 ng/mL, TLR4), and flagellin (FGL; 5 μg/mL, TLR5). (a) The levels of mRNA were determined by real-time RT-PCR. Values normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA level, represent mean ± SEM from four independent experiments. (b, c) After 4 h of incubation, PPAR proteins were evaluated by western blotting and normalized to the loading control β-tubulin. The example is representative for four independent experiments. (d) PPARβ/δ transcriptional activity was determined, as described in Materials and methods. Results are provided as fold-changes, relative to unstimulated astrocytes. Values represent mean ± SEM from four independent experiments, *p < 0.05 compared with the unstimulated cells, #p < 0.05 compared with astrocytes after LPS treatment.

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A similar difference between the TLR1/2, TLR4, and TLR5 agonists was noticeable at the protein expression levels of PPARβ/δ, as assessed by western blot analysis (Fig. 1b, c). LPS and PGN enhanced the protein expression two-fold, whereas FGL increased the PPARβ/δ protein expression 1.5-fold (Fig. 1b, c). Thus, the changes in mRNA level correlate with the alterations in protein level in the experimental conditions.

We assessed the DNA-binding activity of PPARβ/δ in cells stimulated by the three TLR agonists for 4 h. Nuclear proteins were extracted from astrocytes after the respective treatments. Next, DNA-binding activity was measured and normalized to the value seen with lysates of untreated astrocytes as a reference (see Materials and methods). Changes in the DNA-binding activity agree with the changes in the expression levels (Fig. 1d). All three TLR agonists tested clearly increased the activity levels of PPARβ/δ. These data indicate that the increased expression and activity levels of PPARβ/δ are shared by the entire group of TLR.

Modulation of LPS-induced PPARβ/δ expression and activity by MAPK inhibitors

We next focused on the molecular mechanism that links TLR and PPARβ/δ expression. TLR signaling is initiated at plasma membrane receptors and a set of adaptor proteins that trigger kinase cascades and activate transcription factors, is illustrated in Fig. 2a. For further experiments we concentrated on the TLR4 agonist LPS as this is the most frequently used agonist in astrocytes. LPS-stimulated activation of astrocytes results in MAPK activation and increase of inflammatory markers, primarily PGE2 and TNFα (Carpentier et al. 2005; Font-Nieves et al. 2012). MAPK inhibitors decreased the concentration of these markers (Font-Nieves et al. 2012). It is also known that MAPK inhibitors have a significant potential as modulators of brain inflammation and gliosis in neurological disorders (Borders et al. 2008; Chico et al. 2009). Therefore, we tested SB 203580, U0126, PD 98059, SP600125, which are well-established blockers of the kinase activity of p38, MEK1/2, ERK1/2, and JNK, respectively (Ozog et al. 2004; Ikeda et al. 2012; Steelman et al. 2013). The scheme in Fig. 2a presents our experimental design with agonists, inhibitors and measured parameters.

image

Figure 2. A general scheme of inhibitors used in the toll-like receptors (TLR)-mediated signaling pathways (a) and their effects on PGE2 (b) and tumor necrosis factor α (TNFα) (c) release in naïve and lipopolysaccharide (LPS)-stimulated astrocytes. (a) MAPK inhibitors, TLR agonists and polymyxin are shown in bold and italic. (b, c) Cells were pretreated for 1 h with inhibitors of the MAPK signaling axis that include p38 (SB 203580, 20 μM), MEK1/2 (U0126, 10 μM), extracellular signal-regulated kinases (ERK)1/2 (PD 98059, 20 μM), c-Jun N-terminal Kinase (JNK) (SP600125, 10 μM) or Bay 11-7085 (Bay, 5 μM), an inhibitor of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB), and subsequently exposed for 4 h with lipopolysaccharide (LPS, 100 ng/mL). PGE2 and TNFα concentrations were measured by ELISA in two supernatant samples of three cell cultures. Values represent mean ± SEM from three independent experiments performed in triplicate. *p < 0.05 compared with the unstimulated cells, #p < 0.05 compared with astrocytes after LPS treatment.

