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Nerve growth factor regulates substance P in adult sensory neurons through both TrkA and p75 receptors
Experimental Neurology 197 (2006) 430 – 436 www.elsevier.com/locate/yexnr
Regular Article
Nerve growth factor regulates substance P in adult sensory neurons through both TrkA and p75 receptors Anne M. Skoff a,⁎, Joshua E. Adler b a
Department of Neurology, Wayne State University School of Medicine, 8D University Health Center, 4201 St. Antoine, Detroit, MI 48201, USA Department of Neurology, Veterans Administration Medical Center and Wayne State University School of Medicine, Detroit, MI 48201, USA
b
Received 14 July 2005; revised 29 August 2005; accepted 4 October 2005 Available online 21 November 2005
Abstract Expression of the nociceptive peptide, substance P (SP) is regulated by the neurotrophin, nerve growth factor (NGF), and exogenous exposure to high levels of NGF increases its cellular content and release. NGF utilizes two receptors, the NGF-specific tyrosine kinase receptor, TrkA, and also the non-specific neurotrophin receptor, p75NTR (p75). The purpose of this study is to determine the relative involvement of these receptors in nociception. To investigate the role of TrkA in SP signaling, sensory neurons from adult rats were grown in vitro and exposed to a TrkA-blocking antibody. Pretreatment with the antibody inhibited NGF-induced SP elevation. Furthermore, when neurons were exposed to K252a, a relatively specific TrkA kinase inhibitor, the NGF effect on SP was also inhibited. K252a did not prevent SP up-regulation in cells exposed to forskolin or glial cell line-derived neurotrophic factor (GDNF), two agents which increase SP expression independently of TrkA. When p75 was blocked by antiserum, SP up-regulation by NGF was also inhibited. The antiserum neither impacted neuronal survival or basal levels of SP expression, nor did it inhibit SP up-regulation induced by forskolin. Two other neurotrophins, which are also ligands for p75, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) did not block NGF-induced SP up-regulation, raising the possibility that activated p75 is able to cooperate in SP regulation regardless of which neurotrophin ligand occupies it. Our data suggest that NGF up-regulation of SP expression requires the involvement of both TrkA and p75, although the specific contribution of each receptor to SP signaling remains to be determined. © 2005 Elsevier Inc. All rights reserved. Keywords: NGF; Tyrosine kinase A; P75 neurotrophin receptor; Substance P; Nociception; Regulation
Introduction The neurotrophin, nerve growth factor (NGF), plays a critical role in sensory neuronal development and physiology (LeviMontalcini and Angeletti, 1968; Thoenen and Barde, 1980), supporting survival of nociceptive neurons during critical periods of ontogeny (Johnson et al., 1980). NGF displays significant functionality in adult systems as well, modulating the sensitivity and plasticity of the nociceptive system (Mendell et al., 1999; Petruska and Mendell, 2004). It regulates such phenotypic traits as stimulus responsiveness (Lewin et al., 1993), response to injury (Olson et al., 1994; Rich et al., 1987), and neuropeptide expression (Kessler and Black, 1980; Otten et al., 1980). NGF's role in nociception is complicated. It induces rapid sensitization to noxious mechanical and thermal stimuli ⁎
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via direct sensitization of the heat receptor, TRPV1 (vanilloid receptor 1, Caterina et al., 1997), which does not involve gene transcription (Lewin and Mendell, 1993; Shu and Mendell, 1999), while also contributing to a slower, central sensitization, likely to involve the synthesis and release nociceptive peptides, including SP and CGRP (Mendell et al., 1999). Exogenous exposure to high levels of the growth factor, both in vivo and in vitro, increases the intracellular content and release of SP (Adler et al., 1984; Lindsay and Harmar, 1989; Malcangio et al., 1997a,b, 2000; Otten et al., 1980; Skoff et al., 2003; Vedder et al., 1993; Woolf et al., 1994). NGF effects are transduced through two transmembrane receptors, the tyrosine kinase receptor, TrkA, for which NGF is the primary ligand, and the non-specific neurotrophin receptor, p75, which binds all known neurotrophins, including NGF, BDNF, NT-3, and NT-4/5 with low affinity (Hempstead, 2002). p75 has no intrinsic catalytic activity but is a member of the tumor necrosis factor receptor (TNFR) superfamily, with an
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intracellular domain homologous to the “death domain” of TNFR1(Liepinsh et al., 1997). It has the capacity to autonomously activate apoptotic and survival promoting signal pathways and is required for high affinity binding of NGF to TrkA (for review, see Huang and Reichardt, 2003). TrkA signaling largely relates to neuronal survival and neurite outgrowth during development but later plays a significant role in the induction of hyperalgesia induced by inflammation (Cho et al., 1996; Woolf, 1996), likely through induction of TRPV1 expression (Mendell, 2002). When the two receptors are coexpressed, each can modulate the signaling of the other (reviewed in Hempstead, 2002; Roux and Barker, 2002). Development of neuropathic pain following nerve injury may be accompanied by alterations in level or distribution of the nociceptive peptide substance P (SP) in the dorsal horn of the spinal cord (Cameron et al., 1997; Malmberg and Basbaum, 1998; Munglani et al., 1995; Swamydas et al., 2004). Understanding the role NGF plays in SP expression would set the stage for uncovering the mechanisms underlying the induction and modulation of chronic pain. We have employed an in vitro culture system of adult rat dorsal root ganglion neurons to address the involvement of the NGF receptors in SP regulation. Adult sensory neurons survive well in culture without exogenous trophic factors but express only low levels of SP. Addition of NGF to the culture media increases expression of SP several fold (Lindsay and Harmar, 1989; Vedder et al., 1993). To determine the contribution of each of the NGF receptors to substance P expression, cultured neurons were exposed to a number of agents that block the activities of p75 or TrkA. Inhibition of either receptor reduced the NGF-induced increase of SP, suggesting that both of the receptors are required. Furthermore, while neither brainderived neurotrophic factor (BDNF) nor neurotrophin-3 (NT3), two other neurotrophin ligands for p75, was effective in increasing SP alone, their addition to the culture medium potentiated, rather than blocked the NGF-induced SP response. These data have been presented in preliminary form (Skoff and Adler, 2004).
