Hydrocortisone induces changes in gene expression and ...

Am J Physiol Gastrointest Liver Physiol 300: G425–G432, 2011. First published December 9, 2010; doi:10.1152/ajpgi.00011.2010.

Hydrocortisone induces changes in gene expression and differentiation in immature human enterocytes Lei Lu,1 Tiantian Li,1 Graham Williams,2 Elizabeth Petit,1 Mark Borowsky,3 and W. Allan Walker1 1

Developmental Gastroenterology Laboratory, Massachusetts General Hospital for Children, Boston; 3Department of Molecular Biology, Massachusetts General Hospital and Department of Genetics, Harvard Medical School, Boston, Massachusetts; and 2DePauw University, Greencastle, Indiana

Submitted 19 January 2010; accepted in final form 29 November 2010

Lu L, Li T, Williams G, Petit E, Borowsky M, Walker WA. Hydrocortisone induces changes in gene expression and differentiation in immature human enterocytes. Am J Physiol Gastrointest Liver Physiol 300: G425–G432, 2011. First published December 9, 2010; doi:10.1152/ajpgi.00011.2010.—It is known that functional maturation of the small intestine occurring during the weaning period is facilitated by glucocorticoids (such as hydrocortisone, HC), including an increased expression of digestive hydrolases. However, the molecular mechanisms are not well understood, particularly in the human gut. Here we report a microarray analysis of HC-induced changes in gene expression in H4 cells (a well-characterized human fetal small intestinal epithelial cell line). This study identified a large number of HC-regulated genes, some involved in metabolism, cell cycle regulation, cell-cell or cell-extracellular matrix communication. HC also regulates the expression of genes important for cell maturation such as development of cell polarity, tight junction formation, and interactions with extracellular matrices. Using human small intestinal xenografts, we also show that HC can regulate the expression of genes important for intestinal epithelial cell maturation. Our dataset may serve as a useful resource for understanding and dissecting the molecular mechanisms of intestinal epithelial cell maturation. microarray; immature enterocyte development; hydrocortisone; polarity-associated genes; tight junction-associated genes PREMATURE INFANTS ARE AT RISK for developing serious gastrointestinal diseases, such as necrotizing enterocolitis and certain bacteria-induced diarrheas (6, 10) due to developmental immaturity of intestinal host defense. Amniotic fluid and breast milk contain trophic factors that interact with the gut to stimulate the development of host defenses and attenuate the severity of these disease states (reviewed in Ref. 33). Among many endogenous biological mediators, hydrocortisone (HC), epidermal growth factor, and transforming growth factor-␤ (TGF-␤) promote growth and differentiation (34), modulate the inflammatory response (9), and reduce an excessive secretory response to cholera toxin in human and rat immature enterocytes (7, 19, 20). In rodents, the functional maturation of the small intestine during weaning is facilitated by glucocorticoids (4, 25). In vivo, HC treatment of suckling rats increases galactosylation and fucosylation of glycoproteins, which are biochemical markers associated with cellular maturation in the small intestine (4). In addition, the suppression of glucocorticoid secretion by adrenalectomy reduces the level of glycosylation in the small intestine at weaning (22); in vitro, HC induces functional

Address for reprint requests and other correspondence: L. Lu, Developmental Gastroenterology Laboratory, Pediatric Gastroenterology and Nutrition, Harvard Medical School, Massachusetts General Hospital, 114 16th St. (1143503), Charlestown, MA 02129-4404 (e-mail: [email protected]). http://www.ajpgi.org

