Increase In Transepithelial Electrical Resistance Research Articles

IQGAP1 controls tight junction formation through differential regulation of claudin recruitment.

IQGAP1 is a scaffolding protein previously implicated in adherens junction formation. However, its role in the establishment or maintenance of tight junctions (TJs) has not been explored. We hypothesized that IQGAP1 could regulate TJ formation by modulating the expression and/or localization of junctional proteins, and we systematically tested this hypothesis in the model Madin-Darby canine kidney (MDCK) cell line. We find that IQGAP1 silencing enhances a transient increase in transepithelial electrical resistance (TER) observed during the early stages of TJ formation (Cereijido et al., 1978). Quantitative microscopy and biochemical experiments suggest that this effect of IQGAP1 on TJ assembly is accounted for by reduced expression and TJ recruitment of claudin 2, and increased TJ recruitment of claudin 4. Furthermore, we show that IQGAP1 also regulates TJ formation through its interactor CDC42, because IQGAP1 knockdown increases the activity of the CDC42 effector JNK and dominant-negative CDC42 prevents the increase in TER caused by IQGAP1 silencing. Hence, we provide evidence that IQGAP1 modulates TJ formation by a twofold mechanism: (1) controlling the expression and recruitment of claudin 2 and recruitment of claudin 4 to the TJ, and (2) transient inhibition of the CDC42-JNK pathway.

P-183 Kindlin 1and Kindlin 2 Regulate Adhesion and Migration of Colonic Epithelial Cells

The Intestinal epithelial barrier plays an important role in the pathophysiology of inflammatory bowel disease (IBD). Integrity and barrier properties of the intestinal epithelium are determined by specialized adhesive structures such as intercellular junctions and focal adhesions, which respectively mediate cell-cell and cell-matrix interactions. Disassembly of epithelial junctions and matrix adhesion mediates disruption of the gut barrier during acute inflammation, whereas restoration of these adhesion structures is critical for epithelial restitution. Kindlins are unique cytosolic adaptor proteins that are enriched in both epithelial junctions and matrix adhesions. They have multiple binding partners including major classes of integrins and different cytoskeletal regulators. Importantly, mutations in the kindlin-1 gene result in Kindler syndrome, a genetic disorder associated with severe colitis, and deletion of kindlin-2 gene in mice is embryonically lethal. Despite these dramatic phenotypes caused by kindlin dysfunctions, little is known regarding the role of these adaptor proteins in regulation of the intestinal mucosal barrier disruption and restitution. This study was performed using a well-differentiated clone of HT29 human colonic epithelial cell line (HT29cF8). Transient downregulation of kindlin isoforms was achieved using RNA interference. Stable overexpression of kindlins 1 and 2 was achieved using a lentiviral expression system. Cell lines were cultured either on collagen-coated, permeable polycarbonate filters (0.4 mm pore size), or on 6-well plastic plates, for permeability, adhesion and motility assays. Barrier function of epithelial cell monolayers was determined via transepithelial electrical resistance (TEER) measurements Focal adhesions and cytoskeleton were visualized by immunofluorescence labeling and confocal microscopy. Overexpression of either kindlin 1 and/or kindlin 2 resulted in a significant increase in TEER, while siRNA-mediated knockdown of either isoform caused a substantial increase in paracellular permeability. Interestingly, both overexpression and depletion of kindlin 1 attenuated collective cell migration as determined by wound closure assay. By contrast, downregulation of kindlin 2 expression did not affect cell motility. Analysis of cell adhesion and spreading on a collagen matrix revealed significant attenuation of matrix adhesion and spreading of kindlin 1 and kindlin 2 overexpressing cells. These effects correlate well with a marked reduction of the phosphorylated form of focal adhesion protein, paxillin. Additionally, kindlin 1 overexpression regulated an increased level of non-muscle myosin IIB, which is indicative of increased contractile forces. Our data highlights kindlins as important regulators of barrier properties and restitution of human intestinal epithelium.

Investigation of effects of Giardia duodenalis on transcellular and paracellular transport in enterocytes using in vitro Ussing chamber experiments.

