Keratinization and Cornification (including diseases) Flashcards
Describe keratinization
Process of cytodiferentiation that the keratinocyte undergoes when proceeding from their post germinative state (stratum basale) to the finally differentiated, hardened cells of the stratum corneum
Describe the mammalian cytoskeleton
- Primary regulator of cell shape due to its intrinsic mechanical properties allowing it to resist deformation as well as provide contractility
- Generates forces on the surface of the cell to influence cell morphology
- Comprised of actin filaments, intermediate filaments, and microtubules
Describe actin filaments
Also called microfilaments
7 nm in diameter
Describe intermediate filaments
Provide the scaffold
Keratins 7-12 nm diameter
Span the cytoplasm and attach at desmosomes
Describe microtubules
A and B tubulin
20 nm in diameter
Describe the size, charge, and solubility of keratins
- Heterogenous in size (40 to 70 kDa)
- Heterogenous in charge (4.7 to 8.4 pI)
- All of them are insoluble
How many functional genes for keratins have been identified in humans?
54 (28 Type I and 26 Type II)
How well are keratins conserved in mammals?
Nearly perfectly
Where can you find high numbers of keratins?
Most surface exposed squamous epithelium
Describe the rim and spoke hypothesis
Keratins make a pan-cytoplasmic network from the nucleus surface to the periphery of the cytoplasm where they are membrane anchored at the sire of cell-matrix and cell-cell adhesion (ex. hemidesmosomes and desmosomes)
Where are keratins concentrated in polarized epithelial cells?
They are asymmetrically organized with keratins concentrated at the cytoplasmic periphery at the apical pole
Describe Type I keratins
- Slightly smaller and more acidic than Type II
- K9-19 in the epidermis
- HA 1-8 in hair follicles/nails
Describe Type II keratins
- Slightly larger and more basic/neutral than Type I
- K1-8 in the epidermis
- Hb 1-6 in the hair follicle/nail
What is the ratio of Type I to Type II keratins in mature intermediate filaments?
1:1 molar ratio so most Type I and Type II keratin genes are regulated in a pairwise, tissue type-related, and differentiation related fashion
Elaborate on the nomeclature of keratins (old vs new)
- Original naming scheme from 1982 and listed keratins 1-19, Ha, and Hb keratins
- Now there is a new scheme for humans that divides the genes into three categories (numbers go much higher and divide out non-human ones)
Where is the mutation in curly coat/sphynx/Devon rexes?
Keratin 71
What are keratin 71 mutations associated with?
Wavy/curly/wooly phenotype in dogs, cats, and humans
What are keratin 74 mutations associated with?
Wavy/curly/wooly phenotype in humans
What are keratin 25 mutations associated with?
Wavy/curly/wooly phenotype in horses, humans, and mice
What are keratin 27 mutations associated with?
Wavy/curly/wooly phenotype in cattle and mice
What are the primary keratins associated with the stratum basale?
K5/K14 (also K1/K6 in dogs)
Describe the structure of keratin (singular, not yet an intermediate filament)
- Central alpha-helical rod domain
- Amino (N)-terminal head and carboxy (C)-terminal tail that exhibit “glycine loops”
- Head and tail are cystine rich and protease accessible to allow for interactions
- Some middle parts of the rod section are glycine and proline rich for flexibility
Describe the formation of an intermediate filament aka polymerization
- A type I and type II keratin combine to form parallel dimers (“coiled-coil”)
- Two of these combine to form staggered, antiparallel tetramers (“protofibril”)
- Two protofibrils combine (stacking end to end) to form a protofilament
- Four protofilaments form a grouping that is crossed linked by disulfide bonds and is called a keratin intermediate filament
What is filaggrin?
- Filament aggregating protein
- Cationic
- Histidine rich
- Interacts with intermediate filaments but not other components of the cytoskeleton
- Organizes keratin intermediate filaments into tight bundles
What is profilaggrin?
- The precursor to filaggrin
- Giant, heavily phosphorylated, and insoluble
- Main constituent of keratohyalin granules
- Has filaggrin repeats flanked by two truncated filaggrin repeats by a C-terminal and an N-terminal (that has two Ca2+ binding motifs)
- Synthesis starts in stratum spinosum shortly after expression of K1 and K10 start
What is filaggrin degraded into?
Degraded in the stratum corneum by caspase-14 –> hygroscopic free amino acids –> natural moisturizing factors pyrrolidone carboxylic acid (PCA = moisture) and urocanic acid (UCA = photoprotection)
Which keratin regulates protein synthesis and cell size in wound-proximal keratinocytes?
K17
Which keratins are primary present in the suprabasal layers?
K1/K10 (dogs also have K4, K15/16)
- keratin intermediate filaments are organized with more bundling when these appear
Structure of keratin
Think about the structure of keratin intermediate filaments
What is the cubic rod-packing and membrane templating model?
- Disputes keratin self-assembly
- Proposes membrane template organizes keratin assembly
- Explains strength and water-holding capacity of epidermis
- Explains findings with cryotransmission electron microscopy on fully-hydrated epidermis
Which Type II keratin is expressed at later stages of differentiation (ie. starting in the granular layer)?
K2e (and K11?)
Which keratins are normally restricted to wound repair but may be upregulated in psoriasis and other hyperplastic disorders?
K6a, K6b, K16, and/or K17
Which keratin is present in the stress bearing ridges of the palm/sole and provides a more resilient cytoskeleton?
Type I K9
Which keratins have more pliability than K1, K9, and K10; function as a hinge; and are found in the secondary ridges of the human palm/sole?
K6a, K16, and K17
What are the functions of filaggrin?
Aligns KIFs and flattens corneocyte
Contributes to hydration once degraded to NMF
What are the epithelial keratins/genes in the new human nomenclature?
- Type I is 9-28
- Type II is 1-8 and 71-80
What are the hair keratins/genes in the human nomenclature?
- Type I is 31-40
- Type II is 81-86
What are the keratin pseudogenes in the new nomenclature?
