INTRODUCTION:

Tight Junctional Complex:

Tight junction occurs as the paracellular environment in both epithelial and endothelial cells conferring the barrier function through occludin and claudin (CLDN) protein family which are together referred as integral TJ proteins constituents¹. These transmembrane proteins in the tight junction, spanning in the intercellular space confines for its target to the paracellular zone, necessary for its proper function². Precisely, the transmembrane proteins were divided into two main groups, occludin and claudin based on their homology; spanning four times on the membrane. Cellular interactions by these membrane proteins were made via ECL loops, JAMs (junction-adhesion molecules) and CAR (Coxsackievirus and Adenovirus Receptor) contain extracellular IgG-like domains for extracellular adhesion³.

When observed under a freeze facture electron microscopy, TJ is presented as a set of continuous, intramembraneous fibrils known as tight junction strands in P-face(protoplasmic)⁴. Ultrathin section EM study helps in the wide understanding of morphology of tight junction, making adhesive contact with the adjacent cells⁵, further confirming its network association among the TJ intramembranous fibrils constituting the complementary grooves⁶. Tight junctions were observed to possess both the barrier and fence functions in multicellularorganism⁵. TJ protein complex has its distinct functions in three different aspects; based on its localization in the plasma membrane. I) Integral TJ protein: Either mediates or it serves as a scaffold for intercellular environment inciting the barrier function. Thus, integral TJ protein has its capability to function differentially. II) Plaque TJ protein: Expressing PDZ domains (PSd/SAP90 and ZO-1) are the connection between the integral TJ and actin cytoskeleton furthermore recruits molecules in the cytoplasm for cell flagging or signalling. III) Nuclear, cytosolic and regulatory protein, post transcriptional, tumor suppressors along with plaque TJ proteins: co-functions to involve in regulation of permeability, cell proliferation, tumour suppression. The tight junction facilitates the cell-to-cell adhesion in endothelial and epithelial cellular sheets where it serves as a border for its polarization between the domains of apical and basolateral membrane. Polarized epithelial cells were also noted to be located at the interfacial region of the lumen and inner cellular layer of different organs⁶. The cell adhesion molecules are the sole cause for progression of cancer cells in which the later, predominantly increase as an effect of the deterioration of one of the junctional complex constituent TJs in epithelial cells leading to malignant carcinomas⁷. Tight junction conformational changes have contributed its major role in case of diarrhoea, supporting the leak-flux mechanism where water has undergone passive diffusion and have collapsed the intestinal lumen². The existence of TJ protein complex was found to be concentrated at the region upstream of the desmosomes or at the apex of the lateral membranes of the stratum granulosum as confirmed by ultrathin sectional study by Hashimoto,1971. Subsequently, the combined function of claudin membrane protein determine the functionality of TJs among the interactions between the 27 claudin subtypes 8. Sequentially, three individual junctions, a tight junction, adherens, desmosome associate to constitute the apical junction complex at apical membranes in the intercellular regions⁵. Apical junction complex forms interaction with cytoskeleton for stability and develop junctional belts that regulates the numerous signal transduction. Apical junction complex was reported to be composed of transmembrane proteins, cytoskeletal elements bridged by the cytoplasmic scaffolding proteins³. Into the scaffolding molecules, the cytoplasm are involved in strong bondage with these junctions was observed to regulate physiological functionalities in cell proliferation, polarity and diffusion. Occurrence of many diseases was due to the disruption of the TJ barrier proteins as reported by Carola Forster., 2008. Hence, study of the complexity of the TJ and morphological changes in Claudin, a transmembrane protein that is involved in cell to cell adhesion have become the hotspot for treating the cancer.

Claudins:

Claudins are tetraspanic membrane proteins (20–27 kDa) defining the structure for tight junctions distributed in the basolateral membrane proteins in which the later was found to bound with Extra cellular matrix (ECM). The basolateral membrane proteins are also referred as integrins^9,1⁰. Claudins accounts for its existence in unique species of 18 (claudin-1 to -18) as reported by Morita K et al.⁹. Further research on claudin brought the fact into light that there are 27 claudinsubtypes as membrane proteins⁸. The structure of claudin comprises of four transmembrane domains and also two extracellular loops ECL1, ECL2 where the loops were observed to bind with -NH2 and -COOH termini embedded in the cytoplasm. The claudins have shown its homology similarity to an eye specific membrane protein (MP20), peripheral myelin protein 22 (PMP22) which was prevalent for its existence in the liver and intestine, epithelial membrane proteins (EMP 1, 2, 3)¹¹. Cellular receptor for food poison induction in human population is due to the action of combined function of an enterotoxin (CPE) produced by Clostridium perfringens was observed. CPE-R has been identified in claudin-4 that has its binding with terminal -COOH of C-CPE toxin where half of the -NH2 terminal was responsible for the permeability across the plasma membrane via pores. Claudin-3 also accounts for the same action of recepting CPE that has been first observed in RVP1(Rat Ventral Prostate -1)¹².

