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• W2008979188 abstract "Mutations in the SLC26A3 (DRA (down-regulated in adenoma)) gene constitute the molecular etiology of congenital chloride-losing diarrhea in humans. To ascertain its role in intestinal physiology, gene targeting was used to prepare mice lacking slc26a3. slc26a3-deficient animals displayed postpartum lethality at low penetrance. Surviving dra-deficient mice exhibited high chloride content diarrhea, volume depletion, and growth retardation. In addition, the large intestinal loops were distended, with colonic mucosa exhibiting an aberrant growth pattern and the colonic crypt proliferative zone being greatly expanded in slc26a3-null mice. Apical membrane chloride/base exchange activity was sharply reduced, and luminal content was more acidic in slc26a3-null mouse colon. The epithelial cells in the colon displayed unique adaptive regulation of ion transporters; NHE3 expression was enhanced in the proximal and distal colon, whereas colonic H,K-ATPase and the epithelial sodium channel showed massive up-regulation in the distal colon. Plasma aldosterone was increased in slc26a3-null mice. We conclude that slc26a3 is the major apical chloride/base exchanger and is essential for the absorption of chloride in the colon. In addition, slc26a3 regulates colonic crypt proliferation. Deletion of slc26a3 results in chloride-rich diarrhea and is associated with compensatory adaptive up-regulation of ion-absorbing transporters. Mutations in the SLC26A3 (DRA (down-regulated in adenoma)) gene constitute the molecular etiology of congenital chloride-losing diarrhea in humans. To ascertain its role in intestinal physiology, gene targeting was used to prepare mice lacking slc26a3. slc26a3-deficient animals displayed postpartum lethality at low penetrance. Surviving dra-deficient mice exhibited high chloride content diarrhea, volume depletion, and growth retardation. In addition, the large intestinal loops were distended, with colonic mucosa exhibiting an aberrant growth pattern and the colonic crypt proliferative zone being greatly expanded in slc26a3-null mice. Apical membrane chloride/base exchange activity was sharply reduced, and luminal content was more acidic in slc26a3-null mouse colon. The epithelial cells in the colon displayed unique adaptive regulation of ion transporters; NHE3 expression was enhanced in the proximal and distal colon, whereas colonic H,K-ATPase and the epithelial sodium channel showed massive up-regulation in the distal colon. Plasma aldosterone was increased in slc26a3-null mice. We conclude that slc26a3 is the major apical chloride/base exchanger and is essential for the absorption of chloride in the colon. In addition, slc26a3 regulates colonic crypt proliferation. Deletion of slc26a3 results in chloride-rich diarrhea and is associated with compensatory adaptive up-regulation of ion-absorbing transporters. The SLC26A3 or DRA (down-regulated in adenoma) gene was originally identified in a subtractive hybridization screen comparing the mRNAs expressed in colon cancer and normal colon tissue (1Schweinfest C.W. Henderson K.W. Suster S. Kondoh N. Papas T.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4166-4170Crossref PubMed Scopus (193) Google Scholar). DRA is expressed in normal colonic epithelium, but is absent or reduced in adenomas and adenocarcinomas (1Schweinfest C.W. Henderson K.W. Suster S. Kondoh N. Papas T.S. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 4166-4170Crossref PubMed Scopus (193) Google Scholar). Subsequent studies identified SLC26A3 (DRA) as a member of a large conserved family of anion exchangers (SLC26) that encompass at least 10 distinct genes (2Bissig M. Hagenbuch B. Stieger B. Koller T. Meier P.J. J. Biol. Chem. 1994; 269: 3017-3021Abstract Full Text PDF PubMed Google Scholar, 20Xu J. Henriksnas J. Barone S. Witte D. Shull G.E. Forte J.G. Holm L. Soleimani M. Am. J. Physiol. 