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Am J Physiol Cell Physiol 295: C679-C691, 2008. First published July 2, 2008; doi:10.1152/ajpcell.00042.2008
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VASCULAR BIOLOGY

Neurokinin A engages neurokinin-1 receptor to induce NF-{kappa}B-dependent gene expression in murine macrophages: implications of ERK1/2 and PI 3-kinase/Akt pathways

Jia Sun, Raina Devi Ramnath, Ramasamy Tamizhselvi, and Madhav Bhatia

Department of Pharmacology, National University of Singapore, Singapore

Submitted 29 January 2008 ; accepted in final form 2 July 2008


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Neurokinin A (NKA) belongs to the tachykinin neuropeptide family. Its biological functions are primarily mediated by the neurokinin (NK)-2 receptor. NKA has been implicated in several inflammatory conditions. However, there are limited data about the mechanism of its pathogenetic action. Here, we investigated proinflammatory effects of NKA on peripheral immune cells using the mouse macrophage/monocyte cell line RAW 264.7 and primary peritoneal macrophages. The signaling mechanistic pathways involved were also studied. In mouse macrophages with no detectable NK-2 receptors, NKA induces the upregulation of NK-1 but not NK-2 receptor expression. Furthermore, NKA engages this NK-1 receptor, resulting in inflammatory-like responses involving activation of the transcription factor nuclear factor (NF)-{kappa}B and induction of NF-{kappa}B-responsive proinflammatory chemokine expression. NKA activates NF-{kappa}B as evidenced by induced phosphorylation (leading to degradation) of its inhibitory protein I{kappa}B{alpha}, increased cellular levels of the transactivation-active phospho(Ser276)-p65 and its nuclear translocation, as well as enhanced DNA-binding activity of NF-{kappa}B. These responses are specifically inhibited by selective NK-1 receptor antagonists but not NK-2 receptor antagonists, thereby excluding the role of NK-2 receptor. Further investigation on the upstream signaling mechanisms suggests that two NF-{kappa}B-activating pathways (extracellular signal-regulated kinase 1/2 and phosphatidylinositol 3-kinase/protein kinase B) are activated by NKA. Specific inhibitors of the two pathways block NF-{kappa}B-dependent chemokine expression. The inhibitory effects are mediated through regulation of nuclear translocation, DNA-binding activity, and/or transactivation activity of NF-{kappa}B. Together, we provide novel evidence that NKA engages NK-1 receptors on mouse macrophages to elicit NF-{kappa}B-dependent cellular responses. The findings reveal cellular mechanisms that may underlie NKA-mediated inflammatory and immunological conditions.

neuroimmunomodulation; tachykinins; leukocytes; chemokines; signaling transduction pathways

NEUROKININ A (NKA) belongs to the tachykinin family of neuropeptides, which also includes substance P (SP), neurokinin B, and the newly discovered hemokinins and endokinins. They are widely distributed within the central and peripheral nervous system as neurotransmitters and also expressed in nonneuronal cells contributing to the interactions between nervous and peripheral organ systems under both normal and pathological conditions (36). Tachykinins exert their biological actions through at least three distinct transmembrane G protein-coupled receptors: neurokinin (NK)-1, NK-2, and NK-3 receptors. SP, NKA, and NKB are the preferential ligands for NK-1, NK-2, and NK-3 receptors, respectively. However, there is a high level of promiscuity among the tachykinins and their receptors. All ligands can bind to each of the NK receptors with varying affinity (43).

SP and NKA are the two best-characterized members of the tachykinin family that have a broad spectrum of actions, including neuronal excitation, smooth muscle contraction, vasodilatation, plasma extravasation, salivation, nociception, and proinflammatory actions on immune and inflammatory cells (10). NKA, importantly, is a potent smooth muscle spasmogen in the respiratory, gastrointestinal, cardiovascular, and urinary system (10, 40) and has been implicated in airway inflammatory conditions such as smoking and asthma and gastrointestinal disorders (3, 6, 14, 27). NKA has been demonstrated to have regulatory effects on immune cells. It induces histamine release from mast cells (15) and primes neutrophils for increased superoxide anion production in response to formyl-methionyl-leucyl-phenylalanine (50). It stimulates superoxide anion production and tumor necrosis factor-{alpha} (TNF-{alpha}) mRNA expression from human monocytes (9, 29) and also induces de novo protein synthesis and release of interleukin (IL)-1, TNF-{alpha}, and IL-6 from human blood monocytes (30).

The biological functions of NKA are primarily mediated by NK-2 receptors. However, a number of studies show that NKA is also a functional ligand for the NK-1 receptor in vivo and in vitro (1, 5, 8, 17, 45). NKA is a high-affinity ligand for the NK-1 receptors expressed in transfected cell lines, despite its weak ability to displace SP-NK-1 receptor binding (17). NKA is found to mediate some central effects of NK-1 receptors in rat brain, which can be selectively blocked by NK-1 receptor antagonists (5, 17). NKA binds to NK-1 receptors in rat submandibular gland and elicits NK-1 receptor-mediated physiological responses, including salivation (1, 8). In cells such as spinal cord neurons that do not express NK-2 receptors, NKA and SP are found to activate NK-1 receptors at the same concentration (45).

