Stress Signaling Cascades Research Articles

Temozolomide, irradiation and hypoxia promote homing of hematopoietic progenitor cells towards gliomas

20044 Background: Temozolomide and irradiation are essential parts of the standard therapy and hypoxia is a critical aspect of the microenvironment of gliomas. IN the present study, we aimed at investigating the impact of these stimuli on the previously defined transforming growth factor beta (TGF-β)- and stromal cell-derived factor-1/CXC chemokine ligand 12 (SDF-1α/CXCL12)-dependent migration of adult hematopoietic stem and progenitor cells (HPC) towards glioma cells in vitro and the homing to experimental gliomas in vivo. Hyperthermia served as control. Methods and Results: Cerebral irradiation of nude mice at 21 days after intracerebral implantation of LNT-229 glioma induces tumor satellite formation and enhances the glioma tropism of HPC in vivo. Supernatants of temozolomide-treated, irradiated or hypoxic LNT-229 glioma cells promote HPC migration in vitro. Reporter assays reveal that the CXCL12 promoter activity is enhanced in LNT-229 glioma cells at 24 h after irradiation at 8 Gy or after exposure to 1% oxygen for 12 h. The irradiation- and hypoxia-induced release of CXCL12 depends on hypoxia inducible factor-1 alpha (HIF-1α), but not on p53. Induction of transcriptional activity of HIF-1α by hypoxia and irradiation requires an intact signaling cascade of TGF-β. Conclusions: Thus, we delineate a novel stress signaling cascade in glioma cells involving TGF-β, HIF-1α and CXCL12. Stress stimuli can be temozolomide, irradiation and hypoxia but not hyperthermia. These data suggest that the use of HPC as cellular vectors in the treatment of glioblastoma may well be combined with anti-angiogenic therapies which induce tumor hypoxia. [Table: see text]

Advance in Antitumor Agents Targeting Glutathione-S-Transferase

Glutathione-S-transferases (GSTs; EC 2.5.1.18), a family of phase II detoxification enzymes, catalyze the conjugation of glutathione with broad substrates including chemotherapeutic agents, and are involved in cell protection against apoptotic signals by inhibiting the stress-signaling cascade mediated by ASK1 (Apoptosis signal-regulating kinase)-JNK (c-Jun N-terminal kinase). As GSTs are overexpressed in some malignant tumors, GSTs is a promising therapeutic target. This article reviews the progress in the development of GSTs inhibitors and GSTs activated prodrugs.

Purinergic receptor antagonists inhibit odorant‐induced heat shock protein 25 induction in mouse olfactory epithelium

Heat shock proteins (HSPs) accumulate in cells exposed to a variety of physiological and environmental factors, such as heat shock, oxidative stress, toxicants, and odorants. Ischemic, stressed, and injured cells release ATP in large amounts. Our hypothesis is that noxious stimulation (in this case, strong odorant) evokes the release of ATP in the olfactory epithelium (OE). Extracellular ATP, a signal of cellular stress, induces the expression of HSPs via purinergic receptors. In the present study, in vivo odorant exposure (heptanal or R-carvone) led to a selective induction of HSP25 in glia-like sustentacular cells in the Swiss Webster mouse OE, as previously shown in rats (Carr et al., 2001). Furthermore, in vitro and in vivo administration of purinergic receptor antagonists suramin and pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) blocked the expression of HSP25 immunoreactivity in sustentacular cells. ATP released by acutely injured cells could act as an early signal of cell and tissue damage, causing HSP expression and initiating a stress signaling cascade to protect against further damage. Sustentacular cells have a high capacity to detoxify xenobiotics and thereby protect the olfactory epithelium from airborne pollutants. Thus, the robust, rapid induction of HSPs in sustentacular cells may help maintain the integrity of the OE during exposure to toxicants.

