Increased MAPK Phosphorylation Research Articles

Endothelial knockdown of the tumor suppressor, WWOX, increases inflammation in ventilator-induced lung injury.

Chronic cigarette smoke exposure decreases lung expression of WWOX which is known to protect the endothelial barrier during infectious models of acute respiratory distress syndrome (ARDS). Proteomic analysis of WWOX-silenced endothelial cells (ECs) was done using tandem mass tag mass spectrometry (TMT-MS). WWOX-silenced ECs as well as those isolated from endothelial cell Wwox knockout (EC Wwox KO) mice were subjected to cyclic stretch (18% elongation, 0.5 Hz, 4 h). Cellular lysates and media supernatant were harvested for assays of cellular signaling, protein expression, and cytokine release. These were repeated with dual silencing of WWOX and zyxin. Control and EC Wwox KO mice were subjected to high tidal volume ventilation. Bronchoalveolar lavage fluid and mouse lung tissue were harvested for cellular signaling, cytokine secretion, and histological assays. TMT-MS revealed upregulation of zyxin expression during WWOX knockdown which predicted a heightened inflammatory response to mechanical stretch. WWOX-silenced ECs and ECs isolated from EC Wwox mice displayed significantly increased cyclic stretch-mediated secretion of various cytokines (IL-6, KC/IL-8, IL-1β, and MCP-1) relative to controls. This was associated with increased ERK and JNK phosphorylation but decreased p38 mitogen-activated kinases (MAPK) phosphorylation. EC Wwox KO mice subjected to VILI sustained a greater degree of injury than corresponding controls. Silencing of zyxin during WWOX knockdown abrogated stretch-induced increases in IL-8 secretion but not in IL-6. Loss of WWOX function in ECs is associated with a heightened inflammatory response during mechanical stretch that is associated with increased MAPK phosphorylation and appears, in part, to be dependent on the upregulation of zyxin.NEW & NOTEWORTHY Prior tobacco smoke exposure is associated with an increased risk of acute respiratory distress syndrome (ARDS) during critical illness. Our laboratory is investigating one of the gene expression changes that occurs in the lung following smoke exposure: WWOX downregulation. Here we describe changes in protein expression associated with WWOX knockdown and its influence on ventilator-induced ARDS in a mouse model.

A study on specific factors related to inflammation and autophagy in BEAS-2B cells induced by urban particulate matter (PM, 1648a) and histological evaluation of PM-induced bronchial asthma model in mice

As particulate matter (PM) poses an increasing risk, research on its correlation with diseases is active. However, researchers often use their own PM, making it difficult to determine its components. To address this, we investigated the effects of PM with known constituents on BEAS-2B cells, examining cytokine levels, reactive oxygen species ROS production, DNA damage, and MAPK phosphorylation. Additionally, we evaluated the effects of PM on normal and OVA-induced asthmatic mice by measuring organ weight, cytokine levels, and inflammatory cells in bronchoalveolar lavage fluid, and examining histological changes. PM markedly increased levels of IL-6, GM-CSF, TNF-α, ROS, nitric oxide, and DNA damage, while surprisingly reducing IL-8 and MCP-1. Moreover, PM increased MAPK phosphorylation and inhibited mTOR and AKT phosphorylation. In vivo, lung and spleen weights, IgE, OVA-specific IgE, IL-4, IL-13, total cells, macrophages, lymphocytes, mucus generation, and LC3II were higher in the asthma group. PM treatment in asthmatic mice increased lung weight and macrophage infiltration, but decreased IL-4 and IL-13 in BALF. Meanwhile, PM treatment in the Nor group increased total cells, macrophages, lymphocytes, and mucus generation. Our study suggests that PM may induce and exacerbate lung disease by causing immune imbalance via the MAPK and autophagy pathways, resulting in decreased lung function due to increased smooth muscle thickness and mucus generation.

