Stabilization of HIF-2α impacts pancreas growth

Abstract

Hypoxia inducible factors (HIFs) are critical regulators of the response to oxygen deficiency by activating target genes involved in a variety of biological functions. HIFs have been implicated in the pathophysiology of numerous pathologies including cancer. Patients with mutations in the von Hippel-Lindau (VHL) gene, an essential regulator of HIF activity, develop tumors in several organs including the pancreas. Previous functional studies of HIF activation in the pancreas have used Vhlh (the murine homolog of VHL) deficient mice. However, the role of each specific HIF transcription factors in the pancreas has not been thoroughly examined. We derived mice that constitutively express a normoxia-stable form of HIF2α in the pancreas. Activation of HIF2α in the pancreas severely impairs postnatal exocrine pancreas. Mice with pancreas-specific activation of HIF2α develop histological features reminiscent of pancreatitis including loss of acinar cells, ductal dilation and fibrosis. Moreover, we provide evidence that signaling pathways important for acinar cell homeostasis are altered in HIF2α-overexpressing pancreata.

Figure 1

HIF2α stabilization results in exocrine cell atrophy and expansion of duct-like tubular structures. (A) Body weight (left panel), pancreas weight (middle panel) and body/pancreas weight ratio (right panel) in Pdx1-Cre;HIF2dPA and control mice at 2 and 8 weeks of age. Data are presented as mean ± SD. (B) HIF2α accumulation in Pdx1-Cre;HIF2dPA analyzed by Western blot with anti-HA antibody. Two independent two-week-old control and mutant mice are shown. ß-actin protein was used for loading control. Full-length blots are presented in Supplementary Fig. 2. (D) Immunofluorescence analysis of HIF2α in two-week-old control pancreata. Endogenous HIF2α expression is observed in islets (marked by an white asterisk) but not in exocrine tissue. (D) Robust HIF2α accumulation in the pancreas of two-week-old Pdx1-Cre;HIF2dPA mice. Hematoxylin/Eosin-stained pancreatic sections from P0 (E,F), two- (I,J) and eight-week-old (M,N) Pdx1-Cre;HIF2dPA and control mice. Inset in N shows an area with adipose tissue in Pdx1-Cre;HIF2dPA pancreata. Immunofluorescence of amylase and KRT19 shows no differences between Pdx1-Cre;HIF2dPA and control mice at P0 (G,H). Duct-like tubular structures and loss of amylase immunoreactivity in two- (K,L) and eight-week-old (O,P) Pdx1-Cre;HIF2dPA mice compared to control mice. Note areas with normal acini in 8-week-old Pdx1-Cre;HIF2dPA mice (white asterisk in O). Insets in (H,L and P) show higher magnification pictures. DAPI staining is shown in blue in (C,D,G,H,K,L,O and P). Scale bars = 50 µm for (C,D); 100 µm for (E–P). ***P < 0.001.

Figure 2

HIF2α stabilization causes pancreatic abnormalities reminiscent of pancreatitis. Gomori trichome staining reveals increased connective tissue (stained in blue) in Pdx1-Cre;HIF2dPA pancreata (B) compared to control pancreata (A) at 8 weeks of age. Increase in smooth muscle actin (SMA) positive cells (white arrowhead) in Pdx1-Cre;HIF2dPA pancreata (D) compared to control pancreata (C) at 8 weeks of age. Ducts are marked by staining with lectin Dolichos biflorus agglutinin (DBA). Increase of the mesenchymal marker vimentin in Pdx1-Cre;HIF2dPA pancreata (F, black arrowheads) at 8 weeks of age. Only a few scattered cells are positive for vimentin in control pancreata (E, black arrowhead). (G) Quantification of vimentin-positive area per total pancreatic area. CD11b immunohistochemistry in control (H) and Pdx1-Cre;HIF2dPA pancreata (I, black arrowheads) at eight weeks of age. (J) Quantification of CD11b-positive cells per field. F4/80 immunohistochemistry in control (K) and Pdx1-Cre;HIF2dPA pancreata (L, black arrowheads) at eight weeks of age. (M) Quantification of F4/80-positive cells per field. Data points represent values for each individual mouse. The mean value is indicated as a horizontal line. Scale bars = 50 µm. **P < 0.01; ***P < 0.001.

