Background Infants undergoing ECMO may have elevated serum ferritin and iron levels, raising concerns about iron overload. Recent studies question the utility of these markers for acute vs. chronic iron overload during ECMO. This study evaluates iron content and localization in autopsy tissues from deceased infants who received or were considered for ECMO. Methods This retrospective single-center case-control study analyzed paraffin-embedded tissues from the basal ganglia, liver, spleen, pancreas, and kidney. Tissue sections were stained to quantify iron deposition and an independent pathologist reviewed samples for iron accumulation. Results Eighteen deceased infants' tissues were analyzed: nine underwent ECMO, and nine were considered for it. Both groups showed multi-organ iron accumulation with no significant difference between ECMO and non-ECMO cohorts. Red blood cell transfusions were linked to increased iron content in adrenal ( p = 0.004), hepatic ( p = 0.042), and splenic ( p = 0.013) tissues. Conclusions ECMO exposure alone does not independently increase iron content in infants' organs. Multi-organ iron accumulation in both groups suggests iron deposition in critically ill pediatric patients irrespective of ECMO exposure. Further research is needed to understand the mechanisms and implications.

Zonula occludens-1 (ZO-1) is an essential TJ component contributing to junctional formation by mediating interactions between junctional proteins and the actomyosin cytoskeleton. MDCK II cells, a model epithelial cell line, with ZO-1 knocked-out (ZO-1 KO) have observable phenotypic changes such as alterations in the actin cytoskeletal organization and physiological barrier properties. This study focuses on the ZO-1-ZU5 domain by rescuing ZO-1 with full-length and a panel of ZO-1-ZU5 mutants. ZO-1-ZU-5 is known to bind cingulin and through this interaction ZO-1 adopts an extended conformation. We examined actomyosin cytoskeleton organization, ZO-1 mobility, gene expression and barrier function to determine whether the ZO-1-ZU5 mutants rescue full-length activity. We found that the ZO-1- ZU5 truncation mutant cell line rescued transepithelial electrical resistance (TER) but did not rescue tight junction distribution or the amount of total gamma-actin. We will present evaluation of additional ZU5 mutants. This study expands our understanding of how extended ZO-1 regulates the mechanical, genetic, and phenotypic properties of epithelial cells. Trinity University Biology Summer Undergraduate Research Fellowship. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Background ECMO plays a crucial role in treating severe respiratory and cardiac failure in pediatric patients. However, its impact on the regulation of erythropoietin (EPO) and erythropoiesis remains poorly understood. Factors such as improved oxygenation, inflammation, and hemodilution associated with ECMO treatment may influence EPO production and erythropoiesis. This study aimed to examine the effects of ECMO on EPO regulation and erythropoiesis in pediatric patients. Methods This retrospective study serially quantified EPO serum levels, measured markers of erythropoiesis, and tabulated clinical outcomes of pediatric ECMO patients. Descriptive statistics and Pearson correlation coefficients were used to identify associations between biomarkers and clinical care parameters. Results Preliminary findings suggest a disconnection between elevated EPO levels and reduced markers of erythropoiesis or iron metabolism, indicating ineffective erythropoiesis. Patients receiving more than 10 mL/kg/day of RBC transfusions had higher reticulocyte counts. Non-survivors had sustained elevations of EPO serum levels but reduced erythropoietic activity. Conclusion In ECMO-treated pediatric patients, ineffective erythropoiesis is a significant concern and may be associated with higher mortality rates. Understanding the mechanisms behind this pathology could better inform clinical approaches and optimize management strategies. Further research is imperative to uncover the factors resulting in ineffective erythropoiesis in these patients and to develop targeted interventions.

