neurokinin a engages neurokinin-1 receptor to induce nf-b...

Neurokinin A engages neurokinin-1 receptor to induce NF-�B-dependent geneexpression in murine macrophages: implications of ERK1/2 and PI3-kinase/Akt pathways

Jia Sun, Raina Devi Ramnath, Ramasamy Tamizhselvi, and Madhav BhatiaDepartment of Pharmacology, National University of Singapore, Singapore

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

Sun J, Ramnath RD, Tamizhselvi R, Bhatia M. Neurokinin Aengages neurokinin-1 receptor to induce NF-�B-dependent gene ex-pression in murine macrophages: implications of ERK1/2 and PI3-kinase/Akt pathways. Am J Physiol Cell Physiol 295: C679–C691,2008. First published July 2, 2008; doi:10.1152/ajpcell.00042.2008.—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 inflammatoryconditions. However, there are limited data about the mechanism ofits pathogenetic action. Here, we investigated proinflammatory effectsof 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. Inmouse macrophages with no detectable NK-2 receptors, NKA inducesthe upregulation of NK-1 but not NK-2 receptor expression. Further-more, NKA engages this NK-1 receptor, resulting in inflammatory-like responses involving activation of the transcription factor nuclearfactor (NF)-�B and induction of NF-�B-responsive proinflammatorychemokine expression. NKA activates NF-�B as evidenced by in-duced phosphorylation (leading to degradation) of its inhibitory pro-tein I�B�, increased cellular levels of the transactivation-active phos-pho(Ser276)-p65 and its nuclear translocation, as well as enhancedDNA-binding activity of NF-�B. These responses are specificallyinhibited by selective NK-1 receptor antagonists but not NK-2 recep-tor antagonists, thereby excluding the role of NK-2 receptor. Furtherinvestigation on the upstream signaling mechanisms suggests that twoNF-�B-activating pathways (extracellular signal-regulated kinase 1/2and phosphatidylinositol 3-kinase/protein kinase B) are activated byNKA. Specific inhibitors of the two pathways block NF-�B-depen-dent chemokine expression. The inhibitory effects are mediatedthrough regulation of nuclear translocation, DNA-binding activity,and/or transactivation activity of NF-�B. Together, we provide novelevidence that NKA engages NK-1 receptors on mouse macrophagesto elicit NF-�B-dependent cellular responses. The findings revealcellular mechanisms that may underlie NKA-mediated inflammatoryand immunological conditions.

neuroimmunomodulation; tachykinins; leukocytes; chemokines; sig-naling transduction pathways

NEUROKININ A (NKA) belongs to the tachykinin family ofneuropeptides, which also includes substance P (SP), neu-rokinin B, and the newly discovered hemokinins and endo-kinins. They are widely distributed within the central andperipheral nervous system as neurotransmitters and alsoexpressed in nonneuronal cells contributing to the interac-tions between nervous and peripheral organ systems under

both normal and pathological conditions (36). Tachykininsexert their biological actions through at least three distincttransmembrane G protein-coupled receptors: neurokinin(NK)-1, NK-2, and NK-3 receptors. SP, NKA, and NKB arethe preferential ligands for NK-1, NK-2, and NK-3 recep-tors, respectively. However, there is a high level of promis-cuity among the tachykinins and their receptors. All ligandscan bind to each of the NK receptors with varying affinity (43).

SP and NKA are the two best-characterized members of thetachykinin family that have a broad spectrum of actions,including neuronal excitation, smooth muscle contraction, va-sodilatation, plasma extravasation, salivation, nociception, andproinflammatory actions on immune and inflammatory cells(10). NKA, importantly, is a potent smooth muscle spasmogenin the respiratory, gastrointestinal, cardiovascular, and urinarysystem (10, 40) and has been implicated in airway inflamma-tory conditions such as smoking and asthma and gastrointes-tinal disorders (3, 6, 14, 27). NKA has been demonstrated tohave regulatory effects on immune cells. It induces histaminerelease from mast cells (15) and primes neutrophils forincreased superoxide anion production in response toformyl-methionyl-leucyl-phenylalanine (50). It stimulatessuperoxide anion production and tumor necrosis factor-�(TNF-�) mRNA expression from human monocytes (9, 29)and also induces de novo protein synthesis and release ofinterleukin (IL)-1, TNF-�, and IL-6 from human bloodmonocytes (30).

The biological functions of NKA are primarily mediated byNK-2 receptors. However, a number of studies show that NKAis also a functional ligand for the NK-1 receptor in vivo andin vitro (1, 5, 8, 17, 45). NKA is a high-affinity ligand for theNK-1 receptors expressed in transfected cell lines, despite itsweak ability to displace SP-NK-1 receptor binding (17). NKAis found to mediate some central effects of NK-1 receptors inrat brain, which can be selectively blocked by NK-1 receptorantagonists (5, 17). NKA binds to NK-1 receptors in ratsubmandibular gland and elicits NK-1 receptor-mediated phys-iological responses, including salivation (1, 8). In cells such asspinal cord neurons that do not express NK-2 receptors, NKAand SP are found to activate NK-1 receptors at the sameconcentration (45).

