Tight Junction Function Research Articles

Abstract 2387: Targeting PVT1 exon 9 transcript is not pro-apoptotic but induces claudin 4 expression and inhibits migration in triple negative breast cancer cells

Abstract Triple negative breast cancer (TNBC) is a lethal molecular subtype of invasive breast cancer (BC) that is ER-, PR- and HER2-. Claudin low (CL) TNBC, a distinct molecular subtype of TNBC, has the worst prognosis compared to other BC subtypes. Aberrant expression of claudin proteins disrupts the function of tight junctions. Consequently, inducing epithelial-to-mesenchymal transition (EMT) in cancers, an important factor for enhanced motility and metastasis. Therefore, understanding the molecular mechanisms that modulate claudin expression in TNBC is crucial. The 8q24 genomic locus is an important cancer susceptibility locus that is associated with poor clinical outcomes in cancer due to common amplification events that occur in many malignant diseases. This locus contains the PVT1 gene, which encodes a long noncoding RNA (lncRNA) that has been implicated in multiple cancers including BC. PVT1 consists of 12 exons that are alternatively spliced to generate lncRNAs. Although previous research has implicated PVT1 as an important player in BC, the underlying molecular mechanisms of PVT1 in CL TNBC is unknown. To examine the role of PVT1 in CL TNBC, we assessed PVT1 expression in T47D (ER+), MDA MB 231 (CL) and MDA MB 468 (claudin high (CH)) BC cells. We observed that PVT1 exons 4A, 4B, and 9 are significantly overexpressed in CL MDA MB 231 and significantly underexpressed in CH MDA MB 468 in comparison to T47D cells, suggesting a protumorigenic role of PVT1 in CL TNBC. We analyzed the functional consequences of siRNA targeting of PVT1 exon 9 expression in CL TNBC cells. siRNA targeting of PVT1 exon 9 expression in the MDA MB 231 CL TNBC cells led to a significant reduction in migration and the re-expression of claudin 4. To determine the role of PVT1 on EMT, we assessed the expression of EMT markers (vimentin, fibronectin, and E-cadherin) in MDA MB 231 cells. We observed no changes in the expression of EMT markers when PVT1 exon 9 is knocked down. However, our data show that mesenchymal markers are more highly expressed in MDA MB 231 cells in comparison to MDA MB 468 cells. Further, knockdown of PVT1 exon 9 did not induce apoptosis in MDA MB 231 cells as assessed by caspase 3 and caspase 9 expression. Taken together, our data indicate that PVT1 exon 9 regulates claudin expression and migration in CL TNBC, and may have implications for clinical outcomes in TNBC. Citation Format: Fayola A. Levine, Olorunseun O. Ogunwobi. Targeting PVT1 exon 9 transcript is not pro-apoptotic but induces claudin 4 expression and inhibits migration in triple negative breast cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 2387.

Abstract LB070: Development of Claudin 18.2/CD3 bispecific antibody maintaining effector functions for better cancer therapy

Abstract Background: Tumors are heterogeneous in nature with highly suppressive microenvironment. The heterogeneity is distinguished by the presence of distinct cell populations, where many tumors are partial suppressed, thus lead to tumor relapse and develop drug-resistance after initial therapy. Most importantly, the heterogeneity in tumors raises considerable challenges to design effective treatment strategy with predictable outcomes. Paracellular permeability at tight junctions is largely determined by Claudins. Under aberrant condition, Claudins disrupt tight junction functions and promote tumor-initiation. Claudin 18.2 is significantly expressed in gastric and pancreatic cancers, while normal tissue expression is limited to the epithelium of the stomach, making it a promising target for cancer therapy. Although great efficacy has been observed for T-cell engagers, balance of safety-efficacy remains a major concern. Bispecific antibody (BsAb) with lower CD3-binding arm could have a huge potential to minimize unspecific T-cell activation while maintaining better-efficacy. Here, we designed Claudin 18.2/CD3 BsAb with weaker CD3-binding arm and maintain Fc-effector functions to broaden mechanisms of action including antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC). Methods: Claudin 18.2 expressing and T-cells were used for Claudin 18.2 and CD3 binding analysis, respectively. T-cell activation was performed by co-culturing T-cells with Claudin 18.2+ and Chaudin 18.2- cells. Pancreatic cancer cell was co-cultured with human PBMCs for ADCC, T-cell activation and T-cell cytotoxicity studies. Co-culture of Jurkat and CD16+ NK cells were used for Fc-mediated T-cell activation study. Claudin 18.2+ and T-cell cells were used for CDC studies in presence of human complement serum. Results: While comparable Claudin 18.2 binding affinity was maintained, a significant reduction in CD3 binding was observed by Claudin 18.2/CD3 BsAb compared to a benchmark antibody. Although weaker CD3 binding, BsAb induced equivalent Claudin 18.2 specific T-cell activation in presence of Jurkat and/or human PBMCs compared to the benchmark. Moreover, it also induced ADCC of pancreatic cancer cell and T-cell activation in presence of PBMCs in dose-dependent manner. Claudin 18.2/CD3 BsAb induced CDC of Claudin 18.2+ cells but not T-cell cells in presence of human complement serum. While Claudin 18.2/CD3 BsAb maintained the Fc-mediated ADCC and CDC functions, it had no significant effect on Fc-mediated T-cell activation compared to the benchmark antibody in presence of CD16+ NK cells. Conclusion: We designed Claudin 18.2/CD3 BsAb with weaker CD3 binding to minimize unspecific T-cell activation while maintaining Fc-mediated-effector functions. These data suggest the development of Claudin 18.2/CD3 BsAb as a drug target for patients with gastric and pancreatic cancers. Citation Format: Amit K. Chaudhary, Mohd S. Zaman, Cai Huang, Jianbo Dong, Yue Liu. Development of Claudin 18.2/CD3 bispecific antibody maintaining effector functions for better cancer therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB070.

