P38 Mitogen-activated Protein Kinase Inhibitor SB203580 Research Articles

P38 MAPK regulates the expression of ether à go-go potassium channel in human osteosarcoma cells

BackgroundThe ether à go-go (Eag) channel has been shown to be overexpressed in a variety of cancers. However, the expression and function of Eag in osteosarcoma are poorly understood. In addition, the molecular mechanisms responsible for Eag overexpression in cancer cells remain unclear.MethodsThe expression of Eag in human osteosarcoma cell line MG-63 was detected by reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. The effect of Eag inhibition on MG-63 cell proliferation was assessed in vitro. The effect of short hairpin RNA (shRNA) mediated knockdown of Eag on osteosarcoma growth was evaluated in xenograft model in vivo. The activation of mitogen-activated protein kinase (MAPK) pathway and p53 in MG-63 cells was detected by Western blot analysis.ResultsEag was overexpressed in MG-63 cells. Imipramine or Eag shRNA significantly suppressed the proliferation of MG-63 cells in vitro and in vivo. MG-63 cell proliferation was specifically inhibited by p38 MAPK inhibitor SB203580 or small interference RNA (siRNA). The inhibition of p38 MAPK activation by SB203580 or siRNA reduced Eag protein level but increased p53 protein level. Moreover, the activation of p53 by nutlin-3 induced cell growth arrest in MG-63 cells and reduced Eag protein level, while the inactivation of p53 by pifithrin-alpha (PFT-α) promoted MG-63 cell growth and increased Eag protein expression.ConclusionsEag channel functions as an oncogene to promote the proliferation of human osteosarocma cells. Furthermore, the high expression of Eag in osteosarcoma cells is regulated by p38 MAPK/p53 pathway.

Pharmacological Inhibition of p38 Mitogen-Activated Protein Kinases Affects KC/CXCL1-Induced Intraluminal Crawling, Transendothelial Migration, and Chemotaxis of NeutrophilsIn Vivo

p38 mitogen-activated protein kinase (MAPK) signalling is critical in the pathophysiology of a variety of inflammatory processes. Leukocyte recruitment to the site of inflammation is a multistep process governed by specific signalling cascades. After adhesion in the lumen, many leukocytes crawl to optimal sites at endothelial junctions and transmigrate to extravascular tissue in a Mac-1-dependent manner. The signalling mechanisms that regulate postadhesion steps of intraluminal crawling, transmigration, and chemotaxis in tissue remain incompletely understood. The present study explored the effect of p38 MAPK inhibitor SB203580 on various parameters of neutrophil recruitment triggered by chemokine KC (CXCL1) gradient. Neutrophil-endothelial interactions in microvasculature of murine cremaster muscle were determined using intravital microscopy and time-lapsed video analysis. SB203580 (100 nM) did not change leukocyte rolling but significantly attenuated neutrophil adhesion, emigration, and transmigration and impaired the initiation of neutrophil crawling and transmigration. In response to KC chemotactic gradient, SB203580 significantly reduced the velocity of migration and chemotaxis index of neutrophils in tissue. The upregulation of Mac-1 expression in neutrophils stimulated by KC was significantly blunted by SB203580 in vitro. Collectively, our findings demonstrate that pharmacological suppression of p38 MAPK significantly impairs multiple steps of neutrophil recruitment in vivo.

Metformin-mediated downregulation of p38 mitogen-activated protein kinase-dependent excision repair cross-complementing 1 decreases DNA repair capacity and sensitizes human lung cancer cells to paclitaxel

Metformin, an extensively used and well-tolerated drug for treating individuals with type 2 diabetes, has recently gained significant attention as an anticancer drug. On the other hand, paclitaxel (Taxol) is a new antineoplastic drug that has shown promise in the treatment of non-small cell lung cancer (NSCLC). High expression levels of excision repair cross-complementary 1 (ERCC1) in cancers have been positively associated with the DNA repair capacity and a poor prognosis in NSCLC patients treated with platinum-containing chemotherapy. In this current study, paclitaxel was found to increase phosphorylation of mitogen-activated protein kinase (MAPK) kinase 3/6 (MKK3/6)-p38 MAPK as well as protein and mRNA levels of ERCC1 in H1650 and H1703 cells. Moreover, paclitaxel-induced ERCC1 protein and mRNA levels significantly decreased via the downregulation of p38 activity by either a p38 MAPK inhibitor SB202190 or p38 knockdown with specific small interfering RNA (siRNA). Specific inhibition of ERCC1 with siRNA was found to enhance the paclitaxel-induced cytotoxic effect and growth inhibition. Furthermore, metformin was able to not only decrease the paclitaxel-induced p38 MAPK-mediated ERCC1 expression, but also augment the cytotoxic effect induced by paclitaxel. Finally, expression of constitutive activate MKK6 or HA-p38 MAPK vectors in lung cancer cells was able to abrogate ERCC1 downregulation by metformin and paclitaxel as well as cell viability and DNA repair capacity. Overall, our results suggest that inhibition of the p38 MAPK signaling by metformin coupled with paclitaxel therapy in human NSCLC cells may be a clinically useful combination, which however will require further validation.