2005; 289: C493-C505Crossref PubMed Scopus (103) Google Scholar). Except for SLC26A5 (prestin), all function as anion exchangers with versatility with respect to transported anions (2Bissig M. Hagenbuch B. Stieger B. Koller T. Meier P.J. J. Biol. Chem. 1994; 269: 3017-3021Abstract Full Text PDF PubMed Google Scholar, 20Xu J. Henriksnas J. Barone S. Witte D. Shull G.E. Forte J.G. Holm L. Soleimani M. Am. J. Physiol. 2005; 289: C493-C505Crossref PubMed Scopus (103) Google Scholar). Immunohistochemical studies localized SLC26A3 on the apical membrane of colonic mucosa, with lower levels in the small intestine (4Hoglund P. Haila S. Socha J. Tomaszewski L. Saarialho-Kere U. Karjalainen-Lindsberg M.L. Airola K. de la Holmberg C. Chapelle A. Kere J. Nat. Genet. 1996; 14: 316-319Crossref PubMed Scopus (355) Google Scholar, 25Silberg D.G. Wang W. Moseley R.H. Traber P.G. J. Biol. Chem. 1995; 270: 11897-11902Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). In humans, SLC26A3 encodes a 764-amino acid protein and is located on chromosome 7 in a head-to-tail arrangement with SLC26A4 (pendrin), indicating ancient gene duplication. Genetic analysis studies linked mutations in DRA (SLC26A3) to congenital chloride-losing diarrhea (CLD 5The abbreviations used are: CLD, chloride-losing diarrhea; ES, embryonic stem; ENaC, epithelial sodium channel; KO, knock-out. ; OMIM accession number 214700), a disease manifested by enhanced chloride loss in the stool and volume depletion (4Hoglund P. Haila S. Socha J. Tomaszewski L. Saarialho-Kere U. Karjalainen-Lindsberg M.L. Airola K. de la Holmberg C. Chapelle A. Kere J. Nat. Genet. 1996; 14: 316-319Crossref PubMed Scopus (355) Google Scholar). Functional studies in vitro have demonstrated that SLC26A3 can mediate multiple anion exchange modes, including Cl-/HCO3- , Cl-/oxalate, and Cl-/hydroxyl, and possibly sulfate/hydroxyl exchanges (6Melvin J.E. Park K. Richardson L. Schultheis P.J. Shull G.E. J. Biol. Chem. 1999; 274: 22855-22861Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 21Greeley T. Shumaker H. Wang Z. Schweinfest C.W. Soleimani M. Am. J. Physiol. 2001; 281: G1301-G1308PubMed Google Scholar, 26Byeon M.K. Frankel A. Papas T.S. Henderson K.W. Schweinfest C.W. Protein Expr. Purif. 1998; 12: 67-74Crossref PubMed Scopus (43) Google Scholar). Similar anion exchange activities have been described previously in apical membranes of the colon (27Rajendran V.M. Binder H.J. Am. J. Physiol. 1999; 276: G132-G137PubMed Google Scholar, 28Tyagi S. Kavilaveettil R.J. Alrefai W.A. Alsafwah S. Ramaswamy K. Dudeja P.K. Exp. Biol. Med. (Maywood). 2001; 226: 912-918Crossref PubMed Google Scholar), the site of abundant DRA expression. To initiate an investigation into the role of DRA in an in vivo model, we created slc26a3 (dra) gene-targeted mice that are null for expression of the slc26a3 gene. This mouse model closely resembles the clinicopathological presentation of human CLD. Functional studies demonstrated that slc26a3-null mice have significantly reduced levels of apical chloride/base exchange activity in the colon and display unique and distinct adaptive regulation of ion transporters in the proximal and distal colon. In addition, the loss of slc26a3 produces an expansion of the proliferative zone of the colonic crypt epithelium, suggesting a role for loss of slc26a3 expression in colon tumor progression. Isolation of Mouse slc26a3 Genomic Clones—A mouse 129S6/SvEv strain genomic