Remodeling Of Adherens Junctions Research Articles

Proximity interactome of lymphatic VE-cadherin reveals mechanisms of junctional remodeling and reelin secretion

The adhesion receptor vascular endothelial (VE)-cadherin transduces an array of signals that modulate crucial lymphatic cell behaviors including permeability and cytoskeletal remodeling. Consequently, VE-cadherin must interact with a multitude of intracellular proteins to exert these functions. Yet, the full protein interactome of VE-cadherin in endothelial cells remains a mystery. Here, we use proximity proteomics to illuminate how the VE-cadherin interactome changes during junctional reorganization from dis-continuous to continuous junctions, triggered by the lymphangiogenic factor adrenomedullin. These analyses identified interactors that reveal roles for ADP ribosylation factor 6 (ARF6) and the exocyst complex in VE-cadherin trafficking and recycling. We also identify a requisite role for VE-cadherin in the in vitro and in vivo control of secretion of reelin—a lymphangiocrine glycoprotein with recently appreciated roles in governing heart development and injury repair. This VE-cadherin protein interactome shines light on mechanisms that control adherens junction remodeling and secretion from lymphatic endothelial cells.

Hydrostatic pressure drives sprouting angiogenesis via adherens junction remodelling and YAP signalling

Endothelial cell physiology is governed by its unique microenvironment at the interface between blood and tissue. A major contributor to the endothelial biophysical environment is blood hydrostatic pressure, which in mechanical terms applies isotropic compressive stress on the cells. While other mechanical factors, such as shear stress and circumferential stretch, have been extensively studied, little is known about the role of hydrostatic pressure in the regulation of endothelial cell behavior. Here we show that hydrostatic pressure triggers partial and transient endothelial-to-mesenchymal transition in endothelial monolayers of different vascular beds. Values mimicking microvascular pressure environments promote proliferative and migratory behavior and impair barrier properties that are characteristic of a mesenchymal transition, resulting in increased sprouting angiogenesis in 3D organotypic model systems ex vivo and in vitro. Mechanistically, this response is linked to differential cadherin expression at the adherens junctions, and to an increased YAP expression, nuclear localization, and transcriptional activity. Inhibition of YAP transcriptional activity prevents pressure-induced sprouting angiogenesis. Together, this work establishes hydrostatic pressure as a key modulator of endothelial homeostasis and as a crucial component of the endothelial mechanical niche.

Rap1 coordinates cell-cell adhesion and cytoskeletal reorganization to drive collective cell migration in vivo

Collective cell movements contribute to tissue development and repair and spread metastatic disease. In epithelia, cohesive cell movements require reorganization of adherens junctions and the actomyosin cytoskeleton. However, the mechanisms that coordinate cell-cell adhesion and cytoskeletal remodeling during collective cell migration invivo are unclear. We investigated the mechanisms of collective cell migration during epidermal wound healing in Drosophila embryos. Upon wounding, the cells adjacent to the wound internalize cell-cell adhesion molecules and polarize actin and the motor protein non-muscle myosin II to form a supracellular cable around the wound that coordinates cell movements. The cable anchors at former tricellular junctions (TCJs) along the wound edge, and TCJs are reinforced during wound closure. We found that the small GTPase Rap1 was necessary and sufficient for rapid wound repair. Rap1 promoted myosin polarization to the wound edge and E-cadherin accumulation at TCJs. Using embryos expressing a mutant form of the Rap1 effector Canoe/Afadin that cannot bind Rap1, we found that Rap1 signals through Canoe for adherens junction remodeling, but not for actomyosin cable assembly. Instead, Rap1 was necessary and sufficient for RhoA/Rho1 activation at the wound edge. The RhoGEF Ephexin localized to the wound edge in a Rap1-dependent manner, and Ephexin was necessary for myosin polarization and rapid wound repair, but not for E-cadherin redistribution. Together, our data show that Rap1 coordinates the molecular rearrangements that drive embryonic wound healing, promoting actomyosin cable assembly through Ephexin-Rho1, and E-cadherin redistribution through Canoe, thus enabling rapid collective cell migration invivo.

ROR2/PCP a New Pathway Controlling Endothelial Cell Polarity Under Flow Conditions.

