Intercellular Junctional Complexes Research Articles

Overview of the compromised mucosal integrity in celiac disease.

Intestinal epithelium is a dynamic cellular layer that lines the small-bowel and makes a relatively impenetrable barrier to macromolecules. Intestinal epithelial cell polarity is crucial in coordinating signalling pathways within cells and mainly regulated by three conserved polarity protein complexes, the Crumbs (Crb) complex, partitioning defective (PAR) complex, and Scribble (Scrib) complex. Polarity proteins regulate the proper establishment of the intercellular junctional complexes including tight junctions (TJs), adherence junctions (AJs), and desmosomes which hold epithelial cells together and play a major role in maintaining intestinal barrier integrity. Impaired intestinal epithelial cell polarity and barrier integrity result in irreversible immune responses, the host- microbial imbalance and intestinal inflammatory disorders. Disassembling the epithelial tight junction and augmented paracellular permeability is a conspicuous hallmark of celiac disease (CD) pathogenesis. There are several dietary components that can improve intestinal integrity and function. The aim of this review article is to summarize current information about the association of polarity proteins and AJC damages with pathogenesis of CD.

In Vitro and In Vivo Models for Drug Transport Across the Blood-Testis Barrier.

The blood-testis barrier (BTB) is a selectively permeable membrane barrier formed by adjacent Sertoli cells (SCs) in the seminiferous tubules of the testes that develops intercellular junctional complexes to protect developing germ cells from external pressures. However, due to this inherent defense mechanism, the seminiferous tubule lumen can act as a pharmacological sanctuary site for latent viruses (e.g., Ebola, Zika) and cancers (e.g., leukemia). Therefore, it is critical to identify and evaluate BTB carrier-mediated drug delivery pathways to successfully treat these viruses and cancers. Many drugs are unable to effectively cross cell membranes without assistance from carrier proteins like transporters because they are large, polar, and often carry a charge at physiologic pH. SCs express transporters that selectively permit endogenous compounds, such as carnitine or nucleosides, across the BTB to support normal physiologic activity, although reproductive toxicants can also use these pathways, thereby circumventing the BTB. Certain xenobiotics, including select cancer therapeutics, antivirals, contraceptives, and environmental toxicants, are known to accumulate within the male genital tract and cause testicular toxicity; however, the transport pathways by which these compounds circumvent the BTB are largely unknown. Consequently, there is a need to identify the clinically relevant BTB transport pathways in in vitro and in vivo BTB models that recapitulate human pharmacokinetics and pharmacodynamics for these xenobiotics. This review summarizes the various in vitro and in vivo models of the BTB reported in the literature and highlights the strengths and weaknesses of certain models for drug disposition studies. SIGNIFICANCE STATEMENT: Drug disposition to the testes is influenced by the physical, physiological, and immunological components of the blood-testis barrier (BTB). But many compounds are known to cross the BTB by transporters, resulting in pharmacological and/or toxicological effects in the testes. Therefore, models that assess drug transport across the human BTB must adequately account for these confounding factors. This review identifies and discusses the benefits and limitations of various in vitro and in vivo BTB models for preclinical drug disposition studies.

The state of the intestinal microbial-tissue complex in patients with chronic kidney disease

