Derm 5 - Epidermis

Atopic dermatitis (AD)

"*Eczema*;" very common condition that varies in severity from mildly xerotic and pruritic (itchy) skin to a debilitating condition requiring systemic immunosuppressives for control - Often associated w/ *asthma and allergic rhinitis*, and these three conditions are known as the "*atopic triad*." Triad tends to be a *familial condition, w/ family members manifesting all or part of the triad* -*loss of function filaggrin mutations causing an AD phenotype* in a significant number of affected individuals --> mutations lead to *defective cell envelope formation and a dysfunctional barrier, w/ subsequent inflammatory sequellae*

Desmosomal Proteins (Lecture)

*Cadherins*: specific expression varies by tissue - Desmogleins 1, 2, 3, 4 - Desmocollins 1, 2, 4 *Armadillo family*: joins cadherins to plakins - Plakoglobin - Plakophilins *Plakins*: join cadherins to keratins - *Desmoplakins* I, II - Othersenvoplakin, periplakin

Contribution of lipids to the formation of the stratum corneum: Ceramids

*Ceramides*: type of sphingolipid; essential for formation of permeability barrier - function to align lipids in intercellular spaces into layers and contribute to cornified cell envelope

4 Distinctive Differentiation Stages (*Keratinization/Cornification*) give rise to Keratinocytes with Different Structural Features

*Cornified Layer*: detach and desquamate at skin surface *Granular Layer* *Spinous Layer* *Basal Layer*: keratinocytes attached to the dermis at the DEJ --> differentiate to form Spinous/Suprabasal

Skin Tissue Layers

*Epidermis*: formed from *Ectoderm* *Dermis*: CT, mostly collagen Subcutis: from Mesoderm

Case 1: Pt presents with areas of hyperpigmentation and blisters, histo shows split BM

*Epidermolysis Bullosa Simplex * - Inherited blistering diseases - Defined - level of split in skin Simplex - within epidermis

Xerosis: Exogenous and Endogenous Factors

*Exogenous*: include an arid environment, repeated wetting (and subsequent drying), and frequent detergent (including soap) exposure. *Endogenous*: include a genetic propensity for dry and/or hypersensitive skin, certain systemic conditions (e.g. renal failure, hypothyroidism, malnutrition), and certain meds (e.g. lipid lowering agents)

Filaggrin

*Keratin filament aggregating protein*; integrally involved in process of keratinization - derived from a large precursor protein, profilaggrin, that is synthesized in granular cell - *keratohyalin granules define the granular layer* - *Profilaggrin* accumulates in the cytoplasm of granular cell as the *main component of keratohyalin granules* - hematoxylin-staining (visible by light microscopy), intracellular granules, not bound by a membrane, w/ keratin intermediate filaments coursing through them

Epidermis Cell Types

*Melanocytes*: pigmentation *Langerhans Cells*: Antigen Presenting Cells (APCs) - Melanocytes and Langerhans originate elsewhere and migrate to Epidermis *Merkel Cells*: neuroendocrine *Keratinocytes*: primary cells of epidermis that undergo differentiation

Keratin Production

*Only a few keratins are produced by any one epithelial cell type. Furthermore, different keratins are produced depending upon the stage of differentiation of that cell*. - Synthesis of type I and the type II keratin molecules varies in the different cell layers of the epidermis - *Basal cell layer: keratins 5 and 14* form intermediate filaments in cytoskeleton network. These keratin proteins account for ~15% to 25% of the total protein in the basal layer

Transglutaminases

*ca-dependent enzymes* that in keratinocyte; *catalyze cross- linking of involucrin, loricrin and other proteins that act as substrates to form the cornified cell envelope* - catalyze *formation of covalent isodipeptide bonds between glutamine and lysine residues via formation of ε-amino (γ~glutamyl) lysine bonds* - activated by influx of extracellular Ca ions - In the keratinocyte, there are at least 4 distinct transglutaminase enzymes

S100 proteins

- A 3rd family of genes, also located in long arm of chromosome 1 (1q21), code for a group of small calcium binding proteins, the S100 proteins - Because of the profound changes in [Ca2+] seen at the interface of the granular and cornified layers and the Ca dependence of many of the enzymes active in epidermal differentiation, these proteins may be important in the process.

Case 4: Pt has congenital caling skin disease. Scales are "dirty brown" and located everywhere but accentuated on extensor surfaces of his skin. - Histo shows that his granular layer varies, stratum corneum is thickened and compacted *What is necessary for desquamation?*

- Cornified envelope - Desmosomes - Proteases - Lipids

What holds the Epidermis together?

