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Inhibition of protein tyrosine phosphatases in spinal dorsal horn attenuated inflammatory pain by repressing Src signaling
Neuropharmacology 70 (2013) 122e130
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Inhibition of protein tyrosine phosphatases in spinal dorsal horn attenuated inflammatory pain by repressing Src signaling Zhan-Wei Suo, Xian Yang, Lu Li, Yan-Ni Liu, Lei Shi, Xiao-Dong Hu* Department of Molecular Pharmacology, School of Pharmacy, Lanzhou University, Donggang West Road 199, Lanzhou, Gansu 730000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 July 2012 Received in revised form 21 November 2012 Accepted 16 January 2013
Tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) subtype glutamate receptors by Src-family protein tyrosine kinases (SFKs) plays a critical role in spinal sensitization. Besides SFKs, the tyrosine phosphorylation levels of proteins are also determined by protein tyrosine phosphatases (PTPs). However, whether PTPs are involved in spinal nociceptive processing is largely unknown. The present study found that intrathecal application of broad-spectrum PTPs inhibitors orthovanadate or Bpv (phen) generated little effects on the paw withdrawal thresholds of intact rats to Von Frey filament stimuli. Although the basal nociceptive responses didn’t require the involvement of PTPs, the mechanical allodynia evoked by intrathecal injection of NMDA was greatly attenuated by orthovanadate and Bpv (phen), suggesting that PTPs activity, once stimulated by NMDA receptors, became essential for spinal sensitization. Biochemical analysis demonstrated that PTPs functioned to activate SFKs member Src and promote Src interaction with NR2B subunit-containing NMDA receptors (NR2B receptors). As a result, PTPs inhibition largely suppressed Src-mediated NR2B phosphorylation at Tyr1472 and reduced the synaptic concentration of NR2B receptors in spinal dorsal horn of NMDA-treated rats. Importantly, intraplantar injection of Complete Freund’s Adjuvant (CFA) naturally activated spinal PTPs to initiate Src signaling, because PTPs inhibition significantly repressed Src activity, reduced Src phosphorylation of NR2B, decreased NR2B synaptic accumulation and eventually ameliorated inflammatory pain. These data indicated an important role played by spinal PTPs in inducing Src-dependent NR2B receptor hyperfunction and suggested that PTPs inhibition might represent an effective strategy for the treatment of inflammatory pain. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Protein tyrosine phosphatases Protein tyrosine kinases Inflammatory pain Orthovanadate NMDA receptors
1. Introduction Activation of Src-Family Protein Tyrosine Kinases (SFKs) in spinal dorsal horn is essential for the initiation and development of central sensitization after peripheral tissue and nerve injury (Guo et al., 2002; Liu et al., 2008; Yang et al., 2011). The active SFKs integrate multiple intracellular signaling cascades to modulate the nociceptive conveyance and neuronal excitability (Guo et al., 2004; Slack et al., 2008; Yang et al., 2011). One of the best-characterized SFKs substrates involved in pain processing is NMDA subtype glutamate receptors, especially NR2B subunit-containing NMDA receptors (NR2B receptors) (Guo et al., 2002; Liu et al., 2008). Tyrosine phosphorylation by SFKs increases the synaptic accumulation of NR2B receptors (Li et al., 2011; Prybylowski et al., 2005; Yang et al., 2011), boosts NR2B receptor-triggered signaling
* Corresponding author. Tel.: þ86 0931 3585591. E-mail address: [email protected] (X.-D. Hu). 0028-3908/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2013.01.015
transduction (Katano et al., 2011; Matsumura et al., 2010), and causes NR2B receptor-dependent neuronal plasticity and pain sensitization (Katano et al., 2011; Liu et al., 2008; Matsumura et al., 2010). A growing body of evidence has indicated that SFKs-mediated protein tyrosine phosphorylation is also subjected to the control by Protein Tyrosine Phosphatases (PTPs), which dephosphorylate the tyrosine residues on SFKs per se or on SFKs substrates (Alonso et al., 2004; Frank et al., 2004; Roskoski, 2005). A fine balance between tyrosine kinases and phosphatases is critical for many intracellular responses. To date, both receptor-like and nonreceptor PTPs have been detected in postsynaptic density (PSD), where they form macromolecular complex with signaling proteins, scaffolding proteins and substrates (Lei et al., 2002; Peng et al., 2012; Yang et al., 2011). A subpopulation of PTPs has been shown to dephosphorylate SFKs at the conserved autophosphorylation site within the catalytic motif (corresponding to Tyr418 at Src), leading to SFKs inactivation (Roskoski, 2005). Nevertheless, more PTPs dephosphorylate SFKs at their carboxyl-terminal inhibitory
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tyrosine residue (corresponding to Tyr529 at Src), leading to SFKs activation (Roskoski, 2005). Of particular importance is that PTPs activities are dynamically regulated by a wide range of extracellular stimuli, which either change PTPs expression (den Hertog et al., 2008), or modulate PTPs binding to substrates (den Hertog et al., 2008; Yang et al., 2011). The altered catalytic efficacy of PTPs produces a profound impact on synaptic transmission and plasticity, which correlates with an array of physiological and pathological processes such as learning and memory and Alzheimer disease (Lei et al., 2002; Snyder et al., 2005). Although more than 100 members of PTPs superfamily have been identified in mammalian tissues (Alonso et al., 2004), the functional significance of PTPs in pain-related spinal dorsal horn remains largely unknown. The present study found that intrathecal application of broad-spectrum PTPs inhibitors orthovanadate and Bpv (phen) alleviated the mechanical allodynia induced by spinal administration of NMDA or intradermal injection of Complete Freund’s Adjuvant (CFA). PTPs inhibition operated to repress Src activity and decrease the tyrosine phosphorylation and synaptic accumulation of NR2B receptors in NMDA- and CFA-injected rats, implicating that PTPs contributed to spinal sensitization by evoking Src-dependent NR2B receptor hyperfunction.
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2.3. Subcellular fractionation The rats were deeply anesthetized with sodium pentobarbital (30 mg/kg, i.p.) and the spinal cords were quickly removed into ice-cold artificial cerebrospinal fluid (ACSF) (119.0 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 1.3 mM MgCl2, 1.0 mM NaH2PO4, 26.0 mM NaHCO3, 11.0 mM D-glucose, bubbled with 95% O2 þ 5% CO2, pH 7.4). The dorsal quadrant of L4e5 spinal cord was dissected out and homogenized in sucrosecontaining Lysis Buffer [10.0 mM TriseHCl, pH 7.6, 320.0 mM sucrose, 5.0 mM EDTA, plus the inhibitors of proteases and phosphatases (5.0 mM EGTA, 10.0 mM NaF, 1.0 mM Na3VO4, 1.0 mM phenylmethylsulfonyl fluoride, 1.0 mg/ml each of aprotinin, chymostatin, leupeptin, antipain and pepstatin)]. The homogenates were centrifuged at 1000 g for 10 min at 4 C to remove the nuclei and large debris (P1). The supernatant was further centrifuged at 10, 000 g for 15 min to yield the crude synaptosomal fraction (P2). The P2 pellet was resuspended and incubated for 15 min in the Lysis Buffer containing 0.5% Triton X-100, and then centrifuged at 32,000 g for 20 min to obtain the synaptosomal membrane fraction (P3). The final P3 was homogenized in SDS sample buffer and boiled at 95 C for 5 min before processing. We and others have previously demonstrated that P3 fraction is enriched with postsynaptic density (PSD) marker, PSD-95 (Cao et al., 2011; Goebel-Goody et al., 2009; Smith et al., 2006; Yang et al., 2009). The P3 fraction was used instead of the classic PSD fraction because of the limited amount of the starting materials. To assay the tyrosine phosphorylation of NR2B (Guo et al., 2002), the spinal dorsal horn was homogenized in Radio-Immunoprecipitation Assay (RIPA) buffer (50.0 mM Tris$HCl, pH 8.0, 150.0 mM NaCl, 1.0 mM EDTA, 1.0% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, and proteases/phosphatases inhibitors). After centrifugation at 14,000 g for 10 min, the supernatant was harvested and the protein concentration was measured by using BCA protein assay kit (Pierce, Rockford, IL, USA). 2.4. Immunoprecipitation and co-immunoprecipitation
2. Materials and methods 2.1. Animals Male adult SpragueeDawley rats weighing 200e250 g were purchased from the Experimental Animal Center of Lanzhou University and housed on a 12 h light/dark cycle with free access to food and water. The animals were acclimatized to the testing environments for at least 3 days before any experiments were conducted. The inflammatory pain was induced by subcutaneous injection of Complete Freund’s Adjuvant (CFA; 100 ml; Sigma, St. Louis, MO) into the plantar surface of hind paws under light halothane anesthesia. Control animals received identical volumes of saline. All experimental procedures were carried out with the approval of the Animal Care and Use Committee of Lanzhou University. Every effort was made to minimize the number of animals used and their sufferings.
