Signaling In Cells Research Articles

Mineralocorticoid Receptor Signaling in Peripheral Blood Cells in Patients with Multiple Sclerosis.

Multiple sclerosis (MS) is associated with alterations in neuroendocrine function, primarily the hypothalamic-pituitary-adrenal axis, including lower expression of the glucocorticoid receptor (GR) and its target genes in peripheral blood mononuclear cells (PBMC) or full blood. We previously found reduced mineralocorticoid receptor (MR) expression in MS patients' peripheral blood. MS is being treated with a widening variety of disease-modifying treatments (DMT), some of which have similar efficacy but different mechanisms of action; body-fluid biomarkers to support the choice of the optimal initial DMT and/or to indicate an unsatisfactory response before clinical activity are unavailable. Using cell culture of volunteers' PBMCs and subsequent gene expression analysis (microarray and qPCR validation), we identified the mRNA expression of OTUD1 to represent MR signaling. The MR and MR target gene expression levels were then measured in full blood samples. In 119 MS (or CIS) patients, the expression of both MR and OTUD1 was lower than in 42 controls. The expression pattern was related to treatment, with the MR expression being particularly low in patients treated with fingolimod. While MR signaling may be involved in the therapeutic effects of some disease-modifying treatments, MR and OTUD1 expression can complement the neuroendocrine assessment of MS disease course. If confirmed, such assessment may support clinical decision-making.

Insulin-like growth factor 2 targets IGF1R signaling transduction to facilitate metastasis and imatinib resistance in gastrointestinal stromal tumors

BACKGROUND Gastrointestinal stromal tumors (GISTs) are typical gastrointestinal tract neoplasms. Imatinib is the first-line therapy for GIST patients. Drug resistance limits the long-term effectiveness of imatinib. The regulatory effect of insulin-like growth factor 2 (IGF2) has been confirmed in various cancers and is related to resistance to chemotherapy and a worse prognosis. AIM To further investigate the mechanism of IGF2 specific to GISTs. METHODS IGF2 was screened and analyzed using Gene Expression Omnibus (GEO: GSE225819) data. After IGF2 knockdown or overexpression by transfection, the phenotypes (proliferation, migration, invasion, apoptosis) of GIST cells were characterized by cell counting kit 8, Transwell, and flow cytometry assays. We used western blotting to evaluate pathway-associated and epithelial-mesenchymal transition (EMT)-associated proteins. We injected transfected cells into nude mice to establish a tumor xenograft model and observed the occurrence and metastasis of GIST. RESULTS Data from the GEO indicated that IGF2 expression is high in GISTs, associated with liver metastasis, and closely related to drug resistance. GIST cells with high expression of IGF2 had increased proliferation and migration, invasiveness and EMT. Knockdown of IGF2 significantly inhibited those activities. In addition, OE-IGF2 promoted GIST metastasis in vivo in nude mice. IGF2 activated IGF1R signaling in GIST cells, and IGF2/IGF1R-mediated glycolysis was required for GIST with liver metastasis. GIST cells with IGF2 knockdown were sensitive to imatinib treatment when IGF2 overexpression significantly raised imatinib resistance. Moreover, 2-deoxy-D-glucose (a glycolysis inhibitor) treatment reversed IGF2 overexpression-mediated imatinib resistance in GISTs. CONCLUSION IGF2 targeting of IGF1R signaling inhibited metastasis and decreased imatinib resistance by driving glycolysis in GISTs.

Intracellular pH differentially regulates transcription of metabolic and signaling pathways in normal epithelial cells

