Impaired Gastric Gland Differentiation in Peutz-Jeghers Syndrome
2010; Elsevier BV; Volume: 176; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2010.090519
ISSN1525-2191
AutoresLina Udd, Pekka Katajisto, Marika Kyyrönen, Ari Ristimäki, Tomi P. Mäkelä,
Tópico(s)Colorectal Cancer Screening and Detection
ResumoGastrointestinal hamartomatous polyps in the Peutz-Jeghers cancer predisposition syndrome and its mouse model (Lkb1+/−) are presumed to contain all cell types native to the site of their occurrence. This study aimed to explore the pathogenesis of Peutz-Jeghers syndrome polyposis by characterizing cell types and differentiation of the epithelium of gastric polyps and predisposed mucosa. Both antral and fundic polyps were characterized by a deficit of pepsinogen C-expressing differentiated gland cells (antral gland, mucopeptic, and chief cells); in large fundic polyps, parietal cells were also absent. Gland cell loss was associated with an increase in precursor neck cells, an expansion of the proliferative zone, and an increase in smooth muscle α-actin expressing myofibroblasts in the polyp stroma. Lack of pepsinogen C-positive gland cells identified incipient polyps, and even the unaffected mucosa of young predisposed mice displayed an increase in pepsinogen C negative glands (25%; P = 0045). In addition, in small intestinal polyps, gland cell differentiation was defective, with the absence of Paneth cells. There were no signs of metaplastic differentiation in any of the tissues studied, and both the gastric and small intestinal defects were seen in Lkb1+/− mice, as well as polyps from patients with Peutz-Jeghers syndrome. These results identify impaired epithelial differentiation as the earliest pathological sign likely to contribute to tumorigenesis in individuals with inherited Lkb1 mutations. Gastrointestinal hamartomatous polyps in the Peutz-Jeghers cancer predisposition syndrome and its mouse model (Lkb1+/−) are presumed to contain all cell types native to the site of their occurrence. This study aimed to explore the pathogenesis of Peutz-Jeghers syndrome polyposis by characterizing cell types and differentiation of the epithelium of gastric polyps and predisposed mucosa. Both antral and fundic polyps were characterized by a deficit of pepsinogen C-expressing differentiated gland cells (antral gland, mucopeptic, and chief cells); in large fundic polyps, parietal cells were also absent. Gland cell loss was associated with an increase in precursor neck cells, an expansion of the proliferative zone, and an increase in smooth muscle α-actin expressing myofibroblasts in the polyp stroma. Lack of pepsinogen C-positive gland cells identified incipient polyps, and even the unaffected mucosa of young predisposed mice displayed an increase in pepsinogen C negative glands (25%; P = 0045). In addition, in small intestinal polyps, gland cell differentiation was defective, with the absence of Paneth cells. There were no signs of metaplastic differentiation in any of the tissues studied, and both the gastric and small intestinal defects were seen in Lkb1+/− mice, as well as polyps from patients with Peutz-Jeghers syndrome. These results identify impaired epithelial differentiation as the earliest pathological sign likely to contribute to tumorigenesis in individuals with inherited Lkb1 mutations. Peutz-Jeghers syndrome (PJS), presents as a triad of characteristic polyps with branching smooth muscle core, mucocutaneous pigmentations, and a predisposition to cancer development.1Hemminki A Markie D Tomlinson I Avizienyte E Roth S Loukola A Bignell G Warren W Aminoff M Hoglund P Jarvinen H Kristo P Pelin K Ridanpaa M Salovaara R Toro T Bodmer W Olschwang S Olsen AS Stratton MR de la Chapelle A Aaltonen LA A serine/threonine kinase gene defective in Peutz-Jeghers syndrome.Nature. 