Morphology of Claudin in tight junction:

The paracellular barrier constitute a biological system of 4 Å radial minute pores which are charge selective regulated by claudin and also a discontinuous barrier, which was charge and size independent and are regulated by other proteins and kinases¹³. The normal physiology of the cellular environment suffices the condition for claudin family associated multiple expression where the exception lies for certain claudin for its tissue specificity. The ECL-1 containing approximately 52 residues was responsible for paracellular charge selectivity¹³ with the signature motif (Gly-Leu-Trp-x-x-Cys-(8-10aa)-Cys) being extremely conserved was related closely to the epithelial membrane proteins (EMPs, PMP22, MP20)¹⁴ where protein conformation was stabilized by intramolecular disulfide bond of cysteines. ECL-2 which is of 16–33 residues with helix-turn-helix protein conformation involves in claudin - claudin interactions¹⁵. The ECL differ in the amino acid composition in which ECL-1 was found to be built with 61% glycine and tyrosine amino acid residues and ECL-2 only with 18% of tyrosine residues¹⁶. Isoelectric point (pKi) of first ECL was reported to fall in the range of 4.17 in case of claudin -1, 10.49 in claudin-14 and 4.05 in ECL-2 of claudin-10, -2, -14, -7 and 10.5 for claudin-13¹⁷. Golgi apparatus was the prospective organelle for claudin oligomerization. E.coli cytotoxic necrotizing factor -1, H. Pylori associated factors and CPE induce claudin internalization¹⁸. Di-cysteine palmitoylation motifs induce claudin localisation into tight junction¹⁹ and occurs systematically in detergent resistant membranes of palmitoylated mutants; placing the role of lipid in TJ integrity. The role of tyrosine kinase c-Yes, in regulation of TJ integrity have also been proved by the immunoprecipitation of c-Yes, which was a receptor independent tyrosine kinase, by the researchers²⁰. As evolution has occurred in mammals, stable barrier was formed in stratum granulosum of skin and stratum corneum of cornified envelope or lipid lamellae in Tight Junctional complex. This was experimentally analysed by the research group of in Cln1 -/- mice(claudin negative mice) skin that promotes the lethality at the postnatal period where the presence of transglutaminase-1 becomes the negative critical issue which helps us to realize the fact of functioning of stratum granulosum of skin and stratum corneum occurring coevally. Interestingly, tight junction protein, Claudin-4 and its oligomeric structure was partially stablilzed by perfluoro-octanoic acid. On subjectation to sucrose velocity centrifugation in Sf9 cells and this effect was due to the lipid constituent in the cell line, paved the way for the connexin to get oligomerized for stabilization²¹. Single point mutation in claudin-3 ECL-1 domain converts Asn to the analogous amino acid (Thr) in claudin-4, forming heterotypic linkage to claudin-4 with unaltered interaction with claudin-1 and claudin-5²². Figure 1 shows the general structure of claudin and the arrangement of four transmembrane domains with –NH2 and –COOH termini in the cytosol and the extracellular loops in the paracellular space.

Figure 1: General structure of Claudin with four transmembrane domains I, II, III, IV and two extracellular loops (ECL-1 and ECL-2), that protrudes in the paracellular space where ECL-1 is specific for its disulphide bond. ECL-2 facilitates the Clostridium perfringens enterotoxin binding via Claudin-3 and 4. The loop connecting the II and III transmembrane domains; free end at the IV transmembrane domain contain both palmitoylation and phosphorylation sitesGünzel et al., 2013²³.

Other proteins in tight junction protein family:

Occludin:

Occludin, a 65-kDa integral membrane protein was observed at the tight junction complex with 522 amino acidat q13.1 arm inchromosome 5. The thrust on occludin was due to the barrier properties of the tight junction being maintained by occludin, whose absence or mutation may increase epithelial membrane to be leaky, causing metastasis. This has been proved by the observation of reduced occludin expression in the metastatic patients affected by breast cancer²⁴. The function of the Occludin was disrupted by the change in the composition of the extracellular loop 2 (ECL-2), succeeded by either the addition of a peptide or the deletion of the COOH group from the ECL; which resulted in the ceasing of the fence function, that led to the facilitation of the development of the leaky property of the occludin²⁵. Interestingly, the devoiding or re-concealing of the occludin was not found to show any alteration in the phenotype of tight junction. This helps us to conclude that occludin is one of the integral part of the TJ complex constitution but has no involvement in the structural modification of the complex. Occludin have enlightened its greater trans-epithelial electrical resistance (TEER) by influencing the second ECL loop in epithelial cells though itself was not recognized to involve as necessary determinant for the formation of TJ²⁶. Moreover, the overexpression of occludin have increased the tight junction integrity to trans-epithelial resistance (TER) in MDCK cell line. But the major drawback is that there is no continuity of the distribution of the occludin to the whole TJ strands in the total membrane protein complex in the epidermis²⁷. Occludin phosphorylation was observed to be induced by p160ROCK, an important effector of RhoA in endothelial cells. Ubiquitination of occludin at its -NH2 terminus was influenced by the E3-ubiquitin protein ligase²⁸. Thus, the findings of Morita et al and Traweger A.et al suggests that occludin is expressed in both epithelial and endothelial cells. Occludin being specific for its association with TJ strands, can be incorporated for studies in cancer cell line study, that expresses membrane proteins and can be used as a biomarker that stains the regions at the stratum granulosum. Serine or threonine phosphorylation in occludin has been confirmed by the analysis of the phospho aminoacid and the digestion of the phosphatase in the membrane²⁹. Occludin also involves in the apoptotic function where the C-terminus of occludin receives and transmits cell surviving signals whose loss may initiate apoptosis via extrinsic pathwaysas in the same case of breast cancer research performed by Martin et al.Occludin was found to be concentrated in the cytoplasmic vesicles, provided that it plays its crucial role in representing itself as adhesive structures; occurring as a result of phosphorylation of its own aminoacid²⁸.