2.2. Drug preparation, intrathecal injection and behavioral tests To achieve the maximum inhibition of PTPs activities, sodium orthovanadate (Sigma) was activated according to the following procedure (Gordon, 1991). A stock solution of orthovanadate (100 mM) was prepared in water with pH value adjusted to 10. This yellow solution was boiled until it turned colorless. After cooling down at room temperature, the solution was re-adjusted with NaOH to pH 10 and boiled again to be colorless. After three to four cycles of activation procedure, the solution was stabilized at colorless state and then stored at 20 C. Just before use, the stocking solution was diluted to the desired working concentration. Another PTPs inhibitor, potassium Bisperoxo (1,10-phenanthroline) oxovanadate (V) [bpv(phen); Calbiochem, MA, U.S.A], and NMDA (TCI, Tokyo, Japan) were dissolved in saline. SFKs inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-D] pyrimidine (PP2; Calbiochem) was dissolved in dimethyl sulfoxide, which was diluted with saline before use. The final concentration of dimethyl sulfoxide was less than 0.5%. All the chemical reagents in 10-ml volume were intrathecally injected by direct lumbar puncture as previously reported (Mestre et al., 1994). Rats were briefly anesthetized with halothane and a 30-gauge needle attached to a 25 ml microsyringe was inserted between L5eL6 vertebrae. A sudden advancement of the needle accompanied by a slight tail flick was used as the indicator for the proper insertion of the needle tip into the subarachnoid space. The drug vehicles, saline and dimethyl sulfoxide, were used as control, which had no discernable effects on the nociceptive behavioral responses after intrathecal application. The Von Frey test was performed as previously described (Chaplan et al., 1994). In brief, the rats were placed in a cage with a wire mesh floor for at least 30 min to adapt to the environment. A series of calibrated Von Frey filaments (Stoelting, Wood Dale, IL, USA), which increased in force with approximately equal logarithmic value (d; 0.22), were applied perpendicularly to the plantar surface for 6 s. The pattern of positive and negative responses was converted to 50% threshold according to the following formula: 50% threshold (g) ¼ (10[xfþKd])/10,000, in which Xf represented the value of the final Von Frey filament used and K was the tabular value for the pattern of positive/negative responses. These behavioral tests were conducted blindly by experimenters without knowledge of the manipulations that had been performed by others on the animals.
To assay Src phosphorylation in spinal dorsal horn, the crude synaptosomal fraction (P2) was lysed in RIPA buffer for 30 min at 4 C (Yang et al., 2011). After centrifugation at 14,000 g for 10 min, the supernatant was incubated overnight with specific monoclonal antibody against the unique N-terminus of Src (Millipore, Temecula, CA, USA) at 4 C under gentle rotation. Src was immunoprecipitated by incubation with Protein A/G agarose beads for 4 h at 4 C. The beads were washed with RIPA buffer three times, and boiled in SDS sample buffer to elute proteins. For co-immunoprecipitation of proteins associated with NR2B, the P2 fraction was extracted in 50.0 mM TriseHCl, pH 9.0, 1.0% sodium deoxycholate, 10.0 mM EDTA, and proteases/phosphatases inhibitors at 37 C for 30 min (Huang et al., 2001). This extract was mixed with equal volume of Tris buffer (50.0 mM Trise HCl, pH 7.4, 150.0 mM NaCl, 1.0% Triton X-100, 0.1% SDS, proteases/phosphatases inhibitors) and then gently rocked overnight at 4 C. After centrifugation at 10,000 g for 10 min, the supernatant was harvested and incubated with anti-NR2B antibody overnight at 4 C. The immune complex was isolated by the addition of Protein A/G agarose beads as described above. Non-specific IgG was utilized for immunoprecipitation as negative control. 2.5. Western blot The equal amounts of protein samples (20 mg) were resolved on SDSPolyacrylamide Gel Electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk for 30 min at room temperature before incubation overnight with appropriate primary antibody at 4 C. After three times washes with PBST for 10 min each, the membranes were incubated with horseradish peroxidaseconjugated secondary antibody (1:10,000 dilution, goat anti-rabbit and goat antimouse; Jackson ImmunoResearch Laboratories, Baltimore, PA, USA) for 60 min at room temperature. The blots were visualized by Enhanced Chemiluminescence (Beyotime Institute of Biotechnology, Jiangsu, China). The primary antibodies used in the present study included: the rabbit polyclonal anti-NR2B antibody (1:1000), rabbit polyclonal anti-NR2A antibody (1:1000), mouse monoclonal anti-Src antibody (1:500) and rabbit polyclonal antibody against phosphorylated NR2B at Tyr 1472 (1:1000) purchased from Millipore; the rabbit polyclonal antibody against phosphorylated Src at Tyr418 (1:500) from Invitrogen (Camarillo, CA, USA); the mouse monoclonal anti-NR1 antibody (1:1000) from BD Pharmingen (San Diego, CA, USA); the rabbit polyclonal antibody against phosphorylated Src at Tyr529 (1:1000) from BioSource (Camarillo, CA, USA), and the mouse monoclonal anti-b-actin antibody (1:600) from Sigma. In some cases, the PVDF membrane was stripped with the stripping buffer (5.0 mM TriseHCl, pH 6.8, 2.0% SDS, 0.5% b-mercaptoethanol) for 30 min at 60 C and re-probed with distinct antibodies (Cao et al., 2011; Yang et al., 2011). 2.6. Motor function tests The reflexes for surface righting, placing/stepping and grasping/climbing were tested to evaluate the influence of intrathecal drug application on the motor functions (Garraway et al., 2007; Park et al., 2009). In the surface righting test, we had the rats to lie on the back and observed whether they could resume the normal upright
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position within 1.5 s. In the placing/stepping test, the dorsal surfaces of hind paws were drawn over the edge of a table to see if the rats could reflexively step onto the table top. In the grasping/climbing test, the rats were placed on a wire grid inclined at 90 . Failure to grasp and climb on the grid for 30 s indicated the motor impairment. 2.7. Statistical analysis All the data were expressed as mean SEM. For western blot analysis, the scanned digital images were quantified by NIH Image J software. The relative immunoreactive density of each NMDA receptor subunit was calculated by the ratio of its signal to b-actin signal, whereas the phosphorylation level was determined by the ratio of the phosphorylation signal to the corresponding protein signal. These ratios were used for statistical comparisons. For display purpose, these ratios were normalized to control values. The statistical significance was set at p < 0.05 by using one-way ANOVA followed by post hoc Tukey HSD test.
3. Results 3.1. PTPs inhibition in spinal dorsal horn attenuated NMDA-induced pain hypersensitivity To investigate the possible role of PTPs in spinal processing of nociceptive signals, we first injected PTPs inhibitor orthovanadate intrathecally in intact rats, and the Paw Withdrawal Thresholds (PWT) to Von Frey filament stimuli were measured. Our data demonstrated that orthovanadate had no significant effects on the mechanical thresholds under physiological conditions (Fig. 1A). At each dose tested (0.1, 0.2, 0.4 mg), the PWT values were comparable to the pre-drug ones over the entire observation period (Fig. 1A), suggesting that spinal PTPs might be inactive or dispensable for the modification of the basal nociceptive behaviors. Cumulating evidence has indicated that Ca2þ influx via NMDA receptors (NMDARs), a critical player in central sensitization, is capable of stimulating PTPs activity to induce NMDARs-dependent neuronal plasticity (Coussens et al., 2000; Paul et al., 2003). We therefore investigated whether PTPs were recruited to nociceptive processing in a NMDARs-dependent way. For this purpose, NMDA was spinally injected in intact rats. As previously reported (Sato et al., 2003), NMDA at the subconvulsive dose of 1.0 mg elicited a rapid reduction in PWT values, which lasted for at least 70 min (Fig. 1B). At 10 min post-NMDA injection, orthovanadate was intrathecally superimposed. In contrast to the basal nociceptive responses, NMDA-induced pain hypersensitivity was alleviated by orthovanadate in a dose-dependent manner (Fig. 1B). At each dose, orthovanadate had no discernable effects on motor functions. The animals displayed normal reflexes for surface righting, placing/ stepping and grasping/climbing. To confirm the role of PTPs in nociceptive modification, we intrathecally applied another potent PTPs inhibitor, Bpv(phen), finding that Bpv(phen) attenuated the reduction of PWT values evoked by NMDA (Fig. 1C) but devoid of any influence on the basal nociceptive responses and locomotor activity (data not shown). These data implicated that NMDARs activation could stimulate PTPs activity in spinal dorsal horn, which was important for pain sensitization. 3.2. PTPs inhibition repressed Src activity in spinal dorsal horn of NMDA-injected rats Previous studies have described the critical role of SFKs in NMDARs-dependent central sensitization (Liu et al., 2008; Sato et al., 2003). Given that PTPs serve as an intrinsic regulator of SFKs activity (Roskoski, 2005), we wanted to know whether PTPs inhibition ameliorated NMDA-induced mechanical allodynia via SFKs signaling. To this end, NMDA (1.0 mg) was intrathecally applied for 40 min to elicit a maximum decrease in PWT values (Fig. 1B). Immediately after behavioral tests, SFKs member Src kinase was
Fig. 1. Inhibition of protein tyrosine phosphatases (PTPs) in spinal dorsal horn alleviated the mechanical allodynia induced by intrathecal (i.t.) injection of NMDA (1.0 mg) in rats. (A) PTPs inhibitor Orthovanadate (Ortho; 0.1 mg, 0.2 mg, 0.4 mg) didn’t affect the basal nociceptive responses of intact rats to Von Frey filament stimuli. The alteration in Paw Withdrawal Threshold (PWT) was plotted against time. (B) Orthovanadate dosedependently attenuated the reduction of PWT values induced by NMDA. The upward and downward arrows indicated the time points when distinct drugs were spinally given. (C) Another PTPs inhibitor Bpv(phen), when intrathecally applied at 10 min post-NMDA injection, also ameliorated the pain hypersensitivity in a dose-dependent manner. *p < 0.05 when compared with saline-injected control rats. #p < 0.05 when compared with NMDA-injected, saline-treated rats. n ¼ 6 rats in each group.
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immunoprecipitated from the crude synaptosomal fraction of spinal dorsal horn. Src phosphorylation was probed with a phosphotyrosine antibody against its inhibitory tyrosine residue 529 (Tyr529), a well-characterized dephosphorylation site by PTPs (Roskoski, 2005). Compared with saline vehicle, NMDA treatment reduced Tyr529 phosphorylation by 44.8 4.2% (p < 0.05, n ¼ 6) (Fig. 2A), indicating the activation of Src. When orthovanadate (0.4 mg) was spinally applied for 30 min to inhibit the mechanical allodynia in NMDA-injected rats (Fig. 1B), Tyr529 phosphorylation level was simultaneously elevated, which became indistinguishable from that of saline-injected control rats (Fig. 2A). Because phosphorylated Tyr529 negatively controls Src activity by blunting Tyr418 autophosphorylation within the activation loop (Salter and Kalia, 2004), we further performed immunoblotting with a phosphotyrosine antibody against Tyr418 to consolidate the change in Src enzymatic activity. Our data revealed that NMDA injection induced a significant increase in Tyr418 phosphorylation relative to saline vehicle (Fig. 2B), a result consistent with the corresponding decrease in Tyr529 phosphorylation (Fig. 2A).
