Chapter 53. Epidermal and Epidermal–Dermal Adhesion

The cell–cell and cell–basement membrane adhesion in the epidermis provides the skin with its resistance against environmental influences; epidermal integrity is required for protection of the entire organism against mechanical, physical, or microbial insults. The major cellular structures involved are the desmosomes at cell–cell junctions in the epidermis and the hemidesmosome–basement membrane adhesion complexes and related structures at the dermal–epidermal junction. Ultrastructurally, the hemidesmosome closely resembles one-half of the desmosome; however, at the molecular level, these two structures are distinct. Both represent specifically organized assemblies of intracellular and transmembrane molecules. The desmosome anchors cytoskeletal filaments to cell–cell junctions, and the hemidesmosome anchors cytoskeletal filaments of basal epithelial cells to the basement membrane. Our knowledge of the desmosomal, hemidesmosomal, and basement membrane molecules has expanded drastically in recent years due to the great power of both molecular genetics and proteomics. After keratinocyte transmembrane proteins were initially identified as autoantigens in pemphigus and pemphigoid, a multitude of molecules have now been characterized at both protein and gene levels, and their expression, regulation, and functions have been discerned. The antigenic epitopes in different autoimmune blistering skin diseases have been carefully mapped and, to date, mutations in at least 24 different genes have been shown to underlie heritable disorders of epidermal or epidermal–dermal adhesion in humans and mice. Morphologic, molecular, and functional aspects of these adhesion structures are delineated in this chapter.

Ultrastructure of Desmosomes

The desmosome (or macula adherens) is the major cell adhesion junction of the epidermis, serving to anchor apposing keratinocyte cell surface membranes to the intracellular keratin intermediate filament network. Desmosomes are present in almost all epithelial tissues, including the oropharynx, gut, liver, heart, lung, bladder, kidney, prostate, thymus, cornea, and central nervous system, although the desmosomal protein isoforms and intermediate filament proteins vary by cell type.1 The primary role of desmosomes in epidermal cell adhesion is evidenced by the histologic findings in epidermal spongiosis, or intercellular edema, in which adjacent keratinocytes remain attached to each other only at desmosomal junctions (Fig. 53-1). These “intercellular bridges” served as the earliest description of desmosomes in tissues, their observation made possible with the advent of light microscopy in the nineteenth century.2 The development of electron microscopy techniques in the mid-twentieth century allowed for higher resolution micrographs that revealed the ultrastructure of these intercellular junctions. Even in the twenty-first century, the desmosome still remains best defined by its electron micrographic appearance, with an electron-dense midline in the intercellular space halfway between apposing plasma membranes, sandwiched by two pairs of electron-dense cytoplasmic plaques (Fig. 53-2A).3 The intercellular space between plasma membranes was called the desmoglea (from the Greek for “desmosomal glue”), because it was presumed to provide the adhesion that kept cells together.4

Structure and Function of Desmosomal Proteins

In anchoring the cell surface to the intermediate filament network, desmosomes create a three-dimensional scaffolding of proteins that extend from the cell surface all the way to the nuclear envelope. This scaffolding is critical to stabilize epithelia in the face of shear stress or external trauma. Early morphologic studies led to the perception of the desmosome as a static structure, a “spot weld” functioning only to maintain intercellular adhesion.5 Over the last three decades, the individual proteins comprising the desmosome have been biochemically characterized and cloned, shedding light on both the dynamic nature of the desmosome structure and the diversity of desmosomal protein function.

Desmosomal proteins fall into three major categories: (1) desmosomal cadherins (desmogleins and desmocollins), (2) armadillo family proteins (plakoglobin and plakophilins), and (3) plakins (desmoplakin, envoplakin, and periplakin). Additional proteins, such as Perp, ninein, kazrin, and corneodesmosin, have also been localized to epidermal desmosomes.6–9 Immunogold electron microscopy labeling studies have further refined our understanding of how these molecular components of desmosomes are ordered within the desmosome ultrastructure10 (Fig. 53-2B). The desmosomal cadherins are transmembrane proteins whose extracellular amino-terminal domains interact to form the trans-adhesive interface between cells, represented by the electron-dense midline of the desmoglea. Intracellularly, approximately 10–20 nm from the plasma membrane, the outer dense plaque contains the desmosomal cadherin cytoplasmic tails, plakoglobin, the desmoplakin amino-terminal domain, and plakophilin. Approximately 40–50 nm from the plasma membrane, the desmoplakin carboxyl-terminus interacts with keratin intermediate filaments, producing the inner dense plaque. The biochemistry, expression pattern, and diseases of each desmosomal component are discussed in further detail below. Although the specific physiologic roles and pathophysiologic mechanisms affecting many of the desmosomal proteins remain under active investigation, current knowledge clearly indicates the importance of desmosomes and their components beyond just cell adhesion.

Desmosomal Cadherins

Desmogleins and desmocollins are part of the cadherin superfamily of transmembrane glycoproteins. Members of this superfamily, which includes the adherens junction protein E-cadherin, mediate calcium-dependent adhesion in a variety of epithelial tissues. In humans, there are four desmoglein (Dsg) isoforms and three desmocollin (Dsc) isoforms, each with varying expression patterns within and among epithelia.11–13 Within normal human epidermis, Dsg1, Dsg4, and Dsc1 are expressed predominantly in the differentiated cells of the superficial epidermis, while Dsg2, Dsg3, Dsc2, and Dsc3 are expressed more strongly in the basal and/or suprabasal layers14,15 (Fig. 53-3). Among different epithelial tissues, Dsg1 and Dsg3 expression are largely limited to stratified squamous epithelia in the skin and oropharynx, as well as thymic epithelial cells. Dsg3 is also strongly expressed in squamous cell carcinomas and other head and neck cancers, where it has been proposed as a potential molecular target for therapy.16 Dsg2 is the major desmoglein isoform in most simple and transitional epithelia, as well as cardiac myocytes.17 Dsg4 is prominent in desmosomes of the hair follicle, testis, and prostate.18,19 The expression pattern of the human desmocollins is less well characterized. In normal tissues, Dsc1 expression is largely limited to skin and oral epithelia, while Dsc2 is more widely expressed in most desmosome-containing epithelia and is the only desmocollin isoform in cardiac tissue.11 Dsc3, like Dsg3, is most strongly expressed in the stratified squamous epithelia of the skin and oropharynx,20 although UniGene data suggest weaker expression in a variety of other epithelial tissues. Desmocollin switching has been described in colorectal cancer, with downregulation of Dsc2 and upregulation of Dsc1 and Dsc3.21

