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

Thomas J. Oesterreicher1 and Susan J. Henning1,2

Departments of 1Pediatrics and 2Molecular and Cellular Biology, Baylor College of Medicine, Houston, Texas 77030

Submitted 6 November 2003 ; accepted in final form 14 January 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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.

trehalase promoter; primary response; secondary response


MATURATION OF THE INTESTINAL epithelium is critical for the acquisition of digestive and absorptive functions and thus for the survival of the young mammal. In rodents, the final phase of intestinal maturation occurs during the third postnatal week coincident with weaning (11, 14). Among numerous functional changes that occur at this time are those of enzymes responsible for the terminal digestion of carbohydrates. Most significantly, the {alpha}-glucosidases (sucrase-isomaltase, maltase-glucoamylase, and trehalase) display markedly increased expression (11, 14). Although these changes are essential for the transition to solid food, it is well established that they are not actually caused by weaning (14). Instead, studies from many laboratories over the past three decades (11, 15) have shown that the two major controls are an intrinsic timing mechanism (which cues the onset of normal maturation) and a potent effect of glucocorticoid hormones (causing precocious maturation). The involvement of glucocorticoids has interesting evolutionary implications, because it may have provided an adaptive advantage to the young in the face of loss of the dam (15). In addition, the ability of glucocorticoids to elicit intestinal maturation has clinical relevance, specifically in the management of preterm infants (27).

To date, the exploration of the molecular mechanisms of glucocorticoid action on the developing intestine has focused primarily on sucrase-isomaltase and trehalase as marker enzymes. For both of these {alpha}-glucosidases, the increase in enzyme activity following glucocorticoid administration to suckling rodents is paralleled by a dramatic increase in the steady-state levels of their respective mRNAs (16, 21, 25, 28). Furthermore, there is evidence that for both sucrase-isomaltase mRNA (32) and trehalase mRNA (13), the increase elicited by glucocorticoids reflects increased gene transcription. However, for both genes the increased transcription does not appear to be a direct response to glucocorticoids. This conclusion is based primarily on the time course of the mRNA increase following glucocorticoid treatment. Unlike direct response genes whose mRNAs typically peak between 1 and 8 h following hormone administration (7, 26), the mRNAs for sucrase-isomaltase and trehalase display a slow increase, being first detectable between 12 and 24 h after glucocorticoid injection (13, 21, 25) and reaching plateau levels only after ~4 days (13, 21, 25). Because this time course is typical of secondary response genes (7), our long-term goals are to identify the factor(s) that mediates glucocorticoid action on the developing intestine. In the current study, we hypothesized that the primary response to glucocorticoids includes induction of one or more intestinal transcription factors. To explore our hypothesis, we chose to study the proximal promoter of the mouse trehalase gene.

Based on the fact that sucrase-isomaltase and trehalase exhibit similar patterns of expression during development and following glucocorticoid administration, we reasoned that these genes may have similar regulatory elements. The sucrase-isomaltase promoter is well characterized and is currently understood to have two elements that are critical for its expression: a proximal element known as sucrase-isomaltase footprint 1 (SIF1), which binds the homeodomain proteins Cdx-1 and Cdx-2, and a more distal element known as SIF3, which binds HNF-1 factors (3, 30). The activity of these transcription factors is enhanced by proteins of the GATA family, although this effect does not appear to require GATA binding to DNA (3). Our first goal was to compare the sequence of the mouse trehalase promoter with that of sucrase-isomaltase. We found an element homologous to SIF1 but also more complex, because it has potential binding sites for C/EBP factors and GATA factors as well as Cdx proteins. In view of the known role of the latter two in expression of intestinal genes (3, 810, 18, 19, 30) together with the fact that C/EBP proteins have been implicated as mediators of glucocorticoid action in other tissues (5, 26), we felt that this region of the trehalase promoter (designated Treh1) would be a fruitful one with which to investigate our hypothesis. The general approach was to treat suckling mice with dexamethasone (DEX) for a brief period (4 h) in which primary glucocorticoid response genes should be activated. If these genes include transcription factors capable of binding to the Treh1 element, such factors should be detectable by performing EMSAs with nuclear extracts from DEX-treated mice. Our initial experiments indicated that there was a slowly migrating DNA-binding complex elicited by DEX, and our subsequent experiments identified GATA-4 and GATA-6 proteins as being induced by DEX and responsible for this gel shift.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals, treatments, and tissue collection. Timed pregnant C57BL/6J dams were received from Jackson Laboratory (Bar Harbor, ME) on days 1113 of gestation. They were housed individually and provided food (5001 Rodent Diet, PMI Nutrition International, Brentwood, MO) and acidified tap water ad libitum. All animal housing and protocol details were approved by our Institutional Animal Care and Use Committee. On postnatal day 8, four experimental pups from two litters were injected subcutaneously with 1 µg/g body wt of DEX. Four uninjected littermate control pups were killed immediately, whereas injected pups were killed 4 h after treatment. Jejunums were collected, and nuclear extracts were prepared. The experiment was subsequently repeated with an additional litter of 8-day-old mice in which two pups were untreated, two received DEX, and one received vehicle (0.8% ethanol in 0.15 M NaCl) injection. In this experiment, jejunums were cut into 0.5-cm lengths and alternate pieces were used for preparation of 1) whole cell lysates and 2) crude nuclear and cystosol fractions. Although only one vehicle-injected animal was included in this study (because of the size of the litter), other experiments in our laboratory on immediate early effects of glucocorticoids consistently found no significant differences between untreated and vehicle-treated mice.