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Treatment of NMDA-injected rats with orthovanadate, however, repressed Tyr418 phosphorylation level from 188.1 29.9% of vehicle control to 116.8 23.6% of vehicle control (p < 0.05, n ¼ 6) (Fig. 2B). Behavioral tests demonstrated that direct application of SFKs inhibitor PP2 (10.0 mg) noticeably ameliorated NMDA-induced allodynia (Fig. 2C). More importantly, SFKs inhibition by PP2 completely occluded the analgesic action of orthovanadate (0.4 mg) (Fig. 2C), suggesting that Src was a key target for PTPs to regulate NMDARs-dependent spinal sensitization. 3.3. PTPs inhibition suppressed the hyperfunction of NR2B receptors in spinal dorsal horn of NMDA-injected rats The molecular mechanisms for the active Src to exacerbate nociceptive conveyance involve the tyrosine phosphorylation and synaptic retention of NR2B subunit-containing NMDA receptors (NR2B receptors) in spinal dorsal horn (Li et al., 2011; Liu et al., 2008). Here we found that NMDA treatment, in addition to increase Src activity (Fig. 2A and B), also promoted the molecular
Fig. 2. Intrathecal injection of NMDA (1.0 mg) in rats activated Src in PTPs-dependent manner. Src was immunoprecipitated from the crude synaptosomal fraction of spinal dorsal horn at 40 min after NMDA injection, followed by immunoblotting with phosphotyrosine antibodies. (A) PTPs inhibitor orthovanadate (Ortho; 0.4 mg), when intrathecally superimposed at 10 min after NMDA injection, eliminated the decrease of Src phosphorylation at Tyr529 (Src-Y529p) induced by NMDA. The immunoreactivity of phosphorylated Src was shown (up). The graph summarized the percentage change in Src-Y529p (down). (B) Orthovanadate (0.4 mg) also abolished NMDA-induced increase in Src phosphorylation at Tyr418 (Src-Y418p). (C) SFKs inhibitor PP2 (10 mg), when intrathecally superimposed at 10 min post-NMDA injection, attenuated the reduction of PWT values, which also occluded the analgesic action of the subsequently applied orthovanadate (0.4 mg). The upward and downward arrows indicated the time points when distinct reagents were spinally given. *p < 0.05 when compared with saline-injected control rats. #p < 0.05 when compared with NMDA-injected rats. n ¼ 6 in each group.
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interaction of Src with NR2B. As shown in Fig. 3A, the specific antibody against NR2B subunit was able to co-immunoprecipitate Src from the crude synaptosomal fraction of spinal dorsal horn in intact rats, whereas non-specific IgG failed to do so. When NMDA (1.0 mg) was intrathecally applied for 40 min to evoke pronounced allodynia (Fig. 1B), the contents of Src pulled down by anti-NR2B antibody were significantly higher than those in vehicle group (Fig. 3A), suggesting that NMDARs activation simultaneously positioned Src at the proximity of its substrates. Immunoblotting analysis demonstrated that NR2B phosphorylation at Tyr1472, a catalytic site by Src, was enhanced by 113.9 40.3% in NMDAinjected rats (p < 0.05 vs. saline, n ¼ 6) (Fig. 3B). Because one of the most important functions of Tyr1472 phosphorylation is to prevent NR2B endocytosis (Li et al., 2011), we further assayed the synaptic expression of NR2B receptors, finding that NMDA application specifically increased the content of NR2B and NR1 subunits at synaptosomal membrane fraction, with that of NR2A subunit unaltered (Fig. 3C). Interestingly, NMDA-induced Src targeting to NR2B depended on PTPs activities. Intrathecal application of orthovanadate (0.4 mg) for 30 min reduced the amount of Src precipitated by anti-NR2B antibody from NMDA-injected rats (Fig. 3A). Such a dissociation of NR2B/Src complex following PTPs inhibition was concurrent with the repression of NMDA-induced NR2B tyrosine phosphorylation (Fig. 3B). Meanwhile, the increase in the synaptic concentration of NR2B and NR1 subunit
evoked by NMDA was also abolished by orthovanadate (Fig. 3C). These data implicated that active PTPs following NMDARs stimulation facilitated NR2B interaction with Src, leading to NR2B receptor hyperfunction in spinal dorsal horn. 3.4. PTPs inhibition in spinal dorsal horn alleviated inflammatory pain Convincing evidence has established that peripheral tissue injury naturally stimulates spinal NMDARs to trigger multiple signaling pathways essential for inflammatory pain. Since PTPs acted downstream of NMDARs and contributed to spinal sensitization, we next explored the possible role of PTPs in persistent inflammatory pain. Our data demonstrated that intraplantar injection of Complete Freund’s Adjuvant (CFA) induced a rapid decrease in PWT values of the affected hind paws (Fig. 4A). Pretreatment with orthovanadate for 15 min, however, dose-dependently delayed the onset and attenuated the magnitude of reduction in the mechanical thresholds (Fig. 4A). In addition to the blocking effect on the induction of inflammatory pain, orthovanadate application one day after CFA injection also alleviated the established allodynia in a dosedependent manner. As shown in Fig. 4B, post-treatment with a low dose of orthovanadate (0.1 mg) produced minimal effect on the PWT values of inflamed rats. Increasing the dose of orthovanadate to 0.2 mg transiently elevated the PWT values at 20 min and 30 min,
Fig. 3. PTPs inhibitor orthovanadate repressed NMDA-induced NR2B receptor hyperfunction in spinal dorsal horn of rats. (A) Intrathecal application of NMDA (1.0 mg) for 40 min enhanced the amount of Src co-immunoprecipitated (Co-IP) by anti-NR2B antibody from the crude synaptosomal fraction of spinal dorsal horn. Note that orthovanadate (Ortho; 0.4 mg), when spinally superimposed at 10 min post-NMDA injection, reversed NR2B interaction with Src. Non-specific IgG was utilized as negative control for coimmunoprecipitation. IB: Immunoblotting. (B) Intrathecal NMDA application enhanced the phosphorylation of NR2B subunit at Tyr1472 (NR2B-Y1472p) in spinal dorsal horn, which could be abolished by orthovanadate. The membrane was stripped and re-probed with anti-NR2B antibody. The graph summarized the percentage change in NR2B-Y1472p level. (C) NMDA injection increased the content of NMDA receptor NR1 and NR2B subunit at the synaptosomal membrane fraction, which, however, could be abolished by orthovanadate. The immunoreactivity of each NMDARs subunit was shown (left). Equal protein loadings were indicated by b-actin intensity. The graph summarized the percentage changes in NMDARs subunit immunoreactivity (right). *p < 0.05 compared with saline-injected control rats. #p < 0.05 compared with NMDA-injected rats. n ¼ 6 experiments.
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which then returned to the levels comparable to vehicle-treated inflamed rats (Fig. 4B). When the dose of orthovanadate was further increased to 0.4 mg, a long-lasting attenuation of inflammatory pain was observed (Fig. 4B). At 60 min after the drug treatment, the PWT values were still higher than those of saline-treated inflamed rats (orthovanadate vs. saline: 7.35 1.39 g vs. 2.69 0.19 g, p < 0.05, n ¼ 6 in each group). A similar alleviation of inflammatory pain was also observed when PTPs inhibitor Bpv (phen) was spinally given one day after CFA injection (Fig. 4C). These data suggested that PTPs activity in spinal dorsal horn was involved in both the induction and maintenance of inflammatory pain. 3.5. PTPs inhibition repressed Src hyperactivity in spinal dorsal horn of rats with inflammatory pain Next, we examined whether PTPs inhibition alleviated inflammatory pain by modulating spinal Src activity. One day after CFA injection, Src was immunoprecipitated from the crude synaptosomal fractions of spinal dorsal horn. Western blot illustrated that peripheral inflammation reduced Src-Tyr529 phosphorylation level to 47.4 4.9% of vehicle control (p < 0.05, n ¼ 6) (Fig. 5A). This result was supported by a concomitant increase in Tyr418 phosphorylation, which was 197.6 18.9% of vehicle control (p < 0.05, n ¼ 6) (Fig. 5B). Notably, CFA-induced Src activation required PTPs activity. When orthovanadate (0.4 mg) was intrathecally applied in inflamed rats for 30 min, Src phosphorylation at Tyr529 was elevated to 75.0 8.7% of vehicle control (p > 0.05, n ¼ 6) (Fig. 5A), while Tyr418 phosphorylation was repressed to 97.8 11.9% of vehicle control (p > 0.05, n ¼ 6) (Fig. 5B). These data suggested that the analgesic action of orthovanadate was closely correlated with the suppression of Src activity. 3.6. PTPs inhibition reversed NMDA receptor hyperfunction in spinal dorsal horn induced by peripheral inflammation
Fig. 4. PTPs inhibition in spinal dorsal horn blocked the induction and maintenance of inflammatory pain induced by intradermal (i.d.) injection of Complete Freund’s Adjuvant (CFA) in rats. (A) PTPs inhibitor Orthovanadate (Ortho) was intrathecally (i.t.) given 15 min prior to CFA injection. The changes in PWT values were plotted versus time. The upward and downward arrows indicated the time points when intradermal and intrathecal injection were performed, respectively. (B) Orthovanadate (0.1 mg, 0.2 mg, 0.4 mg) was spinally given one day after CFA injection. The time-dependent changes in PWT values were plotted. (C) Intrathecal administration of PTPs inhibitor Bpv (phen) one day after CFA injection attenuated the inflammatory pain in a dosedependent manner. *p < 0.05 when compared with saline-injected control rats. # p < 0.05 when compared with saline-treated inflamed rats. n ¼ 6 rats in each group.
