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Involvement of class II phosphoinositide 3-kinase α-isoform in antigen-induced degranulation in RBL-2H3 cells.

Nigorikawa K, Hazeki K, Guo Y, Hazeki O - PLoS ONE (2014)

Bottom Line: The release of mRFP-tagged neuropeptide-Y, a reporter for the regulated exocytosis, was also decreased in the PI3K-C2α-deficient cells.The release was increased significantly by the expression of the siRNA-resistant version of PI3K-C2α.These results indicated that PI3K-C2α and its product PtdIns(3,4)P2 may play roles in the secretory process.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan.

ABSTRACT
In this study, we present findings that suggest that PI3K-C2α, a member of the class II phosphoinositide 3-kinase (PI3K) subfamily, regulates the process of FcεRI-triggered degranulation. RBL-2H3 cells were transfected with shRNA targeting PI3K-C2α. The knockdown impaired the FcεRI-induced release of a lysosome enzyme, β-hexosaminidase, without affecting the intracellular Ca2+ mobilization. The release of mRFP-tagged neuropeptide-Y, a reporter for the regulated exocytosis, was also decreased in the PI3K-C2α-deficient cells. The release was increased significantly by the expression of the siRNA-resistant version of PI3K-C2α. In wild-type cells, FcεRI stimulation induced the formation of large vesicles, which were associated with CD63, a marker protein of secretory granules. On the vesicles, the existence of PI3K-C2α and PtdIns(3,4)P2 was observed. These results indicated that PI3K-C2α and its product PtdIns(3,4)P2 may play roles in the secretory process.

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Neuropeptide Y release from PI3K-C2α-knockdown cells.(A) Effect of PI3K-C2α overexpression. The control or PI3K-C2α-knockdown (seq2) cells were transfected with NPY-mRFP. EGFP or shRNA-resistant EGFP-PI3K-C2α was transfected simultaneously. After 48 h, the cells were sensitized with anti-DNP IgE and then stimulated with 1 µM DNP-BSA. After stimulation, the red fluorescence in the cells showing the green fluorescence of EGFP was monitored. Scale bar = 5 µm. (B) Quantification of NPY-mRFP in cells. The intensities of mRFP fluorescence in control and PI3K-C2α-knockdown (seq2) cells were quantified and are shown relative to that at time zero. The data were obtained from six separate experiments (42 cells were examined in total) and are shown as the means ± s.e.m. *P<0.05, **P<0.01; the effect of PI3K-C2α expression is significant.
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pone-0111698-g003: Neuropeptide Y release from PI3K-C2α-knockdown cells.(A) Effect of PI3K-C2α overexpression. The control or PI3K-C2α-knockdown (seq2) cells were transfected with NPY-mRFP. EGFP or shRNA-resistant EGFP-PI3K-C2α was transfected simultaneously. After 48 h, the cells were sensitized with anti-DNP IgE and then stimulated with 1 µM DNP-BSA. After stimulation, the red fluorescence in the cells showing the green fluorescence of EGFP was monitored. Scale bar = 5 µm. (B) Quantification of NPY-mRFP in cells. The intensities of mRFP fluorescence in control and PI3K-C2α-knockdown (seq2) cells were quantified and are shown relative to that at time zero. The data were obtained from six separate experiments (42 cells were examined in total) and are shown as the means ± s.e.m. *P<0.05, **P<0.01; the effect of PI3K-C2α expression is significant.

Mentions: Neuropeptide Y (NPY) is a genuine reporter for the regulated exocytosis of mast cells [17]. In the experiments shown in Figure 3A, NPY-mRFP and EGFP- PI3K-C2α were transfected into RBL-2H3 cells. Upon stimulation, the mRFP fluorescence of the control cells decreased gradually, indicating the release of NPY from the cells. The NPY release from the PI3K-C2α-knockdown cells (shPI3K-C2α cells) was markedly slowed. The NPY release was then examined in the cells transfected with the shRNA-resistant PI3K-C2α construct. The overexpression of PI3K-C2α significantly increased the NPY-mRFP release from the control and shPI3K-C2α cells (Figure 3B), confirming the role of PI3K-C2α as a positive regulator of degranulation. A slight decrease in the mRFP fluorescence in the unstimulated cells (Figure 3B) may be due to the spontaneous release of NPY and fluorescence bleaching.


