Serge Paschoud

The cytoplasmic plaque of tight junctions: a scaffolding and signalling center.

The region of cytoplasm underlying the tight junction (TJ) contains several multimolecular protein complexes, which are involved in scaffolding of membrane proteins, regulation of cytoskeletal organization, establishment of polarity, and signalling to and from the nucleus. In this review, we summarize some of the most recent advances in understanding the identity of these proteins, their domain organization, their protein interactions, and their functions in vertebrate organisms. Analysis of knockdown and knockout model systems shows that several TJ proteins are essential for the formation of epithelial tissues and early embryonic development, whereas others appear to have redundant functions.

Claudin-1 and claudin-5 expression patterns differentiate lung squamous cell carcinomas from adenocarcinomas.

We investigated the expression of tight junction proteins in human lung squamous cell carcinomas and adenocarcinomas by immunohistochemistry and quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). We found a statistically significant correlation between diagnosis and positivity of tumors with either claudin (CLDN)-1 or CLDN-5. Squamous cell carcinomas and basal cells of bronchial epithelium were positive for CLDN-1 and negative for CLDN-5, whereas adenocarcinomas, normal cylindrical cells and pneumocytes were positive for CLDN-5 and negative for CLDN-1, suggesting different pathways in tumor development and progression. CLDN-4 and ZO-1 staining were detected in both types of tumors, whereas cingulin (CGN) was not detected in squamous cell carcinomas. Quantitative RT-PCR was used to evaluate changes in transcript levels for a large panel of tight junction proteins. In squamous cell carcinomas, we observed statistically significant decreases in the mRNA levels of JAM-1, occludin, CLDN-3, CLDN-4, CLDN-7, CGN, ZO-2 and ZO-3, and an increase in CLDN-1 mRNA. In adenocarcinomas, when transcript levels were compared with bronchial cells, we observed statistically significant decreases in the mRNA levels of CLDN-1, CLDN-3, CLDN-4, CLDN-7, ZO-2 and ZO-3. These results indicate that characterization of tight junction protein expression in human lung tumors can be an additional diagnostic tool and provide new insights on their histogenesis.

Paracingulin Regulates the Activity of Rac1 and RhoA GTPases by Recruiting Tiam1 and GEF-H1 to Epithelial Junctions

Small GTPases control key cellular events, including formation of cell-cell junctions and gene expression, and are regulated by activating and inhibiting factors. Here, we characterize the junctional protein paracingulin as a novel regulator of the activity of two small GTPases, Rac1 and RhoA, through the functional interaction with their respective activators, Tiam1 and GEF-H1. In confluent epithelial monolayers, paracingulin depletion leads to increased RhoA activity and increased expression of mRNA for the tight junction protein claudin-2. During tight junction assembly by the calcium-switch, Rac1 shows two transient peaks of activity, at earlier (10-20 min) and later (3-8 h) time points. Paracingulin depletion reduces such peaks of Rac1 activation in a Tiam1-dependent manner, resulting in a delay in junction formation. Paracingulin physically interacts with GEF-H1 and Tiam1 in vivo and in vitro, and it is required for their efficient recruitment to junctions, based on immunofluorescence and biochemical experiments. Our results provide the first description of a junctional protein that interacts with GEFs for both Rac1 and RhoA, and identify a novel molecular mechanism whereby Rac1 is activated during junction formation.

Inducible overexpression of cingulin in stably transfected MDCK cells does not affect tight junction organization and gene expression

Cingulin is a component of the cytoplasmic domain of vertebrate tight junctions (TJ). Mutation or down-regulation of cingulin in cultured cells results in changes in gene expression. Some of these changes are dependent on RhoA, whose activity is regulated by GEF-H1, which is inactivated by binding to cingulin at junctions. To gain further insights on the function of cingulin through dominant-negative effects, we cloned and sequenced canine cingulin, and developed stable MDCK cell lines where either full-length cingulin, or head or rod+tail domains were inducibly overexpressed. Surprisingly, analysis of these clones by immunoblotting, microarray, immunofluorescence, measurement of transepithelial resistance, and cell density showed that the overexpression of either full-length cingulin or its domains does not significantly affect TJ protein levels, gene expression, RhoA activity, cell density, doubling time, and the organization and function of TJ. These results suggest that compensatory mechanisms prevent dominant-negative effects in this model system, and that modulation of cellular functions by cingulin occurs within physiological protein levels.

