Involvement of the delayed rectifier outward potassium channel Kv2.1 in methamphetamine‐induced neuronal apoptosis via the p38 mitogen‐activated protein kinase signaling pathway

Methamphetamine (Meth) is an illicit psychostimulant with high abuse potential and severe neurotoxicity. Recent studies have shown that dysfunctions in learning and memory induced by Meth may partially reveal the mechanisms of neuronal channelopathies. Kv2.1, the primary delayed rectifying potassium channel in neurons, is responsible for mediating apoptotic current surge. However, whether Kv2.1 is involved in Meth‐mediated neural injury remains unknown. In the present study, the treatment of primary cultured hippocampal neurons with Meth indicated that Meth induced a time‐ and dose‐dependent augmentation of Kv2.1 protein expression, accompanied by elevated cleaved‐caspase 3 and declined bcl‐2/bax ratio. The blockage of Kv2.1 with the inhibitor GxTx‐1E or the knockdown of the channel noticeably abrogated the pro‐apoptotic effects mediated by Meth, demonstrating the specific roles of Kv2.1 in Meth‐mediated neural damage. Additionally, the p38 mitogen‐activated protein kinase (MAPK) signaling was demonstrated to be involved in Meth‐mediated Kv2.1 upregulation and in the subsequent pro‐apoptotic effects, as treatment with a p38 MAPK inhibitor significantly attenuated Meth‐mediated Kv2.1 upregulation and cell apoptosis. Of note, PRE‐084, a sigma‐1 receptor agonist, obviously attenuated Meth‐induced upregulation of Kv2.1 expression, neural apoptosis and p38 MAPK activation. Taken together, these results reveal a novel mechanism involved in Meth‐induced neural death with implications for therapeutic interventions for Meth users.     

PM2.5 upregulates rat mesenteric arteries 5‐HT2A receptor via inflammatory‐mediated mitogen‐activated protein kinases signaling pathway

Fine particulate matter (PM_2.5) is an important environmental risk factor for cardiovascular diseases. However, little is known about the effects of PM_2.5 on arteries. The present study investigated whether PM_2.5 alters 5‐hydroxytryptamine (5‐HT) receptor expression and inflammatory mediators on rat mesenteric arteries, and examined the underlying mechanisms. Isolated rat mesenteric arteries segments were cultured with PM_2.5 in the presence or absence of ERK1/2, JNK, and p38 pathway inhibitors. Contractile reactivity was monitored by a sensitive myograph. The expression of 5‐HT_2A/1B receptors and inflammatory mediators were studied by a real‐time polymerase chain reaction and/or by immunohistochemistry. The phosphorylation of mitogen‐activated protein kinases (MAPK) pathway was detected by Western blot. Compared with the fresh or culture alone groups, 1.0 μg/mL PM_2.5 cultured for 16 hours significantly enhanced contractile response induced by 5‐HT and increased 5‐HT_2A receptor mRNA and protein expressions, indicating PM_2.5 upregulates 5‐HT_2A receptor. SB203580 (p38 inhibitor) and U0126 (ERK1/2 inhibitor) significantly decreased PM_2.5‐induced elevated contraction and mRNA and protein expression of 5‐HT_2A receptor. Cultured with PM_2.5 significantly increased the mRNA expression of inflammatory mediators (NOS2, IL‐1β, and TNF‐α), while SB203580 decreased mRNA expression level of NOS2, IL‐1β, and TNF‐α. SP600125 (JNK inhibitor) decreased mRNA expression level of TNF‐α and IL‐1β. After PM_2.5 exposure, the phosphorylation of p38 and ERK1/2 protein were increased. SB203580 and U0126 inhibited the PM_2.5 caused increased phosphorylation protein of p38 and ERK1/2. In conclusion, PM_2.5 induces inflammatory‐mediated MAPK pathway in artery which subsequently results in enhanced vascular contraction responding to 5‐HT via the upregulated 5‐HT_2A receptors.     

