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.

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.

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.

Distinct domains of paracingulin are involved in its targeting to the actin cytoskeleton and regulation of apical junction assembly.

Paracingulin is an M(r) 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.

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.

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

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.

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.

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.

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.

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.

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.

Destabilization of interleukin-6 mRNA requires a putative RNA stem-loop structure, an AU-rich element, and the RNA-binding protein AUF1.

Interleukin-6 mRNA is unstable and degraded with a half-life of 30 min. Instability determinants can entirely be attributed to the 3' untranslated region. By grafting segments of this region to stable green fluorescent protein mRNA and subsequent scanning mutagenesis, we have identified two conserved elements, which together account for most of the instability.

Independent regulation of chitin synthase and chitinase activity in Candida albicans and Saccharomyces cerevisiae.

Chitin is an essential structural polysaccharide in fungi that is required for cell shape and morphogenesis. One model for wall synthesis at the growing cell surface suggests that the compliance that is necessary for turgor-driven expansion of the cell wall involves a delicate balance of wall synthesis and lysis. Accordingly, de novo chitin synthesis may involve coordinated regulation of members of the CHS chitin synthase and CHT chitinase gene families.