Formation of hemidesmosomes in vitro by a transformed rat bladder cell line

Two hemidesmosomal plaque components of 230 and 180 kD have recently been characterized using autoantibodies in the serum samples of bullous pemphigoid (BP) patients (Klatte, D. H., M. A. Kurpakus, K. A. Grelling, and J. C. R. Jones. 1989, J. Cell Biol. 109:3377-3390). These BP autoantibodies generate the type of staining patterns that one would predict for formed hemidesmosomes, i.e., a punctate staining pattern towards the substratum; in less than 50% of various primary epithelial and transformed epidermal cell lines even when such cells are maintained in culture for prolonged periods. In contrast, affinity- purified human autoantibodies against the 230-kD hemidesmosomal plaque component generate intense immunofluorescence staining along the region of cell-substratum interaction in the rat bladder tumor cell line 804G maintained on uncoated glass cover-slips. This pattern is distinct from that observed in the 804G cells using an antibody preparation directed against vinculin, a component of adhesion plaques. Ultrastructural analyses of the 804G cells reveals that hemidesmosome-like structures occur along the basal surface of cells where they abut the substratum. These structures are present in 804G cells maintained in culture in reduced levels of Ca2+ and are recognized by autoantibodies directed against the 230-kD hemidesmosomal plaque component as determined by immunogold ultrastructural localization. To study hemidesmosome appearance in this cell line, 804G cells were trypsinized and then allowed to readhere to glass coverslips. In rounded, unattached 804G cells, hemidesmosome-like plaque structures occur along the cell surface. These structures are recognized by the 230-kD autoantibodies. At 1 h after plating, hemidesmosomes are observed along the substratum attached surface of cells. Protein synthesis is not required for the appearance of these hemidesmosomes. Within 4 h of plating, autoantibody staining and hemidesmosomes appear towards the cell periphery. Subsequently, the polypeptide recognized by the BP autoantibodies becomes concentrated in the perinuclear region, where there are numerous hemidesmosomes. We propose that the hemidesmosomes in 804G cells are involved in cell-substratum adhesion. We discuss possible mechanisms of assembly of hemidesmosomes in the 804G cells. Indeed, the 804G cells should prove an invaluable cell line for the biochemical and molecular dissection of hemidesmosome structure, function, and assembly.     

Protein kinase C-dependent mobilization of the alpha6beta4 integrin from hemidesmosomes and its association with actin-rich cell protrusions drive the chemotactic migration of carcinoma cells.

We explored the hypothesis that the chemotactic migration of carcinoma cells that assemble hemidesmosomes involves the activation of a signaling pathway that releases the alpha6beta4 integrin from these stable adhesion complexes and promotes its association with F-actin in cell protrusions enabling it to function in migration. Squamous carcinoma-derived A431 cells were used because they express alpha6beta4 and migrate in response to EGF stimulation. Using function-blocking antibodies, we show that the alpha6beta4 integrin participates in EGF-stimulated chemotaxis and is required for lamellae formation on laminin-1. At concentrations of EGF that stimulate A431 chemotaxis ( approximately 1 ng/ml), the alpha6beta4 integrin is mobilized from hemidesmosomes as evidenced by indirect immunofluorescence microscopy using mAbs specific for this integrin and hemidesmosomal components and its loss from a cytokeratin fraction obtained by detergent extraction. EGF stimulation also increased the formation of lamellipodia and membrane ruffles that contained alpha6beta4 in association with F-actin. Importantly, we demonstrate that this mobilization of alpha6beta4 from hemidesmosomes and its redistribution to cell protrusions occurs by a mechanism that involves activation of protein kinase C-alpha and that it is associated with the phosphorylation of the beta4 integrin subunit on serine residues. Thus, the chemotactic migration of A431 cells on laminin-1 requires not only the formation of F-actin-rich cell protrusions that mediate alpha6beta4-dependent cell movement but also the disruption of alpha6beta4-containing hemidesmosomes by protein kinase C.     

