Downregulated In Adenoma Expression Research Articles

Intestinal Anion Exchanger Down-regulated in Adenoma (DRA) Is Inhibited by Intracellular Calcium

The Na/H exchanger 3 (NHE3) and the Cl/HCO(3) exchanger down-regulated in adenoma (DRA) together facilitate intestinal electroneutral NaCl absorption. Elevated Ca(2+)(i) inhibits NHE3 through mechanisms involving the PDZ domain proteins NHE3 kinase A regulatory protein (E3KARP) or PDZ kidney 1 (PDZK1). DRA also possesses a PDZ-binding motif, but the roles of interactions with E3KARP or PDZK1 and Ca(2+)(i) in DRA regulation are unknown. Wild type DRA and a mutant lacking the PDZ interaction motif (DRA-ETKFminus) were expressed constitutively in human embryonic kidney (HEK) and inducibly in Caco-2/BBE cells. DRA-mediated Cl/HCO(3) exchange was measured as intracellular pH changes. Ca(2+)(i) was assessed fluorometrically. DRA was induced 8-16-fold and was delivered to the apical surface of polarized Caco-2 cells. Putative anion transporter 1 and cystic fibrosis transmembrane regulator did not contribute to Cl/HCO(3) exchange in transfected Caco-2 cells. The calcium ionophore 4Br-A23187 inhibited DRA and DRA-ETKFminus in HEK cells, but only full-length DRA was inhibited in Caco-2 cells. In contrast, 100 microm UTP, which increased Ca(2+)(i), inhibited full-length DRA but not DRA-ETKFminus in Caco-2 and HEK cells. In HEK cells, which express little PDZK1, additional transfection of PDZK1 was required for UTP to inhibit DRA. As HEK cells do not express cystic fibrosis transmembrane regulator or NHE3, the data indicate that Ca(2+)(i)-dependent DRA inhibition is not because of modulation of other transport activities. In polarized epithelium, this inhibition requires interaction of DRA with PDZK1. Together with data from PDZK1(-/-) mice, these data underscore the prominent role of PDZK1 in Ca(2+)(i)-mediated inhibition of colonic NaCl absorption.

Regulation of the Intestinal Anion Exchanger DRA (Downregulated in Adenoma)

Barrier function and transepithelial transport are intimately linked and are sometimes disturbed in parallel. DRA (downregulated in adenoma) is an intestinal chloride/bicarbonate exchanger that is functionally coupled to CFTR (cystic fibrosis transmembrane regulator) in the upper gastrointestinal tract to mediate chloride and bicarbonate secretion and to NHE3 (Na/H exchanger- isoform 3) in the lower gastrointestinal tract to mediate electroneutral NaCl absorption. All three transport proteins possess PDZ domain binding motifs that facilitate binding to members of the NHERF (Na/H exchanger regulatory factor) family of adapter proteins [NHERF, E3KARP (NHE3 kinase A regulatory protein), PDZK1 (PDZ protein kidney 1) and IKEPP (intestinal and kidney enriched PDZ protein)]. Regulation of DRA appears to depend on the presence of a partner transport protein, and this may involve the assembly of different complexes of transporters, adapter proteins, and signaling molecules. We have established stable expression of DRA in HEK cells. In these cells, that do not express significant amounts of CFTR or NHE3, DRA is inhibited by intracellular calcium but not by protein kinase C or protein kinase A. At high calcium concentrations induced by 4Br-A23187 this inhibition is independent of the PDZ interaction of DRA. These data show that DRA can be individually regulated and may be confirmed in a more physiologically relevant expression system (i.e., Caco-2/BBE cells) using natural agonists of the intracellular calcium signal.

