Dissecting the formation, structure and barrier function of the stratum corneum (original) (raw)

Abstract

The skin is the largest organ of the mammalian body. The outermost layer of mammalian skin, the stratum corneum (SC) of the epidermis, consists of piles of dead corneocytes that are the end-products of terminal differentiation of epidermal keratinocytes. The SC performs a crucial barrier function of epidermis. Langerhans cells, when activated, extend their dendrites through tight junctions just beneath the SC to capture external antigens. Recently, knowledge of the biology of corneocytes (‘corneobiology’) has progressed rapidly and many key factors that modulate its barrier function have been identified and characterized. In this review article on the SC, we summarize its evolution, formation, structure and function. Cornification is an important step of SC formation at the conversion of living epithelial cells to dead corneocytes, and consists of three major steps: formation of the intracellular keratin network, cornified envelopes and intercellular lipids. After cornification, the SC undergoes chemical reactions to form the mature SC with different functional layers. Finally, the SC is shed off at the surface (‘desquamation’), mediated by a cascade of several proteases. This review will be helpful to understand our expanding knowledge of the biology of the SC, where immunity meets external antigens.

Introduction

Our body organs are covered with various epithelial cell sheets. These sheets mainly consist of two kinds of tissues: simple (mono-layered) and stratified (multi-layered) epithelia. Simple epithelium covers internal organs, such as lungs, stomach, intestine, colon, liver, kidney and so on. These organs mediate absorption and secretion. On the other hand, stratified squamous epithelium is formed in the tissues that undertake physical protection, such as skin, esophagus, vagina and so on.

Owing to these two kinds of epithelia, all the organs are compartmentalized and perform their own characteristic physiological functions. Epidermis, a major type of stratified squamous epithelium, is found in the body surface of ‘terrestrial vertebrates’. Because epidermis is located at the outermost side of the body, it is always threatened by the harmful outside environment. Evolution thus gave epidermis several strong protective functions that form the ‘epidermal barrier’, which includes both cell- and molecular-based and molecular barrier functions. Among various skin barrier functions, the stratum corneum (SC) provides one of the key factors to regulate cutaneous sensitization.

Understanding of the epidermal barrier is important for understanding self-defense mechanisms of terrestrial vertebrates because its function is closely linked to a part of the skin-associated innate and adaptive immune system (1, 2). Furthermore, accumulating evidence indicates that enhanced cutaneous sensitization to external antigens is one of the major causes of many allergic disorders, including atopic dermatitis (AD), asthma, food allergy and anaphylaxis (3).

In this review, to allow a better understanding of the cutaneous immune system, especially cutaneous sensitization, we rather focus on the field of corneobiology (the biology of the SC) and summarize recent progress in understanding the barrier mechanism of the SC. Reviews on immunological aspects of skin barrier dysfunction in AD or other allergic diseases are found elsewhere (4–6).

The evolutionary origin of the air–liquid interface barrier: the SC

The first terrestrial vertebrates, known as Icthyostega, appeared 360 million years ago, during the late Devonian period. Icthyostega are classified as amphibians. Although there is less information about the surface skin of Icthyostega from fossils, the surface of many present-day amphibians is covered with mucus-rich epidermis with SC (7, 8). The unique feature of amphibian epidermal development is metamorphosis. In response to the thyroid hormone, amphibian larvae remodel many organs for adaptation to terrestrial life. During embryonic stages, amphibian tadpoles are covered with larval epidermis consisting of apical, skein and basal cells, which does not resemble adult epidermis. During metamorphosis, basal cells differentiate into epidermal stem cells and form keratinized stratified squamous epithelium (9, 10).

Thus, it is likely that at the moment of adaptation to the air, these creatures evolved their surface epithelia into a multilayered epithelium that forms a dead but functional air–liquid interface barrier called the SC. This tissue covering the surface of the adult amphibian body might be the origin of the skin of terrestrial vertebrates (7, 8).

The first reptiles appeared in the Carboniferous period (340 million years ago). Then, adaptive radiation formed the major reptilian groups at the end of Palaeozoic era. In the Mesozoic era, they become dominant terrestrial vertebrates. By the end of the Mesozoic era, most of them became extinct, resulting in three groups—Crocodylians, Chelonians and Lepidosaurians. The reptiles were the first terrestrial vertebrates able to survive away from an aqueous environment, for example reproduction became independent of water due to the appearance of an ‘amniotic’ egg and skin evolved to be a dehydration-resistant.

