Search

Simple Summary: It has remained obscure whether microglia proliferate in the diseased or injured brain. This study analyzed the mitotic ability of microglia in vivo in a rat facial nerve axotomy model in which the blood cells do not enter the parenchyma. Microglia were found to proliferate in the axotomized facial nucleus at 5–7 days post-insult. They enhanced the levels of macrophage colony-stimulating factor (M-CSF) as a mitotic factor, its receptor cFms, and cell-cycle-related proteins, such as proliferating cell nuclear antigen (PCNA) and cyclins in vivo. In the M-CSF-dependent proliferation system in vitro, c-Jun N-terminal kinase (JNK) and p38 were shown to function in the induction of PCNA/cyclins and cFms in microglia, respectively. These analyses revealed that some specific signaling cascades are linked to the proliferative reaction of microglia. Microglia in the axotomized facial nucleus were considered to exhibit a mitotic ability to rescue injured motoneurons as neurotrophic cells. Although microglia exist as a minor glial cell type in the normal state of the brain, they increase in number in response to various disorders and insults. However, it remains unclear whether microglia proliferate in the affected area, and the mechanism of the proliferation has long attracted the attention of researchers. We analyzed microglial mitosis using a facial nerve transection model in which the blood–brain barrier is left unimpaired when the nerves are axotomized. Our results showed that the levels of macrophage colony-stimulating factor (M-CSF), cFms (the receptor for M-CSF), cyclin A/D, and proliferating cell nuclear antigen (PCNA) were increased in microglia in the axotomized facial nucleus (axotFN). In vitro experiments revealed that M-CSF induced cFms, cyclin A/D, and PCNA in microglia, suggesting that microglia proliferate in response to M-CSF in vivo. In addition, M-CSF caused the activation of c-Jun N-terminal kinase (JNK) and p38, and the specific inhibitors of JNK and p38 arrested the microglial mitosis. JNK and p38 were shown to play roles in the induction of cyclins/PCNA and cFms, respectively. cFms was suggested to be induced through a signaling cascade of p38-mitogen- and stress-activated kinase-1 (MSK1)-cAMP-responsive element binding protein (CREB) and/or p38-activating transcription factor 2 (ATF2). Microglia proliferating in the axotFN are anticipated to serve as neuroprotective cells by supplying neurotrophic factors and/or scavenging excite toxins and reactive oxygen radicals. [ABSTRACT FROM AUTHOR]

Bambusae caulis in Liquamen (BCL), which is extracted from heat-treated fresh bamboo stems, is a traditional herbal medicine widely used in Eastern countries. Recently, it has been reported to have anti-inflammatory and whitening effects. However, the protective effect of BCL on hepatocytes has not yet been elucidated. The present study aimed to determine whether BCL prevents oxidative stress induced by tert-butyl hydroperoxide (t-BHP) and exerts cytoprotective effects on hepatocytes. High-performance liquid chromatography and liquid chromatography with tandem mass spectroscopy were performed to analyze the type of polyphenols present in BCL. The activities of antioxidant enzymes and hepatocyte viability were assessed. The benzoic acid content was the highest among polyphenols present in BCL. Benzoic acid acts as a scavenger of free radicals, including reactive oxygen species. BCL increased the expression of antioxidant enzymes (glutamate–cysteine ligase and NADPH quinone dehydrogenase (1)) by activating nuclear factor erythroid 2-related factor 2 and reduced tBHP-induced cell death by inhibiting oxidative stress. BCL inhibited tBHP-induced phosphorylation of p38 and c-Jun N-terminal kinase but not that of extracellular signal-regulated kinase. In conclusion, BCL is a promising therapeutic candidate for treating oxidative-stress-induced hepatocyte damage. [ABSTRACT FROM AUTHOR]

Cisplatin is a potent chemotherapeutic drug for ovarian cancer (OC) treatment. However, its efficacy is significantly limited due to the development of cisplatin resistance. Although the acquisition of cisplatin resistance is a complex process involving various molecular alterations within cancer cells, the increased reliance of cisplatin-resistant cells on glycolysis has gained increasing attention. Isoalantolactone, a sesquiterpene lactone isolated from Inula helenium L., possesses various pharmacological properties, including anticancer activity. In this study, isoalantolactone was investigated as a potential glycolysis inhibitor to overcome cisplatin resistance in OC. Isoalantolactone effectively targeted key glycolytic enzymes (e.g., lactate dehydrogenase A, phosphofructokinase liver type, and hexokinase 2), reducing glucose consumption and lactate production in cisplatin-resistant OC cells (specifically A2780 and SNU-8). Importantly, it also sensitized these cells to cisplatin-induced apoptosis. Isoalantolactone–cisplatin treatment regulated mitogen-activated protein kinase and AKT pathways more effectively in cisplatin-resistant cells than individual treatments. In vivo studies using cisplatin-sensitive and resistant OC xenograft models revealed that isoalantolactone, either alone or in combination with cisplatin, significantly suppressed tumor growth in cisplatin-resistant tumors. These findings highlight the potential of isoalantolactone as a novel glycolysis inhibitor for treating cisplatin-resistant OC. By targeting the dysregulated glycolytic pathway, isoalantolactone offers a promising approach to overcoming drug resistance and enhancing the efficacy of cisplatin-based therapies. [ABSTRACT FROM AUTHOR]

