Sulfur dioxide inhibits vascular smooth muscle cell proliferation via suppressing the Erk/MAP kinase pathway mediated by cAMP/PKA signaling

PubMed Central

Liu, D; Huang, Y; Bu, D; Liu, A D; Holmberg, L; Jia, Y; Tang, C; Du, J; Jin, H

2014-01-01

The present study was designed to investigate the role of endogenous sulfur dioxide (SO2) in vascular smooth muscle cell (VSMC) proliferation, and explore the possible role of cross-talk between cyclic adenosine monophosphate (cAMP)/protein kinase A (PKA) and extracellular signal-regulated kinase (Erk)/mitogen-activated protein kinase (MAPK) pathways in this action. By cell counting, growth curve depict, flow cytometry and bromodeoxyuridine (BrdU) labeling assays, we found that SO2 inhibited VSMC proliferation by preventing cell cycle progression from G1 to S phase and by reducing DNA synthesis. SO2 synthase aspartate aminotransferase (AAT1 and AAT2) overexpression significantly inhibited serum-induced proliferating cell nuclear antigen (PCNA) protein expression in VSMCs, demonstrated by western blot analysis. Moreover, overexpression of AAT1 or AAT2 markedly reduced incorporation of BrdU in serum-treated VSMCs. By contrast, either AAT1 or AAT2 knockdown significantly exacerbated serum-stimulated VSMC proliferation. Thus, both exogenous- and endogenous-derived SO2 suppressed serum-induced VSMC proliferation. However, annexin V-propidium iodide (PI) staining and cell cycle analysis demonstrated that SO2 did not influence VSMC apoptosis in the serum-induced proliferation model. In a platelet-derived growth factor (PDGF)-BB-stimulated VSMC proliferation model, SO2 dephosphorylated the active sites of Erk1/2, MAPK kinase 1/2 and RAF proto-oncogene serine/threonine-protein kinase (c-Raf) induced by PDGF-BB. However, the inactivation of the three kinases of the Erk/MAPK pathway was not due to the separate interferences on them by SO2 simultaneously, but a consequence of the influence on the upstream activity of the c-Raf molecule. Hence, we examined the cAMP/PKA pathway, which could inhibit Erk/MAPK transduction in VSMCs. The results showed that SO2 could stimulate the cAMP/PKA pathway to block c-Raf activation, whereas the Ser259 site on c-Raf had an important role in SO2-induced suppression of Erk/MAPK pathway. The present study firstly demonstrated that SO2 exerted a negative regulation of VSMC proliferation via suppressing the Erk/MAPK pathway mediated by cAMP/PKA signaling. PMID:24853429

Low-Dose Radiation Induces Cell Proliferation in Human Embryonic Lung Fibroblasts but not in Lung Cancer Cells: Importance of ERK1/2 and AKT Signaling Pathways.

PubMed

Liang, Xinyue; Gu, Junlian; Yu, Dehai; Wang, Guanjun; Zhou, Lei; Zhang, Xiaoying; Zhao, Yuguang; Chen, Xiao; Zheng, Shirong; Liu, Qiang; Cai, Lu; Cui, Jiuwei; Li, Wei

2016-01-01

Hormesis and adaptive responses are 2 important biological effects of low-dose ionizing radiation (LDR). In normal tissue, LDR induces hormesis as evinced by increased cell proliferation; however, whether LDR also increases tumor cell proliferation needs to be investigated. In this study, cell proliferation was assayed by total cell numbers and the Cell Counting Kit 8 assay. Mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3' -kinase(PI3K)-Akt (PI3K/AKT) phosphorylation were determined by Western blot analysis. Human embryonic lung fibroblast 2BS and lung cancer NCI-H446 cell lines were irradiated with LDR at different doses (20-100 mGy). In response to 20 to 75 mGy X-rays, cell proliferation was significantly increased in 2BS but not in NCI-H446 cells. In 2BS cells, LDR at 20 to 75 mGy also stimulated phosphorylation of MAPK/ERK pathway proteins including ERK, MEK, and Raf and of the PI3K/AKT pathway protein AKT. To test whether ERK1/2 and AKT pathway activation was involved in the stimulation of cell proliferation in 2BS cells, the MAPK/ERK and PI3K/AKT pathways were inhibited using their specific inhibitors, U0126 and LY294002. U0126 decreased the phosphorylation of ERK1/2, and LY294002 decreased the phosphorylation of AKT; each could significantly inhibit LDR-induced 2BS cell proliferation. However, LDR did not stimulate these kinases, and kinase inhibitors also did not affect cell proliferation in the NCI-H446 cells. These results suggest that LDR stimulates cell proliferation via the activation of both MAPK/ERK and PI3K/AKT signaling pathways in 2BS but not in NCI-H446 cells. This finding implies the potential for applying LDR to protect normal tissues from radiotherapy without diminishing the efficacy of tumor therapy.

Reactive oxygen species mediate nitric oxide production through ERK/JNK MAPK signaling in HAPI microglia after PFOS exposure

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wang, Cheng; Nie, Xiaoke; Zhang, Yan

2015-10-15

Perfluorooctane sulfonate (PFOS), an emerging persistent contaminant that is commonly encountered during daily life, has been shown to exert toxic effects on the central nervous system (CNS). However, the molecular mechanisms underlying the neurotoxicity of PFOS remain largely unknown. It has been widely acknowledged that the inflammatory mediators released by hyper-activated microglia play vital roles in the pathogenesis of various neurological diseases. In the present study, we examined the impact of PFOS exposure on microglial activation and the release of proinflammatory mediators, including nitric oxide (NO) and reactive oxidative species (ROS). We found that PFOS exposure led to concentration-dependent NOmore » and ROS production by rat HAPI microglia. We also discovered that there was rapid activation of the ERK/JNK MAPK signaling pathway in the HAPI microglia following PFOS treatment. Moreover, the PFOS-induced iNOS expression and NO production were attenuated after the inhibition of ERK or JNK MAPK by their corresponding inhibitors, PD98059 and SP600125. Interestingly, NAC, a ROS inhibitor, blocked iNOS expression, NO production, and activation of ERK and JNK MAPKs, which suggested that PFOS-mediated microglial NO production occurs via a ROS/ERK/JNK MAPK signaling pathway. Finally, by exposing SH-SY5Y cells to PFOS-treated microglia-conditioned medium, we demonstrated that NO was responsible for PFOS-mediated neuronal apoptosis. - Highlights: • PFOS exposure induced expression of iNOS and production of NO in HAPI microglia. • PFOS induced the production of ROS in HAPI microglia. • ERK/JNK MAPK pathways were activated following PFOS exposure in HAPI microglia. • NO released by HAPI microglia participated in the apoptosis of SH-SY5Y cells.« less

Schisandrin B elicits a glutathione antioxidant response and protects against apoptosis via the redox-sensitive ERK/Nrf2 pathway in H9c2 cells.

PubMed

Chiu, Po Yee; Chen, Na; Leong, Po Kuan; Leung, Hoi Yan; Ko, Kam Ming

2011-04-01

This study investigated the signal transduction pathway involved in the cytoprotective action of (-)schisandrin B [(-)Sch B, a stereoisomer of Sch B]. Using H9c2 cells, the authors examined the effects of (-)Sch B on MAPK and Nrf2 activation, as well as the subsequent eliciting of glutathione response and protection against apoptosis. Pharmacological tools, such as cytochrome P-450 (CYP) inhibitor, antioxidant, MAPK inhibitor, and Nrf2 RNAi, were used to delineate the signaling pathway. (-)Sch B caused a time-dependent activation of MAPK in H9c2 cells, with the degree of ERK activation being much larger than that of p38 or JNK. The MAPK activation was followed by an increase in the level of nuclear Nrf2, an indirect measure of Nrf2 activation, and the eliciting of a glutathione antioxidant response. The activation of MAPK and Nrf2 seemed to involve oxidants generated from a CYP-catalyzed reaction with (-)Sch B. Both ERK inhibition by U0126 and Nrf2 suppression by Nrf2 RNAi transfection largely abolished the cytoprotection against hypoxia/reoxygenation-induced apoptosis in (-)Sch B-pretreated cells. (-)Sch B pretreatment potentiated the reoxygenation-induced ERK activation, whereas both p38 and JNK activations were suppressed. Under the condition of ERK inhibition, Sch B treatment did not protect against ischemia/reperfusion injury in an ex vivo rat heart model. The results indicate that (-)Sch B triggers a redox-sensitive ERK/Nrf2 signaling, which then elicits a cellular glutathione antioxidant response and protects against hypoxia/reoxygenation-induced apoptosis in H9c2 cells. The ERK-mediated signaling is also likely involved in the cardioprotection afforded by Sch B in vivo.

Implication of the ERK/MAPK pathway in antipsychotics-induced dopamine D2 receptor upregulation and in the preventive effects of (±)-α-lipoic acid in SH-SY5Y neuroblastoma cells.

PubMed

Deslauriers, Jessica; Desmarais, Christian; Sarret, Philippe; Grignon, Sylvain

2014-03-01

Chronic administration of antipsychotics (APs) has been associated with dopamine D2 receptor (D2R) upregulation and tardive dyskinesia. We previously showed that haloperidol, a first-generation AP, exerted a more robust increase in D2R expression than amisulpride, a second-generation AP and that (±)-α-lipoic acid pre-treatment reversed the AP-induced D2R upregulation. We also demonstrated that the Akt/GSK-3β/β-catenin pathway is involved in the control of D2R expression levels, but is unlikely implicated in the preventive effects of (±)-α-lipoic acid since co-treatment with haloperidol and (±)-α-lipoic acid exerts synergistic effects on Akt/GSK-3β activation. These findings led us to examine whether the ERK/MAPK signaling pathway may be involved in D2R upregulation elicited by APs, and in its reversal by (±)-α-lipoic acid, in SH-SY5Y human neuroblastoma cells. Our results revealed that haloperidol, in parallel with an elevation in D2R mRNA levels, induced a larger increase of ERK (p42/p44) phosphorylation than amisulpride. Pre-treatment with the selective ERK inhibitor U0126 attenuated haloperidol-induced increase in D2R upregulation. Furthermore, (±)-α-lipoic acid prevented AP-induced ERK activation. These results show that (1) the ERK/MAPK pathway is involved in haloperidol-induced D2R upregulation; (2) the preventive effect of (±)-α-lipoic acid on haloperidol-induced D2R upregulation is in part mediated by an ERK/MAPK-dependent signaling cascade. Taken together, our data suggest that (±)-α-lipoic acid exerts synergistic effects with haloperidol on the Akt/GSK-3β pathway, potentially involved in the therapeutic effects of APs, and antagonism of ERK activation and D2R upregulation, potentially involved in tardive dyskinesia and treatment resistance.

Regulation of Schistosoma mansoni development and reproduction by the mitogen-activated protein kinase signaling pathway.

PubMed

Andrade, Luiza Freire de; Mourão, Marina de Moraes; Geraldo, Juliana Assis; Coelho, Fernanda Sales; Silva, Larissa Lopes; Neves, Renata Heisler; Volpini, Angela; Machado-Silva, José Roberto; Araujo, Neusa; Nacif-Pimenta, Rafael; Caffrey, Conor R; Oliveira, Guilherme

2014-06-01

Protein kinases are proven targets for drug development with an increasing number of eukaryotic Protein Kinase (ePK) inhibitors now approved as drugs. Mitogen-activated protein kinase (MAPK) family members connect cell-surface receptors to regulatory targets within cells and influence a number of tissue-specific biological activities such as cell proliferation, differentiation and survival. However, the contributions of members of the MAPK pathway to schistosome development and survival are unclear. We employed RNA interference (RNAi) to elucidate the functional roles of five S. mansoni genes (SmCaMK2, SmJNK, SmERK1, SmERK2 and SmRas) involved in MAPK signaling pathway. Mice were injected with post-infective larvae (schistosomula) subsequent to RNAi and the development of adult worms observed. The data demonstrate that SmJNK participates in parasite maturation and survival of the parasites, whereas SmERK are involved in egg production as infected mice had significantly lower egg burdens with female worms presenting underdeveloped ovaries. Furthermore, it was shown that the c-fos transcription factor was overexpressed in parasites submitted to RNAi of SmERK1, SmJNK and SmCaMK2 indicating its putative involvement in gene regulation in this parasite's MAPK signaling cascade. We conclude that MAPKs proteins play important roles in the parasite in vivo survival, being essential for normal development and successful survival and reproduction of the schistosome parasite. Moreover SmERK and SmJNK are potential targets for drug development.

Tanshinone IIA inhibits AGEs-induced proliferation and migration of cultured vascular smooth muscle cells by suppressing ERK1/2 MAPK signaling.

PubMed

Lu, Ming; Luo, Ying; Hu, Pengfei; Dou, Liping; Huang, Shuwei

2018-01-01

Vascular smooth muscle cells (VSMCs) play a key role in the pathogenesis of diabetic vascular disease. Our current study sought to explore the effects of tanshinone IIA on the proliferation and migration of VSMCs induced by advanced glycation end products (AGEs). In this study, we examined the effects of tanshinone IIA by cell proliferation assay and cell migration assay. And we explored the underlying mechanism by Western blotting. AGEs significantly induced the proliferation and migration of VSMCs, but treatment with tanshinone IIA attenuated these effects. AGEs could increase the activity of the ERK1/2 and p38 pathways but not the JNK pathway. Treatment with tanshinone IIA inhibited the AGEs-induced activation of the ERK1/2 pathway but not the p38 pathway. Tanshinone IIA inhibits AGEs-induced proliferation and migration of VSMCs by suppressing the ERK1/2 MAPK signaling pathway.

Mineral trioxide aggregate upregulates odonto/osteogenic capacity of bone marrow stromal cells from craniofacial bones via JNK and ERK MAPK signalling pathways.

PubMed

Wang, Y; Li, J; Song, W; Yu, J

2014-06-01

The aim of this study was to investigate effects of mineral trioxide aggregate (MTA) on odonto/osteogenic differentiation of bone marrow stromal cells (BMSCs) from craniofacial bones. Craniofacial BMSCs were isolated from rat mandible and effects of MTA on their proliferation, differentiation and MAPK pathway involvement were subsequently investigated, in vitro. MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2,5-tetrazoliumbromide) assay was performed to evaluate proliferation of the MTA-treated cells. Alkaline phosphatase (ALP) activity, alizarin red staining, real-time reverse transcription polymerase chain reaction and western blot assays were used to assess differentiation capacity as well as MAPK pathway involvement. 0.02 mg/ml MTA-treated BMSCs had significantly higher ALP activity and formed more mineralized nodules than the untreated group. Odonto/osteoblastic marker genes/proteins (Alp, Runx2/RUNX2, Osx/OSX, Ocn/OCN and Dspp/DSP respectively) in MTA-treated cells were remarkably upregulated compared to untreated ones. Mechanistically, phosphorylated Jun N-terminal kinase (P-JNK) and phosphorylated extracellular regulated protein kinases (P-ERK) in MTA-treated BMSCs increased significantly in a time-dependent manner, while inhibition of JNK and ERK MAPK pathways dramatically blocked MTA-induced odonto/osteoblastic differentiation, as indicated by reduced ALP levels, weakened mineralization capacity and downregulated levels of odonto/osteoblastic marker genes (Alp, Runx2, Osx, Ocn and Dspp). Mineral trioxide aggregate promoted odonto/osteogenic capacity of craniofacial BMSCs via JNK and ERK MAPK signalling pathways. © 2014 John Wiley & Sons Ltd.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Song, Xiulong, E-mail: songxiulong@hotmail.com; Wei, Zhengxi; Shaikh, Zahir A., E-mail: zshaikh@uri.edu

Cadmium (Cd) is a common environmental toxicant and an established carcinogen. Epidemiological studies implicate Cd with human breast cancer. Low micromolar concentrations of Cd promote proliferation of human breast cancer cells in vitro. The growth promotion of breast cancer cells is associated with the activation of MAPK/ERK pathway. This study explores the mechanism of Cd-induced activation of MAPK/ERK pathway. Specifically, the role of cell surface receptors ERα, EGFR, and Src kinase was evaluated in human breast cancer MCF-7 cells treated with 1–3 μM Cd. The activation of ERK was studied using a serum response element (SRE) luciferase reporter assay. Receptormore » phosphorylation was detected by Western blot analyses. Cd treatment increased both the SRE reporter activity and ERK1/2 phosphorylation in a concentration-dependent manner. Cd treatment had no effect on reactive oxygen species (ROS) generation. Also, blocking the entry of Cd into the cells with manganese did not diminish Cd-induced activation of MAPK/ERK. These results suggest that the effect of Cd was likely not caused by intracellular ROS generation, but through interaction with the membrane receptors. While Cd did not appear to activate either EGFR or Src kinase, their inhibition completely blocked the Cd-induced activation of ERK as well as cell proliferation. Similarly, silencing ERα with siRNA or use of ERα antagonist blocked the effects of Cd. Based on these results, it is concluded that not only ERα, but also basal activities of EGFR and Src kinase are essential for Cd-induced signal transduction and activation of MAPK/ERK pathway for breast cancer cell proliferation. - Highlights: • Low micromolar concentrations of Cd rapidly activate ERK1/2 in MCF-7 cells. • Signal transduction and resulting cell proliferation require EGFR, ERα, and Src. • These findings implicate Cd in promotion of breast cancer.« less

WNK4 inhibits NCC protein expression through MAPK ERK1/2 signaling pathway.

PubMed

Zhou, Bo; Wang, Dexuan; Feng, Xiuyan; Zhang, Yiqian; Wang, Yanhui; Zhuang, Jieqiu; Zhang, Xuemei; Chen, Guangping; Delpire, Eric; Gu, Dingying; Cai, Hui

2012-03-01

WNK [with no lysine (K)] kinase is a subfamily of serine/threonine kinases. Mutations in two members of this family (WNK1 and WNK4) cause pseudohypoaldosteronism type II featuring hypertension, hyperkalemia, and metabolic acidosis. WNK1 and WNK4 were shown to regulate sodium chloride cotransporter (NCC) activity through phosphorylating SPAK and OSR1. Previous studies including ours have also shown that WNK4 inhibits NCC function and its protein expression. A recent study reported that a phorbol ester inhibits NCC function via activation of extracellular signal-regulated kinase (ERK) 1/2 kinase. In the current study, we investigated whether WNK4 affects NCC via the MAPK ERK1/2 signaling pathway. We found that WNK4 increased ERK1/2 phosphorylation in a dose-dependent manner in mouse distal convoluted tubule (mDCT) cells, whereas WNK4 mutants with the PHA II mutations (E562K and R1185C) lost the ability to increase the ERK1/2 phosphorylation. Hypertonicity significantly increased ERK1/2 phosphorylation in mDCT cells. Knock-down of WNK4 expression by siRNA resulted in a decrease of ERK1/2 phosphorylation. We further showed that WNK4 knock-down significantly increases the cell surface and total NCC protein expressions and ERK1/2 knock-down also significantly increases cell surface and total NCC expression. These data suggest that WNK4 inhibits NCC through activating the MAPK ERK1/2 signaling pathway.

Schwann Cell Migration Induced by Earthworm Extract via Activation of PAs and MMP2/9 Mediated through ERK1/2 and p38

PubMed Central

Chang, Yung-Ming; Shih, Ying-Ting; Chen, Yueh-Sheng; Liu, Chien-Liang; Fang, Wen-Kuei; Tsai, Chang-Hai; Tsai, Fuu-Jen; Kuo, Wei-Wen; Lai, Tung-Yuan; Huang, Chih-Yang

2011-01-01

The earthworm, which has stasis removal and wound-healing functions, is a widely used Chinese herbal medicine in China. Schwann cell migration is critical for the regeneration of injured nerves. Schwann cells provide an essentially supportive activity for neuron regeneration. However, the molecular migration mechanisms induced by earthworms in Schwann cells remain unclear. Here, we investigate the roles of MAPK (ERK1/2, JNK and p38) pathways for earthworm-induced matrix-degrading proteolytic enzyme (PAs and MMP2/9) production in Schwann cells. Moreover, earthworm induced phosphorylation of ERK1/2 and p38, but not JNK, activate the downstream signaling expression of PAs and MMPs in a time-dependent manner. Earthworm-stimulated ERK1/2 and p38 phosphorylation was attenuated by pretreatment with U0126 and SB203580, resulting in migration and uPA-related signal pathway inhibition. The results were confirmed using small interfering ERK1/2 and p38 RNA. These results demonstrated that earthworms can stimulate Schwann cell migration and up-regulate PAs and MMP2/9 expression mediated through the MAPK pathways, ERK1/2 and p38. Taken together, our data suggests the MAPKs (ERK1/2, p38)-, PAs (uPA, tPA)-, MMP (MMP2, MMP9) signaling pathway of Schwann cells regulated by earthworms might play a major role in Schwann cell migration and nerve regeneration. PMID:19808845

Low-Dose Radiation Induces Cell Proliferation in Human Embryonic Lung Fibroblasts but not in Lung Cancer Cells

PubMed Central

Liang, Xinyue; Gu, Junlian; Yu, Dehai; Wang, Guanjun; Zhou, Lei; Zhang, Xiaoying; Zhao, Yuguang; Chen, Xiao; Zheng, Shirong; Liu, Qiang; Cai, Lu

2016-01-01

Hormesis and adaptive responses are 2 important biological effects of low-dose ionizing radiation (LDR). In normal tissue, LDR induces hormesis as evinced by increased cell proliferation; however, whether LDR also increases tumor cell proliferation needs to be investigated. In this study, cell proliferation was assayed by total cell numbers and the Cell Counting Kit 8 assay. Mitogen-activated protein kinases (MAPK)/extracellular signal-regulated kinase (ERK) and phosphatidylinositol 3′ -kinase(PI3K)-Akt (PI3K/AKT) phosphorylation were determined by Western blot analysis. Human embryonic lung fibroblast 2BS and lung cancer NCI-H446 cell lines were irradiated with LDR at different doses (20-100 mGy). In response to 20 to 75 mGy X-rays, cell proliferation was significantly increased in 2BS but not in NCI-H446 cells. In 2BS cells, LDR at 20 to 75 mGy also stimulated phosphorylation of MAPK/ERK pathway proteins including ERK, MEK, and Raf and of the PI3K/AKT pathway protein AKT. To test whether ERK1/2 and AKT pathway activation was involved in the stimulation of cell proliferation in 2BS cells, the MAPK/ERK and PI3K/AKT pathways were inhibited using their specific inhibitors, U0126 and LY294002. U0126 decreased the phosphorylation of ERK1/2, and LY294002 decreased the phosphorylation of AKT; each could significantly inhibit LDR-induced 2BS cell proliferation. However, LDR did not stimulate these kinases, and kinase inhibitors also did not affect cell proliferation in the NCI-H446 cells. These results suggest that LDR stimulates cell proliferation via the activation of both MAPK/ERK and PI3K/AKT signaling pathways in 2BS but not in NCI-H446 cells. This finding implies the potential for applying LDR to protect normal tissues from radiotherapy without diminishing the efficacy of tumor therapy. PMID:26788032

Effects of cadmium on MAPK signalling pathways and HSP70 expression in a human trophoblast cell line.

PubMed

Valbonesi, P; Ricci, L; Franzellitti, S; Biondi, C; Fabbri, E

2008-08-01

The aim of this work was to provide a greater insight into the possible effects of Cd on signal transduction and stress-related pathways in reproductive tissues. Cd is a known placental toxin in both animals and humans. Our experiments were designed to study the influence of Cd on MAPK (ERK1/2, JNK1/2 and p38MAPK) activation in the extravillous trophoblast cell line, HTR-8/SVneo, used as an experimental model. We also studied the HSP70 response in cells exposed to Cd, since these proteins may have an important role in conferring protection and tolerance against teratogenic concentrations of the metal. The effects of Cd were compared with those of a well-known toxic agent, H2O2. The metal triggered MAPK activation in a dose- and time-dependent manner. At 30 microM Cd, stimulations of about 300%, 550% and 250% were observed for ERK1/2, JNK1/2, and p38MAPK, respectively. Phosphorylation of ERK1/2 and JNK1/2 was significantly induced after a 1-h exposure to 30 microM Cd, while that of p38MAPK occurred only after 8h. Similarly, H2O2 caused dose- and time-dependent activation of MAPK pathways. Cd potently stimulated HSP70 expression and that of related genes HSP70 A, B and C. H2O2 did not increase HSP70 and HSP70 A and B expression, while temporarily increasing HSP70C transcript levels. In conclusion, Cd triggers different stress responses in trophoblast cells involving HSP70 and SAPK, and also enhances ERK1/2 phosphorylation. Since MAPK dependent pathways play a crucial role during pregnancy, non-physiological activation by Cd exposure may disrupt normal functions in trophoblast cells.

Interactions between Sirt1 and MAPKs regulate astrocyte activation induced by brain injury in vitro and in vivo.

PubMed

Li, Dan; Liu, Nan; Zhao, Hai-Hua; Zhang, Xu; Kawano, Hitoshi; Liu, Lu; Zhao, Liang; Li, Hong-Peng

2017-03-29

Astrocyte activation is a hallmark of traumatic brain injury resulting in neurological dysfunction or death for an overproduction of inflammatory cytokines and glial scar formation. Both the silent mating type information (Sirt1) expression and mitogen-activated protein kinase (MAPK) signal pathway activation represent a promising therapeutic target for several models of neurodegenerative diseases. We investigated the potential effects of Sirt1 upregulation and MAPK pathway pharmacological inhibition on astrocyte activation in vitro and in vivo. Moreover, we attempted to confirm the underlying interactions between Sirt1 and MAPK pathways in astrocyte activation after brain injury. The present study employs an interleukin-1β (IL-1β) stimulated primary cortical astrocyte model in vitro and a nigrostriatal pathway injury model in vivo to mimic the astrocyte activation induced by traumatic brain injury. The activation of GFAP, Sirt1, and MAPK pathways were detected by Western blot; astrocyte morphological hypertrophy was assessed using immunofluorescence staining; in order to explore the neuroprotective effect of regulation Sirt1 expression and MAPK pathway activation, the motor and neurological function tests were assessed after injury. GFAP level and morphological hypertrophy of astrocytes are elevated after injury in vitro or in vivo. Furthermore, the expressions of phosphorylated extracellular regulated protein kinases (p-ERK), phosphorylated c-Jun N-terminal kinase (p-JNK), and phosphorylated p38 activation (p-p38) are upregulated, but the Sirt1 expression is downregulated. Overexpression of Sirt1 significantly increases the p-ERK expression and reduces the p-JNK and p-p38 expressions. Inhibition of ERK, JNK, or p38 activation respectively with their inhibitors significantly elevated the Sirt1 expression and attenuated the astrocyte activation. Both the overproduction of Sirt1 and inhibition of ERK, JNK, or p38 activation can alleviate the astrocyte activation, thereby improving the neurobehavioral function according to the modified neurological severity scores (mNSS) and balance latency test. Thus, Sirt1 plays a protective role against astrocyte activation, which may be associated with the regulation of the MAPK pathway activation induced by brain injury in vitro and in vivo.

Time-dependent activation of MAPK/Erk1/2 and Akt/GSK3 cascades: modulation by agomelatine.

PubMed

Musazzi, Laura; Seguini, Mara; Mallei, Alessandra; Treccani, Giulia; Pelizzari, Mariagrazia; Tornese, Paolo; Racagni, Giorgio; Tardito, Daniela

2014-10-21

The novel antidepressant agomelatine, a melatonergic MT1/MT2 agonist combined with 5-HT2c serotonin antagonist properties, showed antidepressant action in preclinical and clinical studies. There is a general agreement that the therapeutic action of antidepressants needs the activation of slow-onset adaptations in downstream signalling pathways finally regulating neuroplasticity. In the last several years, particular attention was given to cAMP-responsive element binding protein (CREB)-related pathways, since it was shown that chronic antidepressants increase CREB phosphorylation and transcriptional activity, through the activation of calcium/calmodulin-dependent (CaM) and mitogen activated protein kinase cascades (MAPK/Erk1/2). Aim of this work was to analyse possible effects of chronic agomelatine on time-dependent changes of different intracellular signalling pathways in hippocampus and prefrontal/frontal cortex of male rats. To this end, measurements were performed 1 h or 16 h after the last agomelatine or vehicle injection. We have found that in naïve rats chronic agomelatine, contrary to traditional antidepressants, did not increase CREB phosphorylation, but modulates the time-dependent regulation of MAPK/Erk1/2 and Akt/glycogen synthase kinase-3 (GSK-3) pathways. Our results suggest that the intracellular molecular mechanisms modulated by chronic agomelatine may be partly different from those of traditional antidepressants and involve the time-dependent regulation of MAPK/Erk1/2 and Akt/GSK-3 signalling pathways. This could exert a role in the antidepressant efficacy of the drug.

Effects of Curcumin on Tobacco Smoke-induced Hepatic MAPK Pathway Activation and Epithelial-Mesenchymal Transition In Vivo.

PubMed

Liang, Zhaofeng; Wu, Rui; Xie, Wei; Xie, Chunfeng; Wu, Jieshu; Geng, Shanshan; Li, Xiaoting; Zhu, Mingming; Zhu, Weiwei; Zhu, Jianyun; Huang, Cong; Ma, Xiao; Xu, Wenrong; Zhong, Caiyun; Han, Hongyu

2017-08-01

Tobacco smoke is a major risk factor for hepatic cancer. Epithelial-mesenchymal transition (EMT) induced by tobacco smoke is crucially involved in the initiation and development of cancer. Mitogen-activated protein kinase (MAPK) pathways play important roles in tobacco smoke-associated carcinogenesis including EMT process. The chemopreventive effect of curcumin supplementation against cancers has been reported. In this study, we investigated the effects of tobacco smoke on MAPK pathway activation and EMT alterations, and then the preventive effect of curcumin was examined in the liver of BALB/c mice. Our results indicated that exposure of mice to tobacco smoke for 12 weeks led to activation of ERK1/2, JNK, p38 and ERK5 pathways as well as activator protein-1 (AP-1) proteins in liver tissue. Exposure of mice to tobacco smoke reduced the hepatic mRNA and protein expression of the epithelial markers, while the hepatic mRNA and protein levels of the mesenchymal markers were increased. Treatment of curcumin effectively attenuated tobacco smoke-induced activation of ERK1/2 and JNK MAPK pathways, AP-1 proteins and EMT alterations in the mice liver. Our data suggested the protective effect of curcumin in tobacco smoke-triggered MAPK pathway activation and EMT in the liver of BALB/c mice, thus providing new insights into the chemoprevention of tobacco smoke-associated hepatic cancer. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Ca2+ -stimulated adenylyl cyclases regulate ERK-dependent activation of MSK1 during fear conditioning.

PubMed

Sindreu, Carlos Balet; Scheiner, Zachary S; Storm, Daniel R

2007-01-04

The cAMP and ERK/MAP kinase (MAPK) signal transduction pathways are critical for hippocampus-dependent memory, a process that depends on CREB-mediated transcription. However, the extent of crosstalk between these pathways and the downstream CREB kinase activated during memory formation has not been elucidated. Here we report that PKA, MAPK, and MSK1, a CREB kinase, are coactivated in a subset of hippocampal CA1 pyramidal neurons following contextual fear conditioning. Activation of PKA, MAPK, MSK1, and CREB is absolutely dependent on Ca(2+)-stimulated adenylyl cyclase activity. We conclude that adenylyl cyclase activity supports the activation of MAPK, and that MSK1 is the major CREB kinase activated during training for contextual memory.

Ca2+-Stimulated Adenylyl Cyclases Regulate ERK-Dependent Activation of MSK1 During Fear Conditioning

PubMed Central

Sindreu, Carlos Balet; Scheiner, Zachary S.; Storm, Daniel R.

2007-01-01

The cAMP and ERK/MAP kinase (MAPK) signal transduction pathways are critical for hippocampus-dependent memory, a process that depends on CREB-mediated transcription. However, the extent of crosstalk between these pathways and the downstream CREB kinase activated during memory formation have not been elucidated. Here we report that PKA, MAPK, and MSK1, a CREB kinase, are co-activated in a subset of hippocampal CA1 pyramidal neurons following contextual fear conditioning. Activation of PKA, MAPK, MSK1, and CREB is absolutely dependent on Ca2+-stimulated adenylyl cyclase activity. We conclude that adenylyl cyclase activity supports the activation of MAPK, and that MSK1 is the major CREB kinase activated during training for contextual memory. PMID:17196532

JWA gene regulates PANC-1 pancreatic cancer cell behaviors through MEK-ERK1/2 of the MAPK signaling pathway.

PubMed

Wu, Yuan-Yuan; Ma, Tie-Liang; Ge, Zhi-Jun; Lin, Jie; Ding, Wei-Liang; Feng, Jia-Ke; Zhou, Su-Jun; Chen, Guo-Chang; Tan, Yong-Fei; Cui, Guo-Xing

2014-10-01

The present study aimed to investigate the role of JWA gene in the proliferation, apoptosis, invasion and migration of PANC-1 pancreatic cancer cells and the effect on the MAPK signaling pathway. Human PANC-1 pancreatic cancer cells were cultured in vitro , and small interfering RNA (siRNA) was designed for the JWA gene. The siRNA was transfected into PANC-1 cells. Subsequently, the cell proliferation was measured by MTT assay; cell apoptosis was detected by analyzing BAX and Bcl-2 protein expression; cell migration and invasion were measured using Transwell ® chambers; and the protein expression of JWA and ERK1/2, JNK and p38 and their phosphorylated forms were measured by western blotting. By utilizing the MTT assay, the results showed that when JWA protein expression was inhibited, the proliferation of PANC-1 cells was enhanced. In addition, the expression of apoptosis-associated protein (AAP) BAX was substantially decreased, while the expression of the apoptosis inhibitor gene, Bcl-2 , was significantly enhanced. Using Transwell chambers, it was found that the number of penetrating PANC-1 cells was significantly increased after transfection with JWA siRNA, suggesting that the migration and invasion of the cells was substantially increased. By studying the association between JWA and the MAPK pathway in PANC-1 cells, it was found that the expression of p-ERK1/2 of the MAPK pathway was significantly downregulated following JWA siRNA transfection. However, the expression levels of ERK1/2, JNK, p38, p-JNK and p-p38 showed no significant differences. In conclusion, it was shown that JWA affects the proliferation, apoptosis, invasion and migration of PANC-1 pancreatic cancer cells which could be attributed to effects on the expression of ERK1/2 in the MAPK pathway.

Tanshinone IIA inhibits AGEs-induced proliferation and migration of cultured vascular smooth muscle cells by suppressing ERK1/2 MAPK signaling

PubMed Central

Lu, Ming; Luo, Ying; Hu, Pengfei; Dou, Liping; Huang, Shuwei

2018-01-01

Objective(s): Vascular smooth muscle cells (VSMCs) play a key role in the pathogenesis of diabetic vascular disease. Our current study sought to explore the effects of tanshinone IIA on the proliferation and migration of VSMCs induced by advanced glycation end products (AGEs). Materials and Methods: In this study, we examined the effects of tanshinone IIA by cell proliferation assay and cell migration assay. And we explored the underlying mechanism by Western blotting. Results: AGEs significantly induced the proliferation and migration of VSMCs, but treatment with tanshinone IIA attenuated these effects. AGEs could increase the activity of the ERK1/2 and p38 pathways but not the JNK pathway. Treatment with tanshinone IIA inhibited the AGEs-induced activation of the ERK1/2 pathway but not the p38 pathway. Conclusion: Tanshinone IIA inhibits AGEs-induced proliferation and migration of VSMCs by suppressing the ERK1/2 MAPK signaling pathway. PMID:29372041

Multiple division cycles and long-term survival of hepatocytes are distinctly regulated by extracellular signal-regulated kinases ERK1 and ERK2.

PubMed

Frémin, Christophe; Bessard, Anne; Ezan, Frédéric; Gailhouste, Luc; Régeard, Morgane; Le Seyec, Jacques; Gilot, David; Pagès, Gilles; Pouysségur, Jacques; Langouët, Sophie; Baffet, Georges

2009-03-01

We investigated the specific role of the mitogen-activated protein kinase (MAPK) extracellular signal-regulated kinase 1 (ERK1)/ERK2 pathway in the regulation of multiple cell cycles and long-term survival of normal hepatocytes. An early and sustained epidermal growth factor (EGF)-dependent MAPK activation greatly improved the potential of cell proliferation. In this condition, almost 100% of the hepatocytes proliferated, and targeting ERK1 or ERK2 via RNA interference revealed the specific involvement of ERK2 in this regulation. However, once their first cell cycle was performed, hepatocytes failed to undergo a second round of replication and stayed blocked in G1 phase. We demonstrated that sustained EGF-dependent activation of the MAPK/ERK kinase (MEK)/ERK pathway was involved in this blockage as specific transient inhibition of the cascade repotentiated hepatocytes to perform a new wave of replication and multiple cell cycles. We identified this mechanism by showing that this blockage was in part supported by ERK2-dependent p21 expression. Moreover, continuous MEK inhibition was associated with a lower apoptotic engagement, leading to an improvement of survival up to 3 weeks. Using RNA interference and ERK1 knockout mice, we extended these results by showing that this improved survival was due to the specific inhibition of ERK1 expression/phosphorylation and did not involve ERK2. Our results emphasize that transient MAPK inhibition allows multiple cell cycles in primary cultures of hepatocytes and that ERK2 has a key role in the regulation of S phase entry. Moreover, we revealed a major and distinct role of ERK1 in the regulation of hepatocyte survival. Taken together, our results represent an important advance in understanding long-term survival and cell cycle regulation of hepatocytes.

Inhibitors of stress-activated protein/mitogen-activated protein kinase pathways.

PubMed

Malemud, Charles J

2007-06-01

The importance of stress-activated protein/mitogen-activated protein kinase (SAP/MAPK) pathway signalling (involving c-Jun-N-terminal kinase [JNK], extracellular signal-regulated kinase [ERK] and p38 kinase) in normal cellular proliferation, differentiation and programmed cell death has led to significant recent advances in our understanding of the role of SAP/MAPK signaling in inflammatory disorders such as arthritis and cardiovascular disease, cancer, and pulmonary and neurogenerative diseases. The discovery that several natural products such as resveratrol, tangeretin and ligustilide non-specifically inhibit SAP/MAPK signalling in vitro should now be logically extended to studies designed to determine how agents in these natural products regulate SAP/MAPK pathways in animal models of disease. A new generation of small-molecule SAP/MAPK inhibitors that demonstrate increasing specificity for each of the JNK, ERK and p38 kinase isoforms has shown promise in animal studies and could eventually prove effective for treating human diseases. Several of these compounds are already being tested in human subjects to assess their oral bioavailability, pharmacokinetics and toxicity.

Metabolic Respiration Induces AMPK- and Ire1p-Dependent Activation of the p38-Type HOG MAPK Pathway

PubMed Central

Adhikari, Hema; Cullen, Paul J.

2014-01-01

Evolutionarily conserved mitogen activated protein kinase (MAPK) pathways regulate the response to stress as well as cell differentiation. In Saccharomyces cerevisiae, growth in non-preferred carbon sources (like galactose) induces differentiation to the filamentous cell type through an extracellular-signal regulated kinase (ERK)-type MAPK pathway. The filamentous growth MAPK pathway shares components with a p38-type High Osmolarity Glycerol response (HOG) pathway, which regulates the response to changes in osmolarity. To determine the extent of functional overlap between the MAPK pathways, comparative RNA sequencing was performed, which uncovered an unexpected role for the HOG pathway in regulating the response to growth in galactose. The HOG pathway was induced during growth in galactose, which required the nutrient regulatory AMP-dependent protein kinase (AMPK) Snf1p, an intact respiratory chain, and a functional tricarboxylic acid (TCA) cycle. The unfolded protein response (UPR) kinase Ire1p was also required for HOG pathway activation in this context. Thus, the filamentous growth and HOG pathways are both active during growth in galactose. The two pathways redundantly promoted growth in galactose, but paradoxically, they also inhibited each other's activities. Such cross-modulation was critical to optimize the differentiation response. The human fungal pathogen Candida albicans showed a similar regulatory circuit. Thus, an evolutionarily conserved regulatory axis links metabolic respiration and AMPK to Ire1p, which regulates a differentiation response involving the modulated activity of ERK and p38 MAPK pathways. PMID:25356552

Structural and Dynamic Insights into the Mechanism of Allosteric Signal Transmission in ERK2-Mediated MKP3 Activation.

PubMed

Lu, Chang; Liu, Xin; Zhang, Chen-Song; Gong, Haipeng; Wu, Jia-Wei; Wang, Zhi-Xin

2017-11-21

The mitogen-activated protein kinases (MAPKs) are key components of cellular signal transduction pathways, which are down-regulated by the MAPK phosphatases (MKPs). Catalytic activity of the MKPs is controlled both by their ability to recognize selective MAPKs and by allosteric activation upon binding to MAPK substrates. Here, we use a combination of experimental and computational techniques to elucidate the molecular mechanism for the ERK2-induced MKP3 activation. Mutational and kinetic study shows that the 334 FNFM 337 motif in the MKP3 catalytic domain is essential for MKP3-mediated ERK2 inactivation and is responsible for ERK2-mediated MKP3 activation. The long-term molecular dynamics (MD) simulations further reveal a complete dynamic process in which the catalytic domain of MKP3 gradually changes to a conformation that resembles an active MKP catalytic domain over the time scale of the simulation, providing a direct time-dependent observation of allosteric signal transmission in ERK2-induced MKP3 activation.

CXCL1 inhibits airway smooth muscle cell migration through the decoy receptor Duffy antigen receptor for chemokines.

PubMed

Al-Alwan, Laila A; Chang, Ying; Rousseau, Simon; Martin, James G; Eidelman, David H; Hamid, Qutayba

2014-08-01

Airway smooth muscle cell (ASMC) migration is an important mechanism postulated to play a role in airway remodeling in asthma. CXCL1 chemokine has been linked to tissue growth and metastasis. In this study, we present a detailed examination of the inhibitory effect of CXCL1 on human primary ASMC migration and the role of the decoy receptor, Duffy AgR for chemokines (DARC), in this inhibition. Western blots and pathway inhibitors showed that this phenomenon was mediated by activation of the ERK-1/2 MAPK pathway, but not p38 MAPK or PI3K, suggesting a biased selection in the signaling mechanism. Despite being known as a nonsignaling receptor, small interference RNA knockdown of DARC showed that ERK-1/2 MAPK activation was significantly dependent on DARC functionality, which, in turn, was dependent on the presence of heat shock protein 90 subunit α. Interestingly, DARC- or heat shock protein 90 subunit α-deficient ASMCs responded to CXCL1 stimulation by enhancing p38 MAPK activation and ASMC migration through the CXCR2 receptor. In conclusion, we demonstrated DARC's ability to facilitate CXCL1 inhibition of ASMC migration through modulation of the ERK-1/2 MAPK-signaling pathway. Copyright © 2014 by The American Association of Immunologists, Inc.

The frequencies of calcium oscillations are optimized for efficient calcium-mediated activation of Ras and the ERK/MAPK cascade.

PubMed

Kupzig, Sabine; Walker, Simon A; Cullen, Peter J

2005-05-24

Ras proteins are binary switches that, by cycling through inactive GDP- and active GTP-bound conformations, regulate multiple cellular signaling pathways, including those that control growth and differentiation. For some time, it has been known that receptor-mediated increases in the concentration of intracellular free calcium ([Ca(2+)](i)) can modulate Ras activation. Increases in [Ca(2+)](i) often occur as repetitive Ca(2+) spikes or oscillations. Induced by electrical or receptor stimuli, these repetitive Ca(2+) oscillations increase in frequency with the amplitude of receptor stimuli, a phenomenon critical for the induction of selective cellular functions. Here, we show that Ca(2+) oscillations are optimized for Ca(2+)-mediated activation of Ras and signaling through the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) cascade. We present additional evidence that Ca(2+) oscillations reduce the effective Ca(2+) threshold for the activation of Ras and that the oscillatory frequency is optimized for activation of Ras and the ERK/MAPK pathway. Our results describe a hitherto unrecognized link between complex Ca(2+) signals and the modulation of the Ras/ERK/MAPK signaling cascade.

The role of MAPK signaling pathway in the Her-2-positive meningiomas

PubMed Central

Wang, Zhaoyin; Wang, Weijia; Xu, Shan; Wang, Shanshan; Tu, Yi; Xiong, Yifeng; Mei, Jinhong; Wang, Chunliang

2016-01-01

Meningiomas are common types of adult nerve system tumors. Although most cases are considered benign, due to its high rate of recurrence and easy malignant progression to anaplastic meningioma they present a puzzle for the current treatment. The HER-2 oncogene has important value for meningioma cells development and progression. So far, little is known about the effect on the exact underlying signal pathway and molecular mechanisms of HER-2-positive meningioma cells. The goal of the present study was to determine the effects of HER-2 gene and possible involvement of MAPK signal pathway in human malignant meningioma. We applied q-PCR analysis, immunofluorescence (IF) staining, western blot analysis, animal model, MAPK inhibition, MTT assay and cell invasion analysis for the investigation. The results demonstrated that the downregulation of the expression of HER-2 significantly inhibited cell motility and proliferation of human meningioma cells in vivo. Accordingly, in the HER-2-overexpression meningioma cells with the inhibition of ERK1/2, ERK5, JNK, in the cells with the ERK1/2, ERK5 inhibition, protein expression was markedly suppressed as well as the cell proliferation resistance. No difference was observed in the HER-2-overexpression meningioma cells with the inhibition of JNK. These findings suggest that HER-2 gene can affect the proliferation ability of human meningioma cells in vivo and MAPK signal pathway may contribute to the carcinogenesis and development of human meningiomas combinating with HER-2. PMID:27279438

Alpinia oxyphylla Miquel fruit extract activates MAPK-mediated signaling of PAs and MMP2/9 to induce Schwann cell migration and nerve regeneration.

PubMed

Chang, Yung-Ming; Ye, Chi-Xin; Ho, Tsung-Jung; Tsai, Te-Neng; Chiu, Ping-Ling; Tsai, Chin-Chuan; Lin, Yueh-Min; Kuo, Chia-Hua; Tsai, Fuu-Jen; Tsai, Chang-Hai; Huang, Chih-Yang

2014-05-01

This study investigates the molecular mechanisms by which Alpiniae oxyphyllae fructus (AOF) promotes neuron regeneration. A piece of silicone rubber was guided across a 15 mm gap in the sciatic nerve of a rat. This nerve gap was then filled with different concentrations of AOF extract (0-200 mg/ml). We investigated the role of MAPK (ERK1/2, JNK and p38) pathways for AOF-induced matrix-degrading proteolytic enzyme (PAs and MMP2/9) production in RSC96 Schwann cells. The results showed that AOF increased the expressions of uPA, tPA, MMP-9, and MAPKs in vivo. In vitro, our results show that treatment with AOF extract induces ERK1/2, JNK, and p38 phosphorylation to activate the downstream PAs and MMPs signaling expression. AOF-stimulated ERK1/2, JNK, and p38 phosphorylation attenuated by individual pretreatment with siRNAs or inhibitors (U0126, SP600125 and SB203580), resulting in migration and uPA-related signal pathway inhibition. Taken together our data suggests the MAPKs (ERK1/2, JNK and p38), PAs (uPA, tPA), MMP (MMP2, MMP9) regenerative and migration signaling pathway of Schwann cells regulated by AOF extract might play a major role in Schwann cell migration and damaged peripheral nerve regeneration.

Protocatechuic Acid from Alpinia oxyphylla Induces Schwann Cell Migration via ERK1/2, JNK and p38 Activation.

PubMed

Ju, Da-Tong; Kuo, Wei-Wen; Ho, Tsung-Jung; Paul, Catherine Reena; Kuo, Chia-Hua; Viswanadha, Vijaya Padma; Lin, Chien-Chung; Chen, Yueh-Sheng; Chang, Yung-Ming; Huang, Chih-Yang

2015-01-01

Alpinia oxyphylla MIQ (Alpinate Oxyphyllae Fructus, AOF) is an important traditional Chinese medicinal herb whose fruits is widely used to prepare tonics and is used as an aphrodisiac, anti salivary, anti diuretic and nerve-protective agent. Protocatechuic acid (PCA), a simple phenolic compound was isolated from the kernels of AOF. This study investigated the role of PCA in promoting neural regeneration and the underlying molecular mechanisms. Nerve regeneration is a complex physiological response that takes place after injury. Schwann cells play a crucial role in the endogenous repair of peripheral nerves due to their ability to proliferate and migrate. The role of PCA in Schwann cell migration was determined by assessing the induced migration potential of RSC96 Schwann cells. PCA induced changes in the expression of proteins of three MAPK pathways, as determined using Western blot analysis. In order to determine the roles of MAPK (ERK1/2, JNK, and p38) pathways in PCA-induced matrix-degrading proteolytic enzyme (PAs and MMP2/9) production, the expression of several MAPK-associated proteins was analyzed after siRNA-mediated inhibition assays. Treatment with PCA-induced ERK1/2, JNK, and p38 phosphorylation that activated the downstream expression of PAs and MMPs. PCA-stimulated ERK1/2, JNK and p38 phosphorylation was attenuated by individual pretreatment with siRNAs or MAPK inhibitors (U0126, SP600125, and SB203580), resulting in the inhibition of migration and the uPA-related signal pathway. Taken together, our data suggest that PCA extract regulate the MAPK (ERK1/2, JNK, and p38)/PA (uPA, tPA)/MMP (MMP2, MMP9) mediated regeneration and migration signaling pathways in Schwann cells. Therefore, PCA plays a major role in Schwann cell migration and the regeneration of damaged peripheral nerve.

PKCalpha-mediated ERK, JNK and p38 activation regulates the myogenic program in human rhabdomyosarcoma cells.

PubMed

Mauro, Annunziata; Ciccarelli, Carmela; De Cesaris, Paola; Scoglio, Arianna; Bouché, Marina; Molinaro, Mario; Aquino, Angelo; Zani, Bianca Maria

2002-09-15

We have previously suggested that PKCalpha has a role in 12-O-Tetradecanoylphorbol-13-acetate (TPA)-mediated growth arrest and myogenic differentiation in human embryonal rhabdomyosarcoma cells (RD). Here, by monitoring the signalling pathways triggered by TPA, we demonstrate that PKCalpha mediates these effects by inducing transient activation of c-Jun N-terminal protein kinases (JNKs) and sustained activation of both p38 kinase and extracellular signal-regulated kinases (ERKs) (all referred to as MAPKs). Activation of MAPKs following ectopic expression of constitutively active PKCalpha, but not its dominant-negative form, is also demonstrated. We investigated the selective contribution of MAPKs to growth arrest and myogenic differentiation by monitoring the activation of MAPK pathways, as well as by dissecting MAPK pathways using MEK1/2 inhibitor (UO126), p38 inhibitor (SB203580) and JNK and p38 agonist (anisomycin) treatments. Growth-arresting signals are triggered either by transient and sustained JNK activation (by TPA and anisomycin, respectively) or by preventing both ERK and JNK activation (UO126) and are maintained, rather than induced, by p38. We therefore suggest a key role for JNK in controlling ERK-mediated mitogenic activity. Notably, sarcomeric myosin expression is induced by both TPA and UO126 but is abrogated by the p38 inhibitor. This finding indicates a pivotal role for p38 in controlling the myogenic program. Anisomycin persistently activates p38 and JNKs but prevents myosin expression induced by TPA. In accordance with this negative role, reactivation of JNKs by anisomycin, in UO126-pre-treated cells, also prevents myosin expression. This indicates that, unlike the transient JNK activation that occurs in the TPA-mediated myogenic process, long-lasting JNK activation supports the growth-arrest state but antagonises p38-mediated myosin expression. Lastly, our results with the MEK inhibitor suggest a key role of the ERK pathway in regulating myogenic-related morphology in differentiated RD cells.

Benzo[a]pyrene activates an AhR/Src/ERK axis that contributes to CYP1A1 induction and stable DNA adducts formation in lung cells.

PubMed

Vázquez-Gómez, G; Rocha-Zavaleta, L; Rodríguez-Sosa, M; Petrosyan, P; Rubio-Lightbourn, J

2018-06-01

Benzo[a]pyrene (B[a]P), the most extensively studied carcinogen in cigarette smoke, has been regarded as a critical mediator of lung cancer. It is known that B[a]P-mediated Aryl hydrocarbon Receptor (AhR) activation stimulates the mitogen activated protein kinases (MAPK) signaling cascade in different cell models. MAPK pathway disturbances drive alterations in cellular processes, such as differentiation, proliferation, and apoptosis, and the disturbances may also modify the AhR pathway itself. However, MAPK involvement in B[a]P metabolic activation and toxicity in lung tissues is not well understood. Here, we used a non-transformed human bronchial epithelial lung cell line, BEAS-2B, to study the participation of ERK 1/2 kinases in the metabolic activation of B[a]P and in its related genotoxic effects. Our results indicate that B[a]P is not cytotoxic to BEAS-2B cells at relatively low concentrations, but it enhances CYP1A1 gene transcription and protein induction. Additionally, B[a]P promotes Src and ERK 1/2 phosphorylation. Accordingly, inhibition of both Src and ERK 1/2 phosphorylation decreases CYP1A1 protein induction, AhR nuclear translocation and production of B[a]P adducts. Together, these data suggest a crosstalk between AhR and the members of the MAPK pathway, ERK 1/2 mediated by Src kinase. This interaction is important for the adequate AhR pathway signaling that in turn induces transcription and protein induction of CYP1A1 and B[a]P-induced DNA damage in BEAS-2B cells. Copyright © 2018 Elsevier B.V. All rights reserved.

Function-specific intracellular signaling pathways downstream of heparin-binding EGF-like growth factor utilized by human trophoblasts.

PubMed

Jessmon, Philip; Kilburn, Brian A; Romero, Roberto; Leach, Richard E; Armant, D Randall

2010-05-01

Heparin-binding EGF-like growth factor (HBEGF) is expressed by trophoblast cells throughout gestation. First-trimester cytotrophoblast cells are protected from hypoxia-induced apoptosis because of the accumulation of HBEGF through a posttranscriptional autocrine mechanism. Exogenous application of HBEGF is cytoprotective in a hypoxia/reoxygenation (H/R) injury model and initiates trophoblast extravillous differentiation to an invasive phenotype. The downstream signaling pathways induced by HBEGF that mediate these various cellular activities were identified using two human first-trimester cytotrophoblast cell lines, HTR-8/SVneo and SW.71, with similar results. Recombinant HBEGF (1 nM) induced transient phosphorylation of MAPK3/1 (ERK), MAPK14 (p38), and AKT within 15 min and JNK after 1-2 h. To determine which downstream pathways regulate the various functions of HBEGF, cells were treated with specific inhibitors of the ERK upstream regulator MEK (U0126), the AKT upstream regulator phosphoinositide-3 (PI3)-kinase (LY294002), MAPK14 (SB203580), and JNK (SP600125), as well as with inactive structural analogues. Only SB203580 specifically prevented HBEGF-mediated rescue during H/R, while each inhibitor attenuated HBEGF-stimulated cell migration. Accumulation of HBEGF at reduced oxygen was blocked only by a combination of U0126, SB203580, and SP600125. We conclude that HBEGF advances trophoblast extravillous differentiation through coordinate activation of PI3 kinase, ERK, MAPK14, and JNK, while only MAPK14 is required for its antiapoptotic activity. Additionally, hypoxia induces an autocrine increase in HBEGF protein levels through MAPK14, JNK or ERK. These experiments reveal a complexity of the intracellular signaling circuitry that regulates trophoblast functions critical for implantation and placentation.

Function-Specific Intracellular Signaling Pathways Downstream of Heparin-Binding EGF-Like Growth Factor Utilized by Human Trophoblasts1

PubMed Central

Jessmon, Philip; Kilburn, Brian A.; Romero, Roberto; Leach, Richard E.; Armant, D. Randall

2010-01-01

Heparin-binding EGF-like growth factor (HBEGF) is expressed by trophoblast cells throughout gestation. First-trimester cytotrophoblast cells are protected from hypoxia-induced apoptosis because of the accumulation of HBEGF through a posttranscriptional autocrine mechanism. Exogenous application of HBEGF is cytoprotective in a hypoxia/reoxygenation (H/R) injury model and initiates trophoblast extravillous differentiation to an invasive phenotype. The downstream signaling pathways induced by HBEGF that mediate these various cellular activities were identified using two human first-trimester cytotrophoblast cell lines, HTR-8/SVneo and SW.71, with similar results. Recombinant HBEGF (1 nM) induced transient phosphorylation of MAPK3/1 (ERK), MAPK14 (p38), and AKT within 15 min and JNK after 1–2 h. To determine which downstream pathways regulate the various functions of HBEGF, cells were treated with specific inhibitors of the ERK upstream regulator MEK (U0126), the AKT upstream regulator phosphoinositide-3 (PI3)-kinase (LY294002), MAPK14 (SB203580), and JNK (SP600125), as well as with inactive structural analogues. Only SB203580 specifically prevented HBEGF-mediated rescue during H/R, while each inhibitor attenuated HBEGF-stimulated cell migration. Accumulation of HBEGF at reduced oxygen was blocked only by a combination of U0126, SB203580, and SP600125. We conclude that HBEGF advances trophoblast extravillous differentiation through coordinate activation of PI3 kinase, ERK, MAPK14, and JNK, while only MAPK14 is required for its antiapoptotic activity. Additionally, hypoxia induces an autocrine increase in HBEGF protein levels through MAPK14, JNK or ERK. These experiments reveal a complexity of the intracellular signaling circuitry that regulates trophoblast functions critical for implantation and placentation. PMID:20130271

Significance of decoy receptor 3 (Dcr3) and external-signal regulated kinase 1/2 (Erk1/2) in gastric cancer.

PubMed

Yang, Donghai; Fan, Xin; Yin, Ping; Wen, Qiang; Yan, Feng; Yuan, Sibo; Liu, Bin; Zhuang, Guohong; Liu, Zhongchen

2012-06-06

Decoy receptor 3 (DcR3), a member of the tumor necrosis factor receptor (TNFR) superfamily, is associated with anti-tumor immunity suppression. It is highly expressed in many tumors, and its expression can be regulated by the MAPK/MEK/ERK signaling pathway. The MAPK/MEK/ERK pathway has been reported to be a regulator in tumor occurrence, development and clonal expansion. External-signal regulated kinase (ERK) is a vital member of this pathway. The expression of DcR3 and ERK1/2 in tumor tissues of gastric cancer patients was significantly higher than the non-cancerous group (P < 0.05). There was no statistical difference among tumor tissues from patients with different ages or gender, and even of different differentiation (P > 0.05). However, in patients with stage I gastric cancer, the DcR3 and ERK1/2 levels were significantly lower than patients with more advanced stages. DcR3 and ERK1/2 play a vital role in the development of gastric cancer, and they may be new markers for indicating the efficiency of gastric cancer treatment in the future.

Sodium appetite elicited by low-sodium diet is dependent on p44/42 mitogen-activated protein kinase (extracellular signal-regulated kinase 1/2) activation in the brain.

PubMed

Monteiro, L R N; Marangon, P B; Elias, L L K; Reis, L C; Antunes-Rodrigues, J; Mecawi, A S

2017-09-01

Sodium appetite is regulated by several signalling molecules, among which angiotensin II (Ang II) serves as a key driver of robust salt intake by binding to Ang II type 1 receptors (AT1R) in several regions in the brain. The activation of these receptors recruits the mitogen-activated protein kinase (MAPK) pathway, which has previously been linked to Ang II-induced increases in sodium appetite. Thus, we addressed the involvement of MAPK signalling in the induction of sodium appetite after 4 days of low-sodium diet consumption. An increase in extracellular signal-regulated kinase (ERK) phosphorylation in the laminae terminalis and mediobasal hypothalamus was observed after low-sodium diet consumption. This response was reduced by i.c.v. microinjection of an AT1R antagonist into the laminae terminalis but not the hypothalamus. This result indicates that low-sodium diet consumption activates the MAPK pathway via Ang II/AT1R signalling on the laminae terminalis. On the other hand, activation of the MAPK pathway in the mediobasal hypothalamus after low-sodium diet consumption appears to involve another extracellular mediator. We also evaluated whether a low-sodium diet could increase the sensitivity for Ang II in the brain and activate the MAPK pathway. However, i.c.v. injection of Ang II increased ERK phosphorylation on the laminae terminalis and mediobasal hypothalamus; this increase achieved a response magnitude similar to those observed in both the normal and low-sodium diet groups. These data indicate that low-sodium diet consumption for 4 days is insufficient to change the ERK phosphorylation response to Ang II in the brain. To investigate whether the MAPK pathway is involved in sodium appetite after low-sodium diet consumption, we performed i.c.v. microinjections of a MAPK pathway inhibitor (PD98059). PD98059 inhibited both saline and water intake after low-sodium diet consumption. Thus, the MAPK pathway is involved in promoting the sodium appetite after low-sodium diet consumption. © 2017 British Society for Neuroendocrinology.

Molecular analysis of pediatric brain tumors identifies microRNAs in pilocytic astrocytomas that target the MAPK and NF-κB pathways.

PubMed

Jones, Tania A; Jeyapalan, Jennie N; Forshew, Tim; Tatevossian, Ruth G; Lawson, Andrew R J; Patel, Sheena N; Doctor, Gabriel T; Mumin, Muhammad A; Picker, Simon R; Phipps, Kim P; Michalski, Antony; Jacques, Thomas S; Sheer, Denise

2015-12-18

Pilocytic astrocytomas are slow-growing tumors that usually occur in the cerebellum or in the midline along the hypothalamic/optic pathways. The most common genetic alterations in pilocytic astrocytomas activate the ERK/MAPK signal transduction pathway, which is a major driver of proliferation but is also believed to induce senescence in these tumors. Here, we have conducted a detailed investigation of microRNA and gene expression, together with pathway analysis, to improve our understanding of the regulatory mechanisms in pilocytic astrocytomas. Pilocytic astrocytomas were found to have distinctive microRNA and gene expression profiles compared to normal brain tissue and a selection of other pediatric brain tumors. Several microRNAs found to be up-regulated in pilocytic astrocytomas are predicted to target the ERK/MAPK and NF-κB signaling pathways as well as genes involved in senescence-associated inflammation and cell cycle control. Furthermore, IGFBP7 and CEBPB, which are transcriptional inducers of the senescence-associated secretory phenotype (SASP), were also up-regulated together with the markers of senescence and inflammation, CDKN1A (p21), CDKN2A (p16) and IL1B. These findings provide further evidence of a senescent phenotype in pilocytic astrocytomas. In addition, they suggest that the ERK/MAPK pathway, which is considered the major driver of these tumors, is regulated not only by genetic aberrations but also by microRNAs.

Acrolein increases 5-lipoxygenase expression in murine macrophages through activation of ERK pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Chae E.; Lee, Seung J.; Seo, Kyo W.

2010-05-15

Episodic exposure to acrolein-rich pollutants has been linked to acute myocardial infarction, and 5-lipoxygenase (5-LO) is involved in the production of matrix metalloproteinase-9 (MMP-9), which destabilizes atherosclerotic plaques. Thus, the present study determined the effect of acrolein on 5-LO/leukotriene B{sub 4} (LTB{sub 4}) production in murine macrophages. Stimulation of J774A.1 cells with acrolein led to increased LTB{sub 4} production in association with increased 5-LO expression. Acrolein-evoked 5-LO expression was blocked by pharmacological inhibition of the ERK pathway, but not by inhibitors for JNK and p38 MAPK pathways. In line with these results, acrolein exclusively increased the phosphorylation of ERK amongmore » these MAPK, suggesting a role for the ERK pathway in acrolein-induced 5-LO expression with subsequent production of LTB{sub 4}. Among the receptor tyrosine kinases including epidermal growth factor receptor (EGFR) and platelet derived growth factor receptor (PDGFR), acrolein-evoked ERK phosphorylation was attenuated by AG1478, an EGFR inhibitor, but not by AG1295, a PDGFR inhibitor. In addition, acrolein-evoked 5-LO expression was also inhibited by inhibition of EGFR pathway, but not by inhibition of PDGFR pathway. These observations suggest that acrolein has a profound effect on the 5-LO pathway via an EGFR-mediated activation of ERK pathway, leading to acute ischemic syndromes through the generation of LTB{sub 4}, subsequent MMP-9 production and plaque rupture.« less

Downstream components of RhoA required for signal pathway of superoxide formation during phagocytosis of serum opsonized zymosans in macrophages.

PubMed

Kim, Jun Sub; Kim, Jae Gyu; Jeon, Chan Young; Won, Ha Young; Moon, Mi Young; Seo, Ji Yeon; Kim, Jong Il; Kim, Jaebong; Lee, Jae Yong; Choi, Soo Young; Park, Jinseu; Yoon Park, Jung Han; Ha, Kwon Soo; Kim, Pyeung Hyeun; Park, Jae Bong

2005-12-31

Rac1 and Rac2 are essential for the control of oxidative burst catalyzed by NADPH oxidase. It was also documented that Rho is associated with the superoxide burst reaction during phagocytosis of serum- (SOZ) and IgG-opsonized zymosan particles (IOZ). In this study, we attempted to reveal the signal pathway components in the superoxide formation regulated by Rho GTPase. Tat-C3 blocked superoxide production, suggesting that RhoA is essentially involved in superoxide formation during phagocytosis of SOZ. Conversely SOZ activated both RhoA and Rac1/2. Inhibition of RhoA-activated kinase (ROCK), an important downstream effector of RhoA, by Y27632 and myosin light chain kinase (MLCK) by ML-7 abrogated superoxide production by SOZ. Extracellular signaling-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) were activated during phagocytosis of SOZ, and Tat-C3 and SB203580 reduced ERK1/2 and p38 MAPK activation, suggesting that RhoA and p38 MAPK may be upstream regulators of ERK1/2. Inhibition of ERK1/2, p38 MAPK, phosphatidyl inositol 3-kinase did not block translocation of RhoA to membranes, suggesting that RhoA is upstream to these kinases. Inhibition of RhoA by Tat-C3 blocked phosphorylation of p47(PHOX). Taken together, RhoA, ROCK, p38MAPK, ERK1/2, and p47(PHOX) may be subsequently activated, leading to activation of NADPH oxidase to produce superoxide.

Osmolarity affects matrix synthesis in the nucleus pulposus associated with the involvement of MAPK pathways: A study of ex vivo disc organ culture system.

PubMed

Li, Pei; Gan, Yibo; Xu, Yuan; Li, Songtao; Song, Lei; Li, Sukai; Li, Huijuan; Zhou, Qiang

2016-06-01

Matrix homeostasis within the nucleus pulposus (NP) is important for disc function. Unfortunately, the effects of osmolarity on NP matrix synthesis in a disc organ culture system and the underlying mechanisms are largely unknown. The present study was to investigate the effects of different osmolarity modes (constant and cyclic) and osmolarity levels (hypo-, iso-, and hyper-) on NP matrix synthesis using a disc organ culture system and determine whether ERK1/2 or p38MAPK pathway has a role in this process. Porcine discs were cultured for 7 days in various osmotic media, including constant hypo-, iso-, hyper-osmolarity (330, 430, and 550 mOsm/kg, respectively) and cyclic-osmolarity (430 mOsm/kg for 8 h, followed by 550 mOsm/kg for 16 h). The role of ERK1/2 and p38MAPK pathways were determined by their inhibitors U0126 and SB202190 respectively. The expression of SOX9 and downstream aggrecan and collagen II, biochemical content, and histology were used to assess NP matrix synthesis. The findings revealed that NP matrix synthesis was promoted in iso- and cyclic-osmolarity cultures compared to hypo- or hyper-osmolarity culture although the level of matrix synthesis in cyclic-osmolarity culture did not reach that in iso-osmolarity culture. Further analysis suggested that inhibition of the ERK1/2 or p38MAPK pathway in iso- and cyclic-osmolarity cultures reduced NP matrix production. Therefore, we concluded that the effects of osmolarity on NP matrix synthesis depend on osmolarity level (hypo-, iso-, or hyper-) and osmolarity mode (constant or cyclic), and the ERK1/2 and p38MAPK pathways may participate in this process. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 34:1092-1100, 2016. © 2015 Orthopaedic Research Society. Published by Wiley Periodicals, Inc.

Fructose-1,6-bisphosphatase Inhibits ERK Activation and Bypasses Gemcitabine Resistance in Pancreatic Cancer by Blocking IQGAP1–MAPK Interaction

PubMed Central

Jin, Xin; Pan, Yunqian; Wang, Liguo; Ma, Tao; Zhang, Lizhi; Tang, Amy H.; Billadeau, Daniel D.; Wu, Heshui; Huang, Haojie

2017-01-01

Dysregulation of the MAPK pathway correlates with progression of pancreatic ductal adenocarcinoma (PDAC) progression. IQ motif containing GTPase-activating protein 1 (IQGAP1) is a MAPK scaffold that directly regulates the activation of RAF, MEK, and ERK. Fructose-1,6-bisphosphatase (FBP1), a key enzyme in gluconeogenesis, is transcriptionally downregulated in various cancers, including PDAC. Here, we demonstrate that FBP1 acts as a negative modulator of the IQGAP1–MAPK signaling axis in PDAC cells. FBP1 binding to the WW domain of IQGAP1 impeded IQGAP1-dependent ERK1/2 phosphorylation (pERK1/2) in a manner independent of FBP1 enzymatic activity. Conversely, decreased FBP1 expression induced pERK1/2 levels in PDAC cell lines and correlated with increased pERK1/2 levels in patient specimens. Treatment with gemcitabine caused undesirable activation of ERK1/2 in PDAC cells, but cotreatment with the FBP1-derived small peptide inhibitor FBP1 E4 overcame gemcitabine-induced ERK activation, thereby increasing the anticancer efficacy of gemcitabine in PDAC. These findings identify a primary mechanism of resistance of PDAC to standard therapy and suggest that the FBP1–IQGAP1–ERK1/2 signaling axis can be targeted for effective treatment of PDAC. PMID:28720574

Strategies of biochemical adaptation for hibernation in a South American marsupial Dromiciops gliroides: 1. Mitogen-activated protein kinases and the cell stress response.

PubMed

Wijenayake, Sanoji; Luu, Bryan E; Zhang, Jing; Tessier, Shannon N; Quintero-Galvis, Julian F; Gaitán-Espitia, Juan Diego; Nespolo, Roberto F; Storey, Kenneth B

2017-12-14

Hibernation is a period of torpor and heterothermy that is typically associated with a strong reduction in metabolic rate, global suppression of transcription and translation, and upregulation of various genes/proteins that are central to the cellular stress response such as protein kinases, antioxidants, and heat shock proteins. The current study examined cell signaling cascades in hibernating monito del monte, Dromiciops gliroides, a South American marsupial of the Order Microbiotheria. Responses to hibernation by members of the mitogen-activated protein kinase (MAPK) pathways, and their roles in coordinating hibernator metabolism were examined in liver, kidney, heart and brain of control and versus hibernating (4days continuous torpor) D. gliroides. The targets evaluated included key protein kinases in their activated phosphorylated forms (p-ERK/MAPK 1/2, p-MEK1, p-MSK1, p-p38, p-JNK) and related target proteins (p-CREB 2, p-ATF2, p-c-Jun and p-p53). Liver exhibited a strong coordinated response by MAPK members to hibernation with significant increases in protein phosphorylation levels of p-MEK1, p-ERK/MAPK1/2, p-MSK1, p-JNK and target proteins c-Jun, and p-ATF2, all combining to signify a strong activation of MAPK signaling during hibernation. Kidney also showed activation of MAPK cascades with significant increases in p-MEK1, p-ERK/MAPK1/2, p-p38, and p-c-Jun levels in hibernating animals. By contrast, responses by heart and brain indicated reduced MAPK pathway function during torpor with reduced phosphorylation of targets including p-ERK/MAPK 1/2 in both tissues as well as lower p-p38 and p-JNK content in heart. Overall, the data indicate a vital role for MAPK signaling in regulating the cell stress response during marsupial hibernation. Copyright © 2017 Elsevier Inc. All rights reserved.

Hyperglycemia regulates TXNIP/TRX/ROS axis via p38 MAPK and ERK pathways in pancreatic cancer.

PubMed

Li, Wei; Wu, Zheng; Ma, Qingyong; Liu, Jiangbo; Xu, Qinhong; Han, Liang; Duan, Wanxing; Lv, Yunfu; Wang, Fengfei; Reindl, Katie M; Wu, Erxi

2014-01-01

Approximately 85% of pancreatic cancer patients suffer from glucose intolerance or even diabetes because high glucose levels can contribute to oxidative stress which promotes tumor development. As one of the reactive oxygen species (ROS)-regulating factors, thioredoxin-interacting protein (TXNIP), is involved in the maintenance of thioredoxin (TRX)-mediated redox regulation. In this study, we demonstrated that high glucose levels increased the expression of TXNIP in time- and concentration-dependent manners and modulated the activity of TRX and ROS production in pancreatic cancer cells, BxPC-3 and Panc-1. We also found that glucose activated both p38 MAPK and ERK pathways and inhibitors of these pathways impaired the TXNIP/TRX/ROS axis. Knockdown of TXNIP restored TRX activity and decreased ROS production under high glucose conditions. Moreover, we observed that the integrated optical density (IOD) of TXNIP staining as well as the protein and mRNA expression levels of TXNIP were higher in the tumor tissues of pancreatic cancer patients with diabetes. Taken together, these results indicate that hyperglycemia-induced TXNIP expression is involved in diabetes-mediated oxidative stress in pancreatic cancer via p38 MAPK and ERK pathways.

Stimulation of the p38 Mitogen-activated Protein Kinase Pathway in Neonatal Rat Ventricular Myocytes by the G Protein–coupled Receptor Agonists, Endothelin-1 and Phenylephrine: A Role in Cardiac Myocyte Hypertrophy?

PubMed Central

Clerk, Angela; Michael, Ashour; Sugden, Peter H.

1998-01-01

We examined the activation of the p38 mitogen-activated protein kinase (p38-MAPK) pathway by the G protein–coupled receptor agonists, endothelin-1 and phenylephrine in primary cultures of cardiac myocytes from neonatal rat hearts. Both agonists increased the phosphorylation (activation) of p38-MAPK by ∼12-fold. A p38-MAPK substrate, MAPK-activated protein kinase 2 (MAPKAPK2), was activated approximately fourfold and 10 μM SB203580, a p38-MAPK inhibitor, abolished this activation. Phosphorylation of the MAPKAPK2 substrate, heat shock protein 25/27, was also increased. Using selective inhibitors, activation of the p38-MAPK pathway by endothelin-1 was shown to involve protein kinase C but not Gi/Go nor the extracellularly responsive kinase (ERK) pathway. SB203580 failed to inhibit the morphological changes associated with cardiac myocyte hypertrophy induced by endothelin-1 or phenylephrine between 4 and 24 h. However, it decreased the myofibrillar organization and cell profile at 48 h. In contrast, inhibition of the ERK cascade with PD98059 prevented the increase in myofibrillar organization but not cell profile. These data are not consistent with a role for the p38-MAPK pathway in the immediate induction of the morphological changes of hypertrophy but suggest that it may be necessary over a longer period to maintain the response. PMID:9679149

IL-12 and IL-23 Production in Toxoplasma gondii- or LPS-Treated Jurkat T Cells via PI3K and MAPK Signaling Pathways.

PubMed

Ismail, Hassan Ahmed Hassan Ahmed; Kang, Byung-Hun; Kim, Jae-Su; Lee, Jae-Hyung; Choi, In-Wook; Cha, Guang-Ho; Yuk, Jae-Min; Lee, Young-Ha

2017-12-01

IL-12 and IL-23 are closely related in structure, and have been shown to play crucial roles in regulation of immune responses. However, little is known about the regulation of these cytokines in T cells. Here, we investigated the roles of PI3K and MAPK pathways in IL-12 and IL-23 production in human Jurkat T cells in response to Toxoplasma gondii and LPS. IL-12 and IL-23 production was significantly increased in T cells after stimulation with T. gondii or LPS. T. gondii and LPS increased the phosphorylation of AKT, ERK1/2, p38 MAPK, and JNK1/2 in T cells from 10 min post-stimulation, and peaked at 30-60 min. Inhibition of the PI3K pathway reduced IL-12 and IL-23 production in T. gondii-infected cells, but increased in LPS-stimulated cells. IL-12 and IL-23 production was significantly reduced by ERK1/2 and p38 MAPK inhibitors in T. gondii- and LPS-stimulated cells, but not in cells treated with a JNK1/2 inhibitor. Collectively, IL-12 and IL-23 production was positively regulated by PI3K and JNK1/2 in T. gondii-infected Jurkat cells, but negatively regulated in LPS-stimulated cells. And ERK1/2 and p38 MAPK positively regulated IL-12 and IL-23 production in Jurkat T cells. These data indicate that T. gondii and LPS induced IL-12 and IL-23 production in Jurkat T cells through the regulation of the PI3K and MAPK pathways; however, the mechanism underlying the stimulation of IL-12 and IL-23 production by T. gondii in Jurkat T cells is different from that of LPS.

Upregulation of CD147 promotes cell invasion, epithelial-to-mesenchymal transition and activates MAPK/ERK signaling pathway in colorectal cancer.

PubMed

Xu, Tao; Zhou, Mingliang; Peng, Lipan; Kong, Shuai; Miao, Ruizheng; Shi, Yulong; Sheng, Hongguang; Li, Leping

2014-01-01

Colorectal cancer (CRC) is one of the most common cancers in the world. CD147, a transmembrane protein, has been reported to be correlated with various cancers. In this study, we aimed to investigate the mechanism of CD147 in regulating drug resistance, cell invasion and epithelial-to-mesenchymal transition (EMT) in CRC cells. qRT-PCR and western blotting were used to evaluated the expression of CD147 in 40 CRC cases and 4 cell lines. Increased expression of CD147 at both mRNA and protein levels was found in CRC samples, and the level of CD147 was correlated with lymph node metastasis. CD147 overexpression increased the 5-Fluorouracil (5-FU) resistance, enhanced the invasion and EMT of CRC cells by regulating EMT markers and MMPs. Adverse results were obtained in CD147 knockdown CRC cell line. Further investigation revealed that CD147 activated MAPK/ERK pathway, ERK inhibitor U0126 suppressed the CD147-induced cell invasion, migration and MMP-2, MMP-9 expression. Taken together, our study indicates that CD147 promotes the 5-FU resistance, and MAPK/ERK signaling pathway is involved in CD147-promoted invasion and EMT of CRC cells.

Upregulation of CD147 promotes cell invasion, epithelial-to-mesenchymal transition and activates MAPK/ERK signaling pathway in colorectal cancer

PubMed Central

Xu, Tao; Zhou, Mingliang; Peng, Lipan; Kong, Shuai; Miao, Ruizheng; Shi, Yulong; Sheng, Hongguang; Li, Leping

2014-01-01

Colorectal cancer (CRC) is one of the most common cancers in the world. CD147, a transmembrane protein, has been reported to be correlated with various cancers. In this study, we aimed to investigate the mechanism of CD147 in regulating drug resistance, cell invasion and epithelial-to-mesenchymal transition (EMT) in CRC cells. qRT-PCR and western blotting were used to evaluated the expression of CD147 in 40 CRC cases and 4 cell lines. Increased expression of CD147 at both mRNA and protein levels was found in CRC samples, and the level of CD147 was correlated with lymph node metastasis. CD147 overexpression increased the 5-Fluorouracil (5-FU) resistance, enhanced the invasion and EMT of CRC cells by regulating EMT markers and MMPs. Adverse results were obtained in CD147 knockdown CRC cell line. Further investigation revealed that CD147 activated MAPK/ERK pathway, ERK inhibitor U0126 suppressed the CD147-induced cell invasion, migration and MMP-2, MMP-9 expression. Taken together, our study indicates that CD147 promotes the 5-FU resistance, and MAPK/ERK signaling pathway is involved in CD147-promoted invasion and EMT of CRC cells. PMID:25550778

Comparative study of the efficacy of pulsed electromagnetic field and low level laser therapy on mitogen-activated protein kinases.

PubMed

El-Makakey, Ayman M; El-Sharaby, Radwa M; Hassan, Mohammed H; Balbaa, Alaa

2017-03-01

Mitogen-Activated Protein Kinases (MAPKs) consist of three major signaling members: extracellular signal-regulated kinase (ERK), p38 and C-JUN N-terminal kinase (JNK). We investigated physiological effects of Pulsed Electromagnetic Field Therapy (PEMFT) and Low Level Laser Therapy (LLLT) on human body, adopting the expression level of mitogen-activated protein kinases as an indicator via assessment of the activation levels of three major families of MAPKS, ERK, p38 and JNK in the peripheral lymphocytes of patients before and after the therapies. Assessment for the expression levels of MAPKs families' were done, in the peripheral lymphocytes of patients recently have appendectomy, using flow cytometric analysis of multiple signaling pathways, pre and post LLLT and PEMFT application (twice daily for 6 successive days) on the appendectomy wound. There were non-significant differences in the expression levels of MAPKs families' pre- therapies application. But there were significant increase in the ERK expression levels post application of LLLT compared to its pre application (p<0.01). Also, there was significant increase in the ERK, p38 and C-Jun N terminal expression level values post application of PEMFT compared to its pre application expression levels (p<0.01 for each). The present study demonstrates that PEMFT has a powerful healing effect more than LLLT as it increase the activation of ERK, P38 and C-Jun-N Terminal while LLLT only increase the activation of ERK. LLLT has more potent pain decreasing effect than PEMFT as it does not activate P38 pathway like PEMFT.

Power Frequency Magnetic Fields Affect the p38 MAPK-Mediated Regulation of NB69 Cell Proliferation Implication of Free Radicals.

PubMed

Martínez, María Antonia; Úbeda, Alejandro; Moreno, Jorge; Trillo, María Ángeles

2016-04-06

The proliferative response of the neuroblastoma line NB69 to a 100 µT, 50 Hz magnetic field (MF) has been shown mediated by activation of the MAPK-ERK1/2 pathway. This work investigates the MF effect on the cell cycle of NB69, the participation of p38 and c-Jun N-terminal (JNK) kinases in the field-induced proliferative response and the potential involvement of reactive oxygen species (ROS) in the activation of the MAPK-ERK1/2 and -p38 signaling pathways. NB69 cultures were exposed to the 100 µT MF, either intermittently for 24, 42 or 63 h, or continuously for periods of 15 to 120 min, in the presence or absence of p38 or JNK inhibitors: SB203580 and SP600125, respectively. Antioxidant N-acetylcysteine (NAC) was used as ROS scavenger. Field exposure induced transient activation of p38, JNK and ERK1/2. The MF proliferative effect, which was mediated by changes in the cell cycle, was blocked by the p38 inhibitor, but not by the JNK inhibitor. NAC blocked the field effects on cell proliferation and p38 activation, but not those on ERK1/2 activation. The MF-induced proliferative effects are exerted through sequential upregulation of MAPK-p38 and -ERK1/2 activation, and they are likely mediated by a ROS-dependent activation of p38.

Power Frequency Magnetic Fields Affect the p38 MAPK-Mediated Regulation of NB69 Cell Proliferation Implication of Free Radicals

PubMed Central

Martínez, María Antonia; Úbeda, Alejandro; Moreno, Jorge; Trillo, María Ángeles

2016-01-01

The proliferative response of the neuroblastoma line NB69 to a 100 µT, 50 Hz magnetic field (MF) has been shown mediated by activation of the MAPK-ERK1/2 pathway. This work investigates the MF effect on the cell cycle of NB69, the participation of p38 and c-Jun N-terminal (JNK) kinases in the field-induced proliferative response and the potential involvement of reactive oxygen species (ROS) in the activation of the MAPK-ERK1/2 and -p38 signaling pathways. NB69 cultures were exposed to the 100 µT MF, either intermittently for 24, 42 or 63 h, or continuously for periods of 15 to 120 min, in the presence or absence of p38 or JNK inhibitors: SB203580 and SP600125, respectively. Antioxidant N-acetylcysteine (NAC) was used as ROS scavenger. Field exposure induced transient activation of p38, JNK and ERK1/2. The MF proliferative effect, which was mediated by changes in the cell cycle, was blocked by the p38 inhibitor, but not by the JNK inhibitor. NAC blocked the field effects on cell proliferation and p38 activation, but not those on ERK1/2 activation. The MF-induced proliferative effects are exerted through sequential upregulation of MAPK-p38 and -ERK1/2 activation, and they are likely mediated by a ROS-dependent activation of p38. PMID:27058530

Ultrasound Stimulation of Different Dental Stem Cell Populations: Role of Mitogen-activated Protein Kinase Signaling.

PubMed

Gao, Qianhua; Walmsley, A Damien; Cooper, Paul R; Scheven, Ben A

2016-03-01

Mesenchymal stem cells (MSCs) from dental tissues may respond to low-intensity pulsed ultrasound (LIPUS) treatment, potentially providing a therapeutic approach to promoting dental tissue regeneration. This work aimed to compare LIPUS effects on the proliferation and MAPK signaling in MSCs from rodent dental pulp stem cells (DPSCs) compared with MSCs from periodontal ligament stem cells (PDLSCs) and bone marrow stem cells (BMSCs). Isolated MSCs were treated with 1-MHz LIPUS at an intensity of 250 or 750 mW/cm2 for 5 or 20 minutes. Cell proliferation was evaluated by 5-bromo-2-deoxyuridine (BrdU) staining after 24 hours of culture following a single LIPUS treatment. Specific ELISAs were used to determine the total and activated p38, ERK1/2, and JNK MAPK signaling proteins up to 4 hours after treatment. Selective MAPK inhibitors PD98059 (ERK1/2), SB203580 (p38), and SP600125 (JNK) were used to determine the role of activation of the particular MAPK pathways. The proliferation of all MSC types was significantly increased after LIPUS treatment. LIPUS at a 750-mW/cm2 dose induced the greatest effects on DPSCs. BMSC proliferation was stimulated in equal measures by both intensities, whereas 250 mW/cm2 LIPUS exposure exerted maximum effects on PDLSCs. ERK1/2 was activated immediately in DPSCs after treatment. Concomitantly, DPSC proliferation was specifically modulated by ERK1/2 inhibition, whereas p38 and JNK inhibition exerted no effects. In BMSCs, JNK MAPK signaling was LIPUS activated, and the increase in proliferation was blocked by specific inhibition of the JNK pathway. In PDLSCs, JNK MAPK signaling was activated immediately after LIPUS, whereas p-p38 MAPK increased significantly in these cells 4 hours after exposure. Correspondingly, JNK and p38 inhibition modulated LIPUS-stimulated PDLSC proliferation. LIPUS promoted MSC proliferation in an intensity and cell-specific dependent manner via activation of distinct MAPK pathways. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

ROS generation and MAPKs activation contribute to the Ni-induced testosterone synthesis disturbance in rat Leydig cells.

PubMed

Han, Aijie; Zou, Lingyue; Gan, Xiaoqin; Li, Yu; Liu, Fangfang; Chang, Xuhong; Zhang, Xiaotian; Tian, Minmin; Li, Sheng; Su, Li; Sun, Yingbiao

2018-06-15

Nickel (Ni) can disorder testosterone synthesis in rat Leydig cells, whereas the mechanisms remain unclear. The aim of this study was to investigate the role of reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) in Ni-induced disturbance of testosterone synthesis in rat Leydig cells. The testosterone production and ROS levels were detected in Leydig cells. The mRNA and protein levels of testosterone synthetase, including StAR, CYP11A1, 3β-HSD, CYP17A1 and 17β-HSD, were determined. Effects of Ni on the ERK1/2, p38 and JNK MAPKs were also investigated. The results showed that Ni triggered ROS generation, consequently resulted in the decrease of testosterone synthetase expression and testosterone production in Leydig cells, which were then attenuated by ROS scavengers of N-acetylcysteine (NAC) and 2,2,6,6-tetramethyl-1-piperidinyloxy (TEMPO), indicating that ROS are involved in the Ni-induced testosterone biosynthesis disturbance. Meanwhile Ni activated the ERK1/2, p38 and JNK MAPKs. Furthermore, Ni-inhibited testosterone synthetase expression levels and testosterone secretion were all alleviated by co-treatment with MAPK specific inhibitors (U0126 and SB203580, respectively), implying that Ni inhibited testosterone synthesis through activating ERK1/2 and p38 MAPK signal pathways in Leydig cells. In conclusion, these findings suggest that Ni causes testosterone synthesis disorder, partly, via ROS and MAPK signal pathways. Copyright © 2018 Elsevier B.V. All rights reserved.

Adverse effects of MWCNTs on life parameters, antioxidant systems, and activation of MAPK signaling pathways in the copepod Paracyclopina nana.

PubMed

Kim, Duck-Hyun; Puthumana, Jayesh; Kang, Hye-Min; Lee, Min-Chul; Jeong, Chang-Bum; Han, Jeonghoon; Hwang, Dae-Sik; Kim, Il-Chan; Lee, Jin Wuk; Lee, Jae-Seong

2016-10-01

Engineered multi-walled carbon nanotubes (MWCNTs) have received widespread applications in a broad variety of commercial products due to low production cost. Despite their significant commercial applications, CNTs are being discharged to aquatic ecosystem, leading a threat to aquatic life. Thus, we investigated the adverse effect of CNTs on the marine copepod Paracyclopina nana. Additional to the study on the uptake of CNTs and acute toxicity, adverse effects on life parameters (e.g. growth, fecundity, and size) were analyzed in response to various concentrations of CNTs. Also, as a measurement of cellular damage, oxidative stress-related markers were examined in a time-dependent manner. Moreover, activation of redox-sensitive mitogen-activated protein kinase (MAPK) signaling pathways along with the phosphorylation pattern of extracellular signal-regulated kinase (ERK), p38, and c-Jun-N-terminal kinases (JNK) were analyzed to obtain a better understanding of molecular mechanism of oxidative stress-induced toxicity in the copepod P. nana. As a result, significant inhibition on life parameters and evoked antioxidant systems were observed without ROS induction. In addition, CNTs activated MAPK signaling pathway via ERK, suggesting that phosphorylated ERK (p-ERK)-mediated adverse effects are the primary cause of in vitro and in vivo endpoints in response to CNTs exposure. Moreover, ROS-independent activation of MAPK signaling pathway was observed. These findings will provide a better understanding of the mode of action of CNTs on the copepod P. nana at cellular and molecular level and insight on possible ecotoxicological implications in the marine environment. Copyright © 2016 Elsevier B.V. All rights reserved.

Antiviral Potential of ERK/MAPK and PI3K/AKT/mTOR Signaling Modulation for Middle East Respiratory Syndrome Coronavirus Infection as Identified by Temporal Kinome Analysis

PubMed Central

Ork, Britini; Hart, Brit J.; Holbrook, Michael R.; Frieman, Matthew B.; Traynor, Dawn; Johnson, Reed F.; Dyall, Julie; Olinger, Gene G.; Hensley, Lisa E.

2014-01-01

Middle East respiratory syndrome coronavirus (MERS-CoV) is a lineage C betacoronavirus, and infections with this virus can result in acute respiratory syndrome with renal failure. Globally, MERS-CoV has been responsible for 877 laboratory-confirmed infections, including 317 deaths, since September 2012. As there is a paucity of information regarding the molecular pathogenesis associated with this virus or the identities of novel antiviral drug targets, we performed temporal kinome analysis on human hepatocytes infected with the Erasmus isolate of MERS-CoV with peptide kinome arrays. bioinformatics analysis of our kinome data, including pathway overrepresentation analysis (ORA) and functional network analysis, suggested that extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) and phosphoinositol 3-kinase (PI3K)/serine-threonine kinase (AKT)/mammalian target of rapamycin (mTOR) signaling responses were specifically modulated in response to MERS-CoV infection in vitro throughout the course of infection. The overrepresentation of specific intermediates within these pathways determined by pathway and functional network analysis of our kinome data correlated with similar patterns of phosphorylation determined through Western blot array analysis. In addition, analysis of the effects of specific kinase inhibitors on MERS-CoV infection in tissue culture models confirmed these cellular response observations. Further, we have demonstrated that a subset of licensed kinase inhibitors targeting the ERK/MAPK and PI3K/AKT/mTOR pathways significantly inhibited MERS-CoV replication in vitro whether they were added before or after viral infection. Taken together, our data suggest that ERK/MAPK and PI3K/AKT/mTOR signaling responses play important roles in MERS-CoV infection and may represent novel drug targets for therapeutic intervention strategies. PMID:25487801

Zinc transporter ZnT-3 regulates presynaptic Erk1/2 signaling and hippocampus-dependent memory.

PubMed

Sindreu, Carlos; Palmiter, Richard D; Storm, Daniel R

2011-02-22

The physiological role of vesicular zinc at central glutamatergic synapses remains poorly understood. Here we show that mice lacking the synapse-specific vesicular zinc transporter ZnT3 (ZnT3KO mice) have reduced activation of the Erk1/2 MAPK in hippocampal mossy fiber terminals, disinhibition of zinc-sensitive MAPK tyrosine phosphatase activity, and impaired MAPK signaling during hippocampus-dependent learning. Activity-dependent exocytosis is required for the effect of zinc on presynaptic MAPK and phosphatase activity. ZnT3KO mice have complete deficits in contextual discrimination and spatial working memory. Local blockade of zinc or MAPK in the mossy fiber pathway of wild-type mice impairs contextual discrimination. We conclude that ZnT3 is important for zinc homeostasis modulating presynaptic MAPK signaling and is required for hippocampus-dependent memory.

Zinc transporter ZnT-3 regulates presynaptic Erk1/2 signaling and hippocampus-dependent memory

PubMed Central

Sindreu, Carlos; Palmiter, Richard D.; Storm, Daniel R.

2011-01-01

The physiological role of vesicular zinc at central glutamatergic synapses remains poorly understood. Here we show that mice lacking the synapse-specific vesicular zinc transporter ZnT3 (ZnT3KO mice) have reduced activation of the Erk1/2 MAPK in hippocampal mossy fiber terminals, disinhibition of zinc-sensitive MAPK tyrosine phosphatase activity, and impaired MAPK signaling during hippocampus-dependent learning. Activity-dependent exocytosis is required for the effect of zinc on presynaptic MAPK and phosphatase activity. ZnT3KO mice have complete deficits in contextual discrimination and spatial working memory. Local blockade of zinc or MAPK in the mossy fiber pathway of wild-type mice impairs contextual discrimination. We conclude that ZnT3 is important for zinc homeostasis modulating presynaptic MAPK signaling and is required for hippocampus-dependent memory. PMID:21245308

Cigarette smoke induced urocystic epithelial mesenchymal transition via MAPK pathways.

PubMed

Yu, Dexin; Geng, Hao; Liu, Zhiqi; Zhao, Li; Liang, Zhaofeng; Zhang, Zhiqiang; Xie, Dongdong; Wang, Yi; Zhang, Tao; Min, Jie; Zhong, Caiyun

2017-01-31

Cigarette smoke has been shown to be a major risk factor for bladder cancer. Epithelial-mesenchymal transition (EMT) is a crucial process in cancer development. The role of MAPK pathways in regulating cigarette smoke-triggered urocystic EMT remains to be elucidated. Human normal urothelial cells and BALB/c mice were used as in vitro and in vivo cigarette smoke exposure models. Exposure of human normal urothelial cells to cigarette smoke induced morphological change, enhanced migratory and invasive capacities, reduced epithelial marker expression and increased mesenchymal marker expression, along with the activation of MAPK pathways. Moreover, we revealed that ERK1/2 and p38 inhibitors, but rather JNK inhibitor, effectively attenuated cigarette smoke-induced urocystic EMT. Importantly, the regulatory function of ERK1/2 and p38 pathways in cigarette smoke-triggered urocystic EMT was further confirmed in mice exposed to CS for 12 weeks. These findings could provide new insight into the molecular mechanisms of cigarette smoke-associated bladder cancer development as well as its potential intervention.

Antimelanogenic effect of c-phycocyanin through modulation of tyrosinase expression by upregulation of ERK and downregulation of p38 MAPK signaling pathways

PubMed Central

2011-01-01

Background Pigmentation is one of the essential defense mechanisms against oxidative stress or UV irradiation; however, abnormal hyperpigmentation in human skin may pose a serious aesthetic problem. C-phycocyanin (Cpc) is a phycobiliprotein from spirulina and functions as an antioxidant and a light harvesting protein. Though it is known that spirulina has been used to reduce hyperpigmentation, little literature addresses the antimelanogenic mechanism of Cpc. Herein, we investigated the rationale for the Cpc-induced inhibitory mechanism on melanin synthesis in B16F10 melanoma cells. Methods Cpc-induced inhibitory effects on melanin synthesis and tyrosinase expression were evaluated. The activity of MAPK pathways-associated molecules such as MAPK/ERK and p38 MAPK, were also examined to explore Cpc-induced antimelanogenic mechanisms. Additionally, the intracellular localization of Cpc was investigated by confocal microscopic analysis to observe the migration of Cpc. Results Cpc significantly (P < 0.05) reduced both tyrosinase activity and melanin production in a dose-dependent manner. This phycobiliprotein elevated the abundance of intracellular cAMP leading to the promotion of downstream ERK1/2 phosphorylation and the subsequent MITF (the transcription factor of tyrosinase) degradation. Further, Cpc also suppressed the activation of p38 causing the consequent disturbed activation of CREB (the transcription factor of MITF). As a result, Cpc negatively regulated tyrosinase gene expression resulting in the suppression of melanin synthesis. Moreover, the entry of Cpc into B16F10 cells was revealed by confocal immunofluorescence localization and immunoblot analysis. Conclusions Cpc exerted dual antimelanogenic mechanisms by upregulation of MAPK/ERK-dependent degradation of MITF and downregulation of p38 MAPK-regulated CREB activation to modulate melanin formation. Cpc may have potential applications in biomedicine, food, and cosmetic industries. PMID:21988805

The ERK pathway regulates Na(+)-HCO(3)(-) cotransport activity in adult rat cardiomyocytes.

PubMed

Baetz, Delphine; Haworth, Robert S; Avkiran, Metin; Feuvray, Danielle

2002-11-01

The sarcolemmal Na(+)-HCO cotransporter (NBC) is stimulated by intracellular acidification and acts as an acid extruder. We examined the role of the ERK pathway of the MAPK cascade as a potential mediator of NBC activation by intracellular acidification in the presence and absence of angiotensin II (ANG II) in adult rat ventricular myocytes. Intracellular pH (pH(i)) was recorded with the use of seminaphthorhodafluor-1. The NH method was used to induce an intracellular acid load. NBC activation was significantly decreased with the ERK inhibitors PD-98059 and U-0126. NBC activity after acidification was increased in the presence of ANG II (pH(i) range of 6.75-7.00). ANG II plus PD-123319 (AT(2) antagonist) still increased NBC activity, whereas ANG II plus losartan (AT(1) antagonist) did not affect it. ERK phosphorylation (measured by immunoblot analysis) during intracellular acidification was increased by ANG II, an effect that was abolished by losartan and U-0126. In conclusion, the MAPK(ERK)-dependent pathway facilitates the rate of pH(i) recovery from acid load through NBC activity and is involved in the AT(1) receptor-mediated stimulation of such activity by ANG II.

OCD-like behavior is caused by dysfunction of thalamo-amygdala circuits and upregulated TrkB/ERK-MAPK signaling as a result of SPRED2 deficiency

PubMed Central

Ullrich, M; Weber, M; Post, A M; Popp, S; Grein, J; Zechner, M; Guerrero González, H; Kreis, A; Schmitt, A G; Üçeyler, N; Lesch, K-P; Schuh, K

2018-01-01

Obsessive-compulsive disorder (OCD) is a common neuropsychiatric disease affecting about 2% of the general population. It is characterized by persistent intrusive thoughts and repetitive ritualized behaviors. While gene variations, malfunction of cortico-striato-thalamo-cortical (CSTC) circuits, and dysregulated synaptic transmission have been implicated in the pathogenesis of OCD, the underlying mechanisms remain largely unknown. Here we show that OCD-like behavior in mice is caused by deficiency of SPRED2, a protein expressed in various brain regions and a potent inhibitor of Ras/ERK-MAPK signaling. Excessive self-grooming, reflecting OCD-like behavior in rodents, resulted in facial skin lesions in SPRED2 knockout (KO) mice. This was alleviated by treatment with the selective serotonin reuptake inhibitor fluoxetine. In addition to the previously suggested involvement of cortico-striatal circuits, electrophysiological measurements revealed altered transmission at thalamo-amygdala synapses and morphological differences in lateral amygdala neurons of SPRED2 KO mice. Changes in synaptic function were accompanied by dysregulated expression of various pre- and postsynaptic proteins in the amygdala. This was a result of altered gene transcription and triggered upstream by upregulated tropomyosin receptor kinase B (TrkB)/ERK-MAPK signaling in the amygdala of SPRED2 KO mice. Pathway overactivation was mediated by increased activity of TrkB, Ras, and ERK as a specific result of SPRED2 deficiency and not elicited by elevated brain-derived neurotrophic factor levels. Using the MEK inhibitor selumetinib, we suppressed TrkB/ERK-MAPK pathway activity in vivo and reduced OCD-like grooming in SPRED2 KO mice. Altogether, this study identifies SPRED2 as a promising new regulator, TrkB/ERK-MAPK signaling as a novel mediating mechanism, and thalamo-amygdala synapses as critical circuitry involved in the pathogenesis of OCD. PMID:28070119

Non-thermal plasma inhibits human cervical cancer HeLa cells invasiveness by suppressing the MAPK pathway and decreasing matrix metalloproteinase-9 expression

NASA Astrophysics Data System (ADS)

Li, Wei; Yu, K. N.; Bao, Lingzhi; Shen, Jie; Cheng, Cheng; Han, Wei

2016-01-01

Non-thermal plasma (NTP) has been proposed as a novel therapeutic method for anticancer treatment. However, the mechanism underlying its biological effects remains unclear. In this study, we investigated the inhibitory effect of NTP on the invasion of HeLa cells, and explored the possible mechanism. Our results showed that NTP exposure for 20 or 40 s significantly suppressed the migration and invasion of HeLa cells on the basis of matrigel invasion assay and wound healing assay, respectively. Moreover, NTP reduced the activity and protein expression of the matrix metalloproteinase (MMP)-9 enzyme. Western blot analysis indicated that NTP exposure effectively decreased phosphorylation level of both ERK1/2 and JNK, but not p38 MAPK. Furthermore, treatment with MAPK signal pathway inhibitors or NTP all exhibited significant depression of HeLa cells migration and MMP-9 expression. The result showed that NTP synergistically suppressed migration and MMP-9 expression in the presence of ERK1/2 inhibitor and JNK inhibitor, but not p38 MAPK inhibitor. Taken together, these findings suggested that NTP exposure inhibited the migration and invasion of HeLa cells via down-regulating MMP-9 expression in ERK1/2 and JNK signaling pathways dependent manner. These findings provide hints to the potential clinical research and therapy of NTP on cervical cancer metastasis.

Mitogen-activated protein kinase inhibition reduces mucin 2 production and mucinous tumor growth.

PubMed

Dilly, Ashok K; Song, Xinxin; Zeh, Herbert J; Guo, Zong S; Lee, Yong J; Bartlett, David L; Choudry, Haroon A

2015-10-01

Excessive accumulation of mucin 2 (MUC2) protein (a gel-forming secreted mucin) within the peritoneal cavity is the major cause of morbidity and mortality in pseudomyxoma peritonei (PMP), a unique mucinous malignancy of the appendix. Mitogen-activated protein kinase (MAPK) signaling pathway is upregulated in PMP and has been shown to modulate MUC2 promoter activity. We hypothesized that targeted inhibition of the MAPK pathway would be a novel, effective, and safe therapeutic strategy to reduce MUC2 production and mucinous tumor growth. We tested RDEA119, a specific MEK1/2 (MAPK extracellular signal-regulated kinase [ERK] kinase) inhibitor, in MUC2-secreting LS174T cells, human PMP explant tissue, and in a unique intraperitoneal murine xenograft model of PMP. RDEA119 reduced ERK1/2 phosphorylation and inhibited MUC2 messenger RNA and protein expression in vitro. In the xenograft model, chronic oral therapy with RDEA119 inhibited mucinous tumor growth in an MAPK pathway-dependent manner and this translated into a significant improvement in survival. RDEA119 downregulated phosphorylated ERK1/2 and nuclear factor κB p65 protein signaling and reduced activating protein 1 (AP1) transcription factor binding to the MUC2 promoter in LS174T cells. This study provides a preclinical rationale for the use of MEK inhibitors to treat patients with PMP. Copyright © 2015 Elsevier Inc. All rights reserved.

Gomisin N Inhibits Melanogenesis through Regulating the PI3K/Akt and MAPK/ERK Signaling Pathways in Melanocytes

PubMed Central

Chae, Jae Kyoung; Subedi, Lalita; Jeong, Minsun; Park, Yong Un; Kim, Chul Young; Kim, Hakwon; Kim, Sun Yeou

2017-01-01

Gomisin N, one of the lignan compounds found in Schisandra chinensis has been shown to possess anti-oxidative, anti-tumorigenic, and anti-inflammatory activities in various studies. Here we report, for the first time, the anti-melenogenic efficacy of Gomisin N in mammalian cells as well as in zebrafish embryos. Gomisin N significantly reduced the melanin content without cellular toxicity. Although it was not capable of modulating the catalytic activity of mushroom tyrosinase in vitro, Gomisin N downregulated the expression levels of key proteins that function in melanogenesis. Gomisin N downregulated melanocortin 1 receptor (MC1R), adenylyl cyclase 2, microphthalmia-associated transcription factor (MITF), tyrosinase, tyrosinase-related protein-1 (TRP-1), and tyrosinase-related protein-2 (TRP-2). In addition, Gomisin N-treated Melan-A cells exhibited increased p-Akt and p-ERK levels, which implies that the activation of the PI3K/Akt and MAPK/ERK pathways may function to inhibit melanogenesis. We also validated that Gomisin N reduced melanin production by repressing the expression of MITF, tyrosinase, TRP-1, and TRP-2 in mouse and human cells as well as in developing zebrafish embryos. Collectively, we conclude that Gomisin N inhibits melanin synthesis by repressing the expression of MITF and melanogenic enzymes, probably through modulating the PI3K/Akt and MAPK/ERK pathways. PMID:28241436

c-Raf/MEK/ERK pathway controls protein kinase C-mediated p70S6K activation in adult cardiac muscle cells.

