Astrocyte Resilience to Oxidative Stress Induced by Insulin-like Growth Factor I (IGF-I) Involves Preserved AKT (Protein Kinase B) Activity

Disruption of insulin-like growth factor I (IGF-I) signaling is a key step in the development of cancer or neurodegeneration. For example, interference of the prosurvival IGF-I/AKT/FOXO3 pathway by redox activation of the stress kinases p38 and JNK is instrumental in neuronal death by oxidative stress. However, in astrocytes, IGF-I retains its protective action against oxidative stress. The molecular mechanisms underlying this cell-specific protection remain obscure but may be relevant to unveil new ways to combat IGF-I/insulin resistance. Here, we describe that, in astrocytes exposed to oxidative stress by hydrogen peroxide (H₂O₂), p38 activation did not inhibit AKT (protein kinase B) activation by IGF-I, which is in contrast to our previous observations in neurons. Rather, stimulation of AKT by IGF-I was significantly higher and more sustained in astrocytes than in neurons either under normal or oxidative conditions. This may be explained by phosphorylation of the phosphatase PTEN at the plasma membrane in response to IGF-I, inducing its cytosolic translocation and preserving in this way AKT activity. Stimulation of AKT by IGF-I, mimicked also by a constitutively active AKT mutant, reduced oxidative stress levels and cell death in H₂O₂-exposed astrocytes, boosting their neuroprotective action in co-cultured neurons. These results indicate that armoring of AKT activation by IGF-I is crucial to preserve its cytoprotective effect in astrocytes and may form part of the brain defense mechanism against oxidative stress injury.     

Grape seed proanthocyanidins inhibit UV-radiation-induced oxidative stress and activation of MAPK and NF-kappaB signaling in human epidermal keratinocytes

Solar ultraviolet (UV) radiation-induced oxidative stress has been implicated in various skin diseases. Here, we report the photoprotective effect of grape seed proanthocyanidins (GSPs) on UV-induced oxidative stress and activation of mitogen-activated protein kinase (MAPK) and NF-kappaB signaling pathways using normal human epidermal keratinocytes (NHEK). Treatment of NHEK with GSPs inhibited UVB-induced hydrogen peroxide (H2O2), lipid peroxidation, protein oxidation, and DNA damage in NHEK and scavenged hydroxyl radicals and superoxide anions in a cell-free system. GSPs also inhibited UVB-induced depletion of antioxidant defense components, such as glutathione peroxidase, catalase, superoxide dismutase, and glutathione. As UV-induced oxidative stress mediates activation of MAPK and NF-kappaB signaling pathways, we determined the effects of GSPs on these pathways. Treatment of NHEK with GSPs inhibited UVB-induced phosphorylation of ERK1/2, JNK, and p38 proteins of the MAPK family at the various time points studied. As UV-induced H2O2 plays a major role in activation of MAPK proteins, NHEK were treated with H2O2 with or without GSPs and other known antioxidants, viz. (-)-epigallocatechin-3-gallate, silymarin, ascorbic acid, and N-acetylcysteine. It was observed that H2O2-induced phosphorylation of ERK1/2, JNK, and p38 was decreased by these antioxidants. Under identical conditions, GSPs also inhibited UVB-induced activation of NF-kappaB/p65, which was mediated through inhibition of degradation and activation of IkappaBalpha and IKKalpha, respectively. Together, these results suggest that GSPs could be useful in the attenuation of UV-radiation-induced oxidative stress-mediated skin diseases in human skin.     

AKT/FOXO signaling enforces reversible differentiation blockade in myeloid leukemias

AKT activation is associated with many malignancies, where AKT acts, in part, by inhibiting FOXO tumor suppressors. We show a converse role for AKT/FOXOs in acute myeloid leukemia (AML). Rather than decreased FOXO activity, we observed that FOXOs are active in ～40% of AML patient samples regardless of genetic subtype. We also observe this activity in human MLL-AF9 leukemia allele-induced AML in mice, where either activation of Akt or compound deletion of FoxO1/3/4 reduced leukemic cell growth, with the latter markedly diminishing leukemia-initiating cell (LIC) function in vivo and improving animal survival. FOXO inhibition resulted in myeloid maturation and subsequent AML cell death. FOXO activation inversely correlated with JNK/c-JUN signaling, and leukemic cells resistant to FOXO inhibition responded to JNK inhibition. These data reveal a molecular role for AKT/FOXO and JNK/c-JUN in maintaining a differentiation blockade that can be targeted to inhibit leukemias with a range of genetic lesions.     