Effect of sodium butyrate on slaughter performance, serum indexes and intestinal barrier of rabbits.

The purpose of this study was to investigate the effect of sodium butyrate on slaughter performance, serum indexes and the intestinal barrier in rabbits. Six hundred healthy weaned rabbits were randomly divided into three groups (5 replicates per group, 40 rabbits per replicate): control (fed a basal diet), sodium butyrate (fed a basal diet containing 0.5% sodium butyrate) and antibiotic (fed a basal diet containing 0.004% antibiotic). The trial lasted 35days, including 7days of pretesting and 28days of formal testing. The results showed that dietary sodium butyrate supplementation increased the full-bore weight, the half-bore weight and the half-bore rate of rabbits. Meanwhile, the content of aspartate aminotransferase (AST) in serum was increased in rabbits fed the sodium butyrate diet. According to the intestinal barrier, after adding sodium butyrate to feed, the tight junction function of the rabbit intestine is enhanced, and the intestinal microbial composition is also improved. To sum up, after sodium butyrate was added to feed instead of antibiotics, slaughter performance was significantly enhanced, serum indexes were improved, and intestinal barrier function was also enhanced. Therefore, sodium butyrate can be added to feed as an additive and can replace antibiotics.

Regulatory roles of claudin-1 in cell adhesion and microvilli formation

Aberrant expression of tight junction proteins has recently been focused on in the cancer research field. We previously showed that claudin-1 is aberrantly expressed from an early stage of uterine cervical adenocarcinoma and contributes to malignant potentials. To elucidate the molecular mechanisms underlying tumor-promoting roles of claudin-1, we established and analyzed claudin-1 knockout cells. Knockout of claudin-1 suppressed conventional tight junctional functions, barrier and fence functions, and expression of cell adhesion-associated proteins including E-cadherin. Comparative proteome analysis revealed that expression of claudin-1 affected expression of a wide range of proteins, especially proteins that are associated with cell adhesion and actin cytoskeleton remodeling. Interactome analysis of the identified proteins revealed that E-cadherin and focal adhesion kinase play central roles in the claudin-1-dependently affected protein network. Moreover, knockout of claudin-1 significantly suppressed microvilli formation and activity of Ezrin/Radixin/Moesin. Taken together, the results indicate that expression of claudin-1 affects not only conventional tight junction function but also expression and activity of a wide range of proteins, especially proteins that are associated with cell adhesion and actin cytoskeleton remodeling, to contribute to malignant potentials and microvilli formation in cervical adenocarcinoma cells.

Chimeric Claudins: A New Tool to Study Tight Junction Structure and Function.

The tight junction (TJ) is a structure composed of multiple proteins, both cytosolic and membranal, responsible for cell–cell adhesion in polarized endothelium and epithelium. The TJ is intimately connected to the cytoskeleton and plays a role in development and homeostasis. Among the TJ’s membrane proteins, claudins (CLDNs) are key to establishing blood–tissue barriers that protect organismal physiology. Recently, several crystal structures have been reported for detergent extracted recombinant CLDNs. These structural advances lack direct evidence to support quaternary structure of CLDNs. In this article, we have employed protein-engineering principles to create detergent-independent chimeric CLDNs, a combination of a 4-helix bundle soluble monomeric protein (PDB ID: 2jua) and the apical—50% of human CLDN1, the extracellular domain that is responsible for cell–cell adhesion. Maltose-binding protein-fused chimeric CLDNs (MBP-CCs) used in this study are soluble proteins that retain structural and functional aspects of native CLDNs. Here, we report the biophysical characterization of the structure and function of MBP-CCs. MBP-fused epithelial cadherin (MBP-eCAD) is used as a control and point of comparison of a well-characterized cell-adhesion molecule. Our synthetic strategy may benefit other families of 4-α-helix membrane proteins, including tetraspanins, connexins, pannexins, innexins, and more.