- Type I is 221+
-Type II is 121-220
What is the structure of keratin proteins?
Central alpha-helical rod domain
Amino (N)-terminal head and carboxy (C)-terminal tail that exhibit “glycine loops”
What are the non-human epithelial and hair genes in the new nomenclature?
- Type I 41-70
- Type II which is 87-120
What is the classic model of keratin filament assembly?
Type I and II keratins→
Staggered, antiparallel heterodimers x 2→
Tetramers end to end→
Protofilaments x 2→
Protofibrils x 4→
KIF
How many protofilaments go into a KIF?
8
How are keratins cross-linked?
Disulfide bonds
(also bundled by filaggrin)
Where is profilaggrin made?
In the stratum spinosum after starting production of K1/K10
How many filaggrin repeats does profilaggrin have in dogs?
4
How many filaggrin repeats does profilaggrin have in humans?
10-12
How many filaggrin repeats does profilaggrin have in mice?
12-20
What happens to filaggrin in the stratum granulosum?
Profilaggrin is released from keratohyalin granules –>
Profilaggrin cleaved into filaggrin units –>
Filaggrin bundles KIFs into tight arrangement
What happens to filaggrin in the stratum corneum?
it is enzymatically degraded into NMFs
What do SASPase and Prss8 do?
Degrade profilaggrin to filaggrin molecules
What does peptidylarginine deiminase do?
Takes filaggrin off of KIFs
What happens in SG1?
Keratin intermediate filament assembly
Cornified cell envelope construction
Lamellar body exocytosis and creation of corneocyte lipid envelope and intercellular lipid lamellae
What do the linker domains in the keratin rod region have that helps to confer flexibility?
increased amounts of glycine and proline
What part of the structure of K5 and K14 have been found to have mutations that are linked to EBS?
linker domains (specifically L12 - very middle) in the rod
How do acidic keratins differ from others?
Don’t have the H2 region in their tail
How do the “head” and “tail” of hair and claw/nail keratins differ from others?
Are cysteine rich so that they can have more disulfide bonds for stability
What parts of the keratin structure are most important for protein interactions?
Head and tail
What happens if there is a mutation in the “tail” of K1?
Keratin doesn’t bundle or retract the cytoskeleton from the nucleus
What happens to non-paired keratins?
They degrade quickly
What keratins are expressed in the basal layer?
K5/14
What keratins are expressed in the spinous layer?
K1/10
Has low amount of K6/16 unless there is a wound or inflammation
What is cornification?
The process by which keratinocytes undergo terminal differentiation
- Nucleus and cytoplasmic organelles are hydrolyzed
- Marked reduction of cytosolic water
- Flattened shape
What is the cornified envelope?
Proteinaceous wrapper over a dense core of keratin
What is the cornecyte lipid envelope?
Overlying monolayer of ceramides
What is filaggrin degraded into?
Natural moiusturizing factor
Free amino acids (glutamine, arginine, histidine)
Urocanic acid - UV protection
Pyrrolidonecaroboxylic acid (moisturizing factor)
Lactic acid, citrate, urea
What keratins are in the stratum basale?
K5 and K14 (humans and dogs)
K1 and 6 in dogs
What is the epidermal proliferative unit?
10 basal cells: 1 stem cell surrounded by rapidly proliferating transit amplifying cells
What makes up the “spines” of the stratum spinosum
Desmosomes
What keratins are expressed in the stratum spinosum
K1 and 10
K4 and K15/16
What proteins do the cells of the spinous layer produce
Involucrin and (pro)fillagrin
+ form lamellar bodies which contain lipids, proteins and enzymes
What layers is the stratum granulosum split into?
SG1
SG2 - tight junctions seal intercellular spaces
SG3
At what layer do tight junctions seal intracellular spaces?
Stratum granulosum SG2
Where in the epidermis would you find keratohyalin granules?
Stratum granulosum
What are keratohyalin granules
These basophilic granules are accumulations of the synthesized proteins, primarily profilaggrin, loricrin and keratin filaments, needed for the construction of the stratum corneum.
What is filaggrin
protein involved in the aggregation of keratin
What is loricrin
cysteine rich flexible protein that is made in the granular layer and is a key component of the corneocyte cornified envelope
What change triggers the assembly of the keratin intermediate filaments and construction of the cornified envelope?
Increased intracellular calcium
Where do the keratin intermediate filaments and cornified envelop develop in the epidermis?
Stratum granulosum
Where do epidermal lamellar bodies secrete their contents?
Extracellular spaces at SG1 and the apical surfaces of SG2 cells
What does the cornified cell envelope replace
Plasma membrane
What happens to the keratinocyte as it transitions from the stratum granulosum to the stratum corneum
1) Organelle activities cease
2) Cell Flattens with keratin intermediate filament bundling
3) Degradation of nucleus and organelles
What are corneocytes
Dead, flattened, terminally differentiated keratinocytes that have lost all nuclei and organelles
What makes up the CLE?
ceramides
free fatty acids
cholesterol
What are corneocytes
flattened, keratin-filled cells, lacking organelles and plasma membranes, that are coated with an insoluble proteinaceous layer known as the cornified cell envelope (CE).
What makes up the tripartite structure of keratin proteins?
α-helical rod domain
an amino-terminal head
a carboxy-terminal tail
What are features of Type 1 keratins?
acidic
smaller than type II
include K9-19 in the epidermis
What are features of Type 2 keratins?
basic
larger than type I
include K9-19
How many filaggrin monomers are present in dogs
4
Where does enzymatic cleavage of profilaggrin into filaggrin occur?
Transition of granular to cornified layer
What enzymes cleave filaggrin?
