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Although glucocorticoids are known to elicit functional maturation of the gastrointestinal tract, the molecular mechanisms of glucocorticoid action on the developing intestine have not been fully elucidated. Our previous microarray studies identified 66 transcripts as being rapidly induced in the jejunum following dexamethasone (Dex) administration to suckling mice. Now we report the specific cellular location of a subset of these transcripts. Mouse pups at P8 received Dex or vehicle and intestinal segments were collected 3-4 h later. Robotic-based in situ hybridization (ISH) was performed with digoxygenin-labeled riboprobes. Transcripts studied included Ndrg1, Sgk1, Fos, and two unknown genes (Gene 9 and Gene 36). As predicted, ISH revealed marked diversity of cellular expression. In small intestinal segments, Sgk1 mRNA was in all epithelial cells; Fos mRNA was confined to epithelial cells at the villus tip; and Ndrg1 and Gene 36 mRNAs were localized to epithelial cells of the upper crypt and villus base. The remaining transcript (Gene 9) was induced modestly in villus stroma and strongly in the muscle layers. In the colon, Ndrg1, Sgk1, and Gene 36 were induced in all epithelial cells; Gene 9 was in muscle layers only; and Fos was not detectable. For jejunal segments, quantitation of ISH signals in tissue from Dex-treated and vehicle-treated mice demonstrated mRNA increases very similar to those measured by Northern blotting. We conclude that glucocorticoid action in the intestine reflects diverse molecular mechanisms operating in different cell types and that quantitative ISH is a valuable tool for studying hormone action in this tissue.

Prior studies have demonstrated that glucocorticoid hormones elicit functional maturation of the small intestine as evidenced by their ability to induce increases in the expression of various digestive hydrolases, such as sucrase-isomaltase and trehalase. However, these increases have a lag time of approximately 24 h, suggesting that they are secondary effects of hormone action. To identify candidate primary response genes, we performed microarray analysis on pooled RNA from jejunums of untreated postnatal day 8 mouse pups and from littermates who earlier received dexamethasone 2 h. Fluorescent dye-labeled samples were hybridized in quadruplicate to glass-spotted cDNA microarrays containing 15,000 cDNA clones from the National Institute of Aging cDNA clone set. Analysis of the resulting signals using relatively stringent criteria identified 66 transcripts upregulated and 36 downregulated by 2 h of glucocorticoid treatment. Among the upregulated transcripts, the magnitude of the increase detected by microarray ranged from 1.4- to 16-fold. Selected mRNAs from throughout the range were subsequently analyzed by Northern blot analysis. Of 11 mRNAs chosen all were confirmed, and there was a strong correlation between the magnitude of the increase observed from the microarray analysis and from Northern blot analysis. Additional time points showed that these transcripts peaked between 2 and 6 h and had returned to baseline by 24 h. Gene ontology analysis showed pleiotropic effects of dexamethasone on the developing intestine and pointed to genes in the development category as being likely candidates for mediation of glucocorticoid-induced maturation of intestinal function.

In the developing intestine, transcription of alpha-glucosidase genes such as sucrase-isomaltase and trehalase is stimulated by glucocorticoid administration. The consequent increase of their respective mRNAs is characterized by a 12-h lag, suggesting that the response to glucocorticoids represents a secondary effect. We hypothesized that the primary response of the tissue to glucocorticoids includes induction of one or more intestinal transcription factors. To investigate this hypothesis, we identified a region in the mouse trehalase promoter (located at nucleotides -406 to -377 from the transcription start site) with potential binding sites for three transcription factors: Cdx-2, GATA, and C/EBP. Gel shifts were performed using labeled oligonucleotides from this region with nuclear extracts from jejunums of either control 8-day-old mouse pups or littermates treated with dexamethasone (DEX) 4 h before death. A specific shifted band was observed with DEX extracts but not with control extracts. Supershift assays indicated the presence of GATA-4 and GATA-6 but not GATA-5 nor Cdx-2, C/EBP alpha, C/EBP beta, or C/EBP delta. GATA binding was further implicated by competition studies with mutated oligonucleotides. Finally, Western blot analysis showed GATA-4 and GATA-6 proteins in DEX but not control nuclear extracts. For GATA-4, the same pattern was demonstrated with whole cell extracts and with the cytosol fraction. We conclude that expression of GATA-4 and GATA-6 proteins in the suckling mouse jejunum is stimulated by DEX. This novel finding constitutes an important first step in understanding the molecular mechanism of glucocorticoid action on the developing intestine.

