scholarly journals ZO-1 mRNA and protein expression during tight junction assembly in Caco-2 cells.

1989 ◽  
Vol 109 (3) ◽  
pp. 1047-1056 ◽  
Author(s):  
J M Anderson ◽  
C M Van Itallie ◽  
M D Peterson ◽  
B R Stevenson ◽  
E A Carew ◽  
...  
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Polyclonal Antibodies ◽  
Epithelial Cell Line ◽  
Cell Contact ◽  
Mrna Levels ◽  
Expression Library ◽  
Cell Contacts ◽  
Protein Levels ◽  
Cell Cell

We previously identified and characterized ZO-1 as a peripheral membrane protein specifically associated with the cytoplasmic surface of tight junctions. Here we describe the identification of partial cDNA sequences encoding rat and human ZO-1 and their use to study the assembly of tight junctions in the Caco-2 human intestinal epithelial cell line. A rat cDNA was isolated from a lambda-gtll expression library by screening with mAbs. Polyclonal antibodies were raised to cDNA-encoded fusion protein; several properties of these antibodies support this cDNA as encoding ZO-1. Expression of ZO-1 mRNA occurs in the rat and Caco-2 cells with a major transcript of approximately 7.5 kb. To disrupt tight junctions and study the subsequent process of assembly, Caco-2 cells were grown in suspension for 48 h in Ca++/Mg++-free spinner medium during which time they lose cell-cell contacts, become round, and by immunofluorescence microscopy show diffuse and speckled localization of ZO-1. Within hours of replating at confluent density in Ca++/Mg++-containing media, attached cells show discrete localization of ZO-1 at cell-cell contacts. Within 2 d, fully confluent monolayers form, and ZO-1 localizes in a continuous gasket-like fashion circumscribing all cells. ZO-1 mRNA levels are highest in cells in spinner culture, and upon replating rapidly fall and plateau at approximately 10% of initial levels after 2-3 wk in culture. ZO-1 protein levels are lowest in contact-free cells and rise five- to eightfold over the same period. In contrast, mRNA levels for sucrase-isomaltase, an apical membrane hydrolase, increase only after a confluent monolayer forms. Thus, in this model of contact-dependent assembly of the tight junction, there is both a rapid assembly beginning upon cell-cell contact, as well as a long-term modulation involving changes in expression of ZO-1 mRNA and protein levels.

Characterization of huJAM: evidence for involvement in cell-cell contact and tight junction regulation

2000 ◽  
Vol 279 (6) ◽  
pp. C1733-C1743 ◽  
Author(s):  
Tony W. Liang ◽  
Richard A. DeMarco ◽  
Randy J. Mrsny ◽  
Austin Gurney ◽  
Alane Gray ◽  
...  
Keyword(s):  
Cell Interactions ◽  
Epithelial Barrier ◽  
Epithelial Cell Line ◽  
Cell Contact ◽  
Subsequent Formation ◽  
Epithelial Barrier Function ◽  
Cell Contacts ◽  
Organ Systems ◽  
Tight Junction Regulation ◽  
Cell Cell

Cell-cell interactions of the mucosal epithelia are important for the maintenance and establishment of epithelial barrier function. During events of inflammation, such cell-cell interactions are often disrupted, resulting in a leaky epithelial barrier, which in turn can lead to various inflammatory and infective dysfunctions. Human junctional adhesion molecule (huJAM), found on the mucosal epithelia and vascular endothelia of many major organ systems, is a membrane glycoprotein which resolves to a doublet band of ∼40 and ∼37 kDa under SDS-PAGE analysis, representing differentially glycosylated forms of the same protein. huJAM was localized to the lateral membrane of Caco-2 cells (a human colonic epithelial cell line) monolayers, in an area basolateral of the epithelial tight junctions (TJ). Through functional and biochemical assays, we show huJAM to be able to homotypically associate and to participate in TJ restitution after trypsin-EDTA disruption. Furthermore, we also observed a migration of huJAM expression toward areas of cell-cell contacts during events of cell adhesion and monolayer formation. These qualities makes huJAM a likely player in the regulation of cell-cell contacts and the subsequent formation of TJs.

