Altered Mitogen-activated Protein Kinase Research Articles

Beta-cyfluthrin impairs implantation process by inducing mitochondrial defects and changes in reactive oxygen species-mediated signaling pathways in porcine trophectoderm and uterine luminal epithelial cells

Pyrethroid insecticides, such as beta-cyfluthrin, are used extensively globally, including in households and agriculture, and have been detected in the milk and urine of humans and cattle. Beta-cyfluthrin exhibits toxic effects, including neurotoxicity and male reproductive toxicity; however, few studies have investigated female reproductive toxicity despite its wide environmental distribution. The present study investigates effects of beta-cyfluthrin on implantation in porcine cells (pTr from the trophectoderm and pLE from the endometrial luminal epithelium). To identify the various physiological changes induced by beta-cyfluthrin, such as apoptosis and lipid peroxidation, flow cytometry analysis and immunofluorescence were performed with various reagents. In addition, the expression of genes and proteins associated with intracellular changes was confirmed using qRT-PCR and western blotting. Beta-cyfluthrin induced cell-cycle arrest and altered intracellular calcium flux. It also disrupted the mitochondrial function and promoted reactive oxygen species (ROS) production, leading to lipid peroxidation. Moreover, ROS induced by beta-cyfluthrin altered mitogen-activated protein kinase (MAPK) pathways and decreased cell migration capability. The expression levels of genes that are significant during early pregnancy were altered by beta-cyfluthrin in both cell lines. The changes resulted in apoptosis and diminished cell proliferation of pTr and pLE. Collectively, the results imply that beta-cyfluthrin disrupts the implantation process by affecting the physiology of the trophectoderm and endometrial luminal epithelial cells. The present study is the first to reveal the cellular mechanisms of beta-cyfluthrin on the female reproductive system and highlights the need for further in-depth research into its hazards.

Suppression of the SLC7A11/glutathione axis causes ferroptosis and apoptosis and alters the mitogen-activated protein kinase pathway in nasopharyngeal carcinoma

SLC7A11 is a unit of the glutamate cystine antiporter Xc− system. It functions to import cystine for glutathione biosynthesis and maintains the redox balance in cells. Sorafenib inhibits the transporter activity of SLC7A11. The use of sorafenib has been approved in the treatment of multiple cancers. However, at present, our understanding of the mechanism of SLC7A11 and sorafenib in nasopharyngeal carcinoma (NPC) remains limited. We found that the expression of SLC7A11 was upregulated in NPC. A high SLC7A11 expression was associated with poor prognosis, metastasis, and an advanced T stage, which can be used as an independent prognostic indicator of NPC. In vitro, we observed that NPC cells relied on cystine for survival. Targeting SLC7A11 resulted in glutathione biosynthesis limitation, intracellular reactive oxygen species accumulation, lipid peroxides, ferroptosis, and apoptosis. Meanwhile, it altered mitogen activated protein kinase pathway, including p38 activation but ERK inhibition in NPC. This limited the proliferation of NPC cells. Sorafenib inhibited the proliferation and induced the death of NPC cells in vivo. In conclusion, SLC7A11 plays an important role in the occurrence and progression of NPC and may be a novel target for NPC treatment.

Bifenthrin induces cell death in bovine mammary epithelial cells via ROS generation, calcium ion homeostasis disruption, and MAPK signaling cascade alteration

Bifenthrin is one of the widely used synthetic pyrethroid insecticides, employed for various purposes worldwide. As lipophilic pyrethroids can easily bind to soil particles, which is why their residues are detected in various environments. Consequently, the toxicity of bifenthrin to non-target organisms can be regarded as an environmental concern. The toxic effects of bifenthrin have been studied in various animal models and cell lines; however, its toxic effects on cattle remain unclear. In particular, gaining insights into the toxic effects of bifenthrin on the mammary lactation system is crucial for the dairy industry. Therefore, we proceeded to investigate the toxic effects of bifenthrin on the bovine mammary epithelial cells (MAC-T cells). We established that bifenthrin inhibited cell proliferation and triggered apoptosis in MAC-T cells. Additionally, bifenthrin induced mitochondrial dysfunction and altered inflammatory gene expression by disrupting mitochondrial membrane potential (MMP) and generating excessive reactive oxygen species (ROS). We also demonstrated that bifenthrin disrupted both cytosolic and mitochondrial calcium ion homeostasis. Furthermore, bifenthrin altered mitogen-activated protein kinase (MAPK) signaling cascades and downregulated casein-related genes. Collectively, we confirmed the multiple toxic effects of bifenthrin on MAC-T cells, which could potentially reduce milk yield and quality.