Exposure to Mancozeb results in increased MAPK phosphorylation and locomotor deficits in zebrafish larvae

Mancozeb is a widely used fungicide whose toxicity has been reported in non-target organisms, being considered to have high or very high acute toxicity to aquatic organisms. However, the toxicity of this compound is not well characterized in the developmental stages of fish. In this study, Danio rerio with 4-, 5-, and 6-days post fertilization (dpf) was exposed to MZ at non-lethal concentrations for 24, 48, or 72 h and subsequently, behavioral alterations, oxidative stress parameters and ERK, p38MAPK, and Akt phosphorylation were analyzed. MZ exposure during the larval period decreased motor performance evaluated by traveled distance, immobile time, and time spent in the peripheral area. In parallel, MZ induced ROS levels and increased the number of cells in apoptosis, causing severe DNA damage, inducing Acetylcholinesterase and Superoxide dismutase activities, and inhibiting Glutathione peroxidase and thioredoxin reductase. Additionally, phosphorylation levels of the proteins p38MAPK, ERK2, and Akt were stimulated. These findings are relevant considering the ecological implications of MZ exposure to fishes in different developmental stages and the role of the MAPK pathway in events like development and cell death.

The ATR Inhibitor VE-821 Enhances the Radiosensitivity and Suppresses DNA Repair Mechanisms of Human Chondrosarcoma Cells.

To overcome the resistance to radiotherapy in chondrosarcomas, the prevention of efficient DNA repair with an additional treatment was explored for particle beams as well as reference X-ray irradiation. The combined treatment with DNA repair inhibitors-with a focus on ATRi VE-821-and proton or carbon ions irradiation was investigated regarding cell viability, proliferation, cell cycle distribution, MAPK phosphorylation, and the expression of key DNA repair genes in two human chondrosarcoma cell lines. Pre-treatment with the PARPis Olaparib or Veliparib, the ATMi Ku-55933, and the ATRi VE-821 resulted in a dose-dependent reduction in viability, whereas VE-821 has the most efficient response. Quantification of γH2AX phosphorylation and protein expression of the DNA repair pathways showed a reduced regenerative capacity after irradiation. Furthermore, combined treatment with VE-821 and particle irradiation increased MAPK phosphorylation and the expression of apoptosis markers. At the gene expression and at the protein expression/phosphorylation level, we were able to demonstrate the preservation of DNA damage after combined treatment. The present data showed that the combined treatment with ATMi VE-821 increases the radiosensitivity of human chondrosarcoma cells in vitro and significantly suppresses efficient DNA repair mechanisms, thus improving the efficiency of radiotherapy.

Hatakabb, a herbal extract, contains pyrogallol as the novel mediator inhibiting LPS-induced TNF-α production by NF-κB inactivation and HMOX-1 upregulation

• Hatakabb inhibits LPS-induced TNF-α , IL-6 , COX-2 gene expression in macrophages. • LPS-induced IκB and NF-κB phosphorylation is deviated linking to NF-κB inactivation. • HMOX-1 expression is upregulated in association with increased MAPK phosphorylation. • Pyrogallol acts as the anti-inflammatory mediator inhibiting TNF-α production. Hatakabb is a herbal extract traditionally consumed to ameliorate inflammation from pharynx infections. However, the anti-inflammatory function of Hatakabb was not investigated. This study demonstrated the anti-inflammatory potential, and underlying mechanism of Hatakabb, and its functional ingredient. In LPS-treated macrophages, Hatakabb treatment attenuated TNF-α production. A decrease in TNF-α , IL-6 , and COX-2 expression was detected. In regulatory pathway analysis, Hatakabb suppressed IκB-α phosphorylation in accordance with a decrease in IκB-α degradation, and nucleus NF-κB translocation, while enhanced NF-κB phosphorylation. In addition, MAPK phosphorylation was enhanced in association with HMOX-1 upregulation. Our metabolomic analysis showed gallic acid, and pyrogallol contained in the anti-inflammatory subfraction. Compared to gallic acid, pyrogallol strongly diminished TNF-α production. Significant changes in inflammatory gene expression, and regulatory protein phosphorylation were responded to pyrogallol in similar to Hatakabb treatment. Our study, for the first time, reveals the anti-inflammatory effect, and mechanism of Hatakabb in which pyrogallol is the novel mediator inhibiting LPS-induced inflammation.

