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DEVELOPMENTAL DYNAMICS 212:437–447 (1998)
Identification, Distribution, and Tissular Origin
of the a5(IV) and a6(IV) Collagen Chains
in the Developing Human Intestine
ALINE SIMONEAU,1 F. ELIZABETH HERRING-GILLAM,1 PIERRE H. VACHON,1
NATHALIE PERREAULT,1 NURIA BASORA,1 YAMINA BOUATROUSS,1
LOUIS-PHILLIPE PAGEOT,1 JING ZHOU,2 AND JEAN-FRANÇOIS BEAULIEU1*
1Centre de Recherche en Biologie du Développement des Épithéliums et Unité de Physiopathologie Digestive du
Centre de Recherche Clinique du CUSE, Département d’Anatomie et de Biologie Cellulaire, Faculté de Médecine,
Université de Sherbrooke, Sherbrooke, Québec, Canada
2Renal Division, Brigham and Women’s Hospital, Boston, Massachusetts
ABSTRACT
The basement membrane type
IV collagen is a family composed of six genetically
distinct but structurally similar polypeptide chains,
a1–a6. The a1(IV) and a2(IV) chains are ubiquitous
components of all BMs whereas the other four have
a restricted tissue distribution. In the present study,
we have analyzed the expression, distribution, and
cellular origin of the a5(IV) and a6(IV) chains in the
developing and adult human small intestine and in
well-characterized in vitro models by indirect immunofluorescence, Western blot, and RT-PCR. We have
found that in the fetal small intestine, a5(IV) and
a6(IV) are present in the epithelial BM and, in
contrast to a1(IV) and a2(IV), are produced by both
epithelial and mesenchymal cells. A distinct tissular
origin for the a1/a2(IV) and a5/a6(IV) chains suggests that a5(IV) and a6(IV) associate as a heterotrimer in this organ. We have also found that a particular situation of a5(IV)/a6(IV) chain expression occurs
in the adult intestine. Indeed, as compared with the
fetal intestine, a6(IV) chain production is maintained while the expression of the a5(IV) chain is
substantially reduced. Altered expression of the
a5(IV) chain was also observed in the differentiating
enterocytic-like Caco-2/15 cells, suggesting that in
the intestinal model, the a5(IV) chain is subject to a
regulated expression. Taken together, these observations indicate that the human intestinal epithelial
BM contains up to four type IV collagen chains: the
classical a1(IV)/a2(IV) chains, which originate from
mesenchymal cells, and the a5(IV)/a6(IV) chains,
which are of both epithelial and mesenchymal origin and have their expression regulated throughout
development. Dev. Dyn. 1998; 212:437–447.
r 1998 Wiley-Liss, Inc.
Key words: type IV collagen; intestine; development; intestinal cell lines; human
INTRODUCTION
Type IV collagen is one of the major components of
the basement membrane (BM). Through its supramolecular organization, collagen IV forms an intricate
r 1998 WILEY-LISS, INC.
framework on which other basement membrane macromolecules associate to form a bioactive matrix involved
in the regulation of a number of cellular activities
including attachment/migration, growth, apoptosis, and
gene expression (Hynes, 1992; Paulsson, 1992; Adams
and Watt, 1993; Rosekelly et al., 1995; Sheppard 1996;
Timpl and Brown, 1996). The classical form of type IV
collagen, [a1(IV)]2a2(IV), is found in all BMs. Its a1(IV)
and a2(IV) chains are genetically distinct but structurally related polypeptides of ,185 kD. In the BM, the
heterotrimeric molecules are assembled head to head
through the C-terminal NC1 domain, tail to tail through
the N-terminal triple helix 7S domain into tetrameric
aggregates, and by helix-helix lateral interactions, providing the supramolecular structure of the nonfibrillar
collagen network (reviewed in Burgeson and Nimmi,
1992; Hudson et al., 1993; Yurchenco, 1994; Timpl and
Brown, 1996).
In recent years, four additional type IV collagen
chains, a3(IV)–a6(IV), have been identified and characterized (Pihlajaniemi et al., 1990; Hostikka et al., 1990;
Morrison et al., 1991; Zhou et al., 1992, 1993, 1994;
Mariyama et al., 1994; Sugimoto et al., 1994; Leinonen
et al., 1994; Oohashi et al., 1994). While the primary
structure of these chains and their genomic organization show great similarities to those of the a1(IV) and
a2(IV) chains, they differ considerably with respect to
their tissue distribution, which is much more restricted
(Hostikka et al., 1990; Mariyama et al., 1994; Leinonen
et al., 1994; Peissel et al., 1995). The a3(IV) and a4(IV)
chains, consistently co-expressed, have been identified
in the specialized BM of the kidney, eye, cochlea, lung,
and brain (Butkowski et al., 1987; Kleppel et al.,
1989a,b). The a5(IV) chain has been found to accompany the a3(IV) and a4(IV) chains at some sites, such
Grant sponsor: Medical Research Council of Canada; Grant numbers: MT-11289, MT-12904; Grant sponsor: Fonds pour la Formation
des Chercheurs et l’Aide à la Recherche.