Zona occludens:

ZO-1 (Zonula occludens-1), a 225 kDa membrane protein was first identified TJ protein localized in adherens junctions lacking TJs³⁰. ZO-1, ZO-2, and ZO-3, belongs to the family of membrane associated guanylate kinase with distinct motifs and thus has its specification in its function relating to nuclear localization, signal transduction³¹. ZO-1 has 2 isoforms: ZO-1 alpha+, ZO-1 alpha– which lacks 80 aa residue domain present in ZO-1 alpha+. ZO-1 involve in Cadherin-related cell cell adhesion and its distribution was wide among the keratinocytes²⁷.ZO-1 has three domains in PDZ: a Src oncogene homology region 3 (SH3) domain that binds to the ZO-1 associated kinase (ZAK), ZO-1 nucleic acid binding protein (ZONAB), a serine protein kinase, an inactive guanylate kinase (GUK) at its -NH2 termini. ZO-1, -2, and -3 proteins with PDZ domain was found to be interlinked with claudin containing YV site and COOH terminal domain except that of the claudin 12⁵⁹. PDZ expressing system also constitute the MAGI-1, -2, and -3 in the TJ plaque that also lacks the SH3 domain. MAGI-2 and MAGI-3 were also found to form interaction with tumor suppressor PTEN (phosphatase and tensin)³². PDZ1 domain of ZO-1 binds to claudin and as the same way, PDZ2 domain with ZO-2 and ZO-3, connexin43³². GUK domain of ZO-1 was bound to the COOH terminal where the occludin and actin cytoskeleton bondage through proline-rich COOH terminus was bridged by ZO-1³³. These studies depict the interrelation of occluding, claudin and ZO-1 and occludin also requires the association with ZO-1, for its COOH termini contacting with the actin cytoskeleton. Moreover, Claudin-1 and -2, the transmembrane proteins were reported to be in association with occludin and thus constitutes the integral part of the tight junctional complex. This has been proved after the study and analysis of the researchers that IGF-1 have induced both the claudin-1 and occludin expression in enterocytes, and attenuated tight junction dysfunction in intestine of cirrhotic ^rats34. The localisation of the tight junctions and the protein are mentioned in Table 1.

Table 1 Tight Junction Proteins and their localisation in epithelium and endothelial in varied organs

Tight junction protein	Epithelial Localisation	Endothelial localisation	Organ location	Reference
1	Yes	Not known	Hepatocytes	[38]
5	Not known	Yes	Artries of kidney	[27]
Occludin	Not known	Yes	Brain	[59]
JAM	Yes	Yes	-	[35]
CLDN-16	Yes	Not known	Kidney	[60]
CLDN-14	Yes	Not known	Sensory epithelium	[61]
ZO-1 alpha +	Yes	Not known	-	[62]
ZO-1 alpha -	Yes	Yes	Glomerular podocytes	[64]
MUPP-1	Yes	Not known	-	[65]

JAM (Junction adhesion molecule):

JAM-1 is a 43-kDa protein in IgG (Immunoglobulin G) superfamily and comprises V-type Ig, a single TM (transmembrane) and intracellular COOH terminal domain³⁵. JAM-1 forms homophilic interaction between the opposite JAM-1 molecules at its domain site of first V-type Ig. JAM-1 binds to PDZ3 of ZO-1 and PDZ9 domain of multi-PDZ domain protein 1(MUPP-1). Migration of lymphocytes between endothelial cells via T cells that have acted as counter receptor was controlled by JAM-3; a reserve habitat adhesion molecule in TJ complex³⁶. The barrier function in TJ depends on two pathways basically; I) The transcellular: being piloted by energy-dependent transporters and channels on the membranes (apical and basolateral). II) The paracellular : that controls the passive diffusion³⁷. Steady state analysis of epithelial cells that were subjected to freeze fractured immunolabelling have brought a conclusion about the JAM-1 that these adhesion molecules are localized at TJ strands but has no association in TJ integrity. Junction adhesion molecule (JAM), was stumble upon to be expressed in epithelial and endothelial cells in the tight junction³⁵.