We have earlier demonstrated that SP stimulation of mousemacrophagaes leads to selective chemokine induction via anNK-1 receptor-mediated, nuclear factor (NF)-�B-dependentmechanism. This observation, together with other earlier re-

Address for reprint requests and other correspondence: M. Bhatia, Dept. ofPharmacology, National Univ. of Singapore, Yong Loo Lin School of Medi-cine, Centre for Life Sciences, 28 Medical Dr., Singapore 117456 (e-mail:[email protected]).

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

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

0363-6143/08 $8.00 Copyright © 2008 the American Physiological Societyhttp://www.ajpcell.org C679

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ports suggesting similar proinflammatory actions shared by SPand NKA, prompted us to investigate the possible effects ofNKA in mouse macrophages, in particular pertaining to SP-elicited inflammatory responses, including activation of NF-�B

and induction of NF-�B-dependent chemokine gene expres-sion. The receptor specificity was examined using selectiveNK-1 and -2 receptor antagonists. Additionally, upstreammechanistic pathways were studied by investigating the role of

Fig. 1. Neurokinin (NK) A upregulates NK-1 receptor mRNA and protein expression in mouse macrophages. Cells were left untreated or treated with 1 �M ofNKA for various time periods as indicated. NK-1 receptor mRNA expression was determined by RT-PCR. NK-1 receptor protein expression was determinedby Western blotting and immunofluorescence. A: RT-PCR detection of NK-1 receptor and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNAexpression. B: densitometric analysis of NK-1 receptor mRNA expression relative to GAPDH. C: Western blots of NK-1 receptor and the housekeeping proteinhypoxanthine-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-1receptor protein expression in untreated and NKA-treated cells. Representative micrographs from three separate experiments are shown. Originalmagnification, �100.

C680 NEUROKININ A IN MOUSE MACROPHAGES

AJP-Cell Physiol • VOL 295 • SEPTEMBER 2008 • www.ajpcell.org

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multiple signaling protein kinases suggested previously tomediate signal transduction from SP/NK-1 receptor to NF-�B.

MATERIALS AND METHODS

Cell culture and chemicals. RAW 264.7 cells were obtained fromthe American Type Culture Collection (ATCC, Manassas, VA),grown in DMEM (Invitrogen, Gaithersburg, MD) supplemented with10% (vol/vol) FBS (Invitrogen), 2 mM L-glutamine, 100 U/ml peni-cillin, and 100 �g/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 salthydrate, and the NK-2 receptor antagonist L-659,877 were purchasedfrom Sigma-Aldrich. The NK-1 receptor antagonist CP-96,345 was agift from Pfizer Diagnostics. NK-1 and NK-2 receptor antibodies werepurchased 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, Ger-many).

Isolation of primary peritoneal macrophages. Closed peritoneallavage was performed on anesthetized mice by injection of 10 mlice-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 macrophageswere allowed to adhere in 12- or 24-well plates at 37°C in ahumidified 5% CO2 incubator for 2 h, after which nonadherentcontaminating cells were removed and the primary macrophage cul-tures were maintained for an additional 2 h before they were subjectedto subsequent treatment and experiments. The preparations routinelycontain �95% macrophages, as verified by microscopic examinationwith Turk’s staining.

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

Whole cell lysate preparation and Western blot analysis. At the endof designated treatment, cells were lysed with chilled radioimmuno-

precipitation assay lysis buffer supplemented with protease inhibitorcocktail (Roche, Basel, Switzerland) and phosphatase inhibitor cock-tails (Sigma-Aldrich). Protein concentrations were determined by theBradford protein assay (Bio-Rad Laboratories, Hercules, CA). Proteinsamples (50–100 �g) were separated on Novex 10% Tris-glycinepolyacrylamide gel (Invitrogen) and transferred to polyvinylidenedifluoride membrane by electroblotting in Novex transfer buffer(Invitrogen) containing 20% (vol/vol) methanol. Membranes werethen washed, blocked, and probed overnight with rabbit phospho-I�B� (Ser32), phospho-NF-�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 SignalingTechnology Danvers, MA), NK-1R (1:500 dilution; Santa Cruz Bio-technology), or hypoxanthine-guanine phosphoribosyltransferase an-tibody (HPRT, 1:2,000; Santa Cruz Biotechnology), followed byhorseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG sec-ondary antibody (1:2,000; Santa Cruz Biotechnology) for 2 h. Mem-branes were washed and then incubated in SuperSignal West Picochemiluminescent substrate (Pierce, Rockford, IL) before exposure toX-ray films (CL-XPosure; Pierce). The intensity of bands was quan-tified using LabWorksImage Analysis software (UVP). HPRT wasused as the housekeeping protein.

