Epithelial Adherens Junctions Research Articles

SCUBE2 regulates adherens junction dynamics and vascular barrier function during inflammation.

SCUBE2 (Signal peptide-CUB-epidermal growth factor-like domain-containing protein 2) is a secreted or membrane-bound protein originally identified from endothelial cells (ECs). Our previous work showed that SCUBE2 forms a complex with E-cadherin and stabilizes epithelial adherens junctions (AJs) to promote epithelial phenotypes. However, it remains unclear whether SCUBE2 also interacts with vascular endothelial (VE)-cadherin and modulates EC barrier function. In this study, we investigated whether and how SCUBE2 in ECs regulates vascular barrier maintenance. We showed that SCUBE2 colocalized and interacted with VE-cadherin and VE-protein tyrosine phosphatase (VE-PTP) within EC AJs. Furthermore, SCUBE2 knockdown disrupted EC AJs and increased EC permeability. Expression of EC SCUBE2 was suppressed at both mRNA and protein levels via the nuclear factor-κB (NF-κB) signaling pathway in response to pro-inflammatory cytokines or permeability-inducing agents. In line with these findings, EC-specific deletion of Scube2 (EC-KO) in mice impaired baseline barrier function and worsened vascular leakiness of peripheral capillaries after local injection of histamine or vascular endothelial growth factor. EC-KO mice were also sensitive to pulmonary vascular hyperpermeability and leukocyte infiltration in response to acute endotoxin- or influenza virus-induced systemic inflammation. Meanwhile, EC-specific SCUBE2-overexpressing mice were protected from these effects. Molecular studies suggested that SCUBE2 acts as a scaffold molecule enabling VE-PTP to dephosphorylate VE-cadherin, which prevents VE-cadherin internalization and stabilizes EC AJs. As such, loss of SCUBE2 resulted in hyperphosphorylation of VE-cadherin at tyrosine 685, which led to its endocytosis, thus destabilizing EC AJs and reducing barrier function. All of these effects were exacerbated by inflammatory insults. We found that SCUBE2 contributes to vascular integrity by recruiting VE-PTP to dephosphorylate VE-cadherin and stabilize AJs, thereby promoting EC barrier function. Moreover, our data suggest that genetic overexpression or pharmacological upregulation of SCUBE2 may help to prevent vascular leakage and edema in inflammatory diseases.

The epithelial adherens junction component PLEKHA7 regulates ECM remodeling and cell behavior through miRNA-mediated regulation of MMP1 and LOX.

Epithelial cells instruct ECM remodeling to modulate their behavior, as a result of adherens junction and miRNA disruption.

Repeat DNA methylation is modulated by adherens junction signaling

Through its involvement in gene transcription and heterochromatin formation, DNA methylation regulates how cells interact with their environment. Nevertheless, the extracellular signaling cues that modulate the distribution of this central chromatin modification are largely unclear. DNA methylation is highly abundant at repetitive elements, but its investigation in live cells has been complicated by methodological challenges. Utilizing a CRISPR/dCas9 biosensor that reads DNA methylation of human α-satellite repeats in live cells, we here uncover a signaling pathway linking the chromatin and transcriptional state of repetitive elements to epithelial adherens junction integrity. Specifically, we find that in confluent breast epithelial cell monolayers, α-satellite repeat methylation is reduced by comparison to low density cultures. This is coupled with increased transcriptional activity at repeats. Through comprehensive perturbation experiments, we identify the junctional protein E-cadherin, which links to the actin cytoskeleton, as a central molecular player for signal relay into the nucleus. Furthermore, we find that this pathway is impaired in cancer cells that lack E-cadherin and are not contact-inhibited. This suggests that the molecular connection between cell density and repetitive element methylation could play a role in the maintenance of epithelial tissue homeostasis.

Intestinal mRNA expression analysis of polarity-related genes identified the discriminatory ability of CRB3 as a diagnostic marker for celiac disease.

