ARTICLE |
Correspondence to: Cécile Huin, Laboratoire de Biologie cellulaire du Développement, UPRES 2402 “Proliférateurs de Peroxysomes,” Faculté des Sciences, BP 239, 54506 Vandoeuvre-les-Nancy Cedex, France.
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Summary |
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We investigated the spatiotemporal distributions of the different peroxisome proliferator-activated receptor (PPAR) isotypes (, ß, and
) during development (Week 7 to Week 22 of gestation) of the human fetal digestive tract by immunohistochemistry using specific polyclonal antibodies. The PPAR subtypes, including PPAR
, are expressed as early as 7 weeks of development in cell types of endodermal and mesodermal origin. The presence of PPAR
was also found by Western blotting and nuclease-S1 protection assay, confirming that this subtype is not adipocyte-specific. PPAR
, PPARß, and PPAR
exhibit different patterns of expression during morphogenesis of the digestive tract. Whatever the stage and the gut region (except the stomach) examined, PPAR
is expressed at a high level, suggesting some fundamental role for this receptor in development and/or physiology of the human digestive tract. (J Histochem Cytochem 48:603611, 2000)
Key Words: PPARs, development, differentiation, fetus, digestive tract
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Introduction |
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PEROXISOME PROLIFERATOR-ACTIVATED RECEPTORS (PPARs) are transcription factors belonging to the nuclear receptor superfamily and have been initially described as molecular targets for compounds that cause peroxisome proliferation (for review see (
(
(
mRNA, transcribed from three different promoters, which give rise to two proteins, PPAR
1 and
2, as the protein encoded by PPAR
3 mRNA is indistinguishable from PPAR
1 (
In humans, PPAR is present mainly in liver, heart, and kidney, whose tissues exhibit high fatty acid metabolism and high peroxisome-dependent activity. PPARß is ubiquitously expressed in all tissues tested, whereas PPAR
predominates in adipose tissue, large intestine, and macrophages and monocytes (
regulates the transcription of several target genes involved in lipid metabolism and homeostasis (
controls adipocyte (
Detailed descriptions of the morphological and functional changes occurring during the development of the human gastrointestinal tract are available (
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Materials and Methods |
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Tissue Specimens
Samples of esophagus, stomach, intestine, and colon from 23 fetuses ranging from 7 to 22 weeks of age were obtained from normal elective pregnancy terminations. The project was performed in accordance with the requirements of the Institutional Human Subject Review Board (University of Sherbrooke) for the use of human tissues. The latter were embedded in Polyfreeze Tissue Freezing Medium (Polysciences; Warrington, PA) and frozen in liquid nitrogen.
Production of Anti-PPAR Antibodies
As shown in Fig 1, the anti-PPAR antibody was raised against the amino acid sequence 45SSGSFGFTEYQY56 of human PPAR
(
1/
2 antiserum was raised against the amino acid sequence EMPFWPTNFGISSVD common to PPAR
1 and PPAR
2 [amino acids 519 of mouse PPAR
1 (
2 (
isoforms (
2 differs from the PPAR
1 by an additional specific N-terminal amino acid region, the sequence of the hapten used to produce the anti-human PPAR
2 antibody was mapped at that region and corresponded to 2GETLGDSPIDPESDS16 of human PPAR
2 (
antiserum (Interchim; Montluçon, France) directed against the amino acid sequence MMGEDKIKFKHITPL common to PPAR
1 and PPAR
2 (amino acids 256270 of human PPAR
1, 284298 of human PPAR
2 ).
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Characterization of the Antibodies
The polyclonal antibodies produced were characterized by immunoprecipitation and Western blotting assays. In vitro transcription and translation of mouse PPAR/pSG5, PPARß/pSG5, PPAR
1 /pSG5, and human PPAR
2/pBluescript IIKS+ plasmids (gift of Prof. W. Wahli; University of Lausanne, Switzerland) were performed using reticulocyte lysate (Promega; Charbonnières, France) and L-[35 S]-methionine. Translated products were either immunoprecipitated with the antibodies and analyzed by SDS-PAGE, followed by autoradiography, or directly submitted to Western blotting and enhanced chemiluminescence (ECL) in crossreaction assays.
