Tenascin/hexabrachion in human skin: biochemical identification and localization by light and electron microscopy

Tenascin/hexabrachion is a large glycoprotein of the extracellular matrix. Previous reports have demonstrated that tenascin is associated with epithelial-mesenchymal interfaces during embryogenesis and is prominent in the matrix of many tumors. However, the distribution of tenascin is more restricted in adult tissues. We have found tenascin to be present in normal human skin in a distribution distinct from other matrix proteins. Immunohistochemical studies showed staining of the papillary dermis immediately beneath the basal lamina. Examination of skin that had been split within the lamina lucida of the basement membrane suggested a localization of tenascin beneath the lamina lucida. In addition, there was finely localized staining within the walls of blood vessels and in the smooth muscle bundles of the arrectori pilorem. Very prominent staining was seen around the cuboidal cells that formed the basal layer of sweat gland ducts. The sweat glands themselves did not stain. The distribution of tenascin in the papillary dermis was studied at high resolution by immunoelectron microscopy. Staining was concentrated in small amorphous patches scattered amongst the collagen fibers beneath the basal lamina. These patches were not associated with cell structures, collagen, or elastic fibers. Tenascin could be partially extracted from the papillary dermis by urea, guanidine hydrochloride, or high pH solution. The extracted protein showed a 320-kD subunit similar to that purified from fibroblast or glioma cell cultures. We have developed a sensitive ELISA assay that can quantitate tenascin at concentrations as low as 5 ng/ml. Tests on extracts of the papillary dermis showed tenascin constituted about 0.02-0.05% of the protein extracted.     

Induction of tenascin in healing wounds

The distribution of the extracellular matrix glycoprotein, tenascin, in normal skin and healing skin wounds in rats, has been investigated by immunohistochemistry. In normal skin, tenascin was sparsely distributed, predominantly in association with basement membranes. In wounds, there was a marked increase in the expression of tenascin at the wound edge in all levels of the skin. There was also particularly strong tenascin staining at the dermal-epidermal junction beneath migrating, proliferating epidermis. Tenascin was present throughout the matrix of the granulation tissue, which filled full-thickness wounds, but was not detectable in the scar after wound contraction was complete. The distribution of tenascin was spatially and temporally different from that of fibronectin, and tenascin appeared before laminin beneath migrating epidermis. Tenascin was not entirely codistributed with myofibroblasts, the contractile wound fibroblasts. In EM studies of wounds, tenascin was localized in the basal lamina at the dermal-epidermal junction, as well as in the extracellular matrix of the adjacent dermal stroma, where it was either distributed homogeneously or bound to the surface of collagen fibers. In cultured skin explants, in which epidermis migrated over the cut edge of the dermis, tenascin, but not fibronectin, appeared in the dermis underlying the migrating epithelium. This demonstrates that migrating, proliferating epidermis induces the production of tenascin. The results presented here suggest that tenascin is important in wound healing and is subject to quite different regulatory mechanisms than is fibronectin.     

Tenascin is associated with chondrogenic and osteogenic differentiation in vivo and promotes chondrogenesis in vitro

The tissue distribution of the extracellular matrix glycoprotein, tenascin, during cartilage and bone development in rodents has been investigated by immunohistochemistry. Tenascin was present in condensing mesenchyme of cartilage anlagen, but not in the surrounding mesenchyme. In fully differentiated cartilages, tenascin was only present in the perichondrium. In bones that form by endochondral ossification, tenascin reappeared around the osteogenic cells invading the cartilage model. Tenascin was also present in the condensing mesenchyme of developing bones that form by intramembranous ossification and later was present around the spicules of forming bone. Tenascin was absent from mature bone matrix but persisted on periosteal and endosteal surfaces. Immunofluorescent staining of wing bud cultures from chick embryos showed large amounts of tenascin in the forming cartilage nodules. Cultures grown on a substrate of tenascin produced more cartilage nodules than cultures grown on tissue culture plastic. Tenascin in the culture medium inhibited the attachment of wing bud cells to fibronectin-coated substrates. We propose that tenascin plays an important role in chondrogenesis by modulating fibronectin-cell interactions and causing cell rounding and condensation.     