Intracellular pH (pHi) dynamics regulate normal cell function, and dysregulated pHi dynamics is an emerging hallmark of cancer (constitutively increased pHi) and neurodegeneration (constitutively decreased pHi). However, the molecular mechanisms by which pHi dynamics regulate cell biology are poorly understood. Here, we discovered that altering pHi in normal human breast epithelial cells triggers global transcriptional changes. We identified 176 genes differentially regulated by pHi, with pHi-dependent genes clustering in signaling and glycolytic pathways. Using various normal epithelial cell models, we showed pH-dependent Notch homolog 1 protein expression, with increased protein abundance at high pHi. This resulted in pH-dependent downstream signaling, with increased Notch homolog 1 signaling at high pHi. We also found that high pHi increased the expression of glycolytic enzymes and regulators of pyruvate fate, including lactate dehydrogenase and pyruvate dehydrogenase kinase. These transcriptional changes were sufficient to alter lactate production, with high pHi shifting these normal epithelial cells toward a glycolytic metabolism and increasing lactate production. Thus, pHi dynamics transcriptionally regulate signaling and metabolic pathways in normal epithelial cells. Our data reveal new molecular regulators of pHi-dependent biology and a role for increased pHi in driving the acquisition of cancer-associated signaling and metabolic changes in normal human epithelial cells.

SFRP4 promotes autophagy and blunts FSH responsiveness through inhibition of AKT signaling in ovarian granulosa cells

BackgroundSecreted frizzled-related proteins (SFRPs) comprise a family of WNT signaling antagonists whose roles in the ovary are poorly understood. Sfrp4-null mice were previously found to be hyperfertile due to an enhanced granulosa cell response to gonadotropins, leading to decreased antral follicle atresia and enhanced ovulation rates. The present study aimed to elucidate the mechanisms whereby SFRP4 antagonizes FSH action.MethodsPrimary cultures of granulosa cells from wild-type mice were treated with FSH and/or SFRP4, and effects of treatment on gene expression were evaluated by RT-qPCR and RNAseq. Bioinformatic analyses were conducted to analyse the effects of SFRP4 on the transcriptome, and compare them to those of FSH or a constitutively active mutant of FOXO1. Additional granulosa cell cultures from wild-type or Sfrp4-null mice, some pretreated with pharmacologic inhibitors of specific signaling effectors, were used to examine the effects of FSH and/or SFRP4 on signaling pathways, autophagy and apoptosis by western blotting and TUNEL.ResultsTreatment of cultured granulosa cells with recombinant SFRP4 was found to decrease basal and FSH-stimulated mRNA levels of FSH target genes. Unexpectedly, this effect was found to occur neither via a canonical (CTNNB1-dependent) nor non-canonical WNT signaling mechanism, but was found to be GSK3β-dependent. Rather, SFRP4 was found to antognize AKT activity via a mechanism involving AMPK. This lead to the hypophosphorylation of FOXO1 and a decrease in the expression of a portion of the FSH and FOXO1 transcriptomes. Conversely, FSH-stimulated AMPK, AKT and FOXO1 phosphorylation levels were found to be increased in the granulosa cells of Sfrp4-null mice relative to wild-type controls. SFRP4 treatement of granulosa cells also induced autophagy by signaling via AKT-mTORC1-ULK1, as well as apoptosis.ConclusionsThis study identifies a novel GSK3β-AMPK-AKT signaling mechanism through which SFPR4 antagonizes FSH action, and further identifies SFRP4 as a novel regulator of granulosa cell autophagy. These findings provide a mechanistic basis for the phenotypic changes previously observed in Sfrp4-null mice, and broaden our understanding of the physiological roles of WNT signaling processes in the ovary.

The epithelial Na+ channel (ENaC) in ovarian granulosa cells modulates Ca2+ mobilization and gonadotrophin signaling for estrogen homeostasis and female fertility

Ovarian granulosa cells are essential to gonadotrophin-regulated estrogen production, female cycle maintenance and fertility. The epithelial Na+ channel (ENaC) is associated with female fertility; however, whether and how it plays a role in ovarian cell function(s) remained unexplored. Here, we report patch-clamp and Na+ imaging detection of ENaC expression and channel activity in both human and mouse ovarian granulosa cells, which are promoted by pituitary gonadotrophins, follicle stimulating hormone (FSH) or luteinizing hormone (LH). Cre-recombinase- and CRISPR-Cas9-based granulosa-specific knockout of ENaC α subunit (Scnn1a) in mice resulted in failed estrogen elevation at early estrus, reduced number of corpus luteum, abnormally extended estrus phase, reduced litter size and subfertility in adult female mice. Further analysis using technologies including RNA sequencing and Ca2+ imaging revealed that pharmacological inhibition, shRNA-based knockdown or the knockout of ENaC diminished spontaneous or stimulated Ca2+ oscillations, lowered the capacity of intracellular Ca2+ stores and impaired FSH/LH-stimulated transcriptome changes for estrogen production in mouse and/or human granulosa cells. Together, these results have revealed a previously undefined role of ENaC in modulating gonadotrophin signaling in granulosa cells for estrogen homeostasis and thus female fertility.