PubMed

Iijima, Yoshihiro; Laser, Martin; Shiraishi, Hirokazu; Willey, Christopher D; Sundaravadivel, Balasubramanian; Xu, Lin; McDermott, Paul J; Kuppuswamy, Dhandapani

2002-06-21

p70S6 kinase (S6K1) plays a pivotal role in hypertrophic cardiac growth via ribosomal biogenesis. In pressure-overloaded myocardium, we show S6K1 activation accompanied by activation of protein kinase C (PKC), c-Raf, and mitogen-activated protein kinases (MAPKs). To explore the importance of the c-Raf/MAPK kinase (MEK)/MAPK pathway, we stimulated adult feline cardiomyocytes with 12-O-tetradecanoylphorbol-13-acetate (TPA), insulin, or forskolin to activate PKC, phosphatidylinositol-3-OH kinase, or protein kinase A (PKA), respectively. These treatments resulted in S6K1 activation with Thr-389 phosphorylation as well as mammalian target of rapamycin (mTOR) and S6 protein phosphorylation. Thr-421/Ser-424 phosphorylation of S6K1 was observed predominantly in TPA-treated cells. Dominant negative c-Raf expression or a MEK1/2 inhibitor (U0126) treatment showed a profound blocking effect only on the TPA-stimulated phosphorylation of S6K1 and mTOR. Whereas p38 MAPK inhibitors exhibited only partial effect, MAPK-phosphatase-3 expression significantly blocked the TPA-stimulated S6K1 and mTOR phosphorylation. Inhibition of mTOR with rapamycin blocked the Thr-389 but not the Thr-421/Ser-424 phosphorylation of S6K1. Therefore, during PKC activation, the c-Raf/MEK/extracellular signal-regulated kinase-1/2 (ERK1/2) pathway mediates both the Thr-421/Ser-424 and the Thr-389 phosphorylation in an mTOR-independent and -dependent manner, respectively. Together, our in vivo and in vitro studies indicate that the PKC/c-Raf/MEK/ERK pathway plays a major role in the S6K1 activation in hypertrophic cardiac growth.

Hindlimb unweighting induces changes in the p38MAPK contractile pathway of the rat abdominal aorta.

PubMed

Summers, Scott Matthew; Hayashi, Yuichiro; Nguyen, Steven Vu; Nguyen, Thu Minh; Purdy, Ralph Earl

2009-07-01

Hindlimb unweighting (HLU) of rats is a model used to mimic the cephalic fluid shift potentially involved in the orthostatic intolerance experienced by astronauts. Certain arteries in these rats exhibit a decreased contractile response to adrenergic agonists. It was shown previously that this may be caused by changes in thick filament regulation (Summers et al., Vascul Pharmacol 48: 208-214, 2008). In the present study, it was hypothesized that HLU also modifies thin filament regulation by effects on p38(MAPK) and ERK. Abdominal aorta rings from 20-day HLU rats and untreated controls were subjected to phenylephrine and phorbol 12,13-dibutyrate (PDBU) concentration response curves in the presence and absence of two inhibitors: the p38(MAPK) inhibitor SB-203580 and the MEK inhibitor U-0126. SB-203580 decreased control sensitivity to both agonists, but HLU sensitivity was not significantly affected. U-0126, which blocks enzymes immediately upstream of ERK, affected sensitivity to both agonists equally between control and HLU. Western blot analysis revealed no change in total levels of p38(MAPK) and its downstream target heat shock protein 27 but did reveal a decrease in phosphorylated levels of both after stimulation with PDBU and phenylephrine after HLU treatment. Neither total ERK nor phosphorylated levels after stimulation were affected by HLU. Total levels of caldesmon, a molecule downstream of both pathways, were decreased, but phosphorylated levels after stimulation were decreased by roughly twice as much. The results of this study demonstrate that HLU downregulates p38(MAPK), but not ERK, signaling. In turn, this may decrease actin availability for contraction.

Entire mitogen activated protein kinase (MAPK) pathway is present in preimplantation mouse embryos.

PubMed

Wang, Yingchun; Wang, Fangfei; Sun, Tong; Trostinskaia, Anna; Wygle, Dana; Puscheck, Elizabeth; Rappolee, Daniel A

2004-09-01

To understand how mitogenic signals are transduced into the trophoblasts in preimplantation embryos, the expression of mitogen-activated protein kinase (MAPK) pathway molecules was tested. We used immunocytochemical means and reverse transcriptase-polymerase chain reaction to test whether MAPK pathway molecule gene products exist at the protein and phosphoprotein level in the zygote and the RNA level in the egg and zygote. In addition, all antibodies detected the correct-sized major band in Westerns of placental cell lines representing the most prevalent cell type in preimplantation embryos. A majority of mRNA transcripts of MAPK pathway genes were detected in unfertilized eggs, and all were expressed in the zygote. We found that the MAPK pathway protein set consisting of the following gene products was present: FRS2 alpha, GRB2, GAB1, SOS1, Ha-ras, Raf1/RafB, MEK1,2,5, MAPK/ERK1,2, MAPK/ERK5, and RSK1,2,3 (see abbreviations). These proteins were detected in trophoblasts in embryonic day (E) 3.5 embryos when they could mediate mitogenic fibroblast growth factor signals from the embryo or colony stimulating factor-1 signals from the uterus. The phosphorylation state and position of the phosphoproteins in the cells suggested that they might function in mediating mitogenic signals. Interestingly, a subtle transition from maternal MAPK function to zygotic function was suggested by the localization for three MAPK pathway enzymes between E2.5 and E3.5, Raf1 phospho is largely cell membrane-localized at E2.5 and E3.5, and MEK1,2 phospho accumulates in the nucleus on E2.5 and E3.5. However, MAPK phospho shifts from nuclear accumulation at E2.5 to cytoplasmic accumulation at E3.5. This finding is similar to the cytoplasmic MAPK phospho localization reported in fibroblast growth factor signaling fields in postimplantation embryos (Corson et al. [2003] Development 130:4527-4537). This spatial and temporal expression study lays a foundation to plan and analyze perturbation studies aimed at understanding the role of the major mitogenic pathway in preimplantation mouse embryos.

Activation of multiple mitogen-activated protein kinases by recombinant calcitonin gene-related peptide receptor.

PubMed

Parameswaran, N; Disa, J; Spielman, W S; Brooks, D P; Nambi, P; Aiyar, N

2000-02-18

Calcitonin gene-related peptide is a 37-amino-acid neuropeptide and a potent vasodilator. Although calcitonin gene-related peptide has been shown to have a number of effects in a variety of systems, the mechanisms of action and the intracellular signaling pathways, especially the regulation of mitogen-activated protien kinase (MAPK) pathway, is not known. In the present study we investigated the role of calcitonin gene-related peptide in the regulation of MAPKs in human embryonic kidney (HEK) 293 cells stably transfected with a recombinant porcine calcitonin gene-related peptide-1 receptor. Calcitonin gene-related peptide caused a significant dose-dependent increase in cAMP response and the effect was inhibited by calcitonin gene-related peptide(8-37), the calcitonin gene-related peptide-receptor antagonist. Calcitonin gene-related peptide also caused a time- and concentration-dependent increase in extracellular signal-regulated kinase (ERK) and P38 mitogen-activated protein kinase (P38 MAPK) activities, with apparently no significant change in cjun-N-terminal kinase (JNK) activity. Forskolin, a direct activator of adenylyl cyclase also stimulated ERK and P38 activities in these cells suggesting the invovement of cAMP in this process. Calcitonin gene-related peptide-stimulated ERK and P38 MAPK activities were inhibited significantly by calcitonin gene-related peptide receptor antagonist, calcitonin gene-related peptide-(8-37) suggesting the involvement of calcitonin gene-related peptide-1 receptor. Preincubation of the cells with the cAMP-dependent protein kinase inhibitor, H89 [¿N-[2-((p-bromocinnamyl)amino)ethyl]-5-isoquinolinesulfonamide, hydrochloride¿] inhibited calcitonin gene-related peptide-mediated activation of ERK and p38 kinases. On the other hand, preincubation of the cells with wortmannin ¿[1S-(1alpha,6balpha,9abeta,11alpha, 11bbeta)]-11-(acetyloxy)-1,6b,7,8,9a,10,11, 11b-octahydro-1-(methoxymethyl)-9a,11b-dimethyl-3H-furo[4,3, 2-de]indeno[4,5-h]-2-benzopyran-3,6,9-trione¿, a PI3-kinase inhibitor, attenuated only calcitonin gene-related peptide-induced ERK and not P38 MAPK activation. Thus, these data suggest that activation of ERK by calcitonin gene-related peptide involves a H89-sensitive protein kinase A and a wortmannin-sensitive PI3-kinase while activation of p38 MAPK by calcitonin gene-related peptide involves only the H89 sensitive pathway and is independent of PI3 kinase. This also suggests that although both ERK and P38 can be activated by protein kinase A, the distal signaling components to protein kinase A in the activation of these two kinases (ERK and P38) are different.

MEK5-ERK5 Signaling in Cancer: Implications for Targeted Therapy

PubMed Central

Hoang, Van T.; Yan, Thomas J.; Cavanaugh, Jane E.; Flaherty, Patrick T.; Beckman, Barbara S.; Burow, Matthew E.

2017-01-01

Mitogen-activated protein kinases (MAPKs) regulate diverse cellular processes including proliferation, cell survival, differentiation, and apoptosis. While conventional MAPK constituents have well-defined roles in oncogenesis, the MAPK kinase 5-extracellular signal-regulated kinase 5 (MEK5-ERK5) pathway has only recently emerged in cancer research. In this review, we consider the MEK5 signaling cascade, focusing specifically on its involvement in drug resistance and regulation of aggressive cancer phenotypes. Moreover, we explore the role of MEK5 in tumorigenesis and metastatic progression, discussing the discrepancies in preclinical studies and assessing its viability as a therapeutic target for anti-cancer agents. PMID:28153789

Pirfenidone Induces G1 Arrest in Human Tenon's Fibroblasts In Vitro Involving AKT and MAPK Signaling Pathways.

PubMed

Guo, Xiujuan; Yang, Yangfan; Liu, Liling; Liu, Xiaoan; Xu, Jiangang; Wu, Kaili; Yu, Minbin

2017-06-01

To investigate the underlying mechanism by which pirfenidone blocks the transition from the G1 to S phase in primary human Tenon's fibroblasts. Primary human Tenon's fibroblasts were characterized by immunocytofluorescence staining with vimentin, fibroblast surface protein, and cytokeratin. After treating Tenon's fibroblasts with pirfenidone under proliferation conditions (10% fetal bovine serum), cell proliferation was measured using a WST-1 assay. Progression through the cell cycle was analyzed by flow cytometry. The expression of CDK2, CDK6, cyclinD1, cyclinD3, and cyclinE and the phosphorylation of AKT, ERK1/2/MAPK, JNK/MAPK, and p38 MAPK were estimated using western blot analysis. Under proliferative conditions, pirfenidone inhibited Tenon's fibroblasts proliferation and arrested the cell cycle at the G1 phase; decreased the phosphorylation of AKT, GSK3β, ERK1/2/MAPK, and JNK/MAPK; increased the phosphorylation of p38 MAPK; and inhibited CDK2, CDK6, cyclin D1, cyclin D3, and cyclin E in a dose-dependent manner. Inhibitors of AKT (LY294002), ERK1/2 (U0126), and JNK (SP600125) arrested the G1/S transition, similar to the effect of pirfenidone. The p38 inhibitor (SB202190) decreased the G1-blocking effect of pirfenidone. The expression of CDK2, CDK6, cyclin D1, and cyclin D3 were inhibited by LY294002, U0126, and SP600125. SB202190 attenuated the pirfenidone-induced reduction of CDK2, CDK6, cyclin D1, cyclin D3, and cyclin E. Pirfenidone inhibited HTFs proliferation and induced G1 arrest by downregulating CDKs and cyclins involving the AKT/GSK3β and MAPK signaling pathways.

Monocyte 15-lipoxygenase gene expression requires ERK1/2 MAPK activity.

PubMed

Bhattacharjee, Ashish; Mulya, Anny; Pal, Srabani; Roy, Biswajit; Feldman, Gerald M; Cathcart, Martha K

2010-11-01

IL-13 induces profound expression of 15-lipoxygenase (15-LO) in primary human monocytes. Our studies have defined the functional IL-13R complex, association of Jaks with the receptor components, and the tyrosine phosphorylation of several Stat molecules in response to IL-13. Furthermore, we identified both p38MAPK and protein kinase Cδ as critical regulators of 15-LO expression. In this study, we report an ERK1/2-dependent signaling cascade that regulates IL-13-mediated 15-LO gene expression. We show the rapid phosphorylation/activation of ERK1/2 upon IL-13 exposure. Our results indicate that Tyk2 kinase is required for the activation of ERK1/2, which is independent of the Jak2, p38MAPK, and protein kinase Cδ pathways, suggesting bifurcating parallel regulatory pathways downstream of the receptor. To investigate the signaling mechanisms associated with the ERK1/2-dependent expression of 15-LO, we explored the involvement of transcription factors, with predicted binding sites in the 15-LO promoter, in this process including Elk1, early growth response-1 (Egr-1), and CREB. Our findings indicate that IL-13 induces Egr-1 nuclear accumulation and CREB serine phosphorylation and that both are markedly attenuated by inhibition of ERK1/2 activity. We further show that ERK1/2 activity is required for both Egr-1 and CREB DNA binding to their cognate sequences identified within the 15-LO promoter. Furthermore, by transfecting monocytes with the decoy oligodeoxyribonucleotides specific for Egr-1 and CREB, we discovered that Egr-1 and CREB are directly involved in regulating 15-LO gene expression. These studies characterize an important regulatory role for ERK1/2 in mediating IL-13-induced monocyte 15-LO expression via the transcription factors Egr-1 and CREB.

Mechanism of short-term ERK activation by electromagnetic fields at mobile phone frequencies.

PubMed

Friedman, Joseph; Kraus, Sarah; Hauptman, Yirmi; Schiff, Yoni; Seger, Rony

2007-08-01

The exposure to non-thermal microwave electromagnetic fields generated by mobile phones affects the expression of many proteins. This effect on transcription and protein stability can be mediated by the MAPK (mitogen-activated protein kinase) cascades, which serve as central signalling pathways and govern essentially all stimulated cellular processes. Indeed, long-term exposure of cells to mobile phone irradiation results in the activation of p38 as well as the ERK (extracellular-signal-regulated kinase) MAPKs. In the present study, we have studied the immediate effect of irradiation on the MAPK cascades, and found that ERKs, but not stress-related MAPKs, are rapidly activated in response to various frequencies and intensities. Using signalling inhibitors, we delineated the mechanism that is involved in this activation. We found that the first step is mediated in the plasma membrane by NADH oxidase, which rapidly generates ROS (reactive oxygen species). These ROS then directly stimulate MMPs (matrix metalloproteinases) and allow them to cleave and release Hb-EGF [heparin-binding EGF (epidermal growth factor)]. This secreted factor activates the EGF receptor, which in turn further activates the ERK cascade. Thus this study demonstrates for the first time a detailed molecular mechanism by which electromagnetic irradiation from mobile phones induces the activation of the ERK cascade and thereby induces transcription and other cellular processes.

Overcoming cetuximab resistance in HNSCC: the role of AURKB and DUSP proteins.

PubMed

Boeckx, Carolien; Op de Beeck, Ken; Wouters, An; Deschoolmeester, Vanessa; Limame, Ridha; Zwaenepoel, Karen; Specenier, Pol; Pauwels, Patrick; Vermorken, Jan B; Peeters, Marc; Van Camp, Guy; Baay, Marc; Lardon, Filip

2014-11-28

Unraveling the underlying mechanisms of cetuximab resistance in head and neck squamous cell carcinoma (HNSCC) is of major importance as many tumors remain non-responsive or become resistant. Our microarray results suggest that "resistant" cells still exhibit RAS-MAPK pathway signaling contributing to drug resistance, as witnessed by low expression of DUSP5 and DUSP6, negative regulators of ERK1/2, and increased expression of AURKB, a key regulator of mitosis. Therefore, interrupting the RAS-MAPK pathway by an ERK1/2 inhibitor (apigenin) or an AURKB inhibitor (barasertib) might be a new strategy for overcoming cetuximab resistance in HNSCC. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

Differential effects of fluoxetine and venlafaxine on memory recognition: possible mechanisms of action.

PubMed

Carlini, Valeria Paola; Poretti, María Belén; Rask-Andersen, Mathias; Chavan, Rohit A; Ponzio, Marina F; Sawant, Rahul S; de Barioglio, Susana Rubiales; Schiöth, Helgi B; de Cuneo, Marta Fiol

2012-08-07

Serotonin-specific reuptake inhibitors (SSRI) and serotonin-norepinephrine reuptake inhibitors (SNRI) are antidepressant drugs commonly used to treat a wide spectrum of mood disorders (Wong and Licinio, 2001). Although they have been clinically used for more than 50 years, the molecular and cellular basis for the action of SSRIs and SNRIs is not clear. Considering that the changes in gene expression involved in the action of antidepressant drugs on memory have not been identified, in this study we investigated the impact of chronic treatment with a SSRI (fluoxetine) and a SNRI (venlafaxine) on the mRNA expression of genes related to memory cascade in the mouse hippocampus, namely, α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), nitric oxide synthase 1 (NOS1), neurotrophic tyrosine kinase receptor type 2 (TrKB), mitogen-activated protein kinases (MAPK/ERK) and serotonin transporter (SERT). Animals treated with fluoxetine 10 mg/Kg/day for 28 days showed a significant decrease in the percentage of time spent in the novel object recognition test (p≤0.005) and induced MAPK1/ERK2 down-regulation (p=0.005). Our results suggest that the effect on cognition could probably be explained by fluoxetine interference in the MAPK/ERK memory pathway. In contrast, chronic treatment with venlafaxine did not reduce MAPK1/ERK2 expression, suggesting that MAPK1/ERK2 down-regulation is not a common effect of all antidepressant drugs. Further studies are needed to examine the effect of chronic fluoxetine treatment on the ERK-CREB system, and to determine whether there is a causal relationship between the disruption of the ERK-CREB system and the effect of this antidepressant on memory performance. Copyright © 2012 Elsevier Inc. All rights reserved.

Serotonin disrupts esophageal mucosal integrity: an investigation using a stratified squamous epithelial model.

PubMed

Wu, Liping; Oshima, Tadayuki; Tomita, Toshihiko; Ohda, Yoshio; Fukui, Hirokazu; Watari, Jiro; Miwa, Hiroto

2016-11-01

Serotonin regulates gastrointestinal function, and mast cells are a potential nonneuronal source of serotonin in the esophagus. Tight junction (TJ) proteins in the esophageal epithelium contribute to the barrier function, and the serotonin signaling pathway may contribute to epithelial leakage in gastroesophageal reflux disease. Therefore, the aim of this study was to investigate the role of serotonin on barrier function, TJ proteins, and related signaling pathways. Normal primary human esophageal epithelial cells were cultured with use of an air-liquid interface system. Serotonin was added to the basolateral compartment, and transepithelial electrical resistance (TEER) was measured. The expression of TJ proteins and serotonin receptor 7 (5-HT 7 ) was assessed by Western blotting. The involvement of 5-HT 7 was assessed with use of an antagonist and an agonist. The underlying cellular signaling pathways were examined with use of specific blockers. Serotonin decreased TEER and reduced the expression of TJ proteins ZO-1, occludin, and claudin 1, but not claudin 4. A 5-HT 7 antagonist blocked the serotonin-induced decrease in TEER, and a 5-HT 7 agonist decreased TEER. Inhibition of p38 mitogen-activated protein kinase (MAPK) reduced the serotonin-induced decrease in TEER. Inhibition of p38 MAPK blocked the decrease of ZO-1 levels, whereas extracellular-signal-regulated kinase (ERK) inhibition blocked the decrease in occludin levels. Cell signaling pathway inhibitors had no effect on serotonin-induced alterations in claudin 1 and claudin 4 levels. Serotonin induced phosphorylation of p38 MAPK and ERK, and a 5-HT 7 antagonist partially blocked serotonin-induced phosphorylation of p38 MAPK but not that of ERK. Serotonin disrupted esophageal squamous epithelial barrier function by modulating the levels of TJ proteins. Serotonin signaling pathways may mediate the pathogenesis of gastroesophageal reflux disease.

Mitogen-activated protein kinase signaling pathways promote low-density lipoprotein receptor-related protein 1-mediated internalization of beta-amyloid protein in primary cortical neurons.

PubMed

Yang, Wei-Na; Ma, Kai-Ge; Qian, Yi-Hua; Zhang, Jian-Shui; Feng, Gai-Feng; Shi, Li-Li; Zhang, Zhi-Chao; Liu, Zhao-Hui

2015-07-01

Mounting evidence suggests that the pathological hallmarks of Alzheimer's disease (AD) are caused by the intraneuronal accumulation of beta-amyloid protein (Aβ). Reuptake of extracellular Aβ is believed to contribute significantly to the intraneuronal Aβ pool in the early stages of AD. Published reports have claimed that the low-density lipoprotein receptor-related protein 1 (LRP1) mediates Aβ1-42 uptake and lysosomal trafficking in GT1-7 neuronal cells and mouse embryonic fibroblast non-neuronal cells. However, there is no direct evidence supporting the role of LRP1 in Aβ internalization in primary neurons. Our recent study indicated that p38 MAPK and ERK1/2 signaling pathways are involved in regulating α7 nicotinic acetylcholine receptor (α7nAChR)-mediated Aβ1-42 uptake in SH-SY5Y cells. This study was designed to explore the regulation of MAPK signaling pathways on LRP1-mediated Aβ internalization in neurons. We found that extracellular Aβ1-42 oligomers could be internalized into endosomes/lysosomes and mitochondria in cortical neurons. Aβ1-42 and LRP1 were also found co-localized in neurons during Aβ1-42 internalization, and they could form Aβ1-42-LRP1 complex. Knockdown of LRP1 expression significantly decreased neuronal Aβ1-42 internalization. Finally, we identified that p38 MAPK and ERK1/2 signaling pathways regulated the internalization of Aβ1-42 via LRP1. Therefore, these results demonstrated that LRP1, p38 MAPK and ERK1/2 mediated the internalization of Aβ1-42 in neurons and provided evidence that blockade of LRP1 or inhibitions of MAPK signaling pathways might be a potential approach to lowering brain Aβ levels and served a potential therapeutic target for AD. Copyright © 2015 Elsevier Ltd. All rights reserved.

An Inhibitory Role of Osthole in Rat MSCs Osteogenic Differentiation and Proliferation via Wnt/β-Catenin and Erk1/2-MAPK Pathways.

PubMed

Hu, Hongyang; Chen, Min; Dai, Guangzu; Du, Guoqing; Wang, Xuezong; He, Jie; Zhao, Yongfang; Han, Dapeng; Cao, Yuelong; Zheng, Yuxin; Ding, Daofang

2016-01-01

Bone marrow-derived mesenchymal stem cells (MSCs) are responsible for new bone formation during adulthood. Accumulating evidences showed that Osthole promotes the osteogenic differentiation in primary osteoblasts. The aim of this study was to investigate whether Osthole exhibits a potential to stimulate the osteogenic differentiation of MSCs and the underlying mechanism. MSCs were treated with a gradient concentration of Osthole (6.25 µM, 12.5 µM, and 25 µM). Cell proliferation was assessed by western blotting with the proliferating cell nuclear antigen (PCNA) and Cyclin D1 antibodies, fluorescence activated cell sorting (FACS), and cell counting kit 8 (CCK8). MSCs were cultured in osteogenesis-induced medium for one or two weeks. The osteogenic differentiation of MSCs was estimated by Alkaline Phosphatase (ALP) staining, Alizarin red staining, Calcium influx, and quantitative PCR (qPCR). The underlying mechanism of Osthole-induced osteogenesis was further evaluated by western blotting with antibodies in Wnt/β-catenin, PI3K/Akt, BMPs/smad1/5/8, and MAPK signaling pathways. Osthole inhibited proliferation of rat MSCs in a dose-dependent manner. Osthole suppressed osteogenic differentiation of rat MSCs by down-regulating the activities of Wnt/β-catenin and Erk1/2-MAPK signaling. Osthole inhibits the proliferation and osteogenic differentiation of rat MSCs, which might be mediated through blocking the Wnt/β-catenin and Erk1/2-MAPK signaling pathways. © 2016 The Author(s) Published by S. Karger AG, Basel.

NMDA receptor dependent PGC-1alpha up-regulation protects the cortical neuron against oxygen-glucose deprivation/reperfusion injury.

PubMed

Luo, Yun; Zhu, Wenjing; Jia, Jia; Zhang, Chenyu; Xu, Yun

2009-09-01

The peroxisome proliferator activated receptor coactivator 1 alpha (PGC-1alpha) is a nuclear transcriptional coactivator that is widely expressed in the brain areas. Over-expression of PGC-1alpha can protect neuronal cells from oxidant-induced injury. The purpose of the current study is to investigate the role of PGC-1alpha in the oxygen (anoxia) deprivation (OGD) neurons. The PGC-1alpha mRNA and protein level between control and OGD neurons were examined by real-time PCR and Western blot. More PGC-1alpha expression was found in the OGD neurons compared with the normal group. Over-expression of PGC-1alpha suppressed cell apoptosis while inhibition of the PGC-1alpha expression induced cell apoptosis in OGD neurons. Furthermore, increase of PGC-1alpha resulted in activation of N-methyl-D-aspartate (NMDA) receptor, p38, and ERK mitogen-activated protein kinase (MAPK) pathway. The blocking of the NMDA receptor by its antagonists MK-801 reduced PGC-1alpha mRNA expression in OGD neurons, while NMDA itself can directly induce the expression of PGC-1alpha in neuronal cells. At the same time, PD98059 (ERK MAPK inhibitor) and SB203580 (P38 MAPK inhibitor) also prevented the up-regulation of PGC-1alpha in OGD neurons and MK801 can inhibit the expression of P38 and ERK MAPK. These data suggested that the expression of PGC-1alpha was up-regulated in OGD mice cortical neurons, which protected the neurons against OGD injury. Moreover, this effect was correlated to the NMDA receptor and the ERK and P38 MAPK pathway. The protective effect of PGC-1alpha on OGD cortical neurons may be useful for stroke therapy.

A SPRY2 mutation leading to MAPK/ERK pathway inhibition is associated with an autosomal dominant form of IgA nephropathy.

PubMed

Milillo, Annamaria; La Carpia, Francesca; Costanzi, Stefano; D'Urbano, Vanessa; Martini, Maurizio; Lanuti, Paola; Vischini, Gisella; Larocca, Luigi M; Marchisio, Marco; Miscia, Sebastiano; Amoroso, Antonio; Gurrieri, Fiorella; Sangiorgi, Eugenio

2015-12-01

IgA nephropathy (IgAN) represents the most common primary glomerulonephritis worldwide with a prevalence of 25-50% among patients with primary glomerulopathies. In ~5-10% of the patients the disease segregates with an autosomal dominant (AD) pattern. Association studies identified loci on chromosomes 1q32, 6p21, 8p23, 17p13, 22q12, whereas classical linkage studies on AD families identified loci on chromosomes 2q36, 4q26-31, 6q22, 17q12-22. We have studied a large Sicilian family where IgAN segregates with an AD transmission. To identify the causal gene, the exomes of two affected and one unaffected individual have been sequenced. From the bioinformatics analysis a p.(Arg119Trp) variant in the SPRY2 gene was identified as the probable disease-causing mutation. Moreover, functional characterization of this variant showed that it is responsible for the inhibition of the MAPK/ERK1/2 pathway. The same effect was observed in two sporadic IgAN patients carriers of wild-type SPRY2, suggesting that downregulation of the MAPK/ERK1/2 pathway represents a common mechanism leading to IgAN.

Inhibition of mitogen-activated protein kinase Erk1/2 promotes protein degradation of ATP binding cassette transporters A1 and G1 in CHO and HuH7 cells.

PubMed

Mulay, Vishwaroop; Wood, Peta; Manetsch, Melanie; Darabi, Masoud; Cairns, Rose; Hoque, Monira; Chan, Karen Cecilia; Reverter, Meritxell; Alvarez-Guaita, Anna; Rye, Kerry-Anne; Rentero, Carles; Heeren, Joerg; Enrich, Carlos; Grewal, Thomas

2013-01-01

Signal transduction modulates expression and activity of cholesterol transporters. We recently demonstrated that the Ras/mitogen-activated protein kinase (MAPK) signaling cascade regulates protein stability of Scavenger Receptor BI (SR-BI) through Proliferator Activator Receptor (PPARα) -dependent degradation pathways. In addition, MAPK (Mek/Erk 1/2) inhibition has been shown to influence liver X receptor (LXR) -inducible ATP Binding Cassette (ABC) transporter ABCA1 expression in macrophages. Here we investigated if Ras/MAPK signaling could alter expression and activity of ABCA1 and ABCG1 in steroidogenic and hepatic cell lines. We demonstrate that in Chinese Hamster Ovary (CHO) cells and human hepatic HuH7 cells, extracellular signal-regulated kinase 1/2 (Erk1/2) inhibition reduces PPARα-inducible ABCA1 protein levels, while ectopic expression of constitutively active H-Ras, K-Ras and MAPK/Erk kinase 1 (Mek1) increases ABCA1 protein expression, respectively. Furthermore, Mek1/2 inhibitors reduce ABCG1 protein levels in ABCG1 overexpressing CHO cells (CHO-ABCG1) and human embryonic kidney 293 (HEK293) cells treated with LXR agonist. This correlates with Mek1/2 inhibition reducing ABCG1 cell surface expression and decreasing cholesterol efflux onto High Density Lipoproteins (HDL). Real Time reverse transcriptase polymerase chain reaction (RT-PCR) and protein turnover studies reveal that Mek1/2 inhibitors do not target transcriptional regulation of ABCA1 and ABCG1, but promote ABCA1 and ABCG1 protein degradation in HuH7 and CHO cells, respectively. In line with published data from mouse macrophages, blocking Mek1/2 activity upregulates ABCA1 and ABCG1 protein levels in human THP1 macrophages, indicating opposite roles for the Ras/MAPK pathway in the regulation of ABC transporter activity in macrophages compared to steroidogenic and hepatic cell types. In summary, this study suggests that Ras/MAPK signaling modulates PPARα- and LXR-dependent protein degradation pathways in a cell-specific manner to regulate the expression levels of ABCA1 and ABCG1 transporters.

Injury-induced rapid activation of MAPK signaling in dechorionated eggs and larvae of the silkworm Bombyx mori.

PubMed

Gu, Shi-Hong; Chen, Chien-Hung

2017-04-01

Previous study showed that diapause in Bombyx mori eggs can be terminated by dechorionation and that activation in the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) in dechorionated cultured eggs is involved in diapause termination. In the present study, the possible mechanism underlying activation of ERK upon dechorionation was further investigated. Results showed that mechanical injury of diapause eggs without medium incubation also resulted in rapid increase in the phospho-ERK levels and that injury increased the phospho-ERK levels at different stages of both diapause eggs and eggs in which diapause initiation was prevented by HCl. Effects of anaerobiosis on dechorionation-stimulated phospho-ERK levels showed that the mechanical injury itself but not the dramatic increase in oxygen uptake upon injury is involved in a rapid activation of ERK. Chemical anaerobiosis on dechorionation-stimulated phospho-ERK levels and the in vivo effect of anaerobiosis showed that the supply of oxygen also plays a role in ERK signaling. In addition, injury induced the phosphorylation of c-jun N-terminal kinases (JNKs) and p38 kinase, components of two parallel MAPK pathways. A kinase assay showed a dramatic increase in JNK kinase activity in egg lysates upon injury. When newly hatched first instar larvae were injured, an increase in the phospho-ERK levels similar to that in dechorionated eggs was observed. From the results, we hypothesize that the injury-induced rapid activation of MAPK signaling, which serves as a natural signal for embryonic development, is related to diapause termination in dechorionated eggs. © 2015 Institute of Zoology, Chinese Academy of Sciences.

Phenotypic and evolutionary implications of modulating the ERK-MAPK cascade using the dentition as a model

NASA Astrophysics Data System (ADS)

Marangoni, Pauline; Charles, Cyril; Tafforeau, Paul; Laugel-Haushalter, Virginie; Joo, Adriane; Bloch-Zupan, Agnès; Klein, Ophir D.; Viriot, Laurent

2015-06-01

The question of phenotypic convergence across a signalling pathway has important implications for both developmental and evolutionary biology. The ERK-MAPK cascade is known to play a central role in dental development, but the relative roles of its components remain unknown. Here we investigate the diversity of dental phenotypes in Spry2-/-, Spry4-/-, and Rsk2-/Y mice, including the incidence of extra teeth, which were lost in the mouse lineage 45 million years ago (Ma). In addition, Sprouty-specific anomalies mimic a phenotype that is absent in extant mice but present in mouse ancestors prior to 9 Ma. Although the mutant lines studied display convergent phenotypes, each gene has a specific role in tooth number determination and crown patterning. The similarities found between teeth in fossils and mutants highlight the pivotal role of the ERK-MAPK cascade during the evolution of the dentition in rodents.

Nox4 NADPH oxidase-derived reactive oxygen species, via endogenous carbon monoxide, promote survival of brain endothelial cells during TNF-α-induced apoptosis

PubMed Central

Basuroy, Shyamali; Tcheranova, Dilyara; Bhattacharya, Sujoy; Leffler, Charles W.

2011-01-01

We investigated the role of reactive oxygen species (ROS) in promoting cell survival during oxidative stress induced by the inflammatory mediator tumor necrosis factor-α (TNF-α) in cerebral microvascular endothelial cells (CMVEC) from newborn piglets. Nox4 is the major isoform of NADPH oxidase responsible for TNF-α-induced oxidative stress and apoptosis in CMVEC. We present novel data that Nox4 NADPH oxidase-derived ROS also initiate a cell survival mechanism by increasing production of a gaseous antioxidant mediator carbon monoxide (CO) by constitutive heme oxygenase-2 (HO-2). TNF-α rapidly enhanced endogenous CO production in a superoxide- and NADPH oxidase-dependent manner in CMVEC with innate, but not with small interfering RNA (siRNA)-downregulated Nox4 activity. CORM-A1, a CO-releasing compound, inhibited Nox4-mediated ROS production and enhanced cell survival in TNF-α-challenged CMVEC. The ROS-induced CO-mediated survival mechanism requires functional interactions between the protein kinase B/Akt and extracellular signal-related kinase (ERK)/p38 MAPK signaling pathways activated by TNF-α. In Akt siRNA-transfected CMVEC and in cells with pharmacologically inhibited Akt, Erk1/2, and p38 mitogen-activated protein kinase (MAPK) activities, CORM-A1 was no longer capable of blocking Nox4 activation and apoptosis caused by TNF-α. Overall, Nox4 NADPH oxidase-derived ROS initiate both death and survival pathways in TNF-α-challenged CMVEC. The ROS-dependent cell survival pathway is mediated by an endogenous antioxidant CO, which inhibits Nox4 activation via a mechanism that includes Akt, ERK1/2, and p38 MAPK signaling pathways. The ability of CO to inhibit TNF-α-induced ERK1/2 and p38 MAPK activities in an Akt-dependent manner appears to be the key element in ROS-dependent survival of endothelial cells during TNF-α-mediated brain inflammatory disease. PMID:21123734

Branched-chain amino acids enhance cyst development in autosomal dominant polycystic kidney disease.

PubMed

Yamamoto, Junya; Nishio, Saori; Hattanda, Fumihiko; Nakazawa, Daigo; Kimura, Toru; Sata, Michio; Makita, Minoru; Ishikawa, Yasunobu; Atsumi, Tatsuya

2017-08-01

Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the progressive development of kidney and liver cysts. The mammalian target of rapamycin (mTOR) cascade is one of the important pathways regulating cyst growth in ADPKD. Branched-chain amino acids (BCAAs), including leucine, play a crucial role to activate mTOR pathway. Therefore, we administered BCAA dissolved in the drinking water to Pkd1 flox/flox :Mx1-Cre (cystic) mice from four to 22 weeks of age after polyinosinic-polycytidylic acid-induced conditional Pkd1 knockout at two weeks of age. The BCAA group showed significantly greater kidney/body weight ratio and higher cystic index in both the kidney and liver compared to the placebo-treated mice. We found that the L-type amino acid transporter 1 that facilitates BCAA entry into cells is strongly expressed in cells lining the cysts. We also found increased cyst-lining cell proliferation and upregulation of mTOR and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) pathways in the BCAA group. In vitro, we cultured renal epithelial cell lines from Pkd1 null mice with or without leucine. Leucine was found to stimulate cell proliferation, as well as activate mTOR and MAPK/ERK pathways in these cells. Thus, BCAA accelerated disease progression by mTOR and MAPK/ERK pathways. Hence, BCAA may be harmful to patients with ADPKD. Copyright © 2017 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.

Potential role of insulin signaling on vascular smooth muscle cell migration, proliferation, and inflammation pathways.

PubMed

Cersosimo, Eugenio; Xu, Xiaojing; Musi, Nicolas

2012-02-15

To investigate the role of insulin signaling pathways in migration, proliferation, and inflammation of vascular smooth muscle cells (VSMCs), we examined the expression of active components of the phosphatidyl inositol 3 (PI-3) kinase (p-Akt) and mitogen-activated protein kinase (MAPK) (p-Erk) in primary cultures of VSMCs from human coronary arteries. VSMCs were treated in a dose-response manner with insulin (0, 1, 10, and 100 nM) for 20 min, and Akt and Erk phosphorylation were measured by Western blot analysis. In separate experiments, we evaluated the effect of 200 μM palmitate, in the presence and absence of 8 μM pioglitazone, on insulin-stimulated (100 nM for 20 min) Akt and Erk phosphorylation. The phosphorylation of Akt and Erk in VSMCs exhibited a dose dependency with a three- to fourfold increase, respectively, at the highest dose (100 nM). In the presence of palmitate, insulin-induced Akt phosphorylation was completely abolished, and there was a threefold increase in p-Erk. With addition of pioglitazone, the phosphorylation of Akt by insulin remained unchanged, whereas insulin-stimulated Erk phosphorylation was reduced by pioglitazone. These data in VSMCs indicate that high palmitate decreases insulin-stimulated Akt phosphorylation and stimulates MAPK, whereas preexposure peroxisome proliferator-activated receptor-γ agonist pioglitazone preserves Akt phosphorylation and simultaneously attenuates MAPK signaling. Our results suggest that metabolic and mitogenic insulin signals have different sensitivity, are independently regulated, and may play a role in arterial smooth muscle cells migration, proliferation, and inflammation in conditions of acute hyperinsulinemia.

Invasive ability of human renal cell carcinoma cell line Caki-2 is accelerated by gamma-aminobutyric acid, via sustained activation of ERK1/2 inducible matrix metalloproteinases.

PubMed

Inamoto, Teruo; Azuma, Haruhito; Sakamoto, Takeshi; Kiyama, Satoshi; Ubai, Takanobu; Kotake, Yatsugu; Watanabe, Masahito; Katsuoka, Yoji

2007-10-01

Gamma-aminobutyric acid (GABA) was first discovered as an inhibitory neurotransmitter in the central nervous system (CNS) and has been reported to have a variety of functions, including regulation of cell division, cell differentiation and maturation, and to be involved in the development of certain cancers outside the CNS. In the present study, using the human renal cell carcinoma cell line Caki-2, we demonstrated that GABA stimulation significantly increased the expression of MMP-2 and -9 and subsequently increased the invasive activity of the cancer cells. Because MAPK signaling is one of the key regulators of MMP expression, we further evaluated MAPK signaling after stimulation with GABA. It was found that GABA stimulation promoted the phosphorylation of MAPKs, including ERK1/2, JNK, and p38. ERK1/2 phosphorylation was sustained for up to 12 h, while phosphorylation of JNK and p38 returned to the endogenous level by 30 min. It was noteworthy that the ras/raf/MEK/ERK pathway inhibitor PD98059 attenuated GABA-induced MMP-9 expression and that both PD98059 and MMP inhibitors attenuated the GABA-induced invasive activity of Caki-2 cells. Moreover, data obtained by depletion of the MEK/ERK pathway using interfering RNA transfection of Caki-2 cells clearly corroborated the above results, as both MMP-9 expression and GABA-induced invasive ability were decreased significantly. We also demonstrated that the GABA-induced increase in invasive ability via ERK1/2 up-regulation was mediated mainly through the GABA-B receptor. These results indicate that GABA stimulation promotes cancer cell invasion and that the effect is partly due to ERK1/2-dependent up-regulation of MMPs.

Changes in mitogen-activated protein kinase in cerebellar granule neurons by polybrominated diphenyl ethers and polychlorinated biphenyls

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fan Chunyang; Curriculum in Toxicology, University of North Carolina, Chapel Hill, NC 27599; Besas, Jonathan

2010-05-15

Polybrominated diphenyl ethers (PBDEs) are used as additive flame retardants and have been detected in human blood, adipose tissue, and breast milk. Both in vitro and in vivo studies have shown that the effects of PBDEs are similar to the known human developmental neurotoxicants such as polychlorinated biphenyls (PCBs) on a molar basis. Previously, we reported that PBDE mixtures and congeners, perturbed calcium homeostasis which is critical for the development and function of the nervous system. In the present study, we tested whether environmentally relevant PBDE/PCB mixtures and congeners affected mitogen-activated protein kinase (MAPK) pathways, which are down-stream events ofmore » calcium signaling in cerebellar granule neuronal cultures. In this study, phosphorylated extracellular signal-regulated kinase (pERK)1/2, a widely studied MAPK cascade and known to be involved in learning and memory, levels were quantitated using western blot technique with phospho-specific antibodies. Glutamate (a positive control) increased pERK1/2 in a time- and concentration-dependent manner reaching maximum activation at 5-30 min of exposure and at doses >= 10 muM. Both Aroclor 1254 (a commercial penta PCB mixture) and DE-71 (a commercial penta PBDE mixture) elevated phospho-ERK1/2, producing maximum stimulation at 30 min and at concentrations >= 3 mug/ml; Aroclor 1254 was more efficacious than DE-71. DE-79 (an octabrominated diphenyl ether mixture) also elevated phospho-ERK1/2, but to a lesser extent than that of DE-71. PBDE congeners 47, 77, 99, and 153 also increased phospo-ERK1/2 in a concentration-dependent manner. The data indicated that PBDE congeners are more potent than the commercial mixtures. PCB 47 also increased phospho-ERK1/2 like its structural analog PBDE 47, but to a lesser extent, suggesting that these chemicals affect similar pathways. Cytotoxicity, measured as %LDH release, data showed that higher concentrations (> 30 muM) and longer exposures (> 30 min) are required to see cell death. These results show that PBDE mixtures and congeners activate MAPK pathway at concentrations where no significant cytotoxicity was observed, suggesting that perturbed intracellular signaling including MAPK pathway might be involved in the initiation of adverse effects, including learning and memory, related to these persistent chemicals.« less

Triiodothyronine promotes the proliferation of epicardial progenitor cells through the MAPK/ERK pathway.

PubMed

Deng, Song-Bai; Jing, Xiao-Dong; Wei, Xiao-Ming; Du, Jian-Lin; Liu, Ya-Jie; Qin, Qin; She, Qiang

2017-04-29

Thyroid hormone has important functions in the development and physiological function of the heart. The aim of this study was to determine whether 3,5,3'-Triiodothyronine (T3) can promote the proliferation of epicardial progenitor cells (EPCs) and to investigate the potential underlying mechanism. Our results showed that T3 significantly promoted the proliferation of EPCs in a concentration- and time-dependent manner. The thyroid hormone nuclear receptor inhibitor bisphenol A (100 μmol/L) did not affect T3's ability to induce proliferation. Further studies showed that the mRNA expression levels of mitogen-activated protein kinase 1 (MAPK1), MAPK3, and Ki67 in EPCs in the T3 group (10 nmol/L) increased 2.9-, 3-, and 4.1-fold, respectively, compared with those in the control group (P < 0.05). In addition, the mRNA expression of the cell cycle protein cyclin D1 in the T3 group increased approximately 2-fold compared with the control group (P < 0.05), and there were more EPCs in the S phase of the cell cycle (20.6% vs. 12.0%, P < 0.05). The mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathway inhibitor U0126 (10 μmol/L) significantly inhibited the ability of T3 to promote the proliferation of EPCs and to alter cell cycle progression. This study suggested that T3 significantly promotes the proliferation of EPCs, and this effect may be achieved through activation of the MAPK/ERK signaling pathway. Copyright © 2017 Elsevier Inc. All rights reserved.

Testicular Lumicrine Factors Regulate ERK, STAT, and NFKB Pathways in the Initial Segment of the Rat Epididymis to Prevent Apoptosis1

PubMed Central

Xu, Bingfang; Abdel-Fattah, Rana; Yang, Ling; Crenshaw, Sallie A.; Black, Michael B.; Hinton, Barry T.

2011-01-01

The initial segment of the epididymis is vital for male fertility; therefore, it is important to understand the mechanisms that regulate this important region. Deprival of testicular luminal fluid factors/lumicrine factors from the epididymis results in a wave of apoptosis in the initial segment. In this study, a combination of protein array and microarray analyses was used to examine the early changes in downstream signal transduction pathways following loss of lumicrine factors. We discovered the following cascade of events leading to the loss of protection and eventual apoptosis: in the first 6 h after loss of lumicrine factors, down-regulation of the ERK pathway components was observed at the mRNA expression and protein activity levels. Microarray analysis revealed that mRNA levels of several key components of the ERK pathway, Dusp6, Dusp5, and Etv5, decreased sharply, while the analysis from the protein array revealed a decline in the activities of MAP2K1/2 and MAPK1. Immunostaining of phospho-MAPK3/1 indicated that down-regulation of the ERK pathway was specific to the epithelial cells of the initial segment. Subsequently, after 12 h of loss of lumicrine factors, levels of mRNA expression of STAT and NFKB pathway components increased, mRNA levels of several genes encoding cell cycle inhibitors increased, and levels of protein expression of several proapoptotic phosphatases increased. Finally, after 18 h of loss of protection from lumicrine factors, apoptosis was observed. In conclusion, testicular lumicrine factors protect the cells of the initial segment by activating the ERK pathway, repressing STAT and NFKB pathways, and thereby preventing apoptosis. PMID:21311037

Absence of suppressor of cytokine signalling 3 reduces self-renewal and promotes differentiation in murine embryonic stem cells.

PubMed

Forrai, Ariel; Boyle, Kristy; Hart, Adam H; Hartley, Lynne; Rakar, Steven; Willson, Tracy A; Simpson, Ken M; Roberts, Andrew W; Alexander, Warren S; Voss, Anne K; Robb, Lorraine

2006-03-01

Leukemia inhibitory factor (LIF) is required to maintain pluripotency and permit self-renewal of murine embryonic stem (ES) cells. LIF binds to a receptor complex of LIFR-beta and gp130 and signals via the Janus kinase-signal transducer and activator of transcription (JAK-STAT) pathway, with signalling attenuated by suppressor of cytokine signalling (SOCS) proteins. Recent in vivo studies have highlighted the role of SOCS-3 in the negative regulation of signalling via gp130. To determine the role of SOCS-3 in ES cell biology, SOCS-3-null ES cell lines were generated. When cultured in LIF levels that sustain self-renewal of wild-type cells, SOCS-3-null ES cell lines exhibited less self-renewal and greater differentiation into primitive endoderm. The absence of SOCS-3 enhanced JAK-STAT and extracellular signal-related kinase 1/2 (ERK-1/2)-mitogen-activated protein kinase (MAPK) signal transduction via gp130, with higher levels of phosphorylated STAT-1, STAT-3, SH-2 domain-containing cytoplasmic protein tyrosine phosphatase 2 (SHP-2), and ERK-1/2 in steady state and in response to LIF stimulation. Attenuation of ERK signalling by the addition of MAPK/ERK kinase (MEK) inhibitors to SOCS-3-null ES cell cultures rescued the differentiation phenotype, but did not restore proliferation to wild-type levels. In summary, SOCS-3 plays a crucial role in the regulation of the LIF signalling pathway in murine ES cells. Its absence perturbs the balance between activation of the JAK-STAT and SHP-2-ERK-1/2-MAPK pathways, resulting in less self-renewal and a greater potential for differentiation into the primitive endoderm lineage.

Role played by Disabled-2 in albumin induced MAP Kinase signalling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Diwakar, Ramaswamy; Pearson, Alexander L.; Colville-Nash, Paul

2008-02-15

Albumin has been shown to activate the mitogen activated protein kinase (MAPK) pathway in proximal tubular cells (PTECs) of the kidney. Megalin, the putative receptor for albumin has potential signalling properties. However, the mechanisms by which megalin signals are unclear. The adaptor phosphoprotein Disabled-2 (Dab2) is known to interact with the cytoplasmic tail of megalin and may be involved in albumin-mediated MAPK signalling. In this study, we investigated the role of Dab2 in albumin-mediated MAPK signalling and further studied the role of Dab2 in albumin-induced TGF{beta}-1 secretion, a MAPK dependent event. We used RNA interference to knockdown Dab2 protein abundancemore » in HKC-8 cells a model of human PTECs. Albumin activated ERK1,2 and Elk-1 in a MEK-1 dependent manner and resulted in secretion of TGF{beta}-1. In the absence of albumin, knockdown of Dab2 resulted in a trend towards increase in pERK1,2 consistent with its putative role as an inhibitor of cell proliferation. However albumin-induced ERK1,2 activation was completely abolished by Dab2 knockdown. Dab2 knockdown did not however result in inhibition of albumin-induced TGF{beta}-1 secretion. These results suggest that Dab2 is a ligand dependent bi-directional regulator of ERK1,2 activity by demonstrating that in addition to its more traditional role as an inhibitor of ERK1,2 it may also activate ERK1,2.« less

Intracellular Networks of the PI3K/AKT and MAPK Pathways for Regulating Toxoplasma gondii-Induced IL-23 and IL-12 Production in Human THP-1 Cells

PubMed Central

Choi, In-Wook; Ismail, Hassan Ahmed Hassan Ahmed; Zhou, Wei; Cha, Guang-Ho; Zhou, Yu; Yuk, Jae-Min; Jo, Eun-Kyeong; Lee, Young-Ha

2015-01-01

Interleukin (IL)-23 and IL-12 are closely related in structure, and these cytokines regulate both innate and adaptive immunity. However, the precise signaling networks that regulate the production of each in Toxoplasma gondii-infected THP-1 monocytic cells, particularly the PI3K/AKT and MAPK signaling pathways, remain unknown. In the present study, T. gondii infection upregulated the expression of IL-23 and IL-12 in THP-1 cells, and both cytokines increased with parasite dose. IL-23 secretion was strongly inhibited by TLR2 monoclonal antibody (mAb) treatment in a dose-dependent manner and by TLR2 siRNA transfection, whereas IL-12 secretion was strongly inhibited by TLR4 mAb treatment dose-dependently and by TLR4 siRNA transfection. IL-23 production was dose-dependently inhibited by the PI3K inhibitors LY294002 and wortmannin, whereas IL-12 production increased dose-dependently. THP-1 cells exposed to live T. gondii tachyzoites underwent rapid p38 MAPK, ERK1/2 and JNK activation. IL-23 production was significantly upregulated by the p38 MAPK inhibitor SB203580 dose-dependently, whereas pretreatment with 10 μM SB203580 significantly downregulated IL-12 production. ERK1/2 inhibition by PD98059 was significantly downregulated IL-23 production but upregulated IL-12 production. JNK inhibition by SP600125 upregulated IL-23 production, but IL-12 production was significantly downregulated dose-dependently. T. gondii infection resulted in AKT activation, and AKT phosphorylation was inhibited dose-dependently after pretreatment with PI3K inhibitors. In T. gondii-infected THP-1 cells, ERK1/2 activation was regulated by PI3K; however, the phosphorylation of p38 MAPK and JNK was negatively modulated by the PI3K signaling pathway. Collectively, these results indicate that IL-23 production in T. gondii-infected THP-1 cells was regulated mainly by TLR2 and then by PI3K and ERK1/2; however, IL-12 production was mainly regulated by TLR4 and then by p38 MAPK and JNK. Our findings provide new insight concerning the intracellular networks of the PI3K/AKT and MAPK signaling cascades for regulating T. gondii-induced IL-23 and IL-12 secretion in human monocytic cells. PMID:26528819

Mechanism of short-term ERK activation by electromagnetic fields at mobile phone frequencies

PubMed Central

Friedman, Joseph; Kraus, Sarah; Hauptman, Yirmi; Schiff, Yoni; Seger, Rony

2007-01-01

The exposure to non-thermal microwave electromagnetic fields generated by mobile phones affects the expression of many proteins. This effect on transcription and protein stability can be mediated by the MAPK (mitogen-activated protein kinase) cascades, which serve as central signalling pathways and govern essentially all stimulated cellular processes. Indeed, long-term exposure of cells to mobile phone irradiation results in the activation of p38 as well as the ERK (extracellular-signal-regulated kinase) MAPKs. In the present study, we have studied the immediate effect of irradiation on the MAPK cascades, and found that ERKs, but not stress-related MAPKs, are rapidly activated in response to various frequencies and intensities. Using signalling inhibitors, we delineated the mechanism that is involved in this activation. We found that the first step is mediated in the plasma membrane by NADH oxidase, which rapidly generates ROS (reactive oxygen species). These ROS then directly stimulate MMPs (matrix metalloproteinases) and allow them to cleave and release Hb-EGF [heparin-binding EGF (epidermal growth factor)]. This secreted factor activates the EGF receptor, which in turn further activates the ERK cascade. Thus this study demonstrates for the first time a detailed molecular mechanism by which electromagnetic irradiation from mobile phones induces the activation of the ERK cascade and thereby induces transcription and other cellular processes. PMID:17456048

Vitamin D protects endothelial cells from irradiation-induced senescence and apoptosis by modulating MAPK/SirT1 axis.

PubMed

Marampon, F; Gravina, G L; Festuccia, C; Popov, V M; Colapietro, E A; Sanità, P; Musio, D; De Felice, F; Lenzi, A; Jannini, E A; Di Cesare, E; Tombolini, V

2016-04-01

Radiotherapy toxicity is related to oxidative stress-mediated endothelial dysfunction. Here, we investigated on radioprotective properties of Vitamin D (Vit.D) on human endothelial cells (HUVEC). HUVEC, pre-treated with Vit.D, were exposed to ionizing radiation (IR): ROS production, cellular viability, apoptosis, senescence and western blot for protein detection were performed. The role of MAPKs pathway was investigated by using U0126 (10 μM) MEKs/ERKs-, SB203580 (2.5 μM) p38-inhibitor or by over/expressing MKK6 p38-upstream activator. Vit.D reduced IR-induced ROS production protecting proliferating and quiescent HUVEC from cellular apoptosis or senescence, respectively, by regulating MAPKs pathways. In proliferating HUVEC, Vit.D prevented IR-induced apoptosis by activating ERKs while in quiescent HUVEC counteracted IR-induced senescence by inhibiting the p38-IR-induced activation. MEKs&ERKs inhibition in proliferating or MKK6/mediated p38 activation in quiescent HUVEC, respectively, reverted anti-apoptotic or anti-senescent Vit.D properties. SirT1 protein expression levels were up-regulated by Vit.D. ERKs inhibition blocked Vit.D-induced SirT1 protein up-regulation in proliferating cells. In quiescent HUVEC cells, p38 inhibition counteracted the IR-induced SirT1 protein down-regulation, while MKK6 transfection abrogated the Vit.D positive effects on SirT1 protein levels after irradiation. SirT1 inhibition by sirtinol blocked the Vit.D radioprotective effects. Vit.D protects HUVEC from IR induced/oxidative stress by positively regulating the MAPKs/SirT1 axis.

Mitogen activated protein kinase (MAPK) pathway regulates heme oxygenase-1 gene expression by hypoxia in vascular cells.

PubMed

Ryter, Stefan W; Xi, Sichuan; Hartsfield, Cynthia L; Choi, Augustine M K

2002-08-01

Hypoxia induces the stress protein heme oxygenase-1 (HO-1), which participates in cellular adaptation. The molecular pathways that regulate ho-1 gene expression under hypoxia may involve mitogen activated protein kinase (MAPK) signaling and reactive oxygen. Hypoxia (8 h) increased HO-1 mRNA in rat pulmonary aortic endothelial cells (PAEC), and also activated both extracellular signal-regulated kinase 1 (ERK1)/ERK2 and p38 MAPK pathways. The role of these kinases in hypoxia-induced ho-1 gene expression was examined using chemical inhibitors of these pathways. Surprisingly, SB203580, an inhibitor of p38 MAPK, and PD98059, an inhibitor of mitogen-activated protein kinase kinase (MEK1), strongly enhanced hypoxia-induced HO-1 mRNA expression in PAEC. UO126, a MEK1/2 inhibitor, enhanced HO-1 expression in PAEC under normoxia, but not hypoxia. Diphenylene iodonium, an inhibitor of NADPH oxidase, also induced the expression of HO-1 in PAEC under both normoxia and hypoxia. Similar results were observed in aortic vascular smooth muscle cells. Furthermore, hypoxia induced activator protein (AP-1) DNA-binding activity in PAEC. Pretreatment with SB203580 and PD98059 enhanced AP-1 binding activity under hypoxia in PAEC; UO126 stimulated AP-1 binding under normoxia, whereas diphenylene iodonium stimulated AP-1 binding under normoxia and hypoxia. These results suggest a relationship between MAPK and hypoxic regulation of ho-1 in vascular cells, involving AP-1.

Critical role for ERK1/2 in bone marrow and fetal liver–derived primary megakaryocyte differentiation, motility, and proplatelet formation

PubMed Central

Mazharian, Alexandra; Watson, Steve P.; Séverin, Sonia

2009-01-01

Objective Megakaryopoiesis and platelet formation is a multistep process through which hematopoietic progenitor cells develop into mature megakaryocytes (MKs) and form proplatelets. The present study investigates the regulation of different steps of megakaryopoiesis (i.e., differentiation, migration, and proplatelet formation) by extracellar signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) in two models of primary murine MKs derived from bone marrow (BM) cells and fetal liver (FL) cells. Materials and Methods A preparation of MKs was generated from BM obtained from femora and tibiae of C57BL6 mice. FL-derived MKs were obtained from the liver of mouse fetuses aged 13 to 15 days. Results For both cell populations, activation of MEK-ERK1/2 pathway by thrombopoietin was found to have a critical role in MK differentiation, regulating polyploidy and surface expression of CD34, GPIIb, and GPIb. The MEK-ERK1/2 pathway plays a major role in migration of BM-derived MKs toward a stromal-cell−derived factor 1α (SDF1α) gradient, whereas unexpectedly, FL-derived cells fail to migrate in response to the chemokine due to negligible expression of its receptor, CXCR4. The MEK-ERK1/2 pathway also plays a critical role in the generation of proplatelets. In contrast, p38MAPK pathway was not involved in any of these processes. Conclusion This report demonstrates a critical role of MEK-ERK1/2 pathway in MK differentiation, motility, and proplatelet formation. This study highlights several differences between BM- and FL-derived MKs, which are discussed. PMID:19619605

S-Nitrosylation of Ras Mediates Nitric Oxide-Dependent Post-Injury Neurogenesis in a Seizure Model.

PubMed

Santos, Ana Isabel; Carreira, Bruno Pereira; Izquierdo-Álvarez, Alicia; Ramos, Elena; Lourenço, Ana Sofia; Filipa Santos, Daniela; Morte, Maria Inês; Ribeiro, Luís Filipe; Marreiros, Ana; Sánchez-López, Nuria; Marina, Anabel; Carvalho, Caetana Monteiro; Martínez-Ruiz, Antonio; Araújo, Inês Maria

2018-01-01

Nitric oxide (NO) is involved in the upregulation of endogenous neurogenesis in the subventricular zone and in the hippocampus after injury. One of the main neurogenic pathways activated by NO is the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway, downstream of the epidermal growth factor receptor. However, the mechanism by which NO stimulates cell proliferation through activation of the ERK/MAPK pathway remains unknown, although p21Ras seems to be one of the earliest targets of NO. Here, we aimed at studying the possible neurogenic action of NO by post-translational modification of p21Ras as a relevant target for early neurogenic events promoted by NO in neural stem cells (NSCs). We show that NO caused S-nitrosylation (SNO) of p21Ras in Cys118, which triggered downstream activation of the ERK/MAPK pathway and proliferation of NSC. Moreover, in cells overexpressing a mutant Ras in which Cys118 was replaced by a serine-C118S-, cells were insensitive to NO, and no increase in SNO, in ERK phosphorylation, or in cell proliferation was observed. We also show that, after seizures, in the presence of NO derived from inducible nitric oxide synthase, there was an increase in p21Ras cysteine modification that was concomitant with the previously described stimulation of proliferation in the dentate gyrus. Our work identifies p21Ras and its SNO as an early target of NO during signaling events that lead to NSC proliferation and neurogenesis. Our data highlight Ras SNO as an early event leading to NSC proliferation, and they may provide a target for NO-induced stimulation of neurogenesis with implications for brain repair. Antioxid. Redox Signal. 28, 15-30.

Clinical responses to ERK inhibition in BRAFV600E-mutant colorectal cancer predicted using a computational model.

PubMed

Kirouac, Daniel C; Schaefer, Gabriele; Chan, Jocelyn; Merchant, Mark; Orr, Christine; Huang, Shih-Min A; Moffat, John; Liu, Lichuan; Gadkar, Kapil; Ramanujan, Saroja

2017-01-01

Approximately 10% of colorectal cancers harbor BRAF V600E mutations, which constitutively activate the MAPK signaling pathway. We sought to determine whether ERK inhibitor (GDC-0994)-containing regimens may be of clinical benefit to these patients based on data from in vitro (cell line) and in vivo (cell- and patient-derived xenograft) studies of cetuximab (EGFR), vemurafenib (BRAF), cobimetinib (MEK), and GDC-0994 (ERK) combinations. Preclinical data was used to develop a mechanism-based computational model linking cell surface receptor (EGFR) activation, the MAPK signaling pathway, and tumor growth. Clinical predictions of anti-tumor activity were enabled by the use of tumor response data from three Phase 1 clinical trials testing combinations of EGFR, BRAF, and MEK inhibitors. Simulated responses to GDC-0994 monotherapy (overall response rate = 17%) accurately predicted results from a Phase 1 clinical trial regarding the number of responding patients (2/18) and the distribution of tumor size changes ("waterfall plot"). Prospective simulations were then used to evaluate potential drug combinations and predictive biomarkers for increasing responsiveness to MEK/ERK inhibitors in these patients.

Quercetin suppresses the chymotrypsin-like activity of proteasome via inhibition of MEK1/ERK1/2 signaling pathway in hepatocellular carcinoma HepG2 cells.

PubMed

Ding, Youming; Chen, Xiaoyan; Wang, Bin; Yu, Bin; Ge, Jianhui; Shi, Xiaokang

2018-05-01

The proteasomal system is a promising target for cancer treatment. Quercetin (Que), a flavonoid compound with antitumor ability, displays the inhibitory effect on proteasome activity. However, the underlying molecular mechanisms are ill defined. The present study found that Que treatment significantly reduced the chymotrypsin-like protease activity of proteasome whereas the trypsin- and caspase-like protease activities remained unchanged in HepG2 cancer cells, along with activation of p38 MAPK and JNK and reduction of ERK1/2 phosphorylation. Que-reduced proteasome activity could not be reverted by inhibition of p38 MAPK and JNK signaling pathway. In addition, MEK1 overexpression or knockdown upregulated or downregulated the chymotrypsin-like protease activity of proteasome, respectively. Both Que and MEK1/ERK1/2 inhibitor attenuated the expression levels of proteasome β subunits. These results indicate that Que-induced suppression of MEK1/ERK1/2 signaling and subsequent reduction of proteasome β subunits is responsible for its inhibitory impacts on proteasome activity.

TEC protein tyrosine kinase is involved in the Erk signaling pathway induced by HGF

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Feifei; Jiang, Yinan; Zheng, Qiping

Research highlights: {yields} TEC is rapidly tyrosine-phosphorylated and activated by HGF-stimulation in vivo or after partial hepatectomy in mice. {yields} TEC enhances the activity of Elk and serum response element (SRE) in HGF signaling pathway in hepatocyte. {yields} TEC promotes hepatocyte proliferation through the Erk-MAPK pathway. -- Abstract: Background/aims: TEC, a member of the TEC family of non-receptor type protein tyrosine kinases, has recently been suggested to play a role in hepatocyte proliferation and liver regeneration. This study aims to investigate the putative mechanisms of TEC kinase regulation of hepatocyte differentiation, i.e. to explore which signaling pathway TEC is involvedmore » in, and how TEC is activated in hepatocyte after hepatectomy and hepatocyte growth factor (HGF) stimulation. Methods: We performed immunoprecipitation (IP) and immunoblotting (IB) to examine TEC tyrosine phosphorylation after partial hepatectomy in mice and HGF stimulation in WB F-344 hepatic cells. The TEC kinase activity was determined by in vitro kinase assay. Reporter gene assay, antisense oligonucleotide and TEC dominant negative mutant (TEC{sup KM}) were used to examine the possible signaling pathways in which TEC is involved. The cell proliferation rate was evaluated by {sup 3}H-TdR incorporation. Results: TEC phosphorylation and kinase activity were increased in 1 h after hepatectomy or HGF treatment. TEC enhanced the activity of Elk and serum response element (SRE). Inhibition of MEK1 suppressed TEC phosphorylation. Blocking TEC activity dramatically decreased the activation of Erk. Reduced TEC kinase activity also suppressed the proliferation of WB F-344 cells. These results suggest TEC is involved in the Ras-MAPK pathway and acts between MEK1 and Erk. Conclusions: TEC promotes hepatocyte proliferation and regeneration and is involved in HGF-induced Erk signaling pathway.« less

Effect of aromatase inhibitor letrozole on the proliferation of spermatogonia by regulating the MAPK pathway.

PubMed

Wang, Shunde; Wang, Shuhong; Li, Hang; Li, Xiaoxia; Xie, Menglin; Wen, Jiayu; Li, Meicai; Long, Tengbo

2018-06-01

The molecular mechanism of the aromatase inhibitor letrozole was investigated. It promotes the proliferation of spermatogonia by regulating the mitogen-activated protein kinase (MAPK) pathway. Six different concentrations were selected for letrozole in order to incubate mouse spermatogonia [GC-1 spermatogonia (spg)] for 24, 48 and 72 h, respectively. Cell Counting Kit-8 (CCK-8) was used to observe the effect of letrozole on the proliferation of GC-1 spg cells, and the effect was further verified by cell plate clone formation assay. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot analysis were used to detect the effects of letrozole on MAPK signaling pathways [Ras/extracellular signal-regulated kinase 1 (ERK1)/c-Myc], proliferation indexes [Ki-67 and proliferating cell nuclear antigen (PCNA)]. Bromodeoxyuridine (BrdU) staining was used to study the effects of letrozole and MAPK signaling pathways on cell proliferation. The results of CCK-8 showed that the proliferation rate of GC-1 spg cells was improved. Study results also revealed a significant increase in letrozole concentration along with the time of action. The results of plate clone formation assay further indicated that letrozole could significantly promote the proliferation capacity of GC-1 spg cells (p<0.05). The results of RT-PCR and western blot analysis confirmed letrozole significantly activated the expression of Ras/ERK1/c-Myc in the classical MAPK pathway. A significant increase was noted in the protein levels of Ki-67 and PCNA (p<0.05). By contrast, inhibition of the MAPK pathway resulted in a significant decrease in the levels of the above indexes (p<0.05). The number of BrdU cells in the letrozole group was also higher than that of the control group, while the number of BrdU-stained cells in the letrozole + MAPK inhibition group showed a significant decrease in comparison to the letrozole group. In conclusion, letrozole activated the MAPK signaling pathway and promoted the proliferation of mouse spermatogonia GC-1 spg cells. The present study provides a theoretical basis for the clinical application of letrozole.

Ras-sensitive IMP modulation of the Raf/MEK/ERK cascade through KSR1.

PubMed

Matheny, Sharon A; White, Michael A

2006-01-01

The E3 ubiquitin ligase IMP (impedes mitogenic signal propagation) was isolated as a novel Ras effector that negatively regulates ERK1/2 activation. Current evidence suggests that IMP limits the functional assembly of Raf/MEK complexes by inactivation of the KSR1 adaptor/scaffold protein. Interaction with Ras-GTP stimulates IMP autoubiquitination to relieve limitations on KSR function. The elevated sensitivity of IMP-depleted cells to ERK1/2 pathway activation suggests IMP acts as a signal threshold regulator by imposing reversible restrictions on the assembly of functional Raf/MEK/ERK kinase modules. These observations challenge commonly held concepts of signal transmission by Ras to the MAPK pathway and provide evidence for the role of amplitude modulation in tuning cellular responses to ERK1/2 pathway engagement. Here we describe details of the methods, including RNA interference, ubiquitin ligase assays, and protein complex analysis, that can be used to display the Ras-sensitive contribution of IMP to KSR-dependent modulation of the Raf/MEK/ERK pathway.

Curcumin analog EF24 induces apoptosis and downregulates the mitogen activated protein kinase/extracellular signal-regulated signaling pathway in oral squamous cell carcinoma.

PubMed

Lin, Chongxiang; Tu, Chengwei; Ma, Yike; Ye, Pengcheng; Shao, Xia; Yang, Zhaoan; Fang, Yiming

2017-10-01

Oral squamous cell carcinoma (OSCC) is one of the most common malignancies worldwide. Diphenyldifluoroketone (EF24) is a curcumin analog that has been demonstrated to improve anticancer activity; however, its therapeutic potential and mechanisms in oral cancer remain unknown. In the present study, the effect of EF24 on apoptosis induction and its potential underlying mechanism in the CAL‑27 human OSCC cell line was investigated. To achieve this, various concentrations of cisplatin or EF24 were administrated to CAL‑27 cells for 24 h, and cell viability, apoptotic DNA fragmentation, and cleaved caspase 3 and 9 levels were evaluated. To investigate the potential underlying mechanism, the levels of mitogen‑activated protein kinase kinase 1 (MEK1) and extracellular signal‑regulated kinase (ERK), two key proteins in the mitogen‑activated protein kinase/ERK signaling pathway, were additionally examined. The results indicated that EF24 and cisplatin treatment decreased cell viability. EF24 treatment increased the levels of activated caspase 3 and 9, and decreased the phosphorylated forms of MEK1 and ERK. Sequential treatments of EF24 and 12‑phorbol‑13‑myristate acetate, a MAPK/ERK activator, resulted in a significant increase of activated MEK1 and ERK, and reversed cell viability. These results suggested that EF24 has potent anti‑tumor activity in OSCC via deactivation of the MAPK/ERK signaling pathway. Further analyses using animal models are required to confirm these findings in vivo.

Allicin Alleviates Reticuloendotheliosis Virus-Induced Immunosuppression via ERK/Mitogen-Activated Protein Kinase Pathway in Specific Pathogen-Free Chickens

PubMed Central

Wang, Liyuan; Jiao, Hongchao; Zhao, Jingpeng; Wang, Xiaojuan; Sun, Shuhong; Lin, Hai

2017-01-01

Reticuloendotheliosis virus (REV), a gammaretrovirus in the Retroviridae family, causes an immunosuppressive, oncogenic, and runting–stunting syndrome in multiple avian hosts. Allicin, the main effective component of garlic, has a broad spectrum of pharmacological properties. The hypothesis that allicin could relieve REV-induced immune dysfunction was investigated in vivo and in vitro in the present study. The results showed that dietary allicin supplementation ameliorated REV-induced dysplasia and immune dysfunction in REV-infected chickens. Compared with the control groups, REV infection promoted the expression of inflammatory cytokines including interleukin (IL)-1β, IL-6, IL-10, interferon (IFN)-γ, and tumor necrosis factor-α (TNF-α), whereas, allicin reversed these changes induced by REV infection. The decreased levels of IFN-α, IFN-β, and IL-2 were observed in REV-infected chickens, which were significantly improved by allicin. Allicin suppressed the REV-induced high expression of toll-like receptors (TLRs) as well as melanoma differentiation-associated gene 5 (MDA5) and the activation of mitogen-activated protein kinase (MAPK) and the nuclear factor kappa B p65. REV stimulated the phosphorylation of JNK, ERK, and p38, the downstream key signaling molecules of MAPK pathway, while allicin retarded the augmented phosphorylation level induced by REV infection. The decreased phosphorylation level of ERK was associated with REV replication, suggesting that ERK signaling is involved in REV replication, and allicin can alleviate the REV-induced immune dysfunction by inhibiting the activation of ERK. In addition, REV infection induced oxidative damage in thymus and spleen, whereas allicin treatment significantly decreased the oxidative stress induced by REV infection, suggesting that the antioxidant effect of allicin should be at least partially responsible for the harmful effect of REV infection. In conclusion, the findings suggest that allicin alleviates the inflammation and oxidative damage caused by REV infection and exerts the potential anti-REV effect by blocking the ERK/MAPK pathway. PMID:29312337

Caffeine Inhibits the Activation of Hepatic Stellate Cells Induced by Acetaldehyde via Adenosine A2A Receptor Mediated by the cAMP/PKA/SRC/ERK1/2/P38 MAPK Signal Pathway

PubMed Central

Yang, Wanzhi; Wang, Qi; Zhao, Han; Yang, Feng; Lv, Xiongwen; Li, Jun

2014-01-01

Hepatic stellate cell (HSC) activation is an essential event during alcoholic liver fibrosis. Evidence suggests that adenosine aggravates liver fibrosis via the adenosine A2A receptor (A2AR). Caffeine, which is being widely consumed during daily life, inhibits the action of adenosine. In this study, we attempted to validate the hypothesis that caffeine influences acetaldehyde-induced HSC activation by acting on A2AR. Acetaldehyde at 50, 100, 200, and 400 μM significantly increased HSC-T6 cells proliferation, and cell proliferation reached a maximum at 48 h after exposure to 200 μM acetaldehyde. Caffeine and the A2AR antagonist ZM241385 decreased the cell viability and inhibited the expression of procollagen type I and type III in acetaldehyde-induced HSC-T6 cells. In addition, the inhibitory effect of caffeine on the expression of procollagen type I was regulated by A2AR-mediated signal pathway involving cAMP, PKA, SRC, and ERK1/2. Interestingly, caffeine’s inhibitory effect on the expression of procollagen type III may depend upon the A2AR-mediated P38 MAPK-dependent pathway. Conclusions: Caffeine significantly inhibited acetaldehyde-induced HSC-T6 cells activation by distinct A2AR mediated signal pathway via inhibition of cAMP-PKA-SRC-ERK1/2 for procollagen type I and via P38 MAPK for procollagen type III. PMID:24682220

Monocyte 15-Lipoxygenase Gene Expression Requires ERK1/2 MAPK Activity

PubMed Central

Bhattacharjee, Ashish; Mulya, Anny; Pal, Srabani; Roy, Biswajit; Feldman, Gerald M.; Cathcart, Martha K.

2011-01-01

IL-13 induces profound expression of 15-lipoxygenase (15-LO) in primary human monocytes. Our studies have defined the functional IL-13R complex, association of Jaks with the receptor components, and the tyrosine phosphorylation of several Stat molecules in response to IL-13. Furthermore, we identified both p38MAPK and protein kinase Cδ as critical regulators of 15-LO expression. In this study, we report an ERK1/2-dependent signaling cascade that regulates IL-13–mediated 15-LO gene expression. We show the rapid phosphorylation/activation of ERK1/2 upon IL-13 exposure. Our results indicate that Tyk2 kinase is required for the activation of ERK1/2, which is independent of the Jak2, p38MAPK, and protein kinase Cδ pathways, suggesting bifurcating parallel regulatory pathways downstream of the receptor. To investigate the signaling mechanisms associated with the ERK1/2-dependent expression of 15-LO, we explored the involvement of transcription factors, with predicted binding sites in the 15-LO promoter, in this process including Elk1, early growth response-1 (Egr-1), and CREB. Our findings indicate that IL-13 induces Egr-1 nuclear accumulation and CREB serine phosphorylation and that both are markedly attenuated by inhibition of ERK1/2 activity. We further show that ERK1/2 activity is required for both Egr-1 and CREB DNA binding to their cognate sequences identified within the 15-LO promoter. Furthermore, by transfecting monocytes with the decoy oligodeoxyribonucleotides specific for Egr-1 and CREB, we discovered that Egr-1 and CREB are directly involved in regulating 15-LO gene expression. These studies characterize an important regulatory role for ERK1/2 in mediating IL-13–induced monocyte 15-LO expression via the transcription factors Egr-1 and CREB. PMID:20861348

Therapeutical relevance of MAP-kinase inhibitors in renal diseases: current knowledge and future clinical perspectives.

PubMed

Grande, M Teresa; López-Novoa, José M

2008-01-01

Renal failure, both acute and chronic, represents an important health problem by its social, sanitary and economic aspects. Mitogen-activated protein kinases (MAPK) are a family of mediators involved in the transduction of extracellular stimuli to intracellular responses. The best studied members of this family are extracellular signal-regulated kinases 1 and 2 (ERK1 and ERK2), Jun NH(2)-terminal kinase (JNK), p38 kinase and extracellular signal regulated kinases 5 (ERK5) also known as big MAP Kinase 1 (BMK1). MAPKs plays a role in regulating renal function and all these pathways have been demonstrated to be activated in many "in vivo" and cellular models or renal failure. As MAP kinases are key regulators in the control of cell proliferation and cell death, many more or less specific inhibitors of these pathways are being developed for the treatment of tumors. The purpose of this review is to examine the data available on the role of MAPKs activation in "in vivo" models of renal failure, as well as in different renal cell types (especially in mesangial cells, podocytes, tubular epithelial cells and fibroblasts) subjected to stress or damage. We have also reviewed the effect of MAPKs inhibition on renal damage, both "in vivo" and "in vitro". Data collected allow to suggest that therapy of chronic and acute renal disease with MAPKs inhibitors is a promising therapeutic area, although much more basic and clinical studies are necessary before this kind of therapy can be used in the everyday clinic.

Upregulation of MAPK/Erk and PI3K/Akt pathways in ulcerative colitis-associated colon cancer.

PubMed

Setia, Shruti; Nehru, Bimla; Sanyal, Sankar Nath

2014-10-01

An extracellular signal like a cytokine or chemokine, secreted in the inflammatory microenvironment can activate the mitogen activated protein kinase (MAPK) pathway by binding to a cytokine receptor tyrosine kinase, which further activates tyrosine kinases such as Janus Kinase-3 (Jak-3). This signal is transferred from Jak-3 to the DNA in the nucleus of the cell by a chain of kinases, ultimately activating extracellular receptor kinase (Erk/MAPK). The latter phosphorylates c-myc, an oncogene, which alters the levels and activities of many transcription factors leading to cell survival, proliferation and invasion. The oncogenic PI3K pathway plays a similar role by activating c-myc, leading to cell survival and proliferation. The present study explores the role of ulcerative colitis in colon cancer by investigating the activities of tyrosine kinase activated MAPK pathway and various components of the PI3K pathway including PI3K, PTEN, PDK1, GSK3β, Akt, mTOR, Wnt and β-catenin. This was done by western blot and fluorescent immunohistochemical analysis of the above-mentioned proteins. Also, the morphological and histological investigation of the colonic samples from various animal groups revealed significant alterations as compared to the control in both inflammatory as well as carcinogenic conditions. These effects were reduced to a large extent by the co-administration of celecoxib, a second-generation non-steroidal anti-inflammatory drug (NSAID). Copyright © 2014 Elsevier Masson SAS. All rights reserved.

Effects of FGF-23-mediated ERK/MAPK signaling pathway on parathyroid hormone secretion of parathyroid cells in rats with secondary hyperparathyroidism.

PubMed

Chen, Xiao-Jun; Chen, Xiong; Wu, Wen-Jun; Zhou, Qi; Gong, Xiao-Hua; Shi, Bi-Min

2018-04-10

This study is supposed to investigate the effect of FGF-23 on parathyroid hormone (PTH) secretion through ERK/MAPK signaling pathway in secondary hyperparathyroidism (SHPT) rat model. Thirty rats were equally served as the normal and SHPT groups. After transfection, parathyroid cells was assigned into blank, NC, pcDNA3.1-FGF-23, siRNA-FGF-23, U0126, and siRNA-FGF-23 + U0126 groups. The serum levels of Calcium (Ca), Phosphorus (P), alkaline phosphatase (ALP), and PTH were detected. HE and immunohistochemical (IHC) staining were used for the histopathological changes and the FGF-23, EKR1/2, and pEKR1/2 expressions. qRT-PCR and Western blotting were performed to determine the mRNA and protein expression of FGF-23, PTH, MAPK, EKR1/2, and Klotho. The proliferation, apoptosis, and cell cycle were all measured for parathyroid cells by CCK-8 assay, TUNEL staining and Flow cytometry. Compared with the normal group, the SHPT group showed increased serum levels PTH, P, ALP, and FGF-23 and mRNA and protein expressions of FGF-23 and PTH, whereas declined Ca and p-ERK1/2 expression, mRNA and protein expression of Klotho, cell apoptosis rate was reduced. Furthermore, compared to the blank and NC groups, the pcDNA3.1-FGF-23 and U0126 groups had a decreased mRNA expression of Klotho, protein expression of EKR1/2 and Klotho, and cell apoptosis rate was down-regulated, whereas the RNA and protein expressions of FGF-23 and PTH were up-regulated, and cell proliferation was elevated. The opposite results were observed in the siRNA-FGF-23 group. Our study demonstrated that FGF-23 could inhibit signaling transduction of ERK/MAPK pathway and accelerate the secretion of PTH in rats with SHPT. © 2018 Wiley Periodicals, Inc.

Phenotypic and evolutionary implications of modulating the ERK-MAPK cascade using the dentition as a model

PubMed Central

Marangoni, Pauline; Charles, Cyril; Tafforeau, Paul; Laugel-Haushalter, Virginie; Joo, Adriane; Bloch-Zupan, Agnès; Klein, Ophir D.; Viriot, Laurent

2015-01-01

The question of phenotypic convergence across a signalling pathway has important implications for both developmental and evolutionary biology. The ERK-MAPK cascade is known to play a central role in dental development, but the relative roles of its components remain unknown. Here we investigate the diversity of dental phenotypes in Spry2−/−, Spry4−/−, and Rsk2−/Y mice, including the incidence of extra teeth, which were lost in the mouse lineage 45 million years ago (Ma). In addition, Sprouty-specific anomalies mimic a phenotype that is absent in extant mice but present in mouse ancestors prior to 9 Ma. Although the mutant lines studied display convergent phenotypes, each gene has a specific role in tooth number determination and crown patterning. The similarities found between teeth in fossils and mutants highlight the pivotal role of the ERK-MAPK cascade during the evolution of the dentition in rodents. PMID:26123406

Role of Phosphatidylinositol-3 Kinase Pathway in NMDA Preconditioning: Different Mechanisms for Seizures and Hippocampal Neuronal Degeneration Induced by Quinolinic Acid.

PubMed

Constantino, Leandra C; Binder, Luisa B; Vandresen-Filho, Samuel; Viola, Giordano G; Ludka, Fabiana K; Lopes, Mark W; Leal, Rodrigo B; Tasca, Carla I

2018-04-20

N-methyl D-aspartate (NMDA) preconditioning is evoked by the administration of a subtoxic dose of NMDA and is protective against neuronal excitotoxicity. This effect may involve a diversity of targets and cell signaling cascades associated to neuroprotection. Phosphatidylinositol-3 kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases (MAPKs) such as extracellular regulated protein kinase 1/2 (ERK1/2) and p38 MAPK pathways play a major role in neuroprotective mechanisms. However, their involvement in NMDA preconditioning was not yet fully investigated. The present study aimed to evaluate the effect of NMDA preconditioning on PI3K/Akt, ERK1/2, and p38 MAPK pathways in the hippocampus of mice and characterize the involvement of PI3K on NMDA preconditioning-evoked prevention of seizures and hippocampal cell damage induced by quinolinic acid (QA). Thus, mice received wortmannin (a PI3K inhibitor) and 15 min later a subconvulsant dose of NMDA (preconditioning) or saline. After 24 h of this treatment, an intracerebroventricular QA infusion was administered. Phosphorylation levels and total content of Akt, glycogen synthase protein kinase-3β (GSK-3β), ERK1/2, and p38 MAPK were not altered after 24 h of NMDA preconditioning with or without wortmmanin pretreatment. Moreover, after QA administration, behavioral seizures, hippocampal neuronal degeneration, and Akt activation were evaluated. Inhibition of PI3K pathway was effective in abolishing the protective effect of NMDA preconditioning against QA-induced seizures, but did not modify neuronal protection promoted by preconditioning as evaluated by Fluoro-Jade B staining. The study confirms that PI3K participates in the mechanism of protection induced by NMDA preconditioning against QA-induced seizures. Conversely, NMDA preconditioning-evoked protection against neuronal degeneration is not altered by PI3K signaling pathway inhibition. These results point to differential mechanisms regarding protection against a behavioral and cellular manifestation of neural damage.

Astaxanthin Inhibits Acetaldehyde-Induced Cytotoxicity in SH-SY5Y Cells by Modulating Akt/CREB and p38MAPK/ERK Signaling Pathways.

PubMed

Yan, Tingting; Zhao, Yan; Zhang, Xia; Lin, Xiaotong

2016-03-10

Excessive alcohol consumption can lead to brain tissue damage and cognitive dysfunction. Acetaldehyde, the most toxic metabolite of ethanol, mediates the brain tissue damage and cognitive dysfunction induced by chronic excessive alcohol consumption. In this study, the effect of astaxanthin, a marine bioactive compound, on acetaldehyde-induced cytotoxicity was investigated in SH-SY5Y cells. It was found that astaxanthin protected cells from apoptosis by ameliorating the effect of acetaldehyde on the expression of Bcl-2 family proteins, preventing the reduction of anti-apoptotic protein Bcl-2 and the increase of pro-apoptotic protein Bak induced by acetaldehyde. Further analyses showed that astaxanthin treatment inhibited acetaldehyde-induced reduction of the levels of activated Akt and cyclic AMP-responsive element binding protein (CREB). Astaxanthin treatment also prevented acetaldehyde-induced increase of the level of activated p38 mitogen-activated protein kinase (MAPK) and decrease of the level of activated extracellular signal-regulated kinases (ERKs). Activation of Akt/CREB pathway promotes cell survival and is involved in the upregulation of Bcl-2 gene. P38MAPK plays a critical role in apoptotic events while ERKs mediates the inhibition of apoptosis. Thus, astaxanthin may inhibit acetaldehyde-induced apoptosis through promoting the activation of Akt/CREB and ERKs and blocking the activation of p38MAPK. In addition, astaxanthin treatment suppressed the oxidative stress induced by acetaldehyde and restored the antioxidative capacity of SH-SY5Y cells. Therefore, astaxanthin may protect cells against acetaldehyde-induced cytotoxicity through maintaining redox balance and modulating apoptotic and survival signals. The results suggest that astaxanthin treatment may be beneficial for preventing neurotoxicity associated with acetaldehyde and excessive alcohol consumption.

Desmoglein-1/Erbin interaction suppresses ERK activation to support epidermal differentiation

PubMed Central

Harmon, Robert M.; Simpson, Cory L.; Johnson, Jodi L.; Koetsier, Jennifer L.; Dubash, Adi D.; Najor, Nicole A.; Sarig, Ofer; Sprecher, Eli; Green, Kathleen J.

2013-01-01

Genetic disorders of the Ras/MAPK pathway, termed RASopathies, produce numerous abnormalities, including cutaneous keratodermas. The desmosomal cadherin, desmoglein-1 (DSG1), promotes keratinocyte differentiation by attenuating MAPK/ERK signaling and is linked to striate palmoplantar keratoderma (SPPK). This raises the possibility that cutaneous defects associated with SPPK and RASopathies share certain molecular faults. To identify intermediates responsible for executing the inhibition of ERK by DSG1, we conducted a yeast 2-hybrid screen. The screen revealed that Erbin (also known as ERBB2IP), a known ERK regulator, binds DSG1. Erbin silencing disrupted keratinocyte differentiation in culture, mimicking aspects of DSG1 deficiency. Furthermore, ERK inhibition and the induction of differentiation markers by DSG1 required both Erbin and DSG1 domains that participate in binding Erbin. Erbin blocks ERK signaling by interacting with and disrupting Ras-Raf scaffolds mediated by SHOC2, a protein genetically linked to the RASopathy, Noonan-like syndrome with loose anagen hair (NS/LAH). DSG1 overexpression enhanced this inhibitory function, increasing Erbin-SHOC2 interactions and decreasing Ras-SHOC2 interactions. Conversely, analysis of epidermis from DSG1-deficient patients with SPPK demonstrated increased Ras-SHOC2 colocalization and decreased Erbin-SHOC2 colocalization, offering a possible explanation for the observed epidermal defects. These findings suggest a mechanism by which DSG1 and Erbin cooperate to repress MAPK signaling and promote keratinocyte differentiation. PMID:23524970

Sika pilose antler type I collagen promotes BMSC differentiation via the ERK1/2 and p38-MAPK signal pathways.

PubMed

Wang, Yanshuang; Luo, Su; Zhang, Dafang; Qu, Xiaobo; Tan, Yinfeng

2017-12-01

Sika pilose antler type I collagen (SPC-I) have been reported to promote bone marrow mesenchymal stem cell (BMSC) proliferation and differentiation. However, the underlying mechanism is still unclear. This study investigates the molecular mechanisms of SPC-I on the BMSC proliferation and differentiation of osteoblast (OB) in vitro. The primary rat BMSC was cultured and exposed to SPC-I at different concentrations (2.5, 5.0 and 10.0 mg/mL) for 20 days. The effect of SPC-I on the differentiation of BMSCs was evaluated through detecting the activity of alkaline phosphatase (ALP), ALP staining, collagen I (Col-I) content, and calcified nodules. The markers of osteoblastic differentiation were evaluated using RT-PCR and Western-blot analysis. SPC-I treatment (2.5 mg/mL) significantly increased the proliferation of BMSCs (p < 0.01), whereas, SPC-I (5.0 and 10.0 mg/mL) significantly inhibited the proliferation of BMSCs (p < 0.01). SPC-I (2.5 mg/mL) significantly increased ALP activity and Col-I content (p < 0.01), and increased positive cells in ALP staining and the formation of calcified nodules. Additionally, the gene expression of ALP, Col-I, Osteocalcin (OC), Runx2, Osterix (Osx), ERK1/2, BMP2 and p38-MAPK, along with the protein expression of ERK1/2, p-ERK1/2, p-p38 MAPK were markedly increased in the SPC-I (5.0 mg/mL) treatment group (p < 0.01) compared to the control group. SPC-I can induce BMSC differentiation into OBs and enhance the function of osteogenesis through ERK1/2 and p38-MAPK signal transduction pathways and regulating the gene expression of osteogenesis-specific transcription factors.

Resistance to MEK inhibitors: should we co-target upstream?

PubMed

Poulikakos, Poulikos I; Solit, David B

2011-03-29

Aberrant activation of the ERK pathway is common in human tumors. This pathway consists of a three-tiered kinase module [comprising the kinases RAF, mitogen-activated protein kinase (MAPK) kinase (MEK), and extracellular signal-regulated kinase (ERK)] that functions as a negative feedback amplifier to confer robustness and stabilization of pathway output. Because this pathway is frequently dysregulated in human cancers, intense efforts are under way to develop selective inhibitors of the ERK pathway as anticancer drugs. Although promising results have been reported in early trials for inhibitors of RAF or MEK, resistance invariably occurs. Amplification of the upstream oncogenic driver of ERK signaling has been identified as a mechanism for MEK inhibitor resistance in cells with mutant BRAF or KRAS. Increased abundance of the oncogenic driver (either KRAS or BRAF in the appropriate cellular context) in response to prolonged drug treatment results in increased flux through the ERK pathway and restoration of ERK activity above the threshold required for cell growth. For patients with BRAF mutant tumors, the results suggest that the addition of a RAF inhibitor to a MEK inhibitor may delay or overcome drug resistance. The data thus provide a mechanistic basis for ongoing trials testing concurrent treatment with RAF and MEK inhibitors.

Naja nigricollis CMS-9 enhances the mitochondria-mediated death pathway in adaphostin-treated human leukaemia U937 cells.

PubMed

Chen, Ying-Jung; Wang, Jeh-Jeng; Chang, Long-Sen

2011-11-01

1. The aim of the present study was to explore the effect of the Naja nigricollis phospholipase A(2) CMS-9 on adaphostin-induced death of human leukaemia U937 cells. 2. Leukaemia U937 cells (Bcr/Abl-negative cells) were treated with adaphostin (0-10 μmol/L) and CMS-9 (0-1 μmol/L). The effects of CMS-9, adaphostin and their combination on cell viability, the generation reactive oxygen species (ROS), [Ca(2+) ](i) , p38 mitogen-activated protein kinase (MAPK) activation, Akt and extracellular signal-regulated kinase (ERK) inactivation, mitochondrial membrane potential (ΔΨ(m) ) and Bcl-2 family proteins were analysed. 3. Both adaphostin and CMS-9 induced U937 cell apoptosis, characterized by dissipation of ΔΨ(m) and ROS generation. Combined treatment further increased ΔΨ(m) loss and reduced the viability of adaphostin-treated cells. Unlike in CMS-9-treated cells, in adaphostin-treated cells ROS-induced increases in [Ca(2+) ](i) were observed. CMS-9-induced ROS generation resulted in p38 MAPK activation, whereas adaphostin treatment elicited ROS/Ca(2+) -mediated inactivation of Akt and ERK. Moreover, Akt was found to be involved in ERK phosphorylation. Suppression of p38 MAPK activation blocked CMS-9-induced ΔΨ(m) loss and Bcl-xL downregulation. Overexpression of constitutively active Akt and mitogen-activated protein kinase kinase (MEK) 1 rescued adaphostin-induced ΔΨ(m) loss and Bcl-2 downregulation. Similarly, CMS-9 augmented adaphostin toxicity in human leukaemia K562 cells via increased mitochondrial alterations. 4. The results suggest that two distinct pathways mediate adaphostin- and CMS-9-induced mitochondrial damage (i.e. the ROS-Ca(2+) -Akt-ERK and ROS-p38 MAPK pathways, respectively). These distinct pathway explain the augmentation by CMS-9 of ΔΨ(m) loss and apoptosis in adaphostin-treated U937 cells. © 2011 The Authors. Clinical and Experimental Pharmacology and Physiology © 2011 Blackwell Publishing Asia Pty Ltd.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Seung J.; Kim, Chae E.; Yun, Mi R.

Exaggerated levels of 4-hydroxynonenal (HNE) and 5-lipoxygenase (5-LO) co-exist in macrophages in atherosclerotic lesions, and activated macrophages produce MMP-9 that degrades atherosclerotic plaque constituents. This study investigated the effects of HNE on MMP-9 production, and the potential role for 5-LO derivatives in MMP-9 production in murine macrophages. Stimulation of J774A.1 cells with HNE led to activation of 5-LO, as measured by leukotriene B{sub 4} (LTB{sub 4}) production. This was associated with an increased production of MMP-9, which was blunted by inhibition of 5-LO with MK886, a 5-LO inhibitor or with 5-LO siRNA. A cysteinyl-LT{sub 1} (cysLT{sub 1}) receptor antagonist, REV-5901more » as well as a BLT{sub 1} receptor antagonist, U-75302, also attenuated MMP-9 production induced by HNE. Furthermore, LTB{sub 4} and cysLT (LTC{sub 4} and LTD{sub 4}) enhanced MMP-9 production in macrophages, suggesting a pivotal role for 5-LO in HNE-mediated production of MMP-9. Among the MAPK pathways, LTB{sub 4} and cysLT enhanced phosphorylation of ERK and p38 MAPK, but not JNK. Linked to these results, a p38 MAPK inhibitor as well as an ERK inhibitor blunted MMP-9 production induced by LT. Collectively, these data suggest that 5-LO-derived LT mediates HNE-induced MMP-9 production via activation of ERK and p38 MAPK pathways, consequently leading to plaque instability in atherosclerosis.« less

Helicobacter pylori neutrophil-activating protein induces release of histamine and interleukin-6 through G protein-mediated MAPKs and PI3K/Akt pathways in HMC-1 cells.

PubMed

Tsai, Chung-Che; Kuo, Ting-Yu; Hong, Zhi-Wei; Yeh, Ying-Chieh; Shih, Kuo-Shun; Du, Shin-Yi; Fu, Hua-Wen

2015-01-01

Helicobacter pylori neutrophil-activating protein (HP-NAP) activates several innate leukocytes including neutrophils, monocytes, and mast cells. It has been reported that HP-NAP induces degranulation and interleukin-6 (IL-6) secretion of rat peritoneal mast cells. However, the molecular mechanism is not very clear. Here, we show that HP-NAP activates human mast cell line-1 (HMC-1) cells to secrete histamine and IL-6. The secretion depends on pertussis toxin (PTX)-sensitive heterotrimeric G proteins but not on Toll-like receptor 2. Moreover, HP-NAP induces PTX-sensitive G protein-mediated activation of extracellular signal-regulated kinase 1/2 (ERK1/2), p38-mitogen-activated protein kinase (p38 MAPK), and Akt in HMC-1 cells. Inhibition of ERK1/2, p38 MAPK, or phosphatidylinositol 3-kinase (PI3K) suppresses HP-NAP-induced release of histamine and IL-6 from HMC-1 cells. Thus, the activation of HMC-1 cells by HP-NAP is through Gi-linked G protein-coupled receptor-mediated MAPKs and PI3K/Akt pathways.

The Asian-American variant of human papillomavirus type 16 exhibits higher activation of MAPK and PI3K/AKT signaling pathways, transformation, migration and invasion of primary human keratinocytes.

PubMed

Hochmann, Jimena; Sobrinho, João S; Villa, Luisa L; Sichero, Laura

2016-05-01

Asian-American (AA) HPV-16 variants are associated with higher risk of cancer. Abnormal activation of intracellular signaling play a critical role in cancer development and progression. Our aim was to elucidate mechanisms underlying the higher oncogenic potential attributed to AA variant. We evaluated activation of MAPK and PI3K/AKT pathways in primary human keratinocytes (PHKs) transduced with E6/E7 of three HPV-16 variants: E-P, AA, E-350G. Phenotypes examined included migration, anchorage independent growth and invasion. AA PHKs presented the highest levels of active proteins involved in all cascades analyzed: MAPK-ERK, MAPK-p38 and PI3K-AKT. AA PHKs were more efficient in promoting anchorage independent growth, and in stimulating cell migration and invasion. MEK1 inhibition decreased migration. The mesenchymal phenotype marker vimentin was increased in AA PHKs. Our results suggest that MEK1, ERK2, AKT2 hyperactivation influence cellular behavior by means of GSK-3b inactivation and EMT induction prompting AA immortalized PHKs to more efficiently surpass carcinogenesis steps. Copyright © 2016 Elsevier Inc. All rights reserved.

Ischemic time impacts biological integrity of phospho-proteins in PI3K/Akt, Erk/MAPK, and p38 MAPK signaling networks.

PubMed

Holzer, Timothy R; Fulford, Angie D; Arkins, Austin M; Grondin, Janet M; Mundy, Christopher W; Nasir, Aejaz; Schade, Andrew E

2011-06-01

Post-translational modifications of proteins, such as phosphorylation, are labile events dynamically regulated by opposing kinase and phosphatase activities. Preanalytical factors, such as ischemic time before fixation, affect these activities and can have a significant impact on the ability to elucidate signaling pathways in tissue. Immunohistochemical analysis of phosphorylated proteins involved in PI3K/Akt, Erk/MAPK, and p38 MAPK signaling networks was performed in human cell line xenografts from lung, brain, ovary, and prostate tumors. In order to replicate real-world practices, the tissues were subjected to ischemic times of 0 (baseline), 1, 4, and 24 hours before fixation in formalin. Two key concepts emerge from this analysis: (1) the stability of different phospho-epitopes within a given tumor type is variable (e.g. phospho-PRAS40 is more labile than phospho-S6 ribosomal protein) and (2) the stability of a given phospho-epitope (e.g. phospho-MAPKAPK2) varies significantly across different tumor types. These results highlight the importance of proper tissue acquisition and rapid fixation to preserve the biological integrity of signal transduction pathways that may guide therapeutic decision making.

Active lipids of Ganoderma lucidum spores-induced apoptosis in human leukemia THP-1 cells via MAPK and PI3K pathways.

PubMed

Wang, Jia-He; Zhou, Yi-Jun; Zhang, Meng; Kan, Liang; He, Ping

2012-01-31

Ganoderma lucidum (Lingzhi) is traditionally drug, which has been traditionally effective used in the treatment of chronic hepatopathy, hypertension, hyperglycemia and cancer. THP-1 and HL-60 apoptosis induced by active lipids of Ganoderma lucidum spores was quantified by flow cytometry using FITC-conjugated annexin V and PI; MAPK and Akt were measured by Western blot, and caspase-3, -8 and -9 activities were also detected by spectrophotometric assay. Our results showed that active lipids of Ganoderma lucidum spores decreased phosphorylation-ERK1/2 (P-ERK1/2), P-Akt and increased P-JNK1/2, but did not affect expressions of P-p38 MAPK in THP-1 cells. Moreover, treatment of THP-1 cells with active lipids of Ganoderma lucidum spores resulted in activation of caspase-3, -8 and -9. Furthermore, LY294002 (Akt inhibitor) or PD98059 (ERK1/2 inhibitor) significantly enhanced active lipids of Ganoderma lucidum spores-induced apoptosis in THP-1 cells, whereas caspase inhibitors or SP600125 (JNK inhibitor), decreased apoptosis in THP-1 cells. Taken together, our study for the first time suggests that active lipids of Ganoderma lucidum spores is able to enhance apoptosis in THP-1 cells, at least in part, through inhibition of ERK1/2, Akt and activation of JNK1/2 signaling pathways. Moreover, it also triggers caspase-3, -8 and -9 activation mediated apoptotic induction. Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.

p38 mitogen-activated protein kinase (MAPK) first regulates filamentous actin at the 8-16-cell stage during preimplantation development.

PubMed

Paliga, Andrew J M; Natale, David R; Watson, Andrew J

2005-08-01

The MAPK (mitogen-activated protein kinase) superfamily of proteins consists of four separate signalling cascades: the c-Jun N-terminal kinase or stress-activated protein kinases (JNK/SAPK); the ERKs (extracellular-signal-regulated kinases); the ERK5 or big MAPK1; and the p38 MAPK group of protein kinases, all of which are highly conserved. To date, our studies have focused on defining the role of the p38 MAPK pathway during preimplantation development. p38 MAPK regulates actin filament formation through the downstream kinases MAPKAPK2/3 (MAPK-activated protein kinase 2/3) or MAPKAPK5 [PRAK (p38 regulated/activated kinase)] and subsequently through HSP25/27 (heat-shock protein 25/27). We recently reported that 2-cell-stage murine embryos treated with cytokine-suppressive anti-inflammatory drugs (CSAIDtrade mark; SB203580 and SB220025) display a reversible blockade of development at the 8-16-cell stage, indicating that p38 (MAPK) activity is required to complete murine preimplantation development. In the present study, we have investigated the stage-specific action and role of p38 MAPK in regulating filamentous actin during murine preimplantation development. Treatment of 8-cell-stage embryos with SB203580 and SB220025 (CSAIDtrade mark) resulted in a blockade of preimplantation development, loss of rhodamine phalloidin fluorescence, MK-p (phosphorylated MAPKAPK2/3), HSP-p (phosphorylated HSP25/27) and a redistribution of alpha-catenin immunofluorescence by 12 h of treatment. In contrast, treatment of 2- and 4-cell-stage embryos with CSAIDtrade mark drugs resulted in a loss of MK-p and HSP-p, but did not result in a loss of rhodamine phalloidin fluorescence. All these effects of p38 MAPK inhibition were reversed upon removal of the inhibitor, and development resumed in a delayed but normal manner to the blastocyst stage. Treatment of 8-cell embryos with PD098059 (ERK pathway inhibitor) did not affect development or fluorescence of MK-p, HSP-p or rhodamine phalloidin. Murine preimplantation development becomes dependent on p38 MAPK at the 8-16-cell stage, which corresponds to the stage when p38 MAPK first regulates filamentous actin during early development.

γ-Oryzanol suppresses COX-2 expression by inhibiting reactive oxygen species-mediated Erk1/2 and Egr-1 signaling in LPS-stimulated RAW264.7 macrophages.

PubMed

Shin, Soon Young; Kim, Heon-Woong; Jang, Hwan-Hee; Hwang, Yu-Jin; Choe, Jeong-Sook; Kim, Jung-Bong; Lim, Yoongho; Lee, Young Han

2017-09-16

Cyclooxygenase (COX)-2 produces prostanoids, which contribute to inflammatory responses. Nuclear factor (NF)-κB is a key transcription factor mediating COX-2 expression. γ-Oryzanol is an active component in rice bran oil, which inhibits lipopolysaccharide (LPS)-mediated COX-2 expression by inhibiting NF-κB. However, the inhibition of COX-2 expression by γ-oryzanol independently of NF-κB is poorly understood. We found that LPS upregulated Egr-1 expression at the transcriptional level. Forced expression of Egr-1 trans-activated the Cox-2 promoter independently of NF-κB. In contrast, silencing of Egr-1 abrogated LPS-mediated COX-2 expression. LPS produced reactive oxygen species (ROS), which, in turn, induced Egr-1 expression via the Erk1/2 MAPK pathway. ROS scavenging activity of γ-oryzanol suppressed Egr-1 expression by inhibiting the Erk1/2 MAPK pathway. Our results suggest that γ-oryzanol inhibits LPS-mediated COX-2 expression by suppressing Erk1/2-mediated Egr-1 expression. This study supports that γ-oryzanol may be useful for ameliorating LPS-mediated inflammatory responses. Copyright © 2017 Elsevier Inc. All rights reserved.

