Subepithelial fibroblast cell lines from different levels of gut axis display regional characteristics

Michelina Plateroti1, Deborah C. Rubin2, Isabelle Duluc1, Renu Singh2, Charlotte Foltzer-Jourdainne1, Jean-Noël Freund1, and Michèle Kedinger1

1 Institut National de la Santé et de la Recherche Médicale Unité 381, 67200 Strasbourg, France; and 2 Washington University School of Medicine, St. Louis, Missouri 63110

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The intestine is characterized by morphofunctional differences along the proximodistal axis. The aim of this study was to derive mesenchymal cell lines representative of the gut axis. We isolated and cloned rat intestinal subepithelial myofibroblasts raised from 8-day proximal jejunum, distal ileum, and proximal colon lamina propria. Two clonal cell lines from each level of the gut were characterized. They 1) express the specific markers vimentin, smooth muscle alpha -actin, and smooth muscle myosin heavy chain, revealed by immunofluorescence microscopy and 2) distinctly support endodermal cell growth in a coculture model, depending on their regional origin, and 3) the clones raised from the various proximodistal regions maintain the same pattern of morphogenetic and growth and/or differentiation factor gene expression as in vivo: hepatocyte growth and/or scatter factor and transforming growth factor-beta 1 mRNAs analyzed by RT-PCR were more abundant, in the colon and ileal clones and mucosal connective tissue, respectively. In addition, epimorphin mRNA studied by Northern blot was also the highest in one ileal clone, in which it was selectively upregulated by all-trans retinoic acid (RA) treatment. Epimorphin expression in isolated 8-day intestinal lamina propria was higher in the distal small intestine and proximal colon than in the proximal small intestine. In conclusion, we isolated and characterized homogeneous cell subtypes that can now be used to approach the molecular regulation of the epithelium-mesenchyme-dependent regional specificity along the gut.

intestinal mesenchyme cell lines; proximodistal axis; epithelium-mesenchyme interactions; growth and/or differentiation factors

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

THE GUT IS CHARACTERIZED by a proximodistal (PD) gradient in its form and function, assessed by important differences in the expression of terminal differentiation markers along the duodenum to the colon axis (22, 23, 28). Intestinal morphogenesis and differentiation is achieved by the cross talk between the epithelium and the underlying mesenchyme during both fetal and adult animal life (for reviews see Refs. 5, 52, and 60). Mesenchyme supports growth and differentiation of the adjacent epithelia that cannot undergo normal development in the absence of mesenchyme-derived cells (37). In addition, mesenchyme-epithelium signaling plays a role in the establishment of regional specificity during intestinal development. This is exemplified by association-grafting experiments in which the mesenchyme dictates the form of the organ (10, 29) and in some cases the differentiation fate of the associated endoderm (10, 21, 60).

During intestinal development, the fetal mesenchyme differentiates into various structures: the outer muscle layers, the submucosal connective tissue, and the lamina propria or mucosal connective tissue. Various fibroblast cell phenotypes can be observed in vivo in the lamina propria as subepithelial myofibroblasts (for reviews see Refs. 48 and 59) that form a regular network of stellate cells (32). Despite the role attributed to the mesenchyme in epithelial differentiation and regionalization along the gut axis and in mucosal immunophysiology, little is known about individual characteristics of the various phenotypic subtypes. As an example, subepithelial fibroblasts have been shown to express, or not express, intercellular adhesion molecule 1, according to their localization beneath the follicle-associated epithelium or the villus epithelium, respectively (16).

Attempts to define the molecular nature of the cell interactions involved in epithelium cell differentiation have emphasized the role of basement membrane (BM) molecules produced by both the epithelium and mesenchyme compartments (53), cell surface-associated molecules, and soluble factors (5). To analyze the specific microenvironment originating from the mesenchyme cells, we focused our attention on the expression of signal molecules other than BM components potentially involved in mesenchyme-epithelium cross talk. Among these latter components, epimorphin and hepatocyte growth and/or scatter factor (HGF/SF), expressed by mesenchyme cells in various organs, have been described as potential candidates in the process of morphogenesis and differentiation (24, 46). HGF/SF is a heparin-binding glycoprotein consisting of a 60-kDa alpha -chain and a 30-kDa beta -chain. Its action as mitogen, morphogen, and scatter factor is mediated by the c-met protooncogene, a transmembrane tyrosine kinase receptor predominantly present on epithelial cells (46). The presence of both HGF/SF and c-met in the intestinal mucosa has been described during mouse development (54). Epimorphin, also known as syntaxin-2, is a 150-kDa membrane-bound protein described as a modulator of epithelial morphogenesis in embryonic skin and lung epithelia. It is expressed in 17-day mouse and rat embryonic kidney and intestine (Ref. 24 and D. C. Rubin, unpublished observations). The mechanism involved in epimorphin signaling is not known. It may play a role in targeting secretory vesicles to the appropriate membrane compartment (2, 18). Transforming growth factor-beta 1 (TGF-beta 1) is a homodimeric protein of 25 kDa and a member of the multifunctional TGF-beta proteins involved in morphogenesis, differentiation, proliferation, and immunomodulation (41). It is present in the intestinal mucosa, where it controls extracellular matrix synthesis and remodeling (41) and inhibits epithelial proliferation (31). Its biological activity is mediated by transmembrane receptors (38).