Probiotics Stimulate DRA Expression in Human Intestinal Caco‐2 Cells

DRA (Down Regulated in Adenoma) plays a major role in apical Cl−/OH− (HCO3−) exchange process in the human intestine. Dysregulation of DRA has been implicated in diarrheal conditions, such as congenital chloride diarrhea and enteropathogenic infections. However, little is known about the regulation of DRA by probiotics, currently used as an alternative treatment for diarrhea. Therefore, the present studies were undertaken to determine the regulation of DRA expression and function by Lactobacilli in human intestinal Caco2 cells. Culture supernatant (CS) from an overnight grown Lactobacilli culture of ~1.5X107 CFU/ml was obtained and used at 1:50 dilution. L. rhamnosus or L. acidophilus CS treatment of Caco2 cells for 24h significantly increased (~2 fold) the relative abundance of DRA mRNA. To determine whether Lactobacilli alter the rate of DRA gene transcription, DRA promoter activity was assessed. Parallel to the increased mRNA expression, luciferase reporter gene assays showed ~2.5 fold increase in DRA promoter activity. Both L. rhamnosus or L. acidophilus stimulated Cl−/OH− exchange activity as measured by DIDS‐sensitive 36Cl− uptake (~ 50 %, P<0.05). These findings suggest that the soluble factors from Lactobacilli stimulate DRA expression and Cl− absorption and may aid in the development of novel therapeutic interventions for treatment of diarrheal disorders. (Supported by NIDDK and Dept of Veteran Affairs).

Molecular cloning and promoter analysis of downregulated in adenoma (DRA)

Downregulated in adenoma (DRA), also referred to as SLC26A3, is an intestinal anion transporter essential for intestinal chloride absorption. Mutations in DRA result in congenital chloride diarrhea. DRA expression has been shown to be induced by differentiation and to be modulated by cytokines. However, mechanisms of DRA gene transcription and its tissue-specific targeting have not yet been investigated. In this study, we cloned a 3,765-bp promoter fragment of human DRA gene and characterized its activity in human colonic LS174T and Caco-2 human colon cell lines. Primer extension identified a single transcriptional initiation site that was identical in both colon cancer cell lines and normal colon. Although hepatic nuclear factor HNF-4 is involved in the basal activity of DRA promoter, sodium butyrate induces its activity in LS174T cells via the binding of Yin Yang 1 (YY1) and GATA transcription factors to their respective cis-elements in promoter region. We also demonstrated a reduction in DRA promoter activity in Caco-2 cells by IFN-gamma, suggesting that regulation of DRA promoter by IFN-gamma may contribute to the pathophysiology of intestinal inflammation. Furthermore, we showed that the DRA promoter fragment is sufficient to drive human growth hormone transgene expression specifically in villus epithelial cells of the small intestine and in differentiated upper crypt and surface epithelial cells of the colon. Our studies provide evidence for the involvement of HNF-4, YY1, and GATA transcription factors in DRA expression in intestinal differentiated epithelial cells.

PAT-1 (Slc26a6) is the predominant apical membrane Cl−/HCO3− exchanger in the upper villous epithelium of the murine duodenum

Basal HCO(3)(-) secretion across the duodenum has been shown in several species to principally involve the activity of apical membrane Cl(-)/HCO(3)(-) exchanger(s). To investigate the identity of relevant anion exchanger(s), experiments were performed using wild-type (WT) mice and mice with gene-targeted deletion of the following Cl(-)/HCO(3)(-) exchangers localized to the apical membrane of murine duodenal villi: Slc26a3 [down-regulated in adenoma (DRA)], Slc26a6 [putative anion transporter 1 (PAT-1)], and Slc4a9 [anion exchanger 4 (AE4)]. RT-PCR of the isolated villous epithelium demonstrated PAT-1, DRA, and AE4 mRNA expression. Using the pH-sensitive dye BCECF, anion exchange rates were measured across the apical membrane of epithelial cells in the upper villus of the intact duodenal mucosa. Under basal conditions, Cl(-)/HCO(3)(-) exchange activity was reduced by 65-80% in the PAT-1(-) duodenum, 30-40% in the DRA(-) duodenum, and <5% in the AE4(-) duodenum compared with the WT duodenum. SO(4)(2-)/HCO(3)(-) exchange was eliminated in the PAT-1(-) duodenum but was not affected in the DRA(-) and AE4(-) duodenum relative to the WT duodenum. Intracellular pH (pH(i)) was reduced in the PAT-1(-) villous epithelium but increased to WT levels in the absence of CO(2)/HCO(3)(-) or during methazolamide treatment. Further experiments under physiological conditions indicated active pH(i) compensation in the PAT-1(-) villous epithelium by combined activities of Na(+)/H(+) exchanger 1 and Cl(-)-dependent transport processes at the basolateral membrane. We conclude that 1) PAT-1 is the major contributor to basal Cl(-)/HCO(3)(-) and SO(4)(2-)/HCO(3)(-) exchange across the apical membrane and 2) PAT-1 plays a role in pH(i) regulation in the upper villous epithelium of the murine duodenum.

