PI3K γ Regulatory Protein p84 Determines Mast Cell Sensitivity to Ras Inhibition-Moving Towards Cell Specific PI3K Targeting?

Abstract

Mast cells are the major effector cells in immunoglobulin E (IgE)-mediated allergy. The high affinity IgE receptor FcεRI, as well as G protein-coupled receptors (GPCRs) on the mast cell surface signals to phosphoinositide 3-kinase γ (PI3Kγ) to initiate degranulation, cytokine release, and chemotaxis. PI3Kγ is therefore considered as a target for treatment of allergic disorders. However, leukocyte PI3Kγ is key to many functions in innate and adaptive immunity, and attenuation of host defense mechanisms is an expected adverse effect that complicates treatment of chronic illnesses. PI3Kγ operates as a p110γ/p84 or p110γ/p101 complex, where p110γ/p84 requires Ras activation. Here we investigated if modulation of Ras-isoprenylation could target PI3Kγ activity to attenuate PI3Kγ-dependent mast cell responses without impairment of macrophage functions. In murine bone marrow-derived mast cells, GPCR stimulation triggers activation of N-Ras and H-Ras isoforms, which is followed by the phosphorylation of protein kinase B (PKB/Akt) relayed through PI3Kγ. Although K-Ras is normally not activated in Ras wild-type cells, it is able to compensate for genetically deleted N- and H-Ras isoforms. Inhibition of Ras isoprenylation with farnesyltransferase inhibitor FTI-277 leads to a significant reduction of mast cell degranulation, cytokine production, and migration. Complementation experiments expressing PI3Kγ adaptor proteins p84 or p101 demonstrated a differential sensitivity towards Ras-inhibition depending on PI3Kγ complex composition. Mast cell responses are exclusively p84-dependent and were effectively controlled by FTI-277. Similar results were obtained when GTP-Ras was inactivated by overexpression of the GAP-domain of Neurofibromin-1 (NF-1). Unlike mast cells, macrophages express p84 and p101 but are p101-dominated and thus remain functional under treatment with FTI-277. Our work demonstrates that p101 and p84 have distinct physiological roles, and that Ras dependence of PI3Kγ signaling differs between cell types. FTI-277 reduces GPCR-activated PI3Kγ responses in p84-expressing but not p101-containing bone marrow derived cells. However, prenylation inhibitors have pleiotropic effects beyond Ras and non-tolerable side-effects that disfavor further clinical validation. Statins are, however, clinically well-established drugs that have previously been proposed to block mast cell degranulation by interference with protein prenylation. We show here that Simvastatin inhibits mast cell degranulation, but that this does not occur via Ras-PI3Kγ pathway alterations.

Keywords: IgE (Immunoglobulin E); PIK3CG; Ras family proteins; allergy; inflammation; p101; p84; phosphoinositide-3-kinase (PI3K).

Figure 1

Ras activation by GPCR ligands. (A) Relative mRNA abundance of N-Ras, K-Ras 4A, K-Ras 4B, H-Ras, R-Ras, R-Ras2 and M-Ras was assessed in BMMCs and BMMØs by qPCR and normalized to GAPDH expression level (n = 5–6). (B) N-Ras, K-Ras, H-Ras and R-Ras proteins were detected in BMMC and BMMØ lysates by Western blotting using corresponding isoform-specific antibodies. (C) Quantification from n = 7 measurements of Ras isoforms in BMMCs and BMMØs, with N-Ras expression used as a reference point (=1). (D) Plasmids encoding HA-tagged codon-optimized N-, K-, and H-Ras were transfected into HEK293 cells. Protein expression levels of the three isoforms in HEK293 lysates were equalized using anti-HA antibodies. These lysates were then used as standards for quantification of relative amount of Ras isoforms in (C). (E–H) Ras activation assay in BMMCs and BMMØs. Cells were starved in the corresponding starvation medium for 4 h before stimulation with 4 µM adenosine (Ade) (BMMC) or 10 nM C5a (BMMØ). GST-tagged Ras binding domain (RBD) of Raf1A was used to pull down GTP-loaded activated Ras. N-Ras, K-Ras, and H-Ras were subsequently detected in the pulled down fraction by Western blotting using isoform-specific antibodies and presented as a percentage of total amount of the corresponding isoform in the lysate used for the pull-down experiment (input). (E, F) show quantifications of n = 5–14 pull-down assays. Representative Western blot images are presented in (G, H). Statistical analysis was performed with one-way ANOVA and Bonferroni correction. Significance levels in all figures are indicated as non-significant (ns): p > 0.05; *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001; ****p ≤ 0.0001.

