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SIP30 Is Regulated by ERK in Peripheral Nerve Injury-induced Neuropathic Pain

2009; Elsevier BV; Volume: 284; Issue: 44 Linguagem: Inglês

10.1074/jbc.m109.036756

ISSN

1083-351X

Autores

Guangdun Peng, Mei Han, Yimin Du, Anning Lin, Lei Yu, Yu‐Qiu Zhang, Naihe Jing,

Tópico(s)

Nerve injury and regeneration

Resumo

ERK plays an important role in chronic neuropathic pain. However, the underlying mechanism is largely unknown. Here we show that in chronic constriction injury-treated rat spinal cords, up-regulation of SIP30 (SNAP25-interacting protein 30), which is involved in the development and maintenance of chronic constriction injury-induced neuropathic pain, correlates with ERK activation and that the up-regulation of SIP30 is suppressed by intrathecal delivery of the MEK inhibitor U0126. In PC12 cells, up-regulation of SIP30 by nerve growth factor is also dependent on ERK activation. We found that there is an ERK-responsive region in the rat sip30 promoter. Activation of ERK promotes the recruitment of the transcription factor cyclic AMP-response element-binding protein to the sip30 gene promoter. Taken together, our results provide a potential downstream target of ERK activation-mediated neuropathic pain. ERK plays an important role in chronic neuropathic pain. However, the underlying mechanism is largely unknown. Here we show that in chronic constriction injury-treated rat spinal cords, up-regulation of SIP30 (SNAP25-interacting protein 30), which is involved in the development and maintenance of chronic constriction injury-induced neuropathic pain, correlates with ERK activation and that the up-regulation of SIP30 is suppressed by intrathecal delivery of the MEK inhibitor U0126. In PC12 cells, up-regulation of SIP30 by nerve growth factor is also dependent on ERK activation. We found that there is an ERK-responsive region in the rat sip30 promoter. Activation of ERK promotes the recruitment of the transcription factor cyclic AMP-response element-binding protein to the sip30 gene promoter. Taken together, our results provide a potential downstream target of ERK activation-mediated neuropathic pain. Neuropathic pain is a chronic painful condition due to nerve injury caused by trauma, disease, or surgical accidents. This kind of chronic pain brings severe distress, which disrupts the quality of life. There is a lack of effective treatment for neuropathic pain, and the underlying mechanism is poorly understood. Recently, attention has been focused on the central sensitization of spinal dorsal horn neurons, which are responsible for the modulation of neuropathic pain transmission. Central sensitization is a consequence of increased neuron excitability (1Ji R.R. Kohno T. Moore K.A. Woolf C.J. Trends Neurosci. 2003; 26: 696-705Abstract Full Text Full Text PDF PubMed Scopus (1096) Google Scholar, 2Campbell J.N. Meyer R.A. Neuron. 2006; 52: 77-92Abstract Full Text Full Text PDF PubMed Scopus (947) Google Scholar). It has been shown that MAPKs 4The abbreviations used are: MAPKmitogen-activated protein kinaseCREBcyclic AMP-response element-binding proteinERKextracellular signal-regulated kinaseNGFnerve growth factorCCIchronic constriction injuryMEKMAPK/ERK kinaseUTRuntranslated regionChIPchromatin immunoprecipitationsiRNAsmall interfering RNAPWTpaw withdrawal thresholdPWLpaw withdrawal latencyLTPlong term potentiationDNdominant negativeCAconstitutively active. 4The abbreviations used are: MAPKmitogen-activated protein kinaseCREBcyclic AMP-response element-binding proteinERKextracellular signal-regulated kinaseNGFnerve growth factorCCIchronic constriction injuryMEKMAPK/ERK kinaseUTRuntranslated regionChIPchromatin immunoprecipitationsiRNAsmall interfering RNAPWTpaw withdrawal thresholdPWLpaw withdrawal latencyLTPlong term potentiationDNdominant negativeCAconstitutively active. play a critical role in this sensitization and are responsible for the transduction of nociceptive signals (3Lim G. Sung B. Ji R.R. Mao J. Pain. 2003; 105: 275-283Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). MAPKs are activated in the damaged neurons, and their inhibition can suppress or reverse neuropathic pain (4Zhuang Z.Y. Gerner P. Woolf C.J. Ji R.R. Pain. 2005; 114: 149-159Abstract Full Text Full Text PDF PubMed Scopus (639) Google Scholar, 5Obata K. Noguchi K. Life Sci. 2004; 74: 2643-2653Crossref PubMed Scopus (296) Google Scholar, 6Obata K. Katsura H. Mizushima T. Sakurai J. Kobayashi K. Yamanaka H. Dai Y. Fukuoka T. Noguchi K. J. Neurochem. 