Demonstration by in situ hybridization of the zonal modulation of rat liver cytochrome P-450b and P-450e gene expression after phenobarbital.

The various physiological processes that constitute liver function are compartmentalized within the hepatic acinus. The molecular mechanisms modulating the development and maintenance of this hepatocyte heterogeneity have not been defined. The objective of this study was to determine whether transcriptional or posttranscriptional zonal modulation of cytochromes P-450b,e gene expression was responsible for the heterogeneous induction of the P-450 proteins, which is observed after phenobarbital (PB) administration. The exact localization in liver tissue of hepatocytes responding to PB with induction of either P-450b,e mRNA or proteins was established by in situ hybridization and by immunofluorescence, respectively. As demonstrated by quantitative assessment of autoradiographs of approximately 20 hepatocytes located between a terminal portal venule and a hepatic venule, PB induced the P-450b,e mRNA up to sixfold in the 12-15 hepatocytes located closer to the hepatic venules (zones 2 and 3). In contrast, there was only a twofold induction in the 4-6 hepatocytes surrounding the terminal portal venules (zone 1). Quantitative immunofluorescence using an MAb showed that the acinar distribution of PB-induced P-450b,e proteins was similar to that of the mRNA. This combined approach indicated that, most likely, an increased rate of transcription of cytochromes P-450b,e genes in hepatocytes of zones 2 and 3 concomitantly, with a relative lack of activation, or repression, of these genes in hepatocytes of zone 1, were responsible for the heterogeneous phenotype observed after PB administration. Therefore, modulation of gene expression among hepatocytes of the liver acinus is one mechanism by which the functional heterogeneity of hepatocytes is attained. Experiments in which the induction of cytochromes P-450b,e genes was studied after administration of either PB or para-hydroxyphenobarbital, a main hepatic metabolite of PB, suggested that the species involved in the inductive process is the parent PB molecule rather than para-hydroxyphenobarbital.

An intestine-specific homeobox gene regulates proliferation and differentiation.

Precise regulation of cellular proliferation, differentiation, and senescence results in the continuous renewal of the intestinal epithelium with maintenance of a highly ordered tissue architecture. Here we show that an intestine-specific homeobox gene, Cdx2, is a transcription factor that regulates both proliferation and differentiation in intestinal epithelial cells. Conditional expression of Cdx2 in IEC-6 cells, an undifferentiated intestinal cell line, led to arrest of proliferation for several days followed by a period of growth resulting in multicellular structures containing a well-formed columnar layer of cells. The columnar cells had multiple morphological characteristics of intestinal epithelial cells. Enterocyte-like cells were polarized with tight junctions, lateral membrane interdigitations, and well-organized microvilli with associated glycocalyx located at the apical pole. Remarkably, there were also cells with a goblet cell-like ultrastructure, suggesting that two of the four intestinal epithelial cell lineages may arise from IEC-6 cells. Molecular evidence for differentiation was shown by demonstrating that cells expressing high levels of Cdx2 expressed sucrase-isomaltase, an enterocyte-specific gene which is a well-defined target for the Cdx2 protein. Taken together, our data suggest that Cdx2 may play a role in directing early processes in intestinal cell morphogenesis and in the maintenance of the differentiated phenotype by supporting transcription of differentiated gene products. We propose that Cdx2 is part of a regulatory network that orchestrates a developmental program of proliferation, morphogenesis, and gene expression in the intestinal epithelium.

Novel DNA-binding proteins regulate intestine-specific transcription of the sucrase-isomaltase gene.

Sucrase-isomaltase (SI) is an enterocyte-specific gene which exhibits a complex pattern of expression during intestinal development and in the adult intestinal mucosa. In the studies described in this report, we demonstrate that enterocyte-specific transcription of the SI gene is regulated by an evolutionarily conserved promoter that extends approximately 180 bp upstream of the transcription start site. DNase I footprint analysis allowed the identification of three nuclear protein-binding sites within the SI promoter (SIF1, SIF2, and SIF3 [SI footprint]), each of which acted as a positive regulatory element for transcription in intestinal cell lines. SIF1 was shown to bind nuclear protein complexes present in primary mouse small intestinal cell and in an intestinal cell line (Caco-2). However, SIF1-binding proteins were absent in a variety of other epithelial and nonepithelial cells. In vitro mutagenesis experiments demonstrated that the SIF1 site is required for high-level promoter activity in intestinal cells. The SIF3 element formed prominent binding complexes with intestinal and liver nuclear extracts, whereas nuclear proteins from other epithelial and nonepithelial cells formed weaker complexes of different mobilities. The SIF2 element bound nuclear proteins in a pattern similar to that of SIF3, and cross-competition studies suggested that SIF2 and SIF3 may bind the same nuclear proteins. Taken together, these data have allowed the identification of novel DNA-binding proteins that play an important role in regulating intestine-specific transcription of the SI gene.