Activation of stress-responsive mitogen-activated protein kinase pathways in hybrid poplar (Populus trichocarpa x Populus deltoides)

Plant mitogen-activated protein kinase (MAPK) cascades are important amplifying modules that can rapidly transduce stress signals into various appropriate intracellular responses. Several extracellular regulated kinase (ERK)-type MAPKs involved in plant defense signaling have been identified in herbaceous species, but no MAPK cascade has yet been characterized in a tree species. We examined the signal transduction events that lead to activation of defense mechanisms in poplar, a major forest species of economic and ecological importance which is becoming the model tree system for studying stress and adaptation responses. We show that, in poplar cell suspensions and leaf tissue, chitosan, a non-host-specific elicitor, and ozone, a strong oxidant and atmospheric pollutant, induce rapid and transient activation of at least two myelin basic protein (MBP) kinases with apparent molecular masses of 44 and 47 kD. The chitosan- and ozone-activated kinases have characteristics of MAPKs-they preferentially phosphorylate MBP, require tyrosine and threonine phosphorylation to be activated and are specifically recognized by anti-ERK and anti-pERK antibodies. Moreover, activation of these poplar MAPKs by chitosan or ozone is dependent on the production of reactive oxygen species; the influx of calcium ions via membrane channels; the activation of an upstream, membrane-localized component; and a cognate MAPK kinase (MAPKK). These data suggest that biotic and abiotic challenges activate MAPKs in poplar, as in herbaceous species, which then function as a convergence point for pathogen defense and oxidant stress signaling cascades.

Mitogen‐ and stress‐activated protein kinase 1: Convergence of the ERK and p38 pathways in Alzheimer's disease

Two of the earliest manifestations of the selective neurodegeneration that occurs in Alzheimer's disease (AD) involve the oxidative modification of various biomacromolecules and the reexpression of a multitude of cell cycle-related proteins. Taken together with the proximal and ectopic increases in activated components of the ERK and p38 pathways, involved in mitotic and cellular stress signaling, respectively, there is a clear and important role for mitotic and oxidative insults in the pathogenesis of AD. Despite the mounting evidence, however, for the causal role of mitogenic abnormalities and oxidative stress in AD pathogenesis, the effect of the converging relevant pathways due to chronic stimulation in AD remains largely unknown. To delineate further the mechanism by which mitogenic and stress signaling cascades converge, we focused on one of the downstream effectors of activated ERK and p38, mitogen- and stress-activated kinase 1 (MSK1). Activated MSK1, phosphorylated at residues Ser376 and Thr581, was upregulated in vulnerable neurons in AD when compared to that in age-matched controls, whereas MSK1 phosphorylated at residue Ser360 was not increased in AD. Furthermore, activated MSK1 phosphorylated at Thr581 colocalized strongly with activated p38 but only weakly with activated ERK, whereas MSK1 phosphorylated at Ser376 colocalized strongly with activated ERK but only weakly with activated p38, suggesting potential preferential phosphorylation sites for the two upstream effectors.

Amyloid Aβ1−40preconditions non‐apoptotic signalsin vivoand protects fetal rat brain from intrauterine ischemic stress

Abstract The dualistic activities of the amyloid beta (Abeta) peptide as a pro-oxidant and ubiquitous constituent of amyloid deposits in Alzheimer's disease plaques and as an antioxidant of purported physiological function has been suggested but the mechanisms are far from being understood. In this report we measure several oxidative stress parameters and signaling cascades in brains of fetal rats subjected to global ischemia in order to evaluate the putative bifunctional properties of the Abeta(1-40) peptide. Intraperitoneal injection of 6 microg Abeta(1-40) into 18-days-old rat fetuses (approximately 3 g body weight) resulted after 24 h in the appearance of the peptide in various fetal organs including brain where it enhanced the levels of glutathione (GSH), glutathione reductase, glutathione peroxidase, and stimulated the levels of pro-survival signaling activities such as Akt serine/threonine kinase, extracellular signal-regulated kinase (ERK) and protein kinase C enzymes. Moreover, pretreatment with Abeta(1-40) reversed the consequences of a transient hypovolemic/hypotensive oxidative stress by restoring GSH levels via its recycling enzymes and by lowering the production of lipid peroxides presumably by activating the aforementioned pro-survival signaling cascades. It also caused a reduction in the number of DAPI-enhanced reactive cells and a decrease in p38 kinase phosphorylation and caspase-9 and -3 activity. These data suggest that pre-exposure to Abeta(1-40) stimulates fetal tolerance to ischemia via regulation of GSH metabolism and as such may be considered as neuroprotective.