Celiac disease (CD) is a chronic autoimmune disorder characterized by an abnormal immune response to gluten, a protein found in wheat, barley, and rye. It is well established that the integrity of epithelial tight junctions (TJs) and adherens junctions (AJs) plays a crucial role in the pathogenesis of CD. These junctional complexes contribute to the apical-basal polarity of the intestinal epithelial cells, which is crucial for their proper functioning. Sixty CD subjects, and 50 controls were enrolled in the current study. Mucosal samples were obtained from the distal duodenum, total RNA was extracted and complementary DNA was synthesized. The relative expression levels of the desired genes were evaluated by quantitative real-time polymerase chain reaction based on ΔΔCt method. The gene-gene interaction network was also constructed using GeneMANIA. CRB3 (p = .0005), LKB1 (p < .0001), and SCRIB (p = .0005) had lower expression in CD patients compared to controls, while PRKCZ expression did not differ between groups (p > .05). CRB3 represented a significant diagnostic value for differentiating CD patients from the control group (p = .02). The aim of the current study was to evaluate the changes in the mRNA expression levels of SCRIB, PRKCZ, LKB1, and CRB3 genes in the small intestinal biopsy samples of CD patients in comparison to the healthy control subjects. Our data uncover the importance of polarity-related genes (especially CRB3) in CD pahtomechanism, that may facilitate the planning of the future studies looking for finding innovative diagnostic and therapeutic strategies for CD.

Spatially distinct molecular patterns of gene expression in idiopathic pulmonary fibrosis

BackgroundIdiopathic pulmonary fibrosis (IPF) is a heterogeneous disease that is pathologically characterized by areas of normal-appearing lung parenchyma, active fibrosis (transition zones including fibroblastic foci) and dense fibrosis. Defining transcriptional differences between these pathologically heterogeneous regions of the IPF lung is critical to understanding the distribution and extent of fibrotic lung disease and identifying potential therapeutic targets. Application of a spatial transcriptomics platform would provide more detailed spatial resolution of transcriptional signals compared to previous single cell or bulk RNA-Seq studies.MethodsWe performed spatial transcriptomics using GeoMx Nanostring Digital Spatial Profiling on formalin-fixed paraffin-embedded (FFPE) tissue from 32 IPF and 12 control subjects and identified 231 regions of interest (ROIs). We compared normal-appearing lung parenchyma and airways between IPF and controls with histologically normal lung tissue, as well as histologically distinct regions within IPF (normal-appearing lung parenchyma, transition zones containing fibroblastic foci, areas of dense fibrosis, and honeycomb epithelium metaplasia).ResultsWe identified 254 differentially expressed genes (DEGs) between IPF and controls in histologically normal-appearing regions of lung parenchyma; pathway analysis identified disease processes such as EIF2 signaling (important for cap-dependent mRNA translation), epithelial adherens junction signaling, HIF1α signaling, and integrin signaling. Within IPF, we identified 173 DEGs between transition and normal-appearing lung parenchyma and 198 DEGs between dense fibrosis and normal lung parenchyma; pathways dysregulated in both transition and dense fibrotic areas include EIF2 signaling pathway activation (upstream of endoplasmic reticulum (ER) stress proteins ATF4 and CHOP) and wound healing signaling pathway deactivation. Through cell deconvolution of transcriptome data and immunofluorescence staining, we confirmed loss of alveolar parenchymal signals (AGER, SFTPB, SFTPC), gain of secretory cell markers (SCGB3A2, MUC5B) as well as dysregulation of the upstream regulator ATF4, in histologically normal-appearing tissue in IPF.ConclusionsOur findings demonstrate that histologically normal-appearing regions from the IPF lung are transcriptionally distinct when compared to similar lung tissue from controls with histologically normal lung tissue, and that transition zones and areas of dense fibrosis within the IPF lung demonstrate activation of ER stress and deactivation of wound healing pathways.

Syndecan-1 shedding destroys epithelial adherens junctions through STAT3 after renal ischemia/reperfusion injury

Syndecan-1 shedding destroys epithelial adherens junctions through STAT3 after renal ischemia/reperfusion injury

Notch1 cortical signaling regulates epithelial architecture and cell-cell adhesion.