A homeodomain protein related to caudal regulates intestine-specific gene transcription.

The continually renewing epithelium of the intestinal tract arises from the visceral endoderm by a series of complex developmental transitions. The mechanisms that establish and maintain the processes of cellular renewal, cell lineage allocation, and tissue restriction and spatial assignment of gene expression in this epithelium are unknown. An understanding of the regulation of intestine-specific gene regulation may provide information on the molecular mechanisms that direct these processes. In this regard, we show that intestine-specific transcription of sucrase-isomaltase, a gene that is expressed exclusively in differentiated enterocytes, is dependent on binding of a tissue-specific homeodomain protein (mouse Cdx-2) to an evolutionarily conserved promoter element in the sucrase-isomaltase gene. This protein is a member of the caudal family of homeodomain genes which appear to function in early developmental events in Drosophila melanogaster, during gastrulation in many species, and in intestinal endoderm. Unique for this homeodomain gene family, we show that mouse Cdx-2 binds as a dimer to its regulatory element and that dimerization in vitro is dependent on redox potential. These characteristics of the interaction of Cdx-2 with its regulatory element provide for a number of potential mechanisms for transcriptional regulation. Taken together, these findings suggest that members of the Cdx gene family play a fundamental role both in the establishment of the intestinal phenotype during development and in maintenance of this phenotype via transcriptional activation of differentiated intestinal genes.

Randomised clinical study: GR-MD-02, a galectin-3 inhibitor, vs. placebo in patients having non-alcoholic steatohepatitis with advanced fibrosis.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) and resultant liver fibrosis is a major health problem without approved pharmacotherapy. Pre-clinical results of GR-MD-02, a galectin-3 inhibitor, suggested potential efficacy in NASH with advanced fibrosis/cirrhosis and prompted initiation of a clinical development programme in NASH with advanced fibrosis. AIM: To evaluate the safety, pharmacokinetics and exploratory pharmacodynamic markers of GR-MD-02 in subjects having NASH with bridging fibrosis. METHODS: The GT-020 study was a first-in-human, sequential dose-ranging, placebo controlled, double-blinded study with the primary objective to assess the safety, tolerability and dose limiting toxicity of GR-MD-02, in subjects with biopsy-proven NASH with advanced fibrosis (Brunt stage 3). The secondary objectives were to characterise first-dose and multiple-dose pharmacokinetic profiles and to evaluate changes in potential serum biomarkers and liver stiffness as assessed by FibroScan. RESULTS: GR-MD-02 single and three weekly repeated of 2, 4 and 8 mg/kg revealed no meaningful clinical differences in treatment emergent adverse events, vital signs, electrocardiographic findings or laboratory tests. Pharmokinetic parameters showed a dose-dependent relationship with evidence of drug accumulation following 8 mg/kg (~twofold). CONCLUSIONS: GR-MD-02 doses were in the upper range of the targeted therapeutic dose determined from pre-clinical data and were safe and well tolerated with evidence of a pharmacodynamic effect. These results provide support for a Phase 2 development programme in advanced fibrosis due to NASH.

Expression and regulation of cytochrome P-450I genes (CYP1A1 and CYP1A2) in the rat alimentary tract.