Keratinocyte-Targeted Expression of Human Laminin γ2 Rescues Skin Blistering and Early Lethality of Laminin γ2 Deficient Mice

Laminin-332 is a heterotrimeric basement membrane component comprised of the α3, ß3, and γ2 laminin chains. Laminin-332 modulates epithelial cell processes, such as adhesion, migration, and differentiation and is prominent in many embryonic and adult tissues. In skin, laminin-332 is secreted by keratinocytes and is a key component of hemidesmosomes connecting the keratinocytes to the underlying dermis. In mice, lack of expression of any of the three Laminin-332 chains result in impaired anchorage and detachment of the epidermis, similar to that seen in human junctional epidermolysis bullosa, and death occurs within a few days after birth. To bypass the early lethality of laminin-332 deficiency caused by the knockout of the mouse laminin γ2 chain, we expressed a dox-controllable human laminin γ2 transgene under a keratinocyte-specific promoter on the laminin γ2 (Lamc2) knockout background. These mice appear similar to their wild-type littermates, do not develop skin blisters, are fertile, and survive >1.5 years. Immunofluorescence analyses of the skin showed that human laminin γ2 colocalized with mouse laminin α3 and ß3 in the basement membrane zone underlying the epidermis. Furthermore, the presence of “humanized” laminin-332 in the epidermal basement membrane zone rescued the alterations in the deposition of hemidesmosomal components, such as plectin, collagen type XVII/BP180, and integrin α6 and ß4 chains, seen in conventional Lamc2 knockout mice, leading to restored formation of hemidesmosomes. These mice will be a valuable tool for studies of organs deficient in laminin-332 and the role of laminin-332 in skin, including wound healing.     

Spatial and temporal control of laminin-332 (5) and -511 (10) expression during induction of anagen hair growth.

Basement membrane plays important roles in hair growth. We characterized changes in laminin isoform expression during hair cycling. At the mRNA level, laminin-511 (10) expression underwent a steady increase during anagen stages. In contrast, laminin-332 (5) expression was initially upregulated in outer root sheath (ORS) keratinocytes at anagen II and then transiently downregulated. Laminin-332 significantly increased coincident with the signal in inner root sheath and hair matrix cells after anagen IV. Levels of laminin-332 proteins were also upregulated at late anagen I-III but dropped after anagen IV. This decrease coincided with increased levels of mRNA encoding the two proteases, membrane type 1 metalloproteinase and bone morphogenetic protein 1, involved in laminin-332 processing. Immunohistochemistry demonstrated that laminin-332 and alpha6 beta4 integrin were well colocalized, but their signals were remarkably decreased in the lower half of follicles after anagen VI. Consistent with these data, ultrastructurally mature hemidesmosomes were seen in ORS keratinocytes at anagen II, whereas at anagen VI, only fragmental hemidesmosomes were present. In hair follicle culture, laminin-511 (10)/521 (11)-rich human placental laminin enhanced hair growth, whereas recombinant laminin-332 antagonized hair growth induced by laminin-511. Our results indicate a positive role for laminin-511 and a negative role for laminin-332 on hair growth.     

The tetraspan molecule CD151, a novel constituent of hemidesmosomes, associates with the integrin alpha6beta4 and may regulate the spatial organization of hemidesmosomes