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were seeded onto 12-well culture plates (24 mm/well) coated with poly D-lysine (100 μg/ml) and laminin (1 μg/cm2). The cells were plated at a density of approximately 10,000 viable DRG neurons per well. Ganglia from one rat provided sufficient cells for 12 to 16 wells. Cultures were incubated at 37°C in 95% air–5% CO2 at nearly 100% humidity. Incubation medium consisted of a 50/50 mixture of Ham's nutrient mixture F-12/ Dulbecco's modified Eagle's medium supplemented with penicillin (50 U/ml), streptomycin (50 mg/ml), 5% rat serum, glucose (3 mg/ml), glutamine (2 mM), ascorbic acid (300 μM), glutathione (3.5 μM), imidazole (100 μM), and p-amino benzoic acid (1 μg/ml). Cytosine arabinofuranoside (10 μM) was added to suppress Schwann cell and fibroblast proliferation. These non-neuronal cells comprised approximately 20% of the total population. One third of the medium was exchanged every other day. 2.5S NGF was obtained from Harlan Bioproducts for Science, Inc. (Madison, WI). Culture medium and supplements were obtained from Sigma and Gibco BRL. Following dissociation, the cells were resuspended in culture medium, plated into wells in 100 μl aliquots, and allowed to attach for 30–60 min. The cells were then preincubated with culture medium (control) or test agent for 1 h, after NGF (1, 10, or 50 ng/ml), forskolin (1.0 μM), or GDNF (10 ng/ml) was added. Cultures were grown for 1 week, at which time they were harvested for SP content. The cells from one well served as a single sample. Groups of 6 wells were exposed to each agent. Each experiment was repeated at least twice. NGF receptor blockade
Materials and methods
The blocking agents tested were anti-p75 NTR #9651 (αp75), a rabbit polyclonal antiserum to the extracellular domain of mouse p75 (diluted 1:500) (Huber and Chao, 1995) (gift of Moses Chao), and a blocking antibody to TrkA (αtrkA) (ab8771, Abcam, Cambridge, MA, used at 2 μg/ml). Also tested were two additional neurotrophins, brain-derived neurotrophic factor (BDNF) (Calbiochem, La Jolla, CA) and recombinant human neurotrophin-3 (NT-3) (R&D Systems, Minneapolis, MD). Both were used at 25 ng/ml.
Animals/dissection
TrkA receptor inhibition
Adult (N 3 months), female Sprague–Dawley rats (Charles River, Wilmington MA) were used as the source of sensory neurons. After rats were sacrificed by exposure to CO2, dorsal root ganglia were removed from all levels (approximately 50– 52 ganglia/rat) and maintained in calcium and magnesium-free Puck's Saline G (SalG) (Puck et al., 1958) for a maximum of 2 h before dissociation. The animal protocols used in this study have been approved by the Wayne State University Animal Investigation Committee and conform to NIH guidelines.
The protocol for TrkA receptor inhibition was identical to the blockade protocol above except that the TrkA inhibitor, K-252a (Nocardiopsis, Calbiochem; 400 nM), was used.
Dissociated cell culture Dorsal root ganglia (DRG) were dissociated and plated as described previously (Adler, 1998; Adler et al., 1984). Neurons
Extraction of substance P Peptide extraction was performed as described by Adler et al. (1984) utilizing a cell scraper adapted to fit the bottom of a 24-mm well plate. Briefly, after draining the medium and rinsing each well with 500 μl cold Puck's SalG containing calcium and magnesium, 250 μl of 0.01N HCl was added to each well and the cells scraped from the bottom. The samples were placed in a boiling water bath for 5 min, frozen, and lyophilized.
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A.M. Skoff, J.E. Adler / Experimental Neurology 197 (2006) 430–436
Radioimmunoassay for substance P Substance P was assayed according to the method of Powell et al. (1973). (125 I)-labeled substance P (New England Nuclear, Boston, MA) was used as the tracer. Sensitivity of the assay was typically 2 pg per sample. Statistics Statistical analysis was performed using one-way ANOVA with a Newman–Keuls post-test, and the analyses were performed separately for each experiment. Results NGF regulates such diverse cellular responses and functions as survival, neurite outgrowth, myelination, and apoptosis through well-defined signaling pathways involving one or both of its receptors. Most identified NGF-mediated effects are related to development or are involved in injury response. However, the potential role of NGF and its receptors in nociception remains unclear. To better define the roles of these receptors, we examined them individually. Through the
Fig. 2. GDNF and forskolin signaling of substance P up-regulation is independent of the TrkA receptor. Dissociated cells were preincubated with k252A (400 nM) for 1 h after which they were exposed to either 10 ng/ml GDNF (k252a + GDNF) or 1 M forskolin (k252a + forskolin). Control wells, and wells exposed to GDNF, forskolin, or k252a alone were included for comparison. *P b 0.01 compared to control and k252a.