1998; 391: 184-187Crossref PubMed Scopus (1335) Google Scholar, 2Mehenni H Resta N Park JG Miyaki M Guanti G Costanza MC Cancer risks in LKB1 germline mutation carriers.Gut. 2006; 55: 984-990Crossref PubMed Scopus (98) Google Scholar, 3Hearle N Schumacher V Menko FH Olschwang S Boardman LA Gille JJ Keller JJ Westerman AM Scott RJ Lim W Trimbath JD Giardiello FM Gruber SB Offerhaus GJ de Rooij FW Wilson JH Hansmann A Moslein G Royer-Pokora B Vogel T Phillips RK Spigelman AD Houlston RS Frequency and spectrum of cancers in the Peutz-Jeghers syndrome.Clin Cancer Res. 2006; 12: 3209-3215Crossref PubMed Scopus (581) Google Scholar Peutz-Jeghers polyps have a properly polarized and apparently well-differentiated epithelium4Baas AF Smit L Clevers H LKB1 tumor suppressor protein: PARtaker in cell polarity.Trends Cell Biol. 2004; 14: 312-319Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar native to the site of occurrence.5Giardiello FM Brensinger JD Tersmette AC Goodman SN Petersen GM Booker SV Cruz-Correa M Offerhaus JA Very high risk of cancer in familial Peutz-Jeghers syndrome.Gastroenterology. 2000; 119: 1447-1453Abstract Full Text Full Text PDF PubMed Scopus (1052) Google Scholar, 6Rintala A The histological appearance of gastrointestinal polyps in the Peutz-Jeghers syndrome.Acta Chir Scand. 1959; 117: 366-373PubMed Google Scholar Although both polyps and carcinomas commonly arise in the gastrointestinal tract of PJS patients, the relationship between the two remains unclear. Inactivating mutations in the gene encoding for the LKB1 kinase cause PJS,1Hemminki A Markie D Tomlinson I Avizienyte E Roth S Loukola A Bignell G Warren W Aminoff M Hoglund P Jarvinen H Kristo P Pelin K Ridanpaa M Salovaara R Toro T Bodmer W Olschwang S Olsen AS Stratton MR de la Chapelle A Aaltonen LA A serine/threonine kinase gene defective in Peutz-Jeghers syndrome.Nature. 1998; 391: 184-187Crossref PubMed Scopus (1335) Google Scholar and mice heterozygous for Lkb1 model the disease by developing multiple characteristic Peutz-Jeghers polyps, particularly in their gastric mucosa.7Rossi DJ Ylikorkala A Korsisaari N Salovaara R Luukko K Launonen V Henkemeyer M Ristimaki A Aaltonen LA Makela TP Induction of cyclooxygenase-2 in a mouse model of Peutz-Jeghers polyposis.Proc Natl Acad Sci USA. 2002; 99: 12327-12332Crossref PubMed Scopus (106) Google Scholar The mechanism by which Lkb1 deficiency leads to polyps appears to involve the underlying stroma, based on polyps arising even when Lkb1 mutations are limited to the stromal smooth muscle lineage.8Katajisto P Vaahtomeri K Ekman N Ventela E Ristimaki A Bardeesy N Feil R DePinho RA Makela TP LKB1 signaling in mesenchymal cells required for suppression of gastrointestinal polyposis.Nat Genet. 2008; 40: 455-459Crossref PubMed Scopus (85) Google Scholar The normal gastric mucosa consists of parallel bipartite units of basal glands and luminal pits. The appropriate cell type for each position in the unit is thought to depend on gradients of morphogens like Wnt, Hedgehog, bone morphogenetic protein, and transforming growth factor-ß,9Bleuming SA He XC Kodach LL Hardwick JC Koopman FA Ten Kate FJ van Deventer SJ Hommes DW Peppelenbosch MP Offerhaus GJ Li L van den Brink GR Bone morphogenetic protein signaling suppresses tumorigenesis at gastric epithelial transition zones in mice.Cancer Res. 2007; 67: 8149-8155Crossref PubMed Scopus (96) Google Scholar, 10Brittan M Wright NA Stem cell in gastrointestinal structure and neoplastic development.Gut. 2004; 53: 899-910Crossref PubMed Scopus (125) Google Scholar, 11Mishra L Shetty K Tang Y Stuart A Byers SW The role of TGF-beta and Wnt signaling in gastrointestinal stem cells and cancer.Oncogene. 