To further address whether Src inhibition by orthovanadate led to the reversal of NMDARs hyperfunction in inflamed rats, we analyzed the tyrosine phosphorylation of NR2B in spinal dorsal horn one day after CFA injection. Our data illustrated that peripheral inflammation significantly increased Tyr1472 phosphorylation to 176.9 26.3% of saline control (p < 0.05, n ¼ 6) (Fig. 6A). Nevertheless, intrathecal application of orthovanadate (0.4 mg) for 30 min depressed the NR2B tyrosine phosphorylation level to 105.0 14.7% of saline control (p > 0.05, n ¼ 6) (Fig. 6A). NR2B dephosphorylation by orthovanadate might result from not only Src inhibition (Fig. 5A and B), but also Src dissociation with NR2B. As shown in Fig. 6B, anti-NR2B antibody precipitated much more Src from the crude synaptosomal fraction of inflamed rats than control ones, which, however, could be completely ruled out by orthovanadate. Consistent with NR2B tyrosine dephosphorylation, orthovanadate also abolished the CFA-induced increase in the immunoreactive intensities of NR2B and NR1 subunits at synaptosomal membrane fraction (Fig. 6C). Neither CFA nor orthovanadate altered the synaptic content of NR2A subunit (Fig. 6C). These data suggested that PTPs inhibition ameliorated inflammatory pain by suppressing Src-dependent NR2B receptor hyperfunction in spinal dorsal horn. 4. Discussion Protein tyrosine phosphorylation serves as an important mechanism for the regulation of nociceptive transmission and integration (Pelkey et al., 2002; Salter and Kalia, 2004). Compared with Src-family protein tyrosine kinases (SFKs), protein tyrosine phosphatases (PTPs) have recently been implicated to play equal or
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Fig. 5. Intrathecal application of PTPs inhibitor orthovanadate (Ortho; 0.4 mg) for 30 min repressed Src activity in spinal dorsal horn of rats one day after CFA injection. Src was immunoprecipitated from the crude synaptosomal fraction of spinal dorsal horn. (A) The immunoreactivity of phosphorylated Src at Tyr529 (Src-Y529p) was shown (up). The graph summarized the percentage change in Src phosphorylation (down). *p < 0.05 when compared with saline-injected control rats. #p < 0.05 when compared with CFA-injected rats (n ¼ 6 experiments). (B) CFA injection increased Src phosphorylation at Tyr418 (Src-Y418p), which could be abolished by orthovanadate.
even dominant roles in setting protein tyrosine phosphorylation levels (Alonso et al., 2004). However, whether and how PTPs modulate spinal processing of nociceptive information is largely unknown as yet. The present study demonstrated that PTPs activation was critical for NMDA- and CFA-induced pain hypersensitivity. The active PTPs operated to initiate Src signaling in spinal dorsal horn, leading to NR2B tyrosine phosphorylation and synaptic accumulation. Consequently, PTPs inhibition effectively alleviated spinal sensitization by reversing Src and NR2B hyperactivities in NMDA- and CFA-injected rats. It is well known that Src kinase contains two critical regulatory tyrosine residues, one within its activation loop (Tyr418) and the other at the extreme carboxyl-terminal tail (Tyr529) (Salter and
Kalia, 2004). Under normal conditions, Tyr529 is phosphorylated by C-terminal Src kinase (CSK) (Chong et al., 2005; Roskoski, 2005; Salter and Kalia, 2004). The Tyr529-phosphorylated C-terminal tail interacts intramolecularly with SH2 (Src Homology 2) domain to keep Src in a closed and inactive conformation. When Tyr529 is dephosphorylated by PTPs, the closed conformation is opened, allowing for Tyr418 autophosphorylation and Src activation. The active Src can then be dephosphorylated by specific PTPs that target Tyr418, restricting Src from excessive activation. The present study showed that intrathecal NMDA and intraplantar CFA injection led to a decrease of Tyr529 phosphorylation and a concomitant increase of Tyr418 phosphorylation in a PTPs-dependent manner. Possibly, NMDA and CFA preferentially stimulated PTPs targeting Tyr529 dephosphorylation. Alternatively, the endogenous PTPs responsible for Tyr529 dephosphorylation became to dominate over those for Tyr418 dephosphorylation after NMDA and CFA injection. As a result, the net role of active PTPs seemed to be “excitatory”, which catalyzed Tyr529 dephosphorylation to cause Src hyperactivity and pain sensitization. The functional imbalance between these two populations of PTPs might explain why orthovanadate and Bpv (phen) selectively attenuated NMDA- and CFA-induced mechanical allodynia without any influence on the basal nociceptive behaviors. In agreement with these data, recent studies have indicated that a subpopulation of “excitatory” PTPs actively governs SFKsmediated intracellular responses in a myriad of processes such as neuronal plasticity (Lei et al., 2002; Peng et al., 2012). NMDARs hyperfunction has been widely described as a key contributor to pathological pain. The hyperactive NMDARs might result from the receptor phosphorylation, which augments the receptor channel conductance and synaptic accumulation (Li et al., 2011; Yang et al., 2011, 2009). One of the best-characterized phosphorylation events after peripheral lesions occurs on NR2B subunit at Tyr1472. Genetic knockdown of NR2B or knock-in mutation of Tyr1472 potently alleviates pathological pain symptoms (Katano et al., 2011; Matsumura et al., 2010; Tan et al., 2005). As a critical determinant of protein tyrosine phosphorylation, PTPs have been shown to exert a negative control over NR2B subunit in hippocampal and striatal neurons, because inhibition of PTPs activity significantly increases NR2B tyrosine phosphorylation levels in these brain regions (Chen et al., 2003; Goebel et al., 2005; Hallett et al., 2006; Wang and Salter, 1994). However, the present study in vivo illustrated that intrathecal application of PTPs inhibitor repressed, rather than boosted, the tyrosine phosphorylation of NR2B in NMDA- and CFA-injected rats, suggesting a stimulatory effect of PTPs on NR2B phosphorylation in spinal dorsal horn. This tissue-specific action might be indirect, possibly through downstream Src kinase. We found that spinal PTPs not only increased Src activity but also promoted Src association with NR2B. Because Src activation is always accompanied by the exposure of its substratebinding SH2 domain (Salter and Kalia, 2004), it was likely that the free SH2 domain physically interacted with postsynaptic scaffolding proteins such as PSD-95, through which Src gained access to NR2B and exerted full catalytic action (Kalia and Salter, 2003). Tyr1472 phosphorylation has been illustrated to disrupt NR2B interaction with clathrin adaptor protein AP-2, which prevents the internalization of NR2B receptors from postsynaptic membrane (Prybylowski et al., 2005). Our biochemical analysis also found that PTPs-dependent Tyr1472 phosphorylation was closely correlated with the synaptic accumulation of NR2B receptors in spinal dorsal horn of NMDA- and CFA-injected rats. No detectable change in NR2A synaptic content supported the notion that tyrosine phosphorylation is dispensable for the synaptic trafficking of NR2A subunit (Prybylowski et al., 2005). The selective removal of NR2B receptors from excitatory synapses by PTPs inhibition presumably attenuated NR2B receptors-mediated nociceptive transmission and
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Fig. 6. PTPs inhibitor orthovanadate reversed peripheral inflammation-induced NR2B receptor hyperfunction in spinal dorsal horn of rats. (A) The phosphorylation level of NR2B subunit at Tyr1472 (NR2B-Y1472p) significantly increased one day after CFA injection, which could be abolished by intrathecal application of orthovanadate (Ortho; 0.4 mg) for 30 min. The membrane was stripped and re-probed with anti-NR2B antibody. The graph summarized the percentage change in NR2B-Y1472p level. *p < 0.05 compared with salineinjected control rats; #p < 0.05 compared with CFA-injected rats; n ¼ 6 experiments. (B) Src was co-immunoprecipitated (Co-IP) by anti-NR2B antibody from the crude synaptosomal fraction of spinal dorsal horn one day after CFA injection. Note that treatment with orthovanadate reduced the content of precipitated Src in inflamed rats. Non-specific IgG was utilized as negative control for co-immunoprecipitation. IB: Immunoblotting. (C) The concentrations of NMDA receptor NR1 and NR2B subunits at the synaptosomal membrane fraction significantly increased one day after CFA injection, which could be abolished by orthovanadate. The immunoreactivity of each NMDARs subunit was shown (left). Equal protein loadings were indicated by b-actin intensity. The graph summarized the percentage change in NMDARs subunit immunoreactivity (right).
intracellular signaling cascades, eventually leading to the alleviation of pain hypersensitivity. Taken together, the present study demonstrated that pharmacological inhibition of PTPs ameliorated NMDA- and CFA-induced mechanical allodynia by disturbing Src/NR2B signaling pathway. These data provided an important clue as to how spinal PTPs activity was dynamically modulated to alter the pain sensitivity. However, the number of the identified PTPs superfamily members is still growing (Alonso et al., 2004). Distinct PTPs members might exert different regulations of Src- and NMDARs-dependent synaptic transmission and plasticity (Roskoski, 2005). Which PTPs family member plays a dominant role in the induction and development of pathological pain requires the further identification and detailed investigation. Acknowledgment This work was supported by the National Natural Science Foundation of China (N . 81072626) and by the Fundamental Research Funds for the Central Universities (N . lzujbky-2012-k50; lzujbky-2012-190). We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work.