Deficiency in Embryonic Stem Cell Marker Reduced Expression 1 Activates Mitogen-Activated Protein Kinase Kinase 6-Dependent p38 Mitogen-Activated Protein Kinase Signaling to Drive Hepatocarcinogenesis.

Embryonic stem-cell-related transcription factors are central to the establishment and maintenance of stemness and pluripotency, and their altered expression plays key roles in tumors, including hepatocellular carcinoma (HCC), a malignancy with no effective treatment. Here, we report on the embryonic stem cell marker, reduced expression 1 (REX1; also known as zinc finger protein 42), to be selectively down-regulated in HCC tumors. Deficiency of REX1 in HCC was attributed to a combination of hypermethylation at its promoter as well as histone modification by methylation and acetylation. Clinically, hypermethylation of REX1 was closely associated with neoplastic transition and advanced tumor stage in humans. Functionally, silencing of REX1 potentiated the tumor-initiating and metastasis potential of HCC cell lines and xenografted tumors. Lentivirus-mediated Rex1 ablation in liver of male immunocompetent mice with HCC, induced by hydrodynamic tail vein injection of proto-oncogenes, enhanced HCC development. Transcriptome profiling studies revealed REX1 deficiency in HCC cells to be enriched with genes implicated in focal adhesion and mitogen-activated protein kinase (MAPK) signaling. From this lead, we subsequently found REX1 to bind to the promoter region of mitogen-activated protein kinase kinase 6 (MKK6), thereby obstructing its transcription, resulting in altered p38 MAPK signaling. Our work describes a critical repressive function of REX1 in maintenance of HCC cells by regulating MKK6 binding and p38 MAPK signaling. REX1 deficiency induced enhancement of p38 MAPK signaling, leading to F-actin reorganization and activation of nuclear factor erythroid 2-related factor 2-mediated oxidative stress response, which collectively contributed to enhanced stemness and metastatic capabilities of HCC cells.

Horse Oil Mitigates Oxidative Damage to Human HaCaT Keratinocytes Caused by Ultraviolet B Irradiation.

Horse oil products have been used in skin care for a long time in traditional medicine, but the biological effects of horse oil on the skin remain unclear. This study was conducted to evaluate the protective effect of horse oil on ultraviolet B (UVB)-induced oxidative stress in human HaCaT keratinocytes. Horse oil significantly reduced UVB-induced intracellular reactive oxygen species and intracellular oxidative damage to lipids, proteins, and DNA. Horse oil absorbed light in the UVB range of the electromagnetic spectrum and suppressed the generation of cyclobutane pyrimidine dimers, a photoproduct of UVB irradiation. Western blotting showed that horse oil increased the UVB-induced Bcl-2/Bax ratio, inhibited mitochondria-mediated apoptosis and matrix metalloproteinase expression, and altered mitogen-activated protein kinase signaling-related proteins. These effects were conferred by increased phosphorylation of extracellular signal-regulated kinase 1/2 and decreased phosphorylation of p38 and c-Jun N-terminal kinase 1/2. Additionally, horse oil reduced UVB-induced binding of activator protein 1 to the matrix metalloproteinase-1 promoter site. These results indicate that horse oil protects human HaCaT keratinocytes from UVB-induced oxidative stress by absorbing UVB radiation and removing reactive oxygen species, thereby protecting cells from structural damage and preventing cell death and aging. In conclusion, horse oil is a potential skin protectant against skin damage involving oxidative stress.

Abstract PD2-05: Biomarker analysis by baseline circulating tumor DNA alterations in the MONALEESA-3 study