scholarly journals The Ras Target AF-6 Interacts with ZO-1 and Serves as a Peripheral Component of Tight Junctions in Epithelial Cells

1997 ◽  
Vol 139 (3) ◽  
pp. 785-795 ◽  
Author(s):  
Takaharu Yamamoto ◽  
Naozumi Harada ◽  
Kyoko Kano ◽  
Shin-ichiro Taya ◽  
Eli Canaani ◽  
...  
Keyword(s):  
Epithelial Cells ◽  
Tight Junctions ◽  
Adherens Junctions ◽  
Pdz Domain ◽  
Cell Junctions ◽  
Cell Contact ◽  
Fusion Partner ◽  
Cell Contacts ◽  
Contact Sites ◽  
Cell Cell

The dynamic rearrangement of cell–cell junctions such as tight junctions and adherens junctions is a critical step in various cellular processes, including establishment of epithelial cell polarity and developmental patterning. Tight junctions are mediated by molecules such as occludin and its associated ZO-1 and ZO-2, and adherens junctions are mediated by adhesion molecules such as cadherin and its associated catenins. The transformation of epithelial cells by activated Ras results in the perturbation of cell–cell contacts. We previously identified the ALL-1 fusion partner from chromosome 6 (AF-6) as a Ras target. AF-6 has the PDZ domain, which is thought to localize AF-6 at the specialized sites of plasma membranes such as cell–cell contact sites. We investigated roles of Ras and AF-6 in the regulation of cell–cell contacts and found that AF-6 accumulated at the cell–cell contact sites of polarized MDCKII epithelial cells and had a distribution similar to that of ZO-1 but somewhat different from those of catenins. Immunoelectron microscopy revealed a close association between AF-6 and ZO-1 at the tight junctions of MDCKII cells. Native and recombinant AF-6 interacted with ZO-1 in vitro. ZO-1 interacted with the Ras-binding domain of AF-6, and this interaction was inhibited by activated Ras. AF-6 accumulated with ZO-1 at the cell–cell contact sites in cells lacking tight junctions such as Rat1 fibroblasts and PC12 rat pheochromocytoma cells. The overexpression of activated Ras in Rat1 cells resulted in the perturbation of cell–cell contacts, followed by a decrease of the accumulation of AF-6 and ZO-1 at the cell surface. These results indicate that AF-6 serves as one of the peripheral components of tight junctions in epithelial cells and cell–cell adhesions in nonepithelial cells, and that AF-6 may participate in the regulation of cell–cell contacts, including tight junctions, via direct interaction with ZO-1 downstream of Ras.

Distribution of the tight junction proteins ZO-1, occludin, and claudin-4, -8, and -12 in bladder epithelium

2004 ◽  
Vol 287 (2) ◽  
pp. F305-F318 ◽  
Author(s):  
Prasad Acharya ◽  
Jonathan Beckel ◽  
Wily G. Ruiz ◽  
Edward Wang ◽  
Raul Rojas ◽  
...  
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
High Resistance ◽  
Cell Contact ◽  
Tight Junction Proteins ◽  
Bladder Epithelium ◽  
Cell Layers ◽  
Umbrella Cells ◽  
Junction Proteins ◽  
Cell Cell

