Ribosome Collisions Trigger General Stress Responses to Regulate Cell Fate.

Problems arising during translation of mRNAs lead to ribosome stalling and collisions that trigger a series of quality control events. However, the global cellular response to ribosome collisions has not been explored. Here, we uncover a function for ribosome collisions in signal transduction. Using translation elongation inhibitors and general cellular stress conditions, including amino acid starvation and UV irradiation, we show that ribosome collisions activate the stress-activated protein kinase (SAPK) and GCN2-mediated stress response pathways. We show that the MAPKKK ZAK functions as the sentinel for ribosome collisions and is required for immediate early activation of both SAPK (p38/JNK) and GCN2 signaling pathways. Selective ribosome profiling and biochemistry demonstrate that although ZAK generally associates with elongating ribosomes on polysomal mRNAs, it specifically auto-phosphorylates on the minimal unit of colliding ribosomes, the disome. Together, these results provide molecular insights into how perturbation of translational homeostasis regulates cell fate.

Disome and Trisome Profiling Reveal Genome-wide Targets of Ribosome Quality Control.

The ribosome-associated protein quality control (RQC) system that resolves stalled translation events is activated when ribosomes collide and form disome, trisome, or higher-order complexes. However, it is unclear whether this system distinguishes collision complexes formed on defective mRNAs from those with functional roles on endogenous transcripts. Here, we performed disome and trisome footprint profiling in yeast and found collisions were enriched on diverse sequence motifs known to slow translation. When 60S recycling was inhibited, disomes accumulated at stop codons and could move into the 3' UTR to reinitiate translation. The ubiquitin ligase and RQC factor Hel2/ZNF598 generally recognized collisions but did not induce degradation of endogenous transcripts. However, loss of Hel2 triggered the integrated stress response, via phosphorylation of eIF2α, thus linking these pathways. Our results suggest that Hel2 has a role in sensing ribosome collisions on endogenous mRNAs, and such events may be important for cellular homeostasis.

Cellular stress induces non-canonical activation of the receptor tyrosine kinase EphA2 through the p38-MK2-RSK signaling pathway.

The receptor tyrosine kinase ephrin type-A receptor 2 (EphA2) is overexpressed in malignant tumors. We previously reported that non-canonical EphA2 phosphorylation at Ser-897 was catalyzed by p90 ribosomal S6 kinase (RSK) via the MEK-ERK pathway in ligand- and tyrosine kinase-independent manners. Non-canonical EphA2 activation plays a key role in tumor progression; however, its activation mechanism remains unclear. In the present study, we focused on cellular stress signaling as a novel inducer of non-canonical EphA2 activation. p38, instead of ERK in the case of epidermal growth factor signaling, activated RSK-EphA2 under cellular stress conditions, including anisomycin, cisplatin, and high osmotic stress. Notably, p38 activated the RSK-EphA2 axis via downstream MAPK-activated protein kinase 2 (MK2). Furthermore, MK2 directly phosphorylated both RSK1 Ser-380 and RSK2 Ser-386, critical residues for the activation of their N-terminal kinases, which is consistent with the result showing that the C-terminal kinase domain of RSK1 was dispensable for MK2-mediated EphA2 phosphorylation. Moreover, the p38-MK2-RSK-EphA2 axis promoted glioblastoma cell migration induced by temozolomide, a chemotherapeutic agent for the treatment of glioblastoma patients. Collectively, the present results reveal a novel molecular mechanism for non-canonical EphA2 activation under stress conditions in the tumor microenvironment.

Anisomycin inhibits the activity of human ovarian cancer stem cells via regulating antisense RNA NCBP2-AS2/MEK/ERK/STAT3 signaling.

