Journal of Histochemistry and Cytochemistry, Vol. 48, 603-612, May 2000, Copyright © 2000, The Histochemical Society, Inc.


ARTICLE

Differential Expression of Peroxisome Proliferator-activated Receptors (PPARs) in the Developing Human Fetal Digestive Tract

Cécile Huina, Lina Corriveaub, Arnaud Bianchia, Jean Marie Kellera, Philippe Colleta, Pascaline Krémarik-Bouillauda, Lionel Domenjouda, Philippe Bécuwea, Hervé Schohna, Daniel Ménardb, and Michel Dauçaa
a Laboratoire de Biologie Cellulaire du Développement, EA 2402 "Proliférateurs de Peroxysomes," Faculté des Sciences, Vandoeuvre-les-Nancy, France
b Groupe CRM de Recherche sur le Développement Fonctionnel et la Physiopathologie du Tube Digestif, Département Anatomie et Biologie Cellulaire, Faculté de Médecine, Sherbrooke, Québec, Canada

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.


  Summary
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We investigated the spatiotemporal distributions of the different peroxisome proliferator-activated receptor (PPAR) isotypes ({alpha}, ß, and {gamma}) 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{gamma}, are expressed as early as 7 weeks of development in cell types of endodermal and mesodermal origin. The presence of PPAR{gamma} was also found by Western blotting and nuclease-S1 protection assay, confirming that this subtype is not adipocyte-specific. PPAR{alpha}, PPARß, and PPAR{gamma} exhibit different patterns of expression during morphogenesis of the digestive tract. Whatever the stage and the gut region (except the stomach) examined, PPAR{gamma} 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 48:603–611, 2000)

Key Words: PPARs, development, differentiation, fetus, digestive tract


  Introduction
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Introduction
Materials and Methods
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Discussion
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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 Sorensen et al. 1998 ). To date, three isotypes of PPAR have been described in humans: {alpha} (Sher et al. 1993 ); NUC1, also called ß or {delta} (Schmidt et al. 1992 ); and {gamma} (Elbrecht et al. 1996 ; Fajas et al. 1997 ). There are three subtypes of PPAR{gamma} mRNA, transcribed from three different promoters, which give rise to two proteins, PPAR{gamma}1 and {gamma}2, as the protein encoded by PPAR{gamma}3 mRNA is indistinguishable from PPAR{gamma} 1 (Fajas et al. 1998 ).

In humans, PPAR{alpha} 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{gamma} predominates in adipose tissue, large intestine, and macrophages and monocytes (Mukherjee et al. 1994 ; Auboeuf et al. 1997 ; Marx et al. 1998 ; Ricote et al. 1998 ; Spiegelman 1998 ). Consistent with their localization, the PPAR subtypes play different roles. After binding to specific elements (PPREs), PPAR{alpha} regulates the transcription of several target genes involved in lipid metabolism and homeostasis (Desvergne and Wahli 1995 ; Wahli et al. 1995 ; Lemberger et al. 1996 ; Sorensen et al. 1998 ). PPAR{gamma} controls adipocyte ( Chawla et al. 1994 ; Tontonoz et al. 1995 ; Schoonjans et al. 1996 ; Spiegelman et al. 1997 ) and monocyte/macrophage ( Marx et al. 1998 ; Ricote et al. 1998 ; Tontonoz et al. 1998 ) differentiation. Until now, the precise role of PPARß has not yet been elucidated.

Detailed descriptions of the morphological and functional changes occurring during the development of the human gastrointestinal tract are available (Menard and Calvert 1991 ; Menard 1994 , Menard 1995 ; Montgomery et al. 1999 ). Several lines of evidence suggest an early role of the fetal digestive tract in fat digestion. We have reported that peroxisomes with fatty acid ß-oxidation capacity are present in the fetal human gut (Dauca et al. 1996 ). The latter exhibits functional mechanisms to synthesize all the lipid classes and to secrete them in the form of lipoproteins ( Levy et al. 1992 ; Basque et al. 1998 ). Thus far, no studies have been devoted to the expression of PPARs in the developing human fetal digestive tract. Because the localization of these receptors may help to clarify the physiological roles of PPARs, this analysis was undertaken by immunohistochemistry in esophagus, stomach, small intestine, and colon of human fetuses from Week 7 to Week 23 of development (WD). In this study we demonstrated a spatial and temporal distribution of PPARs in developing digestive tract that suggests a differential role for these receptors during morphogenesis and cell differentiation.