*Correspondence to: Jean-François Beaulieu, Département
d’Anatomie et de Biologie Cellulaire, Faculté de Médecine, Université
de Sherbrooke, Sherbrooke, Québec, Canada, J1H 5N4. E-mail:
jf.beaul@courrier.usherb.ca
Received 24 October 1997; Accepted 19 March 1998
438
SIMONEAU ET AL.
as the glomerular and tubular BMs, but is also present
alone at other sites, such as the epidermal BM, which
lacks the a3(IV) and a4(IV) chains (Butkowski et al.,
1987; Kleppel et al., 1989b; Kashtan et al., 1989). The
expression of the a6(IV) chain has been recently investigated and it has been found to colocalize with the a5(IV)
chain in the BM of many tissues with some exceptions,
for example the glomerular BM that does not express
the a6(IV) chain (Ninomiya et al., 1995; Peissel et al.,
1995). As described for the a1(IV) and a2(IV) genes on
chromosome 13 (Poschl et al., 1988; Soininen et al.,
1988) and most likely for the a3(IV) and a4(IV) genes
on chromosome 2 (Hudson et al., 1993), the head-tohead genomic pairing of the a5(IV) and a6(IV) genes on
chromosome X sharing a common promoter implies
coordinated transcription of the two genes (Sugimoto et
al., 1994; Zhou et al., 1994). Therefore, the mechanism
of expression of the a5(IV) and a6(IV) chains, which is
not always coordinated such as in the glomerular BM,
remains to be elucidated (Ninomiya et al., 1995; Peissel
et al., 1995) but may involve the presence of alternative
promoters (Sugimoto et al., 1994).
The small intestinal epithelium has been proven to
be a useful model for analyzing cell-extracellular matrix interactions in relation to the cell state in both the
developing and adult organ (Beaulieu, 1997a,b). Compositional analysis of the human intestinal epithelial BM
has revealed that it contains all the major macromolecules found in conventional BMs, as well as some
interstitial extracellular matrix components, such as
tenascin-C and cellular fibronectin, and other constituents not yet fully characterized. The relative complexity
of the intestinal BM appears consistent with its potential implication in the regulation of cell proliferation,
migration, and differentiation, which occurs rapidly in
this renewing epithelium.
In a recent study, the expression of the a1–a5(IV)
collagen chains was examined in the human small
intestine (Beaulieu et al., 1994). As expected from
previous studies in this organ (Beaulieu et al., 1991;
Beaulieu, 1992), the a1(IV) and a2(IV) chains were
ubiquitously expressed in the epithelial BM at all
stages. On the other hand, the a3(IV) and a4(IV) chains
were not detected (Beaulieu et al., 1994). Surprisingly,
the a5(IV) chain was identified in the fetal small
intestinal mucosa while its adult counterpart appeared
mostly devoid of this molecule (Beaulieu et al., 1994;
Bouatrouss et al., 1998). An anti-a6(IV) chain antibody
was not available at the time of the original study but
Peissel et al. (1995) reported its presence in the human
fetal small intestine. The observation that both a5(IV)
and a6(IV) chains are present in the epithelial BM of
the fetal intestine suggests that these two molecules
can possibly associate and form a distinct network of
minor type IV collagen. In the present study, as a first
step to examine this hypothesis, we have studied the
distribution and cellular origin of the a5(IV) and a6(IV)
chains in the developing human intestine and in wellcharacterized in vitro models. We have also analyzed
the a6(IV) chain in the adult intestine to determine
whether or not its expression parallels that of the
a5(IV) chain. We have found that in the fetal small
intestine, the a5(IV) and a6(IV) chains are present in
the epithelial BM and, in contrast to a1(IV) and a2(IV),
appear to be of dual epithelial and mesenchymal origin,
suggesting that a5(IV) and a6(IV) from epithelial cells
associate as a heterotrimer. We have also observed that
a particular situation of a5(IV)/a6(IV) chain expression
exists in the adult intestine. Indeed, as compared to the
fetal intestine, the a6(IV) chain production appears to
be maintained while a repression of a5(IV) chain expression occurs. Altered expression of the a5(IV) chain was
also observed in differentiating enterocytic-like Caco2/15 cells, suggesting that in the intestinal model, the
a5(IV) chain is subject to a regulated expression.