use of competitive blockers and/or receptor inhibitors of TrkA and p75 in vitro, we investigated receptor involvement in SP expression and determined that both receptors play a role. TrkA
Fig. 1. NGF up-regulation of substance P expression requires the TrkA receptor. (A) Receptor blockade: dissociated, mature, dorsal root ganglion neurons were grown in the presence of medium alone (Control), 10 ng/ml NGF (NGF), 2 μg/ ml of a blocking antibody to the TrkA receptor (αTrkA), or were preincubated with αTrkA for one hour prior to the addition of NGF (10 ng/ml) (NGF + αTrkA). *P b 0.001 compared with control and αTrkA, P b 0.01 compared with NGF + αTrkA; **P b 0.01 compared with control and αTrkA). (B) Receptor inhibition: cells were treated as in panel A except that NGF was increased to 50 ng/ml and k-252a (400 nM) was used in place of αTrkA (k252a + NGF). (*P b 0.001) In this (and all figures), substance P content was determined after 1 week. Bars represent mean substance P content/well, in picograms, from groups of six wells ±SEM.
Many of the NGF-mediated effects on sensory neurons are signaled through the TrkA receptor, either alone, or in cooperation with p75. To investigate its role in SP regulation, a TrkA-targeted blocking antibody, ab8771 (αTrkA) was added to cultures of mature spinal sensory neurons (Fig. 1A). After a week in culture, NGF increased SP expression four-fold. The αTrkA antibody reduced this by more than 25%. Antibody alone had no effect. A similar but more dramatic reduction was achieved by using the TrkA inhibitor, k252a, to block downstream signaling. It reduced SP to baseline levels (Fig. 1B). Because the TrkA inhibitor is known to inhibit other kinases which could adversely affect cell function and viability, it was tested against two other agents known to elevate SP: GDNF (Adler, 1998) and forskolin (Adler and Walker, 2000). Both agents increased SP at least four-fold, but addition of k252a to these stimulated neurons had no effect (Fig. 2), indicating that forskolin and GDNF up-regulation of SP involves alternate transduction pathways and that cell function and viability were not compromised by the kinase inhibitor. p75NTR To investigate the potential role of the p75 in SP upregulation, a well-characterized blocking antibody directed against the extracellular domain of this receptor was used to
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block NGF binding. While NGF again increased baseline SP levels more than four-fold, αp75 inhibited the NGF-induced SP level by more than 60% implicating the receptor in SP regulation (Fig. 3A). To confirm that the antibody was acting directly on the extracellular domain of p75 and was not actually damaging the neurons, the antibody was tested against forskolin. αp75 had no affect on SP levels induced by forskolin (Fig. 3B). This is consistent with the site of the αp75 inhibition being upstream of the cAMP second messenger system, which is located near the cytoplasmic interface of the plasma membrane. To determine whether the contribution of p75 to the SP response is specifically due to its interaction with NGF, two additional neurotrophins, BDNF and NT-3, also ligands for p75, were used to block NGF binding. Preincubation with a massive excess of either neurotrophin did not inhibit SP expression (Figs. 4A and B), suggesting activated p75, regardless of the neurotrophin bound to it, can cooperate with NGF to induce SP. Taken together, these data indicate that both NGF receptors, p75 and TrkA, are involved in SP upregulation by NGF.
Fig. 3. NGF up-regulation of substance P expression requires p75. (A) Receptor blockade: cells were grown in the presence of medium alone (Control), 50 ng/ml NGF (NGF), a 1:400 dilution of antiserum against p75 (αp75), or were preincubated with αp75 for 1 h prior to the addition of NGF (αp75 + NGF). *P b 0.001 when compared to all other groups. (B) p75 blockade specificity: cultured cells were grown in the presence of medium alone (Control), 50 ng/ml NGF, 1 μM forskolin, a 1:400 dilution of antiserum against p75 (αp75), or were preincubated with αp75 for 1 h prior to the addition of NGF (αp75 + NGF) or forskolin (αp75 + forskolin). ***P b 0.001 when compared to all other groups; **P b 0.01 compared to control and αp75; *P b 0.001 compared to control and αp75.
