Clifford W. Schweinfest

slc26a3 (dra)-deficient Mice Display Chloride-losing Diarrhea, Enhanced Colonic Proliferation, and Distinct Up-regulation of Ion Transporters in the Colon

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.

Acute regulation of the SLC26A3 congenital chloride diarrhoea anion exchanger (DRA) expressed in Xenopus oocytes

Mutations in the human SLC26A3 gene, also known as down‐regulated in adenoma (hDRA), cause autosomal recessive congenital chloride‐losing diarrhoea (CLD). hDRA expressed in Xenopus oocytes mediated bidirectional Cl−‐Cl− and Cl−‐HCO3− exchange. In contrast, transport of oxalate was low, and transport of sulfate and of butyrate was undetectable. Two CLD missense disease mutants of hDRA were nonfunctional in oocytes. Truncation of up to 44 C‐terminal amino acids from the putatively cytoplasmic C‐terminal hydrophilic domain left transport function unimpaired, but deletion of the adjacent STAS (sulfate transporter anti‐sigma factor antagonist) domain abolished function. hDRA‐mediated Cl− transport was insensitive to changing extracellular pH, but was inhibited by intracellular acidification and activated by NH4+ at acidifying concentrations. These regulatory responses did not require the presence of either hDRAs N‐terminal cytoplasmic tail or its 44 C‐terminal amino acids, but they did require more proximate residues of the C‐terminal cytoplasmic domain. Although only weakly sensitive to inhibition by stilbenes, hDRA was inhibited with two orders of magnitude greater potency by the anti‐inflammatory drugs niflumate and tenidap. cAMP‐insensitive Cl−‐HCO3− exchange mediated by hDRA gained modest cAMP sensitivity when co‐expressed with cystic fibrosis transmembrane conductance regulator (CFTR). Despite the absence of hDRA transcripts in human cell lines derived from CFTR patients, DRA mRNA was present at wild‐type levels in proximal colon and nearly so in the distal ileum of CFTR(‐/‐) mice. Thus, pharmacological modulation of DRA might be a useful adjunct treatment of cystic fibrosis.

Subtraction hybridization cDNA libraries from colon carcinoma and hepatic cancer

cDNA clones of differentially expressed mRNAs in a colon carcinoma and a hepatocellular carcinoma have been isolated by subtractive cDNA cloning. The subtracted material is at least 90 X enriched for differentially expressed sequences and can be used for construction of subtractive cDNA libraries and polymerase chain reaction (PCR) amplification to generate differential probes. Commercially available lambda ZAP II is used for construction of primary libraries since single-stranded phage bearing the cloned cDNA can be excised in vivo and because lambda libraries are convenient for subsequent screening and manipulations. Rare mRNAs (less than 0.01% abundance), which are differentially expressed, can be isolated utilizing this procedure.

ETS2 function is required to maintain the transformed state of human prostate cancer cells.

The contribution of the ETS2 transcription factor to the transformed state in prostate cancer cells has been assessed. Northern blot analysis easily detects ETS2 in DU145 and PC3, high grade human prostate cell lines, but ETS2 is not present in lower grade LNCaP cells. Stable transfection of PC3 and DU145 prostate cell lines with an antisense ETS2 vector or with a dominant negative ETS2 mutant significantly reduced the ability of DU145 and PC3 cells to form large colonies in soft agar. Thus, the presence of ETS2 is positively correlated with a more transformed phenotype and blockage of ETS2 function can reduce transformed properties of prostate cancer cells.

Intestinal inflammation reduces expression of DRA, a transporter responsible for congenital chloride diarrhea

The pathogenesis of diarrhea in intestinal inflammatory states is a multifactorial process involving the effects of inflammatory mediators on epithelial transport function. The effect of colonic inflammation on the gene expression of DRA (downregulated in adenoma), a chloride-sulfate anion transporter that is mutated in patients with congenital chloridorrhea, was examined in vivo as well as in an intestinal epithelial cell line. DRA mRNA expression was diminished five- to sevenfold in the HLA-B27/β2m transgenic rat compared with control. In situ hybridization showed that DRA, which is normally expressed in the upper crypt and surface epithelium of the colon, was dramatically reduced in the surface epithelium of the HLA-B27/β2m transgenic rat, the interleukin-10 (IL-10) knockout mouse with spontaneous colitis, and in patients with ulcerative colitis. Immunohistochemistry demonstrated that mRNA expression of DRA reflected that of protein expression in vivo. IL-1β reduced DRA mRNA expression in vitro by inhibiting gene transcription. The loss of transport function in the surface epithelium of the colon by attenuation of transporter gene expression, perhaps inhibited at the level of gene transcription by proinflammatory cytokines, may play a role in the pathogenesis of diarrhea in colitis.