The mechanisms by which different genotypes of Giardia duodenalis result in different symptoms remain unresolved. In particular, we lack detailed knowledge on which transport mechanisms (transcellular or paracellular) are affected by different Giardia isolates. Using horse radish peroxidase (HRP) and creatinine as transcellular and paracellular probes, respectively, we developed a robust assay that can be used with an Ussing chamber to investigate epithelial transport, as well as short-circuit current as an indicator of net ion transport. We investigated 2 Giardia isolates, both Assemblage A, one a lab-adapted strain and the other a field isolate. Results indicate that products from sonicated Giardia trophozoites increase both transcellular and paracellular transport. A non-significant increase in transepithelial electrical resistance (TEER) and short-circuit current were also noted. The paracellular transport was increased significantly more in the field isolate than in the lab-adapted strain. Our results indicate that while both transcellular and paracellular transport mechanisms may be increased following exposure of cells to Giardia trophozoite sonicate, perhaps by inducing non-specific increases in cellular traffic, it is important that in vitro studies of Giardia pathophysiology are conducted with different Giardia isolates, not just lab-attenuated strains.

Elevated placental adenosine signaling contributes to the pathogenesis of preeclampsia

Purpose: Dephosphorylation of the myosin II regulatory light chain (MLC) promotes barrier integrity of cellular monolayers through relaxation of the actin cytoskeleton. This study has investigated the influence of adenosine (ADO) on MLC phosphorylation in cultured bovine corneal endothelial cells (BCEC).Methods: MLC phosphorylation was assessed by urea-glycerol gel electrophoresis and immunoblotting. Elevation of cAMP in response to agonists of A2b receptors (subtype of P1 purinergic receptors) was confirmed by phosphorylation of the cAMP response element binding protein (CREB), which was determined by Western blotting. Activation of MAP kinases (i.e. activated ERK1 and ERK2) was assessed by Western blotting to examine their influence on MLC phosphorylation. Transepithelial electrical resistance (TER) of cells grown on porous filters was measured to assess the altered barrier integrity.Results: Exposure to ADO (200 μm; 30 min) and N-ethyl (carboxamido) adenosine (NECA; 50 μm; 30 min), known agonists of A2b receptors, induced phosphorylation of CREB similar to forskolin (FSK, 20 μm; 30 min), a direct activator of adenylate cyclase. Exposure to ADO, NECA, and FSK led to dephosphorylation of MLC by 51, 40, and 47%, respectively. ADO-induced dephosphorylation was dose-dependent with as much as 31% dephosphorylation at 1 μm ADO. CGS-21680, a selective A2a agonist, neither induced MLC dephosphorylation nor CREB phosphorylation. ADO phosphorylated MAP kinases which could be prevented by exposure to the MAP kinase-specific inhibitor, U0126 (10 μM). NECA and FSK also induced ERK1 and ERK2 activation similar to ADO. Exposure to U0126 inhibited MLC phosphorylation under basal conditions by 17%. ADO-induced MLC dephosphorylation was enhanced by a simultaneous exposure to U0126 (25% increase in dephosphorylation). Exposure to ADO caused an increase in TER from 17 to 22 ohms cm2.Conclusions: (1) CREB phosphorylation in response to ADO and NECA, which indicates activation of the cAMP-PKA axis, suggests expression of A2b receptors in BCEC. (2) ERK1 and ERK2, activated by cAMP and A2b receptors, promote MLC phosphorylation. However, the net result of cAMP elevation is MLC dephosphorylation, presumably because the competing pathways involving inactivation of MLCK and/or ROCK are dominant (Rho-associated coiled coil-containing protein kinase or Rho kinase). (3) Consistent with MLC dephosphorylation, exposure to ADO increases TER, which suggests increased barrier integrity.

EGF regulates claudin-2 and -4 expression through Src and STAT3 in MDCK cells.

