Figure 1.
Expression of coronin 1B in human endothelial cells.
Human lung microvascular, pulmonary artery, umbilical vein and aortic ECs grown to ∼90% confluence in 60 mm dishes were lysed in cell lysis buffer. Cell lysates (20–40 µg of protein) were subjected to 10% SDS-PAGE, transferred to PVDF membrane and probed with anti-coronin 1B and β-actin antibodies as described under Materials and Methods. (A), Shown is a representative Western blot depicting coronin 1B protein expression in different human ECs. (B), In parallel experiments total RNA was isolated from various human ECs and were analyzed for mRNA expression of Coronin 1B by quantitative real time RT-PCR. The values are mean ± S.E.M for three independent experiments each g002performed in triplicate and normalized to GAPDH mRNA expression.
Figure 2.
Coronin 1B localization in human lung endothelial cells.
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 Materials and Methods. Shown are representative immunofluorescence images from several independent experiments as measured by regular (A) immunofluorescence and (B) confocal microscopy.
Figure 3.
Effect of starvation and serum supplementation on Coronin 1B localization in human lung endothelial cells.
HPAECs grown on slide chambers (∼90% confluence) were incubated in EBM-2 medium containing either 0.1% serum for 3 h or in EBM-2 medium containing 5% serum for 3 h and 48 h. Cells were fixed, permeabilized and Coronin 1B localization was visualized by immunocytochemistry as described in Materials and Methods. Shown are representative immunofluorescence images from several independent experiments.
Figure 4.
S1P stimulates redistribution of coronin 1B and cortactin to lamellipodia in human lung endothelial cells.
HPAECs grown on slide chambers (∼90% confluence) were stimulated with 1 µM S1P for different time interval (2, 5, 15, 30 and 60 min) as indicated. Redistribution of Coronin 1B (B) and Cortactin (C) was visualized by immunocytochemistry and quantified by ImageJ software as described in Materials and Methods. Shown are representative immunofluorescence images from several independent experiments.
Figure 5.
Coronin 1B siRNA attenuates S1P-induced chemotaxis, wound healing and lamellipodia localization of cortactin in HPAECs.
(A), HPAECs grown on transwell inserts were stimulated with different S1P concentration (0.01, 0.1, 1 and 10 µM) for 15 min and chemotaxis was estimated by a Boyden chamber-based trans-well assay as described in Materials and Methods. The values are mean±SEM of three independent experiments. *, p<0.05 compared to cells without S1P. (B) HPAECs were transfected with scrambled (sc) or siRNA for Coronin 1B (50 ng/ml, 72 h), and cell lysates (20 µg of protein) were subjected to 10% SDS-PAGE and probed with Coronin 1B and actin antibodies as indicated. (C) HPAECs grown to 50% confluence in 100-mm dishes were transfected with sc (sc) or Coronin 1B siRNA (50 ng/ml) for 72 h. The cells were trypsinazied and plated on to transwell inserts and S1P-induced chemotaxis was determines as described in (A). The values are mean±SEM of three independent experiments in triplicate. *, p<0.05 compared cells without S1P; **, p<0.001 compared to scrambled siRNA transfected cells plus S1P. (D), HPAECs transfected with scrambled (sc) or Coronin 1B siRNA (50 nM, 72 h) were wounded on the gold electrodes as described under Materials and Methods. Measurement of transendothelial electrical resistance (TER) using an electrical cell substrate impedance-sensing system (ECIS) for 12 h after wounding the cells on the gold electrode and exposure to 1.0 µM S1P was carried out. Shown is a tracing from three independent experiments in triplicate. (E), HPAECs transfected with scrambled (sc) or Coronin 1B siRNA (50 nM, 72 h) were seeded on slide chambers for 24 h prior to stimulation with 1 µM S1P for 15 min. Cells were fixed and Coronin 1B and Cortactin redistribution to cell periphery was visualized by immunocytochemistry as described in Materials and Methods. Shown is a representative immunofluorescence image taken using an X 60 oil objective as described under Materials and Methods.
Figure 6.
Role of PLD2 in S1P-induced chemotaxis, Coronin 1B and actin lamellipodia localization in HPAECs.
