Laminin A


Laminin protein interactions and biological interactions

Mutational analyses of genes that encode components of the anchoring complex underlying the basolateral surface of external epithelia indicate that this anchoring structure is the major element providing for resistance to external friction. Ultrastructurally, laminin 5 (alpha3beta3gamma2; a component of the anchoring filament) appears as a thin filament bridging the hemidesmosome with the anchoring fibrils. Laminin 5 binds the cell surface through hemidesmosomal integrin alpha6beta4. However, the interaction of laminin 5 with the anchoring fibril (type VII collagen) has not been elucidated. Monomeric laminin 5 binds the NH2-terminal NC-1 domain of type VII collagen. The binding is dependent upon the native conformation of both laminin 5 and type VII collagen NC-1. Laminin 6 (alpha3beta1gamma1) has no detectable affinity for type VII collagen NC-1, indicating that the binding is mediated by the beta3 and/or gamma2 chains of laminin 5. Approximately half of the laminin 5 solubilized from human amnion or skin is covalently complexed with laminins 6 or 7 (alpha3beta2gamma1). The adduction occurs between the NH2 terminus of laminin 5 and the branch point of the short arms of laminins 6 or 7. The results are consistent with the presumed orientation of laminin 5, having the COOH-terminal G domain apposed to the hemidesmosomal integrin, and the NH2-terminal domains within the lamina densa. The results also support a model predicting that monomeric laminin 5 constitutes the anchoring filaments and bridges integrin alpha6beta4 with type VII collagen; the laminin 5-6/7 complexes are present within the interhemidesmosomal spaces bound at least by integrin alpha3beta1 where they may mediate basement membrane assembly or stability, but contribute less significantly to epithelial friction resistance (Rousselle, 1997).

Subcutaneous injection of beta 1 integrin-deficient embryonic stem cells in mice causes the formation of teratomas, although they occur with a lower frequency and are smaller than those derived from wild-type cells. Immunofluorescence analysis of the deficient tumors indicates a disorganized deposition of several basement membrane proteins. Electron microscopy demonstrates frequent gaps in cell-associated basement membranes or loss of close contact of the basement membrane to the cells. Further aberrant features are multilaminar structures and amorphous deposits, indicating a strong impairment of correct basement membrane assembly. There is a more than 90% decrease in the content of laminin-1 (alpha 1 beta 1 gamma 1) and a 70% decrease in nidogen in the beta 1 integrin-deficient teratomas. No significant changes are detected for other matrix proteins (perlecan, fibronectin, fibulins). This selective change impairs the formation of laminin-nidogen complex and enhances nidogen degradation. There is also a distinctly reduced expression of laminin alpha 1, beta 1, and gamma 1 chains. Similar reductions are also observed in cultured embryonic stem cells prior to any differentiation. No change, or only small changes, are observed for laminin alpha 2 and beta 2 chain, nidogen, and perlecan mRNA. These data emphasize a distinct role for beta 1 integrins in the correct assembly of basement membranes, which may occur through direct ligand binding and/or regulatory events at the transcriptional level (Sasaki, 1998).

The induction of acetylcholine receptor (AChR) clustering by neurally released agrin is a critical, early step in the formation of the neuromuscular junction. Laminin, a component of the muscle fiber basal lamina, also induces AChR clustering. Induction of AChR clustering in C2 myotubes is specific for laminin-1; neither laminin-2 (merosin) nor laminin-11 (a synapse-specific isoform) are active. Laminin-1 induces AChR clustering by means of a pathway that is independent of the one used by neural agrin. The effects of laminin-1 and agrin are strictly additive and occur with different time courses. Most importantly, laminin- 1-induced clustering does not require MuSK, a receptor tyrosine kinase that is part of the receptor complex for agrin. Laminin-1 does not cause tyrosine phosphorylation of MuSK in C2 myotubes and induces AChR clustering in myotubes from MuSK-/- mice that do not respond to agrin. In contrast to agrin, laminin-1 also does not induce tyrosine phosphorylation of the AChR, demonstrating that AChR tyrosine phosphorylation is not required for clustering in myotubes. Laminin-1 thus acts by a mechanism that is independent of the one used by agrin and may provide a supplemental pathway for AChR clustering during synaptogenesis (Sugiyama, 1997).

