Dose effect of oxidative stress on signal transduction in aging

Reactive oxygen species (ROS) during normal metabolism signal cells to stimulate proliferation or to cause cellular damages, depending on a specific concentration. Energy restriction (ER) increases life span in animals, which can explain an effective modulator for reducing oxidative stress. Oxidative stress can result from a decrease in the protection against ROS. The deleterious effects of oxidative stress generally occur after exposure to a relatively high concentration of ROS. Alternatively, it has been suggested that a low concentration of ROS can exert important physiological roles in cellular signaling and proliferation. Signal pathways are crucial for cell survival or death. It is generally acceptable that aged cells have less response to stresses such as ROS than young cells. Oxidative stresses induce JNK and p38 kinase pathways regulated by redox regulatory proteins: thioredoxin and glutathione s-transferase, respectively. Antioxidants such as selenium block apoptosis induced by ROS through blocking apoptotic signal ASK1 and stimulating survival signal Akt activity. Old hepatocytes are more susceptible to ROS-induced apoptosis than young hepatocytes, which is associated with low expression of ERK and Akt kinases. Pharmacological inhibition of ERK and Akt activation in the young cells markedly increase their sensitivity to H(2)O(2), and ER, by preventing loss of ERK and Akt activities, enhances survival of old hepatocytes to a level similar to those of young cells. Expressions of signal pathways such as survival and apoptotic signals can regulate cells' fate and aging process. Further studies on the interaction of signal pathways may change the scientific direction of the study of aging.     

Evidence that gamma-secretase mediates oxidative stress-induced beta-secretase expression in Alzheimer's disease

Beta-secretase (BACE1), an enzyme responsible for the production of amyloid beta-peptide (Abeta), is increased by oxidative stress and is elevated in the brains of patients with sporadic Alzheimer's disease (AD). Here, we show that oxidative stress fails to induce BACE1 expression in presenilin-1 (gamma-secretase)-deficient cells and in normal cells treated with gamma-secretase inhibitors. Oxidative stress-induced beta-secretase activity and sAPPbeta levels were suppressed by gamma-secretase inhibitors. Levels of gamma- and beta-secretase activities were greater in brain tissue samples from AD patients compared to non-demented control subjects, and the elevated BACE1 level in the brains of 3xTgAD mice was reduced by treatment with a gamma-secretase inhibitor. Our findings suggest that gamma-secretase mediates oxidative stress-induced expression of BACE1 resulting in excessive Abeta production in AD.     

IL-17R activation of human periodontal ligament fibroblasts induces IL-23 p19 production: Differential involvement of NF-κB versus JNK/AP-1 pathways

Interleukin (IL)-23 is an essential cytokine involved in the expansion of a novel CD4⁺ T helper subset known as Th17, which has been implicated in the pathogenesis of periodontitis recently. This study hypothesised that Th17 signature cytokine IL-17 may target specialised human periodontal ligament fibroblasts (hPDLFs) for production of IL-23 p19, a key subunit of IL-23. Primary hPDLFs had steady expression of IL-17 receptor (IL-17R) mRNA and surface-bound protein. IL-17 was capable of stimulating the expression of IL-23 p19 mRNA and protein in cultured hPDLFs, which was attenuated by IL-17 or IL-17R neutralising antibodies. In accordance with the enhanced expression of IL-23 p19, IL-17 stimulation resulted in rapid activation of Akt, p38 mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK) 1/2, c-Jun-N-terminal kinase (JNK), nuclear factor-kappaB (NF-κB), and activator protein-1 (AP-1) in hPDLFs. Inhibitors of Akt, p38 MAPK, ERK 1/2, or NF-κB significantly suppressed, whereas blocking JNK and AP-1 substantially augmented IL-23 p19 production from IL-17-stimulated hPDLFs. Moreover, IL-17-initiated NF-κB activation was blocked by Akt, p38 MAPK, or ERK 1/2 inhibition, while AP-1 activity was specifically abrogated by JNK inhibition. Thus, these results provide evidence that hPDLFs are a target of Th17, and that IL-17 appears to up-regulate the expression of IL-23 p19 via a homeostatic mechanism involving Akt-, p38 MAPK-, and ERK 1/2-dependent NF-κB signalling versus the JNK/AP-1 pathway. Taken together, our findings suggest that disruption of the interaction between IL-17 and IL-23 may be a potential therapeutic approach in the treatment of periodontitis.     