Nuclear extract preparation. Nuclear extracts were prepared atvarious time points after treatment for subsequent NF-�B DNA-binding activity assay or electrophoretic mobility shift assay. Cellnuclear fractions were extracted using a nuclear extract kit (ActiveMotif, Carlsbad, CA). Briefly, cells were washed, collected in ice-coldPBS in the presence of phosphatase inhibitors, and then centrifuged at300 g for 5 min. Cell pellets were resuspended in a hypotonic buffer,treated with detergent, and centrifuged at 14,000 g for 30 s. Aftercollection of the cytoplasmic fraction, the nuclei were lysed, andnuclear proteins were solubilized in lysis buffer containing protea-some inhibitors. Protein concentrations were determined by the Brad-ford protein assay (Bio-Rad Laboratories).

NF-�B DNA-binding activity assay. NF-�B DNA-binding activitywas analyzed using the TransAMNF-�B p65 transcription factorassay kit (Active Motif) following the manufacturer’s instructions.Briefly, nuclear extract (5 �g) was incubated in a 96-well plate coatedwith oligonucleotide containing the NF-�B consensus-binding se-quence 5�-GGGACTTTCC-3�. Bound NF-�B was then detected by aspecific primary antibody. An HRP-conjugated secondary antibodywas then applied to detect the bound primary antibody and providedthe basis for colorimetric quantification. The enzymatic product wasmeasured at 450 nm by a microplate reader (Tecan Systems, San Jose,CA). Specificity of this assay was tested by the addition of wild-typeor mutated NF-�B consensus oligonucleotide in the competitive or

Fig. 2. Lack of detectable NK-2 receptor expressionin mouse macrophages. Untreated or NKA-treatedRAW 264.7 cells were subjected to RT-PCR andimmunofluorescence staining for NK-2 receptormRNA and protein expression. A: NK-2 receptormRNA expression was not detected in RAW 264.7cells. L, 100-bp DNA ladder; S1, unstimulated cells;S2, cells treated with NKA for 150 min; PC, positivecontrol, mouse lung tissues. B: NK-2 receptor pro-tein expression was not found in RAW 264.7 cells.Left: negative control, cells were incubated withnonimmune rabbit sera in place of primary antibody.Middle: cells stained negative for NK-2 receptors.Representative micrographs from three separate ex-periments are shown. Right: 3T3 mouse fibroblastsstained positive for NK-2 receptors. Original mag-nification, �100.

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mutated competitive control wells before the addition of nuclearextracts.

RT-PCR. The effect of NKA on NK-1 and -2 receptor mRNAexpression in RAW 264.7 macrophages was examined by RT-PCR.Briefly, cells were left untreated or treated with 1 �M NKA for theindicated periods described in RESULTS at 37°C before total RNA wasextracted using an RNeasy Mini Kit (Qiagen, Hilden, Germany). RNAwas quantitated spectrophotometrically by absorbance at 260 nm.Total RNA (1 �g) RNA was reverse-transcribed using the iScriptTMcDNA Synthesis Kit (Bio-Rad Laboratories). The cDNA synthesizedwas used as the template for PCR amplification using iQTM Supermix(Bio-Rad Laboratories) in MyCycler (Bio-Rad Laboratories). ThePCR protocol consisted of optimal 30–40 cycles of denaturation at95°C for 50 s, annealing for 60 s, and extension at 72°C for 50 s. Thenumber of amplification cycles was optimized to assure that thereaction was terminated in the linear range of amplification for eachgene. The following specific primer pairs (Proligo; Singapore) wereused: NK-1 receptor sense 5�-CTTGCCTTTTGGAACCGTGTG-3�and antisense 5�-CACTGTCCTCATTCTCTTGTG-3�; NK-2 receptorsense 5�-TGCTGTCATCTGGCTGGTAG-3� and antisense 5�-TCT-TCCTCGGTTGGTGTCCC-3�; glyceraldehyde-3-phosphate dehy-drogenase (GAPDH) sense 5�-GCATCTGAGGGCCCACTGAAG-3�

and antisense 5�-GTCCACCACCCTGTTGCTGTA-3�. Amplificationof the GAPDH gene transcript was used as an internal control ofRT-PCR reactions among samples. PCR products were analyzed on1.5% wt/vol agarose gels containing 0.05 mg/100 ml ethidium bro-mide. Densitometry results from PCR products were normalized to thehousekeeping gene GAPDH.