RESULTS
Expression of the a5(IV) and a6(IV) Chains
in the Developing Small Intestine
The expression and distribution of the two collagen
chains were first studied on cryosections of fetal and
adult small intestine by indirect immunofluorescence,
using well-characterized antisera. At 11–12 weeks of
gestation, the a5(IV) chain was detected in both epithelial and mesenchymal compartments as well as at the
interface between the two tissues (Fig. 1A) while the
a6(IV) chain was found in the epithelium and at the
epithelial-mesenchymal interface but was below detection levels in the mesenchyme (Fig. 1B). At midgestation, staining patterns for the a5(IV) and a6(IV)
chains were comparable in the epithelium and in its
underlying basement membrane (Fig. 1C,D). The stroma
and smooth muscle layers were positive for a5(IV) (Fig.
1C) but, as at earlier stages, remained below detection
levels for a6(IV) (Fig. 1D). In the adult, the staining for
the a5(IV) chain was consistently found to be much
weaker than that observed in the fetal (Fig. 2A) while
for the a6(IV) chain, the staining remained relatively
intense, particularly at the epithelial basement membrane (Fig. 2B). Negative controls for immunofluorescence staining included omission of the primary antibody and its replacement with a non-immune rabbit
serum, used at the same concentration (not shown).
Production of the a5(IV) and a6(IV) Chains
by Epithelial and Mesenchymal Cells
The expression of these two type IV collagen chains
by intestinal chains was investigated by Western blot
and RT-PCR analysis in the intact tissue as well as in
human in vitro models (Fig. 3). These models included
the enterocyte-like Caco-2/15 cell line, the normal
crypt-like cell line HIEC-6, and human intestinal mesenchymal (HIM) cells. As shown in Figure 3A, Western
blot analysis revealed that both the a5(IV) and a6(IV)
chains can be detected as specific ,185 kD bands in the
small intestine (SI), in the two epithelial cell lines
tested, as well as in intestinal mesenchymal cells.
RT-PCR analysis confirmed the presence of both transcripts corresponding to the a5(IV) and a6(IV) chains in
the tissues and cells investigated (Fig. 3B). The absence
COLLAGEN a5(IV) AND a6(IV) CHAINS IN INTESTINE
Fig. 1. Expression and distribution of the a5(IV) and a6(IV) chains of
collagen in the fetal small intestine. Representative immunofluorescence
micrographs of human jejunum at 12 (A,B) and 18 weeks (C,D) of
gestation stained for the detection of the a5(IV) (A,C) and a6(IV) (B,D)
chains. At these stages, the a5(IV) chain, although predominantly detected at the epithelial-mesenchymal interface (arrowheads), was found
widely distributed in the epithelium and mesenchyme (M) as well as in
439
close association with the two layers of the surrounding muscularis
propria (MP). The staining for the detection of the a6(IV) chain was also
predominant at the epithelial-mesenchymal interface (arrowheads). The
epithelium was positive for the a6(IV) chain but, in contrast to a5(IV), the
a6(IV) chain was not detected in the mesenchyme (M) and muscularis
propria (MP). A–D, 3148.
440
SIMONEAU ET AL.
Fig. 2. Expression of the a5(IV) and a6(IV) chains of collagen in the
adult small intestinal mucosa. Representative immunofluorescence micrographs of human jejunum stained for the detection of the a5(IV) (A) and
a6(IV) chains (B). In the adult, the expression of the a5(IV) chain was
consistently found at the limit of detection levels. A weak staining was
observed in the epithelial basement membrane (arrowheads) and in
association with cellular elements of the lamina propria (asterisks). On the
contrary, the a6(IV) chain was detected according to a comparable
staining pattern as that observed in the fetal small intestine at midpregnancy (compare B with Fig. 1D). A,B, 3148.
of contaminating epithelial cells in HIM cultures was
confirmed as described previously (Vachon et al., 1993).
Interestingly, while these observations suggest a dual
epithelial and mesenchymal origin for the a5(IV) and
a6(IV) chains, the production of the two other type IV
collagen chains present in the human small intestine,
a1(IV) and a2(IV), were found to be expressed by the
mesenchymal HIM cells but not by the epithelial Caco2/15 cells (Vachon et al., 1993; see Table 1).
The dual origin of the a5(IV) and a6(IV) chains was
further investigated by RT-PCR on epithelial and mesenchymal fractions freshly isolated from the fetal small
intestine using a new non-enzymatic dissociation
method (Perreault and Beaulieu, 1998). As expected
from in vitro studies previously described, the a5(IV)
and a6(IV) transcripts were present in both preparations, confirming their dual origin (Fig. 4). Purity of
each fraction was confirmed with E-cadherin (epithelial
marker) and tenascin-C (mesenchymal marker).
Caco-2/HIM co-culture model (Vachon et al., 1993).