- Desmosomes - Intermediate Fillaments

Granular-Cornified Layer Transition (lecture)

- Lipids polar --> neutral - Lamellar granules fuse with cell membrane, secrete contents - Profilaggrin expressed & processed to filaggrin - Keratohyalin granules form & disappear - Additional cornified envelope precursors are expressed - Keratin filaments aggregate & cross-linked by disulfide bonds - Cells flatten - Cornified cell envelope forms

Structure of Intermediate Filament Proteins (Lecture)

- Molecule - monomer with a central rod-shaped alpha-helical portion - Globular heads & tails *Dimers - coiled coil* - Helix initiation/ termination regions critical for formation

Pemphigus: Histopath

- Pemphigus Ab's are pathogenic and result in a falling apart of the epidermis - Histologically, one sees *acantholysis of keratinocytes in the spinous layer*

Desmosome vs Hemidesmosome

1 desmosome does NOT = 2 hemidesomsomes *Desmosomes*: intercellular junctions - adhesion between cells *Hemidesmosomes*: adhesion to basement  membrane

Questions to ponder before and after lecture:

1. How did such an elaborate scheme for epidermal differentiation evolve? 2. Why are there so many different keratins, and so few other types of intermediate filament proteins? 3. What skin changes might one expect to see if all keratins were absent from the basal keratinocyte? 4. Why do you think EB simplex and epidermolytic ichthyosis are autosomal dominant disorders? 5. What might one see in the skin of a person treated with a lipid lowering drug? 6. Why are there so many different cornified envelope substrates? 7. What might happen if the cornified envelope were defective?

Keratinization Events

1.) *Basal Layer*: *Adhesion* of epidermis to dermis and *production* of new cells occurs. Keratinocytes adhere to one another. 2.) As they leave the basal layer, keratinocytes stop proliferating and *differentiate*. *Spinous layer* marks this departure, and also signifies beginning of prep for barrier formation 3.) *Granular layer* marks the *final preparations for barrier formation* 4.) *Cornified layer*: *barrier forms and cells are sloughed* Perpetual process assures continuous self-renewal of the epidermal barrier

Case 3: Pt born with a skin disease involving generalized scaling, actropion, eclabium, manifests most on the skin - plate-like scales or lamellae with microfissures between them - Skin biopsy shows hyperkeratosis and acanthosis (back of card) 1.) *Where in the skin is the problem?* 2.) *How could this happen?*

1.) *Stratum corneum* - Hyperkeratosis - thick stratum corneum - Acanthosis - thick spinous & granular layer 2.) *Pt is either making skin too fast, or not sloughing it fast enough* Epidermis is an epithelium: Constantly renewing - basal --> cornified ~14 days - cornified --> out ~14 days ~28 days total

Case 2: Man presents w/ a few year hx of blisters in mouth and skin and large areas of erosions with crusting and flaccid bullae - lots of mucous membrane involvement - Histo shows rounded epidermal cells 1.) *Where in the skin is this person's blister forming?* 2.) *What is the pt's Diagnosis?*

1.) Epidermis (note basal keratinocyte layer still attached to BMZ!!!!) 2.) Pemphigus vulgaris

Case 1: Pt presents with areas of hyperpigmentation and blisters, histo shows split BM - Epidermolysis Bullosa Simplex 1.) *Which keratin is affected?* 2.) *Where in the intermediate filament is the mutation most likely to occur?*

1.) Keratin defects basal - K5/ K14 mutations 2.) helix initiation/ termination regions

Terminal differentiation: Steps

1.) Lipid synthesis and the formation of the permeability barrier B.) Loss of nucleus and cytoplasmic organelles C.) Keratin filament aggregation D.) Cornified envelope formation E.) Desquamation of cornified cells

One model of formation of cornified envelope occurs in a sequential series of covalent cross-linking steps involving the cytoplasmic substrates...