library in the vector Lambda Dash II (Stratagene, La Jolla, CA) was screened by plaque hybridization using human DRA cDNA as a probe. A 19-kbp fragment containing at least exons 2-9 was isolated. The identity of the clones was confirmed by Southern blot hybridization and partial sequencing. Targeting Vector Construction—Restriction enzyme mapping revealed the presence of an AgeI restriction site on exon 2 of the mouse dra genomic DNA that was suitable for insertion of the neo cassette. The neo cassette contains the neomycin phosphotransferase gene under the transcriptional control of the RNA polymerase II promoter and the last exon and polyadenylation signal of the hypoxanthine-guanine phosphoribosyltransferase gene for termination. In addition, the neo cassette is flanked by the loxP/Cre recombinase recognition sequences (kindly provided by K. Thomas). The AgeI site of the neo cassette insertion is located 39 bp downstream of the ATG start codon of the dra gene. The targeting vector contains 5.6 kbp of isogenic genomic DNA upstream and 8 kbp of isogenic genomic DNA downstream of the AgeI site. The targeting vector was linearized with SfiI, which cleaves the vector backbone, prior to electroporation into embryonic stem (ES) cells. ES Cell Electroporation—TC1-10 ES cells (kindly provided by Dr. P. Leder) were grown on feeder layers of mouse fibroblasts in knock-out Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 15% serum replacement medium and made complete as described previously (29Hogan B. Costantini F. Lacy E. Manipulating the Mouse Embryo: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1986Google Scholar). ES cells (2 × 107) were electroporated with 68 μg of linearized vector using a Bio-Rad Gene Pulser at 600 V and 25 microfarads. Transfected cells were treated with G418 (230 μg/ml) for positive selection. Drug-resistant ES cell clones were expanded and screened for homologous recombination by Southern blot analysis of their genomic DNA (see Fig. 1A). Three of 40 drug-resistant clones were generated and identified by this method. Generation of slc26a3 (dra) Knock-out Mice—The ES cells from three of the positive colonies were microinjected into C57BL/6J blastocysts and implanted in the uteruses of pseudopregnant female mice. Chimeric males that demonstrated significant agouti coat color were mated with BL6 females to generate heterozygotes, which were initially identified by the same Southern blot strategy used to identify targeted ES cell lines. Subsequently, the slc26a3 genotyping was conducted by PCR using two slc26a3 exon 2-specific primers and a primer specific for the RNA polymerase II gene (see below). Genotyping of Mice—Genotyping was performed on tail DNA obtained from mice at weaning and placed directly into 250 μl of lysis buffer (10 mm Tris-HCl (pH 8.5), 50 mm KCl, 1.5 mm MgCl2, 0.01% gelatin, 0.45% Nonidet P-40, and 0.45% Tween 20). PCR was performed with the following primers: mouse slc26a3 exon 2, 5′-GGCAAAATGATCGAAGCCATAGGG-3′ (forward) and 5′-GATGGTCCAGGAATGTCTTGTGATGTC-3′ (reverse); and neo cassette RNA polymerase II, 5′-GGAAGTAGCCGTTATTAGTGGAGAGG-3′ (reverse). The PCR conditions were as follows: 95 °C for 3 min, followed by 32 cycles at 94 °C for 30 s, 55 °C for 30 s, and 72 °C for 30 s. The PCR products were electrophoresed on 3% low melting point agarose (see Fig. 1B) in 45 mm Tris borate and 1 mm EDTA (pH 8.0). Luminal Chloride, Sodium, and pH Measurement—The chloride assay was performed using a diagnostic kit obtained from Sigma (catalog no. 461-3) according to the manufacturer's instructions. Briefly, stool samples from the animals were collected, weighed, lyophilized, weighed again, resuspended in water at 5-100 