The Tight Junction Protein Cingulin Regulates Gene Expression and RhoA Signaling

Tight junctions (TJ) regulate the passage of solutes across epithelial sheets, contribute to the establishment and maintenance of epithelial apico‐basal polarity and are involved in the regulation of gene expression and cell proliferation. Cingulin, a Mr 140 kDa protein localized in the cytoplasmic region of TJ, is not directly required for TJ formation and epithelial polarity but regulates RhoA signaling, through its interaction with the RhoA activator GEF‐H1, and gene expression. Here we describe in more detail the effect of cingulin mutation in embryoid bodies (EB) on gene expression, by identifying the genes that show the highest degree of up‐ or downregulation, and the putative canonical pathways that might be affected by cingulin. Furthermore, we show that full‐length canine GEF‐H1, produced in baculovirus‐infected insect cells, interacts with regions both in the cingulin globular head, and in the coiled‐coil rod domain. These results extend our previous studies and provide new perspectives for the mechanistic analysis of cingulin function.

A Role for ZO-1 and PLEKHA7 in Recruiting Paracingulin to Tight and Adherens Junctions of Epithelial Cells

Paracingulin is a 160-kDa protein localized in the cytoplasmic region of epithelial tight and adherens junctions, where it regulates RhoA and Rac1 activities by interacting with guanine nucleotide exchange factors. Here, we investigate the molecular mechanisms that control the recruitment of paracingulin to cell-cell junctions. We show that paracingulin forms a complex with the tight junction protein ZO-1, and the globular head domain of paracingulin interacts directly with ZO-1 through an N-terminal region containing a conserved ZIM (ZO-1-Interaction-Motif) sequence. Recruitment of paracingulin to cadherin-based cell-cell junctions in Rat1 fibroblasts requires the ZIM-containing region, whereas in epithelial cells removal of this region decreases the junctional localization of paracingulin at tight junctions but not at adherens junctions. Depletion of ZO-1, but not ZO-2, reduces paracingulin accumulation at tight junctions. A yeast two-hybrid screen identifies both ZO-1 and the adherens junction protein PLEKHA7 as paracingulin-binding proteins. Paracingulin forms a complex with PLEKHA7 and its interacting partner p120ctn, and the globular head domain of paracingulin interacts directly with a central region of PLEKHA7. Depletion of PLEKHA7 from Madin-Darby canine kidney cells results in the loss of junctional localization of paracingulin and a decrease in its expression. In summary, we characterize ZO-1 and PLEKHA7 as paracingulin-interacting proteins that are involved in its recruitment to epithelial tight and adherens junctions, respectively.

Cingulin, paracingulin, and PLEKHA7: signaling and cytoskeletal adaptors at the apical junctional complex.

Cingulin, paracingulin, and PLEKHA7 are proteins localized in the cytoplasmic region of the apical junctional complex of vertebrate epithelial cells. Cingulin has been detected at tight junctions (TJs), whereas paracingulin has been detected at both TJs and adherens junctions (AJs) and PLEKHA7 has been detected at AJs. One function of cingulin and paracingulin is to regulate the activity of Rho family GTPases at junctions through their direct interaction with guanidine exchange factors of RhoA and Rac1. Cingulin also contributes to the regulation of transcription of several genes in different types of cultured cells, in part through its ability to modulate RhoA activity. PLEKHA7, together with paracingulin, is part of a protein complex that links E‐cadherin to the microtubule cytoskeleton at AJs. In this paper, we review the current knowledge about these proteins, including their discovery, the characterization of their expression, localization, structure, molecular interactions, and their roles in different developmental and disease model systems.