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To confirm that the tested inhibitors have anti-inflammatory effects in astrocytes under LPS treatment, we measured their influence on LPS-induced release of PGE2 (Fig. 2b) and TNFα (Fig. 2c). To determine the synthesis of PGE2, cells were preincubated for 1 h with the inhibitors of MAPK or Bay 11-7085, an inhibitor of NF-κB, and subsequently exposed to LPS for 4 h. Results in Fig. 2b show that unstimulated naive cells synthesized 156 ± 4 ng/mL PGE2 (dotted line in Fig. 2b). Application of LPS increased this synthesis 5-fold to 787 ± 83 ng/mL. All the tested substances at the concentrations used inhibited the LPS-induced PGE2 synthesis (Fig. 2b). LPS-induced responses include the release of TNFα in astrocytes (Shao et al. 2011). We detected no TNFα release in naïve cells, whereas addition of LPS increased the TNFα level to 250 ± 25 pg/mL (Fig. 2c). All the tested MAPK inhibitors significantly decreased the TNFα synthesis level (Fig. 2c). Taken together, these data demonstrate that the LPS-induced activation of astrocytes involves the release of proinflammatory markers, such as PGE2 and TNFα, and the tested inhibitors possess anti-inflammatory features at the concentrations used.

Then, we estimated the role of the MAPK inhibitors in the regulation of PPARβ/δ expression level (Fig. 3a, b) and activity level (Fig. 3d, e). We evaluated the influence of the substances in naïve cells (Fig. 3a, d) and in LPS-stimulated astrocytes (Fig. 3b, e). Firstly, in most treatments there was a clear correlation between the changes in mRNA (white columns) and protein (black column) levels (Fig. 3a, b). We found that mRNA levels are more sensitive to LPS stimulation, as compared with the protein levels, in the presence of SB 203580 (Fig. 3b). This indicates a post-transcriptional regulation.

image

Figure 3. The effects of p38, MEK, extracellular signal-regulated kinases (ERK), and c-Jun N-terminal Kinase (JNK) inhibitors alone or in combination with lipopolysaccharide (LPS) on peroxisome proliferator-activated receptor β/δ (PPARβ/δ) expression. Astrocytes were pretreated for 1 h with inhibitors of the MAPK signaling axis that include p38 (SB 203580, 20 μM), MEK1/2 (U0126, 10 μM), ERK1/2 (PD 98059, 20 μM), JNK (SP600125, 10 μM) and subsequently kept for 4 h without any additional stimulation (a, d) or with lipopolysaccharide (LPS, 100 ng/mL) (b, e). The levels of PPARβ/δ mRNA (a, b – white columns) were determined by real-time RT-PCR. Values were normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA level. PPARβ/δ proteins (a, b – black columns) were evaluated by western blotting and normalized to the loading control β-tubulin. (c) The western blot example is representative for four independent experiments. PPARβ/δ transcriptional activity (d, e) was determined at 0.5 h (black columns) and 5 h (white columns) of incubation, as described in Materials and methods. Values represent mean ± SEM from the three independent experiments. *p < 0.05, compared with the unstimulated cells, #p < 0.05 compared with astrocytes after LPS treatment.

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Secondly, there was a clear difference in sensitivity to the inhibitors in naïve and LPS-stimulated cells. Only a slight modulation of the PPARβ/δ expression at protein level was observed for the p38 inhibitor SB 203580 in naïve cells (Fig. 3a). The JNK inhibitor SP600125 decreases PPARβ/δ expression both at mRNA and protein levels in naïve cells (Fig. 3a). Stimulation of the astrocytes with LPS for 4 h leads to a 2.5-fold increase of PPARβ/δ mRNA expression level, compared to the basal conditions of non-treated cells (Fig. 3b). The p38 inhibitor SB 203580 exerted a more potent effect, elevating the PPARβ/δ mRNA expression levels up to 3.5-fold (Fig. 3b). Notably, all the other tested inhibitors decreased the effect of LPS on the PPARβ/δ expression level (Fig. 3b). Pre-treatment with inhibitors of the MEK/ERK pathway (U0126 and PD 98059) or with an inhibitor of the JNK pathway (SP600125) abolished the effect of LPS nearly to the level of non-treated cells; the ERK inhibitor U0126 was the most effective substance at the tested concentration (Fig. 3b). These changes correlated with protein levels (black columns, Fig. 3b, and western blot example in Fig. 3c). The data show that LPS-mediated PPARβ/δ expression is sensitive to all the MAPK signaling axes.

