MAP Kinase Activation Research Articles

Low-Intensity Sonoporation-Induced Intracellular Signalling of Pancreatic Cancer Cells, Fibroblasts and Endothelial Cells.

The use of ultrasound (US) and microbubbles (MB), usually referred to as sonoporation, has great potential to increase the efficacy of chemotherapy. However, the molecular mechanisms that mediate sonoporation response are not well-known, and recent research suggests that cell stress induced by US + MBs may contribute to the treatment benefit. Furthermore, there is a growing understanding that the effects of US + MBs are beyond only the cancer cells and involves the tumour vasculature and microenvironment. We treated pancreatic cancer cells (MIA PaCa-2) and stromal cells, fibroblasts (BJ) and human umbilical vein endothelial cells (HUVECs), with US ± MB, and investigated the extent of uptake of cell impermeable dye (calcein, by flow cytometry), viability (cell count, Annexin/PI and WST-1 assays) and activation of a number of key proteins in important intracellular signalling pathways immediately and 2 h after sonoporation (phospho flow cytometry). Different cell types responded differently to US ± MBs in all these aspects. In general, sonoporation induces immediate, transient activation of MAP-kinases (p38, ERK1/2), and an increase in phosphorylation of ribosomal protein S6 together with dephosphorylation of 4E-BP1. The sonoporation stress-response resembles cellular responses to electroporation and pore-forming toxins in membrane repair and restoring cellular homeostasis, and may be exploited therapeutically. The stromal cells were more sensitive to sonoporation than tumoural cells, and further efforts in optimising sonoporation-enhanced therapy should be targeted at the microenvironment.

Cholesterol-Rich Microdomains Contribute to PAR1 Signaling in Platelets Despite a Weak Localization of the Receptor in These Microdomains.

Platelet protease-activated receptor 1 (PAR1) is a cell surface G-protein-coupled receptor (GPCR) that acts as a thrombin receptor promoting platelet aggregation. Targeting the PAR1 pathway by vorapaxar, a PAR1 antagonist, leads to a reduction in ischemic events in cardiovascular patients with a history of myocardial infarction or with peripheral arterial disease. In platelets, specialized microdomains highly enriched in cholesterol act as modulators of the activity of several GPCRs and play a pivotal role in the signaling pathway. However, their involvement in platelet PAR1 function remains incompletely characterized. In this context, we aimed to investigate whether activation of PAR1 in human platelets requires its localization in the membrane cholesterol-rich microdomains. Using confocal microscopy, biochemical isolation, and proteomics approaches, we found that PAR1 was not localized in cholesterol-rich microdomains in resting platelets, and only a small fraction of the receptor relocated to the microdomains following its activation. Vorapaxar treatment increased the level of PAR1 at the platelet surface, possibly by reducing its endocytosis, while its colocalization with cholesterol-rich microdomains remained weak. Consistent with a cholesterol-dependent activation of Akt and p38 MAP kinase in thrombin receptor-activating peptide (TRAP)-activated platelets, the proteomic data of cholesterol-rich microdomains isolated from TRAP-activated platelets showed the recruitment of proteins contributing to these signaling pathways. In conclusion, contrary to endothelial cells, we found that PAR1 was only weakly present in cholesterol-rich microdomains in human platelets but used these microdomains for efficient activation of downstream signaling pathways following TRAP activation.

The MAP kinase inhibitor PD98059 reduces chromosomal instability in the autoimmune encephalomyelitis SJL/J-mouse model of multiple sclerosis

Multiple sclerosis (MS), a disease in which the immune system attacks nerve cells, has been associated with both genetic and environmental risk factors. We observed increased micronucleus (MN) formation in SJL/J mouse experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Most of these MN were due to chromosomal loss. Increased activation of MAP kinases, which leads to disruption of the mitotic spindle and improper segregation of chromosomes, is associated with MS. MAP kinase inhibitors, such as PD98059, may therefore be beneficial for MS. In the EAE model, PD98059 treatment reduced adverse effects, including MN formation, lipid peroxidation, and GSH oxidation. Interventions that mitigate chromosomal instability may have therapeutic value in MS.

