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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.

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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
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Fig1: 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

Mentions: To determine whether NK1R plays a role in FcεRΙ-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 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)

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
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3472057&req=5

Fig1: 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
Mentions: To determine whether NK1R plays a role in FcεRΙ-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

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