Introduction: Elevations in serum ferritin and serum iron occur during pediatric extracorporeal membrane oxygenation (ECMO). Previous reports attribute the elevation to frequent red blood cell transfusions and/or hemolysis. Chronic transfusion can cause iron deposition in tissues leading to multisystem organ dysfunction. This study aims identify clinical factors associated with elevated ferritin and iron in pediatric ECMO patients, along with post-decannulation magnetic resonance imaging (MRI) assessment of iron deposition in liver and brain. Methods: Prospective, pilot study, using descriptive statistics to investigate potential associations between patient characteristics, serum ferritin and iron levels, and post-decannulation hepatic and basal ganglia iron deposition. Results: In this study, nine patients (100%) had elevated serum ferritin levels during ECMO. High ferritin levels were more common with veno-arterial than with veno-venous cannulation ( p = 0.026) and were also associated with high plasma free hemoglobin levels ( p < 0.001). Five patients presented with elevated serum iron levels. High serum iron levels were associated with higher daily ( p = 0.016) and cumulative transfusion volumes ( p = 0.013) as well ECMO duration beyond 7 days. MRI scans were performed on three patients with no evidence of abnormal iron deposition detected in the liver or brain. Conclusions: This pilot study shows that during pediatric ECMO, elevations in serum ferritin and serum iron occur and those elevations may be related to the cannulation modality, ECMO duration, amount of hemolysis, and volume of red blood cell transfusions. Further investigation is warranted to fully understand the implications of elevated serum iron and ferritin in pediatric ECMO.

Zonula occluden-1 (ZO-1) has been implicated as a major regulator of tight junction formation, functioning as a scaffolding protein and mediator between the junction and the actomyosin cytoskeleton. MDCK II cells that have ZO-1 knocked-down (ZO-1 KD) or knocked-out (ZO-1 KO) have observable phenotypic changes, including increased apical actin accumulation, increased cell height, and linearized junctions. The apical actin accumulation, resulting from the dysregulation of myosin II, is suspected to cause an increase in membrane tension seen in the ZO-1 KO, which may explain the phenotypic changes. The function of ZO-1 as a regulator of myosin II was confirmed by exposing the ZO-1 KO to blebbistatin, a myosin II inhibitor, which restored the cell height to that of wild-type MDCK II cells (WT) and the curvature of the junctions. Flipper-TR, which is a live cell fluorescent membrane tension probe, along with fluorescence-lifetime imaging microscopy (FLIM) analysis, are being used to test the membrane tension of the ZO-1 KO cells compared to WT cells. The Flipper-TR probe works by inserting itself into a lipid membrane where it undergoes conformational changes induced by membrane tension. The relationship between membrane tension and actin was examined using latrunculin A (LatA), which causes the depolymerization of actin filaments. When both the ZO-1 KO and WT cells were incubated with LatA, the membrane tension of both decreased to a similar extent. The preliminary results of this testing have shown an increased membrane tension in the ZO-1 KO cells. To measure the response of ZO-1 KO cells to increased tension, the expression of tension-related genes is being measured using quantitative real-time polymerase chain reaction (qRT-PCR). These tension-related genes include the ERM (ezrin, radixin, and moesin) proteins, with a primary interest in ezrin given its known activity as a major regulator of membrane tension in epithelial cells. This study aims to understand the mechanism between ZO-1 loss and regulation of membrane tension.

Zonula occludens-1 (ZO-1), the major scaffolding protein of tight junctions (TJs), recruits the cytoskeleton-associated proteins cingulin (CGN) and paracingulin (CGNL1) to TJs by binding to their N-terminal ZO-1 interaction motif. The conformation of ZO-1 can be either folded or extended, depending on cytoskeletal tension and intramolecular and intermolecular interactions, and only ZO-1 in the extended conformation recruits the transcription factor DbpA to TJs. However, the sequences of ZO-1 that interact with CGN and CGNL1 and the role of TJ proteins in ZO-1 TJ assembly are not known. Here, we used glutathione-S-transferase pulldowns and immunofluorescence microscopy to show that CGN and CGNL1 bind to the C-terminal ZU5 domain of ZO-1 and that this domain is required for CGN and CGNL1 recruitment to TJs and to phase-separated ZO-1 condensates in cells. We show that KO of CGN, but not CGNL1, results in decreased accumulation of ZO-1 at TJs. Furthermore, ZO-1 lacking the ZU5 domain showed decreased accumulation at TJs, was detectable along lateral contacts, had a higher mobile fraction than full-length ZO-1 by fluorescence recovery after photo bleaching analysis, and had a folded conformation, as determined by structured illumination microscopy of its N-terminal and C-terminal ends. The CGN-ZU5 interaction promotes the extended conformation of ZO-1, since binding of the CGN- ZO-1 interaction motif region to ZO-1 resulted in its interaction with DbpA in cells and in vitro. Together, these results show that binding of CGN to the ZU5 domain of ZO-1 promotes ZO-1 stabilization and accumulation at TJs by promoting its extended conformation.

