Inhibited MAPK Phosphorylation Research Articles

Chondroitin sulphate modified MoS2 nanoenzyme with multifunctional activities for treatment of Alzheimer's disease

Nano-MoS2 exhibit oxidoreductase-like activities, and has been shown to effectively eliminate excessive intracellular ROS and inhibit Aβ aggregation, thus demonstrating promising potential for anti-Alzheimer's disease (anti-AD) intervention. However, the low water dispersibility and high toxicity of nano-MoS2 limits its further application. In this study, we developed a chondroitin sulphate (CS)-modified MoS2 nanoenzyme (CS@MoS2) by harnessing the excellent biocompatibility of CS and the exceptional activities of nano-MoS2 to explore its potential in anti-AD research. Promisingly, CS@MoS2 significantly inhibited Aβ1–40 aggregation and prevented toxic injury in SH-SY5Y cells caused by Aβ1–40. In addition, CS@MoS2 protected these cells from oxidative stress damage by regulating ROS production, as well as promoting the activities of SOD and GSH-Px. CS@MoS2 also modulated the intracellular Ca2+ imbalance and downregulated Tau hyperphosphorylation by activating GSK-3β. CS@MoS2 suppressed p-NF-κB (p65) translocation to the nucleus by inhibiting MAPK phosphorylation, and modulated the expression of downstream anti- and proinflammatory cytokines. Owing to its multifunctional activities, CS@MoS2 effectively improved spatial learning, memory, and anxiety in D-gal/AlCl3-induced AD mice. Taken together, these results indicate that CS@MoS2 has significant potential for improving the therapeutic efficacy of the prevention and treatment of AD, while also presenting a novel framework for the application of nanoenzymes.

Erythropoietin induces odontoblastic differentiation of human-derived pulp stem cells via EphB4-Mediated MAPK signaling pathway.

Human-derived pulp stem cells play key roles during dentinogenesis. Erythropoietin is reportedly involved in osteoblastogenesis and facilitates bone formation. However, the mechanism is still unknown. This research was to study the potential of erythropoietin in enhancing odontoblastic differentiation of human-derived pulp stem cells and to determine the underlying mechanism. The human-derived pulp stem cells were treated with erythropoietin, EphB4 inhibitor, and MAPK inhibitors, and the odontoblastic differentiation was measured by ALP staining, ALP activity assay, alizarin red S staining, and their quantitative analysis, and RT-qPCR of DSPP, DMP1, OCN, and RUNX2. The direct pulp capping model was established to evaluate the formation of tertiary dentin after treatment with erythropoietin. Western blot assay was conducted to assess relevant protein expressions in the phosphorylated EphB4 and MAPK pathway. The results showed that erythropoietin promoted odontoblastic differentiation of human-derived pulp stem cells at 20 U/ml. Erythropoietin induced tertiary dentin formation in vivo. The potential mechanism of this was upregulating phosphorylated EphB4 and phosphorylated MAPK; furthermore, this effect could be decreased by EphB4 inhibitors, which inhibited MAPK phosphorylation. Blockage of MAPK pathways attenuated human-derived pulp stem cells' odontoblastic differentiation, suggesting that MAPK pathways are involved. Erythropoietin induced tertiary dentin formation in vivo. And erythropoietin enhanced human-derived pulp stem cells' odontoblastic differentiation via the EphB4-mediated MAPK signaling pathway.

Microglial LOX-1/MAPKs/NF-κB positive loop promotes the vicious cycle of neuroinflammation and neural injury

Lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), a member of the scavenger receptor family, recognizes multiple ligands and participates in several inflammatory responses, but its function within the central nervous system (CNS) remains unclear. In this study, we discovered an increased LOX-1 expression in activated microglia in vivo and in vitro. Employing the specific inhibitors, we found that conditioned medium of necrotic neurons (Necrotic-CM) induced microglial LOX-1 expression through the MAPKs/NF-κB pathway. Silencing LOX-1 inhibited MAPK phosphorylation, NF-κB-p65 nuclear transportation, and pro-inflammatory factor production in microglia exposed to Necrotic-CM. Furthermore, utilizing the conditioned medium of activated microglia (MG-CM), we discovered microglial LOX-1 aggravated the neuroinflammation-induced neuronal apoptosis. Collectively, a LOX-1/MPAKs/NF-κB positive loop might promote microglia activation and drive the vicious cycle of neuroinflammation and neuronal injury.