alterations in rat enterocytes, including growth arrest, tight junction formation, microvilli maturation, and reorganization of the endoplasmic reticulum and trans-Golgi network (26). In human studies, Arsenault and Ménard (2) have shown that HC exhibits effects on differentiation and proliferation of fetal small intestinal organ culture, such as brush-border membrane hydrolytic activities (sucrase, lactase, glucoamylase, trehalase, and alkaline phosphatases) and epithelial cell proliferation. Finally, in vivo, prenatal corticosteroids given to mothers at risk for premature delivery or to preterm infants during the first week of life have been shown to prevent or significantly decrease the severity of necrotizing enterocolitis (3, 15). Thus, both endogenous and exogenous sources of glucocorticoids may play an important role in the functional maturation of the small intestine.Microarrays have served as useful tools to monitor global gene expression changes in mouse small intestine. Using a time course of dexamethasone in preweaned mouse small intestine, Agbemafle et al. (1) have identified candidate primary responsive genes. Gene ontology analysis and in situ hybridization performed on these genes revealed that glucocorticoids exhibit pleiotropic effects in multiple cell types (1, 35). Accordingly, using microarray analysis, we report that HC stimulation of immature human enterocytes (H4 cells) induced dramatic changes in the expression of a large number of genes that could play an important role in differentiation. MATERIALS AND METHODS

Materials. The RNeasy Mini kit and SuperScript III Platinum SYBR Green One-Step qRT-PCR kits were obtained from Qiagen (Valencia, CA). All tissue culture media and reagents are obtained from Invitrogen-GIBCO (Carlsbad, CA). All other chemicals, unless specified, were purchased from Sigma-Aldrich (St. Louis, MO). Cell culture and growth conditions. This study employed two human intestinal epithelial cell lines, H4 and T84. H4 is a previously characterized primary human fetal small intestinal cell line that we used in numerous studies defining the role of human enterocytes in the development of intestinal host defenses (8, 9, 13, 19, 20). T84, a well-characterized, human colon carcinoma cell line that comprises highly differentiated crypt-like cells, has been used as a mature human intestinal model (8, 20, 28). H4 cells were routinely maintained in Dulbecco’s modified Eagles medium (DMEM) supplemented with 10% FBS and human recombinant insulin (0.5 U/ml). H4 cells were plated on a 15-cm-diameter tissue culture-treated dish. At 90% confluence, H4 cells were incubated with H4 media containing 1 ␮M HC for 12, 24, or 48 h or 5 days. Separate time course experiments were performed four times. T84 cells (American Type Culture Collection, ATCC, Rockville, MD) were maintained in DMEM-Ham’s F-12 with 5% FBS. In this study, T84 cells were grown on a 10-cm-diameter tissue culture-treated dish and harvested at 90% confluence.

