Laurent Guillemot

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.

Disruption of the cingulin gene does not prevent tight junction formation but alters gene expression

Cingulin, a component of vertebrate tight junctions, contains a head domain that controls its junctional recruitment and protein interactions. To determine whether lack of junctional cingulin affects tight-junction organization and function, we examined the phenotype of embryoid bodies derived from embryonic stem cells carrying one or two alleles of cingulin with a targeted deletion of the exon coding for most of the predicted head domain. In homozygous (–/–) embryoid bodies, no full-length cingulin was detected by immunoblotting and no junctional labeling was detected by immunofluorescence. In hetero- and homozygous (+/– and –/–) embryoid bodies, immunoblotting revealed a Triton-soluble, truncated form of cingulin, increased levels of the tight junction proteins ZO-2, occludin, claudin-6 and Lfc, and decreased levels of ZO-1. The +/– and –/– embryoid bodies contained epithelial cells with normal tight junctions, as determined by freeze-fracture and transmission electron microscopy, and a biotin permeability assay. The localization of ZO-1, occludin and claudin-6 appeared normal in mutant epithelial cells, indicating that cingulin is not required for their junctional recruitment. Real-time quantitative reverse-transcription PCR (real-time qRT-PCR) showed that differentiation of embryonic stem cells into embryoid bodies was associated with up-regulation of mRNAs for several tight junction proteins. Microarray analysis and real-time qRT-PCR showed that cingulin mutation caused a further increase in the transcript levels of occludin, claudin-2, claudin-6 and claudin-7, which were probably due to an increase in expression of GATA-6, GATA-4 and HNF-4α, transcription factors implicated in endodermal differentiation. Thus, lack of junctional cingulin does not prevent tight-junction formation, but gene expression and tight junction protein levels are altered by the cingulin mutation.

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.

Angiotensin II-induced Transcriptional Activation of the Cyclin D1 Gene Is Mediated by Egr-1 in CHO-AT1A Cells

Cyclin D1 protein expression is regulated by mitogenic stimuli and is a critical component in the regulation of G1 to S phase progression of the cell cycle. Angiotensin II (Ang II) binds to specific G protein-coupled receptors and is mitogenic in Chinese hamster ovary cells stably expressing the rat vascular Ang II type 1A receptor (CHO-AT1A). We recently reported that in these cells, Ang II induced cyclin D1 promoter activation and protein expression in a phosphatidylinositol 3-kinase (PI3K)-, SHP-2-, and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK)-dependent manner (Guillemot, L., Levy, A., Zhao, Z. J., Béréziat, G., and Rothhut, B. (2000)J. Biol. Chem. 275, 26349–26358). In this report, transfection studies using a series of deleted cyclin D1 promoters revealed that two regions between base pairs (bp) −136 and −96 and between bp −29 and +139 of the human cyclin D1 promoter contained regulatory elements required for Ang II-mediated induction. Mutational analysis in the −136 to −96 bp region provided evidence that a Sp1/early growth response protein (Egr) motif was responsible for cyclin D1 promoter activation by Ang II. Gel shift and supershift studies showed that Ang II-induced Egr-1 binding involved de novo protein synthesis and correlated well with Egr-1 promoter activation. Both U0126 (an inhibitor of the MAPK/ERK kinase MEK) and wortmannin (an inhibitor of PI3K) abrogated Egr-1 endogenous expression and Egr-1 promoter activity induced by Ang II. Moreover, using a co-transfection approach, we found that Ang II induction of Egr-1 promoter activity was blocked by dominant-negative p21 ras , Raf-1, and tyrosine phosphatase SHP-2 mutants. Identical effects were obtained when inhibitors and dominant negative mutants were tested on the −29 to +139 bp region of the cyclin D1 promoter. Taken together, these findings demonstrate that Ang II-induced cyclin D1 up-regulation is mediated by the activation and specific interaction of Egr-1 with the −136 to −96 bp region of the cyclin D1 promoter and by activation of the −29 to +139 bp region, both in a p21 ras /Raf-1/MEK/ERK-dependent manner, and also involves PI3K and SHP-2.

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.

MgcRacGAP interacts with cingulin and paracingulin to regulate Rac1 activation and development of the tight junction barrier during epithelial junction assembly

The Rac1 inhibitor MgcRacGAP regulates Rac1 activation and TJ barrier development during junction assembly in epithelial cells. CGN and CGNL1 recruit MgcRacGAP to the TJ and interact with MgcRacGAP.