Epidermal Growth Factor (EGF) is a key regulator of epithelial paracellular permeability, a property that depends on tight junctions (TJ) and can be evaluated through the measurement of the transepithelial electrical resistance (TER). EGF increases the TER of MDCK monolayers by inducing ERK1/2-dependent downregulation of claudin-2 (CLDN-2) and upregulation of claudin-4 (CLDN-4). Because either increments or decrements in TER often involve Src activation and epithelial cell differentiation occasionally depends on STAT3, here we investigated whether EGF might control CLDN-2 downregulation and CLDN-4 upregulation through those proteins. We found that EGF induces Src activation necessary for the reduction of CLDN-2 at the TJ, the degradation of this CLDN, the reduction of the cellular levels of its mRNA and the resulting increase of TER. EGF-induced changes on CLDN-2 protein and mRNA also depend on STAT3 activity. This growth factor increases the levels of STAT3 phosphorylated at Y705 in the nucleus, a process that depends on Src activation. Interestingly, Src and STAT3 activation do not exclusively mediate the EGF-induced downregulation of CLDN-2, but they are also implicated in the EGF-induced CLDN-4 transcription, translation, and exocytic fusion into TJ. Our results indicate that EGF controls the levels of CLDN-2 and -4 proteins and mRNAs through Src and STAT3 activity.

Doxorubicin에 의한 치밀결합 강화 및 MMPs의 활성 억제를 통한 LNCap 전립선 암세포의 이동성 및 침윤성의 억제

본 연구에서는 anthracycline 계열 항암항생제인 doxorubicin의 암세포 전이 억제 여부를 LNCap 전립선 암세포를 이용하여 이동성 및 침윤성 억제 효능 측면에서 조사하였다. 본 연구의 결과에 의하면 doxorubicin은 LNCap 세포의 이동성과 침윤성을 현저하게 억제시켰으며, matrix metalloproteinase (MMP)-2 및 -9의 발현과 활성을 저해함과 동시에 tissue inhibitor of metalloproteinase (TIMP)-1 및 -2의 발현은 증가시켰다. Doxorubicin은 또한 tight junctions (TJs)의 전기적 저항성을 증대시켰으며, 이는 TJs의 주요 구성인자인 claudin family 인자들의 발현 억제와 연관성이 있었다. 따라서 LNCap 세포에서 doxorubicin에 의한 전이의 억제에는 최소한 TJ의 견고성 증대와 MMPs의 활성 억제가 관여할 것으로 추정된다. Doxorubicin (trade name adriamycin), an anthracycline antibiotic, is commonly used in the treatment of a wide range of cancers, including hematological malignancies, many types of carcinoma, and soft tissue sarcomas. It is closely related to the natural product daunomycin, and like all anthracyclines, it works by intercalating DNA. Its most serious adverse effect is life-threatening heart damage. Its anti-metastatic mechanisms in human prostate carcinomas are not fully understood. In this study, we used LNCap human prostate carcinoma cells to investigate the inhibitory effects of doxorubicin on cell motility and invasion, two critical cellular processes that are often deregulated during metastasis. Doxorubicin treatment inhibited cell migration and invasiveness of LNCap cells without showing any toxicity. Doxorubicin treatment also suppressed the activity and expression of matrix metalloproteinase (MMP)-2 and MMP-9, which were associated with up-regulated expression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in LNCap cells. Doxorubicin treatment also attenuated the expression levels of claudin family members (claudin-1, -2,-3 and -4), major components of tightening of tight junctions (TJs) and increased the tightening of TJs, as demonstrated by an increase in transepithelial electrical resistance. The present findings demonstrate that doxorubicin reduces the migration and invasion of prostate carcinomas LNCap cells by modulating the activity of TJs and MMPs.