The gut microbiota plays a fundamental role in maintaining normal organism homeostasis, regulating a wide range of metabolic, biosynthetic, and immune functions. This complex ecosystem, together with cellular, and stromal components of the intestinal wall, forms the intestinal microbial-tissue complex, which pathology is considered a universal mechanism for the development of many diseases, including chronic kidney disease. Accumulation of nitrogen metabolism products in the intestine, specific diet, polypharmacy, sedentary lifestyle, limited fluid intake, and violation of gastrointestinal motility in patients with chronic kidney disease lead to a decrease in the number of bacteria synthesizing short-chain fatty acids with crucial physiological effects, along with an increase in the content of anaerobic proteolytic bacteria expressing uricase, urease, and p-cresol-, and indole-forming enzymes. The features of gut dysbiosis depend on the etiology of chronic kidney disease and severity of renal insufficiency and differ significantly in individuals receiving various renal replacement therapies (hemodialysis, peritoneal dialysis, kidney transplant recipients). Abnormal microbial metabolism enhances the production and accumulation of microbial-derived uremic toxins: p-cresyl sulfate, indoxyl sulfate, and trimethylamine-N-oxide. Renal insufficiency, through mechanisms mainly associated with the hydrolysis of accumulated in the lumen of the intestine urea, leads to inflammation, and swelling of the intestinal wall, which is accompanied by disorders of the immune tolerance of the mucous layer and disorganization of intercellular junctional complexes, which are critical modulators of intestinal intercellular transepithelial transport. Uremia-induced impairment of the gut epithelial barrier integrity induces the systemic translocation of numerous immunogenic substances generated by the aberrant microbiota, followed by the development of oxidative stress and chronic subclinical inflammation that causes the progression of chronic kidney disease and related complications. The most pronounced changes were observed in people with end-stage chronic kidney disease. Further study of the bidirectional relationship between the kidneys and intestinal microbial-tissue complex will contribute to the development of new directions of pathogenetic therapy and prevention of adverse outcomes in patients with chronic kidney disease.

Drug Transporters at the Human Blood-Testis Barrier.

Transporters are involved in the movement of many physiologically important molecules across cell membranes and have a substantial impact on the pharmacological and toxicological effect of xenobiotics. Many transporters have been studied in the context of disposition to, or toxicity in, organs such as the kidney and liver; however, transporters in the testes are increasingly gaining recognition for their role in drug transport across the blood-testis barrier (BTB). The BTB is an epithelial membrane barrier formed by adjacent Sertoli cells (SCs) in the seminiferous tubules that form intercellular junctional complexes to protect developing germ cells from the external environment. Consequently, many charged or large polar molecules cannot cross this barrier without assistance from a transporter. SCs express a variety of drug uptake and efflux transporters to control the flux of endogenous and exogenous molecules across the BTB. Recent studies have identified several transport pathways in SCs that allow certain drugs to circumvent the human BTB. These pathways may exist in other species, such as rodents and nonhuman primates; however, there is (1) a lack of information on their expression and/or localization in these species, and (2) conflicting reports on localization of some transporters that have been evaluated in rodents compared with humans. This review outlines the current knowledge on the expression and localization of pharmacologically relevant drug transporters in human testes and calls attention to the insufficient and contradictory understanding of testicular transporters in other species that are commonly used in drug disposition and toxicity studies. SIGNIFICANCE STATEMENT: While the expression, localization, and function of many xenobiotic transporters have been studied in organs such as the kidney and liver, the characterization of transporters in the testes is scarce. This review summarizes the expression and localization of common pharmacologically-relevant transporters in human testes that have significant implications for the development of drugs that can cross the blood-testis barrier. Potential expression differences between humans and rodents highlighted here suggest rodents may be inappropriate for some testicular disposition and toxicity studies.

Classical cadherins in the testis: how are they regulated?

Cadherins (CDH) are crucial intercellular adhesion molecules, contributing to morphogenesis and creating tissue barriers by regulating cells' movement, clustering and differentiation. In the testis, classical cadherins such as CDH1, CDH2 and CDH3 are critical to gonadogenesis by promoting the migration and the subsequent clustering of primordial germ cells with somatic cells. While CDH2 is present in both Sertoli and germ cells in rodents, CDH1 is primarily detected in undifferentiated spermatogonia. As for CDH3, its expression is mainly found in germ and pre-Sertoli cells in developing gonads until the establishment of the blood-testis barrier (BTB). This barrier is made of Sertoli cells forming intercellular junctional complexes. The restructuring of the BTB allows the movement of early spermatocytes toward the apical compartment as they differentiate during a process called spermatogenesis. CDH2 is among many junctional proteins participating in this process and is regulated by several pathways. While cytokines promote the disassembly of the BTB by enhancing junctional protein endocytosis for degradation, testosterone facilitates the assembly of the BTB by increasing the recycling of endocytosed junctional proteins. Mitogen-activated protein kinases (MAPKs) are also mediators of the BTB kinetics in many chemically induced damages in the testis. In addition to regulating Sertoli cell functions, follicle stimulating hormone can also regulate the expression of CDH2. In this review, we discuss the current knowledge on regulatory mechanisms of cadherin localisation and expression in the testis.

