2646 Diabetes Volume 66, October 2017 SMAD3/Stat3 Signaling Mediates b-Cell Epithelial- Mesenchymal Transition in Chronic Pancreatitis–Related Diabetes Xiangwei Xiao,1 Shane Fischbach,1 Tina Zhang,1,2 Congde Chen,1 Qingfeng Sheng,1 Ray Zimmerman,1 Sneha Patnaik,1 Joseph Fusco,1 Yungching Ming,1 Ping Guo,1 Chiyo Shiota,1 Krishna Prasadan,1 Nupur Gangopadhyay,1 Sohail Z. Husain,3 Henry Dong,2 and George K. Gittes1 Diabetes 2017;66:2646–2658 | https://doi.org/10.2337/db17-0537 Many patients with chronic pancreatitis develop diabetes Many patients with chronic pancreatitis develop insulinopenia, (chronic pancreatitis–related diabetes [CPRD]) through an glucose intolerance, insulin resistance, and eventually diabetes undetermined mechanism. Here we used long-term par- (2), largely as a result of the intimate proximity of the tial pancreatic duct ligation (PDL) as a model to study CPRD. endocrine pancreas to the exocrine pancreas (3). Moreover, We found that long-term PDL induced significant b-cell de- patients with chronic pancreatitis often develop a fibrotic differentiation, followed by a time-dependent decrease in pancreas with a reduced b-cell mass and have a 15- to b — fi functional -cell mass all speci cally in the ligated tail 16-fold increased risk for pancreatic cancer (4). To date, the portion of the pancreas (PDL-tail). High levels of transform- understanding of the development and pathogenesis of b b ing growth factor 1(TGF 1) were detected in the PDL-tail chronic pancreatitis–relateddiabetes(CPRD)isverylimited. and were mainly produced by M2 macrophages at the In addition, the mechanisms of b-cell loss in CPRD may in early stage and by activated myofibroblasts at the later part be similar to those in type 2 diabetes (T2D) (5,6) and in stage. Loss of b-cell mass was then found to result from ISLET STUDIES cystic fibrosis (7). Thus, elucidation of the underlying mech- TGFb1-triggered epithelial-mesenchymal transition (EMT) by b-cells, rather than resulting directly from b-cell apo- anisms common to chronic pancreatitis, CPRD, and pancre- ptosis. Mechanistically, TGFb1-treated b-cells activated atic cancer is critical. expression of the EMT regulator gene Snail in a SMAD3/ Among animal models for acute and chronic pancreatitis Stat3-dependent manner. Moreover, forced expression of (8), partial pancreatic duct ligation (PDL) has been used to forkhead box protein O1 (FoxO1), an antagonist for acti- generate chronic pancreatitis in mammals (9,10). Ligation vated Stat3, specifically in b-cells ameliorated b-cell EMT of the pancreatic duct immediately at the beginning of the and b-cell loss and prevented the onset of diabetes in splenic or tail part of the pancreas blocks the drainage of duc- mice undergoing PDL. Together, our data suggest that tal fluid from the distal pancreas, resulting in autodigestion chronic pancreatitis may trigger TGFb1-mediated b-cell of acinar cells and severe inflammation specifically in the EMT to lead to CPRD, which could substantially be pre- ligated tail of the pancreas, although initially the islets are vented by sustained expression of FoxO1 in b-cells. largely unaffected. Acinar cell death in the tail of the pancreas leads to the complete loss of acinar cells, without significant acinarcellregeneration(11).Onthecontrary,thenonligated The prevalence of chronic pancreatitis is roughly 50 per part, or head, of the pancreas appears to be normal, thus of- 100,000 people worldwide (1). Chronic pancreatitis in the fering an excellent internal control (12,13). We recently re- United States results in more than 122,000 outpatient vis- ported an inflammatory molecular signature in PDL, which its and more than 56,000 hospitalizations each year (2). induced b-cell proliferation in a transforming growth factor 1Division of Pediatric Surgery, Department of Surgery, Children’s Hospital of Received 7 May 2017 and accepted 27 July 2017. Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA This article contains Supplementary Data online at http://diabetes 2 ’ Division of Immunogenetics, Department of Pediatrics, Children s Hospital of .diabetesjournals.org/lookup/suppl/doi:10.2337/db17-0537/-/DC1. Pittsburgh, University of Pittsburgh School of Medicine, Pittsburgh, PA © 2017 by the American Diabetes Association. Readers may use this article as 3Department of Pediatrics, Children’s Hospital of Pittsburgh, University of Pitts- long as the work is properly cited, the use is educational and not for profit, and the burgh School of Medicine, Pittsburgh, PA work is not altered. More information is available at http://www.diabetesjournals Corresponding author: Xiangwei Xiao, [email protected], or George K. .org/content/license. Gittes, [email protected]. diabetes.diabetesjournals.org Xiao and Associates 2647 b (TGFb) receptor signaling–dependent manner (12–15). In Vitro Culture and Treatment of Mouse Islets As a strong stimulant of epithelial-mesenchymal transition Mouse islets were cultured in Ham’s F10 medium (Life Tech- (EMT) in epithelial cells (16–18), TGFb1 is highly upregulated nologies, St. Louis, MO) supplemented with 