The stratum corneum: the rampart of the mammalian body
Background – The stratum corneum (SC) is the outermost region of the epidermis and plays key roles in cutaneous barrier function in mammals. The SC is composed of ‘bricks’, represented by flattened, protein-enriched corneocytes, and ‘mortar’, represented by intercellular lipid-enriched layers. As a result of this ‘bricks and mortar’ structure, the SC can be considered as a ‘rampart’ that encloses water and solutes essential for physiological homeostasis and that protects mammals from physical, chemical and biological assaults.
Structures and functions – The corneocyte cytoskeleton contains tight bundles of keratin intermediate filaments aggregated with filaggrin monomers, which are subsequently degraded into natural moisturizing compounds by various proteases, including caspase 14. A cornified cell envelope is formed on the inner surface of the corneocyte plasma membrane by transglutaminase-catalysed cross-linking of involucrin and loricrin. Ceramides form a lipid envelope by covalently binding to the cornified cell envelope, and extracellular lamellar lipids play an important role in permeability barrier function. Corneodesmosomes are the main adhesive structures in the SC and are degraded by certain serine proteases, such as kallikreins, during desquamation.
Clinical relevance – The roles of the different SC components, including the structural proteins in corneocytes, extracellular lipids and some proteins associated with lipid metabolism, have been investigated in genetically engineered mice and in naturally occurring hereditary skin diseases, such as ichthyosis, ichthyosis syndrome and atopic dermatitis in humans, cattle and dogs.
The skin, which covers the entire body surface in mammals, is an anatomical and physiological barrier between the environment and the organism. The stratum corneum (SC) provides a barrier function for the skin. It is the outermost region of the epidermis and is composed of ‘bricks’ (i.e. flattened, protein-enriched corneocytes) and ‘mortar’ (i.e. intercellular lipid-enriched layers).1 As a result of this ‘bricks and mortar’ structure, the SC in the mammalian skin can be considered as a ‘rampart’, which encloses ‘citizens’ (i.e. water and solutes) essential for physiological homeostasis and protects the ‘castle’ (i.e. the host) from physical, chemical and biological ‘assaults’.
Within corneocytes are tight bundles of keratin intermediate filaments aggregated with filaggrin (FLG) monomers to provide a flattened shape and mechanical strength to the cells.2–5 The cornified cell envelope is formed on the inner surface of the corneocyte plasma membrane and provides structural and mechanical integrity to the cells.6,7 Covalent binding of the lipid envelope to the cornified cell envelope proteins provides a scaffold for extracellular lipid lamellae (ELL), which are crucial for maintaining permeability barrier function.8,9 Various enzymes and plasma membrane proteins associated with the metabolism, uptake and secretion of lipids are also crucial in organizing intact ELL. Moreover, corneodesmosomes mediate cell-cell adhesion between corneocytes.10–12 Degradation of corneodesmosome proteins in the uppermost SC is a key step in desquamation.10,11
The roles of the SC components in cutaneous barrier function have been studied in genetically engineered mice and in naturally occurring hereditary skin diseases in humans and animals. The aim of this review is to discuss recent progress in understanding the biological functions of the key SC components via the use of genetically engineered mouse models and to discuss the pathophysiology of spontaneous hereditary skin diseases related to genetic mutations or the altered expression of SC components in humans, cattle and dogs (summarized in Table 1).
Stratum corneum components crucial for barrier function
Intermediate filament proteins.
Keratins (encoded by KRT genes) are fibrous structural proteins synthesized by epithelial cells, including keratinocytes. Keratin monomers assemble into web-like bundles to form intermediate filaments, which are cytoskeletal components that terminate at desmosomes to form a cytoplasmic network. In the mammalian epidermis, type I (acidic) keratins and type II (neutral-basic) keratins form heterodimers via disulphide bonds. Specific keratins are found more prominently in various layers of the epidermis. For example, keratin 5 (K5) and keratin 14 (K14) form heterodimers in basal keratinocytes, whereas keratin 1 (K1) and keratin 10 (K10) form heterodimers in keratinocytes in suprabasal layers; keratin 2 (K2) is expressed in the stratum granulosum (SG).13
The functional importance of K1, K2 and K10 to cutaneous barrier function has been studied in mice by genetic engineering of keratin genes (Krt). Mice heterozygous for a mutation in the Krt1 gene or transgenic mice expressing a truncated human KRT1 gene exhibit suprabasalar blistering and skin erosions immediately after birth and develop marked scaling with increasing age.14,15 In addition, mice homozygous for a mutation in the Krt1 gene exhibit severe blistering and widespread desquamation at birth, and die due to severe dehydration.14,15 Likewise, mice heterozygous for mutations in the Krt10 gene or heterozygous transgenic mice generated by introduction of a Krt10 mutation develop hyperkeratosis with age.16–18 Mice homozygous for the gene knockout or transgene exhibit very fragile skin with severe suprabasalar blistering and erosions, and die shortly after birth.16–18 In addition, a point mutation in the Krt2 gene (T500P) in mice with dark skin (Dsk2) causes scaling on the tail, feet and ears owing to impaired intermediate filament assembly, in both homozygous and heterozygous mice, even though abnormalities in cutaneous barrier function in relation to the Krt2 mutation have not been documented in the literature.19
These findings imply that keratins are crucial for the structural integrity of epidermal keratinocytes, and targeted ablation of some Krt genes causes fragility of the epidermis and/or a scaly phenotype.