mg/ml, and then heated to 65 °C for 30 min. The samples were centrifuged in a tabletop centrifuge, and the supernatant was removed and assayed for chloride. The sodium assay was performed by flame photometry. The pH of the intestinal content was assayed using a pH microelectrode. Histopathological Examination of Mouse Tissues—Mice were killed, and tissues were harvested within 10 min. Tissues were fixed in Amsterdam solution and then processed and embedded in paraffin blocks using standard methods. Sections were cut and stained using hematoxylin/eosin or subjected to immunohistochemical/immunofluorescence labeling as described (13Wang Z. Petrovic S. Mann E. Soleimani M. Am. J. Physiol. 2002; 282: G573-G579Crossref PubMed Scopus (228) Google Scholar, 20Xu J. Henriksnas J. Barone S. Witte D. Shull G.E. Forte J.G. Holm L. Soleimani M. Am. J. Physiol. 2005; 289: C493-C505Crossref PubMed Scopus (103) Google Scholar). The primary antibodies used were anti-SLC26A3, anti-NHE3, and anti-SLC26A6. RNA Isolation and Northern Blot Hybridization—Total cellular RNA was extracted from various mouse tissues, including ileum, proximal and distal colon, duodenum, and kidney, according to established methods; quantitated spectrophotometrically; and stored at -80 °C. Total RNA samples (5-30 μg/lane) were fractionated on a 1.2% agarose gel containing formaldehyde, transferred to Magna NT nylon membranes, cross-linked by UV light, and baked. Hybridization was performed according to established protocols (30Church G.M. Gilbert W. Proc. Natl. Acad. Sci. U. S. A. 1984; 81: 1991-1995Crossref PubMed Scopus (7266) Google Scholar). The membranes were washed, blotted dry, and exposed to a PhosphorImager screen (GE Healthcare). The following DNA fragments were used as specific probes for Northern hybridization: for NHE3, a fragment encoding nucleotides 1883-2217; for SLC26A6, a fragment encoding nucleotides 51-488; for colonic H,K-ATPase, three pooled PCR products from rat (nucleotides 135-515, 2369-2998, and 3098-3678); and for ENaCα, ENaCβ, and ENaCγ, fragments encoding nucleotides 1197-1890, 58-671, and 135-790, respectively. Each Northern hybridization was performed on separate samples from three different animals. Immunoblotting and/or Immunohistochemical Labeling of slc26a3 and nhe3 in Mouse Intestine—slc26a3+/+ and slc26a3-/- mice were killed with a sodium pentobarbital overdose and perfused through the left ventricle with 0.9% saline, followed by cold 4% paraformaldehyde in 0.1 m sodium phosphate buffer (pH 7.4). Intestinal segments were removed, cut into tissue blocks, and fixed overnight in formaldehyde solution at 4 °C. The tissues were frozen on dry ice, and 6-μm sections were cut with a cryostat and stored at -80 °C until used. Immunohistochemical labeling was performed as described (13Wang Z. Petrovic S. Mann E. Soleimani M. Am. J. Physiol. 2002; 282: G573-G579Crossref PubMed Scopus (228) Google Scholar) using SLC26A3-specific antibodies. For immunoblot analysis, microsomal membranes from the proximal colon were resolved by SDS-PAGE, and membranes were blotted with anti-SLC26A3 or anti-NHE3 antibodies. Each blot was performed on three separate samples from three different animals. 36Cl and 22Na Transport Measurement—The uptake of 36Cl or 22Na by luminal membrane vesicle suspensions, pooled from the proximal colons of three mice and prepared according to established methods (17Wang Z. Wang T. Petrovic S. Tuo B. Riederer B. Barone S. Lorenz J.N. Seidler U. Aronson P.S. Soleimani M. Am. J. Physiol. 2005; 288: C957-C965Crossref PubMed Scopus (166) Google Scholar, 27Rajendran V.M. Binder H.J. Am. J. Physiol. 