Laminin ß4 is a constituent of the cutaneous basement membrane zone and additional autoantigen of anti-p200 pemphigoid.

BACKGROUND: Anti-p200 pemphigoid is a subepidermal autoimmune blistering disease (AIBD) characterized by autoantibodies against a 200 kDa protein. Laminin γ1 has been described as target antigen in 70% to 90% of patients. No diagnostic assay is widely available for anti-p200 pemphigoid, which might be due to the unclear pathogenic relevance of anti-laminin γ1 autoantibodies. OBJECTIVE: To identify a target antigen with higher clinical and diagnostic relevance. METHODS: Immunoprecipitation, mass spectrometry, and immunoblotting were employed for analysis of skin extracts and sera of patients with anti-p200 pemphigoid (n = 60), other AIBD (n = 33), and healthy blood donors (n = 29). To localize the new antigen in skin, cultured keratinocytes and fibroblasts, quantitative real-time polymerase chain reaction and immunofluorescence microscopy were performed. RESULTS: Laminin ß4 was identified as target antigen of anti-p200 pemphigoid in all analyzed patients. It was located at the level of the basement membrane zone of the skin with predominant expression in keratinocytes. LIMITATIONS: A higher number of sera needs to be tested to verify that laminin ß4 is the diagnostically relevant antigen of anti-p200 pemphigoid. CONCLUSION: The identification of laminin ß4 as an additional target antigen in anti-p200 pemphigoid will allow its differentiation from other AIBD and as such, improve the management of these rare disorders.

Laminins and interaction partners in the architecture of the basement membrane at the dermal-epidermal junction.

The basement membrane at the dermal-epidermal junction keeps the epidermis attached to the dermis. This anatomical barrier is made up of four categories of extracellular matrix proteins: collagen IV, laminin, nidogen and perlecan. These proteins are precisely arranged in a well-defined architecture through specific interactions between the structural domains of the individual components. Some of the molecular constituents are provided by both fibroblasts and keratinocytes, while others are synthesized exclusively by fibroblasts or keratinocytes. It remains to be determined how the components from the fibroblasts are targeted to the dermal-epidermal junction and correctly organized and integrated with the proteins from the adjacent keratinocytes to form the basement membrane.

New specific HSP47 functions in collagen subfamily chaperoning.

Although collagens are the most abundant proteins implicated in various disease pathways, essential mechanisms required for their proper folding and assembly are poorly understood. Heat-shock protein 47 (HSP47), an ER-resident chaperone, was mainly reported to fulfill key functions in folding and secretion of fibrillar collagens by stabilizing pro-collagen triple-helices. In this study, we demonstrate unique functions of HSP47 for different collagen subfamilies. Our results show that HSP47 binds to the N-terminal region of procollagen I and is essential for its secretion. However, HSP47 ablation does not majorly impact collagen VI secretion, but its lateral assembly. Moreover, specific ablation of Hsp47 in murine keratinocytes revealed a new role for the transmembrane collagen XVII triple-helix formation. Incompletely folded collagen XVII C-termini protruding from isolated HSP47 null keratinocyte membrane vesicles could be fully restored upon the application of recombinant HSP47. Thus, our study expands the current view regarding the client repertoire and function of HSP47, as well as emphasizes its importance for transmembrane collagen folding.

Partial loss of epithelial phenotype in kindlin-1-deficient keratinocytes.