We have earlier demonstrated that SP stimulation of mouse macrophagaes leads to selective chemokine induction via an NK-1 receptor-mediated, nuclear factor (NF)-{kappa}B-dependent mechanism. This observation, together with other earlier reports suggesting similar proinflammatory actions shared by SP and NKA, prompted us to investigate the possible effects of NKA in mouse macrophages, in particular pertaining to SP-elicited inflammatory responses, including activation of NF-{kappa}B and induction of NF-{kappa}B-dependent chemokine gene expression. The receptor specificity was examined using selective NK-1 and -2 receptor antagonists. Additionally, upstream mechanistic pathways were studied by investigating the role of multiple signaling protein kinases suggested previously to mediate signal transduction from SP/NK-1 receptor to NF-{kappa}B.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Cell culture and chemicals. RAW 264.7 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA), grown in DMEM (Invitrogen, Gaithersburg, MD) supplemented with 10% (vol/vol) FBS (Invitrogen), 2 mM L-glutamine, 100 U/ml penicillin, and 100 µg/ml streptomycin (Sigma-Aldrich, St. Louis, MO), and maintained at 37°C in a humidified atmosphere containing 5% CO2. NKA, the NK-1 receptor antagonist L-703,606 oxalate salt hydrate, and the NK-2 receptor antagonist L-659,877 were purchased from Sigma-Aldrich. The NK-1 receptor antagonist CP-96,345 was a gift from Pfizer Diagnostics. NK-1 and NK-2 receptor antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). The extracellular signal-regulated kinase (ERK) 1/2 inhibitor PD-98059 and phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor LY-294002 were obtained from Calbiochem, Merck (Darmstadt, Germany).

Isolation of primary peritoneal macrophages. Closed peritoneal lavage was performed on anesthetized mice by injection of 10 ml ice-cold PBS, followed by gravity drainage through 21-gauge needles. Peritoneal exudate cells were collected, washed one time with PBS, and resuspended in supplemented DMEM. Peritoneal macrophages were allowed to adhere in 12- or 24-well plates at 37°C in a humidified 5% CO2 incubator for 2 h, after which nonadherent contaminating cells were removed and the primary macrophage cultures were maintained for an additional 2 h before they were subjected to subsequent treatment and experiments. The preparations routinely contain >95% macrophages, as verified by microscopic examination with Turk's staining.

Treatment. RAW 264.7 cells in serum-free DMEM were treated with 10, 100, or 1 µM of NKA at 37°C for various durations as indicated in RESULTS. For the receptor antagonist experiments, cells were pretreated with CP-96,345, L-703,606 (100 nM), or L-659,877 (1 µM) for 15 min before NKA (1 µM) stimulation. For the ERK1/2 and PI 3-kinase/protein kinase B (Akt) inhibitor experiments, cells were pretreated with ERK1/2 inhibitor PD-98059 (10, 30, or 50 µM) or PI 3-kinase inhibitor LY-294002 (1, 5, 10, 30 µM) for 1 h before NKA (1 µM) stimulation. Peritoneal macrophages in supplemented DMEM were treated with 100 nM or 1 µM of NKA at 37°C for 12 h. For some experiments, the cells were pretreated with CP-96,345 (100 nM) or L-659,877 (1 µM) for 15 min or PD-98059 (10 µM) or LY-294002 (5 µM) for 1 h before NKA (1 µM) stimulation.

Whole cell lysate preparation and Western blot analysis. At the end of designated treatment, cells were lysed with chilled radioimmunoprecipitation assay lysis buffer supplemented with protease inhibitor cocktail (Roche, Basel, Switzerland) and phosphatase inhibitor cocktails (Sigma-Aldrich). Protein concentrations were determined by the Bradford protein assay (Bio-Rad Laboratories, Hercules, CA). Protein samples (50–100 µg) were separated on Novex 10% Tris-glycine polyacrylamide gel (Invitrogen) and transferred to polyvinylidene difluoride membrane by electroblotting in Novex transfer buffer (Invitrogen) containing 20% (vol/vol) methanol. Membranes were then washed, blocked, and probed overnight with rabbit phospho-I{kappa}B{alpha} (Ser32), phospho-NF-{kappa}B p65 (Ser276), phospho-Akt (Ser473), phospho-p44/42 mitogen-activated protein kinase (MAPK) (phospho-ERK1/2), p44/42 MAPK (ERK1/2) (1:1,000 dilution; Cell Signaling Technology Danvers, MA), NK-1R (1:500 dilution; Santa Cruz Biotechnology), or hypoxanthine-guanine phosphoribosyltransferase antibody (HPRT, 1:2,000; Santa Cruz Biotechnology), followed by horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG secondary antibody (1:2,000; Santa Cruz Biotechnology) for 2 h. Membranes were washed and then incubated in SuperSignal West Pico chemiluminescent substrate (Pierce, Rockford, IL) before exposure to X-ray films (CL-XPosure; Pierce). The intensity of bands was quantified using LabWorksImage Analysis software (UVP). HPRT was used as the housekeeping protein.

Nuclear extract preparation. Nuclear extracts were prepared at various time points after treatment for subsequent NF-{kappa}B DNA-binding activity assay or electrophoretic mobility shift assay. Cell nuclear fractions were extracted using a nuclear extract kit (Active Motif, Carlsbad, CA). Briefly, cells were washed, collected in ice-cold PBS in the presence of phosphatase inhibitors, and then centrifuged at 300 g for 5 min. Cell pellets were resuspended in a hypotonic buffer, treated with detergent, and centrifuged at 14,000 g for 30 s. After collection of the cytoplasmic fraction, the nuclei were lysed, and nuclear proteins were solubilized in lysis buffer containing proteasome inhibitors. Protein concentrations were determined by the Bradford protein assay (Bio-Rad Laboratories).

