Dysregulation of laminins in intestinal inflammation.

Laminins are structural components of basement membranes that regulate and control many cellular functions. Changes in basement membrane composition play significant roles in etiology of diseases. Inflammatory bowel diseases are conditions that lead to defects in the mucosal barrier which includes the basement membrane underlying the epithelium. This review will summarize the main findings related to the involvement of laminins and of the laminin-binding receptors in inflammatory conditions such as Crohn's disease and ulcerative colitis. We will review the current literature devoted to studies in humans (immunolocalisation, genetic factors, microarray data), as well as experimental cell models that show that laminins contribute to the inflammation process probably linked to the deregulation of proinflammatory cytokines.

The role of the basement membrane as a modulator of intestinal epithelial-mesenchymal interactions.

Intestinal development is a process of continuous dynamic bidirectional crosstalk between epithelial and underlying mesenchymal cells. This crosstalk is mediated by well-dissected signaling pathways. Another crucial actor in the epithelio-mesenchymal interactions is the stromal microenvironment, which is composed of extracellular matrix molecules. Among them, the basement membrane (BM) molecules are secreted by the epithelium and mesenchyme in a complementary manner. These molecules signal back to the cells via the integrins or other specific receptors. In this review, we mainly focus on the BM molecules, particularly laminins. The major BM molecules are organized in a complex molecular network, which is highly variable among organs. Cell culture, coculture, and grafting models have been of great interest in understanding the importance of these molecules. Mouse gene ablation of laminin chains are interesting models, which often lead to embryonic death and are frequently accompanied by compensatory processes. Overall, the BM molecules have a crucial role in the careful maintenance of intestinal homeostasis.

Cdx1, a dispensable homeobox gene for gut development with limited effect in intestinal cancer.

The homeobox gene Cdx1 is involved in anteroposterior patterning in embryos and its expression selectively persists in the intestinal epithelium throughout life. In human colon cancers, Cdx1 is overexpressed in few cases and lost in the majority of adenocarcinomas. We used mouse models of gain and loss-of-function to investigate the role of Cdx1 in intestinal development and cancers. Transgenic mice overexpressing Cdx1 and knockout mice exhibited a morphologically normal intestine. Cell proliferation, specification into the four differentiated lineages and migration along the crypt-villus axis were unchanged compared to wild-type mice. Changing Cdx1 caused an inverse and dose-dependent modification of the expression of the paralogous gene Cdx2, indicating that Cdx1 fine-tunes Cdx2 activity. Transgenenic and knockout mice failed to spontaneously develop tumours. Overexpressing Cdx1 was without incidence on the frequency of intestinal tumours induced chemically by azoxymethane treatment or genetically in Apc(Delta14/+) mice. However, it augmented the severity of the tumours in Apc(Delta14/+) mice. Inversely, the loss-of-function of Cdx1 in knockout mice was without incidence on the growth of tumours induced by azoxymethane. We conclude that Cdx1 is dispensable for intestinal development and that its overexpression could increase malignancy in early stages of tumourigenesis.

The intestine-specific homeobox gene Cdx2 decreases mobility and antagonizes dissemination of colon cancer cells.

The gravity of colorectal cancer is mainly due to the capacity of tumor cells to migrate out of the tumor mass to invade the stroma and disseminate as metastases. The acquisition of a migratory phenotype also occurs during wound healing. Here, we show that several features characterizing invasive colon tumor cells are shared by migrating cells during wound repair in vitro. In particular, the expression of the intestine-specific transcription factor Cdx2, a key gene for intestinal identity downregulated in invasive cancer cells, is reduced during wound healing in vitro. Transcription factors involved in epithelial-mesenchymal transition such as Snail and Slug are upregulated during wound healing and are able to repress Cdx2 transcription. In vitro, forced expression of Cdx2 in human colon cancer cell lines retarded wound repair and reduced migration, whereas inhibition of Cdx2 expression by RNA interference enhanced migration. In vivo, forced expression of Cdx2 opposed tumor cells spreading in nude mice xenografted at three different sites. These data provide evidence that Cdx2 antagonizes the process of tumor cell dissemination, and they suggest that this homeobox gene might represent a new therapeutic target against metastatic spreading of colon cancer.

