Target proteins in inherited and acquired blistering skin disorders

Maintenance of an intact epidermis depends on secure adhesion between adjacent keratinocytes, and between basal keratinocytes and the underlying epidermal basement membrane. The major adhesion units that achieve this are the hemidesmosomes and desmosomes, but when these structures are disrupted, e.g., by gene mutations or autoantibodies, the resilience of the epidermis is lost and blisters develop. Recently, there have been considerable advances in our knowledge of the proteins and glycoproteins that contribute to maintaining keratinocyte adhesion via hemidesmosomes and desmosomes, as well as new insights into the molecular pathogenesis of several inherited and autoimmune blistering skin diseases. These new basic scientific data are clinically relevant, helping to improve patient management and to provide a rationale for developing better and more specific treatments for patients with inherited or acquired blistering skin diseases. In addition, there have also been improvements in our understanding of the organization and assembly of these adhesion structures, and their involvement in signalling pathways, intricately linked to skin development, wound healing and tumour invasion. This review provides an update on the structure and organization of hemidesmosomes and desmosomes, and on the molecular pathology of their various components that result in bullous skin diseases.     

Hemidesmosomes and focal contact proteins: functions and cross-talk in keratinocytes, bullous diseases and wound healing

The outer most layer of the skin, the epidermis, is attached to the dermis via a sheet of extracellular matrix proteins termed the basement membrane zone (BMZ). In the intact skin, adhesion of the keratinocytes in the basal layer of the epidermis to the BMZ is facilitated primarily by hemidesmosomes which associate with the keratin cytoskeleton. Cultured keratinocytes do not assemble bona fide hemidesmosomes although hemidesmosome protein clusters (stable anchoring contacts) are found along the substrate-attached surface of the cells and towards the leading edge of keratinocytes repopulating scratch wounds. Actin cytoskeleton-associated matrix adhesion devices termed focal contacts are not thought to play an important role in the adhesion of keratinocytes to the BMZ in intact skin but are prominent in cultured keratinocytes where they are believed to regulate cell migration. We review the molecular components, functions, dynamics and cross-talk of hemidesmosomes and focal contacts in keratinocytes. In addition, we briefly describe what is known about their role in autoimmune and genetic blistering diseases of the skin. We also discuss recent publications which indicate, contrary to expectation, that certain focal contact proteins retard keratinocyte migration while hemidesmosomal proteins regulate directed keratinocyte motility during wound healing.     

Hereditary skin diseases of hemidesmosomes.

Studies of hereditary blistering skin diseases (epidermolysis bullosa) and targeted gene mutation experiments in knockout mice have greatly improved our understanding of hemidesmosomes and their associated structures in the cytoskeleton and basement membrane of the skin and mucous membranes. At least 10 molecules are recruited in hemidesmosome complexes, where they interact in a complex way. Hemidesmosomes are not simple adhesion devices, but also transduce signals for cell spreading, cell proliferation and basement membrane organisation. The dynamics of a hemidesmosome raises the metaphor of a self-assembling suspension bridge which evokes activities on both sides of the river. This review summarises our current knowledge of the molecular pathology of hemidesmosomes caused by hereditary skin disease or gene targeting experiment.     

Expression of integrin alpha 6 beta 4 in junctional epidermolysis bullosa.

The integrin alpha 6 beta 4 is a member of the integrin family of adhesion receptors. The integrin alpha 6 beta 4 is preferentially expressed in stratified squamous epithelia, where it is localized in hemidesmosomes. A reduced number of rudimentary hemidesmosomes is often found in skin from patients with junctional epidermolysis bullosa (JEB). In this study we have investigated the expression of alpha 6 beta 4 in skin specimens of patients with junctional (one non-lethal, two lethal) and dystrophic (two) epidermolysis bullosa, using immunofluorescent (IF) staining with five different monoclonal antibodies against the alpha 6 and beta 4 subunits. The intensity of IF staining of the integrin alpha 6 beta 4 and bullous pemphigoid antigen (BPA) was unreduced along the epidermal basement membrane zone (EBMZ) of all EB patients, compared to that in skin of healthy human controls. However, in the skin of two patients with lethal (Herlitz) JEB, who did not express GB3, IF staining of integrin alpha 6 beta 4 and BPA showed a "stitchy" discontinuous linear pattern along the EBMZ with interruptions at the borders of adjoining basal keratinocytes. The same results were obtained by immunoelectron microscopy. They corresponded with freeze-induced partial cell detachment from the basement membrane at the ultimate baso-lateral edge of the basal keratinocytes in lethal JEB skin. The basal lamellipodia at that location almost completely lacked tonofilaments and hemidesmosomes. Furthermore, in JEB there was a split between the intra- and extracellular epitopes of the integrin alpha 6 beta 4 receptor, whereas the integrin remains intact in salt-split skin. This suggests that the defect is in alpha 6 beta 4 itself or perhaps its ligand.     

