Expression In Epithelial Cells Research Articles

Adenylate kinase immobilized on graphene oxide impairs progression of human lung carcinoma epithelial cells through adenosinergic pathway.

Purinergic signaling, the oldest evolutionary transmitter system, has been increasingly studied as a pivotal target for novel anti-cancer therapies. In the present work, the developed nanobiocatalytic system consisting of adenylate kinase immobilized on graphene oxide (AK-GO) was characterized in terms of its physicochemical and biochemical properties. We put special emphasis on the AK-GO influence on purinergic signaling components, that is, ecto-nucleotides concentration and ecto-enzymes expression and activity in human lung carcinoma epithelial (A549) cells. The immobilization-dependent modification of AK kinetic parameters allowed for the removal of ATP excess while maintaining low ATP concentrations, efficient decrease in adenosine concentration, and control of the nucleotide balance in carcinoma cells. The cyto- and hemocompatibility of developed AK-GO nanobiocatalytic system indicates that it can be successfully harnessed for biomedical applications. In A549 cells treated with AK-GO nanobiocatalytic system, the significantly decreased adenosinergic signaling results in reduction of the proliferation and migration capability of cancer cells. This finding is particularly relevant in regard to AK-GO prospective anti-cancer applications.

Characterization of elements, PAHs, AhR-activity and pro-inflammatory responses of road tunnel-derived particulate matter in human hepatocyte-like and bronchial epithelial cells

The aims were to characterize the content of elements and polycyclic aromatic hydrocarbons (PAHs) in size-separated particulate matter (PM) sampled in a road tunnel, estimate the contribution of PAHs to the toxic potential, and measure the pro-inflammatory potential of PM samples and extracts with increasing polarity. Several elements/metals previously associated with cytokine responses were found. Based on PAHs levels and published PAHs potency, the calculated mutagenic and carcinogenic activities of size-separated samples were somewhat lower for coarse than fine and ultrafine PM. The AhR-activity of the corresponding PM extracts measured in an AhR-luciferase reporter model (human hepatocytes) were more similar. The highest AhR-activity was found in the neutral (parent and alkylated PAHs) and polar (oxy-PAHs) fractions, while the semi-polar fractions (mono-nitrated-PAHs) had only weak activity. The neutral and polar aromatic fractions from coarse and fine PM were also found to induce higher pro-inflammatory responses and CYP1A1 expression in human bronchial epithelial cells (HBEC3-KT) than the semi-polar fractions. Fine PM induced higher pro-inflammatory responses than coarse PM. AhR-inhibition reduced cytokine responses induced by parent PM and extracts of both size fractions. Contributors to the toxic potentials include PAHs and oxy-PAHs, but substantial contributions from other organic compounds and/or metals are likely.

Peroxiredoxin 1 aggravates acute kidney injury by promoting inflammation through Mincle/Syk/NF-κB signaling

Damage-associated molecular patterns (DAMPs) are a cause of acute kidney injury (AKI). Our knowledge of these DAMPs remains incomplete. Here, we report serum peroxiredoxin 1 (Prdx1) as a novel DAMP for AKI. Lipopolysaccharide (LPS) and kidney ischemia/reperfusion injury instigated AKI with concurrent increases in serum Prdx1 and reductions of Prdx1 expression in kidney tubular epithelial cells. Genetic knockout of Prdx1 or use of a Prdx1-neutralizing antibody protected mice from AKI and this protection was impaired by introduction of recombinant Prdx1 (rPrdx1). Mechanistically, lipopolysaccharide increased serum and kidney proinflammatory cytokines, macrophage infiltration, and the content of M1 macrophages. All these events were suppressed in Prdx1-/- mice and renewed upon introduction of rPrdx1. In primary peritoneal macrophages, rPrdx1 induced M1 polarization, activated macrophage-inducible C-type lectin (Mincle) signaling, and enhanced proinflammatory cytokine production. Prdx1 interacted with Mincle to initiate acute kidney inflammation. Of note, rPrdx1 upregulated Mincle and the spleen tyrosine kinase Syk system in the primary peritoneal macrophages, while knockdown of Mincle abolished the increase in activated Syk. Additionally, rPrdx1 treatment enhanced the downstream events of Syk, including transcription factor NF-κB signaling pathways. Furthermore, serum Prdx1 was found to be increased in patients with AKI; the increase of which was associated with kidney function decline and inflammatory biomarkers in patient serum. Thus, kidney-derived serum Prdx1 contributes to AKI at least in part by activating Mincle signaling and downstream pathways.

