Kaempferol induces cell death through ERK and Akt-dependent down-regulation of XIAP and survivin in human glioma cells

The present study was undertaken to determine the molecular mechanism by which kaempferol induces cell death in human glioma cells. Kaempferol resulted in loss of cell viability and inhibition of proliferation in a dose- and time-dependent manner, which were largely attributed to cell death. Kaempferol caused an increase in reactive oxygen species (ROS) generation and the kaempferol-induced cell death was prevented by antioxidants, suggesting that ROS generation is involved in kaempferol-induced cell death. Kaempferol caused depolarization of mitochondrial membrane potential. Western blot analysis showed that kaempferol treatment caused a rapid reduction in phosphorylation of extracellular signal-regulated kinase (ERK) and Akt. The ERK inhibitor U0126 and the Akt inhibitor LY984002 increased the kaempferol-induced cell death and overexpression of MEK, the upstream kinase of ERK, and Akt prevented the cell death. The expression of anti-apoptotic proteins XIAP and survivin was down-regulated by kaempferol and its effect was prevented by overexpression of MEK and Akt. Kaempferol induced activation of caspase-3 and kaempferol-induced cell death was prevented by caspase inhibitors. Taken together, these findings suggest that kaempferol results in human glioma cell death through caspase-dependent mechanisms involving down-regulation of XIAP and survivin regulating by ERK and Akt.     

A novel cellular survival factor--the B2 subunit of vacuolar H+-ATPase inhibits apoptosis

The ubiquitous vacuolar H(+)-ATPase, a multisubunit proton pump, is essential for intraorganellar acidification. Disruption of its function leads to disturbances of organelle function and cell death. Here, we report that overexpression of the B2 subunit of the H(+)-ATPase inhibits apoptosis. This antiapoptotic effect is not mediated by an increase in H(+)-ATPase activity but through activation of the Ras-mitogen-activated protein kinase (MAPK)-signaling pathway that results in the serine phosphorylation of Bad at residues 112 and 155. Increased Bad phosphorylation reduces its translocation to mitochondria, limits the release of mitochondrial cytochrome c and apoptosis-inducing factor and increases the resistance of the B2 overexpressing cells to apoptosis. Screening experiments of kinase inhibitors, including inhibitors of cAMP-activated protein kinase, protein kinase C, protein kinase B, (MAPK/extracellular signal-regulated (ERK) kinase) MEK and Ste-MEK1(13), a cell permeable ERK activation inhibitor peptide, revealed that the B2 subunit of H(+)-ATPase acts upstream of MEK activation in the MEK/ERK pathway to ameliorate apoptosis.     

Estrogens down-regulate p27Kip1 in breast cancer cells through Skp2 and through nuclear export mediated by the ERK pathway

The cyclin-dependent kinase (CDK) inhibitor p27Kip1 plays a key role in growth and development of the mammary epithelium and in breast cancer. p27Kip1 levels are regulated through ubiquitin/proteasome-mediated proteolysis, promoted by CDK2 and the F box protein Skp2 at the G1/S transition, and independent of Skp2 in mid-G1. We investigated the respective roles of Skp2 and subcellular localization of p27Kip1 in down-regulation of p27Kip1 induced in MCF-7 cells by estrogens. 17beta-Estradiol treatment increased Skp2 expression in MCF-7 cells; however, this increase was prevented by G1 blockade mediated by p16Ink4a or the CDK inhibitor roscovitine, whereas down-regulation of p27Kip1 was maintained. Exogenous Skp2 prevented growth arrest of MCF-7 cells by antiestrogen, coinciding with decreased p27Kip1 expression. Under conditions of G1 blockade, p27Kip1 was stabilized by inhibition of CRM1-dependent nuclear export with leptomycin B or by mutation of p27Kip1 (Ser10 --> Ala; S10A) interfering with CRM1/p27Kip1 interaction. Antisense Skp2 oligonucleotides and a dominant-interfering Cul-1(1-452) mutant prevented down-regulation of p27Kip1S10A, whereas Skp2 overexpression elicited its destruction in mitogen-deprived cells. Active mediators of the extracellular signal-regulated kinase (ERK) pathway including Raf-1caax induced cytoplasmic localization of p27Kip1 in antiestrogen-treated cells and prevented accumulation of p27Kip1 in these cells independent of Skp2 expression and coinciding with ERK activation. Genetic or chemical blockade of the ERK pathway prevented down-regulation and cytoplasmic localization of p27Kip1 in response to estrogen. Our studies indicate that estrogens elicit down-regulation of p27Kip1 in MCF-7 cells through Skp2-dependent and -independent mechanisms that depend upon subcellular localization of p27Kip1 and require the participation of mediators of the Ras/Raf-1/ERK signaling pathway.     