Kindlin-1 is an adaptor protein that is expressed by most epithelial cells and has been implicated in integrin bidirectional signaling. Mutations in the gene encoding kindlin-1 are associated with Kindler syndrome, a recessively inherited disorder that is characterized by fragile skin. Functionally, a loss of kindlin-1 impairs the adhesion of basal keratinocytes to the extracellular matrix both in vivo and in vitro. In this study, we show that the phenotype of mutant keratinocytes deficient in kindlin-1 is characterized by the modification of the cortical actin network and increased plasticity of the plasma membrane. At the molecular level, expression of several proteins associated with an epithelial phenotype, such as α6ß4 integrin, collagen XVII, E-cadherin, and desmoglein-3, is strongly reduced, whereas, surprisingly, laminin 332 is synthesized in larger amounts than in control keratinocytes. In contrast, mesenchymal markers such as vimentin and fibronectin are increased in keratinocytes lacking kindlin-1. The switch in cell plasticity and protein expression was confirmed by siRNA-mediated down-regulation of kindlin-1 in HaCaT epithelial cells. Furthermore, there was up-regulation of matrix metalloproteinases and pro-inflammatory cytokines in kindlin-1-deficient keratinocytes. These results provide new insights into the pathogenic mechanisms that take place in Kindler syndrome. Moreover, the constellation of molecular defects associated with the loss of kindlin-1 may explain the higher incidence of skin cancer observed in patients affected with this disorder.

Integrin-linked kinase is required for epidermal and hair follicle morphogenesis.

Integrin-linked kinase (ILK) links integrins to the actin cytoskeleton and is believed to phosphorylate several target proteins. We report that a keratinocyte-restricted deletion of the ILK gene leads to epidermal defects and hair loss. ILK-deficient epidermal keratinocytes exhibited a pronounced integrin-mediated adhesion defect leading to epidermal detachment and blister formation, disruption of the epidermal-dermal basement membrane, and the translocation of proliferating, integrin-expressing keratinocytes to suprabasal epidermal cell layers. The mutant hair follicles were capable of producing hair shaft and inner root sheath cells and contained stem cells and generated proliferating progenitor cells, which were impaired in their downward migration and hence accumulated in the outer root sheath and failed to replenish the hair matrix. In vitro studies with primary ILK-deficient keratinocytes attributed the migration defect to a reduced migration velocity and an impaired stabilization of the leading-edge lamellipodia, which compromised directional and persistent migration. We conclude that ILK plays important roles for epidermis and hair follicle morphogenesis by modulating integrin-mediated adhesion, actin reorganization, and plasma membrane dynamics in keratinocytes.

The extracellular matrix of the dermis: flexible structures with dynamic functions.

The current understanding of the role of extracellular matrix proteins is mainly based on their structural properties and their assembly into complex networks. The multiplicity of interactions between cells, cytokines and growth factors within the networks determines functional units dictating the biophysical properties of tissues. This review focuses on the understanding how alterations in the genes, modifying enzymes or biological functions of extracellular matrix molecules, lead to inborn or acquired skin disorders. Analysis of the disease mechanisms provides the basis for the emerging concept that not solely structural defects of single extracellular matrix proteins are at fault, but rather that the functional unit as a whole is not working properly, causing similar clinical symptoms although the causative genes are entirely different. The understanding of these disease-causing pathways has already led to surprising new therapeutic developments applied to rare inborn disorders. They now permit to design new concepts for the treatment of more common diseases associated with the accumulation of connective tissue and alterations of the biomechanical properties of the extracellular matrix.

Laminin 332 Is Indispensable for Homeostatic Epidermal Differentiation Programs.

The skin epidermis is attached to the underlying dermis by a laminin 332 (Lm332)-rich basement membrane. Consequently, loss of Lm332 leads to the severe blistering disorder epidermolysis bullosa junctionalis in humans and animals. Owing to the indispensable role of Lm332 in keratinocyte adhesion in vivo, the severity of the disease has limited research into other functions of the protein. We have conditionally disrupted Lm332 expression in basal keratinocytes of adult mice. Although blisters develop along the interfollicular epidermis, hair follicle basal cells provide sufficient anchorage of the epidermis to the dermis, making inducible deletion of the Lama3 gene compatible with life. Loss of Lm332 promoted the thickening of the epidermis and exaggerated desquamation. Global RNA expression analysis revealed major changes in the expression of keratins, cornified envelope proteins, and cellular stress markers. These modifications of the keratinocyte genetic program are accompanied by changes in cell shape and disorganization of the actin cytoskeleton. These data indicate that loss of Lm332-mediated progenitor cell adhesion alters cell fate and disturbs epidermal homeostasis.