Endothelial cells (ECs) are sensitive to physical forces created by blood flow, especially to laminar shear stress. Among the cell responses to laminar flow, EC polarization against the flow direction emerges as a key event, particularly during the development and remodeling of the vascular network. EC adopt an elongated planar cell shape with an asymmetrical distribution of intracellular organelles along the axis of blood flow. This study aimed to investigate the involvement of planar cell polarity via the receptor ROR2 (receptor tyrosine kinase-like orphan receptor 2) in endothelial responses to laminar shear stress. We generated a genetic mouse model with EC-specific deletion of Ror2, in combination with in vitro approaches involving loss- and gain-of-function experiments. During the first 2 weeks of life, the endothelium of the mouse aorta undergoes a rapid remodeling associated with a loss of EC polarization against the flow direction. Notably, we found a correlation between ROR2 expression and endothelial polarization levels. Our findings demonstrate that deletion of Ror2 in murine ECs impaired their polarization during the postnatal development of the aorta. In vitro experiments further validated the essential role of ROR2 in both EC collective polarization and directed migration under laminar flow conditions. Exposure to laminar shear stress triggered the relocalization of ROR2 to cell-cell junctions where it formed a complex with VE-Cadherin and β-catenin, thereby regulating adherens junctions remodeling at the rear and front poles of ECs. Finally, we showed that adherens junctions remodeling and cell polarity induced by ROR2 were dependent on the activation of the small GTPase Cdc42. This study identified ROR2/planar cell polarity pathway as a new mechanism controlling and coordinating collective polarity patterns of EC during shear stress response.

Abstract 2017: A liquid biopsy approach to gastric cancer reveals a signature reflective of epithelial to mesenchymal transition

Abstract Comprehensive proteomic profiling of plasmas collected before and after neo-adjuvant treatment for gastric cancer (GC) yielded 522 proteins exhibiting higher pre-treatment levels compared to healthy controls and among them, 345 proteins decreased after the treatment. Increased levels of cytoskeleton-related proteins were manifest in the plasmas of GC patients. Cytoskeletal proteins are known to play integral roles in modulating epithelial plasticity by altering mechanical strength and structural design that is critical for cancer cell motility, epithelial to mesenchymal transition (EMT), metastasis, and invasion. Ingenuity Pathway Analysis of highly expressed circulating proteins identified key oncogenic upstream transcriptional regulators and proteins that promote EMT, including KRAS, MYC and TGFB. Additionally, pathways involved in remodeling of epithelial adherens junctions, actin cytoskeleton signaling and VEGF signaling that also play a key role in EMT were upregulated. Additional datasets from proteomic profiling of multiple (N=6) GC cell lines were intersected with GC patient plasma profiles provided supporting evidence of a cancer cell origin of circulating cytoskeletal features. ACTC1, ACTN1, ACTR3, MYH9, TUBA1A, TUBA4A and TLN1 were highly expressed in both conditioned media released from GC cell lines and plasma samples of GC patients. Our findings are indicative of a blood-based signature of cytoskeletal proteins that is reflective of EMT in the context of gastric cancer. Citation Format: Ali H. Abdel Sater, Yihui Chen, Johannes F. Fahrmann, Ehsan Irajizad, Yining Cai, Fuchung Hsiao, Jody V. Vykoukal, Hiroyuki Katayama, Ricardo A. Léon-Letelier, Rongzhang Dou, Edwin J. Ostrin, Melissa Pizzi, Kohei Yamashita, Matheus Sewastjanow Da Silva, Shumei Song, Jaffer Ajani, Samir Hanash. A liquid biopsy approach to gastric cancer reveals a signature reflective of epithelial to mesenchymal transition [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 2017.