Anti-inflammatory and Antioxidative Effects of Tetrahedral DNA Nanostructures via the Modulation of Macrophage Responses.

Tetrahedral DNA nanostructures (TDNs) are a new type of nanomaterials that have recently attracted attention in the field of biomedicine. However, the practical application of nanomaterials is often limited owing to the host immune response. Here, the response of RAW264.7 macrophages to TDNs was comprehensively evaluated. The results showed that TDNs had no observable cytotoxicity and could induce polarization of RAW264.7 cells to the M1 type. TDNs attenuated the expression of NO IL-1β (interleukin-1β), IL-6 (interleukin-6), and TNF-α (tumor necrosis factor-α) in LPS-induced RAW264.7 cells by inhibiting MAPK phosphorylation. In addition, TDNs inhibited LPS-induced reactive oxygen species (ROS) production and cell apoptosis by up-regulating the mRNA expression of antioxidative enzyme heme oxygenase-1 (HO-1). The findings of this study demonstrated that TDNs have great potential as a novel theranostic agent because of their anti-inflammatory and antioxidant activities, high bioavailability, and ease of targeting.

Therapeutic Potential and Molecular Mechanisms of Emblica officinalis Gaertn in Countering Nephrotoxicity in Rats Induced by the Chemotherapeutic Agent Cisplatin.

Emblica officinalis Gaertn. belonging to family Euphorbiaceae is commonly known as Indian gooseberry or “Amla” in India. It is used as a ‘rejuvenating herb’ in traditional system of Indian medicine. It has been shown to possess antioxidant, anti-inflammatory and anti-apoptotic effects. Thus, on the basis of its biological effects, the present study was undertaken to evaluate the protective effect of the dried fruit extract of the E. Officinalis (EO) in cisplatin-induced nephrotoxicity in rats and also to evaluate the mechanism of its nephroprotection. The study was done on male albino Wistar rats. They were divided into six groups (n = 6) viz. control, cisplatin-control, cisplatin and EO (150, 300, and 600 mg/kg; p.o. respectively in different groups) and EO only (600 mg/kg; p.o. only). EO was administered orally to the rats for a period of 10 days and on the 7th day, a single injection of cisplatin (8 mg/kg; i.p.) was administered to the cisplatin-control and EO treatment groups. The rats were sacrificed on the 10th day. Cisplatin-control rats had deranged renal function parameters and the kidney histology confirmed the presence of acute tubular necrosis. Furthermore, there were increased oxidative stress, apoptosis and inflammation along with higher expression of MAPK pathway proteins in the rat kidney from the cisplatin-control group. Contrary to this, EO (600 mg/kg) significantly normalized renal function, bolstered antioxidant status and ameliorated histological alterations. The inflammation and apoptosis were markedly lower in comparison to cisplatin-control rats. Furthermore, EO (600 mg/kg) inhibited MAPK phosphorylation which was instrumental in preserving renal function and morphology. In conclusion, the results of our study demonstrated that EO attenuated cisplatin-induced nephrotoxicity in rats through suppression of MAPK induced inflammation and apoptosis.

Isovitexin Exerts Anti-Inflammatory and Anti-Oxidant Activities on Lipopolysaccharide-Induced Acute Lung Injury by Inhibiting MAPK and NF-κB and Activating HO-1/Nrf2 Pathways.