Geese have strong brooding abilities, which severely affect their egg-laying performance. Phosphorylation is widely involved in regulating reproductive activities, but its role in goose brooding behavior is unclear. In this study, we investigated differences in the phosphoprotein composition of ovarian tissue between laying and brooding geese. Brooding geese exhibited ovarian and follicular atrophy, as well as significant oxidative stress and granulosa cell apoptosis. We identified 578 highly phosphorylated proteins and 281 lowly phosphorylated proteins, and a KEGG pathway analysis showed that these differentially phosphorylated proteins were mainly involved in cell apoptosis, adhesion junctions, and other signaling pathways related to goose brooding behavior. The extracellular regulated protein kinase (ERK)–B-Cell Lymphoma 2(BCL2) signaling pathway was identified as playing an important role in regulating cell apoptosis. The phosphorylation levels of ERK proteins were significantly lower in brooding geese than in laying geese, and the expression of mitogen-activated protein kinase kinase (MEK) was downregulated. Overexpression of MEK led to a significant increase in ERK phosphorylation and BCL2 transcription in H2O2-induced granulosa cells (p < 0.05), partially rescuing cell death. Conversely, granulosa cells receiving MEK siRNA exhibited the opposite trend. In conclusion, geese experience significant oxidative stress and granulosa cell apoptosis during brooding, with downregulated MEK expression, decreased phosphorylation of ERK protein, and inhibited expression of BCL2. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) has a high incidence and is one of the leading causes of cancer-related death. The accumulation of cancer-associated fibroblasts (CAF) induces an aggressive, stem-like phenotype in tumor cells, and it indicates a poor prognosis. However, cellular heterogeneity among CAFs and the targeting of both stromal and CRC cells are not yet well resolved. Here, we identified CD142high fibroblasts with a higher stimulating effect on CRC cell proliferation via secreting more hepatocyte growth factor (HGF) compared to CD142low CAFs. We also found that combinations of inhibitors that had either a promising effect in other cancer types or are more active in CRC compared to normal colonic epithelium acted synergistically in CRC cells. Importantly, heat shock protein 90 (HSP90) inhibitor selected against CD142high fibroblasts, and both CRC cells and CAFs were sensitive to a BCL-xL inhibitor. However, targeting mitogen-activated protein kinase kinase (MEK) was ineffective in fibroblasts, and an epigenetic inhibitor selected for a tumor cell population with markers of aggressive behavior. Thus, we suggest BCL-xL and HSP90 inhibitors to eliminate cancer cells and decrease the tumor-promoting CD142high CAF population. This may be the basis of a strategy to target both CRC cells and stromal fibroblasts, resulting in the inhibition of tumor relapse. [ABSTRACT FROM AUTHOR]

The mammalian Ccr4–Not complex, carbon catabolite repression 4 (Ccr4)-negative on TATA-less (Not), is a large, highly conserved, multifunctional assembly of proteins that acts at different cellular levels to regulate gene expression. It is involved in the control of the cell cycle, chromatin modification, activation and inhibition of transcription initiation, control of transcription elongation, RNA export, and nuclear RNA surveillance; the Ccr4–Not complex also plays a central role in the regulation of mRNA decay. Growing evidence suggests that gene transcription has a vital role in shaping the landscape of genome replication and is also a potent source of replication stress and genome instability. Here, we have examined the effects of the inactivation of the Ccr4–Not complex, via the depletion of the scaffold subunit CNOT1, on DNA replication and genome integrity in mammalian cells. In CNOT1-depleted cells, the elevated expression of the general transcription factor TATA-box binding protein (TBP) leads to increased RNA synthesis, which, together with R-loop accumulation, results in replication fork slowing, DNA damage, and senescence. Furthermore, we have shown that the stability of TBP mRNA increases in the absence of CNOT1, which may explain its elevated protein expression in CNOT1-depleted cells. Finally, we have shown the activation of mitogen-activated protein kinase signalling as evidenced by ERK1/2 phosphorylation in the absence of CNOT1, which may be responsible for the observed cell cycle arrest at the border of G1/S. [ABSTRACT FROM AUTHOR]

Rheumatoid arthritis (RA) is a heterogeneous inflammatory disease with an autoimmune origin and an incompletely elucidated pathophysiological mechanism. RA pharmacotherapy is based on chemically or biologically active substances that provide clinical alleviation and remission, but the disease is still incurable. As a result, there remains a need for significant therapeutic development, and adjuvant therapies may play an essential role in the search for novel RA treatment strategies. The aim of the present study was to investigate potential phytocompounds and phytocompound derivates as RA treatment agents, using in silico methodologies. In this regard, five phytoconstituents identified in different structures of Embelia ribes were evaluated by in silico methods for their potential action on target proteins of therapeutic interest in RA. The methodology involved identifying the phytocompound with the highest binding toward the target protein via molecular docking using AutoDock Vina 1.5.7, followed by a ligand-based virtual screening based on the structure of the most promising phytocompound using SwissSimilarity. This process led to the identification of ligands that are not currently utilized in medical practice, but that might have the potential to be used in the management of RA after further extensive experimental endorsements. ZINC000004024651 showed the highest binding affinity for the Bruton's tyrosine kinase protein, followed by ZINC000000434197 for p38 mitogen-activated protein kinases, ZINC000087606977 for interleukin-1 receptor-associated kinase 4, and ZINC000014728393 for matrix metallopeptidase 9, the latter two showing higher affinity than the co-crystallized compound. The relatively high affinities to target proteins and the pharmacokinetic data obtained by in silico studies using SwisADME suggest a first step for the inclusion of promising new compounds in various more advanced studies, leading to the evaluation of efficacy and safety profiles. [ABSTRACT FROM AUTHOR]

Inflammation is triggered by a variety of danger signals and is now a worldwide concern. Resveratrol, a natural nonflavonoid polyphenol found in naturally consumed plants and foods, has a wide spectrum of bioactive potency. We successfully generated resveratrol-enriched rice by introducing the resveratrol biosynthesis gene into Dongjin rice. In this study, resveratrol- and piceid-enriched rice (DJ526) was investigated for its anti-inflammatory activity in lipopolysaccharide (LPS)-stimulated RAW264.7 cells compared to normal rice (DJ). In addition, the 5-day-old germinated DJ526 (DJ526_5) was tested for its anti-inflammatory effects. The piceid and resveratrol amounts increased in DJ526_5 by germination. Treatment of LPS-stimulated RAW264.7 cells with resveratrol-enriched rice seed extracts (DJ526_0 and DJ526_5) significantly decreased the production of nitric oxide (NO) and the inflammatory mediator prostaglandin E2 (PGE2), downregulated proinflammatory gene expression, and inhibited nuclear factor kappa B (NF-κB) p65, p38 mitogen-activated protein kinase, and extracellular signal-regulated kinase 1/2 (ERK 1/2) phosphorylation. These findings demonstrated the anti-inflammatory mechanisms of resveratrol-enriched rice in LPS-stimulated RAW264.7 cells. Furthermore, resveratrol-enriched rice could be a potential source of anti-inflammatory agents. [ABSTRACT FROM AUTHOR]