Effects of butyltins on mitogen‐activated‐protein kinase kinase kinase and Ras activity in human natural killer cells

Butyltins (BTs) contaminate the environment and are found in human blood. BTs, tributyltin (TBT) and dibutyltin (DBT) diminish the cytotoxic function and levels of key proteins of human natural killer (NK) cells. NK cells are an initial immune defense against tumors, virally infected cells and antibody‐coated cells and thus critical to human health. The signaling pathways that regulate NK cell functions include mitogen‐activated protein kinases (MAPKs). Studies have shown that exposure to BTs leads to activation of specific MAPKs and MAPK kinases (MAP2Ks) in human NK cells. MAP2K kinases (MAP3Ks) are upstream activators of MAP2Ks, which then activate MAPKs. The current study examined if BT‐induced activation of MAP3Ks was responsible for MAP2K and thus, MAPK activation. This study examines the effects of TBT and DBT on the total levels of two MAP3Ks, c‐Raf and ASK1, as well as activating and inhibitory phosphorylation sites on these MAP3Ks. In addition, the immediate upstream activator of c‐Raf, Ras, was examined for BT‐induced alterations. Our results show significant activation of the MAP3K, c‐Raf, in human NK cells within 10 min of TBT exposure and the MAP3K, ASK1, after 1 h exposures to TBT. In addition, our results suggest that both TBT and DBT affect the regulation of c‐Raf. Copyright © 2013 John Wiley & Sons, Ltd.     

Activation of mitogen activated protein kinase (MAPK) during carbon tetrachloride intoxication in the rat liver

Carbon tetrachloride (CCl₄: 4 ml/kg body weight as a 1:1 mixture of CCl₄ and mineral oil) was orally administered to rats. After 12 h the activity of plasma AST (aspartate aminotransferase) and ALT (alanine aminotransferase) was significantly higher than that of the control group and plasma AST and ALT activities increased thereafter. These results indicated that the necrotic process was active at about 12 h and developed thereafter. After 2–24 h of CCl₄ administration, the hepatic level of vitamin C, the most sensitive indicator of oxidative stress, decreased significantly, indicating that oxidative stress was significantly enhanced as early as 2 h after CCl₄ intoxication and thereafter. Phosphorylated JNK (c-Jun NH₂-terminal kinase) and phospho-ERK1/2 (extracellular signal-regulated kinase1/2) were significantly increased transiently 1–3 h after treatment with CCl₄, while phosphorylated p38 decreased significantly 1–24 h after CCl₄ treatment. These results indicated that the change in MAPKs (mitogen activated protein kinases) slightly preceded that in vitamin C, the most sensitive chemical indicator of oxidative stress.     

Manganese modulation of MAPK pathways: effects on upstream mitogen activated protein kinase kinases and mitogen activated kinase phosphatase‐1 in microglial cells

Multiple studies demonstrate that manganese (Mn) exposure potentiates inflammatory mediator output from activated glia; this increased output is associated with enhanced mitogen activated protein kinase (MAPK: p38, ERK and JNK) activity. We hypothesized that Mn activates MAPK by activating the kinases upstream of MAPK, i.e. MKK‐3/6, MKK‐1/2 and MKK‐4 (responsible for activation of p38, ERK, and JNK, respectively), and/or by inhibiting a major phosphatase responsible for MAPK inactivation, MKP‐1. Exposure of N9 microglia to Mn (250 µm ), LPS (100 ng ml^?1) or Mn + LPS increased MKK‐3/6 and MKK‐4 activity at 1 h; the effect of Mn + LPS on MKK‐4 activation was greater than the rest. At 4 h, Mn, LPS, and Mn + LPS increased MKK‐3/6 and MKK‐1/2 phosphorylation, whereas MKK‐4 was activated only by Mn and Mn + LPS. Besides activating MKK‐4 via Ser257/Thr261 phosphorylation, Mn (4 h) prevented MKK‐4's phosphorylation on Ser80, which negatively regulates MKK‐4 activity. Exposure to Mn or Mn + LPS (1 h) decreased both mRNA and protein expression of MKP‐1, the negative MAPK regulator. In addition, we observed that at 4 h, but not at 1 h, a time point coinciding with increased MAPK activity, Mn + LPS markedly increased TNF‐α, IL‐6 and Cox‐2 mRNA, suggesting a delayed effect. The fact that all three major groups of MKKs, MKK‐1/2, MKK‐3/6 and MKK‐4, are activated by Mn suggests that Mn‐induced activation of MAPK occurs via traditional mechanisms, which perhaps involve the MAPKs furthest upstream, MKKKs (MAP3Ks). In addition, for all MKKs, Mn‐induced activation was persistent at least for 4 h, indicating a long‐term effect. Copyright © 2010 John Wiley & Sons, Ltd.     