Preparation of cellular extracts. In the first experiment, for preparation of nuclear extracts, immediately after collection jejunums were chopped into small pieces and Dounce homogenized in ice-cold buffer A, which comprised (in mM) 25 Tris·HCl, pH 7.5, 50 KCl, 2 MgCl2, 1 EDTA, and 5 dithiothreitol, with the following proteinase inhibitors (in µg/ml): 25 leupeptin, 5 aprotonin, 40 phenylmethylsulfonlyl fluoride, 50 benzamidine, and 0.5 pepstatin A. Nuclei were pelleted by centrifugation at 9,000 g for 5 min, resuspended in ice-cold buffer B (25 mM Tris·HCl, pH 7.5, 0.42 M NaCl, 1.5 mM MgCl2, 1 mM dithiothreitol, 0.5 mM EDTA with the proteinase inhibitors as in buffer A), and incubated on ice for 10 min. Samples were recentrifuged, and the nuclear extract supernatant was aliquoted and stored at –80°C. In the second experiment, nuclear extracts were prepared just as in the first experiment and the supernatant from the first centrifugation was considered to be the crude cytosol fraction; whole cell lysates were prepared (from the alternating pieces of jejunum) by direct homogenization in buffer B. Protein concentrations were determined using the Bio-Rad protein assay (Bio-Rad Life Sciences, Hercules, CA) with bovine gamma globulin as the reference.

Sequence homology analysis. To determine conserved regions of the disaccharidase promoters, the sequence for mouse SIF1 (5'-ggtgcaataaaactttatgagta-3') (30) was compared with ~2.5 kb of the mouse trehalase promoter (www.ensembl.org) using the Wisconsin Sequence Analysis Package (Genetics Computer Group, Madison, WI). The most highly homologous region, designated Treh1 (5'-CAGGGAGTTTGATAAAGCTTTGGAGAAGAC-3'), was used for gel shift analysis.

EMSAs. A double-strand DNA probe (Treh1) was made by annealing complementary single-strand oligonucleotides (30 bp) and end-labeling with 32P using T4 polynucleotide kinase. In the experiments reported, the specific activity of the probe ranged from 2 to 4 million cpm/pmol. Nuclear extracts (15 µg protein) were incubated at room temperature for 30 min with 2 µg poly(dI/dC) in gel shift binding buffer (Promega, Madison, WI) and 50,000 cpm of probe in a total volume of 20 µl. The entire reaction volume was loaded onto a 5% polyacrylamide/0.5x Tris-borate-EDTA gel for electrophoresis (175 V for 3–4 h). Afterward, the gels were dried and DNA-protein complexes were detected by autoradiography. For standard cold competition analysis, the gel shift assay was performed using 5-, 50-, and 500-fold molar excess of unlabeled Treh1 or SIF1 probe. For mutation cold competition, four double-strand probes were designed with point mutations made at various positions (shown in bold) along the Treh1 probe: Mut1 5'-CAGGGCTGGTGATAAAGCTTTGGAGAAGAC-3'; Mut2 5'-CAGGGAGTTTTCGCAAGCTTTGGAGAAGAC-3'; Mut3 5'-CAGGGAGTTTGATAACTAGTTGGAGAAGAC-3'; Mut4 5'-CAGGGAGTTTGATAAAGCTTTGGCTCCGAC-3'. These mutated cold probes were added in 1,000-fold molar excess into the reaction. Supershift assays were performed by adding 1 µl of antibody to the reaction 15 min before the addition of the labeled probe. The antibodies used were Cdx-2 (courtesy Dr. E. Sibley, Stanford University) and C/EBP{alpha} (14AA)X sc-61X, C/EBP{beta} (C-19)X sc-150X, C/EBP{delta} (C-22)X sc-151X, GATA-4 (C-20) sc-1237X, GATA-5 (M-20)X sc-7280X, GATA-6 (N-18)X sc-7245X, and an additional GATA-6 (H-92) sc-9055X (Santa Cruz Biotechnology, Santa Cruz, CA). Each of these antibodies was shown to be capable of eliciting a supershift of the respective factor in control experiments using consensus oligonucleotide probes with either in vitro-synthesized proteins (GATA-4, -5, and -6) or with tissue or cell extracts known to express the factor (Cdx-2; C/EBP{alpha}, {beta}, and {delta}).