Adult and fetal colon mucosae were homogenized in 25 mM Hepes buffer, pH 7.4, containing 0.4 M KCl, 1 mM EDTA, 2 mM dithiothreitol, and a cocktail of protease inhibitors (Complete; Roche, Mannheim, Germany). The homogenates were centrifuged at 15,000 x g for 20 min (4C). The protein concentration of the supernatant was determined (
Nuclease Protection Assay
Partial human PPAR2 cDNA corresponding to the 5'UTR sequence (
2 vector. A pBIIKS+/hG3PDH clone containing a 380-bp DNA fragment of the human G3PDH encoding sequence (
2. Total RNA (5 µg) was hybridized overnight with 32P single-stranded DNA probes (105 cpm/sample) at 60C. After incubation, nonhybridized cDNA was digested by nuclease S1 (50 U/sample) for 60 min at 37C. The DNA/RNA hybrids were resolved by electrophoresis and the gel was exposed to Kodak film for 24 hr.
Immunohistochemical Analysis
Cryostat sections (3 µm thick) were fixed in 2% formaldehyde in PBS for 45 min at 4C and rinsed in PBS. They were immersed in 100 mM glycine in PBS for 45 min at 4C, then washed in PBS. The sections were preincubated with a blocking solution containing 0.1% fish gelatin, 0.8% bovine serum albumin, and Tween-80 (2 µl/100 ml PBS) for 30 min at room temperature (RT). They were first exposed to the primary antibody (diluted 1:250 in PBS/defatted dry milk 5% w/v) for 60 min at RT. After two washes in PBS, sections were exposed to the secondary antibody (1:50 in PBSBSA 2%), fluorescein-conjugated goat anti-rabbit IgG (Boehringer Mannheim), for 60 min at RT. Negative controls were performed by replacing the primary antibody with PBS or with preimmune serum. Sections were then mounted in Vectashield medium and photographed with a ReichertJung Polyvar microscope (Vienna, Austria).
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Results |
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Antibody Specificity
In vitro-translated mouse PPAR, ß, and
1 and human PPAR
2 were used for immunoprecipitation assays, taking advantage of the fact that the human peptide sequences chosen for immunization are well-conserved in the corresponding rodent sequences. Fig 2 shows that each antibody recognized the PPAR subtype against which it was raised. When preimmune serum was used as a control, no signal was obtained. Crossreaction between each anti-PPAR antibody against the other PPAR subtypes was absent or very low, as demonstrated by Western blotting assays (Fig 3 ). The anti-PPAR antibodies produced were also characterized by Western blotting using cytosolic extracts from human adult and fetal colon mucosae. The presence of the different PPAR subtypes was detected in both samples examined (Fig 4). However, our results indicated a higher expression of PPAR
2 in fetal colon compared to adult colon. In addition, the anti-PPAR
antiserum provided by Interchim recognizes both human PPAR
1 and PPAR
2, as attested by the manufacturer.
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PPAR Expression
The average immunohistochemical intensity values, as estimated by two independent investigators in four tissue sections from different fetuses, are summarized in Table 1 for the different PPARs. No immunoreactivity was found in control sections when the primary antibody was omitted or replaced by preimmune serum (not shown).
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Fig 5A shows that the PPAR subtype was expressed as early as 7WD in the stratified columnar epithelium. At this stage, the tissue exhibited a cytoplasmic and nuclear staining. At 14WD ( Fig 5B), a lower intensity of fluorescence was observed for this tissue. The decrease was much more pronounced at 20WD because the immunoreaction was mainly restricted to the nuclei of epithelial cells (Fig 5C). Faint staining was observed in the nuclei of mesenchymal and muscular cells throughout development of the fetal esophagus.
