PPARβ/δ Regulates Paneth Cell Differentiation Via Controlling the Hedgehog Signaling Pathway
2006; Elsevier BV; Volume: 131; Issue: 2 Linguagem: Inglês
10.1053/j.gastro.2006.05.004
ISSN1528-0012
AutoresFrédéric Varnat, Béatrice Bordier–Ten Heggeler, Philippe Grisel, Nathalie Boucard, Irène Corthésy–Theulaz, Walter Wahli, Béatrice Desvergne,
Tópico(s)Cancer-related gene regulation
ResumoBackground & Aims: All 4 differentiated epithelial cell types found in the intestinal epithelium derive from the intestinal epithelial stem cells present in the crypt unit, in a process whose molecular clues are intensely scrutinized. Peroxisome proliferator–activated receptor β (PPARβ) is a nuclear hormone receptor activated by fatty acids and is highly expressed in the digestive tract. However, its function in intestinal epithelium homeostasis is understood poorly.Methods: To assess the role of PPARβ in the small intestinal epithelium, we combined various cellular and molecular approaches in wild-type and PPARβ-mutant mice.Results: We show that the expression of PPARβ is particularly remarkable at the bottom of the crypt of the small intestine where Paneth cells reside. These cells, which have an important role in the innate immunity, are strikingly affected in PPARβ-null mice. We then show that Indian hedgehog (Ihh) is a signal sent by mature Paneth cells to their precursors, negatively regulating their differentiation. Importantly, PPARβ acts on Paneth cell homeostasis by down-regulating the expression of Ihh, an effect that can be mimicked by cyclopamine, a known inhibitor of the hedgehog signaling pathway.Conclusions: We unraveled the Ihh-dependent regulatory loop that controls mature Paneth cell homeostasis and its modulation by PPARβ. PPARβ currently is being assessed as a drug target for metabolic diseases; these results reveal some important clues with respect to the signals controlling epithelial cell fate in the small intestine. Background & Aims: All 4 differentiated epithelial cell types found in the intestinal epithelium derive from the intestinal epithelial stem cells present in the crypt unit, in a process whose molecular clues are intensely scrutinized. Peroxisome proliferator–activated receptor β (PPARβ) is a nuclear hormone receptor activated by fatty acids and is highly expressed in the digestive tract. However, its function in intestinal epithelium homeostasis is understood poorly. Methods: To assess the role of PPARβ in the small intestinal epithelium, we combined various cellular and molecular approaches in wild-type and PPARβ-mutant mice. Results: We show that the expression of PPARβ is particularly remarkable at the bottom of the crypt of the small intestine where Paneth cells reside. These cells, which have an important role in the innate immunity, are strikingly affected in PPARβ-null mice. We then show that Indian hedgehog (Ihh) is a signal sent by mature Paneth cells to their precursors, negatively regulating their differentiation. Importantly, PPARβ acts on Paneth cell homeostasis by down-regulating the expression of Ihh, an effect that can be mimicked by cyclopamine, a known inhibitor of the hedgehog signaling pathway. Conclusions: We unraveled the Ihh-dependent regulatory loop that controls mature Paneth cell homeostasis and its modulation by PPARβ. PPARβ currently is being assessed as a drug target for metabolic diseases; these results reveal some important clues with respect to the signals controlling epithelial cell fate in the small intestine. An innate immune defense against microbes is critical to host survival. In this context, the mammalian intestinal mucosa constitutes a critical barrier against pathogens and commensal flora. In the small intestine, Paneth cells, which secrete a variety of microbicidal