Control of Gene Expression by the Retinoic Acid-Related Orphan Receptor Alpha in HepG2 Human Hepatoma Cells

Retinoic acid-related Orphan Receptor alpha (ROR alpha; NR1F1) is a widely distributed nuclear receptor involved in several (patho) physiological functions including lipid metabolism, inflammation, angiogenesis, and circadian rhythm. To better understand the role of this nuclear receptor in liver, we aimed at displaying genes controlled by ROR alpha in liver cells by generating HepG2 human hepatoma cells stably over-expressing ROR alpha. Genes whose expression was altered in these cells versus control cells were displayed using micro-arrays followed by qRT-PCR analysis. Expression of these genes was also altered in cells in which ROR alpha was transiently over-expressed after adenoviral infection. A number of the genes found were involved in known pathways controlled by ROR alpha, for instance LPA, NR1D2 and ADIPOQ in lipid metabolism, ADIPOQ and PLG in inflammation, PLG in fibrinolysis and NR1D2 and NR1D1 in circadian rhythm. This study also revealed that genes such as G6PC, involved in glucose homeostasis, and AGRP, involved in the control of body weight, are also controlled by ROR alpha. Lastly, SPARC, involved in cell growth and adhesion, and associated with liver carcinogenesis, was up-regulated by ROR alpha. SPARC was found to be a new putative ROR alpha target gene since it possesses, in its promoter, a functional RORE as evidenced by EMSAs and transfection experiments. Most of the other genes that we found regulated by ROR alpha also contained putative ROREs in their regulatory regions. Chromatin immunoprecipitation (ChIP) confirmed that the ROREs present in the SPARC, PLG, G6PC, NR1D2 and AGRP genes were occupied by ROR alpha in HepG2 cells. Therefore these genes must now be considered as direct ROR alpha targets. Our results open new routes on the roles of ROR alpha in glucose metabolism and carcinogenesis within cells of hepatic origin.