ABA- and cADPR-mediated effects on respiration and filtration downstream of the temperature-signaling cascade in sponges.

Recently, the thermosensing pathway in sponges (Porifera) was elucidated. The thermosensor triggering this cascade is a heat-activated cation channel, with the phytohormone abscisic acid (ABA), cyclic ADP-ribose (cADPR) and calcium acting as intracellular messengers, similarly to the drought-stress signaling cascade in higher plants. Here, we investigated the functional effects downstream of the temperature-signaling pathway in Axinella polypoides (Porifera, Demonspongiae). Short-term stimulation followed by long-term depression of amino acid incorporation, oxygen consumption and water filtration were observed after exposure of the sponge to a brief heat stress or to micromolar ABA. These effects could be prevented by the targeted interruption of the signaling pathway either at the level of the cation channel thermosensor or at the level of the cADPR-induced intracellular calcium increase. Moreover, release of cyclase activity into the sea water and generation of extracellular cADPR were observed following brief heat stress. Intact sponge cells were sensitive to extracellular cADPR and addition of purified cyclase increased sponge respiration similarly to heat stress. This is the first observation of functional effects exerted on Metazoa by the phytohormone ABA: conservation of the ABA/cADPR stress-signaling cascade points to its early evolution in a common precursor of modern Metazoa and Metaphyta. The functional effects induced by extracellular cyclase/cADPR suggest an evolutionary origin of cADPR as an ancient stress hormone in Porifera.

Stress-induced decrease in TRAF2 stability is mediated by Siah2.

TRAF2 serves as a central regulator of the cellular response to stress and cytokines through the regulation of key stress-signaling cascades. Here we demonstrate that wild-type, but not RING mutant, Siah2 targets TRAF2 for ubiquitylation and degradation in vitro. Siah2 mediates equally efficient ubiquitylation of RING mutant TRAF2. In vivo, Siah2 primarily targets TRAF2 for degradation under stress conditions. Tumor necrosis factor-alpha (TNF-alpha) and actinomycin D treatment results in accelerated TRAF2 degradation in wild-type mouse embryo fibroblasts (MEFs), as compared with Siah2(-/-) cells. Similarly, TRAF2 half-life is prolonged in Siah2(-/-) compared with wild-type MEFs subjected to stress stimuli. Siah2 efficiently decreases TNF-alpha-dependent induction of JNK activity and transcriptional activation of NF-kappaB. Apoptosis induced by TNF-alpha and actinomycin D treatment is increased upon expression of Siah2, or attenuated upon expression of TRAF2 or RING mutant Siah2. Identifying Siah2 as a regulator of TRAF2 stability reveals its role in the regulation of TRAF2 signaling following exposure to stress.

P52rIPK regulates the molecular cochaperone P58IPK to mediate control of the RNA-dependent protein kinase in response to cytoplasmic stress.

