Biomedical subjects

Karl Matter

Publications and source records attributed to Karl Matter.

16 recordsLinked to original sources

PubMed2006

Functional interaction between the ZO-1-interacting transcription factor ZONAB/DbpA and the RNA processing factor symplekin.

Epithelial tight junctions participate in the regulation of gene expression by controlling the activity of transcription factors that can interact with junctional components. One such protein is the Y-box transcription factor ZONAB/DbpA that binds to ZO-1, a component of the junctional plaque. Symplekin, another nuclear protein that can associate with tight junctions, functions in the regulation of polyadenylation and thereby promotes gene expression. Here, we addressed the question of whether these two proteins interact and whether this is of functional relevance. We demonstrate that ZONAB/DbpA and symplekin form a complex in kidney and intestinal epithelial cells that can be immunoprecipitated and that exists in the nucleus. The interaction between ZONAB/DbpA and symplekin can be reconstituted with recombinant proteins. In reporter gene assays in which ZONAB/DbpA functions as a repressor, symplekin functionally interacts with ZONAB/DbpA, indicating that symplekin can also promote transcriptional repression. RNAi experiments indicate that symplekin depletion reduces the nuclear accumulation and the transcriptional activity of ZONAB/DbpA in colon adenocarcinoma cells, resulting in inhibition of proliferation and reduced expression of the ZONAB/DbpA-target gene cyclin D1. Our data thus indicate that symplekin and ZONAB/DbpA cooperate in the regulation of transcription, and that they promote epithelial proliferation and cyclin D1 expression.

Animals↗

PubMed2006

The heat-shock protein Apg-2 binds to the tight junction protein ZO-1 and regulates transcriptional activity of ZONAB.

The tight junction adaptor protein ZO-1 regulates intracellular signaling and cell proliferation. Its Src homology 3 (SH3) domain is required for the regulation of proliferation and binds to the Y-box transcription factor ZO-1-associated nucleic acid binding protein (ZONAB). Binding of ZO-1 to ZONAB results in cytoplasmic sequestration and hence inhibition of ZONAB's transcriptional activity. Here, we identify a new binding partner of the SH3 domain that modulates ZO-1-ZONAB signaling. Expression screening of a cDNA library with a fusion protein containing the SH3 domain yielded a cDNA coding for Apg-2, a member of the heat-shock protein 110 (Hsp 110) subfamily of Hsp70 heat-shock proteins, which is overexpressed in carcinomas. Regulated depletion of Apg-2 in Madin-Darby canine kidney cells inhibits G(1)/S phase progression. Apg-2 coimmunoprecipitates with ZO-1 and partially localizes to intercellular junctions. Junctional recruitment and coimmunoprecipitation with ZO-1 are stimulated by heat shock. Apg-2 competes with ZONAB for binding to the SH3 domain in vitro and regulates ZONAB's transcriptional activity in reporter gene assays. Our data hence support a model in which Apg-2 regulates ZONAB function by competing for binding to the SH3 domain of ZO-1 and suggest that Apg-2 functions as a regulator of ZO-1-ZONAB signaling in epithelial cells in response to cellular stress.

Animals↗

PubMed2006

Tight junctions: molecular architecture and function.

Tight junctions are the most apical component of the epithelial junctional complex and are crucial for the formation and functioning of epithelial and endothelial barriers. They regulate selective diffusion of ions and solutes along the paracellular pathway and restrict apical/basolateral intramembrane diffusion of lipids. Research over the past years provided much insight into the molecular composition of tight junctions, and we are starting to understand the mechanisms that permit selective paracellular diffusion. Moreover, a complex network of proteins has been identified at tight junctions that is based on cytoskeleton-linked adaptors that recruit and thereby often regulate different types of signaling components that regulate epithelial proliferation, differentiation, and polarization.

Animals↗

PubMed2006

Regulation of PCNA and cyclin D1 expression and epithelial morphogenesis by the ZO-1-regulated transcription factor ZONAB/DbpA.