2007; 102: 1569-1584Crossref PubMed Scopus (53) Google Scholar, 7Ji R.R. Kawasaki Y. Zhuang Z.Y. Wen Y.R. Zhang Y.Q. Handb. Exp. Pharmacol. 2007; 177: 359-389Crossref PubMed Scopus (88) Google Scholar, 8Zhuang Z.Y. Wen Y.R. Zhang D.R. Borsello T. Bonny C. Strichartz G.R. Decosterd I. Ji R.R. J. Neurosci. 2006; 26: 3551-3560Crossref PubMed Scopus (448) Google Scholar, 9Ji R.R. Kawasaki Y. Zhuang Z.Y. Wen Y.R. Decosterd I. Neuron Glia Biol. 2006; 2: 259-269Crossref PubMed Scopus (163) Google Scholar). In various pain models, ERK, a member of the MAPK family, is specifically activated in the superficial dorsal horn neurons by noxious but not innocuous stimulation. Inhibition of ERK activation alleviates pain hypersensitivity, indicating that ERK may play an important role in neuropathic pain (10Dai Y. Iwata K. Fukuoka T. Kondo E. Tokunaga A. Yamanaka H. Tachibana T. Liu Y. Noguchi K. J. Neurosci. 2002; 22: 7737-7745Crossref PubMed Google Scholar, 11Ji R.R. Samad T.A. Jin S.X. Schmoll R. Woolf C.J. Neuron. 2002; 36: 57-68Abstract Full Text Full Text PDF PubMed Scopus (1038) Google Scholar, 12Ji R.R. Befort K. Brenner G.J. Woolf C.J. J. Neurosci. 2002; 22: 478-485Crossref PubMed Google Scholar, 13Galan A. Lopez-Garcia J.A. Cervero F. Laird J.M. Neurosci. Lett. 2002; 322: 37-40Crossref PubMed Scopus (77) Google Scholar, 14Ciruela A. Dixon A.K. Bramwell S. Gonzalez M.I. Pinnock R.D. Lee K. Br. J. 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Neurol. 2006; 199: 397-407Crossref PubMed Scopus (159) Google Scholar). mitogen-activated protein kinase cyclic AMP-response element-binding protein extracellular signal-regulated kinase nerve growth factor chronic constriction injury MAPK/ERK kinase untranslated region chromatin immunoprecipitation small interfering RNA paw withdrawal threshold paw withdrawal latency long term potentiation dominant negative constitutively active. mitogen-activated protein kinase cyclic AMP-response element-binding protein extracellular signal-regulated kinase nerve growth factor chronic constriction injury MAPK/ERK kinase untranslated region chromatin immunoprecipitation small interfering RNA paw withdrawal threshold paw withdrawal latency long term potentiation dominant negative constitutively active. ERK appears to regulate pain hypersensitivity in various aspects. It produces not only short term functional changes by post-translational processes, such as phosphorylation of membrane receptors and channels, but also long term adaptive alterations by changing gene expression (10Dai Y. Iwata K. Fukuoka T. Kondo E. Tokunaga A. Yamanaka H. Tachibana T. Liu Y. Noguchi K. J. Neurosci. 2002; 22: 7737-7745Crossref PubMed Google Scholar, 12Ji R.R. Befort K. Brenner G.J. Woolf C.J. J. Neurosci. 2002; 22: 478-485Crossref PubMed Google Scholar, 18Ji R.R. Baba H. Brenner G.J. Woolf C.J. Nat. Neurosci. 1999; 2: 1114-1119Crossref PubMed Scopus (648) Google Scholar, 19Karim F. Wang C.C. Gereau 4th, R.W. J. Neurosci. 2001; 21: 3771-3779Crossref PubMed Google Scholar). Neuropathic pain is a chronic painful situation. It is believed that the long term gene expression regulated by ERK in the spinal cord plays a central role in the development of the disease (14Ciruela A. Dixon A.K. Bramwell S. Gonzalez M.I. Pinnock R.D. Lee K. Br. J. Pharmacol. 2003; 138: 751-756Crossref PubMed Scopus (91) Google Scholar). Thus, understanding of the molecular mechanism by which ERK regulates neuropathic pain is important to understand the pathology of central sensitization. SIP30 (SNAP25-interacting protein 30) was first reported as a SNAP25-interacting protein of 30 kDa that functioned in vesicle trafficking (20Lee H.K. Safieddine S. Petralia R.S. Wenthold R.J. J. Neurochem. 