Elevation of NR4A3 Expression and Its Possible Role in Modulating Insulin Expression in the Pancreatic Beta Cell

Weina Gao,Yong Liu,Shunke Wang,Yuchang Fu,Xia Li,Yuchao Zhang,Cong Yu,Chen Zong,Tongfu Xu,Xiangdong Wang,Bin He

doi:10.1371/journal.pone.0091462

Abstract

BackgroundNR4A3/NOR-1 is a member of the NR4A orphan nuclear receptor subfamily, which contains early response genes that sense and respond to a variety of stimuli in the cellular environment. The role of NR4A3 in insulin expression in pancreatic beta cells remains unknown.MethodsDynamic changes in NR4A3 were examined in a pancreatic beta-cell line, MIN6, treated with thapsigargin (TG), palmitate (PA), tunicamycin (TM), and dithiothreitol (DTT), chemicals that produce cell stress and even apoptosis. We exploited virus infection techniques to induce expression of NR4A3 or three deletion mutants, and determined expression of insulin and insulin regulatory genes in MIN6 cells.ResultsTG and PA, two endoplasmic reticulum (ER) stress inducers, were able to induce unfolded protein response (UPR) activation and elevation of NR4A3 expression in MIN6 cells, whereas TM and DTT, two other ER stress inducers, were able to induce UPR activation but not NR4A3 elevation. MIN6 cells over-expressing NR4A3 protein after adenoviral infection exhibited reduced transcription of the insulin genes Ins1 and Ins2, and reduced insulin protein secretion, which were negatively correlated with NR4A3 expression levels. Functional analysis of different deletion mutants of NR4A3 showed that deleting the activation domain AF1 or the DNA-binding domain abolished the down-regulation of insulin transcription by NR4A3 in MIN6 cells, indicating that this down-regulative role was closely related to the NR4A3 trans-activation activity. Over-expression of NR4A3 in MIN6 cells resulted in reduced mRNA transcription of the insulin positive-regulation genes, Pdx1 and NeuroD1.ConclusionSome ER stress inducers, such as TG or PA, are able to elevate NR4A3 expression in MIN6 cells, while others, such as TM or DTT, are not. Over-expression of NR4A3 in MIN6 cells results in down-regulation of insulin gene transcription and insulin secretion. NR4A3 reduces insulin gene expression by modulating the expression of Pdx1 and NeuroD1.

Highlights

Nuclear receptors make up a large family of ligand-dependent transcription factors [1]
TG treatment resulted in NR4A3 expression and unfolded protein response (UPR) activation in MIN6 cells
MIN6 cells were incubated with different concentrations of TG for 2 h, resulting in NR4A3 mRNA increases, predominantly at doses of 0.1–1 mM (Figure 1A)

Summary

Introduction

Nuclear receptors make up a large family of ligand-dependent transcription factors [1]. They have common structural motifs: a highly variable NH3-terminal region that contains a ligandindependent activation domain called AF1; the central DNAbinding domain (DBD), consisting of two highly conserved zincfinger motifs, which targets the receptor to specific DNA sequences called hormone response elements (HRE); and the Cterminal ligand-binding domain (LBD), which functions in ligand recognition, receptor dimerization, and cofactor interaction [2]. NR4A3/NOR-1 is a member of the NR4A orphan nuclear receptor subfamily, which contains early response genes that sense and respond to a variety of stimuli in the cellular environment. The role of NR4A3 in insulin expression in pancreatic beta cells remains unknown

Methods

Results

Conclusion