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PubMed1997

The Ras target AF-6 interacts with ZO-1 and serves as a peripheral component of tight junctions in epithelial cells.

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.

Animals↗
PubMed1996

Involvement of a heterotrimeric G protein alpha subunit in tight junction biogenesis.

The tight junction (TJ) of polarized epithelial cells is critical for maintaining an impermeant barrier and epithelial cell polarity. The signaling events important for TJ assembly require regulated calcium stores and protein kinase C (PKC), but the earliest signaling events in the cascade have not been well defined. We now show that Galphai2 in Madin Darby canine kidney (MDCK) cells localizes to a region overlapping with the TJ. To further analyze the localization of Galpha subunits in epithelial cells, rat Galphao, Q205Lalphao (Galphao "activated" by point mutation) and plasmid without insert (PC) were transfected into MDCK cells and localized by immunofluorescence and confocal microscopy. Similar to endogenous Galphai2, Galphao-MDCK cells localize Galphao, (84% similar to Galphai2) in the subapical region overlapping with ZO-1 (zona occludens-I), a key component of the TJ. PC-MDCK cells have no detectable Galphao. In Galphao-MDCK cells, a physical association of Galphao with components of the TJ was detectable by immunoprecipitation of ZO-1. Immunoprecipitates of ZO-1 from Galphao-MDCK cells consistently coprecipitated Galphao. Constitutively active Q205LGalphao localized to the subapical lateral membrane similar to wild-type Galphao. To determine if constitutively activated Galpha subunits can affect TJ biogenesis, the formation of tight junctions in PC, Galphao, and Q205Lalphao-MDCK cells was followed by measurement of transepithelial resistance (TER) during the Ca2+ switch, a model widely used to study mechanisms of junctional assembly. Baseline and post Ca2+ switch TER values did not differ among the cell lines. However, constitutively activated Q205Lalphao-MDCK cells developed TER significantly faster than PC and Galphao cells in the early phase (0-4 h) (54 +/- 4 versus 23 +/- 3 (PC); 12 +/- 1 (Galphao) Omega.m2/h) and late phase (4-h peak) (117 +/- 10 versus 45 +/- 5 (PC); 66 +/- 7 (Galphao) Omega.m2/h) after Ca2+ switch. Peak TER values were significantly higher in Q205Lalphao-MDCK cells (1168 +/- 107 versus 437 +/- 37 (PC); 548 +/- 54 (Galphao) Omega.cm2). These results indicate that Galphao and Q205Lalphao expressed in MDCK cells are localized near the junctional complex, associate with at least one TJ protein, and that activated Galphao accelerates TJ biogenesis without significantly affecting the maintenance of the TJ. Together, these results suggest an important role for heterotrimeric G proteins in TJ assembly.

Animals↗
PubMed1999

A dominant mutant of occludin disrupts tight junction structure and function.

The tight junction is the most apical intercellular junction of epithelial cells and forms a diffusion barrier between individual cells. Occludin is an integral membrane protein specifically associated with the tight junction which may contribute to the function or regulation of this intercellular seal. In order to elucidate the role of occludin at the tight junction, a full length and an N-terminally truncated murine occludin construct, both FLAG-tagged at the N terminus, were stably introduced into the murine epithelial cell line CSG 120/7. Both constructs were correctly targeted to the tight junction, as defined by colocalization with another tight junction protein, ZO-1. The construct lacking the N terminus and extracellular domains of occludin was found to exert a dramatic effect on tight junction integrity. Cell monolayers failed to develop an efficient permeability barrier, as demonstrated by low transcellular electrical resistance values and an increased paracellular flux to small molecular mass tracers. Furthermore, gaps were found to have been induced in the P-face associated tight junction strands, as visualized by freeze-fracture electron microscopy. These findings demonstrate an important role for the N-terminal half of occludin in tight junction assembly and maintaining the barrier function of the tight junction.