CD151 is a cell surface protein that belongs to the tetraspan superfamily. It associates with other tetraspan molecules and certain integrins to form large complexes at the cell surface. CD151 is expressed by a variety of epithelia and mesenchymal cells. We demonstrate here that in human skin CD151 is codistributed with alpha3beta1 and alpha6beta4 at the basolateral surface of basal keratinocytes. Immunoelectron microscopy showed that CD151 is concentrated in hemidesmosomes. By immunoprecipitation from transfected K562 cells, we established that CD151 associates with alpha3beta1 and alpha6beta4. In beta4-deficient pyloric atresia associated with junctional epidermolysis bullosa (PA-JEB) keratinocytes, CD151 and alpha3beta1 are clustered together at the basal cell surface in association with patches of laminin-5. Focal adhesions are present at the periphery of these clusters, connected with actin filaments, and they contain both CD151 and alpha3beta1. Transient transfection studies of PA-JEB cells with beta4 revealed that the integrin alpha6beta4 becomes incorporated into the alpha3beta1-CD151 clusters where it induces the formation of hemidesmosomes. As a result, the amount of alpha3beta1 in the clusters diminishes and the protein becomes restricted to the peripheral focal adhesions. Furthermore, CD151 becomes predominantly associated with alpha6beta4 in hemidesmosomes, whereas its codistribution with alpha3beta1 in focal adhesions becomes partial. The localization of alpha6beta4 in the pre-hemidesmosomal clusters is accompanied by a strong upregulation of CD151, which is at least partly due to increased cell surface expression. Using beta4 chimeras containing the extracellular and transmembrane domain of the IL-2 receptor and the cytoplasmic domain of beta4, we found that for recruitment of CD151 into hemidesmosomes, the beta4 subunit must be associated with alpha6, confirming that integrins associate with tetraspans via their alpha subunits. CD151 is the only tetraspan identified in hemidesmosomal structures. Others, such as CD9 and CD81, remain diffusely distributed at the cell surface.In conclusion, we show that CD151 is a major component of (pre)-hemidesmosomal structures and that its recruitment into hemidesmosomes is regulated by the integrin alpha6beta4. We suggest that CD151 plays a role in the formation and stability of hemidesmosomes by providing a framework for the spatial organization of the different hemidesmosomal components.     

The C-terminus of the gamma 2 chain but not of the beta 3 chain of laminin-332 is indirectly but indispensably necessary for integrin-mediated cell reactions

Using a recombinant mini-laminin-332, we showed that truncation of the three C-terminal amino acids of the gamma 2 chain, but not of the C-terminal amino acid of the beta 3 chain, completely abolished alpha 3 beta 1 integrin binding and its cellular functions, such as attachment and spreading. However, a synthetic peptide mimicking the gamma 2 chain C-terminus did not interfere with alpha 3 beta 1 integrin binding or cell adhesion and spreading on laminin-332 as measured by protein interaction assays and electric cell-substrate impedance sensing. Nor was the soluble peptide able to restore the loss of integrin-mediated cell adhesiveness to mini-laminin-332 after deletion of the gamma 2 chain C-terminus. These findings spoke against the hypothesis that the gamma 2 chain C-terminus of laminin-332 is a part of the alpha 3 beta 1 integrin interaction site. In addition, structural studies with electron microscopy showed that truncation of the gamma 2 chain C-terminus opened up the compact supradomain structure of LG1-3 domains. Thus, by inducing or stabilizing an integrin binding-competent conformation or array of the LG1-3 domains, the gamma 2 chain C-terminus plays an indirect but essential role in laminin-332 recognition by alpha 3 beta 1 integrin and, hence, its cellular functions.     

Integrin alpha 6/beta 4 complex is located in hemidesmosomes, suggesting a major role in epidermal cell-basement membrane adhesion

The alpha 6/beta 4 complex is a member of the integrin family of adhesion receptors. It is found on a variety of epithelial cell types, but is most strongly expressed on stratified squamous epithelia. Fluorescent antibody staining of human epidermis suggests that the beta 4 subunit is strongly localized to the basal region showing a similar distribution to that of the 230-kD bullous pemphigoid antigen. The alpha 6 subunit is also strongly localized to the basal region but in addition is present over the entire surfaces of basal cells and some cells in the immediate suprabasal region. By contrast staining for beta 1, alpha 2, and alpha 3 subunits was very weak basally, but strong on all other surfaces of basal epidermal cells. These results suggest that different integrin complexes play differing roles in cell-cell and cell-matrix adhesion in the epidermis. Immunoelectron microscopy showed that the alpha 6/beta 4 complex at the basal epidermal surface is strongly localized to hemidesmosomes. This result provides the first well-characterized monoclonal antibody markers for hemidesmosomes and suggests that the alpha 6/beta 4 complex plays a major role in epidermal cell-basement membrane adhesion. We suggest that the cytoplasmic domains of these transmembrane glycoproteins may contribute to the structure of hemidesmosomal plaques. Immunoultrastructural localization of the BP antigen suggests that it may be involved in bridging between hemidesmosomal plaques and keratin intermediate filaments of the cytoskeleton.     

