Keratin and Keratinocyte Differentiation พญ. จันทณ้ชธน์/ อ. ธนวัฒน์ 11 เม.ย. 2557

EPIDERMIS Stratified, cornified epidermis Continually renewing structure that gives rise to appendages (pilosebaceous units, nails, and sweat glands) Thickness 0.4 to 1.5 mm (1.5- to 4.0-mm full-thickness skin) Other immigrant resident cells—melanocytes, Langerhans cells, and Merkel cells

EPIDERMIS Keratinocyte is an ectodermally derived cell and primary cell type in the epidermis (80%) Keratinocyte differentiation = keratinization From basal cells to the terminally keratinized stratum corneum (corneocyte) Corneocyte contains keratin filaments matrix protein protein-reinforced plasma membrane with surface-associated lipids ultimate fate of these cells is to contribute the components for the epidermal barrier as the stratum corneum. Thus, much of the function of the epidermis can be gleaned from the study of the structure and development of the keratinocyte.

BASAL LAYER Epidermal stem cells - multipotent epidermal stem cells within the bulge region of the hair follicle - keratinocytes are organized into vertical columns of progressively differentiating cells “epidermal proliferating units”

BASAL LAYER 2. Transit amplifying cells - subset of daughter cells produced by the infrequent division of stem cells - provide the bulk of the cell divisions needed for stable self-renewal - are the most common cells in the basal compartment

BASAL LAYER 3. Postmitotic cells - undergo terminal differentiation - In humans, the normal transit time basal cell- SC= at least 14 days SC subsequent desquamation 14 days - these periods of time can be altered in hyperproliferative or growth-arrested states

SPINOUS LAYER Midepidermis Spine-like appearance of the cell margins which are abundant desmosomes (calcium-dependent cell surface modifications)

Flatter and develop lamellar granules SPINOUS LAYER Polyhedral in shape with a rounded nucleus Flatter and develop lamellar granules Also contain large bundles of keratin filaments, organized around the nucleus and inserted into desmosomes peripherally Differentiate + move upward Lamellar granules (LG), also known as keratinosomes, lamellar bodies, membrane-coating granules, and Odland bodies

EM: round or oblong, membrane-delimitated, lamellate organelles Lamellar granules EM: round or oblong, membrane-delimitated, lamellate organelles

Lamellar granules LG are produced as discrete granules in the stratum spinosum, probably from the Golgi apparatus, and then migrate to the cell surface, fuse with the plasma membrane, extruding their contents in the outer stratum granulosum Precursors of stratum corneum lipids Genetic diseases demonstrate the importance of steroid and lipid metabolism for sloughing of cornified cells—in recessive X-linked ichthyosis, for example, mutation of steroid sulfatase results in a retention hyperkeratosis

Lamellar granules glucosylceramides (GlcCer) and other lipids Components include glucosylceramides (GlcCer) and other lipids (glycolipids, phospholipids, free sterols) various hydrolytic enzymes, such as proteases, acid phosphatases, glucosidases, and lipases other proteins including corneodesmosin (Cdsn), glycoproteins glucosylceramides; - precursors to ceramides - dominant component of the stratum corneum lipids

SPINOUS LAYER retain the stable K5/K14 keratins + K1/K10 keratin Differentiation or keratinization-specific keratins Spinous cells retain the stable K5/K14 keratins that are produced in the basal layer and only synthesize new messenger RNA (mRNA) for these proteins in hyperproliferative disorders. retain the stable K5/K14 keratins + K1/K10 keratin pair occurs in this epidermal layer. These keratins are characteristic of an epidermal pattern of differentiation. However, in hyperproliferative conditions such as psoriasis, actinic keratoses, and wound healing, synthesis of K1 and K10 mRNA and protein is downregulated, and the synthesis and translation of messages for K6 and K16 are favored.