The extracellular domains of the desmosomal cadherins consist of four cadherin repeats plus an extracellular anchor domain, each separated by a calcium-binding motif. All cadherins are synthesized as preproproteins, which include an amino-terminal signal sequence and propeptide. The propeptide is thought to prevent intracellular aggregation of newly synthesized cadherins within the secretory pathway of the cell. Proprotein convertases in the late Golgi network cleave the cadherin propeptide, thereby producing the mature adhesive protein.22 Although the crystal structure of the desmosomal cadherins remains unsolved, studies comparing the structure of desmosomes analyzed by cryo-electron tomography of vitreous sections with the solution structure of the classical cadherins suggest common mechanisms of intercellular adhesion, in which a conserved amino-terminal tryptophan residue on one cadherin molecule interacts with a hydrophobic acceptor pocket in the first extracellular domain of another cadherin molecule on a neighboring cell to form the trans-adhesive interface.23–26 Additionally, cadherins may participate in “cis” interactions with cadherin molecules on the same cell through their membrane proximal domains, which may facilitate desmosome assembly. Desmosomal cadherins can engage in both homophilic (i.e., Dsg–Dsg or Dsc–Dsc) as well as heterophilic (i.e., Dsg–Dsc) interactions, although heterophilic interactions are thought to contribute most to strong intercellular adhesion.27–29

The cytoplasmic domains of the desmosomal cadherins are less conserved. Desmocollins have a full length “a” and a shorter “b” splice isoform. The cytoplasmic domains of desmoglein and desmocollin “a” isoforms bind plakoglobin, and some desmoglein and desmocollin isoforms may also directly bind plakophilins30,31 (Fig. 53-2B). Increasing data suggest that desmosomal cadherins are not just adhesive molecules but may also actively regulate intracellular signaling, transcription, and other cellular processes.1 Consistent with this, desmocollin 3-deficiency in mice causes embryonic lethality at day 2.5 before implantation, indicating a central role for desmocollin 3 in early tissue morphogenesis independent of its desmosomal adhesive function.32

In dermatology, the desmosomal cadherins are best known for their role as autoantigens in the immunobullous disease pemphigus (see Chapter 54). The desmoglein 3 gene was originally discovered and cloned because it was the autoantigen in pemphigus vulgaris33 (Fig. 53-4A). Since then, all of the desmosomal cadherins have been associated with human autoimmune, infectious, and/or genetic diseases (summarized below and in Table 53-1).

Desmoglein 1 is the target of pathologic proteolytic cleavage in the infectious disorders bullous impetigo and staphylococcal scalded skin syndrome, as well as the inherited ichthyosis associated with Netherton syndrome (see Chapter 49).34,35 Cleavage of desmoglein 1 by staphylococcal exfoliative toxin occurs between extracellular domains 3 and 4.36 Pathogenic autoantibodies to desmoglein 1 are found in pemphigus foliaceus, mucocutaneous pemphigus vulgaris, and paraneoplastic pemphigus (see Chapters 54 and 55). Most pathogenic pemphigus foliaceus autoantibodies bind the first two extracellular domains of desmoglein 1, overlapping sites that are critical for desmoglein trans-adhesion.37–39 Autosomal dominant mutations causing haploinsufficiency of desmoglein 1 result in palmoplantar keratoderma (PPK; see Chapter 50). The PPK is classically striate (Fig. 53-4B), occurring at sites of greatest trauma or friction, but focal and diffuse forms have also been described.40–42

Desmoglein 2 has been implicated in human cardiovascular disease as a cause of autosomal dominant arrhythmogenic right ventricular cardiomyopathy (ARVC).43 The lack of skin phenotypes in affected patients indicates that desmoglein 2 is not required for epidermal adhesion, likely due to compensatory adhesion from other more highly expressed epidermal desmosomal cadherin isoforms.

Desmoglein 3 is the target of pathogenic autoantibodies in mucosal and mucocutaneous pemphigus vulgaris and paraneoplastic pemphigus (see Chapters 54 and 55.) Most pathogenic autoantibodies in pemphigus vulgaris target the amino-terminal extracellular (EC1–2) domains of desmoglein 3.37,44,45 Because desmoglein 3 deficiency in mice phenotypically resembles autoimmunity to desmoglein 3 in mucosal pemphigus vulgaris with oral suprabasal erosions, pemphigus autoantibodies are thought to cause loss of desmosomal cadherin function.46 More recent research has focused on signaling pathways activated after binding of pemphigus vulgaris autoantibodies to keratinocytes, as several biochemical inhibitors have been shown to prevent blistering in a neonatal mouse passive transfer model47 (discussed in further detail in Chapter 54).

Desmoglein 4 mutations have been described in rare autosomal recessive forms of hypotrichosis and monilethrix.48–51 One patient demonstrated transient scalp erosions during the first 2 weeks of life. Most of the mutations in Dsg4 are frameshift or nonsense mutations that would be predicted to lead to haploinsufficiency, although missense mutations have also been reported,52 Desmoglein 4 immunoreactivity is observed in pemphigus vulgaris and pemphigus foliaceus sera,51 but subsequent studies have attributed this to cross-reactivity from Dsg1 autoantibodies.53

Desmocollin 1 is the target of autoantibodies in the subcorneal pustular dermatosis of IgA pemphigus (see Chapter 54.)

