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Background: Ginsenoside Rg1 (Rg1) is one of the main active components in Chinese medicines, Panax ginseng and Panax notoginseng. Research has shown that Rg1 has a protective effect on the cardiovascular system, including anti-myocardial ischemia-reperfusion injury, anti-apoptosis, and promotion of myocardial angiogenesis, suggesting it a potential cardiovascular agent. However, the protective mechanism involved is still not fully understood. Methods: Based on network pharmacology, ligand-based protein docking, proteomics, Western blot, protein recombination and spectroscopic analysis (UV–Vis and fluorescence spectra) techniques, potential targets and pathways for Rg1 against myocardial ischemia (MI) were screened and explored. Results: An important target set containing 19 proteins was constructed. Two target proteins with more favorable binding activity for Rg1 against MI were further identified by molecular docking, including mitogen-activated protein kinase 1 (MAPK1) and adenosine kinase (ADK). Meanwhile, Rg1 intervention on H9c2 cells injured by H2O2 showed an inhibitory oxidative phosphorylation (OXPHOS) pathway. The inhibition of Rg1 on MAPK1 and OXPHOS pathway was confirmed by Western blot assay. By protein recombination and spectroscopic analysis, the binding reaction between ADK and Rg1 was also evaluated. Conclusion: Rg1 can effectively alleviate cardiomyocytes oxidative stress injury via targeting MAPK1 and ADK, and inhibiting oxidative phosphorylation (OXPHOS) pathway. The present study provides scientific basis for the clinical application of the natural active ingredient, Rg1, and also gives rise to a methodological reference to the searching of action targets and pathways of other natural active ingredients.

Crosstalk between the Hedgehog and MAPK signaling pathways occurs in several types of cancer and contributes to clinical resistance to Hedgehog pathway inhibitors. Here we show that MAP kinase-mediated phosphorylation weakens the binding of the GLI1 transcription factor to its negative regulator SUFU. ERK2 phosphorylates GLI1 on three evolutionarily conserved target sites (S102, S116, and S130) located near the high-affinity binding site for SUFU; these phosphorylations cooperate to weaken the affinity of GLI1-SUFU binding by over 25-fold. Phosphorylation of any one, or even any two, of the three sites does not result in the level of SUFU release seen when all three sites are phosphorylated. Tumor-derived mutations in R100 and S105, residues bordering S102, also diminish SUFU binding, collectively defining a novel evolutionarily conserved SUFU affinity-modulating region. In cultured mammalian cells, GLI1 variants containing phosphomimetic substitutions of S102, S116, and S130 displayed an increased ability to drive transcription. We conclude that multisite phosphorylation of GLI1 by ERK2 or other MAP kinases weakens GLI1-SUFU binding, thereby facilitating GLI1 activation and contributing to both physiological and pathological crosstalk.

Purpose: Vascular endothelial growth factor tyrosine kinase inhibitors (TKIs) have been the standard of care for advanced and metastatic clear cell renal cell carcinoma (ccRCC). However, the therapeutic effect of TKI monotherapy remains unsatisfactory given the high rates of acquired resistance to TKI therapy despite favorable initial tumor response. Materials and Methods: To define the TKI-resistance mechanism and identify new therapeutic target for TKI-resistant ccRCC, an integrative differential gene expression analysis was performed using acquired resistant cohort and a public dataset. Sunitinib-resistant RCC cell lines were established and used to test their malignant behaviors of TKI resistance through in vitro and in vivo studies. Immunohistochemistry was conducted to compare expression between the tumor and normal kidney and verify expression of pathway-related proteins. Results: Integrated differential gene expression analysis revealed increased interferon-induced transmembrane protein 3 (IFITM3) expression in post-TKI samples. IFITM3 expression was increased in ccRCC compared with the normal kidney. TKI-resistant RCC cells showed high expression of IFITM3 compared with TKI-sensitive cells and displayed aggressive biologic features such as higher proliferative ability, clonogenic survival, migration, and invasion while being treated with sunitinib. These aggressive features were suppressed by the inhibition of IFITM3 expression and promoted by IFITM3 overexpression, and these findings were confirmed in a xenograft model. IFITM3-mediated TKI resistance was associated with the activation of TRAF6 and MAPK/AP-1 pathways. Conclusions: These results demonstrate IFITM3-mediated activation of the TRAF6/MAPK/AP-1 pathways as a mechanism of acquired TKI resistance, and suggest IFITM3 as a new target for TKI-resistant ccRCC.

The endocannabinoid system (ECS) exerts immunosuppressive effects, which are mostly mediated by cannabinoid receptor 2 (CBR2), whose expression on leukocytes is higher than CBR1, mainly localized in the brain. Targeted CBR2 activation could limit inflammation, avoiding CBR1-related psychoactive effects. Herein, we evaluated in vitro the biological activity of a novel, selective and high-affinity CBR2 agonist, called JT11, studying its potential CBR2-mediated anti-inflammatory effect. Trypan Blue and MTT assays were used to test the cytotoxic and anti-proliferative effect of JT11 in Jurkat cells. Its pro-apoptotic activity was investigated analyzing both cell cycle and poly PARP cleavage. Finally, we evaluated its impact on LPS-induced ERK1/2 and NF-kB-p65 activation, TNF-α, IL-1β, IL-6 and IL-8 release in peripheral blood mononuclear cells (PBMCs) from healthy donors. Selective CB2R antagonist SR144528 and CBR2 knockdown were used to further verify the selectivity of JT11. We confirmed selective CBR2 activation by JT11. JT11 regulated cell viability and proliferation through a CBR2-dependent mechanism in Jurkat cells, exhibiting a mild pro-apoptotic activity. Finally, it reduced LPS-induced ERK1/2 and NF-kB-p65 phosphorylation and pro-inflammatory cytokines release in human PBMCs, proving to possess in vitro anti-inflammatory properties. JT11 as CBR2 ligands could enhance ECS immunoregulatory activity and our results support the view that therapeutic strategies targeting CBR2 signaling could be promising for the treatment of chronic inflammatory diseases.

BackgroundEmpagliflozin is a selective sodium-glucose cotransporter 2 (SGLT2) inhibitor used to lower blood sugar in adults with type 2 diabetes. Empagliflozin also exerts cardioprotective effects independent from glucose control, but its benefits on arrhythmogenesis and sudden cardiac death are not known. The purpose of this study was to examine the effect of empagliflozin on myocardial ischemia/reperfusion-provoked cardiac arrhythmia and sudden cardiac death in vivo.MethodsMale Sprague Dawley rats were randomly assigned to sham-operated, control or empagliflozin groups. All except for the sham-operated rats were subjected to 5-min left main coronary artery ligation followed by 20-min reperfusion. A standard limb lead II electrocardiogram was continuously measured throughout the experiment. Coronary artery reperfusion-induced ventricular arrhythmogenesis and empagliflozin therapy were evaluated. The hearts were used for protein phosphorylation analysis and immunohistological assessment.ResultsEmpagliflozin did not alter baseline cardiac normal conduction activity. However, empagliflozin eliminated myocardial vulnerability to sudden cardiac death (from 69.2% mortality rate in the control group to 0% in the empagliflozin group) and reduced the susceptibility to reperfusion-induced arrhythmias post I/R injury. Empagliflozin increased phosphorylation of cardiac ERK1/2 after reperfusion injury. Furthermore, inhibition of ERK1/2 using U0126 abolished the anti-arrhythmic action of empagliflozin and ERK1/2 phosphorylation.ConclusionsPretreatment with empagliflozin protects the heart from subsequent severe lethal ventricular arrhythmia induced by myocardial ischemia and reperfusion injury. These protective benefits may occur as a consequence of activation of the ERK1/2-dependent cell-survival signaling pathway in a glucose-independent manner.

Objective To explore the effective ingredients and action targets of Bufei decoction in the treatment of chronic obstructive pulmonary disease (COPD) based on network pharmacology. Methods The active ingredients and action targets of Bufei decoction were analyzed in the previous literatures based on the Traditional Chinese Medicine Sys‐ tems Pharmacology Database and Analysis Platform (TCMSP). COPD-related targets were screened based on Gene Expres‐ sion Omnibus (GEO), Drugbank and Genecards databases, and were intersected with the targets of Bufei decoction to ob‐ tain the potential genes of Bufei decoction in treatment of COPD. GO function and KEGG signaling pathway enrichment analysis were performed on the potential genes. The protein-protein interaction (PPI) network diagram was established, and topological analysis was used to identify the action targets of Bufei decoction on COPD. Results The active ingredi‐ ents were 40 kinds in ginseng, 27 kinds in Astragalus, 9 kinds in Shudi, 19 kinds in Schisandra chinensis, 22 kinds in Aster, and 36 kinds in Morus alba； there were 141 action target . The top 10 most important active ingredients in the drugactive ingredient-target network included quercetin, astragalus isoflavanoside, and kaempferol, etc There were a total of 1 730 COPD-related targets, of which 80 were the potential therapeutic targets of Bufei decoction for COPD. The GO function analysis of the potential genes of Bufei decoction in treating COPD showed that it mainly involved inflammation, apoptosis, cell proliferation, REDOX and so on. KEGG signaling pathway analysis results showed that it mainly involved cell senescence pathway, MAPK signaling pathway, cell cycle cycling pathway, neurotrophin signaling pathway, apoptosis pathway, Th17 cell differentiation pathway, FoxO signaling pathway, PI3K/AKT signaling pathway, Rap1 signaling path‐ way, and Ras signaling pathway. The core targets after PPI network screening included mitogen-activated protein kinase 1 (MAPK1), heat shock protein 90α family class A member 1(HSP90AA1), and v-jun avian sarcona virus 17 oncogene ho‐ molog (JUN), etc. Conclusion The potential effective ingredients of Bufei decoction in treatment of COPD are querce‐ tin, kaempferol, etc., which may play a role in regulating cellular senescence pathway, MAPK signaling pathway and apoptosis pathway by acting on MAPK1, HSP90AA1, JUN and other targets. [ABSTRACT FROM AUTHOR]

