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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -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-
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -chain and a 30-kDa
-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-
1 (TGF-
1) is a
homodimeric protein of 25 kDa and a member of the multifunctional
TGF-
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-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-
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-
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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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, 10Freshly 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 atCoculture 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 -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-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', TGF
1
5'-GAAGTCACCCGCGTGCTAATGG-3', TGF
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).
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 · min1 · ml
1
of radiolabeled epimorphin cDNA probe at 42°C overnight. The probe
was labeled with high-specific-activity
[
-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -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 -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
-actin (Fig.
1E in MIC 101-2 cells).
Interestingly, immunodetection of the various antigens on intestinal
cryosections revealed that desmin (not shown), smooth muscle
-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
-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.
|
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-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-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.
|
|
Interestingly, the analysis of HGF/SF and TGF-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-
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).
|
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-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-
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
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 -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-
1, displaying
specific patterns: the highest expression of HGF/SF was found in the
colon cell lines, and the highest expression of TGF-
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-
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-1 and
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
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-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-
1 and
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-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 |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Basson, M. D.,
G. Turowski,
and
N. J. Emenaker.
Regulation of human Caco-2 intestinal epithelial cell differentiation by extracellular matrix proteins.
Exp. Cell Res.
225:
301-305,
1996[Medline].
2.
Bennett, M. K.,
J. E. Garcia-Arraras,
L. A. Elferink,
K. Peterson,
A. M. Fleming,
C. D. Hazuka,
and
R. H. Scheller.
The syntaxin family of vesicular transport receptors.
Cell
74:
863-873,
1993[Medline].
3.
Benzonana, G.,
O. Skalli,
and
G. Gabbiani.
Correlation between the distribution of smooth muscle or non muscle myosins and -smooth muscle actin in normal and pathological soft tissues.
Cell Motil. Cytoskeleton
11:
260-274,
1988[Medline].
4.
Berschneider, H. M.,
and
D. W. Powell.
Fibroblasts modulate intestinal secretory responses to inflammatory mediators.
J. Clin. Invest.
89:
484-489,
1992[Medline].
5.
Birchmeier, C.,
and
W. Birchmeier.
Molecular aspects of mesenchymal-epithelial interactions.
Annu. Rev. Cell Biol.
9:
511-540,
1993.
6.
Chawengsaksophak, K.,
R. James,
V. E. Hammond,
F. Kontgen,
and
F. Beck.
Homeosis and intestinal tumours in Cdx2 mutant mice.
Am. J. Surg.
385:
84-87,
1997.
7.
De Arcangelis, A.,
P. Neuville,
R. Boukamel,
O. Lefebvre,
M. Kedinger,
and
P. Simon-Assmann.
Inhibition of laminin alpha 1-chain expression leads to alteration of basement membrane assembly and cell differentiation.
J. Cell Biol.
133:
417-430,
1996[Abstract].
8.
Di Renzo, M. F.,
R. P. Narsimhan,
M. Olivero,
S. Bretti,
S. Giordano,
E. Medico,
P. Gaglia,
P. Zara,
and
P. M. Comoglio.
Expression of the Met/HGF receptor in normal and neoplastic human tissues.
Oncogene
6:
1997-2003,
1991[Medline].
9.
Duband, J.-L.,
M. Gimona,
M. Scatena,
S. Sartore,
and
J. V. Small.
Calponin and SM22 as differentiation markers of smooth muscle: spatiotemporal distribution during avian embryonic development.
Differentiation
55:
1-11,
1993[Medline].
10.
Duluc, I.,
J. N. Freund,
C. Leberquier,
and
M. Kedinger.
Fetal endoderm primarily holds the temporal and positional information required for mammalian intestinal development.
J. Cell Biol.
126:
211-221,
1994[Abstract].
11.
Duluc, I.,
O. Lorentz,
C. Fritsch,
C. Leberquier,
M. Kedinger,
and
J. N. Freund.
Changing intestinal connective tissue interactions alters homeobox gene expression in epithelial cells.
J. Cell Sci.
110:
1317-1324,
1997
12.
Evans, G. S.,
N. Flint,
A. S. Somers,
B. Eyden,
and
C. S. Potten.
The development of a method for the preparation of rat intestinal epithelial cell primary cultures.
