Activation Of Stress Response Pathways Research Articles

286: Protein kinase Cδ mediated-oxidative stress in diabetic embryopathy

Embryonic malformation in diabetic pregnancy, including neural tube defects, are associated with oxidative stress and activation of stress-response pathways including the c-Jun N-terminal kinase (JNK), Akt, and Toll Like Receptor 4 (TLR4) pathways. Protein kinase C (PKC) family members, including PKCδ, have been implicated in diabetic embryopathy.

The Hypomethylating Agent 5-Azacytidine Can Target Leukaemic Stem and Progenitor Cells In Acute Myeloid Leukemia (AML) by Inducing DNA Damage and Stress Response Pathways

AbstractAbstract 292Acute Myeloid Leukemia (AML) is a heterogeneous disease that arises from malignant leukaemic stem and progenitor cells (LSPC). LSPC are relatively resistant to current chemotherapy and considered to contribute to disease progression and relapse in patients due to their ability to re-initiate long-term leukaemic cell growth following initial treatment. We have previously shown that CD34+CD38−CD123+ LSPC are less sensitive to cytarabine and to the FLT3 inhibitor AG1296 whilst exhibiting differential sensitivity to Mylotarg (Gemtuzumab Ozogamicin) when grown under niche support, compared to LSPC with no niche support. As the hypomethylating agent and nucleoside analog 5-Azacytidine (Aza) is showing promise clinically in AML and MDS patients with effects, including induction of ATM, seen as early as 5 days post chemotherapy, we assessed this agent in our model. LSPC from AML samples treated for 72hrs with Aza (1microM) were found to be relatively cytotoxic, resulting in 33% cell kill (n=15) compared to 15% AML bulk cell kill (n=19; p=0.031). We used a protein profiler assay to depict the expression of 46 known human phosphokinase proteins when treated with Aza using the CD34+CD38− TF1A cell line. Two phosphokinase proteins were highly activated upon Aza treatment and these were the cell cycle checkpoint regulator and putative tumor suppressor Chk2 and the stress-induced p38 mitogen-activated protein kinase (p38MAPK). Drug-induced DNA damaged cells are known to stall the cell cycle to allow DNA repair by activating the traditional ATM/Chk2/ATR/Chk1 pathway and the more recently described ATM/ATR/p38MAPK/MK2 checkpoint pathway, known to be essential in p53 deficient cells. Thus, we next aimed to examine the DNA damage and stress pathways activated by Aza treatment. Using several AML cell lines, including the CD34+CD38− KG1A and TF1A cells, we demonstrate that Aza treatment eliminates S-phase cells while inducing massive DNA damage response (increase in gH2AX), leading to the activation of the ATM/Chk2 and Chk1 and thus triggering G2/M cell cycle arrest. To date 3 primary AML samples have been treated in vitro with Aza and downstream effects analysed: only 1/3 was sensitive to Aza and this was the only sample that showed an increase in ATM and Chk2 in response to the Aza treatment. As we wanted to investigate whether activation of the stress response pathway encourages any additional cell survival, we pre-treated the same cell lines with the p38MAPK inhibitor SB203580 for 30minutes prior to Aza treatment. Our data shows that there is enhanced cell kill if the p38MAPK pathway is inhibited first and then Aza treated later. We are currently in the process of assessing induction of downstream effector proteins involved in the p38MAPK pathways after Aza treatment. In conclusion, the success of Aza treatment in AML is maybe due to the specific targeting of the relapse-causing LSPCs that is achieved by the activation of DNA damage and stress response pathways leading to G2/M cell cycle arrest. We are currently assessing the efficacy of Aza in in vivo animal models and investigating any dysregulated methylation patterns in Chk2 and p38MAPK in AML cells. Disclosures:No relevant conflicts of interest to declare.

