Keratinisation.pptx

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 d ense basophilic cytoplasm with ribosomes and other cytoplasmic organelles Attach to basement membrane zone via K5/K14 keratins at hemidesmosomes and to each o ther via desmosomes that expresses Dsg3,Pkp2 Mitotically active-cell divisions occur every 18-19 th 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

7. 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

8. 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

9. 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

10. 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

11. 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

12. 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

13. 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

14. 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

15. 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

16. 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

17. 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 )

18. 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

19. 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

20. 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

21. 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

22. 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

23. 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

24. 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

25. 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

26. 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

27. 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

28. 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

29. Keratin expression in embryonic epidermis

30. 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

31. 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

32. 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

33. 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

34. 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 Granula r layer Present absent Keratohyaline granules Present Absent Type of keratin Soft Hard Endpoint Desquamation via enzymatic action Periodic cutting required

35. 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

36. 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

37. 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

38. 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

39. Outermost layer of IRS- Henles layer keratinises 1 st -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)

40. 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

41. 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

42. 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

43. Applied aspects 21 of 54 keratins have been linked to monogenic genetic disorders 1 st 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

44. 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

45. References Moschella and Hurley textbook of dermatology 3 rd edition IADVL textbook of dermatology 5 th edition Rooks textbook of dermatology 9 th edition Fitzpatricks textbook of dermatology 9 th edition Levers histopathology of skin 11 th 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