Induction of neurite outgrowth in PC12 cells by artemisinin through activation of ERK and p38 MAPK signaling pathways

Growth of neurite processes is a critical step in neuronal development, regeneration, differentiation, and response to injury. The discovery of compounds that can stimulate neurite formation would be important for developing new therapeutics against both neurodegenerative disorders and trauma-induced neuronal injuries. Semisynthetic derivatives of artemisinin, an active compound in Artemisia annua, have been effectively used in malaria treatment, but they have been shown to possess neurotoxic potential. In this study, we found unexpectedly that artemisinin and its derivatives induced neurite outgrowth of PC12 cells. Artemisinins containing an endoperoxide bridge such as artemisinin and dihydroartemisinin induced growth of neurite processes at concentrations that were slightly cytotoxic, artemisinin having the most potent maximal effect among them. Deoxyartemisinin, which lacks the endoperoxide bridge, was ineffective. Artemisinin-treated cells expressed increased levels of the neuronal marker βIII-tubulin. Artemisinin upregulated phosphorylation of extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (MAPK), critical signaling molecules in neuronal differentiation. Consistent with activation of the two MAPKs, neurite outgrowth induced by artemisinin was inhibited by the MAPK/ERK kinase inhibitor PD98059 and the p38 MAPK inhibitor SB203580. Artemisinin also induced phosphorylation of cyclic AMP response element-binding protein (CREB) that was almost completely attenuated by PD98059 but not by SB203580. Taken together, our results indicate that artemisinin and its derivatives containing the endoperoxide bridge induced differentiation of PC12 cells toward a neuronal phenotype and suggest that both activation of ERK signaling pathway, which leads to CREB phosphorylation, and activation of p38 MAPK signaling pathway are involved in this process.

Role of JNK and p38 MAPK in Taiwanin A-induced cell death

AimThe lignan compound Taiwanin A is cytotoxic for human cancer cells. Taiwanin A has been previously shown to damage microtubules, induce mitotic arrest and cause apoptosis in cancer cells. The goal of the current study is to identify intracellular signaling pathways that are involved in Taiwanin A-mediated apoptosis. Main methodsWe examined the activation of three mitogen-activated protein kinases (MAPKs), extracellular signal-regulated kinase (ERK), p38 and c-Jun N-terminal kinase (JNK), in HepG2 cells after Taiwanin A treatment. The role of MAPK activation in Taiwanin A-induced apoptosis was examined using Western blotting, caspase activity assays combined with specific MAPK inhibitors and shRNA treatment to knockdown JNK. Key findingsTaiwanin A activated all three MAPKs (ERK, p38 and JNK). Cytotoxicity was blocked by the p38 MAPK inhibitor SB203580 and the JNK inhibitor SP600125 but not by the ERK inhibitor PD98059. A combined treatment of SB203580 and SP600125 showed increased effects on the inhibition of Taiwanin A cytotoxicity, suggesting that both JNK and p38 play a role in Taiwanin A-induced apoptosis. Inhibition of p38 activity reduced Taiwanin A-induced p53 phosphorylation on Ser15. Direct interaction of Taiwanin A-activated p38 and p53 was demonstrated by immunoprecipitation. In addition, inhibition of JNK by SP600125 or silencing of the JNK scaffold protein JIP2 reduced phosphorylation of Bcl-2, which may help to promote anti-apoptotic pathways. SignificanceWe demonstrated for the first time that two distinct apoptotic pathways, the p38-p53 and JNK-Bcl-2 pathways, were triggered by the anti-microtubule compound Taiwanin A.