Androgens modulate autophagy and cell death via regulation of the endoplasmic reticulum chaperone glucose-regulated protein 78/BiP in prostate cancer cells

Pro-survival signalling mediated by the androgen receptor (AR) is implicated as a key contributor to prostate carcinogenesis. As prostate tumours are characterized by nutrient-poor, hypoxic and acidified microenvironments, one mechanism whereby AR signalling may contribute to survival is by promoting adaptation to cellular stress. Here we have identified a novel role for AR in the inhibition of autophagy induced by serum withdrawal. This blockade is attributed to AR-mediated upregulation of the endoplasmic reticulum (ER) chaperone glucose-regulated protein 78/BiP (Grp78/BiP), and occurs independently of ER stress response pathway activation. Interestingly, AR activation did not affect serum starvation-induced mammalian target of rapamycin inhibition, illustrating that the adaptive role for androgens lies not in the ability to modulate nutrient sensing, but in the promotion of ER stability. Finally, we show that the adaptive advantage conferred by AR-mediated Grp78/BiP upregulation is temporary, as upon chronic serum starvation, AR activation delayed but did not suppress the onset of autophagy and cell death. This study reveals a novel mechanism whereby maintained AR signalling promotes temporary adaptation to cellular stress and in turn may contribute to the evasion of prostate tumour cell death.

Açai palm fruit ( Euterpe oleracea Mart.) pulp improves survival of flies on a high fat diet

Reducing oxidative damage is thought to be an effective aging intervention. Açai, a fruit indigenous to the Amazon, is rich in phytochemicals that possesses high anti-oxidant activities, and has anti-inflammatory, anti-cancer and anti-cardiovascular disease properties. However, little is known about its potential anti-aging properties especially at the organismal level. Here we evaluated the effect of açai pulp on modulating lifespan in Drosophila melanogaster. We found that açai supplementation at 2% in the food increased the lifespan of female flies fed a high fat diet compared to the non-supplemented control. We measured transcript changes induced by açai for age-related genes. Although transcript levels of most genes tested were not altered, açai increased the transcript level of l(2)efl, a small heat-shock-related protein, and two detoxification genes, GstD1 and MtnA, while decreasing the transcript level of phosphoenolpyruvate carboxykinase ( Pepck), a key gene involved in gluconeogenesis. Furthermore, açai increased the lifespan of oxidative stressed females caused by sod1 RNAi. This suggests that açai improves survival of flies fed a high fat diet through activation of stress response pathways and suppression of Pepck expression. Açai has the potential to antagonize the detrimental effect of fat in the diet and alleviate oxidative stress in aging.

The MAP kinase-activated protein kinase 2 (MK2) contributes to the Shiga toxin-induced inflammatory response

Infection with Shiga toxin (STx)-producing bacteria can progress to a toxemic, extraintestinal injury cascade known as haemolytic uremic syndrome (HUS), the leading cause of acute renal failure in children. Mounting evidence suggests that STx activates stress response pathways in susceptible cells and has implicated the p38 mitogen-activated protein kinase (MAPK) pathway. More importantly, some of the pathology associated with HUS is believed to be a result of a STx-induced inflammatory response. From a siRNA screen of the human kinome adapted to a high-throughput format, we found that knock-down of the MAPK-activated protein kinase 2 (MK2), a downstream target of the p38 MAPK, protected against Shiga toxicity. Further characterization of the in vitro role of MK2 revealed that STx activates the p38-MK2 stress response pathway in both p38- and MK2-dependent manners in two distinct cell lines. MK2 activation was specific to damage to the ribosome by an enzymatically active toxin and did not result from translational inhibition per se. Genetic and chemical inhibition of MK2 significantly decreased the inflammatory response to STx. These findings suggest that MK2 inhibition might play a valuable role in decreasing the immuopathological component of STx-mediated disease.

Glycinebetaine‐induced water‐stress tolerance in codA‐expressing transgenic indica rice is associated with up‐regulation of several stress responsive genes

Rice (Oryza sativa L.), a non-accumulator of glycinebetaine (GB), is highly susceptible to abiotic stress. Transgenic rice with chloroplast-targeted choline oxidase encoded by the codA gene from Arthrobacter globiformis has been evaluated for inheritance of transgene up to R5 generation and water-stress tolerance. During seedling, vegetative and reproductive stages, transgenic plants could maintain higher activity of photosystem II and they show better physiological performance, for example, enhanced detoxification of reactive oxygen species compared to wild-type plants under water-stress. Survival rate and agronomic performance of transgenic plants is also better than wild-type following prolonged water-stress. Choline oxidase converts choline into GB and H2O2 in a single step. It is possible that H2O2/GB might activate stress response pathways and prepare transgenic plants to mitigate stress. To check this possibility, microarray-based transcriptome analysis of transgenic rice has been done. It unravelled altered expression of many genes involved in stress responses, signal transduction, gene regulation, hormone signalling and cellular metabolism. Overall, 165 genes show more than two-fold up-regulation at P-value < 0.01 in transgenic rice. Out of these, at least 50 genes are known to be involved in plant stress response. Exogenous application of H2O2 or GB to wild-type plants also induces such genes. Our data show that metabolic engineering for GB is a promising strategy for introducing stress tolerance in crop plants and which could be imparted, in part, by H2O2- and/or GB-induced stress response genes.