Then, we evaluated the modulation of PPARβ/δ DNA-binding activity by the tested MAPK inhibitors (Fig. 3 d, e). All the inhibitors increased the PPARβ/δ activity 2- to 3-fold after 5 h of treatment (white columns, Fig. 3d, e). It is known that 0.5 h is a sufficient time period to block the MAPK activity in astrocytes (Ikeda et al. 2012). Therefore, we checked the effects of SB 203580 and SP600125 on PPARβ/δ DNA-binding activity also at this time point (black columns, Fig. 3d). At 0.5 h, these inhibitors did not influence the activity of PPARβ/δ. Thus, there is no direct influence of the inhibitors on the PPARβ/δ binding activity, while in naïve cells the activity is sensitive to the tested substances during a long-term treatment. Meanwhile, the levels of PPARβ/δ activity in naïve cells treated with the MAPK inhibitors were comparable with levels after LPS treatment (compare values in Fig. 3d, e), but none of the tested MAPK inhibitors influenced the LPS-induced increase in PPARβ/δ DNA-binding activity (Fig. 3e).

Characteristics of LPS-induced PPARβ/δ and COX-2 expression

Stimulation of cells with LPS causes the expression of several proinflammatory genes. Among these genes, COX-2 has been thoroughly investigated as a marker gene of inflammation. Moreover, the time course of COX-2 expression level in astrocytes has been well characterized (Font-Nieves et al. 2012). Therefore, we compared the kinetics of LPS-induced COX-2 and PPARβ/δ mRNA expression (Fig. 4a, b). Expression of COX-2 in our experimental condition was induced maximally at 2 h and returned to its basal value at 6 h. The latter is consistent with previously published data (Font-Nieves et al. 2012). We detected that PPARβ/δ expression had its maximum at 4 h, and then it returned to the control level at 24 h. This shows that PPARβ/δ expression, like COX-2 expression, is regulated in the course of cellular responses upon the activation by LPS. The expression of both genes has phases of induction and resolution.

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Figure 4. Characterization of lipopolysaccharide (LPS)-mediated peroxisome proliferator-activated receptor β/δ (PPARβ/δ) and cyclooxygenase 2 (COX-2) mRNA expression. Cells were stimulated for 4 h with lipopolysaccharide (LPS, 100 ng/mL), peptidoglycan [PGN; 5 μg/mL, toll-like receptors (TLR)1/2], and flagellin (FGL; 5 μg/mL, TLR5). The levels of PPARβ/δ and COX-2 mRNA were determined by real-time RT-PCR at indicated time (a, b) or after 4 h of exposition with LPS and tested substances (c, d). (a) Kinetics of expression PPARβ/δ. (b) Kinetics of expression COX-2. (c) Modulation of expression PPARβ/δ by polymyxin (Pmx, 50 μg/mL). (d) Modulation of expression COX-2 by Bay 11-7085 (5 μM), an inhibitor of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB). (e) Modulation of expression of PPARβ/δ by Bay 11-7085 (Bay, 5 μM), an inhibitor of NF-κB. Values represent mean ± SEM from three independent experiments. *p < 0.05, compared with the unstimulated cells, #p < 0.05, compared with the LPS-stimulated cells. Abbreviation: C, unstimulated cells.

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We confirmed that the effect of LPS observed on PPARβ/δ expression is not a result of a contaminating compound that might activate some other pathway. Previously published data show that polymyxin B could be used for neutralizing the effects of LPS in vitro (Storm et al. 1977) because it directly binds to the anionic lipid A portion of LPS (Morrison and Jacobs 1976). Therefore, a cotreatment of astrocytes with polymyxin (50 μg/mL) and LPS was performed and PPARβ/δ mRNA was measured after 4 h of incubation (Fig. 4c). Polymyxin itself did not influence the PPARβ/δ expression, but effectively reversed the LPS-induced activation of PPARβ/δ expression (Fig. 4c). Thus, the effect of LPS on the PPARβ/δ expression is specifically mediated by LPS.

TLR-induced signaling pathways include activation of the transcription factor NF-κB (see the scheme in Fig. 2a). Application of Bay 11-7085, an inhibitor of NF-κB activation and phosphorylation of IκBα (Pierce et al. 1997), blocks PPARβ/δ mRNA expression induced by agonists of TLR4, TLR1/2 and TLR5 receptors (Fig. 4e). These data indicate a similarity in the mechanisms for PPARβ/δ mRNA expression regulation by TLR agonists in inflammation. Notably, LPS-induced COX-2 expression was also reversed by the NF-κB inhibitor (Fig. 4d). We therefore compared LPS-induced COX-2 and PPARβ/δ expression.

