bluuebunny/crossref_metadata_2025_split huggingface dataset:
Leptin Activates Transient Receptor Potential Melastatin 7 (TRPM7) Channels in Leptin‐Receptor Expressing Pheochromocytoma (PC12) Cells
Authors: Liu Xiao‐Ru , Paudel Omkar , Shin Mi‐Kyung , Tang Wan‐Yee , Shirahata Machiko , Polotsky Vsevolod Y. , Sham James S.K. 
Published: November 2023

Obesity is a leading cause of high cardiovascular morbidity and mortality. It is characterized by an increased leptin level, which has been implicated in increased sympathetic activity and the pathogenesis of hypertension. However, the mechanism by which leptin enhances sympathetic activity is unclear. Leptin receptors (LEPRs) are expressed in the carotid bodies, the most important arterial chemoreceptors for the cardiovascular‐respiratory chemoreflex. We have previously demonstrated that leptin enhances carotid sinus nerve activity in response to hypoxia; this effect is abolished by non‐selective blockers of transient receptor potential (TRP) channels; and the TRPM7 channel is the most abundant TRP subtype expressed in CB (Shirahata et al., Adv Exp Med Biol. 2015;860:153–9). Since leptin is known to regulate many different ion channels, we hypothesize that leptin may exert its effects through modulation of TRPM7 channel activity. To test this hypothesis, we used LEPR (LEPRb) expressing pheochromocytoma (PC12LEPRb) cells, which express a high level of TRPM7 channels, as the cell model. Non‐selective cation channels were recorded under amphotericin‐B perforated‐patch techniques with K+ replaced by Cs+ in the presence of voltage‐gated Ca2+ channel and Cl− channel blockers. Voltage‐ramp from −100 to 100 mV activated an outward‐rectifying current. Removal of extracellular divalent ions enhanced the inward current, consistent with the inward divalent ion selectivity of TRPM7. The specific TRPM7 agonist naltriben caused significant increase of the current, which was abolished by the TRPM7 antagonist NS8593 and FTY720, indicating that functional TRPM7 channels are present in PC12LEPRb cells. More importantly, leptin at concentration of 10–100 ng/ml caused concentration‐dependent increase in the outward rectifying current. The enhanced current was completely blocked by NS8593 and FTY720. These results for the first time demonstrated that stimulation of leptin receptor is capable of activating TRPM7 channels, and suggest that leptin may exert its physiological effects through modulation of TRPM7 activity.Support or Funding InformationThis work is supported by the NIH grant HL133100
Molecular probes to study the subcellular localization and dynamics of phospholipids and cholesterol
Authors: Fairn Gregory D. 
Published: November 2023

Cellular lipids are critical components of the membrane bilayers that delineate individual organelles and separate the cell from its environment. Additionally, many classes of lipids serve important roles in signal transduction and vesicular transport. To complement traditional biochemical techniques used to quantify individual classes and species of lipids, researchers have developed a number of genetically encoded probes and antibodies to investigate the subcellular localization and dynamics of lipids of interest. This presentation will highlight the use of specific probes to study the inhomogeneous distribution and dynamics of phospholipids and cholesterol within the cell and during the process of phagocytosis in macrophage.Support or Funding InformationSupported by the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, the Government of Ontario Early Researcher Award, and the Canadian Consortium on Neurodegeneration in Aging.
Cryptosporidium parvum infection disrupts intestinal epithelial barrier function via decreasing expression of specific tight junction and adherens junction proteins
Authors: KUMAR ANOOP , Chaterjee Ishita , Anbazhagan Arivarasu Natarajan , Priyamvada Shubha , Jayawardena Dulari , Alrefai Waddah , Borthakur Alip , Dudeja Pradeep Kumar 
Published: November 2023

