Keratinisation

1. Keratinisation
Dr.Nileena

2. Introduction
 A specific form of cellular differentiation in epidermis resulting
in formation of keratin rich permeability barrier
 Starting with basal keratinocytes of epidermis and adnexa
 End product –corneocytes
 It is a modified form of programmed cell death
 Progeny of epidermal stem cells undergo a series of molecular
and biochemical changes that lead to morphologically different
strata of epidermis
 Consists of 2 aspects
 Synthesis of distinctive proteins(keratin,filaggrin,involucrin)
 Alterations of nuclei,cytoplasmic organelles,plasma
membrane and desmosomes

3. Stratum basale
 Single cell layer
 Small,cuboidal with large dark
staining nuclei and dense basophilic
cytoplasm with ribosomes and other
cytoplasmic organelles
 Attach to basement membrane zone
via K5/K14 keratins at
hemidesmosomes and to each other
via desmosomes that expresses
Dsg3,Pkp2
 Mitotically active-cell divisions
occur every 18-19th day

4. Stratum spinosum
 Aka prickle cell layer due to spine like appearance of cell
margin due to desmosomes that expresses more of DSg1,Pkp1
 8-10 cell layers
 Polyhedral with round nucleus
 Larger,more flattened cells with more organelles in upper
spinous layer
 Conspicuous bundles of K1/K10 keratins inserted to
desmosomes peripherally
 Upper most layers-lamellar granules

5. Stratum granulosum
 2-5 cells thick
 due to intracellular basophilic keratohyaline granules-contains
keratin,profilaggrin,loricrin and other proteins which is
responsible for cornified envelope
 Filaggrin and loricrin are commonly used markers of
differentiation for this layer
 Lamellated granules present
 Tight junctions are prominent in this layer
 Stratum lucidum-only present over palmoplantar epidermis

6. Stratum corneum
 20-25 cell thick
 Flattened,lost nuclei and
organelles
 Contain high molecular mass
keratin stabilised by high
molecular disulphide bonds,
surrounded by crosslinked
networks of proteins which
is surrounded by crosslinked
specialised lipids

8. Basal keratinocytes Corneocytes
 Mitotically active
 Vertically oriented-
columnar/cuboidal
 Basophilic
 Presence of nucleus and
cytoplasmic organelles
 K5/K14(aligned along major
axis of cell)
 50-58kDa keratin
 Desmoglein3
 Terminally differentiated
 Horizontally oriented-
flattened
 eosinophilic
 Covers 25 basal cells in area
 Absent organelles
 K1/10 (higher molecular
mass) forms a bundle
 67kDa
 Desmoglein1

10.  It starts with withdrawal from cell cycle
 Switches off laminin and integrin expression
 Undergoes terminal differentiation resulting in change their
structure,shape and composition
 Keratin aggregation
 Cornified layer formation
 Intercellular Lipid envelope formation
 Loss of nuclear and cytoplasmic organelles
 Loss of cell surface receptors via loss of integrin expression
 Cells increase in size and metabolic activity as it moves from
basal layer to spinous layer and metabolic activity wanes as it
further moves up and loses cytoplasmic organelles

11.  Cells of basal and lower
spinous layer produce
tonofilaments which becomes
aggregated into bundles-alpha
keratin embedded in sulphur
rich amorphous matrix as cells
ascend.
 These do not possess
disulphide crosslinks
 Provides flexibility and
elasticity to cornified layer

12. Keratohyaline granules
 First appear in upper spinous layers- prominent in mid to upper
granular zone and disappears in stratum corneum
 In embryonic epidermis-appears in upper portion of intermediate
layers
 Biochemically complex-Electron dense globular structures
 2nm in length-occurs randomly in rows or lattices
 Consists of
 Loricrin is a cystine-rich proteins whose disulphide bonds
contributing to chemical inertness and strength typical of
cornified layer
Forms major component (70% by mass) of cornified
envelope and contributes to water resistance

13.  Source of amorphous protein-profilaggrin that is degraded into
filaggrin(stratum corneum basic protein/histidine rich protein
II)
 Profilaggrin –heavily phosphorylated insoluble,neutral
protein
 degraded in transitional zone between granular and
cornified layers to filaggrin-1 forms 12 protein
 Filaggrin is a histidine rich,cationic protein functions to
aggregate and align keratin filaments within cornified cells

14.  Filaggrin undergoes rapid proteolysis
soon after into urocanic acid and
pyrrolidone carboxylic acid which
contribute to hydration “Natural
moisturising factor(NMF)” and
filters UV radiation
 Filaggrin acts as matrix protein for
keratin filament aggregation and
precise,parallel alignment into
disulphide cross-linked macrofibrils