BACKGROUND: Ovarian cancer stem cells (OCSCs) are the main cause of relapse and drug resistance in patients with ovarian cancer. Anisomycin has been shown to be an effective antitumor agent, but its mechanism of action in ovarian cancer remains elusive. METHODS: CD44+/CD133+ human OCSCs were isolated from human ovarian cancer tissues. OCSCs were interfered with using anisomycin and specific small-interfering RNA (siRNA). Microarray assay, MTT, in vivo tumorigenic experiments, transwell assay, cell cycle assay, colony formation assay, angiogenesis assay, and hematoxylin and eosin staining were used to detect the mechanism of anisomycin with respect to inhibiting the activity of OCSCs. Expression of the NCBP2-AS2/mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)/signal transducer and activator of transcription 3 (STAT3) pathway was examined using western blotting, a quantitative real-time PCR (RT-qPCR) and immunofluorescence staining. Bioinformatics analysis was used for predictive analysis of NCBP2-AS2 expression in urogenital tumors. RESULTS: Microarray analysis showed that treatment with anisomycin significantly decreased the expression of antisense RNA NCBP2-AS2 in OCSCs. In vitro cellular experiments showed that interfering with endogenous antisense RNA NCBP2-AS2 using siRNA distinctly inhibited the proliferation, migration and angiogenesis of OCSCs, whereas in vivo animal experiments revealed decreased tumorigenesis in nude mice. Moreover, the results of RT-qPCR and western blotting demonstrated that both anisomycin treatment and NCBP2-AS2 silencing led to significant reductions in the mRNA and protein expression levels of NCBP2-AS2, MEK, ERK and STAT3. From a bioinformatic point of view, antisense RNA NCBP2-AS2 exhibited significantly differential expression between urogenital tumors and normal controls, and a similar expression pattern was found in the genes NCBP2, RPL35A, DNAJC19 and ECE2, which have similarity to NCBP2-AS2. CONCLUSIONS: Anisomycin suppresses the in vivo and in vitro activity of human OCSCs by downregulating the antisense RNA NCBP2-AS2/MEK/ERK/STAT3 signaling pathway, whereas the antisense RNA NCBP2-AS2 and genes with similarity have the potential to serve as markers for clinical diagnosis and prognosis of urogenital tumors.

Integration of genetically regulated gene expression and pharmacological library provides therapeutic drug candidates.

Approaches toward new therapeutics using disease genomics, such as genome-wide association study (GWAS), are anticipated. Here, we developed Trans-Phar [integration of transcriptome-wide association study (TWAS) and pharmacological database], achieving in silico screening of compounds from a large-scale pharmacological database (L1000 Connectivity Map), which have inverse expression profiles compared with tissue-specific genetically regulated gene expression. Firstly we confirmed the statistical robustness by the application of the null GWAS data and enrichment in the true-positive drug-disease relationships by the application of UK-Biobank GWAS summary statistics in broad disease categories, then we applied the GWAS summary statistics of large-scale European meta-analysis (17 traits; naverage = 201 849) and the hospitalized COVID-19 (n = 900 687), which has urgent need for drug development. We detected potential therapeutic compounds as well as anisomycin in schizophrenia (false discovery rate (FDR)-q = 0.056) and verapamil in hospitalized COVID-19 (FDR-q = 0.068) as top-associated compounds. This approach could be effective in disease genomics-driven drug development.

Cordycepin buffers anisomycin-induced fear memory deficit by restoring hippocampal BDNF.

The process of memory consolidation involves the synthesis of new proteins, and interfering with protein synthesis through anisomycin can impair memory. Memory deficits due to aging and sleep disorders may also result from a reduction in protein synthesis. Rescuing memory deficits caused by protein synthesis deficiency is therefore an important issue that needs to be addressed. Our study focused on the effects of cordycepin on fear memory deficits induced by anisomycin using contextual fear conditioning. We observed that cordycepin was able to attenuate these deficits and restore BDNF levels in the hippocampus. The behavioral effects of cordycepin were dependent on the BDNF/TrkB pathway, as demonstrated by the use of ANA-12. Cordycepin had no significant impact on locomotor activity, anxiety or fear memory. Our findings provide the first evidence that cordycepin can prevent anisomycin-induced memory deficits by regulating BDNF expression in the hippocampus.

Combined Exogenous Activation of Bovine Oocytes: Effects on Maturation-Promoting Factor, Mitogen-Activated Protein Kinases, and Embryonic Competence.