Cytochrome P-450 (P450) enzymes in the mucosa of the alimentary tract may be involved in the activation and/or inactivation of ingested xenobiotics and procarcinogens. Since the multiple P450 enzymes have overlapping substrate specificities and, in some cases, similar antigenic determinants, definitive identification of P450 genes that are expressed in various tissues requires molecular analysis. In this study, a sensitive and specific polymerase chain reaction assay along with hybridization analysis was used to examine the expression of the CYP1A gene family in the rat alimentary tract. CYP1A1 mRNA was expressed throughout the alimentary tract in untreated rats, as determined by the polymerase chain reaction. However, Northern blot analysis detected CYP1A1 mRNA and enzymatic activity only in small intestine and liver, with greater amounts in intestine. After treatment with beta-naphthoflavone, CYP1A1 mRNA and enzymatic activity was markedly induced in each alimentary tissue including esophagus, fore-stomach, glandular stomach, small intestine and colon. A single dose of inducer resulted in a rapid rise in CYP1A1 mRNA which was shown by nuclear run-on assays to be primarily due to an increase in transcription of the CYP1A1 gene. CYP1A2 mRNA was detected in significant amounts only in glandular stomach following induction although the polymerase chain reaction assay identified low levels of CYP1A2 mRNA in several other tissues. The definitive identification of the CYP1A genes that are expressed in alimentary tissue will allow the design of experiments to investigate the importance of these enzymes in the metabolism of carcinogens, and ultimately carcinogenesis, in the gastrointestinal tract. In addition, these data suggest that the aromatic hydrocarbon receptor, which mediates transcriptional induction of multiple genes by xenobiotics, is expressed through the alimentary tract.

Comparison of intestinal phospholipase A/lysophospholipase and sucrase-isomaltase genes suggest a common structure for enterocyte-specific promoters.

Intestinal phospholipase A/lysophospholipase (IPAL) is an intestine-specific brush-border enzyme expressed during development and along the intestinal crypt-villus axis in a pattern similar to another well characterized brush-border enzyme, sucrase-isomaltase (SI). A tissue-specific DNase I hypersensitive site was identified in chromatin from intestinal nuclei immediately upstream from the transcriptional start site of the IPAL gene. Footprinting analysis showed that two DNA elements within the IPAL promoter were protected by intestinal nuclear proteins. The IPAL-FP1 element was shown to be a monomer binding site for Cdx1 and Cdx2, intestine-specific homeobox proteins. Moreover, this site was important for transcriptional activation of the promoter in intestinal cell lines via interaction with Cdx proteins. Nuclear proteins from both liver and intestine interacted with the IPAL-FP2 element, forming a complex consistent with binding to HNF1. Cdx and HNF1 binding sites have also been shown to be the two major regulatory elements responsible for transcriptional activation of the SI gene promoter, which directs intestine-specific transcription in transgenic mice. These findings suggest that enterocyte genes that are expressed in similar developmental patterns may be regulated by the interaction of common DNA elements and their associated transcription factors.

Molecular pathogenesis of colorectal cancer.

The enormous progress made in the identification of genes that are involved in colon carcinogenesis has provided the foundation for further understanding the biology of both normal and cancer cells and for targeted therapeutic strategies. In one sense, the genes described in this review are only the building blocks of a larger puzzle that constitutes the integrated metabolic function of a cell. The current challenge is to understand the functional role of these genes in normal cellular physiology and make the connections between pathways that knit together integrated cellular homeostasis. A complete understanding of the regulatory pathways, and the synthesis and modifications of the proteins involved, will provide novel targets for therapeutic agents.

Cancer in inflammatory bowel disease.

Patients with inflammatory bowel disease, including both ulcerative colitis (UC) and Crohn's disease, are at increased risk for the development of gastrointestinal carcinoma, particularly colorectal adenocarcinoma. The current options to reduce this cancer risk include prophylactic colectomy, periodic endoscopic screening with colectomy performed in those patients found to have dysplastic colonic mucosa, or expectant management with no routine surveillance regimen. Despite the lack of data demonstrating effectiveness of surveillance colonoscopy, this approach has become the standard of care in most communities in the United States. Although it has fallen out of fashion in recent years, prophylactic colectomy remains a good option for reducing cancer risk for select patients with UC.

Transcriptional regulation in intestinal development. Implications for colorectal cancer.

