Classical Signaling Pathways Research Articles

Neuregulin-1β Alleviates Sepsis-Induced Skeletal Muscle Atrophy by Inhibiting Autophagy via AKT/mTOR Signaling Pathway in Rats.

Several studies have shown that excessive protein degradation is a major cause of skeletal muscle atrophy induced by sepsis, and autophagy is the main pathway participating in protein degradation. However, the role of autophagy in sepsis is still controversial. Previously, we found that neuregulin-1β (NRG-1β) alleviated sepsis-induced diaphragm atrophy through the phosphatidylinositol-3 kinase signaling pathway. Akt/mechanistic target of rapamycin (mTOR) is a classic signaling pathway to regulate autophagy, which maintains intracellular homeostasis. This study aimed to investigate whether NRG-1β could alleviate sepsis-induced skeletal muscle atrophy by regulating autophagy. L6 rat myoblast cells were differentiated using 2% fetal bovine serum into myotubes, which were divided into four groups: Con group treated with normal serum; Sep group treated with septic serum to form a sepsis cell model; septic serum + NRG-1β (SN) group treated with septic serum for 24 h followed by injection with NRG-1β and incubation for another 48 h; and serum+NRG-1β+LY294002 group, in which the PI3K inhibitor LY294002 was added 30 min before NRG-1β, and other treatments were similar to those in SN group. Effects of NRG-1β were also evaluated in vivo using Sprague-Dawley (SD) rats, in which sepsis was induced by cecal ligation and puncture (CLP). In L6 myotubes treated with septic serum, the expression of autophagy-related proteins UNC-51 like kinase 1, p-Beclin-1, and Beclin-1, and the ratio of LC3B II/I were highly increased, while protein p62 expression was decreased, indicating that autophagy was excessively activated. Moreover, NRG-1 expression was decreased, as detected by confocal immunofluorescence and western blotting. Upon exogenous addition of NRG-1β, autophagy was inhibited by the activation of Akt/mTOR signaling pathway, and cell viability was also increased. These effects disappeared in the presence of LY294002. In SD rats, sepsis was induced by CLP. NRG-1β was shown to inhibit autophagy in these rats via the Akt/mTOR pathway, leading to increased body weight of the septic SD rats and alleviation of atrophy of the tibialis anterior muscle. NRG-1β could alleviate sepsis-induced skeletal muscle atrophy by inhibiting autophagy via the AKT/mTOR signaling pathway.

AK2 Promotes the Migration and Invasion of Lung Adenocarcinoma by Activating TGF-β/Smad Pathway In vitro and In vivo.

Adenylate kinase 2 (AK2) is a wide-spread and highly conserved protein kinase whose main function is to catalyze the exchange of nucleotide phosphate groups. In this study, we showed that AK2 regulated tumor cell metastasis in lung adenocarcinoma. Positive expression of AK2 is related to lung adenocarcinoma progression and poor survival of patients. Knockdown or knockout of AK2 inhibited, while overexpression of AK2 promoted, human lung adenocarcinoma cell migration and invasion ability. Differential proteomics results showed that AK2 might be closely related to epithelial-mesenchymal transition (EMT). Further research indicated that AK2 regulated EMT occurrence through the Smad-dependent classical signaling pathways as measured by western blot and qPCR assays. Additionally, in vivo experiments showed that AK2-knockout in human lung tumor cells reduced their EMT-like features and formed fewer metastatic nodules both in liver and in lung tissues. In conclusion, we uncover a cancer metastasis-promoting role for AK2 and provide a rationale for targeting AK2 as a potential therapeutic approach for lung cancer.

Acute and chronic effects of a light-activated FGF receptor in keratinocytes in vitro and in mice.

FGFs and their high-affinity receptors (FGFRs) play key roles in development, tissue repair, and disease. Because FGFRs bind overlapping sets of ligands, their individual functions cannot be determined using ligand stimulation. Here, we generated a light-activated FGFR2 variant (OptoR2) to selectively activate signaling by the major FGFR in keratinocytes. Illumination of OptoR2-expressing HEK 293T cells activated FGFR signaling with remarkable temporal precision and promoted cell migration and proliferation. In murine and human keratinocytes, OptoR2 activation rapidly induced the classical FGFR signaling pathways and expression of FGF target genes. Surprisingly, multi-level counter-regulation occurred in keratinocytes in vitro and in transgenic mice in vivo, including OptoR2 down-regulation and loss of responsiveness to light activation. These results demonstrate unexpected cell type-specific limitations of optogenetic FGFRs in long-term in vitro and in vivo settings and highlight the complex consequences of transferring optogenetic cell signaling tools into their relevant cellular contexts.