Abstract Background: In the Phase III MONALEESA-3 (NCT02422615) study, ribociclib (RIB; cyclin-dependent kinase 4/6 inhibitor) + fulvestrant (FUL) significantly prolonged progression-free survival (PFS) vs placebo (PBO) + FUL in postmenopausal women with hormone receptor-positive (HR+), HER2-negative (HER2–) advanced breast cancer (ABC). Here we present MONALEESA-3 efficacy data by molecular alterations detected in circulating tumor DNA (ctDNA) at baseline. Methods: Postmenopausal women (N=726) with HR+, HER2– ABC (treatment naïve or received ≤1 line of prior endocrine therapy for ABC) were randomized 2:1 to RIB or PBO (600 mg/day; 3-weeks-on/1-week-off) + FUL (500 mg per label). Biomarker analysis of baseline ctDNA was an exploratory endpoint. Baseline plasma samples were collected from 692 patients (pts), and plasma ctDNA was analyzed using next-generation sequencing with a targeted panel of ˜550 genes. The subgroup analysis was performed in 600 pts (RIB + FUL n=400; PBO + FUL n=200) with evaluable ctDNA data. Results were not adjusted for multiple testing. Results: Alterations (frequency) were observed in the following genes: PIK3CA (35%), ESR1 (14%), TP53 (18%), CDH1 (12%), FGFR1 (5%), FGFR1/ZNF703/WHSC1L1 (11%), cell cycle-related (CCC) genes (16%), and genes involved in receptor tyrosine kinase (RTK) signaling (20%), and the mitogen-activated protein kinase (MAPK) pathway (10%). PFS hazard ratios favored RIB vs PBO regardless of baseline ctDNA gene alteration status (Table). Events, n/NMedian PFS, months Gene(s)RIB + FULPBO + FULRIB + FULPBO + FULHazard ratio; 95% confidence intervalPIK3CAWT108/26570/12422.3416.490.67; 0.49–0.91Altered75/13549/7616.3611.100.75; 0.52–1.08ESR1WT141/33899/17622.3414.880.66; 0.51–0.85Altered42/6220/249.236.110.71; 0.41–1.23TP53WT132/32598/17022.3414.880.62; 0.47–0.80Altered51/7521/309.177.390.75; 0.44–1.27CDH1WT156/35697/17320.6316.490.70; 0.54–0.90Altered27/4422/2712.095.130.46; 0.24–0.86FGFR1WT165/378113/19221.3214.550.65; 0.51–0.83Altered18/226/88.346.110.64; 0.23–1.75FGFR1/ZNF703/WHSC1L1WT155/356105/18121.3214.650.67; 0.52–0.85Altered28/4414/1910.976.670.73; 0.37–1.43CCCWT138/331103/17422.0814.650.61; 0.47–0.79Altered45/6916/2612.659.130.92; 0.51–1.65RTKWT126/31595/16622.3414.820.61; 0.47–0.80Altered57/8524/349.175.780.82; 0.50–1.35MAPKWT158/357105/18320.0414.820.69; 0.54–0.89Altered25/4314/1714.723.610.47; 0.22–1.00 Numerically shorter PFS was observed in pts with altered vs wildtype (WT) genes, irrespective of treatment. A slight trend towards limited RIB benefit vs PBO was observed in pts with altered CCC and RTK genes. RIB benefit was more pronounced vs PBO in pts with altered CDH1 and MAPK genes. PIK3CA and ESR1 analyses for pts receiving treatment in the first- or second-line setting will be presented at the meeting. Conclusions: Generally consistent PFS benefit for RIB + FUL vs PBO + FUL was observed irrespective of baseline ctDNA alterations; altered status trended to correlate with a shorter PFS. Results are hypothesis generating and should be interpreted with caution for some subgroups due to small sample sizes. Citation Format: Neven P, Petrakova K, Val Bianchi G, De la Cruz-Merino L, Jerusalem G, Sonke GS, Nusch A, Beck JT, Chia S, Solovieff N, Rodriguez Lorenc K, Miller M, Su F, Lm S-A. Biomarker analysis by baseline circulating tumor DNA alterations in the MONALEESA-3 study [abstract]. In: Proceedings of the 2018 San Antonio Breast Cancer Symposium; 2018 Dec 4-8; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2019;79(4 Suppl):Abstract nr PD2-05.

Different phosphorylation patterns regulate α1D-adrenoceptor signaling and desensitization

Human α1D-adrenoceptors (α1D-ARs) are a group of the seven transmembrane-spanning proteins that mediate many of the physiological and pathophysiological actions of adrenaline and noradrenaline. Although it is known that α1D-ARs are phosphoproteins, their specific phosphorylation sites and the kinases involved in their phosphorylation remain largely unknown. Using a combination of in silico analysis, mass spectrometry and site directed mutagenesis, we identified distinct α1D-AR phosphorylation patterns during noradrenaline- or phorbol ester-mediated desensitizations. We found that the G protein coupled receptor kinase, GRK2, and conventional protein kinases C isoforms α/β, phosphorylate α1D-AR during these processes. Furthermore, we showed that the phosphorylated residues are located in the receptor's third intracellular loop (S300, S323, T328, S331, S332, S334) and carboxyl region (S441, T442, T477, S486, S492, T507, S515, S516, S518, S543) and are conserved among orthologues but are not conserved among the other human α1-adrenoceptor subtypes. Additionally, we found that phosphorylation in either the third intracellular loop or carboxyl tail was sufficient to regulate calcium signaling desensitization. By contrast, mutations in either of these two domains significantly altered mitogen activated protein kinase (ERK) pathway and receptor internalization, suggesting that they have differential regulatory mechanisms. Our data provide new insights into the functional repercussions of these posttranslational modifications in signaling outcomes and desensitization.

