Establishment Of Tight Junctions Research Articles

3D MORPHOGENESIS AND BACTERIAL CO-CULTURE IN A CANINE GUT-ON-A-CHIP USING BIOPSY-DERIVED INTESTINAL ORGANOIDS FROM HEALTHY AND IBD PATIENTS

Abstract The understanding of the role of intestinal bacteria in various intestinal diseases has been limited due to the lack of suitable in vitro models. However, recent advancements in Gut-on-a-Chip culture technology provide a promising avenue for exploring complex interactions between intestinal epithelium and bacteria. In this study, we aimed to create a microfluidic Gut-on-a-Chip co-culture system using dogs as a model. Dogs share similarities with humans in terms of intestinal disease pathophysiology, clinical presentations, and intestinal microbiome, making them a relevant model for various intestinal diseases, including inflammatory bowel disease (IBD). We cultured colonoids from both healthy dogs and dogs with IBD on the Gut-on-a-Chip platform to create villus-like structures under dynamic conditions. Co-cultures with non-pathogenic Escherichia coli (NPE) were established in the Gut-on-a-Chip with healthy colonoids, and we conducted a static Transwell (TW) co-culture for comparison. To assess epithelial barrier integrity, we measured transepithelial electrical resistance (TEER), and we evaluated the expression of ZO-1, a tight junction protein, through immunofluorescence staining (IF). Following 6-9 days of uninterrupted medium flow in the Gut-on-a-Chip models, we observed sporadic clusters of villus-like structures (i.e., 3D morphogenesis) in canine intestinal epithelial cells in both the models derived from healthy dogs and those with IBD. The IF confirmed the presence of ZO-1, indicating the establishment of tight junctions. Infection with NPE in the TW system resulted in a significant reduction in TEER (48.2±4.4% at 12 hours and 7.3±5.3% at 24 hours). In contrast, the Gut-on-a-Chip maintained TEER levels at 100.3±12.8% at 12 hours, 94.9±2.2% at 24 hours, and 88.8±15.0% at 48 hours, preserving the 3D structure. The innovative canine Gut-on-a-Chip model, capable of reproducing villus-like structures and preserving a 3D configuration using both healthy and diseased organoids (specifically, IBD), presents a valuable platform for investigating bacterial-epithelium interactions in both human and veterinary medicine. This dynamic microfluidic culture system enables the co-cultivation of living bacteria while maintaining epithelial integrity, facilitating the study of intricate interactions between intestinal bacteria and epithelial crosstalk.

The Role of ZIP9 and Androgen Receptor in the Establishment of Tight Junctions between Adult Rat Sertoli Cells.

The blood-testis barrier (BTB) is formed from tight junctions (TJs) between Sertoli cells. This dynamic structure, which establishes an immune-privileged environment protecting haploid germ cells formed in puberty from cells of the innate immune system, protects male fertility. Testosterone produced in Leydig cells is one of the main regulators of TJ protein expression and BTB dynamics. Nevertheless, although it has been assumed that testosterone effects on TJs and BTB are mediated through the classical androgen receptor (AR), newer results call the importance of this receptor into question. ZIP9, a recently identified androgen receptor of plasma membranes, mediates testosterone effects that promote the expression of TJ proteins and TJ formation in a rat Sertoli cell line that lacks the classical AR. Although these findings suggest that ZIP9 mediates these testosterone effects, participation of the classical AR in these events cannot be excluded. Here we used immortalized adult rat Sertoli cells that express both ZIP9 and AR and addressed the involvement of these receptors in the stimulation of TJ protein expression and TJ formation in response to testosterone and to the androgenic peptide IAPG that acts via ZIP9. We find that both testosterone and IAPG trigger the so-called non-classical signaling pathway of testosterone and stimulate the expression of TJ-associated proteins and TJ formation. Silencing classical AR expression had no effect on the responses, whereas silencing of ZIP9 expression completely blocked them. Our results demonstrate that ZIP9 is the sole androgen receptor involved in the regulation of TJ protein expression and TJ formation at the BTB.

