Sodium-dependent glucose transporter reduces peroxynitrite and cell injury caused by cisplatin in renal tubular epithelial cells

Cisplatin causes nephropathy accompanied by two types of cell death, necrosis and apoptosis, according to its dosage. The mechanisms of necrosis are still unclear. In this study, we examined how high doses of cisplatin induce cell injury and whether a high affinity sodium-dependent glucose transporter (SGLT1) has a cytoprotective function in renal epithelial LLC-PK(1) cells. Cisplatin decreased in transepithelial electrical resistance (TER) and increased in the number of necrotic dead cells in a time dependent manner. Phloridzin, a potent SGLT1 inhibitor, enhanced both TER decrease and increase of necrotic dead cells caused by cisplatin. Cisplatin increased in the intracellular nitric oxide, superoxide anion and peroxynitrite productions. Phloridzin enhanced the peroxynitrite production caused by cisplatin. The intracellular diffusion of ZO-1 and TER decrease caused by cisplatin were inhibited by N-nitro-l-arginine methyl ester, a nitric oxide synthase inhibitor. Protein kinase C was not involved in the cisplatin-induced injury. 5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrinato iron (III) and reduced glutathione, peroxynitrite scavengers, inhibited the cisplatin-induced ZO-1 diffusion, TER decrease, and increase of necrotic dead cells. These results suggest that peroxynitrite is a key mediator in the nephrotoxicity caused by high doses of cisplatin. SGLT1 endogenously carries out the cytoprotective function by the reduction of peroxynitrite production.     

Sodium-dependent glucose transporter reduces peroxynitrite and cell injury caused by cisplatin in renal tubular epithelial cells

Cisplatin causes nephropathy accompanied by two types of cell death, necrosis and apoptosis, according to its dosage. The mechanisms of necrosis are still unclear. In this study, we examined how high doses of cisplatin induce cell injury and whether a high affinity sodium-dependent glucose transporter (SGLT1) has a cytoprotective function in renal epithelial LLC-PK₁ cells. Cisplatin decreased in transepithelial electrical resistance (TER) and increased in the number of necrotic dead cells in a time dependent manner. Phloridzin, a potent SGLT1 inhibitor, enhanced both TER decrease and increase of necrotic dead cells caused by cisplatin. Cisplatin increased in the intracellular nitric oxide, superoxide anion and peroxynitrite productions. Phloridzin enhanced the peroxynitrite production caused by cisplatin. The intracellular diffusion of ZO-1 and TER decrease caused by cisplatin were inhibited by N-nitro-l-arginine methyl ester, a nitric oxide synthase inhibitor. Protein kinase C was not involved in the cisplatin-induced injury. 5,10,15,20-tetrakis-(4-sulfonatophenyl)-porphyrinato iron (III) and reduced glutathione, peroxynitrite scavengers, inhibited the cisplatin-induced ZO-1 diffusion, TER decrease, and increase of necrotic dead cells. These results suggest that peroxynitrite is a key mediator in the nephrotoxicity caused by high doses of cisplatin. SGLT1 endogenously carries out the cytoprotective function by the reduction of peroxynitrite production.     

Activation of a polyvalent cation-sensing receptor decreases magnesium transport via claudin-16

Renal magnesium is mainly reabsorbed by a paracellular pathway in the thick ascending limb of Henle. The expression of claudin-16 increased magnesium transport in Madin-Darby canine kidney (MDCK) cells. Little is known about the regulatory mechanism of magnesium transport via claudin-16. Here we examined the effect of a polyvalent cation-sensing receptor (CaSR) on the intracellular distribution of and transport of magnesium by claudin-16. FLAG-tagged claudin-16 was stably expressed in MDCK cells using a Tet-OFF system. The activation of CaSR by magnesium, calcium, neomycin, and gadolinium did not affect the expression of FLAG-tagged claudin-16, CaSR, or ZO-1, a tight junctional scaffolding protein. These activators decreased the phosphoserine level of FLAG-tagged claudin-16 and the association of FLAG-tagged claudin-16 with ZO-1. The activation of CaSR induced a decrease in PKA activity. Immunofluorescence microscopy revealed that FLAG-tagged claudin-16 is distributed at the cell-cell border under unstimulated conditions, whereas it translocates to the intracellular compartment, mainly lysosome, with the activation of CaSR. In contrast, the distribution of ZO-1 was unaffected by the activation. The expression of FLAG-tagged claudin-16 increased transepithelial electrical resistance (TER) and transepithelial magnesium transport without affecting FITC-dextran (MW 4000) flux. The activation of CaSR decreased TER and magnesium transport, which were recovered by co-treatment with dibutyryl cAMP, a membrane-permeable cAMP analogue. Taken together, CaSR activation may decrease PKA activity, resulting in a decrease in phosphorylated claudin-16, the translocation of claudin-16 to lysosome and a decrease in magnesium reabsorption.     

