Diverse patterns of cell-specific gene expression in response to glucocorticoid in the developing small intestine.

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.

Immediate early genes of glucocorticoid action on the developing intestine.

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.

Rapid induction of GATA transcription factors in developing mouse intestine following glucocorticoid administration.

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.

Development of the fetal intestine in mice lacking the glucocorticoid receptor (GR).

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.

Developmental expression of trehalase: role of transcriptional activation.

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.