Immunohistochemical Localization of Laminin and Cytokeratin in Embryonic Alligator Gonads

Gonadal sex differentiation is temperature-dependent in Alligator mississippiensis; testis differentiation occurs in embryos incubated at 33°C and ovary differentiation occurs in embryos incubated at 30°C. Laminin and cytokeratin were examined immunohistochemically in the gonads of alligator embryos incubated at these temperatures. The aim of this study was to determine whether these structural proteins show the same sex-specific expression patterns reported for mammalian embryos, and to assess their usefulness as early markers of gonadal differentiation in species with temperature-dependent sex determination. Laminin delineated enlarged seminiferous cords in differentiating testes from developmental stage 23 to hatching. Laminin distribution was more diffuse and revealed smaller cords of cells in differentiating ovaries. Cytokeratin was also detected in developing gonads of both sexes. Cytokeratin became concentrated in the basal cytoplasm of differentiating Sertoli cells in developing testes. In developing ovaries, prefollicular cells of the ovarian cortex and cell cords in the medulla stained strongly for cytokeratin. Cytokeratin did not show the same basal distribution in female medullary cord cells as seen in the Sertoli cells of testes, however. These sex-specific patterns of laminin and cytokeratin distribution in embryonic alligator gonads may serve as early markers of sexual differentiation.     

Rodent myoblast interactions with laminin require cell surface glycoconjugates but not laminin glycosyl groups

Laminin glycosyl groups are necessary for the spreading of murine melanoma cells which become attached to this glycoprotein. Laminin has been implicated in myogenesis but the potential role of its glycosyl groups in this process has not been examined. In this study we report the effects of the carbohydrate moieties of laminin on myoblast adhesion, spreading, and differentiation. Unglycosylated laminin from tunicamycin-treated cultures of a mouse cell line, M1536 B3, was used in the experiments. Glycosylated laminin from a murine tumor and from cultures of M1563 B3 cells served as controls. Cell binding experiments with C2C12 mouse myoblasts showed that the cells preferred a laminin-coated surface, compared to the uncoated plastic surface (nontissue culture wells). Myoblasts did not distinguish between glycosylated and unglycosylated laminin substrates. Both glycosylated and unglycosylated forms of laminin promoted myoblast growth and differentiation. In contrast, cells on uncoated plastic surfaces grew very slowly and did not further differentiate. The L6 rat myoblast response to glycosylated and unglycosylated laminin was the same. These results indicate that although rodent myoblasts in culture require a laminin substratum for spreading, growth, and differentiation on a proprietary plastic surface, laminin carbohydrates are not implicated in those cellular responses. In contrast, parallel studies using the lectin, Con A, indicate that cell surface glycoconjugates of myoblasts are implicated in the response of these cells to a laminin substratum.     

Synthesis and effects of basement membrane components in cultured rat Schwann cells

Schwann cells, the myelin-forming cells of the peripheral nervous system, are surrounded by a basement membrane. Whether cultured rat Schwann cells synthesize the basement membrane-specific components, laminin and collagen type IV, and whether these components influence the adhesion, morphology, and growth of these cells have been investigated. Both laminin and collagen type IV were detected in the cytoplasm of Schwann cells by immunofluorescence. After ascorbate treatment, laminin and collagen type IV were both found in an extracellular fibrillar matrix bound to the Schwann cell surface. Laminin was further localized on the Schwann cell surface by electron microscopy using gold immunolabeling. Anti-laminin IgG-labeled gold particles were scattered over the cell surface, and linear rows of particles and small aggregates were found along the cell edges and at points of contact with other cells. When added to the culture medium, laminin acted as a potent adhesion factor, stimulating Schwann cell adhesion as much as eightfold above control levels on type IV collagen. In the presence of laminin, the cells became stellate and by 24 hr had extended long, thin processes. Laminin also stimulated cell growth in a dose-dependent manner and anti-laminin IgG completely inhibited cell attachment and growth in the absence of exogenous laminin. Thus, cultured Schwann cells synthesize laminin and collagen type IV, two major components of basement membrane, and laminin may trigger Schwann cell differentiation in vivo during early stages of axon-Schwann cell interaction before myelination.     

Mononuclear phagocyte adherence in the presence of laminin : A possible marker of cellular differentiation

The effect of laminin on the vitro adhesive behavior of mononuclear phagocytes was investigated. Laminin significantly inhibited the adhesion of guinea pig, mouse, and rat alveolar or peritoneal macrophages and of human peripheral blood monocytes. Adhesion of these cells was unaffected by similar concentrations of fibronectin. Experiments performed with monocytes maintained in culture showed that the degree of laminin-mediated inhibition of adherence was dependent on the state of differentiation of the cells: the less mature the monocytes, the greater the degree of inhibition. Laminin also reduced the attachment capacity of polymorphonuclear leukocytes isolated from human peripheral blood. These results suggest a possible role for laminin in the regulation of the passage of cells across the basement membrane during inflammation.     