The epidermis of present-day lizard scales also has a similar epidermal structure consisting of the stratum germinativum, which is the same as the stratum basale (SB; see later), an intermediate zone and the SC; note that they have two main types of SC—a softer region (α-layer) and a stiff region (β-layer). The β-layer gives a mechanical strength to the scale epidermis, whereas the α-layer allows stretching and the barrier function. Reptile SC generally possesses a highly impermeable barrier based on the stiff epidermis. The first avians are thought to appeared around 145 million years ago (11). Present avian epidermis is more simple but is similar to that of reptiles, being classified into two layers—the SC and the stratum germinativum. The avian stratum germinativum is further classified into the stratum transitivum, stratum intermedium and SB.

After the Permian-Triassic mass extinction (250 million years ago), the Late Triassic period (about 220 million years ago) featured the origin and radiation of mammals from non-mammalian synapsids (12). Mammals share many characteristics—the presence of mammalian glands, fur, the single bone in the lower jaw, the neocortex of the forebrain, the placenta, diaphragm, secondary palate, mammary glands and auditory ossicles (13). Does our skin have a mammal-specific character? Even though mammals became evolutionally distant from amphibians, during the embryonic development of mammals, the dynamic surface changes are somewhat reminiscent of the ancient evolutionary history of adaptation to life on the land. Similarly to amphibian tadpoles changing their surface into stratified squamous epithelium during metamorphosis, mouse embryonic ectoderm (simple epithelial cells that cover the surface of the embryo) begins to express skin-specific genes and stratify at embryonic day 15.5 (E15.5) to form epidermis. Just before birth (at E18.5), an almost complete form of epidermis is formed with a functional SC. Thus, the formation of the mammalian SC in embryonic development is an ‘intrinsic’ and programed phenomenon.

The mammalian epidermis has highly specialized characteristics that amphibians, reptiles and avians do not have (7, 8). In particular, the SC becomes highly moistened, resulting in the acquisition of a soft epidermis. In concomminant to the soft epidermis, the frequency of innervation by peripheral neurons becomes very high, which enables mammals to be sensitive to the outside environment (14). Therefore, most of the present mammalian epidermis is thought to have both an ancestral barrier system [the SC barrier, the tight-junction (TJ) barrier and the immune barrier; see later] and a soft and moistened SC, which is a mammal-specific trait.

The innate immune system has been known as a semi-specific defense system and the fundamental self-defense mechanism developed in eukaryotes, fungi, plants, invertebrates and vertebrates (2). Recently, it was also recognized to have specific elements, for example mediated via various pattern recognition receptors, which are thought to have evolved before the acquisition of adaptive evolution (15). Mammalian epidermis has several additional elements of the innate immune system: secretion of cytokines (IL-1, IL-6, TNF-α, etc.), desquamation (shedding off of the SC), the weakly acidic pH condition in the SC, and the presence of commensal bacteria and of anti-microbial peptides (defensins, cathelicidins, etc.)

The adaptive immune system is thought to have developed in addition to the innate immune system in ancestral primitive vertebrates (fishes) by acquiring the major histocompatibility complex, T-cell receptors and immunoglobulin superfamily proteins (1, 16–18). Especially, immunoglobulins have differentially changed their repertoire during the evolution of vertebrates, such as in fishes [which have the isotypes IgM, IgT (also called IgZ) and IgD], amphibians (IgM, IgX, IgY, IgD and IgF), reptiles (IgM, IgY, IgA and IgD), birds (IgM, IgY and IgA) and mammals [IgM, IgG, IgA, IgD and IgE (16, 19)]. Cells similar to mammalian Langerhans cells (LCs) are also observed in amphibians, reptiles and birds, suggesting that cutaneous sensitization via LCs was firstly acquired in ancestral amphibians and this coincided with the acquisition of the SC and epidermis (2).

Compared with reptiles’ epidermis, the mammalian epidermis is composed of a moisturized and soft SC. Thus, our skin is susceptible to infections and/or allergy with a complicated crosstalk between innate and adaptive immune systems (20, 21). In the next section, we describe how the fragile but functional mammalian SC is formed and how it can be destroyed via loss of SC-related genes in humans and mice, resulting in barrier defects and occasionally inflammation.