The Signaling Pathways Involved in Ovarian Follicle Development.

The follicle is the functional unit of the ovary, which is composed of three types of cells: oocytes, granulosa cells, and theca cells. Ovarian follicle development and the subsequent ovulation process are coordinated by highly complex interplay between endocrine, paracrine, and autocrine signals, which coordinate steroidogenesis and gametogenesis. Follicle development is regulated mainly by three organs, the hypothalamus, anterior pituitary, and gonad, which make up the hypothalamic-pituitary-gonadal axis. Steroid hormones and their receptors play pivotal roles in follicle development and participate in a series of classical signaling pathways. In this review, we summarize and compare the role of classical signaling pathways, such as the WNT, insulin, Notch, and Hedgehog pathways, in ovarian follicle development and the underlying regulatory mechanism. We have also found that these four signaling pathways all interact with FOXO3, a transcription factor that is widely known to be under control of the PI3K/AKT signaling pathway and has been implicated as a major signaling pathway in the regulation of dormancy and initial follicular activation in the ovary. Although some of these interactions with FOXO3 have not been verified in ovarian follicle cells, there is a high possibility that FOXO3 plays a core role in follicular development and is regulated by classical signaling pathways. In this review, we present these signaling pathways from a comprehensive perspective to obtain a better understanding of the follicular development process.

RNA-Binding Proteins CLK1 and POP7 as Biomarkers for Diagnosis and Prognosis of Esophageal Squamous Cell Carcinoma.

The abnormality of RNA-binding proteins (RBPs) is closely related to the tumorigenesis and development of esophageal squamous cell carcinoma (ESCC), and has been an area of interest for research recently. In this study, 162 tumors and 11 normal samples are obtained from The Cancer Genome Atlas database, among which 218 differentially expressed RBPs are screened. Finally, a prognostic model including seven RBPs (CLK1, DDX39A, EEF2, ELAC1, NKRF, POP7, and SMN1) is established. Further analysis reveals that the overall survival (OS) rate of the high-risk group is lower than that of the low-risk group. The area under the receiver operating characteristic (ROC) curve (AUC) of the training group and testing group is significant (AUCs of 3 years are 0.815 and 0.694, respectively, AUCs of 5 years are 0.737 and 0.725, respectively). In addition, a comprehensive analysis of seven identified RBPs shows that most RBPs are related to OS in patients with ESCC, among which EEF2 and ELCA1 are differentially expressed at the protein level of ESCC and control tissues. CLK1 and POP7 expressions in esophageal cancer tumor samples are undertaken using the tissue microarray, and show that CLK1 mRNA levels are relatively lower, and POP7 mRNA levels are higher compared with non-cancerous esophageal tissues. Survival analysis reveals that a higher expression of CLK1 predicts a significant worse prognosis, and a lower expression of POP7 predicts a worse prognosis in esophageal cancer. These results suggest that CLK1 may promote tumor progression, and POP7 may hinder the development of esophageal cancer. In addition, gene set enrichment analysis reveals that abnormal biological processes related to ribosomes and abnormalities in classic tumor signaling pathways such as TGF-β are important driving forces for the occurrence and development of ESCC. Our results provide new insights into the pathogenesis of ESCC, and seven RBPs have potential application value in the clinical prognosis prediction of ESCC.

Cordycepin inhibits cell senescence by ameliorating lysosomal dysfunction and inducing autophagy through the AMPK and mTOR-p70S6K pathway.

Cell senescence is closely related to autophagy. In this article, we identified a natural nucleoside analogue, cordycepin, that has the ability to significantly improve lysosomal function, enhance the activity of the lysosomal representative protease cathepsin B (CTSB), and promote the expression of the functional protein lysosomal‐associated membrane protein 2 (LAMP2) on the lysosomal membrane. Cordycepin then restores the damaged autophagy level of aging cells by activating the classic AMPK and mTOR–p70S6K signaling pathways, thus inhibiting cell senescence in an H2O2‐induced stress‐induced premature senescence (SIPS) cell model. This study provides new theoretical support for the further development of cordycepin and clinical antiaging drugs to inhibit cell senescence and suggests that the regulatory mechanisms of lysosomes in senescent cells should be considered when treating age‐related diseases.