Antibacterial and antitubercular evaluation of dihydronaphthalenone‐indole hybrid analogs

A new series of indole appended dihydronaphthalenone hybrid analogs ( 5a–t ) have been synthesized through the Lewis acid catalyzed Michael addition of indoles to the arylidene/hetero arylidene ketones. All the synthesized derivatives are well characterized through the ¹H‐NMR, ¹³C‐NMR, HRMS spectroscopic techniques, compound 5r was further confirmed through single crystal X‐ray analysis and screened for antibacterial and antitubercular activities. Among the synthesized compounds, the minimum inhibition concentration of 5l (against Escherichia coli) and 5o & 5p (against E. coli & Staphylococcus aureus) was found to be as low as 3.12 μg/ml as compared to the standard antibacterial drug ciprofloxacin 2.5 μg/ml. In antitubercular activity, compounds 5o and 5p with minimum inhibition concentration 6.25 μg/ml were found to be comparable with that of the drugs Pyrazinamide 5 μg/ml and Streptomycin 5 μg/ml. Compounds 5i , 5j , 5m , 5n , 5q , and 5r also showed promising activity against group of organisms tested.     

Allopurinol induces innate immune responses through mitogen‐activated protein kinase signaling pathways in HL‐60 cells

Allopurinol, an inhibitor of xanthine oxidase, is a frequent cause of severe cutaneous adverse reactions (SCARs) in humans, including drug rash with eosinophilia and systemic symptoms, Stevens–Johnson syndrome and toxic epidermal necrolysis. Although SCARs have been suspected to be immune‐mediated, the mechanisms of allopurinol‐induced SCARs remain unclear. In this study, we examined whether allopurinol has the ability to induce innate immune responses in vitro using human dendritic cell (DC)‐like cell lines, including HL‐60, THP‐1 and K562, and a human keratinocyte cell line, HaCaT. In this study, we demonstrate that treatment of HL‐60 cells with allopurinol significantly increased the mRNA expression levels of interleukin‐8, monocyte chemotactic protein‐1 and tumor necrosis factor α in a time‐ and concentration‐dependent manner. Furthermore, allopurinol induced the phosphorylation of mitogen‐activated protein kinases (MAPK), such as c‐Jun N‐terminal kinase and extracellular signal‐regulated kinase, which regulate cytokine production in DC. In addition, allopurinol‐induced increases in cytokine expression were inhibited by co‐treatment with the MAPK inhibitors. Collectively, these results suggest that allopurinol has the ability to induce innate immune responses in a DC‐like cell line through activation of the MAPK signaling pathways. These results indicate that innate immune responses induced by allopurinol might be involved in the development of allopurinol‐induced SCARs. Copyright © 2015 John Wiley & Sons, Ltd.     

Mitogen-activated protein kinase (MAPK) pathway mediates the oestrogen-like activities of ginsenoside Rg1 in human breast cancer (MCF-7) cells

Background and purpose:

The present study was designed to determine how ginsenoside Rg1, an active ingredient in ginseng root, exerts its oestrogenic effects. We hypothesize that Rg1 may exert oestrogen-like actions in MCF-7 cells by activating the mitogen-activated protein kinase (MAPK) pathway in a ligand-independent manner.