Epidermal differentiation, keratinization and barrier function

Epidermal differentiation and keratinization

The epidermis—keratinized stratified squamous epithelium—covers the body surface of terrestrial vertebrates and serves to protect from entry of pathogens, allergens or toxic substances and to prevent water loss. The mammalian epidermis mainly consists of four cell layers: the SB, the stratum spinosum (SS), the stratum granulosum (SG) and the SC (Fig. 1). Keratinocytes proliferate in the SB and they differentiate and migrate upward into the SS (21, 22). In each layer, keratinocytes express different sets of keratin intermediate filaments. Keratins are elastic fibrous proteins differentially expressed during keratinocyte differentiation. Keratins form heterodimers between acidic (type I) and basic (type II) proteins. Once assembled, they form a three-dimensional cytoskeleton located in the cytoplasm and around the nucleus. These keratin filaments are anchored to desmosomes, which are junctional complexes utilized for cell–cell adhesion.

Fig. 1.

Structure of mammalian epidermis and its three barrier elements. Mammalian epidermis is composed of three barrier elements: the ‘SC’: air–liquid interface barrier; the ‘TJ’: liquid–liquid interface barrier; and the ‘LC network’ which is the immunological barrier. Activated LCs extend their dendrites above the TJ strands to capture external antigens.

At the SB of the epidermis, keratinocytes express keratin 5 (basic, type II) and keratin 14 (acidic, type I). During differentiation from the SB into the SS, keratinocytes dramatically switch the expression of keratins into keratin 1 (type II, basic) and keratin 10 (type I, acidic). After the several layers of SS, numerous keratohyaline granules (KHGs), which are an amorphous protein complex of keratin and keratin-binding proteins [such as filaggrin or loricrin] are formed in the SG. This SG consists of three cell layers, designated as SG1, SG2 and SG3, from the apical to the basal side (3). At the uppermost layer, SG1 cells undergo cell death to form the dead cell layer—the SC. This layer serves as the air–liquid interface barrier.

The TJ barrier system of mammalian epidermis

In addition to the air–liquid interface barrier in the SC, the TJ strand is also important as a ‘liquid–liquid’ interface barrier’ in the epidermis. TJs are commonly observed on the apical side of simple epithelial cell sheets in vertebrates. They are responsible for sealing epithelial cells to form a functional barrier of the paracellular pathway (which otherwise allows passage between cells) and compartmentalize each organ for its physiological functions. It was experimentally demonstrated that the SG2 cell layer is sealed together by a functional TJ in epidermis (3, 23, 24). From these morphological data, SG1 cells are located above the TJ and below the SC barrier (Fig. 1).

The immune barrier system of mammalian epidermis

Among the several subsets of dendritic cells (DCs), epidermis-specific DCs are called LCs and are thought to be involved in induction of antigen-specific Th2 responses as well as maintenance of peripheral tolerance (25, 26). No other immune cells are resident in the epidermis in the normal state. Recently, the whole-mount staining method [Fig. 1; (27)] has shown that, in the resting state, the tips of LC dendrites are aimed at the apical side below the TJ of the SG2 cell layer. Once LCs are activated, they extend their dendrites through the TJ just beneath the SC [Fig. 1; (28)]. When LCs extend their dendrites, they do not break the TJ, but form a different TJ between the extended dendrites and adjacent keratinocytes to maintain the TJ barrier (28). It was also observed that external antigens are taken up by the tips of the extended dendrites of LCs.

These three elements of the skin barrier (the SC as an air–liquid interface barrier, the TJ as a liquid–liquid interface barrier and LCs as a frontline player of the immune barrier) beautifully coordinate and protect living bodies as the first line of defense. And these three barrier elements are found in probably most terrestrial animals, whose life is constantly threatened by the outside environment in the air. When we think of cutaneous sensitization, external antigens need to penetrate SC layers to be taken up by the extended dendrites of LCs. Therefore, the function and dysfunction of the SC determines the nature and quality of cutaneous sensitization. Now, we will discuss how much we know about the mammalian SC.