Location of Retinal Pigment Epithelial Cells in the Eye Is Critical to Their Function

The eye is an extension of the brain and a highly sophisticated sensory organ. The intricacy of the eye is well appreciated on examination of the exquisite posterior pole, composed of numerous interconnected cell and tissue types. These types broadly include the multicellular neural retina; a single layer of epithelial cells providing support to the retina, called the retinal pigment epithelium; a multilaminar extracellular matrix called the Bruch membrane; and the choroid, a thin, trilaminar connective tissue adjacent to the Bruch membrane, composed of a dense web of vessels, pigments, and immune cells. 1 Choudhary M. Malek G. Potential therapeutic targets for age-related macular degeneration: the nuclear option. Prog Retin Eye Res. 2022; 101130 PubMed Google Scholar The posterior pole can be partitioned into two regions, the macula and the periphery, adding another tier of complexity to the eye. The anatomic macula, when evaluated histologically, is an oval-shaped region approximately 5 mm in diameter, centrally located in the posterior pole of eye. Though covering approximately 4% of the retina, it is densely composed of cone photoreceptors, facilitating high-resolution color vision. The rest of the retina is rich in rod photoreceptors, involved in side, or peripheral, and night vision. Other regional differences include, but are not limited to, variances in the structural architecture of the retinal layers, cellular distribution, and blood flow. 2 Johnson M. Dabholkar A. Huang J.D. Presley J.B. Chimento M.F. Curcio C.A. Comparison of morphology of human macular and peripheral Bruch's membrane in older eyes. Curr Eye Res. 2007; 32: 791-799 Crossref PubMed Scopus (35) Google Scholar , 3 Quinn N. Csincsik L. Flynn E. Curcio C.A. Kiss S. Sadda S.R. Hogg R. Peto T. Lengyel I. The clinical relevance of visualising the peripheral retina. Prog Retin Eye Res. 2019; 68: 83-109 Crossref PubMed Scopus (70) Google Scholar , 4 Sabeti F. Lane J. Rohan E.M.F. Rai B.B. Essex R.W. McKone E. Maddess T. Correlation of central versus peripheral macular structure-function with acuity in age-related macular degeneration. Transl Vis Sci Technol. 2021; 10: 10 Crossref PubMed Scopus (9) Google Scholar Thus, it may come as no surprise that human retinal diseases demonstrate regional selectivity, in which a cell or tissue type is particularly vulnerable in one area while in adjacent zones may remain mostly unaffected. In this issue of the American Journal of Pathology, Mullin et al 5 Mullin N.K. Voigt A.P. Boese E.A. Liu X. Stone E.M. Tucker B.A. Mullins R.F. Transcriptomic and chromatin accessibility analysis of the human macular and peripheral retinal pigment epithelium at the single-cell level. Am J Pathol. 2023; Abstract Full Text Full Text PDF PubMed Google Scholar explore several essential questions in this realm: Is there region-associated heterogeneity in retinal pigment epithelial cells, whose dysfunction is central to the development of several vision-debilitating diseases?; What are the differences in the expression profiles of retinal pigment epithelial cells in the macula versus the periphery?; and Could regional differences in the levels of expression in retinal pigment epithelial cells be linked to susceptibility to disease? Transcriptomic and Chromatin Accessibility Analysis of the Human Macular and Peripheral Retinal Pigment Epithelium at the Single-Cell LevelThe American Journal of PathologyPreviewSome human retinal diseases are characterized by pathology that is restricted to specific cell types and to specific regions of the eye. Several disease entities either selectively affect or spare the macula, the retina region at the center of the posterior pole. Photoreceptor cells in the macula are involved in high-acuity vision and require metabolic support from non-neuron cell types. Some macular diseases involve the retinal pigment epithelium (RPE), an epithelial cell layer with several metabolic-support functions essential for the overlying photoreceptors. Full-Text PDF

Protective Effects of Piceatannol on DNA Damage in Benzo[a]pyrene-Induced Human Colon Epithelial Cells

Evidence shows that the dietary intake of polycyclic aromatic hydrocarbons (PAHs) from food processing induces the cellular DNA damage response and leads to the development of colorectal cancer (CRC). Therefore, protecting from cellular DNA damage might be an effective strategy in preventing CRC. Benzo[a]pyrene (B[a]P) was used as a CRC initiator in the present study. Compared with other stilbenoids, piceatannol (PIC) showed the most effective inhibition of B[a]P-induced cytochrome P450 1B1 (CYP1B1) protein expression in NCM460 normal human colon epithelial cells. PIC treatment alleviated DNA migration and enhanced the expression of DNA-repair-related proteins, including histone 2AX (H2AX), checkpoint kinase 1 (Chk1), and p53, in B[a]P-induced NCM460 cells. The 1,1-diphenyl-2-picrylhydrazyl (DPPH) assay, flow cytometry, and enzyme-linked immunosorbent assay (ELISA) revealed that PIC exerted antioxidative effects on NCM460 cells by increasing the glutathione (GSH) content and scavenging the excess intracellular reactive oxygen species (ROS) induced by B[a]P. Furthermore, PIC suppressed B[a]P-induced CYP1B1 protein expression and stimulated miR-27b-3p expression. The upregulation of phase II detoxification enzymes, such as nicotinamide adenine dinucleotide phosphate (NADPH) and quinone oxidoreductase 1 (NQO1), and the antioxidative enzyme, heme oxygenase 1 (HO-1), via the activation of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway was observed in the PIC-treated group. Our results suggest that PIC is a potential CRC-blocking agent due to its ability to alleviate DNA damage, decrease intracellular ROS production, modulate the metabolism and detoxification of B[a]P, and activate the Nrf2 signaling pathway in B[a]P-induced NCM460 cells.