Synthesis and Anticancer Properties of Water-Soluble Zinc Ionophores

Several water-solubilized versions of the zinc ionophore 1-hydroxypyridine-2-thione (ZnHPT), synthesized as part of the present study, have been found both to increase the intracellular concentrations of free zinc and to produce an antiproliferative activity in exponential phase A549 human lung cancer cultures. Gene expression profiles of A549 cultures treated with one of these water-soluble zinc ionophores, PCI-5002, reveal the activation of stress response pathways under the control of metal-responsive transcription factor 1 (MTF-1), hypoxia-inducible transcription factor 1 (HIF-1), and heat shock transcription factors. Additional oxidative stress response and apoptotic pathways were activated in cultures grown in zinc-supplemented media. We also show that these water-soluble zinc ionophores can be given to mice at 100 micromol/kg (300 micromol/m(2)) with no observable toxicity and inhibit the growth of A549 lung and PC3 prostate cancer cells grown in xenograft models. Gene expression profiles of tumor specimens harvested from mice 4 h after treatment confirmed the in vivo activation of MTF-1-responsive genes. Overall, we propose that water-solubilized zinc ionophores represent a potential new class of anticancer agents.

Hos2p/Set3p Deacetylase Complex Signals Secretory Stress through the Mpk1p Cell Integrity Pathway

Perturbations in secretory function activate stress response pathways critical for yeast survival. Here we report the identification of the Hos2p/Set3p deacetylase complex (SET3C) as an essential component of the secretory stress response. Strains lacking core components of the Hos2p/Set3p complex exhibit hypersensitivity to secretory stress. Although not required for the unfolded protein response (UPR) and ribosomal gene repression, the Hos2p complex is required for proper activation of the Mpk1p/Slt2p cell integrity kinase cascade. Disruption of the Hos2p complex results in abrogated Mpk1p phosphorylation, whereas constitutive activation of the Mpk1p pathway rescues the hos2Delta mutant growth defect in response to secretory stress. Furthermore, Hos2p activity is required for the Mpk1p-mediated activation of stress-responsive transcription factor Rlm1p, but not for the stress-induced degradation of the C-type cyclin Ssn8p. Our results identify the Hos2p complex as a critical component of the secretory stress response and support the existence a coordinated stress response consisting of the UPR, ribosomal gene repression, and mitogen-activated protein kinase signaling in response to defects in secretory function.

Aberrant activation of stress-response pathways leads to TNF-α oversecretion in Fanconi anemia

Fanconi anemia (FA), an inherited syndrome that associates bone marrow failure, cancer predisposition, and genetic instability, is characterized by an overproduction of the myelosuppressive cytokine TNF-alpha through unknown mechanisms. We demonstrate here that FANC pathway loss-of-function results in the aberrant activation of 2 major stress-signaling pathways: NF-kappaB and MAPKs. These responses are independent on TNF-alpha expression. On the contrary, inhibition of the MAPK pathways normalizes TNF-alpha oversecretion in FA. Moreover, our data show that the overexpression of the matrix metalloproteinase MMP-7 is the key event directly responsible for the high rate of TNF-alpha shedding and release from the cytoplasmic membrane in FA. TNF-alpha overproduction is, indeed, normalized by MMP-7 inhibition. Finally, MAPK inhibition impacts on MMP-7 overexpression. Evidence is provided of the existence of a linear pathway in which FANC mutations activate MAPK signaling that induces MMP-7 overexpression leading, in fine, to TNF-alpha oversecretion. TNF-alpha may, in turn, sustain or amplify both MAPKs and NF-kappaB activation. Aberrant expression or activity of NF-kappaB and/or MAPKs has been already involved in bone marrow failure and leukemia, and their inhibition offered clinical benefit for patients. In conclusion, our data provide a strong rationale for new clinical trials on FA patients.

