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Intestinal Bile Acid Transport: Biology, Physiology, and Pathophysiology

2001; Lippincott Williams & Wilkins; Volume: 32; Issue: 4 Linguagem: Inglês

10.1097/00005176-200104000-00002

1536-4801

Benjamin L. Shneider,

Pharmacological Effects and Toxicity Studies

Bile acid homeostasis, which is a critical part of human health, involves hepatic biosynthesis and vectorial transport of bile acids in a number of organs (Fig. 1). Bile acids are synthesized from cholesterol via a process that is found exclusively in the liver and involves a number of cytosolic and mitochondrial enzymes (1). Inborn errors in this process lead to neonatal cholestatic liver disease (2–4). Similarly, genetic disruption of cholesterol 7α-hydroxylase, a rate-limiting enzyme in bile acid biosynthesis, leads to a fatal form of cholestasis, which can be reversed by primary bile acid administration (5). Up-regulation of bile acid biosynthesis, which can be induced by bile acid wasting, has been used as a means of treating hypercholesterolemia (6). Bile acids, after being synthesized in the liver, are secreted into bile by an adenosine triphosphate–dependent transporter referred to as the bile salt excretory pump. Once in the intestine, bile salts play a crucial role in activating digestive enzymes and solubilizing fats and fat-soluble vitamins. A small percentage of bile salts may be reabsorbed in the proximal intestine by either passive or carrier-mediated transport processes (7–9). Most bile salts are reclaimed in the distal ileum by a sodium-dependent apically located bile acid transporter (10,11), referred to as apical sodium-dependent bile acid transporter (ASBT). Inside the enterocyte, bile salts may be bound to intracellular binding proteins (eg, ileal lipid binding protein (12)). At the basolateral surface of the enterocyte, a truncated version of ASBT is involved in vectorial transfer of bile acids into the portal circulation. Completion of the enterohepatic circulation occurs at the basolateral surface of the hepatocyte by a transport process that is primarily mediated by a sodium-dependent bile acid transporter (13). The enterohepatic circulation of bile salts is modified by bile acid transport processes in the bile duct epithelium and renal tubules.FIG. 1.: Enterohepatic circulation of bile salts. Bile salts are synthesized in the liver and excreted into the bile ducts by an ATP-driven transporter (bile salt excretory pump = 2). Most bile salts are reabsorbed in the terminal ileum by a sodium-dependent transporter (apical sodium dependent bile acid transporter [ASBT] = 3). ASBT is also expressed on the apical surface of cholangiocytes and renal proximal tubules. An organic anion transporter (5) is expressed on the apical surface of jejunum and ileum and mediated sodium-independent absorption of bile salts. At the basolateral surface of a truncated version of tASBT (4) mediates transport of bile salts into the portal circulation. tASBT is also expressed in cholangiocytes and renal tubule cells. The enterohepatic circulation of bile salts is completed at the basolateral surface of hepatocytes by a sodium-dependent transport process (sodium-dependent taurocholate transporting polypeptide [ntcp] = 1).Intestinal bile acid transport plays a key role in the enterohepatic circulation of bile salts. Molecular analysis of this process has recently led to important advances in our understanding of the biology, physiology and pathophysiology of intestinal bile acid transport. Apical sodiumdependent bile acid transporter is the major carrier protein involved in intestinal reclamation of bile salts. Complete genetic disruption of ASBT activity leads to pathologic bile acid induced diarrhea, whereas partial inhibition of ASBT-mediated transport can be used to treat hypercholesterolemia and cholestasis. Apical sodiumdependent bile acid transporter is expressed on the apical surface of ileal enterocytes, renal tubule cells, and cholangiocytes. A truncated alternatively spliced form of ASBT is expressed on the basolateral surface of cholangiocytes. Apical sodium-dependent bile acid transporter undergoes a biphasic pattern of expression in the rat ileum during normal development. The bile acid responsiveness of the ASBT gene is species specific and also depends on the experimental method used to perturb bile acid homeostasis. ASBT expression is up-regulated by corticosteroids and down-regulated in animal models by ileal inflammation. Specific patterns of adaptation of ASBT expression have been observed in various models of intestinal resection. The molecular basis of the regulation of ASBT in various