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

Fang X, Hu H, Xie J, Zhu H, Zhang D, Mo W, Zhang R, Yu M - Inflamm. Res. (2012)

Bottom Line: 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.NK1R is required for the activation of RBL-2H3 cells following FcεRΙ engagement and involved in the regulation of MAPK signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai 200032, People's Republic of China.

ABSTRACT

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.

Show MeSH
NK1R contributes to the phosphorylation of MAPKs following FcεRI aggregation in RBL-2H3 cells. Sensitized RBL-2H3 cells were starved and stimulated with or not with DNP-BSA (10 ng/ml) for 5 min. The whole cell lysates were immunoblotted with phospho-specific antibodies for Erk1/2 (a), JNK (b), and p38 (c), respectively. To control for loading, the blots were stripped and reprobed with anti-total 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
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC3472057&req=5

Fig3: NK1R contributes to the phosphorylation of MAPKs following FcεRI aggregation in RBL-2H3 cells. Sensitized RBL-2H3 cells were starved and stimulated with or not with DNP-BSA (10 ng/ml) for 5 min. The whole cell lysates were immunoblotted with phospho-specific antibodies for Erk1/2 (a), JNK (b), and p38 (c), respectively. To control for loading, the blots were stripped and reprobed with anti-total 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

Mentions: Efforts were made to explore the effect of NK1R on MAPK signaling downstream of the FcεRΙ in RBL-2H3 cells. After FcεRΙ 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 FcεRΙ aggregation in RBL-2H3 cells.Fig. 3


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

Fang X, Hu H, Xie J, Zhu H, Zhang D, Mo W, Zhang R, Yu M - Inflamm. Res. (2012)

NK1R contributes to the phosphorylation of MAPKs following FcεRI aggregation in RBL-2H3 cells. Sensitized RBL-2H3 cells were starved and stimulated with or not with DNP-BSA (10 ng/ml) for 5 min. The whole cell lysates were immunoblotted with phospho-specific antibodies for Erk1/2 (a), JNK (b), and p38 (c), respectively. To control for loading, the blots were stripped and reprobed with anti-total 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
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3472057&req=5

Fig3: NK1R contributes to the phosphorylation of MAPKs following FcεRI aggregation in RBL-2H3 cells. Sensitized RBL-2H3 cells were starved and stimulated with or not with DNP-BSA (10 ng/ml) for 5 min. The whole cell lysates were immunoblotted with phospho-specific antibodies for Erk1/2 (a), JNK (b), and p38 (c), respectively. To control for loading, the blots were stripped and reprobed with anti-total 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
Mentions: Efforts were made to explore the effect of NK1R on MAPK signaling downstream of the FcεRΙ in RBL-2H3 cells. After FcεRΙ 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 FcεRΙ aggregation in RBL-2H3 cells.Fig. 3

Bottom Line: 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.NK1R is required for the activation of RBL-2H3 cells following FcεRΙ engagement and involved in the regulation of MAPK signaling pathways.

View Article: PubMed Central - PubMed

Affiliation: The Key Laboratory of Molecular Medicine, Ministry of Education, Shanghai 200032, People's Republic of China.

ABSTRACT

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.

Show MeSH