Patients with chronic lymphocytic leukemia (CLL) have high plasma-levels of Wnt5a, which can induce phosphorylation of ERK1/2 and enhance CLL-cell proliferation. Such effects could be inhibited by treatment with an ERK1/2 inhibitor, ERK1/2-specific siRNA, or cirmtuzumab, an anti-ROR1 mAb. The CLL-derived line, MEC1, expresses Wnt5a, but not ROR1. MEC1 cells transfected to express ROR1 (MEC1-ROR1) had higher levels of phosphorylated ERK1/2 than parental MEC1, or MEC1 transfected with ROR1ΔPRD, a truncated ROR1 lacking the cytoplasmic proline-rich domain (PRD), or ROR1P808A a mutant ROR1 with a P→A substitution at 808, which is required for complexing with the Rac-specific-guanine-nucleotide-exchange factor DOCK2 upon stimulation with Wnt5a. We silenced DOCK2 with siRNA and found this repressed the capacity of Wnt5a to induce ERK1/2 phosphorylation in MEC1-ROR1 or CLL cells. CLL cells that expressed ROR1 had higher levels of phosphorylated ERK1/2 or DOCK2 than CLL cells lacking ROR1. Although we found ibrutinib could inhibit the phosphorylation of ERK1/2 and DOCK2 induced by B-cell-receptor ligation, we found that this drug was unable to inhibit Wnt5a-induced, ROR1-dependent phosphorylation of ERK1/2 or DOCK2. This study demonstrates that Wnt5a can induce activation of ERK1/2 and enhance CLL-cell proliferation via a ROR1/DOCK2-dependent pathway independent of BTK.

T cells experience complex temporal patterns of stimulus via receptor-ligand-binding interactions with surrounding cells. From these temporal patterns, T cells are able to pick out antigenic signals while establishing self-tolerance. Although features such as duration of antigen binding have been examined, our understanding of how T cells interpret signals with different frequencies or temporal stimulation patterns is relatively unexplored. We engineered T cells to respond to light as a stimulus by building an optogenetically controlled chimeric antigen receptor (optoCAR). We discovered that T cells respond to minute-scale oscillations of activation signal by stimulating optoCAR T cells with tunable pulse trains of light. Systematically scanning signal oscillation period from 1 to 150 min revealed that expression of CD69, a T cell activation marker, reached a local minimum at a period of ∼25 min (corresponding to 5 to 15 min pulse widths). A combination of inhibitors and genetic knockouts suggest that this frequency filtering mechanism lies downstream of the Erk signaling branch of the T cell response network and may involve a negative feedback loop that diminishes Erk activity. The timescale of CD69 filtering corresponds with the duration of T cell encounters with self-peptide-presenting APCs observed via intravital imaging in mice, indicating a potential functional role for temporal filtering in vivo. This study illustrates that the T cell signaling machinery is tuned to temporally filter and interpret time-variant input signals in discriminatory ways.

AbstractEndothelial cells play a major role in inflammatory responses to infection and sterile injury. Endothelial cells express Toll-like receptor 4 (TLR4) and are activated by LPS to express inflammatory cytokines/chemokines, and to undergo functional changes, including increased permeability. The extracellular signal-regulated kinase 1/2 (ERK1/2) mediates pro-inflammatory signaling in monocytes and macrophages, but the role of ERK1/2 in LPS-induced activation of microvascular endothelial cells has not been defined. We therefore studied the role of ERK1/2 in LPS-induced inflammatory activation and permeability of primary human lung microvascular endothelial cells (HMVEC). Inhibition of ERK1/2 augmented LPS-induced IL-6 and vascular cell adhesion protein (VCAM-1) production by HMVEC. ERK1/2 siRNA knockdown also augmented IL-6 production by LPS-treated HMVEC. Conversely, ERK1/2 inhibition abrogated permeability and restored cell-cell junctions of LPS-treated HMVEC. Consistent with the previously described pro-inflammatory role for ERK1/2 in leukocytes, inhibition of ERK1/2 reduced LPS-induced cytokine/chemokine production by primary human monocytes. Our study identifies a complex role for ERK1/2 in TLR4-activation of HMVEC, independent of myeloid differentiation primary response gene (MyD88) and TIR domain-containing adaptor inducing IFN-β (TRIF) signaling pathways. The activation of ERK1/2 limits LPS-induced IL-6 production by HMVEC, while at the same time promoting HMVEC permeability. Conversely, ERK1/2 activation promotes IL-6 production by human monocytes. Our results suggest that ERK1/2 may play an important role in the nuanced regulation of endothelial cell inflammation and vascular permeability in sepsis and injury.

Human cell division is a highly regulated process that relies on the accurate capture and movement of chromosomes to the metaphase plate. Errors in the fidelity of chromosome congression and alignment can lead to improper chromosome segregation, which is correlated with aneuploidy and tumorigenesis. These processes are known to be regulated by extracellular signal-regulated kinase 2 (ERK2) in other species, but the role of ERK2 in mitosis in mammals remains unclear. Here, we have identified the dual-specificity phosphatase 7 (DUSP7), known to display selectivity for ERK2, as important in regulating chromosome alignment. During mitosis, DUSP7 bound to ERK2 and regulated the abundance of active phospho-ERK2 through its phosphatase activity. Overexpression of DUSP7, but not catalytically inactive mutants, led to a decrease in the levels of phospho-ERK2 and mitotic chromosome misalignment, while knockdown of DUSP7 also led to defective chromosome congression that resulted in a prolonged mitosis. Consistently, knockdown or chemical inhibition of ERK2 or chemical inhibition of the MEK kinase that phosphorylates ERK2 led to chromosome alignment defects. Our results support a model wherein MEK-mediated phosphorylation and DUSP7-mediated dephosphorylation regulate the levels of active phospho-ERK2 to promote proper cell division.

Background and aims: Mitogen-activated protein kinases (MAPKs) have been reported to respond to various stimuli including heat stress. This research aimed to investigate whether StMAPK1 is implicated in the transduction of the heat stress signal to adapt heat stress as a thermos-tolerant gene. Materials and methods: Potato plants were cultivated under mild (30°C) and acute (35°C) heat stress conditions to analyze mRNA expression of StMAPKs and physiological indicators. StMAPK1 was up-regulated and down-regulated by transfection. Subcellular localization of StMAPK1 protein was observed by fluorescence microscope. The transgenic potato plants were assayed for physiological indexes, photosynthesis, cellular membrane integrity, and heat stress response gene expression. Results: Heat stress altered the expression prolife of StMAPKs. StMAPK1 overexpression changed the physiological characteristics and phenotypes of potato plants under heat stresses. StMAPK1 mediates photosynthesis and maintains membrane integrity of potato plants in response to heat stress. Stress response genes (StP5CS, StCAT, StSOD, and StPOD) in potato plants were altered by StMAPK1 dysregulation. mRNA expression of heat stress genes (StHSP90, StHSP70, StHSP20, and StHSFA3) was affected by StMAPK1. Conclusions: StMAPK1 overexpression increases the heat-tolerant capacity of potato plants at the morphological, physiological, molecular, and genetic levels. [ABSTRACT FROM AUTHOR]

Epidermal growth factor receptor tyrosine kinase inhibitors (EGFr TKIs) are first-line therapies for various cancers, and cause dose-limiting severe diarrhea in many patients. We hypothesized that diarrhea caused by EGFr TKIs might reflect actions on epithelial transport, barrier function, or both, which we tested using cell cultures including murine and human enteroid-derived monolayers (EDMs), analyzed using electrophysiological and other relevant methods. EGFr TKIs (such as afatinib, erlotinib, and osimertinib) reversed the acute inhibitory effect of EGF on chloride secretion induced by carbachol (CCh) across T84 human colonic epithelial cells, which correlated with the diarrhea-inducing effect of each agent clinically. EGFr TKIs also reduced transepithelial electrical resistance (TEER), whereas co-treatment with CCh delayed the decrease in TEER compared with that of cells co-treated with EGF. Furthermore, afatinib and erlotinib prevented EGF- or CCh-induced EGFr phosphorylation. EGFr TKIs also suppressed phosphorylation of extracellular signal-regulated kinase (Erk)1/2 in response to EGF, whereas they had weaker effects on CCh-induced Erk1/2 phosphorylation. In human EDMs, EGF potentiated ion transport induced by CCh, whereas afatinib reversed this effect. The ability of EGFr TKIs to reverse the effects of EGF on calcium-dependent chloride secretion could contribute to the diarrheal side effects of these agents, and their disruption of epithelial barrier dysfunction is likely also pathophysiologically significant. CCh-activated Erk1/2 phosphorylation was relatively insensitive to EGFr TKIs and delayed the deleterious effects of EGFr TKIs on barrier function. These findings confirm and extend those of other authors, and may be relevant to designing strategies to overcome the diarrheal side effects of EGFr TKIs.

