33 research outputs found
PI3-Kinase γ Promotes Rap1a-Mediated Activation of Myeloid Cell Integrin α4β1, Leading to Tumor Inflammation and Growth
<div><p>Tumor inflammation, the recruitment of myeloid lineage cells into the tumor microenvironment, promotes angiogenesis, immunosuppression and metastasis. CD11b+Gr1lo monocytic lineage cells and CD11b+Gr1hi granulocytic lineage cells are recruited from the circulation by tumor-derived chemoattractants, which stimulate PI3-kinase γ (PI3Kγ)-mediated integrin α4 activation and extravasation. We show here that PI3Kγ activates PLCγ, leading to RasGrp/CalDAG-GEF-I&II mediated, Rap1a-dependent activation of integrin α4β1, extravasation of monocytes and granulocytes, and inflammation-associated tumor progression. Genetic depletion of PLCγ, CalDAG-GEFI or II, Rap1a, or the Rap1 effector RIAM was sufficient to prevent integrin α4 activation by chemoattractants or activated PI3Kγ (p110γCAAX), while activated Rap (RapV12) promoted constitutive integrin activation and cell adhesion that could only be blocked by inhibition of RIAM or integrin α4β1. Similar to blockade of PI3Kγ or integrin α4β1, blockade of Rap1a suppressed both the recruitment of monocytes and granulocytes to tumors and tumor progression. These results demonstrate critical roles for a PI3Kγ-Rap1a-dependent pathway in integrin activation during tumor inflammation and suggest novel avenues for cancer therapy.</p> </div
RIAM is necessary for PI3Kγ-mediated integrin α4β1 activation.
<p>(A) Adhesion to VCAM-1 of chemoattractant-treated myeloid cells after transfection with RIAM or control siRNA (n = 3), *P<0.01 vs RIAM vs. non-silencing siRNA. (B) Adhesion to VCAM-1 of RIAM or non-silencing siRNA-transfected myeloid cells that were also transfected with p110γCAAX, pRapV12 or control plasmid (n = 3), *P<0.01 RIAM vs non-silencing siRNA (C) VCAM-1/Fc binding to RIAM or non-silencing siRNA-transfected myeloid cells that were also transfected with p110γCAAX, pRapV12 or control plasmid (n = 3), *P<0.01 RIAM vs non-silencing siRNA. (D) Immunoprecipitates of integrin α4β1 from WT, α4Y991A, and p110γ−/− bone marrow derived cells with (+) or without (−) SDF-1α stimulation and immunoblotted to detect the integrin α4 subunit, talin, and paxillin. Histograms: densitometry of talin and paxillin levels normalized to integrin α4 levels. (E) Trafficking to LLC tumors of WT or Rap1a−/− myeloid cells, myeloid cells treated with 10 µM GGTI-2147, and myeloid cells transfected with Rap1a or non-silencing siRNAs, (n = 3), *P<0.01 Rap1a−/− or 10 µM GGTI-2147 vs WT and Rap1a vs non-silencing siRNAs.</p
PI3Kγ-mediated integrin α4β1 adhesion requires Rap1a.
<p>(A) Adhesion to VCAM-1 of p110γCAAX, pRapV12 or pcDNA expressing CD11b+ myeloid cells that were also transfected with either integrin α4 or control siRNA, *P<0.01, integrin α4 vs non-silencing siRNA (n = 3). (B) Adhesion to VCAM-1 of p110γCAAX, pRapV12 and pcDNA (control transfected) WT or Rap1a−/− CD11b+ myeloid cells (n = 3), *P<0.01, p110γCAAX or pRapV12 vs pcDNA. (c) Adhesion to VCAM-1 of pRapV12 and pcDNA chemoattractant-stimulated WT, TG100-115 (PI3Kγ inhibitor)-treated, p110γ−/−, non-silencing, p110γ and integrin α4 siRNA-transfected CD11b+ myeloid cells (n = 3), *P<0.01 Rap1a vs pcDNA.</p
Myeloid cell Rap1a promotes tumor inflammation and growth.
