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Extensive Pancreas Regeneration Following Acinar-Specific GASTROENTEROLOGY 2011;141:1463–1472 Extensive Pancreas Regeneration Following Acinar-Specific Disruption of Xbp1 in Mice DAVID A. HESS,* SEAN E. HUMPHREY,* JEFF ISHIBASHI,* BARBARA DAMSZ,* ANN–HWEE LEE,‡ LAURIE H. GLIMCHER,‡ and STEPHEN F. KONIECZNY* *Department of Biological Sciences and the Purdue Center for Cancer Research, Purdue University, West Lafayette, Indiana; and ‡Department of Immunology and Infectious Diseases, Harvard School of Public Health, and Department of Medicine, Harvard Medical School, Boston, Massachusetts and survival of acinar cells is the management of their See editorial on page 1155. extensive protein synthesis/processing machinery, which uses regulatory pathways involving the Golgi, intermediate BACKGROUND & AIMS: Progression of diseases of the transport vesicles, plasma membrane components, and the exocrine pancreas, which include pancreatitis and cancer, is endoplasmic reticulum (ER). Among these organelles, the associated with increased levels of cell stress. Pancreatic aci- ER ensures that newly synthesized secretory and transmem- nar cells are involved in development of these diseases and, brane proteins are properly folded and modified before ex- because of their high level of protein output, they require an portation. In cells with high protein synthesis demands,2,3 efficient, unfolded protein response (UPR) that mediates with specific differentiation requirements,2,4,5 or that are recovery from endoplasmic reticulum (ER) stress following subject to environmental or physiologic alterations, the abil- the accumulation of misfolded proteins. METHODS: To ity of the protein-folding machinery to handle the synthetic study recovery from ER stress in the exocrine organ, we load can reach an imbalance, setting in motion a series of generated mice with conditional disruption of Xbp1 (a prin- intracellular signaling pathways collectively known as the cipal component of the UPR) in most adult pancreatic aci- unfolded protein response (UPR).6–8 Activation of the UPR nar cells (Xbp1fl/fl). We monitored the effects of constitutive allows cells to adjust to high protein demands by transcription- ER stress in the exocrine pancreas of these mice. RESULTS: ally activating genes that increase protein-folding capacity. Xbp1-null acinar cells underwent extensive apoptosis, fol- The UPR consists of 3 distinct signaling arms (inositol- lowed by a rapid phase of recovery in the pancreas that requiring transmembrane kinase/endonuclease 1 [IRE1]/ included expansion of the centroacinar cell compartment, X-box binding protein 1 (XBP1), activating transcription formation of tubular complexes that contained Hes1- and factor 6 [ATF6], and protein kinase RNA-like ER kinase Sox9-expressing cells, and regeneration of acinar cells that [PERK]), each of which activates unique downstream target expressed Mist1 from the residual, surviving Xbp1ϩ cell genes and pathways (eg, XBP1–protein disulfide isomerase population. CONCLUSIONS: XBP1 is required for ho- [PDI], subunit of the Sec61 transport protein complex meostasis of acinar cells in mice; ER stress induces a [Sec61a]; ATF6-nATF6␣; PERK-p-eIF2␣).8 The ER inositol- regenerative response in the pancreas that involves aci- requiring transmembrane kinase/endonuclease 1 (IRE1) nar and centroacinar cells, providing the needed capacity branch activates multiple classes of molecules, includ- for organ recovery from exocrine pancreas disease. ing chaperones (PDI), protein transporters (Sec61a), and Keywords: Endoplasmic Reticulum Stress; Pancreatic Pro- growth/DNA damage regulators (GADD153–C/EBP-ho- genitor Cells; Protein Folding; Tissue Regeneration. mologous protein [CHOP]). IRE1 oligomerizes and auto- phosphorylates in response to unfolded proteins in the ER lumen, resulting in activation of its novel endoribo- he pancreas is a complex secretory organ tasked with nuclease domain. Activated IRE1 excises a 26-nucleotide Tendocrine-mediated maintenance of blood glucose levels and exocrine production of key enzymatic compo- fragment within the basal Xbp1 transcript, producing a frame shift and consequent translation of a 371–amino PANCREAS nents of the digestive system. Defects or damage to vari- BASIC AND ous pancreatic cells contribute to multiple disease states, TRANSLATIONAL including diabetes, pancreatitis, and pancreatic cancer, Abbreviations used in this paper: ATF6, activating transcription factor 6; the latter two being diseases associated with the exocrine ␤-gal, ␤-galactosidase; CHOP, C/EBP-homologous protein; CPA, carboxy- peptidase; ER, endoplasmic reticulum; IRE1, inositol-requiring transmem- pancreas. An understanding of the response of pancreatic brane kinase/endonuclease 1; PERK, protein kinase RNA-like ER kinase; tissues to damage and