PPARβ/δ expression is regulated via the mechanism of superinduction

Convincing data show that some genes, such as COX-2 or interleukin 6, whose expression levels are activated in the course of the inflammatory response, are regulated via the superinduction mechanism (Newton et al. 1997; Hershko et al. 2004). Superinduction is a phenomenon of gene expression increase observed in the presence of cycloheximide, an inhibitor of protein synthesis (Siegel and Sisler 1963). Induction of mRNA expression means that there is a labile protein (therefore the process is sensitive to protein synthesis inhibition) that normally suppresses the gene expression via promoter activity or is responsible for the stabilization of mRNA (Edwards and Mahadevan 1992; Rypka and Vesely 2010).

In the next experiments, we added cycloheximide to the LPS-stimulated and naïve cells and determined the PPARβ/δ mRNA expression at 4 h (Fig. 5). We observed that administration of cycloheximide alone induced a 6-fold increase of the PPARβ/δ expression level (Fig. 5). The PPARβ/δ expression level after simultaneous addition of LPS with cycloheximide was one-third lower than the level after treatment with cycloheximide alone (Fig. 5, compare column 5 and 6). The application of Bay 11-7085, an inhibitor of the transcription factor NF-kB blocks the LPS-induced expression of PPARβ/δ (compare columns 1 and 2), but has no effect in case of cycloheximide application to the LPS-stimulated cells. These data suggest that the molecular mechanism of PPARβ/δ mRNA expression regulation by cycloheximide differs from the regulation via LPS/NF-kB.

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Figure 5. Superinduction of peroxisome proliferator-activated receptor β/δ (PPARβ/δ). Lipopolysaccharide (LPS) and cycloheximide influence on PPARβ/δ expression and comparison of sensitivity to nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) inhibition. Astrocytes were exposed to the protein synthesis inhibitor cycloheximide (Chx, 5 μg/mL), lipopolysaccharide (LPS, 100 ng/mL), Bay 11-7085 (Bay, 5 μM), an inhibitor of NF-κB inhibitor or combination Chx ± LPS for 4 h. The levels of PPARβ/δ mRNA were determined by real-time RT-PCR. Values normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA level, represent mean ± SEM from three independent experiments. *p < 0.05 compared with the unstimulated cells. #p < 0.05 compared with astrocytes after LPS treatment. ^p < 0.05, compare indicated bars.

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To further characterize the molecular mechanisms of this superinduction, we compared the effect of cycloheximide on PPARβ/δ and COX-2 expression (Fig. 6). Bay 11-7085 blocks the effect of cycloheximide on COX-2 expression (Fig. 6). These data are in accordance with previously published results (Newton et al. 1997; Bansal et al. 2009). Bay 11-7085 did not influence the effect of cycloheximide on PPARβ/δ expression (Fig. 6). Both genes (COX-2 and PPARβ/δ) lost their sensitivity to cycloheximide in the presence of SB 203580, an inhibitor of p38 and were insensitive to SP600125, an inhibitor of JNK (Fig. 6). Thus, superinduction for PPARβ/δ and COX-2 has common features regarding their sensitivity to MAPK inhibitors.

image

Figure 6. The difference in the mechanisms of MAPK and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) participation in superinduction of peroxisome proliferator-activated receptor β/δ (PPARβ/δ) and cyclooxygenase 2 (COX-2) genes. Astrocytes were pre-treated for 1 h with inhibitors of the MAPK p38 (SB 203580, 20 μM), c-Jun N-terminal Kinase (JNK) (SP600125, 10 μM) and Bay 11-7085 (Bay, 5 μM), an inhibitor of NF-κB inhibitor and subsequently kept for 4 h with protein synthesis inhibitor cycloheximide (Chx, 5 μg/mL) (this level of normalized mRNA expression was taken for 100%). The levels of PPARβ/δ and COX-2 mRNA were determined by real-time RT-PCR. Values normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA level, represent mean ± SEM from three independent experiments. *p < 0.05 compared with the unstimulated cells. #p < 0.05 compared with Chx treated cells. Abbreviations: Bay, Bay 11-7085; SB, SB 203580; SP, SP600125.