Animals↗
PubMed1995

Epidermal growth factor induces tyrosine phosphorylation and reorganization of the tight junction protein ZO-1 in A431 cells.

Addition of epidermal growth factor (EGF) to A431 human epidermal carcinoma cells results in actin reorganization and phosphorylation of several cytoskeletal proteins. In the present study, we found that EGF treatment of this cell line also results in the redistribution and tyrosine phosphorylation of ZO-1. In normal polarized epithelial cells, ZO-1 is restricted to the cytoplasmic surface of the most apical of the intercellular junctions, the tight junction. In contrast, ZO-1 in the majority of unstimulated A431 cells in small subconfluent islands colocalizes with actin along the lateral cell membranes and in rare microspikes and membrane ruffles. Exposure to EGF results in a transient redistribution of actin into an apically positioned ring. ZO-1 becomes highly focused at apical sites of cell contact and co-localizes with the newly formed band of perijunctional actin. Coincidently, ZO-1 and another tight junction protein, ZO-2, become transiently phosphorylated on tyrosine residues, as determined by anti-phosphotyrosine immunoblotting. Pre-treatment of A431 cells with cytochalasin D, which disrupts normal microfilament organization, does not affect EGF-dependent phosphorylation of the EGF receptor. However, cytochalasin D pretreatment blocks both the EGF-induced ZO-1 rearrangement and tyrosine phosphorylation, suggesting that these responses are dependent on an intact actin microfilament system. We speculate that the transient tyrosine phosphorylation of ZO-1 in response to EGF treatment may be involved in remodeling of intercellular junctions in A431 cells.

Actins↗
PubMed2002

Claudin-2 expression induces cation-selective channels in tight junctions of epithelial cells.

Tight junctions seal the paracellular pathway of epithelia but, in leaky tissues, also exhibit specific permeability. In order to characterize the contribution of claudin-2 to barrier and permeability properties of the tight junction in detail, we studied two strains of Madin-Darby canine kidney cells (MDCK-C7 and MDCK-C11) with different tight junctional permeabilities. Monolayers of C7 cells exhibited a high transepithelial resistance (>1 kOhms cm(2)), compared with C11 cells (<100 Ohms cm(2)). Genuine expression of claudin-1 and claudin-2, but not of occludin or claudin-3, was reciprocal to transepithelial resistance. However, confocal microscopy revealed a marked subjunctional localization of claudin-1 in C11 cells, indicating that claudin-1 is not functionally related to the low tight junctional resistance of C11 cells. Strain MDCK-C7, which endogenously does not express junctional claudin-2, was transfected with claudin-2 cDNA. In transfected cells, but not in vector controls, the protein was detected in colocalization with junctional occludin by means of immunohistochemical analyses. Overexpression of claudin-2 in the originally tight epithelium with claudin-2 cDNA resulted in a 5.6-fold higher paracellular conductivity and relative ion permeabilities of Na(+) identical with 1, K(+)=1.02, NMDG(+)=0.79, choline(+)=0.71, Cl(-)=0.12, Br(-)=0.10 (vector control, 1:1.04:0.95:0.94:0.85:0.83). By contrast, fluxes of (radioactively labeled) mannitol and lactulose and (fluorescence labeled) 4 kDa dextran were not changed. Hence, with regular Ringer's, Na(+) conductivity was 0.2 mS cm(-2) in vector controls and 1.7 mS cm(-2) in claudin-2-transfected cells, while Cl(-) conductivity was 0.2 mS cm(-2) in both cells. Thus, presence of junctional claudin-2 causes the formation of cation-selective channels sufficient to transform a 'tight' tight junction into a leaky one.

Animals↗
PubMed1992

Structural domains of the tight junctional intramembrane fibrils.

