‘ Cell junctions of oral mucosa ’-in a nutshell

Junctional complexes are specialized contacts between neighboring cells and between cells and the extracellular matrix. They play an important role in embryogenesis, growth and development, as well as being the cause of pathologies. These contacts lead to a number of different interactions that have a profound effect on cellular biology. Cell junctions are best visualized using conventional or freezefracture electron microscopy, which reveals the interacting plasma membranes are highly specialized in these regions. Cell adhesion molecules (CAMs) are proteins responsible for homophillic and heterophillic adhesions. They consist of various groups, including cadherins, selectins and intergrins and they facilitate cell adhesion, cell signaling, and motility. Dysregulation of these molecules can lead to various pathologies, for example mucocutaneous diseases and invasion of cancer. This review focuses on the pathophysiology of cell junctions and related diseases.


INTRODUCTION
Oral mucosa is formed from multiple cell types attached together and to an extracellular matrix by junctional complexes.They are made of protein complexes and form "specialized contact" areas between neighboring cells and the extracellular matrix 1 .Such contacts lead to a number of different interactions that affect shape, position, differentiation, metabolism, morphogenesis and proliferation 2 .
Initially these attachments were thought to be simply adhesive substances present between cells 2 .After the introduction of the electron microscope, it was revealed that these attachments were highly specialized structures, now known as "junctional complexes" 2,3 .These junctional complexes determine the architecture of epithelium by mediating the adhesion of cells to one another in an orderly manner.They help in forming communication channels that allow the passage of signals and metabolites between cells 4 .
Junctional complexes are formed by the binding of cell to cell or cell to extracellular matrix by protein molecules collectively called cell adhesion molecules (CAM)-i.e.integrins, cadherins, catenins, laminins 4 .These are best visualized using conventional or freeze-fracture electron microscopy 2 .
Cell junctions play a crucial role, beginning from physiological processes in embryogenesis, such as differentiation, migration, proliferation and morphogenesis to the causation of pathologies such as cancers and mucocutaneous diseases (e.g.pemphigus, pemphigoid).Studies into alterations in morphological as well as functional changes in junctional complexes within tissue will assist in diagnosis and prognosis of the ailment.

A. TYPES OF JUNCTIONAL COMPLEXES
Junctional complexes consist of two important structures 5 .

A. Cell junctions
Classifications of cell junctions are shown in Figure 1.Cell junctions can be divided according to specialization or according to morphology.i.According to specialization 2 (1) Unspecialized contact: Contacts that do not show any specialized features upon examination under an electron microscope.(2) Specialized junctional contact: These junctional contacts have specialized characters.

iii. Gap junctions
On a molecular level, intercellular junctions typically consist of three components 6 (Figure 2)a transmembrane adhesive protein, a cytoplasmic adapter protein and a cytoskeletal filament.