1999; 276: G132-G137PubMed Google Scholar, 28Tyagi S. Kavilaveettil R.J. Alrefai W.A. Alsafwah S. Ramaswamy K. Dudeja P.K. Exp. Biol. Med. (Maywood). 2001; 226: 912-918Crossref PubMed Google Scholar), was assayed at room temperature in triplicate by the rapid filtration technique (32Jacob P. Rossmann H. Lamprecht G. Kretz A. Neff C. Lin-Wu E. Gregor M. Groneberg D.A. Kere J. Seidler U. Gastroenterology. 2002; 122: 709-724Abstract Full Text Full Text PDF PubMed Scopus (150) Google Scholar). The reaction was stopped by ice-cold medium. The radioactivity in each filter was assayed by scintillation spectroscopy. Vesicles and all experimental media were continuously gassed with 100% N2 or 5% CO2 and 95% N2. The uptake of 4 mm measured under three different conditions: no pH gradient (pHi/pHo 7.5/7.5 without CO2/HCO3- ), outward pH gradient (pHi 7.5/pHo 6.0 without CO2/HCO3- ), and outward pH and bicarbonate gradients (pHi 7.5/pHo 6.0 with CO2/HCO3- ). The bicarbonate concentrations were 25 mm at pH 7.5 and 0 mm at pH 6.0. The uptake of 1 mm 22Na was measured under two different conditions: no pH gradient (pHi 7.5/pHo 7.5) and inward pH gradient (pHi 6.0/pHo 7.5) without CO2/HCO3- . Each radioactive uptake experiment was repeated on three different samples. Materials—[32P]dCTP, 36Cl, and 22Na were purchased from Perkin-Elmer Life Sciences. Nitrocellulose filters and other chemicals were purchased from Sigma. The RadPrime DNA labeling kit was purchased from Invitrogen. Anti-SLC26A6 antibodies was raised in our laboratories (13Wang Z. Petrovic S. Mann E. Soleimani M. Am. J. Physiol. 2002; 282: G573-G579Crossref PubMed Scopus (228) Google Scholar). Antibodies against SLC26A3 peptides were custom-generated by Zymed Laboratories Inc. (South San Francisco, CA) as described (33Byeon M.K. Westerman M.A. Maroulakou I.G. Henderson K.W. Suster S. Zhang X.K. Papas T.S. Vesely J. Willingham M.C. Green J.E. Schweinfest C.W. Oncogene. 1996; 12: 387-396PubMed Google Scholar). Anti-NHE3 polyclonal antibodies were purchased from Chemicon. Statistical Analyses—Values are expressed as the means ± S.E. Statistical analysis was examined using Student's t test or analysis of variance. p < 0.05 was considered statistically significant. An slc26a3 gene-targeted allele was generated in ES cells using a targeting vector containing the neo cassette inserted into exon 2 of an isogenic 129 strain dra genomic clone as shown in Fig. 1A. Three germ line chimeric founder males were generated from dra gene-targeted ES cells and were used to breed in a C57BL6 background. Genotype analysis of offspring was performed by a three-primer PCR using the strategy shown in Fig. 1B. The targeted allele produced a 220-bp PCR fragment, whereas the wild-type allele produced a 115-bp fragment. Fig. 1B shows a typical genotyping demonstrating wild-type, heterozygous, and homozygous knock-out (KO) animals. DRA is expressed in a limited subset of tissues, including small and large intestines, intraprostatic seminal vesicles, and eccrine sweat glands (4Hoglund P. Haila S. Socha J. Tomaszewski L. Saarialho-Kere U. Karjalainen-Lindsberg M.L. Airola K. de la Holmberg C. Chapelle A. Kere J. Nat. Genet. 1996; 14: 316-319Crossref PubMed Scopus (355) Google Scholar, 6Melvin J.E. Park K. Richardson L. Schultheis P.J. Shull G.E. J. Biol. Chem. 1999; 274: 22855-22861Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 33Byeon M.K. Westerman M.A. Maroulakou I.G. Henderson K.W. Suster S. Zhang X.K. Papas T.S. Vesely J. Willingham M.C. Green J.E. Schweinfest C.W. Oncogene. 