Role of Cadherin-mediated Cell-Cell Adhesion in Pancreatic Exocrine-to-Endocrine Transdifferentiation
2008; Elsevier BV; Volume: 283; Issue: 20 Linguagem: Inglês
10.1074/jbc.m710034200
ISSN1083-351X
AutoresKohtaro Minami, Hirotoshi Okano, Akinori Okumachi, Susumu Seino,
Tópico(s)Diabetes and associated disorders
ResumoAlthough pancreatic exocrine acinar cells have the potential to transdifferentiate into pancreatic endocrine cells, the mechanisms are poorly understood. Here we report that intracellular signaling pathways, including those involving MAPK and phosphatidylinositol 3 (PI3)-kinase, are activated by enzymatic dissociation of pancreatic acinar cells and that spherical cell clusters are formed by cadherin-mediated cell-cell adhesion during transdifferentiation. Inhibition of PI3-kinase by LY294002 prevents spheroid formation by degrading E-cadherin and β-catenin, blocking transdifferentiation into insulin-secreting cells. In addition, neutralizing antibody against E-cadherin suppresses the induction of genes characteristic of pancreatic β-cells. We also show that loss of cadherin-mediated cell-cell adhesion induces and maintains a dedifferentiated state in isolated pancreatic acinar cells. Thus, disruption and remodeling of cadherin-mediated cell-cell adhesion is critical in pancreatic exocrine-to-endocrine transdifferentiation, in which the PI3-kinase pathway plays an essential role. Although pancreatic exocrine acinar cells have the potential to transdifferentiate into pancreatic endocrine cells, the mechanisms are poorly understood. Here we report that intracellular signaling pathways, including those involving MAPK and phosphatidylinositol 3 (PI3)-kinase, are activated by enzymatic dissociation of pancreatic acinar cells and that spherical cell clusters are formed by cadherin-mediated cell-cell adhesion during transdifferentiation. Inhibition of PI3-kinase by LY294002 prevents spheroid formation by degrading E-cadherin and β-catenin, blocking transdifferentiation into insulin-secreting cells. In addition, neutralizing antibody against E-cadherin suppresses the induction of genes characteristic of pancreatic β-cells. We also show that loss of cadherin-mediated cell-cell adhesion induces and maintains a dedifferentiated state in isolated pancreatic acinar cells. Thus, disruption and remodeling of cadherin-mediated cell-cell adhesion is critical in pancreatic exocrine-to-endocrine transdifferentiation, in which the PI3-kinase pathway plays an essential role. Although it has long been thought that the terminally differentiated state of cells is fixed, accumulating evidence has demonstrated that even functionally differentiated cells in adults can change their phenotype under certain conditions (1Tosh D. Slack J.M. Nat. Rev. Mol. Cell. Biol. 2002; 3: 187-194Crossref PubMed Scopus (365) Google Scholar, 2Eguchi G. Kodama R. Curr. Opin. Cell Biol. 1993; 5: 1023-1028Crossref PubMed Scopus (194) Google Scholar, 3Tsonis P.A. Washabaugh C.H. Del Rio-Tsonis K. Semin. Cell Biol. 1995; 6: 127-135Crossref PubMed Scopus (20) Google Scholar, 4Bouwens L. Microsc. Res. Tech. 1998; 43: 332-336Crossref PubMed Scopus (140) Google Scholar). Transdifferentiation, the conversion of one already differentiated cell type to another, is a prominent example of phenotypic plasticity of adult cells. In general, such phenotypic changes occur in tissues with chronic damage and in situations of tissue regeneration (2Eguchi G. Kodama R. Curr. Opin. Cell Biol. 1993; 5: 1023-1028Crossref PubMed Scopus (194) Google Scholar, 3Tsonis P.A. Washabaugh C.H. Del Rio-Tsonis K. Semin. Cell Biol. 1995; 6: 127-135Crossref PubMed Scopus (20) Google Scholar, 4Bouwens L. Microsc. Res. Tech. 1998; 43: 332-336Crossref PubMed Scopus (140) Google Scholar, 5Shen C.N. Slack J.M. Tosh D. Nat. Cell Biol. 2000; 2: 879-887Crossref PubMed Scopus (365) Google Scholar). However, pancreas is the organ in which metaplasia, a pathological phenomenon involving transdifferentiation (6Slack J.M. Tosh D. Curr. Opin. Genet. Dev. 2001; 11: 581-586Crossref PubMed Scopus (158) Google Scholar), frequently occurs. In addition, hepatocyte-like cells (liver metaplasia) appear in human pancreatic cancer in some cases (7Yeung R.S. Weese J.L. Hoffman J.P. Solin L.J. Paul A.R. Engstrom P.F. Litwin S. Kowalyshyn M.J. Eisenberg B.L. Cancer. 