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Discussion In this study, we have investigated the mechanism by which NGF regulates the expression of SP, a nociceptive peptide likely to be involved in the development of neuropathic pain. We have utilized cultured adult DRG neurons to investigate the involvement of the two NGF receptors, TrkA and p75, in SP regulation. Both receptors can modulate a variety of cellular responses, and the signals each generates can augment or oppose the other (Kaplan and Miller, 2000). By utilizing targeted blocking antibodies and inhibitors, we have determined that both receptors are required for NGF to up-regulate SP in vitro. We also provide evidence that the critical events initiated by ligand binding to p75 occur regardless of the neurotrophin engaging it. We have demonstrated a critical role for p75 in SP regulation in adult rats. While early data suggested that the majority of NGF effects were mediated through TrkA, more recent reports describe an expanded role for p75 in neuronal development and in the cellular response to injury. p75, acting independently of Trk, can activate signaling cascades affecting apoptosis or survival (reviewed by Roux and Barker, 2002), myelination (Cosgaya et al., 2002), and neurite outgrowth (Wang et al., 2002; Wong et al., 2002). While the functions of p75 have been largely defined in developing systems, it plays a role in adult systems as well. Expression of bradykinin receptors is induced by NGF in adult mouse DRG (Petersen et al., 1998). Also, Zhang and Nicol (2004) recently reported that NGF excitation of adult rat nociceptive neurons is mediated through p75 and its downstream effector, ceramide. Taken together, these studies suggest a role for p75 in nociceptive pathways. Our observation that p75 blockade markedly reduces expression of SP supports this involvement, although the effect does not appears to be mediated through C2-ceramide (data not shown). The observation that other p75 neurotrophin ligands do not inhibit NGF up-regulation of SP, even in large excess, indicates that the process of p75 signaling within this pathway is not solely dependent on NGF. It is likely that activation of p75 by any of its neurotrophin ligands triggers the same sequence of events that enables it to cooperate with TrkA to regulate SP expression. This is evident in the case of BDNF, which, by itself, has no effect on SP expression (Adler, 1998), but which activates p75 as well as its own tyrosine kinase receptor, TrkB. However, the failure of NT-3 to inhibit NGFstimulated SP elevation is more perplexing since, in addition to activating p75 and its own specific tyrosine kinase receptor, TrkC, it can also bind to and activate TrkA (Belliveau et al., 1997; Cordon-Cardo et al., 1991). This raises two questions: if NT-3 activates both p75 and TrkA, why does it not increase SP by itself, and, why does it not prevent the NGF-induced SP increase? One possible explanation is that NGF induces expression of p75 (Mearow and Kril, 1995) which then inhibits activation of TrkA by NT3 but not NGF (Mischel et al., 2001). A second plausible scenario is provided by Kuruvilla and colleagues, who report that, unlike NGF/TrkA dimers, which are endocytosed and
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Fig. 4. Occupation of p75 by other neurotrophins does not inhibit up-regulation of Substance P by NGF. Dissociated cells were preincubated with 25 ng/ml of BDNF (A) or NT-3 (B) before the addition of 1.0 ng/ml of NGF. Control wells containing culture medium, and NGF-containing wells (1.0 ng/ml) were also included. (A) *P b 0.001 compared with control and BDNF; (B) *P b 0.001 compared to control and NT-3, P b 0.05 compared with NGF.
retrogradely transported to the cell body, NT-3/TrkA dimers remain on the cell surface (Kuruvilla et al., 2004). While little is known about the downstream events associated with NGFinduced SP elevation, it is possible that it may require receptor internalization and retrograde transport to accomplish this. The site of critical interaction between p75 and TrkA controlling SP expression remains unknown. However, our observations are consistent with the hypothesis that it occurs extracellularly, with p75 altering the activity of TrkA. If the interaction was intracellular, one would expect to see much more potentiation of SP expression when other neurotrophins are co-administered with NGF. The possibility exists that in long-term culture, TrkB and TrkC cells could express SP. There is precedent for this since Marchand et al. (1994) demonstrated that abnormal expression of PPT mRNA can be induced in nonpeptidergic neurons in vivo at late post-operative times following nerve injury. However, in the time frame of these experiments, no such alterations in expression were observed. There was an apparent difference in the efficacy of the wellcharacterized antisera to alter SP induced by NGF. While αp75 inhibited SP induced by 50 ng/ml of NGF by approximately 60%, αTrkA was ineffective against 50 ng of NGF (not shown) but reduced SP induced by 10 ng/ml NGF approximately 25%. The optimal dilution for efficient blocking by αp75 was empirically determined by titration (not shown), while the dilution used for the αTrkA experiments was obtained from the
literature (Mamet et al., 2003) and tested against 10 and 50 ng/ ml NGF. These differences are most likely due to innate differences in the affinity and/or avidity of each antibody for its specific ligand. The question could be raised whether the effect of NGF on SP could be mediated in part by p75 alone, without invoking TrkA. It is well known that p75 cooperates with many different proteins, including both the pro- and mature forms of neurotrophins, to evoke a variety of responses (reviewed in Lu et al., 2005). Mature neurotrophins preferentially bind to both p75 and their cognate Trk receptors to signal survival-related effects, while the pro form binds with high affinity to p75 alone and evokes death. While it is possible that p75 signaling is being activated by a small amount of proNGF present in the preparation used in this study (Chao and Bothwell, 2002), such signaling would be likely to induce cell death (Lee et al., 2001) with TrkA blockade, which we have not observed. The TrkA inhibitor, k252a, was used to confirm the involvement of that receptor in NGF regulation of SP. The inhibitor was also tested against GDNF, which increases SP by a mechanism independent of NGF (Adler, 1998) and, as expected, did not affect SP levels. When it was tested against forskolin, which increases SP by elevating intracellular cAMP downstream of the TrkA receptor (Adler and Walker, 2000), it also did not inhibit expression. However, k252a is also known to inhibit other kinases, including protein kinase A (pKA), the downstream effector of cAMP. This raises the issue of why it did not inhibit the forskolin-induced increase of SP. There are several plausible explanations for this observation (Tasken and Aandahl, 2004). Phosphorylation of pKA transduces a variety of cell responses induced by cAMP, activating or inhibiting many different proteins and enzymes. There are several different pKA isotypes, each with its own anchoring protein contributing specificity to its effect. Binding of a ligand to its transmembrane receptor activates adenylyl cyclase, causing cAMP to be locally released in proximity to its specific pkA and associated effectors (Tasken and Aandahl, 2004). When forskolin non-specifically elevates cAMP, it triggers widespread release of the messenger and activates many different pKAmediated downstream signaling events, each with its own effect. Furthermore, although pKA is by far the most common downstream effector of cAMP, it is not the only one (Tasken and Aandahl, 2004). The cAMP production up-regulated by forskolin would likely activate these transducers as well. Because of its involvement in nociceptive pathways, understanding the role NGF and its receptors plays in the expression of SP would help set the stage for uncovering the mechanisms underlying the induction and modulation of chronic pain and in the development of neuropathic pain following nerve injury. Defining the specific pathways the NGF receptors utilize to regulate SP would provide an opportunity to modulate this process and develop novel approaches for management and treatment.