0193-1857/11 Copyright © 2011 the American Physiological Society

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HYDROCORTISONE EFFECTS ON GENE EXPRESSION IN FETAL ENTEROCYTES

Fetal small intestinal xenograft transplant model. Human fetal small intestine xenografts were supplied by the Xenograft Core Facility in our laboratory (detailed in Refs. 24 and 29). Five weeks after transplant, mice were injected subcutaneously with HC (50 mg/kg body wt, n ⫽ 2) or an equal volume of normal saline (n ⫽ 2). Tissues were obtained 1 wk later by carefully removing all the serosal and muscle tissues surrounding the intestinal mucosa and homogenized with a hand-held Polytron. Fetal tissue was obtained with the approval from the Partners Human Study Committee and with signed permission (IRB no. 1999p003833). This study has been approved by the Subcommittee on Research Animal Care at Massachusetts General Hospital. RNA isolation and amplification. H4 cells and xenografted human fetal small intestinal mucosa were lysed in RLT buffer (containing guanidine isothiocyanate) from QIAGEN. Total RNA was isolated using an RNeasy kit following the manufacturer’s instruction. RNA concentrations were measured using a spectrophotometer with a ration of absorbance at 260 to 280 nm of 1.8 –2.0, and the final concentration was brought up to 1–2 ␮g/ml. The RNA samples were subjected to an Agilent 2100 Bioanalyzer to assess RNA quality. The bioanalyzer provides a visual inspection of RNA integrity and generates 28S-to18S ribosomal RNA ratios and an RNA Integrity Number (RIN) with an RIN of 10 corresponding to pure, undegraded sample and a 1 corresponding to a completely degraded sample. All RNA samples submitted for standard Affymetrix expression had an RIN of six or better by our core facility of the Harvard Medical School-Partners HealthCare Center for Genetics and Genomics. Human Genome U133 Plus 2.0 GeneChip microarrays were purchased from Affymetrix (Affymetrix, Santa Clara, CA). Preparation of labeled cRNA, hybridization, and scanning of microarray analysis was performed using standard protocols and reagents as described in the Affymetrix Technical Manual (Revision 3). Data filtering and analysis. The database was comprised of 6 expression measurements of 54,675 genes and is submitted to the GEO repository (accession code: GSE22106). Data were analyzed with linear modeling of microarray data (AffylmGUI), and the fluorescence intensity of each spot was normalized by Robust Multiarray Averaging using R/Bioconducter. We defined differentially expressed genes as those whose expression value changed at least twofold (greater than or equal to ⫾ 2-fold change) in response to HC by microarray analysis. These differentially expressed genes were used for further analysis. A Venn diagram was used to analyze the common and unique genes that were affected by HC among the groups using a fold change greater than or equal to ⫾ 2 as cutoff. MetaCore (GeneGo) was used to perform the gene enrichment analysis among the groups and identify canonical pathways and networks that were most significant to the dataset. Fischer’s exact test was used to calculate a P value determining the probability that the association between the genes in the dataset and the canonical pathway was due to chance. Hierarchical clustering analysis was done with Multi-expression viewer (MEV)

software on genes that are associated with tight junction formation, epithelial polarization, and WNT signaling (MetaCore). qRT-PCR and target validation. Select gene profiles were validated using semiquantitative RT-PCR (real-time RT-PCR) from RNA isolated as described above. All RT-PCR reactions were done using the SuperScript III One-Step RT-PCR Kit and following the manufacturer’s protocol. Total RNA (10 ng) was added to each reaction in duplicate with the annealing temperature set at 60°C and 40 cycles. The primer sets were selected and obtained from the Harvard primer Bank (http://pga.mgh. harvard.edu/primerbank) and Partner’s Genomic DNA core facility at Massachusetts General Hospital. Electron microscopic studies. H4 and T84 cells were grown on tissue culture-treated plates to confluence and fixed in 4% paraformaldehyde and embedded in 2% agarose in PBS and finally infiltrated with LR white resin and embedded in gelatin capsules. Thin sections were collected on formvar-coated nickel grids, stained for 5 min with drops of aqueous uranyl acetate (EMS), and examined in a Philips CM 10 transmission electron microscope at 80 kV (see Refs. 12 and 32 for details). Two-dimensional cell culture and confocal microscopy. H4 cells were plated on a permeable filter insert that was either coated with extracellular matrix mixture (matrigel; BD Biosciences) or collagen. After 2–5 days, cells were incubated with or without HC for 5 days. For immunofluorescence staining, cells grown on collagen or matrigel were treated with 0.05% collagenase A alone or with hyaluronidase (50 units) in PBS, fixed with 4% paraformaldyhide at 4°C for 30 min, and blocked and permeablized with 10% gelatin and 0.05% saponin in PBS for 1 h. Cells were then incubated with anti-ZO-1 (monoclonal antibody, 1:1,000) overnight, washed, and incubated with Alexa 595-conjugated goat anti-mouse IgG and examined in a Bio-Rad Axiophot fluorescence photomicroscope and a confocal microscope (Bio-Rad, Hercules, CA). Statistics. The cycles above threshold were used to calculate the expression levels of amplified genes and were normalized with expression levels of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Using the statistical computing program R, we performed ANOVA on the time-course expression data. P ⬍ 0.05 was considered statistically significant. See reference 5 for detailed methodology. RESULTS