Expression patterns and biological function of Specc1 during mouse preimplantation development

Two unique features occur during preimplantation embryo development: 1) initiation of calcium-dependent adhesion and establishment of apicobasal polarity in the morula, and 2) formation of the blastocoel by establishment of tight junctions (TJs), ion channels, and water channels in the outer blastomeres. Although several key genes involved in morula and blastocyst formation have been identified, most remain unknown. Sperm antigen with calponin homology and coiled-coil domains 1(SPECC1) is highly expressed in testis and tumor cells, and is involved in diverse cellular processes such as ribosome biogenesis, rRNA transcription, mitosis, cell growth, and apoptosis in tumor cells. However, spatiotemporal expressions of Specc1 during mouse preimplantation development have not yet been investigated. Here, we examined the expression patterns of Specc1 using qRT-PCR and immunocytochemistry, and its biological function using siRNA injection into 1-cell zygotes. Specc1 was detectable throughout preimplantation development and markedly increased from the morula stage onwards. It was particularly observed in trophectoderm cells, rather than the inner cell mass of blastocyst. Maternal and zygotic Specc1 transcripts were abolished using RNA interference. There were no significant differences in development between Specc1 knock down (KD) and control embryos until the morula stage, but was significantly reduced blastocyst development and increased tight junction permeability in KD embryos, as assessed by FITC uptake. In summary, elevated expression of Specc1 in the morula and blastocyst may affect blastocyst formation, including tight junction complex during the morula to blastocyst transition.

Shear-Enhanced Dynamic Adhesion of Lactobacillus rhamnosus GG on Intestinal Epithelia: Correlative Effect of Protein Expression and Interface Mechanics.

The oral uptake of probiotic microorganisms as food additives is one widely used strategy to sustain and improve the homeostasis of intestinal microbiota that protect the intestinal epithelia from attack by pathogenic bacteria. Once delivered to the ileum and colon, probiotics must adhere and form colonies on mucus that coats the surface of intestinal epithelial cells. Although an increasing amount of knowledge about the genetic and molecular level mechanisms of probiotics-mucus interactions has been accumulated, little is known about the physicochemical aspects of probiotics-mucus interactions under physiological shear in intestines. In this study, we established well-defined models of intestinal epithelial cell monolayers based on two major constituents of gut epithelia, enterocytes and goblet cells. First, the formation of a polarized cell monolayer sealed by tight junctions was monitored by transepithelial electrical resistance over time. The establishment of tight junctions and secretion of mucus proteins (mucin) was confirmed by immunofluorescence staining. In the next step, we measured the elasticity of cell monolayer surfaces by indentation using particle-assisted atomic force microscopy. The effective elastic modulus of goblet cell-like cells was 30 times smaller compared to that of enterocyte-like cells, which can be attributed to the secretion of a 3 μm thick mucin layer. As probiotics, we used Lactobacillus rhamnosus GG (LGG), which is one of the most widely used strains as food additives. To investigate the dynamic adhesion of LGG to the intestine model surface, we transferred the epithelial cell monolayer into a microfluidic chamber. A distinct difference in dynamic adhesion between two cell types was observed, which could be attributed to the difference in the mucin expression amount. Remarkably, we found that the dynamic LGG adhesion is enhanced by the increase in shear stress, showing a maximum binding efficiency at 0.3 Pa. Finally, we examined the persistence of LGG adhesion by a stepwise increase in the shear stress exerted on adherent LGG, demonstrating that LGG could withstand high shear stress even beyond that of physiological stress. The obtained results present a large potential to quantitatively understand the influence of engineered foods and probiotics on the homeostasis of microbiota on the surface of intestinal epithelia.

Regulation of Tight Junction Assembly and Epithelial Polarity by a Resident Protein of Apical Endosomes

The establishment of tight junctions and cell polarity is an essential process in all epithelia. Endotubin is an integral membrane protein found in apical endosomes of developing epithelia when tight junctions and epithelial polarity first arise. We found that the disruption of endotubin function in cells in culture by siRNA or overexpression of the C-terminal cytoplasmic domain of endotubin causes defects in organization and function of tight junctions. We observe defects in localization of tight junction proteins, reduced transepithelial resistance, increased lanthanum penetration between cells and reduced ability of cells to form cysts in three-dimensional culture. In addition, in cells overexpressing the C-terminal domain of endotubin, we observe a delay in re-establishing the normal distribution of endosomes after calcium switch. These results suggest that endotubin regulates trafficking of polarity proteins and tight junction components out of the endosomal compartment, thereby providing a critical link between a resident protein of apical endosomes and tight junctions.