MP01-02 PROTEOMIC ANALYSIS OF SEMINAL FLUID IN RHESUS MACAQUES EXPOSED TO DELTA-9-TETRAHYDROCANNABINOL

You have accessJournal of UrologyCME1 Apr 2023MP01-02 PROTEOMIC ANALYSIS OF SEMINAL FLUID IN RHESUS MACAQUES EXPOSED TO DELTA-9-TETRAHYDROCANNABINOL Jasper C. Bash, Lyndsey E. Shorey-Kendrick, Carol B. Hannah, Charles A. Easley, Eliot Spindel, Jamie O. Lo, and Jason C. Hedges Jasper C. BashJasper C. Bash More articles by this author , Lyndsey E. Shorey-KendrickLyndsey E. Shorey-Kendrick More articles by this author , Carol B. HannahCarol B. Hannah More articles by this author , Charles A. EasleyCharles A. Easley More articles by this author , Eliot SpindelEliot Spindel More articles by this author , Jamie O. LoJamie O. Lo More articles by this author , and Jason C. HedgesJason C. Hedges More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003212.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Cannabis use has been associated with male infertility, but the etiology is not well understood. Seminal fluid is comprised of multiple factors (e.g. proteins), that influence fertility. Our objective was to determine the impact of chronic delta-9-tetrahydrocannabinol (THC) use on the seminal fluid proteome in a rhesus macaque (RM) model. METHODS: 6 adult RMs were given a daily THC edible over 280 days (∼4 spermatogenesis cycles) with serial non-sedated semen collection. Differential expression of proteins in the seminal fluid at baseline (pre-THC), high-THC dose (2.5 mg/7 kg/day), and post-THC discontinuation for 140 days (∼2 spermatogenesis cycles) was evaluated. Proteomic analysis was performed by liquid chromatography tandem mass spectrometry followed by statistical analysis to identify differentially expressed proteins. RESULTS: Proteomic analysis resulted in 1,395 quantifiable proteins (Figure 1A). The average percent identity was 88.6% (SD=14.9%). All RM proteins were matched to human proteins with 99.4% of the RM proteins (1,386) exceeding an identity cutoff of 44.3% (average minus 3 SDs). 7 proteins reached FDR significance in any contrast, thus we focused downstream analyses on nominally significant candidate proteins. Ingenuity Pathway Analysis (Figure 1B) revealed that high-THC exposure was associated with dysregulation of pathways related to “Remodeling of Epithelial Adherens Junctions”, “Axonal Guidance Signaling”, and “Glucocorticoid Receptor Signaling”. THC discontinuation for 140 days was associated with decreased “LXR/RXR Activation”, “Acute Phase Response Signaling”, and “Production of Nitric Oxide and ROS in Macrophages”. 5 proteins dysregulated with THC and significantly restored to baseline included products of CHIT1, FGG, MMP9, TGFBI, and CFB genes (Figure 1D). CHIT1 has been associated with oligozoospermia and MMP9 protein with sperm motility. CONCLUSIONS: This study demonstrated the translational strength of the RM model to study male reproductive health; there was significant seminal fluid proteome homology between humans and RMs. THC-exposed RMs are likely to exhibit proteins in their seminal ejaculate that are involved in regulatory processes that could potentially contribute to male infertility. Source of Funding: OHSU Faculty SEED Award and ASRM Pilot and Exploratory Grant © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e1 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Jasper C. Bash More articles by this author Lyndsey E. Shorey-Kendrick More articles by this author Carol B. Hannah More articles by this author Charles A. Easley More articles by this author Eliot Spindel More articles by this author Jamie O. Lo More articles by this author Jason C. Hedges More articles by this author Expand All Advertisement PDF downloadLoading ...

In colon cancer cells fascin1 regulates adherens junction remodeling.

Adherens junctions (AJs) are a defining feature of all epithelial cells. They regulate epithelial tissue architecture and integrity, and their dysregulation is a key step in tumor metastasis. AJ remodeling is crucial for cancer progression, and it plays a key role in tumor cell survival, growth, and dissemination. Few studies have examined AJ remodeling in cancer cells consequently, it remains poorly understood and unleveraged in the treatment of metastatic carcinomas. Fascin1 is an actin-bundling protein that is absent from the normal epithelium but its expression in colon cancer is linked to metastasis and increased mortality. Here, we provide the molecular mechanism of AJ remodeling in colon cancer cells and identify for the first time, fascin1's function in AJ remodeling. We show that in colon cancer cells fascin1 remodels junctional actin and actomyosin contractility which makes AJs less stable but more dynamic. By remodeling AJs fascin1 drives mechanoactivation of WNT/β-catenin signaling and generates "collective plasticity" which influences the behavior of cells during cell migration. The impact of mechanical inputs on WNT/β-catenin activation in cancer cells remains poorly understood. Our findings highlight the role of AJ remodeling and mechanosensitive WNT/β-catenin signaling in the growth and dissemination of colorectal carcinomas.

