Laminin 332 processing impacts cellular behavior

Laminin 332, composed of the α3, β3 and γ2 chains, is an epithelial-basement membrane specific laminin variant. Its main role in normal tissues is the maintenance of epithelial-mesenchymal cohesion in tissues exposed to external forces, including skin and stratified squamous mucosa. After being secreted and deposited in the extracellular matrix, laminin 332 undergoes physiological maturation processes consisting in the proteolytic processing of domains located within the α3 and the γ2 chains. These maturation events are essential for laminin 332 integration into the basement membrane where it plays an important function in the nucleation and maintenance of anchoring structures. Studies in normal and pathological situations have revealed that laminin 332 can trigger distinct cellular events depending on the level of its proteolytic cleavages. In this review, the biological and structural characteristics of laminin 332 domains are presented and we discuss whether they trigger specific functions.     

Laminin 5 mutations in junctional epidermolysis bullosa: molecular basis of Herlitz vs non-Herlitz phenotypes

Junctional epidermolysis bullosa (JEB) is a group of heritable blistering diseases in which tissue separation occurs within the lamina lucida of the cutaneous basement membrane zone. Clinically, two broad subcategories have been recognized: The Herlitz variant (H-JEB; OMIM 226700) is characterized by early demise of the affected individuals, usually within the first year of life, while non-Herlitz (nH-JEB; OMIM 226650) patients show a milder phenotype with life-long blistering, yet with normal lifespan. In this study, we have examined a cohort of 27 families, 15 with Herlitz and 12 with non-Herlitz JEB, for mutations in the candidate genes, LAMA3, LAMB3, and LAMC2, encoding the subunit polypeptides of laminin 5. The mutation detection strategy consisted of PCR amplification of all exons in these genes, followed by heteroduplex scanning and nucleotide sequencing. We were able to identify pathogenic mutations in both alleles of each proband, the majority of the mutations being in the LAMB3 gene. Examination of the mutation database revealed that most cases with Herlitz JEB harbored premature termination codon (PTC) mutations in both alleles. In non-Herlitz cases, the PTC mutation was frequently associated with a missense mutation or a putative splicing mutation in trans. In three cases with putative splicing mutations, RT-PCR analysis revealed a repertoire of splice variants in-frame, predicting the synthesis of either shortened or lengthened, yet partly functional, polypeptides. These observations would explain the relatively mild phenotype in cases with splicing mutations. Collectively, these findings, together with the global laminin 5 mutation database, contribute to our understanding of the genotype/phenotype correlations explaining the Herlitz vs non-Herlitz phenotypes.     

Laminin 5 can promote assembly of the lamina densa in the skin equivalent model

Skin equivalents were prepared by culturing human keratinocytes on the surface of type I collagen gel contracted by human skin fibroblasts (dermal equivalents) and by raising the gel to an air-liquid interface. A stratified squamous epithelium was formed with a well-differentiated cornified layer at the top of keratinocyte layers within 7 days after plating of the keratinocytes on the dermal equivalents. Although major basement membrane components such as collagens IV and VII and laminin 5 were detected immunohistochemically at the dermal-epidermal junction, a lamina densa was rarely observed by electron microscopy even in 14-day skin equivalents. When laminin 5 (1, 5 or 20 μg/ml) was added to the culture medium on day 7 through day 14, types IV and VII collagens at the dermal-epidermal junction stained more strongly by immunohistochemistry compared with the control. Patches of lamina densa were present along the epidermal-dermal junction, and vesicles containing electron-opaque sheets approximately 0.6 μm in diameter that reacted with anti-collagen IV antibody were also observed in basal keratinocytes in 14-day skin equivalents by electron microscopy. Morphometric analysis showed that the total length of lamina densa along the dermal-epidermal junction as well as in the vesicles increased up to 180%, 230% or 520% of control cultures by the addition of laminin 5 (1, 5 or 20 μg/ml, respectively). These results suggest that laminin 5 accelerates formation of the lamina densa along dermal-epidermal junction of the skin equivalents, depending on the concentration of laminin 5 supplemented exogenously.     