settings suggests that transcriptional and posttranscriptional mechanisms both are operational. This review will summarize our current understanding of the biology, physiology, and pathophysiology of the apical sodium-dependent bile acid transporter. PHYSIOLOGY AND MOLECULAR BASIS OF INTESTINAL BILE ACID TRANSPORT For more than a century it has been known that intestinal reclamation of bile salts occurs in the ileum (1). Various experimental techniques have been used to study intestinal bile acid transport, including surgical exclusion and functional analysis of isolated segments of intestine and transport assays using everted intestine, isolated enterocytes, and brush border membrane vesicles (2–4). Evidence indicates that the majority of bile acid reclamation occurs in the terminal ileum (5–7). The physiologic relevance of jejunal transport of bile salts to the enterohepatic circulation is not adequately characterized (8,9). Kinetic analysis of ileal bile acid transport showed that it was a sodium-dependent carrier-mediated process (4,10). Using an expression cloning strategy based on this finding, Dawson et al. (11) cloned the hamster ileal sodium-dependent bile acid transporter. Northern blotting analysis showed a similarly sized transcript in kidneys, in which apical sodium-dependent bile acid transport was previously observed (12). Subsequently, the ASBT complement DNA (cDNA) has been cloned in human, rat, mouse, and rabbit ileum (13–16). The substrate specificity of human ASBT expressed in COS cells includes conjugated and unconjugated bile salts but not sulfated bile acids or estrogen (14). Western blotting and indirect immunofluorescence analysis has showed that the ASBT gene product is a 48-kd protein localized to the apical surface of ileal enterocytes and proximal renal convoluted tubules (15). It is also expressed on the apical surface of large bile duct epithelial cells (16,17). A novel truncated 19-kDa form of ASBT has been identified in ileum, kidneys, and cholangiocytes (18). It is the consequence of exon-2 skipping that results in a frameshift change and truncation of the ASBT peptide from 348 to 154 amino acids. Alteration of the carboxy-terminal amino acid sequence may permit sorting to the basolateral membrane (19). This truncated protein seemss to be functional and is most likely involved in facilitating efflux of bile salts at the basolateral membrane. Two additional proteins have been implicated in the process of intestinal bile acid transport. OATP3 is a member of the organic anion transport protein family and may facilitate carrier-mediated bile acid transport in the proximal intestine (9). The ileal lipid binding protein (also known as the ileal bile acid binding protein or ILBP) is a cytosolic protein, which binds bile salts and is localized in the terminal ileum (20). It is presumed to be the ileal intracellular binder of bile salts; however, there is no direct evidence that this protein is physiologically relevant to the process of intestinal bile salt transport. ILBP is not expressed in cholangiocytes or renal tubule cells (B. Shneider, unpublished data, January 1997). Therefore, ILBP does not seem to be essential to the process of bile acid transport. The function of ASBT is known from transfection studies and analysis of human disease. Transfection of a number of different cell lines and Xenopus laevis oocytes shows that the ASBT protein functions as a sodium-dependent conjugated bile acid transporter with an apparent 2:1 Na+: bile acid coupled electrogenic stoichiometry (10,11,19,21). In the terminal ileum, it is clear that ASBT plays a key role in bile acid reclamation. Physiologic and molecular analysis of two children with congenital ileal bile acid malabsorption and intractable diarrhea showed that the children had a compound heterozygote defect in the human ASBT gene (22,23). Additional transporters for bile salts, including OATP, may exist in the proximal intestine; however they seem to be of reduced quantitative importance overall in the reclamation of intestinal bile salts (8,9,24). The physiologic role of ASBT in kidney and bile duct epithelia is not certain. Presumably, ASBT in the kidney serves to facilitate tubular reabsorption of bile salts and explains the relatively low concentration of bile acids in the urine of healthy persons. Expression of ASBT in cholangiocytes may subserve cholehepatic shunting of bile acids, although the physiologic relevance of this process is unknown. Interestingly, ASBT has recently been identified in the gallbladder of humans (25) and rabbits (B. Shneider, unpublished data, November 2000), and may play a role in modifying bile composition