BackgroundEnteric infection with the protozoan parasite Cryptosporidium parvum (CP) causes cryptosporidiosis, a widespread diarrheal disease. CP sporozoites released in the lumen invade epithelial cells of the small intestine and complete the infectious cycle therein. However, mechanisms of CP infection‐induced diarrhea are not fully understood. Decreased barrier function and increased epithelial permeability are known to contribute to the pathophysiology of diarrheal diseases caused by enteric infections. Intestinal epithelial barrier integrity is maintained by tight junctions (TJ) and adherens junctions (AJ). However, there are virtually no studies on CP effects on the expression of TJ and AJ proteins and their relevance to cryptosporidiosis.AimThe current studies were performed to examine the effects of CP infection on paracellular permeability and on the expression of occludin and claudins (key proteins of TJ assembly), and E‐cadherin (a major AJ protein) utilizing Caco‐2 cell monolayers, ex vivo mouse enteroids and in vivo mouse model.MethodsTransepithelial resistance (TER) was measured by a Volt Ohm Meter, paracellular permeability was measured by apical to basolateral flux of FITC‐dextran, mRNA and protein levels of the TJ/AJ components were measured by real‐time RT‐PCR and immunoblotting/immunofluorescence, respectively.ResultsCP infection (0.5 × 106 oocysts/well) for 24 h decreased TER and increased paracellular permeability in Caco‐2 cell monolayers. CP infection significantly decreased protein levels of occludin, claudin‐4 and E‐cadherin with no effects on other claudins in Caco‐2 monolayers, mouse‐enteroid‐derived monolayers, and in the mucosa of ileum and jejunum in C57BL/6 mice (infected with 1.0 × 107 oocysts/mouse for 24 hr). mRNA levels of these three proteins were also significantly decreased in CP infected mouse ileum and jejunum, however, CP infection of Caco‐2 cells did not alter occludin and claudin‐4 mRNA levels. Further, bafilomycin, an inhibitor of lysosomal proton pump, partially abrogated CP‐induced downregulation of occludin expression, suggesting a potential role of post‐translational mechanisms such as protein degradation in CP‐induced downregulation of occludin in Caco‐2 cells.ConclusionOur studies suggest that disruption of barrier function via downregulation of specific key components of TJ and AJ could contribute to CP infection‐induced diarrhea.Support or Funding InformationSupported by NIDDK and Department of Veterans Affairs
An Inherent Defect in Tight Junction Structure and Permeability is Apparent in Colonoids from Crohn's Disease Patients
Authors: Gadeock Safina , Schultz Michael , Butt Grant 
Published: November 2023

An increase in intestinal permeability is associated with inflammatory bowel disease (IBD). However, whether the increased permeability is a cause or consequence of the inflammation is unknown. Here we have used epithelial derived colonoids from healthy individuals and Crohn's (CD) patients to determine whether there is an inherent defect in intestinal epithelial permeability associated with CD. Colonoids were grown for 15 days from biopsies from the transverse colon of healthy patients with no known history of IBD and non‐inflamed regions of the transverse colon of CD patients. The epithelial structure was characterised with both conventional and electron microscopy, and the expression of the tight junction proteins occludin, ZO‐1 and claudin 2, quantified with qPCR and western blot. The distribution of the proteins was determined with immunohistochemistry, while the relative permeability of the colonoids was measured by adding 0.1 mg ml−1 of FITC labelled dextran 4000 (FD4) to the growth media for 24 h and then measuring the fluorescence intensity in the organoid lumen by confocal microscopy. Surface area and volume of the organoids was calculated from the measured radius to allow calculation of the FD4 flux (μg cm−2 24 h−1). Macroscopically there were no obvious differences between the control and CD colonoids but, although the epithelium in the CD colonoids consisted of columnar epithelial cells with basal nuclei, the cells in the CD colonoids had extensive apical protrusions and ≈20% of the CD colonoids were inside out. Comparison of the control and CD colonoids indicated there was no difference in the levels of expression of either transcript or protein of occludin, ZO1 and claudin 2. There was, however, a marked redistribution of the tight junction proteins in the CD colonoids. In the control colonoids, ZO‐1 and occludin were concentrated at the apical pole of the epithelial cells, whereas in the CD colonoids they were located in the basolateral membrane and cytoplasm. In contrast, in control colonoids claudin 2 was located in the basolateral membrane, whereas in the CD colonoids it was concentrated at the apical pole. Associated with this redistribution of the tight junction proteins was a marked increase (P<0.001) in the FD4 flux from 0.071±0.07 to 0.425±0.081 μg cm−2 24 h−1 in control (80 colonoids from 5 patients) and CD (98 colonoids from 5 patients) colonoids, respectively. Collectively, these data suggest that an inherent defect develops in the CD organoids independent of inflammatory or bacterial effects. This supports the proposal that in vivo an increased permeability contributes to the development of Crohn's disease.Support or Funding InformationSupported by a University of Otago Research Grant and Grants from the Department of Physiology and the Dean's Funds, Otago School of Biomedical Sciences and Dunedin School of Medicine
Claudin‐4 Disrupts Claudin‐2 Anchoring and Pore Function via Cis, but not Trans, Interactions at the Tight Junction
Authors: France Marion , Turner Jerrold 
Published: November 2023