Activation of p38 MAP kinase and stress signalling in fibroblasts from the progeroid Rothmund–Thomson syndrome

Rothmund–Thomson fibroblasts had replicative lifespans and growth rates within the range for normal fibroblasts; however, they show elevated levels of the stress-associated p38 MAP kinase, suggestive of stress during growth. Treatment with the p38 MAP kinase inhibitor SB203580 increased both lifespan and growth rate, as did reduction of oxidative stress using low oxygen in some strains. At replicative senescence p53, p21WAF1 and p16INK4A levels were elevated, and abrogation of p53 using shRNA knockdown allowed the cells to bypass senescence. Ectopic expression of human telomerase allowed Rothmund–Thomson fibroblasts to bypass senescence. However, activated p38 was still present, and continuous growth for some telomerised clones required either a reduction in oxidative stress or SB203580 treatment. Overall, the evidence suggests that replicative senescence in Rothmund–Thomson cells resembles normal senescence in that it is telomere driven and p53 dependent. However, the lack of RECQL4 leads to enhanced levels of stress during cell growth that may lead to moderate levels of stress-induced premature senescence. As replicative senescence is believed to underlie human ageing, a moderate level of stress-induced premature senescence and p38 activity may play a role in the relatively mild ageing phenotype seen in Rothmund–Thomson.Electronic supplementary materialThe online version of this article (doi:10.1007/s11357-012-9476-9) contains supplementary material, which is available to authorized users.

Puerarin stimulates osteoblasts differentiation and bone formation through estrogen receptor, p38 MAPK, and Wnt/β-catenin pathways

Puerarin is an isoflavone extracted from Radix Puerariae, a traditional Chinese herb used to treat many diseases such as osteoporosis. In this study, puerarin was shown to stimulate alkaline phosphatase (ALP) activity, type I collagen (Col I) secretion, and mineralized nodules formation of primary osteoblasts. Whereas the estrogen receptor (ER) antagonist ICI 182780 was able to reduce the increase in ALP activity and Col I secretion induced by puerarin. Furthermore, puerarin was shown to elevate levels of phospho-p38 mitogen-activated protein kinase (MAPK) and β-catenin proteins in a time-dependent manner. Pretreatment of osteoblasts with ICI 182780 can reduce this elevation, whereas pretreatment with p38 MAPK inhibitor SB 203580 did not affect the increase of β-catenin protein. Meanwhile, intragastric administration of puerarin protected against reduction in bone mineral density and bone mineral content in ovariectomized rats, and improved femur trabecular bone structure. Taken together, ER, p38 MAPK, and Wnt/β-catenin pathways were involved in puerarin-stimulated osteoblasts differentiation and bone formation.

P38 MAP kinase and ERK play an important role in nitric oxide-induced apoptosis of the mouse embryonic stem cells

Previous study showed that nitric oxide (NO) induces apoptosis in mouse embryonic stem (mES) cells, but the precise mechanism governing NO-induced apoptosis in mES remains unclear. This study investigated the mechanism of NO-induced apoptosis of mES cells via MAP kinase signaling pathway. Sodium nitroprusside (SNP), a NO donor, induced apoptosis in mES cells with enhanced production of reactive oxygen species (ROS). In addition, treatment with SNP induced the activation of caspase-3, -8 and -9 as well as mitogen-activated protein (MAP) kinases (JNK, p38 MAP kinase and ERK). However, pretreatment with the p38 MAP kinase inhibitor SB203580 and ERK inhibitor U0126 attenuated NO-induced cell toxicity, ROS production, and caspase-3 activation. Moreover, SB203580 inhibited the translocation of Bax from the cytosol to the mitochondria. Taken together, these results suggest that NO-induced apoptosis in mES cells was mediated through p38 MAP kinase/ERK signaling pathway by triggering caspases activation and Bax translocation from the cytosol to the mitochondria.

Accumulating microglia phagocytose injured neurons in hippocampal slice cultures: involvement of p38 MAP kinase.

