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.