Activation of a polyvalent cation-sensing receptor decreases magnesium transport via claudin-16

Renal magnesium is mainly reabsorbed by a paracellular pathway in the thick ascending limb of Henle. The expression of claudin-16 increased magnesium transport in Madin-Darby canine kidney (MDCK) cells. Little is known about the regulatory mechanism of magnesium transport via claudin-16. Here we examined the effect of a polyvalent cation-sensing receptor (CaSR) on the intracellular distribution of and transport of magnesium by claudin-16. FLAG-tagged claudin-16 was stably expressed in MDCK cells using a Tet-OFF system. The activation of CaSR by magnesium, calcium, neomycin, and gadolinium did not affect the expression of FLAG-tagged claudin-16, CaSR, or ZO-1, a tight junctional scaffolding protein. These activators decreased the phosphoserine level of FLAG-tagged claudin-16 and the association of FLAG-tagged claudin-16 with ZO-1. The activation of CaSR induced a decrease in PKA activity. Immunofluorescence microscopy revealed that FLAG-tagged claudin-16 is distributed at the cell-cell border under unstimulated conditions, whereas it translocates to the intracellular compartment, mainly lysosome, with the activation of CaSR. In contrast, the distribution of ZO-1 was unaffected by the activation. The expression of FLAG-tagged claudin-16 increased transepithelial electrical resistance (TER) and transepithelial magnesium transport without affecting FITC-dextran (MW 4000) flux. The activation of CaSR decreased TER and magnesium transport, which were recovered by co-treatment with dibutyryl cAMP, a membrane-permeable cAMP analogue. Taken together, CaSR activation may decrease PKA activity, resulting in a decrease in phosphorylated claudin-16, the translocation of claudin-16 to lysosome and a decrease in magnesium reabsorption.     

Pseudomonas aeruginosa quorum sensing molecule N-(3 oxododecanoyl)-l-homoserine lactone disrupts epithelial barrier integrity of Caco-2 cells

Acyl-homoserine lactone (HSL) quorum sensing molecules play an important role in regulation of virulence gene expression in Pseudomonas aeruginosa. Here, we show that 3O-C(12)-HSL can disrupt barrier integrity in human epithelial Caco-2 cells as evidenced by decreased transepithelial electrical resistance (TER), increased paracellular flux, reduction in the expression and distribution of ZO-1 and occludin, and reorganization of F-actin. P. aeruginosa 3O-C(12)-HSL activate p38 and p42/44 kinases, and inhibition of these kinases partly prevented 3O-C(12)-HSL-induced changes in TER, paracellular flux and expression of occludin and ZO-1. These findings demonstrate that P. aeruginosa 3O-C(12)-HSL can modulate tight junction integrity of Caco-2 cells.     

Claudin-1 contributes to the epithelial barrier function in MDCK cells

Tight junctions (TJs) create a paracellular permeability barrier and also act as a fence preventing intermixing of proteins and lipids between the apical and basolateral plasma membranes. Recently, claudin-1 has been identified as an integral membrane protein localizing at TJs, and introduced claudin-1 can form TJ-like networks in fibroblasts. To investigate the function of claudin-1, MDCK cells were transfected with a mammalian expression vector containing myc-tagged mouse claudin-1, and four stable clones were obtained. The myc-tagged claudin-1 precisely colocalized with both occludin and ZO-1 at cell-cell contact sites, indicating that exogenous claudin-1 was properly targeted to the TJs. Immunoblot analysis revealed that overexpression of claudin-1 increased expression of ZO-1 but not of occludin or ZO-2. The barrier functions of these cells were evaluated by transepithelial electrical resistance (TER) and paracellular flux. Claudin-1-expressing cells exhibited about four times higher TER than wild-type MDCK cells. Consistent with the increase of TER, the cells overexpressing claudin-1 showed reduced paracellular flux, estimated at 4 and 40 kD FITC-dextrans. These results suggest that claudin-1 is involved in the barrier function at TJs.     

