An involvement of neurokinin-1 receptor in FcεRΙ-mediated RBL-2H3 mast cell activation

Objective and design To determine whether the neurokinin-1 receptor (NK1R) plays a role in the activation of RBL-2H3 mast cells after FcεRΙ aggregation. Materials and methods NK1R expression in RBL-2H3 cells was inhibited by small hairpin RNA (shRNA) against NK1R, and determined by western blotting. For activation, both NK1R knockdown and control RBL-2H3 cells were sensitized by dinitrophenol (DNP)-specific IgE and stimulated with the antigen DNP-bovine serum albumin (BSA). Following the activation of RBL-2H3 cells, monocyte chemoattractant protein (MCP-1) production and intracellular calcium flux were monitored by ELISA and confocal microscopy assay, respectively. For investigation of the signaling mechanism, phosphorylation of mitogen-activated protein kinases (MAPKs) after RBL-2H3 cell activation was assessed by western blotting. Results shRNA-NK1R mediated an effective inhibition of NK1R expression in RBL-2H3 cells. Protein production of MCP-1 was reduced by more than 55 % in NK1R knockdown RBL-2H3 cells compared with control RBL-2H3 cells. In addition, both calcium mobilization and phosphorylation levels of MAPKs (Erk1/2, JNK, and p38) after DNP-BSA stimulation (via FcεRΙ) were decreased due to the inhibition of NK1R expression. Conclusion NK1R is required for the activation of RBL-2H3 cells following FcεRΙ engagement and involved in the regulation of MAPK signaling pathways. Electronic supplementary material The online version of this article (doi:10.1007/s00011-012-0523-x) contains supplementary material, which is available to authorized users.


Introduction
Mast cells are major effectors in allergic diseases. The activation of mast cells leads to the release of various inflammatory mediators, including vasoactive agents, cytokines, and chemokines, contributing to the disease [1].
The key receptor distributed on the surface of mast cells and responsible for their activation is the IgE receptor, FceRI [2]. In a typical allergic response, polyvalent antigens cross-link IgE-bearing FceRI molecules on mast cells. This causes the activation of immunoreceptor tyrosine-based activation motifs (ITAMs) in tails of FceRI and thus initiates intracellular signaling cascades that ultimately result in the activation of mast cells [1,3]. Many signal molecules have been shown to be essential for FceRImediated mast cell activation, such as Src family kinases Lyn and Fyn [4][5][6][7]. However, the precise mechanism underlying mast cell activation remains far from fully understood.
NK1R is a widely expressed G protein-coupled receptor (GPCR) and the preferred receptor for neuropeptide substance P (SP) [8]. It has been revealed to be involved in various biologic responses, including pain transmission, neuroimmune modulation, exocrine secretion, and inflammation [9][10][11][12][13]. The close relationship between NK1R and inflammation has evidenced by the marked elevation of NK1R expression in several kinds of inflammatory diseases, such as asthma [14], acute pancreatitis [15][16][17][18], and abdominal cell adhesion formation [19,20]. Functional expression of NK1R on bone marrow-derived mast cells (BMMCs) induced by IL4 and SCF further suggests the implications of NK1R in inflammation [21]. However, the detailed role of NK1R in allergic disorders remains unclear. Given that previous reports have demonstrated that NK1R is expressed in the RBL-2H3 cell line, a rat mast cell line with the properties of mucosal mast cells [22,23], determining whether NK1R plays a role in FceRI-induced activation of RBL-2H3 cells, will be of significance.
In this work, by inhibiting the expression of NK1R in RBL-2H3 cells, we explored potential functions that NK1R exerts in FceRI-induced RBL-2H3 cell activation in an attempt to provide new insights into the mechanism underlying allergic diseases.

Materials and methods
Cell culture conditions RBL-2H3 cell line [24] was maintained as monolayer cultures in Earle's modified Eagle's medium (MEM), supplemented with 10 % heat-inactivated fetal bovine serum (FBS; HyClone), 2 mM glutamine, 100 U/ml penicillin, and 100 lg/ml streptomycin at 37°C in a humidified incubator under 5 % CO 2 . All cell culture reagents were obtained from Gibco except where otherwise indicated.
MAPK assays DNP-BSA-induced phosphorylation of mitogen-activated protein kinases (MAPKs) was determined by western blotting. RBL-2H3 cells were sensitized and starved overnight in serum-free MEM containing anti-DNP-IgE (0.1 lg/ml) and BSA (2 %). Stimulation was initiated by the addition of DNP-BSA (10 ng/ml) in Tyrode's buffer at 37°C. The reaction was stopped by placing the cells on ice. To determine the activities of MAPK molecules, the whole cell lysates were prepared and subjected to western blotting as described above. Additional protease inhibitors including 60 mM b-glycerophosphate, 2 mM sodium orthovanadate, and 10 mM sodium fluoride were added in the lysates. Anti-phospho-Erk1/2, JNK, and p38 antibodies (phospho-MAPKs) were used. To control for loading, membranes were stripped and reprobed with antibodies to total Erk1/2, JNK, and p38 (MAPKs). The intensities of the phospho-MAPKs were normalized to total MAPKs to correct for any differences in loading. All antibodies were purchased from Cell Signaling Technology unless otherwise noted and used at recommended concentrations. Aprotinin, leupeptin, pepstatin, antipain, phenylmethylsulfonyl fluoride, sodium orthovanadate, and sodium fluoride were from Sigma-Aldrich.