peptides such as α-defensins in response to luminal micro-organisms, are an important component of this defense. Paneth cells derive from the epithelial stem cells located in the crypt of Lieberkühn, which also produce 3 other specialized epithelial cells: enterocytes, goblet cells, and enteroendocrine cells. Unlike these latter 3, Paneth cells differentiate while migrating toward the crypt base, where they reside for about 20 days1Bry L. Falk P. Huttner K. Ouellette A. Midtvedt T. Gordon J.I. Paneth cell differentiation in the developing intestine of normal and transgenic mice.Proc Natl Acad Sci U S A. 1994; 91: 10335-10339Crossref PubMed Scopus (225) Google Scholar and are cleared by phagocytosis.2Garabedian E.M. Roberts L.J. McNevin M.S. Gordon J.I. Examining the role of Paneth cells in the small intestine by lineage ablation in transgenic mice.J Biol Chem. 1997; 272: 23729-23740Crossref PubMed Scopus (222) Google Scholar In addition to their role in innate defense, Paneth cells have been implicated in intestinal angiogenesis3Stappenbeck T.S. Hooper L.V. Gordon J.I. Developmental regulation of intestinal angiogenesis by indigenous microbes via Paneth cells.Proc Natl Acad Sci U S A. 2002; 99: 15451-15455Crossref PubMed Scopus (860) Google Scholar and are abnormally present in intestinal tumors and in inflammatory bowel diseases.4Porter E.M. Bevins C.L. Ghosh D. Ganz T. The multifaceted Paneth cell.Cell Mol Life Sci. 2002; 59: 156-170Crossref PubMed Scopus (335) Google Scholar Interestingly, Paneth cells that express CD15Lacasse J. Martin L.H. Detection of CD1 mRNA in Paneth cells of the mouse intestine by in situ hybridization.J Histochem Cytochem. 1992; 40: 1527-1534Crossref PubMed Scopus (28) Google Scholar and produce various mediators of inflammation such as granulocyte-macrophage colony–stimulating factor,6Fukuzawa H. Sawada M. Kayahara T. Morita-Fujisawa Y. Suzuki K. Seno H. Takaishi S. Chiba T. Identification of GM-CSF in Paneth cells using single-cell RT-PCR.Biochem Biophys Res Commun. 2003; 312: 897-902Crossref PubMed Scopus (47) Google Scholar tumor necrosis factor-α,7Schmauder-Chock E.A. Chock S.P. Patchen M.L. Ultrastructural localization of tumour necrosis factor-alpha.Histochem J. 1994; 26: 142-151Crossref PubMed Scopus (32) Google Scholar prostaglandin E2,8Schmauder-Chock E.A. Chock S.P. Prostaglandin E2 localization in the rat ileum.Histochem J. 1992; 24: 663-672Crossref PubMed Scopus (3) Google Scholar and Fas ligand9Moller P. Walczak H. Reidl S. Strater J. Krammer P.H. Paneth cells express high levels of CD95 ligand transcripts a unique property among gastrointestinal epithelia.Am J Pathol. 1996; 149: 9-13PubMed Google Scholar also might coordinate an innate and adaptive immune response. In mice, Paneth cells appear during the early postnatal period and increase strongly in number during the third postnatal week at weaning time.1Bry L. Falk P. Huttner K. Ouellette A. Midtvedt T. Gordon J.I. Paneth cell differentiation in the developing intestine of normal and transgenic mice.Proc Natl Acad Sci U S A. 1994; 91: 10335-10339Crossref PubMed Scopus (225) Google Scholar Recent observations made on mutant mouse lines suggest that Notch, Wnt, and Hedgehog signaling pathways are involved in intestinal epithelial cell proliferation and differentiation. For example, Math1−/− mice show a total absence of Paneth, goblet, and enteroendocrine cells.10Yang Q. Bermingham N.A. Finegold M.J. Zoghbi H.Y. Requirement of Math1 for secretory cell lineage commitment in the mouse intestine.Science. 