Effect of malignant-associated pleural effusion on endothelial viability, motility and angiogenesis in lung cancer.

Malignant pleural effusion (MPE) and paramalignant pleural effusion (PPE) remain debilitating complications in lung cancer patients with poor prognosis and limited treatment options. The role of vascular endothelial cells has not been explored in the pleural environment of lung cancer. By integrating MPE and PPE as malignant‐associated pleural fluid (MAPF), the current study aimed to evaluate the effect of MAPF on cell proliferation, migration and angiogenesis of HUVEC. First, increased capillaries were identified in the subpleural layer of lung adenocarcinoma. Compatible with pathological observations, the ubiquitous elevation of HUVEC survival was identified in MAPF culture regardless of the underlying cancer type, the driver gene mutation, prior treatments and evidence of malignant cells in pleural fluid. Moreover, MAPF enhanced HUVEC motility with the formation of lamellipodia and filopodia and focal adhesion complex. Tube formation assay revealed angiogenic behavior with the observation of sheet‐like structures. HUVEC cultured with MAPF resulted in a significant increase in MAPK phosphorylation. Accompanied with VEGFR2 upregulation in MAPF culture, there was increased expressions of p‐STAT3, HIF‐1α and Nf‐kB. VEGF/VEGFR2 blockade regressed endothelial migration and angiogenesis but not cell proliferation. Our data indicate the angiogenic activities of MAPF on vascular endothelial cells that revealed increased pleural capillaries in lung cancer. Targeting the VEGF/VEGFR2 pathway might modulate the angiogenic propensity of MAPF in future clinical investigations.

Creatinine metabolite, HMH (5-hydroxy-1-methylhydantoin; NZ-419), modulates bradykinin-induced changes in vascular smooth muscle cells

A creatinine metabolite, 5-hydroxy-1-methylhydantoin (HMH: NZ-419), a hydroxyl radical scavenger, has previously been shown to confer renoprotection by inhibiting the progression of chronic kidney disease in rats. In the current study, we demonstrate that HMH modulates the effects of glucose and bradykinin (BK) in vascular smooth muscle cell (VSMC). HMH a novel anti-oxidant drug completely suppressed the expression of B2-kinin receptors (B2KR) in response to high glucose (25 mM) stimulation in VSMC and was also shown to attenuate the effects of BK on VSMC remodeling. HMH inhibited the BK-induced increase in MAPK phosphorylation and attenuated the increase in connective tissue growth factor (CTGF) protein levels in VSMC. These findings suggest that HMH may confer vascular protection against high glucose concentrations and BK-stimulation to ameliorate vascular injury and remodeling through its anti-oxidant properties.

