Extracellular Signal-regulated Kinase Mitogen-activated Protein Research Articles

Methoxylated Cinnamic Esters with Antiproliferative and Antimetastatic Effects on Human Lung Adenocarcinoma Cells.

Lung cancer is the leading cause of cancer mortality worldwide, and malignant melanomas are highly lethal owing to their elevated metastatic potential. Despite improvements in therapeutic approaches, cancer treatments are not completely effective. Thus, new drug candidates are continuously sought. We synthesized mono- and di-methoxylated cinnamic acid esters and investigated their antitumor potential. A cell viability assay was performed to identify promising substances against A549 (non-small-cell lung cancer) and SK-MEL-147 (melanoma) cells. (E)-2,5-dimethoxybenzyl 3-(4-methoxyphenyl)acrylate (4m), a monomethoxylated cinnamic acid derivative, was identified as the lead antitumor compound, and its antitumor potential was deeply investigated. Various approaches were employed to investigate the antiproliferative (clonogenic assay and cell cycle analysis), proapoptotic (annexin V assay), and antimigratory (wound-healing and adhesion assays) activities of 4m on A549 cells. In addition, western blotting was performed to explore its mechanism of action. We demonstrated that 4m inhibits the proliferation of A549 by promoting cyclin B downregulation and cell cycle arrest at G2/M. Antimigratory and proapoptotic activities of 4m on A549 were also observed. The antitumor potential of 4m involved its ability to modulate the mitogen-activated protein kinases/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway once phosphorylated-ERK expression was considerably reduced in response to treatment. Our findings demonstrate that 4m is a promising anticancer drug candidate.

Ovalbumin’s potential as a wound-healing medicament in tooth extraction socket by induction of cell proliferation through the ERK2 pathway in silico

Background: The trend of studies on dental medicaments is increasing rapidly. Antibacterial or anti-inflammatory activity is most frequently studied. Ovalbumin is one of the proteins whose benefits have been studied, but these benefits are still limited because of ovalbumin’s potential for proliferative bioactivity. Purpose: The aim of this study is to examine ovalbumin’s potential as a woundhealing medicament through molecular docking analysis on a protein related to the extracellular signal-regulated kinases/mitogenactivated protein kinase (ERK/MAPK) signaling pathway. Methods: Ovalbumin was hydrolyzed through BIOPEP-UWM (The BIOPEPUWM™ database of bioactive peptides). Protein target and interaction were predicted using Similarity Ensemble Approach target prediction webserver, SuperPred webserver, STRING webserver, and Cytoscape version 3.9.1. Selected fragments were docked using Autodock Vina in PyRx 0.8 with Tukey’s multiple comparison test and Biovia Discovery Studio version 19.1.0.18287 for visualization. Results: This study found that ovalbumin has the potential to positively regulate cell proliferation, angiogenesis, and fibroblast growth factor production. Six of the 131 fragments of ovalbumin could interact with 73 proteins, and the 20 proteins with the highest probability and score of betweenness centrality showed potential for bioactivity. Five fragments and povidone-iodine interacted inside the Adenosine triphosphate (ATP) phosphorylation site of ERK2, whereas fragment 1 (F1) and glycerin interacted outside the site. F1 could decrease the binding energy required for adenosine 5′-[,-methylene]triphosphate or an ATP-analogue chemical compound to interact with ERK2 compared to the control, with a score that was not significant. Conclusion: Ovalbumin has the potential to induce cell proliferation by affecting ERK2-ligand interactions.

A phase 2 basket trial of ulixertinib (BVD-523) in combination with hydroxychloroquine in patients with advanced gastrointestinal malignancies harboring MAPK pathway mutations (BVD-523-HCQ).