2001; 294: 2155-2158Crossref PubMed Scopus (768) Google Scholar A careful dissection of the molecular mechanism led to a model according to which high levels of Notch proteins in undifferentiated progenitor cells up-regulate Hes1 expression, which in turn inhibits Math1 expression and promotes the differentiation of the progenitor cells into enterocytes. Conversely, a low level of Notch protein and thus of Hes1 results in increased Math1 expression and the commitment of the progenitors into the secretory lineage.10Yang Q. Bermingham N.A. Finegold M.J. Zoghbi H.Y. Requirement of Math1 for secretory cell lineage commitment in the mouse intestine.Science. 2001; 294: 2155-2158Crossref PubMed Scopus (768) Google Scholar Wnt signaling is important to maintain the undifferentiated state of intestinal progenitor cells. Inhibition of Wnt signaling by specific overexpression of Dickkopf1, a secreted Wnt inhibitor in the mouse intestinal epithelium, results in the depletion of the secretory cell lineage including Paneth cells.11Pinto D. Gregorieff A. Begthel H. Clevers H. Canonical Wnt signals are essential for homeostasis of the intestinal epithelium.Genes Dev. 2003; 17: 1709-1713Crossref PubMed Scopus (833) Google Scholar In addition, Wnt signaling participates in the final maturation process of Paneth cells.12van Es J.H. Jay P. Gregorieff A. van Gijn M.E. Jonkheer S. Hatzis P. Thiele A. van den Born M. Begthel H. Brabletz T. Taketo M.M. Clevers H. Wnt signalling induces maturation of Paneth cells in intestinal crypts.Nat Cell Biol. 2005; 7: 381-386Crossref PubMed Scopus (529) Google Scholar The role of hedgehog signaling in gut development is less clear. Recent work clearly shows that hedgehog proteins, secreted by epithelial cells, are responsible for crypt genesis.13Madison B.B. Braunstein K. Kuizon E. Portman K. Qiao X.T. Gumucio D.L. Epithelial hedgehog signals pattern the intestinal crypt-villus axis.Development. 2005; 132: 279-289Crossref PubMed Scopus (307) Google Scholar Moreover, in addition to an overt gut malrotation, Indian hedgehog (Ihh)−/− newborn mice show defective stem cell proliferation and differentiation, more particularly affecting enteroendocrine cell number.14Ramalho-Santos M. Melton D.A. McMahon A.P. Hedgehog signals regulate multiple aspects of gastrointestinal development.Development. 2000; 127: 2763-2772Crossref PubMed Google Scholar Peroxisome proliferator–activated receptors (PPARs) are transcription factors activated on ligand binding. They belong to the superfamily of nuclear receptors and their broad spectrum of ligands, most of them fatty acids or derivatives (prostaglandins and leukotrienes), place them as sensors involved in metabolic homeostasis. Three isotypes of PPAR, PPARα, PPARβ (also called δ, NUCI, and FAAR), and PPARγ (ie, NR1C1, NR1C2, and NR1C3, respectively, according to the unified nomenclature of nuclear hormone receptors15Nuclear Receptors Nomenclature CommitteeA unified nomenclature system for the nuclear receptor superfamily.Cell. 1999; 97: 161-163Abstract Full Text Full Text PDF PubMed Scopus (971) Google Scholar) have been cloned in Xenopus, rodents, and human beings.16Dreyer C. Krey G. Keller H. Givel F. Helftenbein G. Wahli W. Control of the peroxisomal beta-oxidation pathway by a novel family of nuclear hormone receptors.Cell. 1992; 68: 879-887Abstract Full Text PDF PubMed Scopus (1263) Google Scholar, 17Issemann I. Green S. Activation of a member of the steroid hormone receptor superfamily by peroxisome proliferators.Nature. 