and in the pathogenesis of gallstones (26). Study of knockout mice will ultimately be instructive in advancing our understanding of the range of physiologic functions of ASBT. APICAL SODIUM-DEPENDENT BILE ACID TRANSPORTER IN HUMAN HEALTH AND DISEASE Analysis of ASBT function in humans can be accomplished using various experimental approaches. Measurement of fecal bile acids, which is the gold standard measurement, is a noninvasive but cumbersome means of assessing intestinal malabsorption of bile salts. Serum bile acid response to a test-meal stimulus is a relatively easy but potentially unreliable in of assessing intestinal bile acid transport function (27). Normal intestinal bile acid transport is suggested by a greater than twofold increase in serum bile salts 2 hours after a standard meal. This approach requires the presence of a gallbladder and relatively normal intestinal transit times. Kinetic analysis of orally or intravenously administered radiolabeled bile acids can be used to determine intestinal bile acid transport and bile acid pool size (28). Direct assessment of ileal sodium-dependent bile acid transport can be performed using either ileal mucosa or brush border membrane vesicles derived from endoscopically obtained ileal biopsy specimens (22,29). 75SeHCAT ([23-75Se]25-homocholyltaurine) is a radiolabeled bile acid analog that can be used to assess the enterohepatic circulation of bile salts. First-pass intestinal clearance of SeHCAT and total body retention of this compound are excellent markers of intestinal bile acid transport function (30,31). This compound has been widely used outside of the United States, but unfortunately has not been licensed for use within the United States. Indirect assessment of bile acid malabsorption can be inferred from measurement of serum 7α-hydroxy-4-cholesten-3-one, a marker of bile acid biosynthesis (32,33) Apical sodium-dependent bile acid transporter plays a key role in a number of physiologic and pathophysiologic processes and thus has a central role in human health and disease (Table 1). The immediately obvious examples are diseases that result from genetic defects in ASBT. As described previously, two children have been described who have severe congenital diarrhea, steatorrhea, and failure to thrive secondary to a compound heterozygote mutation in the ASBT gene (23). Fecal bile acid analysis and kinetic analysis of orally administered radiolabeled bile salts showed marked bile acid malabsorption and a contracted bile acid pool. Transport assays using ileal mucosal biopsy specimens showed markedly reduced uptake of bile salts as compared with biopsy specimens from children with ileostomies and no intrinsic ileal disease. Twenty years after the childrens' initial presentation, analysis of the human ASBT gene was performed in one of the affected children. Using single-stranded conformational polymorphism (SSCP) and direct sequence analysis, one allele was found to contain two separate missense mutations, both of which abrogated sodium-dependent bile acid transport activity. The other allele was found to contain a splice site mutation, which is presumed to lead to exon 3 skipping. The potentially truncated messenger (mRNA) protein, or both is presumed to be unstable, nonfunctional, or both; however, this was not specifically tested in vitro (34). Given the recent description of a functional protein formed after exon 2 skipping, this supposition is open to question (18). The first discovery of a dysfunctional ASBT mutation occurred during the cloning of human ASBT cDNA from an ileal library constructed from the ileum of a patient with Crohn disease (35). There does not seem to be a clinical phenotype associated with heterozygosity for ASBT mutations; however, large population-based studies will be necessary to confirm this supposition. Apical sodium-dependent bile acid transporter knockout mice will be extremely useful in confirming the functional consequences of loss of ASBT function. Interestingly, a recently described hepatocyte nuclear factor (HNF)-1 α knockout mouse has markedly reduced ASBT protein expression and evidence of significant bile acid malabsorption (36).TABLE 1: ASBT in human diseaseAbnormalities in intestinal bile acid transport function have been implicated in various other disorders. A number of investigators have demonstrated that primary bile acid malabsorption is involved in intractable diarrhea (33,37–40). Treatment of bile acid malabsorption may ameliorate symptoms in a number of patients with irritable bowel syndrome (41). In contrast, it has been postulated that increased active ileal bile acid transport might be associated with