2002; 81: 1338-1347Crossref PubMed Scopus (17) Google Scholar). Through a differential screening of a rat brain cDNA library, we found that sip30 was among the genes that were differentially expressed in the spinal cord CCI rats (21Wang X. Zhang Y. Kong L. Xie Z. Lin Z. Guo N. Strong J.A. Meij J.T. Zhao Z. Jing N. Yu L. Eur. J. Neurosci. 2005; 22: 1090-1096Crossref PubMed Scopus (7) Google Scholar). We further showed that sip30 mRNA and protein levels were up-regulated in the spinal cord of CCI-treated rats and that administration of SIP30 antisense oligonucleotides significantly suppressed CCI-induced pain hypersensitivity in both the onset and the continuous manifestation phases, suggesting that SIP30 may be functionally involved in the development and maintenance of chronic neuropathic pain (22Zhang Y.Q. Guo N. Peng G. Han M. Raincrow J. Chiu C. Coolen L.M. Wenthold R.J. Zhao Z.Q. Jing N.H. Yu L. Pain. 2009; Google Scholar). However, the mechanism underlying the regulation of sip30 gene expression in this pathological process is unknown. In this study, we show that SIP30 is regulated by ERK through the recruitment of cyclic AMP-response element-binding protein (CREB) to the promoter of the sip30 gene, providing a novel mechanism by which ERK regulates CCI-induced neuropathic pain. Adult male Sprague-Dawley rats (200–280 g, from the Experimental Animal Center, Fudan University) were housed two per cage with free access to water and standard rat chow, with a 12:12-h day/night cycle and at a constant room temperature of 21 °C. All experimental protocols and animal handling procedures were consistent with the Chinese national standards for laboratory animal quality and the Chinese guidelines for the care and use of laboratory animals. All efforts were made to minimize the number of animals used. A peripheral mononeuropathy was produced by loosely ligating the sciatic nerve according to a method described previously (23Bennett G.J. Xie Y.K. Pain. 1988; 33: 87-107Abstract Full Text PDF PubMed Scopus (4482) Google Scholar). Briefly, under pentobarbital sodium anesthesia (40 mg/kg, intraperitoneally), the right sciatic nerves were exposed at mid-thigh level by blunt dissection through the biceps femoris muscle. For CCI, four loosely constrictive ligatures (4-0 chromic gut suture) were tied around the nerve at a spacing of about 1 mm. The muscle and skin were closed in layers. For sham controls, the operation was performed to expose and mobilize the nerve, but the nerves were not ligated. For intrathecal drug delivery, a polyethylene-10 catheter was implanted into the subarachnoid space of the spinal cord at the lumbar enlargement, and 10 μl of the MEK (ERK kinase) inhibitor U0126 (3 μg; Sigma, dissolved in 20% DMSO) was administered once daily for 4 days with the first application 4 h before CCI. DMSO (20%, Sigma) was injected as vehicle control. Behavioral testing was performed according to the method described previously (21Wang X. Zhang Y. Kong L. Xie Z. Lin Z. Guo N. Strong J.A. Meij J.T. Zhao Z. Jing N. Yu L. Eur. J. Neurosci. 2005; 22: 1090-1096Crossref PubMed Scopus (7) Google Scholar). Briefly, the hind paw withdrawal threshold (PWT) was determined using a calibrated series of von Frey hairs (Stoelting Co., Wood Dale, IL) ranging from 1 to 26 g. Animals were placed individually into Plexiglas chambers with a customized platform that contains 1.5-mm diameter holes in a 5-mm grid of perpendicular rows throughout the entire area of the platform. The protocol used in this study was a variation of that described by Takaishi et al. (24Takaishi K. Eisele Jr., J.H. Carstens E. Pain. 1996; 66: 297-306Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar). After acclimation to the test chambers, a series of nine calibrated von Frey hairs were applied to the central region of the plantar surface of one hind paw in ascending order (1, 1.4, 2, 4, 6, 8, 10, 15, and 26 g). A particular hair was applied until buckling of the hair occurred. This was maintained for ∼2 s. The hair was applied only when the rat was stationary and standing on all four paws. A withdrawal response was considered valid only if the hind paw was completely removed from the customized platform. Each hair was applied five times at 5-s intervals. If withdrawal