YAP promotes AP-1 expression in tubular epithelial cells in the kidney.

Chronic kidney disease (CKD) is a major health problem. Kidney fibrosis is a hallmark and final common pathway of CKD. The Hippo/yes-associated protein (YAP) pathway regulates organ size, inflammation, and tumorigenesis. Our previous study demonstrated tubular YAP activation by tubule-specific double knockout of mammalian STE20-like protein kinase 1/2 (Mst1/2) induced CKD in mice, but the underlying mechanisms remain to be fully elucidated. Activator protein (AP)-1 activation was found to promote tubular atrophy and tubulointerstitial fibrosis. Therefore, we studied whether YAP regulates AP-1 expression in the kidney. We found that expression of various AP-1 components was induced in kidneys subjected to unilateral ureteric obstruction and in Mst1/2 double knockout kidneys, and these inductions were blocked by deletion of Yap in tubular cells, with Fosl1 being most affected compared with other AP-1 genes. Inhibition of Yap also most highly suppressed Fosl1 expression among AP-1 genes in HK-2 and IMCD3 renal tubular cells. YAP bound to the Fosl1 promoter and promoted Fosl1 promoter-luciferase activity. Our results suggest that YAP controls AP-1 expression and that Fosl1 is the primary target of YAP in renal tubular cells.NEW & NOTEWORTHY Yes-associated protein (YAP) activation leads to tubular injury, renal inflammation, and fibrosis, but the underlying mechanisms are not fully understood. We now provide genetic evidence that YAP promotes activator protein-1 expression and that Fosl1 is the primary target of YAP in renal tubular cells.

Global Down-regulation of Gene Expression Induced by Mouse Mammary Tumor Virus (MMTV) in Normal Mammary Epithelial Cells.

Mouse mammary tumor virus (MMTV) is a betaretrovirus that causes breast cancer in mice. The mouse mammary epithelial cells are the most permissive cells for MMTV, expressing the highest levels of virus upon infection and being the ones later transformed by the virus due to repeated rounds of infection/superinfection and integration, leading eventually to mammary tumors. The aim of this study was to identify genes and molecular pathways dysregulated by MMTV expression in mammary epithelial cells. Towards this end, mRNAseq was performed on normal mouse mammary epithelial cells stably expressing MMTV, and expression of host genes was analyzed compared with cells in its absence. The identified differentially expressed genes (DEGs) were grouped on the basis of gene ontology and relevant molecular pathways. Bioinformatics analysis identified 12 hub genes, of which 4 were up-regulated (Angp2, Ccl2, Icam, and Myc) and 8 were down-regulated (Acta2, Cd34, Col1a1, Col1a2, Cxcl12, Eln, Igf1, and Itgam) upon MMTV expression. Further screening of these DEGs showed their involvement in many diseases, especially in breast cancer progression when compared with available data. Gene Set Enrichment Analysis (GSEA) identified 31 molecular pathways dysregulated upon MMTV expression, amongst which the PI3-AKT-mTOR was observed to be the central pathway down-regulated by MMTV. Many of the DEGs and 6 of the 12 hub genes identified in this study showed expression profile similar to that observed in the PyMT mouse model of breast cancer, especially during tumor progression. Interestingly, a global down-regulation of gene expression was observed, where nearly 74% of the DEGs in HC11 cells were repressed by MMTV expression, an observation similar to what was observed in the PyMT mouse model during tumor progression, from hyperplasia to adenoma to early and late carcinomas. Comparison of our results with the Wnt1 mouse model revealed further insights into how MMTV expression could lead to activation of the Wnt1 pathway independent of insertional mutagenesis. Thus, the key pathways, DEGs, and hub genes identified in this study can provide important clues to elucidate the molecular mechanisms involved in MMTV replication, escape from cellular anti-viral response, and potential to cause cell transformation. These data also validate the use of the MMTV-infected HC11 cells as an important model to study early transcriptional changes that could lead to mammary cell transformation.

The RNA-binding protein Mex3B regulates airway remodeling through modulation of TGFBR3 mRNA stability in chronic rhinosinusitis.