NF-{kappa}B DNA-binding activity assay. NF-{kappa}B DNA-binding activity was analyzed using the TransAMNF-{kappa}B p65 transcription factor assay kit (Active Motif) following the manufacturer's instructions. Briefly, nuclear extract (5 µg) was incubated in a 96-well plate coated with oligonucleotide containing the NF-{kappa}B consensus-binding sequence 5'-GGGACTTTCC-3'. Bound NF-{kappa}B was then detected by a specific primary antibody. An HRP-conjugated secondary antibody was then applied to detect the bound primary antibody and provided the basis for colorimetric quantification. The enzymatic product was measured at 450 nm by a microplate reader (Tecan Systems, San Jose, CA). Specificity of this assay was tested by the addition of wild-type or mutated NF-{kappa}B consensus oligonucleotide in the competitive or mutated competitive control wells before the addition of nuclear extracts.

RT-PCR. The effect of NKA on NK-1 and -2 receptor mRNA expression in RAW 264.7 macrophages was examined by RT-PCR. Briefly, cells were left untreated or treated with 1 µM NKA for the indicated periods described in RESULTS at 37°C before total RNA was extracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany). RNA was quantitated spectrophotometrically by absorbance at 260 nm. Total RNA (1 µg) RNA was reverse-transcribed using the iScriptTM cDNA Synthesis Kit (Bio-Rad Laboratories). The cDNA synthesized was used as the template for PCR amplification using iQTM Supermix (Bio-Rad Laboratories) in MyCycler (Bio-Rad Laboratories). The PCR protocol consisted of optimal 30–40 cycles of denaturation at 95°C for 50 s, annealing for 60 s, and extension at 72°C for 50 s. The number of amplification cycles was optimized to assure that the reaction was terminated in the linear range of amplification for each gene. The following specific primer pairs (Proligo; Singapore) were used: NK-1 receptor sense 5'-CTTGCCTTTTGGAACCGTGTG-3' and antisense 5'-CACTGTCCTCATTCTCTTGTG-3'; NK-2 receptor sense 5'-TGCTGTCATCTGGCTGGTAG-3' and antisense 5'-TCTTCCTCGGTTGGTGTCCC-3'; glyceraldehyde-3-phosphate dehydrogenase (GAPDH) sense 5'-GCATCTGAGGGCCCACTGAAG-3' and antisense 5'-GTCCACCACCCTGTTGCTGTA-3'. Amplification of the GAPDH gene transcript was used as an internal control of RT-PCR reactions among samples. PCR products were analyzed on 1.5% wt/vol agarose gels containing 0.05 mg/100 ml ethidium bromide. Densitometry results from PCR products were normalized to the housekeeping gene GAPDH.

Immunofluorescence. After treatment, cells attached to six-well plates were fixed in 3.7% formaldehyde for 10 min at room temperature, washed, and blocked using nonimmune rabbit sera for 10 min. Following blocking, cells were incubated with 1:50-diluted rabbit anti-mouse NK-1 receptor or NK-2 receptor antibodies (Santa Cruz Biotechnology) for 2 h at room temperature. For the negative control, cells were incubated with blocking sera in place of primary antibodies. After being washed with PBS, the cells were incubated with 1:100-diluted rhodamine-conjugated anti-rabbit IgG secondary antibodies (Santa Cruz Biotechnology) for 2 h at room temperature. The cells were washed in PBS and mounted in Gel Mount (Sigma-Aldrich). Staining was analyzed by fluorescence microscopy using a Leica DM IRB microscope.

Enzyme-linked immunosorbent assay. Chemokine concentration in the media of cultured RAW 264.7 cells was determined using murine macrophage inflammatory protein (MIP)-2 and monocyte chemoattractant protein (MCP)-1 Duoset enzyme-linked immunosorbent assay (ELISA) kits (R&D Systems, Minneapolis, MN) as described previously (39) according to the instructions of the manufacturer. Samples were run in triplicates for each condition in three independent experiments. Absorbance was measured at 450 nm by a microplate reader (Tecan Systems). Results were expressed as picograms per milliliter for each chemokine.

Statistical analysis. Data are expressed as means ± SD. Statistical analyses were performed by independent t-test or, when multiple comparisons were made, by one-way ANOVA with post hoc Tukey's test using SPSS program version 13.0 (Chicago, IL). A P value of <0.05 was considered a statistically significant difference.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
NKA upregulates NK-1 receptor expression in mouse macrophages lacking detectable NK-2 receptors. We first evaluated NK-1 and -2 receptor expression in unstimulated or NKA-stimulated RAW 264.7 macrophages. Cells treated with vehicle or NKA for various periods of time were subjected to RT-PCR analysis of the receptor mRNA expression or Western blot and/or immunofluorescence staining to determine the protein expression. NK-1 receptor expression was readily detected in the cells under baseline conditions. NKA treatment further induced NK-1 receptor expression at both mRNA and protein levels (Fig. 1, A–E). The increase in NK-1 receptor mRNA expression was most evident 40 min after NKA treatment and decreased thereafter. The increase in NK-1 receptor protein expression became significant after 90 min of NKA stimulation and lasted until 150 min of stimulation (Fig. 1, C–E). The NK-2 receptor mRNA expression was not found in either unstimulated or NKA-stimulated cells (Fig. 2A). Consistently, the receptor protein expression was not detected in the cells (Fig. 2B). These results indicate that NKA upregulates NK-1 receptor expression in mouse macrophages that lack NK-2 receptor expression. The findings prompted us to investigate the interaction of NKA with this NK-1 receptor and downstream cellular events triggered by the receptor engagement.