Deciphering the complex mechanisms of intestinal epithelial development will require multiple cell and molecular approaches in both in vitro and whole animal systems. Additionally, the use of model organisms such as D. melanogaster, C. elegans, and zebrafish will help describe paradigms that may be investigated in mammals as well as serve as test systems for findings from mammals. This manuscript reviewed only one approach to understanding intestinal development. However, the Cdx story and the information to be mined from an understanding of SI gene transcription is not at an end. As the other pieces of the transcriptional puzzle of the SI gene are assembled there will be new information to generate hypotheses on the relationship of transcriptional mechanisms to cancer pathogenesis.

Epithelial cell growth and differentiation. V. Transcriptional regulation, development, and neoplasia of the intestinal epithelium.

Coordination of gene transcription is a critical regulatory step in orchestrating developmental, differentiation, and adaptation processes in the mammalian intestinal epithelium. An understanding of the regulatory network of nuclear proteins that direct transcriptional initiation of intestinal genes will provide insight into the mechanisms of normal development and differentiation as well as disease processes such as neoplasia.

Control of gene expression in intestinal epithelial cells.

Coordination of gene transcription is a critical regulatory step in orchestrating developmental, differentiation and adaptation processes in the mammalian intestinal epithelium. Insight into these mechanisms has been gained by the study of transcriptional regulation of the sucrase-isomaltase gene. An understanding of the regulatory network of nuclear proteins that direct transcriptional initiation of intestinal genes such as sucrase-isomaltase will provide insight into the mechanisms of normal development and differentiation as well as disease processes such as neoplasia.

Regulation of sucrase-isomaltase gene expression along the crypt-villus axis of rat small intestine.

The expression of sucrase-isomaltase mRNA was investigated along the crypt-villus axis of rat small intestine using differentially isolated cells and in situ hybridization. A partial rat sucrase-isomaltase cDNA was cloned which coded for a protein that was predicted to be 88% homologous to those encoded by the rabbit and human cDNAs. Southern blot analysis of rat genomic DNA indicated that the cDNA hybridized to a single gene. Northern blots of RNA extracted from subpopulations of intestinal epithelial cells that were isolated from villus and crypt compartments showed that this cDNA hybridized to a 6.5 kb band predominantly in villus RNA. In situ hybridization using 35[S]-labeled RNA probes demonstrated that autoradiographic grains were detected over eptithelial cells located on villi with the greatest number of grains located at the crypt-villus junction and in the lower to mid-villus region; from mid-villus to the villus tip there was a decline in sucrase-isomaltase mRNA. We conclude that expression of sucrase-isomaltase as enterocytes emerge from intestinal crypts is regulated primarily at the level of mRNA accumulation which, most likely, is a result of activation of sucrase-isomaltase gene transcription.

Intestine-specific gene transcription.

The diverse cellular lineages that populate the intestinal epithelium are derived from committed stem cells located in intestinal crypts. The complex architecture of the intestinal epithelium results from well-orchestrated processes of cell-line-age allocation, proliferation of immature cells in the crypt compartment, differentiation of various cell lineages, migration of cells in defined patterns, and cell-specific programmed senescence. The patterns of intestinal gene transcription in the context of this complex architecture are regulated by the combinatorial effect of multiple positive and negative regulatory elements. Although the DNA regulatory elements required to recapitulate the pattern of endogenous gene expression appear to be spread over relatively large genomic distances, short promoters of several intestinal genes are sufficient to direct intestine-specific transcription. The sucrase-isomaltase gene promoter has multiple regulatory elements that bind tissue-restricted transcription factors. A critical factor in regulating the sucrase-isomaltase promoter is Cdx2, an intestine-specific homeobox gene related to caudal, that may also have a broader role in intestinal development and morphogenesis. As additional regulatory elements and their cognate DNA-binding proteins are identified, the challenge will be to define their integrated role in the regulation of intestine-specific genes and in the development and maintenance of the intestinal epithelium.

Differential regulation of cytochrome P-450 genes along rat intestinal crypt-villus axis.