Figure 3

Increased acinar cell apoptosis and proliferation in HIF2α-overexpressing pancreata. (A,B) TUNEL staining shows an increase in apoptotic acinar cells in the pancreas of two-week-old Pdx1-Cre;HIF2dPA mice (B, white arrowheads) compared with wild-type (A) mice. (C) Quantification of apoptotic cells measured as TUNEL positive-cells per total number of amylase-positive cells. Individual data points are presented. The mean value is indicated as a horizontal line. (D,E) Immunohistochemistry for cleaved caspase-3 confirms the increase in apoptotic cells in Pdx1-Cre;HIF2dPA pancreata (E, black arrowheads) compared to control pancreata (D). Increased acinar cell proliferation in two-week-old Pdx1-Cre;HIF2dPA pancreata (G) compared to control pancreata (F), as shown by double amylase/Ki-67 immunofluorescence. Increased ductal cell proliferation in two-week-old Pdx1-Cre;HIF2dPA pancreata (I) compared to control pancreata (H), as shown by double KRT19/Ki-67 immunofluorescence. (J) Quantification of acinar (left graph) and ductal (right graph) proliferating cells, measured as the average number of Ki67-positive cells per amylase- or KRT19-positive cells. Data points represent values for each individual mouse. The mean value is indicated as a horizontal line. Scale bars = 50 µm for A,B,D and E; 100 µm for F–I. **P < 0.01; ***P < 0.001.

Figure 4

Acini disorganization in HIF2α-overexpressing mice. No changes in the expression or localization of laminin (A,B), β-catenin (C,D), and E-cadherin (E–L) between acini of two-week-old Pdx1-Cre;HIF2dPA and control mice. Carboxypeptidase A1 (CPA1) is localized close to the lumen of control acini (E). CPA1 does not show a clear apical localization in Pdx1-Cre;HIF2dPA acinar cells (F). Intracellular accumulation of mucin-1 (H), and PKCε (J), in Pdx1-Cre;HIF2dPA acini while markers are normally localized at the apical region in control acini (G,I). Zonula occludens 1 (ZO-1) immunofluorescence show lumen dilation in Pdx1-Cre;HIF2dPA (L) acini compared to control acini (K). Nuclei are stained with DAPI (blue). Individual acini are outlined in yellow. Scale bars = 20 µm.

Figure 5

Increased ductal-like structures and vascularization in HIF2α-overexpressing mice. Staining for Alcian Blue/nuclear fast red (NFR) (A,B) and PAS (C,D) in Pdx1-Cre;HIF2dPA (B,D) and control pancreata (A,C) at 2 weeks of age. (E,F) Primary cilia, marked by acetylated tubulin (Ac-Tub, white arrowheads) are present in pancreatic ducts of control mice (E) and duct-like tubular structures of Pdx1-Cre;HIF2dPA mice (F). Increased vascularization (marked by MECA32 immunostaining) in two-week-old Pdx1-Cre;HIF2dPA pancreata (I,J) compared to control pancreata (G,H). Note MECA32-positive endothelial cells surrounding duct-like structures in Pdx1-Cre;HIF2dPA pancreas (J). (K) qPCR analysis of VEGF expression in two-week-old pancreata. Data points represent values for each individual mouse. The mean value is indicated as a horizontal line. In two-week-old control pancreata, Sox9 is expressed in scattered centroacinar cells (L, white arrowhead). Strong Sox9 expression is found in disorganized acini (M, yellow arrowhead) as well as in duct-like tubular structures (M, asterisk). Nuclei are stained with DAPI (blue) in (E–J and L,M). Scale bars = 50 µm for (A–D,G,I,L and M); 20 µm for (E,F,H and J). *P < 0.05.

Figure 6

Altered components of MAPK and AKT/mTOR signaling pahtways in HIF2α-overexpressing pancreata. (A) Periacinar cells positive for phosphorylated-ERK (pERK) are found in 2-week-old control pancreata but acinar cells are negative for pERK (white arrowheads, top panels). Increased pERK accumulation is observed in 2-week-old Pdx1-Cre;HIF2dPA pancreata (yellow arrowheads, bottom panels). Note that the increased expression was limited to areas with acinar-ductal metaplasia (yellow arrowheads, bottom panels). Acini with preserved morphology do not show extensive pERK accumulation (white arrowheads, bottom panels). pERK-positive stromal cells are also observed in Pdx1-Cre;HIF2dPA pancreata (asterisks, bottom panels). E-cadherin immunofluorescence was performed to visualize the pancreatic epithelium. (B) Double immunofluorescence analysis of phosphorylated RSP6 (pRSP6) and amylase in 2-week-old mice. pRPS6 (yellow arrows) is detected in amylase-positive acinar cells of control pancreata (top panels). In Pdx1-Cre;HIF2dPA pancreata, pRPS6 is detected in well-preserved acini (bottom panels, yellows arrows). However, areas with acinar-ductal metaplasia display decreased expression of pRPS6 (white arrows). (C) Double immunofluorescence analysis of pRSP6 and KRT19 in 2-week-old mice. Ductal cells (white arrows) are not positive for pRPS6 in both control pancreata (top panels) and Pdx1-Cre;HIF2dPA pancreata (bottom panels). (D) Clusterin is not expressed in control pancreas (top panels). Areas with acinar-ductal metaplasia in Pdx1-Cre;HIF2dPA pancreata display decreased expression of pRPS6 and activation of clusterin expression (white arrows). Yellow arrows indicate acini with normal morphology. Nuclei are stained with DAPI (blue). Scale bars = 50 µm.