1993; 72: 2124-2133Crossref PubMed Scopus (165) Google Scholar), and experimental conditions such as copper depletion can lead to the development of pancreatic hepatocytes in rodents (8Rao M.S. Dwivedi R.S. Subbarao V. Usman M.I. Scarpelli D.G. Nemali M.R. Yeldandi A. Thangada S. Kumar S. Reddy J.K. Biochem. Biophys. Res. Commun. 1988; 156: 131-136Crossref PubMed Scopus (92) Google Scholar, 9Dabeva M.D. Hwang S.G. Vasa S.R. Hurston E. Novikoff P.M. Hixson D.C. Gupta S. Shafritz D.A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7356-7361Crossref PubMed Scopus (180) Google Scholar). Moreover, metaplastic hepatocytes in ciprofibrate-treated rat pancreas have been shown to originate from pancreatic exocrine acinar cells (10Reddy J.K. Rao M.S. Qureshi S.A. Reddy M.K. Scarpelli D.G. Lalwani N.D. J. Cell Biol. 1984; 98: 2082-2090Crossref PubMed Scopus (93) Google Scholar). The phenotypic plasticity of pancreatic cells has been shown in many studies (4Bouwens L. Microsc. Res. Tech. 1998; 43: 332-336Crossref PubMed Scopus (140) Google Scholar, 11Gao R. Ustinov J. Pulkkinen M.A. Lundin K. Korsgren O. Otonkoski T. Diabetes. 2003; 52: 2007-2015Crossref PubMed Scopus (234) Google Scholar, 12Gao R. Ustinov J. Korsgren O. Otonkoski T. Diabetologia. 2005; 48: 2296-2304Crossref PubMed Scopus (75) Google Scholar). Pancreatic acinar cells can convert into ductal cells, and direct evidence for acinar-to-ductal transdifferentiation has been provided both in vitro (13Means A.L. Meszoely I.M. Suzuki K. Miyamoto Y. Rustgi A.K. Coffey Jr., R.J. Wright C.V. Stoffers D.A. Leach S.D. Development (Camb.). 2005; 132: 3767-3776Crossref PubMed Scopus (263) Google Scholar, 14Minami K. Okuno M. Miyawaki K. Okumachi A. Ishizaki K. Oyama K. Kawaguchi M. Ishizuka N. Iwanaga T. Seino S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15116-15121Crossref PubMed Scopus (221) Google Scholar) and in vivo (15Miyatsuka T. Kaneto H. Shiraiwa T. Matsuoka T.A. Yamamoto K. Kato K. Nakamura Y. Akira S. Takeda K. Kajimoto Y. Yamasaki Y. Sandgren E.P. Kawaguchi Y. Wright C.V. Fujitani Y. Genes Dev. 2006; 20: 1435-1440Crossref PubMed Scopus (131) Google Scholar). In addition, transdifferentiation of insulin-secreting cells from pancreatic exocrine cells has been demonstrated in vitro (14Minami K. Okuno M. Miyawaki K. Okumachi A. Ishizaki K. Oyama K. Kawaguchi M. Ishizuka N. Iwanaga T. Seino S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15116-15121Crossref PubMed Scopus (221) Google Scholar, 16Baeyens L. De Breuck S. Lardon J. Mfopou J.K. Rooman I. Bouwens L. Diabetologia. 2005; 48: 49-57Crossref PubMed Scopus (255) Google Scholar), and cell lineage tracing revealed the origin of the newly made insulin-secreting cells to be pancreatic acinar cells (14Minami K. Okuno M. Miyawaki K. Okumachi A. Ishizaki K. Oyama K. Kawaguchi M. Ishizuka N. Iwanaga T. Seino S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15116-15121Crossref PubMed Scopus (221) Google Scholar, 17Okuno M. Minami K. Okumachi A. Miyawaki K. Yokoi N. Toyokuni S. Seino S. Am. J. Physiol. 