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Adhesion molecules in skin seminar (2)


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  • 2. INTRODUCTION Cell Adhesion Molecules (CAMs)  Proteins located on cell surface involved in binding with other cells or with the extracellular matrix (ECM)  Provides resistance against environmental influences  Epidermal integrity is required for protection of the entire organism against - mechanical - physical - microbial insult.
  • 3.  Are typically transmembrane receptors and are composed of three domains:1. Intracellular domain that interacts with the cytoskeleton 2. Transmembrane domain 3. Extracellular domain that interacts either with other CAMs of the same kind (homophilic binding) or with other CAMs or the extracellular matrix (heterophilic binding)  Regulates tissue : - growth - differentiation - repair
  • 4. CLASSIFICATION  Classified as: 1.calcium-dependent CAMs 2.calcium-independent CAMs
  • 5.  calcium-dependent CAMs 1.integrin 2.cadherin 3. selectin  calcium-independent CAMs 1. immunoglobulin superfamily
  • 6. INTEGRINS  Large family of heterodimeric transmembrane glycoproteins that attach cells to extracellular matrix proteins of the basement membrane or to ligands on other cells.  Key to: - embryogenesis - tissue development and differentiation - immune responses - leukocyte trafficking - cancer metastasis - tissue homeostasis.  Integrins contain large (α) and small (β) subunits of sizes 120-170 kDa and 90-100 kDa, respectively
  • 7.  The α subunit of the integrin heterodimer especially the αI domain determines the ligand specificity for cell-ECM adhesion.  Contain binding sites for divalent cations Mg2+ and Ca2+, which are necessary for their adhesive function.  Common ECM components that are bound by integrins (with respective recognition sequence) are - Fibronectin (RGD, LDV) - Collagen (triple helical GFOGER) - Laminin - Vitronectin (RGD) - Fibrinogen(RGD) - Thrombospondin - Glycoproteins (e.g. tenascin C, osteopontin, nefronectin)
  • 8.  Ligation of integrins initiates a plethora of signal transduction events which serve to modulate cell behaviour including : - proliferation - apoptosis - up-regulation of genes Involved in inflammation cell polarity and migration  Many integrin stimulated cell signalling pathways are similar to those triggered by growth factor receptors and cytokine/chemokine receptors  Upon activation, integrins are capable of triggering a variety of signal transduction cascades based on the context, ligand (matrix components/growth factors) and substrate stiffness that result in a variety of short-term and long-term responses
  • 9. Cellular responses elicited by integrin signaling. In response to physical/chemical properties of the matrix and growth factors in the environment (outside-in signaling), integrins bind ligands and get activated. Accordingly, a variety of signaling pathways can be triggered mainly through the different kinases as mentioned above. These can bring about changes in one or more cellular events (short term responses) that eventually result in global (long term) responses in cellular behavior
  • 10. Integrin type In vivo function β1 integrins Development αV Vasculogenesis α9β1 Lymphangiogenesis αIIbβ3 Thrombus formation α6β4 Integrity of skin αVβ3 β2 integrins Suppresses tumorigenesis, angiogenesis, wound healing, inflammation and atherosclerosis Immune responses
  • 11. SELECTINS  Calcium-dependent transmembrane glycoproteins with an amino-group terminal portion that binds to sialylated carbohydrate counter-receptors on other cells.  Key transient link between leukocytes and vascular endothelium thereby mediating the initial steps of leukocyte rolling and tethering  There are three subsets of selectins: - P-Selectin - E-Selectin - L-Selectin
  • 12.  P-selectin and E- selectins plays an essential role in the initial recruitment of leukocytes to the site of injury during inflammation  Following stimulation of TNF-α, IL-1 LPS and substance P peak expression of E-selectin occurs within 4 h unlike Pselectin which takes minutes on being stimulated by histamine, IL-4, IL-13, oncostatin M or thrombin.  L-selectin is unique in that it is the only selectin known to be involved in trafficking of lymphocytes into lymphatic tissue.
