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From the Division of Pediatric Gastroenterology,* Hepatology and Nutrition, the Graduate Program in Molecular and Developmental Biology, and the Division of Pediatric Pathology, Children’s Hospital Research Foundation, Children’s Hospital Medical Center and University of Cincinnati, Cincinnati, Ohio
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Abstract |
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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.2-4 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.5-7 Ligand binding to GC-C initiates a signal transduction cascade that culminates in activation of the cystic fibrosis transmembrane conductance regulator (CFTR).8,9 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.10,11
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.12 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.
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Materials and Methods |
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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.13
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 32
) 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.
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, 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.
Northern Analysis
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.14
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 [
-32P] (DuPont-NEN, Boston, MA) using the Random Primer Labeling system (Roche Molecular Biochemicals, Indianapolis, IN) as described previously.15,16
Blots were visualized and quantitated using a Molecular Dynamics PhosphorImager system (Molecular Dynamics, Sunnyvale, CA).
Western Analysis
Animals were sacrificed and tissues collected as described above. Tissue was homogenized and processed as described previously.14 Membranes were immunoblotted using a 1:1000 dilution of antisera that recognizes proguanylin (antibody 2538) and prouroguanylin (antibody 6910).14 These antibodies were generously provided by Dr. Michael Goy of the University of North Carolina and have been validated and described previously.17,18 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).
Histology
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.
cGMP Measurements
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.19
Guanylate Cyclase Assay
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
Crypt depth, used as an estimate of proliferation and apoptosis rates in colonic epithelia,20,21 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.
Epithelial Cell Proliferation
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 x 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.
Terminal Deoxynucleotidyl Transferase-Mediated dNTP-Biotin Nick End Labeling (TUNEL) Assay
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.
Statistical Analysis
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.
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Results |
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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.22
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.
Northern and Western Analysis of Guanylin Null Mice
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.
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, 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.
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. 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).
Histological Examination of Guanylin Null Mice
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.18 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.
cGMP Levels in Intestinal Mucosa of Guanylin Null Mice
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 this guanylate cyclase-activating peptide resulted in lowered basal levels of cGMP in the epithelia of the colon. To further characterize the guanylate cyclase pathway in these animals, we also measured the activity of GC-C in ileal and colonic explants. In the ileum (n = 2), cGMP levels increased 3.5-fold (range, 2.7 to 4.4) in wild-type animals and 2.5-fold (range, 2.2 to 2.7) in null mice on addition of STa. In the colon (n = 2), cGMP levels increased 1.5-fold (range, 1.4 to 1.5) in wild-type and 1.5-fold (range, 1.3 to 1.6) in null mice on addition of STa. Thus the decreased levels of cGMP in the colon are not due to diminished activity of GC-C.
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cGMP signaling has been linked to control of proliferation in some cell types.24-26
Therefore, we tested the hypothesis that loss of guanylin results in proliferative changes in the colon. A general measure of these parameters is the depth of the crypt glands as has been measured in the small intestine.20,21
Alterations in the cellular division rate of intestinal epithelia result in increased or decreased crypt depth and, consequently, are reflected in crypt depth measurements. We adapted this technique for use in the colon and found significant increases in crypt depth in both the proximal and distal colon of guanylin null mice as compared to heterozygotes (Table 1)
. Increases in crypt depth in both proximal and distal colon were approximately 10%. Additional studies have determined that the number of epithelial cells per crypt in the distal colon is increased in the guanylin null mice when compared to heterozygous littermate controls (44.6 ± 1.4 cells versus 39.6 ± 1.5 cells; n = 12 to 20; P = 0.01). This increase, of approximately 10%, is consistent with the quantitative change in crypt depth.
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, Figure 6A
). A change in migration rate was evident in the colon, however, as epithelia in the guanylin null mice had moved much farther from the base of the crypt by 48 hours when compared to heterozygous littermate controls (Figure 5A
, Figure 6B
). These data parallel the observations of cGMP levels in null mice and controls.
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, Figure 7A
). However, distal colonic epithelia of guanylin null mice had significantly more PCNA-stained cells per crypt than did heterozygous littermate controls (Figure 5B
, Figure 7B
). The number of BrdU-stained cells per crypt was similarly increased in the colon of knockout animals (3.10 ± 0.19 versus 3.67 ± 0.18 BrdU-stained cells, heterozygote versus knockout respectively, P = 0.04, 5 to 6 mice per genotype, total of 46 to 52 crypts examined).
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. However, no difference was seen in the apoptotic index between guanylin null mice and littermate controls in either location (Figure 8)
.