2005; 24: 5775-5789Crossref PubMed Scopus (145) Google Scholar whose characteristic expression patterns are disrupted in gastric cancer or by inflammatory stimuli.12Bleuming SA Kodach LL Garcia Leon MJ Richel DJ Peppelenbosch MP Reitsma PH Hardwick JC van den Brink GR Altered bone morphogenetic protein signalling in the Helicobacter pylori-infected stomach.J Pathol. 2006; 209: 190-197Crossref PubMed Scopus (41) Google Scholar, 13Byun T Karimi M Marsh JL Milovanovic T Lin F Holcombe RF Expression of secreted Wnt antagonists in gastrointestinal tissues: potential role in stem cell homeostasis.J Clin Pathol. 2005; 58: 515-519Crossref PubMed Scopus (73) Google Scholar, 14Fukaya M Isohata N Ohta H Aoyagi K Ochiya T Saeki N Yanagihara K Nakanishi Y Taniguchi H Sakamoto H Shimoda T Nimura Y Yoshida T Sasaki H Hedgehog signal activation in gastric pit cell and in diffuse-type gastric cancer.Gastroenterology. 2006; 131: 14-29Abstract Full Text Full Text PDF PubMed Scopus (120) Google Scholar The gastric gland is subdivided into isthmus, neck, and base regions. The isthmus contains stem cells,15Modlin IM Kidd M Lye KD Wright NA Gastric stem cells: an update.Keio J Med. 2003; 52: 134-137Crossref PubMed Scopus (37) Google Scholar and a variety of precursor cells,16Karam SM Lineage commitment and maturation of epithelial cells in the gut.Front Biosci. 1999; 4: D286-D298Crossref PubMed Google Scholar which migrate bidirectionally out of the isthmus during differentiation. Throughout the stomach, pre-pit precursor cells move upwards to become Muc5a-secreting pit (foveolar) cells. Downward from the isthmus, cells migrate to form glands, specialized according to location. In the proximal glandular stomach, the glands are fundic (oxyntic), whereas pyloric antral glands cover the distal stomach. Acid-producing parietal cells17Karam SM Straiton T Hassan WM Leblond CP Defining epithelial cell progenitors in the human oxyntic mucosa.Stem Cells. 2003; 21: 322-336Crossref PubMed Scopus (118) Google Scholar, 18Lee ER Trasler J Dwivedi S Leblond CP Division of the mouse gastric mucosa into zymogenic and mucous regions on the basis of gland features.Am J Anat. 1982; 164: 187-207Crossref PubMed Scopus (75) Google Scholar are only found in fundic glands, whereas enteroendocrine cells producing gastrin are unique to antral glands. The similarities in migration and differentiation of the fundic zymogenic chief cells and antral pyloric gland cells are considerable. Fundic pre-neck cell precursors (corresponding to antral pre-gland cells) differentiate into mucin producing neck/mucous neck cells in the fundus (differentiating gland cells in the antrum),16Karam SM Lineage commitment and maturation of epithelial cells in the gut.Front Biosci. 1999; 4: D286-D298Crossref PubMed Google Scholar and further into mucopeptic (prezymogenic) cells in the fundus (pyloric gland cells in the antrum)16Karam SM Lineage commitment and maturation of epithelial cells in the gut.Front Biosci. 1999; 4: D286-D298Crossref PubMed Google Scholar as they migrate down. The mature, differentiated pyloric gland cells express pepsinogen C,19Furihata C Iwasaki Y Sugimura T Tatematsu M Takahashi M Differentiation of pepsinogen-producing cells in the fundic and pyloric mucosa of developing rats.Cell Differ. 1973; 2: 179-189Crossref PubMed Scopus (39) Google Scholar, 20Samloff IM Slow moving protease and the seven pepsinogens. Electrophoretic demontration of the existence of eight proteolytic fractions in human gastric mucosa.Gastroenterology. 1969; 57: 659-669Abstract Full Text PDF PubMed Google Scholar mucin 6 (Muc6) and trefoil factor 2 (TFF2),21Kouznetsova I Laubinger W Kalbacher H Kalinski T Meyer F Roessner A Hoffmann W Biosynthesis of gastrokine-2 in the human gastric mucosa: restricted spatial expression along the antral gland axis and differential interaction with TFF1, TFF2, and mucins.Cell Physiol Biochem. 