Evidence for a role of claudin 2 as a proximal tubular stress responsive paracellular water channel

Claudins are the major proteins of the tight junctions and the composition of claudin subtypes is decisive for the selective permeability of the paracellular route and thus tissue specific function. Their regulation is complex and subject to interference by several factors, including oxidative stress. Here we show that exposure of cultured human proximal tubule cells (RPTEC/TERT1) to the immunosuppressive drug cyclosporine A (CsA) induces an increase in transepithelial electrical resistance (TEER), a decrease in dome formation (on solid growth supports) and a decrease in water transport (on microporous growth supports). In addition, CsA induced a dramatic decrease in the mRNA for the pore forming claudins -2 and -10, and the main subunits of the Na+/K+ ATPase. Knock down of claudin 2 by shRNA had no discernable effect on TEER or dome formation but severely attenuated apical to basolateral water reabsorption when cultured on microporous filters. Generation of an osmotic gradient in the basolateral compartment rescued water transport in claudin 2 knock down cells. Inhibition of Na+/K+ ATPase with ouabain prevented dome formation in both cell types. Taken together these results provide strong evidence that dome formation is primarily due to transcellular water transport following a solute osmotic gradient. However, in RPTEC/TERT1 cells cultured on filters under iso-osmotic conditions, water transport is primarily paracellular, most likely due to local increases in osmolarity in the intercellular space. In conclusion, this study provides strong evidence that claudin 2 is involved in paracellular water transport and that claudin 2 expression is sensitive to compound induced cellular stress.

Toll-like receptor 2 regulates the barrier function of human bronchial epithelial monolayers through atypical protein kinase C zeta, and an increase in expression of claudin-1

We investigated the role of Toll-like receptor (TLR) 2 in maintaining the integrity of the airway epithelial barrier using the human bronchial epithelial cell line Calu-3. Activation of TLR2 by its ligands, Pam3CysSK4 and Peptidoglycan showed a concentration dependent increase in epithelial barrier function, as measured by transepithelial electrical resistance (TEER). This was confirmed by a decrease in paracellular flux of fluorescein sodium. This TLR2 induced increase in TEER was significantly reduced by pretreatment with polyclonal anti-human TLR2-neutralizing antibody. TLR2 stimulation in Calu-3 cell monolayers resulted in an increased expression of the tight junction proteins claudin-1 and ZO-1, and a decreased expression of occludin, at both the mRNA and protein levels. A pseudosubstrate inhibitor to PKCζ significantly prevented the TLR2 mediated increase in barrier function. It also prevented the increase in claudin-1 in a concentration dependent manner up to 1 µM. TLR2 stimulation led to an increase in phosphorylation of atypical PKC ζ, which was prevented by the pseudosubstrate inhibitor in a concentration dependent manner. Taken together, our observations support a model whereby increased tight junction barrier function induced by activation of TLR2 occurs through increased expression of claudin-1, and through modulation of PKC ζ activity.

Regulation of RPE barrier function by all-trans retinoic acid in myopia

To investigate the effect of all-trans retinoic acid (atRA) on the barrier function of myopia retinal pigment epithelium (RPE), primary RPE cells from guinea pigs were cultured on the filters as monolayer and treated with 10−9M atRA. Epithelial permeability was measured by transepithelial electrical resistance (TER) and HRP concentration, western blot was used to observe the expression of intercellular junction-associated proteins. Treatment of primary cultures of confluent myopia RPE cells with atRA resulted in a progressive increase in TER and a progressive decrease of HRP concentration in the basolateral compartment. The expression of E-cadherin, Occludin and Claudin-1 proteins were up-regulated after treatment with atRA for 6h to 48h. We concluded that atRA could promote the epithelial barrier function of myopia RPE monolayer possibly by regulating expression of intercellular junction-associated proteins.

Immunocompetent 3D Model of Human Upper Airway for Disease Modeling and In Vitro Drug Evaluation

