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Inositol Polyphosphate-4-Phosphatase Type I Negatively Regulates Phagocytosis via Dephosphorylation of Phagosomal PtdIns(3,4)P2.

Nigorikawa K, Hazeki K, Sasaki J, Omori Y, Miyake M, Morioka S, Guo Y, Sasaki T, Hazeki O - PLoS ONE (2015)

Bottom Line: Raw264.7 cells that express shRNA against Inpp4a (shInpp4a cells) showed significantly increased phagocytic activity.PtdIns(3,4)P2 increased on the phagosome of shInpp4a cells, while PtdIns(3)P significantly decreased.The results indicate that Inpp4a negatively regulates the phagocytic activity of macrophages as a member of the sequential dephosphorylation system that metabolizes phagosomal PtdIns(3,4,5)P3 to PtdIns(3)P.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan.

ABSTRACT
Phagocytosis is a highly conserved process whereby phagocytic cells engulf pathogens and apoptotic bodies. The present study focused on the role of inositol polyphosphate-4-phosphatase type I (Inpp4a) in phagocytosis. Raw264.7 cells that express shRNA against Inpp4a (shInpp4a cells) showed significantly increased phagocytic activity. The introduction of shRNA-resistant human Inpp4a abolished this increase. Macrophages from Inpp4a knockout mice showed similar increases in the phagocytic activity. Inpp4a was recruited to the phagosome membrane by a mechanism other than the direct interaction with Rab5. PtdIns(3,4)P2 increased on the phagosome of shInpp4a cells, while PtdIns(3)P significantly decreased. The results indicate that Inpp4a negatively regulates the phagocytic activity of macrophages as a member of the sequential dephosphorylation system that metabolizes phagosomal PtdIns(3,4,5)P3 to PtdIns(3)P.

No MeSH data available.


Phagosomal levels of PtdIns(3,4)P2 and PtdIns(3)P in Inpp4a-deficient cells.(A) Control (cont) or shInpp4a (seq2) cells were transfected with EGFP-[2×PH(TAPP1)]. (B) Control or shInpp4a (seq2) cells were transfected with EGFP-[3×FYVE(EEA1)]. (A, B; upper panels) the cells were challenged with E-IgG. The arrows indicate the phagosome. Bar, 10 μm. (A, B; lower panels) The intensity of the phagosome-associated fluorescence and total cellular fluorescence was quantified as described in the Methods section. The ratio of phagosome-associated fluorescence to total cellular fluorescence was calculated. The combined results from three separate cells are shown as the means ± s.e.m. *P<0.05, **P<0.01
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pone.0142091.g004: Phagosomal levels of PtdIns(3,4)P2 and PtdIns(3)P in Inpp4a-deficient cells.(A) Control (cont) or shInpp4a (seq2) cells were transfected with EGFP-[2×PH(TAPP1)]. (B) Control or shInpp4a (seq2) cells were transfected with EGFP-[3×FYVE(EEA1)]. (A, B; upper panels) the cells were challenged with E-IgG. The arrows indicate the phagosome. Bar, 10 μm. (A, B; lower panels) The intensity of the phagosome-associated fluorescence and total cellular fluorescence was quantified as described in the Methods section. The ratio of phagosome-associated fluorescence to total cellular fluorescence was calculated. The combined results from three separate cells are shown as the means ± s.e.m. *P<0.05, **P<0.01

Mentions: We examined the dynamics of PtdIns(3,4)P2, the preferential substrate of Inpp4a, during the phagocytosis. In the experiment shown in Fig 4A, the cells were transfected with EGFP-[2×PH(TAPP1)], a specific probe for PtdIns(3,4)P2. After the addition of E-IgG, the fluorescence around the engulfed E-IgG was monitored. In the control cells, PtdIns(3,4)P2 accumulated rapidly on the forming phagocytic cup. This accumulation peaked at 3 min and disappeared within 8 min after the start of phagocytosis (Fig 4A). In shInpp4a cells, PtdIns(3,4)P2 accumulated as rapidly as in the control cells, but its breakdown was markedly slowed (Fig 4A). The results suggest that Inpp4a hydrolyzes PtdIns(3,4)P2 on the phagosomal membrane. Similar results were obtained when EGFP-[PH(Akt)], which recognizes PtdIns(3,4)P2 and PtdIns(3,4,5)P3, was introduced to the cells (S2A Fig). We next monitored PtdIns(3)P, the product of Inpp4a, using EGFP-[3×FYVE(EEA1)] as a probe. As expected, the accumulation of PtdIns(3)P on the phagosome was decreased in the shInpp4a cells (Fig 4B). The impaired accumulation of phagosomal PtdIns(3)P was reproduced when the cells were challenged with zymosan particles (S2B Fig).