Tight junctions/adherens junctions: basic structure and function

Adherens and tight junctions are intercellular junctions crucial for epithelial adhesion and barrier function in a wide variety of tissues and organisms. In stratifying epithelia, such as the epidermis, the role of adherens and tight junctions was considered less important owing to the abundance of desmosomes, mediating firm mechanical stability between the cells, and to the barrier function of the stratum corneum, respectively. This view has changed in recent years because of different studies that showed the importance of these structures for proper skin physiology and barrier function. The current review provides an overview of the crucial molecular constituents of these structures and highlights some recent results on their regulation. In particular, I will discuss their importance in skin biology.     

A new C-xylopyranoside derivative induces skin expression of glycosaminoglycans and heparan sulphate proteoglycans

Severe structural changes, including deterioration of the mechanical properties of the dermis, occur during skin aging. It is well known that the degradation of the extracellular matrix contributes to the physical changes in aged skin. Whereas many studies have been devoted to age-related alterations of collagen fibrils, far less attention has been paid to another major family of extracellular matrix components, the glycosaminoglycans (GAGs) and proteoglycans (PGs). Heparan sulphate-proteoglycans, (HS-PGs), a subclass of the PG family that decreases during aging, regulate proliferation and proteolysis as well as matrix adhesion and assembly, and thus, may have important functions in skin. These PGs may represent important targets for dermo-cosmetology in fighting skin aging. The purpose of this study was to demonstrate the potential of a new C-xylopyranoside derivative (C-beta-D-xylopyranoside-2-hydroxy-propane simplified as C-Xyloside) to improve HS-PGs expression in human skin. In an organotypical model of corticosteroid atrophic human skin, characterized by a decrease of PGs expression, treatment with C-Xyloside improved expression of HS-PGs.     

Cell adhesion molecules

Recently it has become recognized that cell adhesion is critical in many different cellular functions. The proteins involved in the cell-cell or cell-matrix interactions are known as cell adhesion molecules (CAMs). This article reviews some of the most important CAMs and emphasizes their importance in dermatology and in skin diseases.     

Need for a well-balanced sunscreen to protect human skin from both Ultraviolet A and Ultraviolet B damage

Skin exposure to sunlight can cause many adverse effects. It is now recognized that both Ultraviolet A (UVA) and UVB wavelengths are responsible for the detrimental effects of solar radiation on skin. With our increasing knowledge on the harmful effects of UVA, the need for effective, well-balanced photoprotection has become more crucial. Numerous clinical studies showed that well-balanced sunscreen, with a SPF/UVAPF ratio ≤ 3, provide the most effective protection against pigmentation (especially on dark skin), DNA damage, UV-induced skin immunosuppression and photodermatoses. The calculation of UVA protection required in Asia revealed its particular importance in India, and gives clear evidence that the SPF value alone is not sufficient to evaluate the efficacy of a sunscreen.     

Epidermale Stammzellen

Current understanding of the biology of epidermal stem cells opens a totally new perspective in the function of the epidermis and adjacent epithelial structures. A number of pathogenetic as well as clinical‐therapeutic approaches against a variety of dermatoses may become possible with knowledge about keratinocyte proliferation, differentiation and regeneration. The reservoir of epidermal stem cells is located in the interfollicular epidermis, the hair follicle area and the germinal hair follicle matrix. Endogenous stem cell clones exist here, giving rise to transient amplifying cells and postmitotic cells. The stem cell clones are organized in clusters and display high expression of adhesion proteins, which guarantee their stability in a specific environment consisting of different cell types and extracellular substrates in the stratum basale. Differentiation is determined by a specific cascade of chemical signals from the stem cell environment and from the genetic program of the cell. The clinical relevance of stem cells lies primarily in their therapeutic potential with reconstruction of epithelia by reimplantation of autologous stem cells or gene therapeutic applications such as targeted transfection. However, the benefit‐to‐risk ratio cannot yet be accurately estimated.     