The tight junction protein ZO-1 inhibits G1/S-phase transition by cytoplasmic sequestration of a complex formed by CDK4 and the transcription factor ZONAB. Canine ZONAB is the homologue of human DbpA, an E2F target gene that is overexpressed in different carcinomas. Since the ZONAB target genes that are involved in G1/S-phase transition are unknown, we employed the mammary epithelial cell line MCF-10A and cDNA arrays to screen for such genes. We identified genes encoding cell cycle and replication proteins whose expression was altered due to increased ZONAB expression. For proliferative cell nuclear antigen and cyclin D1 genes, we show that increased mRNA levels resulted in increased protein levels and we identified ZONAB-responsive elements in their promoters by using different approaches, including chromatin immunoprecipitation assays. RNA interference and overexpression of ZONAB affected the proliferation of both MCF-10A and MDCK cells as well as the differentiation of MDCK cells into polarized cysts in three-dimensional cultures. These results indicate that ZONAB regulates the transcription of genes that are important for G1/S-phase progression and links tight junctions to the transcriptional control of key cell cycle regulators and epithelial cell differentiation.

Animals↗

PubMed2005

Mammalian tight junctions in the regulation of epithelial differentiation and proliferation.

Tight junctions are important for the permeability properties of epithelial and endothelial barriers as they restrict diffusion along the paracellular space. Recent observations have revealed that tight junctions also function in the regulation of epithelial proliferation and differentiation. They harbour evolutionarily conserved protein complexes that regulate polarisation and junction assembly. Tight junctions also recruit signalling proteins that participate in the regulation of cell proliferation and differentiation. These signalling proteins include components that affect established signalling cascades and dual localisation proteins that can associate with junctions as well as travel to the nucleus where they regulate gene expression.

Animals↗

PubMed2005

Binding of GEF-H1 to the tight junction-associated adaptor cingulin results in inhibition of Rho signaling and G1/S phase transition.

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.

Adaptor Proteins, Signal Transducing↗

PubMed2005

Matrix metalloproteinase-1 associates with intracellular organelles and confers resistance to lamin A/C degradation during apoptosis.

Since the first description of matrix metalloproteinase (MMP)-1 as an interstitial collagenase, great importance has been ascribed to this enzyme in extracellular matrix remodeling during tumoral, inflammatory, and angiogenic processes. As more evidence for the role of MMPs in targeting nonmatrix substrates emerges, casual observations that intracellular MMP-1 is found in vitro and in vivo prompt investigation of the role that MMP-1 may play on basic cell functions such as cell division and apoptosis. Here we show for the first time that MMP-1 not only has extracellular functions but that it is strongly associated with mitochondria and nuclei and accumulates within the cells during the mitotic phase of the cell cycle. On induction of apoptosis, MMP-1 co-localized with aggregated mitochondria and accumulated around fragmented nuclei. Inhibition of this enzyme by RNA interference or treatment with a broad MMP inhibitor caused faster degradation of lamin A, activation of caspases, and fragmentation of DNA when compared with untreated cells. These observations strongly suggest that intracellular association of MMP-1 to mitochondria and nuclei confers resistance to apoptosis and may explain the well-known association of this enzyme with tumor cell survival and spreading.

Apoptosis↗

PubMed2004

The polarized expression of Na+,K+-ATPase in epithelia depends on the association between beta-subunits located in neighboring cells.

The polarized distribution of Na+,K+-ATPase plays a paramount physiological role, because either directly or through coupling with co- and countertransporters, it is responsible for the net movement of, for example, glucose, amino acids, Ca2+, K+, Cl-, and CO3H- across the whole epithelium. We report here that the beta-subunit is a key factor in the polarized distribution of this enzyme. 1) Madin-Darby canine kidney (MDCK) cells (epithelial from dog kidney) express the Na+,K+-ATPase over the lateral side, but not on the basal and apical domains, as if the contact with a neighboring cell were crucial for the specific membrane location of this enzyme. 2) MDCK cells cocultured with other epithelial types (derived from human, cat, dog, pig, monkey, rabbit, mouse, hamster, and rat) express the enzyme in all (100%) homotypic MDCK/MDCK borders but rarely in heterotypic ones. 3) Although MDCK cells never express Na+,K+-ATPase at contacts with Chinese hamster ovary (CHO) cells, they do when CHO cells are transfected with beta1-subunit from the dog kidney (CHO-beta). 4) This may be attributed to the adhesive property of the beta1-subunit, because an aggregation assay using CHO (mock-transfected) and CHO-beta cells shows that the expression of dog beta1-subunit in the plasma membrane does increase adhesiveness. 5) This adhesiveness does not involve adherens or tight junctions. 6) Transfection of beta1-subunit forces CHO-beta cells to coexpress endogenous alpha-subunit. Together, our results indicate that MDCK cells express Na+,K+-ATPase at a given border provided the contacting cell expresses the dog beta1-subunit. The cell-cell interaction thus established would suffice to account for the polarized expression and positioning of Na+,K+-ATPase in epithelial cells.

