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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.


Enhanced phagocytosis of zymosan in Inpp4a-deficient cells.(A) Raw264.7 cells deficient in Inpp4a (seq1 and seq2) or control cells were incubated for 15 min with IgG-coated zymosan. (B) Peritoneal macrophages from wild type (WT) or Inpp4a knock out (KO) mice were incubated for 15 min with IgG-coated zymosan. (A, B) The number of fluorescent particles was counted in merged images, and the number of zymosan particles within the cells was calculated. The results from three separate experiments are shown as the means ± s.e.m. **P<0.01
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pone.0142091.g002: Enhanced phagocytosis of zymosan in Inpp4a-deficient cells.(A) Raw264.7 cells deficient in Inpp4a (seq1 and seq2) or control cells were incubated for 15 min with IgG-coated zymosan. (B) Peritoneal macrophages from wild type (WT) or Inpp4a knock out (KO) mice were incubated for 15 min with IgG-coated zymosan. (A, B) The number of fluorescent particles was counted in merged images, and the number of zymosan particles within the cells was calculated. The results from three separate experiments are shown as the means ± s.e.m. **P<0.01

Mentions: We first determined the mRNA expression of Inpp4a and Inpp4b in Raw264.7 cells. Reverse transcriptase-PCR with specific primers showed that Inpp4a mRNA is expressed, while Inpp4b mRNA is not expressed in Raw264.7 cells (Fig 1A and 1B). The performance of the primer pair for Inpp4b mRNA was confirmed using cDNAs from the mouse heart (Fig 1B). Two lines of cells (shInpp4a-seq1 and shInpp4a-seq2 cells) that produce shRNAs targeting different Inpp4a sequences were prepared. The protein level of Inpp4a decreased to 5% and 14% of the wild type cells in seq1- and seq2-expressing cells, respectively (Fig 1C). The uptake of IgG-zymosan particles increased in the shInpp4a cells (Fig 2A). Binding of IgG-opsonized particles to FcγR is known to cause Akt phosphorylation as a result of cell accumulation of PtdIns(3,4,5)P3 and PtdIns(3,4)P2. The FcγR-induced Akt phosphorylation was markedly increased in the knockdown cells (S1A Fig). We next examined the phagocytic activity of macrophages from Inpp4a knockout mice, which were prepared by crossing Inpp4aflox/flox mice with transgenic mice expressing Cre under the control of the CD11b promoter (Fig 1D). The macrophages from knockout mice showed increased uptake of IgG-opsonized zymosan (Fig 2B). FcγR-induced Akt phosphorylation was increased in the Inpp4a-deficient cells (S1B 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)

Enhanced phagocytosis of zymosan in Inpp4a-deficient cells.(A) Raw264.7 cells deficient in Inpp4a (seq1 and seq2) or control cells were incubated for 15 min with IgG-coated zymosan. (B) Peritoneal macrophages from wild type (WT) or Inpp4a knock out (KO) mice were incubated for 15 min with IgG-coated zymosan. (A, B) The number of fluorescent particles was counted in merged images, and the number of zymosan particles within the cells was calculated. The results from three separate experiments are shown as the means ± s.e.m. **P<0.01
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4633150&req=5

pone.0142091.g002: Enhanced phagocytosis of zymosan in Inpp4a-deficient cells.(A) Raw264.7 cells deficient in Inpp4a (seq1 and seq2) or control cells were incubated for 15 min with IgG-coated zymosan. (B) Peritoneal macrophages from wild type (WT) or Inpp4a knock out (KO) mice were incubated for 15 min with IgG-coated zymosan. (A, B) The number of fluorescent particles was counted in merged images, and the number of zymosan particles within the cells was calculated. The results from three separate experiments are shown as the means ± s.e.m. **P<0.01
Mentions: We first determined the mRNA expression of Inpp4a and Inpp4b in Raw264.7 cells. Reverse transcriptase-PCR with specific primers showed that Inpp4a mRNA is expressed, while Inpp4b mRNA is not expressed in Raw264.7 cells (Fig 1A and 1B). The performance of the primer pair for Inpp4b mRNA was confirmed using cDNAs from the mouse heart (Fig 1B). Two lines of cells (shInpp4a-seq1 and shInpp4a-seq2 cells) that produce shRNAs targeting different Inpp4a sequences were prepared. The protein level of Inpp4a decreased to 5% and 14% of the wild type cells in seq1- and seq2-expressing cells, respectively (Fig 1C). The uptake of IgG-zymosan particles increased in the shInpp4a cells (Fig 2A). Binding of IgG-opsonized particles to FcγR is known to cause Akt phosphorylation as a result of cell accumulation of PtdIns(3,4,5)P3 and PtdIns(3,4)P2. The FcγR-induced Akt phosphorylation was markedly increased in the knockdown cells (S1A Fig). We next examined the phagocytic activity of macrophages from Inpp4a knockout mice, which were prepared by crossing Inpp4aflox/flox mice with transgenic mice expressing Cre under the control of the CD11b promoter (Fig 1D). The macrophages from knockout mice showed increased uptake of IgG-opsonized zymosan (Fig 2B). FcγR-induced Akt phosphorylation was increased in the Inpp4a-deficient cells (S1B 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.