Ovarian cancer is a gynecological tumor with an incidence rate lower than those of other gynecological tumor types and the second-highest death rate. CC chemokine 2 (CCL2) is a multifunctional factor associated with the progression of numerous cancers. However, the effect of CCL2 on ovarian cancer progression is unclear. Here, we found that exogenous CCL2 and the overexpression of CCL2 promoted the proliferation and metastasis of ovarian cancer cells. On the other hand, CCL2 knockdown via CRISPR/Cas9 inhibited ovarian cancer cell proliferation, migration, and invasion. The present study demonstrated that mitogen-activated protein three kinase 19 (MAP3K19) was the key CCL2 target for regulating ovarian cancer progression through transcriptome sequencing. Additionally, MAP3K19 knockout inhibited ovarian cancer cell proliferation, migration, and invasion. Furthermore, CCL2 increased MAP3K19 expression by activating the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK) pathway. The present study showed the correlation between CCL2 and ovarian cancer, suggesting that CCL2 may be a novel target for ovarian cancer therapy. [ABSTRACT FROM AUTHOR]

Lansoprazole, a proton pump inhibitor, can exert antioxidant effects through the induction of the nuclear factor erythroid 2-related factor 2 (Nrf2) pathway, independently of the inhibition of acid secretion in the gastrointestinal tract. Lansoprazole has been reported to provide hepatoprotection in a drug-induced hepatitis animal model through the Nrf2/heme oxygenase-1 (HO1) pathway. We sought to investigate the molecular mechanism of cytoprotection by lansoprazole. An in vitro experimental model was conducted using cultured rat hepatic cells treated with lansoprazole to analyze the expression levels of Nrf2 and its downstream genes, the activity of Nrf2 using luciferase reporter assays, cisplatin-induced cytotoxicity, and signaling pathways involved in Nrf2 activation. Lansoprazole treatment of rat liver epithelial RL34 cells induced transactivation of Nrf2 and the expression of the Nrf2-dependent antioxidant genes encoding HO1, NAD(P)H quinone oxidoreductase-1, and glutathione S-transferase A2. Furthermore, cycloheximide chase experiments revealed that lansoprazole prolongs the half-life of the Nrf2 protein. Notably, cell viability was significantly increased by lansoprazole treatment in a cisplatin-induced cytotoxicity model. Moreover, the siRNA knockdown of Nrf2 fully abolished the cytoprotective effect of lansoprazole, whereas the inhibition of HO1 by tin-mesoporphyrin only partially abolished this. Finally, lansoprazole promoted the phosphorylation of p38 mitogen-activated protein kinase (MAPK) but not that of the extracellular signal-regulated kinase or the c-Jun N-terminal kinase. Using SB203580, a specific inhibitor for p38 MAPK, the lansoprazole-induced Nrf2/antioxidant response elements pathway activation and cytoprotective effects were shown to be exclusively p38 MAPK dependent. Lansoprazole was shown by these results to exert a cytoprotective effect on liver epithelial cells against the cisplatin-induced cytotoxicity through the p38 MAPK signaling pathway. This could have potential applications for the prevention and treatment of oxidative injury in the liver. [ABSTRACT FROM AUTHOR]

Background: Tea geometrid Ectropis grisescens (Geometridae: Lepidoptera), is one of the most destructive defoliators in tea plantations in China. The MAPK cascade is known to be an evolutionarily conserved signaling module, acting as pivotal cores of host–pathogen interactions. Although the chromosome-level reference genome of E. grisescens was published, the whole MAPK cascade gene family has not been fully identified yet, especially the expression patterns of MAPK cascade gene family members upon an ecological biopesticide, Metarhizium anisopliae, remains to be understood. Results: In this study, we have identified 19 MAPK cascade gene family members in E. grisescens, including 5 MAPKs, 4 MAP2Ks, 8 MAP3Ks, and 2 MAP4Ks. The molecular evolution characteristics of the whole Eg-MAPK cascade gene family, including gene structures, protein structural organization, chromosomal localization, orthologs construction and gene duplication, were systematically investigated. Our results showed that the members of Eg-MAPK cascade gene family were unevenly distributed in 13 chromosomes, and the clustered members in each group shared similar structures of the genes and proteins. Gene expression data revealed that MAPK cascade genes were expressed in all four developmental stages of E. grisescens and were fairly and evenly distributed in four different larva tissues. Importantly, most of the MAPK cascade genes were induced or constitutively expressed upon M. anisopliae infection. Conclusions: In summary, the present study was one of few studies on MAPK cascade gene in E. grisescens. The characterization and expression profiles of Eg-MAPK cascades genes might help develop new ecofriendly biological insecticides to protect tea trees. [ABSTRACT FROM AUTHOR]