Desmocollin 2, like desmoglein 2, is mutated in both autosomal dominant and recessive forms of ARVC, with no epidermal phenotype in affected patients.54,55

Desmocollin 3 mutations were found in one Pakistani kindred with autosomal recessive hypotrichosis.56 Autoantibodies to desmocollin 3 have also been found in pemphigus vulgaris patients, particularly those with vegetative lesions.57

Plakoglobin

Plakoglobin (also known as γ-catenin) directly binds the cytoplasmic tails of the desmogleins and desmocollin “a” isoforms, as well as E-cadherin, the major transmembrane protein of adherens junctions in epidermal keratinocytes.58,59 It is expressed throughout all layers of the epidermis and is ubiquitously expressed in all epithelia. Plakoglobin, like plakophilin, is a member of the armadillo gene family, characterized by a conserved protein structure with head and tail domains that flank multiple homologous arm repeats.60 Various domains of plakoglobin modulate its binding to the desmosomal cadherins.61–63 Other domains bind to desmoplakin, thus linking desmogleins and desmocollins to desmoplakin.64 Plakoglobin can also localize to the nucleus, where it may modulate gene transcription by TCF/LEF family members.65,66 Although most depictions of the desmosome show both plakoglobin and plakophilin binding to desmoplakin, biochemical studies suggest that these interactions are mutually exclusive.67 Likely, the armadillo family proteins play more dynamic roles in recruitment of desmosomal proteins to the plaque, similar to α-catenin in adherens junctions.68,69

Plakoglobin mutations result in Naxos disease, an autosomal recessive syndrome of diffuse PPK, wooly hair, and arrhythmogenic right ventricular cardiomyopathy, the latter of which may present in late childhood to adolescence.70

Desmoplakin

Desmoplakin exists in two RNA splice variants, desmoplakin I and II.71,72 It is unknown whether different isoforms perform different cellular functions, although human disease mutations suggest that desmoplakin I is required for normal desmosomal function.73 Desmoplakin is part of the plakin gene family,74 which includes the hemidesmosomal proteins bullous pemphigoid antigen 1 and plectin, as well as envoplakin and periplakin. Similar to other desmosomal components, desmoplakin is a modular protein, with different modules fulfilling different functions. The central part of one desmoplakin molecule coils around the central part of another to form a rod-like center. The amino-terminal head domain binds to plakoglobin,64 and the carboxyl-terminal tail binds to keratin.75 Therefore, desmoplakin provides the major link between the keratin filaments and the desmosomal plaque. Desmoplakin also plays a critical role in development independent of its function in desmosomes, as desmoplakin-null mice die early in embryogenesis at E6.5, before desmosomes are formed.76

A broad range of desmoplakin mutations have been associated with human disease, leading to variable phenotypic combinations of PPK (striate or diffuse), dilated cardiomyopathy (left or right), wooly hair, nail abnormalities, and/or skin blisters.52 Haploinsufficiency of desmoplakin leads to autosomal dominant striate PPK.77 Autosomal recessive mutations in desmoplakin were described in three Ecuadorian families with Carvajal syndrome, consisting of diffuse PPK, wooly hair, and arrhythmogenic left ventricular cardiomyopathy.78 A Naxos-like syndrome of PPK, wooly hair, and ARVC occurs with the p.R1267X nonsense mutation, which affects only the desmoplakin I splice isoform, indicating that desmoplakin II is not sufficient to restore normal desmosomal function in epidermis.73 Lethal acantholytic epidermolysis bullosa (widespread epidermolysis, generalized alopecia, anonychia, and neonatal teeth) was attributed to compound heterozygous mutations in desmoplakin that caused loss of the desmoplakin tail.79 Additionally, desmoplakin autoantibodies are observed in paraneoplastic pemphigus sera (see Chapter 55).

Plakophilins

Plakophilins, like plakoglobin, can localize both to the plasma membrane as well as the nucleus, although their function outside of desmosomal adhesion is not well characterized. Plakophilins directly bind to desmoplakin, and may also directly bind keratins and desmosomal cadherins, which is thought to aid in clustering and lateral stability of the desmosomal plaque.30,31,67 Plakophilin 1 also associates with the eukaryotic translation initiation factor eIF4A1 in the mRNA cap complex, where it functions to regulate translation and cell proliferation.80

Mutations in plakophilin 1 cause ectodermal dysplasia-skin fragility syndrome, suggesting a role for plakophilin 1 in epidermal morphogenesis as well as adhesion.81 Plakophilin 2 mutations are the most common cause of autosomal dominant ARVC.82 Currently, there are no known human diseases associated with plakophilin 3.

Other Desmosomal Proteins

Envoplakin and periplakin are desmosomal plaque proteins expressed in the superficial layers of the epidermis. Both proteins incorporate into the corneodesmosomes of the stratum corneum. Mice deficient in envoplakin, periplakin, and involucrin do not demonstrate adhesion defects, but instead show impaired desquamation and epidermal barrier function.83 Envoplakin and periplakin autoantibodies are characteristic of paraneoplastic pemphigus sera (see Chapter 55).

Corneodesmosin is a secreted glycoprotein that incorporates into corneodesmosomes and is also expressed in the inner root sheath of the hair follicle. Heterozygous mutations in corneodesmosin are associated with an autosomal dominant hypotrichosis simplex of the scalp.84 Loss of cohesion in the inner root sheath and aggregates of proteolytically cleaved corneodesmosin around the hair follicle are observed in scalp biopsies of affected patients.

Structural and Functional Characteristics of Basement Membranes

Basement membranes underlie epithelial and endothelial cells and separate them from each other or from the adjacent stroma. Another form of basement membrane surrounds smooth muscle or nerve cells. The physiologic functions of basement membranes are diverse: in the various organ systems they provide support for differentiated cells, maintain tissue architecture during remodeling and repair, and, in some cases, acquire specialized functions, including the ability to serve as selective permeability barriers (e.g., the glomerular basement membrane or the blood–brain barrier) or acquire strong adhesive properties, like the basement membrane at the dermal–epidermal junction, or that surrounding smooth muscle cells, which provide the tissues resistance against shearing forces. All of these characteristics of basement membranes are also used during development and differentiation of multicellular organisms (Box 53-1).

