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Activation of peroxisome proliferator-activated receptor alpha in rat spinal cord after peripheral noxious stimulation
Neuroscience Letters 369 (2004) 59–63
Activation of peroxisome proliferator-activated receptor alpha in rat spinal cord after peripheral noxious stimulation A. Benani, T. Heurtaux, P. Netter, A. Minn∗ Laboratoire de Pharmacologie, Facult´e de M´edecine, UMR 7561 CNRS-Universit´e Henri Poincar´e Nancy I, POB 184-54505 Vandœuvre-les-Nancy, France Received 6 April 2004; received in revised form 22 June 2004; accepted 18 July 2004
Abstract Following recurrent noxious stimulation, both functional modification and structural reorganization such as activation of the arachidonate cascade or axon sprouting occur in the central nervous system (CNS). It has been recently proposed that these alterations observed during chronic pain state were supported by an intensification of the lipid metabolism. In this regard, it has been shown that mRNA coding for several fatty acid metabolizing enzymes are up-regulated in the rat lumbar spinal cord in response to persistent nociception induced by a peripheral inflammation. As peroxisome proliferators-activated receptor (PPAR) could mediate such effects, we therefore investigated the activation of this transcription factor in the rat spinal cord following subcutaneous injection of complete Freund’s adjuvant (CFA) into a hind paw. In this study, we compared the DNA-binding activity of nuclear proteins extracted from healthy and inflamed rats toward a PPAR response element. Using electrophoretic mobility-shift assay (EMSA), we found that only the PPAR␣ isoform was activated in the rat spinal cord after CFA injection. This activation occurred rapidly, as early as 30 min post-CFA injection, and was persistent up to 10 h, reaching a maximum at 6 h after CFA injection. In view of the consequences of PPAR␣ activation in other tissues, these results suggest that fatty acid utilization is enhanced in the CNS during chronic pain state. Although the physiopathological relevance of PPAR␣ activation during hyperalgesia needs further investigation, we provided here a new player in the molecular modeling of pain pathways. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Nociception; Hyperalgesia; PPAR; Transcription factor
Peripheral inflammation is associated with repetitive painful stimulations which can induce dramatic changes in the CNS, leading consecutively to hyperalgesia, allodynia, and/or spontaneous pain [18]. Examination of central dysfunctions related to persistent nociception represents a first step to the discovery of new targets in pain therapy. In this context, spinal metabolism has been investigated in experimental models of inflammatory pain. An increase in glucose utilization has been described in the spinal cord of arthritic rats, suggesting a general increase of the spinal metabolic activity during Abbreviations: ACO, acyl-coenzyme A oxidase; CFA, complete Freund’s adjuvant; EMSA, electrophoretic mobility-shift assay; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR response element; RXR, 9-cis retinoic receptor ∗ Corresponding author. Tel.: +33 383 683 976; fax: +33 383 683 959. E-mail address: [email protected] (A. Minn). 0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.07.056
the development of hyperalgesia [14]. On the other hand, it has been recently proposed that the lipid metabolism was also intensified following peripheral noxious stimulation, as up-regulation of several fatty acid metabolizing enzymes occurs in the rat spinal cord during inflammation-induced hyperalgesia [1]. Together, these data suggest that the spinal metabolism is highly altered following persistent nociception. However, the physiopathological relevance of such perturbations of central metabolism during hyperalgesia remains unclear. Identification of molecular actors involved in this process could clarify it. As peroxisome proliferator-activated receptors (PPAR) has been thought to mediate these effects, we hypothesized that PPAR activation could be an early event in the central response to persistent nociception. In numerous cells, both energy and lipid metabolism homeostasis are mainly regulated by PPAR [7]. PPAR are
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transcription factors belonging to the nuclear hormone receptor superfamily. Normally PPAR are located in the nucleus of cells as heterodimers with the 9-cis retinoic receptor (RXR). Three PPAR isoforms have been identified (␣, , and ␥), which share common ligands, such as fatty acids, and the same molecular mechanism of action. The activated PPAR–RXR complex can bind to PPAR response element (PPRE) to regulate the transcription of genes. In view of the PPRE-containing target genes, PPAR may have a strong impact on fatty acid homeostasis. Here, we examined the effects of a peripheral noxious stimulation on the PPAR activity in the spinal cord. For this purpose, we compared the DNA-binding activity of PPAR by electrophoretic mobility-shift assay (EMSA) in spinal cord of healthy and inflamed rats. All experimental procedures involving rats were carried out in strict accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). The study was performed on 12 male Wistar rats (200–250 g) purchased from Charles River Laboratories (L’Arbresle, France). Peripheral inflammation was induced by subcutaneous injection (100 l) of 1 mg/ml complete Freund’s adjuvant (CFA; Sigma, SaintQuentin Fallavier, France) into a rat’s hind paw as previously described [1]. CFA inoculation produced a persistent unilateral edema and a rapid hyperalgesia as soon as 30 min after injection. Inflamed rats (n = 2 per each time point) were examined after 30 min, 2 h, 6 h, or 10 h post-CFA injection. Control rats (n = 4) did not receive any injection into the hind paw. All animals