In mammals, the bladder stores urine without permitting the passage of urine contents into the bloodstream, a function, in part, of the uroepithelial-associated tight junction complex. The protein constituents that make up this high-resistance barrier in the bladder are currently unknown, although the claudins, a multigene family, are thought to govern paracellular transport in other epithelia. Reverse transcriptase-polymerase chain reaction analysis was used to define that mRNA for claudin-2, -4, -8, -12, and -13 was expressed in mouse bladder tissue. The localization of these claudins, as well as the tight junction-associated proteins zonula occludens-1 (ZO-1) and occludin, within the bladder epithelium was determined by immunofluorescence microscopy. As expected, occludin and ZO-1 were localized to the tight junctions of rat, mouse, and rabbit umbrella cells. Intriguingly, ZO-1 in mouse epithelium, ZO-1 in the dome region of rabbit bladders and occludin in rat and mouse bladders were also expressed in the underlying intermediate and basal cell layers. Claudin-4, -8, and -12 were found in the umbrella cell tight junction; however, additional staining of claudin-4 was observed along the sites of cell-cell contact in the underlying cell layers of rat, mouse, and rabbit tissue. No claudin-2 staining was associated with tight junctions in the uroepithelium. Our results indicate that claudin-4, -8, and -12 are expressed in umbrella cells, where they may impart the high-resistance phenotype associated with this cell type, and that in some instances tight junction proteins are also associated at the sites of cell contact of the underlying cell layers, perhaps playing some role in cell-cell adhesion.

Functional and molecular characterization of NHE3 expression during ontogeny in rat jejunal epithelium

1997 ◽  
Vol 273 (6) ◽  
pp. C1937-C1946 ◽  
Author(s):  
James F. Collins ◽  
Hua Xu ◽  
Pawel R. Kiela ◽  
Jiamin Zeng ◽  
Fayez K. Ghishan
Keyword(s):  
Northern Blot ◽  
Polyclonal Antibodies ◽  
Membrane Vesicle ◽  
Age Groups ◽  
Fold Increase ◽  
Jejunal Mucosa ◽  
Mrna Levels ◽  
Adult Rats ◽  
Intestinal Brush Border Membrane ◽  
Protein Levels

Ontogenic changes occur in intestinal brush-border membrane vesicle (BBMV) Na+/H+exchange activity. The present studies were designed to investigate ontogenic changes in Na+/H+exchanger (NHE) isoform 3 in rat jejunum. pH-dependent Na+ uptake was assayed in four age groups of rats in the presence of 0, 50, or 800 μM HOE-694, a specific NHE inhibitor with differential sensitivities for NHE2 [inhibition constant ( K i) = 5 μM in PS120 fibroblasts] and NHE3 ( K i = 650 μM). Results showed that NHE2 and NHE3 contribute to basal BBMV uptake at all ages. Uptake levels were highest in 6-wk-old rats, lower in adult rats, and lowest in 2-wk-old (suckling) and 3-wk-old (weanling) rats. NHE3 contribution ranged from 92% at 6 wk of age to 59% at 2 and 3 wk of age. NHE3 inhibition by 800 μM HOE-694 was 38–45%. Statistical analysis showed that HOE-694 had a significant effect at both concentrations at all ages and that differences were present between all ages except 2- and 3-wk rats (at all HOE-694 concentrations). Northern blot analyses of jejunal mucosa showed lowest NHE3 mRNA levels in 2-wk animals and higher levels in all other age groups. Polyclonal antibodies were developed against an NHE3 COOH-terminal fusion protein, and antiserum was characterized with NHE3-transfected PS120 cells and by immunohistochemistry. Western blot analyses showed lowest protein levels in 2-wk animals and higher levels in the other ages. Suckling rats were subcutaneously injected with methylprednisone (MP) for 2 days and killed 1 day later. Northern blot analyses showed a twofold increase in NHE3 mRNA expression with MP treatment. Immunoblot analyses showed a 2.5-fold increase in NHE3 immunoreactive protein levels with MP injection. Overall, these data suggest that NHE3 is regulated during ontogeny and that ontogenic changes are most apparent around the time of weaning. Furthermore, the data suggest that NHE3 is regulated at transcriptional and posttranscriptional levels during mammalian intestinal development.

scholarly journals Rab13 regulates PKA signaling during tight junction assembly

2004 ◽  
Vol 165 (2) ◽  
pp. 175-180 ◽  
Author(s):  
Katja Köhler ◽  
Daniel Louvard ◽  
Ahmed Zahraoui
Keyword(s):  
Tight Junction ◽  
Tight Junctions ◽  
Inhibitory Effect ◽  
Cell Junctions ◽  
Unknown Mechanism ◽  
Epithelial Tight Junctions ◽  
Actin Remodelling ◽  
Cell Cell