Endoplasmic reticulum stress and MAPK signaling pathway activation underlie leflunomide-induced toxicity in HepG2 Cells

PubMed Central

Ren, Zhen; Chen, Si; Qing, Tao; Xuan, Jiekun; Couch, Letha; Yu, Dianke; Ning, Baitang; Shi, Leming; Guo, Lei

2017-01-01

Leflunomide, used for the treatment of rheumatoid arthritis, has been reported to cause severe liver problems and liver failure; however, the underlying mechanisms are not clear. In this study, we used multiple approaches including genomic analysis to investigate and characterize the possible molecular mechanisms of the cytotoxicity of leflunomide in hepatic cells. We found that leflunomide caused endoplasmic reticulum (ER) stress and activated an unfolded protein response, as evidenced by increased expression of related genes including CHOP and GADD34; and elevated protein levels of typical ER stress markers including CHOP, ATF-4, p-eIF2α, and spliced XBP1. The secretion of Gaussia luciferase was suppressed in cells treated with leflunomide in an ER stress reporter assay. Inhibition of ER stress with an ER stress inhibitor 4-phenylbutyrate, and knockdown of ATF-4 and CHOP genes partially protected cells upon leflunomide exposure. In addition, both genomic and biochemical analyses revealed that JNK and ERK1/2 of MAPK signaling pathways were activated, and both contributed to the leflunomide-induced cytotoxicity. Inhibiting JNK activation using a JNK inhibitor attenuated the ER stress and cytotoxicity of leflunomide, whereas inhibiting ERK1/2 using an ERK1/2 inhibitor or ERK1/2 siRNA increased the adverse effect caused by leflunomide, suggesting opposite roles for the two pathways. In summary, our data indicate that both ER stress and the activation of JNK and ERK1/2 contribute to leflunomide-induced cytotoxicity. PMID:28988120

Involvement of PI3K/AKT and MAPK Pathways for TNF-α Production in SiHa Cervical Mucosal Epithelial Cells Infected with Trichomonas vaginalis.

PubMed

Yang, Jung-Bo; Quan, Juan-Hua; Kim, Ye-Eun; Rhee, Yun-Ee; Kang, Byung-Hyun; Choi, In-Wook; Cha, Guang-Ho; Yuk, Jae-Min; Lee, Young-Ha

2015-08-01

Trichomonas vaginalis; induces proinflammation in cervicovaginal mucosal epithelium. To investigate the signaling pathways in TNF-α production in cervical mucosal epithelium after T. vaginalis infection, the phosphorylation of PI3K/AKT and MAPK pathways were evaluated in T. vaginalis-infected SiHa cells in the presence and absence of specific inhibitors. T. vaginalis increased TNF-α production in SiHa cells, in a parasite burden-dependent and incubation time-dependent manner. In T. vaginalis-infected SiHa cells, AKT, ERK1/2, p38 MAPK, and JNK were phosphorylated from 1 hr after infection; however, the phosphorylation patterns were different from each other. After pretreatment with inhibitors of the PI3K/AKT and MAPK pathways, TNF-α production was significantly decreased compared to the control; however, TNF-α reduction patterns were different depending on the type of PI3K/MAPK inhibitors. TNF-α production was reduced in a dose-dependent manner by treatment with wortmannin and PD98059, whereas it was increased by SP600125. These data suggested that PI3K/AKT and MAPK signaling pathways are important in regulation of TNF-α production in cervical mucosal epithelial SiHa cells. However, activation patterns of each pathway were different from the types of PI3K/MAPK pathways.

The activation of p38 MAPK limits the abnormal proliferation of vascular smooth muscle cells induced by high sodium concentrations

PubMed Central

WU, YAN; ZHOU, JUAN; WANG, HUAN; WU, YUE; GAO, QIYUE; WANG, LIJUN; ZHAO, QIANG; LIU, PEINING; GAO, SHANSHAN; WEN, WEN; ZHANG, WEIPING; LIU, YAN; YUAN, ZUYI

2016-01-01

The aim of the present study was to ascertain whether high sodium levels can directly promote the proliferation of vascular smooth muscle cells (VSMCs) and to elucidate the underlying mechanisms. Additional sodium chloride (NaCl) was added to the routine culture medium. Cell proliferation was evaluated by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and 5-ethynyl-2′-deoxyuridine (EdU) incorporation assay. The mRNA expression level of proliferating cell nuclear antigen (PCNA) was measured by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The protein expression levels of PCNA and phosphorylated c-Jun amino N-terminal kinase (p-JNK), phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and phosphorylated p38 mitogen-activated protein kinase (p-p38 MAPK) were measured by western blot analysis. Cell proliferation assay revealed that Na+ rather than Cl− or osmotic pressure promoted the proliferation of the VSMCs. The high sodium level upregulated the expression of PCNA and the phosphorylation levels of JNK, ERK1/2 and p38 MAPK. The inhibition of JNK and ERK1/2 decreased PCNA expression. Of note, the inhibition of p38 MAPK using the inhibitor, SB203580, increased PCNA expression. However, when p38 MAPK was activated by anisomycin, PCNA expression was decreased. On the whole, our findings demonstrate that a relatively high sodium level per se directly promotes the proliferation of VSMCs through the JNK/ERK1/2/PCNA pathway. At the same time, this induction of the proliferation of VSMCs due to high sodium levels can be maintained at a low level via the activation of p38 MAPK. PMID:26530729

Primate Torpor: Regulation of Stress-activated Protein Kinases During Daily Torpor in the Gray Mouse Lemur, Microcebus murinus.

PubMed

Biggar, Kyle K; Wu, Cheng-Wei; Tessier, Shannon N; Zhang, Jing; Pifferi, Fabien; Perret, Martine; Storey, Kenneth B

2015-04-01

Very few selected species of primates are known to be capable of entering torpor. This exciting discovery means that the ability to enter a natural state of dormancy is an ancestral trait among primates and, in phylogenetic terms, is very close to the human lineage. To explore the regulatory mechanisms that underlie primate torpor, we analyzed signal transduction cascades to discover those involved in coordinating tissue responses during torpor. The responses of mitogen-activated protein kinase (MAPK) family members to primate torpor were compared in six organs of control (aroused) versus torpid gray mouse lemurs, Microcebus murinus. The proteins examined include extracellular signal-regulated kinases (ERKs), c-jun NH2-terminal kinases (JNKs), MAPK kinase (MEK), and p38, in addition to stress-related proteins p53 and heat shock protein 27 (HSP27). The activation of specific MAPK signal transduction pathways may provide a mechanism to regulate the expression of torpor-responsive genes or the regulation of selected downstream cellular processes. In response to torpor, each MAPK subfamily responded differently during torpor and each showed organ-specific patterns of response. For example, skeletal muscle displayed elevated relative phosphorylation of ERK1/2 during torpor. Interestingly, adipose tissues showed the highest degree of MAPK activation. Brown adipose tissue displayed an activation of ERK1/2 and p38, whereas white adipose tissue showed activation of ERK1/2, p38, MEK, and JNK during torpor. Importantly, both adipose tissues possess specialized functions that are critical for torpor, with brown adipose required for non-shivering thermogenesis and white adipose utilized as the primary source of lipid fuel for torpor. Overall, these data indicate crucial roles of MAPKs in the regulation of primate organs during torpor. Copyright © 2015. Production and hosting by Elsevier Ltd.

Anti-cancer effects of CME-1, a novel polysaccharide, purified from the mycelia of Cordyceps sinensis against B16-F10 melanoma cells.

PubMed

Jayakumar, Thanasekaran; Chiu, Chong-Chi; Wang, Shwu-Huey; Chou, Duen-Suey; Huang, Yung-Kai; Sheu, Joen-Rong

2014-01-01

Matrix metalloproteinases (MMPs) play important roles in the invasion and migration of cancer cells. In melanoma, several signaling pathways are constitutively activated. Among these, the mitogen-activated protein kinase (MAPKs) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Therefore, the inhibition of MAPK signaling might be a crucial role for the treatment of melanoma cancer. We examined the anticancer effect of CME-1, a novel water-soluble polysaccharide fraction, isolated from Cordyceps sinensis mycelia on B16-F10 melanoma cells. B16-F10 cells were exposed to different concentrations of CME-1 (250, 500 and 800 μg/ml) for 24 h in 5% CO² incubator at 37°C. Western blot analysis was performed to detect the expression of MMP-1, p-p38 MAPK, p-ERK1/2, and IkB-α in B16-F10 cells. Cell migration test was performed by wound healing migration assay. CME-1 suppresses cell migration in a concentration-dependent manner. Western blotting analysis revealed that CME-1 led to the reduction on the expression levels of MMP-1 and down regulated the expression of phosphorylated extracellular signal-regulated kinase (ERK1/2 and p38 mitogen-activated protein kinase (p38 MAPK). CME-1 restored the IkB-degradation in B16F10 cells. These results indicate that CME-1 inhibited MMP-1 expressions in B16F10 melanoma cells through either NF-kB or ERK/p38 MAPK down regulation thereby inhibiting B16F10 cell migration. Therefore, we proposed that CME-1 might be developed as a therapeutic potential candidate for the treatment of cancer metastasis.

Isoproterenol Acts as a Biased Agonist of the Alpha-1A-Adrenoceptor that Selectively Activates the MAPK/ERK Pathway

PubMed Central

Copik, Alicja. J.; Baldys, Aleksander; Nguyen, Khanh; Sahdeo, Sunil; Ho, Hoangdung; Kosaka, Alan; Dietrich, Paul J.; Fitch, Bill; Raymond, John R.; Ford, Anthony P. D. W.; Button, Donald; Milla, Marcos E.

2015-01-01

The α1A-AR is thought to couple predominantly to the Gαq/PLC pathway and lead to phosphoinositide hydrolysis and calcium mobilization, although certain agonists acting at this receptor have been reported to trigger activation of arachidonic acid formation and MAPK pathways. For several G protein-coupled receptors (GPCRs) agonists can manifest a bias for activation of particular effector signaling output, i.e. not all agonists of a given GPCR generate responses through utilization of the same signaling cascade(s). Previous work with Gαq coupling-defective variants of α1A-AR, as well as a combination of Ca2+ channel blockers, uncovered cross-talk between α1A-AR and β2-AR that leads to potentiation of a Gαq-independent signaling cascade in response to α1A-AR activation. We hypothesized that molecules exist that act as biased agonists to selectively activate this pathway. In this report, isoproterenol (Iso), typically viewed as β-AR-selective agonist, was examined with respect to activation of α1A-AR. α1A-AR selective antagonists were used to specifically block Iso evoked signaling in different cellular backgrounds and confirm its action at α1A-AR. Iso induced signaling at α1A-AR was further interrogated by probing steps along the Gαq /PLC, Gαs and MAPK/ERK pathways. In HEK-293/EBNA cells transiently transduced with α1A-AR, and CHO_α1A-AR stable cells, Iso evoked low potency ERK activity as well as Ca2+ mobilization that could be blocked by α1A-AR selective antagonists. The kinetics of Iso induced Ca2+ transients differed from typical Gαq- mediated Ca2+ mobilization, lacking both the fast IP3R mediated response and the sustained phase of Ca2+ re-entry. Moreover, no inositol phosphate (IP) accumulation could be detected in either cell line after stimulation with Iso, but activation was accompanied by receptor internalization. Data are presented that indicate that Iso represents a novel type of α1A-AR partial agonist with signaling bias toward MAPK/ERK signaling cascade that is likely independent of coupling to Gαq. PMID:25606852

Coal-induced interleukin-6 gene expression is mediated through ERKs and p38 MAPK pathways.

PubMed

Huang, X; Zhang, Q

2003-08-15

In the present study, we have tested the ability of coal dust to stimulate kinase phosphorylation of activator protein-1 (AP-1) signal transduction pathways and production of interleukin-6 (IL-6) in both mouse epidermal JB6 and human lung epithelial A549 cells. Seven coal samples from three coalmine regions of Pennsylvania (PA), West Virginia (WV), and Utah (UT) with high, medium, and low prevalence of coal workers' pneumoconiosis (CWP), respectively, were investigated. Results from the present study indicate that three PA coals stimulated the mitogen-activated protein kinase (MAPK) family of extracellular signal-regulated kinases (ERKs) and p38 MAPK, but not c-Jun-NH2-terminal kinases (JNKs) in human lung A549 cells. The effects of three UT coals on the kinase phosphorylation were less as compared to those of the PA coals. Coal dusts from three coalmine regions induced IL-6 in a dose-dependent manner in both JB6 and A549 cells. Interestingly, levels of IL-6 in both cells treated with coals from three coalmine regions correlated well with CWP prevalence from that region. To assess the role of AP-1 pathways in coal-mediated transcriptional activation of IL-6, various inhibitors were used in cells treated with one PA coal, which induced a maximal response. It was found that the increase in IL-6 protein and mRNA by the PA coal was completely eliminated by the pretreatment of both cell types with PD98059, a specific MEK1 inhibitor, and SB202190, a p38 kinase inhibitor. Our results indicate that coal dust can stimulate IL-6 release from mouse epidermal JB6 cells and human lung epithelial A549 cells, and the coal-induced IL-6 increase may involve ERKs and p38 MAPK pathways.

PP2B and ERK1/2 regulate hyaluronan synthesis of HT168 and WM35 human melanoma cell lines.

PubMed

Katona, Éva; Juhász, Tamás; Somogyi, Csilla Szűcs; Hajdú, Tibor; Szász, Csaba; Rácz, Kálmán; Kókai, Endre; Gergely, Pál; Zákány, Róza

2016-03-01

Hyaluronan (HA) is the major glycosaminoglycan component of the extracellular matrix in either normal or malignant tissues and it may affect proliferation, motility and differentiation of various cell types. Three isoforms of plasma membrane-bound hyaluronan synthases (HAS 1, 2 and 3) secrete and simultaneously bind pericellular HA. HAS enzymes are subjects of post-translational protein phosphorylation which is believed to regulate their enzymatic activity. In this study, we investigated the HA homeostasis of normal human epidermal melanocytes, HT168 and WM35 human melanoma cell lines and melanoma metastases. HAS2 and HAS3 were detected in all the samples, while the expression of HAS1 was not detectable in any case. Malignant tissue samples and melanoma cell lines contained extra- and intracellular HA abundantly but not normal melanocytes. Applying HA as a chemoattractant facilitated the migration of melanoma cells in Boyden chamber. The amount of HA was reduced upon the inhibition of calcineurin with cyclosporine A (CsA), while the inhibition of ERK1/2 with PD098059 elevated it in both cell lines. The signals of Ser/Thr phosphoproteins at 57 kD were stronger after CsA treatment, while a markedly weaker signal was detected upon inhibition of the MAPK pathway. Our results suggest opposing effects of the two investigated enzymes on the HA homeostasis of melanoma cells. We propose that the dephosphorylation of HAS enzymes targeted by PP2B augments HA production, while their phosphorylation by the activity of MAPK pathway reduces HA synthesis. As the expression of the HA receptor RHAMM was also significantly enhanced by PD098059, the MAPK pathway exerted a complex attenuating effect on HA signalling in the investigated melanoma cells. This observation suggests that the application of MAPK-ERK pathway inhibitors requires a careful therapeutic design in melanoma treatment.

PP2B and ERK1/2 regulate hyaluronan synthesis of HT168 and WM35 human melanoma cell lines

PubMed Central

KATONA, ÉVA; JUHÁSZ, TAMÁS; SOMOGYI, CSILLA SZŰCS; HAJDÚ, TIBOR; SZÁSZ, CSABA; RÁCZ, KÁLMÁN; KÓKAI, ENDRE; GERGELY, PÁL; ZÁKÁNY, RÓZA

2016-01-01

Hyaluronan (HA) is the major glycosaminoglycan component of the extracellular matrix in either normal or malignant tissues and it may affect proliferation, motility and differentiation of various cell types. Three isoforms of plasma membrane-bound hyaluronan synthases (HAS 1, 2 and 3) secrete and simultaneously bind pericellular HA. HAS enzymes are subjects of post-translational protein phosphorylation which is believed to regulate their enzymatic activity. In this study, we investigated the HA homeostasis of normal human epidermal melanocytes, HT168 and WM35 human melanoma cell lines and melanoma metastases. HAS2 and HAS3 were detected in all the samples, while the expression of HAS1 was not detectable in any case. Malignant tissue samples and melanoma cell lines contained extra- and intracellular HA abundantly but not normal melanocytes. Applying HA as a chemoattractant facilitated the migration of melanoma cells in Boyden chamber. The amount of HA was reduced upon the inhibition of calcineurin with cyclosporine A (CsA), while the inhibition of ERK1/2 with PD098059 elevated it in both cell lines. The signals of Ser/Thr phosphoproteins at 57 kD were stronger after CsA treatment, while a markedly weaker signal was detected upon inhibition of the MAPK pathway. Our results suggest opposing effects of the two investigated enzymes on the HA homeostasis of melanoma cells. We propose that the dephosphorylation of HAS enzymes targeted by PP2B augments HA production, while their phosphorylation by the activity of MAPK pathway reduces HA synthesis. As the expression of the HA receptor RHAMM was also significantly enhanced by PD098059, the MAPK pathway exerted a complex attenuating effect on HA signalling in the investigated melanoma cells. This observation suggests that the application of MAPK-ERK pathway inhibitors requires a careful therapeutic design in melanoma treatment. PMID:26717964

Evaluation of the Genetic Response of U937 and Jurkat Cells to 10-Nanosecond Electrical Pulses (nsEP)

DTIC Science & Technology

2016-05-02

signal-regulated kinase (Erk), heat shock 27kDa protein 1 ( HSP27 ), c-Jun N-terminal kinase (JNK), jun proto-oncogene (c-Jun), dual specificity mitogen...the MAPK pathway-associated proteins were significantly increased (Fig 5D). These included ERK1, JNK, ATF2, HSP27 , c-JUN, and p53. At 12 h post

Tanreqing Injection Attenuates Lipopolysaccharide-Induced Airway Inflammation through MAPK/NF-κB Signaling Pathways in Rats Model

PubMed Central

Liu, Wei; Jiang, Hong-li; Cai, Lin-li; Yan, Min; Dong, Shou-jin; Mao, Bing

2016-01-01

Background. Tanreqing injection (TRQ) is a commonly used herbal patent medicine for treating inflammatory airway diseases in view of its outstanding anti-inflammatory properties. In this study, we explored the signaling pathways involved in contributions of TRQ to LPS-induced airway inflammation in rats. Methods/Design. Adult male Sprague Dawley (SD) rats randomly divided into different groups received intratracheal instillation of LPS and/or intraperitoneal injection of TRQ. Bronchoalveolar Lavage Fluid (BALF) and lung samples were collected at 24 h, 48 h, and 96 h after TRQ administration. Protein and mRNA levels of tumor necrosis factor- (TNF-) α, Interleukin- (IL-) 1β, IL-6, and IL-8 in BALF and lung homogenate were observed by ELISA and real-time PCR, respectively. Lung sections were stained for p38 MAPK and NF-κB detection by immunohistochemistry. Phospho-p38 MAPK, phosphor-extracellular signal-regulated kinases ERK1/2, phospho-SAPK/JNK, phospho-NF-κB p65, phospho-IKKα/β, and phospho-IκB-α were measured by western blot analysis. Results. The results showed that TRQ significantly counteracted LPS-stimulated release of TNF-α, IL-1β, IL-6, and IL-8, attenuated cells influx in BALF, mitigated mucus hypersecretion, suppressed phosphorylation of NF-κB p65, IκB-α, ΙKKα/β, ERK1/2, JNK, and p38 MAPK, and inhibited p38 MAPK and NF-κB p65 expression in rat lungs. Conclusions. Results of the current research indicate that TRQ possesses potent exhibitory effects in LPS-induced airway inflammation by, at least partially, suppressing the MAPKs and NF-κB signaling pathways, in a general dose-dependent manner. PMID:27366191

Roles of mitogen-activated protein kinases and angiotensin II in renal development.

PubMed

Balbi, A P C; Francescato, H D C; Marin, E C S; Costa, R S; Coimbra, T M

2009-01-01

Experimental and clinical evidence suggests that angiotensin II (AII) participates in renal development. Renal AII content is several-fold higher in newborn rats and mice than in adult animals. AII receptors are also expressed in higher amounts in the kidneys of newborn rats. The kidneys of fetuses whose mother received a type 1 AII receptor (AT1) antagonist during gestation present several morphological alterations. Mutations in genes that encode components of the renin-angiotensin system are associated with autosomal recessive renal tubular dysgenesis. Morphological changes were detected in the kidneys of 3-week-old angiotensin-deficient mice. Mitogen-activated protein kinases (MAPKs) are important mediators that transduce extracellular stimuli to intracellular responses. The MAPK family comprises three major subgroups, namely extracellular signal-regulated protein kinase (ERK), c-jun N-terminal kinases (JNK), and p38 MAPK (p38). Important events in renal growth during nephrogenesis such as cellular proliferation and differentiation accompanied by apoptosis on a large scale can be mediated by MAPK pathways. A decrease in glomerulus number was observed in embryos cultured for 48 and 120 h with ERK or p38 inhibitors. Many effects of AII are mediated by MAPK pathways. Treatment with losartan during lactation provoked changes in renal function and structure associated with alterations in AT1 and type 2 AII (AT2) receptors and p-JNK and p-p38 expression in the kidney. Several studies have shown that AII and MAPKs play an important role in renal development. However, the relationship between the effects of AII and MAPK activation on renal development is still unclear.

Effects of Cyclic Mechanical Stretch on the Proliferation of L6 Myoblasts and Its Mechanisms: PI3K/Akt and MAPK Signal Pathways Regulated by IGF-1 Receptor.

PubMed

Fu, Shaoting; Yin, Lijun; Lin, Xiaojing; Lu, Jianqiang; Wang, Xiaohui

2018-06-02

Myoblast proliferation is crucial to skeletal muscle hypertrophy and regeneration. Our previous study indicated that mechanical stretch altered the proliferation of C2C12 myoblasts, associated with insulin growth factor 1 (IGF-1)-mediated phosphoinositide 3-kinase (PI3K)/Akt (also known as protein kinase B) and mitogen-activated protein kinase (MAPK) pathways through IGF-1 receptor (IGF-1R). The purpose of this study was to explore the same stretches on the proliferation of L6 myoblasts and its association with IGF-1-regulated PI3K/Akt and MAPK activations. L6 myoblasts were divided into three groups: control, 15% stretch, and 20% stretch. Stretches were achieved using FlexCell Strain Unit. Cell proliferation and IGF-1 concentration were detected by CCK8 and ELISA, respectively. IGF-1R expression, and expressions and activities of PI3K, Akt, and MAPKs (including extracellular signal-regulated kinases 1 and 2 (ERK1/2) and p38) were determined by Western blot. We found that 15% stretch promoted, while 20% stretch inhibited L6 myoblast proliferation. A 15% stretch increased IGF-1R level, although had no effect on IGF-1 secretion of L6 myoblasts, and PI3K/Akt and ERK1/2 (not p38) inhibitors attenuated 15% stretch-induced pro-proliferation. Exogenous IGF-1 reversed 20% stretch-induced anti-proliferation, accompanied with increases in IGF-1R level as well as PI3K/Akt and MAPK (ERK1/2 and p38) activations. In conclusion, stretch regulated L6 myoblasts proliferation, which may be mediated by the changes in PI3K/Akt and MAPK activations regulated by IGF-1R, despite no detectable IGF-1 from stretched L6 myoblasts.

The non-classical MAP kinase ERK3 controls T cell activation.

PubMed

Marquis, Miriam; Boulet, Salix; Mathien, Simon; Rousseau, Justine; Thébault, Paméla; Daudelin, Jean-François; Rooney, Julie; Turgeon, Benjamin; Beauchamp, Claudine; Meloche, Sylvain; Labrecque, Nathalie

2014-01-01

The classical mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 are activated upon stimulation of cells with a broad range of extracellular signals (including antigens) allowing cellular responses to occur. ERK3 is an atypical member of the MAPK family with highest homology to ERK1/2. Therefore, we evaluated the role of ERK3 in mature T cell response. Mouse resting T cells do not transcribe ERK3 but its expression is induced in both CD4⁺ and CD8⁺ T cells following T cell receptor (TCR)-induced T cell activation. This induction of ERK3 expression in T lymphocytes requires activation of the classical MAPK ERK1 and ERK2. Moreover, ERK3 protein is phosphorylated and associates with MK5 in activated primary T cells. We show that ERK3-deficient T cells have a decreased proliferation rate and are impaired in cytokine secretion following in vitro stimulation with low dose of anti-CD3 antibodies. Our findings identify the atypical MAPK ERK3 as a new and important regulator of TCR-induced T cell activation.

The Non-Classical MAP Kinase ERK3 Controls T Cell Activation

PubMed Central

Mathien, Simon; Rousseau, Justine; Thébault, Paméla; Daudelin, Jean-François; Rooney, Julie; Turgeon, Benjamin; Beauchamp, Claudine; Meloche, Sylvain; Labrecque, Nathalie

2014-01-01

The classical mitogen-activated protein kinases (MAPKs) ERK1 and ERK2 are activated upon stimulation of cells with a broad range of extracellular signals (including antigens) allowing cellular responses to occur. ERK3 is an atypical member of the MAPK family with highest homology to ERK1/2. Therefore, we evaluated the role of ERK3 in mature T cell response. Mouse resting T cells do not transcribe ERK3 but its expression is induced in both CD4+ and CD8+ T cells following T cell receptor (TCR)-induced T cell activation. This induction of ERK3 expression in T lymphocytes requires activation of the classical MAPK ERK1 and ERK2. Moreover, ERK3 protein is phosphorylated and associates with MK5 in activated primary T cells. We show that ERK3-deficient T cells have a decreased proliferation rate and are impaired in cytokine secretion following in vitro stimulation with low dose of anti-CD3 antibodies. Our findings identify the atypical MAPK ERK3 as a new and important regulator of TCR-induced T cell activation. PMID:24475167

Cigarette smoke-induced cell cycle arrest in spermatocytes [GC-2spd(ts)] is mediated through crosstalk between Ahr-Nrf2 pathway and MAPK signaling.

PubMed

Esakky, Prabagaran; Hansen, Deborah A; Drury, Andrea M; Moley, Kelle H

2015-02-01

Our earlier studies have demonstrated that the cigarette smoke in the form of cigarette smoke condensate (CSC) causes growth arrest of a mouse spermatocyte cell line [GC-2spd(ts)] through activation of the AHR-NRF2 pathway. The present study demonstrates the CSC-activated p38 and ERK MAPK signaling in GC-2spd(ts) via arylhydrocarbon receptor (AHR). Pharmacological inhibition by using AHR-antagonist, or p38 MAPK and ERK (MEK1) inhibitors significantly abrogates CSC-induced growth arrest by AHR and MAPK inactivation. QRT-PCR, western blot, and immunofluorescence of Ahr-target of Nrf2, and stress-inducible growth suppressive Atf3 and E2f4 following treatments indicate a crosstalk among these pathways. Regulation of Atf3 by Nrf2 and Ahr through RNA interference suggests the existence of a cross-regulatory loop between the targets. CSC induction of E2f4 via Atf3 and its regulation by pharmacological inhibitors reveal a possible regulatory mechanism of growth inhibitory CSC. SiRNA silencing of Ahr, Nrf2, Atf3, and E2f4 genes and downregulation of cyclins by CSC corroborate the growth inhibitory effect of cigarette smoke. Thus, the data obtained suggest that the CSC-mediated MAPKs and AHR-NRF2 crosstalks lay the molecular basis for the growth arrest and cell death of spermatocytes. © The Author (2014). Published by Oxford University Press on behalf of Journal of Molecular Cell Biology, IBCB, SIBS, CAS. All rights reserved.

Effects of sodium fluoride on MAPKs signaling pathway in the gills of a freshwater teleost, Cyprinus carpio.

PubMed

Cao, Jinling; Chen, Jianjie; Wang, Jundong; Klerks, Paul; Xie, Lingtian

2014-07-01

Exposure to elevated levels of fluoride can cause a variety of adverse effects in fish. Previously we showed that fluoride causes injuries and apoptosis in the gills of Cyprinus carpio. In this study, the effects of fluoride on caspase-3 activity and on accumulation of proteins in the MAPKs pathways were evaluated using Western blotting and immunohistochemistry methods in vivo and in vitro. In vivo experiments showed that the caspase-3 activity increased with fluoride exposure level in a dose-dependent pattern Western blotting and immunohistochemistry results indicated that ERK relative activation tended to decrease as a function of fluoride exposure concentration. In contrast, relative activation of JNK increased with fluoride exposure level. Fluoride exposure did not appear to affect p38 activation. Furthermore, pretreatment of branchial cells with MAPK-specific inhibitors effectively prevented JNK induction and ERK inhibition, respectively, as well as reversed caspase-3 activity in fluoride-treated branchial cells. Our results indicate that activation of JNK and inactivation of ERK were caused by increased ROS and decreased antioxidant capacity in the gills of chronically exposed C. carpio described previously, which eventually caused the observed apoptosis in the fluoride-exposed gills and cells in C. carpio. JNK activation and ERK inactivation mechanism play a crucial role in gill impairment induced by chronic fluorosis. These findings contribute to a better understanding of the initial molecular and cellular events in the gill of fish chronically exposed to fluoride. Copyright © 2014 Elsevier B.V. All rights reserved.

Impact of Glutathione Peroxidase-1 Deficiency on Macrophage Foam Cell Formation and Proliferation: Implications for Atherogenesis

PubMed Central

Degreif, Adriana; Rossmann, Heidi; Canisius, Antje; Lackner, Karl J.

2013-01-01

Clinical and experimental evidence suggests a protective role for the antioxidant enzyme glutathione peroxidase-1 (GPx-1) in the atherogenic process. GPx-1 deficiency accelerates atherosclerosis and increases lesion cellularity in ApoE−/− mice. However, the distribution of GPx-1 within the atherosclerotic lesion as well as the mechanisms leading to increased macrophage numbers in lesions is still unknown. Accordingly, the aims of the present study were (1) to analyze which cells express GPx-1 within atherosclerotic lesions and (2) to determine whether a lack of GPx-1 affects macrophage foam cell formation and cellular proliferation. Both in situ-hybridization and immunohistochemistry of lesions of the aortic sinus of ApoE−/− mice after 12 weeks on a Western type diet revealed that both macrophages and – even though to a less extent – smooth muscle cells contribute to GPx-1 expression within atherosclerotic lesions. In isolated mouse peritoneal macrophages differentiated for 3 days with macrophage-colony-stimulating factor (MCSF), GPx-1 deficiency increased oxidized low density-lipoprotein (oxLDL) induced foam cell formation and led to increased proliferative activity of peritoneal macrophages. The MCSF- and oxLDL-induced proliferation of peritoneal macrophages from GPx-1−/−ApoE−/− mice was mediated by the p44/42 MAPK (p44/42 mitogen-activated protein kinase), namely ERK1/2 (extracellular-signal regulated kinase 1/2), signaling pathway as demonstrated by ERK1/2 signaling pathways inhibitors, Western blots on cell lysates with primary antibodies against total and phosphorylated ERK1/2, MEK1/2 (mitogen-activated protein kinase kinase 1/2), p90RSK (p90 ribosomal s6 kinase), p38 MAPK and SAPK/JNK (stress-activated protein kinase/c-Jun N-terminal kinase), and immunohistochemistry of mice atherosclerotic lesions with antibodies against phosphorylated ERK1/2, MEK1/2 and p90RSK. Representative effects of GPx-1 deficiency on both macrophage proliferation and MAPK phosphorylation could be abolished by the GPx mimic ebselen. The present study demonstrates that GPx-1 deficiency has a significant impact on macrophage foam cell formation and proliferation via the p44/42 MAPK (ERK1/2) pathway encouraging further studies on new therapeutic strategies against atherosclerosis. PMID:23991041

Asbestos exposure induces alveolar epithelial cell plasticity through MAPK/Erk signaling.

PubMed

Tamminen, Jenni A; Myllärniemi, Marjukka; Hyytiäinen, Marko; Keski-Oja, Jorma; Koli, Katri

2012-07-01

The inhalation of asbestos fibers is considered to be highly harmful, and lead to fibrotic and/or malignant disease. Epithelial-to-mesenchymal transition (EMT) is a common pathogenic mechanism in asbestos associated fibrotic (asbestosis) and malignant lung diseases. The characterization of molecular pathways contributing to EMT may provide new possibilities for prognostic and therapeutic applications. The role of asbestos as an inducer of EMT has not been previously characterized. We exposed cultured human lung epithelial cells to crocidolite asbestos and analyzed alterations in the expression of epithelial and mesenchymal marker proteins and cell morphology. Asbestos was found to induce downregulation of E-cadherin protein levels in A549 lung carcinoma cells in 2-dimensional (2D) and 3D cultures. Similar findings were made in primary small airway epithelial cells cultured in 3D conditions where the cells retained alveolar type II cell phenotype. A549 cells also exhibited loss of cell-cell contacts, actin reorganization and expression of α-smooth muscle actin (α-SMA) in 2D cultures. These phenotypic changes were not associated with increased transforming growth factor (TGF)-β signaling activity. MAPK/Erk signaling pathway was found to mediate asbestos-induced downregulation of E-cadherin and alterations in cell morphology. Our results suggest that asbestos can induce epithelial plasticity, which can be interfered by blocking the MAPK/Erk kinase activity. Copyright © 2012 Wiley Periodicals, Inc.

MiR-592 Promotes Gastric Cancer Proliferation, Migration, and Invasion Through the PI3K/AKT and MAPK/ERK Signaling Pathways by Targeting Spry2.

PubMed

He, Yu; Ge, Yugang; Jiang, Mingkun; Zhou, Jundong; Luo, Dakui; Fan, Hao; Shi, Liang; Lin, Linling; Yang, Li

2018-06-21

Gastric cancer (GC) is one of the most prevalent digestive malignancies. MicroRNAs (miRNAs) are involved in multiple cellular processes, including oncogenesis, and miR-592 itself participates in many malignancies; however, its role in GC remains unknown. In this study, we investigated the expression and molecular mechanisms of miR-592 in GC. Quantitative real-time PCR and immunohistochemistry were performed to determine the expression of miR-592 and its putative targets in human tissues and cell lines. Proliferation, migration, and invasion were evaluated by Cell Counting Kit-8, population doubling time, colony formation, Transwell, and wound-healing assays in transfected GC cells in vitro. A dual-luciferase reporter assay was used to determine whether miR-592 could directly bind its target. A tumorigenesis assay was used to study whether miR-592 affected GC growth in vivo. Proteins involved in signaling pathways and the epithelial-mesenchymal transition (EMT) were detected with western blot. The ectopic expression of miR-592 promoted GC proliferation, migration, and invasion in vitro and facilitated tumorigenesis in vivo. Spry2 was a direct target of miR-592 and Spry2 overexpression partially counteracted the effects of miR-592. miR-592 induced the EMT and promoted its progression in GC via the PI3K/AKT and MAPK/ERK signaling pathways by inhibiting Spry2. Overexpression of miR-592 promotes GC proliferation, migration, and invasion and induces the EMT via the PI3K/AKT and MAPK/ERK signaling pathways by inhibiting Spry2, suggesting a potential therapeutic target for GC. © 2018 The Author(s). Published by S. Karger AG, Basel.

The predominant protective effect of tianeptine over other antidepressants in models of neuronal apoptosis: the effect blocked by inhibitors of MAPK/ERK1/2 and PI3-K/Akt pathways.

PubMed

Jantas, D; Krawczyk, S; Lason, W

2014-02-01

Tianeptine (Tian) possesses neuroprotective potential, however, little is known about the effect of this drug in models of neuronal apoptosis. In the present study, we aimed (1) to compare the neuroprotective capacities of some antidepressants (ADs) in the models of staurosporine (St)- and doxorubicin (Dox)-evoked cell death, activating the intracellular and the extracellular apoptotic pathway, respectively; (2) to identify the Tian-modulated steps underlying its neuroprotective action; (3) to test the effect of various ADs against Dox-evoked cell damage in glia cells. Primary neuronal and glia cell cultures and retinoic acid-differentiated human neuroblastoma SH-SY5Y (RA-SH-SY5Y) cells were co-treated with imipramine, fluoxetine, citalopram, reboxetine, mirtazapine or Tian and St or Dox. The data showed the predominant neuroprotective effect of Tian over other tested ADs against St- and Dox-induced cell damage in primary neurons and in RA-SH-SY5Y cells. This effect was shown to be caspase-3-independent but connected with attenuation of DNA fragmentation. Moreover, neuroprotection elicited by Tian was blocked by pharmacological inhibitors of MAPK/ERK1/2 and PI3-K/Akt signaling pathways as well by inhibitor of necroptosis, necrostatin-1. Interestingly, the protective effects of all tested ADs were demonstrated in primary glia cells against the Dox-evoked cell damage. The obtained data suggests the glial cells as a common target for protective action of various ADs whereas in relation to neuronal cells only Tian possesses such properties, at least against St- and Dox-induced cell damage. Moreover, this neuroprotective effect of Tian is caspase-3-independent and engages the regulation of survival pathways (MAPK/ERK1/2 and PI3-K/Akt).

Involvement of the MAPK pathway in the pressure-induced synovial metaplasia procedure for the temporomandibular joint.

PubMed

Wu, M J; Lu, H P; Gu, Z Y; Zhou, Y Q

2016-06-20

Abnormal pressure is an important factor that contributes to bone adaptation in the temporomandibular joint (TMJ). We determined the effect of the mitogen-activated protein kinases (MAPK) pathway on the pressure-induced synovial metaplasia procedure for the TMJ, both in vitro and in vivo. Synovial fibroblasts (SFs) were exacted from rat TMJs and exposed to different hydrostatic pressures. The protein extracts were analyzed to determine the activation of ERK1/2, JNK, and p38. Surgical anterior disc displacement (ADD) was also performed on Japanese rabbits, and the proteins of TMJ were isolated to analyze pressure-induced MAPK activation after 1, 2, 4, and 8 weeks. The results showed that the activation of ERK1/2 and JNK in SFs significantly changed with increasing hydrostatic pressure, whereas p38 activation did not change. Moreover, p38 was activated in animals 1 week after surgical ADD. The levels of p38 gradually increased after 2 and 4 weeks, and then slightly decreased but remained higher than in the control 8 weeks after surgical ADD. Nevertheless, JNK was rarely activated after the ADD treatment. Our findings suggest the involvement of MAPK activation in the pressure-induced synovial metaplasia procedure with pressure loading in TMJ.

N-n-butyl Haloperidol Iodide Protects against Hypoxia/Reoxygenation Injury in Cardiac Microvascular Endothelial Cells by Regulating the ROS/MAPK/Egr-1 Pathway

PubMed Central

Lu, Shishi; Zhang, Yanmei; Zhong, Shuping; Gao, Fenfei; Chen, Yicun; Li, Weiqiu; Zheng, Fuchun; Shi, Ganggang

2017-01-01

Endothelium dysfunction induced by reactive oxygen species (ROS) is an important initial event at the onset of myocardial ischemia/reperfusion in which the Egr-1 transcription factor often serves as a master switch for various damage pathways following reperfusion injury. We hypothesized that an intracellular ROS/MAPK/Egr-1 signaling pathway is activated in cardiac microvascular endothelial cells (CMECs) following hypoxia/reoxygenation (H/R). ROS generation, by either H/R or the ROS donor xanthine oxidase-hypoxanthine (XO/HX) activated all three MAPKs (ERK1/2, JNK, p38), and induced Egr-1 expression and Egr-1 DNA-binding activity in CMECs, whereas ROS scavengers (EDA and NAC) had the opposite effect following H/R. Inhibitors of all three MAPKs individually inhibited induction of Egr-1 expression by H/R in CMECs. Moreover, N-n-butyl haloperidol (F2), previously shown to protect cardiomyocytes subjected to I/R, dose-dependently downregulated H/R-induced ROS generation, MAPK activation, and Egr-1 expression and activity in CMECs, whereas XO/HX and MAPK activators (EGF, anisomycin) antagonized the effects of F2. Inhibition of the ROS/MAPK/Egr-1 signaling pathway, by either F2, NAC, or inhibition of MAPK, increased CMEC viability and the GSH/GSSG ratio, and decreased Egr-1 nuclear translocation. These results show that the ROS/MAPK/Egr-1 signaling pathway mediates H/R injury in CMECs, and F2 blocks this pathway to protect against H/R injury and further alleviate myocardial I/R injury. PMID:28111550

MAPK pathway activation by chronic lead-exposure increases vascular reactivity through oxidative stress/cyclooxygenase-2-dependent pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Simões, Maylla Ronacher, E-mail: yllars@hotmail.com; Department of Pharmacology, Universidad Autonoma de Madrid, Instituto de Investigación Hospital Universitario La Paz; Aguado, Andrea

Chronic exposure to low lead concentration produces hypertension; however, the underlying mechanisms remain unclear. We analyzed the role of oxidative stress, cyclooxygenase-2-dependent pathways and MAPK in the vascular alterations induced by chronic lead exposure. Aortas from lead-treated Wistar rats (1st dose: 10 μg/100 g; subsequent doses: 0.125 μg/100 g, intramuscular, 30 days) and cultured aortic vascular smooth muscle cells (VSMCs) from Sprague Dawley rats stimulated with lead (20 μg/dL) were used. Lead blood levels of treated rats attained 21.7 ± 2.38 μg/dL. Lead exposure increased systolic blood pressure and aortic ring contractile response to phenylephrine, reduced acetylcholine-induced relaxation and didmore » not affect sodium nitroprusside relaxation. Endothelium removal and L-NAME left-shifted the response to phenylephrine more in untreated than in lead-treated rats. Apocynin and indomethacin decreased more the response to phenylephrine in treated than in untreated rats. Aortic protein expression of gp91(phox), Cu/Zn-SOD, Mn-SOD and COX-2 increased after lead exposure. In cultured VSMCs lead 1) increased superoxide anion production, NADPH oxidase activity and gene and/or protein levels of NOX-1, NOX-4, Mn-SOD, EC-SOD and COX-2 and 2) activated ERK1/2 and p38 MAPK. Both antioxidants and COX-2 inhibitors normalized superoxide anion production, NADPH oxidase activity and mRNA levels of NOX-1, NOX-4 and COX-2. Blockade of the ERK1/2 and p38 signaling pathways abolished lead-induced NOX-1, NOX-4 and COX-2 expression. Results show that lead activation of the MAPK signaling pathways activates inflammatory proteins such as NADPH oxidase and COX-2, suggesting a reciprocal interplay and contribution to vascular dysfunction as an underlying mechanisms for lead-induced hypertension. - Highlights: • Lead-exposure increases oxidative stress, COX-2 expression and vascular reactivity. • Lead exposure activates MAPK signaling pathway. • ROS and COX-2 activation by MAPK in lead exposure • Relationship between vascular ROS and COX-2 products in lead exposure.« less

Thrombospondin-4 Promotes Neuronal Differentiation of NG2 Cells via the ERK/MAPK Pathway.

PubMed

Yang, Hai Jie; Ma, Shuang Ping; Ju, Fei; Zhang, Ya Ping; Li, Zhi Chao; Zhang, Bin Bin; Lian, Jun Jiang; Wang, Lei; Cheng, Bin Feng; Wang, Mian; Feng, Zhi Wei

2016-12-01

NG2-expressing neural progenitors can produce neurons in the central nervous system, providing a potential cell resource of therapy for neurological disorders. However, the mechanism underlying neuronal differentiation of NG2 cells remains largely unknown. In this report, we found that a thrombospondin (TSP) family member, TSP4, is involved in the neuronal differentiation of NG2 cells. When TSP4 was overexpressed, NG2 cells underwent spontaneous neuronal differentiation, as demonstrated by the induction of various neuronal differentiation markers such as NeuN, Tuj1, and NF200, at the messenger RNA and protein levels. In contrast, TSP4 silencing had an opposite effect on the expression of neuronal differentiation markers in NG2 cells. Next, the signaling pathway responsible for TSP4-mediated NG2 cell differentiation was investigated. We found that ERK but not p38 and AKT signaling was affected by TSP4 overexpression. Furthermore, when ERK signaling was blocked by the inhibitor U0126, the neuronal marker expression of NG2 cells was substantially increased. Together, these findings suggested that TSP4 promoted neuronal differentiation of NG2 cells by inhibiting ERK/MAPK signaling, revealing a novel role of TSP4 in cell fate specification of NG2 cells.

Role of mitogen-activated protein kinase (MAPK) docking sites on Staufen2 protein in dendritic mRNA transport.

PubMed

Nam, Yeon-Ju; Cheon, Hyo-Soon; Choi, Young-Ki; Kim, Seok-Yong; Shin, Eun-Young; Kim, Eung-Gook; Kim, Hyong Kyu

2008-08-08

Although transport and subsequent translation of dendritic mRNA play an important role in neuronal synaptic plasticity, the underlying mechanisms for modulating dendritic mRNA transport are almost completely unknown. In this study, we identified and characterized an interaction between Staufen2 and mitogen-activated protein kinase (MAPK) with co-immunoprecipitation assays. Staufen2 utilized a docking (D) site to interact with ERK1/2; deleting the D-site decreased colocalization of Staufen2 with immunoreactive ERK1/2 in the cell body regions of cultured hippocampal neurons, and it reduced the amount of Staufen2-containing RNP complexes in the distal dendrites. In addition, the deletion completely abolished the depolarization-induced increase of Staufen2-containing RNP complexes. These results suggest that the MAPK pathway could modulate dendritic mRNA transport through its interaction with Staufen2.

Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS.

PubMed

Zuidervaart, W; van Nieuwpoort, F; Stark, M; Dijkman, R; Packer, L; Borgstein, A-M; Pavey, S; van der Velden, P; Out, C; Jager, M J; Hayward, N K; Gruis, N A

2005-06-06

In contrast to cutaneous melanoma, there is no evidence that BRAF mutations are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in uveal melanoma, although there is increasing evidence that this pathway is activated frequently in the latter tumours. In this study, we performed mutation analysis of the RAS and BRAF genes in a panel of 11 uveal melanoma cell lines and 19 primary uveal melanoma tumours. In addition, Western blot and immunohistochemical analyses were performed on downstream members of the MAPK pathway in order to assess the contribution of each of these components. No mutations were found in any of the three RAS gene family members and only one cell line carried a BRAF mutation (V599E). Despite this, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), ERK and ELK were constitutively activated in all samples. These data suggest that activation of the MAPK pathway is commonly involved in the development of uveal melanoma, but occurs through a mechanism different to that of cutaneous melanoma.

Activation of the MAPK pathway is a common event in uveal melanomas although it rarely occurs through mutation of BRAF or RAS

PubMed Central

Zuidervaart, W; van Nieuwpoort, F; Stark, M; Dijkman, R; Packer, L; Borgstein, A-M; Pavey, S; van der Velden, P; Out, C; Jager, M J; Hayward, N K; Gruis, N A

2005-01-01

In contrast to cutaneous melanoma, there is no evidence that BRAF mutations are involved in the activation of the mitogen-activated protein kinase (MAPK) pathway in uveal melanoma, although there is increasing evidence that this pathway is activated frequently in the latter tumours. In this study, we performed mutation analysis of the RAS and BRAF genes in a panel of 11 uveal melanoma cell lines and 19 primary uveal melanoma tumours. In addition, Western blot and immunohistochemical analyses were performed on downstream members of the MAPK pathway in order to assess the contribution of each of these components. No mutations were found in any of the three RAS gene family members and only one cell line carried a BRAF mutation (V599E). Despite this, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase (MEK), ERK and ELK were constitutively activated in all samples. These data suggest that activation of the MAPK pathway is commonly involved in the development of uveal melanoma, but occurs through a mechanism different to that of cutaneous melanoma. PMID:15928660

Dihydrotestosterone activates the MAPK pathway and modulates maximum isometric force through the EGF receptor in isolated intact mouse skeletal muscle fibres.

PubMed

Hamdi, M M; Mutungi, G

2010-02-01

It is generally believed that steroid hormones have both genomic and non-genomic (rapid) actions. Although the latter form an important component of the physiological response of these hormones, little is known about the cellular signalling pathway(s) mediating these effects and their physiological functions in adult mammalian skeletal muscle fibres. Therefore, the primary aim of this study was to investigate the non-genomic actions of dihydrotestosterone (DHT) and their physiological role in isolated intact mammalian skeletal muscle fibre bundles. Our results show that treating the fibre bundles with physiological concentrations of DHT increases both twitch and tetanic contractions in fast twitch fibres. However, it decreases them in slow twitch fibres. These changes in force are accompanied by an increase in the phosphorylation of MAPK/ERK1/2 in both fibre types and that of regulatory myosin light chains in fast twitch fibres. Both effects were insensitive to inhibitors of Src kinase, androgen receptor, insulin-like growth factor 1 receptor and platelet-derived growth factor receptor. However, they were abolished by the MAPK/ERK1/2 kinase inhibitor PD98059 and the epidermal growth factor (EGF) receptor inhibitor tyrphostin AG 1478. In contrast, testosterone had no effect on force and increased the phosphorylation of ERK1/2 in slow twitch fibres only. From these results we conclude that sex steroids have non-genomic actions in isolated intact mammalian skeletal muscle fibres. These are mediated through the EGF receptor and one of their main physiological functions is the enhancement of force production in fast twitch skeletal muscle fibres.

L-Ascorbate Attenuates the Endotoxin-Induced Production of Inflammatory Mediators by Inhibiting MAPK Activation and NF-κB Translocation in Cortical Neurons/Glia Cocultures

PubMed Central

Huang, Ya-Ni; Lai, Chien-Cheng; Chiu, Chien-Tsai; Lin, Jhen-Jhe; Wang, Jia-Yi

2014-01-01

In response to acute insults to the central nervous system, such as pathogen invasion or neuronal injuries, glial cells become activated and secrete inflammatory mediators such as nitric oxide (NO), cytokines, and chemokines. This neuroinflammation plays a crucial role in the pathophysiology of chronic neurodegenerative diseases. Endogenous ascorbate levels are significantly decreased among patients with septic encephalopathy. Using the bacterial endotoxin lipopolysaccharide (LPS) to induce neuroinflammation in primary neuron/glia cocultures, we investigated how L-ascorbate (vitamin C; Vit. C) affected neuroinflammation. LPS (100 ng/ml) induced the expression of inducible NO synthase (iNOS) and the production of NO, interleukin (IL)-6, and macrophage inflammatory protein-2 (MIP-2/CXCL2) in a time-dependent manner; however, cotreatment with Vit. C (5 or 10 mM) attenuated the LPS-induced iNOS expression and production of NO, IL-6, and MIP-2 production. The morphological features revealed after immunocytochemical staining confirmed that Vit. C suppressed LPS-induced astrocytic and microglial activation. Because Vit. C can be transported into neurons and glia via the sodium-dependent Vit. C transporter-2, we examined how Vit. C affected LPS-activated intracellular signaling in neuron/glia cocultures. The results indicated the increased activation (caused by phosphorylation) of mitogen-activated protein kinases (MAPKs), such as p38 at 30 min and extracellular signal-regulated kinases (ERKs) at 180 min after LPS treatment. The inhibition of p38 and ERK MAPK suppressed the LPS-induced production of inflammatory mediators. Vit. C also inhibited the LPS-induced activation of p38 and ERK. Combined treatments of Vit. C and the inhibitors of p38 and ERK yielded no additional inhibition compared with using the inhibitors alone, suggesting that Vit. C functions through the same signaling pathway (i.e., MAPK) as these inhibitors. Vit. C also reduced LPS-induced IκB-α degradation and NF-κB translocation. Thus, Vit. C suppressed the LPS-stimulated production of inflammatory mediators in neuron/glia cocultures by inhibiting the MAPK and NF-κB signaling pathways. PMID:24983461

IL-17A induces eotaxin-1/CC chemokine ligand 11 expression in human airway smooth muscle cells: role of MAPK (Erk1/2, JNK, and p38) pathways.

PubMed

Rahman, Muhammad Shahidur; Yamasaki, Akira; Yang, Jie; Shan, Lianyu; Halayko, Andrew J; Gounni, Abdelilah Soussi

2006-09-15

Recently, IL-17A has been shown to be expressed in higher levels in respiratory secretions from asthmatics and correlated with airway hyperresponsiveness. Although these studies raise the possibility that IL-17A may influence allergic disease, the mechanisms remain unknown. In this study, we investigated the molecular mechanisms involved in IL-17A-mediated CC chemokine (eotaxin-1/CCL11) production from human airway smooth muscle (ASM) cells. We found that incubation of human ASM cells with rIL-17A resulted in a significant increase of eotaxin-1/CCL11 release from ASM cells that was reduced by neutralizing anti-IL-17A mAb. Moreover, IL-17A significantly induced eotaxin-1/CCL11 release and mRNA expression, an effect that was abrogated with cycloheximide and actinomycin D treatment. Furthermore, transfection studies using a luciferase-driven reporter construct containing eotaxin-1/CCL11 proximal promoter showed that IL-17A induced eotaxin-1/CCL11 at the transcriptional level. IL-17A also enhanced significantly IL-1beta-mediated eotaxin-1/CCL11 mRNA, protein release, and promoter activity in ASM cells. Primary human ASM cells pretreated with inhibitors of MAPK p38, p42/p44 ERK, JNK, or JAK but not PI3K, showed a significant decrease in eotaxin-1/CCL11 release upon IL-17A treatment. In addition, IL-17A mediated rapid phosphorylation of MAPK (p38, JNK, and p42/44 ERK) and STAT-3 but not STAT-6 or STAT-5 in ASM cells. Taken together, our data provide the first evidence of IL-17A-induced eotaxin-1/CCL11 expression in ASM cells via MAPK (p38, p42/p44 ERK, JNK) signaling pathways. Our results raise the possibility that IL-17A may play a role in allergic asthma by inducing eotaxin-1/CCL11 production.

Chronic intermittent hypoxia induces liver fibrosis in mice with diet-induced obesity via TLR4/MyD88/MAPK/NF-kB signaling pathways.

PubMed

Kang, Hyeon Hui; Kim, In Kyoung; Lee, Hye In; Joo, Hyonsoo; Lim, Jeong Uk; Lee, Jongmin; Lee, Sang Haak; Moon, Hwa Sik

2017-08-19

Obstructive sleep apnea (OSA) is associated with nonalcoholic fatty liver disease (NAFLD), and causes chronic intermittent hypoxia (CIH) during sleep. Inflammation is associated with the development of metabolic complications induced by CIH. Research suggests that innate immune mechanisms are involved in the pro-inflammatory pathways of liver fibrosis. The purpose of this study was to investigate whether innate immune responses induce liver fibrosis, and to evaluate mechanisms underlying hepatic inflammation related to CIH in a murine diet-induced obesity (DIO) model. Inflammatory and oxidative stress markers, TLR4, MyD88, Toll/interleukin-1-receptor-domain-containing adaptor-inducing interferon-β (TRIF), I-κB, NF-κB, p38 MAPK, c-JNK, and ERK activation, were measured in the serum and liver. As a result, α1(I)-collagen mRNA was significantly higher in DIO mice exposed to CIH than in the control groups. CIH mice exhibited liver fibrosis and significantly higher protein expression of TLR4, MyD88, phosphorylated (phospho-) I-κB, and phospho-ERK1/2 activation in the liver, and higher expression of NF-κB than that in the controls. TRIF, p38 MAPK, and JNK activation did not differ significantly between groups. We conclude that CIH in DIO mice leads to liver fibrosis via TLR4/MyD88/MAPK/NF-kB signaling pathways. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

PDGF-BB induces PRMT1 expression through ERK1/2 dependent STAT1 activation and regulates remodeling in primary human lung fibroblasts.

PubMed

Sun, Qingzhu; Liu, Li; Mandal, Jyotshna; Molino, Antonio; Stolz, Daiana; Tamm, Michael; Lu, Shemin; Roth, Michael

2016-04-01

Tissue remodeling of sub-epithelial mesenchymal cells is a major pathology occurring in chronic obstructive pulmonary disease (COPD) and asthma. Fibroblasts, as a major source of interstitial connective tissue extracellular matrix, contribute to the fibrotic and inflammatory changes in these airways diseases. Previously, we described that protein arginine methyltransferase-1 (PRMT1) participates in airway remodeling in a rat model of pulmonary inflammation. In this study we investigated the mechanism by which PDGF-BB regulates PRMT1 in primary lung fibroblasts, isolated from human lung biopsies. Fibroblasts were stimulated with PDGF-BB for up-to 48h and the regulatory and activation of signaling pathways controlling PRMT1 expression were determined. PRMT1 was localized by immuno-histochemistry in human lung tissue sections and by immunofluorescence in isolated fibroblasts. PRMT1 activity was suppressed by the pan-PRMT inhibitor AMI1. ERK1/2 mitogen activated protein kinase (MAPK) was blocked by PD98059, p38 MAPK by SB203580, and STAT1 by small interference (si) RNA treatment. The results showed that PDGF-BB significantly increased PRMT1 expression after 1h lasting over 48h, through ERK1/2 MAPK and STAT1 signaling. The inhibition of ERK1/2 MAPK or of PRMT1 activity decreased PDGF-BB induced fibroblast proliferation, COX2 production, collagen-1A1 secretion, and fibronectin production. These findings suggest that PRMT1 is a central regulator of tissue remodeling and that the signaling sequence controlling its expression in primary human lung fibroblast is PDGF-ERK-STAT1. Therefore, PRMT1 presents a novel therapeutic and diagnostic target for the control of airway wall remodeling in chronic lung diseases. Copyright © 2016 Elsevier Inc. All rights reserved.

Communication Between the Calcium and cAMP Pathways Regulate the Expression of the TSH Receptor: TRPC2 in the Center of Action

PubMed Central

Löf, Christoffer; Sukumaran, Pramod; Viitanen, Tero; Vainio, Minna; Kemppainen, Kati; Pulli, Ilari; Näsman, Johnny; Kukkonen, Jyrki P.

2012-01-01

Transient receptor potential (TRP) cation channels are widely expressed and function in many physiologically important processes. Perturbations in the expression or mutations of the channels have implications for diseases. Many thyroid disorders, as excessive growth or disturbed thyroid hormone production, can be a result of dysregulated TSH signaling. In the present study, we found that of TRP canonicals (TRPCs), only TRPC2 was expressed in Fischer rat thyroid low-serum 5% cells (FRTL-5 cells). To investigate the physiological importance of the channel, we developed stable TRPC2 knockdown cells using short hairpin RNA (shTRPC2 cells). In these cells, the ATP-evoked entry of calcium was significantly decreased. This led to increased cAMP production, because inhibitory signals from calcium to adenylate cyclase 5/6 were decreased. Enhanced cAMP signaling projected to Ras-related protein 1-MAPK kinase 1 (MAPK/ERK kinase 1) pathway leading to phosphorylation of ERK1/2. The activated ERK1/2 pathway increased the expression of the TSH receptor. In contrast, secretion of thyroglobulin was decreased in shTRPC2 cells, due to improper folding and glycosylation of the protein. We show here a novel role for TRPC2 in regulating thyroid cell function. PMID:23015753

PKI-587 and sorafenib targeting PI3K/AKT/mTOR and Ras/Raf/MAPK pathways synergistically inhibit HCC cell proliferation.

PubMed

Gedaly, Roberto; Angulo, Paul; Hundley, Jonathan; Daily, Michael F; Chen, Changguo; Evers, B Mark

2012-08-01

Deregulated Ras/Raf/MAPK and PI3K/AKT/mTOR signaling pathways are found in hepatocellular carcinoma (HCC). This study aimed to test the inhibitory effects of PKI-587 and sorafenib as single agents or in combination on HCC (Huh7 cell line) proliferation. (3)H-thymidine incorporation and MTT assay were used to assess Huh7 cell proliferation. Phosphorylation of the key enzymes in the Ras/Raf/MAPK and PI3K/AKT/mTOR pathways was detected by Western blot. We found that PKI-587 is a more potent PI3K/mTOR inhibitor than PI-103. Combination of PKI-587 and sorafenib was a more effective inhibitor of Huh7 proliferation than the combination of PI-103 and sorafenib. Combination of PKI-587 and sorafenib synergistically inhibited epidermal growth factor (EGF)-stimulated Huh7 proliferation compared with monodrug therapy. EGF increased phosphorylation of Ras/Raf downstream signaling proteins MEK and ERK; EGF-stimulated activation was inhibited by sorafenib. However, sorafenib, as a single agent, increased AKT (Ser473) phosphorylation. EGF-stimulated AKT (ser473) activation was inhibited by PKI-587. PKI-587 is a potent inhibitor of AKT (Ser473), mTOR (Ser2448), and S6K (Thr389) phosphorylation; in contrast, rapamycin stimulated mTOR complex 2 substrate AKT(Ser473) phosphorylation although it inhibited mTOR complex 1 substrate S6K phosphorylation. PKI-587, as a single agent, stimulated MEK and ERK phosphorylation. However, when PKI-587 and sorafenib were used in combination, they inhibited all the tested kinases in the Ras/Raf /MAPK and PI3K/AKT/mTOR pathways. The combination of PKI-587 and sorafenib has the advantage over monodrug therapy on inhibition of HCC cell proliferation by blocking both PI3K/AKT/mTOR and Ras/Raf/MAPK signaling pathways. Copyright © 2012 Elsevier Inc. All rights reserved.

17betaE2 promotes cell proliferation in endometriosis by decreasing PTEN via NFkappaB-dependent pathway.

PubMed

Zhang, Hui; Zhao, Xingbo; Liu, Shu; Li, Jijun; Wen, Zeqing; Li, Mingjiang

2010-04-12

The objective of this study was to explore the mechanism of phosphatase and tensin homolog (PTEN) loss in endometriosis. We found that aberrant PTEN expression and mitogen-activated protein kinases (MAPK)/ERK, phosphoinositide 3-kinase (PI3K)/AKt, and nuclear factor-kappaB (NFkappaB) signaling overactivities coexisted in endometriosis. In vitro, 17beta-estradiol rapidly activated the 3 pathways in endometriotic cells and specific inhibitions on the 3 pathways respectively blocked 17beta-estradiol-induced cell proliferation. 17beta-estradiol suppressed PTEN transcription and expression in endometriotic cells which was abolished by specific NFkappaB inhibition. Total/nuclear PTEN-loss and MAPK/ERK, PI3K/AKt, and NFkappaB signal overactivities coexist in endometriosis. In vitro, 17beta-estradiol can promotes cell proliferation in endometriosis by activating PI3K/AKt pathway via an NFkappaB/PTEN-dependent pathway. For the first time we propose the possibility of the presence of a positive feedback-loop: 17beta-estradiol-->high NFkappaB-->low PTEN-->high PI3K-->high NFkappaB, in endometriosis, which may finally promote the proliferation of ectopic endometrial epithelial cells and in turn contributes to the progression of the disease.

Neomorphic Mutations in PIK3R1 Confer Sensitivity to MAPK Inhibitors due to Activation of ERK and JNK Pathways | Office of Cancer Genomics

Cancer.gov

In a recent publication in Cancer Cell, CTD2 investigators discovered that a known cancer-associated gain-of-function alteration in phosphoinositide-3-kinase regulatory subunit 1 (PIK3R1) results in novel protein activity that confers sensitivity to mitogen-activated protein kinase (MAPK) inhibitors. The PIK3R1 gene encodes the p85α regulatory subunit of PIK3. Under normal conditions, p85α suppresses PIK3 mediated activation of downstream pathways that promote cell growth and survival.

Dynamics and control of the ERK signaling pathway: Sensitivity, bistability, and oscillations.

PubMed

Arkun, Yaman; Yasemi, Mohammadreza

2018-01-01

Cell signaling is the process by which extracellular information is transmitted into the cell to perform useful biological functions. The ERK (extracellular-signal-regulated kinase) signaling controls several cellular processes such as cell growth, proliferation, differentiation and apoptosis. The ERK signaling pathway considered in this work starts with an extracellular stimulus and ends with activated (double phosphorylated) ERK which gets translocated into the nucleus. We model and analyze this complex pathway by decomposing it into three functional subsystems. The first subsystem spans the initial part of the pathway from the extracellular growth factor to the formation of the SOS complex, ShC-Grb2-SOS. The second subsystem includes the activation of Ras which is mediated by the SOS complex. This is followed by the MAPK subsystem (or the Raf-MEK-ERK pathway) which produces the double phosphorylated ERK upon being activated by Ras. Although separate models exist in the literature at the subsystems level, a comprehensive model for the complete system including the important regulatory feedback loops is missing. Our dynamic model combines the existing subsystem models and studies their steady-state and dynamic interactions under feedback. We establish conditions under which bistability and oscillations exist for this important pathway. In particular, we show how the negative and positive feedback loops affect the dynamic characteristics that determine the cellular outcome.

HGF potentiates extracellular matrix-driven migration of human myoblasts: involvement of matrix metalloproteinases and MAPK/ERK pathway.

PubMed

González, Mariela Natacha; de Mello, Wallace; Butler-Browne, Gillian S; Silva-Barbosa, Suse Dayse; Mouly, Vincent; Savino, Wilson; Riederer, Ingo

2017-10-10

The hepatocyte growth factor (HGF) is required for the activation of muscle progenitor cells called satellite cells (SC), plays a role in the migration of proliferating SC (myoblasts), and is present as a soluble factor during muscle regeneration, along with extracellular matrix (ECM) molecules. In this study, we aimed at determining whether HGF is able to interact with ECM proteins, particularly laminin 111 and fibronectin, and to modulate human myoblast migration. We evaluated the expression of the HGF-receptor c-Met, laminin, and fibronectin receptors by immunoblotting, flow cytometry, or immunofluorescence and used Transwell assays to analyze myoblast migration on laminin 111 and fibronectin in the absence or presence of HGF. Zymography was used to check whether HGF could modulate the production of matrix metalloproteinases by human myoblasts, and the activation of MAPK/ERK pathways was evaluated by immunoblotting. We demonstrated that human myoblasts express c-Met, together with laminin and fibronectin receptors. We observed that human laminin 111 and fibronectin have a chemotactic effect on myoblast migration, and this was synergistically increased when low doses of HGF were added. We detected an increase in MMP-2 activity in myoblasts treated with HGF. Conversely, MMP-2 inhibition decreased the HGF-associated stimulation of cell migration triggered by laminin or fibronectin. HGF treatment also induced in human myoblasts activation of MAPK/ERK pathways, whose specific inhibition decreased the HGF-associated stimulus of cell migration triggered by laminin 111 or fibronectin. We demonstrate that HGF induces ERK phosphorylation and MMP production, thus stimulating human myoblast migration on ECM molecules. Conceptually, these data state that the mechanisms involved in the migration of human myoblasts comprise both soluble and insoluble moieties. This should be taken into account to optimize the design of therapeutic cell transplantation strategies by improving the migration of donor cells within the host tissue, a main issue regarding this approach.

Anti-Proliferative Effects of Rutin on OLETF Rat Vascular Smooth Muscle Cells Stimulated by Glucose Variability

PubMed Central

Yu, Sung Hoon; Yu, Jae Myung; Lee, Seong Jin; Kang, Dong Hyun; Cho, Young Jung; Kim, Doo Man

2016-01-01

Purpose Proliferation of vascular smooth muscle cells (VSMCs) plays a crucial role in atherosclerosis. Rutin is a major representative of the flavonol subclass of flavonoids and has various pharmacological activities. Currently, data are lacking regarding its effects on VSMC proliferation induced by intermittent hyperglycemia. Here, we demonstrate the effects of rutin on VSMC proliferation and migration according to fluctuating glucose levels. Materials and Methods Primary cultures of male Otsuka Long-Evans Tokushima Fatty (OLETF) rat VSMCs were obtained from enzymatically dissociated rat thoracic aortas. VSMCs were incubated for 72 h with alternating normal (5.5 mmol/L) and high (25.0 mmol/L) glucose media every 12 h. Proliferation and migration of VSMCs, the proliferative molecular pathway [including p44/42 mitogen-activated protein kinases (MAPK), mitogen-activated protein kinase kinase 1/2 (MEK1/2), p38 MAPK, phosphoinositide 3-kinase (PI3K), c-Jun N-terminal protein kinase (JNK), nuclear factor kappa B (NF-κB), and Akt], the migratory pathway (big MAPK 1, BMK1), reactive oxygen species (ROS), and apoptotic pathway were analyzed. Results We found enhanced proliferation and migration of VSMCs when cells were incubated in intermittent high glucose conditions, compared to normal glucose. These effects were lowered upon rutin treatment. Intermittent treatment with high glucose for 72 h increased the expression of phospho-p44/42 MAPK (extracellular signal regulated kinase 1/2, ERK1/2), phospho-MEK1/2, phospho-PI3K, phospho-NF-κB, phospho-BMK1, and ROS, compared to treatment with normal glucose. These effects were suppressed by rutin. Phospho-p38 MAPK, phospho-Akt, JNK, and apoptotic pathways [B-cell lymphoma (Bcl)-xL, Bcl-2, phospho-Bad, and caspase-3] were not affected by fluctuations in glucose levels. Conclusion Fluctuating glucose levels increased proliferation and migration of OLETF rat VSMCs via MAPK (ERK1/2), BMK1, PI3K, and NF-κB pathways. These effects were inhibited by the antioxidant rutin. PMID:26847289

The Interaction of Endothelin-1 and TGF-β1 Mediates Vascular Cell Remodeling

PubMed Central

Lambers, Christopher; Roth, Michael; Zhong, Jun; Campregher, Christoph; Binder, Petra; Burian, Bernhard; Petkov, Ventzislav; Block, Lutz-Henning

2013-01-01

Background Pulmonary arterial hypertension is characterized by increased thickness of pulmonary vessel walls due to both increased proliferation of pulmonary arterial smooth muscle cell (PASMC) and deposition of extracellular matrix. In patients suffering from pulmonary arterial hypertension, endothelin-1 (ET-1) synthesis is up-regulated and may increase PASMC activity and vessel wall remodeling through transforming growth factor beta-1 (TGF-β1) and connective tissue growth factor. Objective To assess the signaling pathway leading to ET-1 induced proliferation and extracellular matrix deposition by human PASMC. Methods PASMC were serum starved for 24 hours before stimulation with either ET-1 and/or TGF-β1. ET-1 was inhibited by Bosentan, ERK1/2 mitogen activated protein kinase (MAPK) was inhibited by U0126 and p38 MAPK was inhibited by SB203580. Results ET-1 increased PASMC proliferation when combined with serum. This effect involved the mitogen activated protein kinases (MAPK) ERK1/2 MAPK and was abrogated by Bosentan which caused a G1- arrest through activation of p27(Kip). Regarding the contribution of extracellular matrix deposition in vessel wall remodeling, TGF-β1 increased the deposition of collagen type-I and fibronectin, which was further increased when ET-1 was added mainly through ERK1/2 MAPK. In contrast, collagen type-IV was not affected by ET-1. Bosentan dose-dependently reduced the stimulatory effect of ET-1 on collagen type-I and fibronectin, but had no effect on TGF-β1. Conclusion and Clinical Relevance ET-1 alone does not induce PASMC proliferation and extracellular matrix deposition. However, ET-1 significantly up-regulates serum induced proliferation and TGF-β1 induced extracellular matrix deposition, specifically of collagen type-I and fibronectin. The synergistic effects of ET-1 on serum and TGF-β1 involve ERK1/2 MAPK and may thus present a novel mode of action in the pathogenesis of pulmonary arterial hypertension. PMID:24015303

Overactivation of Mitogen-Activated Protein Kinase and Suppression of Mitofusin-2 Expression Are Two Independent Events in High Mobility Group Box 1 Protein–Mediated T Cell Immune Dysfunction

PubMed Central

Tang, Lu-ming; Zhao, Guang-ju; Zhu, Xiao-mei; Dong, Ning; Yu, Yan

2013-01-01

High mobility group box 1 protein (HMGB1), a critical proinflammatory cytokine, has recently been identified to be an immunostimulatory signal involved in sepsis-related immune dysfunction when released extracellularly, but the potential mechanism involved remains elusive. Here, we showed that the treatment with HMGB1 in vitro inhibited T lymphocyte immune response and expression of mitofusin-2 (Mfn-2; a member of the mitofusin family) in a dose- and time-dependent manner. Upregulation of Mfn-2 expression attenuated the suppressive effect of HMGB1 on T cell immune function. The phosphorylation of both extracellular signal-regulated kinase (ERK)1/2 and p38 mitogen-activated protein kinase (MAPK) was markedly upregulated by treating with high amount of HMGB1, while pretreatment with ERK1/2 and p38 MAPK-specific inhibitors (U0126 and SB203580) could attenuate suppression of T cell immune function and nuclear factor of activated T cell (NFAT) activation induced by HMGB1, respectively. HMGB1-induced activity of ERK1/2 and p38 was not fully inhibited in the presence of U0126 or SB203580. Interestingly, overexpression of Mfn-2 had no marked effect on HMGB1-mediated activation of MAPK, but could attenuate the suppressive effect of HMGB1 on the activity of NFAT. Thus, the mechanisms involved in HMGB1-induced T cell immune dysfunction in vitro at least partly include suppression of Mfn-2 expression, overactivation of ERK1/2, p38 MAPK, and intervention of NFAT activation, while the protective effect of Mfn-2 on T cell immune dysfunction induced by HMGB1 is dependent on other signaling pathway associated with NFAT, but not MAPK. Taken together, we conclude that overactivation of MAPK and suppression of Mfn-2 expression are two independent events in HMGB1-mediated T cell immune dysfunction. PMID:23697559

Role of androgen receptor on cyclic mechanical stretch-regulated proliferation of C2C12 myoblasts and its upstream signals: IGF-1-mediated PI3K/Akt and MAPKs pathways.

PubMed

Ma, Yiming; Fu, Shaoting; Lu, Lin; Wang, Xiaohui

2017-07-15

To detect the effects of androgen receptor (AR) on cyclic mechanical stretch-modulated proliferation of C2C12 myoblasts and its pathways: roles of IGF-1, PI3K and MAPK. C2C12 were randomly divided into five groups: un-stretched control, six or 8 h of fifteen percent stretch, and six or 8 h of twenty percent stretch. Cyclic mechanical stretch of C2C12 were completed using a computer-controlled FlexCell Strain Unit. Cell proliferation and IGF-1 concentration in medium were detected by CCK8 and ELISA, respectively. Expressions of AR and IGF-1R, and expressions and activities of PI3K, p38 and ERK1/2 in stretched C2C12 cells were determined by Western blot. ①The proliferation of C2C12 cells, IGF-1 concentration in medium, expressions of AR and IGF-1R, and activities of PI3K, p38 and ERK1/2 were increased by 6 h of fifteen percent stretch, while decreased by twenty percent stretch for six or 8 h ②The fifteen percent stretch-increased proliferation of C2C12 cells was reversed by AR inhibitor, Flutamide. ③The increases of AR expression, activities of PI3K, p38 and ERK1/2 resulted from fifteen percent stretch were attenuated by IGF-1 neutralizing antibody, while twenty percent stretch-induced decreases of the above indicators were enhanced by recombinant IGF-1. ④Specific inhibitors of p38, ERK1/2 and PI3K all decreased the expression of AR in fifteen percent and twenty percent of stretched C2C12 cells. Cyclic mechanical stretch modulated the proliferation of C2C12 cells, which may be attributed to the alterations of AR via IGF-1-PI3K/Akt and IGF-1-MAPK (p38, ERK1/2) pathways in C2C12 cells. Copyright © 2017 Elsevier B.V. All rights reserved.

[MAPK signaling pathways involved in aluminum-induced apoptosis and necroptosis in SH-SY5Y cells].

PubMed

Jia, Xiaofang; Zhang, Qinli; Niu, Qiao

2014-11-01

To explore the role of MAPK signaling pathway in apoptosis and necroptosis induced by aluminum in SH-SY5Y cells. To imitate neural cell death induced by aluminium, AlCl3 x 6H2O (4 mmol/L) was used to treat SH-SY5Y cells. Necrostatin-1 (Nec-1,60 μmol/L), the specific inhibitor for necroptosis, and zVAD-fmk (20 μmol/L), the specific inhibitor for apoptosis, were added into cultures for inhibiting the occurrence of necroptosis and apoptosis. CCK-8 was performed to measure cell viability, flow cytometry was used to test the difference of apoptosis rate and necrosis rate between groups, and western-blot was used to detect the change of MAPK protein. Compared with blank control group, solvent control group, Nec-1 control group and zVAD-fmk control group, cell viabiligy of Al(3+) exposed group, Al(3+) plus Nec-1 group and Al(3+) plus zVAD-fmk group decreaced (P < 0.05). Compared with Al(3+) exposed group, cell viability of Al(3+) plus Nec-1 group and Al(3+) plus zVAD-fmk group increased (P < 0.05). Necrotic rate and apoptotic rate in Al(3+) exposed group, Al(3+) plus Nec-1 group and Al(3+) plus zVAD-fmk group obviously increased compared with blank control group, solvent control group, Nec-1 control group and zVAD-fmk control group (P < 0.05). Compared with Al(3+) exposed group, necrotic and apaptotic rate of Al(3+) plus zVAD-fmk group and Al(3+) plus Nec-1 group were statistically significant decreased (P < 0.05). Compared with blank control group, solvent control group, Nec-1 control group and zVAD-fmk control group, expression of p-p38 in Al(3+) exposed group, Al(3+) plus Nec-1 group and Al(3+) plus zVAD-fmk group increased obviously (P < 0.05), and expression of p-ERK decreased significantly (P < 0.05). Compared with Al(3+) exposed group, expression of p-p38 decreased (P < 0.05), but p-ERK increased in Al(3+) plus Nec-1 group (P < 0.05). The ERK and p38 MAPK signaling pathways are involved in aluminum-induced necroptosis in SH-SY5Y cells, but only ERK signaling pathway is involved in aluminum-induced apoptosis, and JNK signaling pathway is not involved in aluminum-induced cell death.

Angiotensin II increases CTGF expression via MAPKs/TGF-{beta}1/TRAF6 pathway in atrial fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Gu, Jun; Liu, Xu, E-mail: xkliuxu@yahoo.cn; Wang, Quan-xing, E-mail: shmywqx@126.com

2012-10-01

The activation of transforming growth factor-{beta}1(TGF-{beta}1)/Smad signaling pathway and increased expression of connective tissue growth factor (CTGF) induced by angiotensin II (AngII) have been proposed as a mechanism for atrial fibrosis. However, whether TGF{beta}1/non-Smad signaling pathways involved in AngII-induced fibrogenetic factor expression remained unknown. Recently tumor necrosis factor receptor associated factor 6 (TRAF6)/TGF{beta}-associated kinase 1 (TAK1) has been shown to be crucial for the activation of TGF-{beta}1/non-Smad signaling pathways. In the present study, we explored the role of TGF-{beta}1/TRAF6 pathway in AngII-induced CTGF expression in cultured adult atrial fibroblasts. AngII (1 {mu}M) provoked the activation of P38 mitogen activated proteinmore » kinase (P38 MAPK), extracellular signal-regulated kinase 1/2(ERK1/2) and c-Jun NH(2)-terminal kinase (JNK). AngII (1 {mu}M) also promoted TGF{beta}1, TRAF6, CTGF expression and TAK1 phosphorylation, which were suppressed by angiotensin type I receptor antagonist (Losartan) as well as p38 MAPK inhibitor (SB202190), ERK1/2 inhibitor (PD98059) and JNK inhibitor (SP600125). Meanwhile, both TGF{beta}1 antibody and TRAF6 siRNA decreased the stimulatory effect of AngII on TRAF6, CTGF expression and TAK1 phosphorylation, which also attenuated AngII-induced atrial fibroblasts proliferation. In summary, the MAPKs/TGF{beta}1/TRAF6 pathway is an important signaling pathway in AngII-induced CTGF expression, and inhibition of TRAF6 may therefore represent a new target for reversing Ang II-induced atrial fibrosis. -- Highlights: Black-Right-Pointing-Pointer MAPKs/TGF{beta}1/TRAF6 participates in AngII-induced CTGF expression in atrial fibroblasts. Black-Right-Pointing-Pointer TGF{beta}1/TRAF6 participates in AngII-induced atrial fibroblasts proliferation. Black-Right-Pointing-Pointer TRAF6 may represent a new target for reversing Ang II-induced atrial fibrosis.« less

MEK1 inhibits cardiac PPARα activity by direct interaction and prevents its nuclear localization.

PubMed

el Azzouzi, Hamid; Leptidis, Stefanos; Bourajjaj, Meriem; van Bilsen, Marc; da Costa Martins, Paula A; De Windt, Leon J

2012-01-01

The response of the postnatal heart to growth and stress stimuli includes activation of a network of signal transduction cascades, including the stress activated protein kinases such as p38 mitogen-activated protein kinase (MAPK), c-Jun NH2-terminal kinase (JNK) and the extracellular signal-regulated kinase (ERK1/2) pathways. In response to increased workload, the mitogen-activated protein kinase kinase (MAPKK) MEK1 has been shown to be active. Studies embarking on mitogen-activated protein kinase (MAPK) signaling cascades in the heart have indicated peroxisome-proliferators activated-receptors (PPARs) as downstream effectors that can be regulated by this signaling cascade. Despite the importance of PPARα in controlling cardiac metabolism, little is known about the relationship between MAPK signaling and cardiac PPARα signaling. Using co-immunoprecipitation and immunofluorescence approaches we show a complex formation of PPARα with MEK1 and not with ERK1/2. Binding of PPARα to MEK1 is mediated via a LXXLL motif and results in translocation from the nucleus towards the cytoplasm, hereby disabling the transcriptional activity of PPARα. Mice subjected to voluntary running-wheel exercise showed increased cardiac MEK1 activation and complex formation with PPARα, subsequently resulting in reduced PPARα activity. Inhibition of MEK1, using U0126, blunted this effect. Here we show that activation of the MEK1-ERK1/2 pathway leads to specific inhibition of PPARα transcriptional activity. Furthermore we show that this inhibitory effect is mediated by MEK1, and not by its downstream effector kinase ERK1/2, through a mechanism involving direct binding to PPARα and subsequent stimulation of PPARα export from the nucleus.

Metastatic function of BMP-2 in gastric cancer cells: The role of PI3K/AKT, MAPK, the NF-{kappa}B pathway, and MMP-9 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kang, Myoung Hee; Oh, Sang Cheul; Lee, Hyun Joo

2011-07-15

Bone morphogenetic proteins (BMPs) have been implicated in tumorigenesis and metastatic progression in various types of cancer cells, but the role and cellular mechanism in the invasive phenotype of gastric cancer cells is not known. Herein, we determined the roles of phosphoinositide 3-kinase (PI3K)/AKT, extracellular signal-regulated protein kinase (ERK), nuclear factor (NF)-{kappa}B, and matrix metalloproteinase (MMP) expression in BMP-2-mediated metastatic function in gastric cancer. We found that stimulation of BMP-2 in gastric cancer cells enhanced the phosphorylation of AKT and ERK. Accompanying activation of AKT and ERK kinase, BMP-2 also enhanced phosphorylation/degradation of I{kappa}B{alpha} and the nuclear translocation/activation of NF-{kappa}B.more » Interestingly, blockade of PI3K/AKT and ERK signaling using LY294002 and PD98059, respectively, significantly inhibited BMP-2-induced motility and invasiveness in association with the activation of NF-{kappa}B. Furthermore, BMP-2-induced MMP-9 expression and enzymatic activity was also significantly blocked by treatment with PI3K/AKT, ERK, or NF-{kappa}B inhibitors. Immunohistochemistry staining of 178 gastric tumor biopsies indicated that expression of BMP-2 and MMP-9 had a significant positive correlation with lymph node metastasis and a poor prognosis. These results indicate that the BMP-2 signaling pathway enhances tumor metastasis in gastric cancer by sequential activation of the PI3K/AKT or MAPK pathway followed by the induction of NF-{kappa}B and MMP-9 activity, indicating that BMP-2 has the potential to be a therapeutic molecular target to decrease metastasis.« less

Dobesilate diminishes activation of the mitogen - activated protein kinase ERK1/2 in glioma cells

PubMed Central

Cuevas, P; Diaz-González, Diana; Garcia-Martin-Córdova, C; Sánchez, I; Lozano, Rosa Maria; Giménez-Gallego, G; Dujovny, M

2006-01-01

Fibroblast growth factors (FGFs) and their receptors, regularly expressed at high levels in gliomas, are further upregulated during the transition of the tumor from low- to high-grade malignancy, and are essential for glioma progression. FGFs induce upregulation of the mitogen-activated protein kinase (MAPK) signaling cascade in cultured glioma cells, which suggests that MAPK pathway participates in the FGF-dependent glioma development. Recently, it has been shown that dobesilate, an inhibitor of FGF mitogenic activity, shows antiproliferative and proapoptotic activities in glioma cell cultures. Accordingly, it should be expected this new synthetic FGF inhibitor to affect the activation levels of MAPK. Here we report that immunocytochemical and Western blot data unequivocally show that treatment of cell cultures with dobesilate causes a significant decrease of the intracellular levels of ERK1/2 activation, one of the components of the MAPK signalling cascade. This finding supports an important role for dobesilate in glioma growth, suggesting that dobesilate should be a treatment to be born in mind for glioma management. PMID:16563234

Voltage-gated Na+ Channel Activity Increases Colon Cancer Transcriptional Activity and Invasion Via Persistent MAPK Signaling

NASA Astrophysics Data System (ADS)

House, Carrie D.; Wang, Bi-Dar; Ceniccola, Kristin; Williams, Russell; Simaan, May; Olender, Jacqueline; Patel, Vyomesh; Baptista-Hon, Daniel T.; Annunziata, Christina M.; Silvio Gutkind, J.; Hales, Tim G.; Lee, Norman H.

2015-06-01

Functional expression of voltage-gated Na+ channels (VGSCs) has been demonstrated in multiple cancer cell types where channel activity induces invasive activity. The signaling mechanisms by which VGSCs promote oncogenesis remain poorly understood. We explored the signal transduction process critical to VGSC-mediated invasion on the basis of reports linking channel activity to gene expression changes in excitable cells. Coincidentally, many genes transcriptionally regulated by the SCN5A isoform in colon cancer have an over-representation of cis-acting sites for transcription factors phosphorylated by ERK1/2 MAPK. We hypothesized that VGSC activity promotes MAPK activation to induce transcriptional changes in invasion-related genes. Using pharmacological inhibitors/activators and siRNA-mediated gene knockdowns, we correlated channel activity with Rap1-dependent persistent MAPK activation in the SW620 human colon cancer cell line. We further demonstrated that VGSC activity induces downstream changes in invasion-related gene expression via a PKA/ERK/c-JUN/ELK-1/ETS-1 transcriptional pathway. This is the first study illustrating a molecular mechanism linking functional activity of VGSCs to transcriptional activation of invasion-related genes.

Regulation of RAW 264.7 cell-mediated immunity by polysaccharides from Agaricus blazei Murill via the MAPK signal transduction pathway.

PubMed

Cheng, Feier; Yan, Xiaoyan; Zhang, Miaoqing; Chang, Mingchang; Yun, Shaojun; Meng, Junlong; Liu, Jingyu; Feng, Cui-Ping

2017-04-19

Agaricus blazei Murill (ABM) is a common anticancer folk remedy. Its active ingredients, i.e., polysaccharides, have been isolated and exhibit indirect tumor-suppressing activity via immunological activation. The effects of polysaccharides derived from A. blazei Murill (ABMP) on RAW 264.7 cells were examined by western blotting and real-time reverse transcription polymerase chain reaction (RT-PCR). The effects of 500, 1000, and 2000 μg mL -1 ABMP on the growth of RAW 264.7 cells were evaluated by measuring the OD 490 value; the optimum concentration was found to be 1000 μg mL -1 . Based on the RT-PCR results, the expression levels of JNK, ERK, and p38 decreased substantially in lipopolysaccharide (LPS)-induced RAW 264.7 cells treated with ABMP. In RAW 264.7 cells treated with LPS, the protein expression levels of JNK, ERK, and p38 were decreased, as were the levels of phosphorylated JNK, ERK, and p38. These results indicate that the MAPK signal transduction pathway is a potential mechanism by which ABMP regulates the cell-mediated immunity of RAW 264.7 cells.

Mitogen-activated protein kinase signaling in the heart: angels versus demons in a heart-breaking tale.

PubMed

Rose, Beth A; Force, Thomas; Wang, Yibin

2010-10-01

Among the myriad of intracellular signaling networks that govern the cardiac development and pathogenesis, mitogen-activated protein kinases (MAPKs) are prominent players that have been the focus of extensive investigations in the past decades. The four best characterized MAPK subfamilies, ERK1/2, JNK, p38, and ERK5, are the targets of pharmacological and genetic manipulations to uncover their roles in cardiac development, function, and diseases. However, information reported in the literature from these efforts has not yet resulted in a clear view about the roles of specific MAPK pathways in heart. Rather, controversies from contradictive results have led to a perception that MAPKs are ambiguous characters in heart with both protective and detrimental effects. The primary object of this review is to provide a comprehensive overview of the current progress, in an effort to highlight the areas where consensus is established verses the ones where controversy remains. MAPKs in cardiac development, cardiac hypertrophy, ischemia/reperfusion injury, and pathological remodeling are the main focuses of this review as these represent the most critical issues for evaluating MAPKs as viable targets of therapeutic development. The studies presented in this review will help to reveal the major challenges in the field and the limitations of current approaches and point to a critical need in future studies to gain better understanding of the fundamental mechanisms of MAPK function and regulation in the heart.

Tributyltin induces disruption of microfilament in HL7702 cells via MAPK-mediated hyperphosphorylation of VASP.

PubMed

Tu, Wei-Wei; Ji, Lin-Dan; Qian, Hai-Xia; Zhou, Mi; Zhao, Jin-Shun; Xu, Jin

2016-11-01

Tributyltin (TBT) has been widely used for various industrial purposes, and it has toxic effects on multiple organs and tissues. Previous studies have found that TBT could induce cytoskeletal disruption, especially of the actin filaments. However, the underlying mechanisms remain unclear. The aim of the present study was to determine whether TBT could induce microfilament disruption using HL7702 cells and then to assess for the total levels of various microfilament-associated proteins; finally, the involvement of the MAPK pathway was investigated. The results showed that after TBT treatment, F-actin began to depolymerize and lost its characteristic filamentous structure. The protein levels of Ezrin and Cofilin remained unchanged, the actin-related protein (ARP) 2/3 levels decreased slightly, and the vasodilator-stimulated phosphoprotein (VASP) decreased dramatically. However, the phosphorylation levels of VASP increased 2.5-fold, and the ratio of phosphorylated-VASP/unphosphorylated-VASP increased 31-fold. The mitogen-activated protein kinases (MAPKs) ERK and JNK were discovered to be activated. Inhibition of ERK and JNK not only largely diminished the TBT-induced hyperphosphorylation of VASP but also recovered the cellular morphology and rescued the cells from death. In summary, this study demonstrates that TBT-induced disruption of actin filaments is caused by the hyperphosphorylation of VASP through MAPK pathways. © 2015 Wiley Periodicals, Inc. Environ Toxicol 31: 1530-1538, 2016. © 2015 Wiley Periodicals, Inc.

Aqueous extract of Taxus chinensis (Pilger) Rehd inhibits lung carcinoma A549 cells through the epidermal growth factor receptor/mitogen-activated protein kinase pathway in vitro and in vivo.

PubMed

Shu, Qijin; Shen, Minhe; Wang, Binbin; Cui, Qingli; Zhou, Xiaoying; Zhu, Luming

2014-06-01

To explore the anticancer mechanism of aqueous extract of Taxus Chinensis (Pilger) Rehd (AETC). The serum pharmacological method was used to avoid interference from administration of the crude medicinal herbs. Eight purebred New Zealand rabbits were used for preparation of serum containing various concentrations of AETC. Forty-eight Balb/c-nu mice were used for in vivo experiments. The effects of serum containing AETC on the proliferation of A549 cells and expression levels of the epidermal growth factor receptor/mitogen-activated protein kinase (EGFR/MAPK) pathway-related proteins in vitro were investigated. Additionally, the effects on the growth of A549 xenografts in nude mice, and expression levels of the EGFR/MAPK pathway-related proteins in the xenografts, were investigated. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay revealed that the serum containing AETC significantly decreased the viability of A549 cells in a dose-dependent manner. Western blot showed that the serum containing various concentrations of AETC strongly reduced the levels of phospho-Jun N-terminal kinase (p-JNK) and phospho-extracellular signal-regulated kinasel/2 (ERK1/2) while it increased the level of p-p38. However, no significant effects on the expression levels of JNK, ERK1/2, and p38 MAPK were found. In addition, an anticancer effect from AETC was observed in vivo in the Balb/c-nu mice bearing A549 xenografts. AETC has significant effects on the growth of A549 xenografts and on the activity of the EGFR/MAPK pathway. Therefore, AETC may be beneficial in lung carcinoma treatment.

Spatiotemporal regulation of ERK2 by dual specificity phosphatases.

PubMed

Caunt, Christopher J; Armstrong, Stephen P; Rivers, Caroline A; Norman, Michael R; McArdle, Craig A

2008-09-26

Although many stimuli activate extracellular signal-regulated kinases 1 and 2 (ERK1/2), the kinetics and compartmentalization of ERK1/2 signals are stimulus-dependent and dictate physiological consequences. ERKs can be inactivated by dual specificity phosphatases (DUSPs), notably the MAPK phosphatases (MKPs) and atypical DUSPs, that can both dephosphorylate and scaffold ERK1/2. Using a cell imaging model (based on knockdown of endogenous ERKs and add-back of wild-type or mutated ERK2-GFP reporters), we explored possible effects of DUSPs on responses to transient or sustained ERK2 activators (epidermal growth factor and phorbol 12,13-dibutyrate, respectively). For both stimuli, a D319N mutation (which impairs DUSP binding) increased ERK2 activity and reduced nuclear accumulation. These stimuli also increased mRNA levels for eight DUSPs. In a short inhibitory RNA screen, 12 of 16 DUSPs influenced ERK2 responses. These effects were evident among nuclear inducible MKP, cytoplasmic ERK MKP, JNK/p38 MKP, and atypical DUSP subtypes and, with the exception of the nuclear inducible MKPs, were paralleled by corresponding changes in Egr-1 luciferase activation. Simultaneous removal of all JNK/p38 MKPs or nuclear inducible MKPs revealed them as positive and negative regulators of ERK2 signaling, respectively. The effects of JNK/p38 MKP short inhibitory RNAs were not dependent on protein neosynthesis but were reversed in the presence of JNK and p38 kinase inhibitors, indicating DUSP-mediated cross-talk between MAPK pathways. Overall, our data reveal that a large number of DUSPs influence ERK2 signaling. Together with the known tissue-specific expression of DUSPs and the importance of ERK1/2 in cell regulation, our data support the potential value of DUSPs as targets for drug therapy.

ERK1/2/MAPK pathway-dependent regulation of the telomeric factor TRF2

PubMed Central

Picco, Vincent; Coste, Isabelle; Giraud-Panis, Marie-Josèphe; Renno, Toufic; Gilson, Eric; Pagès, Gilles

2016-01-01

Telomere stability is a hallmark of immortalized cells, including cancer cells. While the telomere length is maintained in most cases by the telomerase, the activity of a protein complex called Shelterin is required to protect telomeres against unsuitable activation of the DNA damage response pathway. Within this complex, telomeric repeat binding factor 2 (TRF2) plays an essential role by blocking the ataxia telangiectasia-mutated protein (ATM) signaling pathway at telomeres and preventing chromosome end fusion. We showed that TRF2 was phosphorylated in vitro and in vivo on serine 323 by extracellular signal-regulated kinase (ERK1/2) in both normal and cancer cells. Moreover, TRF2 and activated ERK1/2 unexpectedly interacted in the cytoplasm of tumor cells and human tumor tissues. The expression of non-phosphorylatable forms of TRF2 in melanoma cells induced the DNA damage response, leading to growth arrest and tumor reversion. These findings revealed that the telomere stability is under direct control of one of the major pro-oncogenic signaling pathways (RAS/RAF/MEK/ERK) via TRF2 phosphorylation. PMID:27366950

Betulinic acid exerts anti-hepatitis C virus activity via the suppression of NF-κB- and MAPK-ERK1/2-mediated COX-2 expression.

PubMed

Lin, Chun-Kuang; Tseng, Chin-Kai; Chen, Kai-Hsun; Wu, Shih-Hsiung; Liaw, Chih-Chuang; Lee, Jin-Ching

2015-06-23

This study was designed to evaluate the effect of betulinic acid (BA), extracted from Avicennia marina, on the replication of hepatitis C virus (HCV) and to investigate the mechanism of this BA-mediated anti-HCV activity. HCV replicon and infectious systems were used to evaluate the anti-HCV activity of BA. Exogenous COX-2 or knock-down of COX-2 expression was used to investigate the role of COX-2 in the anti-HCV activity of BA. The effects of BA on the phosphorylation of NF-κB and on kinases in the MAPK signalling pathway were determined. The anti-HCV activity of BA in combination with other HCV inhibitors was also determined to assess its use as an anti-HCV supplement. BA inhibited HCV replication in both Ava5 replicon cells and in a cell culture-derived infectious HCV particle system. Treatment with a combination of BA and IFN-α, the protease inhibitor telaprevir or the NS5B polymerase inhibitor sofosbuvir resulted in the synergistic suppression of HCV RNA replication. Exogenous overexpression of COX-2 gradually attenuated the inhibitory effect of BA on HCV replication, suggesting that BA reduces HCV replication by suppressing the expression of COX-2. In particular, BA down-regulated HCV-induced COX-2 expression by reducing the phosphorylation of NF-κB and ERK1/2 of the MAPK signalling pathway. BA inhibits HCV replication by suppressing the NF-κB- and ERK1/2-mediated COX-2 pathway and may serve as a promising compound for drug development or as a potential supplement for use in the treatment of HCV-infected patients. © 2015 The British Pharmacological Society.

Discovery and validation of the tumor-suppressive function of long noncoding RNA PANDA in human diffuse large B-cell lymphoma through the inactivation of MAPK/ERK signaling pathway.

PubMed

Wang, Yingjun; Zhang, Mingzhi; Xu, Huanan; Wang, Yifei; Li, Zhaoming; Chang, Yu; Wang, Xinhuan; Fu, Xiaorui; Zhou, Zhiyuan; Yang, Siyuan; Wang, Bei; Shang, Yufeng

2017-09-22

Diffuse large B-cell lymphoma (DLBCL) is one of the leading causes of cancer-related mortality, and responds badly to existing treatment. Thus, it is of urgent need to identify novel prognostic markers and therapeutic targets of DLBCL. Recent studies have shown that long non-coding RNAs (lncRNAs) play an important role in the development of cancer. By using the next generation HiSeq sequencing assay, we determined lncRNAs exhibiting differential expression between DLBCL patients and healthy controls. Then, RT-qPCR was performed for identification in clinical samples and cell materials, and lncRNA PANDA was verified to be down-regulated in DLBCL patients and have considerable diagnostic potential. In addition, decreased serum PANDA level was correlated to poorer clinical outcome and lower overall survival in DLBCL patients. Subsequently, we determined the experimental role of lncRNA PANDA in DLBCL progression. Luciferase reporter assay and chromatin immunoprecipitation assay suggested that lncRNA PANDA was induced by p53 and p53 interacts with the promoter region of PANDA. Cell functional assay further indicated that PANDA functioned as a tumor suppressor gene through the suppression of cell growth by a G0/G1 cell cycle arrest in DLBCL. More importantly, Cignal Signal Transduction Reporter Array and western blot assay showed that lncRNA PANDA inactivated the MAPK/ERK signaling pathway. In conclusion, our integrated approach demonstrates that PANDA in DLBCL confers a tumor suppressive function through inhibiting cell proliferation and silencing MAPK/ERK signaling pathway. Thus, PANDA may be a promising therapeutic target for patients with DLBCL.

Oxyfadichalcone C inhibits melanoma A375 cell proliferation and metastasis via suppressing PI3K/Akt and MAPK/ERK pathways.

PubMed

Peng, Xiaolin; Wang, Zhengming; Liu, Yang; Peng, Xin; Liu, Yao; Zhu, Shan; Zhang, Zhe; Qiu, Yuling; Jin, Meihua; Wang, Ran; Zhang, Qingying; Kong, Dexin

2018-08-01

Melanoma remains to be one of the most incurable cancers. Discovery of novel antitumor agent for melanoma therapy is expected. We recently isolated Oxyfadichalcone C from Oxytropis falcate and investigated the anti-proliferative and anti-metastatic activity on human melanoma A375 cells in vitro. Cell viability was determined using MTT assay and soft agar cloning formation assay. The effect of Oxyfadichalcone C on cell cycle distribution and apoptosis were analyzed by flow cytometry. Cell metastasis was determined by wound healing assay, Transwell assay and Gelatin zymography assay. The effect of Oxyfadichalcone C on signal proteins of PI3K/Akt and MAPK/ERK pathways was examined by western blot analysis. Synergism assay was employed to determine whether combination of Oxyfadichalcone C with Vemurafenib would enhance the anti-proliferative effect. Oxyfadichalcone C potently inhibited proliferation, induced G1 phase arrest and weak apoptosis in A375 cells. Anti-migration and anti-invasion activities were also indicated. Such effects were associated with upregulation of p27, reduction of cyclin D1, p-pRb, p-Integrin β1, as well as the proteolytic activity of metalloproteinase (MMP)-2/9. Meanwhile, key molecules of PI3K/Akt and MAPK/ERK pathways were downregulated, which might be involved in the inhibition against proliferation and metastasis of A375 cells by Oxyfadichalcone C. In addition, combination of Oxyfadichalcone C with Vemurafenib at a ratio of IC50 Oxyfadichalcone C : 5 × IC 50 Vemurafenib exhibited synergistic anti-proliferative effect on A375 cells. Our findings suggest that Oxyfadichalcone C has the potential to be developed as a promising drug candidate for the treatment of melanoma. Copyright © 2018 Elsevier Inc. All rights reserved.

Oncogenic KIAA1549-BRAF fusion with activation of the MAPK/ERK pathway in pediatric oligodendrogliomas.

PubMed

Kumar, Anupam; Pathak, Pankaj; Purkait, Suvendu; Faruq, Mohammed; Jha, Prerana; Mallick, Supriya; Suri, Vaishali; Sharma, Mehar C; Suri, Ashish; Sarkar, Chitra

2015-03-01

Pediatric oligodendrogliomas (pODGs) are rare central nervous system tumors, and comparatively little is known about their molecular pathogenesis. Co-deletion of 1p/19q; and IDH1, CIC, and FUBP1 mutations, which are molecular signatures of adult oligodendrogliomas, are extremely rare in pODGs. In this report, two pODGs, one each of grade II and grade III, were evaluated using clinical, radiological, histopathologic, and follow-up methods. IDH1, TP53, CIC, H3F3A, and BRAF-V600 E mutations were analyzed by Sanger sequencing and immunohistochemical methods, and 1p/19q co-deletion was analyzed by fluorescence in situ hybridization. PDGFRA amplification, BRAF gain, intragenic duplication of FGFR-TKD, and KIAA1549-BRAF fusion (validated by Sanger sequencing) were analyzed by real-time reverse transcription PCR. Notably, both cases showed the oncogenic KIAA1549_Ex15-BRAF_Ex9 fusion transcript. Further, immunohistochemical analysis showed activation of the MAPK/ERK pathway in both of these cases. However, neither 1p/19q co-deletion; IDH1, TP53, CIC, H3F3A, nor BRAF-V600 E mutation; PDGFRA amplification; BRAF gain; nor duplication of FGFR-TKD was identified. Overall, this study highlights that pODGs can harbor the KIAA1549-BRAF fusion with aberrant MAPK/ERK signaling, and there exists an option of targeting these pathways in such patients. These results indicate that pODGs with the KIAA1549-BRAF fusion may represent a subset of this rare tumor that shares molecular and genetic features of pilocytic astrocytomas. These findings will increase our understanding of pODGs and may have clinical implications. Copyright © 2015 Elsevier Inc. All rights reserved.

Tentacle extract from the jellyfish Cyanea capillata increases proliferation and migration of human umbilical vein endothelial cells through the ERK1/2 signaling pathway

PubMed Central

Wang, Qianqian; Zhang, Hui; Liu, Guoyan; He, Qian; Zhang, Liming

2017-01-01

Wound healing is a complex biological process, and current research finds that jellyfish have a great capacity for promoting growth and healing. However, the underlying mechanisms remain unclear. Thus, this study was conducted to investigate the molecular mechanisms and effects of a tentacle extract (TE) from the jellyfish Cyanea capillata (C. capillata) on cell proliferation and migration in human umbilical vein endothelial cells (HUVECs). First, our results showed that TE at the concentration of 1 μg/ml could promote cell proliferation over various durations, induce a transition of the cells from the G1-phase to the S/G2-phase of the cell cycle, and increase the expression of cell cycle proteins (CyclinB1 and CyclinD1). Second, we found that TE could activate the PI3K/Akt, ERK1/2 and JNK MAPK signaling pathways but not the NF-κB signaling pathway or the apoptosis signaling cascade. Finally, we demonstrated that the TE-induced expression of cell cycle proteins was decreased by ERK1/2 inhibitor PD98059 but not by PI3K inhibitor LY294002 or JNK inhibitor SP600125. Similarly, the TE-enhanced migration ability of HUVECs was also markedly attenuated by PD98059. Taken together, our findings indicate that TE-induced proliferation and migration in HUVECs mainly occurred through the ERK1/2 MAPK signaling pathway. These results are instructively important for further research on the isolation and purification of growth-promoting factors from C. capillata and are hopeful as a means to improve human wound repair in unfavorable conditions. PMID:29261770

Tentacle extract from the jellyfish Cyanea capillata increases proliferation and migration of human umbilical vein endothelial cells through the ERK1/2 signaling pathway.

PubMed

Wang, Beilei; Liu, Dan; Wang, Chao; Wang, Qianqian; Zhang, Hui; Liu, Guoyan; He, Qian; Zhang, Liming

2017-01-01

Wound healing is a complex biological process, and current research finds that jellyfish have a great capacity for promoting growth and healing. However, the underlying mechanisms remain unclear. Thus, this study was conducted to investigate the molecular mechanisms and effects of a tentacle extract (TE) from the jellyfish Cyanea capillata (C. capillata) on cell proliferation and migration in human umbilical vein endothelial cells (HUVECs). First, our results showed that TE at the concentration of 1 μg/ml could promote cell proliferation over various durations, induce a transition of the cells from the G1-phase to the S/G2-phase of the cell cycle, and increase the expression of cell cycle proteins (CyclinB1 and CyclinD1). Second, we found that TE could activate the PI3K/Akt, ERK1/2 and JNK MAPK signaling pathways but not the NF-κB signaling pathway or the apoptosis signaling cascade. Finally, we demonstrated that the TE-induced expression of cell cycle proteins was decreased by ERK1/2 inhibitor PD98059 but not by PI3K inhibitor LY294002 or JNK inhibitor SP600125. Similarly, the TE-enhanced migration ability of HUVECs was also markedly attenuated by PD98059. Taken together, our findings indicate that TE-induced proliferation and migration in HUVECs mainly occurred through the ERK1/2 MAPK signaling pathway. These results are instructively important for further research on the isolation and purification of growth-promoting factors from C. capillata and are hopeful as a means to improve human wound repair in unfavorable conditions.

