Objectives : NR4A1 belongs to a superfamily of orphan nuclear receptors known as immediate-early response genes. We reported the regulation of pituitary TSHβ gene expression in TRH knockout mice by NR4A1, the mRNA level was stimulated by TRH in thyrotrophs. We determined whether NR4A1 mRNA is regulated by thyroid hormone.
Methods : We examined the effects of the thyroid status on pituitary NR4A1 mRNA levels in vivo. We investigated whether thyroid hormone stimulated the promoter activity of the NR4A1 gene in vitro using CV-1 cells. To identify the region responsible for the T3-mediated suppression of the promoter activity, we generated a series of deletion mutants of the NR4A1 promoter and examined the effects of T3. We determined whether TRβ binds to the region responsible for the T3-induced suppression of the promoter activity using EMSA and ChIP assay.
Results : Pituitary NR4A1 mRNA levels were decreased to 50% of control levels within 24 hr after single peritoneal injection of T4 in mice. In an in vitro experiment, the promoter activity of the NR4A1 gene was increased by the overexpression of TRβ, and 10 nM T3 decreased its activity. An examination of a deletion mutant of the promoter region revealed that the region from ~27 to +152 bp was responsible for the T3-induced suppression. An EMSA showed the lack of TRβ binding, whereas a ChIP assay demonstrated the recruitment of TRβ and a co-repressor, NCoR, in the region -147~+148 bp in the absence of T3, and T3 induced the release of TRβ and NCoR. Experiments on the overexpression and knockdown of NCoR, and using the mutant TRs supported the involvement of NCoR in the TR-induced stimulation.
Conclusion : These results demonstrate that thyroid hormone down-regulated basal NR4A1 mRNA levels in the pituitary, and the direct binding of TR was not required.