Regulation of N-formyl peptide receptor trafficking and signaling by arrestins

This work focuses on how arrestin regulates trafficking and signaling of the N-formyl peptide receptor (FPR), a G protein-coupled receptor (GPCR). GPCRs are involved in almost all physiologic processes and numerous pathologic processes. There is an intimate relationship between GPCR trafficking and signaling that controls many cellular processes. However, the protein-protein interactions that control post-endocytic trafficking and signaling of GPCRs are poorly understood. Our previous reports demonstrated that three events take place upon FPR activation in the absence of arrestins: accumulation of FPR in the perinuclear recycling endosome, lack of FPR recycling and apoptosis. All of these phenotypes were rescued by reintroduction of arrestin-2 cDNA. We therefore hypothesized that 1) FPR trafficking and signaling defects were linked and causal and 2) specific regions of arrestin-2 regulate normal FPR trafficking and signaling. To address these hypotheses, we generated mutants of arrestin-2 that were previously described or changed regions of similar amino acids to alanine. We then screened these mutants for the ability to rescue FPR-mediated apoptosis. Subsequently, we examined the role of these arrestin mutants in FPR trafficking. We found that two arrestin-2 mutants demonstrated altered binding to adaptor protein (AP)-2. Furthermore, FPR recycling was inhibited in the presence of either arrestin-2 mutant or the absence of AP-2. We also examined the role of Src kinase in FPR trafficking and signaling and determined that Src kinase has two independent roles in FPR-arrestin-2 regulation: one that controls FPR trafficking and one that mediated FPR signaling. Finally, we found that different SH3-binding domains of arrestin-2 regulate FPR trafficking and signaling independently. An arrestin-2 mutant did not rescue FPR-mediated apoptosis, but did mediate normal FPR trafficking. These results indicate that FPR trafficking and signaling are coordinated processes, but may also be regulated independently. These studies have revealed novel aspects of arrestin-2 that regulate FPR signaling and trafficking. We hope they will serve as a model for the regulation of other GPCRs. Furthermore, we hope these data are used to create small molecule inhibitors to serve as experimental tools and chemotherapeutics to better understand and treat diseases caused by defects in GPCR trafficking and signaling

HMGB1 Accelerates Alveolar Epithelial Repair via an IL-1β- and αvβ6 Integrin-dependent Activation of TGF-β1

High mobility group box 1 (HMGB1) protein is a danger-signaling molecule, known to activate an inflammatory response via TLR4 and RAGE. HMGB1 can be either actively secreted or passively released from damaged alveolar epithelial cells. Previous studies have shown that IL-1β, a critical mediator acute lung injury in humans that is activated by HMGB1, enhances alveolar epithelial repair, although the mechanisms are not fully understood. Herein, we tested the hypothesis that HMGB1 released by wounded alveolar epithelial cells would increase primary rat and human alveolar type II cell monolayer wound repair via an IL-1β-dependent activation of TGF-β1. HMGB1 induced in primary cultures of rat alveolar epithelial cells results in the release of IL-1β that caused the activation of TGF-β1 via a p38 MAPK-, RhoA- and αvβ6 integrin-dependent mechanism. Furthermore, active TGF-β1 accelerated the wound closure of primary rat epithelial cell monolayers via a PI3 kinase α-dependent mechanism. In conclusion, this study demonstrates that HMGB1 released by wounded epithelial cell monolayers, accelerates wound closure in the distal lung epithelium via the IL-1β-mediated αvβ6-dependent activation of TGF-β1, and thus could play an important role in the resolution of acute lung injury by promoting repair of the injured alveolar epithelium

COVID-19 and Long-Term Outcomes: Lessons from Other Critical Care Illnesses and Potential Mechanisms

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that is currently causing a pandemic and has been termed coronavirus disease (COVID-19). The elderly or those with preexisting conditions like diabetes, hypertension, coronary heart disease, chronic obstructive pulmonary disease, cerebrovascular disease, or kidney dysfunction are more likely to develop severe cases when infected. Patients with COVID-19 admitted to the ICU have higher mortality than non-ICU patients. Critical illness has consistently posed a challenge not only in terms of mortality but also in regard to long-term outcomes of survivors. Patients who survive acute critical illness including, but not limited to, pulmonary and systemic insults associated with acute respiratory distress syndrome, pneumonia, systemic inflammation, and mechanical ventilation, will likely suffer from post-ICU syndrome, a phenomenon of cognitive, psychiatric, and/or physical disability after treatment in the ICU. Post-ICU morbidity and mortality continue to be a cause for concern when considering large-scale studies showing 12-month mortality risks of 11.8-21%. Previous studies have demonstrated that multiple mechanisms, including cytokine release, mitochondrial dysfunction, and even amyloids, may lead to end-organ dysfunction in patients. We hypothesize that COVID-19 infection will lead to post-ICU syndrome via potentially similar mechanisms as other chronic critical illnesses and cause long-term morbidity and mortality in patients. We consider a variety of mechanisms and questions that not only consider the short-term impact of the COVID-19 pandemic but its long-term effects that may not yet be imagined

Altered response to Toll-like receptor 4 activation in fibromyalgia: A low-dose, human experimental endotoxemia pilot study

In this pilot study, a human intravenous injection of low-dose endotoxin (lipopolysaccharide, LPS) model was used to test if fibromyalgia is associated with altered immune responses to Toll-like receptor 4 (TLR4) activation. Eight women with moderately-severe fibromyalgia and eight healthy women were administered LPS at 0.1 ng/kg in session one and 0.4 ng/kg in session two. Blood draws were collected hourly to characterize the immune response. The primary analytes of interest, leptin and fractalkine, were assayed via commercial radioimmunoassay and enzyme-linked immunosorbent assay kits, respectively. Exploratory analyses were performed on 20 secreted cytokine assays by multiplex cytokine panels, collected hourly. Exploratory analyses were also performed on testosterone, estrogen, and cortisol levels, collected hourly. Additionally, standard clinical complete blood counts with differential (CBC-D) were collected before LPS administration and at the end of the session. The fibromyalgia group demonstrated enhanced leptin and suppressed fractalkine responses to LPS administration. In the exploratory analyses, the fibromyalgia group showed a lower release of IFN-γ, CXCL10, IL-17A, and IL-12 and higher release of IL-15, TARC, MDC, and eotaxin than the healthy group. The results of this study suggest that fibromyalgia may involve an altered immune response to TLR4 activation

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-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

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