The expression of the genes for entactin, laminin A, laminin B1 and laminin B2 in murine lens morphogenesis and eye development

The temporal expression of the genes for the excellular matrix proteins entactin and the A, B1, and B2 chains of laminin was examined in the eye of the developing mouse embryo by in situ hybridization of their messenger RNAs. Entactin messenger RNA was found in abundance in specific cells. In the 25 somite embryo entactin message was synthesized by mesenchymal cells and, at later stages, by hyalocytes and lens cells in addition. The message was not detectable in corneal epithelium at embryonic stages E15 and E18.5 and at birth but was present in adjacent stromal cells. At the 28 and 38 somite stages, before pigment granules interfered with the detection of silver grains, no entactin message was detected in pigmented epithelial cells, in contrast to the messages for laminin B1 and B2. Entactin was not found in the neural epithelium at any time during development. The distribution of the laminin B1, B2 and A chain messenger RNAs was distinctly different from that of entactin. In particular, during the early stages of development B1 and B2 messages were synthesized by ectodermal, lens, corneal, pigment epithelial and hyaloid cells. In the older embryos cells in the ganglion layer of the retina synthesized B1 and B2 messages but undetectable amounts of entactin or the A chain messages. In general the A chain message was in lower abundance throughout development. The distribution of laminin and entactin messages suggested that the extracellular matrices, which contained both proteins, can be derived either from a single cell type or from the contributions of multiple cell types. The data demonstrate the complexity of extracellular matrix synthesis and assembly in the diverse structures of the developing eye where the temporal expression of specific molecules are tailored to the specific developmental requirements of particular structures.     

Laminin B1 expression is required for laminin deposition into the extracellular matrix of PC12 cells.

The extracellular matrix of rat pheochromocytoma PC12 cells was shown by indirect immunofluorescence to consist of a network of fibronectin. The matrix did not contain laminin. The cells synthesized messenger RNA for fibronectin, laminin B2, and s-laminin but not for entactin or the B1 and A chains of laminin. Laminin B2 but not laminin B1 was detectable in the culture medium and in cell lysates. A full-length cDNA clone for the B1 chain of laminin was constructed in the plasmid p-444, which contains the neomycin-resistance marker and human beta-actin promoter. PC12 cells were transfected with this recombinant plasmid, and stable neomycin-resistant clones were isolated and characterized. Clones that synthesized laminin B1 messenger RNA were found to deposit a laminin-containing matrix. In many of these clones the deposition of the fibronectin matrix was greatly diminished. The laminin matrix was predominantly localized in the intercellular spaces forming a honeycomb pattern. The morphology of the laminin-synthesizing transfected cells was markedly different from the parental cells. The cells grew in tight clusters that were resistant to dissociating agents. It is concluded that the B1 chain of laminin contains information that is required for the formation of a stable laminin-containing extracellular matrix network either by interaction with cell surface receptors or other extracellular matrix components. Furthermore, expression of the laminin B1 gene may be a central regulatory point in determining extracellular matrix composition during embryogenesis.     

Basement membrane components secreted by mouse yolk sac carcinoma cell lines

Three new cell lines (NE, ME, LRD) were cloned from mouse-embryo-derived teratocarcinomas and characterized on the basis of developmental, ultrastructural, and cytochemical criteria as nullipotent embryonal carcinoma (EC), pure parietal yolk sac (PYS) carcinoma and mixed parieto-visceral yolk sac carcinoma respectively. Cell lines NE and ME were composed of a monomorphous cell population; however, the morphology of ME was growth-medium-dependent. LRD was composed of a heterogeneous cell population and formed embryoid bodies. NE secreted soluble laminin, osteonectin, entactin and fibronectin but did not form visible pericellular matrix. ME formed pericellular matrix which was composed of laminin and entactin, but did not contain fibronectin. The LRD cells formed pericellular matrix which was composed of laminin, entactin and fibronectin. Whereas laminin from ME and LRD reacted with polyclonal antibodies and a monoclonal antibody to parietal yolk sac laminin, the laminin from NE cells was unreactive with the monoclonal antibody. Osteonectin was found in the supernatant of LRD and ME, but could not be demonstrated immunohistochemically in the extracellular matrix. We conclude that some extracellular matrix components, such as laminin and fibronectin, are produced not only by yolk sac carcinoma cells but by nullipotent EC as well, although the latter do not assemble them into extracellular matrix. Laminin produced by EC is immunochemically different from laminin secreted by yolk sac carcinoma. The extracellular matrix produced by mixed parieto-visceral yolk sac carcinoma is different from the matrix laid down by the pure PYS in that the latter does not contain fibronectin. The lack of osteonectin in the extracellular matrix of yolk sac carcinoma cells indicates that not all polypeptides secreted by these cell lines are incorporated into the extracellular matrix. The new cell lines described in this paper differ with regard to their capacity to form extracellular matrix and secrete its various components. Hence they could be used for further studies of basement membrane assembly in vitro.     