Ultrastructurally, basement membranes most often appear as trilaminar structures, consisting of a central electron-dense region, known as the lamina densa, adjacent on either side to an apparently less-dense area, known as the lamina lucida or lamina rara. The lamina lucida directly abuts the plasma membranes of the adherent cells. The relative size of each of these regions varies in different tissues, among the basement membranes of the same tissue at different ages, and as a consequence of diseases. For example, the trilaminar glomerular basement membrane in humans varies from 240 nm to 340 nm in width, whereas the bilaminar basement membrane of the dermal–epidermal junction measures 50 nm to 90 nm. This ultrastructure demonstrates that basement membranes serve as substrates for the attachment of cells and fix their polarity. Their continuity throughout the various organ systems stabilizes the tissue orientations and provides a template for orderly repair after traumatic injury. Major disruptions in the basement membrane result in the formation of scar tissue and the loss of function in that area.

Different basement membranes contain both common and unique components. All share a basic network structure to which specific macromolecules have been appended. These molecules are responsible for the specialized functions of different basement membranes. The basic constituents of these structures are collagen IV, laminins, nidogens, and proteoglycans of the perlecan type, which all are highly conserved, although the isoforms, the number of the polypeptide subunits, and their individual structures vary among species.84,85 The nearly ubiquitous distribution of heparan sulfate proteoglycans in all basement membranes suggests that these serve as selective permeability barriers in multiple locations, including the kidney and the blood–brain barrier. Ultrafiltration may be especially important during development and morphogenesis of all tissues.

Basement membranes also provide physical separation between epithelia and their underlying extracellular matrices. This barrier is especially important in the containment of tumors. With the exception of certain cells of the immune system, nonmalignant cells seldom cross a basement membrane. In contrast, malignant cells bind the basement membrane, regionally disrupt its structure, and migrate through the rupture. Laminins and integrins mediate the tumor-cell binding, and the basement membrane dissolution is catalyzed at least in part by metalloproteases produced by the tumor cell. The absence of distinguishable basement membranes in tumor biopsies is used as an indicator of malignancy, and there appears to be a high correlation between metastasis and basement membrane disruption. These observations underline the importance of the basal lamina as an obstacle to cell migration.

By binding biologically active signaling molecules, basement membranes regulate a multitude of biologic events. The constituent proteoglycans can bind growth factors that can be released from the complexes. Thus, the basement membranes are potent regulators of cell adhesion and migration, cytoskeleton and cell form, cell division, differentiation and polarization, and apoptosis.85

The dermal–epidermal junction is an example of a highly complex form of basement membrane,86,87 which underlies the basal cells and extends into the upper layers of the dermis (Fig. 53-5A). This basement membrane is continuous along the epidermis and skin appendages, including sweat glands, hair follicles, and sebaceous glands. The dermal–epidermal junction can be divided into three distinct zones. The first zone contains the keratin filament–hemidesmosome complex of the basal cells and extends through the lamina lucida to the lamina densa. The plasma membranes of the basal cells in this region contain numerous electron-dense plates known as hemidesmosomes. The intracellular architecture and organization of the basal cells are maintained by keratin intermediate filaments, 7 nm to 10 nm in diameter that course through the basal cells and insert into the desmosomes and hemidesmosomes. External to the plasma membrane is a 25-nm to 50-nm-wide lamina lucida that contains anchoring filaments, 2 nm to 8 nm in diameter, originating in the plasma membrane and inserting into the lamina densa. The anchoring filaments can be seen throughout the lamina lucida but they are concentrated in the regions of the hemidesmosomes. Thus, the anchoring filaments appear to secure the epithelial cells to the lamina densa.

The existence of the lamina lucida in vivo has been questioned.86,88 When the ultrastructure of the basement membrane is evaluated after high-pressure preservation techniques, the lamina densa appears intimately associated with the epithelial cell surface. When the dermal–epidermal junction is similarly prepared, no distinct lamina lucida is seen (see Fig. 53-5B). This suggests that the lamina lucida may result from shrinkage of the cell surface away from the lamina densa due to dehydration. The appearance of anchoring filaments spanning the lamina lucida may then result from the firm attachment of constituents of the lamina densa at the hemidesmosome that is subsequently pulled from the lamina densa by shrinkage. Other components that are also tightly fixed to the keratinocyte plasma membrane, either at the hemidesmosomes or at other sites along the membrane, may similarly become displaced into the shrinkage space. Regardless of its actual occurrence in vivo, the evaluation of the lamina lucida by standard electron microscopy techniques has allowed identification of specific structures that would otherwise have been difficult to detect. In addition, the morphologic term lamina lucida remains practical in the scientific communication and continues to be used.

The second zone, the lamina densa, appears as an electron-dense amorphous structure 20 nm to 50 nm in width. At high magnification, it has a granular–fibrous appearance.86 The major molecular components of the lamina densa are collagen IV, nidogens, perlecan, and laminins, which all can polymerize to networks of variable thickness.84,86

The subbasal lamina contains microfibrillar structures. Two of these are readily distinguishable. Anchoring fibrils appear as condensed fibrous aggregates 20 nm to 75 nm in diameter.89 At high resolution, they appear to have a cross-striated banding pattern (see Fig. 53-5A). The length of the anchoring fibril is difficult to measure because of its random orientation in relation to the plane of the section. In toad skin, these structures have lengths of approximately 800 nm. The anchoring fibrils in human skin appear to be somewhat shorter. The ends of the fibrils appear less tightly packed, giving a somewhat frayed appearance. The proximal end inserts into the basal lamina, and the distal end is integrated into the fibrous network of the dermis.90 Many of the anchoring fibrils originating at the lamina densa loop back into the lamina densa in a horseshoe-like manner; others insert their opposite ends into amorphous-appearing structures, termed anchoring plaques.90 These structures are believed to be independent “islands” of electron-dense material, although some controversy exists in the literature.91 Anchoring fibrils are primarily aggregates of collagen VII.