Inositol Polyphosphate-4-Phosphatase Type I Negatively Regulates Phagocytosis via Dephosphorylation of Phagosomal PtdIns(3,4)P2.

Nigorikawa K, Hazeki K, Sasaki J, Omori Y, Miyake M, Morioka S, Guo Y, Sasaki T, Hazeki O - PLoS ONE (2015)

Phagosomal levels of PtdIns(3,4)P2 and PtdIns(3)P in Inpp4a-deficient cells.(A) Control (cont) or shInpp4a (seq2) cells were transfected with EGFP-[2×PH(TAPP1)]. (B) Control or shInpp4a (seq2) cells were transfected with EGFP-[3×FYVE(EEA1)]. (A, B; upper panels) the cells were challenged with E-IgG. The arrows indicate the phagosome. Bar, 10 μm. (A, B; lower panels) The intensity of the phagosome-associated fluorescence and total cellular fluorescence was quantified as described in the Methods section. The ratio of phagosome-associated fluorescence to total cellular fluorescence was calculated. The combined results from three separate cells are shown as the means ± s.e.m. *P<0.05, **P<0.01
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pone.0142091.g004: Phagosomal levels of PtdIns(3,4)P2 and PtdIns(3)P in Inpp4a-deficient cells.(A) Control (cont) or shInpp4a (seq2) cells were transfected with EGFP-[2×PH(TAPP1)]. (B) Control or shInpp4a (seq2) cells were transfected with EGFP-[3×FYVE(EEA1)]. (A, B; upper panels) the cells were challenged with E-IgG. The arrows indicate the phagosome. Bar, 10 μm. (A, B; lower panels) The intensity of the phagosome-associated fluorescence and total cellular fluorescence was quantified as described in the Methods section. The ratio of phagosome-associated fluorescence to total cellular fluorescence was calculated. The combined results from three separate cells are shown as the means ± s.e.m. *P<0.05, **P<0.01
Mentions: We examined the dynamics of PtdIns(3,4)P2, the preferential substrate of Inpp4a, during the phagocytosis. In the experiment shown in Fig 4A, the cells were transfected with EGFP-[2×PH(TAPP1)], a specific probe for PtdIns(3,4)P2. After the addition of E-IgG, the fluorescence around the engulfed E-IgG was monitored. In the control cells, PtdIns(3,4)P2 accumulated rapidly on the forming phagocytic cup. This accumulation peaked at 3 min and disappeared within 8 min after the start of phagocytosis (Fig 4A). In shInpp4a cells, PtdIns(3,4)P2 accumulated as rapidly as in the control cells, but its breakdown was markedly slowed (Fig 4A). The results suggest that Inpp4a hydrolyzes PtdIns(3,4)P2 on the phagosomal membrane. Similar results were obtained when EGFP-[PH(Akt)], which recognizes PtdIns(3,4)P2 and PtdIns(3,4,5)P3, was introduced to the cells (S2A Fig). We next monitored PtdIns(3)P, the product of Inpp4a, using EGFP-[3×FYVE(EEA1)] as a probe. As expected, the accumulation of PtdIns(3)P on the phagosome was decreased in the shInpp4a cells (Fig 4B). The impaired accumulation of phagosomal PtdIns(3)P was reproduced when the cells were challenged with zymosan particles (S2B Fig).

Bottom Line: Raw264.7 cells that express shRNA against Inpp4a (shInpp4a cells) showed significantly increased phagocytic activity.PtdIns(3,4)P2 increased on the phagosome of shInpp4a cells, while PtdIns(3)P significantly decreased.The results indicate that Inpp4a negatively regulates the phagocytic activity of macrophages as a member of the sequential dephosphorylation system that metabolizes phagosomal PtdIns(3,4,5)P3 to PtdIns(3)P.

View Article: PubMed Central - PubMed

Affiliation: Graduate School of Biomedical & Health Sciences, Hiroshima University, Hiroshima 734-8553, Japan.

ABSTRACT
Phagocytosis is a highly conserved process whereby phagocytic cells engulf pathogens and apoptotic bodies. The present study focused on the role of inositol polyphosphate-4-phosphatase type I (Inpp4a) in phagocytosis. Raw264.7 cells that express shRNA against Inpp4a (shInpp4a cells) showed significantly increased phagocytic activity. The introduction of shRNA-resistant human Inpp4a abolished this increase. Macrophages from Inpp4a knockout mice showed similar increases in the phagocytic activity. Inpp4a was recruited to the phagosome membrane by a mechanism other than the direct interaction with Rab5. PtdIns(3,4)P2 increased on the phagosome of shInpp4a cells, while PtdIns(3)P significantly decreased. The results indicate that Inpp4a negatively regulates the phagocytic activity of macrophages as a member of the sequential dephosphorylation system that metabolizes phagosomal PtdIns(3,4,5)P3 to PtdIns(3)P.

No MeSH data available.