‘Cornification’: formation of the SC by a unique cell death mechanism

The SC is generated from cell-death products of the SG1 layer, and consists of 10–20 piled-up layers of dead cells, 10–30 μm in diameter and 1 μm thick (29, 30). It is composed of keratin intermediate filaments, water-soluble lipid, proteins (enzymes) and the humectants [also called ‘natural moisturizing factors’ (NMF)]. The SC is often described as resembling ‘bricks and mortar’ in which corneocytes are the bricks and intercellular lipid lamellae are the mortar (31, 32).

At the final layer of the SG, SG1 cells undergo a cell death program that is not a classical ‘apoptosis’ but is classified as ‘cornification’ (33, 34). Cornification is similar to the cell death of lens epithelial cells and red blood cells in the respect that typical apoptosis-related proteins (e.g. caspase-3) are not activated. However, SG1 cells do not possess a stress-induced cell death program, whereas lens epithelial cells and red blood cells still retain the ability to undergo cell death by stresses, such as ultraviolet light (33, 34).

Several mouse and human genes have been reported to be involved in this formation of the SC in both humans and mice (Table 1). Most of the knockout mice show an epidermal barrier defect and some of them induced inflammation. In the next section, we will describe how the dead bricks-and-mortar layer is formed above the living layers, which is responsible for epidermal barrier function. The major key events of cornification consist of three events, as detailed below: formation of the intracellular keratin network; formation of cornified envelopes (CE) (crosslinking of lipids and proteins) and formation of intercellular lipids [secretion from lamellar body (LB) contents] (Fig. 2).

Table 1.

Examples of genes involved in SC formation

Table 1.

Examples of genes involved in SC formation

Fig. 2.

Major events of SC formation. The formation of the SC (cornification) is composed of three major events: (i) the formation of the intracellular keratin network, (ii) the formation of the CE (crosslinking of lipids and proteins) and (iii) the formation of intercellular lipids (secretion from lamellar body contents). Finally, the surface of the SC is shed off by degradation of corneodesmosomes via the activity of several proteases. CER, ceramide; CHOL, cholesterol; Evpl, envoplakin; FFA, free fatty acid; Lor, loricrin; Ppl, periplakin; SG, stratum granulosum; SPRR, small proline-rich protein family; SS, stratum spinosum; TGase, transglutaminase.

Formation of the intracellular keratin network

Prior to cornification, many KHGs are gradually formed inside the cytoplasm of SG3 cells. The KHGs consist of amorphous, electron-dense materials. KHGs are thought to consist of a complex of keratin (mainly from keratin 1 and 10) and a keratin-binding protein, such as profilaggrin (F-granules) or loricrin [L-granules; only found in rodents (35–37); Table 1].

Profilaggrin is a mammal-specific, insoluble, highly phosphorylated protein [>400kDa in humans (38, 39)]. Profilaggrin consists of an amino-terminal Ca2+-binding protein of the S-100 family, tandemly linked to 10–12 filaggrin monomers and a carboxy-terminal domain (38, 40, 41) (Fig. 3). Filaggrin is the major keratin-binding protein in the mammalian SC. Loss-of-function mutations of filaggrin have been reported as a major predisposing factor for AD, possibly because of enhanced penetration of external antigens through the SC in that disease (43–47) (Table 1).

Fig. 3.

Summary of functional SC zones. The SC layer is reported to be divided into two or three zones. Isolated cornified cells are classified into two morphologies; stratum compactum (CEf) and stratum disjunctum [CEr] (42). The SC is divided into three zones according to the profilaggrin processing pathway: in the lower SC, monomer filaggrin bundles keratin filaments; in the middle SC, keratin-bound filaggrins are citrullinated (Cit) and released from keratin filaments; in the upper SC, filaggrin is degraded into amino acids to produce most of the NMFs (43). Cryo-electron microscopic observation of swelled SC revealed three distinct zones (37, 44). TOF-SIMS analysis revealed three zones: sponge-like upper SC (Khigh, Argininehigh), middle SC (Klow, Argininehigh) and lower SC [Klow, Argininelow (45)]. SASPase, skin aspartic protease; SC, stratum corneum; SG, stratum granulosum.

