AP-1 accumulates in perinuclear endosomes with FPR-arrestin complexes in the absence of Src associating with arrestin.

<p>Arr-2^-/-/-3^-/- FPR cells were transiently co-transfected with the indicated RFP-fused arrestins and the GFP-fused γ-subunit of AP-1. Cells were either treated with vehicle (unstimulated) (<b>A</b>) or stimulated with 10 nM 633-6pep for 1 hour (<b>B</b>). Transfected cells were imaged by confocal fluorescence microscopy. Images are representative of three independent experiments. Scale bars equal 10 μm. <b>C)</b> Quantitation of the percentage of cells with extraperinuclear receptor ligand-arrestin/AP-1 complexes following ligand stimulation. Cells were evaluated from three independent experiments and the results are expressed as the mean percentage ± SEM of cells displaying extraperinuclear colocalized ligand/arrestin/AP-1 clusters. * p<0.001 vs. EGFP-transfected cells; # p<0.001 vs. arr2-WT-transfected cells.</p

Inhibition of Src kinase, but not expression of Arr2-P91G/P121E, prevents FPR-mediated apoptosis.

<p>Arr-2^-/-/-3^-/- FPR cells were transiently transfected with the indicated constructs, stimulated with 10 nM fMLF and stained with PI. Random fields were viewed by fluorescence microscopy until 100–300 GFP-expressing cells were evaluated for PI staining. <b>A)</b> Arr-2^-/-/-3^-/- FPR cells were transiently transfected with the indicated GFP-fused arrestins or vector only (EGFP) and treated with 10 nM fMLF (stimulated) or vehicle (unstimulated). <b>B)</b> Arr-2^-/-/-3^-/- FPR cells were transiently transfected with the indicated GFP plasmids as in <b>A</b> and incubated with DMSO or zVAD-FMK (10 nM, 30 min) before and during stimulation with 10 nM fMLF. <b>C)</b> Arr-2^-/-/-3^-/- FPR cells were transiently transfected with the indicated plasmids and incubated with DMSO or PP2 (10 nM, 30 min) before and during stimulation with 10 nM fMLF. <b>D)</b> Arr-2^-/-/-3^-/- FPR cells were transiently co-transfected with the indicated arrestin plasmids and either wild type Src kinase or kinase dead (K298M) Src kinase, followed by treatment with 10 nM fMLF (stimulated) or vehicle (unstimulated). Data are expressed as the mean percentage of PI positive/GFP cell +/- SEM from three independent experiments. * p<0.001 vs. unstimulated (A, D) or DMSO-treated (B, C) cells; # p<0.001 vs. EGFP-transfected cells (A-C), EGFP-transfected Src WT cells (D) or arr2-P91G/P121E-transfected cells (D) as appropriate; + p<0.001 vs. arr2-WT-transfected cells.</p

Defective FPR localization and recycling in the presence of arr2-P91G/P121E.

<p><b>A)</b> Arr-2^-/-/-3^-/- FPR cells were transiently co-transfected with GFP-fused Rab11 and RFP-fused arrestin constructs. Cells were stimulated with 10 nM 633-6pep for 1 hour and imaged by confocal fluorescence microscopy. Images are representative of three independent experiments. See Suppl. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147442#pone.0147442.s001" target="_blank">S1A Fig</a> for unstimulated controls. <b>B)</b> Quantitation of the percent of cells displaying extraperinuclear (defined by perinuclear Rab11 localization) ligand-arrestin clusters. <b>C)</b> FPR recycling was assessed in arr-2^-/-/-3^-/- FPR cells transfected with EGFP vector, arr2-WT-GFP or arr2-P91G/P121E-GFP. Data are expressed as mean recycling as a percentage of the internalized FPR +/- SEM from three independent experiments. * p<0.001 vs. RFP- (B) or p<0.01 vs. EGFP-transfected cells (C); # p<0.001 (B) or p<0.01 (C) vs. arr2-WT-transfected cells.</p

AP-2 accumulates in perinuclear endosomes with FPR-arrestin complexes in the absence of Src associating with arrestin.

