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1. 1
Good Morning

2. KERATIN AND KERATINIZATION
DR. SUMAN MUKHERJEE
MDS 2ND YEAR
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3. Contents
• Introduction
• Keratin
• Structure of keratin and keratin
filaments
• Classification
• Factors regulating keratinocyte
differentiation
• Function of keratin
• Distribution of major keratins
• Different stains used for keratin
• Pathology of keratinization
• Keratinized epithelia
• Non-Keratinized epithelia
• Oral keratinization disorders
• Conclusion
• References
3

4. Introduction
• Epithelia function to protect the underlying tissues from
environmental influences such as physical damage, infection,
desiccation, UV radiation, heat loss, and to maintain homeostasis.
• Oral epithelium is classified into three types based on their
morphology and specific pattern of differentiation:
• Keratinized stratified squamous epithelium (masticatory mucosa
distributed in hard palate and gingiva), non-keratinized stratified
squamous epithelium (buccal mucosa, labial mucosa) and
specialized mucosa (dorsal surface of the tongue).
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5. 5

6. • An important aspect of stratified squamous epithelia is that the cells
undergo a terminal differentiation program that results in the formation
of a mechanically resistant and toughened surface composed of
cornified cells that are filled with keratin filaments and lack nuclei and
cytoplasmic organelles.
• In these squames, the cell membrane is replaced by a proteinaceous
cornified envelope that is covalently cross linked to the keratin
filaments, providing a highly insoluble yet flexible structure that
protects the underlying epithelial cells.
6

7. Keratin
• Keratin is a multigene family of proteins. The word Kera is derived
from the Greek word meaning horn. Historically the term “keratin”
stood for all of the proteins extracted from skin modifications, such
as horns, claws and hooves.
• Subsequently, it was realized that keratin is actually a mixture of
keratins, keratin filament-associated proteins and other enzyme
proteins derived from epithelial cells.
• These keratins are characteristically found only in the epithelial
cells. In humans, keratins are encoded by 54 genes.
7

8. • Keratinization, also termed as cornification, is a process of
cytodifferentiation which the keratinocytes undergo when
proceeding from their post germinative state (stratum basale) to
finally differentiated, hardened cell filled with protein, constituting
a structurally and functionally distinct keratin containing surface
layer such as stratum corneum.
8

9. • Most of the eukaryotic cell is composed of cytoskeleton which is
made of three components classified on the basis of their diameter
and physicochemical properties into microfilaments, intermediate
filaments, and microtubules.
• Microfilaments are the smallest filaments of cytoskeleton with a
diameter of 7 nm while microtubules are the largest filamentous
structures with a diameter of about 20 nm.
9

10. Structure of keratins and keratin
filaments
10

11. Primary structure of keratins
• Each keratin is characterized by a chain of amino acids as the primary
structure, which varies in the number and sequence of amino acid as well as
in polarity, charge and size.
• The amino acid sequence of a keratin influences the properties and function
of the keratin filament.
• The primary amino acid sequence is slightly longer than that of the mature
keratin which indicates a post-translational modification of the keratin prior to
the formation of keratin filaments.
• Post translational modifications - the formation of disulphide bonds,
phosphorylation, glycosylation, deamination or inter- and intra-chain peptide
11

12. Secondary structure of keratins
• Secondary structure of keratin filaments consisting of an N-terminal
head domain, a central α-helical rod domain and C-terminal tail
domain.
• Each of these domains is divided into sub-domains.
• The head domain consists of sub-domains V1 and H1.
• The central α-helical rod domain is composed of subdomains 1A, 1B,
2A, and 2B connected by linkers L1, L12 and L2
• The tail domain is made of sub-domains H2 and V2.
12

13. • The α-helical rod domain is critical for the assembly of
intermediate filament proteins into filaments and contains all
the information necessary for assembly, while the variant end
domains play accessory roles in filament assembly and
interaction with other proteins and cell structures.
13

14. Tertiary structure of keratins: heterodimer
• The tertiary structure of
keratins is a
heterodimer that is
formed by the rod
domains of one acidic
and one basic keratin
in parallel orientation.
14

15. 15

16. Classification
• Based on the preferential synthesis
1. Primary keratins – keratins which are always synthesized by the
epithelial cells on a regular basis.
For example, – K 8/18 in simple epithelia and K 5/14 in the stratified
epithelia
2. Secondary keratins – Produced by epithelial cells in addition to
primary keratins, K 7/19 in the simple epithelia and 6/16 in the
stratified epithelia
16