ACTIVATION OF AKT, MAPK3/1 AND mTOR SIGNALING PATHWAYS IS ASSOCIATED WITH THE SUCCESS OF CAVITY FORMATION DURING HUMAN BLASTOID GENERATION

Cavitation is a crucial process during human blastocyst development that involves combined actions of ion gradient pumps, water channels, and intercellular junction complexes that assemble on the apical and basolateral membranes of trophectoderm cells, which are dependent on a series of metabolic signaling pathways that are poorly understood in humans. Here, we utilized the blastoid model derived from human naïve pluripotent stem cells to examine which metabolic signaling pathways are important for blastoid cavitation.

Intestinal mucosal barrier in functional constipation: Dose it change?

BACKGROUNDThe intestinal mucosal barrier is the first line of defense against numerous harmful substances, and it contributes to the maintenance of intestinal homeostasis. Recent studies reported that structural and functional changes in the intestinal mucosal barrier were involved in the pathogenesis of several intestinal diseases. However, no study thoroughly evaluated this barrier in patients with functional constipation (FC).AIMTo investigate the intestinal mucosal barrier in FC, including the mucus barrier, intercellular junctions, mucosal immunity and gut permeability.METHODSForty FC patients who fulfilled the Rome IV criteria and 24 healthy controls were recruited in the Department of Gastroenterology of China-Japan Friendship Hospital. The colonic mucus barrier, intercellular junctions in the colonic epithelium, mucosal immune state and gut permeability in FC patients were comprehensively examined. Goblet cells were stained with Alcian Blue/Periodic acid Schiff (AB/PAS) and counted. The ultrastructure of intercellular junctional complexes was observed under an electron microscope. Occludin and zonula occludens-1 (ZO-1) in the colonic mucosa were located and quantified using immunohistochemistry and quantitative real-time polymerase chain reaction. Colonic CD3+ intraepithelial lymphocytes (IELs) and CD3+ lymphocytes in the lamina propria were identified and counted using immunofluorescence. The serum levels of D-lactic acid and zonulin were detected using enzyme-linked immunosorbent assay.RESULTSCompared to healthy controls, the staining of mucus secreted by goblet cells was darker in FC patients, and the number of goblet cells per upper crypt in the colonic mucosa was significantly increased in FC patients (control, 18.67 ± 2.99; FC, 22.42 ± 4.09; P = 0.001). The intercellular junctional complexes in the colonic epithelium were integral in FC patients. The distribution of mucosal occludin and ZO-1 was not altered in FC patients. No significant differences were found in occludin (control, 5.76E-2 ± 1.62E-2; FC, 5.17E-2 ± 1.80E-2; P = 0.240) and ZO-1 (control, 2.29E-2 ± 0.93E-2; FC, 2.68E-2 ± 1.60E-2; P = 0.333) protein expression between the two groups. The mRNA levels in occludin and ZO-1 were not modified in FC patients compared to healthy controls (P = 0.145, P = 0.451, respectively). No significant differences were observed in the number of CD3+ IELs per 100 epithelial cells (control, 5.62 ± 2.06; FC, 4.50 ± 2.16; P = 0.070) and CD3+ lamina propria lymphocytes (control, 19.69 ± 6.04/mm2; FC, 22.70 ± 11.38/mm2; P = 0.273). There were no significant differences in serum D-lactic acid [control, 5.21 (4.46, 5.49) mmol/L; FC, 4.63 (4.31, 5.42) mmol/L; P = 0.112] or zonulin [control, 1.36 (0.53, 2.15) ng/mL; FC, 0.94 (0.47, 1.56) ng/mL; P = 0.185] levels between FC patients and healthy controls.CONCLUSIONThe intestinal mucosal barrier in FC patients exhibits a compensatory increase in goblet cells and integral intercellular junctions without activation of mucosal immunity or increased gut permeability.