SIRT1 deficiency downregulates PTEN/JNK/FOXO1 pathway to block reactive oxygen species-induced apoptosis in mouse embryonic stem cells

Silent mating type information regulation 2 homolog 1 (SIRT1) plays a critical role in reactive oxygen species-triggered apoptosis in mouse embryonic stem (mES) cells. Here, we investigated a possible role for the PTEN/Akt/JNK pathway in the SIRT1-mediated apoptosis pathway in mES cells. Akt was activated by removal of anti-oxidant 2-mercaptoethanol in SIRT1(-/-) mES cells. Since PTEN is a negative regulator of Akt and its activity can be modulated by acetylation, we investigated if SIRT1 deacetylated PTEN to downregulate Akt to trigger apoptosis in anti-oxidant-free culture conditions. PTEN was hyperacetylated and excluded from the nucleus in SIRT1(-/-) mES cells, consistent with enhanced Akt activity. SIRT1 deficiency enhanced the acetylation/phosphorylation level of FOXO1 and subsequently inhibited the nuclear localization of FOXO1. Cellular acetylation levels were enhanced by DNA-damaging agent, not by removal of anti-oxidant. c-Jun NH2-terminal kinase (JNK) was activated by removal of anti-oxidant in SIRT1-dependent manner. Although p53 acetylation was stronger in SIRT1(-/-) mES cells, DNA-damaging stress activated phosphorylation and enhanced cellular levels of p53 irrespective of SIRT1, whereas removal of anti-oxidant slightly activated p53 only with SIRT1. Expression levels of Bim and Puma were increased in anti-oxidant-free culture conditions in an SIRT1-dependent manner and treatment with JNK inhibitor blocked induction of Bim expression. DNA-damaging agent activated caspase3 regardless of SIRT1. Our data support an important role for SIRT1 in preparing the PTEN/JNK/FOXO1 pathway to respond to cellular reactive oxygen species.     

Bisdemethoxycurcumin protects endothelial cells against t-BHP-induced cell damage by regulating the phosphorylation level of ERK1/2 and Akt

Curcuminoids are the major active components extracted from Curcuma longa and are well known for their antioxidant effects. Previous studies have reported that the antioxidant properties of curcuminoids are mainly attributed to their free radical scavenging abilities. However, whether there are other mechanisms besides the non-enzymatic process and how they are involved, still remains unknown. In the present study, we explored the protective effects of bisdemethoxycurcumin (Cur3) against tert-butyl hydroperoxide (t-BHP)-induced cytotoxicity in human umbilical vein endothelial cells (HUVECs), focusing on the effect of Cur3 on the regulation of the phosphatidylinositol 3-kinase (PI3K)/Akt and the mitogen-activated protein kinase (MAPK) pathways. The pre-treatment with Cur3 inhibited t-BHP-induced cell damage dose-dependently, which was evident by the increased cell viability and the corresponding decrease in lactate dehydrogenase release. The pre-treatment with Cur3 also attenuated t-BHP-induced cell morphological changes and apoptosis. MAPKs, including p38, c-Jun N-terminal kinase (JNK), extracellular signal-regulated protein kinase 1/2 (ERK1/2), as well as PI3K/Akt have been reported to be involved in proliferation, apoptosis and differentiation under various stress stimulations. The pre-treatment with Cur3 decreased t-BHP-induced ERK1/2 phosphorylation and increased t-BHP-induced Akt phosporylation but did not affect the phosphorylation of p38 or JNK. In addition, the Cur3-induced increase in cell viability was attenuated by the treatment with wortmannin or LY294002, the upstream inhibitors of Akt, and was enhanced by the treatment with 2-[2'-amino-3'-methoxyphenyl]-oxanaphthalen-4-one (PD98059), an upstream inhibitor of ERK1/2. These results suggest that the ERK1/2 and PI3K/Akt signaling pathways could be involved in the protective effects of Cur3 against t-BHP-induced damage in HUVECs.     