  • 13.  Levels of soluble E-selectin in plasma are increased in - scleroderma - polyarteritis nodosa - SLE - psoriasis - atopic dermatitis  Correlation between levels of soluble E-selectin and disease activity has been observed in - atopic and allergic dermatitis - psoriasis and palmoplantar pustulosis - eczema - Kawasaki disease  E-selectin may be of potential therapeutic value in inflammatory diseases and cancer by virtue of its unique temporal and spatial expression profile
  • 14.  Adhesion is a key step in the metastatic cascade  E-selectin expressed on activated endothelium can facilitate tumor cell seeding  Expression of Lewis carbohydrates is often more pronounced on the surface of metastatic tumor cells  E-selectin expression is often enhanced on the surface of endothelial vessels at the site proximal to or directly at tumor metastases.
  • 15. Schematic presentation of selectin- and integrin-mediated cancer cell interactions with several blood constituents (e.g., platelets, leukocytes, and endothelial cells) during hematogenous metastasis
  • 16. The medical potential of antagonists to human selectins  Intravenous administration of the CDP850 humanized anti-E- selectin antibody reduces levels of soluble E-selectin in plasma and of E-selectin expressed on lesional psoriatic skin in patients with moderate-to-severe chronic plaque psoriasis  Pan selectin competitive inhibitors, including sLeX oligosaccharide, sLeX mimetics , multivalent sLeX ligands or diverse molecular weight species of heparin have been developed.  A few such carbohydrates have shown some success in the treatment of psoriasis (Bimosiamose or TBC-1269 and Efomycine M) or asthma (Bimosiamose)
  • 17. IMMUNOGLOBULIN SUPERFAMILY  Large group of cell surface and soluble proteins that are involved in - recognition - binding - adhesion processes of cells  These molecules have been associated with cancer progression and a metastatic phenotype  Includes members such as: - vascular cell adhesion molecules (VCAM) - neural cell adhesion molecules (NCAM) - intercellular adhesion molecules (ICAM) - nectin and nectin-like (Necl) family - platelet endothelial cell adhesion molecules (PECAM)
  • 18.  ICAM-1 contributes to leukocyte adhesion via the LFA-1 and Mac-1 β2 integrins  The related VCAM-1 molecule expression is increased after cytokine stimulation, and it binds the α4β1 integrin mediating extravascular migration, principally of monocytes and eosinophils.  Platelet–endothelial cell adhesion molecule (PECAM-1 or CD31), is found in platelets and at the intercellular junctions of endothelium,where it contributes to leukocyte passage from blood vessels  sICAM-1 is significantly elevated in the serum of a number of dermatological diseases including atopic dermatitis, psoriasis and metastatic melanoma and correlates with clinical severity in untreated disease.  sICAM-3 is increased in the sera of patients with psoriasis and atopic dermatitis but is less relevant than sICAM-1 to disease activity
  • 19.  sVCAM are elevated in advanced cutaneous melanoma, localized scleroderma, systemic sclerosis and SLE  CD99participates in the process of transendothelial migration of monocytes and neutrophils, can be a potential therapeutic target in skin inflammation  Recent studies demonstrated L1CAM expression in various types of cancer, predominantly at the invasive front of tumours and in metastases
  • 20. Neutrophil emigration. (a) Rolling: cells roll over the endothelium by brief non-adherent contacts via the selectins,particularly L-selectin. The integrins are non-binding. (b) Arrest: on contact with activated endothelium, selectin binding brieflyincreases, integrins become avid and intercellular adhesion molecule1 (ICAM-1) increases on the endothelial surface. (Monocytes and eosinophils also bind via α4β1 to vascular cell adhesion molecule 1 (VCAM-1).) (c) Adherence: the neutrophil fl attens on the endothelium, affinity increases and motility response to chemokines is initiated. (d) Emigration: neutrophil leaves vessel through interendothelial cell junction, regulated by integrins, platelet/endothelium cell adhesion molecule 1 (PECAM-1) and chemokines
  • 21. Pleiotropic anti-inflammatory effect of dPGS diminishes the inflammatory response and reduces leukocyte extravasation. Molecular targets of dPGS are the adhesion molecules L- and P-selectin. dPGS prevents leukocyte extravasation by shielding the selectins. Binding to complement factors C3 and C5 inhibits the formation of the proinflammatory anaphylatoxins. Reduction of the C5a level decreases vascular permeability and further leukocyte extravasation. By addressing these inflammatory targets simultaneously, dPGS promote the resolution of inflammation.