ROCK-mediated contractility, tight junctions and channels contribute to the conversion of a preapical patch into apical surface during isochoric lumen initiation

Epithelial cells assemble into three-dimensional aggregates to generate lumen-containing organ substructures. Cells therein contact the extracellular matrix with their basal surface, neighbouring cells with their contact surface and the lumen with their apical surface. We investigated the development of single MDCK cells into aggregates with lumen using quantitative live-cell imaging to identify morphogenetic rules for lumen formation. In two-cell aggregates, membrane insertion into the contact surface established a preapical patch (PAP) characterized by the presence of the apical marker gp135, microvilli and the absence of E-cadherin. This PAP originated from a compartment that had hallmarks of an apical recycling endosome, and matured through Brefeldin-A-sensitive membrane trafficking and the establishment of tight junctions around itself. As a result of the activity of water and ion channels, an optically resolvable lumen formed. Initially, this lumen enlarged without changes in aggregate volume or cell number but with decreasing cell volumes. Additionally, the ROCK1/2-myosin-II pathway counteracted PAP and lumen formation. Thus, lumen formation results from PAP establishment, PAP maturation, lumen initiation and lumen enlargement. These phases correlate with distinct cell surface and volume patterns, which suggests that such morphometric parameters are regulated by trafficking, ROCK-mediated contractility and hydrostatic pressure or vice versa.

Salmonella entericaserovar Typhimurium regulates intercellular junction proteins and facilitates transepithelial neutrophil and bacterial passage

The establishment of tight junctions (TJ) between columnar epithelial cells defines the functional barrier, which enteroinvasive pathogens have to overcome. Salmonella enterica serovar Typhimurium (S. typhimurium) directly invades intestinal epithelial cells but it is not well understood how the pathogen is able to overcome the intestinal barrier and gains access to the circulation. Therefore, we sought to determine whether infection with S. typhimurium could regulate the molecular composition of the TJ and, if so, whether these modifications would influence bacterial translocation and polymorphonuclear leukocyte (PMN) movement across model intestinal epithelium. We found that infection of a model intestinal epithelium with S. typhimurium over 2 h resulted in an approximately 80% loss of transepithelial electrical resistance. Western blot analysis of epithelial cell lysates demonstrated that S. typhimurium regulated the distribution of the TJ complex proteins claudin-1, zonula occludens (ZO)-2, and E-cadherin in Triton X-100-soluble and insoluble fractions. In addition, S. typhimurium was specifically able to dephosphorylate occludin and degrade ZO-1. This TJ alteration in the epithelial monolayer resulted in 10-fold increase in bacterial translocation and a 75% increase in N-formylmethionin-leucyl-phenyalanine-induced PMN transepithelial migration. Our data demonstrate that infection with S. typhimurium is associated with the rapid targeting of the tight junctional complex and loss of barrier function. This results in enhanced bacterial translocation and initiation of PMN migration across the intestinal barrier. Therefore, the ability to regulate the molecular composition of TJs facilitates the pathogenicity of S. typhimurium by aiding its uptake and distribution within the host.

Normal Establishment of Epithelial Tight Junctions in Mice and Cultured Cells Lacking Expression of ZO-3, a Tight-Junction MAGUK Protein

ZO-1, ZO-2, and ZO-3 are closely related MAGUK family proteins that localize at the cytoplasmic surface of tight junctions (TJs). ZO-1 and ZO-2 are expressed in both epithelia and endothelia, whereas ZO-3 is exclusively expressed in epithelia. In spite of intensive studies of these TJ MAGUKs, our knowledge of their functions in vivo, especially those of ZO-3, is still fragmentary. Here, we have generated mice, as well as F9 teratocarcinoma cell lines, that do not express ZO-3 by homologous recombination. Unexpectedly, ZO-3(-/-) mice were viable and fertile, and rigorous phenotypic analyses identified no significant abnormalities. Moreover, ZO-3-deficient F9 teratocarcinoma cells differentiated normally into visceral endoderm epithelium-like cells in the presence of retinoic acid. These cells had a normal epithelial appearance, and the molecular architecture of their TJs did not appear to be affected, except that TJ localization of ZO-2 was upregulated. Suppression of ZO-2 expression by RNA interference in ZO-3(-/-) cells, however, did not affect the architecture of TJs. Furthermore, the speed with which TJs formed after a Ca(2+) switch was indistinguishable between wild-type and ZO-3(-/-) cells. These findings indicate that ZO-3 is dispensable in vivo in terms of individual viability, epithelial differentiation, and the establishment of TJs, at least in the laboratory environment.

