The role of C/EBPβ phosphorylation in modulating membrane phospholipids repairing in LPS-induced human lung/bronchial epithelial cells

Abstract

Acute lung injury/acute respiratory distress syndrome (ALI/ARDS) is a common critical emergency with high mortality in clinical practice. The key mechanism of ALI/ARDS is that the excessive inflammatory response damages the integrity of alveolar and bronchial cell membrane and thus affects their basic function. Phospholipids are the main component of cell membranes. Phospholipase A2 (PLA2), which catalyzes the cleavage of membrane phospholipids, is the most important inflammatory mediator of ALI. However, clara cell secretory protein 1 (CCSP1), an endogenous PLA2 inhibitor can increase the self-defense of membrane phospholipids. Thus, CCSP1 up-regulation and PLA2 inhibition constitutes an effective method for ensuring the stability of membrane phospholipids and for the treatment of ALI/ARDS. In the present study, we developed an in vitro model of ALI via lipopolysaccharide (LPS) stimulation of a human bronchial epithelial cell line, BEAS-2B, and assessed the mRNA and protein levels of CCSP1 and PLA2 in the model cells. The results demonstrated LPS induction inhibited the transcription and protein expression of CCSP1, but only the protein level of membrane associated PLA2 was increased, suggesting that in the in vitro ALI model, abnormally regulated CCSP1 transcription plays a crucial role in the damage of cell membrane. To find out the reason that CCSP1 expression was decreased in the ALI model, we predicted, by means of bioinformatics, putative transcription factors which would bind to CCSP1 promoter, examined their background and expression, and found that a transcription factor, CCAAT/enhancer binding protein β (C/EBP β), was correlated with the transcription of CCSP1 in the in vitro ALI model, and its phosphorylation in the model was decreased. CHIP-PCR and luciferase reporter assay revealed that C/EBP β bound to CCSP1 promoter and facilitated its transcription. Therefore, we conclude that there is a C/EBP β/CCSP1/PLA2 pathway in the in vitro ALI model. The study of underlying mechanism show that the activity of C/EBP β depends on its phosphorylation:LPS stimulation reduced C/EBP β phosphorylation and suppressed the transcription of CCSP1 in BEAS-2B cells, which resulted in enhanced PLA2 and the consequent membrane damage. And further study shows that overexpression of CDK2(Cyclindependent kinase 2), promoted the phosphorylation of C/EBP β and inhibited PLA2 through the C/EBP β/CCSP1/PLA2 pathway, so as to attenuate membrane damage. The significance of this study lies in that artificial C/EBP β phosphorylation regulation may ease the membrane damage in ALI and improve membrane repair.

Keywords: ALI; ARDS; C/EBP β; CCSP1; CDK2; PLA2.

Fig. 1

Infection efficiency of BEAS-2B cells and gene intervention efficiency of CDK2, C/EBP β and CCSP1. A. Left, cells viewed by bright-field microscopy; right, cells viewed by fluorescent microscopy. The infection efficiency was estimated by dividing the number of cells expressing the fluorescent marker with the total number of cells in the same view, five fields randomly selected for the estimation. BEAS-2B infected with Lv-Control for 72 h, MOI = 10. B. Expression of CDK2, C/EBP β and CCSP1 measured by western blotting. Lower: image of target blots, upper: analysis on the optical density of three proteins, β actin serving as the reference. Data are means ± SD from at least three separate replicates. **, p < 0.01, vs cell group or cell infected with Lv-Control group.

Fig. 2

Verification of ALI model and measurement of relevant proteins. A. TNFα, IL-1β, and IL-10 in BEAS-2B cells were examined using ELISA 12 h, 24 h or 48 h after treatment of 10 mg/ml LPS. The vertical coordinate indicates the concentration of target protein, and the horizontal coordinate indicates the experiment group. B. PAF and AA contents in BEAS-2B cells were measured using ELISA 12 h, 24 h or 48 h after treatment of 10 mg/ml LPS. The vertical coordinate indicates the concentration of target protein, and the horizontal coordinate indicates the experiment group. C. CDK2, CCSP1 and PLA2 expression levels, as well as C/EBP β phosphorylation, were assessed by western blotting 12 h, 24 h or 48 h after treatment of 10 mg/ml LPS. Lower: image of target blots, upper: analysis on the optical density of three proteins, β actin serving as the reference, and for the phosphorylated protein, the total protein of itself serving as the reference. Data are means ± SD from at least three separate replicates.*, p < 0.05; **, p < 0.01. vs cell group.

Fig. 3

Overexpression and knockdown of CDK2 and CCSP1 and their effects on C/EBP β phosphorylation. BEAS-2B cells were infected with the indicated lentivirus, and CDK2, CCSP1 and phosphorylated C/EBP β and total C/EBP β were measured by western blotting 72 h later. Upper: representative blots of CDK2, CCSP1 and phosphorylated C/EBP β and total C/EBP β and β-actin are shown. Lower: the optical density of the target band divided by the optical density of the β-actin band. Data are means ± SD from at least three separate replicates.*, p < 0.05; **, p < 0.01 vs cell group or cell + Lv-NC group.

Fig. 4

Verification of C/EBP β binding site on CCSP1 promoter. A. BEAS-2B cells were infected with the indicated lentivirus, and CCSP1 mRNA was measured by real time quantitative PCR 72 h later. The vertical coordinate indicates the relative amount of CCSP1 mRNA and the horizontal coordinate indicates the experiment group, β actin serving as the reference. B. Verification of C/EBP β binding site on CCSP1 promoter by CHIP-PCR. Upper: relatively quantitative analysis on DNA segments, the vertical coordinate being the ratio of gene copy number of the sample to the control gene copy number of Input, and the horizontal coordinate being experiment group. Lower: western blotting results of C/EBP β protein, Input is the negative control without antibody, and IgG is the control group with a primary antibody against GAPDH. C. Measurements of luciferase activity. The vertical coordinate indicates the luciferase activity expressed as the firefly luciferase/Renilla luciferase, and the horizontal coordinate indicates the experiment group. The determination of the luciferase activity was made 48 h after transfection of 293 cells. Data are means ± SD from at least three separate replicates. *, p < 0.05; **, p < 0.01.

Fig. 5

Verifying the specificity of the pathway and examining membrane damage A. BEAS-2B cells were infected with the indicated lentivirus, and PLA2 expression was measured by western blotting. Upper: representative blots and lower: the optical density of the target band divided by the optical density of the β-actin band. B. BEAS-2B cells were infected with the indicated lentivirus, and treated with LPS 48 h later, and PAF and AA were measured using ELISA. Data are means ± SD from at least three separate replicates.*, p < 0.05; **, p < 0.01.