2007; 292: E158-E165Crossref PubMed Scopus (39) Google Scholar). Regarding the mechanism of transdifferentiation of pancreatic acinar cells, EGF, 2The abbreviations used are: EGF, epidermal growth factor; PI3, phosphatidylinositol 3; MAP, mitogen-activated protein; MAPK, MAP kinase; JNK, c-Jun NH2-terminal kinase; RT-PCR, reverse transcription-PCR; HNF, hepatocyte nuclear factor; NCAM, neural cell adhesion molecule. Notch, and/or leukocyte inhibitory factor/signal transducers and activators of transcription (LIF/STAT) signals are thought to be involved in the process (13Means A.L. Meszoely I.M. Suzuki K. Miyamoto Y. Rustgi A.K. Coffey Jr., R.J. Wright C.V. Stoffers D.A. Leach S.D. Development (Camb.). 2005; 132: 3767-3776Crossref PubMed Scopus (263) Google Scholar, 14Minami K. Okuno M. Miyawaki K. Okumachi A. Ishizaki K. Oyama K. Kawaguchi M. Ishizuka N. Iwanaga T. Seino S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15116-15121Crossref PubMed Scopus (221) Google Scholar, 16Baeyens L. De Breuck S. Lardon J. Mfopou J.K. Rooman I. Bouwens L. Diabetologia. 2005; 48: 49-57Crossref PubMed Scopus (255) Google Scholar, 18Miyamoto Y. Maitra A. Ghosh B. Zechner U. Argani P. Iacobuzio-Donahue C.A. Sriuranpong V. Iso T. Meszoely I.M. Wolfe M.S. Hruban R.H. Ball D.W. Schmid R.M. Leach S.D. Cancer Cell. 2003; 3: 565-576Abstract Full Text Full Text PDF PubMed Scopus (576) Google Scholar), based mainly on studies using signaling inhibitor compounds. However, the precise roles of these signals in transdifferentiation are not clear. In addition, putative progenitor cell markers such as nestin (13Means A.L. Meszoely I.M. Suzuki K. Miyamoto Y. Rustgi A.K. Coffey Jr., R.J. Wright C.V. Stoffers D.A. Leach S.D. Development (Camb.). 2005; 132: 3767-3776Crossref PubMed Scopus (263) Google Scholar) and PGP9.5 (14Minami K. Okuno M. Miyawaki K. Okumachi A. Ishizaki K. Oyama K. Kawaguchi M. Ishizuka N. Iwanaga T. Seino S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15116-15121Crossref PubMed Scopus (221) Google Scholar, 19Rooman I. Heremans Y. Heimberg H. Bouwens L. Diabetologia. 2000; 43: 907-914.17Crossref PubMed Scopus (165) Google Scholar) have been detected in transdifferentiating pancreatic acinar cells, suggesting that dedifferentiated intermediate cells were generated in the course of the transdifferentiation, as seen in lens or tail regeneration in amphibia (2Eguchi G. Kodama R. Curr. Opin. Cell Biol. 1993; 5: 1023-1028Crossref PubMed Scopus (194) Google Scholar, 20Echeverri K. Tanaka E.M. Semin. Cell Dev. Biol. 2002; 13: 353-360Crossref PubMed Scopus (60) Google Scholar). However, such dedifferentiated states are transient, and acinar cell-derived intermediate cells have not been obtained and characterized. Thus, the details of the process of transdifferentiation of pancreatic acinar cells are largely still unknown. Clarification of the signaling mechanism of the transdifferentiation of pancreatic acinar cells should both provide new insight into the mechanism of normal development of pancreas and cell type specification and facilitate the development of cell-based therapy in diabetes mellitus. In the present study, we investigated the mechanism of pancreatic exocrine-to-endocrine transdifferentiation in adult mice in vitro. We found that formation of spherical three-dimensional structures by cadherin-mediated cell-cell adhesion plays a critical role in the induction of β-cell-specific gene expression in acinar-derived cells and that PI3-kinase activity is involved in both the formation of spheroids and the completion of transdifferentiation into insulin-secreting cells. We also show that a dedifferentiated state can be induced in isolated pancreatic acinar cells by inhibiting the restoration of cadherin-mediated cell-cell adhesion in the presence of PI3-kinase inhibitor. Isolation and Culture of Pancreatic Acinar Cells—Pancreatic acinar cells were isolated from streptozotocin-injected 8-week-old male C57Bl/6 mice as described (14Minami K. Okuno M. Miyawaki K. Okumachi A. Ishizaki K. Oyama K. Kawaguchi M. Ishizuka N. Iwanaga T. Seino S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15116-15121Crossref PubMed Scopus (221) Google Scholar, 17Okuno M. Minami K. Okumachi A. Miyawaki K. Yokoi N. Toyokuni S. Seino S. Am. J. Physiol. 