Effects of alcohols on mouse embryonal carcinoma-substrate adhesion

The effects of ethanol and closely related alcohols on the cell-substrate adhesion of embryonal carcinoma cells were studied in microtiter wells using the enzyme cytochemical alkaline phosphatase technique and an ELISA reader. Three embryonal carcinoma cell lines (NF-1, NE and F9) were used. Prior to plating of cells the wells were coated with laminin, fibronectin or collagen type I. NF-1 cells adhered only to laminin; NE adhered to all substrata and uncoated wells equally well; F9 adhered only to fibronectin and laminin coated wells. Ethanol reduced the binding of cells to laminin and collagen type I but did not affect the binding of NE or F9 cells to fibronectin. The effect of ethanols was dose dependent; it lasted as long as an adequate concentration of this alcohol was maintained in vitro, and it was reversible. Other short chain alcohols inhibited the binding of cells to laminin proportionately to their membrane/buffer partition coefficients. These data show that various embryonal carcinoma cells differ with regards to their capacity to adhere to different extracellular matrix components. Cell adhesion to some but not all substrates can be prevented by ethanol and related short chain alcohols. The effects of alcohols on the adhesion of embryonal carcinoma cells to various substrates may be relevant for the elucidation of the fetal alcohol syndrome.     

Basement membrane components produced by a mouse ascites teratocarcinoma TB 24 : Analysis with monoclonal and polyclonal antibodies

Monoclonal antibodies were produced against two basement membrane glycoproteins, laminin and entactin. The specificity of the antibodies and the absence of cross-reactivity were established by radio-immunoprecipitation of mouse ascites teratocarcinoma cell lysates. This teratocarcinoma, TB24, formed large embryoid bodies filled with extracellular matrix. The major component of the matrix was laminin. Abundant entactin, substantial amounts of fibronectin and rather little collagen type IV were also found in the matrix by biochemical and immunofluorescence analyses. TB24 teratocarcinoma appears to be a good model to test anti-basement membrane antibodies and to isolate certain extracellular matrix components in preparative quantities.     

Effects of alcohols on mouse embryonal carcinoma-substrate adhesion

Summary The effects of ethanol and closely related alcohols on the cell-substrate adhesion of embryonal carcinoma cells were studied in microtiter wells using the enzyme cytochemical alkaline phosphatase technique and an ELISA reader. Three embryonal carcinoma cell lines (NF-1, NE and F9) were used. Prior to plating of cells the wells were coated with laminin, fibronectin or collagen type I. NF-1 cells adhered only to laminin; NE adhered to all substrata and uncoated wells equally well; F9 adhered only to fibronectin and laminin coated wells. Ethanol reduced the binding of cells to laminin and collagen type I but did not affect the binding of NE or F9 cells to fibronectin. The effect of ethanols was dose dependent; it lasted as long as an adequate concentration of this alcohol was maintained in vitro, and it was reversible. Other short chain alcohols inhibited the binding of cells to laminin proportionately to their membrane/buffer partition coefficients. These data show that various embryonal carcinoma cells differ with regards to their capacity to adhere to different extracellular matrix components. Cell adhesion to some but not all substrates can be prevented by ethanol and related short chain alcohols. The effects of alcohols on the adhesion of embryonal carcinoma cells to various substrates may be relevant for the elucidation of the fetal alcohol syndrome.     