Tenascin induction in tenascin nonproducing carcinoma cell lines in vivo and by TGF-β1 in vitro

Tenascin, a novel six-armed extracellular-matrix glycoprotein, is expressed in a temporally and spatially restricted pattern during carcinogenesis in association with stromal-epithelial interactions. In this study, we have tested the hypothesis that tenascin expression depends upon the change of the cellular environment from in vitro to in vivo. The distribution and alterations in the expression of tenascin were compared between in vitro and in vivo studies in a variety of human epithelial- and nonepithelial-derived cell lines. When cell lines were transplanted into nude mice, all xenografts induced host-mouse-stroma-derived tenascin. Four carcinoma-derived cell lines and all sarcoma-derived lines, which secreted tenascin in vitro, were found to produce human tenascin after transplantation. Furthermore, three carcinoma-derived cell lines, A431, HEp-2, and MCF7, which did not synthesize tenascin in vitro, did synthesize human tenascin after transplantation. These tenascin nonproducing carcinoma cell lines did not express tenascin mRNA in vitro. The addition of TGF-β1 to the culture medium induced the synthesis and secretion of tenascin, but TGF-β2 and bFGF were less effective. TGF-β1 also induced other extracellular-matrix components, fibronectin and laminin. TGF-β1 did not induce tenascin in tenascin nonproducing carcinoma cell lines, such as WiDr and A549, in which human tenascin was not induced after transplantation. We have established an in vitro system in which tenascin is induced by the diffusible factor TGF-β1. This system could shed light on the mechanism of induction of human tenascin observed in vivo in tenascin nonproducing carcinoma cell lines. © 1994 wiley-Liss, Inc.     

Epithelial induction of stromal tenascin in the mouse mammary gland: from embryogenesis to carcinogenesis

The distribution of the extracellular matrix glycoprotein tenascin was studied by immunofluorescence in the developmental history of the mouse mammary gland from embryogenesis to carcinogenesis. Tenascin appeared only in the mesenchyme immediately surrounding the epithelia just starting morphogenesis, that is, in embryonic mammary glands from 13th to 16th day of gestation, in mammary endbuds which are a characteristic structure starting development during maturation of the mammary gland, and in the stroma of malignant mammary tumors. However, tenascin was absent in the elongating ducts of embryonic, adult, proliferating, and involuting mammary glands and preneoplastic hyperplastic alveolar nodules. The transplantation of embryonic submandibular mesenchyme into adult mammary glands induces the development of duct-alveolus nodules, which morphologically resemble developing endbuds. Tenascin reappeared around those nodules during the initial stages of their development. Tenascin expression could be induced experimentally in several ways. First, tenascin was detected at the site where the first mammary tumor cells GMT-L metastasized. Second, tenascin was detected in the connective tissue in the tumors derived from the injected C3H mammary tumor cell line CMT315 into Balb/c nude mouse. Cross-strain marker anti-CSA antiserum clearly showed that the tenascin-positive fibroblasts were of Balb/c origin. Third, when embryonic mammary epithelium was explanted on to embryonic mammary fat pad cultures, the mesenchymal cells condensed immediately surrounding the epithelium. Tenascin was detected in these condensed cells. From these three observations we conclude that both embryonic and neoplastic epithelium induced tenascin synthesis in their surrounding mesenchyme.     