Figure 1
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  		Fig. 1. Neurokinin (NK) A upregulates NK-1 receptor mRNA and protein expression in mouse macrophages. Cells were left untreated or treated with 1 µM of NKA for various time periods as indicated. NK-1 receptor mRNA expression was determined by RT-PCR. NK-1 receptor protein expression was determined by Western blotting and immunofluorescence. A: RT-PCR detection of NK-1 receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA expression. B: densitometric analysis of NK-1 receptor mRNA expression relative to GAPDH. C: Western blots of NK-1 receptor and the housekeeping protein hypoxanthine-guanine phosphoribosyltransferase (HPRT). D: densitometric analysis of NK-1 receptor protein expression relative to HPRT. Results are means ± SD for duplicate measurements and from three separate experiments. *P < 0.05, compared with basal level (0 min). E: immunofluorescence staining of NK-1 receptor protein expression in untreated and NKA-treated cells. Representative micrographs from three separate experiments are shown. Original magnification, x100.

 

Figure 2
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  		Fig. 2. Lack of detectable NK-2 receptor expression in mouse macrophages. Untreated or NKA-treated RAW 264.7 cells were subjected to RT-PCR and immunofluorescence staining for NK-2 receptor mRNA and protein expression. A: NK-2 receptor mRNA expression was not detected in RAW 264.7 cells. L, 100-bp DNA ladder; S1, unstimulated cells; S2, cells treated with NKA for 150 min; PC, positive control, mouse lung tissues. B: NK-2 receptor protein expression was not found in RAW 264.7 cells. Left: negative control, cells were incubated with nonimmune rabbit sera in place of primary antibody. Middle: cells stained negative for NK-2 receptors. Representative micrographs from three separate experiments are shown. Right: 3T3 mouse fibroblasts stained positive for NK-2 receptors. Original magnification, x100.

 
NKA triggers NF-{kappa}B activation in mouse macrophages via the NK-1 receptor. One key event observed in the cells after NKA stimulation was the activation of transcription factor NF-{kappa}B. NF-{kappa}B activation was evidenced by the phosphorylation of NF-{kappa}B inhibitory protein I{kappa}B (which leads to degradation of I{kappa}B by proteasome) and phosphorylation of p65, the subunit responsible for the transactivation activity of NF-{kappa}B. Western blotting using specific antibodies that recognize only the phosphorylated form of I{kappa}B protein indicated the phosphorylation of I{kappa}B protein starting 5 min after NKA treatment until 30 min (Fig. 3, A and B). This was accompanied by enhanced phosphorylation (Ser276) of p65 subunit in the whole cell lysate and the nuclear extract, indicating the increased cellular phosphorylation status of p65 subunit (Fig. 3, A and C) and nuclear translocation of this transactivation active phospho-p65 protein (Fig. 3, D and E). We further measured the effect of NKA on the ability of NF-{kappa}B to bind DNA using NF-{kappa}B DNA-binding assay. The result revealed that NKA treatment led to a notable increase in the DNA-binding activity of NF-{kappa}B. Kinetic analysis showed a biphasic increase of NF-{kappa}B activity, with the first peak at 30–60 min and the second after 150 min following NKA treatment (Fig. 4A). Furthermore, the NKA-induced increase in NF-{kappa}B activity was NK-1 receptor mediated. Pretreatment of cells with selective NK-1 receptor antagonist CP-96,345 or L-703,606 before NKA treatment significantly inhibited NKA-induced NF-{kappa}B activity. The selective nonpeptide NK-2 receptor antagonist L-659,877 did not have any effect (Fig. 4B).


Figure 3
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  		Fig. 3. NKA activates the transcription factor nuclear factor (NF)-{kappa}B. A: NKA induction of phosphorylation of NF-{kappa}B inhibitory protein I{kappa}B and NF-{kappa}B p65 subunit. Cells were stimulated with 1 µM NKA for 0–60 min before whole cell lysates were prepared for immunoblotting analysis of phospho-I{kappa}B and phospho(Ser276)-p65 protein expression. Equal sample loading was determined by internal control HPRT. B: densitometric analysis of phospho-I{kappa}B expression relative to HPRT. C: densitometric analysis of phospho-p65 expression relative to HPRT. D: time course of nuclear translocation of the transactivation-active subunit phospho(Ser276)-p65 following NKA stimulation. Cells were treated with NKA (1 µM) for 0–180 min. Nuclear fractions were then extracted for Western blot analysis of nuclear levels of phospho-p65. E: densitometry analysis of nuclear phospho-p65. Results are means ± SD of three separate experiments. P < 0.05 compared with basal level (0 min; *) and compared with the corresponding time point control (+).