Different effects of the Cdx1 and Cdx2 homeobox genes in a murine model of intestinal inflammation.

AIMS: The CDX1 and CDX2 homeoproteins are intestine-specific transcription factors regulating homeostasis. We investigated their relevance in experimentally-induced intestinal inflammation. METHODS: The response to intestinal inflammation induced by dextran sodium sulfate (DSS) was compared in wild type, Cdx1(-/-) and Cdx2(+/-) mice. Intestinal permeability was determined in wild type and Cdx2(+/-) mice. Protein-protein interactions were investigated by co-immunoprecipitation and GST-pulldown, and their functional consequences were assessed using Luciferase reporter systems. RESULTS: Heterozygous Cdx2(+/-) mice, but not Cdx1(-/-) mice, were hypersensitive to DSS-induced acute inflammation as all these mice showed blood in the stools at day 1 of DSS treatment. Hypersensitivity was associated to a 50% higher intestinal permeability. In Cdx2(+/-) mice, the colonic epithelium was repaired during the week after the end of DSS treatment, whereas two weeks were required for wild type animals. Subsequently, no colonic tumour was observed in Cdx2(+/-) mice subjected to 5 repeated cycles of DSS, in contrast to the 2.7 tumours found per wild type mouse. Based on the fact that Smad3(+/-) mice, like Cdx2(+/-) mice, better repair the damaged intestinal epithelium, we found that the CDX2 protein interacts with SMAD3, independently of SMAD4, resulting in a 5-fold stimulation of SMAD3 transcriptional activity. CDX1 also interacted with SMAD3 but it inhibited by 10-fold the SMAD3/SMAD4-dependent transcription. CONCLUSION: The Cdx1 and Cdx2 homeobox genes have distinct effects on the outcome of a pro-inflammatory challenge. This is mirrored by different functional interactions of the CDX1 and CDX2 proteins with SMAD3, a major element of the TGFbeta signalling pathway.

Sprouty2 inhibits BDNF-induced signaling and modulates neuronal differentiation and survival.

Sprouty (Spry) proteins are ligand-inducible inhibitors of receptor tyrosine kinases-dependent signaling pathways, which control various biological processes, including proliferation, differentiation and survival. Here, we investigated the regulation and the role of Spry2 in cells of the central nervous system (CNS). In primary cultures of immature neurons, the neurotrophic factor BDNF (brain-derived neurotrophic factor) regulates spry2 expression. We identified the transcription factors CREB and SP1 as important regulators of the BDNF activation of the spry2 promoter. In immature neurons, we show that overexpression of wild-type Spry2 blocks neurite formation and neurofilament light chain expression, whereas inhibition of Spry2 by a dominant-negative mutant or small interfering RNA favors sprouting of multiple neurites. In mature neurons that exhibit an extensive neurite network, spry2 expression is sustained by BDNF and is downregulated during neuronal apoptosis. Interestingly, in these differentiated neurons, overexpression of Spry2 induces neuronal cell death, whereas its inhibition favors neuronal survival. Together, our results imply that Spry2 is involved in the development of the CNS by inhibiting both neuronal differentiation and survival through a negative-feedback loop that downregulates neurotrophic factors-driven signaling pathways.

In vitro models of intestinal epithelial cell differentiation.

The intestinal epithelium is a particularly interesting tissue as (1) it is in a constant cell renewal from a stem cell pool located in the crypts which form, with the underlying fibroblasts, a stem cell niche and (2) the pluripotent stem cells give rise to four main cell types: enterocytes, mucus, endocrine, and Paneth cells. The mechanisms leading to the determination of phenotype commitment and cell-specific expressions are still poorly understood. Although transgenic mouse models are powerful tools for elucidating the molecular cascades implicated in these processes, cell culture approaches bring easy and elegant ways to study cellular behavior, cell interactions, and cell signaling pathways for example. In the present review, we will describe the major tissue culture technologies that allow differentiation of epithelial cells from undifferentiated embryonic or crypt cells. We will point to the necessity of the re-creation of a complex microenvironment that allows full differentiation process to occur. We will also summarize the characteristics and interesting properties of the cell lines established from human colorectal tumors.