responses did not occur more than twice during five applications of a particular hair, the next ascending hair in the series was applied in a similar manner. Once the hind paw was withdrawn from a particular hair in three of the five consecutive applications, the rat was considered responsive to that hair. The PWT was defined as the lowest hair force in grams that produced at least three withdrawal responses in five tests. After the threshold was determined for one hind paw, the same testing procedure was repeated on the other hind paw at 5-min intervals. Thermal hyperalgesia was assessed by measuring the latency of paw withdrawal (PWL) in response to a radiant heat source. Rats were placed individually into Plexiglas chambers on an elevated glass platform, under which a radiant heat source (model 336 combination unit, IITC/Life Science Instruments, Woodland Hill, CA) was applied to the glabrous surface of the paw through the glass plate. The heat source was turned off when the rat lifted the foot, allowing the measurement of time from onset of radiant heat application to withdrawal of the hind paw. This time was defined as the hind paw withdrawal latency. The heat was maintained at a constant intensity, which produced a stable PWL of ∼10–12 s in the absence of arthritis. A 20-s cutoff was used to prevent tissue damage in the absence of a response. Both hind paws were tested for three trials at each time period with 10-min intervals between each trial. The average of the three trials was then determined. Immunostaining was performed as described previously (25Gao X. Bian W. Yang J. Tang K. Kitani H. Atsumi T. Jing N. Biochem. Biophys. Res. Commun. 2001; 284: 1098-1103Crossref PubMed Scopus (84) Google Scholar). Briefly, CCI rats were deeply anesthetized with sodium pentobarbital and transcardially perfused with 50–100 ml of warm saline with heparin sodium, followed by 400 ml of 4% paraformaldehyde. Transverse spinal cord sections (30 μm) were cut and processed for immunostaining. The primary antibodies were rabbit anti-SIP30 (1:2000 (22Zhang Y.Q. Guo N. Peng G. Han M. Raincrow J. Chiu C. Coolen L.M. Wenthold R.J. Zhao Z.Q. Jing N.H. Yu L. Pain. 2009; Google Scholar)), mouse anti-phospho-ERK (1:500, Cell Signaling Technology), mouse anti-NeuN (neuronal marker, 1:500, Chemico), GFAP (astrocytic marker, 1:1000, Sigma), or OX-42 (microglial marker, 1:500, Serotec). Secondary antibodies were fluorescein isothiocyanate-conjugated donkey anti-rabbit/mouse IgG (1:500, Jackson ImmunoResearch) and rhodamine-conjugated donkey anti-rabbit IgG (1:500, Jackson ImmunoResearch), respectively. Normal mouse and rabbit IgGs (1:500, Zymed Laboratories Inc.) were used as the negative control. The images were captured with a Leica SP2 confocal laser-scanning microscope. The specificity of immunostaining was verified by omitting the primary antibodies (supplemental Fig. S1C). The specificity of the primary antibody was examined in HEK 293-T cells in which SIP30 is not internally expressed by Western blot and immunostaining (supplemental Fig. S1, A and B). PC12 cells were cultured in Dulbecco's modified Eagle's medium containing 5% fetal bovine serum, 10% horse serum, 4 mm glutamine, 100 units/ml penicillin, and 0.2 mg/ml streptomycin under humidified conditions in 95% air and 5% CO2 at 37 °C. To examine SIP30 expression in NGF-induced PC12 cells, cells were plated onto 12-well 0.2% gelatin-coated plates for 24 h and serum-starved for at least 12 h before experiments. PC12 cells were pretreated with vehicle (0.2% DMSO) or MEK inhibitor U0126 (20 μm; Sigma, dissolved in 0.2% DMSO) for 1 h and then stimulated with NGF (50 ng/ml; Sigma) for the indicated times. Phosphorylated ERK and SIP30 protein levels were determined by immunoblotting, and sip30 mRNA expression was examined by real time PCR. For transfection, cells were plated onto gelatin-coated 6-well plates at 1 × 106 cells per well. The constitutively active form of MEK1 (CA-MEK1, S218E/S222E) or dominant negative MEK1 (DN-MEK1, K97M) was transfected into PC12 cells and maintained in low serum medium (1% fetal bovine serum/Dulbecco's modified Eagle's medium) in the presence of 50 ng/ml NGF