Acknowledgment Grant sponsor: Department of Veterans Affairs.
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Regular Article
Nerve growth factor regulates substance P in adult sensory neurons through both TrkA and p75 receptors Anne M. Skoff a,⁎, Joshua E. Adler b a
Department of Neurology, Wayne State University School of Medicine, 8D University Health Center, 4201 St. Antoine, Detroit, MI 48201, USA Department of Neurology, Veterans Administration Medical Center and Wayne State University School of Medicine, Detroit, MI 48201, USA
b
Received 14 July 2005; revised 29 August 2005; accepted 4 October 2005 Available online 21 November 2005
Abstract Expression of the nociceptive peptide, substance P (SP) is regulated by the neurotrophin, nerve growth factor (NGF), and exogenous exposure to high levels of NGF increases its cellular content and release. NGF utilizes two receptors, the NGF-specific tyrosine kinase receptor, TrkA, and also the non-specific neurotrophin receptor, p75NTR (p75). The purpose of this study is to determine the relative involvement of these receptors in nociception. To investigate the role of TrkA in SP signaling, sensory neurons from adult rats were grown in vitro and exposed to a TrkA-blocking antibody. Pretreatment with the antibody inhibited NGF-induced SP elevation. Furthermore, when neurons were exposed to K252a, a relatively specific TrkA kinase inhibitor, the NGF effect on SP was also inhibited. K252a did not prevent SP up-regulation in cells exposed to forskolin or glial cell line-derived neurotrophic factor (GDNF), two agents which increase SP expression independently of TrkA. When p75 was blocked by antiserum, SP up-regulation by NGF was also inhibited. The antiserum neither impacted neuronal survival or basal levels of SP expression, nor did it inhibit SP up-regulation induced by forskolin. Two other neurotrophins, which are also ligands for p75, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) did not block NGF-induced SP up-regulation, raising the possibility that activated p75 is able to cooperate in SP regulation regardless of which neurotrophin ligand occupies it. Our data suggest that NGF up-regulation of SP expression requires the involvement of both TrkA and p75, although the specific contribution of each receptor to SP signaling remains to be determined. © 2005 Elsevier Inc. All rights reserved. Keywords: NGF; Tyrosine kinase A; P75 neurotrophin receptor; Substance P; Nociception; Regulation
Introduction The neurotrophin, nerve growth factor (NGF), plays a critical role in sensory neuronal development and physiology (LeviMontalcini and Angeletti, 1968; Thoenen and Barde, 1980), supporting survival of nociceptive neurons during critical periods of ontogeny (Johnson et al., 1980). NGF displays significant functionality in adult systems as well, modulating the sensitivity and plasticity of the nociceptive system (Mendell et al., 1999; Petruska and Mendell, 2004). It regulates such phenotypic traits as stimulus responsiveness (Lewin et al., 1993), response to injury (Olson et al., 1994; Rich et al., 1987), and neuropeptide expression (Kessler and Black, 1980; Otten et al., 1980). NGF's role in nociception is complicated. It induces rapid sensitization to noxious mechanical and thermal stimuli ⁎
Corresponding author. Fax: +1 313 5761112. E-mail address: [email protected] (A.M. Skoff).
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via direct sensitization of the heat receptor, TRPV1 (vanilloid receptor 1, Caterina et al., 1997), which does not involve gene transcription (Lewin and Mendell, 1993; Shu and Mendell, 1999), while also contributing to a slower, central sensitization, likely to involve the synthesis and release nociceptive peptides, including SP and CGRP (Mendell et al., 1999). Exogenous exposure to high levels of the growth factor, both in vivo and in vitro, increases the intracellular content and release of SP (Adler et al., 1984; Lindsay and Harmar, 1989; Malcangio et al., 1997a,b, 2000; Otten et al., 1980; Skoff et al., 2003; Vedder et al., 1993; Woolf et al., 1994). NGF effects are transduced through two transmembrane receptors, the tyrosine kinase receptor, TrkA, for which NGF is the primary ligand, and the non-specific neurotrophin receptor, p75, which binds all known neurotrophins, including NGF, BDNF, NT-3, and NT-4/5 with low affinity (Hempstead, 2002). p75 has no intrinsic catalytic activity but is a member of the tumor necrosis factor receptor (TNFR) superfamily, with an
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intracellular domain homologous to the “death domain” of TNFR1(Liepinsh et al., 1997). It has the capacity to autonomously activate apoptotic and survival promoting signal pathways and is required for high affinity binding of NGF to TrkA (for review, see Huang and Reichardt, 2003). TrkA signaling largely relates to neuronal survival and neurite outgrowth during development but later plays a significant role in the induction of hyperalgesia induced by inflammation (Cho et al., 1996; Woolf, 1996), likely through induction of TRPV1 expression (Mendell, 2002). When the two receptors are coexpressed, each can modulate the signaling of the other (reviewed in Hempstead, 2002; Roux and Barker, 2002). Development of neuropathic pain following nerve injury may be accompanied by alterations in level or distribution of the nociceptive peptide substance P (SP) in the dorsal horn of the spinal cord (Cameron et al., 1997; Malmberg and Basbaum, 1998; Munglani et al., 1995; Swamydas et al., 2004). Understanding the role NGF plays in SP expression would set the stage for uncovering the mechanisms underlying the induction and modulation of chronic pain. We have employed an in vitro culture system of adult rat dorsal root ganglion neurons to address the involvement of the NGF receptors in SP regulation. Adult sensory neurons survive well in culture without exogenous trophic factors but express only low levels of SP. Addition of NGF to the culture media increases expression of SP several fold (Lindsay and Harmar, 1989; Vedder et al., 1993). To determine the contribution of each of the NGF receptors to substance P expression, cultured neurons were exposed to a number of agents that block the activities of p75 or TrkA. Inhibition of either receptor reduced the NGF-induced increase of SP, suggesting that both of the receptors are required. Furthermore, while neither brainderived neurotrophic factor (BDNF) nor neurotrophin-3 (NT3), two other neurotrophin ligands for p75, was effective in increasing SP alone, their addition to the culture medium potentiated, rather than blocked the NGF-induced SP response. These data have been presented in preliminary form (Skoff and Adler, 2004).