Immunofluorescence. After treatment, cells attached to six-wellplates were fixed in 3.7% formaldehyde for 10 min at room temper-ature, washed, and blocked using nonimmune rabbit sera for 10 min.Following blocking, cells were incubated with 1:50-diluted rabbitanti-mouse NK-1 receptor or NK-2 receptor antibodies (Santa CruzBiotechnology) 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 cellswere washed in PBS and mounted in Gel Mount (Sigma-Aldrich).Staining was analyzed by fluorescence microscopy using a Leica DMIRB microscope.

Enzyme-linked immunosorbent assay. Chemokine concentration inthe media of cultured RAW 264.7 cells was determined using murinemacrophage inflammatory protein (MIP)-2 and monocyte chemoat-

Fig. 3. NKA activates the transcription factor nuclear factor (NF)-�B. A: NKA induction of phosphorylation of NF-�B inhibitory protein I�B and NF-�B p65subunit. Cells were stimulated with 1 �M NKA for 0–60 min before whole cell lysates were prepared for immunoblotting analysis of phospho-I�B andphospho(Ser276)-p65 protein expression. Equal sample loading was determined by internal control HPRT. B: densitometric analysis of phospho-I�B expressionrelative to HPRT. C: densitometric analysis of phospho-p65 expression relative to HPRT. D: time course of nuclear translocation of the transactivation-activesubunit phospho(Ser276)-p65 following NKA stimulation. Cells were treated with NKA (1 �M) for 0–180 min. Nuclear fractions were then extracted forWestern 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 ().



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tractant protein (MCP)-1 Duoset enzyme-linked immunosorbent assay(ELISA) kits (R&D Systems, Minneapolis, MN) as described previ-ously (39) according to the instructions of the manufacturer. Sampleswere run in triplicates for each condition in three independent exper-iments. Absorbance was measured at 450 nm by a microplate reader(Tecan Systems). Results were expressed as picograms per milliliterfor each chemokine.

Statistical analysis. Data are expressed as means � SD. Statisticalanalyses were performed by independent t-test or, when multiplecomparisons were made, by one-way ANOVA with post hoc Tukey’stest using SPSS program version 13.0 (Chicago, IL). A P valueof �0.05 was considered a statistically significant difference.

RESULTS

NKA upregulates NK-1 receptor expression in mouse mac-rophages lacking detectable NK-2 receptors. We first evalu-ated NK-1 and -2 receptor expression in unstimulated orNKA-stimulated RAW 264.7 macrophages. Cells treated withvehicle or NKA for various periods of time were subjected toRT-PCR analysis of the receptor mRNA expression or Westernblot and/or immunofluorescence staining to determine theprotein expression. NK-1 receptor expression was readily de-tected in the cells under baseline conditions. NKA treatmentfurther induced NK-1 receptor expression at both mRNA andprotein levels (Fig. 1, A–E). The increase in NK-1 receptormRNA expression was most evident 40 min after NKA treat-ment and decreased thereafter. The increase in NK-1 receptorprotein expression became significant after 90 min of NKAstimulation and lasted until 150 min of stimulation (Fig. 1,C–E). The NK-2 receptor mRNA expression was not found ineither unstimulated or NKA-stimulated cells (Fig. 2A). Con-sistently, the receptor protein expression was not detected inthe cells (Fig. 2B). These results indicate that NKA upregulatesNK-1 receptor expression in mouse macrophages that lackNK-2 receptor expression. The findings prompted us to inves-tigate the interaction of NKA with this NK-1 receptor anddownstream cellular events triggered by the receptor engage-ment.

NKA triggers NF-�B activation in mouse macrophages viathe NK-1 receptor. One key event observed in the cells afterNKA stimulation was the activation of transcription factorNF-�B. NF-�B activation was evidenced by the phosphoryla-tion of NF-�B inhibitory protein I�B (which leads to degrada-tion of I�B by proteasome) and phosphorylation of p65, thesubunit responsible for the transactivation activity of NF-�B.Western blotting using specific antibodies that recognize onlythe phosphorylated form of I�B protein indicated the phos-phorylation of I�B protein starting 5 min after NKA treatmentuntil 30 min (Fig. 3, A and B). This was accompanied byenhanced phosphorylation (Ser276) of p65 subunit in the wholecell lysate and the nuclear extract, indicating the increasedcellular phosphorylation status of p65 subunit (Fig. 3, A and C)and nuclear translocation of this transactivation active phos-pho-p65 protein (Fig. 3, D and E). We further measured theeffect of NKA on the ability of NF-�B to bind DNA usingNF-�B DNA-binding assay. The result revealed that NKAtreatment led to a notable increase in the DNA-binding activityof NF-�B. Kinetic analysis showed a biphasic increase ofNF-�B activity, with the first peak at 30–60 min and thesecond after 150 min following NKA treatment (Fig. 4A).Furthermore, the NKA-induced increase in NF-�B activity was

NK-1 receptor mediated. Pretreatment of cells with selectiveNK-1 receptor antagonist CP-96,345 or L-703,606 before NKAtreatment significantly inhibited NKA-induced NF-�B activity.The selective nonpeptide NK-2 receptor antagonist L-659,877 didnot have any effect (Fig. 4B).

