Regulation of FGF21 expression and secretion by retinoic acid receptor-related orphan receptor alpha

Abstract

Fibroblast growth factor 21 (FGF21) is a hormone produced by fat and the liver that plays an important role in lipid metabolism. FGF21 expression is induced by peroxisome proliferator-actived receptor alpha in response to physiological conditions requiring increased fatty acid oxidation. Retinoic acid receptor-related receptor alpha (RORalpha) is another nuclear receptor that plays a critical role in lipid metabolism as well as in regulation of the circadian rhythm. In this study we demonstrate that RORalpha directly regulates the expression and secretion of FGF21. A canonical ROR response element was identified in the proximal promoter of the FGF21 gene and shown to exhibit functional activity. Overexpression of RORalpha in HepG2 cells resulted in increased expression and secretion of FGF21. Suppression of RORalpha expression caused a decrease in FGF21 expression and secretion, suggesting that RORalpha contributes to the basal expression of FGF21. These data suggest that one mechanism by which RORalpha regulates lipid metabolism may be by modulation of FGF21 secretion. Furthermore, this study identifies a clear link between RORalpha, a key regulator of the mammalian clock, and FGF21, an important hormone regulating glucose and lipid homeostasis.

FIGURE 1.

The FGF21 promoter contains an evolutionarily conserved RORE. A, schematic of the FGF21 promoter. The FGF21 promoter contains a RORE in its proximal promoter region and the two recently identified REV-ERB response elements (REVRE) in its distal region. B, the RORE is evolutionarily conserved between humans and mice. C, sequence of the human FGF21 promoter region. The first underlined sequence depicts the RORE that we have identified. The second and third underlined sequences are the two REV-ERB response elements that were recently identified by Estall et al. (24).

FIGURE 2.

RORα and RORγ increase FGF21 promoter-driven transcription. A, RORα and RORγ increase the transcriptional activity of a FGF21 promoter-driven luciferase construct. HEK293 cells were transfected with RORα or RORγ along with the FGF21 reporter construct. Luciferase values were normalized using Renilla luciferase. *, p < 0.05 versus control vector. B, FGF21 transcriptional activity is ROR-specific. HEK293 cells were transfected with RORα or RORγ along with the FGF21 reporter construct containing the mutated RORE. The data are presented as the means ± S.E. *, p < 0.05 versus the mutant construct. wt, wild type; mt, mutant.

FIGURE 3.

Overexpression of RORα stimulates FGF21 expression. A, adenoviral overexpression of RORα in HepG2 cells increases FGF21 expression at the mRNA level as measured by reverse transcription-PCR. B, RORα binds the RORE within the promoter region of FGF21. IgG was used as a negative control, and α-acetylated histone H3 was used as a positive control. A representative gel is shown. C, adenoviral overexpression of RORα in HepG2 cells increases the expression of secreted FGF21 protein as measured by ELISA. The data are presented as the means ± S.E. *, p < 0.05 versus Ad-LacZ control.

FIGURE 4.

Knock down of RORα results in decreased FGF21 expression and secretion. A, HepG2 cells were treated with either control siRNA or RORα siRNA. Western blot analysis confirms the decrease in RORα protein expression. The blot was then reprobed with α-tubulin to confirm loading. A representative blot is shown. B, decreased expression of RORα in HepG2 cells leads to decreased expression of FGF21 as measured by reverse transcription-PCR. C, decreased expression of RORα in HepG2 cells leads to decreased secretion of FGF21. The data are presented as the means ± S.E. *, indicates p < 0.05 versus control siRNA.

FIGURE 5.

7α-Hydroxycholesterol represses FGF21 promoter-driven transcription. The ability of RORα (A) and RORγ (B) to activate FGF21 promoter-driven transcription is repressed by 7α-OHC in HEK293 cells. 7α-OHC was evaluated at a concentration of 10 μm. The data are presented as the means ± S.E. *, p < 0.05 versus dimethyl sulfoxide (DMSO).