Hyaluronan is apically secreted and expressed by proliferating or regenerating renal tubular cells

Hyaluronan has diverse biologic functions in the body, varying from structural tasks to cell stress-induced CD44-mediated activation of intracellular signaling pathways. Hyaluronan biology is relatively unexplored in the kidney. Previously, we identified hyaluronan as binding molecule for crystals in the renal tubules. Crystal retention is a crucial early event in the etiology of kidney stones. The present study was performed to determine the polarized distribution of hyaluronan and CD44 by renal tubular cells. Madin-Darby canine kidney (MDCK) strain I and primary cultures of human renal tubular cells were grown on permeable supports in a two-compartment culture system. Studies were performed during growth and after scrape-injury. Metabolic labeling studies and an enzyme-linked hyaluronan -binding assay were used to measure the molecular mass and the amount of secreted hyaluronan in apical and basal medium. Confocal microscopy was applied to detect membrane hyaluronan and CD44. Hyaluronan synthase (HAS) mRNA expression was studied with reverse transcriptase-polymerase chain reaction (RT-PCR). The in vitro expression profile of hyaluronan was compared with that in biopsies of transplanted human kidneys with acute tubular necrosis. Proliferating cells produced more hyaluronan (M(r) > 10(6) Da) than growth-inhibited cells in intact monolayers and up to 85% was targeted to the apical compartment, which was accompanied by increased HAS2 mRNA expression and slightly decreased HAS3 mRNA, while HAS1 mRNA remained undetectable. Hyaluronan and CD44 were exclusively expressed at the apical surface of proliferating/regenerating cells. After (re)establishment of tight junctions, hyaluronan was no longer detectable while CD44 was targeted to basolateral membrane domains. In vivo in inflamed human kidneys hyaluronan was abundantly expressed in the cortical tubulointerstitial space as well as at the luminal surface of regenerating renal tubular cells. These results demonstrate that the production of hyaluronan by renal tubular cells is activated during proliferation and in response to mechanical injury and that hyaluronan and CD44 expression is highly polarized. The targeted delivery of hyaluronan to the apical compartment suggests that hyaluronan produced by renal tubular cells supports proliferation/regeneration in the renal tubules, but that it does not contribute to hyaluronan accumulation in the renal interstitium. These data further support the concept that mitogen/stress-induced hyaluronan deposition in the renal tubules increases the risk for crystal retention and stone formation.

Establishment of tight junctions between cells from different animal species and different sealing capacities

Epithelial cells establish tight junctions (TJs) that offer an ample range of transepithelial electrical resistances (TER), in adjustment to physiological requirements. In the present work, we demonstrate that cells from different animal origins, co-cultured in monolayers, can make sealed TJs, suggesting that this structure has a basic universal structure. TJs cannot be established, however, if one of the partners does not normally express TJs, indicating that each neighbor has to contribute its moiety. Furthermore, we observe that clones of the same cell line, with widely different values of TER, do not differ in the number and length of their junctional strands, suggesting that the difference is due to their ability to express ionic channels traversing their strands. The value of TER achieved in mixed monolayers of cells of the same or different lines is the one that may be expected by taking into account the proportion of each type in the mixture and adding in parallel the electrical resistance that they exhibit in pure monolayers. Therefore, epithelial TJs appear to behave as parallel resistances.

Establishment of tight junctions between epithelial cells.

Epithelia serve as barriers to the diffusion of solutes between body compartments, and must do so despite the frequent loss of cells. When single cells are experimentally removed from the Necturus gallbladder epithelium, contiguous cells migrate to fill the defect within 30 min. Electrophysiological measurements show that the local electrical resistance across the epithelium in the region of a wound returns to normal in the same period of time; electron microscopy demonstrates that tight junctions are formed concurrently. Physiologically functional and morphologically recognizable tight junctions can thus be established within 30 min, demonstrating a mechanism for the rapid restoration of epithelial integrity after cell loss.