Targeted Inactivation of the Mouse Guanylin Gene Results in Altered Dynamics of Colonic Epithelial Proliferation
2002; Elsevier BV; Volume: 161; Issue: 6 Linguagem: Inglês
10.1016/s0002-9440(10)64494-x
ISSN1525-2191
AutoresKris A. Steinbrecher, Steve A. Wowk, Jeffrey A. Rudolph, David P. Witte, Mitchell B. Cohen,
Tópico(s)Bacterial Genetics and Biotechnology
ResumoHeat-stable enterotoxin (STa), elaborated by enterotoxigenic Echerichia coli, is a worldwide cause of secretory diarrhea in infants and travelers. Both STa and guanylin, a peptide structurally similar to STa, increase intracellular cGMP levels after binding to the same intestinal receptor, guanylate cyclase C (GC-C). Distinct from its role as an intestinal secretagogue, guanylin may also have a role in intestinal proliferation, as guanylin expression is lost in intestinal adenomas. To determine the function of guanylin in intestinal epithelia, guanylin null mice were generated using a Cre/loxP-based targeting vector. Guanylin null mice grew normally, were fertile and showed no signs of malabsorption. However, the levels of cGMP in colonic mucosa of guanylin null mice were significantly reduced. The colonic epithelial cell migration rate was increased and increased numbers of colonocytes expressing proliferating cell nuclear antigen (PCNA) were present in crypts of guanylin null mice as well. The apoptotic index was similar in guanylin null mice and littermate controls. We conclude from these studies that loss of guanylin results in increased proliferation of colonic epithelia. We speculate that the increase in colonocyte number is related to decreased levels of cGMP and that this increase in proliferation plays a role in susceptibility to intestinal adenoma formation and/or progression. Heat-stable enterotoxin (STa), elaborated by enterotoxigenic Echerichia coli, is a worldwide cause of secretory diarrhea in infants and travelers. Both STa and guanylin, a peptide structurally similar to STa, increase intracellular cGMP levels after binding to the same intestinal receptor, guanylate cyclase C (GC-C). Distinct from its role as an intestinal secretagogue, guanylin may also have a role in intestinal proliferation, as guanylin expression is lost in intestinal adenomas. To determine the function of guanylin in intestinal epithelia, guanylin null mice were generated using a Cre/loxP-based targeting vector. Guanylin null mice grew normally, were fertile and showed no signs of malabsorption. However, the levels of cGMP in colonic mucosa of guanylin null mice were significantly reduced. The colonic epithelial cell migration rate was increased and increased numbers of colonocytes expressing proliferating cell nuclear antigen (PCNA) were present in crypts of guanylin null mice as well. The apoptotic index was similar in guanylin null mice and littermate controls. We conclude from these studies that loss of guanylin results in increased proliferation of colonic epithelia. We speculate that the increase in colonocyte number is related to decreased levels of cGMP and that this increase in proliferation plays a role in susceptibility to intestinal adenoma formation and/or progression. Ion flow across epithelial cell membranes is tightly controlled by various factors, including hormones and the enteric nervous system. Dysregulation of this system by bacterial pathogens is the basis for many secretory diarrheal diseases. Enterotoxigenic Escherichia coli that elaborate heat-stable enterotoxin (STa) are a worldwide cause of secretory diarrhea, especially in infants and travelers.1Cohen MB Giannella RA Enterotoxigenic Escherichia coli.in: Blaser MJ Smith PD Ravdin JI Greenberg HB Infections of the Gastrointestinal Tract. ed 2. Raven Press, New York2002: 579-594Google Scholar The low-molecular-weight peptide guanylin is highly homologous to STa and is secreted from the epithelial cell layer of the small and large intestine.2Currie MG Fok KF Kato J Moore RJ Hamra FK Duffin KL Smith CE Guanylin: an endogenous activator of intestinal guanylate cyclase.Proc Natl Acad Sci USA. 1992; 89: 947-951Crossref PubMed Scopus (510) Google Scholar, 3Forte LR Currie MG Guanylin: a peptide regulator of epithelial transport.EMBO J. 1995; 9: 643-650Google Scholar, 4Cohen MB Witte DP Hawkins JA Currie MG Immunohistochemical localization of guanylin in the rat small intestine and colon.Biochem Biophys Res Commun. 