IAP insertion in the murine LamB3 gene results in junctional epidermolysis bullosa

The laminin-5 molecule functions in the attachment of various epithelia to basement membranes. Mutations in the laminin-5-coding genes have been associated with Herlitz junctional epidermolysis bullosa (HJEB), a severe and often lethal blistering disease of humans. Here we report the characterization of a spontaneous mouse mutant with an autosomal recessive blistering disease. These mice exhibit sub-epithelial blisters of the skin and mucosal surfaces and abnormal hemidesmosomes lacking sub-basal dense plates. By linkage analysis the genetic defect was localized to a 2-cM region on distal Chromosome (Chr) 1 where a laminin-5 subunit gene, LamB3, was previously localized. LamB3 mRNA and laminin-5 protein were undetectable by Northern blot analysis and immunohistochemical methods, respectively. DNA sequence analysis indicated that the LamB3 genetic defect resulted from disruption of the coding sequence by insertion of an intracisternal-A particle (IAP) at an exon/intron junction. These findings suggest a role for laminin-5 in hemidesmosome formation and indicate that the LamB3 ^IAP mutant is a useful mouse model for HJEB. Received: 27 March 1997 / Accepted: 14 May 1997     

Laminin carbohydrates are implicated in cell signaling

We have examined how laminin carbohydrates participate in cellular responses and have focused upon cell spreading and neurite outgrowth. Our earlier studies showed that unglycosylated laminin fully supported cell adhesion but did not promote subsequent spreading of mouse melanoma cells or neurite outgrowth of rat pheochromocytoma cells (Dean et al. (1990): J Biol Chem 265:12553-12562). In the present experiments, we determined whether those cellular responses could be restored to adherent cells. When a mixture of unglycosylated and glycosylated laminins was used as a substratum for mouse melanoma cells, some cells began to spread when 30% glycosylated laminin was present. At least 65% glycosylated laminin was required to elicit a maximal spreading response by the majority of the cells. In separate experiments, we found that cell spreading was fully restored by a pronase digest of glycosylated laminin; a similar digest of unglycosylated laminin had no effect. These results indicate that laminin carbohydrates, rather than polypeptide sequences, were responsible for cell spreading. We also conclude that substrate attachment of the carbohydrate moieties was not essential. In other experiments, laminins containing immature oligosaccharides were produced using two glycosylation pathway inhibitors, swainsonine or castanospermine. When such laminins were used to study cell spreading or neurite outgrowth, laminin containing immature oligosaccharides was as effective as laminin which contains fully processed oligosaccharides. In contrast, laminin with partially processed oligosaccharides had incomplete activity. These composite reconstitution experiments show that laminin carbohydrates provide essential information to responsive cells, enabling them to progress from an adherent state to a spread form or to extend neurite processes.     

Roles for Laminin in Embryogenesis: Exencephaly,Syndactyly, and Placentopathy in Mice Lacking the Laminin α5 Chain

Laminins are the major noncollagenous glycoproteins of all basal laminae (BLs). They are α/β/γ heterotrimers assembled from 10 known chains, and they subserve both structural and signaling roles. Previously described mutations in laminin chain genes result in diverse disorders that are manifested postnatally and therefore provide little insight into laminin''s roles in embryonic development. Here, we show that the laminin α5 chain is required during embryogenesis. The α5 chain is present in virtually all BLs of early somite stage embryos and then becomes restricted to specific BLs as development proceeds, including those of the surface ectoderm and placental vasculature. BLs that lose α5 retain or acquire other α chains. Embryos lacking laminin α5 die late in embryogenesis. They exhibit multiple developmental defects, including failure of anterior neural tube closure (exencephaly), failure of digit septation (syndactyly), and dysmorphogenesis of the placental labyrinth. These defects are all attributable to defects in BLs that are α5 positive in controls and that appear ultrastructurally abnormal in its absence. Other laminin α chains accumulate in these BLs, but this compensation is apparently functionally inadequate. Our results identify new roles for laminins and BLs in diverse developmental processes.     

To form and function: on the role of basement membrane mechanics in tissue development,homeostasis and disease

The basement membrane (BM) is a special type of extracellular matrix that lines the basal side of epithelial and endothelial tissues. Functionally, the BM is important for providing physical and biochemical cues to the overlying cells, sculpting the tissue into its correct size and shape. In this review, we focus on recent studies that have unveiled the complex mechanical properties of the BM. We discuss how these properties can change during development, homeostasis and disease via different molecular mechanisms, and the subsequent impact on tissue form and function in a variety of organisms. We also explore how better characterization of BM mechanics can contribute to disease diagnosis and treatment, as well as development of better in silico and in vitro models that not only impact the fields of tissue engineering and regenerative medicine, but can also reduce the use of animals in research.     