Filaggrin and related proteins.
Profilaggrin, the precursor protein of FLG, is the major constituent of keratohyalin granules in the SG.2,4,5 Profilaggrin (encoded by the FLG gene) is an insoluble, large, highly phosphorylated, histidine-rich protein, which contains tandemly arranged FLG repeats (10-12 repeats in humans) that are flanked on either side by two partial FLG repeats and N- and C-terminal domains.4,5 The N- and C-terminal domains in profilaggrin are thought to be important in processing of profilaggrin to FLG monomers during epidermal differentiation.4,20,21
In humans, each FLG repeat consists of 324 amino acids and shows significant amino acid homology.22–24 Meanwhile, two types of FLG repeats are distributed randomly in the mouse profilaggrin precursor protein.25–27 The precursor protein itself has no keratinocyte-binding activity.4 During the differentiation of keratinocytes, keratohyalin granules degranulate in response to increased Ca2+ levels, and profilaggrin is dephosphorylated and proteolysed into FLG monomers at the border between the SG and SC.2–5 Filaggrin monomers specifically aggregate the keratin intermediate filament cytoskeleton into tight bundles (Figure 1a), thereby collapsing the cells into a flattened shape.2–5 In the upper SC, FLG monomers undergo subsequent degradation into hygroscopic peptides (e.g. pyrrolidone carboxylic acid and urocanic acid), which are natural moisturizing factors (NMFs), by a variety of proteases, including caspase 14.4,28,29
The role of FLG in cutaneous barrier function has been studied by the use of two lines of mutant mice. Flaky tail mice, which have the two spontaneous genetic mutations ft and ma, have been reported to develop spontaneous atopic dermatitis (AD)-like disease.30 Recently, it was found that a 1 bp deletion (5303delA) in the murine Flg gene is associated with the ft genotype in flaky tail mice.27 Moreover, topical application of ovalbumin or Dermatophagoides pteronyssinus to ft/ft mice causes percutaneous allergen sensitization, with allergen-specific IgE production, increased transepidermal water loss (TEWL) and enhanced AD-like phenotyes.27,31 The association of the ma mutation with the AD-like dermatitis phenotype, however, has not yet been demonstrated. Recently generated filaggrin gene knockout mice (Flg−/−) exhibit dry skin and increased desquamation under mechanical stress.32 Furthermore, Flg−/− mice exhibit enhanced penetration of foreign materials into the SC, leading to hapten-induced contact hypersensitivity and allergen-specific humoral immune responses.32 In these mice, SC hydration and TEWL are normal, despite the fact that the NMF level is decreased in the SC of Flg−/− mice.32
Caspase 14, which belongs to a family of cysteine-dependent aspartate-directed proteases, is known to degrade profilaggrin and FLG monomers directly into NMF. Caspase-14-deficient mice show shiny and lichenified phenotypes, with decreased skin hydration and increased TEWL.28,29
In summary, FLG appears to be responsible for hampering a foreign invasion (e.g. penetration of allergens) through the SC rather than performing a water-holding function. In flaky tail mice and Flg−/− mice, data suggest that allergens passing through the SC may be captured by the dendrites of Langerhans cells that penetrate tight junctions located immediately beneath the SC, as previously demonstrated by three-dimensional visualization of mouse epidermis.33 However, the water-holding capacity of FLG-degraded NMF products is debatable and needs to be evaluated further.
Cornified cell envelope proteins.