Differential pathway coupling efficiency of the activated insulin receptor drives signaling selectivity by XMetA, an allosteric partial agonist antibody

USDA-ARS?s Scientific Manuscript database

XMetA, an anti-insulin receptor (IR) monoclonal antibody, is an allosteric partial agonist of the IR. We have previously reported that XMetA activates the “metabolic-biased” Akt kinase signaling pathway while having little or no effect on the “mitogenic” MAPK signaling pathwayof ERK 1/2. To inves...

Differential pathway coupling efficiency of the activated insulin receptor drives signaling selectivity by xmeta, an allosteric partial agonist antibody

USDA-ARS?s Scientific Manuscript database

XMetA, an anti-insulin receptor (IR) monoclonal antibody, is an allosteric partial agonist of the IR. We have previously reported that XMetA activates the “metabolic-biased” Akt kinase signaling pathway while having little or no effect on the “mitogenic” MAPK signaling pathwayof ERK 1/2. To inves...

Fisetin Inhibits Human Melanoma Cell Invasion through Promotion of Mesenchymal to Epithelial Transition and by Targeting MAPK and NFκB Signaling Pathways

PubMed Central

Pal, Harish Chandra; Sharma, Samriti; Strickland, Leah Ray; Katiyar, Santosh K.; Ballestas, Mary E.; Athar, Mohammad; Elmets, Craig A.; Afaq, Farrukh

2014-01-01

Malignant melanoma is responsible for approximately 75% of skin cancer-related deaths. BRAF plays an important role in regulating the mitogen-activated protein kinase (MAPK) signaling cascade in melanoma with activating mutations in the serine/threonine kinase BRAF occurring in 60–70% of malignant melanomas. The BRAF-MEK-ERK (MAPK) pathway is a key regulator of melanoma cell invasion. In addition, activation of NFκB via the MAPK pathway is regulated through MEK-induced activation of IKK. These pathways are potential targets for prevention and treatment of melanoma. In this study, we investigated the effect of fisetin, a phytochemical present in fruits and vegetables, on melanoma cell invasion and epithelial-mesenchymal transition, and delineated the underlying molecular mechanism. Treatment of multiple human malignant melanoma cell lines with fisetin (5–20 µM) resulted in inhibition of cell invasion. BRAF mutated melanoma cells were more sensitive to fisetin treatment, and this was associated with a decrease in the phosphorylation of MEK1/2 and ERK1/2. In addition, fisetin inhibited the activation of IKK leading to a reduction in the activation of the NFκB signaling pathway. Treatment of cells with an inhibitor of MEK1/2 (PD98059) or of NFκB (caffeic acid phenethyl ester) also reduced melanoma cell invasion. Furthermore, treatment of fisetin promoted mesenchymal to epithelial transition in melanoma cells, which was associated with a decrease in mesenchymal markers (N-cadherin, vimentin, snail and fibronectin) and an increase in epithelial markers (E-cadherin and desmoglein). Employing three dimensional skin equivalents consisting of A375 cells admixed with normal human keratinocytes embedded onto a collagen-constricted fibroblast matrix, we found that treatment of fisetin reduced the invasive potential of melanoma cells into the dermis and increased the expression of E-cadherin with a concomitant decrease in vimentin. These results indicate that fisetin inhibits melanoma cell invasion through promotion of mesenchymal to epithelial transition and by targeting MAPK and NFκB signaling pathways. PMID:24466036

Effects of 17β-estradiol on the release of monocyte chemotactic protein-1 and MAPK activity in monocytes stimulated with peritoneal fluid from endometriosis patients.

PubMed

Lee, Dong-Hyung; Kim, Seung-Chul; Joo, Jong-Kil; Kim, Hwi-Gon; Na, Young-Jin; Kwak, Jong-Young; Lee, Kyu-Sup

2012-03-01

Hormones and inflammation have been implicated in the pathological process of endometriosis; therefore, we investigated the combined effects of 17β-estradiol (E2) and peritoneal fluid obtained from patients with endometriosis (ePF) or a control peritoneal fluid (cPF) obtained from patients without endometriosis on the release of monocyte chemotactic protein-1 (MCP-1) by monocytes and the role of signaling pathways. Monocytes were cultured with ePF and cPF in the presence of E2; the MCP-1 levels in the supernatants were then measured by ELISA. In addition, mitogen activated protein kinase (MAPK) activation was measured by Western blotting of phosphorylated proteins. E2 down-regulated MCP-1 release by lipopolysaccharide- or cPF-treated monocytes, but failed to suppress its release by ePF-treated monocytes. The release of MCP-1 by ePF- and cPF-treated monocytes was efficiently abrogated by p38 mitogen activated protein kinase (MAPK) inhibitors; however, the MCP-1 release by cPF-treated monocytes, but not by ePF-treated monocytes, was blocked by a MAPK kinase inhibitor. In addition, ePF and cPF induced the phosphorylation of extracellular stress regulated kinase (ERK)1/2, p38 MAPK and c-Jun N-terminal kinase (JNK). E2 decreased the phosphorylation of p38 MAPK, but not ERK1/2 in ePF-treated monocytes; however, E2 decreased the phosphorylation of p38 MAPK, ERK1/2 and JNK in cPF-treated monocytes. The ability of E2 to modulate MCP-1 production is impaired in ePF-treated monocytes, which may be related to regulation of MAPK activity. These findings suggest that the failure of E2 to suppress ePF-treated production of MCP-1 may be involved in the pathogenesis of endometriosis. © 2012 The Authors. Journal of Obstetrics and Gynaecology Research © 2012 Japan Society of Obstetrics and Gynecology.

Macrophages produce IL-33 by activating MAPK signaling pathway during RSV infection.

PubMed

Qi, Feifei; Bai, Song; Wang, Dandan; Xu, Lei; Hu, Haiyan; Zeng, Sheng; Chai, Ruonan; Liu, Beixing

2017-07-01

It has been reported that RSV infection can enhance IL-33 production in lung macrophages. However, little is known about specific signaling pathways for activation of macrophages during RSV infection. In the present study, by using real-time RT-PCR as well as western blot assay, it became clear that RSV infection can enhance not only the expression of mRNAs for MAPK molecules (including p38, JNK1/2, and ERK1/2), but also the levels of MAPK proteins in lung macrophages as well as RAW264.7 cells. Furthermore, infection with RSV resulted in an increased level of phosphorylated MAPK proteins in RAW264.7 cells, suggesting that MAPK signaling pathway may participate in the process of RSV-induced IL-33 secretion by macrophages. In fact, the elevated production of IL-33 in RAW264.7 was attenuated significantly by pretreatment of the cells with special MAPK inhibitor before RSV infection, further confirming the function of MAPKs pathway in RSV-induced IL-33 production in macrophages. In contrast, the expression of NF-κB mRNA as well as the production of NF-κB protein in lung macrophages and RAW264.7 cells was not enhanced markedly after RSV infection. Moreover, RSV infection failed to induce the phosphorylation of NF-κB in RAW264.7 cells, suggesting that NF-κB signaling pathway may be not involved in RSV-induced IL-33 production in macrophages. Conclusion, these results indicate that RSV-induced production of IL-33 in macrophages is dependent on the activation of MAPK signaling pathway. Copyright © 2017 Elsevier Ltd. All rights reserved.

Stimulation of neuronal neurite outgrowth using functionalized carbon nanotubes

NASA Astrophysics Data System (ADS)

Matsumoto, K.; Sato, C.; Naka, Y.; Whitby, R.; Shimizu, N.

2010-03-01

Low concentrations (0.11-1.7 µg ml - 1) of functionalized carbon nanotubes (CNTs), which are multi-walled CNTs modified by amino groups, when added with nerve growth factor (NGF), promoted outgrowth of neuronal neurites in dorsal root ganglion (DRG) neurons and rat pheochromocytoma cell line PC12h cells in culture media. The quantity of active extracellular signal-regulated kinase (ERK) was higher after the addition of both 0.85 µg ml - 1 CNTs and NGF than that with NGF alone. CNTs increased the number of cells with neurite outgrowth in DRG neurons and PC12h cells after the inhibition of the ERK signaling pathway using a mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor. Active ERK proteins were detected in MEK inhibitor-treated neurons after the addition of CNTs to the culture medium. These results demonstrate that CNTs may stimulate neurite outgrowth by activation of the ERK signaling pathway. Thus, CNTs are biocompatible and are promising candidates for biological applications and devices.

Tumor suppressor activity of the ERK/MAPK pathway by promoting selective protein degradation

PubMed Central

Deschênes-Simard, Xavier; Gaumont-Leclerc, Marie-France; Bourdeau, Véronique; Lessard, Frédéric; Moiseeva, Olga; Forest, Valérie; Igelmann, Sebastian; Mallette, Frédérick A.; Saba-El-Leil, Marc K.; Meloche, Sylvain; Saad, Fred; Mes-Masson, Anne-Marie; Ferbeyre, Gerardo

2013-01-01

Constitutive activation of growth factor signaling pathways paradoxically triggers a cell cycle arrest known as cellular senescence. In primary cells expressing oncogenic ras, this mechanism effectively prevents cell transformation. Surprisingly, attenuation of ERK/MAP kinase signaling by genetic inactivation of Erk2, RNAi-mediated knockdown of ERK1 or ERK2, or MEK inhibitors prevented the activation of the senescence mechanism, allowing oncogenic ras to transform primary cells. Mechanistically, ERK-mediated senescence involved the proteasome-dependent degradation of proteins required for cell cycle progression, mitochondrial functions, cell migration, RNA metabolism, and cell signaling. This senescence-associated protein degradation (SAPD) was observed not only in cells expressing ectopic ras, but also in cells that senesced due to short telomeres. Individual RNAi-mediated inactivation of SAPD targets was sufficient to restore senescence in cells transformed by oncogenic ras or trigger senescence in normal cells. Conversely, the anti-senescence viral oncoproteins E1A, E6, and E7 prevented SAPD. In human prostate neoplasms, high levels of phosphorylated ERK were found in benign lesions, correlating with other senescence markers and low levels of STAT3, one of the SAPD targets. We thus identified a mechanism that links aberrant activation of growth signaling pathways and short telomeres to protein degradation and cellular senescence. PMID:23599344

MAPK signaling pathways and HDAC3 activity are disrupted during differentiation of emerin-null myogenic progenitor cells

PubMed Central

Collins, Carol M.; Ellis, Joseph A.

2017-01-01

ABSTRACT Mutations in the gene encoding emerin cause Emery–Dreifuss muscular dystrophy (EDMD). Emerin is an integral inner nuclear membrane protein and a component of the nuclear lamina. EDMD is characterized by skeletal muscle wasting, cardiac conduction defects and tendon contractures. The failure to regenerate skeletal muscle is predicted to contribute to the skeletal muscle pathology of EDMD. We hypothesize that muscle regeneration defects are caused by impaired muscle stem cell differentiation. Myogenic progenitors derived from emerin-null mice were used to confirm their impaired differentiation and analyze selected myogenic molecular pathways. Emerin-null progenitors were delayed in their cell cycle exit, had decreased myosin heavy chain (MyHC) expression and formed fewer myotubes. Emerin binds to and activates histone deacetylase 3 (HDAC3). Here, we show that theophylline, an HDAC3-specific activator, improved myotube formation in emerin-null cells. Addition of the HDAC3-specific inhibitor RGFP966 blocked myotube formation and MyHC expression in wild-type and emerin-null myogenic progenitors, but did not affect cell cycle exit. Downregulation of emerin was previously shown to affect the p38 MAPK and ERK/MAPK pathways in C2C12 myoblast differentiation. Using a pure population of myogenic progenitors completely lacking emerin expression, we show that these pathways are also disrupted. ERK inhibition improved MyHC expression in emerin-null cells, but failed to rescue myotube formation or cell cycle exit. Inhibition of p38 MAPK prevented differentiation in both wild-type and emerin-null progenitors. These results show that each of these molecular pathways specifically regulates a particular stage of myogenic differentiation in an emerin-dependent manner. Thus, pharmacological targeting of multiple pathways acting at specific differentiation stages may be a better therapeutic approach in the future to rescue muscle regeneration in vivo. PMID:28188262

Trichomonas vaginalis Induces Production of Proinflammatory Cytokines in Mouse Macrophages Through Activation of MAPK and NF-κB Pathways Partially Mediated by TLR2

PubMed Central

Li, Ling; Li, Xin; Gong, Pengtao; Zhang, Xichen; Yang, Zhengtao; Yang, Ju; Li, Jianhua

2018-01-01

Trichomoniasis, caused by Trichomonas vaginalis infection, is the most prevalent sexually transmitted disease in female and male globally. However, the mechanisms by innate immunity against T. vaginalis infection have not been fully elucidated. Toll-like receptor2 (TLR2) has been shown to be involved in pathogen recognition, innate immunity activation, and inflammatory response to the pathogens. Nonetheless, the function of TLR2 against T. vaginalis remains unclear. In the present study, we investigated the role of TLR2 in mouse macrophages against T. vaginalis. RT-qPCR analysis revealed that T. vaginalis stimulation increased the gene expression of TLR2 in wild-type (WT) mouse macrophages. T. vaginalis also induced the secretion of IL-6, TNF-α, and IFN-γ in WT mouse macrophages, and the expression of these cytokines significantly decreased in TLR2-/- mouse macrophages and in WT mouse macrophages pretreated with MAPK inhibitors SB203580 (p38) and PD98059 (ERK). Western blot analysis demonstrated that T. vaginalis stimulation induced the activation of p38, ERK, and p65 NF-κB signal pathways in WT mouse macrophages, and the phosphorylation of p38, ERK, and p65 NF-κB significantly decreased in TLR2-/- mouse macrophages. Taken together, our data suggested that T. vaginalis may regulates proinflammatory cytokines production by activation of p38, ERK, and NF-κB p65 signal pathways via TLR2 in mouse macrophages. TLR2 might be involved in the defense and elimination of T. vaginalis infection. PMID:29692771

Trichomonas vaginalis Induces Production of Proinflammatory Cytokines in Mouse Macrophages Through Activation of MAPK and NF-κB Pathways Partially Mediated by TLR2.

PubMed

Li, Ling; Li, Xin; Gong, Pengtao; Zhang, Xichen; Yang, Zhengtao; Yang, Ju; Li, Jianhua

2018-01-01

Trichomoniasis, caused by Trichomonas vaginalis infection, is the most prevalent sexually transmitted disease in female and male globally. However, the mechanisms by innate immunity against T. vaginalis infection have not been fully elucidated. Toll-like receptor2 (TLR2) has been shown to be involved in pathogen recognition, innate immunity activation, and inflammatory response to the pathogens. Nonetheless, the function of TLR2 against T. vaginalis remains unclear. In the present study, we investigated the role of TLR2 in mouse macrophages against T. vaginalis . RT-qPCR analysis revealed that T. vaginalis stimulation increased the gene expression of TLR2 in wild-type (WT) mouse macrophages. T. vaginalis also induced the secretion of IL-6, TNF-α, and IFN-γ in WT mouse macrophages, and the expression of these cytokines significantly decreased in TLR 2-/- mouse macrophages and in WT mouse macrophages pretreated with MAPK inhibitors SB203580 (p38) and PD98059 (ERK). Western blot analysis demonstrated that T. vaginalis stimulation induced the activation of p38, ERK, and p65 NF-κB signal pathways in WT mouse macrophages, and the phosphorylation of p38, ERK, and p65 NF-κB significantly decreased in TLR2 -/- mouse macrophages. Taken together, our data suggested that T. vaginalis may regulates proinflammatory cytokines production by activation of p38, ERK, and NF-κB p65 signal pathways via TLR2 in mouse macrophages. TLR2 might be involved in the defense and elimination of T. vaginalis infection.

Formononetin-induced apoptosis of human prostate cancer cells through ERK1/2 mitogen-activated protein kinase inactivation.

PubMed

Ye, Y; Hou, R; Chen, J; Mo, L; Zhang, J; Huang, Y; Mo, Z

2012-04-01

Formononetin is a main active component of red clover plants (Trifolium pratense L.), and is considered as a phytoestrogen. Our previous studies demonstrated that formononetin caused cell cycle arrest at the G0/G1 phase by inactivating insulin-like growth factor 1(IGF1)/IGF1R-phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway in MCF-7 cells. In the present study, we investigated the molecular mechanisms involved in the effect of formononetin on prostate cancer cells. Our results suggested that higher concentrations of formononetin inhibited the proliferation of prostate cancer cells (LNCaP and PC-3), while the most striking effect was observed in LNCaP cells. We further found that formononetin inactivated extracellular signal-regulated kinase1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signaling pathway in a dose-dependent manner, which resulted in increased the expression levels of BCL2-associated X (Bax) mRNA and protein, and induced apoptosis in LNCaP cells. Thus, we concluded that the induced apoptosis effect of formononetin on human prostate cancer cells was related to ERK1/2 MAPK-Bax pathway. Considering that red clover plants were widely used clinically, our results provided the foundation for future development of different concentrations formononetin for treatment of prostate cancer. © Georg Thieme Verlag KG Stuttgart · New York.

The dual-specificity phosphatase MKP-1 limits the cardiac hypertrophic response in vitro and in vivo.

PubMed

Bueno, O F; De Windt, L J; Lim, H W; Tymitz, K M; Witt, S A; Kimball, T R; Molkentin, J D

2001-01-19

Mitogen-activated protein kinase (MAPK) signaling pathways are important regulators of cell growth, proliferation, and stress responsiveness. A family of dual-specificity MAP kinase phosphatases (MKPs) act as critical counteracting factors that directly regulate the magnitude and duration of p38, c-Jun N-terminal kinase (JNK), and extracellular signal-regulated kinase (ERK) activation. Here we show that constitutive expression of MKP-1 in cultured primary cardiomyocytes using adenovirus-mediated gene transfer blocked the activation of p38, JNK1/2, and ERK1/2 and prevented agonist-induced hypertrophy. Transgenic mice expressing physiological levels of MKP-1 in the heart showed (1) no activation of p38, JNK1/2, or ERK1/2; (2) diminished developmental myocardial growth; and (3) attenuated hypertrophy in response to aortic banding and catecholamine infusion. These results provide further evidence implicating MAPK signaling factors as obligate regulators of cardiac growth and hypertrophy and demonstrate the importance of dual-specificity phosphatases as counterbalancing regulatory factors in the heart.

Porcine circovirus type 2 activates PI3K/Akt and p38 MAPK pathways to promote interleukin-10 production in macrophages via Cap interaction of gC1qR

PubMed Central

Wang, Tongtong; Zhang, Xiujuan; Chen, Yu; Cui, Beibei; Li, Delong; Zhao, Xiaomin; Zhang, Wenlong; Chang, Lingling; Tong, Dewen

2016-01-01

Porcine circovirus type 2 (PCV2) infection caused PCV2-associated diseases (PCVAD) is one of the major emerging immunosuppression diseases in pig industry. In this study, we investigated how PCV2 inoculation increases interleukin (IL)-10 expression in porcine alveolar macrophages (PAMs). PCV2 inoculation significantly upregulated IL-10 expression compared with PCV1. Upon initial PCV2 inoculation, PI3K/Akt cooperated with NF-κB pathways to promote IL-10 transcription via p50, CREB and Ap1 transcription factors, whereas inhibition of PI3K/Akt activation blocked Ap1 and CREB binding to the il10 promoter, and decreased the binding level of NF-κB1 p50 with il10 promoter, leading to great reduction in early IL-10 transcription. In the later phase of inoculation, PCV2 further activated p38 MAPK and ERK pathways to enhance IL-10 production by promoting Sp1 binding to the il10 promoter. For PCV2-induced IL-10 production in macrophages, PCV2 capsid protein Cap, but not the replicase Rep or ORF3, was the critical component. Cap activated PI3K/Akt, p38 MAPK, and ERK signaling pathways to enhance IL-10 expression. In the whole process, gC1qR mediated PCV2-induced PI3K/Akt and p38 MAPK activation to enhance IL-10 induction by interaction with Cap. Depletion of gC1qR blocked PI3K/Akt and p38 MAPK activation, resulting in significant decrease in IL-10 production in PCV2-inoculated cells. Thus, gC1qR might be a critical functional receptor for PCV2-induced IL-10 production. Taken together, these data demonstrated that Cap protein binding with host gC1qR induction of PI3K/Akt and p38 MAPK signalings activation is a critical process in enhancing PCV2-induced IL-10 production in porcine alveolar macrophages. PMID:26883107

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Guang-Lin; Department of Pharmacology, University of Michigan, Ann Arbor; Du, Yi-Fang

KYKZL-1, a newly synthesized compound with COX/5-LOX dual inhibition, was subjected to the anti-inflammatory activity test focusing on its modulation of inflammatory mediators as well as intracellular MAPK and NF-κB signaling pathways. In acute ear edema model, pretreatment with KYKZL-1 (p.o.) dose-dependently inhibited the xylene-induced ear edema in mice with a higher inhibition than diclofenac. In a three-day TPA-induced inflammation, KYKZL-1 also showed significant anti-inflammatory activity with inhibition ranging between 20% and 64%. In gastric lesion test, KYKZL-1 elicited markedly fewer stomach lesions with a low index of ulcer as compared to diclofenac in rats. In further studies, KYKZL-1 wasmore » found to significantly inhibit the production of NO, PGE{sub 2}, LTB{sub 4} in LPS challenged RAW264.7, which is parallel to its attenuation of the expression of iNOS, COX-2, 5-LOX mRNAs or proteins and inhibition of phosphorylation of p38 and ERK MAPKs and activation of NF-κB. Taken together, our data indicate that KYKZL-1 comprises dual inhibition of COX and 5-LOX and exerts an obvious anti-inflammatory activity with an enhanced gastric safety profile via simultaneous inhibition of phosphorylation of p38 and ERK MAPKs and activation of NF-κB. - Highlights: • KYKZL-1 is designed to exhibit COX/5-LOX dual inhibition. • KYKZL-1 inhibits NO, PGE{sub 2} and LTB{sub 4} and iNOS, COX-2 and 5-LOX mRNAs and MAPKs. • KYKZL-1 inhibits phosphorylation of MAPKs. • KYKZL-1 inactivates NF-κB pathway.« less

Electromagnetic-pulse-induced activation of p38 MAPK pathway and disruption of blood-retinal barrier.

PubMed

Li, Hai-Juan; Guo, Liang-Mei; Yang, Long-Long; Zhou, Yong-Chun; Zhang, Yan-Jun; Guo, Juan; Xie, Xue-Jun; Guo, Guo-Zhen

2013-06-20

The blood-retinal barrier (BRB) is critical for maintaining retina homeostasis and low permeability. In this study, we evaluated the effects of electromagnetic pulse (EMP) exposure on the permeability of BRB, alterations of tight junction (TJ) proteins of BRB and if any, involvement of mitogen-activated protein kinase (MAPK) pathway. Male Sprague-Dawley (SD) rats and RF/6A cells which were pretreated with or without MAPKs inhibitors were sham exposed or exposed to EMP at 200kV/m for 200 pulses. The alteration of BRB permeability was examined through fluorescence microscope and quantitatively assessed using Evans blue (EB) and endogenous albumin as tracers. The expressions of TJ proteins and some signaling molecules of MAPK pathway were measured by Western blots. The observations were that EMP exposure resulted in increased BRB permeability concurrent with the decreased expressions of occludin and claudin-5, which were correlated with the increased expressions of phospho-p38, phospho-JNK and phospho-ERK and could be blocked when pretreated with p38 MAPK inhibitor. Thus, the results suggested that the alterations of occludin and claudin-5 may play an important role in the disruption of TJs, which may lead to the transient breakdown of BRB after EMP exposure with the involvement of p38 MAPK pathway through phosphorylation of signaling molecules. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

PAK1 is a breast cancer oncogene that coordinately activates MAPK and MET signaling

PubMed Central

Shrestha, Yashaswi; Schafer, Eric J.; Boehm, Jesse S.; Thomas, Sapana R.; He, Frank; Du, Jinyan; Wang, Shumei; Barretina, Jordi; Weir, Barbara A.; Zhao, Jean J.; Polyak, Kornelia; Golub, Todd R.; Beroukhim, Rameen; Hahn, William C.

2011-01-01

Activating mutations in the RAS family or BRAF frequently occur in many types of human cancers but are rarely detected in breast tumors. However, activation of the RAS-RAF-MEK-ERK Mitogen-Activated Protein Kinase (MAPK) pathway is commonly observed in human breast cancers, suggesting that other genetic alterations lead to activation of this signaling pathway. To identify breast cancer oncogenes that activate the MAPK pathway, we screened a library of human kinases for their ability to induce anchorage-independent growth in a derivative of immortalized human mammary epithelial cells (HMLE). We identified PAK1 as a kinase that permitted HMLE cells to form anchorage-independent colonies. PAK1 is amplified in several human cancer types, including 33% of breast tumor samples and cancer cell lines. The kinase activity of PAK1 is necessary for PAK1-induced transformation. Moreover, we show that PAK1 simultaneously activates MAPK and MET signaling; the latter via inhibition of Merlin. Disruption of these activities inhibits PAK1-driven anchorage-independent growth. These observations establish PAK1 amplification as an alternative mechanism for MAPK activation in human breast cancer and credential PAK1 as a breast cancer oncogene that coordinately regulates multiple signaling pathways, the cooperation of which leads to malignant transformation. PMID:22105362

PAK1 is a breast cancer oncogene that coordinately activates MAPK and MET signaling.

PubMed

Shrestha, Y; Schafer, E J; Boehm, J S; Thomas, S R; He, F; Du, J; Wang, S; Barretina, J; Weir, B A; Zhao, J J; Polyak, K; Golub, T R; Beroukhim, R; Hahn, W C

2012-07-19

Activating mutations in the RAS family or BRAF frequently occur in many types of human cancers but are rarely detected in breast tumors. However, activation of the RAS-RAF-MEK-ERK MAPK pathway is commonly observed in human breast cancers, suggesting that other genetic alterations lead to activation of this signaling pathway. To identify breast cancer oncogenes that activate the MAPK pathway, we screened a library of human kinases for their ability to induce anchorage-independent growth in a derivative of immortalized human mammary epithelial cells (HMLE). We identified p21-activated kinase 1 (PAK1) as a kinase that permitted HMLE cells to form anchorage-independent colonies. PAK1 is amplified in several human cancer types, including 30--33% of breast tumor samples and cancer cell lines. The kinase activity of PAK1 is necessary for PAK1-induced transformation. Moreover, we show that PAK1 simultaneously activates MAPK and MET signaling; the latter via inhibition of merlin. Disruption of these activities inhibits PAK1-driven anchorage-independent growth. These observations establish PAK1 amplification as an alternative mechanism for MAPK activation in human breast cancer and credential PAK1 as a breast cancer oncogene that coordinately regulates multiple signaling pathways, the cooperation of which leads to malignant transformation.

ACTIVATION OF THE EGF RECEPTOR SIGNALING PATHWAY IN HUMAN AIRWAY EPITHELIAL CELLS EXPOSED TO METALS

EPA Science Inventory

We have previously shown that exposure to combustion-derived metals rapidly (within 20 min) activated mitogen-activated protein kinases (MAPK), including extracellular signal-regulated kinase (ERK), in the human bronchial epithelial cell line BEAS. To study the mechanisms respons...

CD40 engagement on dendritic cells induces cyclooxygenase-2 and EP2 receptor via p38 and ERK MAPKs.

PubMed

Harizi, Hedi; Limem, Ilef; Gualde, Norbert

2011-02-01

We have previously reported that cyclooxygenase (COX)-2-derived prostaglandin (PG)E2 critically regulates dendritic cell (DC) inflammatory phenotype and function through EP2/EP4 receptor subtypes. As genes activated by CD40 engagement are directly relevant to inflammation, we examined the effects of CD40 activation on inflammatory PGs in murine bone marrow-derived DC (mBM-DC). We showed for the first time that activation of mBM-DC with agonist anti-CD40 monoclonal antibody (anti-CD40 mAb) dose dependently induces the synthesis of significant amounts of PGE2 via inducible expression of COX-2 enzyme, as NS-398, a COX-2-selective inhibitor reduces this upregulation. In contrast to lipopolysaccharide, which upregulates mBM-DC surface levels of EP2 and EP4 receptors, CD40 crosslinking on mBM-DC increases EP2, but not EP4, receptor expression. Flow cytometry analysis and radioligand-binding assay showed that EP2 was the major EP receptor subtype, which binds to PGE2 at the surface of anti-CD40-activated mBM-DC. Upregulation of COX-2 and EP2 levels by CD40 engagement was accompanied by dose-dependent phosphorylation of p38 and ERK1/2 mitogen-activated protein kinase (MAPK) and was abrogated by inhibitors of both pathways. Collectively, we demonstrated that CD40 engagement on mBM-DC upregulates COX-2 and EP2 receptor expression through activation of p38 and ERK1/2 MAPK signaling. Triggering the PGE2/EP2 pathway by anti-CD40 mAb resulted on the induction of Th2 immune response. Thus, CD40-induced production of PGE2 by mBM-DC could represent a negative feedback mechanism involving EP2 receptor and limiting the propagation of Th1 responses. Blocking CD40 pathway may represent a novel therapeutic pathway of inhibiting COX-2-derived prostanoids in chronically inflamed tissues (that is, arthritis).

The A2B Adenosine Receptor Modulates the Epithelial– Mesenchymal Transition through the Balance of cAMP/PKA and MAPK/ERK Pathway Activation in Human Epithelial Lung Cells

PubMed Central

Giacomelli, Chiara; Daniele, Simona; Romei, Chiara; Tavanti, Laura; Neri, Tommaso; Piano, Ilaria; Celi, Alessandro; Martini, Claudia; Trincavelli, Maria L.

2018-01-01

The epithelial-mesenchymal transition (EMT) is a complex process in which cell phenotype switches from the epithelial to mesenchymal one. The deregulations of this process have been related with the occurrence of different diseases such as lung cancer and fibrosis. In the last decade, several efforts have been devoted in understanding the mechanisms that trigger and sustain this transition process. Adenosine is a purinergic signaling molecule that has been involved in the onset and progression of chronic lung diseases and cancer through the A2B adenosine receptor subtype activation, too. However, the relationship between A2BAR and EMT has not been investigated, yet. Herein, the A2BAR characterization was carried out in human epithelial lung cells. Moreover, the effects of receptor activation on EMT were investigated in the absence and presence of transforming growth factor-beta (TGF-β1), which has been known to promote the transition. The A2BAR activation alone decreased and increased the expression of epithelial markers (E-cadherin) and the mesenchymal one (Vimentin, N-cadherin), respectively, nevertheless a complete EMT was not observed. Surprisingly, the receptor activation counteracted the EMT induced by TGF-β1. Several intracellular pathways regulate the EMT: high levels of cAMP and ERK1/2 phosphorylation has been demonstrated to counteract and promote the transition, respectively. The A2BAR stimulation was able to modulated these two pathways, cAMP/PKA and MAPK/ERK, shifting the fine balance toward activation or inhibition of EMT. In fact, using a selective PKA inhibitor, which blocks the cAMP pathway, the A2BAR-mediated EMT promotion were exacerbated, and conversely the selective inhibition of MAPK/ERK counteracted the receptor-induced transition. These results highlighted the A2BAR as one of the receptors involved in the modulation of EMT process. Nevertheless, its activation is not enough to trigger a complete transition, its ability to affect different intracellular pathways could represent a mechanism at the basis of EMT maintenance/inhibition based on the extracellular microenvironment. Despite further investigations are needed, herein for the first time the A2BAR has been related to the EMT process, and therefore to the different EMT-related pathologies. PMID:29445342

The estrogen-dependent baroreflex dysfunction caused by nicotine in female rats is mediated via NOS/HO inhibition: Role of sGC/PI3K/MAPK{sub ERK}

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fouda, Mohamed A.; El-Gowelli, Hanan M.; El-Gowilly, Sahar M.

We have previously reported that estrogen (E2) exacerbates the depressant effect of chronic nicotine on arterial baroreceptor activity in female rats. Here, we tested the hypothesis that this nicotine effect is modulated by nitric oxide synthase (NOS) and/or heme oxygenase (HO) and their downstream soluble guanylate cyclase (sGC)/phosphatidylinositol 3-kinase (PI3K)/mitogen-activated protein kinases (MAPKs) signaling. We investigated the effects of (i) inhibition or facilitation of NOS or HO on the interaction of nicotine (2 mg/kg/day i.p., 2 weeks) with reflex bradycardic responses to phenylephrine in ovariectomized (OVX) rats treated with E2 or vehicle, and (ii) central pharmacologic inhibition of sGC, PI3K,more » or MAPKs on the interaction. The data showed that the attenuation by nicotine of reflex bradycardia in OVXE2 rats was abolished after treatment with hemin (HO inducer) or L-arginine (NOS substrate). The hemin or L-arginine effect disappeared after inhibition of NOS (Nω-Nitro-L-arginine methyl ester hydrochloride, L-NAME) and HO (zinc protoporphyrin IX, ZnPP), respectively, denoting the interaction between the two enzymatic pathways. E2-receptor blockade (ICI 182,780) reduced baroreflexes in OVXE2 rats but had no effect on baroreflex improvement induced by hemin or L-arginine. Moreover, baroreflex enhancement by hemin was eliminated following intracisternal (i.c.) administration of wortmannin, ODQ, or PD98059 (inhibitors of PI3K, sGC, and extracellular signal-regulated kinases, MAPK{sub ERK}, respectively). In contrast, the hemin effect was preserved after inhibition of MAPK{sub p38} (SB203580) or MAPK{sub JNK} (SP600125). Overall, NOS/HO interruption underlies baroreflex dysfunction caused by nicotine in female rats and the facilitation of NOS/HO-coupled sGC/PI3K/MAPK{sub ERK} signaling might rectify the nicotine effect. - Highlights: • Hemin or L-arginine blunts baroreflex dysfunction caused by nicotine in OVXE2 rats. • NO/CO crosstalk mediates favorable baroreflex effect of hemin or L-arginine. • Central sGC/PI3K/MAPK{sub ERK} is required for hemin facilitation of baroreflexes. • The favorable baroreflex action of hemin is independent of estrogen receptors.« less

Dusuqing granules (DSQ) suppress inflammation in Klebsiella pneumonia rat via NF-κB/MAPK signaling.

PubMed

Mei, Xue; Wang, Hao-Xun; Li, Jian-Sheng; Liu, Xiao-Hui; Lu, Xiao-Fan; Li, Ya; Zhang, Wei-Yu; Tian, Yan-Ge

2017-04-17

Dusuqing granules (DSQ) have been used in the treatment of bacterial pneumonia clinically, with remarkable benefits. This study was initiated to explore the effects of DSQ on pulmonary inflammation by regulating nuclear factor (NF)-κB/mitogen-activated protein kinase (MAPK) signaling in bacterial pneumonia rats. Rat model was duplicated with Klebsiella pneumonia by a one-time intratracheal injection. Rats were randomized into control, model, DSQ and levofloxacin (LVX) groups. After administrated with appropriate medicines for 7 days, lung tissues were harvested and prepared for pathological analysis, and interleukin (IL)-1, IL-6, monocyte chemotactic protein (MCP)-1and macrophage inflammatory protein (MIP)-2 detections. NF-κB mRNA was measured by real-time qPCR, and the phosphorylation and total proteins of P38MAPK, JNK46/54, ERK42/44 were determined by Western blotting. Marked pathological impairments were observed in model rats, whereas were improved in DSQ group. The cytokines levels, NF-κB mRNA expression and the phosphorylation of P38MAPK, JNK46/54 and ERK42/44 proteins were significantly higher in model group, and were significantly depressed in DSQ group. The protective effects of DSQ on Klebsiella pneumonia might be attributed to its inactivative effects of NF-κB/ MAPK pathway.

Two distinct roles of mitogen-activated protein kinases in platelets and a novel Rac1-MAPK–dependent integrin outside-in retractile signaling pathway

PubMed Central

Flevaris, Panagiotis; Li, Zhenyu; Zhang, Guoying; Zheng, Yi; Liu, Junling

2009-01-01

Mitogen-activated protein kinases (MAPK), p38, and extracellular stimuli-responsive kinase (ERK), are acutely but transiently activated in platelets by platelet agonists, and the agonist-induced platelet MAPK activation is inhibited by ligand binding to the integrin αIIbβ3. Here we show that, although the activation of MAPK, as indicated by MAPK phosphorylation, is initially inhibited after ligand binding to integrin αIIbβ3, integrin outside-insignaling results in a late but sustained activation of MAPKs in platelets. Furthermore, we show that the early agonist-induced MAPK activation and the late integrin-mediated MAPK activation play distinct roles in different stages of platelet activation. Agonist-induced MAPK activation primarily plays an important role in stimulating secretion of platelet granules, while integrin-mediated MAPK activation is important in facilitating clot retraction. The stimulatory role of MAPK in clot retraction is mediated by stimulating myosin light chain (MLC) phosphorylation. Importantly, integrin-dependent MAPK activation, MAPK-dependent MLC phosphorylation, and clot retraction are inhibited by a Rac1 inhibitor and in Rac1 knockout platelets, indicating that integrin-induced activation of MAPK and MLC and subsequent clot retraction is Rac1-dependent. Thus, our results reveal 2 different activation mechanisms of MAPKs that are involved in distinct aspects of platelet function and a novel Rac1-MAPK–dependent cell retractile signaling pathway. PMID:18957688

Substance P Receptor Antagonist Suppresses Inflammatory Cytokine Expression in Human Disc Cells.

PubMed

Kepler, Christopher K; Markova, Dessislava Z; Koerner, John D; Mendelis, Joseph; Chen, Chiu-Ming; Vaccaro, Alexander R; Risbud, Makarand V; Albert, Todd J; Anderson, D Greg

2015-08-15

Laboratory study. To evaluate whether blockade of the Substance P (SP) NK1R attenuates its proinflammatory effect on human intervertebral disc cells (IVD), and to evaluate the signaling pathways associated with SP. SP and its receptors are expressed in human IVD cells, and cause upregulation of inflammatory mediators; however, the effects of blocking these receptors have not been studied in human IVD cells. Human annulus fibrosus (AF) and nucleus pulposus (NP) cells were expanded in monolayer, and then suspended in alginate beads. The alginate beads were treated with culture medium first containing a high affinity NK1R antagonist (L-760735) at different concentrations, and then with medium containing both NK1R antagonist and SP at 2 concentrations. Ribonucleic acid was isolated and transcribed into cDNA. Quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) was performed to evaluate expression of interleukin (IL)-1β, IL-6, and IL-8. Western blot analysis was performed to examine levels of the phosphorylated p38 mitogen-activated protein kinase (MAPK), extracellular signal regulated kinase 1/2 (ERK1/2) and nuclear factor kappa-light-chain-enhancer of activated B cells (NFκB p65). The cells were pretreated with specific inhibitors of p38 (SB203580), ERK1/2 (PD98059), and p65 (SM7368) and then stimulated with SP. We detected expression of NK1R, neurokinin receptor 2 (NK2R), and neurokinin receptor 3 (NK3R) in AF and NP cells. Treatment of disc cells with the NK1R antagonist was able to suppress expression of IL-1β, IL-6, and IL-8 in a dose-dependent manner. SP stimulation increased phosphorylation of p38-MAPK and ERK1/2, but not of NFκB p65. This indicates that p38-MAPK and ERK1/2 control SP-induced cytokine expression independently from NF-kB p65. Inhibition of p38 and ERK1/2 activation reduced SP-induced IL-6 production in human disc cells. NK1R is responsible for the proinflammatory effect of SP on IVD cells and this effect can be blocked by preventing binding of SP to NK1R. This study shows for the first time that SP mediates signaling in disc cells through NK1R and that SP activates the proinflammatory p38-MAPK and ERK1/2 pathways. 4.

Cholesterol depletion in cell membranes of human airway epithelial cells suppresses MUC5AC gene expression.

PubMed

Song, Kee Jae; Kim, Na Hyun; Lee, Gi Bong; Kim, Ji Hoon; Kwon, Jin Ho; Kim, Kyung-Su

2013-05-01

If cholesterol in the cell membrane is depleted by treating cells with methyl-β-cyclodextrin (MβCD), the activities of transmembrane receptors are altered in a cell-specific and/or receptor-specific manner. The proinflammatory cytokines, IL-1β is potent inducers of MUC5AC mRNA and protein synthesis in human airway epithelial cells. Cells activated by IL-1β showed increased phosphorylation of extracellular signal regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK). Thus, we investigated the effects of cholesterol depletion on the expression of MUC5AC in human airway epithelial cells and whether these alterations to MUC5AC expression were related to MAPK activity. After NCI-H292 cells were pretreated with 1% MβCD before adding IL-1β for 24 hours, MUC5AC mRNA expression was determined by reverse transcription- polymerase chain reaction (RT-PCR) and real time-PCR. Cholesterol depletion by MβCD was measured by modified microenzymatic fluorescence assay and filipin staining. The phosphorylation of IL-1 receptor, ERK and p38 MAPK, was analyzed by western blot. Cholesterol in the cell membrane was significantly depleted by treatment with MβCD on cells. IL-1β-induced MUC5AC mRNA expression was decreased by MβCD and this decrease occurred IL-1-receptor- specifically. Moreover, we have shown that MβCD suppressed the activation of ERK1/2 and p38 MAPK in cells activated with IL-1β. This result suggests that MβCD-mediated suppression of IL-1β-induced MUC5AC mRNA operated via the ERK- and p38 MAPK-dependent pathway. Cholesterol depletion in NCI-H292 cell membrane may be considered an anti-hypersecretory method since it effectively inhibits mucus secretion of respiratory epithelial cells.

Mutant human tumor suppressor p53 modulates the activation of mitogen-activated protein kinase and nuclear factor-kappaB, but not c-Jun N-terminal kinase and activated protein-1.

PubMed

Gulati, Anthony P; Yang, Yang-Ming; Harter, David; Mukhopadhyay, Asok; Aggarwal, Bharat B; Aggarwal, Bharat A; Benzil, Deborah L; Whysner, John; Albino, Anthony P; Murali, Raj; Jhanwar-Uniyal, Meena

2006-01-01

The roles of the mitogen-activated kinase protein (MAPK) pathway, nuclear factor-kappa B (NF-kappaB), and activator protein-1 (AP-1) in cellular responses to growth factors and mitogen are well established. However, the manner by which these proliferative pathways are affected by the tumor suppressor protein p53 is not fully understood. We report here the results of an investigation of the status of p53 on two human melanoma cell lines with wild-type p53 (SK-Mel-186) or mutant p53 (SK-Mel-110). The basal levels of the activated extracellular-signal regulated kinases 1 and 2 (ERK1/2) were high in cells with wild-type p53, but low in cells with mutant p53. The 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced activation of ERK1/2 through the phosphorylation of threonine and tyrosine at 202 and 204, respectively, was demonstrated in both cell lines, however, in a discrete manner. TPA-induced activation of ERK1/2 was sustained in wild-type p53 cells, while only a transient activation was seen in mutant p53 cells. Inhibition of MAPK kinase (MEK), an upstream kinase, by U0126, blocked TPA-induced activation of ERK1/2 in wild-type p53 cells and in mutant p53 cells. Treatment of wild-type p53 (SK-Mel 186) cells with small interfering RNA (siRNA) of p53 displayed a transient induction of activation of ERK1/2 following TPA treatment, indicating that p53 has a role in the regulation of the activation of ERK1/2. NF-kappaB activity decreased significantly in cells with wild-type p53, while enhanced NF-kappaB activity was evident in cells with mutant p53. The expression of either wild-type or mutant p53 had a similar effect on TPA-induced Jun N-terminal kinase (JNK) activation, indicating specificity for the ERK pathway. Similarly, AP-1 binding activity showed a transient variation in both cell lines after TPA treatment but with different kinetics. These observations suggest that both wild-type and mutant p53 can modulate the activation pathways for ERK1/2, and NF-kappaB distinctively, while modulating the pathways of JNK and AP-1 similarly. These differences may influence cellular processes such as proliferation, differentiation, and apoptosis. 2005 Wiley-Liss, Inc.

Lysophosphatidylcholine up-regulates human endothelial nitric oxide synthase gene transactivity by c-Jun N-terminal kinase signalling pathway

PubMed Central

Xing, Feiyue; Liu, Jing; Mo, Yongyan; Liu, Zhifeng; Qin, Qinghe; Wang, Jingzhen; Fan, Zhenhua; Long, Yutian; Liu, Na; Zhao, Kesen; Jiang, Yong

2009-01-01

Human endothelial nitric oxide synthase (eNOS) plays a pivotal role in maintaining blood pressure homeostasis and vascular integrity. It has recently been reported that mitogen-activated protein kinases (MAPKs) are intimately implicated in expression of eNOS. However detailed mechanism mediated by them remains to be clarified. In this study, eNOS gene transactivity in human umbilical vein endothelial cells was up-regulated by stimulation of lysophosphatidylcholine (LPC). The stimulation of LPC highly activated both extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinase (JNK), with differences in the dynamic processes of activation between them. Unexpectedly, p38 MAPK could not be activated by the stimulation of LPC. The activation of JNK signalling pathway by overexpression of JNK or its upstream kinase active mutant up-regulated the transactivity of eNOS significantly, but the activation of p38 signalling pathway down-regulated it largely. The inhibition of either ERK1/2 or JNK signalling pathway by kinase-selective inhibitors could markedly block the induction of the transactivity by LPC. It was observed by electrophoretic mobility shift assay that LPC stimulated both SP1 and AP1 DNA binding activity to go up. Additionally using decoy oligonucleotides proved that SP1 was necessary for maintaining the basal or stimulated transactivity, whereas AP1 contributed mainly to the increase of the stimulated transactivity. These findings indicate that the up-regulation of the eNOS gene transactivity by LPC involves the enhancement of SP1 transcription factor by the activation of JNK and ERK1/2 signalling pathways and AP1 transcription factor by the activation of JNK signalling pathway. PMID:18624763

Spinal glucocorticoid receptor‑regulated chronic morphine tolerance may be through extracellular signal‑regulated kinase 1/2.

PubMed

Zhai, Mei-Li; Chen, Yi; Liu, Chong; Wang, Jian-Bo; Yu, Yong-Hao

2018-05-23

Opioid use has been limited in the treatment of chronic pain due to their side effects, including analgesic tolerance. Previous studies demonstrated that glucocorticoid receptors (GRs) may be involved in the development of chronic morphine tolerance; however, the mechanism remains unknown. It was hypothesized that the expression of spinal phosphorylated mitogen‑activated protein kinase [MAPK; phosphorylated extracellular signal‑regulated kinase (ERK)] is regulated through the spinal GRs, following chronic treatment with morphine. In the first experiment, the experimental rats were randomly divided into four groups: Control, morphine, morphine+GR antagonist mifepristone (RU38486) and morphine+GR agonist dexamethasone (Dex). Each group was treated with continuous intrathecal (IT) injection of the drugs for 6 days. The expression of GRs and MAPK 3/1 (p‑ERK 1/2) in the spinal dorsal horn was detected by western blot analysis and immunofluorescence staining. In the second experiment, the MAPK inhibitor PD98059 was added and the rats were randomly divided into four groups: Control, morphine, PD98059+morphine and PD98059+morphine+Dex. The continuous IT injection lasted for 7 days in each group. For all experiments, the tail flick test was conducted 30 min following administration every day to assess the thermal hyperalgesia of the rats. The experimental results demonstrated that there was a co‑existence of GRs and p‑ERK 1/2 in the spinal cord dorsal horn by double immunofluorescence staining. The GR antagonist RU38486 attenuated the morphine analgesia tolerance by inhibiting the expression of GR and increasing the expression of p‑ERK. The MAPK inhibitor PD98059 increased the effect of morphine tolerance and prolonged the duration of morphine tolerance. The present results suggest that spinal GRs may serve an important role in the development of morphine tolerance through the ERK signaling pathway.

Arsenic-induced alteration in intracellular calcium homeostasis induces head kidney macrophage apoptosis involving the activation of calpain-2 and ERK in Clarias batrachus

DOE Office of Scientific and Technical Information (OSTI.GOV)

Banerjee, Chaitali; Goswami, Ramansu; Centre for Environmental Studies, Visva-Bharati University, Santiniketan 731 235

2011-10-01

We had earlier shown that exposure to arsenic (0.50 {mu}M) caused caspase-3 mediated head kidney macrophage (HKM) apoptosis involving the p38-JNK pathway in Clarias batrachus. Here we examined the roles of calcium (Ca{sup 2+}) and extra-cellular signal-regulated protein kinase (ERK), the other member of MAPK-pathway on arsenic-induced HKM apoptosis. Arsenic-induced HKM apoptosis involved increased expression of ERK and calpain-2. Nifedipine, verapamil and EGTA pre-treatment inhibited the activation of calpain-2, ERK and reduced arsenic-induced HKM apoptosis as evidenced from reduced caspase-3 activity, Annexin V-FITC-propidium iodide and Hoechst 33342 staining. Pre-incubation with ERK inhibitor U 0126 inhibited the activation of calpain-2 andmore » interfered with arsenic-induced HKM apoptosis. Additionally, pre-incubation with calpain-2 inhibitor also interfered with the activation of ERK and inhibited arsenic-induced HKM apoptosis. The NADPH oxidase inhibitor apocynin and diphenyleneiodonium chloride also inhibited ERK activation indicating activation of ERK in arsenic-exposed HKM also depends on signals from NADPH oxidase pathway. Our study demonstrates the critical role of Ca{sup 2+} homeostasis on arsenic-induced HKM apoptosis. We suggest that arsenic-induced alteration in intracellular Ca{sup 2+} levels initiates pro-apoptotic ERK and calpain-2; the two pathways influence each other positively and induce caspase-3 mediated HKM apoptosis. Besides, our study also indicates the role of ROS in the activation of ERK pathway in arsenic-induced HKM apoptosis in C. batrachus. - Highlights: > Altered Ca{sup 2+} homeostasis leads to arsenic-induced HKM apoptosis. > Calpain-2 plays a critical role in the process. > ERK is pro-apoptotic in arsenic-induced HKM apoptosis. > Arsenic-induced HKM apoptosis involves cross talk between calpain-2 and ERK.« less

Mitogen-Activated Protein Kinase Signaling in the Heart: Angels Versus Demons in a Heart-Breaking Tale

PubMed Central

ROSE, BETH A.; FORCE, THOMAS; WANG, YIBIN

2013-01-01

Among the myriad of intra-cellular signaling networks that govern the cardiac development and pathogenesis, mitogen-activated protein kinases (MAPKs) are prominent players that have been the focus of extensive investigations in the past decades. The four best characterized MAPK subfamilies, ERK1/2, JNK, p38, and ERK5, are the targets of pharmacological and genetic manipulations to uncover their roles in cardiac development, function, and diseases. However, information reported in the literature from these efforts has not yet resulted in a clear view about the roles of specific MAPK pathways in heart. Rather, controversies from contradictive results have led to a perception that MAPKs are ambiguous characters in heart with both protective and detrimental effects. The primary object of this review is to provide a comprehensive overview of the current progress, in an effort to highlight the areas where consensus is established verses the ones where controversy remains. MAPKs in cardiac development, cardiac hypertrophy, ischemia/reperfusion injury, and pathological remodeling are the main focuses of this review as these represent the most critical issues for evaluating MAPKs as viable targets of therapeutic development. The studies presented in this review will help to reveal the major challenges in the field and the limitations of current approaches and point to a critical need in future studies to gain better understanding of the fundamental mechanisms of MAPK function and regulation in the heart. PMID:20959622

Andrographolide Antagonizes TNF-α-Induced IL-8 via Inhibition of NADPH Oxidase/ROS/NF-κB and Src/MAPKs/AP-1 Axis in Human Colorectal Cancer HCT116 Cells.

PubMed

Yuan, Miaomiao; Meng, Wei; Liao, Wenzhen; Lian, Sen

2018-05-14

Andrographis paniculata Nees is used as a functional food in Japan, Korea, India, and China. Andrographolide, a naturally occurring phytochemical identified in Andrographis paniculata, has been discovered to present anti-inflammatory and anticancer activities. Highly expressed interleukin (IL-8) has been detected in colorectal cancer and is implicated in angiogenesis. However, the effect and molecular mechanisms of IL-8 expression by andrographolide remain obscure in human colorectal cancer cells. The present study was aimed to investigate the effects of andrographolide on TNF-α-induced IL-8 expression and its underlying mechanisms. We found that andrographolide concentration-dependently inhibited TNF-α-induced IL-8 mRNA (2.23 ± 0.15 fold at 20 μM) and protein expression (4.78 ± 0.31 fold at 20 μM) and reduced the IL-8 transcriptional activity (2.59 ± 0.25 fold at 20 μM). TNF-α stimulated the membrane translocation of p47 phox to activate reactive oxygen species (ROS)-producing NADPH oxidase (NOX). Furthermore, TNF-α induced Src and MAPKs (Erk1/2, p38 MAPK) phosphorylation, as well as NF-κB and AP-1 binding activities. We found that NF-κB and AP-1 were the critical transcription factors for TNF-α-induced IL-8 expression. Specific inhibitors and mutagenesis studies indicated that Src, Erk1/2, and p38 MAPK are related to TNF-α-induced IL-8. NOX-derived ROS and Src/MAPKs (Erk1/2 and p38 MAPK) functioned as upstream activators of NF-κB and AP-1, respectively. Taken together, andrographolide antagonizes TNF-α-induced IL-8 via inhibition of NADPH oxidase/ROS/NF-κB and Src/MAPKs/AP-1 signaling pathways in HCT116 colorectal cancer cells and then suppresses angiogenesis in the tumor microenvironment.

Activation and Function of the MAPKs and Their Substrates, the MAPK-Activated Protein Kinases

PubMed Central

Cargnello, Marie; Roux, Philippe P.

2011-01-01

Summary: The mitogen-activated protein kinases (MAPKs) regulate diverse cellular programs by relaying extracellular signals to intracellular responses. In mammals, there are more than a dozen MAPK enzymes that coordinately regulate cell proliferation, differentiation, motility, and survival. The best known are the conventional MAPKs, which include the extracellular signal-regulated kinases 1 and 2 (ERK1/2), c-Jun amino-terminal kinases 1 to 3 (JNK1 to -3), p38 (α, β, γ, and δ), and ERK5 families. There are additional, atypical MAPK enzymes, including ERK3/4, ERK7/8, and Nemo-like kinase (NLK), which have distinct regulation and functions. Together, the MAPKs regulate a large number of substrates, including members of a family of protein Ser/Thr kinases termed MAPK-activated protein kinases (MAPKAPKs). The MAPKAPKs are related enzymes that respond to extracellular stimulation through direct MAPK-dependent activation loop phosphorylation and kinase activation. There are five MAPKAPK subfamilies: the p90 ribosomal S6 kinase (RSK), the mitogen- and stress-activated kinase (MSK), the MAPK-interacting kinase (MNK), the MAPK-activated protein kinase 2/3 (MK2/3), and MK5 (also known as p38-regulated/activated protein kinase [PRAK]). These enzymes have diverse biological functions, including regulation of nucleosome and gene expression, mRNA stability and translation, and cell proliferation and survival. Here we review the mechanisms of MAPKAPK activation by the different MAPKs and discuss their physiological roles based on established substrates and recent discoveries. PMID:21372320

Curcumin produces neuroprotective effects via activating brain-derived neurotrophic factor/TrkB-dependent MAPK and PI-3K cascades in rodent cortical neurons.

PubMed

Wang, Rui; Li, Yu-Hua; Xu, Ying; Li, Ying-Bo; Wu, Hong-Li; Guo, Hao; Zhang, Jian-Zhao; Zhang, Jing-Jie; Pan, Xue-Yang; Li, Xue-Jun

2010-02-01

Curcumin is a major constituent of curcuma longa, a traditional medicine used to manage mental disorders effectively in China. The neuroprotective effects of curcumin have been demonstrated in our previous studies. In the present research, we confirmed this effect by showing that curcumin application promoted the viability of cultured rodent cortical neurons. Moreover, when neurons were pretreated with tyrosine kinase B (TrkB) antibody, known to inhibit the activity of brain-derived neurotrophic factor (BDNF), the protective effect of curcumin was blocked. Additionally, treatment of curcumin increased BDNF and phosphor-TrkB and both of these enhancements can be suppressed by ERK and PI-3K inhibitors. The administration of curcumin led to increased levels of phosphor-ERK and AKT, which were each blocked by MAPK and PI-3K inhibitors. Furthermore, the curcumin-induced increase in phosphorylated cyclic AMP response element binding protein (CREB), which has been implicated as a possible mediator of antidepressant actions, was prevented by MAPK and PI-3K inhibitors. Therefore, we hypothesize the neuroprotection of curcumin might be mediated via BDNF/TrkB-MAPK/PI-3K-CREB signaling pathway. Copyright 2009. Published by Elsevier Inc.

Autophagy and KRT8/keratin 8 protect degeneration of retinal pigment epithelium under oxidative stress.

PubMed

Baek, Ahruem; Yoon, Soojin; Kim, Jean; Baek, Yu Mi; Park, Hanna; Lim, Daehan; Chung, Hyewon; Kim, Dong-Eun

2017-02-01

Contribution of autophagy and regulation of related proteins to the degeneration of retinal pigment epithelium (RPE) in age-related macular degeneration (AMD) remain unknown. We report that upregulation of KRT8 (keratin 8) as well as its phosphorylation are accompanied with autophagy and attenuated with the inhibition of autophagy in RPE cells under oxidative stress. KRT8 appears to have a dual role in RPE pathophysiology. While increased expression of KRT8 following autophagy provides a cytoprotective role in RPE, phosphorylation of KRT8 induces pathologic epithelial-mesenchymal transition (EMT) of RPE cells under oxidative stress, which is mediated by MAPK1/ERK2 (mitogen-activated protein kinase 1) and MAPK3/ERK1. Inhibition of autophagy further promotes EMT, which can be reversed by inhibition of MAPK. Thus, regulated enhancement of autophagy with concurrent increased expression of KRT8 and the inhibition of KRT8 phosphorylation serve to inhibit oxidative stress-induced EMT of RPE cells as well as to prevent cell death, suggesting that pharmacological manipulation of KRT8 upregulation through autophagy with combined inhibition of the MAPK1/3 pathway may be attractive therapeutic strategies for the treatment of AMD.

ISA virus regulates the generation of reactive oxygen species and p47phox expression in a p38 MAPK-dependent manner in Salmo salar.

PubMed

Olavarría, Víctor H; Valdivia, Sharin; Salas, Boris; Villalba, Melina; Sandoval, Rodrigo; Oliva, Harold; Valdebenito, Samuel; Yañez, Alejandro

2015-02-01

Several viruses, including Orthomyxovirus, utilize cellular reactive oxygen species (ROS) for viral genomic replication and survival within host cells. However, the role of ROS in early events of viral entry and signal induction has not been elucidated. Here, we show that ISA virus (ISAV) induces ROS production very early during infection of CHSE-214 and SHK-1Ycells, and that production is sustained over the observed 24h post-infection. The mitogen-activated protein kinase (MAPK) family is responsible for important signaling pathways. In this study, we report that ISAV activates ERK and p38 in Salmo salar. In salmonid macrophages, while ERK was required for SOD, GLURED, p47phox expression, p38 regulated the ROS production by the NADPH oxidase complex activation. These results, together with the presence of several consensus target motifs for p38 MAPK in the promoter of the S. salar p47phox gene, suggest that p38 MAPK regulates p47phox gene expression in fish through the activation of this key transcription factor. Copyright © 2014 Elsevier Ltd. All rights reserved.

Asclepiasterol, a novel C21 steroidal glycoside derived from Asclepias curassavica, reverses tumor multidrug resistance by down-regulating P-glycoprotein expression

PubMed Central

Wang, Jun; Ma, Yan; Li, Wen-Xue; Jiang, Ren-Wang; Cai, Shao-Hui

2016-01-01

Multidrug resistance (MDR) mediated by P-glycoprotein (P-gp) is a major cause of cancer therapy failure. In this study, we identified a novel C21 steroidal glycoside, asclepiasterol, capable of reversing P-gp-mediated MDR. Asclepiasterol (2.5 and 5.0μM) enhanced the cytotoxity of P-gp substrate anticancer drugs in MCF-7/ADR and HepG-2/ADM cells. MDR cells were more responsive to paclitaxel in the presence of asclepiasterol, and colony formation of MDR cells was only reduced upon treatment with a combination of asclepiasterol and doxorubicin. Consistent with these findings, asclepiasterol treatment increased the intracellular accumulation of doxorubicin and rhodamine 123 (Rh123) in MDR cells. Asclepiasterol decreased expression of P-gp protein without stimulating or suppressing MDR1 mRNA levels. Asclepiasterol-mediated P-gp suppression caused inhibition of ERK1/2 phosphorylation in two MDR cell types, and EGF, an activator of the MAPK/ERK pathway, reversed the P-gp down-regulation, implicating the MAPK/ERK pathway in asclepiasterol-mediated P-gp down-regulation. These results suggest that asclepiasterol could be developed as a modulator for reversing P-gp-mediated MDR in P-gp-overexpressing cancer variants. PMID:27129170

Asclepiasterol, a novel C21 steroidal glycoside derived from Asclepias curassavica, reverses tumor multidrug resistance by down-regulating P-glycoprotein expression.

PubMed

Yuan, Wei-Qi; Zhang, Rong-Rong; Wang, Jun; Ma, Yan; Li, Wen-Xue; Jiang, Ren-Wang; Cai, Shao-Hui

2016-05-24

Multidrug resistance (MDR) mediated by P-glycoprotein (P-gp) is a major cause of cancer therapy failure. In this study, we identified a novel C21 steroidal glycoside, asclepiasterol, capable of reversing P-gp-mediated MDR. Asclepiasterol (2.5 and 5.0μM) enhanced the cytotoxity of P-gp substrate anticancer drugs in MCF-7/ADR and HepG-2/ADM cells. MDR cells were more responsive to paclitaxel in the presence of asclepiasterol, and colony formation of MDR cells was only reduced upon treatment with a combination of asclepiasterol and doxorubicin. Consistent with these findings, asclepiasterol treatment increased the intracellular accumulation of doxorubicin and rhodamine 123 (Rh123) in MDR cells. Asclepiasterol decreased expression of P-gp protein without stimulating or suppressing MDR1 mRNA levels. Asclepiasterol-mediated P-gp suppression caused inhibition of ERK1/2 phosphorylation in two MDR cell types, and EGF, an activator of the MAPK/ERK pathway, reversed the P-gp down-regulation, implicating the MAPK/ERK pathway in asclepiasterol-mediated P-gp down-regulation. These results suggest that asclepiasterol could be developed as a modulator for reversing P-gp-mediated MDR in P-gp-overexpressing cancer variants.

Dabrafenib; Preclinical Characterization, Increased Efficacy when Combined with Trametinib, while BRAF/MEK Tool Combination Reduced Skin Lesions

PubMed Central

King, Alastair J.; Arnone, Marc R.; Bleam, Maureen R.; Moss, Katherine G.; Yang, Jingsong; Fedorowicz, Kelly E.; Smitheman, Kimberly N.; Erhardt, Joseph A.; Hughes-Earle, Angela; Kane-Carson, Laurie S.; Sinnamon, Robert H.; Qi, Hongwei; Rheault, Tara R.; Uehling, David E.; Laquerre, Sylvie G.

2013-01-01

Mitogen-Activated Protein Kinase (MAPK) pathway activation has been implicated in many types of human cancer. BRAF mutations that constitutively activate MAPK signalling and bypass the need for upstream stimuli occur with high prevalence in melanoma, colorectal carcinoma, ovarian cancer, papillary thyroid carcinoma, and cholangiocarcinoma. In this report we characterize the novel, potent, and selective BRAF inhibitor, dabrafenib (GSK2118436). Cellular inhibition of BRAFV600E kinase activity by dabrafenib resulted in decreased MEK and ERK phosphorylation and inhibition of cell proliferation through an initial G1 cell cycle arrest, followed by cell death. In a BRAFV600E-containing xenograft model of human melanoma, orally administered dabrafenib inhibited ERK activation, downregulated Ki67, and upregulated p27, leading to tumor growth inhibition. However, as reported for other BRAF inhibitors, dabrafenib also induced MAPK pathway activation in wild-type BRAF cells through CRAF (RAF1) signalling, potentially explaining the squamous cell carcinomas and keratoacanthomas arising in patients treated with BRAF inhibitors. In addressing this issue, we showed that concomitant administration of BRAF and MEK inhibitors abrogated paradoxical BRAF inhibitor-induced MAPK signalling in cells, reduced the occurrence of skin lesions in rats, and enhanced the inhibition of human tumor xenograft growth in mouse models. Taken together, our findings offer preclinical proof of concept for dabrafenib as a specific and highly efficacious BRAF inhibitor and provide evidence for its potential clinical benefits when used in combination with a MEK inhibitor. PMID:23844038

Urotensin II contributes to collagen synthesis and up-regulates Egr-1 expression in cultured pulmonary arterial smooth muscle cells through the ERK1/2 pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Wei; Cai, Zhifeng; Liu, Mengmeng

Aim: The objective of this study was to investigate the effects of urotensin II (UII) treatment on the proliferation and collagen synthesis of cultured rat pulmonary arterial smooth muscle cells (PASMCs) and to explore whether these effects are mediated by mitogen-activated protein kinase (MAPK) signaling pathways and early growth response 1 (Egr-1). Methods: The proliferation of cultured PASMCs stimulated with different doses of UII was detected by BrdU incorporation. The mRNA expression levels of procollagen I (procol I), procollagen III (procol III), extracellular regulated protein kinase 1/2 (ERK1/2), stress-stimulated protein kinase (Sapk), p38 MAPK (p38), and Egr-1 mRNA in culturedmore » PASMCs after treatment with UII, the UII-specific antagonist urantide, and the ERK1/2 inhibitor PD98059 were detected by real-time polymerase chain reaction (PCR), and the protein expression levels of procol I, procol III, phosphorylated (p)-ERK1/2, p-Sapk, p-p38, and Egr-1 were detected by Western blotting. Results: Treatment with UII increased the proliferation of cultured PASMCs in a dose-dependent manner (P < 0.05). However, treatment with urantide and PD98059 inhibited the promoting effect of UII on PASMC proliferation (P < 0.05). Real-time PCR analysis showed that UII up-regulated the expression of procol I, procol III, ERK1/2, Sapk, and Egr-1 mRNA (P < 0.05), but not p38 mRNA. However, the up-regulating effect of UII was inhibited by PD98059 and urantide. Western blotting analysis showed that UII increased the synthesis of collagen I, collagen III, p-ERK1/2, p-Sapk, and Egr-1, and these effects also were inhibited by PD98059 and urantide (P < 0.05). Conclusions: Egr-1 participates in the UII-mediated proliferation and collagen synthesis of cultured rat PASMCs via activation of the ERK1/2 signaling pathway.« less

The Antiviral Alkaloid Berberine Reduces Chikungunya Virus-Induced Mitogen-Activated Protein Kinase Signaling

PubMed Central

Thaa, Bastian; Amrun, Siti Naqiah; Simarmata, Diane; Rausalu, Kai; Nyman, Tuula A.; Merits, Andres; McInerney, Gerald M.; Ng, Lisa F. P.

2016-01-01

ABSTRACT Chikungunya virus (CHIKV) has infected millions of people in the tropical and subtropical regions since its reemergence in the last decade. We recently identified the nontoxic plant alkaloid berberine as an antiviral substance against CHIKV in a high-throughput screen. Here, we show that berberine is effective in multiple cell types against a variety of CHIKV strains, also at a high multiplicity of infection, consolidating the potential of berberine as an antiviral drug. We excluded any effect of this compound on virus entry or on the activity of the viral replicase. A human phosphokinase array revealed that CHIKV infection specifically activated the major mitogen-activated protein kinase (MAPK) signaling pathways extracellular signal-related kinase (ERK), p38 and c-Jun NH2-terminal kinase (JNK). Upon treatment with berberine, this virus-induced MAPK activation was markedly reduced. Subsequent analyses with specific inhibitors of these kinases indicated that the ERK and JNK signaling cascades are important for the generation of progeny virions. In contrast to specific MAPK inhibitors, berberine lowered virus-induced activation of all major MAPK pathways and resulted in a stronger reduction in viral titers. Further, we assessed the in vivo efficacy of berberine in a mouse model and measured a significant reduction of CHIKV-induced inflammatory disease. In summary, we demonstrate the efficacy of berberine as a drug against CHIKV and highlight the importance of the MAPK signaling pathways in the alphavirus infectious cycle. IMPORTANCE Chikungunya virus (CHIKV) is a mosquito-borne virus that causes severe and persistent muscle and joint pain and has recently spread to the Americas. No licensed drug exists to counter this virus. In this study, we report that the alkaloid berberine is antiviral against different CHIKV strains and in multiple human cell lines. We demonstrate that berberine collectively reduced the virus-induced activation of cellular mitogen-activated protein kinase signaling. The relevance of these signaling cascades in the viral life cycle was emphasized by specific inhibitors of these kinase pathways, which decreased the production of progeny virions. Berberine significantly reduced CHIKV-induced inflammatory disease in a mouse model, demonstrating efficacy of the drug in vivo. Overall, this work makes a strong case for pursuing berberine as a potential anti-CHIKV therapeutic compound and for exploring the MAPK signaling pathways as antiviral targets against alphavirus infections. PMID:27535052

Immunolocalization of dually phosphorylated MAPKs in dividing root meristem cells of Vicia faba, Pisum sativum, Lupinus luteus and Lycopersicon esculentum.

PubMed

Winnicki, Konrad; Żabka, Aneta; Bernasińska, Joanna; Matczak, Karolina; Maszewski, Janusz

2015-06-01

In plants, phosphorylated MAPKs display constitutive nuclear localization; however, not all studied plant species show co-localization of activated MAPKs to mitotic microtubules. The mitogen-activated protein kinase (MAPK) signaling pathway is involved not only in the cellular response to biotic and abiotic stress but also in the regulation of cell cycle and plant development. The role of MAPKs in the formation of a mitotic spindle has been widely studied and the MAPK signaling pathway was found to be indispensable for the unperturbed course of cell division. Here we show cellular localization of activated MAPKs (dually phosphorylated at their TXY motifs) in both interphase and mitotic root meristem cells of Lupinus luteus, Pisum sativum, Vicia faba (Fabaceae) and Lycopersicon esculentum (Solanaceae). Nuclear localization of activated MAPKs has been found in all species. Co-localization of these kinases to mitotic microtubules was most evident in L. esculentum, while only about 50% of mitotic cells in the root meristems of P. sativum and V. faba displayed activated MAPKs localized to microtubules during mitosis. Unexpectedly, no evident immunofluorescence signals at spindle microtubules and phragmoplast were noted in L. luteus. Considering immunocytochemical analyses and studies on the impact of FR180204 (an inhibitor of animal ERK1/2) on mitotic cells, we hypothesize that MAPKs may not play prominent role in the regulation of microtubule dynamics in all plant species.

Substance P activates both contractile and inflammatory pathways in lymphatics through the neurokinin receptors NK1R and NK3R.

PubMed

Chakraborty, Sanjukta; Nepiyushchikh, Zhanna; Davis, Michael J; Zawieja, David C; Muthuchamy, Mariappan

2011-01-01

The aim of this study was to elucidate the molecular signaling mechanisms by which substance P (SP) modulates lymphatic muscle contraction and to determine whether SP stimulates both contractile as well as inflammatory pathways in the lymphatics. A rat mesenteric lymphatic muscle cell culture model (RMLMCs) and known specific pharmacological inhibitors were utilized to delineate SP-mediated signaling pathways in lymphatics. We detected expression of neurokinin receptor 1 (NK1R) and neurokinin receptor 3 (NK3R) in RMLMCs. SP stimulation increased phosphorylation of myosin light chain 20 (MLC₂₀) as well as p38 mitogen associated protein kinase (p38-MAPK) and extracellular signal regulated kinase (ERK1/2) indicating activation of both a contractile and a pro-inflammatory MAPK pathway. Pharmacological inhibition of both NK1R and NK3R significantly affected the downstream SP signaling. We further examined whether there was any crosstalk between the two pathways upon SP stimulation. Inhibition of ERK1/2 decreased levels of p-MLC₂₀ after SP activation, in a PKC dependent manner, indicating a potential crosstalk between these two pathways. These data provide the first evidence that SP-mediated crosstalk between pro-inflammatory and contractile signaling mechanisms exists in the lymphatic system and may be an important bridge between lymphatic function modulation and inflammation. © 2010 John Wiley & Sons Ltd.

Substance P activates both contractile and inflammatory pathways in lymphatics through the neurokinin receptors NK1R and NK3R

PubMed Central

Chakraborty, Sanjukta; Nepiyushchikh, Zhanna; Davis, Michael J.; Zawieja, David C.; Muthuchamy, Mariappan

2010-01-01

Objective The aim of this study was to elucidate the molecular signaling mechanisms by which substance P (SP) modulates lymphatic muscle contraction and to determine whether SP stimulates both contractile as well as inflammatory pathways in the lymphatics. Methods A rat mesenteric lymphatic muscle cell culture model (RMLMCs) and known specific pharmacological inhibitors were utilized to delineate SP mediated signaling pathways in lymphatics. Results We detected expression of neurokinin receptor 1 (NK1R) and neurokinin receptor 3 (NK3R) in RMLMCs. SP stimulation increased phosphorylation of myosin light chain 20 (MLC20) as well as p38 mitogen associated protein kinase (p38-MAPK) and extracellular signal regulated kinase (ERK1/2) indicating activation of both a contractile and a pro-inflammatory MAPK pathway. Pharmacological inhibition of both NK1R and NK3R significantly affected the downstream SP signaling. We further examined whether there was any crosstalk between the two pathways upon SP stimulation. Inhibition of ERK1/2 decreased levels of p-MLC20 after SP activation, in a PKC dependent manner, indicating a potential crosstalk between these two pathways. Conclusions These data provide the first evidence that SP mediated crosstalk between pro-inflammatory and contractile signaling mechanisms exists in the lymphatic system and may be an important bridge between lymphatic function modulation and inflammation. PMID:21166923

Fasting up-regulates ferroportin 1 expression via a Ghrelin/GHSR/MAPK signaling pathway.

PubMed

Luo, Qian-Qian; Zhou, Yu-Fu; Chen, Mesona Yung-Jin; Liu, Li; Ma, Juan; Zhang, Meng-Wan; Zhang, Fa-Li; Ke, Ya; Qian, Zhong-Ming

2018-01-01

The significant positive correlation between ghrelin and iron and hepcidin levels in the plasma of children with iron deficiency anemia prompted us to hypothesize that ghrelin may affect iron metabolism. Here, we investigated the effects of fasting or ghrelin on the expression of hepcidin, ferroportin 1 (Fpn1), transferrin receptor 1 (TfR1), ferritin light chain (Ft-L) proteins, and ghrelin, and also hormone secretagogue receptor 1 alpha (GHSR1α) and ghrelin O-acyltransferase (GOAT) mRNAs in the spleen and/or macrophage. We demonstrated that fasting induces a significant increase in the expression of ghrelin, GHSR1α, GOAT, and hepcidin mRNAs, as well as Ft-L and Fpn1 but not TfR1 proteins in the spleens of mice in vivo. Similar to the effects of fasting on the spleen, ghrelin induced a significant increase in the expression of Ft-L and Fpn1 but not TfR1 proteins in macrophages in vitro. In addition, ghrelin was found to induce a significant enhancement in phosphorylation of ERK as well as translocation of pERK from the cytosol to nuclei. Furthermore, the increased pERK and Fpn1 induced by ghrelin was demonstrated to be preventable by pre-treatment with either GHSR1α antagonist or pERK inhibitor. Our findings support the hypothesis that fasting upregulates Fpn1 expression, probably via a ghrelin/GHSR/MAPK signaling pathway. © 2017 Wiley Periodicals, Inc.

Sulforaphane attenuates microglia-mediated neuronal necroptosis through down-regulation of MAPK/NF-κB signaling pathways in LPS-activated BV-2 microglia.

PubMed

Qin, Sisi; Yang, Canhong; Huang, Weihua; Du, Shuhua; Mai, Hantao; Xiao, Jijie; Lü, Tianming

2018-01-31

Sulforaphane (SFN), a natural dietary isothiocyanate in cruciferous vegetables such as broccoli and cabbage, has very strong anti-inflammatory activity. Activation of microglia leads to overexpression of a series of pro-inflammatory mediators, which play a vital role in neuronal damage. SFN may have neuroprotective effects in different neurodegenerative diseases related to inflammation. However, the mechanisms underlying SFN's protection of neurons against microglia-mediated neuronal damage are not fully understood. Here, we investigated how SFN attenuated microglia-mediated neuronal damage. Our results showed that SFN could not directly protect the viability of neurons following pro-inflammatory mediators, but increased the viability of BV-2 microglia and down-regulated the mRNA and protein levels of pro-inflammatory mediators including TNF-α, IL-1β, IL-6 and iNOS in a concentration-dependent manner in BV-2 cells. SFN also significantly blocked the phosphorylation of MAPKs (p38, JNK, and ERK1/2) and NF-κB p65, both by itself and with MAPK inhibitors (SB203580, SP 600125, and U0126) or an NF-κB inhibitor (PDTC). The expression of pro-inflammatory proteins was also blocked by SFN with or without inhibitors. Further, SFN indirectly increased the viability and maintained the morphology of neurons, and the protein expression of RIPK3 and MLKL was significantly suppressed by SFN in neuronal necroptosis through p38, JNK, and NF-κB p65 but not ERK1/2 signaling pathways. Together, our results demonstrate that SFN attenuates LPS-induced pro-inflammatory responses through down-regulation of MAPK/NF-κB signaling pathway in BV-2 microglia and thus indirectly suppresses microglia-mediated neuronal damage. Copyright © 2018 Elsevier Ltd. All rights reserved.

Hypotonic stress upregulates β- and γ-ENaC expression through suppression of ERK by inducing MKP-1

PubMed Central

Niisato, Naomi; Ohta, Mariko; Eaton, Douglas C.

2012-01-01

We investigated a physiological role for ERK, a member of the MAPK family, in the hypotonic stimulation of epithelial Na+ channel (ENaC)-mediated Na+ reabsorption in renal epithelial A6 cells. We show that hypotonic stress causes a major dephosphorylation of ERK following a rapid transient phosphorylation. PD98059 (a MEK inhibitor) increases dephosphorylated ERK and enhances the hypotonic-stress-stimulated Na+ reabsorption. ERK dephosphorylation is mediated by MAPK phosphatase (MKP). Hypotonic stress activates p38, which in turn induces MKP-1 and to a lesser extent MKP-3 mRNA expression. Inhibition of p38 suppresses MKP-1 induction, preventing hypotonic stress from dephosphorylating ERK. Inhibition of MKP-1 and -3 by the inhibitor NSC95397 also suppresses the hypotonicity-induced dephosphorylation of ERK. NSC95397 reduces both β- and γ-ENaC mRNA expression and ENaC-mediated Na+ reabsorption stimulated by hypotonic stress. In contrast, pretreatment with PD98059 significantly enhances mRNA and protein expression of β- and γ-ENaC even under isotonic conditions. However, PD98059 only stimulates Na+ reabsorption in response to hypotonic stress, suggesting that ERK inactivation by itself (i.e., under isotonic conditions) is not sufficient to stimulate Na+ reabsorption, even though ERK inactivation enhances β- and γ-ENaC expression. Based on these results, we conclude that hypotonic stress stimulates Na+ reabsorption through at least two signaling pathways: 1) induction of MKP-1 that suppresses ERK activity and induces β- and γ-ENaC expression, and 2) promotion of translocation of the newly synthesized ENaC to the apical membrane. PMID:22573375

Tumour compartment transcriptomics demonstrates the activation of inflammatory and odontogenic programmes in human adamantinomatous craniopharyngioma and identifies the MAPK/ERK pathway as a novel therapeutic target.

PubMed

Apps, John R; Carreno, Gabriela; Gonzalez-Meljem, Jose Mario; Haston, Scott; Guiho, Romain; Cooper, Julie E; Manshaei, Saba; Jani, Nital; Hölsken, Annett; Pettorini, Benedetta; Beynon, Robert J; Simpson, Deborah M; Fraser, Helen C; Hong, Ying; Hallang, Shirleen; Stone, Thomas J; Virasami, Alex; Donson, Andrew M; Jones, David; Aquilina, Kristian; Spoudeas, Helen; Joshi, Abhijit R; Grundy, Richard; Storer, Lisa C D; Korbonits, Márta; Hilton, David A; Tossell, Kyoko; Thavaraj, Selvam; Ungless, Mark A; Gil, Jesus; Buslei, Rolf; Hankinson, Todd; Hargrave, Darren; Goding, Colin; Andoniadou, Cynthia L; Brogan, Paul; Jacques, Thomas S; Williams, Hywel J; Martinez-Barbera, Juan Pedro

2018-05-01

Adamantinomatous craniopharyngiomas (ACPs) are clinically challenging tumours, the majority of which have activating mutations in CTNNB1. They are histologically complex, showing cystic and solid components, the latter comprised of different morphological cell types (e.g. β-catenin-accumulating cluster cells and palisading epithelium), surrounded by a florid glial reaction with immune cells. Here, we have carried out RNA sequencing on 18 ACP samples and integrated these data with an existing ACP transcriptomic dataset. No studies so far have examined the patterns of gene expression within the different cellular compartments of the tumour. To achieve this goal, we have combined laser capture microdissection with computational analyses to reveal groups of genes that are associated with either epithelial tumour cells (clusters and palisading epithelium), glial tissue or immune infiltrate. We use these human ACP molecular signatures and RNA-Seq data from two ACP mouse models to reveal that cell clusters are molecularly analogous to the enamel knot, a critical signalling centre controlling normal tooth morphogenesis. Supporting this finding, we show that human cluster cells express high levels of several members of the FGF, TGFB and BMP families of secreted factors, which signal to neighbouring cells as evidenced by immunostaining against the phosphorylated proteins pERK1/2, pSMAD3 and pSMAD1/5/9 in both human and mouse ACP. We reveal that inhibiting the MAPK/ERK pathway with trametinib, a clinically approved MEK inhibitor, results in reduced proliferation and increased apoptosis in explant cultures of human and mouse ACP. Finally, we analyse a prominent molecular signature in the glial reactive tissue to characterise the inflammatory microenvironment and uncover the activation of inflammasomes in human ACP. We validate these results by immunostaining against immune cell markers, cytokine ELISA and proteome analysis in both solid tumour and cystic fluid from ACP patients. Our data support a new molecular paradigm for understanding ACP tumorigenesis as an aberrant mimic of natural tooth development and opens new therapeutic opportunities by revealing the activation of the MAPK/ERK and inflammasome pathways in human ACP.

Early immune response and regulation of IL-2 receptor subunits

NASA Technical Reports Server (NTRS)

Hughes-Fulford, Millie; Sugano, Eiko; Schopper, Thomas; Li, Chai-Fei; Boonyaratanakornkit, J. B.; Cogoli, Augusto

2005-01-01

Affymetrix oligonucleotide arrays were used to monitor expression of 8796 genes and probe sets in activated T-cells; analysis revealed that 217 genes were significantly upregulated within 4 h. Induced genes included transcription factors, cytokines and their receptor genes. Analysis by semi-quantitative RT-PCR confirmed the significant induction of IL-2, IL-2R(gamma) and IL-2R(alpha). Forty-eight of the 217 induced genes are known to or predicted to be regulated by a CRE promoter/enhancer. We found that T-cell activation caused a significant increase in CREB phosphorylation furthermore, inhibition of the PKC pathway by GF109203 reduced CREB activation by 50% and inhibition of the PKA pathway caused a total block of CREB phosphorylation and significantly reduced IFN(gamma), IL-2 and IL-2R(alpha) gene expression by approximately 40% (p<0.001). PKC(theta) plays a major role in T-cell activation: inhibition of PKC significantly reduced the expression of IFN(gamma), IL-2 and IL-2R(alpha). Since PKC blocked activation of CREB, we studied potential cross-talk between the PKC and the PKA/MAPK pathways, PMA-stimulated Jurkat cells were studied with specific signal pathway inhibitors. Extracellular signal-regulated kinase-2 (ERK2) pathway was found to be significantly activated greater than seven-fold within 30 min; however, there was little activation of ERK-1 and no activation of JNK or p38 MAPK. Inhibition of the PKA pathway, but not the PKC pathway, resulted in inhibition of ERK1/2 activation at all time points, inhibition of MEK1 and 2 significantly blocked expression of IL-2 and IL-2R(alpha). Gene expression of IL-2R(alpha) and IFN(gamma) was dependent on PKA in S49 wt cells but not in kin- mutants. Using gel shift analysis, we found that forskolin activation of T-cells resulted in activation of AP1 sites; this increase in nuclear extract AP1 was significantly blocked by MEK1 inhibitor U0126. Taken together, these results suggest that the PKA in addition to PKC and MAPK pathways plays a role in early T-cell activation and induction of IL-2, IL-2R(alpha) and IFN(gamma) gene expression.

Early immune response and regulation of IL-2 receptor subunits.

PubMed

Hughes-Fulford, Millie; Sugano, Eiko; Schopper, Thomas; Li, Chai-Fei; Boonyaratanakornkit, J B; Cogoli, Augusto

2005-09-01

Affymetrix oligonucleotide arrays were used to monitor expression of 8796 genes and probe sets in activated T-cells; analysis revealed that 217 genes were significantly upregulated within 4 h. Induced genes included transcription factors, cytokines and their receptor genes. Analysis by semi-quantitative RT-PCR confirmed the significant induction of IL-2, IL-2R(gamma) and IL-2R(alpha). Forty-eight of the 217 induced genes are known to or predicted to be regulated by a CRE promoter/enhancer. We found that T-cell activation caused a significant increase in CREB phosphorylation furthermore, inhibition of the PKC pathway by GF109203 reduced CREB activation by 50% and inhibition of the PKA pathway caused a total block of CREB phosphorylation and significantly reduced IFN(gamma), IL-2 and IL-2R(alpha) gene expression by approximately 40% (p<0.001). PKC(theta) plays a major role in T-cell activation: inhibition of PKC significantly reduced the expression of IFN(gamma), IL-2 and IL-2R(alpha). Since PKC blocked activation of CREB, we studied potential cross-talk between the PKC and the PKA/MAPK pathways, PMA-stimulated Jurkat cells were studied with specific signal pathway inhibitors. Extracellular signal-regulated kinase-2 (ERK2) pathway was found to be significantly activated greater than seven-fold within 30 min; however, there was little activation of ERK-1 and no activation of JNK or p38 MAPK. Inhibition of the PKA pathway, but not the PKC pathway, resulted in inhibition of ERK1/2 activation at all time points, inhibition of MEK1 and 2 significantly blocked expression of IL-2 and IL-2R(alpha). Gene expression of IL-2R(alpha) and IFN(gamma) was dependent on PKA in S49 wt cells but not in kin- mutants. Using gel shift analysis, we found that forskolin activation of T-cells resulted in activation of AP1 sites; this increase in nuclear extract AP1 was significantly blocked by MEK1 inhibitor U0126. Taken together, these results suggest that the PKA in addition to PKC and MAPK pathways plays a role in early T-cell activation and induction of IL-2, IL-2R(alpha) and IFN(gamma) gene expression.

Control of adipogenesis by the autocrine interplays between angiotensin 1-7/Mas receptor and angiotensin II/AT1 receptor signaling pathways.

PubMed

Than, Aung; Leow, Melvin Khee-Shing; Chen, Peng

2013-05-31

Angiotensin II (AngII), a peptide hormone released by adipocytes, can be catabolized by adipose angiotensin-converting enzyme 2 (ACE2) to form Ang(1-7). Co-expression of AngII receptors (AT1 and AT2) and Ang(1-7) receptors (Mas) in adipocytes implies the autocrine regulation of the local angiotensin system upon adipocyte functions, through yet unknown interactive mechanisms. In the present study, we reveal the adipogenic effects of Ang(1-7) through activation of Mas receptor and its subtle interplays with the antiadipogenic AngII-AT1 signaling pathways. Specifically, in human and 3T3-L1 preadipocytes, Ang(1-7)-Mas signaling promotes adipogenesis via activation of PI3K/Akt and inhibition of MAPK kinase/ERK pathways, and Ang(1-7)-Mas antagonizes the antiadipogenic effect of AngII-AT1 by inhibiting the AngII-AT1-triggered MAPK kinase/ERK pathway. The autocrine regulation of the AngII/AT1-ACE2-Ang(1-7)/Mas axis upon adipogenesis has also been revealed. This study suggests the importance of the local regulation of the delicately balanced angiotensin system upon adipogenesis and its potential as a novel therapeutic target for obesity and related metabolic disorders.

JNK signaling pathway regulates sorbitol-induced Tau proteolysis and apoptosis in SH-SY5Y cells by targeting caspase-3.

PubMed

Olivera Santa-Catalina, Marta; Caballero Bermejo, Montaña; Argent, Ricardo; Alonso, Juan C; Centeno, Francisco; Lorenzo, María J

2017-12-15

Growing evidence suggests that Diabetes Mellitus increases the risk of developing Alzheimer's disease. It is well known that hyperglycemia, a key feature of Diabetes Mellitus, may induce plasma osmolarity disturbances. Both hyperglycemia and hyperosmolarity promote the altered post-translational regulation of microtubule-associated protein Tau. Interestingly, abnormal hyperphosphorylation and cleavage of Tau have been proven to lead to the genesis of filamentous structures referred to as neurofibrillary tangles, the main pathological hallmark of Alzheimer's disease. We have previously described that hyperosmotic stress induced by sorbitol promotes Tau proteolysis and apoptosis in SH-SY5Y cells via caspase-3 activation. In order to gain insights into the regulatory mechanisms of such processes, in this work we explored the intracellular signaling pathways that regulate these events. We found that sorbitol treatment significantly enhanced the activation of conventional families of MAPK in SH-SY5Y cells. Tau proteolysis was completely prevented by JNK inhibition but not affected by either ERK1/2 or p38 MAPK blockade. Moreover, inhibition of JNK, but not ERK1/2 or p38 MAPK, efficiently prevented sorbitol-induced apoptosis and caspase-3 activation. In summary, we provide evidence that JNK signaling pathway is an upstream regulator of hyperosmotic stress-induced Tau cleavage and apoptosis in SH-SY5Y through the control of caspase-3 activation. Copyright © 2017 Elsevier Inc. All rights reserved.

Inhibition of macrophage-derived foam cell formation by ezetimibe via the caveolin-1/MAPK pathway.

PubMed

Qin, Li; Yang, Yun-Bo; Yang, Yi-Xin; Zhu, Neng; Liu, Zheng; Ni, Ya-Guang; Li, Shun-Xiang; Zheng, Xi-Long; Liao, Duan-Fang

2016-02-01

Ezetimibe, a selective inhibitor of intestinal cholesterol absorption, effectively reduces plasma cholesterol, but its effect on atherosclerosis is unclear. Foam cell formation has been implicated as a key mediator during the development of atherosclerosis. The purpose of this study was to investigate the effects of ezetimibe on foam cell formation and explore the underlying mechanism. The results presented here show that ezetimibe reduces atherosclerotic lesions in apolipoprotein E deficient (apoE-/-) mice by lowering cholesterol levels. Treatment of macrophages with Chol:MβCD resulted in foam cell formation, which was concentration-dependently inhibited by the presence of ezetimibe. Mechanically, ezetimibe treatment downregulated the expression of CD36 and scavenger receptor class B1 (SR-B1), but upregulated the expression of apoE and caveolin-1 in macrophage-derived foam cells, which kept consistent with our microarray results. Moreover, treatment with ezetimibe abrogated the increase of phospho-extracellular signal regulated kinase (ERK) 1/2 and their nuclear accumulation in foam cells. Inhibition of the MAPK pathway by the MEK inhibitor PD98059 attenuated the inhibitory effect of ezetimibe on the expression of p-ERK1/2 and caveolin-1. Taken together, these results showed that ezetimibe suppressed foam cell formation via the caveolin-1/MAPK signalling pathway, suggesting that inhibition of foam cell formation might be a novel mechanism underlying the anti-atherosclerotic effect of ezetimibe. © 2016 John Wiley & Sons Australia, Ltd.

Burns, inflammation, and intestinal injury: protective effects of an anti-inflammatory resuscitation strategy.

PubMed

Costantini, Todd W; Peterson, Carrie Y; Kroll, Lauren; Loomis, William H; Putnam, James G; Wolf, Paul; Eliceiri, Brian P; Baird, Andrew; Bansal, Vishal; Coimbra, Raul

2009-12-01

Intestinal barrier breakdown after severe burn can lead to intestinal inflammation, which may act as the source of the systemic inflammatory response. In vitro intestinal cell studies have shown that mitogen-activated protein kinase (MAPK) signaling is an important modulator of intestinal inflammation. We have previously observed that pentoxifylline (PTX) attenuates burn-induced intestinal permeability and tight junction breakdown. We hypothesized that PTX would limit intestinal barrier breakdown and attenuate inflammatory signaling via the MAPK pathway. Male balb/c mice underwent 30% total body surface area full-thickness steam burn. Immediately after burn, animals received an intraperitoneal injection of PTX (12.5 mg/kg) in normal saline or normal saline alone. In vivo intestinal permeability to 4 kDa fluorescein isothiocyanate-dextran was measured. Intestinal extracts were obtained to measure interleukin-6 by enzyme-linked immunosorbent assay, and phosphorylated p38 MAPK, p38 MAPK, phosphorylated extracellular signal-related kinase (1/2) (ERK (1/2)), and ERK (1/2) by immunoblotting. Acute lung injury was assessed by histology at 24 hours after burn. Administration of PTX immediately after injury attenuated burn-induced intestinal permeability. PTX also decreased the burn-induced phosphorylation of p38 MAPK and decreased phosphorylation of ERK (1/2) at 2 hours and 24 hours after injury. Animals given PTX had decreased intestinal interleukin-6 levels. A single dose of PTX also decreased histologic lung injury at 24 hours after burn. PTX attenuates burn-induced intestinal permeability and subsequent intestinal inflammation. Use of PTX after burn was also associated with decreased acute lung injury. Because of its compelling anti-inflammatory effects, PTX may be an ideal candidate for use as an immunomodulatory adjunct to resuscitation fluid.

Effect and mechanism of PAR-2 on the proliferation of esophageal cancer cells.

PubMed

Quanjun, D; Qingyu, Z; Qiliang, Z; Liqun, X; Jinmei, C; Ziquan, L; Shike, H

2016-11-01

Esophageal Cancer (EC) is a common malignant tumor occurred in the digestive tract. In this study, we investigated the mechanism of Protease Activated Receptor 2 (PAR-2) on the proliferation of esophageal cancer cell. Transfected esophageal cancer (EC) cell (PAR-2shRNA EC109) was established with low stable PAR-2 expression. EC109 cell was treated with PAR-2 agonist, PAR-2 anti-agonist and MAPK inhibitor respectively; Untreated EC109 cell (blank control) and PAR-2shRNA EC109 cell were used for analysis also. The mRNA expressions of PAR-2, ERK1, Cyclin D1, and c-fos in each group were detected by reverse transcript and polymerase chain reaction. Western blot was used to detect the protein expressions in each group. The cell growth curves were drawn to compare the cell growth. Compared with the blank control, the mRNA and protein expressions of PAR-2, Cyclin D1, and c-fos in PAR-2 agonist group increased significantly (p < 0.05), while decreased significantly in PAR-2shRNA EC109 cell and MAPK inhibitor group (p < 0.05). The mRNA expression of ERK1 and protein expression of p-ERK1 increased in PAR-2 agonist group, decreased in PAR-2shRNA EC109 cell and MAPK inhibitor group when compared with blank control (p < 0.05). The growth of cells was upward in PAR-2 agonist group at cell growth phase when compared with blank control, while decreased in PAR-2 shRNA EC109 cell and MAPK inhibitor group with statistical difference (p < 0.05). PAR-2 regulate cell proliferation through the MAPK pathway in esophageal carcinoma cell, and Cyclin D1, c-fos are involved in this process.

Up-regulation of IL-23 expression in human dental pulp fibroblasts by IL-17 via activation of the NF-κB and MAPK pathways.

PubMed

Wei, L; Liu, M; Xiong, H; Peng, B

2017-11-06

To investigate the effects of the pro-inflammatory and Th17-polarizing mediator IL-17 on HDPFs-mediated IL-23 production and the molecular mechanism involved. Interleukin (IL)-17R expression was determined by semi-quantitative reverse transcriptase-polymerase chain reaction and Western blot in cultured human dental pulp fibroblasts (HDPFs). Quantitative real-time polymerase chain reaction and enzyme-linked immunosorbent assay were used to determine IL-23 mRNA and protein levels in IL-17-stimulated HDPFs, respectively. The nuclear factor-kappa B (NF-κB) and mitogen-activated protein kinases (MAPKs) signalling pathways that mediate the IL-17-stimulated production of IL-23 was investigated using Western blot and specific signalling inhibitor analyses. Statistical analyses were performed using Kruskal-Wallis tests followed by the Mann-Whitney U-test. Statistical significance was considered when the P value < 0.05. Primary HDPFs steadily expressed IL-17R mRNA and surface-bound protein. IL-17 stimulated the expression of IL-23 mRNA and protein in cultured human dental pulp fibroblasts, which was attenuated by IL-17 or IL-17R neutralizing antibodies. In accordance with the enhanced expression of IL-23, IL-17 stimulation resulted in rapid activation of p38 MAPK, extracellular signal-regulated kinase (ERK) 1/2, c-Jun-N-terminal kinase (JNK) and NF-κB in HDPFs. Inhibitors of p38 MAPK, ERK 1/2 or NF-κB significantly suppressed, whereas blocking JNK substantially augmented IL-23 production from IL-17-stimulated HDPFs. HDPFs expressed IL-17R and responded to IL-17 to produce IL-23 via the activation of the NF-κB and MAPK signalling pathways. The findings provide insights into the cellular mechanisms of the participation of IL-17 in the activation of HDPFs in inflamed pulp tissue. © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.

Astaxanthin inhibits NF-κB and Wnt/β-catenin signaling pathways via inactivation of Erk/MAPK and PI3K/Akt to induce intrinsic apoptosis in a hamster model of oral cancer.

PubMed

Kavitha, K; Kowshik, J; Kishore, T Kranthi Kiran; Baba, Abdul Basit; Nagini, S

2013-10-01

The oncogenic transcription factors NF-κB and β-catenin, constitutively activated by upstream serine/threonine kinases control several cellular processes implicated in malignant transformation including apoptosis evasion. The aim of this study was to investigate the chemopreventive effects of astaxanthin, an antioxidant carotenoid, in the hamster buccal pouch (HBP) carcinogenesis model based on its ability to modulate NF-κB and Wnt signaling pathways and induce apoptosis. We determined the effect of dietary supplementation of astaxanthin on the oncogenic signaling pathways - NF-κB and Wnt/β-catenin, their upstream activator kinases - Erk/MAPK and PI-3K/Akt, and the downstream event - apoptosis evasion by real-time quantitative RT-PCR, western blot, and immunohistochemical analyses. We found that astaxanthin inhibits NF-κB and Wnt signaling by downregulating the key regulatory enzymes IKKβ and GSK-3β. Analysis of gene expression and docking interactions revealed that inhibition of these pathways may be mediated via inactivation of the upstream signaling kinases Erk/Akt by astaxanthin. Astaxanthin also induced caspase-mediated mitochondrial apoptosis by downregulating the expression of antiapoptotic Bcl-2, p-Bad, and survivin and upregulating proapoptotic Bax and Bad, accompanied by efflux of Smac/Diablo and cytochrome-c into the cytosol, and induced cleavage of poly (ADP-ribose) polymerase (PARP). The results provide compelling evidence that astaxanthin exerts chemopreventive effects by concurrently inhibiting phosphorylation of transcription factors and signaling kinases and inducing intrinsic apoptosis. Astaxanthin targets key molecules in oncogenic signaling pathways and induces apoptosis and is a promising candidate agent for cancer prevention and therapy. Copyright © 2013 Elsevier B.V. All rights reserved.

INTERPLAY OF SORBITOL PATHWAY OF GLUCOSE METABOLISM, 12/15-LIPOXYGENASE, AND MITOGEN-ACTIVATED PROTEIN KINASES IN THE PATHOGENESIS OF DIABETIC PERIPHERAL NEUROPATHY

PubMed Central

Stavniichuk, Roman; Shevalye, Hanna; Hirooka, Hiroko; Nadler, Jerry L.; Obrosova, Irina G.

2012-01-01

The interactions among multiple pathogenetic mechanisms of diabetic peripheral neuropathy largely remain unexplored. Increased activity of aldose reductase, the first enzyme of the sorbitol pathway, leads to accumulation of cytosolic Ca++, essentially required for 12/15-lipoxygenase activation. The latter, in turn, causes oxidative-nitrosative stress, an important trigger of MAPK phosphorylation. This study therefore evaluated the interplay of aldose reductase, 12/15-lipoxygenase, and MAPKs in diabetic peripheral neuropathy. In experiment 1, male control and streptozotocin-diabetic mice were maintained with or without the aldose reductase inhibitor fidarestat, 16 mg kg−1 d−1, for 12 weeks. In experiment 2, male control and streptozotocin-diabetic wild-type (C57Bl6/J) and 12/15-lipoxygenase-deficient mice were used. Fidarestat treatment did not affect diabetes-induced increase in glucose concentrations, but normalized sorbitol and fructose concentrations (enzymatic spectrofluorometric assays) as well as 12(S) hydroxyeicosatetraenoic concentration (ELISA), a measure of 12/15-lipoxygenase activity, in the sciatic nerve and spinal cord. 12/15-lipoxygenase expression in these two tissues (Western blot analysis) as well as dorsal root ganglia (immunohistochemistry) was similarly elevated in untreated and fidarestat-treated diabetic mice. 12/15-lipoxygenase gene deficiency prevented diabetesassociated p38 MAPK and ERK, but not SAPK/JNK, activation in the sciatic nerve (Western blot analysis) and all three MAPK activation in the dorsal root ganglia (immunohistochemistry). In contrast, spinal cord p38 MAPK, ERK, and SAPK/JNK were similarly activated in diabetic wild-type and 12/15-lipoxygenase−/− mice. These findings identify the nature and tissue specificity of interactions among three major mechanisms of diabetic peripheral neuropathy, and suggest that combination treatments, rather than monotherapies, can sometimes be an optimal choice for its management. PMID:22285226

Phosphorylation of the Grb2- and phosphatidylinositol 3-kinase p85-binding p36/38 by Syk in Lck-negative T cells.

PubMed

von Willebrand, M; Williams, S; Tailor, P; Mustelin, T

1998-06-01

Activation of the mitogen-activated protein kinase (MAPK) pathway by the T-cell antigen receptor (TCR) in T cells involves a positive role for phosphatidylinositol 3-kinase (PI3K) activity. We recently reported that over-expression of the Syk protein tyrosine kinase in the Lck-negative JCaM1 cells enabled the TCR to induce a normal activation of the Erk2 MAPK and enhanced transcription of a reporter gene driven by the nuclear factor of activated T cells and AP-1. Because this system allows us to analyse the targets for Syk in receptor-mediated signalling, we examined the role of PI3K in signalling events between the TCR-regulated Syk and the downstream activation of Erk2. We report that inhibition of PI3K by wortmannin or an inhibitory p85 construct, p85deltaiSH2, reduced the TCR-induced Syk-dependent activation of Erk2, as well as the appearance of phospho-Erk and phospho-Mek. At the same time, expression of Syk resulted in the activation-dependent phosphorylation of three proteins that bound to the src homology 2 (SH2) domains of PI3K p85. The strongest of these bands had an apparent molecular mass of 36-38 kDa on SDS gels, and it was quantitatively removed from the lysates by adsorption to a fusion protein containing the SH2 domain of Grb2. The appearance of this band was Syk dependent, and it was seen only upon triggering of the TCR complex. Thus, p36/38 was phosphorylated by Syk or a Syk-regulated kinase, and this protein may provide a link to the recruitment and activation of PI3K, as well as to the Ras-MAPK pathway, in TCR-triggered T cells.

Transcriptional Modulation of the ERK1/2 MAPK and NF-kB pathways in Human Urothelial cells after trivalent arsenical exposure: Implications for urinary bladder cancer

EPA Science Inventory

Chronic exposure to drinking water contaminated with inorganic arsenic (iAs) is associated with an increased risk ofurinary bladder (DB) cancers in humans. Rodent models administered particular arsenicals have indicated urothelial necrosis followed by regenerative proliferation i...

DIESEL EXHAUST PARTICLES SUPRESS LPS-STIMULATED PRODUCTION OF PGE2 IN HUMAN ALVEOLAR MACHROPHAGES: ROLE OF P38 MAPK AND ERK PATHWAYS

EPA Science Inventory

Numerous studies have reported association between exposure to ambient levels of particulate matter (PM) and adverse health effects, which include respiratory and cardiovascular effects. Diesel exhaust particles (DEP) compose a significant fraction of PM in some areas. Alveolar m...

Characterizing SHP2 as a Novel Therapeutic Target in Breast Cancer

DTIC Science & Technology

2013-02-01

attempted to elucidate interactions with molecular docking (5). The peptide was docked into the SH2 active site of 2SHP.pdb (with SH2 domains...activated protein kinase (MAPK) pathway, which is read as a drop in phosphorylated ERK protein(3). 5 First, the problem of cell permeability

Synergetic topography and chemistry cues guiding osteogenic differentiation in bone marrow stromal cells through ERK1/2 and p38 MAPK signaling pathway.