Oxidative damage and inflammation are closely associated with the pathogenesis of acute lung injury (ALI). Thus, we explored the protective effect of isovitexin (IV), a glycosylflavonoid, in the context of ALI. To accomplish this, we created in vitro and in vivo models by respectively exposing macrophages to lipopolysaccharide (LPS) and using LPS to induce ALI in mice. In vitro, our results showed that IV treatment reduced LPS-induced pro-inflammatory cytokine secretion, iNOS and COX-2 expression and decreased the generation of ROS. Consistent findings were obtained in vivo. Additionally, IV inhibited H2O2-induced cytotoxicity and apoptosis. However, these effects were partially reversed following the use of an HO-1 inhibitor in vitro. Further studies revealed that IV significantly inhibited MAPK phosphorylation, reduced NF-κB nuclear translocation, and upregulated nuclear factor erythroid 2-related factor 2 (Nrf2) and heme oxygenase 1 (HO-1) expression in RAW 264.7 cells. In vivo, pretreatment with IV attenuated histopathological changes, infiltration of polymorphonuclear granulocytes and endothelial activation, decreased the expression of ICAM-1 and VCAM-1, reduced the levels of MPO and MDA, and increased the content of GSH and SOD in ALI. Furthermore, IV treatment effectively increased Nrf2 and HO-1 expression in lung tissues. Therefore, IV may offer a protective role against LPS-induced ALI by inhibiting MAPK and NF-κB and activating HO-1/Nrf2 pathways.

Matrix metalloproteinase-8 plays a pivotal role in neuroinflammation by modulating TNF-α activation.

Matrix metalloproteinases (MMPs) play important roles in normal brain development and synaptic plasticity, although aberrant expression of MMPs leads to brain damage, including blood-brain barrier disruption, inflammation, demyelination, and neuronal cell death. In this article, we report that MMP-8 is upregulated in LPS-stimulated BV2 microglial cells and primary cultured microglia, and treatment of MMP-8 inhibitor (M8I) or MMP-8 short hairpin RNA suppresses proinflammatory molecules, particularly TNF-α secretion. Subsequent experiments showed that MMP-8 exhibits TNF-α-converting enzyme (TACE) activity by cleaving the prodomain of TNF-α (A(74)/Q(75), A(76)/V(77) residues) and, furthermore, that M8I inhibits TACE activity more efficiently than TAPI-0, a general TACE inhibitor. Biochemical analysis of the underlying anti-inflammatory mechanisms of M8I revealed that it inhibits MAPK phosphorylation, NF-κB/AP-1 activity, and reactive oxygen species production. Further support for the proinflammatory role of microglial MMP-8 was obtained from an in vivo animal model of neuroinflammatory disorder. MMP-8 is upregulated in septic conditions, particularly in microglia. Administration of M8I or MMP-8 short hairpin RNA significantly inhibits microglial activation and expression/secretion of TNF-α in brain tissue, serum, and cerebrospinal fluid of LPS-induced septic mice. These results demonstrate that MMP-8 critically mediates microglial activation by modulating TNF-α activity, which may explain neuroinflammation in septic mouse brain.

S52 The role of the Receptor for Advanced End Products (RAGE) in Acute Lung Injury (ALI)

IntroductionRAGE is expressed by both alveolar epithelial and endothelial cells. The receptor can bind to pro-inflammatory ligands, including HMGB1 which is elevated in ALI. RAGE negative mice show a reduced...

SARM inhibits both TRIF‐ and MyD88‐mediated AP‐1 activation

SARM (sterile alpha- and armadillo-motif-containing protein), the fifth identified TIR (Toll-interleukin 1 receptor (IL-1R)) domain-containing adaptors in humans, downregulates NF-kappaB and IRF3 (interferon-regulatory factor 3)-mediated TLR3 and TLR4 signaling. SARM was characterized as a negative regulator of the TRIF (TIR-domain-containing adaptor protein inducing IFN-beta)-dependent pathway via its interaction with TRIF. However, the precise mechanism of action of SARM remains unclear. Here, we demonstrate that SARM inhibits MAPK activation in human embryonic kidney 293 cells, and U937 cells. Both the TRIF- and MyD88-mediated, as well as basal MAPK activity, were repressed, indicating that SARM-mediated inhibition may not be exclusively directed at TRIF or MyD88, but that SARM may also directly inhibit MAPK phosphorylation. The MAPK inhibition effect was verified by RNAi, which increased the basal level of AP-1. Furthermore, LPS challenge upregulated SARM at both the mRNA and protein levels. Finally, we provide evidence to show that truncated SARM changes its subcellular localization, suggesting the importance of the N-terminal and sterile alpha motif domains in the autoregulation of SARM activity.