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Discussion |
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Loss of guanylin activity did not cause severe phenotypic changes and these animals have remained healthy to at least 6 months of age. We noted few unusual deaths of adult guanylin null mice. Similarly, when raised on a standard chow diet, guanylin null mice gained weight normally and, unlike CFTR null mice, they do not demonstrate signs of intestinal obstruction,28 presumably because there are other compensatory mechanisms in this portion of the CFTR-mediated secretory pathway. As part of a separate study, we are currently investigating the changes in colonic sodium and chloride flux and the response to secretagogues as a result of targeted deletion of the guanylin gene.
Histological examination revealed no gross changes in crypt-villus morphology or cell-type representation. However, closer examination revealed a significant decrease in cGMP levels in the colonic but not ileal mucosa of guanylin null mice. Uroguanylin levels are very high throughout the small intestine, but not the colon, and we speculate that it is the presence of uroguanylin and its subsequent activation of GC-C in the ileum that results in sustained levels of cGMP in this intestinal segment.
Changes in the level of cGMP in intestinal epithelia could be expected to influence the rate of epithelial cell proliferation.25 We therefore used a number of different assays to determine the rate of cell proliferation in intestinal mucosa of guanylin null mice. The increase in colonic crypt depth, crypt cell number, PCNA staining, and cell migration all indicated that there was a significant increase in the rate of cell division in the colonic crypts of guanylin null mice. These data strongly support a role for guanylin in epithelial cell proliferation in the large intestine and are consistent with cGMP as a signaling molecule that is central to this process. Although the mechanism through which cGMP influences proliferation is unclear, there is substantial evidence to suggests that it regulates the rate of cell division.25,29 Consistent loss of guanylin in mouse and human adenomatous tissue and its absence in the vast majority of intestinal cell culture lines suggest selective pressure against its presence in rapidly proliferating, transformed epithelia.10,11,30 Guanylin and uroguanylin are located in chromosomal regions that harbor modifiers of intestinal adenoma formation susceptibility.10,13 In addition, a recent study in human colon carcinoma cell lines indicates that the guanylin ligand family may be cytostatic, causing a slowing of cell cycle progression in a cGMP-dependent manner.25 Collectively, these reports and the data presented here suggest that guanylin signaling has an antiproliferative effect on normal intestinal epithelia and that loss of guanylin and subsequent decreases in intracellular cGMP remove this cytostatic block and allow increased cell division. As suggested by Pitari et al,25 these data also imply that the guanylin ligand family may play a role in regulating the transition between intestinal stem cell proliferation and differentiation into mature enterocytes. Additionally, they support a potential role for these ligands as cytostatic agents in the prevention or treatment of colorectal cancer.
There are conflicting data regarding the role of guanylin, uroguanylin, and GC-C as mediators of apoptosis. For example, cGMP is reduced in intestinal cancers due to increased cGMP-hydrolyzing phosphodiesterase activity and use of cGMP-specific phosphodiestase inhibitors in combination with guanylin causes apoptosis in transformed colonic cell lines.31 Uroguanylin was shown to elicit profound apoptosis in human cancer cell lines.29 However, other in vitro studies demonstrate no pro-apoptotic effect of STa or uroguanylin.25 Similarly, measurement of the apoptotic index in intestinal epithelia of guanylin null mice showed no change in the number of apoptotic cells when compared to heterozygous littermates. Experimental variability that is inherent in this index prevents us from knowing whether a small increase in apoptosis is present and counterbalances the observed increase in proliferation to maintain a steady state.
Several other genes in the guanylin-signaling pathway have also been targeted. CFTR null mice exhibit a phenotype that is very similar to the guanylin null mice with respect to epithelial proliferation.32 CFTR inactivation results in increased epithelial cell proliferation in the small intestine, a tissue where we speculate that uroguanylin compensates for the loss of guanylin and thereby diminishes the effect of guanylin loss. Data concerning the effect of loss of GC-C in mouse intestinal epithelia with regard to proliferation and apoptosis have not been reported.33,34 Although the only known receptor for guanylin is GC-C, the existence of other receptors is strongly indicated by multiple data sets.35,36 If the phenotype of the guanylin null mouse is mediated by GC-C, then we would predict a similar phenotype in the GC-C null mouse. The possibility remains, however, that the changes in epithelial cell proliferation and the decrease in intracellular cGMP levels are not connected via GC-C. Future experiments are planned to address these questions.
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Acknowledgements |
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Footnotes |
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Supported by grant DK 47318 from the National Institutes of Health.
Accepted for publication September 9, 2002.
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