Elevated serum ferritin, or hyperferritinemia, is frequently used as a surrogate to represent iron overload in patients who undergo chronic transfusions. Recently, increased ferritin and iron levels have been found in pediatric Extracorporeal Life Support (ECLS) patients. While elevated ferritin may represent an acute inflammatory phase reactant alone, it can also represent both acute or chronic iron overload due to large volume or chronic transfusion. Recognition of iron overload is crucial because when untreated it …

BACKGROUND:Obesity and diabetes mellitus are directly implicated in many adverse health consequences in adults as well as in the offspring of obese and diabetic mothers. Hispanic Americans are particularly at risk for obesity, diabetes, and end-stage renal disease. Maternal obesity and/or diabetes through prenatal programming may alter the fetal epigenome increasing the risk of metabolic disease in their offspring. The aims of this study were to determine if maternal obesity or diabetes mellitus during pregnancy results in a change in infant methylation of CpG islands adjacent to targeted genes specific for obesity or diabetes disease pathways in a largely Hispanic population.METHODS:Methylation levels in the cord blood of 69 newborns were determined using the Illumina Infinium MethylationEPIC BeadChip. Over 850,000 different probe sites were analyzed to determine whether maternal obesity and/or diabetes mellitus directly attributed to differential methylation; epigenome-wide and regional analyses were performed for significant CpG sites.RESULTS:Following quality control, agranular leukocyte samples from 69 newborns (23 normal term (NT), 14 diabetes (DM), 23 obese (OB), 9 DM/OB) were analyzed for over 850,000 different probe sites. Contrasts between the NT, DM, OB, and DM/OB were considered. After correction for multiple testing, 15 CpGs showed differential methylation from the NT, associated with 10 differentially methylated genes between the diabetic and non-diabetic subgroups, CCDC110, KALRN, PAG1, GNRH1, SLC2A9, CSRP2BP, HIVEP1, RALGDS, DHX37, and SCNN1D. The effects of diabetes were partly mediated by the altered methylation of HOOK2, LCE3C, and TMEM63B. The effects of obesity were partly mediated by the differential methylation of LTF and DUSP22.CONCLUSIONS:The presented data highlights the associated altered methylation patterns potentially mediated by maternal diabetes and/or obesity. Larger studies are warranted to investigate the role of both the identified differentially methylated loci and the effects on newborn body composition and future health risk factors for metabolic disease. Additional future consideration should be targeted to the role of Hispanic inheritance. Potential future targeting of transgenerational propagation and developmental programming may reduce population obesity and diabetes risk.

BackgroundZonula occludens (ZO‐1) is a tight junction cytosolic scaffolding protein known to bind the actin cytoskeleton in epithelial cells. ARHGEF11 is a Rho guanine nucleotide exchange factor known to activate the Rho family of small GTPases which are regulators of the actin cytoskeleton of the cell. ARHGEF11 associates with ZO‐1 by binding to the ZU5 domain in the C‐terminal tail. We are interested in understanding the interplay between ZO‐1, ARHGEF11 and the actin cytoskeleton in confluent MDCK II cells.MethodsConfluent monolayers stably transfected with EGFP‐ZO‐1 or EGFP‐ZO‐1Δ ZU5 were treated with latrunculin A to observe the actin depolymerization protection capacity of ZO‐1 with and without the ZU5 domain. Additionally, studies were performed to investigate the localization of EGFP‐ZO‐1 or EGFP‐ZO‐1ΔZU5 proteins following recovery from latrunculin A exposure. Colocalization studies were performed in mCherry‐ARHGEF11/EGFP‐ZO‐1 co‐transfected cells. Pharmacological inhibition of Rho‐kinase was used to target the ARHGEF11 pathway. Electrophysiological studies were conducted to evaluate junctional permeability.ResultsFull‐length EGFP‐ZO‐1 protects against latrunculin A induced actin depolymerization more robustly than EGFP‐ZO‐1ΔZU5. In the actin polymerization recovery paradigm EGFP‐ZO‐1ΔZU5 exhibited enhanced recovery rate when compared to full‐length EGFP‐ZO‐1 following latrunculin A exposure. Enhanced actin networks are observed in mCherry‐ARHGEF11 positive cells in both wildtype and ZO‐1 knockdown MDCK II cells.ConclusionsThe increased recovery rate exhibited by EGFP‐ZO‐1ΔZU5 in the latrunculin A recovery assay coupled with the greater actin protection capacity of full‐length ZO‐1 suggests that truncated ZO‐1 is more mobile. ARHGEF11 elicits actin cytoskeleton reorganization in both wildtype and ZO‐1 knockdown cells that was partially reversed by inhibition of Rho kinase. There was limited accumulation of ARHGEF11 at the apical junctional complex in confluent monolayers.Support or Funding InformationSan Antonio Medical Foundation, Trinity University Biology Department, Mach Research AwardThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

