A vitamin D receptor/SMAD genomic circuit gates hepatic fibrotic response - PubMed (original) (raw)

. 2013 Apr 25;153(3):601-13.

doi: 10.1016/j.cell.2013.03.028.

Ruth T Yu, Nanthakumar Subramaniam, Mara H Sherman, Caroline Wilson, Renuka Rao, Mathias Leblanc, Sally Coulter, Mingxiao He, Christopher Scott, Sue L Lau, Annette R Atkins, Grant D Barish, Jenny E Gunton, Christopher Liddle, Michael Downes, Ronald M Evans

Affiliations

A vitamin D receptor/SMAD genomic circuit gates hepatic fibrotic response

Ning Ding et al. Cell. 2013.

Abstract

Liver fibrosis is a reversible wound-healing response involving TGFβ1/SMAD activation of hepatic stellate cells (HSCs). It results from excessive deposition of extracellular matrix components and can lead to impairment of liver function. Here, we show that vitamin D receptor (VDR) ligands inhibit HSC activation by TGFβ1 and abrogate liver fibrosis, whereas Vdr knockout mice spontaneously develop hepatic fibrosis. Mechanistically, we show that TGFβ1 signaling causes a redistribution of genome-wide VDR-binding sites (VDR cistrome) in HSCs and facilitates VDR binding at SMAD3 profibrotic target genes via TGFβ1-dependent chromatin remodeling. In the presence of VDR ligands, VDR binding to the coregulated genes reduces SMAD3 occupancy at these sites, inhibiting fibrosis. These results reveal an intersecting VDR/SMAD genomic circuit that regulates hepatic fibrogenesis and define a role for VDR as an endocrine checkpoint to modulate the wound-healing response in liver. Furthermore, the findings suggest VDR ligands as a potential therapy for liver fibrosis.

Copyright © 2013 Elsevier Inc. All rights reserved.

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Figures

Figure 1

Figure 1. Systemic Administration of Calcipotriol Attenuates Liver Fibrosis in CCl4-Treated Mice while Genetic Abrogation of Vdr Results in Spontaneous Liver Fibrosis

(A) Livers from 4 wk-treated C57BL/6J mice (vehicle (DMSO) (n=3), carbon tetrachloride (CCl4, 0.5ml/kg i.p., n=6), calcipotriol (Cal, 20 μg/kg oral gavage, n=3) and CCl4 plus calcipotriol (n=6)) stained with Sirius red (left) and H&E (right). Scale bar, 200μm. Fibrosis quantified by (B) Sirius red staining, (C) hydroxyproline content and (D) H&E staining (Ishak score). Asterisks denote statistically significant differences (Student’s unpaired t-test, **p < 0.01, ***p < 0.001). (E)–(G) RT-qPCR measurement of hepatic gene expression levels of Col1a1, Tgfβ1 and Timp1. Data represents the mean ± SEM. Asterisks denote statistically significant differences (Student’s unpaired t-test, **p < 0.01, ***p < 0.001). (H) Sirius red (top) and H&E (bottom) stained liver sections from Vdr+/+ (n=3), Vdr+/- (n=4) and Vdr−/− (n=2 of 4) mice maintained on a calcium- and phosphate-supplemented rescue diet (2% Calcium, 1.25% Phosphorus, 20% Lactose) for 6 months prior to sacrifice. Arrows indicate peri-sinusoidal fibrosis (Vdr+/− mice) and inflammatory cell infiltrate (Vdr−/− mice), respectively. Scale bar, 50μm. (I) Fibrosis quantified by hydroxyproline content and (J) Col1a1 mRNA expression using the two of four livers from Vdr−/− mice exhibiting the least fibrosis on Sirius red staining (refer to results). Data represents the mean ± SEM. Asterisks denote statistically significant differences (Student’s unpaired t-test, *p < 0.05). See also Fig. S1 & S2.

Figure 2

Figure 2. VDR Signaling Suppresses TGFβ-induced Pro-Fibrotic Genes

(A) Heat map comparing 519 differentially expressed genes in freshly isolated rat HSCs (quiescent HSCs, Q-HSCs), activated HSCs (A-HSCs, 3 days culture on plastic) and cells cultures in the presence of 10nM 1,25(OH)2D3 (A-HSCs+1,25(OH)2D3). Euclidean clustering of both rows and columns using log2 transformed microarray expression data, n=2 per treatment group. (B) Heat map of fold change of genes involved in fibrosis in primary rat HSCs treated with TGFβ1 (1ng/ml) and TGFβ1 plus 1,25(OH)2D3 (100nM) for 24 hours, n=2 per treatment group. (C) Fibrotic gene expression in control (siCNTL) or VDR-specific (siVDR) siRNA transfected LX-2 cells treated with Vehicle (DMSO), calcipotriol (Cal, 100nM), TGFβ1 (1ng/ml), or TGFβ1+Cal for 16 hours. Data represents the mean ±SEM of at least three independent experiments performed in triplicate. Asterisks denote statistically significant differences (Student’s unpaired t-test, *p < 0.05, **p < 0.01).

