Origin and deposition of basement membrane heparan sulfate proteoglycan in the developing intestine

The deposition of intestinal heparan sulfate proteoglycan (HSPG) at the epithelial-mesenchymal interface and its cellular source have been studied by immunocytochemistry at various developmental stages and in rat/chick interspecies hybrid intestines. Polyclonal heparan sulfate antibodies were produced by immunizing rabbits with HSPG purified from the Engelbreth-Holm-Swarm mouse tumor; these antibodies stained rat intestinal basement membranes. A monoclonal antibody (mAb 4C1) produced against lens capsule of 11-d-old chick embryo reacted with embryonic or adult chick basement membranes, but did not stain that of rat tissues. Immunoprecipitation experiments indicated that mAb 4C1 recognized the chicken basement membrane HSPG. Immunofluorescent staining with these antibodies allowed us to demonstrate that distribution of HSPG at the epithelial-mesenchymal interface varied with the stages of intestinal development, suggesting that remodeling of this proteoglycan is essential for regulating cell behavior during morphogenesis. The immunofluorescence pattern obtained with the two species-specific HSPG antibodies in rat/chick epithelial/mesenchymal hybrid intestines developed as grafts (into the coelomic cavity of chick embryos or under the kidney capsule of adult mice) led to the conclusion that HSPG molecules located in the basement membrane of the developing intestine were produced exclusively by the epithelial cells. These data emphasize the notion already gained from previous studies, in which type IV collagen has been shown to be produced by mesenchymal cells (Simon- Assmann, P., F. Bouziges, C. Arnold, K. Haffen, and M. Kedinger. 1988. Development (Camb.). 102:339-347), that epithelial-mesenchymal interactions play an important role in the formation of a complete basement membrane.     

Early mouse molar root development: Cellular changes and distribution of fibronectin, laminin and type-IV collagen

We analysed epithelial-mesenchymal interactions that occur during the early stages of the formation of mouse molar roots using light and electron microscopy. Morphological changes observed in the cells of Hertwig's epithelial sheath, the pulp and the follicular mesenchyme are described. The Hertwig's epithelial cells lose their cuboidal form and become flattened, apparently intermixing with the cells of the follicular mesenchyme. At the light- and electron-microscope levels, immunoperoxidase techniques were used to localize fibronectin, laminin and type-IV collagen. These appear to be closely associated with cell differentiation and matrix deposition in developing tooth roots. In addition, at the ultrastructural level, intracellular immunoreactivity was detected. The rough endoplasmic reticulum and nuclear envelope of some cells of the periodontal ligament facing the acellular cementum exhibited specific reactivity with laminin and type-IV collagen. Moreover, these periodontal ligament cells express keratin, but not vimentin, filaments. Our results demonstrate that Hertwig's epithelial cells maintain their capacity to synthesize laminin and type-IV collagen, as well as to express keratin filaments, despite basement membrane fragmentation and the disorganization of Hertwig's epithelial sheath. Thus, some Hertwig's epithelial cells remain in the periodontal ligament intermixed with follicular mesenchyme cells.     

Role of muscle fibroblasts in the deposition of type-IV collagen in the basal lamina of myotubes

In cell cultures of quail, chick, or mouse skeletal muscle, both myogenic and fibrogenic cells synthesize and secrete type-IV collagen, a major structural component of the basal lamina. Type-IV collagen, together with laminin, forms characteristic patches and strands on the surface of developing myotubes, marking the onset of basement-membrane formation. The pattern for type-IV collagen and laminin is unique to these proteins and is not paralleled by other matrix proteins, such as fibronectin or type-I or -III collagen. In the present study, we used species-specific antibodies to either mouse or chick type-IV collagen to demonstrate the ability of fibroblast--derived type-IV collagen to incorporate in the basal lamina of myotubes. In combination cultures of embryonic quail skeletal myoblasts and mouse muscle fibroblasts, antibodies specific for mouse type-IV collagen revealed the deposition of type-IV collagen on the surface of quail myotubes in the pattern typical of the beginning of basement-membrane formation. Control cultures consisting of only quail muscle cells containing myoblasts and fibroblasts demonstrated no such reaction with these antibodies. Deposits of mouse type-IV collagen were also observed on the surface of quail myotubes when conditioned medium from mouse muscle fibroblasts was added to quail myoblast cultures. Similarly, in combination cultures of mouse myoblasts and chick muscle fibroblasts, chick type-IV-collagen deposits were identified on the surface of mouse myotubes. These results indicate that type-IV collagen synthesized by muscle fibroblasts may be incorporated into the basal lamina forming on the plasmalemma of myotubes, and may explain ultrastructural studies by Lipton on the contribution of fibroblasts to the formation of basement membranes in skeletal muscle.     