Oocyte activation via dual inhibition of protein synthesis and phosphorylation has improved in vitro embryo production in different mammalian species. In this study, we evaluated the effects of the combination of cycloheximide (CHX), dimethyl amino purine (DMAP), and anisomycin (ANY) on the activation of bovine oocytes, particularly on dynamics of MPF and MAPKs, embryonic developmental potential, and quality. The results showed that the cleavage and blastocyst rates, as well as levels of CCNB1, CDK1, p-CDK1Thr161, and p-CDK1Thr14-Tyr15, were similar among groups; ANY and ANY + CHX reduced the expression of ERK1/2 compared to DMAP-combinations (p < 0.05), whereas ANY + DMAP, CHX + DMAP, and ANY + CHX + DMAP reduced p-ERK1/2 compared to ANY and ANY + CHX treatments (p < 0.05). The quality of blastocysts in terms of cell counts, their allocation, and the numbers of TUNEL-positive cells did not differ among groups. However, transcript levels of POU5F1 were higher in embryos derived from ANY + CHX + DMAP treatment compared to other groups, while expression levels of CDX2 did not show differences. In addition, the BCL2A1/BAX ratio of the ANY + CHX + DMAP treatment was significantly low compared to the ANY treatment (p < 0.05) and did not differ significantly from the other treatments. In conclusion, oocyte activation by dual inhibition of protein synthesis and phosphorylation induces MPF inactivation without degradation of CCNB1, while MAPK inactivation occurs differentially between these inhibitors. Thus, although the combined use of these inhibitors does not affect early developmental competence in vitro, it positively impacts the expression of transcripts associated with embryonic quality.

C-Jun N-Terminal Kinase Post-Translational Regulation of Pain-Related Acid-Sensing Ion Channels 1b and 3.

Neuronal proton-gated acid-sensing ion channels (ASICs) participate in the detection of tissue acidosis, a phenomenon often encountered in painful pathologic diseases. Such conditions often involve in parallel the activation of various signaling pathways such as mitogen activated protein kinases (MAPKs) that ultimately leads to phenotype modifications of sensory neurons. Here, we identify one member of the MAPKs, c-Jun N-terminal kinase (JNK), as a new post-translational positive regulator of ASICs in rodent sensory neurons. Recombinant H+-induced ASIC currents in HEK293 cells are potently inhibited within minutes by the JNK inhibitor SP600125 in a subunit-dependent manner, targeting both rodent and human ASIC1b and ASIC3 subunits (except mouse ASIC3). The regulation by JNK of recombinant ASIC1b- and ASIC3-containing channels (homomers and heteromers) is lost on mutation of a putative phosphorylation site within the intracellular N- and the C-terminal domain of the ASIC1b and ASIC3 subunit, respectively. Moreover, short-term JNK activation regulates the activity of native ASIC1b- and ASIC3-containing channels in rodent sensory neurons and is involved in the rapid potentiation of ASIC activity by the proinflammatory cytokine TNFα. Local JNK activation in vivo in mice induces a short-term potentiation of the acid-induced cutaneous pain in inflammatory conditions that is partially blocked by the ASIC1-specific inhibitor mambalgin-1. Collectively, our data identify pain-related channels as novel physiological JNK substrates in nociceptive neurons and propose JNK-dependent phosphorylation as a fast post-translational mechanism of regulation of sensory-neuron-expressed ASIC1b- and ASIC3-containing channels that may contribute to peripheral sensitization and pain hypersensitivity.SIGNIFICANCE STATEMENT ASICs are a class of excitatory cation channels critical for the detection of tissue acidosis, which is a hallmark of several painful diseases. Previous work in sensory neurons has shown that ASICs containing the ASIC3 or the ASIC1b subunit are important players in different pain models. We combine here functional and pharmacological in vitro and in vivo approaches to demonstrate that the MAP Kinase JNK is a potent post-translational positive regulator, probably via direct phosphorylation, of rodent and human ASIC1b- and ASIC3-containing channels. This JNK-dependent, fast post-translational mechanism of regulation of sensory-neuron-expressed ASICs may contribute to peripheral sensitization and pain hypersensitivity. These data also identify pain-related channels as direct downstream effectors of JNK in nociceptors.

IL-1ß is a key inflammatory cytokine that weakens lactation-specific tight junctions of mammary epithelial cells.