Integrins can exist in different functional states with low or high binding capacity for particular ligands. The integrin alpha6beta1, on mouse eggs and on alpha6-transfected cells, interacts with the disintegrin domain of the sperm surface protein ADAM 2 (fertilin beta). The hypothesis was tested that different states of alpha6beta1 interact with fertilin and laminin, an extracellular matrix ligand for alpha6beta1. Using alpha6-transfected cells it was found that treatments (e.g., with phorbol myristate acetate or MnCl2) that increase adhesion to laminin inhibit sperm binding. Conversely, treatments that inhibit laminin adhesion increase sperm binding. The ability of fluorescent beads coated with either fertilin beta or with the laminin E8 fragment to bind to eggs was examined. In Ca2+-containing media, fertilin beta beads bind to eggs via an interaction mediated by the disintegrin loop of fertilin beta and by the alpha6 integrin subunit. In Ca2+-containing media, laminin E8 beads do not bind to eggs. Treatment of eggs with phorbol myristate acetate or with the actin disrupting agent, latrunculin A, inhibits fertilin bead binding, but does not induce laminin E8 bead binding. Treatment of eggs with Mn2+ dramatically increases laminin E8 bead binding, and inhibits fertilin bead binding. These results provide the first evidence that different states of an integrin (alpha6beta1) can interact with an extracellular matrix ligand (laminin) or a membrane-anchored cell surface ligand (ADAM 2) (Chen, 1999).

Laminin and Development

Laminins, the main components of basement membranes, are heterotrimers consisting of alpha, beta, and gamma polypeptide chains linked together by disulfide bonds. Laminins-1 and -2 are both composed of beta1 and gamma1 chains and differ from one another in their alpha chains, which are alpha1 and alpha2 for laminin-1 and -2, respectively. The present study shows that whereas laminins-1 and -2 are synthesized in the mouse developing lung and in epithelial-mesenchymal cocultures derived from it, epithelial and mesenchymal monocultures lose their ability to synthesize the laminin alpha1 chain. However, synthesis of laminin alpha1 chain returns upon re-establishment of epithelial-mesenchymal contact. Cell-cell contact is critical, since laminin alpha1 chain is not detected in monocultures exposed to coculture-conditioned medium or in epithelial-mesenchymal cocultures in which heterotypic cell-cell contact is prevented by an interposing filter. Immunohistochemical studies on cocultures treated with brefeldin A, an inhibitor of protein secretion, indicates that both epithelial and mesenchymal cells synthesize laminin alpha1 chain upon heterotypic cell- cell contact. In a set of functional studies, embryonic lung explants were cultured in the presence of monoclonal antibodies to laminin alpha1, alpha2, and beta/gamma chains. Lung explants exposed to monoclonal antibodies to laminin alpha1 chain exhibit alterations in peribronchial cell shape and decreased smooth muscle development, as indicated by low levels of smooth muscle alpha actin and desmin. Taken together, these studies suggest that laminin alpha1 chain synthesis is regulated by epithelial-mesenchymal interaction and may play a role in airway smooth muscle development (Schuger, 1997).

Undifferentiated mesenchymal cells were isolated from mouse embryonic lungs and plated at subconfluent and confluent densities. During the first 5 hours in culture, all the cells were negative for smooth muscle markers. After 24 hours in culture, the mesenchymal cells that spread synthesized smooth muscle alpha-actin, muscle myosin, desmin and SM22 in levels comparable to those of mature smooth muscle. The cells that do not spread remain negative for smooth muscle markers. SM differentiation is independent of cell-cell contact or proliferation. In additional studies, undifferentiated lung mesenchymal cells were cocultured with lung embryonic epithelial cells at high density. The epithelial cells aggregate into cysts surrounded by mesenchymal cells and a basement membrane is formed between the two cell types. In these cocultures, the mesenchymal cells in contact with the basement membrane spread and differentiate into smooth muscle. The rest of the mesenchymal cells remain round and negative for smooth muscle markers. Basement membrane assembly, mesenchymal cell spreading and smooth muscle differentiation are all blocked with the inhibition of laminin polymerization by an antibody to the globular regions of laminin beta1/gamma1 chains. These studies indicated that lung embryonic mesenchymal cells have the potential to differentiate into smooth muscle and the process is triggered by their spreading along the airway basement membrane (Yang, 1998).