Freeze-fracture reveals intramembrane fibrils lying along the intermembrane contacts that characterize tight junctions. Tight junctions from a variety of species are reexamined here by rapid freezing prior to freeze-fracture. The tight junction fibril is uprooted alternatively from either the cytoplasmic or the exoplasmic hemibilayer during freeze-cleavage, exposing two distinct but complementary views of its hybrid structure within the same replica. When the transmembrane fibril is uprooted from the exoplasmic hemibilayer it appears on the P-fracture face as a smooth-surfaced cylinder which is sometimes resolved into periodic globular structures. The lack of indication that the P-face cylinder has been pulled out through the opposite membrane half indicates that this domain of the fibril is, in large part, buried in the hydrophobic interior of the membrane. However, when the transmembrane fibril is uprooted from the cytosolic hemibilayer it appears on the E-fracture face as a row of irregular intramembrane particles. The irregular particles on the E-face aspect of the fibril are interpreted as corresponding to transmembrane protein segments that may very well make projections onto the cytosolic surface of the bilayer. En face views of the outermost junction strand between adjacent epithelial cells show periodic lines on the bilayer on each side of the junction which are interpreted as periodic transmembrane protein segments arising from the core structure of the tight junction fibril. If the backbone of the tight junction strand is an inverted cylindrical micelle, it must typically include proteins, which might anchor it to structures outside the membrane bilayer.

Animals↗
PubMed1998

Tight junction regulation in the mammary gland.

Tight junctions form a narrow, continuous seal that surrounds each endothelial and epithelial cell at the apical border, and act to regulate the movement of material through the paracellular pathway. In the mammary gland, the tight junctions of the alveolar epithelial cells are impermeable during lactation, and thus allow milk to be stored between nursing periods without leakage of milk components from the lumen. Nonetheless mammary epithelial tight junctions are dynamic and can be regulated by a number of stimuli. Tight junctions of the mammary gland from the pregnant animal are leaky, undergoing closure around parturition to become the impermeable tight junctions of the lactating animal. Milk stasis, high doses of oxytocin, and mastitis have been shown to increase tight junction permeability. In general changes in tight junction permeability in the mammary gland appear to be the results of a state change and not assembly and disassembly of tight junctions. Both local factors, such as intramammary pressure and TGF-beta, and systemic factors, such as prolactin, progesterone, and glucocorticoids, appear to play a role in the regulation of mammary tight junctions. Finally, the tight junction state appears to be closely linked to milk secretion. An increase in tight junction permeability is accompanied by decrease in the milk secretion rate, and conversely, a decrease in tight junction permeability is accompanied by an increase in the milk secretion rate.

Animals↗
PubMed2003

Immunohistological distribution of the tight junction components ZO-1 and occludin in regenerating human epidermis.

BACKGROUND: Molecular characterization of tight junction proteins during the past few years has provided novel methods for studying these specialized junctions. Tight junctions have recently been characterized in the granular cell layer of human epidermis, and the role of these junctions in the epidermal barrier is now being re-evaluated. OBJECTIVES: To investigate the expression of tight junction components during the re-epithelialization of suction blisters and the regeneration of the corneal layer after tape stripping. METHODS: Suction blisters were induced in eight healthy volunteers, and skin biopsies were taken 4 or 6 days afterwards. The restoration of epidermal barrier function was evaluated by measuring water evaporation (WE) from the wound area. Tape stripping was performed on three volunteers to remove the corneal layer. The tissues were immunolabelled using indirect immunofluorescence or the avidin-biotin method. RESULTS: Prior to the biopsies, WE from the blister wounds was markedly elevated in comparison with normal skin. In the epidermis surrounding the blister, occludin and ZO-1 were expressed in the granular cell layer only. In the hyperproliferative zone adjacent to the border of the blister, the expression of ZO-1 was redistributed into several spinous cell layers, while occludin expression was restricted to the upper epidermis. In the leading edge of migrating keratinocytes, both proteins were expressed exclusively in the most superficial layer of keratinocytes. Double labelling for ZO-1 and involucrin showed expression of both proteins in the same layers of hyperproliferative keratinocytes, while the expression patterns were clearly different in the migrating keratinocytes. CONCLUSIONS: Tight junctions of regenerating epidermis may provide a functional barrier prior to regeneration of the corneal layer.