Background: Polarization of microglia, the resident retinal immune cells, plays important roles in mediating both injury and repair responses post-retinal ischemia–reperfusion (I/R) injury, which is one of the main pathological mechanisms behind ganglion cell apoptosis. Aging could perturb microglial balances, resulting in lowered post-I/R retinal repair. Young bone marrow (BM) stem cell antigen 1-positive (Sca-1+) cells have been demonstrated to have higher reparative capabilities post-I/R retinal injury when transplanted into old mice, where they were able to home and differentiate into retinal microglia. Methods: Exosomes were enriched from young Sca-1+ or Sca-1− cells, and injected into the vitreous humor of old mice post-retinal I/R. Bioinformatics analyses, including miRNA sequencing, was used to analyze exosome contents, which was confirmed by RT-qPCR. Western blot was then performed to examine expression levels of inflammatory factors and underlying signaling pathway proteins, while immunofluorescence staining was used to examine the extent of pro-inflammatory M1 microglial polarization. Fluoro-Gold labelling was then utilized to identify viable ganglion cells, while H&E staining was used to examine retinal morphology post-I/R and exosome treatment. Results: Sca-1+ exosome-injected mice yielded better visual functional preservation and lowered inflammatory factors, compared to Sca-1−, at days 1, 3, and 7 days post-I/R. miRNA sequencing found that Sca-1+ exosomes had higher miR-150-5p levels, compared to Sca-1− exosomes, which was confirmed by RT-qPCR. Mechanistic analysis found that miR-150-5p from Sca-1+ exosomes repressed the mitogen-activated protein kinase kinase kinase 3 (MEKK3)/JNK/c-Jun axis, leading to IL-6 and TNF-α downregulation, and subsequently reduced microglial polarization, all of which contributes to reduced ganglion cell apoptosis and preservation of proper retinal morphology. Conclusion: This study elucidates a potential new therapeutic approach for neuroprotection against I/R injury, via delivering miR-150-5p-enriched Sca-1+ exosomes, which targets the miR-150-5p/MEKK3/JNK/c-Jun axis, thereby serving as a cell-free remedy for treating retinal I/R injury and preserving visual functioning. [ABSTRACT FROM AUTHOR]

In this study, we demonstrated that 2′-hydroxy-3,6′-dimethoxychalcone (3,6′-DMC) alleviated α-MSH-induced melanogenesis and lipopolysaccharides (LPS)-induced inflammation in mouse B16F10 and RAW 264.7 cells. In vitro analysis results showed that the melanin content and intracellular tyrosinase activity were significantly decreased by 3,6′-DMC, without cytotoxicity, via decreases in tyrosinase and the tyrosinase-related protein 1 (TRP-1) and TRP-2 melanogenic proteins, as well as the downregulation of microphthalmia-associated transcription factor (MITF) expression through the upregulation of the phosphorylation of extracellular-signal-regulated kinase (ERK), phosphoinositide 3-kinase (PI3K)/Akt, and glycogen synthase kinase-3β (GSK-3β)/catenin, and downregulation of the phosphorylation of p38, c-Jun N-terminal kinase (JNK), and protein kinase A (PKA). Furthermore, we investigated the effect of 3,6′-DMC on macrophage RAW264.7 cells with LPS stimulation. 3,6′-DMC significantly inhibited LPS-stimulated nitric oxide production. 3,6′-DMC also suppressed the expression of inducible nitric oxide synthase (iNOS) and cyclooxygenase (COX)-2 on the protein level. In addition, 3,6′-DMC decreased the production of the tumor necrosis factor-α and interleukin-6. Successively, our mechanistic studies revealed that 3,6′-DMC also suppressed the LPS-induced phosphorylation of the inhibitor of IκBα, p38MAPK, ERK, and JNK. The Western blot assay results showed that 3,6′-DMC suppresses LPS-induced p65 translocation from cytosol to the nucleus. Finally, the topical applicability of 3,6′-DMC was tested through primary skin irritation, and it was found that 3,6′-DMC, at 5 and 10 μM concentrations, did not cause any adverse effects. Therefore, 3,6′-DMC may provide a potential candidate for preventing and treating melanogenic and inflammatory skin diseases. [ABSTRACT FROM AUTHOR]

The role of ferroptosis in human acute lymphoblastic leukemia and its possible molecular mechanisms of action are still unknown. In this study, harvested Molt-4 cells were exposed to different concentrations of erastin, and their proliferation capacity was tested by using the cell counting kit-8 assay. Lipid peroxidation levels were detected through flow cytometry. Mitochondrial alterations were observed through transmission electron microscopy. The expression levels of SLC7A11, glutathione peroxidase 4 (GPX4), and mitogen-activated protein kinase (MAPK) were detected by using quantitative real-time PCR and Western blot analysis. This study found that erastin inhibited the growth of Molt-4 cells. This inhibitory effect could be partially reversed by the ferroptosis inhibitor Ferrostatin-1 and the p38 MAPK inhibitor. The mitochondria of Molt-4 cells treated with erastin shortened and condensed. Compared with those in the control group, the levels of reactive oxygen species and malondialdehyde had increased, whereas the levels of glutathione had decreased in the treatment group. The treatment of Molt-4 cells with erastin decreased the levels of SLC7A11 and GPX4 mRNA and increased the expression levels of p38 MAPK, extracellular signal–regulated kinase (ERK), and c-Jun N-terminal kinase. These findings suggested that erastin caused the ferroptosis of Molt-4 cells. This process may be correlated with the inhibition of the cystine/glutamate antiporter system and GPX4 and the activation of p38 MAPK and ERK1/2. [ABSTRACT FROM AUTHOR]

The RAS pathway participates in the cascade of proliferation and cell division process, and the activated RAS pathway can lead to tumorigenesis including hepatocellular carcinoma (HCC). However, few studies have explored the effects of genetic variants in the RAS pathway-related genes on the survival of patients with HBV-related HCC. In the present study, we assessed the associations between 11,658 single-nucleotide polymorphisms (SNPs) in 62 RAS pathway genes and the overall survival (OS) of 866 HBV-related HCC individuals, which were randomly split (1:1) into discovery and validation datasets. As a result, three potentially functional SNPs were identified, based on multivariable cox proportional hazards regression analyses, in SOS Ras/Rho guanine nucleotide exchange factor 2 (SOS2, rs4632055 A > G), Ras protein-specific guanine nucleotide releasing factor 2 (RASGRF2, rs26418A > G) and mitogen-activated protein kinase 1 (MAP2K1,rs57120695 C > T), which were significantly and independently associated with OS of HBV-related HCC patients [adjusted hazards ratios (HRs) of 1.42, 1.32 and 1.50, respectively; 95% confidence intervals (CI), 1.14 to 1.76, 1.15 to 1.53 and 1.15 to 1.97, respectively; P = 0.001, < 0.001 and 0.003, respectively]. Additionally, the joint effects as the unfavorable genotypes of these three SNPs showed a significant association with the poor survival of HCC (trend test P < 0.001). The expression quantitative trait loci (eQTL) analysis further revealed that the rs4632055 G allele and the rs26418 A allele were associated with lower mRNA expression levels of SOS2 and RASGRF2, respectively. Collectively, these potentially functional SNPs of RASGRF2, SOS2 and M2PAK1 may become potential prognostic biomarkers for HBV-related HCC after hepatectomy. [ABSTRACT FROM AUTHOR]