The GTPase Rab13 regulates the assembly of functional epithelial tight junctions (TJs) through a yet unknown mechanism. Here, we show that expression of the GTP-bound form of Rab13 inhibits PKA-dependent phosphorylation and TJ recruitment of the vasodilator-stimulated phosphoprotein, an actin remodelling protein. We demonstrate that Rab13GTP directly binds to PKA and inhibits its activity. Interestingly, activation of PKA abrogates the inhibitory effect of Rab13 on the recruitment of vasodilator-stimulated phosphoprotein, ZO-1, and claudin1 to cell–cell junctions. Rab13 is, therefore, the first GTPase that controls PKA activity and provides an unexpected link between PKA signaling and the dynamics of TJ assembly.

scholarly journals Hepatocyte nuclear factor 4α regulates the expression of intestinal epithelial Na+/H+ exchanger isoform 3

2018 ◽  
Vol 314 (1) ◽  
pp. G14-G21 ◽  
Author(s):  
Saminathan Muthusamy ◽  
Jong Jin Jeong ◽  
Ming Cheng ◽  
Jessica A. Bonzo ◽  
Anoop Kumar ◽  
...  
Keyword(s):  
Promoter Activity ◽  
Core Promoter ◽  
Epithelial Cell Line ◽  
Mrna Levels ◽  
Nuclear Factor ◽  
Intestinal Epithelial ◽  
Hepatocyte Nuclear Factor ◽  
Mobility Shift ◽  
Protein Levels ◽  
Hepatocyte Nuclear Factor 4Α

Na+/H+ exchanger isoform 3 (NHE3) plays a key role in coupled electroneutral NaCl absorption in the mammalian intestine. Reduced NHE3 expression or function has been implicated in the pathogenesis of diarrhea associated with inflammatory bowel disease (IBD) or enteric infections. Our previous studies revealed transcriptional regulation of NHE3 by various agents such as TNF-α, IFN-γ, and butyrate involving transcription factors Sp1 and Sp3. In silico analysis revealed that the NHE3 core promoter also contains a hepatocyte nuclear factor 4α (HNF-4α) binding site that is evolutionarily conserved in several species suggesting that HNF-4α has a role in NHE3 regulation. Nhe3 mRNA levels were reduced in intestine-specific Hnf4α-null mice. However, detailed mechanisms of NHE3 regulation by HNF-4α are not known. We investigated the regulation of NHE3 gene expression by HNF-4α in vitro in the human intestinal epithelial cell line C2BBe1 and in vivo in intestine-specific Hnf4α-null ( Hnf4αΔIEpC) and control ( Hnf4αfl/fl) mice. HNF-4α knockdown by short interfering RNA in C2BBe1 cells significantly decreased NHE3 mRNA and NHE3 protein levels. Gel mobility shift and chromatin immunoprecipitation assays revealed that HNF-4α directly interacts with the HNF-4α motif in the NHE3 core promoter. Site-specific mutagenesis on the HNF-4α motif decreased, whereas ectopic overexpression of HNF-4α increased, NHE3 promoter activity. Furthermore, loss of HNF-4α in Hnf4αΔIEpC mice decreased colonic Nhe3 mRNA and NHE3 protein levels. Our results demonstrate a novel role for HNF-4α in basal regulation of NHE3 expression. These studies represent an important and novel target for therapeutic intervention in IBD-associated diarrhea. NEW & NOTEWORTHY Our studies for the first time show that hepatocyte nuclear factor 4α directly regulates NHE3 promoter activity and its basal expression in the intestine.