General HC effects on gene expression in H4 cells. We analyzed the transcriptional profile of genes that were either up- or downregulated at least twofold from each sample and generated a four-way Venn diagram to examine the overlap/ interactions among sets of resulting gene lists as shown in Fig. 1. Among the topmost HC-affected transcripts (greater than or equal to ⫾ 2), there were 61% common upregulated between the 48-h and 5-day group, and 41% common genes between the 12- and 24-h group. However, there were very few common

Fig. 1. Four-way Venn diagram comparison of early and late hydrocortisone (HC)-induced differentially expressed genes in H4 cells. Long-term HC treatment (48 h and 5 days) has a significantly greater effect on gene expression (late genes) than short-term HC treatment (12 and 24 h, early genes). There is very little overlap between early and late HC-induced genes in H4 cells.

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upregulated genes between the 24- and 48-h HC treatment group (5%). There were only 43 out of 1,260 (3%) upregulated and 95 out of 1,336 (7%) downregulated genes common among all time points, suggesting that there are at least two distinct clusters of HC-responsive genes in H4 cells, those whose transcripts are transiently influenced early during HC exposure and those whose transcripts are influenced either after longer HC exposure or throughout the 5-day HC exposure. Moreover, our data indicated that longer HC exposure (48 h) had a greater effect on gene expression, e.g., 1,025 genes had a higher than twofold upregulation and the maximum fold change of 239⫻ compared with a shorter HC treatment (24 h, 89 genes, and a maximum fold change of 17⫻). Using GeneGo MetaSearch, we find 872 potential glucocorticoid target genes, and 152 of those genes were either up- or downregulated. However, HC has no effect on GAPDH expression in H4 cells (data not shown). There are six GAPDH oligonucleotides on U133 Plus 2.0 GeneChip, and the mean GAPDH expression levels were 13.86 ⫾ 0.11, 13.86 ⫾ 0.098, 13.88 ⫾ 0.129,

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13.80 ⫾ 0.095, and 13.78 ⫾ 0.072 (log2) from control to 5-day HC exposure, respectively. Comparison of the effect of HC on gene expression patterns. The differentially expressed genes were divided into up- and downregulated genes and subjected to integrated pathway enrichment analysis by using the knowledge-based canonical pathways and networks in MetaCore to extract patterns of gene expression, including 1) enrichment of the network and 2) relative enrichment of the genes in canonical pathways in H4 cells with HC at different time points. Ranking of relevant integrated pathways was based on hypergeometric P values. The top 10 scored networks and canonical pathways of upregulated genes in early (12 and 24 h HC) are displayed in Fig. 2, A and B, and that of late (48 h and 5 days HC) groups are displayed in Fig. 2, C and D. Gene enrichment analysis of the H4/HC time course separated samples into two groups in many of the networks and canonical pathways. The first group was comprised of samples with short-term HC treatment, and the second group was comprised of samples

Fig. 2. MetaCore gene enrichment analysis of HC effects in H4 cell microarray. Top up- or downregulated genes (fold change ⱖ2) were further analyzed using GeneGo MetaCore gene enrichment analysis. A and B: top 10 canonical pathways and networks that were affected by short-term (12 and 24 h) HC treatment in H4 cells. C and D: top 10 canonical pathways and networks that were affected by long-term (48 h and 5 days) HC treatment in H4 cells. HC treatment, particularly long-term (⬎48 h), has a global effect on gene expression and a large effect on development genes. PDGF, platelet-derived growth factor; EGF, epidermal growth factor; LT, leukotriene; PI3K, phosphatidylinositol 3-kinase; AKT, protein kinase B; MAPK, mitogen-activated protein; TGF-␤, transforming growth factor-␤; CDK5, cyclin-dependent kinase 5; TREM, triggering receptor expressed on myeloid cells; ILK, integrin-linked kinase. AJP-Gastrointest Liver Physiol • VOL