In lactating mammary glands, alveolar mammary epithelial cells (MECs) produce milk and form less-permeable tight junctions (TJs). However, alveolar TJs are weakened with a reduction in milk production in mammary glands due to mastitis or weaning in the presence of high levels of IL-1ß, IL-6, or TNF-α. In this study, using in vitro cultured model of MECs with milk-producing ability and lactation-specific TJs, we investigated whether the aforementioned cytokines affect MEC TJs. The results showed that TNF-α, IL-1ß, and IL-6 affected lactation-specific TJs in different ways. In particular, upon activation of p38 and JNK signalling, IL-1ß caused rapid disruption of TJs at tricellular contact points. IL-1ß treatment led to decreased CLDN3, CLDN4, and OCLN levels and a weakened TJ barrier. The adverse effects of IL-1ß on TJs were mimicked by anisomycin, which is an activator of p38 and JNK signalling, and were blocked by MEC pretreatment with a p38 inhibitor but not a JNK inhibitor. The mislocalization of tricellulin at tricellular contact areas was confirmed in MECs treated with IL-1ß or anisomycin. These results indicate that IL-1ß is a key cytokine that adversely affects the TJs between MECs by activating p38.

Anisomycin inhibits angiogenesis, growth, and survival of triple-negative breast cancer through mitochondrial dysfunction, AMPK activation, and mTOR inhibition.

Aberrant upregulation of mitochondrial biogenesis is observed in breast cancer and holds potential therapeutic option. In our work, we showed that inhibition of mitochondrial function by anisomycin is effective against triple-negative breast cancer (TNBC). Anisomycin inhibits growth and induces caspase-dependent apoptosis in a panel of TNBC cell lines. Of note, anisomycin at a tolerable dose remarkably suppresses growth of TNBC in mice. In addition, anisomycin effectively targets breast cancer angiogenesis through inhibiting capillary network formation, migration, proliferation, and survival. Mechanistic studies show that although anisomycin activates p38 and JNK, their activations are not required for anisomycin's action. In contrast, anisomycin inhibits mitochondrial respiration, and decreases mitochondrial membrane potential and adenosine triphosphate (ATP) level. The inhibitory effect of anisomycin is significantly reversed in mitochondria respiration-deficient ρ0 cells. As a consequence, anisomycin activates AMPK and inhibits mammalian target-of-rapamycin signaling pathways. Our work demonstrated that anisomycin is a useful addition to the treatment armamentarium for TNBC.

Extinction learning with social support depends on protein synthesis in prefrontal cortex but not hippocampus.

Extinction of contextual fear conditioning (CFC) in the presence of a familiar nonfearful conspecific (social support), such as that of others tasks, can occur regardless of whether the original memory is retrieved during the extinction training. Extinction with social support is blocked by the protein synthesis inhibitors anisomycin and rapamycin and by the inhibitor of gene expression 5,6-dichloro-1-ß-d-ribofuranosylbenzimidazole infused immediately after extinction training into the ventromedial prefrontal cortex (vmPFC) but unlike regular CFC extinction not in the CA1 region of the dorsal hippocampus. So social support generates a form of learning that differs from extinction acquired without social support in terms of the brain structures involved. This finding may lead to a better understanding of the brain mechanisms involved in the social support of memories and in therapies for disorders related to dysfunctional fear memories. Thus, here we show that the consolidation of extinction memory with social support relies on vmPFC rather than hippocampus gene expression and ribosomal- and mammalian target of rapamycin-dependent protein synthesis. These results provide additional knowledge about the cellular mechanisms and brain structures involved on the effect of social support in changing behavior and fear extinction memory.

Motor neuron loss in SMA is not associated with somal stress-activated JNK/c-Jun signaling.

A pathological hallmark of spinal muscular atrophy (SMA) is severe motor neuron (MN) loss, which results in muscle weakness and often infantile or childhood mortality. Although it is well established that deficient expression of survival motor neuron (SMN) protein causes SMA, the molecular pathways that execute MN cell death are poorly defined. The c-Jun NH2-terminal kinases (JNKs) are stress-activated kinases with multiple substrates including c-Jun, which can be activated during neuronal injury and neurodegenerative disease leading to neuronal apoptosis. Recently, increased JNK-c-Jun signaling was reported in SMA raising the possibility that JNK inhibitors could be a novel treatment for this disease. We examined JNK-c-Jun activity in SMA mouse and human cultured cells and tissues. Anisomycin treatment of human SMA fibroblasts and sciatic nerve ligation in SMA mice provoked robust phosphorylated-c-Jun (p-c-Jun) expression indicating that SMN-deficiency does not prevent activation of the stress-induced JNK-c-Jun signaling pathway. Despite retained capacity to activate JNK-c-Jun, we observed no basal increase of p-c-Jun levels in SMA compared to control cultured cells, human or mouse spinal cord tissues, or mouse MNs during the period of MN loss in severe SMA model mice. In both controls and SMA, ~50% of α-MN nuclei express p-c-Jun with decreasing expression during the early postnatal period. Together these studies reveal no evidence of stress-activated JNK-c-Jun signaling in MNs of SMA mice or human tissues, but do highlight the important role of JNK-c-Jun activity during normal MN development raising caution about JNK antagonism in this pediatric neuromuscular disease.

