This study establishes the role of nuclear receptors and RA in regulating lipid homeostasis in the liver. In addition, the mechanisms by which nuclear receptors and RA regulate lipid homeostasis were illustrated at the gene, pathway, and systemic levels. Furthermore, relationships between RXRα and PXR, LXR, FXR, as well as PPARα in regulating lipid homeostasis were analyzed. These nuclear receptors depend on RXRα to execute their functions and more than 85% of their bound genes overlap with those bound by RXRα. The nuclear receptor binding data were strengthened by profiling the expression patterns of 576 lipid genes, which showed that RA treatment and RXRα-deficiency had an opposite effect in regulating lipid homeostasis. Nuclear receptor binding data also illustrated extensive cross talk among the studied nuclear receptors. Thus, our novel in vivo data provided extensive evidence showing the role of RA in dictating lipid homeostasis in the liver.
It is intriguing that more than 85% of the PXR, LXR, FXR, and PPARα binding sites overlapped with those of RXRα. In contrast, only 43% of RARα binding sites overlapped with those of RXRα. It has been shown that RARα can form homodimers . It is also possible that RARα may dimerize with RXRβ and RXRγ to exert its function. Although the percentage of overlapping bindings between RARα and RXRα is not as high as others, the number of genes that could be bound by RXRα/RARα (4554) is the highest, followed by RXRα/PPARα (3468), RXRα/FXR (2019), RXRα/LXR (988), and RXRα/PXR (666), implying the relatively extensive role of these nuclear receptors in regulating hepatic gene expression. It is important to further study the role of RARα and other RARs in the liver. By forming partners with other nuclear receptors, RXRα is a master regulator. Our data showed that more than 8000 hepatic genes were bound by RXRα, and 72% of them overlapped with the genes bound by RARα, PXR, LXR, FXR, or PPARα. The remaining 28% of RXRα binding sites might be bound by RXRα homodimer or the heterodimer of RXRα and VDR or CAR. Thus, the five nuclear receptors (RARα, PXR, LXR, FXR, or PPARα) analyzed in the current study account for almost three quarters of RXRα binding genes in the liver. Furthermore, nearly 50% of RXRα bindings overlapped with the bindings of PXR, LXR, FXR, and PPARα (Figure 1B). Hence, lipid regulation should be one of the major functions of RXRα.
Clustering and PCA showed that the genome-wide binding pattern of RARα is not similar to that of PPARα, LXR, PXR, and FXR. Biological function annotation also showed that RARα has some unique features including protein processing, protein localization, and RNA processing. However, the five studied nuclear receptors also demonstrated functional redundancy. For example, there are four pathways, including oxidation reduction, carboxylic acid catabolic process, organic acid catabolic process, and cofactor metabolic process, that can be regulated by more than four nuclear receptors. This finding suggests the importance of these four pathways in the liver, and the role of RARα in them.
Although PPARα, LXR, FXR, and PXR have extensive roles in regulating lipids, they also have specific roles in regulating different types of lipids. RXRα/PPARα prefers to bind to genes that participate in neutral lipids, glycerol ether, and organic ether as well as fatty acid metabolism processes. All of which are either fatty acid-derived products or precursors for the biosynthesis of fatty acids. Another pathway bound by RXRα/PPARα is the acylglycerol metabolic process, which is involved in triglyceride homeostasis. RXRα/LXR tends to regulate genes involved in sterol metabolism, which is consistent with its known role . RXRα/FXR not only binds to the genes participating in steroid metabolism process, but also those involved in lipid transport and carbohydrate metabolism processes. RXRα/PXR binds to the genes involved in regulating the pyruvate metabolic process at the DNA binding level. Pyruvate is a key intersection for fatty acid, carbohydrate, and protein metabolisms. In addition, RXRα/PXR also regulates response to acute phase, inflammatory, and wounding, implying that PXR can be an excellent target for metabolism and inflammation-related health issues. Lastly, FXR binds to the genes involved in monosaccharide metabolism, which shows the intimate relationship between bile acid and glucose homeostasis .
All of the 114 genes that showed differential effects of RA treatment and RXRα deficiency are bounded by RXRα and RARα, PXR, LXR, FXR, and PPARα heterodimers. These findings indicate that those studied nuclear receptors retain RA response in vivo and the effect of RA is dependent upon those nuclear receptors. RA has a broad spectrum of effects including biosynthesis of retinoids, phospholipids, and unsaturated fatty acids. It also has a role in eliminating retinoids, oxidizing saturated fatty acids, and breaking down triglycerides. It seems that RA has extensive beneficial effects in maintaining the health of the liver. Specifically, RA induced the expression of Cyp2c37/38/50/54/70 and Cyp2j5. These genes encode enzymes involved in the generation of epoxyeicosatrienoic acids , which have anti-inflammatory effects . In contrast, RXRα deficiency induces the gene expression of Cyp4f that is responsible for the generation of 20-hydroxyeicosatetraenoic acid, a pro-inflammation molecule . In addition, RA increases mRNA levels of cbr1(carbonyl reductase 1), which is responsible for transforming prostaglandin E2 to prostaglandin F2α. Prostaglandin E2 and F2α have different effects in regulating lipid breakdown. Prostaglandin E2 is a lipolysis inhibitor ; whereas, prostaglandin F2α has not been shown to have the same effect. Thus, the induction of cbr1 gene expression could be a mechanism by which RA induces lipolysis. RA also induces expression levels of gene encoding proteins for phospholipid biosynthesis, but RXRα deficiency increases the expression of the genes that have a role in the degradation of phospholipids. This finding suggests the potential role of RA in maintaining the normal structure of the cell membrane. Formation of the monolayer of lipoprotein or lipid droplet is one of the major ways that phospholipids regulate lipid metabolism . Phosphatidylethanolamine (PE) and phosphatidylcholine (PC) are two important phospholipids that show different effects on lipid metabolism in humans and rodents. Lower PC/PE ratio induces steatosis or even steatohepatitis in humans , however, PE has a greater effect than PC in reducing the cholesterol level in rodents . Our data showed that RA induced the gene expression of ptdss1 (phosphatidylserine synthase 1), which converts PC to PE. Consistently, the ptdss1 gene expression is reduced due to hepatic RXRα deficiency. These gene expression levels and DNA binding data not only showed the underlying mechanism for RA in regulating liver gene expression, but also suggested the biochemical outcome.