SIPA1 Is a Modulator of HGF/MET Induced Tumour Metastasis via the Regulation of Tight Junction-Based Cell to Cell Barrier Function.

Simple SummaryThe role of Signal Induced Proliferation Associated 1 (SIPA1) in lung cancer remains largely unknown. This study aimed to evaluate the importance of SIPA1 in the development and progression of lung cancer, to demonstrate the cellular functions of SIPA1 and the molecular mechanisms involved.Background: Lung cancer is the leading cause of cancer death. SIPA1 is a mitogen induced GTPase activating protein (GAP) and may hamper cell cycle progression. SIPA1 has been shown to be involved in MET signaling and may contribute to tight junction (TJ) function and cancer metastasis. Methods: Human lung tumour cohorts were analyzed. In vitro cell function assays were performed after knock down of SIPA1 in lung cancer cells with/without treatment. Quantitative polymerase chain reaction (qPCR) and western blotting were performed to analyze expression of HGF (hepatocyte growth factor), MET, and their downstream markers. Immunohistochemistry (IHC) and immunofluorescence (IFC) staining were performed. Results: Higher expression of SIPA1 in lung tumours was associated with a poorer prognosis. Knockdown of SIPA1 decreased invasiveness and proliferation of in vitro cell lines, and the SIPA1 knockdown cells demonstrated leaky barriers. Knockdown of SIPA1 decreased tight junction-based barrier function by downregulating MET at the protein but not the transcript level, through silencing of Grb2, SOCS, and PKCμ (Protein kinase Cµ), reducing the internalization and recycling of MET. Elevated levels of SIPA1 protein are correlated with receptor tyrosine kinases (RTKs), especially HGF/MET and TJs. The regulation of HGF on barrier function and invasion required the presence of SIPA1. Conclusions: SIPA1 plays an essential role in lung tumourigenesis and metastasis. SIPA1 may be a diagnostic and prognostic predictive biomarker. SIPA1 may also be a potential therapeutic target for non-small cell lung cancer (NSCLC) patients with aberrant MET expression and drug resistance.

Role of Probiotics in Modulating Human Gut Microbiota Populations and Activities in Patients with Colorectal Cancer-A Systematic Review of Clinical Trials.

Background: Growing attention has been given to the role of nutrition and alterations of microbial diversity of the gut microbiota in colorectal cancer (CRC) pathogenesis. It has been suggested that probiotics and synbiotics modulate enteric microbiota and therefore may be used as an intervention to reduce the risk of CRC. The aim of this study was to evaluate the influence of probiotics/synbiotics administration on gut microbiota in patients with CRC. Methods: PubMed, Scopus, and Web of Science were searched between December 2020 and January 2021. Randomized controlled trials (RCTs) recruiting adults with CRC, who have taken probiotics/synbiotics for at least 6 days were included. Changes in gut microbiota and selected biochemical and inflammatory parameters (i.e., hsCRP, IL-2, hemoglobin) were retrieved. Results: The search resulted in 198 original research articles and a final 6 were selected as being eligible, including 457 subjects. The median age of patients was 65.4 years old and they were characterized by the median BMI value: 23.8 kg/m2. The literature search revealed that probiotic/synbiotic administration improved enteric microbiota by increasing the abundance of beneficial bacteria such as Lactobacillus, Eubacterium, Peptostreptococcus, Bacillus and Bifidobacterium, and decreased the abundance of potentially harmful bacteria such as Fusobacterium, Porhyromonas, Pseudomonas and Enterococcus. Additionally, probiotic/synbiotic intervention improved release of antimicrobials, intestinal permeability, tight junction function in CRC patients. Conclusions: The use of probiotics/synbiotics positively modulates enteric microbiota, improves postoperative outcomes, gut barrier function and reduces inflammatory parameters in patients suffering from CRC.

The Degradation of Airway Epithelial Tight Junctions in Asthma Under High Airway Pressure Is Probably Mediated by Piezo-1.

