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Recently gene expression studies have been multiplied at an accelerated rate by the use of high-density microarrays. By assaying thousands of transcripts at a time, microarrays have led to the discovery of dozens of genes involved in particular biochemical processes, for example, the response of a tissue/organ to a given chemical with therapeutic or toxic properties. The next step in these studies is to focus on the response of a subset of relevant genes to verify or refine potential therapeutic or toxic properties. We have developed a sensitive, high-throughput gene expression assay for this purpose. In this assay, based on the Luminex xMAP system, carefully selected oligonucleotides were covalently linked to fluorescently coded microspheres that are hybridized to biotinylated cRNA followed by amplification of the signal, which results in a rapid, sensitive, multiplexed assay platform. Using this system, we have developed an RNA expression profiling assay specific for 17 estrogen-responsive transcripts and three controls. This assay can evaluate up to 100 distinct analytes simultaneously in a single sample, in a 96-well plate format. This system has improved sensitivity versus existing microsphere-based assays and has sensitivity and precision comparable with or better than microarray technology. We have achieved detection levels down to 1 amol, detecting rare messages in complex cRNA samples, using as little as 2.5 microg starting cRNA. This assay offers increased throughput with decreased costs compared with existing microarray technologies, with the trade-off being in the total number of transcripts that can be analyzed.

Exposure to some compounds with estrogenic activity, during fetal development, has been shown to alter development of reproductive organs, leading to abnormal function and disease either after birth or during adulthood. In order to understand the molecular events associated with the estrogenicity of different chemicals and to determine whether common sets of gene expression changes can be predictive of estrogenic activity, we have used microarray technology to determine the transcriptional program influenced by exposure to this class of compounds during organogenesis and development. Changes in patterns of gene expression were determined in the developing uterus and ovaries of Sprague-Dawley rats on GD 20, exposed to graded dosages (sc) of 17alpha-ethynyl estradiol (EE), genistein, or bisphenol A (BPA) from GD 11 to GD 20. Dose levels were roughly equipotent in estrogenic activity. We compared the transcript profiles between treatment groups and controls, using oligonucleotide arrays to determine the expression level of approximately 7000 rat genes and over 1000 expressed squence tags (ESTs). At the highest tested doses of EE, BPA, or genistein, we determined that less than 2% of the mRNA detected by the array showed a 2-fold or greater change in their expression level (increase or decrease). A dose-dependent analysis of the transcript profile revealed a common set of genes whose expression is significantly and reproducibly modified in the same way by each of the 3 chemicals tested. Additionally, each compound induces changes in the expression of other transcripts that are not in common with the others, which indicated not all compounds with estrogenic activity act alike. The results of this study demonstrate that transplacental exposure to chemicals with estrogenic activity changes the gene expression profile of estrogen-sensitive tissues, and that the analysis of the transcript profile of these tissues could be a valuable approach to determining the estrogenicity of different compounds.

We have investigated the regulation of gene transcription in the intestine using the guanylyl cyclase C (GCC) gene as a model. GCC is expressed in crypts and villi in the small intestine and in crypts and surface epithelium of the colon. DNase I footprint, electrophoretic mobility shift assay (EMSA), transient transfection assays, and mutagenesis experiments demonstrated that GCC transcription is regulated by a critical hepatocyte nuclear factor-4 (HNF-4) binding site between bp -46 and -29 and that bp -38 to -36 were essential for binding. Binding of HNF-4 to the GCC promoter was confirmed by competition EMSA and by supershift EMSA. In Caco-2 and T84 cells, which express both GCC and HNF-4, the activity of GCC promoter and/or luciferase reporter plasmids containing 128 or 1973 bp of 5'-flanking sequence was dependent on the HNF-4 binding site in the proximal promoter. In COLO-DM cells, which express neither GCC nor HNF-4, cotransfection of GCC promoter/luciferase reporter plasmids with an HNF-4 expression vector resulted in 23-fold stimulation of the GCC promoter. Mutation of the HNF-4 binding site abolished this transactivation. Transfection of COLO-DM cells with the HNF-4 expression vector stimulated transcription of the endogenous GCC gene as well. These results indicate that HNF-4 is a key regulator of GCC expression in the intestine.