Distribution of CD44 variant isoforms in human skin: differential expression in components of benign and malignant epithelia

Expression of cell adhesion molecules regulates epithelial cell differentiation and organization of complex tissues such as skin. The CD44 family of adhesion molecules is generated by alternative splicing of up to 10 variant exons encoding inserts into the extracellular domain. Expression of CD44 variant exons has been correlated with metastatic potential of some epithelial malignancies. We studied the distribution of total and variant CD44 isoforms containing exons v4, v6, and v9 in normal skin, basal cell carcinoma, and in control tissues using immunohistologic assays. While normal epidermis and other stratified squamous epithelia reacted strongly with antibodies specific for standard CD44 (CD44S) and CD44 isoforms containing exons v4, v6, and v9, the epithelium of eccrine glands was reactive, often in a polarized distribution, only with antibodies specific for CD44S and isoforms containing exon v9. These studies suggest that differential expression of CD44 variant exons may be important in development and organization of epithelial structures within skin. Malignant cells in basal carcinoma tissues were found to have low reactivity with antibodies specific for CD44S or variant CD44 molecules. The low expression of CD44 molecules in basal cell carcinomas may play a role in the relatively low probability of metastasis of these neoplasms.     

Kindler syndrome

Kindler syndrome is a rare, autosomal recessive skin fragility disorder characterized by blistering in infancy, followed by photosensitivity and progressive poikiloderma. Ultrastructural examination reveals marked basement membrane reduplication and variable levels of cleavage at the dermal-epidermal junction. The molecular pathology underlying Kindler syndrome has recently been shown to involve loss-of-function mutations in a novel gene, KIND1, encoding kindlin-1. Immunofluorescence, gene expression and cell biology studies have shown that kindlin-1 is expressed mainly in basal keratinocytes and plays a role in the attachment of the actin cytoskeleton via focal contacts to the extracellular matrix. Thus, Kindler syndrome is the first genodermatosis caused by a defect in actin-extracellular matrix linkage rather than the classic keratin-extracellular matrix linkage underlying the pathology of other inherited skin fragility disorders such as epidermolysis bullosa. This article reviews the clinical features as well as the molecular and cellular pathology of Kindler syndrome and highlights the importance of the new protein, kindlin-1, in cell-matrix adhesion and its intriguing link to photosensitivity.     

Proteolytic processing of the laminin alpha3 G domain mediates assembly of hemidesmosomes but has no role on keratinocyte migration

Laminin-5 (Lm5), the major adhesion ligand of basal epithelial cells, undergoes complex extracellular proteolytic processing that influences cell adhesion and migration. In tumor cell lines, the proteolytic truncation of the C-terminal G domain of the Lm alpha3 chain induces assembly of hemidesmosomes and downregulates cell migration. To define the biological functions of the alpha3 G domain processing in physiological conditions, we have expressed a series of mutant alpha3 complementary DNA in human primary alpha3-null keratinocytes immortalized by human papillomavirus E6E7 (HKalpha3 cells). Using monolayer and organotypic cell cultures we show that: (1) the hinge region between subdomains G3 and G4 carries the proteolytic cleavage sites; (2) nucleation of the hemidesmosomal proteins is independent of the proteolytic maturation of the alpha3 G domain, whereas formation of mature hemidesmosomes relies on proteolytic cleavage of alpha3; and (3) the proteolytic processing plays no role in cell migration, which suggests that nucleation of hemidesmosomal structures in culture does not reflect the migratory potential of the epithelial cells. Our results also demonstrate that HKalpha3 cells are a unique model system, which will be useful to dissect the functions and molecular interactions of Lm5.     

Kindler syndrome: a focal adhesion genodermatosis

Kindler syndrome (OMIM 173650) is an autosomal recessive genodermatosis characterized by trauma-induced blistering, poikiloderma, skin atrophy, mucosal inflammation and varying degrees of photosensitivity. Although Kindler syndrome is classified as a subtype of epidermolysis bullosa, it has distinct clinicopathological and molecular abnormalities. The molecular pathology of Kindler syndrome involves loss-of-function mutations in a newly recognized actin cytoskeleton-associated protein, now known as fermitin family homologue 1, encoded by the gene FERMT1 . This protein mediates anchorage between the actin cytoskeleton and the extracellular matrix via focal adhesions, and thus the structural pathology differs from other forms of epidermolysis bullosa in which there is a disruption of the keratin intermediate filament–hemidesmosome network and the extracellular matrix. In the skin, fermitin family homologue 1 is mainly expressed in basal keratinocytes and binds to the cytoplasmic tails of β1 and β3 integrins as well as to fermitin family homologue 2 and filamin-binding LIM protein 1. It also plays a crucial role in keratinocyte migration, proliferation and adhesion. In this report, we review the clinical, cellular and molecular pathology of Kindler syndrome and discuss the role of fermitin family homologue 1 in keratinocyte biology.     