Inhibition of JNK by cellular stress- and tumor necrosis factor alpha-induced AKT2 through activation of the NF kappa B pathway in human epithelial Cells

Previous studies have demonstrated that AKT1 and AKT3 are activated by heat shock and oxidative stress via both phosphatidylinositol 3-kinase-dependent and -independent pathways. However, the activation and role of AKT2 in the stress response have not been fully elucidated. In this study, we show that AKT2 in epithelial cells is activated by UV-C irradiation, heat shock, and hyperosmolarity as well as by tumor necrosis factor alpha (TNFalpha) through a phosphatidylinositol 3-kinase-dependent pathway. The activation of AKT2 inhibits UV- and TNF alpha-induced c-Jun N-terminal kinase (JNK) and p38 activities that have been shown to be required for stress- and TNF alpha-induced programmed cell death. Moreover, AKT2 interacts with and phosphorylates I kappa B kinase alpha. The phosphorylation of I kappa B kinase alpha and activation of NF kappa B mediates AKT2 inhibition of JNK but not p38. Furthermore, phosphatidylinositol 3-kinase inhibitor or dominant negative AKT2 significantly enhances UV- and TNF alpha-induced apoptosis, whereas expression of constitutively active AKT2 inhibits programmed cell death in response to UV and TNFalpha -induced apoptosis by inhibition of stress kinases and provide the first evidence that AKT inhibits stress kinase JNK through activation of the NF kappa B pathway.     

Pathological roles of MAPK signaling pathways in human diseases

The mammalian family of mitogen-activated protein kinases (MAPKs) includes extracellular signal-regulated kinase (ERK), p38, and c-Jun NH₂-terminal kinase (JNK), with each MAPK signaling pathway consisting of at least three components, a MAPK kinase kinase (MAP3K), a MAPK kinase (MAP2K), and a MAPK. The MAPK pathways are activated by diverse extracellular and intracellular stimuli including peptide growth factors, cytokines, hormones, and various cellular stressors such as oxidative stress and endoplasmic reticulum stress. These signaling pathways regulate a variety of cellular activities including proliferation, differentiation, survival, and death. Deviation from the strict control of MAPK signaling pathways has been implicated in the development of many human diseases including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and various types of cancers. Persistent activation of the JNK or p38 signaling pathways has been suggested to mediate neuronal apoptosis in AD, PD, and ALS, whereas the ERK signaling pathway plays a key role in several steps of tumorigenesis including cancer cell proliferation, migration, and invasion. In this review, we summarize recent findings on the roles of MAPK signaling pathways in human disorders, focusing on cancer and neurodegenerative diseases including AD, PD, and ALS.     

beta-Amyloid peptide induces formation of actin stress fibers through p38 mitogen-activated protein kinase

Based on the critical role of actin in the maintenance of synaptic function, we examined whether expression of familial beta-amyloid precursor protein APP-V642I (IAPP) or mutant presenilin-1 L286V (mPS1) affects actin polymerization in rat septal neuronal cells. Expression of either IAPP or mPS1 but not wild-type amyloid precursor protein or presenilin-1induced formation of actin stress fibers in SN1 cells, a septal neuronal cell line. Treatment with beta-amyloid (Abeta) peptide also caused formation of actin stress fibers in SN1 cells and primary cultured hippocampal neurons. Treatment with a gamma-secretase inhibitor completely blocked formation of actin stress fibers, indicating that overproduction of Abeta peptide induces actin stress fibers. Because activation of the p38 mitogen-activated protein kinase (p38MAPK)-mitogen-associated protein kinase-associated protein kinase (MAPKAPK)-2-heat-shock protein 27 signaling pathway mediates actin polymerization, we explored whether Abeta peptide activates p38MAPK and MAPKAPK-2. Expression of IAPP or mPS1 induced activation of p38MAPK and MAPKAPK-2. Treatment with a p38MAPK inhibitor completely inhibited formation of actin stress fibers mediated by Abeta peptide, IAPP or mPS1. Moreover, treatment with a gamma-secretase inhibitor completely blocked activation of p38MAPK and MAPKAPK-2. In summary, our data suggest that overproduction of Abeta peptide induces formation of actin stress fibers through activation of the p38MAPK signaling pathway in septal neuronal cells.     