AmphiregulinDownregulates E-cadherin Expressionby Activating YAP/Egr-1/Slug Signaling in SKOV3 Human Ovarian Cancer Cells.

Amphiregulin (AREG) stimulates human epithelial ovarian cancer (EOC) cell invasion by downregulating E-cadherin expression. YAP is a transcriptional cofactor that has been shown to regulate tumorigenesis. This study aimed to examine whether AREG activates YAP in EOC cells and explore the roles of YAP in AREG-induced downregulation of E-cadherin and cell invasion. Analysis of the Cancer Genome Atlas (TCGA) showed that upregulation of AREG and EGFR were associated with poor survival in human EOC. Treatment of SKOV3 human EOC cells with AREG induced the activation of YAP. In addition, AREG downregulated E-cadherin, upregulated Egr-1 and Slug, and stimulated cell invasion. Using gain- and loss-of-function approaches, we showed that YAP was required for the AREG-upregulated Egr-1 and Slug expression. Furthermore, YAP was also involved in AREG-induced downregulation of E-cadherin and cell invasion. This study provides evidence that AREG stimulates human EOC cell invasion by downregulating E-cadherin expression through the YAP/Egr-1/Slug signaling.

Requirement of two simultaneous environmental signals for activation of Arabidopsis ELIP2 promoter in response to high light, cold, and UV-B stresses

ABSTRACT Arabidopsis EARLY LIGH-INDUCIBLE PROTEIN 2 (ELIP2) is a chlorophyll- and carotenoid-binding protein and is involved in photoprotection under stress conditions. Because its expression is induced through high light, cold, or UV-B stressors, its mechanism of induction has been studied. It is known that a functional unit found in the promoter, which is composed of Element B and Element A, is required and sufficient for full activation by these stressors. In this study, the role of each element in the unit was analyzed by introducing weak mutations in each element as synthetic promoters in addition to intensive repeat constructs of each single element. The results suggest that a stressor like cold stress generates two parallel signals in plant cells, and they merge at the promoter region for the activation of ELIP2 expression, which constitutes an “AND” gate and has a potential to realize strong response with high specificity by an environmental trigger.

Oleanonic acid ameliorates mutant Aβ precursor protein-induced oxidative stress, autophagy deficits, ferroptosis, mitochondrial damage, and ER stress in vitro

Accumulation in the brain of amyloid-β (Aβ), derived from cleavage of Aβ precursor protein (APP), is a hallmark of Alzheimer's disease (AD). Oleanonic acid (OA), a phytochemical from several plants, has proven anti-inflammatory effects, but its role in AD remains unknown. Here we found that OA reduced APP expression and inhibited oxidative stress via Nrf2/HO-1 signaling in SH-SY5Y neuroblastoma cells stably overexpressing APP. OA suppressed phosphorylated mTOR but increased autophagy markers ATG5 and LC3-II. Moreover, OA rescued ferroptosis-related factors GPX4, NCOA, and COX2 and ER stress markers GRP78, CHOP, and three main induction pathways of ER stress including IRE1/XBP1s, PERK/EIF2α, and ATF6. OA alleviated mitochondrial damage through MFN1, MFN2, OPA1, FIS1, and DRP1. Furthermore, OA upregulated GDF11 expression and downregulated phosphorylation of ErbB4 and TrkB without affecting BDNF levels. Thus, OA might protect neurons from APP-induced neurotoxicity by inhibiting oxidative stress, autophagy deficits, ferroptosis, mitochondrial damage, and ER stress in AD, providing a new promising therapeutic strategy in patients with AD.