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.

FGFR1 and FGF Coamplification in Breast Cancer.

Abstract Background:FGFR1 is a tyrosine kinase receptor involved in cell proliferation, migration and differentiation. FGFR1 gene is amplified in about 10% of breast cancer. Several studies have previously reported that FGFR1 inhibition could lead to a decrease in cell proliferation. Recent data obtained in prostate cancer have shown that FGFR1 activation could mediate epithelial to mesenchymal transition. In the present study, we evaluated the rational to target FGFR1 in clinical setting.Material and methods:We first assessed whether FGFR1 amplification was associated with a specific breast cancer phenotype. We assessed FGFR1 amplification by CGH array on 235 samples and by FISH (Z-2072, Spec FGFR1/Cen8 dual color probe, Zytovision®) on 260 samples, not included in the CGH part. We then assessed the functional effects of FGFR1 inhibitions in vitro. The antiproliferative effect of E3810 (FGFR1 inhibitor) was assessed both on FGFR1-non amplified (T47D/MCF7) and FGFR1 amplified (SUM44PE/MDA134) cell lines. Effect of FGFR1 inhibition was assessed on gene expression profiling.Results:When assessed by FISH, FGFR1 amplification was observed in 23/260 cases. Most of the samples (62%) exhibited an ER+/PR- phenotype, suggesting activation of intracellular kinase. FGFR1 amplification was observed in 24 out of 235 samples (10%). Interestingly, FGFR1 amplification was associated with gene gain in FGF3/FGF4/FGF19 (11q13) (55% versus 22%, p=0.05). FGFR1 inhibition by E-3810 inhibits MAPk phosphorylation, cell proliferation. A differential IC 50 for cell proliferation was observed between amplified cell lines and control cell lines. Preliminary data on gene expression profiling suggest that FGFR1 inhibition on breast cancer cells could lead to significant decrease in the Sox9 expression, a protein involved in epithelial to mesenchymal transition.Conclusion:FGFR1-amplified breast cancers present a discrete phenotype, including a high rate of ER+/PR- and high rate of FGF3/4/19 gains. Pharmacologic inhibition of FGFR1 leads to cell proliferation decrease and modulation of genes involved in EMT. These data suggest that there is a rational to target FGFR1 in clinical setting in patients with FGFR1-amplified breast cancer. Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 4170.

Involvement of endoplasmic reticulum stress and activation of MAP kinases in beta-lapachone-induced human prostate cancer cell apoptosis.

Beta-lapachone, an o-naphthoquinone, induces various carcinoma cells to undergo apoptosis, but the mechanism is poorly understood. In the present study, we found that the beta-lapachone-induced apoptosis of DU145 human prostate carcinoma cells was associated with endoplasmic reticulum (ER) stress, as shown by increased intracellular calcium levels and induction of GRP-78 and GADD-153 proteins, suggesting that the endoplasmic reticulum is a target of beta-lapachone. Beta-Lapachone-induced DU145 cell apoptosis was dose-dependent and accompanied by cleavage of procaspase-12 and phosphorylation of p38, ERK, and JNK, followed by activation of the executioner caspases, caspase-7 and calpain. However, pretreatment with the general caspase inhibitor, z-VAD-FMK, or calpain inhibitors, including ALLM or ALLN, failed to prevent beta-lapachone-induced apoptotic cell death. Blocking the enzyme activity of NQO1 with dicoumarol, a known NQO1 inhibitor, or preventing an increase in intracellular calcium levels using BAPTA-AM, an intracellular calcium chelator, substantially inhibited MAPK phosphorylation, abolished the activation of calpain, caspase-12 and caspase-7, and provided significant protection of beta-lapachone-treated cells. These findings show that beta-lapachone-induced ER stress and MAP kinase phosphorylation is a novel signaling pathway underlying the molecular mechanism of the anticancer effect of beta-lapachone.