Localization and synthesis of entactin in seminiferous tubules of the mouse.

Localization and synthesis of entactin in seminiferous tubules of mouse testis was studied by immunocytochemistry. Frozen sections from adult mice testes were subjected to anti-entactin and anti-laminin immunofluorescence. Both entactin and laminin were localized within the seminiferous tubule basement membrane and intertubular region of the testis. The addition of excess amount of entactin (but not fibronectin), premixed with anti-entactin antiserum, abolished the immunostain. Western blotting showed that a protein extract from a seminiferous tubule basement membrane preparation was recognized by anti-entactin anti-serum and comigrated with recombinant entactin. Enriched fractions of isolated primary Sertoli cells and peritubular myoid cells cultured for 6 days on a glass coverslip were able to synthesize and secrete entactin as detected by immunofluorescence microscopy. Entactin was also produced by TM3 (Leydig-like) and TM4 (Sertoli-like) cell lines as detected by both immunofluorescence and Western blotting. The distribution of entactin vs. laminin within both the cultured primary cells and the TM3 and TM4 cell lines differed. Entactin appeared mainly localized extracellularly. In contrast, laminin was mainly localized intracellularly. The above findings suggested that 1) entactin existed in the seminiferous tubule basement membrane and intertubular region of adult mice testis, co-localized with laminin; 2) entactin was synthesized by the cultured primary Sertoli cells and peritubular myoid cells and the TM3 and TM4 cell lines; 3) entactin was exocytosed with little intracellular accumulation, in contrast to an intracellular accumulation of laminin.     

Colocalization of laminin and fibronectin in bovinelens epithelial cells in vitro

Summary The distribution and organization of the extracellular matrix (ECM) proteins laminin, fibronectin, entactin, and type IV collagen were investigated in primary colonies and secondary cultures of bovine lens epithelial cells using species-specific antisera and indirect immunofluorescence microscopy. Primary cell colonies fixed in formaldehyde and permeabilized with Triton X-100 displayed diffuse clonies. In contrast, thick bundles of laminin and fibronectin were located on the basal cellsurfaces and in between cells in the densely packed center of the colonies, and as “adhesive plaques” and fine extracellular matrix cords in the sparsely populated (migratory) outer edge of the colonies. The distribution of ECM proteins observed in secondary lens epithelial cell cultures was similar to that observed at the periphery of the primary colony. Extraction of the secondary cell cultures with sodium deoxycholate confirmed that laminin and fibronectin were deposited on the basal cell surface. Indeed, the patterns of laminin and fibronectin deposition suggested that these proteins codistribute. These results establish that lens epithelial cells in culture can be used as a model system to study the synthesis and extracellular deposition of the basement membrane proteins, laminin and fibronectin. Supported by Public Health Service grant EY05570 from the National Eye Institute Bethesda, MD.     

Recombinant entactin promotes mouse primary trophoblast cell adhesion and migration through the Arg-Gly-Asp (RGD) recognition sequence [published erratum appears in J Cell Biol 1993 Jul;122(1):279]

In vitro culture of mouse blastocysts during the period coinciding with implantation has revealed that primary trophoblast cells can adhere and migrate in serum-free medium when provided with certain extracellular matrix components, including fibronectin and laminin. Tightly associated with laminin is the glycoprotein, entactin, that may play an important role in basement membrane assembly and cell attachment. Mouse blastocysts were studied using this in vitro model to determine whether entactin was capable of mediating trophoblast invasive activity. Although entactin has never been shown to promote cell migration, we report here that recombinant entactin supported blastocyst outgrowth in a dose-dependent manner, with a maximal effect at 20-50 micrograms/ml. The ability of trophoblast cells to adhere and migrate on entactin was specifically inhibited by anti-entactin antibody, but not by antibodies raised against laminin. The synthetic peptide, Gly-Arg-Gly-Asp-Ser-Pro, that contains the Arg-Gly-Asp (RGD) integrin recognition site, reversibly inhibited entactin-mediated blastocyst outgrowth in a dose- dependent manner, but had no effect on laminin-mediated outgrowth. The synthetic peptide, Gly-Phe-Arg-Gly-Asp-Gly-Gln, that comprises the actual RGD-containing sequence within entactin, promoted trophoblast outgrowth when immobilized on the substratum. Furthermore, a mutated recombinant entactin, altered to contain a Glu in place of Asp at the RGD site, provided no trophoblast cell adhesive activity. We conclude that entactin promotes trophoblast outgrowth through a mechanism mediated by the RGD recognition site, and that it may play an important role during invasion of the endometrial basement membrane at implantation.     

