Bmi1 is required for regeneration of the exocrine pancreas in mice

Abstract

Background & aims: Bmi1 is a member of the Polycomb protein family and represses transcription by modifying chromatin organization at specific promoters. Bmi1 is implicated in the control of stem cell self-renewal and has been shown to regulate cell proliferation, tissue homeostasis, and differentiation. Bmi1 is present in a subpopulation of self-renewing pancreatic acinar cells and is expressed in response to pancreatic damage. We investigated the role of Bmi1 in regeneration of exocrine pancreas.

Methods: Acute pancreatitis was induced in Bmi1(-/-) mice with cerulein; pancreatic cell regeneration, differentiation, and apoptosis were assessed. Cultured Bmi1(-/-) and wild-type primary acini were analyzed in vitro to determine acinar-specific consequences of Bmi1 deletion. To investigate cell autonomous versus non-cell autonomous roles for Bmi1 in vivo, pancreatitis was induced in Bmi1(-/-) mice reconstituted with a wild-type hematopoietic system.

Results: Bmi1 expression was up-regulated in the exocrine pancreas during regeneration after cerulein-induced pancreatitis. Exocrine regeneration was impaired following administration of cerulein to Bmi1(-/-) mice. Pancreata of Bmi1(-/-) mice were hypoplastic, and the exocrine pancreas was replaced with ductal metaplasia that had increased apoptosis and decreased cell proliferation compared with that of wild-type mice. Expression of Cdkn2a and p53-dependent apoptotic genes was markedly up-regulated in Bmi1(-/-) pancreas compared with wild-type mice after injury. Furthermore, after transplantation of bone marrow from wild-type to Bmi1(-/-) mice, the chimeric mice had intermediate levels of pancreatic hypoplasia and significant but incomplete rescue of impaired exocrine regeneration after cerulein injury.

Conclusions: Bmi1 contributes to regeneration of the exocrine pancreas after cerulein-induced injury through cell autonomous mechanisms, in part by regulating Cdkn2a expression, and non-cell autonomous mechanisms.

Figure 1. Bmi1 expression is upregulated in the exocrine pancreas after caerulein injury

(A) Immunohistochemistry for Bmi1 in the pancreas in adult wild-type mice, 2 days post-PBS injection or 2 and 7 days post-caerulein injection. Inset, arrows indicate Bmi1-positive acinar cells. (B) Immunofluorescence for amylase, GFP, and DAPI in the pancreas of adult Bmi1^GFP/+ mice after PBS injection or 2 and 7 days post-caerulein injection. Arrows indicate GFP-positive acinar cells. (C) Immunofluorescence for DBA lectin, GFP, and DAPI in the pancreas of adult Bmi1^GFP/+ mice after PBS injection or 2 and 7 days post-caerulein injection. (D) Immunofluorescence for vimentin or desmin, GFP, and DAPI in the pancreas in adult Bmi1^GFP/+ mice after PBS injection or 2 and 7 days post-caerulein injection.

Figure 2. Impaired exocrine pancreas regeneration in Bmi1 KO mice after caerulein pancreatitis

Control or Bmi1 KO mice were injected with caerulein and sacrificed 7 days post-injection. (A) Macroscopic views of pancreas (outlined with dashed lines), showing hypoplastic pancreas in Bmi1 KO mice (red) compared to control mice (blue). (B) H&E staining showing impaired exocrine pancreas regeneration with increased duct-like structures and reduced acinar area in Bmi1 KO pancreas compared to control mice. (C) Co-staining for amylase/CK19/DAPI reveals reduced number of amylase-positive cells and increased number of CK19-positive duct-like epithelial cells in Bmi1 KO pancreas. (D) Immunostaining for Hes1, Sox9, and Pdx1. Immunohistochemistry shows Hes1 expression in the duct-like epithelial cells in Bmi1 KO pancreas 7 days post-injection. Co-staining for Sox9/DAPI demonstrates Sox9 expression in the duct-like epithelial cells in Bmi1 KO pancreas 7 days post-injection. Immunohistochemistry shows absence of Pdx1 expression in the duct-like epithelial cells in Bmi1 KO pancreas 7 days post-injection. Pdx1 remains expressed in control and mutant islets. (E) Relative expression levels of Sox9 and Hes1 in control and Bmi1 KO pancreata 7 days post-injection by q-PCR. N = 3 mice. (F) Relative expression levels of Ptf1a and Mist1 in control and Bmi1 KO pancreata 3 days post-injection. N = 3 mice. Means ± SD. * p< 0.05.