Distribution of type-VII collagen in xenografted human carcinomas

The distribution of type-VII collagen, the main molecular component of the anchoring fibrils (AF) attaching the basal lamina (BL, lamina densa of the basement membrane) to the surrounding connective tissue, was investigated in four xenografted human carcinomas of the hypopharynx (H-Stg 1), the lung (L 261), the sigmoid colon (CA 1), and the rectum (R 85). The studies were performed with a recently prepared, affinity-purified and highly specific antibody to type-VII collagen by using the indirect immunofluorescence and the APAAP (alkaline phosphatase anti-alkaline phosphatase) techniques. For comparison, the localization of the intrinsic BL components laminin and type-IV collagen were additionally analyzed in all four carcinomas. It was shown that type-VII collagen usually colocalized to laminin and type-IV collagen and was deposited at the borderline between carcinoma cell clusters and the surrounding strands of connective tissue in a similar, but more diffuse and less continuous distribution than both intrinsic BL components. In the squamous cell carcinoma H-Stg 1 and the adenocarcinoma L 261, type-VII collagen was additionally accumulated in enlarged extracellular spaces between carcinoma cells, away from the contact zone to the connective tissue and again colocalized to laminin and type-IV collagen. Numerous carcinoma cells of both xenografts showed remarkable intracytoplasmic immunoreactivity for the antibody to type-VII collagen. Even in the case of the gastrointestinal carcinomas CA 1 and R 85, faint immunoreactivity for type-VII collagen was found at the contact zone between the mucosal epithelium and the surrounding connective tissue. These results confirm that epithelial carcinoma cells are obviously involved with the synthesis of the main molecular component of AF usally attaching the BL to the adjacent connective tissue and hint at a possible correlation between the localization of type-VII collagen and the observed pattern of the BL. However, it cannot be decided whether there is a direct causal relation between both phenomena or whether they are both the consequence of an independent but common cause, such as abnormal cellular differentiation of carcinoma cells. In no case, can the discontinuities in the distribution of type-VII collagen be explained by active tumor cell invasion since xenografted human carcinomas neither invade nor metastasize.     

Differentiation of pericytes in culture is accompanied by changes in the extracellular matrix

Summary We have previously reported that pericytes derived from retinal and brain microvessels aggregate into nodules soon after reaching confluence. Nodule formation involves a reorganization of the cells resulting in the presence of sparse cells, confluent monolayers, multilayers, sprouts, and nodules within the same culture dish. Extracellular calcification occurs only within the nodules, demonstrating that pericytes are capable of undergoing osteogenic differentiation in culture and that this differentiation is related to nodule formation. Using immunofluorescence we have now studied the distribution of laminin, type IV collagen, type X collagen, and tenascin in pericyte cultures during nodule formation. These matrix macromolecules were also identified by a combination of biochemical techniques, including Northern blot hybridization, immunoblotting and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A molecule that seems to be related to type X collagen was demonstrated by the presence of a pepsin-resistant, collagenase-sensitive polypeptide of molecular weight approximately 45 kDa. The production of laminin, type X-related collagen, and tenascin by pericytes has not been previously reported. Our results suggest that the synthesis or distribution or both of these molecules is dependent on the state of pericyte differentiation. The expression of laminin, type IV collagen, and type X-related collagen was maximal in multilayer areas, sprouts, and nodules. Tenascin appeared homogeneously distributed in monolayer and multilayer areas; when calcified nodules were present, the anti-tenascin serum preferentially decorated a discrete area circumscribing the nodules. Tenascin and type X collagen have been found transiently in vivo preceding calcification; their possible role in this process is not known. Our results also suggest an association between laminin, type IV collagen, and calcification. The in vitro experimental system described here may help to clarify the role of matrix macromolecules in the calcification process.     

Distribution of extracellular matrix proteins in odontogenic tumours and developing teeth

The distribution of two cellular fibronectins (cFn), tenascin, laminin, as well as type VII collagen was studied in 14 benign odontogenic tumours of epithelial (ameloblastoma) and epithelial-ectomesenchymal (ameloblastic fibroma) origins, as well as in developing human teeth by immunocytochemical means using monoclonal antibodies (Mabs). An extradomain sequence-A-containing form of cFn (EDA-cFn) was seen in the extracellular matrix (ECM) of all tumours studied and in the mesenchyme of the developing tooth germs, indicating that cFn in these tissues are predominantly produced locally. A form of cFn containing an oncofetal domain (Onc-cFn), hitherto found only in carcinomas, was detected focally in the stroma of most ameloblastomas but was absent from ameloblastic fibromas and tooth germs. Tenascin was strongly expressed in the basement membrane (BM) zone of all odontogenic tumours and in that of the early tooth germs. Focal absence of laminin and type VII collagen from the BM of some ameloblastomas and the presence of Onc-cFn in the ECM of most ameloblastomas may correlate with their aggressive behaviour. The results also suggest that EDA-cFn and tenascin are involved in epithelial-mesenchymal interactions during tooth development and in odontogenic tumours.     

Distribution of extracellular matrix proteins in odontogenic tumours and developing teeth.