Sprouty-related EVH-1 domain-containing (SPRED) proteins are a family of proteins that negatively regulate theRAS-Mitogen-Activated Protein Kinase (MAPK) pathway, which is involved in the regulation of the mitogenic response and cell proliferation. However, the mechanism by which these proteins affect RASMAPK signaling has not been elucidated. Patients with mutations in SPRED give rise to unique disease phenotypes; thus, we hypothesized that distinct interactions across SPRED proteins may account for alternative nodes of regulation. To characterize the SPREDinteractomeand evaluatehowmembers of the SPRED family function through unique binding partners, we performed affinity purification mass spectrometry. We identified 90-kDa ribosomal S6 kinase 2 (RSK2) as a specific interactor of SPRED2 but not SPRED1 or SPRED3. We identified that the Nterminal kinase domain of RSK2 mediates the interaction between amino acids 123 to 201 of SPRED2. Using X-ray crystallography, we determined the structure of the SPRED2-RSK2 complex and identified the SPRED2 motif, F145A, as critical for interaction. We found that the formation of this interaction is regulated by MAPK signaling events. We also find that this interaction between SPRED2 and RSK2 has functional consequences, whereby the knockdown of SPRED2 resulted in increased phosphorylation of RSK substrates, YB1 and CREB. Furthermore, SPRED2 knockdown hindered phospho-RSK membrane and nuclear subcellular localization. We report that disruption of the SPRED2-RSK complex has effects on RASMAPK signaling dynamics. Our analysis reveals that members of the SPRED family have unique protein binding partners and describes the molecular and functional determinants of SPRED2-RSK2 complex dynamics. [ABSTRACT FROM AUTHOR]

A large number of oocytes in the perinatal ovary in rodents get lost for unknown reasons. The granulosa cell-oocyte mutual communication is pivotal for directing formation of the primordial follicle; however, little is known if paracrine factors participate in modulating programmed oocyte death perinatally. We report here that pregranulosa cell-derived fibroblast growth factor 23 (FGF23) functioned in preventing oocyte apoptosis in the perinatal mouse ovary. Our results showed that FGF23 was exclusively expressed in pregranulosa cells, while fibroblast growth factor receptors (FGFRs) were specifically expressed in the oocytes in perinatal ovaries. FGFR1 was one of the representative receptors in mediating FGF23 signaling during the formation of the primordial follicle. In cultured ovaries, the number of live oocytes declines significantly, accompanied by the activation of the p38 mitogen-activated protein kinase signaling pathway, under the condition of FGFR1 disruption by specific inhibitors of FGFR1 or silencing of Fgf23. As a result, oocyte apoptosis increased and eventually led to a decrease in the number of germ cells in perinatal ovaries following the treatments. In the perinatal mouse ovary, pregranulosa cell-derived FGF23 binds to FGFR1 and activates at least the p38 mitogen-activated protein kinase signaling pathway, thereby regulating the level of apoptosis during primordial follicle formation. This study reemphasizes the importance of granulosa cell-oocyte mutual communication in modulating primordial follicle formation and supporting oocyte survival under physiological conditions. [ABSTRACT FROM AUTHOR]

The present study primarily focuses on the efficacy of Malabaricone C (Mal C) as an anti-inflammatory agent. Mal C inhibited mitogen-induced T-cell proliferation and cytokine secretion. Mal C significantly reduced cellular thiols in lymphocytes. N-acetyl cysteine (NAC) restored cellular thiol levels and abrogated Mal C-mediated inhibition of T-cell proliferation and cytokine secretion. Physical interaction between Mal C and NAC was evinced from HPLC and spectral analysis. Mal C treatment significantly inhibited concanavalin A-induced phosphorylation of ERK/JNK and DNA binding of NF-κB. Administration of Mal C to mice suppressed T-cell proliferation and effector functions ex vivo. Mal C treatment did not alter the homeostatic proliferation of T-cells in vivo but completely abrogated acute graft-versus-host disease (GvHD)-associated morbidity and mortality. Our studies indicate probable use of Mal C for prophylaxis and treatment of immunological disorders caused due to hyper-activation of T-cells. [ABSTRACT FROM AUTHOR]

Osteosarcoma is the most common primary bone cancer that affects adolescents with early metastatic potential and drastically reduces their long‐term survival rate if pulmonary metastases are detected at diagnosis. The natural naphthoquinol compound deoxyshikonin exhibits anticancer properties, so we hypothesized that it has an apoptotic effect on osteosarcoma U2OS and HOS cells and studied its mechanisms. After deoxyshikonin treatment, dose‐dependent decreases in cell viability, induction of cell apoptosis and arrest in the sub‐G1 phase of U2OS and HOS cells were observed. The increases in cleaved caspase 3 expression and the decreases in X‐chromosome‐linked IAP (XIAP) and cellular inhibitors of apoptosis 1 (cIAP‐1) expressions after deoxyshikonin treatment in the human apoptosis array were identified in HOS cells, and dose‐dependent expression changes of IAPs and cleaved caspase 3, 8 and 9 were verified by Western blotting in U2OS and HOS cells. Phosphorylation of extracellular signal‐regulated protein kinases (ERK)1/2, c‐Jun N‐terminal kinases (JNK)1/2 and p38 expressions in U2OS and HOS cells was also increased by deoxyshikonin in a dose‐dependent manner. Subsequently, cotreatment with inhibitors of ERK (U0126), JNK (JNK‐IN‐8) and p38 (SB203580) was performed to show that p38 signalling is responsible for deoxyshikonin‐induced apoptosis in U2OS and HOS cells, but not via the ERK and JNK pathways. These discoveries demonstrate that deoxyshikonin may be a possible chemotherapeutic candidate to induce cell arrest and apoptosis by activating extrinsic and intrinsic pathways through p38 for human osteosarcoma. [ABSTRACT FROM AUTHOR]