Down-regulated in Adenoma Cl/HCO3 Exchanger Couples With Na/H Exchanger 3 for NaCl Absorption in Murine Small Intestine

BACKGROUND & AIMS Electroneutral NaCl absorption across small intestine contributes importantly to systemic fluid balance. Disturbances in this process occur in both obstructive and diarrheal diseases, eg, cystic fibrosis, secretory diarrhea. NaCl absorption involves coupling of Cl(-)/HCO(3)(-) exchanger(s) primarily with Na(+)/H(+) exchanger 3 (Nhe3) at the apical membrane of intestinal epithelia. Identity of the coupling Cl(-)/HCO(3)(-) exchanger(s) was investigated using mice with gene-targeted knockout (KO) of Cl(-)/HCO(3)(-) exchangers: Slc26a3, down-regulated in adenoma (Dra) or Slc26a6, putative anion transporter-1 (Pat-1). METHODS Intracellular pH (pH(i)) of intact jejunal villous epithelium was measured by ratiometric microfluoroscopy. Ussing chambers were used to measure transepithelial (22)Na(36)Cl flux across murine jejunum, a site of electroneutral NaCl absorption. Expression was estimated using immunofluorescence and quantitative polymerase chain reaction. RESULTS Basal pH(i) of DraKO epithelium, but not Pat-1KO epithelium, was alkaline, whereas pH(i) in the Nhe3KO was acidic relative to wild-type. Altered pH(i) was associated with robust Na(+)/H(+) and Cl(-)/HCO(3)(-) exchange activity in the DraKO and Nhe3KO villous epithelium, respectively. Contrary to genetic ablation, pharmacologic inhibition of Nhe3 in wild-type did not alter pH(i) but coordinately inhibited Dra. Flux studies revealed that Cl(-) absorption was essentially abolished (>80%) in the DraKO and little changed (<20%) in the Pat-1KO jejunum. Net Na(+) absorption was unaffected. Immunofluorescence demonstrated modest Dra expression in the jejunum relative to large intestine. Functional and expression studies did not indicate compensatory changes in relevant transporters. CONCLUSIONS These studies provide functional evidence that Dra is the major Cl(-)/HCO(3)(-) exchanger coupled with Nhe3 for electroneutral NaCl absorption across mammalian small intestine.

Role of Down-Regulated in Adenoma Anion Exchanger in HCO3 - Secretion Across Murine Duodenum

BACKGROUND & AIMS The current model of duodenal HCO(3)(-) secretion proposes that basal secretion results from Cl(-)/HCO(3)(-) exchange, whereas cyclic adenosine monophosphate (cAMP)-stimulated secretion depends on a cystic fibrosis transmembrane conductance regulator channel (Cftr)-mediated HCO(3)(-) conductance. However, discrepancies in applying the model suggest that Cl(-)/HCO(3)(-) exchange also contributes to cAMP-stimulated secretion. Of 2 candidate Cl(-)/HCO(3)(-) exchangers, studies of putative anion transporter-1 knockout (KO) mice find little contribution of putative anion transporter-1 to basal or cAMP-stimulated secretion. Therefore, the role of down-regulated in adenoma (Dra) in duodenal HCO(3)(-) secretion was investigated using DraKO mice. METHODS Duodenal HCO(3)(-) secretion was measured by pH stat in Ussing chambers. Apical membrane Cl(-)/HCO(3)(-) exchange was measured by microfluorometry of intracellular pH in intact villous epithelium. Dra expression was assessed by immunofluorescence. RESULTS Basal HCO(3)(-) secretion was reduced approximately 55%-60% in the DraKO duodenum. cAMP-stimulated HCO(3)(-) secretion was reduced approximately 50%, but short-circuit current was unchanged, indicating normal Cftr activity. Microfluorimetry of villi demonstrated that Dra is the dominant Cl(-)/HCO(3)(-) exchanger in the lower villous epithelium. Dra expression increased from villous tip to crypt. DraKO and wild-type villi also demonstrated regulation of apical Na(+)/H(+) exchange by Cftr-dependent cell shrinkage during luminal Cl(-) substitution. CONCLUSIONS In murine duodenum, Dra Cl(-)/HCO(3)(-) exchange is concentrated in the lower crypt-villus axis where it is subject to Cftr regulation. Dra activity contributes most basal HCO(3)(-) secretion and approximately 50% of cAMP-stimulated HCO(3)(-) secretion. Dra Cl(-)/HCO(3)(-) exchange should be considered in efforts to normalize HCO(3)(-) secretion in duodenal disorders such as ulcer disease and cystic fibrosis.

