Secretion Of Laminin Research Articles

Regulation of fibronectin and laminin receptor expression, fibronectin and laminin secretion in human colon cancer cells by transforming growth factor‐β1

Transforming growth factor (TGF)-beta 1 modulates the expression of extracellular matrix (ECM) glycoproteins, fibronectin and laminin and the adhesion of Moser colon cancer cells to these glycoproteins. Since adhesion can be altered through expression of cell-surface receptors, binding affinities of adhesion molecules for receptors, or both, we investigated the effect of TGF-beta 1 on the binding properties of fibronectin and laminin to their cell-surface receptors by saturation binding and Scatchard analyses using radiolabeled fibronectin and laminin. Fibronectin bound to its cell-surface receptor with high affinity (Kd = 1.25 x 10(-9) M), Moser cells had approximately 7.1 x 10(4) fibronectin-binding sites per cell. TGF-beta 1 treatment rapidly up-modulated the number of cell-surface fibronectin-binding sites by 1.9-fold. The binding affinity of fibronectin for the receptor, however, was not altered. Laminin was found to bind to a higher-affinity and a lower-affinity receptor. Moser cells expressed approximately 1.1 x 10(3) higher-affinity laminin-binding sites and approximately 3.1 x 10(4) lower-affinity-binding sites per cell. TGF-beta 1 rapidly increased the expression of the higher-affinity sites 3-fold and the lower-affinity sites 5-fold. The binding affinity of both the higher-affinity and lower-affinity laminin receptors increased 3-fold after 2 and 6 hr of TGF-beta 1 treatment respectively. Concurrent with receptor modulation, TGF-beta 1 induced the secretion of fibronectin and laminin from Moser cells. Northern hybridization analyses showed a concurrent stimulation of the expression of the mRNAs for ligands (fibronectin and laminin) and the mRNAs for the integrin species of the fibronectin and laminin receptors (alpha 5 and alpha 6 subunits). Thus the production of fibronectin and laminin and the expression of their receptors were tightly co-regulated by TGF-beta 1.

Secretion of amyloid precursor protein and laminin by cultured astrocytes is influenced by culture conditions

Although normally quiescent, astrocytes in the adult brain respond to various types of brain injury by rapidly dividing, swelling, extending cellular processes, and expressing increased amounts of glial fibrillary acidic protein (GFAP). These phenomena are collectively referred to as "astrogliosis." Similarly, astroglia in primary culture stop dividing when they attain confluency, yet, as seen in situ, they retain their proliferative capacity for extended periods and resume rapid division when subcultured. To examine the impact of glial division on secretion of neurite-promoting factors, conditioned medium (CM) was removed from subconfluent, newly confluent, and long-term confluent ("aged") neonatal rat astrocyte cultures, and from aged confluent cultures that had been repassaged, "lesioned" (scraping with a rubber policeman), or triturated 3 days before harvest. Secretion of neurite-promoting factor(s) by glial cells into these CM was then assayed by treating neuroblastoma cultures with these various CM and quantitating neurite elaboration. Extensive neurite sprouting was elicited by CM from cultures just reaching confluency and from repassaged, lesioned, or triturated cultures. CM from aged confluent cultures did not induce sprouting. These results indicate that secretion of neurite-promoting factor(s) is regulated by glial division, and suggest that gliosis in situ may contribute to neurite sprouting by similar mechanisms. Immunoblot analysis demonstrated the presence in CM of varying amounts of laminin and amyloid precursor protein (APP), including isoforms containing the Kunitz-type protease inhibitor domain. CM from subconfluent cultures contained trace amounts of these proteins, but CM from cultures just reaching confluency contained significant amounts. Although CM from aged cultures contained barely detectable levels of either protein, trituration or repassage of aged cultures dramatically increased secretion of these proteins. APP- and laminin-enriched CM fractions promoted neuritogenesis to a similar level as respective unfractionated CM; anti-APP and anti-laminin antisera blocked this effect. Purified human brain APP promoted neuritogenesis when added to non-conditioned medium and aged CM. Increased secretion of APP and laminin therefore mediates at least a portion of CM-induced neuronal sprouting; these proteins may perform analogous functions during astrogliosis in situ.

