Article

Binding of GEF-H1 to the Tight Junction-Associated Adaptor Cingulin Results in Inhibition of Rho Signaling and G1/S Phase Transition

Division of Cell Biology, Institute of Ophthalmology, University College London, UK.
Developmental Cell (Impact Factor: 10.37). 06/2005; 8(5):777-86. DOI: 10.1016/j.devcel.2005.03.003
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ABSTRACT The activity of Rho GTPases is carefully timed to control epithelial proliferation and differentiation. RhoA is downregulated when epithelial cells reach confluence, resulting in inhibition of signaling pathways that stimulate proliferation. Here we show that GEF-H1/Lfc, a guanine nucleotide exchange factor for RhoA, directly interacts with cingulin, a junctional adaptor. Cingulin binding inhibits RhoA activation and signaling, suggesting that the increase in cingulin expression in confluent cells causes downregulation of RhoA by inhibiting GEF-H1/Lfc. In agreement, RNA interference of GEF-H1 or transfection of GEF-H1 binding cingulin mutants inhibit G1/S phase transition of MDCK cells, and depletion of cingulin by regulated RNA interference results in irregular monolayers and RhoA activation. These results indicate that forming epithelial tight junctions contribute to the downregulation of RhoA in epithelia by inactivating GEF-H1 in a cingulin-dependent manner, providing a molecular mechanism whereby tight junction formation is linked to inhibition of RhoA signaling.

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Available from: Sandra Citi, Sep 02, 2015
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    • "Tight junctions are specifically relevant for water and small protein containment within the vessel lumens. Although cingulin is known to regulate tight junction redistribution via GEF-H1 (Aijaz et al., 2005), we have not yet identified our direct target structure of our peptide Xib13. We show that the non-perfused area on day 12 is smaller in Xib13- treated animals. "
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    ABSTRACT: Objective : Edema due to capillary leak is a generalized and life threatening event in sepsis and major burns for which there is no causal treatment. Local burn wounds are an ideal model to investigate the impact of a new therapeutic agent on edema formation. We aimed to identify peptide sequences of cingulin that can attenuate stress-induced endothelial cytoskeleton disarrangement in vitro and which reduce burn-induced edema in vivo. Methods : Cingulin-derived peptides were screened in high content cell culture assays monitoring actin displacement and endothelial cell/cell contacts. The ears of male hairless mice (n = 44) were inflicted with full thickness burns using a hot air jet. Mice with and without burn injuries were treated with Xib13 or solvent by continuous intraperitoneal application for 3 days. Edema, microcirculation, leukocyte-endothelial interactions and angiogenesis – measured as non-perfused area - were investigated over a 12-day period using intravital fluorescence microscopy. Results : Xib13 reduced endothelial stress formation and stabilized endothelial tight junctions in cell-cultures. In the burn model, Xib13 improved angiogenesis compared to controls (non-perfused area on day 12: 5.7 ± 1.5% vs. 12.0 ± 2.1%; p < 0.05). Edema was significantly reduced at all observation points in Xib13-treated animals as compared to controls (day 12: 67.6 ± 2.6% vs. 83.2 ± 6.4%). Conclusion : Xib13 improved angiogenesis, reduced edema formation and showed no side effects on other physiological parameters. Since edema formation is a serious parameter for burn conversion and is associated with survival it could provide a new treatment option for patients with burn injuries.
    Microvascular Research 05/2014; 93. DOI:10.1016/j.mvr.2014.04.003 · 2.43 Impact Factor
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    • "On the other hand, in cingulin KD cells, EB1 signals tended to be located end on with respect to the membranes at points of cell–cell adhesion (Videos 4 and 5). Cingulin is also reported to associate with actin filaments (D'Atri and Citi, 2001) as well as with guanine nucleotide exchange factor (GEF)–H1 and p114 RhoGEF, as shown in MDCK and Caco-2 cells, respectively (Aijaz et al., 2005; Terry et al., 2011). There was no difference in actin filament arrangement, myosin light chain phosphorylation, p114 RhoGEF, or GEF-H1 between wild-type Eph4 and cingulin KD Eph4 cells (Fig. S2, B–E). "
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    ABSTRACT: Epithelial cells characteristically have noncentrosomal microtubules that are arranged in the apicobasal direction. In this paper, we examined cell sheets formed by an epithelial (Eph4) cell line by structure illumination microscopy and found a previously not clearly described planar apical network of noncentrosomal microtubules (MTs) in which the sides of the MT bundles were associated with tight junctions (TJs). In a gel overlay assay with taxol-stabilized MTs, cingulin showed strong binding to MTs, and a domain analysis showed that this binding occurred through cingulin's N-terminal region. The association of planar apical MTs with TJs was compromised by cingulin knockdown (KD) or the expression of dephosphomimetic mutants of cingulin at its adenosine monophosphate-activated protein kinase (AMPK) target sites, whereas phosphorylation at these sites facilitated cingulin-tubulin binding. In addition, although wild-type colonies formed spheres in 3D culture, the cingulin KD cells had anisotropic shapes. These findings collectively suggest that the regulated cingulin-MT association has a specific role in TJ-related epithelial morphogenesis that is sensitive to metabolic homeostasis-related AMPK activity.
    The Journal of Cell Biology 11/2013; 203(4):605-14. DOI:10.1083/jcb.201304194 · 9.69 Impact Factor
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    • "Significantly, cingulin depletion in MDCK cells also resulted in increased expression of claudin-2 (Guillemot and Citi, 2006), suggesting that CGN is part of a signaling network that controls claudin-2 expression in different cell culture model systems. In addition, CGN controls RhoA activity in cultured epithelial cells, through its interaction with the RhoA activator GEF-H1, and cingulin depletion results in increased cell proliferation in MDCK cells (Aijaz et al., 2005; Guillemot and Citi, 2006; Citi et al., 2009). Here we investigate the physiological role of CGN in a whole organism, by targeting CGN in mice, and analyzing the phenotype of CGN 2/2 mice. "
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    ABSTRACT: Cingulin (CGN) is a M(r) 140 kDa protein, which is localized in the cytoplasmic region of vertebrate tight junctions (TJ), and regulates gene expression and RhoA signalling 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 labelling at junctions, 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 2-fold 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 sulphate 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 it is embedded in signalling networks that control the expression of claudin-2, and the mucosal response to acute injury in the duodenum.
    Journal of Cell Science 09/2012; 125(21). DOI:10.1242/jcs.101261 · 5.33 Impact Factor
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