N-cadherin in adult rat cardiomyocytes in culture. II. Spatio-temporal appearance of proteins involved in cell-cell contact and communication. Formation of two distinct N-cadherin/catenin complexes

1996 ◽  
Vol 109 (1) ◽  
pp. 11-20 ◽  
Author(s):  
C.M. Hertig ◽  
S. Butz ◽  
S. Koch ◽  
M. Eppenberger-Eberhardt ◽  
R. Kemler ◽  
...  
Keyword(s):  
Plasma Membrane ◽  
Gap Junctions ◽  
Connexin 43 ◽  
Cell Contact ◽  
Beta Catenin ◽  
Rat Cardiomyocytes ◽  
Cell Contacts ◽  
Adult Rat ◽  
Spatio Temporal ◽  
Cell Cell

The spatio-temporal appearance and distribution of proteins forming the intercalated disc were investigated in adult rat cardiomyocytes (ARC). The ‘redifferentiation model’ of ARC involves extensive remodelling of the plasma membrane and of the myofibrillar apparatus. It represents a valuable system to elucidate the formation of cell-cell contact between cardiomyocytes and to assess the mechanisms by which different proteins involved in the cell-cell adhesion process are sorted in a precise manner to the sites of function. Appearance of N-cadherin, the catenins and connexin43 within newly formed adherens and gap junctions was studied. Here first evidence is provided for a formation of two distinct and separable N-cadherin/catenin complexes in cardiomyocytes. Both complexes are composed of N-cadherin and alpha-catenin which bind to either beta-catenin or plakoglobin in a mutually exclusive manner. The two N-cadherin/catenin complexes are assumed to be functionally involved in the formation of cell-cell contacts in ARC; however, the differential appearance and localization of the two types of complexes may also point to a specific role during ARC differentiation. The newly synthesized beta-catenin containing complex is more abundant during the first stages in culture after ARC isolation, while the newly synthesized plakoglobin containing complex progressively accumulates during the morphological changes of ARC. ARC formed a tissue-like pattern in culture whereby the new cell-cell contacts could be dissolved through Ca2+ depletion. Presence of cAMP and replenishment of Ca2+ content in the culture medium not only allowed reformation of cell-cell contacts but also affected the relative protein ratio between the two N-cadherin/catenin complexes, increasing the relative amount of newly synthesized beta-catenin over plakoglobin at a particular stage of ARC differentiation. The clustered N-cadherin/catenin complexes at the plasma membrane appear to be a prerequisite for the following gap junction formation; a temporal sequence of the appearance of adherens junction proteins and of gap junctions forming connexin-43 is suggested.

scholarly journals PLAC-24 Is a Cytoplasmic Dynein-Binding Protein That Is Recruited to Sites of Cell-Cell Contact

2002 ◽  
Vol 13 (5) ◽  
pp. 1722-1734 ◽  
Author(s):  
Sher Karki ◽  
Lee A. Ligon ◽  
Jamison DeSantis ◽  
Mariko Tokito ◽  
Erika L. F. Holzbaur
Keyword(s):  
Epithelial Cells ◽  
Recent Observation ◽  
Polyclonal Antibodies ◽  
Adherens Junction ◽  
Cytoplasmic Dynein ◽  
Cell Contact ◽  
Dynein Intermediate Chain ◽  
Cellular Cytoskeleton ◽  
Cell Cell ◽  
Intermediate Chain

We screened for polypeptides that interact specifically with dynein and identified a novel 24-kDa protein (PLAC-24) that binds directly to dynein intermediate chain (DIC). PLAC-24 is not a dynactin subunit, and the binding of PLAC-24 to the dynein intermediate chain is independent of the association between dynein and dynactin. Immunocytochemistry using PLAC-24–specific polyclonal antibodies revealed a punctate perinuclear distribution of the polypeptide in fibroblasts and isolated epithelial cells. However, as epithelial cells in culture make contact with adjacent cells, PLAC-24 is specifically recruited to the cortex at sites of contact, where the protein colocalizes with components of the adherens junction. Disruption of the cellular cytoskeleton with latrunculin or nocodazole indicates that the localization of PLAC-24 to the cortex is dependent on intact actin filaments but not on microtubules. Overexpression of β-catenin also leads to a loss of PLAC-24 from sites of cell-cell contact. On the basis of these data and the recent observation that cytoplasmic dynein is also localized to sites of cell-cell contact in epithelial cells, we propose that PLAC-24 is part of a multiprotein complex localized to sites of intercellular contact that may function to tether microtubule plus ends to the actin-rich cellular cortex.