Statistical analysis
All data were analyzed with Prism 5 software (GraphPad Software). Data were considered statistically significant when a P value was less than 0.05, obtained with a twotailed t test.

shRNA-mediated knockdown of NK1R expression in RBL-2H3 cells
To determine whether NK1R plays a role in FceRI-mediated RBL-2H3 cell activation, we constructed two shRNAs against NK1R (NK1R-shRNA1 and NK1R-shRNA2) and transfected them into wide-type RBL-2H3 cells to inhibit the expression of NK1R. Negative control plasmids containing scrambled shRNA (Con-shRNA) were also transfected. NK1R protein expression in RBL-2H3 cells after 36 h of transfection was assessed by western blotting (Fig. 1). Expression of NK1R-shRNA1 and NK1R-shRNA2 led to reductions in NK1R expression of 68.43 ± 2.41 and 81.1 ± 2.49 %, respectively, in RBL-2H3 cells compared with Con-shRNA. This indicates an effective knockdown of NK1R expression in RBL-2H3 cells by either NK1R-shRNA1 or NK1R-shRNA2. Hence, we named RBL-2H3 cells expressing NK1R-shRNA1 or NK1R-shRNA2 for NK1R knockdown RBL-2H3 cells and expressing Con-shRNA for control RBL-2H3 cells. Fig. 1 An effective inhibition of NK1R expression mediated by the shRNA in RBL-2H3 cells. Upper panel wild-type RBL-2H3 cells were transfected with Con-shRNA, NK1R-shRNA1, and NK1R-shRNA2 constructs. The transfected cells were harvested 36 h later and the effect of these three shRNA constructs on the expression of NK1R was analyzed by western blotting with anti-NK1R antibody. Representative blots from three independent experiments are shown. Lower panel densitometry analysis of NK1R expression is shown. Values normalized to GAPDH expression are represented as mean ± SD (n = 3) and are expressed in arbitrary densitometry units (ADU). *P \ 0.05 as compared with control RBL-2H3 cells, Student's t test NK1R is required for the FceRI-evoked activation of RBL-2H3 cells Cytokine gene expression and calcium mobilization are two remarkable events in mast cells after FceRI aggregation. To evaluate the effect of NK1R on cytokine gene expression in RBL-2H3 cells following FceRI stimulation, amounts of released MCP-1 were determined by ELISA. As shown in Fig. 2a, a significant reduction of MCP-1 expression was observed in NK1R knockdown RBL-2H3 cells (124.1 ± 17.7 for NK1R-shRNA1, and 99.5 ± 18.6 for NK1R-shRNA2) relative to control RBL-2H3 cells (278.2 ± 23.5 for Con-shRNA). We also monitored calcium flux in both control and NK1R knockdown RBL-2H3 cells following FceRI aggregation (Fig. 2b). RBL-2H3 cells expressing either NK1R-shRNA1 or NK1R-shRNA2 showed decreased calcium mobilization compared with RBL-2H3 cells expressing Con-shRNA. These results strongly indicate an essential role of NK1R in FceRIevoked RBL-2H3 cell activation.

NK1R knockdown results in defective activation of MAPKs
Efforts were made to explore the effect of NK1R on MAPK signaling downstream of the FceRI in RBL-2H3 cells. After FceRI stimulation, phosphorylation levels of MAPKs were determined by western blotting. For phospho-Erk1/2, phosphorylation signals were hardly detected under basal conditions in both control and NK1R knockdown RBL-2H3 cells. After the antigen DNP-BSA treatment for 5 min, control RBL-2H3 cells showed a marked increase in levels of phospho-Erk1/2. However, a much lower elevation of phosphorylation levels of Erk1/2 were observed in NK1R knockdown RBL-2H3 cells compared with control RBL-2H3 cells (Fig. 3a). Similar tendencies were observed on both phospho-JNK (Fig. 3b) and phospho-p38 (Fig. 3c). This significantly inhibited phosphorylation of MAPKs, which is attributable to the down-regulation of NK1R expression, indicates an involvement of NK1R in the regulation of MAPK signaling after FceRI aggregation in RBL-2H3 cells.