The development of more complex in vitro models for the assessment of novel drugs and chemicals is needed because of the limited biological relevance of animal models to humans as well as ethical considerations. Although some human-cell-based assays exist, they are usually 2D, consist of single cell type, and have limited cellular and functional representation of the native tissue. In this study, we have used biomimetic porous electrospun scaffolds to develop an immunocompetent 3D model of the human respiratory tract comprised of three key cell types present in upper airway epithelium. The three cell types, namely, epithelial cells (providing a physical barrier), fibroblasts (extracellular matrix production), and dendritic cells (immune sensing), were initially grown on individual scaffolds and then assembled into the 3D multicell tissue model. The epithelial layer was cultured at the air–liquid interface for up to four weeks, leading to formation of a functional barrier as evidenced by an increase in transepithelial electrical resistance (TEER) and tight junction formation. The response of epithelial cells to allergen exposure was monitored by quantifying changes in TEER readings and by assessment of cellular tight junctions using immunostaining. It was found that epithelial cells cocultured with fibroblasts formed a functional epithelial barrier at a quicker rate than single cultures of epithelial cells and that the recovery from allergen exposure was also more rapid. Also, our data show that dendritic cells within this model remain viable and responsive to external stimulation as evidenced by their migration within the 3D construct in response to allergen challenge. This model provides an easy to assemble and physiologically relevant 3D model of human airway epithelium that can be used for studies aiming at better understanding lung biology, the cross-talk between immune cells, and airborne allergens and pathogens as well as drug delivery.

Glucocorticoids regulate barrier function and claudin expression in intestinal epithelial cells via MKP-1

Barrier dysfunction is pivotal to the pathogenesis of inflammatory bowel diseases (IBD) and collagenous colitis. Glucocorticoids restore barrier function in Crohn's disease, but whether this reflects attenuated inflammation or an epithelial-specific action has not yet been addressed. Using filter-grown Caco-2 monolayers as an in vitro model of the intestinal epithelial barrier, we observed that glucocorticoids induced a time- and dose-dependent increase in transepithelial electrical resistance (TEER) in a glucocorticoid receptor-dependent manner without altering flux of larger solutes or changing principal tight junction architecture. This was accompanied by reduced paracellular cation flux, reduced expression of the pore-forming tight junction component claudin-2, and upregulation of the sealing tight junction protein claudin-4. In contrast, expression of occludin, claudin-1, -7, or -8 was not altered. Dexamethasone increased expression and activity of MAPK phosphatase-1 and inhibition of this phosphatase prevented the glucocorticoid-induced changes in TEER and claudin expression, whereas inhibiting p38 or MEK1/2 was not sufficient to replicate the glucocorticoid effects. Upon exposure to IFN-γ, TNF-α, or IL-1β, TEERs declined in dexamethasone-treated cells but remained consistently higher than in cells not receiving glucocorticoids. Treatment with IFN/TNF resulted in an upregulation of claudin-2 that was significantly attenuated by dexamethasone, whereas increased claudin-2 expression upon IL-1β stimulation was not affected by glucocorticoids. Taken together, barrier augmentation might represent a previously unrecognized mechanism of action, potentially contributing to the therapeutic efficacy of glucocorticoids in IBD and collagenous colitis.

Inhibiting Invasion into Human Bladder Carcinoma 5637 Cells with Diallyl Trisulfide by Inhibiting Matrix Metalloproteinase Activities and Tightening Tight Junctions

Diallyl trisulfide (DATS), an organosulfur compound in garlic, possesses pronounced anti-cancer potential. However, the anti-invasive mechanism of this compound in human bladder carcinoma is not fully understood. In this study, we evaluated the anti-invasive effects of DATS on a human bladder carcinoma (5637) cell line and investigated the underlying mechanism. The results indicated that DATS suppressed migration and invasion of 5637 cells by reducing the activities and expression of matrix metalloproteinase (MMP)-2 and MMP-9 at both the protein and mRNA levels. DATS treatment up-regulated expression of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2 in 5637 cells. The inhibitory effects of DATS on invasiveness were associated with an increase in transepithelial electrical resistance and repression of the levels of claudin family members. Although further studies are needed, our data demonstrate that DATS exhibits anti-invasive effects in 5637 cells by down-regulating the activity of tight junctions and MMPs. DATS may have future utility in clinical applications for treating bladder cancer.