Laminin-1 induces neurite outgrowth in human mesenchymal stem cells in serum/differentiation factors-free conditions through activation of FAK-MEK/ERK signaling pathways

Mesenchymal stem cells (MSCs) can be differentiated into cell types derived from all three germ layers by manipulating culture conditions in vitro. A multitude of growth and differentiation factors have been employed for driving MSCs towards a neuronal phenotype. In the present study, we investigated the potential of extracellular matrix (ECM) proteins—fibronectin, collagen-1, collagen-IV, laminin-1, and laminin-10/11, to induce a neuronal phenotype in bone marrow derived human MSCs in the absence of growth factors/differentiating agents. All of the ECM proteins tested were found to support adhesion of MSCs to different extents. However, direct interaction only with laminin-1 triggered sprouting of neurite-like processes. Cells plated on laminin-1 exhibited neurite out growth as early as 3 h, and by 24 h, the cells developed elaborate neurites with contracted cell bodies and neuronal-like morphology. Function-blocking antibodies directed against α6 and β1 integrin subunits inhibited neurite formation on laminin-1 which confirmed the involvement of integrin α6β1 in neurite outgrowth. Mechanistic studies revealed that cell adhesion to laminin-1 activated focal adhesion kinase (FAK), and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) signaling pathways. Abrogation of FAK phosphorylation by herbimycin-A inhibited neurite formation and also decreased activities of MEK and ERK. Pharmacological inhibitors of MEK (U0126) and ERK (PD98059) also blocked neurite outgrowth in cells plated on laminin-1. Our study demonstrates the involvement of integrin α6β1 and FAK-MEK/ERK signaling pathways in laminin-1-induced neurite outgrowth in MSCs in the absence of serum and differentiation factors.     

The beta3 chain short arm of laminin-332 (laminin-5) induces matrix assembly and cell adhesion activity of laminin-511 (laminin-10)

The basement membrane (BM) protein laminin-332 (Lm332) (laminin-5) has unique activity and structure as compared with other laminins: it strongly promotes cellular adhesion and migration, and its alpha3, beta3, and gamma2 chains are all truncated in their N-terminal regions (short arms). In the present study, we investigated the biological function of the laminin beta3 chain. When the beta3 chain short arm (beta3SA) was overexpressed in HEK293 cells (beta3SA-HEK), they deposited a large amount of beta3SA and a small amount of laminin-511 (Lm511) (laminin-10) on culture plates. Control HEK293 cells secreted Lm511 but failed to deposit it. The extracellular matrix (ECM) deposited by beta3SA-HEK cells strongly promoted cell attachment and spreading. The beta3SA-HEK ECM did not directly bind Lm511, but it stimulated control HEK293 cells to deposit Lm511 on the culture plates. Although purified beta3SA did not support cell adhesion by itself, it enhanced the cell adhesion activity of Lm511. Experiments with anti-integrin antibodies also suggested that the strong cell adhesion activity of the beta3SA-HEK ECM was derived from the synergistic action of beta3SA and Lm511. It has previously been found that beta3SA binds an unknown cell surface receptor. Taken together, the present study suggests that the short arm of the laminin beta3 chain enhances the matrix assembly of Lm511 and its cell adhesion activity by interacting with its receptor.     

The hemidesmosomal plaque

To examine whether constituent proteins of hemidesmosomal structures can be used as markers for certain pathways of epithelial differentiation we have examined the occurrence of the major M- approximately 230,000 plaque protein, the "bullous pemphigoid" (BP) antigen. Several bovine, rat and human tissues and bovine cell culture lines were examined, using different human autoantibody preparations in immunocytochemistry and immunoblotting. We report that this protein, also unequivocally identified by cDNA cloning from expression libraries and DNA sequencing, occurs not only in different stratified epithelia but also, apparently always in hemidesmosomal structures, in urothelium of bladder and the complex epithelia of trachea, bronchus and several glands, notably myoepithelium-containing skin glands, the mammary gland and salivary glands. The protein is absent, however, in all single-layered epithelia and in several tissues reported to have subplasmalemmal densities structurally similar to hemidesmosomes, such as Purkinje fibers of heart, meninges and perineuria. A mammary-gland-derived epithelial cell line (BMGE + H) is particularly rich in hemidesmosomes. This has been used to study the endocytotic uptake of hemidesmosome-containing plasma membrane domains into cytoplasmic vesicles upon detachment of cell sheets during treatment with dispase, a proteolytic enzyme. We propose to use the Mr- approximately 230,000 plaque protein as a marker selective for certain subsets of epithelial cell types and epithelium-derived tumors in studies of fetal and tumor development, including differentiation diagnosis of carcinomas.     