  • 22. EPIDERMAL, DERMO-EPIDERMAL ATTACHMENT COMPLEX  The basement membrane between the epidermis and the dermis contains unique structures that maintains the attachment of the epithelium.  The components of the attachment complex provide links to the intracellular intermediate filament network of basal keratinocytes and to the extracellular matrix of the papillary dermis.  Can be divided into : 1. Epidermal attachment complex 2. Dermo-epidermal attachment complex
  • 23.  Epidermal attachment complex includes: 1. Desmosomes 2. Adherens Junctions 3. Tight Junctions 4. Gap Junctions  Dermo-epidermal attachment complex includes: 1. Intermediate filaments 2. Hemidesmosomes 3. Anchoring Filaments 4. Anchoring Fibrils
  • 24. DESMOSOMES  Desmosomes are small, electron-dense structures that link the intermediate filament within cells to the plasma membrane and to adjacent cells  Found in Epidermis, myocardium, meninges and cortex of lymph nodes  Characteristic ultrastructural appearance, in which the cell membrane of two adjacent cells forms a symmetrical junction with a central intercellular space of 30 nm containing a dense line
  • 25. COMPONENTS OF DESMOSOMES  Transmembranous cadherins - heterophilic associations of desmogleins and desmocolins There are Four main epidermis-specific desmogleins (Dsg1–4) and Three desmocollins (Dsc1–3), which show differentiation-specific expression.  Desmoglein-3 is expressed only in the basal and suprabasal layers of the epidermis  Desmoglein-1 is found throughout the epidermis, particularly in the upper layers  In mucosae, desmoglein-3 is strongly expressed throughout whereas desmoglein-1 is expressed only weakly
  • 26.  Plaque components- Intracellular parts of desmosomal cadherins attached to KF network via-Desmoplakin -Plakoglobin -Armadillo protein: plakophilin -Enovaplakin -Periplakin
  • 27. Molecular model of the desmosome. The desmosomal cadherins desmoglein and desmocollin undergo homophilic and heterophilic binding via interaction with the amino-terminal extracellular (EC) 1 domain of partner molecules on the same (cis) as well as on the neighbouring cell (trans). The cytoplasmic domains are largely embedded in the outer dense plaque (ODP) where they are associated with plakoglobin and plakophilin. In the inner dense plaque (IDP), desmoplakin links these adaptor molecules to the intermediate filament cytoskeleton
  • 28. Autosomal dominant and autosomal recessive mutations in human desmosome genes resulting in skin, hair and cardiac abnormalities
  • 29. Desmosomal components as targets for immunobullous diseases. Antibodies to these proteins may directly lead to blistering skin and mucous membrane disorders.
  • 30.  Disruption of extracellular domain of Dsg1 by bacterial toxin:  Staphylococcal scalded skin syndrome  Bullous impetigo
  • 31. ADHERENS JUNCTIONS  Electron dense,transmembrane structures  Adhesion site that links Actin cytoskeletal components of two cells  Contribute to: - epithelial assembly - adhesion - barrier formation - cell motility - changes in cell shape  Transmembrane component: Cadherins, Nectin  Plaque component : Catenin,Afadin
  • 32.  Charaterized by two opposing membranes separated by approximately 20 nm space and are 0.2–0.5 μm in diameter  Two basic adhesive units: 1. Nectin–afadin complex 2.Cadherin-catenins (α, β,p120 catenin)complex both attach to Actin cytoskeleton.  Cell signalling via β-catenin can activate several Wnt pathways which implicates adherens junctions in coordinating morphogenetic movements with cell fate determination.
  • 33.  Associated with a variety of actin-binding molecules, suggesting multiple dynamic interactions with the cytoskeleton  Spatially coordinate : a.signalling molecules b.polarity cues c.serve as docking sites for vesicle release
  • 34. CLINICAL RELEVANCE OF ADHERENS JUNCTIONS  Mutation in CDH3 gene, which encodes P-cadherin :- Autosomal recessive hypotrichosis with juvenile macular dystrophy.
  • 35. TIGHT JUNCTIONS  Form at the apical end of lateral membranes  Regulate the paracellular flux of water-soluble molecules between adjacent cells  Transmembranous components: 1.Junctional adhesion molecules (JAM) 2.Claudins 3.Occludins  Claudins and occludins bind to the intracellular zonula occudens proteins ZO-1, ZO-2, ZO-3; these proteins can also interact with actin thus providing a direct link with the cytoskeleton.