The metabolic response of HepG2 cells to extracellular vesicles derived from Raphanus sativus L. var. caudatus Alef microgreens probed by chemometrics-assisted LC-MS/MS analysis

Extracellular vesicles (EVs) derived from Thai rat-tailed radish (Raphanus sativus L. var. caudatus Alef) microgreens were previously reported as novel bioactive bioparticles against cancer. This study aimed to investigate the metabolic disruption associated with the antiproliferative effect against HepG2 liver cancer cells, a representative of metabolizing cells and tissue. In this study, the neutral red uptake assay was performed to screen for the antiproliferative effect and determine the cytotoxic concentrations of EVs against HepG2 cells. An untargeted approach to cellular metabolomics was conducted using liquid chromatography coupled with the high-resolution mass spectrometry system with multivariate and univariate analyses to determine the metabolic changes of HepG2 liver cancer cells after EV treatment. EVs showed an antiproliferative effect in HepG2 cells with a half-maximal inhibitory concentration (IC50) of 685.5 ± 26.4 and 139.7 ± 4.2 μg/ml at 24 and 48 h, respectively. In the metabolomics study, 163 metabolites were annotated, with 61 significantly altered metabolites. Among these significant metabolites, 18 were related to glycerophospholipid metabolism. Phosphatidylcholine—the important lipid building blocks for cell membranes, lipid mediators for cell proliferation, and immunosuppressive signaling—was mainly decreased by EV treatment. The alteration of cellular phospholipids in cancer was discussed. This finding suggested the possible mechanism of anticancer action of EVs by disrupting phospholipid metabolism and survival signaling in cancer cells. Further studies should be made to confirm EVs' potential as single and combination therapy in vivo to reduce cancer resistance. This may close the gap between in vitro study and clinical setting.

Icariin alleviates the injury of Sertoli cell junction function by upregulating PKR pathway via ERα/c-fos signaling in aged mice

Ethnopharmacological relevenaceSertoli cells are vital to maintain spermatogenesis and their function decline during aging. Epimedium has the effects of tonifying kidney-yang, strengthening bones and muscles, and expelling wind and dampness, and is commonly used in the treatment of kidney-yang deficiency, impotence and spermatorrhea. Icariin is the main active ingredients from Epimedium exhibiting delaying aging effects and improving male reproductive dysfunction. Whereas, it remains poorly understood how icariin alleviates age-associated decline in testicular function by protecting against the damage of junction function of Sertoli cells. Aim of the studyThis study aimed to evaluate the improvement effect of icariin on Sertoli cell junction function damage and explore the underlying mechanisms. Materials and methodsMale C57BL/6 mice and mouse Sertoli cell line TM4 cells were utilized to assess the improvement effect of icariin on aging-associated Sertoli cell junction function injury. H&E staining, transmission electron microscopy, qPCR, Western blot, molecular docking, siRNA transfection, and immunofluorescence were performed in this study. ResultsDietary administration of icariin remarkly attenuated age-associated deterioration in spermatogenic function as evidenced by elevated testicular weight and index, sperm concentration and sperm viability. In addition, icariin protected Sertoli cell junction function from age-associated damage as proven by increased Sertoli cell numbers, improved tight junction ultrastructure, and upregulated junction-related proteins (ZO-1, Occludin and β-Catenin). Moreover, icariin significantly upregulated ERα/c-fos signaling and PKR pathway in testicular Sertoli cells. Similarly, in vitro studies revealed that deletion of ERα, c-fos or PKR abolished the improvement effects of icariin on Sertoli cell junction function damage. ConclusionsIcariin effectively mitigates age-associated decline in testicular function by diminished Sertoli cell junction function damage through upregulating PKR pathway via ERα/c-fos signaling. Therefore, attenuating Sertoli cell junction function injury by the upregulation of PKR pathway via ERα/c-fos signaling probably indicates an effective target for the prevention and treatment of testicular spermatogenic function with aging.

Ciliary length variations impact cilia-mediated signaling and biological responses.