The Role of Pseudomonas aeruginosa Virulence Factors in Cytoskeletal Dysregulation and Lung Barrier Dysfunction.

Pseudomonas (P.) aeruginosa is an opportunistic pathogen that causes serious infections and hospital-acquired pneumonia in immunocompromised patients. P. aeruginosa accounts for up to 20% of all cases of hospital-acquired pneumonia, with an attributable mortality rate of ~30–40%. The poor clinical outcome of P. aeruginosa-induced pneumonia is ascribed to its ability to disrupt lung barrier integrity, leading to the development of lung edema and bacteremia. Airway epithelial and endothelial cells are important architecture blocks that protect the lung from invading pathogens. P. aeruginosa produces a number of virulence factors that can modulate barrier function, directly or indirectly, through exploiting cytoskeleton networks and intercellular junctional complexes in eukaryotic cells. This review summarizes the current knowledge on P. aeruginosa virulence factors, their effects on the regulation of the cytoskeletal network and associated components, and molecular mechanisms regulating barrier function in airway epithelial and endothelial cells. A better understanding of these processes will help to lay the foundation for new therapeutic approaches against P. aeruginosa-induced pneumonia.

Enigmatic Neural Pathways: Potentiating Viral Neuroinvasion Into the CNS.

Enigmatic Neural Pathways: Potentiating Viral Neuroinvasion Into the CNS.

Abstract 3120: MMP-7 increases migration by decreasing intercellular cell junction complexes in prostate cancer microtumors formed by perlecan/HSPG2

Abstract Background: Polarized epithelium is stabilized by lateral cell-cell interactions via cell adhesion molecules (CAMs) and by cell-matrix interactions with basement membrane including with perlecan/HSPG2 (pln). Prostate cancer (PCa) patients with bone metastases have circulating pln fragments, with inverse correlation between MMP-7 staining and loss of pln in cancer tissue. Cleavage of pln by MMP-7 increases cell-matrix interactions and dysregulates cell signaling, permitting migration. We earlier showed pln domain IV-3 (DmIV-3) drives cell-cell cohesion and, when digested with MMP-7, drives cell dyscohesion. Methods: To evaluate the impact of MMP-7 and pln fragments on microtumor dyscohesion and cell migration, uniformly sized PCa microtumors were pre-formed in a microwell system. Pre-formed microtumors were transferred to Dm IV-3 or full length perlecan (FL pln) coated wells for 16-24 hrs. Microtumors were treated with MMP-7 alone or MMP-7 plus predigested Dm IV-3 fragments or MMP-7 plus FL pln fragments. For live cell imaging, tracking of migratory cells leaving microtumors was performed with Imaris software. Co-location of cell adhesion complex components was assessed with Imaris Spots. Results: Pre-formed microtumors cultured with DmIV-3 cleaved by MMP-7 showed lower Pearson's correlation values at cell boundaries compared to microtumors treated with intact Dm IV-3. Line scan analysis revealed E-cadherin and F-actin fluorescent signals were enriched and co-aligned in microtumors treated with Dm IV-3; enrichment and co-alignment were reduced when DmIV-3 fragments and MMP-7 were present. Track number detected per cell cluster was highest in the presence of FL pln fragments plus MMP-7 along with a measurable change in distribution of track displacement lengths of cells toward high values. Conclusion: Boundary reorganization promotes a migratory cell phenotype in microtumors treated with MMP-7 and DmIV-3 fragments. DmIV-3 fragments generated by MMP-7 cleavage can enhance cell dyscohesion by disrupting interactions between CAMs. Ongoing work will identify DmIV-3 fragment(s) positively associated with tumor dyscohesion that play key roles in secondary metastasis formation. Following patterns of dyscohesion of pre-formed microtumors provides a good model to study dynamic changes in intercellular junction components and quantitate cell migration patterns that foster circulating tumor cell formation and metastasis. Citation Format: Lissette A. Cruz, Tristen V. Tellman, Oleg Igoshin, Daniel Carson, Mary (Cindy) Farach-Carson. MMP-7 increases migration by decreasing intercellular cell junction complexes in prostate cancer microtumors formed by perlecan/HSPG2 [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 3120.

