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. 2015 Nov 1;195(9):4492-502.
doi: 10.4049/jimmunol.1500665. Epub 2015 Sep 21.

Differential Roles of Phospholipase D Proteins in FcεRI-Mediated Signaling and Mast Cell Function

Minghua Zhu  1 , Jianwei Zou  1 , Tieshi Li  1 , Sarah A O'Brien  1 , Yao Zhang  1 , Sarah Ogden  1 , Weiguo Zhang  2
Affiliations

Differential Roles of Phospholipase D Proteins in FcεRI-Mediated Signaling and Mast Cell Function

Minghua Zhu et al. J Immunol. .

Abstract

Phospholipase D (PLD) proteins are enzymes that catalyze the hydrolysis of phosphatidylcholine to generate an important signaling lipid, phosphatidic acid. Phosphatidic acid is a putative second messenger implicated in the regulation of vesicular trafficking and cytoskeletal reorganization. Previous studies using inhibitors and overexpression of PLD proteins indicate that PLD1 and PLD2 play positive roles in FcεRI-mediated signaling and mast cell function. We used mice deficient in PLD1, PLD2, or both to study the function of these enzymes in mast cells. In contrast to published studies, we found that PLD1 deficiency impaired FcεRI-mediated mast cell degranulation; however, PLD2 deficiency enhanced it. Biochemical analysis showed that PLD deficiency affected activation of the PI3K pathway and RhoA. Furthermore, our data indicated that, although PLD1 deficiency impaired F-actin disassembly, PLD2 deficiency enhanced microtubule formation. Together, our results suggested that PLD1 and PLD2, two proteins that catalyze the same enzymatic reaction, regulate different steps in mast cell degranulation.