Characterization of laminin isoforms in human amnion

Epithelial cells of the human amnion have been reported to possess similar functions to many types of cells, such as hepatocytes, neurons, and pancreatic beta-cells. We reported previously that one of the hepatocyte-like functions of human amniotic epithelial cells was reinforced by the presence of basement membrane components. Laminin is one of the main components of the basement membrane; it critically contributes to cell differentiation. Laminin has several heterotrimer isoforms composed of an alpha-, a beta-, and a gamma-chain, and each type of chain has several types of subunit chains: alpha1-5, beta1-3, and gamma1-3. In this study, we characterized the laminin subunit chains in human amnion. Laminin is produced and secreted from adjacent epithelial cells, and therefore, the gene expression of laminin subunit chains in human amniotic epithelial cells was investigated by RT-PCR. Their localization was examined by immunohistochemical staining of frozen sections. The findings suggested that the basement membrane of the human amnion contains a broad spectrum of laminin isoforms, laminin-2, -4, -5, -6, -7, -10, -11. These findings will provide clues not only for understanding the physiological roles of the amnion and hAECs, but also for applying this tissue as a source of donor cells for cell transplantation therapy.     

Synergistic activation of the rat laminin gamma1 chain promoter by the gut-enriched Kruppel-like factor (GKLF/KLF4) and Sp1

Laminin is a multifunctional heterotrimeric protein present in extracellular matrix where it regulates processes that compose tissue architecture including cell differentiation. Laminin γ1 is the most widely expressed laminin chain and its absence causes early lethality in mouse embryos. Laminin γ1 chain gene (LAMC1) promoter contains several GC/GT-rich motifs including the bcn-1 element. Using the bcn-1 element as a bait in the yeast one-hybrid screen, we cloned the gut-enriched Kruppel-like factor (GKLF or KLF4) from a rat mesangial cell library. We show that GKLF binds bcn-1, but this binding is not required for the GKLF-mediated activation of the LAMC1 promoter. The activity of GKLF is dependent on a synergism with another Kruppel-like factor, Sp1. The LAMC1 promoter appears to have multiple GKLF- and Sp1-responsive elements which may account for the synergistic activation. We provide evidence that the synergistic action of GKLF and Sp1 is dependent on the promoter context and the integrity of GKLF activation and DNA-binding domain. GKLF is thought to participate in the switch from cell proliferation to differentiation. Thus, the Sp1–GKLF synergistic activation of the LAMC1 promoter may be one of the avenues for expression of laminin γ1 chain when laminin is needed to regulate cell differentiation.     

Distinct GATA6- and laminin-dependent mechanisms regulate endodermal and ectodermal embryonic stem cell fates

This study investigates the establishment of alternative cell fates during embryoid body differentiation when ES cells diverge into two epithelia simulating the pre-gastrulation endoderm and ectoderm. We report that endoderm differentiation and endoderm-specific gene expression, such as expression of laminin 1 subunits, is controlled by GATA6 induced by FGF. Subsequently, differentiation of the non-polar primitive ectoderm into columnar epithelium of the epiblast is induced by laminin 1. Using GATA6 transformed Lamc1-null endoderm-like cells, we demonstrate that laminin 1 exhibited by the basement membrane induces epiblast differentiation and cavitation by cell-to-matrix/matrix-to-cell interactions that are similar to the in vivo crosstalk in the early embryo. Pharmacological and dominant-negative inhibitors reveal that the cell shape change of epiblast differentiation requires ROCK, the Rho kinase. We also show that pluripotent ES cells display laminin receptors; hence, these stem cells may serve as target for columnar ectoderm differentiation. Laminin is not bound by endoderm derivatives; therefore, the sub-endodermal basement membrane is anchored selectively to the ectoderm, conveying polarity to its assembly and to the differentiation induced by it. Unique to these interactions is their flow through two cell layers connected by laminin 1 and their involvement in the differentiation of two epithelia from the same stem cell pool: one into endoderm controlled by FGF and GATA6; and the other into epiblast regulated by laminin 1 and Rho kinase.     