Gene expression profiling of chromosome 10 in PTEN-knockout (−/−) human neural and mesenchymal stem cells: A system biology study

The present study investigates the effects of PTEN deletion in human neuro and mesenchymal stem cells related to the chromosome 10 gene expression profile. The RNA sequencing (RNA-seq) performed on four neural and four mesenchymal stem cells. DEG analysis outcome revealed 122 genes for neural stem cells (57 up-regulated and 65 down-regulated genes) and 258 genes for mesenchymal stem cells (98 up-regulated and 160 down-regulated genes) that were deferentially expressed in the PTEN (-/-) group compared to the normal group. Gene ontology analysis indicated that in the NSCs upregulated DEGs were significantly enriched in transcriptional activator activity, phosphatidylinositol phosphate phosphatase activity, MAP kinase activity while the downregulated DEGs were mainly involved in glycolytic process through glucose-6-phosphate, canonical glycolysis, glucose catabolic process to pyruvate. Meanwhile, in MSCs, the upregulated DEGs were mainly enriched in positive regulation of cell migration, chromatin silencing complex, RNA polymerase core enzyme binding while the downregulated DEGs phosphofructokinase activity, focal adhesion, glycolytic process through glucose-6-phosphate. On the basis of the PPI network of the DEGs, the following top hub genes were detected: six hub genes (PTEN, MAPK8, VCL, PAX2, ITGB1 and RET) in neural stem cells and 9 hub genes (PTEN, CDK1, KIF11, VCL, HK1, KIF20B, SIRT1, ACTR1A and ITGB1) appeared in mesenchymal stem cells respectively in each of the top 10 gene lists. In conclusion, PTEN, VCL and ITGB are found to be the main hub genes in both neural and mesenchymal stem cells, which involve in cell cycle, tumor progression and metastasis.

Inducible and conditional activation of ERK5 MAP kinase rescues mice from cadmium-induced olfactory memory deficits

Cadmium (Cd) is a heavy metal that is one of the most toxic environmental pollutants throughout the world. We previously reported that Cd exposure impairs olfactory memory in mice. However, the underlying mechanisms for its neurotoxicity for olfactory function are not well understood. Since adult Subventricular zone (SVZ) and Olfactory Bulb (OB) neurogenesis contributes to olfaction, olfactory memory defects caused by Cd may be due to inhibition of neurogenesis. In this study, using bromodeoxyuridine (BrdU) labeling and immunohistochemistry, we found that 0.6 mg/L Cd exposure through drinking water impaired adult SVZ/OB neurogenesis in C57BL/6 mice. To determine if the inhibition of olfactory memory by Cd can be reversed by stimulating adult neurogenesis, we utilized the transgenic caMEK5 mouse strain to conditional stimulate of adult neurogenesis by activating the endogenous ERK5 MAP kinase signaling pathway. This was accomplished by conditionally induced expression of active MEK5 (caMEK5) in adult neural stem/progenitor cells. The caMEK5 mice were exposed to 0.6 mg/L Cd for 38 weeks, and tamoxifen was administered to induce caMEK5 expression and stimulate adult SVZ/OB neurogenesis during Cd exposure. Short-term olfactory memory test and sand-digging based, odor-cued olfactory learning and memory test were conducted after Cd and tamoxifen treatments to examine their effects on olfaction. Here we report that Cd exposure impaired short-term olfactory memory and odor-cued associative learning and memory in mice. Furthermore, the Cd-impaired olfactory memory deficits were rescued by the tamoxifen-induction of caMEK5 expression. This suggests that Cd exposure impairs olfactory function by affecting adult SVZ/OB neurogenesis in mice.

BRAFV600E Overrides NOTCH Signaling in Thyroid Cancer.

Background: Several mechanisms likely cooperate with the mitogen-activated protein (MAP)-kinase pathway to promote cancer progression in the thyroid. One putative pathway is NOTCH signaling, which is implicated in several other malignancies. In thyroid cancer, data regarding the role of the NOTCH pathway are insufficient and even contradictory. Methods: A BRAFV600E-driven papillary thyroid carcinoma (PTC) mouse model was subjected to NOTCH pathway genetic alterations, and the tumor burden was followed by ultrasound. Further analyses were performed on PTC cell lines or noncancerous cells transfected with NOTCHIC or BRAFV600E, which were then subjected to pharmacological treatment with MAP-kinase or NOTCH pathway inhibitors. Results: The presence of the BRAFV600E mutation coupled with overexpression of the NOTCH intracellular domain led to significantly bigger thyroid tumors in mice, to a more aggressive carcinoma, and decreased overall survival. Although more cystic, the tumors did not progress into anaplastic thyroid carcinomas. On the contrary, the deletion of RBP-jκ (a major cofactor involved in NOTCH signaling) did not alter the phenotype in mice. BRAFV600E-mutated PTC cell lines were resistant to pharmacological inhibition of the NOTCH pathway. Inhibition of MEK1/2 uncovered a predominant effect on Hes1/Hey1 transcription compared with NOTCH inhibition in BRAFV600E-mutated cell lines. Finally, γ-secretase activity and γ-secretase subunit transcription levels were dependent on ERK activation. Our findings suggest that MAP-kinase activity overrides the NOTCH pathway in the context of thyroid cancer. Conclusions: The interaction between the BRAF and NOTCH pathways demonstrates that the BRAFV600E mutation might bypass NOTCH and exert a strong positive effect on NOTCH downstream targets in thyroid carcinoma.