글루타민 결핍에 따른 Tight Junction 및 MMPs 활성 조절을 통한 전립선 암세포의 침윤 억제 현상

암세포를 포함한 생체 내 빠른 분열을 요구하는 세포 집단에서 세포 내 구성요소 및 에너지원으로서 글루타민의 요구량이 증대되지만, 종양세포의 글루타민 의존적 대사작용에 관한 기전은 여전히 잘 알려진 바 없다. 본 연구에서는 LnCaP 전립선 암세포의 이동성 및 침윤성에 미치는 글루타민 결핍효능을 조사하였다. 본 연구의 결과에 의하면 LnCaP 세포에서 글루타민 결핍에 의하여 세포의 이동성 및 세포의 침윤성이 현저하게 억제되었으며, 이러한 이동성 및 침윤성 억제는 TIMPs의 발현 증대에 의한 MMPs의 발현 감소 및 그들의 효소적 활성 저하와 연관성이 있었다. 또한 글루타민이 결핍된 조건에서 배양된 LnCaP 세포에서 TER의 현저한 증가가 관찰되었는데, 이는 TJs의 조절인자인 claudin family 발현의 차단에 의한 것으로 생각되어진다. 본 연구의 결과에 의하면 암세포의 증식에서 글루타민의 결핍은 TJ의 결합력 증대와 MMPs의 활성을 저하시킴으로써 암세포 전이에 가장 기본적인 과정인 암세포의 이동성과 침윤성을 억제시킬 수 있을 것으로 생각된다.

Strain-Dependent Augmentation of Tight-Junction Barrier Function in Human Primary Epidermal Keratinocytes by Lactobacillus and Bifidobacterium Lysates

In this study, we investigated whether probiotic lysates can modify the tight-junction function of human primary keratinocytes. The keratinocytes were grown on cell culture inserts and treated with lysates from Bifidobacterium longum, Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus fermentum, or Lactobacillus rhamnosus GG. With the exception of L. fermentum (which decreased cell viability), all strains markedly enhanced tight-junction barrier function within 24 h, as assessed by measurements of transepithelial electrical resistance (TEER). However, B. longum and L. rhamnosus GG were the most efficacious, producing dose-dependent increases in resistance that were maintained for 4 days. These increases in TEER correlated with elevated expression of tight-junction protein components. Neutralization of Toll-like receptor 2 abolished both the increase in TEER and expression of tight-junction proteins induced by B. longum, but not L. rhamnosus GG. These data suggest that some bacterial strains increase tight-junction function via modulation of protein components but the different pathways involved may vary depending on the bacterial strain.

Expression of claudins -2 and -4 and cingulin is coordinated with the start of stratification and differentiation in corneal epithelial cells: retinoic acid reversibly disrupts epithelial barrier

SummaryAlthough tight junctions (TJ) have been extensively studied in simple epithelial cells, it is still unknown whether their organization is coupled to cell differentiation in stratified epithelia. We studied the expression of TJ in RCE1(5T5) cells, an in vitro model which mimics the sequential steps of rabbit corneal epithelial differentiation. RCE1(5T5) cells expressed TJ components which were assembled once cells constituted differentiated epithelia, as suggested by the increase of transepithelial electrical resistance (TER) which followed a similar kinetic to the expression of the early differentiation marker Pax-6. TJ were functional as indicated by the establishment of an epithelial barrier nonpermeable to ruthenium red or a biotin tracer. In immunostaining experiments, TJ were located at the superficial cells from the suprabasal layers; Western blot and RT-PCR suggested that TJ were composed of claudins (cldn) -1, -2, -4, cingulin (cgn), occludin (ocln) and ZO-1. Semi-quantitative RT-PCR and TER measurements showed that TJ became organized when cells began to form a 3–5 layers stratified epithelium; TER increased once cells reached confluence, with a time course comparable to the raise in the expression of cgn, cldn-2 and -4. Nevertheless, cldn-1, -2, ZO-1 and ocln were present in the cells from the beginning of cultivation, suggesting that TER increases mainly depend on TJ assembly. While EGF increased epithelial barrier strength, retinoic acid disrupted it, increasing paracellular flux about 2-fold; this effect was concentration dependent and completely reversible. Our results suggest that TJ assembly is tightly linked to the expression of corneal epithelial terminal phenotype.