NDRG1 enhances the sensitivity of cetuximab by modulating EGFR trafficking in colorectal cancer

N-myc downstream-regulated gene 1 (NDRG1) is a key regulator that interacts with many classic tumor signaling pathways, including some molecules downstream of the epidermal growth factor receptor (EGFR). However, whether NDRG1 is involved in the mechanism of resistance to cetuximab (CTX), the first monoclonal antibody targeting the EGFR has not been reported. Here, we found that NDRG1 enhanced the sensitivity of CTX in colorectal cancer (CRC) cell lines. Afterwards, we determined the underlying mechanism of this phenomenon. We demonstrated that NDRG1 inhibited the expression of EGFR; blocked EGFR phosphorylation and reduced the EGFR distribution in the cell membrane, cytoplasm and nucleus. And then, NDRG1 suppressed the EGFR downstream signaling: RAS/RAF/ERK and PI3k/AKT/mTOR pathways. Moreover, we discovered that NDRG1 attenuated the endocytosis and degradation of EGFR induced by caveolin-1 (Cav1). Additionally, our findings were further observed in an animal model and human tissues. Our results represent a potentially significant discovery that explains the mechanisms of NDRG1 in CTX resistance. NDRG1 could be a promising biomarker to predict optimum responses to CTX, and a key target to enhance CTX activity in the treatment of metastatic CRC (mCRC).

Modaline sulfate promotes Oct4 expression and maintains self-renewal and pluripotency of stem cells through JAK/STAT3 and Wnt signaling pathways

BackgroundStem cells have been extensively explored for a variety of regenerative medical applications and they play an important role in clinical treatment of many diseases. However, the limited amount of stem cells and their tendency to undergo spontaneous differentiation upon extended propagation in vitro restrict their practical application. Octamer-binding transcription factor-4 (Oct4), a transcription factor belongs to the POU transcription factor family Class V, is fundamental for maintaining self-renewal ability and pluripotency of stem cells.MethodsIn the present study, we used the previously constructed luciferase reporters driven by the promoter and 3’-UTR of Oct4 respectively to screen potential activators of Oct4. Colony formation assay, sphere-forming ability assay, alkaline phosphatase (AP) activity assay and teratoma-formation assay were used to assess the role of modaline sulfate (MDLS) in promoting self-renewal and reinforcing pluripotency of P19 cells. Immunofluorescence, RT-PCR, and western blotting were used to measure expression changes of stem-related genes and activation of related signaling pathways.ResultsWe screened 480 commercially available small-molecule compounds and discovered that MDLS greatly promoted the expression of Oct4 at both mRNA and protein levels. Moreover, MDLS significantly promoted the self-renewal capacity of P19 cells. Also, we observed that the expression of pluripotency markers and alkaline phosphatase (AP) increased significantly in MDLS-treated colonies. Furthermore, MDLS could promote teratoma formation and enhanced differentiation potential of P19 cells in vivo. In addition, we found that in the presence of LIF, MDLS could replace feeder cells to maintain the undifferentiated state of OG2-mES cells (Oct4-GFP reporter gene mouse embryonic stem cell line), and the MDLS-expanded OG2-mES cells showed an elevated expression levels of pluripotency markers in vitro. Finally, we found that MDLS promoted Oct4 expression by activating JAK/STAT3 and classic Wnt signaling pathways, and these effects were reversed by treatment with inhibitors of corresponding signaling pathways.ConclusionsThese findings demonstrated, for the first time, that MDLS could maintain self-renewal and pluripotency of stem cells.

Clinicopathological and Molecular Characteristics of Colorectal Signet Ring Cell Carcinoma: A Review.