During cornification, SG1 cells enucleate, lose organelles and change their shapes into a two-dimensional flat polygonal structure. During this transition of SG1 to SC, KHGs also gradually disappear. Although the mechanism of disappearance of KHGs is still unknown, dephosphorylation of profilaggrin is thought to be the initial step of dissociation of the keratin–profilaggrin complex in KHGs (Fig. 2). Dephosphorylated profilaggrins are cleaved to generate filaggrin monomers during the SG1-to-SC transition (47). At this step, several proteases are thought to be involved in the processing of profilaggrin. SASPase/Taps/ASPRV1 (skin aspartic protease/TPA-inducible aspartic proteinase-like gene/aspartic protease, retroviral-like 1) is exclusively expressed in SG1 cells and is a candidate protease of profilaggrin linker-cleavage (48) (Table 1).

Other proteases, such as endopeptidase-1 (PEP-1), μ-calpain, matriptase, prostasin and KLK5 are also reported to cleave profilaggrin-linker peptides (47, 49–54). The filaggrin monomers that are produced (37kDa in human) are thought to strongly bind and bundle keratin filaments in the lower SC (55).

On the basis of cryo-electron microscopic analysis of human vitreous skin sections, it has been proposed that keratin filaments are arranged to form the template for the membrane in the SC, with cube-like rods packed in the structure [Fig. 2; (56)]. This unique three-dimensional structure is considered to be important for the hydration of the SC and gives rigidity to each SC layer. Double-knockout of keratin-1 and keratin-10 in mice causes neonatal lethality due to fragility in the epidermal structure (35). Nevertheless, those mice still had KHGs and a normal profilaggrin-to-filaggrin processing pathway, suggesting that keratin-1 and keratin-10 are not directly involved in KHG formation itself but rather involved in formation of the three-dimensional network of keratin filaments with the aid of filaggrin released from KHGs.

These keratin filament networks are thought to be associated with desmosomes of the SC, and are called ‘corneodesmosomes’; they are slightly modified forms of the desmosomes in living layers. Corneodesmosomes consist of adhesion molecules, such as desmoglein 1 (DSG1) and desmocollin 1 (DSC1), and cytoplasmic anchoring proteins, such as plakoglobin, plakophilin and desmoplakin, which are probably also associated with keratin filaments. Corneodesmosin (CDSN) is secreted from the LBs and localized in the intracellular space of the SC and becomes a component of the corneodesmosome to help adhesion functions (57–59) (Fig. 2; Table 1).

Abnormalities of corneodesmosomal proteins are known to link to several inflammatory diseases in humans and mice. It has been recently reported that a specific form of DSG1 deficiency in humans results in severe dermatitis, multiple allergies and metabolic wasting (SAM syndrome) with increased serum IgE (60). CDSN-deficient mice showed abnormalities in the SC and epidermal barrier function (58, 59). Furthermore, loss-of-function mutation of CDSN in humans causes peeling skin syndrome, ichthyosiform erythroderma (61). In this disease, detachment of the SC layer from epidermis causes chronic dermatitis, asthma, allergic rhinitis, elevation of serum IgE and food allergy (61). This evidence suggests the importance of the corneodesmosome in the maintenance of the epidermal barrier and a link to percutaneous immunization. However, we still don’t know how these diseases cause SC barrier disruption and lead to an immune barrier abnormality because of the lack of knowledge of the SC barrier itself.

Formation of the CE: crosslinking of lipids and proteins

During the SG1-to-SC transition, an intracellular Ca2+ increase induces terminal differentiation. The major target of Ca2+ is transglutaminase (TGase). TGase is a Ca2+-activated enzyme that crosslinks with ε-(γ-glutamyl)lysine isopeptide bonds. Increased activity of intracellular membrane-bound TGase I and cytoplasmic TGase III cross-links protein products of the epidermal differentiation complex (EDC) genes, involucrin, loricrin, envoplakin, periplakin and the small proline-rich protein family (SPRRs), etc. underneath the plasma membrane (62–64) (Table 1).

Most TGase-crosslinking proteins are products of genes located in the locus of human chromosome 1q21 called the EDC. This is a large gene cluster located on chromosome 3 in mice (43, 65, 66). The EDC genes of mammals include the S100A family, loricrin, involucrin, SPRRs late cornified envelope (LCE) protein family and the S100-fused type proteins [e.g. filaggrin]. The antimicrobial peptidoglycan-recognition proteins (PGLYRPs) 3 and 4 are also reported to be localized in the EDC (67, 68).