for 24 h, then followed by immunoblotting or real time PCR analysis. Transient transfection was carried out using the FuGENE HD transfection reagents (Roche Applied Science) according to the manufacturer's instructions. Western blot analysis was performed as described previously (26Shen C. Chen Y. Liu H. Zhang K. Zhang T. Lin A. Jing N. J. Biol. Chem. 2008; 283: 17721-17730Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar). Briefly, the cells or tissues were homogenized and harvested in 100 μl of lysis buffer containing 50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 0.5% sodium deoxycholate, 0.1% SDS, 1% Nonidet P-40, 5 mm EDTA, 0.25 mm phenylmethanesulfonyl fluoride, and a mixture of protease inhibitors. The lysates were subjected to immunoblotting with the following primary antibodies: anti-phospho-ERK1/2 (1:2000, Cell Signaling Technology), anti-ERK (1:1000, Santa Cruz Biotechnology), and anti-SIP30 (22Zhang Y.Q. Guo N. Peng G. Han M. Raincrow J. Chiu C. Coolen L.M. Wenthold R.J. Zhao Z.Q. Jing N.H. Yu L. Pain. 2009; Google Scholar). For each blot, either the mouse monoclonal anti-α-tubulin (1:10,000, Sigma) or the anti-β-actin (1:7000, Sigma) was applied to serve as an internal control. The autoradiography of x-ray films and the band intensity were processed using National Institutes of Health ImageJ software. For reverse transcription, total RNA was extracted from PC12 cells by TRIzol (Invitrogen). Three micrograms of RNA was used as the template for first strand cDNA synthesis. Real time PCR was performed using an Eppendorf Realplex Cycler, as described previously (22Zhang Y.Q. Guo N. Peng G. Han M. Raincrow J. Chiu C. Coolen L.M. Wenthold R.J. Zhao Z.Q. Jing N.H. Yu L. Pain. 2009; Google Scholar). Briefly, the program was run in a final reaction volume of 20 μl containing the following reagents: 5.5 μl of PCR-grade water, 10 μl of 2× Taq mixture (Sigma), 0.5 μl of EvaGreen, 3 μl of cDNA template, and 0.5 μl of forward and reverse primer (20 μm). The amplification protocol included 3 min at 95 °C, followed by 40 cycles of 10 s of denaturation at 95 °C, 15 s of annealing at 58 °C, and 20-s extension at 72 °C. Each sample was run at least in triplicate, and Ct values were averaged from each reaction and then normalized to the gapdh value. The primers are listed in supplemental Table 1. The 2.4-kb rat SIP30-Luc plasmid (5′UTR-sip30-luc) was generated by PCR amplification of the region upstream of the sip30 translational start site, using the rat genomic DNA as a template. The region was then cloned into a pGL3 basic vector (Promega). Sequence fidelity was verified by sequencing analysis. Luciferase assay was performed as described previously (25Gao X. Bian W. Yang J. Tang K. Kitani H. Atsumi T. Jing N. Biochem. Biophys. Res. Commun. 2001; 284: 1098-1103Crossref PubMed Scopus (84) Google Scholar). Briefly, PC12 cells were plated in a 24-well plate 1 day prior to transfection. Each well was transfected with a mixture of 0.8 μg of DNA, containing 0.1 μg of the reporter plasmid and 0.02 μg of pRL-TK (internal control) for 24 h. To detect luciferase activity, 100 μl of passive lysis buffer was added to each well for 1 h. The expression of luciferase and Renilla was determined using the Dual-Luciferase Reporter Assay kit (Promega) according to the manufacturer's instructions. Values were normalized to Renilla and plotted as a percentage relative to transfection with pcDNA3. Silencing of creb or elk was achieved by transfection of siRNAs targeting rat creb mRNA or elk mRNA, respectively. Two different creb siRNAs were introduced, corresponding to different regions of creb as follows: siCREB-1 (5′-GCACUUAAGGACCUUUACUtt-3′) and siCREB-2 (5′-GGAGUCUGUGGAUAGUGUAtt-3′). The siRNA duplexes for elk were siElk-1 (5′-GGTGAGCGGCCAGAAGTTT-3′) and siElk-2 (5′-CCTCTATTCTACCTTCACAAT-3). Scrambled RNA against Thermotoga maritima (5′-CUCCGAACGUGUCACGUtt-3′) was used as a control. All siRNA duplexes were chemically synthesized by Shanghai GeneChem Co. Ltd. PC12 cells were plated in 12-well plates and transfected for 24 h with 50 nm siRNA or co-transfected with reporter plasmids using Lipofectamine 2000 (Invitrogen) for 24 h. The