Fibrillin-containing microfibrils, 10 nm to 12 nm in diameter, are also localized in the sublamina densa region. These are elastic-related fibers, because elastic components of the dermis are formed from microfibrillar and amorphous components.92 The microfibrillar component in the presence of abundant amorphous component is known as the elastic fiber. In the papillary dermis, the microfibrils insert into the basal lamina perpendicular to the basement membrane and extend into the dermis, where they gradually merge with the elastic fibers to form a plexus parallel to the dermal–epidermal junction. These two elastic components appear to be continuous with the elastic fibers present deep within the reticular dermis.92

In summary, the ultrastructure of the dermal–epidermal junction strongly suggests that the lamina densa functions as a structural scaffold for the attachment of the epidermal cells at one surface, secured by anchoring filaments extending from the lamina densa to the hemidesmosomes. The latter also serve as insertion points for intracellular keratin filaments that form scaffolding for the basal cells. On the opposite surface, the extracellular matrix suprastructures of the dermis are firmly attached to the lamina densa. The interaction of collagen-containing dermal fibers with the lamina densa appears to be mediated by the anchoring fibrils. The elastic system of the dermis inserts directly into the basal lamina via the microfibrils. Thus, the dermal–epidermal junction provides a continuous series of attachments between the reticular dermis and the cytoskeleton of the basal cells. These observations suggest four major functions for the epidermal basement membrane: (1) a structural foundation for the secure attachment and polarity of the epidermal basal cells; (2) a barrier separating the epidermis and the dermis; (3) firm attachment of the dermis to the epidermis through a continuous system of structural elements; and (4) modification of cellular functions, such as organization of the cytoskeleton, differentiation, or rescue from apoptotic signaling via outside-in signaling mechanisms.

Basement membranes contain collagenous and noncollagenous glycoproteins and proteoglycans. The content of the collagen-specific amino acids hydroxyproline and hydroxylysine suggests that collagens account for 40% to 65% of the total basement membrane protein. All basement membranes contain certain isoforms of collagen IV, laminin, nidogen, and the heparan sulfate proteoglycan perlecan (Box 53-2). For example, the α3 chain of collagen IV is localized in the basement membrane of the kidney and lung, but not in those of the skin and blood vessels. In contrast, collagens VII and XVII are associated with the squamous epithelia of skin but are not found in glomerular and alveolar basement membranes. In addition, many other tissue-specific components are found in basement membranes, including different collagens, laminins, fibulins, and fibronectin.84,85,92,93 Differences in macromolecular composition are responsible for morphologic and functional variance of basement membranes.

Ubiquitous Components of Basement Membranes

Collagen IV

Collagen IV is a heterotrimer of three α chains.84,94 Each of these contains three distinct domains (Fig. 53-6A): (1) the N-terminal cysteine-rich (7-S) domain, (2) a central triple-helical domain, and (3) a C-terminal globular domain (NC-l). The trimer composition is determined by the NC-l domains, and the α chains are linked to each other by covalent interactions through these domains.95 The triple helix of collagen IV is long and contains several discontinuities, which result in increased flexibility in the collagen IV helix, but also render it susceptible to proteases.

The suprastructure of collagen IV has been partially elucidated by rotary shadowing electron microscopy that indicated that the major interactions among collagen IV molecules occur at their amino- and carboxyl-terminal domains, and by lateral association of their triple helices (see Fig. 53-6A).84,95 Covalent interactions among 7-S regions of different molecules are the basis for the specialized network characteristic of basement membranes (see Fig. 53-6B). The individual 7-S regions overlap in both the parallel and antiparallel directions, producing a characteristic four-legged “spider” form. The NC-l domains at the end of each leg of the spider interact with the NC-l domain of the adjacent aggregates (see Fig. 53-6B). Association is stabilized by covalent bonds. These end-to-end interactions result in an extended two-dimensional network that is the basis of basement membrane organization.

The high flexibility of the basement membrane network structure makes the possibility of interactions with other molecules very attractive. An open meshwork of collagen IV with, for example, laminins or perlecan, can be easily visualized (Fig. 53-7).85 The implied porosity of this structure would then be limited by the size of the pores in the collagen network and by structural elements associated with it. This model of the basement membrane structure allows considerable mechanical stability while retaining physiologic flexibility. These properties of strength and elasticity would be expected for a dynamic surface, such as that seen in the dermal–epidermal junction and surrounding blood vessels.

Collagen IV molecules in different basement membranes contain genetically distinct but structurally homologous α chains. The α1 and α2 chains are ubiquitous, but the α3, α4, α5, and α6 chains show restricted distribution among tissues.93 The chain organization and discriminatory interactions between the NC-1 domains govern network assembly in the basement membranes.95 The six chains of collagen IV are distributed in three major networks, (1) α1–α2, (2) α3–α4–α5, and (3) α1–α2–α5–α6, whose chain composition is determined by the NC-l domains. Two networks, namely α1–α2-containing and α3–α4–α5-containing networks, exist in the glomerular basement membrane. Smooth muscle basement membranes have an α1–α2–α5–α6-containing network in addition to the classic α1–α2 network. Within the dermal–epidermal junction, the α1–α2-containing collagen IV network dominates, but α1–α2–α5–α6-containing network is also likely to be present.96

Mutations in the genes encoding the α1 and α2 chains cause pathologies in different organs, ranging from small-vessel disease, which often underlies ischemic strokes and intracerebral hemorrhages to the eye and the HANAC (hereditary angiopathy with nephropathy, aneurysms, and muscle cramps) syndrome.93,97,98 Interestingly, despite the ubiquitous presence of the α1 and α2 chains of collagen IV in basement membranes, aberrations do not occur in all basement membranes, suggesting varying tissue-specific roles for collagen IV. Structural aberrations in the genes encoding the α3, α4, α5, and α6 chains cause different forms of Alport syndrome, a genetic disease characterized by nephritis and deafness.93 The α3(IV) chain is the antigen recognized by the circulating autoantibodies in the Goodpasture syndrome.93

Laminins

Laminins are very large glycoproteins (600 to 950 kDa) within the lamina lucida/lamina densa of all basement membranes.99 Three types of subunit chains have been designated α, β, and γ chains, and each laminin is a trimeric aggregate of one α, β, and γ chain. The trimers have semirigid and extended structures, which appear as an asymmetric cross in rotary shadowing electron microscopy (see Fig. 53-6C). The long arm of the cross is approximately 125 nm in length; the short arms are variable. Each laminin molecule contains globular and rod-like domains that have been individually implicated in various functions, such as aggregation with itself and with other components of the lamina densa (Fig. 53-8), cell attachment and spreading, neurite outgrowth, or cellular differentiation.85,99 The C-terminal laminin-type globular (LG) domain of the α chain, at the foot of the long arm of the laminin cross, harbors the binding site for integrins.99

To date, 15 laminin isoforms have been identified. These represent different trimeric combinations of five distinct α chains, three β chains, and three γ chains known so far. Historically, laminins were named as laminin-1 to laminin-15, in the order of their discovery, but this classification had grown quite impractical, with the need to memorize the numbers. The current, simplified nomenclature is based on the chain composition and the number of each α, β, and γ chain (Table 53-2).100 For example, the classic “prototype” laminin-1, with α chain composition α1β1γ1, is now called laminin 111. The major laminin of the epidermal basement membrane, the previous laminin-5, with α chain composition α3β3γ2, is now called laminin 332.