Experimental approach:

MCF-7 cells were co-incubated with the MAPK inhibitor PD98059 to determine whether the stimulant effects of Rg1 on cell proliferation, the induction of IGF-IR and pS2, the functional transactivation of oestrogen receptor-α (ERα), as well as ERα phosphorylation are dependent on MAPK. The time-dependent responses of mitogen-activated protein kinase kinase (MEK) and extracellular signal-regulated protein kinase (ERK) to Rg1 in MCF-7 cells were studied. The responses of MEK phosphorylation to Rg1 in oestrogen receptor (ER)-negative HEK293 cells were also determined. The effects of Rg1 on cell proliferation and IGF-IR protein expression were studied in the presence of tyrosine kinase inhibitor genistein to elucidate the involvement of tyrosine kinase in mediating these effects.

Key results:

The oestrogenic effects of Rg1 in MCF-7 cells were abolished in the presence of PD98059. Rg1 could induce MEK protein expression and the phosphorylation level of MEK and ERK significantly in a time- and dose-dependent manner. Rg1 activated MEK phosphorylation in ER-negative HEK293 cells in a time- and dose-dependent manner. Rg1 induction of cell proliferation and IGF-IR protein expression was abolished by co-treatment with genistein.

Conclusions and implications:

Taken together, these results show that the MAPK pathway is involved in mediating the oestrogen-like actions of Rg1 in MCF-7 cells and suggest that Rg1 may activate ERα via MEK/ERK in a ligand-independent manner.     

Phytoglycoprotein (75 kDa) isolated from Cudrania tricuspidata Bureau inhibits expression of interleukin‐4 in the presence of di (2‐ethylhexyl) phthalate via modulation of p38 mitogen‐activated protein kinase in primary‐cultured mouse thymocytes

The purpose of this study is to determine the inhibitory effect of a glycoprotein isolated from Cudrania tricuspidata Bureau (CTB glycoprotein) on di(2‐ethylhexyl) phthalate (DEHP)‐induced differentiation of T helper (Th) type 2 cells in primary cultured thymocytes. The results obtained from this study revealed that the CTB glycoprotein in the presence of DEHP produces an antioxidative activity against intracellular reactive oxygen species production in cells. In addition, the activities of p38 mitogen‐activated protein kinase and GATA‐binding protein 3 were decreased by treatment with the CTB glycoprotein (100 µg ml^?1). The CTB glycoprotein also has an inhibitory effect on the expressions of interleukin (IL)‐4 and IL‐10 induced by DEHP in thymocytes. Hence, we speculate that the CTB glycoprotein might be one component for preparation of health supplements for prevention of Th2 cell response‐related immune diseases. Copyright © 2009 John Wiley & Sons, Ltd.     

Neuroprotective effects of ebselen in traumatic brain injury model: involvement of nitric oxide and p38 mitogen‐activated protein kinase signalling pathway

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Corticosteroid insensitive alveolar macrophages from asthma patients; synergistic interaction with a p38 mitogen-activated protein kinase (MAPK) inhibitor

Aims

Some asthma patients remain symptomatic despite using high doses of inhaled corticosteroids (ICS). We used alveolar macrophages to identify individual patients with insensitivity to corticosteroids and to evaluate the anti-inflammatory effects of a p38 mitogen-activated protein kinase (MAPK) inhibitor combined with a corticosteroid on these cells.

Methods

Alveolar macrophages from 27 asthma patients (classified according to the Global Initiative for Asthma (GINA) treatment stage. Six GINA1, 10 GINA2 and 11 GINA3/4) were stimulated with lipoploysaccharide (LPS) (1 μg ml⁻¹). The effects of dexamethasone (dex 1–1000 nm), the p38 MAPK inhibitor 1-(5-tert-butyl-2-p-tolyl-2Hpyrazol-3-yl)-3(4-(2-morpholin-4-yl-ethoxy)naphthalen-1-yl)urea (BIRB-796 1–1000 nm) and both drugs combined at all concentrations on supernatant TNFα, IL-6 and CXCL-8 concentrations were analyzed by ELISA. Dose-sparing and efficacy enhancing effects of combination treatment were determined.