Claudin- a breakthrough and Claudin-Occludin interaction:

A major breakthrough for the presence of two integral membrane proteins claudin-1 and claudin-2, have been identified to suffice the major concentration of TJ strands, with V23 kDa as their molecular masses³⁷. Development of TJs between adjacent transfected cells in mouse L-fibroblasts was observed after the introduction of claudin-1 or -2, which were devoid of TJs before their introduction³⁸. Further research on claudin-1 and claudin-2, led us to understand the fact that claudin promoters were found to be regulated by the binding capacities of the TFs catenin/Tcf complexand Cdx homeodomain protein/hepatocyte nuclear factor-1³⁹. However, Claudin-1 and Claudin-2 DNA sequences were subjected to radiolabelling with (32P) dCTP which was used as Northern blot probes. Hence, claudin-1 and claudin-2 can be used as drug targeting probes for cells affected by cancer like icthyosis, cervical cancer and also helps in localizing the cancer prone region in case of metastasis and tumor, providing us a vast knowledge about metastasis and tumour. Experimental proof by Furuse et al., in 1998 unravelled the great fact that the treatment of claudin cDNA with L Fibroblast cells, which lacks claudin or tight junction, has resulted in the well-developed stable network of tight junction in the transfected cells, that sustained as outcome of the report that failure of occludin to be the tight junctional protein, constitutes the structural and functional integrity in the endothelial cells. Studies undergone in mouse epidermal layer (MDCK cell line) have also resulted in the breakthrough of the claudin-1 and -4 expression in the Occludin positive region, that helping in the prediction of the complexity of Occludin-Claudin integrity (heteropolymeric interaction) in the TJ strands³⁸. This study provides a clear note about the concept of TJ integrity where the Occludin-Claudin interlinkage was observed. But in this interlinkage, Occludin is not a necessary factor for the TJ integrity which rather supports the claudin to function via its association with claudin. Research by Moldvay et al. have postulated that downregulation of claudin-7 was prominently found in several types of lung cancers that has been confirmed by the expression profile studies by the research team. Claudin-7 either exhibits reduced expression or degrading rate of its prevalence in lung cancer. The role of different claudins in different diseases and their locations are mentioned in Table 2 and Figure 2.

Table 2 Claudin and its role in development of different diseases

Claudin	Action	Disease	Location	Identified by	Organ	References
14	Depletion of CLDN-14	Hereditary deafness	Not known	Positional cloning	Cochlea in corti	[61]
16	Mutation of CLDN-16	Hereditary hypomagnesemia	Not known	Mutation	-	[60]
3	Up regulated	Ovarian cancer	Epithelial	Malignancy	Ovary	[65]
4	Up regulated	Ovarian cancer	Epithelial	Malignancy	Ovary	[65]
1	Down regulated	Cancer	Not known	Not known	-	[32]
3	Down regulated	Colon cancer	Not known	Malignancy	-	[65]
4	Up regulated	Gastric, pancreatic, colon cancer	Not known	Malignancy	-	[65]
7	Down regulated	Breast cancer	Epithelial	Not known	Breast	[66]
4	Up regulated	Invasive intraductal papillary mucinous neoplasias (IPMNs)	Epithelial	Not known	-	[67]
3,4	Receptor for CPE (Clostridium perfringens enterotoxin)	Pancreatic tumor	Not known	Not known	Pancreas	[68]
4	Up regulated	Cervical cancer, acute self limited colitis (ASLC)	Not known	Not known	-	[69]
1,2	Up regulated	Inflammatory bowel disease (IBD)	Not known	Not known	-	[69]
15	Down regulated	Developmental intestinal defect loss of ion transport selectivity	Not known	Not known	Zebrafish (intestine)	[70]
1	Mutation of CLDN-1	Neonatal ichthyosis-sclerosing cholangitis syndrome(NISCH)	Not known	Not known	-	[71]
7	Down regulated	Neck carcinomas	Not known	Not known	-	[72]
4,5	Down regulated	Hepatocellular and renal carcinomas	Not known	Not known	-	[73]
1,7	Up regulated	Cervical neoplasia	Epithelial	Not known	-	[73]
4	Down regulated	Invasive ductal carcinomas	Not known	Not known	-	[74]
16	Down regulated	Breast tumor	Not known	Not known	-	[75]
1,7	Down regulated	Hepatocellular carcinomas	Not known	Not known	-	[76]
1	Up regulated	Icthyosis	Not known	Not known	Liver, kidney, heart, brain, spleen, lung and testis	[71]
3	Up regulated	Williams-Beuren syndrome	Not known	Not known	Ovary, lung, pancreas, salivary gland, kidney, adrenal, small intestine, colon and thyroid	[77]
1	Up regulated	Cervical cancer	Not known	Not known	Cervix	[69]
2	Up regulated	Cervical cancer	Not known	Not known	Cervix	[69]
4	Up regulated	Cervical cancer	Not known	Not known	Cervix	[69]
7	Up regulated	Cervical cancer	Not known	Not known	Cervix	[69]

Figure 2 The tight junction family proteins and their localisation in the cell membrane Brown et al.,2002⁴⁰.