Kindlin-1 Is required for RhoGTPase-mediated lamellipodia formation in keratinocytes.

Kindlin-1 is an epithelial-specific member of the novel kindlin protein family, which are regulators of integrin functions. Mutations in the gene that encodes Kindlin-1, FERMT1 (KIND1), cause the Kindler syndrome (KS), a human disorder characterized by mucocutaneous fragility, progressive skin atrophy, ulcerative colitis, photosensitivity, and propensity to skin cancer. Our previous studies indicated that loss of kindlin-1 resulted in abnormalities associated with integrin functions, such as adhesion, proliferation, polarization, and motility of epidermal cells. Here, we disclosed novel FERMT1 mutations in KS and used them, in combination with small-interfering RNA, protein, and imaging studies, to uncover new functions for kindlin-1 in keratinocytes and to discern the molecular pathology of KS. We show that kindlin-1 forms molecular complexes with beta1 integrin, alpha-actinin, migfilin, and focal adhesion kinase and regulates cell shape and migration by controlling lamellipodia formation. Kindlin-1 governs these processes by signaling via Rho family GTPases, and it is required to maintain the pool of GTP-bound, active Rac1, RhoA and Cdc42, and the phosphorylation of their downstream effectors p21-activated kinase 1, LIM kinase, and cofilin. Loss of these kindlin-1 functions forms the biological basis for the epithelial cell fragility and atrophy in the pathology of KS.

Integrin alpha3 subunit regulates events linked to epithelial repair, including keratinocyte migration and protein expression.

Two integrins, alpha3beta1 and alpha6beta4, are high-affinity receptors for laminin 332, the major laminin isoform of the dermal-epidermal junction, although they are thought to have different functions. Biological and genetic studies have firmly established that the alpha6beta4 integrin is indispensable for the stable anchorage of the epidermis to the underlying dermis. In contrast, the alpha3beta1 integrin is thought to be important for cell migration, although the issue is controversial, and both positive and negative effects have been reported. To address the function of alpha3beta1 integrin, we used small interfering RNA to down-regulate the alpha3 subunit in human keratinocytes. The resulting phenotype indicates that lack of alpha3beta1 integrin compromises intercellular adhesion and collective migration, while it enhances single cell migration with a concomitant increase of both focal adhesion kinase and extracellular signal-regulated kinase. In addition, down-regulation of integrin alpha3 subunit results in an increased expression of fibronectin and precursor laminin 332, two extracellular matrix proteins known to be up-regulated during wound healing. Thus, down-regulation of alpha3beta1 integrin recapitulates crucial events governing keratinocyte migration associated with wound healing and tissue repair.

Keratoepithelin suppresses the progression of experimental human neuroblastomas.

Neuroblastoma is the most common extracranial childhood tumor. High expression of activin A is associated with a favorable prognosis, but the contributing mechanisms have remained unclear. Our previous demonstration of the activin A-mediated up-regulation of keratoepithelin led to the consideration that keratoepithelin could modulate neuroblastoma growth and/or progression. We report here that enhanced keratoepithelin expression in human neuroblastoma cells suppresses neuroblastoma cell cohesion and adhesion to various extracellular matrix proteins and that it inhibits neuroblastoma cell proliferation and invasion in vitro and in vivo. Using microarray analysis, we identified several keratoepithelin-regulated genes that may contribute to these biological changes. Together with the observation that keratoepithelin is expressed in human neuroblastomas in vivo, our data suggest that keratoepithelin could play a beneficial role in neuroblastoma development and/or progression.

Isolation and analysis of laminins.

Laminins are large glycoproteins forming structural and signaling networks with two major physiological roles: one role crucial for the formation and stability of basement membranes and the other role, as crucial as the first, in cell anchorage and signaling. Laminins come in several flavors as 16 different isoforms are known, each with both common and unique functions. Here the most current techniques for purification and identification of laminins in tissues and cultivated cells as well as for testing the cell adhesion-promoting activity of laminins will be described.