Caspase-14
Bleomycin hydrolase
What are the products of filaggrin degradation
Natural moisturizing factors:
- urocanic acid (photoprotection, hydration, antimicrobial)
- pyrrolidone carboxylic acid (hydration, antimicrobial)
Name the main components of the cornified cell envelope
Involucrin
Loricrin
Envoplakin
Periplakin
Small proline-rich peptides
(Also proteins like S100 and trichohyalin, cystatin, elafin and annexin)
What is necessary for transglutimase activity
Increase intracellular calcium
Where is TG1 located
Membrane
Where is TG3 located
Cytoplasm
Which protein is the major structural component of the CE
Loricrin (70% of mass) - cystine-rich highly flexible protein with glycine loops and is a fundamental promoter of terminal differentiation
What is the function of TG3
Crosslinking of loricrin and SPRs
What is the function of TG1?
Cross link oligomers to the developing CE
What is the corneocyte lipid envelope?
Hydrophobic intercellular ω-hydroxyceramides lipids that fill the spaces between the corneocytes
What are the major lipid classes of the stratum corneum extracellular lipid layer?
ceramides (50%)
free fatty acids (15%)
cholesterol (25%
What are ceramides?
amide-linked fatty acids attached to long chain amino alcohols called sphingoid bases
What are the contents of Lamellar bodies?
lipid precursors, including glucosylceramides, sphingomyelin, phospholipids, and cholesterol sulfate,
enzymes: β-glucocerebrosidase, acidic sphingomyelinase, phospholipase A2, and steroid sulfatase, respectively
From what is cholesterol synthesized?
Acetate - from lower epidermis
Which enzyme metabolizes cholesterol suphate to cholesterol?
Steroid sulphatase (contained within lamellar bodies)
What is the role of cholesterol sulfate?
Inhibits serine proteases (kallikreins) involved in desquamation
Which enzymes control desquamation?
Kallikreins (serine proteases)
Cathepsins (cysteine proteases)
What are desmosomes?
Specialized keratinocyte junctions that link KIFs to sites of intercellular adhesion, tethering adjacent cells and providing structural strength for the epidermis
What are the three families of proteins that comprise desmosomes?
❖ Cadherins – Transmembrane molecules that form a calcium-dependent adhesive interface between neighboring keratinocytes
❖ Armadillo protein family – Bind to the cytoplasmic portions of the cadherins
❖ Plakin protein family - Binds KIFs to armadillo family proteins
What molecules are contained within corneodesmosomes?
desmoglein 1
desmocollin 1
corneodesmosin (CDSN)
Which molecules contain corneodesmosin?
Keratins
Loricrin
Corneodemsosomes
CDSN forms structural motifs called “glycine loops” which have been suggested to mediate reversible and adjustable intermolecular adhesion by acting like Velcro.
Where are tight junctions located
SG2
What are tight junctions made up of?
claudins
occludins
junctional adhesion molecules (JAMs)
zonula occludens (ZO) proteins.
What are claudins?
Primary transmebrane molecules that function as a zip lock to seal the intercellular barrier, and claudins 1, 4, and 7 are expressed in the epidermis.
What is desquamation?
process by which corneocytes are shed from the epidermal surface
Which enzymes mediate desquamation?
Promoters:
kallikreins (KLK, a family of serine proteases)
cathepsins (cysteine proteases)
Inhibitors:
KLK inhibitors LEKTI (lymphoepithelial Kazal-type-related inhibitor, encoded by SPINK5 gene)
cholesterol sulfate
At what layer is the concentration of Ca2+ highest in the epidermis
Stratum granulosum (low in basal and spinous layers and decreases again in stratum corneum)
What processes are controlled by high intracellular calcium?
lamellar body secretion
transglutaminase activity
cleavage of profilaggrin to filaggrin
What are the barrier effects of filaggrin mutations?
- decreased corneocyte osmolytes
- decreased organic acids (UA, PCA)
- decreased corneocyte hydration
- increased water loss
- increased pH
Which cytokines down regulate filaggrin expression?
Th2 cytokines
What happens to extracellular lipids in humans with AD?
Composition and architecture are disrupted
◦ Lamellar body secretion abnormal and some retained in corneocytes
◦ Reduction in
- Total lipids in stratum corneum
- Proportion of long chain ceramides
- Chain length of fatty acids in ceramides and free fatty acids
What happens to extracellular lipids in dogs with CAD?
- Similar to humans
- Decreased total lipids, fatty acids, and ceramides in AD
- Abnormal, disorganized, and reduced intercellular lipid lamellae
What happens in the scaffolding stage (1st stage) of cornified envelope construction?
Envoplakin, periplakin, and involucrin move to cell membrane
Transglutaminases link involucrin to other proteins to form scaffold
Stage 1 of CE development, scaffolding
What happens in the lipid envelope stage (2nd stage) of cornified envelope construction?
Lamellar bodies fuse to cell membrane and secrete lipids and enzymes
Stage 2 of CE development, lipid envelope
What happens in the reinforcement stage (3rd stage) of cornified envelope construction?
-Loricrin comes in
- TG1 links loricrin to involucrin scaffold and involucrin to ω-hydroxyceramides to form CLE
- TG3 links loricrin to other cytoplasmic proteins
What are transgluaminase enzymes?
◦ Catalyze formation of N(ε)-(γ-glutamyl)-lysine isopeptide bonds
◦ Highly resistant to proteolytic enzymes
◦ Calcium dependent
Stage 3 of CE development, reinforcement
What happens in the mature corneocyte (final stage of development)?
CE replaces cell membrane with loricrin inside, involucrin outside
KIFs are linked to CE
Mature corneocyte
Put these in order from inside to outside in the mature corneocyte: involucrin, keratin, loricrin
keratin –> loricrin –> involucrin
What is the “mortar” of the stratum corneum?
Composed of ceramides, free
fatty acids, and cholesterol
Extracellular lipid matrix
◦ Corneocyte lipid envelope (CLE)
◦ Intercellular lipid lamellae
What happens in SG1?
Keratin intermediate filament assembly
Cornified cell envelope construction
Lamellar body exocytosis and creation of corneocyte
lipid envelope and intercellular lipid lamellae (at the SC interface)
In which layer of the skin do lamellar bodies deliver lipid precursors and enzymes?
SG1
and stratum corneum interface
What are the contents of lamellar bodies?