Appropriate tension sensitivity of α-catenin ensures rounding morphogenesis of epithelial spheroids

The adherens junction (AJ) is an actin filament-anchoring junction. It plays a central role in epithelial morphogenesis through cadherin-based recognition and adhesion among cells. The stability and plasticity of AJs are required for the morphogenesis. An actin-binding α-catenin is an essential component of the cadherin-catenin complex and functions as a tension transducer that changes its conformation and induces AJ development in response to tension. Despite much progress in understanding molecular mechanisms of tension sensitivity of α-catenin, its significance on epithelial morphogenesis is still unknown. Here we show that the tension sensitivity of α-catenin is essential for epithelial cells to form round spheroids through proper multicellular rearrangement. Using a novel in vitro suspension culture model, we found that epithelial cells form round spheroids even from rectangular-shaped cell masses with high aspect ratios without using high tension and that increased tension sensitivity of α-catenin affected this morphogenesis. Analyses of AJ formation and cellular tracking during rounding morphogenesis showed cellular rearrangement, probably through AJ remodeling. The rearrangement occurs at the cell mass level, but not single-cell level. Hypersensitive α-catenin mutant-expressing cells did not show cellular rearrangement at the cell mass level, suggesting that the appropriate tension sensitivity of α-catenin is crucial for the coordinated round morphogenesis.Key words: α-catenin, vinculin, adherens junction, morphogenesis, mechanotransduction.

Differential procoagulatory response of microvascular, arterial and venous endothelial cells upon inflammation in vitro

IntroductionInflammation induces a procoagulant phenotype of endothelial cells (EC) with the exposure of tissue factor (TF), a potent initiator of the extrinsic coagulation cascade. Although systemic inflammation affects the whole vascular system, thrombotic lesions occur particularly in microcirculation. This raises the question of whether TF-procoagulant activity (TF-PCA) differs between EC from arterial, venous, and microvascular beds. Materials and methodsFunctional coagulation tests, including TF-PCA, and inflammatory responses were investigated on arterial, venous and microvascular endothelial cells. Interleukin-6 (IL-6) and TF-levels were determined in cohort of 59 septic patients. ResultsWe found that tumor necrosis factor alpha (TNFα), lipopolysaccharide, and interleukin-1β induce a solid, dose-dependent increase in TF-PCA, which is highest in microvascular EC. A positive correlation of interleukin-6 (IL-6) with TF levels was observed in a cohort of 59 septic patients. In contrast, TF-PCA was independent of IL-6 concentrations in vitro. Re-analysis of publicly available gene expression data revealed that among the top 50 genes annotated to coagulation, TF is one of three regulated genes common to the three investigated EC subtypes. The response to inflammatory stimuli in terms of exposure of leukocyte-endothelial- and platelet-endothelial adhesion molecules (E-selectin and PECAM-1), remodeling of adherens junctions, co-exposure of negatively charged surfaces nor breakdown of the glycocalyx was comparable between the EC subtypes and did not explain the higher TF-PCA on microvascular cells. We found that the ratio of TF and TFPI exposure on the endothelial membrane significantly differs between the EC subtypes. ConclusionsThese findings indicate that the ratio of TF to its inhibitor TFPI is a determinant of endothelial TF-PCA, which is most pronounced on microvascular endothelial cells and might explain why the microvascular system is particularly susceptible to inflammation-induced thrombosis.

Enhancement of Endothelialization by Topographical Features Is Mediated by PTP1B-Dependent Endothelial Adherens Junctions Remodeling.

Endothelial Cells (ECs) form cohesive cellular lining of the vasculature and play essential roles in both developmental processes and pathological conditions. Collective migration and proliferation of endothelial cells (ECs) are key processes underlying endothelialization of vessels as well as vascular graft, but the complex interplay of mechanical and biochemical signals regulating these processes are still not fully elucidated. While surface topography and biochemical modifications have been used to enhance endothelialization in vitro, thus far such single-modality modifications have met with limited success. As combination therapy that utilizes multiple modalities has shown improvement in addressing various intractable and complex biomedical conditions, here, we explore a combined strategy that utilizes topographical features in conjunction with pharmacological perturbations. We characterized EC behaviors in response to micrometer-scale grating topography in concert with pharmacological perturbations of endothelial adherens junctions (EAJ) regulators. We found that the protein tyrosine phosphatase, PTP1B, serves as a potent regulator of EAJ stability, with PTP1B inhibition synergizing with grating topographies to modulate EAJ rearrangement, thereby augmenting global EC monolayer sheet orientation, proliferation, connectivity, and collective cell migration. Our data delineates the crosstalk between cell-ECM topography sensing and cell-cell junction integrity maintenance and suggests that the combined use of grating topography and PTP1B inhibitor could be a promising strategy for promoting collective EC migration and proliferation.