Notch receptors control tissue morphogenic processes that involve coordinated changes in cell architecture and gene expression, but how a single receptor can produce these diverse biological outputs is unclear. Here, we employ a 3D model of a human ductal epithelium to reveal tissue morphogenic defects result from loss of Notch1, but not Notch1 transcriptional signaling. Instead, defects in duct morphogenesis are driven by dysregulated epithelial cell architecture and mitogenic signaling which result from the loss of a transcription-independent, Notch1 cortical signaling mechanism that ultimately functions to stabilize adherens junctions and cortical actin. We identify that Notch1 localization and cortical signaling are tied to apical-basal cell restructuring and discover that a Notch1-FAM83H interaction underlies control of epithelial adherens junctions and cortical actin. Together, these results offer new insights into Notch1 signaling and regulation and advance a paradigm in which transcriptional and cell adhesive programs might be coordinated by a single receptor.

Degradation of p0071 and p120-catenin during adherens junction disassembly by Leptospira interrogans.

Leptospira interrogans disseminates hematogenously to reach the target organs by disrupting epithelial adherens junctions (AJs), thus causing leptospirosis, which is a globally neglected zoonotic disease. L. interrogans induces E-cadherin (E-cad) endocytosis and cytoskeletal rearrangement during AJ disassembly, but the detailed mechanism remains unknown. Elucidation of AJ disassembly mechanisms will guide new approaches to developing vaccines and diagnostic methods. In this study, we combine proteomic and imaging analysis with chemical inhibition studies to demonstrate that disrupting the AJs of renal proximal tubule epithelial cells involves the degradation of two armadillo repeat-containing proteins, p0071 and p120-catenin, that stabilize E-cad at the plasma membrane. Combining proteasomal and lysosomal inhibitors substantially prevented p120-catenin degradation, and monolayer integrity destruction without preventing p0071 proteolysis. In contrast, the pan-caspase inhibitor Z-VAD-FMK inhibited p0071 proteolysis and displacement of both armadillo repeat-containing proteins from the cell-cell junctions. Our results show that L. interrogans induces p120-catenin and p0071 degradation, which mutually regulates E-cad stability by co-opting multiple cellular degradation pathways. This strategy may allow L. interrogans to disassemble AJs and disseminate through the body efficiently.

Pore-forming activity of S. pneumoniae pneumolysin disrupts the paracellular localization of the epithelial adherens junction protein E-cadherin.

Streptococcus pneumoniae, a common cause of community-acquired bacterial pneumonia, can cross the respiratory epithelial barrier to cause lethal septicemia and meningitis. S. pneumoniae pore-forming toxin pneumolysin (PLY) triggers robust neutrophil (PMN) infiltration that promotes bacterial transepithelial migration in vitro and disseminated disease in mice. Apical infection of polarized respiratory epithelial monolayers by S. pneumoniae at a multiplicity of infection (MOI) of 20 resulted in recruitment of PMNs, loss of 50% of the monolayer, and PMN-dependent bacterial translocation. Reducing the MOI to 2 decreased PMN recruitment two-fold and preserved the monolayer, but apical-to-basolateral translocation of S. pneumoniae remained relatively efficient. At both MOI of 2 and 20, PLY was required for maximal PMN recruitment and bacterial translocation. Co-infection by wild-type S. pneumoniae restored translocation by a PLY-deficient mutant, indicating that PLY can act in trans. Investigating the contribution of S. pneumoniae infection on apical junction complexes in the absence of PMN transmigration, we found that S. pneumoniae infection triggered the cleavage and mislocalization of the adherens junction (AJ) protein E-cadherin. This disruption was PLY-dependent at MOI of 2 and was recapitulated by purified PLY, requiring its pore-forming activity. In contrast, at MOI of 20, E-cadherin disruption was independent of PLY, indicating that S. pneumoniae encodes multiple means to disrupt epithelial integrity. This disruption was insufficient to promote bacterial translocation in the absence of PMNs. Thus, S. pneumoniae triggers cleavage and mislocalization of E-cadherin through PLY-dependent and -independent mechanisms, but maximal bacterial translocation across epithelial monolayers requires PLY-dependent neutrophil transmigration.

Paneth cell dysfunction in radiation injury and radio-mitigation by human α-defensin 5.