Retinoic acid (RA) and its analogs act as morphogens and differentiation inducers in many organs (20, 34). Our previous results indicated that their effect on intestinal epithelial cell differentiation requires heterologous cell contact and is mediated by the mesenchyme cells (40). Although the synthesis and deposition of BM molecules are modified under the influence of RA, it has not been analyzed whether other molecules secreted or expressed on the membrane surface are also involved in this signaling.

The aim of our work was to design a reproducible and well-defined in vitro model to approach the molecular regulation of epithelium-mesenchyme-dependent region-specific properties of the gut. For this purpose, we isolated and cloned subepithelial myofibroblasts from 8-day intestinal lamina propria raised from the proximal jejunum (PJ), the distal ileum (DI), and the proximal colon (PC). We analyzed the expression of HGF/SF, TGF-beta 1, and epimorphin and their regulation by RA treatment. We also recorded the ability of the different clones to support endodermal cell growth in cocultures. For this study, two clones from each PD level of the gut have been used. The results indicated that the PD expression pattern of HGF/SF, TGF-beta 1, and to some extent, of epimorphin, was in accordance with their regional origin: the highest level of HGF/SF mRNA was found in colon derivatives whereas TGF-beta 1 and epimorphin (in 1 of the 2 cell lines considered) mRNAs were more abundant in the ileal derivatives. In addition, epimorphin expression was selectively upregulated by RA treatment in one ileal clone.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Animals

Wistar rats were from our breeding colony and were used at 8 and 15 days after birth as well as at an adult stage. Rat fetuses were delivered by cesarean section at 14 days of gestation (existence of the vaginal plug was designated day 0).

Mesenchyme-Derived Cell Isolation, Primary Cultures, and Cloning

Eight-day rat intestinal tubes were dissected out from the ligament of Treitz to the rectum. The first (PJ) and last (DI) one-fourth of the small intestine (up to the cecum) were used. The PC, characterized by the presence of slanting parallel folds, was also recovered. Each part was then treated separately. The subepithelial mesenchyme cells were isolated and cloned as previously described (36) according to the technique derived from Evans et al. (12). Briefly, 8-day postnatal rat intestinal segments (PJ, DI, and PC) pooled from three animals were first incubated for 10 min with 300 U/ml collagenase XI (Sigma, Saint Quentin Fallavier, France) and 0.1 mg/ml dispase (Boehringer Mannheim, Meylan, France) in Hank's balanced salt solution (HBSS; GIBCO BRL, Cergy-Pontoise, France). The tissues were then cut into small fragments. After a low-speed centrifugation (200 g for 10 min), the supernatant containing isolated cells derived from the lamina propria was discarded; the pellet, which contained mainly intact organoids with attached subepithelial fibroblasts, was mechanically triturated by pipetting. The explants were washed five times in DMEM (GIBCO BRL)-2% sorbitol to eliminate isolated cells, seeded in culture dishes, and cultured in DMEM supplemented with 10% FCS (GIBCO-BRL), 0.25 U/ml insulin (Sigma), 10 µg/ml transferrin (Sigma), 20 ng/ml epidermal growth factor (EGF; Sigma), and 40 µg/ml of gentamycin (Shering-Plough, Segré, France). After 4 days, mesenchyme-derived cells were passaged using 0.01% trypsin (GIBCO BRL)-2 mM EDTA treatment; under these conditions, epithelial cells do not survive. The three populations of subepithelial myofibroblasts (PJ, DI, and PC) were subcultured four to five times and then cloned two times using the dilution limit technique. Several cell lines, named mesenchyme-derived intestinal cell lines (MIC), were obtained. For RA treatment, 10-8 M all-trans RA (Sigma) was added to the culture medium for 48 h; the same volume of solvent was added in control dishes.