Adult↗
PubMed2006

Antibody blockade of junctional adhesion molecule-A in rabbit corneal endothelial tight junctions produces corneal swelling.

PURPOSE: The ultrastructure of tight junctions in the corneal endothelium has been studied extensively, yet little is known about their molecular composition. Junctional adhesion molecule-A (JAM-A) is a tight junction-associated adhesion protein previously implicated in tight junction assembly and regulation of barrier function. In this study, we sought to investigate the expression and function of JAM-A in the corneal endothelium. METHODS: Immunofluorescence confocal microscopy was used to investigate expression of JAM-A and the related proteins JAM-C, CAR, and AF-6 in the rabbit corneal endothelium. Corneal endothelial perfusion specular microscopy was then used to test the effects of antibodies to JAM-A on corneal swelling. RESULTS: The expression of JAM-A was observed in the tight junctions of rabbit corneal endothelium in a localization pattern identical with that of ZO-1, a known marker of the tight junction and binding partner of JAM-A. Expression of related proteins JAM-C and CAR (Coxsackie and adenovirus receptor) was also observed in the corneal endothelium, but their distribution was diffuse and not limited to the tight junction. Expression of AF-6, a known binding partner of JAM-A, was also observed in the tight junction in a pattern similar to ZO-1. Last, functional experiments were performed in which a monoclonal antibody to JAM-A was shown to increase rabbit corneal swelling by 63% compared with the control. CONCLUSIONS: The results provide new evidence that JAM-A and its binding partner AF-6 are expressed in tight junctions of the corneal endothelium and that JAM-A has a major role in maintaining the corneal endothelial barrier function.

Animals↗
PubMed2006

Attaching and effacing pathogen-induced tight junction disruption in vivo.

Diarrhoea is a hallmark of infections by the human attaching and effacing (A/E) pathogens, enterohaemorrhagic Escherichia coli (EHEC) and enteropathogenic E. coli (EPEC). Although the mechanisms underlying diarrhoea induced by these pathogens remain unknown, cell culture results have suggested that these pathogens may target tight junctions. Tight junctions in the colon function as physical intercellular barriers that separate and prevent mixing of the luminal contents with adlumenal regions of the epithelium. Consequently, it is thought that the disruption of intestinal epithelial tight junctions by A/E pathogens could result in a loss of barrier function in the alimentary tract; however, this remains unexamined. Here we demonstrate for the first time that A/E pathogen infection results in the morphological alteration of tight junctions during natural disease. Tight junction alteration, characterized by relocalization of the transmembrane tight junction proteins claudin 1, 3 and 5, is a functional disruption; molecular tracers, which do not normally penetrate uninfected epithelia, pass across pathogen-infected epithelia. Functional junction disruption occurs with a concomitant increase in colon luminal water content. The effects on tissue are dependent upon the bacterial type III effector EspF (E. coli secreted protein F), because bacteria lacking EspF, while able to colonize, are defective for junction disruption and result in decreased proportions of water in the colon compared with wild-type infection. These results suggest that the diarrhoea induced by A/E pathogens occurs as part of functional tight junction disruption.

Animals↗
PubMed2005

Assay of Rab13 in regulating epithelial tight junction assembly.

