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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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β-hexosaminidase release of PI3K-C2α-knockdown cells.(A) FcεRI-mediated β-hexosaminidase release. Control or PI3K-C2α-knockdown cells were sensitized with anti-DNP IgE and then stimulated with the indicated concentrations of DNP-BSA at 37°C for 10 min. After centrifugation, the β-hexosaminidase activity in the supernatant was determined and is shown as a percentage of the activity in the total cell lysate. The data are shown as the means ± s.d. (n = 4). (B) Content of β-hexosaminidase. The cells were solubilized for determination of β-hexosaminidase activity. The activities are expressed as the absorbance change at 405 nm per 1 µg of protein and are shown as the means ± s.d. (n = 4). (C) PMA/A23187-induced β-hexosaminidase release. The cells were pre-treated with 30 nM PMA at 37°C for 10 min and then stimulated with 1 µM A23187 for 10 min. The β-hexosaminidase activity was measured as in (A). The data are shown as the means ± s.d. (n = 4). (D) Effect of wortmannin. Sensitized or non-sensitized RBL-2H3 cells were incubated at 37°C with or without 30 nM PMA for 10 min. When added, 1 µM wortmannin was included during this period. The cells were then incubated with or without 100 ng/mL DNP-BSA or 1 µM A23187 for 10 min. The β-hexosaminidase activity was measured as in (A). The data are shown as the means ± s.d. (n = 3).
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pone-0111698-g002: β-hexosaminidase release of PI3K-C2α-knockdown cells.(A) FcεRI-mediated β-hexosaminidase release. Control or PI3K-C2α-knockdown cells were sensitized with anti-DNP IgE and then stimulated with the indicated concentrations of DNP-BSA at 37°C for 10 min. After centrifugation, the β-hexosaminidase activity in the supernatant was determined and is shown as a percentage of the activity in the total cell lysate. The data are shown as the means ± s.d. (n = 4). (B) Content of β-hexosaminidase. The cells were solubilized for determination of β-hexosaminidase activity. The activities are expressed as the absorbance change at 405 nm per 1 µg of protein and are shown as the means ± s.d. (n = 4). (C) PMA/A23187-induced β-hexosaminidase release. The cells were pre-treated with 30 nM PMA at 37°C for 10 min and then stimulated with 1 µM A23187 for 10 min. The β-hexosaminidase activity was measured as in (A). The data are shown as the means ± s.d. (n = 4). (D) Effect of wortmannin. Sensitized or non-sensitized RBL-2H3 cells were incubated at 37°C with or without 30 nM PMA for 10 min. When added, 1 µM wortmannin was included during this period. The cells were then incubated with or without 100 ng/mL DNP-BSA or 1 µM A23187 for 10 min. The β-hexosaminidase activity was measured as in (A). The data are shown as the means ± s.d. (n = 3).

Mentions: The effect of PI3K-C2α knockdown on the FcεRI-triggered release of a lysosomal enzyme, namely β-hexosaminidase, was examined (Figure 2A). The β-hexosaminidase release was decreased significantly in both PI3K-C2α-knockdown cells. The total granule content of β-hexosaminidase was unchanged by the shRNA transfection (Figure 2B). The results suggested that PI3K-C2α is required for efficient degranulation via FcεRI. When RBL-2H3 cells were treated with calcium ionophore and phorbol ester simultaneously, a significant amount of β-hexosaminidase was released. This response was, however, unaffected by PI3K-C2α knockdown (Figure 2C). The pan-PI3K inhibitor wortmannin, which inhibits PI3K-C2α with an IC50 value of 420 nM [16], efficiently decreased the FcεRI-triggered degranulation at 1 µM but did not alter the calcium ionophore/phorbol ester-induced response (Figure 2D).


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)

β-hexosaminidase release of PI3K-C2α-knockdown cells.(A) FcεRI-mediated β-hexosaminidase release. Control or PI3K-C2α-knockdown cells were sensitized with anti-DNP IgE and then stimulated with the indicated concentrations of DNP-BSA at 37°C for 10 min. After centrifugation, the β-hexosaminidase activity in the supernatant was determined and is shown as a percentage of the activity in the total cell lysate. The data are shown as the means ± s.d. (n = 4). (B) Content of β-hexosaminidase. The cells were solubilized for determination of β-hexosaminidase activity. The activities are expressed as the absorbance change at 405 nm per 1 µg of protein and are shown as the means ± s.d. (n = 4). (C) PMA/A23187-induced β-hexosaminidase release. The cells were pre-treated with 30 nM PMA at 37°C for 10 min and then stimulated with 1 µM A23187 for 10 min. The β-hexosaminidase activity was measured as in (A). The data are shown as the means ± s.d. (n = 4). (D) Effect of wortmannin. Sensitized or non-sensitized RBL-2H3 cells were incubated at 37°C with or without 30 nM PMA for 10 min. When added, 1 µM wortmannin was included during this period. The cells were then incubated with or without 100 ng/mL DNP-BSA or 1 µM A23187 for 10 min. The β-hexosaminidase activity was measured as in (A). The data are shown as the means ± s.d. (n = 3).
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pone-0111698-g002: β-hexosaminidase release of PI3K-C2α-knockdown cells.(A) FcεRI-mediated β-hexosaminidase release. Control or PI3K-C2α-knockdown cells were sensitized with anti-DNP IgE and then stimulated with the indicated concentrations of DNP-BSA at 37°C for 10 min. After centrifugation, the β-hexosaminidase activity in the supernatant was determined and is shown as a percentage of the activity in the total cell lysate. The data are shown as the means ± s.d. (n = 4). (B) Content of β-hexosaminidase. The cells were solubilized for determination of β-hexosaminidase activity. The activities are expressed as the absorbance change at 405 nm per 1 µg of protein and are shown as the means ± s.d. (n = 4). (C) PMA/A23187-induced β-hexosaminidase release. The cells were pre-treated with 30 nM PMA at 37°C for 10 min and then stimulated with 1 µM A23187 for 10 min. The β-hexosaminidase activity was measured as in (A). The data are shown as the means ± s.d. (n = 4). (D) Effect of wortmannin. Sensitized or non-sensitized RBL-2H3 cells were incubated at 37°C with or without 30 nM PMA for 10 min. When added, 1 µM wortmannin was included during this period. The cells were then incubated with or without 100 ng/mL DNP-BSA or 1 µM A23187 for 10 min. The β-hexosaminidase activity was measured as in (A). The data are shown as the means ± s.d. (n = 3).
Mentions: The effect of PI3K-C2α knockdown on the FcεRI-triggered release of a lysosomal enzyme, namely β-hexosaminidase, was examined (Figure 2A). The β-hexosaminidase release was decreased significantly in both PI3K-C2α-knockdown cells. The total granule content of β-hexosaminidase was unchanged by the shRNA transfection (Figure 2B). The results suggested that PI3K-C2α is required for efficient degranulation via FcεRI. When RBL-2H3 cells were treated with calcium ionophore and phorbol ester simultaneously, a significant amount of β-hexosaminidase was released. This response was, however, unaffected by PI3K-C2α knockdown (Figure 2C). The pan-PI3K inhibitor wortmannin, which inhibits PI3K-C2α with an IC50 value of 420 nM [16], efficiently decreased the FcεRI-triggered degranulation at 1 µM but did not alter the calcium ionophore/phorbol ester-induced response (Figure 2D).

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