Promoter Characterization and Role of cAMP/PKA/CREB in the Basal Transcription of the Mouse ORMDL3 Gene

Abstract

Orosomucoid 1-like 3 (ORMDL3) gene was strongly linked with the development of asthma in genetic association studies, and its expression could be significantly induced by allergen in airway epithelial cells of mice. However, the expression mechanism of ORMDL3 was still unclear. Here we have identified and characterized the mouse ORMDL3 gene promoter. Deletion constructs of the 5′ flanking region were fused to a luciferase reporter gene. After transient transfection in mouse fibroblast cell line NIH3T3, a CRE (−27/−20) binding CREB was identified in the core promoter region. Deletion or mutation of the CRE consensus sequence resulted in a significant loss of the promoter activity. EMSA and ChIP assays demonstrated the binding of CREB to the core promoter. Knocking down endogenous CREB led to a reduction in ORMDL3 expression. Conversely, overexpression of CREB up-regulated ORMDL3 expression. Moreover, forskolin, a PKA activator, could facilitate the phosphorylation of CREB, which in turn heightens ORMDL3 expression. H-89, a PKA-specific inhibitor, could significantly inhibit ORMDL3 expression. This study delineates the characterization of mouse ORMDL3 gene promoter and shows signaling pathway cAMP/PKA/CREB plays an important role in regulating ORMDL3 expression, which will be helpful for future animal model studies regarding the regulation or function of ORMDL3 gene.

Expression of the ORMDLS, Modulators of Serine Palmitoyltransferase, Is Regulated by Sphingolipids in Mammalian Cells

“…None of the ORMDL mRNA levels changed significantly after tetracycline-induction of SPT and transcriptional regulation has only been reported for ORMDL3 (7,8,22,23), making it unlikely that a common transcriptional mechanism can account for the regulation we have observed. One possible explanation for these results is that regulation involves alternative splicing or modulation of translational efficiency.…”

“…However, while highly homologous to their yeast counterparts, they lack the major portion of the N-terminal domain of the yeast Orm proteins that is phosphorylated by Ypk1, Ypk2, * This work was supported by Uniformed Services University Grants R071KD and Npr1 and far less is known about the mechanisms by which they regulate SPT activity. Transcriptional regulation of ORMDL3, polymorphisms in which are associated with an increased risk of childhood asthma, has been reported (7,8). In addition, Breslow et al reported that silencing of all three ORMDL mRNAs resulted in increased ceramide levels (2).…”

The role of ORMDL proteins, guardians of cellular sphingolipids, in asthma

“…Wu et al (3) explained ORMDL3 as an orosomucoid 1-like 3 gene. This statement, replicated in several other papers (4)(5)(6)(7)(8)(9)(10), originated from the fact that both the yeast ORM1 gene and human a1-acid glycoprotein (orosomucoid 1) gene share the same name, ORM1. However, the amino acid sequence comparison ( Fig.…”

“…Furthermore, overexpression of ORMDL3 has been described in patients with different asthma pathologies (7,13), and enhanced expression of ORMDL3 was induced by Est-1, p300, CREB (43), STAT6 (44) as well as Th2 cytokines (67,91). CREB-induced ORMDL3 expression is involved in the retinoic acid receptor pathway activated by all trans-retinoic acid (ATRA), which is PKA dependent (8). ORMDL3 is also an allergen-inducible gene.…”

Review of Methods to Study Gene Expression Regulation Applied to Asthma

“…Zhuang et al characterized the mouse ormdl3 gene promoter region by different approaches, including EMSA. In vitro EMSA assays identifi ed a protein -DNA complex and the complex was supershift when it was incubated with the CREB antibody, validating the CREB-binding site in the ormdl3 promoter region [ 51 ].…”