bluuebunny/crossref_metadata_2025_split huggingface dataset:
Role of IGSF3 in Lung Epithelial Barrier Disruption Induced by Cigarette Smoke Exposure
Authors: Schweitzer Kelly , Petrache Irina 
Published: June 2021

RationaleCigarette smoke (CS) exposure disrupts lung epithelial and endothelial cell barrier function which is linked to lung injury and inflammation. We identified a tetraspanin‐interacting protein, IGSF3, with roles in cell proliferation, adhesion, and migration via unclear mechanisms. We have shown that lung IGSF3 is decreased by cigarette smoke (CS) exposure and that loss of IGSF3 can disrupt sphingolipid metabolites. Of these, sphingosine‐1 phosphate (S1P) protects endothelial barrier function but may disrupt epithelial barrier via distinct receptors. We investigated if IGSF3 and S1P are required for maintaining the lung epithelial barrier in response to CS.MethodsHuman bronchial lung epithelial cells (Beas2b) were stably transfected with lentivirus control or IGSF3 shRNA (shIGSF3). Trans‐epithelial resistance (TER) was measured using electric cell‐substrate impedance sensing. CS extract was prepared using research cigarettes (3R4F, University of KY). Phosphatidylinositol lipid strips (Echelon) were used to study IGSF3‐lipid binding.ResultsCompared to control shRNA, knockdown of IGSF3 did not alter epithelial barrier function as measured by TER. Exposure to CS (7.5%), as expected, decreased epithelial cell monolayers TER in control cells by 35%. CS‐induced effects on TER were attenuated by treatment with N‐acetylcysteine or with (R)‐FTY720‐phosphonate (1R), a sphingosine‐1 phosphate receptor 1 (S1PR1) agonist, and were worsened by sphingosine kinase‐1 inhibition using SKI2. The CS‐induced decrease in TER was significantly worsened (by 55%, p<0.01 vs. control cells exposed to CS) in shIGSF3 ‐ transfected cells which responded in turn to the barrier‐enhancing effect of N‐acetylcysteine treatment (by 30%, p<0.05). A putative Pleckstrin‐homology (PH) domain was identified in the C‐terminal cytoplasmic domain of IGSF3 which predicted binding to PI(3,4)P2, which was then experimentally confirmed. Further, site directed mutagenesis within PH domain disrupted binding of IGSF3 with tetraspanin CD81.ConclusionsIGSF3 is required for partial maintenance of the lung epithelial barrier function in response to CS injury. Loss of IGSF3 may worsen CS injury of the airways, contributing to inflammation, an effect that could be alleviated by antioxidants and S1P1‐mediated signaling. The mechanisms of IGSF3 barrier effects may include binding to and tethering PI(3,4)P2 and CD81 at the cell membrane via its PH domain.Support or Funding InformationFunding: Wollowick Chair of Pulmonary Research (IP), Department of Medicine Research Microgrant (KSS) from National Jewish Health.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
The role of calcium in RGS10 regulation of inflammatory signaling in microglia
Authors: Almutairi Faris , Alqinyah Mohammed , Wendimu Menbere , Vella Stephen , Moreno Silvia , Hooks Shelley B. 
Published: June 2021