Many cell- and tissue-level functions are coordinated by intracellular signaling pathways that trigger the expression of context-specific target genes. Yet the input-output relationships that link pathways to the genes they activate are incompletely understood. Mapping the pathway-decoding logic of natural target genes could also provide a basis for engineering novel signal-decoding circuits. Here we report the construction of synthetic immediate-early genes (SynIEGs), target genes of Erk signaling that implement complex, user-defined regulation and can be monitored by using live-cell biosensors to track their transcription and translation. We demonstrate the power of this approach by confirming Erk duration-sensing by FOS, elucidating how the BTG2 gene is differentially regulated by external stimuli, and designing a synthetic immediate-early gene that selectively responds to the combination of growth factor and DNA damage stimuli. SynIEGs pave the way toward engineering molecular circuits that decode signaling dynamics and combinations across a broad range of cellular contexts.

Yes-associated protein (YAP) and its homolog transcriptional coactivator with PDZ-binding motif (TAZ) are key effectors of the Hippo pathway to control cell growth and organ size, of which dysregulation yields to tumorigenesis or hypertrophy. Upon activation, YAP/TAZ translocate into the nucleus and bind to TEAD transcription factors to promote transcriptional programs for proliferation or cell specification. Immediate early genes, represented by AP-1 complex, are rapidly induced and control later-phase transcriptional program to play key roles in tumorigenesis and organ maintenance. Here, we report that YAP/TAZ directly promote FOS transcription that in turn contributes to the biological function of YAP/TAZ. YAP/TAZ bind to the promoter region of FOS to stimulate its transcription. Deletion of YAP/TAZ blocks the induction of immediate early genes in response to mitogenic stimuli. FOS induction contributes to expression of YAP/TAZ downstream target genes. Genetic deletion or chemical inhibition of AP-1 suppresses growth of YAP-driven cancer cells, such as Lats1/2-deficient cancer cells as well as Gαq/11 mutated uveal melanoma. Furthermore, AP-1 inhibition almost completely abrogates the hepatomegaly induced by YAP overexpression. Our findings reveal a feed-forward interplay between immediate early transcription of AP-1 and Hippo pathway function.

Gastric signet ring cell carcinoma (GSRC) is a special subtype of gastric cancer (GC) associated with poor prognosis, but an in-depth and systematic study of GSRC is lacking. Here, we perform single-cell RNA sequencing to assess GC samples. We identify signet ring cell carcinoma (SRCC) cells. Microseminoprotein-beta (MSMB) can be used as a marker gene to guide the identification of moderately/poorly differentiated adenocarcinoma and signet ring cell carcinoma (SRCC). The upregulated differentially expressed genes in SRCC cells are mainly enriched in abnormally activated cancer-related signalling pathways and immune response signalling pathways. SRCC cells are also significantly enriched in mitogen-activated protein kinase and oestrogen signalling pathways, which can interact and promote each other in a positive feedback loop. SRCC cells are shown to have lower cell adhesion and higher immune evasion capabilities as well as an immunosuppressive microenvironment, which may be closely associated with the relatively poor prognosis of GSRC. In summary, GSRC exhibits unique cytological characteristics and a unique immune microenvironment, which may be advantageous for accurate diagnosis and treatment. The tumour microenvironment in signet ring cell carcinoma (SRCC) remains to be characterised. Here, the authors perform single-cell RNA sequencing and reveal distinct features compared to moderately/poorly differentiated adenocarcinoma. [ABSTRACT FROM AUTHOR]

Hepatitis B virus (HBV) infection is a leading cause of hepatocellular carcinoma (HCC), one of the most prevalent malignant tumors worldwide that poses a significant threat to human health. The multifunctional regulator known as Hepatitis B virus X-protein (HBx) interacts with host factors, modulating gene transcription and signaling pathways and contributing to hepatocellular carcinogenesis. The p90 ribosomal S6 kinase 2 (RSK2) is a member of the 90 kDa ribosomal S6 kinase family involved in various intracellular processes and cancer pathogenesis. At present, the role and mechanism of RSK2 in the development of HBx-induced HCC are not yet clear. In this study, we found that HBx upregulates the expression of RSK2 in HBV-HCC tissues, HepG2, and SMMC-7721 cells. We further observed that reducing the expression of RSK2 inhibited HCC cell proliferation. In HCC cell lines with stable HBx expression, RSK2 knockdown impaired the ability of HBx to promote cell proliferation. The extracellularly regulated protein kinases (ERK) 1/2 signaling pathway, rather than the p38 signaling pathway, mediated HBx-induced upregulation of RSK2 expression. Additionally, RSK2 and cyclic adenosine monophosphate (cAMP) response element binding protein (CREB) were highly expressed and positively correlated in HBV-HCC tissues and associated with tumor size. This study showed that HBx upregulates the expression of RSK2 and CREB by activating the ERK1/2 signaling pathway, promoting the proliferation of HCC cells. Furthermore, we identified RSK2 and CREB as potential prognostic markers for HCC patients. [ABSTRACT FROM AUTHOR]