1996; 12: 387-396PubMed Google Scholar, 34Haila S. Saarialho-Kere U. Karjalainen-Lindsberg M.L. Lohi H. Airola K. Holmberg C. Hastbacka J. Kere J. Hoglund P. Histochem. Cell Biol. 2000; 113: 279-286Crossref PubMed Scopus (63) Google Scholar). However, the intestines express DRA to the greatest degree (4Hoglund P. Haila S. Socha J. Tomaszewski L. Saarialho-Kere U. Karjalainen-Lindsberg M.L. Airola K. de la Holmberg C. Chapelle A. Kere J. Nat. Genet. 1996; 14: 316-319Crossref PubMed Scopus (355) Google Scholar, 6Melvin J.E. Park K. Richardson L. Schultheis P.J. Shull G.E. J. Biol. Chem. 1999; 274: 22855-22861Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 33Byeon M.K. Westerman M.A. Maroulakou I.G. Henderson K.W. Suster S. Zhang X.K. Papas T.S. Vesely J. Willingham M.C. Green J.E. Schweinfest C.W. Oncogene. 1996; 12: 387-396PubMed Google Scholar). Therefore, to test for generation of a null allele via gene targeting, we performed Northern blot analysis on RNAs from the ilea and colons of wild-type and homozygous animals (Fig. 1C). The results showed that the homozygous individuals were completely devoid of DRA mRNA expression. Heterozygous intercross mating was performed to determine whether the dra KO allele is transmitted to the subsequent generation in Mendelian ratios. Table 1 shows the results of genotype analysis of 409 such offspring at weaning and reveals a non-Mendelian distribution of genotypes (40.6% wild-type, 43.8% heterozygous, and 15.6% KO). The deviation from Mendelian ratios is highly significant (p < 0.0001) for both heterozygous and homozygous animals, suggesting a dra gene dosage affect on viability, albeit at reduced penetrance. Table 2 shows the results of mating between heterozygous and homozygous KO littermates. Again, the weaned offspring demonstrated a non-Mendelian distribution that is a highly significant departure from expected Mendelian ratios (p = 0.0002).TABLE 1Distribution of genotypes among 409 progeny of dra heterozygotous matingsGenotypeNo. of offspringϰ2p valueObservedExpecteddra+/+166102.25dra+/−179204.50dra−/−64102.25Total40940957.235aϰ2 was calculated as Σ(observed - expected)2/expected.<0.0001bThe p value was determined from an unpaired Student's t test (two-tailed).a ϰ2 was calculated as Σ(observed - expected)2/expected.b The p value was determined from an unpaired Student's t test (two-tailed). Open table in a new tab TABLE 2Distribution of genotypes among 50 progeny of dra+/− × dra−/− matingsGenotypeNo. of offspringϰ2p valueObservedExpecteddra+/+00dra+/−3825dra−/−1225Total505013.520aϰ2 was calculated as Σ (observed - expected)2/expected.0.0002bThe p value was determined from an unpaired Student's t test (two-tailed).a ϰ2 was calculated as Σ (observed - expected)2/expected.b The p value was determined from an unpaired Student's t test (two-tailed). Open table in a new tab To determine whether dra disruption affects the gross morphology of the developing embryo, we examined the intestinal morphology during embryogenesis. We have shown previously that dra gene expression commences in the gut at embryonic day 16.5 postcoitus (33Byeon M.K. Westerman M.A. Maroulakou I.G. Henderson K.W. Suster S. Zhang X.K. Papas T.S. Vesely J. Willingham M.C. Green J.E. Schweinfest C.W. Oncogene. 1996; 12: 387-396PubMed Google Scholar). Therefore, we harvested embryos at day 18.5 postcoitus. There were no detectable differences in the gross morphology or architecture of the intestines of dra-deficient mice and their normal siblings (data not shown). The expected Mendelian ratios for heterozygous mating was observed among the embryos (data not shown), suggesting that postpartum lethality is responsible for the non-Mendelian ratios observed later. Surviving dra-deficient mice exhibited diarrhea with elevated chloride content. Fig. 2A shows that the chloride level of stool specimens from dra-deficient mice (mean ± S.D. of 28.37 ± 6.66 μg/mg (dry