Full functioning of the airway physical barrier depends on cellular integrity, which is coordinated by a series of tight junction (TJ) proteins. Due to airway spasm, edema, and mucus obstruction, positive end-expiratory alveolar pressure (also termed auto-PEEP) is a common pathophysiological phenomenon, especially in acute asthma attack. However, the influence of auto-PEEP on small airway epithelial TJs is currently unclear. We performed studies to investigate the effect of extra pressure on small airway epithelial TJs and its mechanism. The results first confirmed that a novel mechanosensitive receptor, piezo-1, was highly expressed in the airway epithelium of asthmatic mice. Extra pressure induced the degradation of occludin, ZO-1 and claudin-18 in primary human small airway epithelial cells (HSAECs), resulting in a decrease in transepithelial electrical resistance (TER) and an increase in cell layer permeability. Through in vitro investigations, we observed that exogenous pressure stimulation could elevate the intracellular calcium concentration ([Ca2+]i) in HSAECs. Downregulation of piezo-1 with siRNA and pretreatment with BAPTA-AM or ALLN reduced the degradation of TJs and attenuated the impairment of TJ function induced by exogenous pressure. These findings indicate the critical role of piezo-1/[Ca2+]i/calpain signaling in the regulation of small airway TJs under extra pressure stimulation.

Molecular Characterization of the Extracellular Domain of Human Junctional Adhesion Proteins.

The junction adhesion molecule (JAM) family of proteins play central roles in the tight junction (TJ) structure and function. In contrast to claudins (CLDN) and occludin (OCLN), the other membrane proteins of the TJ, whose structure is that of a 4α-helix bundle, JAMs are members of the immunoglobulin superfamily. The JAM family is composed of four members: A, B, C and 4. The crystal structure of the extracellular domain of JAM-A continues to be used as a template to model the secondary and tertiary structure of the other members of the family. In this article, we have expressed the extracellular domains of JAMs fused with maltose-binding protein (MBP). This strategy enabled the work presented here, since JAM-B, JAM-C and JAM4 are more difficult targets due to their more hydrophobic nature. Our results indicate that each member of the JAM family has a unique tertiary structure in spite of having similar secondary structures. Surface plasmon resonance (SPR) revealed that heterotypic interactions among JAM family members can be greatly favored compared to homotypic interactions. We employ the well characterized epithelial cadherin (E-CAD) as a means to evaluate the adhesive properties of JAMs. We present strong evidence that suggests that homotypic or heterotypic interactions among JAMs are stronger than that of E-CADs.

Establishment of a novel probe-based RT-qPCR approach for detection and quantification of tight junctions reveals age-related changes in the gut barriers of broiler chickens.

Tight junctions (TJs) play a dominant role in gut barrier formation, therefore, resolving the structures of TJs in any animal species is crucial but of major importance in fast growing broilers. They are regulated in molecular composition, ultrastructure and function by intracellular proteins and the cytoskeleton. TJ proteins are classified according to their function into barrier-forming, scaffolding and pore-forming types with deductible consequences for permeability. In spite of their importance for gut health and its integrity limited studies have investigated the TJs in chickens, including the comprehensive evaluation of TJs molecular composition and function in the chicken gut. In the actual study sequence-specific probes to target different TJ genes (claudin 1, 3, 5, 7, 10, 19, zonula occludens 1 (ZO1), occludin (OCLN) and tricellulin (MD2)) were designed and probe-based RT-qPCRs were newly developed. Claudin (CLDN) 1, 5, ZO1 and CLDN 3, 7, MD2 were engulfed in multiplex RT-qPCRs, minimizing the number of separate reactions and enabling robust testing of many samples. All RT-qPCRs were standardized for chicken jejunum and caecum samples, which enabled specific detection and quantification of the gene expression. Furthermore, the newly established protocols were used to investigate the age developmental changes in the TJs of broiler chickens from 1–35 days of age in the same organ samples. Results revealed a significant increase in mRNA expression between 14 and 21days of age of all tested TJs in jejunum. However, in caecum, mRNA expression of some TJs decreased after 1 day of age whereas some TJs mRNA remained constant till 35 days of age. Taken together, determining the segment-specific changes in the expression of TJ- proteins by RT-qPCR provides a deeper understanding of the molecular mechanisms underpinning pathophysiological changes in the gut of broiler chickens with various etiologies.