1995; 209: 803-808Crossref PubMed Scopus (59) Google Scholar Once elaborated into the intestinal lumen, guanylin, another similar mammalian peptide, uroguanylin, and STa can all bind to the receptor guanylate cyclase C (GC-C), located on the enterocyte brush border membrane.5Gao Z Yuen PS Garbers DL Interruption of specific guanylyl cyclase signaling pathways.Adv Second Messenger Phosphoprotein Res. 1997; 31: 183-190Crossref PubMed Google Scholar, 6Schulz S Green CK Yuen PS Garbers DL Guanylyl cyclase is a heat-stable enterotoxin receptor.Cell. 1990; 63: 941-948Abstract Full Text PDF PubMed Scopus (521) Google Scholar, 7Garbers DL Guanylyl cyclase receptors and their ligands.Adv Second Messenger Phosphoprotein Res. 1993; 28: 91-95PubMed Google Scholar Ligand binding to GC-C initiates a signal transduction cascade that culminates in activation of the cystic fibrosis transmembrane conductance regulator (CFTR).8Cuthbert AW Hickman ME MacVinish LJ Evans MJ Colledge WH Ratcliff R Seale PW Humphrey PP Chloride secretion in response to guanylin in colonic epithelial from normal and transgenic cystic fibrosis mice.Br J Pharmacol. 1994; 112: 31-36Crossref PubMed Scopus (91) Google Scholar, 9Chao AC de Sauvage FJ Dong YJ Wagner JA Goeddel DV Gardner P Activation of intestinal CFTR Cl-channel by heat-stable enterotoxin and guanylin via cAMP-dependent protein kinase.EMBO J. 1994; 13: 1065-1072Crossref PubMed Scopus (234) Google Scholar In this system, STa acts as a superagonist that results in secretory diarrhea. Similarity in structure between guanylin and STa led to the hypothesis that guanylin contributes to regulation of gastrointestinal fluid homeostasis. However, distinct from its role as an intestinal secretagogue, guanylin may also have a role in intestinal proliferation. For example, we have previously shown that guanylin expression is lost in mouse and human intestinal adenomas.10Steinbrecher KA Tuohy TM Heppner Goss K Scott MC Witte DP Groden J Cohen MB Expression of guanylin is down-regulated in mouse and human intestinal adenomas.Biochem Biophys Res Commun. 2000; 273: 225-230Crossref PubMed Scopus (67) Google Scholar, 11Cohen MB Hawkins JA Witte DP Guanylin mRNA expression in human intestine and colorectal adenocarcinoma.Lab Invest. 1998; 78: 101-108PubMed Google Scholar To determine the effect of loss of guanylin activity in the whole animal, we inactivated the mouse guanylin gene using homologous recombination. We used the Cre/loxP system to target the mouse guanylin gene in a manner that allowed for removal of the targeting selection cassette and minimal alteration of the sequence surrounding the guanylin allele.12Shibata H Toyama K Shioya H Ito M Hirota M Hasegawa S Matsumoto H Takano H Akiyama T Toyoshima K Kanamaru R Kanegae Y Saito I Nakamura Y Shiba K Noda T Rapid colorectal adenoma formation initiated by conditional targeting of the Apc gene.Science. 1997; 278: 120-123Crossref PubMed Scopus (474) Google Scholar We demonstrate that mice lacking guanylin develop normally, are fertile, and display no evidence of intestinal obstruction or obvious loss in intestinal absorptive capacity. However, we demonstrate that levels of cGMP are lower in the colonic epithelia of guanylin null mice. Furthermore, we show that the amount of apoptosis in intestinal epithelia is unchanged but the rate of proliferation of colonic epithelia in these mice is significantly increased. The targeting construct contained the third exon of the guanylin gene, which encodes for the active guanylin peptide, as well as an hypoxanthine-guanine phosphoribosyl transferase (HPRT) selection cassette flanked by loxP sites (Figure 1). Cloning of the complete mouse guanylin gene and surrounding sequence was described previously.13Sciaky D Jenkins NA Gilbert DJ Copeland NG Sonoda G Testa JR Cohen MB Mapping of guanylin to murine chromosome 4 and human chromosome 1p34–p35.Genomics. 