TPS4174 Background: RAS-induced signaling through the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway plays an important role in the pathogenesis of many solid tumors. Ulixertinib (BVD-523) is a first-in-class and best-in-class small molecule inhibitor of ERK 1/2 that is currently being investigated in multiple cancer clinical trials, both as a single agent and as part of combination therapy. Preclinical studies have demonstrated that inhibition of the RAS/MAPK/ERK signaling cascade leads to upregulation of autophagy, a catabolic pathway that includes the lysosomal degradation of proteins to support cellular metabolism during times of cellular stress. Given cancer cells’ increased reliance on autophagy to enhance their survival in this context, this study seeks to evaluate the combination of ulixertinib with hydroxychloroquine, an antimalarial drug known to suppress autophagy, in patients with advanced gastrointestinal malignancies harboring mutations in genes involved in MAPK signaling. Methods: This is an open-label, multicenter, phase II basket study of ulixertinib in combination with hydroxychloroquine in patients with advanced GI malignancies harboring mutations in one of the following MAPK signaling-associated genes: KRAS, NRAS, HRAS, BRAF (non-V600), MEK1/2, or ERK1/2. The completed Phase 1 dose-escalation trial (NCT04145297) established the recommended Phase 2 dose which will be the doses used in this study; ulixertinib 450mg PO BID combined with hydroxychloroquine 600mg PO BID, both drugs administered daily in 28-day cycles, with tumor assessments after every 2 cycles. The trial has five baskets based on primary disease: cholangiocarcinoma, pancreatic adenocarcinoma, colorectal adenocarcinoma, esophageal adenocarcinoma, and gastric adenocarcinoma. A Simon two-stage design will be employed within each of the 5 disease-specific baskets noted above, with the first stage of enrollment consisting of n=13 patients/ basket. To be considered evaluable, a patient must complete at least one cycle of therapy and receive at least 75% of the prescribed dose during that first cycle. If ≥ 4 patients in a specific basket achieve a response (complete or partial), that basket will then open to Stage 2 with enrollment of an additional 30 patients. Primary study endpoints will include safety/toxicity as well as ORR by RECIST 1.1. The secondary endpoint for efficacy is progression-free survival. Exploratory endpoints include pharmacokinetics of both ulixertinib and hydroxychloroquine, as well as predictive biomarkers from blood and tumor tissue-based samples. At the time of abstract submission, 26 of the planned 65 patients in Stage 1 had been enrolled. Clinical trial information: NCT05221320 .

Pro-neurogenic effects of Lilii Bulbus on hippocampal neurogenesis and memory

Lilii Bulbus, the bulb of tiger lily, has anti-oxidant and anti-tumorigenic properties. However, the effects of Lilii Bulbus on learning, memory, and hippocampal neurogenesis remain unknown. This study investigated whether water extract of Lilii Bulbus (WELB) affects memory ability and hippocampal neurogenesis. Behavioral analyses (Morris water maze and passive avoidance test), immunohistochemistry, cell proliferation assay, and immunoblot analysis were performed. WELB (50 and 100 mg/kg; for 14 days) enhanced memory retention and spatial memory in normal mice as well as in scopolamine-treated mice with memory deficits. Furthermore, the administration of WELB significantly increased the number of proliferating cells and surviving newborn cells in the dentate gyrus of the hippocampus in normal mice. We found that WELB has a pro-neurogenic effect by increasing the activation of brain-derived neurotrophic factor (BDNF)/cAMP response element-binding protein (CREB) and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK) in the hippocampus. Moreover, we confirmed that WELB (100 and 200 μg/ml) significantly increased NE-4 C and primary embryonic NSCs proliferation. Inhibition/knockdown of MEK/ERK blocked WELB-induced MEK/ERK phosphorylation and NSCs proliferation. Hence, MEK/ERK activation was required in WELB-induced NSCs proliferation. Our study demonstrates the first evidence for WELB promoting hippocampal neurogenesis and memory; pro-neurogenic activity may enhance brain plasticity, with implications for treating neurodegenerative diseases.

Spinal alarmin HMGB1 and the activation of TLR4 lead to chronic stress-induced nociceptive hypersensitivity in rodents

Chronic stress affects millions of people around the world, and it can trigger different behavioral disorders like nociceptive hypersensitivity and anxiety, among others. However, the mechanisms underlaying these chronic stress-induced behavioral disorders have not been yet elucidated. This study was designed to understand the role of high-mobility group box-1 (HMGB1) and toll-like receptor 4 (TLR4) in chronic stress-induced nociceptive hypersensitivity. Chronic restraint stress induced bilateral tactile allodynia, anxiety-like behaviors, phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38MAPK) and activation of spinal microglia. Moreover, chronic stress enhanced HMGB1 and TLR4 protein expression at the dorsal root ganglion, but not at the spinal cord. Intrathecal injection of HMGB1 or TLR4 antagonists reduced tactile allodynia and anxiety-like behaviors induced by chronic stress. Additionally, deletion of TLR4 diminished the establishment of chronic stress-induced tactile allodynia in male and female mice. Lastly, the antiallodynic effect of HMGB1 and TLR4 antagonists were similar in stressed male and female rats and mice. Our results suggest that chronic restraint stress induces nociceptive hypersensitivity, anxiety-like behaviors, and up-regulation of spinal HMGB1 and TLR4 expression. Blockade of HMGB1 and TLR4 reverses chronic restraint stress-induced nociceptive hypersensitivity and anxiety-like behaviors and restores altered HMGB1 and TLR4 expression. The antiallodynic effects of HMGB1 and TLR4 blockers in this model are sex independent. TLR4 could be a potential pharmacological target for the treatment of the nociceptive hypersensitivity associated with widespread chronic pain.