NKA triggers NF-�B-dependent proinflammatory chemo-kine production in mouse macrophages via the NK-1 receptor.We next investigated whether NKA could induce NF-�B-responsive gene expression. The expression of two chemo-kines, MIP-2 and MCP-1, inducible by NK-1 receptor engage-ment and the transcription of which is NF-�B-dependent wasexamined. RAW 264.7 macrophages were incubated with threedoses of NKA (1 nM, 100 nM, and 1 �M) for 2, 6, 12, and24 h. The supernatants were harvested at different time pointsfor ELISA measurement of the two chemokine levels. Signif-icant increases in MIP-2 and MCP-1 release from RAW 264.7macrophages were observed with 1 �M NKA after 12 and 24 hof incubation. No significant effect was noted for lower doses(Fig. 5, A and B).

Fig. 4. NKA enhances the DNA-binding activity of NF-�B via the NK-1receptor. A: time course of NKA-induced NF-�B DNA-binding activity inRAW 264.7 cells following NKA stimulation. B: NKA induction of NF-�Bp65 activity is NK-1 receptor dependent. Cells were preincubated with selec-tive NK-1 receptor antagonist CP-96,345 or L-703,606 (100 nM) or a selectiveNK-2 receptor antagonist L-659,877 before NKA stimulation. Results aremeans � SD for triplicate measurements and from three separate experiments.P � 0.05, compared with basal level (0 min) or control (*) and compared withNKA (†).



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In addition, we found that NKA-elicited NF-�B-dependentgene expression was also mediated by NK-1 receptor. Prein-cubation of cells with either CP-96,345 or L-703,606 (100 nM)before NKA treatment inhibited NKA-induced chemokine re-lease. Specific NK-2 receptor antagonist L-659,877 caused nodifference in the chemokine production (Fig. 5, C and D).

An inductive effect of NKA on chemokine expression wasalso demonstrated in mouse primary peritoneal macrophages.NKA (1 �M) induced a significant increase of MIP-2 andMCP-1 production after 12 h of treatment (Fig. 6, A and B).NKA-induced chemokine production in peritoneal macro-phages was also NK-1 receptor mediated. This was evidencedby preincubation of primary macrophages with the NK-1receptor antagonist CP-96,345 but not the NK-2 receptorantagonist L-659,877, which significantly attenuated the NKA-

induced increase in both MIP-2 and MCP-1 production by thecells (Fig. 6, C and D).

NKA activates ERK1/2 and Akt signaling kinases upstreamof NF-�B in mouse macrophages. We further examined theupstream NF-�B-activating kinases, including MAPKs andAkt potentially involved in NKA-NK-1 receptor responses inRAW 264.7 macrophages. Cells were treated with 1 �M ofNKA for 0, 5, 15, 30, or 60 min. Subsequently, whole celllysates were subjected to Western blotting using phosphospe-cific antibodies to detect the phosphorylated (and active) ki-nases. Among the MAPK members, only ERK1/2 was found tobe activated by NKA in the cells, whereas p38 MAPK andc-jun NH2-terminal kinase remained low (data not shown). Asshown in Fig. 7, A and B, NKA induced phosphorylation ofERK1/2 in a time-dependent manner without changing total

Fig. 5. NKA induces NF-�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, 100nM, or 1 �M 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 with100 nM CP-96,345 or L-703,606, the specific NK-1 receptor antagonists, or 1 �M L-659,877, the selective NK-2 receptor antagonist, for 15 min before additionof NKA (1 �M) 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 withNKA (†).



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ERK1/2 protein levels, suggesting that NKA activates ERK1/2signaling pathways. Akt, a serine/threonine kinase that has animportant role in regulating cellular growth, differentiation,adhesion, and the inflammatory reaction, was also found to beupregulatable by NKA (Fig. 7, C and D). Both ERK1/2 andAkt were activated early in the cells within 5 min after NKAstimulation, followed by a gradual decrease of the activatedprotein until 60 min.

Blockade of ERK1/2 or PI 3-kinase/Akt activation inhibitsNKA-induced NF-�B activation and NF-�B-dependent geneexpression. Given that NKA induced activation of ERK1/2 andAkt, a PI 3-kinase downstream effector kinase, we furtherdetermined whether ERK1/2 and PI 3-kinase/Akt mediatedNKA-induced NF-�B activation and the resultant gene tran-scription in macrophages. Specific inhibitors of ERK1/2 (PD-98059) and PI 3-kinase/Akt (LY-294002) were added to thecells 1 h before NKA stimulation. The inhibitor effects onNKA-induced NF-�B DNA-binding activity and nuclear trans-location of phospho(Ser276)-p65 were investigated.