Introduction: GEO- and TCGA-based data analysis suggested the differential expression of miR-29c in pancreatic cancer. However, limited data are available on the downstream mechanistic actions of miR-29c, which may fuel the in vitro and in vivo studies of pancreatic cancer. Methods: The downstream target gene of miR-29c and the downstream ERK/MAPK pathway involved in pancreatic cancer were predicted by bioinformatics tools. Next, the expression of miR-29c and MAPK1 was determined in pancreatic cancer tissues and cells. After ectopic expression and depletion experiments in pancreatic cancer cells, oncogenic phenotypes of pancreatic cancer cells were tested by MTS assay, Transwell assay, and flow cytometry. Effects of miR-29c/MAPK1 on tumorigenic ability in vivo were evaluated in pancreatic cancer xenografts in nude mice. Results: Through differential analysis, five pancreatic cancer-related miRNAs (hsa-miR-29c, hsa-miR-107, hsa-miR-324-3p, hsa-miR-375, and hsa-miR-210) were screened out, among which miR-29c was selected as the key miRNA related to prognosis of pancreatic cancer patients. miR-29c could target and inhibit MAPK1 to suppress the activation of ERK/MAPK pathway. miR-29c was downregulated in pancreatic cancer, and its high expression was related to the good prognosis of pancreatic cancer patients. Both in vitro and in vivo experiments demonstrated that restoration of miR-29c inhibited oncogenic phenotypes of pancreatic cancer cells, as well as repressed tumorigenic ability of pancreatic cancer cells in nude mice. Conclusions: Taken together, we unveil a novel miR-29c/MAPK1/ERK/MAPK axis that suppresses pancreatic cancer both in vitro and in vivo. [ABSTRACT FROM AUTHOR]

Background and aimsMitogen-activated protein kinases (MAPKs) have been reported to respond to various stimuli including heat stress. This research aimed to investigate whether StMAPK1 is implicated in the transduction of the heat stress signal to adapt heat stress as a thermos-tolerant gene.Materials and methodsPotato plants were cultivated under mild (30°C) and acute (35°C) heat stress conditions to analyze mRNA expression of StMAPKs and physiological indicators. StMAPK1 was up-regulated and down-regulated by transfection. Subcellular localization of StMAPK1 protein was observed by fluorescence microscope. The transgenic potato plants were assayed for physiological indexes, photosynthesis, cellular membrane integrity, and heat stress response gene expression.ResultsHeat stress altered the expression prolife of StMAPKs. StMAPK1 overexpression changed the physiological characteristics and phenotypes of potato plants under heat stresses. StMAPK1 mediates photosynthesis and maintains membrane integrity of potato plants in response to heat stress. Stress response genes (StP5CS, StCAT, StSOD, and StPOD) in potato plants were altered by StMAPK1 dysregulation. mRNA expression of heat stress genes (StHSP90, StHSP70, StHSP20, and StHSFA3) was affected by StMAPK1.ConclusionsStMAPK1 overexpression increases the heat-tolerant capacity of potato plants at the morphological, physiological, molecular, and genetic levels.

RationaleGq signaling in cardiac myocytes is classically considered toxic. Targeting Gq directly to test this is problematic, because cardiac myocytes have many Gq-coupled receptors.ObjectiveTest whether Gq coupling is required for the cardioprotective effects of an alpha-1A-AR (adrenergic receptor) agonist.Methods and resultsIn recombinant cells, a mouse alpha-1A-AR with a 6-residue substitution in the third intracellular loop does not couple to Gq signaling. Here we studied a knockin mouse with this alpha-1A-AR mutation. Heart alpha-1A receptor levels and antagonist affinity in the knockin were identical to wild-type. In wild-type cardiac myocytes, the selective alpha-1A agonist A61603-stimulated phosphoinositide-phospholipase C and myocyte contraction. In myocytes with the alpha-1A knockin, both A61603 effects were absent, indicating that Gq coupling was absent. Surprisingly, A61603 activation of cardioprotective ERK (extracellular signal-regulated kinase) was markedly impaired in the KI mutant myocytes, and A61603 did not protect mutant myocytes from doxorubicin toxicity in vitro. Similarly, mice with the α1A KI mutation had increased mortality after transverse aortic constriction, and A61603 did not rescue cardiac function in mice with the Gq coupling-defective alpha-1A receptor.ConclusionsGq coupling is required for cardioprotection by an alpha-1A-AR agonist. Gq signaling can be adaptive.

Radiotherapy (RT) is the major modality for control of glioblastoma multiforme (GBM), the most aggressive brain tumor in adults with poor prognosis and low patient survival rate. To improve the RT efficacy on GBM, the mechanism causing tumor adaptive radioresistance which leads to the failure of tumor control and lethal progression needs to be further elucidated. Here, we conducted a comparative analysis of RT-treated recurrent tumors versus primary counterparts in GBM patients, RT-treated orthotopic GBM tumors xenografts versus untreated tumors and radioresistant GBM cells versus wild type cells. The results reveal that activation of STAT3, a well-defined redox-sensitive transcriptional factor, is causally linked with GBM adaptive radioresistance. Database analysis also agrees with the worse prognosis in GBM patients due to the STAT3 expression-associated low RT responsiveness. However, although the radioresistant GBM cells can be resensitized by inhibition of STAT3, a fraction of radioresistant cells can still survive the RT combined with STAT3 inhibition or CRISPR/Cas9-mediated STAT3 knockout. A complementally enhanced activation of ERK1/2 by STAT3 inhibition is identified responsible for the survival of the remaining resistant tumor cells. Dual inhibition of ERK1/2 and STAT3 remarkably eliminates resistant GBM cells and inhibits tumor regrowth. These findings demonstrate a previously unknown feature ofSTAT3-mediated ERK1/2 regulation and an effective combination of two targets in resensitizing GBM to RT.

Sudden cardiac death (SCD) is the leading global cause of mortality. SCD often arises from cardiac ischemia reperfusion (IR) injury, pathologic sequence variants within ion channel genes, or a combination of the two. Alternative approaches are needed to prevent or ameliorate ventricular arrhythmias linked to SCD. Here, we investigated the efficacy of remote ischemic preconditioning (RIPC) of the limb versus the liver in reducing ventricular arrhythmias in a mouse model of SCD. Mice lacking the Kcne2 gene, which encodes a potassium channel β subunit associated with acquired Long QT syndrome were exposed to IR injury via coronary ligation. This resulted in ventricular arrhythmias in all mice (15/15) and SCD in 5/15 mice during reperfusion. Strikingly, prior RIPC (limb or liver) greatly reduced the incidence and severity of all ventricular arrhythmias and completely prevented SCD. Biochemical and pharmacological analysis demonstrated that RIPC cardioprotection required ERK1/2 and/or AKT phosphorylation. A lack of alteration in GSK-3β phosphorylation suggested against conventional reperfusion injury salvage kinase (RISK) signaling pathway protection. If replicated in human studies, limb RIPC could represent a noninvasive, nonpharmacological approach to limit dangerous ventricular arrhythmias associated with ischemia and/or channelopathy-linked SCD.

Colonocytes possess a specific carrier-mediated uptake process for the microbiota-generated thiamin (vitamin B1) pyrophosphate (TPP) that involves the TPP transporter (TPPT; product of the SLC44A4 gene). Little is known about the effect of exogenous factors (including enteric pathogens) on the colonic TPP uptake process. Our aim in this study was to investigate the effect of Enterohemorrhagic Escherichia coli (EHEC) infection on colonic uptake of TPP. We used human-derived colonic epithelial NCM460 cells and mice in our investigation. The results showed that infecting NCM460 cells with live EHEC (but not with heat-killed EHEC, EHEC culture supernatant, or with non-pathogenic E. Coli) to lead to a significant inhibition in carrier-mediated TPP uptake, as well as in level of expression of the TPPT protein and mRNA. Similarly, infecting mice with EHEC led to a significant inhibition in colonic TPP uptake and in level of expression of TPPT protein and mRNA. The inhibitory effect of EHEC on TPP uptake by NCM460 was found to be associated with reduction in the rate of transcription of the SLC44A4 gene as indicated by the significant reduction in the activity of the SLC44A4 promoter transfected into EHEC infected cells. The latter was also associated with a marked reduction in the level of expression of the transcription factors CREB-1 and ELF3, which are known to drive the activity of the SLC44A4 promoter. Finally, blocking the ERK1/2 and NF-kB signaling pathways in NCM460 cells significantly reversed the level of EHEC inhibition in TPP uptake and TPPT expression. Collectively, these findings show, for the first time, that EHEC infection significantly inhibit colonic uptake of TPP, and that this effect appears to be exerted at the level of SLC44A4 transcription and involves the ERK1/2 and NF-kB signaling pathways.