Effects of Media pH on VACM‐1/cul5 Protein Expression in T47D Cancer Cell Line

VACM‐1, a cul‐5 gene product is involved in the ubiquitin ligase complex formation and subsequent protein degradation. When expressed in T47D breast cancer cells, VACM‐1 inhibits growth by inhibiting MAPK phosphorylation (MAPK‐P) and decreasing nuclear estrogen receptors, ERα concentration. Factors that can regulate VACM‐1 protein concentration have not been identified. We have observed that changes in protocol from powder media to a purchased liquid media have led to an increase of endogenous VACM‐1 concentration in control T47D cells. The goal of this project was to examine whether changes in pH will affect VACM‐1 concentration and cell proliferation. Our work has shown that endogenous VACM‐1 protein expression in T47D cells is induced by changes in media pH. Immunocytochemistry data have verified the increased concentration of VACM‐1 in cells grown in liquid media (lower pH) when compared to the powder media (higher pH). The increase in VACM‐1 expression was directly correlated with the decrease in cell growth. These differences were no longer observed when culture media was adjusted to the same pH. These results support the hypothesis that changes in pH may affect VACM‐1 concentrations. This work was supported by a grant from NCI (R15CA104014) and by Dept of Chemistry.

Epidermal Growth Factor Receptor Inhibition by Tyrphostin 51 Induces Apoptosis in Luteinized Granulosa Cells

Growth factors may be involved in the control of ovarian cell fate and could contribute to regulation of ovarian cell apoptosis. Our objective is to test the hypothesis that, in human luteinized granulosa cells, epidermal growth factor (EGF) works through the MAPK signaling pathway and inhibition of EGF receptor by a specific tyrosine kinase inhibitor, tyrphostin 51, will inhibit the activation of MAPK and induce apoptosis. Luteinized granulosa cells from human in vitro fertilization aspirates were treated as follows: 1) vehicle (dimethylsulfoxide:ethanol), 2) EGF, 3) tyrphostin 51, and 4) tyrphostin 51 plus EGF. Blockage of EGF receptor by tyrphostin 51 reduced the MAPK activity and inhibited phosphorylation and nuclear translocation of activated MAPK. Blockage of EGF receptor also induced apoptosis as demonstrated by the activation of caspase-3, an executioner protease of the apoptotic pathway, and by an increased percentage of subdiploid apoptotic nuclei. These results support the hypothesis that in human luteinized granulosa cells, EGF works through the MAPK signaling pathway and that its inhibition by tyrphostin 51 inhibits MAPK phosphorylation and induces apoptotic nuclear changes. Our data thus provide additional information regarding regulation of apoptosis in luteinized granulosa cells.

Heterotrimeric Gαq/Gα11 Proteins Function Upstream of Vascular Endothelial Growth Factor (VEGF) Receptor-2 (KDR) Phosphorylation in Vascular Permeability Factor/VEGF Signaling