NF-�B DNA-binding activity assays demonstrated thatinhibition of PI 3-kinase/Akt significantly blocked NKA-induced NF-�B DNA-binding activity. However, there was

no significant alteration of NKA-induced NF-�B activitywhen cells were pretreated with ERK1/2 inhibitor PD-98059(Fig. 8A). The results suggest a role for PI 3-kinase/Akt butnot ERK1/2 in regulating DNA-binding activity of NF-�B.However, both inhibitors significantly reduced nuclear lev-els of phospho(Ser276)-p65 protein (Fig. 8B). Because phos-phorylation of p65 at Ser276 residue is associated with coacti-vator recruitment to the NF-�B transcription complex andenhanced transactivational activity of the protein, ERK1/2 andPI 3-kinase/Akt may both be implicated in the posttranslationalphosphorylation and regulation of transactivation potential ofthe p65 subunit.

As shown in Fig. 9, A and C, PD-98059 dose dependentlyinhibited both MIP-2 and MCP-1 protein synthesis induced byNKA. Although the inhibition was maximal with 50 �M, theinhibitor was effective at a low dose of 10 �M. LY-294002, atthe dose range of 1–10 �M, also significantly attenuatedNKA-induced MIP-2 and MCP-1 production (Fig. 9, B and D).

ERK1/2 and PI 3-kinase/Akt signaling pathways are alsoimportant in the NKA-induced chemokine production in pri-mary macrophages. PD-98059 (10 �M) and LY-294002(5 �M) pretreatment significantly inhibited NKA-induced in-

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 mouseperitoneal macrophages following NKA stimulation. Cells were treated with 0.1 or 1 �M of NKA for 12 h. C and D: NKA-stimulated MIP-2 and MCP-1production is NK-1 receptor dependent. Cells were preincubated with 100 nM CP-96,345 or 1 �M L-659,877 for 15 min before addition of NKA (1 �M) for12 h. MIP-2 and MCP-1 concentrations were measured by ELISA with the supernatants harvested. Results are expressed as means � SD for triplicatemeasurements and from three separate experiments. P � 0.05, compared with control (*) and compared with NKA (†).



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crease of MIP-2 and MCP-1 production by primary peritonealmacrophages (Fig. 10, A and B).

DISCUSSION

In this study, we report that NKA is able to engage the NK-1receptor on mouse macrophages and induce activation of thetranscription factor NF-�B and expression of NF-�B-drivengenes. Furthermore, PI 3-kinase/Akt and ERK1/2 kinases areidentified to mediate the signal transduction from NKA/NK-1receptor to NF-�B. The findings presented here suggest ahitherto unknown mechanism of the neuropeptide NKA regu-lation of inflammatory responses in immune cells via the NK-1receptor.

Up to date, there are limited data in the literature on theproinflammatory effects of NKA on macrophage functions. Incomparison, the closely related neuropeptide SP has beendemonstrated by convincing data to regulate a variety offunctions of macrophages (35). SP and NKA are both tachy-kinin neuropeptides produced from a single precursor, PPT-Agene, and colocalized and coreleased from primary afferentnociceptors (45). Specific effects of these two neuropeptidesare generally achieved by binding to their differential preferredreceptors (NK-1 and -2 receptors for SP and NKA, respec-tively) on the target cells. Our RT-PCR analysis and immuno-fluorescence staining demonstrate functional NK-2 receptor,the preferential receptor for NKA, is not found in RAW 264.7

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 �M 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 totalERK1/2. C: phospho(Ser473)-Akt and total Akt protein levels. D: densitometric analysis of phospho-Akt protein expression relative to total Akt. Results aremeans � SD of three separate experiments. P � 0.05, compared with basal level (0 min; *) and compared with the corresponding time point control ().



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cells. The NK-1 receptor, however, is upregulated in the cellsafter NKA treatment, which raises the possibility that, in theabsence of NK-2 receptor, NKA may act via NK-1 receptor onRAW 264.7 macrophages to elicit NK-1 receptor-mediatedcellular responses, comparable to SP. This notion has beensupported by earlier studies that suggest NKA can bind to thesame NK-1 receptors as SP (1, 8, 17, 42, 45). The commonCOOH-terminal sequences of SP and NKA are suggested tocontact the same region of NK-1 receptor (7). Convincingevidence demonstrates that NKA is an important ligand forNK-1 receptor in vivo to elicit NK-1 receptor-mediated phys-iological responses such as salivation (8). In spinal cord neu-rons where NK-2 receptor expression is not identified, NKAequally activates NK-1 receptor compared with SP (45). It is

suggested that NKA and SP share some functional character-istics, since the two neuropeptides act in NK-1 receptor sig-naling at the same doses (1, 42). Consistent with these studies,we demonstrate that NKA and SP stimulate similar responsesin macrophages: NF-�B activation and expression of NF-�B-responsive chemokines via the NK-1 receptor. Lack of effectson NKA-induced NF-�B activation by selective NK-2 receptorantagonist at pharmacologically active doses confirmed NK-1receptor is the primary receptor for NKA-mediated effects inmouse macrophages.

