Abstract
Chronic inflammation increases the risk of developing one of several types of cancer. Inflammatory responses are currently thought to be controlled by mechanisms that rely on transcriptional networks that are distinct from those involved in cell differentiation1,2,3. The orphan nuclear receptor NR5A2 participates in a wide variety of processes, including cholesterol and glucose metabolism in the liver, resolution of endoplasmic reticulum stress, intestinal glucocorticoid production, pancreatic development and acinar differentiation4,5,6,7,8. In genome-wide association studies9,10, single nucleotide polymorphisms in the vicinity of NR5A2 have previously been associated with the risk of pancreatic adenocarcinoma. In mice, Nr5a2 heterozygosity sensitizes the pancreas to damage, impairs regeneration and cooperates with mutant Kras in tumour progression11. Here, using a global transcriptomic analysis, we describe an epithelial-cell-autonomous basal pre-inflammatory state in the pancreas of Nr5a2+/− mice that is reminiscent of the early stages of pancreatitis-induced inflammation and is conserved in histologically normal human pancreases with reduced expression of NR5A2 mRNA. In Nr5a2+/−mice, NR5A2 undergoes a marked transcriptional switch, relocating from differentiation-specific to inflammatory genes and thereby promoting gene transcription that is dependent on the AP-1 transcription factor. Pancreatic deletion of Jun rescues the pre-inflammatory phenotype, as well as binding of NR5A2 to inflammatory gene promoters and the defective regenerative response to damage. These findings support the notion that, in the pancreas, the transcriptional networks involved in differentiation-specific functions also suppress inflammatory programmes. Under conditions of genetic or environmental constraint, these networks can be subverted to foster inflammation.
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References
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Acknowledgements
We thank O. Domínguez, J. Herranz, T. Lobato, L. Martínez, and Y. Cecilia, as well as members of the CNIO core facilities, Epithelial Carcinogenesis Group, and Genes, Development and Disease Group; L. Montuenga, C. Rodríguez-Ortigosa, B. Bréant and cited investigators for providing antibodies; and E. Batlle and P. Muñoz-Cánoves for critical comments. This study used the high-performance computational capabilities of the Biowulf Linux cluster (https://hpc.nih.gov/). The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, US National Institutes of Health (NIH), nor does mention of trade names, commercial products or organizations imply endorsement by the US government. This work was supported in part by grants SAF2011-29530 and SAF2015-70553-R from the Ministerio de Economía y Competitividad (co-funded by the ERDF-EU), RTICC from the Instituto de Salud Carlos III (RD12/0036/0034, RD12/0036/0050) and grants 256974 and 289737 from the European Union Seventh Framework Program to F.X.R.; grants BFU 2012-40230 and SAF2015-70857 from the Ministerio de Economía y Competitividad (co-funded by the ERDF-EU) and Worldwide Cancer Research (13-0216) to E.F.W.; grants PI12/00815 and PI1501573 from the Fondo de Investigaciones Sanitarias, Instituto de Salud Carlos III, Spain and EUPancreas COST Action BM1204 to N.M.; grant P30CA008748 from the US NIH, National Cancer Institute to S.H.O.; the Intramural Research Program of the NIH, National Cancer Institute; and Mayo Clinic SPORE in Pancreatic Cancer funded by National Cancer Institute grant P50 CA102701. L.T. and T.B. were supported by the Department of Technology, Norwegian University of Science and Technology, the Central Norway Regional Health Authority and by the European Science Foundation. P.M. and I.C. are recipients of Juan de la Cierva and Beca de Formación del Personal Investigador, respectively, from Ministerio de Economía y Competitividad. I.F. is the recipient of a ‘Juegaterapia-Amigos del CNIO’ Postdoctoral Fellowship. F.X.R. acknowledges the support of Asociación Española Contra el Cáncer.