Regulation of Human (Caco-2) Intestinal Epithelial Cell Differentiation by Extracellular Matrix Proteins

Extracellular matrix regulation of intestinal epithelial differentiation may affect development, differentiation during migration to villus tips, healing, inflammatory bowel disease, and malignant transformation. Cell culture studies of intestinal epithelial biology may also depend on the matrix substrate used. We evaluated matrix effects on differentiation and proliferation in human intestinal Caco-2 epithelial cells, a model for intestinal epithelial differentiation. Proliferation, brush border enzyme specific activity, and spreading were compared in cells cultured on tissue culture plastic with interstitial collagen I and the basement membrane constituents collagen IV and laminin. Each matrix significantly increased alkaline phosphatase, dipeptidyl peptidase, lactase, sucrase-isomaltase, and cell spreading in comparison to plastic. However, the basement membrane proteins collagen IV and laminin further promoted all four brush border enzymes but inhibited spreading compared to collagen I. Proliferation was most rapid on type I collagen and slowest on laminin and tissue culture plastic. Basement membrane matrix proteins may promote intestinal epithelial differentiation and inhibit proliferation compared with interstitial collagen I.     

Collagen-binding proteins secreted by type II pneumocytes in culture

The alveolar epithelial basement membrane is believed to play important roles in lung development, in maintaining normal alveolar architecture, and in guiding repair following lung injury. However, little is known about the formation of this structure, or of the mechanisms which mediate interactions between the epithelium and specific matrix macromolecules. Since type IV collagen is a major structural component of basement membranes, we investigated the production of type IV collagen-binding proteins by primary cultures of rat lung type II pneumocytes. Cultures were labeled for up to 24 h with 3H-labeled amino acids or [3H]mannose. Soluble collagen-binding proteins which accumulated in the culture medium were isolated by chromatography on collagen-Sepharose and examined by SDS-polyacrylamide gel electrophoresis. The major type IV collagen-binding protein (CBP1) was identified as fibronectin. We also identified a novel disulfide-bonded collagen-binding glycoprotein (CBP2; Mr = 45,000, reduced). This protein was not recognized by polyclonal antibodies to fibronectin, and showed no detectable binding to denatured type I collagen. The protein was resolved from fibronectin and partially purified by sequential chromatography on gelatin and type IV collagen-Sepharose. We suggest that type II pneumocyte-derived collagen-binding proteins contribute to the formation of the epithelial basement membrane and/or mediate the attachment of these cells to collagenous components of the extracellular matrix.     

Organization of intestinal epithelial cells into multicellular structures requires laminin and functional actin microfilaments

Epithelial cell organization into multicellular structures is a critical biological process required for both organogenesis and repair following injury. The basement membrane and the cytoskeleton have important roles in this process; however, the functions of individual components of basement membrane and cytoskeleton are poorly understood. We used IEC-6 cells, a rat intestinal crypt cell line, grown on a three-dimensional gel of reconstituted basement membrane as a model system to determine which extracellular matrix and cytoskeletal components mediate intestinal epithelial cell organization. The cells entered the gel and formed hollow, tubular structures that resembled intestinal crypts. These structures were characterized by a single layer of polarized cells with apical tight junctions and microvilli on the luminal surface. Antiserum to laminin and the pentapeptide Tyr-Ile-Gly-Ser-Arg (which prevents cell attachment to laminin) inhibited this organization, but a control pentapeptide (Tyr-Tyr-Gly-Asp-Ala) and antiserum to collagen IV did not. Cytochalasin B, which interferes with actin microfilament polymerization, also inhibited organization of cells into multicellular structures, but vinblastine and Colcemid, which disrupt microtubules, and cycloheximide, which inhibits protein synthesis, did not. We conclude that organization of intestinal epithelial cells on a basement membrane into multicellular structures results from specific interactions between cells and laminin and requires intact actin microfilaments.     

Cells that emerge from embryonic explants produce fibers of type IV collagen

Double immunofluorescence staining experiments designed to examine the synthesis and deposition of collagen types I and IV in cultured explants of embryonic mouse lung revealed the presence of connective tissue-like fibers that were immunoreactive with anti-type IV collagen antibodies. This observation is contrary to the widely accepted belief that type IV collagen is found only in sheet-like arrangements beneath epithelia or as a sheath-like layer enveloping bundles of nerve or muscle cells. The extracellular matrix produced by cells that migrate from embryonic mouse lung rudiments in vitro was examined by double indirect immunofluorescence microscopy. Affinity-purified monospecific polyclonal antibodies were used to examine cells after growth on glass or native collagen substrata. The data show that embryonic mesenchymal cells can produce organized fibers of type IV collagen that are not contained within a basement membrane, and that embryonic epithelial cells deposit fibers and strands of type IV collagen beneath their basal surface when grown on glass; however, when grown on a rat tail collagen substratum the epithelial cells produce a fine meshwork. To our knowledge this work represents the first report that type IV collagen can be organized by cells into a fibrous extracellular matrix that is not a basement membrane.     