The α2 chain containing laminins are present primarily within the basement membranes of the muscle fibers, nerves, neuromuscular junction, and glomerulus. The α3 chain is involved in epithelial adhesion, and the α4 and α5 chains are found in a variety of tissues including endothelia, epithelia, neuromuscular junction, and glomerulus.101 Laminin 511 is present in the basement membrane of the epidermis and the hair follicles, where it is believed to be involved in developmental signaling.99,100 The distribution of the β2 chain is largely restricted to the neuromuscular junction, but it is also found in nonmuscle tissues such as the kidney glomerulus and the capillary basal lamina.99 The β3 chain is involved in epithelial adhesion. Three γ chain variants—γ1, γ2, and γ3—are known. The γ2 chain is found only in laminin 332 in the skin. The γ3 chain, a component of laminins 423 and 523, binds nidogens and is localized in basement membrane zones of adult and embryonic brain, kidney, skin, muscle, and testis. It is present in much lower concentrations than the γ1 chain, a fact that may indicate highly specialized functions.99 The functions of all laminins are not yet fully understood, but by interacting with integrins and other cell surface components laminins control cellular activities such as adhesion, migration, proliferation, and polarity in a wide variety of organs.99,100

Several human congenital diseases are caused by mutations in the laminin chains.99 Since some chains can be components in several different laminins, the mutations can affect functions of multiple laminins in different tissues. For example, mutations in the α2 chain cause congenital muscular dystrophy and a significant decrease in the amount of basement membrane accumulated surrounding muscle cells.99 The absence of the basement membrane leads to progressive degeneration of the muscle due to cell death. Therefore, the prediction is that laminins, and basement membranes in general, are required to prevent apoptosis by the cell types they surround.99 Mutations in the α3, β3, or γ2 chains underlie junctional epidermolysis bullosa, and mutations of the α4 chain are associated with cardiomyopathy. Pierson syndrome, a severe congenital condition affecting mainly the kidney and the eye, is caused by mutations of the β2 chain.

Nidogens

Two nidogens, nidogen 1 and 2, previously known as entactin, are distinct gene products. Both are relatively small molecules (see Fig. 53-6D), which bind laminins at a specific site within the γ1 and the γ3 chain,93,102 but also collagen IV, perlecan, and fibulins. Nidogens are likely to act as connecting elements between the collagen IV and laminin networks and integrate other basement membrane components into this specialized extracellular matrix.84 Targeted ablation of nidogens in mice showed that the loss of either isoform has no effect on basement membrane formation and organ development, but lack of both results in severe defects of the lung and the heart102 that are not compatible with life. Interestingly, despite the ubiquitous presence of nidogens in basement membranes, aberrations did not occur in all basement membranes, suggesting distinct roles for nidogens in different basement membranes.102

Heparan Sulfate Proteoglycans

Another class of ubiquitous integral basement membrane constituents are the proteoglycans. Three proteoglycans are characteristically present in vascular and epithelial basement membranes: (1) perlecan, (2) agrin, and (3) collagen XVIII.103 They consist of a core protein of various lengths, and carry primarily heparan sulfate side chains. The name perlecan is derived from its rotary shadowing appearance reminiscent of a string of pearls. Perlecan represents a complex multidomain proteoglycan with enormous dimensions and a number of posttranslational modifications. Knockout mice lacking perlecan exhibited abnormalities in many tissues, including basement membranes, and embryonic lethality. The basement membranes deteriorated in regions under increased mechanical stress, such as myocardium or skin, resulting in lethal cardiac abnormalities and skin blistering.103 Agrin is a major heparan sulfate proteoglycan of neuromuscular junctions and renal tubular basement membranes. Collagen XVIII is considered to be a hybrid collagen—proteoglycan in various organs.104 In collagen XVIII knockout mice, basement membranes were broadened in different organs, accompanied with mesangial expansion and reduced function of the kidney. The proteoglycans can interact with several other basement membrane components and are believed to contribute to the overall architecture of the basement membrane as well as provide tissue-specific functions. The high sulfate content makes them highly negatively charged and hydrophilic, and the charge density is responsible for providing the selective permeability of the glomerular basement membrane.

Syndecans are transmembrane heparan sulfate proteoglycans present on most cell types, including basal keratinocytes of the epidermis. The extracellular domains have affinity for laminins and, presumably through these interactions, they engage in outside-in signaling and regulate cellular processes ranging from growth factor signaling, cell adhesion, and cytoskeletal organization, to infection of cells with microorganisms.105

Fibulins

Fibulins are a family of six highly conserved, calcium-binding extracellular matrix proteins. They are located in vessel walls, basement membranes, and microfibrillar structures and they have overlapping binding sites for a variety of ligands, both basement membrane proteins and components of the interstitial connective tissues.106 Therefore, fibulins are believed to function as intermolecular bridges that stabilize the supramolecular organization of extracellular membrane structures, such as elastic fibers and basement membranes. Genetic defects of the genes encoding fibulin 4 and 5 cause different forms of cutis laxa.

The dermal–epidermal junction of skin is an excellent example of specific divergence in basement membrane structure. The structural components of hemidesmosomes, anchoring filaments, and anchoring fibrils in the basement membrane zone are quite well characterized.84–87,107,108 A cartoon depicting the relative locations of the proteins found at the dermal–epidermal junction is shown in Fig. 53-9. These proteins are listed in Table 53-3 and discussed in the following sections.