Primary tumour genetic alterations and intra-tumoral heterogeneity are maintained in xenografts of human colon cancers showing chromosome instability.

Evaluation of the role of clonal heterogeneity in colon tumour sensitivity/resistance to drugs and/or in conferring metastatic potential requires an adequate experimental model in which the tumour cells maintain the initial genetic alterations and intra-tumoral heterogeneity through maintenance of the genetic clones present in the initial tumour. Therefore, we xenografted subcutaneously into nude mice seven human colonic tumours (from stages B1 to D) that showed chromosome instability and transplanted them sequentially for up to 14 passages. Maintenance after xenografting of the genetic alterations present in the initial tumours was scored by allelotype studies targeting 45 loci localized on 18 chromosomes. We show that xenografting does not alter the genetic or the histological profiles of the tumours even after 14 passages. Screening of the entire genome of one tumour by comparative genome hybridization also showed overall stability of the alterations between the initial and the xenografted tumour. In addition, intra-tumoral heterogeneity was maintained over time, suggesting that no clonal selection occurred in the nude mice. The observation that some loci showed partial allelic imbalance in the initial tumour but loss of heterozygosity after the first passage in nude mice when all the normal cells were lost may allow identification of interesting genetic defects that could be involved in tumour expansion. Thus, sequential xenografts of colon tumours will provide a powerful model for further study of tumour clonality and for the identification of genetic profiles responsible for differential resistance to therapeutic treatments. Our data also suggest that tumour expansion can result from alterations in several distinct genetic pathways.

Generation of a conditionally null allele of the laminin alpha1 gene.

Laminins are heterotrimeric glycoproteins of the basement membranes. Laminin 1 (alpha1, beta1, gamma1) is the major laminin expressed during early mouse embryogenesis. To gain access to the physiological function of laminin alpha1 chain, we developed a conditionally null allele of its encoding gene (Lama1) using the cre/loxP system. Floxed-allele-carrying mice (Lama1(flox/flox)) display no overt phenotype. Lama1(flox/flox) mice were crossed with transgenic deleter mice (CMV-Cre) to generate Lama1-deficient mice (Lama1(Delta/Delta)). Lama1(Delta/Delta) embryos die during the early postimplantation period after embryonic day 6.5. They lack Reichert's membrane, an extraembryonic basement membrane in which laminin alpha1 is normally highly expressed. In parallel, Lama1(Delta/Delta) embryos display 1) parietal and visceral endoderm differentiation defects with altered expression of cytokeratin 19 and GATA4, respectively, and 2) an induction of apoptosis. This new mouse model is of particular interest as it will allow time- and tissue-specific inactivation of the Lama1 gene in various organs.

The Cdx2 homeobox gene has a tumour suppressor function in the distal colon in addition to a homeotic role during gut development.

BACKGROUND: During development, the homeobox gene Cdx2 exerts a homeotic function, providing the positional information necessary for correct specification of the midgut endoderm. This is illustrated by the non-neoplastic gastric-type heteroplasias present at birth in the pericaecal region of Cdx2(+/-) mice. Furthermore, intestinal expression of Cdx2 continues throughout life but diminishes in colorectal cancers compared with adjacent normal tissue, suggesting a role in tumorigenesis. AIM: To investigate the consequence of altered Cdx2 expression on colon tumour initiation and/or progression. METHODS: Heterozygous Cdx2(+/-) mice were analysed for spontaneous malignant tumours and for tumour development after treatment with a DNA mutagen, azoxymethane. RESULTS: Cdx2(+/-) mice did not spontaneously develop malignant tumours. After azoxymethane treatment, the gastric-like heteroplasias in the pericaecal region did not evolve into cancer indicating that they are not precancerous lesions. However, azoxymethane treated Cdx2(+/-) mice developed tumours specifically in the distal colon 12 weeks after azoxymethane treatment whereas no tumours were found in wild-type littermates at this stage. Histopathological and molecular analyses indicated that these tumours were invasive adenocarcinomas that recapitulated the malignant sequence observed in the majority of sporadic colorectal cancers in human. In addition, we found that the colonic epithelium was less sensitive to radiation induced apoptosis in Cdx2(+/-) than in wild-type mice. CONCLUSION: This study provides the first experimental evidence that Cdx2 is a tumour suppressor gene involved in cancer progression in the distal colon. This action in adults is functionally and geographically distinct from its homeotic role during gut development.