BackgroundThe use of hydrocortisone in association with indomethacin is a known risk factor for spontaneous intestinal perforation in extremely low birthweight infants. Hypoxia, inflammation and non‐steroidal anti‐inflammatory drugs independently alter apical junctional complexes of epithelial cells. We are interested in understanding the combined effects of hypoxia, inflammation and non‐steroidal anti‐inflammatory drugs on the apical junctional complex and barrier integrity in a gastrointestinal epithelial cell model.MethodsThrough the use of CaCo‐2 BBe monolayers, an established in vitro model of the intestinal epithelium, we analyzed the effects of the pro‐inflammatory agents (TNF and IFN‐gamma) and anti‐inflammatory agents (indomethacin and hydrocortisone) on junctional protein expression and localization, cell proliferation using Edu labeling, cellular ATP concentration and barrier function.ResultsProtein expression analyzed by Western blotting techniques demonstrated an alteration of claudin‐1, a tight junction sealing protein. The soluble and insoluble fractions of the cells were analyzed in order to determine the location of these proteins. We determined inflammatory conditions produced a higher concentration in the soluble fraction indicating impaired function. Confocal analysis after 24‐hour exposure to hypoxia showed that the combined use of hypoxia and inflammatory agents elicited no significant change in cell proliferation levels. Cellular ATP levels were analyzed to corroborate proliferation studies. Confocal imaging under the same experimental conditions was also conducted in order to probe for claudin‐1 localization within the cells.ConclusionsThere was no observed significant difference in the cell proliferation rate or marked changes in ATP levels in our model. Therefore, we hypothesize that disturbance in barrier function may be related to specific junctional mechanisms. We found claudin‐1 protein expression and localization to be sensitive to both hypoxia and inflammatory cytokines contributing to impaired barrier integrity. These cellular studies suggest claudin‐1 stability may be a potential target in the progression of spontaneous intestinal perforation.Disclosure: The view(s) expressed herein are those of the author(s) and do not reflect the official policy or position of Brooke Army Medical Center, the U.S. Army Medical Department, the U.S. Army Office of the Surgeon General, the Department of the Air Force and Army, Department of Defense or the U.S. Government.Support or Funding InformationSan Antonio Medical Foundation Grant, Trinity University, US Air Force 59th Medical Wing Clinical Research WingThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Zonula occludens‐1 (ZO‐1) is a cytosolic scaffolding protein involved in tight junction formation and maintenance of barrier function. The carboxy‐terminus contains a unique and poorly characterized ZU5 domain while structural data indicates exposed hydrophobic residues that are putative sites of protein interaction. We hypothesized the ZU5 domain contributes to stabilizing interactions and barrier properties of the tight junction. To determine ZO‐1‐ZU5 domain function, EGFP‐ZO‐1, EGFP‐ZO‐1ΔZU5, and EGFP‐ZO‐1 constructs mutated within the hydrophobic pocket of ZU5 were engineered and stably transfected into low‐resistance MDCK lines. The carboxy‐terminus of ZO‐1 contains an actin binding region (ABR) in close proximity to the ZU5 region that has been found to bind to F‐actin, and we hypothesized that the ZU5 region may have a stabilizing effect on these interactions. Actin protection assays indicated the EGFP‐ZO‐1 provides enhanced protection of perijunctional actin from degradation compared to the EGFP‐ZO‐1ΔZU5. The hydrophobic pocket mutants displayed varying degrees of actin protection. ZO‐1 knockdown cells have an increased paracellular flux, and FITC‐dextran paracellular flux assays in ZO‐1‐ZU5 mutants display enhanced leak pathway permeability. FRAP analyses show that EGFP‐ZO‐1ΔZU5 rapidly recovers and the mobile fraction (Mf) is elevated compared to EGFP‐ZO‐1 suggesting an important role in stabilization. Hydrophobic pocket mutants displayed similar changes suggesting the ZU5 domain participates scaffolding ZO‐1 at the tight junction.Support or Funding InformationR01DK061931