Vascular permeability factor/vascular endothelial growth factor (VPF/VEGF) functions by activating two receptor-tyrosine kinases, Flt-1 (VEGF receptor (VEGFR)-1) and KDR (VEGFR-2), both of which are selectively expressed on primary vascular endothelium. KDR is responsible for VPF/VEGF-stimulated endothelial cell proliferation and migration, whereas Flt-1 down-modulates KDR-mediated endothelial cell proliferation. Our most recent works show that pertussis toxin-sensitive G proteins and Gbetagamma subunits are required for Flt-1-mediated down-regulation of human umbilical vein endothelial cell (HUVEC) proliferation and that Gq/11 proteins are required for KDR-mediated RhoA activation and HUVEC migration. In this study, we demonstrate that Gq/11 proteins are also required for VPF/VEGF-stimulated HUVEC proliferation. Our results further indicate that Gq/11 proteins specifically mediate KDR signaling such as intracellular Ca2+ mobilization rather than Flt-1-induced CDC42 activation and that a Gq/11 antisense oligonucleotide completely inhibits MAPK phosphorylation induced by KDR but has no effect on Flt-1-induced MAPK activation. More importantly, we demonstrate that Gq/11 proteins interact with KDR in vivo, and the interaction of Gq/11 proteins with KDR does not require KDR tyrosine phosphorylation. Surprisingly, the Gq/11 antisense oligonucleotide completely inhibits VPF/VEGF-stimulated KDR phosphorylation. Expression of a constitutively active mutant of G11 but not Gq can cause phosphorylation of KDR and MAPK. In addition, a Gbetagamma minigene, hbetaARK1(495), inhibits VPF/VEGF-stimulated HUVEC proliferation, MAPK phosphorylation, and intracellular Ca2+ mobilization but has no effect on KDR phosphorylation. Taken together, this study demonstrates that Gq/11 proteins mediate KDR tyrosine phosphorylation and KDR-mediated HUVEC proliferation through interaction with KDR.

8-(3-oxo-4,5,6-trihydroxy-3h-xanthen-9-yl)-1-naphthoic acid inhibits MAPK phosphorylation in endothelial cells induced by VEGF and bFGF.

Angiogenesis is regulated by various factors. In particular, VEGF and basic FGF are of much importance. We found that 8-(3-oxo-4,5,6-trihydroxy-3h-xanthen-9-yl)-1-naphthoic acid inhibited the binding of VEGF to KDR/Flk-1 (VEGF receptor-2) or Flt-1 (VEGF receptor-1) and that it inhibited the MAPK phosphorylation in HUVEC induced by VEGF or basic FGF but not by EGF. 8-(3-oxo-4,5,6-trihydroxy-3h-xanthen-9-yl)-1-naphthoic acid might be used as an inhibitor of VEGF and basic FGF signal transduction.

Phosphorylation of the eIF4E-binding protein PHAS-I after exposure of PC12 cells to EGF and NGF

PHAS-I or the eIF4E-binding protein 1 regulates the cap-binding activity of eIF4E by sequestering eIF4E. Binding of eIF4E to PHAS-I is regulated by phosphorylation of PHAS-I. PC12 cells were used to study the signal transduction pathway leading to phosphorylation of PHAS-I. Both EGF and NGF induced phosphorylation of PHAS-I. Wortmannin, a PI-3 kinase inhibitor, staurosporine, a PKC inhibitor, and rapamycin, a FRAP inhibitor all blocked the phosphorylation of PHAS-I. Of the three inhibitors, only wortmannin was able to inhibit MAPK phosphorylation. This excludes a role for MAPK in NGF- and EGF-induced PHAS-I phosphorylation in PC12 cells. Apparently, PHAS-I was phosphorylated in a PI-3 kinase-, PKC-, and FRAP-dependent manner after EGF or NGF stimulation. Only PI-3 kinase and FRAP are involved in the regulation of the basal level of PHAS-I phosphorylation.

The dual EGF/VEGF receptor tyrosine kinase inhibitor AEE788 inhibits growth of human hepatocellular carcinoma xenografts in nude mice

We investigated the effect of AEE788, a novel dual receptor tyrosine kinase inhibitor of the EGF and the VEGF receptor, for treatment of human HCC cell lines and in a subcutaneous xenograft model. Cell viability and apoptosis of HepG2 and Hep3B cells incubated with 0.1-100 microM AEE788 were quantified. In vivo, HepG2 cells were xenografted to NMRI mice and animals were treated orally with 50 mg/kg AEE788 3x/week. Immunohistochemistry and quantitative Western blotting was performed for pathway analysis in vitro and in vivo. AEE788 reduced growth and induced apoptosis of HCC cells by disrupting mitochondrial transmembrane potentials and inhibiting MAPK phosphorylation. In the xenografts, AEE788 lead to a reduced tumor growth by reducing proliferation and vascularisation. Except for a reversible skin reaction and weight loss, no signs of toxicity were observed. AEE788 is a promising new option for the treatment of HCC.