Changes in the rate of laminin and entactin synthesis in F9 embryonal carcinoma cells treated with retinoic acid and cyclic amp

The synthesis of laminin A and B chains, and of entactin, has been measured in murine F9 embryonal carcinoma cells differentiating in response to retinoic acid and cyclic AMP. Undifferentiated cells synthesis low levels of laminin, amounting to approximately 0.02% of the [35S]methionine incorporated into cytoplasmic protein during a 15-min pulse. After 6 days induction, laminin synthesis has increased 15- to 20-fold. Undifferentiated F9 cells synthesise more intracellular laminin B2 chains (Mr 225,000) than B1 chains (Mr 225,000), but the excess B2 chains are apparently not assembled into the secreted laminin molecule. Indirect immunofluorescence shows faint cytoplasmic staining and short fibrils of laminin between the undifferentiated F9 cells.     

The receptor for the basement membrane glycoprotein entactin is the integrin alpha 3/beta 1.

Entactin is a glycoprotein found in basement membranes in complex with laminin, and purified entactin can promote the attachment and spreading of cells. We report here the isolation and identification of the plasma membrane receptor for entactin from PC-3 human prostate carcinoma cells which attach and spread on entactin. The receptor was isolated by affinity chromatography on mouse recombinant entactin-Sepharose of 125I surface-labeled octyl glucoside cell extracts. The receptor, which consisted of two polypeptides of relative molecular masses of 150 and 116 kDa, bound to the entactin-Sepharose matrix in the presence of CaCl2, MgCl2, and MnCl2, and was eluted with EDTA, but not with Arg-Gly-Asp-containing peptides. Utilizing anti-integrin antibodies, the heterodimeric receptor was identified as the integrin alpha 3 beta 1. Purified alpha 3 beta 1 bound to entactin Sepharose in a divalent cation-dependent manner and liposomes prepared with fractions eluted from the entactin-Sepharose matrix, as well as purified alpha 3 beta 1 also bound to entactin. Liposomes prepared with other integrins such as alpha 2 beta 1 did not bind to entactin. Antibody inhibition assays demonstrated that an anti-alpha 3 antibody (P1B5) inhibited the attachment of PC-3 cells to entactin whereas this antibody did not inhibit the attachment of these cells to laminin. Attachment to laminin was, however, blocked by anti-alpha 6 antibody (G0H3). These data demonstrate that the cell surface receptor for entactin on these prostate carcinoma cells is the integrin alpha 3 beta 1 and that these cells utilize alpha 6 beta 1 as the receptor for laminin.     