Structure-function analysis of tetraspanin CD151 reveals distinct requirements for tumor cell behaviors mediated by α3β1 versus α6β4 integrin

The basement membrane protein laminin-332 (laminin-5) mediates both stable cell adhesion and rapid cell migration and thus has the potential to either restrain or promote tumor cell metastasis. The major cellular receptors for laminin-332 are integrin α3β1, which mediates rapid tumor cell migration, and integrin α6β4, which often mediates stable cell attachment. Tetraspanin protein CD151 interacts directly with both α3β1 and α6β4 integrins and with other tetraspanins, thereby promoting α3β1 and α6β4 association with tetraspanin-enriched microdomains on the cell surface. To explore the possibility of selectively modulating tumor cell responses to laminin-332, we re-expressed a series of CD151 mutants in epidermoid carcinoma cells with near total, RNAi-mediated silencing of endogenous CD151. The interactions of CD151 with its integrin partners or its interactions with other tetraspanins were selectively disrupted by specific mutations in the CD151 large extracellular loop (EC2 domain) or in intracellular CD151 palmitoylation sites, respectively. CD151-integrin association and CD151-tetraspanin association were both important for α3β1 integrin-dependent initial adhesion and rapid migration on laminin-332. Remarkably, however, only CD151-integrin association was required for stable, α6β4 integrin-dependent cell attachment on laminin-332. In addition, we found that a QRD amino acid motif in the CD151 EC2 domain, which had been thought to be crucial for CD151-integrin interaction, is not essential for CD151-integrin association or for the ability of CD151 to promote several different integrin functions. These new data suggest potential strategies for selectively modulating migratory cell responses to laminin-332, while leaving stable cell attachment on laminin-332 intact.     

Laminin-5-deficient human keratinocytes: defective adhesion results in a saltatory and inefficient mode of migration

Laminin-5 is a major adhesion protein of the skin basement membrane and crucially involved in integrin-mediated cell substrate attachment of keratinocytes, which is important for hemidesmosomal anchorage as well as for keratinocyte migration during epidermal wound healing. To investigate its role in keratinocyte migration, we analyzed laminin-5-deficient cells of patients with a lethal variant of junctional epidermolysis bullosa. Normal migrating keratinocytes adopted monopolar morphology with a distinct front lamella and employed a continuous mode of translocation. In contrast, laminin-5-deficient cells assumed a stretched bipolar shape with two lamella regions and migrated in a discontinuous, saltatory manner characterized by significantly decreased directional persistence and reduced migration velocity. The distinct morphology as well as the migratory phenotype apparently resulted from a defect in the formation of cell substrate adhesions that were completely missing in the cell body and less stable in the lamella regions. Accordingly in normal keratinocytes, a bipolar shape and a saltatory migration mode were inducible by blocking laminin-5-mediated substrate adhesion. Our findings clearly point to an essential role of laminin-5 in forming dynamic cell substrate adhesion during migration of epidermal keratinocytes and provide an explanation for the cellular mechanisms that underlie the lethal form of junctional epidermolysis bullosa.     

Role of the Bullous Pemphigoid Antigen 180 (BP180) in the Assembly of Hemidesmosomes and Cell Adhesion—Reexpression of BP180 in Generalized Atrophic Benign Epidermolysis Bullosa Keratinocytes