Mitochondrially localized ERK2 regulates mitophagy and autophagic cell stress: implications for Parkinson's disease

Degenerating neurons of Parkinson's disease (PD) patient brains exhibit granules of phosphorylated extracellular signal-regulated protein kinase 1/2 (ERK1/2) that localize to autophagocytosed mitochondria. Here we show that 6-hydroxydopamine (6-OHDA) elicits activity-related localization of ERK1/2 in mitochondria of SH-SY5Y cells, and these events coincide with induction of autophagy and precede mitochondrial degradation. Transient transfection of wildtype (WT) ERK2 or constitutively active MAPK/ERK Kinase 2 (MEK2-CA) was sufficient to induce mitophagy to a degree comparable with that elicited by 6-OHDA, while constitutively active ERK2 (ERK2-CA) had a greater effect. We developed green fluorescent protein (GFP) fusion constructs of WT, CA, and kinase-deficient (KD) ERK2 to study the role of ERK2 localization in regulating mitophagy and cell death. Under basal conditions, cells transfected with GFP-ERK2-WT or GFP-ERK2-CA, but not GFP-ERK2-KD, displayed discrete cytoplasmic ERK2 granules of which a significant fraction colocalized with mitochondria and markers of autophagolysosomal maturation. The colocalizing GFP-ERK2/mitochondria granules are further increased by 6-OHDA and undergo autophagic degradation, as bafilomycin-A, an inhibitor of autolysosomal degradation, robustly increased their detection. Interestingly, increasing ERK2-WT or ERK2-CA expression was sufficient to promote comparable levels of macroautophagy as assessed by analysis of the autophagy marker microtubule-associated protein 1 light chain 3 (LC3). In contrast, the level of mitophagy was more tightly correlated with ERK activity levels, potentially explained by the greater localization of ERK2-CA to mitochondria compared to ERK2-WT. These data indicate that mitochondrial localization of ERK2 activity is sufficient to recapitulate the effects of 6-OHDA on mitophagy and autophagic cell death.     

Role of ERK in Hydrogen Peroxide-Induced Cell Death of Human Glioma Cells

Oxidative stress is known to induce cell death in a wide variety of cell types, apparently by modulating intracellular signaling pathways. Activation of extracellular signal-regulated kinase (ERK) in oxidative stress remains controversial. In some cellular systems, the ERK activation is associated with protection against oxidative stress, while in other system, the ERK activation is involved in apoptotic cell death. The present study was undertaken to examine the role of ERK activation in H₂O₂-induced cell death of human glioma (A172) cells. H₂O₂ resulted in a time- and dose-dependent cell death, which was largely attributed to apoptosis. H₂O₂ treatment caused marked sustained activation of ERK. The ERK activation and cell death induced by H₂O₂ was prevented by catalase, the hydrogen peroxide scavenger, and U0126, an inhibitor of ERK upstream kinase MEK1/2. Transient transfection with constitutive active MEK1, an upstream activator of ERK1/2, increased H₂O₂-induced cell death, whereas transfection with dominant-negative mutants of MEK1 decreased the cell death. The ERK activation and cell death caused by H₂O₂ was inhibited by antioxidants (N-acetylcysteine and trolox), Ras inhibitor, and suramin. H₂O₂ produced depolarization of mitochondrial membrane potential and its effect was prevented by catalase and U0126. Taken together, these findings suggest that growth factor receptor/Ras/MEK/ERK signaling pathway plays an active role in mediating H₂O₂-induced apoptosis of human glioma cells and functions upstream of mitochondria-dependent pathway to initiate the apoptotic signal.     

ERK1/2 regulates two sequential steps promoting monocyte survival to peroxynitrite

Previous studies from our laboratory indicate that cytosolic phospholipase A(2) (cPLA(2))-released arachidonic acid promotes monocyte/macrophage survival in the presence of peroxynitrite. In particular, the lipid messenger is metabolised by 5-lipoxygenase (5-LO) to 5-hydroxyeicosatetraenoic acid and causes the mitochondrial translocation of protein kinase Calpha (PKCalpha), an event associated with the cytosolic accumulation of Bad and Bax. Here we show that phosphorylation reactions driven by extracellular regulated kinase 1/2 (ERK1/2) critically regulate the activation/nuclear translocation of 5-LO. Inhibition of ERK1/2 was invariably associated with the cytosolic localisation of PKCalpha, the mitochondrial accumulation of Bad and Bax and with a rapid mitochondrial permeability transition-dependent necrosis. All these events were prevented by nanomolar concentrations of 5-hydroxyeicosatetraenoic acid. Hence, in addition to the previously characterised effects on cPLA(2), ERK1/2 critically regulates 5-LO activity in the absence of additional downstream targets in the survival signalling preventing peroxynitrite toxicity.     