To explore the role of homeobox genes in the intestine, the human colon adenocarcinoma cell line Caco2-TC7 has been stably transfected with plasmids synthesizing Cdx1 and Cdx2 (Homologs of Drosophila Caudal) sense and antisense RNAs. Cdx1 overexpression or inhibition by antisense RNA does not markedly modify the cell differentiation markers analyzed in this study. In contrast, Cdx2 overexpression stimulates two typical markers of enterocytic differentiation: sucrase-isomaltase and lactase. Cells in which the endogenous expression of Cdx2 is reduced by antisense RNA attach poorly to the substratum. Conversely, Cdx2 overexpression modifies the expression of molecules involved in cell-cell and cell-substratum interactions and in transduction process: indeed, E-cadherin, integrin-beta4 subunit, laminin-gamma2 chain, hemidesmosomal protein, APC, and alpha-actinin are upregulated. Interestingly, most of these molecules are preferentially expressed in vivo in the differentiated villi enterocytes, rather than in crypt cells. Cdx2 overexpression also results in the stimulation of HoxA-9 mRNA expression, a homeobox gene selectively expressed in the colon. In contrast, Cdx2-overexpressing cells display a decline of Cdx1 mRNA, which is mostly found in vivo in crypt cells. When implanted in nude mice, Cdx2-overexpressing cells produce larger tumors than control cells, and form glandular and villus-like structures. Laminin-1 is known to stimulate intestinal cell differentiation in vitro. The differentiating effect of laminin-1 coatings on Caco2-TC7 cells is accompanied by an upregulation of Cdx2. To further document this observation, a series of Caco2 clones was analyzed in which the production of laminin-alpha1 chain was differentially inhibited by antisense RNA. A positive correlation exists between the level of Cdx2 expression, that of endogenous laminin-alpha1 chain mRNA and that of sucrase-isomaltase expression in these cell lines. Taken together, these results suggest (1) that Cdx1 and Cdx2 homeobox genes play distinct roles in the intestinal epithelium; (2) that Cdx2 provokes pleiotropic effects triggering cells towards the phenotype of differentiated villus enterocytes, and (3) that Cdx2 expression is modulated by basement membrane components. It is concluded that Cdx2 plays a key role in the extracellular matrix-mediated intestinal cell differentiation (Lorentz, 1997).

The role of integrin-extracellular matrix interactions in the morphogenesis of ductal structures in vivo has been examined using the developing mouse mammary gland as a model. At puberty, ductal growth from terminal end buds results in an arborescent network that eventually fills the gland, whereupon the buds shrink in size and become mitotically inactive. End buds are surrounded by a basement membrane, which contains laminin-1 and collagen IV. To address the role of cell-matrix interactions in gland development, pellets containing function-perturbing anti-beta1 integrin, anti-alpha6 integrin, and anti-laminin antibodies, respectively, were implanted into mammary glands at puberty. Blocking beta1 integrins dramatically reduces both the number of end buds per gland and the extent of the mammary ductal network, compared with controls. These effects are specific to the end buds since the rest of the gland architecture remains intact. Reduced development is still apparent after 6 days, but end buds subsequently reappear, indicating that the inhibition of beta1 integrins is reversible. Similar results were obtained with anti-laminin antibodies. In contrast, no effect on morphogenesis in vivo was seen with anti-alpha6 integrin antibody, suggesting that alpha6 is not the important partner for beta1 in this system. The studies with beta1 integrin were confirmed in a culture model of ductal morphogenesis, where hepatocyte growth factor (HGF)-induced tubulogenesis is dependent on functional beta1 integrins. Thus integrins and HGF cooperate to regulate ductal morphogenesis. It is proposed that both laminin and beta1 integrins are required to permit cellular traction through the stromal matrix and are therefore essential for maintaining end bud structure and function in normal pubertal mammary gland development (Klinowska, 1999).