Regulator of G‐protein Signaling (RGS10) is a member of a family of proteins that canonically bind and terminate signaling downstream of G proteins. RGS10 is highly expressed in brain and immune cells, with specific enrichment in microglia. Microglial RGS10 serves an anti‐inflammatory and neuroprotective role by suppressing expression of pro‐inflammatory genes, such as tumor necrosis factor‐alpha (TNF‐α), and cyclooxygenase (COX‐2) in response to TLR4 receptor activation. We recently demonstrated that this anti‐inflammatory activity of RGS10 occurs through an undefined G protein‐independent mechanism. Further, RGS10 expression is suppressed by activation of TLR4 receptor, resulting in amplification of inflammatory gene expression. The molecular mechanisms directly linking TLR4 activation to RGS10 suppression are also undefined. In order to exploit the anti‐inflammatory effects of RGS10 in the development of therapeutics for the treatment of neuroinflammation and accompanying neurodegeneration, it is essential to define both the mechanism for RGS10 suppression of inflammatory gene expression, and the mechanism for RGS10 silencing. The current study implicates calcium signaling as playing a critical role in the regulation of both processes. We have identified STIM2 as a novel RGS10 interacting protein in BV2 microglia cells and in RAW macrophages. STIM2 is an ER‐resident calcium sensor that mediates store‐operated Ca2+ entry (SOCE) through Orai channels in the plasma membrane in response to ER calcium depletion. STIM2 activation and its coupling to Orai‐mediated calcium signaling is strongly implicated in regulation of inflammatory signaling in microglia. Therefore, we hypothesized that RGS10 suppression of TLR4‐stimulated inflammatory gene expression reflects regulation of STIM2‐Orai activity. Our results demonstrate that pharmacological inhibition of Orai channels completely blocks the amplification of TLR4‐stimulated COX‐2 and TNFα expression in response to loss of RGS10 expression. Further, RGS10 knockout microglia exhibit markedly enhanced SOCE in response to ER calcium depletion. These results suggest that RGS10 suppresses COX‐2 and TNF‐α expression downstream of TLR4 activation of Orai‐mediated SOCE. Further, we demonstrate that thapsigargin, which triggers transient rise in cytoplasmic calcium and ER calcium depletion, strongly enhanced LPS‐induced RGS10 silencing, suggesting a potential mechanism for feedback regulation of calcium signaling. These findings provide critical insight into the mechanism that RGS10 utilizes to regulate inflammatory signaling. Defining these mechanisms in microglia is essential to develop novel therapeutics that target RGS10 in treatment of neuroinflammatory diseases.Support or Funding InformationNational Institutes of HealthThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Structural and Functional Insights into Cytochromes P450 11B1 and 11B2 Interaction with Ligands and Redox Partner Proteins
Authors: Brixius‐Anderko Simone , Scott Emily E. 
Published: June 2021

Human steroidogenic cytochrome P450 enzymes 11B1 (CYP11B1) and 11B2 (CYP11B2) generate cortisol and aldosterone, controlling stress/immune responses and blood pressure, respectively. Excessive cortisol production by CYP11B1 results in Cushing's disease, while aldosterone overproduction by CYP11B2 causes hypertension and cardiac disease. Treatment of each disease with drugs inhibiting the respective enzyme is impeded because CYP11B1 and CYP11B2 have 93% identical amino acid sequences and no structures of CYP11B1 are available to identify structural differences that could be exploited clinically. A two‐fold strategy is employed herein to support the development of more selective treatment options for Cushing's and hypertension. First, structural differences between human CYP11B1 and CYP11B2 were identified. The first X‐ray structure of human CYP11B1 was solved, doing so in complex with the non‐selective inhibitor fadrozole. Comparison with a previous CYP11B2 structure also binding fadrozole revealed large differences in inhibitor orientation in the respective active sites. The two CYP11B enzymes bind distinct enantiomers of fadrozole, with CYP11B1 binding (S)‐fadrozole and CYP11B2 binding (R)‐fadrozole. Knowledge of the distinct active site architectures will facilitate structure‐based drug design for the treatment of Cushing's and hypertension. A second strategy explores the concept of selective inhibition of CYP11B enzymes interactions with their redox partner protein adrenodoxin (Adx). Binding assays revealed that adrenodoxin binding on the CYP11B2 surface significantly increases CYP11B2 affinity for its substrate in the distant active site, with much less effect on CYP11B1. Any differences in the protein/protein interaction might be exploited to provide an orthogonal approach for selective inhibition of CYP11B enzymes. A combination of structural and functional studies is thus used to identify similarities and differences in CYP11B interactions with Adx and with ligands to facilitate the design of compounds useful in the treatment of Cushing's disease and hypertension.Support or Funding InformationX‐ray data were collected at the Stanford Synchrotron Radiation Lightsource (SSRL). The SSRL Structural Molecular Biology Program is supported by the US Department of Energy Office of Biological and Environmental Research and by the US National Institutes of Health (NIH), National Center for Research Resources, Biomedical Technology Program and the National Institute of General Medical Sciences. This research was funded by R37 GM 076343 and startup funds from the University of MichiganThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Nix induced mitochondrial fission, mitophagy, and myocyte insulin resistance are abrogated by PKA phosphorylation
Authors: Gordon Joseph W. , da Silva Rosa Simone , Field Jared , Martens Matthew , Dolinsky Vernon W. , Aliani Michel 
Published: June 2021