Figure 2

N- or H-Ras deletion without effect on PI3Kγ signaling due to Ras isoform compensation. (A) WT, H-Ras^−/− and N-Ras^−/− BMMCs were starved in IL-3-free medium containing 2% FCS for 4 h and stimulated with 2 µM adenosine for 2 min at 37°C. Phosphorylation of PKB at Ser473 was determined by Western blotting and normalized to the total PKB levels (n = 8–12). (B, C) Migration of wild-type (WT), H-Ras^−/− and N-Ras^−/− cells was assessed in Transwell chambers for 6 h at 37°C with 2 mM Ade in the lower well (n = 5–8). (D) Wild-type (WT), H-Ras^−/−, and N-Ras^−/− BMMCs were loaded overnight with anti-DNP IgE (100 ng/ml) followed by stimulation with DNP-HSA (antigen, Ag, 2 ng/ml) alone or in combination with 2 µM Ade. Release of β-Hexosaminidase was quantified 20 min after stimulation (n = 3–9). (E) K-Ras, N-Ras and H-Ras proteins were detected in the lysates from WT, N-Ras^−/− or H-Ras^−/− BMMCs by Western blotting using isoform-specific antibodies and normalized to the corresponding protein amount in WT cells (n = 8–15). (F) WT, N-Ras^−/− and H-Ras^−/− BMMCs were starved in IL-3-free medium containing 2% FCS for 4 h and stimulated with 4 µM Ade for 1 min at 37°C. Active Ras was pulled down from the lysates using GST-tagged Ras binding domain of Raf1A protein, detected by Western blotting with isoform-specific Ras antibodies and presented as a percentage of the total amount of the corresponding Ras isoform in the lysate used for the pull-down experiment (n = 3–9). Statistical analysis was performed with one-way ANOVA for (A–E), and Student’s t-test for (F).

Figure 3

Action of FTI-277 on Ras membrane localization. BMMCs were transfected with constructs expressing GFP-tagged CAAX-domain (C-terminal 25 amino acids) of H-Ras, N-Ras or K-Ras and treated with 5µM FTI-277 for 72 h. Cells were fixed in 4% p-formaldehyde, and images were acquired by confocal microscopy. The image squares have a length of 10 µm. Left side graphs show distribution of Ras protein along a cross-section of the cell. For each condition n ≥ 18 cells were analyzed. Plotted are mean values ± standard error of mean (SEM).

Figure 4

Prevention of activation of Ras isoforms by FTI-277. (A, B) Effect of FTI-277 on N-Ras and H-Ras activation in BMMCs. Cells were treated with DMSO or 5 µM FTI-277 for 72 h. Activated N-Ras and H-Ras was pulled down with GTP-Raf-RBD and normalized to Ras amount in complete cell lysate. (C–E) Effect of FTI-277 on N-Ras, K-Ras, and H-Ras activation in BMMØs. BMMØs cultured for 5 days were treated with DMSO or 5 µM FTI-277 for 72 h. Every quantification contains n = 3–6 Ras activation assays, and one representative immunoblot is shown for each condition. Student’s t-test was applied for statistical analysis.

Figure 5

Mast cell but not macrophage activation is affected by FTI-277-mediated Ras inhibition. (A) DMSO- or FTI-277-treated WT-BMMCs were loaded overnight with anti-DNP IgE (100 ng/ml) followed by stimulation with DNP-HSA (Ag, 2 ng/ml) alone or in combination with 2µM Ade or 10 ng/ml SCF. Release of β-Hexosaminidase was quantified 20 min after stimulation (n = 6–7). (B) DMSO- or FTI-277-treated WT-BMMCs as well as p110γ^−/− BMMCs were exposed overnight to anti-DNP IgE (100 ng/ml) followed by stimulation with DNP-HSA (Ag, 2 ng/ml) together with 1 µM Ade for 6 h. TNF-α and IL-6 expressions were determined by qPCR and normalized to the level of GAPDH expression. Fold change of expression in FTI-277 treated or p110γ^−/− cells was quantified relative to DMSO-treated control (n = 6). (C, D) BMMCs and BMMØs were treated with either DMSO or 5 µM FTI-277 for 72 h, starved for 4 h, and activated with 2 µM Ade, 10 ng/ml SCF, 10 nM C5a, or 30 ng/ml M-CSF for 2 min at 37°C. Phosphorylation of PKB at Ser473 was determined by Western blot analysis of cell lysates with anti-PKB-Ser473 antibodies and normalized to the total level of PKB (n = 9–18). (E, F) Migration of BMMCs and BMMØs was assessed in Transwell chambers for 6 h at 37°C with indicated stimuli in the lower well, followed by quantification of migrated cells (n = 6–16). (E, F) were assessed with Student’s t-test and (B–D) were subjected to one-way ANOVA with Bonferroni’s post hoc test.