Indirect immunofluorescence analysis of the a1/a2(IV),
a5(IV), and a6(IV) chains in 8-day co-cultures demonstrated a predominant accumulation of these molecules
at the epithelial-mesenchymal interface (Fig. 5). Indeed, as previously observed for a1/a2(IV) collagen
(Vachon et al., 1993), used here as a positive control
(Fig. 5A), the a5(IV) and a6(IV) chains were strongly
detected at the basal aspect of the Caco-2/15 cells lying
on HIM cells (Fig. 5B,C). HIM cells themselves were
moderately stained for these molecules particularly in
the region neighbouring the epithelial-mesenchymal
interface while Caco-2/15 cells were negative for the
a1/a2(IV) chains (Fig. 5A) but stained for the a5(IV)
and a6(IV) chains (Fig. 5B,C), according to a basolateral pattern comparable to that observed in the fetal
intestine (see Fig. 1).
Deposition of the a5(IV) and a6(IV) Chains
at the Epithelial-Mesenchymal Interface
in a Co-Culture Model
To further characterize the a5(IV) and a6(IV) chains
produced by intestinal cells in vitro, we used the
Differential Expression of a5(IV) and a6(IV)
Chains in Differentiating Caco-2/15 Cells
The Caco-2 cell line is currently used as a model for
enterocytic differentiation. The gradual acquisition of
morphological and functional characteristics of mature
intestinal epithelial cells by post-confluent Caco-2 cells
is well documented (Pinto et al., 1983; Vachon et al.,
COLLAGEN a5(IV) AND a6(IV) CHAINS IN INTESTINE
1996). To determine whether the expression of the two
collagen chains is correlated with epithelial differentiation, the expression of the a5(IV) and a6(IV) chains was
analyzed throughout the enterocytic differentiation process of Caco-2/15 cells. As summarized in Figure 6, both
441
TABLE 1. Summary of the Expression
of Type IV Collagen Chainsa
Chain
a1
a2
a3
a4
a5
a6
Intestinal EBM
Fetal
Adult
(IF)
(IF)
111
111
111
111
2
2
2
2
11
6
11
11
Caco-2/15
IF
2
2
2
2
11
11
WB
N.D.
N.D.
n.t.
n.t.
,185 kD
,185 kD
HIM
WB
,185 kD
,185 kD
n.t.
n.t.
,185 kD
,185 kD
aAs determined by indirect immunofluorescence (IF) and
Western blot (WB) with specific antibodies directed to the various
type IV collagen chains. Staining intensity was expressed on a
scale from 2 (negative) to 111 (maximum reaction).
EBM, epithelial basement membrane; N.D., not detected; n.t.,
not tested.
molecules were detected at all stages. When protein
expression levels were compared to those of cytokeratin
18, which in Caco-2/15 cells remains constant relative
to total cellular proteins during enterocytic differentiation (Vachon et al., 1995), levels of the a6(IV) chain
were found to be constant while those of the a5(IV)
chain increased by more than 3-fold (Fig. 6A).
The expression of these collagen chains was further
investigated at the RNA level. RT-PCR was carried out
on total RNA extracted from Caco-2/15 cells at various
stages of confluence to determine the pattern of a5(IV)
and a6(IV) mRNA expression in relation to S14. Statistical analysis of the densitometric data (Fig. 6B) indicated that the relative amounts of each transcript did
not vary significantly throughout the culture period,
although a gradual reduction in the levels of a6(IV)
relative to a5(IV) was observed after confluence
(P , 0.03–0.01).
DISCUSSION
By analyzing the expression of the a5(IV) and a6(IV)
chains of collagen in the human fetal small intestine,
we demonstrated that the two molecules are present at
both the mRNA and protein levels and are predominantly distributed at the epithelial basement membrane where they appear to colocalize. These data,
which are in agreement with previous observations
reported separately from our laboratories (Beaulieu et
al., 1994; Peissel et al., 1995), also suggest, as deduced
Fig. 3. Expression of a5(IV) and a6(IV) chains in intestinal cells. A:
Total protein from fetal small intestine (SI, lane 1), Caco-2/15 cells (lane
2), HIEC-6 cells (lane 3), and human intestinal mesenchymal (HIM) cells
(lane 4) were analyzed by Western blot for the detection of the a5(IV) and
a6(IV) chains. TP: Transferred proteins. Representative remaining portion
of nitrocellulose (60–120 kD range) stained with Ponceau red to compare
the amounts of proteins in samples. B: Representative RT-PCR analysis
of a5(IV) and a6(IV) chain mRNA in the fetal small intestine (SI, lane 1),
Caco-2/15 cells (lane 2), HIEC-6 cells (lane 3), and human intestinal
mesenchymal (HIM) cells (lane 4). 2RT: reverse transcriptase omitted.
