Signal Pathways Of IL-1β-induced COX-2 Expression In A549 Cells And Regulating Mechanism Of AP-2α

Cyclooxygenase is the rate-limiting enzyme of prostagladins synthesis, which play important role in the process.Interlukine-1β(IL-1β) is an important proinflammatory cytokine that induces COX-2 expression.But the signal pathways and regulatory mechenism of IL-1β-induced COX-2 expression are not completely clear.Aim of this study is to investigate the signal pathways and gene regulatory mechanism involved in IL-16-induced COX-2 expression to further understand mechanism of COX-2 expression.Part Ⅰ Cyclooxygenase-2 expression induced by IL-1β in A549 cells and signal pathway.Objective:Protein kinase C and mitogen activated protein(MAP) kinase are important signal pathways,which can be activated by IL-1β.The objective of this part is to determinate whether PKC or MAPK is involved in IL-1β-induced-COX-2 expression.Methods:(1)A549 cells were chosen to be incubated with different concentration (0,0.1,1,5,10ng/ml) of IL-1β or with 5ng/ml IL-1β for different time(0h,3h, 6h, 9h, 12h, 24h) to detect the dose-response and time-dependent expression of COX-2 expression induced by IL-1β in A549 cells.(2)A549 cells were incubated with 5ng/ml IL-1β in presence or absence of H-7,a protein kinase C inhibitor,SB203580,a P38 mitogen activated protein(MAP) kinase . inhibitor and PD098059,a mitogen activated and extracellular regulated kinase kinase(MEK1) inhibitor to determine whether PKC or MAPK signal pathways was involved inIL-16-induced COX-2 expression.(3) The expression of COX-2 mRNA and protein were determined by RT-PCR and Western blot respectively.Results:(1)IL-1B induced COX-2 mRNA and protein expression in a time-and dose-dependent manner in A549 cells.(2)SB203580 and H-7 abrogated COX-2 expression induced by IL-1B.PD098059 had little effect on it.Conclusions:IL-1B induces COX-2 expression in A549 cells in a time -and dose-dependent manner. Signal pathways PKC and P38 MAPK is involved in the process.Part II Effect of IL-1B on COX-2 reporter geneObjective:In part I ,our studies showed that signal pathways PKC and P38 MAPK were involved in IL-IB-induced COX-2 expression.There are important regulatory elements in COX-2 promoter. Signal pathways PKC and P38 MAPK phospholate downstream transcriptional factors which then combine with the corresponding regulatory elements to induce COX-2 gene transcription. The objective of this part is to determine which binding sites are involved in IL-lB-induced COX-2 expression.Methods:(1)A549 cells transiently transfected with COX-2 reporter gene(— 1432/ +59, -327/+59, -220/+59, -124/+59, -52/+59) to determine which binding sites are involved in IL-IB-induced COX-2' expression.(2) COX-2 reporter gene activity was measured by luciferase activity assay system.Results:IL-1B did not affect the transcription of any COX-2 reporter construct.Conclusions:The effect of IL-16 on COX-2 report gene is complicated.lt may be influented by many factors such as cell-type.Part El Role of AP-2 a on COX-2 expressionObjective:AP-2 is a 52KD transcriptional factor that can regulate gene transcription,such as human manganese superoxide dismutase (MnSOD) gene,transforming growth factor gene.There are two binding sites in COX-2 promoter.The role of AP-2 on COX-2 expression is not clear.Aim of this part to determine whether AP-2 a is involved in COX-2 expression. Methods:(1)A549 cells were transiently transfected with adenovirus vector overexpressed AP-2a and AP-2 dominant-negetive mutant plasmid vector.The transfected cells were incubated with or without IL-16 to test the role of AP-2a on COX-2 expression.(2)The expression of COX-2 protein was determined by Western blot.Results:AP-2a adenovirus vector increased COX-2 expression and IL-16-induced COX-2 expression in A549 cells.AP-2 dominant-negetive mutant decreased IL-16-induced COX-2 expression.Conclusion:Transcriptional factor AP-2a may be involved in COX-2 expression and IL-16-induced COX-2 expression in A549 cells.