Antiviral activity of the human endogenous retrovirus‐R envelope protein against SARS‐CoV‐2

AbstractCoronavirus‐induced disease‐19 (COVID‐19), caused by SARS‐CoV‐2, is still a major global health challenge. Human endogenous retroviruses (HERVs) represent retroviral elements that were integrated into the ancestral human genome. HERVs are important in embryonic development as well as in the manifestation of diseases, including cancer, inflammation, and viral infections. Here, we analyze the expression of several HERVs in SARS‐CoV‐2‐infected cells and observe increased activity of HERV‐E, HERV‐V, HERV‐FRD, HERV‐MER34, HERV‐W, and HERV‐K‐HML2. In contrast, the HERV‐R envelope is downregulated in cell‐based models and PBMCs of COVID‐19 patients. Overexpression of HERV‐R inhibits SARS‐CoV‐2 replication, suggesting its antiviral activity. Further analyses demonstrate the role of the extracellular signal‐regulated kinase (ERK) in regulating HERV‐R antiviral activity. Lastly, our data indicate that the crosstalk between ERK and p38 MAPK controls the synthesis of the HERV‐R envelope protein, which in turn modulates SARS‐CoV‐2 replication. These findings suggest the role of the HERV‐R envelope as a prosurvival host factor against SARS‐CoV‐2 and illustrate a possible advantage of integration and evolutionary maintenance of retroviral elements in the human genome.

Anti-Atopic Effect of Isatidis Folium Water Extract in TNF-α/IFN-γ-Induced HaCaT Cells and DNCB-Induced Atopic Dermatitis Mouse Model.

Isatidis folium or Isatis tinctoria L. is a flowering plant of the Brassicaceae family, commonly known as woad, with an ancient and well-documented history as an indigo dye and medicinal plant. This study aimed to evaluate the anti-atopic dermatitis (AD) effects of Isatidis folium water extract (WIF) using a 2,4-dinitrochlorobenzene (DNCB)-induced AD-like mouse model and to investigate the underlying mechanism using tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ)-activated HaCaT cells. Oral administration of WIF reduced spleen weight, decreased serum IgE and TNF-α levels, reduced epidermal and dermal thickness, and inhibited eosinophil and mast cell recruitment to the dermis compared to DNCB-induced control groups. Furthermore, oral WIF administration suppressed extracellular signal-regulated kinase and p38 mitogen-activated protein kinase protein expression levels, p65 translocation from the cytoplasm to the nucleus, and mRNA expression of TNF-α, IFN-γ, interleukin (IL)-6, and IL-13 in skin lesion tissues. In HaCaT cells, WIF suppressed the production of regulated upon activation, normal T cell expressed and secreted (RANTES), thymus and activation-regulated chemokine (TARC), macrophage-derived chemokine (MDC), MCP-1, and MIP-3a, which are inflammatory cytokines and chemokines related to AD, and inhibited the mRNA expression of RANTES, TARC, and MDC in TNF-α/IFN-γ-stimulated HaCaT cells. Overall, the results revealed that WIF ameliorated AD-like skin inflammation by suppressing proinflammatory cytokine and chemokine production via nuclear factor-κB pathway inhibition, suggesting WIF as a potential candidate for AD treatment.

Chronic binge alcohol-induced hippocampal plasticity in SIV-infected female rhesus macaques