Cingulin is dispensable for epithelial barrier function and tight junction structure, and plays a role in the control of claudin-2 expression and response to duodenal mucosa injury

Summary Cingulin (CGN) is a 140 kDa protein, which is localized to the cytoplasmic region of vertebrate tight junctions (TJ), and regulates gene expression and RhoA signaling in cultured cells. To investigate the function of CGN at the organism level, we generated CGN knockout (CGN−/−) mice by homologous recombination. CGN−/− mice are viable and fertile, and are born at the expected mendelian ratios. Immunohistochemistry, immunofluorescence, electron microscopy and permeability assays of epithelial tissues of CGN−/− mice show no cingulin labeling at junctions, a normal localization of TJ proteins, and normal TJ structure and barrier function. Microarray analysis of intestinal cells does not show significant changes in gene expression between CGN−/− and CGN+/+ mice, whereas immunoblotting analysis shows a twofold increase in the levels of claudin-2 protein in the duodenum and the kidney of CGN−/− mice, compared to CGN+/+ littermates. Furthermore, CGN−/− mice show an exacerbated response to the ulcerogenic action of cysteamine, whereas acute injury of the colon by dextran sodium sulfate elicits undistinguishable responses in CGN−/− and CGN+/+ mice. We conclude that at the organism level cingulin is dispensable for the structure and barrier function of TJ, and is embedded in signaling networks that control the expression of claudin-2, and the mucosal response to acute injury in the duodenum.

The junctional proteins cingulin and paracingulin modulate the expression of tight junction protein genes through GATA-4.

The cytoplamic junctional proteins cingulin and paracingulin have been implicated in the regulation of gene expression in different cultured cell models. In renal epithelial MDCK cells, depletion of either protein results in a Rho-dependent increase in the expression of claudin-2. Here we examined MDCK cell clones depleted of both cingulin and paracingulin (double-KD cells), and we found that unexpectedly the expression of claudin-2, and also the expression of ZO-3 and claudin-3, were decreased, while RhoA activity was still higher than in control cells. The decreased expression of claudin-2 and other TJ proteins in double–KD cells correlated with reduced levels of the transcription factor GATA-4, and was rescued by overexpression of GATA-4, but not by inhibiting RhoA activity. These results indicate that in MDCK cells GATA-4 is required for the expression of claudin-2 and other TJ proteins, and that maintenance of GATA-4 expression requires either cingulin or paracingulin. These results and previous studies suggest a model whereby cingulin and paracingulin redundantly control the expression of specific TJ proteins through distinct GATA-4- and RhoA-dependent mechanisms, and that in the absence of sufficient levels of GATA-4 the RhoA-mediated upregulation of claudin-2 is inhibited.

Cingulin, a Cytoskeleton-Associated Protein of the Tight Junction

Cingulin is a Mr ∼140 kDa phosphoprotein component of the cytoplasmic plaque of vertebrate tight junctions (TJ), and was identified as an actomyosin-associated protein. The cingulin sequence predicts that the molecule is a parallel homodimer, each sub-unit consisting of a coiled-coil “rod” domain separating a large globular N-terminal “head” domain from a small globular C-terminal “tail”. The head domain mediates direct in vitro interaction with ZO-1, ZO-2, ZO-3 and actin, and is critical for the junctional recruitment of cingulin in transfected cells, whereas the rod domain mediates dimerization. Cingulin forms complexes with ZO-1, ZO-2 and the immunoglobulin-like adhesion molecule JAM-1. These complexes are only detected in Triton-insoluble cell fractions, suggesting that one function of cingulin is to link TJ-associated proteins to the actomyosin cytoskeleton during TJ biogenesis and regulation. In fact, purified cingulin acts in vitro as an actin-binding and -bundling protein. Cingulin shows a cortical, apical localization in early vertebrate embryos, and in Xenopus embryos is recruited into nascent TJ starting at the 2-cell stage, suggesting that cingulin may play an important role in the development of vertebrate epithelia. Cingulin may represent the first member of a family of proteins, since a novel protein, paracingulin, with similar domain organization and significant sequence homology to cingulin has recently been identified.

Distinct Domains of Paracingulin Are Involved in Its Targeting to the Actin Cytoskeleton and Regulation of Apical Junction Assembly

Background: Paracingulin is a junctional protein that regulates Rho GTPase activities in epithelial cells. Results: Overexpressed mutated paracingulin constructs show different subcellular localizations and effects on junction assembly. Conclusion: The domains of paracingulin involved in targeting to the actin cytoskeleton and in regulation of junction assembly were identified. Significance: The structural basis for the functional differences between cingulin and paracingulin was clarified. Paracingulin is an Mr 150–160 kDa cytoplasmic protein of vertebrate epithelial tight and adherens junctions and comprises globular head, coiled-coil rod, and globular tail domains. Unlike its homologous tight junction protein cingulin, paracingulin has been implicated in the control of junction assembly and has been localized at extrajunctional sites in association with actin filaments. Here we analyze the role of paracingulin domains, and specific regions within the head and rod domains, in the function and localization of paracingulin by inducible overexpression of exogenous proteins in epithelial Madin Darby canine kidney (MDCK) cells and by expression of mutated and chimeric constructs in Rat1 fibroblasts and MDCK cells. The overexpression of the rod + tail domains of paracingulin perturbs the development of the tight junction barrier and Rac1 activation during junction assembly by the calcium switch, indicating that regulation of junction assembly by paracingulin is mediated by these domains. Conversely, only constructs containing the head domain target to junctions in MDCK cells and Rat1 fibroblasts. Furthermore, expression of chimeric cingulin and paracingulin constructs in Rat1 fibroblasts and MDCK cells identifies specific sequences within the head and rod domains of paracingulin as critical for targeting to actin filaments and regulation of junction assembly, respectively. In summary, we characterize the functionally important domains of paracingulin that distinguish it from cingulin.