Mechanism Research On Mouse ORMDL3Gene Transcriptional Regulation

Backgrounds:Genome-wide association studies showed that Orosomucoid1-like3(ORMDL3) gene is a candidate gene of childhood-onset asthma, and the abnormity of ORMDL3expression is associated with the pathophysiologic process of asthma. To data, the regulation mechanism of ORMDL3is poorly understood. CREB is a key transcription factor for human ORMDL3gene transcriptional regulation. cAMP/PKA/CREB pathway plays important roles in airway smooth muscle relaxation, airway repair, et al. Vitamin A deficiency is a risk factor for asthma, and prior studies illustrated that cAMP/PKA/CREB pathway is an integrated part of Vitamin A signal network.Objective:Clone and analysis the promoter of mouse ORMDL3gene, screen out key transcription factors, and explore signaling pathways that control ORMDL3expression. Detect the impacts of Vitamin A on ORMDL3transcription, and define the related mechanisms.Methods:The proximal minimal promoter of mouse ORMDL3gene was identified using a series of5’deletion promoter plasmids in luciferase reporter assays. Transcription factors regulating ORMDL3gene transcription were demonstrated by mutational analysis. ChIP and EMSA assays were carried out to identify the binding of transcription factor to the promoter. RNA interference and overexpression experiments were used to detect the effects of transcription factor on ORMDL3expression. PKA activator Forskolin and PKA specific inhibitor H-89were used to explore the regulatory effects of cAMP/PKA signaling on mouse ORMDL3gene transcription. The major metabolite of Vitamin A in vivo, All-trans retinoic acid (ATRA), and RAR a (retinoic acid recoptor a) agonist Am-80were implored to detect the effects of Vitamin A on ORMDL3expression. Results:The proximal minimal promoter of mouse ORMDL3gene was located in-74/+141bp relative to the transcriptional start site. Web software TFSEARCH ver.1.3showed that the core promoter of mouse ORMDL3gene contained GATA-1、 STAT6and CREB transcription factor binding sites, and all of this three binding sites were involved in maintaining the basal transcriptional activity of the mouse ORMDL3promoter. CREB could directly bind to the core promoter of ORMDL3gene and up-regulate ORMDL3expression. Forskolin and H-89could regulate ORMDL3expression through facilitating and inhibiting CREB phosphorylation, respectively. ATRA could up-regulate mouse ORMDL3gene expression by promoting CREB phosphorylation in a PKA-dependent manner.Conclusion:The core promoter of mouse ORMDL3was located between-74bp and+141bp, and transcriptional factors STAT6, GATA-1and CREB might be involved in ORMDL3transcriptional regulation. It has reported that cAMP/PKA signaling pathway plays a key role in airway relaxation, and mediates the effects of β2agonists on airway smooth muscle relaxation. Here we showed that cAMP/PKA/CREB pathway participated in regulating ORMDL3expression. Our results provide clues for further investigate the mechanisms of ORMDL3in the development of asthma. Vitamin A shows protective effects on asthma, including maintains airway epithelial integrity, inhibits asthma effector cells differentiation, modulates immune response, et al. However, clinical application of Vitamin A in asthma treatment is still controversial. Our study indicated that Vitamin A could up-regulate ORMDL3expression partly through PKA/CREB pathway, which laid the foundation for comprehensive assessment of the clinical value of Vitamin A on asthma treatment.