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Inhibition of protein tyrosine phosphatases in spinal dorsal horn attenuated inflammatory pain by repressing Src signaling Zhan-Wei Suo, Xian Yang, Lu Li, Yan-Ni Liu, Lei Shi, Xiao-Dong Hu* Department of Molecular Pharmacology, School of Pharmacy, Lanzhou University, Donggang West Road 199, Lanzhou, Gansu 730000, PR China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 July 2012 Received in revised form 21 November 2012 Accepted 16 January 2013
Tyrosine phosphorylation of N-methyl-D-aspartate (NMDA) subtype glutamate receptors by Src-family protein tyrosine kinases (SFKs) plays a critical role in spinal sensitization. Besides SFKs, the tyrosine phosphorylation levels of proteins are also determined by protein tyrosine phosphatases (PTPs). However, whether PTPs are involved in spinal nociceptive processing is largely unknown. The present study found that intrathecal application of broad-spectrum PTPs inhibitors orthovanadate or Bpv (phen) generated little effects on the paw withdrawal thresholds of intact rats to Von Frey filament stimuli. Although the basal nociceptive responses didn’t require the involvement of PTPs, the mechanical allodynia evoked by intrathecal injection of NMDA was greatly attenuated by orthovanadate and Bpv (phen), suggesting that PTPs activity, once stimulated by NMDA receptors, became essential for spinal sensitization. Biochemical analysis demonstrated that PTPs functioned to activate SFKs member Src and promote Src interaction with NR2B subunit-containing NMDA receptors (NR2B receptors). As a result, PTPs inhibition largely suppressed Src-mediated NR2B phosphorylation at Tyr1472 and reduced the synaptic concentration of NR2B receptors in spinal dorsal horn of NMDA-treated rats. Importantly, intraplantar injection of Complete Freund’s Adjuvant (CFA) naturally activated spinal PTPs to initiate Src signaling, because PTPs inhibition significantly repressed Src activity, reduced Src phosphorylation of NR2B, decreased NR2B synaptic accumulation and eventually ameliorated inflammatory pain. These data indicated an important role played by spinal PTPs in inducing Src-dependent NR2B receptor hyperfunction and suggested that PTPs inhibition might represent an effective strategy for the treatment of inflammatory pain. Ó 2013 Elsevier Ltd. All rights reserved.
Keywords: Protein tyrosine phosphatases Protein tyrosine kinases Inflammatory pain Orthovanadate NMDA receptors
1. Introduction Activation of Src-Family Protein Tyrosine Kinases (SFKs) in spinal dorsal horn is essential for the initiation and development of central sensitization after peripheral tissue and nerve injury (Guo et al., 2002; Liu et al., 2008; Yang et al., 2011). The active SFKs integrate multiple intracellular signaling cascades to modulate the nociceptive conveyance and neuronal excitability (Guo et al., 2004; Slack et al., 2008; Yang et al., 2011). One of the best-characterized SFKs substrates involved in pain processing is NMDA subtype glutamate receptors, especially NR2B subunit-containing NMDA receptors (NR2B receptors) (Guo et al., 2002; Liu et al., 2008). Tyrosine phosphorylation by SFKs increases the synaptic accumulation of NR2B receptors (Li et al., 2011; Prybylowski et al., 2005; Yang et al., 2011), boosts NR2B receptor-triggered signaling
* Corresponding author. Tel.: þ86 0931 3585591. E-mail address: [email protected] (X.-D. Hu). 0028-3908/$ e see front matter Ó 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neuropharm.2013.01.015
transduction (Katano et al., 2011; Matsumura et al., 2010), and causes NR2B receptor-dependent neuronal plasticity and pain sensitization (Katano et al., 2011; Liu et al., 2008; Matsumura et al., 2010). A growing body of evidence has indicated that SFKs-mediated protein tyrosine phosphorylation is also subjected to the control by Protein Tyrosine Phosphatases (PTPs), which dephosphorylate the tyrosine residues on SFKs per se or on SFKs substrates (Alonso et al., 2004; Frank et al., 2004; Roskoski, 2005). A fine balance between tyrosine kinases and phosphatases is critical for many intracellular responses. To date, both receptor-like and nonreceptor PTPs have been detected in postsynaptic density (PSD), where they form macromolecular complex with signaling proteins, scaffolding proteins and substrates (Lei et al., 2002; Peng et al., 2012; Yang et al., 2011). A subpopulation of PTPs has been shown to dephosphorylate SFKs at the conserved autophosphorylation site within the catalytic motif (corresponding to Tyr418 at Src), leading to SFKs inactivation (Roskoski, 2005). Nevertheless, more PTPs dephosphorylate SFKs at their carboxyl-terminal inhibitory
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tyrosine residue (corresponding to Tyr529 at Src), leading to SFKs activation (Roskoski, 2005). Of particular importance is that PTPs activities are dynamically regulated by a wide range of extracellular stimuli, which either change PTPs expression (den Hertog et al., 2008), or modulate PTPs binding to substrates (den Hertog et al., 2008; Yang et al., 2011). The altered catalytic efficacy of PTPs produces a profound impact on synaptic transmission and plasticity, which correlates with an array of physiological and pathological processes such as learning and memory and Alzheimer disease (Lei et al., 2002; Snyder et al., 2005). Although more than 100 members of PTPs superfamily have been identified in mammalian tissues (Alonso et al., 2004), the functional significance of PTPs in pain-related spinal dorsal horn remains largely unknown. The present study found that intrathecal application of broad-spectrum PTPs inhibitors orthovanadate and Bpv (phen) alleviated the mechanical allodynia induced by spinal administration of NMDA or intradermal injection of Complete Freund’s Adjuvant (CFA). PTPs inhibition operated to repress Src activity and decrease the tyrosine phosphorylation and synaptic accumulation of NR2B receptors in NMDA- and CFA-injected rats, implicating that PTPs contributed to spinal sensitization by evoking Src-dependent NR2B receptor hyperfunction.
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2.3. Subcellular fractionation The rats were deeply anesthetized with sodium pentobarbital (30 mg/kg, i.p.) and the spinal cords were quickly removed into ice-cold artificial cerebrospinal fluid (ACSF) (119.0 mM NaCl, 2.5 mM KCl, 2.5 mM CaCl2, 1.3 mM MgCl2, 1.0 mM NaH2PO4, 26.0 mM NaHCO3, 11.0 mM D-glucose, bubbled with 95% O2 þ 5% CO2, pH 7.4). The dorsal quadrant of L4e5 spinal cord was dissected out and homogenized in sucrosecontaining Lysis Buffer [10.0 mM TriseHCl, pH 7.6, 320.0 mM sucrose, 5.0 mM EDTA, plus the inhibitors of proteases and phosphatases (5.0 mM EGTA, 10.0 mM NaF, 1.0 mM Na3VO4, 1.0 mM phenylmethylsulfonyl fluoride, 1.0 mg/ml each of aprotinin, chymostatin, leupeptin, antipain and pepstatin)]. The homogenates were centrifuged at 1000 g for 10 min at 4 C to remove the nuclei and large debris (P1). The supernatant was further centrifuged at 10, 000 g for 15 min to yield the crude synaptosomal fraction (P2). The P2 pellet was resuspended and incubated for 15 min in the Lysis Buffer containing 0.5% Triton X-100, and then centrifuged at 32,000 g for 20 min to obtain the synaptosomal membrane fraction (P3). The final P3 was homogenized in SDS sample buffer and boiled at 95 C for 5 min before processing. We and others have previously demonstrated that P3 fraction is enriched with postsynaptic density (PSD) marker, PSD-95 (Cao et al., 2011; Goebel-Goody et al., 2009; Smith et al., 2006; Yang et al., 2009). The P3 fraction was used instead of the classic PSD fraction because of the limited amount of the starting materials. To assay the tyrosine phosphorylation of NR2B (Guo et al., 2002), the spinal dorsal horn was homogenized in Radio-Immunoprecipitation Assay (RIPA) buffer (50.0 mM Tris$HCl, pH 8.0, 150.0 mM NaCl, 1.0 mM EDTA, 1.0% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, and proteases/phosphatases inhibitors). After centrifugation at 14,000 g for 10 min, the supernatant was harvested and the protein concentration was measured by using BCA protein assay kit (Pierce, Rockford, IL, USA). 2.4. Immunoprecipitation and co-immunoprecipitation
2. Materials and methods 2.1. Animals Male adult SpragueeDawley rats weighing 200e250 g were purchased from the Experimental Animal Center of Lanzhou University and housed on a 12 h light/dark cycle with free access to food and water. The animals were acclimatized to the testing environments for at least 3 days before any experiments were conducted. The inflammatory pain was induced by subcutaneous injection of Complete Freund’s Adjuvant (CFA; 100 ml; Sigma, St. Louis, MO) into the plantar surface of hind paws under light halothane anesthesia. Control animals received identical volumes of saline. All experimental procedures were carried out with the approval of the Animal Care and Use Committee of Lanzhou University. Every effort was made to minimize the number of animals used and their sufferings.