Type 1 insulin-like growth factor receptor (IGF-IR) signaling inhibits apoptosis signal-regulating kinase 1 (ASK1)

The type 1 insulin-like growth factor receptor (IGF-IR) is a receptor-tyrosine kinase that plays a critical role in signaling cell survival and proliferation. IGF-IR binding to its ligand, insulin-like growth factor (IGF-I) activates phosphoinositide 3-kinase (PI3K), promotes cell proliferation by activating the mitogen-activated protein kinase (MAPK) cascade, and blocks apoptosis by inducing the phosphorylation and inhibition of proapoptotic proteins such as BAD. Apoptosis signal-regulating kinase 1 (ASK1) is a MAP kinase kinase kinase (MAPKKK) that is required for c-Jun N-terminal kinase (JNK) and p38 activation in response to Fas and tumor necrosis factor (TNF) receptor stimulation, and for oxidative stress- and TNFalpha-induced apoptosis. The results presented here indicate that ASK1 forms a complex with the IGF-IR and becomes phosphorylated on tyrosine residue(s) in a manner dependent on IGF-IR activity. IGF-IR signaling inhibited ASK1 irrespective of TNFalpha-induced ASK1 activation and resulted in decreased ASK1-dependent JNK1 stimulation. Signaling through IGF-IR rescued cells from ASK1-induced apoptotic cell death in a manner independent of PI3K activity. These results indicate that IGF-IR signaling suppresses the ASK-1-mediated stimulation of JNK/p38 and the induction of programmed cell death. The simultaneous activation of MAP kinases and the inhibition of the stress-activated arm of the cascade by IGF-IR may constitute a potent proliferative signaling system and is possibly a mechanism by which IGF-I can stimulate growth and inhibit cell death in a wide variety of cell types and biological settings.     

Inhibition of mixed lineage kinase 3 prevents HIV-1 Tat-mediated neurotoxicity and monocyte activation

The HIV-1 gene products Tat and gp120 are toxic to neurons and can activate cells of myeloid origin, properties that are thought to contribute to the clinical manifestations of HIV-1-associated dementia (HAD). To investigate the intracellular signaling mechanisms involved in these events, the effect of Tat and gp120 on mixed lineage kinase (MLK) 3 activation was examined. Tat and gp120 were shown to induce autophosphorylation of MLK3 in primary rat neurons; this was abolished by the addition of an inhibitor of MLK3 (CEP1347). CEP1347 also enhanced survival of both rat and human neurons and inhibited the activation of human monocytes after exposure to Tat and gp120. Furthermore, overexpression of wild-type MLK3 led to the induction of neuronal death, whereas expression of a dominant negative MLK3 mutant protected neurons from the toxic effects of Tat. MLK3-dependent downstream signaling events were implicated in the neuroprotective and monocyte-deactivating pathways triggered by CEP1347. Thus, the inhibition of p38 MAPK and JNK protected neurons from Tat-induced apoptosis, whereas the inhibition of p38 MAPK, but not of JNK, was sufficient to prevent Tat- and gp120-mediated activation of monocytes. These results suggest that the normal function of MLK3 is compromised by HIV-1 neurotoxins (Tat, gp120), resulting in the activation of downstream signaling events that result in neuronal death and monocyte activation (with release of inflammatory cytokines). In aggregate, our data define MLK3 as a promising therapeutic target for intervention in HAD.     