Inhibition of Migration of Neural Crest-Derived Cells by the Abnormal Mesenchyme of the Presumptive Aganglionic Bowel of ls/ls Mice: Analysis with Aggregation and Interspecies Chimeras

The terminal bowel is congenitally aganglionic in ls/ls mice. The condition has been associated with an overabundance of laminin and other matrix molecules. Aggregation ls/ls ↔ C3H chimetic mice and interspecies mouse ↔ quail chimeras were constructed to test the hypothesis that the aganglionosis arises because the ls/ls gut and not the neural crest is abnormal. Demonstration of β-glucuronidase activity permitted genotypically ls/ls and C3H cells to be distinguished in the ls/ls ↔ C3H chimeras. Aganglionosis did not occur in the ls/ls ↔ C3H mice and ls/ls neurons were observed in the terminal bowel. Following backtransplantation of control segments of mouse gut into quail host embryos, mouse cells migrated to host targets normally colonized by cells from the neural crest; moreover, quail crest-derived cells entered the mouse gut. In contrast, cells did not migrate to these targets from presumptive aganglionic ls/ls bowel and quail crest-derived cells neither entered the ls/ls gut nor migrated through it. Laminin immunoreactivity was present in the backgrafts of murine colon and was far more abundant and widespread in those from ls/ls than in those from control animals. These data suggest that the presumptive aganglionic ls/ls bowel does not contain crest-derived cells because these cells, which are normal in ls/ls mice, do not enter it. This failure of colonization may be related to the premature formation of neurons outside the abnormal gut, a response that may be promoted by the excessive secretion of laminin by the ls/ls enteric mesenchyme.

Basal lamina formation by epithelial cell lines correlates with laminin A chain synthesis and secretion

Laminin is a major component of the basal lamina upon which all epithelial cells reside in vivo. The synthesis of basal lamina components and their subsequent assembly into a morphologically distinct basal lamina is a differentiated function of epithelial cells in vivo. Ultrastructural studies in our laboratory show that some epithelial cell lines (P-MDCK) form a basal lamina when cultured on membrane-permeable substrata (Millipore Millicells or type I collagen gels). Under the same conditions other epithelial cell lines (MDCK-AA7, M-mTAL-1P, and T84) do not form a basal lamina. When metabolically labeled with [ 35S]methionine, laminin A and B chains can be immunoprecipitated from the culture medium and culture lysates of P-MDCK cells. In contrast, labeled laminin chains cannot be immunoprecipitated from the culture medium of MDCK-AA7, M-mTAL-1P, and T84 cells. Immunoprecipitates of MDCK-AA7, M-mTAL-1P, and T84 culture lysates demonstrate the presence of one or both B chains but not A chains. These results suggest that laminin B chain synthesis is constitutive in MDCK-AA7, M-mTAL-1P, and T84 cells and that B chains, in the absence of A chains, are not secreted. Furthermore, laminin secretion and basal lamina formation are not required to maintain structural and functional polarity in these cell lines.

Polarized and functional epithelia can form after the targeted inactivation of both mouse keratin 8 alleles.

We have tested the requirement of keratin intermediate filaments for the formation and function of a simple epithelium. We disrupted both alleles of the mouse keratin 8 (mK8) gene in embryonic stem cells, and subsequently analyzed the phenotype in developing embryoid bodies in suspension culture. After the inactivation of the mouse keratin 8 (mK8) gene by a targeted insertion, mK8 protein synthesis was undetectable. In the absence of mK8 its complementary partners mK18 and mK19 were unable to form filaments within differentiated cells. Surprisingly, these ES cells differentiate to both simple and cystic embryoid bodies with apparently normal epithelia. Ultrastructural analysis shows an apparently normal epithelium with microvilli on the apical membrane, tight junctions and desmosomes on the lateral membrane, and an underlying basal membrane. No significant differences in the synthesis or secretion of alpha 1-fetoprotein and laminin were observed between the mK8- or wild-type embryoid bodies. Our data show that mK8 is not required for simple epithelium formation of extraembryonic endoderm.