Tight junction strands are composed of claudin (cldn) proteins. The consensus view is that cldns either form pores that mediate paracellular flux or form seals that prevent such flux. An alternative view is that cldns that reduce paracellular flux do so by competitively disrupting pore‐forming cldn function. Both trans‐ and cis‐interactions between similar (homotypic) and dissimilar (heterotypic) cldns have been implicated in strand assembly and anchoring at the tight junction. We sought to test the cldn competition model and define the topology of cldn‐cldn interactions involved in cldn2 anchoring. mEGFP‐cldn‐2 expression in monolayers of high resistance MDCK I cells, which lack endogenous cldn2, reduced transepithelial electrical resistance (TER) by 38%±3%. Conversely, mCh‐cldn4 expression increased TER of mEGFP‐cldn‐2 expressing monolayers. These data indicate that cldn4 can oppose cldn2‐dependent paracellular pore function, but do not distinguish between pore‐seal and competition models of cldn function. The latter is, however, favored by the observation that cldn4 knockout does not significantly affect TER of MDCK I monolayers. Fluorescence recovery after photobleaching (FRAP) analyses demonstrated an mEGFP‐cldn2 mobile fraction (Mf) of 18%±3%, while mCherry‐cldn4 displayed a much higher Mf of 44%±4%. When mCherry‐cldn4 was expressed with mEGFP‐cldn2 at symmetric tight junctions, mEGFP‐cldn2 FRAP increased markedly, with an overall Mf of 35%±3%. This suggests that cldn4 increases TER by destabilizing cldn2 at the tight junction. Cldn2 stabilization at the tight junction did not require cldn2‐cldn2 trans‐interactions, as the mEGFP‐cldn2 Mf was similar at asymmetric junctions between cells expressing mEGFP‐cldn2 and non‐transfected MDCK I cells (23%±4%). Further, cldn4‐mediated cldn2 destabilization was not dependent on cldn4‐cldn2 trans‐interactions as demonstrated by similar mEGFP‐cldn2 Mf at asymmetric junctions between cells expressing mEGFP‐cldn2 and those expressing mCherry‐cldn4 (25%±3%). Cldn4 therefore modifies cldn2 anchoring at the tight junction via cis‐interactions within individual cells. These data support the cldn competition model and suggest that cldn4 increases TER by disrupting cldn2‐cldn2 cis‐interactions that are required for anchoring and pore function.Support or Funding InformationDK061931
TFF2, CXCR4 and EGF‐R mediated gastric wound repair in vitro in gastric organoids
Authors: Engevik Kristen , Aihara Eitaro , Matthis Andrea , Montrose Marshall 
Published: November 2023

BackgroundTrefoil factor 2 (TFF2), a secreted peptide found in the stomach mucosa, is known to play an important role in promoting gastric epithelial repair following damage. We previously demonstrated in vivo that TFF2 acts via the chemokine C‐X‐C receptor 4 (CXCR4). Due to the complexity of dissecting molecular pathways in vivo, the mechanisms of TFF2 action remain unclear. Epidermal growth factor receptor (EGF‐R) is implicated in repair and has been shown to be involved in the TFF2 pathway outside of the gastrointestinal tract. Utilizing the 3D primary culture of gastric epithelial cells known as gastric organoids, which mimic in vivo gastric epithelium, we tested the role of EGF‐R and TFF2‐driven restitution. We tested the hypothesis that TFF2 activates CXCR4 and transactivates EGF‐R to promote gastric restitution.MethodsGastric organoids were generated from isolated corpus tissue of wild‐type (WT) and TFF2 knockout (TFF2−/−) mice. Gastric organoids were cultured for 4–5 days prior to experiments. Damage was induced in gastric organoids by 5s exposure of single epithelial cells to high intensity two‐photon 720 nm light (photodamage), resulting in damage to the targeted cell and subsequent cell death. Progression of damage and repair was evaluated using confocal/2‐photon microscopy, imaging cell nuclei (10 μg/ml Hoechst33342 vital DNA stain) and measuring the intensity of Lucifer yellow ([LY] 20 μM added to culture medium) in the luminal side and extracellular space outside of the organoid.ResultsUnperturbed organoids maintain the integrity of the epithelial barrier and show no sign of basal cell migration. Photodamage injury in WT organoids resulted in the simultaneous events of dead cell exfoliation and migration of undamaged neighboring cells to restore a continuous epithelium in the damaged site. To assess repair of damage, we measured the rate of cell exfoliation (μm/min) as determined by nuclear (Hoechst) and fluorescence (LY) confocal images. Photodamaged TFF2−/− gastric organoids showed slowed repair of damage (0.09 ± 0.02 μm/min, n=5) compared to WT organoids (0.5 ± 0.045 μm/min, n=5). Addition of 1 μg/mL AMD3100 (CXCR4 inhibitor) to WT organoids similarly slowed repair (0.08 ± 0.01 μm/min, n=6), suggesting the presence of CXCR4 receptor in the gastric organoid model. Moreover, photodamage in WT organoids exposed to 200 nM AG1478 (EGF‐R inhibitor) resulted in a slower repair at 0.14 ± 0.03 μm/min (n=3).ConclusionResults demonstrate that TFF2, CXCR4 and EGF‐R are required for efficient epithelial restitution in gastric organoids. Outcomes suggest that gastric organoids can be a useful tool to elucidate cellular signaling mechanisms involved in epithelial repair of gastric damage.Support or Funding InformationSupported by NIH R01 DK102551
Alteration of colonic motility by protease activated receptors in DSS‐induced colitis
Authors: Sung Tae Sik , Sanders Kenton , Koh Sang Don 
Published: November 2023