Basement membranes of the developing tooth contain laminins, but the nature of these laminins has not been described. The distribution of five different laminin alpha chains was studied during tooth development. Both epithelial and mesenchymal cells produce laminin alpha chains. The mRNAs of three laminin alpha chains, alpha1, alpha2, and alpha4, are expressed in the tooth mesenchyme, whereas two, the alpha3 and alpha5 chain mRNAs, are found in epithelium. Drastic changes in the expression patterns of the two epithelial chains are found during development. The alpha5 mRNA is widely expressed in tooth epithelia, and the corresponding protein is evenly distributed along the tooth basement membrane throughout embryonic development. This suggests a role for alpha5 as a major laminin alpha chain in tooth basement membrane during embryonic stages. The subsequent disappearance of alpha5 and the drastic increase in alpha3A mRNA expression during terminal ameloblast differentiation and enamel secretion suggests that alpha3A acts as an important chain in the enamel matrix after degradation of tooth basement membrane. These studies show that laminin networks in tooth epithelia form as a result of epithelial-mesenchymal interactions and that the molecular composition of the laminin networks varies drastically during development of tooth (Salmivirta, 1997).

Tooth morphogenesis is regulated by epithelial-mesenchymal interactions mediated by the basement membrane (BM). Laminins are major glycoprotein components of the BMs, which are involved in several cellular activities. The expression and localization of the alpha3, beta3, and gamma2 laminin-5 subunits have been analyzed by in situ hybridization and immunohistochemistry during mouse molar development. Initially (E12), mRNAs of all subunits are detected in the entire dental epithelium and the corresponding proteins are located in the BM. During cap formation (E13-14), transcripts for the alpha3 and gamma2 subunits are localized in the outer dental epithelium (ODE), whereas the beta3 subunit mRNA is present in the inner dental epithelium (IDE). During the early bell stage (E16), immunoreactivity for all subunits disappears from the BM along the IDE, although intense signals for beta3 mRNA are detectable in cells of the IDE. Subsequently, when the dentinal matrix is secreted by odontoblasts (E18-19.5), mRNAs of all three subunits are re-expressed by ameloblasts, and the corresponding proteins are detected in ameloblasts and in the enamel matrix. Tissue recombination experiments demonstrate that when E16 IDE or ODE is associated with E18 dental papilla mesenchyme, immunostaining for all laminin-5 subunits disappears from the BM, whereas when cultured with non-dental limb bud mesenchyme, ameloblasts remain positive after 48 hr of culture. These results suggest that the temporospatial expression of laminin-5 subunits in tooth development, which appears to be differentially controlled by the dental mesenchyme, might be related to the enamel organ histo-morphogenesis and the ameloblast differentiation (Yoshiba, 1998).

Presynaptic and postsynaptic membranes directly oppose each other at chemical synapses, minimizing the delay in transmitting information across the synaptic cleft. In contrast, extrasynaptic neuronal surfaces are almost entirely covered by processes from glial cells. The exclusion of glial cells from the synaptic cleft, and the long-term stability of synapses, presumably result in large part from the tight adhesion between presynaptic and postsynaptic elements. There is another requirement for synaptic maintenance: Schwann cells (glial cells of the skeletal neuromuscular synapse) are actively inhibited from entering the synaptic cleft between the motor nerve terminal and the muscle fiber. One inhibitory component is laminin 11, a heterotrimeric glycoprotein that is concentrated in the synaptic cleft. Regulation of an inhibitory interaction between glial cells and synaptic cleft components may contribute to synaptic rearrangements, and loss of this inhibition may underlie the loss of synapses that results from injury to the postsynaptic cell (Patton, 1998).