Concomitant use of hydrocortisone and indomethacin increases the risk for spontaneous intestinal perforation in extremely low birth weight infants. Hypoxia, inflammation, and hydrocortisone alter epithelial tight junction formation/maintenance independently. The concomitant effect of inflammation, hypoxia, indomethacin, …

BACKGROUND Dysregulation of inflammatory processes are associated with intestinal injury, necrotizing enterocolitis, and risk of perforation in the newborn. A reduction of Epidermal Growth Factor (EGF) and impaired function of the Epidermal Growth Factor Receptor (EGFR), have been demonstrated in the preterm gastrointestinal system compared to term. Prostaglandin E2 (PGE2) as well as EGF have independently been shown to have a role in the barrier maintenance, recovery from injury, and regulation of perfusion. OBJECTIVE Evaluate the regulation of EGF/EGFR/PGE2 pathway in a non‐human primate gastrointestinal explant. Additionally evaluating the roles of inflammatory and anti‐inflammatory agents on this pathway. DESIGN/METHODS Tissue explants were created from gastrointestinal regions (ileum through colon) upon necropsy of term and preterm (67% gestation) non‐human primates. Explants were evaluated in conditions of control, pro‐inflammatory (TNF 10 ng/ml), and anti‐inflammatory (indomethacin 50uM) variables. Absolute and relative prostaglandin secretion (PGE2); mRNA expression of Prostanoid (EP) receptor subtypes, EGFR, EPS8, AKT1, AKT2, and MAPK1 were used as markers of EGF/PGE2 pathway regulation at 24 hours post exposure. RESULTS PGE2 secretion significantly increases in term colon compared preterm colonic secretion. In addition, the term gastrointestinal explants are significantly more resistant to indomethacin inhibition of PGE2 secretion, noted across ileum, cecum, and colon. There were no significant differences noted in EP receptor subtype expression. Preterm expression of EGFR and its downstream effectors are elevated when compared to term ileum explants. However, there is markedly more variability in response noted to TNF and indomethacin in the term explants when compared to preterm ileum explants. CONCLUSIONS A positive gradient of PGE2 secretion in the distal gastrointestinal regions is observed in term explants likely correlated to the advanced differentiation status compared to the preterm. In addition, the up regulation of EGFR along with overall increased expression of downstream targets in the preterm explant is consistent with the expected mitogenic status. However, the decreased response to TNF and indomethacin, suggest a limited ability to modulate inflammation.

BackgroundAstrocytes provide support for neurons, however, with age, astrocytes’ ability to provide neuroprotection decreases. This decrease in neuroprotection increases the likelihood of neurodegenerative diseases, that compromise function. One hallmark of neurodegeneration is accumulation of β‐amyloid (βA) plaques, which disrupts cellular function and often leads to cellular death. As astrocytes age, they have been shown to naturally have lower mitochondrial membrane potential (MMP) and more frequent with larger amplitude calcium waves. Treatment with melatonin has been shown to decrease βA production and prevent plaque formation in the brain, suggesting melatonin plays a role during neurodegeneration. The interplay between melatonin and βA effects on astrocyte function was examined by measuring MMP and calcium waves.MethodsPrimary mouse astrocyte cultures obtained from young (4 month) and old (28 month) animals were used in the current study. Astrocytes were cultured for 24hrs with 100μM melatonin before loading with either MitoID or Fluo3. Measures were obtained using galvano or resonant scanning modes on a confocal microscope system, respectively. Astrocytes were then treated with 0.5μM βA (AggreSure) and imaged for an additional 30 minutes. MMP and calcium waves were analyzed to indicate cellular stress and change in cellular function.ResultsMelatonin significantly elevated baseline MMP in young astrocytes while protecting MMP loss due to βA exposure. Baseline MMP levels were unaffected by melatonin in old astrocytes although melatonin preserved MMP levels during βA exposure. This suggests young astrocytes capacity to alter cellular activities due to melatonin exposure is more robust. Calcium waves in young astrocytes persisted for a longer duration during βA exposure.ConclusionsThese results indicate that melatonin has protective effects on preserving MMP in the presence of βA, but produced greater effects in younger astrocytes. These data indicate effects are related to cellular vitality and not due to accumulation of βA.Support or Funding InformationSupport:Trinity University Murchison Undergraduate Research Fellowship (EKD), Trinity University Biology Department and Neuroscience Program.