were killed under deep anesthesia, and the lumbar spinal cord (L3–L5 segment) was removed and washed in a saline buffer. Harvested samples were not pooled and nuclear proteins were extracted independently. Tissues were crushed in 2 ml of hypotonic solution A (10 mM Hepes buffer pH 7.9 containing 10 mM KCl, 1.5 mM MgCl2 , 0.5 mM DTT, and protease inhibitors) using a Dounce homogenizer with a A pestle. Homogenates were incubated on ice for 10 min. After addition of 0.05% Igepal (Sigma), samples were further homogenized and nuclei were pelleted by centrifugation at 250 × g for 10 min at 4 ◦ C. Cytosolic supernatants were removed, and nuclear pellets were resuspended in 0.5 ml of a lysis hypertonic solution B (5 mM Hepes buffer pH 7.9 containing 20% glycerol, 400 mM NaCl, 1.5 mM MgCl2 , 0.5 mM DTT, and protease inhibitors). After incubation on ice for 45 min, extracts were finally centrifuged at 18,000 × g for 30 min at 4 ◦ C to sediment nuclear debris, and supernatants containing nuclear proteins were stored at −70 ◦ C. Determination of PPAR activation was assessed by evaluation of the nuclear extract-binding activity on a DNA probe containing the PPRE consensus sequence. The probe was double-stranded oligonucleotides as follows: sense 5 -gagcttggaccaggacaaaggtcacgttcggga-3 , and antisense 3 acctggtcttgtttccagtgcaagccctccgagg-5 , which were diluted at 10 ng/l, and hybridized by incubation for 10 min at room temperature. The probe was labeled using Klenow enzyme (USB, Cleveland, USA) and [␣-33 P]CTP (Amersham, Saclay, France) according to the manufacturer’s instructions.
For purification, the probe was precipitated for 2 h at −20 ◦ C in absolute ethanol containing 0.3 M sodium acetate (pH 6), and centrifuged at 10,000 × g for 10 min at 4 ◦ C. Finally, the probe was resuspended in water and diluted to obtain a 30 cps radioactive solution. For gel shift analysis, probe (2 l) and nuclear protein extract (10 g) were incubated for 45 min at 4 ◦ C in a binding buffer (100 mM Hepes pH 7.9, 20 mM MgCl2 , 160 mM KCl, 4 mM EDTA, 4 mM DTT, 40% glycerol) containing 1 g poly-dIdC. The DNA–protein complexes were separated by 5% non-denaturing polyacrylamide gel electrophoresis in 0.5× Tris-borate buffer at 150 V for 1 h. The gel was dried and exposed onto storage phosphor screens, revealed and analyzed using a high resolution laser scanner (TyphoonTM , Amersham). For gel supershift analysis, the following antibodies were further added to the reaction mixture: anti-PPAR␣, anti-PPAR, anti-PPAR␥ (generous gifts from Professor M. Dauc¸a) or goat anti-rat IgG (cat.# R 5130, Sigma). We first evaluated the PPAR activity in spinal cord of healthy rats. No shifted signal was detected in none of the four control rats examined, suggesting that no DNA–protein complex was formed when spinal nuclear protein extracts were incubated with a labeled double-stranded oligonucleotide probe containing a PPRE motif (Fig. 1). Therefore, we concluded that PPAR which are constitutively expressed in lumbar spinal cord of healthy rats [2] displayed no basal activity. However, a shifted band corresponding to a protein binding on the DNA probe appeared when nuclear proteins from inflamed rats were used, suggesting that PPAR activation occurred in lumbar spinal cord of inflamed rats. The PPAR activation was detected as early as 30 min after the CFA injection, reached a maximum at 6 h post-inoculation, and decreased at 10 h, as estimated by the band intensity. The shifted band totally disappeared when an excess of unlabeled oligonucleotide was added as competitor in the nuclear protein-PPRE labeled probe mixture, demonstrating the binding specificity. To analyze the nature of the DNA–protein complex observed, different antibodies toward each PPAR isoforms were added separately in the mixture of nuclear extracts from spinal cord of 6 h-inflamed rats. A weak supershifted band appeared only in the presence of the anti-PPAR␣ antibody, which went with a partial decrease of the intensity of the related shifted band (Fig. 2). Neither the presence of anti-PPAR, anti-PPAR␥ nor anti-IgG antibodies was able to induce the formation of such a supershifted band. Therefore, we considered that this weak signal was specific, and we concluded that PPAR␣ was activated in lumbar spinal cord of inflamed rats, and could bind to a PPRE sequence on DNA. Altogether, these data demonstrated that PPAR␣ was rapidly activated in lumbar spinal cord after persistent nociception. This result suggests that this transcription factor could bind to PPAR response elements to regulate the expression of genes during chronic pain state. To test whether PPAR␣ activation could trigger such effect in vivo, we have investigated using RT–PCR analysis, the expression of a PPAR␣ target gene, the acyl CoA oxidase (ACO), in ani-
A. Benani et al. / Neuroscience Letters 369 (2004) 59–63
Fig. 1. Peripheral noxious stimulation induced protein binding on DNAcontaining PPRE sequence. Representative autoradiography showing the time course of PPAR activation in rat spinal cord. PPAR activation was monitored by EMSA analysis. Animals (n = 12) were randomly divided into two groups, one healthy (n = 4 animals) and one inflamed (n = 2 animals per time-point). Peripheral inflammation was induced by subcutaneous injection of CFA into a hind paw. Harvested samples isolated from rat spinal cord were not pooled and several EMSA assays were carried out to analyze each sample. Nuclear proteins extracts were mixed to a radioactive labeled DNA probe containing a PPRE sequence and subjected to non-denaturing electrophoresis. Note the formation of DNA–protein complex when using nuclear extract from inflamed spinal cord harvested after 30 min, 2 h, 6 h, and 10 h post-CFA injection. FP indicates free probe; S, PPAR-related shifted complex; c, competitor consisting in 100-fold unlabeled probe; and (−) no nuclear extract added.