Observation of a tight junction structure generated in LbL-3D skin reconstructed by layer-by-layer cell coating technique.

Tissue-engineered skin equivalents are reconstructed the functions of human skin and can be used as an alternative to animal experiments in basic study or as cultured skin for regenerative medicine. Recent studies confirmed that epidermal tight junctions (TJs), which are complex intercellular junctions formed in the stratum granulosum of human skin, play an important part in the formation of the skin barrier function. In well-formed reconstructed human skin models, there are several reports on the expression of TJ proteins and their localization in epidermal layer, however, the morphological features of TJ, showing tight junctional contacts and the process of TJ formation have yet to be investigated. In this study, we systematically examined and identified TJ-related proteins and TJ structure in three-dimensional (3D) human skin equivalents reconstructed by layer-by-layer (LbL) cell coating technique (LbL-3D Skin). We demonstrate localization of TJ-related proteins and time course of formation of TJ structure with typical junctional morphology in LbL-3D Skin. These data provide evidence that the LbL-3D Skin is an in vitro model with structure and function extremely similar to living skin.

NMR-Guided Repositioning of Non-Steroidal Anti-Inflammatory Drugs into Tight Junction Modulators.

Bioavailability is a major bottleneck in the clinical application of medium molecular weight therapeutics, including protein and peptide drugs. Paracellular transport of these molecules is hampered by intercellular tight junction (TJ) complexes. Therefore, safe chemical regulators for TJ loosening are desired. Here, we showed a potential application of select non-steroidal anti-inflammatory drugs (NSAIDs) as TJ modulators. Based on our previous observation that diclofenac and flufenamic acid directly bound various PDZ domains with a broad specificity, we applied solution nuclear magnetic resonance techniques to examine the interaction of other NSAIDs and the first PDZ domain (PDZ1) of zonula occludens (ZO)-1, ZO-1(PDZ1). Inhibition of ZO-1(PDZ1) is expected to provide loosening of the epithelial barrier function because the domain plays a crucial role in maintaining TJ integrity. Accordingly, diclofenac and indomethacin were found to decrease the subcellular localization of claudin (CLD)-2 but not occludin and ZO-1 at the apicolateral intercellular compartment of Madin–Darby canine kidney (MDCK) II cells. These NSAIDs exhibited 125–155% improved paracellular efflux of fluorescein isothiocyanate insulin for the Caco-2 cell monolayer. We propose that these NSAIDs can be repurposed as drug absorption enhancers for peptide drugs.

Anionic nanoparticles enable the oral delivery of proteins by enhancing intestinal permeability.

The oral delivery of bioactive peptides and proteins is prevented by the intestinal epithelial barrier, in which intercellular tight junction complexes block the uptake of macromolecules. Here we show that anionic nanoparticles induce tight junction relaxation, increasing intestinal permeability and enabling the oral delivery of proteins. This permeation-enhancing effect is a function of nanoparticle size and charge, with smaller (≤ 200 nm) and more negative particles (such as silica) conferring enhanced permeability. In healthy mice, silica nanoparticles enabled the oral delivery of insulin and exenatide, with 10 U kg-1 orally delivered insulin sustaining hypoglycaemia for a few hours longer than a 1 U kg-1 dose of subcutaneously injected insulin. In healthy, hyperglycaemic and diabetic mice, the oral delivery of 10 U kg-1 insulin led to a dose-adjusted bioactivity of, respectively, 35%, 29% and 23% that of the subcutaneous injection of 1 U kg-1 insulin. The permeation-enhancing effect of the nanoparticles was reversible, non-toxic, and attributable to the binding to integrins on the surface of epithelial cells.