Recently, the EDC was reported in non-mammalian vertebrates, such as chickens and green anole lizards (69, 70). Interestingly, chickens and lizards also have similar EDC-homolog genes to mammals that are expressed in an epidermis-specific manner. Comprehensive analysis of EDC genes in chickens and lizards led to the proposal that epidermal barrier proteins were derived from fusion of ancient S100A and PGLYRP genes and also the loss of exons and multiple rounds of fusions, duplications, loss of exons/introns and amino acid substitutions. It is hypothesized that the predicted origin of loricrin derives from a common ancestor among amniotes (70).

Many EDC-genes (the LCE family, SPRR family, S100 family, etc.) show highly homologous sequence similarity, suggesting that the SC is formed by a ‘redundant’ mechanism. Even for single gene products, each EDC-protein shows functional redundancy: mice with single knockouts for EDC-derived involucrin/envoplakin/periplakin did not show any evident aberrant phenotype in the epidermis (71, 72). Triple knockout of these three genes resulted in defective epidermal barrier function assessed by transepidermal water loss measurement, suggesting that the cross-linking of these EDC gene products is essential for corneocyte permeability barrier function (72).

Recently, the redundant mechanism of SC formation has been shown to be dependent on the effect of the amniotic fluid from the uterus. Loricrin-deficient mice have previously shown only a transient abnormality in the neonatal period, even though loricrin makes up 70% of SC protein (73) (Table 1). In these mice, epidermal barrier acquisition is delayed by 24h at E16.5 and increased expression of Sprr2d and Sprr2h mRNAs was observed; resulting in cornified cell envelopes that are composed of the Sprr2 family instead of loricrin (73, 74). Between E14.5 and E16.5 of mouse embryonic development, the composition of the amniotic fluid changes (75), which coincides with the formation of the epidermal barrier, possibly for the protection of the embryo from the effect from E16.5-amnionic fluid. Genetic blocking of antioxidant responses in the early embryo by the Nrf2/Keap1 (NF-E2-related factor 2/Kelch-like ECH-associated protein 1) pathway resulted in the inhibition of compensatory phenotypes of loricrin-deficient mice (75) (Table 1). Those mice showed apparent defects in epidermal barrier function. This mechanism may be a remnant of the preadaptation mechanism of epidermal barrier evolution.

Unlike the conservation of loricrin across the avians and reptiles, filaggrin is a newly acquired EDC gene in the mammalian taxa, suggesting that filaggrin is derived from the EDC of reptiles or mammal-like reptiles (70). Considering that filaggrin is a skin-specific protein and is involved in the formation of the proper keratin network in the SC, it was suggested to be acquired for a mammal-specific SC function, such as moisturization or NMF production. The link of filaggrin-dependent characteristics of the SC in mammals and SC barrier disruption in human AD patients is still a mystery in this field. Other EDC genes that cause human diseases are listed in Table 1.

Formation of intercellular lipids: secretion from LB contents

LBs are organelles derived from the Golgi apparatus and contain phospholipids, glucosylceramides, sphingomyelin and cholesterol; they begin to form in the SS layers (63, 76). During the SG1-to-SC transition, at the apical surface of SG1 cells, LBs secrete their contents into the extracellular space between the SG1 and the lower SC, which includes various kinds of proteases and protease inhibitors as well as lipids like glycosylceramide; these components are involved in the barrier formation by the SC. The ω-hydroxyceramide is also included in LBs and, once secreted from SG1 cells, it is cross-linked to the plasma membrane as a 5-nm monolayer sheet and covers the surface of corneocytes (77). Using this cross-linked lipid as a template, the extracellular space of corneocytes is filled with periodic sheets of lipid lamellae, which serve as an impermeable barrier in the SC.