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Figures

Figure 1
Figure 1
Generation of PLD1−/− and PLD2−/− mice. (A). Targeting constructs. The neo gene was removed by the FLP recombinase. The Cre-loxP system was used to delete exon 11 of PLD1 or exons 11 and 12 of PLD2. These exons were floxed by two LoxP sites. (B). Absence of PLD1 and PLD2 protein in PLD-deficient mice. BMMCs derived from the bone marrow cells of dKO (PLD1−/−PLD2−/−), PLD1−/−, PLD2−/−, and WT mice were analyzed by Western blotting after anti-PLD1 and anti-PLD2 immunoprecipitation.
Figure 2
Figure 2
PLD1 and PLD2 in mast cell function in vivo. (A). Passive systemic anaphylaxis. dKO, PLD1−/−, PLD2−/−, and WT mice were sensitized with 2μg of anti-DNP IgE for 20-24 hours. Anaphylaxis was induced by injection of 500 μg of DNP-HSA. Histamine concentration in the blood was determined by ELISA (n = 11 for each group of mice). The horizontal bars indicate mean values. Statistical analysis was done by two-tailed t test. (B). The levels of IL-6, TNF-α, and MCP-1. Mice were sensitized with 2 μg of anti-DNP IgE for 20-24 hours. Sera were collected 30 and 180 mins after injection of 500 μg of DNP-HSA. The concentrations of IL-6, TNF-α, and MCP-1 were determined by ELISA. (C). Expression of c-Kit and FcεRIα on peritoneal mast cells.
Figure 3
Figure 3
PLD in mast cell function in vitro. Mast cells were derived from the bone marrow cells from dKO, PLD1−/−, PLD2−/−, and WT mice in the presence of IL-3 for three weeks before analysis. (A). Expression of c-Kit and FcεRIα on BMMCs. (B). The PLD deficiency on PLD activity in mast cells. (C). FcεRI-mediated degranulation. BMMCs were sensitized with 1 μg/ml of anti-DNP IgE and then stimulated with various concentrations of DNP-HSA for 10 min or with thapsigargin. Degranulation of BMMCs was determined by measuring the release of β-hexosaminidase in the culture supernatant. (D). FcεRI-induced up-regulation of CD107a surface expression. IgE-sensitized BMMCs were stimulated with 100ng/ml DNP-HSA for 0, 2, 5, and 10min before FACS analysis. (E). FcεRI-mediated degranulation using a poorly cytokinergic IgE. BMMCs were sensitized with 1 μg/ml of anti-TNP IgE (C48-2) and then stimulated with various concentrations of TNP-BSA for 10 min. Degranulation of BMMCs was determined by measuring the release of β-hexosaminidase into the culture supernatant. (F). FcεRI-induced up-regulation of CD107a surface expression. IgE (C48-2)-sensitized BMMCs were stimulated with 100ng/ml TNP-BSA for 5min before FACS analysis.
Figure 4
Figure 4. PLD in FcεRI-mediated cytokine production
(A). Real-time PCR analysis. IL-6 and TNF-α. Sensitized BMMCs were stimulated with 100 ng/ml DNP-HSA for 1 h or left untreated before RNA isolation. Relative levels of IL-6 and TNF-α RNAs were analyzed by real-time PCR. Data shown were normalized by GAPDH and are representative of three independent experiments. (B). Intracellular cytokine production. Sensitized BMMCs were stimulated with DNP-HSA (100 ng/ml) for 4 hours in the presence of monensin before intracellular staining for IL-6 and TNF-α. (C). IL-6 and TNF-α secretion. Sensitized BMMCs were stimulated with different concentration of DNP-HSA for 24 hours. IL-6 and TNF-α concentrations in the supernatant were measured by ELISA.
Figure 5
Figure 5
The effect of PLD deficiency on FcεRI-mediated proximal signaling. (A). Tyrosine phosphorylation of proteins. After sensitization with anti-DNP IgE, dKO, PLD1−/−, PLD2−/−, and WT BMMCs were stimulated with DNP-HSA (100ng/ml) for the indicated time points. Whole cell lysates were analyzed by Western blotting with an anti-pTyr antibody. (B). MAPK activation. (C). PLC-γ activation. The numbers shown were relative intensities for the phosphorylated form of proteins normalized by non-phosphorylated form. (D). FcεRI-mediated calcium flux. BMMCs were sensitized with anti-DNP IgE and then loaded with Indo-1 in the presence of EGTA. DNP-HSA was used to induce intracellular Ca2+ mobilization followed by adding 20mM CaCl2 for extracellular Ca2+ flux. Data shown are representative of three experiments.
Figure 6
Figure 6
The effect of PLD deficiency on the PI3K pathway. dKO, PLD1−/−, PLD2−/−, and WT BMMCs were sensitized with anti-DNP IgE and activated with DNP-HSA before lysis. Whole cell lysates were blotted with antibodies against the phosphorylated and non-phosphorylated forms of PDK1, p70S6K, and AKT. Data shown are representative of three experiments. The numbers shown were relative intensities for the phosphorylated form of proteins normalized by non-phosphorylated form.
Figure 7
Figure 7
PLD in FcεRI-mediated cytoskeletal rearrangement. (A). BMMCs were sensitized with anti-DNP IgE and then stimulated with DNP-HSA for 2 and 5 min. Cells were fixed and stained with anti–α-tubulin (green) and rhodamine-phalloidin (red). Representative images by the confocal microscopy are shown (A). (B). The enlarged images from two representative cells of each genotype.
Figure 8
Figure 8
PLD deficiency affects FcεRI-mediated RhoA activation in mast cells. BMMCs were stimulated with DNP-HSA for 2 and 5 min before lysis. (A). Dephosphorylation of cofilin after FcεRI engagement. Whole cell lysates were immunoprecipitated with anti-cofilin antibody, followed by blotting with anti-p-cofilin and anti-cofilin antibodies, respectively. (B). Normal Vav phosphorylation. (C). RhoA activation. The whole cell lysates were subjected to precipitation with GST-Rhotekin-RBD beads. RhoA in precipitates (top) and input lysates (bottom) was detected by Western blotting with anti-RhoA antibodies. One representative of three independent experiments is shown.
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