S14 transcript was determined to ensure cDNA integrity and to compare
amounts of starting RNA material in the various samples.
442
SIMONEAU ET AL.
Fig. 4. Distribution of a5(IV) and a6(IV) transcripts in fetal small
intestinal tissues. Representative RT-PCR analysis of a5(IV) and a6(IV)
chain mRNA in the intact small intestine (SI, lane 1) and corresponding
isolated mesenchymal (Mes, lane 2) and epithelial fractions (Epi, lane 3).
2RT: reverse transcriptase omitted (lane 4). S14 transcript was determined to ensure cDNA integrity and to compare amounts of starting RNA
material in the various samples. E-cadherin and tenascin-C transcripts
were assassed as specific markers of the intestinal epithelium and
mesenchyme, respectively.
by immunofluorescence staining, that the a6(IV) chain
could be mainly of epithelial origin while the a5(IV)
seems to be produced by both epithelial and mesenchymal cells. This difference may appear surprising at first
glance since the two genes are arranged head-to-head
and may share a bidirectional promoter (Zhou et al.,
1993) and, of the few positive tissues identified by
immunofluorescence staining, most seem to coexpress
the a5(IV) and a6(IV) chains (Ninomiya et al., 1995;
Peissel et al., 1995). However, this is not without
precedent since a differential expression of these two
chains has also been noted in some other tissues
(Ninomiya et al., 1995; Peissel et al., 1995). It should be
pointed out that the mesenchymal origin for a basement membrane molecule such as the a5(IV) chain is
readily conceivable in the light of previous studies that
demonstrated that the a1(IV) and a2(IV) chains of
collagen found in the intestinal epithelial basement
membrane are mainly, if not exclusively, produced by
the mesenchymal compartment (Simon-Assmann et al.,
1990, Hewitt et al., 1992; Vachon et al., 1993).
To further investigate the tissular origin of the a5(IV)
and a6(IV) chains, we first utilized in vitro models.
Although in situ hybridization on tissues was initially
considered, this technique has unfortunately been
proven to be difficult to apply for the detection of
transcripts expressed at relatively low levels such as
those encoding the a5(IV) and a6(IV) chains in the
human intestine (Zhou et al., 1993; Beaulieu et al.,
1994). The use of intestinal cell culture models as an
alternative approach, although indirect, presents the
advantage of examining pure and well-characterized
derivatives from either the epithelium or the mesenchyme of the human intestine, available in relatively
large amounts. The Caco-2 cell line is currently used as
a model for enterocytic differentiation; at postconfluence, they gradually acquire the morphological and
functional characteristics of mature intestinal epithelial cells (Ménard and Beaulieu, 1994; Zweibaum and
Chantret, 1989). The HIEC-6 cell line has been generated from normal fetal human small intestine; these
cells express a number of intestinal crypt cell markers
but no villus cell markers and are thus considered to be
poorly differentiated epithelial cells (Perreault and
Beaulieu, 1996). Finally, the HIM cells are mesenchymal cell cultures obtained from the human fetal intestine, tested for the lack of contaminating epithelial and
endothelial cells, and maintained at low passage levels
(Vachon et al., 1993). Interestingly, the Caco-2 and HIM
cells models were used previously to demonstrate the
exclusive mesenchymal origin of the a1(IV) and a2(IV)
chains of collagen in the human intestine (Vachon et al.,
1993). Herein, with the same models, we have shown
that both the a5(IV) and a6(IV) chains are produced by
epithelial and mesenchymal cells (Table 1). These observations were confirmed at the mRNA level on freshly
dissociated 17–19-week fetal intestinal epithelia and
mesenchymes by using a newly described non-enzymatic procedure (Perreault and Beaulieu, 1998). Although this method does not allow the determination of
the origin of a5(IV) and a6(IV) at the protein level, the
detection of the two transcripts in both tissues is in
good agreement with the in vitro data supporting a dual
origin for these molecules. The lack of a6(IV) chain
detection in the fetal stroma by indirect immunofluorescence (Fig. 1) could thus be explained by differential
tissue masking of epitopes although a post-transcriptional mechanism cannot be excluded. Reminiscent of
the in vivo situation, the a5(IV) and a6(IV) chains
COLLAGEN a5(IV) AND a6(IV) CHAINS IN INTESTINE
443
Fig. 5. Indirect immunofluorescence analysis of type IV
collagen chain expression and distribution in Caco-2/HIM
co-cultures. Caco-2/15 cells were grown on top of confluent
HIM cells for 8 days, embedded in OCT and sectioned for
the detection of the a1/a2(IV) (A), a5(IV) (B), and a6(IV)
(C) collagen chains. As observed for a1/a2(IV) chains, the
a5(IV) and a6(IV) chains were found predominantly at the
interface between Caco-2/15 and HIM cells. Note that the
a6(IV) staining in HIM cells appears weaker in intensity
than that of a5(IV). The a5(IV) and a6(IV) chains were also
detected in Caco-2/15 cells. A–C, 3592.
substantially accumulated at the epithelial-mesenchymal interface and colocalized with the a1(IV)/a2(IV)
chains in co-cultures of Caco-2 cells and HIM cells. It is
noteworthy that under these conditions, the a6(IV)
staining was consistently found to be weaker than
a5(IV) over HIM cells relative to the epithelial layer.