Human immunodeficiency virus (HIV) infection is detrimental to central nervous system (CNS) function and may result in HIV-associated neurocognitive disorder (HAND) over time. HIV-associated cognitive deficits are further exacerbated by chronic alcohol use. With the rising prevalence of alcohol use disorder (AUD) in females and people living with HIV (PLWH), understanding the neurobiological impact of chronic alcohol exposure in these populations is increasingly important. As the hippocampus plays a prominent role in cognition, examining neuroadaptations in this brain region in the context of comorbid HIV infection and alcohol exposure is critical for understanding the mechanisms of neurocognitive impairment. We hypothesized that simian immunodeficiency virus (SIV)-infected female rhesus macaques with a history of chronic binge alcohol (CBA) administration (n=7-8 per group) would exhibit increased phosphorylation of mitogen-activated protein kinase (MAPK) family members, indicative of increased hippocampal activity and potential hyperexcitability. We also measured cognitive performance in macaques via the novel object recognition (NOR) task and further hypothesized a manifestation of cognitive deficits in the CBA-treated group. Finally, we incorporated additional groups of ovariectomized (OVX) females in the design as we hypothesized that the loss of ovarian hormones would reduce cognitive performance. Data were analyzed via two-way ANOVA with CBA treatment and OVX as factors. Western blot analyses demonstrated that CBA significantly increased phosphorylation of MAPK-family members extracellular signal-regulated kinase (ERK; F=8.278, p=0.0093) and p38 mitogen-activated protein kinase (p38 MAPK; F=4.414, p=0.0485). Furthermore, CBA significantly increased both phosphorylated and total levels of Jun N-terminal kinase (JNK; F=5.989, p=0.0233; F=7.158, p=0.0145). While analysis of the NOR task revealed a significant reduction in latency to approach the novel versus familiar object on test day across all groups (F = 11.97, p=0.0013), no significant difference was seen between CBA and control groups. Interestingly, no main effects of OVX or interactive CBA/OVX effects were observed for either molecular or behavioral outcomes. We are currently considering alternative cognitive tasks that may better reveal CBA-associated cognitive deficits, as well as neuroadaptations in nuclear signaling downstream of MAPK activity. Together, these data suggest that chronic alcohol use results in profound changes in hippocampal plasticity, potentially facilitating cognitive decline and other CNS symptoms observed in PLWH. This research was supported by grant # T32AA007577 and #P60AA009803 from the NIAAA at the National Institutes of Health. This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Reciprocity between IL-4 and opioid immune regulation: IL-4 primes human CD4+ T cells for opioid receptor driven MAP kinase signaling

Abstract Opioid receptor (OR) signaling induces transcriptomic and functional changes in human T cell responses at both a molecular and cellular level, yet the signaling pathways initiated by OR engagement in human T cells are poorly characterized. ORs are G-protein coupled receptors with three subtypes: MuOR, KappaOR, and DeltaOR. Using the human lymphoma Jurkat T cell line and negatively selected human peripheral blood CD4 +T cells, the effects of opioid exposure on the activation kinetics of the MAP kinases ERK 1/2, JNK, and p38, scored as their level of phosphorylation, was followed by immunoblot. Opioid-driven phosphorylation of the MAPKs was dependent on IL-4 pretreatment of Jurkat cells, but not blood-derived CD4+ T cells. After Jurkat cells were primed with a 3-day exposure to IL-4, a series of opioid agonists selective for each OR subtype induced the phosphorylation of all three MAPKs (ERK, JNK, p38) within 2 min. Phosphorylation of the MAPKs was independent of OR subtype and opioid agonist. Notably, the kinetics were different than that observed in neurons. When primary human CD4 +T cells were treated with opioids, only p38 MAPK, not ERK or JNK, was activated with similar kinetics and OR subtype independence as that seen in Jurkat cells. This difference between primary cells and Jurkat cells suggests an additional level of regulation of opioid signaling in primary cells not found in Jurkat cells. The requirement for IL-4 pretreatment suggests that opioids may play a role in Th2 biology. Preliminary data indicate that morphine modestly increases the expression of IL-4 mRNA. Overall, these results reveal that p38 functions as a primary effector of opioid receptor signal transduction in human T cells. Supported by grants from NIH / NIDA (R01 DA 043253, )

MAPK family genes' influences on myogenesis in cattle: Genome-wide analysis and identification

The mitogen-activated protein kinase (MAPK) family is highly conserved in mammals, and is involved in a variety of physiological phenomena like regeneration, development, cell proliferation, and differentiation. In this study, 13 MAPK genes were identified in cattle and their corresponding protein properties were characterized using genome-wide identification and analysis. Phylogenetic analysis showed that the 13 BtMAPKs were cluster grouped into eight major evolutionary branches, which were segmented into three large subfamilies: ERK, p38 and JNK MAPK. BtMAPKs from the same subfamily had similar protein motif compositions, but considerably different exon-intron patterns. The heatmap analysis of transcriptome sequencing data showed that the expression of BtMAPKs was tissue-specific, with BtMAPK6 and BtMAPK12 highly expressed in muscle tissues. Furthermore, knockdown of BtMAPK6 and BtMAPK12 revealed that BtMAPK6 had no effect on myogenic cell proliferation, but negatively affected the differentiation of myogenic cells. In contrast, BtMAPK12 improved both the cell proliferation and differentiation. Taken together, these results provide novel insights into the functions of MAPK families in cattle, which could serve as a basis for further studies on the specific mechanisms of the genes in myogenesis.