J. Cell Sci.
101:
219-231,
1992[Abstract].
13.
Feinberg, A. P.,
and
B. Vogelstein.
A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity.
Anal. Biochem.
132:
6-13,
1983[Medline].
14.
Freund, J. N.,
R. Boukamel,
and
A. Benazzouz.
Gradient expression of Cdx along the rat intestine throughout postnatal development.
FEBS Lett.
314:
163-166,
1992[Medline].
15.
Fritsch, C.,
P. Simon-Assmann,
M. Kedinger,
and
G. S. Evans.
Cytokines modulate fibroblast phenotype and epithelial-stroma interactions in rat intestine.
Gastroenterology
112:
826-838,
1997[Medline].
16.
Fujimura, Y.,
and
T. Kihara.
Immunohistochemical localisation of intercellular adhesion molecule-1 in follicle associated epithelium of Peyer's patches.
Gut
35:
46-50,
1994[Abstract].
17.
Furuya, S.,
and
K. Furuya.
Characteristics of cultured subepithelial fibroblasts of rat duodenal villi.
Anat. Embryol. (Berl.)
187:
529-538,
1993[Medline].
18.
Gaisano, H. Y.,
M. Ghai,
P. N. Malkus,
L. Sheu,
A. Bouquillon,
M. K. Bennett,
and
W. S. Trimble.
Distinct cellular locations of the syntaxin family of proteins in rat pancreatic acinar cells.
Mol. Biol. Cell
7:
2019-2027,
1996[Abstract].
19.
Green, R. P.,
S. M. Cohn,
J. C. Sacchettini,
K. E. Jackson,
and
J. I. Gordon.
The mouse intestinal fatty acid binding protein gene: nucleotide sequence, pattern of developmental and regional expression, and proposed structure of its protein product.
DNA Cell Biol.
11:
31-41,
1992[Medline].
20.
Gudas, L. J.
Retinoids and vertebrate development.
J. Biol. Chem.
269:
15399-15402,
1994
21.
Haffen, K.,
M. Kedinger,
F. Bouziges,
and
P. Simon-Assmann.
Mesenchyme-endoderm interactions and enterocyte development.
In: Adaptation and Development of Gastrointestinal Function, edited by M. W. Smith,
and F. V. Sepulveda. Manchester, UK: Manchester University, 1989, p. 92-102.
22.
Henning, S. J.,
D. C. Rubin,
and
J. Shulman.
Ontogeny of the intestinal mucosa.
In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by L. R. Johnson. New York: Raven, 1994, p. 571-601.
23.
Hermiston, M. L.,
T. Simon,
M. W. Grossman,
and
J. I. Gordon.
Model systems for studying cell fate specification and differentiation in the gut epithelium.
In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by L. R. Johnson. New York: Raven, 1994, p. 521-569.
24.
Hirai, Y.,
K. Takebe,
M. Takashina,
S. Kobayashi,
and
M. Takeichi.
Epimorphin: a mesenchymal protein essential for epithelial morphogenesis.
Cell
69:
471-481,
1992[Medline].
25.
James, R.,
and
J. Kazenwadel.
Homeobox gene expression in the intestinal epithelium of adult mice.
J. Biol. Chem.
266:
3246-3251,
1991
26.
Kaestner, K. H.,
D. G. Silberg,
P. G. Traber,
and
G. Schutz.
The mesenchymal winged helix transcription factor Fkh6 is required for the control of gastrointestinal proliferation and differentiation.
Genes Dev.
11:
1583-1595,
1997[Abstract].
27.
Kedinger, M.
Growth and development of intestinal mucosa.
In: Small Bowel Enterocyte Culture and Transplantation, edited by F. C. Campbell. Georgetown, TX: Landes, 1994, p. 1-31.
28.
Kedinger, M.,
and
D. Newgreen.
The gut and enteric nervous system.
In: Embryos, Genes and Birth Defects, edited by P. Thorogood. Chichester, UK: Wiley, 1997, p. 153-196.
29.
Kedinger, M.,
P. M. Simon,
J. F. Grenier,
and
K. Haffen.
Role of epithelial-mesenchymal interactions in the ontogenesis of intestinal brush-border enzymes.
Dev. Biol.