2007; 20: 899-908Crossref PubMed Scopus (51) Google Scholar that also mark early mucopeptic cells. The latter gradually transform from mostly mucin-producing cells into cells mostly producing proteolytic enzymes, reaching the base of the fundic gland as mature chief cells, no longer expressing mucin.16Karam SM Lineage commitment and maturation of epithelial cells in the gut.Front Biosci. 1999; 4: D286-D298Crossref PubMed Google Scholar In mouse, chief cells are also the major producers of intrinsic factor (IF),22Shao J Sartor RB Dial E Lichtenberger LM Schepp W Alpers DH Expression of intrinsic factor in rat and murine gastric mucosal cell lineages is modified by inflammation.Am J Pathol. 2000; 157: 1197-1205Abstract Full Text Full Text PDF PubMed Scopus (20) Google Scholar whereas human IF is expressed mainly, although not exclusively, in parietal cells.23Howard TA Misra DN Grove M Becich MJ Shao JS Gordon M Alpers DH Human gastric intrinsic factor expression is not restricted to parietal cells.J Anat. 1996; 189: 303-313PubMed Google Scholar The signals mediating gland cell migration and differentiation in the distal stomach are only partially understood, but the transition from mucopeptic cells to chief cells requires the activity of transcription factor Mist124Ramsey VG Doherty JM Chen CC Stappenbeck TS Konieczny SF Mills JC The maturation of mucus-secreting gastric epithelial progenitors into digestive-enzyme secreting zymogenic cells requires Mist1.Development. 2007; 134: 211-222Crossref PubMed Scopus (146) Google Scholar and can be inhibited by stimulation of activin II receptor.25Li Q Karam SM Coerver KA Matzuk MM Gordon JI Stimulation of activin receptor II signaling pathways inhibits differentiation of multiple gastric epithelial lineages.Mol Endocrinol. 1998; 12: 181-192Crossref PubMed Google Scholar The development and persistence of pepsinogen-expressing cells, and particularly chief cells, require glucocorticoids,26Tseng CC Schmidt KL Johnson LR Hormonal effects on development of the secretory apparatus of chief cells.Am J Physiol. 1987; 253: G274-G283PubMed Google Scholar, 27Tsukada S Ichinose M Tatematsu M Tezuka N Yonezawa S Kakei N Matsushima M Miki K Kurokawa K Kageyama T Takahashi K Famaci H Glucocorticoids inhibit the proliferation of mucosal cells and enhance the expression of a gene for pepsinogen and other markers of differentiation in the stomach mucosa of the adult rat.Biochem Biophys Res Commun. 1994; 202: 1-9Crossref PubMed Scopus (17) Google Scholar whose homeostatic effects are mediated via the mesenchymal stroma.28Tsukada S Ichinose M Yahagi N Matsubara Y Yonezawa S Shiokawa K Furihata C Miki K Fukamachi H Induction of precocious pepsinogen synthesis by glucocorticoids in fetal rat gastric epithelium in organ culture: importance of mesenchyme for epithelial differentiation.Differentiation. 1998; 62: 239-247Crossref PubMed Google Scholar This stroma, the lamina propria, consisting of fibroblasts, endothelial, and immune cells, is located between and underneath the gastric units. The unit bases, the glands, are surrounded by a sheath of smooth muscle α-actin (SMA)-expressing intestinal subepithelial myofibroblasts,29Leedham SJ Brittan M Preston SL McDonald SA Wright NA The stomach periglandular fibroblast sheath: all present and correct.Gut. 2006; 55: 295-296PubMed Google Scholar which, at least in the small intestine, link with the thin muscular sheath under the basal lamina called muscularis mucosae.30Powell DW Adegboyega PA Di Mari JF Mifflin RC Epithelial cells and their neighbors I. Role of intestinal myofibroblasts in development, repair, and cancer.Am J Physiol Gastrointest Liver Physiol. 