Bufalin prevents the migration and invasion of T24 bladder carcinoma cells through the inactivation of matrix metalloproteinases and modulation of tight junctions

Bufalin, a cardiotonic steroid extracted from toad venom, has generally been known to possess a range of biological activities; however, only a few studies have reported the anti-metastatic activity of bufalin. In the present study, we investigated the inhibitory effects of bufalin on cell migration and invasion, two critical cellular processes that are often deregulated during metastasis, using the human bladder cancer cell line, T24. Within the concentration range that was not cytotoxic, bufalin markedly inhibited the cell motility and invasiveness of T24 cells. The inhibitory effects of bufalin on cell invasiveness were associated with the tightening of tight junctions (TJs), which was demonstrated by an increase in transepithelial electrical resistance (TER). Bufalin treatment also repressed the levels of claudin proteins (claudin-2, -3 and -4) and the major components of TJs that play key roles in the control and selectivity of paracellular transport. Furthermore, the activities of matrix metalloproteinase (MMP)‑2 and -9 in T24 cells were dose‑dependently inhibited by treatment with bufalin and this also correlated with a decrease in their mRNA and protein expression levels; however, the mRNA and protein levels of the tissue inhibitor of metalloproteinase (TIMP)‑1 and -2 were increased. In addition, these effects were related to the increased phosphorylation of the extracellular signal-regulated protein kinase (ERK) pathway. The inhibition of ERK (PD98059) significantly prevented the bufalin‑induced suppression of T24 cell migration. These findings suggest that bufalin inhibits the migration and invasion of T24 cells by modulating the activity of TJs and MMPs, possibly in association with the activation of ERK.

Lactobacillus amylophilus D14 protects tight junction from enteropathogenic bacteria damage in Caco-2 cells

Enterotoxigenic Escherichia coli and Salmonella Typhimurium could adhere to epithelial tissue and destroy cell junctions, leading to intestinal inflammation and diarrhea. Lactobacillus could prevent the adhesion of pathogens to host cells and protect the mucosal barrier. The objective of this study was to investigate the protective effects of Lactobacillus amylophilus D14 on Caco-2 cells against the invasion of enterotoxigenic Escherichia coli K88 and Salmonella Typhimurium SL1344. We found that with a reduction in dextran permeability and an increase in transepithelial electrical resistance, L. amylophilus D14 could ameliorate the damage to cell integrity caused by pathogens. Furthermore, L. amylophilus D14 reduced the expression of phosphorylated extracellular signal-regulated protein kinase and phospho-c-jun N-terminal kinase, and it decreased the secretion of IL-8. The abilities of the Lactobacillus to protect the cell junctions were then evaluated on Caco-2 cells. Increased expression and amelioration distribution of tight junction proteins (zonula occludens-1, claudin-1, and E-cadherin) were observed when the cells were cocultured with pathogens and Lactobacillus simultaneously. Lactobacillus amylophilus D14 may influence the mitogen-activated protein kinase pathway to regulate the correct assembly of the tight junction and adherens junction, protecting the cell junctions and mucosal barrier damaged by enterotoxigenic E. coli K88 or Salmonella Typhimurium SL1344 infection.

GLP-2 enhances barrier formation and attenuates TNFα-induced changes in a Caco-2 cell model of the intestinal barrier