Colorectal signet ring cell carcinoma (SRCC) is a rare subtype of colorectal cancer (CRC) with unique characteristics. Due to the limited researches on it, a comprehensive and in-depth understanding of this subtype is still lacking. In this article, we summarize the clinicopathological features and molecular characteristics of colorectal SRCC based on a literature review. Clinically, SRCC has been associated with young age, proximal site preference, advanced tumor stage, high histological grade, high rate of lymph node involvement, frequent peritoneal metastasis, and a significantly poor prognosis. Regarding molecular characteristics, in SRCC, the mutation burden of the classic signaling pathways that include WNT/β-catenin, RAS/RAF/MAPK, and PI3K/AKT/mTOR signaling pathways are generally reduced. In contrast, some genes related to the “epithelial-mesenchymal transition (EMT) process” and the “stem cell properties”, including RNF43, CDH1, and SMAD4, as well as the related TGF-β signaling pathway have been observed more frequently altered in SRCC than in conventional adenocarcinoma (AC). In many studies but not in others, SRCC showed a higher frequency of BRAF mutation, microsatellite instability-high (MSI-H) and CpG island methylator phenotype (CIMP) positive status compared to AC. It has been proposed that colorectal SRCC consists of two subtypes, in which the MSI+/CIMP+/BRAF +/CD3+/PD-L1+ hypermethylated genotype is more common in the proximal colon, and may represent the potential candidate for immunotherapy. Understanding the special molecular mechanisms related to the aggressive biology of SRCC is of great importance, which may provide a theoretical basis for the development of more targeted and effective treatments for this refractory disease.

Phenotypic and Functional Analyses of B7S1 in Ovarian Cancer.

Background: Although programmed death (PD) ligand 1 (PD-L1)/PD-1 inhibitors show potent and durable antitumor effects in a variety of tumors, their efficacy in patients with OvCa is modest. Thus, additional immunosuppressive mechanisms beyond PD-L1/PD-1 need to be identified. Methods: The mRNA expression profiles of OvCa patients were obtained from The Cancer Genome Atlas (TCGA) database. The expression and clinical characteristics of VTCN1 (encoding B7S1) in OvCa were analyzed. The molecular interaction network, Gene Ontology (GO) analysis and Gene set enrichment analysis (GSEA) were used to functionally annotate and predict signaling pathways of VTCN1 in OvCa. Moreover, 32 treatment-naïve patients with OvCa were recruited to assess B7S1 expression. The cytotoxic immune phenotypes in distinct subgroups were analyzed. Results: B7S1 expression was increased in tumor sections compared with that in normal tissues from OvCa patients at both the mRNA and protein levels. VTCN1 expression was significantly correlated with the mRNA expression levels of several other co-inhibitory immune checkpoints. B7S1 protein was found to be highly expressed in CD45+CD68+ myeloid cells, whereas its putative receptor was expressed in CD8+ tumor-infiltrating lymphocytes (TILs). Furthermore, expression of B7S1 in antigen-presenting cells (APCs) was significantly correlated with the cytolytic function of CD8+ TILs. Functional annotations indicated that VTCN1 was involved in regulating T cell-mediated immune responses and participated in the activation of a variety of classic signaling pathways related to the progression of human cancer. Conclusion: In OvCa, B7S1 was highly expressed and may initiate dysfunction of CD8+ TILs, which could be targeted for cancer immunotherapy.

Driver variations in tumor classical signaling pathways and treatment response of immune checkpoint inhibitors

免疫检查点抑制剂（ICI）在肿瘤治疗中日趋重要，ICI疗效预测分子标志物备受关注。程序性死亡受体1及其配体1（PD-L1）表达、微卫星不稳定（MSI）和肿瘤突变负荷（TMB）作为目前最常见的ICI疗效预测标志物广泛应用于临床实践。此外，越来越多的研究发现维系肿瘤恶性生物学表型的经典信号通路驱动基因变异与ICI疗效密切相关。本文围绕RTK/RAS/MAPK、PI3K/AKT/mTOR、Nrf2、p53、细胞周期、β-catenin/Wnt、Hippo、Notch等典型癌症信号通路，循序介绍经典肿瘤信号通路中驱动基因变异在介导ICI疗效中的作用和可能的机制。.

Promotion Effect of EGCG on the Raised Expression of IL-23 through the Signaling of STAT3-BATF2-c-JUN/ATF2