17. • Based on biochemical properties
• Type I – Acidic (9–20) Type II – Basic (1–8).
• Based on molecular weight
• Low – Glandular and simple
• Intermediate – Stratified epithelia
• High – Keratinized stratified epithelia.
• Based on distribution
• Soft – Skin and mucosa Hard – Nails and hair.
17

18. • Based on X-ray diffraction pattern:
• Alpha: The X-ray diffraction pattern of this type resembles that of
α-helix with a 5.1 Å spacing. The α-helix is right handed and has 3.6
residues per turn. The hydrogen bonding occurs within one
polypeptide chain.
• Beta: In the X-ray diffraction pattern of this type, periodic repeats
were 3.5 and 7 angstroms. The helix is right-handed with an
average of 6 residues. The hydrogen bonding occurs between
neighboring polypeptide chains
• Feather keratins
• Amorphous keratins
18

19. • Based on amino acid sequence, keratins are classified into type I
and type II:
• Type I family includes keratins numbered 9-20 which are
composed of acidic proteins, with a molecular weight 40-56 kDa
• Type II family includes keratins numbered 1-8 which are composed
of basic proteins, with a molecular weight of 52-67 kDa
19

20. Factors regulating keratinocyte differentiation
1. Active metabolites of Vitamin D3 act in an autocrine pathway to
decrease keratinocyte proliferation and to increase cell
differentiation
2. Epidermal growth factor (EGF) and transforming growth factor-α
exert a mitogenic effect on basal cells through interaction with EGF
receptors. The receptor for EGF has been localized in basal cells of
the oral mucosa
3. Keratinocyte growth factor (KGF), a member of the fibroblast
growth factor family, is produced by lamina propria fibroblasts. Acting
in a paracrine pathway, KGF exerts a powerful stimulus for epithelial
cell proliferation.
20

21. 4. Interleukin 1-β and interleukin-6 can increase keratinocyte
proliferation by stimulating the production of KGF.
5. Hepatocyte growth factor is another paracrine factor originating in
connective tissue that elicits keratinocyte proliferation and migration
6. TGF-β inhibits DNA synthesis in basal cells and promotes terminal
differentiation. It is secreted by the basal and suprabasal cells.
21

22. 7. High levels of Vitamin A cause normally cornified epithelia to
undergo mucous metaplasia, a condition wherein cornified
surface cells are replaced by noncornified cells of lining mucosa.
Vitamin A deficiency can cause an opposite effect, i.e., squamous
metaplasia.
8. Calcium plays an important role in keratinocyte differentiation.
22

23. FUNCTIONS OF KERATIN
1. Keratins fundamentally influence the architecture and mitotic
activity of epithelial cells
2. Keratins and associated filaments provide a scaffold for
epithelial cells and tissues to sustain mechanical stress, maintain
their structural integrity, ensure mechanical resilience, protect
against variations in hydrostatic pressure and establish cell
polarity
3. Keratins and its filaments are involved in cell signaling, cell
transport, cell compartmentalization and cell differentiation. 23

24. 4. Keratin proteins regulate the response to pro-apoptotic
signals and have the ability to modulate protein synthesis and
cell size in epithelial cells
5. Keratins also participate in wound healing.
24

25. 25
Distribution Of Major
Keratins

26. 26
Distribution of keratin in oral epithelium
(Reproduced from Presland and Dale 2000)

27. 27
Distribution of
keratin in gingival
epithelium
(Reproduced from
Presland and Dale
2000)

28. DIFFERENT STAINS USED FOR
KERATIN
28

29. PATHOLOGY OF KERATINIZATION
• Pathology of keratinization shows various patterns.
• These pathologies can be due to defect in genes which code for
keratin proteins or there are lesions which demonstrate abnormal
keratinization histopathologically due to different etiological factors.
• There could be increased keratinization, decreased keratinization, or
abnormal keratinization.
29

30. • Hyperkeratinization is the defect of epithelial cells.
• Normally, these epithelial cells shed or de-squamate at regular
intervals. In hyperkeratinization, this process is disturbed because of
an excess of keratin formation and accumulation due to lack of
adequate desquamation.
• It occurs as a secondary reaction to chronic irritation or some
infection or malignancy.
• Hyperkeratinization which occurs because of chronic irritation is due
to higher rate of proliferation of the epithelial cells
30