Alterations in Epithelial Cell Polarity During Endometrial Receptivity: A Systematic Review.

BackgroundAbnormal endometrial receptivity is one of the major causes of embryo implantation failure and infertility. The plasma membrane transformation (PMT) describes the collective morphological and molecular alterations occurring to the endometrial luminal epithelium across the mid-secretory phase of the menstrual cycle to facilitate implantation. Dysregulation of this process directly affects endometrial receptivity and implantation. Multiple parallels between these alterations to confer endometrial receptivity in women have been drawn to those seen during the epithelial-mesenchymal transition (EMT) in tumorigenesis. Understanding these similarities and differences will improve our knowledge of implantation biology, and may provide novel therapeutic targets to manage implantation failure.MethodsA systematic review was performed using the Medline (Ovid), Embase, and Web of Science databases without additional limits. The search terms used were “(plasma membrane* or cell membrane*) and transformation*” and “endometrium or endometrial.” Research studies on the PMT or its regulation in women, discussing either the endometrial epithelium, decidualized stroma, or both, were eligible for inclusion.ResultsA total of 198 articles were identified. Data were extracted from 15 studies that matched the inclusion criteria. Collectively, these included studies confirmed the alterations occurring to the endometrial luminal epithelium during the PMT are similar to those seen during the EMT. Such similarities included alterations to the actin cytoskeleton remodeling of adherens junctions, integrin expression and epithelial-stromal communication. These were also some differences between these processes, such as the regulation of tight junctions and mucins, which need to be further researched.ConclusionsThis review raised the prospect of shared and distinct mechanisms existing in PMT and EMT. Further investigation into similarities between the PMT in the endometrium and the EMT in tumorigenesis may provide new mechanistic insights into PMT and new targets for the management of implantation failure and infertility.

Molecular abnormalities in autopsied brain tissue from the inferior horn of the lateral ventricles of nonagenarians and Alzheimer disease patients

BackgroundThe ventricular system plays a vital role in blood-cerebrospinal fluid (CSF) exchange and interstitial fluid-CSF drainage pathways. CSF is formed in the specialized secretory tissue called the choroid plexus, which consists of epithelial cells, fenestrated capillaries and the highly vascularized stroma. Very little is currently known about the role played by the ventricles and the choroid plexus tissue in aging and Alzheimer’s disease (AD).MethodsIn this study, we used our state-of-the-art proteomic platform, a liquid chromatography/mass spectrometry (LC-MS/MS) approach coupled with Tandem Mass Tag isobaric labeling to conduct a detailed unbiased proteomic analyses of autopsied tissue isolated from the walls of the inferior horn of the lateral ventricles in AD (77.2 ± 0.6 yrs), age-matched controls (77.0 ± 0.5 yrs), and nonagenarian cases (93.2 ± 1.1 yrs).ResultsIngenuity pathway analyses identified phagosome maturation, impaired tight-junction signaling, and glucose/mannose metabolism as top significantly regulated pathways in controls vs nonagenarians. In matched-control vs AD cases we identified alterations in mitochondrial bioenergetics, oxidative stress, remodeling of epithelia adherens junction, macrophage recruitment and phagocytosis, and cytoskeletal dynamics. Nonagenarian vs AD cases demonstrated augmentation of oxidative stress, changes in gluconeogenesis-glycolysis pathways, and cellular effects of choroidal smooth muscle cell vasodilation. Amyloid plaque score uniquely correlated with remodeling of epithelial adherens junctions, Fc γ-receptor mediated phagocytosis, and alterations in RhoA signaling. Braak staging was uniquely correlated with altered iron homeostasis, superoxide radical degradation and phagosome maturation.ConclusionsThese changes provide novel insights to explain the compromise to the physiological properties and function of the ventricles/choroid plexus system in nonagenarian aging and AD pathogenesis. The pathways identified could provide new targets for therapeutic strategies to mitigate the divergent path towards AD.