0.5% BSA (Sigma- in the ligated tail of the pancreas after PDL (PDL-tail) (14). Aldrich, St. Louis, MO), 2 mmol/L glutamine, 2 mmol/L Thus we were prompted to evaluate the effects of PDL- calcium, and 5 mmol/L glucose at 37°C in 95% air and 5% induced TGFb1ontheEMTofb-cells. CO2. After overnight culture, islets were either treated or Forkhead box protein O1 (FoxO1) is a pivotal factor in not treated with 20 ng/mL TGFb1 (with or without SMAD3 orchestrating the response of b-cell mass and function to and Stat3 inhibitors) and harvested after 0, 0.5, 1, 3, 12, overnutrition and obesity (19) and to oxidative stress and48h.TheSMAD3inhibitorSIS3(2mmol/L) or Stat3 (20–22). FoxO1 is predominantly expressed by b-cells in inhibitor cryptotanshinone (CTSN; 5 mmol/L) or control the adult pancreas. We and others have shown that FoxO1 DMSO solution was added with the TGFb1. nuclear translocation increases NeuroD1, MafA, and Nkx6.1 Western Blotting expression in b-cells, contributing to the maintenance of a Western blotting was performed as previously described functional differentiated phenotype to resist stress-induced (14,30). Primary antibodies for Western blotting are rabbit dedifferentiation, dysfunction, and failure (23–25). Never- polyclonal anti-MafA (Abcam), anti-GAPDH, anti-Snail, anti- theless, a role for FoxO1 during the pathogenesis of CPRD Slug, anti-Twist, anti-ZEB1, anti-ZEB2, anti-SMAD3, anti- is unknown. pSMAD3, anti-Stat3, anti-pStat3, anti-NeuroD1, anti-Nkx6.1, Here we studied long-term PDL (12–24 weeks) as a anti-Pdx1, and anti–epithelial cadherin (E-cadherin), all from model of chronic pancreatitis in humans. We further ana- Cell Signaling Technologies, San Jose, CA). The secondary lyzed the molecular mechanisms underlying the gradual antibody was horseradish peroxidase–conjugated antirabbit b-cell loss in this model, which mimics CPRD in humans. (Dako, Carpinteria, CA). RESEARCH DESIGN AND METHODS Virus Production AAV serotype 8 vectors were used to generate AAV-RIP-null Mouse Manipulation and AAV-RIP-FoxO1 viruses, as described before (25). All mouse experiments were approved by the Animal Re- search and Care Committee at the Children’s Hospital of Isolation of RNA and Quantitative RT-PCR Pittsburgh and the University of Pittsburgh Institutional An- RNA extraction and quantitative RT-PCR have been de- imal Care and Use Committee. BAC transgenic rat insulin scribed previously (12,13,27). The following primers all were promoter (RIP) Cre reporter (RIP-Cre) mice, MIP-GFP mice purchased from Qiagen (Valencia, CA): CycloA (QT00247709), (green fluorescent protein reporter under the control of a FoxO1 (QT00116186), Pdx1 (QT00102235), NeuroD1 mouse insulin promoter), and Rosa26CAG-mTmG (mTmG) (QT00251265), MafA (QT01037638), Nkx6.1 (QT00143318), mice have been described before (13). These mice and TGFb1 (QT00145250), vimentin (QT00159670), a-SMA C57BL/6 mice were all purchased from The Jackson Labo- (QT00140119), F4/80 (QT00099617), collagen I (QT00 ratory (Bar Harbor, ME). TGFb receptor II (TBR2) fx/fx 162204), E-cadherin (QT00121163), Snail (QT00240940), mice were generous gifts from Professor Stefan Karlsson Slug (QT00098273), ZEB1 (QT00105385), ZEB2 (QT00148 (University of Lund, Sweden) and have been described pre- 995), and fibronection (QT00135758). Quantitative RT-PCR viously (12). All mice were 10-week-old males and had a values were normalized against CycloA, which proved to be C57BL/6 background. PDL was performed and validated as stable across the samples. Fold changes from the control are described elsewhere (12–15). Intraductal viral infusion was showninthefigures. performed as described previously (26). Adeno-associated Histology and Immunohistochemistry virus (AAV) 8 viruses (titration of 1012 genome copy All pancreas samples were fixed and cryoprotected in 30% particles/mL in a 150-mL volume) were delivered via a cath- sucrose overnight before freezing, as described before (12). eter at a rate of 6 mL/min. BrdU-supplemented drinking Masson trichrome staining was performed using a Trichrome water was given to mice 1 week before the analysis, as Stain (Masson) Kit (Sigma-Aldrich). The fluorescent membrane- previously described (12). targeted Tomato (mT) and membrane-targeted EGFP (mG) Pancreatic Digestion, Islet Isolation, and FACS were detected by direct fluorescence. Primary antibodies for for b-Cells immunostaining were guinea pig polyclonal anti-insulin Pancreas digestion, islet isolation, and b-cell purification (Dako), rabbit polyclonal anti-F4/80 (Invitrogen, CA, Carls- from MIP-GFP mice have been described previously, taking bad), goat polyclonal antiamylase (Santa Cruz Biotechnol- advantage of the specific expression of green fluorescent ogy, Dallas, TX), rabbit polyclonal anti-FoxO1 (made by protein on b-cells (12–14,27–29). Dissociated pancreatic H.D.), goat polyclonal anti-E-cadherin (R&D Systems, Los cells were sorted based on a-smooth muscle actin (SMA) pos- Angeles, CA), rabbit polyclonal anti-a-SMA (Abcam), and itivity after the cells were fixed in 4% formalin and then rat polyclonal anti-CD45 (BD Biosciences).