Adherens Junctions↗

PubMed2004

RalA interacts with ZONAB in a cell density-dependent manner and regulates its transcriptional activity.

Ral proteins are members of the Ras superfamily of small GTPases and are involved in signalling pathways for actin cytoskeleton remodelling, cell cycle control, cellular transformation and vesicle transport. To identify novel RalA effector proteins, we used the reverse Ras recruitment system and found that RalA interacts with a Y-box transcription factor, ZO-1-associated nucleic acid-binding protein (ZONAB), in a GTP-dependent manner. The amount of the RalA-ZONAB complex increases as epithelial cells become more dense and increase cell contacts. The RalA-ZONAB interaction results in a relief of transcriptional repression of a ZONAB-regulated promoter. Additionally, expression of oncogenic Ras alleviates transcriptional repression by ZONAB in a RalA-dependent manner. The data presented here implicate the RalA/ZONAB interaction in the regulation of ZONAB function.

Animals↗

PubMed2003

Organization on the plasma membrane of the retinitis pigmentosa protein RP2: investigation of association with detergent-resistant membranes and polarized sorting.

Mutations in the retinitis pigmentosa protein gene RP2 account for up to 15% of X-linked retinitis pigmentosa. RP2 is a novel protein of unknown function, which is targeted to the plasma membrane by dual N-terminal acyl-modification. Dual-acylated proteins are targeted to lipid rafts, and some are subject to polarized sorting. Therefore we investigated the organization of RP2 on the plasma membrane. Endogenous RP2 protein was predominantly localized at the plasma membrane, and exogenously expressed green-fluorescent-protein-tagged protein was also targeted to the membrane in a wide range of cultured cells. High levels of endogenous RP2 protein were present in HeLa cells and in the retinal pigment epithelium-derived cell line ARPE19. A significant proportion of RP2 in cultured neuroblastoma cells was associated with detergent-resistant membranes (DRMs), but much less than other dually acylated proteins (e.g. Lyn and Fyn). In contrast, the RP2-interacting protein Arl3 (ADP-ribosylation factor-like 3) was not found to be associated with DRMs. The association of RP2 with DRMs was cholesterol-dependent. In polarized epithelial cells in culture and in vivo, RP2 was present in both the apical and basolateral domains of the plasma membrane. These data show that RP2 is not specific to either domain, unlike some other dually acylated proteins. Interestingly, the level of RP2 protein increased in the epithelial cell line Caco-2 with differentiation and polarization. These data show that RP2 is present on the membrane of all cell types examined both in vitro and in vivo, and that RP2 associates with lipid rafts, suggesting a potential role for the protein in signal transduction.

Acylation↗

PubMed2003

Holey barrier: claudins and the regulation of brain endothelial permeability.

Endothelial tight junctions (TJs)* are an important functional part of the blood-brain barrier (BBB). In this issue, Nitta et al. (2003) demonstrate that claudin-5, a transmembrane protein of TJs, is a critical determinant of BBB permeability in mice. Unexpectedly, knockout of claudin-5 did not result in a general breakdown of TJs but in a selective increase in paracellular permeability of small molecules. This suggests that the BBB can be manipulated to allow selective diffusion of small molecules and makes claudin-5 a possible target for the development of drugs for this purpose.

Animals↗

PubMed2003

Identification of a tight junction-associated guanine nucleotide exchange factor that activates Rho and regulates paracellular permeability.