HPAECs (∼50% confluence) were transfected with scrambled (sc), PLD1 or PLD2 siRNA (50 ng/ml) for 72 h. (A) Cell lysates (20–40 µg of protein) were subjected to 10% SDS-PAGE, Western blotted and probed with PLD1 and PLD2 antibodies as indicated; (B) chemotaxis of scrambled (sc) or siRNA transfected cells to S1P (1 µM) for 15 min was carried out in a Boyden chamber-based trans-well assay as described under Materials and Methods. Values are mean±SEM of three independent experiments in triplicate. *, p<0.01 compared cells without S1P; **, p<0.005 compared to scrambled siRNA transfected cells plus S1P; HPAECs transfected with sc, PLD1 (C) or PLD2 (E) siRNA in 100-mm dishes as described under (A) were trypsinazied and seeded onto slide chambers prior to stimulation with S1P (1 µM) for 15 min. Cells were washed, fixed, permeabilized, and probed with anti-Coronin 1B and AlexaFluor Phalloidin antibodies, and redistribution of Coronin 1B and actin due to downregulation of PLD1 (D) or PLD2 (F) was examined by immunofluorescence microscopy using a 60 X oil objective and quantified by ImageJ software as described under “Experimental Procedures”. Shown is an immunofluorescence micrograph from three independent experiments.
Figure 7.
Role of Rac1 in S1P-induced chemotaxis and redistribution of coronin 1B and cortactin to lamellipodia in human lung endothelial cells.
(A), 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) as described under Materials and Methods. Shown is an immunofluorescence micrograph from three independent experiments. (C), In parallel experiments the effect of NSC23766 on chemotaxis was determined by a Boyden chamber-based trans-well assay as described in Materials and Methods. 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.
Figure 8.
PLD2 mutant attenuates S1P-induced lamellipodial localization of Rac1 and Coronin 1B in human lung endothelial cells.
HPAECs (∼50% confluence) grown on slide chambers were infected with vector-control or adenoviral mPLD2 K758R mutant (5 MOI) for 24 h, prior to stimulation with 1 µM S1P for 15 min. Cells were washed, fixed, permeabilized, probed with antibodies, and redistribution of Rac1 and Coronin 1B was examined by immunofluorescence microscopy using a 60 X oil objective and quantified by ImageJ software (B) as described under Materials and Methods. Shown are representative immunofluorescence micrographs from three independent experiments.
Figure 9.
Role of PKC δ, ε, and ζ isoforms on S1P mediated chemotaxis and lamellipodial localization of Coronin 1B in human lung endothelial cells.
HPAECs grown on slide chambers or 35-mm dishes (∼70% confluence) were infected with empty vector or adenoviral vectors encoding PKC dominant negative (dn) δ, ε, and ζ isoforms (5 MOI) in complete EGM-2 medium for 24 h. (A), Cell lysates (20 µg of protein) were subjected to 10% SDS-PAGE, Western blotting and probed with anti-PKC δ, ε, ζ and actin antibodies. In parallel experiments, the effect of dn PKC δ, ε and ζ isoforms on chemotaxis (B) and lamellipodial localization of coronin 1B and actin (C, D and E) was examined as described in Materials and Methods. Values are mean±SEM of three independent experiments. *, p<0.01 compared cells without S1P; **, p<0.005 compared to cells infected with empty vector and stimulated with S1P.
Figure 10.
Proposed signaling mechanisms involved in S1P-induced lamellipodial localization of Coronin 1B, Cortactin and chemotaxis of human lung endothelial cells.
S1P binding to G-protein coupled S1P1-5 receptors activates PLD2 via PKC δ and ε and activation of PLD2 results in hydrolysis of membrane associated phosphatidylcholine (PC) to phosphatidic acid (PA) and phospho-choline. PA can be converted to DAG by PA-phosphatases or can activate PKC ζ via of phosphatidylinositol-4-phosphate-5kinase activation. Activation of PKC ζ results in redistribution of Coronin 1B and Cortactin to cell periphery and localization in lamellipodia of endothelial cells. PA can directly bind to and activate Rac1 and formation of actin stress fibers. Additionally, PLD2 has guanine nucleotide-exchange factor (GEF) activity for Rho and can regulate actin stress fibers in a manner independent of its lipase activity. S1P-induced activation of PKC δ/ε → PLD2/PA → PKC ζ → Rac1 signaling cascade facilitates recruitment of Coronin 1B, Cortactin and Actin to lamellipodia and chemotaxis of endothelial cells.