The 52 kDa protein referred to as P52(rIPK) was first identified as a regulator of P58(IPK), a cellular inhibitor of the RNA-dependent protein kinase (PKR). P52(rIPK) and P58(IPK) each possess structural domains implicated in stress signaling, including the charged domain of P52(rIPK) and the tetratricopeptide repeat (TPR) and DnaJ domains of P58(IPK). The P52(rIPK) charged domain exhibits homology to the charged domains of Hsp90, including the Hsp90 geldanamycin-binding domain. Here we present an in-depth analysis of P52(rIPK) function and expression, which first revealed that the 114 amino acid charged domain was necessary and sufficient for interaction with P58(IPK). This domain bound specifically to P58(IPK) TPR domain 7, the domain adjacent to the TPR motif required for P58(IPK) interaction with PKR, thus providing a mechanism for P52(rIPK) inhibition of P58(IPK) function. Both the charged domain of P52(rIPK) and the TPR 7 domain of P58(IPK) were required for P52(rIPK) to mediate downstream control of PKR activity, eIF2alpha phosphorylation, and cell growth. Furthermore, we found that P52(rIPK) and P58(IPK) formed a stable intracellular complex during the acute response to cytoplasmic stress induced by a variety of stimuli. We propose a model in which the P52(rIPK) charged domain functions as a TPR-specific signaling motif to directly regulate P58(IPK) within a larger cytoplasmic stress signaling cascade culminating in the control of PKR activity and cellular mRNA translation.

ROS, stress‐activated kinases and stress signaling in cancer

Anticancer therapy is frequently efficient in early stages of the disease, whereas advanced tumors are usually resistant to the same treatments. The molecular basis for this change is not entirely understood. Many anticancer agents are DNA- or cytoskeleton-damaging drugs that show some specificity towards dividing cells. However, recent studies show that these agents also activate stress-signaling cascades that may play a role in eliciting the observed therapeutic effects. We discuss recent findings that suggest that induction of stress signaling in oncogenically transformed cells is integrated into apoptotic pathways. Reactive oxygen species (ROS) and stress-activated protein kinases (SAPKs), which are potentiated in recently transformed cells, emerge as key effectors of cell death. In advanced tumors, however, these agents are downregulated and, consequently, death signaling is suppressed. Such changes in ROS and SAPK activity levels during the course of tumor development may underlie the changes in responsiveness to anticancer therapy.

Proteolytic Cleavage of Molecules Involved in Cell Death or Survival Pathways: A Role in the Control of Apoptosis?

Proteolytic modification of certain key regulatory molecules involved in apoptotic and prosurvival pathways may be a feature of the control of programmed cell death. Four molecules of the Bd-2 family (BID, Bcl-2, Bax, Bcl-X(L)) have been reported to be deaved during apoptosis, as has a cellular inhibitor of apoptosis (XIAP). Two proteins involved in NF-kappaB activation, RIP and TRAF1, are cleaved during apoptosis induced by agents that activate both pathways. MEKK1, a molecule involved in a protein kinase stress signaling cascade that contributes to apoptosis and NF-kappaB activation, also undergoes cleavage. In each case, the cleavage products may result in the inactivation of a former function or the gaining of a new function, thus contributing to the delicately balanced regulation of apoptosis.

CEP‐1347 (KT7515), an Inhibitor of JNK Activation, Rescues Sympathetic Neurons and Neuronally Differentiated PC12 Cells from Death Evoked by three Distinct Insults

The c-Jun N-terminal kinase signaling cascade appears to play a role in some cases of cell death, including neuronal apoptosis. CEP-1347 (KT7515), an indolocarbazole of the K252a family, blocks this stress signaling cascade and promotes survival. Here, we used CEP-1347 to probe whether neuronal death pathways activated by distinct insults also possess elements in common. Cultured rat sympathetic neurons and neuronally differentiated PC12 cells were induced to die by withdrawal of nerve growth factor, exposure to ultraviolet irradiation, or subjection to oxidative stress. In each case, death was prevented by 100-200 nM CEP-1347. Moreover, in each of these death paradigms, c-Jun N-terminal kinase 1 activity in neuronally differentiated PC12 cells was elevated by two- or threefold, and this increase was totally blocked by CEP-1347 at concentrations that promoted survival. In contrast, 200 nM CEP-1347 did not block death due to serum withdrawal from undifferentiated PC12 cells or to activation of Fas in Jurkat T cell cultures, even though in each case c-Jun N-terminal kinase 1 activation occurred and was inhibited by CEP-1347. These observations suggest that some but not all death pathways triggered by different insults can include a common mechanistic component, a likely candidate for which is activation of the c-Jun N-terminal kinase signaling cascade.