Results

Dexamethasone reduced LPS-induced TNFα, IL-6 and CXCL-8 in all groups, but maximum inhibition was significantly reduced for GINA3/4 compared with GINA2 and GINA1 (P < 0.01). A subgroup of corticosteroid insensitive patients with a reduced effect of dexamethasone on cytokine secretion were identified. BIRB-796 in combination with dexamethasone significantly increased cytokine inhibition compared with either drug alone (P < 0.001) in all groups. This effect was greater in corticosteroid insensitive compared with sensitive patients. There were significant synergistic dose-sparing effects (P < 0.05) for the combination treatment on inhibition of TNFα, IL-6 and CXCL-8 in all groups. There was also significant efficacy enhancing benefits (P < 0.05) on TNFα and IL-6.

Conclusions

p38 MAPK inhibitors synergistically enhance efficacy of corticosteroids in macrophages from asthma patients. This effect is greater in corticosteroid insensitive asthma patients, suggesting that this class of drug should be targeted to this patient phenotype.     

Interleukin‐6 enhances matrix metalloproteinase‐14 expression via the RAF–mitogen‐activated protein kinase kinase–extracellular signal‐regulated kinase 1/2–activator protein‐1 pathway

1. Interleukin (IL)‐6 is a pivotal cytokine that regulates extracellular matrix (ECM) metabolism by increasing collagen degradation via activation of matrix metalloproteinases (MMPs), such as MMP‐14. In the present study, we investigated the role of IL‐6 in atherosclerotic plaque and signalling pathways in apolipoprotein E‐deficient (ApoE^?/?) mice. 2. Twenty‐five male ApoE^?/? mice were fed a high‐fat diet and atherosclerotic lesions in the right common carotid artery were induced by perivascular placement of a constrictive collar. Immunohistochemical analysis detected expression of IL‐6 and MMP‐14 in atherosclerotic lesions of the right common carotid artery. 3. On silencing activator protein (AP)‐1 expression with a specific small interfering RNA, 75% of the IL‐6‐induced increase in MMP‐14 expression was abolished through the RAF–mitogen‐activated protein kinase kinase–extracellular signal‐regulated kinase 1/2–AP‐1 pathway. 4. These findings suggest a novel molecular pathway for inflammation‐associated ECM dysregulation, which may account for atherosclerotic plaque rupture.     

Silver nanoparticles increase connexin43‐mediated gap junctional intercellular communication in HaCaT cells through activation of reactive oxygen species and mitogen‐activated protein kinase signal pathway

Silver nanoparticles (AgNPs) are widely used in health and consumer products that routinely contact skin. However, the biological effects and possible mechanisms of AgNPs on skin remain unclear. Gap junctional intercellular communication (GJIC) plays a critical role in multicellular organisms to maintain tissue homeostasis. The aim of this study is to examine if non‐coated AgNPs affect GJIC in human keratinocytes (HaCaT cells), and to identify the possible molecular mechanisms responsible for the effects. GJIC, connexin (Cx)43 protein and mRNA expression, and the effect of siRNA‐mediated knockdown of Cx43 on GJIC were assessed. HaCaT cells exposed to non‐coated AgNPs at different doses after a 24 hour exposure. To explore further the underlying mechanism, reactive oxygen species and mitogen‐activated protein kinase pathway were evaluated after 2, 6, 12 and 24 hours. Our results revealed that non‐coated AgNP exposure at subcytotoxic doses increase GJIC partially via Cx43 upregulation. Reactive oxygen species and extracellular signal‐regulated kinase and activation of c‐Jun N‐terminal kinase were involved in the AgNP‐induced upregulation of Cx43. This study provides new insight into the potential mechanism of AgNP biological activity.     

Relevance of mitogen activated protein kinase (MAPK) and phosphotidylinositol-3-kinase/protein kinase B (PI3K/PKB) pathways to induction of apoptosis by curcumin in breast cells