Lipotoxicity is a form of cellular stress caused by the accumulation of lipids resulting in mitochondrial dysfunction and insulin resistance in muscle. Previously, we demonstrated that Nix is a lipotoxicity‐responsive gene that accumulates in response to diacylglycerols induced by high‐fat and sucrose (HFS) feeding and exacerbated by exposure to gestational diabetes (GDM) during fetal development. Genetic inhibition of stimulus‐activated autophagy results in impaired exercise tolerance and reduced glucose uptake, yet reducing excessive mitophagy improves insulin resistance following high‐fat feeding. To provide a better understanding of these observations, we undertook a gene expression array and shot‐gun metabolomics in soleus muscle from rodents on an HFS diet and/or exposed to GDM. Interestingly, we observed a modest reduction in several autophagy‐related genes including Beclin‐1, ATG3, ‐5, and ‐7. Moreover, we observed an alteration in the ratio of cardiolipin to phosphatidic acid and reduced mitofusin‐2 expression, suggesting aberrant mitochondrial dynamics underlies muscle insulin resistance. In a series of gain‐of‐function and loss‐of‐function experiments in rodent and human myotubes, we demonstrate that Nix accumulation triggers mitochondrial depolarization, fragmentation, calcium‐dependent activation of DRP‐1, and mitophagy. In addition, Nix‐induced mitophagy leads to myotube insulin resistance through activation of mTOR‐S6K inhibition of IRS‐1. Finally, we demonstrate that Nix‐induced mitophagy and insulin resistance can be reversed by direct phosphorylation of Nix by PKA, leading to the translocation of Nix from the mitochondria and sarcoplasmic reticulum to the cytosol. These findings provide insight into the role of Nix‐induced mitophagy and muscle insulin resistance during an overfed state when overall autophagy‐related gene expression is reduced. Furthermore, our data suggests a mechanism by which exercise or pharmacological activation of PKA may overcome insulin resistance.Support or Funding InformationSupport by NSERC, DREAM‐CHRIM, and Research Manitoba.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Lipopolysaccharide at low concentration suppressed tight junction assembly in intestinal epithelial cells via AMPK suppression and MAPK pathway
Authors: Meesomboon Roojanaat , Wongkrasant Preedajit , Muanprasat Chatchai 
Published: June 2021

Intestinal tight junction acts as a physical barrier and selective filter in intestine. Effect of low concentration of lipopolysaccharide (LPS) on AMPK activity, which is central to tight junction regulation, and its mechanism in intestinal epithelial cells (IEC) are not well characterized. This study aimed to investigate the effect of low‐concentration of LPS on AMPK suppression and intestinal tight junction permeability. To test whether low‐concentration of LPS inhibits AMPK phosphorylation, western blot analysis was performed. The result revealed that low dose LPS (1 ng/ml) suppresses AMPK activation in T84 cells. In order to investigate whether LPS suppressed intestinal tight junction assembly, calcium switch assays were performed. The result showed that LPS significantly suppressed tight junction assembly at 6 to 24 hours post‐treatment, indicating that low‐concentration of LPS inhibits tight junction assembly in IEC. Moreover, this effect was disappeared in the presence of U0126, a MAPK inhibitor. This data indicate that low‐concentration of LPS inhibits tight junction assembly via MAPK pathways. In conclusion, LPS induced AMPK suppression and tight junction permeability via MAPK pathway in IEC. This study shed light on the mechanism of intestinal diseases involving gram negative bacterial infection.Support or Funding InformationThis work is supported by Tonkla Ramathibodi program and Thailand Research Fund and Mahidol University. (BRG5980008)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Optical tweezers with IRM, TIRF and Widefield: studying cytoskeletalprocesses with or without the need for fluorescence labeling
Authors: Simpson Trey , Raja Ali , Au Ernie , Tafoya Sara , Sitters Gerrit 
Published: June 2021