Ultrastructure of intestinal mucosa in diarrhea-predominant irritable bowel syndrome.

Impaired intestinal barrier function has been demonstrated in the pathophysiology of diarrhea-predominant irritable bowel syndrome (IBS-D). This study aimed to describe the intestinal ultrastructural findings in the intestinal mucosal layer of IBS-D patients. In total, 10 healthy controls and 10 IBS-D patients were analyzed in this study. The mucosa of each patient's rectosigmoid colon was first assessed by confocal laser endomicroscopy (CLE); next, biopsied specimens of these sites were obtained. Intestinal tissues of IBS-D patients and healthy volunteers were examined to observe cellular changes by transmission electron microscopy (TEM). CLE showed no visible epithelial damage or inflammatory changes in the colonic mucosa of IBS-D compared with healthy volunteers. On transmission electron microscopic examination, patients with IBS-D displayed a larger apical intercellular distance with a higher proportion of dilated (>20nm) intercellular junctional complexes, which was indicative of impaired mucosal integrity. In addition, microvillus exfoliation, extracellular vesicle as well as increased presence of multivesicular bodies were visible in IBS-D patients. Single epithelial cells appeared necrotic, as characterized by cytoplasmic vacuolization, cytoplasmic swelling, and presence of autolysosome. A significant association between bowel habit, frequency of abdominal pain, and enlarged intercellular distance was found. This study showed ultrastructural alterations in the architecture of intestinal epithelial cells and intercellular junctional complexes in IBS-D patients, potentially representing a pathophysiological mechanism in IBS-D.

MYCN amplification drives an aggressive form of spinal ependymoma

Spinal ependymal tumors form a histologically and molecularly heterogeneous group of tumors with generally good prognosis. However, their treatment can be challenging if infiltration of the spinal cord or dissemination throughout the central nervous system (CNS) occurs and, in these cases, clinical outcome remains poor. Here, we describe a new and relatively rare subgroup of spinal ependymal tumors identified using DNA methylation profiling that is distinct from other molecular subgroups of ependymoma. Copy number variation plots derived from DNA methylation arrays showed MYCN amplification as a characteristic genetic alteration in all cases of our cohort (n = 13), which was subsequently validated using fluorescence in situ hybridization. The histological diagnosis was anaplastic ependymoma (WHO Grade III) in ten cases and classic ependymoma (WHO Grade II) in three cases. Histological re-evaluation in five primary tumors and seven relapses showed characteristic histological features of ependymoma, namely pseudorosettes, GFAP- and EMA positivity. Electron microscopy revealed cilia, complex intercellular junctions and intermediate filaments in a representative sample. Taking these findings into account, we suggest to designate this molecular subgroup spinal ependymoma with MYCN amplification, SP-EPN-MYCN. SP-EPN-MYCN tumors showed distinct growth patterns with intradural, extramedullary localization mostly within the thoracic and cervical spine, diffuse leptomeningeal spread throughout the whole CNS and infiltrative invasion of the spinal cord. Dissemination was observed in 100% of cases. Despite high-intensity treatment, SP-EPN-MYCN showed significantly worse median progression free survival (PFS) (17 months) and median overall survival (OS) (87 months) than all other previously described molecular spinal ependymoma subgroups. OS and PFS were similar to supratentorial ependymoma with RELA-fusion (ST-EPN-RELA) and posterior fossa ependymoma A (PF-EPN-A), further highlighting the aggressiveness of this distinct new subgroup. We, therefore, propose to establish SP-EPN-MYCN as a new molecular subgroup in ependymoma and advocate for testing newly diagnosed spinal ependymal tumors for MYCN amplification.