Insulin-like growth factor-1 activates Akt and Jun N-terminal kinases (JNKs) in promoting the survival of T lymphocytes

Insulin-like growth factor 1 receptor (IGF-1R) expression is augmented on T cells upon ligation of CD28, and this promotes IGF-1-mediated protection from Fas-induced cell death for up to 6 days. To determine the mechanism of action of IGF-1R in T-cell expansion, we investigated the signalling pathways activated by IGF-1 in T cells and in Jurkat cells. We found that IGF-1 transiently induces Akt, jun N-terminal kinases (JNK), and c-Jun phosphorylation in activated T cells, with JNK and c-Jun phosphorylation occurring faster than Akt phosphorylation. To mimic IGF-1R expression levels in CD28-stimulated Jurkat cells these cells were stably transfected to over-express the IGF-1R. Jurkat/IGF-1R cells exhibited enhanced constitutive Akt phosphorylation compared with mock-transfected controls, but IGF-1 induced transient phosphorylation of MKK4, JNKs, and c-Jun. Inhibition of PI-3 kinase activity and Akt phosphorylation with LY294002 totally suppressed IGF-1-mediated protection from Fas killing in activated T cells, but only partially suppressed IGF-1-mediated protection in Jurkat/IGF-1R cells. However, either dicumarol in T cells or a dominant negative JNK1 (APF) in Jurkat/IGF-1R cells greatly suppressed IGF-1-mediated protection from Fas killing. Together, these data demonstrate that IGF-1-mediated activation of JNKs and PI-3 kinase contributes to normal T-cell survival, whereas the JNK pathway may be more important in Jurkat leukaemia cells.     

MAP kinase subtypes and Akt regulate diosgenin-induced apoptosis of rheumatoid synovial cells in association with COX-2 expression and prostanoid production

In the present study, we investigated the signalling pathways involved in diosgenin-induced apoptosis in human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) in vitro with particular interest on Akt and MAPKs activation in relation to arachidonic acid metabolism via COX-2 pathway. MAPK activation was measured by ELISA quantification in diosgenin-treated human RA FLS. Expression of Akt and phospho-Akt was analyzed by Western blot analysis. Nuclear factor-kappaB (NF-kappaB) translocation was evaluated by electromobility shift assay. The prostanoid production (COX-2 activity) was measured by quantitative ELISA. Diosgenin-induced apoptosis in the presence of MAPK or Akt inhibitors was detected by a quantitative determination of DNA fragmentation. Treatment of human RA FLS with 40 microM diosgenin caused an activation of p38 and JNK and an inhibition of ERK phosphorylation. Akt and NF-kappaB are potentially required for diosgenin-induced apoptosis in human RA FLS because 40 microM diosgenin abrogated Akt phosphorylation which correlated with an inhibition of NF-kappaB nuclear translocation. SB203580 and SP600125 (p38 and JNK inhibitors) reduced diosgenin-induced DNA fragmentation whereas U0126 and LY294002 (MEK and PI3 kinase/Akt inhibitors) caused an amplification of proapoptotic effect of diosgenin. Diosgenin increased COX-2 activity resulting in PGE2 and 6-keto-PGF1alpha overproduction in human RA FLS. All MAPK inhibitors markedly reduced diosgenin-induced PGE2 and 6-keto-PGF1alpha synthesis except for SP600125 on 6-keto-PGF1alpha production. These results provide, for the first time, strong evidence that a combined association implicating a MEK inhibitor (U0126) and diosgenin is the most effective in inducing very strong apoptosis with down-regulation of COX-2 expression and activity in human RA FLS.     