Microglia increase tight-junction permeability in coordination with Müller cells under hypoxic condition in an in vitro model of inner blood-retinal barrier

Microglia and Müller cells (MCs) are believed to be critically involved in hypoxia-induced blood-retinal barrier (BRB) disruption, which is a major pathogenic factor of various retinopathies. However, the underlying mechanism remains poorly defined. The inner BRB (iBRB) is primarily formed of microvascular endothelial cells (ECs) with tight junction (TJ), which are surrounded and supported by retinal glial cells. We developed a novel in vitro iBRB model sheet by sandwiching Transwell membrane with layered mouse brain microvascular ECs (bEnd.3) and mouse retinal MCs (QMMuC-1) on each side of the membrane. Using this model, we tested the hypothesis that under hypoxic condition, activated microglia produce inflammatory cytokines such as interleukin (IL)-1β, which may promote vascular endothelial growth factor (VEGF) production from MCs, leading to TJ disruption. The iBRB model cell sheets were exposed to 1% oxygen for 6 h with or without mouse brain microglia (BV2) or IL-1β. TJ structure and function were examined by zonula occludens (ZO)-1 immunostaining and fluorescein isothiocyanate permeability assay, respectively. Relative gene expression of IL-1β in BV2 under normoxic and hypoxic conditions was examined by real-time reverse transcription-polymerase chain reaction. VEGF protein concentration in QMMuC-1 supernatants was measured by enzyme-linked immunosorbent assay. The bEnd.3 cell sheet incubated with BV2 in hypoxic condition or with IL-1β in normoxic condition showed abnormal localization of ZO-1 and aberrated barrier function. Under normoxic condition, EC-MC iBRB model cell sheet showed lower permeability than bEnd.3 cell sheet. Under hypoxic conditions, the barrier function of EC-MC iBRB model cell sheet was more deteriorated compared to bEnd.3 cell sheet. Under hypoxic condition, incubation of EC-MC iBRB model cell sheet with BV2 cells or IL-1β significantly increased barrier permeability, and hypoxia-treated BV2 cells expressed significantly higher levels of IL-1β mRNA. Incubation of QMMuC-1 with IL-1β increased VEGF production. These results suggest that under hypoxic condition, microglia are activated to release proinflammatory cytokines such as IL-1β that promote VEGF production from MCs, leading to disruption of iBRB function. Modulating microglia and MCs function may be a novel approach to treat hypoxia-induced retinal BRB dysfunction.

Abstract PS4-44: Expression of the claudin transmembrane tight junction protein family (CLDN) and the prediction value of a claudin subset to the clinical outcome of patients with breast cancer

Abstract Background: Tight Junction (TJ) proteins are directly or indirectly involved in breast cancer progression and metastasis where the mutual adhesiveness of cancer cells is significantly weaker than normal cells and so reduced cell-cell interaction results in a loss of normal tissue architecture (1). This adhesiveness is in addition to the role of the proteins in the control of the barrier functions of epithelial and endothelial cells. The claudin family (CLDN) of transmembrane proteins, the largest protein family of the tight junctions, are located in the TJ of cells of epithelial and endothelial origin where they are key in the maintenance of TJ function and have been increasingly shown to have important roles during key steps in the progression of cancer. We have previously demonstrated that the expression of some TJ molecules (occludin, claudins -5, -16, -20) are associated with disease progression in breast cancer (2-5). This study sought to evaluate the possible clinical and prognostic value of the entire CLDN family, in patients with breast cancer. Methods: Members of the claudin family of transmembrane TJ proteins were assessed and correlated with clinical and pathological parameters at the messenger level in a Cardiff breast cancer cohort. Breast cancer primary tumours (n=114) and matched background tissue (n=30) were processed for RNA extraction. RNA was reverse transcribed and quantified before analysis by Q-PCR CLDN-1 to -24. The expression profile was analysed against the clinical and pathological information and most importantly the clinical outcome of the patients. Results. Mammary tissues expressed varying levels of CLDNs. However, the levels of CLDN1, CLDN3, CLDN4, CLDN11, CLDN19, and CLDN22 were found to be significantly aberrant in breast cancer tissues compared with normal tissues. Members of the family, namely CLDN10, CLDN11, and CLDN18 were found to have a significant predictive value to the prognosis when using the Nottingham Prognostic Index. Of the claudin family, we identified eight members whose expression was negatively correlated with long term survival, and three members positively correlated with the survival. A comprehensive bioinformatic analyses of these CLDN members revealed a highly significant signature in predicting the clinical outcome. A favourable CLDN signature predicted a subset of the patients who all survived the ten-year followup, compared with those with unfavourable signature (69.1%), p<0.0001 (Log ranked). Likewise, the CLDN signature also successfully predicted the occurrence of breast cancer related incidence (97.6% vs 61.8%, p<0.0001). Multivariate analyses confirmed that the CLDN signature has a highly significant value as an independent predictor for both overall survival (p=0.001) and disease-free survival (p=0.001). Moreover, the predictive power was more profound for patients with ER negative and Her2 negative tumours. Discussion: Claudins, key tight junctional transmembrane proteins, have important prognostic value in human breast cancer. Together with their pivotal roles in controlling the adhesiveness and permeability of the cell layers, this family is a key player in the development and progression of breast cancer.