Guanosine controls inflammatory pathways to afford neuroprotection of hippocampal slices under oxygen and glucose deprivation conditions

Guanosine (GUO) is an endogenous modulator of glutamatergic excitotoxicity and has been shown to promote neuroprotection in in vivo and in vitro models of neurotoxicity. This study was designed to understand the neuroprotective mechanism of GUO against oxidative damage promoted by oxygen/glucose deprivation and reoxygenation (OGD). GUO (100 μM) reduced reactive oxygen species production and prevented mitochondrial membrane depolarization induced by OGD. GUO also exhibited anti‐inflammatory actions as inhibition of nuclear factor kappa B activation and reduction of inducible nitric oxide synthase induction induced by OGD. These GUO neuroprotective effects were mediated by adenosine A₁ receptor, phosphatidylinositol‐3 kinase and MAPK/ERK. Furthermore, GUO recovered the impairment of glutamate uptake caused by OGD, an effect that occurred via a Pertussis toxin‐sensitive G‐protein‐coupled signaling, blockade of adenosine A_2A receptors (A_2AR), but not via A₁ receptor. The modulation of glutamate uptake by GUO also involved MAPK/ERK activation. In conclusion, GUO, by modulating adenosine receptor function and activating MAPK/ERK, affords neuroprotection of hippocampal slices subjected to OGD by a mechanism that implicates the following: (i) prevention of mitochondrial membrane depolarization, (ii) reduction of oxidative stress, (iii) regulation of inflammation by inhibition of nuclear factor kappa B and inducible nitric oxide synthase, and (iv) promoting glutamate uptake.     

Phosphoprotein Enriched in Astrocytes 15 kDa (PEA-15) Reprograms Growth Factor Signaling by Inhibiting Threonine Phosphorylation of Fibroblast Receptor Substrate 2α

Changes in cellular expression of phosphoprotein enriched in astrocytes of 15 kDa (PEA-15) are linked to insulin resistance, tumor cell invasion, and cellular senescence; these changes alter the activation of the extracellular signal-regulated kinase (ERK)1/2 mitogen-activated protein (MAP) kinase pathway. Here, we define the mechanism whereby increased PEA-15 expression promotes and sustains ERK1/2 activation. PEA-15 binding prevented ERK1/2 membrane recruitment and threonine phosphorylation of fibroblast receptor substrate 2α (FRS2α), a key link in fibroblast growth factor (FGF) receptor activation of ERK1/2. This reduced threonine phosphorylation led to increased FGF-induced tyrosine phosphorylation of FRS2α, thereby enhancing downstream signaling. Conversely, short hairpin RNA-mediated depletion of endogenous PEA-15 led to reduced FRS2α tyrosine phosphorylation. Thus, PEA-15 interrupts a negative feedback loop that terminates growth factor receptor signaling downstream of FRS2α. This is the dominant mechanism by which PEA-15 activates ERK1/2 because genetic deletion of FRS2α blocked the capacity of PEA-15 to activate the MAP kinase pathway. Thus, PEA-15 prevents ERK1/2 localization to the plasma membrane, thereby inhibiting ERK1/2-dependent threonine phosphorylation of FRS2α to promote activation of the ERK1/2 MAP kinase pathway.     

Nicotine activates the extracellular signal-regulated kinase 1/2 via the alpha7 nicotinic acetylcholine receptor and protein kinase A, in SH-SY5Y cells and hippocampal neurones