  • 36. CLINICAL RELEVANCE OF TIGHT JUNCTIONS  Key role in skin barrier integrity and maintaining cell polarity  Mutations in Claudin 1 – Autosomal recessive disease with clinical features of - diffuse ichthyosis with large scales - hypotrichosis - scarring alopecia - sclerosing cholangitis.
  • 37. GAP JUNCTIONS  Clusters of intercellular channels, known as connexons, that directly form connections between the cytoplasm of adjacent keratinocytes  Connexons - assembly of six connexin subunits within the Golgi network that are then transported to plasma membrane, where they associate with other connexons to form a gap junction.
  • 38.  Essential for  Cell synchronization  Differentiation  Cell growth and  Metabolic coordination of avascular organs, including epidermis.  Functions are: 1. sharing of low-molecular-mass metabolites 2. ion exchange between neighbouring cells 3. intercellular coordination 4. tissue/organ homeostasis
  • 39. CLINICAL RELEVANCE OF GAP JUNCTIONS  Inherited abnormalities in genes encoding four different connexins (Cx 26, 30, 30.3 and 31) leads to several forms of keratoderma and/or hearing loss.
  • 40. DERMO-EPIDERMAL JUNCTION  The functions and homeostasis of skin critically depend on stable organization cohesion between epidermis and dermis  The suprastructural entity affording pivotal mechanical stability is the anchoring complex sequentially consists of : 1. hemidesmosomes at the basal surface of the keratinocytes 2. anchoring filaments linking the hemidesmosomes to the basement membrane 3. anchoring fibrils connecting the basement membrane with the underlying dermal stroma
  • 41. MOLECULAR COMPONENTS OF EPIDERMAL BASEMENT MEMBRANE ZONE  Intermediate filament (IF) components - Keratin 5 - Keratin 14  Hemidesmosomal plaque components - 230-kDa bullous pemphigoid antigen(BP230/BPAG1) - Plectin
  • 42.  Transmembrane components - α6β4 integrin - Type XVII collagen (180-kDa bullous pemphigoid antigen/BPAG2) - α3β1 integrin - Type XIII collagen - Syndecans 1 and 4  Lamina lucida/lamina densa components - Laminin 332 (laminin 5) - Laminin 311 (laminin 6) - Laminin 511 (laminin 10)
  • 43.  Lamina densa components - Type IV collagen - Laminin 111 (laminin 1) - Nidogen - Perlecan  Anchoring fibril components - Type VII collagen
  • 44. Fig. - The dermal–epidermal junction. , The structure of the adhesion complex of the basement-membrane zone
  • 45. INTERMEDIATE FILAMENTS  Proteins which form building blocks of cytoskeletal filaments     of cells along with Actin and Tubulin. IF proteins are expressed in a tissue and cell compartment specific manner. Subdivided into 5 major types Types I–IV : Cytoplasmic IFs, Type V(lamins) : Nuclear IFs.