A synthetic peptide containing the IKVAV sequence from the A chain of laminin mediates cell attachment, migration, and neurite outgrowth

Laminin is a basement membrane glycoprotein which consists of A, B1, and B2 chains. Laminin has diverse biological activities including promoting cell adhesion, migration, differentiation, growth, and neurite extension. Synthetic peptides from the active region of the A chain were prepared and tested for their biological activity. A 19-mer peptide (designated PA22-2), from just above the carboxyl globule on the long arm of the A chain, was found to promote cell adhesion, spreading, migration, and neurite outgrowth. By testing smaller sequences within the 19-mer peptide, a constituent pentapeptide, IKVAV (Ile-Lys-Val-Ala-Val), was identified as the active site for cell adhesion and neurite outgrowth. These data suggest that this sequence is one of the principle sites in laminin which regulate cellular behavior.     

Synthetic pentapeptide from the B1 chain of laminin promotes B16F10 melanoma cell migration

Laminin is a basement membrane-specific glycoprotein that promotes cell adhesion, proliferation, differentiation, and tumor cell migration. Synthetic peptides from the amino acid sequence deduced from a cDNA clone of the B1 chain of laminin were tested for their ability to promote the migration of B16F10 melanoma cells. A peptide, CDPGYIGSR, that is able to mediate epithelial cell attachment to laminin was found to promote migration, and the constituent pentapeptide YIGSR was also active but to a lesser degree. This nine-amino acid peptide blocked migration of melanoma cells to laminin but had no effect on migration to fibronectin. These data suggest that the cell-binding site and migration site on laminin share a common sequence that is unique to laminin.     

Signaling site of laminin with mitogenic activity.

Laminin, a basement membrane glycoprotein, has diverse biological activities including cell adhesion, growth, and differentiation. However, little is known concerning the signal transduction and active site involved in cell growth. In this study, we have shown that laminin and a 19-mer peptide (PA22-2) from the carboxyl-terminal end of the long arm of the laminin A chain, which was previously shown to promote cell adhesion and neurite outgrowth, stimulate thymidine incorporation and cell growth of PC12 cells. Laminin and PA22-2 (PA) were also found to induce a rapid and transient mRNA expression of c-fos and c-jun protooncogenes in PC12 cells. Further, both laminin and PA stimulated the DNA binding activity of c-Fos and c-Jun protein complex to the AP-1 site. We have also found that there is a correlation between cell growth, c-fos expression, and the ability of cell attachment to laminin or to PA in different cell types. These results suggest that the PA sequence is a potent site in laminin for both signal transduction and cell growth.     

Embryonic heart mesenchymal cell migration in laminin

Laminin, a large glycoprotein and major component of basement membranes, influences cell adhesion, migration, morphology, and differentiation. A peptide sequence, YIGSR, from the B1 chain of laminin has been found to correspond to an active site for cell adhesion. We report here that cardiac mesenchymal cells migrate vigorously within three-dimensional gels of laminin and that the YIGSR peptide will completely abolish this migratory activity. In contrast, migration of the mesenchymal cells into three-dimensional gels composed of collagen or collagen + laminin is not effected by YIGSR or other peptides (GRGDS, GRGDTP) reported to mediate cellular adhesion.     

The role of laminin in attachment,growth, and differentiation of cultured cells: a brief review

Laminin, a major structural multidomain protein of basement membranes, has been shown to exert a variety of biological activities. Prominent among those are mediation of cell attachment as well as influences on cellular proliferation, differentiation and motility. Distinct domains of laminin have been identified which carry these activities. The active sites on laminin are recognized by cellular receptors, several of which belong to the integrin class of heterodimeric transmembrane proteins. These are in direct contact with intracellular proteins and mediate signals from the extracellular matrix to the cytoskeleton and possibly to other intracellular regulatory systems. The biological activities of laminin may be used to design optimal conditions for the expression of a differentiated phenotype of cells in culture.     

Effects of extracellular matrix components on the differentiation of chick embryo tail bud mesenchyme in culture

The mesenchymal cells of the chick tail bud comprise the remains of Hensen's node and the primitive streak after gastrulation. This mass of cells, situated at the caudal limit of the chick embryo, is morphologically homogeneous but pluripotent, with the ability to differentiate into a variety of tissues that are both ectoderm- and mesoderm-derived elsewhere in the embryo. These tissues include neuroectoderm, neurons, myoblasts and chondrocytes. As the factors regulating the differentiation of tail bud mesenchyme into so many cell types are unclear, and because the extracellular matrix (ECM) is known to have a profound effect on cellular differentiation in many embryonic systems, we studied the differentiation of tail bud mesenchyme explanted onto a variety of different ECM components as substrata. We report that the histogenetic potential of isolated tail buds in culture compares favourably with that in situ. Using various antibody markers, we have demonstrated that tail bud mesenchyme cultured upon different ECM components as substrata is able to differentiate into neurons, neuroepithelium, melanocytes, muscle and cartilage. Laminin and laminin-containing substrata (Matrigel) were found to promote the differentiation of neural crest derivatives (neurons and melanocytes) and neuroepithelial cells; type I collagen promoted both myogenesis and chondrogenesis; while type IV collagen promoted myogenesis only. We have therefore demonstrated that differentiation of tail bud mesenchyme in vitro is substratum-dependent.     