Robust T-cell signaling triggered on soft polydimethylsiloxane-supported lipid-bilayers

2020 ◽  
Author(s):  
Anna H. Lippert ◽  
Ivan B. Dimov ◽  
Alexander Winkel ◽  
James McColl ◽  
Jane Humphrey ◽  
...  
Keyword(s):  
T Cell ◽  
Lipid Bilayers ◽  
T Cell Activation ◽  
Cell Activation ◽  
Tyrosine Phosphatase ◽  
Supported Lipid Bilayers ◽  
Cell Contact ◽  
Mechanical Resistance ◽  
Cell Contacts ◽  
Cell Cell

AbstractThe T-cell receptor (TCR) is thought to be triggered either by mechano-transduction or local tyrosine phosphatase exclusion at cell-cell contacts. However, the effects of the mechanical properties of activating surfaces have only been tested for late-stage T-cell activation, and phosphatase segregation has mostly been studied on glass-supported lipid bilayers that favor imaging but are orders-of-magnitude stiffer than typical cells. We developed a method for attaching lipid bilayers to polydimethylsiloxane polymer supports, producing ‘soft bilayers’ with physiological levels of mechanical resistance (Young’s modulus of 4 kPa). Comparisons of T-cell behavior on soft and glass-supported bilayers revealed that early calcium signaling is unaffected by substrate rigidity, implying that early steps in TCR triggering are not mechanosensitive. Robust phosphatase exclusion was observed on the soft bilayers, however, suggesting it likely occurs at cell-cell contacts. This work sets the stage for an imaging-based exploration of receptor signaling under conditions closely mimicking physiological cell-cell contact.

Modification of biophysical properties of lung epithelial Na+ channels by dexamethasone

2000 ◽  
Vol 279 (3) ◽  
pp. C762-C770 ◽  
Author(s):  
A. Lazrak ◽  
A. Samanta ◽  
K. Venetsanou ◽  
P. Barbry ◽  
S. Matalon
Keyword(s):  
Cell Line ◽  
Single Channel ◽  
Alveolar Epithelial Cell ◽  
A549 Cells ◽  
Alveolar Epithelium ◽  
Housekeeping Gene ◽  
Epithelial Cell Line ◽  
Mrna Levels ◽  
Open Probability ◽  
Protein Levels

There is considerable interest in identifying the basic mechanisms by which dexamethasone alters ion transport across the adult alveolar epithelium. Herein, we incubated synchronized A549 cells, a human alveolar epithelial cell line, with dexamethasone (1 μM) for 24–48 h. When normalized to HPRT (a housekeeping gene), A549 β- and γ-subunit mRNA levels for the human amiloride-sensitive epithelial sodium channel (hENaC), assessed by RT-PCR, increased by 1.6- and 17-fold respectively, compared with control values ( P < 0.05). These changes were abolished by actinomycin D, indicating transcriptional regulation. Western blotting studies revealed that dexamethasone also increased expression of β- and γ-hENaC protein levels. In contrast, α-hENaC mRNA increased by onefold ( P > 0.05) and α-hENaC protein level was unchanged. Incubation of A549 cells with dexamethasone increased their whole cell amiloride-sensitive sodium currents twofold and decreased the K 0.5 for amiloride from 833 ± 69 to 22 ± 5.4 nM (mean ± SE; P < 0.01). Single channel recordings in the cell-attached mode showed that dexamethasone treatment increased single channel open time and open probability threefold and decreased channel conductance from 8.63 ± 0.036 to 4.4 ± 0.027 pS (mean ± SE; P < 0.01). We concluded that dexamethasone modulates the amiloride-sensitive Na+ channels by differentially regulating the expression of β- and γ-subunits at the mRNA and protein levels in the human A549 cell line, with little effect on α-hENaC subunit.

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