constipation, an extremely common and problematic disorder (29). Primary defects in ileal bile acid transport may also be involved in familial hypertriglyceridemia; however, the pathophysiologic mechanism is uncertain (42). Apical sodium-dependent bile acid transporter mRNA and protein levels were reduced in ileal biopsy samples obtained from 13 patients with hypertriglyceridemia (43). In many circumstances, ASBT expression is secondarily affected by intestinal disease and results in significant morbidity. Bile acid malabsorption in these circumstances leads to bile salt-induced diarrhea and depletion of the bile salt pool. More importantly, bile acid malabsorption may be a risk factor for the development of colorectal cancer (44). Various animal studies, including a rat ileal resection model, support this proposition; however, definitive proof of an association in humans is lacking (45). Ileal resection associated with necrotizing enterocolitis, volvulus, and inflammatory bowel disease also leads to bile acid malabsorption and diarrhea (27,46–49). Bile acid malabsorption has been well-described in human immunodeficiency virus (HIV) enteropathy and may lead to significant morbidity (50,51). It is not known whether this is a direct effect of HIV on ASBT or an indirect effect mediated by intestinal injury or inflammatory cytokines. Significant bile acid malabsorption is seen in many acquired immune deficiency syndrome (AIDS) patients in spite of relatively normal ileal histology (52,53). Radiation enteritis has also been shown to induce bile acid malabsorption (54,55). Bile acid malabsorption is a major clinical problem in children with cystic fibrosis (47). Fat malabsorption associated with pancreatic insufficiency is clearly one factor involved in the pathogenesis of bile acid malabsorption; however the degree of fat malabsorption does not always correlate with the degree of bile acid wasting, suggesting that there may be specific abnormalities in ASBT function in cystic fibrosis patients (56). A cellular defect in bile acid transport was suggested by in vitro analysis of taurocholate transport using ileal biopsy specimens from children with cystic fibrosis (57). The role of inflammatory cytokines in bile acid malabsorption is not well understood. Clearly, bile acid malabsorption is commonly seen in Crohn disease, in which ileal inflammation is prominent (41,58). Furthermore, apical sodium-dependent bile acid transport activity and ASBT protein expression are decreased in rabbit models of ileal inflammation (59). THERAPEUTIC MODULATION OF APICAL SODIUM-DEPENDENT BILE ACID TRANSPORTER FUNCTION Although ASBT dysfunction can lead to significant human disease, there are circumstances in which either surgical or pharmacologic reduction in ASBT function has had important therapeutic applications. The most notable of these is the treatment of hypercholesterolemia and related reduction in morbidity and mortality caused by atherosclerosis. Inhibition of ileal bile acid reclamation leads to increased fecal wasting of bile salts and compensatory hepatic up-regulation of bile acid biosynthesis from cholesterol. The result is a significant increase in cholesterol catabolism. Proof that this concept is valid comes from the Program on the Surgical Control of the Hyperlipidemias (POSCH) trial of partial ileal bypass for hypercholesterolemia, in which surgical reduction in ileal bile acid reabsorption lead to a significant reduction in plasma cholesterol levels and coronary artery disease (6). On the basis of these important findings, a number of pharmacologic agents have been developed which interfere with ASBT function (60–63). Animal studies in hamsters, mice, rats, and rabbits have shown that these pharmacologic compounds effectively increase fecal bile acid wasting and decrease serum cholesterol levels (64–66). In addition, decreased atherosclerosis was observed in Watanabe rabbits that were fed one of these inhibitors (64). Surgically induced bile acid wasting has also been used in the treatment of some forms of intrahepatic cholestasis. In particular, children with Byler disease may achieve sustained and remarkable improvement in liver disease after either partial external biliary diversion or partial ileal bypass (67–70). Ultimately, it will be of interest to know whether ASBT inhibitors (60–66) or novel potent bile acid sequestrants will be as effective as these surgical interventions (71). Whether this approach can be extrapolated to other forms of cholestasis is not known. One of the effects of ursodeoxycholic acid, which seems to be effective in cholestatic liver disease, is to interfere with ileal reclamation of bile