The cornified cell envelope is a 15-nm-thick layer of proteins (in humans) located on the inner surface of the keratinocyte plasma membrane. The cornified cell envelope is formed by the assembly of several precursor proteins, including involucrin, loricrin, envoplakin, periplakin and small proline-rich proteins (Figure 1b). Transglutaminases in the epidermis are thought to be responsible for the assembly of the precursor proteins that form the cornified cell envelope.7,13,34,35 Among the three subtypes of transglutaminases, transglutaminase-1 is known to be a membrane-located transglutaminase in the epidermis.36,37 Transglutaminase-1 is synthesized in the epidermis as an inactivated precursor protein, which is later processed by cathepsin D for activation.38
Transglutaminase-1-deficient mice exhibit a defective SC that causes neonatal death owing to increased TEWL. Additionally, cathepsin D-deficient mice exhibit reduced transglutaminase-1 activity and reduced expression of the cornified cell envelope proteins.38 In contrast, loricrin-deficient mice exhibit transient congenital erythroderma, with a shiny, translucent skin at birth and increased susceptibility to mechanical stress.39 However, the neonatal mutant mice do not exhibit increased TEWL, and lose the skin phenotypes at 4- 5 days after birth.39 This phenotypic change may be associated with increased expression of other cornified cell envelope components. Targeted ablation of the murine involucrin gene alone does not cause any histopathological changes in the epidermis which shows an ultrastructurally normal cornified cell envelope.40 However, triple knockout of involucrin, envoplakin and periplakin in mice leads to the development of phenotypes characterized by postnatal hyperkeratosis, defects in the assembled cornified cell envelope and increased susceptibility to mechanical stress.41 Conversely, hyperkeratosis is not evident in the epidermis of involucrin, envoplakin or periplakin single knockout mice.41
Thus, evidence obtained through the use of genetically engineered mice suggests that the assembly of cornified cell envelope proteins is crucial for the normal desquamation process and the structural and mechanical integrity of corneocytes in the epidermis, even though targeted ablation of single protein genes did not cause lethal or lifelong cutaneous abnormalities.
Functional proteins in the stratum corneum
Corneodesmosomes are the main adhesive structure in the SC (Figure 2).10–12 On the cytoplasmic side of corneodesmosomes, desmosomal plaque proteins are incorporated into the cornified cell envelope and separated from tonofilaments attached to the intermediate filament cytoskeleton.6,42 The extracellular part of corneodesmosomes is comprised of desmosomal cadherins, such as desmoglein (Dsg)1 and desmocollin (Dsc)1, as well as corneodesmosin, a unique extracellular component of corneodesmosomes.11,43-46 During desquamation, the extracellular components of corneodesmosomes are degraded by kallikreins (KLKs) and cathepsins.47–49
The KLKs are a family of 15 trypsin- or chymotrypsin-like serine proteases (KLK1-KLK15).50–53 In human skin, at least eight KLKs, including KLK5 and KLK7, are expressed and secreted into the extracellular space between the SG and SC.54 Kallikrein 5 degrades all extracellular components of corneodesmosomes, while KLK7 degrades only Dsc1 and corneodesmosin but not Dsg1.42,53 The enzymatic activities of KLK5 and KLK7 are known to be inhibited by lymphoepithelial Kazal-type 6 serine protease inhibitor (LEKTI), which is encoded by the SPINK5 gene (Figure 2).55,56 The LEKTI is synthesized in the SG and released by lamellar granules into the extracellular space.57
The generation of Spink5 gene knockout mice has provided insight into the detailed biological functions of LEKTI and KLKs in the skin. Spink5−/− newborn mice have very fragile skin, with severe erosions, and die within a few hours of birth.58 A deficiency of LEKTI in the epidermis causes hyperactivity of KLK5 and KLK7, as well as desmosomal separation at the SG-SC interface.58 In addition, Spink5−/− embryo skin grafted onto nude mice exhibits parakeratotic hyperkeratosis and desmosomal cleavage resembling human Netherton syndrome.58,59 It is therefore suggested that regulation of the enzymatic activity of KLKs by LEKTI is crucial for maintaining the normal desquamation process and thus cutaneous barrier formation.
Extracellular lipids and related proteins.