Laminin extraction and disorganization of collagen fibrils in snail muscles by EDTA treatment

Summary— Snail muscles were extracted by a solution of EDTA and electron microscopy showed that the extract contained dispersed, depolymerized collagen fibrils and cross-shaped laminin-like structures. The extracts were purified by ultracentrifugation followed by two different procedures which enriched the content of laminin-like structures. The laminin-related molecules displayed unique properties when analyzed by biochemical, immunological and morphological methods. Electrophoretic patterns of the molecular form purified primarily by ion exchange chromatography, resembled EHS-tumor laminin and displayed a cruciform shape when viewed by electron microscopy. Immunohistology, using antiserum obtained against the agarose gel-purified protein, showed that this laminin was primarily located in the extracellular matrix surrounding muscle fibers. Western blots using anti-EHS laminin antibody showed reaction of a 300 kDa subunit of this snail laminin. The protein obtained by another procedure, initially using gel filtration, followed by ion exchange chromatography, also appeared to be a laminin. It had a collapsed cruciform appearance when viewed by electron microscopy. It contained several different subunits, one of which, ca 300 kDa, reacted with anti-EHS-laminin antibody and with anti-snail laminin antibody. In contrast, EHS laminin did not react with the anti-snail laminin antibody. The composite results suggest that at least two different forms of laminin are extractable from snail muscle and that they share molecular properties and immune determinants with mouse tumor laminin.     

Laminin isoforms in endothelial and perivascular basement membranes

Laminins, one of the major functional components of basement membranes, are found underlying endothelium, and encasing pericytes and smooth muscle cells in the vessel wall. Depending on the type of blood vessel (capillary, venule, postcapillary venule, vein or artery) and their maturation state, both the endothelial and mural cell phenotype vary, with associated changes in laminin isoform expression. Laminins containing the α4 and α5 chains are the major isoforms found in the vessel wall, with the added contribution of laminin α2 in larger vessels. We here summarize current data on the precise localization of these laminin isoforms and their receptors in the different layers of the vessel wall, and their potential contribution to vascular homeostasis.     

Digenic junctional epidermolysis bullosa: mutations in COL17A1 and LAMB3 genes

Junctional epidermolysis bullosa (JEB), a genetically heterogeneous group of blistering skin diseases, can be caused by mutations in the genes encoding laminin 5 or collagen XVII, which are components of the hemidesmosome-anchoring filament complex in the skin. Here, a family with severe nonlethal JEB and with mutations in genes for both proteins was identified. The index patient was compound heterozygous for the COL17A1 mutations L855X and R1226X and was heterozygous for the LAMB3 mutation R635X. As a consequence, two functionally related proteins were affected. Absence of collagen XVII and attenuated laminin 5 expression resulted in rudimentary hemidesmosome structure and separation of the epidermis from the basement membrane, with severe skin blistering as the clinical manifestation. In contrast, single heterozygotes carrying either (1) one or the other of the COL17A1 null alleles or (2) a double heterozygote for a COL17A1 and a LAMB3 null allele did not have a pathological skin phenotype. These observations indicate that the known allelic heterogeneity in JEB is further complicated by interactions between unlinked mutations. They also demonstrate that identification of one mutation in one gene is not sufficient for determination of the genetic basis of JEB in a given family.     

Adhesion of Human Glioma Cell Lines to Fibronectin, Laminin, Vitronectin and Collagen I Is Modulated by Gangliosides in vitro