Identification of a novel senomorphic agent, avenanthramide C, via the suppression of the senescence-associated secretory phenotype

Senescent cells are deeply involved in the induction of tissue damage and aging-related diseases. The identification of factors that eliminate senescent cells or inhibit the senescence-associated secretory phenotype (SASP) in these cells is necessary. Here, we report an avenanthramice C (Avn C) extracted from oat as a new SASP modulator. Treatment with Avn C led to a significant reduction in the levels of markers of senescent cells, with no toxicity observed. The SASP was also inhibited by Avn C treatment, similar to non-senescent cells, and the suppression of cell division by autocrine signals associated with SASP was restored. To investigate the mechanism underlying SASP inhibition by Avn C, we analyzed the effect of Avn C in lipopolysaccharide (LPS)-induced inflammation in non-senescent cells. Avn C inhibited nuclear factor κB (NF-κB) activity and the secretion of inflammatory cytokines before or after LPS treatment. Although the activity of MAP kinases, which are NF-κB upstream signals, was inhibited by Avn C in LPS-induced inflammation, only p38 activity was specifically inhibited in senescent cells. Interestingly, the inhibition of p38 in senescent cells was observed through Avn C-induced 5′-adenosine monophosphate-activated protein kinase (AMPK) activity. Avn C-induced inhibition of the SASP is triggered by senescence-related stress.

Abstract IA03: Systems approaches reveal shared pathways affected in SARS-CoV-2 infection and cancer

Abstract Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the causative agent of the coronavirus disease 2019 (COVID-19) pandemic, has infected millions and killed hundreds of thousands of people worldwide. Clinicians are fighting the battle against the virus with a limited arsenal of drugs that have been shown to be safe and efficacious in treating SARS-CoV-2 infection. We thus rely on gaining a deeper understanding of the molecular mechanisms of this virus, and desperately need new strategies to get drugs to patients in need quickly. To combine both of these aims, my lab has focused much of our recent work on applying systems biology approaches to identify the cellular pathways hijacked by SARS-CoV-2, with the aim of pinpointing promising clinically available drugs for rapid repurposing to treat COVID-19. Identifying which cellular pathways and mechanisms are affected across different diseases will open the existing toolbox of drug treatment for COVID-19. Cancer drugs are a particularly promising set of drugs in this regard: During the process of virus replication, viruses rely on a multitude of interactions with their host cell, and hijack similar pathways that are affected in cancer cells. For example, viruses manipulate the cell cycle for their own benefit, recruit host DNA-damage machinery to viral replication sites, rely on the host translation machinery for viral protein production, and interfere with several signaling pathways, for example, to suppress cellular antiviral defenses. This list, while not comprehensive, makes apparent the commonalities between the exploitation of molecular mechanisms by cancer and virus infection. To get a full picture of cellular pathways targeted by SARS-CoV-2, we recently generated two virus-host interaction networks using systems biology approaches. First, we used affinity-purification mass spectrometry to create a virus-host protein-protein interaction (PPI) map. We cloned, tagged, and expressed 26 of the 29 SARS-CoV-2 proteins in human cells and identified the human proteins and complexes that physically associate with the individual viral proteins. Our map revealed 332 high-confidence PPIs with human proteins involved in a wide spectrum of cell biology, highlighting several oncogenic pathways. We identified 66 druggable human proteins at the virus-host interface, targeted by 69 compounds (of which 29 drugs are approved by the US Food and Drug Administration, 12 are in clinical trials, and 28 are preclinical compounds). We screened a subset of these in multiple viral assays and found that, for example, inhibitors of mRNA translation displayed antiviral activity. In a separate study building on these results, we created a quantitative mass spectrometry-based phosphoproteomics survey of SARS-CoV-2 infection in cell culture. Focusing on phosphorylation events, which are also highly misregulated in cancer, showed stark changes for both host and viral proteins and revealed dramatic rewiring of a number of signaling pathways. For example, SARS-CoV 2 infection promoted casein kinase II (CK2) and p38 MAP kinase activation and shutdown of mitotic kinases. We identified 87 drugs and compounds by mapping global phosphorylation profiles to dysregulated kinases and pathways. Pharmacologic inhibition of p38, CK2, CDKs, AXL, and PIKFYVE kinases possessed antiviral efficacy, representing potential COVID-19 therapies. Clinical trials with some drugs and compounds implicated by our studies are currently being discussed or are already under way. To prepare for future outbreaks, we need to further increase our knowledge of cellular pathways targeted during virus infection. To this end, in addition to a number of infectious agents my lab has studied previously, we are currently performing similar studies on the closely related coronaviruses responsible for outbreaks of SARS and Middle East respiratory syndrome (MERS) in 2003 and 2012, respectively. Combined with the results discussed above, we hope to position the health care community to successfully fight this and potential future outbreaks of novel infectious diseases. Importantly, the input of physicians and experts from other fields will be crucial to leverage the knowledge gained from our studies about the pathways targeted across diseases. Citation Format: Nevan Krogan. Systems approaches reveal shared pathways affected in SARS-CoV-2 infection and cancer [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2020 Jul 20-22. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(18_Suppl):Abstract nr IA03.