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I.C., M.F. and F.X.R. conceived the study. I.C., M.F. and N.d.P. performed animal experiments. I.C., E.C.-d.-S.-P., M.Z. and J.J. conducted bioinformatics analyses. I.C., V.J.S.-A.L. and I.F. conducted in vitro studies using mouse cells. S.H.O., J.S., W.R.B., G.M.P. and N.M. provided samples and information on human subjects. W.R.B., G.M.P., N.M. and L.T.A. designed and performed clinical studies, obtained samples and performed human data analysis. I.M., D.M., L.T. and T.B. were involved in data analysis. L.B., K.S. and E.F.W. provided reagents. P.M., L.B., L.T.A. and E.F.W. had critical input into experimental design, data analysis and interpretation. I.C. and F.X.R. wrote the manuscript with contributions of P.M., L.B., L.T.A. and E.F.W. F.X.R. supervised the overall conduct of the study. All authors read and approved the final manuscript.
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Extended data figures and tables
Extended Data Figure 1 Protein eQTL analysis in human PDAC.
Pancreatic tumours (n = 110) from patients carrying the risk-increasing allele (T) at rs3790844 express lower levels of NR5A2 protein than those carrying the protective allele (C). NR5A2 expression was assessed using immunohistochemistry, and scored based on percentage of reactive cells and intensity of staining. The analysis was performed for mean histoscore (P = 0.097, β = −18.0; two-sided Wilcoxon test) and mean histoscore quantiles (P = 0.028, β = −0.57; two-sided Wilcoxon test).
Extended Data Figure 2 The pancreas of Nr5a2+/− mice is histologically normal but displays increased expression of inflammatory genes.
a, qRT–PCR analysis of the expression of transcripts coding for acinar-related genes in wild-type and Nr5a2+/− mice (n = 7 per group). Data were obtained from a series of mice that was independent of the series used for RNA-seq. b, Immunofluorescence analysis of PTF1a, CDH1 and CPA in the pancreas of wild-type and Nr5a2+/− mice (n = 3 per group). Arrow, acinus. c, Immunohistochemical analysis of expression of C5AR1 and CFD in the pancreas of wild-type and Nr5a2+/− mice shows patchy expression in acinar cells (arrows) (n = 5 per group). d, Percentage of inflammatory cell subtypes (from total cells) in wild-type and Nr5a2+/− pancreases (n ≥ 4 per group) analysed by flow cytometry (two different experiments). e, f, Quantification of periacinar Cd45+ cells in the pancreas of wild-type and Nr5a2+/− mice using immunofluorescence on frozen sections. Broken line delineates a pancreatic lymph node, used as a control. Two independent assessments were performed. e, Quantification of cells expressing PAX5, MAC2 and CD3 in the pancreas of wild-type and Nr5a2+/− mice using immunofluorescence (n ≥ 3 per group). In a, d and e, one-sided Mann–Whitney U test; *P < 0.05, **P < 0.01.
Extended Data Figure 3 The upregulation of inflammatory markers, AP-1 components, p-JUN and p-JNK in Nr5a2+/− pancreases is epithelial-cell-autonomous, as shown by the analysis of isolated primary acinar cells.
a, Expression of inflammatory proteins in primary acinar cells from wild-type and Nr5a2+/− mice shown using western blotting (n = 4 per group). b–f, Primary acinar cell fractions from wild-type and Nr5a2+/− mice largely depleted of DBA+ ductal cells (b, c), show reduced expression of the ductal cell marker HNF1β (d, e) and inflammatory cell markers (f) compared to total pancreas (n = 4 per group). Inset in b, DBA-labelled duct. Two independent experiments were performed. g, h, Expression of AP-1 components and JNK in primary acinar cells from wild type and Nr5a2+/− mice using western blotting. NR5A2 is expressed at reduced levels in Nr5a2+/− pancreases (n = 4/group). In c–f, one-sided Mann–Whitney U test; *P < 0.05, **P < 0.01.
Extended Data Figure 4 The defective pancreatic response to damage is epithelial-cell-autonomous.
a, Constitutive Nr5a2+/− mice display more severe pancreatitis upon administration of seven doses of caerulein (given once per hour). b, d, This severe phenotype is recapitulated at 48 h in mice harbouring a heterozygous deletion of Nr5a2 in pancreatic epithelial cells (b) but not in mice in which both alleles of Nr5a2 are inactivated in myeloid cells by Cre activation from the lysozyme endogenous locus (Lys) (d). This experiment was performed once for the conditional mice; for Nr5a2+/− mice, more than four independent experiments were performed. Representative histological images are shown. Semi-quantitative inflammation scores corresponding to the experiments are shown in a–d (n ≥ 4 per group). c, qRT–PCR analysis of the expression of transcripts coding for AP-1 and inflammatory genes in control and Ptf1acre;Nr5a2lox/+ mice (n = 6 per group). In a–d, one-sided Mann–Whitney U test; *P < 0.05, **P < 0.01.