Abstract Objective: To explore the pathogenic function of Mex3 RNA binding family member B (Mex3b) in chronic rhinosinusitis(CRS). Methods: The expression of Mex3b, TGF-β receptor III (TGFBR3), TGF-β2, p-Smad2 were studied by quantitative RT-PCR, immunohistochemistry, western blotting, and ELISA. The binding of MEX3b with TGFBR3was test by improved RNA immunoprecipitation–coupled high-throughput sequencing (iRIP-Seq). Results: Compared to control subjects, mNRA and protein expression of Mex3b in nasal epithelial cells from CRSwNP patients are upregulated. And Mex3b mRNA and protein levels in cultured human nasal epithelial cells(HNECs) obtained from control subjects were upregulated by LPS, CpG and IL-1β. Incresed Mex3b downregulates TGFBR3expression in airway epithelial cells by binding to TGFBR3mRNA, which results in downregulated level of TGFBR3in CRS compared to control subjects. Loss of TGFBR3 inhibits TGF-β2 induced gene transcription in nasal epithelial cells. And Mex3b suppresses TGF-β2 signaling activation by downregulating TGFBR3 expression in nasal epithelial cells. Mex3b and TGFBR3 expression levels are correlated with tissue edema in CRSwNP. Conclusions: The upregulation of Mex3b in CRSwNP suppresses TGF-β2 signaling activation by downregulating TGFBR3 expression in nasal epithelial cells, and thefore regulates tissue remodeling of CRS.

Epithelial cells activate fibroblasts to promote esophageal cancer development.

Esophageal squamous-cell carcinoma (ESCC) develops through multistage epithelial cancer formation, i.e., from normal epithelium, low- and high-grade intraepithelial neoplasia to invasive carcinoma. However, how the precancerous lesions progress to carcinoma remains elusive. Here, we report a comprehensive single-cell RNA sequencing and spatial transcriptomic study of 79 multistage esophageal lesions from 29 patients with ESCC. We reveal a gradual and significant loss of ANXA1 expression in epithelial cells due to its transcription factor KLF4 suppression along the lesion progression. We demonstrate that ANXA1 is a ligand to formyl peptide receptor type 2 (FPR2) on fibroblasts that maintain fibroblast homeostasis. Loss of ANXA1 leads to uncontrolled transformation of normal fibroblasts into cancer-associated fibroblasts (CAFs), which can be enhanced by secreted TGF-β from malignant epithelial cells. Given the role of CAFs in cancer, our study underscores ANXA1/FPR2 signaling as an important crosstalk mechanism between epithelial cells and fibroblasts in promoting ESCC.

Peroxisome proliferator-activated receptors mediate phytochemical-induced upregulation of farnesoid X receptor expression and activity in colonic epithelial cells

Introduction: The nuclear bile acid receptor, farnesoid X receptor (FXR), is expressed on intestinal epithelial cells where its potential as a new drug target for treatment of intestinal and metabolic disorders, such as chronic diarrhoea, colitis, colon cancer, obesity and diabetes, has been well-established. We have previously shown that pentacyclic triterpenes (PCTs), a class of dietary phytochemical, enhance the expression and activity of colonic epithelial FXR. Here, we investigated if another common class of dietary phytochemical, polyunsaturated fatty acids (PUFAs), also modulate FXR signalling in these cells. Furthermore, since peroxisome proliferator-activated receptors (PPARs) have been shown to regulate FXR expression in other cell types, we investigated their potential in mediating the effects of PCTs and PUFAs on FXR expression in the colonic epithelium. Methods: All experiments were conducted on monolayers of T 84 colonic epithelial cells grown on permeable supports. Alpha-linolenic acid (ALA; 100 μM) and hederagenin (HG; 5 μM) were used as prototypical PUFAs or PCTs, respectively. Rosiglitazone (1 μM) and WY14643 (1 – 20 μM) were employed as PPARγ and PPARα agonists, whereas GW9662 (20 μM) and GW6471 (1 μM) were used as PPARγ and PPARα antagonists, respectively. Levels of FXR, FGF19, an index of FXR activation, and ANGPTL4, a PPAR target gene, were measured by qRT-PCR, western blotting, or ELISA. Results: Similar to our previous studies with HG, treatment of T 84 cells with ALA upregulated FXR mRNA and protein expression to 11.43 ± 2.1 fold (n = 7, p < 0.01) and 6.4 ± 1.2 fold (n = 6, p < 0.05) of that in control cells, respectively. ALA treatment also enhanced the effects of an FXR agonist, GW4064 (5 μM), on FGF19 mRNA and protein expression by 4.4 ± 1.6 (n = 7) and 6.5 ± 1.4 fold (n = 8, p < 0.05), respectively. Treatment with HG and ALA (3 hrs) also increased mRNA expression of the PPAR target gene, ANGPTL4, to 5.7 ± 1.0 (n = 5; p < 0.05) and 66.3 ± 9.5 fold (n = 7; p < 0.01) of that in untreated cells, respectively. Furthermore, the effects of the phytochemicals on FXR expression were mimicked by the PPARγ and PPARα agonists, rosiglitazone and WY14643, respectively. The PPARγ antagonist, GW9662, inhibited ALA-induced FXR mRNA expression by 0.5 ± 0.1 fold (n = 11; p < 0.001) but did not alter responses to HG. In contrast, the PPARα antagonist, GW6471, inhibited both ALA and HG-induced FXR mRNA expression by 0.71 ± 0.1 and 0.58 ± 0.1 fold (n = 4; p < 0.05) of that in control cells, respectively. Conclusion: The dietary phytochemicals, HG and ALA, upregulate colonic epithelial FXR through mechanisms that appear to involve differential activation of PPARs. By virtue of their ability to upregulate FXR expression and activity, foods or food supplements, rich in such PCTs and PUFAs have excellent potential for development as a new class of “FXR-targeted nutraceutical” for treatment and prevention of intestinal and metabolic disorders. Science Foundation Ireland This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