salts (72). REGULATION OF APICAL SODIUM-DEPENDENT BILE ACID TRANSPORTER EXPRESSION In light of the significance of ASBT in human health and disease, there is great value in understanding its regulation. Knowledge of its normal regulation may lead to novel pharmacotherapeutic methods and to optimization of current regimens. In addition, dissection of its regulation may permit new approaches to diseases in which ASBT expression is abnormal. The normal regulation of the expression of ASBT is complex (Table 2). ASBT has a distinct pattern of tissue-specific expression, including terminal ileum, proximal renal convoluted tubule, large cholangiocytes, and gallbladder (10,15–17,25). In the rat intestine, ASBT mRNA, protein and function are restricted to the terminal 30% of the small intestine (73). In adult humans, ASBT expression is primarily limited to the terminal 15% of the small intestine (74), whereas it is localized to the terminal 20% in the hamster small intestine (75).TABLE 2: Regulation of ASBT expressionAnalysis of the factors controlling the region-specific expression in the intestine may lead to methods to induce expression in proximal intestine. This could be especially useful in Crohn disease and necrotizing enterocolitis, conditions in which the terminal ileum is often resected. In fact, the response of ASBT to intestinal resection is complex and is dependent on the type of intestinal resection (73,76). After ileal resection, ASBT expression is restricted to regions of the intestine that natively express ASBT. Oddly, ASBT expression seems to be specifically down-regulated in surgical models of massive intestinal resection, but not in more limited ileal resection (73,76,77). The physiologic consequences of this down-regulation must be assessed. Surgical transposition of the ileum into the proximal intestine does not alter ASBT expression and leads to premature reabsorption of bile salts and cholesterol malabsorption (78). Isolated jejunum is rarely used in small bowel transplantation because it does not have the capacity to express terminal ileal gene products. DEVELOPMENTAL REGULATION Physiologic and molecular studies have shown that intestinal bile acid transport is up-regulated in the intestine during normal development. This phenomenon has been observed in humans, dogs, rats, rabbits, and guinea pigs (79–85). In rats, this process is associated with an up-regulation of intestinal ASBT protein and mRNA expression (11). The mechanisms underlying the developmental stage-specific expression of ASBT are not known. Corticosteroids, thyroid hormone, and bile acid feeding can precociously induce up-regulation of intestinal bile acid transport before weaning (86–88). A potential clue to the mechanisms underlying the ontogenic regulation of ASBT is the finding that the expression of the same gene product in the kidney is unchanged during this developmental period (15). Technical issues have not permitted analysis of the developmental regulation of ASBT in the bile duct epithelium. In rats, the up-regulation of ASBT expression at weaning is in fact a reinduction of prenatal expression that was repressed postnatally (11,89). This pattern is reminiscent of the developmental expression of the ileal lipid binding protein in the mouse (20). Careful assessment of normal rat development strongly indicates that transcriptional and posttranscriptional controls are involved in the regulation of ASBT expression. Measurement of steady state ASBT mRNA levels by northern blotting and ASBT transcription analyses by nuclear run-on assays has been performed in developing rat intestines and kidneys (11,15). On postnatal day 7 there is 10-fold more ASBT mRNA in the intestine than in the kidney, despite identical transcription rates. Between postnatal day 7 and postnatal day 28 there is a 200-to 400-fold increase in ASBT mRNA in the intestine, yet there is only a 10-fold increase in transcription. These two findings strongly suggest that ASBT transcription and mRNA stability change during normal intestinal development. BILE ACID RESPONSIVENESS The bile acid responsiveness of the ASBT gene is an area of controversy. Different investigators have observed that the ASBT gene is up-regulated, down-regulated, or independent of bile acids (71,73,90–92). Differences in experimental models, methods of assessment of ASBT, and species investigated may account for some of the discrepancies that have been observed. Initial investigations of the bile acid responsiveness of intestinal bile acid transport were performed in rats and guinea pigs using cholestyramine or bile acid feeding. Intestinal transport of bile salts was equated with biliary