Involvement of class II phosphoinositide 3-kinase α-isoform in antigen-induced degranulation in RBL-2H3 cells.

Nigorikawa K, Hazeki K, Guo Y, Hazeki O - PLoS ONE (2014)

Neuropeptide Y release from PI3K-C2α-knockdown cells.(A) Effect of PI3K-C2α overexpression. The control or PI3K-C2α-knockdown (seq2) cells were transfected with NPY-mRFP. EGFP or shRNA-resistant EGFP-PI3K-C2α was transfected simultaneously. After 48 h, the cells were sensitized with anti-DNP IgE and then stimulated with 1 µM DNP-BSA. After stimulation, the red fluorescence in the cells showing the green fluorescence of EGFP was monitored. Scale bar = 5 µm. (B) Quantification of NPY-mRFP in cells. The intensities of mRFP fluorescence in control and PI3K-C2α-knockdown (seq2) cells were quantified and are shown relative to that at time zero. The data were obtained from six separate experiments (42 cells were examined in total) and are shown as the means ± s.e.m. *P<0.05, **P<0.01; the effect of PI3K-C2α expression is significant.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4214793&req=5

pone-0111698-g003: Neuropeptide Y release from PI3K-C2α-knockdown cells.(A) Effect of PI3K-C2α overexpression. The control or PI3K-C2α-knockdown (seq2) cells were transfected with NPY-mRFP. EGFP or shRNA-resistant EGFP-PI3K-C2α was transfected simultaneously. After 48 h, the cells were sensitized with anti-DNP IgE and then stimulated with 1 µM DNP-BSA. After stimulation, the red fluorescence in the cells showing the green fluorescence of EGFP was monitored. Scale bar = 5 µm. (B) Quantification of NPY-mRFP in cells. The intensities of mRFP fluorescence in control and PI3K-C2α-knockdown (seq2) cells were quantified and are shown relative to that at time zero. The data were obtained from six separate experiments (42 cells were examined in total) and are shown as the means ± s.e.m. *P<0.05, **P<0.01; the effect of PI3K-C2α expression is significant.
Mentions: Neuropeptide Y (NPY) is a genuine reporter for the regulated exocytosis of mast cells [17]. In the experiments shown in Figure 3A, NPY-mRFP and EGFP- PI3K-C2α were transfected into RBL-2H3 cells. Upon stimulation, the mRFP fluorescence of the control cells decreased gradually, indicating the release of NPY from the cells. The NPY release from the PI3K-C2α-knockdown cells (shPI3K-C2α cells) was markedly slowed. The NPY release was then examined in the cells transfected with the shRNA-resistant PI3K-C2α construct. The overexpression of PI3K-C2α significantly increased the NPY-mRFP release from the control and shPI3K-C2α cells (Figure 3B), confirming the role of PI3K-C2α as a positive regulator of degranulation. A slight decrease in the mRFP fluorescence in the unstimulated cells (Figure 3B) may be due to the spontaneous release of NPY and fluorescence bleaching.

Bottom Line: The release of mRFP-tagged neuropeptide-Y, a reporter for the regulated exocytosis, was also decreased in the PI3K-C2α-deficient cells.The release was increased significantly by the expression of the siRNA-resistant version of PI3K-C2α.These results indicated that PI3K-C2α and its product PtdIns(3,4)P2 may play roles in the secretory process.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima, Japan.

ABSTRACT
In this study, we present findings that suggest that PI3K-C2α, a member of the class II phosphoinositide 3-kinase (PI3K) subfamily, regulates the process of FcεRI-triggered degranulation. RBL-2H3 cells were transfected with shRNA targeting PI3K-C2α. The knockdown impaired the FcεRI-induced release of a lysosome enzyme, β-hexosaminidase, without affecting the intracellular Ca2+ mobilization. The release of mRFP-tagged neuropeptide-Y, a reporter for the regulated exocytosis, was also decreased in the PI3K-C2α-deficient cells. The release was increased significantly by the expression of the siRNA-resistant version of PI3K-C2α. In wild-type cells, FcεRI stimulation induced the formation of large vesicles, which were associated with CD63, a marker protein of secretory granules. On the vesicles, the existence of PI3K-C2α and PtdIns(3,4)P2 was observed. These results indicated that PI3K-C2α and its product PtdIns(3,4)P2 may play roles in the secretory process.

Show MeSH
Related in: MedlinePlus