Lipid precursors
Enzymes
Lipid hydrolases
Steroid sulfatase
Kallikreins
Cathepsins
Corneodesmosin
Antimicrobial peptides
What primarily makes up the corneocyte lipid envelope?
ω-hydroxyceramides
What primarily makes up the intercellular lipid lamellae?
Ceramides, free fatty acids, and cholesterol
What are the sphingoid bases?
- phytosphingosine
- sphingosine
- dihydrosphingosine
6-hydroxylsphingosine
What are the features of the ω-hydroxyceramides that make up the corneocyte lipid envelope?
◦ Unique to stratum corneum
◦ Ultra long chain (ULC) fatty acids
◦ Linoleic acid (ω-6 EFA) is component
◦ Bonded to involucrin in CE
What are the roles of the corneocyte lipid envelope?
Forms scaffold for intercellular lipid layers of stratum corneum
Key structure for skin barrier function and ichthyosis pathogenesis
What links corneocytes to each other?
Corneodesmosomes
How are corneodesmosomes different from desmosomes?
They have corneodesmosin
◦ Serine and glycine rich protein
◦ Forms glycine loops
◦ Secreted from lamellar bodies
◦ Attaches to desmoglea
◦ Key to stratum corneum cohesion
What is desquamation?
Shedding of corneocytes
Regulated enzymatic cleavage of corneodesmosomes
Maintains epidermal homeostasis
Removes microorganisms, preventing colonization
Where do the enzymes and inhibitors involved in desquamation come from?
Lamellar bodies
What are the primary proteases involved in desquamation?
kallikreins (KLK 5, 7) and cathepsins
What are the inhibitors involved in desquamation?
LEKTI (lymphoepithelial Kazal-typerelated inhibitor)
Encoded by SPINK5
What is the role of pH in desquamation?
More desquamation at more acidic (low) pH
- LEKTI-1 binds strongly to KLK at neutral pH (deep stratum corneum)
- pH decreases in the superficial SC which releases KLK from LEKTI
- released KLK can now cause desquamation
pH normally drops more superficially in stratum corneum
What is the genetic variant for ichthyosis in American bulldogs?
Autosomal recessive mutation in NIPAL-4 (ichthyin, involve in fatty acid synthesis, increases non-esterified free fatty acids –> creates cytotoxicity and damage to cell membranes)
CLE absent or attenuated
Appears before weaning
Generalized white scale, ventrally orientated erythema and adherent scale
Histologically looks like that in goldens with more inflammation and can be confused with AD in adults
What is the genetic variant for ichthyosis in Golden retrievers?
Type 1: Autosomal recessive mutation in PNPLA-1 (involved in fatty acid acid synthesis)
CLE absent or attenuated
Usually appears <1yr but can manifest in adults and be transient in puppies
Has large white to grey scale
Type 2: ABHD5 in a family in the US
What is the genetic variant for ichthyosis in Jack Russel terriers?
TG1 (transgluaminase 1)
Autosomal recessive
CE markedly attenuated
What keratins are primarily expressed in the granular cell layer?
K1/10 (and 2/11)
What are lamellar granules?
- Secretory granules secreted into the intercellular space of the stratum granulosum
- Contain precursors of stratum corneum lipids (glycoproteins, glycolipids, phospholipids, free sterols, glycosylceramides, acid hydrolases) and proteins that respond to signals that occur during the transition from the granular to cornified layer
- Are secreted via exocytosis
What does transglutaminase 1 do to plankins and ivolucrin?
Links them together by forming NE-(y-glutamyl) lysine isopeptide crosslinks
- due to increase in Ca2+
How much of the cornified envelope does loricrin make up?
80%
What are the KIFs present in the cornified cell envelope?
K1/10 (and 2e - esp thickened sites)
What percent protein mass of the epidermis do filaggrin and KIFs make up?
80-90%
What enzymes are responsible for the extrusion of lamellar bodies and the release of their contents?
acid hydrolases
What are the primary components involved in the complex changes in the lipid composition after the extrusion of lamellar bodies?
- β glucocerebroside
- acid sphingomyelinase
- phospholipase A
What are the transglutaminases involved in the formation of the cornified envelope?
TG1
TG3
TG5
What mineral are transglutaminases dependent on?
Ca2+
What is the function of S100 in the cornified envelope?
Are Ca binding proteins that transmit Ca dependent regulatory signals
Are substrates for TG1 and 2
What is the main component of the epidermal cornified envelope?
Loricrin (70-85%)
What does loricrin crosslink?
Other loricrin and SRP
Which transglutaminases are involved in loricrin oligomerization by way of interchain crosslinks?
TG1 and TG5 initially then compacted by TG3
Which KIFs are crosslinked by TGs?
Type II (K1, K2e, and K5)
What is involucrin?
Created in spinous layer
Glutamine-rich protein (why it crosslinks with transglutaminase-1)
Connects corneocytes to extracellular lipid layer, scaffold
First protein that is deposited to create the CE so ends up outside
What are the 4 distinct cellular events in the process of cornification?
1) Keratinization
2) Keratohyalin synthesis
3) Formation of the insoluble SC cornified envelope
4) Production of lipid-rich intercellular domains
What is the epidermal differentiation complex?
Cassette of genes important in growth and differentiation of keratinocytes
2Mb region of the human chromosome 1q21
What are small proline-rich proteins?
Members of the epidermal differentiation complex 1q21
Crosslink with one another and loricrin in the CE
SPR1
SPR2 (most rigid, induced with aging)
SPR3 (most flexible)
Where on the body are more small proline-rich proteins found?
oral mucosa
What are plankins?
Envoplakin, Periplakin
Proteins from desmosomal plaque expressed in superficial layers of epidermis –> incorporated into corneodesmosomes in SC
Attachment site for ceramides in SC
What are minor molecules in the cornified envelope that play a role in barrier function?
Cystatin A, elafin, annexin, S100A
What is the function of ceramides?