Microtubules and actin filaments are dynamic cytoskeletal structures that interact with motor proteins and play a fundamental role in many essential biological processes including cell division, cell migration, and mechanosensing.Single‐molecule analysis played a central role in revealing many aspects of these complex and dynamic interactions. During a typical dynamic single‐molecule experiment, cytoskeletal filaments are imaged for an extensive period of time using fluorescence methods. It can be highly desirable to study these individual protein filaments with high contrast and at high temporal resolution but without the need for fluorescence labeling, to make the assay set‐up less laborious, costly and – in some experiments – prevent inducing phototoxicity.Here we present an experimental arrangement that includes optical tweezers in combination with Interference Reflection Microscopy (IRM) and Total Internal Reflection Fluorescence (TIRF) Microscopy. Interference Reflection Microscopy is a recently introduced imaging method that allows visualizing biological structures in 3D without the need of fluorescence labeling and with sensitivity exceeding DIC microscopy. Total Internal Reflection Fluorescence microscopy provides high resolution fluorescent imaging of specimen near the working surface with high signal to noise ratio resulting in improved single‐molecule surface assays. In addition, we demonstrate the faster 2D imaging capabilities obtained – compared to confocal and STED techniques – by introducing widefield microscopy methods to our system.In this work, we will discuss the experimental design and show an overview of the latest results obtained using this single‐molecule approach.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Comprehensive identification of micropeptides encoded by long noncoding RNAs in human tissues
Authors: Cao Huojun , Shao Fan , Li Mark , Sweat Mason , Qian Qingwen , Guo Yuwei , Amendt Brad , Yang Ling 
Published: June 2021

Many poorly characterized long noncoding RNAs (lncRNA) are expressed in human pancreas. Recently, a few lncRNAs have been found encode very small open reading frame, so‐called micropeptides. By regulating larger protein complex, these micropeptides have been shown to have important roles in fundamental biological processes such as muscle performance, mTORC1 activation and cell movement. To determine if any micropeptide play critical role for pancreas functions, we have took a bioinformatics approach to comprehensively analyze protein coding potential for lncRNAs expressed in human pancreas. We found more than 30 lncRNAs that are likely encode micropeptides. We have verified one conserved micropeptide, which we named Beta cell Glucose Regulated Micropeptide 1(BGRM1). We found BGRM1 regulate calcium homoeostasis and insulin secretion in beta cell. We have also performed mass spec to identify its binding proteins. Our result shows that some of lncRNA expressed in human pancreas might actually be translated as micropeptides and they play important roles for pancreas functions. We have also used the same pipeline to analysis lncRNA expressed in other organs. These results provides foundations for future comprehensive analysis of micropeptides functions.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Long Noncoding RNA H19 Plays an Important Role in a4‐Regulated Intestinal Epithelial Barrier Function Through microRNA 675
Authors: Chung Hee K. , Lan Shaoyang , Wang Shelley R. , Kwon Min S. , Xiao Lan , Turner Douglas J. , Wang Jian‐Ying , Jaladanki Rao N. 
Published: June 2021

The mammalian intestinal epithelial barrier protects the subepithelial tissue by preventing the intrusion of luminalnoxious substances and microbiota, and the barrier dysfunction occurs commonly in various pathologies. The protein phosphatase 2A‐associated protein α4 controls the activity and specificity of serine/threonine phosphatases and is thus implicated in many cellular processes. We have recently reported that targeted α4 deletion in intestinal epithelial cells (IECs) disrupts the epithelium homeostasis and impairs the barrier function, but its exact mechanism remains largely unknown. The H19 long noncoding RNA regulates expression of different genes via its encoded microRNA 675 (miR‐675) and is involved in the regulation of gut epithelial barrier. The current study tested the hypothesis that α4 modulates intestinal epithelial barrier function through H19/miR‐675 axis.MethodsStudies were conducted in IEC tissue‐specific α4 knockout (IE‐α4−/−)mice and differentiated IEC‐Cdx2L1 cells. Levels of H19 and miR‐675 were measured by quantitative real‐time RT‐PCR analysis, and various proteins were examined by Western immunoblotting analysis. Gut permeability was measured by FITC‐conjugated dextran in vivo and by transepithelial electrical resistance (TEER) and FITC‐dextran assays in vitro.ResultsConditional deletion of α4 in IECs increased the levels of mucosal H19 and miR‐675 in the small intestine, which was associated with a significant increase in gut permeability in IE‐α4−/− mice relative to control littermates. The levels of several intercellular junction proteins, including ZO‐1 and E‐cadherin, also decreased in the α4‐deficient intestinal epithelium. α4 silencing in cultured IECs by transfection with specific siRNA (siα4) also increased cellular H19 and miR‐675 abundances and disrupted the epithelial barrier function as indicated by a decrease in TEER and an increase in paracellular flux of FITC‐dextran. Both α4‐deficient intestinal epithelium in IE‐α4−/− mice and α4‐silenced IECs exhibited the decreased levels of RNA‐binding protein HuR that prevented miR‐675 processing from H19. Ectopically expressed HuR in α4‐silenced cells not only abolished the increased miR‐675 but also restored the epithelial barrier function to near normal level.ConclusionsThese results indicate that increased H19 in the α4‐deficient intestinal mucosa plays an important role in the epithelial barrier dysfunction through miR‐675 and that the induction in H19/miR‐675 axis by α4 deletion is prevented by HuR overexpression.Support or Funding InformationDepartment of Veterans Affairs Merit Award (RNJ, DJT, JYW) and NIH‐NIDDK (JYW)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
NLRP3 Inflammasome as a Novel Target to Abrogate Nicotine‐Induced Podocyte Injury
Authors: Singh Gurinder Bir , Kshirasagar Naresh , Patibandla Sai , Hussain Tahir , Li Xiang , Li Pin‐Lan , Koka Saisudha , Boini Krishna M 
Published: June 2021