According to morphology
Zonula occludens/Tight junctions (TJ): These consist of transmembrane adhesive proteins, namely claudins and occludins.They acts as the major contributors to cell polarity at apical and basolateral membranes by preventing certain material from moving between domains 4 .At the molecular level, these proteins form sealing strands that stitch the membrane together, controlling the passage of material through intercellular spaces 7 .These proteins are key regulators in transmitting signal pathways.Occludin is one protein that enables transforming growth factor b (TGFb)-dependent tight junction disassembly, shown to be responsible for epithelial-mesenchymal transition in development and tumerogenesis 4 .
Adhesive junctions (AJ): They form a lateral adhesion device in a band or belt-like configuration around the cell.Thus they are often referred to as zonula adherens 8 .In biogenesis, AJ are the first cell junctions to form between two contacting cells 4 .The morphological and functional integrity of the zonula adherens is dependent on calcium molecules.Removal of Ca 2þ ions from epithelia leads to the disruption of adhesive junctions 8 .
i) Cell-to-cell junctions: (a) Zonula adherens (ZA): In ZA, the principle transmembrane proteins are members of the cadherin (Ca) family.Alpha and b catenins are the cytoplasmic adapter proteins 3 and actin filaments the cytoskeletal components 8 .They play a role in cellular signaling using intracellular signaling proteins to enable junction signals to reach the cell interior 4 .Their cytoplasmic component interacts with the cytoskeleton causing changes in cell shape and motility 3 .Loss of cell -cell contact can lead to apoptosis 9 .(b) Macula adherens (Desmosomes): Desmosomes are button-like points of intercellular contact that tie cells together.They serve as anchoring sites for intermediate filaments and form a structural framework, as a result they are responsible for great tensile strength 8 .
Composed of cadherins these junctions form a desmosomal plaque to which keratin filaments bind.Desmoglein 1 and 4 and desmocollin 1 and 3 are found in the human epidermis, the intracellular domain coordinates with adaptor proteins to form the desmosomal plaque 9 .The two families of plaque proteins in desmosomes are: i) plakoglobin and plakophilins, these include b-catenin and p120ctn, and ii) desmoplakin, envoplakin, periplakin and plectin from the plakin family 9 .Desmogleins 1 and 3 are found exclusively in oral squamous epithelium from stratum granulosum to stratum basale 8 (Figure 3).
ii) Cell to matrix: (a) Focal adhesions (FA)-Focal adhesions are sites where integrin and proteoglycan mediated adhesion link the actin cytoskeleton.Located at the cell periphery and in central areas, these junctions are associated with stress fibers, such as actin filaments.Multiple protein-protein interactions take place at FA. Various FA molecules are focal adhesion kinase (FAK), src, paxillin, talin, vinculin and calpain 10 .In cancer, it is evident that an increase in expression of FA is directly associated with an increase in invasive and metastatic properties of that cancer 11 .(b) Hemidesmosome or half-desmosomes-Formed from proteins including laminin and plectin.
Transmembrane proteins present are integrins 12 .The cytoplasmic adapter protein is bullous pemphigoid antigen 230 (BP230), plectin forms a dense plaque on the cytoplasmic surface of hemidesmosomes.These junctions act as rivets to distribute tensile or shearing forces through an epithelium 8 .They are important in various blistering diseases such as epidermolysis bullosa and bullous pemphigoid 10 .In squamous cell carcinoma, cell invasion occurs by an increase in disassembly of hemidesmosomes 11 .
Gap junctions (GJ): Gap junctions are plaque-like regions of the cell membrane.They electrically couple cells 8 .Special transmembrane proteins, called connexins are present in gap junctions, consisting of four membrane-spanning a helical and intracellular C and N terminals.Six of these protein units in a ring formation creates central aqueous pore.GJs are required for rapid signaling between electrically excitable cells and for a slower spread of intercellular, second messenger signals between other cell types 13 .Various studies have shown that other than connexins different transmembrane proteins, for example pannexins and vinnexins, are present in gap junctions 14 .
GJs allow hydrophilic passageways between adjacent cell membranes for the intercellular exchange of ions and small molecules 15  associated with tumerogenesis.Deficiency of connexin expression results in a decrease in GJ assembly in OSCC 16 .
B. Cell adhesion molecules (Figure 4) Cell adhesion molecules (CAMs) are proteins responsible for homophilic adhesion (binding of an adhesion molecule on one cell to the same adhesion molecule on a second cell) and heterophilic adhesion (an adhesion molecule on one cell type binding to a different type of cell adhesion molecule on a second cell) 17 .
Generally the adhesion between CAMs is dependent on the presence of calcium ions, see Table 1 4 .
Cadherins: Cadherins are transmembrane proteins with an external domain of 110 amino acids, and a cytoplasmic domain that binds to p120 catenin and b-catenin 18 .First, they mediate cell-cell adhesion in tissues 19 .Secondly, they reduce surface tension at the cell-cell interface.For reduction in surface tension, a reorganization of actomyosin cytoskeleton signaling is required, controlled from the cadherin adhesion complex.Thirdly, they stabilize cell contact by resisting mechanical forces.
The cadherin superfamily includes cadherins, protocadherins, desmogleins and desmocollins, plus others.Different cell types possess different members of the cadherin family, e.g.N-cadherins in nervous tissue, E-cadherins in epithelia, P-cadherins in the placenta, CDH4 -R-cadherin in the retina, CDH5 -VE-cadherin in vascular endothelium and CDH6 -K-cadherin in the kidneys 18,19 .
Catenins: Cateninsare proteins present in complexes with cadherins.The name "catenin" was originally selected ('catena' means 'chain' in Latin) because catenins link cadherins to the cytoskeleton.There are four types of catenins: Alpha, Beta, Delta and Gamma catenin; alpha and beta catenin being the more important proteins 20 .Beta-catenin is a protein in humans that is encoded by the CTNNB1gene 21 .It is an integral component in Wnt (wingless) signaling that directs cell proliferation, cell polarity and cell fate determination during embryonic development and tissue homeostasis 22 .Mutations result in the formation of congenital abnormalities, cancers and other defects 22 .Importantly they are also responsible for the maintenance of stem cell populations 23 .
Selectins: Selectins are transmembrane lectin glycoproteins that can bind with low affinity to carbohydrate groups on other cell surfaces.The 3 major types of selectins are: L-selectins, E-selectins and P-selectins.L-selectin is concentrated on the tips of microvilli on most leukocytes that promote trafficking on high endothelial venules in lymph nodes and in activated endothelial lining during inflammation.E-selectin is secreted as de novo by endothelial cells.Finally, P selectin is stored in Weibel Palade bodies, present in endothelial cells, and is released after stimulation by cytokines at an inflammatory site 24 .Selectins, with integrins and other molecules, play a role in inflammation by slowly rolling and capturing leukocytes from the blood into inflamed tissues.Defects in selectin ligands are responsible for leukocyte adhesion deficiency type II disease, which leads to bacterial infections in oral mucous membrane 25 .
Integrins: Integrins are hetero dimers made up of a and b subunits, a minimum of14 a and 8 b subunits have been identified.Some integrins have receptors for leukocytes, collagen, RGD, laminin, etc.They are able to transduce signals intracellularly, following ligand binding "outside-in" signaling and "inside-out" signaling 26 .Integrins have a unique role in cell adhesion due to their ability to couple the ECM to the intracellular cytoskeletal network.Integrins are responsible for intracellular cell signaling, and have been shown to regulate signaling cascade in cancer cells that support cancer growth, progress and metastasis.Thus integrins can be considered as a cancer target therapy and used as a drug therapy in prevention of cancer progression 25 .
Syndecan: Syndecans are a family of transmembrane core proteins that contain heparan sulfate and chondroitin sulfate glycosaminoglycan chains.Vertebrates have four syndecan genes (Syndecan-1,-2, -3,-4).The protein has a short cytoplasmic domain, a single-span transmembrane domain (TM) and an extracellular domain 27 .Syndecan is responsible for cell adhesion and regulation of various signaling events 27,28 .Syndecans potentiate signaling by binding fibroblast growth factor2 (FGF2), Heparin-binding EGF-like growth factor (HB-EGF), and vascular endothelial cell growth factor (VEGF) 28 .
Syndecan plays a role in developmental processes and alteration of its expression leads to tumor formation, progression and metastasis 28 .Syndecan 1 and 2 are expressed in epithelial and mesenchymal tissue 27 .It was found that Syndecan 1 is responsible for the maintenance of epithelial morphology, due to anchorage-dependent growth 28 .Thus, down regulation of syndecan is associated with a decrease in cell adhesion and acquisition of cell motility.