Subpopulations of human dendritic cells display a distinct phenotype and bind differentially to proteins of the extracellular matrix.

CD1a(pos) dendritic cells (DCs) and Langerhans cells (LCs) are highly specialized antigen-presenting cells mainly localized in the skin. Various cells have been identified as precursors of cutaneous DCs, but the definitive precursor subpopulations remain to be defined and characterized in detail. In this study, DCs were generated in vitro from monocytes (monocyte-derived DCs, MoDCs) and from CD34(pos) stem cells (CD34(pos) cell-derived DCs, CD34DCs). By virtue of their CD14 and CD1a expression, four CD34DC subpopulations were characterized while MoDCs contain three different subpopulations. Of these, CD14-expressing cells are considered to be precursors of fully differentiated DCs, which themselves are CD14(neg)CD1a(pos). Both, MoDCs and CD34DCs expressed the alpha integrins LFA-1, Mac-1, CR4, VLA-4, VLA-5 and the beta2 integrin CD18. CD34DCs and MoDCs were negative for VLA-3, whereas MoDCs, but not CD34DCs expressed VLA-6. Phenotypic and functional characterization of the cells generated herein at earlier time points revealed that DCs at day 3 of culture may reflect the in vivo situation more closely than at day 7. Adhesion of DC precursors to endothelial cells and to components of the extracellular matrix is a prerequisite for their migration towards the epidermis. To this end, we investigated adhesion of CD34DCs and MoDCs to components of the cutaneous extracellular matrix. Distinct DC subsets showed a differential binding pattern to proteins of the extracellular matrix. MoDCs and CD34DCs bound preferentially to laminin 332 via CD49f and to fibronectin via CD49e, but only weakly to laminin 111 or to collagens. While CD14(pos) cells preferentially bound to laminin 332, CD1a(pos) cells adhered to fibronectin. In summary, subpopulations of CD34DCs and MoDCs are phenotypically related to each other, but not identical and display differential binding to components of the extracellular matrix.

Activities of the matrix metalloproteinase stromelysin-2 (MMP-10) in matrix degradation and keratinocyte organization in wounded skin.

The matrix metalloproteinase stromelysin-2 is expressed in keratinocytes of the epithelial tongue of skin wounds, suggesting a role in keratinocyte migration. Here, we show that stromelysin-2 enhances migration of cultured keratinocytes. To gain insight into the in vivo activities of stromelysin-2 in epithelial repair, we generated transgenic mice expressing a constitutively active stromelysin-2 mutant in keratinocytes. These animals had no alterations in skin architecture, and the healing rate of skin wounds was normal. Histologically, however, we found abnormalities in the organization of the wound epithelium. Keratinocytes at the migrating epidermal tip were scattered in most sections of mice with high expression level, and there was a reduced deposition of new matrix. In particular, the staining pattern of laminin-5 at the wound site was altered. This may be due to proteolytic processing of laminin-5 by stromelysin-2, because degradation of laminin-5 by this enzyme was observed in vitro. The inappropriate matrix contact of keratinocytes was accompanied by aberrant localization of beta1-integrins and phosphorylated focal adhesion kinase, as well as by increased apoptosis of wound keratinocytes. These results suggest that a tightly regulated expression level of stromelysin-2 is required for limited matrix degradation at the wound site, thereby controlling keratinocyte migration.

Targeted Disruption of the Lama3 Gene in Adult Mice Is Sufficient to Induce Skin Inflammation and Fibrosis.

Genetic, clinical, and biochemical studies have shown that integrity of the dermal-epidermal junction requires a particular subset of laminins, that is, those containing the α3 chain encoded by the Lama3 gene. Inherited mutations in the human gene or introduction of constitutive mutations in the mouse gene prevent expression of these laminins, causing junctional epidermolysis bullosa, a very severe, often lethal disorder characterized by detachment of the epidermis from the dermis. This has precluded in vivo functional analysis of α3 chain-containing laminins, and it is still unknown whether and how they contribute to adult skin homeostasis. To address this question, we have disrupted the Lama3 gene in basal keratinocytes of adult mice. This led to the gradual disappearance of α3 chain-containing laminins along the dermal-epidermal junction and formation of subepidermal blisters like in congenital junctional epidermis bullosa. The mice lose their nails and have bullae and erosions on the footpads. Because the blistering is restricted to the interfollicular epidermis, the animals do not lose the epidermis and are viable. There is abundant and scattered deposition of collagen VII on the dermal side of the blisters, inflammation, and development of skin fibrosis with extensive accumulation of interstitial and microfibrillar collagens.