stress is of great interest as a means PDI, protein disulfide isomerase; Sec61a, subunit of the Sec61 transport of exploring the development of disease and as a potential protein complex; TM, tamoxifen; TUNEL, terminal deoxynucleotidyl trans- avenue for therapeutic intervention. ferase–mediated deoxyuridine triphosphate nick-end labeling; UPR, un- As professional secretory cells, pancreatic acinar cells are folded protein response; Xbp1, X-box binding protein 1; Xbp1s, Xbp1 spliced version; ZC, zymogenic cell; ZG, zymogen granule. responsible for the synthesis, storage, and secretion of vast © 2011 by the AGA Institute quantities of digestive hydrolases that assist in food diges- 0016-5085/$36.00 tion.1 One of the key requirements for the proper function doi:10.1053/j.gastro.2011.06.045 1464 HESS ET AL. GASTROENTEROLOGY Vol. 141, No. 4 acid XBP1s (“spliced”) transcription factor that functions ated secondary antibodies were applied for 10 minutes at to up-regulate UPR target genes.2,8 25oC. Visualization was accomplished via 3,3=-diaminobenzi- The high protein synthesis capacity of pancreatic acinar dine tetrahydrochloride peroxidase staining (Vector Labora- cells suggests that they likely rely on an efficient IRE1/ tories) or tertiary, avidin-conjugated fluorescent antibodies. Primary antibodies and conditions are provided in Supple- XBP1 pathway to maintain proper protein processing.9 In mentary Table 2. Terminal deoxynucleotidyl transferase–medi- support of this idea, Iwawaki et al have shown that the ated deoxyuridine triphosphate nick-end labeling (TUNEL) as- IRE1/XBP1 pathway is constitutively active in the exocrine says were performed following digestion in 250 ␮g/mL pancreas.10,11 Similarly, acinar cells undergoing acute pan- proteinase K in 2.5 mmol/L CaCl2, 10 mmol/L Tris-HCl, pH 7.5, creatitis exhibit elevated levels of ER stress accompanied for 1 minute at 25oC using the In Situ Death Detection Kit by activation of the Xbp1 splicing machinery.6,7,10 Indeed, (Roche, Indianapolis, IN). embryonic deletion in Xbp1Ϫ/Ϫ;LivXBP1 mice results in pancreata that are severely devoid of acinar cells, leading Electron Microscopy to neonatal death due to limited food digestion and Pancreata were fragmented and fixed in 3% paraformal- consequent hypoglycemia.3 XBP1 is also essential for dehyde/0.5% glutaraldehyde in phosphate-buffered saline and plasma cell differentiation2 and is critical for tumor an- then gradually dehydrated and embedded in Epon resin (EMS, giogenesis in pancreatic adenocarcinoma,11 revealing a Hatfield, PA). Electron micrographs were obtained from ultra- central role for this factor in many different biologic thin sections using a Philips transmission electron microscope (Philips, Andover, MA). contexts. Because XBP1 possesses additional functions in normal cell development2,4,12 outside ER stress mainte- Protein Immunoblot Assays nance, it has been difficult to assess the importance of the Twenty micrograms of whole cell protein extracts were IRE1/XBP1 pathway in the adult exocrine pancreas. To separated on 12% acrylamide gels, transferred to polyvinylidene circumvent this obstacle, we conditionally inactivated an difluoride membranes, and incubated with primary antibodies Xbp1fl/fl allele in adult acinar cells to address 2 key ques- (antibody conditions are provided in Supplementary Table 3). tions: (1) What is the fate of individual acinar cells un- Immunoblots were developed using an enhanced chemilumines- dergoing chronic ER stress in the absence of the IRE1/ cence kit (Pierce, Rockford, IL). XBP1 pathway? and (2) How does the exocrine organ cope with defective secretory cells? Our results show that loss Reverse-Transcription Quantitative of XBP1 function is lethal to mature acinar cells. However, Polymerase Chain Reaction Gene Expression Analysis the pancreas itself exhibits a remarkable ability to recover from chronic ER stress by eliciting a rapid regeneration Pancreas RNA was isolated using the RNeasy Isolation response from a minority of unrecombined Xbp1fl/fl acinar System (Qiagen, Valencia, CA) and reverse transcribed using the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). Comple- cells as well as from Hes1/Sox9-positive centroacinar pro- mentary DNA reactions were amplified with QPCR SYBR Green genitor cells. We conclude that the IRE1/XBP1 pathway is Mix (Abgene, Epsom, United Kingdom) as described in Supple- critical to maintaining normal ER homeostasis and that mentary Materials and Methods using the primer sets listed in exploiting ER stress represents a novel approach to gen- Supplementary Table 4. erating and studying pancreatic damage in vivo. Results Materials and Methods Acinar-Restricted Deletion of Xbp1 Mouse Strains and Genotyping To examine the importance of XBP1 and the UPR Mist1CreER/ϩ and Xbp1fl/fl
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