In vitro synthesis of GATA proteins. Expression vectors for GATA-4, -5, and -6 (1) were procured from Dr. N. S. Belaguli (Baylor College of Medicine). These vectors were transcribed and translated using a T7 RNA polymerase/reticulocyte lysate system (TNT, Promega) according to the manufacturer's instructions. The binding efficiencies of the resulting proteins were tested by EMSA using a consensus GATA probe (sc 2531, Santa Cruz Biotechnology), and supershift assays were performed with the antibodies described above.

Western blot analysis. Protein samples (30–100 µg) were electrophoresed in 13% SDS-PAGE. After electrophoresis, proteins were transferred onto Immun-Blot PVDF or nitrocellulose membrane (Bio-Rad) by electroblotting. Each gel included a lane with the Full-Range Rainbow Molecular Weight protein marker from Amersham Biosciences (Piscataway, NJ). Membranes were blocked overnight in TTBS (20 mM Tris·HCl, pH 7.5, 150 mM NaCl, and 0.1% Tween 20) with 10% nonfat dried milk and 2% bovine serum albumin. The primary antibodies used were GATA-4 (C-20) sc-1237X, GATA-5 (m-20) sc-7280X, and GATA-6 (N-18)X sc-7245X (Santa Cruz Biotechnology). The blots were probed with either anti-GATA-4 (1:10,000 dilution), anti-GATA-5 (1:2,500), or anti-GATA-6 (1:10,000) in TTBS + 2.5% nonfat dried milk and then incubated with bovine anti-goat IgG-horseradish peroxidase (HRP) sc-2350 (Santa Cruz Biotechnology) serum (1:1,000 dilution). To test for equal loading of the samples, the blots were stripped in 100 mM Tris·HCl (pH 7.5), 2% SDS, and 14 mM {beta}-mercaptoethanol at room temperature for 1 h, blocked, and reprobed with mouse monoclonal anti-{beta}-actin (Sigma, St. Louis, MO) at 1:6,000 dilution. Anti-mouse IgG-HRP (Amersham Life Sciences) was used at a 1:3,000 dilution. Detection was performed by chemiluminescence using the ECL kit (Amersham), and signals were visualized by autoradiography. Specificity of the detected bands was by size and by competition with a fivefold excess of blocking peptides for GATA-4 sc-1237P and GATA-6 sc-7245P (Santa Cruz Biotechnology).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Comparison of sucrase-isomaltase and trehalase promoters shows conserved regions. To find common regulatory elements, we performed sequence homology searches of the mouse sucrase-isomaltase and trehalase promoters. The SIF1 sequence, which is known to include a binding site for Cdx dimers (29, 30), showed homology to a region in the trehalase promoter (Fig. 1). Interestingly, this region also included sequences identified as binding sites for GATA factors and C/EBP factors. A double-strand oligonucleotide probe that included the most highly homologous region and flanking sequences was created for use in EMSAs and was designated Treh1. This sequence was located at nucleotides –406 to –377 from the transcription start site of the mouse trehalase gene (24).



View larger version (10K):
[in this window]
[in a new window]
 
Fig. 1. Sequence of mouse sucrase-isomaltase footprint 1 (SIF1) (30) and a homologous region of the mouse trehalase promoter (Treh1). The location of the Cdx binding sites (29) is shown below the SIF1 sequence. Potential binding sites for C/EBP and GATA transcription factors are shown above the trehalase sequence.

 
DEX induces proteins that bind to Treh1. To test our hypothesis that the primary response genes of glucocorticoid action on the developing intestine include one or more transcription factors capable of binding to the trehalase promoter, we chose to use mice at postnatal day 8. Previous work showed that basal expression of trehalase is quite low at this age and that there is a robust, but delayed, activation of trehalase transcription following glucocorticoid administration (13). In the current study, nuclear extracts were prepared from jejunums of postnatal day 8 mice that were either untreated or injected with DEX 4 h ahead. Figure 2 shows the ability of proteins found in these extracts to bind to the Treh1 probe. As can be seen, the extracts from DEX-treated mice yield a reproducible, slowly migrating DNA-protein complex that is not found in the uninjected controls. There were also differences in faster-migrating complexes, but these were less distinct and less reproducible from animal to animal. To assess the integrity of the nuclear extracts from control animals, the extracts were subjected to EMSA using another probe available in our laboratory, namely the IR-1 consensus (17), which binds the nuclear receptor complex FXR/RXR. Distinct bands of appropriate size were detected in all four control samples (data not shown). This, together with the {beta}-actin data presented below (see Fig. 7), indicates that nuclear extracts are not degraded under the isolation conditions used in this study.