Rho family GTPases are important regulators of epithelial tight junctions (TJs); however, little is known about how the GTPases themselves are controlled during TJ assembly and function. We have identified and cloned a canine guanine nucleotide exchange factor (GEF) of the Dbl family of proto-oncogenes that activates Rho and associates with TJs. Based on sequence similarity searches and immunological and functional data, this protein is the canine homologue of human GEF-H1 and mouse Lfc, two previously identified Rho-specific exchange factors known to associate with microtubules in nonpolarized cells. In agreement with these observations, immunofluorescence of proliferating MDCK cells revealed that the endogenous canine GEF-H1/Lfc associates with mitotic spindles. Functional analysis based on overexpression and RNA interference in polarized MDCK cells revealed that this exchange factor for Rho regulates paracellular permeability of small hydrophilic tracers. Although overexpression resulted in increased size-selective paracellular permeability, such cell lines exhibited a normal overall morphology and formed fully assembled TJs as determined by measuring transepithelial resistance and by immunofluorescence and freeze-fracture analysis. These data indicate that GEF-H1/Lfc is a component of TJs and functions in the regulation of epithelial permeability.

Actin Cytoskeleton↗

PubMed2003

The ZO-1-associated Y-box factor ZONAB regulates epithelial cell proliferation and cell density.

Epithelial tight junctions regulate paracellular permeability, restrict apical/basolateral intramembrane diffusion of lipids, and have been proposed to participate in the control of epithelial cell proliferation and differentiation. Previously, we have identified ZO-1-associated nucleic acid binding proteins (ZONAB), a Y-box transcription factor whose nuclear localization and transcriptional activity is regulated by the tight junction-associated candidate tumor suppressor ZO-1. Now, we found that reduction of ZONAB expression using an antisense approach or by RNA interference strongly reduced proliferation of MDCK cells. Transfection of wild-type or ZONAB-binding fragments of ZO-1 reduced proliferation as well as nuclear ZONAB pools, indicating that promotion of proliferation by ZONAB requires its nuclear accumulation. Overexpression of ZONAB resulted in increased cell density in mature monolayers, and depletion of ZONAB or overexpression of ZO-1 reduced cell density. ZONAB was found to associate with cell division kinase (CDK) 4, and reduction of nuclear ZONAB levels resulted in reduced nuclear CDK4. Thus, our data indicate that tight junctions can regulate epithelial cell proliferation and cell density via a ZONAB/ZO-1-based pathway. Although this regulatory process may also involve regulation of transcription by ZONAB, our data suggest that one mechanism by which ZONAB and ZO-1 influence proliferation is by regulating the nuclear accumulation of CDK4.

Active Transport, Cell Nucleus↗

PubMed2003

Epithelial cell adhesion and the regulation of gene expression.

Epithelial cells adhere to their neighbors and the extracellular matrix by means of adhesion complexes composed of transmembrane components connected to cytoplasmic proteins and the cytoskeleton. Recent results suggest that these complexes not only mediate adhesion but are also engaged in the transmission of signals from the plasma membrane to the nucleus to regulate gene expression. Main mediators of such signal-transduction processes are proteins that localize to sites of adhesion and the nucleus. Here, we review such signal-transduction pathways and discuss how they influence cell proliferation and differentiation.

Animals↗

PubMed2003

Functional analysis of tight junctions.

Epithelial and endothelial cells are joined to each other via a set of intercellular junctions that differ in their morphological appearance, composition, and function. The tight junction or zonula occludens is the intercellular junction that regulates diffusion between cells and therefore allows endothelia and epithelia to form cellular barriers that separate compartments of different composition. This intercellular gate formed by tight junctions is not only highly regulated but is size- and ion-selective and, hence, represents a semipermeable diffusion barrier. In epithelia, tight junctions form a morphological and functional border between the apical and basolateral cell surface domains. They directly contribute to the maintenance of cell surface polarity by forming a fence that prevents apical/basolateral diffusion of lipids in the outer leaflet of the plasma membrane. Here we describe a set of assays that allow the analysis of tight junctions to determine their integrity and functional state.

Cytological Techniques↗

PubMed2003

Signalling to and from tight junctions.

Tight junctions have long been regarded as simple barriers that separate compartments of different compositions, but recent research indicates that different types of signalling proteins and transduction pathways are associated with these junctions. They receive and convert signals from the cell interior to regulate junction assembly and function, and transmit signals to the cell interior to modulate gene expression and cell behaviour.

Animals↗