Elucidating the role of protein synthesis in hippocampus-dependent memory consolidation across the day and night.

It is widely acknowledged that de novo protein synthesis is crucial for the formation and consolidation of long-term memories. While the basal activity of many signaling cascades that modulate protein synthesis fluctuates in a circadian fashion, it is unclear whether the temporal dynamics of protein synthesis-dependent memory consolidation vary depending on the time of day. More specifically, it is unclear whether protein synthesis inhibition affects hippocampus-dependent memory consolidation in rodents differentially across the day (i.e., the inactive phase with an abundance of sleep) and night (i.e., the active phase with little sleep). To address this question, male and female C57Bl6/J mice were trained in a contextual fear conditioning task at the beginning or the end of the light phase. Animals received a single systemic injection with the protein synthesis inhibitor anisomycin or vehicle directly, 4, 8 hr, or 11.5 hr following training, and memory was assessed after 24 hr. Here, we show that protein synthesis inhibition impaired the consolidation of context-fear memories selectively when the protein synthesis inhibitor was administered at the first three time points, irrespective of timing of training. Even though the basal activity of signaling pathways regulating de novo protein synthesis may fluctuate across the 24-hr cycle, these results suggest that the temporal dynamics of protein synthesis-dependent memory consolidation are similar for day-time and night-time learning.

PP1/PP2A phosphatase inhibition-induced metaplasticity in protein synthesis blocker-treated hippocampal slices: LTP and LTD, or There and Back again.

Long-term potentiation (LTP) and long-term depression (LTD) are key forms of synaptic plasticity in the hippocampus. LTP and LTD are believed to underlie the processes occurring during learning and memory. Search of mechanisms responsible for switching from LTP to LTD and vice versa is an important fundamental task. Protein synthesis blockers (PSB) are widely used in models of memory impairment and LTP suppression. Here, we found that blockade of serine/threonine phosphatases 1 (PP1) and 2A (PP2A) with the specific blockers, calyculin A (CalyA) or okadaic acid (OA), and simultaneous blockade of the protein translation by anisomycin or cycloheximide leads to a switch from PSB-impaired LTP to LTD. PP1/PP2A-dependent LTD was extremely sensitive to the intensity of the test stimuli, whose increase restored the field excitatory postsynaptic potentials (fEPSP) to the values corresponding to control LTP in the non-treated slices. PP1/PP2A blockade affected the basal synaptic transmission, increasing the paired-pulse facilitation (PPF) ratio, and restored the PSB-impaired PPF 3 h after tetanus. Prolonged exposure to anisomycin led to the NO synthesis increase (measured using fluorescent dye) both in the dendrites and somata of CA1, CA3, dentate gyrus (DG) hippocampal layers. OA partially prevented the NO production in the CA1 dendrites, as well in the CA3 and DG somas. Direct measurements of changes in serine/threonine phosphatase (STPP) activity revealed importance of the PP1/PP2A-dependent component in the late LTP phase (L-LTP) in anisomycin-treated slices. Thus, serine/threonine phosphatases PP1/PP2A influence both basal synaptic transmission and stimulation-induced synaptic plasticity.

Combined effect of chrysin and apigenin on inhibiting the development and progression of colorectal cancer by suppressing the activity of P38-MAPK/AKT pathway.