Claudin-2:

Research on quercetin becomes an interesting area of research because of its antitumor effect due to apoptosis in prostate cancer. Claudin-2 knockdown inhibited the migration of the cancer cells as like the bioavailability of Quercetin, a flavonoid protein in lung adenocarcinoma A549 cells and further claudin-2 showed greater expression in lung adenocarcinoma cells but there was no effect on claudin-1 by quercetin. Phosphatidylinositol 3-kinase (PI3-K) inhibitor (LY-294002) and MEK inhibitor (U0126) downregulates the expression claudin-2 and also inhibits the Luciferase promoter activity as both of these inhibitions exhibited by LY-294002 and U0126 on chemotherapeutic drugs affects the Akt and ERK1/2 mediated signalling pathway that exist as the targets of PI3-K and MEK, respectively leading to the phosphorylation of Akt and ERK1/2. However, promoter function of claudin-2 was inhibited by LY-294002 and U0126 but not by quercetin. claudin-2 was suppressed because of the negative effect on target of ERK1/2 i.e., p-c-Fos level deterioration. The quercetin affects only the stability of claudin-2 mRNA by inducing the expression level of miR-16 which when blocked with an inhibitor restored the normal expression level of claudin-2 in lung adenocarcinomas and quercetin reduces the content of Cyt c, protein level of Bcl-2⁴¹. The expression of claudin-1 and -7 is up-regulated and that of claudin-2 is down-regulated in human lung adenocarcinoma^40,42,⁴³. Claudin-2 also cause Paget’s disease⁴³. The concentration/level of mRNA decides the expression of protein and claudin-2 expression was decreased by the inhibitory activity of cycloheximide and actinomycin D on translation of the claudin-2 protein expression on a timely basis. PC-3 and ABC-1(lung adenocarcinoma cell lines) was supplemented with quercetin which resulted in the decrease of claudin-2 level whereas STAT3 upregulated this transmembrane protein expression in MDCK cell line and cdx1, cdx2, hepatocyte nuclear factor-1 in intestinal Caco-2 cells³⁹ respectively. Scientists have concluded that research in A549 cells, confirmed that EGFR/MEK/ERK/c-Fos pathway upregulated promoter functioning of claudin-2. Claudin -2 also involves in cell proliferation affecting it positively i.e., enhances the proliferation in A549 cells in its nuclei being influenced by the nucleic acid binding protein and cyclin D1 that has its association with the Zona Occludens⁴³. Quercetin also increases the level of claudin -4 in Caco-2 cells and thus serves as the support for intestinal barrier⁴⁴ and the drugs Genistein and Daidzein helps in the rescue of the damaged endometrial cells by elevating the re-development of lipopolysaccharide⁴⁵. The effect of cordycepin-treaed cells (polyadenylation inhibitor) was found to exhibit a short and unclear band when compared to the PCR amplified 3’UTR (Untranslated region) of quercetin-treated cells. Claudin-2 expression was found to be higher in colon⁴⁶ cancer tissues. Quercetin was not directly indulged to affect the expression of claudin-2 rather it inhibits the ERK1/2 and Akt via U0126 and LY-294002 respectively, subjecting them to phosphorylation. The rate of inflation for carcinoma, Alzheimer’s disease are investigated to be higher in mRNAs associated with binding proteins, because of the stabilization authoritated by the binding proteins to the target mRNAs (TNF- α) at the ARE element of the 3’-UTR site, as in the case of Human antigen R (HuR) in RAW264.7 mouse macrophage cells⁴⁷ and claudin-2 was found to be devoid of ARE and thus, HuR does not decides the stability of mRNA of claudin-2. The causation of continuity in the cell division of the premalignant neoplastic cells has a major effect on development of carcinoma due to the GF and receptor barrier disruption ⁷.Transcription activation of Cdx1⁴⁸ has an indirect influence and LEF-1/β-catenin complex mediated Wnt signalling has a direct effect on claudin-2 expression and promoter regulation respectively. Such a binding may protect mRNA degradation from nucleases. The stabilization occurs due to the RNA binding proteins (trans acting) and the mRNA (cis acting) and it has also been found that quercetin inhibits this action of the binding proteins. Cordycepin did not support the amplification of DNA of claudin-2, but quercetin does it and inhibition of polyadenylation was not linked with association of quercetin with the claudin -2 mRNA as well⁴⁹. Claudin-2 expression was also found to be positively influenced by IL13 and TNF⁵⁰. Expression of claudin-2 expression is highly related to epithelial permeability with claudin-1/7 such that claudin-2 is mislocalized or downregulated due to TER which probably increases in colorectal cancer⁵¹. Urinary claudin-2 was elevated in Necrotizing Enterocolitis(NEC) affected patients than its control samples and thus we can switch onto claudin-2 as a biomarker or potential predictor of NEC. Pregnant C57/BL6 mice being parted on the basis of gestation duration as 12, 16 and 20 days showed greater claudin-1, -2,-4,-5 expressions during transcription. Administration of estrogen receptor antagonist ICI 182,780 and progesterone receptor antagonist RU-486 was shown to express claudin-4 mRNA and its associated protein in higher level. Moreover, the hormonal study made by the researchers have made the clear understanding about the activity of these hormones in regulatory pathways that induce the functional modulation of claudins which serves as effectors for ionic movement at the end of these pathways in the mice placenta. The response of claudins for hormonal factors was determined by Realtime PCR and Western Blotting Table 3.