Most important lipid component for lamellar arrangement in the SC
Barrier function
Plasticizing the SC to allow stretching and bending
What are the types of fatty acids?
esterified ω-hydroxy fatty acids
α-hydroxy fatty acids
nonhydroxy fatty acids
What is the major ceramide in the epidermis?
w-OH ceramide (hydroxyceramide)
What is the rate limiting enzyme for production of ceramides?
serine-palmitoyltransferase (first step)
What is the function of cholesterol in the stratum corneum?
Part of plasma membrane and intercellular lipid lamellae in SC
Cholesterol sulfate inhibits proteases, impairs desquamation
Where is cholesterol synthesized in the epidermis?
Synthesized in lower epidermis from acetate
Then incorporated into lamellar bodies
What is the rate limiting step in cholesterol synthesis?
hydroxymethylglutaryl (HMG)-CoA reductase
What happens to the concentration of cholesterol in the stratum corneum during permeability barrier repair?
It increases
What converts cholesterol to cholesterol sulfate?
Cholesterol sulfotransferase aka sterol sulfatase
What is squalene broken down into?
Cholesterol
What are the two primary products that acetyl-CoA can be broken down into?
Free fatty acids (acetyl-CoA carboxylase starts it)
Cholesterol (HMC-CoA synthase starts it)
What are the recognized essential fatty acids?
Omega 6s:
Dogs, humans, and cats - linoleic acid (LA, 18:2n-6)
Just cats - also arachadonic acid (AA, 20:4n-6) for cats due to limited Δ6-desaturase
Omega 3s:
Only one needed in humans: α-linolenic acid (ALA, 18:3n-3) acids
Dogs and cats: eicosapentaenoic acid (EPA, 20:5) and docosahexaenoic acid (DHA, 22:6) due to poor conversion of ALA to EPA and DHA
What do linoleic acid and α-linolenic acid do when supplied in the diet?
Are converted to long chain PUFA (>20 carbons with >2 double bonds) by desaturase and chain elongation enzymes in endoplasmic reticulum
What do long chain PUFAs do?
Serves as precursor for eicosanoids, leukotrienes or prostanoids as well as structural and functional lipids
What is the rate limiting enzyme in conversion of EFA (18 carbons) to longer PUFAs?
Δ6-desaturase in the ER
First step (also works further down on 24 carbon PUFAs)
*Cats have limited activity = decreased capacity to make arachidonic acid
How is Acetyl CoA made?
by glycolysis of carbohydrates
- instead going into Krebs, citrate is broken down in cytoplasm
How do fatty acids affect the pH of skin?
Acidify the stratum corneum
What is the difference between saturated and non-saturated fatty acids?
Saturated = no double bonds (every carbon is saturated with hydrogen)
Unsaturated = a double bond
What do deficiencies in omega-6 fatty acid do?
Cause skin and coat abnormalities, reproductive problems, and failure to thrive
What happens in ichthyosis vulgaris?
Autosomal semidominant mutation on FLG so filaggrin is absent
Common and typically mild in humans –> named because it is common
Fine scale with superficial fissuring and relative flexural sparing but worse on lower extremities with hyperlinear palms and soles
Histopathology: hyperkeratosis, reduced or absent granular layer
What is lamellar ichthyosis?
Autosomal recessive mutation in TGM1 in a JRT
Poor/no cornified envelope formation
Large dark scale and often have secondary Malassezia infections
Also reported in humans but is now considered a phenotype
At birth in humans, “collodion baby” is encased in coherent scale
Affected individuals may have ectropion, eclabium
What is x-linked ichthyosis?
Recessive, x-linked STS deletion so reduction in cholesterol activity
Fine to large scales that are black and brown, comma shaped corneal opacities, cryptorchidism or delayed labor
Palms and sols are spared
Not reported in animals yet
What gene causes Harlequin ichthyosis?
Autosomal recessive, ABCA12 gene –> defective ATP binding cassette subfamily A member 12 = altered lipid metabolism, membrane transport resulting in lamellar bodies with lipids that are TOO polar
What is the clinical and histopathologic presentation of Harlequin ichthyosis?
Clinical: Markedly thickened skin with geometric, deep fissues; survivors develop severe congenital ichthyosiform erythroderm phenotype, often failure to thrive due to poor feeding, neonatal sepsis
Histopath: markedly thickened orthokeratotic SC with acanthosis
What animals (and breeds) are reported to have Harlequin ichthyosis?
Bovine: Holstein-fresian, brown swiss, norweigan red polls
Greater kudu, llama, koala, wallabies
Harlequin ichthyosis
What causes the basket-weave appearance of the stratum corneum on light microscopy?
an artifact created by the loss of intercellular lipids during tissue processing
What 3 extracellular lipids affect the permeability barrier of the stratum corneum?
cholesterol
free fatty acids
ceramides
What is the current functional model used to understand how genotype drives disease phenotype?
Barrier-driven hypothesis
How does epidermal hyperplasia influence lipid production?
Epidermal hyperplasia ensues to produce more cells, which in turn generate more lipids, restoring hydrophobicity
What happens to lipid production when the skin barrier is disturbed and the epidermis needs to generate repair?
Increases
Which pathogenesis-based therapy has been used in the rare x-linked dominant disorder, congenital hemidysplasia with ichthyosiform erythroderma and limb deficits (CHILD syndrome)?
Topical cholesterol in conjunction with systemic cholesterol lowering drugs (statins) because it is a failure to convert lanosterol to cholesterol
What does premature keratinocyte death release?
DAMPs
Lamellar ichthyosis in a JRT
Mutation in TG1
C= a normal age-matched dog cornified envelope
D= absence of cornified envelope in affected dog
Black arrows show corneodesmosomes
What a mutation in which gene has been found for Norfolk terriers with epidermolytic ichthyosis?
KRT10
Is a mild form that only causes swelling/lysis in the upper spinous/granular cell layer and causes generalized, pigmented hyperkeratosis
What are ceramides converted to in the cytosol of lamellar bodies?