USP18 promotes endometrial receptivity via the JAK/STAT1 and the ISGylation pathway.

Interferon-tau (IFNT), a pregnancy recognition signal in ruminants, promotes the establishment of endometrial receptivity by inducing the expression of interferon-stimulated genes (ISGs) via the Janus kinase/signal transducer and activator of transcription (JAK/STATs) signaling pathway. However, the precise mechanisms remain largely unknown. Ubiquitin-specific protease 18 (USP18) acts specifically on the ISGylation modification system to exert deubiquitination and participates in the regulation of the type I IFN signaling pathway. The purpose of this study was to determine the role and mechanism of USP18 on endometrial receptivity in goat. USP18 was mainly localized in the uterine luminal and glandular epithelium, and its expression levels were significantly increased from days 5-18 of early pregnancy. Progesterone (P4), estradiol (E2), and IFNT significantly stimulated USP18 expression in goat endometrial epithelial cells (gEECs) cultured invitro. Meanwhile, the markers of endometrial receptivity HOXA11, ITGB1, ITGB3, and ITGB5 were significantly upregulated after USP18 overexpression in gEECs. However, USP18 interference significantly inhibited the expression of HOXA10, ITGB1, ITGB3, and ITGB5 in gEECs. In addition, both the phosphorylation levels of STAT1 and the expression of ISGylation-modified proteins were significantly increased after USP18 silencing in gEECs. Furthermore, pretreatment with the STAT1 inhibitor Fludara markedly restored the effect of USP18 interference in gEECs. In summary, USP18 may play an important role in promoting goat endometrial receptivity by regulating the JAK/STAT1 pathway and ISGylation.

Successful editing and maintenance of lactogenic gene expression in primary bovine mammary epithelial cells

In vitro investigation of bovine lactation processes is limited by a lack of physiologically representative cell models. This deficiency is most evident through the minimal or absent expression of lactation-specific genes in cultured bovine mammary tissues. Primary bovine mammary epithelial cells (pbMECs) extracted from lactating mammary tissue and grown in culture initially express milk protein transcripts at relatively representative levels. However, expression drops dramatically after only three or four passages, which greatly reduces the utility of primary cells to model and further examine lactogenesis. To investigate the effects of alternate alleles in pbMECs including effects on transcription, we have developed methods to deliver CRISPR-Cas9 gene editing reagents to primary mammary cells, resulting in very high editing efficiencies. We have also found that culturing the cells on an imitation basement membrane composed of Matrigel, results in the restoration of a more representative lactogenic gene expression profile and the cells forming three-dimensional structures in vitro. Here, we present data from four pbMEC lines recovered from pregnant cows and detail the expression profile of five key milk synthesis genes in these MECs grown on Matrigel. Additionally, we describe an optimised method for preferentially selecting CRISPR-Cas9-edited cells conferring a knock-out of DGAT1, using fluorescence-activated cell sorting (FACS). The combination of these techniques facilitates the use of pbMECs as a model to investigate the effects of gene introgressions and genetic variation in lactating mammary tissue.