Mammalian small intestine contains cytochrome P-450-dependent monooxygenase enzymes that are capable of metabolizing a wide variety of xenobiotics and activating procarcinogens to mutagenic compounds. The epithelial cells lining the small intestine are separated into a proliferating undifferentiated compartment located in crypts and a nonproliferating differentiated compartment located on villi. The constitutive expression and induction by xenobiotics of genes that encode components of the cytochrome P-450-dependent mono-oxygenase system along the rat intestinal crypt-villus axis were investigated using isolated epithelial cells and in situ hybridization. For each gene examined, hybridization analysis of RNA obtained from isolated epithelial cells correlated with findings on in situ RNA hybridization. Cytochrome P-450IA1 mRNA (CYP1A1), the major aromatic hydrocarbon-inducible P-450, and cytochrome P-450IIB1 mRNA (CYP2B1), the major phenobarbital-inducible P-450, were constitutively expressed in villus cells with no detectable mRNA present in crypts. Treatment with several chemical inducers resulted in a marked increase in CYP1A1 mRNA in both crypt and villus cells. In contrast, although CYP2B1 mRNA was inducible in villus cells, CYP2B1 mRNA was not detected in crypts after treatment with chemical inducers. NADPH cytochrome P-450 reductase, a necessary component for the activity of all P-450 enzymes, was expressed constitutively at low levels only in villus cells. Treatment with dexamethasone induced reductase mRNA in both crypt and villus cells. Taken together, these results demonstrate that there is a complex gene-specific pattern of expression of the microsomal monooxygenase system along the crypt-villus axis of rat small intestine.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolation and characterization of the human sucrase-isomaltase gene and demonstration of intestine-specific transcriptional elements.

The molecular mechanisms that regulate intestine-specific gene expression and the transition from proliferating, undifferentiated crypt cells to nonproliferating, differentiated villus cells are unknown. Sucrase-isomaltase is an apical membrane disaccharidase that is found exclusively in enterocytes of adult intestine and is expressed in a complex pattern along the intestinal crypt-villus axis. To investigate the regulation of sucrase-isomaltase, we have cloned and sequenced 3.6 kilobases of the 5'-flanking region of the human sucrase-isomaltase gene. The transcriptional start site was mapped in human small intestine and in a colonic adenocarcinoma cell line (Caco-2) using an anchored polymerase chain reaction, primer extension, and RNase protection assays. The 5'-flanking DNA of the gene was linked to either chloramphenicol acetyltransferase or luciferase reporter genes and used for transfection into Caco-2, HeLa, and HepG2 cells. This analysis demonstrated that intestine-specific transcription of the sucrase-isomaltase gene involves both proximal and distal regulatory elements. Use of sucrase-isomaltase as a model gene will allow investigation of the mechanisms that regulate transcription of enterocyte-specific genes, developmental gene expression in the small intestine and colon, and the process of differentiation as epithelial cells migrate from intestinal crypts onto the villus in adult intestine.

Induction of cytochrome P450IA genes (CYP1A) by omeprazole in the human alimentary tract.

Cytochrome P450 enzymes are capable of converting procarcinogens into either active mutagens or inactive metabolites. Because the distribution of these enzymes may be important for tissue susceptibility to procarcinogens, the expression and induction of CYP1A genes in the human alimentary tract were investigated. Endoscopic biopsy specimens were obtained from buccal mucosa, esophagus, gastric body, antrum, duodenum, and colon of 6 healthy volunteers before and 1 week after taking 20 mg of omeprazole daily. Tissue specimens were analyzed for the presence of CYP1A1 and 1A2 transcripts using hybridization methods and the polymerase chain reaction. P450-dependent enzymatic activity was assessed by deethylation of ethoxyresorufin. CYP1A1 messenger RNA (mRNA) and ethoxyresorufin activity were present constitutively in the duodenum of each volunteer. Omeprazole (20 mg/day for 1 week) induced CYP1A1 mRNA and enzymatic activity in 5 of 6 volunteers. The one individual who did not initially respond had a marked increase in both mRNA and enzymatic activity after receiving 60 mg of omeprazole daily for 1 week. After treatment with omeprazole, two individuals had low levels of CYP1A1 mRNA in several other alimentary tissues as well as low levels of CYP1A2 mRNA in the duodenum. The expression and induction by a pharmaceutical agent of CYP1A genes may have implications for intestinal metabolism of ingested xenobiotics including procarcinogens.

Isolation of intestinal epithelial cells for the study of differential gene expression along the crypt-villus axis.