Table 3Association of different claudin with different cell lines and the cancer expression in the cell lines

Cell line	Cancer expression	Claudin expression	Reference
OVCA432 and OVCA433	Ovarian	3,4	[65]
HT-29 and SW480	Colon	3,4	[65]
PC3	Prostate	3,4	[65]
MCF-7	Breast	3	[65]
OVCA432	Ovarian	3(high level), 4(low level)	[65]
OVCAR-2	Ovarian	3,4	[65]
A2780	Not known	-	[65]
Sf9	Not known	4	[17]

PKCι was a necessary factor for epithelial polarity among TJ structures. PKCι knockdown by aPKC pseudosubstrate zeta inhibitory peptide (ZIP) showed increased rate of transepithelial resistance (TER), declined paracellular permeability and claudin-2 expression, though the protein kinase C knockdown have not affected the physiology of TJ. Research contributions by this team depicted us the direct relation of Rab14 and PKCι which shows stability on the cell, confirmed through ELISA and coimmunoprecipitation assays. They colocalize at both the cytoplasmic and the cell-cell contact regions. Also, PKCι lies as the dependent substrate on Rab14 expression for its intracellular and plasma membrane presentation which confirms the Rab14 and aPKC association to regulate claudin-2 expression. IQGAP1, a scaffold protein and whose knockdown have brought TER increase of TJ, depletion of claudin-2 and transitory blocking of CDC42–JNK pathway and thus IQGAP1 controls the TJ protein expression⁵⁴.

microRNAs (miRNA) in Claudin function:

miRNA functions as gene silencers in RNAi interference and this function depends on the target gene for its repression of the gene or the effect would also be vice versa i.e., it turns out to act as an oncogene and this typical expression of miRNAs are profoundly found in lung cancer⁵⁵. Moreover, this microRNA-16 acts an inhibitory template for nuclear factor-kappa B transcription that is a necessary criterion of EMT in glioma cells⁵⁶. Cell cycle arrest was found to be promoted by miR-16 in claudin-2. miR-16 targets G1 cyclin (G1 phase regulator) and thus induces cell cycle arrest. miR-16 expression was inversely correlated with Bcl-2 expression, which induces apoptosis in leukemic cells. miR-16 plays a depletory function on hepatoma-derived growth factor and thus the former is an oncogene to NSCLC cells. miR-15a and miR-16-1was down-regulated or even deleted in CLL of most cases i.e., these micro RNAs are inversely correlated to Bcl-2 at post transcriptional level, thus acting as antisense interactors with the protein Bcl2 in therapeutic technology to treat tumor. Over expression of miR-155 was reported to find its effect to prevent the occurrence of via downregulatory activity on claudin-1 and moreover, inverse proportionate effect of miR-1303 on claudin-18 influences the gastric cancer⁵⁷. Research by the eminent scientists suggests that Claudin-1 TJ protein was found to be downregulated by miR-155 in colorectal and ovarian cancer cells whereas the effect is upside down in the case of hepatocellular carcinoma cells, being influenced by miR-198⁵⁸. Chronic lymphocytic leukemia (CLL) developed by malignant B cells and these non-dividing cells expresses the protein anti-apoptotic B cell lymphoma 2 (Bcl2). It has also been reported that miR-1303 functioned as a gene silencer for gastric cancer cells in claudin-18⁵⁷Table 4.

CONCLUSION:

Wide research on claudin is necessary because claudins can be employed as targets for drugs in the metastatic cells. Claudins shows its responsiveness for hormones that regulate the pathways and thus claudins are the proteins expressed that depends on the hormones, pathways, phosphorylation, palmitoylation for its expression. Claudins are also used as biomarkers as in the case of claudin-5 in detection of CNS injury. From this review paper, we can conclude that claudin is dependent on integrin β1. Claudin-1, claudin-3, E-cadherin remains unaffected by the KD of claudin-7 by HCC827 cells but level of claudin-4 expression was found to increase, suggesting that claudin-7 and claudin-4 expresses inverse proportionally.Claudin-1 was regulated by Akt, Src signalling pathways as in the same case of β catenin. NF-κB, Snail, HGF-R/ cMet Twist mediated signal transduction produce pro survival signals that prevent anoikis in the cells. Claudin knockdown was observed in BGC-823 and HS-746T cell lines, on treatment with specific shRNA. In malignant samples, c-abl-Ras-Raf-1-ERK1/2 signaling controls claudin-1 expression. Src and PIK3/Akt plays opposing function to c-abl-Ras-Raf-1-ERK1/2. This gives us a clear idea about the claudin and its signalling molecules involved in cancer progression. The signal transduction was inhibited by repressors like E12/47, SIP1 which bind to the promoter E-box region. The receptors are the major players in binding the ligands that access the entry of molecules or viruses to enter into the cells as in the case HCV entry into the plasma membrane, reaching the cytoplasm via C-CPE. This is due to the change in the charge of the extra cellular loops. β catenin regulated by Wnt signalling is necessary for stability and on communication with LEF or TCF that induce polarization and differentiation in epithelial cells also possess anti-apoptotic activity. Moreover, it lightens on our knowledge of IGF-1 in its direct cooperation with the tight junction proteins and the cells, function of Kupffer being dependent on IGF-1 for its normal function and its malfunction can be studied by the TJ proteins claudin-1 and occludin. claudin-1 go about as tumor silencer. Downregulation of claudin depletes the level of integrin β1 but has no support to fall with the same case, in the sense of addition of integrin β1 in claudin suppressed cultures had an effect to stimulate the cell adhesion. Claudin has its interlinking cooperation with integrin for its action in the progression or degradation of cancer cells in various tissues where claudin and integrin express proportionally. In case of claudin-5, its conformational changes cause BBB compromisation resulting on CNS injury. In cases of inflammation, environmental stresses, TJ disassembly and its paracellular permeability facilitation occurs due to the activation of myosin light chain kinase (MLCK) and rho kinase mediated phosphorylation. Results have also been established that C-CPE has its effect on MDCK I cells to deplete claudin-4 level resulting in the reduction of transepithelial resistance of TJs confirming that C-CPE can function as a solution for consequences in drug delivery at aspects of cell and molecular ways in which findings provide a remarkable note that claudin-3 is the specific binding protein to C-CPE. So we can conclude that Studies by researchers reveal that higher concentration of claudin-7 KD cells can be observed in DNA synthesis phase(S phase) and mitosis(G2/M) phase, observed via cell cycle analysis provided that the proliferation of the growth of the cells is probably increasing that finally led to the confirmation of the greater expression of phospho-ERK1/2, phospho-Bcl-2 and survivin and decreasing expression of cleaved PARP in claudin-7 KD cells depicted by the Western Blot analysis. The ability for the tight junction to cause paracellular permeability becomes altered in cases of liver cirrhosis where the occurrence of endotoxemia becomes more prevalent due to liver cirrhosis. Hence, depth knowledge on claudin is required as the claudin-1 became resistant to chemotherapeutic drugs. This may become a disadvantage for patients suffering from cancers, developed due to claudin-1 mutation in NISCH syndrome. claudin-2 promoter inhibition was observed due to the effect of inhibitors LY-294002 and U0126 that cause Akt, ERK 1/2 phosphorylation. This effect causes p-c-Fos level deterioration. Thus claudin-2 serves as a therapeutic target for lung adenocarcinoma. microRNAs were observed to influence the stability of claudin-2 and miR16 acts as the inhibitory template for nuclear factor-kappa B transcription which is a critic for EMT. This causes cell cycle arrest in claudin-2. Claudin-1, -2, -4 and -5 was observed to be expressed at higher rate at their transcriptional level in pregnant C57/BL6 mice during different stages of gestation. IQGAP1 knockdown have also caused decreased claudin-2 expression and temporarily blocks the CDC42–JNK pathway.Claudin-2 due to its over expression in NEC patients can be used as a biomarker for the detection of the infected human. Protein kinase C knock down have declined the paracellular permeability which is regulated by claudin-2. Thus, it may strike us that expression of claudin- 2 has undergone reduction in its expression on subjectation of TJ to aPKC pseudosubstrate ZIP. Thus,protein kinase C knock down in NEC patient samples may control the expression of claudin 2 to reduce the severity of the infection. And also, expression of Rab14 that associates directly with aPKC for claudin-2 expression may also be studied in NEC patients. Estrogen receptor influences the claudin-4 expression via regulatory pathways in mice placenta. Estrogen antagonist ICI 182,780 and progesterone antagonist RU-486 expressing claudin-4 also provides the knowledge of understanding the interrelation of claudin-4 and claudin-2 in NEC patients. Our future area of interest is to identify the association of estrogen receptor with the claudin-2 transmembrane protein in cancer and the drug targeting ability of the claudin-2 in treating cancer since hormones controlling the regulatory pathways in transmembraneprotein expression may serve as a clear area for identifying the cause and effect of the cancer.

Conflicts of interest:

The authors declare no conflict of interest.

ACKNOWLEDGEMENTS:

The authors thank the management of VIT University for providing the facilities to carry out this work.