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/10(6) cells. A large amount of fibronectin (82 ng/10(6) cells) was secreted into the culture medium, while secretion of laminin was not detected.

Organisation and reorganisation of blood vessels in embryonic development

The initial establishment of blood vessels is genetically predetermined but it is subsequently supplemented by local and haemodynamic factors. The earliest embryonic vessels arise independently in the mesenchyme of the yolk sac wall, chorion and embryonic disc. The angioblasts aggregate as cords which canalise by a process which includes intracellular vacuolation. Subsequent extension of the network occurs by a combination of (1) in situ formation of new vessels, (2) sprouting and fusion of established neighbouring vessels and (3) invading angioblasts from other regions. As capillaries mature, a basement membrane is established containing collagen IV, laminin, nidogen/entactin, heparan sulphate proteoglycan and a variable amount of fibronectin. Early capillary basement membrane contains more fibronectin which promotes proliferative and migratory activity of endothelium. As vessels mature, the endothelium switches to laminin secretion which causes differentiation including cell attachment, junction formation and establishment of tubes while cell proliferation is suppressed.

The behavior of the extracellular matrix and the basal lamina during the repair of cryogenic injury in the adult rat cerebral cortex

A well-defined coagulative lesion was produced in the fronto-parietal cortex of adult rats by application of a cold probe, and the chronological sequence of events during the healing process, particularly the relationship between astroglial processes and the newly forming basal lamina (BL) and the behavior of the extracellular matrix (ECM) was examined immunocytochemically and ultrastructurally. By electron microscopy, new BL synthesis was first noted 7 days following injury, and a continuous and well-defined BL was present from 14 days onward. These findings correlated well with the pattern of immunoreactivity for laminin and for type IV collagen. Both laminin and type IV collagen appeared both to be produced primarily by mesenchymal cells within and around the wound as well as those of the blood vessels, and to become an integral part of the new BL. Although there was no immunocytochemical evidence to indicate secretion of laminin, type IV collagen or fibronectin by astrocytes, a well-defined BL appeared to form only in tight apposition with astroglial processes over the pial surface. This suggests that the BL is formed by subpial astrocytes in close interaction with ECM components at the pial surface. Fibronectin appears to contribute significantly to the formation of the BL by providing a suitable substratum for the coordinated cellular interaction necessary for successful regeneration of the BL.

Ascorbic acid phosphate stimulates type IV collagen synthesis and accelerates adipose conversion of 3T3-L1 cells

We showed that the synthesis and secretion of type IV collagen, entactin, and laminin were enhanced when adipose conversion of 3T3-L1 cells at confluence was stimulated by hormones (Y. Aratani and Y. Kitagawa (1988) J. Biol. Chem. 263, 16163–16169) . Ascorbic acid phosphate (Asc-P) stimulated the synthesis and secretion of type IV collagen and other collagens from both 3T3-L1 preadipocytes and adipocytes. The synthesis and secretion of laminin and entactin were not affected by Asc-P. The continuous addition of Asc-P stimulated cell growth and increased cell density at confluence 1.3-fold. Concomitantly, Asc-P remarkably accelerated the emergence of lipoprotein lipase, glycerophosphate dehydrogenase, and Oil Red O-stainable lipid droplets. These findings suggest an important role for type IV collagen in adipocyte differentiation.

Cerebral endothelial cell-derived laminin promotes the outgrowth of neurites in CNS neuronal cultures

The aim of the present study was to elucidate whether the endothelial cells, besides providing the structural basis for the blood-brain barrier (M. Bradbury, Concept for a Blood-Brain Barrier, 1979; F. Joó, Nature321, 197–198, 1986) can also produce laminin and, via its release, regulate the growth of neuntes of neurons originating from the central nervous system (CNS). In immunohistochemical investigations, the presence of laminin-like immunoreactivity could be detected in the cytoplasm and on the surface of endothelial cells derived from brain microvessels. Further, the neurite-promoting effect of laminin on CNS neurons was also noted in a co-culture system designed especially for conditioning nerve cell cultures continuously with materials released from the cerebral endothelial cells. It is suggested that the endothelial cells may be involved via their laminin secretion in the regulation of axonal growth and guidance in the CNS.