1990; 347: 645-650Crossref PubMed Scopus (3139) Google Scholar, 18Desvergne B. Wahli W. Peroxisome proliferator-activated receptors nuclear control of metabolism.Endocr Rev. 1999; 20: 649-688Crossref PubMed Scopus (2780) Google Scholar The ubiquitous expression of PPARβ and, until recent years, the unavailability of a PPARβ-specific ligand were practical reasons that hampered the functional characterization of this isotype. In addition, the invalidation of both PPARβ alleles is embryonic lethal, with high but not total penetrance. The rare mouse embryos that are born alive have allowed the generation of PPARβ-null mutant mouse lines, as obtained by us and others.19Peters J.M. Lee S.S. Li W. Ward J.M. Gavrilova O. Everett C. Reitman M.L. Hudson L.D. Gonzalez F.J. Growth, adipose, brain, and skin alterations resulting from targeted disruption of the mouse peroxisome proliferator-activated receptor beta(delta).Mol Cell Biol. 2000; 20: 5119-5128Crossref PubMed Scopus (606) Google Scholar, 20Barak Y. Liao D. He W. Ong E.S. Nelson M.C. Olefsky J.M. Boland R. Evans R.M. Effects of peroxisome proliferator-activated receptor delta on placentation, adiposity, and colorectal cancer.Proc Natl Acad Sci U S A. 2002; 99: 303-308Crossref PubMed Scopus (538) Google Scholar, 21Nadra K. Anghel S.I. Joye E. Tan N.S. Basu-Modak S. Trono D. Wahli W. Desvergne B. Differentiation of trophoblast giant cells and their metabolic functions are dependent on peroxisome proliferator-activated receptor beta/delta.Mol Cell Biol. 2006; 26: 3266-3281Crossref PubMed Scopus (174) Google Scholar These mice are born smaller than the wild-type controls and are poor breeders, but do not suffer from an overt phenotype. However, more subtle analyses identified a crucial role of PPARβ in wound healing via activation of the Akt1 pathway,22Michalik L. Desvergne B. Tan N.S. Basu-Modak S. Escher P. Rieusset J. Peters J.M. Kaya G. Gonzalez F.J. Zakany J. Metzger D. Chambon P. Duboule D. Wahli W. Impaired skin wound healing in peroxisome proliferator-activated receptor (PPAR)alpha and PPARbeta mutant mice.J Cell Biol. 2001; 154: 799-814Crossref PubMed Scopus (374) Google Scholar, 23Tan N.S. Michalik L. Noy N. Yasmin R. Pacot C. Heim M. Fluhmann B. Desvergne B. Wahli W. Critical roles of PPAR beta/delta in keratinocyte response to inflammation.Genes Dev. 2001; 15: 3263-3277Crossref PubMed Scopus (373) Google Scholar, 24Di-Poi N. Tan N.S. Michalik L. Wahli W. Desvergne B. Antiapoptotic role of PPARbeta in keratinocytes via transcriptional control of the Akt1 signaling pathway.Mol Cell. 2002; 10: 721-733Abstract Full Text Full Text PDF PubMed Scopus (291) Google Scholar as well as in energy homeostasis.25Oliver Jr, W.R. Shenk J.L. Snaith M.R. Russell C.S. Plunket K.D. Bodkin N.L. Lewis M.C. Winegar D.A. Sznaidman M.L. Lambert M.H. Xu H.E. Sternbach D.D. Kliewer S.A. Hansen B.C. Willson T.M. A selective peroxisome proliferator-activated receptor delta agonist promotes reverse cholesterol transport.Proc Natl Acad Sci U S A. 2001; 98: 5306-5311Crossref PubMed Scopus (980) Google Scholar, 26Wang Y.X. Lee C.H. Tiep S. Yu R.T. Ham J. Kang H. Evans R.M. Peroxisome-proliferator-activated receptor delta activates fat metabolism to prevent obesity.Cell. 2003; 113: 159-170Abstract Full Text Full Text PDF PubMed Scopus (1160) Google Scholar The high expression levels of PPARβ in the gut, from the duodenum to the ileum, with a lesser but still detectable expression in the colon,27Braissant O. Foufelle F. Scotto C. Dauca M. Wahli W. Differential expression of peroxisome proliferator-activated receptors (PPARs) tissue distribution of PPAR-alpha, -beta, and -gamma in the adult rat.Endocrinology. 1996; 137: 354-366Crossref PubMed Scopus (1737) Google Scholar, 28Escher P. Braissant O. Basu-Modak S. Michalik L. Wahli W. Desvergne B. Rat PPARs quantitative analysis in adult rat tissues and regulation in fasting and refeeding.Endocrinology. 