Ganglioglioma is the most common epilepsy-associated neoplasm that accounts for approximately 2% of all primary brain tumors. While a subset of gangliogliomas are known to harbor the activating p.V600E mutation in the BRAF oncogene, the genetic alterations responsible for the remainder are largely unknown, as is the spectrum of any additional cooperating gene mutations or copy number alterations. We performed targeted next-generation sequencing that provides comprehensive assessment of mutations, gene fusions, and copy number alterations on a cohort of 40 gangliogliomas. Thirty-six harbored mutations predicted to activate the MAP kinase signaling pathway, including 18 with BRAF p.V600E mutation, 5 with variant BRAF mutation (including 4 cases with novel in-frame insertions at p.R506 in the β3-αC loop of the kinase domain), 4 with BRAF fusion, 2 with KRAS mutation, 1 with RAF1 fusion, 1 with biallelic NF1 mutation, and 5 with FGFR1/2 alterations. Three gangliogliomas with BRAF p.V600E mutation had concurrent CDKN2A homozygous deletion and one additionally harbored a subclonal mutation in PTEN. Otherwise, no additional pathogenic mutations, fusions, amplifications, or deletions were identified in any of the other tumors. Amongst the 4 gangliogliomas without canonical MAP kinase pathway alterations identified, one epilepsy-associated tumor in the temporal lobe of a young child was found to harbor a novel ABL2-GAB2 gene fusion. The underlying genetic alterations did not show significant association with patient age or disease progression/recurrence in this cohort. Together, this study highlights that ganglioglioma is characterized by genetic alterations that activate the MAP kinase pathway, with only a small subset of cases that harbor additional pathogenic alterations such as CDKN2A deletion.

Background: Long non-coding RNA (lncRNA) exhibits a crucial role in multiple human malignancies. The expression of lncRNA LINC00511, reportedly, is aberrantly up-regulated in several types of tumors. Our research was aimed at deciphering the role and mechanism of LINC00511 in the progression of cervical cancer (CC). Method: Quantitative real-time polymerase chain reaction (qRT-PCR) was performed to quantify the expression levels of LINC00511, miR-497-5p and MAPK1 mRNA in CC tissues and cell lines. Cell counting kit-8 (CCK-8), 5-bromo-2'-deoxyuridine (BrdU) and Transwell assays were conducted for detecting the proliferation, migration and invasion of CC cells. Dual-luciferase reporter gene experiments were performed to verify the targeting relationships amongst LINC00511, miR-497-5p and MAPK1. Besides, MAPK1 expression in CC cells was detected via Western blot after LINC00511 and miR-497-5p were selectively regulated. Results: Up-regulation of LINC00511 expression in CC tissues and cell lines was observed, which was in association with tumor size, clinical stage and lymph node metastasis of the patients. LINC00511 overexpression facilitated the proliferation, migration and invasion of CC cells, while opposite effects were observed after knockdown of LINC00511. Mechanistically, LINC00511 was capable of targeting miR-497-5p and up-regulating MAPK1 expression. Conclusion: LINC00511/miR-497-5p/MAPK1 axis regulates CC progression. [ABSTRACT FROM AUTHOR]

The goal of this study was to identify the intrinsic links that explain the effect of a Western diet (WD) on cognitive dysfunction. Specific pathogen-free, wild-type mice were fed either a control diet (CD) or a high-fat, high-sucrose WD after weaning and were euthanized at 10 mo of age to study the pathways that affect cognitive health. The results showed that long-term WD intake reduced hippocampal synaptic plasticity and the level of brain-derived neurotrophic factor mRNA in the brain and isolated microglia. A WD also activated ERK1/2 and reduced postsynaptic density-95 in the brain, suggesting postsynaptic damage. Moreover, WD-fed mice had increased inflammatory signaling in the brain, ileum, liver, adipose tissue, and spleen, which was accompanied by microglia activation. In the brain, as well as in the digestive tract, a WD reduced signaling regulated by retinoic acid and bile acids (BAs), whose receptors form heterodimers to control metabolism and inflammation. Furthermore, a WD intake caused dysbiosis and dysregulated BA synthesis with reduced endogenous ligands for BA receptors, i.e., farnesoid X receptor and G-protein-coupled bile acid receptor in the liver and brain. Together, dysregulated BA synthesis and dysbiosis were accompanied by systemic inflammation, microglial activation, and reduced neuroplasticity induced by WD.-Jena, P. K., Sheng, L., Di Lucente, J., Jin, L.-W., Maezawa, I., Wan, Y.-J. Y. Dysregulated bile acid synthesis and dysbiosis are implicated in Western diet-induced systemic inflammation, microglial activation, and reduced neuroplasticity.

Pancreatic ductal adenocarcinoma (PDAC) is a particularly deadly disease. Chronic conditions, including obesity and type-2 diabetes are risk factors, thus making PDAC amenable to preventive strategies. We aimed to characterize the chemo-preventive effects of metformin, a widely used anti-diabetic drug, on PDAC development using the KrasG12D mouse model subjected to a diet high in fats and calories (HFCD). LSL-KrasG12D/+;p48-Cre (KC) mice were given control diet (CD), HFCD, or HFCD with 5 mg/ml metformin in drinking water for 3 or 9 months. After 3 months, metformin prevented HFCD-induced weight gain, hepatic steatosis, depletion of intact acini, formation of advanced PanIN lesions, and stimulation of ERK and mTORC1 in pancreas. In addition to reversing hepatic and pancreatic histopathology, metformin normalized HFCD-induced hyperinsulinemia and hyperleptinemia among the 9-month cohort. Importantly, the HFCD-increased PDAC incidence was completely abrogated by metformin (p

Subtype-selective antagonists for muscarinic acetylcholine receptors (mAChRs) have long been elusive, owing to the highly conserved orthosteric binding site. However, allosteric sites of these receptors are less conserved, motivating the search for allosteric ligands that modulate agonists or antagonists to confer subtype selectivity. Accordingly, a 4.6 million-molecule library was docked against the structure of the prototypical M2 mAChR, seeking molecules that specifically stabilized antagonist binding. This led us to identify a positive allosteric modulator (PAM) that potentiated the antagonist N-methyl scopolamine (NMS). Structure-based optimization led to compound '628, which enhanced binding of NMS, and the drug scopolamine itself, with a cooperativity factor (α) of 5.5 and a KB of 1.1 μM, while sparing the endogenous agonist acetylcholine. NMR spectral changes determined for methionine residues reflected changes in the allosteric network. Moreover, '628 slowed the dissociation rate of NMS from the M2 mAChR by 50-fold, an effect not observed at the other four mAChR subtypes. The specific PAM effect of '628 on NMS antagonism was conserved in functional assays, including agonist stimulation of [35S]GTPγS binding and ERK 1/2 phosphorylation. Importantly, the selective allostery between '628 and NMS was retained in membranes from adult rat hypothalamus and in neonatal rat cardiomyocytes, supporting the physiological relevance of this PAM/antagonist approach. This study supports the feasibility of discovering PAMs that confer subtype selectivity to antagonists; molecules like '628 can convert an armamentarium of potent but nonselective GPCR antagonist drugs into subtype-selective reagents, thus reducing their off-target effects.

BackgroundConstitutive activation of ERK1/2 occurs in various cancers, and its reactivation is a well-described resistance mechanism to MAPK inhibitors. ERK inhibitors may overcome the limitations of MAPK inhibitor blockade. The dual mechanism inhibitor SCH772984 has shown promising preclinical activity across various BRAFV600/RAS-mutant cancer cell lines and human cancer xenografts.MethodsWe have developed an orally bioavailable ERK inhibitor, MK-8353; conducted preclinical studies to demonstrate activity, pharmacodynamic endpoints, dosing, and schedule; completed a study in healthy volunteers (P07652); and subsequently performed a phase I clinical trial in patients with advanced solid tumors (MK-8353-001). In the P07652 study, MK-8353 was administered as a single dose in 10- to 400-mg dose cohorts, whereas in the MK-8353-001 study, MK-8353 was administered in 100- to 800-mg dose cohorts orally twice daily. Safety, tolerability, pharmacokinetics, pharmacodynamics, and antitumor activity were analyzed.ResultsMK-8353 exhibited comparable potency with SCH772984 across various preclinical cancer models. Forty-eight patients were enrolled in the P07652 study, and twenty-six patients were enrolled in the MK-8353-001 study. Adverse events included diarrhea (44%), fatigue (40%), nausea (32%), and rash (28%). Dose-limiting toxicity was observed in the 400-mg and 800-mg dose cohorts. Sufficient exposure to MK-8353 was noted that correlated with biological activity in preclinical data. Three of fifteen patients evaluable for treatment response in the MK-8353-001 study had partial response, all with BRAFV600-mutant melanomas.ConclusionMK-8353 was well tolerated up to 400 mg twice daily and exhibited antitumor activity in patients with BRAFV600-mutant melanoma. However, antitumor activity was not particularly correlated with pharmacodynamic parameters.Trial registrationClinicalTrials.gov NCT01358331.FundingMerck Sharp & Dohme Corp., a subsidiary of Merck & Co. Inc., and NIH (P01 CA168585 and R35 CA197633).