1995; 26: 427-429Crossref PubMed Scopus (16) Google Scholar A loxP-flanked, or floxed, HPRT selection cassette and Cre recombinase expression plasmid used in these studies were generously provided by Dr. Joanna Groden of the University of Cincinnati. All bacterial transformations and preparations were performed according to standard practices with recombination-deficient STBL2 competent E. coli cells (Life Technologies, Gaithersburg, MD). Construction of the guanylin targeting vector began by cloning a single loxP site into the second intron of guanylin. A 4.5-kb fragment (− 1886 to + 2714; numbering based on Reference 32Gallagher AM Gottlieb RA Proliferation, not apoptosis, alters epithelial cell migration in small intestine of CFTR null mice.Am J Physiol. 2001; 281: G681-G687Google Scholar) that encompassed the entire guanylin gene and the intronic loxP was placed just 5[prime] of the floxed HPRT cassette. Polymerase chain reaction (PCR) was performed using Pfu DNA polymerase to amplify a 4.0-kb region (+2714 to + 6653) downstream of exon 3 of the guanylin gene region. This PCR fragment was then cloned into the 3[prime] end of the floxed HPRT cassette. This vector, pGEM(1E12), containing the floxed third exon of guanylin and a floxed HPRT cassette, was used to target the mouse guanylin gene locus (Figure 1). Before this, the construct was sequenced (University of Cincinnati Sequencing Core) at all ligation junctions and loxP sites to ensure base sequence fidelity and correct orientation of loxP sequence. The University of Cincinnati Transgenic Core Facility performed all embryonic stem (ES) cell targeting and blastocyst injection procedures based on our genotyping of clones as detailed below. The targeting vector pGEM (1E12) was linearized and electroporated into 129/SV strain E14 ES cells. All genotyping of both ES cells and mice was performed using Southern analysis or PCR according to standard protocols. Of 220 ES cell clones screened by Southern analysis, 15 correctly recombined clones were identified. Southern analysis was performed using EcoRI, XbaI, and Hind III restriction enzyme digests with three probes that hybridize to regions inclusive or exclusive of the original targeting vector sequence. Restriction sites are shown in Figure 1, as are two probes used in Southern analysis. Following electroporation with a Cre recombinase expression plasmid, PCR screening and Southern analysis was used to identify ES cells containing either Type I (total recombination between outer loxP sites, Figure 1D) or Type II recombinations (recombination between the loxP sites that flank the HPRT cassette, Figure 1E). The vast majority of clones were Type I deletions but several also contained the Type II, floxed third exon allele. Clones from both the Type I and II deletion ES cells were injected into C57BL/6-derived blastocysts and implanted into pseudopregnant females. All mice used in these studies were cared for under guidelines defined by the University of Cincinnati or the Children's Hospital Medical Center Institutional Animal Care and Use Committee. Mice had access to food and water and were housed in a temperature, humidity, and light-cycle controlled pathogen-free, micro-isolation facility. The studies described here were performed on mice bred 3 to 4 generations into the Balb/C strain and littermate mice were used as control animals. Animals were sacrificed by CO2 asphyxiation; this was performed during a 2-hour window to avoid differences in circadian expression of guanylin and uroguanylin. For protein and mRNA studies, the intestine was flushed with cold saline, separated into segments, and stored at − 80°C. Intestinal segments were defined as follows: proximal jejunum (proximal third of the small intestine); ileum (distal third of the small intestine); cecum; proximal colon (proximal 40% of colon); and distal colon (distal 60% of the colon). Frozen tissue was pulverized in chilled mortars and pestles and RNA was extracted using Trizol reagent (Gibco BRL, Gaithersburg, MD) as described previously.14Steinbrecher KA Mann EA Giannella RA Cohen MB Increases in guanylin and uroguanylin in a mouse model of osmotic diarrhea are guanylate cyclase c-independent.Gastroenterology. 2001; 121: 1191-1202Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar Portions of the guanylin, uroguanylin, guanylate cyclase-C, down-regulated in adenoma (DRA), sodium hydrogen exchanger 3 (NHE3), and aquaporin 4 and 8 cDNAs were radiolabeled with [α-32P] (DuPont-NEN, Boston, MA) using the Random Primer Labeling system (Roche Molecular Biochemicals, Indianapolis, IN) as described previously.15Swenson ES Mann EA Jump ML Witte DP Giannella RA The guanylin/STa receptor is expressed in crypts and apical epithelium throughout the mouse intestine.Biochem Biophys Res Commun. 