Adhesion molecules

BASIC UNDERSTANDING OF ADHESION MOLECULES IN HEALTH AND DISEASE.

1. ADHESION MOLECULES DR.ASRAFUL ALOM DEPARTMENT OF PATHOLOGY GAUHATI MEDICAL COLLEGE

2. BRIEF INTRODUCTION Cells in vivo must form contacts with their neighbours or with extracellular matrix (ECM) in order to form tissues or organ. In tissue cells are organised into very precise and distinctive pattern controlled by specific cell adhesion. Cell adhesion is how cells talk to each other.

3. The evolution of adhesion molecules with specialized structures and functions permits cells to assemble into diverse classes of tissues with varying functions. Cell-cell and cell-ECM interactions are critical for assembling cells into tissues, controlling cell shape and function, and determining the developmental fate of cells and tissues. Diseases may result from abnormalities in the structures or expression of adhesion molecules.

4. Cell adhesion molecules mediate, through their extracellular domains, adhesive interactions between cells of the same type ( homotypic adhesion ) or between cells of different types (heterotypic adhesion ). A CAM on one cell can directly bind to the same kind of CAM on an adjacent cell ( homophilic binding ) or to a different class of CAM or ECM ( heterophilic binding ). Cell-cell adhesions can be tight and long lasting or relatively weak and transient.eg

5. CELL ADHESION MOLECULES(CAMs) CAMs are proteins located on the cell surface involved in binding with other cells or with the ECM in the process called cell adhesion. Can be classified into four families CADHERINS INTEGRINS IMMUNOGLOBULIN SUPERFAMILY( IgSF ) SELECTINS

6. CAMS ARE TYPICALLY TRANSMEMBRANE RECEPTORS AND ARE COMPOSED OF THREE DOMAINS: INTRACELLULAR DOMAIN- interacts with cytoskeleton and intracellular signaling molecules. TRANSMEMBRANE DOMAIN- EXTRACELLULAR DOMAIN- interacts with other CAMs or ECM.

7. Immunobiology, 6 th Edition, Janeway, Travers, Walport, and Shlomchik

8. CAMs PLAY ROLE IN Cell-cell interactions . Embryogenesis . Immunity(migration of immun e cells to the inflamation center) . Cell - tissue - organ development . wound healing . Cancer metastasis .

9. CADHERINS The primary CAMs in adherens junctions and desmosomes belong to the cadherin family. In vertebrates, >100 family members have been identified with diverse protein structures,but all with characteristic extracellular cadherin repeats ,can be grouped into classical and non classical cadherins . The diversity of cadherins arises from the presence of multiple cadherin genes and alternative RNA splicing .

10. The role of cadherins is not limited to mechanical adhesion between cells. Rather, cadherin function extends to multiple aspects of tissue morphogenesis,including cell recognition and sorting, boundary formation and maintenance, coordinated cell movements and the induction and maintenance of structural and functional cell and tissue polarity. Cadherins have been implicated in the formation and maintenance of diverse tissues and organs ranging from polarization of simple epithelia to mechanically linking hair cells in the cochlea to providing an adhesion code for neural circuit formation during wiring of the brain.

11. The cadherin superfamily includes hundreds of different proteins:

12. Table 19-3 Molecular Biology of the Cell (© Garland Science 2008)

13. CLASSICAL CADHERINS The "classical'' cadherins include E-,N-, and P- cadherins , named for the type of tissues in which they were initially identified ( epithelial, neural, and placental ). The adhesiveness of cadherins depends on the presence of extracellular Ca2+, the property that gave rise to their name ( calcium adhering ). Each classical cadherin contains a single transmembrane domain, a relatively short C-terminal cytosolic domain, and five extracellular " cadherin " domains (EC).

14. Figure 19-9a Molecular Biology of the Cell (© Garland Science 2008) Extracellular domains of a classical cadherin (C-cadherin)

15. If the calcium concentration is less the extra cellular domain becomes floppy and rapidly degraded by proteolytic enzymes

16. Different CAMs function in different junctions

17. Adherens junctions coordinate the actinbased motility of adjacent cells : • Allow cells to coordinate the activities of their cytoskeletons • Form a continuous adhesion belt around each of the interacting cells in a sheet of epithelium • Network can contract via myosin motor proteins – Motile force for folding of epithelial sheets.

18. DEVELOPMENTAL ROLES OF CADHERINS E-cadherin is the first cadherin expressed during mammalian development. It helps to cause compaction, an important morphological change that occurs at the eight-cell stage of embryo development . Differential expression of cadherins (either by modulating subtype or expression level) induces sorting out of mixed cell populations. Cadherin subtype switching occurs during coordinated cell movements such as neurulation,where the invaginating neural plate expresses N- cadherin , while the overlying ectoderm expresses E- cadherin .