This process is thought to be performed by 12R-lipoxygenase. Deficiency of the 12R-lipoxygenase gene in mice results in an epidermal barrier defect and a decreased amount of ω-hydroxyceramide-bound protein (78, 79) (Table 1). Mutation of the 12R-lipoxygenase in humans causes non-bullous congenital ichthyosiform erythroderma (NCIE) [Table 1; (80)]. LB-secreted glucosylceramides and sphingomyelins are converted into ceramides by β-glucocerebrosidase and sphingomyelinase, respectively. β-glucocerebrosidase-deficient mice showed decreased ceramides in the SC and showed epidermal barrier defects (81, 82) and deficiency is associated with Type 2 Gaucher disease (83, 84). The ATP-binding cassette subfamily A member 12 (ABCA12) is associated with Harlequin ichthyosis and ABCA12-deficient mice showed neonatal lethality due to epidermal barrier function with hyperkeratosis and accumulation of lipid droplets, suggesting a defect in incorporation of glucosylceramide into LBs (85, 86) (Table 1).

Lamellar lipids are composed of the same molar ratio of ceramides, free fatty acids and cholesterol (87). Recent cryo-electron microscopic analysis has revealed that ceramides are stacked as bilayers of fully extended ceramide side-chains and cholesterol molecules are associated with the ceramide sphingoid moiety (88). This report suggested that the unique arrangement of lipid lamellae in the structure is important for the skin barrier and robustness of hydration as well as responding to environmental and mechanical changes. Several mice with knockouts related to lipid metabolism of the SC were reported to have disrupted epidermal barrier function [reviewed in ref. (76)] (Table 1).

Recently, another EDC gene, transmembrane protein 79/mattrin (Tmem79/Matt), has been reported to be involved in secretion of LB contents and identified as a gene responsible for causing spontaneous dermatitis in mice (89, 90) (Table 1). Analysis of a human single nucleotide polymorphism of the Tmem79 gene revealed a low but significant association with AD (90). Tmem79 is a five-transmembrane protein localized in LBs of the SG1 layer of epidermis and Tmem79-deficient mice showed decreased secretion of the contents of lamellar granules, resulting in aberrant SC formation, suggesting a novel pathway of spontaneous inflammation (91).

Maturation of the SC: the zone hypothesis

After cornification, dead SC layers are piled up and change their properties via various chemical reactions, the components of which were already present in SG1 cells. Thus, the SC zone is not a simple accumulation of homogeneous dead cornified cells. Various chemical reactions occur during the upward (lower-to-upper) migration and maturation of the SC. Several lines of evidence indicated that there are apparent functionally distinct SC zones.

Morphological analysis of each layer of the SC showed that, in the lower SC (often called the ‘stratum compactum’), fragile corneocytes (CEf) are rather small, less hydrophobic and fragile (CEf) than the cells above. In the upper SC, (the ‘stratum disjunctum’), the SC cells become large, hydrophobic and rigid (rigid corneocytes; CEr) (42). This classification suggested that at least two kinds of morphologically different corneocytes are formed.

From the steps of profilaggrin processing, the SC can be roughly divided into three zones. In the lower SC, filaggrin monomers are cleaved out from profilaggrin at the SG-to-SC transitional zone and monomeric filaggrin bundles with keratin filaments. Probably in the middle SC zone, keratin-bound filaggrins are citrullinated by peptidylimidases (91–95). This modification may affect the conformation of filaggrin released from keratin filaments. Released filaggrins are attacked by several proteases, such as caspase-14, elastase and further degraded into amino acids by breomycin hydrolases (Sandilands; 95, 96). These sequential reactions occurring in each SC layer were confirmed by data from immunoelectron-microscopic analysis with an anti-filaggrin antibody (97) (Fig. 3).

By using cryo-fixation and scanning electron microscopic (cryo-SEM) technology against human epidermis, three hydration zones (lower, middle and upper SC) were identified based on their swelling potential after immersion in 5–20% salt solutions (98). After swelling, the center zone remains unchanged, whereas the upper SC swelled massively and the lower SC swelled twice in size. It was proposed that the middle SC zone serves as the major permeability SC barrier. On the contrary, by using isolated human SC, it was demonstrated that middle SC zone swells the most, suggesting that different sample preparation affects the swelling capacity of SC hydration zones (99).