View larger version (79K):
[in this window]
[in a new window]
 
Fig. 2. EMSA using radiolabeled Treh1 probe with nuclear extracts from postnatal day 8 jejunums of 4 pairs of control (C) and dexamethasone (DEX)-treated (D) mice. Lane 1: probe alone.

 


View larger version (44K):
[in this window]
[in a new window]
 
Fig. 7. Western blots of nuclear extracts from postnatal day 8 jejunums of 4 control mice (C) and 4 littermates treated with D. Also shown are results with in vitro-expressed protein (E). In the second lane (M), the solid line shows the position of the 50-kDa band in the marker mixture and the dotted line shows the position of the 35-kDa band. Either 30 or 100 µg of nuclear extracts were blotted and probed with antibodies for GATA-4 or -6 (top and lower middle, respectively). Each blot was reprobed with anti-{beta}-actin to check for equal loading and protein integrity. Arrow in GATA-6 row indicates the position of GATA-6, whereas * designates a nonspecific band. Right: approximate protein sizes.

 
The slowly migrating complex is specific. The specificity of the Treh1-shifted band was tested by competition with increasing amounts of unlabeled Treh1 probe (Fig. 3). As expected, there was no slowly migrating complex detected in nuclear extracts from control animals. Unlabeled Treh1 at fivefold molar excess was able to partially compete away the high band in the DEX-treated sample. Competition was almost complete at 50-fold excess, and probe binding was totally abolished at 500-fold excess. In contrast, there was very weak competition by unlabeled SIF1 oligonucleotide, with some reduction seen at 50-fold excess but competition being incomplete even at 500-fold excess. Thus it appears that the band shift elicited by DEX treatment is specific for Treh1 and the protein responsible does not bind to SIF1. Examination of lower parts of the gel showed a smear in both control and DEX lanes that was weakly competed by both the Treh1- and SIF1-unlabeled oligonucleotides, as well as a fast-migrating band in all lanes that was not competed by either. These observations confirm those of Fig. 2 in which we noted that only the slowly migrating band is specific to the extracts from DEX-treated pups.



View larger version (109K):
[in this window]
[in a new window]
 
Fig. 3. EMSA using radiolabeled Treh1 probe interacting with nuclear extracts from postnatal day 8 jejunum of a C and a D mouse. Cold competition analysis was performed using unlabeled Treh1 (T) and SIF1 (S) oligonucleotides in 5-, 50-, and 500-fold molar excess.

 
Supershift analyses point to GATA proteins in the complex from DEX-treated mice. Because the Treh1 probe includes potential binding sites for proteins of the Cdx, C/EBP, and GATA families, supershift analyses were performed with antibodies to selected members of these families, together with nuclear extracts from jejunums of DEX-treated mice. As shown in Fig. 4, the slowly migrating complex was not affected by antibodies to Cdx-2, C/EBP{alpha}, C/EBP{beta}, or C/EBP{delta}. In contrast, the GATA-4 antibody yielded a clear supershift of the slowly migrating DNA-protein complex. This antibody also caused some dimunition of the smear below the main band, suggesting the presence of partially degraded GATA-4 protein. No shifted band was detectable with the GATA-5 antibody (even at longer exposures). A very weak supershift was observed with one antibody to GATA-6, whereas the other produced a stronger supershift but no ablation of the lower complex. Thus it appears that the slowly migrating complex elicited by DEX includes GATA-4 and possibly GATA-6.



View larger version (82K):
[in this window]
[in a new window]
 
Fig. 4. Supershift analysis using radiolabeled Treh1 probe. Antibodies for Cdx-2, C/EBP{alpha}, {beta}, and {delta}, and GATA-4, -5, and -6 were incubated with nuclear extracts from jejunum of a postnatal day 8 mouse treated with DEX (D) for 4 h. Also shown is the untreated sample (C). Two different antibodies for GATA-6 were used in lanes 10 and 11, namely (N-18)X sc-7245X and (H-92) sc-9055X, respectively.

 
Mutational analysis and band size confirm GATA binding in the complex from DEX-treated mice. To further characterize the slowly migrating complex observed with nuclear extracts from DEX-treated mice, we made oligonucleotides with various mutations in the Treh1 sequence (Fig. 5A) and performed cold competition assays with these. As can be seen in Fig. 5B, only the Mut2 probe was unable to compete the complex. Because this mutation disrupts the putative GATA binding site and a similar mutation has previously been shown to destroy GATA binding to the lactase-phlorizin hydrolase promoter (10), these data are consistent with the supershift analyses in pointing to one or more GATA factors being involved in the slowly migrating complex observed after DEX treatment.