Methods for the differential isolation of intestinal epithelial cells from crypt and villus compartments of small intestine have been used in the study of intestinal functions. However, the use of different methods has resulted in discrepant conclusions as to the localization of expressed genes. Therefore, we undertook a careful comparison of two methods of intestinal epithelial cell isolation, the distended intestinal sac method and the everted intestinal sac method. The isolated cell fractions (distended sac fractions 1-10, everted sac fractions 1-5) were evaluated for the expression of two mRNAs whose localization along the crypt-villus axis had been previously elucidated by in situ hybridization: cytochrome P-450IIB1 (expressed in villus cells) and cryptdin (expressed in crypt cells). Northern blots of total or poly(A)+ RNA from each cell population showed that the first fractions from both methods contained P-450IIB1 mRNA, suggesting that both methods allowed the isolation of cells originating from the villus. Cryptdin mRNA was detected only in cell fractions 5-10 using the distended sac method and was not detected in any fractions from the everted sac method. [3H]thymidine incorporation demonstrated that dividing (crypt) cells were successfully removed by the distended sac method, but remained with the everted sac intestinal remnant. Finally, light and electron microscopy of the isolated cells as well as the intestinal remnants confirmed that while undifferentiated crypt cells were present in distended sac cell fractions 9 and 10, they remained with the everted sac remnant. Thus the distended sac protocol was useful for the isolation of cells from tip and crypt compartments.(ABSTRACT TRUNCATED AT 250 WORDS)

Brain edema in rabbits with galactosamine-induced fulminant hepatitis. Regional differences and effects on intracranial pressure.

Brain edema and intracranial hypertension are major complications of fulminant hepatic failure. We investigated the development of brain edema and monitored intracranial pressure in rabbits with toxic hepatitis induced by galactosamine. Using a gravimetric technique to assay small tissue samples, we found that brain water was increased in cortical grey matter, but not in subcortical, mesencephalic, and pontine white matter, or in the cerebellum. The proportion of water in cerebral grey matter in control animals was 80.96% +/- 0.49% with significant elevations to 81.96% +/- 0.47% and 82.95% +/- 1.49% in mild and severe encephalopathy, respectively. This corresponds to mean increases in tissue volume of 5.5% and 11.7%. The hippocampal grey matter also accumulated water in severe encephalopathy with a 30% increase in mean tissue volume. The regional increase in brain water was confirmed by the wet-dry weight method. Neither hypotension, hypoxia, nor severe hypoglycemia were present to account for the edema. Intracranial pressure was monitored continuously in unanesthetized rabbits via an intraventricular cannula as encephalopathy developed. The pressure was normal in the mild stage, but was intermittently elevated in animals with severe encephalopathy. The normal range of intracranial pressure was 2-9 mmHg and the range of peak values in galactosamine-treated rabbits was 18-55 mmHg. The regional differences in brain water accumulation suggest that cellular swelling and abnormalities in the movement of water across the blood-brain barrier may account for the brain edema in this model.

Sucrase-isomaltase gene transcription requires the hepatocyte nuclear factor-1 (HNF-1) regulatory element and is regulated by the ratio of HNF-1 alpha to HNF-1 beta.

The mouse sucrase-isomaltase (SI) gene is an enterocyte-specific gene expressed in a complex developmental pattern. We previously reported that a short, evolutionarily conserved gene promoter regulates developmental expression of SI in mouse small intestine. Herein, we investigated the role of a hepatocyte nuclear factor-1 (HNF-1) cis-acting element to regulate SI gene expression in vivo. Transgenic SI gene constructs with a mutated HNF-1 element (SIF3) revealed a strong reduction in promoter activity in comparison with a wild-type construct in mice and during Caco-2 cell differentiation. Nuclear proteins isolated from enterocytes showed increased binding of the HNF-1 alpha complex with a concomitant decrease in the HNF-1 beta-containing complex to the SIF3 element both during the suckling-weaning developmental transition and Caco-2 cell differentiation. These changes coincided with a strong induction of SI gene transcription. In transfection experiments, HNF-1 alpha activated the SI promoter via the SIF3 element, and co-expression of HNF-1 beta impaired this transcriptional activation. These findings demonstrate the essential role of the HNF-1 regulatory element to support SI gene transcription in vivo and suggest that the ratio of HNF-1 alpha to HNF-1 beta plays a role in the transcriptional activity of this gene during intestinal development.