The distribution of two cellular fibronectins (cFn), tenascin, laminin, as well as type VII collagen was studied in 14 benign odontogenic tumours of epithelial (ameloblastoma) and epithelial-ectomesenchymal (ameloblastic fibroma) origins, as well as in developing human teeth by immunocytochemical means using monoclonal antibodies (Mabs). An extradomain sequence-A-containing form of cFn (EDA-cFn) was seen in the extracellular matrix (ECM) of all tumours studied and in the mesenchyme of the developing tooth germs, indicating that cFn in these tissues are predominantly produced locally. A form of cFn containing an oncofetal domain (Onc-cFn), hitherto found only in carcinomas, was detected focally in the stroma of most ameloblastomas but was absent from ameloblastic fibromas and tooth germs. Tenascin was strongly expressed in the basement membrane (BM) zone of all odontogenic tumours and in that of the early tooth germs. Focal absence of laminin and type VII collagen from the BM of some ameloblastomas and the presence of Onc-cFn in the ECM of most ameloblastomas may correlate with their aggressive behaviour. The results also suggest that EDA-cFn and tenascin are involved in epithelial-mesenchymal interactions during tooth development and in odontogenic tumours.     

Tenascin distribution in the normal human breast is altered during the menstrual cycle and in carcinoma

Tenascin is a novel extracellular matrix glycoprotein which appears to have a major role in tissue development. Previous studies have stated that tenascin is absent from the normal human, rat and mouse breast, its distribution being restricted to embryonic and malignant mammary tissues. No previous studies have investigated tenascin distribution as a function of the normal menstrual cycle. Therefore this study addresses the cyclical appearance of tenascin in the normal breast and associated changes in distribution in preinvasive cancer (carcinoma-in-situ) and invasive infiltrating ductal carcinoma. Tenascin is present in the normal human adult mammary gland, principally in the basement membrane, sub-basement-membrane zone and delimiting layer of fibroblasts around the ductules. Both the distribution and quantity of tenascin change during the menstrual cycle. In carcinoma-in-situ (preinvasive cancer) tenascin is present in the attenuated basement membrane/sub-basement-membrane zone around the expanded ductules and in small amounts in the stroma. In infiltrating ductal carcinoma, tenascin is absent from the remnants of the basement membrane and sub-basement-membrane zone but greatly increased in the adjacent intralobular and interlobular stroma. Therefore, if tenascin is used as a basement membrane/sub-basement-membrane marker for distinguishing carcinoma-in-situ from invasive ductal carcinoma, the time of the menstrual cycle is of importance in interpreting the biopsy appearance. This study suggests that the optimal time for biopsy is between weeks 3 and 4 of the cycle, to avoid confusion between the normal low levels of tenascin (due to hormonal status) and those due to microinvasive disease.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and partial characterization of Xenopus laevis tenascin from the XTC cell line.

We report here the purification of tenascin, an extracellular matrix molecule involved in the control of morphogenesis, from the conditioned medium of the Xenopus XTC cell line. Tenascin was purified by affinity chromatography on a column of the monoclonal antibody mAb TnM1; the molecule eluted from this column has a relative molecular mass of 210 kDa after reduction. Electrophoretic analysis under non-reducing conditions shows that the purified components are oligomeric disulfide-linked complexes which barely enter a 4% polyacrylamide gel. Upon rotary shadowing these molecules appear to possess a central globular domain to which pairs or triplets of arms are attached. Polyclonal antibodies have been raised against purified Xenopus tenascin. They recognise specifically the antigen on Western blots of XTC conditioned medium and adult brain, by immunofluorescence, these antibodies reveal large amounts of tenascin in the secretory vesicles as well as in the extracellular matrix of XTC cells. In the Xenopus tadpole, they stain the developing cartilage, the basal lamina of skin epidermis, myotendinous ligaments and restricted regions of the central nervous system.     

Origin of laminin in the extracellular matrix of human tumor xenografts in nude mice

Monoclonal antibodies reacting exclusively with laminin of human origin and a polyclonal antibody reacting with both murine and human laminin were used to immunohistochemically study the extracellular matrix of four human tumors grown as xenografts in nude mice: a lung carcinoma and a yolk sac carcinoma because they produced cell associated laminin in vitro; and two hepatocellular carcinomas which did not produce cell associated laminin in vitro. The extracellular matrix of the xenografts of the lung carcinoma and the yolk sac carcinoma contained laminin of both human and murine origin. Xenografts of liver carcinoma contained only laminin of mouse origin. This shows that the malignant cells capable of laminin production in vitro contribute this glycoprotein to the extracellular matrix of the solid tumor formed by them in vivo.     