1.) begins in spinous cells when *envoplakin and periplakin*, members of one of the families of proteins involved in desmosome plaque formation, are *bound near PM between desmosomes* 2.) In upper granular cells. *intracellular [Ca2+] increases* and keratinocyte *transglutaminase catalyzes cross-linking of involucrin w/ envoplakin and periplakin* 3.) Concurrently, *lamellar granules fuse w/ the apical PM of granular cells and secrete their contents into the intercellular space* 4.) *Loricrin molecules are cross-linked w/ themselves and w/ cross-linked involucrin-peri/envoplakin; other cornified envelope substrate proteins are cross-linked to the structure* 5.) *Keratin filaments and intercellular proteins are bound*; aggregation of keratin filaments and formation of cornified envelope results in *"collapse" flattening of keratinocytes and formation of cornified cells in stratum corneum* - Ultimately acylceramide in intercellular space is covalently cross-linked to this structure, replacing PM *Entire process is completed by the time keratinocytes reach lowest layers of cornified layer*

Keratinocyte Morphology

2 things distinguish KC's morphologically - *Keratins (yellow circle) and Desmosomes* Morphology changes as keratinocytes progress through the stages of differentiation Basal Cells: cuboidal to slightly columnar Spinous Cells: becomes more rounded and oval. As these cells move toward the surface, they become flatter and thinner... *Stratum corneum cells*: very thin, flat discs that cover an area that would be occupied by ~20 basal cells

Keratinocyte Proliferation

3 populations of cells w/ different capacities for proliferation: 1.) *Stem cells (S)*: proliferate at a slow rate 2.) *Transient amplifying cells (TA)*: proliferate at a faster rate 3.) *Postmitotic cells (PM)*: do not proliferate at all - heterogeneity of proliferating keratinocytes in epidermis, esp in the basal cell layer *Basal Layer*: *All 3* cell populations *Spinous Layer*: TA and PM *Granular and Cornified Layers*: PM cells

Filaggrin Modification

After covalent disulfide cross linking of cysteine residues in aggregated keratin macrofibrils, filaggrin is broken down into free a.a.'s in upper stratum corneum - free a.a.'s metabolized into compounds that have good water binding properties - modified filaggrin residues thought to assist in *hydrating outer layers of the cornified layer* - Null mutations in profilaggrin underlie the most common form of ichthyosis, ichthyosis vulgaris, and are strongly associated with atopic dermatitis

Keratinocyte Differentiation and Keratin Production

As basal cell moves into spinous layer and begins process of terminal differentiation, different sets of keratin genes are switched on. In suprabasal keratinocytes, *synthesis of K1/K10 proteins progressively increases as the synthesis of K5/K14 progressively diminishes* - upper cells of the spinous layer: K2 is expressed; acidic partner of K2 not known - throughout differentiation, the *amount of protein synthesis devoted to keratin production steadily increases* - In *fully differentiated cornified cell, keratin accounts for ~85% of the total protein in the cell*

Keratin Assembly

Assembly of keratin dimers into keratin filaments occurs *spontaneously* w/out need for scaffolding from microtubules or energy source 1.) 2 heterodimers associate w/ each other to form a tetramer 2.) Tetramers associate w/ each other end to end and side to side to form a helical array of bundles that are assembled into a 10nm diameter keratin filament *Polypeptides in amino and carboxyl terminal ends of the helical rod domain (the "helix initiation" and "helix termination" regions)* adjacent to the nonhelical head and tail domains are most critical in this assembly process - Point mutations in the genes that control structure of these polypeptides lead to changes seen in most inherited keratin disorders, including epidermolysis bullosa simplex and epidermolytic ichthyosis

Case 3: Pt born with a skin disease involving generalized scaling, actropion, eclabium, manifests most on the skin - plate-like scales or lamellae with microfissures between them - Skin biopsy shows hyperkeratosis and acanthosis *What is the diagnosis?*

Autosomal Recessive Congenital Ichthyosis - A form of ichthyosis - autosomal recessive - thick, generalized, plate-like scalered skin - Phenotypic heterogeneity - Genetic heterogeneity - Transglutaminase mutations

Transit Time

Average time it takes a keratinocyte to migrate from one cell layer to the next; observed by progress of labeled cells through cell layers - Ave keratinocyte transit time in normal epidermis from the basal layer through the granular layer is ~2 weeks - from bottom of the cornified layer to top is also ~2 weeks - *Total transit time* from basal layer to desquamation ~4 weeks

Case 2: Pemphigus vulgaris *Why do the basal keratinocytes line the bottom of the blister?*

Because the Hemidesmisomes are still there

Keratinocyte Cell Cycle: Stem Cells and Transient Amplifying Cells

Cell cycle varies between *proliferating keratinocytes, stem cells and transient amplifying cells* - cell cycle times for stem cells much longer than transient amplifying cells. - not all daughter cells of a dividing transient amplifying cell must become postmitotic cells; some of the daughter cells may return to proliferating pool