LXA4 stimulates ZO-1 expression and transepithelial electrical resistance in human airway epithelial (16HBE14o-) cells

Lipoxin A(4) (LXA(4)) is a biologically active eicosanoid produced in human airways that displays anti-inflammatory properties. In cystic fibrosis and severe asthma, LXA(4) production has been reported to be decreased, and, in such diseases, one of the consequences of airway inflammation is disruption of the tight junctions. In the present study, we investigated the possible role of LXA(4) on tight junction formation, using transepithelial electrical resistance (TER) measurements, Western blotting, and immunofluorescence. We observed that exposure to LXA(4) (100 nM) for 2 days significantly increased zonula occludens-1 (ZO-1), claudin-1, and occludin expression at the plasma membrane of confluent human bronchial epithelial 16HBE14o- cells. LXA(4) (100 nM) stimulated the daily increase of the 16HBE14o- cell monolayer TER, and this effect was inhibited by boc-2 (LXA(4) receptor antagonist). LXA(4) also had a rapid effect on ZO-1 immunofluorescence at the plasma membrane and increased TER within 10 min. In conclusion, our experiments provide evidence that LXA(4) plays certainly a new role for the regulation of tight junction formation and stimulation of the localization and expression of ZO-1 at the plasma membrane through a mechanism involving the LXA(4) receptor.     

Effect of acetaminophen on the membrane anchoring of Na+, K+ATPase of rat renal cortical cells

In previous works we reported that the administration of a toxic dose of acetaminophen (APAP) induces acute renal failure (ARF) and promotes changes on Na(+), K(+)ATPase distribution in renal proximal plasma membranes. In the present work, we analyzed if APAP could promote the dissociation of Na(+), K(+)ATPase from its membrane anchorage. The participation of calpain activation was also evaluated. We analyzed the Triton X-100 extractability of Na(+), K(+)ATPase in freshly isolated cortical cell suspensions incubated with different APAP concentrations (0.1, 1, 10 and 100 mM). Both alpha(1) and beta(1) subunits were studied by Western blot. APAP promoted the increment of both subunits abundance in the Triton-soluble fraction. Calpain activation was detected in the membrane fractions of cells incubated with APAP. Incubation with APAP 0.1, 1 and 10 mM did not promote an increment in LDH release compared with controls, while APAP 100 mM promoted an increased LDH release. Our results show that incubation of proximal cells with sublethal and lethal APAP concentrations promotes the detachment of Na(+), K(+)ATPase from its membrane anchoring. Inhibition of calpain activation by SJA 7029 protected against APAP-induced membrane damage but not against APAP-induced increase of the Triton X-100 extractability of Na(+), K(+)ATPase.     

Neutrophil-mediated activation of epithelial protease-activated receptors-1 and -2 regulates barrier function and transepithelial migration

Neutrophil (PMN) infiltration and associated release of serine proteases contribute to epithelial injury during active phases of mucosal disorders such as inflammatory bowel disease. Previous studies have demonstrated that PMN contact with basolateral surfaces of intestinal epithelial cells in the presence of a chemoattractant results in disruption of barrier function even without transmigration. Similarly, serine protease-mediated activation of epithelial protease-activated receptors (PARs) has been shown to increase permeability. In this study, we assessed whether transmigrating PMNs can regulate barrier function through epithelial PAR activation. Transepithelial resistance (TER) decreased significantly after PMN contact with basolateral surfaces of T84 monolayers or after incubation with PMN elastase and proteinase-3, but not cathepsin G. Inhibition of PMN serine proteases, but not selective inhibition of elastase or cathepsin G, prevented the fall in TER induced by PMN contact and blocked PMN transepithelial migration. Basolateral, but not apical, PAR-1 and -2 activation with selective agonists also decreased TER. PAR-1 and -2 were localized intracellularly and in close proximity to lateral surfaces beneath tight junctions, and expression was increased in colonic mucosa from individuals with Crohn's disease. Combined, but not individual, transfection with small interfering RNAs targeted against epithelial PAR-1 and -2, prevented the fall in TER induced by PMN contact. Furthermore, basolateral PAR-1 and -2 activation induced phosphorylation of myosin L chain kinase and regulatory myosin L chain. Lastly, epithelial PAR-1 and -2 knockdown decreased the rate of PMN transepithelial migration. These results suggest that protease-mediated epithelial PAR-1 and -2 activation, by migrating PMNs, induces signaling events that increase epithelial permeability thereby facilitates PMN transepithelial migration.     