Taken together, these data suggest a dual origin for the
two minor chains of type IV collagen, which in absence
of the a3(IV) and a4(IV) chains in this tissue (Beaulieu
et al., 1994), probably associate as a [a5(IV)]2a6(IV)
heterotrimer (Hudson et al., 1993). Further, because of
their codistribution with the major [a1(IV)]2a2(IV)
network at the basement membrane in both the intact
fetal small intestine and Caco-2/HIM co-cultures, the
a5(IV) and a6(IV) chains may constitute a distinct
network of BM collagen in this tissue, as recently
proposed for other tissues as well (Ninomiya et al.,
1995; Peissel et al., 1995).
In the adult small intestine, the situation appears
quite different. The expression of the a5(IV) chain is
downregulated while the a6(IV) chain remains expressed at levels comparable to those observed in the
fetus. The detection of the a6(IV) chain in the epithelial
basement membrane suggests that the integration of
this chain into the BM network does not require the
presence of other minor a(IV) chains, although an
implication for the a5(IV) chain, even present in very
low amounts, cannot be ruled out. The weak signal
observed in the detection of the a5(IV) chain in the
adult small intestine seems to be representative of a
very modest expression of the molecule as supported by
the low level observed for the corresponding transcript
(Beaulieu et al., 1994). To our knowledge, this differential pattern of expression for the a5(IV) and a6(IV)
chains in an adult tissue is unique at the present time.
The only other reported case of relative amounts of the
444
SIMONEAU ET AL.
Fig. 6. Differential expression of the a5(IV) and a6(IV) chains during
enterocytic differentiation. Estimation of the relative abundance of a5(IV)
and a6(IV) proteins (A) and transcripts (B) in Caco-2/15 cells at 22, 0, 3,
8, and 15 days of confluence. Relative amounts of proteins and transcripts
were determined by densitometry as ratios relative to keratin 18 and S14,
respectively. Data represent means 6 SE from four separate experiments. A statistically significant difference in the relative amounts of
a5(IV) as compared to those for a6(IV) was found at 3, 8, and 15 days of
confluence (asterisks).
a6(IV) chain exceeding those of the a5(IV) chain is in
the skeletal muscle, a tissue in which a6(IV) levels are
already presumed to be fairly low (Ninomiya et al.,
1995). The molecular organization of the a6(IV) chain
in the absence of significant levels of a5(IV) and its
mode of integration into the BM remain to be determined. Furthermore, the mechanism controlling differential a5(IV) chain expression shutdown in the adult
intestine requires further investigation. In a first attempt to address this question, the a5(IV) and a6(IV)
chains were analyzed throughout the Caco-2/15 cell
differentiation process. Indeed, previous studies have
shown that the expression of basement membrane
molecules can be regulated during Caco-2/15 cell differentiation as illustrated by a gradual down-regulation of
cellular fibronectin production and an onset of laminin-1 deposition, both occurring after confluence (Vachon
et al., 1995; Vachon and Beaulieu, 1995). When tested
for type IV collagen chain expression, differentiating
Caco-2/15 cells showed a significant accumulation of
the a5(IV) chain while amounts of the a6(IV) chain
remained constant. Corresponding transcripts stayed
statistically constant over the same period but a significant difference in the relative amounts of a5(IV) and
a6(IV) mRNA was noted after confluence, suggesting
that differential expression of the proteins can result,
at least in part, from a differential regulation at the
mRNA levels. It is pertinent to note that it is the
expression of the a5(IV) chain that seems subject to
variations both in the intact intestine during development and in differentiating Caco-2/15 cells, emphasizing the possibility that, although the two genes may
share a common bidirectional promoter (Zhou et al.,
1993; Sugimoto et al., 1994), the modulation of their
transcription, namely that of the COL4A5 gene, relies
on additional regulatory elements in this organ. In this
context, the differential regulation of a5(IV) expression
during transition between fetal and adult stages in the
intestine (decrease) and in differentiating Caco-2/15
cells (increase) could be indicative of a relatively complex transcriptional regulation, which will require further investigation. This situation is, however, not without precedent since a similar differential regulation is
also observed under these specific conditions for lactasephlorizin hydrolase expression (Pinto et al., 1983;
Zweibaum and Chantret, 1989; Ménard and Beaulieu,
1994; Vachon et al., 1996).