Laminin 5 (alpha3beta3gamma2) distribution in the human thymus was investigated by immunofluorescence on frozen sections with anti-alpha3, -beta3, and -gamma2 mAbs. In addition to a linear staining of subcapsular basal laminae, the three mAbs give a disperse staining in the parenchyma restricted to the medullary area on a subset of stellate epithelial cells and vessel structures. Laminin 5 may influence mature human thymocyte expansion. Although bulk laminin and laminin 2, when cross-linked, are comitogenic with a TCR signal, cross-linked laminin 5 has no effect on mitosis. By contrast, soluble laminin 5 inhibits thymocyte proliferation induced by a TCR signal. This is accompanied by a particular pattern of inhibition of early tyrosine kinases, including Zap 70 and p59(fyn) inhibition, but not overall inhibition of p56(lck). Using a mAb specific for alpha6beta4 integrins, it was observed that while alpha3beta1 is known to be uniformly present on all thymocytes, alpha6beta4 expression parallels thymocyte maturation; thus a correspondence exists between laminin 5 in the thymic medulla and alpha6beta4 on mature thymocytes. Moreover, the soluble Ab against alpha6beta4 inhibits thymocyte proliferation and reproduces the same pattern of tyrosine kinase phosphorylation suggesting that alpha6beta4 is involved in laminin 5-induced modulation of T cell activation (Vivinus-Nebot, 1999).

Laminin alpha/beta/gamma heterotrimers are the major noncollagenous components of all basement membranes. To date, five alpha, three beta, and three gamma chains have been identified. Laminin alpha 5 is expressed early in lung development and colocalizes with laminin alpha1. While laminin alpha1 expression in the lung is restricted to the embryonic period, laminin alpha 5 expression persists throughout embryogenesis and adulthood. Targeted mutation of the mouse laminin alpha 5 gene Lama5 causes embryonic lethality at E14-E17 associated with exencephaly, syndactyly, placentopathy, and kidney defects, all attributable to abnormal basement membranes. In this investigation, lung development in Lama5-/- mice up to E16.5 was examined. Normal lung branching morphogenesis and vasculogenesis were observed, accompanied by incomplete lobar septation and absence of the visceral pleura basement membrane. Preservation of branching morphogenesis was associated with ectopic deposition of laminin alpha 4 in the airway basement membrane. Perturbation of pleural basement membrane formation and right lung septation correlates with absence of laminin alpha 5, which is the only laminin alpha chain present in the normal visceral pleura basement membrane. The finding of normal lung branching morphogenesis with abnormal lobar septation demonstrates that these processes are not obligatorily linked (Nguyen, 2002).

Development of the peripheral nervous system requires radial axonal sorting by Schwann cells (SCs). To accomplish sorting, SCs must both proliferate and undergo morphogenetic changes such as process extension. Signaling studies reveal pathways that control either proliferation or morphogenesis, and laminin is essential for SC proliferation. However, it is not clear whether laminin is also required for SC morphogenesis. By using a novel time-lapse live-cell-imaging technique, this study demonstrated that laminins are required for SCs to form a bipolar shape as well as for process extension. These morphological deficits are accompanied by alterations in signaling pathways. Phosphorylation of Schwannomin at serine 518 and activation of Rho GTPase Cdc42 and Rac1 were all significantly decreased in SCs lacking laminins. Inhibiting Rac1 and/or Cdc42 activities in cultured SCs attenuated laminin-induced myelination, whereas forced activation of Rac1 and/or Cdc42 in vivo improved sorting and hypomyelinating phenotypes in SCs lacking laminins. These findings indicate that laminins play a pivotal role in regulating SC cytoskeletal signaling. Coupled with previous results demonstrating that laminin is critical for SC proliferation, this work identifies laminin signaling as a central regulator coordinating the processes of proliferation and morphogenesis in radial axonal sorting (Yu, 2009).

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

The establishment of alternative cell fates during embryoid body differentiation has been investigated, when embryonic stem (ES) cells diverge into two epithelia simulating the pre-gastrulation endoderm and ectoderm. 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, it was demonstrated 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. 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 stem cell flow through two cell layers connected by laminin 1 and stem cell 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 (Li, 2004).