2005; 289: G2-G7Crossref PubMed Scopus (179) Google Scholar In some pathological conditions of the gastric epithelium, like neo- or metaplasia31Mutoh H Sakurai S Satoh K Osawa H Tomiyama T Kita H Yoshida T Tamada K Yamamoto H Isoda N Ido K Sugano K Pericryptal fibroblast sheath in intestinal metaplasia and gastric carcinoma.Gut. 2005; 54: 33-39Crossref PubMed Scopus (23) Google Scholar, 32Nakayama H Enzan H Miyazaki E Toi M Alpha smooth muscle actin positive stromal cells in gastric carcinoma.J Clin Pathol. 2002; 55: 741-744Crossref PubMed Scopus (33) Google Scholar or helicobacter gastritis,33McCaig C Duval C Hemers E Steele I Pritchard DM Przemeck S Dimaline R Ahmed S Bodger K Kerrigan DD Wang TC Dockray GJ Varro A The role of matrix metalloproteinase-7 in redefining the gastric microenvironment in response to Helicobacter pylori.Gastroenterology. 2006; 130: 1754-1763Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar these myofibroblasts increase. In polyps of Peutz-Jeghers patients34Estrada R Spjut HJ Hamartomatous polyps in Peutz-Jeghers syndrome. A light-, histochemical, and electron-microscopic study.Am J Surg Pathol. 1983; 7: 747-754Crossref PubMed Scopus (31) Google Scholar and Lkb1+/− mice,7Rossi DJ Ylikorkala A Korsisaari N Salovaara R Luukko K Launonen V Henkemeyer M Ristimaki A Aaltonen LA Makela TP Induction of cyclooxygenase-2 in a mouse model of Peutz-Jeghers polyposis.Proc Natl Acad Sci USA. 2002; 99: 12327-12332Crossref PubMed Scopus (106) Google Scholar thick disorganized bundles of smooth muscle originating from the muscularis mucosae branch like a cauliflower stalk into polyp lobes. Here we studied the epithelia of gastric polyps and predisposed stomach in Lkb1+/− mice and Peutz-Jeghers patients to explore pathogenesis of Peutz-Jeghers polyps. Lkb1+/−35Ylikorkala A Rossi DJ Korsisaari N Luukko K Alitalo K Henkemeyer M Makela TP Vascular abnormalities and deregulation of VEGF in Lkb1-deficient mice.Science. 2001; 293: 1323-1326Crossref PubMed Scopus (251) Google Scholar and wild-type control mice in F1 and F2 C57BL6; CD1 backgrounds were sacrificed at an age of 4.5 months, or 10 to 11.5 months. Tissue specimens were collected and fixed in 4% paraformaldehyde for paraffin embedding as previously described.36Udd L Katajisto P Rossi DJ Lepisto A Lahesmaa AM Ylikorkala A Jarvinen HJ Ristimaki AP Makela TP Suppression of Peutz-Jeghers polyposis by inhibition of cyclooxygenase-2.Gastroenterology. 2004; 127: 1030-1037Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar All animal experimentation was performed with permission from and in accordance with the recommendations of the local animal welfare committee at the University of Helsinki and State Provincial Office of Southern Finland. Paraffin-embedded samples of polyps and biopsies of normal gastric tissue from Peutz-Jeghers patients were obtained from the archives of the Helsinki University Central Hospital Pathology Department, in accordance with local ethical regulations. All immunohistochemistry was performed on dewaxed and rehydrated sections, with endogenous peroxidase quenched by 3% hydrogen peroxide in either PBS or methanol. For staining against H+/K+-ATPase (HA3-923, Affinity Bioreagent, Golden, CO), Cdx2 (MU392A-U, BioGenex Laboratories, San Ramon, CA), chromogranin A (A0430, DakoCytomation, Carpinteria, CA), Muc6 (NCL-MUC-6, Novocastra, Newcastle On Tyne, UK), or Ki-67 (Clone TEC-3, DakoCytomation), sections were pre-treated with DAKO Target Retrieval (DakoCytomation) at 95°C, 20 minutes. H+/K+-ATPase signal was detected with the ARK-kit (DakoCytomation), whereas Ki-67 and CgA signal with biotinylated anti-rat- and anti-rabbit-antibodies (Vector Laboratories, Burlingame, CA), ABC-reagent (Vector) and diaminobenzidine substrate (Sigma-Aldrich, St. Louis, MO). Muc6 signal was detected with AlexaFluor488 conjugated anti-mouse antibody (Invitrogen, Carlsbad, CA). TFF2 (a kind gift by Dr. George Elia, Histopathology Unit, ICRF, London) and gastrin (A0568, DakoCytomation) antibodies were used without pretreatment, and detected with fluorescein isothiocyanate-conjugated anti-mouse IgM (Vector) and with biotinylated anti-rabbit-antibody (Vector), and ABC-reagent with 3-amino-9-ethylcarbazole substrate (Vector) respectively. For staining with alkaline phosphatase-conjugated α-SMA (A5691, Sigma-Aldrich), sections were incubated at 37°C, 30 minutes in 0.02% Trypsin (Difco Laboratories, Detroit, MI) in 0.05 mol/L Tris-HCl-CaCl2 (pH 7.6) and unspecific antibody binding blocked with blocking reagent in the TSA-kit (Perkin Elmer, Foster City, CA). Signal was detected by addition of Black Alkaline Phosphatase Substrate (Vector). Desmin antibody (DE-R-11, Novocastra, Newcastle, UK) and lysozyme antibody (A0099, DakoCytomation) were used after similar pretreatment as α-SMA. Desmin was detected with the ARK-kit, lysozyme and biotinylated Griffonia simplicifolia (GS) lectin II (B-1215, Vector) were detected with ABC-reagent and 3-amino-9-ethylcarbazole substrate. Staining against pepsinogen C was performed with an antibody against rat Pg-137Tatematsu M Mutai M Aoki T de Camargo JL Furihata C Ito N Proliferation kinetics of pepsinogen altered pyloric gland cells in rats treated with N-methyl-N′-nitro-N-nitrosoguanidine.Carcinogenesis. 1989; 10: 907-911Crossref PubMed Scopus (35) Google Scholar, 38Yamamoto M Furihata C Ogiu T Tsukamoto T Inada K Hirano K Tatematsu M Independent variation in susceptibilities of six different mouse strains to induction of pepsinogen-altered pyloric glands and gastric tumor intestinalization by N-methyl-N-nitrosourea.Cancer Lett. 2002; 179: 121-132Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar kindly provided by Dr. T. Tsukamoto (Aichi Cancer Center Research Institute, Nagoya, Japan) and staining against IF with a polyclonal rabbit antibody kindly contributed by Dr. D.H. Alpers (Washington University School of Medicine, St. Louis, MO) according to the respective recommended protocols. All immunostainings except TFF2, IF, and Ki-67 were counterstained with hematoxylin (Shandon Instant Hematoxylin, Thermo Fisher Scientific, Waltham, MA). Foveolar cells were detected with Alcian blue-Periodic acid-Schiff (AB-PAS) staining. The quantified tissue sections were derived from the proximity of the major curvature. Pepsinogen altered pyloric glands (PAPG) were determined in 4.5-month-old mice by blinded scoring of 115 ± 30 pyloric glands (= bases of antral gastric units) per stained section as either positive or negative for pepsinogen C. The pylorus itself was excluded from the score, and subsequent analysis, as were weakly positive glands (2.8 ± 1.8% of all counted glands in wild-type and 3.4 ± 1.7% in Lkb1+/− mice). Sizes of TFF2 expressing cells were manually scored as the cross section area in micrographs of stained tissue sections, using ImageJ software (). Cell numbers in the fundic stomach of aged (10- to 11.5-month-old mice) were scored from gastric units assessable in their full length. The units were selected 5 to 10 mm from the border to the squamous forestomach (the fundic-most part of murine stomach). The significance of the differences between groups was determined with Mann-Whitney U-tests, using the SPSS-software package (SPSS Inc., Chicago, IL). All values are given as mean ± SD. Tissue samples were prepared from the fundus of 8-month-old littermate mice. Total RNA samples were collected using the RNEasy-kit (Qiagen, Valencia, CA) from pieces of wild-type