In the current study, we identified a mechanism of resveratrol (RES) underlying its anti-cancer properties against human ovarian adenocarcinoma SKOV-3 cells. We investigated its anti-proliferative and apoptosis-inducing effects in combination with cisplatin, using cell viability assay, flow cytometry, immunofluorescence study and Western blot analysis. We discovered that RES suppressed cancer cell proliferation and stimulated apoptosis, especially when combined with cisplatin. This compound also inhibited SKOV-3 cell survival, which may partly be due to its potential to inhibit protein kinase B (AKT) phosphorylation and induce the S-phase cell cycle arrest. RES in combination with cisplatin strongly induced cancer cell apoptosis through activating the caspase-dependent cascade, which was associated with its ability to stimulate nuclear phosphorylation of p38 mitogen-activated protein kinase (MAPK), well recognized to be involved in transducing environmental stress signals. RES-induced p38 phosphorylation was very specific, and the activation status of extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK) was not mainly affected. Taken together, our study provides accumulated evidence that RES represses proliferation and promotes apoptosis in SKOV-3 ovarian cancer cells through activating the p38 MAPK pathway. It is interesting that this active compound may be used as an effective agent to sensitize ovarian cancer to apoptosis induced by standard chemotherapies. [ABSTRACT FROM AUTHOR]

Maternal embryonic leucine-zipper kinase (MELK) plays a significant role in cell cycle progression, mitosis, cell migration, cell renewal, gene expression, embryogenesis, proliferation, apoptosis, and spliceosome assembly. In addition, MELK is known to be overexpressed in multiple types of cancer and is associated with cancer proliferation. Tumorigenesis shares many similarities with wound healing, in which the rate of cell proliferation is a critical factor. Therefore, this study aimed to determine the involvement of MELK in the regulation of cell division in two cell types involved in this process, namely fibroblasts and keratinocytes. We examined how temporal overexpression of wild-type and kinase-dead MELK kinase variants affect the rate of proliferation, viability, cell cycle, and phosphorylation state of other kinases involved in these processes, such as ERK1/2, AKT1, MAPK9, p38, and p53. We explored if MELK could be used as a therapeutic stimulator of accelerated wound healing via increased proliferation. We observed that aberrant expression of MELK results in abnormal proliferation, altered cell cycle distribution, and decreased viability of the cells, which challenge the utility of MELK in accelerated wound healing. Our results indicate that, at least in healthy cells, any deviation from precisely controlled MELK expression is harmful to fibroblasts and keratinocytes. [ABSTRACT FROM AUTHOR]

Oedema disease (OD) in piglets is one of the most important pathologies, as it causes significant losses due to the high mortality because of the Shiga toxin family, which produces Escherichia coli (STEC) strains. The main toxin responsible for the characteristic pathologies in pigs is Shiga toxin 2 subtype e (Stx2e). Moreover, there is growing evidence that Stx's family of toxins also targets immune cells. Therefore, this study evaluated the effect of different concentrations of Stx2e on porcine immune cells. Porcine peripheral blood mononuclear cells were pre-incubated with Stx2e, at three different concentrations (final concentrations of 10, 500, and 5000 CD50/mL) and with a negative control group. Cells were then stimulated with polyclonal mitogens: concanavalin A, phytohemagglutinin, pokeweed mitogen, or lipopolysaccharides. Cell proliferation was assessed by BrdU (or EdU) incorporation into newly created DNA. The activation of the lymphocyte subsets was assessed by the detection of CD25, using flow cytometry. The toxin significantly decreased mitogen-driven proliferation activity, and the effect was partially dose-dependent, with a significant impact on both T and B populations. The percentage of CD25+ cells was slightly lower in the presence of Stx2e in all the defined T cell subpopulations (CD4+, CD8+, and γδTCR+)—in a dose-dependent manner. B cells seemed to be the most affected populations. The negative effects of different concentrations of Stx2e on the immune cells in this study may explain the negative impact of the subclinical course of OD. [ABSTRACT FROM AUTHOR]

Oral squamous cell carcinoma (OSCC) has a high recurrence rate and poor prognosis. Hispolon, a polyphenolic compound with antiviral, antioxidant, and anticancer activities, is a potential chemotherapy agent. However, few studies have investigated the anti‐cancer mechanism of hispolon in oral cancer. This present study used the cell viability assay, clonogenic assay, fluorescent nuclear staining, and flow cytometry assay to analyse the apoptosis‐inducing effects of hispolon in OSCC cells. After hispolon treatment, the apoptotic initiators, cleaved caspase‐3, −8, and − 9, were upregulated, whereas the cellular inhibitor of apoptosis protein‐1 (cIAP1) was downregulated. Furthermore, a proteome profile analysis using a human apoptosis array revealed the overexpression of heme oxygenase‐1 (HO‐1) by hispolon, which was determined to be involved in caspase‐dependent apoptosis. Moreover, cotreatment with hispolon and mitogen‐activated protein kinase (MAPK) inhibitors revealed that hispolon induces apoptosis in OSCC cells through activation of the c‐Jun N‐terminal kinase (JNK) pathway and not the extracellular signal‐regulated kinase (ERK) or p38 pathway. These findings indicate that hispolon may exert an anticancer effect on oral cancer cells by upregulating HO‐1 and inducing caspase‐dependent apoptosis by activating the JNK pathway. [ABSTRACT FROM AUTHOR]