<p>(A) Tumor volume and (B) weight of LLC tumors grown in WT mice transplanted with WT or Rap1a−/− bone marrow (n = 10); *P = 0.001 to 0.003, WT vs Rap1a−/− BM, as determined by ANOVA. (C) Percentage of CD11b+Gr1+ tumor-infiltrating myeloid cells in WT and Rap1a−/− bone marrow transplanted (BMT) tumors, *P<0.01, Rap1a−/− vs WT (ANOVA). (D) Percentage of CD11b+Gr1lo monocytic and CD11b+Gr1hi granulocytic lineage cells in WT and Rap1a−/− BMT tumors, *P<0.01 Rap1a−/− vs WT (ANOVA). (E) Representative FACs profiles from D, with upper quadrant corresponding to CD11b+Gr1hi granulocytic and lower quadrant corresponding to CD11b+Gr1lo monocytic lineage cells. (F) Percentage of F4/80+ tumor-infiltrating myeloid cells in WT and Rap1a−/− BMT tumors, *P<0.01 Rap1a−/− vs WT (ANOVA). (G) Representative images of tumor cryosections that were immunostained to detect F4/80+ macrophages (red) and CD31+ blood vessels (green) and counterstained with Dapi. Scale bar = 40 µM. (H) Quantification of F4/80+ pixels/field in cryosections from G, *P<0.01 Rap1a−/− vs WT. (I) CD31+ pixels/field in cryosections from G, *P<0.01 Rap1a−/− vs WT.</p
Rap1a and PI3Kγ each promote integrin α4β1-dependent myeloid cell adhesion.
<p>(A) Schematic of circulating, unstimulated myeloid cells, indicating inactive receptor tyrosine kinases, chemokine receptors and Toll-like Receptor/IL-1 Receptor family members (TIR family) and inactive downstream PI3Kγ and integrin α4β1. (B) Schematic of stimulated myeloid cells showing all three receptor types that activate PI3Kγ, thereby promoting integrin α4β1 conformational changes, leading to integrin activation. Co-factors that may play roles in activating integrin α4β1 include PKC, Rap1, RIAM, talin and paxillin. (C) Representative immunoblot of GTP-Rap and total Rap in myeloid cells that were stimulated with or without the chemoattractants IL-1β, IL6 or SDF1α and treated with the PI3Kγ inhibitor, TG100-115 (+) or the chemically similar, inert control (-). (D) Adhesion to the integrin α4β1 ligand VCAM-1 (expressed as fluorescence units, F.U.) of chemoattractant-stimulated WT and Rap1a−/− CD11b+ cells and Rap1a or non-silencing siRNA-transfected CD11b+ cells (n = 3), *P<0.01, Rap1a−/− vs WT and Rap1a vs nonsilencing siRNA. (E) Adhesion to VCAM-1 of CD11bGr1lo monocytes sorted from WT, p110γ−/− and Rap1a−/− animals (n = 3), *P<0.01, p110γ−/− and Rap1a−/− vs WT. (F) Adhesion to VCAM-1 of CD11bGr1hi granulocytes sorted from WT, p110γ−/− and Rap1a−/− animals (n = 3), *P<0.01, p110γ−/− and Rap1a−/− vs WT.</p
CalDAG-GEFs are required for PI3Kγ-mediated Rap1 and integrin α4β1 activation in myeloid cells.
<p>(A) Adhesion of chemoattractant-treated CD11b+ myeloid cells to VCAM-1 after transfection with non-silencing, CalDAG-GEFI, CalDAG-GEFII, Epac1, Epac2 and CalDAG-GEFI+CalDAG-GEFII siRNAs (n = 3), *P<0.01, CalDAG-GEFI, CalDAG-GEFII vs non silencing siRNA. (B) Adhesion to VCAM-1 of myeloid cells after transfection with CalDAG-GEFI and II or control siRNAs in combination with pRapV12, p110γCAAX, or empty plasmid (n = 3), *P<0.01 CalDAG-GEFI and II vs control siRNAs. (C) Rap1 GTP and total Rap immunoblot of unstimulated, non-silencing siRNA-transfected (Basal) myeloid cells and SDF-1α stimulated CalDAG-GEFI and II, Epac 1 and 2, and non-silencing siRNA-transfected myeloid cells. (D) Rap1 GTP and total Rap immunoblot of unstimulated non-silencing siRNA-transfected (basal) and SDF-1α-stimulated myeloid cells after transfection with PLCγ1 or control siRNA. (E) Adhesion of PLCγ1 or control siRNA-transfected, chemoattractant-stimulated myeloid cells (n = 3), *P<0.01 PLCγ1 vs non-silencing siRNA. (F) Adhesion to VCAM-1 of myeloid cells after transfection with PLCγ1 or control siRNA in combination with pRapV12, p110γCAAX, or vector control (empty plasmid) (n = 3), *P<0.01 vs PLCγ1 non-silencing siRNA.</p
PI3Kγ mediated activation of integrin α4β1 requires Rap1a and other co-factors.
<p>Schematic of stimulated myeloid cells demonstrating that diverse receptor types activate PI3Kγ, thereby promoting integrin α4β1 conformational changes, leading to integrin activation in a PLCγ1, RAP-GEF, Rap1, RIAM, talin and paxillin-dependent manner.</p
Rap1 promotes myeloid cell integrin α4β1-ligand binding and conformational changes.
