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

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

The Sphingosine Kinase 1 Inhibitor, PF543, Mitigates Pulmonary Fibrosis by Reducing Lung Epithelial Cell mtDNA Damage and Recruitment of Fibrogenic Monocytes

Idiopathic pulmonary fibrosis (IPF) is a chronic disease for which novel approaches are urgently required. We reported increased sphingosine kinase 1 (SPHK1) in IPF lungs and that SPHK1 inhibition using genetic and pharmacologic approaches reduces murine bleomycin-induced pulmonary fibrosis. We determined whether PF543, a specific SPHK1 inhibitor post bleomycin or asbestos challenge mitigates lung fibrosis by reducing mitochondrial (mt) DNA damage and pro-fibrotic monocyte recruitment—both are implicated in the pathobiology of pulmonary fibrosis. Bleomycin (1.5 U/kg), crocidolite asbestos (100 µg/50 µL) or controls was intratracheally instilled in Wild-Type (C57Bl6) mice. PF543 (1 mg/kg) or vehicle was intraperitoneally injected once every two days from day 7−21 following bleomycin and day 14−21 or day 30−60 following asbestos. PF543 reduced bleomycin- and asbestos-induced pulmonary fibrosis at both time points as well as lung expression of profibrotic markers, lung mtDNA damage, and fibrogenic monocyte recruitment. In contrast to human lung fibroblasts, asbestos augmented lung epithelial cell (MLE) mtDNA damage and PF543 was protective. Post-exposure PF543 mitigates pulmonary fibrosis in part by reducing lung epithelial cell mtDNA damage and monocyte recruitment. We reason that SPHK1 signaling may be an innovative therapeutic target for managing patients with IPF and other forms of lung fibrosis

Coronin 1B localization in human lung endothelial cells.

<p>HPAECs grown to ∼90% confluence on slide chambers were fixed, permeabilized and localization of Coronin 1B, actin and co-localization of Coronin 1B with actin was visualized by immunocytochemistry as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063007#s4" target="_blank">Materials and Methods</a>. Shown are representative immunofluorescence images from several independent experiments as measured by regular (<b>A</b>) immunofluorescence and (<b>B</b>) confocal microscopy.</p

Role of Rac1 in S1P-induced chemotaxis and redistribution of coronin 1B and cortactin to lamellipodia in human lung endothelial cells.

<p>(<b>A</b>), HPAECs (∼90% confluence) grown on chamber slides were pretreated for 30 min with NSC23766 (50 µM), a Rac1 inhibitor, prior to stimulation with S1P (1 µM) for 15 min. Cells were washed, fixed, permeabilized, probed with antibodies, and redistribution of Coronin 1B and Cortactin was examined by immunofluorescence microscopy using a 60 X oil objective and quantified by ImageJ software (<b>B</b>) as described under <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063007#s4" target="_blank">Materials and Methods</a>. Shown is an immunofluorescence micrograph from three independent experiments. (<b>C</b>), In parallel experiments the effect of NSC23766 on chemotaxis was determined by a Boyden chamber-based trans-well assay as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0063007#s4" target="_blank">Materials and Methods</a>. Values are mean±SEM of three independent experiments. *, p<0.05 compared cells without S1P; **, p<0.005 compared to cells stimulated with S1P in the absence of NSC23766.</p