In the upper stratum spinosum (SS) and SG, lamellar granules, which originate from a part of the Golgi apparatus, contain precursors of intercellular lipids in the SC, such as phospholipids, glucosylceramides, sphingomyelin and cholesterol.7,60,61 During the differentiation of keratinocytes, lamellar granules move to the apex of granular cells, fuse with the plasma membrane and secrete their contents into the extracellular space at the SG-SC border by exocytosis.34 Two epidermal lipoxygenases, lipoxygenase 3 and 12R-lipoxygenase, are presumed to be associated with lipid metabolism of the lamellar granule contents and/or extracellular lipid layers in the epidermis.35 Targeted ablation of the murine 12R-lipoxygenase gene results in neonatal death, with progressive dehydration, increased TEWL and decreased protein-bound ω-hydroxyceramides.62,63
Secreted lipids are subsequently processed and arranged into ELL in the intercellular space of the SC (Figure 3a).64 Some lipid classes are covalently bound to cornified cell envelope proteins, such as involucrin, envoplakin and periplakin, and form a lipid envelope that acts as a scaffold for the ELL.7,65 Ceramides (CERs), cholesterol and long-chain free-fatty acids are the three major lipid constituents in the SC and critical ingredients to form the ‘mortar’ of the epidermal ‘rampart’.64,66
Ceramides are a class of lipid molecules consisting of sphingoid bases that are amide linked to fatty acids. It is generally accepted that CERs are the main constituents of the SC and play important roles in maintaining permeability barrier function.67,68 In the suprabasal layer, CERs newly synthesized by the de novo pathway are immediately converted to glucosylceramides and sphingomyelins. The glucosylceramides and sphingomyelins are then incorporated into lamellar granules and secreted into the interface of the SG and SC, where they are converted back to CERs by β-glucocerebrosidase and amide sphingomyelinase (Figure 3b).69–73 Deficiency of β-glucocere-brosidase in type 2 Gaucher mice results in increased TEWL, increased glucosylceramides, decreased SC CERs and incompetent structure of ELL.72,74 The ATP-binding cassette subfamily A member 12 (ABCA12) is a lipid transporter that plays a role, at least in part, in the incorporation of glucosylceramides into lamellar granules (Figure 3b).75 Mice homozygous for the Abca12 null allele exhibit neonatal lethality, with skin fissures and severe weight loss that are probably due to increased TEWL.76 In the epidermis of Abca12−/− mice, there was remarkable hyperkeratosis, lipid droplets in the SG suggestive of lipid congestion in lamellar granules, and sparse SC CERs.76
Free extractable CERs in the human SC were formerly divided into eight fractions corresponding to 10 classes.77,78 However, recent studies using liquid chromatography-mass spectrometry revealed that free extractable CERs in human and canine SC could be divided into 11 groups according to their sphingoid and fatty acid structures, as follows: CER[EOH] (combination of ω-hydroxy fatty acids and 6-hydroxylsphingosines), CER[EOP] (combination of ω-hydroxy fatty acids and phytosphingosines), CER[EOS] (combination of ω-hydroxy fatty acids and sphingosines), CER[AH] (combination of α-hydroxy fatty acids and 6-hydroxyl sphingosines), CER[AP] (combination of α-hydroxy fatty acids and phytosphingosines), CER[AS] (combination of α-hydroxy fatty acids and sphingosines), CER[ADS] (combination of α-hydroxy fatty acids and dihydrosphingosines), CER[NH] (combination of nonhydroxy fatty acids and 6-hydroxyl sphingosines), CER[NP] (combination of nonhydroxy fatty acids and phytosphingosines), CER[NS] (combinations of nonhydroxy fatty acids and sphingosines) and CER[NDS] (combination of nonhydroxy fatty acids and dihydrosphingosines; Figure 4).79,80 Among the 11 classes of human SC CERs, seven CER classes, including esterified ω-hydroxyceramides with very long carbon chains (CER[EOS], CER[-EOP] and CER[EOH]), are expressed exclusively in the SC.79,80 It is well recognized that the esterified ω-hydroxyceramides play important roles in epidermal barrier function owing to their extremely long fatty acid chains, although their composition ratios are relatively low among all free extractable SC CERs.79–83 Moreover, nonesterified ω-hydroxyceramides (CER[OS], CER[OP] and CER[OH]), which are degraded products of esterified ω-hydroxyceramides, covalently bind to the cornified cell envelope and form a lipid envelope.84 In human SC, CER[OS] and CER[OH] are two major protein-bound CERs.85 In contrast, CER[OS] and CER[OP] are two major protein-bound CERs in canine SC.86 Ceramides are cleaved to generate sphingosine, which is then phosphorylated to sphingosine-1-phosphate (S1P) by sphingosine kinase. Sphingosine-1-phosphate is irreversibly inactivated by a S1P lyase (SGPL1; Figure 3b).87,88