Rab13 is recruited to tight junctions from a cytosolic pool after cell-cell contact formation. Tight junctions are intercellular junctions that separate apical from basolateral domains and are required for the establishment/maintenance of polarized transport in epithelial cells. They form selective barriers regulating the diffusion of ions and solutes between cells. They also maintain the cell surface asymmetry by forming a "fence" that prevents apical/basolateral diffusion of membrane proteins and lipids in the outer leaflet of the plasma membrane. We generate stable MDCK cell lines expressing inactive (T22N mutant) and constitutively active (Q67L mutant) Rab13 as GFP-Rab13 chimeras. Expression of GFP-Rab13Q67L delays the formation of electrically tight epithelial monolayers, induces the leakage of small nonionic tracers from the apical domain, and disrupts the tight junction fence diffusion barrier. It also alters the tight junction strand structure and delays the localization of the tight junction transmembrane protein, claudin1. In contrast, the inactive Rab13T22N mutant does not disrupt tight junction functions, tight junction strand architecture, or claudin1 localization. Here we describe a set of assays that allows us to investigate the role of Rab13 in modulating tight junction structure and function.

Animals↗
PubMed1983

Formation of misplaced and reflexive tight junction strands in prostate epithelial cells.

Tight junction strands occur at three atypical locations in slices of rat ventral prostate exposed to conditions promoting rapid tight junction assembly: (1) in the basal plasma membranes of the columnar epithelial cells, over 40 microns from the native apical tight junction band (misplaced tight junctions); (2) in the plasma membranes of basal epithelial cells, which never have tight junctions in the native state; and (3) between processes of the same cell (reflexive or autocellular tight junctions) at the basal or lateral portions of the columnar epithelial cell. These findings suggest that tight junction formation is not limited to specific parts of the plasma membrane, even in highly polarized cells such as those in prostate epithelium. Taken together with other new evidence, they also suggest that tight junctions may be very labile.

Animals↗
PubMed1997

A synthetic peptide corresponding to the extracellular domain of occludin perturbs the tight junction permeability barrier.

Occludin, the putative tight junction integral membrane protein, is an attractive candidate for a protein that forms the actual sealing element of the tight junction. To study the role of occludin in the formation of the tight junction seal, synthetic peptides (OCC1 and OCC2) corresponding to the two putative extracellular domains of occludin were assayed for their ability to alter tight junctions in Xenopus kidney epithelial cell line A6. Transepithelial electrical resistance and paracellular tracer flux measurements indicated that the second extracellular domain peptide (OCC2) reversibly disrupted the transepithelial permeability barrier at concentrations of < 5 microM. Despite the increased paracellular permeability, there were no changes in gross epithelial cell morphology as determined by scanning EM. The OCC2 peptide decreased the amount of occludin present at the tight junction, as assessed by indirect immunofluorescence, as well as decreased total cellular content of occludin, as assessed by Western blot analysis. Pulse-labeling and metabolic chase analysis suggested that this decrease in occludin level could be attributed to an increase in turnover of cellular occludin rather than a decrease in occludin synthesis. The effect on occludin was specific because other tight junction components, ZO-1, ZO-2, cingulin, and the adherens junction protein E-cadherin, were unaltered by OCC2 treatment. Therefore, the peptide corresponding to the second extracellular domain of occludin perturbs the tight junction permeability barrier in a very specific manner. The correlation between a decrease in occludin levels and the perturbation of the tight junction permeability barrier provides evidence for a role of occludin in the formation of the tight junction seal.

Amino Acid Sequence↗
PubMed1984

Evidence against the presence of tight junctions in normal endocrine pancreas.

Tight junctional fibrils were absent in freeze-fracture replicas of rat and human islets in situ, but were easily discerned in collagenase-isolated islets. Disruption of the pancreatic gland and its exposure to trypsin were each found to induce tight junction formation in rat islets. The amount of tight junctions between islet cells declined progressively during culture, but tight junctional structures remained detectable after 1 day of culture. It is suggested that rather than being involved in normal islet cell function tight junctions provide an adaptive mechanism intended to seal and hence to protect islet microdomains against sudden perturbations in local interstitial fluid.

Animals↗
PubMed1996

Occludin is a functional component of the tight junction.