Downregulation of tenascin expression by glucocorticoids in bone marrow stromal cells and in fibroblasts

Tenascin, a predominantly mesenchymal extracellular matrix (ECM) glycoprotein has a rather restricted tissue distribution, but until now factors that inhibit its expression have not been identified. Glucocorticoids are known to be beneficial for establishment of myelopoiesis in long-term bone marrow cultures. Tenascin was found to be expressed in the bone marrow, and glucocorticoids were found to affect bone marrow tenascin expression. Both tenascin mRNAs and the mRNA of another ECM protein, laminin B1 chain, were drastically downregulated by glucocorticoids during initiation of bone marrow cultures. However, in already established long-term cultures glucocorticoids did not affect laminin B1 chain mRNA levels although tenascin mRNAs continued to be downregulated. Studies with a stromal cell line (MC3T3-G2/PA6) and fibroblasts (3T3) suggested that glucocorticoids act directly on the stromal cells that produce tenascin. In 3T3 cells this downregulation occurred within 12 h of glucocorticoid-treatment, suggesting that glucocorticoids acted through cis regulatory elements of the tenascin gene. We suggest that glucocorticoids in part regulate hematopoiesis by modifying the ECM. Furthermore, downregulation of tenascin expression by glucocorticoids may in part explain the restricted tissue distribution of tenascin in other tissues.     

Expression of J1/tenascin in the crypt-villus unit of adult mouse small intestine: implications for its role in epithelial cell shedding.

The localization of the extracellular matrix recognition molecule J1/tenascin was investigated in the crypt-villus unit of the adult mouse ileum by immunoelectron microscopic techniques. In the villus region, J1/tenascin was detected strongly in the extracellular matrix (ECM) between fibroblasts of the lamina propria. It was generally absent in the ECM at the interface between subepithelial fibroblasts and intestinal epithelium, except for some restricted areas along the epithelial basal lamina of villi, but not of crypts. These restricted areas corresponded approximately to the basal part of one epithelial cell. In J1/tenascin-positive areas, epithelial cells contacted the basal lamina with numerous microvillus-like processes, whereas in J1/tenascin-negative areas the basal surface membranes of epithelial cells contacted their basal lamina in a smooth and continuous apposition. In order to characterize the functional role of J1/tenascin in the interaction between epithelial cells and ECM, the intestinal epithelial cell line HT-29 was tested for its ability to adhere to different ECM components. Cells adhered to substratum-immobilized fibronectin, laminin and collagen types I to IV, but not to J1/tenascin. When laminin or collagen types I to IV were mixed with J1/tenascin, cell adhesion was as effective as without J1/tenascin. However, adhesion was completely abolished when cells were offered a mixture of fibronectin and J1/tenascin as substratum. The ability of J1/tenascin to reduce the adhesion of intestinal epithelial cells to their fibronectin-containing basal lamina suggests that J1/tenascin may be involved in the process of physiological cell shedding from the villus.     

The extracellular matrix of lip wounds in fetal, neonatal and adult mice.