mals treated with ciprofibrate, a specific PPAR␣ activator which penetrates to the CNS [16]. The RT–PCR analysis was performed as previously described [1]. Here, we have observed an increased expression of ACO in the spinal cord of ciprofibrate treated animals (Fig. 3). In addition, an increased expression of ACO was also detected in the liver but not in the adipose tissue of treated rats, which are high PPAR␣ and high PPAR␥-containing organs, respectively. This result suggests that specific activation of PPAR␣ in the rat spinal cord can induce transcriptional effects. Such regulation has been already established in rat brain in response to PPAR␣ activation induced by ciprofibrate administration [5]. Activation of PPAR␣ induced by persistent inflammation may therefore mediate the up-regulation of several fatty acid metabolizing enzymes which occurs in the rat spinal cord during inflammation-induced hyperalgesia [1], or in neurons of dorsal root ganglia after peripheral nerve injury [6]. In this study, we found that the maximal DNA-binding activity of PPAR␣ occurred later than that of other transcrip-
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Fig. 2. Peripheral noxious stimulation induced PPAR␣ activation. Representative autoradiography showing PPAR␣ activation in rat spinal cord. Nuclear extracts from spinal cord of 6 h inflamed rats were mixed to labeled DNA probe containing a PPRE sequence, with different antibodies. Note the formation of a supershifted DNA–protein complex when using an antiPPAR␣ antibody. FP indicates free probe; S, PPAR-related shifted complex; SS, PPAR-related supershifted complex; (−), no nuclear extract added; n, no antibody added; ␣, , ␥, IgG, indicates addition of anti-PPAR␣, anti-PPAR, anti-PPAR␥, or anti-IgG, respectively, into the nuclear extract- DNA probe mixture.
tion factors previously examined, as maximum PPAR␣ activation was observed at 6 h following the CFA-induced peripheral inflammation, whereas maximal binding of CREB, AP-1, NF-B, and Oct was found at 0.25 h, 0.5 h, 1 h, and 2 h after induction of peripheral inflammation, respectively [4,12]. Time-course of activation of these different transcription factors depends on cellular signaling pathways, and on their own genes inducibility [10]. For instance, following peripheral noxious stimulation, CREB activation observed in the spinal cord is caused by post-transcriptional phosphorylation, whereas AP-1 activation is rather due to rapid induction of expression of the immediate early gene c-fos [8,12]. Concerning PPAR␣ activation, its signaling mechanism in the spinal cord during hyperalgesia remains to be elucidated. Nevertheless, several mechanisms may be suggested: (i) direct activation of constitutively expressed PPAR␣ by either endogenous ligands or by phosphorylation [15], and (ii) an
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of the spinal cord, such as activation of the arachidonate cascade [17], or axon sprouting [11]. In conclusion, we reported that PPAR␣ activation occurred in the spinal cord following persistent nociception. Although the relation between PPAR␣ activation and hyperalgesia needs further investigations, we provided here a new actor in the molecular modeling of pain pathways. In an attempt to reduce pain by blocking the expression of target genes, inhibitors of PPAR␣ activation may be relevant antinociceptive drugs.
Acknowledgments This work is supported by grants from the R´egion Lorraine and from the “Communaut´e Urbaine du Grand Nancy”. We thank Professor M. Dauc¸a for the generous gift of the PPAR antibodies (Laboratoire de Biologie du Peroxysome, EA 3446, Facult´e des Sciences, Vandoeuvre-les-Nancy, France).
References
Fig. 3. Specific PPAR␣ activation induced ACO up-regulation in the spinal cord. Expression of ACO in healthy (n = 3) or ciprofibrate-treated rats (n = 3). Ciprofibrate was orally administrated at 20 mg/kg/day for 5 days. The level of ACO in different organs of rats was monitored using semi-quantitative RT–PCR. Top: representative electrophoretic profile of RT-PCR products after ACO and L27 specific amplification of mRNA from spinal cord, liver, and adipose tissue. Bottom: Densitometric analysis showing the ACO expression quantified using L27 expression as an internal control. Results are expressed in relative arbitrary units (RAU). Statistical analysis was performed to compare treated vs untreated animals using Student’s t-test after analysis of variance (n = 3; *, P < 0.05). L indicates DNA ladder; (−), healthy animals; (+) ciprofibrate-treated animals.