Occludin's role in mammalian tight junction activity was examined by 'labeling' the occludin pool with immunologically detectable chick occludin. This was accomplished by first transfecting MDCK cell with the Lac repressor gene. HygR clones were then transfected with chick occludin cDNA inserted into a Lac operator construct. The resulting HygR/NeoR clones were plated on porous inserts and allowed to form tight junctions. Once steady state transepithelial electrical resistance was achieved, isopropyl- beta-D-thiogalactoside was added to induce chick occludin expression. Confocal laser scanning microscopy of monolayers immunolabeled with Oc-2 monoclonal antibody revealed that chick occludin localized precisely to the preformed tight junctions. When sparse cultures were maintained in low Ca2+ medium, chick occludin and canine ZO-1 co-localized to punctate sites in the cytoplasm suggesting their association within the same vesicular structures. In low calcium medium both proteins also co-localized to contact sites between occasional cell pairs, where a prominent bar was formed at the plasma membrane. Chick occludin was detectable by western blot within two hours of adding isopropyl- beta-D-thiogalactoside to monolayers that had previously achieved steady state transepithelial electrical resistance; this coincided with focal immunofluorescence staining for chick occludin at the cell membrane of some cells. A gradual rise in transepithelial electrical resistance, above control steady state values, began five hours after addition of the inducing agent reaching new steady state values, which were 30-40% above baseline, 31 hours later. Upon removal of isopropyl- beta-D-thiogalactoside chick occludin expression declined slowly until it was no longer detected in western blots 72 hours later; transepithelial electrical resistance also returned to baseline values during this time. While densitometric analysis of western blots indicated that the presence of chick occludin had no detectable effect on E-cadherin or ZO-1 expression, the possibility cannot be excluded that ZO-1 might be a limiting factor in the expression of chick occludin at the cell surface. To test whether expression of chick occludin affected the process of tight junction assembly, monolayers in low Ca2+ medium were treated with isopropyl- beta-D-thiogalactoside for 24 or 48 hours, before Ca2+ was added to stimulate tight junction assembly. Chick occludin did not alter the rate at which transepithelial electrical resistance developed, however, steady state values were 30-40% above control monolayers not supplemented with the inducing agent. By freeze fracture analysis, the number of parallel tight junction strands shifted from a mode of three in controls to four strands in cells expressing chick occludin and the mean width of the tight junction network increased from 175 +/- 11 nm to 248 +/- 16 nm. Two days after plating confluent monolayers that were induced to express chick occludin, mannitol flux was reduced to a variable degree relative to control monolayers. With continued incubation with the inducing agent, mannitol flux increased on day 11 to 50%, and TER rose to 45% above controls. Both of these changes were reversible upon removal of isopropyl- beta-D-thiogalactoside. These data are consistent with the notion that occludin contributes to the electrical barrier function of the tight junction and possibly to the formation of aqueous pores within tight junction strands.

Animals↗
PubMedDate not supplied

Structure and function of tight junctions. Role in intestinal barrier.

The tight junctions are narrow belts that circumferentially surround the upper part of the lateral surfaces of the adjacent epithelial cells to create fusion points or "kisses". They are involved in maintaining the cellular polarity and in the establishment of compositionally distinct fluid compartments in the body. Tight junctions are formed by many specific proteins and are connected with the cytoskeleton. In contrast to what might be expected, the intestinal tight junctions are highly dynamic areas and their permeability can change in response to both external and intracellular stimuli. In fact, the tight junctions play an important role in the regulation of the passive transepithelial movement of molecules. A number of signalling molecules have been implicated in the regulation of tight junction function, including Ca++, protein kinase C, G proteins, phospholipase A2 and C. In many intestinal and systemic diseases, changes in intestinal permeability are related to alteration of tight junctions as an expression of intestinal barrier damage. Moreover, permeability of the tight junctions can be modified by bacterial toxins, cytokines, hormones and drugs. A better understanding of tight junction structure, biogenesis and regulation mechanisms should throw further light on the intestinal barrier functions and suggest innovative therapeutic strategies.

