Nr5a2 maintains acinar cell differentiation and constrains oncogenic Kras-mediated pancreatic neoplastic initiation - PubMed (original) (raw)

Nr5a2 maintains acinar cell differentiation and constrains oncogenic Kras-mediated pancreatic neoplastic initiation

Guido von Figura et al. Gut. 2014 Apr.

Abstract

Objectives: Emerging evidence from mouse models suggests that mutant Kras can drive the development of pancreatic ductal adenocarcinoma (PDA) precursors from acinar cells by enforcing ductal de-differentiation at the expense of acinar identity. Recently, human genome-wide association studies have identified NR5A2, a key regulator of acinar function, as a susceptibility locus for human PDA. We investigated the role of Nr5a2 in exocrine maintenance, regeneration and Kras driven neoplasia.

Design: To investigate the function of Nr5a2 in the pancreas, we generated mice with conditional pancreatic Nr5a2 deletion (PdxCre(late); Nr5a2(c/c)). Using this model, we evaluated acinar differentiation, regeneration after caerulein pancreatitis and Kras driven pancreatic neoplasia in the setting of Nr5a2 deletion.

Results: We show that Nr5a2 is not required for the development of the pancreatic acinar lineage but is important for maintenance of acinar identity. Nr5a2 deletion leads to destabilisation of the mature acinar differentiation state, acinar to ductal metaplasia and loss of regenerative capacity following acute caerulein pancreatitis. Loss of Nr5a2 also dramatically accelerates the development of oncogenic Kras driven acinar to ductal metaplasia and PDA precursor lesions.

Conclusions: Nr5a2 is a key regulator of acinar plasticity. It is required for maintenance of acinar identity and re-establishing acinar fate during regeneration. Nr5a2 also constrains pancreatic neoplasia driven by oncogenic Kras, providing functional evidence supporting a potential role as a susceptibility gene for human PDA.

Keywords: PANCREATIC CANCER; PANCREATIC DAMAGE; PANCREATITIS.

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Conflict of interest statement

Competing interests None.

Figures

Figure 1

Figure 1

Nr5a2 is downregulated in acini expressing oncogenic Kras that undergo acinar to ductal metaplasia (ADM)/pancreatic intraepithelial neoplasia (PanIN) lesion formation and in pancreatic ductal adenocarcinoma (PDA). (A) Nr5a2 staining on pancreata at different time points (day 2 and 7) after pancreatitis induction or PBS injection of Ptf1aCre; KrasG12D mice; arrows mark Nr5a2 positive cells, arrowheads mark Nr5a2 negative cells. Upper panel: Immunohistochemistry staining for Nr5a2. Lower panel: Immunofluorescent co-staining of acinar tissue or ADM/PanIN lesions for CPA1, Clusterin and Nr5a2. Clusterin positive persistent ADM and early PanIN lesions exhibit strong reduction of nuclear Nr5a2. Note that the specific Nr5a2 staining is nuclear, whereas cytoplasmic staining is unspecific background. (B) Nr5a2 staining on pancreata 7 days after caerulein induction or PBS injection of ElastaseCreERT2; KrasG12D; R26REYFP mice. Depicted is an YFP positive PanIN lesion lacking nuclear Nr5a2, which occurred 7 days after caerulein induction; arrows mark Nr5a2 positive cells, arrowheads mark Nr5a2 negative cells. (C) RNA expression of Nr5a2 during indicated time points after pancreatitis induction in Ptf1aCre; KrasG12D mice. p Values are relative to PBS treated animals; values are shown as mean±SEM. (D) Nr5a2 staining of a PDA derived from a Ptf1aCre; KrasG12D mouse. (A) Upper panel: scale bar 50 μm. Lower panel: scale bar 10 μm. (B) Scale bar 10 μm. (D) Scale Bar 500 μm.

Figure 2

Figure 2

Nr5a2 is essential for proper maintenance of acinar cell differentiation. (A) H&E staining and (B) immunofluorescence staining for Nr5a2 and CPA1 (marks acinar cells). (C) Immunofluorescence staining for acinar (CPA1), ductal (CK19) and β cells insulin (INS) of pancreata of the indicated genotypes at 3 weeks of age (arrow marks ductal metaplasia). (D) Pancreas weight to body weight ratio; p values are compared with control, values are shown as mean±SEM. (E) Real-time PCR (RT-PCR) for Nr5a2 relative to Cyclophilin A; p values represent comparison with control mice, values are shown as mean±SEM. All RT-PCR analyses in this figure were performed on 3-week-old animals. (A) Scale bar 100 μm, (B) scale bar 10 μm and (C) scale bar 50 μm.

Figure 3

Figure 3

Loss of Nr5a2 compromises acinar differentiation. (A) Real-time PCR for transcriptions factors and markers of acinar differentiation (Gata6, Hnf1a, Ptf1a, Mist1 and CPA1) and the duct gene Ck19; p values represent comparison with control mice; values are shown as mean±SEM. (B) Acinar culture of isolated acinar clusters; representative picture of acinar culture at day 2 and CPA1/CK19 co-staining; scale bar 25 μm. (C) Quantification of acinar clusters; p values are compared with acini from control mice for each time point, values are shown as mean±SEM. All analyses in this figure were performed on 3-week-old animals.

Figure 4

Figure 4

Loss of Nr5a2 blocks pancreas regeneration. (A) Representative H&E staining at the indicated time points after caerulein-induced acute pancreatitis of control and PdxCrelate; Nr5a2c/c mice. (B) Immunofluorescent co-staining for CPA1 and CK19 and (C) pancreas weight to body weight ratio (n=3 per group) during the time course of pancreatitis of the indicated genotypes; values are shown as mean±SEM. p Values are calculated comparing control and PdxCrelate; Nr5a2c/c mice of each time point. (A) Scale bar 100 μm and (B) scale bar 50 μm.

Figure 5

Figure 5

Loss of Nr5a2 accelerates oncogenic Kras driven neoplastic initiation. (A) H&E staining and immunofluorescence co-staining for CPA1/CK19/insulin/DAPI and for Clusterin/Sox9/DAPI, and immunohistochemical staining for pMAPK and pStat3 in 3-week-old mice of the indicated genotypes. (B) Alcian blue staining in 3-week-old mice of the indicated genotypes. (C) H&E staining and immunofluorescence co-staining for CPA1/CK19/DAPI and YFP/Clusterin/DAPI of the indicated genotypes 2 weeks after tamoxifen induction. (A) H&E staining: scale bar 500 μm; immunofluorescent/immunohistochemical staining: scale bar 50 μM, (B) scale bar 500 μm and (C) scale bar for H&E 500 μm and for immunofluorescence stainings 50 μm.

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