C-Src controls stability of sprouting blood vessels in the developing retina independently of cell-cell adhesion through focal adhesion assembly.

ABSTRACTEndothelial cell adhesion is implicated in blood vessel sprout formation, yet how adhesion controls angiogenesis, and whether it occurs via rapid remodeling of adherens junctions or focal adhesion assembly, or both, remains poorly understood. Furthermore, how endothelial cell adhesion is controlled in particular tissues and under different conditions remains unexplored. Here, we have identified an unexpected role for spatiotemporal c-Src activity in sprouting angiogenesis in the retina, which is in contrast to the dominant focus on the role of c-Src in the maintenance of vascular integrity. Thus, mice specifically deficient in endothelial c-Src displayed significantly reduced blood vessel sprouting and loss in actin-rich filopodial protrusions at the vascular front of the developing retina. In contrast to what has been observed during vascular leakage, endothelial cell-cell adhesion was unaffected by loss of c-Src. Instead, decreased angiogenic sprouting was due to loss of focal adhesion assembly and cell-matrix adhesion, resulting in loss of sprout stability. These results demonstrate that c-Src signaling at specified endothelial cell membrane compartments (adherens junctions or focal adhesions) control vascular processes in a tissue- and context-dependent manner.

Adherens junction remodelling during mitotic rounding of pseudostratified epithelial cells

Epithelial cells undergo cortical rounding at the onset of mitosis to enable spindle orientation in the plane of the epithelium. In cuboidal epithelia in culture, the adherens junction protein E‐cadherin recruits Pins/LGN/GPSM2 and Mud/NuMA to orient the mitotic spindle. In the pseudostratified columnar epithelial cells of Drosophila, septate junctions recruit Mud/NuMA to orient the spindle, while Pins/LGN/GPSM2 is surprisingly dispensable. We show that these pseudostratified epithelial cells downregulate E‐cadherin as they round up for mitosis. Preventing cortical rounding by inhibiting Rho‐kinase‐mediated actomyosin contractility blocks downregulation of E‐cadherin during mitosis. Mitotic activation of Rho‐kinase depends on the RhoGEF ECT2/Pebble and its binding partners RacGAP1/MgcRacGAP/CYK4/Tum and MKLP1/KIF23/ZEN4/Pav. Cell cycle control of these Rho activators is mediated by the Aurora A and B kinases, which act redundantly during mitotic rounding. Thus, in Drosophila pseudostratified epithelia, disruption of adherens junctions during mitosis necessitates planar spindle orientation by septate junctions to maintain epithelial integrity.

Next-generation sequencing identified somatic alterations that may underlie the etiology of Chinese papillary thyroid carcinoma.

To better understand the etiology of papillary thyroid carcinoma, we did next-generation sequencing for the exomes and transcriptomes of a Chinese cohort of 28 pairs of DNA and RNA samples extracted from papillary thyroid carcinoma tumors and adjacent normal thyroid samples. The Chinese papillary thyroid carcinoma tumors harbored somatic mutations in the known driver genes, such as KRAS, TP53, BRAF, ERBB2 , and MET . In addition, we identified novel papillary thyroid carcinoma candidate genes that had not been well studied before. We also identified a gene mutation signature involving SPTA1, MAP2, SYNE1 , and SLIT3 that is significantly associated with survival of papillary thyroid carcinoma patients. Transcriptome analysis using the initial papillary thyroid carcinoma tumor samples and a new Chinese papillary thyroid carcinoma dataset identified six commonly upregulated oncogenic pathways in both datasets including eukaryotic translation initiation factor 2, phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K)/serine/threonine kinase (AKT), Ephrin Receptor, Rho Family GTPase signaling, nuclear factor, erythroid 2 like 2 (NRF2)-mediated oxidative stress response, and remodeling of epithelial adherens junctions. Overall, we identified novel candidate genes and oncogenic pathways important to the etiology of papillary thyroid carcinoma in Chinese patients and found the association of a gene signature with the survival outcome of the thyroid cancer patients. These findings may help in moving toward the more comprehensive and effective personalized treatment of papillary thyroid carcinoma in Chinese.