Transient activation of Jun N-terminal kinases and protection from apoptosis by the insulin-like growth factor I receptor can be suppressed by dicumarol

The insulin-like growth factor I receptor (IGF-IR) activated by its ligands insulin-like growth factor (IGF)-I or IGF-II mediates suppression of apoptosis and contributes to tumorigenesis and cell growth. Here we investigated the activation of the stress-activated protein kinases including Jun N-terminal Kinases and p38 MAPK by IGF-I in interleukin-3-dependent FL5.12 lymphocytic cells that overexpress the IGF-IR (FL5.12/WT). We have shown previously that IGF-I protects these cells from apoptosis induced by interleukin-3 withdrawal but does not promote proliferation. IGF-I induced a rapid and transient activation of JNK that peaked at 40 min that was paralleled by a transient and robust phosphorylation of c-Jun. p38 was constitutively phosphorylated in FL5.12/WT cells. Activation of the JNK pathway by IGF-I occurred in the presence of phosphatidylinositol 3-kinase inhibitors and could be enhanced by anisomycin. Analysis of a series of FL5.12 cells expressing mutated IGF-IRs and analysis of 32D/IGF-IR cells showed that neither the C terminus of the receptor nor IRS-1 and IRS-2 were required for JNK activation, although tyrosine 950 was essential for full activation. The JNK inhibitor dicumarol suppressed IGF-I-mediated activation of JNK and phosphorylation of c-Jun but did not affect p38 and IkappaB phosphorylation or activation of AKT. IGF-I-mediated protection from apoptosis in FL5.12/WT cells was completely suppressed by dicumarol and partially suppressed by a p38 inhibitor. In the breast carcinoma cell line MCF-7, treatment with dicumarol also induced apoptosis. These data indicate that transient activation of JNK by IGF-I is mediated by signals that are distinct from those leading to phosphatidylinositol 3-kinase and AKT activation. The data further suggest that the SAPK pathways contribute to suppression of apoptosis by the IGF-IR.     

The relationship between p53/p21/Rb and MAPK signaling pathways in human endometrium-derived stem cells under oxidative stress

Human endometrium-derived mesenchymal stem cells (hMESC) under the sublethal oxidative stress induced by H₂O₂ activate both the p53/p21/Rb and p38/MAPKAPK-2 pathways that are responsible for the induction of hMESC premature senescence (Borodkina et al., 2014). However, the interrelations between the p53/p21/Rb and MAPK signaling pathways, including ERK1/2, p38, and JNK, remain yet unexplored. Here, we used the specific inhibitors—pifithrin-α (PFT), U0126, SB203580, and SP600125 to “switch off” one of the proteins in these cascades and to evaluate the functional status alterations of the rest of the proteins. Each MAPK suppression significantly increased the p53 phosphorylation level, as well as p21 protein expression followed by Rb hypophosphorylation. On the other hand, PFT-induced p53 inhibition enhanced mostly the ERK1/2 activation rather than p38 and JNK. These results suggest the existence of a reciprocal negative regulation between p53- and MAPK-dependent signaling pathways. By analyzing the possible interactions among the members of the MAPK family, we showed that p38 and JNK can function as ERK antagonists: JNK is able to activate ERK, while p38 may block ERK activation. Together, these results demonstrate the existence of complex links between different signaling cascades in stressed hMESC, implicating ERK, p38, and JNK in regulation of premature senescence via the p53/p21/Rb pathway.     

Calcium signaling is involved in cadmium-induced neuronal apoptosis via induction of reactive oxygen species and activation of MAPK/mTOR network

Cadmium (Cd), a toxic environmental contaminant, induces oxidative stress, leading to neurodegenerative disorders. Recently we have demonstrated that Cd induces neuronal apoptosis in part by activation of the mitogen-activated protein kineses (MAPK) and mammalian target of rapamycin (mTOR) pathways. However, the underlying mechanism remains elusive. Here we show that Cd elevated intracellular calcium ion ([Ca2+](i)) level in PC12, SH-SY5Y cells and primary murine neurons. BAPTA/AM, an intracellular Ca2+ chelator, abolished Cd-induced [Ca2+](i) elevation, and blocked Cd activation of MAKPs including extracellular signal-regulated kinase 1/2 (Erk1/2), c-Jun N-terminal kinase (JNK) and p38, and mTOR-mediated signaling pathways, as well as cell death. Pretreatment with the extracellular Ca2+ chelator EGTA also prevented Cd-induced [Ca2+](i) elevation, MAPK/mTOR activation, as well as cell death, suggesting that Cd-induced extracellular Ca2+ influx plays a critical role in contributing to neuronal apoptosis. In addition, calmodulin (CaM) antagonist trifluoperazine (TFP) or silencing CaM attenuated the effects of Cd on MAPK/mTOR activation and cell death. Furthermore, Cd-induced [Ca2+](i) elevation or CaM activation resulted in induction of reactive oxygen species (ROS). Pretreatment with BAPTA/AM, EGTA or TFP attenuated Cd-induced ROS and cleavage of caspase-3 in the neuronal cells. Our findings indicate that Cd elevates [Ca2+](i), which induces ROS and activates MAPK and mTOR pathways, leading to neuronal apoptosis. The results suggest that regulation of Cd-disrupted [Ca2+](i) homeostasis may be a new strategy for prevention of Cd-induced neurodegenerative diseases.     