Exogenous expression of the amino-terminal half of the tight junction protein ZO-3 perturbs junctional complex assembly

The functional characteristics of the tight junction protein ZO-3 were explored through exogenous expression of mutant protein constructs in MDCK cells. Expression of the amino-terminal, PSD95/dlg/ZO-1 domain-containing half of the molecule (NZO-3) delayed the assembly of both tight and adherens junctions induced by calcium switch treatment or brief exposure to the actin-disrupting drug cytochalasin D. Junction formation was monitored by transepithelial resistance measurements and localization of junction-specific proteins by immunofluorescence. The tight junction components ZO-1, ZO-2, endogenous ZO-3, and occludin were mislocalized during the early stages of tight junction assembly. Similarly, the adherens junction proteins E-cadherin and beta-catenin were also delayed in their recruitment to the cell membrane, and NZO-3 expression had striking effects on actin cytoskeleton dynamics. NZO-3 expression did not alter expression levels of ZO-1, ZO-2, endogenous ZO-3, occludin, or E-cadherin; however, the amount of Triton X-100-soluble, signaling-active beta-catenin was increased in NZO-3-expressing cells during junction assembly. In vitro binding experiments showed that ZO-1 and actin preferentially bind to NZO-3, whereas both NZO-3 and the carboxy-terminal half of the molecule (CZO-3) contain binding sites for occludin and cingulin. We hypothesize that NZO-3 exerts its dominant-negative effects via a mechanism involving the actin cytoskeleton, ZO-1, and/or beta-catenin.     

Entamoeba histolytica disturbs the tight junction complex in human enteric T84 cell layers.

Entamoeba (E.) histolytica trophozoites initiate amebiasis through invasion into the enteric mucosa. It was our aim to understand the molecular interactions between amebic trophozoites and enterocytes during the early steps of invasion. Trophozoites of E. histolytica strain HM1:IMSS were seeded on the apical side of enteric T84 cell layers, which were established on filters in two-compartment culture chambers. Cocultures were analyzed for paracellular permeability by measurement of transepithelial electrical resistance (TER) and for the tight junction proteins ZO-1, ZO-2, occludin, and cingulin by immunocytochemistry and immunoprecipitation. On direct contact with the apical side of the enteric cells, trophozoites caused an increase in paracellular permeability as evidenced by a decrease of TER associated with an increase in [(3)H]mannitol flux. Immunoprecipitation of cocultures revealed dephosphorylation of ZO-2, loss of ZO-1 from ZO-2, and degradation of ZO-1 but less so of ZO-2 and none of occludin or E-cadherin. In conclusion, trophozoite-associated increase in paracellular permeability of enteric cell layers is ascribed to disturbance of the molecular organization of tight junction proteins.     

Activation of dynein 1 adenosine triphosphatase by organic solvents and by Triton X-100

The presence of low concentrations of methanol or isopropyl alcohol (2-5%, v/v) in the assay medium stabilizes the latency of dynein 1 from sea urchin sperm flagella, with about a 50% decrease in ATPase level compared to that in the absence of solvent. Somewhat higher concentrations (10-20%, v/v) of these solvents in the assay give a 5-10-fold activation of ATPase activity. Dioxane, formamide, and dimethylformamide, on the other hand, always activate the ATPase activity, with a 5-10-fold increase observed at about 15% (v/v). The activation of latent ATPase activity by solvents is reversible for short exposures, especially in the presence of ATP and at low temperature, but the activation becomes irreversible upon more prolonged exposure. The rate constant for irreversible activation by 16% methanol at 21 degrees C is 0.08 min-1, compared to rates of 0.44 and 0.02 min-1 for activation by 0.05% Triton X-100 at 21 and 0 degree C, respectively. The slowness of this reversible activation induced by methanol and by Triton X-100 suggests that it is the result of large-scale conformational changes in the structure of the dynein. However, the activation by methanol occurs without the dissociation of the alpha and beta subunits of dynein that is observed with Triton X-100. The presence of 1 mM MgATP, or of 100 microM MgATP and 10 microM vanadate substantially protects latent dynein from activation by 0.05% Triton X-100.     