PubMed

Zhang, Xinran; Li, Haotian; Lin, Chucheng; Ning, Congqin; Lin, Kaili

2018-01-30

Both the topographic surface and chemical composition modification can enhance rapid osteogenic differentiation and bone formation. Till now, the synergetic effects of topography and chemistry cues guiding biological responses have been rarely reported. Herein, the ordered micro-patterned topography and classically essential trace element of strontium (Sr) ion doping were selected to imitate topography and chemistry cues, respectively. The ordered micro-patterned topography on Sr ion-doped bioceramics was successfully duplicated using the nylon sieve as the template. Biological response results revealed that the micro-patterned topography design or Sr doping could promote cell attachment, ALP activity, and osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs). Most importantly, the samples both with micro-patterned topography and Sr doping showed the highest promotion effects, and could synergistically activate the ERK1/2 and p38 MAPK signaling pathways. The results suggested that the grafts with both specific topography and chemistry cues have synergetic effects on osteogenic activity of BMSCs and provide an effective approach to design functional bone grafts and cell culture substrates.

A short pulse of mechanical force induces gene expression and growth in MC3T3-E1 osteoblasts via an ERK 1/2 pathway

NASA Technical Reports Server (NTRS)

Hatton, Jason P.; Pooran, Milad; Li, Chai-Fei; Luzzio, Chris; Hughes-Fulford, Millie

2003-01-01

Physiological mechanical loading is crucial for maintenance of bone integrity and architecture. We have calculated the strain caused by gravity stress on osteoblasts and found that 4-30g corresponds to physiological levels of 40-300 microstrain. Short-term gravity loading (15 minutes) induced a 15-fold increase in expression of growth-related immediate early gene c-fos, a 5-fold increase in egr-1, and a 3-fold increase in autocrine bFGF. The non-growth-related genes EP-1, TGF-beta, and 18s were unaffected by gravity loading. Short-term physiological loading induced extracellular signal-regulated kinase (ERK 1/2) phosphorylation in a dose-dependent manner with maximum phosphorylation saturating at mechanical loading levels of 12g (p < 0.001) with no effect on total ERK. The phosphorylation of focal adhesion kinase (FAK) was unaffected by mechanical force. g-Loading did not activate P38 MAPK or c-jun N-terminal kinase (JNK). Additionally, a gravity pulse resulted in the localization of phosphorylated ERK 1/2 to the nucleus; this did not occur in unloaded cells. The induction of c-fos was inhibited 74% by the MEK1/2 inhibitor U0126 (p < 0.001) but was not affected by MEK1 or p38 MAPK-specific inhibitors. The long-term consequence of a single 15-minute gravity pulse was a 64% increase in cell growth (p < 0.001). U0126 significantly inhibited gravity-induced growth by 50% (p < 0.001). These studies suggest that short periods of physiological mechanical stress induce immediate early gene expression and growth in MC3T3-E1 osteoblasts primarily through an ERK 1/2-mediated pathway.

The diurnal oscillation of MAP (mitogen-activated protein) kinase and adenylyl cyclase activities in the hippocampus depends on the suprachiasmatic nucleus.

PubMed

Phan, Trongha X; Phan, Trongha H; Chan, Guy C-K; Sindreu, Carlos B; Eckel-Mahan, Kristin L; Storm, Daniel R

2011-07-20

Consolidation of hippocampus-dependent memory is dependent on activation of the cAMP/Erk/MAPK (mitogen-activated protein kinase) signal transduction pathway in the hippocampus. Recently, we discovered that adenylyl cyclase and MAPK activities undergo a circadian oscillation in the hippocampus and that inhibition of this oscillation impairs contextual memory. This suggests the interesting possibility that the persistence of hippocampus-dependent memory depends upon the reactivation of MAPK in the hippocampus during the circadian cycle. A key unanswered question is whether the circadian oscillation of this signaling pathway is intrinsic to the hippocampus or is driven by the master circadian clock in the suprachiasmatic nucleus (SCN). To address this question, we ablated the SCN of mice by electrolytic lesion and examined hippocampus-dependent memory as well as adenylyl cyclase and MAPK activities. Electrolytic lesion of the SCN 2 d after training for contextual fear memory reduced contextual memory measured 2 weeks after training, indicating that maintenance of contextual memory depends on the SCN. Spatial memory was also compromised in SCN-lesioned mice. Furthermore, the diurnal oscillation of adenylyl cyclase and MAPK activities in the hippocampus was destroyed by lesioning of the SCN. These data suggest that hippocampus-dependent long-term memory is dependent on the SCN-controlled oscillation of the adenylyl cyclase/MAPK pathway in the hippocampus.

The Diurnal Oscillation of MAP Kinase and Adenylyl Cyclase Activities in the Hippocampus Depends on the SCN

PubMed Central

Phan, Trongha; Chan, Guy; Sindreu, Carlos; Eckel-Mahan, Kristin; Storm, Daniel R.

2011-01-01

Consolidation of hippocampus dependent memory is dependent on activation of the cAMP/ Erk/MAPK signal transduction pathway in the hippocampus. Recently, we discovered that adenylyl cyclase and MAPK activities undergo a circadian oscillation in the hippocampus and that inhibition of this oscillation impairs contextual memory. This suggests the interesting possibility that the persistence of hippocampus-dependent memory depends upon the reactivation of MAPK in the hippocampus during the circadian cycle. A key unanswered question is whether the circadian oscillation of this signaling pathway is intrinsic to the hippocampus or is driven by the master circadian clock in the suprachiasmatic nucleus (SCN). To address this question, we ablated the SCN of mice by electrolytic lesion and examined hippocampus-dependent memory as well as adenylyl cyclase and MAPK activities. Electrolytic lesion of the SCN two days after training for contextual fear memory reduced contextual memory measured two weeks after training indicating that maintenance of contextual memory depends on the SCN. Spatial memory was also compromised in SCN-lesioned mice. Furthermore, the diurnal oscillation of adenylyl cyclase and MAPK activities in the hippocampus was destroyed by lesioning of the SCN. These data suggest that hippocampus-dependent long-term memory is dependent on the SCN-controlled oscillation of the adenylyl cyclase/MAPK pathway in the hippocampus. PMID:21775607

Coactivation of the PI3K/Akt and ERK signaling pathways in PCB153-induced NF-κB activation and caspase inhibition

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Changjiang; Key Lab of Birth Defects and Reproductive Health of National Health and Family Planning Commission, Chongqing Population and Family Planning Science and Technology Research Institute, Chongqing 400020; Yang, Jixin

2014-06-15

Polychlorinated biphenyls (PCBs) are a group of persistent and widely distributed environmental pollutants that have various deleterious effects, e.g., neurotoxicity, endocrine disruption and reproductive abnormalities. In order to verify the hypothesis that the PI3K/Akt and MAPK pathways play important roles in hepatotoxicity induced by PCBs, Sprague–Dawley (SD) rats were dosed with PCB153 intraperitoneally at 0, 4, 16 and 32 mg/kg for five consecutive days; BRL cells (rat liver cell line) were treated with PCB153 (0, 1, 5, and 10 μM) for 24 h. Results indicated that the PI3K/Akt and ERK pathways were activated in vivo and in vitro after exposuremore » to PCB153, and protein levels of phospho-Akt and phospho-ERK were significantly increased. Nuclear factor-κB (NF-κB) activation and caspase-3, -8 and -9 inhibition caused by PCB153 were also observed. Inhibiting the ERK pathway significantly attenuated PCB153-induced NF-κB activation, whereas inhibiting the PI3K/Akt pathway hardly influenced phospho-NF-κB level. However, inhibiting the PI3K/Akt pathway significantly elevated caspase-3, -8 and -9 activities, while the ERK pathway only synergistically regulated caspase-9. Proliferating cell nuclear antigen (PCNA), a reliable indicator of cell proliferation, was also induced. Moreover, PCB153 led to hepatocellular hypertrophy and elevated liver weight. Taken together, PCB153 leads to aberrant proliferation and apoptosis of hepatocytes through NF-κB activation and caspase inhibition, and coactivated PI3K/Akt and ERK pathways play critical roles in PCB153-induced hepatotoxicity. - Highlights: • PCB153 led to hepatotoxicity through NF-κB activation and caspase inhibition. • The PI3K/Akt and ERK pathways were coactivated in vivo and in vitro by PCB153. • The ERK pathway regulated levels of phospho-NF-κB and caspase-9. • The PI3K/Akt pathway regulated levels of caspase-3, -8 and -9.« less

Bacopa monnieri protects SH-SY5Y cells against tert-Butyl hydroperoxide-induced cell death via the ERK and PI3K pathways.

PubMed

Petcharat, Kanoktip; Singh, Meharvan; Ingkaninan, Kornkanok; Attarat, Jongrak; Yasothornsrikul, Sukkid

2015-01-01

Oxidative stress plays an important role in the pathological processes of various neurodegenerative diseases. Bacopa monnieri (BM) has a potent antioxidant property. Therefore, the purpose of this study was to evaluate the neuroprotective potential of BM against SH-SY5Y neuroblastoma cell death induced by the pro-oxidant insult, tert -Butyl hydroperoxide (TBHP), and to identify possible mechanisms related to its neuroprotective action. The neuroprotective effect of BM was evaluated by the degree of protection against TBHP-induced cell death in human SH-SY5Y cells that was measured by calcein-AM assay. ERK1/2 and Akt phosphorylation was evaluated by immunoblotting. We found that BM exhibited protection against TBHP-mediated cytotoxicity. The neuroprotective effect of BM was abolished in the presence of either ERK1/2 or PI3K inhibitors. In addition, western blotting with anti-phospho-ERK1/2 and anti-phospho-Akt antibodies showed that BM increased both ERK1/2 and Akt phosphorylation. These results suggest that BM by activation of ERK/MAPK and PI3K/Akt signaling pathways protects SH-SY5Y cells from TBHP-induced cell death.

A sestrin-dependent Erk/Jnk/p38 MAPK activation complex inhibits immunity during ageing

PubMed Central

Lanna, Alessio; Gomes, Daniel C O; Muller-Durovic, Bojana; McDonnell, Thomas; Escors, David; Gilroy, Derek W; Lee, Jun Hee; Karin, Michael; Akbar, Arne N

2016-01-01

Mitogen activated protein kinases (MAPKs) including Erk, Jnk and p38 regulate diverse cellular functions, and are thought to be controlled by independent upstream activation cascades. Here we show that the sestrins bind to and co-ordinate simultaneous Erk, Jnk and p38 MAPK activation in T lymphocytes within a new immune-inhibitory complex (sestrin-MAPK Activation Complex; sMAC). Whereas sestrin ablation resulted in broad reconstitution of immune function in stressed T cells, inhibition of individual MAPKs only allowed partial functional recovery. T cells from old humans and mice were more likely to form the sMAC, and disruption of this complex restored antigen-specific functional responses in these cells. Correspondingly, sestrin deficiency or simultaneous inhibition of all three MAPKs enhanced vaccine responsiveness in old mice. Thus, disruption of sMAC provides a foundation for rejuvenating immunity during ageing. PMID:28114291

Regulation of mGlu4 metabotropic glutamate receptor signaling by type-2 G-protein coupled receptor kinase (GRK2).

PubMed

Iacovelli, L; Capobianco, L; Iula, M; Di Giorgi Gerevini, V; Picascia, A; Blahos, J; Melchiorri, D; Nicoletti, F; De Blasi, A

2004-05-01

We examined the role of G-protein coupled receptor kinase-2 (GRK2) in the homologous desensitization of mGlu4 metabotropic glutamate receptors transiently expressed in human embryonic kidney (HEK) 293 cells. Receptor activation with the agonist l-2-amino-4-phosphonobutanoate (l-AP4) stimulated at least two distinct signaling pathways: inhibition of cAMP formation and activation of the mitogen-activated protein kinase (MAPK) pathway [assessed by Western blot analysis of phosphorylated extracellular signal-regulated kinase (ERK) 1 and 2]. Activation of both pathways was attenuated by pertussis toxin. Overexpression of GRK2 (but not GRK4) largely attenuated the stimulation of the MAPK pathway by l-AP4, whereas it slightly potentiated the inhibition of FSK-stimulated cAMP formation. Transfection with a kinase-dead mutant of GRK2 (GRK2-K220R) or with the C-terminal fragment of GRK2 also reduced the mGlu4-mediated stimulation of MAPK, suggesting that GRK2 binds to the Gbetagamma subunits to inhibit signal propagation toward the MAPK pathway. This was confirmed by the evidence that GRK2 coimmunoprecipitated with Gbetagamma subunits in an agonist-dependent manner. Finally, neither GRK2 nor its kinase-dead mutant had any effect on agonist-induced mGlu4 receptor internalization in HEK293 cells transiently transfected with GFP-tagged receptors. Agonist-dependent internalization was instead abolished by a negative-dominant mutant of dynamin, which also reduced the stimulation of MAPK pathway by l-AP4. We speculate that GRK2 acts as a "switch molecule" by inhibiting the mGlu4 receptor-mediated stimulation of MAPK and therefore directing the signal propagation toward the inhibition of adenylyl cyclase.

Mitogen-activated protein kinases (MAPKs) regulate IL-6 over-production during concomitant influenza virus and Staphylococcus aureus infection.

PubMed

Klemm, Carolin; Bruchhagen, Christin; van Krüchten, Andre; Niemann, Silke; Löffler, Bettina; Peters, Georg; Ludwig, Stephan; Ehrhardt, Christina

2017-02-14

Bacterial super-infections are a major complication of influenza virus (IV) infections and often lead to severe pneumonia. One hallmark of IV-associated Staphylococcus aureus (S. aureus) infection is rapid progression to a serious disease outcome. Changes in immune and inflammatory host responses increase morbidity and complicate efficient therapy. A key player during inflammation is the multifunctional cytokine IL-6. Although increased IL-6 levels have been observed after severe disease upon IV and/or bacterial super-infection, the underlying molecular mechanisms still remain to be elucidated. In the present study, we focused on cellular signalling pathways regulating IL-6 production upon IV/S. aureus super-infection. Additionally, infection with viable bacteria was mimicked by lipoteichoic acid stimulation in this model. Analyses of cellular signalling mechanisms revealed synergistically increased activation of the MAPK p38 as well as enhanced phosphorylation of the MAPKs ERK1/2 and JNK in the presence of super-infecting bacteria. Interestingly, inhibition of MAPK activity indicated a strong dependence of IL-6 expression on p38 and ERK1/2, while the MAPK JNK seems not to be involved. Thus, our results provide new molecular insights into the regulation of IL-6, a marker of severe disease, which might contribute to the lethal synergism of IV and S. aureus.

Quinacrine induces apoptosis in human leukemia K562 cells via p38 MAPK-elicited BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Changchien, Jung-Jung; Chen, Ying-Jung; Huang, Chia-Hui

2015-04-01

Although previous studies have revealed the anti-cancer activity of quinacrine, its effect on leukemia is not clearly resolved. We sought to explore the cytotoxic effect and mechanism of quinacrine action in human leukemia K562 cells. Quinacrine induced K562 cell apoptosis accompanied with ROS generation, mitochondrial depolarization, and down-regulation of BCL2L1 and BCL2. Upon exposure to quinacrine, ROS-mediated p38 MAPK activation and ERK inactivation were observed in K562 cells. Quinacrine-induced cell death and mitochondrial depolarization were suppressed by the p38MAPK inhibitor SB202190 and constitutively active MEK1 over-expression. Activation of p38 MAPK was shown to promote BCL2 degradation. Further, ERK inactivation suppressedmore » c-Jun-mediated transcriptional expression of BCL2L1. Over-expression of BCL2L1 and BCL2 attenuated quinacrine-evoked mitochondrial depolarization and rescued the viability of quinacrine-treated cells. Taken together, our data indicate that quinacrine-induced K562 cell apoptosis is mediated through mitochondrial alterations triggered by p38 MAPK-mediated BCL2 down-regulation and suppression of ERK/c-Jun-mediated BCL2L1 expression. - Highlights: • Quinacrine induces K562 cell apoptosis via down-regulation of BCL2 and BCL2L1. • Quinacrine induces p38 MAPK activation and ERK inactivation in K562 cells. • Quinacrine elicits p38 MAPK-mediated BCL2 down-regulation. • Quinacrine suppresses ERK/c-Jun-mediated BCL2L1 expression.« less

Hyaluronan up-regulates growth and invasion of trophoblasts in an autocrine manner via PI3K/AKT and MAPK/ERK1/2 pathways in early human pregnancy.

PubMed

Zhu, R; Huang, Y-H; Tao, Y; Wang, S-C; Sun, Ch; Piao, H-L; Wang, X-Q; Du, M-R; Li, D-J

2013-09-01

As one of the key molecules in the extracellular matrix in human conceptus, hyaluronan (HA) has been receiving particular attention. Here, we have investigated the expression and regulation of different molecular weight HA on the biological behaviors of primary human trophoblasts during the first trimester of pregnancy. The expression of HA and HA synthetase (HAS) by human first trimester trophoblasts was analyzed in placentae from normal pregnancy or miscarriage by immunochemistry and real-time RT-PCR, respectively. ELISA was used to measure the secretion of HA by primary trophoblasts. The effects of HA on the proliferation, apoptosis and invasiveness of trophoblasts were examined. We also investigated the signaling pathways involved in HA activation in human trophoblasts. The higher HAS2 expression and HA secretion were observed in normal villi than that of miscarriage, and the primary trophoblasts secreted HA continuously. High molecular weight HA (HMW-HA) and medium molecular weight HA (MMW-HA) promoted proliferation and invasiveness while inhibited apoptosis of trophoblasts. However, low molecular weight HA (LMW-HA) had no obvious effect on the growth or invasiveness of human trophoblasts. In addition, HMW-HA showed more efficiently than MMW-HA on the growth while MMW-HA displayed a more obvious effect on the invasiveness of trophoblasts than HMW-HA. HMW-HA activated PI3K/AKT and MAPK/ERK1/2 signaling pathways in trophoblasts. Blocking PI3K/AKT or MAPK/ERK1/2 signaling inhibited the HA-upregulated growth and invasiveness of human trophoblasts. Our results suggest that higher level and greater molecular mass of HA can promote trophoblast growth and invasion in an autocrine manner, which was beneficial to placentation and maintenance of human early pregnancy. Copyright © 2013 Elsevier Ltd. All rights reserved.

The TORC1/P70S6K and TORC1/4EBP1 signaling pathways have a stronger contribution on skeletal muscle growth than MAPK/ERK in an early vertebrate: Differential involvement of the IGF system and atrogenes.

PubMed

Fuentes, Eduardo N; Einarsdottir, Ingibjörg Eir; Paredes, Rodolfo; Hidalgo, Christian; Valdes, Juan Antonio; Björnsson, Björn Thrandur; Molina, Alfredo

2015-01-01

Knowledge about the underlying mechanisms, particularly the signaling pathways that account for muscle growth in vivo in early vertebrates is still scarce. Fish (Paralichthys adspersus) were fasted for 3weeks to induce a catabolic period of strong muscle atrophy. Subsequently, fish were refed for 2weeks to induce compensatory muscle hypertrophy. During refeeding, the fish were treated daily with either rapamycin (TORC blocker), PD98059 (MEK blocker), or PBS (V; vehicle), or were untreated (C; control). Rapamycin and PD98059 differentially impaired muscle cellularity in vivo, growth performance, and the expression of growth-related genes, and the inhibition of TORC1 had a greater impact on fish muscle growth than the inhibition of MAPK. Blocking TORC1 inhibited the phosphorylation of P70S6K and 4EBP1, two downstream components activated by TORC1, thus affecting protein contents in muscle. Concomitantly, the gene expression in muscle of igf-1, 2 and igfbp-4, 5 was down-regulated while the expression of atrogin-1, murf-1, and igfbp-2, 3 was up-regulated. Muscle hypertrophy was abolished and muscle atrophy was promoted, which finally affected body weight. TORC2 complex was not affected by rapamycin. On the other hand, the PD98059 treatment triggered ERK inactivation, a downstream component activated by MEK. mRNA contents of igf-1 in muscle were down-regulated, and muscle hypertrophy was partially impaired. The present study provides the first direct data on the in vivo contribution of TORC1/P70S6K, TORC1/4EBP1, and MAPK/ERK signaling pathways in the skeletal muscle of an earlier vertebrate, and highlights the transcendental role of TORC1 in growth from the cellular to organism level. Copyright © 2014 Elsevier Inc. All rights reserved.

Synergistic Effect of Combinatorial Treatment with Curcumin and Mitomycin C on the Induction of Apoptosis of Breast Cancer Cells: A cDNA Microarray Analysis

PubMed Central

Zhou, Qian-Mei; Chen, Qi-Long; Du, Jia; Wang, Xiu-Feng; Lu, Yi-Yu; Zhang, Hui; Su, Shi-Bing

2014-01-01

In order to explore the synergistic mechanisms of combinatorial treatment using curcumin and mitomycin C (MMC) for breast cancer, MCF-7 breast cancer xenografts were conducted to observe the synergistic effect of combinatorial treatment using curcumin and MMC at various dosages. The synergistic mechanisms of combinatorial treatment using curcumin and MMC on the inhibition of tumor growth were explored by differential gene expression profile, gene ontology (GO), ingenuity pathway analysis (IPA) and Signal–Net network analysis. The expression levels of selected genes identified by cDNA microarray expression profiling were validated by quantitative RT-PCR (qRT-PCR) and Western blot analysis. Effect of combinatorial treatment on the inhibition of cell growth was observed by MTT assay. Apoptosis was detected by flow cytometric analysis and Hoechst 33258 staining. The combinatorial treatment of 100 mg/kg curcumin and 1.5 mg/kg MMC revealed synergistic inhibition on tumor growth. Among 1501 differentially expressed genes, the expression of 25 genes exhibited an obvious change and a significant difference in 27 signal pathways was observed (p < 0.05). In addition, Mapk1 (ERK) and Mapk14 (MAPK p38) had more cross-interactions with other genes and revealed an increase in expression by 8.14- and 11.84-fold, respectively during the combinatorial treatment by curcumin and MMC when compared with the control. Moreover, curcumin can synergistically improve tumoricidal effect of MMC in another human breast cancer MDA-MB-231 cells. Apoptosis was significantly induced by the combinatorial treatment (p < 0.05) and significantly inhibited by ERK inhibitor (PD98059) in MCF-7 cells (p < 0.05). The synergistic effect of combinatorial treatment by curcumin and MMC on the induction of apoptosis in breast cancer cells may be via the ERK pathway. PMID:25226537

Procyanidin B2 induces Nrf2 translocation and glutathione S-transferase P1 expression via ERKs and p38-MAPK pathways and protect human colonic cells against oxidative stress.

PubMed

Rodríguez-Ramiro, Ildefonso; Ramos, Sonia; Bravo, Laura; Goya, Luis; Martín, Maria Ángeles

2012-10-01

Procyanidin B2 (PB2) is a naturally occurring flavonoid widely found in cocoa, red wine and grape juice. Recent studies have suggested that PB2 could protect against oxidative stress- and chemical-induced injury in colonic cells by modulating the endogenous cellular defence. However, the precise mechanism for this protection is not fully understood. Herein, we examined the effect of PB2 on the expression of one of the major antioxidant/detoxificant enzymes related to intestinal protection, the glutathione S-transferase P1 (GSTP1), and the molecular mechanisms involved. Human colonic Caco-2 cells were treated with PB2 at different times and enzymatic activity, and mRNA and protein levels of GSTP1 were evaluated. The nuclear translocation of the transcription factor NF-erythroid 2-related factor (Nrf2) and the phosphorylation states of specific proteins central to intracellular signalling cascades were also investigated. PB2 induced the expression and activity of GSTP1 and the nuclear translocation of Nrf2. Interestingly, two important signalling proteins involved in Nrf2 translocation, the extracellular signal-regulated protein kinases (ERKs) and the p38 mitogen-activated protein kinase (MAPK) were also activated. Further experiments with specific inhibitors of both pathways confirmed their critical role in the beneficial effects induced by PB2. The present results show that PB2 protects against oxidative injury in colonic cells and up-regulate the expression of GSTP1 via a mechanism that involves ERK and p38 MAPK activation and Nrf2 translocation. These results provide a molecular basis for the potential contribution of PB2 in the prevention of oxidative stress-related intestinal injury and gut pathologies.

Arctigenin Induces an Activation Response in Porcine Alveolar Macrophage Through TLR6-NOX2-MAPKs Signaling Pathway.

PubMed

Lu, Zheng; Chang, Lingling; Du, Qian; Huang, Yong; Zhang, Xiujuan; Wu, Xingchen; Zhang, Jie; Li, Ruizhen; Zhang, Zelin; Zhang, Wenlong; Zhao, Xiaomin; Tong, Dewen

2018-01-01

Arctigenin (ARG), one of the most active ingredients abstracted from seeds of Arctium lappa L. , has been proved to exert promising biological activities such as immunomodulatory, anti-viral, and anti-cancer etc. However, the mechanism behind its immunomodulatory function still remains elusive to be further investigated. In this study, we found that ARG had no significant effects on the cell proliferation in both porcine alveolar macrophage cell line (3D4/21) and primary porcine derived alveolar macrophage. It remarkably increased the expression and secretion of the two cytokines including tumor necrosis factor-alpha (TNF-α) and transforming growth factor beta1 (TGF-β1) in a dose-dependent manner with the concomitant enhancement of phagocytosis, which are the indicators of macrophage activation. ARG also elevated the intracellular reactive oxygen species (ROS) production by activating NOX2-based NADPH oxidase. Furthermore, inhibition of ROS generation by diphenyliodonium and apocynin significantly suppressed ARG-induced cytokine secretion and phagocytosis increase, indicating the requirement of ROS for the porcine alveolar macrophage activation. In addition, TLR6-My88 excitation, p38 MAPK and ERK1/2 phosphorylation were all involved in the process. As blocking TLR6 receptor dramatically attenuated the NOX2 oxidase activation, cytokine secretion and phagocytosis increase. Inhibiting ROS generation almost abolished p38 and ERK1/2 phosphorylation, and the cytokine secretion could also be remarkably reduced by p38 and ERK1/2 inhibitors (SB203580 and UO126). Our finding gave a new insight of understanding that ARG could improve the immune-function of porcine alveolar macrophages through TLR6-NOX2 oxidase-MAPKs signaling pathway.

Arctigenin Induces an Activation Response in Porcine Alveolar Macrophage Through TLR6-NOX2-MAPKs Signaling Pathway

PubMed Central

Lu, Zheng; Chang, Lingling; Du, Qian; Huang, Yong; Zhang, Xiujuan; Wu, Xingchen; Zhang, Jie; Li, Ruizhen; Zhang, Zelin; Zhang, Wenlong; Zhao, Xiaomin; Tong, Dewen

2018-01-01

Arctigenin (ARG), one of the most active ingredients abstracted from seeds of Arctium lappa L., has been proved to exert promising biological activities such as immunomodulatory, anti-viral, and anti-cancer etc. However, the mechanism behind its immunomodulatory function still remains elusive to be further investigated. In this study, we found that ARG had no significant effects on the cell proliferation in both porcine alveolar macrophage cell line (3D4/21) and primary porcine derived alveolar macrophage. It remarkably increased the expression and secretion of the two cytokines including tumor necrosis factor-alpha (TNF-α) and transforming growth factor beta1 (TGF-β1) in a dose-dependent manner with the concomitant enhancement of phagocytosis, which are the indicators of macrophage activation. ARG also elevated the intracellular reactive oxygen species (ROS) production by activating NOX2-based NADPH oxidase. Furthermore, inhibition of ROS generation by diphenyliodonium and apocynin significantly suppressed ARG-induced cytokine secretion and phagocytosis increase, indicating the requirement of ROS for the porcine alveolar macrophage activation. In addition, TLR6-My88 excitation, p38 MAPK and ERK1/2 phosphorylation were all involved in the process. As blocking TLR6 receptor dramatically attenuated the NOX2 oxidase activation, cytokine secretion and phagocytosis increase. Inhibiting ROS generation almost abolished p38 and ERK1/2 phosphorylation, and the cytokine secretion could also be remarkably reduced by p38 and ERK1/2 inhibitors (SB203580 and UO126). Our finding gave a new insight of understanding that ARG could improve the immune-function of porcine alveolar macrophages through TLR6-NOX2 oxidase-MAPKs signaling pathway. PMID:29867481

Upregulation of cell proliferation via Shc and ERK1/2 MAPK signaling in SaOS-2 osteoblasts grown on magnesium alloy surface coating with tricalcium phosphate.

PubMed

Jiang, Tianlong; Guo, Lei; Ni, Shenghui; Zhao, Yuyan

2015-04-01

Magnesium (Mg) alloys have been demonstrated to be viable orthopedic implants because of mechanical and biocompatible properties similar to natural bone. In order to improve its osteogenic properties, a porous β-tricalcium phosphate (β-TCP) was coated on the Mg-3AI-1Zn alloy by alkali-heat treatment technique. The human bone-derived cells (SaOS-2) were cultured on (β-TCP)-Mg-3AI-1Zn in vitro, and the osteoblast response, the morphology and the elements on this alloy surface were investigated. Also, the regulation of key intracellular signalling proteins was investigated in the SaOS-2 cells cultured on alloy surface. The results from scanning electron microscope and immunofluorescence staining demonstrated that (β-TCP)-Mg-3AI-1Zn induced significant osteogenesis. SaOS-2 cell proliferation was improved by β-TCP coating. Moreover, the (β-TCP)-Mg-3AI-1Zn surface induced activation of key intracellular signalling proteins in SaOS-2 cells. We observed an enhanced activation of Src homology and collagen (Shc), a common point of integration between bone morphogenetic protein 2, and the Ras/mitogen-activated protein kinase (MAPK) pathway. ERK1/2 MAP kinase activation was also upregulated, suggesting a role in mediating osteoblastic cell interactions with biomaterials. The signalling pathway involving c-fos (member of the activated protein-1) was also shown to be upregulated in osteoblasts cultured on the (β-TCP)-Mg-3AI-1Zn. These results suggest that β-TCP coating may contribute to successful osteoblast function on Mg alloy surface. (β-TCP)-Mg-3AI-1Zn may upregulate cell proliferation via Shc and ERK1/2 MAPK signaling in SaOS-2 osteoblasts grown on Mg alloy surface.

FGFR2c-mediated ERK-MAPK activity regulates coronal suture development

PubMed Central

Pfaff, Miles J.; Xue, Ke; Li, Li; Horowitz, Mark C.; Steinbacher, Derek M.; Eswarakumar, Jacob V.P.

2017-01-01

Fibroblast growth factor receptor 2 (FGFR2) signaling is critical for proper craniofacial development. A gain-of-function mutation in the 2c splice variant of the receptor’s gene is associated with Crouzon syndrome, which is characterized by craniosynostosis, the premature fusion of one or more of the cranial vault sutures, leading to craniofacial maldevelopment. Insight into the molecular mechanism of craniosynostosis has identified the ERK-MAPK signaling cascade as a critical regulator of suture patency. The aim of this study is to investigate the role of FGFR2c-induced ERK-MAPK activation in the regulation of coronal suture development. Loss-of-function and gain-of-function Fgfr2c mutant mice have overlapping phenotypes, including coronal synostosis and craniofacial dysmorphia. In vivo analysis of coronal sutures in loss-of-function and gain-of-function models demonstrated fundamentally different pathogenesis underlying coronal suture synostosis. Calvarial osteoblasts from gain-of-function mice demonstrated enhanced osteoblastic function and maturation with concomitant increase in ERK-MAPK activation. In vitro inhibition with the ERK protein inhibitor U0126 mitigated ERK protein activation levels with a concomitant reduction in alkaline phosphatase activity. This study identifies FGFR2c-mediated ERK-MAPK signaling as a key mediator of craniofacial growth and coronal suture development. Furthermore, our results solve the apparent paradox between loss-of-function and gain-of-function FGFR2c mutants with respect to coronal suture synostosis. PMID:27034231

FGFR2c-mediated ERK-MAPK activity regulates coronal suture development.

PubMed

Pfaff, Miles J; Xue, Ke; Li, Li; Horowitz, Mark C; Steinbacher, Derek M; Eswarakumar, Jacob V P

2016-07-15

Fibroblast growth factor receptor 2 (FGFR2) signaling is critical for proper craniofacial development. A gain-of-function mutation in the 2c splice variant of the receptor's gene is associated with Crouzon syndrome, which is characterized by craniosynostosis, the premature fusion of one or more of the cranial vault sutures, leading to craniofacial maldevelopment. Insight into the molecular mechanism of craniosynostosis has identified the ERK-MAPK signaling cascade as a critical regulator of suture patency. The aim of this study is to investigate the role of FGFR2c-induced ERK-MAPK activation in the regulation of coronal suture development. Loss-of-function and gain-of-function Fgfr2c mutant mice have overlapping phenotypes, including coronal synostosis and craniofacial dysmorphia. In vivo analysis of coronal sutures in loss-of-function and gain-of-function models demonstrated fundamentally different pathogenesis underlying coronal suture synostosis. Calvarial osteoblasts from gain-of-function mice demonstrated enhanced osteoblastic function and maturation with concomitant increase in ERK-MAPK activation. In vitro inhibition with the ERK protein inhibitor U0126 mitigated ERK protein activation levels with a concomitant reduction in alkaline phosphatase activity. This study identifies FGFR2c-mediated ERK-MAPK signaling as a key mediator of craniofacial growth and coronal suture development. Furthermore, our results solve the apparent paradox between loss-of-function and gain-of-function FGFR2c mutants with respect to coronal suture synostosis. Copyright © 2016 Elsevier Inc. All rights reserved.

Activation of Extracellular Signal-Regulated Kinase but Not of p38 Mitogen-Activated Protein Kinase Pathways in Lymphocytes Requires Allosteric Activation of SOS

PubMed Central

Jun, Jesse E.; Yang, Ming; Chen, Hang; Chakraborty, Arup K.

2013-01-01

Thymocytes convert graded T cell receptor (TCR) signals into positive selection or deletion, and activation of extracellular signal-related kinase (ERK), p38, and Jun N-terminal protein kinase (JNK) mitogen-activated protein kinases (MAPKs) has been postulated to play a discriminatory role. Two families of Ras guanine nucleotide exchange factors (RasGEFs), SOS and RasGRP, activate Ras and the downstream RAF-MEK-ERK pathway. The pathways leading to lymphocyte p38 and JNK activation are less well defined. We previously described how RasGRP alone induces analog Ras-ERK activation while SOS and RasGRP cooperate to establish bimodal ERK activation. Here we employed computational modeling and biochemical experiments with model cell lines and thymocytes to show that TCR-induced ERK activation grows exponentially in thymocytes and that a W729E allosteric pocket mutant, SOS1, can only reconstitute analog ERK signaling. In agreement with RasGRP allosterically priming SOS, exponential ERK activation is severely decreased by pharmacological or genetic perturbation of the phospholipase Cγ (PLCγ)-diacylglycerol-RasGRP1 pathway. In contrast, p38 activation is not sharply thresholded and requires high-level TCR signal input. Rac and p38 activation depends on SOS1 expression but not allosteric activation. Based on computational predictions and experiments exploring whether SOS functions as a RacGEF or adaptor in Rac-p38 activation, we established that the presence of SOS1, but not its enzymatic activity, is critical for p38 activation. PMID:23589333

Activation of VEGF/Flk-1-ERK Pathway Induced Blood-Brain Barrier Injury After Microwave Exposure.

PubMed

Wang, Li-Feng; Li, Xiang; Gao, Ya-Bing; Wang, Shui-Ming; Zhao, Li; Dong, Ji; Yao, Bin-Wei; Xu, Xin-Ping; Chang, Gong-Min; Zhou, Hong-Mei; Hu, Xiang-Jun; Peng, Rui-Yun

2015-08-01

Microwaves have been suggested to induce neuronal injury and increase permeability of the blood-brain barrier (BBB), but the mechanism remains unknown. The role of the vascular endothelial growth factor (VEGF)/Flk-1-Raf/MAPK kinase (MEK)/extracellular-regulated protein kinase (ERK) pathway in structural and functional injury of the blood-brain barrier (BBB) following microwave exposure was examined. An in vitro BBB model composed of the ECV304 cell line and primary rat cerebral astrocytes was exposed to microwave radiation (50 mW/cm(2), 5 min). The structure was observed by scanning electron microscopy (SEM) and the permeability was assessed by measuring transendothelial electrical resistance (TEER) and horseradish peroxidase (HRP) transmission. Activity and expression of VEGF/Flk-1-ERK pathway components and occludin also were examined. Our results showed that microwave radiation caused intercellular tight junctions to broaden and fracture with decreased TEER values and increased HRP permeability. After microwave exposure, activation of the VEGF/Flk-1-ERK pathway and Tyr phosphorylation of occludin were observed, along with down-regulated expression and interaction of occludin with zonula occludens-1 (ZO-1). After Flk-1 (SU5416) and MEK1/2 (U0126) inhibitors were used, the structure and function of the BBB were recovered. The increase in expression of ERK signal transduction molecules was muted, while the expression and the activity of occludin were accelerated, as well as the interactions of occludin with p-ERK and ZO-1 following microwave radiation. Thus, microwave radiation may induce BBB damage by activating the VEGF/Flk-1-ERK pathway, enhancing Tyr phosphorylation of occludin, while partially inhibiting expression and interaction of occludin with ZO-1.

Role of PTEN in the Tumor Microenvironment

DTIC Science & Technology

2008-06-01

van Diest PJ. (2008). Hexokinase III, cyclin A and galectin - 3 are overexpressed in malignant follicular thyroid nodules. Clin Endocrinol (Oxf) 2...Annual 3 . DATES COVERED (From - To) 15 May 2007 – 14 May 2008 4. TITLE AND SUBTITLE Role of PTEN in the Tumor Microenvironment 5a. CONTRACT NUMBER...1998) that impacts several signaling pathways, including phosphoinositide 3 -kinase (PI3K), and Ras-MAPK-Erk1/2 signaling pathways. Pten inactivation

Disorders of dysregulated signal traffic through the RAS-MAPK pathway: phenotypic spectrum and molecular mechanisms.

PubMed

Tartaglia, Marco; Gelb, Bruce D

2010-12-01

RAS GTPases control a major signaling network implicated in several cellular functions, including cell fate determination, proliferation, survival, differentiation, migration, and senescence. Within this network, signal flow through the RAF-MEK-ERK pathway-the first identified mitogen-associated protein kinase (MAPK) cascade-mediates early and late developmental processes controlling morphology determination, organogenesis, synaptic plasticity, and growth. Signaling through the RAS-MAPK cascade is tightly controlled; and its enhanced activation represents a well-known event in oncogenesis. Unexpectedly, in the past few years, inherited dysregulation of this pathway has been recognized as the cause underlying a group of clinically related disorders sharing facial dysmorphism, cardiac defects, reduced postnatal growth, ectodermal anomalies, variable cognitive deficits, and susceptibility to certain malignancies as major features. These disorders are caused by heterozygosity for mutations in genes encoding RAS proteins, regulators of RAS function, modulators of RAS interaction with effectors, or downstream signal transducers. Here, we provide an overview of the phenotypic spectrum associated with germline mutations perturbing RAS-MAPK signaling, the unpredicted molecular mechanisms converging toward the dysregulation of this signaling cascade, and major genotype-phenotype correlations. © 2010 New York Academy of Sciences.

Naproxen Induces Type X Collagen Expression in Human Bone-Marrow-Derived Mesenchymal Stem Cells Through the Upregulation of 5-Lipoxygenase

PubMed Central

Alaseem, Abdulrahman M.; Madiraju, Padma; Aldebeyan, Sultan A.; Noorwali, Hussain; Antoniou, John

2015-01-01

Several studies have shown that type X collagen (COL X), a marker of late-stage chondrocyte hypertrophy, is expressed in mesenchymal stem cells (MSCs) from osteoarthritis (OA) patients. We recently found that Naproxen, but not other nonsteroidal anti-inflammatory drugs (NSAIDs) (Ibuprofen, Celebrex, Diclofenac), can induce type X collagen gene (COL10A1) expression in bone-marrow-derived MSCs from healthy and OA donors. In this study we determined the effect of Naproxen on COL X protein expression and investigated the intracellular signaling pathways that mediate Naproxen-induced COL10A1 expression in normal and OA hMSCs. MSCs of OA patients were isolated from aspirates from the intramedullary canal of donors (50–80 years of age) undergoing hip replacement surgery for OA and were treated with or without Naproxen (100 μg/mL). Protein expression and phosphorylation were determined by immunoblotting using specific antibodies (COL X, p38 mitogen-activated protein kinase [p38], phosphorylated-p38, c-Jun N-terminal kinase [JNK], phosphorylated-JNK, extracellular signal-regulated kinase [ERK], and phosphorylated-ERK). Real-time reverse transcription polymerase chain reaction (RT-PCR) was performed to determine the expression of COL10A1 and Runt-related transcription factor 2 gene (Runx2). Our results show that Naproxen significantly stimulated COL X protein expression after 72 h of exposure both in normal and OA hMSCs. The basal phosphorylation of mitogen-activated protein kinases (MAPKs) (ERK, JNK, and p38) in OA hMSCs was significantly higher than in normal. Naproxen significantly increased the MAPK phosphorylation in normal and OA hMSCs. NSAID cellular effects include cyclooxygenase, 5-lipoxygenase, and p38 MAPK signaling pathways. To investigate the involvement of these pathways in the Naproxen-induced COL10A1 expression, we incubated normal and OA hMSCs with Naproxen with and without inhibitors of ERK (U0126), JNK (BI-78D3), p38 (SB203580), and 5-lipoxygenase (MK-886). Our results showed that increased basal COL10A1 expression in OA hMSCs was significantly suppressed in the presence of JNK and p38 inhibitors, whereas Naproxen-induced COL10A1 expression was suppressed by 5-lipoxygenase inhibitor. This study shows that Naproxen induces COL X both at transcriptional and translational levels in normal and OA hMSCs. Elevated basal COL10A1 expression in OA hMSCs is probably through the activation of MAPK pathway and Naproxen-induced COL10A1 expression is through the increased 5-lipoxygenase signaling. PMID:25091567

Molecular Dynamics Simulations Provide Atomistic Insight into Hydrogen Exchange Mass Spectrometry Experiments.

PubMed

Petruk, Ariel A; Defelipe, Lucas A; Rodríguez Limardo, Ramiro G; Bucci, Hernán; Marti, Marcelo A; Turjanski, Adrian G

2013-01-08

It is now clear that proteins are flexible entities that in solution switch between conformations to achieve their function. Hydrogen/Deuterium Exchange Mass Spectrometry (HX/MS) is an invaluable tool to understand dynamic changes in proteins modulated by cofactor binding, post-transductional modifications, or protein-protein interactions. ERK2MAPK, a protein involved in highly conserved signal transduction pathways of paramount importance for normal cellular function, has been extensively studied by HX/MS. Experiments of the ERK2MAPK in the inactive and active states (in the presence or absence of bound ATP) have provided valuable information on the plasticity of the MAPK domain. However, interpretation of the HX/MS data is difficult, and changes are mostly explained in relation to available X-ray structures, precluding a complete atomic picture of protein dynamics. In the present work, we have used all atom Molecular Dynamics simulations (MD) to provide a theoretical framework for the interpretation of HX/MS data. Our results show that detailed analysis of protein-solvent interaction along the MD simulations allows (i) prediction of the number of protons exchanged for each peptide in the HX/MS experiments, (ii) rationalization of the experimentally observed changes in exchange rates in different protein conditions at the residue level, and (iii) that at least for ERK2MAPK, most of the functionally observed differences in protein dynamics are related to what can be considered the native state conformational ensemble. In summary, the combination of HX/MS experiments with all atom MD simulations emerges as a powerful approach to study protein native state dynamics with atomic resolution.

RAS diseases in children

PubMed Central

Niemeyer, Charlotte M.

2014-01-01

RAS genes encode a family of 21 kDa proteins that are an essential hub for a number of survival, proliferation, differentiation and senescence pathways. Signaling of the RAS-GTPases through the RAF-MEK-ERK pathway, the first identified mitogen-associated protein kinase (MAPK) cascade is essential in development. A group of genetic syndromes, named “RASopathies”, had been identified which are caused by heterozygosity for germline mutations in genes that encode protein components of the RAS/MAPK pathway. Several of these clinically overlapping disorders, including Noonan syndrome, Noonan-like CBL syndrome, Costello syndrome, cardio-facio-cutaneous (CFC) syndrome, neurofibromatosis type I, and Legius syndrome, predispose to cancer and abnormal myelopoiesis in infancy. This review focuses on juvenile myelomonocytic leukemia (JMML), a malignancy of early childhood characterized by initiating germline and/or somatic mutations in five genes of the RAS/MAPK pathway: PTPN11, CBL, NF-1, KRAS and NRAS. Natural courses of these five subtypes differ, although hematopoietic stem cell transplantation remains the only curative therapy option for most children with JMML. With whole-exome sequencing studies revealing few secondary lesions it will be crucial to better understand the RAS/MAPK signaling network with its crosstalks and feed-back loops to carefully design early clinical trials with novel pharmacological agents in this still puzzling leukemia. PMID:25420281

Dual specificity phosphatase 5 and 6 are oppositely regulated in human skeletal muscle by acute exercise.

PubMed

Pourteymour, Shirin; Hjorth, Marit; Lee, Sindre; Holen, Torgeir; Langleite, Torgrim M; Jensen, Jørgen; Birkeland, Kåre I; Drevon, Christian A; Eckardt, Kristin

2017-10-01

Physical activity promotes specific adaptations in most tissues including skeletal muscle. Acute exercise activates numerous signaling cascades including pathways involving mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK)1/2, which returns to pre-exercise level after exercise. The expression of MAPK phosphatases (MKPs) in human skeletal muscle and their regulation by exercise have not been investigated before. In this study, we used mRNA sequencing to monitor regulation of MKPs in human skeletal muscle after acute cycling. In addition, primary human myotubes were used to gain more insights into the regulation of MKPs. The two ERK1/2-specific MKPs, dual specificity phosphatase 5 (DUSP5) and DUSP6, were the most regulated MKPs in skeletal muscle after acute exercise. DUSP5 expression was ninefold higher immediately after exercise and returned to pre-exercise level within 2 h, whereas DUSP6 expression was reduced by 43% just after exercise and remained below pre-exercise level after 2 h recovery. Cultured myotubes express both MKPs, and incubation with dexamethasone (Dex) mimicked the in vivo expression pattern of DUSP5 and DUSP6 caused by exercise. Using a MAPK kinase inhibitor, we showed that stimulation of ERK1/2 activity by Dex was required for induction of DUSP5 However, maintaining basal ERK1/2 activity was required for basal DUSP6 expression suggesting that the effect of Dex on DUSP6 might involve an ERK1/2-independent mechanism. We conclude that the altered expression of DUSP5 and DUSP6 in skeletal muscle after acute endurance exercise might affect ERK1/2 signaling of importance for adaptations in skeletal muscle during exercise. © 2017 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.

Non-Smad signaling pathways.

PubMed

Mu, Yabing; Gudey, Shyam Kumar; Landström, Maréne

2012-01-01

Transforming growth factor-beta (TGFβ) is a key regulator of cell fate during embryogenesis and has also emerged as a potent driver of the epithelial-mesenchymal transition during tumor progression. TGFβ signals are transduced by transmembrane type I and type II serine/threonine kinase receptors (TβRI and TβRII, respectively). The activated TβR complex phosphorylates Smad2 and Smad3, converting them into transcriptional regulators that complex with Smad4. TGFβ also uses non-Smad signaling pathways such as the p38 and Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) pathways to convey its signals. Ubiquitin ligase tumor necrosis factor (TNF)-receptor-associated factor 6 (TRAF6) and TGFβ-associated kinase 1 (TAK1) have recently been shown to be crucial for the activation of the p38 and JNK MAPK pathways. Other TGFβ-induced non-Smad signaling pathways include the phosphoinositide 3-kinase-Akt-mTOR pathway, the small GTPases Rho, Rac, and Cdc42, and the Ras-Erk-MAPK pathway. Signals induced by TGFβ are tightly regulated and specified by post-translational modifications of the signaling components, since they dictate the subcellular localization, activity, and duration of the signal. In this review, we discuss recent findings in the field of TGFβ-induced responses by non-Smad signaling pathways.

Dexamethasone Protects Neonatal Hypoxic-Ischemic Brain Injury via L-PGDS-Dependent PGD2-DP1-pERK Signaling Pathway

PubMed Central

Gonzalez-Rodriguez, Pablo J.; Li, Yong; Martinez, Fabian; Zhang, Lubo

2014-01-01

Background and Purpose Glucocorticoids pretreatment confers protection against neonatal hypoxic-ischemic (HI) brain injury. However, the molecular mechanism remains poorly elucidated. We tested the hypothesis that glucocorticoids protect against HI brain injury in neonatal rat by stimulation of lipocalin-type prostaglandin D synthase (L-PGDS)-induced prostaglandin D2 (PGD2)-DP1-pERK mediated signaling pathway. Methods Dexamethasone and inhibitors were administered via intracerebroventricular (i.c.v) injections into 10-day-old rat brains. Levels of L-PGD2, D prostanoid (DP1) receptor, pERK1/2 and PGD2 were determined by Western immunoblotting and ELISA, respectively. Brain injury was evaluated 48 hours after conduction of HI in 10-day-old rat pups. Results Dexamethasone pretreatment significantly upregulated L-PGDS expression and the biosynthesis of PGD2. Dexamethasone also selectively increased isoform pERK-44 level in the neonatal rat brains. Inhibitors of L-PGDS (SeCl4), DP1 (MK-0524) and MAPK (PD98059) abrogated dexamethasone-induced increases in pERK-44 level, respectively. Of importance, these inhibitors also blocked dexamethasone-mediated neuroprotective effects against HI brain injury in neonatal rat brains. Conclusion Interaction of glucocorticoids-GR signaling and L-PGDS-PGD2-DP1-pERK mediated pathway underlies the neuroprotective effects of dexamethasone pretreatment in neonatal HI brain injury. PMID:25474649

Induction of keratinocyte migration by ECa 233 is mediated through FAK/Akt, ERK, and p38 MAPK signaling.

PubMed

Singkhorn, Sawana; Tantisira, Mayuree H; Tanasawet, Supita; Hutamekalin, Pilaiwanwadee; Wongtawatchai, Tulaporn; Sukketsiri, Wanida

2018-03-13

Centella asiatica is widely considered the most important medicinal plant for treating and relieving skin diseases. Recently developed standardized extract of Centella asiatica ECa 233 has demonstrated positive effects on wound healing of incision and burn wound in rats. However, knowledge associated with wound healing mechanism of ECa 233 was scare. Therefore, this study aimed to investigate the effect and underlying molecular mechanisms of ECa 233 on the migration of a human keratinocyte cell line (HaCaT) using scratch wound healing assay. Formation of filopodia, a key protein in cell migration as well as signaling pathways possibly involved were subsequently assessed. It was found that HaCaT cell migration was significantly enhanced by ECa 233 in a concentration- and time-dependent manner. The filopodia formations were accordingly increased in exposure to ECa 233 at concentrations of 0.1-100 μg/ml. Furthermore, ECa 233 was found to significantly upregulate the expression of Rac1 and RhoA and to induce phosphorylation of FAK and Akt as well as ERK and p38 MAPK. Taken all together, it is suggestive that ECa 233 induces cell migration and subsequently promotes wound healing activity, through the activation of FAK, Akt, and MAPK signaling pathways thereby supporting the role of ECa 233 to be further developed for the clinical treatment of wound. Copyright © 2018 John Wiley & Sons, Ltd.

The Effects of Topographical Patterns and Sizes on Neural Stem Cell Behavior

PubMed Central

Qi, Lin; Li, Ning; Huang, Rong; Song, Qin; Wang, Long; Zhang, Qi; Su, Ruigong; Kong, Tao; Tang, Mingliang; Cheng, Guosheng

2013-01-01

Engineered topographical manipulation, a paralleling approach with conventional biochemical cues, has recently attracted the growing interests in utilizations to control stem cell fate. In this study, effects of topological parameters, pattern and size are emphasized on the proliferation and differentiation of adult neural stem cells (ANSCs). We fabricate micro-scale topographical Si wafers with two different feature sizes. These topographical patterns present linear micro-pattern (LMP), circular micro-pattern (CMP) and dot micro-pattern (DMP). The results show that the three topography substrates are suitable for ANSC growth, while they all depress ANSC proliferation when compared to non-patterned substrates (control). Meanwhile, LMP and CMP with two feature sizes can both significantly enhance ANSC differentiation to neurons compared to control. The smaller the feature size is, the better upregulation applies to ANSC for the differentiated neurons. The underlying mechanisms of topography-enhanced neuronal differentiation are further revealed by directing suppression of mitogen-activated protein kinase/extracellular signaling-regulated kinase (MAPK/Erk) signaling pathway in ANSC using U0126, known to inhibit the activation of Erk. The statistical results suggest MAPK/Erk pathway is partially involved in topography-induced differentiation. These observations provide a better understanding on the different roles of topographical cues on stem cell behavior, especially on the selective differentiation, and facilitate to advance the field of stem cell therapy. PMID:23527077

Tetramethylpyrazine suppresses transient oxygen-glucose deprivation-induced connexin32 expression and cell apoptosis via the ERK1/2 and p38 MAPK pathway in cultured hippocampal neurons.

PubMed

Gong, Gu; Yuan, Libang; Cai, Lin; Ran, Maorong; Zhang, Yulan; Gong, Huaqu; Dai, Xuemei; Wu, Wei; Dong, Hailong

2014-01-01

Tetramethylpyrazine (TMP) has been widely used in China as a drug for the treatment of various diseases. Recent studies have suggested that TMP has a protective effect on ischemic neuronal damage. However, the exact mechanism is still unclear. This study aims to investigate the mechanism of TMP mediated ischemic hippocampal neurons injury induced by oxygen-glucose deprivation (OGD). The effect of TMP on hippocampal neurons viability was detected by MTT assay, LDH release assay and apoptosis rate was measured by flow cytometry. TMP significantly suppressed neuron apoptosis in a concentration-dependent manner. TMP could significantly reduce the elevated levels of connexin32 (Cx32) induced by OGD. Knockdown of Cx32 by siRNA attenuated OGD injury. Moreover, our study showed that viability was increased in siRNA-Cx32-treated-neurons, and neuron apoptosis was suppressed by activating Bcl-2 expression and inhibiting Bax expression. Over expression of Cx32 could decrease neurons viability and increase LDH release. Furthermore, OGD increased phosphorylation of ERK1/2 and p38, whose inhibitors relieved the neuron injury and Cx32 up-regulation. Taken together, TMP can reverse the OGD-induced Cx32 expression and cell apoptosis via the ERK1/2 and p38 MAPK pathways.

[Intracellular signaling mechanisms in thyroid cancer].

PubMed

Mondragón-Terán, Paul; López-Hernández, Luz Berenice; Gutiérrez-Salinas, José; Suárez-Cuenca, Juan Antonio; Luna-Ceballos, Rosa Isela; Erazo Valle-Solís, Aura

2016-01-01

Thyroid cancer is the most common malignancy of the endocrine system, the papillary variant accounts for 80-90% of all diagnosed cases. In the development of papillary thyroid cancer, BRAF and RAS genes are mainly affected, resulting in a modification of the system of intracellular signaling proteins known as «protein kinase mitogen-activated» (MAPK) which consist of «modules» of internal signaling proteins (Receptor/Ras/Raf/MEK/ERK) from the cell membrane to the nucleus. In thyroid cancer, these signanling proteins regulate diverse cellular processes such as differentiation, growth, development and apoptosis. MAPK play an important role in the pathogenesis of thyroid cancer as they are used as molecular biomarkers for diagnostic, prognostic and as possible therapeutic molecular targets. Mutations in BRAF gene have been correlated with poor response to treatment with traditional chemotherapy and as an indicator of poor prognosis. To review the molecular mechanisms involved in intracellular signaling of BRAF and RAS genes in thyroid cancer. Molecular therapy research is in progress for this type of cancer as new molecules have been developed in order to inhibit any of the components of the signaling pathway (RET/PTC)/Ras/Raf/MEK/ERK; with special emphasis on the (RET/PTC)/Ras/Raf section, which is a major effector of ERK pathway. Copyright © 2016 Academia Mexicana de Cirugía A.C. Publicado por Masson Doyma México S.A. All rights reserved.

Phosphorylation of ERK/MAP Kinase Is Required for Long-Term Potentiation in Anatomically Restricted Regions of the Lateral Amygdala in Vivo

ERIC Educational Resources Information Center

Schafe, Glenn E.; Swank, Michael W.; Rodriguez, Sarina M.; Debiec, Jacek; Doyere, Valerie

2008-01-01

We have previously shown that the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/ MAPK) is transiently activated in anatomically restricted regions of the lateral amygdala (LA) following Pavlovian fear conditioning and that blockade of ERK/MAPK activation in the LA impairs both fear memory consolidation and long-term…

Analysis of the ERK1,2 transcriptome in mammary epithelial cells

PubMed Central

Grill, Constance; Gheyas, Ferdous; Dayananth, Priya; Jin, Weihong; Ding, Wei; Qiu, Ping; Wang, Luquan; Doll, Ronald J.; English, Jessie M.

2004-01-01

MAPK (mitogen-activated protein kinase) pathways constitute major regulators of cellular transcriptional programmes. We analysed the ERK1,2 (extracellular-signal-regulated kinase 1,2) transcriptome in a non-transformed MEC (mammary epithelial cell) line, MCF-12A, utilizing rAd MEK1EE, a recombinant adenovirus encoding constitutively active MEK1 (MAPK/ERK kinase 1). rAd MEK1EE infection induced morphological changes and DNA synthesis which were inhibited by the MEK1,2 inhibitor PD184352. Hierarchical clustering of data derived from seven time points over 24 h identified 430 and 305 co-ordinately up-regulated and down-regulated genes respectively. c-Myc binding sites were identified in the promoters of most of these up-regulated genes. A total of 46 candidate effectors of the Raf/MEK/ERK1,2 pathway in MECs were identified by comparing our dataset with previously reported Raf-1-regulated genes. These analyses led to the identification of a suite of growth factors co-ordinately induced by MEK1EE, including multiple ErbB ligands, vascular endothelial growth factor and PHRP (parathyroid hormone-related protein). PHRP is the primary mediator of humoral hypercalcaemia of malignancy, and has been implicated in metastasis to bone. We demonstrate that PHRP is secreted by MEK1EE-expressing cells. This secretion is inhibited by PD184352, but not by ErbB inhibitors. Our results suggest that, in addition to anti-proliferative properties, MEK1,2 inhibitors may be anti-angiogenic and possess therapeutic utility in the treatment of PHRP-positive tumours. PMID:15109307

The MAPK1/3 pathway is essential for the deregulation of autophagy observed in G2019S LRRK2 mutant fibroblasts

PubMed Central

Bravo-San Pedro, José M.; Gómez-Sánchez, Rubén; Niso-Santano, Mireia; Pizarro-Estrella, Elisa; Aiastui-Pujana, Ana; Gorostidi, Ana; Climent, Vicente; López de Maturana, Rakel; Sanchez-Pernaute, Rosario; López de Munain, Adolfo; Fuentes, José M.; González-Polo, Rosa A.

2012-01-01

The link between the deregulation of autophagy and cell death processes can be essential in the development of several neurodegenerative diseases, such as Parkinson disease (PD). However, the molecular mechanism of deregulation of this degradative process in PD patients is unknown. The leucine-rich repeat kinase 2 (LRRK2) gene is related to PD and its implication in autophagy regulation has been described. Our recent work shows that the presence of the G2019S LRRK2 mutation, one of the most prevalent in LRRK2, is accompanied by a deregulation of autophagy basal levels dependent on the MAPK1/3 (ERK2/1) pathway. PMID:22914360

Viral exploitation of the MEK/ERK pathway - A tale of vaccinia virus and other viruses.

PubMed

Bonjardim, Cláudio A

2017-07-01

The VACV replication cycle is remarkable in the sense that it is performed entirely in the cytoplasmic compartment of vertebrate cells, due to its capability to encode enzymes required either for regulating the macromolecular precursor pool or the biosynthetic processes. Although remarkable, this gene repertoire is not sufficient to confer the status of a free-living microorganism to the virus, and, consequently, the virus relies heavily on the host to successfully generate its progeny. During the complex virus-host interaction, viruses must deal not only with the host pathways to accomplish their temporal demands but also with pathways that counteract viral infection, including the inflammatory, innate and acquired immune responses. This review focuses on VACV and other DNA or RNA viruses that stimulate the MEK (MAPK - Mitogen Activated Protein Kinase)/ERK- Extracellular signal-Regulated Kinase) pathway as part of their replication cycle. Copyright © 2016 Elsevier Inc. All rights reserved.

Growth differentiation factor 9 signaling requires ERK1/2 activity in mouse granulosa and cumulus cells.

PubMed

Sasseville, Maxime; Ritter, Lesley J; Nguyen, Thao M; Liu, Fang; Mottershead, David G; Russell, Darryl L; Gilchrist, Robert B

2010-09-15

Ovarian folliculogenesis is driven by the combined action of endocrine cues and paracrine factors. The oocyte secretes powerful mitogens, such as growth differentiation factor 9 (GDF9), that regulate granulosa cell proliferation, metabolism, steroidogenesis and differentiation. This study investigated the role of the epidermal growth factor receptor (EGFR)-extracellular signal-regulated kinase 1 and 2 (ERK1/2; also known as MAPK3/1) signaling pathway on GDF9 action on granulosa cells. Results show that mitogenic action of the oocyte is prevented by pharmacological inhibition of the EGFR-ERK1/2 pathway. Importantly, EGFR-ERK1/2 activity as well as rous sarcoma oncogene family kinases (SFK) are required for signaling through SMADs, mediating GDF9, activin A and TGFbeta1 mitogenic action in granulosa cells. GDF9 could not activate ERK1/2 or affect EGF-stimulated ERK1/2 in granulosa cells. However, induction of the SMAD3-specific CAGA reporter by GDF9 in granulosa cells required active EGFR, SFKs and ERK1/2 as did GDF9-responsive gene expression. Finally, the EGFR-SFKs-ERK1/2 pathway was shown to be required for the maintenance of phosphorylation of the SMAD3 linker region. Together our results suggest that receptivity of granulosa cells to oocyte-secreted factors, including GDF9, is regulated by the level of activation of the EGFR and resulting ERK1/2 activity, through the requisite permissive phosphorylation of SMAD3 in the linker region. Our results indicate that oocyte-secreted TGFbeta-like ligands and EGFR-ERK1/2 signaling are cooperatively required for the unique granulosa cell response to the signal from oocytes mediating granulosa cell survival and proliferation and hence the promotion of follicle growth and ovulation.

Rewiring MAP kinases in Saccharomyces cerevisiae to regulate novel targets through ubiquitination.

PubMed

Groves, Benjamin; Khakhar, Arjun; Nadel, Cory M; Gardner, Richard G; Seelig, Georg

2016-08-15

Evolution has often copied and repurposed the mitogen-activated protein kinase (MAPK) signaling module. Understanding how connections form during evolution, in disease and across individuals requires knowledge of the basic tenets that govern kinase-substrate interactions. We identify criteria sufficient for establishing regulatory links between a MAPK and a non-native substrate. The yeast MAPK Fus3 and human MAPK ERK2 can be functionally redirected if only two conditions are met: the kinase and substrate contain matching interaction domains and the substrate includes a phospho-motif that can be phosphorylated by the kinase and recruit a downstream effector. We used a panel of interaction domains and phosphorylation-activated degradation motifs to demonstrate modular and scalable retargeting. We applied our approach to reshape the signaling behavior of an existing kinase pathway. Together, our results demonstrate that a MAPK can be largely defined by its interaction domains and compatible phospho-motifs and provide insight into how MAPK-substrate connections form.

Novel Reporter for Faithful Monitoring of ERK2 Dynamics in Living Cells and Model Organisms

PubMed Central

Sipieter, François; Cappe, Benjamin; Gonzalez Pisfil, Mariano; Spriet, Corentin; Bodart, Jean-François; Cailliau-Maggio, Katia; Vandenabeele, Peter; Héliot, Laurent; Riquet, Franck B.

2015-01-01

Uncoupling of ERK1/2 phosphorylation from subcellular localization is essential towards the understanding of molecular mechanisms that control ERK1/2-mediated cell-fate decision. ERK1/2 non-catalytic functions and discoveries of new specific anchors responsible of the subcellular compartmentalization of ERK1/2 signaling pathway have been proposed as regulation mechanisms for which dynamic monitoring of ERK1/2 localization is necessary. However, studying the spatiotemporal features of ERK2, for instance, in different cellular processes in living cells and tissues requires a tool that can faithfully report on its subcellular distribution. We developed a novel molecular tool, ERK2-LOC, based on the T2A-mediated coexpression of strictly equimolar levels of eGFP-ERK2 and MEK1, to faithfully visualize ERK2 localization patterns. MEK1 and eGFP-ERK2 were expressed reliably and functionally both in vitro and in single living cells. We then assessed the subcellular distribution and mobility of ERK2-LOC using fluorescence microscopy in non-stimulated conditions and after activation/inhibition of the MAPK/ERK1/2 signaling pathway. Finally, we used our coexpression system in Xenopus laevis embryos during the early stages of development. This is the first report on MEK1/ERK2 T2A-mediated coexpression in living embryos, and we show that there is a strong correlation between the spatiotemporal subcellular distribution of ERK2-LOC and the phosphorylation patterns of ERK1/2. Our approach can be used to study the spatiotemporal localization of ERK2 and its dynamics in a variety of processes in living cells and embryonic tissues. PMID:26517832

The Nuclear Dbf2-Related Kinase COT1 and the Mitogen-Activated Protein Kinases MAK1 and MAK2 Genetically Interact to Regulate Filamentous Growth, Hyphal Fusion and Sexual Development in Neurospora crassa

PubMed Central

Maerz, Sabine; Ziv, Carmit; Vogt, Nico; Helmstaedt, Kerstin; Cohen, Nourit; Gorovits, Rena; Yarden, Oded; Seiler, Stephan

2008-01-01

Ndr kinases, such as Neurospora crassa COT1, are important for cell differentiation and polar morphogenesis, yet their input signals as well as their integration into a cellular signaling context are still elusive. Here, we identify the cot-1 suppressor gul-4 as mak-2 and show that mutants of the gul-4/mak-2 mitogen-activated protein (MAP) kinase pathway suppress cot-1 phenotypes along with a concomitant reduction in protein kinase A (PKA) activity. Furthermore, mak-2 pathway defects are partially overcome in a cot-1 background and are associated with increased MAK1 MAPK signaling. A comparative characterization of N. crassa MAPKs revealed that they act as three distinct modules during vegetative growth and asexual development. In addition, common functions of MAK1 and MAK2 signaling during maintenance of cell-wall integrity distinguished the two ERK-type pathways from the p38-type OS2 osmosensing pathway. In contrast to separate functions during vegetative growth, the concerted activity of the three MAPK pathways is essential for cell fusion and for the subsequent formation of multicellular structures that are required for sexual development. Taken together, our data indicate a functional link between COT1 and MAPK signaling in regulating filamentous growth, hyphal fusion, and sexual development. PMID:18562669

Dynamic regulation of VEGF-inducible genes by an ERK/ERG/p300 transcriptional network.

PubMed

Fish, Jason E; Cantu Gutierrez, Manuel; Dang, Lan T; Khyzha, Nadiya; Chen, Zhiqi; Veitch, Shawn; Cheng, Henry S; Khor, Melvin; Antounians, Lina; Njock, Makon-Sébastien; Boudreau, Emilie; Herman, Alexander M; Rhyner, Alexander M; Ruiz, Oscar E; Eisenhoffer, George T; Medina-Rivera, Alejandra; Wilson, Michael D; Wythe, Joshua D

2017-07-01

The transcriptional pathways activated downstream of vascular endothelial growth factor (VEGF) signaling during angiogenesis remain incompletely characterized. By assessing the signals responsible for induction of the Notch ligand delta-like 4 (DLL4) in endothelial cells, we find that activation of the MAPK/ERK pathway mirrors the rapid and dynamic induction of DLL4 transcription and that this pathway is required for DLL4 expression. Furthermore, VEGF/ERK signaling induces phosphorylation and activation of the ETS transcription factor ERG, a prerequisite for DLL4 induction. Transcription of DLL4 coincides with dynamic ERG-dependent recruitment of the transcriptional co-activator p300. Genome-wide gene expression profiling identified a network of VEGF-responsive and ERG-dependent genes, and ERG chromatin immunoprecipitation (ChIP)-seq revealed the presence of conserved ERG-bound putative enhancer elements near these target genes. Functional experiments performed in vitro and in vivo confirm that this network of genes requires ERK, ERG and p300 activity. Finally, genome-editing and transgenic approaches demonstrate that a highly conserved ERG-bound enhancer located upstream of HLX (which encodes a transcription factor implicated in sprouting angiogenesis) is required for its VEGF-mediated induction. Collectively, these findings elucidate a novel transcriptional pathway contributing to VEGF-dependent angiogenesis. © 2017. Published by The Company of Biologists Ltd.

Tpbpa-Cre-mediated deletion of TFAP2C leads to deregulation of Cdkn1a, Akt1 and the ERK pathway, causing placental growth arrest.

PubMed

Sharma, Neha; Kubaczka, Caroline; Kaiser, Stephanie; Nettersheim, Daniel; Mughal, Sadaf S; Riesenberg, Stefanie; Hölzel, Michael; Winterhager, Elke; Schorle, Hubert

2016-03-01

Loss of TFAP2C in mouse leads to developmental defects in the extra-embryonic compartment with lethality at embryonic day (E)7.5. To investigate the requirement of TFAP2C in later placental development, deletion of TFAP2C was induced throughout extra-embryonic ectoderm at E6.5, leading to severe placental abnormalities caused by reduced trophoblast population and resulting in embryonic retardation by E8.5. Deletion of TFAP2C in TPBPA(+) progenitors at E8.5 results in growth arrest of the junctional zone. TFAP2C regulates its target genes Cdkn1a (previously p21) and Dusp6, which are involved in repression of MAPK signaling. Loss of TFAP2C reduces activation of ERK1/2 in the placenta. Downregulation of Akt1 and reduced activation of phosphorylated AKT in the mutant placenta are accompanied by impaired glycogen synthesis. Loss of TFAP2C led to upregulation of imprinted gene H19 and downregulation of Slc38a4 and Ascl2. The placental insufficiency post E16.5 causes fetal growth restriction, with 19% lighter mutant pups. Knockdown of TFAP2C in human trophoblast choriocarcinoma JAr cells inhibited MAPK and AKT signaling. Thus, we present a model where TFAP2C in trophoblasts controls proliferation by repressing Cdkn1a and activating the MAPK pathway, further supporting differentiation of glycogen cells by activating the AKT pathway. © 2016. Published by The Company of Biologists Ltd.

TWEAK promotes migration and invasion in MEFs through a mechanism dependent on ERKs activation and Fibulin 3 down-regulation.

PubMed

Sequera, Celia; Vázquez-Carballo, Ana; Arechederra, María; Fernández-Veledo, Sonia; Porras, Almudena

2018-02-01

TWEAK regulates multiple physio-pathological processes in fibroblasts such as fibrosis. It also induces migration and invasion in tumors and it can activate p38 MAPK in various cell types. Moreover, p38α MAPK promotes migration and invasion in several cancer cells types and in mouse embryonic fibroblasts (MEFs). However, it remains unknown if TWEAK could promote migration in fibroblasts and whether p38α MAPK might play a role. Our results reveal that TWEAK activates ERKs, Akt, and p38α/β MAPKs and reduces secreted Fibulin 3 in MEFs. TWEAK also increases migration and invasion in wt and p38α deficient MEFs, which indicates that p38α MAPK is not required to mediate these effects. In contrast, ERKs inhibition significantly decreases TWEAK-induced migration and Fibulin 3 knock-down mimics TWEAK effect. These results indicate that both ERKs activation and Fibulin 3 down-regulation would contribute to mediate TWEAK pro-migratory effect. In fact, the additional regulation of ERKs and/or p38β as a consequence of Fibulin 3 decrease might be also involved in the pro-migratory effect of TWEAK in MEFs. In conclusion, our studies uncover novel mechanisms by which TWEAK would favor tissue repair by promoting fibroblasts migration. © 2017 Wiley Periodicals, Inc.

Fragment-Based Discovery of a Potent, Orally Bioavailable Inhibitor That Modulates the Phosphorylation and Catalytic Activity of ERK1/2.

PubMed

Heightman, Tom D; Berdini, Valerio; Braithwaite, Hannah; Buck, Ildiko M; Cassidy, Megan; Castro, Juan; Courtin, Aurélie; Day, James E H; East, Charlotte; Fazal, Lynsey; Graham, Brent; Griffiths-Jones, Charlotte M; Lyons, John F; Martins, Vanessa; Muench, Sandra; Munck, Joanne M; Norton, David; O'Reilly, Marc; Palmer, Nick; Pathuri, Puja; Reader, Michael; Rees, David C; Rich, Sharna J; Richardson, Caroline; Saini, Harpreet; Thompson, Neil T; Wallis, Nicola G; Walton, Hugh; Wilsher, Nicola E; Woolford, Alison J-A; Cooke, Michael; Cousin, David; Onions, Stuart; Shannon, Jonathan; Watts, John; Murray, Christopher W

2018-05-31

Aberrant activation of the MAPK pathway drives cell proliferation in multiple cancers. Inhibitors of BRAF and MEK kinases are approved for the treatment of BRAF mutant melanoma, but resistance frequently emerges, often mediated by increased signaling through ERK1/2. Here, we describe the fragment-based generation of ERK1/2 inhibitors that block catalytic phosphorylation of downstream substrates such as RSK but also modulate phosphorylation of ERK1/2 by MEK without directly inhibiting MEK. X-ray crystallographic and biophysical fragment screening followed by structure-guided optimization and growth from the hinge into a pocket proximal to the C-α helix afforded highly potent ERK1/2 inhibitors with excellent kinome selectivity. In BRAF mutant cells, the lead compound suppresses pRSK and pERK levels and inhibits proliferation at low nanomolar concentrations. The lead exhibits tumor regression upon oral dosing in BRAF mutant xenograft models, providing a promising basis for further optimization toward clinical pERK1/2 modulating ERK1/2 inhibitors.

COT drives resistance to RAF inhibition through MAP kinase pathway reactivation.

PubMed

Johannessen, Cory M; Boehm, Jesse S; Kim, So Young; Thomas, Sapana R; Wardwell, Leslie; Johnson, Laura A; Emery, Caroline M; Stransky, Nicolas; Cogdill, Alexandria P; Barretina, Jordi; Caponigro, Giordano; Hieronymus, Haley; Murray, Ryan R; Salehi-Ashtiani, Kourosh; Hill, David E; Vidal, Marc; Zhao, Jean J; Yang, Xiaoping; Alkan, Ozan; Kim, Sungjoon; Harris, Jennifer L; Wilson, Christopher J; Myer, Vic E; Finan, Peter M; Root, David E; Roberts, Thomas M; Golub, Todd; Flaherty, Keith T; Dummer, Reinhard; Weber, Barbara L; Sellers, William R; Schlegel, Robert; Wargo, Jennifer A; Hahn, William C; Garraway, Levi A

2010-12-16

Oncogenic mutations in the serine/threonine kinase B-RAF (also known as BRAF) are found in 50-70% of malignant melanomas. Pre-clinical studies have demonstrated that the B-RAF(V600E) mutation predicts a dependency on the mitogen-activated protein kinase (MAPK) signalling cascade in melanoma-an observation that has been validated by the success of RAF and MEK inhibitors in clinical trials. However, clinical responses to targeted anticancer therapeutics are frequently confounded by de novo or acquired resistance. Identification of resistance mechanisms in a manner that elucidates alternative 'druggable' targets may inform effective long-term treatment strategies. Here we expressed ∼600 kinase and kinase-related open reading frames (ORFs) in parallel to interrogate resistance to a selective RAF kinase inhibitor. We identified MAP3K8 (the gene encoding COT/Tpl2) as a MAPK pathway agonist that drives resistance to RAF inhibition in B-RAF(V600E) cell lines. COT activates ERK primarily through MEK-dependent mechanisms that do not require RAF signalling. Moreover, COT expression is associated with de novo resistance in B-RAF(V600E) cultured cell lines and acquired resistance in melanoma cells and tissue obtained from relapsing patients following treatment with MEK or RAF inhibitors. We further identify combinatorial MAPK pathway inhibition or targeting of COT kinase activity as possible therapeutic strategies for reducing MAPK pathway activation in this setting. Together, these results provide new insights into resistance mechanisms involving the MAPK pathway and articulate an integrative approach through which high-throughput functional screens may inform the development of novel therapeutic strategies.

Evidence for nucleotide receptor modulation of cross talk between MAP kinase and NF-kappa B signaling pathways in murine RAW 264.7 macrophages.

PubMed

Aga, Mini; Watters, Jyoti J; Pfeiffer, Zachary A; Wiepz, Gregory J; Sommer, Julie A; Bertics, Paul J

2004-04-01

Extracellular nucleotides such as ATP are present in abundance at sites of inflammation and tissue damage, and these agents exert a potent modulatory effect on macrophage/monocyte function via the nucleotide receptor P2X(7). In this regard, after exposure to bacterial LPS, P2X(7) activation augments expression of the inducible nitric oxide (NO) synthase and production of NO in macrophages. Because P2X(7) has been reported to stimulate certain members of the MAP kinase family (ERK1/2) and can enhance the DNA-binding activity of NF-kappa B, we tested the hypothesis that LPS and nucleotides regulate NF-kappa B-dependent inflammatory events via cross talk with MAPK-associated pathways. In this regard, the present studies revealed that cotreatment of macrophages with LPS and the P2X(7)-selective ligand 2'-3'-O-(4-benzoylbenzoyl)adenosine 5'-triphosphate (BzATP) results in the cooperative activation of NF-kappa B DNA-binding activity and a sustained attenuation of levels of the NF-kappa B inhibitory protein I kappa B alpha. Interestingly, a persistent reduction in I kappa B alpha levels is also observed when the MEK1/2 inhibitor U0126 is coadministered with LPS, suggesting that components of the MEK/ERK pathway are involved in regulating I kappa B alpha protein expression and/or turnover. The observation that U0126 and BzATP exhibit overlapping actions with respect to LPS-induced changes in I kappa B alpha levels is supported by the finding that Ras activation, which is upstream of MEK/ERK activation, is reduced upon macrophage cotreatment with BzATP and LPS compared with the effects of BzATP treatment alone. These data are consistent with the concept that the Ras/MEK/ERK pathways are involved in regulating NF-kappa B/I kappa B-dependent inflammatory mediator production and suggest a previously unidentified mechanism by which nucleotides can modulate LPS-induced action via cross talk between NF-kappa B and Ras/MEK/MAPK-associated pathways.

Developmental fluoride exposure influenced rat's splenic development and cell cycle via disruption of the ERK signal pathway.

PubMed

Ma, Yanqin; Zhang, Kankan; Ren, Fengjun; Wang, Jundong

2017-11-01

Excessive fluoride exposure has been reported to cause damage to spleen. Neonatal period is characterized by rapid proliferation and differentiation of lymphocyte in the spleen. Children may be more sensitive to the toxicity of fluoride compared to the adults. The aim of this study was to investigate the effects of postnatal exposure (from neonatal period to early adulthood) to fluoride on the development of spleen on a regular basis and the underlying signal pathway. Results showed a marked decrease in spleen weight index and altered morphology in the spleen of fluoride-treated group on PND-84, which reflected fluoride inhibition of the development of spleen. Fluoride exposure induced cell cycle arrest of splenocytes and decreased the mRNA expression of IL-2, which indicated compromised baseline lymphocyte proliferation in the spleen. Time course research from 3-wk-of-age until 12-wk-of-age showed an adverse and cumulative impact of fluoride on the development of spleen. In view of the key role of MAPK/ERK pathway in lymphocyte development, Raf-1/MEK-1/ERK-2/c-fos mRNA expression and ERK/p-ERK protein expression were detected. Results showed despite a transitory increase in mRNA expression from PND-42 to PND-63 in fluoride-treated group, the expression of these genes on PND-84 decreased significantly compared with PND-42 or PND-63. NaF significantly inhibited the phosphorylation of ERK protein on PND-84. Taken together, these results emphasized the vital role of ERK pathway in the interfered development of spleen induced by a high dose of fluoride exposure in rats. Copyright © 2017 Elsevier Ltd. All rights reserved.

Signaling intermediates (MAPK and PI3K) as therapeutic targets in NSCLC.

PubMed

Ciuffreda, Ludovica; Incani, Ursula Cesta; Steelman, Linda S; Abrams, Stephen L; Falcone, Italia; Curatolo, Anais Del; Chappell, William H; Franklin, Richard A; Vari, Sabrina; Cognetti, Francesco; McCubrey, James A; Milella, Michele

2014-01-01

The RAS/RAF/MEK/ ERK and the PI3K/AKT/mTOR pathways govern fundamental physiological processes, such as cell proliferation, differentiation, metabolism, cytoskeleton reorganization and cell death and survival. Constitutive activation of these signal transduction pathways is a required hallmark of cancer and dysregulation, on either genetic or epigenetic grounds, of these pathways has been implicated in the initiation, progression and metastastic spread of lung cances. Targeting components of the MAPK and PI3K cascades is thus an attractive strategy in the development of novel therapeutic approaches to treat lung cancer, although the use of single pathway inhibitors has met with limited clinical success so far. Indeed, the presence of intra- and inter-pathway compensatory loops that re-activate the very same cascade, either upstream or downstream the point of pharmacological blockade, or activate the alternate pathway following the blockade of one signaling cascade has been demonstrated, potentially driving preclinical (and possibly clinical) resistance. Therefore, the blockade of both pathways with combinations of signaling inhibitors might result in a more efficient anti-tumor effect, and thus potentially overcome and/or delay clinical resistance, as compared with single agent. The current review aims at summarizing the current status of preclinical and clinical research with regard to pathway crosstalks between the MAPK and PI3K cascades in NSCLC and the rationale for combined therapeutic pathway targeting.

Bacopa monnieri protects SH-SY5Y cells against tert-Butyl hydroperoxide-induced cell death via the ERK and PI3K pathways

PubMed Central

Petcharat, Kanoktip; Singh, Meharvan; Ingkaninan, Kornkanok; Attarat, Jongrak; Yasothornsrikul, Sukkid

2017-01-01

Objective Oxidative stress plays an important role in the pathological processes of various neurodegenerative diseases. Bacopa monnieri (BM) has a potent antioxidant property. Therefore, the purpose of this study was to evaluate the neuroprotective potential of BM against SH-SY5Y neuroblastoma cell death induced by the pro-oxidant insult, tert-Butyl hydroperoxide (TBHP), and to identify possible mechanisms related to its neuroprotective action. Methods The neuroprotective effect of BM was evaluated by the degree of protection against TBHP-induced cell death in human SH-SY5Y cells that was measured by calcein-AM assay. ERK1/2 and Akt phosphorylation was evaluated by immunoblotting. Results We found that BM exhibited protection against TBHP-mediated cytotoxicity. The neuroprotective effect of BM was abolished in the presence of either ERK1/2 or PI3K inhibitors. In addition, western blotting with anti-phospho-ERK1/2 and anti-phospho-Akt antibodies showed that BM increased both ERK1/2 and Akt phosphorylation. Conclusion These results suggest that BM by activation of ERK/MAPK and PI3K/Akt signaling pathways protects SH-SY5Y cells from TBHP-induced cell death. PMID:29152617

ERK1/2 activation in human taste bud cells regulates fatty acid signaling and gustatory perception of fat in mice and humans.

PubMed

Subramaniam, Selvakumar; Ozdener, Mehmet Hakan; Abdoul-Azize, Souleymane; Saito, Katsuyoshi; Malik, Bilal; Maquart, Guillaume; Hashimoto, Toshihiro; Marambaud, Philippe; Aribi, Mourad; Tordoff, Michael G; Besnard, Philippe; Khan, Naim Akhtar

2016-10-01

Obesity is a major public health problem. An in-depth knowledge of the molecular mechanisms of oro-sensory detection of dietary lipids may help fight it. Humans and rodents can detect fatty acids via lipido-receptors, such as CD36 and GPR120. We studied the implication of the MAPK pathways, in particular, ERK1/2, in the gustatory detection of fatty acids. Linoleic acid, a dietary fatty acid, induced via CD36 the phosphorylation of MEK1/2-ERK1/2-ETS-like transcription factor-1 cascade, which requires Fyn-Src kinase and lipid rafts in human taste bud cells (TBCs). ERK1/2 cascade was activated by Ca 2+ signaling via opening of the calcium-homeostasis modulator-1 (CALHM1) channel. Furthermore, fatty acid-evoked Ca 2+ signaling and ERK1/2 phosphorylation were decreased in both human TBCs after small interfering RNA knockdown of CALHM1 channel and in TBCs from Calhm1 -/- mice. Targeted knockdown of ERK1/2 by small interfering RNA or PD0325901 (MEK1/2 inhibitor) in the tongue and genetic ablation of Erk1 or Calhm1 genes impaired preference for dietary fat in mice. Lingual inhibition of ERK1/2 in healthy volunteers also decreased orogustatory sensitivity for linoleic acid. Our data demonstrate that ERK1/2-MAPK cascade is regulated by the opening of CALHM1 Ca 2+ channel in TBCs to modulate orogustatory detection of dietary lipids in mice and humans.-Subramaniam, S., Ozdener, M. H., Abdoul-Azize, S., Saito, K., Malik, B., Maquart, G., Hashimoto, T., Marambaud, P., Aribi, M., Tordoff, M. G., Besnard, P., Khan, N. A. ERK1/2 activation in human taste bud cells regulates fatty acid signaling and gustatory perception of fat in mice and humans. © FASEB.

Propofol pretreatment attenuates LPS-induced granulocyte-macrophage colony-stimulating factor production in cultured hepatocytes by suppressing MAPK/ERK activity and NF-{kappa}B translocation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jawan, Bruno; Kao, Y.-H.; Department of Biological Sciences, National Sun Yat-Sen University, 70 Lien-Hai Road, Kaohsiung 804, Taiwan

Propofol (PPF), a widely used intravenous anesthetic for induction and maintenance of anesthesia during surgeries, was found to possess suppressive effect on host immunity. This study aimed at investigating whether PPF plays a modulatory role in the lipopolysaccharide (LPS)-induced inflammatory cytokine expression in a cell line of rat hepatocytes. Morphological observation and viability assay showed that PPF exhibits no cytotoxicity at concentrations up to 300 {mu}M after 48 h incubation. Pretreatment with 100 {mu}M PPF for 24 h prior to LPS stimulation was performed to investigate the modulatory effect on LPS-induced inflammatory gene production. The results of semi-quantitative RT-PCR demonstratedmore » that PPF pretreatment significantly suppressed the LPS-induced toll-like receptor (TLR)-4, CD14, tumor necrosis factor (TNF)-{alpha}, and granulocyte-macrophage colony-stimulating factor (GM-CSF) gene expression. Western blotting analysis showed that PPF pretreatment potentiated the LPS-induced TLR-4 downregulation. Flow cytometrical analysis revealed that PPF pretreatment showed no modulatory effect on the LPS-upregulated CD14 expression on hepatocytes. In addition, PPF pretreatment attenuated the phosphorylation of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and I{kappa}B{alpha}, as well as the nuclear translocation of NF-{kappa}B primed by LPS. Moreover, addition of PD98059, a MAPK kinase inhibitor, significantly suppressed the LPS-induced NF-{kappa}B nuclear translocation and GM-CSF production, suggesting that the PPF-attenuated GM-CSF production in hepatocytes may be attributed to its suppressive effect on MAPK/ERK signaling pathway. In conclusion, PPF as an anesthetic may clinically benefit those patients who are vulnerable to sepsis by alleviating sepsis-related inflammatory response in livers.« less

Advanced oxidative protein products induced human keratinocyte apoptosis through the NOX-MAPK pathway.

PubMed

Sun, Baihui; Ding, Ruoting; Yu, Wenlin; Wu, Yanhong; Wang, Bulin; Li, Qin

2016-07-01

Impaired wound healing is a major diabetes-related complication. Keratinocytes play an important role in wound healing. Multiple factors have been proposed that can induce dysfunction in keratinocytes. The focus of present research is at a more specific molecular level. We investigated the role of advanced oxidative protein products (AOPPs) in inducing human immortalized keratinocyte (HaCaT) cell apoptosis and the cellular mechanism underlying the proapoptotic effect of AOPPs. HaCaT cells were treated with increasing concentrations of AOPP-human serum albumin or for increasing time durations. The cell viability was measured using the thiazolyl blue tetrazolium bromide method, and flow cytometry was used to assess the rate of cell apoptosis. A loss of mitochondrial membrane potential (MMP) and an increase in intracellular reactive oxygen species (ROS) were observed through a confocal laser scanning microscope system, and the level of ROS generation was determined using a microplate reader. Nicotinamide adenine dinucleotide phosphate oxidase (NOX)4, extracellular signal-regulated kinase (ERK)1/2, p38 mitogen-activated protein kinase (MAPK), and apoptosis-related downstream protein interactions were investigated using the Western blot analysis. We found that AOPPs triggered HaCaT cell apoptosis and MMP loss. After AOPP treatment, intracellular ROS generation increased in a time- and dose-dependent manner. Proapoptotic proteins, such as Bax, caspase 9/caspase 3, and poly(ADP-ribose) polymerase (PARP)-1 were activated, whereas anti-apoptotic Bcl-2 protein was downregulated. AOPPs also increased NOX4, ERK1/2, and p38 MAPK expression. Taken together, these findings suggest that extracellular AOPP accumulation triggered NOX-dependent ROS production, which activated ERK1/2 and p38 MAPK, and induced HaCaT cell apoptosis by activating caspase 3 and PARP-1.

Gastrin-releasing peptide induces monocyte adhesion to vascular endothelium by upregulating endothelial adhesion molecules

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Mi-Kyoung; Park, Hyun-Joo; Department of Dental Pharmacology, BK21 PLUS Project, School of Dentistry, Pusan National University, Yangsan 626-870

Gastrin-releasing peptide (GRP) is a neuropeptide that plays roles in various pathophysiological conditions including inflammatory diseases in peripheral tissues; however, little is known about whether GRP can directly regulate endothelial inflammatory processes. In this study, we showed that GRP promotes the adhesion of leukocytes to human umbilical vein endothelial cells (HUVECs) and the aortic endothelium. GRP increased the expression of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1) by activating nuclear factor-κB (NF-κB) in endothelial cells. In addition, GRP activated extracellular signal-regulated kinase 1/2 (ERK1/2), p38MAPK, and AKT, and the inhibition of these signaling pathways significantly reduced GRP-inducedmore » monocyte adhesion to the endothelium. Overall, our results suggested that GRP may cause endothelial dysfunction, which could be of particular relevance in the development of vascular inflammatory disorders. - Highlights: • GRP induces adhesion of monocytes to vascular endothelium. • GRP increases the expression of endothelial adhesion molecules through the activation of NF-κB. • ERK1/2, p38MAPK, and Akt pathways are involved in the GRP-induced leukocyte adhesiveness to endothelium.« less

Role of CRF Receptor Signaling in Stress Vulnerability, Anxiety, and Depression

PubMed Central

Hauger, Richard L.; Risbrough, Victoria; Oakley, Robert H.; Olivares-Reyes, J. Alberto; Dautzenberg, Frank M.

2011-01-01

Markers of hyperactive central corticotropin releasing factor (CRF) systems and CRF-related single nucleotide polymorphisms (SNPs) have been identified in patients with anxiety and depressive disorders. Designing more effective antagonists may now be guided by data showing that small molecules bind to transmembrane domains. Specifically, CRF1 receptor antagonists have been developed as novel anxiolytic and antidepressant treatments. Because CRF1 receptors become rapidly desensitized by G protein-coupled receptor kinase (GRK) and β-arrestin mechanisms in the presence of high agonist concentrations, neuronal hypersecretion of synaptic CRF alone may be insufficient to account for excessive central CRF neurotransmission in stress-induced affective pathophysiology. In addition to desensitizing receptor function, GRK phosphorylation and β-arrestin binding can shift a G protein-coupled receptor (GPCR) to signal selectively via the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK-MAPK) or Akt pathways independent of G proteins. Also, Epac-dependent CRF1 receptor signaling via the ERK-MAPK pathway has been found to potentiate brain-derived neurotrophic factor (BDNF)-stimulated TrkB signaling. Thus, genetic or acquired abnormalities in GRK and β-arrestin function may be involved in the pathophysiology of stress-induced anxiety and depression. PMID:19906236

Interference of silibinin with IGF-1R signalling pathways protects human epidermoid carcinoma A431 cells from UVB-induced apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Weiwei; Otkur, Wuxiyar; Li, Lingzhi

Highlights: ► Silibinin protects A431 cells from UVB irradiation-induced apoptosis. ► Up-regulation of the IGF-1R-JNK/ERK pathways by UVB induces cell apoptosis. ► Silibinin inhibits IGF-1R pathways to repress caspase-8-mediated apoptosis. -- Abstract: Ultraviolet B (UVB) from sunlight is a major cause of cutaneous lesion. Silibinin, a traditional hepatic protectant, elicits protective effects against UVB-induced cellular damage. In A431 cells, the insulin-like growth factor-1 receptor (IGF-1R) was markedly up-regulated by UVB irradiation. The activation of the IGF-1R signalling pathways contributed to apoptosis of the cells rather than rescuing the cells from death. Up-regulated IGF-1R stimulated downstream mitogen-activated protein kinases (MAPKs), suchmore » as c-Jun N-terminal kinases (JNK) and extracellular signal-regulated protein kinases 1/2 (ERK1/2). The subsequent activation of caspase-8 and caspase-3 led to apoptosis. The activation of IGF-1R signalling pathways is the cause of A431 cell death. The pharmacological inhibitors and the small interfering RNA (siRNA) targeting IGF-1R suppressed the downstream activation of JNK/ERK-caspases to help the survival of the UVB-irradiated A431 cells. Indeed, silibinin treatment suppressed the IGF-1R-JNK/ERK pathways and thus protected the cells from UVB-induced apoptosis.« less

Modulation of skeletal muscle fiber type by mitogen-activated protein kinase signaling.

PubMed

Shi, Hao; Scheffler, Jason M; Pleitner, Jonathan M; Zeng, Caiyun; Park, Sungkwon; Hannon, Kevin M; Grant, Alan L; Gerrard, David E

2008-08-01

Skeletal muscle is composed of diverse fiber types, yet the underlying molecular mechanisms responsible for this diversification remain unclear. Herein, we report that the extracellular signal-regulated kinase (ERK) 1/2 pathway, but not p38 or c-Jun NH(2)-terminal kinase (JNK), is preferentially activated in fast-twitch muscles. Pharmacological blocking of ERK1/2 pathway increased slow-twitch fiber type-specific reporter activity and repressed those associated with the fast-twitch fiber phenotype in vitro. Overexpression of a constitutively active ERK2 had an opposite effect. Inhibition of ERK signaling in cultured myotubes increased slow-twitch fiber-specific protein accumulation while repressing those characteristic of fast-twitch fibers. Overexpression of MAP kinase phosphatase-1 (MKP1) in mouse and rat muscle fibers containing almost exclusively type IIb or IIx fast myosin heavy chain (MyHC) isoforms induced de novo synthesis of the slower, more oxidative type IIa and I MyHCs in a time-dependent manner. Conversion to the slower phenotype was confirmed by up-regulation of slow reporter gene activity and down-regulation of fast reporter activities in response to forced MKP1 expression in vivo. In addition, activation of ERK2 signaling induced up-regulation of fast-twitch fiber program in soleus. These data suggest that the MAPK signaling, most likely the ERK1/2 pathway, is necessary to preserve the fast-twitch fiber phenotype with a concomitant repression of slow-twitch fiber program.

MAPK/ERK signal pathway involved expression of COX-2 and VEGF by IL-1β induced in human endometriosis stromal cells in vitro

PubMed Central

Huang, Fengying; Cao, Jing; Liu, Qiuhong; Zou, Ying; Li, Hongyun; Yin, Tuanfang

2013-01-01

Objective: Now there are more and more evidences that Cyclooxygenase-2 (COX-2) plays an important role in angiogenesis of endometriosis (EMs). Vascular endothelial growth factor (VEGF) has a potent angiogenic activity. However, it is worth studying about the regulating mechanism of COX-2/COX-1 and VEGF in the development of human endometriosis in vitro. The current study was designed to investigate the effect of 4 cytokines on COX-2/COX-1 expression and the effect of IL-1β on VEGF release in human endometriosis stromal cells (ESC), and to explore the related signaling pathways involved in vitro. Methods: Isolation, culture and identification of ESC. Cells were treated with 4 cytokines, and the inhibitor mitogen-activated protein-Erk (MEK) and the inhibitor p38 mitogen-activated protein kinase (MAPK) prior to adding cytokine IL-1β. COX-2 protein expression was measured by western blot and VEGF secretion was determined by ELISA. Results: Among four kinds of cytokines, IL-1β treatment increased COX-2 protein expression and VEGF release in three ESC, and TNF-α had the same effect on COX-2 protein level as IL-1β only in ectopic and eutopic ESC, and MCSF had only slight effect on ectopic ESC. In contrast, cytokines had no effect on COX-1 expression. We also demonstrated that MAPK reduced the synthesis of COX-2 by IL-1β induced. COX-2 inhibitor reduced VEGF release by IL-1β induced. Conclusions: i) In human ESC in vitro, IL-1β up-regulated the COX-2 expression through the activation of p38 MAPK pathway, and not to COX-1. ii) Up-regulation of VEGF level by IL-1β treatment was found in human endometriosis stromal cell and COX-2 inhibitor was involved in this process. PMID:24133591

Sampling-based Bayesian approaches reveal the importance of quasi-bistable behavior in cellular decision processes on the example of the MAPK signaling pathway in PC-12 cell lines.

PubMed

Jensch, Antje; Thomaseth, Caterina; Radde, Nicole E

2017-01-25

Positive and negative feedback loops are ubiquitous motifs in biochemical signaling pathways. The mitogen-activated protein kinase (MAPK) pathway module is part of many distinct signaling networks and comprises several of these motifs, whose functioning depends on the cell line at hand and on the particular context. The maintainance of specificity of the response of the MAPK module to distinct stimuli has become a key paradigm especially in PC-12 cells, where the same module leads to different cell fates, depending on the stimulating growth factor. This cell fate is regulated by differences in the ERK (MAPK) activation profile, which shows a transient response upon stimulation with EGF, while the response is sustained in case of NGF. This behavior was explained by different effective network topologies. It is widely believed that this sustained response requires a bistable system. In this study we present a sampling-based Bayesian model analysis on a dataset, in which PC-12 cells have been stimulated with different growth factors. This is combined with novel analysis methods to investigate the role of feedback interconnections to shape ERK response. Results strongly suggest that, besides bistability, an additional effect called quasi-bistability can contribute to explain the observed responses of the system to different stimuli. Quasi-bistability is the ability of a monostable system to maintain two distinct states over a long time period upon a transient signal, which is also related to positive feedback, but cannot be detected by standard steady state analysis methods. Although applied on a specific example, our framework is generic enough to be also relevant for other regulatory network modeling studies that comprise positive feedback to explain cellular decision making processes. Overall, this study advices to focus not only on steady states, but also to take transient behavior into account in the analysis.

Icariin and icaritin stimulate the proliferation of SKBr3 cells through the GPER1-mediated modulation of the EGFR-MAPK signaling pathway.

PubMed

Ma, Hai-Rong; Wang, Jie; Chen, Yiu-Fai; Chen, Hua; Wang, Wei-Shan; Aisa, Haji Akber

2014-06-01

Icariin (ICA) and icaritin (ICT), with a similar structure to genistein, are the important bioactive components of the genus Epimedium, and regulate many cellular processes. In the present study, using the estrogen receptor (ER)-negative breast cancer cell line, SKBr3, as a model, we examined the hypothesis that ICA and ICT at low concentrations stimulate SKBr3 cell proliferation in vitro through the functional membrane, G protein‑coupled estrogen receptor 1 (GPER1), mediated by the epithelial growth factor receptor (EGFR)‑mitogen-activated protein kinase (MAPK) signaling pathway. MTT assay revealed that ICA and ICT at doses of 1 nM to 1 µM markedly stimulated SKBr3 cell proliferation in a dose-dependent manner. The ICA- and ICT-stimulated cell growth was completely suppressed by the GPER1 antagonist, G-15, indicating that the ICA‑ and ICT-stimulated cell proliferation was mediated by GPER1 activation. Semi-quantitative RT-PCR analysis revealed that treatment with ICA and ICT enhanced the transcription of c-fos, a proliferation-related early gene. The ICA- and ICT-stimulated mRNA expression was markedly attenuated by G-15, AG-1478 (an EGFR antagonist) or PD98059 (a MAPK inhibitor). Our data also demonstrated that ICA and ICT increased the phosphorylation of ERK1/2. The ICA- and ICT-stimulated ERK1/2 phosphorylation was blocked by pre-treatment of the cells with G-15 and AG-1478 or PD 98059. Flow cytometric analysis confirmed that the ICA- and ICT-stimulated SKBr3 cell proliferation involved the GPER1-mediated modulation of the EGFR‑MAPK signaling pathway. To the best of our knowledge, our current findings demonstrate for the first time that ICA and ICT promote the progression of ER-negative breast cancer through the activation of membrane GPER1.

Reactivation of Mitogen-activated Protein Kinase (MAPK) Pathway by FGF Receptor 3 (FGFR3)/Ras Mediates Resistance to Vemurafenib in Human B-RAF V600E Mutant Melanoma*

PubMed Central

Yadav, Vipin; Zhang, Xiaoyi; Liu, Jiangang; Estrem, Shawn; Li, Shuyu; Gong, Xue-Qian; Buchanan, Sean; Henry, James R.; Starling, James J.; Peng, Sheng-Bin

2012-01-01

Oncogenic B-RAF V600E mutation is found in 50% of melanomas and drives MEK/ERK pathway and cancer progression. Recently, a selective B-RAF inhibitor, vemurafenib (PLX4032), received clinical approval for treatment of melanoma with B-RAF V600E mutation. However, patients on vemurafenib eventually develop resistance to the drug and demonstrate tumor progression within an average of 7 months. Recent reports indicated that multiple complex and context-dependent mechanisms may confer resistance to B-RAF inhibition. In the study described herein, we generated B-RAF V600E melanoma cell lines of acquired-resistance to vemurafenib, and investigated the underlying mechanism(s) of resistance. Biochemical analysis revealed that MEK/ERK reactivation through Ras is the key resistance mechanism in these cells. Further analysis of total gene expression by microarray confirmed a significant increase of Ras and RTK gene signatures in the vemurafenib-resistant cells. Mechanistically, we found that the enhanced activation of fibroblast growth factor receptor 3 (FGFR3) is linked to Ras and MAPK activation, therefore conferring vemurafenib resistance. Pharmacological or genetic inhibition of the FGFR3/Ras axis restored the sensitivity of vemurafenib-resistant cells to vemurafenib. Additionally, activation of FGFR3 sufficiently reactivated Ras/MAPK signaling and conferred resistance to vemurafenib in the parental B-RAF V600E melanoma cells. Finally, we demonstrated that vemurafenib-resistant cells maintain their addiction to the MAPK pathway, and inhibition of MEK or pan-RAF activities is an effective therapeutic strategy to overcome acquired-resistance to vemurafenib. Together, we describe a novel FGFR3/Ras mediated mechanism for acquired-resistance to B-RAF inhibition. Our results have implications for the development of new therapeutic strategies to improve the outcome of patients with B-RAF V600E melanoma. PMID:22730329

Antitumor activity of taspine by modulating the EGFR signaling pathway of Erk1/2 and Akt in vitro and in vivo.

PubMed

Zhang, Yanmin; Zheng, Lei; Zhang, Jie; Dai, Bingling; Wang, Nan; Chen, Yinnan; He, Langchong

2011-11-01

EGFR, as a critical signaling pathway in many human tumors, has become an important target of cancer drug design. Taspine has shown meaningful angiogenesis activity in previous studies. This paper is to investigate the antitumor action of taspine by modulating the EGFR signaling pathway. The study determined the expression of key signaling molecules of EGFR (EGFR, Akt, p-Akt, Erk, and p-Erk) by Western blot and real-time PCR and analyzed their correlations with subsequent reactions. In addition, the cell proliferation, migration, and EGF production were examined by MTT, transwell system, and ELISA. The antitumor activity in vivo was carried out by xenograft in athymic mice. The results showed that taspine could inhibit A431 and Hek293/EGFR cell proliferation and A431 cell migration as well as EGF production. Compared to the negative control, EGFR, Akt, and phosphorylation of Akt were significantly inhibited by taspine treatment in A431 and HEK293/EGFR cells. Consistent with the inhibition of Akt activity, Erk1/2 and its phosphorylation were reduced. Moreover, taspine inhibited A431 xenograft tumor growth. These results suggest that EGFR activated by EGF and its downstream signaling pathways proteins could be downregulated by taspine in a dose-dependent manner. The antitumor mechanism of taspine through the EGFR pathway lies in the ability to inhibit A431 cell proliferation and migration by reducing EGF secretion. This occurs through the repression of EGFR which mediates not only MAPK (Erk1/2) but also Akt signals. © Georg Thieme Verlag KG Stuttgart · New York.

Colon cancer cells colonize the lung from established liver metastases through p38 MAPK signalling and PTHLH.

PubMed

Urosevic, Jelena; Garcia-Albéniz, Xabier; Planet, Evarist; Real, Sebastián; Céspedes, María Virtudes; Guiu, Marc; Fernandez, Esther; Bellmunt, Anna; Gawrzak, Sylwia; Pavlovic, Milica; Mangues, Ramon; Dolado, Ignacio; Barriga, Francisco M; Nadal, Cristina; Kemeny, Nancy; Batlle, Eduard; Nebreda, Angel R; Gomis, Roger R

2014-07-01

The mechanisms that allow colon cancer cells to form liver and lung metastases, and whether KRAS mutation influences where and when metastasis occurs, are unknown. We provide clinical and molecular evidence showing that different MAPK signalling pathways are implicated in this process. Whereas ERK2 activation provides colon cancer cells with the ability to seed and colonize the liver, reduced p38 MAPK signalling endows cancer cells with the ability to form lung metastasis from previously established liver lesions. Downregulation of p38 MAPK signalling results in increased expression of the cytokine PTHLH, which contributes to colon cancer cell extravasation to the lung by inducing caspase-independent death in endothelial cells of the lung microvasculature. The concerted acquisition of metastatic traits in the colon cancer cells together with the sequential colonization of liver and lung highlights the importance of metastatic lesions as a platform for further dissemination.