Stimulation of the intestinal Cdx2 homeobox gene by butyrate in colon cancer cells.

BACKGROUND: The transcription factor encoded by the intestinal Cdx2 homeobox gene and treatment with sodium butyrate (NaB), a byproduct of fibre fermentation by colonic bacteria, exert similar effects on colon cancer cell lines as they both inhibit cell growth and stimulate cell differentiation and apoptosis. AIM: To investigate whether NaB regulates expression of the Cdx2 gene in colon cancer cell lines. METHODS: Human adenocarcinoma cell lines Caco2 and HT29 were grown in the presence or absence of NaB. Cells were analysed for Cdx2 mRNA expression by reverse transcription-polymerase chain reaction, for protein expression by western blotting and electromobility shift assays, and for transcriptional activity of the Cdx2 promoter by transfection with luciferase reporter plasmids. RESULTS: In HT29 and Caco2 cells, NaB stimulated Cdx2 mRNA and protein expression as well as transcriptional activity of the Cdx2 promoter. Stimulation of the activity of the Cdx2 promoter by NaB was dose and time dependent. The Cdx2 promoter contains discrete regions that participate in or inversely that blunt the stimulatory effect exerted by NaB. In addition, NaB stimulated the transcriptional activity of the Cdx2 promoter downregulated by oncogenic ras. CONCLUSION: This study is the first report of an intestine specific transcription factor, Cdx2, stimulated by butyrate. Thus it provides a new mechanism whereby butyrate controls proliferation and differentiation of colon cancer cells.

In vitro and in vivo efficacy of photofrin and pheophorbide a, a bacteriochlorin, in photodynamic therapy of colonic cancer cells.

This study was designed to investigate the efficacy of photodynamic therapy (PDT) in treating colonic cancer in a preclinical study. Photofrin, a porphyrin mixture, and pheophorbide a (Ph a), a bacteriochlorin, were tested on HT29 human colonic tumor cells in culture and xenografted into athymic mice. Their pharmacokinetics were investigated in vitro, and the PDT efficacy at increasing concentrations was determined with proliferative, cytotoxic and apoptotic assessments. The in vivo distribution and pharmacokinetics of these dyes (30 mg/kg, intraperitoneal) were investigated on HT29 tumor-bearing nude mice. The inhibition of tumor growth after a single 100 J/cm2 PDT session was measured by the changes in tumor volume and by histological analysis of tumor necrosis. PDT inhibited HT29 cell growth in culture. The cell photodamage occurred since the time the concentrations of Ph a and Photofrin reached 5.10(-7) M (or 0.3 microg/mL) and 10 microg/mL, respectively. A photosensitizer dose-dependent DNA fragmentation was observed linked to a cleavage of poly(ADP-ribose) polymerase and associated with an increased expression of mutant-type p53 protein. PDT induced a 3-week delay in tumor growth in vivo. The tumor injury was corroborated by histological observation of necrosis 48 h after treatment, with a correlated loss of specific enzyme expression in most of the tumor cells. In conclusion, PDT has the ability to destroy human colonic tumor cells in vitro and in vivo. This tumoricidal effect is likely associated with a p53-independent apoptosis, as HT29 cells express only mutated p53. The current study suggests a preferential use of Photofrin in PDT of colonic cancer because it should be more effective in vivo than Ph a as a consequence of better tumor uptake.