P38 predicts depression and poor outcome in esophageal cancer.

p38 mitogen-activated protein kinase (MAPK) signaling has been implicated in the cancer development and progression. However, the precise mechanism of this association remains unknown. The aim of the present study was to evaluate the association between p38 and cancer progression, including investigations into the effects on cell proliferation, resistance to thalidomide, indoleamine 2,3-dioxygenase (IDO) expression and prognosis in patients with esophageal cancer. The present retrospective study included patients with stage I–III esophageal cancer. A total of 228 patients with esophageal cancer were recruited to analyze the expression of phosphorylated (p)-p38 and IDO in tumor, and normal tissues through immunohistochemistry. Depression status was measured using the Zung Self-Rating Depression Scale. P38 cDNA was transfected into esophageal cancer cells to assess tumor cell viability, sensitivity to thalidomide treatment and IDO gene expression. Western blotting and flow cytometry was used to analyze protein expression alterations, and apoptosis in esophageal cancer cells. P-p38 protein was expressed in 68.9% of cancer tissues, and was significantly associated with depressive symptoms, tumor recurrence and poor survival of patients. In vitro experiments revealed that the expression of p-p38 induced esophageal cancer Eca-109 and TE-1 cell viability, and resistance to thalidomide treatment, as well as in the expression of IDO without the application of lipopolysaccharides. Further follow-up of patients revealed that depression was also an independent factor for early recurrence and overall survival rate. Altered p38 MAPK expression was associated with poor outcome in patients with esophageal cancer. p38 may be a potential biomarker for the prediction of depressive symptoms and prognosis in patients with esophageal cancer.

Acute exercise activates p38 MAPK and increases the expression of telomere-protective genes in cardiac muscle.

What is the central question of this study? A positive association between telomere length and exercise training has been shown in cardiac tissue of mice. It is currently unknown how each bout of exercise influences telomere-length-regulating proteins. We sought to determine how a bout of exercise altered the expression of telomere-length-regulating genes and a related signalling pathway in cardiac tissue of mice. What is the main finding and its importance? Acute exercise altered the expression of telomere-length-regulating genes in cardiac tissue and might be related to altered mitogen-activated protein kinase signalling. These findings are important in understanding how exercise provides a cardioprotective phenotype with ageing. Age is the greatest risk factor for cardiovascular disease. Telomere length is shorter in the hearts of aged mice compared with young mice, and short telomere length has been associated with an increased risk of cardiovascular disease. One year of voluntary wheel-running exercise attenuates the age-associated loss of telomere length and results in altered gene expression of telomere-length-maintaining and genome-stabilizing proteins in heart tissue of mice. Understanding the early adaptive response of the heart to an endurance exercise bout is paramount to understanding the impact of endurance exercise on heart tissue and cells. To this end, we studied mice before (BL), immediately after (TP1) and 1h after a treadmill running bout (TP2). We measured the changes in expression of telomere-related genes (shelterin components), DNA-damage-sensing (p53 and Chk2) and DNA-repair genes (Ku70 and Ku80) and mitogen-activated protein kinase (MAPK) signalling. The TP1 animals had increased TRF1 and TRF2 protein and mRNA levels, greater expression of DNA-repair and -response genes (Chk2 and Ku80) and greater protein content of phosphorylated p38 MAPK compared with both BL and TP2 animals. These data provide insights into how physiological stressors remodel the heart tissue and how an early adaptive response mediated by exercise may be maintaining telomere length and/or stabilizing the heart genome through the upregulation of telomere-protective genes.

Calf Spleen Extractive Injection (CSEI), a small peptides enriched extraction, induces human hepatocellular carcinoma cell apoptosis via ROS/MAPKs dependent mitochondrial pathway