In this study, microglial migration and phagocytosis were examined in mouse organotypic hippocampal slice cultures, which were treated with N-methyl-D-aspartate (NMDA) to selectively injure neuronal cells. Microglial cells were visualized by the expression of enhanced green fluorescent protein. Daily observation revealed microglial accumulation in the pyramidal cell layer, which peaked 5 to 6 days after NMDA treatment. Time-lapse imaging showed that microglia migrated to the pyramidal cell layer from adjacent and/or remote areas. There was no difference in the number of proliferating microglia between control and NMDA-treated slices in both the pyramidal cell layer and stratum radiatum, suggesting that microglial accumulation in the injured areas is mainly due to microglial migration, not to proliferation. Time-lapse imaging also showed that the injured neurons, which were visualized by propidium iodide (PI), disappeared just after being surrounded by microglia. Daily observation revealed that the intensity of PI fluorescence gradually attenuated, and this attenuation was suppressed by pretreatment with clodronate, a microglia toxin. These findings suggest that accumulating microglia phagocytosed injured neurons, and that PI fluorescence could be a useful indicator for microglial phagocytosis. Using this advantage to examine microglial phagocytosis in living slice cultures, we investigated the involvements of mitogen-activated protein (MAP) kinases in microglial accumulation and phagocytosis. p38 MAP kinase inhibitor SB203580, but not MAP kinase/extracellular signal-regulated kinase inhibitor PD98059 or c-Jun N-terminal kinase inhibitor SP600125, suppressed the attenuation of PI fluorescence. On the other hand, microglial accumulation in the injured areas was not inhibited by any of these inhibitors. These data suggest that p38 MAP kinase plays an important role in microglial phagocytosis of injured neurons.

HGFK1 inhibits bone metastasis in breast cancer through the TAK1/p38 MAPK signaling pathway

Breast cancer metastasis to bone represents a devastating complication of advanced breast cancer, frequently resulting in significant increases in morbidity and mortality. An understanding of the mechanisms that govern breast cancer metastasis at the molecular level should lead to more effective therapies. Recently, the kringle 1 domain of human hepatocyte growth factor (HGFK1) was identified as a candidate metastasis suppressor gene. Here, we investigated whether HGFK1 is a key regulator of breast cancer bone metastasis. Of the 193 human breast carcinoma tissue samples examined, HGFK1 expression was relative higher in 82 (42.4%) by western blot and in 84 (43.5%) by quantitative real-time PCR. The higher expression of HGFK1 was significantly associated with a better prognostic value (P<0.001) and inversely correlated with bone metastasis (P=0.003). The efficacy of adeno-associated virus carrying HGFK1 (AAV-HGFK1) in osteolytic bone metastasis was then evaluated using an in vivo bone metastasis model. AAV-HGFK1 significantly inhibited osteolytic bone metastasis and prolonged the survival of mice in this model (P<0.01). In vitro, HGFK1 expression resulted in significant anti-invasion effects, enhanced the phosphorylation of TAK1 (transforming growth factor-β-activated kinase 1), p38 MAPK (mitogen-activated protein kinase) and MAPKAPK2 (MAPK-activated protein kinase 2) and decreased the expression of receptor activator of nuclear factor-κB (RANK), which was abrogated by the p38 MAPK inhibitor SB203580. This study shows for the first time that HGFK1 significantly inhibits the metastasis of breast cancer to bone by activating the TAK1/p38 MAPK signaling pathway and inhibiting RANK expression. Thus, AAV-HGFK1 treatment represents a potential therapy for bone metastasis in breast cancer.

Evidence That Formation of Vimentin·Mitogen-activated Protein Kinase (MAPK) Complex Mediates Mast Cell Activation following FcεRI/CC Chemokine Receptor 1 Cross-talk

Accumulating evidence points to cross-talk between FcεRI and CC chemokine receptor (CCR)-mediated signaling pathways in mast cells. Here, we propose that vimentin, a protein comprising type III intermediate filament, participates in such cross-talk for CCL2/monocyte chemotactic protein 1 (MCP-1) production in mast cells, which is a mechanism for allergic inflammation. Co-stimulation via FcεRI, using IgE/antigen, and CCR1, using recombinant CCL3/macrophage inflammatory protein-1α (MIP-1α), increased expression of phosphorylated, disassembled, and soluble vimentin in rat basophilic leukemia (RBL)-2H3 cells expressing human CCR1 (RBL-CCR1 cells) and bone marrow-derived murine mast cells, both models of mucosal type mast cells. Furthermore, co-stimulation enhanced production of CCL2 as well as phosphorylation of MAPK. Treating the cells with p38 MAPK inhibitor SB203580, but not with MEK inhibitor PD98058, reduced CCL2 production, suggesting that p38 MAPK, but not ERK1/2, plays a critical role in the chemokine production. Immunoprecipitation analysis showed that vimentin interacts with phosphorylated ERK1/2 and p38 MAPKs in the co-simulated cells. Preventing disassembly of the vimentin by aggregating vimentin filaments using β,β'-iminodipropionitrile reduced the interaction of vimentin with phosphorylated MAPKs as well as CCL2 production in the cells. Taken together, disassembled vimentin interacting with phosphorylated p38 MAPK could mediate CCL2 production in mast cells upon FcεRI and CCR1 activation.