Enhanced synthesis of basement membrane proteins during the differentiation of rat mammary tumour epithelial cells into myoepithelial-like cells in vitro

Rama 25, an epithelial cell line obtained from a dimethylbenzanthracene-induced rat mammary tumour differentiates spontaneously in culture forming elongated myoepithelial-like cells. The elongated cells form multilayered ridge structures from which cultures of elongated cells, relatively uncontaminated by epithelial cells, can be obtained. By using immunofluorescence techniques, both the elongated cells and the cells in ridges, but not undifferentiated Rama 25 cells, have been demonstrated to synthesize three basement membrane proteins, laminin, type IV collagen, and fibronectin. The identity of these basement membrane proteins has been confirmed by immunoprecipitation. These proteins appear to be located in a fibrillar extracellular matrix. We suggest that the ability to synthesize basement membrane proteins by mammary epithelial cells in vitro on plastic is a characteristic of myoepithelial-like cells.     

Regulation of mammary epithelial cell function: a role for stromal and basement membrane matrices

Summary Interactions between epithelial cells and their environment are critical for normal function. Mammary epithelial cells require hormonal and extracellular matrix (ECM) signalling for the expression of tissue specific characteristics. With regard to ECM, cultured mammary epithelial cells synthesize and secrete milk proteins on stromal collagen I matrices. The onset of function coincides both with morphogenesis of a polarized epithelium and with deposition of basement membrane ECM basal to the cell layer. Mammary specific morphogenesis and biochemical differentiation is induced if mammary cells are cultured directly on exogenous basement membrane (EHS). Thus ECM may effect function by the concerted effect of permissivity for cell shape changes and the direct biochemical signalling of basement membrane molecules.A model is discussed where initial ECM control of mammary epithelial cell function originates in the interstitial matrix of stroma and subsequently transfers to the basement membrane when the epithelial cells have accumulated and deposited an organized basement membrane matrix.Dedicated to Professor Stuart Patton on the occasion of his 70th birthday.     

Collagen VI is a basement membrane component that regulates epithelial cell–fibronectin interactions

Collagen VI is a heterotrimer composed of three α chains (α1, α2, α3) widely expressed throughout various interstitial matrices. Collagen VI is also found near the basement membranes of many tissues where it serves as an anchoring meshwork. The aim of this study was to investigate the distribution and role of collagen VI at the epithelial-stromal interface in the intestine. Results showed that collagen VI is a bona fide epithelial basal lamina component and constitutes the major collagen type of epithelial origin in this organ. In vitro, collagen VI co-distributes with fibronectin. Targeted knockdown of collagen VI expression in intestinal epithelial cells was used to investigate its function. Depletion of collagen VI from the matrix led to a significant increase in cell spreading and fibrillar adhesion formation coinciding with an upregulation of fibronectin expression, deposition and organization as well as activation of myosin light chain phosphorylation by the myosin light chain kinase and Rho kinase dependent mechanisms. Plating cells deficient for collagen VI on collagen VI rescued the phenotype. Taken together, these data demonstrate that collagen VI is an important basal lamina component involved in the regulation of epithelial cell behavior most notably as a regulator of epithelial cell-fibronectin interactions.     

Changes in the distribution of type IV collagen,laminin, proteoglycan,and fibronectin during mouse tooth development

The distribution of certain basement membrane (BM) components including type IV collagen, laminin, BM proteoglycan, and fibronectin was studied in developing mouse molar teeth, using antibodies or antisera specific for these substances in indirect immunofluorescence. At the onset of cuspal morphogenesis, type IV collagen, laminin, and BM proteoglycan were found to be present throughout the basement membranes of the tooth. Fibronectin was abundant under the inner enamel epithelium at the region of differentiating odontoblasts and also in the mesenchymal tissues. After the first layer of predentin had been secreted by the odontoblasts at the epithelial-mesenchymal interface, laminin remained in close association with the epithelial cells whereas type IV collagen, BM proteoglycan, and fibronectin were distributed uniformly throughout this area. Later when dentin had been produced and the epithelial cells had differentiated into ameloblasts, basement membrane components disappeared from the cuspal area. These matrix components were not detected in dentin while BM proteoglycan and fibronectin were present in predentin. The observed changes in the collagenous and noncollagenous glycoproteins and the proteoglycan appear to be closely associated with cell differentiation and matrix secretion in the developing tooth.     