Adherens junctions Actin microfilaments at cell–cell interfaces, via a distinct set of cadherins (e.g., E-cadherin) and intracellular catenin adapter molecules

GRANULAR LAYER Lamellar granules Basophilic keratohyalin granules within cells Site of generation of structural components (epidermal barrier) proteins that process these components Keratohyalin granules are composed of profilaggrin, keratin filaments, and loricrin It is in this layer that the cornified cell envelope begins to form, with the conversion of profilaggrin to filaggrin

GRANULAR LAYER Keratin aggregation form macrofilaments Filaggrin is degraded. Urocanic acid and pyrrolidone carboxylic acid Contribute to hydration of the stratum corneum and help filter UV radiation

GRANULAR LAYER Loricrin is a cysteine-rich protein that forms the major protein component of the cornified envelope KH granules ปล่อย loricrinให้ไป binds to desmosomal structures and is subsequently cross-linked to the plasma membrane by tissue transglutaminases (TGMs, primarily TGMs 3 and 1) to form the cornified cell envelope.

GRANULAR LAYER The final stage of granular cell differentiation into a corneocyte involves the cell’s own programed destruction almost all cellular contents are destroyed except keratin filaments and filaggrin matrix

STRATUM CORNEUM Anucleate, flattened cornified cells A two-compartment system of lipid-depleted, protein-enriched corneocytes surrounded by a continuous extracellular lipid matrix “Barrier activity” Provides mechanical protection to the skin and a barrier to water loss and permeation of soluble substances from the environment

Extracellular lipid matrix STRATUM CORNEUM Extracellular lipid matrix Corneocytes Regulation of permeability Mechanical reinforcement Desquamation Hydration AMP activity Protection from UV damage Toxin exclusion Selective chemical absorption Cytokine-mediated initiation of inflammation

STRATUM CORNEUM KC terminal differentiation culminates in the replacement of the plasma membrane with the cornified cell envelope (CE), a composite of several covalently cross-linked proteins Examples of CE components include involucrin, small proline-rich proteins (SPR), 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. For example, mutations in the loricrin and filaggrin genes give rise to palmoplantar keratoderma (PPK) and ichthyosis vulgaris (see below), respectively

STRATUM CORNEUM KC terminal differentiation culminates in the replacement of the plasma membrane with the cornified cell envelope (CE), a composite of several covalently cross-linked proteins Examples of CE components include Proteases  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, congenital hemidysplasia with ichthyosiform erythroderma and limb defects; LI, lamellar ichthyosis; CIE, congenital ichthyosiform erythroderma

Cornified cell envelope (CE)

Cornified lipid envelope (CLE) The extracellular surface of the CE is covered by lipids, which form the cornified lipid envelope (CLE)

STRATUM CORNEUM CE and the CLE are required for a cutaneous water barrier If fail increased transcutaneous water loss + increased susceptibility to infection CE and the CLE are required for a cutaneous water barrier If fail increased transcutaneous water loss + increased susceptibility to infections, a major problem in premature infants, and disorders such as Netherton syndrome.

Cornified cell envelope (CE) Begins within the upper spinous and granular cell layers Proteins are chemically cross-linked, primarily by ε-(γ-glutamyl) lysine isopeptide bonds  This reaction is catalyzed by enzymes 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.

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

Keratinocytes have keratin. So, what is keratin ??

Keratins (Cytokeratins) Structural proteins that belong to the superfamily of intermediate filament (IF) proteins Heterogeneous in size (40–70 kDa), charge (pI 4.7–8.4), and notoriously insoluble 54 functional keratin genes—34 epithelial keratins and 17 hair keratins Keratin They serve a predominantly structural role in the cells

Keratins are a family of intermediate filaments Fifty-four different functional keratin genes—34 epithelial keratins and 17 hair keratins The coexpression of specific keratin pairs is dependent on cell type, tissue type, developmental stage, differentiation stage, and disease condition (Table 7-2) Furthermore, the critical role of these molecules is underscored by the numerous manifestations of disease that arise because of mutations in these genes (see Table 7-2). Thus, knowledge of keratin expression, regulation, and structure provides insight into epidermal differentiation and structure. Keratin They serve a predominantly structural role in the cells