Epac1 is required for cAMP‐mediated endothelial barrier stabilization

Regulation of intercellular junctional complexes is critical for the control of endothelial barrier function and dependent on cAMP‐mediated Rac1 activation. Recently, the exchange protein activated by cAMP (Epac1) has been shown to serve as a tonic stabilizer of the endothelial barrier. Here, we further elucidate the role of Epac1 in cAMP and Rac1‐mediated endothelial barrier regulation.Transendothelial‐electrical‐resistance (TER) measurements in newly generated immortalized myocardial endothelial cells derived from Epac1‐knockout (KO) mice revealed significant reduction in the baseline TER, when compared to wild‐type (WT) cells, indicating that Epac1 is required for maintenance of endothelial barrier function. This effect was associated with fragmented VE‐cadherin staining and reduced localization of tight junctional proteins ZO‐1 and occludin along cell borders. In WT‐cells, application of forskolin (F) and rolipram (R) resulted in increased TER associated with linearization and augmentation of VE‐cadherin immunostaining at adherens junctions. In contrast, Epac1‐KO‐cells did not respond to F/R treatment indicating that Epac1 is crucial for cAMP‐mediated endothelial barrier stabilization. In comparison to WT‐cells, Epac1‐KO‐cells revealed significantly increased protein levels of Rac1, whereas Rac1 basal activity was not enhanced, indicating a defect in Rac1 activation. However, in Epac1‐KOcells, cAMP‐mediated Rac1 activation was not abolished and the basal cAMP‐concentration was significantly increased. Additionally, in Epac1‐deficient cells F/R‐mediated increase in cAMP levels was less compared to WT cells. Taken together, our data show that Epac1 is crucial for cAMP‐mediated barrier stabilization by mechanisms which at least in part are independent of Rac1 regulation.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

P73 regulates ependymal planar cell polarity by modulating actin and microtubule cytoskeleton

Planar cell polarity (PCP) and intercellular junctional complexes establish tissue structure and coordinated behaviors across epithelial sheets. In multiciliated ependymal cells, rotational and translational PCP coordinate cilia beating and direct cerebrospinal fluid circulation. Thus, PCP disruption results in ciliopathies and hydrocephalus. PCP establishment depends on the polarization of cytoskeleton and requires the asymmetric localization of core and global regulatory modules, including membrane proteins like Vangl1/2 or Frizzled. We analyzed the subcellular localization of select proteins that make up these modules in ependymal cells and the effect of Trp73 loss on their localization. We identify a novel function of the Trp73 tumor suppressor gene, the TAp73 isoform in particular, as an essential regulator of PCP through the modulation of actin and microtubule cytoskeleton dynamics, demonstrating that Trp73 is a key player in the organization of ependymal ciliated epithelia. Mechanistically, we show that p73 regulates translational PCP and actin dynamics through TAp73-dependent modulation of non-musclemyosin-II activity. In addition, TAp73 is required for the asymmetric localization of PCP-core and global signaling modules and regulates polarized microtubule dynamics, which in turn set up the rotational PCP. Therefore, TAp73 modulates, directly and/or indirectly, transcriptional programs regulating actin and microtubules dynamics and Golgi organization signaling pathways. These results shed light into the mechanism of ependymal cell planar polarization and reveal p73 as an epithelial architect during development regulating the cellular cytoskeleton.

Characterization of cell-cell junction changes associated with the formation of a strong endothelial barrier