Figure 3. Stromal reaction and persistent inflammation in Bmi1 KO pancreas after caerulein pancreatitis

(A–B) Immunohistochemistry for desmin in control (A) and Bmi1 KO pancreata (B) 7 days after caerulein treatment. (C–D) Immunohistochemistry for phospho-Stat3 in control (C) and Bmi1 KO pancreata (D) 7 days after caerulein treatment. (E) Relative expression levels of IL-6 in control and Bmi1 KO pancreata by q-PCR analysis. N = 3 mice. Means ± SD. ***p< 0.001.

Figure 4. Increased apoptosis and decreased cell proliferation in Bmi1 KO pancreas after caerulein pancreatitis

(A) Co-staining for cleaved Caspase 3 (c-Caspase 3)/E-cadherin/DAPI in control and Bmi1 KO pancreata 3 days post caerulein injection. (B) Co-staining for TUNEL/E-cadherin/DAPI in contro l and Bmi1 KO pancreata 3 days post caerulein injection. (C) Quantification of the number of Cleaved Caspase 3/E-cadherin double positive cells per field in control and Bmi1 KO pancreata 3 days and 7 days post-injection. N = 3 mice. (D) Co-staining for phospho-histone H3 (PHH3)/E-cadherin/DAPI in control and Bmi1 KO pancreata 3 days post caerulein injection. (E) Quantification of the number of PHH3/E-cadherin double positive cells per field in control and Bmi1 KO pancreata 3 days and 7 days post caerulein injection. N = 3 mice. Means ± SD. ** p < 0.01.

Figure 5. p16, p19 and p53 apoptotic pathways are upregulated in Bmi1 KO pancreata after caerulein injury

(A) Relative expression levels of Cdkn2a, p16 and p19 in the pancreas by q-PCR analyses. N = 3 mice. Means ± SD (B) Co-staining for p16/Sox9 in control and Bmi1 KO pancreata 3 days post-injection. The merged image shows increased p16 expression in Sox9-positive duct-like epithelial cells in Bmi1 KO mice 3 days post caerulein injection. (C) Co-staining for phospho-Rb/DAPI in control and Bmi1 KO pancreata 3 days post caerulein injection. (D) Immunohistochemistry for p53 in control and Bmi1 KO pancreata 3 days post caerulein injection. (E) Relative expression levels of Noxa, Apaf1, Bax and p21 in the pancreas by q-PCR analyses. N = 3 mice. Means ± SD. * p < 0.05,** p < 0.01, and ***p< 0.001.

Figure 6. Hypoplastic pancreas and impaired exocrine pancreas regeneration are mediated through a combination of cell autonomous and non-cell autonomous mechanisms in Bmi1 KO mice after caerulein pancreatitis

(A) Experimental outline. Wild-type bone marrow was transplanted into irradiated wild-type or Bmi1 KO mice at 6–8 weeks. 8 weeks after bone marrow transplantation, reconstituted Bmi1 KO chimera and wild type mice were subjected to caerulein pancreatitis and were sacrificed 7 days after treatment. (B) Macroscopic view of the pancreas reveals moderate pancreatic hypoplasia in reconstituted Bmi1 KO chimera mice 7 days after caerulein treatment. H&E stain and Co-staining for amylase/CK19 show exocrine pancreas regeneration is partially impaired in reconstituted Bmi1 KO chimeric mice 7 days after caerulein treatment. (C) Relative pancreatic weight normalized to body weight. N = 3 to 6 mice. Means ± SD. Quantification of acinar regeneration. N = 3 or 4 mice. Means ± SD. Co-staining for cleaved Caspase 3/E-cadherin/DAPI in reconstituted Bmi1 KO chimeric mice reveals presence of apoptotic cells 7 days after caerulein treatment. (D) Relative expression levels of Cdkn2a, Noxa, and Apaf1 in the pancreas by q-PCR analysis. N = 3 or 4 mice. Means ± SD. *p < 0.05, ** p < 0.01. N.P. = not significant p value.

Figure 7. Cdkn2a and Noxa expression is upregulated in a cell-autonomous manner in Bmi1 null cultured acini treated with caerulein in vitro

(A–B) Immunohistochemistry for amylase and DAPI in control (A) and Bmi1 KO cultured acini (B) in vitro. (C–D) Immunohistochemistry for Bmi1 in control (C) and Bmi1 KO cultured acini (D) in vitro. (E) Relative expression levels of Cdkn2a and Noxa in control or Bmi1 null cultured acini treated with BSA or caerulein in vitro by q-PCR analyses. N = 3. Means ± SD. *p < 0.05 and ***p < 0.001.