Freshly Isolated Lamina Propria From 8-Day Rat Intestines

After dissection of the muscular tissue, the epithelial layer was separated from the lamina propria by incubating the PJ, DI, and PC fragments in 5 mM EDTA solution for 15 min at 37°C. The two components were then mechanically separated under a microscope. All tissues were immediately frozen in liquid nitrogen and stored at -80°C until use for RNA extraction. The purity of the lamina propria preparation was assessed by RT-PCR analysis using specific primers for intestinal fatty acid binding protein (I-FABP) mRNA detection (this mRNA is exclusively expressed by the epithelial cells; see Ref. 47).

Coculture Experiments

MIC cell lines were used for coculture experiments with endodermal microexplants prepared from the PJ of 14-day rat fetuses. Briefly, the dissected fetal intestines were incubated in 0.03% collagenase A (Boehringer Mannheim) in CMRL medium (GIBCO BRL) for 1 h at 37°C and then in medium enriched in 50% newborn calf serum for 30 min at room temperature. The endoderm was then separated from the mesenchyme under a microscope. Small fragments of endoderm (<1 mm2) were seeded over confluent fibroblasts and maintained in coculture for 3 days in DMEM supplemented with 2.5% FCS, 0.25 U/ml insulin, 10 µg/ml transferrin, 20 ng/ml EGF, and 40 µg/ml of gentamycin. The mean surface of endodermal cells derived from isolated fragments cocultured on the various mesenchyme cell clones was measured under an inverted microscope. The statistical significance of the differences was evaluated using Student's t-test.

Immunofluorescence Analysis

Cells cultured on glass coverslips were fixed in 2% paraformaldehyde for 15 min at room temperature. Five-micrometer cryosections of 8-day and adult proximal small intestine were fixed in acetone for 10 min. Cells and tissue sections were incubated with the specific primary antibodies for 1 h. After three washes, they were incubated for 30-45 min with FITC-labeled sheep-anti-mouse IgG (Sanofi Diagnostic Pasteur, Marnes-la-Coquette, France) or FITC-labeled goat-anti-rabbit IgG (Nordic Immunological Laboratories, Capistrano Beach, CA). After they were mounted under coverslips in phenylene-PBS-diamine buffer, the preparations were observed under a Zeiss Axiophot fluorescence microscope (Zeiss, Thornwood, NY).

Antibodies. Monoclonal antibodies (MAb) to vimentin were from Amersham (Les Ulis, France), MAb to smooth muscle alpha -actin were from Sigma, and MAb to desmin were from Dako (Trappes, France). The polyclonal antibody to factor VIII-related antigen was from Sigma. The polyclonal antibody to smooth muscle myosin heavy chain was a generous gift from Dr. G. Gabbiani (3), and the monoclonal anti-SM22 antibody (clone 3E11) was a gift from Dr. S. Sartore (9).

Gene Expression Analysis

RNA extraction. RNA was extracted from cell cultures and tissues using Trizol reagent (GIBCO BRL) as recommended by the supplier. The RNA was used both for semiquantitative RT-PCR and Northern blot assays.

RNA analysis by RT-PCR. We analyzed HGF/SF, TGF-beta 1, and I-FABP gene expression by RT-PCR, normalized by glyceraldehyde-3-phosphate dehydrogenase (GAPDH) content in each sample. Single-stranded cDNAs were synthesized for 1 h at 42°C using 3 µg of RNA, 50 pmol oligo(dT)17, 1 mM of all four deoxynucleotide triphosphates, and 15 U of avian myeloblastosis virus reverse transcriptase (Promega, Charbonnieres, France) in 20 µl of 50 mM Tris · HCl pH 8.3, 50 mM KCl, 10 mM MgCl2, and 4 mM sodium pyrophosphate. The sequences of the primers (from Eurogentec, Serain, Belgium) used for PCR analysis were: HGF1 5'-ATGTTTTCCAGCCAGAAACAAAGA-3', HGF2 5'-AATGACACCAAGAACCATTCTCAT-3', TGFbeta 1 5'-GAAGTCACCCGCGTGCTAATGG-3', TGFbeta 2 5'-GTGTGTCCAGGCTCCAAATGTAGG-3', FABP1 5'-ATGAAGAGGAAGCTTGGAGCT-3', FABP2 5'-GGCCTCAACTCCATATGTGTA-3', GAPDH1 5'-GGCTGAGAACGGGAAGCTTGTGATCAATGG-3', and GAPDH2 5'-TGTCGCTGTTGAAGTCAGAGGAGACCACCT-3'. The primers were chosen on the basis of specific sequences that were previously published (19, 42, 49, 56).