Keratin Characterized by a chain of amino acids (1° structure of the keratin protein) Classified as type I (K9–K28, K31–K40); acidic type II (K1–K8, K71–K86); basic

Keratin Most type I and II keratin genes are regulated in a pairwise, tissue type-related, and differentiation-related fashion. Associated with desmosomes, hemidesmosomes and protein complexes within the cornified cell envelope

Keratin 54 human keratin genesthree categories: (1) epithelial keratin genes (2) hair keratin genes (3) keratin pseudogenes

Keratin Soft keratin Hard keratin Epidermis(รวม palms, soles), ORS, some parts of IRS Keratins in the Stratum corneum are cross-linked by intermolecular disulfide bonds Hard keratin Hair cortex/cuticle, IRS, nail plate Intensive concentration of sulfur through the amino acids cysteine and methionine

Keratin Composed of 3 domains Head Central rod domain Tail α-helical: four segments (1A, 1B, 2A, 2B) non-helical segments: linkers Tail In head and tail domains Epithelial keratins: rich of glycine, serine Hair keratins: rich of cysteine, proline ). 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

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 (

Keratin intermediate filaments Intermediate filament proteins Keratins= the largest group provide resilience to keratinocytes, the most abundant cell type in the epidermis. Types of intermediate filaments. GFAP, glial fibrillary acidic protein; L, M and H, low-, medium- and high-molecular weight.

Keratin intermediate filaments Begins with the heterodimerization of one type I and one type II keratin protein Can bind signaling proteins controlling the cytoplasmic + nuclear molecules influence cell cycle progression, metabolic activity and apoptosis provide resilience to keratinocytes, the most abundant cell type in the epidermis.

Fig. 56.4 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

Hair Keratins The medulla: mixture of epithelial (K17, K75) and hair keratins (K33, K34, K36, K37, K81) The cortex: type I hair keratins (K31–K38) and type II hair keratins (K81, K83, K85 and K86) In the cuticle: Hair keratins K32, K35, K82, K85 The three IRS layers: K71, K74, K73. The full thickness of the ORS: epithelial keratins K5, K14 The isthmus and the lower ORS: K6, K16 and K17 Additional keratins expressed in the ORS: K15, K19.

Companion layer is located between the IRS and the ORS. Expression of K6, K16 and K17 is limited to the isthmus and the lower ORS. Additional keratins expressed in the ORS are K15 and K19. The epithelial keratins K5 and K14 are found throughout the full thickness of the ORS,

Fig. 56.6 Complex pattern of hair keratin expression in the human anagen hair follicle. Major type I hair keratins are in blue, and major type II hair keratins are in green. Minor hair keratins are in pink. 1This protein is weakly expressed at this site. 2To date, expression of this protein has only been detected in single cortex cells. 3To date, this protein has only been detected in vellus hairs. Autosomal dominant monilethrix is caused by mutations in K81, K83, and K86.

EPIDERMAL DIFFERENTIATION 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 express the keratin pair K5 and K14. In addition, but less abundantly, K15 is expressed. In the absence of K14, K15 can assemble with K5, thereby providing mechanical stability to the keratinocyte. As keratinocytes move suprabasally to the spinous layer, they withdraw from the cell cycle. This process is associated with a down-regulation of K5 and K14 and an induction of the differentiation-specific keratins, K1 and K10. Further maturation of spinous keratinocytes into granular keratinocytes results in expression of K2, 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.

Granular KC expresses K2, a reinforcement keratin. The mitotically active keratinocytes in the basal compartment of the epidermis express the keratin pair K5 and K14. In addition, but less abundantly, K15 is expressed. In the absence of K14, K15 can assemble with K5, thereby providing mechanical stability to the keratinocyte. e.g., liver, gut, pancreas

K9 is specifically expressed in the suprabasal cells of palmoplantar skin. The mitotically active keratinocytes in the basal compartment of the epidermis express the keratin pair K5 and K14. In addition, but less abundantly, K15 is expressed. In the absence of K14, K15 can assemble with K5, thereby providing mechanical stability to the keratinocyte.