Bullous pemphigoid antigen 180 (BP180) is a transmembrane component of hemidesmosomes (HD), cell–substrate attachment complexes in stratified and complex epithelia. To determine the role of BP180 in the assembly of HD and cell adhesion, using SV40 virions we have immortalized BP180-deficient keratinocytes derived from a patient with the inherited skin blistering disorder generalized atrophic benign epidermolysis bullosa (GABEB). The GABEB keratinocytes form HD-like structures, which contain α6β4 integrin and HD1/plectin, but not the bullous pemphigoid antigen 230 (BP230). The expression of integrin subunits by GABEB keratinocytes was comparable to that of an immortalized normal human keratinocyte cell line (NHK), except for α6 and β4, which were less strongly expressed in GABEB cells. In short-term adhesion assays, both GABEB keratinocytes and NHK bound strongly and to a similar extent to laminin-1, laminin-5, fibronectin, and type IV and V collagens, which suggests that BP180 is not involved in promoting the initial adhesion to these ligands. Transfection of GABEB keratinocytes with cDNAs for wild-type or a mutant of BP180 lacking the collagenous extracellular domain resulted in the expression of recombinant BP180 proteins that were correctly polarized at the basal cell surface together with α6β4. In addition, restored synthesis of BP180 affected the subcellular localization of BP230, which was no longer diffusely distributed in the cytoplasm, but was found in HD-like structures. In contrast, a BP180 mutant with a 36-amino-acid deletion from the amino terminus of the cytoplasmic domain failed to localize to HD-like structures. These results demonstrate that a region within the cytoplasmic domain of BP180 is essential for its localization into HD and that BP180 may play a critical role in coordinating the subcellular distribution of BP230.     

Localization of laminin-5, HD1/plectin, and BP230 in the submandibular glands of developing and adult mice

We studied the distributions of laminin-5 and hemidesmosome components, HD1/plectin and BP230, in the submandibular glands of adult and developing mice. In adult mice, laminin-5 was expressed in the basement membranes of both the myoepithelial cells and excretory ducts. The former expression was predictable because laminin-5 is a ligand for hemidesmosomes, which appear in myoepithelial cells and stratified epithelium. However, the latter expression pattern suggested that the non-stratified epithelium of the excretory duct might also be associated with hemidesmosomes. During fetal development, laminin-5 was found in the basement membrane of developing ducts but not epithelial end buds in which future lobules are formed by epithelial branching. The expression of HD1/plectin but not BP230 was noted in the developing duct at early embryonic stages, indicating the presence of type II hemidesmosomes. Expression of BP230 appeared in the excretory duct epithelium at around the day of birth. At this stage, the typical hemidesmosome was observed in the duct epithelium. Our results suggest that laminin-5 is involved in duct development rather than epithelial branching. The results also suggest that the developing duct epithelium interacts with laminin-5 through the type II hemidesmosome, which later matures into a typical hemidesmosome upon the onset of expression of BP230. Accepted: 12 October 1999     

Extracellular cleavage of bullous pemphigoid antigen 180/type XVII collagen and its involvement in hemidesmosomal disassembly

Bullous pemphigoid antigen 180 (BP180)/type XVII collagen is a transmembrane hemidesmosomal protein. Previously, we demonstrated that the collagenous ectodomain of BP180 can be cleaved within the extracellular non-collagenous (NC) 16A domain adjacent to the cell membrane and released from the cell surface. Here, we report that the BP180 cleavage is mediated by a membrane-associated metalloprotease expressed in epithelial cells. A tissue inhibitor of metalloprotease 1 (TIMP-1), but not TIMP-2, like the synthetic metalloprotease inhibitor KB-R8301, significantly reduced the cleavage. Within epithelial cells cultured for more than 36 h past confluency, antibodies to BP180 showed a reduced hemidesmosomal staining. Observed for the first time, addition of KB-R8301 to the cell culture preserved this staining. To examine the effect of the extracellular cleavage of BP180 on molecular interactions among hemidesmosomal components, we eliminated its collagenous extracellular portion, except for the NC16A domain, by collagenase digestion. Interestingly, this collagenase treatment caused partial disassembly of hemidesmosomal components in cultured human keratinocytes. Moreover, a monoclonal antibody specific for the cleaved extracellular fragment detected a unique tissue distribution of the fragment that might reflect an association of the cleavage process with the mitotic activity of epithelial tissues. Our observations demonstrate that the cleavage of BP180 occurring within the NC16A domain is mediated by a membrane-associated metalloprotease and suggest a possible involvement of the cleavage in hemidesmosomal disassembly.     