16. Odland bodies
 Aka lamellar
granules,keratinosomes,
cementosomes, membrane coating
granules
 100-300nm in diameter
 Highly ordered,internal lamellar
structure
 Near the top of spinous zone-
formed from golgi apparatus of
keratinocytes and stored in
cytoplasm as intracellular vesicles

17.  Contains free sterols,
 polar lipids (phospholipids, glycosphingolipids),
 hydrolytic enzymes (eg lipases, glycosidases and acid
phosphatases),
 protease and protease inhibitors,antimicrobial peptides
 The limiting membrane of lamellar bodies are rich in acyl
ceramides with omega hydroxylated ultra long chain fatty acids
 The fusion increases amount of acylceramides within lipid
bilayer of plasma membrane
 It acts as scaffold for lamellar formation of intercellular lipids

18. These coalesce into crystalline sheets within intercellular space of cornified
layer forming effective waterproof mantle
Lipases and hydrolases remodel polar lipids into more hydrophobic,non
polar,neutral lipid products like ceramides and free fatty acids
Contents are organised into lamallae that constitute structural basis for
barrier to epidermal permeability
Fuses with it and releases contents into intercellular spaces
migrate towards plasma membrane via cytoplasm in granular layer

19.  These acylceramides later is covalently bound to outer surface
of cornified envelope by transglutaminase 1
 These eventually replace plasma membrane
 Forms a 5nm hydrophobic lipid envelope that is a determinant
of cohesiveness of stratum corneum with barrier function that
occurs concurrently with cornified envelope formation
 Process of desquamation involves degradation of intercellular
lamellated lipid and loss of residual desmosomes via steroid
sulfatase,acid phosphatase
 Functions of the lipid layer
 Prevents water loss
 Stratum corneum cohesion

21. Cornified cellular envelope
 Aka marginal band
 Chemically resistant,highly insoluble proteinaceous structure at
inner surface of plasma membrane of cornifying keratinocytes
 15nm thick-Oriented parallel to skin surface
 Formed first at interface between granular zone and cornified
layer
 Contiguous plasma membranes of upper granular layers
become thickened by deposits of dense material on their
internal lamina

22.  Loricrin,involucrin,envoplakin,periplakin are crosslinked via
transglutaminase-1 and form a monomolecular layer along
inner surface of plasma membrane
 Along with varying amounts of small proline rich proteins and
other(eg repetin,trichohyalin,cystatin,elafin)

23.  Formed by epidermal transglutaminase
 Calcium dependent enzyme
 Cytoplasmic side of plasma membrane
 Forms crosslinks with various precursor proteins such as
keratolinin and involucrin via isopeptide bonds and cornified
lipid layer with cellular envelope
 Expression begins in spinous layer,but inactive
 Activated by calcium and cofactors in granular layer

25.  Sources of calcium activating TG
 Permeability of plasma membrane increases allowing
calcium influx
 release of sequestered calcium from degenerating organelles
 release of bound calcium from calcium binding proteins
activating calcium dependent transglutaminases
 Thus ,there is calcium gradient across the layers -lowest in
corneum (~nil) and highest in granulosum.This gradient has to
be maintained for Odland bodies to secrete its contents

26.  In lower stratum corneum,plasma membrane surrounds the
impervious envelope ,whereas in upper stratum
corneum,plasma membrane along with desmosomes become
discontinuous replaced by the insoluble envelope
 In lower stratum corneum,CE is irregularly shaped and fragile
whereas in upper stratum corneum,it is polygonal and rigid
representing different stages of maturation
 Serves as template that orients intercellular neutral lipids from
lamellar granules constitute hydrophobic barrier

27.  Crosslinks chemically in ester linkage with the layer of neutral
lipids
 Insolubility and stability is due to numerous disulphide and
highly resistant bonds
 Properties of cornified layer
 Physical toughness and strength
 Flexibility and elasticity
 High electrical impedence
 Dry surface
 Retards proliferation of microorganisms
 Rate limiting membrane for passage of water,electrolytes and
other molecules

28. Loss of cytoplasmic organelles
 Lysosomal enzymes mediate degradation of cytoplasmic
organelles
 Tonofilaments and keratohyaline granules are resistant
 Nuclear DNA is replaced by histones and other proteins
synthesised in granular zone

29. Intercellular connections
 Desmosomes break and reform continuously as keratinocytes
ascend and mature.They ascend randomly by having contractile
components of actin attach to desmosomes of neighbouring
cells and pulling on them
 In contast,corneocytes are tightly attached to each other and
move in unison
 In the uppermost layer of granular and cornified layers -
desmosomes and marginal band form a continuous layer