PCR was carried out on the cDNA mixture 2 µl using 50 pmol of each oligonucleotide pair. In controls, the cDNA templates were either omitted or replaced by 0.3 µg of RNA. The reaction was performed in 100 µl of 75 mM Tris · HCl at pH 9, 20 mM (NH4)2SO4, 0.01% Tween 20, 1 mM MgCl2, 0.2 mM of each 2-deoxynucleotide-5'-triphosphate, and 1 U of Goldstar DNA polymerase (Eurogentec). PCR was performed in a Thermojet apparatus (Eurogentec) using the following conditions: 30 s at 94°C for denaturation; 45 s at 50°C for annealing in the case of HGF/SF, TGF-beta 1, and I-FABP primers and 60°C for GAPDH; 45 s of elongation at 72°C; and finally 5 min at 72°C. For HGF/SF, TGF-beta 1, I-FABP, and GAPDH, the PCR was conducted up to 32, 30, 32, and 25 cycles, respectively. Semiquantitative RT-PCR analysis was performed (specific mRNA vs. GAPDH mRNA as control). For every oligonucleotide pair and for every RNA species, a preliminary analysis was conducted to define the appropriate range of cycles consistent with an exponential increase of the amount of the DNA product. Ten microliters of the PCR products were separated by electrophoresis on 3% agarose gels, stained with ethidium bromide, and observed under ultraviolet illumination. The expected size of each DNA fragment was (in bp) 478 for HGF/SF, 684 for TGF-beta 1, 297 for I-FABP, and 685 for GAPDH. Densitometric analysis was performed using the Bio-Rad GS-700 imaging densitometer apparatus (Ivry sur Seine, France). The statistical significance of the differences between two parameters was evaluated using Student's t-test.

Every PCR fragment was inserted into the pGEM-T vector (Promega) and sequenced using the T7 sequencing kit (Pharmacia Biotech, Orsay, France).

Northern blot analysis. Epimorphin mRNA expression was analyzed by Northern blot using a rat epimorphin partial cDNA as probe (nt 600-949). Ten micrograms per lane of total RNA were electrophoresed in a formaldehyde-containing agarose gel and transferred to Hybond N (Amersham) nylon membrane overnight. The membrane was then prehybridized in 5× SSC solution containing 30% formamide, 5× Denhardt's solution, 0.5% SDS, and 150 µg/ml salmon sperm DNA. Prehybridization was performed at 42°C overnight. Hybridization was carried out in the same solution with the addition of 1 × 106 counts · min-1 · ml-1 of radiolabeled epimorphin cDNA probe at 42°C overnight. The probe was labeled with high-specific-activity [alpha -32P]dCTP by the random primer method (13). Posthybridization washes were performed in 1× SSC-0.1% SDS at room temperature and 0.1× SSC-0.1% SDS at 55°C. Membranes were exposed to Kodak Biomax film in a Biomax cassette at -70°C for 1 wk. The relative abundance of epimorphin mRNA in each sample was determined by analysis of digitized images with National Institutes of Health Image version 1.55 software (W. Rasband, National Institute of Mental Health, Bethesda, MD), obtained with a UMAX T-S-2400X scanner using Magiscan version 1.2 (UMAX Technologics, Fremont, CA). Results were normalized for differences in RNA loading by digitalized image analysis of 18S rRNA bands, visualized by ethidium bromide staining of the RNA gel.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Subepithelial Myofibroblasts: Isolation and Cloning

The method used for cell isolation allowed us to recover an enriched population of fibroblasts adherent to the epithelial cells known as subepithelial myofibroblasts. The strategic localization of these cells in the close vicinity of the epithelial cells indicates their potential role as effectors in the integrated epithelium-mesenchyme unit. Because of morphological and functional differences along the gut axis (23), we isolated myofibroblasts from different regions: PJ, DI, and PC.

Despite the morphological heterogeneity of the uncloned populations, all cells were vimentin positive, as expected for mesenchyme derivatives, but expressed various levels of smooth muscle alpha -actin, a myofibroblast-specific marker; only a few cells were desmin positive. Cells were cloned two times at passage 5 using the limit-dilution method; subcultures were done only from colonies derived from one cell. Almost 30 clones have been isolated for each intestinal segment (PJ, DI, and PC); however, there was a higher clonal efficiency starting from the ileal primary cultures, contrasting with a lower efficiency for the jejunal cells. The heterogeneity in cell shape (stellate, flat, elongated, and polygonal cells) among the different clones was similar for each level of the gut. These cell lines were subcultured at least 10-fold times and retained a stable phenotype; some of them are used at passage 25. For this study, we analyzed six representative clones, two for each intestinal region: MIC 101-1-derived and MIC 101-2-derived PJ, MIC 216 and 219 from DI, and MIC 307-1 and 316 from PC.