stomach wall (n = 6), Lkb1+/− stomach wall (n = 6) approximately 5 mm in diameter, and from Lkb1+/− fundic polyps (n = 4). Amplification and labeling of mRNA were performed with Superscript II (Qiagen, Valencia, CA) and ENZO (Enzo Life Sciences, Farmingdale, NY) kits, according to the manufacturers' protocols. Expression profiling was conducted with Mg-U74a2 GeneChips (Affymetrix, PaloAlto, CA), also according to the manufacturer's instructions. Expression profiles were normalized using the GC-RMA method within the GeneSpring GX software (Agilent Technologies, Santa Clara, CA). Expression of genes reported to be enriched in foveolar cells,39Mueller A Merrell DS Grimm J Falkow S Profiling of microdissected gastric epithelial cells reveals a cell type-specific response to Helicobacter pylori infection.Gastroenterology. 2004; 127: 1446-1462Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar chief cells,40Mills JC Andersson N Stappenbeck TS Chen CC Gordon JI Molecular characterization of mouse gastric zymogenic cells.J Biol Chem. 2003; 278: 46138-46145Crossref PubMed Scopus (32) Google Scholar and parietal cells41Mills JC Syder AJ Hong CV Guruge JL Raaii F Gordon JI A molecular profile of the mouse gastric parietal cell with and without exposure to Helicobacter pylori.Proc Natl Acad Sci USA. 2001; 98: 13687-13692Crossref PubMed Scopus (70) Google Scholar were compared between wild-type and Lkb1+/− tissues. Identifiers of the published gene lists were translated to Mg-U74a2 probe set identifiers using the NetAffx batch query tool (Affymetrix, Santa Clara, CA). In addition, a random control gene list was generated from all genes containing the letters G and A in their GeneSpring GX annotation (196 identifiers). For each list, the average and standard deviations of the expression fold change were calculated, and a two-way analysis of variance test for significant differences in the expression levels between the experimental groups was performed using the R statistical analysis environment. Posthoc analysis was done with Student's t-test. Analysis of Peutz-Jeghers type gastric polyps from 10- to 11.5-month Lkb1+/− mice revealed a widened proliferative zone as recognized by Ki-67 staining (Figure 1A). Foveolar cell differentiation upwards from the proliferating zone appeared normal based on AB-PAS staining demonstrating neutral mucus-containing cells at the surface of both unaffected normal mucosa and in polyps. Also initial gland cell differentiation appeared normal based on staining with the GS lectin II (GS-lectin) in antral (Figure 1A) and fundic (Figure 1C) mucosa. This was also observed when staining polyps with an antibody against Muc6 (Supplemental Figure S1 at ). While the proportion of foveolar cells to the GS-lectin positive cells in unaffected Lkb1+/− mucosa was comparable with that of controls, it varied considerably within and among polyps. In the proximity of smooth muscle bundles, the epithelium contained a higher proportion of GS-lectin positive cells, while elsewhere the epithelium contained more foveolar cells (Figure 1B). Surprisingly, both antral and fundic polyps demonstrated either weak (n = 26/47) or no (n = 21/47) pepsinogen C staining, indicating a deficiency in antral gland, mucopeptic, and chief cells not observed in the unaffected mucosa; a fundic polyp is shown in Figure 1C. Large fundic polyps also demonstrated absence of parietal cells based on morphological analysis and H+/K+-ATPase-antibody staining (Figure 1C). Concomitantly with lack of terminally differentiated gland cells, their precursors reached the gland base, based on GS-lectin staining (Figure 1C, inset). Apart from large polyps with typical Peutz-Jeghers features, the antral mucosa of Lkb1+/− mice contains areas with varying features of hyperplasia or hyperplastic polyposis characterized as incipient polyps.36Udd L Katajisto P Rossi DJ Lepisto A Lahesmaa AM Ylikorkala A Jarvinen HJ Ristimaki AP Makela TP Suppression of Peutz-Jeghers polyposis by inhibition of cyclooxygenase-2.Gastroenterology. 