PI3K/Akt and ERK1/2 signalling pathways are involved in endometrial cell migration induced by 17beta-estradiol and growth factors

Cell motility and invasion are crucial events for endometrial cells, not only for the establishment of pathological states but also during the physiological tissue remodelling that occurs during the menstrual cycle and embryo implantation. We have characterized these phenomena in endometrial stromal cells evaluating cell migration-specific stimuli and the biochemical pathways involved. Ability of endometrial cells to migrate on collagen type IV substrate was evaluated by means of chemotaxis experiments. Modulation of this phenomenon by different growth factors and steroid hormones and their ability to activate extracellular signal-regulated protein kinase (ERK) and phosphatidylinositol 3 kinase (PI3K)/Akt signalling in this context were examined. Platelet-derived growth factor (PDGF)-BB, epidermal growth factor and fibroblast growth factor-2 as chemoattractant agents stimulated basal migration of endometrial stromal cells through the rapid activation of both ERK1/2 and PI3K/Akt signalling pathways. Experiments using wortmannin and PD98059, specific inhibitors of the PI3K/Akt and ERK1/2 activity, respectively, showed that the activation of both pathways is required for growth-factor-induced cell motility responses. Similarly, 17beta-estradiol (10(-6)-10(-8) M) could enhance both constitutive and PDGF-induced migration of the cells and their rapid treatment with the hormone significantly increased phosphorylation of ERK1/2 and Akt. Conversely, progesterone did not interfere with the basal migration but inhibits the PDGF-induced motility of this cell type. Rapid activation of intracellular signalling cascades ERK1/2 and PI3K/Akt by growth factors and estrogens is involved in the migration of normal endometrial stromal cells.     

Anti-citrullinated Protein Antibodies Activated ERK1/2 and JNK Mitogen-activated Protein Kinases via Binding to Surface-expressed Citrullinated GRP78 on Mononuclear Cells

In a previous study, we found that anti-citrullinated protein antibodies (ACPAs) enhance nuclear factor (NF)-κB activity and tumor necrosis factor (TNF)-α production by normal human peripheral blood mononuclear cells (PBMCs) and U937 cells via binding to surface-expressed citrullinated glucose-regulated protein 78 (cit-GRP78). However, the downstream signaling pathways remain unclear after binding. In the present study, we firstly measured the effects of different kinase inhibitors on ACPA-mediated TNF-α production from normal PBMCs and monocytes. Then, the native and phosphorylated mitogen-activated protein kinases (MAPKs) were detected in ACPA-activated U937 cells by Western blotting. We also explored the role of the phosphoinositide 3-kinase (PI3K)-Akt pathway in activating IκB kinase alpha (IKK-α) in ACPA-stimulated U937 cells. Finally, we measured the amount of cit-GRP78 from PBMC membrane extracts in RA patients and controls. We found that MAPK and Akt inhibitors, but not PI3K inhibitor, remarkably suppressed ACPA-mediated TNF-α production. Interestingly, ACPAs selectively activated extracellular signal-regulated kinase 1/2 (ERK1/2) and c-jun N-terminal kinase (JNK), but not p38 MAPK, in U937 cells. This activation was suppressed by cit-GRP78, but not GRP78. The JNK activation further enhanced the phosphorylation of Akt and IKK-α. The expression of cit-GRP78 on cell membrane was higher in RA than normal PBMCs. Taken together; these results suggest that through binding to surface, over-expressed cit-GRP78 on RA PBMCs, ACPAs selectively activate ERK1/2 and JNK signaling pathways to enhance IKK-α phosphorylation, which leads to the activation of NF-κB and the production of TNF-α .     

Exercise-induced alterations in extracellular signal-regulated kinase 1/2 and mammalian target of rapamycin (mTOR) signalling to regulatory mechanisms of mRNA translation in mouse muscle