Expression of laminin 5 by parental and c-Ha-ras-transformed HaCaT keratinocytes in organotypic cultures.

Tumor cells traverse the basement membrane zone and gain access to the underlying mesenchyme to eventually form metastases. Laminin 5 is a major component of the basement membrane and connects keratinocytes at the level of hemidesmosomes to the mesenchyme. Underneath invading tumor cells anti-laminin 5 staining is diminished, and laminin 5 degradation products can stimulate cell migration and epidermal growth factor (EGF) receptor signaling. To investigate laminin 5 expression in parental HaCaT and tumorigenic c-Ha-ras-transformed HaCaT II-4rt keratinocytes, the cells were cultivated under monolayer and organotypic culture conditions. In monolayer cultures, HaCaT and c-Ha-ras-transformed HaCaT II-4rt keratinocytes secreted comparable amounts of laminin 5. After 7 days of organotypic cultures, collagen IV, beta4-integrin, nidogen and laminin 5 were detected along the epithelial-mesenchymal interface of parental HaCaT keratinocytes, while staining for these proteins was patchy or absent in the organotypic cultures with c-Ha-ras-transformed HaCaT II-4rt cells. Immunoblotting analysis confirmed absence of laminin 5 deposition in organotypic cultures of c-Ha-ras-transformed HaCaT II-4rt while the protein was detected in organotypic cultures of HaCaT keratinocytes. Surprisingly, however, the alpha3 and gamma2 laminin chain transcripts were strongly induced in c-Ha-ras-transformed HaCaT II-4rt cells by organotypic culture conditions, indicating that invasive epidermal tumor cells retain high mRNA levels for laminin 5 chains and suggesting an autocrine/paracrine induction of the laminin chain mRNAs. Moreover, as laminin 5 was absent in organotypic cultures of c-Ha-ras-transformed HaCaT II-4rt cells, it suggests immediate degradation of the protein. Degradation products may further contribute to the malignant phenotype by enhancing cellular migration and EGF-receptor activation.

Molecular basis of inherited skin-blistering disorders, and therapeutic implications.

Epidermolysis bullosa (EB) and associated skin-fragility syndromes are a group of inherited skin diseases characterised by trauma-induced blistering of the skin and mucous membranes. Mutations in at least 14 distinct genes encoding molecular components of the epidermis or the dermal-epidermal junction (DEJ) can cause blistering skin diseases that differ by clinical presentation and severity of the symptoms. Despite great advances in discerning the genetic basis of this group of diseases, the molecular pathways leading to symptoms are not yet fully understood. Unravelling these pathways by molecular analysis of the structure and in vitro assessment of functional properties of the human proteins involved, combined with genetic models in lower organisms, should pave the way for specific cures for inherited skin fragility.

A simplified laminin nomenclature.

A simplification of the laminin nomenclature is presented. Laminins are multidomain heterotrimers composed of alpha, beta and gamma chains. Previously, laminin trimers were numbered with Arabic numerals in the order discovered, that is laminins-1 to -5. We introduce a new identification system for a trimer using three Arabic numerals, based on the alpha, beta and gamma chain numbers. For example, the laminin with the chain composition alpha5beta1gamma1 is termed laminin-511, and not laminin-10. The current practice is also to mix two overlapping domain and module nomenclatures. Instead of the older Roman numeral nomenclature and mixed nomenclature, all modules are now called domains. Some domains are renamed or renumbered. Laminin epidermal growth factor-like (LE) domains are renumbered starting at the N-termini, to be consistent with general protein nomenclature. Domain IVb of alpha chains is named laminin 4a (L4a), domain IVa of alpha chains is named L4b, domain IV of gamma chains is named L4, and domain IV of beta chains is named laminin four (LF). The two coiled-coil domains I and II are now considered one laminin coiled-coil domain (LCC). The interruption in the coiled-coil of beta chains is named laminin beta-knob (Lbeta) domain. The chain origin of a domain is specified by the chain nomenclature, such as alpha1L4a. The abbreviation LM is suggested for laminin. Otherwise, the nomenclature remains unaltered.