secretion measured by biliary drainage. Using this model, negative feedback regulation was observed (90). Kinetic analysis of brush border membrane vesicles prepared from the ileum of rats subjected to either common bile duct ligation or biliary diversion showed positive feedback regulation (91,92). In studies from our laboratory, systematic analysis of rats fed bile salts, cholesterol, or bile acid sequestrants showed no change in sodium-dependent bile acid transport or in ASBT protein expression (71). We have also noted that common bile duct ligation in rats produced no change in ASBT (71). Other studies have shown that oral administration of cholic acid, which can be absorbed in the proximal intestine, leads to an increase in ASBT mRNA and protein in rats (93). A recent study of bile duct ligation or diversion in rats revealed positive feedback regulation of bile acid transport despite unchanged steady state levels of the ileal lipid binding protein, OATP3 and ASBT monomer (94). The limited number of investigations that have been performed in mice seem to show negative feedback regulation of ASBT (95,96). Bile acid feeding leads to down-regulation of expression, whereas decreased presentation of bile salts in cholesterol 7-alpha hydroxylase knockout mice or after cholestyramine feeding leads to up-regulation of transport. Continued systematic examination of this question will be needed to resolve these different observations. The importance of the bile acid responsiveness of ASBT is most evident in the treatment of hypercholesterolemia. If there is a compensatory response to inhibiting ASBT expression or to bile acid wasting, the effectiveness of this pharmacologic approach will be limited. Inhibition of that compensation might optimize the usefulness of ASBT inhibitors. An example of the physiologic significance of the bile acid responsiveness of ASBT can be seen from analysis of differences of the effect of cholesterol feeding in the rat and rabbit (97). Cholesterol feeding in rabbits but not rats leads to marked hypercholesterolemia. In both species, cholesterol feeding leads to increased absorption of cholesterol and increased synthesis of bile acids from cholesterol. In rats, there is no compensatory change in ASBT expression. Because the level of ASBT is rate limiting for expansion of the bile acid pool, there is no increase in the pool size and no mechanism that down-regulates bile acid biosynthesis. The net result is conversion of cholesterol to bile acids and subsequent excretion in stool. In contrast, in the rabbit, ASBT is up-regulated and is not rate limiting for bile acid reabsorption, and the bile acid pool expands. This leads to down-regulation of bile acid biosynthesis and eliminates the major mechanism of cholesterol excretion: bile acid wasting. Thus, the regulatory response of ASBT is a major factor in cholesterol homeostasis. Recent advances in our understanding of the molecular mechanisms of bile acid responsiveness may help to clarify the controversies regarding the bile acid responsiveness of the ASBT gene (98). Bile acids up-regulate the expression of the ileal lipid binding protein gene via activation of a complex consisting of the farnesoid X receptor (FXR) and the 9-cis-retinoic acid receptor (RXR) (99–101). Bile acid-mediated down-regulation of the cholesterol 7-alpha hydroxylase gene is more complex. It involves FXR-mediated induction of transcription of the short heterodimer partner (SHP), which in turn inactivates the liver receptor homolog-1 (LRH-1), which is a positive transacting factor for cholesterol 7-alpha hydroxylase gene transcription (102). Of note, FXR and SHP are expressed in the small intestine (103,104). In contrast, it does not appear that LRH-1 is expressed in the small intestine and if this system is active in ASBT down-regulation, an alternative positive transacting factor is likely to be involved (105). The effects of targeted disruption of the FXR gene in mice on expression of ASBT are not clear (106). Systematic analysis of the effects of bile acids on ASBT transcription, including analysis of the roles of FXR, RXR, and SHP, will likely clarify the bile acid responsiveness of the ASBT gene. INFLAMMATORY AND CORTICOSTEROID RESPONSIVENESS Intestinal bile acid malabsorption has clearly been shown in ileitis associated with Crohn disease (107). Using a rabbit Eimeria magna ileitis model, Sundaram et al. (59) showed that rabbit ASBT is down-regulated by inflammation. Furthermore, corticosteroids restore diminished ASBT expression in this model (108). ASBT also seems to be glucocorticoid responsive in a noninflamed state, with up-regulation seen in both the rat and