Biological function of laminin-5 and pathogenic impact of its deficiency

The basement membrane glycoprotein laminin-5 is a key component of the anchoring complex connecting keratinocytes to the underlying dermis. It is secreted by keratinocytes as a cross-shaped heterotrimer of alpha3, beta3 and gamma2 chains and serves as a ligand of various transmembrane receptors, thereby regulating keratinocyte adhesion, motility and proliferation. In intact skin, laminin-5 provides essential links to both the hemidesmosomal alpha6beta4 integrin and the collagen type VII molecules which form the anchoring fibrils inserting into the dermis. If the basement membrane is injured, laminin-5 production increases rapidly. It then serves as a scaffold for cell migration, initiates the formation of hemidesmosomes and accelerates basement membrane restoration at the dermal-epidermal junction. Mutations of the laminin-5 genes or auto-antibodies against one of the subunits of laminin-5 may lead to a significant lack of this molecule in the epidermal basement membrane zone. The major contributions of laminin-5 to the resistance of the epidermis against frictional stress but also for basement membrane regeneration and repair of damaged skin are reflected by the phenotype of Herlitz junctional epidermolysis bullosa, which is caused by an inherited absence of functional laminin-5. This lethal disease becomes manifest in widespread blistering of skin and mucous membranes, impaired wound healing and chronic erosions containing exuberant granulation tissue. Here, we discuss current understanding of the biological functions of laminin-5, the pathogenic impact of its deficiency and implications on molecular approaches towards a therapy of junctional epidermolysis bullosa.     

Crucial role of the specificity-determining loop of the integrin beta4 subunit in the binding of cells to laminin-5 and outside-in signal transduction

Within each hemidesmosome, alpha6beta4 integrin plays a crucial role in hemidesmosome assembly by binding to laminin-5 in the basement membrane zone of epithelial tissue. Recent analyses have implicated "specificity-determining loops" (SDLs) in the I-like domain of beta integrin in regulating ligand binding. Here, we investigated the function of an SDL-like motif within the extracellular I-like domain of beta4 integrin. We generated point mutations within the SDL of beta4 integrin tagged with green fluorescent protein (GFP-beta4K150A and GFP-beta4Q155L). We also generated a mutation within the I-like domain of the beta4 integrin, lying outside the SDL region (GFP-beta4V284E). We transfected constructs encoding the mutated beta4 integrins and a GFP-conjugated wild type beta4 integrin (GFP-beta4WT) into 804G cells, which assemble hemidesmosomes, and human endothelial cells, which express little endogenous beta4 integrin. In transfected 804G cells, GFP-beta4WT and GFP-beta4V284E colocalize with hemidesmosome proteins, whereas hemidesmosomal components in cells expressing GFP-beta4K150A and GFP-beta4Q155L are aberrantly localized. In endothelial cells, GFP-beta4WT and mutant proteins are co-expressed at the cell surface with alpha6 integrin. When transfected endothelial cells are plated onto laminin-5 matrix, GFP-beta4WT and GFP-beta4V284E localize with laminin-5, whereas GFP-beta4K150A and GFP-beta4Q155L do not. GFP-beta4WT and GFP-beta4V284E expressed in endothelial cells associate with the adaptor protein Shc when the cells are stimulated with laminin-5. However, GFP-beta4K150A and GFP-beta4Q155L fail to associate with Shc even when laminin-5 is present, thus impacting downstream signaling. These results provide evidence that the SDL segment of the beta4 integrin subunit is required for ligand binding and is involved in outside-in signaling.     

Genomic organization, amplification, fine mapping, and intragenic polymorphisms of the human hemidesmosomal tetraspanin CD151 gene

CD151 is a member of the tetraspanin family that is involved in cellular processes including cell adhesion. The protein is expressed in a variety of tissues including vascular endothelium and epidermis, and has been shown to be a component of hemidesmosomes. Mutations in genes encoding other hemidesmosomal proteins give rise to a range of human disorders, characterized by fragility of the skin and/or mucous membranes. It is, therefore, plausible that inherited or acquired mutations in the gene encoding CD151 may be fundamental to the integrity and maturation of basal cell keratinocytes. To aid mutation analysis, we have characterized the intron-exon organization of the CD151 gene which comprises 8 exons spanning approximately 4.3 kb, and have developed a comprehensive PCR-based mutation detection strategy. In addition, to aid linkage analysis of CD151 in genetic disease we have fine-mapped the gene by radiation-hybrid methodology to 11p15.5, and detected a number of intragenic polymorphisms.     