Integrin-linked kinase regulates the niche of quiescent epidermal stem cells.

Stem cells reside in specialized niches that are critical for their function. Quiescent hair follicle stem cells (HFSCs) are confined within the bulge niche, but how the molecular composition of the niche regulates stem cell behaviour is poorly understood. Here we show that integrin-linked kinase (ILK) is a key regulator of the bulge extracellular matrix microenvironment, thereby governing the activation and maintenance of HFSCs. ILK mediates deposition of inverse laminin (LN)-332 and LN-511 gradients within the basement membrane (BM) wrapping the hair follicles. The precise BM composition tunes activities of Wnt and transforming growth factor-ß pathways and subsequently regulates HFSC activation. Notably, reconstituting an optimal LN microenvironment restores the altered signalling in ILK-deficient cells. Aberrant stem cell activation in ILK-deficient epidermis leads to increased replicative stress, predisposing the tissue to carcinogenesis. Overall, our findings uncover a critical role for the BM niche in regulating stem cell activation and thereby skin homeostasis.

Laminin 5 processing and its integration into the ECM.

Laminins are a family of multi-functional basement membrane proteins. Their C-terminal domain binds to cell surface receptors and is thereby responsible for cell anchorage and the initiation of specific outside-in and inside-out signals. With their N-terminal parts, laminins interact with proteins of the extracellular matrix scaffold to secure the basement membrane to the underlying mesenchymal tissue. Laminins 5A (alpha3Abeta3gamma2), 5B (alpha3Bbeta3gamma2) and 6 (alpha3Abeta1gamma1) are isoforms specific of the basement membrane underneath the epidermis and they undergo a sequential series of extracellular proteolytic changes, which might successively turn on and off one or several of their biological and mechanical functions. Under physiological conditions, such as in adult human skin, epithelial laminins have lost part of the C- and N-terminal domains of the alpha3 and gamma2 chains, respectively. In contrast, in cylindromatosis, a rare inherited disease characterised by major ultrastructural alterations of the basement membrane and altered expression/distribution of integrin receptors, laminin processing has not been completed. Together, these results suggest that laminin processing may regulate signalling pathways and the architecture of the basement membrane by restricting the repertoire of interactions with cell surface receptors and extracellular matrix components.

The PDZ domain of TIP-2/GIPC interacts with the C-terminus of the integrin alpha5 and alpha6 subunits.

Different cDNA libraries were screened by the yeast two-hybrid system using as a bait the cytoplasmic sequence of integrin alpha6A or alpha6B subunits. Surprisingly, the same PDZ domain-containing protein, TIP-2/GIPC, was isolated with either of the variants, although their sequences are different. Direct interaction assays with the cytoplasmic domain of the integrin alpha1--7 subunits revealed that in addition to alpha6A and alpha6B, TIP-2/GIPC reacted also with alpha5, but not other alpha integrin subunits. The specificity of the interaction was confirmed by in vitro protein binding assays with purified peptides corresponding to integrin cytoplasmic domains. Further analysis with either truncation fragments of TIP-2/GIPC or mutated integrin cytoplasmic domains indicated that the interaction occurs between the PDZ domain of TIP-2/GIPC and a consensus PDZ domain-binding sequence, SDA, present at the C-terminus of the integrin alpha5 and alpha6A subunits. The integrin alpha6B subunit terminates with a different sequence, SYS, which may represent a new PDZ domain-binding motif.