31. • Decreased keratinization or lack of keratin production is due to failure
of the epithelial cells to undergo complete differentiation and
maturation to the point of keratin formation.
• Dyskeratosis is premature keratinization which occurs in individual
cells or group of cells in different strata of the epithelium, before they
reach the surface. These cells become separated from the adjacent
cells. These dyskeratotic cells are large and round with a deep
eosinophilic cytoplasm and a hyperchromatic nucleus.
• A benign keratin pearl is surrounded by cells which are not dysplastic
in nature; for example, – dyskeratosis. When there is lack of cohesion
among the epithelial cells due to malignant changes, the cells get
arranged in a concentric manner.
31

32. • As the fate of a squamous cell is to form keratin, these cells lay down
keratin in a concentric manner and then appear as keratin pearls which
are known as malignant keratin pearls. Keratin pearls are thus
whorl-shaped accumulations of keratin made by malignant squamous
cells and are present in concentric layers in between the squamous
epithelium.
• These different patterns of keratin formation depend on the amount and
the nature of the inciting stimulus. In frictional keratosis or in mild
leukoplakia if the underlying stimulus is removed, the change in the
mucosa is reverted back to normal; while in squamous cell carcinoma,
there is premature keratinization of the cells before they undergo
32

33. Keratinized
Epithelia has 4
layers:
33

34. Stratum Basale
• Made up of a single layer of cuboidal cells.
• Consists of progenitor cells giving rise to
cells in the epithelial layers.
• Made up of cells that synthesize DNA and
undergo mitosis, thus providing new cells
called keratinocytes.
34

35. • Daughter cells pass towards the surface and, during the process of
maturation, take on the appearance characteristic of the various layers.
• Specialized structure called hemidesmosomes which abut on the basal
lamina are found on the basal surface —attach the epithelium to the
connective tissue.
• The lateral borders of adjacent basal cells are connected by desmosomes
35

36. • Cytoplasm of the basal cells contain widely dispersed
tonofilaments, also referred to as Cytokeratins which are
precurser of keratin.
• Ribosomes & rough endoplasmic reticulum are found -
indicative of protein synthesizing activity.
36

37. Basal cells are made of 2
populations
• 1. Serrated and heavily packed with
Tonofilaments.
• 2. Non-serrated and composed of
slowly cycling stem cells, which give rise
to slowly dividing cells which serve to
protect genetic information to the tissue.
37

38. Stratum Spinosum
• Above the stratum basale, round or ovoid cells form a layer several
cells thick called stratum spinosum.
• These cells show the first stages of maturation, being larger and
rounded than those in the stratum basale.
• The intercellular spaces of spinous cells in keratinized epithelium are
large and distended, thus making desmosomes more prominent and
these cells are given a prickly appearance.
38

39. • Spiny appearance is due to shrinkage of cells causing
them to separate at points where desmosomes do not
anchor them together
39

40. • Of all the layers, spinous cells are most active in protein
synthesis.
• Involucrin which is soluble precursor protein of the cornified
envelope appears first in the spinosum.
• The uppermost cells of spinous layer contains numerous
dense intracellular membrane coating granues
called Keratinosomes or Odland bodies).
40

41. Stratum Granulosum
• Cells of this layer show further increase
in maturation.
• Contains flatter and wider and larger
cells than spinous cells.
• Many organelles are reduced or lost,
such that the cytoplasm is
predominantly occupied by
tonofilaments and tonofibrils.
• The cells contain number of small
granules called keratohyaline granules
41

42. • These granules contain the precursor of filaggrin (Profilaggrin).
• These basophillic granules are 0.5 – 1.0 µm in length and form the
matrix in which tonofilaments are embeded.
• Profilaggrins, decompose into filaggrins by the action of the
protease peptidylarginine deiminase during keratinization.
• Released filaggrin facilitate aggregation and cross links b/w
cytokeratin filaments
42

43. Keratinosomes or Odland bodies or
lamellar granules
• Upper part of spinous and basal part of granular layer .
• They are rich in phospholipids.
• Are approximately 0.25 µm in length
• Originate from golgi apparatus.
• Contain an enzyme, acid phosphotase, involved in the destruction of
organelle membranes
44

44. This barrier is forms at the junction of granular and
cornified cell layer which limits the movement of
substances between the cells
Contributing to the formation of permeability barrier
Discharge their contents into the intercellular space
forming an intercellular lamellar material
Upper part of spinous and basal part of granular
layer →dense granules, keratinosomes or Odland
bodies →modified lysosomes.
45

45. 46
Nuclei and
other
organelles
get
disappeared.
Involucrin becomes cross
linked by enzyme
transgultaminase to form
cornified envelope.