Bullous pemphigoid antigen localization suggests an intracellular association with hemidesmosomes

Autoantibodies to a normal component of stratified squamous epithelia, the bullous pemphigoid antigen (BPA), are synthesized in patients with the disease bullous pemphigoid. We have used these sera to study the distribution of BPA in vivo and in vitro. At low magnification, indirect immunofluorescent staining for BPA is linear at the basement membrane zone (BMZ) of skin and many other epithelial tissues. At higher magnification however, we observed a punctate staining pattern for BPA which was regular in appearance and suggested localization of BPA to discrete structures at the BMZ. Subsequent immunoelectron microscopy using both peroxidase and colloidal gold labeling techniques with patients' sera or IgG, revealed that BPA is associated with hemidesmosomes--putative adhesion structures at the BMZ, based on their similarity in ultrastructure to desmosomes. More specifically BPA was immunolocalized to the cytoplasmic face of hemidesmosomes and was not observed extracellularly in the basement membrane. In stratifying and nonstratifying cultures of rat keratinocytes, BPA is expressed intracellularly and not in the cell-derived matrix, unlike other known basement membrane components. These cells also synthesize BPA in vitro, and immunoprecipitation from metabolically labeled cultures revealed a 220 kD polypeptide under reducing conditions. From these observations we conclude (1) that BPA is a 220 kD polypeptide component either of or associated with hemidesmosomes, and (2) that it is localized intracellularly both in vivo and in vitro. We suggest that BPA is not normally a lamina lucida component, but that it may form part of a linkage between the cytoskeleton and the basement membrane.     

Tight junctions: is there a role in dermatology?

A variety of tight junction (TJ) proteins including claudins, occludin, tricellulin, zonula occludens-proteins and junctional adhesion molecules have been identified in complex localization patterns in mammalian epidermis. Their expression and/or localization is frequently altered in skin diseases including skin tumors. However, our understanding of the function(s) of TJ and TJ proteins in the skin is, even though rapidly increasing, still limited. This review summarizes our current knowledge of the involvement of TJ and TJ proteins in mammalian skin in functions ascribed to TJ in simple epithelia, such as barrier function, polarity, gene expression, proliferation, differentiation, and vesicle transport.     

Laminin-332 and -511 in skin

Abstract:  The extracellular matrix (ECM) was long thought to be merely a structural tissue support and/or a filter. However, recent studies have suggested that ECM proteins regulate many intracellular and extracellular events, including cell growth, cell adhesion, cell division, cell movement, and apoptosis. They do so through activation of several families of cell surface receptor, including the integrins and syndecans. The focus of this review is on two laminin isoforms expressed in the skin. Laminins are an important molecular component of the basement membranes in a variety of tissue types. They have a cruciform shape, and are composed of three chains-α, β, and γ. Keratinocytes of the skin secrete numerous laminin isoforms, including laminin-511 and laminin-332. The latter are known to affect the behaviour of keratinocytes through binding to membrane-penetrating receptors (outside-in signal transduction). Conversely, the expression, secretion and assembly of laminin-rich matrices is regulated by cell surface receptors through inside-out signal transduction. We will review how integrins regulate laminin matrix assembly and the signals elicited by laminins that support either migration or stable adhesion of keratinocytes. We will also discuss recent data indicating that laminins plays key regulatory roles in the development of skin appendages and contribute to the pathogenesis of skin cancer.     

The skin as a signal organ in the development of recent cytostatic drugs and anti-tumor strategies

It has been estimated that of all new cancers diagnosed annually in the USA almost one third originate in the skin such as basal cell carcinoma, squamous cell carcinoma, and melanoma. Much progress has been achieved in our understanding of the pathogenesis and it became clear primarily by studies of chemical carcinogenesis in murine skin that this is a stepwise process comprising at least three distinct stages: initiation, promotion, and malignant conversion or progression. This knowledge of the pathogenesis prompted to the development of chemoprevention of cancer in humans. Large clinical chemoprevention intervention studies with vitamin A, -carotene, vitamin E, and selenium are on the way. The metabolism of chemical carcinogens by cytochrome P-450 isozymes and other xenobiotica metabolizing enzymes has been proven essential in chemocarcinogenesis but it plays also a crucial role in the biotransformation of cancer themotherapeutic agents and may be of importance in influencing the efficacy as well as the side effects of these drugs in the target organ. Although our current knowledge is limited, studies of the xenobiotic metabolizing enzymes in extrahepatic tissues such as the skin demonstrate that they are possible. They may be of special importance in the near future in order to design individual therapies that will avoid unwanted toxicities and enhance therapeutic effectiveness.     