Either apigenin or chrysin alone has been found to exert anti-inflammatory and tumor suppressive effect. However, the combined effect of apigenin and chrysin on colorectal cancer (CRC) has not been fully clarified. We attempted to explore the effect of chrysin and apigenin on CRC and its related mechanism. SW480 and HCT-116 cells were treated with either apigenin or chrysin alone or two-drug combination at different doses of 5, 25, 50, 100 µM for optimal concentration determination. Then, we focused on the individual and combined effect of apigenin and chrysin on clonogenicity, apoptosis, metastasis-related behaviors of CRC cells by colony formation assay, cell scratch assay, flow cytometry, and transwell assay. The changes of the activation of P38-MAPK/AKT pathway were evaluated underlying apigenin and chrysin intervention, further after co-treated with P38-MAPK agonist anisomycin. Apigenin (25 µM) combined with chrysin (25 µM) were determined to be optimal. Treatment with the combination of apigenin (25 µM) and chrysin (25 µM) significantly reduced cell clone numbers, migration, and invasion ability, while increased the cell apoptosis in both CRC cell lines. The combined effect was higher than chrysin or apigenin alone. Meanwhile, p-P38 and p-AKT were significantly downregulated by chrysin and apigenin treatment. The tumor inhibitive effect of apigenin combined with chrysin was obviously reversed by adding P38 agonist, anisomycin. Apigenin (25 µM) combined with chrysin (25 µM) showed synergetic effect in inhibiting the growth and metastasis of CRC cells by suppressing the activity of P38-MAPK/AKT pathway.

Anisomycin is active in preclinical models of pediatric acute myeloid leukemia via specifically inhibiting mitochondrial respiration.

The poor outcomes in acute myeloid leukemia (AML) necessitate new treatments. In this work, we identified that anisomycin is a potential selective anti-AML candidate, particularly for those with FLT3-ITD mutation. We found that anisomycin potently inhibited proliferation and induced apoptosis in multiple AML cell lines. Anisomycin was effective in targeting progenitor cells isolated from all tested pediatric AML patients, while sparing normal counterparts. Using AML xenograft mouse models, anisomycin exhibited inhibitory effect on tumor growth throughout the whole duration without causing toxicity in mice. The combination of anisomycin with standard of care drugs is synergistic and selective in AML cell culture system and mouse model. In addition, FLT3-ITD cells were more sensitive to anisomycin than FLT3 WT cells. Mechanistic studies revealed that anisomycin acted on AML in a p38-independent manner. We found that anisomycin decreased mitochondrial respiration by disrupting complex I activity, leading to intracellular oxidative stress. AML ρ0 cells that lack of mitochondrial respiration exhibited resistance to anisomycin. Finally, we showed that mitochondrial biogenesis contributes to differential sensitivity of FLT3-ITD and FLT3 WT cells to anisomycin. Our work is the first to systematically demonstrate that anisomycin is a useful addition to the treatment armamentarium for AML. Our findings highlight the therapeutic value of mitochondrial respiration inhibition in AML patients harboring FLT3-ITD mutation.

Nucleus reuniens of the thalamus controls fear memory reconsolidation.

The nucleus reuniens has been shown to support the acquisition, consolidation, maintenance, destabilization upon retrieval, and extinction of aversive memories. However, the direct participation of this thalamic subregion in memory reconsolidation is yet to be examined. The present study addressed this question in contextually fear-conditioned rats. Post-reactivation infusion of the GABAA receptor agonist muscimol, the glutamate N2A-containing NMDA receptor antagonist TCN-201, or the protein synthesis inhibitor anisomycin into the NR induced significant impairments in memory reconsolidation. Administering muscimol or TCN-201 and anisomycin locally, or associating locally infused muscimol or TCN-201 with systemically administered clonidine, an α2-receptor adrenergic agonist that attenuates the noradrenergic tonus associated with memory reconsolidation, produced no further reduction in freezing times when compared with the muscimol-vehicle, TCN-201-vehicle, vehicle-anisomycin, and vehicle-clonidine groups. This pattern of results indicates that such treatment combinations produced no additive/synergistic effects on reconsolidation. It is plausible that NR inactivation and antagonism of glutamate N2A-containing NMDA receptors weakened/prevented the subsequent action of anisomycin and clonidine because they disrupted the early stages of signal transduction pathways involved in memory reconsolidation. It is noteworthy that these pharmacological interventions, either alone or combined, induced no contextual memory specificity changes, as assessed in a later test in a novel and unpaired context. Besides, omitting memory reactivation precluded the impairing effects of muscimol, TCN-201, anisomycin, and clonidine on reconsolidation. Together, the present findings demonstrate interacting mechanisms through which the NR can regulate contextual fear memory restabilization.

Temozolomide Induces Endocytosis of EGFRvIII via p38-Mediated Non-canonical Phosphorylation in Glioblastoma Cells.