The mechanism underlying radiation-induced gut microbiota dysbiosis is undefined. This study examined the effect of radiation on the intestinal Paneth cell α-defensin expression and its impact on microbiota composition and mucosal tissue injury and evaluated the radio-mitigative effect of human α-defensin 5 (HD5). Adult mice were subjected to total body irradiation, and Paneth cell α-defensin expression was evaluated by measuring α-defensin mRNA by RT-PCR and α-defensin peptide levels by mass spectrometry. Vascular-to-luminal flux of FITC-inulin was measured to evaluate intestinal mucosal permeability and endotoxemia by measuring plasma lipopolysaccharide. HD5 was administered in a liquid diet 24 hours before or after irradiation. Gut microbiota was analyzed by 16S rRNA sequencing. Intestinal epithelial junctions were analyzed by immunofluorescence confocal microscopy and mucosal inflammatory response by cytokine expression. Systemic inflammation was evaluated by measuring plasma cytokine levels. Ionizing radiation reduced the Paneth cell α-defensin expression and depleted α-defensin peptides in the intestinal lumen. α-Defensin down-regulation was associated with the time-dependent alteration of gut microbiota composition, increased gut permeability, and endotoxemia. Administration of human α-defensin 5 (HD5) in the diet 24 hours before irradiation (prophylactic) significantly blocked radiation-induced gut microbiota dysbiosis, disruption of intestinal epithelial tight junction and adherens junction, mucosal barrier dysfunction, and mucosal inflammatory response. HD5, administered 24 hours after irradiation (treatment), reversed radiation-induced microbiota dysbiosis, tight junction and adherens junction disruption, and barrier dysfunction. Furthermore, HD5 treatment also prevents and reverses radiation-induced endotoxemia and systemic inflammation. These data demonstrate that radiation induces Paneth cell dysfunction in the intestine, and HD5 feeding prevents and mitigates radiation-induced intestinal mucosal injury, endotoxemia, and systemic inflammation.

Novel functional atrial fibrillation risk genes and pathways identified from coexpression analyses in human left atria

Genomewide association studies have associated >100 genetic loci with atrial fibrillation (AF), but establishing causal genes contributing to AF remains challenging. The purpose of this study was to determine candidate novel causal genes and mechanistic pathways associated with AF risk loci by incorporating gene expression and coexpression analyses and to provide a resource for functional studies and targeting of AF-associated genes. Cis-expression quantitative trait loci were identified for candidate genes near AF risk variants in human left atrial tissues. Coexpression partners were identified for each candidate gene. Weighted gene coexpression network analysis (WGCNA) identified modules and modules with overrepresentation of candidate AF genes. Ingenuity pathway analysis (IPA) was applied to the coexpression partners of each candidate gene. IPA and gene set over representation analysis were applied to each WGCNA module. One hundred sixty-six AF-risk single nucleotide polymorphisms were located in 135 loci. Eighty-one novel genes not previously annotated as putative AF risk genes were identified. IPA identified mitochondrial dysfunction, oxidative stress, epithelial adherens junction signaling, and sirtuin signaling as the most frequent significant pathways. WGCNA characterized 64 modules (candidate AF genes overrepresented in 8), represented by cell injury, death, stress, developmental, metabolic/mitochondrial, transcription/translation, and immune activation/inflammation regulatory pathways. Candidate gene coexpression analyses suggest significant roles for cellular stress and remodeling in AF, supporting a dual risk model for AF: Genetic susceptibility to AF may not manifest until later in life, when cellular stressors overwhelm adaptive responses. These analyses also provide a novel resource to guide functional studies on potential causal AF genes.

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 ...

Hereditary Diffuse Gastric Cancer

Hereditary diffuse gastric cancer (HDGC) is a dominantly inherited cancer syndrome characterized by a high incidence of diffuse gastric cancer (DGC) and lobular breast cancer (LBC). HDGC is caused by germline mutations in 2 genes involved in the epithelial adherens junction complex, CDH1 and CTNNA1. We discuss the genetics of HDGC and the variability of its clinical phenotype, in particular the variable penetrance of advanced DGC and LBC, both within and between families. We review the pathology of the disease, the mechanism of tumor initiation, and its natural history. Finally, we describe current best practice for the clinical management of HDGC, including emerging genetic testing criteria for the identification of new families, methods for endoscopic surveillance, the complications associated with prophylactic surgery, postoperative quality of life, and the emerging field of HDGC chemoprevention.