A loss of functional beta cell mass is a final etiological event in the development of frank type 2 diabetes (T2D). To preserve or expand beta cells and therefore treat/prevent T2D, growth factors have been considered therapeutically but have largely failed to achieve robust clinical success. The molecular mechanisms preventing the activation of mitogenic signaling pathways from maintaining functional beta cell mass during the development of T2D remain unknown. We speculated that endogenous negative effectors of mitogenic signaling cascades impede beta cell survival/expansion. Thus, we tested the hypothesis that a stress-inducible epidermal growth factor receptor (EGFR) inhibitor, mitogen-inducible gene 6 (Mig6), regulates beta cell fate in a T2D milieu. To this end, we determined that: (1) glucolipotoxicity (GLT) induces Mig6, thereby blunting EGFR signaling cascades, and (2) Mig6 mediates molecular events regulating beta cell survival/death. We discovered that GLT impairs EGFR activation, and Mig6 is elevated in human islets from T2D donors as well as GLT-treated rodent islets and 832/13 INS-1 beta cells. Mig6 is essential for GLT-induced EGFR desensitization, as Mig6 suppression rescued the GLT-impaired EGFR and ERK1/2 activation. Further, Mig6 mediated EGFR but not insulin-like growth factor-1 receptor nor hepatocyte growth factor receptor activity in beta cells. Finally, we identified that elevated Mig6 augmented beta cell apoptosis, as Mig6 suppression reduced apoptosis during GLT. In conclusion, we established that T2D and GLT induce Mig6 in beta cells; the elevated Mig6 desensitizes EGFR signaling and induces beta cell death, suggesting Mig6 could be a novel therapeutic target for T2D. [ABSTRACT FROM AUTHOR]

The efficiency of inducing human embryonic stem cells into NEUROG3+ pancreatic endocrine cells is a bottleneck in stem cell therapy for diabetes. To understand the cell properties and fate decisions during differentiation, we analyzed the modified induction method using single-cell transcriptome and found that DAPT combined with four factors (4FS): nicotinamide, dexamethasone, forskolin and Alk5 inhibitor II (DAPT + 4FS) increased the expression of NEUROG3 to approximately 34.3%. The increased NEUROG3+ cells were mainly concentrated in Insulin + Glucagon + (INS + GCG+) and SLAC18A1 + Chromogranin A+(SLAC18A1 + CHGA +) populations, indicating that the increased NEUROG3+ cells promoted the differentiation of pancreatic endocrine cells and enterochromaffin-like cells. Single-cell transcriptome analysis provided valuable clues for further screening of pancreatic endocrine cells and differentiation of pancreatic islet cells. The gene set enrichment analysis (GSEA) suggest that we can try to promote the expression of INS + GCG+ population by up-regulating G protein-coupled receptor (GPCR) and mitogen-activated protein kinase signals and down-regulating Wnt, NIK/NF-KappaB and cytokine-mediated signal pathways. We can also try to regulate GPCR signaling through PLCE1, so as to increase the proportion of NEUROG3+ cells in INS+GCG+ populations. To exclude non-pancreatic endocrine cells, ALCAMhigh CD9low could be used as a marker for endocrine populations, and ALCAMhigh CD9lowCDH1low could remove the SLC18A1 + CHGA+ population. [ABSTRACT FROM AUTHOR]

Simple Summary: Excellent quality of early embryonic development contributes to a successful pregnancy. At present, most in vitro cultured embryos can only develop to the blastocyst stage at most, because an in vitro culture (IVC) system cannot replace the physiological environment in vivo. During IVC, excessive accumulated reactive oxygen species in embryos cannot be easily metabolized, which will cause oxidative stress and suppress embryo development. In this study, we found that anti-oxidation capacity of early embryo was improved by adding 6-gingerol to IVC. Moreover, 6-gingerol can also improve blastocyst rate, cell proliferation, mitochondrial function, inhibit cell apoptosis, autophagy, and regulate functional genes expression in blastocyst. These results are helpful to optimize the early embryo culture system, and thus provide a theoretical basis for improving the early embryo quality and the efficiency of subsequent pregnancy. 6-Gingerol, the main active ingredient in ginger, exhibits a variety of biological activities, such as antioxidant, anti-inflammatory, and anticancer activities, and can affect cell development. However, the effects of 6-gingerol on mammalian reproductive processes, especially early embryonic development, are unclear. This study explored whether 6-gingerol can be used to improve the quality of in vitro-cultured porcine embryos. The results showed that 5 μM 6-gingerol significantly increased the blastocyst formation rates of porcine early embryos. 6-Gingerol attenuated intracellular reactive oxygen species accumulation and autophagy, increased intracellular glutathione levels, and increased mitochondrial activity. In addition, 6-gingerol upregulated NANOG, SRY-box transcription factor 2, cytochrome c oxidase subunit II, mechanistic target of rapamycin kinase, and RPTOR independent companion of MTOR complex 2 while downregulating Caspase 3, baculoviral IAP repeat containing 5, autophagy related 12, and Beclin 1. Most importantly, 6-gingerol significantly increased the levels of p-extracellular regulated protein kinase 1/2 while reducing the levels of p-c-Jun N-terminal kinase 1/2/3 and p-p38. These results indicate that 6-gingerol can promote the development of porcine early embryos in vitro. [ABSTRACT FROM AUTHOR]

Oxidative stress regulates many physiological and pathological processes. Indeed, a low increase in the basal level of reactive oxygen species (ROS) is essential for various cellular functions, including signal transduction, gene expression, cell survival or death, as well as antioxidant capacity. However, if the amount of generated ROS overcomes the antioxidant capacity, excessive ROS results in cellular dysfunctions as a consequence of damage to cellular components, including DNA, lipids and proteins, and may eventually lead to cell death or carcinogenesis. Both in vitro and in vivo investigations have shown that activation of the mitogen-activated protein kinase kinase 5/extracellular signal-regulated kinase 5 (MEK5/ERK5) pathway is frequently involved in oxidative stress-elicited effects. In particular, accumulating evidence identified a prominent role of this pathway in the anti-oxidative response. In this respect, activation of krüppel-like factor 2/4 and nuclear factor erythroid 2-related factor 2 emerged among the most frequent events in ERK5-mediated response to oxidative stress. This review summarizes what is known about the role of the MEK5/ERK5 pathway in the response to oxidative stress in pathophysiological contexts within the cardiovascular, respiratory, lymphohematopoietic, urinary and central nervous systems. The possible beneficial or detrimental effects exerted by the MEK5/ERK5 pathway in the above systems are also discussed. [ABSTRACT FROM AUTHOR]