19. Cadherin -dependent Cell Sorting

20. Folding of an epithelial sheet to form an epithelial tube

21. interior interior external effe ctors The changes in cell shape Cell differentation ? Cell- cell Adhesion GTP Small GTPa z Ca 2+ GTP cell cell Cadherins and Catenins participate in transduction of extracellular signals and Mediate various cellular response.

22. WNT-B CATENIN PATHWAY

23. INTEGRINS Integrins are a large family of type I transmembrane heterodimeric glycoprotein receptors that function as the major metazoan receptors for cell adhesion and connect the intracellular and extracellular environments. Integrins exist as two noncovalently bound α and β subunits, which pair to form heterodimers . There are 18 α and 8 β known subunits which combine to form at least 24 distinct integrin heterodimers .

24. Integrins can bind to extracellular matrix (ECM) glycoproteins including collagens, fibronectins , laminins ,and cellular receptors such as vascular cell adhesion molecule-1 (VCAM-1 ) and the intercellular cell adhesion molecule ( ICAM ) family. Integrins also play key roles in the assembly of the actin cytoskeleton as well as in modulating signal transduction pathways that control biological and cellular functions including cell adhesion, migration, proliferation, cell differentiation,and apoptosis .

25. SUBFAMILIES OF INTEGRINS The specificity of integrin binding to ECM components including laminins , collagens,and fibronectin depends on the extracellular domains of the α and β integrin subunits. Integrins α 1 β 1, α 2 β 1, α 10 β 1, and α 11 β 1 represent the primary collagen receptors. integrins α 3 β 1, α 6 β 1, α 6 β 4 and α 7 β 1 are the major laminin receptors. integrins α 5 β 1, α 8 β 1, α IIb β 3 and the α v β 1integrins are the major fibronectin receptors that bind in an RGD-dependent manner.

26. PHENOTYPE OF INTEGRIN KNOCKOUT MICE Integrins play diverse and important roles in most biological processes. Integrins knockout mice have provided much insight into the functions of specific integrin heterodimers , reflecting the unique roles of the various integrins . Mutations in integrin subunits have been found to cause clinical disorders in human and these correlate well with mice in which the same integrins are deleted.

27. There are three well-described inherited autosomal recessive diseases in humans linked to germline mutations in integrin subunits. Mutations in αIIb and β 3 integrin subunits are associated with Glanzmann’s thrombasthenia , a clinical entity associated with platelet dysfunction and bleeding disorders. Point mutations and gene deletion in β 2 integrin in humans and mice, respectively, have been associated with LeukocyteAdhesion Deficiency (LAD). Finally mutations in α 6 and β 4 integrin result in junctional epidermolysis bullosa with skin blistering.

28. STRUCTURE

29. INTEGRIN SIGNALING Integrins are able to transduce signals intracellularly following ligand binding (“ outside-in” signaling). However, unlike most other cell receptors, integrins can shift between high- and low-affinity conformations for ligand binding (“ inside- out”signaling ). Depending on the cell type, integrins can be either basally activated, as with most adherent cells that are attached to a basement membrane, or basally inactive, as with platelets or leukocytes that freely circulate until activated to undergo platelet aggregation or mediate an inflammatory response, respectively.

30. INSIDE OUT SIGNALING In the normal resting, inactive state, integrin extracellular domains are unbound to ligands and exist in a bent conformation. Activation signals from within the cell induce straightening of the extracellular domains and stabilize the extended, active conformation. This conformational change exposes the external ligand -binding site to which ligands bind, allowing the transmission of signals from the outside to the inside. The TM domains play key roles in integrin activation. Separation of integrin TM domains is generally believed to be a requirement for integrins to adopt the high affinity state.

31. OUTSIDE IN SIGNALING Integrins themselves lack intrinsic catalytic activity. Ligand binding to the extracellular domain of integrins results in signal transduction to the cytoplasm in the classical direction from the outside-in. These intracellular signals affect cellular growth,differentiation and apoptosis . Integrin signaling is complex and significantly influenced by crosstalk with growth factor receptors. Further, the intracellular signals generated lead to the assembly of the FA complex , a large, dynamic multiprotein complex involving over 150 intracellular proteins.

32. ROLE OF FEW IMPORTANT INTEGRINS Integrin type  In vivo function β 1 integrins Development α V Vasculogenesis α 9 β 1 Lymphangiogenesis α IIb β 3 Thrombus formation α 6 β 4 Integrity of skin

33. FUNCTION OF INTEGRINS Attachment of cell to ECM Signal transduction from ECM to cell have relation to- -cell growth -cell division -cell survival -cellular differentiation -apoptosis -cell migration during embryogenesis , thrombosis, haemostasis,wound healing etc.