544 Uroguanylin-GUCY2C Brain Gut Axis Regulating Feeding Behavior Is Mediated by Nodose Ganglia HCN2 Ion Channel

Opiates differ in their ability to trigger μOR endocytosis and to promote physiological tolerance, which involve changes at the receptor levels and downstream of the receptor. The aims of this study were to test whether agonists with different internalizing abilities activate MAPK, a μOR downstream signaling, in enteric neurons, and the effect of chronic morphine treatment on opioid-induced activation of MAPK. Cultured rat myenteric neurons were incubated with 1 μM DAMGO, a potent μOR-internalizing agonist or 10 μM morphine, a poor μOR-internalizing agonist for 0'-30' at 37oC. MAPK or extracellular signal-regulated kinases 1 and 2 (ERK1/2) phosphorylation was determined byWestern Blot using membranes from cell lysates and the phospho-p44/p42 MAPK and total ERK1/2 antibodies. Specificity controls included pre-incubation with the opioid receptor antagonist, naloxone (10 μM), tetrodotoxin (TTX; 10μM) to block neurotransmitter release or the MAPK inhibitor, U0126 (10 μM). To determine whether opioid-induced MAPK requires receptor internalization and the role of the intracellular trafficking protein, dynamin, myenteric neurons were treated with hypertonic sucrose solution (0.5 M) to block endocytosis, with the dynamin inhibitor, dynasore (10μM) prior to opioid stimulation, or transfected with a wild-type dynamin or a mutated-dynamin using lentiviral vectors. There was a significant increase in MAPK phosphorylation in neurons exposed to DAMGO at 5' and 10', which returned to control levels at 20', while morphine did not induce MAPK activation. DAMGO-induced MAPK phosphorylation was blocked by naloxone and MAPK inhibitor U0126, and by sucrose, dynasore and mutated-dynamin transfection. TTX did not prevent MAPK phosphorylation. To determine the effect of chronic μOR activation on opioid-induced MAPK phosphorylation, cells were treated with 10 μM morphine for 4 days before exposure to DAMGO or morphine. Both DAMGO and morphine acute stimulation induced MAPK activation in enteric neurons chronically treated with morphine, with a peak at 5' and 10' returning to control levels at 20' as in normal cells. Stimulation with DAMGObut not morphine induces μOR internalization in Naive myenteric neurons, whereas both DAMGO and morphine stimulation trigger μOR internalization in neurons chronically treated with morphine. This study shows that opioid stimulation induces a rapid and transient activation of MAPK/ERK in enteric neurons, which is ligand dependent and mediated by direct interaction of the ligand with μOR. Opioid stimulation of MAPK/ERK pathway in enteric neurons requires μOR internalization and is dynamin-dependent. This suggests that receptor endocytosis regulates MAPK, which in turn could trigger feedback signaling affecting cellular response and adaptations, mechanisms underlying the development of chronic opiate adverse effects.

Lysophosphatidylcholine is not a paracrine factor following stretch in C2C12 myotubes

Lysophospholipids, including lysophosphatidylcholine (LPC) are generated through the action of phospholipase A2. LPC can act as a paracrine factor through G2A receptors and by altering Transient receptor potential channel conductance, but also has non‐specific detergent effects. We have previously reported that stretch‐induced LPC production may mediate mechanotransduction in skeletal myotubes. The purpose of this study was to investigate the mechanism of LPC in MAPK activation in C2C12 myotubes and to examine the influence of LPC structure. Exogenous LPC induced a dose‐dependent increase in MAPK phosphorylation, however this was closely correlated with membrane permeabilization, indicated by Trypan blue uptake, and with cell death. These effects were independent of acyl chain length or saturation. Likewise, incubation with bee venom PLA2 was accompanied by increasing MAP Kinase phosphorylation and cell death. These results suggest that exogenous LPC acts primarily as a detergent and induces MAPK phosphorylation only at cytotoxic concentrations. If LPC participates in mechanotransduction, it must be through a highly localized reaction pathway.

MAPK14 and CNR1 gene variant interactions: effects on brain volume deficits in schizophrenia patients with marijuana misuse

Adolescent marijuana use is associated with increased risk for schizophrenia. We previously reported that marijuana misuse in conjunction with specific cannabinoid receptor 1 (CNR1) genetic variants (rs12720071-G-allele carriers) contributed to white-matter (WM) brain volume deficits in schizophrenia patients. In this study, we assessed the influence of another cannabinoid-related gene, mitogen-activated protein kinase 14 (MAPK14), and potential MAPK14-CNR1 gene-gene interactions in conferring brain volume abnormalities among schizophrenia patients with marijuana abuse/dependence. MAPK14 encodes a member of the MAPK family involved in diverse cellular processes, including CNR1-induced apoptosis. We genotyped 235 schizophrenia patients on nine MAPK14 tag single nucleotide polymorphisms (tSNPs). Approximately one quarter of the sample had marijuana abuse or dependence. Differential effects of MAPK14 tSNPs on brain volumes across patients with versus without marijuana abuse/dependence were examined using ANCOVA. Of the MAPK14 tSNPs, only rs12199654 had significant genotype effects and genotype × marijuana misuse interaction effects on WM volumes. rs12199654-A homozygotes with marijuana abuse/dependence had significantly smaller total cerebral and lobar WM volumes. The effects of MAPK14 rs12199654 on WM volume deficits remained significant even after controlling for the CNR1 rs12720071 genotype. There were significant main effects of the MAPK14 CNR1 diplotype and diplotype × marijuana interaction on WM brain volumes, with both genetic variants having additive contributions to WM volume deficits only in patients with marijuana misuse. Given that CNR1-induced apoptosis is preceded by increased MAPK phosphorylation, our study suggests that potential MAPK14-CNR1 gene-gene interactions may mediate brain morphometric features in schizophrenia patients with heavy marijuana use.