The inner cell mass (ICM) of preimplantation and early postimplantation mammalian embryos contain cells ancestral to the entire individual, that undergo extensive morphological change prior to gastrulation. In the blastocyst and early egg cylinder the ICM consists of an aggregate of non-polar stem cells, which before gastrulation undergo epithelialization and cavitation, creating a pseudostratified columnar epithelium that surrounds a central cavity similar to the proamniotic canal of the early embryo. The pseudostratified columnar epithelium or epiblast attaches to the sub-endodermal basement membrane (BM). This polarized epithelium allows intermingling of clonal derivatives and is thought to be necessary for gastrulation. Much is known about the role of endoderm to ectoderm signalling in anteroposterior patterning of the early embryo. The establishment of major elements of the amniote body plan during gastrulation has been also studied in detail. However, the mechanism that precedes these changes and transforms the non-polar primitive ectoderm into the columnar polar epiblast is little understood (Li, 2004).

Embryonic stem cell derived embryoid bodies (EBs) are similar to the egg cylinder embryo, but, in contrast to it, they can be grown in large quantities, providing a useful model for early embryogenesis. The mechanism of EB differentiation has been set out as a model for pregastrulation development and tube formation by cavitation. EBs have an external endoderm that is similar to the primitive or visceral endoderm of the embryo and is separated from the inner columnar ectoderm by a basement membrane (BM). Using a genetically undefined spontaneous mutation, which fails to form the columnar ectoderm layer, it was proposed that cavitation is regulated by two signals: one emanating from the outer endoderm layer was thought to be responsible for the apoptotic signal/s of cavitation; the second, originating in the BM, was considered necessary for the maintenance and survival of the columnar ectoderm (Li, 2004 and references therein).

The work carried out in this study started as a study of the role of FGF signalling in EB differentiation and led to questions regarding BM assembly that were investigated using ES cells that express truncated Fgfr2 cDNA as a dominant-negative mutation. ES cells expressing dnFgfr fail to develop the two characteristic cell layers of the EB. They display a homogenous aggregate of non-polar cells and form no endoderm or ectoderm-like elements, but survive for weeks during cultivation. EBs formed by dnFgfr ES cells fail to synthesize laminin and collagen IV isotypes, which supply the protein network of the BM. Co-cultivating wild-type and dnFgfr ES cells rescued EB differentiation, suggesting that an FGF-controlled extracellular substance, subsequently identified as laminin 1, is required for epiblast differentiation. Exogenously added laminin 1 partially rescues the EB phenotype and induces epithelial transformation, demonstrating that laminin 1 produced by the endoderm is necessary and sufficient to induce epiblast polarization (Li, 2004).

Laminin 1 has been shown to be required for EB differentiation. Targeted disruption of ß1-integrin, which inhibits laminin alpha1 synthesis, interferes with epiblast differentiation. Disruption of Lamc1 encoding laminin gamma1, one of the three polypeptides of the laminin 1 heterotrimer, leads to a similar phenotype. Significantly, defective epiblast differentiation caused by loss of either gene was rescued by exogenously added laminin 1, which in turn could be inhibited by the E3 fragment of laminin alpha1 containing the heparin and sulfatide binding site of the LG4 globular domain of the laminin alpha1-chain. Recognising the potential importance of these findings for understanding epithelial differentiation and early development, it would help their analysis if the succession and main intermediates of EB differentiation were defined (Li, 2004).

In the present study, attempts were made to obtain a comprehensive view of the developmental interactions that precede gastrulation. To achieve this, several specific questions had to be answered. Is FGF signalling required for the differentiation of both epithelia and the pattern of their arrangement in the EB, or for only an initial step that is necessary for later events? Defective FGF signalling could be partially restored by exogenous laminin 1. The next question is can the same effect be obtained by laminin 1 presented by the BM in a physiological cell-matrix interaction? It was also important to determine whether laminin affects the stem cell directly, or whether it activates precursors after they reached a specific stage of FGF dependent differentiation. To answer these questions, mutant and wild-type ES cell lines were used , and their behaviour was studied as an effect of chemical inhibitors and co-cultivation experiments between mutant and wild-type cells (Li, 2004).