Terminal differentiation

Cellular process in which cells withdraw from cell cycle and begin processes that result in unique features of a particular tissue; in epidermis, terminal differentiation of keratinocyte results in transformation into the *cornified cell of the stratum corneum*

Psoriasis

Common (1-2% affected) Manifests w/ *scaly and hyperkeratotic (adherent thickened stratum corneum) red plaques*, thought to be primarily induced by an immunologic dysfunction - Besides inflammation, involved skin in psoriasis shows *keratinocyte hyperproliferation, w/ keratinocyte transit times much less than normal* - Abnormal keratins are expressed and *keratinocyte nuclei are retained w/in the stratum corneum* (a histologic feature known as *parakeratosis*) - *Barrier dysfunction, hyperproliferation caused protein loss*, are clinical features of the disease, esp when widespread - Most Tx for psoriasis *target the immune system factors that appear to incite this hyperproliferation*

Serum Albumin

Competitive inhibitor of *steroid sulfatase* and other enzymes - in inflammatory conditions e.g. psoriasis and chronic dermatitis, there may be enough leakage of serum proteins into epidermis to diminish actions of steroid sulfatase and thus contribute to scaling

Desquamation of Cornified Cells

Complex, active process in which biochemical changes play an important role - Marked decrease in quantity of cholesterol sulfate at surface of stratum corneum *Steroid sulfatase*: enzyme present in outer layers; catalyzes loss of cholesterol from cholesterol sulfate --> exposes desmosomes so that they become vulnerable to attack of proteolytic enzymes

Desquamation

Corneal layer cells' ability to slough away - just as critical to formation and reformation - think of what would occur if desquamation stopped or was faulty. Alteration of epidermal differentiation occurs in many common skin diseases, e.g., psoriasis, warts, and atopic dermatitis (so-called eczema).

Cytoskeleton - Dynamic or Stable?

Cytoskeleton is a dynamic structure in which keratin filaments and other filaments of cytoskeleton are constantly changing their location within the keratinocyte - Moreover, the desmosomes move from spot to spot along the cell membrane as they form and then disassemble - Desmosomal formation appears to be a Ca-dependent process

Epidermolysis Bullosa Simplex - Why do mutations in a single keratin allele produce disease?

Dominant negative effect

Xerosis

Dry skin; extremely common problem affecting most people by their 60's - Manifests as scaly, rough and often itchy skin, esp during seasons of low ambient humidity *Pathogenesis*: complex, but involves decreased lipids in intercellular spaces, lack of coordinated desquamation, and dehydration of corneocytes --> As a result, stratum corneum is less flexible and prone to microfissure formation and ultimately visible cracking - breach in barrier leads to inflammatory response often associated w/ itch, leading to further trauma to the barrier - breach also provides an opportunity for invasion by pathogenic microbes, furthering inflammatory response

Keratin in the Cell

Elaborate network of keratin filaments builds up in keratinocyte to provide mechanical strength - keratin filaments loop through cytoplasmic portion of desmosomal plaques - certain regions of central rod are believed to associate w/ the carboxyl-terminal ends of desmoplakin I and II, proteins that help make the desmosome Keratin network appears to extend through epidermis as a semicontinuous structure, interrupted by nuclei, desmosomes and plasma membrane of individual keratinocytes - looping of keratin filaments through desmosomes and the opposition of desmosomes on adjacent keratinocytes provides a means to mechanically link keratinocytes together

Epidermis Thickness

Epidermis is remarkably thin and nearly uniform in thickness (0.06-0.1mm) over most of the body surface - epidermis of the *palms and soles* is much thicker; the difference is accounted for largely by the *increased thickness of the stratum corneum*

Case 3: Autosomal Recessive Congenital Ichthyosis *How does disruption of the cornified envelope result in acanthosis and hyperkeratosis?*

Epidermis knows when the barrier is disrupted and responds by proliferaiton and inflammation Point - cell envelope is necessary for barrier function and desquamation of the epidermis

Lipid Synthesis in Keratinocytes - Fatty Acids

FA's are diverted into making an unusual group of glycolipids, *glycosylceramides*, rather than phospholipids - *Glycosylceramide and ceramide synthesis* becomes more important in the *upper spinous and granular cell layers* - *Cholesterol synthesis* increases in the *granular cell* layer as cholesterol sulfate --> Sulfate moiety is cleaved from cholesterol sulfate to form cholesterol in the upper layers of the stratum corneum

