Introduction

The main function of the epidermis is to provide a barrier between the organism and the environment. This barrier is the result of a complex terminal differentiation program in which the progeny of epidermal stem cells undergo a series of molecular and biochemical changes that lead to generation of the morphologically different strata of the epidermis ( Fig. 56.1 ). This chapter reviews key signaling pathways that control this terminal differentiation program, with discussion of inherited skin diseases that are caused by a failure of normal epidermal differentiation.

Epidermal differentiation.

As keratinocytes are transformed from mitotically active cells in the basal layer to fully differentiated, enucleated squames in the cornified layer, specific proteins are expressed at particular locations within the epidermis. Keratohyalin (profilaggrin- and loricrin-containing) and lamellar (lipid-containing) granules extrude their contents in the granular layer, leading to bundling of keratin filaments and replacement of the plasma membrane with the highly cross-linked, lipid-covered cornified cell envelope (see inset and Fig. 56.2 ).

Courtesy, Julie V Schaffer, MD.

The Epidermis: Structure and Function

The epidermis, the outermost layer of the skin, functions as a barrier that provides the organism with protection against harmful substances and water loss. The barrier function is established during embryogenesis and maintained in mature epidermis through a continuous program of self-renewal. Under homeostatic conditions, this self-renewal is mediated by epidermal stem cells located in the basal layer of the interfollicular epidermis . Another population of stem cells, located in the bulge region of the hair follicles, contributes to the interfollicular epidermis in response to injury (see Ch. 2 ). Each division of an interfollicular stem cell gives rise to a daughter stem cell and a transit amplifying cell through a process termed asymmetric cell division. Transit amplifying cells undergo a few cell divisions before beginning a terminal differentiation program, which is associated with irreversible withdrawal from the cell cycle. Terminally differentiating keratinocytes detach from the basement membrane and move suprabasally, a process that is mediated by the inactivation of integrins, extracellular matrix receptors that anchor basal cells to the basement membrane (see Ch. 28 ). They are termed spinous keratinocytes when they reach the first suprabasal cell layer, and further differentiation as they move upward leads to the formation of granular keratinocytes.

Keratinocyte terminal differentiation culminates in the replacement of the plasma membrane with the cornified cell envelope (CE), a composite of several covalently cross-linked proteins ( Fig. 56.2 ) , most of which are encoded by genes located in the epidermal differentiation cluster on chromosome 1. Examples of CE components include involucrin, small proline-rich proteins (SPRR), XP-5/late envelope proteins (LEP), loricrin, cystatin, envoplakin, periplakin, elafin, repetin, filaggrin, S100 proteins, keratins, and desmosomal proteins. Note that mutations in some of the genes that encode these proteins can lead to skin disorders (see below). The extracellular surface of the CE is covered by lipids, which form the cornified lipid envelope (CLE) (see Ch. 124 ).

Formation of the cornified cell envelope (CE).

Terminal differentiation of keratinocytes is triggered by an increase in the intracellular Ca ²⁺ concentration of the suprabasal epidermis. CE assembly is initiated in the upper spinous layer via formation of a cross-linked scaffold composed of envoplakin, periplakin and involucrin along the inner surface of the cell membrane ( 1 ). This is followed by (or perhaps coincident with) extrusion of lamellar granules into the extracellular space ( 2 ). Specialized ω-hydroxyceramides are delivered to and eventually replace the cell membrane, where they become linked to scaffold proteins. Reinforcement occurs via cross-linking and translocation to the cell periphery of loricrin (which accounts for >80% of the mass of the CE) and small proline-rich proteins (SPRRs) ( 3 ). Complexes of keratin and filaggrin also become cross-linked to the CE. In addition, proteases play important roles in processing of CE proteins and the proteolysis of corneodesmosomes that is required for desquamation. A mature, terminally differentiated cornified cell thus consists of keratin filaments covalently attached to the CE, which is composed of protein and lipid envelope components and imbedded in extracellular lipid lamellae. Defects in transglutaminases, lipid metabolism, CE structural proteins and proteases lead to a variety of diseases characterized by ichthyosis and/or keratoderma ( 1–3 ). CHILD, c ongenital h emidysplasia with i chthyosiform erythroderma and l imb d efects; LI, lamellar ichthyosis; CIE, congenital ichthyosiform erythroderma.

Courtesy, Julie V Schaffer, MD.

Keratinocytes that have assembled the CE/CLE and lost their nucleus and cytoplasmic organelles are termed corneocytes . These cell remnants constitute the stratum corneum, which serves to cover the body surface of terrestrial mammals. The CE and CLE are major contributors to the cutaneous water barrier. Failure or impairment of the water barrier leads to increased transcutaneous water loss and an increased susceptibility to infections, a major problem in premature infants and in disorders such as Netherton syndrome.