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At 12WD the gastric epithelium showed the highest labeling with the anti-PPAR antibody compared with the intensity of fluorescence in the extra-epithelial layers. The PPAR
protein was detected in the surface epithelial cells as well as in the epithelial growing buds (Fig 5D). At 15WD no significant change was noted in PPAR
expression (Fig 5E). Four weeks later the staining was barely detectable in the gastric epithelial cells ( Fig 5F). PPAR
was faintly detected at 7WD in the stratified jejunal epithelium (Fig 5G ). At 12WD PPAR
expression remained very low in the villous jejunum (Fig 5H) but was higher in ileum ( Fig 5J). No immunoreaction was detected at 16WD in the jejunal epithelium (Fig 5I). Meanwhile, PPAR
was well expressed in the ileal tissue (Fig 5K). At 22WD (Fig 5L) PPAR
expression was high, particularly in nuclei of ileal cells. At 8WD (Fig 5M) the colon was a simple tube with a slit-like lumen composed of stratified epithelium surrounded by mesenchyme. At this stage the PPAR
subtype was moderately expressed in the epithelial cells (Fig 5M). Nuclei of mesenchymal cells were also stained by the antibody. At 14WD (Fig 5N) the luminal surface of the colon exhibited well-formed villi in which staining was slightly decreased. As mucous goblet cells differentiated, they produced secretory granules in which the PPAR
was not detected. At 20WD (Fig 5O ), the villous structures were present in the different segments of the colon. The specialized cells facing the colon lumen were stained. However, the intensity of fluorescence was faint and diffuse.
PPARß Expression
Between 7WD (Fig 6A) and 14WD (Fig 6B), the PPARß subtype was substantially expressed in the cytoplasm and nucleus of human esophageal epithelial cells. A marked decrease was observed in the intensity of immunoreaction at 20WD (Fig 6C). Whatever the developmental stage examined, PPARß was detected overall in the gastric epithelium ( Fig 6D6F). A peak in fluorescence intensity was observed at 15WD (Fig 6E; Table 1). At later stages the staining was more restricted to nuclei (Fig 6F).
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In the small intestine, the anti-PPARß antibody showed stronger staining in the ileum (Fig 6J6L) than in the jejunum (Fig 6G6I) at all stages studied. The staining in the epithelial cells remained moderate and high throughout development of the jejunum and ileum, respectively. At 22 WD the intensity of fluorescence was stronger in the ileal crypt epithelial cells than in the differentiated villous cells (Fig 6L).
The PPARß subtype was well expressed in the different layers of the human fetal colon at 8WD (Fig 6M) and 20WD ( Fig 6O). The anti-PPARß antibody was located mainly in the nuclei of the epithelial and mesenchymal cells. As for the ileum, stronger staining was observed in the crypt regions. However, it was barely detected at 14WD (Fig 6N ).
PPAR Expression
The different antibodies (anti-PPAR, anti-PPAR
1 /
2, and anti-PPAR
2 antisera) used gave similar results for their distribution throughout the development of the human fetal digestive tract. However, the immunoreactivity was always higher with the anti-PPAR
2 antibody compared to that observed with the two other antibodies. One can explain this difference by a higher level of immunoglobulins in the anti-PPAR
2 antiserum. Therefore, only results obtained with the anti-PPAR
2 antiserum are presented here (Table 1). In addition, the presence of mRNA encoding PPAR
2 was confirmed in intestinal extracts from human fetuses by nuclease S1 protection assay ( Fig 7).
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The presence of the PPAR2 protein was detected at 7WD ( Fig 8A), 14WD (Fig 8B ), and 20WD (Fig 8C) in esophagus. Throughout human fetal esophageal development, the staining was moderate or high and was restricted to nuclei of both epithelial and extra-epithelial cells.
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Expression of PPAR2 was lower in the gastric epithelium ( Fig 8D8F) than in the esophageal tissue. A slight increase was noted at 15WD (Fig 8E ).
Staining with the anti-PPAR2 antibody was particularly prominent in epithelial cell nuclei of jejunum (Fig 8G8I) and ileum (Fig 8J8L). Because nuclei were localized in the basal part of epithelial cells, PPAR
2 staining showed a spotted distribution along the basal plasma membrane. Owing to the abundance of nuclei, the intensity of fluorescence appeared higher in the crypt regions compared with the immunoreactivity in the upper villous regions (Fig 8I, Fig 8K, and Fig 8L).
At 8 WD (Fig 8M) and 14WD (Fig 8N), most nuclei of colon epithelial and mesenchymal cells were labeled with the anti-PPAR2 antibody. At 20WD the immunoreaction was higher and was more restricted to nuclei of specialized cells facing the colon lumen (Fig 8O ).
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Discussion |
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The results described here establish for the first time the presence and the spatiotemporal distribution of three PPAR subtypes in the developing human fetal digestive tract.