Expression of extracellular matrix components is regulated by substratum

Reconstituted basement membranes and extracellular matrices have been demonstrated to affect, positively and dramatically, the production of milk proteins in cultured mammary epithelial cells. Here we show that both the expression and the deposition of extracellular matrix components themselves are regulated by substratum. The steady-state levels of the laminin, type IV collagen, and fibronectin mRNAs in mammary epithelial cells cultured on plastic dishes and on type I collagen gels have been examined, as has the ability of these cells to synthesize, secrete, and deposit laminin and other, extracellular matrix proteins. We demonstrate de novo synthesis of a basement membrane by cells cultured on type I collagen gels which have been floated into the medium. Expression of the mRNA and proteins of basement membranes, however, are quite low in these cultures. In contrast, the levels of laminin, type IV collagen, and fibronectin mRNAs are highest in cells cultured on plastic surfaces, where no basement membrane is deposited. It is suggested that the interaction between epithelial cells and both basement membrane and stromally derived matrices exerts a negative influence on the expression of mRNA for extracellular matrix components. In addition, we show that the capacity for lactational differentiation correlates with conditions that favor the deposition of a continuous basement membrane, and argue that the interaction between specialized epithelial cells and stroma enables them to create their own microenvironment for accurate signal transduction and phenotypic function.     

Synthesis of basement membrane proteins by rat mammary epithelial cells

A mammary epithelial cell line, Rama 25, growing on plastic, deposits fibronectin, type IV collagen, and laminin in punctate structures located beneath the basal surface of the cells. When grown on the surface of collagen gels, Rama 25 cells deposit these basement membrane proteins in a continuous layer between the basal surface of the cells and the surface of the collagen matrix. Rama 25 cells also penetrate the collagen matrix forming rudimentary duct-like structures. These structures are surrounded by a discontinuous layer of basement membrane proteins. The ducts of fetal and neonatal rat mammary glands contain few mature myoepithelial cells and our results suggest that some mammary epithelial cells, in contact with a collagenous stroma, are capable of synthesizing a basal lamina-like structure.     

The role of the arginine-glycine-aspartic acid-directed cellular binding to type I collagen and rat mesenchymal cells in colorectal tumour differentiation

The relationship between the adhesion of five human colorectal carcinoma cell lines to extracellular matrix (ECM) proteins, namely type I collagen, type IV collagen, fibronectin, laminin and basement membrane extract (Matrigel), and the ability of these cells to express morphological differentiation when grown in a basement membrane extract (Matrigel) or on normal rat mesenchymal cells has been examined. Two cell lines, SW1222 and HRA-19, organised into glandular structures, with well-defined polarity when cultured on both substrata as well as in three-dimensional (3D) collagen gel culture as previously shown. The remaining three cell lines (SW620, SW480 and HT29) grew as loose aggregates or as they would normally grow on tissue culture plastic. Addition to the culture medium of a hexapeptide, containing the cell-matrix recognition sequence arginine-glycine-aspartic acid (RGD), inhibited attachment and glandular formation of SW1222 and HRA-19 when these cells were grown on living mesenchymal cells, but not in Matrigel. The morphological differentiation of HRA-19 cells in 3D-collagen was also inhibited by the same RGD-containing peptide, as previously shown for SW1222 cells. Attachment of the remaining three cell lines was inhibited on mesenchyme but not in Matrigel, further supporting the specificity of the peptide effect on epithelial-mesenchymal binding. In conclusion we have shown that colorectal tumour cells are able to bind ECM proteins and that the cellular binding is an essential step in the induction of the morphological differentiation seen on living mesenchymal cells, in basement membrane extracts and in type I collagen gel.(ABSTRACT TRUNCATED AT 250 WORDS)     

Matrix metalloproteinases and their role in the renal epithelial mesenchymal transition

Tubular cell epithelial-mesenchymal transition (EMT) is a fundamental contributor to renal fibrosis. The aim of this study was to investigate the activity of different matrix metalloproteinases by immunohistochemistry and gel-zymography in a model of chronic canine kidney disease. Immunohistochemistry for antibodies against MMP-9, MMP-2, MMP-13, MMP-14 and TIMP-2 was performed on 28 renal biopsy specimens. Selected cases were chosen for gelatin zymography. In moderate and severe tubulo-interstitial damage, increased expression of MMP-2 was noted. A peculiar staining pattern for MMP-2 in variable-sized vesicles, corresponding to the area of basement membrane splitting, was observed. The immunoexpression of MMP-9 and TIMP-2 was reduced in the same cases, compared to control dogs. The splitting of the membrane suggests an active role of this gelatinase in the disruption of type-IV collagen, the main basement membrane component, confirmed by MMP2 gelatinolytic activity by gel-zymography. These data could provide the basis for clinical trials examining the potential benefits of selective MMP-2 inhibitors in dogs with chronic kidney disease.     