Anti-infectious pneumonia target discovery and molecular mechanism study of Jingfang Granules

This study aimed to identify the direct pharmacological targets of Jingfang Granules in treating infectious pneumonia via &quot;target fishing&quot; strategy. Moreover, the molecular mechanism of Jingfang Granules in treating infectious pneumonia was also investigated based on target-related pharmacological signaling pathways. First, the Jingfang Granules extract-bound magnetic nanoparticles were prepared, which were incubated with lipopolysaccharide(LPS)-induced mouse pneumonia tissue lysates. The captured proteins were analyzed by high-resolution mass spectrometry(HRMS), and the target groups with specific binding to the Jingfang Granules extract were screened out. Kyoto Encyclopedia of Genes and Genomes(KEGG) enrichment analysis was used to identify the target protein-associated signaling pathways. On this basis, the LPS-induced mouse model of infectious pneumonia was established. The possible biological functions of target proteins were verified by hematoxylin-eosin(HE) staining and immunohistochemical assay. A total of 186 Jingfang Granules-specific binding proteins were identified from lung tissues. KEGG pathway enrichment analysis showed that the target protein-associated signaling pathways mainly included Salmonella infection, vascular and pulmonary epithelial adherens junction, ribosomal viral replication, viral endocytosis, and fatty acid degradation. The target functions of Jingfang Granules were related to pulmonary inflammation and immunity, pulmonary energy metabolism, pulmonary microcirculation, and viral infection. Based on the in vivo inflammation model, Jingfang Granules significantly improved the alveolar structure of the LPS-induced mouse model of infectious pneumonia and down-regulated the expressions of tumor necrosis factor-α(TNF-α) and interleukin-6(IL-6). Meanwhile, Jingfang Gra-nules significantly up-regulated the expressions of key proteins of mitochondrial function COX Ⅳ and ATP, microcirculation-related proteins CD31 and Occludin, and proteins associated with viral infection DDX21 and DDX3. These results suggest that Jingfang Gra-nules can inhibit lung inflammation, improve lung energy metabolism and pulmonary microcirculation, resist virus infection, thus playing a protective role in the lung. This study systematically explains the molecular mechanism of Jingfang Granules in the treatment of respiratory inflammation from the perspective of target-signaling pathway-pharmacological efficacy, thereby providing key information for clinical rational use of Jingfang Granules and expanding potential pharmacological application.

The Effect of Carnosine on UVA-Induced Changes in Intracellular Signaling of Human Skin Fibroblast Spheroids

Dermis fibroblasts are very sensitive to penetrating UVA radiation and induce photo-damage. To protect skin cells against this environmental damage, there is an urgent need for effective compounds, specifically targeting UVA-induced mitochondrial injury. This study aimed to analyze the effect of carnosine on the proteome of UVA-irradiated human skin fibroblast, cultured in a three-dimensional (3D) biological system recapitulating dermal compartment as a test system to investigate the altered cellular pathways after 48 h and 7 days of culture with or without carnosine treatment. The obtained results indicate that UVA dysregulates Oxidative Phosphorylation, the Fibrosis Signaling Pathway, Glycolysis I and Nrf2-mediated Oxidative Stress Response. Carnosine exercises provide a protective function against the harmful effects of UVA radiation by activating the Nrf2 pathway with the upregulations of some ROS-detoxifying enzymes such as the glutathione S-transferase (GST) protein family. Additionally, carnosine regulates the activation of the Epithelial Adherens Junction and Wound Healing Signaling Pathway by mediating the activation of structural proteins such as vinculin and zyxin as well as fibronectin 1 and collagen type XVIII alpha 1 chain against UVA-induced changes.