  • 46.  Keratins are the largest subgroup of IF proteins .  54 functional human keratin genes have been recognised  Classified as Epithelial keratins : Encoded by 37 genes Hair keratins : Encoded by 17 genes  Genes encoding individual keratins fall into Two families: Type I ( basic ) Type II (acidic)
  • 47.  Intermediate Filament proteins have a conserved central rod domain of helical coiled-coil segments  Keratins heterodimerize with specific pairing partners: One Type I family One Type II family  Heterodimers then oligomerize into longer fibrils  Fibrils continue to assemble untill Intermediate Filament is 10-12 nm in diameter
  • 48.  Keratin filaments extends from the nuclear membrane to desmosomal plaques at the cell membrane  Keratins enable keratinocytes to sustain mechanical and non-mechanical stress
  • 49. CLINICAL RELEVANCE Disease associations with Mutation in keratin genes:  Palmoplantar keratoderma,epidermolytic: K1,K9  Palmoplantar keratoderma,diffuse nonepidermolytic: K1  Palmoplantar keratoderma,focal nonepidermolytic: K16  Epidermolysis Bullosa Simplex: K5, K14  Epidermolytic Hyperkeratosis: K1, K10  Ichthyosis hystrix type Curth-Macklin: K1
  • 50. HEMIDESMOSOMES-ANCHORING FILAMENT COMPLEX  Electron dense attachment complexes, extend from intracellular compartment of basal keratinocytes to lamina lucida in upper portion of D-E BMZ.  Intracellular component of a hemidesmosome attaches to the keratin IF network  Within the lamina lucida ,HD contiguous with anchoring filaments,which are thread-like structures that traverse the lamina lucida and tend to concentrate below the hemidesmosomes
  • 51.  Five components of hemidesmosomes, originally designated as HD1–HD5  HD2 and HD4:- 230-kDa and 180-kDa bullous pemphigoid antigens (BPAG1 and BPAG2), respectively  HD3 and HD5:- β4 and α6 integrin subunit polypeptides, respectively;  HD1 corresponds to plectin
  • 52.  The 180-kDa BP Ag, a transmembrane collagenous protein, also known as type XVII collagen,  It interacts with α6β4 integrin and extends from the intracellular compartment of basal cells to the extracellular space  stabilizes the association of basal keratinocytes to the underlying basement membrane
  • 53. CLINICAL RELEVANCE  Mutations in the BPAG2 gene result in a relatively mild, non-Herlitz type of junctional EB  Mutations in the α6β4 integrin genes (ITGA6, ITGB4) cause skin fragility associated with congenital pyloric atresia  Mutations in the plectin gene (PLEC1) result in skin blistering disorder frequently associated with late-onset muscular dystrophy
  • 54. BASEMENT MEMBRANE  Interface between the lower part of the epidermis and the top layer of the dermis comprises D-E BMZ, consist of various extracellular matrix macromolecules  BMZ by transmission electron microscopy shows two distinct layers with different optical densities: 1) Upper layer- Lamina lucida 2) Lower layer- Lamina densa
  • 55.  Major biochemical components of the BMZ are type IV collagen and a number of collagenous glycoproteins, including laminin 322.  Component of BMZ distributed in LL and LD  Major Laminin in skin Laminin 332 - α3β3γ2 Laminin 311 - α3β1γ1 Laminin 511 - α5β1γ1  Cell binding of Laminins is mediated by Integrins –family of cellular receptors
  • 56.  Laminin 332 is a disulphide-bonded complex of α3, β3, and γ2 chains which associate into a trimeric, cruciform structure  Complete absence of any of the three subunit polypeptides of laminin 332, due to loss-of-function mutations in both alleles of the corresponding gene, results in Herlitz junctional EB
  • 57. In the absence of laminin 332, affected individuals develop erosions and blisters at the level of lamina lucida, manifesting with the Herlitz form of junctional EB
  • 58. Type IV collagen  major component of Lamina densa  Consist of 3 polypeptide subunits (α-chains)  Autoantibodies against α5 and α6 chain epitopes glomerulonephritis and subepidermal blistering
  • 59. ANCHORING FIBRILS  Ultrastructurally recognizable, U-shaped structures, extends from the lower part of lamina densa to upper reticular dermis  Type VII collagen is major component of anchoring fibrils  Synthesized by both dermal fibroblasts and epidermal keratinocytes  The anchoring fibril network forms a scaffold that entraps large numbers of dermal fibrils, securing the lamina densa to the subjacent dermis
  • 60. CLINICAL RELAVANCE  In the acquired form of epidermolysis bullosa, epidermolysis bullosa acquisita and in bullous systemic lupus erythematosus, autoantibodies target mainly the NC-1 domain of coliagen VII.  Mutation causing complete absence of type VII collagen results in severe, generalized, recessive dystrophic EB  Missense mutations, particularly glycine substitution mutations, can result in somewhat milder, dominantly inherited dystrophic EB
  • 61. Schematic representation of the gene/protein systems within the cutaneous basement membrane zone that can harbour mutations
  • 62. Subepidermal immunobullous disorders Autoantibodies to molecules within the anchoring complex of the Basement-membrane zone       Bullous pemphigoid- 230-kDa BP Ag &180-kDa BP Ag Mucous Membrane pemphigoid- all components of BMZ Pemphigoid gestationis- 180-kDa BP Ag & 230 kDaBPAg Linear IgA disease - BP180/ collagen XVII Epidermolysis bullosa acquisita- Type VII collagen Bullous SLE - Type VII collagen