Calf Spleen Extractive Injection (CSEI), a small peptides enriched extraction, performs immunomodulatory activity on cancer patients suffering from radiotherapy or chemotherapy. The present study aims to investigate the anti-hepatocellular carcinoma effects of CSEI in cells and tumor-xenografted mouse models. In HepG2 and SMMC-7721 cells, CSEI reduced cell viability, enhanced apoptosis rate, caused reactive oxygen species (ROS) accumulation, inhibited migration ability, and induced caspases cascade and mitochondrial membrane potential dissipation. CSEI significantly inhibited HepG2-xenografted tumor growth in nude mice. In cell and animal experiments, CSEI increased the activations of pro-apoptotic proteins including caspase 8, caspase 9 and caspase 3; meanwhile, it suppressed the expressions of anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) and anti-oxidation proteins, such as nuclear factor-erythroid 2 related factor 2 (Nrf2) and catalase (CAT). The enhanced phosphorylation of P38 and c-JunN-terminalkinase (JNK), and decreased phosphorylation of extra cellular signal-regulated protein kinase (ERKs) were observed in CSEI-treated cells and tumor tissues. CSEI-induced cell viability reduction was significantly attenuated by N-Acetyl-l-cysteine (a ROS inhibitor) pretreatment. All data demonstrated that the upregulated oxidative stress status and the altered mitogen-activated protein kinases (MAPKs) phosphorylation contributed to CSEI-driven mitochondrial dysfunction. Taken together, CSEI exactly induced apoptosis in human hepatocellular carcinoma cells via ROS/MAPKs dependent mitochondrial pathway.

Coriandrum sativum Suppresses Aβ42-Induced ROS Increases, Glial Cell Proliferation, and ERK Activation.

Alzheimer's disease (AD), the most common neurodegenerative disease, has a complex and widespread pathology that is characterized by the accumulation of amyloid [Formula: see text]-peptide (A[Formula: see text]) in the brain and various cellular abnormalities, including increased oxidative damage, an amplified inflammatory response, and altered mitogen-activated protein kinase signaling. Based on the complex etiology of AD, traditional medicinal plants with multiple effective components are alternative treatments for patients with AD. In the present study, we investigated the neuroprotective effects of an ethanol extract of Coriandrum sativum (C. sativum) leaves on A[Formula: see text] cytotoxicity and examined the molecular mechanisms underlying the beneficial effects. Although recent studies have shown the benefits of the inhalation of C. sativum oil in an animal model of AD, the detailed molecular mechanisms by which C. sativum exerts its neuroprotective effects are unclear. Here, we found that treatment with C. sativum extract increased the survival of both A[Formula: see text]-treated mammalian cells and [Formula: see text]42-expressing flies. Moreover, C. sativum extract intake suppressed [Formula: see text]-induced cell death in the larval imaginal disc and brain without affecting A[Formula: see text]42 expression and accumulation. Interestingly, the increases in reactive oxygen species levels and glial cell number in AD model flies were reduced by C. sativum extract intake. Additionally, C. sativum extract inhibited the epidermal growth factor receptor- and A[Formula: see text]-induced phosphorylation of extracellular signal-regulated kinase (ERK). The constitutively active form of ERK abolished the protective function of C. sativum extract against the [Formula: see text]-induced eye defect phenotype in Drosophila. Taken together, these results suggest that C. sativum leaves have antioxidant, anti-inflammatory, and ERK signaling inhibitory properties that are beneficial for patients with AD.

Advantages and limitations of shot-gun proteomic analyses on Arabidopsis plants with altered MAPK signaling.

The molecular mechanism of signal transduction by mitogen-activated protein kinases (MAPKs) in plants is an intensive research field of contemporary plant biology. Signaling in plants involves perception of changing environment and the intracellular transduction of such generated information leading to the activation of adaptive mechanisms or programmed cell death, as it is in the case of hypersensitive response to some biotrophic pathogen attack. At the molecular level, distinct signaling pathways, such as MAPK modules, involve reversible phosphorylation, enzymatic activation, protein-protein interaction and finally transcriptional activation via nuclear-resident transcription factors or cell reorganization via regulation of cytoplasmic or plasma membrane-associated proteins. All these processes are strictly regulated and subcellularly compartmentalized. Modern OMICS technologies encompassing transcriptomic (Krysan et al., 2002; Frei dit Frey et al., 2014), metabolomic (Lassowskat et al., 2014), proteomic (Miles et al., 2009; Conroy et al., 2013; Lassowskat et al., 2014; Takac et al., 2014) and phosphoproteomic (Hoehenwarter et al., 2013; Lassowskat et al., 2014) approaches have been employed to gain functional information on plant signaling. The main challenges of advanced (phospho)proteomic studies are: reliable identification of all signaling constituents of a particular MAPK pathway as well as identification and validation of respective target proteins. In this opinion article, we aim to briefly evaluate benefits and pitfalls of various proteomic approaches that are used to study MAPK signaling in plants. We emphasize on the advantages of comparative shot-gun proteomic analyses of Arabidopsis mutants and transgenic plants with altered MAPK signaling. The importance of critical data validation and interpretation is also discussed.