the rabbit ileum (108,109). MECHANISMS OF TRANSCRIPTIONAL REGULATION OF INTESTINAL GENES Transcriptional regulation in the intestine has been studied by using three distinct and complementary approaches: in vitro transcription of the gene, analysis of in vitro DNA, and protein interactions and studies of transgenic animals. The first two approaches have identified a cadre of potential interactions between cis-acting and transacting elements in a number of intestinal genes, including sucrase isomaltase, lactase-phlorizin hydrolase, ileal bile acid binding protein, Na+–glucose cotransporter, calbindin-D9k, apolipoprotein B, aminopeptidase N, α-1-antitrypsin, and trefoil factor (101,110–117). The sucrase–isomaltase and lactase–phlorizin genes are the best studied examples, and, in both circumstances, a combination of transcription factors seems to be involved in the control of basal transcription. In vitro transcriptional analysis of the human sucrase–isomaltase (SI) 5' flanking region showed that it functioned as a promoter in cell lines and directed specificity to intestinal-derived cell lines (118). Three distinct DNA-binding proteins were described in the proximal promoter of SI (119). Two of these transacting factors were hepatic nuclear factor-1 α (HNF-1 α) and HNF-1 β, both of which are transcription factors previously found to play a key role in hepatic gene regulation (110). The other transacting factor is Cdx-2, a member of the caudal family of homeodomain proteins (120). This protein seems to be involved in the regulation of various intestinal genes and is also fundamentally involved in intestinal cell line differentiation (111,113,114,121–123). Similarly, the HNF-1 family of transcription factors appears to be involved in the regulation of a variety of intestinal genes (112,116,124). Various transcription factors also seem to be involved in the regulation of the lactase–phlorizin gene, including Cdx-2, GATA-6, HOXC11 and HNF-1 (111,124–128). The precise mechanisms by which these transcription factors direct tissue-, region-and developmental stage-specific expression is not clear. Simple changes in single transacting factors have sufficiently explained observed alterations in intestinal gene expression. Relative levels of one factor compared with another may be one mechanism used to direct the complexities of intestinal gene regulation, including developmental regulation and glucose responsiveness (129,130). Further analysis of the mechanisms of intestinal gene regulation has made complementary transgenic approaches necessary. Transgenic analysis of the regulatory elements of intestinal genes potentially permits more precise analysis of the ability of these elements to direct tissue–, cell type– and developmental stage-specific expression. In vitro analyses, such as those described previously, can suggest that specific cis-acting and transacting elements may be involved in regulatory processes. Unfortunately, no cell line or culture system adequately recapitulates normal physiology. However, transgenic mouse studies permit in vivo testing of potential regulatory control mechanisms. Transgenic analyses have been used in the investigation of a wide range of intestinal genes including, sucrase–isomaltase, lactase–phlorizin hydrolase, apolipoprotein B, apolipoprotein A-I, intestinal and liver fatty acid binding proteins, ileal lipid binding protein, calbindin-D9K, carbamoyl-phosphate synthetase I, alpha-fetoprotein, villin, MUC2, and cryptidin (20,131–143). A number of regulatory themes seem to have evolved from these analyses, although clear understanding of the basic molecular mechanisms of tissue and developmental stage-specific expression is unresolved. Relatively small proximal promoter fragments (144–146) can impart intestine-specific expression in many circumstances (eg. sucrase isomaltase, liver, and intestine fatty acid binding proteins). In sharp contrast, the intestinal specificity of the apolipoprotein B gene is directed by an element that is more than 54,000 base pairs (bp) away from the gene itself (147). Appropriate patterns of ontogenic expression of reporter constructs have been observed to be directed by relatively small promoter fragments of the sucrase, lactase, fatty acid and lipid binding proteins (20,132,136,144,148). POTENTIAL ROLE OF MRNA STABILITY IN THE REGULATION OF INTESTINAL GENES Although mRNA stability has been shown to play a key role in the regulation of the expression of a wide range of genes, little information exists regarding this mechanism of gene regulation in the intestine. It seems that mRNA stability plays a significant role in the regulation of various genes. Supporting