Heat Shock Factor 1 Attenuates 4-Hydroxynonenal-mediated Apoptosis: CRITICAL ROLE FOR HEAT SHOCK PROTEIN 70 INDUCTION AND STABILIZATION OF Bcl-XL

Lipid peroxidation is a consequence of both normal physiology and oxidative stress that generates various reactive metabolites, a principal end product being 4-hydroxynonenal (HNE). As a diffusible electrophile, HNE reacts extensively with cellular nucleophiles. Consequently, HNE alters cellular signaling and activates the intrinsic apoptotic cascade. We have previously demonstrated that in addition to promoting apoptosis, HNE activates stress response pathways, including the antioxidant, endoplasmic reticulum stress, DNA damage, and heat shock responses. Here we demonstrate that activation of the heat shock response by HNE is dependent on the expression and nuclear translocation of heat shock factor 1 (HSF1), which promotes the expression of heat shock protein 40 (Hsp40) and Hsp70-1. Ectopic expression and immunoprecipitation of c-Myc-tagged Hsp70-1 indicates that HNE disrupts the inhibitory interaction between Hsp70-1 and HSF1, leading to the activation heat shock gene expression. Using siRNA to silence HSF1 expression, we observe that HSF1 is necessary for the induction of Hsp40 and Hsp70-1 by HNE, and the lack of Hsp expression is correlated with an increase in apoptosis. Nrf2, the transcription factor that mediates the antioxidant response, was also silenced using siRNA. Silencing Nrf2 also enhanced the cytotoxicity of HNE, but not as effectively as HSF1. Silencing HSF1 expression facilitates the activation of JNK pro-apoptotic signaling and selectively decreases expression of the anti-apoptotic Bcl-2 family member Bcl-X(L). Overexpression of Bcl-X(L) attenuates HNE-mediated apoptosis in HSF1-silenced cells. Overall, activation of HSF1 and stabilization of Bcl-X(L) mediate a protective response that may contribute significantly to the cellular biology of lipid peroxidation.

The growth inhibitory effect of actein on human breast cancer cells is associated with activation of stress response pathways

Previous studies indicate that the triterpene glycoside actein from the herb black cohosh inhibits growth of human breast cancer cells. This study seeks to identify genes altered in human breast cancer cells by treatment with actein, using gene expression analysis. We treated MDA-MB-453 human breast cancer cells with actein at 2 doses, 20 or 40 microg/mL, for 6 or 24 hr. We identified 5 genes that were activated after each of the treatments that are known to play a role in cellular responses to diverse stresses, including the DNA damage and unfolded protein responses. In addition, four genes that mediate the integrated stress response (ISR), including activating transcription factor 4, were induced under at least one of the 4 treatment conditions. We used hierarchical clustering to define clusters comprising patterns of gene expression. Two ISR genes, activating transcription factor 3 (ATF3) and DNA damage- inducible transcript 3, and lipid biosynthetic genes were activated after exposure to actein at 40 microg/mL for 6 hr, whereas the cell cycle genes cyclin E2 and cell division cycle 25A were repressed. Our results suggest that actein induces 2 phases of the ISR, the survival phase and the apoptotic phase, depending on the dose and duration of treatment. We confirmed the results of gene expression analysis with real-time RT-PCR for 18 selected genes and Western blot analysis for ATF3. Since actein activated transcription factors that enhance apoptosis, and repressed cell cycle genes, it may be useful in the prevention and therapy of breast cancer.

A proteome study of the proliferation of cultured Medicago truncatula protoplasts

A proteome study of the first five days of Medicago truncatula protoplast cultures was done to investigate molecular changes taking place during protoplast proliferation. A total of 1556 protein spots were analysed, of which 886 protein spots showed significant (p<0.005) changes in abundance at some time during the first five days of protoplast culture. Of the 886 significantly changing protein spots, 89 proteins were identified by MALDI-TOF MS. The majority of the identified proteins were part of four main cellular processes that may be involved in protoplast proliferation: energy metabolism, defence or stress response, secondary metabolism and protein synthesis and folding. The accumulation pattern of these proteins indicates extensive changes in the energy metabolism of the cells, accompanied by the activation of stress response pathways and modifications of the cell wall. In addition, seven PR10-like (pathogenesis related) proteins were identified. The accumulation pattern of these seven PR10-like proteins suggests that they could have a developmental role during protoplast proliferation.

