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: email@example.com 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. 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