Milk somatic cell derived transcriptome analysis identifies regulatory genes and pathways during lactation in Indian Sahiwal cattle (Bos indicus).

The present study is an effort to understand the genomic drivers of lactation in Sahiwal (Bos indicus), the best milch cattle breed of the tropics. RNA sequencing of four animals from early, mid and late lactation stages was performed using milk somatic cells as source of RNA. The genes encoding the milk casein and whey proteins showed highest expression in early and mid lactation, with a declining trend towards the late stage. The enhanced expression of PLIN2, FABP5 and FABP3 genes in mid lactation suggests enrichment of the PPARα pathway which is linked to fatty acid metabolism. A gradual decline in the percentage of genes involved in metabolism of proteins, mRNA and insulin synthesis from early to late lactation reflected transition from lactogenesis to involution. Major biological pathways maintained throughout lactation were adaptive immune system, FGF signaling, EGFR signaling, activated TLR4 signaling, NFkB and MAP kinases activation mediated by TLR4 signaling repertoire. Differential expression analysis revealed 547, 1010 and 1313 differentially expressed genes (p < 0.05) between early-late, early-mid and mid-late stages, respectively. The topmost regulatory genes identified by network analysis from the differentially expressed genes, were involved in Chemokine receptor, GPCR and EGFR1 pathways. The genes and pathways delineated in this study have regulatory implications in cell morphogenesis, lipid droplet formation and protein synthesis in the course of lactation. The study provides an insight into the expression profile of genes influencing milk properties and lactation in Sahiwal cattle.

Abstract 3750: STEAP2 is upregulated and necessary for hepatocellular carcinoma progression via increased copper levels and stress-activated MAP kinase activity