Abstract A30: MKP-1 negatively regulates Il-1beta in the oral tumor microenvironment

Abstract Head and neck cancer accounts for approximately 6% of diagnosed malignancies in the United States, the most common form being head and neck squamous cell carcinoma (HNSCC). Cytokines and pro-inflammatory factors have been shown to play a critical role in various steps of malignant transformation, many of which are regulated by mitogen-activated protein kinases (MAPK). These kinases are negatively regulated by MAPK phosphatases (MKPs), the founding member of this class being mitogen-activated protein kinase phosphatase-1 (MKP-1). MKP-1 is often over-expressed in early stages of epithelial malignancies with levels of expression falling progressively in tumors of higher histological grade. As MKP-1 is a crucial negative regulator of innate immunity, we hypothesized MKP-1 inhibits tumor-promoting inflammation in the HNSCC microenvironment. MKP-1 deficient animals treated with 4-nitroquinoline 1-oxide to induce HNSCC formation showed a significant increase in tumor development, characterized by tumor onset, size, histological grade, and inflammation. Tumor tissues expressed increased levels of multiple inflammatory cytokines and chemokines by qPCR and altered MAPK activity by immunoblot and immunohistochemistry. In particular, Il-1beta was significantly increased in MKP-1 deficient tumors as well as primary bone marrow-derived macrophages. These data suggest MKP-1 inhibits oral cancer progression by regulating Il-1beta expression. Elucidating the regulation of this inflammatory mediator in the tumor microenvironment may reveal critical tumor suppressive interactions mediated by MKP-1 that are lost during cancer progression. Citation Format: Xiaoyi Zhang, Hong Yu, Keith L. Kirkwood. MKP-1 negatively regulates Il-1beta in the oral tumor microenvironment. [abstract]. In: Abstracts: AACR Special Conference on Cellular Heterogeneity in the Tumor Microenvironment; 2014 Feb 26-Mar 1; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2015;75(1 Suppl):Abstract nr A30. doi:10.1158/1538-7445.CHTME14-A30

Chronic brain inflammation causes a reduction in GluN2A and GluN2B subunits of NMDA receptors and an increase in the phosphorylation of mitogen-activated protein kinases in the hippocampus.

Neuroinflammation plays a key role in the initiation and progression of neurodegeneration in Alzheimer’s disease (AD). Chronic neuroinflammation results in diminished synaptic plasticity and loss of GluN1 N-methyl-D-aspartate (NMDA) receptors in the hippocampus, leading to the cognitive deficits that are the most common symptoms of AD. Therefore, it is suggested that chronic inflammation may alter expression levels of GluN2A and GluN2B subunits of NMDA receptors and associated intracellular signalling. Chronic neuroinflammation was induced by chronic infusion of lipopolysaccharide (LPS) into the fourth ventricle in Fischer-344 rats. The status of hippocampus-dependent memory was evaluated in control rats and rats chronically infused with LPS. Microglial activation in the hippocampus was examined using immunohistochemical staining. Western blot analysis was used to measure membrane levels of GluN2A and GluN2B subunits of NMDA receptors and mitogen-activated protein kinase (MAPK) in the hippocampi of these rats, and immunofluorescent double labeling was used to assess the cellular location of MAPK. Microglial activation was observed in the hippocampi of rats that showed memory impairments with chronic LPS infusion. Chronic LPS infusion reduced the levels of GluN2A and GluN2B and increased the levels of phosphorylated MAPKs in the hippocampus. MAPK-positive immunoreactivity was observed mostly in the neurons and also in non-neuronal cells. Reductions in GluN2A and GluN2B subunits of NMDA receptors coupled with altered MAPK signaling, in response to inflammatory stimuli may be related to the cognitive deficits observed in AD.

Glucose 6-phosphate dehydrogenase deficiency enhances germ cell apoptosis and causes defective embryogenesis in Caenorhabditis elegans