Abstract PD15-05: Sipa1 effects rock pathway in human breast cancer linking to HGF mediated changes in tight junction functions controlling metastasis

Abstract Background: SIPA1 (Signal-induced proliferation-associated gene 1) is a mitogen-inducible gene encoding a GTPase-activating protein for Rap1 and Rap2 and has been suggested to be involved in metastatic progression. We have previously shown SIPA1 may regulate barrier function in breast cancer cells as part of hepatocyte growth factor-determined changes and that this may be via a number of regulatory pathways, most likely via the ROCK pathway. Our aim with this current work was to definitively identify which pathway is involved. Methods: After knockdown of SIPA1 in MDA-MB-231 cells, they were treated to a number of inhibitors to proteins involved in pathways involved in TJ barrier function (ROCK, N-WASP, ARPs, WAVEs, MAPK, ERK, PLC-gamma). Following the identification of suitable inhibitor a large scale in vivo study was carried out using a tumour model of SIPA1 knockdown versus WT control was treated with/without inhibitors and immunohistochemistry (IHC) used to assess changes in tumour growth, architecture and protein expression. We also assessed the expression of SIPA-1 in a cohort of human breast cancer tissues using IHC. Results: After knockdown of SIPA1 in MDA-MB-231 cells, we subjected cells to a number of inhibitors to proteins involved in pathways involved barrier function (ROCK, N-WASP, ARPs, WAVEs, MAPK, ERK, PLC-gamma). Analysis of behaviour showed that knockdown cells no longer responded to the ROCK inhibitor (ROCKi Y-27632, p<0.05, n=10). In vivo tumour samples were assessed for expression of ROCKI, expression of phospho-ROCK, and any differences when treated with ROCKi. Control tissues showed good levels of all three proteins (SIPA1, ROCKI and phosphor-ROCK). Knockdown tissues exhibited significantly lower levels of ROCK and phosphor-ROCK (p<0.01, n=12). In control tumours, phosphor-ROCK was increased after treatment with ROCKi; conversely, in SIPA1 knockdown tumours, phosphor-ROCK was decreased after treatment with ROCKi (p<0.05). In SIPA1 knockdown tissues, ROCK and phosphor-ROCK was diffuse in staining pattern when compared to the control where the staining was located at the cell membrane. Conclusion: Our results confirm our hypothesis that SIPA1 is involved in the control of TJ in breast cancer and that this is through the ROCK pathway. Moreover, these results demonstrate that this may be due to the effect that SIPA1 has on cellular ratios of ROCK to phosphor-ROCK in breast cancer. Citation Format: Chang Liu, Fiona Ruge, Lin Ye, Gregory M. Harrison, Wen G. Jiang, Tracey A. Martin. Sipa1 effects rock pathway in human breast cancer linking to HGF mediated changes in tight junction functions controlling metastasis [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PD15-05.

Mechanism of endocytic regulation of intestinal tight junction remodeling during nutrient starvation in jejunal IPEC-J2 cells.

Intestinal epithelial cells are tightly bound by tight junction proteins (TJP) which are dynamic and sensitive to environmental stress. However, the role of the endocytic pathway in the regulation of TJP abundance and tight junction integrity during nutrient stress is poorly understood. Therefore, this study was conducted to investigate the regulation of TJP abundance during nutrient starvation and the role of the endocytic mechanism in this process. IPEC-J2 cells were subjected to nutrient starvation in Krebs-Ringer bicarbonate buffer (KRB) and abundance of TJP, an indication of tight junction remodeling, was characterized with RT-PCR, western blotting and immunofluorescence. Abundance of TJP was dynamically regulated by nutrient starvation. The protein levels of claudin-1, 3, and 4 were initially downregulated within the first 6 hours of starvation, and then, increased thereafter (P<.01). However, there was no change in occludin and ZO-1. Lysosome and proteasome inhibitors were used to determine the contribution of these protein degradation pathways to the TJP remodeling. Short-term starvation-induced degradation of claudin-1, 3, and 4 was found to be lysosome dependent. Specifically, the downregulation of claudin-3 and 4 was via a dynamin-dependent, but clathrin and caveolae independent, endocytic pathway and this downregulation was partly reversed by amino acids supplementation. Interestingly, the re-synthesis of TJP with prolonged starvation partly depended on proteasome function. Collectively, this study, for the first time, elucidated a major role for dynamin-dependent endocytosis of claudin-3 and 4 during nutrient stress in intestinal epithelial cells. Therefore, transient endocytosis inhibition may be a potential mechanism for preserving tight junction integrity and function in metabolic or pathological states such as inflammatory bowel disease that involves destruction of intestinal epithelial TJP.

