Role Of FOXA1 In The Apoptosis Of Type Ⅱ Alveolar Epithelial Cells During Acute Lung Injury

Acute lung injury(ALI) or acute respiratory distress syndrome (ARDS) is a severe complication and a significant cause of mortality in critically ill patients.Despite decades of research,few therapeutic strategies for clinical ALI/ARDS have emerged,and the current specific treatment options remain limited.ALI/ARDS continues to be an important contributor to prolonged mechanical ventilation in the intensive care unit,and ALI/ARDS-associated mortality remains high at 30-50% despite optimal supportive care.Forkhead box protein A1(FoxA1) is an evolutionarily conserved winged helix transcription factor with diverse regulatory functions in cell growth,proliferation,differentiation,and embryogenesis.However,little is known about the role of FoxA1 in ALI and pulmonary cell injury.Our previously research from cDNA microarray assays revealed that the expression level of FoxA1 was up-regulated in lung tissues at 2 h,and further increased up to more than 10 times of the normal level at 20 h after lipopolysacchride(LPS)-induced mouse ALI.These results indicated that FoxA1 might be an important transcriptional factor involved in ALI.To determine the role of FoxA1 in ALI,an in vivo model was employed whereby rats were administered an injection of oleic acid(OA, 0.1 ml/kg,intravenously),LPS(15mg/kg,intraperitoneally) or a trachinstillation of LPS(2mg/kg).The degree of ALI was evaluated by lung wet/dry ratio and lung injury score.Caspase-3 activity assays and TUNNEL/anti-MNF116 double staining were used to measure alveolar typeâ…¡epithelial cells apoptosis in rat lung tissue following ALI.In vitro, cell injury was induced by the incubation of primary rat alveolar typeâ…¡epithelial cell or A549 human alveolar typeâ…¡epithelial cell line with hydrogen peroxide(H₂O₂,0.5 mM),LPS(1000ng/ml) or TNF-α(10ng/ml).Western blotting and reverse transcription-polymerase chain reaction were performed to determine the changes in FoxA1 expression that occur in lung tissue of the in vivo ALI model and in H₂O₂,LPS or TNF-αchallenged alveolar typeâ…¡epithelial cell.FoxA1 was overexpressed using a full-length FoxA1 cDNA,and inhibited using FoxA1 antisense oligonucleotides,and the degree of apoptosis induced by H₂O₂ was analyzed by flow cytometry.The results showed that injection of OA,LPS,or trachinstillation of LPS resulted in lung injury and alveolar typeâ…¡epithelial cells apoptosis in vivo.FoxA1 mRNA and protein levels were upregulated in lung tissue of the in vivo ALI model and in H₂O₂,LPS or TNF-αchallenged alveolar typeâ…¡epithelial cell.In order to understand the roles of FoxA1 in regulating the alveolar typeâ…¡epithelial cells apoptosis,we first overexpressed FoxA1 cDNA gene(pcDNA3.1-FoxA1) into A549 human alveolar typeâ…¡epithelial cell line by gene transfection,and inhibited the expression of FoxA1 gene in A549 cells by transfection of FoxA1 antisense oligonucleotides.It was showed that overexpression of FoxA1 promoted apoptosis,whereas FoxA1 deficiency decreased the apoptosis induced by H₂O₂ in A549 cells.These results suggest that FoxA1 may play an important role in ALI by promoting apoptosis of pulmonary epithelial cells.Then,according to the screening results from bioinformatics and RT-PCR,we selected bcl2 and UCP2,two representative anti-apoptosis genes,to investigate the effect of FoxA1 on their expression under normal condition and H₂O₂ treatment.The results showed that,FoxA1 overexpression decreased the expression of bcl2 and UCP2,while FoxA1 depletion increased the expression of bcl2 and UCP2 under the normal condition and after H₂O₂ stimulation.Electrophoretic mobility shift assay and chromatin immunoprecipitation revealed that FoxA1 bound to bcl2 and UCP2 promoter,and H₂O₂ promoted its DNA binding activity. Luciferase reporter assay showed that FoxA1 suppressed the transcription activity of bcl2 and UCP2 promoter under normal conditions and oxidative stress.These results indicate that FoxA1 plays a pro-apoptotic role by inhibiting the expression of anti-apoptotic gene bcl2 and UCP2.Taken together,the present studies suggest a novel function of FoxA1,that is,the expression of FoxA1 is upregulated in rat lung tissues of ALI and alveolar typeâ…¡epithelial cells challenged by H₂O₂,LPS and TNF-α,it can bind to the FoxA1 binding sites on the promoters of anti-apoptosis genes bcl2 and UCP2 to suppress the expression of these genes and accelerate apoptosis of alveolar typrâ…¡epithelial cells in ALI. These findings provide novel insights into the pathogenesis of ALI,and also new clues and information for the studies on the biological functions of FoxA1,and the prevention and treatment of ALI.