Taken together, these observations provide the evidence that the a5(IV) and a6(IV) chains are expressed
from both epithelial and mesenchymal cells in the fetal
human small intestine where they appear to accumulate at the epithelial BM. The function of this a5(IV)/
a6(IV) collagen in the developing intestine is unknown
but, likely, differs from that of the mature intestine
where only the a6(IV) chain remains expressed at
significant levels. Further analysis of these collagen
chains in the Caco-2/15, HIEC, and HIM cell models
should help to clarify their roles.
EXPERIMENTAL PROCEDURES
Tissues
Specimens of small intestine from fetuses ranging
from 9 to 20 weeks postfertilization were obtained after
legal abortion. Samples of adult small intestine (jejunum and ileum) were obtained from non-diseased parts
of resected segments. Between five and twelve samples
were studied for each stage. Only specimens obtained
rapidly (less than 60 min) were used. The project was in
accordance with a protocol approved by the Institutional Human Research Review Committee for the use
of human material.
Cell Culture
The human colon carcinoma Caco-2/15 cell line, a
stable clone of the parent Caco-2 cells (HBT 37; ATCC,
Rockville, MD) has been characterized elsewhere (Beaulieu and Quaroni, 1991; Vachon and Beaulieu, 1992;
Vachon et al., 1996). These cells are well known for
their enterocyte-like differentiation properties, similar
COLLAGEN a5(IV) AND a6(IV) CHAINS IN INTESTINE
to those observed in the epithelium of the intact fetal
intestine (Pinto et al., 1983). Cells between passages 54
and 70 were cultured as described (Vachon and Beaulieu, 1992). The human intestinal mesenchymal (HIM)
cells derived from an 18-week-old fetal small intestine
were obtained and grown as described elsewhere
(Vachon et al., 1993). For co-cultures, the seeding of
Caco-2/15 cells on confluent HIM cells and their maintenance was performed as described previously (Vachon
et al., 1993) for a period of 8 days. The HIEC-6 cells
were generated from normal fetal human small intestine. They were used between passages 5 and 10 and
grown as described (Perreault and Beaulieu, 1996).
HIEC cells are considered to be poorly differentiated
crypt cells (Perreault and Beaulieu, 1996; Quaroni and
Beaulieu, 1997).
Epithelial-Mesenchymal Dissociation
Pure epithelial and mesenchymal fractions were
obtained from 17–19-week-old fetal small intestines
according to a non-enzymatic method recently developed (Perreault and Beaulieu, 1998). Briefly, starting
with 5-mm intestinal fragments, the procedure consisted of removing the muscle coating, opening the
fragments longitudinally, and soaking them in ice-cold
Matrisperse (Collaborative Biomedical Products, Becton Dickenson Labware, Mississauga, Ont., Canada)
for 8 hr at 4°C. Then the preparations were gently
shaken, resulting in a complete separation of the
epithelial lining from the mesenchyme. Recovered epithelial suspensions and remaining mesenchymal fragments were both washed twice in PBS before use.
Primary Antibodies
Antibodies used in this work were the monoclonal
M3F7 (DSHB; Foellmer et al., 1983) and the AB748
serum (Chemicon Int., El Segundo, CA; Beaulieu et al.,
1994) directed to the a1(IV) and a2(IV) chains. The
monoclonal Mab17 and an anti-a3(IV) serum directed
to the a3(IV) chain (Butkowski et al., 1987; Saus et al.,
1988; Kleppel et al., 1989a,b) and an anti-a4(IV) serum
(Butkowski et al., 1987) were kindly provided by Dr. J.
Wieslander, BioCarb, Lund, Sweden. The production
and characterization of the specific anti-a5(IV) and
anti-a6(IV) antibodies have been described previously
(Beaulieu et al., 1994; Peissel et al., 1995).
Indirect Immunofluorescence
The preparation and embedding of tissue and cell
samples for cryosectioning was performed as described
previously (Beaulieu, 1992; Vachon et al., 1993). Frozen
sections were fixed in 95% ethanol (10 min, 220°C),
rinsed with phosphate-buffered saline (PBS) and denatured in 6 M urea/0.1 M glycine HCl, pH 3.5, for 1 hr at
4°C, before immunostaining (Peissel et al., 1995). Sections were sequentially incubated with primary antibodies diluted in 10% non-fat powdered milk in PBS and
FITC-conjugated goat anti-rabbit IgG or anti-mouse
IgG (Boehringer Mannheim Canada, Laval, Québec,
445
Canada) used at 1/25 in 2% bovine serum albumin in
PBS. Sections were then stained with 0.01% Evan’s
blue in PBS, mounted in glycerol-PBS (9:1) containing
0.1% paraphenylenediamine, and viewed with a Reichart Polyvar 2 microscope (Leica, Canada, St-Laurent,
Québec) equiped for epifluorescence. In all cases no
specific immunofluorescent staining was observed when
primary antibodies were omitted or replaced by mouse
or rabbit non-immune serum.