Primary cilia are thin hair-like organelles that protrude from the surface of most mammalian cells. They act as specialized cell antennas that can vary widely in response to specific stimuli. However, the effect of changes in cilia length on cellular signaling and behavior remains unclear. Therefore, we aimed to characterize the elongated primary cilia induced by different chemical agents, lithium chloride (LiCl), cobalt chloride (CoCl2), and rotenone, using human retinal pigmented epithelial 1 (hRPE1) cells expressing ciliary G protein-coupled receptor (GPCR), melanin-concentrating hormone (MCH) receptor 1 (MCHR1). MCH induces cilia shortening mainly via MCHR1-mediated Akt phosphorylation. Therefore, we verified the proper functioning of the MCH-MCHR1 axis in elongated cilia. Although MCH shortened cilia that were elongated by LiCl and rotenone, it did not shorten CoCl2-induced elongated cilia, which exhibited lesser Akt phosphorylation. Furthermore, serum readdition was found to delay cilia shortening in CoCl2-induced elongated cilia. In contrast, rotenone-induced elongated cilia rapidly shortened via a chopping mechanism at the tip of the cilia. Conclusively, we found that each chemical exerted different effects on ciliary GPCR signaling and serum-mediated ciliary structure dynamics in cells with elongated cilia. These results provide a basis for understanding the functional consequences of changes in ciliary length.

MTF2 facilitates the advancement of osteosarcoma through mediating EZH2/SFRP1/Wnt signaling

BackgroundOsteosarcoma is a soft tissue neoplasm with elevated recurrence risk and highly metastatic potential. Metal response element binding transcriptional factor 2 (MTF2) has been revealed to exert multiple activities in human tissues. The present research was conducted to explore the functions and related response mechanism of MTF2 in osteosarcoma which have not been introduced yet.MethodsBioinformatics tools identified the differential MTF2 expression in osteosarcoma tissues. MTF2 expression in osteosarcoma cells was examined with Western blot. Cell Counting Kit-8 (CCK-8) assay, 5-Ethynyl-2’-deoxyuridine (EDU) staining, wound healing as well as transwell assays measured cell proliferation, migration and invasion, respectively. Flow cytometry assay detected the cellular apoptotic level. Western blot also measured the expressions of proteins associated with epithelial mesenchymal transition (EMT), apoptosis and enhancer of zeste homolog 2 (EZH2)/secreted frizzled-related protein 1 (SFRP1)/Wnt signaling. Co-immunoprecipitation (Co-IP) assay confirmed MTF2-EZH2 interaction.ResultsMTF2 expression was increased in osteosarcoma tissues and cells. MTF2 interference effectively inhibited the proliferation, migration and invasion of osteosarcoma cells and promoted the cellular apoptotic rate. MTF2 directly bound to EZH2 and MTF2 silence reduced EZH2 expression, activated SFRP1 expression and blocked Wnt signaling in osteosarcoma cells. EZH2 upregulation or SFRP1 antagonist WAY-316606 partly counteracted the impacts of MTF2 down-regulation on the SFRP1/Wnt signaling and the biological phenotypes of osteosarcoma cells.ConclusionsMTF2 might down-regulate SFRP1 to activate Wnt signaling and drive the progression of osteosarcoma via interaction with EZH2 protein.

Therapeutic Senolysis of Axitinib-Induced Senescent Human Lung Cancer Cells.

Tyrosine kinase inhibitors (TKIs) inhibit receptor-mediated signals in cells. Axitinib is a TKI with high specificity for vascular endothelial growth factor receptors (VEGFRs). We determined whether axitinib could induce senescence in human cancer cells and be lysed by the senolytic drug ABT-263. Human lung and breast adenocarcinoma cell lines were used. These cells were cultured with axitinib or a multi-target TKI lenvatinib. The expression of β-galactosidase, VEGFRs, Ki-67, reactive oxygen species (ROS) of cancer cells, and their BrdU uptake were evaluated by flow cytometry. The mRNA expression of p21 and IL-8 was examined by quantitative PCR. The effects of TKIs on phosphorylation of Akt and Erk1/2, as downstream molecules of VEGFR signaling, were examined by immunoblot. The in vivo anti-cancer effect was examined using a xenograft mice model. Axitinib, but not lenvatinib, induced cellular senescence (increased cell size and enhanced expression of β-galactosidase) in all adenocarcinoma cell lines. Axitinib-induced senescence was unrelated to the expression of VEGFRs on cancer cells. ROS were involved in axitinib-induced senescence. Axitinib-induced senescent lung adenocarcinoma A549 cells were drastically lysed by ABT-263. In A549-xenografted mice, combination therapy with axitinib and ABT-263 significantly suppressed tumor growth with the induction of apoptotic cancer cells.