The phosphodiesterase inhibitor, pentoxifylline, alters rat intestinal epithelial cell proliferation via changes in the expression of transforming growth factors.

BACKGROUND: Phosphodiesterase (PDE) inhibitors, among which pentoxifylline (PTX), are candidate molecules for the treatment of TNF-alpha-dependent inflammatory diseases. Based on the controversial effects of PTX observed in experimentally-induced colitis, the aim of this work was to analyse its influence on intestinal epithelial cell proliferation and growth factor expression using the well-established IEC18 cell line. METHODS: The effects of PTX, and of an activation (addition of dibutyryl-cAMP, db-cAMP) or inhibition (by a specific cAMP-protein kinase inhibitor, PKI) of the cAMP pathway, were examined after 3 days of culture. The IEC18 cell proliferation and [3H] thymidine incorporation, as well as the expression of TGF-alpha, TGF-beta1 and -beta2 mRNAs, were analysed in basal culture conditions and in the presence of the pro-inflammatory cytokine, TNF-alpha. RESULTS: PTX, like exogenous db-cAMP, inhibited in a dose-dependent manner the basal and TNF-alpha-modulated IEC18 cell proliferation; this effect was partly prevented by PKI. We confirmed that PTX induced a dose-related increase in intracellular cAMP. Concomitantly, the expression of TGF-alpha mRNA dropped and that of TGF-beta2 increased. Addition of db-cAMP instead of PTX also decreased TGF-alpha mRNA, but did not change TGF-beta2 transcripts. The decrease in the expression of TGF-alpha mRNA caused by PTX and db-cAMP was completely abolished by PKI; in contrast, TGF-beta2 remained unaltered. Yet, anti-TGF-beta2 antibodies partially restored the PTX-inhibited cell proliferation. CONCLUSION: The phosphodiesterase inhibitor, PTX, inhibits IEC18 cell proliferation via a differential modulation of TGF-alpha and TGF-beta2 expression. The drop in TGF-alpha mRNA is related to increasing intracellular cAMP, whereas the effect upon TGF-beta2 appears cAMP-independent.

Overexpression of laminin alpha1 chain in colonic cancer cells induces an increase in tumor growth.

Laminins represent a growing family of glycoproteins constituting the basement membrane. They are known to direct many biological processes. With respect to carcinogenesis, laminins play an important role in cell adhesion, mitogenesis, differentiation and even metastasis. To further study the biological significance of laminin-1 (composed of alpha1, beta1 and gamma1 chains) in intestinal cell differentiation or tumorigenesis, an alpha1-laminin expression vector was introduced into the HT29 colonic cancer cells, in which laminin alpha1 chain is not expressed. Upon transfection of the alpha1 chain, the alpha1beta1gamma1 trimer was found secreted in the media along with free alpha1 chain as assessed by immunoprecipitation. The presence of the laminin alpha1 chain did not significantly modify the levels of the other laminin chains nor the integrins expressed by the HT29 cells. In spite of similar growth properties with the control cells in vitro (plastic dish, soft agar), the laminin alpha1 transfectants showed a significantly increased tumor growth when injected in nude mice. Histologic and immunohistochemic examination of the laminin alpha1-expressing tumors points to an increased recruitment of the host stromal and vascular cells, without modification in the differentiation profile and invasion potential. In parallel, a clear accumulation of laminin-10 (alpha5beta1gamma1) at the carcinoma/stromal interface and a segregation of the integrin beta4 subunit at the basal pole of the cancer cells occurred, compared to control tumors. Overall, our observations emphasize the importance of laminin-1 as a chemoattractant of both stromal and vascular cells and in epithelial/stromal cell interactions for the organization of the basement membrane and segregation of integrins leading to an epithelial cell growth signal. Such a sequence of events is reminiscent of what occurs during development.

Differentially expressed endoderm and mesenchyme genes along the fetal rat intestine.