Heat-induced aggregation of XRCC5 (Ku80) in nontolerant and thermotolerant cells.

XRCC5 (also known as Ku80) is a component of the DNA-dependent protein kinase (DNA-PK), existing as a heterodimer with G22P1 (also known as Ku70). DNA-PK is involved in the nonhomologous end-joining (NHEJ) pathway of DNA double-strand break (DSB) repair, and kinase activity is dependent upon interaction of the Ku subunits with the resultant DNA ends. Nuclear XRCC5 is normally extractable with non-ionic detergent; it is found in the soluble cytoplasmic fraction after nuclear isolation with Triton X-100. In this study, we found that heating at 45.5 degrees C causes a decreased extractability of XRCC5 from the nuclei of human U-1 melanoma or HeLa cells. Such decreases in extractability are indicative of protein aggregation within nuclei. Recovery of extractability of XRCC5 to that of unheated control cells was observed after incubation at 37 degrees C after heat shock. The decrease in extractability and the kinetics of recovery were dependent on dose, although the decrease in extractability reached a plateau after heating for 15 min or more. Thermotolerant U-1 cells also showed decreased extractability of XRCC5, but to a lesser degree compared to nontolerant cells. When a comparable initial reduction of extractability of XRCC5 was induced in both thermotolerant and nontolerant cells, the kinetics of recovery was nearly identical. The kinetics of recovery of the extractability of XRCC5 was different from that of total nuclear protein in nontolerant cells; recovery of extractability of XRCC5 occurred faster initially and returned to the level in unheated cells faster than total nuclear protein. Similar results were obtained for thermotolerant cells, with differences between the initial recovery of the extractability of XRCC5 and total protein being particularly evident after longer heating times. Heat has been shown to inactivate XRCC5. We speculate that inactivation of XRCC5 after heat shock results from protein aggregation, and that changes in XRCC5 may, in part, lead to inhibition of DSB repair through inactivation of the NHEJ pathway.     

Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation

Tight junctions serve as the rate-limiting barrier to passivemovement of hydrophilic solutes across intestinal epithelia. Afteractivation of Na⁺-glucosecotransport, the permeability of intestinal tight junctions isincreased. Because previous analyses of this physiological tightjunction regulation have been restricted to intact mucosae, dissectionof the mechanisms underlying this process has been limited. Tocharacterize this process, we have developed a reductionist modelconsisting of Caco-2 intestinal epithelial cells transfected with theintestinal Na⁺-glucosecotransporter, SGLT1. Monolayers of SGLT1 transfectants demonstratephysiological Na⁺-glucosecotransport. Activation of SGLT1 results in a 22 ± 5% fall intransepithelial resistance (TER) (P < 0.001). Similarly, inactivation of SGLT1 by addition of phloridzinincreases TER by 24 ± 2% (P < 0.001). The increased tight junction permeability is size selective,with increased flux of small nutrient-sized molecules, e.g., mannitol,but not of larger molecules, e.g., inulin. SGLT1-dependent increases intight junction permeability are inhibited by myosin light-chain kinaseinhibitors (20 µM ML-7 or 40 µM ML-9), suggesting that myosinregulatory light-chain (MLC) phosphorylation is involved in tightjunction regulation. Analysis of MLC phosphorylation showed a 2.08-foldincrease after activation of SGLT1 (P < 0.01), which was inhibited by ML-9(P < 0.01). Thus monolayersincubated with glucose and myosin light-chain kinase inhibitors arecomparable to monolayers incubated with phloridzin. ML-9 also inhibitsSGLT1-mediated tight junction regulation in small intestinal mucosa(P < 0.01). These data demonstrate that epithelial cells are the mediators of physiological tight junctionregulation subsequent to SGLT1 activation. The intimate relationshipbetween tight junction regulation and MLC phosphorylation suggests thata critical step in regulation of epithelial tight junction permeabilitymay be myosin ATPase-mediated contraction of the perijunctionalactomyosin ring and subsequent physical tension on the tight junction.