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with a long-term HC treatment. These findings suggested that HC may induce an early transient change in gene expression and also a later persistent change in mRNA expression in H4 cells. Moreover, many of the later responsive genes are involved in intestinal epithelial cell differentiation/maturation, including the organization of extracellular matrix and intracellular components, TGF-␤ and WNT-mediated cytoskeleton remodeling, and cell adhesion and cell-matrix interactions (Fig. 2). HC effects on genes associated with polarization of intestinal epithelial cells. The establishment of a polarized enterocyte membrane and its cytoarchitecture is fundamental to the specialized function of intestinal epithelial cells (16). Recent studies have shown a profound change of transcriptional patterns occurs in Caco-2 cells during development of polarity in vitro (16, 27). These studies suggest that an intrinsic transcriptional program participates in multilayered signaling pathways, including WNT, BMPs, hedgehog, FGF, and Notch for the

development of enterocyte polarity. To investigate whether HC affects the expression of genes that play any role in polarization, a further hierarchical clustering analysis using MEV software was performed to examine genes that are associated with tight junction formation, epithelial polarization, and WNT signaling. Figure 3 depicts the HC-induced differential gene expression pattern in the time-course samples. Hierarchical clustering of organized genes (rows) fell into two HC-responsive cluster groups with the majority of HC-upregulated tight junction and polarity-associated genes seen in the late group (48 h and 5 days). To validate the results obtained from gene enrichment analysis of the microarray data, we performed semiquantitative (real-time) RT-PCRs for specific tight junction/ polarizationrelated genes (reviewed in Ref. 30) in H4 cells treated with HC. The average change of mRNA expression (n ⫽ 4) of these genes in response to HC treatment is shown in Fig. 4. These

Fig. 3. Hierarchical clustering of HC-induced genes encoding polarity, tight junctions, and WNT signaling proteins in H4 cells. HC-affected expression levels of each gene over time relative to untreated H4 cells are displayed as fold changes. Red or green color indicates the fold change of up- or downregulated expression levels compared with unchanged (black). A: HC effects on polarization-associated gene expression. B: HC effects on tight junction-associated gene expression. C: HC effects on WNT signaling-associated gene expression. Long-term HC treatment (ⱖ48 h) has profound effects on genomic regulation of these developmental events in immature enterocytes. AJP-Gastrointest Liver Physiol • VOL

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Fig. 4. HC effects on polarity-, tight junction-, and differentiation-associated gene expressions by qRT-PCR in human fetal enterocytes. A: average HC-induced transcriptional change of polarity- and cell differentiation-associated genes in H4 cells (n ⫽ 4). B: average HC-induced transcriptional change of tight junction-associated genes in H4 cells (n ⫽ 4). C: HC-induced transcriptional change of polarity-, tight junction-, and cell differentiation-associated genes in human fetal small intestinal xenografts (bold letters: same as seen in H4 cells). HC-induced differential expression levels of genes are normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and expressed as fold change over untreated H4 cells (designated as 1).

findings confirm our microarray data that showed treatment with HC, most notably for 48 h, increased expression of genes encoding proteins involved in intestinal epithelial cell maturation markers, polarization (Fig. 4A), and tight junction (Fig. 4B). Expression of representative genes encoding proteins involved in polarity, tight junction, and enterocyte maturation (apical markers) was examined in fetal small intestinal xenografts and as shown in Fig. 4C. Consistent with the gene expression patterns seen in H4 cells, HC increased mRNA expression of tight junction and polarity genes. Moreover, there is a dramatic increase in apical marker gene expression in response to HC treatment. HC modulates H4 interaction with the extracellular matrix to form cell-cell junctions in a two-dimensional culture system. Using microarray analysis of H4 cells, we have shown that HC may induce a transcriptional change in genes involved in enterocyte polarization and differentiation (Fig. 2). To examine H4 cell surface membrane structure in greater detail, we performed preliminary electron microscopy (EM). EM results show, in contrast to the flat cell surface of an undifferentiated intestinal epithelial cell membrane morphology in H4 cells, T84 cells display a typical differentiated morphology with well orderly microvilli on the cell surface. HC-treated H4 cells exhibited an increase in microvilli formation (a differentiation marker of enterocytes), suggesting that HC can potentiate polarization in H4 cells (Fig. 5). Using a two-dimensional culture system, we examined the effect of extracellular matrix and HC on morphological characteristics associated with enAJP-Gastrointest Liver Physiol • VOL