Neuronal nicotinic acetylcholine receptors (nAChR) can modulate many cellular mechanisms, such as cell survival and memory processing, which are also influenced by the serine/threonine protein kinases ERK1/2. In SH-SY5Y cells and hippocampal neurones, nicotine (100 microM) increased the activity of ERK1/2. This effect was Ca2+ dependent, and prevented by the alpha7 nAChR antagonist alpha-bungarotoxin (alpha-Bgt) and an inhibitor (PD98059) of the upstream kinase MEK. To determine the intervening steps linking Ca2+ entry to MEK-ERK1/2 activation, inhibitors of Ca2+-dependent kinases were deployed. In SH-SY5Y cells, selective blockers for PKC (Ro 31-8220), CaM kinase II (KN-62) or PI3 kinase (LY 294002) failed to inhibit the nicotine-evoked increase in ERK1/2 activity. In contrast, two structurally different inhibitors of PKA (KT 5720 and H-89) completely prevented the nicotine-dependent increase in ERK1/2 activity. Inhibition of the nicotine-evoked increase in ERK1/2 activity by H-89 was also observed in hippocampal cultures. Down stream of PKA, the activity of B-Raf was significantly decreased by nicotine in SH-SY5Y cells, as determined by direct measurement of MEK1 phosphorylation or in vitro kinase assays, whereas the modulation of MEK1 phosphorylation by Raf-1 tended to increase. Thus, this study provides evidence for a novel signalling route coupling the stimulation of alpha7 nAChR to the activation of ERK1/2, in a Ca2+ and PKA dependent manner.     

Coupling of M(2) muscarinic receptors to ERK MAP kinases and caldesmon phosphorylation in colonic smooth muscle

Coupling of M(2) and M(3) muscarinic receptors to activation of mitogen-activated protein (MAP) kinases and phosphorylation of caldesmon was studied in canine colonic smooth muscle strips in which M(3) receptors were selectively inactivated by N, N-dimethyl-4-piperidinyl diphenylacetate (4-DAMP) mustard (40 nM). ACh elicited activation of extracellular signal-regulated kinase (ERK) 1, ERK2, and p38 MAP kinases in control muscles and increased phosphorylation of caldesmon (Ser(789)), a putative downstream target of MAP kinases. Alkylation of M(3) receptors with 4-DAMP had only a modest inhibitory effect on ERK activation, p38 MAP kinase activation, and caldesmon phosphorylation. Subsequent treatment with 1 microM AF-DX 116 completely prevented activation of ERK and p38 MAP kinase and prevented caldesmon phosphorylation. Caldesmon phosphorylation was blocked by the MAP kinase/ERK kinase inhibitor PD-98509 but not by the p38 MAP kinase inhibitor SB-203580. These results indicate that colonic smooth muscle M(2) receptors are coupled to ERK and p38 MAP kinases. Activation of ERK, but not p38 MAP kinases, results in phosphorylation of caldesmon in vivo, which is a novel function for M(2) receptor activation in smooth muscle.     

Molecular mechanisms of low intensity pulsed ultrasound in human skin fibroblasts

Soluble factors such as polypeptide growth factors, mitogenic lipids, inflammatory cytokines, and hormones are known regulators of cell proliferation. However, the effect of mechanical stimuli on cell proliferation is less well understood. Here we examined the effect of low intensity pulsed ultrasound (US), which is used to promote wound healing, on the proliferation of primary human foreskin fibroblasts and the underlying signaling mechanisms. We show that a single 6-11-min US stimulation increases bromodeoxyuridine incorporation. In addition, an increase in the total cell number is observed after sequential US stimulation. US induced stress fiber and focal adhesion formation via activation of Rho. We further observed that US selectively induced activation of extracellular signal-regulated kinase (ERK) 1/2. Inhibition of Rho-associated coiled-coil-containing protein kinase (ROCK) prevented US-induced ERK1/2 activation, demonstrating that the Rho/ROCK pathway is an upstream regulator of ERK activation in response to US. Consequently, activation of ROCK and MEK-1 was required for US-induced DNA synthesis. Finally, an integrin beta(1) blocking antibody as well as a RGD peptide prevented US-induced DNA synthesis. In addition, US slightly increased phosphorylation of Src at Tyr(416), and Src activity was found to be required for ERK1/2 activation in response to US. In conclusion, our data demonstrate for the first time that US promotes cell proliferation via activation of integrin receptors and a Rho/ROCK/Src/ERK signaling pathway.     