indirect mechanism involving a prior induction of PPAR␣ expression. Activation of transcription factors is often an early molecular event preceding long-term changes in brain function [9,19]. To date, the precise role of PPAR␣ in the nervous tissue is unknown. As PPAR␣ is constitutively expressed in astrocytes and neurons, it has been proposed that PPAR␣ regulate brain lipid homeostasis during physiological conditions [1,3,13]. During hyperalgesia, PPAR␣ activation may improve fatty acids mobilization supporting both functional modification and structural reorganization in the dorsal horn
[1] A. Benani, C. Vol, T. Heurtaux, C. Asensio, M. Dauca, F. Lapicque, P. Netter, A. Minn, Up-regulation of fatty acid metabolizing-enzymes mRNA in rat spinal cord during persistent peripheral local inflammation, Eur. J. Neurosci. 18 (2003) 1904–1914. [2] A. Benani, P. Kremarik-Bouillaud, A. Bianchi, P. Netter, A. Minn, M. Dauc¸a, Evidence for the presence of both peroxisome proliferatoractivated receptors alpha and beta in the rat spinal cord, J. Chem. Neuroanat. 25 (2003) 29–38. [3] O. Braissant, F. Foufelle, C. Scotto, M. Dauc¸a, W. Wahli, Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat, Endocrinology 137 (1996) 354–366. [4] C.F. Chan, W.Z. Sun, J.K. Lin, S.Y. Lin-Shiau, Activation of transcription factors of nuclear factor kappa B, activator protein-1 and octamer factors in hyperalgesia, Eur. J. Pharmacol. 402 (2000) 61–68. [5] T.E. Cullingford, C.T. Dolphin, H. Sato, The peroxisome proliferatoractivated receptor alpha-selective activator ciprofibrate upregulates expression of genes encoding fatty acid oxidation and ketogenesis enzymes in rat brain, Neuropharmacology 42 (2002) 724–730. [6] M. De Leon, A.A. Welcher, R.H. Nahin, Y. Liu, M.A. Ruda, E.M. Shooter, C.A. Molina, Fatty acid binding protein is induced in neurons of the dorsal root ganglia after peripheral nerve injury, J. Neurosci. Res. 44 (1996) 283–292. [7] B. Desvergne, W. Wahli, Peroxisome proliferator-activated receptors: nuclear control of metabolism, Endocr. Rev. 20 (1999) 649–688. [8] G. Draisci, M.J. Iadarola, Temporal analysis of increases in c-fos, preprodynorphin and preproenkephalin mRNAs in rat spinal cord, Brain Res. Mol. Brain. Res. 6 (1989) 31–37. [9] E. Hinoi, V.J. Balcar, N. Kuramoto, N. Nakamichi, Y. Yoneda, Nuclear transcription factors in the hippocampus, Prog. Neurobiol. 68 (2002) 145–165. [10] P. Hughes, M. Dragunow, Induction of immediate-early genes and the control of neurotransmitter-regulated gene expression within the nervous system, Pharmacol. Rev. 47 (1995) 133–178. [11] Q.P. Ma, L. Tian, Cholera toxin B subunit labeling in lamina II of spinal cord dorsal horn following chronic inflammation in rats, Neurosci. Lett. 327 (2002) 161–164. [12] D.J. Messersmith, D.J. Kim, M.J. Iadarola, Transcription factor regulation of prodynorphin gene expression following rat hind-
A. Benani et al. / Neuroscience Letters 369 (2004) 59–63 paw inflammation, Brain Res. Mol. Brain Res. 53 (1998) 260– 269. [13] S. Moreno, S. Farioli-Vecchioli, M.P. Ceru, Immunolocalization of peroxisome proliferator-activated receptors and retinoid X receptors in the adult rat CNS, Neuroscience 123 (2004) 131–145. [14] J. Schadrack, F.L. Neto, A. Ableitner, J.M. Castro-Lopes, F. Willoch, P. Bartenstein, W. Zieglgansberger, T.R. Tolle, Metabolic activity changes in the rat spinal cord during adjuvant monoarthritis, Neuroscience 94 (1999) 595–605. [15] A. Shalev, C.A. Siegrist-Kaiser, P.M. Yen, W. Wahli, A.G. Burger, W.W. Chin, C.A. Meier, The peroxisome proliferator-activated receptor alpha is a phosphoprotein: regulation by insulin, Endocrinology 137 (1996) 4499–4502.
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[16] I. Singh, O. Lazo, Peroxisomal enzyme activities in brain and liver of pups of lactating mothers treated with ciprofibrate, Neurosci. Lett. 138 (1992) 283–286. [17] C.I. Svensson, T.L. Yaksh, The spinal phospholipasecyclooxygenase-prostanoid cascade in nociceptive processing, Annu. Rev. Pharmacol. Toxicol. 42 (2002) 553–583. [18] C.J. Woolf, M. Costigan, Transcriptional and posttranslational plasticity and the generation of inflammatory pain, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 7723–7730. [19] Y. Yoneda, N. Kuramoto, T. Kitayama, E. Hinoi, Consolidation of transient ionotropic glutamate signals through nuclear transcription factors in the brain, Prog. Neurobiol. 63 (2001) 697– 719.