Discussion
In the present study, we demonstrate that NK1R promotes FceRI-evoked MCP-1 mRNA expression and calcium mobilization in RBL-2H3 cells. Furthermore, our results suggest that NK1R is required for the initiation of rapid, full activation of MAPKs upon FceRI aggregation in RBL-2H3 cells. Previous studies have revealed an involvement of NK1R in various inflammatory disorders, but little is known about the role of NK1R in FceRI-mediated allergic reactions. Our data provide the first evidence for functional roles of NK1R in the activation of RBL-2H3 cells following FceRI aggregation.
MCP-1 is one of chemokines produced by mast cells after FceRI activation [26,27]. Given that the expression of MCP-1 was significantly reduced in Fyn-deficient BMMCs [28], Fyn kinase has been reported to be essential for the production of MCP-1 after FceRI aggregation in mast cells. Our finding that NK1R knockdown RBL-2H3 cells showed decreased MCP-1expression after antigen stimulation (Fig. 2a) suggests that NK1R is also required for the production of MCP-1 and that there perhaps exist cross-talk between NK1R and Fyn in FceRI signaling. Moreover, the decreased phosphorylation levels of JNK (Fig. 3b) and p38MAPK (Fig. 3c) observed in NK1R knockdown RBL-2H3 cells were also similar to that  [28]. Activities of MAPKs are required for the MCP-1 gene expression in mast cells [29]. Based on these results, it is possible that NK1R promotes the FceRI-induced MCP-1 expression through Fyn kinase. Actually, in mouse pancreatic acinar cells, NK1R has been demonstrated to mediate the production of MCP-1 via Src family kinases following the stimulation of SP [30]. More experiments are needed to be carried out to delineate the relationship between NK1R and Fyn in mast cells.
As with the reduced MCP-1 expression, we found that calcium flux was also inhibited as a consequence of the knockdown of NK1R expression in RBL-2H3 cells (Fig. 2b). This is as we expected, because the release of inflammatory mediators is always coupled with the elevation of intracellular calcium concentration following the activation of mast cells [7,26]. FceRI-evoked calcium mobilization in mast cells involves the activation of phospholipase C (PLC) [26,31]. The activated PLC is capable of inducing the production of diacylglycerol (DAG) and inositol 1,4,5-trisphosphate (IP3), both of which are required for calcium mobilization, leading to increased calcium concentration [32]. Previous studies have demonstrated that NK1R, as a GPCR, is coupled to Gq/11 receptor. This receptor is associated with PLC [33]. These clues allow us to assume that the inhibition of NK1R expression in RBL-2H3 cells may impair the interaction of Gq/11 with PLC, resulting in reduced activity of PLC followed by decreased calcium flux after FceRI stimulation. This assumption might be reasonable considering that the NK1R expressed in RBL-2H3 cells is in its full-length form (407aa). In fact, NK1R has two naturally occurring forms: a full-length NK1R (407aa) isoform and a truncated NK1R (311aa) isoform lacking 96-aa residues in the carboxyl terminus [34]. The absent C-terminal domain is required for the coupling to Gq/11 receptor, making the To control for loading, the blots were stripped and reprobed with antitotal Erk1/2, JNK, and p38 antibodies, respectively. Representative blots of at least three independent experiments are shown. The levels of phospho-MAPKs (at 5 min) were normalized to the expression of MAPKs and qualified by densitometry in ADU. The phosphorylation levels of Erk1/2 decreased by 52.99 ± 2.84 % for NK1R-shRNA1 and 60.96 ± 1.88 % for NK1R-shRNA2; of JNK decreased by 73.08 ± 3.66 % for NK1R-shRNA1 and 81.97 ± 1.33 % for NK1R-shRNA2; and of p38 decreased by 48.33 ± 3.51 % for NK1R-shRNA1 and 57 ± 4.58 % for NK1R-shRNA2, as compared with Con-shRNA. Values are expressed as mean ± SD (n = 3). *P \ 0.05 as compared with the control RBL-2H3 cells, Student's t test An involvement of NK1R in FceRI-mediated RBL-2H3 mast cell activation 1261 truncated NK1R disassociated with PLC [8,33] and unable to prime calcium mobilization by itself. The functional role of NK1R in FceRI signaling is further confirmed by the observation that the phosphorylation of Erk1/2 was defective in NK1R knockdown RBL-2H3 cells (Fig. 3a). This, in addition to similar defects found on phospho-JNK (Fig. 3b) and phospho-p38MAPK (Fig. 3c), indicates a critical role of NK1R in MAPK signaling downstream of the FceRI in RBL-2H3 cells. The activation of NK1R by DNP-BSA (via FceRI) is virtually identical to that by SP, resulting in the expression of MCP-1 as well as the phosphorylation of MAPKs [35]. Previous investigations of the signaling mechanism have revealed that SP activation of NK1R induces the phosphorylation of Erk1/2 involves b-arrestin/src or b-arrestin2 [36]. Whether this mechanism is reasonable to explain the role of NK1R in FceRI signaling awaits further exploration. In addition, the question of how NK1R is activated and then involved in the following signaling events after FceRI cross-linking is still elusive and needs more attention in further study.
In conclusion, our data suggest that NK1R functions as a regulator in the activation of RBL-2H3 cells through FceRI signaling. In this way, they contribute to the understanding of the mechanisms underlying allergic diseases.