Wound healing in the fetus occurs rapidly, by a regenerative process and without an inflammatory response, resulting in complete restitution of normal tissue function. By contrast, in the adult, wounds heal with scar formation, which may impair function and inhibit further growth. The cellular mechanisms underlying these differing forms of wound healing are unknown but the extracellular matrix (ECM), through its effects on cell function, may play a key role. We have studied the ECM in upper lip wounds of adult, neonatal and fetal mice at days 14, 16 and 18 of gestation. The spatial and temporal distribution of collagen types I, III, IV, V and VI, fibronectin, tenascin, laminin, chondroitin and heparan sulphates were examined immunohistochemically. Results from the fetal groups were essentially similar whilst there were distinct differences between fetus, neonate and adult. Fibronectin was present at the surface of the wound in all groups at 1 h post-wounding. Tenascin was also present at the wound surface but the time at which it was first present differed between fetus (1 h), neonate (12 h) and adult (24 h). The time of first appearance paralleled the rate of wound healing which was most rapid in the fetus and slowest in the adult. Tenascin inhibits the cell adhesion effect of fibronectin and during development the appearance of tenascin correlates with the initiation of cell migration. During wound healing the appearance of tenascin preceded cell migration and the rapid closure of fetal wounds may be due to the early appearance of tenascin in the wound. Collagen types I, III, IV, V and VI were present in all three wound groups but the timing and pattern of collagen deposition differed, with restoration of the normal collagen pattern in the fetus and a scar pattern in the adult. This confirms that lack of scarring in fetal wounds is due to the organisation of collagen within the wound and not simply lack of collagen formation. The distribution of chondroitin sulphate differed between normal fetal and adult tissues and between fetal and adult wounds. Its presence in the fetal wound may alter collagen fibril formation. No inflammatory response was seen in the fetal wounds. The differences in the ECM of fetal and adult wounds suggests that it may be possible to alter the adult wound so that it heals by a fetal-like process without scar formation, loss of tissue function or restriction of growth.     

Stimulation of tenascin expression in mesenchyme by epithelial-mesenchymal interactions

Tenascin is an extracellular matrix glycoprotein with an unusually restricted tissue distribution in the developing embryo. The protein was independently discovered by several investigators, and has been given many different names. Synonyms of tenascin include cytotactin, J1, hexabrachion and glioma-mesenchymal extracellular matrix antigen. Whereas fibronectin is expressed rather uniformly in matrices of embryonic mesenchyme, tenascin is found in the mesenchyme at sites of epithelial-mesenchymal interactions. Tenascin is thus found close to epithelial basement membranes but it is probably not an integral basement membrane component. The distribution suggests that developing epithelial cells may produce locally active factors that stimulate tenascin synthesis in the nearby mesenchyme. Tenascin is composed of disulfide-bonded subunits of approximate Mr between 200-280 kD. Using monoclonal antibodies to mouse tenascin, we find two major subunits of Mr 260 and 200 kD from mouse fibroblasts. Work from many laboratories suggests that the different subunits arise by differential splicing of one mRNA. Rotary shadowing electron microscopy of the intact molecule suggests a six-armed structure connected by a central region. However, the different subunits are not co-ordinately expressed during embryogenesis, suggesting that tenascin can exist as different isoforms. The different isoforms may serve distinct functions. The function of tenascin is not well known, but it has been suggested that it alters the adhesive properties of cells and causes cell rounding.     

Tenascin C interacts with ecto-5'-nucleotidase (eN) and regulates adenosine generation in cancer cells

Tenascin C is expressed in invasive human solid tumors; however its specific role in cancer biology remains obscure. Previously, we have found that ecto-5'-nucleotidase (eN) is a marker of ER (-) breast carcinoma and elevated expression correlates with invasive mesenchymal cell phenotype. To investigate for the potential relationship between eN and protein components of the extracellular matrix (ECM) we measured adenosine generation from AMP in cells incubated with soluble ECM proteins. We found that tenascin C was the only ECM component that strongly inhibited ecto-5'-nucleotidase (eN) activity in situ and adenosine generation from AMP (75% inhibition, p < 0.01). The inhibition was comparable to that induced by concanavalin A, a well-defined and strong inhibitor of eN. Resin immobilized tenascin C, but not collagen, and only weakly fibronectin, specifically and quantitatively bound cell-extracted eN. We further developed breast cancer cell line with reduced eN expression and tested changes in cell adhesion on different ECM. Breast cancer cells expressing reduced eN attached 56% weaker (p < 0.05) to immobilized tenascin C. This difference was not detected with other ECM proteins. Finally, control breast cancer cells migrated slower on tenascin C when compared with clone with reduced eN expression. These data suggest that eN is a novel and specific receptor for tenascin C and that the interaction between these proteins may influence cell adhesion and migration and also lead to decreased generation of local adenosine.     

Tenascin: a modulator of cell growth.