1996; 225: 1009-1014Crossref PubMed Scopus (58) Google Scholar, 16Whitaker TL Witte DP Scott MC Cohen MB Uroguanylin and guanylin: distinct but overlapping patterns of messenger RNA expression in mouse intestine.Gastroenterology. 1997; 113: 1000-1006Abstract Full Text PDF PubMed Scopus (62) Google Scholar Blots were visualized and quantitated using a Molecular Dynamics PhosphorImager system (Molecular Dynamics, Sunnyvale, CA). Animals were sacrificed and tissues collected as described above. Tissue was homogenized and processed as described previously.14Steinbrecher KA Mann EA Giannella RA Cohen MB Increases in guanylin and uroguanylin in a mouse model of osmotic diarrhea are guanylate cyclase c-independent.Gastroenterology. 2001; 121: 1191-1202Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar Membranes were immunoblotted using a 1:1000 dilution of antisera that recognizes proguanylin (antibody 2538) and prouroguanylin (antibody 6910).14Steinbrecher KA Mann EA Giannella RA Cohen MB Increases in guanylin and uroguanylin in a mouse model of osmotic diarrhea are guanylate cyclase c-independent.Gastroenterology. 2001; 121: 1191-1202Abstract Full Text Full Text PDF PubMed Scopus (16) Google Scholar These antibodies were generously provided by Dr. Michael Goy of the University of North Carolina and have been validated and described previously.17Perkins A Goy MF Li Z Uroguanylin is expressed by enterochromaffin cells in the rat gastrointestinal tract.Gastroenterology. 1997; 113: 1007-1014Abstract Full Text PDF PubMed Scopus (73) Google Scholar, 18Li Z Taylor-Blake B Light AR Goy MF Guanylin, an endogenous ligand for C-type guanylate cyclase, is produced by goblet cells in the rat intestine.Gastroenterology. 1995; 109: 1863-1875Abstract Full Text PDF PubMed Scopus (57) Google Scholar Following incubation with a horseradish peroxidase-conjugated secondary antibody, signal was visualized on Kodak X-OMAT AR film using a commercially available chemiluminescent kit (NEN Life Science Products, Boston, MA). As a control for loading, blots were reevaluated with an actin probe (gift of J.L. Lessard, Children's Hospital Research Foundation, Cincinnati, OH). Guanylin heterozygous and null animal littermates were sacrificed and tissues collected as above except that tissue was initially placed in 10% neutral-buffered formalin. Intestinal tissue was cut longitudinally on foam biopsy sponges and fixed as flat sheets. After fixing for ∼18 hours in formalin, samples were then removed and dehydrated through an increasing ethanol series over an 6-hour period, cleared in the xylene substitute Hemo-D (Fisher Scientific, Pittsburgh, PA) for 2 hours, and infiltrated with two changes of paraffin (Paraplast X-Tra Tissue Embedding media, Fisher Scientific) for 1 hour each. Tissue was then mounted in paraffin blocks and sectioned at 5-μm thickness for subsequent analysis. Initial characterization of both guanylin heterozygous and null mouse intestinal sections was performed using Harris' hematoxylin and eosin staining according to standard protocols. To identify goblet cells, we used periodic acid-Schiff (PAS) reagents to stain the mucin stores that are found in these cells. A commercially available PAS staining system (Sigma Diagnostics, St. Louis, MO) was used according to the manufacturer's protocol. Ileum and colon were dissected from both wild-type and guanylin null mice. These segments were flushed with phosphate-buffered saline and laid flat using a lengthwise incision. A glass slide was used to scrape the mucosal surface of these segments, and the scrapings were immediately placed in liquid nitrogen. The tissue was homogenized in 6% trichloroacetic acid to give a 10% w/v homogenate. The homogenate was washed five times with four volumes of water-saturated diethyl ether. These samples were then dried down under nitrogen for 30 minutes, and resuspended in 0.05 mol/L sodium acetate, pH 6.2. cGMP was then measured in a validated radioimmunoassay and cGMP extractions were normalized per gram of tissue wet weight.19Balint JP Kosiba JL Cohen MB The heat-stable enterotoxin-guanylin receptor is expressed in rat hepatocytes and in a rat hepatoma (H-35) cell line.J Recept Signal Transduct Res. 1997; 17: 609-630Crossref PubMed Scopus (7) Google Scholar Two centimeters of terminal ileum and proximal colon were dissected from both wild-type and guanylin null mice, flushed with phosphate buffered saline, and transected longitudinally. Each segment was divided into equal pieces and submerged in 500 μmol/L 3-isobutyl-1-methylxanthine (IBMX; phosphodiesterase inhibitor) in Hanks' buffered salt solution (HBSS), pH 7.0, for 15 minutes. The explants were removed and placed in IBMX/HBSS in the presence or absence of 5 μmol/L STa and incubated for a further 15 minutes at 37°C. The explant was removed, homogenized in 6% trichloroacetic acid, and cGMP was extracted as described above. cGMP levels were measured by radioimmunoassay. Duplicate segments from each mouse were analyzed for basal activity and STa-stimulated cGMP accumulation. The guanylyl cyclase activity was measured as femtomoles cGMP produced per gram of tissue wet weight per minute incubation at 37°C. Crypt depth, used as an estimate of proliferation and apoptosis rates in colonic epithelia,20Stern LE Falcone Jr, RA Kemp CJ Braun MC Erwin CR Warner BW Salivary epidermal growth factor and intestinal adaptation in male and female mice.Am J Physiol. 2000; 278: G871-G877Google Scholar, 21Helmrath MA VanderKolk WE Can G Erwin CR Warner BW Intestinal adaptation following massive small bowel resection in the mouse.J Am Coll Surg. 1996; 183: 441-449PubMed Google Scholar was measured in proximal and distal colon of heterozygous and null mice as follows. Paraffin-embedded tissue was sectioned at 5.0-μm thickness and stained with hematoxylin and eosin as above. Crypts were measured using image analysis software (NIH Image 1.62, National Institutes of Health, Bethesda, MD) that had been calibrated with a micrometer slide image. The observer was blinded to genotype as all images were captured and measurements were performed. Criteria for selecting which crypts to measure included a clearly seen and continuous cell column on each side of the crypt and a completely visible crypt lumen and opening. Many crypts, from multiple sections prepared from 4 to 5 animals per group were measured to evaluate a total of 40 to 50 crypts per group. Bromo-deoxyuridine (BrdU) incorporation was used as a marker to estimate epithelial cell migration rate, and therefore proliferation, in ileum and distal colon of guanylin heterozygous and null mice. BrdU was injected intraperitoneally (150 μg BrdU in phosphate-buffered saline per gram body weight) and mice were sacrificed at 1 hour, 24 hours, or 48 hours. Intestinal segments were fixed and embedded in paraffin as described above. Sections were processed as per the manufacturer's protocol for BrdU immunohistochemical staining (BrdU Staining kit, Zymed, South San Francisco, CA). Migration distance was measured as follows. Images were digitally captured at × 200. In ileum, the distance from the distal-most Paneth cell to the farthest BrdU-positive cell was determined using NIH Image 1.62. In distal colon, the distance from the base of the crypt to the farthest migrated BrdU-positive cell was used. Immunohistochemistry was used to determine the number of cells expressing proliferating cell nuclear antigen (PCNA) per crypt/villus unit in the ileum and per crypt in the distal colon. PCNA staining was performed on paraffin sections of guanylin heterozygous and guanylin null mouse ileum and distal colon according to manufacturer's protocol (PCNA Staining kit, Zymed). PCNA-positive cells in select, correctly oriented crypts were counted by an observer who was blinded to genotype. Deparaffinized sections of mouse intestine were digested with proteinase K solution (Gibco BRL) (20 μg/ml) for 20 minutes at room temperature. Slides were rinsed in water and treated with 0.5% H2O2 for 10 minutes at room temperature. Test slides were incubated in terminal deoxytransferase (TdT) (Roche) (20 units in 100 μl of buffer with 1 μl of biotin-dUTP) (Roche) for 1 hour at 37°C. Slides were washed in water, incubated with strepavidin-horseradish peroxidase complex (Dako, Carpinteria, CA) for 30 minutes at room temperature, and detected with AEC (3-amino-9-ethylcarbazole) solution (Sigma) for 10 minutes. Positive control slides included sections predigested with