Integrating serum metabolomics analysis and network pharmacology to reveal the potential mechanism of Shengmai Jianghuang San in the treatment of nasopharyngeal carcinoma.

Shengmai Jianghuang San (SMJHS) is a traditional Chinese herbal compound reported to inhibit Nasopharyngeal Carcinoma (NPC) progression and enhance radiosensitivity. However, the specific active ingredients and regulatory mechanisms of SMJHS against NPC, particularly under hypoxic conditions, remain unclear. In this study, Sprague-Dawley (SD) rats were gavaged with Shengmai Jianghuang San (SMJHS), and their blood was collected from the abdominal aorta. UHPLC-Q-Exactive orbitrap MS/MS was used to identify the metabolite profiles of SMJHS drug-containing serum. A molecular network of the active compositions in SMJHS targeting NPC was constructed through network pharmacology and molecular docking. The HIF-1α/VEGF pathway was in key positions. The effects of SMJHS on the proliferation, migration, and radiosensitivity of hypoxic NPC cells were assessed by in vitro experiments. NPC cell lines stably overexpressing HIF-1α were established using a lentivirus to investigate the regulation of HIF-1α/VEGF signaling in hypoxic NPC cells by SMJHS. Through a combination of network pharmacological analysis, cellular biofunctional validation, and molecular biochemical experiments, our study found that SMJHS had an anti-proliferative effect on NPC cells cultured under hypoxic conditions, inhibiting their migration and increasing their radiosensitivity. Additionally, SMJHS suppressed the expression of HIF-1α and VEGFA, exhibiting potential as an effective option for improving NPC treatment.

TNF-α and RPLP0 drive the apoptosis of endothelial cells and increase susceptibility to high-altitude pulmonary edema

High-altitude pulmonary edema (HAPE) is a fatal threat for sojourners who ascend rapidly without sufficient acclimatization. Acclimatized sojourners and adapted natives are both insensitive to HAPE but have different physiological traits and molecular bases. In this study, based on GSE52209, the gene expression profiles of HAPE patients were compared with those of acclimatized sojourners and adapted natives, with the common and divergent differentially expressed genes (DEGs) and their hub genes identified, respectively. Bioinformatic methodologies for functional enrichment analysis, immune infiltration, diagnostic model construction, competing endogenous RNA (ceRNA) analysis and drug prediction were performed to detect potential biological functions and molecular mechanisms. Next, an array of in vivo experiments in a HAPE rat model and in vitro experiments in HUVECs were conducted to verify the results of the bioinformatic analysis. The enriched pathways of DEGs and immune landscapes for HAPE were significantly different between sojourners and natives, and the common DEGs were enriched mainly in the pathways of development and immunity. Nomograms revealed that the upregulation of TNF-α and downregulation of RPLP0 exhibited high diagnostic efficiency for HAPE in both sojourners and natives, which was further validated in the HAPE rat model. The addition of TNF-α and RPLP0 knockdown activated apoptosis signaling in endothelial cells (ECs) and enhanced endothelial permeability. In conclusion, TNF-α and RPLP0 are shared biomarkers and molecular bases for HAPE susceptibility during the acclimatization/adaptation/maladaptation processes in sojourners and natives, inspiring new ideas for predicting and treating HAPE.

Pan-cancer analysis of immune checkpoint receptors and ligands in various cells in the tumor immune microenvironment.