 

Figure 4
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  		Fig. 4. NKA enhances the DNA-binding activity of NF-{kappa}B via the NK-1 receptor. A: time course of NKA-induced NF-{kappa}B DNA-binding activity in RAW 264.7 cells following NKA stimulation. B: NKA induction of NF-{kappa}B p65 activity is NK-1 receptor dependent. Cells were preincubated with selective NK-1 receptor antagonist CP-96,345 or L-703,606 (100 nM) or a selective NK-2 receptor antagonist L-659,877 before NKA stimulation. Results are means ± SD for triplicate measurements and from three separate experiments. P < 0.05, compared with basal level (0 min) or control (*) and compared with NKA ({dagger}).

 
NKA triggers NF-{kappa}B-dependent proinflammatory chemokine production in mouse macrophages via the NK-1 receptor. We next investigated whether NKA could induce NF-{kappa}B-responsive gene expression. The expression of two chemokines, MIP-2 and MCP-1, inducible by NK-1 receptor engagement and the transcription of which is NF-{kappa}B-dependent was examined. RAW 264.7 macrophages were incubated with three doses of NKA (1 nM, 100 nM, and 1 µM) for 2, 6, 12, and 24 h. The supernatants were harvested at different time points for ELISA measurement of the two chemokine levels. Significant increases in MIP-2 and MCP-1 release from RAW 264.7 macrophages were observed with 1 µM NKA after 12 and 24 h of incubation. No significant effect was noted for lower doses (Fig. 5, A and B).


Figure 5
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  		Fig. 5. NKA induces NF-{kappa}B-dependent chemokine expression in RAW 264.7 macrophages via the NK-1 receptor. A and B: macrophage inflammatory protein (MIP)-2 and monocyte chemoattractant protein (MCP)-1 production by RAW 264.7 macrophages following NKA stimulation. Cells were treated with 1 nM, 100 nM, or 1 µM of NKA for 2, 6, 12, or 24 h. C and D: NKA-stimulated MIP-2 and MCP-1 production is NK-1 receptor dependent. Cells were preincubated with 100 nM CP-96,345 or L-703,606, the specific NK-1 receptor antagonists, or 1 µM L-659,877, the selective NK-2 receptor antagonist, for 15 min before addition of NKA (1 µM) for 12 h. MIP-2 and MCP-1 concentrations were measured by enzyme-linked immunosorbent assay (ELISA) with the supernatants harvested. Results are expressed as means ± SD for triplicate measurements and from three separate experiments. P < 0.05, compared with control (*) and compared with NKA ({dagger}).

 
In addition, we found that NKA-elicited NF-{kappa}B-dependent gene expression was also mediated by NK-1 receptor. Preincubation of cells with either CP-96,345 or L-703,606 (100 nM) before NKA treatment inhibited NKA-induced chemokine release. Specific NK-2 receptor antagonist L-659,877 caused no difference in the chemokine production (Fig. 5, C and D).

An inductive effect of NKA on chemokine expression was also demonstrated in mouse primary peritoneal macrophages. NKA (1 µM) induced a significant increase of MIP-2 and MCP-1 production after 12 h of treatment (Fig. 6, A and B). NKA-induced chemokine production in peritoneal macrophages was also NK-1 receptor mediated. This was evidenced by preincubation of primary macrophages with the NK-1 receptor antagonist CP-96,345 but not the NK-2 receptor antagonist L-659,877, which significantly attenuated the NKA-induced increase in both MIP-2 and MCP-1 production by the cells (Fig. 6, C and D).


Figure 6
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  		Fig. 6. NKA induces MIP-2 and MCP-1 production in mouse primary macrophages via the NK-1 receptor. A and B: MIP-2 and MCP-1 production by mouse peritoneal macrophages following NKA stimulation. Cells were treated with 0.1 or 1 µM of NKA for 12 h. C and D: NKA-stimulated MIP-2 and MCP-1 production is NK-1 receptor dependent. Cells were preincubated with 100 nM CP-96,345 or 1 µM L-659,877 for 15 min before addition of NKA (1 µM) for 12 h. MIP-2 and MCP-1 concentrations were measured by ELISA with the supernatants harvested. Results are expressed as means ± SD for triplicate measurements and from three separate experiments. P < 0.05, compared with control (*) and compared with NKA ({dagger}).

 
NKA activates ERK1/2 and Akt signaling kinases upstream of NF-{kappa}B in mouse macrophages. We further examined the upstream NF-{kappa}B-activating kinases, including MAPKs and Akt potentially involved in NKA-NK-1 receptor responses in RAW 264.7 macrophages. Cells were treated with 1 µM of NKA for 0, 5, 15, 30, or 60 min. Subsequently, whole cell lysates were subjected to Western blotting using phosphospecific antibodies to detect the phosphorylated (and active) kinases. Among the MAPK members, only ERK1/2 was found to be activated by NKA in the cells, whereas p38 MAPK and c-jun NH2-terminal kinase remained low (data not shown). As shown in Fig. 7, A and B, NKA induced phosphorylation of ERK1/2 in a time-dependent manner without changing total ERK1/2 protein levels, suggesting that NKA activates ERK1/2 signaling pathways. Akt, a serine/threonine kinase that has an important role in regulating cellular growth, differentiation, adhesion, and the inflammatory reaction, was also found to be upregulatable by NKA (Fig. 7, C and D). Both ERK1/2 and Akt were activated early in the cells within 5 min after NKA stimulation, followed by a gradual decrease of the activated protein until 60 min.