86:
339-347,
1981[Medline].
30.
Kedinger, M.,
P. Simon-Assmann,
F. Bouziges,
C. Arnold,
E. Alexandre,
and
K. Haffen.
Smooth muscle actin expression during rat gut development and induction in fetal skin fibroblastic cells associated with intestinal embryonic epithelium.
Differentiation
43:
87-97,
1990[Medline].
31.
Ko, T. C.,
R. D. Beauchamp,
C. M. Townsend, Jr.,
E. A. Thompson,
and
J. C. Thompson.
Transforming growth factor-beta inhibits rat intestinal cell growth by regulating cell cycle specific gene expression.
Am. J. Surg.
167:
14-19,
1994[Medline].
32.
Komuro, T.,
and
Y. Hashimoto.
Three-dimensional structure of the rat intestinal wall (mucosa and submucosa).
Arch. Histol. Cytol.
53:
1-21,
1990[Medline].
33.
Koshida, S.,
and
Y. Hirai.
Identification of cellular recognition sequence of epimorphin and critical role of cell/epimorphin interaction in lung branching morphogenesis.
Biochem. Biophys. Res. Commun.
234:
522-525,
1997[Medline].
34.
Leid, M.,
P. Kastner,
and
P. Chambon.
Multiplicity generates diversity in the retinoic acid signalling pathways.
Trends Biochem. Sci.
17:
427-433,
1992[Medline].
35.
Leivo, I.,
T. Tani,
L. Laitinen,
R. Bruns,
E. Kivilaakso,
V. P. Lehto,
R. E. Burgeson,
and
I. Virtanen.
Anchoring complex components laminin-5 and type VII collagen in intestine: association with migrating and differentiating enterocytes.
J. Histochem. Cytochem.
44:
1267-1277,
1996
36.
Lorentz, O.,
I. Duluc,
A. De Arcangelis,
P. Simon-Assmann,
M. Kedinger,
and
J. N. Freund.
Key role of the Cdx2 homeobox gene in extracellular matrix-mediated intestinal cell differentiation.
J. Cell Biol.
139:
1553-1565,
1997
37.
Louvard, D.,
M. Kedinger,
and
H. P. Hauri.
The differentiating intestinal epithelial cell: establishment and maintenance of functions through interactions between cellular structures.
Annu. Rev. Cell Biol.
8:
157-195,
1992.
38.
Massague, J.
TGF- signaling: receptors, transducers, and Mad proteins.
Cell
85:
947-950,
1996[Medline].
39.
Orian-Rousseau, V.,
D. Aberdam,
L. Fontao,
L. Chevalier,
G. Meneguzzi,
M. Kedinger,
and
P. Simon-Assmann.
Developmental expression of laminin-5 and HD1 in the intestine: epithelial to mesenchymal shift for the laminin gamma-2 chain subunit deposition.
Dev. Dyn.
206:
12-23,
1996[Medline].
40.
Plateroti, M.,
J. N. Freund,
C. Leberquier,
and
M. Kedinger.
Mesenchyme-mediated effects of retinoic acid during rat intestinal development.
J. Cell Sci.
110:
1227-1238,
1997
41.
Podolsky, D. K.
Peptide growth factors in the gastrointestinal tract.
In: Physiology of the Gastrointestinal Tract (3rd ed.), edited by L. R. Johnson. New York: Raven, 1994, p. 129-160.
42.
Qian, S. W.,
P. Kondaiah,
A. B. Roberts,
and
M. B. Sporn.
cDNA cloning by PCR of rat transforming growth factor beta-1.
Nucleic Acids Res.
18:
3059,
1990[Medline].
43.
Ratineau, C.,
M. Plateroti,
J. Dumortier,
M. Blanc,
M. Kedinger,
J. A. Chayvialle,
and
C. Roche.
Intestinal-type fibroblasts selectively influence proliferation rate and peptide expression in the entero-endocrine cell line STC-1.
Differentiation
62:
139-147,
1997[Medline].
44.
Richman, P. I.,
R. Tilly,
J. R. Jass,
and
W. F. Bodmer.
Colonic pericrypt sheath cells: characterisation of cell type with new monoclonal antibody.
J. Clin. Pathol.
40:
593-600,
1987[Abstract].