  Materials and Methods
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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{alpha} antibody was raised against the amino acid sequence 45SSGSFGFTEYQY56 of human PPAR{alpha} (Sher et al. 1993 ). The sequence 24EGAPELNGGPQHAL37 of human NUC1 (Schmidt et al. 1992 ) was used to produce the anti-PPARß polyclonal antibody. The anti-PPAR{gamma} 1/{gamma}2 antiserum was raised against the amino acid sequence EMPFWPTNFGISSVD common to PPAR{gamma}1 and PPAR{gamma} 2 [amino acids 5–19 of mouse PPAR{gamma}1 ( Zhu et al. 1993 ); 35–49 of PPAR{gamma}2 ( Tontonoz et al. 1994 )]. The sequence is well conserved in the two human PPAR{gamma} isoforms (Elbrecht et al. 1996 ; Fajas et al. 1997 ). Taking advantage of the fact that the PPAR{gamma}2 differs from the PPAR{gamma}1 by an additional specific N-terminal amino acid region, the sequence of the hapten used to produce the anti-human PPAR{gamma}2 antibody was mapped at that region and corresponded to 2GETLGDSPIDPESDS16 of human PPAR{gamma}2 (Elbrecht et al. 1996 ; Fajas et al. 1997 ). The synthetic peptides were coupled to keyhole limpet hemocyanin as a carrier according to the glutaraldehyde method (Avrameas 1969 ). Polyclonal antibodies were raised by SC injections into rabbits using standard procedures. In addition, we used a commercial anti-PPAR{gamma} antiserum (Interchim; Montluçon, France) directed against the amino acid sequence MMGEDKIKFKHITPL common to PPAR{gamma}1 and PPAR{gamma}2 (amino acids 256–270 of human PPAR{gamma}1, 284–298 of human PPAR{gamma}2 ).



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Figure 1. Schematic comparison of the different human PPAR subtypes vs PPAR{alpha}. The positions of the peptides used to produce the polyclonal antibodies are mapped. The DNA (DBD) and ligand (LBD) binding domains are represented.

Characterization of the Antibodies
The polyclonal antibodies produced were characterized by immunoprecipitation and Western blotting assays. In vitro transcription and translation of mouse PPAR{alpha}/pSG5, PPARß/pSG5, PPAR{gamma}1 /pSG5, and human PPAR{gamma}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 ( Bradford 1976 ). Samples were analyzed by Western blotting and ECL according to the manufacturer's protocol (Boehringer Mannheim Biochemica; Mannheim, Germany) using the different antibodies.

Nuclease Protection Assay
Partial human PPAR{gamma}2 cDNA corresponding to the 5'UTR sequence (Fajas et al. 1997 ) was obtained by standard RT-PCR using total RNA extracted from human adipocytes ( Chomzynski and Sacchi 1987 ) and primers up (5'-CCCATCTCTCCCAAATATTT-3') and down (5'-GGGCCAGAATGCGATCTCTGTG-3'). The resulting fragment (282 bp) was cloned into the pBSIIKS+ plasmid (Stratagene; La Jolla, CA), giving rise to the pBIIKS+/hPPAR{gamma}2 vector. A pBIIKS+/hG3PDH clone containing a 380-bp DNA fragment of the human G3PDH encoding sequence (Tso et al. 1985 ) was produced using the same protocol and primers up (5'-CCCATCACCATCTTCCGA-3') and down (5'-CTACAGGCCACAGTTTCC-3'). Nuclease protection assay was carried out according to Sambrook et al. 1989 . Total RNA was extracted from human fetal intestines (14WD) and from 3T3 L1 cells (passage 58) as described above. 3T3 L1 cells were chosen for a positive control because those preadipocytes express PPAR{gamma}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 PBS–BSA 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 Reichert–Jung Polyvar microscope (Vienna, Austria).