Animals↗
PubMed1999

Induction of tight junctions in human connexin 32 (hCx32)-transfected mouse hepatocytes: connexin 32 interacts with occludin.

Small gap junction plaques are associated with tight junction strands in some cell types including hepatocytes and it is thought that they may be closely related to tight junctions and the establishment of cell polarity. In order to examine roles of gap junctions in regulating expression and structure of tight junctions, we transfected human Cx32 cDNA into immortalized mouse hepatocytes (CHST8 cells) which lack endogenous Cx32 and Cx26. Immunocytochemistry revealed that endogenous integral tight junction protein occludin was strongly localized and was colocalized with Cx32 at cell borders in transfectants, whereas neither was detected in parental cells. In Northern blots, mRNAs encoding occludin and the other integral tight junction proteins, claudin-1 and -2, were induced in the transfectants compared to parental cells. In Western blots, occludin protein was increased in the transfectants compared to parental cells, and binding of occludin to Cx32 protein was demonstrated by immunoprecipitation. In freeze fracture of the transfectants, tight junction strands were more numerous and complex compared to parental cells, and small gap junction plaques appeared within induced tight junction strands. Nevertheless, no change in barrier function of tight junctions was observed. These results indicate that in hepatocytes, gap junction, and tight junction expression are closely coordinated, and that Cx32 may play a role in regulating occludin expression.

Animals↗
PubMedDate not supplied

Changes in the expression of junctional and nonjunctional complex component genes when inter-sertoli tight junctions are formed in vitro.

Throughout spermatogenesis, germ cells move progressively from the basal to the adluminal compartment, which is accompanied by continual disassembly and reassembly of intercellular junctions suggesting germ cell movement is composed of intermittent phases of junction disassembly and reassembly. A study was performed to correlate the expression of junctional-complex components (such as zonula occludens-1 [ZO-1], a tight-junction component protein) and nonjunctional complex components (such as urokinase-type plasminogen activator [uPA], a serine protease; cathepsin L, a cysteine protease; alpha2-macroglobulin, a nonspecific protease inhibitor; and cystatin C, a cysteine protease inhibitor) at the time when inter-Sertoli tight junctions were established in vitro. This is an attempt to investigate whether the expression of nonjunctional component genes also correlates with the formation of inter-Sertoli tight junctions in vitro. This is part of an effort to understand the physiologic elements of germ cell movement in the epithelium. Sertoli cells cultured in vitro are known to undergo programmed cell death. To ensure that the changes in target gene expression were not the result of apoptosis, Sertoli cells were cultured in vitro at densities of 0.25, 0.75, and 3 x 10(6) cells/cm2 for up to 7 days on bicameral culture units coated with Matrigel (Collaborative Research) and were assessed by morphologic analysis and agarose gel electrophoresis. It was noted that many of the Sertoli cells cultured at 3 x 10(6) cells/cm2 underwent apoptosis by day 7, in contrast to cultures at 0.25 and 0.75 x 10(6) cells/cm2 illustrating the Sertoli cell number per unit of area may be an important parameter to be considered when studying Sertoli cell function in vitro. Also, it was shown that the expression of ZO-1 increased significantly between days 2 and 3 prior to the establishment of inter-Sertoli tight junctions assessed by transepithelial resistance measurement (TER), which illustrates that ZO-1 can be used as a marker to monitor this cellular event. More interestingly, there was also a transient increase in the expression of uPA and cathepsin L between days 2 and 3 at the time preceding the formation of tight junctions. In Sertoli cells cultured at low density (2 x 10(4) cells/cm2), when a confluent monolayer of cells could not form, there were no changes in the expression of either ZO-1, uPA, or cathepsin L throughout the 7-day culture period. These results show that the establishment of specialized junctions, such as tight junctions between Sertoli cells in vitro, may require the participation of both junctional and nonjunctional complex components.

Animals↗