Coronin 1B Regulates S1P-Induced Human Lung Endothelial Cell Chemotaxis: Role of PLD2, Protein Kinase C and Rac1 Signal Transduction

Coronins are a highly conserved family of actin binding proteins that regulate actin-dependent processes such as cell motility and endocytosis. We found that treatment of human pulmonary artery endothelial cells (HPAECs) with the bioactive lipid, sphingosine-1-phosphate (S1P) rapidly stimulates coronin 1B translocation to lamellipodia at the cell leading edge, which is required for S1P-induced chemotaxis. Further, S1P-induced chemotaxis of HPAECs was attenuated by pretreatment with small interfering RNA (siRNA) targeting coronin 1B (∼36%), PLD2 (∼45%) or Rac1 (∼50%) compared to scrambled siRNA controls. Down regulation PLD2 expression by siRNA also attenuated S1P-induced coronin 1B translocation to the leading edge of the cell periphery while PLD1 silencing had no effect. Also, S1P-induced coronin 1B redistribution to cell periphery and chemotaxis was attenuated by inhibition of Rac1 and over-expression of dominant negative PKC δ, ε and ζ isoforms in HPAECs. These results demonstrate that S1P activation of PLD2, PKC and Rac1 is part of the signaling cascade that regulates coronin 1B translocation to the cell periphery and the ensuing cell chemotaxis

Novel Peptide Nanoparticle Biased Antagonist of CCR3 Blocks Eosinophil Recruitment and Airway Hyperresponsiveness

Background—Chemokine signaling through CCR3 is a key regulatory pathway for eosinophil recruitment into tissues associated with allergic inflammation and asthma. To date, none of the CCR3 antagonists have shown efficacy in clinical trials. One reason may be their unbiased mode of inhibition that prevents receptor internalization, leading to drug tolerance. Objective—We sought to develop a novel peptide nanoparticle CCR3 inhibitor (R321) with a biased mode of inhibition that would block G-protein signaling, but enable or promote receptor internalization. Methods—Self-assembly of R321 peptide into nanoparticles and peptide binding to CCR3 were analyzed by dynamic light scattering and NMR. Inhibitory activity on CCR3 signaling was assessed in vitro using flow cytometry, confocal microscopy, and western blot analysis in a CCR3+ eosinophil cell line and blood eosinophils. In vivo effects of R321 were assessed using a triple allergen mouse asthma model. Results—R321 self-assembles into nanoparticles and binds directly to CCR3, altering receptor function. IC50 values for eotaxin-induced chemotaxis of blood eosinophils are in the low nanomolar range. R321 inhibits only the early phase of ERK1/2 activation and not the late phase generally associated with β-arrestin recruitment and receptor endocytosis, promoting CCR3 internalization and degradation. In vivo, R321 effectively blocks eosinophil recruitment into the lungs and airways and prevents airway hyperresponsiveness in a mouse eosinophilic asthma model. Conclusions—R321 is a potent and selective antagonist of the CCR3 signaling cascade. Inhibition through a biased mode of antagonism may hold significant therapeutic promise by eluding the formation of drug tolerance

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