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Abbreviations:

ARE - Adenylate-uridylate-rich elements

Asn - Aspargine

BBB - Blood Brain Barrier

Bcl - B cell lymphoma

CAIX - Carbonic Anhydrase IX

CAR - Coxsackievirus and Adenovirus Receptor

C-CPE - C-terminal- Clostridium perfringens enterotoxin

Cdx - Caudal-related homeobox

CLDN - Claudin

CLL - Chronic Lymphocytic leukemia

CNS - Central Nervous System

CPE-R - Clostridium perfringens enterotoxin receptor

CRC - Colorectal Cancer

CTR - Copper Influx Transporter

Cys - Cystiene

Cyt c - Cytochrome C

DNA - Deoxyribonucleic acid

ECL - Extracellular Loop

ECM - Extracellular Matrix

EGFR - Epidermal Growth Factor Receptor

EM - Electron Microscopy

EMP - Epithelial Membrane Proteins

EMT - Epithelial to Mesenchymal Transition

ERK - Extracellular signal-regulated kinases

GFAP - Glial Fibrillary Acidic Protein

Gly - Glycine

GUK - Guanylate Kinase

HCV - Hepatitis C virus

H.E - Hematoxylin and Eosin

HDAC - Histone deacetylase

HGF-R - Human Growth Factor Receptor

H-I - Hypoxic-Ischemic

HuR - Human antigen R

Ig -Immunoglobulin

IGF - Insulin-like growth factor

IGF-1 - Insulin-like growth factor 1

JAM - Junction adhesion molecule

KD - knock down

kDa - kilo Dalton

LEF - Lymphoid enhancer factor

LEF-1 - Lymphoid enhancer-binding factor-1

Leu - Leucine

MAGI-1 - Membrane-associated guanylate kinase, WW and PDZ domain-containing protein 1

MAPK - MAP kinase

MBEC1 - Mouse brain endothelial cell 1

MDCK - Madin Darby canine kidney

MEK - mitogen-activated protein kinase

miRNA - micro RNA

MLCK - Myosin light chain kinase

MMP - Matrix metalloproteinases

mRNA - messenger RNA

MSPCR - Methylation Specific Polymerase Chain Reaction

MUPP - multi-PDZ domain protein

MVHP - Microvesicular Hyperplastic Polyps

NF-κB - Nuclear Factor Kappa B

NSCLC - Non-Small Cell Lung Cancer

PET - Positron Emission Tomography

PI3-K - Phosphatidylinositol-3-kinase

PKC - Protein kinase C

pKi - Isoelectric Point

PMP22 - Peripherial myelin protein

PTEN - Phosphatase and Tensin

sh RNA - short hairpin RNA

SH3 - Src Oncogene homology-3

SSA/P - Sessile serrated adenomas/polyps

STAT - Signal transducer and activator of transcription

TCF - T-Cell factor

TEER - Trans-Epithelial Electrical Resistance

TER - Trans-Epithelial Resistance

TEWL - Trans Epidermal Water Lossn

Thr - Threoine

TJ - Tight Junction

TM - Transmembrane

TMVCF - Transmembrane protein deleted in velo-cardio-facial syndrome

Trp - Tryptophan

ZAK - ZO-1 associated kinase

ZO - Zona Occludens

ZONAB - ZO-1 Nucleic acid binding protein

Received on 28.06.2017 Modified on 28.07.2017

Research J. Pharm. and Tech 2018; 11(8): 3561-3571.

DOI: 10.5958/0974-360X.2018.00656.X

Claudin	Position	Size	mRNA transcripts	Function	UNIPROT Id	Reference (for position and mRNA transcripts only)
1	3q28	211	1	Co-receptor for HCV passage into hepatic cells (Evans et al. 2007), (Hadj-Rabia, S.et al.,2004), mediate cell adhesion (weak) (Kubota et al. 1999), TJ-specific obliteration of the intercellular space through Ca2+-independent cell-adhesion activity (Hadj-Rabia, S.et al.,2004)	O95832	[79]
2	Not known	230	Not known	Mediate cell adhesion (weak) (Kubota et al. 1999)	P57739	-
3	7q11	220	1	Mediate cell adhesion (weak) (Kubota et al. 1999),TJ-specific obliteration of the intercellular space through Ca2+-independent cell-adhesion activity(CPE is the natural ligand)(Lameris, A.L.et al.,2010)	O15551	[79]
4	7q11	209	1	TJ-specific obliteration of the intercellular space (CPE is the natural ligand) (Lameris, A.L.et al.,2010)	O14493	[79]
5	22q11	218	2	Target molecule of hypoxia (Morita, K.et al.,1999)	O00501	[79]
6		220		coreceptor for hepatitis C virus (Zheng et al.2007)	P56747	-
7	Not known	211	Not known	TJ-specific obliteration of the intercellular space. Co-localizes with EPCAM at the lateral cell membrane and TJ (Kominsky, S.L.et al.,2003)	O95471	-
8	Not known	225	Not known	Not known	P56748	-
9	Not known	217	Not known	Co-receptor for hepatitis C virus ( Zheng et al.2007)	O95484	-
10	13q31	228	2	Not known	P78369	[79]
11	Not known	207	Not known	Mediate cell cycle regulation (Mazaud-Guittot et al. 2010)	O75508	-