Development of glucocorticoid-responsiveness in mouse intestine.

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.

Cloning, characterization and mapping of the mouse trehalase (Treh) gene.

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 mRNA in rat intestine during normal and glucocorticoid-induced maturation: divergent patterns of expression of alpha and beta subunits.

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.

Rat trehalase: cDNA cloning and mRNA expression in adult rat tissues and during intestinal ontogeny.

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.

Intestinal maturation in mice lacking CCAAT/enhancer-binding protein alpha (C/EPBalpha).

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.