  Results
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Materials and Methods
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Antibody Specificity
In vitro-translated mouse PPAR{alpha}, ß, and {gamma}1 and human PPAR{gamma}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{gamma}2 in fetal colon compared to adult colon. In addition, the anti-PPAR{gamma} antiserum provided by Interchim recognizes both human PPAR{gamma}1 and PPAR{gamma}2, as attested by the manufacturer.



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Figure 2. Recognition of the different PPAR subtypes by their respective antibody. (A) SDS-PAGE analysis of in vitro-translated mouse PPAR{alpha}, ß, and {gamma}1 and human PPAR{gamma}2. Two µl of lysate was loaded in each lane and analyzed by SDS-PAGE on 10% gels. Dried gels were exposed for autoradiography. (B) Immunoprecipitation assay with in vitro-translated mouse PPAR{alpha}, ß, and {gamma}1 and human PPAR{gamma}2. Translation product was incubated with the appropriate primary anti-PPAR antibody (diluted 1:250). The complexes were immunoprecipitated using protein A–Sepharose, then analyzed by SDS-PAGE with 9000 cpm for PPAR{alpha} and PPAR{gamma}2, 13000 cpm for PPARß, and 16000 cpm for PPAR{gamma}1. Exposure was 7 days for PPAR{gamma}2 and 27 days for the other PPARs.



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Figure 3. Specificity of anti-PPAR antibodies by Western blotting. Mouse PPAR{alpha}/pSG5, PPARß/pSG5, PPAR{gamma}1/pSG5, and human PPAR{gamma}2/pBSIIKS+ plasmids were in vitro-translated using reticulocyte lysate and L-[35 S]-methionine. Translated products were submitted to SDS-PAGE (10%). The gels were either subjected to autoradiography (*) or processed by Western blotting and ECL using the anti-PPAR antibody (diluted 1:500) indicated above the different lanes.



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Figure 4. Western blotting analysis of PPAR{alpha}, ß, and {gamma}2 expression in human fetal (A) and adult (B) colon mucosae. Fifty µg of protein was run on 15% SDS-PAGE, then transferred onto a PVDF membrane. Immunoreactivity of the different PPAR subtypes was detected by incubation of the membrane with the appropriate primary antibody (diluted 1:1000 for anti-PPAR{alpha} and anti-PPARß antibodies and 1:5000 for anti-PPAR{gamma}2 antibody). The final reaction was detected by ECL.

PPAR{alpha} 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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Table 1. Differential expression of PPAR{alpha}, PPARß, and PPAR{gamma}2 during fetal development of the human digestive tract a

Fig 5A shows that the PPAR{alpha} 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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Figure 5. Differential expression of PPAR{alpha} in developing human fetal digestive tract by immunohistochemistry. (A–C) Transverse esophageal sections at 7WD (A), 14WD (B), and 20WD (C), showing expression of the PPAR isotype mainly in the epithelial cells. ( D–F) Gastric sections at 12WD (D), 15WD (E), and 19WD (F). Expression of PPAR{alpha} is detected in the cytoplasm of the gastric epithelial cells. (G–L) Small intestine. Expression of the PPAR subtype is faint in jejunum (G, 7WD; H, 12WD; I, 16WD) and higher in ileum (J, 12WD; K, 16WD; L, 22WD). The presence of PPAR{alpha} is also detected in the epithelial colon cells (M–O) at 8WD ( M), 14WD (N), and 20WD (O). Bar = 75 µm.