Cingulin and paracingulin show similar dynamic behaviour, but are recruited independently to junctions

Abstract Cingulin (CGN) and paracingulin (CGNL1) are structurally related proteins that regulate Rho family GTPases by recruiting guanine nucleotide exchange factors to epithelial junctions. Although the subcellular localization of cingulin and paracingulin is likely to be essential for their role as adaptor proteins, nothing is known on their in vivo localization, and their dynamics of exchange with the junctional membrane. To address these questions, we generated stable clones of MDCK cells expressing fluorescently tagged cingulin and paracingulin. By FRAP analysis, cingulin and paracingulin show a very similar dynamic behaviour, with recovery curves and mobile fractions that are distinct from ZO-1, and indicate a rapid exchange with a cytosolic pool. Interestingly, only paracingulin, but not cingulin, is peripherally localized in isolated cells, requires the integrity of the microtubule cytoskeleton to be stably anchored to junctions, and associates with E-cadherin. In contrast, both proteins require the integrity of the actin cytoskeleton to maintain their junctional localization. Although cingulin and paracingulin form a complex and can interact in vitro, the junctional recruitment and the dynamics of membrane exchange of paracingulin is independent of cingulin, and vice-versa. In summary, cingulin and paracingulin show a similar dynamic behaviour, but partially distinct localizations and functional interactions with the cytoskeleton, and are recruited independently to junctions.

PLEKHA7 modulates epithelial tight junction barrier function

PLEKHA7 is a recently identified protein of the epithelial zonula adhaerens (ZA), and is part of a protein complex that stabilizes the ZA, by linking it to microtubules. Since the ZA is important in the assembly and disassembly of tight junctions (TJ), we asked whether PLEKHA7 is involved in modulating epithelial TJ barrier function. We generated clonal MDCK cell lines in which one of four different constructs of PLEKHA7 was inducibly expressed. All constructs were localized at junctions, but constructs lacking the C-terminal region were also distributed diffusely in the cytoplasm. Inducible expression of PLEKHA7 constructs did not affect the expression and localization of TJ proteins, the steady-state value of transepithelial resistance (TER), the development of TER during the calcium switch, and the flux of large molecules across confluent monolayers. In contrast, expression of three out of four constructs resulted both in enhanced recruitment of E-cadherin and associated proteins at the apical ZA and at lateral puncta adherentia (PA), a decreased TER at 18 h after assembly at normal calcium, and an attenuation in the fall in TER after extracellular calcium removal. This latter effect was inhibited when cells were treated with nocodazole. Immunoprecipitation analysis showed that PLEKHA7 forms a complex with the cytoplasmic TJ proteins ZO-1 and cingulin, and this association does not depend on the integrity of microtubules. These results suggest that PLEKHA7 modulates the dynamics of assembly and disassembly of the TJ barrier, through E-cadherin protein complex- and microtubule-dependent mechanisms.

Toll-like receptor 2 regulates the barrier function of human bronchial epithelial monolayers through atypical protein kinase C zeta, and an increase in expression of claudin-1

We investigated the role of Toll-like receptor (TLR) 2 in maintaining the integrity of the airway epithelial barrier using the human bronchial epithelial cell line Calu-3. Activation of TLR2 by its ligands, Pam3CysSK4 and Peptidoglycan showed a concentration dependent increase in epithelial barrier function, as measured by transepithelial electrical resistance (TEER). This was confirmed by a decrease in paracellular flux of fluorescein sodium. This TLR2 induced increase in TEER was significantly reduced by pretreatment with polyclonal anti-human TLR2-neutralizing antibody. TLR2 stimulation in Calu-3 cell monolayers resulted in an increased expression of the tight junction proteins claudin-1 and ZO-1, and a decreased expression of occludin, at both the mRNA and protein levels. A pseudosubstrate inhibitor to PKCζ significantly prevented the TLR2 mediated increase in barrier function. It also prevented the increase in claudin-1 in a concentration dependent manner up to 1 µM. TLR2 stimulation led to an increase in phosphorylation of atypical PKC ζ, which was prevented by the pseudosubstrate inhibitor in a concentration dependent manner. Taken together, our observations support a model whereby increased tight junction barrier function induced by activation of TLR2 occurs through increased expression of claudin-1, and through modulation of PKC ζ activity.