2001; 142: 4195-4202Crossref PubMed Scopus (347) Google Scholar suggest a role in intestinal functions, which stimulated a careful evaluation of the gut phenotype of PPARβ-null mice. Our results show that PPARβ is required for the differentiation of Paneth cells. In addition, we show that PPARβ allows the differentiation of Paneth cell precursors into fully mature Paneth cells by inhibiting the activity of the hedgehog signaling pathway. The PPARβ–selective agonist L-165041 (4-[3-[2-propyl-3-hydroxy-4-acetyl] phenoxy] propyloxyphenoxy acetic acid)29Berger J. Leibowitz M.D. Doebber T.W. Elbrecht A. Zhang B. Zhou G. Biswas C. Cullinan C.A. Hayes N.S. Li Y. Tanen M. Ventre J. Wu M.S. Berger G.D. Mosley R. Marquis R. Santini C. Sahoo S.P. Tolman R.L. Smith R.G. Moller D.E. Novel peroxisome proliferator-activated receptor (PPAR)gamma and PPARdelta ligands produce distinct biological effects.J Biol Chem. 1999; 274: 6718-6725Crossref PubMed Scopus (380) Google Scholar was synthesized in our laboratory but now is available commercially (cat# 422175; Calbiochem; Dormstadt, Germany). All animals had free access to a standard laboratory chow diet in a temperature- and light-controlled environment. PPARβ-null mice and their control wild-type were bred on a mixed genetic background (SV129 and C57BL/6) and were killed at 10 weeks of age. After death by CO2 inhalation the proximal duodenum, jejunum, and distal ileum were dissected out and abundantly flushed with cold phosphate-buffered saline (PBS). Scraped mucosa were frozen in liquid nitrogen for subsequent RNA and protein analysis or processed for histologic analyses. Total RNA was extracted using TRIzol LS Reagent (Gibco, Carlsbad, CA). Chemical treatments were administered to 10-week-old male mice. L-165041 in .5% carboxymethylcellulose was administered orally at a dose of 30 mg/kg per day for 12 days.29Berger J. Leibowitz M.D. Doebber T.W. Elbrecht A. Zhang B. Zhou G. Biswas C. Cullinan C.A. Hayes N.S. Li Y. Tanen M. Ventre J. Wu M.S. Berger G.D. Mosley R. Marquis R. Santini C. Sahoo S.P. Tolman R.L. Smith R.G. Moller D.E. Novel peroxisome proliferator-activated receptor (PPAR)gamma and PPARdelta ligands produce distinct biological effects.J Biol Chem. 1999; 274: 6718-6725Crossref PubMed Scopus (380) Google Scholar, 30Leibowitz M.D. Fievet C. Hennuyer N. Peinado-Onsurbe J. Duez H. Bergera J. Cullinan C.A. Sparrow C.P. Baffic J. Berger G.D. Santini C. Marquis R.W. Tolman R.L. Smith R.G. Moller D.E. Auwerx J. Activation of PPARdelta alters lipid metabolism in db/db mice.FEBS Lett. 2000; 473: 333-336Abstract Full Text Full Text PDF PubMed Scopus (297) Google Scholar, 31Letavernier E. Perez J. Joye E. Bellocq A. Fouqueray B. Haymann J.P. Heudes D. Wahli W. Desvergne B. Baud L. Peroxisome proliferator-activated receptor beta/delta exerts a strong protection from ischemic acute renal failure.J Am Soc Nephrol. 2005; 16: 2395-2402Crossref PubMed Scopus (104) Google Scholar Cyclopamine (Toronto Research Chemicals, North York, Canada) was prepared as described previously and was administered by intraperitoneal injections at a dose of 2 mg/kg per day for 14 days.32van den Brink G.R. Hardwick J.C. Tytgat G.N. Brink M.A. Ten Kate F.J. Van Deventer S.J. Peppelenbosch M.P. Sonic hedgehog regulates gastric gland morphogenesis in man and mouse.Gastroenterology. 2001; 121: 317-328Abstract Full Text Full Text PDF PubMed Scopus (220) Google Scholar All animal procedures were performed with authorization from the cantonal veterinary service of the Canton of Vaud. HT-29 cells were cultured in Dulbecco's modified Eagle medium (Sigma, Buchs, Switzerland) containing 4.5 mg/L glucose supplemented with 10% fetal calf serum, 50 mg/mL penicillin, and 50 mg/mL streptomycin (Gibco). Cell differentiation was induced by adding 2.5 mmol/L of sodium butyrate to the culture medium. Cells were treated with 1 μmol/L L-165041, 2.5 mg/mL recombinant Sonic hedgehog (Shh) (purchased from R&D, Minneapolis, MN), or 2 mg/mL cyclopamine (Toronto Research Chemicals) for the indicated amount of time. Intestines were fixed in 4% paraformaldehyde–PBS 1×, dehydrated, and embedded