weight)) was significantly higher (p < 0.0001) than that from wild-type and heterozygous animals (mean ± S.D. of 1.87 ± 0.79 μg/mg (dry weight)). In addition, stools from the dra-deficient mice had a higher water content compared with those from their control littermates (p < 0.0001) (Fig. 2B), although the stools were not as watery as described in human CLD. In addition, homozygous animals occasionally developed prolapsed rectums. Finally, dra-deficient mice were smaller compared with agematched heterozygous and wild-type animals (p = 0.0007) (Fig. 2C) and had greatly shortened life spans (mean ± S.D. of 121.6 ± 50.8 days). These observations are consistent with the conditions in human CLD, which is characterized by voluminous watery diarrhea with high chloride content and growth retardation in untreated individuals (35Holmberg C. Clin. Gastroenterol. 1986; 15: 583-602Crossref PubMed Google Scholar). Patients treated with electrolyte replacement continue to have watery, high chloride content diarrhea, but have normal life spans (35Holmberg C. Clin. Gastroenterol. 1986; 15: 583-602Crossref PubMed Google Scholar). Mutations in human DRA are the cause of CLD (4Hoglund P. Haila S. Socha J. Tomaszewski L. Saarialho-Kere U. Karjalainen-Lindsberg M.L. Airola K. de la Holmberg C. Chapelle A. Kere J. Nat. Genet. 1996; 14: 316-319Crossref PubMed Scopus (355) Google Scholar, 5Moseley R.H. Hoglund P. Wu G.D. Silberg D.G. de la Haila S. Chappelle A. Holmberg C. Kere J. Am. J. Physiol. 1999; 276: G185-G192PubMed Google Scholar, 34Haila S. Saarialho-Kere U. Karjalainen-Lindsberg M.L. Lohi H. Airola K. Holmberg C. Hastbacka J. Kere J. Hoglund P. Histochem. Cell Biol. 2000; 113: 279-286Crossref PubMed Scopus (63) Google Scholar). Therefore, the dra-deficient mouse model closely resembles the molecular etiology and clinical manifestations of human CLD. Other morphological and histological aspects distinguished the dra KO mice from normal animals. Upon immediate postdeath dissection, dra-deficient mice presented with distension of the large bowel (Fig. 3I, panels A-C) compared with wild-type mice (panel D). The small intestine was not affected in this manner. Such intestinal dilation is consistent with pre- and postpartum observations of human CLD and is due to increased volume of the bowel contents (35Holmberg C. Clin. Gastroenterol. 1986; 15: 583-602Crossref PubMed Google Scholar). We next examined the morphology of the colonic tissue. Whole mount preparations of colons that were sliced open longitudinally, fixed, and stained with methylene blue revealed a starkly altered mucosal morphology in dra-deficient mice compared with those in heterozygous and wild-type mice. Fig. 3II (panels A-C) shows an abnormal growth pattern of the mucosal surface wherein the crypt orifices were conjoined rather than distinct as in normal mucosa (panel D). This conjoined phenotype was observed in sections from both the proximal and distal colons of KO mice, but not in the ilea (data not shown). Fig. 3II (panel E) demonstrates serial sections (8 μm) down through an example of conjoined crypts. It shows that the conjoined crypt phenotype occurred at the very top of the crypt, which is closest to the lumen (panel E). SLC26A3 functions predominantly as a chloride/base exchanger in vitro (6Melvin J.E. Park K. Richardson L. Schultheis P.J. Shull G.E. J. Biol. Chem. 1999; 274: 22855-22861Abstract Full Text Full Text PDF PubMed Scopus (253) Google Scholar, 21Greeley T. Shumaker H. Wang Z. Schweinfest C.W. Soleimani M. Am. J. Physiol. 2001; 281: G1301-G1308PubMed Google Scholar, 23Shcheynikov N. Wang Y. Park M. Ko S.B. Dorwart M. Naruse S. Thomas P.J. Muallem S. J. Gen. Physiol. 