The fact that 4.5% of all human genes encode proteases in the human genome reflects the importance of proteases in the human body under physiological and pathophysiological conditions. Proteases can trigger cell signaling pathways via activation of protease activated receptors (PARs). Activation PARs appear to play a significant role in GI inflammatory bowel diseases (IBD) such as Crohn's disease and ulcerative colitis, and GI motor dysfunction (leading to diarrhea or constipation). Although the role of PARs in GI mucosa and lamina propria has been reported, information of PARs expression and their pathological significance of PAR modulation in GI muscles layer are limited. Inflammation was induced by exposing to DSS for 7 days in C57BL/6 mice. Inflammatory colon showed bloody stool, erosion and inflammatory cell infiltration. PAR1 agonist‐induced initial relaxation was reduced in DSS‐treated colons. PAR2 agonist‐induced responses were not affected on DSS‐treated colitis. DSS‐treated colonic smooth muscle revealed the depolarized the resting membrane potentials and EFS‐induced purinergic inhibitory junction potentials which are mediated by activation of small conductance Ca2+‐activated K+ channels (SK) were also reduced in DSS‐colitis. Quantitative transcriptional analysis showed that expressions of Pdgra and Kcnn3 were reduced in DSS‐treated colons. Since PAR activation can be coupled to Ca2+ sensitization pathways, we examined the changes in protein expression of MYPT1 and CPI‐17. MYPT1 and CPI‐17 phosphorylation were increased in DSS‐treated colons. In conclusion, the alteration of motility by PAR1 is due to changes in Pdgfra and Kcnn3 with Ca2+‐sensitization pathways. These findings suggest a role for PARs in contributing to IBD associated functional dysmotility.Support or Funding InformationP01‐DK41315
Transcriptional Regulation of SLC26A3 (DRA) by CDX2
Authors: Chatterjee Ishita , Kumar Anoop , Madrigal Rosa Mª Castilla , Pellon‐Cardenas Oscar , Borthakur Alip , Gill Ravinder , Alrefai Waddah , Verzi Michael , Dudeja Pradeep Kumar 
Published: November 2023