EXTRACELLULAR MATRIX MOLECULES
Fibronectin: Fibronectin has a dimeric structure composed of two dimeric polypeptide chains joined near their C-terminal by disulfide bonds.Each chain has been identified for cell membrane integrins and a variety of extracellular matrix molecules 29 .
Two types of fibronectin are present 30 .Soluble plasma fibronectin, produced in the liver by hepatocytes.
. Insoluble cellular fibronectin, secreted by various cells, primarily fibroblasts, as a soluble dimer and then assembled into an insoluble matrix in a complex cell-mediated process.
Fibronectin is a significant component of basement membranes in developing organ systems, where it stabilizes cells and allows them to establish polarity and to undergo further differentiation 8 .Interaction occurs during the differentiation of preodontoblasts 5 , and fibronectin plays a crucial role in wound healing.Fibronectin is found in human saliva and prevents adherence to buccal epithelial cells 31 .It is crucial for blood vessel morphogenesis and integration between endothelial and perivascular cells 32 .
Laminin: Laminin is a major constituent of the basal lamina complex.The laminin molecule is a heterodimer of a, b and g subunits.The three chains assemble to form a cross-shaped molecule 29 .Fifteen laminin trimers have been identified.The laminins are combinations of different a, b, g chains: Five forms of a chains: LAMA (1, 2, 3, 4 and 5) four of b chains: LAMB (1, 2, 3, and 4), three g chains: LAMC (1, 2 and 3) 33 .
Laminin is responsible for binding heparin.It promotes cell adhesion of murine melanoma cells, fibrosarcoma and chemotaxis.It also promotes melanoma cell migration.It inhibits the formation of lung tumors and promotes adhesion of endothelial cells 28,34 .Laminin 5 plays a vital role in tumor migration and shows increased expression in areas of tumor-stroma interaction 34 .
Tenascin: Tenascin is also known as Cytotactin and H exabrachion.It is made of six polypeptide chains assembled to form a six-armed structure 29 .Four types of paralogues have been identified as C,R, X, and W. It modulates cell-matrix interactions and influences a state of matrix attachment that promotes motility.It has a role in development as well as in wound healing, neovascularization and tumerogenesis 35 .
Nidogen: Nidogenis also known as entactin.It is a rod shaped protein consisting of a single polypeptide chain approximately 30 nm long with globular domains at each end and one centrally located domain 29 , produced by mesenchymal cells.It is ubiquitously found in the basement membrane.Nidogen is produced by mesenchymal cells and is ubiquitously found in the basement membrane, where it serves as a link between laminin-1 and collagen type IV, here it acts to stabilize the basement membrane.A nidogen 2 molecule covalently present with nidogen 1 plays an important role in organogenesis, at the time of development 36 .