Laminin promotes formation of cord-like structures by Sertoli cells in vitro

Basement membranes are thin extracellular matrices which contact epithelial cells and promote their adhesion, migration, differentiation, and morphogenesis. These matrices are composed of collagen IV, heparan sulfate proteoglycan, laminin, and entactin as well as other minor components. Sertoli cells, like most epithelial cells, are in contact at their basal surface with a basement membrane. When cultured within three-dimensional basement membrane gels (Matrigel), Sertoli cells reorganize into cords that resemble testicular seminiferous cords found in the in vivo differentiating testis. Anti-laminin and anti-entactin antisera inhibit this cord morphogenesis by Sertoli cells whereas antisera against type IV and type I collagen, heparan sulfate proteoglycan, fibronectin, and preimmune sera had no effect. The RGD (RGDS-NH2) sequence, found in the cell binding domain of the integrin family of cell adhesion molecules as well as in the A chain of laminin and in entactin, effectively inhibited Sertoli cell cord formation at a concentration of 1.0 mg/ml but was unable to prevent Sertoli cell attachment at concentrations as high as 2.0 mg/ml. A synthetic pentapeptide from a cell-binding domain of the B1 chain of laminin. YIGSR-NH2, inhibited cord formation at a concentration of 0.25 mg/ml, but Sertoli cells were still adherent to the basement membrane matrix. At concentrations greater than 0.50 mg/ml, Sertoli cells detached. Antiserum against the YIGSR-NH2-containing sequence was also effective in inhibiting cord formation by Sertoli cells. Ligand (YIGSR-NH2 peptide) blot analysis of Sertoli cell lysates revealed an interaction with a major band at 60 kDa and with minor bands at 39 and 127 kDa. Furthermore, in Western blot analysis the anti-67-kDa laminin-binding protein antibody recognized a 59- to 60-kDa protein in Sertoli cells. The data indicate that laminin is involved in both Sertoli cell attachment and migration during formation of histotypic cord structures by these cells in culture. Two separate laminin cell-binding domains appear to be involved in Sertoli cell cord morphogenesis in vitro and are likely to participate in the formation of seminiferous cords in vivo.     

Macromolecular organization of bovine lens capsule

Rabbit antisera to type IV collagen, laminin, entactin, heparan sulfate proteoglycan and fibronectin were used to localize these proteins in cross-sections of bovine anterior lens capsule. The antisera were exposed to (a) 10-micron frozen-thawed sections of formaldehyde-fixed tissue for examination in the light microscope by the indirect immunofluorescence method and (b) formaldehyde-fixed and L. R. White plastic-embedded thin sections for electron microscopic examination by the protein A-gold technique. The intensity of immunofluorescence was both uniform and strong throughout for type IV collagen, laminin and entactin, but patchy and weak for fibronectin. Electron microscopic immunolabeling with protein A-gold showed that all five components were distributed throughout the full thickness of the membrane, albeit the density of gold particles was not identical for all basement membrane proteins. In general, the number of particles per micron2 was greatest for type IV collagen and entactin, moderate for laminin and heparan sulfate proteoglycan and low for fibronectin. The ultrastructure of the lens capsule as examined by the electron microscope revealed a relatively uniform parallel alignment of filaments, thought to be collagenous. Since the distribution of the filaments corresponds well with the observed immunocytochemical pattern it is concluded that type IV collagen, laminin, entactin, heparan sulfate proteoglycan and fibronectin co-localize throughout the cross-section of the anterior lens capsule.     

Localization of fibronectin, laminin-entactin, and entactin in Reichert's membrane by immunoelectron microscopy.

Immunoelectron microscopy using protein A-colloidal gold complexes of different sizes was used to study the relative distribution of extracellular matrix glycoproteins within Reichert's membrane (RM) of 13.5-day mouse embryos. Labelling for fibronectin was distributed asymmetrically; the highest concentration occurring in the outermost layer adjacent to the trophoblast cells and negligible labelling in the inner matrix, beneath the parietal endoderm cells. Within the main body of the membrane, fibronectin was concentrated in discrete electron-opaque deposits. Antibodies raised against the native complex between laminin and entactin , and against entactin alone labelled the RM more uniformly.     

Lack of heparan sulfate proteoglycan in a discontinuous and irregular placental basement membrane

A discontinuous basement membrane of variable width that surrounds spongiotrophoblast cells of rat placenta was examined for the presence of type IV collagen, laminin, a heparan sulfate proteoglycan, entactin, and fibronectin using monospecific antibodies or antisera and the indirect peroxidase technique. At the level of the light microscope, the basement membrane was immunostained for type IV collagen, laminin, entactin, and fibronectin. Heparan sulfate proteoglycan immunostaining, however, was virtually absent even after pretreatment of sections with 0.1 N acetic acid, pepsin (0.1 microgram/ml) or 0.13 M sodium borohydride. Examination in the electron microscope confirmed the lack of immunostaining for heparan sulfate proteoglycan, whereas the other substances were mainly localized to the lamina densa part of the basement membrane. The absence of heparan sulfate proteoglycan in this discontinuous and irregular basement membrane even though type IV collagen, laminin, entactin, and fibronectin are present, suggests that heparan sulfate proteoglycan may have a structural role in the formation of basement membrane.     