The present study examined the effects of an acute bout of treadmill exercise on signalling through the extracellular signal-regulated kinase (ERK)1/2 and mammalian target of rapamycin (mTOR) pathways to regulatory mechanisms involved in mRNA translation in mouse gastrocnemius muscle. Briefly, C57BL/6 male mice were run at 26 m min⁻¹ on a treadmill for periods of 10, 20 or 30 min, then the gastrocnemius was rapidly removed and analysed for phosphorylation and/or association of protein components of signalling pathways and mRNA translation regulatory mechanisms. Repression of global mRNA translation was suggested by disaggregation of polysomes into free ribosomes, which occurred by 10 min and was sustained throughout the time course. Exercise repressed the mTOR signalling pathway, as shown by dephosphorylation of the eukaryotic initiation factor (eIF)4E-binding protein-1 (4E-BP1), enhanced association of the regulatory-associated protein of mTOR with mTOR, and increased assembly of the tuberin–hamartin complex. In contrast, exercise caused no change in phosphorylation of either Akt/PKB or tuberin. Upstream of mTOR, exercise was associated with an increase in cAMP, protein kinase A activity, and AMP-activated protein kinase phosphorylation. Simultaneously, exercise caused a rapid and sustained activation of the MEK1/2–ERK1/2–p90RSK pathway, resulting in increased phosphorylation of downstream targets including eIF4E and the ribosomal protein (rp)S6 on S235/S236. Overall, the data are consistent with exercise-induced repression of mTOR signalling and global rates of mRNA translation, accompanied perhaps by up-regulated translation of selected mRNAs through regulatory mechanisms such as eIF4E and rpS6 phosphorylation, mediated by activation of the ERK1/2 pathway.     

Differential activation of neuronal ERK, JNK/SAPK and p38 in Alzheimer disease: the 'two hit' hypothesis.

There are multiple lines of evidence showing that oxidative stress and aberrant mitogenic signaling play an important role in the pathogenesis of Alzheimer disease. However, the chronological relationship between these and other events associated with disease pathogenesis is not known. Given the important role that mitogen-activated protein kinase (MAPK) pathways play in both mitogenic signaling (ERK) and cellular stress signaling (JNK/SAPK and p38), we investigated the chronological and spatial relationship between activated ERK, JNK/SAPK and p38 during disease progression. While all three kinases are activated in the same susceptible neurons in mild and severe cases (Braak stages III-VI), in non-demented cases with limited pathology (Braak stages I and II), both ERK and JNK/SAPK are activated but p38 is not. However, in non-demented cases lacking any sign of pathology (Braak stage 0), either ERK alone or JNK/SAPK alone can be activated. Taken together, these findings indicate that MAPK pathways are differentially activated during the course of Alzheimer disease and, by inference, suggest that both oxidative stress and abnormalities in mitotic signaling can independently serve to initiate, but both are necessary to propagate, disease pathogenesis. Therefore, we propose that both 'hits', oxidative stress and mitotic alterations, are necessary for the progression of Alzheimer disease.     

Down-regulation of the PI3-kinase/Akt pathway by ERK MAP kinase in growth factor signaling

The ERK MAP kinase and PI3-kinase/Akt pathways are major intracellular signaling modules, which are known to regulate diverse cellular processes including cell proliferation, survival and malignant transformation. However, it has not been fully understood how these two pathways interact with each other. Here, we demonstrate that inhibition of the ERK pathway by the MEK inhibitor U0126 or PD98059 significantly potentiates EGF- and FGF-induced Akt phosphorylation at both Thr308 and Ser473. We also show that hyperactivation of the ERK pathway greatly attenuates EGF- and FGF-induced Akt phosphorylation. Furthermore, the enhanced Akt phosphorylation induced by U0126 is inhibited by the PI3-kinase inhibitor LY294002, and is accompanied by the up-regulation of Ras activity. These results suggest that the ERK pathway inhibition enhances Akt phosphorylation through the Ras/PI3-kinase pathway. Thus, our results demonstrate that the ERK pathway negatively modulates the PI3-kinase/Akt pathway in response to growth factor stimulation.     