View larger version (47K):
[in this window]
[in a new window]
 
Fig. 5. Mutational analysis. A: sequence of wild-type Treh1 compared with double-strand mutations, with mutations shown in bold text. The potential GATA factor binding site is underlined. B: EMSA using radiolabeled Treh1 probe with nuclear extracts from jejunum of a postnatal day 8 mouse treated with DEX (D). Cold competition was performed using 1,000-fold molar excess of the mutated oligonucleotides.

 
To confirm the specificity of GATA binding to Treh1, we performed further EMSAs using in vitro-expressed protein. As can be seen in Fig. 6A, expressed GATA-4 showed a distinct band when incubated with the Treh1 probe. The mobility of this band was similar to that seen with the nuclear extract from the jejunum of a DEX-treated mouse, indicating no additional proteins are present in the latter. Consistent with the original experiment shown in Fig. 2, no slowly migrating complex was observed with nuclear extract from an untreated (control) littermate. In Fig. 6A, we also included a vehicle-injected littermate to confirm that the complex observed after DEX injection was indeed elicited by the hormone. Finally, in Fig. 6B, we demonstrated that nuclear extract from DEX-treated but not from control mice gave a very strong complex with a GATA consensus probe. Furthermore, this complex was supershifted by GATA-4 antibodies.



View larger version (31K):
[in this window]
[in a new window]
 
Fig. 6. A: EMSA with radiolabeled Treh1 probe incubated with in vitro-expressed GATA-4 protein compared with jejunal nuclear extracts from postnatal day 8 mice that were untreated (C), vehicle-treated (V), or DEX-treated (D). B: EMSA with radiolabeled GATA consensus probe incubated with jejunal nuclear extracts from C and D mice. The last lane shows D extract incubated with antibodies for GATA-4.

 
DEX induces GATA proteins in mouse jejunum. The data presented so far indicate that jejunal nuclear extracts from DEX-treated mice have GATA-4 and GATA-6 proteins that are capable of binding to Treh1, whereas no such binding is observed in nuclear extracts from littermate controls. To assess whether this reflects differences in the amounts of these proteins (as compared with their DNA-binding capacity), Western blot analysis of nuclear extracts was performed. With 30 µg of nuclear proteins, we were able to detect GATA-4 protein only in samples from DEX-treated mice and not in those from control mice (Fig. 7, top). A similar pattern was observed for GATA-6 protein (Fig. 7, lower middle), although in this case detection required the use of 100 µg of nuclear protein. In contrast, GATA-5 protein was not detected even at this high loading (data not shown). The blots were reprobed with {beta}-actin antibody to verify that equal amounts of protein were loaded in each lane and that there was no evidence of protein degradation in the control samples. The specificity of the antibodies used was assessed by probing identical blots with antibodies that had been incubated with a fivefold excess of specific blocking peptides. This resulted in complete ablation of the GATA-4 and GATA-6 bands (data not shown). The antibodies were also tested with in vitro-expressed GATA-4 and GATA-6 protein (Fig. 7, lane E).

The DEX-induced appearance of GATA-4 and GATA-6 proteins in nuclear extracts could reflect either stimulation of protein translocation from cytosol to nucleus or increases in whole cell levels of these proteins. To distinguish between these two possibilities, alternate pieces of jejunum were used to generate either whole cell lysates or nuclear extracts plus crude cytosol. Western blot analysis (Fig. 8) shows that GATA-4 protein was undetectable in all fractions from untreated and vehicle-injected pups. In contrast, after 4-h DEX treatment, GATA-4 was found in the whole cell lysate as well as both the crude cytosol and nuclear extract. We conclude that the effect of DEX is to elevate steady-state levels of GATA-4 protein in both the cytoplasm and nucleus.



View larger version (35K):
[in this window]
[in a new window]
 