ROLE OF CAM IN ORAL SQUAMOUS CELL CARCINOMA (OSCC)
The integrity of cadherin-catenin junctions is of the utmost importance as they consist of fundamental components responsible for progression and metastasis of oral cancer.Loss of E-cadherin and increase in N-cadherin underlines a poor prognosis of the disease.At present many studies have proven the role of these junctions in metastasis and invasion.Hanemann et al, used immunostaining E-cadherin studies on various grades of OSCC.They showed that in well-differentiated OSCC the cohesiveness between tumor cells was adequately maintained by E-cadherin and the tumor was less invasive.Cells of poorly differentiated OSCC showed weaker immunostaining for E-cadherin and did not have intactintercellular junctions.Importantly, single cell invasions were more pronounced.E-cadherin signifies the biological potential of both well-differentiated and poorly differentiated OSCC 37 .Epithelial mesenchymal interactions are a key process where there is down-regulation of E-cadherin and epithelial cells assume mesenchymal attribute.Transcription of E-cadherin is controlled by several transcriptional repressors including snail, slug, DeltaEF1/ZEB1, SIP1 (Smad interacting protein 1)/ZEB2 genes 38 .Cox2 is responsible for the carcinogenesis process and subsequent down regulation of E-cadherin.Studies demonstrated that there was an inverse relationship between E-cadherin and Cox2.Fujji et al, showed 'in vitro' that inhibition of Cox2 expression can restore the loss of E-cadherin expression by down-regulating the transcriptional repressor genes 38 .
Catenins are invariably associated with cadherins.Dysregulation of catenins is responsible for the dysfunction of E-cadherin.E-cadherin expression is significantly reduced in advanced OSCC 39 .Its supra molecular components, such as desmoglein, desmoplakin and desmocollin are associated with tumerogenesis and metastatic processes 39 .Chen et al, found that desmoglein (DSG) 3 was highly expressed in head and neck carcinoma.It was explained that DSG3 binds with specific desmosomal proteins and disrupts the stability of desmosomes, resulting in metastasis and invasion 40 .While desmosomal components desmoplakin and desmoglein showed no correlation with survival level 39 .
Another significant CAM molecule is selectin.Research has shown that tumor cells express E-selectin on their cell surface and use it to attach to endothelial cells that aids transmigration of the tumor cells through venules during metastasis, mimicking leukocyte transmigration process in inflammation.Highly metastatic tumor cells up-regulate selectin ligands on their surfaces, such as sLeX or sLea 24 .
The role of integrins has been widely studied, resulting in the conclusion that cancer is associated with defective signaling by integrins.Different integrins, for example a1b1 or agb 1, play a role in the attachment of cancer cells to the basement membrane, via fibronectin and laminin, in head and neck cancer.Thus tumor integrins confer polarity by binding to the ECM 41 .High expression of one integrin heterodimer, a1b2, is directly correlated with the invasiveness of a tumor 42 .Recent studies show highly metastatic head and neck tumors were associated with an increased expression of the a1 integrin.Wang et al, 2012, found that an increased expression of a1 integrin was found in primary tumors with metastasis.They also concluded that a low expression of a1 integrins were directly proportional to the longer, overall survival rate of the patient, hence can be used a prognostic biomarker 43 .
In OSCC, increased expression of avb6 integrin leads to activation of MMP3, an enzyme responsible for the increase in cell migration and invasion on fibronectin.Therefore, integrins can responsible for invasive processes in cancers 44 .Antibody or peptide inhibitors against integrins can act as a therapeutic approach to prevent the metastasis and migration of cancer cells 45 .
The CAM molecule syndecan has a significant role in cancer.Approximately 60% of primary tumor mucosa showed a loss in the expression of syndecan-1 as compared to benign oral mucosa 46 .
Studies have shown that expression of glycosylated oncofetal fibronectin was increased in an invasive phenotype of oral cancer cell lines 40 .In OSCC, increased expression of fibronectin (120kDa fragment) in association with an enhanced activity of MMP-9 is correlated with highly motile phenotype 47 .
Laminin is considered to be the biochemical equivalent to the anchoring filament and is used as a marker for the disintegration of the basement membrane in the carcinogenesis process.Tumor cells attach to laminin receptors and secrete MMP's, which subsequently degrade and invade the basement membrane Shruthy et al, 2013, studied laminin expression in OSCC and found increased aggregation around the basement membrane in well differentiated OSCC 48 .
In head and neck cancer, high expression of tenascin C is correlated with poor prognosis and survival of the patient 49 .Messenger RNA expression and protein levels were found to be elevated in OSCC 50 .In OSCC tenascin acts as an anti-adhesive agent by blocking fibronectin binding to cells, and therefore is responsible for proliferation and migration of cells 51 .