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were seeded onto 12-well culture plates (24 mm/well) coated with poly D-lysine (100 μg/ml) and laminin (1 μg/cm2). The cells were plated at a density of approximately 10,000 viable DRG neurons per well. Ganglia from one rat provided sufficient cells for 12 to 16 wells. Cultures were incubated at 37°C in 95% air–5% CO2 at nearly 100% humidity. Incubation medium consisted of a 50/50 mixture of Ham's nutrient mixture F-12/ Dulbecco's modified Eagle's medium supplemented with penicillin (50 U/ml), streptomycin (50 mg/ml), 5% rat serum, glucose (3 mg/ml), glutamine (2 mM), ascorbic acid (300 μM), glutathione (3.5 μM), imidazole (100 μM), and p-amino benzoic acid (1 μg/ml). Cytosine arabinofuranoside (10 μM) was added to suppress Schwann cell and fibroblast proliferation. These non-neuronal cells comprised approximately 20% of the total population. One third of the medium was exchanged every other day. 2.5S NGF was obtained from Harlan Bioproducts for Science, Inc. (Madison, WI). Culture medium and supplements were obtained from Sigma and Gibco BRL. Following dissociation, the cells were resuspended in culture medium, plated into wells in 100 μl aliquots, and allowed to attach for 30–60 min. The cells were then preincubated with culture medium (control) or test agent for 1 h, after NGF (1, 10, or 50 ng/ml), forskolin (1.0 μM), or GDNF (10 ng/ml) was added. Cultures were grown for 1 week, at which time they were harvested for SP content. The cells from one well served as a single sample. Groups of 6 wells were exposed to each agent. Each experiment was repeated at least twice. NGF receptor blockade
Materials and methods
The blocking agents tested were anti-p75 NTR #9651 (αp75), a rabbit polyclonal antiserum to the extracellular domain of mouse p75 (diluted 1:500) (Huber and Chao, 1995) (gift of Moses Chao), and a blocking antibody to TrkA (αtrkA) (ab8771, Abcam, Cambridge, MA, used at 2 μg/ml). Also tested were two additional neurotrophins, brain-derived neurotrophic factor (BDNF) (Calbiochem, La Jolla, CA) and recombinant human neurotrophin-3 (NT-3) (R&D Systems, Minneapolis, MD). Both were used at 25 ng/ml.
Animals/dissection
TrkA receptor inhibition
Adult (N 3 months), female Sprague–Dawley rats (Charles River, Wilmington MA) were used as the source of sensory neurons. After rats were sacrificed by exposure to CO2, dorsal root ganglia were removed from all levels (approximately 50– 52 ganglia/rat) and maintained in calcium and magnesium-free Puck's Saline G (SalG) (Puck et al., 1958) for a maximum of 2 h before dissociation. The animal protocols used in this study have been approved by the Wayne State University Animal Investigation Committee and conform to NIH guidelines.
The protocol for TrkA receptor inhibition was identical to the blockade protocol above except that the TrkA inhibitor, K-252a (Nocardiopsis, Calbiochem; 400 nM), was used.
Dissociated cell culture Dorsal root ganglia (DRG) were dissociated and plated as described previously (Adler, 1998; Adler et al., 1984). Neurons
Extraction of substance P Peptide extraction was performed as described by Adler et al. (1984) utilizing a cell scraper adapted to fit the bottom of a 24-mm well plate. Briefly, after draining the medium and rinsing each well with 500 μl cold Puck's SalG containing calcium and magnesium, 250 μl of 0.01N HCl was added to each well and the cells scraped from the bottom. The samples were placed in a boiling water bath for 5 min, frozen, and lyophilized.
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Radioimmunoassay for substance P Substance P was assayed according to the method of Powell et al. (1973). (125 I)-labeled substance P (New England Nuclear, Boston, MA) was used as the tracer. Sensitivity of the assay was typically 2 pg per sample. Statistics Statistical analysis was performed using one-way ANOVA with a Newman–Keuls post-test, and the analyses were performed separately for each experiment. Results NGF regulates such diverse cellular responses and functions as survival, neurite outgrowth, myelination, and apoptosis through well-defined signaling pathways involving one or both of its receptors. Most identified NGF-mediated effects are related to development or are involved in injury response. However, the potential role of NGF and its receptors in nociception remains unclear. To better define the roles of these receptors, we examined them individually. Through the
Fig. 2. GDNF and forskolin signaling of substance P up-regulation is independent of the TrkA receptor. Dissociated cells were preincubated with k252A (400 nM) for 1 h after which they were exposed to either 10 ng/ml GDNF (k252a + GDNF) or 1 M forskolin (k252a + forskolin). Control wells, and wells exposed to GDNF, forskolin, or k252a alone were included for comparison. *P b 0.01 compared to control and k252a.