CE assembly begins within the upper spinous and granular cell layers, where proteins are chemically cross-linked, primarily by ε-(γ-glutamyl) lysine isopeptide bonds ( Fig. 56.3 ). This reaction is catalyzed by a class of enzymes termed transglutaminases (TGases). Loss-of-function mutations in the gene that encodes TGase 1 lead to lamellar ichthyosis and congenital ichthyosiform erythroderma, generalized skin disorders resulting from a failure to form proper CEs.

Transglutaminase catalysis of isopeptide bond formation between proteins.

Transglutaminases (TGs) are calcium-dependent enzymes that catalyze the formation of γ-glutamyl lysine isopeptide bonds between proteins. TGs also have a role in the creation of ester bonds between proteins and ω-hydroxyceramides. Such cross-linking is essential for assembly of the CE. Four TGs are expressed in the epidermis: TG1 (keratinocyte TG; membrane-bound), TG2 (tissue TG; basal layer), TG3 (epidermal TG; hair follicle and terminally differentiating keratinocytes) and TG5 (upper epidermis).

Courtesy, Julie V Schaffer, MD.

Because the CE is highly resistant to chemical attack, very harsh conditions (e.g. boiling the epidermal cells in buffers containing high concentrations of detergents) are required to isolate and purify CE proteins. One surprising finding is that a loss of individual CE components in knockout mice does not necessarily have a major impact on barrier function. One possible explanation for this unexpected result is the presence of compensatory gene regulatory pathways that maintain barrier function even in the absence of major CE components . Better characterization of these regulatory pathways will provide insights into how the barrier is established and maintained, hopefully leading to the development of new strategies for the treatment of diseases caused by impaired barrier function.

Keratin Intermediate Filaments

Keratin intermediate filaments provide resilience to keratinocytes, the most abundant cell type in the epidermis. Keratins represent the largest group of intermediate filament proteins ( Table 56.1 ). The current classification system includes 54 human keratin genes ( Table 56.2 ), which can be divided into three categories: (1) epithelial keratin genes; (2) hair keratin genes; and (3) keratin pseudogenes . Based upon their biochemical properties (e.g. isoelectric point, molecular weight), keratins are also classified as either type I (KRT9–KRT28, KRT31–KRT40) or type II (KRT1–KRT8, KRT71–KRT86) (see Table 56.2 ). Type I and type II keratins form obligatory heteropolymers (i.e. pairs composed of one keratin from each group) that become the basic building blocks of epithelial intermediate filaments ( Fig. 56.4 ).

Table 56.2

Previous and current human keratin nomenclature.

Darker shading indicates keratins with names that were changed. irs, inner root sheath; Ha, type I hair keratins; Hb, type II hair keratins.

From Schweizer J, Bowden PE, Coulombe PA, et al. J Cell Biol. 2006;174:169–74.

 

Alignment and assembly of keratin molecules and keratin filament packing.

Intermediate filament assembly takes place in several stages and begins with the heterodimerization of one type I and one type II keratin protein in a coiled-coil fashion. Two heterodimers then associate to form a tetramer. Lateral aggregation of tetramers yields higher-order polymers which eventually make up the filament network of the keratinocyte.

Courtesy, Julie V Schaffer, MD.

Keratins have a common domain structure that they share with other intermediate filament proteins . The central α-helical rod domain consists of 310 amino acid residues and is divided into four segments (1A, 1B, 2A, 2B), which are interrupted by three non-helical segments of variable lengths, termed linkers ( Fig. 56.5 ). The rod domain is composed of seven-residue amino acid sequence repeats (a-b-c-d-e-f-g) _n termed “heptad repeats”, where positions “a” and “d” represent hydrophobic residues that are considered crucial for stabilization of the heterodimer. In the middle of the 2B domain, the heptad pattern is interrupted, giving rise to the “stutter”. This helical segment is highly conserved among intermediate filaments and does not participate in the formation of the coiled-coil dimer that forms the basic building block of intermediate filaments (see Fig. 56.4 ).

Primary sites of keratin mutations and associated skin diseases.

Over 90% of pathogenic alterations in keratins are missense mutations. Genotype-phenotype correlations are particularly well established for EBS. In the generalized severe form of EBS, many patients have a “hot spot” mutation in a highly conserved arginine (Arg125) located within the helix initiation motif of KRT14. The corresponding arginine (Arg156) in KRT10 is also a mutational “hot spot” in epidermolytic ichthyosis. In the generalized intermediate EBS subtype, mutations are more centrally located in the rod domains of KRT5 and KRT14. In contrast, mutations in the localized form of EBS are often outside the rod domain, e.g. in the L12 linker motifs of KRT5 and KRT14 or the H1 homologous subdomain of KRT5. Mutations in the variable head (V1) and tail (V2) domains of KRT1 can lead to non-epidermolytic PPK and ichthyosis hystrix Curth–Macklin. Rather than interfering with keratin intermediate filament assembly, mutations in these domains may cause intracellular maldistribution of loricrin. K or KRT, keratin; PPK, palmoplantar keratoderma.