As an experimental system to elucidate interactions between the endoderm and primitive ectoderm GATA4- or GATA6-transformed endoderm-like cells co-cultivated with mutant ES cell lines were used. This system demonstrated that GATA4 and GATA6 transform ES cells into functional extra-embryonic endoderm that deposits a BM, which in turn mediates epiblast polarization. GATA transformed cells synthesize and later secrete laminin 1 and collagen IV into the culture supernatant, which could be used to rescue epiblast differentiation. Genetic evidence of laminin gamma1 null ES cells has demonstrated the specificity of mutant rescue. This experimental system thus recreated the physiological BM-mediated interaction and allowed the separation of endoderm and epiblast differentiation according to their respective FGF/GATA6 and laminin/Rho kinase-dependent mechanisms (Li, 2004).

Endoderm differentiation depends on FGF signalling, as demonstrated by the targeted disruption of Fgf4. Fgf4 is expressed in the ICM and contributes to the maintenance of the endoderm, where the multiple FGF receptors that read its signals are localized. Expression of GATA4 and GATA6, where GATA4 is regulated by GATA6, is controlled by FGF signalling. Nevertheless, the immediate downstream elements of FGF signalling are insufficiently understood in EB differentiation. In vitro evidence suggests that most FGF dependent signals go through Frs2a, a docking protein, which communicates with the Grb2 adaptor. Interestingly although null mutants of Fgf4 die with defective endoderm development shortly after implantation, Frs2a null embryos survive until advanced gastrulation, indicating that FGF signalling may exhibit unique characteristics in the early embryo. Analysis of signal transduction in dnFgfr ES cells revealed that PI3K-Akt/PKB rather than MAPK-ERK signalling is affected by defective FGF activity. In agreement, this study found that constitutively active Akt/PKB enhances endoderm development and the synthesis of laminin and collagen IV isotypes, indicating that the PI3K-Akt/PKB pathway predominates in FGF-dependent endoderm differentiation (Li, 2004).

GATA6 is an intermediary of FGF signalling. GATA6, which is transcribed already in the ICM, behaves as a master gene for endoderm differentiation. GATA6 activates the synthesis of all three polypeptide chains of laminin 1, which together with collagen IV, nidogen and perlecan assemble into the sub-endodermal BM. GATA factors induce endoderm differentiation and BM assembly even in dnFgfr ES cells, indicating that once activated, these transcription factors induce endoderm differentiation independently from FGF signalling. Because endoderm differentiation requires GATA6 and because cysts of GATA6 transformed cells contain only endoderm-like elements, it is concluded that GATA factors are required and sufficient to induce endoderm development and deposition of the subendodermal BM (Li, 2004).

Additional elements of this pathway are the transcription factors COUP-TFs I and II, which are upregulated by GATA4/6 during endoderm development and induce Lamc1 and Lamb1 expression. It follows that minimal elements of this interaction are, sequentially, Fgf4, multiple Fgfr, PI3K and AKT/PKB, GATA6 and GATA4, COUP-TFs I and II, as well as the genes encoding the three polypeptide chains of laminin 1 (Li, 2004).

Evidence demonstrates that E-cadherin is also required for early EB differentiation. E-cadherin-null ES cells fail to aggregate, do not form a normal ectoderm and do not undergo EB differentiation. Therefore, E-cadherin-dependent ES cell aggregation may be a prerequisite for the restriction of FGF signalling to the outer cells of the developing EB. E-cadherin is connected to the ß-catenin-GSK3-wnt pathway. Patterning events involving cadherin-Wnt/ß-catenin interactions have been shown to be controlled by FGF signalling (Li, 2004).