34. IMMUNOGLOBULIN SUPERFAMILY CAM( IgSF -CAM) IgSF cell adhesion molecules are calcium-independent transmembrane glycoproteins . Each IgSF CAM has an extracellular domain, which contains several Ig -like intra-chain disulfide-bonded loops with conserved cysteine residues , a transmembrane domain, and an intracellular domain that interacts with the cytoskeleton. They are either homophilic or heterophilic and bind integrins or different IgSF CAMs.

35. consists of more than 25 molecules . They includes.. Intercellular cell adhesion molecules (ICAMs) Vascular cell adhesion molecule (VCAM) Platelet-endothelial cell adhesion molecule (PECAM-1) Neural cell adhesion molecules (NCAMs) Endothelial cell-selective adhesion molecule (ESAM) Junctional adhesion molecule (JAMs) Nectins , and other cell adhesion molecules.

36. Intercellular adhesion molecule (ICAM) ICAM-1 (CD54) ICAM-1 is expressed on leukocytes, fibroblasts, epithelial cells and endothelial cells. Expression can be induceed by cytokines (IL-1, TNF-α, IFN-γ), or bacterial endotoxin . IFN-γ selectively induces ICAM-1 expression without affecting expression of other adhesion molecules. Ligands for the most N-terminal domain (first) of ICAM-1 include LFA-1, fibrinogen, and most serotypes of rhinovirus , whereas the third domain is recognized by Mac-1.

37. Linkage with the cytoskeleton may localise ICAM-1 within regions of the endothelial cell membrane in order to facilitate leukocyte adherence and transmigration. The expression of ICAM-1 in primary melanoma is related to the presence of distant metastases. Elevated levels of soluble ICAM-1, which still retains the binding site for LFA-1 in its extracellular domain, have been found in the serum of melanoma and ovarian cancer patients.

38. ICAM-2 (CD102) ICAM-2 also has a similar tissue distribution to ICAM-1, but apparently is expressed constitutively and is not regulated by cytokines. Two Ig -like extracellular domains that possess 34% homology to the first two domains of ICAM-1. Ligand binding site for LFA-1. ICAM-2 does not serve as an endothelial ligand for this leukocyte integrin .

39. ICAM-3 (CD50) Functions as an LFA-1 ligand , αdβ2integrin. Expressed on all leukocytes and on mast cells. ICAM-3 cross-linking results in calcium mobilization, tyrosine phosphorylation , enhanced adhesion, chemokine secretion, and modulation of basophil mediator release.

40. VASCULAR CELL ADHESION MOLECULES VCAM-1 (CD106) VCAM-1, is a 90-110 kDa glycoprotein expressed on the surface of activated endothelium and a variety of other cell types including dendritic cells, tissue macrophages and bone marrow fibroblasts . VCAM-1 expression on endothelial cells can be up-regulated by several cytokines, such as IL-1 β, IL-4, TNF- α and interferon- γ ( IFN- γ) . VCAM-1 interacts with the leukocyte integrin α4β1 (VLA-4) on many different cells including eosinophils , monocytes and with α4β7 on activated peripheral T cells.

41. Thus α4β1/VCAM-1 interactions, like LFA-1/ICAM-1 interactions, may regulate the movement of leukocytes out of blood vessels to inflammatory sites. α4β1/VCAM-1 interaction has been shown to be crucial for the binding of haematopoietic precursor cells to bone marrow stroma . Interaction of VCAM-1 with α4β1 integrin expressed on certain tumour types has also been suggested to be an important mechanism for the development of metastases in cancers such as melanomas, osteosarcomas , neuroblastomas and rhabdomyosarcomas .

42. PLATELET-ENDOTHELIAL CAM PECAM-1 (CD31) Expressed on endothelial cells, platelets and on some leukocytes such as monocytes and neutrophils . PECAM-1 invoved in leukocyte transmigration and particularly in the preferential migration of naive and CD8+ T cells across HEV. PECAM-1 is expressed at intercellular junctions of endothelial cells and the surface expression of PECAM-1 is not increased by treatment with cytokines, such as TNF-α and IL-1.

43. NEURAL CELL ADHESION MOLECULES ( NCAM ) Many Ig superfamily proteins function as adhesion molecules in the nervous system and they have been implicated in various roles during the development of the nervous system. Based on the composition of their extracellular domains, of the Ig superfamily ( IgCAMs ) NCAM can be divided in three groups: containing Ig folds only, containing Ig folds followed by FNIII domain(s), Ig folds linked to protein modules other than an FNIII domain.

