A novel Sp1-related cis element involved in intestinal alkaline phosphatase gene transcription.

We have used sodium butyrate-treated HT-29 cells as an in vitro model system to study the molecular mechanisms underlying intestinal alkaline phosphatase (IAP) gene activation. Transient transfection assays using human IAP-CAT reporter genes along with DNase I footprinting were used to localize a critical cis element (IF-III) corresponding to the sequence 5'-GACTGGGCGGGGTCAAGATGGA-3'. Deletion of the IF-III element resulted in a dramatic reduction in reporter gene activity, and IF-III was shown to function in the context of a heterologous (SV40) promoter in a cell type-specific manner, further supporting its functional role in IAP transactivation. Electrophoretic mobility shift assays revealed that IF-III binds Sp1 and Sp3, but these factors comprise only a portion of the total nuclear binding and appear to mediate only a small portion of its transcriptional activity. IF-III does not correspond to any previously characterized regulatory region from other intestine-specific genes. We have thus identified a novel, Sp1-related cis-regulatory element in the human IAP gene that appears to play a role in its transcriptional activation during differentiation in vitro.

p21(WAF1) is required for butyrate-mediated growth inhibition of human colon cancer cells.

A diet high in fiber is associated with a decreased incidence and growth of colon cancers. Butyrate, a four-carbon short-chain fatty acid product of fiber fermentation within the colon, appears to mediate these salutary effects. We sought to determine the molecular mechanism by which butyrate mediates growth inhibition of colonic cancer cells and thereby to elucidate the molecular link between a high-fiber diet and the arrest of colon carcinogenesis. We show that concomitant with growth arrest, butyrate induces p21 mRNA expression in an immediate-early fashion, through transactivation of a promoter cis-element(s) located within 1.4 kb of the transcriptional start site, independent of p53 binding. Studies using the specific histone hyperacetylating agent, trichostatin A, and histone deacetylase 1 indicate that growth arrest and p21 induction occur through a mechanism involving histone hyperacetylation. We show the critical importance of p21 in butyrate-mediated growth arrest by first confirming that stable overexpression of the p21 gene is able to cause growth arrest in the human colon carcinoma cell line, HT-29. Furthermore, using p21-deleted HCT116 human colon carcinoma cells, we provide convincing evidence that p21 is required for growth arrest to occur in response to histone hyperacetylation, but not for serum starvation nor postconfluent growth. Thus, p21 appears to be a critical effector of butyrate-induced growth arrest in colonic cancer cells, and may be an important molecular link between a high-fiber diet and the prevention of colon carcinogenesis.

Transcriptional activation of the human villin gene during enterocyte differentiation.

Enterocyte differentiation occurs along the crypt-villus axis and is generally thought to involve the transcriptional activation of cell-specific genes, among which is the brush-border structural protein villin. We have examined the molecular mechanisms of villin induction using both in vivo and in vitro systems. Total RNA was purified from rat tissues or cultured cells by the guanidinium thiocyanate method and Northern blot analyses carried out using radiolabeled complementary DNA probes specific for villin or the actin control. Transient transfection (calcium/phosphate method) assays were performed using a luciferase reporter gene containing 2 kb of the human villin gene 5'-flanking region. We have found that the villin mRNA was expressed at high levels in the small intestine, to a lesser degree in the colon, and was not detected in the brain or liver. In HT-29 cells, villin mRNA levels increased 2.5-fold (P<0.001) after 24 hours of sodium butyrate treatment, consistent with the process of enterocyte differentiation. Similarly, villin gene expression was induced in Caco-2 cells during postconfluence differentiation. Transient transfection assays demonstrated marked reporter gene activation (fourfold, P<0.001) in response to sodium butyrate in HT-29 cells, but no activation in the liver cell line HepG2. The effects of sodium butyrate were dose dependent, reaching a maximum at a concentration of 5 mmol/L. We conclude that a 2 kb region of the human villin gene is able to mediate its transcriptional activation during HT-29 cell differentiation. This DNA regulatory region appears to function in a cell type-specific (gut) manner.

Thyroid hormone and the gut: selective transcriptional activation of a villus-enterocyte marker.