Respective contribution of neutrophil elastase and matrix metalloproteinase 9 in the degradation of BP180 (type XVII collagen) in human bullous pemphigoid.

Bullous pemphigoid is a blistering disorder associated with autoantibodies directed against two components of hemidesmosomes, BP180 and BP230. Autoantibodies to the extracellular collagenous domain of BP180 are thought to play a key role in the pathogenesis of the disease. In a murine model of bullous pemphigoid, neutrophil elastase and 92 kDa gelatinase (matrix metalloproteinase 9) have been implicated in subepidermal blister formation via proteolytic degradation of BP180. In this study we sought to elucidate the contribution of these two enzymes to subepidermal blister formation by assessing the expression, localization, and activity of the two proteases in lesional skin, serum samples, and blister fluids obtained from 17 patients with bullous pemphigoid. The results indicate that (i) neutrophil elastase is found in skin biopsy specimens from bullous pemphigoid lesions and is recovered as active enzyme in blister fluids, and (ii) although proform of matrix metalloproteinase 9 is present in lesional skin, it is present only as proenzyme in blister fluids, which also contain high levels of tissue inhibitor of metalloproteinase-1. Next, the capacity of matrix metalloproteinase 9 and neutrophil elastase to degrade a recombinant protein corresponding to the extracellular collagenous domain of the BP180 was studied. Our data illustrate that (i) recombinant matrix metalloproteinase 9, neutrophil elastase, and blister fluid from bullous pemphigoid patients are all able to hydrolyze recombinant BP180; (ii) the pattern of recombinant BP180 proteolysis with blister fluid was similar to that obtained with neutrophil elastase; and (iii) recombinant BP180 degradation by blister fluid could be inhibited by chloromethylketone, a specific elastase inhibitor, but not by batimastat, a wide spectrum matrix metalloproteinase inhibitor. Our results confirm the importance of neutrophil elastase but not matrix metalloproteinase 9 in the direct cleavage of BP180 autoantigen and subepidermal blister formation in human bullous pemphigoid.     

Upregulation of cathepsin S in psoriatic keratinocytes

Please cite this paper as: Upregulation of cathepsin S in psoriatic keratinocytes. Experimental Dermatology 2010; 19 : e80–e88. Abstract: Cathepsin S (CATS) is a cysteine protease, well known for its role in MHC class II‐mediated antigen presentation and extracellular matrix degradation. Disturbance of the expression or metabolism of this protease is a concomitant feature of several diseases. Given this importance we studied the localization and regulation of CATS expression in normal and pathological human/mouse skin. In normal human skin CATS‐immunostaining is mainly present in the dermis and is localized in macrophages, Langerhans, T‐ and endothelial cells, but absent in keratinocytes. In all analyzed pathological skin biopsies, i.e. atopic dermatitis, actinic keratosis and psoriasis, CATS staining is strongly increased in the dermis. But only in psoriasis, CATS‐immunostaining is also detectable in keratinocytes. We show that cocultivation with T‐cells as well as treatment with cytokines can trigger expression and secretion of CATS, which is involved in MHC II processing in keratinocytes. Our data provide first evidence that CATS expression (i) is selectively induced in psoriatic keratinocytes, (ii) is triggered by T‐cells and (iii) might be involved in keratinocytic MHC class II expression, the processing of the MHC class II‐associated invariant chain and remodeling of the extracellular matrix. This paper expands our knowledge on the important role of keratinocytes in dermatological disease.     

Skin aging: the dermal perspective

The world population of adults aged 60 years or more is increasing globally, and this development can impact skin disease morbidity and mortality, as well as being reflected in the health care system organization. There is substantial evidence that the burden from a remarkable number of skin nonmalignant and malignant conditions is greater in the elderly. Dermatologic research and clinical education in dermatology should focus on both challenges and opportunities created by aging. Skin aging due to intrinsic and extrinsic factors can alter significantly epidermal and dermal structure and functions. Dermal aging can be linked to a great number of complications in routine dermatologic conditions, with slow healing as an example of a severe complication in the elderly. This may be attributed to aged dermal fibroblasts modifying the tissue microenvironment via a shift in their soluble factors and extracellular matrix repertoire. This senescence-associated secretory phenotype can explain the particular proclivity of aged skin to develop malignancies.