Glycosylceramides, sphingomyelin and phospholipids
but then the first two get converted back once expelled
What converts phospholipids to free fatty acids?
phospholipase A2 (increased activation with inflammatory stimuli)
What is the ratio of ceramides, free fatty acids, and cholesterol in the SC lipid lamellae?
equimolar
What is the pH of dog skin?
highly variable and typically neutral (6.8–7.7) and therefore more basic than humans
What is the pH of human and hairless mouse skin?
acidic surface (5–5.5)
What are primary scaling disorders caused by?
anomalies in genes that encode enzymes and structural proteins (e.g. TG1), or processes involved in lipid metabolism or lipid transport
What are secondary scaling disorders caused by?
any insult that drives hyperkeratosis
What is the onion skin approach?
A way to diagnose disorders of cornification in humans
(i) clinical features
(ii) skin localization with or without involvement of other organs
(iii) heritability
(iv) mutation
(v) loss of the target protein or enzyme
What is one of the few instances that electron microscopy can be helpful as the sole diagnostic tool for pathogenesis of a disorder of cornification?
thinning of cornified envelopes in TGM1-deficient lamellar ichthyosis
Which form of ichthyosis is more common?
Non-epidermolytic
What is epidermolytic ichthyosis correlated with?
Defects in keratin formation (aka keratinopathic ichthyosis)
from Mauldin and Elias
A= NIPAL4 and FATP4
B= PNPLA1
C= NIPAL4 and FATP4
What pattern of inheritance do most ichthyoses in dogs have?
Autosomal recessive
What is the most severe form of ichthyosis in humans?
Harlequin (potentially fatal)
What is the genetic variant for ichthyosis in great Danes?
Autosomal recessive mutation in SLC27a4 (encodes FATP4 synthesis of very long chain fatty acids)
Appears at birth
Greasy scale, wrinkling of face and extremities
Non-viable into adulthood
histological lesions are typical of nonepidermolytic ichthyosis, but have eosinophilic material (glycosaminoglycan)
in hair follicles
Autosomal recessive mutation in SLC27a4 (great Dane ichthyosis)
What is the genetic variant for ichthyosis in Cavalier King Charles spaniels?
Autosomal recessive mutation in FAM83H (unknown function)
Appears at birth/first few weeks of life
Keratoconjunctivitis sicca from eyelid opening, scaling, abdominal pigmentation, curly pelage, pawpad hyperkeratosis, nail dystrophy
What is the genetic variant for ichthyosis in German shepherd dogs?
A de novo mutation in ASPRV (profilaggrin processing)
Generalized scale since birth
What is the genetic variant for the cornification disorder in Labrador retrievers?
x-linked semidominant mutation in NSDHL (cholesterol processing)
Appears when a puppy
Linear hyperkeratotic plaques and follicular fronds and pawpad hyperkeratosis
What is the genetic variant for the cornification disorder in Chihuahuas?
probable x-linked semidominant mutation in NSDHL (cholesterol processing)
Appears when a puppy
Linear verrucous nevi
What are the unique histopathologic features of ichthyosis in Norfolk terriers?
Unique histological features are a combination of acanthosis with hyperkeratosis, cytolysis in the granular layer, coarse keratohyalin granules and eosinophilic perinuclear bodies which represent clumped keratin filaments
Skin of a Norfolk terrier
Epidermolytic ichthyosis (KRT10 mutation)
* vesicle in stratum granulosum
Arrowhead – coarse KHG
Arrows (pink) – keratin clumps
What genetic mutation cause hereditary footpad hyperkeratosis in douges de Bordeaux aka plamoplantar keratoderma?
Spontaneous, autosomal recessive KRT16 mutation
Lesions develop between 10 wks and 1 yr and become fissures/cracks
Histologically, the SC is expanded by tremendous orthokeratotic hyperkeratosis with mild parakeratosis at the tips of villae
What causes hereditary nasal parakeratosis (HNPK) arises in Labrador retrievers and greyhounds?
mutation in SUV39H2 (decreases the expression of loricrin in the SC)
develop thick, brown scale on the nasal planum with variable depigmentation
How do you diagnose ichthyoses in veterinary medicine?
Signalment
◦ Young patient
◦ Breed predilections
Clinical signs
◦ Scale, hyperkeratosis
◦ Non-pruritic (unless secondary infections)
Rule out secondary disorders of cornification
◦ Skin scrapes, cytology, histopathology
Breeders
+/- DNA testing
How do you treat ichthyoses in veterinary medicine?
Topical “soak and slather” regimens
◦Management of barrier defects
◦ Lifelong treatments
Maybe there will be more targeted therapies eventually
What are the patterns of the stratum corneum that can be seen on histopathology?
Basket weave (lacy) **normal
Lamellar/laminated (condensed, but separates)
Compact (dense, does not separate)
Parakeratotic (retained nuclei) versus normal orthokeratotic
What is parakeratosis indicate on histopathology?
Incomplete keratinocyte maturation or a defect in filaggrin conversion
What is primary, idiopathic seborrhea?
A defect in cornification
Clinical excessive scaling, crusting, +/ greasiness (oleosa) or dryness (sicca)
No known cause, usually juvenile onset
Typically trunk, pinna with ceruminous otitis externa common
Cocker Spaniels, Persian kittens, horses (mane/tail) most represented
Young Cocker spaniel
Epidermal hyperplasia (mild to papillated
Alternating vertical tiers of ortho- and parakeratosis)
Follicular plugging and follicular ostia w/ parakeratotic caps/epaulettes
Suggestive of primary seborrhea (ddx: allergy, Vit A-responsive)
What is Vitamin A responsive dermatosis?
Do not actually have a measurable vitamin A deficiency
Cocker spaniels, miniature Schnauzers, and labs at risk
Usually adult-onset
Alopecic plaques w/ follicular casts esp lateroventral trunk
Ceruminous otitis also common
Confirm by response to vit A supplementation in 3 to 8 weeks
Which animals get true vitamin A deficiency?
Birds (on a seed-rich diet)
- White plaques on mucosa, rhinitis, blepharitis
- Rhinolith
Reptiles (esp box turtles)
- Squamous metaplasia, conjunctivitis, aural “abscesses”…
What are the active metabolites of vitamin A?