KC in nail bed, hair follicle, sebaceous and sweat glands K6, K16 and K17: - Palmoplantar KC in nail bed, hair follicle, sebaceous and sweat glands This group of keratins is rapidly induced by injury, ultraviolet radiation, wounding, hyperproliferative conditions The mitotically active keratinocytes in the basal compartment of the epidermis express the keratin pair K5 and K14. In addition, but less abundantly, K15 is expressed. In the absence of K14, K15 can assemble with K5, thereby providing mechanical stability to the keratinocyte. นอกจากนี้ K6a, K6b, K16, K17 ยังพบใน recruited KC ตรงขอบแผล for restoration of epi barrier following injury.

FUNCTION OF KERATIN IN THE EPIDERMIS AND OTHER SKIN EPITHELIA Enhance the cell’s ability to withstand trauma (by IF networks) Attachment of IFs to adhesion complexes (desmosomes, hemidesmosomes), and to F-actin and microtubules Loss of this function leads to fragile cells and unable to sustain mechanical stress.

FUNCTION OF KERATIN IN THE EPIDERMIS AND OTHER SKIN EPITHELIA Nonmechanical functions: In hair follicles, K17 promotes the anagen (growth) phase by attenuating TNF-α-induced apoptosis in matrix keratinocytes In the epidermis the suprabasally expressed K10 regulate proliferation in the basal layer of epidermis and in sebaceous glands while K17 cell autonomously regulates protein synthesis and cell size in wound-proximal keratinocytes

FUNCTION OF KERATIN IN THE EPIDERMIS AND OTHER SKIN EPITHELIA Keratins influence the melanin pigment distribution and, thus, skin pigmentation Ex. - Dowling-Degos dz K5 mutation aberrations in skin pigmentation - Naegeli–Franceschetti–Jadassohn syndrome and dermatopathia pigmentosa reticularis - EBS with mottled pigmentation

Regulatory Pathways Involved in Epidermal Development and Differentiation The regulatory pathways necessary for normal keratinocyte differentiation: (1) establish and maintain basal keratinocytes (2) initiate and execute terminal differentiation (3) form the stratum corneum

Genes required for establishing/maintaining basal KC Regulatory Pathways Involved in Epidermal Development and Differentiation Genes required for establishing/maintaining basal KC The p63 gene encodes six different proteins, each of which can function as a transcriptional activator or repressor. The p63 is required for both the initial induction of K5/K14 expression in embryonic basal keratinocytes and the maintenance of K5/K14 expression in the basal layer of mature epidermis. Also maintain the proliferative state of basal KC by repress the expression of cell cycle inhibitors prevent the onset of terminal differentiation transcription factor p63. In mice, completely p63-deficient fails to initiate epidermal morphogenesis a single-layered epithelium covering their bodies rather than a stratified epidermis. Rapid death due to dehydration.Consistent with this hypothesis, ectopic p63 expression was shown to induce expression of the epidermal keratins K5 and K14.

Becomimg spinous KC is controlled by Regulatory Pathways Involved in Epidermal Development and Differentiation Genes required for terminal differentiation in mature epidermis Becomimg spinous KC is controlled by an isoform of p63, ΔNp63α the Notch signaling pathway ΔNp63α synergizes with Notch signaling to induce K1 expression cell cycle withdrawal  terminal differentiation In addition, ΔNp63α mediates cell cycle exit by inducing cell cycle inhibitors and by repressing genes required for cell cycle progression. The importance of p63 for normal epidermal development and differentiation is further underscored by the finding that p63 mutations underlie a subset of ectodermal dysplasias, which are characterized by abnormalities in the skin and skin appendages (see Ch. 63). Ablation of Notch  an extremely thin spinous layer Active Notch signaling resulted in an expansion of the spinous layer