Characterization of D3 dopamine receptor signaling in a PD mouse model

The dopamine D3 receptor is known to couple to many signal transduction pathways and has been implicated in Parkinson's disease (PD). The D3 receptor exhibits a tolerance property wherein repeated stimulation elicits a progressively decreasing response. In vitro studies have shown that among others, the tolerance is observed in the D3 receptor‐Mitogen Activated Protein Kinase (MAPK) signal transduction pathway. PD patients on chronic levodopa treatment develop Levodopa Induced Dyskinesia (LID). In this study our objective was to test whether the D3 tolerance property exists in the 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) mouse model of PD. Drd3‐EGFP mice that report the expression of endogenous D3 receptor were used in this study. D3 receptor induced activation of the MAPK kinase pathway was determined in the striatum of control mice or mice treated chronically with levodopa, MPTP or MPTP + levodopa. MPTP treated mice exhibited increased MAPK phosphorylation when challenged with a D3 selective dose of PD128907. However, the MPTP group chronically treated with L‐dopa showed a decrease in phospho‐MAPK after administration of PD128907. These results suggest that in the mouse PD model chronically treated with levodopa, the tolerance property of D3 receptor might be involved.Supported by: Parkinson's Disease Foundation, NIH (MH82376) and the FM Kirby foundation grants

Stereoselective Effects of 4-Hydroxynonenal in Cultured Mouse Hepatocytes

4-Hydroxynonenal (HNE) is produced from arachidonic acid or linoleic acid during oxidative stress. Although HNE is formed in tissues as a racemate, enantiospecific HNE effects have not been widely documented, nor considered. Therefore, a panel of cellular responses was compared after treatment with (R)-HNE, (S)-HNE, or racemic HNE. The phosphorylation status of Jun kinase (JNK) or Akt increased 28-fold or 2-3-fold, respectively, after treatment with 100 μM (S)-HNE and racemic HNE compared to (R)-HNE. In contrast, the increase in phosphorylation of MAPK was greatest for (R)-HNE. Caspase-3-dependent cleavage of the glutamate cysteine ligase (GCL) catalytic subunit and focal adhesion kinase (FAK) were greater in cells treated with (S)-HNE at 48 h. (S)-HNE also caused a greater number of subG1 nuclei, a hallmark of apoptosis, at 30 h after treatment. Together, the results demonstrate different dose- and time-dependent responses to (R)-HNE and (S)-HNE. The results further suggest that HNE enantiomers could differentially contribute to the progression of different diseases or contribute by different mechanisms.

Functional expression and intracellular signaling of UTP-sensitive P2Y receptors in theca-interstitial cells