Activation of peroxisome proliferator-activated receptor alpha in rat spinal cord after peripheral noxious stimulation A. Benani, T. Heurtaux, P. Netter, A. Minn∗ Laboratoire de Pharmacologie, Facult´e de M´edecine, UMR 7561 CNRS-Universit´e Henri Poincar´e Nancy I, POB 184-54505 Vandœuvre-les-Nancy, France Received 6 April 2004; received in revised form 22 June 2004; accepted 18 July 2004
Abstract Following recurrent noxious stimulation, both functional modification and structural reorganization such as activation of the arachidonate cascade or axon sprouting occur in the central nervous system (CNS). It has been recently proposed that these alterations observed during chronic pain state were supported by an intensification of the lipid metabolism. In this regard, it has been shown that mRNA coding for several fatty acid metabolizing enzymes are up-regulated in the rat lumbar spinal cord in response to persistent nociception induced by a peripheral inflammation. As peroxisome proliferators-activated receptor (PPAR) could mediate such effects, we therefore investigated the activation of this transcription factor in the rat spinal cord following subcutaneous injection of complete Freund’s adjuvant (CFA) into a hind paw. In this study, we compared the DNA-binding activity of nuclear proteins extracted from healthy and inflamed rats toward a PPAR response element. Using electrophoretic mobility-shift assay (EMSA), we found that only the PPAR␣ isoform was activated in the rat spinal cord after CFA injection. This activation occurred rapidly, as early as 30 min post-CFA injection, and was persistent up to 10 h, reaching a maximum at 6 h after CFA injection. In view of the consequences of PPAR␣ activation in other tissues, these results suggest that fatty acid utilization is enhanced in the CNS during chronic pain state. Although the physiopathological relevance of PPAR␣ activation during hyperalgesia needs further investigation, we provided here a new player in the molecular modeling of pain pathways. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: Nociception; Hyperalgesia; PPAR; Transcription factor
Peripheral inflammation is associated with repetitive painful stimulations which can induce dramatic changes in the CNS, leading consecutively to hyperalgesia, allodynia, and/or spontaneous pain [18]. Examination of central dysfunctions related to persistent nociception represents a first step to the discovery of new targets in pain therapy. In this context, spinal metabolism has been investigated in experimental models of inflammatory pain. An increase in glucose utilization has been described in the spinal cord of arthritic rats, suggesting a general increase of the spinal metabolic activity during Abbreviations: ACO, acyl-coenzyme A oxidase; CFA, complete Freund’s adjuvant; EMSA, electrophoretic mobility-shift assay; PPAR, peroxisome proliferator-activated receptor; PPRE, PPAR response element; RXR, 9-cis retinoic receptor ∗ Corresponding author. Tel.: +33 383 683 976; fax: +33 383 683 959. E-mail address: [email protected] (A. Minn). 0304-3940/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2004.07.056
the development of hyperalgesia [14]. On the other hand, it has been recently proposed that the lipid metabolism was also intensified following peripheral noxious stimulation, as up-regulation of several fatty acid metabolizing enzymes occurs in the rat spinal cord during inflammation-induced hyperalgesia [1]. Together, these data suggest that the spinal metabolism is highly altered following persistent nociception. However, the physiopathological relevance of such perturbations of central metabolism during hyperalgesia remains unclear. Identification of molecular actors involved in this process could clarify it. As peroxisome proliferator-activated receptors (PPAR) has been thought to mediate these effects, we hypothesized that PPAR activation could be an early event in the central response to persistent nociception. In numerous cells, both energy and lipid metabolism homeostasis are mainly regulated by PPAR [7]. PPAR are
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transcription factors belonging to the nuclear hormone receptor superfamily. Normally PPAR are located in the nucleus of cells as heterodimers with the 9-cis retinoic receptor (RXR). Three PPAR isoforms have been identified (␣, , and ␥), which share common ligands, such as fatty acids, and the same molecular mechanism of action. The activated PPAR–RXR complex can bind to PPAR response element (PPRE) to regulate the transcription of genes. In view of the PPRE-containing target genes, PPAR may have a strong impact on fatty acid homeostasis. Here, we examined the effects of a peripheral noxious stimulation on the PPAR activity in the spinal cord. For this purpose, we compared the DNA-binding activity of PPAR by electrophoretic mobility-shift assay (EMSA) in spinal cord of healthy and inflamed rats. All experimental procedures involving rats were carried out in strict accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC). The study was performed on 12 male Wistar rats (200–250 g) purchased from Charles River Laboratories (L’Arbresle, France). Peripheral inflammation was induced by subcutaneous injection (100 l) of 1 mg/ml complete Freund’s adjuvant (CFA; Sigma, SaintQuentin Fallavier, France) into a rat’s hind paw as previously described [1]. CFA inoculation produced a persistent unilateral edema and a rapid hyperalgesia as soon as 30 min after injection. Inflamed rats (n = 2 per each time point) were examined after 30 min, 2 h, 6 h, or 10 h post-CFA injection. Control rats (n = 4) did not receive any injection into the hind paw. All animals were killed under deep anesthesia, and the lumbar spinal cord (L3–L5 segment) was removed and