Fig. 8. Western blot of whole cell lysates (W), crude cytosol (Ct), and nuclear extract (NE) from postnatal day 8 jejunums of mice that were either untreated (Control), vehicle-injected (Veh), or DEX-injected (DEX). Each lane was loaded with 50 µg of protein. The blot was probed with GATA-4 antibody and then reprobed with anti-{beta}-actin.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
To date, understanding the molecular details of glucocorticoid action on expression of digestive hydrolases in the developing intestine has remained an important but elusive goal. As noted in the introduction, the importance lies in the fact that the ability of glucocorticoids to elicit expression of these enzymes has both evolutionary (15) and clinical (27) significance. Interestingly, although glucocorticoid-induced precocious maturation was initially viewed as a simple advancement of normal maturation, subsequent studies (23, 31) showed that the glucocorticoid pathway is distinct from that of normal maturation. Furthermore, direct activation of intestinal hydrolase promoters via the glucocorticoid receptor seems unlikely in view of the significant delay in the rise of the mRNAs for sucrase-isomaltase and trehalase following treatment (13, 21, 25). These findings led to our hypothesis that glucocorticoid action on such genes represents a secondary effect mediated by one or more transcription factors. The data presented in this paper using a region of the mouse trehalase promoter as a probe for EMSA show that, indeed, DEX elicits the appearance of a DNA-binding protein in the jejunal nuclei. Subsequent analyses indicated that the binding protein is a member of the GATA family of transcription factors. Both supershift assays and Western blot analyses indicated that GATA-4 is the predominant species elicited by DEX but that some GATA-6 is also elicited.

Although GATA factors are now recognized as being critical regulators of transcription of numerous intestinal genes (3, 810, 18, 19), there is surprisingly little information about the expression of GATA proteins at various stages of intestinal development. In the chicken intestine, GATA-4, GATA-5, and GATA-6 are all expressed in the villus epithelium (12) and the abundance of all three mRNAs increases markedly at the time of hatching (20). Adult mouse intestine has been reported to express GATA-4, GATA-5, and GATA-6 mRNA with distinct patterns along the longitudinal axis (9). GATA-4 and GATA-5 have a reciprocal pattern such that GATA-4 is the most abundant isoform in the duodenum and jejunum, whereas GATA-5 is most abundant in the ileum. In contrast, GATA-6 mRNA is expressed at low levels throughout the small intestine. The only study of GATA expression in the postnatal mouse intestine is that by Boudreau et al. (3). These authors showed, by immunohistochemical staining, that in the jejunum, GATA-4 protein is detectable in nuclei of villus epithelial cells at postnatal days 12, 15, 17, and 21. Earlier ages were not reported. Although immunohistochemistry is not quantitative, there appears to be distinctly stronger staining at day 15 than at day 12 (3). This finding, together with our Western blots, suggests that GATA-4 is either not expressed or minimally expressed in the mouse jejunum at postnatal day 8 but can be induced precociously by glucocorticoid treatment (as in Figs. 7 and 8) and displays a normal developmental onset of expression by day 12 (3). A more complete study of GATA-4, GATA-5, and GATA-6 in the developing intestine is clearly warranted.

In the current study, we have no evidence that the GATA proteins induced in response to glucocorticoid actually mediate hormone effects on transcription of the {alpha}-glucosidase genes. Transient transfections of constructs including the Treh1 portion of the trehalase promoter in Caco-2 cells have failed to show functional activation by GATA-4 (data not shown). We interpret this as indicating that transcription from the trehalase promoter requires the presence of one or more accessory factors that are not present in Caco-2 cells. It is well recognized that neither Caco-2 cells, nor any other intestinal epithelial cell line currently available, accurately recapitulates in vivo gene expression. Thus the ideal approach to assess the functionality of the GATA site in the trehalase promoter would be to perform transgenic studies in which the activities of constructs including the native GATA site were compared with those of constructs having this site mutated. This approach has given unequivocal evidence of the importance of GATA binding for the intestinal expression of adenosine deaminase (9). Analogous studies with the trehalase and sucrase-isomaltase promoters will be an important future direction to assess the functional role of GATA factors as mediators of glucocorticoid effects on {alpha}-glucosidase genes.

To our knowledge, there has been only one prior study examining the possibility that glucocorticoids elicit the expression of one or more transcription factors in the developing intestine. In that study, by Levenson et al. (22), nuclear extracts from intestines of 6-day-old DEX-treated and control rats were used in EMSAs with two different portions of the proximal promoter of cysteine-rich intestinal peptide. A distinct band shift was observed in the extracts from the DEX-treated animals. However, the nature of the binding protein was not further investigated. Interestingly, both probes used included two GATA sites. Thus these investigators may have been observing the same response to glucocorticoids in the developing rat intestine as we are reporting in the current study for mouse intestine. The only other report in the literature of glucocorticoid induction of intestinal transcription factors is an in vitro study by Boudreau et al. (2) in which the rat intestinal epithelial crypt cell line IEC-6 was shown to have rapid induction of C/EBP{delta} and C/EBP{beta} following treatment with DEX. Although these authors did not examine the same phenomenon in vivo, their findings point to the possibility that glucocorticoid action on the developing intestine may be mediated by induction of a combination of factors including GATA-4 and GATA-6, as well as selected C/EBP isoforms. In addition to the C/EBP family, GATA factors may act in a combinatorial fashion with other endodermal transcription factors such as Cdx, Pdx, HNF-1, and HNF-4 (3, 8, 9, 19). Alternate plausible scenarios would include interaction with generic transcription factors such as the Sp family (18) or serum response factor (1) or with coactivators such as p300 (6). A particularly appealing possibility is that, as in early liver development (4), the role of GATA factors may be to open the nucleosomal domain of compacted chromatin to allow cooperating transcription factors to bind to the DNA. Although there is much work to be done to complete our understanding of glucocorticoid action in this complex tissue, the demonstration that GATA-4 and GATA-6 proteins are rapidly induced following hormone treatment constitutes a novel finding and provides a useful basis for further studies.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study was supported by National Institutes of Health Grant R01-HD-14094.