deoxyribonuclease and negative control slides were run in parallel without TdT. All values are presented as mean ± SE. Unless otherwise stated, all comparisons are made between wild-type or heterozygous and null mice using the unpaired t-test. Differences were considered statistically significant at P < 0.05. Following gene targeting of the mouse guanylin allele, mice were obtained that harbored the Type II deletion (Figure 1E), ie, the guanylin allele containing the floxed third exon but not the HPRT selection cassette. Unfortunately, no chimeric mice that produced Type I deletion (Figure 1D) agouti pups were obtained during these studies. To ablate the guanylin gene, we bred Type II deletion mice with cytomegalovirus (CMV)-Cre transgenic mice of the BALB/c genetic background strain. These transgenic mice express the Cre recombinase under the control of a human cytomegalovirus minimal promoter in all cells at the pre-implantation stage and beyond.22Schwenk F Baron U Rajewsky K A cre-transgenic mouse strain for the ubiquitous deletion of loxP-flanked gene segments including deletion in germ cells.Nucleic Acids Res. 1995; 23: 5080-5081Crossref PubMed Scopus (1013) Google Scholar This allowed us to breed double transgenics, ie, animals with both the guanylin Type II allele and the CMV-Cre transgene. This generates a Type I deletion (Figure 1D) and this occurs before germline specification, resulting in animals that can pass this guanylin null allele to their offspring. We were able to breed mice that were homozygous guanylin null, suggesting the guanylin gene was not critical for development. Guanylin null mice grew normally to adulthood and were of similar weight as heterozygous controls. We continued breeding the guanylin null mice into the BALB/c strain and selected mice that did not contain the CMV-Cre transgene. This was the basis for the generation of the guanylin null animals used in the studies that are described here. To confirm inactivation of the guanylin gene, we compared expression of guanylin mRNA and protein in guanylin null mice with that of guanylin wild-type and guanylin heterozygous mice. It was possible that the guanylin promoter was still active and that the first portion of the gene was still being transcribed as no direct changes were made to these regions during the initial targeting event or in subsequent Cre-mediated recombinations. To determine this, we hybridized a full-length guanylin cDNA probe to Northern blots of ileum, proximal colon, and distal colon RNA from wild-type, heterozygous, and null mice. No signal was found in guanylin null mice (Figure 2, top). This suggests that the guanylin promoter is inactive or that the partial guanylin transcripts that are produced are highly unstable. During our investigations, we noted no consistent difference between the level of guanylin mRNA expression in wild-type versus heterozygous littermates (Figure 2 and data not shown). Consequently, we used guanylin heterozygotes as littermate control animals in the experiments described here. The guanylin and uroguanylin genes are located extremely close to each other at both mouse and human chromosomal loci (intragenic gap approximately 7 kb) and their mRNA expression patterns and functional similarity suggests coordinate regulation in a manner that allows for the presence of a GC-C binding ligand in all regions of the intestinal tract.13Sciaky D Jenkins NA Gilbert DJ Copeland NG Sonoda G Testa JR Cohen MB Mapping of guanylin to murine chromosome 4 and human chromosome 1p34–p35.Genomics. 1995; 26: 427-429Crossref PubMed Scopus (16) Google Scholar, 16Whitaker TL Witte DP Scott MC Cohen MB Uroguanylin and guanylin: distinct but overlapping patterns of messenger RNA expression in mouse intestine.Gastroenterology. 1997; 113: 1000-1006Abstract Full Text PDF PubMed Scopus (62) Google Scholar, 23Whitaker TL Steinbrecher KA Copeland NG Gilbert DJ Jenkins NA Cohen MB The uroguanylin gene (Guca1b) is linked to guanylin (Guca2) on mouse chromosome 4.Genomics. 