Biphasic response to retinoic acid dose in differentiation of F9 cells into primitive endoderm-like cells

The differentiation of F9 cells, as monitored by the secretion of basement membrane proteins, was biphasic depending on the dose of retinoic acid (RA) used. Secretion of laminin A reached a plateau at about 10 n M and increased again at more than 100 n M RA. A similar biphasic response was observed in the secretion of tissue-type plasminogen activator as well. In the presence of RA alone, this biphasic pattern of differentiation was stable for a long period of time, but the addition of 1 m M dibutyryl cAMP changed it to monophasic after 12 days of exposure.

Developmental regulation of laminin accumulation in the extracellular matrix of a mouse muscle cell line

We have investigated the synthesis, accumulation, and secretion of laminin, an extracellular matrix glycoprotein, during differentiation of the C2 mouse skeletal muscle cell line in culture. Myoblasts actively synthesized laminin, as measured by incorporation of [ 35S]methionine and by a dot-immunobinding assay. In myoblast cultures laminin accumulated in an intracellular compartment and could be extracted with a physiological salt solution containing the detergent Triton X-100. After the culture medium was replaced to promote differentiation of myoblasts to myotubes, laminin synthesis was increased, and laminin began to accumulate in the medium in soluble form. During differentiation, laminin also accumulated in an insoluble cell-associated fraction that required guanidinium chloride for extraction. Indirect immunofluorescence and immunobinding assays showed that myotubes but not myoblasts contained laminin on their external surface. The time course of increase in surface laminin paralleled that of the accumulation of insoluble laminin. These results suggest that the insoluble fraction represents laminin bound to the extracellular matrix at the cell surface. Our experiments demonstrate, contrary to previous observations, that myotube cultures synthesize and accumulate laminin, and further, that the differentiation of proliferating myoblasts to multinucleated myotubes is accompanied by increased laminin synthesis, by secretion of laminin into the medium, and by the deposition of laminin into an extracellular matrix on the myotube surface.

The biosynthesis, processing, and secretion of laminin by human choriocarcinoma cells.

Laminin, a glycoprotein component of basal laminae, is synthesized and secreted in culture by a human malignant cell line (JAR) derived from gestational choriocarcinoma. Biosynthetically labeled human laminin subunits A (Mr approximately 400,000) and B (Mr = 200,000 doublet) are glycoslyated with asparagine-linked high mannose oligosaccharides that are processed to complex oligosaccharides before the laminin molecule is externalized by the cell. The rate-limiting step in the processing of the asparagine-linked glycans of laminin is at the point of action of alpha-mannosidase I since the principal laminin forms that accumulate in JAR cells contain Man9GlcNAc2 and Man8GlcNAc2 oligosaccharide units. The combination of subunits to form the disulfide-linked laminin molecule (Mr approximately 950,000) occurs rapidly within the cell at a time when the subunits contain these high mannose oligosaccharides. The production of laminin is limited by the availability of the A subunit such that excess B subunit forms accumulate intracellularly as uncombined B and a disulfide-linked B dimer. Pulse-chase kinetic studies establish these B forms as intermediates in the assembly of the laminin molecule. The fully assembled laminin undergoes further oligosaccharide processing and translocation to the cell surface, but uncombined B and B dimer are neither processed nor secreted to any significant extent. Therefore, laminin subunit combination appears to be a prerequisite for intracellular translocation, processing, and secretion. The mature laminin that contains complex oligosaccharides does not accumulate intracellularly but is rapidly externalized upon completion, either secreted into the culture medium (25%) or associated with the cell surface (75%) as determined by susceptibility to degradation by trypsin. About one-third of the laminin molecules secreted or shed by JAR cells into the chase medium contain a smaller A subunit form that appears to have been modified by limited proteolytic cleavage. The putative proteolytic event is closely timed to the release of the laminin into the culture medium.