During rodent development there are two surges of circulating corticosterone: one just prior to birth and then one in the third postnatal week. Prior studies have shown that the latter controls the rate of intestinal development in the postnatal period. To date, a role for the earlier surge in the prenatal phase of intestinal development has not been investigated. We hypothesized that the late fetal surge of circulating corticosterone is involved in both morphologic and functional maturation of the intestinal epithelium, and thus that such maturation would be delayed if glucocorticoid action was abrogated. The hypothesis was tested by studying intestinal development in mice lacking a functional glucocorticoid receptor (GR). After GR+/- mice were bred onto a C57Bl/6 background, heterozygote matings yielded the expected ratios of -/-, +/-, and +/+ offspring. Analysis of GR mRNA in intestines of +/+ and -/- fetuses confirmed expression in wild-type mice but not in the GR-null mice. Intestinal histology of GR+/+ and -/- littermates at E13.5, E15.5, and E18.5 showed no effect of GR genotype on morphologic development. Further studies at E18.5 showed that GR-/- mice have normal functional maturation of the intestinal epithelium as assessed by: lactase activity in the enterocyte lineage, normal numbers of goblet and enteroendocrine cells, and normal numbers of proliferating cells in the intestinal crypts. Neither the minerolocorticoid receptor (MR) nor the pregnane X receptor (PXR) showed compensatory up-regulation in GR-/- mice. We conclude that, in contrast to our original hypothesis, the rodent intestine passes through a phase of glucocorticoid independence (late fetal) prior to becoming responsive to glucocorticoids in the postnatal period. These findings have implications for the clinical use of corticosteroids to enhance intestinal maturation in preterm infants., (Copyright 2002 Wiley-Liss, Inc.)

The third postnatal week of mouse development is characterized by dramatic changes of gene expression in the small intestine. Although these changes are often assumed to reflect regulation at the level of transcription, to date there have been no direct investigations of this. In the current study we have used trehalase as a marker of intestinal maturation. Highly sensitive reverse transcriptase-polymerase chain reaction methods were developed for semi-quantitative analysis of both initial and mature transcripts, i.e., hnRNA and mRNA. Jejunums collected during normal development (specifically from postnatal days 8-21) showed parallel increases in the levels of trehalase hnRNA and mRNA. Likewise, when precocious gut maturation was elicited by dexamethasone administration on days 8-10, both initial and mature trehalase transcripts were significantly increased, although with a relatively slow time course. We conclude that both normal and glucocorticoid-induced maturation of trehalase expression reflect transcriptional activation. However, the slow time course of the glucocorticoid effect suggests that trehalase may not be a primary response gene.

There are conflicting data from human studies regarding the ability of exogenous glucocorticoids to stimulate maturation of the small intestine. The discrepancies may relate to differences in hormone doses and age administered. To explore this general concept, we have used a mouse model to determine intestinal responsiveness to dexamethasone (DEX) at various times during development. We first showed that trehalase mRNA is a sensitive marker of intestinal maturation in the mouse; being undetectable (by Northern blotting) in the prenatal period, expressed at low levels during the first 2 postnatal weeks and then displaying a marked increase in the 3rd postnatal week. DEX was unable to elicit detectable trehalase mRNA in fetal mice, but caused significant increases in the postnatal period. The use of a range of DEX doses (0.0125-2.5 nmol/g BW per day) established that there is no change in sensitivity between the 1st and 2nd postnatal weeks, but there is a significant increase in maximal responsiveness of trehalase mRNA to the hormone. Similar results were obtained when sucrase-isomaltase mRNA was assayed in the same animals. Thus, in this rodent model, there appears to be at least three phases in the DEX responsiveness of the developing intestine: an early phase (prenatal) when DEX is unable to elicit intestinal maturation; then a phase (first postnatal week) of modest responsiveness; then a transition to increased responsiveness. These findings point to the need for careful attention to dose and age in analyses of glucocorticoid effects in human infants.