Zonula occludens (ZO‐1) is a cytoplasmic scaffold protein that structurally and functionally tethers cytoskeletal components and membrane proteins at the tight junction. Interestingly, ZO‐1 has a poorly characterized ZU5 domain at the carboxy‐terminus that ZO‐2 and ZO‐3 proteins lack. The goal of this study is to examine ZO‐1‐ZU‐5 function in a structurally defined manner. To determine ZO‐1‐ZU5 domain function, EGFP‐ZO‐1, EGFP‐ZO‐1ΔZU5 and EGFP‐ZO‐1 constructs mutated within the ZU5 domain hydrophobic pocket were engineered and stably transfected into MDCK II cells. FRAP analyses show that EGFP‐ZO‐1ΔZU5 rapidly recovers and the mobile fraction (Mf) is elevated compared to EGFP‐ZO‐1. The Mf increases by 30% in EGFP‐ZO‐1ΔZU5 as a 50% decrease in t1/2 is observed. Disruption of the ZU5‐hydrophobic pocket caused similar changes suggesting the ZU5 domain participates in stabilizing ZO‐1 at the tight junction. Macromolecular flux studies demonstrated that ZO‐1ΔZU5 expression enhances leak pathway permeability compared to EGFP‐ZO‐1. These data indicate that the ZU5 domain stabilizes ZO‐1 at the tight junction and contributes to development of the barrier to paracellular flux of small solutes.

Endometrial cancer is the most common female reproductive cancer in the United States and is associated with deregulated tight junction protein expression. Given the highly estrogen-responsive nature of this tissue, we investigated the effects of estrogen and its agonist, 4-OH TAM, on the expression and subcellular localization of the tight junction protein claudin-4 (CLDN-4), in HEC-1A endometrial cancer cells. In untreated HEC-1A cells, we observed dramatic overexpression of claudin-4 protein. In addition, differential detergent extraction analysis indicated that claudin-4 was localized primarily in the membrane but also found in the cytosolic, nuclear and cytoskeletal fractions. Upon exposure of HEC-1A to estradiol (E-2), we observed a biphasic effect both on the overall expression of claudin-4 protein and on its cytosolic and cytoskeletal presence as demonstrated by immunoblot analysis. Immunofluorescence analysis also revealed a biphasic effect of E-2, on claudin-4 expression. In contrast, we observed no changes in expression levels nor in the subcellular distribution patterns of claudin-4 in HEC-1A cells treated with different concentrations of 4-OH TAM. The intracellular presence of CLDN-4 coupled with the biphasic effects of E-2 on CLDN-4 expression in the cytoskeleton suggest that this protein may be involved in cell signaling to and from TJs.

Tumor-specific deregulated expression of claudins, integral membrane proteins found in tight junctions (TJs), has indicated a possible role for TJ disruption in cancer progression. The current study demonstrates the marked overexpression of claudin-3 protein in two breast cancer cell lines of metastatic origin (MCF-7 and MDA-MB-415). Immunofluorescence and differential detergent fractionation analyses revealed that, although claudin-3 was primarily localized at cell junctions, it was also detected intracellularly. Similarly, the siRNA-mediated suppression of claudin-3 did not considerably affect its pattern of subcellular distribution relative to mock-transfected cells. However, there appeared to be a preferential loss of claudin-3 signal in the cytoskeletal fraction. Wound-healing assays were conducted to assess the effect of endogenous overexpression versus siRNA-mediated suppression of claudin-3 on cellular motility in MCF-7 cells. Suppression of claudin-3 protein levels resulted in a marked decrease in the rate of cellular motility relative to mock-transfected cells. These findings suggest that overexpression of claudin-3 may be important in disrupting TJ integrity and thus contribute to enhanced cellular motility, a key component of tumor progression.