Identification and partial characterization of laminin binding proteins in immature rat Sertoli cells

Laminin, a major component of basement membrane extracellular matrices, promotes differentiation in a number of cell types, including Sertoli cells. We have identified and characterized Sertoli cells. We have identified and characterized Sertoli cell surface molecules which interact with laminin. Using laminin-Sepharose affinity chromatography and [125I]laminin binding to Sertoli cell plasma membranes, binding proteins have been identified with the Mr 110,000, 67,000, 55,000, 45,000, 36,000, and 25,000. In addition, the Mr 110,000 and 67,000 laminin binding proteins were phosphorylated. The 67,000, 45,000, and 36,000 react with antibodies to the previously characterized laminin receptor and these antibodies stain the basolateral surface of Sertoli cells in vivo. Cultured Sertoli cells stain for laminin receptor both on the cell surface and within the cells. Antiserum to the 32,000 and 67,000 laminin binding proteins partially inhibited spreading of Sertoli cells on a laminin-coated culture dish, suggesting a functional importance of those proteins in Sertoli cell differentiation. The 25,000 and 45,000 laminin binding proteins reacted with integrin antibodies, but no high-molecular-weight forms could be detected. Integrin was localized to the cell surface and intracellularly but antibodies did not block Sertoli cell spreading on laminin. This work represents the first identification and characterization of extracellular matrix binding proteins in an endocrine organ and suggests an important role for the nonintegrin 32/67 laminin binding proteins.     

Thioredoxin inhibits microvascular endothelial capillary tubule formation

Thioredoxin (Trx) inhibited human HMEC-1 dermal microvascular endothelial cell capillary tubule forming capacity in a Matrigel based assay in vitro. Inhibition of capillary tubule formation was Trx catalytic site and thioredoxin reductase (TrxR) dependent, mediated at the Matrigel matrix level, and associated with a shift from morphological differentiation to continuous proliferation, with enhanced cell spreading resulting in eventual monolayer formation. Soluble complex carbohydrates, which inhibited capillary tubule formation on Matrigel without induction of cell spreading or monolayer formation, failed to impair Trx promotion of cell spreading and mono-layer formation, suggesting a shift away from carbohydrate-mediated cell/matrix adhesive interactions. Laminin peptides YIGRS and SIKVAV, which impaired tubule formation on Matrigel without inducing cell spreading or monolayer formation, partially impaired cell spreading upon Trx-treated Matrigel without restoring tubule formation, consistent with a potential role for laminin in Trx-mediated effects. Trx reduced laminin and destabilised laminin/galectin-3 complexes within Matrigel. Native purified EHS Laminin (also containing galectin-3), but not recombinant galectin-3, restored HMEC-1 capillary tubule formation on Trx-treated Matrigel. These data highlight a novel deregulatory effect of extracellular Trx upon morphological capillary differentiation that appears to depend upon the reduction of laminin and destabilisation of its interaction with galectin-3, possibly leading to galectin-3 neutralisation that shifts cell/matrix adhesive interactions away from being carbohydrate mediated and results in loss of proliferation-inhibiting and differentiation promoting cues from this tumor basement membrane matrix.     

Transient and locally restricted expression of laminin A chain mRNA by developing epithelial cells during kidney organogenesis

Three polypeptide chains, A, B1, and B2, have been described for mouse laminin, a basement membrane protein. We studied expression of laminin A, B1, and B2 mRNA in the developing mouse kidney. Induction of kidney mesenchyme differentiation in vitro led to an increased expression of B1 and B2 chain mRNA on day 1 of development. In contrast, expression of A chain mRNA increased on day 2, when epithelial cell polarization begins. Laminin A mRNA and polypeptide were expressed only by epithelia during in vivo development as well. Some polarized cell types producing basement membrane (endothelium, some adult epithelia) lacked the A chain mRNA and polypeptide, although they did express B chains. Laminin with the 400 kd A chain is therefore a transient form appearing at specific sites of kidney morphogenesis, whereas isoforms with a different A chain or without it have a more widespread distribution.     