Oxymatrine Inhibits Influenza A Virus Replication and Inflammation via TLR4, p38 MAPK and NF-κB Pathways.

PubMed

Dai, Jian-Ping; Wang, Qian-Wen; Su, Yun; Gu, Li-Ming; Deng, Hui-Xiong; Chen, Xiao-Xuan; Li, Wei-Zhong; Li, Kang-Sheng

2018-03-23

Oxymatrine (OMT) is a strong immunosuppressive agent that has been used in the clinic for many years. In the present study, by using plaque inhibition, luciferase reporter plasmids, qRT-PCR, western blotting, and ELISA assays, we have investigated the effect and mechanism of OMT on influenza A virus (IAV) replication and IAV-induced inflammation in vitro and in vivo. The results showed that OMT had excellent anti-IAV activity on eight IAV strains in vitro. OMT could significantly decrease the promoter activity of TLR3, TLR4, TLR7, MyD88, and TRAF6 genes, inhibit IAV-induced activations of Akt, ERK1/2, p38 MAPK, and NF-κB pathways, and suppress the expressions of inflammatory cytokines and MMP-2/-9. Activators of TLR4, p38 MAPK and NF-κB pathways could significantly antagonize the anti-IAV activity of OMT in vitro, including IAV replication and IAV-induced cytopathogenic effect (CPE). Furthermore, OMT could reduce the loss of body weight, significantly increase the survival rate of IAV-infected mice, decrease the lung index, pulmonary inflammation and lung viral titter, and improve pulmonary histopathological changes. In conclusion, OMT possesses anti-IAV and anti-inflammatory activities, the mechanism of action may be linked to its ability to inhibit IAV-induced activations of TLR4, p38 MAPK, and NF-κB pathways.

Costunolide ameliorates lipoteichoic acid-induced acute lung injury via attenuating MAPK signaling pathway.

PubMed

Chen, Zhengxu; Zhang, Dan; Li, Man; Wang, Baolong

2018-06-12

Lipoteichoic acid (LTA)-induced acute lung injury (ALI) is an experimental model for mimicking Gram-positive bacteria-induced pneumonia that is a refractory disease with lack of effective medicines. Here, we reported that costunolide, a sesquiterpene lactone, ameliorated LTA-induced ALI. Costunolide treatment reduced LTA-induced neutrophil lung infiltration, cytokine and chemokine production (TNF-α, IL-6 and KC), and pulmonary edema. In response to LTA challenge, treatment with costunolide resulted less iNOS expression and produced less inflammatory cytokines in bone marrow derived macrophages (BMDMs). Pretreatment with costunolide also attenuated the LTA-induced the phosphorylation of p38 MAPK and ERK in BMDMs. Furthermore, costunolide treatment reduced the phosphorylation of TAK1 and inhibited the interaction of TAK1 with Tab1. In conclusion, we have demonstrated that costunolide protects against LTA-induced ALI via inhibiting TAK1-mediated MAPK signaling pathway, and our studies suggest that costunolide is a promising agent for treatment of Gram-positive bacteria-mediated pneumonia. Copyright © 2018 Elsevier B.V. All rights reserved.

Arctigenin induces the apoptosis of primary effusion lymphoma cells under conditions of glucose deprivation.

PubMed

Baba, Yusuke; Shigemi, Zenpei; Hara, Naoko; Moriguchi, Misato; Ikeda, Marina; Watanabe, Tadashi; Fujimuro, Masahiro

2018-02-01

Kaposi's sarcoma-associated herpesvirus (KSHV) is the causative agent of primary effusion lymphoma (PEL) and Kaposi's sarcoma. PEL is a type of non-Hodgkin's B-cell lymphoma, affecting immunosuppressed individuals, such as post-transplant or AIDS patients. However, since PEL is resistant to chemotherapeutic regimens, new effective treatment strategies are required. Arctigenin, a natural lignan compound found in the plant Arctium lappa, has been widely investigated as a potential anticancer agent in the clinical setting. In the present study, we examined the cytotoxic effects of arctigenin by cell viability assay and found that arctigenin markedly inhibited the proliferation of PEL cells compared with KSHV-uninfected B-lymphoma cells under conditions of glucose deprivation. Arctigenin decreased cellular ATP levels, disrupted mitochondrial membrane potential and triggered caspase-9-mediated apoptosis in the glucose-deprived PEL cells. In addition, western blot analysis using phospho-specific antibodies were used to evaluate activity changes in the signaling pathways of interest. As a result, arctigenin suppressed the activation of the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) signaling pathways by inhibiting ERK and p38 MAPK phosphorylation in the glucose-deprived PEL cells. We confirmed that an inhibitor of ERK (U0126) or p38 MAPK (SB202190 and SB203580) suppressed the proliferation of the BC3 PEL cells compared with the KSHV-negative DG75 cells. Moreover, RT-PCR and luciferase reporter assay revealed that arctigenin and p38 MAPK inhibition by SB202190 or SB203580 downregulated the transcriptional expression of unfolded protein response (UPR)‑related molecules, including GRP78 and ATF6α under conditions of glucose deprivation. Finally, we confirmed that arctigenin did not affect KSHV replication in PEL cells, suggesting that arctigenin treatment for PEL does not contribute to the risk of de novo KSHV production. These data thus indicate that arctigenin may serve as a lead compound for the development of novel and effective drugs for the treatment of PEL.

Contribution of nonesterified fatty acids to mitogen-activated protein kinase activation in human skeletal muscle during endurance exercise.

PubMed

Zbinden-Foncea, Hermann; van Loon, Luc J C; Raymackers, Jean-Marc; Francaux, Marc; Deldicque, Louise

2013-06-01

Mitogen-activated protein kinase (MAPK) pathways are activated in skeletal muscle during endurance exercise, but the upstream molecular events are incompletely resolved. As an increase in plasma nonesterified fatty acids (NEFA) is a common feature of long-lasting exercise, the authors tested the hypothesis that NEFA contribute to the activation of MAPK during endurance exercise. Acipimox was used before and during endurance exercise to prevent the elevation of plasma NEFA levels in healthy subjects and patients with diabetes. In 2 separate studies, healthy subjects cycled for 2 hr and patients with diabetes for 1 hr at 50% Wmax. In control conditions, plasma NEFA concentrations increased from 0.35 to 0.90 mM during exercise in healthy subjects and from 0.55 to 0.70 mM in patients with diabetes (p < .05). Phosphorylation states of extracellularly regulated kinase 1 and 2 (ERK1/2), p38, and c-Jun NH2-terminal kinases (JNK) were significantly increased after exercise in the vastus lateralis in both groups. Acipimox blocked the increase in plasma NEFA concentrations and almost completely repressed any rise in ERK1/2 and p38 but not in JNK. In conclusion, the data support a role for plasma NEFA in the activation of p38 and ERK1/2 in skeletal-muscle tissue of healthy and diabetic subjects during endurance exercise. Further investigation will be required to determine the molecular link between NEFA and MAPK activation during exercise in human skeletal muscle.

Beta1 integrin inhibits apoptosis induced by cyclic stretch in annulus fibrosus cells via ERK1/2 MAPK pathway.

PubMed

Zhang, Kai; Ding, Wei; Sun, Wei; Sun, Xiao-jiang; Xie, You-zhuan; Zhao, Chang-qing; Zhao, Jie

2016-01-01

Low back pain is associated with intervertebral disc degeneration (IVDD) due to cellular loss through apoptosis. Mechanical factors play an important role in maintaining the survival of the annulus fibrosus (AF) cells and the deposition of extracellular matrix. However, the mechanisms that excessive mechanical forces lead to AF cell apoptosis are not clear. The present study was to look for how AF cells sense mechanical changes. In vivo experiments, the involvement of mechanoreceptors in apoptosis was examined by RT-PCR and/or immunoblotting in the lumbar spine of rats subjected to unbalanced dynamic and static forces. In vitro experiments, we investigated apoptotic signaling pathways in untransfected and transfected AF cells with the lentivirus vector for rat β1 integrin overexpression after cyclic stretch. Apoptosis in AF cells was assessed using flow cytometry, Hoechst 33258 nuclear staining. Western blotting was used to analyze expression of β1 integrin and caspase-3 and ERK1/2 MAPK signaling molecules. In the rat IVDD model, unbalanced dynamic and static forces induced apoptosis of disc cells, which corresponded to decreased expression of β1 integrin. Cyclic stretch-induced apoptosis in rat AF cells correlated with the activation of caspase-3 and with decreased levels of β1 integrin and the phosphorylation levels of ERK1/2 activation level. However, the overexpression of β1 integrin in AF cells ameliorated cyclic stretch-induced apoptosis and decreased caspase-3 activation. Furthermore, ERK1/2-specific inhibitor promotes apoptosis in vector β1-infected AF cells. These results suggest that the disruption of β1 integrin signaling may underlie disc cell apoptosis induced by mechanical stress. Further work is necessary to fully elucidate the pathophysiological mechanisms that underlie IVDD caused by unbalanced dynamic and static forces.

Role of the protein kinase BRAF in the pathogenesis of endometriosis.

PubMed

Santulli, Pietro; Marcellin, Louis; Chouzenoux, Sandrine; Boulard, Veronique; Just, Pierre-Alexandre; Nicco, Carole; Chereau, Christiane; Tosti, Claudia; Chapron, Charles; Batteux, Frédéric

2016-08-01

Mitogen-activated protein kinases (MAPKs) are involved in the proliferation and survival of endometriotic lesions. Vemurafenib (PLX4032) is a novel protein kinase inhibitor that targets BRAF, a member of the MAPK pathway. The present study tested the in vitro and in vivo effects of PLX4032 on endometriotic cells. We conducted a laboratory study in a tertiary-care university hospital from January 2013 to September 2013. We enrolled a cohort of 40 patients: 20 with histologically proven endometriosis and 20 unaffected women. A thorough surgical examination of the abdominopelvic cavity was performed on all of the study participants. Ex vivo stromal and epithelial cells were extracted from endometrial and endometriotic biopsies from both sets of patients. Proliferation, apoptosis, pERK/ERK ratio, cell cycle regulation (Cyclin D1 and CDK4) and inflammation (PTGS2) were explored with and without PLX4032 treatment. Human endometriotic lesions were implanted into 40 nude mice that were separated into two groups according to PLX4032 or vehicle treatment, which they received for four weeks, before sacrifice and histological examination. Treating endometriotic cells with PLX4032 abrogated the phosphorylation of ERK, significantly reducing the pERK/ERK ratio in both epithelial and stromal cells from endometriotic women compared to the controls (p < 0.05). In addition, treatment with PLX4032 significantly decreased proliferation in both stromal and epithelial cells with a concomitant decrease in Cyclin D1/CDK4 complex and PTGS2 levels. Using a murine model of endometriosis, we observed that PLX4032-treated mice displayed a significant decrease in implant volume compared to the initial size; a slight, but non-significant, increase in size was observed in the vehicle-treated mice. Our data suggest that MAPKs and BRAF are involved in the pathogenesis of endometriosis. PLX4032-induced inhibition of BRAF controlled endometriotic growth, both in vitro and in vivo, and could constitute a promising target for the treatment of endometriosis.

Identification of a novel human kinase supporter of Ras (hKSR-2) that functions as a negative regulator of Cot (Tpl2) signaling.

PubMed

Channavajhala, Padma L; Wu, Leeying; Cuozzo, John W; Hall, J Perry; Liu, Wei; Lin, Lih-Ling; Zhang, Yuhua

2003-11-21

Kinase suppressor of Ras (KSR) is an integral and conserved component of the Ras signaling pathway. Although KSR is a positive regulator of the Ras/mitogen-activated protein (MAP) kinase pathway, the role of KSR in Cot-mediated MAPK activation has not been identified. The serine/threonine kinase Cot (also known as Tpl2) is a member of the MAP kinase kinase kinase (MAP3K) family that is known to regulate oncogenic and inflammatory pathways; however, the mechanism(s) of its regulation are not precisely known. In this report, we identify an 830-amino acid novel human KSR, designated hKSR-2, using predictions from genomic data base mining based on the structural profile of the KSR kinase domain. We show that, similar to the known human KSR, hKSR-2 co-immunoprecipitates with many signaling components of the Ras/MAPK pathway, including Ras, Raf, MEK-1, and ERK-1/2. In addition, we demonstrate that hKSR-2 co-immunoprecipitates with Cot and that co-expression of hKSR-2 with Cot significantly reduces Cot-mediated MAPK and NF-kappaB activation. This inhibition is specific to Cot, because Ras-induced ERK and IkappaB kinase-induced NF-kappaB activation are not significantly affected by hKSR-2 co-expression. Moreover, Cot-induced interleukin-8 production in HeLa cells is almost completely inhibited by the concurrent expression of hKSR-2, whereas transforming growth factor beta-activated kinase 1 (TAK1)/TAK1-binding protein 1 (TAB1)-induced interleukin-8 production is not affected by hKSR-2 co-expression. Taken together, these results indicate that hKSR-2, a new member of the KSR family, negatively regulates Cot-mediated MAP kinase and NF-kappaB pathway signaling.

Formononetin accelerates wound repair by the regulation of early growth response factor-1 transcription factor through the phosphorylation of the ERK and p38 MAPK pathways.

PubMed

Huh, Jeong-Eun; Nam, Dong-Woo; Baek, Young-Hyun; Kang, Jung Won; Park, Dong-Suk; Choi, Do-Young; Lee, Jae-Dong

2011-01-01

Formononetin, a phytoestrogen from the root of Astragalus membranaceus, is used as a blood enhancer and to improve blood microcirculation in complementary and alternative medicine. The present study investigated the influence of formononetin on the expression of early growth response factor-1 (Egr-1) and growth factors contributing to wound healing. Formononetin significantly increased growth factors such as transforming growth factor-beta 1 (TGF-β1), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) in human umbilical vein endothelial cells (HUVECs). Formononetin also increased the expression of Egr-1 transcription factor by 3.2- and 10.5-fold, compared with recombinant VEGF(125) in HUVECs. The formononetin-mediated 12%-43% increase induced endothelial cell proliferation and recovered the migration of wounded HUVECs. In an ex vivo angiogenesis assay, formononetin produced a larger capillary sprouting area than produced using recombinant VEGF(125). Cell proliferation and migration of HUVECs were also greater in the presence of formonectin than VEGF(125). Western blot analysis of scratch-wounded confluent HUVECs showed that formononetin induced the phosphorylation of extracellular signal-regulated kinase (ERK) and slightly inhibited the phosphorylation of p38 mitogen-activated protein kinase (MAPK). The formononetin-mediated sustained activation of Egr-1 was suppressed by the ERK inhibitor PD98059 and the p38 inhibitor SB203580. PD98059 inhibited the formononetin-induced endothelial proliferation and repair in scratch-wounded HUVECs, SB203580 increased the cell proliferation and wound healing. Formononetin accelerate wound closure rate as early as day 3 after surgery and consistently observed until day 10 after in wound animal model. These data suggest that formononetin promotes endothelial repair and wound healing in a process involving the over-expression of Egr-1 transcription factor through the regulation of the ERK1/2 and p38 MAPK pathways. Crown Copyright © 2010. Published by Elsevier B.V. All rights reserved.

Differential Regulation of ERK1/2 and mTORC1 Through T1R1/T1R3 in MIN6 Cells

PubMed Central

Wauson, Eric M.; Guerra, Marcy L.; Dyachok, Julia; McGlynn, Kathleen; Giles, Jennifer; Ross, Elliott M.

2015-01-01

The MAPKs ERK1/2 respond to nutrients and other insulin secretagogues in pancreatic β-cells and mediate nutrient-dependent insulin gene transcription. Nutrients also stimulate the mechanistic target of rapamycin complex 1 (mTORC1) to regulate protein synthesis. We showed previously that activation of both ERK1/2 and mTORC1 in the MIN6 pancreatic β-cell-derived line by extracellular amino acids (AAs) is at least in part mediated by the heterodimeric T1R1/T1R3, a G protein-coupled receptor. We show here that AAs differentially activate these two signaling pathways in MIN6 cells. Pretreatment with pertussis toxin did not prevent the activation of either ERK1/2 or mTORC1 by AAs, indicating that Gi is not central to either pathway. Although glucagon-like peptide 1, an agonist for a Gs-coupled receptor, activated ERK1/2 well and mTORC1 to a small extent, AAs had no effect on cytosolic cAMP accumulation. Ca2+ entry is required for ERK1/2 activation by AAs but is dispensable for AA activation of mTORC1. Pretreatment with UBO-QIC, a selective Gq inhibitor, reduced the activation of ERK1/2 but had little effect on the activation of mTORC1 by AAs, suggesting a differential requirement for Gq. Inhibition of G12/13 by the overexpression of the regulator of G protein signaling domain of p115 ρ-guanine nucleotide exchange factor had no effect on mTORC1 activation by AAs, suggesting that these G proteins are also not involved. We conclude that AAs regulate ERK1/2 and mTORC1 through distinct signaling pathways. PMID:26168033

Differential Regulation of ERK1/2 and mTORC1 Through T1R1/T1R3 in MIN6 Cells.

PubMed

Wauson, Eric M; Guerra, Marcy L; Dyachok, Julia; McGlynn, Kathleen; Giles, Jennifer; Ross, Elliott M; Cobb, Melanie H

2015-08-01

The MAPKs ERK1/2 respond to nutrients and other insulin secretagogues in pancreatic β-cells and mediate nutrient-dependent insulin gene transcription. Nutrients also stimulate the mechanistic target of rapamycin complex 1 (mTORC1) to regulate protein synthesis. We showed previously that activation of both ERK1/2 and mTORC1 in the MIN6 pancreatic β-cell-derived line by extracellular amino acids (AAs) is at least in part mediated by the heterodimeric T1R1/T1R3, a G protein-coupled receptor. We show here that AAs differentially activate these two signaling pathways in MIN6 cells. Pretreatment with pertussis toxin did not prevent the activation of either ERK1/2 or mTORC1 by AAs, indicating that G(I) is not central to either pathway. Although glucagon-like peptide 1, an agonist for a G(s-)coupled receptor, activated ERK1/2 well and mTORC1 to a small extent, AAs had no effect on cytosolic cAMP accumulation. Ca(2+) entry is required for ERK1/2 activation by AAs but is dispensable for AA activation of mTORC1. Pretreatment with UBO-QIC, a selective G(q) inhibitor, reduced the activation of ERK1/2 but had little effect on the activation of mTORC1 by AAs, suggesting a differential requirement for G(q). Inhibition of G(12/13) by the overexpression of the regulator of G protein signaling domain of p115 ρ-guanine nucleotide exchange factor had no effect on mTORC1 activation by AAs, suggesting that these G proteins are also not involved. We conclude that AAs regulate ERK1/2 and mTORC1 through distinct signaling pathways.

Mechanism of uptake of ZnO nanoparticles and inflammatory responses in macrophages require PI3K mediated MAPKs signaling.

PubMed

Roy, Ruchi; Parashar, Vyom; Chauhan, L K S; Shanker, Rishi; Das, Mukul; Tripathi, Anurag; Dwivedi, Premendra Dhar

2014-04-01

The inflammatory responses after exposure to zinc oxide nanoparticles (ZNPs) are known, however, the molecular mechanisms and direct consequences of particle uptake are still unclear. Dose and time-dependent increase in the uptake of ZNPs by macrophages has been observed by flow cytometry. Macrophages treated with ZNPs showed a significantly enhanced phagocytic activity. Inhibition of different internalization receptors caused a reduction in uptake of ZNPs in macrophages. The strongest inhibition in internalization was observed by blocking clathrin, caveolae and scavenger receptor mediated endocytic pathways. However, FcR and complement receptor-mediated phagocytic pathways also contributed significantly to control. Further, exposure of primary macrophages to ZNPs (2.5 μg/ml) caused (i) significant enhancement of Ras, PI3K, (ii) enhanced phosphorylation and subsequent activation of its downstream signaling pathways via ERK1/2, p38 and JNK MAPKs (iii) overexpression of c-Jun, c-Fos and NF-κB. Our results demonstrate that ZNPs induce the generation of reactive nitrogen species and overexpression of Cox-2, iNOS, pro-inflammatory cytokines (IL-6, IFN-γ, TNF-α, IL-17 and regulatory cytokine IL-10) and MAPKs which were found to be inhibited after blocking internalization of ZNPs through caveolae receptor pathway. These results indicate that ZNPs are internalized through caveolae pathway and the inflammatory responses involve PI3K mediated MAPKs signaling cascade. Copyright © 2013 Elsevier Ltd. All rights reserved.

CXCR3 chemokine receptor-induced chemotaxis in human airway epithelial cells: role of p38 MAPK and PI3K signaling pathways.

PubMed

Shahabuddin, Syed; Ji, Rong; Wang, Ping; Brailoiu, Eugene; Dun, Na; Yang, Yi; Aksoy, Mark O; Kelsen, Steven G

2006-07-01

Human airway epithelial cells (HAEC) constitutively express the CXC chemokine receptor CXCR3, which regulates epithelial cell movement. In diseases such as chronic obstructive pulmonary disease and asthma, characterized by denudation of the epithelial lining, epithelial cell migration may contribute to airway repair and reconstitution. This study compared the potency and efficacy of three CXCR3 ligands, I-TAC/CXCL11, IP-10/CXCL10, and Mig/CXCL9, as inducers of chemotaxis in HAEC and examined the underlying signaling pathways involved. Studies were performed in cultured HAEC from normal subjects and the 16-HBE cell line. In normal HAEC, the efficacy of I-TAC-induced chemotaxis was 349 +/- 88% (mean +/- SE) of the medium control and approximately one-half the response to epidermal growth factor, a highly potent chemoattractant. In normal HAEC, Mig, IP-10, and I-TAC induced chemotaxis with similar potency and a rank order of efficacy of I-TAC = IP-10 > Mig. Preincubation with pertussis toxin completely blocked CXCR3-induced migration. Of interest, intracellular [Ca(2+)] did not rise in response to I-TAC, IP-10, or Mig. I-TAC induced a rapid phosphorylation (5-10 min) of two of the three MAPKs, i.e., p38 and ERK1/2. Pretreatment of HAEC with the p38 inhibitor SB 20358 or the PI3K inhibitor wortmannin dose-dependently inhibited the chemotactic response to I-TAC. In contrast, the ERK1/2 inhibitor U0126 had no effect on chemotaxis. These data indicate that in HAEC, CXCR3-mediated chemotaxis involves a G protein, which activates both the p38 MAPK and PI3K pathways in a calcium-independent fashion.

Mercury induces proliferation and reduces cell size in vascular smooth muscle cells through MAPK, oxidative stress and cyclooxygenase-2 pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Aguado, Andrea; Galán, María; Zhenyukh, Olha

2013-04-15

Mercury exposure is known to increase cardiovascular risk but the underlying cellular mechanisms remain undetermined. We analyzed whether chronic exposure to HgCl{sub 2} affects vascular structure and the functional properties of vascular smooth muscle cells (VSMC) through oxidative stress/cyclooxygenase-2 dependent pathways. Mesenteric resistance arteries and aortas from Wistar rats treated with HgCl{sub 2} (first dose 4.6 mg kg{sup −1}, subsequent doses 0.07 mg kg{sup −1} day{sup −1}, 30 days) and cultured aortic VSMC stimulated with HgCl{sub 2} (0.05–5 μg/ml) were used. Treatment of rats with HgCl{sub 2} decreased wall thickness of the resistance and conductance vasculature, increased the number ofmore » SMC within the media and decreased SMC nucleus size. In VSMCs, exposure to HgCl{sub 2}: 1) induced a proliferative response and a reduction in cell size; 2) increased superoxide anion production, NADPH oxidase activity, gene and/or protein levels of the NADPH oxidase subunit NOX-1, the EC- and Mn-superoxide dismutases and cyclooxygenase-2 (COX-2); 3) induced activation of ERK1/2 and p38 MAPK. Both antioxidants and COX-2 inhibitors normalized the proliferative response and the altered cell size induced by HgCl{sub 2}. Blockade of ERK1/2 and p38 signaling pathways abolished the HgCl{sub 2}-induced Nox1 and COX-2 expression and normalized the alterations induced by mercury in cell proliferation and size. In conclusion, long exposure of VSMC to low doses of mercury activates MAPK signaling pathways that result in activation of inflammatory proteins such as NADPH oxidase and COX-2 that in turn induce proliferation of VSMC and changes in cell size. These findings offer further evidence that mercury might be considered an environmental risk factor for cardiovascular disease. - Highlights: ► Chronic HgCl{sub 2} exposure induces vascular remodeling. ► HgCl{sub 2} induces proliferation and decreased cell size in vascular smooth muscle cells. ► HgCl{sub 2} induces MAPK activation, oxidative stress and COX-2 expression. ► Inhibition of MAPK reduces HgCl{sub 2}-induced oxidative stress and COX-2 expression. ► Inhibition of MAPK, oxidative stress and COX-2 restores the altered cell proliferation and size.« less

Targeting Hodgkin and Reed–Sternberg Cells with an Inhibitor of Heat-Shock Protein 90: Molecular Pathways of Response and Potential Mechanisms of Resistance

PubMed Central

Corrêa, Stephany; Du Rocher, Bárbara; Krsticevic, Flavia; Arce, Debora; Sternberg, Cinthya; Abdelhay, Eliana

2018-01-01

Classical Hodgkin lymphoma (cHL) cells overexpress heat-shock protein 90 (HSP90), an important intracellular signaling hub regulating cell survival, which is emerging as a promising therapeutic target. Here, we report the antitumor effect of celastrol, an anti-inflammatory compound and a recognized HSP90 inhibitor, in Hodgkin and Reed–Sternberg cell lines. Two disparate responses were recorded. In KM-H2 cells, celastrol inhibited cell proliferation, induced G0/G1 arrest, and triggered apoptosis through the activation of caspase-3/7. Conversely, L428 cells exhibited resistance to the compound. A proteomic screening identified a total of 262 differentially expressed proteins in sensitive KM-H2 cells and revealed that celastrol’s toxicity involved the suppression of the MAPK/ERK (extracellular signal regulated kinase/mitogen activated protein kinase) pathway. The apoptotic effects were preceded by a decrease in RAS (proto-oncogene protein Ras), p-ERK1/2 (phospho-extracellular signal-regulated Kinase-1/2), and c-Fos (proto-oncogene protein c-Fos) protein levels, as validated by immunoblot analysis. The L428 resistant cells exhibited a marked induction of HSP27 mRNA and protein after celastrol treatment. Our results provide the first evidence that celastrol has antitumor effects in cHL cells through the suppression of the MAPK/ERK pathway. Resistance to celastrol has rarely been described, and our results suggest that in cHL it may be mediated by the upregulation of HSP27. The antitumor properties of celastrol against cHL and whether the disparate responses observed in vitro have clinical correlates deserve further research. PMID:29534015

Targeting Hodgkin and Reed-Sternberg Cells with an Inhibitor of Heat-Shock Protein 90: Molecular Pathways of Response and Potential Mechanisms of Resistance.

PubMed

Segges, Priscilla; Corrêa, Stephany; Du Rocher, Bárbara; Vera-Lozada, Gabriela; Krsticevic, Flavia; Arce, Debora; Sternberg, Cinthya; Abdelhay, Eliana; Hassan, Rocio

2018-03-13

Classical Hodgkin lymphoma (cHL) cells overexpress heat-shock protein 90 (HSP90), an important intracellular signaling hub regulating cell survival, which is emerging as a promising therapeutic target. Here, we report the antitumor effect of celastrol, an anti-inflammatory compound and a recognized HSP90 inhibitor, in Hodgkin and Reed-Sternberg cell lines. Two disparate responses were recorded. In KM-H2 cells, celastrol inhibited cell proliferation, induced G0/G1 arrest, and triggered apoptosis through the activation of caspase-3/7. Conversely, L428 cells exhibited resistance to the compound. A proteomic screening identified a total of 262 differentially expressed proteins in sensitive KM-H2 cells and revealed that celastrol's toxicity involved the suppression of the MAPK/ERK (extracellular signal regulated kinase/mitogen activated protein kinase) pathway. The apoptotic effects were preceded by a decrease in RAS (proto-oncogene protein Ras), p-ERK1/2 (phospho-extracellular signal-regulated Kinase-1/2), and c-Fos (proto-oncogene protein c-Fos) protein levels, as validated by immunoblot analysis. The L428 resistant cells exhibited a marked induction of HSP27 mRNA and protein after celastrol treatment. Our results provide the first evidence that celastrol has antitumor effects in cHL cells through the suppression of the MAPK/ERK pathway. Resistance to celastrol has rarely been described, and our results suggest that in cHL it may be mediated by the upregulation of HSP27. The antitumor properties of celastrol against cHL and whether the disparate responses observed in vitro have clinical correlates deserve further research.

Brain-Derived Neurotrophic Factor Increases Synaptic Protein Levels via the MAPK/Erk Signaling Pathway and Nrf2/Trx Axis Following the Transplantation of Neural Stem Cells in a Rat Model of Traumatic Brain Injury.

PubMed

Chen, Tao; Wu, Yu; Wang, Yuzi; Zhu, Jigao; Chu, Haiying; Kong, Li; Yin, Liangwei; Ma, Haiying

2017-11-01

Brain-derived neurotrophic factor (BDNF) plays an important role in promoting the growth, differentiation, survival and synaptic stability of neurons. Presently, the transplantation of neural stem cells (NSCs) is known to induce neural repair to some extent after injury or disease. In this study, to investigate whether NSCs genetically modified to encode the BDNF gene (BDNF/NSCs) would further enhance synaptogenesis, BDNF/NSCs or naive NSCs were directly engrafted into lesions in a rat model of traumatic brain injury (TBI). Immunohistochemistry, western blotting and RT-PCR were performed to detect synaptic proteins, BDNF-TrkB and its downstream signaling pathways, at 1, 2, 3 or 4 weeks after transplantation. Our results showed that BDNF significantly increased the expression levels of the TrkB receptor gene and the phosphorylation of the TrkB protein in the lesions. The expression levels of Ras, phosphorylated Erk1/2 and postsynaptic density protein-95 were elevated in the BDNF/NSCs-transplanted groups compared with those in the NSCs-transplanted groups throughout the experimental period. Moreover, the nuclear factor (erythroid-derived 2)-like 2/Thioredoxin (Nrf2/Trx) axis, which is a specific therapeutic target for the treatment of injury or cell death, was upregulated by BDNF overexpression. Therefore, we determined that the increased synaptic proteins level implicated in synaptogenesis might be associated with the activation of the MAPK/Erk1/2 signaling pathway and the upregulation of the antioxidant agent Trx modified by BDNF-TrkB following the BDNF/NSCs transplantation after TBI.

Therapeutic potential of MEK inhibition in acute myelogenous leukemia: rationale for "vertical" and "lateral" combination strategies.

PubMed

Ricciardi, Maria Rosaria; Scerpa, Maria Cristina; Bergamo, Paola; Ciuffreda, Ludovica; Petrucci, Maria Teresa; Chiaretti, Sabina; Tavolaro, Simona; Mascolo, Maria Grazia; Abrams, Stephen L; Steelman, Linda S; Tsao, Twee; Marchetti, Antonio; Konopleva, Marina; Del Bufalo, Donatella; Cognetti, Francesco; Foà, Robin; Andreeff, Michael; McCubrey, James A; Tafuri, Agostino; Milella, Michele

2012-10-01

In hematological malignancies, constitutive activation of the RAF/MEK/ERK pathway is frequently observed, conveys a poor prognosis, and constitutes a promising target for therapeutic intervention. Here, we investigated the molecular and functional effects of pharmacological MEK inhibition in cell line models of acute myeloid leukemia (AML) and freshly isolated primary AML samples. The small-molecule, ATP-non-competitive, MEK inhibitor PD0325901 markedly inhibited ERK phosphorylation and growth of several AML cell lines and approximately 70 % of primary AML samples. Growth inhibition was due to G(1)-phase arrest and induction of apoptosis. Transformation by constitutively active upstream pathway elements (HRAS, RAF-1, and MEK) rendered FDC-P1 cells exquisitely prone to PD0325901-induced apoptosis. Gene and protein expression profiling revealed a selective effect of PD0325901 on ERK phosphorylation and compensatory upregulation of the RAF/MEK and AKT/p70( S6K ) kinase modules, potentially mediating resistance to drug-induced growth inhibition. Consequently, in appropriate cellular contexts, both "vertical" (i.e., inhibition of RAF and MEK along the MAPK pathway) and "lateral" (i.e., simultaneous inhibition of the MEK/ERK and mTOR pathways) combination strategies may result in synergistic anti-leukemic effects. Overall, MEK inhibition exerts potent growth inhibitory and proapoptotic activity in preclinical models of AML, particularly in combination with other pathway inhibitors. Deeper understanding of the molecular mechanisms of action of MEK inhibitors will likely translate into more effective targeted strategies for the treatment of AML.

ERK and p38 Upregulation versus Bcl-6 Downregulation in Rat Kidney Epithelial Cells Exposed to Prolonged Hypoxia.

PubMed

Luo, Fengbao; Shi, Jian; Shi, Qianqian; He, Xiaozhou; Xia, Ying

2017-08-01

Hypoxia is a common cause of kidney injury and a major issue in kidney transplantation. Mitogen-activated protein kinases (MAPKs) are involved in the cellular response to hypoxia, but the precise roles of MAPKs in renal cell reactions to hypoxic stress are not well known yet. This work was conducted to investigate the regulation of extracellular signal-regulated kinase-1 and -2 (ERK1/2) and p38 and their signaling-relevant molecules in kidney epithelial cells exposed to prolonged hypoxia. Rat kidney epithelial cells Normal Rat Kidney (NRK)-52E were exposed to hypoxic conditions (1% O 2 ) for 24 to 72 h. Cell morphology was examined by light microscopy, and cell viability was checked by 3-[4,5-dimethylthiazol-2-yl]-5-[3-carboxymethoxypheny]-2-[4-sulfophenyl]-2H-tetrazolium (MTS). The expression of ERK1/2 and p38 MAPK, as well as their signaling-related molecules, was measured by Western blot and real-time polymerase chain (RT-PCR) reaction. At the 1% oxygen level, cell morphology had no appreciable changes compared to the control up to 72 h of exposure under light microscopy, whereas the results of MTS showed a slight but significant reduction in cell viability after 72 h of hypoxia. On the other hand, ERK1/2 and p38 phosphorylation remarkably increased in these cells after 24 to 72 h of hypoxia. In sharp contrast, the expression of transcription factor B-cell lymphoma 6 (Bcl-6) was significantly downregulated in response to hypoxic stress. Other intracellular molecules relevant to the ERK1/2 and p38 signaling pathway, such as protein kinase A, protein kinase C, Bcl-2, nuclear factor erythroid 2-related factor 2, tristetraprolin, and interleukin-10(IL-10), had no significant alterations after 24 to 72 h of hypoxic exposure. We conclude that hypoxic stress increases the phosphorylation of both ERK1/2 and p38 but decreases the level of Bcl-6 in rat kidney epithelial cells.

Novel agents for advanced pancreatic cancer

PubMed Central

Akinleye, Akintunde; Iragavarapu, Chaitanya; Furqan, Muhammad; Cang, Shundong; Liu, Delong

2015-01-01

Pancreatic cancer is relatively insensitive to conventional chemotherapy. Therefore, novel agents targeting dysregulated pathways (MAPK/ERK, EGFR, TGF-β, HEDGEHOG, NOTCH, IGF, PARP, PI3K/AKT, RAS, and Src) are being explored in clinical trials as monotherapy or in combination with cytotoxic chemotherapy. This review summarizes the most recent advances with the targeted therapies in the treatment of patients with advanced pancreatic cancer. PMID:26369833

Molecularly defined diffuse leptomeningeal glioneuronal tumor (DLGNT) comprises two subgroups with distinct clinical and genetic features.

PubMed

Deng, Maximilian Y; Sill, Martin; Chiang, Jason; Schittenhelm, Jens; Ebinger, Martin; Schuhmann, Martin U; Monoranu, Camelia-Maria; Milde, Till; Wittmann, Andrea; Hartmann, Christian; Sommer, Clemens; Paulus, Werner; Gärtner, Jutta; Brück, Wolfgang; Rüdiger, Thomas; Leipold, Alfred; Jaunmuktane, Zane; Brandner, Sebastian; Giangaspero, Felice; Nozza, Paolo; Mora, Jaume; Morales la Madrid, Andres; Cruz Martinez, Ofelia; Hansford, Jordan R; Pietsch, Torsten; Tietze, Anna; Hernáiz-Driever, Pablo; Stoler, Iris; Capper, David; Korshunov, Andrey; Ellison, David W; von Deimling, Andreas; Pfister, Stefan M; Sahm, Felix; Jones, David T W

2018-05-15

Diffuse leptomeningeal glioneuronal tumors (DLGNT) represent rare CNS neoplasms which have been included in the 2016 update of the WHO classification. The wide spectrum of histopathological and radiological features can make this enigmatic tumor entity difficult to diagnose. In recent years, large-scale genomic and epigenomic analyses have afforded insight into key genetic alterations occurring in multiple types of brain tumors and provide unbiased, complementary tools to improve diagnostic accuracy. Through genome-wide DNA methylation screening of > 25,000 tumors, we discovered a molecularly distinct class comprising 30 tumors, mostly diagnosed histologically as DLGNTs. Copy-number profiles derived from the methylation arrays revealed unifying characteristics, including loss of chromosomal arm 1p in all cases. Furthermore, this molecular DLGNT class can be subdivided into two subgroups [DLGNT methylation class (MC)-1 and DLGNT methylation class (MC)-2], with all DLGNT-MC-2 additionally displaying a gain of chromosomal arm 1q. Co-deletion of 1p/19q, commonly seen in IDH-mutant oligodendroglioma, was frequently observed in DLGNT, especially in DLGNT-MC-1 cases. Both subgroups also had recurrent genetic alterations leading to an aberrant MAPK/ERK pathway, with KIAA1549:BRAF fusion being the most frequent event. Other alterations included fusions of NTRK1/2/3 and TRIM33:RAF1, adding up to an MAPK/ERK pathway activation identified in 80% of cases. In the DLGNT-MC-1 group, age at diagnosis was significantly lower (median 5 vs 14 years, p < 0.01) and clinical course less aggressive (5-year OS 100, vs 43% in DLGNT-MC-2). Our study proposes an additional molecular layer to the current histopathological classification of DLGNT, of particular use for cases without typical morphological or radiological characteristics, such as diffuse growth and radiologic leptomeningeal dissemination. Recurrent 1p deletion and MAPK/ERK pathway activation represent diagnostic biomarkers and therapeutic targets, respectively-laying the foundation for future clinical trials with, e.g., MEK inhibitors that may improve the clinical outcome of patients with DLGNT.

Formononetin upregulates nitric oxide synthase in arterial endothelium through estrogen receptors and MAPK pathways.

PubMed

Sun, Tao; Cao, Lei; Ping, Na-Na; Wu, Yue; Liu, Dong-Zheng; Cao, Yong-Xiao

2016-03-01

Formononetin, a phytoestrogen, can improve arterial endothelial cell function by upregulating endothelial nitric oxide synthase (eNOS). The estrogen receptor plays an important role in the regulation of eNOS. This study investigated the hypothesis that formononetin upregulates eNOS through estrogen receptors and MAPK pathways. The rat superior mesenteric arteries were cultured with formononetin or formononetin plus inhibitors for 24 h. The isometric tension of the arteries was measured using a myograph system. The mRNA and protein expression levels of eNOS were determined by real-time PCR and immunohistochemistry, respectively. Acetylcholine (ACh) relaxed the mesenteric arteries precontracted with 5-hydroxytryptamine. This relaxation could be enhanced by formononetin. The removal of endothelium or incubation with l-NAME (a NOS inhibitor) completely abolished the formononetin-enhanced relaxation induced by ACh, suggesting that the formononetin-enhanced vasodilatation is dependent on endothelium and NO pathway. The estrogen receptor inhibitor ICI 182780 attenuated the formononetin-enhanced vasodilatation induced by ACh, suggesting that the formononetin-enhanced arterial relaxation is mediated by the estrogen receptor. Formononetin increased the mRNA and protein expression levels of eNOS. ICI 182780, U0126 (an ERK1/2 inhibitor) and SP600125 (a JNK inhibitor) prevented the increases in arterial relaxation and eNOS levels. Formononetin upregulates eNOS expression in mesenteric arteries via estrogen receptors, ERK1/2 and JNK pathways. © 2016 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology.

Src Family Kinases and p38 Mitogen-Activated Protein Kinases Regulate Pluripotent Cell Differentiation in Culture

PubMed Central

Tan, Boon Siang Nicholas; Kwek, Joly; Wong, Chong Kum Edwin; Saner, Nicholas J.; Yap, Charlotte; Felquer, Fernando; Morris, Michael B.; Gardner, David K.; Rathjen, Peter D.; Rathjen, Joy

2016-01-01

Multiple pluripotent cell populations, which together comprise the pluripotent cell lineage, have been identified. The mechanisms that control the progression between these populations are still poorly understood. The formation of early primitive ectoderm-like (EPL) cells from mouse embryonic stem (mES) cells provides a model to understand how one such transition is regulated. EPL cells form from mES cells in response to l-proline uptake through the transporter Slc38a2. Using inhibitors of cell signaling we have shown that Src family kinases, p38 MAPK, ERK1/2 and GSK3β are required for the transition between mES and EPL cells. ERK1/2, c-Src and GSK3β are likely to be enforcing a receptive, primed state in mES cells, while Src family kinases and p38 MAPK are involved in the establishment of EPL cells. Inhibition of these pathways prevented the acquisition of most, but not all, features of EPL cells, suggesting that other pathways are required. L-proline activation of differentiation is mediated through metabolism and changes to intracellular metabolite levels, specifically reactive oxygen species. The implication of multiple signaling pathways in the process suggests a model in which the context of Src family kinase activation determines the outcomes of pluripotent cell differentiation. PMID:27723793

EMODIN EFFICACY ON THE AKT, MAPK, ERK AND DNMT EXPRESSION PATTERN DURING DMBA-INDUCED ORAL CARCINOMA IN GOLDEN SYRIAN HAMSTERS.

PubMed

Manimaran, Asokan; Manoharan, Shanmugam; Neelakandan, Mani

2016-01-01

The present study has evaluated the Emodin efficacy on the Akt, MAPK, ERK and DNMT expression pattern during 7,12-dimethylbenz[a]anthracene (DMBA)-induced oral carcinoma in golden Syrian hamsters, in order to explore its antitumor potential. Oral tumors were developed in the buccal pouches of golden Syrian hamsters using the carcinogen, DMBA. While the incidence of tumor formation was 100% in hamsters treated with DMBA alone, the tumor formation was not noticed in DMBA+ Emodin treated hamsters. Also, Emodin reduced the severity of precancerous pathological lesions such as dysplasia, in the hamsters treated with DMBA. Emodin administration corrected the abnormalities in the expression pattern of Akt, MAPK, ERK and DNMT in the buccal mucosa of hamsters treated with DMBA. The present study thus suggests that the tumor preventive potential of Emodin is partly related to its modulating effect on the Akt, MAPK, ERK and DNMT expression pattern, as these molecular markers have a pivotal role in the process of cell proliferation, inflammation, invasion, and apoptosis.

Erythrocyte plasma membrane-bound ERK1/2 activation promotes ICAM-4-mediated sickle red cell adhesion to endothelium.

PubMed

Zennadi, Rahima; Whalen, Erin J; Soderblom, Erik J; Alexander, Susan C; Thompson, J Will; Dubois, Laura G; Moseley, M Arthur; Telen, Marilyn J

2012-02-02

The core pathology of sickle cell disease (SCD) starts with the erythrocyte (RBC). Aberration in MAPK/ERK1/2 signaling, which can regulate cell adhesion, occurs in diverse pathologies. Because RBCs contain abundant ERK1/2, we predicted that ERK1/2 is functional in sickle (SS) RBCs and promotes adherence, a hallmark of SCD. ERK1/2 remained active in SS but not normal RBCs. β(2)-adrenergic receptor stimulation by epinephrine can enhance ERK1/2 activity only in SS RBCs via PKA- and tyrosine kinase p72(syk)-dependent pathways. ERK signaling is implicated in RBC ICAM-4 phosphorylation, promoting SS RBC adhesion to the endothelium. SS RBC adhesion and phosphorylation of both ERK and ICAM-4 all decreased with continued cell exposure to epinephrine, implying that activation of ICAM-4-mediated SS RBC adhesion is temporally associated with ERK1/2 activation. Furthermore, recombinant ERK2 phosphorylated α- and β-adducins and dematin at the ERK consensus motif. Cytoskeletal protein 4.1 also showed dynamic phosphorylation but not at the ERK consensus motif. These results demonstrate that ERK activation induces phosphorylation of cytoskeletal proteins and the adhesion molecule ICAM-4, promoting SS RBC adhesion to the endothelium. Thus, blocking RBC ERK1/2 activation, such as that promoted by catecholamine stress hormones, could ameliorate SCD pathophysiology.

Insulin-like Growth Factor 1 Rescues R28 Retinal Neurons from Apoptotic Death through ERK-mediated BimEL Phosphorylation Independent of Akt

PubMed Central

Kong, Dejuan; Gong, Lijie; Arnold, Edith; Shanmugam, Sumathi; Fort, Patrice E.; Gardner, Thomas W.; Abcouwer, Steven F.

2016-01-01

Insulin-like growth factor 1 (IGF-1) can provide long-term neurotrophic support by activation of Akt, inhibition of FoxO nuclear localization and suppression of Bim gene transcription in multiple neuronal systems. However, MEK/ERK activation can also promote neuron survival through phosphorylation of BimEL. We explored the contribution of the PI3K/Akt/FoxO and MEK/ERK/BimEL pathways in IGF-1 stimulated survival after serum deprivation (SD) of R28 cells differentiated to model retinal neurons. IGF-1 caused rapid activation of Akt leading to FoxO1/3-T32/T24 phosphorylation, and prevented FoxO1/3 nuclear translocation and Bim mRNA upregulation in response to SD. IGF-1 also caused MAPK/MEK pathway activation as indicated by ERK1/2-T202/Y204 and Bim-S65 phosphorylation. Overexpression of FoxO1 increased Bim mRNA expression and amplified the apoptotic response to SD without shifting the serum response curve. Inhibition of Akt activation with LY294002 or by Rictor knockdown did not block the protective effect of IGF-1, while inhibition of MEK activity with PD98059 prevented Bim phosphorylation and blocked IGF-1 protection. In addition, knockdown of Bim expression was protective during SD, while co-silencing of FoxO1 and Fox03 expression had little effect. Thus, the PI3K/Akt/FoxO pathway was not essential for protection from SD-induced apoptosis by IGF-1 in R28 cells. Instead, IGF-1 protection was dependent on activation of the MEK/ERK pathway leading to BimEL phosphorylation, which is known to prevent Bax/Bak oligomerization and activation of the intrinsic mitochondrial apoptosis pathway. These studies demonstrate the requirement of the MEK/ERK pathway in a model of retinal neuron cell survival and highlight the cell specificity for IGF-1 signaling in this response. PMID:27511131

Roles of MAPK pathway activation during cytokine induction in BEAS-2B cells exposed to fine World Trade Center (WTC) dust.

PubMed

Wang, Shang; Prophete, Colette; Soukup, Joleen M; Chen, Lung-Chi; Costa, Max; Ghio, Andrew; Qu, Qingshan; Cohen, Mitchell D; Chen, Haobin

2010-01-01

The World Trade Center (WTC) collapse on September 11, 2001 released copious amounts of particulate matter (PM) into the atmosphere of New York City. Follow-up studies on persons exposed to the dusts have revealed a severely increased rate for asthma and other respiratory illnesses. There have only been a few studies that have sought to discern the possible mechanisms underlying these untoward pathologies. In one study, an increased cytokine release was detected in cells exposed to WTC fine dusts (PM₂.₅ fraction or WTC₂.₅). However, the mechanism(s) for these increases has yet to be fully defined. Because activation of the mitogen-activated protein kinase (MAPK) signaling pathways is known to cause cytokine induction, the current study was undertaken to analyze the possible involvement of these pathways in any increased cytokine formation by lung epithelial cells (as BEAS-2B cells) exposed to WTC₂.₅. Our results showed that exposure to WTC₂.₅ for 5 hr increased interleukin-6 (IL-6) mRNA expression in BEAS-2B cells, as well as its protein levels in the culture media, in a dose-dependent manner. Besides IL-6, cytokine multiplex analyses revealed that formation of IL-8 and -10 was also elevated by the exposure. Both extracellular signal-regulated kinase (ERK) and p38, but not c-Jun N-terminal protein kinase, signaling pathways were found to be activated in cells exposed to WTC₂.₅. Inactivation of ERK signaling pathways by PD98059 effectively blocked IL-6, -8, and -10 induction by WTC₂.₅; the p38 kinase inhibitor SB203580 significantly decreased induction of IL-8 and -10. Together, our data demonstrated activation of MAPK signaling pathway(s) likely played an important role in the WTC₂.₅-induced formation of several inflammatory (and, subsequently, anti-inflammatory) cytokines. The results are important in that they help to define one mechanism via which the WTC dusts may have acted to cause the documented increases in asthma and other inflammation-associated respiratory dysfunctions in the individuals exposed to the dusts released from the WTC collapse.

Activated STAT5 proteins induce activation of the PI 3-kinase/Akt and Ras/MAPK pathways via the Gab2 scaffolding adapter.

PubMed

Nyga, Rémy; Pecquet, Christian; Harir, Noria; Gu, Haihua; Dhennin-Duthille, Isabelle; Régnier, Aline; Gouilleux-Gruart, Valérie; Lassoued, Kaïss; Gouilleux, Fabrice

2005-08-15

The active forms of STAT5A (signal transducer and activator of transcription 5A) and STAT5B are able to relieve the cytokine dependence of haematopoietic cells and to induce leukaemia in mice. We have demonstrated previously that activation of the PI3K (phosphoinositide 3-kinase) signalling cascade plays a major role in cell growth and survival induced by these proteins. Interaction between STAT5 and p85, the regulatory subunit of the PI3K, has been suggested to be required for this activation. We show in the present study that the scaffolding protein Gab2 [Grb2 (growth-factor-receptor-bound protein 2)-associated binder-2] is an essential component of this interaction. Gab2 is persistently tyrosine-phosphorylated in Ba/F3 cells expressing caSTAT5 (constitutively activated STAT5), independent of JAK2 (Janus kinase 2) activation where it interacts with STAT5, p85 and Grb2, but not with Shp2 [SH2 (Src homology 2)-domain-containing tyrosine phosphatase] proteins. Interaction of STAT5 with Gab2 was also observed in Ba/F3 cells stimulated with interleukin-3 or expressing the oncogenic fusion protein Tel-JAK2. The MAPKs (mitogen-activated protein kinases) ERK1 (extracellular-signal-regulated kinase 1) and ERK2 were constitutively activated in the caSTAT5-expressing cells and were found to be required for caSTAT5-induced cell proliferation. Overexpression of Gab2-3YF, a mutant of Gab2 incapable of binding PI3K, inhibited the proliferation and survival of caSTAT5-expressing cells as well as ERK1/2 and Akt/protein kinase B phosphorylation. Taken together, our results indicate that Gab2 is required for caSTAT5-induced cell proliferation by regulating both the PI3K/Akt and the Ras/MAPK pathways.

Activated STAT5 proteins induce activation of the PI 3-kinase/Akt and Ras/MAPK pathways via the Gab2 scaffolding adapter

PubMed Central

2005-01-01

The active forms of STAT5A (signal transducer and activator of transcription 5A) and STAT5B are able to relieve the cytokine dependence of haematopoietic cells and to induce leukaemia in mice. We have demonstrated previously that activation of the PI3K (phosphoinositide 3-kinase) signalling cascade plays a major role in cell growth and survival induced by these proteins. Interaction between STAT5 and p85, the regulatory subunit of the PI3K, has been suggested to be required for this activation. We show in the present study that the scaffolding protein Gab2 [Grb2 (growth-factor-receptor-bound protein 2)-associated binder-2] is an essential component of this interaction. Gab2 is persistently tyrosine-phosphorylated in Ba/F3 cells expressing caSTAT5 (constitutively activated STAT5), independent of JAK2 (Janus kinase 2) activation where it interacts with STAT5, p85 and Grb2, but not with Shp2 [SH2 (Src homology 2)-domain-containing tyrosine phosphatase] proteins. Interaction of STAT5 with Gab2 was also observed in Ba/F3 cells stimulated with interleukin-3 or expressing the oncogenic fusion protein Tel–JAK2. The MAPKs (mitogen-activated protein kinases) ERK1 (extracellular-signal-regulated kinase 1) and ERK2 were constitutively activated in the caSTAT5-expressing cells and were found to be required for caSTAT5-induced cell proliferation. Overexpression of Gab2-3YF, a mutant of Gab2 incapable of binding PI3K, inhibited the proliferation and survival of caSTAT5-expressing cells as well as ERK1/2 and Akt/protein kinase B phosphorylation. Taken together, our results indicate that Gab2 is required for caSTAT5-induced cell proliferation by regulating both the PI3K/Akt and the Ras/MAPK pathways. PMID:15833084

Fas signaling induces stemness properties in colorectal cancer by regulation of Bmi1.

PubMed

Chen, Jiaxuan; Wang, Yadong; Zhuo, Linghao; Liu, Zhizhong; Liu, Tao; Li, Wenjing; Cai, Yidong; Zheng, Haoxuan

2017-10-01

Fas signaling promotes colorectal cancer (CRC) metastasis by inducing epithelial-mesenchymal transition (EMT). The acquisition of EMT properties in turn induces stemness but the mechanism by which Fas signaling contributes to it still remains unclear. Hence, the aim of this study was to investigate how Fas signaling regulates CRC stemness. For this purpose, soft agar assay, sphere formation assay, cell survival analysis, immunoblot, qRT-PCR, chromatin immunoprecipitation, and luciferase reporter assay were performed. Expression of FasL, Bmi1, and the miR-200c in CRC specimens was examined through immunohistochemistry, qRT-PCR, and immunoblot. In our study, Fas signaling induced stem cell properties in CRC specimens, relying on ERK1/2 MAPK pathway, with Bmi1 being mainly responsible for FasL-induced stemness. FasL treatment promoted Bmi1 expression by inhibiting miR-200c, which targets Bmi1 3'UTR region. Furthermore, FasL-induced Zeb1 binded with miR-200c promoter and inhibited its expression. Moreover, FasL-induced β-catenin nuclear expression promoted Zeb1 expression by binding with Zeb1 promoter. GSK-3β, which regulates β-catenin, was inhibited by FasL-induced ERK1/2 MAPK signaling. Finally, FasL and Bmi1 expression in clinical samples increased during CRC progression, and a positive correlation between them was observed. Patients with high FasL and Bmi1 expression had a worse prognosis than patients with low expression. In conclusion, our results showed that Fas signaling can promote stemness in CRC through the modulation of Bmi1 expression via the ERK1/2 MAPK/GSK-3β/β-catenin/Zeb1/miR-200c axis, suggesting that Fas signaling-based cancer therapies should be administered cautiously, as the activation of this pathway not only leads to apoptosis but also induces stemness in CRC. © 2017 Wiley Periodicals, Inc.

Comprehensive gene- and pathway-based analysis of depressive symptoms in older adults.

PubMed

Nho, Kwangsik; Ramanan, Vijay K; Horgusluoglu, Emrin; Kim, Sungeun; Inlow, Mark H; Risacher, Shannon L; McDonald, Brenna C; Farlow, Martin R; Foroud, Tatiana M; Gao, Sujuan; Callahan, Christopher M; Hendrie, Hugh C; Niculescu, Alexander B; Saykin, Andrew J

2015-01-01

Depressive symptoms are common in older adults and are particularly prevalent in those with or at elevated risk for dementia. Although the heritability of depression is estimated to be substantial, single nucleotide polymorphism-based genome-wide association studies of depressive symptoms have had limited success. In this study, we performed genome-wide gene- and pathway-based analyses of depressive symptom burden. Study participants included non-Hispanic Caucasian subjects (n = 6,884) from three independent cohorts, the Alzheimer's Disease Neuroimaging Initiative (ADNI), the Health and Retirement Study (HRS), and the Indiana Memory and Aging Study (IMAS). Gene-based meta-analysis identified genome-wide significant associations (ANGPT4 and FAM110A, q-value = 0.026; GRM7-AS3 and LRFN5, q-value = 0.042). Pathway analysis revealed enrichment of association in 105 pathways, including multiple pathways related to ERK/MAPK signaling, GSK3 signaling in bipolar disorder, cell development, and immune activation and inflammation. GRM7, ANGPT4, and LRFN5 have been previously implicated in psychiatric disorders, including the GRM7 region displaying association with major depressive disorder. The ERK/MAPK signaling pathway is a known target of antidepressant drugs and has important roles in neuronal plasticity, and GSK3 signaling has been previously implicated in Alzheimer's disease and as a promising therapeutic target for depression. Our results warrant further investigation in independent and larger cohorts and add to the growing understanding of the genetics and pathobiology of depressive symptoms in aging and neurodegenerative disorders. In particular, the genes and pathways demonstrating association with depressive symptoms may be potential therapeutic targets for these symptoms in older adults.

Ezetimibe suppresses cholesterol accumulation in lipid-loaded vascular smooth muscle cells in vitro via MAPK signaling

PubMed Central

Qin, Li; Yang, Yun-bo; Yang, Yi-xin; Zhu, Neng; Gong, Yong-zhen; Zhang, Cai-ping; Li, Shun-xiang; Liao, Duan-fang

2014-01-01

Aim: To investigate the mechanisms of anti-atherosclerotic action of ezetimibe in rat vascular smooth muscle cells (VSMCs) in vitro. Methods: VSMCs of SD rats were cultured in the presence of Chol:MβCD (10 μg/mL) for 72 h, and intracellular lipid droplets and cholesterol levels were evaluated using Oil Red O staining, HPLC and Enzymatic Fluorescence Assay, respectively. The expression of caveolin-1, sterol response element-binding protein-1 (SREBP-1) and ERK1/2 were analyzed using Western blot assays. Translocation of SREBP-1 and ERK1/2 was detected with immunofluorescence. Results: Treatment with Chol:MβCD dramatically increased the cellular levels of total cholesterol (TC), cholesterol ester (CE) and free cholesterol (FC) in VSMCs, which led to the formation of foam cells. Furthermore, Chol:MβCD treatment significantly decreased the expression of caveolin-1, and stimulated the expression and nuclear translocation of SREBP-1 in VSMCs. Co-treatment with ezetimibe (3 μmol/L) significantly decreased the cellular levels of TC, CE and FC, which was accompanied by elevation of caveolin-1 expression, and by a reduction of SREBP-1 expression and nuclear translocation. Co-treatment with ezetimibe dose-dependently decreased the expression of phosphor-ERK1/2 (p-ERK1/2) in VSMCs. The ERK1/2 inhibitor PD98059 (50 μmol/L) altered the cholesterol level and the expression of p-ERK1/2, SREBP-1 and caveolin-1 in the same manner as ezetimibe did. Conclusion: Ezetimibe suppresses cholesterol accumulation in rat VSMCs in vitro by regulating SREBP-1 and caveolin-1 expression, possibly via the MAPK signaling pathway. PMID:25087996

Negative feedback via RSK modulates Erk-dependent progression from naïve pluripotency.

PubMed

Nett, Isabelle Re; Mulas, Carla; Gatto, Laurent; Lilley, Kathryn S; Smith, Austin

2018-06-12

Mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) signalling is implicated in initiation of embryonic stem (ES) cell differentiation. The pathway is subject to complex feedback regulation. Here, we examined the ERK-responsive phosphoproteome in ES cells and identified the negative regulator RSK1 as a prominent target. We used CRISPR/Cas9 to create combinatorial mutations in RSK family genes. Genotypes that included homozygous null mutations in Rps6ka1, encoding RSK1, resulted in elevated ERK phosphorylation. These RSK-depleted ES cells exhibit altered kinetics of transition into differentiation, with accelerated downregulation of naïve pluripotency factors, precocious expression of transitional epiblast markers and early onset of lineage specification. We further show that chemical inhibition of RSK increases ERK phosphorylation and expedites ES cell transition without compromising multilineage potential. These findings demonstrate that the ERK activation profile influences the dynamics of pluripotency progression and highlight the role of signalling feedback in temporal control of cell state transitions. © 2018 The Authors. Published under the terms of the CC BY 4.0 license.

Functional Toll-like Receptor 4 Overexpression in Papillary Thyroid Cancer by MAPK/ERK-Induced ETS1 Transcriptional Activity.

PubMed

Peyret, Victoria; Nazar, Magalí; Martín, Mariano; Quintar, Amado A; Fernandez, Elmer A; Geysels, Romina C; Fuziwara, Cesar S; Montesinos, María M; Maldonado, Cristina A; Santisteban, Pilar; Kimura, Edna T; Pellizas, Claudia G; Nicola, Juan P; Masini-Repiso, Ana M

2018-05-01

Emerging evidence suggests that unregulated Toll-like receptor (TLR) signaling promotes tumor survival signals, thus favoring tumor progression. Here, the mechanism underlying TLR4 overexpression in papillary thyroid carcinomas (PTC) mainly harboring the BRAF V600E mutation was studied. TLR4 was overexpressed in PTC compared with nonneoplastic thyroid tissue. Moreover, paired clinical specimens of primary PTC and its lymph node metastasis showed a significant upregulation of TLR4 levels in the metastatic tissues. In agreement, conditional BRAF V600E expression in normal rat thyroid cells and mouse thyroid tissue upregulated TLR4 expression levels. Furthermore, functional TLR4 expression was demonstrated in PTC cells by increased NF-κB transcriptional activity in response to the exogenous TLR4-agonist lipopolysaccharide. Of note, The Cancer Genome Atlas data analysis revealed that BRAF V600E -positive tumors with high TLR4 expression were associated with shorter disease-free survival. Transcriptomic data analysis indicated a positive correlation between TLR4 expression levels and MAPK/ERK signaling activation. Consistently, chemical blockade of MAPK/ERK signaling abrogated BRAF V600E -induced TLR4 expression. A detailed study of the TLR4 promoter revealed a critical MAPK/ERK-sensitive Ets-binding site involved in BRAF V600E responsiveness. Subsequent investigation revealed that the Ets-binding factor ETS1 is critical for BRAF V600E -induced MAPK/ERK signaling-dependent TLR4 gene expression. Together, these data indicate that functional TLR4 overexpression in PTCs is a consequence of thyroid tumor-oncogenic driver dysregulation of MAPK/ERK/ETS1 signaling. Implications: Considering the participation of aberrant NF-κB signaling activation in the promotion of thyroid tumor growth and the association of high TLR4 expression with more aggressive tumors, this study suggests a prooncogenic potential of TLR4 downstream signaling in thyroid tumorigenesis. Mol Cancer Res; 16(5); 833-45. ©2018 AACR . ©2018 American Association for Cancer Research.

The MAP kinase ERK and its scaffold protein MP1 interact with the chromatin regulator Corto during Drosophila wing tissue development

PubMed Central

2011-01-01

Background Mitogen-activated protein kinase (MAPK) cascades (p38, JNK, ERK pathways) are involved in cell fate acquisition during development. These kinase modules are associated with scaffold proteins that control their activity. In Drosophila, dMP1, that encodes an ERK scaffold protein, regulates ERK signaling during wing development and contributes to intervein and vein cell differentiation. Functional relationships during wing development between a chromatin regulator, the Enhancer of Trithorax and Polycomb Corto, ERK and its scaffold protein dMP1, are examined here. Results Genetic interactions show that corto and dMP1 act together to antagonize rolled (which encodes ERK) in the future intervein cells, thus promoting intervein fate. Although Corto, ERK and dMP1 are present in both cytoplasmic and nucleus compartments, they interact exclusively in nucleus extracts. Furthermore, Corto, ERK and dMP1 co-localize on several sites on polytene chromosomes, suggesting that they regulate gene expression directly on chromatin. Finally, Corto is phosphorylated. Interestingly, its phosphorylation pattern differs between cytoplasm and nucleus and changes upon ERK activation. Conclusions Our data therefore suggest that the Enhancer of Trithorax and Polycomb Corto could participate in regulating vein and intervein genes during wing tissue development in response to ERK signaling. PMID:21401930

Role of mitogen-activated protein kinases and Mcl-1 in apoptosis induction by withaferin A in human breast cancer cells.

PubMed

Hahm, Eun-Ryeong; Lee, Joomin; Singh, Shivendra V

2014-11-01

Withaferin A (WA), a bioactive constituent of Ayurvedic medicine plant Withania somnifera, is a potent apoptosis inducer in cancer cells but the mechanism of cell death induction is not fully characterized. The present study was undertaken to determine the role of mitogen-activated protein kinases (MAPK), including c-jun NH2 -terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and p38 MAPK, and anti-apoptotic protein myeloid cell leukemia-1 (Mcl-1) in regulation of WA-induced apoptosis using human breast cancer cells. Exposure of MCF-7 (estrogen responsive) and SUM159 (triple negative) human breast cancer cells to WA resulted in increased phosphorylation of ERK, JNK, and p38 MAPK, but these effects were relatively more pronounced in the former cell line than in SUM159. Overexpression of manganese-superoxide dismutase conferred partial protection against WA-mediated hyperphosphorylation of ERK, but not JNK or p38 MAPK. Cell death resulting from WA treatment in MCF-7 cells was significantly augmented by pharmacological inhibition of ERK and p38 MAPK. Interestingly, the WA-induced apoptosis in MCF-7 cells was partially but significantly blocked in the presence of a JNK-specific inhibitor. Pharmacological inhibition of ERK or JNK had no effect on WA-induced apoptosis in SUM159 cells. The WA-treated cells exhibited induction of long and short forms of Mcl-1. RNA interference of Mcl-1 alone triggered apoptosis. Furthermore, the WA-induced cell death in MCF-7 cells was modestly but significantly augmented by knockdown of the Mcl-1 protein. These observations indicate that: MAPK have cell line-specific role in cell death by WA, and Mcl-1 induction confers modest protection against WA-induced apoptosis. © 2013 Wiley Periodicals, Inc.

Transcriptional and translational control of ornithine decarboxylase during Ras transformation.

PubMed Central

Shantz, Lisa M

2004-01-01

ODC (ornithine decarboxylase) activity is induced following ras activation. However, the Ras effector pathways responsible are unknown. These experiments used NIH-3T3 cells expressing partial-loss-of-function Ras mutants to activate selectively pathways downstream of Ras and examined the contribution of each pathway to ODC induction. Overexpression of Ras12V, a constitutively active mutant, resulted in ODC activities up to 20-fold higher than controls. Stable transfections of Ras partial-loss-of-function mutants and constitutively active forms of MEK (MAPK kinase) and Akt indicated that activation of more than one Ras effector pathway is necessary for the complete induction of ODC activity. The increase in ODC activity in Ras12V-transformed cells is not owing to a substantial change in ODC protein half-life, which increased by <2-fold. Northern-blot analysis and reporter assays suggested that the mechanism of ODC induction involves both a modest increase in the transcription of ODC mRNA and a much more considerable increase in the translation of mRNA into protein. ODC transcription was controlled through a pathway dependent on Raf/MEK/ERK (where ERK stands for extracellular-signal-regulated kinase) activation, whereas activation of the phosphoinositide 3-kinase and the Raf/MEK/ERK pathways were necessary for translational regulation of ODC. The increase in ODC synthesis was accompanied by changes in phosphorylation of eukaryotic initiation factor 4E and its binding protein 4E-BP1. Results show that the phosphoinositide 3-kinase pathway regulates phosphorylation of both proteins, whereas the Raf/MEK/ERK pathway affects only the eukaryotic initiation factor 4E phosphorylation. PMID:14519103

Cyclic Tensile Strain Upregulates Pro-Inflammatory Cytokine Expression Via FAK-MAPK Signaling in Chondrocytes.

PubMed

Yanoshita, Makoto; Hirose, Naoto; Okamoto, Yuki; Sumi, Chikako; Takano, Mami; Nishiyama, Sayuri; Asakawa-Tanne, Yuki; Horie, Kayo; Onishi, Azusa; Yamauchi, Yuka; Mitsuyoshi, Tomomi; Kunimatsu, Ryo; Tanimoto, Kotaro

2018-05-08

Excessive mechanical stimulation is considered an important factor in the destruction of chondrocytes. Focal adhesion kinase (FAK) is non-receptor tyrosine kinase related to a number of different signaling proteins. Little is known about the function of FAK in chondrocytes under mechanical stimulation. In the present study, we investigated the function of FAK in mechanical signal transduction and the mechanism through which cyclic tensile strain (CTS) induces expression of inflammation-related factors. Mouse ATDC5 chondrogenic cells were subjected to CTS of 0.5 Hz to 10% cell elongation with an FAK inhibitor. The expression of genes encoding COX-2, IL-1β, and TNF-α was examined using real-time RT-PCR after CTS application with FAK inhibitor. Phosphorylation of p-38, ERK, and JNK was analyzed by Western blotting. Differences in COX-2 expression following pretreatment with FAK, p-38, ERK, and JNK inhibitors were compared by Western blotting. We found that CTS increased the expression of genes encoding COX-2, IL-1β, and TNF-α and activated the phosphorylation of FAK, p-38, ERK, and JNK. Pretreatment with an FAK inhibitor for 2 h reduced the expression of genes encoding COX-2, IL-1β, and TNF-α induced by CTS-associated inflammation and decreased phosphorylation of FAK, p-38, ERK, and JNK. Pretreatment with FAK, p-38, ERK, and JNK inhibitors markedly suppressed COX-2 and IL-1β protein expression. In conclusion, FAK appears to regulate inflammation in chondrocytes under CTS via MAPK pathways.

Oxymatrine lightened the inflammatory response of LPS-induced mastitis in mice through affecting NF-κB and MAPKs signaling pathways.

PubMed

Yang, Zhengtao; Yin, Ronglan; Cong, Yunfeng; Yang, Zhanqing; Zhou, Ershun; Wei, Zhengkai; Liu, Zhicheng; Cao, Yongguo; Zhang, Naisheng

2014-12-01

Mastitis, an inflammatory reaction of the mammary gland, is recognized as one of the most costly diseases in dairy cattle. Oxymatrine, one of the alkaloids extracted from Chinese herb Sophora flavescens Ait, has been reported to have many biological activities, such as anti-inflammatory, anti-virus, and anti-hepatic fibrosis properties. The aim of this study was to investigate the protective effect and the anti-inflammatory mechanism of oxymatrine on lipopolysaccharide (LPS)-induced mastitis in mice. The mouse mastitis was induced by 10 μg of LPS for 24 h. Oxymatrine was intraperitoneally administered with the dose of 30, 60, and 120 mg/kg 1 h before and 12 h after LPS induction. The results showed that oxymatrine significantly attenuated the damage of the mammary gland induced by LPS. Oxymatrine inhibited the phosphorylation of NF-κB p65 and IκB in NF-κB signal pathway and reduced the phosphorylation of p38, ERK, and JNK in mitogen-activated protein kinase (MAPKs) signal pathway. The results showed that oxymatrine had a protective effect on LPS-induced mastitis, and the anti-inflammatory mechanism of oxymatrine was related to the inhibition of NF-κB and MAPKs signal pathways.

Phyllanthus Suppresses Prostate Cancer Cell, PC-3, Proliferation and Induces Apoptosis through Multiple Signalling Pathways (MAPKs, PI3K/Akt, NFκB, and Hypoxia).

PubMed

Tang, Yin-Quan; Jaganath, Indubala; Manikam, Rishya; Sekaran, Shamala Devi

2013-01-01

Phyllanthus is a traditional medicinal plant that has been found to have antihepatitis, antibacterial, and anticancer properties. The present studies were to investigate the in vitro molecular mechanisms of anticancer effects of Phyllanthus (P. amarus, P. niruri, P. urinaria, and P. watsonii) plant extracts in human prostate adenocarcinoma. The cancer ten-pathway reporter array was performed and revealed that the expression of six pathway reporters were significantly decreased (Wnt, NFκB, Myc/Max, hypoxia, MAPK/ERK, and MAPK/JNK) in PC-3 cells after treatment with Phyllanthus extracts. Western blot was conducted and identified several signalling molecules that were affected in the signalling pathways including pan-Ras, c-Raf, RSK, Elk1, c-Jun, JNK1/2, p38 MAPK, c-myc, DSH, β-catenin, Akt, HIF-1α, GSK3β, NFκB p50 and p52, Bcl-2, Bax, and VEGF, in treated PC-3 cells. A proteomics-based approach, 2D gel electrophoresis, was performed, and mass spectrometry (MS/MS) results revealed that there were 72 differentially expressed proteins identified in treated PC-3 cells and were involved in tumour cell adhesion, apoptosis, glycogenesis and glycolysis, metastasis, angiogenesis, and protein synthesis and energy metabolism. Overall, these findings suggest that Phyllanthus can interfere with multiple signalling cascades involved in tumorigenesis and be used as a potential therapeutic candidate for treatment of cancer.

Involvement of IGF-1 and MEOX2 in PI3K/Akt1/2 and ERK1/2 pathways mediated proliferation and differentiation of perivascular adipocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Liu, Ping, E-mail: lping@sdu.edu.cn; Kong, Feng; Wang, Jue

Perivascular adipocyte (PVAC) proliferation and differentiation were closely involved in cardiovascular disease. We aimed to investigate whether phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways enhance PVAC functions activated by insulin-like growth factor 1(IGF-1) and suppressed by mesenchyme homeobox 2 (MEOX2). In this study, PVACs from primary culture were cultured and induced to differentiate. Cell viability assays demonstrated that IGF-1 promoted PVAC proliferation and differentiation. However MEOX2 counteracted these IGF-1-mediated actions. Flow Cytometry revealed that IGF-1 increased S phase cells and decreased apoptosis; however, MEOX2 decreased S phase cells, increased G0–G1 phase cells, and promoted apoptosis. During PVACmore » proliferation and differentiation, IGF-1 activated PI3K/Akt1/2 and ERK1/2 signaling pathways, upregulated the expression of these signaling proteins and FAS, and increased PVAC lipid content. In contrast, MEOX2 constrained the phosphorylation of ERK1/2 and Akt1/2 protein, down-regulated these signaling molecules and FAS, and decreased PVAC lipid content. Instead, MEOX2 knockdown enhanced the ERK1/2 and Akt1/2 phosphorylation, augmented the expression of these signaling molecules and FAS, and increased PVAC lipid content. Our findings suggested that PI3K/Akt1/2 and ERK1/2 activation mediated by IGF-1 is essential for PVAC proliferation and differentiation, and MEOX2 is a promising therapeutic gene to intervene in the signaling pathways and inhibit PVAC functions. - Highlights: • IGF-1 activated PI3K/Akt2 and ERK1/2 pathways to mediate PVAC proliferation and differentiation. • The expression of ERK1, ERK 2, PI3K, Akt1 and Akt2 showed different change trends between PVAC proliferation and differentiation. • MEOX2 effectively expressed in PVAC, increased early and late cellular apoptosis, and inhibited its proliferation. • MEOX2 depressed PVAC differentiation and FAS expression, and decreased lipid content in PVAC. • MEOX2 repressed the effects of IGF-1 on PVAC by restraining the activation of PI3K/Akt1/2 and ERK1/2 signaling pathways.« less

Anti-inflammatory effects of ursodeoxycholic acid by lipopolysaccharide-stimulated inflammatory responses in RAW 264.7 macrophages

PubMed Central

Ko, Wan-Kyu; Lee, Soo-Hong; Kim, Sung Jun; Jo, Min-Jae; Kumar, Hemant; Han, In-Bo; Sohn, Seil

2017-01-01

Purpose The aim of this study was to investigate the anti-inflammatory effects of Ursodeoxycholic acid (UDCA) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. Methods We induced an inflammatory process in RAW 264.7 macrophages using LPS. The anti-inflammatory effects of UDCA on LPS-stimulated RAW 264.7 macrophages were analyzed using nitric oxide (NO). Pro-inflammatory and anti-inflammatory cytokines were analyzed by quantitative real time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). The phosphorylations of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 in mitogen-activated protein kinase (MAPK) signaling pathways and nuclear factor kappa-light polypeptide gene enhancer in B-cells inhibitor, alpha (IκBα) signaling pathways were evaluated by western blot assays. Results UDCA decreased the LPS-stimulated release of the inflammatory mediator NO. UDCA also decreased the pro-inflammatory cytokines tumor necrosis factor-α (TNF-α), interleukin 1-α (IL-1α), interleukin 1-β (IL-1β), and interleukin 6 (IL-6) in mRNA and protein levels. In addition, UDCA increased an anti-inflammatory cytokine interleukin 10 (IL-10) in the LPS-stimulated RAW 264.7 macrophages. UDCA inhibited the expression of inflammatory transcription factor nuclear factor kappa B (NF-κB) in LPS-stimulated RAW 264.7 macrophages. Furthermore, UDCA suppressed the phosphorylation of ERK, JNK, and p38 signals related to inflammatory pathways. In addition, the phosphorylation of IκBα, the inhibitor of NF-κB, also inhibited by UDCA. Conclusion UDCA inhibits the pro-inflammatory responses by LPS in RAW 264.7 macrophages. UDCA also suppresses the phosphorylation by LPS on ERK, JNK, and p38 in MAPKs and NF-κB pathway. These results suggest that UDCA can serve as a useful anti-inflammatory drug. PMID:28665991

Fisetin induces apoptosis and endoplasmic reticulum stress in human non-small cell lung cancer through inhibition of the MAPK signaling pathway.

PubMed

Kang, Kyoung Ah; Piao, Mei Jing; Madduma Hewage, Susara Ruwan Kumara; Ryu, Yea Seong; Oh, Min Chang; Kwon, Taeg Kyu; Chae, Sungwook; Hyun, Jin Won

2016-07-01

Fisetin (3,3',4',7-tetrahydroxyflavone), a dietary flavonoid compound, is currently being investigated for its anticancer effect in various cancer models, including lung cancer. Recent studies show that fisetin induces cell growth inhibition and apoptosis in the human non-small cell lung cancer line NCI-H460. In this study, we investigated whether fisetin can induce endoplasmic reticulum (ER) stress-mediated apoptosis in NCI-H460 cells. Fisetin induced mitochondrial reactive oxygen species (ROS) and characteristic signs of ER stress: ER staining; mitochondrial Ca(2+) overload; expression of ER stress-related proteins; glucose-regulated protein (GRP)-78, phosphorylation of protein kinase RNA (PKR)-like endoplasmic reticulum kinase (PERK) and phosphorylation of eukaryotic initiation factor-2 α subunit; cleavage of activating transcription factor-6; phosphorylation of inositol-requiring kinase-1 and splicing of X-box transcription factor-1; induction of C/EBP homologous protein and cleaved caspase-12. siRNA-mediated knockdown of CHOP and ATF-6 attenuated fisetin-induced apoptotic cell death. In addition, fisetin induced phosphorylation of ERK, JNK, and p38 MAPK. Moreover, silencing of the MAPK signaling pathway prevented apoptotic cell death. In summary, our results indicate that, in NCI-H460 cells, fisetin induces apoptosis and ER stress that is mediated by induction of the MAPK signaling pathway.

Persistent increases in inflammatory cytokines, Akt, and MAPK/ERK pathways after inhalation exposure of rats to Libby amphibole (LA) or amosite: comparison to effects after intratracheal exposure to LA or naturally occurring asbestos.

EPA Science Inventory

Human exposure to LA and other mined or processed asbestos increases risk of lung inflammation, fibrosis, and cancer. Health risks from exposure to naturally occurring asbestos (NOA) are not as well-understood. Mechanisms of long-term toxicity were compared in male F344 rats expo...

Patented natural avocado sugar modulates the HBD-2 and HBD-3 expression in human keratinocytes through toll-like receptor-2 and ERK/MAPK activation.

PubMed

Paoletti, Iole; Buommino, Elisabetta; Fusco, Alessandra; Baudouin, Caroline; Msika, Philippe; Tufano, Maria Antonietta; Baroni, Adone; Donnarumma, Giovanna

2012-10-01

Keratinocytes stimulated by microbial organisms secrete not only a variety of cytokines, chemokines and growth factors, but also antimicrobial peptides such as beta-defensins (HBDs) such as HBD-2 and HBD-3. AV119, a patented blend of avocado sugar, triggers the up-regulation of HBD-2 in skin epithelia upon contact with AV119, but the signalling mechanisms involved are not completely understood. The purpose of this study was to determine if AV119 was able to induce also the expression of HBD-3 in human keratinocytes. In addition, the receptor and intracellular pathways involved in the AV119 up-regulation of HBD-2 and HBD-3 were investigated. Our results demonstrated that AV119 induces a significantly increase of the expression of HBD-3. In addition, the HBD-2 and HBD-3 AV119-induced gene expression and release are TLR-2 dependent. Finally, we demonstrated that AV119 induced ERK/MAPK phosphorylation in human keratinocytes, thus providing evidence that HBD-2 and HBD-3 secretion is through the same transductional pathway. The ability of AV119 to induce also HBD-3 may amplify its therapeutic potential against a broader spectrum of bacterial and yeast strains responsible for human skin disorders.

Fine chalk dust induces inflammatory response via p38 and ERK MAPK pathway in rat lung.

PubMed

Zhang, Yuexia; Yang, Zhenhua; Chen, Yunzhu; Li, Ruijin; Geng, Hong; Dong, Wenjuan; Cai, Zongwei; Dong, Chuan

2018-01-01

Chalk teaching is widely used in the world due to low cost, especially in some developing countries. During teaching with chalks, a large amount of fine chalk dust is produced. Although exposure to chalk dust is associated with respiratory diseases, the mechanism underlying the correlation between chalk dust exposure and adverse effects has not fully been elucidated. In this study, inflammation and its signal pathway in rat lungs exposed to fine chalk dust were examined through histopathology analyses; pro-inflammatory gene transcription; and protein levels measured by HE staining, RT-PCR, and western blot analysis. The results demonstrated that fine chalk dust increased neutrophils and up-regulated inflammatory gene mRNA levels (TNF-α, IL-6, TGF-β1, iNOS, and ICAM-1), and oxidative stress marker (HO-1) level, leading to the increase of inflammatory cell infiltration and inflammatory injury on the lungs. These inflammation responses were mediated, at least in part, via p38 and extracellular regulated proteinase (ERK) mitogen-activated protein kinase (MAPK) signaling mechanisms. In contrast, N-acetyl-L-cysteine (NAC) supplement significantly ameliorated these changes in inflammatory responses. Our results support the hypothesis that fine chalk dust can damage rat lungs and the NAC supplement may attenuate fine chalk dust-associated lung inflammation.

Pathways Impacted by Genomic Alterations in Pulmonary Carcinoid Tumors.

PubMed

Asiedu, Michael K; Thomas, Charles F; Dong, Jie; Schulte, Sandra C; Khadka, Prasidda; Sun, Zhifu; Kosari, Farhad; Jen, Jin; Molina, Julian; Vasmatzis, George; Kuang, Ray; Aubry, Marie Christine; Yang, Ping; Wigle, Dennis A

2018-04-01

Purpose: Pulmonary carcinoid tumors account for up to 5% of all lung malignancies in adults, comprise 30% of all carcinoid malignancies, and are defined histologically as typical carcinoid (TC) and atypical carcinoid (AC) tumors. The role of specific genomic alterations in the pathogenesis of pulmonary carcinoid tumors remains poorly understood. We sought to identify genomic alterations and pathways that are deregulated in these tumors to find novel therapeutic targets for pulmonary carcinoid tumors. Experimental Design: We performed integrated genomic analysis of carcinoid tumors comprising whole genome and exome sequencing, mRNA expression profiling and SNP genotyping of specimens from normal lung, TC and AC, and small cell lung carcinoma (SCLC) to fully represent the lung neuroendocrine tumor spectrum. Results: Analysis of sequencing data found recurrent mutations in cancer genes including ATP1A2, CNNM1, MACF1, RAB38, NF1, RAD51C, TAF1L, EPHB2, POLR3B , and AGFG1 The mutated genes are involved in biological processes including cellular metabolism, cell division cycle, cell death, apoptosis, and immune regulation. The top most significantly mutated genes were TMEM41B, DEFB127, WDYHV1, and TBPL1 Pathway analysis of significantly mutated and cancer driver genes implicated MAPK/ERK and amyloid beta precursor protein (APP) pathways whereas analysis of CNV and gene expression data suggested deregulation of the NF-κB and MAPK/ERK pathways. The mutation signature was predominantly C>T and T>C transitions with a minor contribution of T>G transversions. Conclusions: This study identified mutated genes affecting cancer relevant pathways and biological processes that could provide opportunities for developing targeted therapies for pulmonary carcinoid tumors. Clin Cancer Res; 24(7); 1691-704. ©2018 AACR . ©2018 American Association for Cancer Research.

BRAFV600E negatively regulates the AKT pathway in melanoma cell lines.

PubMed

Chen, Brenden; Tardell, Christine; Higgins, Brian; Packman, Kathryn; Boylan, John F; Niu, Huifeng

2012-01-01

Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167) or mTORC1 inhibitor (rapamycin) induced AKT phosphorylation (pAKT) and downstream signal activation. Treatment-induced pAKT elevation is found in BRAF wild type melanoma cells but not in a subset of melanoma cell lines harboring BRAFV600E. Knock-down of BRAFV600E in these melanoma cells elevates basal pAKT and downstream signals, whereas knock-down of CRAF, MEK1/2 or ERK1/2 or treatment with a BRAF inhibitor have no impact on pAKT. Mechanistically, we show that BRAFV600E interacts with rictor complex (mTORC2) and regulates pAKT through mTORC2. BRAFV600E is identified in mTORC2 after immunoprecipitation of rictor. Knock-down of rictor abrogates BRAFV600E depletion induced pAKT. Knock-down of BRAFV600E enhances cellular enzyme activity of mTORC2. Aberrant activation of AKT pathway by PTEN loss appears to override the negative impact of BRAFV600E on pAKT. Taken together, our findings suggest that in a subset of BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT pathway in a rictor-dependent, MEK/ERK and BRAF kinase-independent manner. Our study reveals a novel molecular mechanism underlying the regulation of feedback loops between the MAPK and AKT pathways.

Effects of TGF-β signaling blockade on human A549 lung adenocarcinoma cell lines.

PubMed

Xu, Cheng-Cheng; Wu, Lei-Ming; Sun, Wei; Zhang, Ni; Chen, Wen-Shu; Fu, Xiang-Ning

2011-01-01

Transforming growth factor β (TGF-β) is overexpressed in a wide variety of cancer types including lung adenocarcinoma (LAC), and the TGF-β signaling pathway plays an important role in tumor development. To determine whether blockade of the TGF-β signaling pathway can inhibit the malignant biological behavior of LAC, RNA interference (RNAi) technology was used to silence the expression of TGF-β receptor, type II (TGFβRII) in the LAC cell line, A549, and its effects on cell proliferation, invasion and metastasis were examined. Three specific small interfering RNAs (siRNAs) designed for targeting human TGFβRII were transfected into A549 cells. The expression of TGFβRII was detected by Western blot analysis. Cell proliferation was measured by MTT and clonogenic assays. Cell apoptosis was assessed by flow cytometry. The invasion and metastasis of A549 cells were investigated using the wound healing and Matrigel invasion assays. The expression of PI3K, phosphorylated Smad2, Smad4, Akt, Erk1/2, P38 and MMPs was detected by Western blot analysis. The TGFβRII siRNA significantly reduced the expression of TGFβRII in A549 cells. The knockdown of TGFβRII in A549 cells resulted in the suppression of cell proliferation, invasion and metastasis and induced cell apoptosis. In addition to the Smad-dependent pathway, independent pathways including the Erk MAPK, PI3K/Akt and p38 MAPK pathways, as well as the expression of MMPs and VEGF, were inhibited. In conclusion, TGF-β signaling is required for LAC progression. Therefore, the blockade of this signaling pathway by the down-regulation of TGFβRII using SiRNA may provide a potential gene therapy for LAC.

Fisetin regulates TPA-induced breast cell invasion by suppressing matrix metalloproteinase-9 activation via the PKC/ROS/MAPK pathways.

PubMed

Noh, Eun-Mi; Park, Yeon-Ju; Kim, Jeong-Mi; Kim, Mi-Seong; Kim, Ha-Rim; Song, Hyun-Kyung; Hong, On-Yu; So, Hong-Seob; Yang, Sei-Hoon; Kim, Jong-Suk; Park, Samg Hyun; Youn, Hyun-Jo; You, Yong-Ouk; Choi, Ki-Bang; Kwon, Kang-Beom; Lee, Young-Rae

2015-10-05

Invasion and metastasis are among the main causes of death in patients with malignant tumors. Fisetin (3,3',4',7-tetrahydroxyflavone), a natural flavonoid found in the smoke tree (Cotinus coggygria), is known to have antimetastatic effects on prostate and lung cancers; however, the effect of fisetin on breast cancer metastasis is unknown. The aim of this study was to determine the anti-invasive activity of fisetin in human breast cancer cells. Matrix metalloproteinase (MMP)-9 is a major component facilitating the invasion of many cancer tumor cell types, and thus the inhibitory effect of fisetin on MMP-9 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated human breast cancer cells was investigated in this study. Fisetin significantly attenuated TPA-induced cell invasion in MCF-7 human breast cancer cells, and was found to inhibit the activation of the PKCα/ROS/ERK1/2 and p38 MAPK signaling pathways. This effect was furthermore associated with reduced NF-κB activation, suggesting that the anti-invasive effect of fisetin on MCF-7 cells may result from inhibited TPA activation of NF-κB and reduced TPA activation of PKCα/ROS/ERK1/2 and p38 MAPK signals, ultimately leading to the downregulation of MMP-9 expression. Our findings indicate the role of fisetin in MCF-7 cell invasion, and clarify the underlying molecular mechanisms of this role, suggesting fisetin as a potential chemopreventive agent for breast cancer metastasis. Copyright © 2015 Elsevier B.V. All rights reserved.

Cinnamophilin isolated from Cinnamomum philippinense protects against collagen degradation in human chondrocytes.

PubMed

Lu, Yung-Chang; Hsiao, George; Lin, Kuan-Hung; Hsieh, Ming-Shium; Jayakumar, Thanasekaran; Wu, Tian-Shung; Sheu, Joen-Rong

2013-06-01

To investigate the therapeutic potential of naturally occurring cinnamophilin against cartilage degradation and its action mechanisms, its effects on matrix metalloproteinase (MMP)-1 and -13 induction were examined in the human SW1353 chondrocytic cell line. Human chondrocytes (SW1353) were stimulated with interleukin (IL)-1β, and then mitogen-activated protein kinase (MAPK) and c-Jun activations, inhibitory κB-α (IκB-α) degradation, and MMP-1, and 13 expressions were assayed by a Western blot analysis. Cinnamophilin strongly inhibited MMP-1 and -13 induction in IL-1β-treated (30 ng/mL) SW1353 cells in a concentration-dependent manner, and it also reduced MAPK family members including extracellular signal-regulated kinase (ERK), p38 MAPKs, and c-Jun N-terminal kinase. Moreover, nuclear factor (NF)-κB signaling activation through IκB-α degradation, IκB kinase (IKK)-α/β, and p-65 phosphorylation was restored by cinnamophilin upon IL-1β stimulation. Importantly, results showed that IL-1β-induced activation of phosphorylated (p)-c-Jun in chondrocytes was significantly inhibited by cinnamophilin. These results indicate that cinnamophilin inhibited MMP-1 and -13 expressions in IL-1β-treated chondrocytes through either NF-κB or ERK/p38 MAPK downregulation and/or suppressing p-c-Jun pathways. Furthermore, these findings suggest that cinnamophilin may have the potential for chondroprotection against collagen matrix breakdown in cartilage of diseased tissues such as those found in arthritic disorders. Copyright © 2012 John Wiley & Sons, Ltd.

Rat mesothelioma cell proliferation requires p38δ mitogen activated protein kinase and C/EBP-α.

PubMed

Zhong, Jun; Lardinois, Didier; Szilard, John; Tamm, Michael; Roth, Michael

2011-08-01

Pleural malignant mesothelioma is a rare but deadly tumour mainly induced by asbestos inhalation. Despite the ban of asbestos in 1990 in 52 countries, mesothelioma cases still increase worldwide. In pleural mesothelioma, p38 mitogen activated protein kinases (MAPK) have been suggested to play a major role in carcinogenesis and aggressiveness of tumours. The aim of this study was to determine the role of the different four p38 MAPK isoforms and their effect on proliferation together with the underlying signalling pathways in a rat pleural mesothelioma cell line. Rat pleural mesothelioma cells were stimulated with platelet-derived growth factor (PDGF)-BB and/or transforming growth factor beta (TGF)-β. MAPK and transcription factor expression and activation was monitored in the cytosol and nucleus by immuno-blotting. Proliferation was determined by manual cell count and siRNAs were used to control MAPK and transcription factor expression and action. Only PDGF-BB, but not TGF-β1 induced proliferation via activated Erk1/2 and p38 MAPK. The p38α and δ isoforms were expressed in the cytosol, and upon activation p38δ translocated into the nucleus, while p38α remained in the cytosol. No other p38 isoform was expressed by rat mesothelioma cells. C/EBP-α was found in both the cytosol and the nucleus, while C/EBP-β was not expressed at all. PDGF-BB induced proliferation was suppressed by down-regulation of either Erk1/2, or p38δ MAPK, or C/EBP-α. Furthermore, TGF-β inhibited PDGF-BB induced proliferation by interruption of p38 MAPK signalling. From this rat model, we conclude that in pleural mesothelioma, p38δ in C/EBP-α mediate proliferation and thus may represent new targets in mesothelioma therapy. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

Titanium dioxide nanoparticles stimulate sea urchin immune cell phagocytic activity involving TLR/p38 MAPK-mediated signalling pathway

PubMed Central

Pinsino, Annalisa; Russo, Roberta; Bonaventura, Rosa; Brunelli, Andrea; Marcomini, Antonio; Matranga, Valeria

2015-01-01

Titanium dioxide nanoparticles (TiO2NPs) are one of the most widespread-engineered particles in use for drug delivery, cosmetics, and electronics. However, TiO2NP safety is still an open issue, even for ethical reasons. In this work, we investigated the sea urchin Paracentrotus lividus immune cell model as a proxy to humans, to elucidate a potential pathway that can be involved in the persistent TiO2NP-immune cell interaction in vivo. Morphology, phagocytic ability, changes in activation/inactivation of a few mitogen-activated protein kinases (p38 MAPK, ERK), variations of other key proteins triggering immune response (Toll-like receptor 4-like, Heat shock protein 70, Interleukin-6) and modifications in the expression of related immune response genes were investigated. Our findings indicate that TiO2NPs influence the signal transduction downstream targets of p38 MAPK without eliciting an inflammatory response or other harmful effects on biological functions. We strongly recommend sea urchin immune cells as a new powerful model for nano-safety/nano-toxicity investigations without the ethical normative issue. PMID:26412401

Uridine 5′-Triphosphate Promotes In Vitro Schwannoma Cell Migration through Matrix Metalloproteinase-2 Activation

PubMed Central

Martiañez, Tania; Segura, Mònica; Figueiro-Silva, Joana; Grijota-Martinez, Carmen; Trullas, Ramón; Casals, Núria

2014-01-01

In response to peripheral nerve injury, Schwann cells adopt a migratory phenotype and modify the extracellular matrix to make it permissive for cell migration and axonal re-growth. Uridine 5′-triphosphate (UTP) and other nucleotides are released during nerve injury and activate purinergic receptors expressed on the Schwann cell surface, but little is known about the involvement of purine signalling in wound healing. We studied the effect of UTP on Schwannoma cell migration and wound closure and the intracellular signaling pathways involved. We found that UTP treatment induced Schwannoma cell migration through activation of P2Y2 receptors and through the increase of extracellular matrix metalloproteinase-2 (MMP-2) activation and expression. Knockdown P2Y2 receptor or MMP-2 expression greatly reduced wound closure and MMP-2 activation induced by UTP. MMP-2 activation evoked by injury or UTP was also mediated by phosphorylation of all 3 major mitogen-activated protein kinases (MAPKs): JNK, ERK1/2, and p38. Inhibition of these MAPK pathways decreased both MMP-2 activation and cell migration. Interestingly, MAPK phosphorylation evoked by UTP exhibited a biphasic pattern, with an early transient phosphorylation 5 min after treatment, and a late and sustained phosphorylation that appeared at 6 h and lasted up to 24 h. Inhibition of MMP-2 activity selectively blocked the late, but not the transient, phase of MAPK activation. These results suggest that MMP-2 activation and late MAPK phosphorylation are part of a positive feedback mechanism to maintain the migratory phenotype for wound healing. In conclusion, our findings show that treatment with UTP stimulates in vitro Schwannoma cell migration and wound repair through a MMP-2-dependent mechanism via P2Y2 receptors and MAPK pathway activation. PMID:24905332

CFTR-regulated MAPK/NF-κB signaling in pulmonary inflammation in thermal inhalation injury.

PubMed

Dong, Zhi Wei; Chen, Jing; Ruan, Ye Chun; Zhou, Tao; Chen, Yu; Chen, YaJie; Tsang, Lai Ling; Chan, Hsiao Chang; Peng, Yi Zhi

2015-10-30

The mechanism underlying pulmonary inflammation in thermal inhalation injury remains elusive. Cystic fibrosis, also hallmarked with pulmonary inflammation, is caused by mutations in CFTR, the expression of which is temperature-sensitive. We investigated whether CFTR is involved in heat-induced pulmonary inflammation. We applied heat-treatment in 16HBE14o- cells with CFTR knockdown or overexpression and heat-inhalation in rats in vivo. Heat-treatment caused significant reduction in CFTR and, reciprocally, increase in COX-2 at early stages both in vitro and in vivo. Activation of ERK/JNK, NF-κB and COX-2/PGE2 were detected in heat-treated cells, which were mimicked by knockdown, and reversed by overexpression of CFTR or VX-809, a reported CFTR mutation corrector. JNK/ERK inhibition reversed heat-/CFTR-knockdown-induced NF-κB activation, whereas NF-κB inhibitor showed no effect on JNK/ERK. IL-8 was augmented by heat-treatment or CFTR-knockdown, which was abolished by inhibition of NF-κB, JNK/ERK or COX-2. Moreover, in vitro or in vivo treatment with curcumin, a natural phenolic compound, significantly enhanced CFTR expression and reversed the heat-induced increases in COX-2/PGE2/IL-8, neutrophil infiltration and tissue damage in the airway. These results have revealed a CFTR-regulated MAPK/NF-κB pathway leading to COX-2/PGE2/IL-8 activation in thermal inhalation injury, and demonstrated therapeutic potential of curcumin for alleviating heat-induced pulmonary inflammation.

Arctic ground squirrel (Spermophilus parryii) hippocampal neurons tolerate prolonged oxygen– glucose deprivation and maintain baseline ERK1/2 and JNK activation despite drastic ATP loss

PubMed Central

Christian, Sherri L; Ross, Austin P; Zhao, Huiwen W; Kristenson, Heidi J; Zhan, Xinhua; Rasley, Brian T; Bickler, Philip E; Drew, Kelly L

2009-01-01

Oxygen–glucose deprivation (OGD) initiates a cascade of intracellular responses that culminates in cell death in sensitive species. Neurons from Arctic ground squirrels (AGS), a hibernating species, tolerate OGD in vitro and global ischemia in vivo independent of temperature or torpor. Regulation of energy stores and activation of mitogen-activated protein kinase (MAPK) signaling pathways can regulate neuronal survival. We used acute hippocampal slices to investigate the role of ATP stores and extracellular signal-regulated kinase (ERK)1/2 and Jun NH2-terminal kinase (JNK) MAPKs in promoting survival. Acute hippocampal slices from AGS tolerated 30 mins of OGD and showed a small but significant increase in cell death with 2 h OGD at 37°C. This tolerance is independent of hibernation state or season. Neurons from AGS survive OGD despite rapid ATP depletion by 3 mins in interbout euthermic AGS and 10 mins in hibernating AGS. Oxygen–glucose deprivation does not induce JNK activation in AGS and baseline ERK1/2 and JNK activation is maintained even after drastic depletion of ATP. Surprisingly, inhibition of ERK1/2 or JNK during OGD had no effect on survival, whereas inhibition of JNK increased cell death during normoxia. Thus, protective mechanisms promoting tolerance to OGD by AGS are downstream from ATP loss and are independent of hibernation state or season. PMID:18398417

Apigenin Reduces Survival of Choriocarcinoma Cells by Inducing Apoptosis via the PI3K/AKT and ERK1/2 MAPK Pathways.

PubMed

Lim, Whasun; Park, Sunwoo; Bazer, Fuller W; Song, Gwonhwa

2016-12-01

Apigenin is a flavonoid found in parsley, onions, oranges, tea, chamomile, wheat, and sprouts. It has a variety of biological properties including anti-oxidant, anti-mutagenic, anti-carcinogenic, anti-inflammatory, anti-proliferative, and anti-spasmodic effects. Based on epidemiological and case-control studies, apigenin is regarded as a novel chemotherapeutic agent against various cancer types. However, little is known about the effects of apigenin on choriocarcinoma cells. Therefore, we investigated the anti-cancer effects of apigenin on choriocarcinoma cells (JAR and JEG3) in the present study. Apigenin reduced viability and migratory properties, increased apoptosis, and suppressed mitochondrial membrane potential in both the JAR and JEG3 cells. In addition, apigenin predominantly decreased phosphorylation of AKT, P70RSK, and S6 whereas the phosphorylation of ERK1/2 and P90RSK was increased by apigenin treatment of JAR and JEG3 cells in a dose-dependent manner. Moreover, treatment of JAR and JEG3 cells with both apigenin and pharmacological inhibitors of PI3K/AKT (LY294002) and ERK1/2 (U0126) revealed synergistic anti-proliferative effects. Collectively, these results indicated that the apigenin is an invaluable chemopreventive agent that inhibits progression and metastasis of choriocarcinoma cells through regulation of PI3K/AKT and ERK1/2 MAPK signal transduction mechanism. J. Cell. Physiol. 231: 2690-2699, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

ERK5 signaling gets XIAPed: a role for ubiquitin in the disassembly of a MAPK cascade

PubMed Central

Klein, Aileen M; Cobb, Melanie H

2014-01-01

Mitogen-activated protein kinase (MAPK) cascades are tightly controlled through a series of well-characterized phospho-regulatory events. In this issue, Takeda et al (2014) identify the inhibitor of apoptosis protein, XIAP, as a key regulator of ERK5 activation via uncoupling of upstream kinase activity by non-degradative ubiquitination. PMID:25012518

Patient-derived iPSCs show premature neural differentiation and neuron-type specific phenotypes relevant to neurodevelopment

PubMed Central

Yeh, Erika; Dao, Dang Q.; Wu, Zhi Y.; Kandalam, Santoshi M.; Camacho, Federico M.; Tom, Curtis; Zhang, Wandong; Krencik, Robert; Rauen, Katherine A.; Ullian, Erik M.; Weiss, Lauren A.

2017-01-01

Ras/MAPK pathway signaling is a major participant in neurodevelopment, and evidence suggests that BRAF, a key Ras signal mediator, influences human behavior. We studied the role of the mutation BRAFQ257R, the most common cause of cardiofaciocutaneous syndrome (CFC), in an induced pluripotent stem cell (iPSC)-derived model of human neurodevelopment. In iPSC-derived neuronal cultures from CFC subjects, we observed decreased p-AKT and p-ERK1/2 compared to controls, as well as a depleted neural progenitor pool and rapid neuronal maturation. Pharmacological PI3K/AKT pathway manipulation recapitulated cellular phenotypes in control cells and attenuated them in CFC cells. CFC cultures displayed altered cellular subtype ratios and increased intrinsic excitability. Moreover, in CFC cells, Ras/MAPK pathway activation and morphological abnormalities exhibited cell subtype-specific differences. Our results highlight the importance of exploring specific cellular subtypes and of using iPSC models to reveal relevant human-specific neurodevelopmental events. PMID:29158583

Fluoxetine signature on hippocampal MAPK signalling in sex-dependent manner.

PubMed

Mitic, Milos; Lukic, Iva; Bozovic, Natalija; Djordjevic, Jelena; Adzic, Miroslav

2015-02-01

A growing body of evidence indicates that mitogen-activated protein kinase (MAPK) participates in various stress-induced responses and is considered to be one of the pathophysiological mechanisms in depression. Surprisingly, the effect of antidepressants on MAPKs is almost unexplored, particularly from the perspective of sexes. The present study investigates the cytoplasm-nuclear distribution of MAPK family, c-Jun N-terminal kinases (JNKs) 1, 2 and 3; extracellular signal-regulated kinases (ERKs) 1 and 2; and p38 kinases, as well as their phosphoisoforms in the hippocampus of chronically stressed female and male rats and upon chronic fluoxetine treatment. Additionally, we analysed crosstalk between MAPK signalling and depressive-like behaviour which correlated with brain-derived neurotrophic factor (BDNF) expression. Our results emphasize a gender-specific and compartment-dependent response of MAPKs to stress and fluoxetine. In females, stress decreased pp38 and pJNK and induced cytosolic retention of pERKs which reduced all nuclear pMAPKs. These changes correlated with altered BDNF expression and behaviour. Similarly, in males, stress decreased pp38 but promoted nuclear translocation of pJNKs and pERKs. These stress alterations of pMAPKs in males were not associated with BDNF expression and depressive-like behaviour. Fluoxetine treatment in stressed females upregulated whole pMAPK signalling particularly those in nucleus which was followed with BDNF expression and normalization of behaviour. In stressed males, fluoxetine affected only cytosolic pJNKs, while nuclear pMAPK signalling and BDNF expression were unaffected even though fluoxetine normalized behaviour. Overall, our results suggest existence of gender-specific mechanism of fluoxetine on nuclear pMAPK/BDNF signalling and depressive-like behaviour and reinforce the antidepressant dogma that females and males respond differently to certain antidepressants.

Genome-wide genetic analyses highlight mitogen-activated protein kinase (MAPK) signaling in the pathogenesis of endometriosis.