Reversible effects of dibutyryl cAMP on laminin and type IV collagen secretion from retinoic acid treated F9 cells.

The effects of dibutyryl cAMP on the differentiation of embryonal carcinoma F9 cells were studied mainly using the secretion of laminin and type IV collagen as the marker. For this purpose, F9 cells were labeled with 35S-methionine and radioactive proteins in the medium were analyzed by SDS-polyacrylamide gel electrophoresis. Treatment of F9 cells with retinoic acid alone induced differentiation into cells secreting type IV collagen. The combination of retinoic acid and dibutyryl cAMP stimulated laminin secretion in addition to type IV collagen secretion. This effect of dibutyryl cAMP was observed only 16 h after adding dibutyryl cAMP. Immunofluorescence staining demonstrated that the majority of the cells in culture were converted into cells secreting laminin under these conditions. In contrast to the irreversible effect of retinoic acid, the effect of dibutyryl cAMP on laminin and type IV collagen secretion was reversible at least during the first 5 days of maintaining cells in the medium containing retinoic acid plus dibutyryl cAMP. Removal of dibutyryl cAMP from the culture medium decreased the protein secretion to the basal levels within 2 days. This reversibility was not due to a change in cell number. An in vitro translation assay also suggested the reversible effect of dibutyryl cAMP on the levels of laminin mRNA. Coinciding with variations of the protein secretion, a reversible and homogeneous change in the morphology of retinoic acid generated F9 cells was observed by dibutyryl cAMP.

Expression of nidogen and laminin in basement membranes during mouse embryogenesis and in teratocarcinoma cells

Nidogen and laminin were localized at preimplantation stages of mouse development by immunofluorescence. Laminin was already present on the cell surface at the 2-cell stage, while nidogen was first detectable on compacted 8- to 16-cell stage morulae. Nidogen and laminin colocalized at the blastocyst stage and in postimplantation basement membranes. Immunoblot analyses of tissue extracts and cell culture media indicated the 150-kDa form of nidogen as the largest and predominant form in all tissues examined. Radiolabeled nidogen and laminin synthesized by Reichert's membrane were coprecipitated by antibodies against each antigen, indicating complex formation in situ. Equimolar amounts of laminin and nidogen were determined in 6 M guanidine·HCl extracts of tissues by radioimmunoassays, further indicating stoichiometric complexes. However, lower levels of nidogen than laminin were found in tissue and cell culture media. A less than 2-fold increase in nidogen was found when F9 cells were stimulated to differentiate with retinoic acid and dibutyryl cAMP, compared to a 30-fold increase in laminin secretion.

Abnormal Laminin Secretion from Parietal Endoderm-Like F9 Cells in the Presence of Monensin1

We studied the effects of monensin on post-translational modification and intracellular transport of precursors of laminin subunits in parietal endoderm-like F9 cells. At concentrations higher than 0.1 microM, monensin inhibited the processing of high-mannose type precursors for all three subunits and caused their cytoplasmic accumulation. Furthermore, the secretion of mature subunits of laminin was inhibited. Instead, polypeptides with similar molecular weights to those of intracellular precursors were secreted. These polypeptides were immunologically related to laminin subunits and were sensitive to digestion with beta-N-acetylglucosaminidase H (Endo H). This indicated that Golgi complexes of the cells can transport the precursors of laminin subunits even with their terminal glycosylation inactivated by monensin. Tunicamycin induced the accumulation of unglycosylated precursors and strongly reduced their secretion into the medium.

Modulation of type-IV procollagen and laminin production in A431 human squamous epidermoid carcinoma cells by 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF).