Recent in vivo and in vitro studies have established the role of nicotine in chronic kidney diseases. However, the molecular mechanisms of how nicotine induces chronic kidney disease are still unclear. The present study tested whether nicotine induces NLRP3 inflammasome formation and activation and thereby contributes to podocyte injury and dysfunction. Confocal microscopic analysis showed that nicotine treatment increased the colocalization of NLRP3 with Asc in podocytes compared to control cells. Pretreatment with caspase‐1 inhibitor, WEHD abolished the nicotine‐induced colocalization of NLRP3 with Asc, suggesting the formation of Nlrp3 inflammasomes. Correspondingly, nicotine treatment significantly increased the caspase‐1 activity and IL‐1β production compared to control cells. The prior treatment with WEHD significantly attenuated the nicotine‐induced caspase‐1 activity and IL‐1β production. Immunofluorescence analysis showed that nicotine treatment significantly decreased the podocin and nephrin expression compared to control cells. However, prior treatment with WEHD attenuated the nicotine‐induced podocin and nephrin reduction. In addition, prior treatment with ROS inhibitor, NAC significantly attenuated the nicotine‐induced caspase‐1 activity, IL‐1β production and podocin and nephrin reduction in podocytes. Based on these results, it is concluded that nicotine‐induced the NLRP3 inflammasome activation in podocytes and thereby resulting in podocyte injury and dysfunction (supported by NIH grant, DK104031).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.
Orbital shaking drives differential changes in OK proximal tubule cell metabolism and endocytosis
Authors: Ren Qidong , Gliozzi Megan L. , Rittenhouse Natalie L. , Edmunds Lia R. , Rbaibi Youssef , Poholek Amanda C. , Locker Joseph D. , Jurczak Michael J. , Baty Catherine J. , Weisz Ora A. 
Published: June 2021

Kidney proximal tubule (PT) cells have high metabolic demands to drive the extraordinary ion and solute transport, water reabsorption, and endocytic uptake that occur in this nephron segment. Increases in renal blood flow alter glomerular filtration rate (GFR) and lead to rapid mechanosensitive adaptations in PT transport, impacting metabolic demand. Although the PT reabsorbs essentially all of the filtered glucose, PT cells rely primarily on oxidative metabolism rather than glycolysis to meet their energy demands. We lack an understanding of how PT metabolism is impacted by changes in O2 availability via cortical capillaries and mechanosensitive signaling in response to alterations in luminal flow. Previously, we found that opossum kidney (OK) cells recapitulate key features of PT cells in vivo, including enhanced endocytic uptake and ion transport, when exposed to mechanical stimulation by culture on an orbital shaker. We hypothesized that increased oxygenation resulting from orbital shaking also contributes to this more physiologic phenotype. RNA Seq of OK cells maintained under static conditions or exposed to orbital shaking for up to 96h revealed significant time‐ and culture‐dependent changes in gene expression. Transcriptional and metabolomics data were consistent with a decrease in glycolytic flux and with an increased utilization of aerobic metabolic pathways in cells exposed to orbital shaking. Moreover, we found spatial differences in the pattern of mitogenesis and endocytic capacity in our culture system that highlight the complexity of O2‐dependent and mechanosensitive crosstalk to regulate PT cell function.Support or Funding InformationTsinghua MD Scholars ProgramNIH R01 DK101484NIH P30 DK079307This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.