All selected clones expressed vimentin. A representative picture is shown in Fig. 1 for the clones MIC 101-2 (A), MIC 216 (B), and MIC 316 (C), also illustrating the difference in cell shape: MIC 101-2, 216, and 316 displayed, respectively, elongated, polygonal/epithelioid, and stellate morphology. In accordance with the heterogeneity observed in the uncloned population, the different clones displayed various immunofluorescence intensities of smooth muscle alpha -actin (Fig. 1D, representative illustration in MIC 101-2 cells). Similarly, every clone tested displayed a positive smooth muscle myosin staining with variations in the intensity among the clones identical to those of smooth muscle alpha -actin (Fig. 1E in MIC 101-2 cells). Interestingly, immunodetection of the various antigens on intestinal cryosections revealed that desmin (not shown), smooth muscle alpha -actin (not shown), and myosin heavy chain (Fig. 1, F and G), which are smooth muscle-specific markers, are also expressed in the subepithelial fibroblasts. This has already been reported by Richman et al. (44) for desmin and by Kedinger et al. (30) for smooth muscle alpha -actin (see also Ref. 48). In contrast, none of the six clones expressed either desmin or the SM22 antigen, which is a specific marker of smooth muscle layers (9), or factor VIII-related antigen, an endothelial cell marker (not shown). The phenotypic characteristics of the cell lines, as well as the technique used to prepare the primary cultures, bring substantial evidence that we have raised subepithelial myofibroblast clonal cell lines.


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Fig. 1.   Immunofluorescence staining for mesenchyme markers in mesenchyme-derived intestinal cell line (MIC) 101-2 (A, D, and E), MIC 216 (B), and MIC 316 (C). Cells represented in A are elongated, in B they are polygonal or epithelioid, and in C they are stellate. Cells cultured on coverslips were fixed in paraformaldehyde and labeled with antibodies against vimentin (A-C) and smooth muscle alpha -actin (D). Expression of smooth muscle myosin heavy chain is illustrated in MIC 101-2 cells (E) and in muscle layers and crypt zone of intestinal tissue (F and G, respectively). ml, External muscle layers; mm, muscularis mucosae; c, crypt epithelium. Arrows indicate subepithelial myofibroblasts in crypts. Bar = 30 (A-D), 38 (E), 69 (F), and 26 µm (G).

Clones Raised From Different Anteroposterior Levels of Gut Exhibit Differential Pattern of Growth and/or Differentiation Factor Expression Similar to Tissues In Vivo

To characterize the mesenchyme cells representative of the different levels of the gut, the expression of three growth, differentiation, and/or morphogenetic factors produced by two clonal cell lines per region was compared with the pattern expressed by the corresponding freshly isolated tissue from each PD segment. HGF/SF and TGF-beta 1 mRNA expression was recorded by semiquantitative RT-PCR; epimorphin gene expression was analyzed by Northern blot hybridization.

HGF/SF was expressed by every clone analyzed (Fig. 2, A and B). A significant difference was observed in the two colon cell lines (MIC 307-1 and 316) that expressed higher levels of HGF/SF mRNA than the jejunal (MIC 101-1 and 101-2) or ileal (MIC 216 and 219) mesenchyme cell lines. MIC 101-2 cells displayed the lowest amount of HGF/SF mRNA. TGF-beta 1 mRNA was also present in every clone; it was expressed at the highest level in the ileal clones MIC 216 and 219 (Fig. 2, A and C). Northern blot analysis showed that epimorphin was expressed by all clonal cell lines analyzed; the ileum-derived clone MIC 216 exhibited the highest level of epimorphin transcript (Fig. 3, A and B). No correlation between the level of expression of the three factors studied and the morphology of the cells could be made.