use of competitive blockers and/or receptor inhibitors of TrkA and p75 in vitro, we investigated receptor involvement in SP expression and determined that both receptors play a role. TrkA
Fig. 1. NGF up-regulation of substance P expression requires the TrkA receptor. (A) Receptor blockade: dissociated, mature, dorsal root ganglion neurons were grown in the presence of medium alone (Control), 10 ng/ml NGF (NGF), 2 μg/ ml of a blocking antibody to the TrkA receptor (αTrkA), or were preincubated with αTrkA for one hour prior to the addition of NGF (10 ng/ml) (NGF + αTrkA). *P b 0.001 compared with control and αTrkA, P b 0.01 compared with NGF + αTrkA; **P b 0.01 compared with control and αTrkA). (B) Receptor inhibition: cells were treated as in panel A except that NGF was increased to 50 ng/ml and k-252a (400 nM) was used in place of αTrkA (k252a + NGF). (*P b 0.001) In this (and all figures), substance P content was determined after 1 week. Bars represent mean substance P content/well, in picograms, from groups of six wells ±SEM.
Many of the NGF-mediated effects on sensory neurons are signaled through the TrkA receptor, either alone, or in cooperation with p75. To investigate its role in SP regulation, a TrkA-targeted blocking antibody, ab8771 (αTrkA) was added to cultures of mature spinal sensory neurons (Fig. 1A). After a week in culture, NGF increased SP expression four-fold. The αTrkA antibody reduced this by more than 25%. Antibody alone had no effect. A similar but more dramatic reduction was achieved by using the TrkA inhibitor, k252a, to block downstream signaling. It reduced SP to baseline levels (Fig. 1B). Because the TrkA inhibitor is known to inhibit other kinases which could adversely affect cell function and viability, it was tested against two other agents known to elevate SP: GDNF (Adler, 1998) and forskolin (Adler and Walker, 2000). Both agents increased SP at least four-fold, but addition of k252a to these stimulated neurons had no effect (Fig. 2), indicating that forskolin and GDNF up-regulation of SP involves alternate transduction pathways and that cell function and viability were not compromised by the kinase inhibitor. p75NTR To investigate the potential role of the p75 in SP upregulation, a well-characterized blocking antibody directed against the extracellular domain of this receptor was used to
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block NGF binding. While NGF again increased baseline SP levels more than four-fold, αp75 inhibited the NGF-induced SP level by more than 60% implicating the receptor in SP regulation (Fig. 3A). To confirm that the antibody was acting directly on the extracellular domain of p75 and was not actually damaging the neurons, the antibody was tested against forskolin. αp75 had no affect on SP levels induced by forskolin (Fig. 3B). This is consistent with the site of the αp75 inhibition being upstream of the cAMP second messenger system, which is located near the cytoplasmic interface of the plasma membrane. To determine whether the contribution of p75 to the SP response is specifically due to its interaction with NGF, two additional neurotrophins, BDNF and NT-3, also ligands for p75, were used to block NGF binding. Preincubation with a massive excess of either neurotrophin did not inhibit SP expression (Figs. 4A and B), suggesting activated p75, regardless of the neurotrophin bound to it, can cooperate with NGF to induce SP. Taken together, these data indicate that both NGF receptors, p75 and TrkA, are involved in SP upregulation by NGF.
Fig. 3. NGF up-regulation of substance P expression requires p75. (A) Receptor blockade: cells were grown in the presence of medium alone (Control), 50 ng/ml NGF (NGF), a 1:400 dilution of antiserum against p75 (αp75), or were preincubated with αp75 for 1 h prior to the addition of NGF (αp75 + NGF). *P b 0.001 when compared to all other groups. (B) p75 blockade specificity: cultured cells were grown in the presence of medium alone (Control), 50 ng/ml NGF, 1 μM forskolin, a 1:400 dilution of antiserum against p75 (αp75), or were preincubated with αp75 for 1 h prior to the addition of NGF (αp75 + NGF) or forskolin (αp75 + forskolin). ***P b 0.001 when compared to all other groups; **P b 0.01 compared to control and αp75; *P b 0.001 compared to control and αp75.
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Discussion In this study, we have investigated the mechanism by which NGF regulates the expression of SP, a nociceptive peptide likely to be involved in the development of neuropathic pain. We have utilized cultured adult DRG neurons to investigate the involvement of the two NGF receptors, TrkA and p75, in SP regulation. Both receptors can modulate a variety of cellular responses, and the signals each generates can augment or oppose the other (Kaplan and Miller, 2000). By utilizing targeted blocking antibodies and inhibitors, we have determined that both receptors are required for NGF to up-regulate SP in vitro. We also provide evidence that the critical events initiated by ligand binding to p75 occur regardless of the neurotrophin engaging it. We have demonstrated a critical role for p75 in SP regulation in adult rats. While early data suggested that the majority of NGF effects were mediated through TrkA, more recent reports describe an expanded role for p75 in neuronal development and in the cellular response to injury. p75, acting independently of Trk, can activate signaling cascades affecting apoptosis or survival (reviewed by Roux and Barker, 2002), myelination (Cosgaya et al., 2002), and neurite outgrowth (Wang et al., 2002; Wong et al., 2002). While the functions of p75 have been largely defined in developing systems, it plays a role in adult systems as well. Expression of bradykinin receptors is induced by NGF in adult mouse DRG (Petersen et al., 1998). Also, Zhang and Nicol (2004) recently reported that NGF excitation of adult rat nociceptive neurons is mediated through p75 and its downstream effector, ceramide. Taken together, these studies suggest a role for p75 in nociceptive pathways. Our observation that p75 blockade markedly reduces expression of SP supports this involvement, although the effect does not appears to be mediated through C2-ceramide (data not shown). The observation that other p75 neurotrophin ligands do not inhibit NGF up-regulation of SP, even in large excess, indicates that the process of p75 signaling within this pathway is not solely dependent on NGF. It is likely that activation of p75 by any of its neurotrophin ligands triggers the same sequence of events that enables it to cooperate with TrkA to regulate SP expression. This is evident in the case of BDNF, which, by itself, has no effect on SP expression (Adler, 1998), but which activates p75 as well as its own tyrosine kinase receptor, TrkB. However, the failure of NT-3 to inhibit NGFstimulated SP elevation is more perplexing since, in addition to activating p75 and its own specific tyrosine kinase receptor, TrkC, it can also bind to and activate TrkA (Belliveau et al., 1997; Cordon-Cardo et al., 1991). This raises two questions: if NT-3 activates both p75 and TrkA, why does it not increase SP by itself, and, why does it not prevent the NGF-induced SP increase? One possible explanation is that NGF induces expression of p75 (Mearow and Kril, 1995) which then inhibits activation of TrkA by NT3 but not NGF (Mischel et al., 2001). A second plausible scenario is provided by Kuruvilla and colleagues, who report that, unlike NGF/TrkA dimers, which are endocytosed and
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Fig. 4. Occupation of p75 by other neurotrophins does not inhibit up-regulation of Substance P by NGF. Dissociated cells were preincubated with 25 ng/ml of BDNF (A) or NT-3 (B) before the addition of 1.0 ng/ml of NGF. Control wells containing culture medium, and NGF-containing wells (1.0 ng/ml) were also included. (A) *P b 0.001 compared with control and BDNF; (B) *P b 0.001 compared to control and NT-3, P b 0.05 compared with NGF.