Mitogen-activated protein kinases-dependent induction of hepatocyte growth factor production in human dermal fibroblasts by the antibiotic polymyxin B

Hepatocyte growth factor (HGF) stimulates migration and proliferation of keratinocytes and has been suggested to be involved in wound healing. The cationic antibiotic polymyxin B (PMB) is commonly used as a topical antibiotic for wound care. If PMB possesses an HGF-inducing activity, the antibiotic is potentially beneficial for wound healing in addition to minimizing chances of infection. In this study, we found that PMB markedly induced HGF production from various types of cells including human dermal fibroblasts. Its effect was stronger than the effects of epidermal growth factor and cholera toxin and was comparable to the effect of 8-bromo-cAMP. Among the polymyxin family and polymyxin derivatives, colistin was also effective, whereas colistin methanesulfonate had only a marginal effect and PMB nonapeptide was ineffective. The stimulatory effect of PMB was accompanied by upregulation of HGF gene expression. Increase in phosphorylation of extracellular signal-regulated protein kinase (ERK) and c-Jun N-terminal kinase (JNK) was observed from 0.25h to 6h after the addition of PMB, while increase in phosphorylation of p38 mitogen-activated protein kinase (MAPK) was detected from 24h to 60h after PMB addition. The MAPK/ERK kinase inhibitor PD98059, the JNK inhibitor SP600125 and the p38 MAPK inhibitor SB203580 all potently inhibited PMB-induced HGF production. Lastly, proliferation of human dermal fibroblasts was significantly stimulated by PMB. These results indicate that PMB-induced HGF production and proliferation of human dermal fibroblasts and suggest that activation of MAPKs is involved in the induction of HGF production.

Sphingosine kinase 1 enhances colon cancer cell proliferation and invasion by upregulating the production of MMP-2/9 and uPA via MAPK pathways

Sphingosine kinase (SphK) 1 is an oncogenic enzyme promoting transformation, proliferation, and survival of a number of human tumor cells. However, its effect on colon cancer cell behavior has not been fully clarified. SphK1 plasmid or SphK1 shRNA transfection and N,N-dimethylsphingosine (DMS) was used to regulate the expression and activity of SphK1 in colon cancer line LOVO. Cell proliferation, apoptosis, invasion, and protein expression were detected by MTT, flow cytometry, transwell chambers model, and western blot. The levels of metalloproteinases-2/9 (MMP-2/9) and urokinase plasminogen activator (uPA) were detected by ELISA. Overexpression of SphK1 after plasmid transfection markedly enhanced LOVO cell viability and invasiveness and reduced cell apoptosis. In contrast, inhibition of SphK1 by DMS and shRNA significantly suppressed cell viability and invasiveness but promoted cell apoptosis. SphK1 increased the constitutive expression of extracellular signal-regulated kinase1/2 (ERK1/2) but reduced the constitutive expression of p38 mitogen-activated protein kinase (MAPK). Blocking ERK1/2 pathway inhibited the biological effects induced by overexpression of SphK1. Blocking p38 MAPK pathway reversed the effects of DMS and SphK1 shRNA. Moreover, SphK1 was required for the production of MMP-2/9 and uPA in tumor cells, which was suppressed by ERK1/2 inhibitor U0126, but enhanced by the p38 MAPK inhibitor SB203580. SphK1 enhances colon cancer cell proliferation and invasiveness, meanwhile suppressing cell apoptosis. SphK1 promoting the secretion of MMP-2/9 and uPA via activation of ERK1/2 and suppression of p38 MAPK pathways maybe the molecular mechanisms for its regulation of the malignant behavior of colon cancer cell.

