Chronic activation of FXR-induced liver growth with tissue-specific targeting Cyclin D1

Abstract

The nuclear receptor (FXR) plays essential roles in maintaining bile acid and lipid homeostasis by regulating diverse target genes. And its agonists were promising agents for treating various liver diseases. Nevertheless, the potential side effect of chronic FXR activation by specific agonists is not fully understood. In this study, we investigated the mechanism of FXR agonist WAY-362450 induced liver enlargement during treating liver diseases. We demonstrated that chronic ingestion of WAY-362450 induced liver hypertrophy instead of hyperplasia in mouse. Global transcriptional pattern was also examined in mouse livers after treatment with WAY-362450 by RNA-seq assay. Through GO and KEGG enrichment analyses, we demonstrated that the expression of Cyclin D1 (Ccnd1) among the cell cycle-regulating genes was notably increased in WAY-362450-treated mouse liver. Activation of FXR-induced Ccnd1 expression in hepatocyte in a time-dependent manner in vivo and in vitro. Through bioinformatics analysis and ChIP assay, we identified FXR as a direct transcriptional activator of Ccnd1 through binding to a potential enhancer, which was specifically active in livers. We also found active histone acetylation was essential for Ccnd1 induction by FXR. Thus, our study indicated that activation of FXR-induced harmless liver hypertrophy with spatiotemporal modulation of Ccnd1. With a better understanding of the mechanism of tissue-specific gene regulation by FXR, it is beneficial for development and appropriate application of its specific agonist in preventing hepatic diseases.

Keywords: Farnesoid x receptor; WAY-362450; cyclin D1; histone modification; hypertrophy; transcription regulation.

Figure 1.

WAY-362450 promoted liver growth by induction of hepatocyte hypertrophy in mouse. (a-b) Wild type (WT) or FXR deficient (FXR-KO) mice (female; 8–10-week-old) treated with 17α-ethynylestradiol (EE2; 5 mg/kg, s.c.) were simultaneously gavaged with (EE2-WAY) or without (EE2-Veh) WAY-362450 for four weeks. (a) The ratios of liver/body weight in each group were shown; (b) Representative images were shown for staining with H&E (upper panels) or immunohistochemistry of Ki67 (lower panels) in WT liver tissues (Original magnification 200 x, bar: 100 μm). (C-H) WT mice (male; 8 weeks old) were administrated with either vehicle (Veh) or WAY-362450 (WAY) once daily for one-week. (c) The liver weight and ratios of liver/body weight in vehicle (Veh) or WAY-362450 (WAY)-treated mice were shown; (d) Plasma membrane of hepatocytes was immuno-stained by pan-cadherin (red), and the nucleus was stained by DAPI (blue). Original magnification 200 x, bar: 25 μm. The number of hepatocytes per microscope field. (e) The expression of Mki67 and Pcna in liver tissues was determined by RT-qPCR assay. (f) Representative images are shown for immunohistochemistry staining of Ki-67 in liver tissues from vehicle- or WAY-362,450-treated mice; Original magnification 200 x, bar: 100 μm for upper panel. The proportions of Ki67-positive hepatocytes in livers of vehicle or WAY-362,450 treated mice were shown in the right panel. (g) Nuclear DNA content in hepatocytes was semi-quantified in vehicle and WAY-362,450- treated mice; 1,500 nuclei were considered in hepatocytes. (h) The proportions of mitotic figure (as indicated by phospho-histone-H3 (Ser10) positive hepatocytes) in livers of vehicle or WAY-362450 treated mice were shown. (Data are represented as mean ± SEM; *p < 0.05, ***p < 0.001, ns: not significant (p > 0.05)).

Figure 2.

Activation of FXR by WAY-362450 markedly induced Ccnd1 expression in mouse liver. Male mice (8 weeks old) were administrated with either vehicle (Veh) or WAY-362450 (WAY) once daily for one-week. Gene expression pattern in mouse livers was analysis by RNA-seq assay. (a-b) Volcano plot (a) and heatmap (b) for identification of differentially expressed genes (DEGs, |fold change| ≥2, FDR < 0.05; n = 3 in each group) in mouse liver tissues upon WAY-362450 treatment. (c-d) Gene ontology (GO; C) and KEGG pathway (d) enrichment analysis of DEGs in mouse liver after WAY-362450 treatment was shown (p < 0.05). (e) The expression of cell cycle-regulating genes in the RNA-seq data was displayed as a heatmap. (f-g) Liver tissues from mice treated with vehicle or WAY-362450 were subjected to RT-qPCR assays for determination mRNA expression of the well-known FXR-target genes (Nr0b2, Abcb11 and Cyp7a1) and cell cycle genes (Ccna2, Ccnb1, Ccnb2, Ccnd1, Ccne1, Cdkn1a, Cdkn1b, Cdkn2c, Cdk1, Cdk2, Cdk4 and Cdk6). (n = 8; *p < 0.05, ***p < 0.001; ns, not significant).

Figure 3.