Transport properties of the human intestinal anion exchanger DRA (down-regulated in adenoma) in transfected HEK293 cells.

Electroneutral NaCl absorption in the intestine is mediated by parallel Na+/H+ and Cl-/HCO3- exchange. Mutations in the down-regulated in adenoma (DRA) gene cause congenital chloride diarrhoea but the transport characteristics of human DRA have not been studied in a heterologous human expression system. A N-terminal enhanced green fluorescence protein (EGFP)-tagged human DRA construct was therefore expressed stably in HEK293 cells. Cl-/HCO3- exchange was assessed by measuring intracellular pH and intracellular Cl- using fluorescent dyes. Expression of DRA resulted in the appearance of EGFP fluorescence and DRA immunoreactivity consistent with a location in the plasma membrane and possibly structures below the plasma membrane. DRA mediated electroneutral Cl-/HCO3- exchange but OH- was not transported and SO4(2-)/HCO3- exchange was minimal. In the presence of 5% CO2/HCO3- the apparent affinity of DRA for Cl- in transfected HEK cells was 23-36 mM, which is lower than that reported for rabbit ileal brush border membrane vesicles and for oocytes injected with human DRA. DRA was inhibited by 4 mM DIDS (45+/-11%), by 50 microM tenidap (71+/-8%) and by 100 microM glibenclamide (59+/-22% inhibition of HCO3- transport and 79+/-3% inhibition of Cl- transport). The effects of DIDS and tenidap were not additive to those of glibenclamide.

Downregulated in adenoma and putative anion transporter are regulated by CFTR in cultured pancreatic duct cells

The mechanism of the pancreatic ductal HCO secretion defect in cystic fibrosis (CF) is not well defined. However, a lack of apical Cl(-)/HCO exchange may exist in CF. To test this hypothesis, we examined the expression of Cl(-)/HCO exchangers in cultured pancreatic duct epithelial cells with physiological features prototypical of CF [CFPAC-1 cells lacking a functional CF transmembrane conductance regulator (CFTR)] or normal duct cells (CFPAC-1 cells transfected with functional wild-type CFTR, CFPAC-WT). Cl(-)/HCO exchange activity, assayed with the pH-sensitive dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein in cells grown on coverslips, increased about twofold in cells transfected with functional CFTR. This correlated with increased apical (36)Cl influx in cells expressing functional CFTR and grown on permeable support. Northern hybridizations indicated the induction of downregulated in adenoma (DRA) in cells expressing functional CFTR. The expression of putative anion transporter PAT1 also increased significantly in cells expressing functional CFTR. DRA was detected at high levels in native mouse pancreas by Northern hybridization and localized to the apical domain of the duct cells by immunohistochemical studies. In conclusion, CFTR upregulates DRA and PAT1 expression in cultured pancreatic duct cells. We propose that the pancreatic HCO secretion defect in CF patients is partly due to the downregulation of apical Cl(-)/HCO exchange activity mediated by DRA (and possibly PAT1).

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/beta2m 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/beta2m 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-1beta 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.

Mutations of the Down-regulated in adenoma (DRA) gene cause congenital chloride diarrhoea.

A major transport function of the human intestine involves the absorption of chloride in exchange for bicarbonate. We have studied a recessively inherited defect of this exchange, congenital chloride diarrhoea (CLD; MIM 214700). The clinical presentation of CLD is a lifetime, potentially fatal diarrhoea with a high chloride content. The CLD locus was previously mapped to 7q3 adjacent to the cystic fibrosis gene (CFTR). By refined genetic and physical mapping, a cloned gene having anion transport function, Down-regulated in adenoma (DRA), was implicated as a positional and functional candidate for CLD. In this study, we report segregation of two missense mutations, delta V317 and H124L, and one frameshift mutation, 344delT, of DRA in 32 Finnish and four Polish CLD patients. The disease-causing nature of delta V317 is supported by genetic data in relation to the population history of Finland. By mRNA in situ hybridization, we demonstrate that the expression of DRA occurs preferentially in highly differentiated colonic epithelial cells, is unchanged in Finnish CLD patients with delta V317, and is low in undifferentiated (including neoplastic) cells. We conclude that DRA is an intestinal anion transport molecule that causes chloride diarrhoea when mutated.