2.2. Drug preparation, intrathecal injection and behavioral tests To achieve the maximum inhibition of PTPs activities, sodium orthovanadate (Sigma) was activated according to the following procedure (Gordon, 1991). A stock solution of orthovanadate (100 mM) was prepared in water with pH value adjusted to 10. This yellow solution was boiled until it turned colorless. After cooling down at room temperature, the solution was re-adjusted with NaOH to pH 10 and boiled again to be colorless. After three to four cycles of activation procedure, the solution was stabilized at colorless state and then stored at 20 C. Just before use, the stocking solution was diluted to the desired working concentration. Another PTPs inhibitor, potassium Bisperoxo (1,10-phenanthroline) oxovanadate (V) [bpv(phen); Calbiochem, MA, U.S.A], and NMDA (TCI, Tokyo, Japan) were dissolved in saline. SFKs inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl) pyrazolo [3,4-D] pyrimidine (PP2; Calbiochem) was dissolved in dimethyl sulfoxide, which was diluted with saline before use. The final concentration of dimethyl sulfoxide was less than 0.5%. All the chemical reagents in 10-ml volume were intrathecally injected by direct lumbar puncture as previously reported (Mestre et al., 1994). Rats were briefly anesthetized with halothane and a 30-gauge needle attached to a 25 ml microsyringe was inserted between L5eL6 vertebrae. A sudden advancement of the needle accompanied by a slight tail flick was used as the indicator for the proper insertion of the needle tip into the subarachnoid space. The drug vehicles, saline and dimethyl sulfoxide, were used as control, which had no discernable effects on the nociceptive behavioral responses after intrathecal application. The Von Frey test was performed as previously described (Chaplan et al., 1994). In brief, the rats were placed in a cage with a wire mesh floor for at least 30 min to adapt to the environment. A series of calibrated Von Frey filaments (Stoelting, Wood Dale, IL, USA), which increased in force with approximately equal logarithmic value (d; 0.22), were applied perpendicularly to the plantar surface for 6 s. The pattern of positive and negative responses was converted to 50% threshold according to the following formula: 50% threshold (g) ¼ (10[xfþKd])/10,000, in which Xf represented the value of the final Von Frey filament used and K was the tabular value for the pattern of positive/negative responses. These behavioral tests were conducted blindly by experimenters without knowledge of the manipulations that had been performed by others on the animals.
To assay Src phosphorylation in spinal dorsal horn, the crude synaptosomal fraction (P2) was lysed in RIPA buffer for 30 min at 4 C (Yang et al., 2011). After centrifugation at 14,000 g for 10 min, the supernatant was incubated overnight with specific monoclonal antibody against the unique N-terminus of Src (Millipore, Temecula, CA, USA) at 4 C under gentle rotation. Src was immunoprecipitated by incubation with Protein A/G agarose beads for 4 h at 4 C. The beads were washed with RIPA buffer three times, and boiled in SDS sample buffer to elute proteins. For co-immunoprecipitation of proteins associated with NR2B, the P2 fraction was extracted in 50.0 mM TriseHCl, pH 9.0, 1.0% sodium deoxycholate, 10.0 mM EDTA, and proteases/phosphatases inhibitors at 37 C for 30 min (Huang et al., 2001). This extract was mixed with equal volume of Tris buffer (50.0 mM Trise HCl, pH 7.4, 150.0 mM NaCl, 1.0% Triton X-100, 0.1% SDS, proteases/phosphatases inhibitors) and then gently rocked overnight at 4 C. After centrifugation at 10,000 g for 10 min, the supernatant was harvested and incubated with anti-NR2B antibody overnight at 4 C. The immune complex was isolated by the addition of Protein A/G agarose beads as described above. Non-specific IgG was utilized for immunoprecipitation as negative control. 2.5. Western blot The equal amounts of protein samples (20 mg) were resolved on SDSPolyacrylamide Gel Electrophoresis (PAGE) and transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were blocked with 5% non-fat milk for 30 min at room temperature before incubation overnight with appropriate primary antibody at 4 C. After three times washes with PBST for 10 min each, the membranes were incubated with horseradish peroxidaseconjugated secondary antibody (1:10,000 dilution, goat anti-rabbit and goat antimouse; Jackson ImmunoResearch Laboratories, Baltimore, PA, USA) for 60 min at room temperature. The blots were visualized by Enhanced Chemiluminescence (Beyotime Institute of Biotechnology, Jiangsu, China). The primary antibodies used in the present study included: the rabbit polyclonal anti-NR2B antibody (1:1000), rabbit polyclonal anti-NR2A antibody (1:1000), mouse monoclonal anti-Src antibody (1:500) and rabbit polyclonal antibody against phosphorylated NR2B at Tyr 1472 (1:1000) purchased from Millipore; the rabbit polyclonal antibody against phosphorylated Src at Tyr418 (1:500) from Invitrogen (Camarillo, CA, USA); the mouse monoclonal anti-NR1 antibody (1:1000) from BD Pharmingen (San Diego, CA, USA); the rabbit polyclonal antibody against phosphorylated Src at Tyr529 (1:1000) from BioSource (Camarillo, CA, USA), and the mouse monoclonal anti-b-actin antibody (1:600) from Sigma. In some cases, the PVDF membrane was stripped with the stripping buffer (5.0 mM TriseHCl, pH 6.8, 2.0% SDS, 0.5% b-mercaptoethanol) for 30 min at 60 C and re-probed with distinct antibodies (Cao et al., 2011; Yang et al., 2011). 2.6. Motor function tests The reflexes for surface righting, placing/stepping and grasping/climbing were tested to evaluate the influence of intrathecal drug application on the motor functions (Garraway et al., 2007; Park et al., 2009). In the surface righting test, we had the rats to lie on the back and observed whether they could resume the normal upright
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position within 1.5 s. In the placing/stepping test, the dorsal surfaces of hind paws were drawn over the edge of a table to see if the rats could reflexively step onto the table top. In the grasping/climbing test, the rats were placed on a wire grid inclined at 90 . Failure to grasp and climb on the grid for 30 s indicated the motor impairment. 2.7. Statistical analysis All the data were expressed as mean SEM. For western blot analysis, the scanned digital images were quantified by NIH Image J software. The relative immunoreactive density of each NMDA receptor subunit was calculated by the ratio of its signal to b-actin signal, whereas the phosphorylation level was determined by the ratio of the phosphorylation signal to the corresponding protein signal. These ratios were used for statistical comparisons. For display purpose, these ratios were normalized to control values. The statistical significance was set at p < 0.05 by using one-way ANOVA followed by post hoc Tukey HSD test.
3. Results 3.1. PTPs inhibition in spinal dorsal horn attenuated NMDA-induced pain hypersensitivity To investigate the possible role of PTPs in spinal processing of nociceptive signals, we first injected PTPs inhibitor orthovanadate intrathecally in intact rats, and the Paw Withdrawal Thresholds (PWT) to Von Frey filament stimuli were measured. Our data demonstrated that orthovanadate had no significant effects on the mechanical thresholds under physiological conditions (Fig. 1A). At each dose tested (0.1, 0.2, 0.4 mg), the PWT values were comparable to the pre-drug ones over the entire observation period (Fig. 1A), suggesting that spinal PTPs might be inactive or dispensable for the modification of the basal nociceptive behaviors. Cumulating evidence has indicated that Ca2þ influx via NMDA receptors (NMDARs), a critical player in central sensitization, is capable of stimulating PTPs activity to induce NMDARs-dependent neuronal plasticity (Coussens et al., 2000; Paul et al., 2003). We therefore investigated whether PTPs were recruited to nociceptive processing in a NMDARs-dependent way. For this purpose, NMDA was spinally injected in intact rats. As previously reported (Sato et al., 2003), NMDA at the subconvulsive dose of 1.0 mg elicited a rapid reduction in PWT values, which lasted for at least 70 min (Fig. 1B). At 10 min post-NMDA injection, orthovanadate was intrathecally superimposed. In contrast to the basal nociceptive responses, NMDA-induced pain hypersensitivity was alleviated by orthovanadate in a dose-dependent manner (Fig. 1B). At each dose, orthovanadate had no discernable effects on motor functions. The animals displayed normal reflexes for surface righting, placing/ stepping and grasping/climbing. To confirm the role of PTPs in nociceptive modification, we intrathecally applied another potent PTPs inhibitor, Bpv(phen), finding that Bpv(phen) attenuated the reduction of PWT values evoked by NMDA (Fig. 1C) but devoid of any influence on the basal nociceptive responses and locomotor activity (data not shown). These data implicated that NMDARs activation could stimulate PTPs activity in spinal dorsal horn, which was important for pain sensitization. 3.2. PTPs inhibition repressed Src activity in spinal dorsal horn of NMDA-injected rats Previous studies have described the critical role of SFKs in NMDARs-dependent central sensitization (Liu et al., 2008; Sato et al., 2003). Given that PTPs serve as an intrinsic regulator of SFKs activity (Roskoski, 2005), we wanted to know whether PTPs inhibition ameliorated NMDA-induced mechanical allodynia via SFKs signaling. To this end, NMDA (1.0 mg) was intrathecally applied for 40 min to elicit a maximum decrease in PWT values (Fig. 1B). Immediately after behavioral tests, SFKs member Src kinase was
Fig. 1. Inhibition of protein tyrosine phosphatases (PTPs) in spinal dorsal horn alleviated the mechanical allodynia induced by intrathecal (i.t.) injection of NMDA (1.0 mg) in rats. (A) PTPs inhibitor Orthovanadate (Ortho; 0.1 mg, 0.2 mg, 0.4 mg) didn’t affect the basal nociceptive responses of intact rats to Von Frey filament stimuli. The alteration in Paw Withdrawal Threshold (PWT) was plotted against time. (B) Orthovanadate dosedependently attenuated the reduction of PWT values induced by NMDA. The upward and downward arrows indicated the time points when distinct drugs were spinally given. (C) Another PTPs inhibitor Bpv(phen), when intrathecally applied at 10 min post-NMDA injection, also ameliorated the pain hypersensitivity in a dose-dependent manner. *p < 0.05 when compared with saline-injected control rats. #p < 0.05 when compared with NMDA-injected, saline-treated rats. n ¼ 6 rats in each group.