Positive feedback on gonadotropin release requires not only estrogen but also progesterone to activate neural circuits. In rodents, ovarian estradiol (E2) stimulates progesterone synthesis in hypothalamic astrocytes (neuroP), needed for the luteinizing hormone (LH) surge. Kisspeptin (kiss) neurons are the principal stimulators of gonadotropin-releasing hormone neurons, and disruption of kiss signaling abrogates the LH surge. Similarly, blocking steroid synthesis in the hypothalamus or deleting classical progesterone receptor (PGR) selectively in kiss neurons prevents the LH surge. These results suggest a synergistic action of E2 and progesterone in kiss neurons to affect gonadotropin release. The mHypoA51, immortalized kiss-expressing neuronal cell line derived from adult female mice, is a tractable model for examining integration of steroid signaling underlying estrogen positive feedback. Here, we report that kiss neurons in vitro integrate E2 and progesterone signaling to increase levels of kiss translation and release. mHypoA51 neurons expressed nonclassical membrane progesterone receptors (mPRα and mPRβ) and E2-inducible PGR, required for progesterone-augmentation of E2-induced kiss expression. With astrocyte-conditioned media or in mHypoA51-astrocyte co-culture, neuroP augmented stimulatory effects of E2 on kiss protein. Progesterone activation of classical, membrane-localized PGR led to activation of MAPK and Src kinases. Importantly, progesterone or Src activation induced release of kiss from E2-primed mHypoA51 neurons. Consistent with previous studies, the present results provide compelling evidence that the interaction of E2 and progesterone stimulates kiss expression and release. Further, these results demonstrate a mechanism though which peripheral E2 may prime kiss neurons to respond to neuroP, mediating estrogen positive feedback.

Hair follicle stem cells (HFSCs) are responsible for hair growth and hair follicle regeneration. MicroRNAs have been demonstrated to be involved in the differentiation of HFSCs. Thus, this study aimed to explore the potential role of miR-149 in the differentiation of HFSCs. The isolated HFSCs were identified by flow cytometric sorting. miR-149 expression was determined during differentiation of HFSCs. Gain- and loss-of-function approaches were conducted to explore the roles of miR-149, MAPK1/ERK2, and FGF2/c-MYC in colony formation and proliferation of HFSCs. Furthermore, in vivo assays were undertaken in miR-149 knockout mice to confirm their roles in HFSC differentiation. miR-149 was found to be downregulated during HFSC differentiation, and overexpressed miR-149 restricted the proliferation and differentiation of HFSCs. miR-149 was confirmed to target and inhibit MAPK1/ERK2, which was highly expressed in and positively associated with HFSC differentiation. The MAPK1/ERK2 promotion in HFSC differentiation was achieved by augmenting expression of FGF2 and c-MYC. The in vitro effects of miR-149 were validated in in vivo experiments. Taken together, upregulated miR-149 restricted HFSC differentiation and hair growth by targeting MAPK1/ERK2 to reduce expression of FGF2 and c-MYC, which sheds light on the underlying molecular mechanism of hair growth. [ABSTRACT FROM AUTHOR]

Epidemiological studies found an increased risk for kidney cancer in hypertensive patients, of which a subgroup has high aldosterone (Ald) levels. We recently showed that Ald is genotoxic both in kidney tubular cells and in rats with mineralocorticoid-mediated hypertension. The present work investigated in vitro and in vivo, if the oxidative stress-mediated activation of the ERK1/2 pathway, and its downstream target STAT3, could be one mechanism involved in the potential oncogenic capability of excess Ald exposure. The effects of excess Ald were investigated in LLC-PK1 cells and in Ald-induced hypertensive rats. Ald caused cRaf, MEK1/2, and ERK1/2 phosphorylation both in LLC-PK1 cells and in rat kidneys. ERK1/2 activation led to an increased phosphorylation of MSK1, p90RSK, and STAT3. The involvement of ERK1/2 in the activation of STAT3 was evidenced by the capacity of the MEK inhibitor U0126 to prevent Ald-mediated ERK1/2 and STAT3 phosphorylation. Both in vitro and in vivo, the activation of ERK1/2 and STAT3 by Ald was dependent on the mineralocorticoid receptor and was triggered by an increase in cellular oxidants. Ald-mediated oxidant increase was in part due to the activation of the enzymes NADPH oxidase and NO synthase. Proliferation was significantly enhanced and apoptosis decreased in Ald-treated rat kidneys and/or LLC-PK1 cells. Results support the concept that the oxidant-mediated long-term activation of ERK1/2/STAT3 by persistently high Ald levels could trigger proliferative and prosurvival events. Ald-mediated promotion of cell survival and DNA damage could result in kidney cell transformation and initiation of cancer in hypertensive patients with hyperaldosteronism.

Unlike prototypical IκB proteins, which are inhibitors of NF-κB RelA, cRel, and RelB dimers, the atypical IκB protein Bcl3 is primarily a transcriptional coregulator of p52 and p50 homodimers. Bcl3 exists as phospho-protein in many cancer cells. Unphosphorylated Bcl3 acts as a classical IκB-like inhibitor and removes p50 and p52 from bound DNA. Neither the phosphorylation site(s) nor the kinase(s) phosphorylating Bcl3 is known. Here we show that Akt, Erk2, and IKK1/2 phosphorylate Bcl3. Phosphorylation of Ser33 by Akt induces switching of K48 ubiquitination to K63 ubiquitination and thus promotes nuclear localization and stabilization of Bcl3. Phosphorylation by Erk2 and IKK1/2 of Ser114 and Ser446 converts Bcl3 into a transcriptional coregulator by facilitating its recruitment to DNA. Cells expressing the S114A/S446A mutant have cellular proliferation and migration defects. This work links Akt and MAPK pathways to NF-κB through Bcl3 and provides mechanistic insight into how Bcl3 functions as an oncoprotein through collaboration with IKK1/2, Akt, and Erk2.

Mitogen-activated protein kinase (MAPK) scaffold proteins, such as IQ motif containing GTPase activating protein 1 (IQGAP1), are promising targets for novel therapies against cancer and other diseases. Such approaches require accurate information about which domains on the scaffold protein bind to the kinases in the MAPK cascade. Results from previous studies have suggested that the WW domain of IQGAP1 binds to the cancer-associated MAPKs ERK1 and ERK2, and that this domain might thus offer a new tool to selectively inhibit MAPK activation in cancer cells. The goal of this work was therefore to critically evaluate which IQGAP1 domains bind to ERK1/2. Here, using quantitative in vitro binding assays, we show that the IQ domain of IQGAP1 is both necessary and sufficient for binding to ERK1 and ERK2, as well as to the MAPK kinases MEK1 and MEK2. Furthermore, we show that the WW domain is not required for ERK-IQGAP1 binding, and contributes little or no binding energy to this interaction, challenging previous models of how WW-based peptides might inhibit tumorigenesis. Finally, we show that the ERK2-IQGAP1 interaction does not require ERK2 phosphorylation or catalytic activity and does not involve known docking recruitment sites on ERK2, and we obtain an estimate of the dissociation constant (Kd ) for this interaction of 8 μm These results prompt a re-evaluation of published findings and a refined model of IQGAP scaffolding.

Non-specific cytotoxins, including paclitaxel and sirolimus analogues, currently utilized as anti-restenotic therapeutics, affect not only smooth muscle cells (SMCs) but also neighbouring vascular endothelial cells (ECs). These drugs inhibit the formation of an intact endothelium following vessel injury, thus emphasizing the critical need for new candidate therapeutics. Utilizing our in vitro models, including EC monolayers and both hyperplastic and quiescent EC-SMC co-cultures, we investigated the ability of DS-SILY20 , a decorin mimic, to promote EC health. DS-SILY20 increased EC proliferation and migration by 1.5- and 2-fold, respectively, which corresponded to increased phosphorylation of ERK-1/2. Interestingly, IL-6 secretion and the production of both E-selectin and P-selectin were reduced in the presence of 10 μm DS-SILY20 , even in the presence of the potent pro-inflammatory cytokine platelet-derived growth factor (PDGF). In hyperplastic and quiescent EC-SMC co-cultures, DS-SILY20 treatment reduced the secretion of IFNγ, IL-1β, IL-6 and TNFα, corresponding to a 23% decrease in p38 phosphorylation. E-selectin and P-selectin expression was further reduced following DS-SILY20 treatment in both co-culture models. These results indicate that DS-SILY20 promotes EC health and that this decorin mimic could serve as a potential therapeutic to promote vessel healing following percutaneous coronary intervention (PCI). Copyright © 2015 John Wiley & Sons, Ltd.