PubMed

Uimari, Outi; Rahmioglu, Nilufer; Nyholt, Dale R; Vincent, Katy; Missmer, Stacey A; Becker, Christian; Morris, Andrew P; Montgomery, Grant W; Zondervan, Krina T

2017-04-01

Do genome-wide association study (GWAS) data for endometriosis provide insight into novel biological pathways associated with its pathogenesis? GWAS analysis uncovered multiple pathways that are statistically enriched for genetic association signals, analysis of Stage A disease highlighted a novel variant in MAP3K4, while top pathways significantly associated with all endometriosis and Stage A disease included several mitogen-activated protein kinase (MAPK)-related pathways. Endometriosis is a complex disease with an estimated heritability of 50%. To date, GWAS revealed 10 genomic regions associated with endometriosis, explaining <4% of heritability, while half of the heritability is estimated to be due to common risk variants. Pathway analyses combine the evidence of single variants into gene-based measures, leveraging the aggregate effect of variants in genes and uncovering biological pathways involved in disease pathogenesis. Pathway analysis was conducted utilizing the International Endogene Consortium GWAS data, comprising 3194 surgically confirmed endometriosis cases and 7060 controls of European ancestry with genotype data imputed up to 1000 Genomes Phase three reference panel. GWAS was performed for all endometriosis cases and for Stage A (revised American Fertility Society (rAFS) I/II, n = 1686) and B (rAFS III/IV, n = 1364) cases separately. The identified significant pathways were compared with pathways previously investigated in the literature through candidate association studies. The most comprehensive biological pathway databases, MSigDB (including BioCarta, KEGG, PID, SA, SIG, ST and GO) and PANTHER were utilized to test for enrichment of genetic variants associated with endometriosis. Statistical enrichment analysis was performed using the MAGENTA (Meta-Analysis Gene-set Enrichment of variaNT Associations) software. The first genome-wide association analysis for Stage A endometriosis revealed a novel locus, rs144240142 (P = 6.45 × 10-8, OR = 1.71, 95% CI = 1.23-2.37), an intronic single-nucleotide polymorphism (SNP) within MAP3K4. This SNP was not associated with Stage B disease (P = 0.086). MAP3K4 was also shown to be differentially expressed in eutopic endometrium between Stage A endometriosis cases and controls (P = 3.8 × 10-4), but not with Stage B disease (P = 0.26). A total of 14 pathways enriched with genetic endometriosis associations were identified (false discovery rate (FDR)-P < 0.05). The pathways associated with any endometriosis were Grb2-Sos provides linkage to MAPK signaling for integrins pathway (P = 2.8 × 10-5, FDR-P = 3.0 × 10-3), Wnt signaling (P = 0.026, FDR-P = 0.026) and p130Cas linkage to MAPK signaling for integrins pathway (P = 6.0 × 10-4, FDR-P = 0.029); with Stage A endometriosis: extracellular signal-regulated kinase (ERK)1 ERK2 MAPK (P = 5.0 × 10-4, FDR-P = 5.0 × 10-4) and with Stage B endometriosis: two overlapping pathways that related to extracellular matrix biology-Core matrisome (P = 1.4 × 10-3, FDR-P = 0.013) and ECM glycoproteins (P = 1.8 × 10-3, FDR-P = 7.1 × 10-3). Genes arising from endometriosis candidate gene studies performed to date were enriched for Interleukin signaling pathway (P = 2.3 × 10-12), Apoptosis signaling pathway (P = 9.7 × 10-9) and Gonadotropin releasing hormone receptor pathway (P = 1.2 × 10-6); however, these pathways did not feature in the results based on GWAS data. Not applicable. The analysis is restricted to (i) variants in/near genes that can be assigned to pathways, excluding intergenic variants; (ii) the gene-based pathway definition as registered in the databases; (iii) women of European ancestry. The top ranked pathways associated with overall and Stage A endometriosis in particular involve integrin-mediated MAPK activation and intracellular ERK/MAPK acting downstream in the MAPK cascade, both acting in the control of cell division, gene expression, cell movement and survival. Other top enriched pathways in Stage B disease include ECM glycoprotein pathways important for extracellular structure and biochemical support. The results highlight the need for increased efforts to understand the functional role of these pathways in endometriosis pathogenesis, including the investigation of the biological effects of the genetic variants on downstream molecular processes in tissue relevant to endometriosis. Additionally, our results offer further support for the hypothesis of at least partially distinct causal pathophysiology for minimal/mild (rAFS I/II) vs. moderate/severe (rAFS III/IV) endometriosis. The genome-wide association data and Wellcome Trust Case Control Consortium (WTCCC) were generated through funding from the Wellcome Trust (WT084766/Z/08/Z, 076113 and 085475) and the National Health and Medical Research Council (NHMRC) of Australia (241944, 339462, 389927, 389875, 389891, 389892, 389938, 443036, 442915, 442981, 496610, 496739, 552485 and 552498). N.R. was funded by a grant from the Medical Research Council UK (MR/K011480/1). A.P.M. is a Wellcome Trust Senior Fellow in Basic Biomedical Science (grant WT098017). All authors declare there are no conflicts of interest. © The Author 2017. Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology.

Genome-wide genetic analyses highlight mitogen-activated protein kinase (MAPK) signaling in the pathogenesis of endometriosis

PubMed Central

Uimari, Outi; Rahmioglu, Nilufer; Nyholt, Dale R.; Vincent, Katy; Missmer, Stacey A.; Becker, Christian; Morris, Andrew P.; Montgomery, Grant W.

2017-01-01

Abstract STUDY QUESTION Do genome-wide association study (GWAS) data for endometriosis provide insight into novel biological pathways associated with its pathogenesis? SUMMARY ANSWER GWAS analysis uncovered multiple pathways that are statistically enriched for genetic association signals, analysis of Stage A disease highlighted a novel variant in MAP3K4, while top pathways significantly associated with all endometriosis and Stage A disease included several mitogen-activated protein kinase (MAPK)-related pathways. WHAT IS KNOWN ALREADY Endometriosis is a complex disease with an estimated heritability of 50%. To date, GWAS revealed 10 genomic regions associated with endometriosis, explaining <4% of heritability, while half of the heritability is estimated to be due to common risk variants. Pathway analyses combine the evidence of single variants into gene-based measures, leveraging the aggregate effect of variants in genes and uncovering biological pathways involved in disease pathogenesis. STUDY DESIGN, SIZE, DURATION Pathway analysis was conducted utilizing the International Endogene Consortium GWAS data, comprising 3194 surgically confirmed endometriosis cases and 7060 controls of European ancestry with genotype data imputed up to 1000 Genomes Phase three reference panel. GWAS was performed for all endometriosis cases and for Stage A (revised American Fertility Society (rAFS) I/II, n = 1686) and B (rAFS III/IV, n = 1364) cases separately. The identified significant pathways were compared with pathways previously investigated in the literature through candidate association studies. PARTICIPANTS/MATERIALS, SETTING, METHODS The most comprehensive biological pathway databases, MSigDB (including BioCarta, KEGG, PID, SA, SIG, ST and GO) and PANTHER were utilized to test for enrichment of genetic variants associated with endometriosis. Statistical enrichment analysis was performed using the MAGENTA (Meta-Analysis Gene-set Enrichment of variaNT Associations) software. MAIN RESULTS AND THE ROLE OF CHANCE The first genome-wide association analysis for Stage A endometriosis revealed a novel locus, rs144240142 (P = 6.45 × 10−8, OR = 1.71, 95% CI = 1.23–2.37), an intronic single-nucleotide polymorphism (SNP) within MAP3K4. This SNP was not associated with Stage B disease (P = 0.086). MAP3K4 was also shown to be differentially expressed in eutopic endometrium between Stage A endometriosis cases and controls (P = 3.8 × 10−4), but not with Stage B disease (P = 0.26). A total of 14 pathways enriched with genetic endometriosis associations were identified (false discovery rate (FDR)-P < 0.05). The pathways associated with any endometriosis were Grb2-Sos provides linkage to MAPK signaling for integrins pathway (P = 2.8 × 10−5, FDR-P = 3.0 × 10−3), Wnt signaling (P = 0.026, FDR-P = 0.026) and p130Cas linkage to MAPK signaling for integrins pathway (P = 6.0 × 10−4, FDR-P = 0.029); with Stage A endometriosis: extracellular signal-regulated kinase (ERK)1 ERK2 MAPK (P = 5.0 × 10−4, FDR-P = 5.0 × 10−4) and with Stage B endometriosis: two overlapping pathways that related to extracellular matrix biology—Core matrisome (P = 1.4 × 10−3, FDR-P = 0.013) and ECM glycoproteins (P = 1.8 × 10−3, FDR-P = 7.1 × 10−3). Genes arising from endometriosis candidate gene studies performed to date were enriched for Interleukin signaling pathway (P = 2.3 × 10−12), Apoptosis signaling pathway (P = 9.7 × 10−9) and Gonadotropin releasing hormone receptor pathway (P = 1.2 × 10−6); however, these pathways did not feature in the results based on GWAS data. LARGE SCALE DATA Not applicable. LIMITATIONS, REASONS FOR CAUTION The analysis is restricted to (i) variants in/near genes that can be assigned to pathways, excluding intergenic variants; (ii) the gene-based pathway definition as registered in the databases; (iii) women of European ancestry. WIDER IMPLICATIONS OF THE FINDINGS The top ranked pathways associated with overall and Stage A endometriosis in particular involve integrin-mediated MAPK activation and intracellular ERK/MAPK acting downstream in the MAPK cascade, both acting in the control of cell division, gene expression, cell movement and survival. Other top enriched pathways in Stage B disease include ECM glycoprotein pathways important for extracellular structure and biochemical support. The results highlight the need for increased efforts to understand the functional role of these pathways in endometriosis pathogenesis, including the investigation of the biological effects of the genetic variants on downstream molecular processes in tissue relevant to endometriosis. Additionally, our results offer further support for the hypothesis of at least partially distinct causal pathophysiology for minimal/mild (rAFS I/II) vs. moderate/severe (rAFS III/IV) endometriosis. STUDY FUNDING/COMPETING INTEREST(S) The genome-wide association data and Wellcome Trust Case Control Consortium (WTCCC) were generated through funding from the Wellcome Trust (WT084766/Z/08/Z, 076113 and 085475) and the National Health and Medical Research Council (NHMRC) of Australia (241944, 339462, 389927, 389875, 389891, 389892, 389938, 443036, 442915, 442981, 496610, 496739, 552485 and 552498). N.R. was funded by a grant from the Medical Research Council UK (MR/K011480/1). A.P.M. is a Wellcome Trust Senior Fellow in Basic Biomedical Science (grant WT098017). All authors declare there are no conflicts of interest. PMID:28333195

Betalactam antibiotics affect human dendritic cells maturation through MAPK/NF-kB systems. Role in allergic reactions to drugs

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lopez, Soledad; Department of Medical Biochemistry, Molecular Biology and Immunology, The University of Seville Medical School, Seville; Gomez, Enrique

The mechanisms leading to drug allergy in predisposed patients, especially those related to T-cell-mediated drug hypersensitivity, are not well understood. A key event in allergic reactions to drugs is the maturation process undergone by dendritic cells (DCs). Although amoxicillin (AX) has been reported to interact and maturate DCs from patients with AX-induced delayed-type hypersensitivity, the cell signaling pathways related to AX-mediated DC maturation have not been elucidated. We sought to determine the role of the MAPK and NF-κΒ pathways on AX-induced DC maturation and functional status. For that purpose, in monocyte-derived-DCs from AX-delayed allergic patients and tolerant subjects, we analyzedmore » the activation pattern of p38MAPK, JNK, and ERK signaling and the NF-κB, maturation markers as well as endocytosis and allostimulatory capacities driven by AX-stimulated-DCs. Our data reveal that AX induces an increase in the phosphorylation levels of the three MAPKsand activated NF-κB in DCs from allergic patients. Moreover, the inhibition of these pathways prevents the up-regulation of surface molecules induced by AX. Additionally, we observed that the allostimulatory capacity and the endocytosis down-regulation in AX-stimulated-DCs from allergic patients depend on JNK and NF-κB activities. Taken together, our data shed light for the first time on the main signaling pathways involved in DC maturation from AX-delayed allergic patient. - Highlights: • The cell signaling pathways related to drug-mediated DC maturation were tested. • Amoxicillin induces activation of MAPK and NF-κB in DCs from allergic patients. • The inhibition of these pathways prevents the up-regulation of DC surface molecules. • Their allostimulatory and endocytosis capacities depend on JNK and NF-κB activities. • The low involvement of p38-MAPK could be the cause of an incomplete DC maturation.« less

DJ-1/PARK7, But Not Its L166P Mutant Linked to Autosomal Recessive Parkinsonism, Modulates the Transcriptional Activity of the Orphan Nuclear Receptor Nurr1 In Vitro and In Vivo.

PubMed

Lu, Lingling; Zhao, Shasha; Gao, Ge; Sun, Xiaohong; Zhao, Huanying; Yang, Hui

2016-12-01

Although mutations of DJ-1 have been linked to autosomal recessive Parkinsonism for years, its physiological function and the pathological mechanism of its mutants are not well understood. We report for the first time that exogenous application of DJ-1, but not its L166P mutant, enhances the nuclear translocation and the transcriptional activity of Nurr1, a transcription factor essential for dopaminergic neuron development and maturation, both in vitro and in vivo. Knockdown of DJ-1 attenuates Nurr1 activity. Further investigation showed that signaling of Raf/MEK/ERK MAPKs is involved in this regulatory process and that activation induced by exogenous DJ-1 is antagonized by U0126, an ERK pathway inhibitor, indicating that DJ-1 modulates Nurr1 activity via the Raf/MEK/ERK pathway. Our findings shed light on the novel function of DJ-1 to enhance Nurr1 activity and provide the first insight into the molecular mechanism by which DJ-1 enhances Nurr1 activity.

Anti-oxidizing effect of the dichloromethane and hexane fractions from Orostachys japonicus in LPS-stimulated RAW 264.7 cells via upregulation of Nrf2 expression and activation of MAPK signaling pathway.

PubMed

Lee, Hyeong-Seon; Lee, Gyeong-Seon; Kim, Seon-Hee; Kim, Hyun-Kyung; Suk, Dong-Hee; Lee, Dong-Seok

2014-02-01

Orostachys japonicus shows various biological activities. However, the molecular mechanisms remain unknown in LPS-stimulated macrophages. Here, we investigated the anti-oxidizing effect of the dichloromethane (DCM) and hexane fractions from O. japonicus (OJD and OJH) against oxidative stress in RAW 264.7 cells stimulated by LPS. OJD and OJH significantly increased the expression of heme oxygenase-1 (HO-1) in a dose- and time-dependent manner. Additionally, it was found that the expression of HO-1 was stimulated by Nrf2 activated via degradation of Keap1. ERK and p38 inhibitors repressed HO-1 induced by OJD and OJH in LPS-stimulated cells, respectively. In conclusion, these results suggest that OJD and OJH may block oxidative damage stimulated by LPS, via increasing the expression of HO-1 and Nrf2, and MAPK signaling pathway.

MEK-1 Activates C-Raf Through a Ras-Independent Mechanism

PubMed Central

Leicht, Deborah T.; Balan, Vitaly; Zhu, Jun; Kaplun, Alexander; Bronisz, Agnieszka; Rana, Ajay; Tzivion, Guri

2013-01-01

C-Raf is a member of the Ras-Raf-MEK-ERK mitogen-activated protein kinase (MAPK) signaling pathway that plays key roles in diverse physiological processes and is upregulated in many human cancers. C-Raf activation involves binding to Ras, increased phosphorylation and interactions with co-factors. Here, we describe a Ras-independent in vivo pathway for C-Raf activation by its downstream target MEK. Using 32P-metabolic labeling and 2D-phosphopeptide mapping experiments, we show that MEK increases C-Raf phosphorylation by up-to 10-fold. This increase was associated with C-Raf kinase activation, matching the activity seen with growth factor stimulation. Consequently, coexpression of wildtype C-Raf and MEK was sufficient for full and constitutive activation of ERK. Notably, the ability of MEK to activate C-Raf was completely Ras independent, since mutants impaired in Ras binding that are irresponsive to growth factors or Ras were fully activated by MEK. The ability of MEK to activate C-Raf was only partially dependent on MEK kinase activity but required MEK binding to C-Raf, suggesting that the binding results in a conformational change that increases C-Raf susceptibility to phosphorylation and activation or in the stabilization of the phosphorylated-active form. These findings propose a novel Ras-independent mechanism for activating C-Raf and the MAPK pathway without the need for mutations in the pathway. This mechanism could be of significance in pathological conditions or cancers overexpressing C-Raf and MEK or in conditions where C-Raf-MEK interaction is enhanced due to the downregulation of RKIP and MST2. PMID:23360980

1,25-Dihydroxyvitamin D3 attenuates endotoxin-induced production of inflammatory mediators by inhibiting MAPK activation in primary cortical neuron-glia cultures.

PubMed

Huang, Ya-Ni; Ho, Yi-Jung; Lai, Chien-Cheng; Chiu, Chien-Tsai; Wang, Jia-Yi

2015-08-12

Neuroinflammation occurs in insulted regions of the brain and may be due to reactive oxygen species (ROS), nitric oxide (NO), cytokines, and chemokines produced by activated glia. Excessive production of neurotoxic molecules causes further neuronal damage. Low levels of vitamin D3 are a risk factor for various brain diseases. Using the bacterial endotoxin, lipopolysaccharide (LPS), to induce neuroinflammation in primary cortical neuron-glia cultures, we investigated how 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) affected neuroinflammation. LPS (100 ng/ml) induced the accumulation of nitrite and the production of ROS, interleukin (IL)-6, and macrophage inflammatory protein (MIP)-2 in time-dependent manners. Inhibition of p38 and extracellular signal-regulated kinase (ERK) but not c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase (MAPK) by 20 μM of SB203580, PD98059, and SP600125, significantly reduced LPS-induced ROS production, NO accumulation, and inducible NO synthase (iNOS) expression, respectively. LPS-induced IL-6 and MIP-2 were significantly attenuated by inhibition of p38, ERK, and JNK MAPK. Cotreatment with 1,25(OH)2D3 attenuated LPS-induced ROS production, NO accumulation, and iNOS expression in concentration-dependent manners. 1,25(OH)2D3 also reduced LPS-induced production of IL-6 and MIP-2. Similarly, iNOS, IL-6, and MIP-2 mRNA expression in cells treated with LPS significantly increased, whereas this effect was attenuated by 1,25(OH)2D3. Moreover, LPS-induced phosphorylation of p38, ERK, and JNK MAPK was significantly inhibited by 1,25(OH)2D3. Our findings indicate that 1,25(OH)2D3 reduced the LPS-stimulated production of inflammatory molecules in neuron-glia cultures by inhibiting MAPK pathways and the production of downstream inflammatory molecules. We suggest that 1,25(OH)2D3 can be used to alleviate neuroinflammation in various brain injuries.

Involvement of IGF-1 and MEOX2 in PI3K/Akt1/2 and ERK1/2 pathways mediated proliferation and differentiation of perivascular adipocytes.

PubMed

Liu, Ping; Kong, Feng; Wang, Jue; Lu, Qinghua; Xu, Haijia; Qi, Tonggang; Meng, Juan

2015-02-01

Perivascular adipocyte (PVAC) proliferation and differentiation were closely involved in cardiovascular disease. We aimed to investigate whether phosphatidylinositol 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signaling pathways enhance PVAC functions activated by insulin-like growth factor 1(IGF-1) and suppressed by mesenchyme homeobox 2 (MEOX2). In this study, PVACs from primary culture were cultured and induced to differentiate. Cell viability assays demonstrated that IGF-1 promoted PVAC proliferation and differentiation. However MEOX2 counteracted these IGF-1-mediated actions. Flow Cytometry revealed that IGF-1 increased S phase cells and decreased apoptosis; however, MEOX2 decreased S phase cells, increased G0-G1 phase cells, and promoted apoptosis. During PVAC proliferation and differentiation, IGF-1 activated PI3K/Akt1/2 and ERK1/2 signaling pathways, upregulated the expression of these signaling proteins and FAS, and increased PVAC lipid content. In contrast, MEOX2 constrained the phosphorylation of ERK1/2 and Akt1/2 protein, down-regulated these signaling molecules and FAS, and decreased PVAC lipid content. Instead, MEOX2 knockdown enhanced the ERK1/2 and Akt1/2 phosphorylation, augmented the expression of these signaling molecules and FAS, and increased PVAC lipid content. Our findings suggested that PI3K/Akt1/2 and ERK1/2 activation mediated by IGF-1 is essential for PVAC proliferation and differentiation, and MEOX2 is a promising therapeutic gene to intervene in the signaling pathways and inhibit PVAC functions. Copyright © 2014 Elsevier Inc. All rights reserved.

The inhibitory effect of dexamethasone on platelet-derived growth factor-induced vascular smooth muscle cell migration through up-regulating PGC-1{alpha} expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xu, Wei; Department of cardiology, the Second Affiliated Hospital of Harbin Medical University, 246 Xuefu Road, Harbin 150081; Guo, Ting

2011-05-01

Dexamethasone has been shown to inhibit vascular smooth muscle cell (VSMC) migration, which is required for preventing restenosis. However, the mechanism underlying effect of dexamethasone remains unknown. We have previously demonstrated that peroxisome proliferator-activated receptor gamma (PPAR{gamma}) coactivator-1 alpha (PGC-1{alpha}) can inhibit VSMC migration and proliferation. Here, we investigated the role of PGC-1{alpha} in dexamethasone-reduced VSMC migration and explored the possible mechanism. We first examined PGC-1{alpha} expression in cultured rat aortic VSMCs. The results revealed that incubation of VSMCs with dexamethasone could significantly elevate PGC-1{alpha} mRNA expression. In contrast, platelet-derived growth factor (PDGF) decreased PGC-1{alpha} expression while stimulating VSMC migration.more » Mechanistic study showed that suppression of PGC-1{alpha} by small interfering RNA strongly abrogated the inhibitory effect of dexamethasone on VSMC migration, whereas overexpression of PGC-1{alpha} had the opposite effect. Furthermore, an analysis of MAPK signal pathways showed that dexamethasone inhibited ERK and p38 MAPK phosphorylation in VSMCs. Overexpression of PGC-1{alpha} decreased both basal and PDGF-induced p38 MAPK phosphorylation, but it had no effect on ERK phosphorylation. Finally, inhibition of PPAR{gamma} activation by a PPAR{gamma} antagonist GW9662 abolished the suppressive effects of PGC-1{alpha} on p38 MAPK phosphorylation and VSMC migration. These effects of PGC-1{alpha} were enhanced by a PPAR{gamma} agonist troglitazone. Collectively, our data indicated for the first time that one of the anti-migrated mechanisms of dexamethasone is due to the induction of PGC-1{alpha} expression. PGC-1{alpha} suppresses PDGF-induced VSMC migration through PPAR{gamma} coactivation and, consequently, p38 MAPK inhibition.« less

Lipopolysaccharide-regulated production of bone sialoprotein and interleukin-8 in human periodontal ligament fibroblasts: the role of toll-like receptors 2 and 4 and the MAPK pathway.

PubMed

Zhang, Y; Li, X

2015-04-01

Lipopolysaccharide (LPS) on the cell wall of periodontal pathogens is a major mediator of the inflammatory response and can enhance alveolar bone resorption in periodontitis. Bone sialoprotein is an early marker of osteoblast differentiation. The proinflammatory cytokine, interleukin-8 (IL-8), induces osteoclast differentiation, maturation and maintenance of bone resorption activity. However, the effects of LPS from periodontal pathogens on the expression of bone sialoprotein and IL-8 in human osteoblasts and the mechanism of periodontal bone metabolism regulation are rather unclear. The objectives of this study were to determine the effects of Porphyromonas gingivalis LPS on the production of bone sialoprotein and IL-8 in human periodontal ligament fibroblasts (hPDLFs), and to investigate whether toll-like receptor (TLR) 2, TLR4 and MAPKs pathways are involved in the regulation of production of bone sialoprotein and IL-8 by P. gingivalis LPS. The third-generation of hPDLFs were cultured with mineralization-inducing culture medium. After hPDLFs were treated with P. gingivalis LPS, bone sialoprotein and IL-8 mRNA expression were detected using Real time PCR. Then hPDLFs were transiently transfected with siTLR2 or siTLR4 (20 nm) or inhibited by MAPK signaling pathways inhibitors, and then bone sialoprotein and IL-8 mRNA and protein expression were also detected using Real time PCR and western blotting. Treatments with 0.01 and 0.1 mg/L of P. gingivalis LPS for 8 h up-regulated bone sialoprotein mRNA expression, whereas 10 and 100 mg/L of P. gingivalis LPS induced a significant decrease in the expression of bone sialoprotein mRNA. In contrast, IL8 mRNA levels were increased significantly by 10 mg/L of P. gingivalis LPS. Interestingly, small interfering RNA (siRNA) knock down of the TLR2 and ERK1/2 inhibitor, PD98059, abolished the effects of P. gingivalis LPS on the bone sialoprotein mRNA level, whereas siRNA knock down of the TLR2 and p38 MAPK inhibitor, SB203580, blocked the effect of P. gingivalis LPS on IL-8 in hPDLFs. This study suggests that in hPDLFs, P. gingivalis LPS suppresses bone sialoprotein and enhances IL-8 gene and protein expression via TLR2 and ERK1/2 or the p38 MAPK signaling pathway, respectively. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Hydroxysafflor Yellow A Attenuates Neuron Damage by Suppressing the Lipopolysaccharide-Induced TLR4 Pathway in Activated Microglial Cells.

PubMed

Lv, Yanni; Qian, Yisong; Ou-Yang, Aijun; Fu, Longsheng

2016-11-01

Microglia activation initiates a neurological deficit cascade that contributes to substantial neuronal damage and impairment following ischemia stroke. Toll-like receptor 4 (TLR4) has been demonstrated to play a critical role in this cascade. In the current study, we tested the hypothesis that hydroxysafflor yellow A (HSYA), an active ingredient extracted from Flos Carthami tinctorii, alleviated inflammatory damage, and mediated neurotrophic effects in neurons by inducing the TLR4 pathway in microglia. A non-contact Transwell co-culture system comprised microglia and neurons was treated with HSYA followed by a 1 mg/mL lipopolysaccharide (LPS) stimulation. The microglia were activated prior to neuronal apoptosis, which were induced by increasing TLR4 expression in the activated microglia. However, HSYA suppressed TLR4 expression in the activated microglia, resulting in less neuronal damage at the early stage of LPS stimulation. Western blot analysis and immunofluorescence indicated that dose-dependently HSYA down-regulated TLR4-induced downstream effectors myeloid differentiation factor 88 (MyD88), nuclear factor kappa b (NF-κB), and the mitogen-activated protein kinases (MAPK)-regulated proteins c-Jun NH2-terminal protein kinase (JNK), protein kinase (ERK) 1/2 (ERK1/2), p38 MAPK (p38), as well as the LPS-induced inflammatory cytokine release. However, HSYA up-regulated brain-derived neurotrophic factor (BDNF) expression. Our data suggest that HSYA could exert neurotrophic and anti-inflammatory functions in response to LPS stimulation by inhibiting TLR4 pathway-mediated signaling.

E4orf1 induction in adipose tissue promotes insulin-independent signaling in the adipocyte.

PubMed

Kusminski, Christine M; Gallardo-Montejano, Violeta I; Wang, Zhao V; Hegde, Vijay; Bickel, Perry E; Dhurandhar, Nikhil V; Scherer, Philipp E

2015-10-01

Type 2 diabetes remains a worldwide epidemic with major pathophysiological changes as a result of chronic insulin resistance. Insulin regulates numerous biochemical pathways related to carbohydrate and lipid metabolism. We have generated a novel mouse model that allows us to constitutively activate, in an inducible fashion, the distal branch of the insulin signaling transduction pathway specifically in adipocytes. Using the adenoviral 36 E4orf1 protein, we chronically stimulate locally the Ras-ERK-MAPK signaling pathway. At the whole body level, this leads to reduced body-weight gain under a high fat diet challenge. Despite overlapping glucose tolerance curves, there is a reduced requirement for insulin action under these conditions. The mice further exhibit reduced circulating adiponectin levels that ultimately lead to impaired lipid clearance, and inflamed and fibrotic white adipose tissues. Nevertheless, they are protected from diet-induced hepatic steatosis. As we observe constitutively elevated p-Akt levels in the adipocytes, even under conditions of low insulin levels, this pinpoints enhanced Ras-ERK-MAPK signaling in transgenic adipocytes as a potential alternative route to bypass proximal insulin signaling events. We conclude that E4orf1 expression in the adipocyte leads to enhanced baseline activation of the distal insulin signaling node, yet impaired insulin receptor stimulation in the presence of insulin, with important implications for the regulation of adiponectin secretion. The resulting systemic phenotype is complex, yet highlights the powerful nature of manipulating selective branches of the insulin signaling network within the adipocyte.

Essential Roles of Raf/Extracellular Signal-regulated Kinase/Mitogen-activated Protein Kinase Pathway, YY1, and Ca2+ Influx in Growth Arrest of Human Vascular Smooth Muscle Cells by Bilirubin*

PubMed Central

Stoeckius, Marlon; Erat, Anna; Fujikawa, Tatsuya; Hiromura, Makoto; Koulova, Anna; Otterbein, Leo; Bianchi, Cesario; Tobiasch, Edda; Dagon, Yossi; Sellke, Frank W.; Usheva, Anny

2012-01-01

The biological effects of bilirubin, still poorly understood, are concentration-dependent ranging from cell protection to toxicity. Here we present data that at high nontoxic physiological concentrations, bilirubin inhibits growth of proliferating human coronary artery smooth muscle cells by three events. It impairs the activation of Raf/ERK/MAPK pathway and the cellular Raf and cyclin D1 content that results in retinoblastoma protein hypophosphorylation on amino acids S608 and S780. These events impede the release of YY1 to the nuclei and its availability to regulate the expression of genes and to support cellular proliferation. Moreover, altered calcium influx and calpain II protease activation leads to proteolytical degradation of transcription factor YY1. We conclude that in the serum-stimulated human vascular smooth muscle primary cell cultures, bilirubin favors growth arrest, and we propose that this activity is regulated by its interaction with the Raf/ERK/MAPK pathway, effect on cyclin D1 and Raf content, altered retinoblastoma protein profile of hypophosphorylation, calcium influx, and YY1 proteolysis. We propose that these activities together culminate in diminished 5 S and 45 S ribosomal RNA synthesis and cell growth arrest. The observations provide important mechanistic insight into the molecular mechanisms underlying the transition of human vascular smooth muscle cells from proliferative to contractile phenotype and the role of bilirubin in this transition. PMID:22262839

Immature osteoblastic MG63 cells possess two calcitonin gene-related peptide receptor subtypes that respond differently to [Cys(Acm)(2,7)] calcitonin gene-related peptide and CGRP(8-37).

PubMed

Kawase, Tomoyuki; Okuda, Kazuhiro; Burns, Douglas M

2005-10-01

Calcitonin gene-related peptide (CGRP) is clearly an anabolic factor in skeletal tissue, but the distribution of CGRP receptor (CGRPR) subtypes in osteoblastic cells is poorly understood. We previously demonstrated that the CGRPR expressed in osteoblastic MG63 cells does not match exactly the known characteristics of the classic subtype 1 receptor (CGRPR1). The aim of the present study was to further characterize the MG63 CGRPR using a selective agonist of the putative CGRPR2, [Cys(Acm)(2,7)]CGRP, and a relatively specific antagonist of CGRPR1, CGRP(8-37). [Cys(Acm)(2,7)]CGRP acted as a significant agonist only upon ERK dephosphorylation, whereas this analog effectively antagonized CGRP-induced cAMP production and phosphorylation of cAMP response element-binding protein (CREB) and p38 MAPK. Although it had no agonistic action when used alone, CGRP(8-37) potently blocked CGRP actions on cAMP, CREB, and p38 MAPK but had less of an effect on ERK. Schild plot analysis of the latter data revealed that the apparent pA2 value for ERK is clearly distinguishable from those of the other three plots as judged using the 95% confidence intervals. Additional assays using 3-isobutyl-1-methylxanthine or the PKA inhibitor N-(2-[p-bromocinnamylamino]ethyl)-5-isoquinolinesulfonamide hydrochloride (H-89) indicated that the cAMP-dependent pathway was predominantly responsible for CREB phosphorylation, partially involved in ERK dephosphorylation, and not involved in p38 MAPK phosphorylation. Considering previous data from Scatchard analysis of [125I]CGRP binding in connection with these results, these findings suggest that MG63 cells possess two functionally distinct CGRPR subtypes that show almost identical affinity for CGRP but different sensitivity to CGRP analogs: one is best characterized as a variation of CGRPR1, and the second may be a novel variant of CGRPR2.

Cypermethrin Induces Macrophages Death through Cell Cycle Arrest and Oxidative Stress-Mediated JNK/ERK Signaling Regulated Apoptosis

PubMed Central

Huang, Fang; Liu, Qiaoyun; Xie, Shujun; Xu, Jian; Huang, Bo; Wu, Yihua; Xia, Dajing

2016-01-01

Cypermethrin is one of the most highly effective synthetic pyrethroid insecticides. The toxicity of cypermethrin to the reproductive and nervous systems has been well studied. However, little is known about the toxic effect of cypermethrin on immune cells such as macrophages. Here, we investigated the cytotoxicity of cypermethrin on macrophages and the underlying molecular mechanisms. We found that cypermethrin reduced cell viability and induced apoptosis in RAW 264.7 cells. Cypermethrin also increased reactive oxygen species (ROS) production and DNA damage in a dose-dependent manner. Moreover, cypermethrin-induced G1 cell cycle arrest was associated with an enhanced expression of p21, wild-type p53, and down-regulation of cyclin D1, cyclin E and CDK4. In addition, cypermethrin treatment activated MAPK signal pathways by inducing c-Jun N-terminal kinase (JNK) and extracellular regulated protein kinases 1/2 ERK1/2 phosphorylation, and increased the cleaved poly ADP-ribose polymerase (PARP). Further, pretreatment with antioxidant N-acetylcysteine (NAC) effectively abrogated cypermethrin-induced cell cytotoxicity, G1 cell cycle arrest, DNA damage, PARP activity, and JNK and ERK1/2 activation. The specific JNK inhibitor (SP600125) and ERK1/2 inhibitor (PD98059) effectively reversed the phosphorylation level of JNK and ERK1/2, and attenuated the apoptosis. Taken together, these data suggested that cypermethrin caused immune cell death via inducing cell cycle arrest and apoptosis regulated by ROS-mediated JNK/ERK pathway. PMID:27322250

Sulforaphane, a natural constituent of broccoli, prevents cell death and inflammation in nephropathy.

PubMed

Guerrero-Beltrán, Carlos Enrique; Mukhopadhyay, Partha; Horváth, Béla; Rajesh, Mohanraj; Tapia, Edilia; García-Torres, Itzhel; Pedraza-Chaverri, José; Pacher, Pál

2012-05-01

Cisplatin (cis-diamminedichloroplatinum II, CIS) is a potent and widely used chemotherapeutic agent to treat various malignancies, but its therapeutic use is limited because of dose-dependent nephrotoxicity. Cell death and inflammation play a key role in the development and progression of CIS-induced nephropathy. Sulforaphane (SFN), a natural constituent of cruciferous vegetables such as broccoli, Brussels sprouts, etc., has been shown to exert various protective effects in models of tissue injury and cancer. In this study, we have investigated the role of prosurvival, cell death and inflammatory signaling pathways using a rodent model of CIS-induced nephropathy, and explored the effects of SFN on these processes. Cisplatin triggered marked activation of stress signaling pathways [p53, Jun N-terminal kinase (JNK), and p38-α mitogen-activated protein kinase (MAPK)] and promoted cell death in the kidneys (increased DNA fragmentation, caspases-3/7 activity, terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick-end labeling), associated with attenuation of various prosurvival signaling pathways [e.g., extracellular signal-regulated kinase (ERK) and p38-β MAPK]. Cisplatin also markedly enhanced inflammation in the kidneys [promoted NF-κB activation, increased expression of adhesion molecules ICAM and VCAM, enhanced tumor necrosis factor-α (TNF-α) levels and inflammatory cell infiltration]. These effects were significantly attenuated by pretreatment of rodents with SFN. Thus, the cisplatin-induced nephropathy is associated with activation of various cell death and proinflammatory pathways (p53, JNK, p38-α, TNF-α and NF-κB) and impairments of key prosurvival signaling mechanisms (ERK and p38-β). SFN is able to prevent the CIS-induced renal injury by modulating these pathways, providing a novel approach for preventing this devastating complication of chemotherapy. Published by Elsevier Inc.

Sulforaphane, a natural constituent of broccoli, prevents cell death and inflammation in nephropathy

PubMed Central

Guerrero-Beltrán, Carlos Enrique; Mukhopadhyay, Partha; Horváth, Béla; Rajesh, Mohanraj; Tapia, Edilia; García-Torres, Itzhel; Pedraza-Chaverri, José; Pacher, Pál

2011-01-01

Cisplatin (cis-diamminedichloroplatinum II, CIS) is a potent and widely used chemotherapeutic agent to treat various malignancies, but its therapeutic use is limited because of the dose-dependent nephrotoxicity. Cell death and inflammation play key role in the development and progression of CIS-induced nephropathy. Sulforaphane (SFN), a natural constituent of cruciferous vegetables such as broccoli, Brussels sprouts, etc., has been shown to exert various protective effects in models of tissue injury and cancer. In this study, we have investigated the role of pro-survival, cell death and inflammatory signaling pathways using a rodent model of CIS-induced nephropathy, and explored the effects of SFN on these processes. Cisplatin triggered marked activation of stress signaling pathways (p53, Jun N-terminal kinase (JNK), and p38-α MAPK) and promoted cell death in the kidneys (increased DNA fragmentation, caspases-3/7 activity, TUNEL), associated with attenuation of various pro-survival signaling pathways (e.g. extracellular signal-regulated kinase (ERK) and p38-β MAPK). Cisplatin also markedly enhanced inflammation in the kidneys (promoted NF-κB activation, increased expression of adhesion molecules ICAM and VCAM, enhanced tumor necrosis factor-alpha (TNF-α) levels, and inflammatory cell infiltration). These effects were significantly attenuated by pre-treatment of rodents with SFN. Cisplatin-induced nephropathy is associated with activation of various cell death and pro-inflammatory pathways (p53, JNK, p38-α, TNF-α, and NF-κB) and impairments of key pro-survival signaling mechanisms (ERK and p38-β). SFN is able to prevent the CIS-induced renal injury by modulating these pathways, providing a novel approach for preventing this devastating complication of the chemotherapy. PMID:21684138

Essential role of protein kinase C delta in platelet signaling, alpha IIb beta 3 activation, and thromboxane A2 release.

PubMed

Yacoub, Daniel; Théorêt, Jean-François; Villeneuve, Louis; Abou-Saleh, Haissam; Mourad, Walid; Allen, Bruce G; Merhi, Yahye

2006-10-06

The protein kinase C (PKC) family is an essential signaling mediator in platelet activation and aggregation. However, the relative importance of the major platelet PKC isoforms and their downstream effectors in platelet signaling and function remain unclear. Using isolated human platelets, we report that PKCdelta, but not PKCalpha or PKCbeta, is required for collagen-induced phospholipase C-dependent signaling, activation of alpha(IIb)beta(3), and platelet aggregation. Analysis of PKCdelta phosphorylation and translocation to the membrane following activation by both collagen and thrombin indicates that it is positively regulated by alpha(IIb)beta(3) outside-in signaling. Moreover, PKCdelta triggers activation of the mitogen-activated protein kinase-kinase (MEK)/extracellular-signal regulated kinase (ERK) and the p38 MAPK signaling. This leads to the subsequent release of thromboxane A(2), which is essential for collagen-induced but not thrombin-induced platelet activation and aggregation. This study adds new insight to the role of PKCs in platelet function, where PKCdelta signaling, via the MEK/ERK and p38 MAPK pathways, is required for the secretion of thromboxane A(2).

Dusp5 negatively regulates IL-33-mediated eosinophil survival and function

PubMed Central

Holmes, Derek A; Yeh, Jung-Hua; Yan, Donghong; Xu, Min; Chan, Andrew C

2015-01-01

Mitogen-activated protein kinase (MAPK) activation controls diverse cellular functions including cellular survival, proliferation, and apoptosis. Tuning of MAPK activation is counter-regulated by a family of dual-specificity phosphatases (DUSPs). IL-33 is a recently described cytokine that initiates Th2 immune responses through binding to a heterodimeric IL-33Rα (ST2L)/IL-1α accessory protein (IL-1RAcP) receptor that coordinates activation of ERK and NF-κB pathways. We demonstrate here that DUSP5 is expressed in eosinophils, is upregulated following IL-33 stimulation and regulates IL-33 signaling. Dusp5−/− mice have prolonged eosinophil survival and enhanced eosinophil effector functions following infection with the helminth Nippostrongylus brasiliensis. IL-33-activated Dusp5−/− eosinophils exhibit increased cellular ERK1/2 activation and BCL-XL expression that results in enhanced eosinophil survival. In addition, Dusp5−/− eosinophils demonstrate enhanced IL-33-mediated activation and effector functions. Together, these data support a role for DUSP5 as a novel negative regulator of IL-33-dependent eosinophil function and survival. PMID:25398911

Role of Piezo Channels in Ultrasound-stimulated Dental Stem Cells.

PubMed

Gao, Qianhua; Cooper, Paul R; Walmsley, A Damien; Scheven, Ben A

2017-07-01

Piezo1 and Piezo2 are mechanosensitive membrane ion channels. We hypothesized that Piezo proteins may play a role in transducing ultrasound-associated mechanical signals and activate downstream mitogen-activated protein kinase (MAPK) signaling processes in dental cells. In this study, the expression and role of Piezo channels were investigated in dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) after treatment with low-intensity pulsed ultrasound (LIPUS). Cell proliferation was evaluated by bromodeoxyuridine incorporation. Western blots were used to analyze the proliferating cell nuclear antigen as well as the transcription factors c-fos and c-jun. Enzyme-linked immunosorbent assay and Western blotting were used to determine the activation of MAPK after LIPUS treatment. Ruthenium red (RR), a Piezo ion channel blocker, was applied to determine the functional role of Piezo proteins in LIPUS-stimulated cell proliferation and MAPK signaling. Western blotting showed the presence of Piezo1 and Piezo2 in both dental cell types. LIPUS treatment significantly increased the level of the Piezo proteins in DPSCs after 24 hours; however, no significant effects were observed in PDLSCs. Treatment with RR significantly inhibited LIPUS-stimulated DPSC proliferation but not PDLSC proliferation. Extracellular signal-related kinase (ERK) 1/2 MAPK was consistently activated in DPSCs over a 24-hour time period after LIPUS exposure, whereas phosphorylated c-Jun N-terminal kinase and p38 mitogen-activated protein kinase MAPK were mainly increased in PDLSCs. RR affected MAPK signaling in both dental cell types with its most prominent effects on ERK1/2/MAPK phosphorylation levels; the significant inhibition of LIPUS-induced stimulation of ERK1/2 activation in DPSCs by RR suggests that stimulation of DPSC proliferation by LIPUS involves Piezo-mediated regulation of ERK1/2 MAPK signaling. This study for the first time supports the role of Piezo ion channels in transducing the LIPUS response in dental stem cells. Copyright © 2017 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Role of a cysteine residue in the active site of ERK and the MAPKK family

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ohori, Makoto; Kinoshita, Takayoshi; Yoshimura, Seiji

2007-02-16

Kinases of mitogen-activated protein kinase (MAPK) cascades, including extracellular signal-regulated protein kinase (ERK), represent likely targets for pharmacological intervention in proliferative diseases. Here, we report that FR148083 inhibits ERK2 enzyme activity and TGF{beta}-induced AP-1-dependent luciferase expression with respective IC{sub 50} values of 0.08 and 0.05 {mu}M. FR265083 (1'-2' dihydro form) and FR263574 (1'-2' and 7'-8' tetrahydro form) exhibited 5.5-fold less and no activity, respectively, indicating that both the {alpha},{beta}-unsaturated ketone and the conformation of the lactone ring contribute to this inhibitory activity. The X-ray crystal structure of the ERK2/FR148083 complex revealed that the compound binds to the ATP binding sitemore » of ERK2, involving a covalent bond to S{gamma} of ERK2 Cys166, hydrogen bonds with the backbone NH of Met108, N{zeta} of Lys114, backbone C=O of Ser153, N{delta}2 of Asn154, and hydrophobic interactions with the side chains of Ile31, Val39, Ala52, and Leu156. The covalent bond motif in the ERK2/FR148083 complex assures that the inhibitor has high activity for ERK2 and no activity for other MAPKs such as JNK1 and p38MAPK{alpha}/{beta}/{gamma}/{delta} which have leucine residues at the site corresponding to Cys166 in ERK2. On the other hand, MEK1 and MKK7, kinases of the MAPKK family which also can be inhibited by FR148083, contain a cysteine residue corresponding to Cys166 of ERK2. The covalent binding to the common cysteine residue in the ATP-binding site is therefore likely to play a crucial role in the inhibitory activity for these MAP kinases. These findings on the molecular recognition mechanisms of FR148083 for kinases with Cys166 should provide a novel strategy for the pharmacological intervention of MAPK cascades.« less

Immunomodulatory effect of tea saponin in immune T-cells and T-lymphoma cells via regulation of Th1, Th2 immune response and MAPK/ERK2 signaling pathway.

PubMed

Bhardwaj, Jyoti; Chaudhary, Narendra; Seo, Hyo-Jin; Kim, Min-Yong; Shin, Tai-Sun; Kim, Jong-Deog

2014-06-01

The anti-cancer activity of saponins and phenolic compounds present in green tea was previously reported. However, the immunomodulatory and adjuvanticity activity of tea saponin has never been studied. In this study, we investigated the immunomodulatory effect of tea saponin in T-lymphocytes and EL4 cells via regulation of cytokine response and mitogen-activated protein kinases (MAPK) signaling pathway. Quantitative analysis of mRNA expression level of cytokines were performed by reverse transcription polymerase chain reaction following stimulation with tea saponin, ovalbumin (OVA) alone or tea saponin in combination with OVA. Tea saponin inhibited the proliferation of EL4 cells measured in a dose-dependent manner. No cytotoxicity effect of tea saponin was detected in T-lymphocytes; rather, tea saponin enhanced the proliferation of T-lymphocytes. Tea saponin with OVA increased the expression of interleukin (IL)-1, IL-2, IL-12, interferon-γ and tumor necrosis factor (TNF)-α and decreased the expression level of IL-10 and IL-8 in T-lymphocytes. Furthermore, tea saponin, in the presence of OVA, downregulated the MAPK signaling pathway via inhibition of IL-4, IL-8 and nuclear factor kappaB (NF-κB) in EL4 cells. Th1 cytokines enhancer and Th2 cytokines and NF-κB inhibitor, tea saponin can markedly inhibit the proliferation and invasiveness of T-lymphoma (EL4) cells, possibly due to TNF-α- and NF-κB-mediated regulation of MAPK signaling pathway.

[Effect of Echinococcus multilocularis Cyst Fluid on the Expression of Five MAPK-pathway Genes of Rat Hepatic Stellate Cells].

PubMed

Ren, Bin; Fan, Hai-ning; Deng, Yong; Wang, Hai-jiu; Ren, Li

2015-04-01

To investigate the effect of Echinococcus multilocularis cyst fluid on five MAPK (mitogen-activated protein kinase)-pathway genes of rat hepatic stellate cell. Rat hepatic stellate cell line, HSC-T6 cells were co-cultured with different protein concentrations of E. multilocularis cyst fluid (0.01, 0.025, 0.05, 0.1, 0.2, 0.4, 0.9, 1.7, 3.4, 6.8, and 13.5 mg/ml) for 24 h. HSC-T6 cells cultured with complete medium served as control group. The morphological change of cells was observed under the microscope. The expression of extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK) and mitogen-activated protein kinase(p38) in HSC-T6 cells was detected by real time fluorescent quantitative PCR. After co-cultured for 24 h, most HSC-T6 cells in 13.5 mg/ml group shrank as a precursor to slough off; In 6.8 mg/ml group, some HSC-T6 cells shrank and changed to long fusiform shape with many slender pseudopodia; In 3.4 mg/ml group, most HSC-T6 cells showed as adherent cells with an irregular polygon shape, formed a sheet with short pseudopodia. There was no difference in cell morphology between < 1.7 mg/ml groups and control group. When the protein concentration was above 1.7 mg/ml, the mRNA level of ERK1/2, JNK1/2, and P38 increased significantly increased. In 6.8 mg/ml cyst fluid group, the mRNA level of ERK1/2, JNK1/2, and P38 was higher than that of the control (P < 0.05). 6.8 mg/ml Echinococcus multilocularis cyst fluid can have a significant impact on mRNA levels of ERK1/2, JNK1/2 and p38 in rat hepatic stellate cells.

Differential Regulation of MAPK Phosphorylation in the Dorsal Hippocampus in Response to Prolonged Morphine Withdrawal-Induced Depressive-Like Symptoms in Mice

PubMed Central

Shi, Jianguo; Wu, Bin; Dang, Wei; Du, Ying; Zhou, Qiong; Wang, Jianhua; Zhang, Rui

2013-01-01

Depression is one of the most frequent neuropsychiatric comorbidities associated with opiate addiction. Mitogen activated protein kinase (MAPK) and MAPK phosphatase (MKP) are involved in drug addiction and depression. However, the potential role of MAPK and MKP in depression caused by morphine withdrawal remains unclear. We utilized a mouse model of repeated morphine administration to examine the molecular mechanisms that contribute to prolonged withdrawal induced depressive-like behaviors. Depressive-like behaviors were significant at 1 week after withdrawal and worsened over time. Phospho-ERK (extracellular signal-regulated protein kinase) was decreased and MKP-1 was elevated in the hippocampus, and JNK (c-Jun N-terminal protein kinase), p38 (p38 protein kinase) and MKP-3 were unaffected. A pharmacological blockade of MKP-1 by intra-hippocampal sanguinarine (SA) infusion prevented the development of depressive-like behaviors and resulted in relatively normal levels of MKP-1 and phospho-ERK after withdrawal. Our findings support the association between hippocampal MAPK phosphorylation and prolonged morphine withdrawal-induced depression, and emphasize the MKP-1 as an negative regulator of the ERK phosphorylation that contributes to depression. PMID:23823128

Src is a major signaling component for CTGF induction by TGF-β1 in osteoblasts

PubMed Central

X, Zhang; JA, Arnott; S, Rehman; WG, DeLong; A, Sanjay; FF, Safadi; SN, Popoff

2010-01-01

Connective tissue growth factor (CTGF/CCN2) is induced by transforming growth factor beta 1(TGF-β1) where it acts as a downstream mediator of TGF-β1 induced matrix production in osteoblasts. We have shown the requirement of Src, Erk and Smad signaling for CTGF induction by TGF-β1 in osteoblasts, however the potential interaction among these signaling pathways remains undetermined. In this study we demonstrate that TGF-β1 activates Src kinase in ROS17/2.8 cells and that treatment with the Src family kinase inhibitor PP2 prevents Src activation and CTGF induction by TGF-β1. Additionally, inhibiting Src activation prevented Erk activation, Smad 2 & 3 activation and nuclear translocation by TGF-β1, demonstrating that Src is an essential upstream signaling partner of both Erk and Smads in osteoblasts. MAPKs such as Erk can modulate the Smad pathway through directly mediating the phosphorylation of Smads or indirectly through activation/inactivation of required nuclear co-activators that mediate Smad DNA binding. When we treated cells with the Erk inhibitor, PD98059 it inhibited TGF-β1-induced CTGF protein expression but had no effect on Src activation, Smad activation or Smad nuclear translocation. However PD98059 impaired transcriptional complex formation on the Smad binding element (SBE) on the CTGF promoter, demonstrating that Erk activation was required for SBE transactivation. This data demonstrates that Src is an essential upstream signaling transducer of Erk and Smad signaling with respect to TGF-β1 in osteoblasts and that Smads and Erk function independently but are both essential for forming a transcriptionally active complex on the CTGF promoter in osteoblasts. PMID:20432467

BDE-47 induces oxidative stress, activates MAPK signaling pathway, and elevates de novo lipogenesis in the copepod Paracyclopina nana.

PubMed

Lee, Min-Chul; Puthumana, Jayesh; Lee, Seung-Hwi; Kang, Hye-Min; Park, Jun Chul; Jeong, Chang-Bum; Han, Jeonghoon; Hwang, Dae-Sik; Seo, Jung Soo; Park, Heum Gi; Om, Ae-Son; Lee, Jae-Seong

2016-12-01

Brominated flame retardant, 2, 2', 4, 4'-tetrabromodiphenyl ether (BDE-47), has received grave concerns as a persistent organic pollutant, which is toxic to marine organisms, and a suspected link to endocrine abnormalities. Despite the wide distribution in the marine ecosystem, very little is known about the toxic impairments on marine organisms, particularly on invertebrates. Thus, we examined the adverse effects of BDE-47 on life history trait (development), oxidative markers, fatty acid composition, and lipid accumulation in response to BDE-47-induced stress in the marine copepod Paracyclopina nana. Also, activation level of mitogen-activated protein kinase (MAPK) signaling pathways along with the gene expression profile of de novo lipogenesis (DNL) pathways were addressed. As a result, BDE-47 induced oxidative stress (e.g. reactive oxygen species, ROS) mediated activation of extracellular signal-regulated kinase (ERK) and c-Jun-N-terminal kinase (JNK) signaling cascades in MAPK pathways. Activated MAPK pathways, in turn, induced signal molecules that bind to the transcription factors (TFs) responsible for lipogenesis to EcR, SREBP, ChREBP promoters. Also, the stress stimulated the conversion of saturated fatty acids (SFAs) to polyunsaturated fatty acids (PUFAs), a preparedness of the organism to adapt the observed stress, which could be correlated with the elongase and desaturase gene (e.g. ELO3, Δ5-DES, Δ9-DES) expressions, and then extended to the delayed early post-embryonic development and increased accumulation of lipid droplets in P. nana. This study will provide a better understanding of how BDE-47 effects on marine invertebrates particularly on the copepods, an important link in the marine food chain. Copyright © 2016 Elsevier B.V. All rights reserved.

Calcium alters monoamine oxidase-A parameters in human cerebellar and rat glial C6 cell extracts: possible influence by distinct signalling pathways.

PubMed

Cao, Xia; Li, Xin-Min; Mousseau, Darrell D

2009-07-31

Calcium (Ca(2+)) is known to augment monoamine oxidase-A (MAO-A) activity in cell cultures as well as in brain extracts from several species. This association between Ca(2+) and MAO-A could contribute to their respective roles in cytotoxicity. However, the effect of Ca(2+) on MAO-A function in human brain has as yet to be examined as does the contribution of specific signalling cascades. We examined the effects of Ca(2+) on MAO-A activity and on [(3)H]Ro 41-1049 binding to MAO-A in human cerebellar extracts, and compared this to its effects on MAO-A activity in glial C6 cells following the targeting of signalling pathways using specific chemical inhibitors. Ca(2+) enhances MAO-A activity as well as the association of [(3)H]Ro 41-1049 to MAO-A in human cerebellar extracts. The screening of neuronal and glial cell cultures reveals that MAO-A activity does not always correlate with the expression of either mao-A mRNA or MAO-A protein. Inhibition of the individual PI3K/Akt, ERK and p38(MAPK) signalling pathways in glial C6 cells all augment basal MAO-A activity. Inhibition of the p38(MAPK) pathway also augments Ca(2+)-sensitive MAO-A activity. We also observe the inverse relation between p38(MAPK) activation and MAO-A function in C6 cultures grown to full confluence. The Ca(2+)-sensitive component to MAO-A activity is present in human brain and in vitro studies link it to the p38(MAPK) pathway. This means of influencing MAO-A function could explain its role in pathologies as diverse as neurodegeneration and cancers.

A tissue microarray study of toll-like receptor 4, decoy receptor 3, and external signal regulated kinase 1/2 expressions in astrocytoma.

PubMed

Lin, Chih-Kung; Ting, Chun-Chieh; Tsai, Wen-Chiuan; Chen, Yuan-Wu; Hueng, Dueng-Yuan

2016-01-01

Decoy receptor 3 (DcR3) functions as a death decoy inhibiting apoptosis mediated by the tumor necrosis factor receptor family. It is highly expressed in many tumors and its expression can be regulated by the MAPK/ERK signaling pathway and ERK is a vital member of this pathway. Toll-like receptor 4 (TLR4) is expressed on immune cells. Increased TLR4 expression has been associated with various types of cancers. The study was conducted to investigate the expression of DcR3, ERK1/2, and TLR4 in astrocytomas and evaluate if they are validating markers for discriminating glioblastoma from anaplastic astrocytoma in limited surgical specimen. Expression of DcR3, ERK1/2, and TLR4 was determined by immunohistochemical staining of tissue microarray from 48 paraffin-embedded tissues. A binary logistic regression method was used to generate functions that discriminate between anaplastic astrocytomas and glioblastomas. The expression of TLR4 and DcR3 was significantly higher in glioblastomas than in anaplastic astrocytomas. DcR3 could discriminate anaplastic astrocytomas from glioblastomas with high sensitivity (93.8%), specificity (90%), and accuracy (92.3%). Our results suggest that DcR3 may be a useful marker for discriminating anaplastic astrocytomas from glioblastomas.

Pirfenidone attenuates the IL-1β-induced hyaluronic acid increase in orbital fibroblasts from patients with thyroid-associated ophthalmopathy.

PubMed

Chung, Seung Ah; Jeon, Bo Kyung; Choi, Youn-Hee; Back, Keum Ok; Lee, Jong Bok; Kook, Koung Hoon

2014-04-09

This study aimed to investigate the effect of pirfenidone on the IL-1β-induced hyaluronic acid (HA) increase in orbital fibroblasts from patients with thyroid-associated ophthalmopathy (TAO). Primary cultured orbital fibroblasts were obtained from patients with TAO, and the excreted levels of HA from IL-1β-treated cells with or without pirfenidone were measured. The effect of pirfenidone on IL-1β-induced hyaluronic acid synthase (HAS) expression was evaluated. The relevance of the mitogen-activated protein kinase (MAPK)-mediated signaling pathway in IL-1β-induced HAS expression was assessed using specific inhibitors to p38, extracellular signal-regulated kinase (ERK), or c-Jun N-terminal kinase (JNK). The phosphorylation level of each MAPK in IL-1β-treated cells with or without pirfenidone and the level of AP-1 DNA binding were measured. The inhibitory potency of pirfenidone on HA production was evaluated using dexamethasone as a reference agent. Pirfenidone strongly attenuated the IL-1β-induced HA release in a dose-dependent manner. The IL-1β-induced HAS expression was decreased significantly following cotreatment with pirfenidone at the mRNA and protein levels. The production of mRNAs was halted by cotreatment with inhibitors of ERK and p38, but not by inhibitors of JNK. The IL-1β-induced ERK and p38 phosphorylation, and AP-1 DNA binding were attenuated in the presence of pirfenidone. Pirfenidone showed greater potency than dexamethasone in inhibiting increases in IL-1β-induced HA. Pirfenidone attenuates the IL-1β-induced HA production in orbital fibroblasts from patients with TAO, at least in part, through suppression of the MAPK-mediated HAS expression. These results support the potential use of pirfenidone for treatment of patients with TAO.

[Effect of Guanmaitong Tablet on ERK and p38 Protein of TLR2 Pathway Expression in Cerebral Ischemia/Reperfusion Rats: an Experimental Study].

PubMed

Zhang, Cui-xiang; Liu, Jian-xun; Li, Dan; Li, Lei; Fu, Jian-hua; Hou, Jin-cai; Du, Xue-mei; Zhang, Fa-chang

2015-06-01

To explore the inflammatory cascade mechanism through Toll like receptor 2 (TLR2) pathway after cerebral ischemia/reperfusion, and to study molecular mechanisms of Guanmaitong (GMT) Tablet for protecting brain damage. We used bolt-line method to block/release the middle cerebral artery, causing cerebral ischemia/reperfusion (I/R) injury model. GMT Tablet was given by gastrogavage. Rats were then divided into the high dose GMT group (1200 mg/kg), the middle dose GMT group (600 mg/kg), the low dose GMT group (300 mg/kg), the positive control group (Tanakan, 20 mg/kg). Their right brain tissues were fixed in 10% neutral formalin. TLR2 expressions were detected by immunofluorescence staining. The total protein was extracted from right brain tissues by ultrasonica- tion. Expression levels of extracellular regulated protein kinases (ERK), phospho-extracellular regulated protein kinases (p-ERK), p38-mitogen activated protein kinases (p-ERK), phospho-p38-mitogen activated protein kinases [p-p38-MAPKs(p-p38)] were assessed by Western blot. Abdominal aortic blood was withdrawn. IL-6 and IL-1β levels were detected by ELISA in brain tissues and serum. Compared with the sham-oepration group, expression levels of TLR2, ERK, p-ERK, p38, p-p38 protein were up-regulated (P < 0.05, P < 0.01), and contents of IL-6 and IL-1β in brain tissues and serum were increased in the model group (P < 0.01). Expression levels of TLR2, ERK, p-ERK, p38, p-p38 were down-regulated (P < 0.05, P < 0.01), and contents of IL-6 and IL-1β were reduced in brain tissues and serum in middle and high dose GMT groups (P < 0.05, P < 0.01). TLR2 pathway was involved in cerebral I/R injury. GMT protected neurons by down-regulating protein expressions of TLR2, ERK, p-ERK, p38, p-p38 and contents of IL-1β and IL-6.

Insulin-like growth factor 1 rescues R28 retinal neurons from apoptotic death through ERK-mediated BimEL phosphorylation independent of Akt.

PubMed

Kong, Dejuan; Gong, Lijie; Arnold, Edith; Shanmugam, Sumathi; Fort, Patrice E; Gardner, Thomas W; Abcouwer, Steven F

2016-10-01

Insulin-like growth factor 1 (IGF-1) can provide long-term neurotrophic support by activation of Akt, inhibition of FoxO nuclear localization and suppression of Bim gene transcription in multiple neuronal systems. However, MEK/ERK activation can also promote neuron survival through phosphorylation of BimEL. We explored the contribution of the PI3K/Akt/FoxO and MEK/ERK/BimEL pathways in IGF-1 stimulated survival after serum deprivation (SD) of R28 cells differentiated to model retinal neurons. IGF-1 caused rapid activation of Akt leading to FoxO1/3-T32/T24 phosphorylation, and prevented FoxO1/3 nuclear translocation and Bim mRNA upregulation in response to SD. IGF-1 also caused MAPK/MEK pathway activation as indicated by ERK1/2-T202/Y204 and Bim-S65 phosphorylation. Overexpression of FoxO1 increased Bim mRNA expression and amplified the apoptotic response to SD without shifting the serum response curve. Inhibition of Akt activation with LY294002 or by Rictor knockdown did not block the protective effect of IGF-1, while inhibition of MEK activity with PD98059 prevented Bim phosphorylation and blocked IGF-1 protection. In addition, knockdown of Bim expression was protective during SD, while co-silencing of FoxO1 and Fox03 expression had little effect. Thus, the PI3K/Akt/FoxO pathway was not essential for protection from SD-induced apoptosis by IGF-1 in R28 cells. Instead, IGF-1 protection was dependent on activation of the MEK/ERK pathway leading to BimEL phosphorylation, which is known to prevent Bax/Bak oligomerization and activation of the intrinsic mitochondrial apoptosis pathway. These studies demonstrate the requirement of the MEK/ERK pathway in a model of retinal neuron cell survival and highlight the cell specificity for IGF-1 signaling in this response. Copyright © 2016 Elsevier Ltd. All rights reserved.

Evidence that simulated microgravity may alter the vascular nonreceptor tyrosine kinase second messenger pathway

NASA Technical Reports Server (NTRS)

Kahwaji, C. I.; Sheibani, S.; Han, S.; Siu, W. O.; Kaka, A. H.; Fathy, T. M.; el-Abbadi, N. H.; Purdy, R. E.

2000-01-01

Simulated microgravity (hind limb unweighting; HU) reduces maximal contractile capacity to norepinephrine (NE) but not 5-hydroxytryptamine (5-HT) in the rat abdominal aorta of male Wistar rats. Our earlier study showed that voltage-operated calcium channels, the MAPK pathway [1], and vasoconstrictive prostaglandins contribute to the NE-induced contraction of control (C) but not HU, aorta rings. Genistein, a general tyrosine kinase inhibitor, caused a significant reduction in vascular contractility in C but not HU arteries. The present study explored the role of protein kinase C (PKC) and extracellular receptor-activated kinase 1 and 2 (ERK1/2) in the HU-induced vascular hyporesponsiveness to NE. Microgravity was simulated in Wistar rats by 20 day HU. The abdominal aorta was removed from control and HU rats, cut into 3 mm rings, and mounted in tissue baths to measure isometric contraction. Protein levels were determined using Western blot analysis. PD98059, a selective MAPKK inhibitor, caused a marked inhibition of NE-induced contraction in both C and HU arteries. Calphostin C, a PKC inhibitor, completely abolished the contractile response to NE in both C and HU tissues. Phosphorylated (activated) ERK1/2 protein mass was greater in C, compared to HU, aortas, and was reduced by genistein only in C tissues. MAPK total protein levels in the rat aorta were increased in the HU-treated, compared to C, animals. These results indicate that PKC represents an early transduction step in the contractile response to NE in the rat abdominal aorta. That inhibition of the step immediately before activation of MAPK reduced contraction in both C and HU tissues, while general tyrosine kinase inhibition with genistein blocked only the control responses, suggests that a nonreceptor tyrosine kinase may be involved in HU-induced vascular hyporesponsiveness to NE.

Hyperoside attenuates hydrogen peroxide-induced L02 cell damage via MAPK-dependent Keap{sub 1}-Nrf{sub 2}-ARE signaling pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Xing, Hai-Yan; Liu, Yao; Chen, Jian-Hong

Highlights: {yields} Hyperoside attenuated H{sub 2}O{sub 2}-induced L02 cell damage. {yields} Hyperoside up-regulated HO-1 expression at both mRNA and protein levels. {yields} Hyperoside activated both Nrf{sub 2} nuclear translocation and gene expression. {yields} Hyperoside may inhibit Keap{sub 1} mRNA translation or protein degradation. {yields} Phosphorylation of ERK and p38 is involved in hyperoside-mediated Nrf{sub 2} activation. -- Abstract: The flavonoid hyperoside has been reported to elicit cytoprotection against oxidative stress partly by increasing the activity of antioxidant enzymes, such as glutathione peroxidase, superoxide dismutase and catalase. However, the cellular and molecular mechanisms underlying this effect remain unclear. Here, hepatic L02more » cells exposed to H{sub 2}O{sub 2} (100 {mu}M) were used to demonstrate that hyperoside protected cells by significantly inhibiting overproduction of intracellular ROS, depletion of the mitochondrial membrane potential and leakage of lactate dehydrogenase. Hyperoside further enhanced the cellular antioxidant defense system through increasing the activity of heme oxygenase-1 (HO-1), and by up-regulating HO-1 expression. Meanwhile, real time PCR, western blot and immunofluorescence studies revealed that hyperoside stimulated nuclear translocation of the Nrf{sub 2} transcription factor in a dose-dependent manner, and this effect was significantly suppressed by pharmacological inhibition of the mitogen-activated protein kinases (MAPK) p38 and ERK. Collectively, our data provide the first description of the mechanism underlying hyperoside's ability to attenuate H{sub 2}O{sub 2}-induced cell damage, namely this compound interacts with the MAPK-dependent Keap{sub 1}-Nrf{sub 2}-ARE signaling pathway to up-regulate HO-1 expression and enhance intracellular antioxidant activity.« less

Role of Spm-Cer-S1P signalling pathway in MMP-2 mediated U46619-induced proliferation of pulmonary artery smooth muscle cells: protective role of epigallocatechin-3-gallate.

PubMed

Chowdhury, Animesh; Sarkar, Jaganmay; Chakraborti, Tapati; Chakraborti, Sajal

2015-10-01

During remodelling of pulmonary artery, marked proliferation of pulmonary artery smooth muscle cells (PASMCs) occurs, which contributes to pulmonary hypertension. Thromboxane A2 (TxA2) has been shown to produce pulmonary hypertension. The present study investigates the inhibitory effect of epigallocatechin-3-gallate (EGCG) on the TxA2 mimetic, U46619-induced proliferation of PASMCs. U46619 at a concentration of 10 nM induces maximum proliferation of bovine PASMCs. Both pharmacological and genetic inhibitors of p(38)MAPK, NF-κB and MMP-2 significantly inhibit U46619-induced cell proliferation. EGCG markedly abrogate U46619-induced p(38)MAPK phosphorylation, NF-κB activation, proMMP-2 expression and activation, and also the cell proliferation. U46619 causes an increase in the activation of sphingomyelinase (SMase) and sphingosine kinase (SPHK) and also increase sphingosine 1 phosphate (S1P) level. U46619 also induces phosphorylation of ERK1/2, which phosphorylates SPHK leading to an increase in S1P level. Both pharmacological and genetic inhibitors of SMase and SPHK markedly inhibit U46619-induced cell proliferation. Additionally, pharmacological and genetic inhibitors of MMP-2 markedly abrogate U46619-induced SMase activity and S1P level. EGCG markedly inhibit U46619-induced SMase activity, ERK1/2 and SPHK phosphorylation and S1P level in the cells. Overall, Sphingomyeline-Ceramide-Sphingosine-1-phosphate (Spm-Cer-S1P) signalling axis plays an important role in MMP-2 mediated U46619-induced proliferation of PASMCs. Importantly, EGCG inhibits U46619 induced increase in MMP-2 activation by modulating p(38)MAPK-NFκB pathway and subsequently prevents the cell proliferation. Copyright © 2015 John Wiley & Sons, Ltd.

KIR2DL4 differentially signals downstream functions in human NK cells through distinct structural modules.

PubMed

Miah, S M Shahjahan; Hughes, Tracey L; Campbell, Kerry S

2008-03-01

KIR2DL4 (2DL4) is a member of the killer cell Ig-like receptor (KIR) family in human NK cells. It can stimulate potent cytokine production and weak cytolytic activity in resting NK cells, but the mechanism for 2DL4-mediated signaling remains unclear. In this study we characterized the signaling pathways stimulated by 2DL4 engagement. In a human NK-like cell line, KHYG-1, cross-linking of 2DL4 activated MAPKs including JNK, ERK, and p38. Furthermore, 2DL4 cross-linking resulted in phosphorylation of IkappaB kinase beta (IKKbeta) and the phosphorylation and degradation of IkappaBalpha, which indicate activation of the classical NF-kappaB pathway. Engagement of 2DL4 was also shown to activate the transcription and translation of a variety of cytokine genes, including TNF-alpha, IFN-gamma, MIP1alpha, MIP1beta, and IL-8. Pharmacological inhibitors of JNK, MEK1/2 and p38, blocked IFN-gamma, IL-8, and MIP1alpha production, suggesting that MAPKs are regulating 2DL4-mediated cytokine production in a nonredundant manner. Activation of both p38 and ERK appear to be upstream of the stimulation of NF-kappaB. Mutation of a transmembrane arginine in 2DL4 to glycine (R/G mutant) abrogated FcepsilonRI-gamma association, as well as receptor-mediated cytolytic activity and calcium responses. Surprisingly, the R/G mutant still activated MAPKs and the NF-kappaB pathway and selectively stimulated the production of MIP1alpha, but not that of IFN-gamma or IL-8. In conclusion, we provide evidence that the activating functions of 2DL4 can be compartmentalized into two distinct structural modules: 1) through transmembrane association with FcepsilonRI-gamma; and 2) through another receptor domain independent of the transmembrane arginine.

Anti-CD20 antibody induces the improvement of cytokine-induced killer cell activity via the STAT and MAPK/ERK signaling pathways

PubMed Central

DENG, QI; BAI, XUE; LV, HAI-RONG; XIAO, XIA; ZHAO, MING-FENG; LI, YU-MING

2015-01-01

There is a current requirement for novel therapeutic strategies for the treatment of hematopoietic tumors. Residual tumor cells are the main origin of tumor relapse. The aim of this study was to eliminate the residual tumor cells of hematopoietic tumors. Cytokine-induced killer (CIK) cells are used in immunotherapy to deplete the residual cells. However, it is necessary to increase the antitumor activity and clinical applicability of CIK cells. The present study investigated the antitumor activity of CIK cells to the SU-DHL2 human B-cell lymphoma and K562 human chronic myelogenous leukemia cell lines. CD3+CD56+ cells from healthy donors were expanded in culture with cytokines and anti-CD20 monoclonal antibody (mAb; rituximab) to generate CIK cells. A preliminary investigation of their mechanism was then performed. The increase in the cytotoxicity of the CIK cells induced by the anti-CD20 mAb was associated with an increase in the expression of cytotoxic factors. The expression of components of the signal transducer and activator of transcription (STAT) and mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) signaling pathways was found to increase. Upregulation of the expression of STAT1, STAT3 and STAT5 is important as these co-stimulatory molecules enhance T-cell proliferation. Activation of the MAPK signaling pathway is a possible mechanism for the anti-apoptosis effect on the proliferation of CIK cells. In conclusion, anti-CD20 mAb may play an important role in the improvement of CIK-mediated cytotoxicity to tumor cells. These observations may aid in the improvement of the effects of immunotherapy in depleting the residual cells of hematopoietic tumors. Thus, the use of CIK cells cultured with anti-CD20 mAb could be a novel therapeutic strategy for the depletion of chemotherapy-resistant or residual cells in anaplastic large and B-cell lymphoma. PMID:25780412

MEK-ERK pathway modulation ameliorates disease phenotypes in a mouse model of Noonan syndrome associated with the Raf1L613V mutation

PubMed Central

Wu, Xue; Simpson, Jeremy; Hong, Jenny H.; Kim, Kyoung-Han; Thavarajah, Nirusha K.; Backx, Peter H.; Neel, Benjamin G.; Araki, Toshiyuki

2011-01-01

Hypertrophic cardiomyopathy (HCM) is a leading cause of sudden death in children and young adults. Abnormalities in several signaling pathways are implicated in the pathogenesis of HCM, but the role of the RAS-RAF-MEK-ERK MAPK pathway has been controversial. Noonan syndrome (NS) is one of several autosomal-dominant conditions known as RASopathies, which are caused by mutations in different components of this pathway. Germline mutations in RAF1 (which encodes the serine-threonine kinase RAF1) account for approximately 3%–5% of cases of NS. Unlike other NS alleles, RAF1 mutations that confer increased kinase activity are highly associated with HCM. To explore the pathogenesis of such mutations, we generated knockin mice expressing the NS-associated Raf1L613V mutation. Like NS patients, mice heterozygous for this mutation (referred to herein as L613V/+ mice) had short stature, craniofacial dysmorphia, and hematologic abnormalities. Valvuloseptal development was normal, but L613V/+ mice exhibited eccentric cardiac hypertrophy and aberrant cardiac fetal gene expression, and decompensated following pressure overload. Agonist-evoked MEK-ERK activation was enhanced in multiple cell types, and postnatal MEK inhibition normalized the growth, facial, and cardiac defects in L613V/+ mice. These data show that different NS genes have intrinsically distinct pathological effects, demonstrate that enhanced MEK-ERK activity is critical for causing HCM and other RAF1-mutant NS phenotypes, and suggest a mutation-specific approach to the treatment of RASopathies. PMID:21339642

A Tumor Cell-Selective Inhibitor of Mitogen-Activated Protein Kinase Phosphatases Sensitizes Breast Cancer Cells to Lymphokine-Activated Killer Cell Activity

PubMed Central

Kaltenmeier, Christof T.; Vollmer, Laura L.; Vernetti, Lawrence A.; Caprio, Lindsay; Davis, Keanu; Korotchenko, Vasiliy N.; Day, Billy W.; Tsang, Michael; Hulkower, Keren I.; Lotze, Michael T.

2017-01-01

Dual specificity mitogen-activated protein kinase (MAPK) phosphatases [dual specificity phosphatase/MAP kinase phosphatase (DUSP-MKP)] have been hypothesized to maintain cancer cell survival by buffering excessive MAPK signaling caused by upstream activating oncogenic products. A large and diverse body of literature suggests that genetic depletion of DUSP-MKPs can reduce tumorigenicity, suggesting that hyperactivating MAPK signaling by DUSP-MKP inhibitors could be a novel strategy to selectively affect the transformed phenotype. Through in vivo structure-activity relationship studies in transgenic zebrafish we recently identified a hyperactivator of fibroblast growth factor signaling [(E)-2-benzylidene-5-bromo-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one (BCI-215)] that is devoid of developmental toxicity and restores defective MAPK activity caused by overexpression of DUSP1 and DUSP6 in mammalian cells. Here, we hypothesized that BCI-215 could selectively affect survival of transformed cells. In MDA-MB-231 human breast cancer cells, BCI-215 inhibited cell motility, caused apoptosis but not primary necrosis, and sensitized cells to lymphokine-activated killer cell activity. Mechanistically, BCI-215 induced rapid and sustained phosphorylation of extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK) in the absence of reactive oxygen species, and its toxicity was partially rescued by inhibition of p38 but not JNK or ERK. BCI-215 also hyperactivated MKK4/SEK1, suggesting activation of stress responses. Kinase phosphorylation profiling documented BCI-215 selectively activated MAPKs and their downstream substrates, but not receptor tyrosine kinases, SRC family kinases, AKT, mTOR, or DNA damage pathways. Our findings support the hypothesis that BCI-215 causes selective cancer cell cytotoxicity in part through non-redox-mediated activation of MAPK signaling, and the findings also identify an intersection with immune cell killing that is worthy of further exploration. PMID:28154014

Excessive L-cysteine induces vacuole-like cell death by activating endoplasmic reticulum stress and mitogen-activated protein kinase signaling in intestinal porcine epithelial cells.

PubMed

Ji, Yun; Wu, Zhenlong; Dai, Zhaolai; Sun, Kaiji; Zhang, Qing; Wu, Guoyao

2016-01-01

High intake of dietary cysteine is extremely toxic to animals and the underlying mechanism remains largely unknown. This study was conducted to test the hypothesis that excessive L-cysteine induces cell death by activating endoplasmic reticulum (ER) stress and mitogen-activated protein kinase (MAPK) signaling in intestinal porcine epithelial cells. Jejunal enterocytes were cultured in the presence of 0-10 mmol/L L-cysteine. Cell viability, morphologic alterations, mRNA levels for genes involved in ER stress, protein abundances for glucose-regulated protein 78, C/EBP homologous protein (CHOP), alpha subunit of eukaryotic initiation factor-2 (eIF2α), extracellular signal-regulated kinase (ERK1/2), p38 MAPK, and c-Jun N-terminal protein kinase (JNK1/2) were determined. The results showed that L-cysteine (5-10 mmol/L) reduced cell viability (P < 0.05) and led to vacuole-like cell death in intestinal porcine epithelial cells. These adverse effects of L-cysteine were not affected by the autophagy inhibitor 3-methyladenine. The protein abundances for CHOP, phosphorylated (p)-eIF2α, p-JNK1/2, p-p38 MAPK, and the spliced form of XBP-1 mRNA were enhanced (P < 0.05), whereas those for p-ERK1/2 were reduced (P < 0.05). Collectively, excessive L-cysteine induces vacuole-like cell death via the activation of ER stress and MAPK signaling in small intestinal epithelial cells. These signaling pathways may be potential targets for developing effective strategies to prevent the toxicity of dietary cysteine.

Effect of mitogen-activated protein kinases on chemokine synthesis induced by substance P in mouse pancreatic acinar cells

PubMed Central

Ramnath, Raina Devi; Sun, Jia; Adhikari, Sharmila; Bhatia, Madhav

2007-01-01

Abstract Substance P, acting via its neurokinin 1 receptor (NK1 R), plays an important role in mediating a variety of inflammatory processes. Its interaction with chemokines is known to play a crucial role in the pathogenesis of acute pancreatitis. In pancreatic acinar cells, substance P stimulates the release of NFκB-driven chemokines. However, the signal transduction pathways by which substance P-NK1 R interaction induces chemokine production are still unclear. To that end, we went on to examine the participation of mitogen-activated protein kinases (MAPKs) in substance P-induced synthesis of pro-inflammatory chemokines, monocyte chemoanractant protein-1 (MCP-I), macrophage inflammatory protein-lα (MIP-lα) and macrophage inflammatory protein-2 (MIP-2), in pancreatic acini. In this study, we observed a time-dependent activation of ERK1/2, c-Jun N-terminal kinase (JNK), NFκB and activator protein-1 (AP-1) when pancreatic acini were stimulated with substance P. Moreover, substance P-induced ERK 1/2, JNK, NFκB and AP-1 activation as well as chemokine synthesis were blocked by pre-treatment with either extracellular signal-regulated protein kinase kinase 1 (MEK1) inhibitor or JNK inhibitor. In addition, substance P-induced activation of ERK 112, JNK, NFκB and AP-1-driven chemokine production were attenuated by CP96345, a selective NK1 R antagonist, in pancreatic acinar cells. Taken together, these results suggest that substance P-NK1 R induced chemokine production depends on the activation of MAPKs-mediated NFκB and AP-1 signalling pathways in mouse pancreatic acini. PMID:18205703

Globular adiponectin induces a pro-inflammatory response in human astrocytic cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wan, Zhongxiao; Mah, Dorrian; Simtchouk, Svetlana

Highlights: • Adiponectin receptors are expressed in human astrocytes. • Globular adiponectin induces secretion of IL-6 and MCP-1 from cultured astrocytes. • Adiponectin may play a pro-inflammatory role in astrocytes. - Abstract: Neuroinflammation, mediated in part by activated brain astrocytes, plays a critical role in the development of neurodegenerative disorders, including Alzheimer’s disease (AD). Adiponectin is the most abundant adipokine secreted from adipose tissue and has been reported to exert both anti- and pro-inflammatory effects in peripheral tissues; however, the effects of adiponectin on astrocytes remain unknown. Shifts in peripheral concentrations of adipokines, including adiponectin, could contribute to the observedmore » link between midlife adiposity and increased AD risk. The aim of the present study was to characterize the effects of globular adiponectin (gAd) on pro-inflammatory cytokine mRNA expression and secretion in human U373 MG astrocytic cells and to explore the potential involvement of nuclear factor (NF)-κB, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK)1/2, c-Jun N-terminal kinase (JNK) and phosphatidylinositide 3-kinases (PI3 K) signaling pathways in these processes. We demonstrated expression of adiponectin receptor 1 (adipoR1) and adipoR2 in U373 MG cells and primary human astrocytes. gAd induced secretion of interleukin (IL)-6 and monocyte chemoattractant protein (MCP)-1, and gene expression of IL-6, MCP-1, IL-1β and IL-8 in U373 MG cells. Using specific inhibitors, we found that NF-κB, p38MAPK and ERK1/2 pathways are involved in gAd-induced induction of cytokines with ERK1/2 contributing the most. These findings provide evidence that gAd may induce a pro-inflammatory phenotype in human astrocytes.« less

Conserved Regulation of MAP Kinase Expression by PUF RNA-Binding Proteins

PubMed Central

Lee, Myon-Hee; Hook, Brad; Pan, Guangjin; Kershner, Aaron M; Merritt, Christopher; Seydoux, Geraldine; Thomson, James A; Wickens, Marvin; Kimble, Judith

2007-01-01

Mitogen-activated protein kinase (MAPK) and PUF (for Pumilio and FBF [fem-3 binding factor]) RNA-binding proteins control many cellular processes critical for animal development and tissue homeostasis. In the present work, we report that PUF proteins act directly on MAPK/ERK-encoding mRNAs to downregulate their expression in both the Caenorhabditis elegans germline and human embryonic stem cells. In C. elegans, FBF/PUF binds regulatory elements in the mpk-1 3′ untranslated region (3′ UTR) and coprecipitates with mpk-1 mRNA; moreover, mpk-1 expression increases dramatically in FBF mutants. In human embryonic stem cells, PUM2/PUF binds 3′UTR elements in both Erk2 and p38α mRNAs, and PUM2 represses reporter constructs carrying either Erk2 or p38α 3′ UTRs. Therefore, the PUF control of MAPK expression is conserved. Its biological function was explored in nematodes, where FBF promotes the self-renewal of germline stem cells, and MPK-1 promotes oocyte maturation and germ cell apoptosis. We found that FBF acts redundantly with LIP-1, the C. elegans homolog of MAPK phosphatase (MKP), to restrict MAPK activity and prevent apoptosis. In mammals, activated MAPK can promote apoptosis of cancer cells and restrict stem cell self-renewal, and MKP is upregulated in cancer cells. We propose that the dual negative regulation of MAPK by both PUF repression and MKP inhibition may be a conserved mechanism that influences both stem cell maintenance and tumor progression. PMID:18166083

Molecular Analysis of Neurotoxin-Induced Apoptosis

DTIC Science & Technology

2006-03-01

Chem. 270, 27489–27494. Anderson C. N. and Tolkovsky A. M. (1999) A role for MAPK/ERK in sympathetic neuron survival: protection against a p53 ...Kaplan D. (1999) Ras regulates sympathetic neuron survival by suppressing the p53 -mediated cell death pathway. J. Neurosci. 19, 9716–9727. Mikula M...for 30min. ProteinA /G-agarose beadswere added to themixture, and further incubation was carried out at 4 °C overnight. After incuba- tion, beads were

Connexin 32 and its derived homotypic gap junctional intercellular communication inhibit the migration and invasion of transfected HeLa cells via enhancement of intercellular adhesion.

PubMed

Yang, Jie; Liu, Bing; Wang, Qin; Yuan, Dongdong; Hong, Xiaoting; Yang, Yan; Tao, Liang

2011-01-01

The effects of connexin (Cx) and its derived homotypic gap junctional intercellular communication (GJIC) between tumor cells on the invasion of metastatic cancers and the underlying mechanisms remain unclear. In this study, we investigated the influence of Cx32 and the homotypic GJIC mediated by this Cx on the migration, invasion and intercellular adhesion of transfected HeLa cells. The expression of Cx32 significantly increased cell adhesion and inhibited migration and invasion. The inhibition of GJIC by oleamide, a widely used GJIC inhibitor, reduced the enhanced adhesion and partly reversed the decreased migration and invasion that had been induced by Cx32 expression. Blockage of the p38 and extracellular signal-regulated kinase 1 and 2 mitogen-activated protein kinase (ERK1/2 MAPKs) pathways using their specific inhibitors attenuated the effects of Cx32, but not those of GJIC, on cell adhesion, migration and invasion. These results indicate that the homotypic GJIC mediated by Cx32, as well as the Cx itself, inhibit cell migration and invasion, most likely through the elevation of intercellular adhesion. The suppressive effect of Cx32 on the migration and invasion of cancer cells, but not that of its derived homotypic GJIC, partly depends on the activation of the p38 and the ERK1/2 MAPKs pathways.

Apigenin Induces the Apoptosis and Regulates MAPK Signaling Pathways in Mouse Macrophage ANA-1 Cells

PubMed Central

Liao, Yuexia; Shen, Weigan; Kong, Guimei; Lv, Houning; Tao, Wenhua; Bo, Ping

2014-01-01

Apigenin is a naturally occurring plant flavonoid that possesses antioxidant, anti-cancer and anti-inflammatory properties. However, there are few reports has been done on the ability of apigenin to induce apoptosis in macrophages. In this study, mouse macrophage ANA-1 cells were incubated with different concentrations of apigenin. The cell viability was determined by an MTT assay. The cell apoptosis were analyzed by flow cytometric analysis. Apoptosis were also analyzed using a TUNEL assay and a DNA ladder. The level of intracellular ROS was detected using a dichlorofluorescein -diacetate probe. The expression levels of apoptosis-related proteins were detected by western blot analysis. The results showed that apigenin decreased the viability of ANA-1 cells and induced apoptosis in a dose- and time-dependent manner. Apigenin increased the level of intracellular ROS, downregulated the expression of Bcl-2 and upregulated the expression of caspase-3 and caspase-8 in ANA-1 cells. Furthermore, apigenin downregulated the expression of phospho-ERK and phospho-JNK, upregulated the expression of phospho-p38 and had no significant effect on the expression of Bax, ERK, JNK and p38. The results suggested that apigenin induced cell apoptosis in mouse macrophage ANA-1 cells may via increasing intracellular ROS, regulating the MAPK pathway, and then inhibiting Bcl-2 expression. PMID:24646936

Astragaloside IV inhibits progression of glioma via blocking MAPK/ERK signaling pathway.

PubMed

Li, Bin; Wang, Fei; Liu, Ningtao; Shen, Wen; Huang, Tao

2017-09-09

Glioma is one of the most common primary brain tumors in adults with a high mortality rate and relapse rate. Thus, finding better effective approaches to treat glioma has become very urgent. Astragaloside IV (AS-IV), the major active triterpenoid in Radix Astragali, has shown anti-tumorigenic properties in certain cancers. However, its role in glioma remains unclear. Here, we studied the effects of AS-IV on glioma in vitro and in vivo, and explored the underlying mechanisms. Our results revealed that AS-IV dose-dependently inhibited the proliferation of U251 cells in vitro and attenuated tumor growth in vivo. In addition, the migration and invasion ability of U251 cell has been suppressed in presence of AS-IV. The levels of proliferating cell nuclear antigen (PCNA), Ki67, matrix metallopeptidase (MMP) -2, MMP-9 and vascular endothelial growth factor (VEGF) were decreased significantly by the treatment of different concentrations AS-IV. Furthermore, AS-IV also significantly weakened the activation of Mitogen-activated protein kinase/Extracellular regulated protein kinase (MAPK/ERK) signaling pathway in vitro and in vivo. Taken together our study has identified a novel function of AS-IV and provided a molecular basis for AS-IV potential applications in the treatment of glioma and other cancers. Copyright © 2017 Elsevier Inc. All rights reserved.

Kaempferol Attenuates Myocardial Ischemic Injury via Inhibition of MAPK Signaling Pathway in Experimental Model of Myocardial Ischemia-Reperfusion Injury.

PubMed

Suchal, Kapil; Malik, Salma; Gamad, Nanda; Malhotra, Rajiv Kumar; Goyal, Sameer N; Chaudhary, Uma; Bhatia, Jagriti; Ojha, Shreesh; Arya, Dharamvir Singh

2016-01-01

Kaempferol (KMP), a dietary flavonoid, has antioxidant, anti-inflammatory, and antiapoptotic effects. Hence, we investigated the effect of KMP in ischemia-reperfusion (IR) model of myocardial injury in rats. We studied male albino Wistar rats that were divided into sham, IR-control, KMP-20 + IR, and KMP 20 per se groups. KMP (20 mg/kg; i.p.) was administered daily to rats for the period of 15 days, and, on the 15th day, ischemia was produced by one-stage ligation of left anterior descending coronary artery for 45 min followed by reperfusion for 60 min. After completion of surgery, rats were sacrificed; heart was removed and processed for biochemical, morphological, and molecular studies. KMP pretreatment significantly ameliorated IR injury by maintaining cardiac function, normalizing oxidative stress, and preserving morphological alterations. Furthermore, there was a decrease in the level of inflammatory markers (TNF-α, IL-6, and NFκB), inhibition of active JNK and p38 proteins, and activation of ERK1/ERK2, a prosurvival kinase. Additionally, it also attenuated apoptosis by reducing the expression of proapoptotic proteins (Bax and Caspase-3), TUNEL positive cells, and increased level of antiapoptotic proteins (Bcl-2). In conclusion, KMP protected against IR injury by attenuating inflammation and apoptosis through the modulation of MAPK pathway.

Tormentic acid inhibits LPS-induced inflammatory response in human gingival fibroblasts via inhibition of TLR4-mediated NF-κB and MAPK signalling pathway.

PubMed

Jian, Cong-Xiang; Li, Ming-Zhe; Zheng, Wei-Yin; He, Yong; Ren, Yu; Wu, Zhong-Min; Fan, Quan-Shui; Hu, Yong-He; Li, Chen-Jun

2015-09-01

Periodontal disease is one of the most prevalent oral diseases, which is associated with inflammation of the tooth-supporting tissues. Tormentic acid (TA), a triterpene isolated from Rosa rugosa, has been reported to exert anti-inflammatory effects. The aim of this study was to investigate the anti-inflammatory effects of TA on lipopolysaccharide (LPS)-stimulated human gingival fibroblasts (HGFs). The levels of inflammatory cytokines such as interleukin (IL)-6 and chemokines such as IL-8 were detected by enzyme-linked immunosorbent assay (ELISA). The expression of Toll-like receptor 4 (TLR4), nuclear factor kappa B (NF-κB), IκBα, p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) was determined by Western blotting. The results showed that Porphyromonas gingivalis LPS significantly upregulated the expression of IL-6 and IL-8. TA inhibited the LPS-induced production of IL-6 and IL-8 in a dose-dependent manner. Furthermore, TA inhibited LPS-induced TLR4 expression; NF-κB activation; IκBα degradation; and phosphorylation of ERK, JNK, and P38. TA inhibits the LPS-induced inflammatory response in HGFs by suppressing the TLR4-mediated NF-κB and mitogen-activated protein kinase (MAPK) signalling pathway. Copyright © 2015 Elsevier Ltd. All rights reserved.

l-Theanine prevents ETEC-induced liver damage by reducing intrinsic apoptotic response and inhibiting ERK1/2 and JNK1/2 signaling pathways.

PubMed

Gong, Zhihua; Liu, Qiuling; Lin, Ling; Deng, Yanli; Cai, Shuxian; Liu, Zunying; Zhang, Sheng; Xiao, Wenjun; Xiong, Shuo; Chen, Dong

2018-01-05

l-Theanine (LTA; γ-glutamylethylamide), a peculiar non-protein-derived amino acid isolated from tea, is widely used as a functional ingredient and dietary supplement. l-Theanine has been confirmed to have hepatoprotective effects, but the underlying mechanism remains unknown. This study investigated the protective effect of l-Theanine-in vivo, using an enterotoxigenic Escherichia coli (ETEC)-infected mouse model. l-Theanine significantly decreased the elevated serum activities of both aspartate aminotransferase (AST) and alanine aminotransferase (ALT), two biomarkers of hepatic impairment. This was consistent with histopathological images from the microscopic observation of liver tissue. In addition, l-theanine significantly increased the mRNA and protein expression of Bcl-2 and decreased the expression of Bax, anti- and pro-apoptotic molecules, respectively, compared with levels in the ETEC control group. The expression of cleaved caspase-3 protein in the group pre-treated with l-theanine was significantly lower than that in the ETEC group. Additionally, decreases in extracellular signal-regulated kinase (ERK1/2) and c-Jun NH 2 -terminal kinase(JNK1/2) MAPK phosphorylation were observed in the l-theanine pre-treated group. Our study demonstrates that l-theanine possesses anti-apoptotic activity, which can be attributed to suppression of the intrinsic mitochondria-mediated apoptosis and MAPK phosphorylation signaling pathways. Copyright © 2017 Elsevier B.V. All rights reserved.

The Effects of Aronia melanocarpa 'Viking' Extracts in Attenuating RANKL-Induced Osteoclastic Differentiation by Inhibiting ROS Generation and c-FOS/NFATc1 Signaling.

PubMed

Ghosh, Mithun; Kim, In Sook; Lee, Young Min; Hong, Seong Min; Lee, Taek Hwan; Lim, Ji Hong; Debnath, Trishna; Lim, Beong Ou

2018-03-08

This study aimed to determine the anti-osteoclastogenic effects of extracts from Aronia melanocarpa 'Viking' (AM) and identify the underlying mechanisms in vitro. Reactive oxygen species (ROS) are signal mediators in osteoclast differentiation. AM extracts inhibited ROS production in RAW 264.7 cells in a dose-dependent manner and exhibited strong radical scavenging activity. The extracts also attenuated the number of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated osteoclasts. To attain molecular insights, the effect of the extracts on the signaling pathways induced by receptor activator of nuclear factor kappa B ligand (RANKL) were also investigated. RANKL triggers many transcription factors through the activation of mitogen-activated protein kinase (MAPK) and ROS, leading to the induction of osteoclast-specific genes. The extracts significantly suppressed RANKL-induced activation of MAPKs, such as extracellular signal-regulated kinase (ERK), c-Jun- N -terminal kinase (JNK) and p38 and consequently led to the downregulation of c-Fos and nuclear factor of activated T cells 1 (NFATc1) protein expression which ultimately suppress the activation of the osteoclast-specific genes, cathepsin K, TRAP, calcitonin receptor and integrin β₃. In conclusion, our findings suggest that AM extracts inhibited RANKL-induced osteoclast differentiation by downregulating ROS generation and inactivating JNK/ERK/p38, nuclear factor kappa B (NF-κB)-mediated c-Fos and NFATc1 signaling pathway.

Enterolactone modulates the ERK/NF-κB/Snail signaling pathway in triple-negative breast cancer cell line MDA-MB-231 to revert the TGF-β-induced epithelial-mesenchymal transition.

PubMed

Mali, Aniket V; Joshi, Asavari A; Hegde, Mahabaleshwar V; Kadam, Shivajirao S

2018-05-01

Triple-negative breast cancer (TNBC) is highly metastatic, and there is an urgent unmet need to develop novel therapeutic strategies leading to the new drug discoveries against metastasis. The transforming growth factor-β (TGF-β) is known to promote the invasive and migratory potential of breast cancer cells through induction of epithelial-mesenchymal transition (EMT) via the ERK/NF-κB/Snail signaling pathway, leading to breast cancer metastasis. Targeting this pathway to revert the EMT would be an attractive, novel therapeutic strategy to halt breast cancer metastasis. Effects of enterolactone (EL) on the cell cycle and apoptosis were investigated using flow cytometry and a cleaved caspase-3 enzyme-linked immunosorbent assay (ELISA), respectively. Effects of TGF-β induction and EL treatment on the functional malignancy of MDA-MB-231 breast cancer cells were investigated using migration and chemo-invasion assays. The effects of EL on EMT markers and the ERK/NF-κB/Snail signaling pathway after TGF-β induction were studied using confocal microscopy, quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot, and flow cytometry. Herein, we report that EL exhibits a significant antimetastatic effect on MDA-MB-231 cells by almost reverting the TGF-β-induced EMT in vitro . EL downregulates the mesenchymal markers N-cadherin and vimentin, and upregulates the epithelial markers E-cadherin and occludin. It represses actin stress fiber formation via inhibition of mitogen-activated protein kinase p-38 (MAPK-p38) and cluster of differentiation 44 (CD44). EL also suppresses ERK-1/2, NF-κB, and Snail at the mRNA and protein levels. Briefly, EL was found to inhibit TGF-β-induced EMT by blocking the ERK/NF-κB/Snail signaling pathway, which is a promising target for breast cancer metastasis therapy.

Involvement of the MAPK and PI3K pathways in chitinase 3-like 1-regulated hyperoxia-induced airway epithelial cell death

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kim, Mi Na; Lee, Kyung Eun; Hong, Jung Yeon

Highlights: Black-Right-Pointing-Pointer Hyperoxia induces apoptosis and chitinase 3-like 1 expression in human airway epithelial cells. Black-Right-Pointing-Pointer Presence of chitinase 3-like 1 affects airway epithelial cell death after hyperoxic exposure. Black-Right-Pointing-Pointer Silencing chitinase 3-like 1 manipulate the phosphorylation of ERK, p38 and Akt. -- Abstract: Background: Exposure to 100% oxygen causes hyperoxic acute lung injury characterized by cell death and injury of alveolar epithelial cells. Recently, the role of chitinase 3-like 1 (CHI3L1), a member of the glycosyl hydrolase 18 family that lacks chitinase activity, in oxidative stress was demonstrated in murine models. High levels of serum CHI3L1 have been associatedmore » with various diseases of the lung, such as asthma, chronic obstructive pulmonary disease, and cancer. However, the role of CHI3L1 in human airway epithelial cells undergoing oxidative stress remains unknown. In addition, the signaling pathways associated with CHI3L1 in this process are poorly understood. Purpose: In this study, we demonstrate the role of CHI3L1, along with the MAPK and PI3K signaling pathways, in hyperoxia-exposed airway epithelial cells. Method: The human airway epithelial cell line, BEAS-2B, was exposed to >95% oxygen (hyperoxia) for up to 72 h. Hyperoxia-induced cell death was determined by assessing cell viability, Annexin-V FITC staining, caspase-3 and -7 expression, and electron microscopy. CHI3L1 knockdown and overexpression studies were conducted in BEAS-2B cells to examine the role of CHI3L1 in hyperoxia-induced apoptosis. Activation of the MAPK and PI3K pathways was also investigated to determine the role of these signaling cascades in this process. Results: Hyperoxia exposure increased CHI3L1 expression and apoptosis in a time-dependent manner. CHI3L1 knockdown protected cells from hyperoxia-induced apoptosis. In contrast, CHI3L1 overexpression promoted cell death after hyperoxia exposure. Finally, phosphorylation of ERK1/2, p38, and Akt were affected by CHI3L1 knockdown. Conclusion: This study indicates that CHI3L1 is involved in hyperoxia-induced cell death, suggesting that CHI3L1 may be one of several cell death regulators influencing the MAPK and PI3K pathways during oxidative stress in human airway epithelial cells.« less

Calcium Signaling Is Involved in Cadmium-Induced Neuronal Apoptosis via Induction of Reactive Oxygen Species and Activation of MAPK/mTOR Network

PubMed Central

Luo, Yan; Chen, Zi; Liu, Lei; Zhou, Hongyu; Chen, Wenxing; Shen, Tao; Han, Xiuzhen; Chen, Long; Huang, Shile

2011-01-01

Cadmium (Cd), a toxic environmental contaminant, induces oxidative stress, leading to neurodegenerative disorders. Recently we have demonstrated that Cd induces neuronal apoptosis in part by activation of the mitogen-activated protein kineses (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains elusive. Here we show that Cd elevated intracellular calcium ion ([Ca2+]i) level in PC12, SH-SY5Y cells and primary murine neurons. BAPTA/AM, an intracellular Ca2+ chelator, abolished Cd-induced [Ca2+]i elevation, and blocked Cd activation of MAKPs including extracellular signal-regulated kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK) and p38, and mTOR-mediated signaling pathways, as well as cell death. Pretreatment with the extracellular Ca2+ chelator EGTA also prevented Cd-induced [Ca2+]i elevation, MAPK/mTOR activation, as well as cell death, suggesting that Cd-induced extracellular Ca2+ influx plays a critical role in contributing to neuronal apoptosis. In addition, calmodulin (CaM) antagonist trifluoperazine (TFP) or silencing CaM attenuated the effects of Cd on MAPK/mTOR activation and cell death. Furthermore, Cd-induced [Ca2+]i elevation or CaM activation resulted in induction of reactive oxygen species (ROS). Pretreatment with BAPTA/AM, EGTA or TFP attenuated Cd-induced ROS and cleavage of caspase-3 in the neuronal cells. Our findings indicate that Cd elevates [Ca2+]i, which induces ROS and activates MAPK and mTOR pathways, leading to neuronal apoptosis. The results suggest that regulation of Cd-disrupted [Ca2+]i homeostasis may be a new strategy for prevention of Cd-induced neurodegenerative diseases. PMID:21544200

Role of the MAPK pathway in the observed bystander effect in lymphocytes co-cultured with macrophages irradiated with γ-rays or carbon ions.

PubMed

Dong, Chen; He, Mingyuan; Ren, Ruiping; Xie, Yuexia; Yuan, Dexiao; Dang, Bingrong; Li, Wenjian; Shao, Chunlin

2015-04-15

The radiation-induced bystander effect (RIBE) has potential implications in cancer risks from space particle radiation; however, the mechanisms underlying RIBE are unclear. The role of the MAPK pathway in the RIBEs of different linear energy transfer (LET) was investigated. Human macrophage U937 cells were irradiated with γ-rays or carbon ions and then co-cultured with nonirradiated HMy2.CIR (HMy) lymphocytes for different periods. The activation of MAPK proteins and the generation of intracellular nitric oxide (NO) and reactive oxygen species (ROS) in the irradiated U937 cells were measured. Micronuclei (MN) formation in the HMy cells was applied to evaluate the bystander damage. Some U937 cells were pretreated with different MAPK inhibitors before irradiation. Additional MN formation was induced in the HMy cells after co-culturing with irradiated U937 cells, and the yield of this bystander MN formation was dependent on the co-culture period with γ-ray irradiation but remained high after 1h of co-culture with carbon irradiation. Further investigations disclosed that the time response of the RIBEs had a relationship with LET, where ERK played a different role from JNK and p38 in regulating RIBEs by regulating the generation of the bystander signaling factors NO and ROS. The finding that the RIBE of high-LET radiation could persist for a much longer period than that of γ-rays implies that particle radiation during space flight could have a high risk of long-term harmful effects. An appropriate intervention targeting the MAPK pathway may have significant implications in reducing this risk. Copyright © 2015 Elsevier Inc. All rights reserved.

Structural assembly of the signaling competent ERK2–RSK1 heterodimeric protein kinase complex

PubMed Central

Alexa, Anita; Gógl, Gergő; Glatz, Gábor; Garai, Ágnes; Zeke, András; Varga, János; Dudás, Erika; Jeszenői, Norbert; Bodor, Andrea; Hetényi, Csaba; Reményi, Attila

2015-01-01

Mitogen-activated protein kinases (MAPKs) bind and activate their downstream kinase substrates, MAPK-activated protein kinases (MAPKAPKs). Notably, extracellular signal regulated kinase 2 (ERK2) phosphorylates ribosomal S6 kinase 1 (RSK1), which promotes cellular growth. Here, we determined the crystal structure of an RSK1 construct in complex with its activator kinase. The structure captures the kinase–kinase complex in a precatalytic state where the activation loop of the downstream kinase (RSK1) faces the enzyme's (ERK2) catalytic site. Molecular dynamics simulation was used to show how this heterodimer could shift into a signaling-competent state. This structural analysis combined with biochemical and cellular studies on MAPK→MAPKAPK signaling showed that the interaction between the MAPK binding linear motif (residing in a disordered kinase domain extension) and the ERK2 “docking” groove plays the major role in making an encounter complex. This interaction holds kinase domains proximal as they “readjust,” whereas generic kinase domain surface contacts bring them into a catalytically competent state. PMID:25730857

Role of Sigma-1 Receptor/p38 MAPK Inhibition in Acupoint Catgut Embedding-Mediated Analgesic Effects in Complete Freund's Adjuvant-Induced Inflammatory Pain.