At 12WD the gastric epithelium showed the highest labeling with the anti-PPAR{alpha} antibody compared with the intensity of fluorescence in the extra-epithelial layers. The PPAR{alpha} 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{alpha} expression (Fig 5E). Four weeks later the staining was barely detectable in the gastric epithelial cells ( Fig 5F). PPAR{alpha} was faintly detected at 7WD in the stratified jejunal epithelium (Fig 5G ). At 12WD PPAR{alpha} 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{alpha} was well expressed in the ileal tissue (Fig 5K). At 22WD (Fig 5L) PPAR{alpha} 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{alpha} 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{alpha} 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 6D–6F). 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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Figure 6. Differential expression of PPARß in the developing human fetal digestive tract by immunohistochemistry. (A–C) Esophagus at 7WD (A), 14WD (B), and 20WD (C). A decrease in PPARß expression is observed at 20WD. (D–F) Stomach at 12WD (D), 15WD (E), and 19WD (F). (G–I) Jejunum. Throughout jejunum morphogenesis (G, 7WD; H, 12WD; I, 16WD), PPARß is detected in nuclei of mesenchymal and epithelial cells. However, the intensity of fluorescence is lower than that observed during ileal development (J, 12WD; K, 16WD; L, 22WD). (M–O) Colon (M, 8WD; N, 14WD; O, 20WD). The immunoreactivity is mainly detected in nuclei of epithelial and mesenchymal cells. Note that the reaction is absent in mucous secretory granules. Bars: A J,MO = 75 µm; K, L = 120 µm.

In the small intestine, the anti-PPARß antibody showed stronger staining in the ileum (Fig 6J–6L) than in the jejunum (Fig 6G–6I) 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{gamma} Expression
The different antibodies (anti-PPAR{gamma}, anti-PPAR{gamma}1 /{gamma}2, and anti-PPAR{gamma}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{gamma}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{gamma}2 antiserum. Therefore, only results obtained with the anti-PPAR{gamma}2 antiserum are presented here (Table 1). In addition, the presence of mRNA encoding PPAR{gamma}2 was confirmed in intestinal extracts from human fetuses by nuclease S1 protection assay ( Fig 7).



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Figure 7. Detection of PPAR{gamma}2 mRNA in human fetal intestine (14WD) by nuclease protection assay. 32P-labeled DNA probes to detect either G3PDH or PPAR{gamma}2 mRNA were prepared as described in Materials and Methods. 32P-labeled G3PDH (Lane 1) and PPAR{gamma} 2 (Lane 2) DNA probes (105 cpm) were run on a 5% polyacrylamide gel. Total RNA (5 µg) isolated from 3T3 L1 cells (Lane 3) or from human fetal intestine (14WD) (Lane 4) were hybridized with the two radiolabeled probes. After nuclease S1 digestion, the resulting products were electrophoresed and the gels were autoradiographed. The presence of PPAR{gamma}2 mRNA is obvious in the two models examined.

The presence of the PPAR{gamma}2 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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Figure 8. Differential expression of PPAR{gamma}2 in developing human fetal digestive tract by immunohistochemistry. (A–C) Esophagus (A, 7WD; B, 14WD; C, 20WD). The PPAR{gamma} isotype is expressed throughout fetal esophageal development. ( D–F) Stomach at 12WD (D), 15WD (E), and 19WD ( F), showing immunoreactivity in nuclei of growing pit cells. ( G–I) Jejunum (G, 7WD; H, 12WD; I , 16WD). (J–L) Ileum (J, 12WD; K, 16WD; L, 22WD). (M–O) Colon (M, 8WD; N, 14WD; O, 20WD). In the small intestine and colon, PPAR{gamma} exhibits a nuclear localization in both mesenchymal and epithelial cells. In the epithelium, PPAR{gamma} is expressed in the different regions of the crypt–villous axis. Bars: A J,MO = 75 µm; K, L = 120 µm.

Expression of PPAR{gamma}2 was lower in the gastric epithelium ( Fig 8D–8F) than in the esophageal tissue. A slight increase was noted at 15WD (Fig 8E ).

Staining with the anti-PPAR{gamma}2 antibody was particularly prominent in epithelial cell nuclei of jejunum (Fig 8G–8I) and ileum (Fig 8J–8L). Because nuclei were localized in the basal part of epithelial cells, PPAR{gamma}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-PPAR{gamma}2 antibody. At 20WD the immunoreaction was higher and was more restricted to nuclei of specialized cells facing the colon lumen (Fig 8O ).


