Fibrates are widely used hypolipidemic drugs which activate the nuclear peroxisome proliferator-activated receptor (PPAR) ␣ and thereby alter the transcription of genes controlling lipoprotein metabolism. Fibrates influence plasma high density lipoprotein and its major protein, apolipoprotein (apo) A-I, in an opposite manner in man (increase) versus rodents (decrease). In the present study we studied the molecular mechanisms of this species-specific regulation of apoA-I expression by fibrates. In primary rat and human hepatocytes fenofibric acid, respectively, decreased and increased apoA-I mRNA levels. The absence of induction of rat apoA-I gene expression by fibrates is due to 3 nucleotide differences between the rat and the human apoA-I promoter A site, rendering a positive PPAR-response element in the human apoA-I promoter nonfunctional in rats. In contrast, rat, but not human, apoA-I transcription is repressed by the nuclear receptor Rev-erb␣, which binds to a negative response element adjacent to the TATA box of the rat apoA-I promoter. In rats fibrates increase liver Rev-erb␣ mRNA levels >10-fold. In conclusion, the opposite regulation of rat and human apoA-I gene expression by fibrates is linked to differences in cis-elements in their respective promoters leading to repression by Rev-erb␣ of rat apoA-I and activation by PPAR␣ of human apoA-I. Finally, Rev-erb␣ is identified as a novel fibrate target gene, suggesting a role for this nuclear receptor in lipid and lipoprotein metabolism.

Fibrates are widely used hypolipidemic drugs that act by modulating the expression of genes involved in lipid and lipoprotein metabolism. Whereas the activation of gene transcription by fibrates occurs via the nuclear receptor peroxisome proliferator-activated receptor-alpha (PPARalpha) interacting with response elements consisting of a direct repeat of the AGGTCA motif spaced by one nucleotide (DR1), the mechanisms of negative gene regulation by fibrates and PPARalpha are largely unknown. In the present study, we demonstrate that fibrates induce the expression of the nuclear receptor Rev-erbalpha, a negative regulator of gene transcription. Fibrates increase Rev-erbalpha mRNA levels both in primary human hepatocytes and in HepG2 hepatoblastoma cells. In HepG2 cells, fibrates furthermore induce Rev-erbalpha protein synthesis rates. Transfection studies with reporter constructs driven by the human Rev-erbalpha promoter revealed that fibrates induce Rev-erbalpha expression at the transcriptional level via PPARalpha. Site-directed mutagenesis experiments identified a PPAR response element that coincides with the previously identified Rev-erbalpha negative autoregulatory Rev-DR2 element. Electromobility shift assay experiments indicated that PPARalpha binds as heterodimer with 9-cis-retinoic acid receptor to a subset of DR2 elements 5' flanked by an A/T-rich sequence such as in the Rev-DR2. PPARalpha and Rev-erbalpha bind with similar affinities to the Rev-DR2 site. In conclusion, these data demonstrate human Rev-erbalpha as a PPARalpha target gene and identify a subset of DR2 sites as novel PPARalpha response elements. Finally, the PPARalpha and Rev-erbalpha signaling pathways cross-talk through competition for binding to those response elements.

Estrogen receptor-related receptor alpha (ERR alpha) is an orphan nuclear receptor closely related to the estrogen receptor (ER), whose expression covers various stages of embryonic development and persists in certain adult tissues. We show that ERR alpha binds as a homodimer on a specific target sequence, the SFRE (SF-1 response element), already known to respond to the orphan nuclear receptor SF-1. Target sequences that are related to the SFRE and that discriminate between ERR alpha and SF-1 were identified. We have also analyzed the transcriptional properties of the ERR alpha originating from various species. All ERR alpha orthologs act as potent transactivators through the consensus SFRE. ERR alpha activity depends on the putative AF2AD domain, as well as on a serum compound that is withdrawn by charcoal treatment, suggesting the existence of a critical regulating factor brought by serum.

The orphan receptors Rev-erbα and Rev-erbβ are members of the nuclear receptors superfamily and act as transcriptional repressors. Rev-erbα is expressed with a robust circadian rhythm and is involved in liver metabolism through repression of the ApoA1 gene, but no role has been yet defined for Rev-erbβ. To gain better understanding of their function and mode of action, we characterized the proteins encoded by these two genes. Both Rev-erbα and Rev-erbβ proteins were nuclear when transiently transfected in COS-1 cells. The major nuclear location signal (NLS) of Rev-erbα is in the amino-terminal region of the protein. Fusion of green fluorescent protein (GFP) to the amino terminus of Rev-erbα deletion mutants showed that the NLS is located whithin a 53 amino acid segment of the DNA binding domain (DBD). The homologous region of Rev-erbβ fused to GFP also targeted the fusion protein to the nucleus, suggesting that the location of this NLS is conserved among all the Rev-erb group members. Interestingly, members of the phylogenetically closest nuclear orphan receptor group (ROR), which exhibit 58% amino acid identity with Rev-erb in the DBD, do not have their NLS located whithin the DBD. GFP/DBD.RORα or GFP/DBD.RORβ remained cytoplasmic, in contrast to GFP/DBD.Rev-erb fusion proteins. Alignment of human Rev-erb and ROR DBD amino acid sequences predicted that the two basic residues, K167 and R168, located just upstream from the second zinc finger, could play a critical part in the nuclear localization of Rev-erb proteins. Substitution of these two residues with those found in ROR, in the GFP/DBD.Rev-erb context, resulted in cytoplasmic proteins. In contrast, the reverse mutation of the GFP/DBD.RORα towards the Rev-erbα residues targeted the fusion protein to the nucleus. Our data demonstrate that Rev-erb proteins contain a functional NLS in the DBD. Its location is unusual within the nuclear receptor superfamily and suggests that Rev-erb orphan receptors control their intracellular localization via a mechanism different from that of other nuclear receptors.