Glucose 6-phosphate dehydrogenase (G6PD) deficiency, known as favism, is classically manifested by hemolytic anemia in human. More recently, it has been shown that mild G6PD deficiency moderately affects cardiac function, whereas severe G6PD deficiency leads to embryonic lethality in mice. How G6PD deficiency affects organisms has not been fully elucidated due to the lack of a suitable animal model. In this study, G6PD-deficient Caenorhabditis elegans was established by RNA interference (RNAi) knockdown to delineate the role of G6PD in animal physiology. Upon G6PD RNAi knockdown, G6PD activity was significantly hampered in C. elegans in parallel with increased oxidative stress and DNA oxidative damage. Phenotypically, G6PD-knockdown enhanced germ cell apoptosis (2-fold increase), reduced egg production (65% of mock), and hatching (10% of mock). To determine whether oxidative stress is associated with G6PD knockdown-induced reproduction defects, C. elegans was challenged with a short-term hydrogen peroxide (H2O2). The early phase egg production of both mock and G6PD-knockdown C. elegans were significantly affected by H2O2. However, H2O2-induced germ cell apoptosis was more dramatic in mock than that in G6PD-deficient C. elegans. To investigate the signaling pathways involved in defective oogenesis and embryogenesis caused by G6PD knockdown, mutants of p53 and mitogen-activated protein kinase (MAPK) pathways were examined. Despite the upregulation of CEP-1 (p53), cep-1 mutation did not affect egg production and hatching in G6PD-deficient C. elegans. Neither pmk-1 nor mek-1 mutation significantly affected egg production, whereas sek-1 mutation further decreased egg production in G6PD-deficient C. elegans. Intriguingly, loss of function of sek-1 or mek-1 dramatically rescued defective hatching (8.3- and 9.6-fold increase, respectively) induced by G6PD knockdown. Taken together, these findings show that G6PD knockdown reduces egg production and hatching in C. elegans, which are possibly associated with enhanced oxidative stress and altered MAPK pathways, respectively.

The Fibrin-Derived Peptide Bβ15-42 Attenuates Liver Damage in a Rat Model of Liver Ischemia/Reperfusion Injury

The inflammatory response after liver ischemia/reperfusion (I/R) contributes to increased risk of liver failure after liver surgery. Strategies aimed to preventing inflammation could be beneficial in reducing liver I/R injury. Recent studies have demonstrated that peptide Bβ15-42 is able to decrease the injury of I/R in heart and kidney by inhibition of leukocyte migration and preserving endothelial barrier function. Prompted by these results, we hypothesized that Bβ15-42 could also possess anti-inflammatory abilities to protect from or reduce hepatic I/R injury. Therefore, in this study, we aimed to evaluate the effects of Bβ15-42 in a model of liver I/R injury in rats. Rats were treated with Bβ15-42 at initiation of reperfusion and 2 h thereafter. Rats were killed at 0.5, 6, 24, and 48 h after reperfusion. Hepatic mRNA levels of fibrinogen-α (Fgα), Fgβ, Fgγ were significantly increased after I/R. Treatment with Fg-derived Bβ15-42 ameliorated liver I/R injury, as indicated by lower serum aminotransferase levels and fewer I/R-associated histopathologic changes. Bβ15-42 treatment decreased leukocyte infiltration and expression of hepatic inflammatory cytokines. Moreover, Bβ15-42 significantly reduced high-mobility group box 1 release and altered mitogen-activated protein kinase activation. In conclusion, Bβ15-42 treatment protected against liver warm I/R injury. The mechanism of protective action of Bβ15-42 seemed to involve its ability to reduce hepatic inflammatory response through preventing high-mobility group box 1 release and altering mitogen-activated protein kinase activation.

Altered MAPK Signaling in Progressive Deterioration of Endothelial Function in Diabetic Mice

We aimed to investigate specific roles of mitogen-activated protein kinases (MAPK) in the deterioration of endothelial function during the progression of diabetes and the potential therapeutic effects of MAPK inhibitors and agonists in the amelioration of endothelial function. Protein expression and phosphorylation of p38, c-Jun NH2-terminal kinase (JNK), and extracellular signal–regulated kinase (Erk) were assessed in mesenteric arteries of 3- (3M) and 9-month-old (9M) male diabetic and control mice. The expression of p38, JNK, and Erk was comparable in all groups of mice, but the phosphorylation of p38 and JNK was increased in 3M and further increased in 9M diabetic mice, whereas the phosphorylation of Erk was substantially reduced in 9M diabetic mice. NADPH oxidase–dependent superoxide production was significantly increased in vessels of two ages of diabetic mice. Inhibition of either p38 with SB203580 or JNK with SP600125 reduced superoxide production and improved shear stress–induced dilation (SSID) in 3M, but not in 9M, diabetic mice. Treating the vessels of 9M diabetic mice with resveratrol increased Erk phosphorylation and shear stress–induced endothelial nitric oxide synthase (eNOS) phosphorylation and activity, but resveratrol alone did not improve SSID. Administration of resveratrol and SB203580 or resveratrol and SP600125 together significantly improved SSID in vessels of 9M diabetic mice. The improved response was prevented by U0126, an Erk inhibitor. Thus, p38/JNK-dependent increase in oxidative stress diminished nitric oxide–mediated dilation in vessels of 3M diabetic mice. Oxidative stress and impaired Erk-dependent activation of eNOS exacerbates endothelial dysfunction in the advanced stage of diabetes.