Drugs that target immune checkpoint have become the most popular weapon in cancer immunotherapy, yet only have practical benefits for a small percentage of patients. Tumor cells constantly interact with their microenvironment, which is made up of a variety of immune cells as well as endothelial cells and fibroblasts. Immune checkpoint expression and blocked signaling of immune cells in the tumor microenvironment (TME) are key to tumor progression. In this study, we perform deliberation convolution on the TCGA database for human lung, breast, and colorectal cancer to infer crosstalk between immune checkpoint receptors (ICRs) and ligands (ICLs) in TME of pan-carcinogenic solid tumor types, validated by flow cytometry. Analysis of immune checkpoints showed that there was little variation between different tumor types. It showed that CD160, LAG3, TIGIT were found to be highly expressed in CD8+ T cells instead of CD4+ T cells, PD-L1, PD-L2, CD86, LGALS9, TNFRSF14, LILRB4 and other ligands were highly expressed on macrophages, FVR, NECTIN2, FGL1 were highly expressed on Epithelial cells, CD200 was highly expressed in Endothelial cells, and CD80 was highly expressed in CD8 High expression on T cells. Overall, our study provides a new resource for the expression of immune checkpoints in TME on various types of cells. Significance: This study provides immune checkpoint expression of immune cells of multiple cancer types to infer immune mechanisms in the tumor microenvironment and provide ideas for the development of new immune checkpoint-blocking drugs.

Interleukin-17F suppressed colon cancer by enhancing caspase 4 mediated pyroptosis of endothelial cells

The combination of anti-angiogenic treatment and immunotherapy presents a promising strategy against colon cancer. Interleukin-17F (IL-17F) emerges as a critical immune cell cytokine expressed in colonic epithelial cells, demonstrating potential in inhibiting angiogenesis. In order to clarify the roles of IL-17F in the colon cancer microenvironment and elucidate its mechanism, we established a mouse colon carcinoma cell line CT26 overexpressing IL-17F and transplanted it subcutaneously into syngeneic BALB/c mice. We also analyzed induced colon tumor in IL-17F knockout and wild type mice. Our results demonstrated that IL-17F could suppress colon tumor growth in vivo with inhibited angiogenesis and enhanced recruitment of cysteine-cysteine motif chemokine receptor 6 (CCR6) positive immune cells. Additionally, IL-17F suppressed the tube formation, cell growth and migration of endothelial cells EOMA in vitro. Comprehensive bioinformatics analysis of transcriptome profiles between EOMA cells and those treated with three different concentrations of IL-17F identified 109 differentially expressed genes. Notably, a potential new target, Caspase 4, showed increased expressions after IL-17F treatment in endothelial cells. Further molecular validation revealed a novel downstream signaling for IL-17F: IL-17F enhanced Caspase 4/GSDMD signaling of endothelial cells, CT26 cells and CT26 transplanted tumors, while IL-17F knockout colon tumors exhibited decreased Caspase 4/GSDMD signaling. The heightened expression of the GSDMD N-terminus, coupled with increased cellular propidium iodide (PI) uptake and lactate dehydrogenase (LDH) release, revealed that IL-17F promoted pyroptosis of endothelial cells. Altogether, IL-17F could modulate the colon tumor microenvironment with inhibited angiogenesis, underscoring its potential as a therapeutic target for colon cancer.

Developmental exposure to nonylphenol leads to depletion of the neural precursor cell pool in the hippocampal dentate gyrus

Developmental exposure to nonylphenol (NP) results in irreversible impairments of the central nervous system (CNS). The neural precursor cell (NPC) pool located in the subgranular zone (SGZ), a substructure of the hippocampal dentate gyrus, is critical for the development of hippocampal circuits and some hippocampal functions such as learning and memory. However, the effects of developmental exposure to NP on this pool remain unclear. Thus, our aim was to clarify the impacts of developmental exposure to NP on this pool and to explore the potential mechanisms. Animal models of developmental exposure to NP were created by treating Wistar rats with NP during pregnancy and lactation. Our data showed that developmental exposure to NP decreased Sox2-and Ki67-positive cells in the SGZ of offspring. Inhibited activation of Shh signaling and decreased levels of its downstream mediators, E2F1 and cyclins, were also observed in pups developmentally exposed to NP. Moreover, we established the in vitro model in the NE-4C cells, a neural precursor cell line, to further investigate the effect of NP exposure on NPCs and the underlying mechanisms. Purmorphamine, a small purine-derived hedgehog agonist, was used to specifically modulate the Shh signaling. Consistent with the in vivo results, exposure to NP reduced cell proliferation by inhibiting the Shh signaling in NE-4C cells, and purmorphamine alleviated this reduction in cell proliferation by restoring this signaling. Altogether, our findings support the idea that developmental exposure to NP leads to inhibition of the NPC proliferation and the NPC pool depletion in the SGZ located in the dentate gyrus. Furthermore, we also provided the evidence that suppressed activation of Shh signaling may contribute to the effects of developmental exposure to NP on the NPC pool.