Hemidesmosome protein dynamics in live epithelial cells

Hemidesmosomes mediate stable anchorage of epithelial cells to laminin-5 in the basement membrane zone and have been likened to spot-welds. Indeed, it has been assumed that hemidesmosomes are not dynamic, at least when compared to other matrix adhesion sites including focal contacts. We tested this notion by monitoring the fate of green fluorescent protein (GFP)-tagged human integrin beta4 subunit (GFP-hbeta4) and GFP-tagged 180-kD human bullous pemphigoid (BP) autoantigen (GFP-BP180) in live cultures of 804G cells that assemble numerous mature hemidesmosomes. In subconfluent 804G cells, both GFP-hbeta4 and GFP-BP180 protein clusters are not stable but assemble into and disassemble out of cat paw-like arrays at a relatively rapid rate. In confluent populations of 804G cells, although some cat paw-like clusters of both GFP-hbeta4 and GFP-BP180 are stable over periods of >60 min, other GFP-hbeta4 and GFP-BP180 protein arrays form and/or disappear during the same time period. Moreover, individual labeled particles show considerable motility in the plane of the membrane. Fluorescence recovery after photobleaching analyses provide a further indication of the dynamics of hemidesmosome proteins. In particular, bleached GFP-hbeta4 protein clusters in confluent cells recover signal within about 30 min, indicating that there is a relatively rapid turnover of hemidesmosome components in protein arrays clustered along the substratum attached surface of a cell. The rate of recovery is dependent on an intact microfilament system. In sharp contrast, bleached GFP-BP180 protein clusters in confluent cells fail to recover signal even when observed for longer than 60 min. To evaluate hemidesmosome protein dynamics in motile cells, we monitored GFP-hbeta4 and GFP-BP180 in 804G cells populating scrape wound sites in vitro. In these migratory cells, which lack mature hemidesmosomes, integrin beta4 subunit and BP180 protein clusters progressively assemble and disassemble into linear and cat-paw arrays. In summary, hemidesmosome protein clusters, like their counterparts in focal contacts, are dynamic. We discuss these results in relation to hemidesmosome functions.     

H-Ras activation and fibroblast-induced TGF-β signaling promote laminin-332 accumulation and invasion in cutaneous squamous cell carcinoma

Cutaneous squamous cell carcinoma (cSCC) is the most common metastatic skin cancer, with increasing incidence worldwide. The molecular basis of cSCC progression to invasive and metastatic disease is still incompletely understood. Here, we show that fibroblasts and transforming growth factor-β (TGF-β) signaling promote laminin-332 synthesis in cancer cells in an activated H-Ras-dependent manner, which in turn promotes cancer cell invasion. Immunohistochemical analysis of sporadic UV-induced invasive human cSCCs (n = 208) revealed prominent cSCC cell specific immunostaining for laminin-332 γ2 chain, located in the majority of cases (90%, n = 173) in the invasive edge of the tumors. To mimic the progression of cSCC we established 3D spheroid cocultures using primary skin fibroblasts and HaCaT/ras-HaCaT human keratinocytes. Our results indicate that in 3D spheroids, unlike in monolayer cultures, TGF-β upregulates laminin-332 production, but only in cells that harbour oncogenic H-Ras. Accumulation of laminin-332 was prevented by both H-Ras knock down and inhibition of TGF-β signaling by SB431542 or RAdKD-ALK5 kinase-defective adenovirus. Furthermore, fibroblasts accelerated the invasion of ras-HaCaT cells through collagen I gels in a Ras/TGF-β signaling dependent manner. In conclusion, we demonstrate the presence of laminin-332 in the invasive front of cSCC tumors and report a new Ras/TGF-β-dependent mechanism that promotes laminin-332 accumulation and cancer cell invasion.