<p>Arr-2^-/-/-3^-/- FPR cells were transiently co-transfected with the indicated RFP-fused arrestins and the GFP-fused α-subunit of AP-2. Cells were either treated with vehicle (unstimulated) (<b>A</b>) or stimulated with 10 nM 633-6pep for 1 hour (<b>B</b>) and imaged by confocal fluorescence microscopy. Images are representative of three independent experiments. Scale bars equal 10 μm. <b>C)</b> Quantitation of the percentage of cells with extraperinuclear receptor ligand-arrestin/AP-2 complexes following ligand stimulation. Cells were counted from three independent experiments and data are expressed as the mean percentage ± SEM of cells displaying extraperinuclear colocalized ligand/arrestin/AP-2 clusters. * p<0.001 vs. RFP-transfected cells; # p<0.001 vs. arr2-WT-transfected cells.</p

Regulation of <i>N</i>-Formyl Peptide Receptor Signaling and Trafficking by Arrestin-Src Kinase Interaction

<div><p>Arrestins were originally described as proteins recruited to ligand-activated, phosphorylated G protein-coupled receptors (GPCRs) to attenuate G protein-mediated signaling. It was later revealed that arrestins also mediate GPCR internalization and recruit a number of signaling proteins including, but not limited to, Src family kinases, ERK1/2, and JNK3. GPCR-arrestin binding and trafficking control the spatial and temporal activity of these multi-protein complexes. In previous reports, we concluded that <i>N</i>-formyl peptide receptor (FPR)-mediated apoptosis, which occurs upon receptor stimulation in the absence of arrestins, is associated with FPR accumulation in perinuclear recycling endosomes. Under these conditions, inhibition of Src kinase and ERK1/2 prevented FPR-mediated apoptosis. To better understand the role of Src kinase in this process, in the current study we employed a previously described arrestin-2 (arr2) mutant deficient in Src kinase binding (arr2-P91G/P121E). Unlike wild type arrestin, arr2-P91G/P121E did not inhibit FPR-mediated apoptosis, suggesting that Src binding to arrestin-2 prevents apoptotic signaling. However, in cells expressing this mutant, FPR-mediated apoptosis was still blocked by inhibition of Src kinase activity, suggesting that activation of Src independent of arrestin-2 binding is involved in FPR-mediated apoptosis. Finally, while Src kinase inhibition prevented FPR-mediated-apoptosis in the presence of arr2-P91G/P121E, it did not prevent FPR-arr2-P91G/P121E accumulation in the perinuclear recycling endosome. On the contrary, inhibition of Src kinase activity mediated the accumulation of activated FPR-wild type arrestin-2 in recycling endosomes without initiating FPR-mediated apoptosis. Based on these observations, we conclude that Src kinase has two independent roles following FPR activation that regulate both FPR-arrestin-2 signaling and trafficking.</p></div

FPR-mediated apoptosis in the presence of arr2-P91G/P121E is sensitive to ERK inhibition.

<p>Arr-2^-/-/-3^-/- FPR cells were transiently transfected with GFP-fused arrestins and pre-incubated with U0126 (10 μM, 30 min), PD98059 (25 μM, 30 min) or vehicle (DMSO). Cells were stimulated with 10 nM fMLF and stained with PI. Five random fields were viewed by fluorescence microscopy until 100–300 cells GFP cells were evaluated and GFP-expressing cells were scored for the presence of PI staining. Data are expressed as the mean PI positive/GFP cell +/- SEM from three independent experiments. * p<0.001 vs. corresponding DMSO-treated cells; # p<0.001 vs. EGFP-transfected cells.</p

FPR-arrestin complexes exhibit differential trafficking in response to Src inhibition.