Extracellular Release of Citrullinated Vimentin Directly Acts on Osteoclasts to Promote Bone Resorption in a Mouse Model of Periodontitis.

Elevated osteoclast (OC)-mediated bone resorption, a common pathological feature between periodontitis and rheumatoid arthritis (RA), implicates a possible mutually shared pathogenesis. The autoantibody to citrullinated vimentin (CV), a representative biomarker of RA, is reported to promote osteoclastogenesis (OC-genesis). However, its effect on OC-genesis in the context of periodontitis remains to be elucidated. In an in vitro experiment, the addition of exogenous CV upregulated the development of Tartrate-resistant acid phosphatase (TRAP)-positive multinuclear OCs from mouse bone marrow cells and increased the formation of resorption pits. However, Cl-amidine, an irreversible pan-peptidyl arginine deiminase (PAD) inhibitor, suppressed the production and secretion of CV from RANKL-stimulated OC precursors, suggesting that the citrullination of vimentin occurs in OC precursors. On the other hand, the anti-vimentin neutralizing antibody suppressed in vitro Receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced OC-genesis. The CV-induced upregulation of OC-genesis was abrogated by the Protein kinase C (PKC)-δ inhibitor Rottlerin, accompanied by the downmodulation of OC-genesis-related genes, including Osteoclast stimulatory transmembrane protein (OC-STAMP), TRAP and Matrix Metallopeptidase 9 (MMP9) as well as extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP)-kinase phosphorylation. Elevated levels of soluble CV and vimentin-bearing mononuclear cells were found in the bone resorption lesions of periodontitis induced in mice in the absence of an anti-CV antibody. Finally, local injection of anti-vimentin neutralizing antibody suppressed the periodontal bone loss induced in mice. Collectively, these results indicated that the extracellular release of CV promoted OC-genesis and bone resorption in periodontitis.

A tri-herb formulation protects against ethanol-induced mouse liver injury and downregulates mitogen-activated protein kinase phosphatase 1

BackgroundA tri-herb formulation comprising Ganoderma (the dried fruiting body of Ganoderma lucidum), Puerariae Thomsonii Radix (the dried root of Pueraria thomsonii) and Hoveniae Semen (the dried mature seed of Hovenia acerba) —GPH for short— has been using for treating liver injury; however, the pharmacological basis of this application of GPH is unknown. This study aimed to investigate the liver protective effects and mechanisms of action of an ethanolic extract of GPH (GPHE) in mice. MethodsTo control the quality of GPHE, the contents of ganodermanontriol, puerarin and kaempferol in the extract were quantified by ultra-performance liquid chromatography. An ethanol (6 ml/kg, i.g.)-induced liver injury ICR mouse model was employed to investigate the hepatoprotective effects of GPHE. RNA-sequencing analysis and bioassays were performed to reveal the mechanisms of action of GPHE. ResultsThe contents of ganodermanontriol, puerarin and kaempferol in GPHE were 0.0632%, 3.627% and 0.0149%, respectively. Daily i.g. administration of 0.25, 0.5 or 1 g/kg of GPHE for 15 consecutive days suppressed ethanol (6 ml/kg, i.g., at day 15)-induced upregulation of serum AST and ALT levels and improved histological conditions in mouse livers, indicating that GPHE protects mice from ethanol-induced liver injury. Mechanistically, GPHE downregulated the mRNA level of Dusp1 (encoding MKP1 protein, an inhibitor of the mitogen-activated protein kinases JNK, p38 and ERK), and upregulated expression and phosphorylation of JNK, p38 and ERK, which are involved in cell survival in mouse liver tissues. Also, GPHE increased PCNA (a cell proliferation marker) expression and reduced TUNEL-positive (apoptotic) cells in mouse livers. ConclusionGPHE protects against ethanol-induced liver injury, and this effect of GPHE is associated with regulation of the MKP1/MAPK pathway. This study provides pharmacological justifications for the use of GPH in treating liver injury, and suggests that GPHE has potential to be developed into a modern medication for managing liver injury.

ACAGT-007a, an anti-cancer compound that modulates ERK MAPK signaling, induces nuclear enrichment of phosphorylated ERK in T3M4 pancreatic cancer cells.