Tea polyphenol of epigallocatechin-3-gallate (EGCG) has been verified to possess multiple biological activities. Interleukin-23 (IL-23) is a heterodimeric cytokine consisting of two subunits of IL-23p19 and IL-12p40, with the functionality in regulating the production of cytokines under physiological or pathological conditions. By serendipity, the raised expression of IL-23 was observed after treating cells with EGCG, whereas the detailed mechanism remains poorly understood. This study was proposed to investigate the signaling related to EGCG-induced IL-23. The raised expression of IL-23 was confirmed primarily by intraperitoneally injecting with different concentrations of EGCG (0, 20, 50, 80 mg/kg) into BALB/c mice, and the raised expression was confirmed by quantitative real-time polymerase chain reaction (qRT-PCR) and western blot. Results from enzyme-linked immunosorbent assay (ELISA) revealed the increase of IL-23 in serum from 116.09 to 153.90 pg/mL after treating with EGCG. The same results were also observed in RAW264.7 and peritoneal macrophages after treating with EGCG (0, 1, 5, 10, 25 μM) with the increased tendency of IL-23 in cultural medium (7.98 to 25.38 pg/mL for RAW264.7; 3.64 to 260.93 pg/mL for peritoneal macrophages). After preliminary exploration of the signaling related to the increased IL-23, the classical signaling pathways and key transcription factors, such as nuclear factor kappa-B (NF-κB), mitogen-activated protein kinase (MAPK) signaling pathways, and interferon regulatory factor 5 (IRF5), were demonstrated with no relevant contribution. A further study revealed the involvement of the key transcription factor of BATF2, which could antagonistically modulate the transcription and translation of IL-23. The signaling of STAT3-BATF2-c-JUN/ATF2-IL-23 has been further verified in RAW264.7 macrophages using the STAT3 inhibitor of AG490 and the activator of Colivelin TFA. The results indicated that EGCG inhibits the phosphorylation of STAT3 to facilitate the decreased level of BATF2, which contributed to the increased level of IL-23 by the enhancing heterodimerization of c-JUN and ATF2.

Abstract 1267: Combination therapy of JNJ-67856633, a novel, first-in-class MALT1 protease inhibitor, and JNJ-64264681, a novel BTK inhibitor, for the treatment of B-cell lymphomas

Abstract Background: Constitutive activation of the classical nuclear factor kappa-light-chain-enhancer of activated B-cells (NF-κB) pathway is a clear driver of B-cell non-Hodgkin lymphomas (NHL). Bruton's Tyrosine Kinase (BTK) and Mucosa-associated lymphoid tissue lymphoma translocation protein 1 (MALT1), which lies downstream of BTK, are key mediators of the classical NF-κB signaling pathway activated by BCR and TCR receptors. JNJ-67856633 is a first-in-class MALT1 protease inhibitor. JNJ-64264681 is a BTK inhibitor with improved selectivity against BTK. Blocking the BCR pathway at multiple points using these two orally bioavailable compounds could enhance clinical activity in B-cell lymphoma patients. Methods: JNJ-67856633 and JNJ-64264681 are currently being evaluated in phase 1 clinical trials designed to establish safety, PK, PD and the Recommended Phase 2 Dose (RP2D) of each agent. Results: JNJ-67856633 is a potent, selective, orally bioavailable, allosteric inhibitor of MALT1 protease activity. The compound inhibits proliferation of activated B-cell diffuse large B-cell lymphoma (ABC-DLBCL) cell lines bearing CD79b or CARD11 mutations as well as models mimicking resistance to covalent BTK inhibitors. JNJ-67856633 exhibits potent tumor growth inhibition in two human DLBCL xenograft models, OCI-Ly3 and OCI-Ly10, and mutation selected patient derived DLBCL xenografts. Furthermore, treatment with JNJ-67856633 leads to dose dependent inhibition of the generation of Tregs (CD4+CD25+FoxP3+) following CD3/28 stimulation in vitro, suggesting a potential immune modulatory role of MALT1 inhibition. JNJ-64264681 is an orally active small molecule that is a potent, selective, and irreversible covalent BTK inhibitor. JNJ-64264681 inhibits the growth of CD79b-mutant DLBCL cell lines in vitro and potently inhibits tumor growth in xenograft- or patient-derived DLBCL models in vivo. Treatment with JNJ-64264681 and JNJ-67856633 administered together demonstrated statistically significant tumor growth inhibition compared with vehicle control in two CD79b mutant mouse lymphoma models, one based on a DLBCL cell line (OCI-Ly10) and one based on a patient-derived DLBCL model (LY2298). In both models, the combination showed increased growth inhibition compared with single agents and tumor regression in the combination arm. Synergistic anti-proliferative activity was observed in three DLBCL cell lines carrying CD79b mutations and one MCL cell line. Conclusions: Taken together, the in vitro and in vivo data for JNJ-67856633 and JNJ-64264681 suggest that combination therapy can increase the anti-tumor effect of the monotherapies and provide a more sustained response, offering strong support for clinical investigation of the combination of these two novel agents. A phase 1b combination study is scheduled to initiate. Citation Format: Ulrike Philippar, Lorena Fontan, Ivo Cornelissen, Haopeng Rui, Sriram Balasubramanian, Marcello Gaudiano, Mariette Bekkers, Luc Van Nuffel, Tianbao Lu, Tianbao Lu, John Wiener, Mark Tichenor, Tony Greway, Kathryn Packman, Bie Verbist, Yusri Elsayed, Ricardo Attar, Jacqueline Bussolari, John Gerecitano. Combination therapy of JNJ-67856633, a novel, first-in-class MALT1 protease inhibitor, and JNJ-64264681, a novel BTK inhibitor, for the treatment of B-cell lymphomas [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1267.