Gel Electrophoresis and Immunoblotting
Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) on 12% acrylamide gels and immunoblotting were performed as described previously (Vachon
et al., 1993; Beaulieu et al., 1993; Vachon and Beaulieu,
1995). Intestinal cells grown to different stages of
confluence were washed twice in PBS and were harvested in 1 3 solubilization buffer (2.3% SDS, 10%
glycerol, 0.001% bromophenol blue in 62.5 mM TrisHCl pH 6.8, containing 5% b-mercaptoethanol). Total
proteins from whole small intestinal homogenates (in
20 mM Tris-HCl, pH 6.8, containing 0.1 mM phenylmethylsulfonyl fluoride, 50 µg/ml leupeptine, 50 µg/ml
antipain, and 0.1 mg/ml aprotinin) were rapidly processed for solubilization in 2 3 solubilization buffer 1:1.
Samples were boiled for 5 min, cleared by centrifugation (13,000g, 5 min), and aliquoted for storage at
280°C. Separated proteins were transferred onto nitrocellulose (Bio-Rad, Mississauga, Ontario, Canada) and
blocked in PBS containing 10% powdered skim milk,
then incubated overnight at room temperature with
primary antibodies (anti-a5 and anti-a6 antibodies,
1/500). Alkaline phosphatase (Bio-Rad) detection was
used according to the manufacturer’s instructions. Band
intensities were quantified using an LKB Ultroscan XL
densitometer (Pharmacia, Piscataway, NJ).
RNA Isolation and RT-PCR
Total RNA was isolated from cell lines or tissue
homogenates using TriZOL (Gibco-BRL, Burlington,
Ontario, Canada). For the reverse transcription reaction, reverse transcriptase SuperScript II (Gibco-BRL)
and 0.5 µg of oligo-(dT)12-18 primer (Pharmacia, Baie
d’Urfe, Quebec, Canada) were added to 5 µg of total
RNA, as described elsewhere (Beaulieu et al., 1994).
For PCR amplification, we used the sense primer col5-3
58-CTTCCTGGATTTCCAGGGAC-38 and the antisense
primer col5-4 58-CCCTGAAGACCTTGCTCAACC-38
specific for the a5(IV) chain (Beaulieu et al., 1994) and
the sense primer JZ3F1 58-CTTATTGGAAAGCCATGTGGG-38 and the antisense JZ/R11 58-CGGACCTTCCGGATACTCTG-38 specific for the a6(IV) chain (Zhou
et al., 1994). Single-stranded cDNA was amplified in
PCR buffer (Pharmacia) containing 1 µM of both sense
and antisense primers for 25 cycles of denaturation (1
min at 94°C) and annealing/extension (a5(IV): 1 min at
63°C/1 min at 72°C; a6(IV): 1 min at 60°C/1.5 min at
72°C) in a thermal cycler (Perkin-Elmer, Branchburg,
NJ, DNA Thermal Cycler Model 480) in the presence of
446
SIMONEAU ET AL.
250 µM dNTPs and 2 µCi [32P]dCTP (Amersham,
Oatville, Ontario, Canada) and 2.5 U of Taq (Roche;
obtained from Pharmacia). Conditions for amplification
of S14, used as an endogenous control, and E-cadherin
and tenascin-C have been described elsewhere (Jumarie et al., 1996; Perreault and Beaulieu, unpublished
data). The products were separated on a 5% acrylamide/
TBE or a 2% agarose/TAE gel and visualized by either
autoradiography (Kodak Biomax MR films, Kodak,
Rochester, NY) or ethidium bromide staining.
ACKNOWLEDGMENTS
The authors thank Dr. J. Wieslander (BioCarb, Lund,
Sweden) for the generous gift of antibodies directed to
the a3(IV) and a4(IV) chains and Drs. A. Bilodeau, C.
Poulin, M. Morin, F. Jacot, L. Lemieux, and J. Poisson
of the CUSE of Sherbrooke for their cooperation in
providing specimens for this study. P.H.V. and N.B.
were supported by a post-doctoral fellowship and a
studentship, respectively, from the MRC of Canada,
and N.P. and Y.B. were supported by a studentship from
the FCAR and the Centre de recherche clinique du
CUSE, respectively. The M3F7 antibody was obtained
from the Developmental Studies Hybridoma Bank maintained by the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, Baltimore, MD, and the Department of Biology,
University of Iowa, Iowa City, IA, under contract N01HD-2-3144 from the NICHD.
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