Wood kraft pulp supplementation alters the rumen fermentation characteristics and epithelial transcriptomes in Holstein cattle during the high-grain diet challenge

We investigated the effects of wood kraft pulp (WP) supplementation on ruminal pH, fermentation, and epithelial transcriptomic dynamics in Holstein cattle during the high-grain diet challenge. Five rumen-fistulated Holstein bulls (215.2 ± 20.4 kg of body weight; 7.4 ± 0.3 months of age) were used in a cross-over design without a wash-out period. After two weeks of adaptation period (100% forage feeding ad libitum), cattle were fed a high-grain diet containing corn flakes (control diet) or the WP as a half replacement of corn flakes in the control diet (WP diet) during the first 14 days, and then, the diets were switched for last 14 days. Ruminal pH was measured continuously, and the rumen fluid and epithelium (RE) were collected on the last days of each feeding period simultaneously with blood samples. mRNA expression profiles of the RE were examined by one-color microarray. The differentially expressed genes (DEGs) were investigated using the Ingenuity Pathway Analysis (IPA). The 24 h mean ruminal pH was not different between the control (n = 5) and WP (n = 5) groups (6.39 vs. 6.46, respectively), being higher in the WP group. The proportion of acetic acid tended to be higher in the WP group, and blood urea nitrogen concentration was significantly higher in the WP group compared with the control group. The top differentially expressed candidate genes were involved in inhibition of the immune and inflammatory responses by suppressing the expressions of M-SAA3.2 and IL-36 cytokine signaling pathway (IL36G, IL36A, and IL36RN) in the WP group. In silico analysis revealed that “Remodeling of Epithelial Adherens Junctions” was the most significant signaling pathway, and epidermal growth factor receptor was the most significant upstream regulators, which were involved in the adaptation of the RE to high-grain diet challenge. Furthermore, upstream regulator analysis suggested that microRNAs may be involved in a central role in the RE cellular regulations. These results suggested that WP supplementation as 50% replacement of corn flakes in a high-grain diet did not cause significant contrast in the rumen fermentation and blood metabolite profiles, preferably leading the activation of rumen epithelial transcriptomes to facilitate the development or adaptation of the RE in Holstein cattle during the high-grain diet challenge.

N-Terminomics/TAILS Profiling of Proteases and Their Substrates in Ulcerative Colitis.

Dysregulated protease activity is often implicated in the initiation of inflammation and immune cell recruitment in gastrointestinal inflammatory diseases. Using N-terminomics/TAILS (terminal amine isotopic labeling of substrates), we compared proteases, along with their substrates and inhibitors, between colonic mucosal biopsies of healthy patients and those with ulcerative colitis (UC). Among the 1642 N-termini enriched using TAILS, increased endogenous processing of proteins was identified in UC compared to healthy patients. Changes in the reactome pathways for proteins associated with metabolism, adherens junction proteins (E-cadherin, liver-intestinal cadherin, catenin alpha-1, and catenin delta-1), and neutrophil degranulation were identified between the two groups. Increased neutrophil infiltration and distinct proteases observed in ulcerative colitis may result in extensive break down, altered processing, or increased remodeling of adherens junctions and other cellular functions. Analysis of the preferred proteolytic cleavage sites indicated that the majority of proteolytic activity and processing comes from host proteases, but that key microbial proteases may also play a role in maintaining homeostasis. Thus, the identification of distinct proteases and processing of their substrates improves the understanding of dysregulated proteolysis in normal intestinal physiology and ulcerative colitis.

The Drosophila Afadin and ZO-1 homologues Canoe and Polychaetoid act in parallel to maintain epithelial integrity when challenged by adherens junction remodeling.

During morphogenesis, cells must change shape and move without disrupting tissue integrity. This requires cell–cell junctions to allow dynamic remodeling while resisting forces generated by the actomyosin cytoskeleton. Multiple proteins play roles in junctional–cytoskeletal linkage, but the mechanisms by which they act remain unclear. Drosophila Canoe maintains adherens junction–cytoskeletal linkage during gastrulation. Canoe’s mammalian homologue Afadin plays similar roles in cultured cells, working in parallel with ZO-1 proteins, particularly at multicellular junctions. We take these insights back to the fly embryo, exploring how cells maintain epithelial integrity when challenged by adherens junction remodeling during germband extension and dorsal closure. We found that Canoe helps cells maintain junctional–cytoskeletal linkage when challenged by the junctional remodeling inherent in mitosis, cell intercalation, and neuroblast invagination or by forces generated by the actomyosin cable at the leading edge. However, even in the absence of Canoe, many cells retain epithelial integrity. This is explained by a parallel role played by the ZO-1 homologue Polychaetoid. In embryos lacking both Canoe and Polychaetoid, cell junctions fail early, with multicellular junctions especially sensitive, leading to widespread loss of epithelial integrity. Our data suggest that Canoe and Polychaetoid stabilize Bazooka/Par3 at cell–cell junctions, helping maintain balanced apical contractility and tissue integrity.