2007; 292: E158-E165Crossref PubMed Scopus (39) Google Scholar). To remove almost all the β-cells, we used an excessive dosage of streptozotocin (200 mg/kg, intraperitoneal) for this strain, by which more than a half of the mice die within a week without insulin treatment. Mice with blood glucose concentration above 350 mg/dl were used for the present study. Pancreas was digested with 1 mg/ml collagenase P (Roche Diagnostics) and subjected to discontinuous Ficoll (GE Healthcare) gradient centrifugation. The exocrine acinar cell-enriched fraction recovered as a pellet was stained with sterilized dithizone solution (0.1 mg/ml) to confirm that there was no contamination of pre-existing pancreatic β-cells. The fraction contained >90% of acinar cells and ∼5% of ductal cells. Only a trace amount (<0.001% of normal pancreas) of mRNA expressions of both insulin 1 and insulin 2 were detected by quantitative real-time RT-PCR analysis, indicating that contamination of pancreatic β-cells in this fraction was negligible. The purified acinar-enriched exocrine cells were precultured in RPMI 1640 medium (Sigma) with 10% fetal calf serum for 6 h. Floating cells were then collected and replated on low cell-binding dishes (Nalge Nunc) in RPMI 1640 medium supplemented with 0.5% fetal calf serum, 20 ng/ml EGF (R&D Systems) at a density of 1 × 104 cells/cm2. As found in previous studies (14Minami K. Okuno M. Miyawaki K. Okumachi A. Ishizaki K. Oyama K. Kawaguchi M. Ishizuka N. Iwanaga T. Seino S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15116-15121Crossref PubMed Scopus (221) Google Scholar, 17Okuno M. Minami K. Okumachi A. Miyawaki K. Yokoi N. Toyokuni S. Seino S. Am. J. Physiol. 2007; 292: E158-E165Crossref PubMed Scopus (39) Google Scholar), the total cell number decreased to ∼25% of that in the initial preparation, and cell proliferation was rarely detected under these conditions. To inhibit spheroid formation of isolated pancreatic acinar cells, the neutralizing antibody against E-cadherin (ECCD-1: Takara) was added to the culture at a concentration of 200 μg/ml. Rat IgG2b isotype control (R&D Systems) was used as a negative control. All animal experiments were approved by the Animal Research Committees of Kyoto University Graduate School of Medicine and Kobe University Graduate School of Medicine. Generation and Infection of Recombinant Adenoviruses—Mouse inhibitor of β-catenin and TCF4 (ICAT) cDNA (obtained by RT-PCR) and Myc-His-tagged mouse myr-Akt1 cDNA (purchased from Upstate Biotechnology) were cloned into pENTR vector (Invitrogen). Adenoviruses were generated by using ViraPower adenoviral expression system (Invitrogen) according to the manufacturer's instructions. Cells were infected with these adenoviruses on day 0 (the day of cell isolation) for 6 h. Multiplicity of infection is indicated in the legend for Fig. 1. Adenovirus expressing Cre-recombinase was used as a negative control. Immunofluorescence—Cells were pelleted and fixed with 4% paraformaldehyde. Frozen sections were stained with antibodies against E-cadherin (ECCD-2: Takara) (1:200), β-catenin (Cell Signaling Technologies) (1:100), or neural cell adhesion molecule (NCAM) (Chemicon) (1:500). Secondary antibodies used were Alexa Fluor 488- or 546-conjugated IgGs (Molecular Probes) (1:400). For F-actin staining, Alexa Fluor 488-conjugated phalloidin (Molecular Probes) was used. Images were obtained by a fluorescent microscope (IX-71; Olympus) equipped with an objective lens (UPLFLN ×20 and ×40) and a CCD camera (ORCA-ER; Hamamatsu Photonics). Deconvoluted images were obtained by BZ9000 microscope (Keyence). Figures were then generated using PhotoShop 7.0 and Illustrator 10 (Adobe). Subcellular Fractionation and Immunoblotting—Total cellular proteins were extracted with buffer containing 50 mm Tris-HCl (pH 8.0), 150 mm NaCl, 100 mm NaF, 10 mm sodium pyrophosphate, 1 mm Na3VO4, 0.1% SDS, 0.5% sodium deoxysholate, 1% Nonidet P-40, and protease inhibitor mixture (Nacalai). Subcellular fractionation of the cells was performed by using a ProteoExtract subcellular extraction kit (Calbiochem) according to the manufacturer's instructions. Protein concentration was determined by BCA protein assay system (Pierce), and equivalent amounts of proteins were separated by SDS-PAGE and blotted onto polyvinylidene difluoride membrane (Millipore). Membranes were probed with antibodies against E-cadherin (H-108, Santa Cruz Biotechnology; RDI-ECADHERabm: Fitzgerald); β-catenin (catalog number 9587 (C-terminal antigen); Cell Signaling Technology; ab2982 (full-length antigen, AbCam); NCAM (AB5032, Chemicon); Erk1/2 (catalog number 9102, Cell Signaling Technology); phospho-Erk1/2 (catalog number 9101, Cell Signaling Technology); p38 MAP kinase (catalog number 9212, Cell Signaling Technology); phospho-p38 (catalog number 9211, Cell Signaling Technology); JNK (catalog number 9252, Cell Signaling Technology); phospho-JNK (catalog number 9251, Cell Signaling Technology); Akt (catalog number 9272, Cell Signaling Technology); or phosph-Akt (catalog number 9271 and catalog number 9275, Cell Signaling Technology). RNA Analyses—Total RNA was isolated with RNeasy mini kit (Qiagen) according to the manufacturer's instructions. After treatment with DNase I (Qiagen), the RNA was reverse-transcribed by ReverTra Ace (Toyobo), and the resultant cDNA was subjected to PCR using AmpliTaq Gold DNA polymerase (Applied Biosystems). PCR primers were designed such that the amplified regions spanned introns in the gene, except for Kir6.2, which has no intron in the protein-coding region. The PCR conditions were as follows: denaturation at 95 °C for 30 s, annealing at 59 °C for 30 s, and extension at 72 °C for 45 s, with hot start at 95 °C for 10 min. Nucleotide sequences of the primers were described previously (14Minami K. Okuno M. Miyawaki K. Okumachi A. Ishizaki K. Oyama K. Kawaguchi M. Ishizuka N. Iwanaga T. Seino S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15116-15121Crossref PubMed Scopus (221) Google Scholar). Real-time PCR was performed using TaqMan probes (Applied Biosystems) with a model 7300 real-time thermal cycler (Applied Biosystems). Measurement of the expression levels of 18 S ribosomal RNA was used as internal control. Measurement of Insulin Secretion—Cells in the culture were harvested and resuspended with the Krebs-HEPES buffer (119 mm NaCl, 4.74 mm KCl, 2.54 mm CaCl2, 1.19 mm MgCl2, 1.19 mm KH2PO4, 25 mm NaHCO3, and 10 mm HEPES, pH 7.4) containing 0.1% bovine serum albumin. The cells were preincubated in the buffer with 3 mm glucose for 30 min at 37 °C. Subsequently, aliquots of the cell suspension were incubated for a further 60 min with various secretagogues as indicated. After incubation, the solutions were collected to measure insulin concentration by enzyme-linked immunosorbent assay (Shibayagi). Total protein was extracted in 1 n NaOH, and the concentration was determined using Coomassie Brilliant Blue-G250 reagent (Bio-Rad Laboratories). Intracellular Signals Essential for Transdifferentiation of Pancreatic Acinar Cells—We have previously shown that enzymatic dissociation of pancreas causes tyrosine phosphorylation of the EGF receptor in pancreatic acinar cells, leading to activation of its downstream intracellular signals and