cells were harvested for real time PCR analysis or assessment of luciferase activity. ChIP assay was performed according to the method described by Impey et al. (27Impey S. McCorkle S.R. Cha-Molstad H. Dwyer J.M. Yochum G.S. Boss J.M. McWeeney S. Dunn J.J. Mandel G. Goodman R.H. Cell. 2004; 119: 1041-1054Abstract Full Text Full Text PDF PubMed Scopus (628) Google Scholar). Briefly, PC12 cells were serum-starved for 12–16 h and stimulated with or without NGF for 1 h. Cells were fixed for 10 min with 1% formaldehyde at room temperature, and the cross-linking was stopped by the addition of 0.125 m glycine. Cells were washed and harvested with ice-cold phosphate-buffered saline and sonicated by a Bioruptor (Diagenode). Supernatants were transferred to fresh tubes and precleared with protein A-Sepharose. Immunoprecipitation was performed overnight at 4 °C with 10 μg of anti-CREB antibody (Millipore). Control groups were incubated with normal rabbit IgG. Immune complexes were captured with 30 μl of 50% protein A-agarose slurry (Santa Cruz Biotechnology) for 3 h at 4 °C. CREB-DNA complexes were then washed and eluted for reverse cross-linking. DNA fragments were purified by ethanol precipitation and quantified by real time PCR. Primers for ChIP assay are listed in supplemental Table 2. Total and phosphorylated ERK and SIP30 levels were quantified by scanning immunoblots with an HP2840 scanner. Data were calculated as the ratio of arbitrary densitometric units of phosphorylated ERK and SIP30 normalized to values obtained for total ERK or α-tubulin immunoreactive bands from the same immunoblots. The experiments were repeated at least three times. For the quantification of immunoreactive signals, six nonadjacent sections (30 μm) through the spinal laminae IV–V were randomly selected. The numbers of SIP30-labeled cells were counted in laminae I and II and laminae IV–VI under ×20 magnification, using a computerized image analysis system (Leica Qwin 500, Germany). Spinal regions were defined according to Molander et al. (28Molander C. Xu Q. Grant G. J. Comp. Neurol. 1984; 230: 133-141Crossref PubMed Scopus (697) Google Scholar). Four to five rats were included in each group for quantification of immunohistochemistry results. All data are presented as mean ± S.D. or S.E. Parametric statistical analysis was performed using Student's t test (to compare two treatment groups) or a randomized one-way analysis of variance followed by Dunnett's test (for multiple group comparisons) to determine which groups were significantly different. Mean values were considered different if p < 0.05 (*, for p < 0.05; **, for p < 0.01). CCI causes persistent pain hypersensitivity (23Bennett G.J. Xie Y.K. Pain. 1988; 33: 87-107Abstract Full Text PDF PubMed Scopus (4482) Google Scholar, 29Moalem G. Xu K. Yu L. Neuroscience. 2004; 129: 767-777Crossref PubMed Scopus (225) Google Scholar). Previously, we found that SIP30 is functionally involved in CCI-induced neuropathic pain (22Zhang Y.Q. Guo N. Peng G. Han M. Raincrow J. Chiu C. Coolen L.M. Wenthold R.J. Zhao Z.Q. Jing N.H. Yu L. Pain. 2009; Google Scholar). To understand how SIP30 expression is regulated in neuropathic pain, we examined spinal cord SIP30 expression in response to CCI by immunostaining. Prior to CCI treatment, SIP30-positive cells were mainly detected in laminae I and II and IV–VI of the dorsal horn where sensation fibers project. After unilateral CCI treatment, SIP30-positive cells increased significantly both in laminae I and II and IV–VI neurons of the ipsilateral spinal cord but not in the contralateral side (Fig. 1A). This increase occurred as early as the 1st day after CCI treatment and was sustained up to 3 weeks (Fig. 1, A–C). There are significant differences in SIP30-positive cells between ipsilateral sides and contralateral sides on both superficial layer (laminae I and II) and deep layer (laminae IV–VI) in the dorsal horn neurons of CCI rats (Fig. 1, B and C). Double immunofluorescence showed that almost all the SIP30-positive cells on the spinal cord were neurons, because they co-expressed the neuronal marker NeuN (supplemental Fig. S2A). Consistent with these findings, immunoblotting