The ligand-induced internalization of epidermal growth factor receptor (EGFR) is generally considered to attenuate downstream signaling via its endosomal degradation. However, the endocytosis of an oncogenic EGFR variant III (EGFRvIII) is impaired, which leads to persistent signaling from the cell surface, thereby promoting the proliferation and survival of glioblastoma multiforme (GBM) cells. Cellular stress triggers the non-canonical endocytosis-recycling of EGFR by p38-mediated phosphorylation. In the present study, we used temozolomide (TMZ), the standard chemotherapeutic agent for the treatment of GBM patients, to examine whether EGFRvIII is controlled by a non-canonical mechanism. TMZ triggered the endocytic trafficking of serine phosphorylated EGFRvIII. Moreover, phosphorylation and endocytosis were abrogated by the selective p38 inhibitor SB203580, but not gefitinib, indicating that EGFRvIII is recruited to p38-mediated non-canonical endocytosis. The combination of TMZ and SB203580 also showed potential inhibitory effects on the proliferation and motility of glioblastoma cells.

Lipoxin A4-Mediated p38 MAPK Signaling Pathway Protects Mice Against Collagen-Induced Arthritis.

The aim of the article was to study the mechanism of Lipoxin A4 (LXA4)-mediated p38 MAPK pathway protecting mice against collagen-induced arthritis (CIA). The impact of LXA4 (0, 5, 10, 15 nM) on synoviocytes proliferation of CIA mice was detected using 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. CIA mice were treated with LXA4, SB203580 (a p38 inhibitor), and/or anisomycin (a p38 agonist), and the arthritis severity score in each mouse was determined. The gene or protein expressions were detected with Western Blotting, ELISA, or qRT-PCR. LXA4 inhibited the synoviocytes proliferation of CIA mice with decreased levels of TNF-α, IL-6, IL-1ß, and IFN-γ and reduced p-p38/total p38 expression in synoviocytes in a dose-dependent manner. LXA4 levels were decreased in synovial tissues and plasma of CIA mice, but p-p38/total p38 expression was increased in synovial tissues. LXA4 could downregulate p-p38/total p38 expression in synovial tissues of CIA mice. Both LXA4 and SB203580 reduced arthritis severity score of CIA mice with the reduction of synovial tissue hyperplasia and inflammatory cell infiltration. CIA mice treated with LXA4 and SB203580 had lower levels of TNF-α, IL-6, IL-1ß, and IFN-γ, accompanying decreased MDA as well as increased SOD, CAT,and GPx. However, anisomycin could reverse the protect effects of LXA4 on CIA mice regarding the abovementioned inflammatory factors and oxidative stress indexes. LXA4 protected mice against collagen-induced arthritis via inhibiting p38 MAPK signaling pathway, which may be a potential new therapeutic target for rheumatoid arthritis.

Serine/Threonine Phosphatases in LTP: Two B or Not to Be the Protein Synthesis Blocker-Induced Impairment of Early Phase.

Dephosphorylation of target proteins at serine/threonine residues is one of the most crucial mechanisms regulating their activity and, consequently, the cellular functions. The role of phosphatases in synaptic plasticity, especially in long-term depression or depotentiation, has been reported. We studied serine/threonine phosphatase activity during the protein synthesis blocker (PSB)-induced impairment of long-term potentiation (LTP). Established protein phosphatase 2B (PP2B, calcineurin) inhibitor cyclosporin A prevented the LTP early phase (E-LTP) decline produced by pretreatment of hippocampal slices with cycloheximide or anisomycin. For the first time, we directly measured serine/threonine phosphatase activity during E-LTP, and its significant increase in PSB-treated slices was demonstrated. Nitric oxide (NO) donor SNAP also heightened phosphatase activity in the same manner as PSB, and simultaneous application of anisomycin + SNAP had no synergistic effect. Direct measurement of the NO production in hippocampal slices by the NO-specific fluorescent probe DAF-FM revealed that PSBs strongly stimulate the NO concentration in all studied brain areas: CA1, CA3, and dentate gyrus (DG). Cyclosporin A fully abolished the PSB-induced NO production in the hippocampus, suggesting a close relationship between nNOS and PP2B activity. Surprisingly, cyclosporin A alone impaired short-term plasticity in CA1 by decreasing paired-pulse facilitation, which suggests bi-directionality of the influences of PP2B in the hippocampus. In conclusion, we proposed a minimal model of signaling events that occur during LTP induction in normal conditions and the PSB-treated slices.