Apoptosis induced by domoic acid in mouse cerebellar granule neurons involves activation of p38 and JNK MAP kinases

In mouse cerebellar granule neurons (CGNs) the marine neurotoxin domoic acid (DomA) induces neuronal cell death, either by apoptosis or by necrosis, depending on its concentration, with apoptotic damage predominating in response to low concentrations (100 nM). DomA-induced apoptosis is due to selective activation of AMPA/kainate receptors, and is mediated by DomA-induced oxidative stress, leading to mitochondrial dysfunction and activation of caspase-3. The p38 MAP kinase and the c-Jun NH2-terminal protein kinase (JNK) have been shown to be preferentially activated by oxidative stress. Here we report that DomA increases p38 MAP kinase and JNK phosphorylation, and that this effect is more pronounced in CGNs from Gclm (-/-) mice, which lack the modifier subunit of glutamate-cysteine ligase, have very low glutathione (GSH) levels, and are more sensitive to DomA-induced apoptosis than CGNs from wild-type mice. The increased phosphorylation of JNK and p38 kinase was paralleled by a decreased phosphorylation of Erk 1/2. The AMPA/kainate receptor antagonist NBQX, but not the NMDA receptor antagonist MK-801, prevents DomA-induced activation of p38 and JNK kinases. Several antioxidants (GSH ethyl ester, catalase and phenylbutylnitrone) also prevent DomA-induced phosphorylation of JNK and p38 MAP kinases. Inhibitors of p38 (SB203580) and of JNK (SP600125) antagonize DomA-induced apoptosis. These results indicate the importance of oxidative stress-activated JNK and p38 MAP kinase pathways in DomA-induced apoptosis in CGNs.     

Activation of c-Jun-N-terminal kinase is required for apoptosis triggered by glutathione disulfide in neuroblastoma cells

Changes in intracellular redox status are crucial events that trigger downstream proliferation or death responses through activation of specific signaling pathways. Moreover, cell responses to oxidative challenge may depend on the pattern of redox-sensitive molecular factors. The stress-activated protein kinases c-Jun-N-terminal kinase (JNK) and p38 MAP kinase (p38MAPK) are implicated in different forms of apoptotic neuronal cell death. Here, we investigated the effects, on neuroblastoma cells, of the prooxidant molecule GSSG, which we previously demonstrated to be an efficient proapoptotic compound able to activate the p38MAPK death pathway in promonocytic cells. We found that neuroblastoma cells are not prone to GSSG-induced apoptosis, although the treatment slightly induced growth arrest through the accumulation of p53 and its downstream target gene, p21. However, GSSG treatment became cytotoxic when cells were previously depleted of intracellular GSH content. Under this condition, apoptosis was triggered by an increased production of superoxide that led to a specific activation of the JNK-dependent pathway. The involvement of superoxide and JNK was demonstrated by cell death inhibition in experiments carried out in the presence of Cu,Zn superoxide dismutase or with specific inhibitors of JNK activity. Our data give support to the studies that indicate preferential requirements for the involvement of stress-activated kinases in apoptotic neuronal cells.     