<p>(A) Representative histogram of soluble, fluorescently labeled VCAM-1/Fc bound to CD11b+ myeloid cells 3 min after treatment with (black line) or without (grey line) IL-1β. (B) Mean fluorescence intensity (MFI) of VCAM-1/Fc bound to WT, p110γ−/− and Rap1a−/− myeloid cells in the absence (basal) or presence of chemoattractants or the positive control stimulus Mn2+ (n = 3), *P<0.01, p110γ−/− or Rap1a−/− vs WT. (C) MFI of VCAM-1/Fc bound to pRapV12- and control-transfected WT and p110γ−/− myeloid cells (n = 3), *P<0.01, pRapV12 vs control. (D) MFI of VCAM-1/Fc bound to WT and Rap1a−/− myeloid cells transfected with p110γCAAX, pRapV12 or control plasmid or treated with Mn2+ (n = 3), *P<0.01, Rap1a−/− vs WT. (E) Representative histogram of HUTS21 antibody binding to unstimulated (grey line) or SDF-1α stimulated (black line) human CD11b+ myeloid cells. (F) MFI of HUTS21 binding to unstimulated or SDF-1α stimulated human myeloid cells in the presence of absence of 10 µM geranylgeranyltransferase inhibitor (GGTI-2147) or Mn2+ (n = 3), *P<0.01, +GGTI vs -GGTI.</p
p110γ/δ Double-Deficiency Induces Eosinophilia and IgE Production but Protects from OVA-Induced Airway Inflammation
<div><p>The catalytical isoforms p110γ and p110δ of phosphatidylinositide 3-kinase γ (PI3Kγ) and PI3Kδ play an important role in the pathogenesis of asthma. Two key elements in allergic asthma are increased levels of eosinophils and IgE. Dual pharmacological inhibition of p110γ and p110δ reduces asthma-associated eosinophilic lung infiltration and ameliorates disease symptoms, whereas the absence of enzymatic activity in p110γ<sup>KO</sup>δ<sup>D910A</sup> mice increases IgE and basal eosinophil counts. This suggests that long-term inhibition of p110γ and p110δ might exacerbate asthma. Here, we analysed mice genetically deficient for both catalytical subunits (p110γ/δ<sup>-/-</sup>) and determined basal IgE and eosinophil levels and the immune response to ovalbumin-induced asthma. Serum concentrations of IgE, IL-5 and eosinophil numbers were significantly increased in p110γ/δ<sup>-/-</sup> mice compared to single knock-out and wildtype mice. However, p110γ/δ<sup>-/-</sup> mice were protected against OVA-induced infiltration of eosinophils, neutrophils, T and B cells into lung tissue and bronchoalveolar space. Moreover, p110γ/δ<sup>-/-</sup> mice, but not single knock-out mice, showed a reduced bronchial hyperresponsiveness. We conclude that increased levels of eosinophils and IgE in p110γ/δ<sup>-/-</sup> mice do not abolish the protective effect of p110γ/δ-deficiency against OVA-induced allergic airway inflammation.</p></div
Bronchoalveolar infiltration of eosinophils, neutrophils, T and B cells is reduced in OVA-treated p110γ<sup>-/-</sup>, p110δ<sup>-/-</sup>, and p110γ/δ<sup>-/-</sup> mice.
<p>To determine the number of eosinophils, neutrophils, T and B cells in the BALF from OVA-treated and PBS-treated KO and corresponding WT mice, cells were collected, and analysed by flow cytometry. Cell counts were normalised as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159310#pone.0159310.g002" target="_blank">Fig 2</a>. (<b>A</b>) Eosinophils (eos) in BALF from p110γ<sup>-/-</sup> and WT mice (left), from p110δ<sup>-/-</sup> and WT mice (middle), and from p110γ/δ<sup>-/-</sup> and WT mice (right). (<b>B</b>) Neutrophils (neutros) in BALF from p110γ<sup>-/-</sup> and WT mice (left), p110δ<sup>-/-</sup> and WT mice (middle), and p110γ/δ<sup>-/-</sup> and WT mice (right). (<b>C</b>) T cells in BALF from p110γ<sup>-/-</sup> and WT mice (left), p110δ<sup>-/-</sup> and WT mice (middle), and p110γ/δ<sup>-/-</sup> and WT mice (right). (<b>D</b>) B cells in BALF from p110γ<sup>-/-</sup> and WT mice (left), p110δ<sup>-/-</sup> and WT mice (middle), and p110γ/δ<sup>-/-</sup> and WT mice (right). Data (n = 3–6) are presented as means + SD. Data were analysed by One-way ANOVA followed by Bonferroni’s comparison tests for selected pairs of columns. <sup>+++</sup> P < 0.001, <sup>++</sup> P < 0.01, <sup>+</sup> P < 0.05. <sup>+</sup> indicate differences between WT PBS and WT OVA groups. ***P < 0.001, **P < 0.01, *P < 0.05. Asterisks indicate differences between OVA-treated groups.</p