Hemidesmosomes

Ultrastructurally, the hemidesmosome closely resembles one-half of the desmosome at cell–cell junctions in the epidermis. However, the components of these two structures are distinct. The 230-kDa BPAG1 (bullous pemphigoid antigen 1, or BP230) is a coiled-coil dimeric protein with homology to plakins, which bind intermediate filaments. BPAG1 is the major component of the hemidesmosomal inner dense plaque. Ablation of BPAG1 is associated with epidermolysis bullosa simplex in mice and humans.72,109 The 180-kDa BPAG2 or BP180, and the major antigen in bullous pemphigoid (Fig. 53-10), is a transmembrane collagen now known as collagen XVII, where the collagenous domain is extracellular. In fact, collagen XVII is a prototype of the novel protein family of collagenous transmembrane proteins, type II transmembrane molecules with one or more collagenous stretches in the extracellular domain.110 The intracellular ligands of collagen XVII are plectin, BPAG1 and β4 integrin, and the extracellular ligands α6 integrin and laminin 332.107,108 The 120-kDa ectodomain of collagen XVII is shed from the cell surface by proteinases of the ADAM (a disintegrin-like and metalloproteinase-containing) family through cleavage within the juxtamembranous NC-16 domain (Fig. 53-11).111 Mutations in collagen XVII cause junctional epidermolysis bullosa (see Chapter 62), indicating that it stabilizes interactions of basal keratinocytes with the basement membrane.112 Ablation of the mouse Col17a1 gene resulted in moderate skin blistering, dental anomalies, and graying hair.113,114 Plectin, another dimeric plakin homolog, is also a component of the hemidesmosome. However, its tissue distribution is not limited to hemidesmosome-containing basement membranes. Mutations of plectin result in epidermolysis bullosa simplex and progressive muscular dystrophy and, in some cases, epidermolysis bullosa simplex with pyloric atresia,115 indicating a role for plectin in the stability of cell–basement membrane adhesion in a variety of tissues.

One key component of the hemidesmosome is the integrin α6β4.107 It has a high affinity for laminin 332 and is essential to integration of the hemidesmosome with the underlying basement membrane and stroma.107,108 Mutations in either the α6 or β4 chains result in junctional epidermolysis bullosa associated with pyloric atresia.108,112

A member of the widely expressed cell surface transmembrane proteins of the tetraspanin family, CD151, is also a component of the hemidesmosome. It forms complexes with α3β1 and α6β4 integrins at the basolateral surface of basal keratinocytes and stabilizes their functions.116 CD151-null mice have apparently normal hemidesmosomes, but exhibit some functional aberrations, for example, poor keratinocyte migration in explant cultures. A very rare human genetic condition delivered indirect information on the functions of CD151. In addition to the expression of CD151 in several tissues like the kidney and the skin, its gene also encodes the MER2 blood group antigen on erythrocytes. Homozygous CD151-null mutations were identified in three MER2-negative patients, who also presented with hereditary nephritis, sensorineural deafness, pretibial epidermolysis bullosa, and β-thalassemia minor.117 These symptoms suggest that CD151 is important for the assembly of the basement membrane in the kidney, skin, and inner ear and plays a role in erythropoiesis.

Other Epidermal Adhesion Complexes

In addition to the hemidesmosomal components, other adhesion molecules are known to be present at the basolateral aspect of basal keratinocytes, for example, integrin α3β1, the receptor for the laminin 332–311 complex in the basement membrane between the hemidesmosomes,118 and another transmembrane collagen, type XIII.94 Since these proteins are localized to focal contacts in vitro, together with vinculin and talin, they are predicted to function as the link between the basement membrane and the epithelial cortical actin network. A novel intracellular component of this complex, kindlin-1, or fermitin-family-homolog 1, was identified by the genetic studies. The kindlin-1 gene, FERMT1, is mutated in the Kindler syndrome,119,120 a disorder with skin blistering in infancy, progressive poikiloderma, skin atrophy, pigment anomalies, and, occasionally, skin cancer. In the epidermis, kindlin-1 is expressed at the basolateral surface of basal keratinocytes and has important functions in β1 integrin-mediated outside-in signaling that regulates cell–matrix adhesions, cell migration, and polarity.121,122 Thus, kindlin-1 is necessary for the stability of the dermal–epidermal junction and that, in addition to hemidesmosomes, tethering the actin cytoskeleton to cell–matrix adhesions offers alternative means to anchor basal epithelial cells to the basement membrane.

Anchoring Filaments

The anchoring filaments contain laminin 332 and the ectodomain of collagen XVII, two ligands that interact with each other through noncovalent bonds.99,100,108 The ectodomain of collagen XVII, which protrudes from the plasma membrane into the lamina lucida, has a loop structure consistent with its role as an anchoring filament protein.123 Laminin 332 is a disulfide-bonded complex of α3, β3, and γ2 chains. The two splice variants of the α3 chain, α3A and α3B, associate with the α3 and γ2 chains to form laminin 3A32 and 3B32 (see Fig. 53-8).99,100 Rotary shadowing imaging indicates that laminin 332 has a rod-like structure terminating in the globular regions (see Fig. 53-6F), a shape consistent with its role as an anchoring filament protein. The individual chains are considerably truncated relative to other laminin chains, and this truncation is reflected in the loss of the structures equivalent to the short arms of other laminins. Additionally, the α3 and γ2 chains are proteolytically processed after secretion from the keratinocyte, further trimming the short arms.99,100 The C-terminus of the α chain, longer than that of the β and γ chains, comprises five globular LG modules, LG1 through LG5, which interact with cell surface receptors. The α3β1 and α6β4 integrins have affinity for the LG1-3 domains, whereas the LG4-5 tandem has affinity for syndecans and β-dystroglycan on the keratinocyte surface.99,105 The LG4-5 modules are proteolytically cleaved in most laminins, a process which may modulate interactions with cell surface receptors.99,100 Laminin 332 forms covalent complexes with laminin 311 (α3β1γ1), binds to the NC-1 domain of collagen VII, the anchoring fibril protein.124 and to the distal ectodomain of collagen XVII.123 Genetic evidence demonstrates that laminin 332 is essential in keratinocyte adhesion, as null mutations in any of its component α3, β3, or γ2 chains result in severe Herlitz junctional epidermolysis bullosa112 (see Chapter 62). Targeted disruption of the mouse lama3 gene prevented the synthesis of both laminin 332 and 311 molecules and resulted in abnormal hemidesmosomes, severe junctional blistering, abnormalities of ameloblast differentiation in developing teeth, and perinatal lethality.125 Therefore, the severity of the laminin 332 null phenotype indirectly emphasizes its functional importance in bridging the hemidesmosomes and the anchoring fibrils.