Keratinocyte Differentiation: Gene Control

Genes that control keratin synthesis are influenced by *Ca concentration, VitD, retinoids and other tissue-specific factors* - e.g. the mechanisms that regulate keratinocyte differentiation cause a switch in keratin expression from K5/K14 in the basal cells to K1/K10 in the suprabasal cells

Pemphigus

Groun of autoimmune blistering diseases of the epidermis --> Superficial blisters of epidermis and oral mucosa that heal w/out scarring *acantholysis* - Before treatments, pts usually died of sepsis because of loss of barrier function - *Antibodies that bind to desmosomal proteins (desmoglein 3)* can be identified in serum of pts, and *direct binding of Ab's can be detected in affected skin* - Serum from affected pts was used to identify cadherins found in desmosomes

Epidermolysis bullosa (EB)

Group of genetic disorders in which skin blisters after mild to moderate shearing trauma - different syndromes of these disorders range from mild blister formation on feet and hands to severe and fatal blister formation over extensive areas of body and oral mucosa - Classified into 3 groups according to the location of blistering process *EB Simplex*: Blisters in Epidermis (basal layer) *Junctional EB*: Blisters in Dermal-Epidermal Junction (BMZ) *Dystrophic EB*: Blisters in Papillary (superficial) Dermis

Autosomal recessive congenital ichthyosis (ARCI).

Group of ichthyoses in which *thick, plate-like scales and red skin* are found in a *generalized* distribution - *Mutations in keratinocyte transglutaminase (TG1)* have been identified in a subset of pts w/ ARCI - *Defects in cornified cell envelope* are associated w/ these mutations, demonstrating necessity of an intact cornified envelope in epidermal function - clinical and genetic heterogeneity are found in these disorders

Epidermolytic Ichtyosis

Group of inherited disorders characterized by generalized erythema (red skin) and blister formation beginning at birth and extending into early childhood - skin is often inflamed, followed in later childhood and adulthood by extensive scaling - disorder is a form of ichthyosis, a large, heterogeneous group of inherited skin conditions - Autosomal recessive *Transglutaminase mutations* OR - Genetic defects in these disorders occur in *keratins K1, K10, and K2 of the suprabasal keratinocytes*. In a manner similar to EB simplex, the mutations occur primarily in the helix initiation and helix termination regions of the molecules.

Keratin

Heterogeneous: >50 keratin proteins, each coded for by its own gene Classified into 2 subfamilies of intermediate filaments according to charge (isoelectric point) and molecular weight: - Type I keratins: acidic - Type II: neutral to basic Basic unit of keratin intermediate filaments is a *pair of type I and type II keratin protein molecules joined together to form a heterodimer* made up of a *type I intermediate filament protein monomer wrapped around a type II intermediate filament protein monomer* - Monomers are helical, so when they are coiled around each other, they form a *coiled coil structure*

Packaging of lipids in lamellar granules

High proportion of lipid synthesized in ER and Golgi apparatus of upper spinous and granular cells appears in special transport vesicles, called *lamellar granules*: - *membrane bound, ovoid cytoplasmic organelles*, ~100X300nm diameter - *internal structure of flattened lipid vesicles that take on appearance of stacks of discs* or lamellae, containing *lipids, hydrolytic enzymes, and other proteins such as antimicrobial peptides*

Hyperproliferation

Hyperproliferation and altered differentiation of suprabasal keratinocytes in psoriasis and squamous cell carcinoma and other disorders of epidermal differentiation are associated with a *switch from K1/K10 to K6/K16 keratins*

Contribution of lipids to the formation of the stratum corneum: *Acylglucosylceramide*

Important ceramide in epidermis 1.) when contents of lamellar granule are extruded into the extracellular space, *glucose is cleaved from acylglucosylceramide* to form *acylceramide* 2.) *Acylceramide* is *covalently bonded to a cell envelope protein on surface of the cornified cells* to help form the *cornified cell envelope* This compound membrane *replaces PM in cornified layer*. The outer coating by the acylceramide molecule is thought to protect the cornified cell from attack by proteolytic enzymes secreted by microorganisms.

