the epithelial tissue in various organs ( e.g. , lungs , intestines , and skin ) is composed of a seat of epithelial cells that separate the biological compartments in the body with different internal environments . the intercellular adhesion complex between epithelial cells consists of tight junctions , adherens junctions , and desmosomes and is fundamental to the construction of the epithelial cell seat and the maintenance of cellular polarity [ 1 , 2 ] .   within the intercellular adhesion complex , tight junctions possess two distinct functions in the epithelium tissue ( figure 1(a ) ) . they function as a barrier controlling molecular penetration of ions , solutes , water , and cells through intercellular space and act as a fence dividing apical and basolateral domains to compartmentalize the plasma membrane . these characteristics of tight junctions allow epithelium to prevent pathogens and foreign substances from invading and to facilitate directional exchange of materials . tight junctions comprised 4 kinds of transmembrane proteins : occludin , claudins , junctional adhesion molecules ( jams ) , and tricellulin as well as numerous cytosolic proteins ( figure 1(b ) ) . the cytosolic proteins are roughly divided into two groups depending on the presence or absence of pdz ( psd-95 , dlg , and zo-1 ) domains : the pdz proteins ( zo-1 , -2 , -3 , par-3 , -6 , and membrane - associated guanylase kinase protein [ magi]-1 , -2 , -3 , etc . ) and the non - pdz proteins ( cingulin , heteromeric g proteins , atypical pkc [ apkc ] , rab-3b , -13 , pten , etc . ) [ 5 , 6 ] . thus , a growing body of studies has clarified the molecular components of tight junctions , but it is still obscure how they accumulate and form tight junctions .   the intercellular adhesion complex and cell polarity should be established during differentiation of stem cells into epithelial cells . in this paper , we focus on transmembrane proteins of tight junctions and highlight the participation of tight junctions , extracellular matrix , and nuclear receptors in epithelial differentiation . occludin is a tetraspan membrane protein with two extracellular loops ( ec1 and ec2 ) , a short intracellular turn , and n- and c - terminal cytoplasmic domains . among these domains , the long c - terminal domain is phosphorylated at serine , threonine , and tyrosine residues by various protein kinases including src family kinase and ck2 [ 8 , 9 ] . the c - terminal region of occludin also directly binds to zo-1 , and the phosphorylation of some tyrosine residues prevents both the interaction with zo-1 and the assembly at tight junctions . in addition , the phosphorylation of some threonine and serine residues enhances occludin trafficking to tight junctions and the barrier function . although there are four splicing variants in occludin , the biological significance of each variant in tight junctions is unclear . overexpression of full - length and mutated occludin gene in madin - darby canine kidney cells or xenopus cells   suggests that occludin contributes to the barrier function of tight junctions . by contrast , occludin - deficient embryonic stem cells differentiate into polarized epithelial cells with well - developed tight junctions . moreover , occludin - null mice are born with normal structure and barrier function of tight junctions in the intestinal epithelial tissue , but they exhibit various phenotypes , such as growth retardation , hyperplasia of gastric mucosa , absence of cytoplasmic granules in striated duct cells of the salivary gland , thinning of the compact bone , brain calcification , and testicular atrophy . these phenotypes imply that occludin may be involved in neither epithelial differentiation nor the barrier function but in other unknown roles . occludin interacts with a variety of cellular signaling molecules and may contribute to the signal transduction . for example , occludin binds to transforming growth factor [ tgf]- type i receptor and regulates tgf--dependent disassembly of tight junctions during epithelial - to - mesenchymal transition . it also associates with nonreceptor tyrosine kinase c - yes , apkc , pi-3 kinase , and protein phosphatases 2a [ 6 , 17 ] . furthermore , occludin regulates the organization of actin and the directional migration in epithelial cells . in addition , it is reported that occludin is concerned with apoptosis via activation of the small gtpase rhoa , mitogen - activated protein kinase ( mapk ) , and akt signaling pathways [ 19 , 20 ] . the claudin family consists of 24 distinct members in human and mice with three others recently identified [ 4 , 21 , 22 ] . they are 18- to 27-kda tetraspan proteins