Early Interleukin-6 Mediated Activation of SGK1 is Primarily via Variant 6

Hypertension is associated with inflammation, including elevated interleukin-6 (IL-6) levels. IL-6 is an inflammatory cytokine, which we have previously shown to activate the mineralocorticoid receptor (MR) and the downstream sodium (Na) transporters- Na-chloride cotransporter (NCC) and the epithelial Na channel (ENaC). However, how IL-6 activates the MR is still unknown. IL-6 is also linked to Serum and Glucocorticoid-regulated Kinase 1 (SGK1), which is a downstream effector of the MR. SGK1 regulates both NCC and ENaC, increasing activity and tubular reabsorption of Na. This increased Na reabsorption favors water retention, thus increasing blood pressure. Previously, IL-6 has also been linked with SGK1 activation. We hypothesize that IL-6 increases SGK1 mRNA and protein expression in renal epithelial cells. To test this, we used in vitro renal epithelial cell models of both the late distal convoluted tubule (DCT2) and inner medullary collecting duct (IMCD), mDCT15 and IMCD3, respectively. Cells were treated with an IL-6 time-course (100ng/ml for 1, 3 and 6 hours). There are 6 known transcriptional variants for SGK1. We designed variant-specific PCR primers and, a pilot study based on regular PCR concluded that expression levels of only variants 5 and 6 (V5, V6) are significant. Using Real Time quantitative RT-PCR, we were then able to quantify transcript levels of both SGK1 V5 and V6 variants. With IL-6 treatment, we observed a 160%-211% increase at early time points (30mins-1hr) in SKG1 V6 and a lesser response (26%-88% increase) with V5 as compared to vehicle treated mDCT15 cells. Since SGK1 has been observed in the membrane fraction and in the cytoplasm, protein extracts from cells were fractionated (mPER kit) to assess membrane and cytosolic SGK1 protein levels using immunoblots. Densitometric analysis of immunoblots demonstrated a 5.5- and 3.1-fold increase in SGK1 protein expression at 1hr in mDCT15 and IMCD3 cells, respectively. These data suggest that IL-6 induces increases in SGK1 transcription and translation. Interestingly, we also found that majority of SGK1 was in the membrane fraction and the level was consistent. Together, these data suggest that SGK1 V6 may be responsible for the increased sodium reabsorption in response to IL-6 stimulation in renal epithelial cells. NIH funding This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Intestinal Epithelial Cell PTPN2 Restricts Intestinal Permeability and Tight-Junction Remodelling Caused by Adherent-invasive E. coli in Mice

Background: Alterations in epithelial tight junction proteins can compromise the ability of the intestinal mucosa to act as a barrier between luminal contents and underlying tissues. The resulting increase in intestinal permeability can give rise to conditions such as inflammatory bowel disease (IBD). Additionally, expansion of pathobionts, such as adherent-invasive Escherichia coli (AIEC), are associated with IBD. Mice deficient in the IBD gene candidate, Ptpn2, exhibit increased intestinal permeability and a pronounced expansion of a novel murine AIEC ( mAIEC). This study aimed to investigate how PTPN2 expression in intestinal epithelial cells protects intestinal barrier properties after mAIEC infection. Methods: Tamoxifen-inducible, intestinal epithelial cell (IEC)-specific knockout mice ( Ptpn2 ΔIEC ) and control littermates ( Ptpn2 fl/fl ) were infected with either PBS, non-invasive E. coli K12, or mcherry-tagged mAIEC ( mAIEC red ). FITC-Dextran (FD4) and Rhodamine-Dextran (RD70) permeability probes were administered by oral-gavage and serum collected after 5hrs. Results: Ptpn2 ΔIEC mice exhibited intestinal region-specific higher mAIEC red - but not K12 - bacterial load compared to Ptpn2 fl/fl mice ( P=0.038). E. coli K12 increased FD4 but not RD70 permeability in Ptpn2 ΔIEC mice compared with Ptpn2 fl/fl mice ( P=0.0025). mAIEC red also had no effect on RD70, but caused an even greater increase in FD4 permeability in Ptpn2 ΔIEC vs. Ptpn2 fl/fl mice ( P< 0.0001). Ptpn2 fl/fl mice infected with mAIEC red also exhibited increased FD4 permeability compared to PBS-treated littermates ( P =0.0210). To identify if increased FD4 permeability was due to changes in expression of relevant tight-junction proteins, we performed western blot analysis on isolated IECs. Ptpn2 ΔIEC + mAIEC red mice had decreased expression of occludin ( P=0.0030) and the adherens junction protein, E-cadherin ( P=0.0142). Expression of the cation pore, claudin-2, was increased in PBS and K12-infected Ptpn2 ΔIEC vs. Ptpn2 fl/fl mice ( P=0.015). Interestingly, mAIEC red increased claudin-2 expression in Ptpn2 fl/fl mice compared to PBS controls ( P=0.0105) however, Ptpn2 ΔIEC + mAIEC red mice exhibited decreased claudin-2 levels compared to Ptpn2 fl/fl + mAIEC red mice ( P=0.0436). Immunofluorescence staining of claudin-2 confirmed reduced claudin-2 in Ptpn2 ΔIEC + mAIEC red mice. Furthermore, we observed gaps in ZO-1 membrane localization in Ptpn2 ΔIEC mice treated with PBS and K12 but not in their Ptpn2 fl/fl controls. ZO-1 membrane localization was dramatically depleted in Ptpn2 ΔIEC + mAIEC red mice compared to the Ptpn2 fl/fl + mAIEC red controls. Conclusion: Intestinal epithelial PTPN2 exhibits a major role in maintaining intestinal homeostasis by preserving intestinal barrier function and the integrity of tight-junction protein localization. Given that loss of PTPN2 in vivo also provokes expansion of adherent-invasive E. coli, these studies indicate that PTPN2 is an important mediator of host-microbiota crosstalk. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Antifibrotic treatment reduces NOX4 expression in bronchial epithelial cells of patients with pulmonary fibrosis