in paraffin according to the standard procedure. Sections (5 μm) were cut and processed for staining or immunohistology techniques. For electron microscopy, small pieces of duodenum (2 mm2) were fixed in a mixture of paraformaldehyde (2% wt/vol) and glutaraldehyde (2% vol/vol) in sodium cacodylate buffer (.1 mol/L, pH 7.4) for 2 hours at room temperature. Samples subsequently were submitted to 2 hours of postfixation in 1.5% osmium tetroxide, stained for 2 hours at 4°C with uranyl acetate (2%) in sodium maleate buffer (.1 mol/L, pH 6.0), dehydrated in graded alcohols, and embedded in Epon 812 (Shell Chemical Corp, Cleveland, OH). The size of Paneth cell granules was assessed by light microscopy on 1-μm Epon sections stained with methylene blue, azure II, and fuchsin. Electron microscopy was performed on 60-nm ultrathin sections contrasted with uranyl acetate and lead citrate with a Philips CM 10 electron microscope (FEI Company, Eindhoven, the Netherlands). After dissection, intestinal pieces were washed and flushed with cold PBS, embedded in a tissue-freezing medium (Tissue-Tek O.C.T., Digitana AG, Yverdon-les-bains, Switzerland), and stored at −70°C until processing. Cryosections (8 μm) were obtained with a Frigocut 2800/Reichert–Jung cryostat (Microm International, Walldorf, Germany). Slides were fixed in 4% paraformaldehyde PBS and washed in active PBS diethylpyrocarbonate (DEPC) and sodium and sodium citrate (SSC) 4×. Sections were prehybridized at 60°C for 2 hours in prehybridization buffer (50% deionized formamide, SSC 4×, Denhardt 1×, and .2 ng/mL salmon sperm DNA in a box containing 50% deionized formamide, 25% SSC 20×, and 25% H2O DEPC). Sections subsequently were hybridized for 48 hours at 60°C in the prehybridization buffer supplemented with 500 ng/mL of uridine triphosphate–digoxygenin–labeled sense or antisense riboprobes. After washing in SSC 4×, sections were incubated for 2 hours in Tris/NaCl 100/150 mmol/L, pH 7.5, .5% casein, and anti–digoxygenin (DIG) antibody coupled to alkaline phosphatase (1:3000, Roche, Basel, Switzerland). Finally, sections were incubated with alkaline phosphatase substrates that formed a blue precipitate (Tris/NaCl/MgCl2 100/150/50 mmol/L, pH 9.5, nitro blue tetrazolium (NBT)-blue 4.5 μL/mL, X-phosphate 3.5 μL/mL), allowing the visualization of the messenger RNA (mRNA) localization. The gene-specific probe corresponding to PPARβ (nucleotides 42–426 from the start codon ATG) were cloned in the pGEM-3Zf(+) vector (Promega, Madison, WI). Shh (5–183), Ihh (638–952), Ptch-1 (909–1199), BMP-4 (250–541), L27 (152–308), cryptdin-1 (119–432), L-FABP (63–378), c-myc (1741–2028), EphB3 (665–1015), and math-1 (417–741) were cloned into a pGEM-T Easy vector (Promega). Gene-specific antisense riboprobes were synthesized by in vitro transcription with either T7 or Sp6 RNA polymerase (Ambion, Austin, TX). RNase Protection Assay (RPA) on total RNA extracted with TRIzol LS reagent (Gibco) was performed as described by the manufacturer (Direct Protect Lysate RPA, Ambion). Ten micrograms of total RNA was mixed with 1 ng of gene-specific riboprobes and 10 ng of L27 probe. RPA products were resolved in a 6% denaturing polyacrylamide gel. Gels were dried and exposed to radiograph film or to Phosphor screens (GE Health Care, Fairfield, CT). Tissues were lysed in ice-cold lysis buffer (20 mmol/L Na2H2PO4, 250 mmol/L NaCl, 1% Triton X-100, and .1% sodium dodecyl sulfate) supplemented with complete protease inhibitor (Roche). Equal amounts of protein extracts were resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and electrotransferred onto polyvinylidene difluoride membranes. Membranes were processed as described by the manufacturer (Cell Signaling, Beverly, MA) and detected by chemiluminescence (Pierce, Rockford, IL). Lysozyme, β-tubulin, hedgehog proteins, Ihh, and BMP-4 were detected using a rabbit polyclonal antibody (Dako, Glostrup, Denmark), the 5H1 monoclonal antibody (Pharmingen, San Diego, CA), the 5E1 monoclonal antibody (Developmental Studies Hybridoma Bank, Iowa City, IA), and the goat polyclonal antibodies I-19 and N-16 (Santa Cruz Biotechnology, Santa Cruz, CA), respectively. Villin was detected with a monoclonal antibody from BD Transduction Laboratories (V34420; Franklin Lakes, NJ). Paraffin sections were hydrated and boiled for 10 minutes in .01 mol/L sodium citrate, pH 6. For immunohistochemistry, endogenous peroxidase activity was inhibited by 3% H2O2 in PBS. Sections were incubated in 5% normal serum in PBS–1% Triton X-100 before primary antibody incubation. Sections subsequently were incubated with the appropriate biotinylated secondary antibody and detection was performed with the Vectastain ABC kit (Vector Laboratories, Burlingame, CA), followed by incubation with diaminobenzidin (DAB) peroxidase substrate alone or with metal enhancer (Sigma). For immunofluorescence, a fluorescein isothiocyanate–coupled secondary antibody was used. For double immunofluorescence, sections were incubated in 5% normal goat serum in PBS–.1% Triton X-100 before primary antibody incubation. Sections were subsequently incubated with secondary antibodies (fluorescein isothiocyanate–coupled goat anti-rabbit [Sigma] and rhodamine-coupled donkey anti-goat [Santa Cruz Biotechnology]). Primary anti–Ki-67 (NCL-Ki67p) antibodies were purchased from Novocastra (Newcastle Upon Tyne, UK). Antilysozyme antibodies were purchased from Dako. Anti–chromogranin A (18-0094) was purchased from Zymed (San Francisco, CA). Anti–Ptch-1 (G-19), anti–Hedgehog interacting protein (Hip) (M-17), and anti-Ihh (C-15) primary antibodies were purchased from Santa Cruz Biotechnology. Anti-PPARβ primary antibodies (PA1-823) were purchased from ABR Affinity Bioreagents (Golden, CO). Scrapped duodenal mucosa were lysed and mechanically homogenized with a douncer in an NP40 lysis buffer (20 mmol/L Tris pH 7.4, 150 mmol/L NaCl, 2.5 mmol/L KCl, 5 mmol/L ethylenediaminetetraacetic acid, glycerol [5% vol/vol], NP40 [1% vol/vol]) supplemented with Complete protease inhibitor (Roche). In triplicates, 60 μg of duodenal mucosa lysates were incubated for 60 minutes at 37°C in 10 mmol/L piperazine N,N'bis [2-ethanesulfonic acid] (PIPES) buffer (pH 7.4) containing exponentially growing Escherichia coli K12 (106 colony-forming units (CFU)/mL). A 20-μL sample subsequently was diluted 1:1000 in 10 mmol/L PIPES buffer (pH 7.4) and 50 μL of this dilution was plated on Luria broth (LB)-agar solid medium. Surviving bacteria were quantitated as CFU on plates after incubation overnight at 37°C. Duodenum, jejunum, and ileum segments were obtained under sterile conditions. Samples were analyzed immediately by serial dilution and plated on semiselective media.33Guigoz Y. Rochat F. Perruisseau-Carrier G. Rochat I. Schiffrin E.J. Effects of oligosaccharide on the faecal flora and non-specific immune system in elderly people.Nutr Res. 2002; 22: 13-25Abstract Full Text Full Text PDF Scopus (180) Google Scholar Lactobacilli, bifidobacteria, and bacteroides were incubated anaerobically at 37°C for 48 hours. Enterobacteria, staphylococci, and enterococci were incubated aerobically at 37°C for 24 hours. The staphylococci were plated in Chapman Medium (BioMérieux, Marcy L'Etoile, France). Bacterial populations were estimated by counting the CFUs and the counts were expressed in CFU log10 per gram of tissue. The detection limit is 3.00 log10 CFU/g. Scraped duodenal mucosa were lysed with a douncer in an NP40 lysis buffer (20 mmol/L Tris pH 7.4, 150 mmol/L NaCl, 2.5 mmol/L KCl, 5 mmol/L