Following observations that curcumin inhibited proliferation (IC(50)=1-5 microM), invasiveness and progression through S/G2/M phases of the cell cycle in the non-tumourigenic HBL100 and tumourigenic MDA-MB-468 human breast cell lines, it was noted that apoptosis was much more pronounced in the tumour line. Therefore, the ability of curcumin to modulate signalling pathways which might contribute to cell survival was investigated. After pre-treatment of cells for 20 min, curcumin (40 microM) inhibited EGF-stimulated phosphorylation of the EGFR in MDA-MB-468 cells and phosphorylation of extracellular signal regulated kinases (ERKs) 1 and 2, as well as ERK activity and levels of nuclear c-fos in both cell lines. At a lower dose (10 microM), it also inhibited the ability of anisomycin to activate JNK, resulting in decreased c-jun phosphorylation, although it did not inhibit JNK activity directly. In contrast, the activation of p38 mitogen activated protein kinase (MAPK) by anisomycin was not inhibited. Curcumin inhibited basal phosphorylation of Akt/protein kinase B (PKB) in both cell lines, but more consistently and to a greater extent in the MDA-MB-468 cells. The MAPK kinase (MKK) inhibitor U0126 (10 microM), while preventing ERK phosphorylation in MDA-MB-468 cells, did not induce apoptosis. The PI3K inhibitor LY294002 (50 microM) inhibited PKB phosphorylation in both cells lines, but only induced apoptosis in the MDA-MB-468 line. These results suggest that while curcumin has several different molecular targets within the MAPK and PI3K/PKB signalling pathways that could contribute to inhibition of proliferation and induction of apoptosis, inhibition of basal activity of Akt/PKB, but not ERK, may facilitate apoptosis in the tumour cell line.     

Mitogen-activated protein kinase (MEK) inhibitors to treat melanoma alone or in combination with other kinase inhibitors

Introduction: Malignant melanoma (MM) is an aggressive disease with a rapidly rising incidence due to neoplasm of melanocytes. Molecular targeted therapies have demonstrated lower toxicity and improved overall survival versus conventional therapies of MM. The revealing of mutations in the BRAF/MEK/ERK pathway has led to the development of BRAF inhibitors such as vemurafenib and dabrafenib for the treatment of cutaneous MM. Though, progression of resistance to these agents has prompted attempts to target downstream proteins in this pathway. Trametinib, a MEK1/2 inhibitor, was approved in 2013 for the treatment of BRAF V600E/K mutation-positive unresectable or metastatic cutaneous melanoma patients.

Areas covered: The aim of the current review is to present an update on the role of MEK in progressive melanomas and summarize latest results of clinical studies with innovative MEK inhibitors and/or combined approaches with other kinase inhibitors such as BRAF inhibitors in the treatment of MM.

Expert opinion: Two combined treatments (i.e. trametinib plus dabrafenib and vemurafenib plus cobimetinib) target two different kinases in the BRAF/MEK/ERK pathway. The simultaneous prohibition of both MEK and BRAF is associated with more durable response rate than BRAF monotherapy and can overcome acquired resistance.     

Fish multigeneration test with preliminary short‐term reproduction assay for estrone using Japanese medaka (Oryzias latipes)

The most potent chemicals potentially causing adverse effects on fish species are estrogens in human waste. Sewage is a source of these estrogens and it is difficult to reduce. In particular, although the bioactivity of estrone is estimated to be about half of that of estradiol, multiple studies report that more than 100 ng l^–1 of estrone can be detected in urban rivers, including discharges from sewage treatment works; approximately two times as high as estradiol. Few studies have been conducted to investigate the long‐term effects of estrone on wildlife; therefore, we conducted fish multigeneration test using Japanese medaka (Oryzias latipes). Medaka were exposed to estrone for 27 weeks across three generations in environmentally relevant concentrations, being 5.74, 11.4, 24.0, 47.1 and 91.4 ng l^–1. No effects on reproduction were observed in the first generation; however, a decline in egg production and fertility was observed in the second generation exposed to 91.4 ng l^–1 estrone, which is lower than some known environmental concentrations in urban environments. Furthermore, histopathological abnormalities were observed in the third generation exposed to both 47.1 and 91.4 ng l^–1, suggesting that estrone possibly exerts severe effects on the third or later generations. However, appearances of testis–ova were observed in the second and third generation they were not consistent with actual effects on reproduction, notwithstanding the testis‐ova is regarded as the key evidence for endocrine disruption. Accordingly, we consider that qualitative measurement of abnormalities using histopathological observations is required for appropriate evaluation of endocrine disruption. Copyright © 2014 John Wiley & Sons, Ltd.