<p>Arr-2^-/-/-3^-/- FPR cells were transiently transfected with the constructs indicated below. Cells were stimulated with 10 nM 633-6pep for 1 hour and imaged by confocal fluorescence microscopy. <b>A)</b> Arr-2^-/-/-3^-/- FPR cells were transiently co-transfected with the indicated RFP-fused arrestin construct and GFP-fused Rab11 and incubated with PP2 (10 nM, 30 min) before and during ligand stimulation. See Suppl. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147442#pone.0147442.s001" target="_blank">S1B and S1C Fig</a> for unstimulated controls and Suppl. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147442#pone.0147442.s002" target="_blank">S2A Fig</a> for stimulated vehicle (no PP2) controls. <b>B)</b> Arr-2^-/-/-3^-/- FPR cells were transiently transfected with GFP-fused Rab11, RFP-fused arrestins and kinase dead Src kinase. See Suppl. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147442#pone.0147442.s001" target="_blank">S1D–S1F Fig</a> for unstimulated controls transfected with Src plasmid vector only (pUSE), wild type Src and kinase dead Src. See Suppl. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0147442#pone.0147442.s002" target="_blank">S2B and S2C Fig</a> for stimulated controls of cells transfected with Src plasmid vector only (pUSE) and wild type Src, respectively. Images are representative of three independent experiments. <b>C, D)</b> Quantitation of the percent of cells displaying extraperinuclear receptor ligand-arrestin complexes following treatment with PP2 (<b>C</b>) or transfection with kinase dead Src (<b>D</b>). Cells were counted from three independent experiments and data are expressed as the mean percentage ± SEM of cells displaying ligand-arrestin complexes outside the perinuclear region. Scale bars in images equal 10 μm. * p<0.001 vs. RFP-transfected cells; + p<0.001 vs. arr2-WT-transfected cells; # p<0.001 vs. corresponding DMSO-treated cells (C) or Src-WT-transfected cells (D) as appropriate.</p

IL-1β increases wound closure via a TGF-β1-dependent mechanism in primary human ATII cell monolayers.

<p>(<b>A</b>) IL-1β increases the rate of wound closure in primary human ATII cell monolayers. IL-1β (10 ng/ml) and/or TGF-βscRII or their respective vehicles were added to the monolayers after the scratch. Phase contrast microscopy (20X magnification) immediately after wounding (left panels, t = 0 h) and after 36 h (right panels t = 36 h). Scale bar: 100 µm. (<b>B</b>) TGF-β1 soluble receptor (TGF-βscRII) prevents IL-1β-dependent increase in rate of wound closure of primary human ATII cell monolayers. IL-1β (10 ng/ml) and/or TGF-βscRII or their respective vehicles were added to the monolayers after the scratch. Rate of wound closure was expressed as percent of control 16 h after wounding. *<i>p</i><0.05 from monolayers exposed to IL-1β vehicle. **<i>p</i><0.05 from monolayers exposed to IL-1β.</p

Endogenous HMGB1 released by primary rat ATII cell monolayers after scratch wounds increases alveolar epithelial wound closure via an IL-1β-dependent mechanism.

<p>(<b>A</b>) Supernatant from primary rat ATII monolayers collected 6 hours after multiple scratches (MS Cell Sup) induces an increase in the secretion of IL-1β by primary rat ATII cell monolayers via a TLR4-dependent pathway. MS Cell Sup, a blocking TLR4 antibody or its isotype control IgG were added to the monolayers after the scratch. HMGB1 was depleted from MS Cell Sup by immunoprecipitation using 30 µg/ml of HMGB1 specific Ab (MS Cell Sup IP w/HMGB1 Ab). Controls were MS Cell Sup immunoprecipitated with a control IgG (MS Cell Sup IP w/Cont Ab). IL-1β was measured by ELISA (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063907#s2" target="_blank">methods</a>) in the cell supernatant. (<b>B</b>) IL-1β receptor antagonist (IL-1RA) prevented the MS Cell Sup-dependent increase in the rate of wound closure of primary rat ATII cell monolayers. MS Cell Sup, and IL-1β receptor antagonist (IL-1RA, 1 µg/ml) or its vehicle were added to the monolayers after the scratch. Rate of wound closure is expressed as percent of control 16 h after wounding. *<i>p</i><0.05 from monolayers exposed to condition media. **<i>p</i><0.05 from monolayers exposed to MS Cell Sup.</p

List of class I PI3K isoform inhibitors and their respective in vitro IC₅₀.

<p>Assays were conducted side by side with 10 µM ATP using the method described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063907#pone.0063907-Knight1" target="_blank">[22]</a>.</p