Decreased expression of colonic Slc26a3 and carbonic anhydrase iv as a cause of fatal infectious diarrhea in mice.

ABSTRACT Citrobacter rodentium causes epithelial hyperplasia and colitis and is used as a model for enteropathogenic and enterohemorrhagic Escherichia coli infections. Little or no mortality develops in most inbred strains of mice, but C3H and FVB/N mice exhibit fatal outcomes of infection. Here we test the hypothesis that decreased intestinal transport activity during C. rodentium infection results in fatality in C3H/HeOu and FVB/N mice. Susceptible strains were compared to resistant C57BL/6 mice and to inbred strains SWR and SJL of Swiss origin, which have not been previously characterized for outcomes of C. rodentium infection. Mortality in susceptible strains C3H/HeOu and FVB/N was associated with significant fluid loss in feces, a remarkable downregulation of Slc26a3 and carbonic anhydrase IV (CAIV) message and protein expression, retention of chloride in stool, and hypochloremia, suggesting defects in intestinal chloride absorption. SWR, SJL, and C57BL/6 mice were resistant and survived the infection. Fluid therapy fully prevented mortality in C3H/HeOu and FVB/N mice without affecting clinical disease. Common pathogenic mechanisms, such as decreased levels of expression of Slc26a3 and CAIV, affect intestinal ion transport in C. rodentium-infected FVB and C3H mice, resulting in profound electrolyte loss, dehydration, and mortality. Intestinal chloride absorption pathways are likely a potential target for the treatment of infectious diarrhea.

A heat-shock-inducible eukaryotic expression vecto

Abstract We describe a construct, pHS/Cla, containing the Drosophila melanogaster hsp70 promoter which serves as an inducible expression vector in mammalian cells. The construct was made in the plasmid pAT 153, a derivative of pBR322. In transient transfections of human H9 T-cells, the transactivator of transcription protein of the human AIDS virus HIV-1 was functionally expressed in response to heat shock. The promoter is very tightly regulated in that no expression can be detected at 37°C. Expression is highly induced upon heat shock and lasts at least 4 h. The construct contains a unique Cla I site for cloning and expressing potentially any gene.

Characterization of human N8 protein

We have shown before that the N8 mRNA is expressed at higher levels in lung tumor and lung tumor-derived cell lines than normal lung cells. In this paper, we have characterized the N8 protein, and studied its properties. The N8 gene encodes a major 24 kDa protein and its expression correlates well with the N8 mRNA expression pattern observed in different cell lines. N8 protein is capable of forming a homodimer or multimeter in vitro. It is a phosphorylated cytoplasmic protein and phosphorylation occurs mainly at serine residues. N8 protein is expressed at higher levels in epithelial cells than in mesenchymal cells. N8 protein expression is induced in a fibroblast cell line expressing adenoviral E1a protein, which acquired epithelial-like characteristics. Furthermore, ectopic expression of N8 protein in NIH3T3 cells converts them into a spheroid form. These spheroids also have some of the characteristic features of epithelial cells. Taken together, these results suggest that the N8 protein may be associated with the development or maintenance of epithelial cell phenotype.