Figure 7
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  		Fig. 7. NKA induces activation of extracellular signal-regulated kinase (ERK) 1/2 and protein kinase B (Akt) signaling kinases in RAW 264.7 macrophages. The cells were treated with 1 µM NKA for 0–60 min. Whole cell lysates were then prepared for Western blotting to detect the phosphorylated (and thus active) forms of the kinases. A: phospho-ERK1/2 and total ERK1/2 protein levels. B: densitometric analysis of phospho-ERK1/2 protein expression relative to total ERK1/2. C: phospho(Ser473)-Akt and total Akt protein levels. D: densitometric analysis of phospho-Akt protein expression relative to total Akt. Results are means ± SD of three separate experiments. P < 0.05, compared with basal level (0 min; *) and compared with the corresponding time point control (+).

 
Blockade of ERK1/2 or PI 3-kinase/Akt activation inhibits NKA-induced NF-{kappa}B activation and NF-{kappa}B-dependent gene expression. Given that NKA induced activation of ERK1/2 and Akt, a PI 3-kinase downstream effector kinase, we further determined whether ERK1/2 and PI 3-kinase/Akt mediated NKA-induced NF-{kappa}B activation and the resultant gene transcription in macrophages. Specific inhibitors of ERK1/2 (PD-98059) and PI 3-kinase/Akt (LY-294002) were added to the cells 1 h before NKA stimulation. The inhibitor effects on NKA-induced NF-{kappa}B DNA-binding activity and nuclear translocation of phospho(Ser276)-p65 were investigated.

NF-{kappa}B DNA-binding activity assays demonstrated that inhibition of PI 3-kinase/Akt significantly blocked NKA-induced NF-{kappa}B DNA-binding activity. However, there was no significant alteration of NKA-induced NF-{kappa}B activity when cells were pretreated with ERK1/2 inhibitor PD-98059 (Fig. 8A). The results suggest a role for PI 3-kinase/Akt but not ERK1/2 in regulating DNA-binding activity of NF-{kappa}B. However, both inhibitors significantly reduced nuclear levels of phospho(Ser276)-p65 protein (Fig. 8B). Because phosphorylation of p65 at Ser276 residue is associated with coactivator recruitment to the NF-{kappa}B transcription complex and enhanced transactivational activity of the protein, ERK1/2 and PI 3-kinase/Akt may both be implicated in the posttranslational phosphorylation and regulation of transactivation potential of the p65 subunit.


Figure 8
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  		Fig. 8. PI 3-kinase/Akt and ERK1/2 mediate NKA-induced signal transduction, leading to NF-{kappa}B activation. RAW 264.7 cells were either left untreated (control) or pretreated with the specific inhibitor of PI 3-kinase/Akt (LY-294002) or of ERK1/2 (PD-98059) and subsequently stimulated with 1 µM NKA for 30 min. Nuclear fractions were prepared and analyzed for the DNA-binding activity of NF-{kappa}B and phospho-p65 expression. A: effects of PI 3-kinase/Akt and ERK inhibitors on NKA-induced DNA binding of NF-{kappa}B. B: effects of PI 3-kinase/Akt and ERK inhibitors on nuclear phospho-p65 levels. C: densitometric analysis of nuclear phospho-p65 levels. Results are means ± SD of three separate experiments. P < 0.05, compared with control (*) and compared with NKA ({dagger}).

 
As shown in Fig. 9, A and C, PD-98059 dose dependently inhibited both MIP-2 and MCP-1 protein synthesis induced by NKA. Although the inhibition was maximal with 50 µM, the inhibitor was effective at a low dose of 10 µM. LY-294002, at the dose range of 1–10 µM, also significantly attenuated NKA-induced MIP-2 and MCP-1 production (Fig. 9, B and D).


Figure 9
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  		Fig. 9. Blockade of ERK1/2 or PI 3-kinase/Akt pathway inhibits NKA-induced chemokine synthesis in RAW 264.7 cells. The cells were either left untreated or were pretreated with ascending concentrations of the ERK1/2 inhibitor PD-98059 or the PI 3-kinase/Akt inhibitor LY-294002 and subsequently stimulated with 1 µM NKA for 12 h. MIP-2 (A and B) and MCP-1 (C and D) levels were measured in cell supernatants by ELISA. Results are means ± SD for triplicate measurements and from three separate experiments. P < 0.05, compared with control (*) and compared with NKA ({dagger}).

 
ERK1/2 and PI 3-kinase/Akt signaling pathways are also important in the NKA-induced chemokine production in primary macrophages. PD-98059 (10 µM) and LY-294002 (5 µM) pretreatment significantly inhibited NKA-induced increase of MIP-2 and MCP-1 production by primary peritoneal macrophages (Fig. 10, A and B).


Figure 10
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  		Fig. 10. ERK1/2 or PI 3-kinase/Akt pathways are important for NKA-induced chemokine production in mouse primary macrophages. Isolated cells were either left untreated or were pretreated with PD-98059 (10 µM) or LY-294002 (5 µM) and subsequently stimulated with 1 µM NKA for 12 h. MIP-2 (A) and MCP-1 (B) levels were measured in cell supernatants by ELISA. Results are means ± SD for triplicate measurements and from three separate experiments. P < 0.05, compared with control (*) and compared with NKA ({dagger}).