Intermediate Filaments

Intermediate filaments are present in most animal cell types as an important constituent of cytoskeleton - *Keratins* are intermediate filaments of epithelial cells - classified as intermediate because their diameter of *10nm* is between that of the microtubule (28 nm) and the actin filament (4 nm) - primary structure of intermediate filament proteins is the *monomer*, which consists of a *central, alpha helical, rod-shaped domain with non-helical ends*

Desmosomes

Intermediate filaments attach to the cell membrane in special cell to cell junctions called desmosomes. - can be thought of as temporary spot *welds holding keratinocytes together* - junctions are found in epithelial cells, along w/ cells of the heart and meninges and follicular dendritic cells of lymph nodes

Mutations in the K5 and K14 Intermediate Keratin Filament Genes

K5 and K14 genes in the basal keratinocytes mutated in EB simplex - affects normal structure of the keratin heterodimer, resulting in cell fragility Many mutations localized to particular regions of keratin heterodimer: the amino-terminal and carboxy-terminal ends of the α helical rod, the so- called helix initiation and helix termination sites - Structural integrity of these regions is believed to be important for the formation of tetramers and the higher order multimers of the 10nm keratin intermediate filament.

Keratinocyte Cell Cycle: Cell Cycle Time and Consequences

Keratinocyte stem cells = slow cycling cells w/ long intervals between mitoses and long-lived DNA precursor pools - has several consequences: - slow cycling stem cells better able to preserve the genome of their cell line by minimizing copying errors that occur w/ frequent mitoses - suggests that DNA in stem cells may be exposed to potential carcinogens for longer time before replication so stem cells may be vulnerable to mutagenesis - possibility also exists of correcting an inherited gene defect by inserting a desired gene into the stem cell population

Lipid Synthesis and the Formation of the Permeability Barrier

Keratinocytes synthesize lipids that form the intercellular permeability barrier of the stratum corneum. The high level of lipid synthesis is reflected by the progressive increase in lipid content of each keratinocyte as it passes through successive stages of differentiation. The level of lipid synthesis in the spinous and granular cells is closely controlled by the physical state of the permeability barrier. 1.) Lipid synthesis and degradation 2.) Packaging of lipids in lamellar granules 3.) Secretion of lamellar granule contents into the extracellular space 4.) Contribution of lipids to the formation of the stratum corneum 5.) Feedback mechanisms controlling lipid synthesis

Microtubules

Labile structures that can rapidly assemble/disassemble - Form mitotic spindle. - Provide "highways" for movement of organelles and transport vesicles in cytoplasm - Provide framework for building actin filaments

Secretion of lamellar granule lipids into the extracellular space

Lamellar granules in granular cell fuse to inner portion of cell membrane on side of the granular cell closest to cornified cells - lamellar granules secrete their lipids and proteins into the extracellular space between the granular cell and the cornified cell layers - Stacks of lamellae from individual granules unfold and are joined to create continuous *layers of lipids encircling the cornified cells throughout stratum corneum --> form much of the permeability barrier of the stratum corneum*

Desmosomal Cadherins

Link keratinocytes through the intercellular space - are closely associated w/ 2 other families of proteins w/in the cytoplasm of keratinocytes that form an intracellular plaque - intermediate filaments w/in keratinocytes are observed "looping" their ends through this electron dense plaque inside the cell adjacent to the cytoplasmic face of the cell membrane. In epithelial cells, the intermediate filaments are the keratin filaments

Lipid Synthesis

Lipid synthesis in epidermis in normal physiological conditions takes place mostly independent of influence of other body tissues. Only pathological conditions, e.g. severe hormone imbalance or essential fatty acid deficiency affect keratinocyte lipid synthesis - e.g. cholesterol synthesis in epidermis does not depend on circulating lipoproteins. *Autonomy of keratinocyte lipid synthesis* is vital to the *maintenance of a functional permeability barrier*

Keratinocyte

Majority (~80%) of cells in epidermis are keratinocytes 2 structural features, with biochemical correlates define keratinocytes: keratin intermediate filaments and desmosomes - wide range of biological activities. Along w/ *fibroblasts and endothelial cells* in dermis and *Merkel cells*, a keratinocyte constitutes a *permanent, resident cell of the skin* - these *4 cells form foundation of the skin from the earliest stages of embryonic development*

The epidermal differentiation complex

Many genes expressed in late stages of epidermal differentiation are grouped at one location on *long arm of chromosome 1 (1q21)* - include genes for intermediate filament associated proteins *profilaggrin, trichohyalin (seen in hair follicles)* and a group of similar repeating proteins - Also found in the same area are genes for many proteins that act as substrates for cornified envelope - A 3rd family of genes, also located in this region, code for a group of small calcium binding proteins, the *S100 proteins*; bc of *profound changes in [Ca2+] seen at the interface of granular and cornified layers and the Ca dependence of many of the enzymes active in epidermal differentiation, these proteins may be important in the process* - Speculation that this area may coordinately regulated, in a fashion similar to the locus control region seen on chromosome 6 in the region that controls T cell receptor rearrangement