Cyclosporin A induces apoptosis in H9c2 cardiomyoblast cells through calcium-sensing receptor-mediated activation of the ERK MAPK and p38 MAPK pathways

The cardiotoxicity of cyclosporine A (CsA) limits its clinical application in extensive and long-term therapies. Our group has shown that CsA induces myocardium cell apoptosis in vivo and increases calcium-sensing receptor (CaSR) expression. However, its molecular mechanism remains unknown. The purpose of this study was to determine whether CaSR plays an essential role in CsA-induced apoptosis in H9c2 cells and to investigate the role of the mitogen-activated protein kinase (MAPK) signaling cascade in this process. H9c2 cells were treated with CsA in a dose-dependent manner, and decreased Bcl-2 expression, increased Bax expression, and caspase-3 activation were observed. In a time-dependent manner, CsA increased CaSR expression, activated the extracellularly regulated kinase (ERK) and p38 MAPK pathways, and inactivated the c-Jun N-terminal kinase (JNK) MAPK signaling pathway. When H9c2 cardiomyoblast cells pretreated with gadolinium chloride (GdCl(3)), a CaSR activator, were treated with CsA, decreased phosphorylation of ERK1/2, increased phosphorylation of p38, decreased Bcl-2 expression, increased Bax expression, and activated caspase-3 were observed. Cells pretreated with the CaSR inhibitor NPS2390 inhibited this process. Furthermore, the MEK1/2 inhibitor U0126 and the p38 MAPK inhibitor SB203580 markedly blocked the effect of CsA on cell apoptosis, apoptotic-related protein expression, and caspase-3 activation. These findings showed that CsA induced apoptosis in H9c2 cells in vitro, and CaSR mediated the degradation of ERK MAPK and the upregulation of the p38 MAPK pathway involved in CsA-induced H9c2 cardiomyoblast cell apoptosis.

Interaction of vimentin and phosphorylated mitogen-activated protein kinases in FcϵRI and CC chemokine receptor 1 engaged mast cells (44.18)

Abstract Accumulating evidence points to cross-talk between FcϵRI and CC chemokine receptor (CCR)-mediated signaling pathways in mast cells. Here, we propose that vimentin, a protein composing type III intermediate filament, participates in such cross-talk for CCL2/ monocyte chemotactic protein 1 production in mast cells. Co-stimulation via FcϵRI, with IgE/antigen, and CCR1, with recombinant CCL3/macrophage inflammatory protein-1α increased expression of phosphorylated, disassembled and soluble vimentin in rat basophilic leukemia (RBL)-2H3 cells expressing human CCR1 (RBL-CCR1 cells), a model of mucosal type mast cells. Furthermore, the co-stimulation enhanced production of CCL2 and phosphorylation of mitogen-activated protein (MAP) kinases. Treating the cells with p38 MAP kinase inhibitor SB203580, but not with MAP kinase kinase (MEK) inhibitor PD98058, reduced CCL2 production, suggesting that p38 MAP kinase, but not extracellular-signal-regulated kinase 1/2 (ERK1/2), plays a critical role in the chemokine production. Immunoprecipitation analysis showed association of vimentin with phosphorylated ERK1/2 and p38 MAP kinases in the co-simulated cells. Preventing formation of the soluble vimentin by aggregating vimentin filaments using beta,beta'-iminodipropionitrile reduced the association of vimentin with phospholylated MAP kinases, and CCL2 production in the cells. Taken together, soluble vimentin could need optimal CCL2 production in mast cells.

Concomitant Induction of Heme Oxygenase‐1 Attenuates the Cytotoxicity of Arsenic Species from Lumbricus Extract in Human Liver HepG2 Cells

Heme oxygenase-1 (HO-1) is an inducible antioxidant enzyme that degrades heme to three products, biliverdin, carbon monoxide (CO), and iron ion. The present study was originally designed to characterize the HO-1 induction by Lumbricus extract as a potential cytoprotective mechanism. Through bioactivity-guided fractionation, with human HepG2 cells as the cellular detector, surprisingly, we found that arsenic was enriched in the active fractions isolated from Lumbricus extract. Arsenic speciation was further carried out by liquid chromatography with inductively coupled plasma mass spectrometry (LC/ICP-MS). Our results showed that Lumbricus extract contained two major arsenic species, arsenite (As(III) ; 53.7%) and arsenate (As(V) ; 34.2%), and six minor arsenic species. Commercial sodium arsenite (NaAsO(2) ) was used to verify the effects of Lumbricus extract on HO-1 expression and related intracellular signaling pathways. Both p38 MAP kinase and NF-E2-related factor 2 (Nrf2) pathways were found to modulate HO-1 induction by Lumbricus extract and NaAsO(2) . The cytotoxicity of arsenite was augmented by p38 MAP kinase inhibitor SB202190 and HO-1 inhibitor tin protoporphyrin IX (SnPP), whereas p38 MAP kinase inhibitor SB202190 also inhibited HO-1 induction by NaAsO(2) . These results suggest that arsenic-containing compounds are responsible for HO-1 induction by Lumbricus extract. Although the exact role of toxic arsenic compounds in the treatment of oxidative injury remains unclear, concomitant HO-1 induction may be a key mechanism to antagonize the cytotoxicity of arsenic compounds in human cells.