Extracellular matrix stiffness sensing by living cells is known to play a major role in a variety of cell mechanobiological processes, such as migration and differentiation. Various membrane and cytoplasmic proteins are involved in transmitting and transducing environmental signals to biochemical cascades. Protein kinases play a key role in regulating the activity of focal adhesion proteins. Recently, an interaction between mitogen-activated protein kinase (MAPK1) and vinculin was experimentally shown to mediate this process. Here, we adopt a molecular modeling approach to further investigate this interaction and its possible regulatory effects. Using a combination of data-driven flexible docking and molecular dynamics simulations guided by previous experimental studies, we predict the structure of the MAPK1-vinculin complex. Furthermore, by comparing the association of MAPK1 with open versus closed vinculin, we demonstrate that MAPK1 exhibits preferential binding toward the open conformation of vinculin, suggesting that the MAPK1-vinculin interaction is conformationally selective. Finally, we demonstrate that changes in the size of the D3-D4 cleft provide a structural basis for the conformational selectivity of the interaction.

In the peripheral nervous system, Schwann cells (SCs) demonstrate surveillance activity, detecting injury and undergoing trans-differentiation to support repair. SC receptors that detect peripheral nervous system injury remain incompletely understood. We used RT-PCR to profile ionotropic glutamate receptor expression in cultured SCs. We identified subunits required for assembly of N-methyl-d-aspartic acid (NMDA) receptors (NMDA-Rs), α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors, and kainate receptors. Treatment of SCs with 40-100 µM glutamate or with 0.5-1.0 µM NMDA robustly activated Akt and ERK1/2. The response was transient and bimodal; glutamate concentrations that exceeded 250 µM failed to activate cell signaling. Phosphoprotein profiling identified diverse phosphorylated proteins in glutamate-treated SCs in addition to ERK1/2 and Akt, including p70 S6-kinase, glycogen synthase kinase-3, ribosomal S6 kinase, c-Jun, and cAMP response element binding protein. Activation of SC signaling by glutamate was blocked by EGTA and dizocilpine and by silencing expression of the NMDA-R NR1 subunit. Phosphoinositide 3-kinase/PI3K functioned as an essential upstream activator of Akt and ERK1/2 in glutamate-treated SCs. When glutamate or NMDA was injected directly into crush-injured rat sciatic nerves, ERK1/2 phosphorylation was observed in myelinated and nonmyelinating SCs. Glutamate promoted SC migration by a pathway that required PI3K and ERK1/2. These results identified ionotropic glutamate receptors and NMDA-Rs, specifically, as potentially important cell signaling receptors in SCs.-Campana, W. M., Mantuano, E., Azmoon, P., Henry, K., Banki, M. A., Kim, J. H., Pizzo, D. P., Gonias, S. L. Ionotropic glutamate receptors activate cell signaling in response to glutamate in Schwann cells.

Canine oral mucosal melanoma is an aggressive malignant neoplasm and is characterized by local infiltration and a high metastatic potential. The disease progression is similar to that of human oral melanomas. Whereas human cutaneous melanoma is primarily driven by activating mutations in Braf (60%) or Nras (20%), human mucosal melanoma harbors these mutations much less frequently. This makes therapeutic targeting and research modeling of the oral form potentially different from that of the cutaneous form in humans. Similarly, research has found only rare Nras mutations and no activating Braf mutations in canine oral melanomas, but they are still reliant on MAPK signaling. IQGAP1 is a signaling scaffold that regulates oncogenic ERK1/2 MAPK signaling in human Ras- and Raf- driven cancers, including melanomas. To investigate whether IQGAP1 is a potential target in canine melanoma, we examined the expression and localization of IQGAP1 in primary canine melanomas and canine oral melanoma cell lines obtained from the University of California-Davis. Using CRISPR/Cas9 knockout of IQGAP1, we examined effects on downstream ERK1/2 pathway activity and assayed proliferation of cell lines when treated with a peptide that blocks the interaction between IQGAP1 and ERK1/2. We observed that canine IQGAP1 is expressed and localizes to a similar extent in both human and canine melanoma by qPCR, Western blot, and immunofluorescence. Deletion of IQGAP1 reduces MAPK pathway activation in cell lines, similar to effects seen in human BrafV600E cell lines. Additionally, we demonstrated reduced proliferation when these cells are treated with a blocking peptide in vitro.

BackgroundBrain inflammation is a crucial component of demyelinating diseases such as multiple sclerosis. Although the initiation of inflammatory processes by the production of cytokines and chemokines by immune cells is well characterized, the processes of inflammatory aggravation of demyelinating diseases remain obscure. Here, we examined the contribution of Erk2, one of the isoforms of the extracellular signal-regulated kinase, to demyelinating inflammation.MethodsWe used the cuprizone-induced demyelinating mouse model. To examine the role of Erk2, we used Nestin-cre-driven Erk2-deficient mice. We also established primary culture of microglia or astrocytes in order to reveal the crosstalk between two cell types and to determine the downstream cascades of Erk2 in astrocytes.ResultsFirst, we found that Erk is especially activated in astrocytes within the corpus callosum before the peak of demyelination (at 4 weeks after the start of cuprizone feeding). Then, we found that in our model, genetic ablation of Erk2 from neural cells markedly preserved myelin structure and motor function as measured by the rota-rod test. While the initial activation of microglia was not altered in Erk2-deficient mice, these mice showed reduced expression of inflammatory mediators at 3-4 model weeks. Furthermore, the subsequent inflammatory glial responses, characterized by accumulation of microglia and reactive astrocytes, were significantly attenuated in Erk2-deficient mice. These data indicate that Erk2 in astrocytes is involved in augmentation of inflammation and gliosis. We also found that activated, cultured microglia could induce Erk2 activation in cultured astrocytes and subsequent production of inflammatory mediators such as Ccl-2.ConclusionsOur results suggest that Erk2 activation in astrocytes plays a crucial role in aggravating demyelinating inflammation by inducing inflammatory mediators and gliosis. Thus, therapies targeting Erk2 function in glial cells may be a promising approach to the treatment of distinct demyelinating diseases.

Fibroblast Growth Factors and their receptors (FGFRs) comprise a cell signaling module that can stimulate signaling by Ras and the kinases Raf, MEK, and ERK to regulate animal development and homeostatic functions. In Caenorhabditis elegans, the sole FGFR ortholog EGL-15 acts with the GRB2 ortholog SEM-5 to promote chemoattraction and migration by the sex myoblasts (SMs) and fluid homeostasis by the hypodermis (Hyp7). Cell-specific differences in EGL-15 signaling were suggested by the phenotypes caused by egl-15(n1457), an allele that removes a region of its C-terminal domain (CTD) known to bind SEM-5. To determine how mutations altered EGL-15 activity in the SMs and Hyp7, we used the kinase reporter ERK-KTR to measure activation of the ERK ortholog MPK-1. Consequences of egl-15(n1457) were cell-specific, resulting in loss of MPK-1 activity in the SMs and elevated activity in Hyp7. Previous studies of Hyp7 showed that loss of the CLR-1 phosphatase causes a fluid homeostasis defect termed "Clear" that is suppressed by reduction of EGL-15 signaling, a phenotype termed "Suppressor of Clear" (Soc). To identify mechanisms that permit EGL-15 signaling in Hyp7, we conducted a genetic screen for Soc mutants in the clr-1; egl-15(n1457) genotype. We report the identification of SOC-3, a protein with putative SEM-5-binding motifs and PH and PTB domains similar to DOK and IRS proteins. In combination with the egl-15(n1457) mutation, loss of either soc-3, the GAB1 ortholog soc-1, or the SHP2 ortholog ptp-2, reduced MPK-1 activation. We generated alleles of soc-3 to test the requirement for the SEM-5-binding motifs, finding that residue Tyr 356 is required for function. We propose that EGL-15-mediated SM chemoattraction relies solely on the direct interaction between SEM-5 and the EGL-15 CTD. In Hyp7, EGL-15 signaling uses two mechanisms: the direct SEM-5 binding mechanism; and an alternative, CTD-independent mechanism involving SOC-3, SOC-1, and PTP-2. This work demonstrates that FGF signaling uses distinct, tissue-specific mechanisms in development, and identifies SOC-3 as a potential adaptor that facilitates Ras pathway activation by FGFR., (Copyright © 2024 Elsevier Inc. All rights reserved.)

LDL Receptor-related Protein-1 (LRP1) is an endocytic receptor for diverse ligands. In neurons and neuron-like cells, ligand-binding to LRP1 initiates cell-signaling. Herein, we show that in PC12 and N2a neuron-like cells, LRP1 distributes into lipid rafts and non-raft plasma membrane fractions. When lipid rafts were disrupted, using methyl-β-cyclodextrin or fumonisin B1, activation of Src family kinases and ERK1/2 by the LRP1 ligands, tissue-type plasminogen activator and activated α2-macroglobulin, was blocked. Biological consequences of activated LRP1 signaling, including neurite outgrowth and cell growth, also were blocked. The effects of lipid raft disruption on ERK1/2 activation and neurite outgrowth, in response to LRP1 ligands, were reproduced in experiments with cerebellar granule neurons in primary culture. Because the reagents used to disrupt lipid rafts may have effects on the composition of the plasma membrane outside lipid rafts, we studied the effects of these reagents on LRP1 activities unrelated to cell-signaling. Lipid raft disruption did not affect the total ligand binding capacity of LRP1, the affinity of LRP1 for its ligands, or its endocytic activity. These results demonstrate that well described activities of LRP1 require localization of this receptor to distinct plasma membrane microdomains.