washed in a saline buffer. Harvested samples were not pooled and nuclear proteins were extracted independently. Tissues were crushed in 2 ml of hypotonic solution A (10 mM Hepes buffer pH 7.9 containing 10 mM KCl, 1.5 mM MgCl2 , 0.5 mM DTT, and protease inhibitors) using a Dounce homogenizer with a A pestle. Homogenates were incubated on ice for 10 min. After addition of 0.05% Igepal (Sigma), samples were further homogenized and nuclei were pelleted by centrifugation at 250 × g for 10 min at 4 ◦ C. Cytosolic supernatants were removed, and nuclear pellets were resuspended in 0.5 ml of a lysis hypertonic solution B (5 mM Hepes buffer pH 7.9 containing 20% glycerol, 400 mM NaCl, 1.5 mM MgCl2 , 0.5 mM DTT, and protease inhibitors). After incubation on ice for 45 min, extracts were finally centrifuged at 18,000 × g for 30 min at 4 ◦ C to sediment nuclear debris, and supernatants containing nuclear proteins were stored at −70 ◦ C. Determination of PPAR activation was assessed by evaluation of the nuclear extract-binding activity on a DNA probe containing the PPRE consensus sequence. The probe was double-stranded oligonucleotides as follows: sense 5 -gagcttggaccaggacaaaggtcacgttcggga-3 , and antisense 3 acctggtcttgtttccagtgcaagccctccgagg-5 , which were diluted at 10 ng/l, and hybridized by incubation for 10 min at room temperature. The probe was labeled using Klenow enzyme (USB, Cleveland, USA) and [␣-33 P]CTP (Amersham, Saclay, France) according to the manufacturer’s instructions.
For purification, the probe was precipitated for 2 h at −20 ◦ C in absolute ethanol containing 0.3 M sodium acetate (pH 6), and centrifuged at 10,000 × g for 10 min at 4 ◦ C. Finally, the probe was resuspended in water and diluted to obtain a 30 cps radioactive solution. For gel shift analysis, probe (2 l) and nuclear protein extract (10 g) were incubated for 45 min at 4 ◦ C in a binding buffer (100 mM Hepes pH 7.9, 20 mM MgCl2 , 160 mM KCl, 4 mM EDTA, 4 mM DTT, 40% glycerol) containing 1 g poly-dIdC. The DNA–protein complexes were separated by 5% non-denaturing polyacrylamide gel electrophoresis in 0.5× Tris-borate buffer at 150 V for 1 h. The gel was dried and exposed onto storage phosphor screens, revealed and analyzed using a high resolution laser scanner (TyphoonTM , Amersham). For gel supershift analysis, the following antibodies were further added to the reaction mixture: anti-PPAR␣, anti-PPAR, anti-PPAR␥ (generous gifts from Professor M. Dauc¸a) or goat anti-rat IgG (cat.# R 5130, Sigma). We first evaluated the PPAR activity in spinal cord of healthy rats. No shifted signal was detected in none of the four control rats examined, suggesting that no DNA–protein complex was formed when spinal nuclear protein extracts were incubated with a labeled double-stranded oligonucleotide probe containing a PPRE motif (Fig. 1). Therefore, we concluded that PPAR which are constitutively expressed in lumbar spinal cord of healthy rats [2] displayed no basal activity. However, a shifted band corresponding to a protein binding on the DNA probe appeared when nuclear proteins from inflamed rats were used, suggesting that PPAR activation occurred in lumbar spinal cord of inflamed rats. The PPAR activation was detected as early as 30 min after the CFA injection, reached a maximum at 6 h post-inoculation, and decreased at 10 h, as estimated by the band intensity. The shifted band totally disappeared when an excess of unlabeled oligonucleotide was added as competitor in the nuclear protein-PPRE labeled probe mixture, demonstrating the binding specificity. To analyze the nature of the DNA–protein complex observed, different antibodies toward each PPAR isoforms were added separately in the mixture of nuclear extracts from spinal cord of 6 h-inflamed rats. A weak supershifted band appeared only in the presence of the anti-PPAR␣ antibody, which went with a partial decrease of the intensity of the related shifted band (Fig. 2). Neither the presence of anti-PPAR, anti-PPAR␥ nor anti-IgG antibodies was able to induce the formation of such a supershifted band. Therefore, we considered that this weak signal was specific, and we concluded that PPAR␣ was activated in lumbar spinal cord of inflamed rats, and could bind to a PPRE sequence on DNA. Altogether, these data demonstrated that PPAR␣ was rapidly activated in lumbar spinal cord after persistent nociception. This result suggests that this transcription factor could bind to PPAR response elements to regulate the expression of genes during chronic pain state. To test whether PPAR␣ activation could trigger such effect in vivo, we have investigated using RT–PCR analysis, the expression of a PPAR␣ target gene, the acyl CoA oxidase (ACO), in ani-
A. Benani et al. / Neuroscience Letters 369 (2004) 59–63
Fig. 1. Peripheral noxious stimulation induced protein binding on DNAcontaining PPRE sequence. Representative autoradiography showing the time course of PPAR activation in rat spinal cord. PPAR activation was monitored by EMSA analysis. Animals (n = 12) were randomly divided into two groups, one healthy (n = 4 animals) and one inflamed (n = 2 animals per time-point). Peripheral inflammation was induced by subcutaneous injection of CFA into a hind paw. Harvested samples isolated from rat spinal cord were not pooled and several EMSA assays were carried out to analyze each sample. Nuclear proteins extracts were mixed to a radioactive labeled DNA probe containing a PPRE sequence and subjected to non-denaturing electrophoresis. Note the formation of DNA–protein complex when using nuclear extract from inflamed spinal cord harvested after 30 min, 2 h, 6 h, and 10 h post-CFA injection. FP indicates free probe; S, PPAR-related shifted complex; c, competitor consisting in 100-fold unlabeled probe; and (−) no nuclear extract added.