retrogradely transported to the cell body, NT-3/TrkA dimers remain on the cell surface (Kuruvilla et al., 2004). While little is known about the downstream events associated with NGFinduced SP elevation, it is possible that it may require receptor internalization and retrograde transport to accomplish this. The site of critical interaction between p75 and TrkA controlling SP expression remains unknown. However, our observations are consistent with the hypothesis that it occurs extracellularly, with p75 altering the activity of TrkA. If the interaction was intracellular, one would expect to see much more potentiation of SP expression when other neurotrophins are co-administered with NGF. The possibility exists that in long-term culture, TrkB and TrkC cells could express SP. There is precedent for this since Marchand et al. (1994) demonstrated that abnormal expression of PPT mRNA can be induced in nonpeptidergic neurons in vivo at late post-operative times following nerve injury. However, in the time frame of these experiments, no such alterations in expression were observed. There was an apparent difference in the efficacy of the wellcharacterized antisera to alter SP induced by NGF. While αp75 inhibited SP induced by 50 ng/ml of NGF by approximately 60%, αTrkA was ineffective against 50 ng of NGF (not shown) but reduced SP induced by 10 ng/ml NGF approximately 25%. The optimal dilution for efficient blocking by αp75 was empirically determined by titration (not shown), while the dilution used for the αTrkA experiments was obtained from the
literature (Mamet et al., 2003) and tested against 10 and 50 ng/ ml NGF. These differences are most likely due to innate differences in the affinity and/or avidity of each antibody for its specific ligand. The question could be raised whether the effect of NGF on SP could be mediated in part by p75 alone, without invoking TrkA. It is well known that p75 cooperates with many different proteins, including both the pro- and mature forms of neurotrophins, to evoke a variety of responses (reviewed in Lu et al., 2005). Mature neurotrophins preferentially bind to both p75 and their cognate Trk receptors to signal survival-related effects, while the pro form binds with high affinity to p75 alone and evokes death. While it is possible that p75 signaling is being activated by a small amount of proNGF present in the preparation used in this study (Chao and Bothwell, 2002), such signaling would be likely to induce cell death (Lee et al., 2001) with TrkA blockade, which we have not observed. The TrkA inhibitor, k252a, was used to confirm the involvement of that receptor in NGF regulation of SP. The inhibitor was also tested against GDNF, which increases SP by a mechanism independent of NGF (Adler, 1998) and, as expected, did not affect SP levels. When it was tested against forskolin, which increases SP by elevating intracellular cAMP downstream of the TrkA receptor (Adler and Walker, 2000), it also did not inhibit expression. However, k252a is also known to inhibit other kinases, including protein kinase A (pKA), the downstream effector of cAMP. This raises the issue of why it did not inhibit the forskolin-induced increase of SP. There are several plausible explanations for this observation (Tasken and Aandahl, 2004). Phosphorylation of pKA transduces a variety of cell responses induced by cAMP, activating or inhibiting many different proteins and enzymes. There are several different pKA isotypes, each with its own anchoring protein contributing specificity to its effect. Binding of a ligand to its transmembrane receptor activates adenylyl cyclase, causing cAMP to be locally released in proximity to its specific pkA and associated effectors (Tasken and Aandahl, 2004). When forskolin non-specifically elevates cAMP, it triggers widespread release of the messenger and activates many different pKAmediated downstream signaling events, each with its own effect. Furthermore, although pKA is by far the most common downstream effector of cAMP, it is not the only one (Tasken and Aandahl, 2004). The cAMP production up-regulated by forskolin would likely activate these transducers as well. Because of its involvement in nociceptive pathways, understanding the role NGF and its receptors plays in the expression of SP would help set the stage for uncovering the mechanisms underlying the induction and modulation of chronic pain and in the development of neuropathic pain following nerve injury. Defining the specific pathways the NGF receptors utilize to regulate SP would provide an opportunity to modulate this process and develop novel approaches for management and treatment.
Acknowledgment Grant sponsor: Department of Veterans Affairs.
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