Expression of PPARs in Human Digestive Tract
With the use of polyclonal antibodies specifically directed against PPAR, PPARß, and PPAR
(
1/
2 and
2), our immunohistochemical data show that the different PPAR subtypes are expressed as early as 7WD in human fetal digestive tract and in different cell types of endodermal and mesodermal origin. The presence of PPAR
, PPARß, and PPAR
has already been reported in adult rat esophagus, stomach, small intestine and colon by in situ hybridization (
and PPARß in adult mouse colon compared to small intestine. In contrast, PPAR
expression was higher in the intestinal mucosa (
is reported to be also expressed in human (
1 and
2 proteins are detected in fat tissues, it is believed that expression of the PPAR
2 isoform remains mainly adipocyte-specific, whereas the PPAR
1 isoform expression may be extra-adipocytic. However, in two recent studies the presence of the two PPAR
isoforms is obvious in human (
1 and PPAR
2. Our data from immunohistochemical, Western blotting, and nuclease S1 protection assays agree with these results. They are somewhat at variance with those of
and PPARß mRNA, but not PPAR
mRNA, in rat fetal intestine. The discrepancy probably reflects differences in models examined or in timing of functionality of the gut in fetal life, or in the techniques used, as we mostly analyzed protein levels.
Spatiotemporal Distribution of PPARs
PPARs are expressed at different levels in cell types of endodermal and mesodermal origin during development of the human fetal digestive tract.
At early stages (7-15WD) of esophageal and stomach development, PPAR and PPARß are more localized in the cytoplasm than in the nucleus, whereas at later stages they become predominantly nuclear. This expression pattern overlaps for the esophagus, with replacement of the columnar ciliated epithelium by adult squamous tissue exhibiting flattened cells with microvillous processes in their apical membrane (
is involved in lipid metabolism and homeostasis (
expression in knockout mice prevents the PP inducibility of genes encoding peroxisomal and microsomal lipid-metabolizing enzymes (
expression in the esophagus and stomach suggests the involvement of this subtype in establishment of epithelial lipid metabolism. The decrease of PPARß expression in the esophageal epithelium is likely due to a shift in the physiology of this tissue. A role of PPARß in the onset of gastric cell differentiation is possible because
is predominantly nuclear throughout esophageal and stomach development. This subtype is well expressed during esophageal morphogenesis, suggesting a role for PPAR
during this process. On the other hand, PPAR
expression is low or moderate during stomach formation.
In the intestine as a whole and whatever the fetal stage examined, the different PPAR subtypes are more expressed in ileum and, to a lesser extent, in colon than in duodenum. The different spatiotemporal expression of PPAR, PPARß, and PPAR
during development of the human fetal intestine and their ligand specificity (
ligands induce the expression of genes involved in lipid absorption and transport in the rat small intestine (
ligands have been shown to inhibit proliferation and to induce differentiation of human colon cancer cells (
is expressed along the intestinal cryptvillous region in both proliferating and differentiated cells. At present, it is difficult to speculate about the precise role played by PPAR
in intestinal cell life.
In summary, the spatiotemporal distribution of the PPAR subtypes has been described during development of the human fetal digestive tract. The different PPARs are predominantly expressed in epithelial cells, although their presence is also detected in nuclei of cells of mesodermal origin. The three PPAR subtypes exhibit different patterns of expression in relation to the morphogenesis of the digestive tract. They are expressed very early, suggesting that these receptors play major roles in the development and/or the physiology of the digestive tract. Furthermore, the fact that PPAR is expressed at a high level whatever the region considered (except the stomach) and the stage studied argues for a prominent role of this receptor in human digestive tract.
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Acknowledgments |
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Supported by the Association de la Recherche contre le Cancer (Contrat ARC no. 9233), the Ligue contre le Cancer (Comité de Meuthe et Moselle), the Fondation de la Recherche Médicale (Comité de Lorraine), and the Conseil de Recherches Médicales du Canada.
We are grateful to W. Wahli (University of Lausanne) for the mPPAR/pSG5, mPPARß/pSG5, mPPAR
1/pSG5, and hPPAR
2/pBSIIKS+ plasmids, to M. Donner (UPRES 2402, Nancy) for the 3T3 L1 cells, and to A. Stoekel for her skillful assistance.
Received for publication August 9, 1999; accepted January 5, 2000.
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