Ca2+ in epidermal differentiation Regulatory Pathways Involved in Epidermal Development and Differentiation Ca2+ in epidermal differentiation An increase in extracellular Ca2+ trigger of KC differentiation  formation of the granular cell layer Several Ca2+-responsive proteins in the epidermis that are involved in the formation of the granular layer The protein kinase C (PKC)  down-regulate K1 and K10 expression as well as to the induct markers of granular KCs, including loricrin, filaggrin and transglutaminases The calcium-sensing receptor (undergo conformational changes upon binding to Ca2+) is expressed in granular KC. The protein kinase C (PKC) family of proteins is activated by Ca2+ signaling and functions specifically in the transition from spinous to granular cells. Interestingly, mice lacking the full-length form of the calcium-sensing receptor fail to properly form a granular layer, while overexpression of the calcium-sensing receptor in basal keratinocytes causes expanded spinous and granular cell layers17.

Genes required for terminal differentiation in embryonic epidermis Regulatory Pathways Involved in Epidermal Development and Differentiation Genes required for terminal differentiation in embryonic epidermis The molecular mechanisms for development of a spinous layer during epidermal morphogenesis appear to be different Basal KCs initially differentiate into intermediate keratinocytes, which express K1undergo proliferation differentiate into spinous and granular cells then, terminal differentiation The intermediate cell layer exists only transiently during epidermal morphogenesis, and intermediate cells ultimately

Genes required for terminal differentiation in embryonic epidermis Regulatory Pathways Involved in Epidermal Development and Differentiation Genes required for terminal differentiation in embryonic epidermis intermediate cells fail to mature into spinous and granular cells. Such a block in differentiation occurs in mice lacking expression of inhibitor of κB kinase-α (IKKα), interferon regulatory factor 6 (IRF6) or ovo-like 1 (Ovol1), as well as in mice expressing a mutant form of 14–3–3σ18. The latter mutant protein was identified in repeated epilation (Er) mutant mice. In all instances, an expanded intermediate cell layer develops, further terminal differentiation is disrupted, and the consequent failure to establish barrier function results in neonatal lethality. The intermediate cell layer exists only transiently during epidermal morphogenesis, and intermediate cells ultimately

Aggregation of disorganized keratin bundles Keratin Disorders Usually with an autosomal dominant; typically dominant-negative fashion interfering with normal intermediate filament assembly Aggregation of disorganized keratin bundles Cell fragility mutations in the helix initiation and termination motifs are generally associated with relatively severe disease phenotypes, whereas mutations affecting other keratin domains usually cause milder disease

Keratin Disorders White sponge nevus of Cannon white plaques involving the oral mucosa +/-other mucosal surfaces (esophagus, vagina, rectum and nasal cavity) wax and wane over time suprabasal cytolysis and keratin clumping mutations in K4 and K13, which are specifically expressed in mucosal keratinocytes mutations in the helix initiation and termination motifs are generally associated with relatively severe disease phenotypes, whereas mutations affecting other keratin domains usually cause milder disease

Keratin Disorders Gastrointestinal disorders K8 and K18 are the major keratins that are expressed in gastrointestinal epithelia, including the liver, pancreas and gut.. K8 and K18 mutations  risk factors for developing liver and GI disorders (e.g. cirrhosis, inflammatory bowel disease), with additional genetic and environmental alterations likely required for disease development. The mutation-associated predisposition to tissue injury is likely related to mechanical and non-mechanical keratin functions, including maintenance of cell integrity and protection from oxidative injury and apoptosis. Mutations in these simple keratins are typically located within the head and tail domains and do not involve the highly conserved helix boundary regions

X-linked dominant Charcot–Marie–Tooth disease loss of connexin 32 (Cx32) impaired diffusion of nutrients and signaling molecules into peripheral nerves. recessive mutations in Cx26 the single most important cause of non-syndromic congenital hearing impairment (with carrier frequencies ranging from 3% to 10% in the general population) autosomal dominant in Cx26 range from Vohwinkel syndrome (mutilating PPK with deafness) to keratitis–ichthyosis–deafness (KID) syndrome (which also features a “stippled” PPK