Structure and function of matrix components in the cruciate ligaments. An immunohistochemical, electron-microscopic, and immunoelectron-microscopic study.

In the present study, the matrix components of 100 cruciate ligaments were analyzed by conventional electron microscopy, immunohistology, morphometry, and immunoelectron microscopy. The anterior (ACL) and the posterior (PCL) cruciate ligaments contained collagen types III, IV, and VI. Several structural glycoproteins, like fibronectin, laminin, entactin, tenascin, and undulin were detected using monoclonal antibodies. Whereas laminin and entactin were higher concentrated in the PCL, type VI collagen was more frequently found in the ACL. The ACL had a critical nourishment in its distal and middle thirds. In all ligament parts the PCL revealed a better vascular supply with strong correlation to type IV collagen expression. The normal matrix of the cruciate ligaments represented a complicated regulatory network of proteins, glycoproteins, elastic systems, and glycosaminoglycans with multiple functional interactions.     

Light microscopic immunolocalization of type IV collagen, laminin, heparan sulfate proteoglycan, and fibronectin in the basement membranes of a variety of rat organs

Immunohistochemical methods were used to determine whether type IV collagen, laminin, fibronectin, and heparan sulfate proteoglycan were present in diverse basement membranes. Antisera or antibodies against each substance were prepared, tested by enzyme-linked immunosorbent assay, and exposed to frozen sections of duodenum, trachea, kidney, spinal cord, cerebrum, and incisor tooth from rats aged 20 days to 34 months. Bound antibodies were then localized by indirect or direct peroxidase methods for examination in the light microscope. Immunostaining for type IV collagen, laminin, fibronectin, and heparan sulfate proteoglycan was observed in all of the basement membranes encountered. Fibronectin was also found in connective tissue. In general, the intensity of immunostaining was strong for type IV collagen and laminin, moderate for heparan sulfate proteoglycan, and weak for fibronectin. The pattern was similar in the age groups under study. Very recently the sulfated glycoprotein, entactin, was also detected in the basement membranes of the listed tissues in 20-day-old rats. It is accordingly proposed that, at least in the organs examined, type IV collagen, laminin, fibronectin, heparan sulfate proteoglycan, and entactin are present together in basement membranes.     

Expression of fibronectin and laminin by different types of mouse glial cells cultured in a serum-free medium

The expression of fibronectin and laminin by cultured glial cells was studied. The glial culture from neonatal mouse cerebra maintained in a chemically defined, serum-free medium consisted of type-1 astrocytes, oligodendrocyte-type-2 astrocyte (O-2A) progenitor cells, oligodendrocytes and type-2 astrocytes. Double-labelling immunofluorescent experiments performed using the mixed glial culture indicated that fibronectin and laminin are expressed in different patterns among the glial subtypes. The staining intensities with anti-fibronectin or anti-laminin antibodies decreased in the order: type-1 astrocytes, O-2A progenitor cells and type-2 astrocytes. Both molecules were deposited in a fibrillar matrix underneath type-1 astrocytes, whereas only intracytoplasmic localization of these molecules was observed with O-2A progenitor cells and type-2 astrocytes. Western blot analysis showed that glial fibronectin has a slightly higher molecular weight than mouse plasma fibronectin (230 kDa) and that glial laminin is a variant with a 220 kDa B chain present and the 400 kDa A chain missing. Using enzyme-linked immunosorbent assays (ELISA), these molecules were detected in the glial extracellular matrix at the concentration of 4 ng/10⁶ cells. A large amount of fibronectin (82 ng/10⁶ cells) was secreted into the culture medium, while secretion of laminin was not detected.