The extracellular-signal-regulated-kinase (ERK) signaling pathway is essential for cell proliferation and is frequently deregulated in human tumors such as pancreatic cancers. ACAGT-007a (GT-7), an anti-cancer compound, stimulates ERK phosphorylation, thereby inducing growth inhibition and apoptosis in T3M4 pancreatic cancer cells. However, how GT-7 stimulates ERK phosphorylation and induces apoptosis in ERK-active T3M4 cells remains unclear. To look into the mechanism, we performed a spatiotemporal analysis of ERK phosphorylation mediated by GT-7 in T3M4 cells. The immunoblotting showed that GT-7 stimulates ERK phosphorylation within 1 h, which was more remarkable after 2 h. Importantly, apoptosis induction as evaluated by the cleaved Caspase-3 was observed only after 2-h incubation with GT-7. The immunofluorescence staining revealed the enrichment of phosphorylated ERK (phospho-ERK) in the nucleus upon 1-h incubation with GT-7. Fractionation experiments showed that GT-7 increases phospho-ERK levels in the cytoplasm within 1 h, whereas nuclear phospho-ERK accumulation is observed after 2-h incubation with GT-7. MEK inhibition by U0126 significantly diminishes nuclear phospho-ERK distribution and apoptosis induction stimulated by GT-7. Thus, GT-7 may initiate the induction of ERK phosphorylation in the cytoplasm, which leads to phospho-ERK enrichment in the nucleus. This nuclear phospho-ERK accumulation by GT-7 precedes and may underlie apoptosis induction in T3M4.

Abstract 5831: CD200R signaling contributes to unfavorable tumor microenvironment through regulating production of chemokines by tumor associated macrophages

Abstract CD200 is overexpressed in human solid tumors such as neuroblastoma and melanoma and is considered as an immune checkpoint molecule for dampening cancer immunity. However, how CD200 affects tumor microenvironment (TME) and host immunity is incompletely understood. In the present study, we evaluated the role of the CD200-CD200R pathway in TME using loss-of-function CD200R-/- mice, along with two clinically relevant, CD200-positive murine tumor models: the mouse neuroblastoma NB9464D and melanoma Yummer1.7. We found that comparing with the wild type mice, CD200R-/- mice were significantly more potent in rejecting these CD200+ tumors. Transcriptome analysis by single cell RNA sequencing demonstrated that tumors from CD200R-/- mice had more infiltration of CD45+ immune cells, including CD4+ and CD8+ T cells, and NK cells but less infiltration of neutrophils. Antibody depletion experiments revealed that immune effectors including CD8+ T cells and NK cells in combination, are crucial in inhibiting tumor growth in CD200R-/- mice. Mechanistically, we found that CD200R signaling differentially regulates the expression of chemokines in tumor-associated macrophages (TAMs). In the absence of CD200R, TAMs up-regulate CCL24 and CCL8 and down-regulate CXCL3, CXCL2, and CCL3 via activation of ERK and/or p38 MAP kinases. Increased expression of CCL24 in CD200R-deficient tumors resulted in increased infiltration of eosinophils, which also contributes to anti-tumor activity. Overall, we conclude that CD200R-mediated signaling, via interaction with CD200 on tumor cells, contributes to unfavorable TME primarily through chemokine-dependent recruitment of immune suppressive neutrophils and exclusion of anti-cancer immune effectors. Our study has significant implications in developing CD200-targeted immunotherapy of solid tumors. Citation Format: Cho-Hao Lin, Fatemeh Talebian, Yang Li, Jianmin Zhu, Bolin Zhao, Jin-Qing Liu, Sujit Basu, Xueling Pan, Xi Chen, Pearlly Yan, William E. Carson, Gang Xin, Haitao Wen, Ruoning Wang, Zihai Li, Qin Ma, Xue-Feng Bai. CD200R signaling contributes to unfavorable tumor microenvironment through regulating production of chemokines by tumor associated macrophages [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5831.