Trehalase is the least studied of the membrane-bound alpha- glucosidase enzymes. Here we report the isolation and characterization of the mouse trehalase (Treh) gene. Initially, PCR using primers based on published rat cDNA sequence was used to clone a partial mouse cDNA. This allowed design of mouse primers which identified a single positive clone in a bacterial artificial chromosome (BAC) library of mouse genomic DNA. Analysis of BAC subclones showed that the Treh structural gene spans approximately 13 kb and comprises 15 exons. Data from genomic Southern blotting were consistent with mouse Treh being a single copy gene. The transcription initiation site was determined by both S1 nuclease mapping and 5' rapid amplification of cDNA ends (5' RACE) to be located 25 nt upstream of the ATG in exon 1. The mouse Treh exons were found to have an open reading frame of 1728 nt and the encoded protein of 576 amino acids showed 81, 82 and 93% amino acid sequence identity with rabbit, human and rat trehalase, respectively. The trehalase signature sequence found at amino acids 162 to 175 had 100% identity with the corresponding region of rabbit, human and rat and 79% identity with that for yeast trehalase. When a mouse Treh cDNA was used for Northern blot analysis of RNA from 12 mouse tissues, Treh mRNA expression was detected only in kidney and small intestine. The size of the mRNA in both of these tissues was estimated to be approximately 2.1 kb, furthermore both tissues appear to have the same transcription initiation site as determined by nuclease protection. Using the T31 radiation hybrid panel, mouse Treh was shown to be located on Chromosome 9 in a broad region that is orthologous with human Chromosome 11q23. The human trehalase gene (TREH) was identified in the latter location via database searching, which also revealed the overall structure of the human gene as being similar to that of the mouse.

Meprin is a zinc-metalloendopeptidase expressed in intestinal epithelial cells. In rat jejunum collected from postnatal day 4 (P4) through P25 meprins alpha mRNA exhibited uniform levels for the first three postnatal weeks and then declined, whereas meprin beta mRNA showed a biphasic pattern with high levels in the first postnatal week followed by low levels from P7 through P19 and then a marked rise at P22 and P25. Dexamethasone treatment beginning at P10 had no significant effect on levels of meprins a mRNA, whereas this treatment caused a precocious increase in expression of meprin beta mRNA. These divergent patterns of expression of meprins alpha and beta mRNA suggest distinct roles for the two subunits during the suckling and weaning phases of rodent intestinal development.

A partial rat trehalase cDNA has been cloned and used to examine trehalase mRNA expression. Northern blotting with total RNA from 11 adult rat tissues showed a trehalase transcript only in small intestine, where it was abundant in proximal regions but declined steeply toward the ileum. During development, trehalase mRNA was not detectable in jejunum until postnatal day 19 and then increased markedly through day 25. Modest levels in trehalase mRNA were induced precociously by administration of dexamethasone, with increasing responsiveness evident between the first and second postnatal weeks. In contrast, analysis of sucrase-isomaltase mRNA on the same blots showed maximal induction at both ages. In adrenalectomized animals, the ontogenic increase of trehalase mRNA began as usual but proceeded more slowly than in control animals. Overall, trehalase mRNA expression in the rat displayed both similarities and differences compared with rabbit. Moreover, the differences revealed in glucocorticoid responsiveness of trehalase mRNA and sucrase-isomaltase mRNA suggest that the actions of these hormones on the developing intestine may be more complex than previously recognized.

In rodents, there is a surge of intestinal expression of CCAAT/enhancer-binding protein alpha (C/EBPalpha) in the late fetal phase just before morphological maturation and the onset of expression of numerous epithelial genes. To investigate directly the hypothesis that C/EBPalpha plays a causal role in the latter phenomena, we have assessed both structural and functional maturation in neonatal intestine from C/EBPalpha-null mice and their littermates. No effects of C/EBPalpha genotype were observed on mucosal architecture or on the size of the proliferative zone in the intestinal crypts. Likewise, the mRNA levels for the glucose transporter 2 (GLUT2), intestinal and liver fatty acid-binding proteins, and apolipoprotein A-IV in newborn intestine were similar in all genotypes. Paradoxically, Na+/glucose co-transporter (SGLT1), lactase phlorizin-hydrolase and apolipoprotein B mRNAs were more abundant in the C/EBPalpha-deficient animals. In wild-type intestines, C/EBPbeta and C/EBPdelta mRNAs were detectable throughout the late fetal period and increased toward term in parallel with C/EBPalpha mRNA. In newborn intestine, there was no compensatory up-regulation of these isoforms in the C/EBPalpha-deficient mice. We conclude that C/EBPalpha has no essential role in morphological maturation of the intestine, the pattern of proliferation of the epithelium, or the onset of expression of this cluster of epithelial mRNAs. However, since other C/EBP isoforms are present in the developing intestine, it is possible that there is a generic requirement for a member of the C/EBP family.