BackgroundPurinergic receptors are expressed in the ovary of different species; their physiological roles remain to be elucidated. UTP-sensitive P2Y receptor activity may regulate cell proliferation. The aim of the present work was to study the functional expression of these receptors in theca/interstitial cells (TIC).MethodsTIC were isolated by centrifugation in a Percoll gradient. P2Y receptors and cellular markers in TIC were detected by RT-PCR and Western blot. Intracellular calcium mobilization induced by purinergic drugs was evaluated by fluorescence microscopy, phosphorylation of MAPK p44/p42 and of cAMP response element binding protein (CREB) was determined by Western blot and proliferation was quantified by [3H]-thymidine incorporation into DNA.ResultsRT-PCR showed expression of p2y2r and p2y6r transcripts, expression of the corresponding proteins was confirmed. UTP and UDP, agonists for P2Y2 and P2Y6 receptors, induced an intracellular calcium increase with a maximum of more than 400% and 200% of basal level, respectively. The response elicited by UTP had an EC50 of 3.5 +/- 1.01 μM, while that for UDP was 3.24 +/- 0.82 μM. To explore components of the pathway activated by these receptors, we evaluated the phosphorylation induced by UTP or UDP of MAPK p44 and p42. It was found that UTP increased MAPK phosphorylation by up to 550% with an EC50 of 3.34 +/- 0.92 and 1.41 +/- 0.67 μM, for p44 and p42, respectively; these increases were blocked by suramin. UDP also induced p44/p42 phosphorylation, but at high concentrations. Phosphorylation of p44/p42 was dependent on PKC and intracellular calcium. To explore possible roles of this pathway in cell physiology, cell proliferation and hCG-induced CREB-phosphorylation assays were performed; results showed that agonists increased cell proliferation and prevented CREB-phosphorylation.ConclusionHere, it is shown that UTP-sensitive P2Y receptors are expressed in cultured TIC and that these receptors had the ability to activate mitogenic signaling pathways and to promote cell proliferation, as well as to prevent CREB-phosphorylation by hCG. Regulation of TIC proliferation and steroidogenesis is relevant in ovarian pathophysiology since theca hyperplasia is involved in polycystic ovarian syndrome. Purinergic receptors described might represent an important new set of molecular therapeutic targets.

Mouse CD84 is a pan-leukocyte cell-surface molecule that modulates LPS-induced cytokine secretion by macrophages

CD84 is 1 of the 9 SLAM family cell-surface receptors involved in leukocyte activation. The CD84 ectodomain is highly glycosylated, and its cytoplasmic tail contains 2 copies of an ITSM, which can be phosphorylated. Here, we report that although mouse CD84 was present on all BM HSCs, its expression declined in developing thymic and BM lymphocytes. However, CD84 expression levels did increase significantly during the later maturation stages and were expressed abundantly on mature B and T cells. Among lymphocyte subsets, the highest expression was found on innate-like lymphocytes; specifically, on NKT and marginal zone B cells. Splenic CD4+ T(FH) cells exhibited higher levels of CD84 compared with the other CD4+ T cell subsets. CD84 was expressed abundantly on monocytes, macrophages, granulocytes, and DCs. Moreover, as the function of CD84 in myeloid cells remains unknown, we focused on the role this receptor plays in mouse macrophage activation. Transfection of CD84 in RAW-264.7 macrophages led to an increase in MAPK phosphorylation and NF-κB activation upon LPS stimulation. Concomitantly, the presence of CD84 increased the LPS-induced secretion of TNF-α and MCP-1 but lowered IL-10 and IL-6 production significantly. This modulatory effect was mediated by Y(300) within the second ITSM of CD84. Additionally, CD84 knock-down decreased TNF-α and IL-6 production in LPS-activated BMDMs. Taken together, these results show that mouse CD84 is a pan-leukocyte receptor, able to modulate signaling pathways downstream of TLR4, and regulates macrophage cell-fate decisions and effector functions.

Protection of Cells in Physiological Oxygen Tensions against DNA Damage-induced Apoptosis

Oxygen availability has important effects on cell physiology. Although hyperoxic and hypoxic stresses have been well characterized, little is known about cellular functions in the oxygen levels commonly found in vivo. Here, we show that p53-dependent apoptosis in response to different DNA-damaging agents was reduced when normal and cancer cells were cultured at physiological oxygen tensions instead of the usual atmospheric levels. Different from what has been described in hypoxia, this was neither determined by decreases in p53 induction or its transactivation activity, nor by differences in the intracellular accumulation of reactive oxygen species. At these physiological oxygen levels, we found a constitutive activation of the ERK1/2 MAPK in all the models studied. Inhibition of this signaling pathway reversed the protective effect in some but not all cell lines. We conclude that a stress-independent constitutive activation of prosurvival pathways, including but probably not limited to MAPK, can protect cells in physiological oxygen tensions against genotoxic stress. Our results underscore the need of considering the impact of oxygen levels present in the tissue microenvironment when studying cell sensitivity to treatments such as chemotherapy and radiotherapy.