One observation of this study at variance with some earlierdata is the dose difference between SP and NKA in inducingNK-1 receptor-mediated effects (1, 42, 45). Whereas othersreport similar concentrations of NKA and SP in eliciting NK-1receptor-mediated effect, we find the effective dose of NKA (1�M) is higher than that of SP (10–100 nM). That NKA is a lesspotent stimulator of macrophages than SP may be due to itsrelatively lower affinity for NK-1 receptor or possible differ-ences in signaling mechanisms. In fact, earlier studies alsosuggest 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 extracellularloop of the NK-1 receptor is identified to be necessary for thebinding and biological activity of NKA but not SP (48). Thishypothesis is reinforced by the ability of some NK-1 receptorantagonists such as GR-205171 to selectively block NKA-mediated NK-1 receptor responses. It is likely that GR-205171more 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 thanof SP (31, 45), which is consistent with our finding thatCP-96,345 inhibits effects of NKA at lower doses than of SP.Other evidence supporting this hypothesis shows that, whereasSP increases levels of both cAMP and inositol trisphosphate(IP3) in NK-1 receptor-transfected CHO cells, NKA onlyaffects IP3 (42).

Neuropeptide regulation of various functions of immunecells provides a mechanism for neural control of immune andinflammatory responses. NKA, in addition to SP, is an impor-tant neuropeptide involved. In addition to earlier findings (9,15, 29, 30, 50), our study, for the first time, shows that NKAstimulation of murine macrophages leads to activation ofNF-�B, a transcription factor that has a crucial modulatory rolein inflammation, immunity, cell proliferation, and apoptosis(53). NF-�B can be activated by three mechanisms: the clas-sical pathway dependent on NF-�B inhibitory protein I�Bdegradation and two atypical pathways [one is through theprocessing of p100 and release of p52/RelB in the nucleus; theother is through the phosphorylation of p65 at multiple serinesites by some protein kinases (34, 47)]. The classical pathwayand posttranslational modifications (phosphorylation) of p65subunit are involved in NKA activation of NF-�B in the cells.This is evidenced by NKA-induced phosphorylation (leadingto subsequent degradation) of the inhibitory protein of NF-�B,I�B�, and nuclear translocation of phospho(Ser276)-NF-�Bp65. Also, the DNA-binding activity of NF-�B is enhanced inmacrophages after exposure to NKA. Furthermore, NKA in-duces NF-�B-regulated chemokine (MIP-2 and MCP-1) ex-pression in murine macrophages. Consistently, stimulation ofNK-1 receptor on immune cells resulting in activation ofNF-�B and proinflammatory gene expression has been de-

Fig. 8. PI 3-kinase/Akt and ERK1/2 mediate NKA-induced signal transduc-tion, leading to NF-�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 �MNKA for 30 min. Nuclear fractions were prepared and analyzed for theDNA-binding activity of NF-�B and phospho-p65 expression. A: effects of PI3-kinase/Akt and ERK inhibitors on NKA-induced DNA binding of NF-�B.B: effects of PI 3-kinase/Akt and ERK inhibitors on nuclear phospho-p65levels. C: densitometric analysis of nuclear phospho-p65 levels. Results aremeans � SD of three separate experiments. P � 0.05, compared with control(*) and compared with NKA (†).



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scribed in a number of in vitro systems (2, 4, 44, 49). Becauseboth MCP-1 and MIP-2 have been implicated in the develop-ment of clinical and experimental asthma to which NKA has astrong pathophysiological association (13, 21, 33, 46), ourstudy suggests that alveolar macrophages present in the bron-chioles and large airways heavily innervated by NKA- andSP-containing nerves (6, 27) might be the cellular sources ofthese chemokines in asthma.

Further study on the inductive mechanisms for NKA-elicitedNF-�B responses reveals two important signaling pathways(ERK1/2 MAPK and PI 3-kinase/Akt) involved in mediatingthe responses.