BackgroundSLC26A3 (Down Regulated in Adenoma, DRA) plays a key role in mammalian intestinal NaCl absorption via mediating apical membrane Cl−/HCO3− exchange. DRA function and expression is significantly decreased in diarrheal diseases associated with infections and inflammatory bowel diseases (IBD). Since, DRA is now being touted as an important therapeutic target for infectious and IBD associated diarrhea, it is critical to understand the detailed mechanisms of regulation of DRA expression at the molecular and cellular level in health and disease. Earlier studies showed that, the intestinal epithelial caudal‐related homeobox transcription factor 2 (CDX2) is an essential regulator of intestinal epithelial homeostasis and is also involved in differentiation. Interestingly, DRA expression is higher in differentiated cells and in silico analysis of DRA promoter showed 2 consensus sites for CDX2 binding.AimCurrent studies were, therefore, undertaken to test the hypothesis that CDX2 regulates DRA transcription.MethodsCdx2Fl/Fl;Villin‐CreERT2 mice, [where tamoxifen (TAM) deletes Cdx2 in intestine], were used for these studies and Caco2 cells were used as an in vitro model to study the overexpression and silencing effects of CDX2 by transfecting the cDNA and siRNA, respectively. mRNA and protein levels were assessed by real time RT‐PCR and immunoblotting/immuno‐staining, respectively. DRA promoter activity was determined by luciferase assay. Direct binding of CDX2 to DRA promoter region was assessed by chromatin immuno‐precipitation (CHIP) and electrophoretic mobility shift assay (EMSA).ResultsCDX2− /− mice showed marked reduction in DRA mRNA in ileum (Arbitrary units: WT=2.73 ± 1.04, KO=0.029 ± 0.02 (P<0.05), proximal colon (PC) WT=1.87 ± 0.53, KO=0.51 ± 0.08; P <0.05) and distal colon (DC) (WT=1.75 ± 0.46, KO=0.02 ±0.02, P<0.05) as well as in protein levels as compared to wild type mice. Similarly, silencing of CDX2 in Caco2 cells resulted in a marked (~ 50%) decrease in DRA mRNA and protein levels, whereas, ectopic over‐expression of CDX2 significantly up‐regulated DRA expression. CDX2 overexpression also stimulated DRA promoter activity by ~10‐fold, indicating its role in transcriptional regulation. We next utilized EMSA and ChIP assays to demonstrate direct binding of CDX2 to one putative cis elements identified in the 645 to 663 (+) region of the DRA promoter.ConclusionOur studies, for the first time, demonstrate a direct transcriptional regulation of DRA by CDX2. Given that CDX2 has been shown to be downregulated in UC (ulcerative colitis) and CD (crohn's disease) patients, CDX2 may at least partly be responsible for the observed decrease in DRA expression in IBD.Support or Funding InformationSupported by NIDDK/Department of Veteran Affairs
Desmoglein 2 regulates PI3‐kinase/AKT‐dependent expression of Claudin 2 in intestinal epithelial cells
Authors: Burkard Natalie , Meir Michael , Flemming Sven , Germer Christoph‐Thomas , Waschke Jens , Schlegel Nicolas 
Published: November 2023

Desmoglein 2 (Dsg2) is a desmosomal junctional protein expressed in all epithelia including the intestinal mucosa, and has been recently proposed to contribute in the pathogenesis of inflammatory bowel disease. In patients suffering from Crohn's disease (CD) it has been reported that reduced expression of intestinal Dsg2 is associated with impaired intestinal epithelial barrier function and accompanied with changes of tight junction (TJ) integrity. A previous study has provided that the application of a Dsg‐specific tandem peptide (TP) linking extracellular domains of adjacent desmogleins increases Dsg2‐mediated intercellular adhesion and strongly reduced TNFα‐induced expression of claudin2 (Cld2), which is referred to as “leaky” TJ protein. However the signaling pathways involved in the regulation of Cld2 by Dsg2 under inflammatory conditions are not elucidated yet.The incubation of differentiated CaCo2 cells with TNFα led to an impaired epithelial barrier function as revealed by decreased trans‐epithelial electric resistance (TER) and loss of intercellular adhesion in dispase‐based enterocyte dissociation assay. Simultaneously, Dsg2 was reduced at the cell borders and claudin2 expression was significantly augmented as confirmed in Western blot and RT‐PCR analyses. Interestingly, the application of TP restored all TNFα‐induced effects.Further analyses on signaling pathways demonstrated that both activation of the p38 mitogen‐activated protein kinase (p38MAPK) and phosphatidylinositol 3‐kinase (PI3K) with consecutive AKT phosphorylation are required for TNFα‐induced up‐regulation of Cld2 since the PI3‐kinase inhibitor LY2914002 and the p38MAPK inhibitor SB2021290 blocked up‐regulation of Cld2. Sequential experiments using these inhibitors determined that p38MAPK appears to be upstream of PI3K/AKT signaling. Interestingly, TP blocked both TNF‐induced activation of p38MAPK and PI3K/AKT signaling. By immunoprecipitation we found a direct interaction between Dsg2 and PI3‐kinase which was abrogated after TNFα incubation conforming to the observation in immunostaining that PI3‐kinase co‐localized with Dsg2 under steady state but dissociated from the cell borders to the cytoplasm under inflammatory conditions. These results were strengthened by knock‐down experiments using Dsg2‐siRNA where neither LY294002 nor TP were able to inhibit the PI3‐kinase signaling pathway and consecutive up‐regulation of Cld2.Our findings provide new insights into the contribution of Dsg2 in regulating TJ composition under inflammatory conditions. Based on our in vitro observations Dsg2 regulates Cld2 expression in a p38MAPK/PI3K/AKT‐dependent manner.