ROLE OF DESMOSOMES IN AUTOIMMUNE BLISTERING DISEASES
Pemphigus is a potentially fatal autoimmune disease 52 , where autoantibodies are directed against desmosomal glycoproteins that result in the loss of cell-cell junctions.Of the two variants of pemphigus-P Vulgaris (PV) and P Foleacae (PF), P Vulgaris is more common in oral mucosa.Desmogleins act as the antigens, specifically, desmoglein 1(Dsg1) is associated with foleacae and desmoglein 3(Dsg3) is associated with vulgaris 53 .Desmocollin (DSC) 3 is an isoform of desmosomal cadherin predominantly expressed in the basal cell layer 52 it acts as an antigen in some pemphigus vulgaris patients 52 .Deletion of mouse Dsc3 causes suprabasillar blisters similar to those seen in PV.DSC 1 is associated with IgA pemphigus, specifically in subcorneal pustular dermatosis-type (SPD-type) 54 .
A study by Abe ´T et al, 2014, showed that not only Dsg3 played a part, but that IgG4 was also involved in the pathogenesis of PV.Concluding both should be evaluated in PV cases 55 .
Mucous membrane pemphigoid is a chronic autoimmune disorder in which hemidesmosomes are targeted by auto-antibodies 55 .Other antigens involved are laminin 5 and b4 integrin 56 .Subepithelial vesiculobullous disorders present as blisters and erosions, diagnosis is established through biopsy 57 .
CAM and Diseases: Cell adhesion molecules are complex structure and are targeted in the following common diseases 58 -60 CONCLUSION Junctional complexes are present ubiquitously.TJ, AJ, and GJ are unique type of cellular junctions and bear their own exceptional characteristics and functions.They play a significant role in development, embryogenesis and normal physiological processes.CAMs, such as cadherins, catenin, integrins, are present at the cell surface for attachment and signal transmission.Lack of these complexes can result in an absence of intercellular communication and thus a lack of cell survival.Recently, research on these molecules has validated using them in diagnosing various pathologies.Dysregulation results in dreadful diseases, an example being oral squamous cell carcinoma.Desmosomes and the supramolecules associated with them are involved in mucocutaneous diseases, such as pemphigus and pemphigoid.Deciphering the biophysical properties and consequences of cell junction dysfunction will aid in clinical intervention for the practitioner.

Figure 1 .
Figure 1.Types of cell junctions

Figure 3 .
Figure 3. Types and distribution of desmosomes in oral epithelium.

Table 1 .
Summary of cell adhesion molecules, calcium dependency and cellular expression.