terocyte maturation. Our findings show that H4 cells, when grown on collagen, display a flat cellular morphology and cytosolic expression of ZO-1, a tight junction marker (Fig. 6, A and D). When plated on matrigel, H4 cells exhibit more differentiated characteristics, which include increased expression of ZO-1, an increased cell height, and the formation of tight cell-cell adhesion (Fig. 6, B and E). Moreover, HC treatment of H4 cells further increased the maturational morphology of H4 cells on matrigel (Fig. 6C) with a further increase in cell height (further away from the filter where cells were originally seeded), a cell surface expression of ZO-1 protein, and cell-cell junction formations (Fig. 6, C and F). DISCUSSION

The differentiation of mammalian intestinal epithelium is regulated by glucocorticoids from the pituitary-adrenal axis and occurs in several pre- and postnatal phases characterized by distinct changes in enterocyte morphology and expression of biochemical markers (reviewed in Ref. 11). In fact, in rodents, adrenalectomy during the suckling period retards enterocyte development in the small intestine, whereas the early administration of exogenous HC may cause a precocious maturation of the gut (14, 23, 31). The molecular mechanisms of HC action(s) in these systems are not well understood. Currently, several groups are actively involved in identifying glucocorticoid-responsive genes in mammalian intestinal cells. However, most published studies have 300 • MARCH 2011 •

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Fig. 5. HC effect on H4 plasma membrane morphology by electron microscopy (EM). A: freeze-fracture EM demonstrated a typical smooth basolateral phenotype of plasma membrane (PM) in H4 cells and a typical apical PM phenotype in T84 cells (C). HC treatment of H4 cells induced a phenotypic change in plasma membrane with increased appearance of microvilli structure (B). Morphology studies using EM also depict a smooth plasma membrane phenotype in H4 cells (D) and increased microvilli formation in H4 cells treated with HC (5 days) (E, black arrow) and T84 (F).

used a rodent-derived lineage (1, 35), and less is known about the effects of glucocorticoids on gene expression in immature human intestinal cells. Here we report the use of microarray analysis to investigate the effects of HC on gene expression in the H4 immature human enterocyte cell line. These data comprise the first characterization of the HC effect on gene expression in human intestinal cells. In general, the effects of HC on H4 gene expression are consistent with the molecular and morphological changes associated with HC-induced differentiation of mammalian enterocytes previously reported in vivo and in vitro (1, 31, 35). In these studies, gene transcripts were identified to be up- or downregulated rapidly (2–24 h) or slowly (⬎48 h) (1, 35). Thus a better determination of the short- and long-term effects of HC exposure on gene expression patterns in developing human enterocytes is required for the understanding of the HC response. In this study, we have used

microarray analysis to identify transcripts that are differentially up- and downregulated in response to the duration of HC exposure in H4 cells. The observed pattern of H4 cell responsiveness to HC indicated an early and a late phase of transcription regulation (Figs. 1 and 2). Given the well-known fact that HC has pleiotropic effects on cells (26), it was not surprising that our gene enrichment analysis showed HC-affected transcripts to be distributed through various gene categories. Many transcripts in H4 cells that were most affected by HC treatment were found in cell adhesion, TGF-␤⫺ and WNT-regulated cell proliferation, cytoskeleton reorganization, and extracellular matrix remodeling categories (Fig. 2). Furthermore, several early HC-responsive gene transcripts that we detected are known to be involved in regulating cell cycle stages (G2-M) and cell cycle core. In addition, several late-responsive genes are known to play a role in the growth factor regulation of cell-cycle stages (G1-S), implicating effects of HC on regula-