46. Stratum Corneum
• In keratinized epithelium, the final
stage in the maturation of the
epithelial cells is the loss of
organelles (including nuclei and
keratohyaline granules).
• Nuclei and other organelles such
as mitochondria and ribosome
get disappeared.
47

47. • The cells of the stratum corneum become filled entirely with closely
packed tonofilaments surrounded by the matrix protein filaggrin.
• This mixture of protein is collectively called keratin.
• The cells of the stratum corneum may be termed epithelial squames
: it is these cells that are shed (the process of desquamation) ,
necessitating the constant turnover of the epithelial cells.
• Desmosomes weaken and disappear to allow for this
desquammation.
48

48. • The stratum corneum provides the mechanical protective
function to the mucosa.
49

49. the corneocyte.
cells filled with
tonofilaments in filaggrin-
matrix
the keratohylin granules
disappear
that is cornified envelope
to form a thin highly
resistant electron dense
envelope beneath the
plasma membrane
Involucrin becomes cross
linked by enzyme
transgultaminase.,
Thus,corneocytes are mainly formed by bundles of keratin
tonofilaments embedded in an amorphous matrix of filaggrin
and surrounded by a resistant envelope under the cell
membrane. 50

50. PARAKERATINIZ ATION:
• the stratum corneum retains pyknotic nuclei and
• the keratohyaline granules are dispersed, not giving
rise to stratum granulosum.
ORTHOKERATINIZATION:
 a well-defined stratum granulosum with no nuclei in
the stratum corneum.
NON-KERATINIZED EPITHELIUM
• Has neither granulosum nor corneum strata
• superficial cells have viable nuclei.
51

51. Non- keratinized epithelium
• Differ from keratinizing epithelia
primarily because they do not
produce a cornified surface layer
• Layers present are- stratum
basale, stratum intermedium,
stratum superficial.
• There is no stratum granulosum
• Have high rate of mitosis
52

52. ORAL KERATINIZATION DISORDERS
• A number of lesions occur in the oral cavity which show an abnormal
pattern of keratinization. These include oral geno-dermatosis to mild self-
limiting lesions to cysts and tumors. All these lesions show some defect in
keratinization
• The genetic disorders of keratin formation are due to mutations in genes
which code for different keratin proteins. These lesions and the associated
mutations are mentioned in next slide.
53

53. 54

54. Keratin Disorders
Genetic
• White sponge Nevus
• Pachyonychia congenita
• Dyskeratosis congenita
• Hereditary benign intraepithelial
dyskeratosis
• Pemphigus
• Epidermolysis bullosa
Acquired
Reactive Lesions
Frictional keratosis
Oral hairy
leukoplakia
Nicotina stomatitis
Immune mediated
Lesions
Lichen Planus
Psoriasis
Graft vs Host disease
DLE
Infections
Verruca vulgaris
Verruciform
- Xanthoma
Sq. papilloma
Pre neoplastic
Leukoplakia
Erythroplakia
Sq. cell carcinoma
OSMF
Verrucous carcinoma
KCOT
Cysts containing
keratin
Dermoid and
epidermoid cysts
Oral lymphoepithelial
cyst
Ortho keratinized
Odontogenic cyst
Others
Exfoliative Chelitis
Leukoedema
Vit. A deficiency
states
Erythema Migrans
Traumatic
Ulcerations
Deo and Deshmukh: Pathophysiology of
keratinization
55

55. Conclusion
• A cell synthesizes different subsets of keratin during the process of
maturation, for example., basal cells of keratinized epithelia express
K5 and 14, while suprabasal cells express K1 and K10.
• These epithelia can be classified according to CK expression.
• This pattern of keratin expression of a particular cell allows one to
identify the origin of the cell and its stage of differentiation and thus
helps to characterize the neoplasm.
56

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