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immunoprecipitated from the crude synaptosomal fraction of spinal dorsal horn. Src phosphorylation was probed with a phosphotyrosine antibody against its inhibitory tyrosine residue 529 (Tyr529), a well-characterized dephosphorylation site by PTPs (Roskoski, 2005). Compared with saline vehicle, NMDA treatment reduced Tyr529 phosphorylation by 44.8 4.2% (p < 0.05, n ¼ 6) (Fig. 2A), indicating the activation of Src. When orthovanadate (0.4 mg) was spinally applied for 30 min to inhibit the mechanical allodynia in NMDA-injected rats (Fig. 1B), Tyr529 phosphorylation level was simultaneously elevated, which became indistinguishable from that of saline-injected control rats (Fig. 2A). Because phosphorylated Tyr529 negatively controls Src activity by blunting Tyr418 autophosphorylation within the activation loop (Salter and Kalia, 2004), we further performed immunoblotting with a phosphotyrosine antibody against Tyr418 to consolidate the change in Src enzymatic activity. Our data revealed that NMDA injection induced a significant increase in Tyr418 phosphorylation relative to saline vehicle (Fig. 2B), a result consistent with the corresponding decrease in Tyr529 phosphorylation (Fig. 2A).
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Treatment of NMDA-injected rats with orthovanadate, however, repressed Tyr418 phosphorylation level from 188.1 29.9% of vehicle control to 116.8 23.6% of vehicle control (p < 0.05, n ¼ 6) (Fig. 2B). Behavioral tests demonstrated that direct application of SFKs inhibitor PP2 (10.0 mg) noticeably ameliorated NMDA-induced allodynia (Fig. 2C). More importantly, SFKs inhibition by PP2 completely occluded the analgesic action of orthovanadate (0.4 mg) (Fig. 2C), suggesting that Src was a key target for PTPs to regulate NMDARs-dependent spinal sensitization. 3.3. PTPs inhibition suppressed the hyperfunction of NR2B receptors in spinal dorsal horn of NMDA-injected rats The molecular mechanisms for the active Src to exacerbate nociceptive conveyance involve the tyrosine phosphorylation and synaptic retention of NR2B subunit-containing NMDA receptors (NR2B receptors) in spinal dorsal horn (Li et al., 2011; Liu et al., 2008). Here we found that NMDA treatment, in addition to increase Src activity (Fig. 2A and B), also promoted the molecular
Fig. 2. Intrathecal injection of NMDA (1.0 mg) in rats activated Src in PTPs-dependent manner. Src was immunoprecipitated from the crude synaptosomal fraction of spinal dorsal horn at 40 min after NMDA injection, followed by immunoblotting with phosphotyrosine antibodies. (A) PTPs inhibitor orthovanadate (Ortho; 0.4 mg), when intrathecally superimposed at 10 min after NMDA injection, eliminated the decrease of Src phosphorylation at Tyr529 (Src-Y529p) induced by NMDA. The immunoreactivity of phosphorylated Src was shown (up). The graph summarized the percentage change in Src-Y529p (down). (B) Orthovanadate (0.4 mg) also abolished NMDA-induced increase in Src phosphorylation at Tyr418 (Src-Y418p). (C) SFKs inhibitor PP2 (10 mg), when intrathecally superimposed at 10 min post-NMDA injection, attenuated the reduction of PWT values, which also occluded the analgesic action of the subsequently applied orthovanadate (0.4 mg). The upward and downward arrows indicated the time points when distinct reagents were spinally given. *p < 0.05 when compared with saline-injected control rats. #p < 0.05 when compared with NMDA-injected rats. n ¼ 6 in each group.
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interaction of Src with NR2B. As shown in Fig. 3A, the specific antibody against NR2B subunit was able to co-immunoprecipitate Src from the crude synaptosomal fraction of spinal dorsal horn in intact rats, whereas non-specific IgG failed to do so. When NMDA (1.0 mg) was intrathecally applied for 40 min to evoke pronounced allodynia (Fig. 1B), the contents of Src pulled down by anti-NR2B antibody were significantly higher than those in vehicle group (Fig. 3A), suggesting that NMDARs activation simultaneously positioned Src at the proximity of its substrates. Immunoblotting analysis demonstrated that NR2B phosphorylation at Tyr1472, a catalytic site by Src, was enhanced by 113.9 40.3% in NMDAinjected rats (p < 0.05 vs. saline, n ¼ 6) (Fig. 3B). Because one of the most important functions of Tyr1472 phosphorylation is to prevent NR2B endocytosis (Li et al., 2011), we further assayed the synaptic expression of NR2B receptors, finding that NMDA application specifically increased the content of NR2B and NR1 subunits at synaptosomal membrane fraction, with that of NR2A subunit unaltered (Fig. 3C). Interestingly, NMDA-induced Src targeting to NR2B depended on PTPs activities. Intrathecal application of orthovanadate (0.4 mg) for 30 min reduced the amount of Src precipitated by anti-NR2B antibody from NMDA-injected rats (Fig. 3A). Such a dissociation of NR2B/Src complex following PTPs inhibition was concurrent with the repression of NMDA-induced NR2B tyrosine phosphorylation (Fig. 3B). Meanwhile, the increase in the synaptic concentration of NR2B and NR1 subunit
evoked by NMDA was also abolished by orthovanadate (Fig. 3C). These data implicated that active PTPs following NMDARs stimulation facilitated NR2B interaction with Src, leading to NR2B receptor hyperfunction in spinal dorsal horn. 3.4. PTPs inhibition in spinal dorsal horn alleviated inflammatory pain Convincing evidence has established that peripheral tissue injury naturally stimulates spinal NMDARs to trigger multiple signaling pathways essential for inflammatory pain. Since PTPs acted downstream of NMDARs and contributed to spinal sensitization, we next explored the possible role of PTPs in persistent inflammatory pain. Our data demonstrated that intraplantar injection of Complete Freund’s Adjuvant (CFA) induced a rapid decrease in PWT values of the affected hind paws (Fig. 4A). Pretreatment with orthovanadate for 15 min, however, dose-dependently delayed the onset and attenuated the magnitude of reduction in the mechanical thresholds (Fig. 4A). In addition to the blocking effect on the induction of inflammatory pain, orthovanadate application one day after CFA injection also alleviated the established allodynia in a dosedependent manner. As shown in Fig. 4B, post-treatment with a low dose of orthovanadate (0.1 mg) produced minimal effect on the PWT values of inflamed rats. Increasing the dose of orthovanadate to 0.2 mg transiently elevated the PWT values at 20 min and 30 min,
Fig. 3. PTPs inhibitor orthovanadate repressed NMDA-induced NR2B receptor hyperfunction in spinal dorsal horn of rats. (A) Intrathecal application of NMDA (1.0 mg) for 40 min enhanced the amount of Src co-immunoprecipitated (Co-IP) by anti-NR2B antibody from the crude synaptosomal fraction of spinal dorsal horn. Note that orthovanadate (Ortho; 0.4 mg), when spinally superimposed at 10 min post-NMDA injection, reversed NR2B interaction with Src. Non-specific IgG was utilized as negative control for coimmunoprecipitation. IB: Immunoblotting. (B) Intrathecal NMDA application enhanced the phosphorylation of NR2B subunit at Tyr1472 (NR2B-Y1472p) in spinal dorsal horn, which could be abolished by orthovanadate. The membrane was stripped and re-probed with anti-NR2B antibody. The graph summarized the percentage change in NR2B-Y1472p level. (C) NMDA injection increased the content of NMDA receptor NR1 and NR2B subunit at the synaptosomal membrane fraction, which, however, could be abolished by orthovanadate. The immunoreactivity of each NMDARs subunit was shown (left). Equal protein loadings were indicated by b-actin intensity. The graph summarized the percentage changes in NMDARs subunit immunoreactivity (right). *p < 0.05 compared with saline-injected control rats. #p < 0.05 compared with NMDA-injected rats. n ¼ 6 experiments.
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which then returned to the levels comparable to vehicle-treated inflamed rats (Fig. 4B). When the dose of orthovanadate was further increased to 0.4 mg, a long-lasting attenuation of inflammatory pain was observed (Fig. 4B). At 60 min after the drug treatment, the PWT values were still higher than those of saline-treated inflamed rats (orthovanadate vs. saline: 7.35 1.39 g vs. 2.69 0.19 g, p < 0.05, n ¼ 6 in each group). A similar alleviation of inflammatory pain was also observed when PTPs inhibitor Bpv (phen) was spinally given one day after CFA injection (Fig. 4C). These data suggested that PTPs activity in spinal dorsal horn was involved in both the induction and maintenance of inflammatory pain. 3.5. PTPs inhibition repressed Src hyperactivity in spinal dorsal horn of rats with inflammatory pain Next, we examined whether PTPs inhibition alleviated inflammatory pain by modulating spinal Src activity. One day after CFA injection, Src was immunoprecipitated from the crude synaptosomal fractions of spinal dorsal horn. Western blot illustrated that peripheral inflammation reduced Src-Tyr529 phosphorylation level to 47.4 4.9% of vehicle control (p < 0.05, n ¼ 6) (Fig. 5A). This result was supported by a concomitant increase in Tyr418 phosphorylation, which was 197.6 18.9% of vehicle control (p < 0.05, n ¼ 6) (Fig. 5B). Notably, CFA-induced Src activation required PTPs activity. When orthovanadate (0.4 mg) was intrathecally applied in inflamed rats for 30 min, Src phosphorylation at Tyr529 was elevated to 75.0 8.7% of vehicle control (p > 0.05, n ¼ 6) (Fig. 5A), while Tyr418 phosphorylation was repressed to 97.8 11.9% of vehicle control (p > 0.05, n ¼ 6) (Fig. 5B). These data suggested that the analgesic action of orthovanadate was closely correlated with the suppression of Src activity. 3.6. PTPs inhibition reversed NMDA receptor hyperfunction in spinal dorsal horn induced by peripheral inflammation
Fig. 4. PTPs inhibition in spinal dorsal horn blocked the induction and maintenance of inflammatory pain induced by intradermal (i.d.) injection of Complete Freund’s Adjuvant (CFA) in rats. (A) PTPs inhibitor Orthovanadate (Ortho) was intrathecally (i.t.) given 15 min prior to CFA injection. The changes in PWT values were plotted versus time. The upward and downward arrows indicated the time points when intradermal and intrathecal injection were performed, respectively. (B) Orthovanadate (0.1 mg, 0.2 mg, 0.4 mg) was spinally given one day after CFA injection. The time-dependent changes in PWT values were plotted. (C) Intrathecal administration of PTPs inhibitor Bpv (phen) one day after CFA injection attenuated the inflammatory pain in a dosedependent manner. *p < 0.05 when compared with saline-injected control rats. # p < 0.05 when compared with saline-treated inflamed rats. n ¼ 6 rats in each group.