Fig. 6. HC facilitates H4 cell differentiation on matrigel 2-dimensional culture system. A and D: H4 cells, when grown on collagen, displayed a scattered growth and cytosolic expression of ZO-1. B and E: H4 cells, when grown on matrigel, displayed cell-cell adhesion and increased ZO-1 expression. C and F: H4 cells, when grown on matrigel and incubated with HC (1 ␮M, 5 days), exhibited a cell-cell junction formation, increased cell surface ZO-1 expression, and an increase in cell height. XZ view of H4 cells probed with anti-ZO1 (green). Black arrow indicates ZO-1 staining in the cell and dashed arrow indicates the filter surface. AJP-Gastrointest Liver Physiol • VOL

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tion of differentiation and possibly induction of a mitotic process in immature human enterocytes. The development of epithelial cell polarity, expressed by structurally and functionally distinct apical and basolateral plasma membranes, is critical for the formation of many organs and tissues (37). This process requires protein trafficking, cytoskeleton rearrangement, and changes in both cell-cell and cell-matrix interactions (16, 21, 36). The transcriptional regulation of these processes has not been widely studied at the genomic level in human enterocytes. Here we report results of microarray analysis to examine the transcriptional changes induced by HC in H4 cells that may be associated with epithelial cell polarization. Our results uncovered a complex pattern of change in gene expression that is associated with cell-cell junctions (e.g., apical and tight junction markers). We found that HC differentially influenced the expression levels of transcripts encoding components of several cell adhesion complexes. As shown in Fig. 4, HC induces genes encoding distinct protein complexes, such as those involved in cell adhesion, tight junctions, and polarization. For example, HC-treated H4 cells show increased expression of mRNA encoding tight junction proteins (TJP-1,2: ZO-1,2), claudins (1, 4, 15, etc.), and polarization complex proteins (PAR3/PAR6/atypical protein kinase C, PRKCi, PRKCz) that regulate formation of the apical domain in mammalian epithelial cells (17, 18). In general, there is a correlation between HC-regulated gene expression and HCinduced differentiation in immature H4 cells. Under EM, T84 cells exhibited the typical apical membrane structure, studded with microvilli, while H4 cells manifested the smooth basolateral membranes characteristic of undifferentiated enterocytes (Fig. 5). After HC exposure, the H4 membrane developed with some microvilli that mimic an apical membrane. Our results suggest that HC treatment of immature human enterocytes dramatically alters the expression of many genes, some of which have an established role in enterocyte differentiation and maturation. This study shows that HC may not modulate a specific cellular function robustly but instead modulates many important biological pathways in developing cells. The differentiation of intestinal epithelium also involves posttranscriptional/ posttranslational modifications affecting protein structure, function, and distribution within the cell. Thus HC may also influence maturation of the immature enterocyte by modulating transcription of genes encoding posttranslational modifiers (kinases, phosphatases, etc). Our data will be invaluable for future investigation of the regulation of human intestinal cell maturation. Additional exploration of the complexity of the HC effect on the developing human intestine should provide insights into studying alterations of intestinal pathophysiology in immature human enterocytes that may result in age-related gastrointestinal diseases. GRANTS This work was supported by National Institutes of Health Grants R01-HD12437, R01-DK-70260, P01-DK-33506, and P30-DK-40561 (W. A. Walker). DISCLOSURES No conflicts of interest are declared by the authors. REFERENCES 1. Agbemafle BM, Oesterreicher TJ, Shaw CA, Henning SJ. Immediate early genes of glucocorticoid action on the developing intestine. Am J Physiol Gastrointest Liver Physiol 288: G897–G906, 2005. AJP-Gastrointest Liver Physiol • VOL

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