with n- and c - terminal cytoplasmic domains and two extracellular loop domains , and are expressed in tissue- and cell type - specific manners [ 3 , 23 ] . in addition , isoforms of some claudins ( e.g. , claudin-10 and -18 ) are generated by alternative splicing and exhibit different expression patterns and functions [ 24 , 25 ] . claudin genes have few introns , and several pairs of them are located in close proximity in human and mouse genome . for example , in humans , claudin-3 and claudi-4 are located on chromosome 7 , claudin-6 and claudin-9 on chromosome 16 , claudin-8 and claudin-17 on chromosome 21 , and claudin-22 and claudin-24 on chromosome 4 . gene duplication is assumed in these claudins , and the coordinated expression is reported at least for claudin-3 and claudin-4 genes . claudins are indeed the backbone of tight junctions , since they can reconstitute tight - junction strands even in fibroblasts . all claudins except for claudin-12 possess a pdz - binding motif and are capable of direct interaction with the proteins containing pdz domain such as zo-1 , -2 , -3 , multi - pdz domain protein ( mupp-1 ) , and pals-1 associated tj protein via the cytoplasmic tail ( patj ) [ 2932 ] . hence , claudins are linked to the actin cytoskeleton through binding to the scaffolding proteins ( zo-1 and zo-2 ) , stabilizing tight junctions . it should be noted that the posttranslational modification of claudins influences their localization and function . first , the phosphorylation of claudins is associated with tight - junctions assembly and paracellular permeability . phosphorylation of claudin-3 , -5 , and -16 by protein kinase a facilitates tight - junctions assembly and functionality [ 34 , 35 ] . second , palmitoylation of claudins at the dicysteine motif , which is conserved through the claudin family , is thought to increase claudin accumulation in tight junctions and enable their translocation to detergent - resistant plasma membranes ( lipid rafts ) . third , o - glycosylation in some claudins , claudin-1 , -3 , and -4 by o - linked n - acetylglucosamine transferase is predicted , but the function of this modification is unknown . physiological roles of claudins have been clarified from studies of transgenic and knockout mice or human diseases . mutations in claudin-1 gene cause neonatal ichthyosis and sclerosing cholangitis in humans , and claudin-1-deficient mice result in neonatal death due to the disturbance of skin permeability [ 40 , 41 ] . claudin-2 knockout mice show no obvious phenotypic changes ( e.g. , of growth and behavior ) . claudin-7-null mice die within 12 days of birth owing to severe salt wasting , chronic dehydration , and growth retardation , which are caused by the breakdown of nacl homeostasis . claudin-11 ( also known as oligodendrocyte - specific protein ) ko mice exhibit slowed cns nerve conduction , markedly hindlimb weakness , and sterility in males due to the absence of tight - junction strands in cns myelin and between sertoli cells . mutations of claudin-14 gene lead to autosomal recessive deafness in mice and humans , suggesting that claudin-14 is associated with the cation - restrictive barrier in outer hair cells of the cochlea in the ear [ 46 , 47 ] . claudin-15-deficient mice show uneventful birth and growth but manifest an enlarged upper small intestine due to increased proliferation of normal cryptic cells . moreover , in the adult small intestine of claudin-15-deficient mice , luminal na and k homeostasis is disturbed , and glucose absorption is significantly decreased . various mutations in claudin-16 gene are seen in patients of familial hypomagnesemia with hypercalciuria and nephrocalcinosis , an autosomal recessive disease with severe mg and ca wasting . in fact , claudin-16 is expressed in epithelial cells of the thick ascending loop of henle . taken together with the finding that mutations in claudin-19 gene also show similar phenotypes with abnormal mg reabsorption , it appears that claudin-16 and -19 modulate paracellular absorption of mg and ca in the kidney . jams belong to the immunoglobulin ( ig ) superfamily and are n - glycosylated transmembrane proteins of tight junctions [ 5 , 52 ] . they consist of two extracellular ig - like domains , a single transmembrane region , and a c - terminal cytoplasmic domain that ends in a canonical pdz domain - binding site . jam - a ( also referred as jam / jam-1/106 antigen / f11r ) , jam - b ( also known as ve - jam / mjam-3/hjam-2 ) , and jam - c ( also known as mjam-2/hjam-3 ) have a class ii pdz - binding domain at c - terminal ends , which directly interact with proteins containing pdz domains : zo-1 , af-6/afadin , mupp1 , and par-3 . on the other hand , coxsackie and adenovirus receptor ( car ) , endothelial cell - selective adhesion molecule ( esam ) , and jam4 contain a class i pdz domain - binding motif at their c - terminal ends , associate with ligand - of - numb protein x1 and magi-1 [ 5659 ] . in addition , it has been reported that jam - a , jam - b , and jam - c interact with integrins l2 ( lfa-1 ) , 41 ( val-4 ) , and m2 ( mac-1 ) , respectively , via the extracellular domains [ 60 , 61 ] . jam - a contributes to the formation of tight junctions and cell polarity through homophilic binding and interaction with par-3/apkc / par-6 complex in epithelial cells [ 62 , 63 ] . jam - b and jam - c also associate with par-3 via pdz - binding domain . jam - b and jam - c are expressed in sertoli cells and spermatids , and jam - c is essential for the polarization of round spermatids . by contrast , esam is observed in endothelial cells and platelets , and the deletion of esam gene increases vegf - induced permeability in mice endothelial cells [ 6567 ] . interestingly , jam - c is expressed in undifferentiated embryonic stem cells more abundantly than in differentiated cells . tricellulin , a tetraspan protein that concentrates at tricellular contacts of epithelial cells , was identified by using gene chip analysis to compare parental epithelial cells and cells undergoing emt . it is phosphorylated by ck1 , and its expression is repressed by snai1 , a zinc - finger type transcription factor that plays an important role in emt . in addition , tricellulin concentrates at tricellular tight junctions in cochlear vestibular epithelial cells and the recessive mutations of tricellulin gene cause nonsyndromic deafness . moreover , tricellulin is related to the epithelial barrier and organization of both bicellular and tricellular tight junctions . interestingly , tricellulin is incorporated into claudin - based tight junctions independently of zo-1 binding and is translocated from bicellular to tricellular tight junctions in the presence of occludin . during embryonic development in mice , claudin-6 is first detected in the epithelial zone of embryonic bodies at least in part via the bone morphogenic protein - signaling pathway , and afterwards observed in several types of epithelial tissues such as the epiblast and hypoblast ( to e7.5 ) , the definitive endoderm ( to e8.5 ) , the entire gut , optic vesicles , and a small region of the forebrain ( at e9.5 ) . although these results imply that claudin-6 is involved in epithelialization , the examined claudin-6-null mice show no abnormality . the epithelial - mesenchymal transition ( emt ) is a process during which epithelial cells convert to mesenchymal cell morphology . this occurs in normal developmental processes including mesoderm and neural - crest formation and in the invasive process in tumors of epithelial origin . emt has two steps : loss of intercellular adhesion ( adherens and tight junctions ) and acquiring cell motility . snail directly binds to the promoter of e - cadherin , claudin-3 , claudin-4 , and claudin-7 and represses the expression of those genes , thereby inducing emt . therefore , not only e - cadherin , but also claudins are possibly related to emt .   in the development of gut tube , endodermal cells differentiate into gut epithelial cells and form a lumen as the cells polarize . claudin-15 associates with single lumen formation and forms ion - permeable pores from the study of knockdown of it using morpholino in zebrafish . these findings also suggest that claudins directly relate to epithelial differentiation . retinoids have numerous biological effects on vertebrate development , differentiation , proliferation , and homeostasis through two types of nuclear receptors , retinoic acid receptors ( rars ) and retinoid x receptors ( rxrs ) [ 78 , 79 ] . using a mouse f9 stem - cell line , we previously showed that various rxr / rar heterodimers exerted both specific and redundant functions in endodermal ( epithelium - like cells ) differentiation . in addition , we found that the rxr/rar pair mediated the induced expression of tight - junctions molecules ( occludin , claudin-6 , and claudin-7 ) and the establishment of both polarized epithelial morphology and functional tight junctions ( table 1 ) . it is also reported that retinoid receptors and the kinase ikk1 cooperatively regulate claudin-23 expression in keratinocytes and participate in the formation of the epidermal barrier ( table 1 ) . hepatocyte nuclear factor 4 ( hnf4 ) , another member of the nuclear receptor superfamily , transcriptionally controls the expression of a large number of target genes involved in nutrient