Effect of hyperthermia and retinol treatment in vitro on HTC cell adhesiveness to laminin and fibronectin]

Since cell adhesiveness is very important in the metastatic process and because both hyperthermia and treatment with Retinol can modify the fluidity of the lipid components of the plasma membrane (and therefore its receptor distribution), we investigated if a hyperthermic treatment (at 42 degrees C or 44 degrees C, for one hour) of HTC hepatoma cells, preceded or followed by treatment with 5 microM Retinol, could alter cell adhesiveness to Laminin or to Fibronectin-coated substrata. Hepatoma cells, after such treatments, were collected and processed by Auerbach's method. In the control cells thermal treatment alone caused a decrease of adhesiveness to Laminin but no change in that to Fibronectin. When treatment with Retinol was carried out before hyperthermia, the cells showed an increased adhesiveness to Laminin and a decreased adhesiveness to Fibronectin. Instead, when treatment with Retinol was performed in cells previously thermo-selected, a decrease of adhesiveness to both tested ligands was observed.     

Laminin in cultured mouse C1300 neuroblastoma cells: immunocytochemical localization by pre- and postembedding electron microscope procedures

Laminin was localized in cultured mouse C1300 neuroblastoma cells by applying the peroxidase-antiperoxidase technique in preembedding electron microscopy. The results were compared to those obtained by indirect immunofluorescence and by the colloidal gold second antibody method on Epon-embedded ultrathin sections. Laminin was found in the cell membranes and within the rough endoplasmic reticulum as well as in intracytoplasmic vacuoles. Plasma membranes of the neuroblastoma cells showed a patchy localization of laminin that was apparently involved in cell-to-substrate attachment and in gap junction-like intercellular connections. Under normal conditions, the Golgi cisternae contained no laminin. Pretreatment of cells with micromolar concentrations of monensin, however, lead to an accumulation of laminin within the Golgi cisternae. These results support a role for laminin as an adhesion protein in cultured neuroblastoma cells and indicate that laminin is transported through the Golgi complex.     

Effects of laminin, fibronectin and type IV collagen on liver cell cultures

The effects on mouse liver cells of laminin, fibronectin and type IV collagen, all of which are the main matrix of the basement membrane, were studied. Laminin, a glycoprotein isolated from cultures of rat yolk sac carcinoma cells, promoted the attachment of mouse fetal liver cells to laminin-coated dishes, but did not have a strong influence upon the attachment of normal adult liver cells. On the other hand, fibronectin which was purified from mouse plasma promoted the attachment of adult liver cells but not that of fetal liver cells. The number of neonatal liver cells attached to the surfaces coated was intermediate between those of fetal and adult liver cells in each matrix. DNA synthesis and cell proliferation during the culture of full-term fetal liver cells in laminin-coated dishes were higher than those in fibronectin- or type IV collagen-coated dishes. The amount of alpha-fetoprotein secreted in the laminin-coated dishes was more than in other groups. No differences in secretion of albumin into media, however, were observed in either group. These results suggest that laminin may be necessary for cell growth, tissue organization and cell differentiation during the normal development of liver in vivo.     

Inhibition of Endothelial Cell Differentiation on a Glycosylated Reconstituted Basement Membrane Complex

The vascular basement membrane is involved in the regulation of endothelial cell differentiation. The accumulation of advanced glycosylation endproducts (AGEs) has been demonstrated on these basement membranes in patients with diabetes. We examined the effect of AGEs on endothelial cell behavior on reconstituted basement membrane, Matrigel. Human umbilical vein-derived endothelial cells (HUVECs) stopped proliferating and differentiated into capillary-like tube-shaped structures on Matrigel. Laminin antibody partially blocked this process. HUVECs cultured on glycosylated Matrigel, however, proliferated and formed a monolayer without tube formation. The inclusion of aminoguanidine, an inhibitor of AGE formation, during the glycosylation of Matrigel restored HUVEC differentiation. Although the laminin adsorbed onto the plastic culture wells promoted HUVEC attachment and spreading, glycosylated laminin reduced HUVEC attachment by 50% and abolished cellular spreading. These effects were restored by aminoguanidine. HUVEC attachment to glycosylated laminin was further reduced by AGE-modified albumin, poly I, acetylated low-density lipoprotein, or maleylated albumin, ligands for a scavenger receptor. Coating the culture dishes with the laminin peptides RGD, YIGSR, and SIKVAV supported the attachment of HUVECs that was unaffected by glycosylation. Results suggest that AGE accumulation on the basement membranes inhibits endothelial cell differentiation by impairing the normal interactions of endothelial cell receptors with their specific matrix ligands. This process may be involved in diabetic angiopathy.