Gas6-mediated survival in NIH3T3 cells activates stress signalling cascade and is independent of Ras.

Gas6 is a growth factor membrane of the vitamin K-dependent family of proteins which is preferentially expressed in quiescent cells. Gas6 was identified as the ligand for Axl tyrosine kinase receptor family. Consistent with this, Gas6 was previously reported to induce cell cycle re-entry of serum-starved NIH3T3 cells and to prevent cell death after complete growth factor withdrawal, the survival effect being uncoupled from Gas6-induced mitogenesis. We have previously demonstrated that both Gas6 mitogenic and survival effects are mediated by Src and the phosphatidylinositol3-OH kinase (PI3K). Here we report that Ras is required for Gas6 mitogenesis but is dispensable for its survival effect. Gas6-induced survival requires the activity of the small GTPases of the Rho family, Rac and Rho, together with the downstream kinase Pak. Overexpression of the respective dominant negative constructs abrogates Gas6-mediated survival functions. Addition of Gas6 to serum starved cells results in the activation of AKT/PKB and in the phosphorylation of the Bcl-2 family member, Bad. By ectopic expression of a catalytically inactive form of AKT/PKB, we demonstrate that AKT/PKB is necessary for Gas6-mediated survival functions. We further show evidence that Gas6 stimulation of serum starved NIH3T3 cells results in a transient ERK, JNK/SAPK and p38 MAPK activation. Blocking ERK activation did not influence Gas6-induced survival, suggesting that such pathway is not involved in Gas6 protection from cell death. On the contrary we found that the late constitutive increase of p38 MAPK activity associated with cell death was downregulated in Gas6-treated NIH3T3 cells thus suggesting that Gas6 might promote survival by interfering with this pathway. Taken together the evidence here provided identity elements involved in Gas6 signalling more specifically elucidating the pathway responsible for Gas6-induced cell survival under conditions that do not allow cell proliferation.

JNK and p53 stress signaling cascades are altered in MCF-7 cells resistant to tumor necrosis factor-mediated apoptosis.

Tumor necrosis factor (TNF) signal transduction is a complex process involving activation of receptor-linked and stress-sensitive signaling cascades that stimulate apoptosis in some tumor cell lines. Initial studies suggested that these signaling events cooperatively induced TNF responses, but recent studies suggest that some of these signals antagonize the apoptotic response or play no discernible role in cell death. As TNF induces cellular stress and activates several stress-sensitive cascades that may play a role in apoptosis, TNF-induced stress signaling was examined in MCF-7 cells and compared with a variant MCF-7 cell line resistant to TNF-mediated apoptosis (MCF-7/3E9). TNF rapidly stimulated both NF-kappaB and JNK activation in MCF-7 and MCF-7/3E9 cells, but JNK activation was significantly reduced (threefold) in apoptotically resistant cells. TNF also stimulated p53, p21WAF1, and Bax accumulation with subsequent PARP cleavage and nucleosomal DNA laddering in MCF-7 cells but did not stimulate these processes in MCF-7/3E9 cells. Importantly, 3E9 cells retained wild-type p53 function, induced p21WAF1 in response to DNA damage, and expressed almost equal sensitivity to other stress stimuli (gamma-radiation, chemotherapeutic agents) as parental MCF-7 cells. These results suggest that selective defects in TNF-activated stress cascades are associated with reduced sensitivity to TNF but not other cell death stimuli. Loss of potent TNF-mediated activation of JNK and p53 cascades may permit tumor cells to evade receptor-mediated apoptosis but have only limited influence on cellular sensitivity to other agents that effectively engage these stress pathways.