Abstract B144: NVP-BEZ235, a dual inhibitor of PI3K and mTOR, induces MAPK phosphorylation in HER2-positive cells

Abstract The PI3K pathway is highly deregulated in human cancer with the most common alterations being amplification of the receptor tyrosine kinases (RTKs) EGFR and HER2 and mutations in PI3K itself or inactivation of the tumor suppressor PTEN. Thus, the rationale exists to develop therapeutic agents targeting the PI3K/AKT pathway. Several small molecule inhibitors targeting PI3K, AKT and mTOR are now in clinical trials. However, these novel compounds may interfere with regulatory feedback loops. One such example involves the activation of AKT following rapamycin analogues treatment, such as everolimus, an allosteric mTOR inhibitor. Blocking mTOR signaling also alters the regulation of other signaling pathways, like the MAPK cascade. The aim of this study was to investigate the mechanism/s by which inhibition of the PI3K pathway leads to activation of MAPK kinase signaling in breast cancer cell lines and its relevance in tumor treatment. We have observed an increased MAPK phosphorylation in HER2 positive BT474, SKBR-3, MDA-361 and MCF-7HER2 cells when treated with NVP-BEZ235, a dual inhibitor of both mTOR and p110, the catalytic component of PI3K. The increase of P-MAPK was observed by western blot and correlated with hyperphosphorylation of several RTKs. PD325901, a MEK1/2 inhibitor, or lapatinib, a HER1/2 tyrosine kinase inhibitor, reverted the NVP-BEZ235-induced MAPK phosphorylation. On the other hand, NVP-AEW541, an IGFR-1 tyrosine kinase inhibitor, did not prevent NVP-BEZ235-dependent HER2 phosphorylation and downstream MAPK activation. Activation of MAPK upon administration of NVP-BEZ235 was also observed in vivo. We are currently investigating the possible involvement of known regulatory feedback loops as well as the individual RTK contribution. Activation of parallel pathways in a condition of PI3K inhibition may reduce potential benefits of these anti-cancer treatments. For this reason, we propose the combination of an anti-HER2 therapy together with PI3K inhibitors for the treatment of HER2 positive tumors. Citation Information: Mol Cancer Ther 2009;8(12 Suppl):B144.

Differential involvement of H- and K-Ras in Raf-1 activation determines the role of calmodulin in MAPK signaling

We have previously demonstrated that inhibition of calmodulin (CaM) and the concomitant reduction of PI3K interfere with H-Ras-mediated activation of Raf-1 [1]. In the present study, we show that CaM has completely opposite effects on K-Ras-mediated Raf-1 activation. The differential contribution of CaM in the regulation of Raf-1 kinase activity via K- or H-Ras correlates with the stimulatory or inhibitory effect of CaM on MAPK phosphorylation depending on the cell type analyzed. FRET microscopy and biochemical analysis show that inhibition of CaM increases K-Ras-GTP levels and consequently its association with Raf-1. Though inhibition of CaM, using the CaM antagonist W-13, significantly increased Raf-1 activation by K-Ras-GTP, MAPK activation downstream K-Ras/Raf-1 was strongly reduced in COS-1 and several other cell lines. In contrast, in other cell lines such as NIH3T3-wt8, W-13-mediated inhibition of CaM increased Raf-1 activity, but resulted in an increase in MAPK phosphorylation. These findings suggest that modulation of K-Ras activity via CaM regulates MAPK signaling only in certain cell types. In support of this hypothesis, the comparison of H- and K-Ras expression, GTP loading and Raf-1 interaction in COS-1 and NIH3T3-wt8 suggests that the overall role of CaM in MAPK signal output is determined by the ratio of activated H- and K-Ras and the cell-specific contribution of each isoform in Raf-1 activation.