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Quantitative determination of rate of PPARβ/δ mRNA degradation

Recently, it became obvious that the rate of mRNA decay regulates many genes, which are involved in inflammatory responses (Anderson 2010). The rate of PPARβ/δ mRNA decay in the presence of TLR agonists has not been previously investigated. Therefore, for estimation of the PPARβ/δ mRNA stability in the presence of LPS, astrocytes were stimulated with 100 ng/mL LPS for 1 h, and then de novo LPS-induced PPARβ/δ mRNA transcription was inhibited by the addition of the transcriptional inhibitor actinomycin D (5 μg/mL; Fig. 7). Only pre-existing mRNA is detected after actinomycin D treatment and, thus, it is possible to estimate the lifetime of the mRNA. We observed that within 4 h the LPS-induced PPARβ/δ mRNA level decayed after treatment with actinomycin D; in naïve cells the PPARβ/δ mRNA level was not significantly changed and in LPS-treated cells the PPARβ/δ mRNA level continued to grow (Fig. 7). The half-life of PPARβ/δ mRNA in LPS-treated cells was 50 ± 4 min (Fig. 7). Such rate of mRNA decay allows to connect this gene to others that are induced in the course of inflammatory responses and are regulated via mRNA decay (Anderson 2010).

image

Figure 7. Stability of lipopolysaccharide (LPS)-induced peroxisome proliferator-activated receptor β/δ (PPARβ/δ) mRNA. Astrocyte cultures were pre-treated with LPS (100 ng/mL), then cells were follow incubated without (black line) or with actinomycin D (Act, 5 μg/mL) (dotted line). The levels of PPARβ/δ mRNA were determined by real-time RT-PCR. Values normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA level, represent mean ± SEM from three independent experiments. #p < 0.05 compared with actinomycin D untreated cells at 1 h of LPS-stimulated level. *p < 0.05 compared with the unstimulated cells. Abbreviation: () Control; (●) LPS; (♦) LPS with actinomycin D.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

PPARβ/δ agonists are promising substances for the treatment of various brain pathologies (Hall et al. 2008; Schnegg and Robbins 2011; Aleshin et al. 2013). Nevertheless, little is known about the regulation of PPARβ/δ expression in brain cells, which is important given that the concentration of PPARβ/δ influences the effects of corresponding agonists. The present report had the aim to fill this gap in knowledge studying how PPARβ/δ levels are regulated by TLR agonists. We found that (i) agonists of TLR1/2, TLR4, and TLR5 induced the PPARβ/δ expression and activity levels; (ii) PPARβ/δ expression and activity levels are controlled via different mechanisms, which could be differentiated by application of MAPK inhibitors; (iii) stimulation of astrocytes by the TLR4 agonist LPS enhances the PPARβ/δ mRNA degradation rate compared to unstimulated cells; (iv) comparison of PPARβ/δ expression levels with those of the proinflammatory marker COX-2 reveals that the time course of expression of both genes is bell shaped; and (v) the COX-2 and PPARβ/δ genes are up-regulated after inhibition of protein synthesis by cycloheximide. Thus, we suggest that PPARβ/δ belongs to inflammation-related genes.

The TLR/PPARβ/δ signaling pathway in glial cells

Previously, it was shown that astrocytes express plasma membrane TLR of types 1, 2, 4, and 5 (Borysiewicz et al. 2009; van Noort and Bsibsi 2009). From the known TLR signaling pathways a similarity between TLR types could be supposed (Fig. 2a), and we prove this hypothesis by showing that application of all tested TLR agonists NF-κB-dependently up-regulates the PPARβ/δ expression levels. Our data are consistent with previous results showing that PGN and LPS signal via the founding family member of the MyD88 family, which predominantly activates NF-kB-mediated responses (Borysiewicz et al. 2009; van Noort and Bsibsi 2009). For a more detailed characterization of the TLR/PPARβ/δ signaling we used LPS, which is the best studied TLR agonist in astrocytes (Borysiewicz et al. 2009; Aleshin et al. 2009). Meanwhile, it is interesting to note that the TLR5 agonist FGL induces changes in PPARβ/δ expression and activity level. Expression of TLR5 in astrocytes was already shown (Bowman et al. 2003; Gurley et al. 2008), but its role is still under-investigated. Up-regulation of PPARβ/δ expression levels by the TLR5 agonist suggests a hitherto unknown role of TLR5 in astrocytes. Further investigations will clarify the exact role of TLR5 in astrocytes. Taken together, our data add the TLR/PPARβ/δ signaling pathway to the inflammatory response system in glial cells.