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Fig. 2.   A: representative results of RT-PCR analysis of hepatocyte growth and/or scatter factor (HGF/SF) and transforming growth factor-beta 1 (TGF-beta 1) mRNAs on control (lanes a, c, and e) and retinoic acid (RA)-treated (lanes b, d, and f ) MIC 101-2 [proximal jejunum (PJ), lanes a and b] MIC 216 [distal ileum (DI), lanes c and d], and MIC 316 [proximal colon (PC), lanes e and f ] cells. B and C: densitometric analysis of HGF/SF (B) and TGF-beta 1 (C) signals (from 3 independent experiments) after scanning of each specific band normalized to value of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) internal control. Relative values obtained in 2 different clonal cell lines from each level of gut are represented. Statistical analysis done by paired comparisons of 1 control cell line vs. each other individual control clone and by paired comparisons of each RA-treated clone vs. its control. * P < 0.05, ** P < 0.005 by Student's t-test.


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Fig. 3.   A: representative Northern hybridization of the 32P-labeled cDNA fragment of epimorphin in control (lanes a, c, and e) and RA-treated (lanes b, d, and f ) MIC 101-2 (lanes a and b), MIC 216 (lanes c and d), and MIC 316 (lanes e and f ) cells. B: relative mRNA levels generated from digitized image analysis of autoradiographs normalized to 18S rRNA in 2 different clonal cell lines from each level of gut. Each column represents mean of 2 or 3 independent experiments. Statistical analysis done as indicated in Fig. 2. * P < 0.05 MIC 216 RA treatment vs. control, ** P < 0.005 MIC 216 control compared with each other control clonal cell line. C: epimorphin mRNA expression in freshly isolated jejunal (a), ileal (b), and colon (c) lamina propria.

Interestingly, the analysis of HGF/SF and TGF-beta 1 mRNAs in freshly isolated lamina propria prepared from 8-day rat PJ, DI, and PC pointed to variations in the PD expression patterns similar to those found in the cell lines. Figure 4, A and B, shows that HGF/SF mRNA was more abundant in the colon than in the small intestine (jejunal or ileal segments). TGF-beta 1 mRNA exhibited the highest signal in the ileum (Fig. 4, A and C). The maximal expression of epimorphin mRNA in one of the ileal clones analyzed also agrees partially with its in vivo expression. Indeed, the highest level of epimorphin expression was observed in the distal intestinal regions at this specific stage (Fig. 3C). Interestingly, during fetal development, there is more epimorphin mRNA in the distal small bowel than in the proximal small intestine or in the colon (D. C. Rubin, personal communication).


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Fig. 4.   A: RT-PCR analysis of HGF/SF and TGF-beta 1 mRNAs on freshly isolated intestinal lamina propria from 8-day PJ (a), DI (b), and PC (c). B and C: densitometric analysis of HGF/SF (B) and TGF-beta 1 (C) bands normalized to values of GAPDH as internal control, from 2 independent experiments. * P < 0.05, ** P < 0.005 vs. other intestinal regions by Student's t-test. Mean obtained for PC (B) or DI (C) compared with mean obtained in other intestinal regions.

RA Selectively Stimulates Epimorphin Gene Transcription in Ileal Mesenchyme Clones

Previous results showed that the differentiating effect of RA may be mediated by the intestinal myofibroblasts that respond to RA treatment by increasing their levels of retinoid binding proteins as well as laminin-alpha 1 and -beta 1 constituent chains (40). In an attempt to define other molecular targets of RA possibly involved in the epithelium-mesenchyme-dependent morphogenesis and/or differentiation, we analyzed the effect of RA on the expression of HGF/SF, TGF-beta 1, and epimorphin at the mRNA level. The results did not show any differences in HGF/SF and TGF-beta 1 mRNA levels between control and RA culture conditions in the various clones used (Fig. 2). Interestingly, epimorphin mRNA level was specifically upregulated in the two ileal clones MIC 216 and 219 (40 and 20%, respectively; Fig. 3, A and B). The difference is statistically significant only in the MIC 216 cell line.

Mesenchyme Cell Lines Differentially Support Endodermal Spreading and Growth

Because the final aim of this model will be to further study the mesenchyme-mediated epithelial response according to the PD characteristics, we analyzed the ability of the cloned cell lines to support endodermal cell spreading and growth. For this purpose, 14-day fetal intestine endodermal microexplants were seeded on confluent monolayers of MIC cells. After an overnight period, we observed that the adhesion and/or spreading of endodermal fragments to the fibroblast cell layer varied among the clones used (shown in Fig. 5, A and C). The enlargement of the explants over a 3-day period was also dependent on the clone used (Fig. 5, B and D). Endoderms cocultured on a feeder layer of jejunal MIC 101-2 adhere and spread more rapidly and exhibit a higher proliferation ability than on a feeder layer of ileal MIC 216 (Fig. 5, A vs. C and B vs. D). The extension of the endodermal areas on the fibroblast layers has been evaluated by measuring their mean surface on the various mesenchymal cell clones after 3 days in culture. The size of the endodermal areas was significantly lower in the cocultures composed of the two MIC ileal clones compared with those composed of jejunal and colonic clones (Table 1). It is worth noting that the ileal clones express the highest level of TGF-beta 1. These data agree with the well-known effect of TGF-beta 1 on the arrest of epithelial cell proliferation in the gut (31). Again, no correlation between the ability to support growth and the cell shape could be made.