45.
Rosen, E. M.,
and
I. D. Goldberg.
Regulation of scatter factor (hepatocyte growth factor) production by tumor-stroma interaction.
EXS
74:
17-31,
1995[Medline].
46.
Rosen, E. M.,
S. K. Nigam,
and
I. D. Goldberg.
Scatter factor and the c-met receptor: a paradigm for mesenchymal/epithelial interaction.
J. Cell Biol.
127:
1783-1787,
1994[Abstract].
47.
Rubin, D. C.,
D. E. Ong,
and
J. I. Gordon.
Cellular differentiation in the emerging fetal rat small intestinal epithelium: mosaic patterns of gene expression.
Proc. Natl. Acad. Sci. USA
86:
1278-1282,
1989[Abstract].
48.
Sappino, A. P.,
W. Schurch,
and
G. Gabbiani.
Differentiation repertoire of fibroblastic cells: expression of cytoskeletal proteins as marker of phenotypic modulations.
Lab. Invest.
63:
144-161,
1990[Medline].
49.
Sasaki, M.,
M. Nishio,
T. Sasaki,
and
J. Enami.
Identification of mouse mammary fibroblast-derived mammary growth factor as hepatocyte growth factor.
Biochem. Biophys. Res. Commun.
199:
772-779,
1994[Medline].
50.
Simo, P.,
F. Bouziges,
J. C. Lissitzky,
L. Sorokin,
M. Kedinger,
and
P. Simon-Assmann.
Dual and asynchronous deposition of laminin chains at the epithelial-mesenchymal interface in the gut.
Gastroenterology
102:
1835-1845,
1992[Medline].
51.
Simo, P.,
P. Simon-Assmann,
C. Arnold,
and
M. Kedinger.
Mesenchyme-mediated effect of dexamethasone on laminin in cocultures of embryonic gut epithelial cells and mesenchyme-derived cells.
J. Cell Sci.
101:
161-171,
1992[Abstract].
52.
Simon-Assmann, P.,
and
M. Kedinger.
Heterotypic cellular cooperation in gut morphogenesis and differentiation.
Semin. Cell Biol.
4:
221-230,
1993[Medline].
53.
Simon-Assmann, P.,
M. Kedinger,
A. De Arcangelis,
V. Orian-Rousseau,
and
P. Simo.
Extracellular matrix components in intestinal development.
Experientia
51:
883-900,
1995[Medline].
54.
Sonnenberg, E.,
D. Meyer,
K. M. Weidner,
and
C. Birchmeier.
Scatter factor/hepatocyte growth factor and its receptor, the c-met tyrosine kinase, can mediate a signal exchange between mesenchyme and epithelia during mouse development.
J. Cell Biol.
123:
223-235,
1993[Abstract].
55.
Traber, P. G.
Differentiation of intestinal epithelial cells: lessons from the study of intestine-specific gene expression.
J. Lab. Clin. Med.
123:
467-477,
1994[Medline].
56.
Tso, J. Y.,
X. H. Sun,
T. H. Kao,
K. S. Reece,
and
R. Wu.
Isolation and characterization of rat and human glyceraldehyde-3-phosphate dehydrogenase cDNAs: genomic complexity and molecular evolution of the gene.
Nucleic Acids Res.
13:
2485-2502,
1985[Abstract].
57.
Vachon, P. H.,
and
J. F. Beaulieu.
Extracellular heterotrimeric laminin promotes differentiation in human enterocytes.
Am. J. Physiol.
268 (Gastrointest. Liver Physiol. 31):
G857-G867,
1995
58.
Valentich, J. D.,
V. Popov,
J. I. Saada,
and
D. W. Powell.
Phenotypic characterization of an intestinal subepithelial myofibroblast cell line.
Am. J. Physiol.
272 (Cell Physiol. 41):
C1513-C1524,
1997
59.
Valentich, J. D.,
and
D. W. Powell.
Intestinal subepithelial myofibroblasts and mucosal immunophysiology.
Curr. Opin. Gastroenterol.
10:
645-651,
1994.
60.
Yasugi, S.
Role of epithelial-mesenchymal interactions in differentiation of epithelium of vertebrate digestive organs.
Dev. Growth & Differ.
35:
1-9,
1993.