The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) and epidermal growth factor (EGF) on type-IV-procollagen (basement-membrane collagen) and laminin synthesis, turnover, and secretion was studied in human A431 squamous epidermoid carcinoma cells. Type-IV procollagen and laminin were biochemically and immunologically identified in the medium and cell extracts using immuno-precipitation followed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. EGF or TPA produced a sixfold increase in type-IV-procollagen and laminin secretion within 2 h; this was accompanied by a three- to fourfold increase in the levels of cell-associated type-IV procollagen and laminin, respectively. The level of type-IV-procollagen and laminin synthesis and secretion remained elevated for at least 16 h after the administration of either EGF or TPA. A combination of EGF and TPA was more effective than either agent alone in promoting the secretion of laminin but not of type-IV procollagen. EGF and/or TPA did not, however, produce a selective increase in the synthesis of collagen and laminin, since total protein synthesis was also increased to the same degree by these agents. As determined by labeling and chase studies, neither EGF nor TPA had any appreciable effect upon type-IV-procollagen or laminin degradation. These results indicate that the synthesis of components associated with the basement membrane in A431 cells (i.e., type-IV procollagen and laminin) can be rapidly modulated by EGF and a tumor promoter, i.e., TPA.(ABSTRACT TRUNCATED AT 250 WORDS)

Selection and characterization of F9 teratocarcinoma stem cell mutants with altered responses to retinoic acid.

Retinoic acid induces the differentiation of many murine teratocarcinoma stem cell lines. To elucidate the molecular mechanism of action of retinoic acid, we have selected a series of mutants which exhibit altered differentiation responses to retinoic acid. All of the mutants display abnormal morphology following addition of 5 X 10(-7) M retinoic acid (RA) and dibutyryl cAMP. In addition, none of the mutants are resistant to the cytotoxic effects of higher concentrations of retinoic acid (greater than 75 microM). After the addition of retinoic acid, one mutant, RA-3-10, does not differentiate by any of the biochemical criteria we have used; this mutant also possesses less than 5% of the wild type level of cellular retinoic acid binding protein (CRABP). Other mutants, such as RA-3-3, RA-3-4, and RA-5-1, contain the same amount of CRABP as wild type F9 cells. However, the mutants RA-3-3 and RA-3-4 exhibit lower levels of plasminogen activator activity, and RA-3-4 also exhibits only 10-20% of the wild type synthesis and secretion of laminin and collagen IV following treatment with RA. After RA treatment of the mutant RA-5-1, laminin and collagen IV are synthesized and secreted at reduced rates relative to wild type cells, and the secreted collagen IV has a lower molecular weight than that of wild type; this suggests that RA-5-1 cells have a mutation in one of the enzymes responsible for post-translational modification of collagen IV. None of the mutants tested exhibits alterations in either cytosolic or membrane bound cAMP-dependent protein kinase activity. These studies provide genetic evidence that the CRABP is required for the differentiation of F9 teratocarcinoma stem cells by retinoic acid. However, even in the presence of CRABP, other types of alterations, such as synthesis of collagen IV with an abnormal molecular weight, appear to cause alterations in the differentiation response of cells to retinoic acid.

Functional role of laminin carbohydrate.

Previous work showed that tunicamycin suppresses glycosylation of laminin. In the present work, the role of glycosylation in the secretion of laminin and in the disulfide bonding of laminin subunits was studied, using tunicamycin to inhibit glycosylation. Tunicamycin inhibited extensively the secretion of laminin into culture medium and extracellular matrix even though the treated cells contained higher concentrations of laminin than the control cells. The laminin subunits synthesized in the presence of tunicamycin were disulfide bonded. Thus, suppression of glycosylation did not adversely affect disulfide bonding of the subunits, but did decrease the secretion of laminin. Glycosidases were also used to remove the carbohydrate of laminin to study the role of carbohydrate in the stability of laminin and in its interaction with another extracellular matrix component, heparin. The glycosidases removed about 73% of [3H]glucosamine. Both glycosidase-treated and untreated laminin were stable when incubated with cell lysate or culture medium. The glycosidase-treated laminin bound as efficiently as the untreated laminin to heparin. These results suggest that the presence of a carbohydrate moiety, at least at the level found in untreated laminin, is not essential in binding to heparin or in protecting laminin from proteolytic degradation in the cell or culture medium.