Abstract To understand potential molecular mechanisms that drive hepatocellular carcinoma (HCC) progression, we performed RNA sequencing from paired adjacent non-tumor liver and HCC tumor tissues of nine local HCC patients which revealed significant involvement of a gene coding for Six Transmembrane Epithelial Antigen of Prostate 2 (STEAP2) protein. STEAP2 is a metalloreductase involved in the reduction and indirectly, uptake, of iron and copper ions. We found significantly higher levels of total copper in HCC tissues than those in paired adjacent non-tumor tissues. The upregulation of STEAP2 expression in HCC was confirmed at its protein level and in TCGA and other published HCC gene expression datasets. Hepatic copper overload is associated with Wilson's disease and a known risk factor for HCC, therefore, we hypothesize that STEAP2 upregulation and copper accumulation contribute to HCC progression. Paired HCC and adjacent non-tumor tissues were collected for RNA sequencing, metal ion measurement, and measurements of STEAP2 levels with RT-qPCR and Western blot. Public HCC datasets were queried for STEAP2 expression in HCC and non-tumor tissues. HCC cell lines with knockdown (KD) and overexpression (OE) of STEAP2 were created to perform mechanistic studies including measurements of copper levels and MAP kinase activities, cell proliferation, migration, and anchorage independent growth in vitro and tumor growth in vivo. STEAP2 is significantly upregulated in various HCC gene expression datasets; its expression is positively associated with tumor grade. STEAP2 KD in HCC cell lines decreased cell growth, migration, invasion, and xenograft tumor growth, while STEAP2 OE showed opposite effects. STEAP2 KD in HCC cells also reduced intracellular copper levels and activation of stress-activated MAP kinases including p38 and JNK. Treatment with copper rescued the reduced HCC cell migration due to STEATP2 KD and activated p38 and JNK. Furthermore, treatment with p38 or JNK inhibitors significantly inhibited cell migration. Thus, STEAP2 appears to play a malignant-promoting role in HCC cells by driving migration/invasion via increased copper levels and MAP kinase activities. Our study uncovered a novel molecular mechanism contributing to HCC malignancy and a potential therapeutic target for HCC treatment. Citation Format: Carla R. Zeballos, Acarizia Easley, Hakim Bouamar, Guixi Zheng, Xiang Gu, Yang Junhua, Yidong Chen, Francisco Cigarroa, LuZhe Sun. STEAP2 is upregulated and necessary for hepatocellular carcinoma progression via increased copper levels and stress-activated MAP kinase activity [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3750.

Abstract 4143: Identification of molecular targets for conservative management of early stage, low-grade endometrial cancer through compartment-specific transcriptome profiling

Abstract Understanding tumor microenvironment is critical to improving conservative management (non-surgical) strategies for early stage, low grade endometrial cancer (EC). This remains an unmet clinical need for women who wish to maintain fertility and those who cannot undergo surgery due to medical co-morbidities. Current regimens rely on progestin treatment, but resistance and early relapse require improved therapeutic strategies. We sought to evaluate alterations in gene expression in the epithelial and stromal compartment of EC compared to normal endometrium to identify targets for combination therapies. RNA was extracted from microdissected epithelial and stromal compartments of formalin fixed paraffin embedded early stage, low grade EC and normal endometrium (NE). Comprehensive transcriptome profiling was performed with Affymetrix Clariom D assay and pathway analysis was performed. Immunohistochemistry (IHC) was used for validation. Functional studies of differentially expressed genes were performed in EC cell lines, ECC-1 and Hec1A. Focusing on differentially expressed genes in the epithelial compartment of EC vs NE, multiple activated signaling pathways were identified, including eIF2 signaling, eIF4 and p70S6K signaling, PI3K/AKT/mTOR, and hormone signaling. Top gene expression changes with significantly lower expression in EC included RBPMS (RNA-binding protein with multiple splicing), SORD (sorbitol dehydrogenase), DDX17 (DEAD-box helicase 17), and TPT1 (translationally-controlled tumor protein) gene expression, while MTDH (metadherin) was significantly increased in EC. Differential expression of proteins coded by these genes was confirmed by IHC. Additionally, RBPMS silencing enhanced AKT and S6 protein phosphorylation in Hec1A cells and forced expression of RBPMS in ECC-1 cells attenuated Erk1/2 MAP kinase activation. Expression of RBPMS inhibited cell proliferation and knockdown of RBPMS increased proliferation. Treatment of EC cells with everolimus (mTOR inhibitor) resulted in differential inhibition of proliferation based on RBPMS expression. Communication between tumor and stroma is also being evaluated. While estrogen-regulated changes were prominent in early stage low-grade EC, the discovery of alterations in other signaling pathways suggest additional targets for conservative management of the disease. The identification of signaling nodes shared between hormone-regulated pathways and mTOR signaling provides further justification for the ongoing combination therapy trial (progestin plus everolimus). Additionally, biomarkers from these pathways, such as RBPMS, can be studied to guide the selection of progestin-only therapy versus combination therapy. Citation Format: Qian Zhang, Xiaoping Su, Joseph Celestino, Ying Yuan, Samuel C. Mok, Karen H. Lu, Melinda S. Yates. Identification of molecular targets for conservative management of early stage, low-grade endometrial cancer through compartment-specific transcriptome profiling [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 4143.

FGF5 Regulates Schwann Cell Migration and Adhesion.