30.  Adhesion in cornified layer involves direct contact between
lipid envelopes of adjacent corneocytes at discrete sites where
intercellular lipid lamallae is absent
 Cholesteryl sulfate is thought to be intercellular cement
substance,hydrolysis of which to cholesterol coincides with
desquamation of corneocytes

31. Regulation of differentiation
 Epidermal differentiation complex
 Chromosome 1q21 with 25 genes
 Cluster of genes coding for proteins in differentiation like
loricrin,involucrin,SPRPs,filaggrin,trichohyaline.
 Roles in structural integrity,signal transduction,cell cycle
progression
 Regulators include aryl hydrocarbon
receptora(AhR’s),nuclear factor erythroid 2 related factor 2
(Nrf-2)-upregulate cornified envelope transcription
 Increase in extracellular calcium is important trigger for
terminal differentiation

32.  P63 has role in induction of
differetiation via transcription
factors Klf4 via ZNF750
which upregulates lipid
modifying enzymes and
proteins for cornified
envelope
 NOTCH signalling is present
in spinous cells ,absent in
basal cells-commitment factor
for transition, promotes K10
through other factors
 GRHL3 is required for
transglutaminase-1 expression

33. Cell kinetics
 Mean turnover of epidermis is ~39 days with
 13 days:proliferation (in lower 2 rows)
 12 days:differentiation-transit time from basal layer to
cornified layer
 14 days:transit time within cornified layer prior to shedding

34. Keratins
 Keratins are markers for keratinocyte differentiation and are
required to maintain epithelial integrity
 Intermediate filaments (7-10nm in diameter)
 Forms filamentous cytoskeleton of all mammalian cells
 54 human keratins of Molecular weight 40-67 kDa
 Keratins form obligate heteropolymers with a member of each
family (acidic and basic) of identical size ranks coexpressed to
form filamentous structure eg K1/K10
 Expression is dependent on cell type,tissue type,stage of
embryonic development,degree of differentiation,disease stats

35. Keratin expression in embryonic epidermis

36. Classified based on
 pH
 Type 1 or acidic keratins (4.9 -5.4)
 Type 2 basic keratins (6.5-8.5)
 distribution –
 epithelial/soft keratins –skin and mucosa
 hard/trichocyte keratins – hair ,nail apparatus,filiform papillae
of tongue,thymic epithelium
 Preferential synthesis
 Primary keratins-synthesised by epithelial cells on regular
basis. Eg:K8/K18 in simple epithelia and K5/K14 in stratified
epithelia
 Secondary keratins-produced in addition to primary keratins eg
K7/K19 in simple epithelia,K6/K16 in stratified epithelia
Soft keratins Hard keratins
Type II(basic) K1-8,K71-80 K81-86
Type I(acidic) K9-K28 K31-K40

37. Type 2 (basic) Type 1 (acidic)
Epidermis Basal cells K5 K14/K15
Suprabasal K1
K2(granulosum)
K10/K9
Cornea K3 K12
Oral mucosa K4,K76 K13
Epithelial appendages K6 K16/k17
Simple epithelia K7/K8 K18
Hair Inner root sheath K71-74 K25-28
Outer root sheath
(isthmus)
K5
K6
K14/K15
K16/K17
Companion layer K75
Matrix K85 K35
Cortex K81,83,85,86 K31-38
Cuticle K82,K85 K32,K35,K39,K40
Medulla K6
K81/K85
K16,17,25-28
K33-39

38.  During keratinisation,
keratins are expressed highly
specific for the state of
differentiation
 Stratum basale-K5/14
 Stratum spinosum
 Downregulation of K5/K14
 Expression of K1/K10 –
”differentiation specific
/keratinisation specific”
 Stratum granulosum-K2
 Stratum corneum- forms
bundles parallel to surface

39.  Keratins of increasing molecular weight are synthesised as
cells migrate from basal zone to cornified layer
 50-58kDA in basal cells to 65-67 kDa in suprabasal
keratinocytes
 This is a reflection of epidermal stratification and extent of
differentiation
 Functions of keratin
 Provide flexibility and elasticity
 Mechanical strength to cornified layer
 Modulate shape of keratinocyte
 Promote centralisation of nucleus
 Mediates cell to cell contact via desmosomes
 May transfer information between nucleus to cell surface and
vice versa

40. Nail and hair keratinisation
 Keratinisation in hair and nail unit is distinct
 Hard keratins consist of 9 type I and 6 type II
 Consists of 2 types of keratinisation in different components of
the unit
Epidermal type Onycho/tricholemmal
Granular layer Present absent
Keratohyaline
granules
Present Absent
Type of keratin Soft Hard
Endpoint Desquamation via
enzymatic action
Periodic cutting required