Figure 6

GGTI-298 affects activation of mast cells, but not macrophages. BMMCs (A, C) and BMMØs (B, D) were treated with either DMSO or 5 µM GGTI-298 for 24 h, starved for 4 h and activated with 2 µM Ade, 10 ng/ml SCF, 10 nM C5a or 30 ng/ml M-CSF for 2 min at 37°C. Phosphorylation of PKB (Ser473) and MAPK was determined by immunoblot blot analysis of cell lysates and normalized to the total level of corresponding proteins (n = 4–10). Statistical significances were assessed by Student’s t-test.

Figure 7

(A) Simvastatin inhibits mast cell degranulation without interfering with PI3Kγ signaling. BMMCs were treated with indicated statins at 5 µM for 16 h and then loaded with anti-DNP IgE (100 ng/ml). The following day, cells were stimulated with DNP-HSA (Ag, 2 ng/ml) plus 2 µM adenosine. Release of β-Hexosaminidase was quantified 20 min after stimulation and normalized to untreated control (ctrl= DMSO 0.05% for Lovastatin and Simvastatin; PBS 0.001% MetOH for Atorvastatin). Lovastatin (n = 4–7) and Simvastatin (n = 10–16) are pro-drugs, while Atorvastatin (n = 4), Simvastatin-Na (n = 10–16) and Lovastatin-Na (n = 4–7) are active drugs. (B) Dose dependency for Simvastatin-Na was tested in three independent experiments with total n = 9 biological replicates. Degranulation was assessed with IgE/antigen-stimulation alone (Ag, DNP-HSA 2 ng/ml) and IgE/antigen co-stimulation with adenosine (2 µM). (C, D) Western blot analysis of BMMCs treated with 5µM Simvastatin-Na for 16 h and starved for 4 h. Adenosine (2 µM) was used as stimulus. Phosphorylated PKB and MAPK was quantified from n = 3–9 experiments. Statistical significance was tested with one-way ANOVA applying Bonferroni correction.

Figure 8

Sensitivity to Ras inhibition is defined by PI3Kγ adaptor subunit. (A) Expression of p110γ, p84, and p101 in BMMCs and BMMØs was assessed by qPCR and normalized to GAPDH expression in BMMCs. (B) p84, p101, and p110γ proteins were detected in BMMC and BMMØ lysates by Western blotting and quantified using recombinant p84/p110γ and p101/p110γ complexes with known protein concentration. (C) p110γ and HA-p84 or HA-p101 were co-expressed with or without Flag-NF1 (GAP domain) in p110γ^−/− BMMCs; additionally, GFP-expressing plasmid was used to select for transfected cells. Migration of GFP-positive BMMCs was assayed in Transwell chambers for 6 h in the presence of 2 µM Ade in the lower well. Subsequently, GFP-positive cells were quantified by fluorescence microscopy. The panel contains n = 5 biological replicates from two independent experiments. (D, E) p110γ^−/− BMMCs were transfected with plasmids encoding functional p110γ and either HA-tagged p84 or p101. 5 h after transfection cells were put in fresh medium containing DMSO or 5 µM FTI-277. The next day, cells were starved in IL-3-free medium containing 2% FCS and stimulated with 2 µM Ade for 2 min at 37°C. Phosphorylation of PKB at Ser473 and MAPK was determined by Western blotting and normalized to the total PKB or MAPK levels, correspondingly. Transient expression of p110γ was assessed with anti-p110γ antibodies, while p84 and p101 were detected with anti-HA antibodies (n = 5–6). Student’s t-test was performed to test for statistical relationships.