Retinoic acid and retinal
What is the primary effect of vitamin A on the skin?
Increases cell turn over
10 yr MC Cocker spaniel with alopecic plaques and follicular casts
Severe follicular keratosis
Distended ostia with plug protrusion (follicular fronds)
Suggestive of vitamin A-responsive dermatosis
What do you expect to see on histopathology of non-epidermolytic ichthyosis?
Lamellar / laminated to compact orthohyperkeratosis (mild to severe)
- “filo dough effect”
- Goldens: abnormal keratin rather than ↑
- Focal parakeratosis can happen
Perinuclear clear spaces in granular layer
What do you expect to see on histopathology of epidermolytic ichthyosis?
Lamellar / laminated to compact orthohyperkeratosis (mild to severe)
Lysis of upper epidermal layers
Hypergranulosis with abnormal keratohyalin granules
Diffuse keratinocyte ballooning
What form of ichthyosis is this?
Non-epidermolytic
What form of ichthyosis is this?
Epidermolytic
What is free arachidonic acid a substrate for?
cyclo-oxygenase (COX) –> prostaglandins and thromboxanes
lipoxygenase (LOX) –> leukotrienes
cytochrome P450 enzymes
Describe the stratum basale/stratum germativum/basal layer.
- Single row of columnar to cuboidal cells resting on the basement membrane zone
- Most of the cells are mitotically active keratinocytes
- small polar, undifferentiated cells
- attach to the basement membrane at hemidesmosomes
Which are the primary keratins expressed in the stratum basale?
K5/K14 (but dogs also have K1/K6)
How thick is the stratum spinosum?
- Typically 1-2 layers thick in haired skin
- Thicker at foot pads, nasal planum, and mucocutaneous junctions (can be 20 cells thick)
What are the primary keratins in the stratum spinosum and how does this vary in hyperproliferative disorders?
- Retain stable K5/K14 with new synthesis of K1/K10
- dogs also have K4 and K15/16
- Hyperproliferative disorders have down regulation of K1/K10 mRNA and protein while K6/K16 are favored
In what layer of the skin are lamellar granules (aka keratinosomes/membrane-coated granules/Odland bodies) formed?
Upper stratum spinosum but more prominent in granulosum
What are lamellar granules?
- Secretory granules secreted into the intercellular space of the stratum granulosum
- Contain precursors of stratum corneum lipids (glycoproteins, glycolipids, phospholipids, free sterols, glycosylceramides, acid hydrolases) and proteins that respond to signals that occur during the transition from the granular to cornified layer
- Are secreted via exocytosis
When is profilaggrin produced?
Stratum spinosum (cleaved into filaggrin in the granular layer)
What is involucrin?
- First major component to be activated that will be cross-linked in the emerging cornified envelope
- Initiated in the spinous layer soon after K1/K10 expression
- Glutamine-rich
- Largest protein in the stratum corneum
- Synthesized in the spinous layer
- Cross-linked in the granular layer by transglutaminases
- Is rigid and firm, forming an insoluble cell boundary and connecting corneocytes to the extracellular lipid layer
What are the two primary categories of things that occur in the stratum spinosum?
1) keratinocytes start differentiating
2) things that will be needed later start to be made
Describe the histopathologic appearance of the spinous layer.
- Cells are lightly basophilic to eosinophilic, nucleated, and polyhedral to flattened cuboidal
- Have a spine-like appearance of the cell margins which are abundant desmosomes
What is the final layer of the epidermis in which keratinocytes are alive?
Stratum granulosum
What is the primary roles of the stratum granulosum?
- A final packaging stage which generates a number of structural components that will form the epidermal barrier
- The final stage of granular cell differentiation into a corneocyte is the cell’s programed destruction, destroying almost all cellular contents with the exception of keratin filaments and filaggrin matrix
What are keratohyalin granules composed of?
- Profilaggrin, keratin filaments, and loricrin
- But not true granules as they lack a membrane
What are the two types of keratohyalin granules?
- F granules
- L granules
What are F granules?
- Named for filaggrin
- A type of keratohyalin granules
- Irregularly shaped
What are L granules?
- Contain loricrin
- Are smaller and more rounded
Describe the fate of profilaggrin.
- Profilaggrin becomes dephosphorylated and undergoes Ca2+- dependent proteolytic cleavage
- Filaggrin is later degraded into the natural moisturizing factors, urocanic acid (UCA) and pyrrolidone carboxylic acid (PCA)
What is loricirin?
- A cystine-rich, highly flexible protein with glycine loops
- A major structural component of the cornified envelope, binding keratin filaments together in the corneocyte and anchoring them to the cross-linked envelope
- Most prevalent structural protein in the stratum corneum (~80% of volume of cornified envelope)
- Synthesized in the upper spinous layer and throughout the granular layers
How is keratin expression modified in the stratum granulosum?
Modified from K1/K10 to also include K2/K11
What is the stratum lucidum/stratum conjunctum?
- A fully keratinized, compact, thin layer of dead cells which is anuclear, homogenous, and hyaline-like, containing refractile droplets and semifluid eleidin
- Present in canine footpads (sometimes nasal planum) and coronary band of horses but absent elsewhere
What is the stratum corneum?
Outer layer of terminally differentiated keratinocytes that are constantly being shed (balanced by proliferation of basal layer)
- Multilayer zone of anucleate, flattened, lipid-depleted, protein-enriched corneocytes as the bricks and a continuous extracellular lipid matrix comprised of ceramides, FFAs, and cholesterol as the mortar
What does the lipid matrix of the stratum corneum do?
- Serves as the mortar
- Mechanical protection
- Barrier to water loss
- Regulates permeation of soluble substances
What keratins are primarily expressed in the stratum corneum?
K1/K10/K2e
What are the major proteins of the stratum corneum?
- Involucrin
- Loricrin
- Envoplankin
- Periplankin
- Small protein rich proteins (SPRPs)
What do envoplankin and periplankin do in stratum corneum?