Courtesy, Julie V Schaffer, MD.

The beginning and end of the α-helical rod domain, referred to as the helix initiation and helix termination motifs, are highly conserved among the different keratins and play a pivotal role in keratin intermediate filament assembly (e.g. filament elongation). These helix boundary peptides represent genetic “hot spots” for mutations in many of the hereditary keratin disorders (see Fig. 56.5 ). The severity and other phenotypic features of genodermatoses caused by keratin defects often correlate with the position of the underlying mutation, with more severe disease resulting from mutations in the helix initiation and termination motifs (see Table 56.4 ).The head and tail regions that flank the rod domain are subdivided into extreme end (E), variable (V), and (in type II keratins) homologous (H) domains. Whereas epithelial keratins possess glycine- and serine-rich head and tail domains, these regions have a high content of cysteine and proline in hair keratins. Variations in the head and tail domains account for much of the diversity among different keratin proteins, which suggests that these domains play an important role in cell type-specific functions.

Hair Keratins

The mature hair is the differentiation product of trichocytes and is found within the central core of the hair follicle. The anagen (growing) follicle is a complex structure consisting of eight distinct, concentrically arranged cell layers (see Ch. 68 ). The innermost compartment is the hair shaft, which is composed of a medulla, cortex, and one-layered cuticle; the latter serves as a protective coat for the hair shaft. The innermost living cell layer of the hair follicle is the inner root sheath (IRS); it surrounds the growing hair shaft and consists of the IRS cuticle as well as Huxley and Henle layers. The outermost compartment is the outer root sheath (ORS), which is continuous with the interfollicular epidermis. The companion layer is located between the IRS and the ORS.

The medulla contains a mixture of epithelial keratins (KRT17, KRT75) and hair keratins (KRT33, KRT34, KRT36, KRT37, KRT81), whereas the cortex contains type I hair keratins (KRT31–KRT38) and type II hair keratins (KRT81, KRT83, KRT85 and KRT86) ( Fig. 56.6 ). Hair keratins KRT32 and KRT35 and their partners KRT82 and KRT85 are found in the cuticle, and the three IRS layers can be detected with antibodies against KRT71, KRT74, and KRT73. The epithelial keratins KRT5 and KRT14 are found throughout the full thickness of the ORS, while expression of KRT6, KRT16, and KRT17 is limited to the isthmus and the lower ORS. Additional keratins expressed in the ORS are KRT15 and KRT19.

Complex pattern of hair keratin expression in the human anagen hair shaft.

Major type I hair keratins are in blue, and major type II hair keratins are in green. Minor hair keratins are in pink. ^a This protein is weakly expressed at this site. ^b To date, expression of this protein has only been detected in single cortex cells. ^c To date, this protein has only been detected in vellus hairs. Autosomal dominant monilethrix is caused by mutations in KRT81, KRT83, and KRT86.

Redrawn from Langbein L, et al. The catalog of human hair keratins. J Biol Chem. 2001;276:35123–32.

Epidermal Differentiation

Epidermal differentiation is a tightly regulated process that involves the transformation of proliferating cells in the basal layer into the dead corneocytes of the stratum corneum. During this process, the keratins that are expressed are highly specific for the state of differentiation ( Fig. 56.7 ). The mitotically active keratinocytes in the basal compartment of the epidermis primarily express the keratin pair KRT5 and KRT14, with less abundant expression of KRT15. In the absence of KRT14, KRT15 can assemble with KRT5, thereby providing mechanical stability to the keratinocyte.

Cytokeratin expression.

Type II (basic) keratins are in yellow (KRT1–KRT8; KRT76) and type I (acidic) keratins are in blue (KRT9–KRT20). Keratin disorders are presented in Fig. 56.5 and Tables 56.3 & 56.5 .

As keratinocytes move suprabasally to the spinous layer, they withdraw from the cell cycle. This process is associated with a down-regulation of KRT5 and KRT14 and an induction of the differentiation-specific keratins, KRT1 and KRT10. Further maturation of spinous keratinocytes into granular keratinocytes results in expression of KRT2, a reinforcement keratin. With further maturation, filaments containing the suprabasal keratins are bundled parallel to the surface and, eventually, keratinocytes lose their cytoplasmic organelles and differentiate into lifeless corneocytes that are shed into the environment.

Interestingly, a number of epidermal keratins have a more restricted anatomic distribution pattern. For example, KRT9 is specifically expressed in the suprabasal cells of palmoplantar skin. KRT6, KRT16, and KRT17 are expressed not only in the palmoplantar epidermis, but also in keratinocytes of the nail bed, hair follicle, and sebaceous and sweat glands. In addition, this group of keratins is rapidly induced by injury and ultraviolet radiation, as well as in hyperproliferative conditions .