P38 mitogen‐activated protein kinase signaling, ERCC1 expression, and viability of lung cancer cells from never or light smoker patients

Expression of DNA-repair proteins and activated mitogen-activated protein kinases (MAPKs) may differ according to smoking status. The authors investigated whether p38 MAPK activity contributed to the viability of cisplatin in lung cancer cell lines from never or light smokers and to ERCC1 mRNA expression. Activated p38 MAPK was tested as a predictor for ERCC1 levels in 117 lung adenocarcinomas. Cell viabilities of NCI-H1975, NCI-H1793, NCI-H1650, and NCI-H1651 cell lines, derived from never or light smokers, were measured after treatment with the p38 MAPK inhibitor SB202190 and cisplatin. The role of p38α (MAPK14) and p38β (MAPK11) isoforms and ERCC1 was evaluated using RNA interference. ERCC1 protein-level expression was predicted by activated p38 MAPK in lung adenocarcinoma tissues. The p38-specific inhibitor SB202190 strongly decreased cell viability (43%-63%). SB202190 plus cisplatin significantly decreased cell viability in every cell line, including cisplatin-resistant NCI-H1793. Genetic inhibition, targeting both MAPK11 and MAPK14, reduced the viability of the different cell lines: down-regulation of p38β accounted for most of this effect. Cisplatin's effect was greater after MAPK11 down-regulation for NCI-H1651, and MAPK14 down-regulation for NCI-H1650. In addition, both SB202190 and MAPK11 inhibition reduced excision repair cross-complementing 1 mRNA levels. Lung cancer cells from never or light smokers rely on p38 MAPK signaling for survival. MAPK11 is involved in that pathway and might contribute to ERCC1 expression. Sensitization to cisplatin can be achieved by pharmacological inhibition of p38 MAPK signaling.

Nicotine stimulates urokinase-type plasminogen activator receptor expression and cell invasiveness through mitogen-activated protein kinase and reactive oxygen species signaling in ECV304 endothelial cells

Urokinase-type plasminogen activator receptor (uPAR) expression is elevated during inflammation, tissue remodeling and in many human cancers. This study investigated the effect of nicotine, a major alkaloid in tobacco, on uPAR expression and cell invasiveness in ECV304 endothelial cells. Nicotine stimulated uPAR expression in a dose-dependent manner and activated extracellular signal-regulated kinases-1/2 (Erk-1/2), c-Jun amino-terminal kinase (JNK) and p38 mitogen activated protein kinase (MAPK). Specific inhibitors of MEK-1 (PD98059) and JNK (SP600125) inhibited the nicotine-induced uPAR expression, while the p38 MAPK inhibitor SB203580 did not. Expression vectors encoding dominant negative MEK-1 (pMCL-K97M) and JNK (TAM67) also prevented nicotine-induced uPAR promoter activity. The intracellular hydrogen peroxide (H2O2) content was increased by nicotine treatment. The antioxidant N-acetylcysteine prevented nicotine-activated production of reactive oxygen species (ROS) and uPAR expression. Furthermore, exogenous H2O2 increased uPAR mRNA expression. Deleted and site-directed mutagenesis demonstrated the involvement of the binding sites of transcription factor nuclear factor-kappaB (NF-κB) and activator protein (AP)-1 in the nicotine-induced uPAR expression. Studies with expression vectors encoding mutated NF-κB signaling molecules and AP-1 decoy confirmed that NF-κB and AP-1 were essential for the nicotine-stimulated uPAR expression. MAPK (Erk-1/2 and JNK) and ROS functioned as upstream signaling molecules in the activation of AP-1 and NF-κB, respectively. In addition, ECV304 endothelial cells treated with nicotine displayed markedly enhanced invasiveness, which was partially abrogated by uPAR neutralizing antibodies. The data indicate that nicotine induces uPAR expression via the MAPK/AP-1 and ROS/NF-κB signaling pathways and, in turn, stimulates invasiveness in human ECV304 endothelial cells.