The fibroblast growth factor (FGF) family polypeptides play key roles in promoting tissue regeneration and repair. FGF5 is strongly up-regulated in Schwann cells of the peripheral nervous system following injury; however, a role for FGF5 in peripheral nerve regeneration has not been shown up to now. In this report, we examined the expression of FGF5 and its receptors FGFR1-4 in Schwann cells of the mouse sciatic nerve following injury, and then measured the effects of FGF5 treatment upon cultured primary rat Schwann cells. By microarray and mRNA sequencing data analysis, RT-PCR, qPCR, western blotting and immunostaining, we show that FGF5 is highly up-regulated in Schwann cells of the mouse distal sciatic nerve following injury, and FGFR1 and FGFR2 are highly expressed in Schwann cells of the peripheral nerve both before and following injury. Using cultured primary rat Schwann cells, we show that FGF5 inhibits ERK1/2 MAP kinase activity but promotes rapid Schwann cell migration and adhesion via the upregulation of N-cadherin. Thus, FGF5 is an autocrine regulator of Schwann cells to regulate Schwann cell migration and adhesion.

Key phosphorylation sites in GPCR s orchestrate the contribution of β‐Arrestin 1 in ERK 1/2 activation

β-arrestins (βarrs) are key regulators of G protein-coupled receptor (GPCR) signaling and trafficking, and their knockdown typically leads to a decrease in agonist-induced ERK1/2 MAP kinase activation. Interestingly, for some GPCRs, knockdown of βarr1 augments agonist-induced ERK1/2 phosphorylation although a mechanistic basis for this intriguing phenomenon is unclear. Here, we use selected GPCRs to explore a possible correlation between the spatial positioning of receptor phosphorylation sites and the contribution of βarr1 in ERK1/2 activation. We discover that engineering a spatially positioned double-phosphorylation-site cluster in the bradykinin receptor (B2 R), analogous to that present in the vasopressin receptor (V2 R), reverses the contribution of βarr1 in ERK1/2 activation from inhibitory to promotive. An intrabody sensor suggests a conformational mechanism for this role reversal of βarr1, and molecular dynamics simulation reveals a bifurcated salt bridge between this double-phosphorylation site cluster and Lys294 in the lariat loop of βarr1, which directs the orientation of the lariat loop. Our findings provide novel insights into the opposite roles of βarr1 in ERK1/2 activation for different GPCRs with a direct relevance to biased agonism and novel therapeutics.

Hypoxic induction of CCN2 mRNA through p38 MAP kinase activation in the human chondrosarcoma‐derived cell line, HCS‐2/8

AbstractCCN2/CTGF (cellular communication network factor 2/connective tissue growth factor) plays critical roles in cartilage development, maintenance, and regeneration. Hypoxia‐induced expression of CCN2 mRNA is regulated post‐transcriptionally in the human chondrosarcoma‐derived cell line, HCS‐2/8. In the present study, the hypoxia‐induced increase in CCN2 mRNA expression was assessed with quantitative real‐time PCR in HCS‐2/8 cells in the presence of inhibitors of mitogen‐activated protein kinases (MAPKs). Subsequently, the stability of CCN2 mRNA in hypoxia was evaluated with mRNA degradation assays in the presence or absence of the selective p38 MAPK inhibitor, SB203580. We detected phosphorylation of p38 MAPK by immunoblot analysis within 30 minutes in hypoxia, and we observed ~twofold higher CCN2 mRNA levels in hypoxia compared to normoxia. Blockade of p38 MAPK activation with 10 µmol/L SB203580 abolished this CCN2 induction. Furthermore, we found that inhibition of p38 MAPK suppressed the elongation of CCN2 mRNA half‐life in hypoxia. Taken together, these findings suggest that the molecular mechanism, by which CCN2 mRNA expression is increased in hypoxia, induces the activation of p38 MAPK leading to the post‐transcriptional regulation of the stability of CCN2 mRNA.