Impact of Radiation on Tumor Derived Exosome Production, Proteomics and Biodistribution

Exosomes are cell derived nanoparticles implicated in malignant processes, including the determination of organ specific metastasis. This study aims to test the hypothesis that radiation interferes with the ability of tumor-derived exosomes to establish a pre-metastatic niche by affecting exosome production and proteomics. To understand the effect of radiation across tumors with different radiation sensitivities, breast cancer cell lines with known organotropism (MDA-MB-231 parental and the lung tropic subline, 4175) were compared to melanoma cancer cell line (B16F10) and exposed to 2 Gy vs. 10 Gy radiation. Exosomes were harvested from conditioned media using differential centrifugation and characterized using protein BCA, NanoSight, and electron microscopy. Exosome size was further characterized using a novel asymmetric flow fractionator. DNA packaging was quantified with Qubit ds DNA assay kit and proteomics analyzed through mass spectrometry. Biodistribution was assessed in vivo through retro-orbital sinus injection of fluorescently labeled exosomes and subsequent imaging of organ distribution. Radiation increased the quantity of exosomes produced in a dose dependent manner. Across all cell lines, 2 Gy vs. 10 Gy radiation increased exosome yield (ug/cell) by an average of 2.1 ± 0.4 and 7.8 ± 2.7-fold, respectively. In radioresistant melanoma cells, 2 Gy and 10 Gy radiation increased DNA content per exosome by 1.9 and 8.2-fold, respectively, whereas in more radiosensitive breast cancer cells, 2 Gy and 10 Gy only moderately increased DNA packaging by 1.1 and 1.4-fold. In general, radiation decreased exosome particle size. In breast cancer cells, mean particle size decreased from 135±6.9 nm to 125.7±1.8 and 124.3±3.9 nm for 2 Gy and 10 Gy radiation, respectively. Radiation also decreased the abundance of the smallest exosome subparticles, exomeres, which preferentially traffic to the liver to effect changes in metabolomics. For breast cancer cell lines, radiated exosomes retained the ability to traffic to the lung and liver 24 hours after injection. Pathway analysis of proteomic changes across radiation subgroups implicate significant changes in integrin signaling and remodeling of epithelial adherens junctions, including integrins and metalloproteases. Radiation significantly increased the quantity and DNA packaging of exosomes across all tumor cell lines. Although radiated exosomes trafficked to potential metastatic sites 24 hours after injection, their function in establishing a pre-metastatic niche remains to be explored. Changes in proteomics and exosome subpopulations suggest differences in migration and liver education. These changes trended with radiation dose and intrinsic radiation sensitivity, offering a promising platform for monitoring treatment response and underlying tumor biology in patients.

α-Catenin Controls the Anisotropy of Force Distribution at Cell-Cell Junctions during Collective Cell Migration

Adherens junctions (AJs) control epithelial cell behavior, such as collective movement and morphological changes, during development and in disease. However, the molecular mechanism of AJ remodeling remains incompletely understood. Here, we report that the conformational activation of α-catenin is the key event in the dynamic regulation of AJ remodeling. α-catenin activates RhoA to increase actomyosin contractility at cell-cell junctions. This leads to the stabilization of activated α-catenin, in part through the recruitment of the actin-binding proteins, vinculin and afadin. In this way, α-catenin regulates force sensing, as well as force transmission, through a Rho-mediated feedback mechanism. We further show that this is important for stable directional alignment of multiple cells during collective cell movement by both experimental observation and mathematical modeling. Taken together, our findings demonstrate that α-catenin controls the establishment of anisotropic force distribution at cell junctions to enable cooperative movement of the epithelial cell sheet.