The large, multidomain extracellular matrix protein tenascin displays a markedly restricted tissue distribution during embryogenesis and remains present only in a few adult tissues. The protein is reexpressed, however, during wound healing and in the stroma of malignant tumours. While a variety of studies have dealt with the important role of tenascin in the development of neural and non-neural tissues, there is growing evidence that tenascin expression may be associated with proliferation of cells lining these tissues. The presence of repeating domains in tenascin similar to those in epidermal growth factor prompted us to investigate the ability of tenascin to modulate the growth of different cell types. Tenascin was actually found to be mitogenic for several cell types. This mitogenic activity, however, appears to be associated with a region in the fibronectin type III domains. The mitogenic mechanism is clearly distinct from pathways used by peptide growth factors such as epidermal growth factor and platelet-derived growth factor, which activate the intrinsic tyrosine kinase activity of their cell-surface receptors. However, we show that this large extracellular matrix molecule is efficiently internalised and may be processed by responding cells.     

The matrix form of collagen and basal microporosity influence basal lamina deposition and laminin synthesis/secretion by stratified human keratinocytes in vitro

Summary The ability of the collagen matrix form to support the formation of a basal lamina by cultured normal human epidermal keratinocytes (NHEK) was determined using transmission electron microscopy. The collagen matrix forms tested in this study were a) a dry type I collagen film and b) a type I collagen gel. NHEK were grown for 14 days on the following five different substrates: plain plastic culture dishes without the addition of collagen (PP); plain plastic culture dishes overlaid with a dry, aldehyde-crosslinked type I collagen film (DCF-P); plain plastic culture dishes overlaid with an aldehyde-crosslinked type I collagen gel (GEL-P); Millipore Millicell CM microporous membranes overlaid with a dry, aldehyde-crosslinked type I collagen film (DCF-CM); and Millipore Millicell CM microporous membranes overlaid with an aldehyde-crosslinked type I collagen gel (GEL-CM). NHEK maintained for 2 wk on PP and DCF-P were unable to secrete a basal lamina. NHEK grown for 2 wk on the GEL-P and GEL-CM substrates, however, secreted a contiguous basal lamina at the GEL-NHEK interface. To determine if the appearance of this basal lamina correlated with laminin synthesis, laminin was immunoprecipitated from cellular extracts, as well as media from the apical and basal chambers. NHEK grown on the GEL-P substrate synthesized more laminin than did NHEK grown on the other four alternative substrates. In addition, NHEK grown on GEL-CM were able to direct more laminin to the basal compartment than NHEK grown on DCF-CM substrates. Taken together, the data indicate that the matrix form of collagen can influence basal lamina deposition, laminin synthesis, and laminin trafficking in NHEK.     

Tenascin Expression in Vitro and in Vivo: Comparison between Epithelial and Nonepithelial Rat Cell Lines

Tenascin is a novel six-armed extracellular-matrix glycoprotein expressed in association with mesenchymal-epithelial interactions, and its expression is temporally and spatially restricted during organogenesis and carcinogenesis. The distribution and alterations in the expression of fibronectin, laminin, and especially of tenascin, were compared between in vitro and in vivo studies with rat epithelial (hepatocyte-derived) and nonepithelial (sarcoma-derived) cell lines. Immunoprecipitation studies revealed that the production of extracellular-matrix glycoproteins varied among the cell lines. Two ascites-hepatoma-derived cell lines and one sarcoma-derived line were found to synthesize tenascin in vitro. Their major tenascin isoform yielded a molecular weight of 220 kDa under reducing conditions. The other cell lines examined, including all of those derived from normal hepatocytes, were negative for the expression of tenascin. Coculture studies were performed between epithelial and nonepithelial cell lines. No drastic change in tenascin expression was found after coculturing the cells. As an in vivo study, cell lines were transplanted into nude mice. All xenografts of the epithelial lines were associated with a strong positive reaction for extracellular-matrix glycoproteins, and especially for tenasein, in the mouse fibrous stroma adjacent to them. This represents the epithelial induction of stromal tenascin. Whether or not they produced tenascin in vitro, after transplantation none of the epithelial cell lines themselves produced tenascin, whereas both of the nonepithelial cell lines prominently produced tenascin. These findings suggest that, in the process of interactions between epithelial and nonepithelial cells, the expression of tenascin depends on the switch from in vitro to in vivo.