Abstract 16962: Angiotensin II Limits NO Production by Upregulating Arginase through a Mitogen Activated Protein Kinase-Activating Transcription Factor-2 pathway

Nitric oxide (NO) produced by endothelial nitric oxide synthase (NOS) is needed for normal vascular function. During hypertension, diabetes or aging, elevated levels of arginase (ARG) can compete with NOS for available L-arginine thus reducing vascular NO production. Elevated angiotensin II (Ang II) is a key participant of endothelial dysfunction in many cardiovascular diseases and has been linked to elevated ARG activity. In this study we explored the signaling pathway leading to increased ARG expression/activity in responses to Ang II in bovine aortic endothelial cells (BAEC). Treatment of BAEC with Ang II (10 -7 M, 24 hrs) caused a 40±6% increase (p&lt;0.05) in ARG activity. This was accompanied by 30±8% decrease (p&lt;0.05) in NO production. Our studies indicate involvement of p38 mitogen activated protein kinase (MAPK) in Ang II-induced ARG upregulation and reduced NO production as p38 MAPK inhibitor SB202190 (2 μM) prevented the Ang II-induced increase in ARG activity. Our mouse studies also show involvement of p38 MAPK in Ang II-induced vascular dysfunction associated with elevated ARG activity and ARG expression. Ang II (42 µg/kg/h, sc, 2 wks) caused impaired EC-dependent vasorelaxation in mouse aorta (55±7% vs. 75±8% for control). This impairment was prevented by treatment with p38 inhibitor SB203580 (5µg/kg/day). Ang II also caused a 6.2 fold increase in vascular ARG activity/expression that was completely prevented by p38 MAPK inhibition. Additionally, treatment of BAEC with Ang II causes phosphorylation of activating transcription factor-2 (ATF-2) by 10 minutes with a peak at 30 minutes (3.9 fold). Transfection of BAEC with ATF-2 siRNA prevents Ang II-induced increases in ARG activity/expression and maintains NO production. These results indicate that ATF-2 is necessary for enhanced expression of ARG by Ang II. Collectively our results indicate that Ang II increases endothelial ARG activity/expression through a p38 MAPK-ATF-2 pathway leading to reduced NO production and endothelial dysfunction. Targeting these signaling steps might be therapeutic points for preventing vascular endothelial dysfunction associated with elevated ARG activity/expression.

Tissue factor/activated factor VIIa induces matrix metalloproteinase-7 expression through activation of c-Fos via ERK1/2 and p38 MAPK signaling pathways in human colon cancer cell

Increased expression of tissue factor (TF) is associated with tumor invasion and metastasis in human colorectal cancer. We have previously observed that TF/FVIIa upregulates matrix metalloproteinase-7 (MMP-7) expression at the transcriptional level in colon cancer cells. MMP-7 overexpression is believed to play an important role in tumor invasion and metastasis. The aim of this study is to elucidate the molecular mechanisms by which TF/FVIIa induced MMP-7 expression and cell invasion in vitro. Reverse transcription polymerase chain reaction, Western blot, luciferase assay, and chromatin immunoprecipitation (ChIP) were used to determine the potential mechanism and signaling pathways by which TF/FVIIa induced MMP-7 expression and cell invasion in LoVo cells. Small interfering RNA (siRNA) and cell invasion assay was used to examine whether blocking c-Fos expression could abolish FVIIa-mediated upregulation of MMP-7 and cell invasion in vitro. The results showed that FVIIa induced the upregulation of MMP-7 both at the mRNA and protein levels in a time- and dose-dependent manner and increased the invasive behavior of LoVo cells. FVIIa enhanced the promoter activity of MMP-7, and the activator protein-1 (AP-1) binding site was responsible for the activation. Site mutation of the AP-1 binding site in the promoter almost completely abolished FVIIa-mediated response. Furthermore, ChIP assay confirmed that FVIIa promoted the direct binding of c-Fos with the MMP-7 promoter in vivo. FVIIa also induced the expression and nuclear accumulation of the AP-1 subunit c-Fos. siRNA-mediated knockdown of c-Fos eliminated FVIIa-stimulated MMP-7 expression and cell migration in vitro. In addition, selective mitogen-activated protein kinase (MAPK) kinase (MEK1/2) inhibitor (PD98059) and p38 MAPK inhibitor SB203580 suppressed MMP-7 upregulation induced by FVIIa. Our data suggest that a novel TF/FVIIa/MAPK/c-Fos/MMP-7 axis plays an important role in modulating the invasion of colon cancer cells and blockage of this pathway holds promise to treat colon cancer metastasis.