     

Connexin 43 interacts with zona occludens-1 and -2 proteins in a cell cycle stage-specific manner

Gap junction channels play an important role in cell growth control, secretion and embryonic development. Gap junctional communication and channel assembly can be regulated by protein-protein interaction with kinases and phosphatases. We have utilized tandem mass spectrometry (MS/MS) sequence analysis as a screen to identify proteins from cell lysates that interact with the C-terminal cytoplasmic region of connexin 43 (Cx43). MS/MS analysis of tryptic fragments yielded several proteins including zona occludens-1 (ZO-1), a structural protein previously identified to interact with Cx43, and ZO-2, a potential novel interacting partner. We confirmed the interaction of ZO-2 with Cx43 by using a combination of fusion protein "pull down," co-immunoprecipitation, and co-localization experiments. We show that the C-terminal region of Cx43 is necessary for interaction with the PDZ2 domain of ZO-2. Far Western analysis revealed that ZO-2 can directly bind to Cx43 independent of other interacting partners. Immunofluorescence studies indicate that both ZO-1 and ZO-2 can co-localize with Cx43 within the plasma membrane at apparent gap junctional structures. We examined Cx43 interaction with ZO-1 and ZO-2 at different stages of the cell cycle and found that Cx43 had a strong preference for interaction with ZO-1 during G0, whereas ZO-2 interaction occurred approximately equally during G0 and S phases. Since essentially all of the Cx43 in G0 cells is assembled into Triton X-100-resistant junctions, Cx43-ZO-1 interaction may contribute to their stability.     

Checkpoint Kinase 1 Activation Enhances Intestinal Epithelial Barrier Function via Regulation of Claudin-5 Expression

Several stressors are known to influence epithelial tight junction (TJ) integrity, but the association between DNA damage and TJ integrity remains unclear. Here we examined the effects of daunorubicin and rebeccamycin, two anti-tumor chemicals that induce DNA damage, on TJ integrity in human intestinal epithelial cells. Daunorubicin and rebeccamycin dose-dependently enhanced transepithelial electrical resistance (TER) and decreased flux of the 4 kDa FITC-dextran in Caco-2 cell monolayer. Daunorubicin- or rebeccamycin-induced enhancement of the TJ barrier function partly rescued attenuation of the barrier function by the inflammatory cytokines TNF-α and IFN-γ. Daunorubicin and rebeccamycin increased claudin-5 expression and the product was distributed in the actin cytoskeleton fraction, which was enriched with TJ proteins. Caffeine, which is an inhibitor of ataxia telangiectasia mutated protein (ATM) and ataxia telangiectasia mutated and Rad3-related protein (ATR), and the Chk1 inhibitor inhibited the TER increases induced by daunorubicin and rebeccamycin, whereas a Chk2 inhibitor did not. Treatment with Chk1 siRNA also significantly inhibited the TER increases. Induction of claudin-5 expression was inhibited by Chk1 inhibitor and by siRNA treatment. Our results suggest that Chk1 activation by daunorubicin and rebeccamycin induced claudin-5 expression and enhanced TJ barrier function in Caco-2 cell monolayer, which suggests a link between DNA damage and TJ integrity in the human intestine.     