Muscarinic receptor activation protects cells from apoptotic effects of DNA damage,oxidative stress,and mitochondrial inhibition

The impact of muscarinic receptor stimulation was examined on apoptotic signaling induced by DNA damage, oxidative stress, and mitochondrial impairment. Exposure of human neuroblastoma SH-SY5Y cells to the DNA-damaging agent camptothecin increased p53 levels, activated caspase-3, and caused cell death. Pretreatment with oxotremorine-M, a selective agonist of muscarinic receptors that are expressed endogenously in these cells, did not affect the accumulation of p53 but greatly attenuated caspase-3 activation and protected from cell death to nearly the same extent as treatment with a general caspase inhibitor. Treatment with 50-200 microm H(2)O(2) caused the activation of caspase-3 beginning after 2-3 h, followed by eventual cell death. Oxotremorine-M pretreatment protected cells from H(2)O(2)-induced caspase-3 activation and death, and this was equivalent to protection afforded by a caspase inhibitor. Muscarinic receptor stimulation also protected cells from caspase-3 activation induced by exposure to rotenone, a mitochondrial complex 1 inhibitor, but no protection was evident from staurosporine-induced caspase-3 activation. The mechanism of protection afforded by muscarinic receptor activation from camptothecin-induced apoptotic signaling involved blockade of mitochondrial cytochrome c release associated with a bolstering of mitochondrial bcl-2 levels and blockade of the translocation of Bax to mitochondria. Likely the most proximal of these events to muscarinic receptor activation, mitochondrial Bax accumulation, also was attenuated by oxotremorine-M treatment after treatment with H(2)O(2) or rotenone. These results demonstrate that stimulation of muscarinic receptors provides substantial protection from DNA damage, oxidative stress, and mitochondrial impairment, insults that may be encountered by neurons in development, aging, or neurodegenerative diseases. These findings suggest that neurotransmitter-induced signaling bolsters survival mechanisms, and inadequate neurotransmission may exacerbate neuronal loss.     

JNK3 contributes to c-Jun activation and apoptosis but not oxidative stress in nerve growth factor-deprived sympathetic neurons

The stress activated protein kinase pathway culminates in c-Jun phosphorylation mediated by the Jun Kinases (JNKs). The role of the JNK pathway in sympathetic neuronal death is unclear in that apoptosis is not inhibited by a dominant negative protein of one JNK kinase, SEK1, but is inhibited by CEP-1347, a compound known to inhibit this overall pathway but not JNKs per se. To evaluate directly the apoptotic role of the JNK isoform that is selectively expressed in neurons, JNK3, we isolated sympathetic neurons from JNK3-deficient mice and quantified nerve growth factor (NGF) deprivation-induced neuronal death, oxidative stress, c-Jun phosphorylation, and c-jun induction. Here, we report that oxidative stress in neurons from JNK3-deficient mice is normal after NGF deprivation. In contrast, NGF-deprivation-induced increases in the levels of phosphorylated c-Jun, c-jun, and apoptosis are each inhibited in JNK3-deficient mice. Overall, these results indicate that JNK3 plays a critical role in activation of c-Jun and apoptosis in a classic model of cell-autonomous programmed neuron death.     

Modification of collagen by 3-deoxyglucosone alters wound healing through differential regulation of p38 MAP kinase

Background

Wound healing is a highly dynamic process that requires signaling from the extracellular matrix to the fibroblasts for migration and proliferation, and closure of the wound. This rate of wound closure is impaired in diabetes, which may be due to the increased levels of the precursor for advanced glycation end products, 3-deoxyglucosone (3DG). Previous studies suggest a differential role for p38 mitogen-activated kinase (MAPK) during wound healing; whereby, p38 MAPK acts as a growth kinase during normal wound healing, but acts as a stress kinase during diabetic wound repair. Therefore, we investigated the signaling cross-talk by which p38 MAPK mediates wound healing in fibroblasts cultured on native collagen and 3DG-collagen.

Methodology/Principal Findings

Using human dermal fibroblasts cultured on 3DG-collagen as a model of diabetic wounds, we demonstrated that p38 MAPK can promote either cell growth or cell death, and this was dependent on the activation of AKT and ERK1/2. Wound closure on native collagen was dependent on p38 MAPK phosphorylation of AKT and ERK1/2. Furthermore, proliferation and collagen production in fibroblasts cultured on native collagen was dependent on p38 MAPK regulation of AKT and ERK1/2. In contrast, 3DG-collagen decreased fibroblast migration, proliferation, and collagen expression through ERK1/2 and AKT downregulation via p38 MAPK.

Conclusions/Significance

Taken together, the present study shows that p38 MAPK is a key signaling molecule that plays a significantly opposite role during times of cellular growth and cellular stress, which may account for the differing rates of wound closure seen in diabetic populations.