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
In this study, we report that NKA is able to engage the NK-1 receptor on mouse macrophages and induce activation of the transcription factor NF-{kappa}B and expression of NF-{kappa}B-driven genes. Furthermore, PI 3-kinase/Akt and ERK1/2 kinases are identified to mediate the signal transduction from NKA/NK-1 receptor to NF-{kappa}B. The findings presented here suggest a hitherto unknown mechanism of the neuropeptide NKA regulation of inflammatory responses in immune cells via the NK-1 receptor.

Up to date, there are limited data in the literature on the proinflammatory effects of NKA on macrophage functions. In comparison, the closely related neuropeptide SP has been demonstrated by convincing data to regulate a variety of functions of macrophages (35). SP and NKA are both tachykinin neuropeptides produced from a single precursor, PPT-A gene, and colocalized and coreleased from primary afferent nociceptors (45). Specific effects of these two neuropeptides are generally achieved by binding to their differential preferred receptors (NK-1 and -2 receptors for SP and NKA, respectively) on the target cells. Our RT-PCR analysis and immunofluorescence staining demonstrate functional NK-2 receptor, the preferential receptor for NKA, is not found in RAW 264.7 cells. The NK-1 receptor, however, is upregulated in the cells after NKA treatment, which raises the possibility that, in the absence of NK-2 receptor, NKA may act via NK-1 receptor on RAW 264.7 macrophages to elicit NK-1 receptor-mediated cellular responses, comparable to SP. This notion has been supported by earlier studies that suggest NKA can bind to the same NK-1 receptors as SP (1, 8, 17, 42, 45). The common COOH-terminal sequences of SP and NKA are suggested to contact the same region of NK-1 receptor (7). Convincing evidence demonstrates that NKA is an important ligand for NK-1 receptor in vivo to elicit NK-1 receptor-mediated physiological responses such as salivation (8). In spinal cord neurons where NK-2 receptor expression is not identified, NKA equally activates NK-1 receptor compared with SP (45). It is suggested that NKA and SP share some functional characteristics, since the two neuropeptides act in NK-1 receptor signaling at the same doses (1, 42). Consistent with these studies, we demonstrate that NKA and SP stimulate similar responses in macrophages: NF-{kappa}B activation and expression of NF-{kappa}B-responsive chemokines via the NK-1 receptor. Lack of effects on NKA-induced NF-{kappa}B activation by selective NK-2 receptor antagonist at pharmacologically active doses confirmed NK-1 receptor is the primary receptor for NKA-mediated effects in mouse macrophages.

One observation of this study at variance with some earlier data is the dose difference between SP and NKA in inducing NK-1 receptor-mediated effects (1, 42, 45). Whereas others report similar concentrations of NKA and SP in eliciting NK-1 receptor-mediated effect, we find the effective dose of NKA (1 µM) is higher than that of SP (10–100 nM). That NKA is a less potent stimulator of macrophages than SP may be due to its relatively lower affinity for NK-1 receptor or possible differences in signaling mechanisms. In fact, earlier studies also suggest that, despite some comparable effects of NKA and SP, the two neuropeptides do not bind to NK-1 receptor identically (45). A domain located at the end of the second extracellular loop of the NK-1 receptor is identified to be necessary for the binding and biological activity of NKA but not SP (48). This hypothesis is reinforced by the ability of some NK-1 receptor antagonists such as GR-205171 to selectively block NKA-mediated NK-1 receptor responses. It is likely that GR-205171 more directly inhibits the NKA binding portion of the receptor. Additionally, other NK-1 receptor antagonists, including CP-96,345, have been shown to be better inhibitors of NKA than of SP (31, 45), which is consistent with our finding that CP-96,345 inhibits effects of NKA at lower doses than of SP. Other evidence supporting this hypothesis shows that, whereas SP increases levels of both cAMP and inositol trisphosphate (IP3) in NK-1 receptor-transfected CHO cells, NKA only affects IP3 (42).

Neuropeptide regulation of various functions of immune cells provides a mechanism for neural control of immune and inflammatory responses. NKA, in addition to SP, is an important neuropeptide involved. In addition to earlier findings (9, 15, 29, 30, 50), our study, for the first time, shows that NKA stimulation of murine macrophages leads to activation of NF-{kappa}B, a transcription factor that has a crucial modulatory role in inflammation, immunity, cell proliferation, and apoptosis (53). NF-{kappa}B can be activated by three mechanisms: the classical pathway dependent on NF-{kappa}B inhibitory protein I{kappa}B degradation and two atypical pathways [one is through the processing of p100 and release of p52/RelB in the nucleus; the other is through the phosphorylation of p65 at multiple serine sites by some protein kinases (34, 47)]. The classical pathway and posttranslational modifications (phosphorylation) of p65 subunit are involved in NKA activation of NF-{kappa}B in the cells. This is evidenced by NKA-induced phosphorylation (leading to subsequent degradation) of the inhibitory protein of NF-{kappa}B, I{kappa}B{alpha}, and nuclear translocation of phospho(Ser276)-NF-{kappa}B p65. Also, the DNA-binding activity of NF-{kappa}B is enhanced in macrophages after exposure to NKA. Furthermore, NKA induces NF-{kappa}B-regulated chemokine (MIP-2 and MCP-1) expression in murine macrophages. Consistently, stimulation of NK-1 receptor on immune cells resulting in activation of NF-{kappa}B and proinflammatory gene expression has been described in a number of in vitro systems (2, 4, 44, 49). Because both MCP-1 and MIP-2 have been implicated in the development of clinical and experimental asthma to which NKA has a strong pathophysiological association (13, 21, 33, 46), our study suggests that alveolar macrophages present in the bronchioles and large airways heavily innervated by NKA- and SP-containing nerves (6, 27) might be the cellular sources of these chemokines in asthma.