1997; 45: 348-354Crossref PubMed Scopus (22) Google Scholar Therefore, we determined the effect of loss of guanylin on uroguanylin levels. Uroguanylin mRNA levels are not affected by the manipulation of the mouse guanylin gene locus as expression is similar in both the small and large intestine of guanylin null mice when compared to that seen in guanylin wild-type and heterozygous animals and normalized by GAPDH expression (Figure 2, middle). GC-C mRNA levels were also unaffected (data not shown). Western analysis of intestinal tissue from guanylin null mice was performed to confirm the loss of guanylin prohormone. We used antisera that was specific for the prohormone portion of the guanylin gene and expected to see the complete loss of proguanylin as suggested by the absence of even partial mRNA transcripts (Figure 2, top). Western blotting showed no traces of proguanylin found in ileum (Figure 3A, top) or in distal colon (Figure 3A, bottom) of guanylin null mice. Taken collectively, these Northern and Western data confirm the complete inactivation of the mouse guanylin gene. We next determined that cellular levels of prouroguanylin were not affected by loss of proguanylin. Western analysis of small and large intestinal tissue homogenate demonstrated equal amounts of prouroguanylin peptide in null and heterozygous mice (Figure 3B). These data suggest that loss of the guanylin gene and activity does not affect levels of uroguanylin mRNA or prohormone in enterocytes, although further studies will be needed to determine whether levels of uroguanylin secretion into the intestine are altered in guanylin null mice. We next sought to determine the mRNA levels of several genes known to be instrumental in fluid homeostasis in the mouse intestine. We found no difference in the mRNA levels of several ion and water channels such as the sodium-hydrogen exchanger 3 (NHE3), the chloride diarrhea anion exchanger (CLD), and aquaporins 4 and 8 (AQP4, AQP8) in guanylin null mice as compared to heterozygous littermate controls (data not shown). We used hematoxylin and eosin staining to determine the small and large intestinal morphology of heterozygous and null animals. We noted no obvious abnormalities in crypt-villus structure in the ileum and in crypt morphology in the colon in nullizygous mice as compared to control. Guanylin is expressed at especially high levels in goblet cells of the intestine.18Li Z Taylor-Blake B Light AR Goy MF Guanylin, an endogenous ligand for C-type guanylate cyclase, is produced by goblet cells in the rat intestine.Gastroenterology. 1995; 109: 1863-1875Abstract Full Text PDF PubMed Scopus (57) Google Scholar We use periodic acid-Schiff (PAS) staining to identify goblet cells and determine whether loss of guanylin affected either goblet cell number or storage of mucin in these cells as judged by stain intensity. PAS reagents showed strong staining of goblet cell mucin in heterozygous and null mice and suggested no difference in goblet cell number or mucin storage (data not shown). Similarly, no difference was seen in apparent numbers of Paneth cells in the ileum of guanylin heterozygous and guanylin null mice (data not shown). Uroguanylin expression is very robust in the small intestine and guanylin expression is highest in the large intestine of wild-type mice. Because it seems likely that these genes have similar functions, we speculated that the phenotype generated from the loss of the guanylin gene might be found in the large intestine due to compensatory actions of uroguanylin in the small intestine. Therefore the studies described here were designed to measure the effects of guanylin loss on the colon, with small intestinal segments often included as examples of tissues that are not devoid of a GC-C-binding peptide. GC-C is the major transmembrane guanylate cyclase in the intestine and the only known endogenous ligands of GC-C are guanylin and uroguanylin. Loss of guanylin might be expected to result in diminished cGMP levels in regions of the intestine that do not have appreciable levels of uroguanylin. We determined cGMP content in mucosal scrapings from ileum and colon of wild-type and null mice by radioimmunoassay. The amount of cGMP in ileum of wild-type littermates and guanylin null mice was similar, although there was significant animal-to-animal variation (Figure 4A). Colonic cGMP, however, were markedly different in each genotype. cGMP levels in the colon were decreased considerably in guanylin null mice as compared to wild-type littermates (Figure 4B), suggesting the loss of thi
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