False responses of Renilla luciferase reporter control to nuclear receptor TR4

Abstract

Renilla luciferase reporter is a widely used internal control in dual luciferase reporter assay system, where its transcription is driven by a constitutively active promoter. However, the authenticity of the Renilla luciferase response in some experimental settings has recently been questioned. Testicular receptor 4 (TR4, also known as NR2C2) belongs to the subfamily 2 of nuclear receptors. TR4 binds to a direct repeat regulatory element in the promoter of a variety of target genes and plays a key role in tumorigenesis, lipoprotein regulation, and central nervous system development. In our experimental system using murine pituitary corticotroph tumor AtT20 cells to investigate TR4 actions on POMC transcription, we found that overexpression of TR4 resulted in reduced Renilla luciferase expression whereas knockdown TR4 increased Renilla luciferase expression. The TR4 inhibitory effect was mediated by the TR4 DNA-binding domain and behaved similarly to the GR and its agonist, Dexamethasone. We further demonstrated that the chimeric intron, commonly present in various Renilla plasmid backbones such as pRL-Null, pRL-SV40, and pRL-TK, was responsible for TR4's inhibitory effect. The results suggest that an intron-free Renilla luciferase reporter may provide a satisfactory internal control for TR4 at certain dose range. Our findings advocate caution on the use of Renilla luciferase as an internal control in TR4-directed studies to avoid misleading data interpretation.

Assessment of NR4A Ligands That Directly Bind and Modulate the Orphan Nuclear Receptor Nurr1

“…However, their use is implicit on the premise that ligand treatment does not influence cell viability, which is the case for amodiaquine and chloroquine. Ligand concentration-dependent cell death will cause a decrease in β-galactosidase or Renilla luciferase internal control activity. − Normalization of luciferase reporter activity by the internal control will artifactually influence the dose response curves, often causing a steep dose-responsive increase in normalized luciferase activity. For amodiaquine and chloroquine, normalization of luciferase reporter data to an internal control would mask the real impacts on Nurr1-mediated transcription.…”

The role of hyperuricemia on vascular endothelium dysfunction

“…Transfection of human umbilical vein ECs was performed using the nucleofection device and solutions to deliver electrical stimuli to the ECs (Lonza Group, Basel, Switzerland) (13). Following 24 h transfection, 10 mg/dl UA was used to treat the cells for 3 h and luciferase activity was examined by using a Dual Luciferase Reporter Assay System and a luminometer (Promega Corporation, Madison, WI, USA) as per the manufacturer's protocol (14).…”