Developmental studies have shown that morphological and functional regionalization occurring along the mammalian intestine is defined at early fetal stages and that some aspects of this patterning are dependent on epithelial-mesenchymal cell interactions. The molecular basis of these processes are largely unknown. In this study, a differential display approach was used to identify genes differentially expressed along the longitudinal axis in the intestinal endoderm and mesenchyme moieties of 14-day-old rat fetuses at a stage prior to morpho-functional differentiation of the gut. Fifty-eight genes were identified, 36 being identical or similar to known genes and 13 corresponding to ESTs or genome sequences with unknown function. Nine cDNAs could not be assigned to any previously described nucleotide sequence. The selected genes are involved in several aspects of cell physiology, including metabolic pathways, cytoskeleton organization, signal transduction, protein biosynthesis, and regulation of gene transcription.

Cytokine gene expression during postnatal small intestinal development: regulation by glucocorticoids.

BACKGROUND: In the intestinal mucosa, numerous cytokines produced by the epithelium, fibroblasts, and immune cells were shown to affect epithelial differentiation and proliferation through epithelial-mesenchymal and epithelial-immune cell interactions. To date, the importance of cytokines in postnatal development of the rat small intestine has not been established. AIM: To investigate the developmental changes in expression of mucosal cytokines in the postnatal maturation of the rat small intestinal epithelium and their regulation by glucocorticoids (GC). METHODS: Mucosal maturation was assessed by the onset of sucrase-isomaltase (SI) mRNA, analysed by in situ hybridisation. The amount of transforming growth factor beta1 (TGF-beta1), beta2 (TGF-beta2), tumour necrosis factor alpha (TNF-alpha), interleukin 1beta (IL-1beta), and TGF-alpha was analysed by reverse transcription-polymerase chain reaction (RT-PCR) in mucosal extracts from weaning (14-21 days old) and adult rats, or one day after an injection of hydrocortisone (HC) in 11 day old rats. Similarly, expression of cytokines and the regulatory effect of GC were studied on cultured subepithelial myofibroblasts cloned from postnatal jejunum and ileum cultured in the absence or presence of dexamethasone (DX). RESULTS: TGF-beta1, TGF-beta2, and IL-1beta decreased during the third week of life while levels of TNF-alpha increased and TGF-alpha remained constant. In parallel, SI transcripts increased and showed a progressive accumulation in the apical part of the enterocytes first localised at the base of the villi from 18 days onwards. Interestingly, precocious induction of SI mRNA by HC paralleled the decrease in expression of TGF-beta isoforms and of IL-1beta. All cytokines were expressed in the myofibroblast cell lines. In addition, the results showed that TNF-alpha was differentially expressed in basal culture conditions and after DX stimulation in jejunal and ileal myofibroblasts. DX decreased IL-1beta but not the TGF-beta isoforms, similar to that in vivo. CONCLUSIONS: This study shows that mucosal cytokines are developmentally regulated and that GC are potentially involved in this regulation in parallel with maturation of the gut mucosa at weaning.

Involvement of activator protein 1 complexes in the epithelium-specific activation of the laminin gamma2-chain gene promoter by hepatocyte growth factor (scatter factor).

Laminin-5 is a trimer of laminin alpha3, beta3 and gamma2 chains that is found in the intestinal basement membrane. Deposition of the laminin gamma2 chain at the basement membrane is of great interest because it undergoes a developmental shift in its cellular expression. Here we study the regulatory elements that control basal and cytokine-activated transcriptional expression of the LAMC2 gene, which encodes the laminin gamma2 chain. By using transient transfection experiments we demonstrated the presence of constitutive and cytokine-responsive cis-elements. Comparison of the transcriptional activity of the LAMC2 promoter in the epithelial HT29mtx cells with that in small-intestinal fibroblastic cells (C20 cells) led us to conclude that two regions with constitutive epithelium-specific activity are present between positions -1.2 and -0.12 kb. This was further validated by transfections of primary foetal intestinal endoderm and mesenchyme. A 2.5 kb portion of the LAMC2 5' flanking region was equally responsive to PMA and hepatocyte growth factor (HGF), whereas it was less responsive to transforming growth factor beta1. A minimal promoter limited to the initial 120 bp upstream of the transcriptional start site maintained inducibility by PMA and HGF. This short promoter fragment contains two activator protein 1 (AP-1) elements and the 5'-most of these is a composite AP-1/Sp1 element. The 5'AP-1 element is crucial to the HGF-mediated activity of the promoter; analysis of interacting nuclear proteins demonstrated that AP-1 proteins containing JunD mediate the response to HGF.