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Fig. 5.   Representative morphological features of cocultures composed of fetal rat intestinal endodermal cells (e) seeded over confluent mesenchyme cell lines (m). Phase-contrast micrographs show living cocultures at 24 h (A and C) and 3 days (B and D). Endodermal microexplants were seeded on MIC 101-2 (A and B) or MIC 216 (C and D). Bar = 90 µm.

                              
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Table 1.   Endodermal areas in 3-day cocultures

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

The intestinal subepithelial fibroblastic network consists of myofibroblasts underlying the crypt, villus, and surface epithelium in both small intestine and colon (32, 59). There is growing evidence that this cell population plays an important role in the development and maintenance of the morphological and functional steady state of the gut. Studies concerning the regulation of the differentiation of embryonic epithelial cells or postnatal crypt cell lines showed that a mesenchyme support is required to achieve morphogenesis as well as functional development and cytodifferentiation (27, 28, 37). More recently, divergent morphogenetic influences (crypt-villus vs. gland morphogenesis) of two mesenchyme-derived intestinal clonal cell lines have been described (15). A human colonic myofibroblastic cell line is also able to modulate the secretory response of the epithelial cells to inflammatory mediators, indicating their involvement in the physiopathology of the gut (4, 58, 59). Despite the existence of much experimental evidence of a functional epithelium-mesenchyme unit, the molecular mechanisms involved in fibroblast-mediated epithelial differentiation and functional regionalization along the PD axis are far from being understood. The poorness of suitable in vitro models may, at least in part, explain the slow progress in this subject.

In this study, we raised subepithelial fibroblast clonal cell lines derived from the intestinal lamina propria of 8-day rats taken from the PJ, DI, and PC. The various cell lines expressed mesenchyme-derived myofibroblast markers such as vimentin, smooth muscle alpha -actin, and myosin. This study, conducted on two clonal cell lines from each level of the gut, pointed to interesting specific characteristics. 1) The various clonal cell lines expressed the mRNAs for three morphogenetic and/or differentiation factors, HGF/SF, epimorphin, and TGF-beta 1, displaying specific patterns: the highest expression of HGF/SF was found in the colon cell lines, and the highest expression of TGF-beta 1 and epimorphin was found in the ileal ones. Interestingly, the regional differences in these transcripts reflected the expression profiles of the three genes in freshly isolated tissues. 2) RA treatment of the cell lines induced an increased expression of epimorphin only in the ileal cells; no detectable effect of RA treatment was observed on HGF/SF and TGF-beta 1 expression. 3) When used as feeder layers for cocultures, the various cell lines differentially supported the growth of the juxtaposed fetal endodermal microexplants: the jejunal and colon clones were more effective than the ileal ones.

Former work performed in the gut system emphasized the role of BM molecules, in particular laminins, in the mesenchyme-epithelium signaling; epithelial differentiation requires a BM at the heterologous cell interface (1, 7, 51, 57). Interestingly, intestinal mesenchyme cells are responsible for the production of some key BM proteins. This is the case constitutively for type IV collagen and nidogen, and with a peculiar chronology for laminin-alpha 1 and gamma 2 constituent chains, only when the subepithelial fibroblasts have differentiated into myofibroblasts under the influence of the epithelial cells (39, 50). Despite the fact that the requirement and role of the BM in the epithelium-mesenchyme-dependent differentiation of the gut have been demonstrated, the BM composition shows only little variation, except for collagen VII, along the PD axis (35, 53). Thus other effectors must act in concert with the BM molecules to influence morphology and differentiation characteristics along the gut axis. Here we show that three potential effectors, whose role as morphogens and growth and/or differentiation factors has been suggested in several organs, displayed a differential expression in the three segments of the gut analyzed; in addition, these specific patterns are maintained in the cloned cell lines corresponding to the different PD levels. These molecules, mostly expressed in situ by the mesenchyme compartment, could act on the juxtaposed epithelial cells via receptor-mediated signaling.

