Integration of the nuclear receptor REV-ERBα linked with circadian oscillators in the expressions of Alas1, Ppargc1a, and Il6 genes in rat granulosa cells

Chronobiol Int. 2015;32(6):739-49. doi: 10.3109/07420528.2015.1042582. Epub 2015 Jun 23.

Abstract

The nuclear receptor REV-ERBα links circadian rhythms and numerous physiological processes, but its physiological role in ovaries remains largely unknown. The aim of this study was to determine the potential role of REV-ERBα in the regulation of the transcription of its putative target genes in granulosa cells (GCs) prepared from Per2-destablized luciferase (dLuc) reporter gene transgenic rats. Alas1, Ppargc1a, and Il6 were chosen as representatives for genes analysis. A real-time monitoring system of Per2 promoter activity was performed to detect Per2-dLuc circadian oscillations. Two agonists (GSK4112, heme) and an antagonist (SR8278) of REV-ERBα as well as Rev-erbα siRNA knockdown were used to identify its target genes. Clear Per2-dLuc circadian oscillations were generated in matured GCs after synchronization with GSK4112 or SR8278. GSK4112 treatment lengthened and SR8278 treatment shortened the period of circadian oscillations in matured GCs stimulated with or without luteinizing hormone (LH). GSK4112 showed an inhibitory effect on the amplitude of circadian oscillations and caused an arrhythmic expression of canonical clock genes. SR8278 also had a subtle effect on their daily expression profiles, but the treatment resulted only in the arrhythmic expression of Rev-erbα. These findings indicate the functional biological activity of REV-ERBα in response to its ligands. Its natural ligand heme further elongated the period of circadian oscillations and alleviated their amplitudes in GCs cultured with LH. Heme treatment also repressed the expressions of clock genes, Alas1, Il6, and Ppargc1a. Rev-erbα knockdown up-regulated these transcript levels. Collectively, these data extend the recent finding to rat GCs and demonstrate that REV-ERBα represses the expressions of Alas1, Ppargc1a, and Il6, providing novel insights into the physiological significance of REV-ERBα in ovarian circadian oscillators.

Keywords: Clock-controlled genes; GSK4112; REV-ERBα; SR8278; ovarian circadian oscillators.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • 5-Aminolevulinate Synthetase / metabolism*
  • Animals
  • Animals, Genetically Modified
  • Circadian Rhythm
  • Female
  • Gene Expression Regulation*
  • Glycine / analogs & derivatives
  • Glycine / chemistry
  • Granulosa Cells / metabolism*
  • Heme / chemistry
  • Interleukin-6 / metabolism*
  • Isoquinolines / chemistry
  • Ligands
  • Nuclear Receptor Subfamily 1, Group D, Member 1 / metabolism*
  • Ovary / metabolism
  • Period Circadian Proteins / metabolism
  • Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha
  • RNA, Small Interfering / metabolism
  • Rats
  • Thiophenes / chemistry
  • Transcription Factors / metabolism*

Substances

  • GSK4112
  • Interleukin-6
  • Isoquinolines
  • Ligands
  • Nr1d1 protein, rat
  • Nuclear Receptor Subfamily 1, Group D, Member 1
  • Per2 protein, rat
  • Period Circadian Proteins
  • Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha
  • Ppargc1a protein, rat
  • RNA, Small Interfering
  • SR 8278
  • Thiophenes
  • Transcription Factors
  • Heme
  • 5-Aminolevulinate Synthetase
  • Glycine