enabling the isolated acinar cells to transdifferentiate into insulin-secreting cells even without the addition of EGF (14Minami K. Okuno M. Miyawaki K. Okumachi A. Ishizaki K. Oyama K. Kawaguchi M. Ishizuka N. Iwanaga T. Seino S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15116-15121Crossref PubMed Scopus (221) Google Scholar). Although activation of the EGF receptor is essential for the transdifferentiation of acinar cells into insulin-secreting cells, it is not clear which intracellular signals are responsible for the transdifferentiation. To further confirm and quantify signal activations by isolation of pancreatic acinar cells, we investigated phosphorylation of the major signaling molecules downstream of the EGF receptor using phospho-specific antibodies. Phosphorylated forms of Erk1/2, p38 MAPK, JNK, and Akt were found to be increased in dissociated pancreatic acinar cells (Fig. 1A). Quantification by densitometric analysis of the blots showed that the increments in phosphorylation all were statistically significant (Fig. 1A). These results indicate that the MAPK and PI3-kinase/Akt pathways are activated during the acinar cell isolation procedure. The addition of EGF in the culture enhanced and/or prolonged activation of the signals (data not shown). We then determined which signals are critical for transdifferentiation of acinar cells into insulin-secreting cells by utilizing inhibitors specific for each pathway (Fig. 1B). Among the inhibitors used, LY294002, a specific PI3-kinase inhibitor, showed the strongest effect on transdifferentiation (Fig. 1C). Gene expressions of insulins (both 1 and 2), glucokinase, SUR1, and Kir6.2 were not induced in the presence of LY294002 (Fig. 1C). Pdx1 expression was markedly diminished under these conditions (Fig. 1C). No insulin secretion was observed in acinar-derived cells cultured with LY294002 (data not shown). In addition, introduction of constitutively activated Akt (myrAkt) (21Kohn A.D. Summers S.A. Birnbaum M.J. Roth R.A. J. Biol. Chem. 1996; 271: 31372-31378Abstract Full Text Full Text PDF PubMed Scopus (1093) Google Scholar) by adenovirus under inhibition of PI3-kinase induced expressions of insulins, glucokinase, and pdx1 (Fig. 1D), demonstrating that the expressions of these genes are regulated by PI3-kinase/Akt signaling in transdifferentiating pancreatic acinar cells. Indeed, Akt inhibitor IV, an Akt-specific inhibitor, prevented induction of these genes (Fig. 1E). Because the expressions of SUR1 and Kir6.2 were suppressed by the Akt inhibitor (Fig. 1E) and were not recovered by gene transfer of constitutively activated Akt (Fig. 1D), activation of Akt is necessary but not sufficient for the expressions of SUR1 and Kir6.2. In addition, these results demonstrate that activation of the PI3-kinase/Akt pathway is essential for transdifferentiation of pancreatic acinar cells into insulin-secreting cells. Destruction and Restoration of Intercellular Contacts in Isolated Pancreatic Acinar Cells—Collagenase digestion resulted in dissociation of pancreatic acinus into single cells or small clumps (Fig. 2A, left panel). When cultured in suspension with the addition of EGF, the conditions under which transdifferentiation into insulin-secreting cells occurs, the isolated acinar cells formed three-dimensional spherical structures within a few days (Fig. 2A, middle panel), as described previously (14Minami K. Okuno M. Miyawaki K. Okumachi A. Ishizaki K. Oyama K. Kawaguchi M. Ishizuka N. Iwanaga T. Seino S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15116-15121Crossref PubMed Scopus (221) Google Scholar). Since cadherin-mediated cell-cell adhesion is essential for intercellular contact of epithelial cells (22Nagafuchi A. Curr. Opin. Cell Biol. 2001; 13: 600-603Crossref PubMed Scopus (258) Google Scholar), the expression and subcellular localization of E-cadherin and β-catenin, the major components of adherens junctions, were examined. In native pancreatic acinar cells, both E-cadherin and β-catenin were localized exclusively in the intercellular region of plasma membranes (Fig. 2B, left panels). Some loss of E-cadherin was detected in isolated acinar cells, and strikingly, most β-catenin was dissociated from E-cadherin and distributed throughout the cytoplasm and the nucleus (Fig. 2B, left middle panels). However, after 4 days of culture in the presence of EGF, cadherin-mediated cell-cell adhesion recovered, as evidenced by membrane distribution of both E-cadherin and β-catenin in these acinar-derived cells (Fig. 2B, right middle panels). Pancreatic acinar cells are known to be highly polarized. Indeed, F-actin is predominantly localized to apical membrane in native pancreatic acinar cells (Fig. 2C, upper panels). However, the cultured acinar cells that formed spheroids exhibited uniform membrane distribution of F-actin (Fig. 2C, right upper panel), indicating alteration of the cell polarity during the transdifferentiation. The honey-comb appearance of F-actin staining in transdifferentiated acinar cell clusters resembles that in islet cells (Fig. 2C, upper panels). The difference in cell polarity between endocrine (islet) and exocrine (acinar) cells has been shown to be caused, at least in part, by the expression of NCAM (23Esni F. Taljedal I.B. Perl A.K. Cremer H. Christofori G. Semb H. J. Cell Biol. 1999; 144: 325-337Crossref PubMed Scopus (140) Google Scholar). Pancreatic islet cells express NCAM, but acinar cells do not (Fig. 2C, lower panels). Interestingly, NCAM expression was induced in isolated pancreatic acinar cells by culture (Fig. 2, D and E), and many of the transdifferentiated acinar cells were positive for NCAM (Fig. 2C, right lower panel). These results demonstrate that the cell polarity seen in native pancreatic acinar cells is first lost and then remodeled to islet-like membrane organization and suggest that induction of NCAM expression has a role in this alteration of cell polarity. Role of PI3-Kinase in Cadherin-mediated Cell-Cell Adhesion— Pancreatic acinar cells cultured in the presence of LY294002 did not form spherical structures (Fig. 2B, right panels). Immunocytochemical analysis showed that E-cadherin was expressed at the intercellular contacts of some cell clumps, but many of the cells were negative for E-cadherin 4 days after culture with LY294002 (Fig. 2B, right panels). Expression of β-catenin was rarely detected (Fig. 2B, right panels). These results demonstrate that recovery of cadherin-mediated cell-cell adhesion in isolated pancreatic acinar cells is dependent on PI3-kinase activity. In the absence of PI3-kinase inhibitor, expressions of E-cadherin and β-catenin were increased by culture at both protein and mRNA levels (Fig. 3, A–D). In contrast, although mRNA levels of E-cadherin and β-catenin were not decreased, both proteins were decreased to almost undetectable levels by culture in the presence of LY294002 (Fig. 3, E–I), suggesting the involvement of posttranscriptional events in the loss of these molecules. Interestingly, as the full-length form of E-cadherin was decreased, a low molecular weight band was increased (Fig. 3F; arrow). Using a different antibody (purchased from Fitzgerald Industries International), it became clear that the full-length form of E-cadherin decreased, whereas lower molecular weight bands gradually increased in the plasma membrane fraction (Fig. 3G), indicating that E-cadherin was degraded during culture. In addition, although only the full-length form of β-catenin was detected by the antibody against the C-terminal peptide of β-catenin (catalog number 