Orphan nuclear hormone receptor RevErbalpha modulates expression from the promoter of the hydratase-dehydrogenase gene by inhibiting peroxisome proliferator-activated receptor alpha-dependent transactivation.

Peroxisome proliferator-activated receptor alpha (PPARalpha) heterodimerizes with the 9-cis-retinoic acid receptor (RXRalpha) to bind to peroxisome proliferator-response elements (PPRE) present in the upstream regions of a number of genes involved in metabolic homeostasis. Among these genes are those encoding fatty acyl-CoA oxidase (AOx) and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase (HD), the first two enzymes of the peroxisomal beta-oxidation pathway. Here we demonstrate that the orphan nuclear hormone receptor, RevErbalpha, modulates PPARalpha/RXRalpha- dependent transactivation in a response element-specific manner. In vitro binding analysis showed that RevErbalpha bound the HD-PPRE but not the AOx-PPRE. Determinants within the HD-PPRE required for RevErbalpha binding were distinct from those required for PPARalpha/RXRalpha binding. In transient transfections, RevErbalpha antagonized transactivation by PPARalpha/RXRalpha from an HD-PPRE luciferase reporter construct, whereas no effects were observed with an AOx-PPRE reporter construct. These data identify the HD gene as a target for RevErbalpha and illustrate cross-talk between the RevErbalpha and PPARalpha signaling pathways on the HD-PPRE. Our results suggest a novel role for RevErbalpha in regulating peroxisomal beta-oxidation.     

The atypical interaction of peroxisome proliferator-activated receptor alpha with liver X receptor alpha antagonizes the stimulatory effect of their respective ligands on the murine cholesterol 7alpha-hydroxylase gene promoter

Cholesterol 7alpha-hydroxylase (cyp7a) mediates cholesterol elimination in the liver by catalyzing the first and rate-limiting step in the conversion of cholesterol into bile acids. Peroxisome proliferator-activated receptor alpha (PPARalpha; NR1C1) and liver X receptor alpha (LXRalpha; NR1H3) are two nuclear receptors that stimulate the murine Cyp7a1 gene. Here we report that co-expression of PPARalpha and LXRalpha in hepatoma cells abolishes the stimulation of Cyp7a1 gene promoter in response to their respective agonists. PPARalpha and LXRalpha form an atypical heterodimer that binds to two directly adjacent hexameric sequences localized within overlapping PPARalpha and LXRalpha response elements (termed Site I), antagonizing the interaction of PPARalpha:retinoid X receptor alpha (RXRalpha) or RXRalpha:LXRalpha with the Cyp7a1 gene promoter. Mutations within either hexameric sequences that specifically abolished LXRalpha:PPARalpha heterodimer binding to the murine Cyp7a1 Site I also relieved promoter inhibition. The LXRalpha:PPARalpha heterodimer may be important in coordinating the expression of genes that encode proteins involved in metabolism of fats and cholesterol.     

The expression of PPAR-associated genes is modulated through postnatal development of PPAR subtypes in the small intestine

In this study, we found that the mRNA level of peroxisome proliferator-activated receptor (PPAR) alpha, but not of PPARdelta, was elevated in the jejunum during the postnatal development of the rat. Moreover, we found that the expressions of PPAR-dependent genes, such as acyl-CoA oxidase, L-FABP, and I-FABP, were also increased during the postnatal development of the small intestine. Electrophoretic mobility shift assay revealed that both the PPARalpha-9-cis-retinoic acid receptor alpha (RXRalpha) heterodimer and the PPARdelta-RXRalpha heterodimer bound to the peroxisome proliferator response element (PPRE) of acyl-CoA oxidase and L-FABP genes. The binding of the PPARalpha-RXRalpha heterodimer to the PPREs of the various genes was enhanced by the addition of PPARalpha, with a concomitant reduction of the binding of PPARdelta-RXRalpha to the PPREs. Furthermore, the binding activity of PPARalpha-RXRalpha, but not PPARdelta-RXRalpha, to the PPREs was enhanced by the addition of a PPAR ligand, WY14,643. The GAL4-PPAR-chimera reporter assay showed that WY14,643 transactivated the reporter gene through action of PPARalpha, but not through PPARdelta, in Caco-2 cells. Furthermore, oral administration of a PPAR ligand, clofibrate, during 3 consecutive days of the weanling period caused a parallel increase in the mRNA levels of these PPAR-dependent genes. These results suggest that acyl-CoA oxidase, L-FABP and the other PPAR-dependent genes in the small intestine may be coordinately modulated during postnatal development by the disproportional expression of PPARalpha over PPARdelta.     