and drug metabolism , hematopoiesis , and blood coagulation [ 9294 ] . it is initially detected in primitive endoderm and subsequently in visceral endoderm ( ve ) during early mouse development . in adults , hnf4 is expressed in a variety of epithelial cells of the liver , kidney , intestine , pancreas , and stomach . hnf4 also participates in the differentiation of ve cells and hepatocytes [ 96 , 97 ] . interestingly , we demonstrated that hnf4 triggered the expression of tight - junction molecules ( occludin , claudin-6 , and claudin-7 ) and translocated tight junction proteins ( zo-1 , -2 , -3 , jam - b , and jam - c ) and cell polarity proteins ( par-3 , par6 , apkc , crb3 , pals1 , and patj ) to tight junctions as well as the formation of functional tight - junctions and epithelial polarity [ 85 , 86 ] ( figure 2 and table 1 ) . hnf4 also induced the expression of ezrin / radixin / moesin- ( erm- ) binding phosphoprotein 50 and phosphorylation and apical concentration of erm proteins   ( figure 2 ) . thus , hnf4 initiates the formation of tight - junctions and microvillus and induces differentiation in epithelial cells . androgen and estrogen receptors induce the differentiation of prostate and mammary gland epithelium , respectively . several other nuclear receptors , including those of progesterone , corticoid , vitamin d , and ppargamma , induce the expression of claudins in various cells ( table 1 ) [ 83 , 84 , 8991 ] . taken collectively , it is strongly suggested that nuclear receptors induce the expression of tight junctions and cell - polarity proteins and provoke epithelial differentiation with the formation of cell junctions and cell polarity . extracellular matrix ( ecm , also known as basement membrane ) is composed of various proteins , such as collagen , laminin , fibronectin , heparan sulfate proteoglycan , and nidogen , and these proteins are expressed in an organ- and development - dependent manner [ 100 , 101 ] . in epithelial tissue , for instance , laminin and collagen type iv promote the differentiation of intestinal epithelial cells , and ecm functions to increase the ability of proliferation and differentiation of human renal cells and to maintain the differentiated epithelial cells for a long term . concerning the relationship between ecm and tight junctions , ecm proteins , especially fibronectin , increase the expression of claudin-18 and occludin along the plasma membrane in alveolar epithelial cells , and enhance the barrier function . in turn , claudins recruit certain types of matrix metalloproteinases ( mmps , which serve as proteinases for ecm ) onto cell surfaces and enhance the activation of mmp . thus , ecm and tight junctions influence the expression and function of each other 's components in ways that suggest exciting avenues for further research . for instance , marveld3 has similar structure to occludin and tricellulin and is able to partially compensate for the deletion of those genes . bves / pop1a , which belongs to the popeye family popdc , modulates rhoa and zonab / dbpa , a y - box transcription factor , and associates with tight - junctions proteins via zo-1 in epithelial cells [ 4 , 108110 ] . in addition , a novel rhogef ( p114rhogef ) is identified to be involved in maturation of tight junctions via restricted activation of rhoa . thus , a variety of extracellular and intracellular proteins appear to participate in the formation and function of tight junctions in epithelial cells . ( 1 ) where are tight - junctions proteins assembled ( e.g. , cytoplasmic versus junctional assembly ) ? ( 2 ) how are they recruited into the junctions ( e.g. , do cargo proteins exit ) ? ( 3 ) what accounts for the selectivity of heteromeric or homomeric claudin - claudin interactions ? ( 4 ) what is the functional relevance of posttranslational modification of tight - junctions proteins ? in addition , it should also be determined how tight - junctions proteins regulate epithelial differentiation .
tight junctions are intercellular junctions localized at the most apical end of the lateral plasma membrane . 
 they consist of four kinds of transmembrane proteins ( occludin , claudins , junctional adhesion molecules , and tricellulin ) and huge numbers of scaffolding proteins and contribute to the paracellular barrier and fence function . 
 the mutation and deletion of these proteins impair the functions of tight junctions and cause various human diseases . in this paper 
 , we provide an overview of recent studies on transmembrane proteins of tight junctions and highlight the functional significance of tight junctions , extracellular matrix , and nuclear receptors in epithelial differentiation .