mals treated with ciprofibrate, a specific PPAR␣ activator which penetrates to the CNS [16]. The RT–PCR analysis was performed as previously described [1]. Here, we have observed an increased expression of ACO in the spinal cord of ciprofibrate treated animals (Fig. 3). In addition, an increased expression of ACO was also detected in the liver but not in the adipose tissue of treated rats, which are high PPAR␣ and high PPAR␥-containing organs, respectively. This result suggests that specific activation of PPAR␣ in the rat spinal cord can induce transcriptional effects. Such regulation has been already established in rat brain in response to PPAR␣ activation induced by ciprofibrate administration [5]. Activation of PPAR␣ induced by persistent inflammation may therefore mediate the up-regulation of several fatty acid metabolizing enzymes which occurs in the rat spinal cord during inflammation-induced hyperalgesia [1], or in neurons of dorsal root ganglia after peripheral nerve injury [6]. In this study, we found that the maximal DNA-binding activity of PPAR␣ occurred later than that of other transcrip-
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Fig. 2. Peripheral noxious stimulation induced PPAR␣ activation. Representative autoradiography showing PPAR␣ activation in rat spinal cord. Nuclear extracts from spinal cord of 6 h inflamed rats were mixed to labeled DNA probe containing a PPRE sequence, with different antibodies. Note the formation of a supershifted DNA–protein complex when using an antiPPAR␣ antibody. FP indicates free probe; S, PPAR-related shifted complex; SS, PPAR-related supershifted complex; (−), no nuclear extract added; n, no antibody added; ␣, , ␥, IgG, indicates addition of anti-PPAR␣, anti-PPAR, anti-PPAR␥, or anti-IgG, respectively, into the nuclear extract- DNA probe mixture.
tion factors previously examined, as maximum PPAR␣ activation was observed at 6 h following the CFA-induced peripheral inflammation, whereas maximal binding of CREB, AP-1, NF-B, and Oct was found at 0.25 h, 0.5 h, 1 h, and 2 h after induction of peripheral inflammation, respectively [4,12]. Time-course of activation of these different transcription factors depends on cellular signaling pathways, and on their own genes inducibility [10]. For instance, following peripheral noxious stimulation, CREB activation observed in the spinal cord is caused by post-transcriptional phosphorylation, whereas AP-1 activation is rather due to rapid induction of expression of the immediate early gene c-fos [8,12]. Concerning PPAR␣ activation, its signaling mechanism in the spinal cord during hyperalgesia remains to be elucidated. Nevertheless, several mechanisms may be suggested: (i) direct activation of constitutively expressed PPAR␣ by either endogenous ligands or by phosphorylation [15], and (ii) an
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of the spinal cord, such as activation of the arachidonate cascade [17], or axon sprouting [11]. In conclusion, we reported that PPAR␣ activation occurred in the spinal cord following persistent nociception. Although the relation between PPAR␣ activation and hyperalgesia needs further investigations, we provided here a new actor in the molecular modeling of pain pathways. In an attempt to reduce pain by blocking the expression of target genes, inhibitors of PPAR␣ activation may be relevant antinociceptive drugs.
Acknowledgments This work is supported by grants from the R´egion Lorraine and from the “Communaut´e Urbaine du Grand Nancy”. We thank Professor M. Dauc¸a for the generous gift of the PPAR antibodies (Laboratoire de Biologie du Peroxysome, EA 3446, Facult´e des Sciences, Vandoeuvre-les-Nancy, France).
References
Fig. 3. Specific PPAR␣ activation induced ACO up-regulation in the spinal cord. Expression of ACO in healthy (n = 3) or ciprofibrate-treated rats (n = 3). Ciprofibrate was orally administrated at 20 mg/kg/day for 5 days. The level of ACO in different organs of rats was monitored using semi-quantitative RT–PCR. Top: representative electrophoretic profile of RT-PCR products after ACO and L27 specific amplification of mRNA from spinal cord, liver, and adipose tissue. Bottom: Densitometric analysis showing the ACO expression quantified using L27 expression as an internal control. Results are expressed in relative arbitrary units (RAU). Statistical analysis was performed to compare treated vs untreated animals using Student’s t-test after analysis of variance (n = 3; *, P < 0.05). L indicates DNA ladder; (−), healthy animals; (+) ciprofibrate-treated animals.