Extended Data Figure 5 Nr5a2 haploinsufficiency causes a basal pre-inflammatory state similar to that associated with the early stages of pancreatitis.
a, Comparative expression (wild-type versus Nr5a2+/− mice) of the upregulated (left), downregulated (middle) or control (right) genes over time, after induction of pancreatitis. RNA-seq analysis was performed once. One-sided Student’s t-test. b, Immunohistochemical analysis shows persistent overexpression of AP-1 components during the recovery period after induction of acute pancreatitis (one dose per hour for seven hours). Representative results of one of five pancreases analysed are shown.
Extended Data Figure 6 A single dose of caerulein does not cause inflammation but does induce an upregulation of AP-1 and p-JUN that precedes STAT3 phosphorylation both in wild-type and Nr5a2+/− mice.
a, Quantification of infiltration by Cd45+ cells in the pancreas of wild-type and Nr5a2+/− mice after administration of one dose of caerulein (n = 1). b, Immunohistochemical analysis of expression of JUN, FOS, p-JUN and phospho-STAT3 (p-STAT3) in wild-type and Nr5a2+/− mice at various time points after caerulein administration (n = 4 per group). c, qRT–PCR analysis of expression of a panel of inflammatory genes in isolated acini treated with PBS or caerulein (100 pM). Data are shown relative to values of wild-type acini incubated with PBS (n = 4 per group). Two independent experiments were performed. In c, one-sided Mann–Whitney U test; *P < 0.05, **P < 0.01.
Extended Data Figure 7 NR5A2 cooperates with AP-1 to regulate inflammatory gene expression.
a, Analysis of putative NR5A2 and AP-1 binding sites in the proximal promoter of C1qb, Ccc7 and Ccl8 using the JASPAR algorithm (http://jaspar.genereg.net/search?q=&collection=CORE&tax_group=vertebrates). Sequence matrices for NR5A2, FOS:JUN, FOS, JUN, JUN (var.2), FOSL1, FOSL2 and BATF:JUN were computed. Motifs with a score of greater than 7.5 for NR5A2 (blue) and AP-1 (orange) are highlighted. Additionally, a manual search for the NR5A2 binding motif CAAGGNCA was performed. Purple, genomic regions amplified in the sequential ChIP–qPCR experiments shown in Fig. 3f. The sequences of the 400 nucleotides upstream and downstream of each amplicon are also shown. b, Ccl8 luciferase promoter–reporter activity (−1960 to −655) using HEK293 cells and increasing concentrations of JUN-coding plasmid in the absence (left) or presence (right) of NR5A2. Data shown corresponds to the mean of six independent experiments. In b, one-sided Mann–Whitney U test; *P < 0.05, **P < 0.01.
Extended Data Figure 8 NR5A2 regulates AP-1 expression, in part through the modulation of NR0B2 and its recruitment to AP-1 gene promoters.
a, Expression of Nr0b1 and Nr0b2 transcripts in total pancreas and isolated acini of wild-type and Nr5a2+/− mice (n = 4 per group). Arrow, acinus; broken line delineates an islet. b, Immunohistochemical and double immunofluorescence analysis showing acinar distribution of NR0B2 in wild-type pancreas, and reduced expression in Nr5a2+/− pancreases. Acinar cells are delineated with anti-CDH1 antibodies (n = 5 per group). c, Reduced expression of Nr0b2 mRNA and corresponding protein in total pancreas and isolated acini of wild-type and Nr5a2+/− mice. Densitometric quantification of NR0B2 expression relative to vinculin (n ≥ 4 per group). d, e, Expression of Nr0b2 mRNA in wild-type mice on induction of mild acute pancreatitis (d) (n = 3 per group) or on administration of a single dose of caerulein (e) (n ≥ 3 per group). f, Correlation of NR5A2 and NR0B2 mRNA expression in normal human pancreas using RNA-seq. g, ChIP–qPCR analysis of the occupancy by NR0B2 at the AP-1 (left) and inflammatory gene promoters (right panel) in wild-type and Nr5a2+/− mice. In the left and right panels, data are shown relative to control IgG and an unrelated genomic region (n = 3 per group). ChIP–qPCR analysis of NR0B2 on the promoter of AP-1 genes shows reduced occupancy in wild-type mice 1 h after administration of one dose of caerulein. Results in the middle panel are normalized to enrichment in wild-type mice (n ≥ 6 per group). h, ChIP–qPCR analysis of the occupancy of the Nr0b2 promoter by NR5A2 in wild-type and Nr5a2+/− mice. Data are shown relative to control IgG and an unrelated genomic region (n ≥ 5 per group). i, Co-immunoprecipitation of NR5A2 and NR0B2 in wild-type and Nr5a2+/− pancreases under basal conditions or 1 h after administration of a single dose of caerulein. Densitometric quantification of NR0B2 bands (right) (n = 3 per group). At least two independent experiments were performed. In a–i, one-sided Mann–Whitney U test; *P < 0.05, **P < 0.01.