Abstract 25: MIF and NR3C2 interactively regulate glucose metabolism in pancreatic cancer

Abstract Inflammation and altered cellular metabolism are described as hallmarks of cancer. Although inflammatory signaling and metabolic reprogramming are highly integrated and play critical role in the development and progression of pancreatic ductal adenocarcinoma (PDAC). The role of inflammatory mediators in the regulation of metabolic reprogramming is not adequately studied in pancreatic cancer and remains to be explored. Here we show that macrophage migration inhibitory factor (MIF) collaborates with its downstream transcriptional factor nuclear-receptor-subfamily-3, group-C, member-2 (NR3C2) regulate metabolic reprogramming to support cancer growth and progression in PDAC. MIF enhances glucose uptake and lactate efflux through enhancing HK1 (hexokinase 1), HK2 (hexokinase 2) and LDHA (lactate dehydrogenase) expression in pancreatic cancer cell in vitro and in an orthotopic mouse model. Furthermore, a decreased HK1, HK2, LDHA expression, pyruvate and lactate production are found in patient tumors with lower MIF expression and MIF-deficiency tumors from genetically engineered KPC mouse model. Conversely, NR3C2 suppresses HK1, HK2, and LDHA expression and decreases glucose uptake and lactate efflux in pancreatic cancer. Mechanistically, MIF-mediated regulation of glycolytic metabolism involves MAPK-ERK Kinase pathway while NR3C2 interacts with AP-1 transcriptional factor in regulation of glycolysis metabolism. Taken together, our result demonstrates an interactive role of MIF and NR3C2 signaling in regulation cancer metabolism and therapeutic approaches target this axis may improve disease outcome in patients with PDAC. Citation Format: Shouhui Yang, Wei Tang, Azadeh Azizian, Philipp Ströbel, Yuuki Ohara, Helen Cawley, Nader Hanna, B. Michael Ghadimi, Nagireddy Putluri, S. Perwez Hussain. MIF and NR3C2 interactively regulate glucose metabolism in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 25.

The Modulation of Immune Responses in Tilapinevirus tilapiae-Infected Fish Cells through MAPK/ERK Signalling.

Tilapia lake virus (TiLV) is a novel RNA virus that has been causing substantial economic losses across the global tilapia industry. Despite extensive research on potential vaccines and disease control methods, the understanding of this viral infection and the associated host cell responses remains incomplete. In this study, the involvement of the mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathway in the early stages of TiLV infection was investigated. The results showed a distinct pattern of ERK phosphorylation (p-ERK) upon TiLV infection in two fish cell lines, E-11 and TiB. Specifically, the p-ERK levels in the TiB cells decreased substantially, while the p-ERK levels in the E-11 cells remained constant. Interestingly, a large number of cytopathic effects were observed in the infected E-11 cells but none in the infected TiB cells. Furthermore, when p-ERK was suppressed using the inhibitor PD0325901, a significant reduction in the TiLV load and decrease in the mx and rsad2 gene expression levels were observed in the TiB cells in days 1-7 following infection. These findings highlight the role of the MAPK/ERK signalling pathway and provide new insights into the cellular mechanisms during TiLV infection that could be useful in developing new strategies to control this virus.

Edible O. fragrans flower ameliorates LPS-induced inflammatory responses through suppressing NF-κB and AP-1 pathways

Osmanthus fragrans (Thunb.) Lour. is one of the most common edible plants in China. Its flower (O. fragrans flower) is widely utilized as a food ingredient and traditional Chinese medicine for hundreds of years. In lipopolysaccharide (LPS)-activated macrophages, OFE suppressed the pro-inflammatory mediators (iNOS, MCP-1, IL-6 and IL-1β) at the transcriptional and translational levels, showing a potent anti-inflammatory effect. Mechanism study indicated that, OFE inhibited IκBα phosphorylation and prevented the nuclear translocation of p65, thereby inactivating nuclear factor-κB (NF-κB) signaling. Concurrently, OFE also suppressed AP-1 signaling through inhibiting the phosphorylation of c-Jun N-terminal kinase (JNK), extracellular signal-regulated kinase (ERK) and p38 MAPK. In endotoxemia mice, a single intraperitoneal administration of OFE significantly decreased serum IL-6 and MCP-1 levels. Our study demonstrates that O. fragrans flower may be the beneficial food for inflammatory diseases.