Xerosis: Treatment

Minimizing exogenous factors contributing to xerosis and whenever possible, correcting endogenous contributors *Emollient*: helps temporarily restore a barrier to water loss and provide hydration of stratum corneum to improve pliability to prevent fissuring *Keratolytics*: contain ammonium lactate or urea to enhance outer corneocyte desquamation, and humectants which attract moisture into the stratum corneum *Topical steroids and antihistamines*: used to suppress inflammatory processes that contribute to itch and barrier disruption Tx of xerosis is a HUGE industry, though in many cases the factors caulesing it are wholly preventable

Keratinocyte Cohesion

Most of stratum corneum, the keratinocytes remain tightly bind each other, but in outermost stratum corneum, keratinocytes separate from each other and desquamate at the surface Several structures and molecular substances contribute to cellular cohesion: - *Desmosomal attachment structures*: between keratinocytes; maintained until they are degraded closest to the surface of epidermis - *Intercellular lipid lamellae*: contribute to keratinocyte cohesion - *Cholesterol sulfate*: helps bond keratinocytes together; its presence in cornified envelope may protect desmosomes from proteolytic enzymes

Terminal differentiation - aka Cornification/Keratinization

Numerous cellular/biochemical processes that culminate in formation of the anucleate stratum corneum: *outward migration, loss of ability to proliferate, synthesis and secretion of lipids into extracellular space, loss of nucleus and cytoplasmic organelles, aggregation of keratin intermediate filaments, formation of a thickened cell envelope, and the ultimate sloughing* of cornified epidermal cells - Although mitoses are observed in suprabasal keratinocytes, most of the cells in the spinous cell layer have entered terminal differentiation

Cornified Envelope Formation

One important change seen in keratinization is *alteration in cell membrane permeability leads to rapid changes in intracellular ion concentrations* - e.g. change in influx of Ca2+ ions that activate a number of enzymes, including *transglutaminases* These *enzymes cross link structural proteins in cytoplasm to proteins at cell periphery, creating a thickened structure* called the *cornified cell envelope*

EB Simplex

One sees trauma-induced blisters - blisters heal w/ minimal scarring, but pigmentary changes are often seen. The blisters form in the *basal layer of the epidermis*. Electron microscopy of the epidermis shows *clumped intermediate filament proteins within the basal keratinocytes and lysis of these cells*

Keratinocyte Cell Cycle: Postmitotic Cells

Postmitotic cells are the only cells in the upper spinous, granular and cornified cell layers - mitoses not observed in these layers - Postmitotic cells that may be present in basal layer cannot be distinguished from non-dividing transient amplifying cells or stem cells Many keratinocytes in basal and lower spinous layers are transient amplifying cells - cell division does take place in these locations

Keratin Filament Aggregation

Process of differentiating keratinocytes in which keratin intermediate filaments and filaggrin (filament aggregating protein) are separately synthesized in viable cell layers and then aggregate together in the cornified cell - Intermediate filament-associated proteins interact w/ intermediate filaments, including keratins - One function of these proteins is to organize the filaments into macromolecular structures called *Macrofibrils*: prime example of this is association of filaggrin w/ keratin filaments - in lower part of cornified cell layer, filaggrin aggregates keratin filaments into bundles w/ noncovalent bonds. The aggregated intermediate filaments are subsequently covalantly cross linked by disulfide bonds.

Keratinocyte Cytoskeleton

Provides mechanical strength and means for locomotion, cellular division and rapid communication between cell membrane and nucleus Made up of *microtubules, actin microfilaments and intermediate filaments* - In keratinocytes intermediate filaments are keratins

Case 4: Pt has congenital caling skin disease. Scales are "dirty brown" and located everywhere but accentuated on extensor surfaces of his skin. - Histo shows that his granular layer varies, stratum corneum is thickened and compacted *How does this happen?*

Pt is not sloughing skin fast enough

Cornified Cell Envelope

Rigid structure of cross-linked molecules that forms at periphery of the cornified cell, internal to the PM, and ultimately replaces PM of cornified cell as it is covalently crosslinked to acylceramide in the intercellular space - rigidity makes cornified cell more durable because of its resistance to external mechanical forces and to the chemical effects of detergents, strong alkali, organic solvents and reducing agents - may also contribute to the permeability barrier