41. Nail unit keratinisation
 Epidermal type seen in
 Proximal nail fold
 Lateral nail folds
 Hyponychium
 Separated from zone of
onycholemmal type by
grooves and onychodermal
band seperating nail plate
from hyponychium

42.  Proximal nail fold-2 layers of
epidermis with granular layer
showing keratohyaline granules
produceing soft keratin
 Onycholemmal keratinisation
seen nail matrix and nail bed
 Nail matrix-80% of nail plate
 Distal matrix-ventral nail
plate- lunula
 Proximal matrix-dorsal nail
plate
 Multilayered basal layer
below broad keratogenous
zone,no granular layer

43.  Nail bed-20% of nail plate
 Thinner epidermis than that of
matrix with less prominent
basal layer
 As epidermis is
thin,differentiation occurs
within 1-2 cell layers
Nail plate is a fully keratinised structure consisting of closely
packed,adherent , interdigitating onychocytes that lack nuclei or
organelles
Hard keratins ,rich in sulphur containing aminoacids like cystiene
and lack of extended glycine residues is responsible for mechanical
resilience of nail plate

44. Hair keratinisation
 Pluripotent cells of hair
matrix in hair bulb gives rise
to hair shaft and IRS which
as they move upwards,
differentiate into several
layers that keratinises at
different levels
 Epidermal type- IRS
ORS in infundibulum part
 Trichilemmal part-Hair
shaft,ORS in isthmus

45.  Outermost layer of IRS-
Henles layer keratinises 1st-
thus forming a firm coat
around soft central parts
 2 opposing cuticles-cuticle of
hair shaft and cuticle of IRS
 Huxleys layer
 Hair cortex
 Medulla (last)

46.  Medulla-partially keratinised
 Cortex cells keratinise to
form tightly packed,fusiform
cells
 hard keratins along long
axis of cells
 Without keratohyaline
granules or their
counterparts
Cuticle -has overlapping cells that are pointed upwards in hair
and downwards in IRS that interlock resulting in attachment of
hair to IRS such that they move together

47.  Inner root sheath-keratinise with
trichohyaline granules-eosinophilic (vs
basophilic nature of keratohyaline
granules)
 filaments oriented in direction of hair
growth
 Few in cuticle,numerous in Huxleys
layer,many in Henles present n
emergence from matrix
 After complete keratinisation,all 3 layers
disintegrate on reaching isthmus by
abrupt desquamation
 Do not contribute to emerging hair

48.  Outer root sheath
 Thinnest at level of hair
bulb ,thickest at isthmus
 Epidermal keratinisation
in infundibulum part
 Trichilemmal
keratinisation in Isthmus
 Below Isthmus,ORS is
covered by IRS and do not
undergo keratinisation

49. Applied aspects
 21 of 54 keratins have been linked to monogenic genetic
disorders
 1st keratinisation associated genetic disorder was EBS with
K5/K14 mutation
 Keratinisation disorders associated with hair are
Monilethrix,Hair-Nail ectodermal dysplasia,pseudofoliculitis
barbae,Woolly hair
 Filaggrin gene mutation-Ichthyosis vulgaris,Atopic dermatitis
 Loricrin defect-Vohwinkel syndrome
 Transglutaminase defect-Acral peeling skin syndrome,lamellar
ichthyosis,congenital icthyosiform erythroderma

50. X linked Ichthyosis Steroid sulfatase deficiency
Netherton syndrome Premature Odland body secretion
Harlequin fetus ABCA 12 mutations-complete
loss of Odland bodies
Lamellar Ichthyosis ABCA 12 dysfunction- loss of
function of Odland bodies
Epidermolytic hyperkeratosis,bullous
congenital ichthyosiform
erythroderma
K1/K10
Epidermolytic bullosa Simplex
Dowling degos disease
K5/K14
Epidermolytic palmoplantar
keratoderma
K9/K1
Non epidermolytic PPK K1
Paronychia Congenita K6/K16,17
White sponge nevus K4/K13

54. References
 Moschella and Hurley textbook of dermatology 3rd edition
 IADVL textbook of dermatology 5th edition
 Rooks textbook of dermatology 9th edition
 Fitzpatricks textbook of dermatology 9th edition
 Levers histopathology of skin 11th edition
 Smack DP,Korge BP,James WD J AM ACAD DERMATOL
1994;30:85-102
 Wertz P.”Epidermal Lamellar Granules”.Skin Pharmacology
and Physiology,vol 31,no.5,Aug.2018,pp 262+
 Mclean WHI,Irvine AD Disorders of keratinisation:from rare to
common genetic diseases of skin and other epithelial tissues
Ulster MedJ 2007 May;76(2):72-82