indirect mechanism involving a prior induction of PPAR␣ expression. Activation of transcription factors is often an early molecular event preceding long-term changes in brain function [9,19]. To date, the precise role of PPAR␣ in the nervous tissue is unknown. As PPAR␣ is constitutively expressed in astrocytes and neurons, it has been proposed that PPAR␣ regulate brain lipid homeostasis during physiological conditions [1,3,13]. During hyperalgesia, PPAR␣ activation may improve fatty acids mobilization supporting both functional modification and structural reorganization in the dorsal horn
[1] A. Benani, C. Vol, T. Heurtaux, C. Asensio, M. Dauca, F. Lapicque, P. Netter, A. Minn, Up-regulation of fatty acid metabolizing-enzymes mRNA in rat spinal cord during persistent peripheral local inflammation, Eur. J. Neurosci. 18 (2003) 1904–1914. [2] A. Benani, P. Kremarik-Bouillaud, A. Bianchi, P. Netter, A. Minn, M. Dauc¸a, Evidence for the presence of both peroxisome proliferatoractivated receptors alpha and beta in the rat spinal cord, J. Chem. Neuroanat. 25 (2003) 29–38. [3] O. Braissant, F. Foufelle, C. Scotto, M. Dauc¸a, W. Wahli, Differential expression of peroxisome proliferator-activated receptors (PPARs): tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat, Endocrinology 137 (1996) 354–366. [4] C.F. Chan, W.Z. Sun, J.K. Lin, S.Y. Lin-Shiau, Activation of transcription factors of nuclear factor kappa B, activator protein-1 and octamer factors in hyperalgesia, Eur. J. Pharmacol. 402 (2000) 61–68. [5] T.E. Cullingford, C.T. Dolphin, H. Sato, The peroxisome proliferatoractivated receptor alpha-selective activator ciprofibrate upregulates expression of genes encoding fatty acid oxidation and ketogenesis enzymes in rat brain, Neuropharmacology 42 (2002) 724–730. [6] M. De Leon, A.A. Welcher, R.H. Nahin, Y. Liu, M.A. Ruda, E.M. Shooter, C.A. Molina, Fatty acid binding protein is induced in neurons of the dorsal root ganglia after peripheral nerve injury, J. Neurosci. Res. 44 (1996) 283–292. [7] B. Desvergne, W. Wahli, Peroxisome proliferator-activated receptors: nuclear control of metabolism, Endocr. Rev. 20 (1999) 649–688. [8] G. Draisci, M.J. Iadarola, Temporal analysis of increases in c-fos, preprodynorphin and preproenkephalin mRNAs in rat spinal cord, Brain Res. Mol. Brain. Res. 6 (1989) 31–37. [9] E. Hinoi, V.J. Balcar, N. Kuramoto, N. Nakamichi, Y. Yoneda, Nuclear transcription factors in the hippocampus, Prog. Neurobiol. 68 (2002) 145–165. [10] P. Hughes, M. Dragunow, Induction of immediate-early genes and the control of neurotransmitter-regulated gene expression within the nervous system, Pharmacol. Rev. 47 (1995) 133–178. [11] Q.P. Ma, L. Tian, Cholera toxin B subunit labeling in lamina II of spinal cord dorsal horn following chronic inflammation in rats, Neurosci. Lett. 327 (2002) 161–164. [12] D.J. Messersmith, D.J. Kim, M.J. Iadarola, Transcription factor regulation of prodynorphin gene expression following rat hind-
A. Benani et al. / Neuroscience Letters 369 (2004) 59–63 paw inflammation, Brain Res. Mol. Brain Res. 53 (1998) 260– 269. [13] S. Moreno, S. Farioli-Vecchioli, M.P. Ceru, Immunolocalization of peroxisome proliferator-activated receptors and retinoid X receptors in the adult rat CNS, Neuroscience 123 (2004) 131–145. [14] J. Schadrack, F.L. Neto, A. Ableitner, J.M. Castro-Lopes, F. Willoch, P. Bartenstein, W. Zieglgansberger, T.R. Tolle, Metabolic activity changes in the rat spinal cord during adjuvant monoarthritis, Neuroscience 94 (1999) 595–605. [15] A. Shalev, C.A. Siegrist-Kaiser, P.M. Yen, W. Wahli, A.G. Burger, W.W. Chin, C.A. Meier, The peroxisome proliferator-activated receptor alpha is a phosphoprotein: regulation by insulin, Endocrinology 137 (1996) 4499–4502.
63
[16] I. Singh, O. Lazo, Peroxisomal enzyme activities in brain and liver of pups of lactating mothers treated with ciprofibrate, Neurosci. Lett. 138 (1992) 283–286. [17] C.I. Svensson, T.L. Yaksh, The spinal phospholipasecyclooxygenase-prostanoid cascade in nociceptive processing, Annu. Rev. Pharmacol. Toxicol. 42 (2002) 553–583. [18] C.J. Woolf, M. Costigan, Transcriptional and posttranslational plasticity and the generation of inflammatory pain, Proc. Natl. Acad. Sci. U.S.A. 96 (1999) 7723–7730. [19] Y. Yoneda, N. Kuramoto, T. Kitayama, E. Hinoi, Consolidation of transient ionotropic glutamate signals through nuclear transcription factors in the brain, Prog. Neurobiol. 63 (2001) 697– 719.