Altered mitogen‐activated protein kinase signaling in dystrophic (mdx) muscle

Duchenne muscular dystrophy (DMD) results from a deficiency in the protein, dystrophin. Dystrophic myotubes are susceptible to stressful stimuli. This may be partly due to altered regulation of pro-survival signaling pathways, but a role for mitogen-activated protein (MAP) kinases has not been investigated. We examined patterns of phosphorylation of key MAP kinase proteins in cultured myotubes responding to oxidative stress, and in muscle tissue in vivo. Dystrophic (mdx) myotubes have an increased susceptibility to oxidant-induced death compared with wild-type (C57Bl/10ScSn) myotubes. This correlates with late phosphorylation of c-Jun N-terminal kinase (JNK), and persistently high p38 MAP kinase phosphorylation in mdx myotubes. JNK and extracellular signal-regulated kinase 1/2 (ERK1/2) also showed altered phosphorylation levels in mdx muscle tissue. We show altered patterns of MAP kinase protein phosphorylation in dystrophic muscle in vitro and in vivo. These pathways may be novel pharmacological targets for treating DMD.

Role Of Rostral Ventrolateral Medullary ERK/JNK/p38 MAPK Signaling In The Pressor Effects Of Ethanol And Its Oxidative Product Acetaldehyde In SHRs

The mitogen‐activated protein kinases (MAPKs) modulate sympathetic activity within the rostral ventrolateral medulla (RVLM). As intra‐RVLM ethanol causes sympathoexcitation and hypertension in SHRs, we tested the hypotheses: (i) altered MAPKs phosphorylation/dephosphorylation in RVLM underlies ethanol hypertension, and (ii) acetaldehyde, oxidative product of ethanol, replicates molecular and related BP effects of ethanol. The BP effect of either drug was evaluated in presence of inhibitors of JNK (SP600125), ERK1/2 (PD98059), p38 (SB203580), or phosphatase (okadaic acid, OKA). Intra‐RVLM ethanol (10 μg) or acetaldehyde (2 μg) caused similar increases in BP. The pressor action is probably mediated via enhanced ERK1/2 activity because it was (i) paralleled with increased RVLM p‐ERK1/2, and (ii) abolished in PD98059‐treated SHRs. SP600125 abrogated ethanol‐but not acetaldehyde‐evoked hypertension, thus implicating JNK in ethanol effects. Despite increased p‐p38, pharmacological intervention revealed no role for p38 in ethanol hypertension. Although phosphatase activity was not altered by either drug, OKA increased baseline BP and abrogated the pressor effect of ethanol or acetaldehyde. Collectively, enhanced ERK1/2 phosphorylation, without any changes in the dephosphorylation profile, accounts for the pressor effect of ethanol or acetaldehyde in SHRs.

HIV-1 Infection Abrogates CD8+T Cell Mitogen-Activated Protein Kinase Signaling Responses

Mitogen-activated protein kinase (MAPK) signaling pathways are dynamic and sensitive regulators of T cell function and differentiation. Altered MAPK signaling has been associated with the inflammatory and autoimmune diseases lupus and arthritis and with some pathogenic viral infections. HIV-1 infection is characterized by chronic immune inflammation, aberrantly heightened CD8(+) T cell activation levels, and altered T cell function. The relationship between MAPK pathway function, HIV-1-induced activation (CD38 and HLA-DR), and exhaustion (Tim-3) markers in circulating CD8(+) T cells remains unknown. Phosphorylation of the MAPK effector proteins ERK and p38 was examined by "phosflow" flow cytometry in 79 recently HIV-1-infected, antiretroviral-treatment-naïve adults and 21 risk-matched HIV-1-negative controls. We identified a subset of CD8(+) T cells refractory to phorbol 12-myristate 13-acetate plus ionomycin-induced ERK1/2 phosphorylation (referred to as p-ERK1/2-refractory cells) that was greatly expanded in HIV-1-infected adults. The CD8(+) p-ERK1/2-refractory cells were highly activated (CD38(+) HLA-DR(+)) but not exhausted (Tim-3 negative), tended to have low CD8 expression, and were enriched in intermediate and late transitional memory states of differentiation (CD45RA(-) CD28(-) CD27(+/-)). Targeting MAPK pathways to restore ERK1/2 signaling may normalize immune inflammation levels and restore CD8(+) T cell function during HIV-1 infection.