Extended Data Figure 9 NR0B2 has an important role in the dynamic regulation of inflammatory genes by NR5A2.
a–c, Validation of 266-6 cells as a model for mechanistic studies. a, Dose-dependent effects of caerulein on ERK activation, AP-1 expression and NR0B2 expression shown using western blotting. b, qRT–PCR analysis showing caerulein-induced changes in expression of Nr5a2, AP-1 and inflammatory genes. c, ChIP–qPCR analysis showing changes in NR5A2 occupancy of the promoters of acinar (Ctrb1, Cpa and Nr0b2), Jun and inflammatory genes (C1qb, Ccl7 and Ccl8) 30 min after treatment with caerulein (4 independent experiments) These findings largely recapitulate the observations made in the mouse pancreas. d, Forced overexpression of NR0B2 leads to reduced expression of Jun mRNA but does not affect expression of inflammatory genes (four independent experiments). e, Effects of NR0B2 knockdown on NR5A2 binding to the promoter of acinar, Jun and inflammatory genes (three independent experiments). f, Combined NR5A2 knockdown and NR0B2 overexpression showing that higher levels of NR0B2 are associated with reduced expression of inflammatory gene transcripts, a situation that mimics normal pancreas under basal conditions in wild-type mice (four independent experiments). At least two independent experiments were performed. In c–f, one-sided Mann–Whitney U test; *P < 0.05, **P < 0.01.
Extended Data Figure 10 JUN is required for the overactivation of AP-1 that is observed in Nr5a2+/− mice during caerulein-mediated pancreatitis.
Immunohistochemical analysis of the expression of JUN, JUNB, JUND, FOS, FOSL1 and FOSL2 in the pancreas of control (Nr5a2+/+, Nr5a2+/− and Nr5a2+/+;JunΔP) and Nr5a2+/−;JunΔP mice 48 h after the initiation of pancreatitis (n = 4 per group). One experiment was performed. Arrowhead, acinus; arrow, mesothelial cell.
Supplementary information
Supplementary Figure 1
This file contains the original experimental plots data and the original western blot gel images. (PDF 1994 kb)
Supplementary Table 1
This file contains association of NR5A2 expression in PDAC with gender, body mass index, and prior medical history of diabetes and chronic pancreatitis. (PDF 83 kb)
Supplementary Figure 2-5
This file contains Supplementary Table 2: Gene Set Enrichment Analysis for the pre-ranked list of differentially expressed genes (DEG) (Nr5a2+/- vs. Nr5a2+/+) in basal conditions, Supplementary Table 3: Gene Set Enrichment Analysis for the genes up-regulated in Nr5a2+/- mice in basal conditions. Top-20 most significant gene sets when computing with the Molecular Signature tool of GSEA, using the Biological Processes dataset, Supplementary Table 4: Primers used for RT-qPCR and Supplementary Table 5: Primers used for ChIP-qPCR. (XLSX 19 kb)
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Cobo, I., Martinelli, P., Flández, M. et al. Transcriptional regulation by NR5A2 links differentiation and inflammation in the pancreas. Nature 554, 533–537 (2018). https://doi.org/10.1038/nature25751
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DOI: https://doi.org/10.1038/nature25751
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