    ACKNOWLEDGMENTS
 
The authors thank Drs. N. S. Belaguli and E. Sibley for providing critical reagents and Drs. S. T. Hwang and S. Thevananther for constructive input.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. J. Henning, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030 (E-mail: shenning{at}bcm.tmc.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Belaguli NS, Sepulveda JL, Nigam V, Charron F, Nemer M, and Schwartz RJ. Cardiac tissue enriched factors serum response factor and GATA-4 are mutual coregulators. Mol Cell Biol 20: 7550–7558, 2000.[Abstract/Free Full Text]
  2. Boudreau F, Blais S, and Asselin C. Regulation of CCAAT/enhancer binding protein isoforms by serum and glucocorticoids in the rat intestinal epithelial crypt cell line IEC-6. Exp Cell Res 222: 1–9, 1996.[CrossRef][ISI][Medline]
  3. Boudreau F, Rings EH, van Wering HM, Kim RK, Swain GP, Krasinski SD, Moffett J, Grand RJ, Suh ER, and Traber PG. Hepatocyte nuclear factor-1{alpha}, GATA-4, and caudal related homeodomain protein Cdx2 interact functionally to modulate intestinal gene transcription. Implication for the developmental regulation of the sucrase-isomaltase gene. J Biol Chem 277: 31909–31917, 2002.[Abstract/Free Full Text]
  4. Cirillo LA, Lin FR, Cuesta I, Friedman D, Jarnik M, and Zaret KS. Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4. Mol Cell 9: 279–289, 2002.[ISI][Medline]
  5. Crosson SM and Roesler WJ. Hormonal regulation of the phosphoenolpyruvate carboxykinase gene. Role of specific CCAAT/enhancer-binding protein isoforms. J Biol Chem 275: 5804–5809, 2000.[Abstract/Free Full Text]
  6. Dai YS and Markham BE. p300 Functions as a coactivator of transcription factor GATA-4. J Biol Chem 276: 37178–37185, 2001.[Abstract/Free Full Text]
  7. Dean DM and Sanders MM. Ten years after: reclassification of steroid-responsive genes. Mol Endocrinol 10: 1489–1495, 1996.[Abstract]
  8. Divine JK, McCaul SP, and Simon TC. HNF-1{alpha} and endodermal transcription factors cooperatively activate Fabpl: MODY3 mutations abrogate cooperativity. Am J Physiol Gastrointest Liver Physiol 285: G62–G72, 2003.[Abstract/Free Full Text]
  9. Dusing MR, Florence EA, and Wiginton DA. High-level activation by a duodenum-specific enhancer requires functional GATA binding sites. Am J Physiol Gastrointest Liver Physiol 284: G1053–G1065, 2003.[Abstract/Free Full Text]
  10. Fang R, Olds LC, Santiago NA, and Sibley E. GATA family transcription factors activate lactase gene promoter in intestinal Caco-2 cells. Am J Physiol Gastrointest Liver Physiol 280: G58–G67, 2001.[Abstract/Free Full Text]
  11. Galand G. Brush border membrane sucrase-isomaltase, maltase-glucoamylase and trehalase in mammals. Comparative development, effects of glucocorticoids, molecular mechanisms, and phylogenetic implications. Comp Biochem Physiol B 94: 1–11, 1989.[ISI][Medline]
  12. Gao X, Sedgwick T, Shi YB, and Evans T. Distinct functions are implicated for the GATA-4, -5, and -6 transcription factors in the regulation of intestine epithelial cell differentiation. Mol Cell Biol 18: 2901–2911, 1998.[Abstract/Free Full Text]
  13. Gartner H, Shukla P, Markesich DC, Solomon NS, Oesterreicher TJ, and Henning SJ. Developmental expression of trehalase: role of transcriptional activation. Biochim Biophys Acta 1574: 329–336, 2002.[ISI][Medline]
  14. Henning SJ. Functional development of the gastrointestinal tract. In: Physiology of the Gastrointestinal Tract, edited by Johnson LR. New York: Raven, 1987, p. 285–300.
  15. Henning SJ. Gastrointestinal development: an overview. In: Falk Symposium 94: The Gut as a Model in Cell and Molecular Biology, edited by Halter F, Winton D, and Wright NA. Dordrecht, The Netherlands: Kluwer Academic Publishers, 1997, p. 49–60.