The cardioprotective effect and mechanism of bioactive glass on myocardial reperfusion injury

Myocardial reperfusion treatment for ischemic infarction may cause lethal injury of cardiomyocytes, which is known as ischemia/reperfusion (I/R) injury. As a kind of prospective biomaterial with superior properties, the application of bioactive glasses (BGs) in myocardial tissue engineering have received great interests. In this study, the cardioprotective effect and relevant mechanism of BG on myocardial reperfusion injury were investigated in vitro. H9c2 cardiomyocytes were pretreated with BG extracts and then cultured in hypoxic environment for 30 min followed by reoxygenation for 1 h. The activity of released lactate dehydrogenase (LDH) and the content of malondialdehyde (MDA) in H9c2 cells were tested by assay kits. Cell viability was analyzed by Live/Dead staining assay and the number of living cells was detected by Cell Counting Kit-8 (CCK-8) assay. The cytoskeletal protein F-actin was stained and observed under inverted fluorescence microscope. Mitochondrial membrane potential (MMP) level, reactive oxygen species (ROS) production and apoptosis ratio were evaluated by fluorescent observation and flow cytometry simultaneously. The gene expressions relevant to apoptosis were detected by quantitative real time polymerase chain reaction (qRT-PCR) analysis. The results showed that BG extracts effectively inhibited hypoxia/reoxygenation (H/R)-induced cell injury by suppressing oxidative stress and mitochondrial permeability transition (MPT) within H9c2 cells. Meanwhile, apoptosis caused by H/R injury was alleviated and three classic apoptotic signaling pathways were proved to be regulated by BG extracts. Further analysis showed that phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signaling pathway was up-regulated in H/R-induced H9c2 cells by BG extracts, leading to relieved cellular apoptosis. These results indicated that BG might exert cardioprotective effect in reperfusion injury when applied in myocardial tissue regeneration and repair.

Bergenin Monohydrate Attenuates Inflammatory Response via MAPK and NF-κB Pathways Against Klebsiella pneumonia Infection.

Background: Klebsiella pneumonia has emerged as a critical pathogen causing severe clinical problems, such as pneumonia and sepsis. Meanwhile, intensified drug resistance induced by antibiotic therapy necessitates discovering novel and active molecules from Traditional Chinese Medicine (TCM) for treatment. Methods and results: In this study, the isolated Bergenin monohydrate showed an anti-inflammatory effect in Klebsiella-infected mice. We initially investigated the anti-inflammatory effects and cytoprotection against oxidative stress in vitro and in vivo. Interestingly, a specific dose of Bm can effectively ameliorate lung injury and suppress the expression of inflammatory cytokines such as TNF-α, IL-6, IL-1β and PEG2. Moreover, Bm was also shown to reduced the levels of MPO, MDA and increased SOD and GSH activities. Moreover, we assessed the intracellular signaling molecules including p38, ERK, JNK, IκB, NF-κB-p65 by western blotting and verified through MAPK and NF-κB pathways inhibition experiments. These results reveal that Bm executed its effects via the classical MAPK signaling pathway and NF-κB pathway. Conclusion: Given its underlying anti-inflammatory effect, Bm may be used as a promising therapeutic against Klebsiella-induced infection, thus providing a benefit for the future clinical therapy of pneumonia and medicine design.