Link the cornified envelope to desmosomes and keratin filaments
What do small protein rich proteins do in the stratum corneum?
Cross-link with loricrin in the cornified envelope
What is desquamation?
- A process of enzymatic cleavage of corneodesmosomes
- Balance between KLKs, LEKTI
- Promoted by acidic pH, decreases in Ca, increased natural moisturizing factors, and decreased water
What is KLK7?
- Stratum corneum chymotryptic enzyme aka kallikrein 7
- One of the two serine proteases of the kallikrein family that are implicated in dequamation
- Degrades corneodesmosin and desmocollin 1
What is KLK5?
- Stratum corneum tryptic enzyme aka kallikrein 5
- One of the two serine proteases of the kallikrein family that are implicated in dequamation
- Degrades corneodesmosin, desmocollin 1, and desmoglein 1
What is corneodesmosin?
- Synthesized as a glycine- and serine-rich glycoprotein in lamellar bodies then released from the lamellar bodies and associated with desmosomal desmogleins and desmocollines until activated by chemotryptic enzymes
What is LEKTI?
- Lymphoepithelial Kazal-type inhibitor
- Inhibits desquamation
- A serine protease inhibitor secreted from lamellar bodies
- Most effective at neutral pH
Rank the layers of the epidermis based on Ca2+ concentration.
Granulosum>spinosum>basale>corneum
What are the roles of Ca2+ on keratinocyte differentiation?
- Depletion regulates lamellar body exocytosis
- Regulator of protein synthesis (primarily transglutaminase 1 activity)
- Important for cell-to-cell adhesion
What is Darier disease (keratosis follicularis)?
- A disease with an increase in transepidermal water loss, altered calcium regulation, loss of adhesion between suprabasalar epidermal cells, and abnormal keratinization
- Caused by one gene involved in Ca transport, SERCA2
What is Hailey-Hailey disease (benign chronic pemphigus)?
- Loss of cell-cell adhesion
- Caused by a calcium-regulating gene, ATP2C1
Describe holoclones?
- Epidermal stem cells
- Slow dividing cells
- Proposed to maintain an “immortal strand” of DNA at each division
- May divide symmetrically (useful when skin needs to be stabilized) or asymmetrically
What are meroclones?
- Transient amplifying cells
- Rapidly dividing
- Typically divide symmetrically
What are paraclones?
Terminally differentiated/daughter cells
How does calcium regulate stem cell fate and maintenance?
- Low calcium environment promotes maintenance and prevents differentiation, thereby stimulating proliferation
- High calcium environment inhibits it
What are the categories of regulatory mediators which play a role in keratinocyte stem cell differentiation?
- Stimuli that direct progenitor cells toward a particular type of terminal differentiation
- Molecules and pathways characteristic of the microenvironment and stem cell homeostasis
- Molecules that differently alter stem cells and transient amplifying cell proliferation
- Positive and negative regulators involved in commitment and terminal differentiation
What stimuli are involved in directing progenitor epithelial cells into hair follicle cells?
- Positively by Wnt/β-catenin
- Negatively by DiKK1 and Lef1/Tcf
What molecules are involved in maintenance of stem cell quiescence in the epidermis?
Lrig1
What molecules are involved in modifying the differentiation of stem cells into transient amplifying cells and stem cell renewal?
- Myc
- p63
- miR203 microRNA
- Histone
What are negative regulators of terminal differentiation?
- Extracellular matrix
- pcG repression
What are markers for stem cells in the epidermis?
- β-integrin
- CD71 (transferrin receptor)
- LRIG1
What markers of proliferation are upregulated in psoriasis?
- Ki67
- C-myc
What is the [interfollicular] epidermal proliferative unit?
- A proliferative unit arranged in a hexagon consisting of approximately 10 basal cells with a clonogenic cell in the center overlaid by the suprabasal and corneocyte progeny
- Functionally independent and self-renewing
What is the epidermal differentiation complex in humans?
- Genes present in a 2mb region of the human chromosome 1q21
- Epigenetic mechanisms play an important role in growth and differentiation of keratinocytes by regulating expression of the cassette of genes in this region
- Encode structural proteins involucrin, loricrin, and SPRPs as well as a number of Ca-binding proteins including profilaggrin, trichohyalin and several S100A proteins
How many layers does the stratum granulosum have?
3 - numbering starts superficial to deep
What components start to be produced in the stratum spinosum?
◦ Involucrin
◦ Profilaggrin
◦ Lamellar bodies
What are the “spines” in the stratum spinosum?
They represent desmosomes
What happens in SG2?
It creates a permeability barrier due to tight junctions holding water inside and preventing entry of antigens
Where are tight junctions found in the epidermis?
SG2
What happens in SG1?
- Keratin filament assembly
- Cornified envelope construction
- lamellar body exocytosis –> creation of lipid envelope and intracellular lamellae
What links corneocytes together?
Corneodesmosomes
How far do activated Langerhans cells reach?
SG1, they now can get through tight junction barrier
(normally can only get to SG2)
What promotes the transition from spinous to granular layer cells?
Protein kinase C
What role does calcium play in terminal keratinocyte differentiation?
low calcium = high proliferation (less differentiation)
high calcium = promotes terminal differentiation
Where are S100 proteins primary expressed in the normal epidermis?
Stratum basale and spinosum
Why does the 2018 report of the Nomenclature Committee on Cell Death (NCCD) recommend that programmed cell death and terminal differentiation of keratinocytes be conceptually well discriminated?
terminal differentiation of keratinocytes involves caspase 14, but not executioner caspases
How long does it take for keratinization and cornification to occur in healthy skin?
21 days
What adhesion method is the main block to the penetration of external agents into the epidermis?
tight junctions
What is the pH of equine skin?
7-7.4
What are the layers of the avian epidermis?
Stratum germinativum
- stratum basale
- stratum intermedium
- stratum transitivum
Stratum corneum (vacuolated and flattened cells that only retain hard keratin)