  16. Henning SJ, Rubin DC, and Shulman RJ. Ontogeny of the intestinal mucosa. In: Physiology of the Gastrointestinal Tract, edited by Johnson LR. New York: Raven, 1994, p. 571–610.
  17. Hwang ST, Urizar NL, Moore DD, and Henning SJ. Bile acids regulate the ontogenic expression of ileal bile acid binding protein in the rat via the farnesoid X receptor. Gastroenterology 122: 1483–1492, 2002.[ISI][Medline]
  18. Kiela PR, LeSueur J, Collins JF, and Ghishan FK. Transcriptional regulation of the rat NHE3 gene. Functional interactions between GATA-5 and Sp family transcription factors. J Biol Chem 278: 5659–5668, 2003.[Abstract/Free Full Text]
  19. Krasinski SD, van Wering HM, Tannemaat MR, and Grand RJ. Differential activation of intestinal gene promoters: functional interactions between GATA-5 and HNF-1{alpha}. Am J Physiol Gastrointest Liver Physiol 281: G69–G84, 2001.[Abstract/Free Full Text]
  20. Laverriere AC, MacNeill C, Mueller C, Poelmann RE, Burch JB, and Evans T. GATA-4/5/6, a subfamily of three transcription factors transcribed in developing heart and gut. J Biol Chem 269: 23177–23184, 1994.[Abstract/Free Full Text]
  21. Leeper LL and Henning SJ. Development and tissue distribution of sucrase-isomaltase mRNA in rats. Am J Physiol Gastrointest Liver Physiol 258: G52–G58, 1990.[Abstract/Free Full Text]
  22. Levenson CW, Shay NF, and Cousins RJ. Cloning and initial characterization of the promoter region of the rat cysteine-rich intestinal protein gene. Biochem J 303: 731–736, 1994.[ISI][Medline]
  23. Nanthakumar NN and Henning SJ. Distinguishing normal and glucocorticoid-induced maturation of intestine using bromodeoxyuridine. Am J Physiol Gastrointest Liver Physiol 268: G139–G145, 1995.[Abstract/Free Full Text]
  24. Oesterreicher TJ, Markesich DC, and Henning SJ. Cloning, characterization and mapping of the mouse trehalase (Treh) gene. Gene 270: 211–220, 2001.[CrossRef][ISI][Medline]
  25. Oesterreicher TJ, Nanthakumar NN, Winston JH, and Henning SJ. Rat trehalase: cDNA cloning and mRNA expression in adult rat tissues and during intestinal ontogeny. Am J Physiol Regul Integr Comp Physiol 274: R1220–R1227, 1998.[Abstract/Free Full Text]
  26. Shi XM, Blair HC, Yang X, McDonald JM, and Cao X. Tandem repeat of C/EBP binding sites mediates PPAR{gamma}2 gene transcription in glucocorticoid-induced adipocyte differentiation. J Cell Biochem 76: 518–527, 2000.[CrossRef][ISI][Medline]
  27. Shulman RJ, Schanler RJ, Lau C, Heitkemper MA, Ou C, and Smith EO. Early feeding, antenatal glucocorticoids, and human milk decrease intestinal permeability in preterm infants. Pediatr Res 44: 519–523, 1998.[Abstract]
  28. Solomon NS, Gartner H, Oesterreicher TJ, and Henning SJ. Development of glucocorticoid-responsiveness in mouse intestine. Pediatr Res 49: 782–788, 2001.[Abstract/Free Full Text]
  29. Suh E, Chen L, Taylor J, and Traber PG. A homeodomain protein related to caudal regulates intestine-specific gene transcription. Mol Cell Biol 14: 7340–7351, 1994.[Abstract]
  30. Traber PG and Silberg DG. Intestine-specific gene transcription. Annu Rev Physiol 58: 275–297, 1996.[CrossRef][ISI][Medline]
  31. Tung J, Markowitz AJ, Silberg DG, and Traber PG. Developmental expression of SI is regulated in transgenic mice by an evolutionarily conserved promoter. Am J Physiol Gastrointest Liver Physiol 273: G83–G92, 1997.[Abstract/Free Full Text]
  32. Yeh KY, Yeh M, and Glass J. Glucocorticoids and dietary iron regulate postnatal intestinal heavy and light ferritin expression in rats. Am J Physiol Gastrointest Liver Physiol 278: G217–G226, 2000.[Abstract/Free Full Text]