Contribution of lipids to the formation of the stratum corneum: Enzymes

Several hydrolytic enzymes are packaged in the lamellar granule which help modify cholesterol, cholesterol esters, free fatty acids, and ceramide molecules to help form the permeability barrier of the stratum corneum. - When these lipids are dissolved out of stratum corneum by acetone or other solvents, the permeability barrier of the epidermis is lost

Feedback mechanisms controlling lipid synthesis

Sophisticated feedback mechanisms control synthesis of lipids - Function of the permeability barrier directly controls production of cholesterol and sphingolipids - also influences DNA synthesis and cytokine release in viable keratinocytes - *When permeability barrier is impaired or disrupted, DNA synthesis increases and cytokines are released from keratinocytes, initiating an inflammatory cascade*

Epidermis

Stratified squamous, keratinized epithelium w/ upper layers containing anucleare stratum corneum cells - separated from demis by BM at Dermal-Epidermal Junction (DEJ)

Lipid Synthesis in Stages

Synthesis of lipids changes significantly as keratinocytes progress through each stage of differentiation - *Polar lipids are replaced by neutral lipids creating a more hydrophobic barrier*

Barrier Formation of the Epidermis

Think of epidermis as forming a series of proteins that are metabolized that bind epidermal cells and then allow them to desquamate - in the process lipids are formed that help to form the barrier and shed epidermis - all orchestrated in elaborate series of events

X-linked recessive ichthyosis

Type of ichthyosis - Incidence: ~1:2,000-9,500 males - Large, dark scales, tacked down in center, often seen on trunk, extremities, and sides of neck - NO Erythema (redness) - Cornified layer is thickened - *Deficiency in enzyme steroid sulfatase* --> *accumulation of cholesterol sulfate in stratum corneum that results in failure to desquamate the cornified cells*

Actin

Ubiquitous protein found in cytoplasm of all cells; Actin filaments provide *mechanical means for cell migration in wound healing and separation of daughter cells during mitosis* - Similar to the microtubules, actin filaments are *labile structures*

Loss of Nucleus and Cytoplasmic Organelles

Viable granular cell is abruptly transformed into nonviable cornified cell: -* nucleus and cytoplasmic organelles are swept away by activated enzymes* that facilitate degradation of the structural and functional proteins, lipids and carbs of the nucleus and organelles in cytoplasm; Result is *formation of a normal stratum corneum* - In this process *keratin cytoskeleton, precursors of filaggrin , and cell envelope that is created during these changes are NOT destroyed* - unknown what protects these

Actin: Keratinocyte Phenotype Change

When skin is wounded w/ loss of the epidermis, keratinocytes adjacent to wound edges change their phenotype - Keratin filaments retract toward the nucleus, the desmosomal attachments between keratinocytes disappear and actin filaments appear in cortical region of cytoplasm adjacent to cell membrane - Keratinocyte in this phenotype is able to migrate over the wound surface

Case 4: Pt has congenital caling skin disease. Scales are "dirty brown" and located everywhere but accentuated on extensor surfaces of his skin. - Histo shows that his granular layer varies, stratum corneum is thickened and compacted *What is the diagnosis?*

X-Linked Recessive Ichthyosis

Epidermolytic Ichtyosis: Histopathology

disruption of pattern of differentiation of suprabasal keratinocytes w/ *large, irregular keratohyalin granules and clumping of the keratin intermediate filaments*

Lipid Synthesis in Keratinocytes - Phospholipids

most dramatic changes occur in *phospholipids, w/ rapid decline in granular cell layer and disappearance in cornified cell layer* --> as granular layer differentiates into cornified layer, *phospholipids are replaced by cholesterol, fatty acids and other neutral lipids* - *Total absence of phospholipids in cornified layer* reflects *loss of plasma membrane and metabolic machinery of viable keratinocytes*

Keratinocyte junctions

specialized adhesion structures to anchor epi to dermis and maintain cohesion between keratinocytes - closely opposed keratinocytes must be able to resist shearing forces *Hemidesmosomes and Integrin family* adhesion molecules: attach basal layer of epidermis to dermis *Desmosomes and Adherense Junctions*: keratinocytes in all layers of epidermis maintain strong attachments to each other w/ desmosomes and adherens junctions - Both of these junctions rely on members of cadherin (calcium-dependent adherence molecules) adhesion molecules - Tight (occluding) and communicating (gap) junctions not discussed