MAPKs, important NF-�B-activating kinases, are proline-directed protein serine/threonine kinases activated by a cascadeof intracellular phosphorylation events (11, 12, 16, 18, 51).Their substrates, located in the cytoplasm as well as in thenucleus, include other kinases, transcription factors, phospho-lipases, and cytoskeletal proteins (25, 28, 37, 41). Among all

MAPK members, only ERK1/2 are found to be activated byNKA in RAW 264.7 macrophages. The role of ERK1/2 inNKA-triggered intracellular signaling is further characterizedusing a potent selective inhibitor of ERK1/2 (PD-98059) thatinhibits the phosphorylation and activation of ERK by itsimmediate upstream activator mitogen/extracellular signal-reg-ulated kinase. Pretreatment of cells with PD-98059 dose de-pendently attenuates chemokine production induced by NKA.Moreover, inhibition of ERK1/2 results in diminished nuclearlevels of phospho(Ser276)-p65, a transactivation-active subunitof NF-�B, although it exhibits minimal effect on the DNA-binding activity of NF-�B. These results suggest that ERK1/2mediate NKA-induced NF-�B activation and proinflammatorychemokine 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-ki-nase. PI 3-kinase/Akt has an important role in regulating

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 untreatedor were pretreated with ascending concentrations of the ERK1/2 inhibitor PD-98059 or the PI 3-kinase/Akt inhibitor LY-294002 and subsequently stimulatedwith 1 �M 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 triplicatemeasurements and from three separate experiments. P � 0.05, compared with control (*) and compared with NKA (†).



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cellular growth, differentiation, adhesion, and the inflamma-tory reaction (19, 24). Although the role of PI 3-kinase in theinflammatory and immunological responses has been contro-versial (38), evidence has suggested that PI 3-kinase/Akt reg-ulates NF-�B activity in response to a variety of extracellularstimuli (22, 23, 32). Given that NKA treatment stimulatesactivation of Akt in macrophages as shown here, we hypoth-esize that PI 3-kinase/Akt might be involved in NKA-inducedNF-�B responses in RAW 264.7 cells. Supporting this hypoth-esis, it is found that inactivation of PI 3-kinase with the specificpharmacological inhibitor LY-294002 inhibits NKA inductionof phosphorylation(Ser276) of the transactivation subunit p65and production of the two NF-�B-inducible chemokines. Theinhibitor also blocks NKA-induced NF-�B DNA binding, aneffect not observed with the ERK1/2 inhibitor PD-98059.Ser276 is inducibly phosphorylated by protein kinase A andmitogen- and stress-activated protein kinase-1 to enhance thetransactivational potential of the subunit p65, which is mainlyresponsible for transactivaiton activity. These data suggest thatPI 3-kinase and ERK1/2 lie upstream of NF-�B activation inresponse to NKA in RAW 264.7 cells. It is at present unclear,however, how NKA treatment triggers PI 3-kinase/Akt andERK1/2 activation in RAW cells. NK-1 receptor-mediated-NF-�B activation is frequently associated with activation ofreceptor-associated G proteins and/or their effectors, includingsmall GTPases, phospholipase C, calcium, and protein kinaseC (20, 26, 49, 52). The role of these signaling molecules in thecontext of NKA/NK-1 receptor signaling remains to be inves-tigated.

In conclusion, we demonstrate for the first time that NKAstimulates NK-1 receptor-mediated cellular signaling in mousemacrophages. NKA/NK-1 receptor signaling leads to activa-tion of the transcription factor NF-�B and the resultant proin-flammatory chemokine production in the cells. NF-�B activa-tion in this system involves at least two signaling pathways:ERK1/2 and PI 3-kinase/Akt (Fig. 11). The findings presentedby this study suggest NKA as an important mediator ofneuroimmunomodulatory activity and that macrophages andproinflammatory chemokines may be implicated in NKA-

Fig. 10. ERK1/2 or PI 3-kinase/Akt pathways are important for NKA-inducedchemokine production in mouse primary macrophages. Isolated cells wereeither left untreated or were pretreated with PD-98059 (10 �M) or LY-294002(5 �M) and subsequently stimulated with 1 �M NKA for 12 h. MIP-2 (A) andMCP-1 (B) levels were measured in cell supernatants by ELISA. Results aremeans � SD for triplicate measurements and from three separate experiments.P � 0.05, compared with control (*) and compared with NKA (†).

Fig. 11. NKA-induced signal transduction cascadein mouse macrophages. NKA engaging the NK-1receptor activates both ERK1/2 and PI 3-kinase/Aktsignaling pathways, which activate NF-�B by en-hancing phosphorylation of NF-�B p65 and its nu-clear translocation. NF-�B activation leads to sub-sequent expression of proinflammatory chemokinegenes.



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mediated inflammatory and immunological conditions. Furtherelucidation of key signaling molecules involved may haveimportant implications in controlling the conditions.

ACKNOWLEDGMENTS

We thank Akhil Kumar Hegde Rama for reading the manuscript.

GRANTS

This work was supported by Academic Research Fund Grant R-184-000-054-112, Office of Life Sciences Cardiovascular Biology Program GrantR-184-000-074-712, and the Bridging Grant R-184-000-139-101.

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