PubMed

Du, Kairong; Wang, Xue; Chi, Laiting; Li, Wenzhi

2017-08-01

The endoplasmic reticulum chaperone protein Sigma-1 receptor (Sig-1 R) and mitogen-activated protein kinases (MAPKs) are involved in the mechanism of pain. Acupoint stimulation exerts an exact antihyperalgesic effect in inflammatory pain. However, whether Sig-1 R and MAPKs are associated with the acupoint stimulation-induced analgesic effects is not clear. This study investigated the analgesic effect of acupoint catgut embedding (ACE) and the inhibition of Sig-1 R and MAPKs in ACE analgesia. Rats were prepared with intrathecal catheter implantation. ACE was applied to bilateral "Kunlun" (BL60), "Zusanli" (ST36), and "Sanyinjiao" (SP6) acupoints in the rat model of inflammatory pain (complete Freund's adjuvant [CFA] intraplantar injection). Then, Sig-1R agonist PRE084 or saline was intrathecally given daily. The paw withdrawal thresholds and paw edema were measured before CFA injection and at 1, 3, and 5 day after CFA injection. Western bolt was used to evaluate the protein expression of spinal Sig-1R, p38MAPK, and extracellular signal-regulated kinase (ERK), and immunohistochemistry of Sig-1R was detected at 1, 3, and 5 days after CFA injection. ACE exhibited specific analgesic effects. ACE increased paw withdrawal thresholds and markedly decreased CFA-induced paw edema at 1, 3, and 5 days. ACE downregulated the protein expression of Sig-1R, which was increased significantly at 1, 3, and 5 days after CFA injection. ACE decreased the expression of p38 MAPK and ERK at 1 and 3 days but not at 5 days. However, an injection of Sig-1R agonist PRE084 markedly reversed these alterations, except ERK expression. The present study demonstrated that ACE exhibited antihyperalgesic effects via the inhibition of the Sig-1R that modulated p38 MAPK, but not ERK, expression in the CFA-induced inflammatory pain model in rats.

CpG- and LPS-activated MAPK signaling in in vitro cultured salmon (Salmo salar) mononuclear phagocytes.

PubMed

Iliev, Dimitar B; Hansen, Tom; Jørgensen, Sven Martin; Krasnov, Aleksei; Jørgensen, Jorunn B

2013-10-01

The Mitogen-activated protein kinases (MAPK) are involved in transmitting intracellular signals downstream of diverse cell surface receptors and mediate the response to ligands such as growth factors, hormones and cytokines. In addition, MAPK are critically involved in the innate immune response to pathogen-derived substances, commonly referred to as pathogen-associated molecular patterns (PAMPs), such as bacterial lipopolysaccharide (LPS) and bacterial DNA rich in CpG dinucleotides. Currently, a great deal of knowledge is available about the involvement of MAPK in the innate immune response to PAMPs in mammals; however, little is known about the role of the different MAPK classes in the immune response to PAMPs in lower vertebrates. In the current study, p38 phosphorylation was induced by CpG oligonucleotides (ODNs) and LPS in primary salmon mononuclear phagocytes. Pre-treatment of the cells with a p38 inhibitor (SB203580) blocked the PAMP-induced p38 activity and suppressed the upregulation of most of the CpG- and LPS-induced transcripts highlighting the role of this kinase in the salmon innate immune response to PAMPs. In contrast to p38, the phosphorylation of extracellular signal-regulated kinase (ERK), a MAPK involved primarily in response to mitogens, was high in resting cells and, surprisingly, incubation with both CpG and control ODNs downregulated the phospho-ERK levels independently of p38 activation. The basal phospho-ERK level and the CpG-inducible p38 phosphorylation were greatly influenced by the length of in vitro incubation. The basal phospho-ERK level increased gradually throughout a 5-day culture period and was PI3K-dependent as demonstrated by its sensitivity to Wortmannin suggesting it is influenced by growth factors. Overall these data indicate that both basal and PAMP-induced activity of MAPKs might be greatly influenced by the differentiation status of salmon mononuclear phagocytes. Copyright © 2013. Published by Elsevier Ltd.

Involvement of PI3K/Akt, ERK and p38 signaling pathways in emodin-mediated extrinsic and intrinsic human hepatoblastoma cell apoptosis.

PubMed

Cui, Yuting; Lu, Peiran; Song, Ge; Liu, Qian; Zhu, Di; Liu, Xuebo

2016-06-01

As a natural anthraquinone derivative, 1,3,8-trihydroxy-6-methylanthraquinone, known as emodin, has recently been reported to possess potential chemopreventive capacity, but the underlying molecular mechanism of its hepatocyte toxicity remains poorly clarified. The present research indicated that emodin targeted HepG2 cells without being cytotoxic to primary human hepatocyte cells in comparison with chrysophanol and rhein. The anti-proliferative effect of emodin was ascribed to occurrence of apoptosis, which characterized by higher ethidium bromide signal, brighter DAPI fluorescence, cleavages of procaspase-3 and poly (ADP-ribose) polymerase as well as quantitative result from Annexin V-FITC/PI double staining. Furthermore, emodin improved Bax/Bcl-2 ratio, elicited disruption of mitochondrial membrane potential and promoted efflux of cytochrome c to cytosol, indicative of features of mitochondria-dependent apoptotic signals. Emodin concurrently led to activations of Fas, Fas-L, caspase-8 and tBid, which provoked death receptor apoptotic signals. Notably, activated tBid relayed the Fas apoptotic signal to the mitochondrial pathway. Besides, emodin effectively attenuated phosphorylations of Akt and ERK and promoted phosphorylation of p38. Inhibitions of PI3K/Akt and ERK and activation of p38 mediated emodin-induced apoptosis through modulating the mitochondrial pathway and/or death receptor pathway. Additionally, there was a cross-talk between PI3K/Akt and MAPKs pathways in emodin-induced apoptosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

ERK3 signals through SRC-3 coactivator to promote human lung cancer cell invasion

PubMed Central

Long, Weiwen; Foulds, Charles E.; Qin, Jun; Liu, Jian; Ding, Chen; Lonard, David M.; Solis, Luisa M.; Wistuba, Ignacio I.; Qin, Jun; Tsai, Sophia Y.; Tsai, Ming-Jer; O’Malley, Bert W.

2012-01-01

In contrast to the well-studied classic MAPKs, such as ERK1/2, little is known concerning the regulation and substrates of the atypical MAPK ERK3 signaling cascade and its function in cancer progression. Here, we report that ERK3 interacted with and phosphorylated steroid receptor coactivator 3 (SRC-3), an oncogenic protein overexpressed in multiple human cancers at serine 857 (S857). This ERK3-mediated phosphorylation at S857 was essential for interaction of SRC-3 with the ETS transcription factor PEA3, which promotes upregulation of MMP gene expression and proinvasive activity in lung cancer cells. Importantly, knockdown of ERK3 or SRC-3 inhibited the ability of lung cancer cells to invade and form tumors in the lung in a xenograft mouse model. In addition, ERK3 was found to be highly upregulated in human lung carcinomas. Our study identifies a previously unknown role for ERK3 in promoting lung cancer cell invasiveness by phosphorylating SRC-3 and regulating SRC-3 proinvasive activity by site-specific phosphorylation. As such, ERK3 protein kinase may be an attractive target for therapeutic treatment of invasive lung cancer. PMID:22505454

TGF-β induction of FGF-2 expression in stromal cells requires integrated smad3 and MAPK pathways.

PubMed

Strand, Douglas W; Liang, Yao-Yun; Yang, Feng; Barron, David A; Ressler, Steven J; Schauer, Isaiah G; Feng, Xin-Hua; Rowley, David R

2014-01-01

Transforming Growth Factor-β (TGF-β) regulates the reactive stroma microenvironment associated with most carcinomas and mediates expression of many stromal derived factors important for tumor progression, including FGF-2 and CTGF. TGF-β is over-expressed in most carcinomas, and FGF-2 action is important in tumor-induced angiogenesis. The signaling mechanisms of how TGF-β regulates FGF-2 expression in the reactive stroma microenvironment are not understood. Accordingly, we have assessed key signaling pathways that mediate TGF-β1-induced FGF-2 expression in prostate stromal fibroblasts and mouse embryo fibroblasts (MEFs) null for Smad2 and Smad3. TGF-β1 induced phosphorylation of Smad2, Smad3, p38 and ERK1/2 proteins in both control MEFs and prostate fibroblasts. Of these, Smad3, but not Smad2 was found to be required for TGF-β1 induction of FGF-2 expression in stromal cells. ChIP analysis revealed a Smad3/Smad4 complex was associated with the -1.9 to -2.3 kb upstream proximal promoter of the FGF-2 gene, further suggesting a Smad3-specific regulation. In addition, chemical inhibition of p38 or ERK1/2 MAPK activity also blocked TGF-β1-induced FGF-2 expression in a Smad3-independent manner. Conversely, inhibition of JNK signaling enhanced FGF-2 expression. Together, these data indicate that expression of FGF-2 in fibroblasts in the tumor stromal cell microenvironment is coordinately dependent on both intact Smad3 and MAP kinase signaling pathways. These pathways and key downstream mediators of TGF-β action in the tumor reactive stroma microenvironment, may evolve as putative targets for therapeutic intervention.

MiR-103 alleviates autophagy and apoptosis by regulating SOX2 in LPS-injured PC12 cells and SCI rats.

PubMed

Li, Guowei; Chen, Tao; Zhu, Yingxian; Xiao, Xiaoyu; Bu, Juyuan; Huang, Zongwen

2018-03-01

Recent studies revealed that microRNAs (miRNAs) may play crucial roles in the responses and pathologic processes of spinal cord injury (SCI). This study aimed to investigate the effect and the molecular basis of miR-103 on LPS-induced injuries in PC12 cells in vitro and SCI rats in vivo . PC12 cells were exposed to LPS to induce cell injuries to mimic the in vitro model of SCI. The expression of miR-103 and SOX2 in PC12 cells were altered by transient transfections. Cell viability and apoptotic cell rate were measured by CCK-8 assay and flow cytometry assay. Furthermore, Western blot analysis was performed to detect the expression levels of apoptosis- and autophagy- related proteins, MAPK/ERK pathway- and JAK/STAT pathway-related proteins. In addition, we also assessed the effect of miR-103 agomir on SCI rats. LPS exposure induced cell injuries in PC12 cells. miR-103 overexpression significantly increased cell viability, reduced cell apoptosis and autophagy, and opposite results were observed in miR-103 inhibition. miR-103 attenuated LPS-induced injuries by indirect upregulation of SOX2. SOX2 overexpression protected PC12 cells against LPS-induced injuries, while SOX2 inhibition expedited LPS-induced cell injuries. Furthermore, miR-103 overexpression inhibited MAPK/ERK pathway and JAK/STAT pathway through upregulation of SOX2. We also found that miR-103 agomir inhibited cell apoptosis and autophagy in SCI rats. This study demonstrates that miR-103 may represent a protective effect against cell apoptosis and autophagy in LPS-injured PC12 cells and SCI rats by upregulation of SOX2 expression.

Distinct MAPK signaling pathways, p21 up-regulation and caspase-mediated p21 cleavage establishes the fate of U937 cells exposed to 3-hydrogenkwadaphnin: Differentiation versus apoptosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Moosavi, Mohammad Amin; Yazdanparast, Razieh

2008-07-01

Despite the depth of knowledge concerning the pathogenesis of acute myeloblastic leukemia (AML), long-term survival remains unresolved. Therefore, new agents that act more selectively and more potently are required. In that line, we have recently characterized a novel diterpene ester, called 3-hydrogenkwadaphnin (3-HK), with capability to induce both differentiation and apoptosis in various leukemia cell lines. These effects of 3-HK were mediated through inhibition of inosine 5'-monophosphate dehydrogenase, a selective up-regulated enzyme in cancerous cells, especially leukemia. However, it remains elusive to understand how cells display different fates in response to 3-HK. Here, we report the distinct molecular signaling pathwaysmore » involved in forcing of 3-HK-treated U937 cells to undergo differentiation and apoptosis. After 3-HK (15 nM) treatment, a portion of U937 cells adhered to the culture plates and showed macrophage criteria while others remained in suspension and underwent apoptosis. The differentiated cells arrested in G{sub 0}/G{sub 1} phase of cell cycle and showed early activation of ERK1/2 pathway (3 h) along with ERK-dependent p21{sup Cip/WAF1} (p21) up-regulation and expression of p27{sup Kip1} and Bcl-2. In contrast, the suspension cells underwent apoptosis through Fas/FasL and mitochondrial pathways. The occurrence of apoptosis in these cells were accompanied with caspase-8-mediated p21 cleavage and delayed activation (24 h) of JNK1/2 and p38 MAPK. Taken together, these results suggest that distinct signaling pathways play a pivotal role in fates of drug-treated leukemia cells, thus this may pave some novel therapeutical utilities.« less

Macrophage migration inhibitory factor promotes osteosarcoma growth and lung metastasis through activating the RAS/MAPK pathway.

PubMed

Wang, Chen; Zhou, Xing; Li, Wentao; Li, Mingyue; Tu, Tingyue; Ba, Ximing; Wu, Yinyu; Huang, Zhen; Fan, Gentao; Zhou, Guangxin; Wu, Sujia; Zhao, Jianning; Zhang, Junfeng; Chen, Jiangning

2017-09-10

Emerging evidence suggests that the tumour microenvironment plays a critical role in osteosarcoma (OS) development. Thus, cytokine immunotherapy could be a novel strategy for OS treatment. In this study, we explored the role of macrophage migration inhibitory factor (MIF), an important cytokine in OS progression, and investigated the anti-tumour effects of targeting MIF in OS. The results showed that MIF significantly increased in the tissue and serum samples of OS patients and was associated with tumour size, pulmonary metastasis and the survival rate of OS patients. We verified a positive correlation between MIF and p-ERK1/2 in OS patients. The in vitro results indicated that MIF could activate the RAS/MAPK pathway in a time- and dose-dependent manner, thereby promoting cell proliferation and migration. Furthermore, shRNA targeting MIF significantly inhibited tumour growth and lung metastasis in a mouse xenograft model and orthotopic model of OS. Additionally, inhibition of MIF significantly enhanced the sensitivity of OS cells to cisplatin and doxorubicin. Our findings suggest that immunotherapy targeting MIF to block the RAS/MAPK kinase cascade may represent a feasible and promising approach for OS treatment. Copyright © 2017 Elsevier B.V. All rights reserved.

Hypoxia-induced Bcl-2 expression in endothelial cells via p38 MAPK pathway

DOE Office of Scientific and Technical Information (OSTI.GOV)

Zhang, Cui-Li, E-mail: zhangcuili@hotmail.com; Song, Fei; Zhang, Jing

Angiogenesis and apoptosis are reciprocal processes in endothelial cells. Bcl-2, an anti-apoptotic protein, has been found to have angiogenic activities. The purpose of this study was to determine the role of Bcl-2 in hypoxia-induced angiogenesis in endothelial cells and to investigate the underlying mechanisms. Human aortic endothelial cells (HAECs) were exposed to hypoxia followed by reoxygenation. Myocardial ischemia and reperfusion mouse model was used and Bcl-2 expression was assessed. Bcl-2 expression increased in a time-dependent manner in response to hypoxia from 2 to 72 h. Peak expression occurred at 12 h (3- to 4-fold, p < 0.05). p38 inhibitor (SB203580)more » blocked hypoxia-induced Bcl-2 expression, whereas PKC, ERK1/2 and PI3K inhibitors did not. Knockdown of Bcl-2 resulted in decreased HAECs' proliferation and migration. Over-expression of Bcl-2 increased HAECs' tubule formation, whereas knockdown of Bcl-2 inhibited this process. In this model of myocardial ischemia and reperfusion, Bcl-2 expression was increased and was associated with increased p38 MAPK activation. Our results showed that hypoxia induces Bcl-2 expression in HAECs via p38 MAPK pathway.« less

[The mRNA expression of mitogen-activated protein kinase signal pathway related genes in the blood of arseniasis patients caused by burning coal].

PubMed

Luo, Peng; Zhang, Ai-hua; Xiao, Yun; Pan, Xue-li; Dong, Xue-xin; Huang, Xiao-xin

2013-09-01

To detect the mRNA expression of ERK1, ERK2, JNK1 and P38 gene in mitogen-activated protein kinase(MAPK) path way in the arseniasis patients caused by burning coal. 70 arseniasis patients caused by burning coal at Jiaole village XingRen county in December 2006 were selected as case group, and another 30 villagers with similar living habits, matched gender and age, healthy physical condition without history of burning high arsenic coal were selected as control group from 12 km nearby the same village.Silver diethyl dithiocarbamate method (Ag-DDC) was taken to detect the arsenic contents in the environmental media, food, and arsenic level in the urine and hair of arseniasis patients.On the principle of informed consent, the peripheral blood was collected from the patients. The total RNA was extracted with Trizol method and cDNA was reversed from it. The mRNA expression of ERK1, ERK2, JNK1 and P38 gene in MAPK path way were tested by real-time fluorescent quantitative PCR (QT-PCR). A total of 70 cases of arseniasis patients (31 cases of mild, 25 cases of moderate and 14 cases of severe) and 30 cases of control were chosen. The median (quartile) of arsenic contents in the indoor air, outdoor air, coal, chili and corn were 0.079 (0.053-0.117) mg/m(3) ,0.007 (0.002-0.015) mg/m(3) , 93.010 (39.460-211.740) mg/kg, 3.460(0.550-16.760) mg/kg and 1.500(0.300-4.140) mg/kg respectively. They were above the national health standards. The median (quartile) of arsenic contents in the soil, rice and drinking water were separately 12.130(4.230-24.820) mg/kg, 0.650(0.300-0.980) mg/kg and 0.043(0.012-0.089)mg/kg, which were within the national health standards. Compared with the control group ((26.97 ± 9.71)µg/g Cr), arsenic level in the patients' urine ((71.48 ± 22.74)µg/g Cr) increased significantly, the differences were significant (F = 90.38, P < 0.01). Compared with the control group ((1.58 ± 1.07)µg/g), arsenic level in the patients' hair ((4.45 ± 2.78) µg/g) increased significantly, the differences were significant (F = 48.22, P < 0.01). The relative expression amount of the median(quartile) for ERK2, JNK1 mRNA were 0.0667 (0.0378-0.1371) and 0.0013 (0.0009-0.0025), respectively. Compared with the control group 0.1744 (0.1009-0.1985) and 0.0022 (0.0017-0.0030) , only the decreases of ERK2, JNK1 mRNA expression was significant (χ(2) = 15.10, 14.25, P < 0.01), and no significance in the other index. ERK2 mRNA relative expression for mild, medium and severe groups were separately 0.0818 (0.0408-0.1509) ,0.0582 (0.0154-0.1699) and 0.0588 (0.0399-0.1034) . Compared with the control group (0.1744 (0.1099-0.1985) ), there was significant difference (Z = -2.89, -3.19, -2.67, P < 0.01). JNK1 mRNA relative expression were 0.0012 (0.0007-0.001 57), 0.0019 (0.0011-0.0035), 0.0013 (0.0010-0.0026), respectively. Compared with the control group (0.0022 (0.0017-0.0030) ), significances were found in the mild groups (Z = -3.72, P < 0.01). Arsenic could induce the changes of ERK2 and JNK1mRNA expression in the MAPK path way in arseniasis patients.It suggests that the MAPK signaling pathway take part in the occurrence and development process of arseniasis caused by burning coal.

Icariin inhibits TNF-α/IFN-γ induced inflammatory response via inhibition of the substance P and p38-MAPK signaling pathway in human keratinocytes.

PubMed

Kong, Lingwen; Liu, Jiaqi; Wang, Jia; Luo, Qingli; Zhang, Hongying; Liu, Baojun; Xu, Fei; Pang, Qi; Liu, Yingchao; Dong, Jingcheng

2015-12-01

Pro-inflammatory cytokines play a crucial role in the etiology of atopic dermatitis. We demonstrated that Herba Epimedii has anti-inflammatory potential in an atopic dermatitis mouse model; however, limited research has been conducted on the anti-inflammatory effects and mechanism of icariin, the major active ingredient in Herba Epimedii, in human keratinocytes. In this study, we evaluated the anti-inflammatory potential and mechanisms of icariin in the tumor necrosis factor-α (TNF-α)/interferon-γ (IFN-γ)-induced inflammatory response in human keratinocytes (HaCaT cells) by observing these cells in the presence or absence of icariin. We measured IL-6, IL-8, IL-1β, MCP-1 and GRO-α production by ELISA; IL-6, IL-8, IL-1β, intercellular adhesion molecule-1 (ICAM-1) and tachykinin receptor 1 (TACR1) mRNA expression by real-time PCR; and P38-MAPK, P-ERK and P-JNK signaling expression by western blot in TNF-α/IFN-γ-stimulated HaCaT cells before and after icariin treatment. The expression of TNF-α-R1 and IFN-γ-R1 during the stimulation of the cell models was also evaluated before and after icariin treatment. We investigated the effect of icariin on these pro-inflammatory cytokines and detected whether this effect occurred via the mitogen-activated protein kinase (MAPK) signal transduction pathways. We further specifically inhibited the activity of two kinases with 20μM SB203580 (a p38 kinase inhibitor) and 50μM PD98059 (an ERK1/2 kinase inhibitor) to determine the roles of the two signal pathways involved in the inflammatory response. We found that icariin inhibited TNF-α/IFN-γ-induced IL-6, IL-8, IL-1β, and MCP-1 production in a dose-dependent manner; meanwhile, the icariin treatment inhibited the gene expression of IL-8, IL-1β, ICAM-1 and TACR1 in HaCaT cells in a time- and dose-dependent manner. Icariin treatment resulted in a reduced expression of p-P38 and p-ERK signal activation induced by TNF-α/IFN-γ; however, only SB203580, the p38 alpha/beta inhibitor, inhibited the secretion of inflammatory cytokines induced by TNF-α/IFN-γ in cultured HaCaT cells. The differential expression of TNF-α-R1 and IFN-γ-R1 was also observed after the stimulation of TNF-α/IFN-γ, which was significantly normalized after the icariin treatment. Collectively, we illustrated the anti-inflammatory property of icariin in human keratinocytes. These effects were mediated, at least partially, via the inhibition of substance P and the p38-MAPK signaling pathway, as well as by the regulation of the TNF-α-R1 and IFN-γ-R1 signals. Copyright © 2015 Elsevier B.V. All rights reserved.

Leptin induces osteocalcin expression in ATDC5 cells through activation of the MAPK-ERK1/2 signaling pathway.

PubMed

Han, Yingchao; Xu, Guanghui; Zhang, Jingjie; Yan, Meijun; Li, Xinhua; Ma, Bin; Jun, Lili; Wang, Shan-Jin; Tan, Jun

2016-09-27

Both leptin and osteocalcin have been found to affect growth-plate cartilage development through regulation of the physiologic processes of endochondral bone formation. Leptin mediates bone development and osteocalcin secreted in the late stage of osteoblast differentiation. The relationship between leptin and osteocalcin expression in the chondrogenic cells line is still not clear. Thus, the aim of this study was to explore the effect of leptin on the expression of osteocalcin in chondrocytes. We used clonal mouse chondrogenic ATDC5 cells to investigate the relationship between leptin and osteocalcin. We found that both leptin and osteocalcin expression were dynamically expressed during ATDC5 cell differentiation from 4 to 21 days. We also found that leptin significantly upregulated osteocalcin mRNA and protein levels 24 h after leptin stimulation. However, different concentrations and exposure times of osteocalcin did not affect the levels of leptin protein. Furthermore, we confirmed that leptin augmented the phosphorylation of extracellular signal-regulated kinase 1/2 (ERK1/2) in a time-dependent manner but not p38 or AKT. Inhibition of pERK1/2 expression by a specific ERK1/2 inhibitor U0126 and a special small interfering RNA attenuated levels of leptin-induced osteocalcin expression, indicating that ERK1/2 mediates, in part, the effects of leptin on osteocalcin. Taken together, our results suggest that leptin regulates the expression of osteocalcin in growth plate chondrocytes via the ERK1/2 signaling pathway, while there is no effect on the phosphorylation of either p38 or AKT.

E4orf1 induction in adipose tissue promotes insulin-independent signaling in the adipocyte

PubMed Central

Kusminski, Christine M.; Gallardo-Montejano, Violeta I.; Wang, Zhao V.; Hegde, Vijay; Bickel, Perry E.; Dhurandhar, Nikhil V.; Scherer, Philipp E.

2015-01-01

Background/Purpose Type 2 diabetes remains a worldwide epidemic with major pathophysiological changes as a result of chronic insulin resistance. Insulin regulates numerous biochemical pathways related to carbohydrate and lipid metabolism. Methods We have generated a novel mouse model that allows us to constitutively activate, in an inducible fashion, the distal branch of the insulin signaling transduction pathway specifically in adipocytes. Results Using the adenoviral 36 E4orf1 protein, we chronically stimulate locally the Ras-ERK-MAPK signaling pathway. At the whole body level, this leads to reduced body-weight gain under a high fat diet challenge. Despite overlapping glucose tolerance curves, there is a reduced requirement for insulin action under these conditions. The mice further exhibit reduced circulating adiponectin levels that ultimately lead to impaired lipid clearance, and inflamed and fibrotic white adipose tissues. Nevertheless, they are protected from diet-induced hepatic steatosis. As we observe constitutively elevated p-Akt levels in the adipocytes, even under conditions of low insulin levels, this pinpoints enhanced Ras-ERK-MAPK signaling in transgenic adipocytes as a potential alternative route to bypass proximal insulin signaling events. Conclusion We conclude that E4orf1 expression in the adipocyte leads to enhanced baseline activation of the distal insulin signaling node, yet impaired insulin receptor stimulation in the presence of insulin, with important implications for the regulation of adiponectin secretion. The resulting systemic phenotype is complex, yet highlights the powerful nature of manipulating selective branches of the insulin signaling network within the adipocyte. PMID:26500839

Smad4 inactivation promotes malignancy and drug resistance of colon cancer

PubMed Central

Papageorgis, Panagiotis; Cheng, Kuanghung; Ozturk, Sait; Gong, Yi; Lambert, Arthur W.; Abdolmaleky, Hamid M.; Zhou, Jin-Rong; Thiagalingam, Sam

2010-01-01

SMAD4 is localized to chromosome 18q21, a frequent site for loss of heterozygosity (LOH) in advanced stage colon cancers. Although Smad4 is regarded as a signaling mediator of the TGFβ signaling pathway, its role as a major suppressor of colorectal cancer progression and the molecular events underlying this phenomenon, remain elusive. Here, we describe the establishment and use of colon cancer cell line model systems to dissect the functional roles of TGFβ and Smad4 inactivation in the manifestation of a malignant phenotype. We found that loss of SMAD4 and retention of intact TGFβ receptors could synergistically increase the levels of VEGF, a major pro-angiogenic factor. Pharmacological inhibition studies suggest that overactivation of the TGFβ-induced MEK-Erk and p38-MAPK auxiliary pathways are involved in the induction of VEGF expression in SMAD4 null cells. Overall, SMAD4 deficiency was responsible for the enhanced migration of colon cancer cells with a corresponding increase in MMP9, enhanced hypoxia-induced GLUT1 expression, increased aerobic glycolysis and resistance to 5′-fluoruracil-mediated apoptosis. Interestingly, Smad4 specifically interacts with HIF1α under hypoxic conditions providing a molecular basis for the differential regulation of target genes to suppress a malignant phenotype. In summary, our results define a molecular mechanism that explains how loss of the tumor suppressor Smad4 promotes colorectal cancer progression. These findings are also consistent with targeting TGFβ-induced auxiliary pathways, such as MEK-ERK, p38-MAPK and the glycolytic cascade, in SMAD4-deficient tumors as attractive strategies for therapeutic intervention. PMID:21245094

[Inhibitory effect of apatinib on HCT-116 cells and its mechanism].

PubMed

Yin, Liang; Wang, Jin; Huang, Feng-Chang; Zhang, Yun-Fei; Xu, Ning; Wen, Zheng-Qi; Li, Wen-Liang; Dong, Jian

2017-03-20

To investigate the inhibitory effects of apatinib on colorectal carcinoma HCT-116 cells in vitro and the signaling pathways involved. The cytotoxicity of different concentrations (0, 0.5, 1, 1.5, and 2 µmol/L) of apatinib in HCT-116 cells was assessed by MTT assay, using capecitabine as the positive control. The apoptosis rate of apatinib-treated HCT-116 cells was detected using flow cytometry, and the expressions of Bcl-2, Bax, and caspase-3 were determined with quantitative real-time PCR and Western blotting. The effect of apatinib on the expressions of Akt, pAkt, Erk1/2 and pErk1/2 in HCT-116 cells was evaluated using Western blotting. Apatinib significantly inhibited the proliferation of HCT-116 cells in a concentration-dependent manner with an IC 50 value of 1.335 µmol/L. Flow cytometric analysis showed that apatinib significantly increased the apoptotic rate of HCT-116 cells dose-dependently. Apatinib induced the expression of the pro-apoptotic genes Bax and caspase-3 at both the mRNA and protein levels while inhibited the expression of the anti- apoptotic gene Bcl-2. The expressions of p-Akt and p-Erk1/2 were decreased in HCT-116 cells after apatinib treatment, but the total protein levels did not undergo obvious changes. Apatinib inhibits the proliferation and induces apoptosis of HCT-116 cells by suppressing the phosphorylation of Erk1/2 and Akt in the MAPK/Erk and PI3K/Akt signaling pathways.

Resveratrol inhibits LPS-induced mice mastitis through attenuating the MAPK and NF-κB signaling pathway.

PubMed

Zhang, Xu; Wang, Yanan; Xiao, Chong; Wei, Zhengkai; Wang, Jingjing; Yang, Zhengtao; Fu, Yunhe

2017-06-01

Resveratrol is a natural polyphenol extracted from mangy plants. It has been reported that resveratrol show multitudinous positive role in biology such as anti-oxidant, anti-nociception and anti-inflammatory effects. Therefore, the present study devotes to test the effect of resveratrol on LPS-induced mastitis in mice. Resveratrol was administered intraperitoneally 1 h before LPS treatment. And the anti-inflammatory effect of resveratrol was measured by histopathological examination, MPO assay, real-time PCR and western blotting analysis. The results showed that resveratrol significantly reduced the LPS-induced mammary histopathological changes. Meanwhile, it sharply attenuated the activity of MPO. The result also indicated that the resveratrol can decrease the expression of pro-inflammatory cytokines TNF-α and IL-1β. From the results of western blotting, resveratrol suppressed the expression of phosphorylation of p65 and IκB from NF-κB signal pathway and phosphorylation of p38 and ERK from MAPK signal pathway. These findings suggested that resveratrol may inhibit the inflammatory response in the mastitis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Relaxation oscillations and hierarchy of feedbacks in MAPK signaling

NASA Astrophysics Data System (ADS)

Kochańczyk, Marek; Kocieniewski, Paweł; Kozłowska, Emilia; Jaruszewicz-Błońska, Joanna; Sparta, Breanne; Pargett, Michael; Albeck, John G.; Hlavacek, William S.; Lipniacki, Tomasz

2017-01-01

We formulated a computational model for a MAPK signaling cascade downstream of the EGF receptor to investigate how interlinked positive and negative feedback loops process EGF signals into ERK pulses of constant amplitude but dose-dependent duration and frequency. A positive feedback loop involving RAS and SOS, which leads to bistability and allows for switch-like responses to inputs, is nested within a negative feedback loop that encompasses RAS and RAF, MEK, and ERK that inhibits SOS via phosphorylation. This negative feedback, operating on a longer time scale, changes switch-like behavior into oscillations having a period of 1 hour or longer. Two auxiliary negative feedback loops, from ERK to MEK and RAF, placed downstream of the positive feedback, shape the temporal ERK activity profile but are dispensable for oscillations. Thus, the positive feedback introduces a hierarchy among negative feedback loops, such that the effect of a negative feedback depends on its position with respect to the positive feedback loop. Furthermore, a combination of the fast positive feedback involving slow-diffusing membrane components with slower negative feedbacks involving faster diffusing cytoplasmic components leads to local excitation/global inhibition dynamics, which allows the MAPK cascade to transmit paracrine EGF signals into spatially non-uniform ERK activity pulses.

Stimulation of Innate Immune Function by Panax ginseng after Heat Processing.

PubMed

Shin, Myoung-Sook; Song, Ji Hoon; Choi, Pilju; Lee, Jong Hun; Kim, Song-Yi; Shin, Kwang-Soon; Ham, Jungyeob; Kang, Ki Sung

2018-05-09

Panax ginseng Meyer has been used for the treatment of immune diseases and for strengthening the immune function. In this study, we evaluated the innate immune-stimulating functions and action mechanisms of white ginseng (WG) and heat-processed ginseng (HPG) in RAW264.7 cells. According to LC-MS analysis results, WG contained typical ginsenosides, such as Rb1, Rc, Rb2, Rd, and Rg1, whereas HPG contained Rg3, Rk1, and Rg5 as well as typical ginsenosides. HPG, not WG, enhanced NF-κB transcriptional activity, cytokine production (IL-6 and TNF-α), and MHC class I and II expression in RAW264.7 cells. In addition, HPG phosphorylated MAPKs and NF-kB pathways. In experiments with inhibitors, the ERK inhibitor completely suppressed the effect of HPG on IL-6 and TNF-α production. HPG-induced c-Jun activation was suppressed by an ERK inhibitor and partially suppressed by JNK, p38, and IκBα inhibitors. Collectively, these results suggested that HPG containing Rg3, Rg5, and Rk1 increased macrophage activation which was regulated by the ERK/c-Jun pathway in RAW264.7 cells.

Hsa-let-7c controls the committed differentiation of IGF-1-treated mesenchymal stem cells derived from dental pulps by targeting IGF-1R via the MAPK pathways.

PubMed

Liu, Gen-Xia; Ma, Shu; Li, Yao; Yu, Yan; Zhou, Yi-Xiang; Lu, Ya-Die; Jin, Lin; Wang, Zi-Lu; Yu, Jin-Hua

2018-04-13

The putative tumor suppressor microRNA let-7c is extensively associated with the biological properties of cancer cells. However, the potential involvement of let-7c in the differentiation of mesenchymal stem cells has not been fully explored. In this study, we investigated the influence of hsa-let-7c (let-7c) on the proliferation and differentiation of human dental pulp-derived mesenchymal stem cells (DPMSCs) treated with insulin-like growth factor 1 (IGF-1) via flow cytometry, CCK-8 assays, alizarin red staining, real-time RT-PCR, and western blotting. In general, the proliferative capabilities and cell viability of DPMSCs were not significantly affected by the overexpression or deletion of let-7c. However, overexpression of let-7c significantly inhibited the expression of IGF-1 receptor (IGF-1R) and downregulated the osteo/odontogenic differentiation of DPMSCs, as indicated by decreased levels of several osteo/odontogenic markers (osteocalcin, osterix, runt-related transcription factor 2, dentin sialophosphoprotein, dentin sialoprotein, alkaline phosphatase, type 1 collagen, and dentin matrix protein 1) in IGF-1-treated DPMSCs. Inversely, deletion of let-7c resulted in increased IGF-1R levels and enhanced osteo/odontogenic differentiation. Furthermore, the ERK, JNK, and P38 MAPK pathways were significantly inhibited following the overexpression of let-7c in DPMSCs. Deletion of let-7c promoted the activation of the JNK and P38 MAPK pathways. Our cumulative findings indicate that Let-7c can inhibit the osteo/odontogenic differentiation of IGF-1-treated DPMSCs by targeting IGF-1R via the JNK/P38 MAPK signaling pathways.

The roles of DNA damage-dependent signals and MAPK cascades in tributyltin-induced germline apoptosis in Caenorhabditis elegans.

PubMed

Wang, Yun; Wang, Shunchang; Luo, Xun; Yang, Yanan; Jian, Fenglei; Wang, Xuemin; Xie, Lucheng

2014-08-01

The induction of apoptosis is recognized to be a major mechanism of tributyltin (TBT) toxicity. However, the underlying signaling pathways for TBT-induced apoptosis remain unclear. In this study, using the nematode Caenorhabditis elegans, we examined whether DNA damage response (DDR) pathway and mitogen-activated protein kinase (MAPK) signaling cascades are involved in TBT-induced germline apoptosis and cell cycle arrest. Our results demonstrated that exposing worms to TBT at the dose of 10nM for 6h significantly increased germline apoptosis in N2 strain. Germline apoptosis was absent in strains that carried ced-3 or ced-4 loss-of-function alleles, indicating that both caspase protein CED-3 and Apaf-1 protein CED-4 were required for TBT-induced apoptosis. TBT-induced apoptosis was blocked in the Bcl-2 gain-of-function strain ced-9(n1950), whereas TBT induced a minor increase in the BH3-only protein EGL-1 mutated strain egl-1(n1084n3082). Checkpoint proteins HUS-1 and CLK-2 exerted proapoptotic effects, and the null mutation of cep-1, the homologue of tumor suppressor gene p53, significantly inhibited TBT-induced apoptosis. Apoptosis in the loss-of-function strains of ERK, JNK and p38 MAPK signaling pathways were completely or mildly suppressed under TBT stress. These results were supported by the results of mRNA expression levels of corresponding genes. The present study indicated that TBT-induced apoptosis required the core apoptotic machinery, and that DDR genes and MAPK pathways played essential roles in signaling the processes. Copyright © 2014 Elsevier Ltd. All rights reserved.

ERK and p38 MAPK-Activated Protein Kinases: a Family of Protein Kinases with Diverse Biological Functions

PubMed Central

Roux, Philippe P.; Blenis, John

2004-01-01

Conserved signaling pathways that activate the mitogen-activated protein kinases (MAPKs) are involved in relaying extracellular stimulations to intracellular responses. The MAPKs coordinately regulate cell proliferation, differentiation, motility, and survival, which are functions also known to be mediated by members of a growing family of MAPK-activated protein kinases (MKs; formerly known as MAPKAP kinases). The MKs are related serine/threonine kinases that respond to mitogenic and stress stimuli through proline-directed phosphorylation and activation of the kinase domain by extracellular signal-regulated kinases 1 and 2 and p38 MAPKs. There are currently 11 vertebrate MKs in five subfamilies based on primary sequence homology: the ribosomal S6 kinases, the mitogen- and stress-activated kinases, the MAPK-interacting kinases, MAPK-activated protein kinases 2 and 3, and MK5. In the last 5 years, several MK substrates have been identified, which has helped tremendously to identify the biological role of the members of this family. Together with data from the study of MK-knockout mice, the identities of the MK substrates indicate that they play important roles in diverse biological processes, including mRNA translation, cell proliferation and survival, and the nuclear genomic response to mitogens and cellular stresses. In this article, we review the existing data on the MKs and discuss their physiological functions based on recent discoveries. PMID:15187187

Culture medium, gas atmosphere and MAPK inhibition affect regulation of RNA-binding protein targets during mouse preimplantation development.

PubMed

Calder, Michele D; Watson, Patricia H; Watson, Andrew J

2011-11-01

During oogenesis, mammalian oocytes accumulate maternal mRNAs that support the embryo until embryonic genome activation. RNA-binding proteins (RBP) may regulate the stability and turnover of maternal and embryonic mRNAs. We hypothesised that varying embryo culture conditions, such as culture medium, oxygen tension and MAPK inhibition, affects regulation of RBPs and their targets during preimplantation development. STAU1, ELAVL1, KHSRP and ZFP36 proteins and mRNAs were detected throughout mouse preimplantation development, whereas Elavl2 mRNA decreased after the two-cell stage. Potential target mRNAs of RBP regulation, Gclc, Slc2a1 and Slc7a1 were detected during mouse preimplantation development. Gclc mRNA was significantly elevated in embryos cultured in Whitten's medium compared with embryos cultured in KSOMaa, and Gclc mRNA was elevated under high-oxygen conditions. Inhibition of the p38 MAPK pathway reduced Slc7a1 mRNA expression while inhibition of ERK increased Slc2a1 mRNA expression. The half-lives of the potential RBP mRNA targets are not regulated in parallel; Slc2a1 mRNA displayed the longest half-life. Our results indicate that mRNAs and proteins encoding five RBPs are present during preimplantation development and more importantly, demonstrate that expression of RBP target mRNAs are regulated by culture medium, gas atmosphere and MAPK pathways.

Combination Kinase Inhibitor Treatment Suppresses Rift Valley Fever Virus Replication.

PubMed

Bell, Todd M; Espina, Virginia; Lundberg, Lindsay; Pinkham, Chelsea; Brahms, Ashwini; Carey, Brian D; Lin, Shih-Chao; Dahal, Bibha; Woodson, Caitlin; de la Fuente, Cynthia; Liotta, Lance A; Bailey, Charles L; Kehn-Hall, Kylene

2018-04-13

Viruses must parasitize host cell translational machinery in order to make proteins for viral progeny. In this study, we sought to use this signal transduction conduit against them by inhibiting multiple kinases that influence translation. Previous work indicated that several kinases involved in translation, including p70 S6K, p90RSK, ERK, and p38 MAPK, are phosphorylated following Rift Valley fever virus (RVFV) infection. Furthermore, inhibiting p70 S6K through treatment with the FDA approved drug rapamycin prevents RVFV pathogenesis in a mouse model of infection. We hypothesized that inhibiting either p70 S6K, p90RSK, or p90RSK’s upstream kinases, ERK and p38 MAPK, would decrease translation and subsequent viral replication. Treatment with the p70 S6K inhibitor PF-4708671 resulted in decreased phosphorylation of translational proteins and reduced RVFV titers. In contrast, treatment with the p90RSK inhibitor BI-D1870, p38MAPK inhibitor SB203580, or the ERK inhibitor PD0325901 alone had minimal influence on RVFV titers. The combination of PF-4708671 and BI-D1870 treatment resulted in robust inhibition of RVFV replication. Likewise, a synergistic inhibition of RVFV replication was observed with p38MAPK inhibitor SB203580 or the ERK inhibitor PD0325901 combined with rapamycin treatment. These findings serve as a proof of concept regarding combination kinase inhibitor treatment for RVFV infection.

Combination Kinase Inhibitor Treatment Suppresses Rift Valley Fever Virus Replication

PubMed Central

Bell, Todd M.; Espina, Virginia; Lundberg, Lindsay; Pinkham, Chelsea; Brahms, Ashwini; Dahal, Bibha; Woodson, Caitlin; de la Fuente, Cynthia; Liotta, Lance A.; Bailey, Charles L.

2018-01-01

Viruses must parasitize host cell translational machinery in order to make proteins for viral progeny. In this study, we sought to use this signal transduction conduit against them by inhibiting multiple kinases that influence translation. Previous work indicated that several kinases involved in translation, including p70 S6K, p90RSK, ERK, and p38 MAPK, are phosphorylated following Rift Valley fever virus (RVFV) infection. Furthermore, inhibiting p70 S6K through treatment with the FDA approved drug rapamycin prevents RVFV pathogenesis in a mouse model of infection. We hypothesized that inhibiting either p70 S6K, p90RSK, or p90RSK’s upstream kinases, ERK and p38 MAPK, would decrease translation and subsequent viral replication. Treatment with the p70 S6K inhibitor PF-4708671 resulted in decreased phosphorylation of translational proteins and reduced RVFV titers. In contrast, treatment with the p90RSK inhibitor BI-D1870, p38MAPK inhibitor SB203580, or the ERK inhibitor PD0325901 alone had minimal influence on RVFV titers. The combination of PF-4708671 and BI-D1870 treatment resulted in robust inhibition of RVFV replication. Likewise, a synergistic inhibition of RVFV replication was observed with p38MAPK inhibitor SB203580 or the ERK inhibitor PD0325901 combined with rapamycin treatment. These findings serve as a proof of concept regarding combination kinase inhibitor treatment for RVFV infection. PMID:29652799

Characterization of p38 MAPK isoforms for drug resistance study using systems biology approach.

PubMed

Peng, Huiming; Peng, Tao; Wen, Jianguo; Engler, David A; Matsunami, Risë K; Su, Jing; Zhang, Le; Chang, Chung-Che Jeff; Zhou, Xiaobo

2014-07-01

p38 mitogen-activated protein kinase activation plays an important role in resistance to chemotherapeutic cytotoxic drugs in treating multiple myeloma (MM). However, how the p38 mitogen-activated protein kinase signaling pathway is involved in drug resistance, in particular the roles that the various p38 isoforms play, remains largely unknown. To explore the underlying mechanisms, we developed a novel systems biology approach by integrating liquid chromatography-mass spectrometry and reverse phase protein array data from human MM cell lines with computational pathway models in which the unknown parameters were inferred using a proposed novel algorithm called modularized factor graph. New mechanisms predicted by our models suggest that combined activation of various p38 isoforms may result in drug resistance in MM via regulating the related pathways including extracellular signal-regulated kinase (ERK) pathway and NFкB pathway. ERK pathway regulating cell growth is synergistically regulated by p38δ isoform, whereas nuclear factor kappa B (NFкB) pathway regulating cell apoptosis is synergistically regulated by p38α isoform. This finding that p38δ isoform promotes the phosphorylation of ERK1/2 in MM cells treated with bortezomib was validated by western blotting. Based on the predicted mechanisms, we further screened drug combinations in silico and found that a promising drug combination targeting ERK1/2 and NFκB might reduce the effects of drug resistance in MM cells. This study provides a framework of a systems biology approach to studying drug resistance and drug combination selection. RPPA experimental Data and Matlab source codes of modularized factor graph for parameter estimation are freely available online at http://ctsb.is.wfubmc.edu/publications/modularized-factor-graph.php. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

LPS Increases 5-LO Expression on Monocytes via an Activation of Akt-Sp1/NF-κB Pathways.

PubMed

Lee, Seung Jin; Seo, Kyo Won; Kim, Chi Dae

2015-05-01

5-Lipoxygenase (5-LO) plays a pivotal role in the progression of atherosclerosis. Therefore, this study investigated the molecular mechanisms involved in 5-LO expression on monocytes induced by LPS. Stimulation of THP-1 monocytes with LPS (0~3 µg/ml) increased 5-LO promoter activity and 5-LO protein expression in a concentration-dependent manner. LPS-induced 5-LO expression was blocked by pharmacological inhibition of the Akt pathway, but not by inhibitors of MAPK pathways including the ERK, JNK, and p38 MAPK pathways. In line with these results, LPS increased the phosphorylation of Akt, suggesting a role for the Akt pathway in LPS-induced 5-LO expression. In a promoter activity assay conducted to identify transcription factors, both Sp1 and NF-κB were found to play central roles in 5-LO expression in LPS-treated monocytes. The LPS-enhanced activities of Sp1 and NF-κB were attenuated by an Akt inhibitor. Moreover, the LPS-enhanced phosphorylation of Akt was significantly attenuated in cells pretreated with an anti-TLR4 antibody. Taken together, 5-LO expression in LPS-stimulated monocytes is regulated at the transcriptional level via TLR4/Akt-mediated activations of Sp1 and NF-κB pathways in monocytes.

Postsynaptic localization of PSD-95 is regulated by all three pathways downstream of TrkB signaling.

PubMed

Yoshii, Akira; Constantine-Paton, Martha

2014-01-01

Brain-derived neurotrophic factor (BDNF) and its receptor TrkB regulate synaptic plasticity. TrkB triggers three downstream signaling pathways; Phosphatidylinositol 3-kinase (PI3K), Phospholipase Cγ (PLCγ) and Mitogen activated protein kinases/Extracellular signal-regulated kinases (MAPK/ERK). We previously showed two distinct mechanisms whereby BDNF-TrkB pathway controls trafficking of PSD-95, which is the major scaffold at excitatory synapses and is critical for synapse maturation. BDNF activates the PI3K-Akt pathway and regulates synaptic delivery of PSD-95 via vesicular transport (Yoshii and Constantine-Paton, 2007). BDNF-TrkB signaling also triggers PSD-95 palmitoylation and its transport to synapses through the phosphorylation of the palmitoylation enzyme ZDHHC8 by a protein kinase C (PKC; Yoshii etal., 2011). The second study used PKC inhibitors chelerythrine as well as a synthetic zeta inhibitory peptide (ZIP) which was originally designed to block the brain-specific PKC isoform protein kinase Mϖ (PKMϖ). However, recent studies raise concerns about specificity of ZIP. Here, we assessed the contribution of TrkB and its three downstream pathways to the synaptic distribution of endogenous PSD-95 in cultured neurons using chemical and genetic interventions. We confirmed that TrkB, PLC, and PI3K were critical for the postsynaptic distribution of PSD-95. Furthermore, suppression of MAPK/ERK also disrupted PSD-95 expression. Next, we examined the contribution of PKC. While both chelerythrine and ZIP suppressed the postsynaptic localization of PSD-95, RNA interference for PKMϖ did not have a significant effect. This result suggests that the ZIP peptide, widely used as the "specific" PKMϖ antagonist by many investigators may block a PKC variant other than PKMϖ such as PKCλ/ι. Our results indicate that TrkB regulates postsynaptic localization of PSD-95 through all three downstream pathways, but also recommend further work to identify other PKC variants that regulate palmitoylation and synaptic localization of PSD-95.

BRAFV600E Negatively Regulates the AKT Pathway in Melanoma Cell Lines

PubMed Central

Chen, Brenden; Tardell, Christine; Higgins, Brian; Packman, Kathryn; Boylan, John F.; Niu, Huifeng

2012-01-01

Cross-feedback activation of MAPK and AKT pathways is implicated as a resistance mechanism for cancer therapeutic agents targeting either RAF/MEK or PI3K/AKT/mTOR. It is thus important to have a better understanding of the molecular resistance mechanisms to improve patient survival benefit from these agents. Here we show that BRAFV600E is a negative regulator of the AKT pathway. Expression of BRAFV600E in NIH3T3 cells significantly suppresses MEK inhibitor (RG7167) or mTORC1 inhibitor (rapamycin) induced AKT phosphorylation (pAKT) and downstream signal activation. Treatment-induced pAKT elevation is found in BRAF wild type melanoma cells but not in a subset of melanoma cell lines harboring BRAFV600E. Knock-down of BRAFV600E in these melanoma cells elevates basal pAKT and downstream signals, whereas knock-down of CRAF, MEK1/2 or ERK1/2 or treatment with a BRAF inhibitor have no impact on pAKT. Mechanistically, we show that BRAFV600E interacts with rictor complex (mTORC2) and regulates pAKT through mTORC2. BRAFV600E is identified in mTORC2 after immunoprecipitation of rictor. Knock-down of rictor abrogates BRAFV600E depletion induced pAKT. Knock-down of BRAFV600E enhances cellular enzyme activity of mTORC2. Aberrant activation of AKT pathway by PTEN loss appears to override the negative impact of BRAFV600E on pAKT. Taken together, our findings suggest that in a subset of BRAFV600E melanoma cells, BRAFV600E negatively regulates AKT pathway in a rictor-dependent, MEK/ERK and BRAF kinase-independent manner. Our study reveals a novel molecular mechanism underlying the regulation of feedback loops between the MAPK and AKT pathways. PMID:22880048

Immunomodulatory Activity of Ganoderma atrum Polysaccharide on Purified T Lymphocytes through Ca2+/CaN and Mitogen-Activated Protein Kinase Pathway Based on RNA Sequencing.

PubMed

Xiang, Quan-Dan; Yu, Qiang; Wang, Hui; Zhao, Ming-Ming; Liu, Shi-Yu; Nie, Shao-Ping; Xie, Ming-Yong

2017-07-05

Our previous study has demonstrated that Ganoderma atrum polysaccharide (PSG-1) has immunomodulatory activity on spleen lymphocytes. However, how PSG-1 exerts its effect on purified lymphocytes is still obscure. Thus, this study aimed to investigate the immunomodulatory activity of PSG-1 on purified T lymphocytes and further elucidate the underlying mechanism based on RNA sequencing (RNA-seq). Our results showed that PSG-1 promoted T lymphocytes proliferation and increased the production of IL-2, IFN-γ, and IL-12. Meanwhile, RNA-seq analysis found 394 differentially expressed genes. KEGG pathway analysis identified 20 significant canonical pathways and seven biological functions. Furthermore, PSG-1 elevated intracellular Ca 2+ concentration and calcineurin (CaN) activity and raised the p-ERK, p-JNK, and p-p38 expression levels. T lymphocytes proliferation and the production of IL-2, IFN-γ, and IL-12 were decreased by the inhibitors of calcium channel and mitogen-activated protein kinases (MAPKs). These results indicated that PSG-1 possesses immunomodulatory activity on purified T lymphocytes, in which Ca 2+ /CaN and MAPK pathways play essential roles.

Integrating patterning signals: Wnt/GSK3 regulates the duration of the BMP/Smad1 signal.

PubMed

Fuentealba, Luis C; Eivers, Edward; Ikeda, Atsushi; Hurtado, Cecilia; Kuroda, Hiroki; Pera, Edgar M; De Robertis, Edward M

2007-11-30

BMP receptors determine the intensity of BMP signals via Smad1 C-terminal phosphorylations. Here we show that a finely controlled cell biological pathway terminates this activity. The duration of the activated pSmad1(Cter) signal was regulated by sequential Smad1 linker region phosphorylations at conserved MAPK and GSK3 sites required for its polyubiquitinylation and transport to the centrosome. Proteasomal degradation of activated Smad1 and total polyubiquitinated proteins took place in the centrosome. Inhibitors of the Erk, p38, and JNK MAPKs, as well as GSK3 inhibitors, prolonged the duration of a pulse of BMP7. Wnt signaling decreased pSmad1(GSK3) antigen levels and redistributed it from the centrosome to cytoplasmic LRP6 signalosomes. In Xenopus embryos, it was found that Wnts induce epidermis and that this required an active BMP-Smad pathway. Epistatic experiments suggested that the dorsoventral (BMP) and anteroposterior (Wnt/GSK3) patterning gradients are integrated at the level of Smad1 phosphorylations during embryonic pattern formation.

Follistatin-like protein 1 induction of matrix metalloproteinase 1, 3 and 13 gene expression in rheumatoid arthritis synoviocytes requires MAPK, JAK/STAT3 and NF-κB pathways.

PubMed

Ni, Su; Li, Chenkai; Xu, Nanwei; Liu, Xi; Wang, Wei; Chen, Wenyang; Wang, Yuji; van Wijnen, Andre J

2018-06-22

Elevated levels of follistatin-like protein 1 (FSTL1) have been found both in mouse models for human rheumatoid arthritis (RA) and collagen-induced arthritis (CIA). In this study, we elucidated the potential mechanisms by which FSTL1 contributes to the pathogenesis of RA. Fibroblast-like synoviocytes (FLSs) were established from synovial tissues of RA patients and stimulated with human recombinant FSTL1. Protein and mRNA expression levels of select matrix metalloproteinases (i.e., MMP1, MMP3, MMP13) in FLS were measured by, respectively, real-time RT-qPCR and ELISA. Activation of MAPK and other pathways that affect MMPs were evaluated by Western blotting. We also compared concentrations of MMPs in plasma in RA patients versus healthy controls (HC). Expression levels of MMP1, MMP3, and MMP13 were clearly stimulated by FSTL1 in vitro. FSTL1 activated the inflammation-related NF-κB signaling pathway, as well as all three mitogen-activated protein kinase (MAPK) pathways and the JAK/STAT3 pathway. Moreover, select chemical inhibitors that target p38 (SB203580), Erk1/2 (SP600125), JNK (SCH772984), STAT3 (AG490), and NF-κB (BAY 11-7082) significantly attenuated MMP expression. Inhibition of Toll-like receptor 4 by compound TAK-242 significantly abolished those effects of FSTL1. Importantly, elevated plasma concentrations of MMP3 were found to correlate with plasma FSTL1 levels in RA patients. These findings suggest that FSTL1 accelerates RA progression by activating MAPK, JAK/STAT3, and NF-κB pathways to enhance secretion of different MMPs and this enhancement is via TLR4. Targeting FSTL1 may provide a promising pharmacological drug therapy to ameliorate RA symptoms and perhaps reverse disease progression. © 2018 Wiley Periodicals, Inc.

p53 constrains progression to anaplastic thyroid carcinoma in a Braf-mutant mouse model of papillary thyroid cancer

PubMed Central

McFadden, David G.; Vernon, Amanda; Santiago, Philip M.; Martinez-McFaline, Raul; Bhutkar, Arjun; Crowley, Denise M.; McMahon, Martin; Sadow, Peter M.; Jacks, Tyler

2014-01-01

Anaplastic thyroid carcinoma (ATC) has among the worst prognoses of any solid malignancy. The low incidence of the disease has in part precluded systematic clinical trials and tissue collection, and there has been little progress in developing effective therapies. v-raf murine sarcoma viral oncogene homolog B (BRAF) and tumor protein p53 (TP53) mutations cooccur in a high proportion of ATCs, particularly those associated with a precursor papillary thyroid carcinoma (PTC). To develop an adult-onset model of BRAF-mutant ATC, we generated a thyroid-specific CreER transgenic mouse. We used a Cre-regulated BrafV600E mouse and a conditional Trp53 allelic series to demonstrate that p53 constrains progression from PTC to ATC. Gene expression and immunohistochemical analyses of murine tumors identified the cardinal features of human ATC including loss of differentiation, local invasion, distant metastasis, and rapid lethality. We used small-animal ultrasound imaging to monitor autochthonous tumors and showed that treatment with the selective BRAF inhibitor PLX4720 improved survival but did not lead to tumor regression or suppress signaling through the MAPK pathway. The combination of PLX4720 and the mapk/Erk kinase (MEK) inhibitor PD0325901 more completely suppressed MAPK pathway activation in mouse and human ATC cell lines and improved the structural response and survival of ATC-bearing animals. This model expands the limited repertoire of autochthonous models of clinically aggressive thyroid cancer, and these data suggest that small-molecule MAPK pathway inhibitors hold clinical promise in the treatment of advanced thyroid carcinoma. PMID:24711431

Lasiodin inhibits proliferation of human nasopharyngeal carcinoma cells by simultaneous modulation of the Apaf-1/caspase, AKT/MAPK and COX-2/NF-κB signaling pathways.

PubMed

Lin, Lianzhu; Deng, Wuguo; Tian, Yun; Chen, Wangbing; Wang, Jingshu; Fu, Lingyi; Shi, Dingbo; Zhao, Mouming; Luo, Wei

2014-01-01

Rabdosia serra has been widely used for the treatment of the various human diseases. However, the antiproliferative effects and underlying mechanisms of the compounds in this herb remain largely unknown. In this study, an antiproliferative compound against human nasopharyngeal carcinoma (NPC) cells from Rabdosia serra was purified and identified as lasiodin (a diterpenoid). The treatment with lasiodin inhibited cell viability and migration. Lasiodin also mediated the cell morphology change and induced apoptosis in NPC cells. The treatment with lasiodin induced the Apaf-1 expression, triggered the cytochrome-C release, and stimulated the PARP, caspase-3 and caspase-9 cleavages, thereby activating the apoptotic pathways. The treatment with lasiodin also significantly inhibited the phosphorylations of the AKT, ERK1/2, p38 and JNK proteins. The pretreatment with the AKT or MAPK-selective inhibitors considerably blocked the lasiodin-mediated inhibition of cell proliferation. Moreover, the treatment with lasiodin inhibited the COX-2 expression, abrogated NF-κB binding to the COX-2 promoter, and promoted the NF-κB translocation from cell nuclei to cytosol. The pretreatment with a COX-2-selective inhibitor abrogated the lasiodin-induced inhibition of cell proliferation. These results indicated that lasiodin simultaneously activated the Apaf-1/caspase-dependent apoptotic pathways and suppressed the AKT/MAPK and COX-2/NF-κB signaling pathways. This study also suggested that lasiodin could be a promising natural compound for the prevention and treatment of NPC.

Major role of the PI3K/Akt pathway in ischemic tolerance induced by sublethal oxygen-glucose deprivation in cortical neurons in vitro.

PubMed

Bhuiyan, Mohammad Iqbal Hossain; Jung, Seo Yun; Kim, Hyoung Ja; Lee, Yong Sup; Jin, Changbae

2011-06-01

Ischemic preconditioning can provide protection to neurons from subsequent lethal ischemia. The molecular mechanisms of neuronal ischemic tolerance, however, are still not well-known. The present study, therefore, examined the role of MAPK and PI3K/Akt pathways in ischemic tolerance induced by preconditioning with sublethal oxygen-glucose deprivation (OGD) in cultured rat cortical neurons. Ischemic tolerance was simulated by preconditioning of the neurons with sublethal 1-h OGD imposed 12 h before lethal 3-h OGD. The time-course studies of relative phosphorylation and expression levels of ERK1/2, JNK and p38 MAPK showed lack of their involvement in ischemic tolerance. However, there were significant increases in Akt phosphorylation levels during the reperfusion period following preconditioned lethal OGD. In addition, Bcl-2 associated death promoter (Bad) and GSK-3β were also found to be inactivated during that reperfusion period. Finally, treatment with an inhibitor of PI3K, wortmannin, applied from 15 min before and during lethal OGD abolished not only the preconditioning-induced neuroprotection but also the Akt activation. Concomitant with blockade of the Akt activation, PI3K inhibition also resulted in activation of Bad and GSK-3β. The results suggest that ischemic tolerance induced by sublethal OGD preconditioning is primarily mediated through activation of the PI3K/Akt pathway, but not the MAPK pathway, in rat cortical neurons.

Basic fibroblast growth factor (bFGF) facilitates differentiation of adult dorsal root ganglia-derived neural stem cells toward Schwann cells by binding to FGFR-1 through MAPK/ERK activation.

PubMed

Gu, Yun; Xue, Chenbin; Zhu, Jianbin; Sun, Hualin; Ding, Fei; Cao, Zheng; Gu, Xiaosong

2014-04-01

Considerable research has been devoted to unraveling the regulation of neural stem cell (NSC) differentiation. The responses of NSCs to various differentiation-inducing stimuli, however, are still difficult to estimate. In this study, we aimed to search for a potent growth factor that was able to effectively induce differentiation of NSCs toward Schwann cells. NSCs were isolated from dorsal root ganglia (DRGs) of adult rats and identified by immunostaining. Three different growth factors were used to stimulate the differentiation of DRG-derived NSCs (DRG-NSCs). We found that among these three growth factors, bFGF was the strongest inducer for the glial differentiation of DRG-NSCs, and bFGF induced the generation of an increased number of Schwann cell-like cells as compared to nerve growth factor (NGF) and neuregulin1-β (NRG). These Schwann cell-like cells demonstrated the same characteristics as those of primary Schwann cells. Furthermore, we noted that bFGF-induced differentiation of DRG-NSCs toward Schwann cells might be mediated by binding to fibroblast growth factor receptor-1 (FGFR-1) through activation of MAPK/ERK signal pathway.

Downregulation of miR-133 via MAPK/ERK signaling pathway involved in nicotine-induced cardiomyocyte apoptosis.

PubMed

Wang, Lu; Li, Xuelian; Zhou, Yuhong; Shi, Hui; Xu, Chaoqian; He, Hua; Wang, Shuxuan; Xiong, Xuehui; Zhang, Yong; Du, Zhimin; Zhang, Ruixue; Lu, Yanjie; Yang, Baofeng; Shan, Hongli

2014-02-01

Tobacco smoking is a risk factor for many diseases, and nicotine is a major component of tobacco. Our previous work revealed that nicotine can induce myocardial fibrosis. This study aimed to investigate whether nicotine can induce cardiomyocyte apoptosis and to explore the mechanisms involved. Cardiomyocytes were exposed to different nicotine concentrations for 48 h. MTT assay showed that the viability of cardiomyocytes was significantly inhibited by nicotine in a dose- and time-dependent manner. Loss of mitochondrial membrane potential, nuclear and DNA defragmentation determined by TUNEL and ELISA assays, and morphological alterations all revealed the pro-apoptotic property of nicotine. Meanwhile, miR-133, a muscle-specific microRNA, was markedly downregulated by nicotine. Consistently, caspase-9, a target gene for miR-133, was significantly upregulated, leading to an increase in caspase-3, in nicotine-treated cardiomyocytes compared to non-treated cells. Furthermore, ERK1/2 protein levels were considerably downregulated, along with reduction of serum response factor (SRF), which is a downstream target protein of ERK1/2 and an upstream transactivator of miR-133 as well. Our findings therefore revealed that inhibition of the ERK1/2-SRF-miR-133 signaling pathway to increase caspases-9 and -3 is a novel mechanism for nicotine to induce cardiomyocyte apoptosis and these tobacco smokers.

Smad, but not MAPK, pathway mediates the expression of type I collagen in radiation induced fibrosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yano, Hiroyuki; Division of Radioisotope Research, Department of Research Support, Research Promotion Project, Oita University, 1-1 Idaigaoka Hasama-machi, Yufu, Oita 879-5593; Hamanaka, Ryoji

Highlights: Black-Right-Pointing-Pointer We examine how radiation affects the expression level and signal pathway of collagen. Black-Right-Pointing-Pointer TGF-{beta}1 mRNA is elevated earlier than those of collagen genes after irradiation. Black-Right-Pointing-Pointer Smad pathway mediates the expression of collagen in radiation induced fibrosis. Black-Right-Pointing-Pointer MAPK pathways are not affected in the expression of collagen after irradiation. -- Abstract: Radiation induced fibrosis occurs following a therapeutic or accidental radiation exposure in normal tissues. Tissue fibrosis is the excessive accumulation of collagen and other extracellular matrix components. This study investigated how ionizing radiation affects the expression level and signal pathway of type I collagen. Realmore » time RT-RCR showed that both {alpha}1and {alpha}2 chain of type I collagen mRNA were elevated from 48 h after irradiation with 10 Gy in NIH3T3 cells. The relative luciferase activities of both genes and type I collagen marker were elevated at 72 h. TGF-{beta}1 mRNA was elevated earlier than those of type I collagen genes. A Western blot analysis showed the elevation of Smad phosphorylation at 72 h. Conversely, treatment with TGF-{beta} receptor inhibitor inhibited the mRNA and relative luciferase activity of type I collagen. The phosphorylation of Smad was repressed with the inhibitor, and the luciferase activity was cancelled using a mutant construct of Smad binding site of {alpha}2(I) collagen gene. However, the MAPK pathways, p38, ERK1/2 and JNK, were not affected with specific inhibitors or siRNA. The data showed that the Smad pathway mediated the expression of type I collagen in radiation induced fibrosis.« less

PSMA redirects cell survival signaling from the MAPK to the PI3K-AKT pathways to promote the progression of prostate cancer

PubMed Central

Caromile, Leslie Ann; Dortche, Kristina; Rahman, M. Mamunur; Grant, Christina L.; Stoddard, Christopher; Ferrer, Fernando A.; Shapiro, Linda H.

2017-01-01

Increased abundance of the prostate-specific membrane antigen (PSMA) on prostate epithelium is a hallmark of advanced metastatic prostate cancer (PCa) and correlates negatively with prognosis. However, direct evidence that PSMA functionally contributes to PCa progression remains elusive. We generated mice bearing PSMA-positive or PSMA-negative PCa by crossing PSMA-deficient mice with transgenic PCa (TRAMP) models, enabling direct assessment of PCa incidence and progression in the presence or absence of PSMA. Compared with PSMA-positive tumors, PSMA-negative tumors were smaller, lower-grade, and more apoptotic with fewer blood vessels, consistent with the recognized proangiogenic function of PSMA. Relative to PSMA-positive tumors, tumors lacking PSMA had less than half the abundance of type 1 insulin-like growth factor receptor (IGF-1R), less activity in the survival pathway mediated by PI3K-AKT signaling, and more activity in the proliferative pathway mediated by MAPK-ERK1/2 signaling. Biochemically, PSMA interacted with the scaffolding protein RACK1, disrupting signaling between the β1 integrin and IGF-1R complex to the MAPK pathway, enabling activation of the AKT pathway instead. Manipulation of PSMA abundance in PCa cell lines recapitulated this signaling pathway switch. Analysis of published databases indicated that IGF-1R abundance, cell proliferation, and expression of transcripts for antiapoptotic markers positively correlated with PSMA abundance in patients, suggesting that this switch may be relevant to human PCa. Our findings suggest that increase in PSMA in prostate tumors contributes to progression by altering normal signal transduction pathways to drive PCa progression and that enhanced signaling through the IGF-1R/β1 integrin axis may occur in other tumors. PMID:28292957

Tunicamycin promotes apoptosis in leukemia cells through ROS generation and downregulation of survivin expression.

PubMed

Lim, Eun Jin; Heo, Jeonghoon; Kim, Young-Ho

2015-08-01

Tunicamycin (TN), one of the endoplasmic reticulum stress inducers, has been reported to inhibit tumor cell growth and exhibit anticarcinogenic activity. However, the mechanism by which TN initiates apoptosis remains poorly understood. In the present study, we investigated the effect of TN on the apoptotic pathway in U937 cells. We show that TN induces apoptosis in association with caspase-3 activation, generation of reactive oxygen species (ROS), and downregulation of survivin expression. P38 MAPK (mitogen-activated protein kinase) and the generation of ROS signaling pathway play crucial roles in TN-induced apoptosis in U937 cells. We hypothesized that TN-induced activation of p38 MAPK signaling pathway is responsible for cell death. To test this hypothesis, we selectively inhibited MAPK during treatment with TN. Our data demonstrated that inhibitor of p38 (SB), but not ERK (PD) or JNK (SP), partially maintained apoptosis during treatment with TN. Pre-treatment with NAC and GSH markedly prevented cell death, suggesting a role for ROS in this process. Ectopic expression of survivin in U937 cells attenuated TN-induced apoptosis by suppression of caspase-3 cleavage, mitochondrial membrane potential, and cytochrome c release in U937 cells. Taken together, our results show that TN modulates multiple components of the apoptotic response of human leukemia cells and raise the possibility of a novel therapeutic strategy for hematological malignancies.

Orphan nuclear receptor NR4A2 inhibits hepatic stellate cell proliferation through MAPK pathway in liver fibrosis.

PubMed

Chen, Pengguo; Li, Jie; Huo, Yan; Lu, Jin; Wan, Lili; Li, Bin; Gan, Run; Guo, Cheng

2015-01-01

Hepatic stellate cells (HSCs) play a crucial role in liver fibrosis, which is a pathological process characterized by extracellular matrix accumulation. NR4A2 is a nuclear receptor belonging to the NR4A subfamily and vital in regulating cell growth, metabolism, inflammation and other biological functions. However, its role in HSCs is unclear. We analyzed NR4A2 expression in fibrotic liver and stimulated HSCs compared with control group and studied the influence on cell proliferation, cell cycle, cell apoptosis and MAPK pathway after NR4A2 knockdown. NR4A2 expression was examined by real-time polymerase chain reaction, Western blotting, immunohistochemistry and immunofluorescence analyses. NR4A2 expression was significantly lower in fibrotic liver tissues and PDGF BB or TGF-β stimulated HSCs compared with control group. After NR4A2 knockdown α-smooth muscle actin and Col1 expression increased. In addition, NR4A2 silencing led to the promotion of cell proliferation, increase of cell percentage in S phase and reduced phosphorylation of ERK1/2, P38 and JNK in HSCs. These results indicate that NR4A2 can inhibit HSC proliferation through MAPK pathway and decrease extracellular matrix in liver fibrogenesis. NR4A2 may be a promising therapeutic target for liver fibrosis.

Protective activity of salidroside against ethanol-induced gastric ulcer via the MAPK/NF-κB pathway in vivo and in vitro.

PubMed

Chang, Xiayun; Luo, Fen; Jiang, Wenjiao; Zhu, Lingpeng; Gao, Jin; He, He; Wei, Tingting; Gong, Shilin; Yan, Tianhua

2015-09-01

Salidroside (Sal) is a traditional Chinese medicine with various pharmacological effects. The present study aimed to investigate the protective effect of Sal on ethanol-induced acute gastric ulcer and H2O2-induced gastric epithelial cell damage. 0.2 ml ethanol and 400 μM H2O2 were applied to establish a gastric ulcer model in vivo and in vitro respectively. The production of interleukin (IL)-6, interleukin (IL)-1β and tumor necrosis factor (TNF)-α was analyzed, as well as myeloperoxidase (MPO), malondialdehyde (MDA) and superoxide dismutase (SOD). MTT assay was used to detect cell viability. In addition, MAPK/NF-κB signal pathway-related proteins p-ERK, p-JNK, p-p38, p-IκBα and p-NF-κBp65 were analyzed to determine the underlying protective mechanism. Downstream genes such as cyclooxygenase-2 (COX-2), 5-lipoxygenase (5-LOX) and leukotrienes B4 (LTB4) were also measured. Obtained data indicated that Sal inhibited the overproduction of pro-inflammatory cytokines and enhanced antioxidant activity. Collectively, it is assumed that Sal could alleviate ethanol-induced acute gastric ulcer and H2O2-induced gastric epithelial cell damage through the MAPK/NF-κB pathway. Copyright © 2015 Elsevier B.V. All rights reserved.

Arctigenin, a dietary phytoestrogen, induces apoptosis of estrogen receptor-negative breast cancer cells through the ROS/p38 MAPK pathway and epigenetic regulation.

PubMed

Hsieh, Chia-Jung; Kuo, Po-Lin; Hsu, Ying-Chan; Huang, Ya-Fang; Tsai, Eing-Mei; Hsu, Ya-Ling

2014-02-01

This study investigates the anticancer effect of arctigenin (ATG), a natural lignan product of Arctium lappa L., in human breast cancer MDA-MB-231 cells. Results indicate that ATG inhibits MDA-MB-231 cell growth by inducing apoptosis in vitro and in vivo. ATG triggers the mitochondrial caspase-independent pathways, as indicated by changes in Bax/Bcl-2 ratio, resulting in AIF and EndoG nuclear translocation. ATG increased cellular reactive oxygen species (ROS) production by increasing p22(phox)/NADPH oxidase 1 interaction and decreasing glutathione level. ATG clearly increases the activation of p38 MAPK, but not JNK and ERK1/2. Antioxidant EUK-8, a synthetic catalytic superoxide and hydrogen peroxide scavenger, significantly decreases ATG-mediated p38 activation and apoptosis. Blocking p38 with a specific inhibitor suppresses ATG-mediated Bcl-2 downregulation and apoptosis. Moreover, ATG activates ATF-2, a transcription factor activated by p38, and then upregulates histone H3K9 trimethylation in the Bcl-2 gene promoter region, resulting in Bcl-2 downregulation. Taken together, the results demonstrate that ATG induces apoptosis of MDA-MB-231 cells via the ROS/p38 MAPK pathway and epigenetic regulation of Bcl-2 by upregulation of histone H3K9 trimethylation. Crown Copyright © 2013. Published by Elsevier Inc. All rights reserved.

PfPK7, an atypical MEK-related protein kinase, reflects the absence of classical three-component MAPK pathways in the human malaria parasite Plasmodium falciparum.

PubMed

Dorin, Dominique; Semblat, Jean-Philippe; Poullet, Patrick; Alano, Pietro; Goldring, J P Dean; Whittle, Christina; Patterson, Shelley; Chakrabarti, Debopam; Doerig, Christian

2005-01-01

Two members of the mitogen-activated protein kinase (MAPK) family have been previously characterized in Plasmodium falciparum, but in vitro attempts at identifying MAP kinase kinase (MAPKK) homologues have failed. Here we report the characterization of a novel plasmodial protein kinase, PfPK7, whose top scores in blastp analysis belong to the MAPKK3/6 subgroup of MAPKKs. However, homology to MAPKKs is restricted to regions of the C-terminal lobe of the kinase domain, whereas the N-terminal region is closer to fungal protein kinase A enzymes (PKA, members of the AGC group of protein kinases). Hence, PfPK7 is a 'composite' enzyme displaying regions of similarity to more than one protein kinase family, similar to a few other plasmodial protein kinases. PfPK7 is expressed in several developmental stages of the parasite, both in the mosquito vector and in the human host. Recombinant PfPK7 displayed kinase activity towards a variety of substrates, but was unable to phosphorylate the two P. falciparum MAPK homologues in vitro, and was insensitive to PKA and MEK inhibitors. Together with the absence of a typical MAPKK activation site in its T-loop, this suggests that PfPK7 is not a MAPKK orthologue, despite the fact that this enzyme is the most 'MAPKK-like' enzyme encoded in the P. falciparum genome. This is consistent with recent observations that the plasmodial MAPKs are not true orthologues of the ERK1/2, p38 or JNK MAPKs, and strengthens the evidence that classical three-component module-dependent MAPK signalling pathways do not operate in malaria parasites, a feature that has not been described in any other eukaryote.

Reactive oxygen species regulated mitochondria-mediated apoptosis in PC12 cells exposed to chlorpyrifos

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Jeong Eun; Hanyang Biomedical Research Institute, Seoul; Park, Jae Hyeon

2012-09-01

Reactive oxidative species (ROS) generated by environmental toxicants including pesticides could be one of the factors underlying the neuronal cell damage in neurodegenerative diseases. In this study we found that chlorpyrifos (CPF) induced apoptosis in dopaminergic neuronal components of PC12 cells as demonstrated by the activation of caspases and nuclear condensation. Furthermore, CPF also reduced the tyrosine hydroxylase-positive immunoreactivity in substantia nigra of the rat. In addition, CPF induced inhibition of mitochondrial complex I activity. Importantly, N-acetyl cysteine (NAC) treatment effectively blocked apoptosis via the caspase-9 and caspase-3 pathways while NAC attenuated the inhibition of mitochondrial complex I activity asmore » well as the oxidative metabolism of dopamine (DA). These results demonstrated that CPF-induced apoptosis was involved in mitochondrial dysfunction through the production of ROS. In the response of cellular antioxidant systems to CPF, we found that CPF treatment increased HO-1 expression while the expression of CuZnSOD and MnSOD was reduced. In addition, we found that CPF treatment activated MAPK pathways, including ERK 1/2, the JNK, and the p38 MAP kinase in a time-dependent manner. NAC treatment abolished MAPK phosphorylation caused by CPF, indicating that ROS are upstream signals of MAPK. Interestingly, MAPK inhibitors abolished cytotoxicity and reduced ROS generation by CPF treatment. Our results demonstrate that CPF induced neuronal cell death in part through MAPK activation via ROS generation, suggesting its potential to generate oxidative stress via mitochondrial damage and its involvement in oxidative stress-related neurodegenerative disease. -- Highlights: ► Chlorpyrifos induces apoptosis. ► Chlorpyrifos inhibits mitochondrial complex I activity. ► ROS is involved in chlorpyrifos-induced apoptosis. ► Chlorpyrifos affects cellular antioxidant systems. ► Chlorpyrifos-induced apoptosis mediates activation of MAPK.« less

Dusp6 attenuates Ras/MAPK signaling to limit zebrafish heart regeneration.

PubMed

Missinato, Maria A; Saydmohammed, Manush; Zuppo, Daniel A; Rao, Krithika S; Opie, Graham W; Kühn, Bernhard; Tsang, Michael

2018-03-06

Zebrafish regenerate cardiac tissue through proliferation of pre-existing cardiomyocytes and neovascularization. Secreted growth factors such as FGFs, IGF, PDGFs and Neuregulin play essential roles in stimulating cardiomyocyte proliferation. These factors activate the Ras/MAPK pathway, which is tightly controlled by the feedback attenuator Dual specificity phosphatase 6 (Dusp6), an ERK phosphatase. Here, we show that suppressing Dusp6 function enhances cardiac regeneration. Inactivation of Dusp6 by small molecules or by gene inactivation increased cardiomyocyte proliferation, coronary angiogenesis, and reduced fibrosis after ventricular resection. Inhibition of Erbb or PDGF receptor signaling suppressed cardiac regeneration in wild-type zebrafish, but had a milder effect on regeneration in dusp6 mutants. Moreover, in rat primary cardiomyocytes, NRG1-stimulated proliferation can be enhanced upon chemical inhibition of Dusp6 with BCI. Our results suggest that Dusp6 attenuates Ras/MAPK signaling during regeneration and that suppressing Dusp6 can enhance cardiac repair. © 2018. Published by The Company of Biologists Ltd.

MAPK signaling is required for LPS-induced VEGF in pulp stem cells.

PubMed

Botero, T M; Son, J S; Vodopyanov, D; Hasegawa, M; Shelburne, C E; Nör, J E

2010-03-01

Caries-induced pulpitis is typically accompanied by an increase in dental pulp microvascular density. However, the mechanisms by which dental pulp cells recognize lipopolysaccharides (LPSs) remain unclear. We hypothesized that Porphyromonas endodontalis and Escherichia coli LPSs induce vascular endothelial growth factor (VEGF) expression in dental pulp stem cells (DPSC) and human dental pulp fibroblasts (HDPF) through mitogen-activated protein kinase (MAPK) signaling. ELISA, semi-quantitative RT-PCR, immunofluorescence, and Western blots were used. Here, we observed that LPSs induced VEGF expression in DPSC and HDPF cells, and both cell types express Toll-like receptor 4 (TLR- 4). Notably, LPS-induced VEGF is associated with phosphorylation of protein kinase C (PKC zeta) and extracellular signal-regulator kinase (ERK1/2) and is dependent upon MAPK activation. Analysis of these data, collectively, unveils a signaling pathway responsible for synthesis of VEGF by pulp cells and suggests a novel therapeutic target for the management of vascular responses in teeth with pulpitis.

IL17A augments autophagy in Mycobacterium tuberculosis-infected monocytes from patients with active tuberculosis in association with the severity of the disease.

PubMed

Tateosian, Nancy Liliana; Pellegrini, Joaquín Miguel; Amiano, Nicolás Oscar; Rolandelli, Agustín; Casco, Nicolás; Palmero, Domingo Juan; Colombo, María Isabel; García, Verónica Edith

2017-07-03

During mycobacterial infection, macroautophagy/autophagy, a process modulated by cytokines, is essential for mounting successful host responses. Autophagy collaborates with human immune responses against Mycobacterium tuberculosis (Mt) in association with specific IFNG secreted against the pathogen. However, IFNG alone is not sufficient to the complete bacterial eradication, and other cytokines might be required. Actually, induction of Th1 and Th17 immune responses are required for protection against Mt. Accordingly, we showed that IL17A and IFNG expression in lymphocytes from tuberculosis patients correlates with disease severity. Here we investigate the role of IFNG and IL17A during autophagy in monocytes infected with Mt H37Rv or the mutant MtΔRD1. Patients with active disease were classified as high responder (HR) or low responder (LR) according to their T cell responses against Mt. IL17A augmented autophagy in infected monocytes from HR patients through a mechanism that activated MAPK1/ERK2-MAPK3/ERK1 but, during infection of monocytes from LR patients, IL17A had no effect on the autophagic response. In contrast, addition of IFNG to infected monocytes, increased autophagy by activating MAPK14/p38 α both in HR and LR patients. Interestingly, proteins codified in the RD1 region did not interfere with IFNG and IL17A autophagy induction. Therefore, in severe tuberculosis patients' monocytes, IL17A was unable to augment autophagy because of a defect in the MAPK1/3 signaling pathway. In contrast, both IFNG and IL17A increased autophagy levels in patients with strong immunity to Mt, promoting mycobacterial killing. Our findings might contribute to recognize new targets for the development of novel therapeutic tools to fight the pathogen.

Differential interaction of the tyrosine phosphatases PTP-SL, STEP and HePTP with the mitogen-activated protein kinases ERK1/2 and p38alpha is determined by a kinase specificity sequence and influenced by reducing agents.

PubMed Central

Muñoz, Juan José; Tárrega, Céline; Blanco-Aparicio, Carmen; Pulido, Rafael

2003-01-01

The protein tyrosine phosphatases (PTPs) PTP-SL, STEP and HePTP are mitogen-activated protein kinase (MAPK) substrates and regulators that bind to MAPKs through a kinase-interaction motif (KIM) located in their non-catalytic regulatory domains. We have found that the binding of these PTPs to the MAPKs extracellular-signal-regulated kinase 1 and 2 (ERK1/2), and p38alpha is differentially determined by the KIM-adjacent C-terminal regions of the PTPs, which have been termed kinase-specificity sequences, and is influenced by reducing agents. Under control conditions, PTP-SL bound preferentially to ERK1/2, whereas STEP and HePTP bound preferentially to p38alpha. Under reducing conditions, the association of p38alpha with STEP or HePTP was impaired, whereas the association with PTP-SL was unaffected. On the other hand, the association of ERK1/2 with HePTP was increased under reducing conditions, whereas the association with STEP or PTP-SL was unaffected. In intact cells, PTP-SL and STEP distinctively regulated the kinase activity and the nuclear translocation of ERK1/2 and p38alpha. Our results suggest that intracellular redox conditions could modulate the activity and subcellular location of ERK1/2 and p38alpha by controlling their association with their regulatory PTPs. PMID:12583813

Mitogen-activated protein kinase pathway mediates DBP-maf-induced apoptosis in RAW 264.7 macrophages.

PubMed

Gumireddy, Kiranmai; Reddy, C Damodar; Swamy, Narasimha

2003-09-01

Vitamin D-binding protein-macrophage-activating factor (DBP-maf) is derived from serum vitamin D binding protein (DBP) by selective deglycosylation during inflammation. In the present study, we investigated the effect of DBP-maf on RAW 264.7 macrophages and the underlying intracellular signal transduction pathways. DBP-maf increased proapoptotic caspase-3, -8, and -9 activities and induced apoptosis in RAW 264.7 cells. However, DBP, the precursor to DBP-maf did not induce apoptosis in these cells. Cell cycle analysis of DBP-maf-treated RAW 264.7 cells revealed growth arrest with accumulation of cells in sub-G(0)/G(1) phase. We also investigated the role of mitogen-activated protein kinase (MAPK) pathways in the DBP-maf-induced apoptosis of RAW264.7 cells. DBP-maf increased the phosphorylation of p38 and JNK1/2, while it decreased the ERK1/2 phosphorylation. Treatment with the p38 MAPK inhibitor, SB202190, attenuated DBP-maf-induced apoptosis. PD98059, a MEK specific inhibitor, did not show a significant inhibition of apoptosis induced by DBP-maf. Taken together, these results suggest that the p38 MAPK pathway plays a crucial role in DBP-maf-mediated apoptosis of macrophages. Our studies indicate that, during inflammation DBP-maf may function positively by causing death of the macrophages when activated macrophages are no longer needed at the site of inflammation. In summary, we report for the first time that DBP-maf induces apoptosis in macrophages via p38 and JNK1/2 pathway. Copyright 2003 Wiley-Liss, Inc.

FGFR2IIIb-MAPK Activity Is Required for Epithelial Cell Fate Decision in the Lower Müllerian Duct

PubMed Central

Terakawa, Jumpei; Rocchi, Altea; Serna, Vanida A.; Bottinger, Erwin P.; Graff, Jonathan M.

2016-01-01

Cell fate of lower Müllerian duct epithelium (MDE), to become uterine or vaginal epithelium, is determined by the absence or presence of ΔNp63 expression, respectively. Previously, we showed that SMAD4 and runt-related transcription factor 1 (RUNX1) were independently required for MDE to express ΔNp63. Here, we report that vaginal mesenchyme directs vaginal epithelial cell fate in MDE through paracrine activation of fibroblast growth factor (FGF) receptor-MAPK pathway. In the developing reproductive tract, FGF7 and FGF10 were enriched in vaginal mesenchyme, whereas FGF receptor 2IIIb was expressed in epithelia of both the uterus and vagina. When Fgfr2 was inactivated, vaginal MDE underwent uterine cell fate, and this differentiation defect was corrected by activation of MEK-ERK pathway. In vitro, FGF10 in combination with bone morphogenetic protein 4 and activin A (ActA) was sufficient to induce ΔNp63 in MDE, and ActA was essential for induction of RUNX1 through SMAD-independent pathways. Accordingly, inhibition of type 1 receptors for activin in neonatal mice induced uterine differentiation in vaginal epithelium by down-regulating RUNX1, whereas conditional deletion of Smad2 and Smad3 had no effect on vaginal epithelial differentiation. In conclusion, vaginal epithelial cell fate in MDE is induced by FGF7/10-MAPK, bone morphogenetic protein 4-SMAD, and ActA-RUNX1 pathway activities, and the disruption in any one of these pathways results in conversion from vaginal to uterine epithelial cell fate. PMID:27164167

β-Hydroxy-β-Methylbutyrate (HMB) Promotes Neurite Outgrowth in Neuro2a Cells.

PubMed

Salto, Rafael; Vílchez, Jose D; Girón, María D; Cabrera, Elena; Campos, Nefertiti; Manzano, Manuel; Rueda, Ricardo; López-Pedrosa, Jose M

2015-01-01

β-Hydroxy-β-methylbutyrate (HMB) has been shown to enhance cell survival, differentiation and protein turnover in muscle, mainly activating phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases/ extracellular-signal-regulated kinases (MAPK/ERK) signaling pathways. Since these two pathways are related to neuronal survival and differentiation, in this study, we have investigated the neurotrophic effects of HMB in mouse neuroblastoma Neuro2a cells. In Neuro2a cells, HMB promotes differentiation to neurites independent from any effects on proliferation. These effects are mediated by activation of both the PI3K/Akt and the extracellular-signal-regulated kinases (ERK1/2) signaling as demonstrated by the use of specific inhibitors of these two pathways. As myocyte-enhancer factor 2 (MEF2) family of transcription factors are involved in neuronal survival and plasticity, the transcriptional activity and protein levels of MEF2 were also evaluated. HMB promoted MEF2-dependent transcriptional activity mediated by the activation of Akt and ERK1/2 pathways. Furthermore, HMB increases the expression of brain glucose transporters 1 (GLUT1) and 3 (GLUT3), and mTOR phosphorylation, which translates in a higher protein synthesis in Neuro2a cells. Furthermore, Torin1 and rapamycin effects on MEF2 transcriptional activity and HMB-dependent neurite outgrowth support that HMB acts through mTORC2. Together, these findings provide clear evidence to support an important role of HMB in neurite outgrowth.

β-Hydroxy-β-Methylbutyrate (HMB) Promotes Neurite Outgrowth in Neuro2a Cells

PubMed Central

Girón, María D.; Cabrera, Elena; Campos, Nefertiti; Manzano, Manuel; Rueda, Ricardo; López-Pedrosa, Jose M.

2015-01-01

β-Hydroxy-β-methylbutyrate (HMB) has been shown to enhance cell survival, differentiation and protein turnover in muscle, mainly activating phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases/ extracellular-signal-regulated kinases (MAPK/ERK) signaling pathways. Since these two pathways are related to neuronal survival and differentiation, in this study, we have investigated the neurotrophic effects of HMB in mouse neuroblastoma Neuro2a cells. In Neuro2a cells, HMB promotes differentiation to neurites independent from any effects on proliferation. These effects are mediated by activation of both the PI3K/Akt and the extracellular-signal-regulated kinases (ERK1/2) signaling as demonstrated by the use of specific inhibitors of these two pathways. As myocyte-enhancer factor 2 (MEF2) family of transcription factors are involved in neuronal survival and plasticity, the transcriptional activity and protein levels of MEF2 were also evaluated. HMB promoted MEF2-dependent transcriptional activity mediated by the activation of Akt and ERK1/2 pathways. Furthermore, HMB increases the expression of brain glucose transporters 1 (GLUT1) and 3 (GLUT3), and mTOR phosphorylation, which translates in a higher protein synthesis in Neuro2a cells. Furthermore, Torin1 and rapamycin effects on MEF2 transcriptional activity and HMB-dependent neurite outgrowth support that HMB acts through mTORC2. Together, these findings provide clear evidence to support an important role of HMB in neurite outgrowth. PMID:26267903

Tamoxifen inhibits tumor cell invasion and metastasis in mouse melanoma through suppression of PKC/MEK/ERK and PKC/PI3K/Akt pathways

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matsuoka, Hiroshi; Department of Pharmacy, Nara Hospital, Kinki University School of Medicine, 1248-1 Ikoma, Nara 630-0293; Tsubaki, Masanobu

2009-07-15

In melanoma, several signaling pathways are constitutively activated. Among these, the protein kinase C (PKC) signaling pathways are activated through multiple signal transduction molecules and appear to play major roles in melanoma progression. Recently, it has been reported that tamoxifen, an anti-estrogen reagent, inhibits PKC signaling in estrogen-negative and estrogen-independent cancer cell lines. Thus, we investigated whether tamoxifen inhibited tumor cell invasion and metastasis in mouse melanoma cell line B16BL6. Tamoxifen significantly inhibited lung metastasis, cell migration, and invasion at concentrations that did not show anti-proliferative effects on B16BL6 cells. Tamoxifen also inhibited the mRNA expressions and protein activities ofmore » matrix metalloproteinases (MMPs). Furthermore, tamoxifen suppressed phosphorylated extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt through the inhibition of PKC{alpha} and PKC{delta} phosphorylation. However, other signal transduction factor, such as p38 mitogen-activated protein kinase (p38MAPK) was unaffected. The results indicate that tamoxifen suppresses the PKC/mitogen-activated protein kinase kinase (MEK)/ERK and PKC/phosphatidylinositol-3 kinase (PI3K)/Akt pathways, thereby inhibiting B16BL6 cell migration, invasion, and metastasis. Moreover, tamoxifen markedly inhibited not only developing but also clinically evident metastasis. These findings suggest that tamoxifen has potential clinical applications for the treatment of tumor cell metastasis.« less

Anxa5 mediates the in vitro malignant behaviours of murine hepatocarcinoma Hca-F cells with high lymph node metastasis potential preferentially via ERK2/p-ERK2/c-Jun/p-c-Jun(Ser73) and E-cadherin.

PubMed

Sun, Xujuan; Wei, Bin; Liu, Shuqing; Guo, Chunmei; Wu, Na; Liu, Qinlong; Sun, Ming-Zhong

2016-12-01

Annexin A5 (Anxa5) is associated with the progression of some cancers, while its role and regulation mechanism in tumor lymphatic metastasis is rarely reported. This study aims to investigate the influence of Anxa5 knockdown on the malignant behaviours of murine hepatocarcinoma Hca-F cell line with high lymph node metastatic (LNM) potential and the underlying regulation mechanism. RNA interfering was performed to silence Anxa5 in Hca-F. Monoclonal shRNA-Anxa5- Hca-F cells were obtained via G418 screening by limited dilution method. Quantitative real-time RT-PCR (qRT-PCR) and Western blotting (WB) were applied to measure Anxa5 expression levels. CCK-8, Boyden transwell-chamber and in situ LN adhesion assays were performed to explore the effects of Anxa5 on the proliferation, migration, invasion and adhesion capacities of Hca-F. WB and qRT-PCR were used to detect the level changes of key molecules in corresponding signal pathways. We obtained two monoclonal shRNA-Anxa5-transfected Hca-F cell lines with stable knockdowns of Anxa5. Anxa5 knockdown resulted in significantly reduced proliferation, migration, invasion and in situ LN adhesion potentials of Hca-F in proportion to its knockdown extent. Anxa5 downregulation enhanced E-cadherin levels in Hca-F. Moreover, Anxa5 affected Hca-F behaviours specifically via ERK2/p-ERK2/c-Jun/p-c-Jun(Ser73) instead of p38MAPK/c-Jun, Jnk/c-Jun and AKT/c-Jun pathways. Anxa5 mediates the in vitro malignant behaviours of murine hepatocarcinoma Hca-F cells via ERK2/c-Jun/p-c-Jun(Ser73) and ERK2/E-cadherin pathways. It is an important molecule in metastasis (especially LNM) and a potential therapeutic target for hepatocarcinoma. Copyright © 2016 Elsevier Masson SAS. All rights reserved.

Genome-wide gene expression profiling reveals aberrant MAPK and Wnt signaling pathways associated with early parthenogenesis.

PubMed

Liu, Na; Enkemann, Steven A; Liang, Ping; Hersmus, Remko; Zanazzi, Claudia; Huang, Junjiu; Wu, Chao; Chen, Zhisheng; Looijenga, Leendert H J; Keefe, David L; Liu, Lin

2010-12-01

Mammalian parthenogenesis could not survive but aborted during mid-gestation, presumably because of lack of paternal gene expression. To understand the molecular mechanisms underlying the failure of parthenogenesis at early stages of development, we performed global gene expression profiling and functional analysis of parthenogenetic blastocysts in comparison with those of blastocysts from normally fertilized embryos. Parthenogenetic blastocysts exhibited changes in the expression of 749 genes, of which 214 had lower expression and 535 showed higher expressions than fertilized embryos using a minimal 1.8-fold change as a cutoff. Genes important for placenta development were decreased in their expression in parthenote blastocysts. Some maternally expressed genes were up-regulated and paternal-related genes were down-regulated. Moreover, aberrantly increased Wnt signaling and reduced mitogen-activated protein kinase (MAPK) signaling were associated with early parthenogenesis. The protein level of extracellular signal-regulated kinase 2 (ERK2) was low in parthenogenetic blastocysts compared with that of fertilized blastocysts 120 h after fertilization. 6-Bromoindirubin-3'-oxime, a specific glycogen synthase kinase-3 (GSK-3) inhibitor, significantly decreased embryo hatching. The expression of several imprinted genes was altered in parthenote blastocysts. Gene expression also linked reduced expression of Xist to activation of X chromosome. Our findings suggest that failed X inactivation, aberrant imprinting, decreased ERK/MAPK signaling and possibly elevated Wnt signaling, and reduced expression of genes for placental development collectively may contribute to abnormal placenta formation and failed fetal development in parthenogenetic embryos.

The Alternative Epac/cAMP Pathway and the MAPK Pathway Mediate hCG Induction of Leptin in Placental Cells

PubMed Central

Maymó, Julieta Lorena; Pérez Pérez, Antonio; Maskin, Bernardo; Dueñas, José Luis; Calvo, Juan Carlos; Sánchez Margalet, Víctor; Varone, Cecilia Laura

2012-01-01

Pleiotropic effects of leptin have been identified in reproduction and pregnancy, particularly in the placenta, where it works as an autocrine hormone. In this work, we demonstrated that human chorionic gonadotropin (hCG) added to JEG-3 cell line or to placental explants induces endogenous leptin expression. We also found that hCG increased cAMP intracellular levels in BeWo cells in a dose-dependent manner, stimulated cAMP response element (CRE) activity and the cotransfection with an expression plasmid of a dominant negative mutant of CREB caused a significant inhibition of hCG stimulation of leptin promoter activity. These results demonstrate that hCG indeed activates cAMP/PKA pathway, and that this pathway is involved in leptin expression. Nevertheless, we found leptin induction by hCG is dependent on cAMP levels. Treatment with (Bu)2cAMP in combination with low and non stimulatory hCG concentrations led to an increase in leptin expression, whereas stimulatory concentrations showed the opposite effect. We found that specific PKA inhibition by H89 caused a significant increase of hCG leptin induction, suggesting that probably high cAMP levels might inhibit hCG effect. It was found that hCG enhancement of leptin mRNA expression involved the MAPK pathway. In this work, we demonstrated that hCG leptin induction through the MAPK signaling pathway is inhibited by PKA. We observed that ERK1/2 phosphorylation increased when hCG treatment was combined with H89. In view of these results, the involvement of the alternative cAMP/Epac signaling pathway was studied. We observed that a cAMP analogue that specifically activates Epac (CPT-OMe) stimulated leptin expression by hCG. In addition, the overexpression of Epac and Rap1 proteins increased leptin promoter activity and enhanced hCG. In conclusion, we provide evidence suggesting that hCG induction of leptin gene expression in placenta is mediated not only by activation of the MAPK signaling pathway but also by the alternative cAMP/Epac signaling pathway. PMID:23056265

ML-7 amplifies the quinocetone-induced cell death through akt and MAPK-mediated apoptosis on HepG2 cell line.

PubMed

Zhou, Yan; Zhang, Shen; Deng, Sijun; Dai, Chongshan; Tang, Shusheng; Yang, Xiayun; Li, Daowen; Zhao, Kena; Xiao, Xilong

2016-01-01

The study aims at evaluating the combination of the quinocetone and the ML-7 in preclinical hepatocellular carcinoma models. To this end, the effect of quinocetone and ML-7 on apoptosis induction and signaling pathways was analyzed on HepG2 cell lines. Here, we report that ML-7, in a nontoxic concentration, sensitized the HepG2 cells to quinocetone-induced cytotoxicity. Also, ML-7 profoundly enhances quinocetone-induced apoptosis in HepG2 cell line. Mechanistic investigations revealed that ML-7 and quinocetone act in concert to trigger the cleavage of caspase-8 as well as Bax/Bcl-2 ratio up-regulation and subsequent cleavage of Bid, capsases-9 and -3. Importantly, ML-7 weakened the quinocetone-induced Akt pathway activation, but strengthened the phosphorylation of p-38, ERK and JNK. Further treatment of Akt activator and p-38 inhibitor almost completely abolished the ML-7/quinocetone-induced apoptosis. In contrast, the ERK and JNK inhibitor aggravated the ML-7/quinocetone-induced apoptosis, indicating that the synergism critically depended on p-38 phosphorylation and HepG2 cells provoke Akt, ERK and JNK signaling pathways to against apoptosis. In conclusion, the rational combination of quinocetone and ML-7 presents a promising approach to trigger apoptosis in hepatocellular carcinoma, which warrants further investigation.

Monosodium iodoacetate-induced joint pain is associated with increased phosphorylation of mitogen activated protein kinases in the rat spinal cord.

PubMed

Lee, Younglim; Pai, Madhavi; Brederson, Jill-Desiree; Wilcox, Denise; Hsieh, Gin; Jarvis, Michael F; Bitner, Robert S

2011-05-20

Intra-articular injection of monosodium iodoacetate (MIA) in the knee joint of rats disrupts chondrocyte metabolism resulting in cartilage degeneration and subsequent nociceptive behavior that has been described as a model of osteoarthritis (OA) pain. Central sensitization through activation of mitogen activated protein kinases (MAPKs) is recognized as a pathogenic mechanism in chronic pain. In the present studies, induction of central sensitization as indicated by spinal dorsal horn MAPK activation, specifically ERK and p38 phosphorylation, was assessed in the MIA-OA model. Behaviorally, MIA-injected rats displayed reduced hind limb grip force 1, 2, and 3 weeks post-MIA treatment. In the same animals, activation of phospho ERK1/2 was gradually increased, reaching a significant level at post injection week 3. Conversely, phosphorylation of p38 MAPK was enhanced maximally at post injection week 1 and decreased, but remained elevated, thereafter. Double labeling from 3-wk MIA rats demonstrated spinal pERK1/2 expression in neurons, but not glia. In contrast, p-p38 was expressed by microglia and a subpopulation of neurons, but not astrocytes. Additionally, there was increased ipsilateral expression of microglia, but not astrocytes, in 3-wk MIA-OA rats. Consistent with increased MAPK immunoreactivity in the contralateral dorsal horn, mechanical allodynia to the contralateral hind-limb was observed 3-wk following MIA. Finally, intrathecal injection of the MEK1 inhibitor PD98059 blocked both reduced hind-limb grip force and pERK1/2 induction in MIA-OA rats. Results of these studies support the role of MAPK activation in the progression and maintenance of central sensitization in the MIA-OA experimental pain model.

Stromal cell-derived factor 1 protects human periodontal ligament stem cells against hydrogen peroxide-induced apoptosis.

PubMed

Feng, Yimiao; Fu, Xiaohui; Lou, Xintian; Fu, Baiping

2017-10-01

Periodontal ligament stem cells (PDLSCs) are considered a promising cell source for dental tissue regeneration. Stromal cell-derived factor 1 [SDF‑1, also known as chemokine (C‑X‑C motif) ligand 12] is regarded as a critical cytokine involved in stem/progenitor cell chemotaxis and homing during tissue regeneration. The present study described a previously unsuspected role for SDF‑1 in the protection of PDLSCs against oxidative stress‑induced apoptosis. In the present study, apoptosis was induced by exposure of PDLSCs to various concentrations of H2O2 for 12 h, following which cell viability was assessed, and cleaved caspase‑3 and ‑9 expression levels were evaluated. To investigate the potential mechanism underlying this protection, the protein expression levels of total and phosphorylated extracellular signal‑regulated kinase (ERK), a key protein of the mitogen‑activated protein kinase (MAPK) signaling pathway, were examined. The results of the present study revealed that SDF‑1 pretreatment increased cell viability following H2O2 administration, and downregulated protein expression levels of activated caspase‑3 and ‑9. Furthermore, treatment with SDF‑1 increased the phosphorylation of ERK. The protective effect of SDF‑1 was partially inhibited by treatment with PD98059, a MAPK/ERK inhibitor, which decreased cell viability. The results of the present study suggested that SDF‑1 treatment is a potential strategy to improve the survival of PDLSCs, which may be beneficial for dental tissue regeneration.

Beta-hydroxy-beta-methylbutyrate (HMB) stimulates myogenic cell proliferation, differentiation and survival via the MAPK/ERK and PI3K/Akt pathways.

PubMed

Kornasio, Reut; Riederer, Ingo; Butler-Browne, Gillian; Mouly, Vincent; Uni, Zehava; Halevy, Orna

2009-05-01

Beta-hydroxy-beta-methylbutyrate (HMB), a leucine catabolite, has been shown to prevent exercise-induced protein degradation and muscle damage. We hypothesized that HMB would directly regulate muscle-cell proliferation and differentiation and would attenuate apoptosis, the latter presumably underlying satellite-cell depletion during muscle degradation or atrophy. Adding various concentrations of HMB to serum-starved myoblasts induced cell proliferation and MyoD expression as well as the phosphorylation of MAPK/ERK. HMB induced differentiation-specific markers, increased IGF-I mRNA levels and accelerated cell fusion. Its inhibition of serum-starvation- or staurosporine-induced apoptosis was reflected by less apoptotic cells, reduced BAX expression and increased levels of Bcl-2 and Bcl-X. Annexin V staining and flow cytometry analysis showed reduced staurosporine-induced apoptosis in human myoblasts in response to HMB. HMB enhanced the association of the p85 subunit of PI3K with tyrosine-phosphorylated proteins. HMB elevated Akt phosphorylation on Thr308 and Ser473 and this was inhibited by Wortmannin, suggesting that HMB acts via Class I PI3K. Blocking of the PI3K/Akt pathway with specific inhibitors revealed its requirement in mediating the promotive effects of HMB on muscle cell differentiation and fusion. These direct effects of HMB on myoblast differentiation and survival resembling those of IGF-I, at least in culture, suggest its positive influence in preventing muscle wasting.