ethylenediaminetetraacetic acid, glycerol [5% vol/vol], NP40 [1% vol/vol]) supplemented with Complete protease inhibitor (Roche). One microgram of each sample was electrophoresed in a 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis containing .1% casein (Fluka, Buchs, Switzerland). After electrophoresis, the gels were washed twice with 2.5% Triton X-100 for 60 minutes to remove the sodium dodecyl sulfate. The gels then were incubated in 50 mmol/L Tris-Cl (pH 7.5), 5 mmol/L CaCl2, 1 μmol/L ZnCl2 at 37°C for 20 hours, stained with Coomassie brilliant blue, and destained with a solution at 10% acetatic acid and 30% isopropanol. One microliter of trypsin–ethylenediaminetetraacetic acid (Gibco-BRL) was used as a positive control. PPARβ is expressed in all parts of the small intestine in the mouse, with the highest expression in the duodenal mucosa (Figure 1A). It is present mainly in the epithelial cells, with a decreasing gradient of expression from the bottom to the top of the villi (Figure 1B). The highest PPARβ expression is at the bottom of the crypts, with a marked decrease from cell position 9 upward (Figure 1C). In addition to Paneth cells, stem cells (for which the lack of a reliable marker limits determination of their number and position) are present in the lower part of each small intestinal crypt, where PPARβ is highly expressed. In addition to a moderate impairment in duodenal epithelial cell proliferation (Table 1), the most remarkable feature of the gut epithelium in PPARβ-null mutant mice was the low number of Paneth cells in adult PPARβ-null mice compared with the wild-type controls. This defect was most pronounced in the duodenum (Figure 2A and Table 1). Consistent with this, lysozyme protein, α-defensin cryptdin-1 mRNA, matrix metalloproteinase-7, and trypsin activity, used as Paneth cell markers,34Wilson C.L. Ouellette A.J. Satchell D.P. Ayabe T. Lopez-Boado Y.S. Stratman J.L. Hultgren S.J. Matrisian L.M. Parks W.C. Regulation of intestinal alpha-defensin activation by the metalloproteinase matrilysin in innate host defense.Science. 1999; 286: 113-117Crossref PubMed Scopus (930) Google Scholar, 35Wilson C.L. Heppner K.J. Rudolph L.A. Matrisian L.M. The metalloproteinase matrilysin is preferentially expressed by epithelial cells in a tissue-restricted pattern in the mouse.Mol Biol Cell. 1995; 6: 851-869Crossref PubMed Scopus (134) Google Scholar, 36Ghosh D. Porter E. Shen B. Lee S.K. Wilk D. Drazba J. Yadav S.P. Crabb J.W. Ganz T. Bevins C.L. Paneth cell trypsin is the processing enzyme for human defensin-5.Nat Immunol. 2002; 3: 583-590Crossref PubMed Scopus (370) Google Scholar were reduced significantly in PPARβ-null mice (Figure 2B and C). In electron microscopy, Paneth cells from PPARβ−/− mice showed a quasi-absence of large granules (diameter, >5 μm), but an increased proportion of small granules (diameter, <2 μm) (Figure 2D). In addition, the rough endoplasmic reticulum appeared less compact in the PPARβ−/− mice compared with the wild-type control (Figure 2E), suggesting a role of PPARβ in the proper organization of the secretory pathway of the Paneth cells. This relative deficiency of Paneth cells is not caused by apoptotic events because they were not detected in the crypts of wild-type and PPARβ-null mice as assessed by terminal deoxynucleotidyl transferase–mediated deoxyuridine triphosphate nick-end labeling assay (data not shown).Table 1Main Characteristics of PPARβ−/− Small-Intestine EpitheliumCell type and parametersGenotypeDuodenumJejunumIleumEpithelial cell proliferation in the cryptsPPARβ+/+30.2 ± 1.918.8 ± 1.112.5 ± .2PPARβ−/−25.7 ± 1.719.6 ± .511.6 ± .3P = .01P = .5P = .4Goblet cellsPPARβ+/+.10 ± .01.10 ± .01.11 ± .004PPARβ−/−.11 ± .01.11 ± .003.13 ± .01P = .2P = .3P = .1Enteroendo
Referência(s)