Cell Morphology-Based Screening Identified Vitetrifolin D from &lt;i&gt;Vitex Rotundifolia&lt;/i&gt; as an Inhibitor of Phorbol Ester–Induced Downregulation of E-Cadherin in HHUA Endometrial Cells

The epithelial–mesenchymal transition (EMT) of endometrial cells contributes to the development of endometrial cancer, endometriosis, and adenomyosis. We have recently reported that 12-O-Tetradecanoylphorbol 13-acetate (TPA), a protein kinase C activator, induces EMT in HHUA endometrial cells cultured on collagen type I gels. HHUA cells showed an obvious morphological change from spheroids to scattered spindle cells during the TPA-induced EMT. In this study, we searched for inhibitors of the TPA-induced morphological change, and isolated vitetrifolin D from Vitex rotundifolia leaves. Vitetrifolin D suppressed the TPA-induced downregulation of E-cadherin, a major component of the epithelial adherens junction. Since vitetrifolin D showed a modest cytotoxicity at the concentration required to inhibit E-cadherin downregulation, it would not be suitable as a drug to treat endometrial disorders. However, our cell morphology-based screening could contribute to the discovery of new compounds that inhibit the loss of epithelial cell junctions.

S-27-3: HYPERTENSIVE NEPHROPATHY: GLOMERULAR PROTEOMIC PROFILING OF KIDNEY BIOPSIES REVEALS A SIGNATURE OF DISEASE PROGRESSION

Objective: Hypertensive nephropathy, a major cause of chronic kidney disease (CKD) worldwide, requires kidney biopsy as the diagnostic gold-standard. However, histological findings are unspecific and specific prognostic markers are missing. Accordingly, our objective was to detect candidate markers based on glomerular protein signatures from the diagnostic kidney biopsy that predict subsequent disease progression. Design and method: We studied adult patients (n = 17) with an eGFR &gt; 30 ml/min/1.73m2 and proteinuria &lt; 3 g/d from the Norwegian Kidney Biopsy Registry, including stable non-progressing patients (n = 9) and patients progressing (n = 8) to end-stage kidney disease (ESKD) within 20 years. Glomerular cross-sections from archival kidney biopsy sections were microdissected and processed for protein extraction. Proteomic analyses were performed using Q-exactive HF mass spectrometer and relative glomerular protein abundances were compared between progressive and non-progressive patients. Results: Amongst n = 1870 quality filtered proteins, n = 58 were differentially expressed in progressive and non-progressive glomerular samples, with absolute fold changes ≥ 1.5, p ≤ 0.05. However, only by using n = 17 glomerular proteins with absolute fold changes ≥ 2 and p ≤ 0.05, the hierarchical clustering and principal component analysis (PCA) effectively separated progressor and non-progressor patient samples. Supervised K nearest neighbor (KNN) analysis with leave-one-out internal cross validation was employed to develop a prognostic five protein classifier signature of hypertensive nephropathy progression. Thereby, Gamma-butyrobetaine dioxygenase (BBOX1, O75936) and Cadherin 16 (CDH16, O75309), overexpressed in progressors, and Eosinophil peroxidase (EPX, P11678), DnaJ homolog subfamily B member 1 (DNAJB1, P25685) and Alpha-1-syntrophin (SNTA1, Q13424), overexpressed in non-progressive glomeruli, correctly classified 16/17 samples into progressors and non-progressors. Respective classifier results, including area under the ROC curve of 0.99, hierarchical cluster analysis and PCA, are depicted in Figure 1 A-D (P = progressors; NP = non-progressors). Immunohistochemistry confirmed results of BBOX1 and CDH16. Geneset Enrichment Analysis (GSEA) showed that metabolic pathways were enriched in progressors, and structural cell pathways in non-progressors. Pathway analysis identified Epithelial Adherens Junction Signaling as most affected canonical pathway. We then evaluated protein signatures associated with hypertensive nephropathy progression with those associated with IgA nephropathy progression from our published dataset (Paunas T. Clin Proteom 2017). The expression of only three proteins were altered in a similar direction in both datasets. KNN classifier proteins of progression of HN were either not detected in IgAN or were not significantly altered during progression of IgAN. Conclusion: Glomerular proteomic profiling from the initial diagnostic kidney biopsy can be used to discriminate progressive from non-progressive patients with hypertensive nephropathy.