Further study on the inductive mechanisms for NKA-elicited NF-{kappa}B responses reveals two important signaling pathways (ERK1/2 MAPK and PI 3-kinase/Akt) involved in mediating the responses.

MAPKs, important NF-{kappa}B-activating kinases, are proline-directed protein serine/threonine kinases activated by a cascade of intracellular phosphorylation events (11, 12, 16, 18, 51). Their substrates, located in the cytoplasm as well as in the nucleus, include other kinases, transcription factors, phospholipases, and cytoskeletal proteins (25, 28, 37, 41). Among all MAPK members, only ERK1/2 are found to be activated by NKA in RAW 264.7 macrophages. The role of ERK1/2 in NKA-triggered intracellular signaling is further characterized using a potent selective inhibitor of ERK1/2 (PD-98059) that inhibits the phosphorylation and activation of ERK by its immediate upstream activator mitogen/extracellular signal-regulated kinase. Pretreatment of cells with PD-98059 dose dependently attenuates chemokine production induced by NKA. Moreover, inhibition of ERK1/2 results in diminished nuclear levels of phospho(Ser276)-p65, a transactivation-active subunit of NF-{kappa}B, although it exhibits minimal effect on the DNA-binding activity of NF-{kappa}B. These results suggest that ERK1/2 mediate NKA-induced NF-{kappa}B activation and proinflammatory chemokine expression.

In addition to ERK1/2, another signaling protein kinase (Akt) is also found to be activated by NKA. Akt, a serine/threonine kinase, is a direct downstream effector of PI 3-kinase. PI 3-kinase/Akt has an important role in regulating cellular growth, differentiation, adhesion, and the inflammatory reaction (19, 24). Although the role of PI 3-kinase in the inflammatory and immunological responses has been controversial (38), evidence has suggested that PI 3-kinase/Akt regulates NF-{kappa}B activity in response to a variety of extracellular stimuli (22, 23, 32). Given that NKA treatment stimulates activation of Akt in macrophages as shown here, we hypothesize that PI 3-kinase/Akt might be involved in NKA-induced NF-{kappa}B responses in RAW 264.7 cells. Supporting this hypothesis, it is found that inactivation of PI 3-kinase with the specific pharmacological inhibitor LY-294002 inhibits NKA induction of phosphorylation(Ser276) of the transactivation subunit p65 and production of the two NF-{kappa}B-inducible chemokines. The inhibitor also blocks NKA-induced NF-{kappa}B DNA binding, an effect not observed with the ERK1/2 inhibitor PD-98059. Ser276 is inducibly phosphorylated by protein kinase A and mitogen- and stress-activated protein kinase-1 to enhance the transactivational potential of the subunit p65, which is mainly responsible for transactivaiton activity. These data suggest that PI 3-kinase and ERK1/2 lie upstream of NF-{kappa}B activation in response to NKA in RAW 264.7 cells. It is at present unclear, however, how NKA treatment triggers PI 3-kinase/Akt and ERK1/2 activation in RAW cells. NK-1 receptor-mediated-NF-{kappa}B activation is frequently associated with activation of receptor-associated G proteins and/or their effectors, including small GTPases, phospholipase C, calcium, and protein kinase C (20, 26, 49, 52). The role of these signaling molecules in the context of NKA/NK-1 receptor signaling remains to be investigated.

In conclusion, we demonstrate for the first time that NKA stimulates NK-1 receptor-mediated cellular signaling in mouse macrophages. NKA/NK-1 receptor signaling leads to activation of the transcription factor NF-{kappa}B and the resultant proinflammatory chemokine production in the cells. NF-{kappa}B activation in this system involves at least two signaling pathways: ERK1/2 and PI 3-kinase/Akt (Fig. 11). The findings presented by this study suggest NKA as an important mediator of neuroimmunomodulatory activity and that macrophages and proinflammatory chemokines may be implicated in NKA-mediated inflammatory and immunological conditions. Further elucidation of key signaling molecules involved may have important implications in controlling the conditions.


Figure 11
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  		Fig. 11. NKA-induced signal transduction cascade in mouse macrophages. NKA engaging the NK-1 receptor activates both ERK1/2 and PI 3-kinase/Akt signaling pathways, which activate NF-{kappa}B by enhancing phosphorylation of NF-{kappa}B p65 and its nuclear translocation. NF-{kappa}B activation leads to subsequent expression of proinflammatory chemokine genes.

 

   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Academic Research Fund Grant R-184-000-054-112, Office of Life Sciences Cardiovascular Biology Program Grant R-184-000-074-712, and the Bridging Grant R-184-000-139-101.


   ACKNOWLEDGMENTS
 
We thank Akhil Kumar Hegde Rama for reading the manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Bhatia, Dept. of Pharmacology, National Univ. of Singapore, Yong Loo Lin School of Medicine, Centre for Life Sciences, 28 Medical Dr., Singapore 117456 (e-mail: mbhatia{at}nus.edu.sg)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 DISCUSSION
 GRANTS
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