The similarity in the regulation of PPARβ/δ and COX-2 expression

The assumption that the TLR/PPARβ/δ signaling pathway plays a role in the inflammatory process has led us to a comparison of PPARβ/δ with COX-2, a well-known marker of inflammation with dual features, i.e., pro- and anti-inflammatory. The comparison reveals that PPARβ/δ shares a number of regulatory mechanisms with COX-2. We have shown that LPS-induced PPARβ/δ expression reaches a maximum at 4 h and then returns to baseline level after 24 h. This time curve is similar to the LPS-induced kinetics of COX-2 expression, which was described in the literature (Font-Nieves et al. 2012) and in the present report. We further compared the pathways involved in the regulation of induction of PPARβ/δ and COX-2 expression by TLR agonists. We found that the expression was blocked by an NF-κB inhibitor.

The next common feature of COX-2 and PPARβ/δ is superinduction. This phenomenon means induction of mRNA expression levels after blockade of protein synthesis (Newton et al. 1997; Hershko et al. 2004). Superinduction has been explained by the involvement of a labile protein, which blocks the expression of the gene of interest or increases the speed of mRNA decay. For COX-2, superinduction was described in macrophages (Newton et al. 1997). In the present report, we show that superinduction of COX-2 is abolished by NF-κB and p38 MAPK inhibitors. These data are in agreement with results obtained with the A549 cell line (Bansal et al. 2009).

In the present report, we show that also PPARβ/δ expression may be regulated via superinduction. The mechanism of PPARβ/δ superinduction was similar, but not identical to that of COX-2. Superinduction of both PPARβ/δ and COX-2 was blocked by p38 MAPK inhibitor, but in contrast to COX-2, superinduction of PPARβ/δ in astrocytes was not sensitive to NF-kB inhibition. Thus, we conclude that the labile protein responsible for PPARβ/δ superinduction differs from the labile protein involved in COX-2 regulation. Further investigations will shed light on the molecular nature of such labile proteins. Our data reveal short-term regulation of PPARβ/δ expression, which stresses the importance of PPARβ/δ in cellular responses during the acute phase of inflammation.

MAPK inhibitors as regulators of PPARβ/δ activity

A major finding of our study was the MAPK inhibitor-dependent regulation of the PPARβ/δ activity and expression levels. While numerous data have demonstrated that PPAR proteins contain several protein kinase phosphorylation sites and are sensitive to MAPK inhibitors (Burns and Vanden Heuvel 2007; Burgermeister and Seger 2008), little is known about the influence of these inhibitors on the PPAR activity levels in the course of cellular inflammatory responses. It was shown that modulators of protein kinase A increase basal and ligand-induced PPARβ/δ activity (Hansen et al. 2001; Krogsdam et al. 2002). Our data add MAPK inhibitors to the list of substances that modulate the activity of PPARβ/δ. We showed that after 4 h of incubation all tested MAPK-related substances increase the PPARβ/δ activity in naïve astrocytes. This effect was also observed in LPS-stimulated astrocytes and it was NF-kB independent. Thus, we suggest a parallel involvement of MAPK- and NF-kB-dependent pathways. To our knowledge, this is the first work estimating the role of MAPK inhibitors in the regulation of PPARβ/δ isotype expression and activity levels. Further investigations should target the regulation of PPARβ/δ-mediated processed by MAPK inhibitors simultaneously with an addition of synthetic PPARβ/δ agonists.

General considerations

Our data indicate three independent pathways of regulation of PPARβ/δ expression and activity: (i) TLR/NF-kB; (ii) p38, ERK/MEK, JNK MAPKs; and (iii) a labile protein that increases the mRNA turnover. These pathways are prominent targets for separate regulation of PPARβ/δ expression and activity levels and PPARβ/δ-dependent genes. Our results also point to the future usage of specific MAPK inhibitors to manage PPARβ/δ expression in TLR-mediated signaling pathways.

Acknowledgments

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgments
  7. References

This work was supported by grants RU08/002 and RU09/030 from Bundesministerium für Bildung und Forschung (BMBF; G.R, S.A.) and by grants from the Russian Foundation for Basic Research (13-04-00833a (M.G.S., D.Ch), 12-04-32133 (D.Ch). Conflict of interest statement: None to declare.

All experiments were conducted in compliance with the ARRIVE guidelines.

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  6. Acknowledgments
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