IntroductionTight junctions are intercellular permeability seals that regulate paracellular transport across epithelia. Tight junction function, expression and localisation of constituent proteins are significantly altered by cytokines such as TNFα. Glucagon-like peptide-2 (GLP-2) is an intestinotrophic enteroendocrine peptide. It is not known whether GLP-2 regulates the barrier or tight junctions. The aim of this study was to investigate whether GLP-2 has an effect on tight junction function or protein expression, alone or in response to TNFα exposure. MethodsCaco-2 cells were grown to confluence on filters in the presence or absence of GLP-2. The time course of transepithelial electrical resistance developing across the monolayer was measured; tight junction protein expression was quantified by immunoblotting. At day 20, TNFα in the presence or absence of GLP-2 was added. Changes in TEER and tight junction proteins expression were quantified. Both TNFα and GLP-2 were added on the basolateral side. ResultsGLP-2 exposed Caco-2 cell monolayers showed a significant increase in transepithelial electrical resistance compared to that in untreated control cells. At the same time, expression of the tight junction proteins occludin and zona occludens-1 (ZO-1) was increased at day 17 post-seeding (1.6-fold; p=0.037 and 4.7 fold; p=0.039 respectively). Subsequent TNFα exposure induced a significant 9.3-fold (p<0.001) decrease in transepithelial electrical resistance and a corresponding reduction in the expression of ZO-1 (5.3 fold; p<0.01). However, the TNFα-induced reduction in transepithelial electrical resistance in GLP-2-exposed cells was highly attenuated to 1.8-fold (p<0.01). No change in tight junction protein expression was noted in GLP-2 exposed cells after cytokine exposure. ConclusionGLP-2 enhances formation of the epithelial barrier and its constituent proteins in Caco-2 cells, and diminishes the effects of TNFα. If these effects are replicated in vivo the GLP-2 receptor may present a therapeutic target in intestinal inflammation.

Anti-invasive effects of decitabine, a DNA methyltransferase inhibitor, through tightening of tight junctions and inhibition of matrix metalloproteinase activities in AGS human gastric carcinoma cells

The DNA methyltransferase inhibitor decitabine, 5-Aza-2'-deoxycytidine, possesses anti-metabolic and anticancer activities in various cancer cells. However, the biochemical mechanisms underlying decitabine-induced inhibition of invasiveness and metastasis have not been thoroughly studied. In this study, we investigated the effect of decitabine on the correlation between tightening of tight junctions (TJs) and anti-invasive activity in AGS human gastric cancer cells. Our data indicated that the inhibitory effects of decitabine on cell motility and invasiveness were associated with increased tightness of the TJ, which was demonstrated by an increase in transepithelial electrical resistance (TER). Immunoblotting results indicated that decitabine repressed the levels of the claudin proteins, major components of TJs that play a key role in the control and selectivity of paracellular transport. Furthermore, matrix metalloproteinase (MMP)-2 and -9 activity in the AGS cells was dose-dependently inhibited by treatment with decitabine, and this was correlated with a decrease in mRNA and protein expression. In addition, these effects were related to inactivation of the phosphoinositide 3-kinase (PI3K)/Akt pathway in AGS cells. In conclusion, this study suggests that TJs and MMPs are critical targets of decitabine-induced inhibition of invasiveness in AGS human gastric cancer cells.

Inhibition of migration and invasion of LNCaP human prostate carcinoma cells by cordycepin through inactivation of Akt

Cordycepin (3'-deoxyadenosine), a major bioactive compound of Cordyceps militaris, has many pharmacological actions, such as anti-inflammatory and anticancer activities. In this study, the relationship between inhibition of cell motility and anti-invasive activity by cordycepin in LNCaP human prostate carcinoma cells was investigated. Within the concentration range that was not cytotoxic, cordycepin time-dependently inhibited cell motility and invasiveness of LNCaP cells. The inhibitory effects of cordycepin on cell invasiveness were associated with tightening of tight junctions (TJs), which was demonstrated by an increase in transepithelial electrical resistance (TER). Immunoblotting indicated that cordycepin decreases levels of claudin proteins, which are major components of TJs that play a key role in control and selectivity of paracellular transport. Furthermore, cordycepin inhibited the expression and activity of matrix metalloproteinase (MMP)-2 and MMP-9, and simultaneously increased levels of tissue inhibitor of metalloproteinase (TIMP)-1 and TIMP-2. These effects were related to inactivation of the phosphoinositide 3-kinase (PI3K)/Akt pathway in LNCaP cells. These findings suggest that cordycepin inhibits the migration and invasion of LNCaP cells by downregulating the activity of TJs and MMPs, possibly in association with suppression of Akt activation.