Glomerular proteomic profiling of kidney biopsies with hypertensive nephropathy reveals a signature of disease progression.

Hypertensive nephropathy (HN) requires a kidney biopsy as diagnostic gold-standard but histological findings are unspecific and specific prognostic markers are missing. We aimed at identifying candidate prognostic markers based on glomerular protein signatures. We studied adult patients (n = 17) with eGFR >30 ml/min/1.73m2 and proteinuria <3 g/d from the Norwegian Kidney Biopsy Registry, including subjects non progressing (NP, n = 9), or progressing (P, n = 8) to end-stage renal disease (ESRD) within an average follow-up of 22 years. Glomerular cross-sections from archival kidney biopsy sections were microdissected and processed for protein extraction. Proteomic analyses were performed using Q-exactive HF mass spectrometer and relative glomerular protein abundances were compared between P and NP patients. Immunohistochemistry (IHC) was used to validate selected data. Amongst 1870 quality filtered proteins, 58 were differentially expressed in P and NP patients' glomeruli, with absolute fold changes (FC) ≥1.5, p ≤ 0.05. Supervised classifier analysis (K nearest neighbor) identified a set of five proteins, including Gamma-butyrobetaine dioxygenase (BBOX1, O75936) and Cadherin 16 (CDH16, O75309), overexpressed in P, and Eosinophil peroxidase (EPX, P11678), DnaJ homolog subfamily B member 1 (DNAJB1, P25685) and Alpha-1-syntrophin (SNTA1, Q13424), overexpressed in NP glomeruli, correctly classifying 16/17 kidney biopsy samples. Geneset Enrichment Analysis (GSEA), showed that metabolic pathways were generally enriched in P, and structural cell pathways in NP. Pathway analysis identified Epithelial Adherens Junction Signaling as most affected canonical pathway. IHC analysis confirmed overexpression of BBOX1 and Cadherin 16 in glomeruli from P patients. In conclusion, glomerular proteomic profiling can be used to discriminate P from NP HN patients.

Clostridiumnovyi's Alpha-Toxin Changes Proteome and Phosphoproteome of HEp-2 Cells.

C. novyi type A produces the alpha-toxin (TcnA) that belongs to the large clostridial glucosylating toxins (LCGTs) and is able to modify small GTPases by N-acetylglucosamination on conserved threonine residues. In contrast, other LCGTs including Clostridioides difficile toxin A and toxin B (TcdA; TcdB) modify small GTPases by mono-o-glucosylation. Both modifications inactivate the GTPases and cause strong effects on GTPase-dependent signal transduction pathways and the consequent reorganization of the actin cytoskeleton leading to cell rounding and finally cell death. However, the effect of TcnA on target cells is largely unexplored. Therefore, we performed a comprehensive screening approach of TcnA treated HEp-2 cells and analyzed their proteome and their phosphoproteome using LC-MS-based methods. With this data-dependent acquisition (DDA) approach, 5086 proteins and 9427 phosphosites could be identified and quantified. Of these, 35 proteins were found to be significantly altered after toxin treatment, and 1832 phosphosites were responsive to TcnA treatment. By analyzing the TcnA-induced proteomic effects of HEp-2 cells, 23 common signaling pathways were identified to be altered, including Actin Cytoskeleton Signaling, Epithelial Adherens Junction Signaling, and Signaling by Rho Family GTPases. All these pathways are also regulated after application of TcdA or TcdB of C. difficile. After TcnA treatment the regulation on phosphorylation level was much stronger compared to the proteome level, in terms of both strength of regulation and the number of regulated phosphosites. Interestingly, various signaling pathways such as Signaling by Rho Family GTPases or Integrin Signaling were activated on proteome level while being inhibited on phosphorylation level or vice versa as observed for the Role of BRCA1 in DNA Damage Response. ZIP kinase, as well as Calmodulin-dependent protein kinases IV & II, were observed as activated while Aurora-A kinase and CDK kinases tended to be inhibited in cells treated with TcnA based on their substrate regulation pattern.