2006; 127: 511-524Crossref PubMed Scopus (153) Google Scholar). To ascertain the role of SLC26A3 in mediating chloride/base exchange in vivo, apical membrane vesicles were isolated from the proximal colons of wild-type and slc26a3-null mice and assayed for chloride/base exchange using the 36Cl influx method. As demonstrated in Fig. 4A, the influx of radiolabeled chloride mediated via Cl-/OH- and Cl-/HCO3- exchange decreased by 76 and 69%, respectively, in apical membrane vesicles from the colons of slc26a3-null mice (p < 0.001 versus wild-type mice). The reduction in apical chloride influx correlated with results from immunoblot analysis demonstrating the complete absence of slc26a3 protein in the colons of KO mice (Fig. 4B).FIGURE 4Expression and activities of ion transporters in slc26a3 KO mouse colon. A, 36Cl influx ( Cl-/OH-/HCO3- exchange) in colonic apical membrane vesicles from normal and slc26a3-null mice. B, immunoblot analysis of dra in normal (slc26a3+/+) and slc26a3-/- mice. C, NHE3 mRNA expression in normal and slc26a3-null mice. D, immunoblot analysis of NHE3 in the colons of normal and slc26a3 KO mice. E, 22Na influx (the Na+/H+ exchanger activity) in colonic apical membrane vesicles in normal and slc26a3-null mice. EIPA, ethylisopropyl amiloride. F, colonic H,K-ATPase (HKA) mRNA expression in normal and slc26a3-null mice. G, expression of electrogenic Na+ channel subunits in normal and slc26a3-null mice.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Despite a very watery cecal content, the final stools were less watery, suggesting an increased absorptive capacity of the colons in slc26a3 KO mice. To ascertain the molecular mechanism of enhanced electrolyte absorption in the colon, the expression and/or activities of the apical Na+/H+ exchanger NHE3, the electrogenic sodium channel, and colonic H,K-ATPase were examined. NHE3 mRNA expression was increased by 2- and 3-fold in the proximal and distal colons, respectively, from slc26a3-null mice (p < 0.001 versus wild-type mice; n = 4) (Fig. 4C). Immunoblotting confirmed the up-regulation of NHE3 in the proximal colons of slc26a3 KO mice (Fig. 4D). The enhanced expression of NHE3 was associated with increased Na+/H+ exchange activity in luminal membrane vesicles isolated from the colons of slc26a3 KO mice (Fig. 4E). Colonic H,K-ATPase is expressed in the distal colon and is predominantly responsible for the absorption of potassium. Colonic H,K-ATPase mRNA was increased by ∼4-fold in the distal colons from slc26a3 KO mice (p < 0.001 versus wild-type mice; n = 4) (Fig. 4F). In addition to NHE3 and colonic H,K-ATPase, an electrogenic Na+ channel is expressed on the apical membranes of the distal colon and is involved in the absorption of sodium. Northern analysis showed significant up-regulation of the ENaC α-, β-, and γ-subunits in the distal colons of slc26a3 KO mice (p < 0.001 versus wild-type mice; n = 4) (Fig. 4G). Histological and immunohistochemical analyses of the colonic crypts further distinguished slc26a3-deficient animals from their normal littermates. By these methods, we determined the expression patterns for dra and the retinoblastoma protein and p53 tumor suppressor genes, the proliferating cell nuclear antigen proliferation marker, and incorporated bromodeoxyuridine. In addition, the expression of cyclins A, B1, D1, E, and H, and cyclin-dependent kinase inhibitory proteins p27, p21, and p16 was examined. Most of these proteins showed similar expression patterns in all three dra genotypes (data not shown). However, as shown in Fig. 5, several had notable differences that distinguished the homozygous mice from the heterozygous and" @default.
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