44. SELECTINS Selectins are a unique family of 3 adhesion molecules that are expressed and function only on cells in the vasculature. The term selectin was originally proposed to highlight the presence of the lectin domain , as well as to emphasize the selective nature of the expression and function of these molecules. Selectins mediate the first step required for emigration of leukocytes from the bloodstream , tethering of a leukocyte in flow.

45. Selectins have an N-terminal C-type (Ca2-binding) lectin domain, followed by a single EGF domain, 2–9 CRP domains, a single transmembrane domain, and a short cytoplasmic domain.

46. L- SELECTIN: - OR CD62L Located on tip of microvillus projection of leukocytes facilitating its interaction with ligands of endothelium. L- selectin is constitutively expressed at the leukocyte surface rapidly shed this selectin following activation. It is involved in binding of circulating T-lymphocytes to the high endothelial postcapillary venules in lymph nodes and mucosal lymphoid tissues and as such acts as homing receptors for lymphocytes to these tissues. In addition to its role in lymphocyte homing L- selectin appears to participate in the adhesion of neutrophils , monocytes , and lymphocytes to activated endothelium.

47. E-SELECTIN OR CD62E : The expression of E- selectin appears to be largely restricted to activated endothelial cells. Previously known as endothelial leukocyte adhesion molecule-1 (ELAM-1). This expression peaks in - 4-6 h, declines to basal levels by 24-48 h, and requires de novo RNA and protein synthesis. E- Selectin recognizes complex sialylated carbohydrate groups related to the Lewis X or Lewis A family found on various surface proteins of granulocytes, monocytes , and previously activated effector and memory T cells.

48. E- selectin is important in the homing of effector and memory T cells to some peripheral sites of inflammation, particularly in the skin. Endothelial cell expression of E- selectin is a hallmark of acute cytokine-mediated inflammation , and antibodies to E- selectin can block leukocyte accumulation in vivo. E xpressed only on cytokine activated endothelial cells. Recent study suggests that the antiinflammatory effects of corticosteroids may, at least in part, involve such a mechanism : dexamethasone can inhibit endothelial cell expression of E- selectin stimulated by LPS and IL- I.

49. 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.

50. P-SELECTIN (CD62P) Unlike E- selectin , P- selectin is synthesized constitutively and stored intracellularly in both platelets and endothelial cells. After synthesis, it appears to be targeted to storage/ secretory granules by virtue of a sorting signal present in its cytoplasmic domain. From these intracellular pools, P- selectin can be rapidly mobilized to the cell surface, where it binds leukocytes. A variety of mediators, including thrombin,histamine , terminal complement components, and H₂0₂ have been shown to induce rapid surface expression of P- selectin .

51. Was first identified in the secretory granules ( α granule ) of platelets, hence the designation P. Also found in secretory granules of endothelial cells, called Weibel -Palade bodies. The expression of P- selectin at the cell surface is short lived , declining substantially within minutes. Recent studies suggest that new P- selectin synthesis may be induced by cytokines such as IL- 1 and TNF in a manner similar to that of E- selectin

52. Leukocyte migration and CAM

53. CAMs in leukocyte adhesion deficiency Leukocyte adhesion deficiency  (LAD), is a rare  autosomal   recessive disorder characterized by   immunodeficiency  resulting in recurrent  infections . LAD is currently divided into three subtypes:  LAD1 ,  LAD2 , and the recently described LAD3, also known as LAD-1/variant. In LAD3, the immune defects are supplemented by a  Glanzmann thrombasthenia -like bleeding tendency.

54. CAMs in CANCER and METASTASIS The function of epithelial (E)- cadherin is decreased in most epithelial tumors during cancer progression. Loss of E- cadherin function elicits active signals that support tumor-cell migration, invasion and metastasis . Loss of E- cadherin can be accompanied by increased level of other cadherins such as N- cadherin which promotes tumor cell motility and migration. E cadherin functions as a Tumor Suppressor. E cadherin loss enables disaggregation of cancer cells from one another.

55. CDH1 mutations involved with several cancers: Breast, Liver, Prostate, Stomach, Endometrium , Ovary, and Lung . Loss of E cadherin Function Correlates with Poor Prognosis. Accumulating evidence from several preclinical models confirms that tumor cell interactions through selectins and integrins actively contribute to the metastatic spread of tumor cell.