Epithelial Lamina Densa

The basement membrane beneath and between the hemidesmosomes contains the α1–α2-containing collagen IV network, probably some α1–α2–α5–α6-containing collagen IV network, as well as nidogen, perlecan, and α3 and α5-chain containing laminins.93 The laminin α3 chain can associate with the β1 and γ1 chains of laminin 311, which has the unique property of forming disulfide-bonded dimers with laminin 332.99,100 The major α3-containing laminin in the lamina densa between hemidesmosomes is probably the laminin 332–311 complex.99,100,108 As the laminin α3 chain is a ligand for integrin α3β1 present between hemidesmosomes, binding of laminin 332–311 complex to the intracellular actin cytoskeleton is likely to be mediated by this integrin. This is consistent with studies in mice in which targeted ablation of the integrin α3 chain causes loss of the basement membrane between hemidesmosomes but not beneath them.93,99 The N-termini of laminin 332 bind to collagen VII, the main component of the anchoring fibrils in the sublamina densa, so that anchoring filaments and fibrils are directly connected.93 The laminin 332–311 complex and laminin 511 (α5β1γ1) contain a γ1 chain and can therefore bind nidogens and the collagen IV network.93,99 Further, nidogen 1 and fibulin 1 and 2 were shown to be ligands for the laminin γ2 chain.93 These interactions are important for the integration of laminin 332 into the extracellular matrix before the maturation of the γ2 chain, as a substantial portion of N-terminus of the γ2 chain is cleaved in human adult skin.108 Yet another link, which strengthens dermal–epidermal cohesion, is provided by molecular interactions between perlecan within the lamina densa and fibrillin 1 in the microfibrils.126

Anchoring Fibrils

Collagen VII is the major component of the anchoring fibrils.90,124 The collagen VII molecule is distinguished from other collagens in that it has a very long triple-helical domain, 450 nm in length. Globular domains exist at both ends of the triple helix, and the N-terminal domain NC-1 is very large and trident-like (see Fig. 53-6E). The smaller C-propeptide, NC-2, is believed to facilitate the formation of the antiparallel, centrosymmetric dimers, before it is removed by the metalloproteinase bone morphogenetic protein 1127 to yield a mature collagen VII. The dimers are covalently cross-linked through disulfide bonds at the carboxyl terminus, and they aggregate laterally to form the anchoring fibrils. The fibrils are further stabilized by tissue transglutaminase, which catalyzes the formation of covalent γ-glutamyl-ϵ-lysine cross-links.128

The NC-1 domain of collagen VII binds to laminin 332 and collagen IV within the lamina densa (see Fig. 53-5A).124 The triple helical domains of an antiparallel collagen VII dimer make the length of the anchoring fibril. It extends perpendicularly from the lamina densa and either loops back into the lamina densa or inserts into the anchoring plaques.90,91 The anchoring plaques are electron-dense structures that contain collagen IV and laminin 332, and perhaps other basement membrane components, but which are believed to be independent of the lamina densa itself.90 They are distributed randomly in the papillary dermis below the lamina densa and are interrelated by additional anchoring fibrils. The anchoring fibril network forms a scaffold that entraps large numbers of dermal fibrils and, most probably, binds them through covalent cross-links between collagen VII and collagen I,129 thus securing the lamina densa to the subjacent dermis.124 In the acquired form of epidermolysis bullosa, epidermolysis bullosa acquisita (see Chapter 60), and in bullous systemic lupus erythematosus (see Chapter 155), autoantibodies target mainly the NC-1 domain of collagen VII.130

Mutations in COL7A1, the gene encoding collagen VII, result in dystrophic epidermolysis bullosa (see Chapter 62). Almost 500 COL7A1 mutations have been found in both recessive and dominant forms of dystrophic epidermolysis bullosa (Fig. 53-12), and the spectrum of biologic and clinical phenotypes is very broad.131 In mouse models, complete or partial deficiency of collagen VII recapitulated the clinical and morphologic characteristics of recessive dystrophic epidermolysis bullosa in humans.132,133 These mice have been useful for testing of molecular therapy strategies for dystrophic epidermolysis bullosa. Both cell therapy approaches using fibroblasts134 or bone marrow-derived mesenchymal stem cells135 and protein136 therapy have shown promise in terms of increasing collagen VII and stabilizing the dermal–epidermal junction. A human clinical trial to treat recessive dystrophic epidermolysis bullosa using bone marrow transplantation is currently ongoing.137

Cellular Origin of the Dermal–Epidermal Basement Membrane

The basement membrane constituents are products of both epithelial and mesenchymal cells. In vitro modeling of basement membrane formation using different skin equivalent culture models has demonstrated that a tight interplay between fibroblasts and keratinocytes contributes to the dermal–epidermal basement membrane. Differentiated fibroblasts adjacent to epithelia in vivo produce basement membrane components and assist in basement membrane assembly.138–141 Of the known basement membrane components, only laminins 332 and 311A are exclusively produced by the epidermis. Conditional knockout of collagen VII has demonstrated that both epithelial and mesenchymal cells manufacture collagen VII,142 whereas mainly mesenchymal cells synthesize collagen IV, nidogen, perlecan, and the laminin α2 chain.93 Because the mesenchymal products are translocated to the baso-lateral epithelial surface where they condense, that surface must provide the localization cues. Integrins α6β4 and α3β1 and collagen XVII have been implicated in this process, suggesting that the laminins coordinate basement membrane polymerization.99 An interesting regulatory step may be added by dermal enzymes (e.g., bone morphologic protein 1), which process epithelial cell products, such as laminin 332 and procollagen VII, to mature basement membrane molecules.99,127