analysis revealed that although SIP30 protein was detectable at the contralateral side of spinal cord, its expression level was significantly up-regulated at the ipsilateral side upon CCI treatment. Interestingly, ERK phosphorylation was also significantly increased at the ipsilateral side in comparison with the contralateral side of spinal cord (Fig. 2A). In the contralateral spinal cord, phosphorylated ERK and SIP30 protein expression levels were not affected by CCI. Furthermore, double immunostaining revealed that CCI-induced phosphorylated ERK was co-localized with SIP30 in the same dorsal horn neurons (Fig. 2B and supplemental Fig. S2B). The parallel increase of SIP30 protein expression and ERK phosphorylation in the same spinal dorsal horn neurons suggests that activation of ERK may be involved in the up-regulation of SIP30 expression in CCI-induced pain induction and development. To explore whether ERK activation is involved in the SIP30 up-regulation in CCI-induced neuropathic pain, we applied the MEK-specific inhibitor U0126 to CCI rats, and we examined mechanical sensitivity by von Frey hair test and thermal sensitivity by Hargreave's test, as well as SIP30 expression in the spinal cord (Fig. 3). Intrathecal administration of this inhibitor significantly suppressed CCI-induced mechanical allodynia and thermal hyperalgesia in the ipsilateral hind paw for several days. U0126, however, produced no significant change on the PWT to von Frey hairs and PWL to thermal stimulation of the contralateral hind paw (Fig. 3A), suggesting that ERK may play a critical role in CCI-induced neuropathic pain, but it does not interfere with the normal pain sensation (30Garry E.M. Delaney A. Blackburn-Munro G. Dickinson T. Moss A. Nakalembe I. Robertson D.C. Rosie R. Robberecht P. Mitchell R. Fleetwood-Walker S.M. Mol. Cell. Neurosci. 2005; 30: 523-537Crossref PubMed Scopus (38) Google Scholar, 31Obata K. Yamanaka H. Dai Y. Mizushima T. Fukuoka T. Tokunaga A. Noguchi K. Eur. J. Neurosci. 2004; 20: 2881-2895Crossref PubMed Scopus (127) Google Scholar). To examine SIP30 expression after U0126 treatment, immunostaining and Western blot analysis were performed. The MEK inhibitor significantly suppressed CCI-induced increases in SIP30-positive neurons in the laminae I and II when compared with normal CCI or DMSO controls (Fig. 3, B and C). The suppression of SIP30 expression by U0126 was not obvious in the contralateral side of the spinal dorsal horn (Fig. 3C), suggesting that the interference of SIP30 expression caused by U0126 was constrained within the CCI-treated superficial dorsal horn of the spinal cord. Under the same conditions, the ipsilateral side of the spinal cord displayed a significant increase in ERK phosphorylation 3 days after CCI (Fig. 3D, pERK, lanes 1 and 2), which was blocked by administering the MEK inhibitor U0126 (Fig. 3D, pERK, lanes 5 and 6 versus lanes 1 and 2). CCI also induced a robust up-regulation of SIP30 protein (Fig. 3D, SIP30, lanes 1 and 2), which was inhibited by the MEK inhibitor U0126 as well (Fig. 3D, SIP30, lanes 5 and 6 versus lanes 1 and 2; also see Fig. 3E). The intrathecal injection of the DMSO (control) had no detectable inhibitory effects (Fig. 3D, SIP30, lanes 3 and 4 versus lanes 5 and 6). On the contralateral side of the spinal cord, the MEK inhibitor U0126 exerted no detectable effects on SIP30 expression (Fig. 3D, SIP30, lanes 1 and 3 versus lane 5; Fig. 3E). Because the MEK inhibitor U0126 was delivered intrathecally, it should exert its effects on both ipsilateral and contralateral sides. Thus, U0126 inhibited CCI-induced up-regulation but not basal expression of SIP30. Taken together, these results show that CCI-induced up-regulation of SIP30 depends on ERK activation and that SIP30 may be a downstream target in the ERK signaling pathway that is involved in neuropathic pain. When grown in the presence of NGF, rat pheochromocytoma PC12 cells can differentiate into a sympathetic neuron-like phenotype (32Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U.S.A. 1976; 73: 2424-2428Crossref PubMed Scopus (4853) Google Scholar). 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