Roles of Src and epidermal growth factor receptor transactivation in transient and sustained ERK1/2 responses to gonadotropin-releasing hormone receptor activation

The duration as well as the magnitude of mitogen-activated protein kinase activation has been proposed to regulate gene expression and other specific intracellular responses in individual cell types. Activation of ERK1/2 by the hypothalamic neuropeptide gonadotropin-releasing hormone (GnRH) is relatively sustained in alpha T3-1 pituitary gonadotropes and HEK293 cells but is transient in immortalized GT1-7 neurons. Each of these cell types expresses the epidermal growth factor receptor (EGFR) and responds to EGF stimulation with significant but transient ERK1/2 phosphorylation. However, GnRH-induced ERK1/2 phosphorylation caused by EGFR transactivation was confined to GT1-7 cells and was attenuated by EGFR kinase inhibition. Neither EGF nor GnRH receptor activation caused translocation of phospho-ERK1/2 into the nucleus in GT1-7 cells. In contrast, agonist stimulation of GnRH receptors expressed in HEK293 cells caused sustained phosphorylation and nuclear translocation of ERK1/2 by a protein kinase C-dependent but EGFR-independent pathway. GnRH-induced activation of ERK1/2 was attenuated by the selective Src kinase inhibitor PP2 and the negative regulatory C-terminal Src kinase in GT1-7 cells but not in HEK293 cells. In GT1-7 cells, GnRH stimulated phosphorylation and nuclear translocation of the ERK1/2-dependent protein, p90RSK-1 (RSK-1). These results indicate that the duration of ERK1/2 activation depends on the signaling pathways utilized by GnRH in specific target cells. Whereas activation of the Gq/protein kinase C pathway in HEK293 cells causes sustained phosphorylation and translocation of ERK1/2 to the nucleus, transactivation of the EGFR by GnRH in GT1-7 cells elicits transient ERK1/2 signals without nuclear accumulation. These findings suggest that transactivation of the tightly regulated EGFR can account for the transient ERK1/2 responses that are elicited by stimulation of certain G protein-coupled receptors.     

Epidermal growth factor receptor and protein kinase C signaling to ERK2: spatiotemporal regulation of ERK2 by dual specificity phosphatases

Spatiotemporal aspects of ERK activation are stimulus-specific and dictate cellular consequences. They are dependent upon dual specificity phosphatases (DUSPs) that bind ERK via docking domains and can both inactivate and anchor ERK in cellular compartments. Using high throughput fluorescence microscopy in combination with a system where endogenous ERKs are removed and replaced with wild-type or mutated ERK2-green fluorescent protein (GFP), we show that ERK2 activation responses to epidermal growth factor (EGF) and protein kinase C (PKC) are transient and sustained, respectively. PKC-mediated ERK2 activation is associated with prolonged nuclear localization in the dephosphorylated form, whereas EGF-stimulated ERK2 activation mediates only transient nuclear accumulation. By using short inhibitory RNAs to nuclear inducible DUSP1, -2, or -4 (alone or in combination), we demonstrate that all three of these enzymes contribute to the dephosphorylation of PKC (but not EGF)-activated ERK2 in the nucleus but that they have opposing effects on localization. DUSP2 and -4 inactivate and anchor ERK2, whereas DUSP1 dephosphorylates ERK in the nucleus but allows its traffic back to the cytoplasm. Overexpression of DUSP1, -2, or -4 prevented ERK2 activation, but only DUSP2 and -4 caused ERK2-GFP nuclear accumulation or could be immunoprecipitated with ERK2. Furthermore, protein synthesis inhibition or replacement of wild-type ERK2-GFP with docking domain mutants selectively increased PKC effects on ERK activity and altered ERK2-GFP localization. These mutations also impaired the ability of ERK2-GFP to bind DUSP2 and -4. Together, our data reveal a novel, stimulus-specific, and phosphatase-specific mechanism of ERK2 regulation in the nucleus by DUSP1, -2, and -4.     

Phosphatidylinositol-3 kinase dependent MAP kinase activation via p21ras in endothelial cells exposed to cyclic strain

Hemodynamic forces play a key role in the modulation of the morphology and function of the endothelium by activating several kinases. We have previously shown that cyclic strain, a repetitive mechanical stretch, induces activation of extracellular signal-regulated protein kinases 1 and 2 (ERK1/2), members of the mitogen activated protein (MAP) kinase family. In order to investigate the upstream pathway of strain-induced ERK1/2 activation, we examined p21ras activation by cyclic strain and the effect of wortmannin and LY294002, phosphatidylinositol-3 kinase (PI 3-kinase) inhibitors on ERK1/2 phosphorylation. Cyclic strain induced a transient and rapid activation of p21ras at 1 min after strain. Wortmannin inhibited strain-induced ERK1/2 activation by 56.3 and 86.3 %, respectively. LY294002 inhibited ERK1 activation completely and ERK2 activation by 42.9%. These results suggest a possible involvement of p21ras and PI 3-kinase in the signal transduction pathway leading to the strain-induced ERK1/2 activation.