KIF15 Supports Spermatogenesis Via Its Effects on Sertoli Cell Microtubule, Actin, Vimentin, and Septin Cytoskeletons

Throughout spermatogenesis, cellular cargoes including haploid spermatids are required to be transported across the seminiferous epithelium, either toward the microtubule (MT) plus (+) end near the basement membrane at stage V, or to the MT minus (-) end near the tubule lumen at stages VI to VIII of the epithelial cycle. Furthermore, preleptotene spermatocytes, differentiated from type B spermatogonia, are transported across the Sertoli cell blood-testis barrier (BTB) to enter the adluminal compartment. Few studies, however, have been conducted to explore the function of MT-dependent motor proteins to support spermatid transport during spermiogenesis. Herein, we examined the role of MT-dependent and microtubule plus (+) end-directed motor protein kinesin 15 (KIF15) in the testis. KIF15 displayed a stage-specific expression across the seminiferous epithelium, associated with MTs, and appeared as aggregates on the MT tracks that aligned perpendicular to the basement membrane and laid across the entire epithelium. KIF15 also tightly associated with apical ectoplasmic specialization, displaying strict stage-specific distribution, apparently to support spermatid transport across the epithelium. We used a loss-of-function approach by RNAi to examine the role of KIF15 in Sertoli cell epithelium in vitro to examine its role in cytoskeletal-dependent Sertoli cell function. It was noted that KIF15 knockdown by RNAi that reduced KIF15 expression by ~70% in Sertoli cells with an established functional tight junction barrier impeded the barrier function. This effect was mediated through remarkable changes in the cytoskeletal organization of MTs, but also actin-, vimentin-, and septin-based cytoskeletons, illustrating that KIF15 exerts its regulatory effects well beyond microtubules.

Molecular mechanism mediating enteric bacterial translocation after severe burn: the role of cystic fibrosis transmembrane conductance regulator.

BackgroundGut ischemia and hypoxia post severe burn leads to breakdown of intestinal epithelial barrier and enteric bacterial translocation (EBT), resulting in serious complications, such as systemic inflammatory response syndrome, sepsis and multiple organ failure. Cystic fibrosis transmembrane conductance regulator (CFTR) is known to be downregulated by hypoxia and modulate junctional complexes, which are crucial structures maintaining the intestinal barrier. This study aimed to investigate whether CFTR plays a role in both regulating the intestinal barrier and mediating EBT post severe burn, as well as the signaling pathways involved in these processes.MethodsAn in vitro Caco-2 cell model subjected to hypoxic injury and an in vivo mouse model with a 30% total body surface area full-thickness dermal burn were established. DF 508 mice (mice with F508del CFTR gene mutation) were used as an in vivo model to further demonstrate the role of CFTR in maintaining normal intestinal barrier function. QRT-PCR, western blot, ELISA, TER assay and immunofluorescence staining were used to detect the expression and localization of CFTR and tight junction proteins, as well as the function of tight junctions.ResultsOur data indicated that, in Caco-2 cells, the hypoxia condition significantly reduced CFTR expression; activated extracellular signal-regulated kinase and nuclear factor-κB signaling; elevated secretion of inflammatory factors (tumor necrosis factor-α, interleukin-1β and interleukin-8); downregulated zonula occludens-1, occludin and E-cadherin expression; decreased transepithelial electrical resistance values; and led to a cellular mislocation of ZO-1. More importantly, knockdown of CFTR caused similar alterations. The upregulation of inflammatory factors and downregulation of tight junction proteins (ZO-1 and occludin) induced by knockdown of CFTR could be reversed by specific extracellular signal-regulated kinase or nuclear factor-κB inhibition. In support of the in vitro data, exuberant secretion of pro-inflammatory mediators and EBT was observed in the intestine of severely burnt mice in vivo. EBT occurred in DF508 mice (mice with the F508del CFTR gene mutation), accompanied by augmented tumor necrosis factor-α, interleukin-1β and interleukin-8 levels in the ileum compared to wildtype mice. In addition, vitamin D3 was shown to protect the intestinal epithelial barrier from hypoxic injury.ConclusionsCollectively, the present study illustrated that CFTR and downstream signaling were critical in modulating the intestinal epithelial junction and EBT post severe burn.

Feed your microbiome and your heart: The gut-heart axis.