Induction of oxidative stress and DNA damage in human renal proximal tubular cells by aristolochic acid

Aristolochic acid 1 (AAI) is found primarily in the plant Aristolochia. Consumption of products containing AAI has been linked with permanent kidney damage and urothelial carcinoma. This study applied human proximal tubule epithelial cell line (HK-2) to examine the relationship among AAI-induced intracellular oxidative stress, DNA damage and MAP kinase activation. High concentrations of AAI caused a decrease in cell viability and an increase in the activity of caspase 3. AAI treatment also led to a dose-dependent increase of reactive oxygen species (ROS) in HK-2 cells, and the presence of antioxidant glutathione (GSH) effectively inhibited ROS generation. Stimulating HK-2 cultures with AAI also led to GSH depletion. Results from single cell gel electrophoresis (SCGE) assays demonstrated that AAI showed the ability to increase the levels of DNA strand breaks in HK-2 cells. Up-regulation of luciferase activity driven by the Nrf2 binding element was also observed after 200 μM AAI treatment. Exposure of HK-2 cells with AAI activated both ERK1/2 and p38 kinase phosphorylation, while only the MEK1/2 inhibitor, U0126, significantly decreased the levels of AAI-mediated ROS. In addition, both U0126 and SB202190 effectively reversed the levels of DNA damage triggered by AAI. This suggests that AAI treatment of HK-2 results in ROS formation and DNA damage. Furthermore, ROS generation occurs via the MEK/ERK1/2 signaling pathway, whereas DNA damage occurs via both the ERK1/2 and p38 pathways.

Diffuse leptomeningeal glioneuronal tumor: a double misnomer? A report of two cases

Diffuse leptomeningeal glioneuronal tumor (DLGNT) was introduced, for the first time, as a provisional entity in the 2016 WHO classification of central nervous system tumors. DLGNT mainly occur in children and characterized by a widespread leptomeningeal growth occasionally associated with intraspinal tumor nodules, an oligodendroglial-like cytology, glioneuronal differentiation and MAP-Kinase activation associated with either solitary 1p deletion or 1p/19q codeletion in the absence of IDH mutation.We report here two unexpected DLGNTs adult cases, characterized by a unique supratentorial circumscribed intraparenchymal tumor without leptomeningeal involvement in spite of long follow-up. In both cases, the diagnosis of DLGNT was made after DNA-methylation profiling which demonstrated that one case belonged to the DLGNT class whereas the other remained not classifiable but showed on CNV the characteristic genetic findings recorded in DLGNT. Both cases harbored 1p/19q codeletion associated with KIAA1549:BRAF fusion in one case and with BRAF V600E and PIK3CA E545A mutations, in the other.Our study enlarges the clinical and molecular spectrum of DLGNTs, and points out that the terminology of DLGNTs is not fully appropriate since some cases could have neither diffuse growth nor leptomeningeal dissemination. This suggests that DLGNTs encompass a wide spectrum of tumors that has yet to be fully clarified.

Network pharmacology-based study on material basis and mechanism of Qingfei Paidu Decoction against COVID-19

This study used the method of network pharmacology to study the multi-component,multi-target and multi-pathway mechanism of Qingfei Paidu Decoction in the treatment of COVID-19,in order to provide basis for related basic research and clinical application. First of all, according to the screening conditions of OB 30% and DL 0.18,302 active components and 148 related targets in Qingfei Paidu Decoction were screened by TCMSP database, screening of 362 COVID-19 -related targets by GeneCards database,23 intersection targets were obtained by Venn analysis. Then 10 significant effective compounds and 5 key targets were obtained by using the CentiScaPe plug-in of Cytoscape software, Further construct the network topology diagram. The results of molecular docking of significant effective compounds and key targets show that the binding ability and interaction ability between molecules are strong. Finally, the GO and KEGG pathway enrichment analysis of the key targets were done through the ClusterProfiler package of R software, key compounds such as quercetin,luteolin,naringin, kaempferol and baicalein have antitussive,expectorant,anti-inflammatory and antiviral effects to varying degrees, the key targets are mainly concentrated in 144 related signal pathways such as IL-17,Tuberculosis,TNF,MAPK,Th17,Pertussis,etc.It involves 28 biological functions such as phosphatase binding, MAP kinase activity and cytokine receptor binding to regulate metabolism, immune regulation, inflammation and other physiological processes. According to the above results,it is considered that the active components of Qingfei Paidu decoction have multi-target and multi-pathway regulating effect on the treatment of COVID-19.

Evidence of capacitation in the parasitoid wasp, Nasonia vitripennis, and its potential role in sex allocation.