  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{alpha}, PPARß, and PPAR{gamma} ({gamma}1/{gamma}2 and {gamma}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{alpha}, PPARß, and PPAR{gamma} has already been reported in adult rat esophagus, stomach, small intestine and colon by in situ hybridization (Braissant et al. 1996 ). In another recent study, quantification of PPAR mRNA by ribonuclease protection assay revealed relatively high expression of PPAR{gamma} and PPARß in adult mouse colon compared to small intestine. In contrast, PPAR{alpha} expression was higher in the intestinal mucosa (Mansen et al. 1996 ). Furthermore, PPAR{gamma} is reported to be also expressed in human ( Brockman et al. 1998 ; Sarraf et al. 1998 ) and rodent (Lefebvre et al. 1998 ; Saez et al. 1998 ) colon tumor cells. Although both PPAR{gamma} 1 and {gamma}2 proteins are detected in fat tissues, it is believed that expression of the PPAR{gamma}2 isoform remains mainly adipocyte-specific, whereas the PPAR{gamma}1 isoform expression may be extra-adipocytic. However, in two recent studies the presence of the two PPAR{gamma} isoforms is obvious in human ( Dubois et al. 1998 ) and rodent ( Lefebvre et al. 1998 ) colon cells as shown by the results obtained in Western blotting using polyclonal antibodies reactive with both PPAR{gamma}1 and PPAR{gamma}2. Our data from immunohistochemical, Western blotting, and nuclease S1 protection assays agree with these results. They are somewhat at variance with those of Braissant and Wahli 1998 , who detected by in situ hybridization the presence of PPAR{alpha} and PPARß mRNA, but not PPAR{gamma} 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{alpha} 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 ( Johns 1952 ). For the stomach, it coincides with the acquisition of adult features, characterized by differentiation of gastric cells appearing at the base of the evolving pits ( Salenius 1962 ), followed by differentiation of enterochromaffin cells and mucous neck cells (De Lomos 1977 ). PPAR{alpha} is involved in lipid metabolism and homeostasis ( Wahli et al. 1995 ; Schoonjans et al. 1996 ). Indeed, the absence of PPAR{alpha} expression in knockout mice prevents the PP inducibility of genes encoding peroxisomal and microsomal lipid-metabolizing enzymes (Lee et al. 1995 ). The early onset of PPAR{alpha} 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 Braissant and Wahli 1998 have found a correlation between the peak of ubiquitous expression of PPARß during mouse embryogenesis and the period of greatest cell differentiation. Our results also show that PPAR{gamma} is predominantly nuclear throughout esophageal and stomach development. This subtype is well expressed during esophageal morphogenesis, suggesting a role for PPAR{gamma} during this process. On the other hand, PPAR{gamma} 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{alpha}, PPARß, and PPAR{gamma} during development of the human fetal intestine and their ligand specificity ( Sorensen et al. 1998 ) suggest that these receptors are involved in different intestinal functions. PPAR{alpha} ligands induce the expression of genes involved in lipid absorption and transport in the rat small intestine (Martin et al. 1997 ; Motojima et al. 1998 ). The physiological role of PPARß in the small and large intestine remains unknown. PPAR{gamma} ligands have been shown to inhibit proliferation and to induce differentiation of human colon cancer cells (Brockman et al. 1998 ; Sarraf et al. 1998 ). On the other hand, the same ligands enhance colon polyp and tumor formation in the min/+ mouse model ( Lefebvre et al. 1998 ; Saez et al. 1998 ). It is evident from our results that PPAR{gamma} is expressed along the intestinal crypt–villous region in both proliferating and differentiated cells. At present, it is difficult to speculate about the precise role played by PPAR{gamma} 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{gamma} 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.


  Acknowledgments

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{alpha}/pSG5, mPPARß/pSG5, mPPAR{gamma}1/pSG5, and hPPAR{gamma}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.


  Literature Cited
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Summary
Introduction
Materials and Methods
Results
Discussion
Literature Cited

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