There is strong evidence for the epithelialization of ES cells by exogenous laminin 1. Laminin 1 can induce epiblast differentiation as part of the BM that mediates the physiological interaction of the endoderm with the epiblast. While laminin 1 binds to ES cells and their ectodermal derivatives, it does not associate with the primitive endoderm. Thus, the cell-binding domains of the laminin alpha1 chain determine the location of the subendodermal BM by interacting with their receptors displayed by the stem cells localized below the endoderm layer. This therefore defines the direction of laminin-mediated signalling, thereby determining the topographical relationship of endoderm and ectoderm (Li, 2004).

Besides inducing epiblast polarization, the BM affects the simple two-cell layer pattern of the EB and egg cylinder embryo. Since cell-to-matrix interactions take place through direct contact, epithelialization of residual stem cells is precluded, and a single epiblast monolayer develops from cells immediately adjacent to the BM. It has been proposed that the residual stem cells are removed by programmed cell death induced by factors derived from the endoderm, to form a central cavity. Investigation of the role of BMP signalling in cavitation indicates that BMP2 synthesizes in the endoderm, and BMP4 in the primitive ectoderm can both contribute to cavitation, although BMP4 is expressed only for a short period. The data indicate that cavitation and columnar ectoderm differentiation do not require the endoderm, provided that exogenous laminin 1 is presented. It is therefore possible that the developing ectoderm itself secretes the necessary apoptotic factors, such as BMP4, although inhibition of ROCK activity uncouples cavitation from full epithelialization of the primitive ectoderm and argues that cavitation may be either not different from necrosis, or it might be due to mechanical separation of the columnar ectoderm from the residual stem cells. This issue requires further study (Li, 2004).

Dominant-negative ROCK abolishes epiblast polarization without affecting endoderm differentiation, suggesting that it may be regulated separately in the two cell lineages. This assumption was supported by observing that ROCK expression and epiblast polarization does not require the endoderm for the laminin-induced differentiation of dnFgfr ES cells. Although ROCK is required for the epithelialization of the primitive ectoderm, it is not sufficient to induce this process, as suggested by the observation that dominant-active ROCK does not rescue dnFgfr differentiation. Although in the epiblast ROCK activity may be induced by laminin, in the endoderm it appears to be under FGF control and the resistance of endodermal differentiation to ROCK inhibition is consistent with the possibility that RAC1 or Cdc42, which are co-expressed in the endoderm, may have a role in endodermal differentiation (Li, 2004).

Separation of endoderm and epiblast differentiation has been repeatedly observed in this study. FGF signalling is shown to be required for endoderm differentiation but not for epiblast polarization, which is independently induced by laminin 1 of the sub-endodermal BM. The two lineages are also distinguished by laminin binding. ES cells and their ectodermal derivatives bind laminin, while the primitive and visceral endoderm do not, which defines the direction of laminin-induced differentiation. It follows that the extra-embryonic and embryonic epithelium of the EB and egg cylinder embryo develop by distinct mechanisms, which are connected by the inductive activity of the laminin component of their common BM. Future research will have to clarify whether other epithelial transitions are also controlled by laminin-dependent mechanisms (Li, 2004).

Laminin and cell death

Excess excitatory amino acids can provoke neuronal death in the hippocampus, and the extracellular proteases tissue plasminogen activator (tPA) and plasmin (ogen) have been implicated in this death. To investigate substrates for plasmin that might influence neuronal degeneration, extracellular matrix (ECM) protein expression was examined. Laminin is expressed in the hippocampus and disappears after excitotoxin injection. Laminin disappearance precedes neuronal death, is spatially coincident with regions that exhibit neuronal loss, and is blocked by either tPA-deficiency or infusion of a plasmin inhibitor, both of which also block neuronal degeneration. Preventing neuron-laminin interaction by infusion of anti-laminin antibodies into tPA-deficient mice restores excitotoxic sensitivity to their hippocampal neurons. These results indicate that disruption of neuron-ECM interaction via tPA/plasmin catalyzed degradation of laminin, sensitizes hippocampal neurons to cell death. tPA is approved for treatment of thromotic stroke due to its fibrinolytic activity. However, based on their involvement in neuronal degeneration, tPA/plasmin use for stroke might have deleterious consequences (Chen, 1997).

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Laminin A: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

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