Antifibrotic treatment reduces NOX4 expression in bronchial epithelial cells of patients with pulmonary fibrosis

Butyrate silences MUC5ACexpression in airway epithelial cells by mediating histone deacetylation at its transcription start site

Abstract Secreted airway mucins form a mucous gel consisting of MUC5AC and MUC5B and maintain airway homeostasis for appropriate mucociliary clearance and airway defense. There is conceivably a global mechanism to counteract excessive mucin production in response to incessant exposure to external and internal stimuli, which otherwise leads to goblet metaplasia and mucus hyperproduction. Here, we report that butyrate, a short-chain fatty acid (SCFA), strongly suppresses MUC5AC production at as low as sub-to micromolar concentrations in NCI-H292, airway epithelial cells. Butyrate transcriptionally inhibits MUC5AC mRNA and protein that are induced by a variety of stimuli including EGF and LPS. In line, butyrate upregulates FOXA2, which is known to block goblet cell metaplasia. The mode of the inhibition by butyrate appears to be independent of the known receptors of butyrate. In addition, butyrate does not alter the EGFR signaling pathway. Chromatin immunoprecipitation analysis demonstrates that histone H3K27 acetylation level is increased upon stimulation with EGF and returns to baseline in the presence of butyrate. We propose that butyrate, a metabolic product of gut microbiota, plays a central role in maintaining homeostasis of airway mucous gel by counteracting mucin production resulting from repeated provocation of airway epithelium. National Research Foundation of Korea (NRF) funded by the Korean government (MSIT), NRF-2022R1A2B5B01002127.

Characterizing purinergic receptors in the gastrointestinal epithelium and immune cells

Background: Extracellular release of nucleotides (e.g., ATP, ADP, UTP, UDP) are well established as potent intercellular signaling molecules during homeostasis but are also released from cells during pathogenic microbial infection and tissue injury to serve as danger signals. These important extracellular signaling molecules activate purinergic receptors, of which there are 19 different purinergic receptor subtypes that are classified into P1 and P2 receptors. P1 receptor subtypes are activated by adenosine, while P2 receptors are differentially activated by ATP, ADP, UTP, UDP, and UP-glucose. P2 receptors are further divided into P2Y receptors (P2YRs) and P2X receptors (P2XRs) based on their structures and distinct signal-transduction mechanisms. Several studies have revealed crucial roles for P2 receptors during inflammatory and infectious diseases, yet the majority of work to date has focused on purinergic signaling in immune cells. Our group has previously identified a role for P2Y1 in mediating ADP-driven calcium waves during rotavirus infection. While purinergic signaling is a well-established mode of signaling in the enteric nervous system, the functional roles of purinergic signaling in the gastrointestinal tract remain incompletely understood. Hypothesis: Since epithelial cells serve as the first barrier against irritants and infection, we hypothesized that gut epithelium express multiple purinergic receptors that regulate cellular responses to extracellular nucleotide signals. Methods & Results: Using the Human Protein Atlas, we queried single cell RNA sequencing data for the P2 purinergic receptors. We examined these receptors along the length of the gastrointestinal tract, including esophagus, small intestine, colon, and rectum as well as in flow-sorted immune cells, peripheral blood mononuclear cells and and bone marrow derived immune cells. In silico analysis revealed high expression of P2Y1, P2Y2, P2Y11 and P2X4 among the purinergic receptors throughout the gastrointestinal tract. Interestingly, P2Y1 showed highest expression in esophageal epithelial cells, small intestinal enterocytes, and goblet cells. P2Y6 was also observed in promixal enterocytes of the small intestine, but was largely absent from the colon and rectum. In contrast, we found that immune cells from bone marrow, peripheral blood and flow sorted cells had relatively high expression of P2X1 and P2X4. We noted P2X5 expression in plasma cells and B -cells in all sets. Pathway analysis from the Reactome database revealed P2Y receptors are associated with signal transduction, metabolism and hemostasis pathways, while P2X receptors were primarily involved in immune response and infectious disease pathways. Conclusions: These findings confirm that the gastrointestinal tract possess robust P2Y1, P2Y2, P2Y11 and P2X4 receptor expression and can participate in purinergic signaling pathways. R01 DK115507 (Hyser); R01 AI158683 (Hyser); APS Postdoctoral Fellowship (Engevik); F32 DK130288 (Engevik) This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Nicotine promotes e-cigarette vapour-induced lung inflammation and structural alterations.