Specific p38 inhibition in stimulated endothelial cells: A possible new anti-inflammatory strategy after hypothermia and rewarming

To protect immature organ systems during corrective cardiac surgery, patients are cooled to a minimal temperature of 17 °C during cardiopulmonary bypass (CPB). However hypothermic CPB triggers the whole body inflammatory response and results in unwanted prolonged inflammation. The present study was designed to clarify the hypothermia and rewarming induced mechanisms and examine interventional pharmacological strategies that could prevent prolonged inflammation. Stimulated primary human umbilical vein endothelial cells (HUVECs) were exposed to a dynamic temperature protocol analogous to clinical settings. Furthermore endothelial cells were pretreated with methylprednisolone and/or tacrolimus as well as with MAPK inhibitors (SB203580, U0126 and SP600125). Cell viability, expression of IL-6 and ERK 1/2, p38 and SAPK/JNK were investigated. Stimulated endothelial cells secreted significantly higher IL-6 protein 2 h after rewarming in comparison to normothermic control cells. Moreover, dynamic temperature changes lead to increased MAPK phosphorylation. Only the combined pre-treatment with MP and TAC served to inhibit the IL-6 secretion. As intracellular signalling pathway we could demonstrate that SB203580 as specific p38 inhibitor most effectively down regulated the unwanted IL-6 release after cooling and rewarming. Therefore inhibition of p38 or components of the p38 pathway could be a promising and selective antiinflammatory therapeutic target after hypothermic CPB.

Insulin increase in MAP kinase phosphorylation is shifted to early time-points by overexpressing APS, while Akt phosphorylation is not influenced

Upon insulin stimulation, the adaptor protein APS is recruited to the insulin receptor and tyrosine phosphorylated. APS initiates the insulin-induced TC10 cascade which participates to GLUT4 translocation to the plasma membrane. Nevertheless, the molecular mechanism that governs APS and its SH2 and PH domains action on the insulin transduction cascade is not yet fully understood. Here, we show that APS co-immunoprecipitates with the class I PI 3-kinase regulatory subunit p85, through its SH2 domain but that APS does not modulate neither PtdIns(3,4,5)P3 levels nor Akt phosphorylation provoked by insulin. We have confirmed a previously described positive effect of APS overexpression on insulin-induced MAPK phosphorylation upregulation. Consequently, we analyzed the role of SH2 and PH domains of APS in the APS increased MAPK phosphorylation observed upon insulin stimulation and correlated this with the membrane localization of the protein. The effect observed on MAPK phosphorylation requires the intact PH binding domain of APS as well as its SH2 domain.

Selenium Inhibits Proliferation Signaling and Restores Sodium/Potassium Pump Function of Diabetic Rat Aorta

Diabetes is characterized with increased oxidant stress, vasculopathy, and neuropathy. In diabetic vasculopathy, the observed thickening of the media and intima is not only a result of vascular smooth muscle cell proliferation but also due to modification of the extracellular matrix by these cells. Also, there is hampered membrane function and a reduction in sodium pump expression in the vessels of the diabetic animals. Selenium, being a trace element, has both insulinomimetic and antioxidant effects. Thus, we hypothesized that selenium treatment will reduce proliferation, restore physiology, and correct increased proliferation signaling of diabetic aorta. Diabetes was induced by streptozotocin (50 mg/kg body weight), and rats were then treated with sodium selenate (15 mumol/kg body weight/day) for 4 weeks. Our data from diabetic rats showed an increase in proliferation rate and matrix metalloproteinase activity in aortic cell cultures. We observed marked increases in MAPK phosphorylation and caveolin 1 expression but a decrease in Na(+)/K(+) ATPase activity in diabetic rat aorta homogenates. Selenium treatment resulted in complete normalization of the above parameters to control level, while it increased Na(+)/K(+) pump activity by 40%. Our results suggest that selenium treatment of diabetics can play beneficial role in protecting vascular architecture and function against diabetes-induced pathology.