Mechanisms of age-related Treg dysfunction in an arthritic environment

Rheumatoid arthritis (RA) is an autoimmune disease characterized by a polyarticular synovitis. In recent years, elderly onset rheumatoid arthritis (EORA) has been increasing. Treg cells in RA have been reported to be dysfunctional, but the relationship between aging and their functional changes is unclear. Here, we found that Treg cells from EORA patients had increased percentages, but decreased activity compared to those from younger onset RA (YORA) patients. In experiments using arthritis model mice, decreased suppressive function and oxygen consumption rate (OCR) were observed in Treg cells only from old arthritic mice. Furthermore, type I interferon (IFN) signaling was upregulated in Treg cells from old GIA mice, and IFN-β decreased the suppressive function of Treg cells. Our findings demonstrate that increased type I IFN signaling in old Treg cells is induced only in the arthritic environment and relates to decreased suppressive function of Treg cells, gets involved in EORA.

Abstract Mo046: GJA1-20k Restores Connexin-43 Trafficking and β-Catenin Signaling in Myocytes Lacking Desmoplakin

Background: Arrhythmogenic Cardiomyopathy (ACM) is an insidious hereditary heart disease that results in structural and electrical cardiac abnormalities leading to arrhythmogenesis and heart failure. ACM is characterized by cardiomyocyte loss, decreased cellular coupling, and fibrofatty replacement. The disease arises from mutations in desmosomal genes, such as plakophilin (PKP), desmoglein (DSG), and desmoplakin (DSP). The pathogenesis of this disease is linked to dysfunction at the level of several cellular processes and pathways, including Wnt/β-catenin signaling. Recent evidence has shown that GJA1-20k, a truncated isoform of Connexin-43 (Cx43) generated by internal translation, has a therapeutic role in preserving Cx43 trafficking and cell-cell coupling in ACM of DSG origin. This study aims to investigate the role of GJA1-20k in protecting against ACM of DSP origin by evaluating its effect in cell models on Cx43 trafficking and Wnt/β-catenin signaling. Methods: DSP knockdown was performed on HEK cells and mouse neonatal cardiomyocytes. The role of GJA1-20k was determined using imaging, biochemical, and molecular biology techniques. Results: Protein levels of Cx43 and β-catenin were decreased on Western blot (WB) upon DSP knockdown. These findings were corroborated through imaging wherein the signal intensity for Cx43 at cellular junctions and β-catenin at the membrane was significantly decreased compared to the control group. β-catenin signal intensity was also decreased within the nucleus. Upon administration of GJA1-20k, protein levels of Cx43 were normalized on WB. Moreover, the ratio of phosphorylated β-catenin (inactive) to total β-catenin was decreased. Imaging studies identified a significant increase in Cx43 and β-catenin at the membrane in ACM models treated with GJA1-20k. This was also accompanied by an increase in β-catenin signal intensity within the nucleus. A TOP-flash luciferase assay was performed and revealed a significant increase in β-catenin transcriptional activity in the presence of GJA1-20k despite DSP knockdown. Conclusions: These results suggest that GJA1-20k is able to rescue Cx43 trafficking and recover Wnt/β-catenin signaling in cells and cardiomyocytes lacking DSP. The ability of GJA1-20k to restore dysfunctional genetic pathways involved in arrhythmogenesis and fibro-adipogenesis make it an attractive potential candidate for targeted therapy against ACM.