Adhesion of eight cell lines, derived from human gliomas of different histological types, to fibronectin, collagen I, vitronectin, and laminin was investigated in vitro. The glioma cell lines were found to attach to these substrates to different extents. Interestingly, all cell lines strongly attached to laminin. In addition, glioma cell adhesion was found to be dose dependent. Moreover, adhesion of three cell lines to fibronectin and collagen I was partially inhibited and to vitronectin completely prevented by GRGDTP peptide, indicating the involvement of integrin receptors in glioma cell adhesion. We have demonstrated, recently, that gangliosides play an important role in promoting glioma cell invasion of the reconstituted basement membrane, Matrigel, in vitro. In order to study the mechanism of action of gangliosides in this process, the role of six gangliosides (GM1, GM3, GD3, GD1a, GD1b, and GT1b) in cell adhesion to the four proteins was investigated in three cell lines. Although all gangliosides, with the exception of GM3, were found to enhance cell adhesion to these proteins to different extents, GD3 proved to be the most effective adhesion-promoting ganglioside in all three cell lines. GM3 was found to inhibit cell adhesion to the four proteins in one cell line but enhanced cell adhesion in two other cell lines. The three cell lines were found to express both GD3 and gangliosides recognised by the A2B5 antibody. Furthermore, adhesion of the three cell lines to fibronectin, vitronectin, laminin, and collagen I was inhibited by incubation with A2B5, demonstrating the involvement of intrinsic cell membrane gangliosides in adhesion of glioma cells to these proteins. Taken together with the observation that gangliosides modulate integrin receptor function, these data suggest that gangliosides may play a central role in the control of the adhesive and invasive properties of human glioma cells.     

Partial deletion of the LAMA3 gene is responsible for hereditary junctional epidermolysis bullosa in the American Saddlebred Horse

Laminin 5 is a heterotrimeric basement membrane protein integral to the structure and function of the dermal–epidermal junction. It consists of three glycoprotein subunits: the α3, β3 and γ2 chains, which are encoded by the LAMA3 , LAMB3 and LAMC2 genes respectively. A mutation in any of these genes results in the condition known as hereditary junctional epidermolysis bullosa (JEB). A 6589-bp deletion spanning exons 24–27 was found in the LAMA3 gene in American Saddlebred foals born with the skin-blistering condition epitheliogenesis imperfecta. The deletion confirms that this autosomal recessive condition in the American Saddlebred Horse can indeed be classified as JEB and corresponds to Herlitz JEB in humans. A diagnostic test was developed and nine of 175 randomly selected American Saddlebred foals from the 2007 foal crop were found to be carriers of the mutation (frequency of 0.026).     

Laminin Production and Basement Membrane Deposition by Mesenchymal Stem Cells upon Adipogenic Differentiation

The aim was to study laminin (LM) synthesis, integration, and deposition into the basement membrane (BM) during adipogenesis. Human bone marrow-derived mesenchymal stromal cells (MSCs) were induced along the adipogenic lineage. LM chain mRNA and protein levels were followed using quantitative real-time polymerase chain reaction (qRT-PCR), immunofluorescence (IF) staining, transmission electron microscopy (TEM), and immunoprecipitation. MSCs produced low levels of LM mRNAs but were not surrounded by BM in IF and TEM imaging. LM-α4, LM-β1, and LM-γ1 mRNAs increased during adipogenesis 3.9-, 5.8-, and 2.8-fold by day 28. LM-411 was immunoprecipitated from the ECM of the differentiated cells. Immunostaining suggested deposition of LM-411 and some LM-421. BM build-up was probably organized in part by integrin (Int) α₆β₁. At day 28, TEM images revealed BM-like structures around fat droplet-containing cells. The first signs of BM formation and Int α₆β₁ were seen using IF imaging at day 14. Laminin-411 and Int α₆β₁ were expressed in vivo in mature human subcutaneous fat tissue. Undifferentiated human MSCs did not organize LM subunits into BM, whereas LM-411 and some LM-421 are precipitated in the BM around adipocytes. This is the first demonstration of LM-411 precipitation during hMSC adipogenesis around adipocytes as a structural scaffold and Int-regulated signaling element.     

Laminin alpha 5 is required for lobar septation and visceral pleural basement membrane formation in the developing mouse lung

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. We observed normal lung branching morphogenesis and vasculogenesis, but 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 correlated with absence of laminin alpha 5, which was found to be the only laminin alpha chain present in the normal visceral pleura basement membrane. Our finding of normal lung branching morphogenesis with abnormal lobar septation demonstrates that these processes are not obligatorily linked.     