9587), bands of some smaller molecules were detected when the antibody against full-length β-catenin (purchased from AbCam) was used (Fig. 3J), indicating that β-catenin also was degraded during culture under these conditions. Degradation products of β-catenin also were detected in the presence of EGF without LY294002 when the antibody from AbCam was used, but the full-length protein was predominant at day 4 (data not shown). These findings suggest that the recovery of cadherin-mediated cell-cell adhesion, which was disrupted by the isolation of the pancreatic acinar cells, depends on PI3-kinase activity, most probably because of its prevention of the degradation of both E-cadherin and β-catenin. Suppressed Induction of β-Cell-specific Genes by Loss of Cadherin-mediated Cell-Cell Adhesion—Since intercellular contact and communication are involved in both cellular functions and differentiation (24Potter E. Bergwitz C. Brabant G. Endocr. Rev. 1999; 20: 207-239Crossref PubMed Scopus (110) Google Scholar), we examined the relationship between cadherin-mediated cell-cell adhesion and transdifferentiation in pancreatic acinar cells. ECCD-1, a monoclonal antibody to mouse E-cadherin, has been shown to inhibit cadherin-mediated cell-cell adhesion (25Kanno Y. Sasaki Y. Shiba Y. Yoshida-Noro C. Takeichi M. Exp. Cell Res. 1984; 152: 270-274Crossref PubMed Scopus (44) Google Scholar). In the presence of ECCD-1 (200 μg/ml), isolated pancreatic acinar cells did not form spherical structures (Fig. 4A), and the inductions of genes characteristic of pancreatic β-cells, including insulins, glucokinase, SUR1, and Kir6.2, were significantly reduced (Fig. 4B). In contrast, transcription factors seen in the early developing pancreas, such as Pdx1, HNF6, Foxa2, and Nkx2.2, were similarly up-regulated regardless of the presence of the neutralizing antibody (Fig. 4B). Amylase expression was drastically decreased in all conditions. Induction of CK19 tended to be increased in the presence of ECCD-1, but the difference was not significant (Fig. 4B). Rat IgG2b, used as a negative control, had no effect on morphology and gene expressions (Fig. 4, A and B). These results indicate that spheroid formation by cadherin-mediated cell-cell adhesion facilitates transdifferentiation of pancreatic acinar cells into insulin-secreting cells. Dedifferentiation of Pancreatic Acinar Cells—Although the expressions of β-cell-specific genes were markedly suppressed by the loss of cadherin-mediated cell-cell adhesion caused by the addition of LY294002 or ECCD-1, transcription factors such as Foxa2, HNF6, and Nkx2.2 were induced regardless of the presence or absence of the inhibitors (Figs. 1C and 4B). This suggests that isolated pancreatic acinar cells dedifferentiate but do not undergo complete transdifferentiation under the condition of disrupted cadherin-mediated cell-cell adhesion. To investigate this possibility, pancreatic acinar cells cultured in the presence of LY294002 were subjected to gene expression profiling by quantitative real-time RT-PCR analysis. Because the addition of the PI3-kinase inhibitor damages the cells, nicotinamide (10 mm) was added to improve cell survival. Nicotinamide has been shown to prevent cell damage due to activation of poly(ADP-ribose) synthetase/polymerase (26Yamamoto H. Uchigata Y. Okamoto H. Nature. 1981; 294: 284-286Crossref PubMed Scopus (476) Google Scholar). Indeed, the addition of nicotinamide doubled viable cell number in our culture system (14Minami K. Okuno M. Miyawaki K. Okumachi A. Ishizaki K. Oyama K. Kawaguchi M. Ishizuka N. Iwanaga T. Seino S. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 15116-15121Crossref PubMed Scopus (221) Google Scholar). As shown in Fig. 5, amylase e
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