Figure 3

Figure 3. VDR and SMAD3 Cistromes in Hepatic Stellate Cells

(A) and (E) Pie charts illustrating genomic locations of VDR and SMAD3 binding sites in treated LX-2 cells (calcipotriol (100nM) and TGFβ1 (1ng/ml) for 4 hours following 16 hours calcipotriol (100nM) pretreatment, FDR<0.0001). Promoter regions, < 2kb from TSS; intergenic regions, not promoter, intron or exon. (B) and (F) Representative ChIP-Seq reads for VDR and SMAD3 aligned to the CYP24A1 and ID1 genes, respectively. (C) and (G) Gene ontology (GO) classification of genes annotated with VDR and SMAD3 binding sites. (D) and (H) De novo motif analysis performed on sequences located within 100bp of VDR and SMAD3 peaks (FDR<0.0001). See also Fig. S3 & S4.

Figure 4

Figure 4. Antagonism of TGFβ Signaling via VDR/SMAD3 Genomic Crosstalk

(A) Venn diagram depicting overlap of VDR and SMAD3 genomic binding sites in LX-2 cells treated as in Figure 3. (B) Intensity plots showing hierarchical clustering of ChIP-fragment densities as a function of distance from the center of statistically significant SMAD3 binding peaks (23,532 peaks, FDR=0.0001). Intensity around position 0 of VDR (blue) indicates overlapping VDR/SMAD3 sites with SMAD3 (red) acting as a positive control. (C) ChIP-re-ChIP of treated LX-2 cells analyzed by qPCR at VDR and SMAD3 co-bound sites. Occupancy is expressed relative to input chromatin. (D) Common human phenotypes enriched in genes co-occupied by VDR and SMAD3. (E) The number of TGFβ1/VDR-coregulated pro-fibrotic genes harboring genomic sites co-occupied by VDR and SMAD3. (F) The number of VDR/SMAD3 co-occupied sites observed in pro-fibrotic genes coregulated by TGFβ1 and VDR. LX-2 cells treated as in Figure 3. Data represents the mean ± SEM of at least three independent experiments performed in triplicate. Asterisks denote statistically significant differences (Student’s t-test, *p < 0.05, **p < 0.01). See also Suppl Table 2 & Fig. S5.

Figure 5

Figure 5. Genomic Antagonism between VDR and SMAD

(A) and (B) Plots of VDR and SMAD3 ChIP-Seq signal intensity relative to the center of VDR/SMAD3 co-occupied sites in LX-2 cells (TGFβ1 (1ng/ml) ± calcipotriol (100nM) for 4 hours). (C) Representative ChIP-Seq reads aligned to COL1A1 for VDR and SMAD3 in treated LX-2 cells (Vehicle (DMSO), Calcipotriol (Cal, 100nM), TGFβ1 (1ng/ml), or TGFβ1+calcipotriol). The three co-occupied sites are designated as 1, 2 and 3. (D) and (F) ChIP-qPCR at COL1A1 regulatory region #1 co-bound by VDR and SMAD3 in LX-2 cells treated as above. (E) and (G) ChIP-qPCR at COL1A1 regulatory region #1 of control (siCNTL), VDR-specific (siVDR), or SMAD3-specific (siSMAD3) siRNA transfected LX-2 cells treated as above. Occupancy is expressed relative to input chromatin. Data represents the mean ± SEM of at least three independent experiments performed in triplicate. Asterisks denote statistically significant differences (Student’s t-test, *p < 0.05, **p < 0.01). See also Fig. S6 & S7.

Figure 6

Figure 6. TGFβ Unmasks a Signal Dependent VDR Citrome

(A) Venn diagram displaying overlapping VDR cistromes in treated LX-2 cells (FDR<0.0001). (B) Plot of VDR ChIP-Seq peak locations depicted in (A) categorized as VDR Cal/TGFβ1+Cal (3,537 overlapping), VDR Cal only (2,744 calcipotriol-only), or VDR TGFβ1+Cal only (21,447 calcipotriol+TGFβ1-only) relative to the center of SMAD3 binding sites in LX-2 cells. (C) Plot of VDR ChIP-Seq signal intensity relative to the center of VDR/SMAD3 co-occupied sites in LX-2 cells treated as indicated. (D) Western blot for VDR in nuclear and whole cell extracts (NE, WCE) from LX-2 cells treated as above. TFIIH (p89) was used as a loading control. (E) The percentages of calcipotriol-only, calcipotriol+TGFβ1-only or calcipotriol/calcipotriol+TGFβ1-overlapping VDR ChIP-Seq peaks containing VDREs. (F) Plot of histone H3 ChIP-Seq signal intensity relative to the center of VDR/SMAD3 co-occupied sites in LX-2 cells treated as indicated.

Figure 7

Figure 7. VDR/SMAD Genomic Circuit

(A) and (B) Time course of VDR and SMAD3 binding at the COL1A1 regulatory region #1 in treated LX-2 cells (vehicle (DMSO), calcipotriol (100nM), TGFβ1 (1ng/ml), TGFβ1 (1ng/ml) + calcipotriol (100nM)) determined by ChIP-qPCR. LX-2 cells were pretreated with calcipotriol (100nM) for 16 hours prior to time course assay and occupancy is expressed relative to input chromatin. Data represents the mean ± SEM of at least three independent experiments performed in triplicate. Asterisks denote statistically significant differences compared to calcipotriol–induced VDR occupancy or TGFβ1-induced SMAD3 occupancy of corresponding time point (Student’s unpaired t-test, *p < 0.05, **p < 0.01). (C) Time course of TGFβ1+calcipotriol-induced VDR and SMAD3 binding, normalized to calcipotriol alone or TGFβ1 alone, respectively. Data represents the mean ± SEM of at least three independent experiments performed in triplicate. (D) Model depicting proposed VDR/SMAD genomic circuit controlling pro-fibrogenic responses in HSCs.

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