UnlabelledBipolar disorder (BD) is characterized by depression, mania, and circadian rhythm abnormalities. Lithium, a treatment for BD stabilizes mood and increases circadian rhythm amplitude. However, in fibroblasts grown from BD patients, lithium has weak effects on rhythm amplitude compared to healthy controls. To understand the mechanism by which lithium differentially affects rhythm amplitude in BD cells, we investigated the extracellular-signal-regulated kinase (ERK) and related signaling molecules linked to BD and circadian rhythms. In fibroblasts from BD patients, controls and mice, we assessed the contribution of the ERK pathway to lithium-induced circadian rhythm amplification. Protein analyses revealed low phospho-ERK1/2 (p-ERK) content in fibroblasts from BD patients vs.ControlsPharmacological inhibition of ERK1/2 by PD98059 attenuated the rhythm amplification effect of lithium, while inhibition of two related kinases, c-Jun N-terminal kinase (JNK), and P38 did not. Knockdown of the transcription factors CREB and EGR-1, downstream effectors of ERK1/2, reduced baseline rhythm amplitude, but did not alter rhythm amplification by lithium. In contrast, ELK-1 knockdown amplified rhythms, an effect that was not increased further by the addition of lithium, suggesting this transcription factor may regulate the effect of lithium on amplitude. Augmentation of ERK1/2 signaling through DUSP6 knockdown sensitized NIH3T3 cells to rhythm amplification by lithium. In BD fibroblasts, DUSP6 knockdown reversed the BD rhythm phenotype, restoring the ability of lithium to increase amplitude in these cells. We conclude that the inability of lithium to regulate circadian rhythms in BD may reflect reduced ERK activity, and signaling through ELK-1.

Understanding of the biology of oral squamous cell carcinoma (SCC) has not progressed significantly in the past 60 years, with 5-year survival remaining at approximately 50%. The epidemic of Human Papilloma Virus and its associated SCC warrants a renewed emphasis on fully understanding this disease. We previously used the 3-dimensional multicellular spheroid (MCS) model system to evaluate SCC behavior more accurately. In this study, we determined that SCC growth in MCS approximates epithelial to mesenchymal transition. Organization of an MCS requires the full-length β6 integrin subunit and its maintenance requires mitogen-activated protein kinase (MAPK). Limiting FYN kinase activation results in the down-regulation of E-cadherin, β-catenin and an increase in expression of N-cadherin and SNAIL. These results indicate that the microenvironment and growth patterns in an MCS are complex and require MAPK and FYN kinase.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have not been effective in unselected head and neck squamous cell carcinoma (HNSCC) populations. We previously reported an exceptional response to a brief course of erlotinib in a patient with advanced HNSCC whose tumor harbored a MAPK1E322K somatic mutation. MAPK1E322Kwas associated with increased p-EGFR, increased EGFR downstream signaling and increased sensitivity to erlotinib. In this study, we investigated the mechanism of MAPK1E322K-mediated EGFR activation in the context of erlotinib sensitivity. We demonstrated increased AREG secretion in HNSCC cell lines harboring endogenous or exogenous MAPK1E322K compared to wild type MAPK1. We found inhibition or knockdown of MAPK1 with siRNA resulted in reduced secretion of AREG and decreased sensitivity to erlotinib in the setting of MAPK1E322K. MAPK1E322K was associated with increased AREG secretion leading to an autocrine feedback loop involving AREG, EGFR and downstream signaling. Knockdown of AREG in HNSCC cells harboring MAPK1E322K abrogated EGFR signaling and decreased sensitivity to erlotinib in vitro and in vivo. These cumulative findings implicate increased AREG secretion and EGFR activation as contributing to increased erlotinib sensitivity in MAPK1E322K HNSCC.

Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) have not been effective in unselected head and neck squamous cell carcinoma (HNSCC) populations. We previously reported an exceptional response to a brief course of erlotinib in a patient with advanced HNSCC whose tumor harbored a MAPK1E322K somatic mutation. MAPK1E322Kwas associated with increased p-EGFR, increased EGFR downstream signaling and increased sensitivity to erlotinib. In this study, we investigated the mechanism of MAPK1E322K-mediated EGFR activation in the context of erlotinib sensitivity. We demonstrated increased AREG secretion in HNSCC cell lines harboring endogenous or exogenous MAPK1E322K compared to wild type MAPK1. We found inhibition or knockdown of MAPK1 with siRNA resulted in reduced secretion of AREG and decreased sensitivity to erlotinib in the setting of MAPK1E322K. MAPK1E322K was associated with increased AREG secretion leading to an autocrine feedback loop involving AREG, EGFR and downstream signaling. Knockdown of AREG in HNSCC cells harboring MAPK1E322K abrogated EGFR signaling and decreased sensitivity to erlotinib in vitro and in vivo. These cumulative findings implicate increased AREG secretion and EGFR activation as contributing to increased erlotinib sensitivity in MAPK1E322K HNSCC.

Arrestins are cytosolic proteins that regulate G-protein-coupled receptor (GPCR) desensitization, internalization, trafficking and signalling. Arrestin recruitment uncouples GPCRs from heterotrimeric G proteins, and targets the proteins for internalization via clathrin-coated pits. Arrestins also function as ligand-regulated scaffolds that recruit multiple non-G-protein effectors into GPCR-based 'signalsomes'. Although the dominant function(s) of arrestins vary between receptors, the mechanism whereby different GPCRs specify these divergent functions is unclear. Using a panel of intramolecular fluorescein arsenical hairpin (FlAsH) bioluminescence resonance energy transfer (BRET) reporters to monitor conformational changes in β-arrestin2, here we show that GPCRs impose distinctive arrestin 'conformational signatures' that reflect the stability of the receptor-arrestin complex and role of β-arrestin2 in activating or dampening downstream signalling events. The predictive value of these signatures extends to structurally distinct ligands activating the same GPCR, such that the innate properties of the ligand are reflected as changes in β-arrestin2 conformation. Our findings demonstrate that information about ligand-receptor conformation is encoded within the population average β-arrestin2 conformation, and provide insight into how different GPCRs can use a common effector for different purposes. This approach may have application in the characterization and development of functionally selective GPCR ligands and in identifying factors that dictate arrestin conformation and function.

Serum amyloid A (SAA) is an evolutionary highly conserved acute phase protein that is predominantly secreted by hepatocytes. However, its role in liver injury and fibrogenesis has not been elucidated so far. In this study, we determined the effects of SAA on hepatic stellate cells (HSCs), the main fibrogenic cell type of the liver. Serum amyloid A potently activated IκB kinase, c-Jun N-terminal kinase (JNK), Erk and Akt and enhanced NF-κB-dependent luciferase activity in primary human and rat HSCs. Serum amyloid A induced the transcription of MCP-1, RANTES and MMP9 in an NF-κB- and JNK-dependent manner. Blockade of NF-κB revealed cytotoxic effects of SAA in primary HSCs with signs of apoptosis such as caspase 3 and PARP cleavage and Annexin V staining. Serum amyloid A induced HSC proliferation, which depended on JNK, Erk and Akt activity. In primary hepatocytes, SAA also activated MAP kinases, but did not induce relevant cell death after NF-κB inhibition. In two models of hepatic fibrogenesis, CCl4 treatment and bile duct ligation, hepatic mRNA levels of SAA1 and SAA3 were strongly increased. In conclusion, SAA may modulate fibrogenic responses in the liver in a positive and negative fashion by inducing inflammation, proliferation and cell death in HSCs.

Abstract Background Dysfunction of the DNA methylation was associated with stem cell reprogramming. Moreover, DNA methyltransferase 1 (DNMT1) deficiency was involved in the differentiation of hair follicle stem cell (HFSc), but the molecular mechanisms remain unknown. Methods HFSc from human scalp tissues were isolated and cultured. The oil red O staining was used to observe the adipogenesis. The interaction relationship between microRNA (miR)-214-3p and mitogen-activated protein kinase 1 (MAPK1) was accessed by dual-luciferase reporter gene assay. The methylation level of miR-214-3p promoter was detected by methylation-specific PCR and the enrichment of DNMT1 in miR-214-3p promoter by chromatin immunoprecipitation assay. A mouse model of trauma was established to observe the skin regeneration at 0, 6, and 14 days. Results Expression of DNMT1 and MAPK1 was increased in the HFSc, while the expression of miR-214-3p was reduced. Moreover, DNMT1 inhibited the expression of miR-214-3p by promoting the promoter methylation of miR-214-3p. Overexpression of DNMT1 could reduce the expression of miR-214-3p, but increase the expression of MAPK1 and the extent of extracellular signal regulated kinase (ERK)1/2 phosphorylation, leading to enhanced adipogenic differentiation. Importantly, DNMT1 promoted skin regeneration in vivo. Conversely, overexpression of miR-214-3p could reverse the effects of DNMT1 on adipogenesis of HFSc. Conclusion DNMT1 promotes adipogenesis of HFSc by mediating miR-214-3p/MAPK1/p-ERK1/2 axis. This study may provide novel biomarkers for the potential application in stem cell therapy.