White-spotted flower chafer (Protaetia brevitarsis) ameliorates inflammatory responses in LPS-stimulated RAW 264.7 macrophages

White-spotted flower chafer (Protaetia brevitarsis) is an edible insect andP. brevitarsis larvae are used in traditional Oriental medicine. However, the molecular mechanisms behind the anti-inflammatory action ofP. brevitarsis larvae are unknown. We investigated the effects ofP. brevitarsis larvae ethanol extract (PBLE) on the inflammatory response as well as the molecular mechanisms underpinning this effect in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. PBLE effectively inhibited the synthesis of nitric oxide (NO) through inducible NO synthase (iNOS) and cyclooxygenase 2 protein expression and 20 μM PBLE suppressed the expression of pro-inflammatory cytokines, including interleukin (IL)-6 and IL-8, at a dose of. Further investigation of the mechanisms revealed that PBLE attenuated phosphorylation of nuclear factor kappa B (NF-κB) inhibitor alpha (IκB-α) and NF-κB p65 and interfered selectively with extracellular signal-regulated kinase (ERK)1/2 and p38 phosphorylation. These findings provide the first molecular basis that PBLE could be a potential candidate for attenuating proinflammatory NO induction via selective inhibition of the NF-κB, ERK, and p38 mitogen-activated protein kinase signalling pathways. It is necessary to verify the anti-inflammatory action of PBLE and to ascertain the related mechanisms, both inin vivo models and in clinical studies, to enable the application of PBLE as a potential therapeutic agent for inflammatory diseases.

PTD-FGF2 Attenuates Elastase Induced Emphysema in Mice and Alveolar Epithelial Cell Injury

Aberrant communication in alveolar epithelium is a major feature of inflammatory response for the airway remodeling leading to chronic obstructive pulmonary disease (COPD). In this study, we investigated the effect of protein transduction domains (PTD) conjugated Basic Fibroblast Growth Factor (FGF2) (PTD-FGF2) in response to cigarette smoke extract (CSE) in MLE-12 cells and porcine pancreatic elastase (PPE)-induced emphysematous mice. When PPE-induced mice were intraperitoneally treated with 0.1–0.5 mg/kg PTD-FGF2 or FGF2, the linear intercept, infiltration of inflammatory cells into alveoli and pro-inflammatory cytokines were significantly decreased. In western blot analysis, phosphorylated protein levels of c-Jun N-terminal Kinase 1/2 (JNK1/2), extracellular signal-regulated kinase (ERK1/2) and p38 mitogen-activated protein kinases (MAPK) were decreased in PPE-induced mice treated PTD-FGF2. In MLE-12 cells, PTD-FGF2 treatment decreased reactive oxygen species (ROS) production and further decreased Interleukin-6 (IL-6) and IL-1b cytokines in response to CSE. In addition, phosphorylated protein levels of ERK1/2, JNK1/2 and p38 MAPK were reduced. We next determined microRNA expression in the isolated exosomes of MLE-12 cells. In reverse transcription-polymerase chain reaction (RT-PCR) analysis, level of let-7c miRNA was significantly increased while levels of miR-9 and miR-155 were decreased in response to CSE. These data suggest that PTD-FGF2 treatment plays a protective role in regulation of let-7c, miR-9 and miR-155 miRNA expressions and MAPK signaling pathways in CSE-induced MLE-12 cells and PPE-induced emphysematous mice.

Caspase 3-specific cleavage of MEK1 suppresses ERK signaling and sensitizes cells to stress-induced apoptosis.

Proper regulation of apoptotic cell death is crucial for normal development and homeostasis in multicellular organisms and is achieved by the balance between pro-apoptotic processes, such as caspase activation, and pro-survival signaling, such as extracellular signal-regulated kinase (ERK) activation. However, the functional interplay between these opposing signaling pathways remains incompletely understood. Here, we identified MAPK/ERK kinase (MEK) 1, a central component of the ERK pathway, as a specific substrate for the executioner caspase-3. During apoptosis, MEK1 is cleaved at an evolutionarily conserved Asp282 residue in the kinase domain, thereby losing its catalytic activity. Gene knockout experiments showed that MEK1 cleavage was mediated by caspase-3, but not by the other executioner caspases, caspase-6 or -7. Following exposure of cells to osmotic stress, elevated ERK activity gradually decreased, and this was accompanied by increased cleavage of MEK1. In contrast, the expression of a caspase-uncleavable MEK1(D282N) mutant in cells maintained stress-induced ERK activity and thereby attenuated apoptotic cell death. Thus, caspase-3-mediated, proteolytic inhibition of MEK1 sensitizes cells to apoptosis by suppressing pro-survival ERK signaling. Furthermore, we found that a RASopathy-associated MEK1(Y130C) mutation prevented this caspase-3-mediated proteolytic inactivation of MEK1 and efficiently protected cells from stress-induced apoptosis. Our data reveal the functional crosstalk between ERK-mediated cell survival and caspase-mediated cell death pathways and suggest that its dysregulation by a disease-associated MEK1 mutation is at least partly involved in the pathophysiology of congenital RASopathies.