data include finding a discrepancy between steady-state mRNA levels and transcription rates (as measured by nuclear run-on assays) and by finding inducible changes in mRNA half-lives in vitro or in vivo (149–156). Experimental analysis of the role of mRNA stability in intestinal gene regulation is limited. Polyamine depletion leads to marked increases in transglutaminase mRNA stability in Caco-2 cells (157). The human small intestine peptide transporter mRNA is stabilized to a small extent via induction of its substrate Gly-Gln (158). In HT-29 cells, decay-accelerating factor mRNA is stabilized after treatment with tumor necrosis factor-α(159). Nearly all the investigations of mRNA stability have been performed using in vitro systems. A limited series of investigations of in vivo hepatic mRNA stability studies have been accomplished using coadministration of the transcriptional inhibitors actinomycin D and α-amanitin (160). The mechanisms of alterations of mRNA stability are mediated by interactions between 3´ untranslated RNA sequences and specific RNA-binding binding proteins with endonucleolytic and RNase activities. Messenger RNA degradation is a regulated process. Polyadenylation and associated polyadenylation binding proteins increase the stability of mRNA by interfering with direct 3´ to 5´ exonucleolytic decay. Specific RNA endonucleases can cleave mRNA at specific sites, which then promulgates both 5´ to 3´ and 3´ to 5´ exonucleolytic degradation. Finally, the 5´ cap of mRNA imparts stability and its removal, which may be facilitated by deadenylation, promotes exonucleolytic degradation (161–163). The best characterized eukaryotic examples of this process include lymphokines, cytokines, and transcription factors, which have short half-lives. Specific RNA sequences, most notably those that are adenylate and uridylate rich impart relative RNA instability (164). Two specific RNA binding proteins, HuR and hnRNP D, bind to these elements and may influence relative mRNA stability (165,166). This type of mechanistic detail is unknown for intestinal genes. Relative mRNA stability can be assessed by evaluating the half-life of a particular RNA transcript. Unstable transcripts will often have half-lives in the range of 10 to 30 minutes, whereas stable transcripts may decay over periods as long as 12 to 24 hours. Typically, RNA half-life is assessed by cell culture, using timed quantitation of RNA levels after transcription has been discontinued. Discontinuation of transcription can be induced by several means. A number of transcriptional inhibitors have been used in this regard and include actinomycin D, amanitin, and DRB (167–170). The major drawback of these agents is their potential nonspecific toxic effects, which can result in spurious information regarding mRNA half-lives (171). An alternative approach is to transiently induce mRNA production and then follow the rate of RNA disappearance. Serum starvation followed by serum induction of the c-fos promoter has been classically used for this approach (172). Recently, the use of a tetracycline repressible system has been described for the analysis of mRNA half-lives (173,174). This system may be the least "toxic" and may be the optimal method for in vitro analysis of mRNA stability. Cell-free systems for the analysis of RNA stability have also recently been characterized and potentially permit insight into the physiologic relevance of in vitro studies (175). SUMMARY Intestinal reabsorption of bile salts plays a crucial role in human health and disease. This process is primarily localized to the terminal ileum and is mediated by a 48-kd sodium-dependent bile acid cotransporter (SLC10A2 = ASBT). ASBT is also expressed in renal tubule cells, cholangiocytes, and the gallbladder. Exon skipping leads to a truncated version of ASBT, which sorts to the basolateral surface and mediates efflux of bile salts. Inherited mutation of ASBT leads to congenital diarrhea secondary to bile acid malabsorption. Partial inhibition of ASBT may be useful in the treatment of hypercholesterolemia and intrahepatic cholestasis. During normal development in the rat ileum, ASBT undergoes a biphasic pattern of expression with a prenatal onset, postnatal repression, and reinduction at the time of weaning. The bile acid responsiveness of the ASBT gene is not clear and may be dependent on both the experimental model used and the species being investigated. Future studies of the transcriptional and posttranscriptional regulation of the ASBT gene and analysis of ASBT knockout mice will provide further insight into the biology, physiology, and pathophysiology of intestinal bile acid transport.