Small Synthetic Peptides Homologous to Segments of the First External Loop of Occludin Impair Tight Junction Resealing

This study shows that resealing of opened tight junctions (TJs) is impaired by interaction with oligopeptides homologous to the external domain of chick occludin. The experiments were carried out with confluent A6 cell monolayers grown on collagen supports under stable transepithelial electrical resistance (TER). The monolayers were bathed on the apical side with a 75 mm KCl solution and on the basolateral side by NaCl-Ringer's solution. TJ opening was induced by basolateral Ca²⁺ removal and was characterized by a marked drop of TER. The reintroduction of Ca²⁺ triggered junction resealing as indicated by an elevation of TER to control values. Custom-made peptides SNYYGSGLSY (corresponding to the residues 100 to 109) and SNYYGSGLS (residues 100 to 108), homologous to segments of the first external loop of chick occludin molecule, impaired junction resealing when the peptides were included in the apical bathing fluid (concentrations in the range of 0.5 to 1.5 mg/ml). Peptide removal from the apical solution usually triggered a slow recovery of TER, indicating a slow recovery of the TJ seal. Changes in localization of ZO-1, a cytoplasmic protein that underlies the membrane at the TJs, were evaluated immunocytochemically following Ca²⁺ removal and reintroduction. The presence or absence of the oligopeptides showed no influence on the pattern of change of ZO-1 localization. These observations support the hypothesis that the TJ seal results from the interaction of specific homologous segments of occludin on the surface of adjacent cells. Additionally, our results show that small peptides homologous to segments of the occludin first external loop can be used as specific reagents to manipulate the permeability of tight junctions. Received: 4 December 1998/Revised: 22 January 1999     

Requirement for Ras and phosphatidylinositol 3-kinase signaling uncouples the glucocorticoid-induced junctional organization and transepithelial electrical resistance in mammary tumor cells.

In Con8 rat mammary epithelial tumor cells, the synthetic glucocorticoid dexamethasone stimulates the remodeling of the apical junction (tight and adherens junctions) and the transepithelial electrical resistance (TER), which reflects tight junction sealing. Indirect immunofluorescence revealed that dexamethasone induced the recruitment of endogenous Ras and the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase to regions of cell-cell contact, concurrently with the stimulation of TER. Expression of dominant-negative RasN17 abolished the dexamethasone stimulation in TER, whereas, dexamethasone induced the reorganization of tight junction and adherens junction proteins, ZO-1 and beta-catenin, as well as F-actin, to precise regions of cell-cell contact in a Ras-independent manner. Confocal microscopy revealed that RasN17 and the p85 regulatory subunit of PI 3-kinase co-localized with ZO-1 and F-actin at the tight junction and adherens junction, respectively. Treatment with either of the PI 3-kinase inhibitors, wortmannin or LY294002, or the MEK inhibitor PD 098059, which prevents MAPK signaling, attenuated the dexamethasone stimulation of TER without affecting apical junction remodeling. Similar to dominant-negative RasN17, disruption of both Ras effector pathways using a combination of inhibitors abolished the glucocorticoid stimulation of TER. Thus, the glucocorticoiddependent remodeling of the apical junction and tight junction sealing can be uncoupled by their dependence on Ras and/or PI 3-kinase-dependent pathways, implicating a new role for Ras and PI 3-kinase cell signaling events in the steroid control of cell-cell interactions.     

Structural and Functional Regulation of Tight Junctions by RhoA and Rac1 Small GTPases

Tight junctions (TJ) govern ion and solute diffusion through the paracellular space (gate function), and restrict mixing of membrane proteins and lipids between membrane domains (fence function) of polarized epithelial cells. We examined roles of the RhoA and Rac1 GTPases in regulating TJ structure and function in MDCK cells using the tetracycline repressible transactivator to regulate RhoAV14, RhoAN19, Rac1V12, and Rac1N17 expression. Both constitutively active and dominant negative RhoA or Rac1 perturbed TJ gate function (transepithelial electrical resistance, tracer diffusion) in a dose-dependent and reversible manner. Freeze-fracture EM and immunofluoresence microscopy revealed abnormal TJ strand morphology and protein (occludin, ZO-1) localization in RhoAV14 and Rac1V12 cells. However, TJ strand morphology and protein localization appeared normal in RhoAN19 and Rac1N17 cells. All mutant GTPases disrupted the fence function of the TJ (interdomain diffusion of a fluorescent lipid), but targeting and organization of a membrane protein in the apical membrane were unaffected. Expression levels and protein complexes of occludin and ZO-1 appeared normal in all mutant cells, although ZO-1 was more readily solubilized from RhoAV14-expressing cells with Triton X-100. These results show that RhoA and Rac1 regulate gate and fence functions of the TJ, and play a role in the spatial organization of TJ proteins at the apex of the lateral membrane.