Homeobox genes are good candidates as upstream regulators of the epithelium-mesenchyme cell interactions in the gut system. Hox and Cdx homeobox genes display a PD gradient of expression in the adult and developing intestine (11, 14, 25, 55) and can potentially play an important role through their control of the expression of various cell-cell or cell-matrix molecules. This is exemplified by the strong inductive effect of Cdx2 on the expression of the beta 4-integrin subunit in the colonic cancer cell line Caco-2 and the fact that in these cells Cdx2 is regulated by a BM component, laminin (36). Interestingly, Cdx2 expression in the epithelial cells can be modulated by the associated mesenchyme compartment (11), and Cdx2 heterozygous knockout mice develop colon adenocarcinomas (6). Another interesting gene involved in mesenchyme-to-epithelium signaling is the mesenchyme winged helix transcription factor Fkh6, which is localized in the gastrointestinal mesenchyme adjacent to the endoderm-epithelium. A mutation in the Fkh6 gene results in profound dysregulation of the proliferation, cytodifferentiation, and morphogenesis of the gut and in a reduced expression of Bmp2 and Bmp4 growth factors (26). On the basis of these results, it will be interesting to evaluate the reciprocal dialogue between the mesenchyme cell lines and the epithelial cells in the expression of signaling molecules, their receptors, and various homeobox genes or specific transcription factors.

The physio(patho)logical importance of the epithelium-mesenchyme cell interactions is emphasized by the fact that several regulatory molecules induce phenotypic variations in intestinal mesenchyme cells: growth arrest or stimulation by cytokines like TGF-beta 1 or interleukin-2 (15), changes in the form (flat to stellate or vice versa) under the influence of cAMP concentration or of endothelin (17), modulation of prostaglandin secretion in response to inflammatory mediators (4, 58), and increase in synthesis of laminin-alpha 1 and beta 1 constituent chains and mesenchyme-dependent epithelial differentiation by glucocorticoids and RA (40, 51). Moreover, various inflammatory cytokines stimulate human fibroblast lines to produce HGF/SF; this can lead to abnormal epithelium-stroma interactions (45). It has been reported that HGF/SF receptor c-met expression is upregulated in cancer colonic mucosa (8). In this study, we additionally showed that the expression of epimorphin can be stimulated by RA; this observation is in accordance with the postulated morphogenetic role of this component (24). During fetal life, epimorphin distribution in the gut seems to correlate with villus morphogenesis and colonic crypt formation, i.e., more expression in the region that is undergoing morphogenesis (D. C. Rubin, personal communication). Unfortunately, the putative ligand or receptor of epimorphin on epithelial cells is still not known, nor is its signaling pathway. A recent study by Koshida and Hirai (33) suggests the presence of a unique cellular recognition sequence in the central portion of epimorphin.

Most of the data describing HGF/SF, TGF-beta 1, and epimorphin expression derive from studies on organs that do not display regional organization (5). This is the first report on PD variations in the expression of these molecules; at present it is difficult to understand their physiological significance. The maintenance of characteristics similar to those found in vivo in our homogeneous cell cultures should help the study of their action in vitro. Previous successful studies using the MIC cell lines presented here illustrated the role of MIC cells on small intestine endodermal differentiation in cocultures upon RA treatment (40) and the effect of MIC cell-conditioned media on the up- or downregulation of neuropeptides preferentially expressed in the proximal small intestine or in the colon in the intestinal endocrine cell line STC-1 (43).

In conclusion, the cell lines established in this work represent interesting in vitro models mimicking the in vivo situation that will allow the facilitation of analysis of the molecular mechanisms underlying region-specific gut morphogenesis and differentiation as well as cancer-related misregulation.

    ACKNOWLEDGEMENTS

We gratefully acknowledge C. Leberquier and C. Arnold for excellent technical support and I. Gillot and L. Mathern for help in the preparation of the manuscript and illustrations. We thank Dr. G. Gabbiani (Geneva, Switzerland) for the helpful discussion and for the anti-myosin antibodies and Dr. S. Sartore (Padua, Italy) for anti-SM22 antibodies.

    FOOTNOTES

Financial support comes from Institut National de la Santé et de la Recherche Médicale, Association pour la Recherche contre le Cancer (Grant no. 1251), Ligue Nationale contre le Cancer, and Association François Aupetit. M. Plateroti has a fellowship from the European Science Foundation (Developmental Biology Program).

Address for reprint requests: M. Kedinger, INSERM Unité 381, 3 Avenue Molière, 67200 Strasbourg, France.

Received 18 August 1997; accepted in final form 15 January 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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