氧自由基对3T3-L1脂肪细胞表达PAI-1的调节及其可能机制

目的： 研究脂肪细胞中氧自由基（ROS）对纤溶酶原激活物抑制物-1（PAI-1）表达的调节,并探讨其机制。方法：培养3T3-L1细胞,并诱导其分化成为脂肪细胞,以MTT比色法检测细胞的活性。分别以定量PCR、多重免疫分析及夹心ELISA法检测PAI-1 mRNA和蛋白表达的水平,并采用多重磷酸化蛋白分析系统检测细胞内多种信号分子的蛋白磷酸化水平。结果：H2O2可剂量依赖性地增加PAI-1的产生。并且激活了3T3-L1脂肪细胞中多种信号转导通路,包括ERK1/2、JNK、Akt、p70 S6K及JAK/STAT,其中Akt、JAK/STAT及ERK1/2的活化可能参与到H2O2对于PAI-1的调节过程中。结论： H2O2可能通过磷酸化激活Akt、JAK/STAT及ERK1/2,上调脂肪细胞PAI-1的表达。     

Opposing role of JNK-p38 kinase and ERK1/2 in hydrogen peroxide-induced oxidative damage of human trophoblast-like JEG-3 cells

Trophoblasts play a crucial role in embryo implantation and maintenance of normal pregnancy. Recently, oxidative stress has been considered as one important factor in the pathogenesis of spontaneous abortion and preeclampsia. Many studies have reported that the plasma levels of hydrogen peroxide (H₂O₂) are significantly increased in women with preeclampsia, but the mechanisms involved in H₂O₂-induced cell cytotoxicity in trophoblasts are still not completely explained. Our present study was undertaken to provide a united understanding of the role of oxidative stress generated by H₂O₂ on human trophoblasts and the underlying intracellular signaling pathways. Exposure to H₂O₂ resulted in a concentration-dependent growth decrease and apoptosis in human trophoblast-like JEG-3 cells. H₂O₂ treatment also caused intracellular reactive oxygen species (ROS) production and concomitant dissipation of the mitochondrial membrane potential. The three MAPK subfamilies, ERK1/2, JNK and p38 kinase, were all activated under H₂O₂-induced oxidative stress. Blocking the activation of JNK and p38 kinase increased cell viability and decreased apoptosis induced by H₂O₂ with their respective inhibitors, SP600125 and SB203580. However, preventing ERK1/2 activation further increased H₂O₂-induced cell death with U0126, an inhibitor of ERK upstream kinase MEK1/2. Taken together, these findings suggest that the mitochondria-dependent pathways and JNK-p38 kinase pathways are involved in H₂O₂-induced oxidative damage of human trophoblast-like JEG-3 cells, while ERK1/2 pathway may play an active role in cell survival following oxidant injury.     

Fractalkine functions as a chemoattractant for osteoarthritis synovial fibroblasts and stimulates phosphorylation of mitogen‐activated protein kinases and Akt

Fractalkine (FKN/CX3CL1) has been detected in synovial fluids from osteoarthritis (OA) patients. Additionally, low‐level expression of the FKN receptor, CX3CR1, has been demonstrated in OA synovial lining. This study aimed to determine a biological function for this ligand/receptor pair in OA and to assess a potential signalling mechanism for FKN in this predominant synovial lining cell type, using chemotaxis assays, Western blotting and F‐actin staining. Chemotaxis assays demonstrate that the chemokine domain of FKN effectively induces migration of OA fibroblasts. Consistent with this finding, visualization of F‐actin demonstrates that 1 or 10 nM FKN induces noticeable reorganization of cytoskeletal structure in OA fibroblasts after 30 min stimulation with a maximal enhancement at approximately 2 h. In addition, Western blotting analysis demonstrates that FKN stimulates phosphorylation of the mitogen‐activated protein (MAP) kinases p38, c‐Jun N‐terminal kinase (JNK) and extracellular‐regulated kinase (ERK) 1/2 as well as the serine‐threonine kinase Akt at Ser 473 and Thr 308. All these phosphorylation events occur in a time‐dependent manner, with little or no activation within 1 min, and maximal activation occurring typically between 5 and 30 min. Moreover, inhibition of ERK 1/2 significantly reduces FKN‐induced OA fibroblast migration. These results suggest that FKN is a novel chemoattractant for OA fibroblasts, consistent with FKN‐induced alterations in cytoskeletal structure. In addition, FKN induces OA fibroblast signalling via the MAP kinases p38, JNK and ERK 1/2, as well as Akt.