The three-hydration-zone hypothesis was also confirmed by recent observations from time-of-flight secondary ion mass spectrometry (TOF-SIMS) against the SC of mouse tail epidermis and revealed that the SC has three functionally distinct layers with different properties (100). Firstly, the lower SC has a high Na+, low arginine, low K+ concentration and an impermeable barrier against Cr (VI) ions. Secondly, the middle SC layer has a high arginine, low K+ concentration and an impermeable barrier against Cr (III) ions. A high arginine concentration suggests that the zone of filaggrin degradation results in the production of free amino acids, which make up almost half of the NMFs. NMFs are hygroscopic substances and serve as natural humectants in the SC (42, 101). Thirdly, the upper SC layer has a sponge-like layer (Khigh, Argininehigh), where external solutes easily flow in and out. In the case of mice, a high K+ ion concentration was observed due to smears of urine. In the upper SC, a high concentration of Na+ ions is also observed within corneocytes but could be washed out, suggesting the presence of a pathway of trans-corneocyte infiltration.

Interestingly, TOF-SIMS analysis of the epidermis of filaggrin-deficient mice demonstrated loss of the arginine-high layer and increased Cr (III) penetration in the impermeable barrier present in the lower SC. In support of this observation, calcein liposomes easily penetrate into the SC in filaggrin-knockout epidermis (44). These results suggested that filaggrin deficiency causes a certain abnormality in the formation of the three SC zones, which may affect the SC barrier and cutaneous sensitization. Detailed classification of SC zones and the functional abnormalities of knockout mouse models or human diseases would be the logical next research field of corneobiology and cutaneous immune responses.

Desquamation of SC: the end of the SC barrier

At the final uppermost layer, each corneocyte must shed off, a process called ‘desquamation’. This continuous shedding-off process is part of the physical innate immune system in the epidermis, which is useful to remove harmful microorganisms or infectious viruses. Various proteases are involved in this process. The weakly acidic condition of the upper SC is the important factor in maintaining the protease activity. Among the proteases, the KLK-related peptidase family (KLK5/KLK7) is known as a regulator of desquamation (102). These proteases are tryptic or chymotryptic serine proteases having a neutral optimum pH and are secreted from LBs of SG1 cells into the intercellular space between the SG1 and SC. However, lymphoepithelial Kazal type–related inhibitor [LEKTI; encoded by the serine peptidase inhibitor, Kazal type 5 (SPINK5) gene] is also secreted from LBs of the SG1 and inhibits KLK5 and KLK7 (Table 1). In the upper SC, a decreased pH towards weak acidity induces the release of KLK5 and KLK7 from LEKTI. Even in the weakly acidic pH, the activity of KLK5/KLK 7 is thought to be enough to cleave the extracellular domain of DSG1, DSC1, CDSN, etc. and finally the uppermost corneocytes are shed off.

Several studies have demonstrated genetic linkage between the SPINK5 gene and dermatitis (Table 1). Polymorphism of SPINK5 is related to AD, asthma and an increased level of serum IgE (103–105). Netherton syndrome, caused by a loss-of-function mutation of SPINK5, is a severe autosomal recessive ichthyosis with chronic dermatitis, asthma and allergic rhinitis (106). Electron microscopic analysis of patient skin revealed detachment of the SC from the SG accompanied by the degradation of corneodesmosomes (107–109). Consistent with these reports, SPINK5-knockout mice showed an elevated activity of KLK5 activity in the SC and increased degradation of CDSN, resulting in defective epidermal barrier function (110–112). This evidence suggested that the desquamation mechanism itself has some link to cutaneous sensitization.

Conclusion

Considering that most terrestrial animals have an LC network, a TJ barrier and an SC barrier in their epidermis, how they evolved and what is acquired in the mammalian epidermis may be the key to understand the complex pathophysiology of cutaneous sensitization, the upstream event of the many allergic disorders. The link of filaggrin mutation in human AD patients and the mammal-specific acquisition of the filaggrin gene still has some missing pieces. A combination of SC-zone analysis by recent advanced microscopic techniques and genetically engineered mice, together with the characterization of human disorders with genetic causes, would further advance the field of corneobiology.

Funding

This work was supported by Grants-in-Aid for Scientific Research funding from the Ministry of Education, Culture, Sports, Science and Technology of Japan, by Health Labour Sciences Research Grants for Research on Allergic Diseases and Immunology from the Ministry of Health, Labour, and Welfare of Japan and by the Takeda Science Foundation.

Conflict of interest statement: The authors declared no conflict of interests.

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Author notes

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