A nonsense variant in the KRT14 gene in a domestic shorthair cat with epidermolysis bullosa simplex

Epidermolysis bullosa simplex (EBS) is a hereditary blistering disease affecting the skin and mucous membranes. It has been reported in humans, cattle, buffaloes and dogs, but so far not in cats. In humans, EBS is most frequently caused by variants in the KRT5 or KRT14 genes. Here, we report a case of feline epidermolysis bullosa simplex and describe the causative genetic variant. An 11-month-old male domestic shorthair cat presented with a history of sloughed paw pads and ulcerations in the oral cavity and inner aspect of the pinnae, starting a few weeks after birth. Clinical and histopathological findings suggested a congenital blistering disease with a split formation within the basal cell layer of the epidermis and oral mucous epithelium. The genetic investigation revealed a homozygous nonsense variant in the KRT14 gene (c.979C>T, p.Gln327*). Immunohistochemistry showed a complete absence of keratin 14 staining in all epithelia present in the biopsy. To the best of our knowledge, this is the first report of feline EBS, and the first report of a spontaneous pathogenic KRT14 variant in a non-human species. The homozygous genotype in the affected cat suggests an autosomal recessive mode of inheritance.     

Sustained phenotypic reversion of junctional epidermolysis bullosa dog keratinocytes: Establishment of an immunocompetent animal model for cutaneous gene therapy

Gene transfer represents the unique therapeutic issue for a number of inherited skin disorders including junctional epidermolysis bullosa (JEB), an untreatable genodermatose caused by mutations in the adhesion ligand laminin 5 (alpha3beta3gamma2) that is secreted in the extracellular matrix by the epidermal basal keratinocytes. Because gene therapy protocols require validation in animal models, we have phenotypically reverted by oncoretroviral transfer of the curative gene the keratinocytes isolated from dogs with a spontaneous form of JEB associated with a genetic mutation in the alpha3 chain of laminin 5. We show that the transduced dog JEB keratinocytes: (1) display a sustained secretion of laminin 5 in the extracellular matrix; (2) recover the adhesion, proliferation, and clonogenic capacity of wild-type keratinocytes; (3) generate fully differentiated stratified epithelia that after grafting on immunocompromised mice produce phenotypically normal skin and sustain permanent expression of the transgene. We validate an animal model that appears particularly suitable to demonstrate feasibility, efficacy, and safety of genetic therapeutic strategies for cutaneous disorders before undertaking human clinical trials.     

A mutation in the LAMC2 gene causes the Herlitz junctional epidermolysis bullosa (H-JEB) in two French draft horse breeds

Epidermolysis bullosa (EB) is a heterogeneous group of inherited diseases characterised by skin blistering and fragility. In humans, one of the most severe forms of EB known as Herlitz-junctional EB (H-JEB), is caused by mutations in the laminin 5 genes. EB has been described in several species, like cattle, sheep, dogs, cats and horses where the mutation, a cytosine insertion in exon 10 of the LAMC2 gene, was very recently identified in Belgian horses as the mutation responsible for JEB. In this study, the same mutation was found to be totally associated with the JEB phenotype in two French draft horse breeds, Trait Breton and Trait Comtois. This result provides breeders a molecular test to better manage their breeding strategies by genetic counselling.     

A frameshift mutation within LAMC2 is responsible for Herlitz type junctional epidermolysis bullosa (HJEB) in black headed mutton sheep

Junctional epidermolysis bullosa (JEB) is a hereditary mechanobullous skin disease in humans and animals. A Herlitz type JEB was identified in German Black Headed Mutton (BHM) sheep and affected lambs were reproduced in a breeding trial. Affected lambs showed skin and mucous membranes blistering and all affected lambs died within the first weeks of life. The pedigree data were consistent with a monogenic autosomal recessive inheritance. Immunofluorescence showed a reduced expression of laminin 5 protein which consists of 3 subunits encoded by the genes LAMA3, LAMB3 and LAMC2. We screened these genes for polymorphisms. Linkage and genome-wide association analyses identified LAMC2 as the most likely candidate for HJEB. A two base pair deletion within exon 18 of the LAMC2 gene (FM872310:c.2746delCA) causes a frameshift mutation resulting in a premature stop codon (p.A928*) 13 triplets downstream of this mutation and in addition, introduces an alternative splicing of exon 18 LAMC2. This deletion showed a perfect co-segregation with HJEB in all 740 analysed BHM sheep. Identification of the LAMC2 deletion means an animal model for HJEB is now available to develop therapeutic approaches of relevance to the human form of this disease.