2004; 127: 1030-1037Abstract Full Text Full Text PDF PubMed Scopus (75) Google Scholar Also in these, a deficiency in pepsinogen C staining was observed, together with a widened proliferative zone (Figure 2A, incipient polyp). Indeed, reduced pepsinogen C staining identified small incipient polyps from gastric folds, sometimes similar in appearance in tissue sections (Figure 2A, wild-type). The interglandular stroma of the incipient polyps showed increased SMA, compared with surrounding mucosa (seen in 42/72 polyps with a 0.2 to 2.0 mm diameter) associated with an increase in GS-lectin positive epithelial cells (Figure 2B) demonstrating a correlation between stromal and epithelial alterations already in incipient Peutz-Jeghers polyps. A small fraction (ca. 6%) of normal gastric pyloric glands have been found to contain strongly reduced or undetectable pepsinogen C,37Tatematsu M Mutai M Aoki T de Camargo JL Furihata C Ito N Proliferation kinetics of pepsinogen altered pyloric gland cells in rats treated with N-methyl-N′-nitro-N-nitrosoguanidine.Carcinogenesis. 1989; 10: 907-911Crossref PubMed Scopus (35) Google Scholar and these PAPGs increase following carcinogen exposure.37Tatematsu M Mutai M Aoki T de Camargo JL Furihata C Ito N Proliferation kinetics of pepsinogen altered pyloric gland cells in rats treated with N-methyl-N′-nitro-N-nitrosoguanidine.Carcinogenesis. 1989; 10: 907-911Crossref PubMed Scopus (35) Google Scholar, 38Yamamoto M Furihata C Ogiu T Tsukamoto T Inada K Hirano K Tatematsu M Independent variation in susceptibilities of six different mouse strains to induction of pepsinogen-altered pyloric glands and gastric tumor intestinalization by N-methyl-N-nitrosourea.Cancer Lett. 2002; 179: 121-132Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar To investigate possible alterations in the frequency of these altered glands in Lkb1+/− mice before polyposis, antral mucosa of young (4.5 mo) mice without detectable hyperplasia or polyps was analyzed for PAPGs (Figure 2C). The frequency was significantly increased in Lkb1+/− glands, compared with wild-type littermates (8.6 ± 2.5% vs. 6.4 ± 2.3%; P = 0045). Furthermore, concomitant staining with Ki-67 demonstrated that some pepsinogen C negative glands contained proliferative cells at the gland base (Figure 2D, arrowheads), and the frequency was significantly higher in Lkb1+/− mice 19/47 (40%) compared with controls (2/13; 15%). Excluding these focal alterations, proliferation in the unaffected antral mucosa in these young mice was not altered between Lkb1+/− (11.0 ± 2.5 Ki-67 positive cells per gland) and wild-type (11.2 ± 2.22 cells) mice. These results indicate that the unaffected but predisposed Lkb1+/− antral epithelium in young mice contains differentiation defects similar to those detected in polyps of older mice. Interestingly, the lack of pepsinogen C and expansion of Ki-67 were not associated with any detectable increase in SMA-positive stromal cells (Figure 2D), suggesting that the epithelial defects seen in the young mice would precede stromal defects observed in incipient and large polyps. On the other hand, in mice with a conditional ablation of Lkb1 only in stromal Tagln-CRE positive cells (Lkb1lox/+; TaglnCre/+ mice8Katajisto P Vaahtomeri K Ekman N Ventela E Ristimaki A Bardeesy N Feil R DePinho RA Makela TP LKB1 signaling in mesenchymal cells required for suppression of gastrointestinal polyposis.Nat Genet. 2008; 40: 455-459Crossref PubMed Scopus
Referência(s)