Phytanic acid and docosahexaenoic acid increase the metabolism of all-trans-retinoic acid and CYP26 gene expression in intestinal cells.

Retinoids are essential for growth and cell differentiation of epithelial tissues. The effects of the food compounds phytol, the phytol metabolite phytanic acid, and the fatty acid docosahexaenoic acid (DHA) on the retinoid signaling pathway in intestinal cells were studied. Phytol inhibited the formation of all-trans-retinoic acid (RA) from dietary retinol in intestinal cells. Phytanic acid, a known retinoic X receptor (RXRalpha) and peroxisome proliferator activating receptor (PPARalpha) activator, also activated PPARdelta, and to a lesser degree PPARgamma, in a transactivation assay. Phytanic acid had no effect on intestinal RA hydroxylase CYP26 (also named P450RAI) gene expression and metabolism of all-trans-RA in intestinal Caco-2 cells. However, in combination with retinoic acid receptor (RAR)-ligands (all-trans-RA or synthetic Am580) phytanic acid enhanced the induction of CYP26 and RA-metabolism in comparison to treatments with all-trans-RA or Am580 alone. Also treatment with DHA did not affect CYP26 gene expression and RA-metabolism but cotreatment of the cells with DHA and all-trans-RA or Am580 enhanced the induction of CYP26, in comparison to the induction caused by all-trans-RA or Am580 alone. This study indicates that food compounds such as phytanic acid and DHA that are RXR-agonists and have an impact on intestinal CYP26 gene expression and metabolism of all-trans-RA in intestinal cells.     

Stimulated release of arachidonic acid by agonists of the peroxisome proliferator-activated receptor and retinoic acid receptors.

Release of arachidonic acid from rat liver cells is stimulated after a 6-hour incubation with 9-cis retinoic acid, all trans retinoic acid, the selective peroxisome proliferator-activated receptor-gamma synthetic thiazolidinedione, ciglitazone, the cyclopentenones, 15-deoxy-Delta(12,14) PGJ2 and PGA1 and the non-steroidal anti-inflammatory drugs, celecoxib and indomethacin. The rates of the release stimulated by 15-deoxy-Delta(12,14) PGJ2 differ from those observed with celecoxib. Arachidonic acid release by9-cis retinoic acid in the presence of either ciglitazone or trans retinoic acid is synergistic. It is additive in the presence of celecoxib. Cycloheximide and actinomycin inhibit the release of arachidonic acid stimulated by 15-deoxy-Delta(12,14) PGJ2 but not by celecoxib. The findings indicate that agonists of the peroxisome proliferator-activated receptor-gamma and retinoic acid receptors stimulate the release of arachidonic acid. The mechanisms involved may differ in the cases of 15-deoxy-Delta(12,14) PGJ2 and celecoxib.     

Retinoic acid activates human inducible nitric oxide synthase gene through binding of RARalpha/RXRalpha heterodimer to a novel retinoic acid response element in the promoter

Human inducible nitric oxide synthase (hiNOS) catalyzes nitric oxide (NO) which has a significant effect on tumor suppression and cancer therapy. Here we revealed the detailed molecular mechanism involved in the regulation of hiNOS expression induced by retinoic acid (RA). We showed that RARalpha/RXRalpha heterodimer was important in hiNOS promoter activation, hiNOS protein expression, and NO production. Serial deletion and site-directed mutation analysis revealed two half-sites of retinoic acid response element (RARE) spaced by 5bp located at -172 to -156 in the hiNOS promoter. EMSA and ChIP assays demonstrated that RARalpha/RXRalpha directly bound to this RARE of hiNOS promoter. Our results suggested the identification of a novel RARE in the hiNOS promoter and the roles of the nuclear receptors (RARalpha/RXRalpha) in the induction of hiNOS by RA.