Pseudomonas aeruginosa Outer Membrane Vesicles (OMVs) Cause Inflammation and Cardiomyocyte Contractile Dysfunction

Abstract P. aeruginosa (P.a) infection causes severe pneumonia, which can lead to cardiac inflammation and severe cardiac complication. Growing evidence indicates that cardiac resident macrophages are important regulators of inflammation and steady state heart function. On the other hand, pathogen secreted OMVs carry bacterial immunogens that activate the host immune system and alter the homeostasis of normal organ function. Our published results showed that P.a. infection causes cardiac inflammation, fibrosis, and cardiac dysfunction without the bacteria disseminating into heart tissue. However, the mechanism of P.a. induced cardiac inflammation and dysfunction is not known and understanding this is critical for the development of host directed therapy. Thus, we hypothesize that cardiac damage caused by P.a infection is mainly due to the release of bacterial toxins and/or OMVs or by inflammatory mediators released by immune cells. To test our hypothesis we have used human monocyte derived macrophages (hMDMs) and human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Our results show that conditioned media from P.a. infected hMDMs causes contractile dysfunction in hiPSC-CMs. In addition, we found that P.a. OMVs induce similar contractile dysfunction in hiPSC-CMs. Notably, we also found that OMV stimulation activates classical inflammatory signaling pathways and enhances hMDM inflammatory cytokine production. These findings demonstrated that P.a OMVs induce inflammatory cytokine production and subsequent myocyte contractile dysfunction. Studies are ongoing to determine the mechanism of OMV induced contractile dysfunction as well as the impact of OMVs on cardiac dysfunction in a mouse model.

Molecular Mechanism of 73HOXC-AS1-Activated Wntβ-Catenin Signaling and eIF4AIII in Promoting Progression of Gastric Cancer.

Objective This study is aimed at exploring the regulatory mechanism of 73HOXC-AS1 overexpression plasmid-activated Wntβ-catenin classic signaling pathway and eukaryotic initiation factor 4A (eIF4AIII) expression increased by lentivirus-eIF4AIII-RNAi (44682-1) (LV-eIF4AIII-RNAi (44682-1)). Methods Focusing on the occurrence and progression of gastric cancer, the human gastric cancer cell line BGC823 (University Experimental Center) was taken as the research object and was transfected after subculture. According to the different ways of transfection, the cells were divided into the P1 group (LV-eIF4AIII-RNAi (44682-1) overexpressed plasmid), the P2 group (pcDNA-HOXC-AS1 overexpressed plasmid), the P3 group (LV-eIF4AIII-RNAi (44682-1) + pcDNA-HOXC-AS1), and the P4 group (no transfection, control group). Cell proliferation was detected by CCK-8 (Cell Counting Kit-8) assay, Western blotting was adopted to detect Wnt3a and P-GSK3β proteins, Transwell assay was adopted to detect the ability of cell migration and invasion, and cell cycle and apoptosis were detected by flow cytometry. Results The results show that the protein expression levels of Wnt3a and P-GSK3β (glycogen synthase kinase-3β) in the P1 and P4 groups were lower than those in the P2 and P3 groups (P < 0.05). The cell activity and clone number of BGC823 in the P3 group were higher than those in the P1, P2, and P4 groups (P < 0.05). The apoptosis rate of BGC823 cells in the P3 group was significantly higher than those in the P1, P2, and P4 groups (P < 0.05). The proportion of BGC823 cells in the P3 group at the S phase was significantly higher than those in the P1, P2, and P4 groups, while the proportion in the G2 phase was significantly lower than those in the P1, P2, and P4 groups (P < 0.05). The number of migrating and invading BGC823 cells in the P3 group was significantly higher than those in the P1, P2, and P4 groups, while the number of migrating BGC823 cells in the P4 group was significantly lower than those in the P1 and P2 groups (P < 0.05). Conclusion The 73HOXC-AS1 overexpression plasmid-activated Wntβ-catenin classic signaling pathway and eIF4AIII expression increased by LV-eIF4AIII-RNAi (44682-1) could act together on BGC823 cells to improve cell proliferation activity, migration, and invasion; inhibit cell apoptosis; and prevent cells from entering the S phase.

Distal Enhancer Potentiates Activin- and GnRH-Induced Transcription of FSHB.