WAY-362450 enhanced Ccnd1 expression time-dependently in mouse livers. (a) FXR-deficient mice were treated with either vehicle (Veh) or WAY-362450 (WAY) for 24 h. The mRNA levels of Nr0b2 and Ccnd1 in mouse livers were determined by RT-qPCR (n = 6 each). (b) The mRNA levels of Nr0b2 and D-type cyclin family members (Ccnd1, Ccnd2, and Ccnd3) in mouse livers after WAY-362450 treatment for 4 h were determined by RT-qPCR (n = 6). (c) The hepatic mRNA levels of Nr0b2 and Ccnd1 in wild-type male mice treated with vehicle or WAY-362450 for 1–3 days were determined by RT-qPCR. (d-e) Male mice were treated with WAY-362450 for one-week. (d) Representative images are shown for immunohistochemistry staining of Cyclin D1 in liver tissues from vehicle- or WAY-362,450-treated mice. Original magnification 200 x, bar: 100 μm for upper panel. (e) The protein level of Cyclin D1 in mouse livers was determined by Western blot assay. Densitometric analysis of Cyclin D1 band density with normalization to β-Actin was shown in the lower panel. (f) The protein level of Cyclin D1 in female mouse livers treated with WAY-362450 for four weeks was determined by Western blot. (g) The expression levels of Nr0b2 and Ccnd1 in the intestine and kidney tissues from mice treated with vehicle or WAY-362450 for one-week were determined by RT-qPCR. (Mean ± SEM, ***p < 0.001; ns, not significant).

Figure 4.

FXR promoted Ccnd1 expression time-dependently in hepatocyte in vitro. (a-c) Mouse primary hepatocytes (A; mPH) and human hepatocellular carcinoma cell lines HepG2 (b) and Hep3B (c) were treated with DMSO (Veh), WAY-362450 (WAY, 2 μM) or GW4064 (GW, 2 μM) for 24 h. (d) HepG2 cells were treated with an FXR antagonist (z-guggulsterone, 20 μM) for 24 h. The mRNA expression levels of Nr0b2 (or NR0B2) and Ccnd1 (or CCND1) were determined by RT-qPCR. (e) HepG2 cells treated with DMSO, WAY-362450 (2 μM) or GW4064 (2 μM) were harvested at indicated time points and subjected to RT-qPCR analysis of NR0B2 and CCND1 mRNA levels. (f) Western blot analysis of CCND1 protein levels in 2 μM WAY-362450-treated HepG2 cells for 12, 24, 48 and 72 h. Densitometric analysis of Western blot result with normalization to β-Actin was shown in the right panel. (Mean ± SEM; *p < 0.05, *p < 0.01, ***p < 0.001).

Figure 5.

FXR regulates Ccnd1 transcription through direct binding to a downstream potential enhancer. (a) Public data, including FXR ChIP-seq (GEO: GSE73624), and H3K4me1, H3K4me3 and H3K27ac ChIP-seq (ENCODE: ENCSR854HXZ), on adult mouse livers were analyzed for potential FXR-binding sites and enhancer signature on mouse Ccnd1 gene. The results were exhibited on IGV 2.4 software. (b) Sequence alignment of the putative FXR responsive element (inverted repeat-1, IR-1) downstream Ccnd1 gene in mouse, rat, guinea pig, dog, horse, bushbaby, marmoset, rhesus, chimp, orangutan, and human. The consensus sequence in IR-1 elements was shown in red. (c) HEK293T cells were transfected with constructs containing FXR response element (FXRE)-approximal region (Ccnd1-FXRE) in Ccnd1, Ccnd1-FXRE region with IR1 deletion (Ccnd1-FXRE-ΔIR1), or Ccnd1 IR1 core element (Ccnd1-IR1). The construct containing the mouse Nr0b2 FXRE-containing region was used as a positive control (Nr0b2-FXRE). After treatment with vehicle (Veh) or WAY-362450 (WAY), the cells were subjected to dual-luciferase reporter assays. (d) HEK-293T Cells transfected with constructs containing Ccnd1-FXRE or the FXR response element (FXRE) from the mouse Nr0b2 gene (Nr0b2-FXRE) were treated with 2 μM WAY-362450 for the indicated time period. The activity of IR1 element of Ccnd1 was determined by dual-luciferase reporter assay. (e) A chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assay was conducted using liver tissues from the vehicle or WAY-362450-treated mice (n = 3 each). ChIP signals are expressed as the ratio of immunoprecipitated DNA to the total input chromatin. A region of the Actb promoter was used as a negative control. The proximal region of the Nr0b2 promoter containing FXRE was used as the positive control. (Mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001; ns, not significant).

Figure 6.

The signature of downstream enhancer of Ccnd1 was tissue-specific and developmental stage-dependent in the liver. (a) ChIP-qPCR analysis of FXR binding on Ccnd1 gene in vehicle or WAY-362450 treated liver and intestine tissues (n = 4). (b) The levels of H3K4me1 and H3K27ac at FXR-binding region of Ccnd1 gene in liver and intestine tissues (n = 4) were examined. (c) ChIP-seq data of histone modifications (H3K4me1, H3K4me3, H3K27ac, and H3K27me3) and chromatin accessibility (DNase-seq) on Ccnd1 gene from ENCODE were visualized using IGV 2.4 software. Data on liver, small intestine, kidney and heart tissues in adult mouse (8 weeks) were analyzed. (d-e) Mouse primary hepatocytes (d) and HepG3 cells (e) were treated with 2 μM WAY-362450 with or without 3 μM (+)-JQ1 and 10 μM C646 for 4 h. The mRNA levels of NR0B2 and CCND1 genes were determined by RT-qPCR assay. (Mean ± SEM, ***p < 0.001) (f) Schematic diagram for FXR agonist in the induction of liver and hepatocyte hypertrophy by regulating Ccnd1 expression through binding to a potential enhancer region (H3K4me1/H3K27ac positive and H3K4me3 negative). Red oval, H3K4me1; Green oval, H3K27ac; Blue oval, H3K4me3.