Electronic cigarette (e-cigarette) vapour is gaining popularity as an alternative to tobacco smoking and can induce acute lung injury. However, the specific role of nicotine in e-cigarette vapour and its long-term effects on the airways, lung parenchyma and vasculature remain unclear. In vitro exposure to nicotine-containing e-cigarette vapour extract (ECVE) or to nicotine-free e-cigarette vapour extract (NF ECVE) induced changes in gene expression of epithelial cells and pulmonary arterial smooth muscle cells (PASMCs), but ECVE in particular caused functional alterations (e.g. a decrease in human and mouse PASMC proliferation by 29.3±5.3% and 44.3±8.4%, respectively). Additionally, acute inhalation of nicotine-containing e-cigarette vapour (ECV) but not nicotine-free e-cigarette vapour (NF ECV) increased pulmonary endothelial permeability in isolated lungs. Long-term in vivo exposure of mice to ECV for 8 months significantly increased the number of inflammatory cells, in particular lymphocytes, compared to control and NF ECV in the bronchoalveolar fluid (BALF) (ECV: 853.4±150.8 cells·mL-1; control: 37.0±21.1 cells·mL-1; NF ECV: 198.6±94.9 cells·mL-1) and in lung tissue (ECV: 25.7±3.3 cells·mm-3; control: 4.8±1.1 cells·mm-3; NF ECV: 14.1±2.2 cells·mm-3). BALF cytokines were predominantly increased by ECV. Moreover, ECV caused significant changes in lung structure and function (e.g. increase in airspace by 17.5±1.4% compared to control), similar to mild tobacco smoke-induced alterations, which also could be detected in the NF ECV group, albeit to a lesser degree. In contrast, the pulmonary vasculature was not significantly affected by ECV or NF ECV. NF ECV components induce cell type-specific effects and mild pulmonary alterations, while inclusion of nicotine induces significant endothelial damage, inflammation and parenchymal alterations.

Integrin β1 is a key determinant of the expression of angiotensin-converting enzyme 2 (ACE2) in the kidney epithelial cells.

The expression of the angiotensin-converting enzyme 2 (ACE2) is altered in multiple chronic kidney diseases like hypertension and renal fibrosis, where the signaling from the basal membrane proteins is critical for the development and progression of the various pathologies. Integrins are heterodimeric cell surface receptors that have important roles in the progression of these chronic kidney diseases by altering various cell signaling pathways in response to changes in the basement membrane proteins. It is unclear whether integrin or integrin-mediated signaling affects the ACE2 expression in the kidney. The current study tests the hypothesis that integrin β1 regulates the expression of ACE2 in kidney epithelial cells. The role of integrin β1 in ACE2 expression in renal epithelial cells was investigated by shRNA-mediated knockdown and pharmacological inhibition. In vivo studies were carried out using epithelial cell-specific deletion of integrin β1 in the kidneys. Deletion of integrin β1 from the mouse renal epithelial cells reduced the expression of ACE2 in the kidney. Furthermore, the downregulation of integrin β1 using shRNA decreased ACE2 expression in human renal epithelial cells. ACE2 expression levels were also decreased in renal epithelial cells and cancer cells when treated with an integrin α2β1 antagonist, BTT 3033. SARS-CoV-2 viral entry to human renal epithelial cells and cancer cells was also inhibited by BTT 3033. This study demonstrates that integrin β1 positively regulates the expression of ACE2, which is required for the entry of SARS-CoV-2 into kidney cells.

Proteolytic Activity of the Paracaspase MALT1 Is Involved in Epithelial Restitution and Mucosal Healing.

The paracaspase MALT1 is a crucial regulator of immune responses in various cellular contexts. Recently, there is increasing evidence suggesting that MALT1 might represent a novel key player in mucosal inflammation. However, the molecular mechanisms underlying this process and the targeted cell population remain unclear. In this study, we investigate the role of MALT1 proteolytic activity in the context of mucosal inflammation. We demonstrate a significant enrichment of MALT1 gene and protein expression in colonic epithelial cells of UC patients, as well as in the context of experimental colitis. Mechanistically we demonstrate that MALT1 protease function inhibits ferroptosis, a form of iron-dependent cell death, upstream of NF-κB signaling, which can promote inflammation and tissue damage in IBD. We further show that MALT1 activity contributes to STAT3 signaling, which is essential for the regeneration of the intestinal epithelium after injury. In summary, our data strongly suggests that the protease function of MALT1 plays a critical role in the regulation of immune and inflammatory responses, as well as mucosal healing. Understanding the mechanisms by which MALT1 protease function regulates these processes may offer novel therapeutic targets for the treatment of IBD and other inflammatory diseases.