ABSTRACTA principal function of endothelial cells is the formation of a barrier between the blood and tissues. This barrier arises from the physical connections at cell-cell junctions, which includes cytoskeletal tight junction and adherens junction proteins. Methods that alter barrier function must therefore affect these cell-cell connections. The blood brain barrier (BBB) represents perhaps the most selective endothelial barrier, which arises from endothelial cell interactions with astrocytes and pericytes. Even in non-central nervous system (CNS) endothelial cells, barrier properties can be enhanced, mimicking the BBB, through induction of intercellular junctions, by either direct co-culture with astrocytes, supplementation with astrocyte conditioned medium (ACM) and/or pharmacologic enhancement of cAMP. To understand how cell-cell junctions change during endothelial barrier enhancement, we examined the effects of ACM and/or cAMP donors added to standard media on human umbilical vein endothelial cells (HUVEC). HUVEC cultured with cAMP-elevating agents had the most enhanced barrier function as measured by Electric Cell-substrate Impedance Sensing (ECIS®), a real-time, label-free, impedance based method of studying cell barrier properties. However, subtle differences in actin and cell-cell junction proteins were seen across all four culture conditions. cAMP-elevating agents also triggered the redistribution of ZO-1 and VE-cadherin to cell-cell junctions, and intensified the actin microfilament network at the cell cortex. Using a VE-cadherin FRET-force sensor, we observed a decrease in VE-cadherin force in HUVEC cultured with ACM with cAMP donors. Our data indicate cAMP elevation induces both junctional strengthening and reduced VE-cadherin forces. Additionally, treatment with an inhibitor of formin, which reduced actin stress fibers, enhanced barrier function. These data suggest that barrier function is modulated both through the trafficking of proteins to cell-cell junctions, and through the modulation and a relaxation of mechanical force through adherens junctions as intercellular junctional complexes become established.

Phospholipase Cγ1 links inflammation and tumorigenesis in colitis-associated cancer.

Colorectal cancer (CRC) is the third diagnosed cancer and the second leading cause of cancer-related deaths in the United States. Colorectal cancer is linked to inflammation and phospholipase Cγ1 (PLCγ1) is associated with tumorigenesis and the development of colorectal cancer; however, evidence of mechanisms connecting them remains unclear. The tight junctions (TJ), as intercellular junctional complexes, have an important role for integrity of the epithelial barrier to regulate the cellular permeability. Here we found that PLCγ1 regulated colitis and tumorigenesis in intestinal epithelial cells (IEC). To induce the colitis-associated cancer (CAC), we used the AOM/DSS model. Mice were sacrificed at 100 days (DSS three cycles) and 120 days (DSS one cycle). In a CAC model, we showed that the deletion of PLCγ1 in IEC decreased the incidence of tumors by enhancing apoptosis and inhibiting proliferation during tumor development. Accordingly, the deletion of PLCγ1 in IEC reduced colitis-induced epithelial inflammation via inhibition of pro-inflammatory cytokines and mediators. The PLCγ1 pathway in IEC accelerated colitis-induced epithelial damage via regulation of TJ proteins. Conclusions: Our findings suggest that PLCγ1 is a critical regulator of colitis and colorectal cancer and could further help in the development of therapy for colitis-associated cancer.

The Human Blood-Nerve Barrier Transcriptome

The blood-nerve barrier (BNB), formed by tight junction-forming microvessels within peripheral nerve endoneurium, exists to regulate its internal microenvironment essential for effective axonal signal transduction. Relatively little is known about the unique human BNB molecular composition. Such knowledge is crucial to comprehend the relationships between the systemic circulation and peripheral nerves in health, adaptations to intrinsic or extrinsic perturbations and alterations that may result in disease. We performed RNA-sequencing on cultured early- and late-passage adult primary human endoneurial endothelial cells and laser-capture microdissected endoneurial microvessels from four cryopreserved normal adult human sural nerves referenced to the Genome Reference Consortium Human Reference 37 genome browser, using predefined criteria guided by known transcript or protein expression in vitro and in situ. We identified 12881 common transcripts associated by 125 independent biological networks, defined as the normal adult BNB transcriptome, including a comprehensive array of transporters and specialized intercellular junctional complex components. These identified transcripts and their interacting networks provide insights into peripheral nerve microvascular morphogenesis, restrictive barrier formation, influx and efflux transporters with relevance to understanding peripheral nerve homeostasis and pharmacology, including targeted drug delivery and the mediators of leukocyte trafficking in peripheral nerves during normal immunosurveillance.