BACKGROUND: Thyroid hormone (T3) is an important regulator of gut mucosal growth, differentiation, and barrier function, but its mechanism of action in the gastrointestinal tract is largely unknown. The present studies were carried out to define the molecular mechanisms by which T3 alters gut gene expression. METHODS: In vivo: Adult, male, Sprague-Dawley rats were given three daily injections (intraperitoneal) of either saline solution or 30 micrograms/kg triiodothyronine. Small intestinal tissues were harvested, and Northern blot analyses were performed by using specific radiolabeled cDNA probes. In vitro: HT-29 cells were transfected with reporter plasmids and treated with or without T3, and chloramphenicol acetyltransferase activity was measured. RESULTS: The T3-induced changes in enterocyte gene expression occurred in villus enterocytes and not in crypt cells and were independent of food intake. Northern analyses with an intron-specific probe revealed that the T3 induction in intestinal alkaline phosphatase (IAP) expression occurs at the level of transcription. Transient transfection assays revealed no T3-induced changes under basal conditions but marked increases (sixfold, p < 0.001) when a T3-receptor (TR beta-1) plasmid was cotransfected. Furthermore, T3 was found to induce greater IAP reporter gene activity in differentiated (+ sodium butyrate) compared with undifferentiated HT-29 cells. CONCLUSIONS: T3 induces IAP expression at the level of gene transcription. Both in vivo and in vitro, IAP transcriptional activation occurs to a greater extent in differentiated enterocytes than in undifferentiated crypt cells. Transactivation of the IAP gene by T3 is mediated via a DNA cis-element(s) located within the 2.4 kb segment present in the reporter gene.

Differential cloning of novel intestine-specific genes whose expression is altered under conditions of villus atrophy.

Atrophy of the small intestinal villi occurs in a variety of disease states and is associated with diarrhea, malabsorption, and impaired barrier function. We have previously demonstrated that villus atrophy is associated with an increase in lactase and a decrease in intestinal alkaline phosphatase gene expression. Given these changes in enterocyte phenotype with villus atrophy, we speculated that there may be other intestine-specific genes whose expression is altered as a function of epithelial growth state. We have employed two molecular techniques in order to identify and clone complementary DNAs (cDNA) which are differentially expressed in atrophic compared to normal small intestinal mucosa. In differential cDNA library (+/-) screening, duplicate filters of a normal jejunal cDNA library are hybridized with radiolabeled cDNA probes from either atrophic or control tissues. Comparisons of the intensities of hybridized clones allows for the identification of differentially expressed gene products. In the mRNA differential display system, RT-PCR is used to randomly amplify mRNA species. Similar to cDNA library screening, comparisons of radiolabeled bands on a polyacrylamide sequencing gel allow for the identification of differentially expressed genes. Using these methods, we have identified a novel cDNA, called D9, which appears to be expressed exclusively in the intestinal mucosa. Northern analyses have confirmed that the expression of the D9 mRNA is dramatically decreased under conditions of villus atrophy, suggesting an underlying relationship with epithelial growth state. DNA sequence analysis (GenBank) reveals no identity to previously cloned genes.(ABSTRACT TRUNCATED AT 250 WORDS)

Bombesin maintains enterocyte phenotype in fasted rats.

BACKGROUND: Previous studies have suggested a relationship between enterocyte phenotype and the growth state of the epithelium; under atrophic conditions, lactase gene expression is high, whereas intestinal alkaline phosphatase (IAP) expression is low, and vice versa. On the basis of this model, we hypothesized that the intestinal trophic factor bombesin would alter brush-border enzyme gene expression in a predictable way. METHODS: Adult rats were fasted for 48 hours and treated (intraperitoneally) with either 10 micrograms/kg bombesin or the saline control every 8 hours. Small intestinal mucosal scrapings were taken, total RNA purified, and Northern blot analyses performed with radiolabeled cDNA probes corresponding to lactase, IAP, villin, and actin. Tissue samples were also taken for measurement of mucosal thickness. RESULTS: Bombesin administration caused an increase in jejunal mucosal thickness, thereby confirming its trophic effects. Bombesin resulted in a decrease in lactase mRNA levels and an increase in IAP mRNA levels along the length of the small intestine. No changes occurred in the expression of either villin or actin. The pattern of enterocyte gene expression in the bombesin-treated animals was similar to that in control-fed rats. CONCLUSIONS: Bombesin differentially regulates rat enterocyte gene expression, decreasing lactase and increasing IAP mRNA levels. These results lend further support to the hypothesis that a close relationship exists between enterocyte phenotype and epithelial growth state.