Activated stress response pathways within multicellular aggregates utilize an autocrine component

Multicellular aggregates (spheroids) of primary human foreskin fibroblasts (HFF-2) and a glioblastoma cell line (T98G) entered and exited from long term (2 weeks) metabolic arrest utilizing an autocrine response. Cytokine production (specifically IFN-γ) activated a Gadd45α/p38 pathway that led to increased AP-1 (c- jun and ATF3) transcription factor levels, augmenting cytokine production in an autocrine fashion. Whereas HFF-2 aggregates were capable of surviving long term arrest and recovery during NF-κB inhibition independent of JNK activation, T98G aggregates were not. Such endogenous processes are not easily observed with adherent monolayer cell culturing systems, strongly suggesting that more emphasis needs to be placed on determining the operational signal transduction cascades within multicellular aggregates. Extracellular inputs such as spheroid formation, arrest, and regrowth as monolayers invoke intracellular signaling responses converging at the AP-1 transcription factor level. Variations in responses are both cell type and transformation state dependent and require an autocrine cytokine component. The data are discussed in relation to the wounding response and avascular tumor growth mechanisms.

Activation of MAP kinases by hexavalent chromium, manganese and nickel in human lung epithelial cells

Epidemiological studies indicate that workers who perform welding operations are at increased risk for bronchitis, siderosis, occupational asthma and lung cancer due to fume exposure. Welding fumes are a complex chemical mixture, and the metal composition is hypothesized to be an etiological factor in respiratory disease due to this exposure. In the present study, human lung epithelial cells in vitro responded to hexavalent chromium, manganese and nickel over a concentration range of 0.2–200 μM with a significant increase in intracellular phosphoprotein (a measure of stress response pathway activation). The mitogen-activated protein kinases ERK1/2, SAPK/JNK and p38 were activated via phosphorylation following 1-h exposures. Hexavalent chromium up-regulated p-38 phosphorylation 23-fold and SAPK/JNK phosphorylation 17-fold, with a comparatively modest 4-fold increase in ERK1/2 phosphorylation. Manganese caused a two- to four-fold increase in SAPK/JNK and ERK 1/2 phosphorylation, with no observed effects on p38 kinase. Nickel caused increased (two-fold) phosphorylation of ERK 1/2 only, and was not cytotoxic over the tested concentration range. The observed effects of welding fume metals on cellular signaling in lung epithelium demonstrate a potentially significant interplay between stress-response signaling (p38 and SAPK/JNK) and anti-apototic signaling (ERK1/2) that is dependant on the specific metal or combination of metals involved.

Viewing a Stressful Episode of ER

See related article, pages 1186–1193 How cardiac cells sense, respond, and adapt to acute and chronic changes of their metabolic and environmental milieu remain among the most enigmatic and fundamental questions in contemporary cardiovascular biology and medicine. With more than 700 000 deaths and 6 million hospital discharges annually in the U.S., ischemic heart disease remains a major public health problem. If prompt therapy is delayed, myocardial injury from depletion of high-energy phosphates from inhibition of oxidative phosphorylation is inevitable. Ischemia/reperfusion (I/R) also unleashes a cascade of cellular and molecular events whose sequalae may sustain organ function at diminished capacity but, beyond a critical balance, threatens the organism’s survival. Reactive oxygen and nitrogen species (ROS; RNS), released from the mitochondria and other sources, alter the tertiary and quaternary structures of proteins, exposing their hydrophobic residues to allow conformational tendencies toward protein misfolding and aggregation.1 Nevertheless, highly sophisticated schemes equipped with molecular sensors, rapid response pathways, and adaptor mechanisms have evolved to mitigate the accumulation of unfolded proteins in a compartment specific manner, principally the cytoplasm and endoplasmic reticulum (ER). Although the cardiovascular field has paid considerably more attention to the cytoprotective mechanisms of heat shock proteins in response to I/R, termed the “classical” heat shock response, a parallel series of events has been unfolding at a breathtaking pace in the ER, with arguably considerable excitement to warrant a sneak preview of the article published in this issue of Circulation Research . To place into perspective the current buzz being garnered by the ER requires a brief discussion of more familiar themes in the field, which were developed thanks to the pioneering efforts of cardiovascular scientists, and others, working on the complementary stress response network, best exemplified by heat shock “stress” proteins and their transcriptional regulators. First discovered by …

Peroxynitrite: just an oxidative/nitrosative stressor or a physiological regulator as well?