Heat-stable enterotoxin (STa) is an important causative agent of diarrheal disease throughout the world. STa is known to bind specifically to receptors in the intestine, provoking intense intestinal secretion. Binding of STa, or of the mammalian endogenous ligands guanylin and uroguanylin, activates the guanylyl cyclase C receptor (GC-C); the resulting elevation of cGMP levels stimulates chloride secretion via CFTR. We have generated knockout mice which completely lack the GC-C receptor. These mice are viable and show no obvious alteration in intestinal fluidity. However, GC-C null mice are refractory to the secretory action of STa, proving that the GC-C receptor is necessary for the diarrheal response induced by STa., (Copyright 1997 Academic Press.)

Guanylyl cyclase C (GC-C), a transmembrane receptor for E. coli heat-stable enterotoxin (STa) and for the endogenous peptides guanylin and uroguanylin, catalyzes formation of cGMP and influences fluid and electrolyte flux in the gut. We characterized the expression of GC-C in the mouse by Northern blot, in situ hybridization, and ligand binding studies. GC-C mRNA was present in mouse intestine by embryonic day 12, and was expressed at high levels in both crypts and villus or surface epithelium of adult small intestine and colon, respectively. Radiolabeled STa binding to membranes from several tissues correlated with the presence of GC-C mRNA. Extraintestinal GC-C expression was detected only in neonatal mouse liver. The presence of GC-C in mouse intestinal crypts supports the putative role of GC-C in fluid and electrolyte homeostasis and resembles the pattern in human tissues.

The guanylyl cyclase C protein, expressed primarily in the intestine, is the receptor for the heat-stable enterotoxin of Escherichia coli. We have isolated and sequenced the promoter region and the first exon of human guanylyl cyclase C and determined the major site of transcription initiation. Transfection of a -1973/+124 promoter/luciferase gene fusion construct in the Caco-2 intestinal cell line resulted in a high level of expression; results with deletion constructs indicate the presence of multiple positive-acting sequence elements. These promoter elements were not active upon transfection into NIH/3T3 and LLC-PK1 cell lines which do not express GC-C.

Expression of the Na(+)-K(+)-adenosinetriphosphatase (ATPase) gene family in rat intestinal epithelial cells was examined using RNA blot hybridization analyses. Rat intestinal epithelial cells express only the alpha 1- and beta 1-subunit mRNAs. A gradient in expression of alpha 1- and beta 1-subunit mRNA was seen along the villus-crypt unit in both jejunum and ileum, i.e., villus tip >> crypt cells. Regional differences in expression were observed along the intestine. alpha 1- and beta 1-subunit mRNA abundance was similar in jejunum, ileum, and colon while enzymatic activity was highest in the jejunum and lowest in the ileum. Administration of thyroid hormone to thyroidectomized rats increased the expression of alpha 1- and beta 1-subunit mRNAs in jejunum but not in colon. Hypothyroidism had no effect on subunit mRNA expression. The human intestinal cell line Caco-2 was also studied. These cells also expressed only the alpha 1- and beta 1-isoform mRNAs and demonstrated a developmental profile in both mRNA and enzymatic activity. Furthermore, in Caco-2 cells both alpha 1- and beta 1-mRNAs and Na(+)-K(+)-ATPase enzymatic activity were stimulated by thyroid hormone. Caco-2 cells transfected with 5' flanking regions of the human Na(+)-K(+)-ATPase beta 1-gene linked to the chloramphenicol acetyltransferase (CAT) reporter gene responded to 3,5,3'-triiodothyronine (T3) treatment with increased expression of CAT activity. This suggests that the 5' flanking region of the beta 1-gene contains a thyroid hormone response element and that T3 upregulation occurs at the transcriptional level.(ABSTRACT TRUNCATED AT 250 WORDS)