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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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mRNA and protein expression levels of PI3K-C2α in control and shRNA-transfected RBL-2H3 cells.(A) Real-time PCR analysis of PI3K-C2α and PI3K-C2β in cells transfected with the control or shRNA expression vector. The mRNA levels are normalized against that of actin, and values relative to those of the control cells are shown as the means ± s.d. (n = 4). (B) Western blotting analysis of PI3K-C2α in cells transfected with control or shRNA expression vector. Representative blots from four separate experiments are shown. The protein expression levels are normalized against that of actin, and values relative to those of the control cells are shown as the means ± s.d. (n = 4).
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pone-0111698-g001: mRNA and protein expression levels of PI3K-C2α in control and shRNA-transfected RBL-2H3 cells.(A) Real-time PCR analysis of PI3K-C2α and PI3K-C2β in cells transfected with the control or shRNA expression vector. The mRNA levels are normalized against that of actin, and values relative to those of the control cells are shown as the means ± s.d. (n = 4). (B) Western blotting analysis of PI3K-C2α in cells transfected with control or shRNA expression vector. Representative blots from four separate experiments are shown. The protein expression levels are normalized against that of actin, and values relative to those of the control cells are shown as the means ± s.d. (n = 4).

Mentions: We first examined the expression of PI3K-C2α and PI3K-C2β mRNA in RBL-2H3 cells. Reverse transcriptase-PCR with specific primers showed that PI3K-C2α and PI3K-C2β mRNA is expressed in RBL-2H3 cells (Figure S1). Because PI3K-C2β has been reported to regulate the FcεRI-induced Ca2+ influx and degranulation in BMMCs [8], we examined whether PI3K-C2α plays any role in the cells. To this end, we prepared RBL-2H3 cells expressing shRNA against PI3K-C2α. Two lines of cells that produce shRNA against the different sequences (seq1 or seq2) of PI3K-C2α were prepared. In the seq1- and seq2-targeted cells, the levels of PI3K-C2α mRNA were 37% and 27%, respectively, of the level observed in the control vector-transfected cells (Figure 1A). The PI3K-C2β mRNA was unaffected by the shRNA (Figure 1A). The protein levels of PI3K-C2α in the seq1- and seq2-targeted cells, as determined by western blotting with a specific antibody, were 20% and 9.9%, respectively, of the levels observed in the control cells (Figure 1B). The protein levels of PI3K-C2β were not significantly affected by the shRNAs (Figure 1B).


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)

mRNA and protein expression levels of PI3K-C2α in control and shRNA-transfected RBL-2H3 cells.(A) Real-time PCR analysis of PI3K-C2α and PI3K-C2β in cells transfected with the control or shRNA expression vector. The mRNA levels are normalized against that of actin, and values relative to those of the control cells are shown as the means ± s.d. (n = 4). (B) Western blotting analysis of PI3K-C2α in cells transfected with control or shRNA expression vector. Representative blots from four separate experiments are shown. The protein expression levels are normalized against that of actin, and values relative to those of the control cells are shown as the means ± s.d. (n = 4).
© Copyright Policy
Related In: Results  -  Collection

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

pone-0111698-g001: mRNA and protein expression levels of PI3K-C2α in control and shRNA-transfected RBL-2H3 cells.(A) Real-time PCR analysis of PI3K-C2α and PI3K-C2β in cells transfected with the control or shRNA expression vector. The mRNA levels are normalized against that of actin, and values relative to those of the control cells are shown as the means ± s.d. (n = 4). (B) Western blotting analysis of PI3K-C2α in cells transfected with control or shRNA expression vector. Representative blots from four separate experiments are shown. The protein expression levels are normalized against that of actin, and values relative to those of the control cells are shown as the means ± s.d. (n = 4).
Mentions: We first examined the expression of PI3K-C2α and PI3K-C2β mRNA in RBL-2H3 cells. Reverse transcriptase-PCR with specific primers showed that PI3K-C2α and PI3K-C2β mRNA is expressed in RBL-2H3 cells (Figure S1). Because PI3K-C2β has been reported to regulate the FcεRI-induced Ca2+ influx and degranulation in BMMCs [8], we examined whether PI3K-C2α plays any role in the cells. To this end, we prepared RBL-2H3 cells expressing shRNA against PI3K-C2α. Two lines of cells that produce shRNA against the different sequences (seq1 or seq2) of PI3K-C2α were prepared. In the seq1- and seq2-targeted cells, the levels of PI3K-C2α mRNA were 37% and 27%, respectively, of the level observed in the control vector-transfected cells (Figure 1A). The PI3K-C2β mRNA was unaffected by the shRNA (Figure 1A). The protein levels of PI3K-C2α in the seq1- and seq2-targeted cells, as determined by western blotting with a specific antibody, were 20% and 9.9%, respectively, of the levels observed in the control cells (Figure 1B). The protein levels of PI3K-C2β were not significantly affected by the shRNAs (Figure 1B).

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