British Journal of Pharmacology (2006) 148, 1–3. doi:10.1038/sj.bjp.0706693

The Golgi Ca2+-ATPase KlPmr1p Function Is Required for Oxidative Stress Response by Controlling the Expression of the Heat-Shock ElementHSP60inKluyveromyces lactis

The Golgi P-type Ca2+-ATPase, Pmr1p, is the major player for calcium homeostasis in yeast. The inactivation of KlPMR1 in Kluyveromyces lactis leads to high pleiotropic phenotypes that include reduced glycosylation, cell wall defects, and alterations of mitochondrial metabolism. In this article we found that cells lacking KlPmr1p have a morphologically altered mitochondrial network and that mitochondria (m) from Klpmr1delta cells accumulate Ca2+ more slowly and reach a lower [Ca2+]m level, when exposed to [Ca2+] < 5 microM, than wild-type cells. The Klpmr1delta cells also exhibit traits of ongoing oxidative stress and present hyperphosphorylation of KlHog1p, the hallmark for the activation of stress response pathways. The mitochondrial chaperone KlHsp60 acts as a multicopy suppressor of phenotypes that occur in cells lacking the Ca2+-ATPase, including relief from oxidative stress and recovery of cell wall thickness and functionality. Inhibition of KlPMR1 function decreases KlHSP60 expression at both mRNA and protein levels. Moreover, KlPRM1 loss of function correlates with both decreases in HSF DNA binding activity and KlHSP60 expression. We suggest a role for KlPMR1 in HSF DNA binding activity, which is required for proper KlHSP60 expression, a key step in oxidative stress response.

Activation of stress-responsive pathways by the sympathetic nervous system in burn trauma.

We have shown previously that bum trauma activates the stress responsive proteins, p38 mitogen-activated protein kinase (MAPK), c-jun NH2-terminal kinase (JNK), and NF-kappaB, and we have shown further that p38 MAPK is an important mediator of cardiomyocyte TNF-alpha secretion and cardiac dysfunction in burn trauma. Since burn trauma causes a rise in circulating catecholamine levels, we hypothesized that this increased sympathetic activity may function as an upstream activator of the p38 MARK pathway in burn trauma. This study determined whether the alpha1-adrenergic receptor ligand phenylephrine could mimic burn trauma activation of p38 MAPK, JNK, and NF-kappaB nuclear translocation; and the effect of the alpha1-adrenergic receptor antagonist prazosin on either phenylephrine or burn-mediated activation of the stress response pathway was examined. Sprague Dawley rats were divided into seven groups: Group 1, controls; Group 2, phenylephrine-treated (2 microg/kg, i.v.) control rats; Group 3, phenylephrine-treated plus prazosin-treated (1 mg/kg, i.v.) control rats; additional rats were given burn over 40% total body surface area (TBSA) and received vehicle (1 mL of 2% sucrose, p.o.) plus fluid resuscitation (Group 4), while in Group 5, burn rats were given prazosin (1 mg/kg, p.o.) plus fluid resuscitation. In Groups 6 and 7, sham-burned rats were given either vehicle (1 mL of 2% sucrose, p.o.) or prazosin (1 mg/kg, p.o.) to provide appropriate controls. Administration of phenylephrine to rats caused a significant activation of cardiac p38 MAPK/JNK activities (Western blot) and cardiac NF-kappaB nuclear translocation (electrophoretic mobility shift assay, EMSA). Prazosin blocked phenylephrine mediated changes in p38 MAPK/JNK activities. Burn trauma activated cardiac p38 MAPK/JNK and NF-kappaB, increased TNF-alpha secretion by cardiomyocytes, and impaired cardiac function. Prazosin treatment in burns interrupted the burn-mediated signaling cascade, decreasing TNF-alpha secretion by cardiomyocytes and preventing post-burn cardiac contractile dysfunction. Thus, burn trauma-related sympathetic activity likely activates the stress-responsive cascade, which regulates myocardial TNF-alpha transcription/translation and culminates in cardiac contraction and relaxation defects.