Distribution and synthesis of type V collagen in the rat mammary gland

In the 100-day-old virgin and lactating rat mammary glands, type V collagen is mainly present in the interstitial connective tissue and in association with blood vessels. It is not present in the basement membrane region surrounding the ducts in mature virgin glands but is present in this region in neonatal and lactating glands. Ultrastructural localization of type V collagen reveals that it is mainly located on the basal surface (i.e., the surface in contact with the basement membrane) of epithelial but not myoepithelial cells. In addition, type V collagen is located on some interstitial collagen fibers and on a large number of granules that are in close proximity to the basal surface of both epithelial and myoepithelial cells. Immunofluorescence and biochemical studies indicate that several clonal mammary fibroblastic cell lines synthesize type V collagen in vitro. In some cell lines, type V collagen is secreted as an extensive fibrillar meshwork on the surface of the cells, whereas in other cell lines, it is secreted beneath the cells around their periphery. A number of mammary epithelial and myoepithelial-like cells, however, do not synthesize type V collagen in vitro.     

Cellular and molecular partners involved in gut morphogenesis and differentiation.

The intestinal mucosa represents an interesting model to study the cellular and molecular basis of epithelial-mesenchymal cross-talk participating in the development and maintenance of the digestive function. This cross-talk involves extracellular matrix molecules, cell-cell and cell-matrix adhesion molecules as well as paracrine factors and their receptors. The cellular and molecular unit is additionally regulated by hormonal, immune and neural inputs. Such integrated cell interactions are involved in pattern formation, in proximodistal regionalization, in maintenance of a gradient of epithelial proliferation and differentiation, and in epithelial cell migration. We focus predominantly on two aspects of these integrated interactions in this paper: (i) the role of basement membrane molecules, namely laminins, in the developmental and spatial epithelial behaviour; and (ii) the importance of the mesenchymal cell compartment in these processes.     

Immunocytochemical localization of extracellular-matrix proteins in relation to rat intestinal morphogenesis

Various extracellular-matrix proteins were detected by indirect immunofluorescence in rat intestine at various stages of development ranging from 14 days of gestation to the adult stage. At the earliest stage studied, laminin, nidogen and type-IV collagen were present at the epithelial/mesenchymal interface, whereas fibronectin and type-III procollagen were found throughout the whole mesenchyme. We were able to relate some changes in the staining patterns of extracellular-matrix proteins to morphogenetic processes. As early as 15 days of gestation, i.e. before villus formation, modifications in the distribution or in the staining intensity of all of the antigens within the mesenchyme paralleled the orientation and segregation of mesenchymal cells in the region surrounding the basal membrane and in the presumptive peripheral muscular layers. During villus outgrowth, the transient disappearance of fibronectin and particularly type-III procollagen from the top of the protruding villus core was evident. During the perinatal period, i.e. when crypts develop, the linear staining for the basal-membrane proteins became restricted to the base of the villi, their labelling along the remaining portion of the villi being more irregular. In mature rat intestine, no major modifications in matrix proteins along the crypt-villus axis in relation to epithelial differentiation were found, except that the labelling for fibronectin and type-III procollagen, which are at this stage more closely related to the basement membrane, was less pronounced in the upper part of villi.     

Distribution of entactin in the basement membrane of the rat mammary gland. Evidence for a non-epithelial origin

Entactin, a sulfated glycoprotein with a molecular weight (MW) of about 150 kD, is present in vascular basement membranes and in the interstitial connective tissue of the mammary glands of virgin rats. It does not appear to be present in the basement membrane surrounding the mammary ductal system. However, in lactating mammary glands entactin is also present in the basement membrane region surrounding the secretory alveoli. Ultrastructural localisation of entactin reveals that it is present on the basal surface of epithelial cells, with patchy staining in the lamina lucida and lamina densa. Entactin also appears to be associated with interstitial collagen fibres. Mammary fibroblastic cells in culture are able to produce entactin, whereas mammary epithelial and myoepithelial cells, which synthesise the basement membrane proteins laminin and type IV collagen, fail to synthesise entactin.