Developmental expression and cellular origin of the laminin alpha2, alpha4, and alpha5 chains in the intestine.

Laminins are extracellular matrix glycoproteins that are involved in various cellular functions, including adhesion, proliferation, and differentiation. In this study, we examine the expression patterns and the cellular origins of the laminin alpha2, alpha4, and alpha5 chains in the developing mouse intestine and in in vitro mouse/chick or chick/mouse interspecies hybrid intestines. In situ hybridization and Northern blot analysis revealed that mRNA levels for all three laminin alpha chains are highest in the fetal intestine undergoing intense morphogenetic movements. Laminin alpha4 mRNA and polypeptide are associated with mesenchyme-derived cell populations such as endothelium and smooth muscle. In contrast, laminin alpha2 and alpha5 chains participate in the structural organization of the subepithelial basement membrane and, in the mature intestine, show a complementary pattern of expression. All three laminin alpha chains occur in the smooth muscle basement membrane, with a differential expression of laminin alpha5 chain in the circular and longitudinal smooth muscle layers. The cellular origin of laminin alpha2 and alpha5 chains found in the subepithelial cell basement membrane was studied by immunocytochemical analysis of mouse/chick or chick/mouse interspecies hybrid intestines at various stages of development using mouse-specific antibodies. Laminin alpha2 was found to be deposited into the basement membrane exclusively by mesenchymal cells, while the laminin alpha5 chain was deposited by both epithelial and mesenchymal cells in an apparently developmentally regulated pattern. We conclude that (1) multiple laminin alpha chains are expressed in the intestine, implying specific roles for individual laminin isoforms during intestinal development, and (2) reciprocal epithelial/mesenchymal interactions are essential for the formation of a structured subepithelial basement membrane.

Characterization of human intestinal stromal cell lines: response to cytokines and interactions with epithelial cells.

The maintenance of the physiological homeostasis of the gut mucosa characterized by continuous proliferation and differentiation processes results from epithelial-mesenchymal cell cross-talk. To set out stable and homogeneous models for the study of the (dys)regulation of various morphofunctional aspects, we established and characterized three clonal cell lines (C9, C11, and C20) derived from human duodenal mucosal connective tissue. We defined the expression of (i) cytoskeletal proteins; (ii) basement membrane molecules (laminins, collagen IV, nidogen) which have been shown formerly to be deposited at the epithelial/mesenchymal interface in situ by the mesenchymal compartment; and (iii) soluble factors, HGF, and TGFbeta1. The three cell lines display common but also specific proliferative responses to cytokines (IL1beta, IL2, IL8, TNFalpha, IFNgamma, TGFbeta1, and HGF). When cocultured with embryonic intestinal endoderms or with human colonic Caco2 or HT29 cancer cells, C9 versus C11 and C20 cell lines induced limited versus extensive growth of the associated epithelial cells. In addition C20 cells allowed spreading of HT29 cells with the formation of a basement membrane at the heterologous interface. Morphogenesis obtained by intracoelomic grafts of associations comprising the mesenchymal cell lines and intestinal endoderms was also different among those composed of C9 cells or of C11 or C20 cells. In conclusion, these data indicate that the mucosal connective tissue is heterogeneous and comprises several phenotypically different mesenchyme-derived cells whose equilibrium may be important in the gut homeostasis. These cells can now be used to define tissue-specific factors which may be involved in the physiopathology of the intestinal epithelium.