FSH is critical for fertility. Transcription of FSHB, the gene encoding the beta subunit, is rate-limiting in FSH production and is regulated by both GnRH and activin. Activin signals through SMAD transcription factors. Although the mechanisms and importance of activin signaling in mouse Fshb transcription are well-established, activin regulation of human FSHB is less well understood. We previously reported a novel enhancer of FSHB that contains a fertility-associated single nucleotide polymorphism (rs10031006) and requires a region resembling a full (8 base-pair) SMAD binding element (SBE). Here, we investigated the role of the putative SBE within the enhancer in activin and GnRH regulation of FSHB. In mouse gonadotrope-derived LβT2 cells, the upstream enhancer potentiated activin induction of both the human and mouse FSHB proximal promoters and conferred activin responsiveness to a minimal promoter. Activin induction of the enhancer required the SBE and was blocked by the inhibitory SMAD7, confirming involvement of the classical SMAD signaling pathway. GnRH induction of FSHB was also potentiated by the enhancer and dependent on the SBE, consistent with known activin/GnRH synergy regulating FSHB transcription. In DNA pull-down, the enhancer SBE bound SMAD4, and chromatin immunoprecipitation demonstrated SMAD4 enrichment at the enhancer in native chromatin. Combined activin/GnRH treatment elevated levels of the active transcriptional histone marker, histone 3 lysine 27 acetylation, at the enhancer. Overall, this study indicates that the enhancer is directly targeted by activin signaling and identifies a novel, evolutionarily conserved mechanism by which activin and GnRH can regulate FSHB transcription.

Are We Benign? What Can Wnt Signaling Pathway and Epithelial to Mesenchymal Transition Tell Us about Intracranial Meningioma Progression.

Simple SummaryIntracranial meningiomas are one of the most common primary brain tumors. Although mostly benign, a small portion may exhibit aggressive and malignant characteristics leading to higher recurrence and mortality rate. Detecting the molecular profiles and genes that are involved in meningioma progression can lead to better stratification of patients and more efficient and targeted treatments. The results of this study reveal the role of main actors of the Wnt signaling pathway (β-catenin) and epithelial to mesenchymal transition (E-cadherin, N-cadherin, TWIST1, SNAIL and SLUG) in progression of these tumors, potentially bringing novel biomarkers in diagnostics and molecular targets for therapeutic interventions.Epithelial to mesenchymal transition (EMT), which is characterized by the reduced expression of E-cadherin and increased expression of N-cadherin, plays an important role in the tumor invasion and metastasis. Classical Wnt signaling pathway has a tight link with EMT and it has been shown that nuclear translocation of β-catenin can induce EMT. This research has showed that genes that are involved in cadherin switch, CDH1 and CDH2, play a role in meningioma progression. Increased N-cadherin expression in relation to E-cadherin was recorded. In meningioma, transcription factors SNAIL, SLUG, and TWIST1 demonstrated strong expression in relation to E- and N-cadherin. The expression of SNAIL and SLUG was significantly associated with higher grades (p = 0.001), indicating their role in meningioma progression. Higher grades also recorded an increased expression of total β-catenin followed by an increased expression of its active form (p = 0.000). This research brings the results of genetic and protein analyzes of important molecules that are involved in Wnt and EMT signaling pathways and reveals their role in intracranial meningioma. The results of this study offer guidelines and new markers of progression for future research and reveal new molecular targets of therapeutic interventions.

Development and validation of a RNA binding protein-associated prognostic model for head and neck squamous cell carcinoma.

Evidence shows that defects in RNA-binding proteins (RBPs) are closely related to the occurrence and development of HNSCC. We obtained 502 tumors and 44 normal samples from the TCGA database, among which 190 differentially expressed RBPs were screened. Finally, a prognostic model containing nine RBPs (CELF2, CPEB1, DDX39B, EIF3L, EZH2, KHDRBS3, RNASE10, RNASE3 and SIDT1) was produced. Further analysis showed that the overall survival rate in the high-risk group was lower than that in the low-risk group. The area under the ROC curve (AUC) in the training and testing groups was significant (3-year AUC, 0.735 vs 0.796; 5-year AUC, 0.821 vs 0.804). In addition, a comprehensive analysis of nine identified RBPs showed that most of them were related to the OS of HNSCC patients, and three of them (CELF2, EZH2, and SIDT1) were differentially expressed in HNSCC and control tissues at the protein level. In addition, our data revealed that the identified RBPs are highly interconnected, with high frequency copy number changes in HNSCC samples. GSEA indicated that the abnormal biological processes related to RNA and the activation of some classical tumor signaling pathways were important driving forces for the development of HNSCC. Our results provide novel insights into the pathogenesis of HNSCC, among which nine RBP markers have potential application value in clinical decision-making and individualized treatment of HNSCC.