Nuclear translocation of cysteinyl leukotriene receptor 1 is involved in oxygen-glucose deprivation-induced damage to endothelial cells

Abstract

Aim: Cysteinyl leukotriene receptor 1 (CysLT(1) receptor) is located in epithelial cells, and translocates from the plasma membrane to the nucleus in a ligand-dependent manner. Here, we investigated whether CysLT(1) receptors translocated to the nucleus in endothelial cells after ischemic insult in vitro and whether it was involved in ischemic injury to endothelial cells.

Methods: EA.hy926 cell line, derived from human umbilical vein endothelial cells, was subjected to oxygen-glucose deprivation (OGD). The expression and distribution of CysLT(1) receptors were detected by immunofluorescent staining, immunogold labeling and immunoblotting analyses. Cell viability was evaluated using MTT reduction assay. Necrosis and apoptosis were determined by double fluorescent staining with propidium iodide and Hoechst 33342.

Results: CysLT(1) receptors were primarily distributed in the cytoplasm and nucleus in EA.hy926 cells, and few was found in the cell membrane. OGD induced the translocation of CysLT(1) receptors from the cytoplasm to the nucleus in a time-depen dent manner, with a peak reached at 6 h. OGD-induced nuclear translocation of CysLT(1) receptors was inhibited by pretreatment with the CysLT(1) receptor antagonist pranlukast (10 μmol/L), or by preincubation with NLS-pep, a peptide corresponding to the nuclear localization sequence of CysLT(1) receptor (10 μg/mL). However, zileuton, an inhibitor of 5-lipoxygenase that was a key enzyme in cysteinyl leukotriene generation, did not inhibit the nuclear translocation of CysLT(1) receptors. Moreover, preincubation with NLS-pep (0.4 μg/mL) significantly ameliorated OGD-induced cell viability reduction and necrosis.

Conclusion: CysLT(1) receptors in endothelial cells translocate to the nucleus in a ligand-independent manner after ischemic insult in vitro, and it is involved in the ischemic injury.

Figure 1

Nuclear localization of the CysLT₁ receptor in EA.hy926 cells. Confocal microscopy assay showing CysLT₁ receptor distribution after immunostaining and DAPI counterstaining (A). Immunogold detection of nuclear CysLT₁ receptors (B). EA.hy926 cells were immunostained with antibody against the CysLT₁ receptor followed by gold-particle conjugated secondary antibody. Arrows indicate gold particles, and arrow heads indicate the nuclear envelope. Immunoblotting analysis of CysLT₁ receptor expression (C). The membrane (M), cytosolic (C), and nuclear (N) fractions were extracted, and equal amounts of proteins were used for SDS polyacrylamide gel analysis. The purity of subcellular fractions was verified by immunoblotting for CD44 for the plasma membrane, GAPDH for the cytoplasm, and lamin B for the nucleus. Scale bar=5 μm in Figure 1A and 0.5 μm in Figure 1B.

Figure 2

OGD time-dependently induces nuclear translocation of the CysLT₁ receptor in EA.hy926 cells. Intracellular distribution of the CysLT₁ receptor (A), immunoblotting analysis of CysLT₁ receptor expression (B), immunoblotting analysis of the CysLT₁ receptor in the cytoplasmic fraction (C), and immunoblotting analysis of the CysLT₁ receptor in the nuclear fraction (D) after OGD are shown. OGD did not change the CysLT₁ receptor expression level, but induced nuclear translocation of the CysLT₁ receptor in a time-dependent manner. Data are expressed as mean±SEM, n=4 experiments and ^bP<0.05 vs control. Data were analyzed by one-way ANOVA. Ctrl, control; OGD, oxygen-glucose deprivation. Scale bar=50 μm.

Figure 3

NLS-pep and pranlukast inhibit OGD-induced nuclear translocation of the CysLT₁ receptor in EA.hy926 cells. Six hours of OGD significantly induced nuclear translocation of the CysLT₁ receptor. The translocation was reduced by NLS-pep (a peptide corresponding to the NLS of the CysLT₁ receptor) and pranlukast (a CysLT₁ receptor antagonist), but not by NLS-mut or zileuton (a 5-lipoxygenase inhibitor). Data are expressed as mean±SEM, n=4, ^bP<0.05 vs control, ^eP<0.05. Data were analyzed by one-way ANOVA. Ctrl, control; OGD, oxygen-glucose deprivation; Pran, pranlukast; Zileu, zileuton. Scale bar=50 μm.

Figure 4

NLS-pep does not inhibit OGD-induced nuclear translocation of NF-κB in EA.hy926 cells. Eight hours of OGD significantly induced nuclear translocation of NF-κB. The translocation was reduced by PDTC (a specific NF-κB inhibitor), but not by NLS-pep or NLS-mut. Ctrl, control; OGD, oxygen-glucose deprivation; PDTC, pyrrolidine dithiocarbamate. Scale bar=50 μm.

Figure 5

NLS-pep protects EA.hy926 cells from OGD-induced damage. Cell viability was determined by MTT reduction assay (A), and cell death by PI (red) and Hoechst 33342 (blue) staining (B and C). Necrosis was the primary cause of OGD-induced cell death. OGD-induced cell viability reduction and cell necrosis were ameliorated by NLS-pep, but not by NLS-mut. Data are expressed as mean±SEM, n=8, ^bP<0.05 vs control, and ^eP<0.05 vs OGD at 8 h. Data were analyzed by one-way ANOVA. Scale bar=50 μm.