Background: Ginsenoside Rg 1 (Rg 1 ) is one of the main active components in Chinese medicines, Panax ginseng and Panax notoginseng . Research has shown that Rg 1 has a protective effect on the cardiovascular system, including anti-myocardial ischemia-reperfusion injury, anti-apoptosis, and promotion of myocardial angiogenesis, suggesting it a potential cardiovascular agent. However, the protective mechanism involved is still not fully understood., Methods: Based on network pharmacology, ligand-based protein docking, proteomics, Western blot, protein recombination and spectroscopic analysis (UV-Vis and fluorescence spectra) techniques, potential targets and pathways for Rg 1 against myocardial ischemia (MI) were screened and explored., Results: An important target set containing 19 proteins was constructed. Two target proteins with more favorable binding activity for Rg 1 against MI were further identified by molecular docking, including mitogen-activated protein kinase 1 (MAPK1) and adenosine kinase (ADK). Meanwhile, Rg 1 intervention on H9c2 cells injured by H 2 O 2 showed an inhibitory oxidative phosphorylation (OXPHOS) pathway. The inhibition of Rg 1 on MAPK1 and OXPHOS pathway was confirmed by Western blot assay. By protein recombination and spectroscopic analysis, the binding reaction between ADK and Rg 1 was also evaluated., Conclusion: Rg 1 can effectively alleviate cardiomyocytes oxidative stress injury via targeting MAPK1 and ADK, and inhibiting oxidative phosphorylation (OXPHOS) pathway. The present study provides scientific basis for the clinical application of the natural active ingredient, Rg 1 , and also gives rise to a methodological reference to the searching of action targets and pathways of other natural active ingredients., Competing Interests: The authors declare no conflict of interest., (© 2024 The Korean Society of Ginseng. Publishing services by Elsevier B.V.)

Background: The activation of G protein-coupled receptors (GPCR) signaling by external stimuli has been implicated in inducing cardiac stress and stress responses. GPR22 is an orphan GPCR expressed in brains and hearts, while its expression level is associated with cardiovascular damage in diabetes. Previous studies have suggested a protective role of GPR22 in mechanical cardiac stress, as loss of its expression increases susceptibility to heart failure post-ventricular pressure overload. However, the involvement and underlying signaling of GPR22 in cardiac stress response to ischemic stress remains unexplored., Methods: In this study, we used cultured cells and a transgenic mouse model with cardiomyocyte-specific GPR22 overexpression to investigate the impact of ischemic stress on GPR22 expression and to elucidate its role in myocardial ischemic injury. Acute myocardial infarction (AMI) was induced by left coronary artery ligation in eight-week-old male GPR22 transgenic mice, followed by histopathological and biochemical examination four weeks post-AMI induction., Results: GPR22 expression in H9C2 and RL-14 cells, two cardiomyocyte cell lines, was decreased by cobalt chloride (CoCl 2 ) treatment. Similarly, reduced expression of myocardial GPR22 was observed in mice with AMI. Histopathological examinations revealed a protective effect of GPR22 overexpression in attenuating myocardial infarction in mice with AMI. Furthermore, myocardial levels of Bcl-2 and activation of PI3K-Akt signaling were downregulated by ischemic stress and upregulated by GPR22 overexpression. Conversely, the expression levels of caspase-3 and phosphorylated ERK1/2 in the infarcted myocardium were downregulated with GPR22 overexpression., Conclusion: Myocardial ischemic stress downregulates cardiac expression of GPR22, whereas overexpression of GPR22 in cardiomyocytes upregulates Akt signaling, downregulates ERK activation, and mitigates ischemia-induced myocardial injury., (© 2024. The Author(s).)

MAPKs bind to many of their upstream regulators and downstream substrates via a short docking motif (the D-site) on their binding partner. MAPKs that are in different families (e.g. ERK, JNK, and p38) can bind selectively to D-sites in their authentic substrates and regulators while discriminating against D-sites in other pathways. Here we demonstrate that the short hydrophobic region at the distal end of the D-site plays a critical role in determining the high selectivity of JNK MAPKs for docking sites in their cognate MAPK kinases. Changing just 1 or 2 key hydrophobic residues in this submotif is sufficient to turn a weak JNK-binding D-site into a strong one, or vice versa. These specificity-determining differences are also found in the D-sites of the ETS family transcription factors Elk-1 and Net. Moreover, swapping two hydrophobic residues between these D-sites switches the relative efficiency of Elk-1 and Net as substrates for ERK versus JNK, as predicted. These results provide new insights into docking specificity and suggest that this specificity can evolve rapidly by changes to just 1 or 2 amino acids.

BackgroundRELM-β has been implicated in airways inflammation and remodelling in murine models. Its possible functions in human airways are largely unknown. The aim was to address the hypothesis that RELM-β plays a role in extracellular matrix deposition in asthmatic airways.MethodsThe effects of RELM-β gene deficiency were studied in a model of allergen exposure in mice sensitised and challenged with Aspergillus fumigatus (Af). RELM-β expression was investigated in bronchial biopsies from asthmatic patients. Direct regulatory effects of RELM-β on human lung fibroblasts were examined using primary cultures and the MRC5 cell line in vitro.ResultsSensitisation and challenge of wild-type mice with Af-induced release of RELM-β with a time course coincident with that of procollagen in the airways. Af-induced expression of mRNA encoding some, but not all ECM in the lung parenchyma was attenuated in RELM-β-/- mice. RELM-β expression was significantly increased in the bronchial submucosa of human asthmatics compared with controls, and its expression correlated positively with that of fibronectin and α-smooth muscle actin. In addition to epithelial cells, macrophages, fibroblasts and vascular endothelial cells formed the majority of cells expressing RELM-β in the submucosa. Exposure to RELM-β increased TGF-β1, TGF-β2, collagen I, fibronectin, smooth muscle α-actin, laminin α1, and hyaluronan and proteoglycan link protein 1 (Hapl1) production as well as proliferation by human lung fibroblasts in vitro. These changes were associated with activation of ERK1/2 in MRC5 cells.ConclusionThe data are consistent with the hypothesis that elevated RELM-β expression in asthmatic airways contributes to airways remodelling at least partly by increasing fibroblast proliferation and differentiation with resulting deposition of extracellular matrix proteins.

ImportanceRandomized clinical trials demonstrate no benefit for epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors in unselected patients with head and neck squamous cell carcinoma (HNSCC). However, a patient with stage IVA HNSCC received 13 days of neoadjuvant erlotinib and experienced a near-complete histologic response.ObjectiveTo determine a mechanism of exceptional response to erlotinib therapy in HNSCC.Design, setting, and participantsSingle patient with locally advanced HNSCC who received erlotinib monotherapy in a window-of-opportunity clinical trial (patients scheduled to undergo primary cancer surgery are treated briefly with an investigational agent). Whole-exome sequencing of pretreatment tumor and germline patient samples was performed at a quaternary care academic medical center, and a candidate somatic variant was experimentally investigated for mediating erlotinib response.InterventionA brief course of erlotinib monotherapy followed by surgical resection.Main outcomes and measuresIdentification of pretreatment tumor somatic alterations that may contribute to the exceptional response to erlotinib. Hypotheses were formulated regarding enhanced erlotinib response in preclinical models harboring the patient tumor somatic variant MAPK1 E322K following the identification of tumor somatic variants.ResultsNo EGFR alterations were observed in the pretreatment tumor DNA. Paradoxically, the tumor harbored an activating MAPK1 E322K mutation (allelic fraction 0.13), which predicts ERK activation and erlotinib resistance in EGFR-mutant lung cancer. The HNSCC cells with MAPK1 E322K exhibited enhanced EGFR phosphorylation and erlotinib sensitivity compared with wild-type MAPK1 cells.Conclusions and relevanceSelective erlotinib use in HNSCC may be informed by precision oncology approaches.

PurposeAcquired resistance to erlotinib in patients with EGFR-mutant non-small cell lung cancer can result from aberrant activation of alternative receptor tyrosine kinases, such as the HGF-driven c-MET receptor. We sought to determine whether inhibition of AKT signaling could augment erlotinib activity and abrogate HGF-mediated resistance.MethodsThe effects of MK-2206, a selective AKT inhibitor, were evaluated in combination with erlotinib on a large panel of 13 lung cancer cell lines containing different EGFR or KRAS abnormalities. The activity of the combination was assessed using proliferation assays, flow cytometry and immunoblotting. The MEK inhibitor PD0325901 was used to determine the role of the MAP kinase pathway in erlotinib resistance.ResultsThe combination of MK-2206 and erlotinib resulted in synergistic growth inhibition independent of EGFR mutation status. In cell lines where HGF blocked the anti-proliferative and cytotoxic effects of erlotinib, MK-2206 could restore cell cycle arrest, but MEK inhibition was required for erlotinib-dependent apoptosis. Both AKT and MEK inhibition contributed to cell death independent of erlotinib in the T790M-containing H1975 and the EGFR-WT cell lines tested.ConclusionsThese findings illustrate the potential advantages and challenges of combined signal transduction inhibition as a generalized strategy to circumvent acquired erlotinib resistance.