56. CAMs IN INFLAMMATORY BOWEL DISEASE VCAM- 1 concentration were higher in patients with active ulcerative colitis compare to inactive ulcerative colitis. VCAM-1 concentration were also greater in patients with both active and inactive crohns disease than controls. Patients with active crohns disease had higher ICAM-1 concentration than control.

57. CAMs in CARDIOVASCULAR PATHOLOGY A. Coronary Artery disease: T he infiltration of monocytes and T- lymphocytes, which initiates the atherosclerotic process is mediated by adhesion receptors. There are increased level of β -1 integrin , VCAM-1, ICAM-1 and E- selectin in patients with atherosclerosis. B. Thrombosis: Activation of α IIb β 3 receptor can result in platelet adhesion even if Arachidonic Acid pathway is blocked.

58. Neuropathologically , AD is primarily characterized by intraneuronal neurofibrillary tangles (NFTs) of hyperphosphorylated tau and extracellular deposits of mainly aggregated Aβ peptide, known as senile or neuritic plaques. The role of CAMs in events considered central to the pathogenesis and progression of Alzheimer’s disease (AD) such as amyloid -β ( Aβ ) metabolism, neuronal plasticity, inflammation, and vascular changes. CAMS IN ALZHEIMER DISEASE

59. THERAPEUTIC UTILITY OF CAMs C ancer : selectins can be used as nanodevices to treat cancer. Researchers are trying to create a device capable of killing cancer cells circulating in the blood. 2 . Osteoporosis: osteoporosis is a disease that occurs when bone forming cells called osteoblasts become to scarce. Osteoblast developed from stem cells can be able to treat osteoporosis by adding stem cells to a patient’s bone marrow . E- selectins are constitutively expressed in the bone marrow, and researchers have shown that tagging stem cells with a certain glycoprotein causes those cells to migrate to bone marrow.

60. 3.Ischemic heart disease : GPIIb / IIIa antagonists (like abciximab,tirofiban,eptifibatide )are effective antithrombotic agents. As compared to alone thrombolytic agent, combination of GPIIb / IIIa antagonists and a thrombolytic agent produces more rapid and extensive clot lysis,reduces the risk of re-occlusion and dimnishes infarct size. Trials of these antagonists along with other thrombolytic agents and anticoagulants are currently ongoing in MI and unstable angina. Other conditions which can be benefited are stroke, thrombotic thrombocytopenic purpura and microvascular surgery etc.

61. 4.Inflammatory diseases: Overzealous accumulation of leukocytes in tissues contributes to a wide variety of diseases. Atherosclerosis Asthma and COPD Chronic IBD Rheumatoid arthritis Multiple sclerosis Juvenile diabetes etc Therapeutic strategies are thus directed to reduce or prevent leukocyte-endothelial cell interactions and communication, in order to limit the progression of inflammatory diseases.

62. TENSIN IN HEALTH AND DISEASE Tensin is a cytoplasmic phosphoprotein that localized to integrin -mediated focal adhesions. It binds to actin filaments( at ABD domain ) and contains a phosphotyrosine -binding ( PTB) domain, which interacts with the cytoplasmic tails of integrin . In addition, tensin has an Src Homology 2 (SH2) domain capable of interacting with tyrosine- phosphorylated proteins. Furthermore, several factors induce tyrosine phosphorylation of tensin .

63. Thus, tensin functions as a platform for dis /assembly of signaling complexes at focal adhesions by recruiting tyrosine- phosphorylated signaling molecules through the SH2 domain, and also by providing interaction sites for other SH2-containing proteins. Analysis of knockout mice has demonstrated critical roles of tensin in renal function, muscle regeneration, and cell migration. Therefore, tensin and its downstream signaling molecules may be targets for therapeutic interventions in renal disease, wound healing and cancer.

64. ROLE OF TENSIN IN CARCINOGENESIS Tensins are deregulated in cancer although their function is tissue dependent. Cten ( Tensin 4) is the most extensively studied tensin molecule and this act as a tumour suppressor in the prostate but as an oncogene in colon,breast and lung neoplasia . Tensin 1 and 3 are found to be downregulated in those of the prostate,breast,kidney and skin cancer. Tensin 2 is downregulated in kidney and lung cancer but over expressed in hepatocellular carcinoma.

65. CONCLUSION Cell adhesion and adhesion molecules have been shown to contribute to the pathogenesis of a large number of common human disorders and tumor cell metastasis in cancer. Recent studies have demostrated that CAM are involved in signal transduction pathways. These molecules transmit signals from the extracellular matrix to the cell interior (outside-in) and from the inside of the cell to the outside (inside-out) similar to those transduced by growth factors, hormones and cytokines. These results are extremely significant in metastatic spread and the treatment of a large number of human disorders.