The allocation of resources to the production of one sex or another has been observed in a large variety of animals. Its theoretical basis allows accurate predictions of offspring sex ratios in many species, but the mechanisms by which sex allocation is controlled are poorly understood. Using previously published data, we investigated whether alternative splicing, combined with differential gene expression, was involved with sex allocation in the parasitoid wasp, Nasonia vitripennis. We found that sex allocation is not controlled by alternative splicing but changes in gene and transcript‐specific expression, which were identified to be involved with oviposition, were shown to be similar to those involved in sperm motility and capacitation. Genes involved in cholesterol efflux, a key component of capacitation, along with calcium transport, neurotransmission, trypsin, and MAPKinase activity were regulated in ovipositing wasps. The results show evidence for regulation of sperm motility and of capacitation in an insect which, in the context of the physiology of the N. vitripennis spermatheca, could be important for sex allocation.

P-Coumaric Acid Enriched-Peanut Sprout Improves High Fat Diet-Induced Obesity and Insulin Resistance by Inhibiting Macrophage Recruitment

Abstract Objectives We have previously reported that Peanut Sprout Extract (PSE) inhibited adipogenesis through augmentation of mitochondrial fatty acid oxidation. However, it is unknown whether PS consumption reduced visceral obesity and obesity-mediated metabolic complications in vivo. Methods To test this question, obesity-prone C57BL/6 male mice were randomly assigned into three different group; 1) low-fat (LF) diets (11% calories from fat), 2) high-fat (HF) diets (60% calories from fat) or 2) HF diets plus PS (4% in diet, HF-P). Results HF diet for 8 weeks resulted in a significant increase in body weight, liver, and adipose tissue mass compared to LF alone. PS supplementation reduced 1) body weight gain, 2) lipid profile, 3) up-regulated glucose levels, and 4) recruitment of adipose tissue macrophage in HF diet-induced obese C57BL6 mice. In parallel, lipopolysaccharide (LPS)-mediated induction of inflammation was reversed by PSE treatment in macrophages and adipocytes with significant suppression of MAP kinase and NF-kB activation. PSE also reduced LPS-mediated M1 macrophage polarization in bone marrow stem cells. Additionally, we found that, among constitutes of PSE, p-coumaric acid has been identified as responsible polyphenol that exclusively showed a similar trends as PSE. Conclusions Collectively, these data suggest that p-coumaric acid enriched-Peanut Sprout attenuates visceral obesity and obesity-mediated metabolic complications by inhibiting adipose inflammation. Funding Sources This work has supported by the National Research Foundation of Korea(NRF) grant funded by the Korea government (MSIT).

CDC20 is a novel potential therapeutic target in NF1-mutant melanoma.

e22075 Background: NF1-mutant melanoma (NF1-MT) is a distinct molecular subtype with worse overall prognosis than NF1-wild melanoma (NF1-WT). Despite its prevalence in 20% of patients and associated clinical disadvantage, there is no specific targeted therapy available to treat NF1-MT as its biological impact remains understudied. We tested the hypothesis that NF1-MT possess distinct genomic and transcriptomic characteristics from NF1-WT, which can potentially inform therapeutic innovation. Methods: Tumor DNA from metastatic melanoma (MM) patients treated with checkpoint inhibition at NYU was genotyped using exome sequencing. Microenvironment-related transcripts (n = 770) were then compared in NF1-MT versus NF1-WT using unsupervised clustering, differential gene expression, and gene set enrichment analyses. Verification of genomic and transcriptional findings was performed using qPCR and immunohistochemistry (IHC). TCGA melanoma (SKCM) data were analyzed as an independent cohort. Results: Pathogenic NF1 mutations were detected in 31/121 (27%) cases and were significantly associated with higher tumor mutational burden (P = 0.02). NF1-MT showed recurrent homozygous deletion in the membrane regulatory gene SYT1 and amplifications in oncogenes including CCND1 and MDM2, verified by qPCR. NF1-MT showed upregulation of proliferation with significantly increased expression of Ki-67 and cell division cycle protein 20 CDC20 (P = 0.03) validated by IHC (P = 0.03). Cell cycle was the top enriched pathway in NF1-MT (P = 0.003, FDR = 0.04). Analysis of TCGA melanoma data (n = 440) corroborated upregulation of MKI67 and CDC20 in NF1-MT melanoma (P = 0.01, P = 0.03). Conclusions: NF1-MT is associated with a hyper-proliferative tumor phenotype that cannot be only attributed to MAP kinase activation. Our data suggest that NF1-MT may be vulnerable to pharmacological inhibitors of cell cycle progression and proliferation, such as those targeting CDC20 or CDK4/6.