Cardiovascular and metabolic diseases are the leading causes of disability, morbidity, and mortality worldwide. Genetics plays an important role, but environmental factors change the game and hold the potential for prevention, reversibility, and applied therapy. Nutrition, phenotype, and behavior of microorganisms, intestinal eco-events, and intestinal permeability play a crucial role in the induction of diseases. The present mini-review summarizes nutrients, diets, microbial manipulations, and tight junction function modifiers that might prevent, modulate, or treat certain diseases.

Trifostigmanoside I, an Active Compound from Sweet Potato, Restores the Activity of MUC2 and Protects the Tight Junctions through PKCα/β to Maintain Intestinal Barrier Function.

Sweet potato (Ipomoea batata) is considered a superfood among vegetables and has been consumed for centuries. Traditionally, sweet potato is used to treat several illnesses, including diarrhea and stomach disorders. This study aimed to explore the protective effect of sweet potato on intestinal barrier function, and to identify the active compounds of sweet potato and their underlying mechanism of action. To this purpose, bioactivity-guided isolation, Western blotting, and immunostaining assays were applied. Interestingly, our bioactivity-guided approach enabled the first isolation and identification of trifostigmanoside I (TS I) from sweet potato. TS I induced mucin production and promoted the phosphorylation of PKCα/β in LS174T human colon cancer cells. In addition, it protected the function of tight junctions in the Caco-2 cell line. These findings suggest that TS I rescued the impaired abilities of MUC2, and protected the tight junctions through PKCα/β, to maintain intestinal barrier function.

Cell Sheets Restore Secretory Function in Wounded Mouse Submandibular Glands.

Thermoresponsive cell culture plates release cells as confluent living sheets in response to small changes in temperature, with recovered cell sheets retaining functional extracellular matrix proteins and tight junctions, both of which indicate formation of intact and functional tissue. Our recent studies demonstrated that cell sheets are highly effective in promoting mouse submandibular gland (SMG) cell differentiation and recovering tissue integrity. However, these studies were performed only at early time points and extension of the observation period is needed to investigate duration of the cell sheets. Thus, the goal of this study was to demonstrate that treatment of wounded mouse SMG with cell sheets is capable of increasing salivary epithelial integrity over extended time periods. The results indicate that cell sheets promote tissue organization as early as eight days after transplantation and that these effects endure through Day 20. Furthermore, cell sheet transplantation in wounded SMG induces a significant time-dependent enhancement of cell polarization, differentiation and ion transporter expression. Finally, this treatment restored saliva quantity to pre-wounding levels at both eight and twenty days post-surgery and significantly improved saliva quality at twenty days post-surgery. These data indicate that cell sheets engineered with thermoresponsive cell culture plates are useful for salivary gland regeneration and provide evidence for the long-term stability of cell sheets, thereby offering a potential new therapeutic strategy for treating hyposalivation.

PSIII-36 The endocytic pathway plays a major role in the regulation of tight junction remodeling during nutrient starvation in jejunal IPEC-J2 cells

Abstract Postweaning pigs are subjected to nutrient deprivation during which intestinal epithelial cells undergo increased turnover. To preserve intestinal function, intestinal epithelial cells must activate adaptive mechanisms that allow them to cope with starvation-induced stress; most importantly, the preservation of intestinal barrier function. The objective of this study was to investigate the underlying mechanisms involved in starvation-induced alteration of tight junction protein abundance and function in IPEC-J2 cells. Cells were subjected to total nutrient starvation in Krebs-Ringer bicarbonate (KRB) buffer for 0, 3, 6, 12 and 24 h. Abundance of tight junction proteins was determined by RT-PCR, western blotting and immunofluorescence. Compared with control group (0 h), the protein expression of claudin 1, claudin 3 and claudin 4 protein was downregulated up to 6 h of starvation and then increased thereafter (P &amp;lt; 0.01). However, there was no change in the protein level of occludin and ZO-1. To determine the contribution of the lysosome and the ubiquitin proteasome pathways to regulation of tight junction protein abundance, the lysosome (Bafilomycin A1) and the proteasome (MG132) inhibitors were used in nutrient starved cells. Results showed the degradation of claudin 1, 3 and 4 up to 6 h of starvation was through the lysosomal pathway. Surprisingly, re-synthesis of claudins 4 and claudin 3 after prolonged starvation (12 and 24 h) was prevented when cells were treated with bafilomycin A1 and MG132, respectively. The autophagy-lysosome pathway inhibitors (Wortmannin and MHY1485) and endosome-lysosome pathway inhibitors (Dynasore and Pitstop 2) were further used to determine the specific roles of these pathways. In summary, the degradation of claudin 3 and claudin 4 during short-term starvation (up to 6 h) was through the dynamin-dependent endocytic pathway. However, re-synthesis of these proteins after prolonged starvation relies on both the lysosome and proteasome pathways.