To further address whether Src inhibition by orthovanadate led to the reversal of NMDARs hyperfunction in inflamed rats, we analyzed the tyrosine phosphorylation of NR2B in spinal dorsal horn one day after CFA injection. Our data illustrated that peripheral inflammation significantly increased Tyr1472 phosphorylation to 176.9 26.3% of saline control (p < 0.05, n ¼ 6) (Fig. 6A). Nevertheless, intrathecal application of orthovanadate (0.4 mg) for 30 min depressed the NR2B tyrosine phosphorylation level to 105.0 14.7% of saline control (p > 0.05, n ¼ 6) (Fig. 6A). NR2B dephosphorylation by orthovanadate might result from not only Src inhibition (Fig. 5A and B), but also Src dissociation with NR2B. As shown in Fig. 6B, anti-NR2B antibody precipitated much more Src from the crude synaptosomal fraction of inflamed rats than control ones, which, however, could be completely ruled out by orthovanadate. Consistent with NR2B tyrosine dephosphorylation, orthovanadate also abolished the CFA-induced increase in the immunoreactive intensities of NR2B and NR1 subunits at synaptosomal membrane fraction (Fig. 6C). Neither CFA nor orthovanadate altered the synaptic content of NR2A subunit (Fig. 6C). These data suggested that PTPs inhibition ameliorated inflammatory pain by suppressing Src-dependent NR2B receptor hyperfunction in spinal dorsal horn. 4. Discussion Protein tyrosine phosphorylation serves as an important mechanism for the regulation of nociceptive transmission and integration (Pelkey et al., 2002; Salter and Kalia, 2004). Compared with Src-family protein tyrosine kinases (SFKs), protein tyrosine phosphatases (PTPs) have recently been implicated to play equal or
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Fig. 5. Intrathecal application of PTPs inhibitor orthovanadate (Ortho; 0.4 mg) for 30 min repressed Src activity in spinal dorsal horn of rats one day after CFA injection. Src was immunoprecipitated from the crude synaptosomal fraction of spinal dorsal horn. (A) The immunoreactivity of phosphorylated Src at Tyr529 (Src-Y529p) was shown (up). The graph summarized the percentage change in Src phosphorylation (down). *p < 0.05 when compared with saline-injected control rats. #p < 0.05 when compared with CFA-injected rats (n ¼ 6 experiments). (B) CFA injection increased Src phosphorylation at Tyr418 (Src-Y418p), which could be abolished by orthovanadate.
even dominant roles in setting protein tyrosine phosphorylation levels (Alonso et al., 2004). However, whether and how PTPs modulate spinal processing of nociceptive information is largely unknown as yet. The present study demonstrated that PTPs activation was critical for NMDA- and CFA-induced pain hypersensitivity. The active PTPs operated to initiate Src signaling in spinal dorsal horn, leading to NR2B tyrosine phosphorylation and synaptic accumulation. Consequently, PTPs inhibition effectively alleviated spinal sensitization by reversing Src and NR2B hyperactivities in NMDA- and CFA-injected rats. It is well known that Src kinase contains two critical regulatory tyrosine residues, one within its activation loop (Tyr418) and the other at the extreme carboxyl-terminal tail (Tyr529) (Salter and
Kalia, 2004). Under normal conditions, Tyr529 is phosphorylated by C-terminal Src kinase (CSK) (Chong et al., 2005; Roskoski, 2005; Salter and Kalia, 2004). The Tyr529-phosphorylated C-terminal tail interacts intramolecularly with SH2 (Src Homology 2) domain to keep Src in a closed and inactive conformation. When Tyr529 is dephosphorylated by PTPs, the closed conformation is opened, allowing for Tyr418 autophosphorylation and Src activation. The active Src can then be dephosphorylated by specific PTPs that target Tyr418, restricting Src from excessive activation. The present study showed that intrathecal NMDA and intraplantar CFA injection led to a decrease of Tyr529 phosphorylation and a concomitant increase of Tyr418 phosphorylation in a PTPs-dependent manner. Possibly, NMDA and CFA preferentially stimulated PTPs targeting Tyr529 dephosphorylation. Alternatively, the endogenous PTPs responsible for Tyr529 dephosphorylation became to dominate over those for Tyr418 dephosphorylation after NMDA and CFA injection. As a result, the net role of active PTPs seemed to be “excitatory”, which catalyzed Tyr529 dephosphorylation to cause Src hyperactivity and pain sensitization. The functional imbalance between these two populations of PTPs might explain why orthovanadate and Bpv (phen) selectively attenuated NMDA- and CFA-induced mechanical allodynia without any influence on the basal nociceptive behaviors. In agreement with these data, recent studies have indicated that a subpopulation of “excitatory” PTPs actively governs SFKsmediated intracellular responses in a myriad of processes such as neuronal plasticity (Lei et al., 2002; Peng et al., 2012). NMDARs hyperfunction has been widely described as a key contributor to pathological pain. The hyperactive NMDARs might result from the receptor phosphorylation, which augments the receptor channel conductance and synaptic accumulation (Li et al., 2011; Yang et al., 2011, 2009). One of the best-characterized phosphorylation events after peripheral lesions occurs on NR2B subunit at Tyr1472. Genetic knockdown of NR2B or knock-in mutation of Tyr1472 potently alleviates pathological pain symptoms (Katano et al., 2011; Matsumura et al., 2010; Tan et al., 2005). As a critical determinant of protein tyrosine phosphorylation, PTPs have been shown to exert a negative control over NR2B subunit in hippocampal and striatal neurons, because inhibition of PTPs activity significantly increases NR2B tyrosine phosphorylation levels in these brain regions (Chen et al., 2003; Goebel et al., 2005; Hallett et al., 2006; Wang and Salter, 1994). However, the present study in vivo illustrated that intrathecal application of PTPs inhibitor repressed, rather than boosted, the tyrosine phosphorylation of NR2B in NMDA- and CFA-injected rats, suggesting a stimulatory effect of PTPs on NR2B phosphorylation in spinal dorsal horn. This tissue-specific action might be indirect, possibly through downstream Src kinase. We found that spinal PTPs not only increased Src activity but also promoted Src association with NR2B. Because Src activation is always accompanied by the exposure of its substratebinding SH2 domain (Salter and Kalia, 2004), it was likely that the free SH2 domain physically interacted with postsynaptic scaffolding proteins such as PSD-95, through which Src gained access to NR2B and exerted full catalytic action (Kalia and Salter, 2003). Tyr1472 phosphorylation has been illustrated to disrupt NR2B interaction with clathrin adaptor protein AP-2, which prevents the internalization of NR2B receptors from postsynaptic membrane (Prybylowski et al., 2005). Our biochemical analysis also found that PTPs-dependent Tyr1472 phosphorylation was closely correlated with the synaptic accumulation of NR2B receptors in spinal dorsal horn of NMDA- and CFA-injected rats. No detectable change in NR2A synaptic content supported the notion that tyrosine phosphorylation is dispensable for the synaptic trafficking of NR2A subunit (Prybylowski et al., 2005). The selective removal of NR2B receptors from excitatory synapses by PTPs inhibition presumably attenuated NR2B receptors-mediated nociceptive transmission and
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Fig. 6. PTPs inhibitor orthovanadate reversed peripheral inflammation-induced NR2B receptor hyperfunction in spinal dorsal horn of rats. (A) The phosphorylation level of NR2B subunit at Tyr1472 (NR2B-Y1472p) significantly increased one day after CFA injection, which could be abolished by intrathecal application of orthovanadate (Ortho; 0.4 mg) for 30 min. The membrane was stripped and re-probed with anti-NR2B antibody. The graph summarized the percentage change in NR2B-Y1472p level. *p < 0.05 compared with salineinjected control rats; #p < 0.05 compared with CFA-injected rats; n ¼ 6 experiments. (B) Src was co-immunoprecipitated (Co-IP) by anti-NR2B antibody from the crude synaptosomal fraction of spinal dorsal horn one day after CFA injection. Note that treatment with orthovanadate reduced the content of precipitated Src in inflamed rats. Non-specific IgG was utilized as negative control for co-immunoprecipitation. IB: Immunoblotting. (C) The concentrations of NMDA receptor NR1 and NR2B subunits at the synaptosomal membrane fraction significantly increased one day after CFA injection, which could be abolished by orthovanadate. The immunoreactivity of each NMDARs subunit was shown (left). Equal protein loadings were indicated by b-actin intensity. The graph summarized the percentage change in NMDARs subunit immunoreactivity (right).
intracellular signaling cascades, eventually leading to the alleviation of pain hypersensitivity. Taken together, the present study demonstrated that pharmacological inhibition of PTPs ameliorated NMDA- and CFA-induced mechanical allodynia by disturbing Src/NR2B signaling pathway. These data provided an important clue as to how spinal PTPs activity was dynamically modulated to alter the pain sensitivity. However, the number of the identified PTPs superfamily members is still growing (Alonso et al., 2004). Distinct PTPs members might exert different regulations of Src- and NMDARs-dependent synaptic transmission and plasticity (Roskoski, 2005). Which PTPs family member plays a dominant role in the induction and development of pathological pain requires the further identification and detailed investigation. Acknowledgment This work was supported by the National Natural Science Foundation of China (N . 81072626) and by the Fundamental Research Funds for the Central Universities (N . lzujbky-2012-k50; lzujbky-2012-190). We declare that we have no financial and personal relationships with other people or organizations that can inappropriately influence our work.
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