Activation Of Stress-activated Protein Kinase Research Articles

Drosophila Toll-9 is induced by aging and neurodegeneration to modulate stress signaling and its deficiency exacerbates tau-mediated neurodegeneration

Drosophila Toll-9 is most closely related to mammalian Toll-like receptors; however, physiological functions of Toll-9 remain elusive. We examined the roles of Toll-9 in fly brains in aging and neurodegeneration. Toll-9 mRNA levels were increased in aged fly heads accompanied by activation of nuclear factor-kappa B (NF-kB) and stress-activated protein kinase (SAPK) signaling, and many of these changes were modulated by Toll-9 in glial cells. The loss of Toll-9 did not affect lifespan or brain integrity, whereas it exacerbated hydrogen peroxide-induced lethality. Toll-9 expression was also induced by nerve injury but did not affect acute stress response or glial engulfment activity, suggesting Toll-9 may modulate subsequent neurodegeneration. In a fly tauopathy model, Toll-9 deficiency enhanced neurodegeneration and disease-related tau phosphorylation with reduced SAPK activity, and blocking SAPK enhanced tau phosphorylation and neurodegeneration. In sum, Toll-9 is induced upon aging and nerve injury and affects neurodegeneration by modulating stress kinase signaling.

The HOG pathway and the regulation of osmoadaptive responses in yeast.

Cells coordinate intracellular activities in response to changes in the extracellular environment to maximize their probability of survival and proliferation. Eukaryotic cells need to adapt to constant changes in the osmolarity of their environment. In yeast, the high-osmolarity glycerol (HOG) pathway is responsible for the response to high osmolarity. Activation of the Hog1 stress-activated protein kinase (SAPK) induces a complex program required for cellular adaptation that includes temporary arrest of cell cycle progression, adjustment of transcription and translation patterns, and the regulation of metabolism, including the synthesis and retention of the compatible osmolyte glycerol. Hog1 is a member of the family of p38 SAPKs, which are present across eukaryotes. Many of the properties of the HOG pathway and downstream-regulated proteins are conserved from yeast to mammals. This review addresses the global view of this signaling pathway in yeast, as well as the contribution of Dr Hohmann's group to its understanding.

Dual-Specificity Phosphatase 1 (DUSP1) Has a Central Role in Redox Homeostasis and Inflammation in the Mouse Cochlea.

Stress-activated protein kinases (SAPK) are associated with sensorineural hearing loss (SNHL) of multiple etiologies. Their activity is tightly regulated by dual-specificity phosphatase 1 (DUSP1), whose loss of function leads to sustained SAPK activation. Dusp1 gene knockout in mice accelerates SNHL progression and triggers inflammation, redox imbalance and hair cell (HC) death. To better understand the link between inflammation and redox imbalance, we analyzed the cochlear transcriptome in Dusp1−/− mice. RNA sequencing analysis (GSE176114) indicated that Dusp1−/− cochleae can be defined by a distinct profile of key cellular expression programs, including genes of the inflammatory response and glutathione (GSH) metabolism. To dissociate the two components, we treated Dusp1−/− mice with N-acetylcysteine, and hearing was followed-up longitudinally by auditory brainstem response recordings. A combination of immunofluorescence, Western blotting, enzymatic activity, GSH levels measurements and RT-qPCR techniques were used. N-acetylcysteine treatment delayed the onset of SNHL and mitigated cochlear damage, with fewer TUNEL+ HC and lower numbers of spiral ganglion neurons with p-H2AX foci. N-acetylcysteine not only improved the redox balance in Dusp1−/− mice but also inhibited cytokine production and reduced macrophage recruitment. Our data point to a critical role for DUSP1 in controlling the cross-talk between oxidative stress and inflammation.

Stress-activated protein kinases are involved in the replication of porcine deltacoronavirus

This study was conducted to examine the role of stress-activated protein kinases (SAPKs), including c-Jun NH2-terminal kinases (JNK1/2) and p38 mitogen-activated protein kinase (MAPK), in porcine deltacoronavirus (PDCoV) infection. Results demonstrated the activation of JNK1/2 and p38 MAPK in PDCoV-infected cells, which occurred concomitant with viral biosynthesis and irrespective of cell type. Pharmacological inhibition or knockdown of either SAPK significantly attenuated PDCoV replication, whereas addition of a signaling activator augmented virus infectivity. Moreover, pharmacological inhibition of JNK1/2 or p38 MAPK activation was innocuous to viral entry but significantly detrimental to post uncoating stages of the replication cycle. Remarkably, cytokine gene expression in PDCoV-infected IPEC-J2 cells was modified by inhibiting the activation of either SAPK. Collectively, these data indicate that JNK1/2 and p38 MAPK signaling pathways contribute to viral biosynthesis and regulate immune responses, thereby favoring the replication of PDCoV.

The role of stress kinases in metabolic disease.

Obesity is a health condition that has reached pandemic levels and is implicated in the development and progression of type 2 diabetes mellitus, cancer and heart failure. A key characteristic of obesity is the activation of stress-activated protein kinases (SAPKs), such as the p38 and JNK stress kinases, in several organs, including adipose tissue, liver, skeletal muscle, immune organs and the central nervous system. The correct timing, intensity and duration of SAPK activation contributes to cellular metabolic adaptation. By contrast, uncontrolled SAPK activation has been proposed to contribute to the complications of obesity. The stress kinase signalling pathways have therefore been identified as potential targets for the development of novel therapeutic approaches for metabolic syndrome. The past few decades have seen intense research efforts to determine how these kinases are regulated in a cell-specific manner and to define their contribution to the development of obesity and insulin resistance. Several studies have uncovered new and unexpected functions of the non-classical members of both pathways. Here, we provide an overview of the role of SAPKs in metabolic control and highlight important discoveries in the field.

Ribosome Collisions Trigger General Stress Responses to Regulate Cell Fate

Problems arising during translation of mRNAs lead to ribosome stalling and collisions that trigger a series of quality control events. However, the global cellular response to ribosome collisions has not been explored. Here, we uncover a function for ribosome collisions in signal transduction. Using translation elongation inhibitors and general cellular stress conditions, including amino acid starvation and UV irradiation, we show that ribosome collisions activate the stress-activated protein kinase (SAPK) and GCN2-mediated stress response pathways. We show that the MAPKKK ZAK functions as the sentinel for ribosome collisions and is required for immediate early activation of both SAPK (p38/JNK) and GCN2 signaling pathways. Selective ribosome profiling and biochemistry demonstrate that although ZAK generally associates with elongating ribosomes on polysomal mRNAs, it specifically auto-phosphorylates on the minimal unit of colliding ribosomes, the disome. Together, these results provide molecular insights into how perturbation of translational homeostasis regulates cell fate.

Activation of p38α stress-activated protein kinase drives the formation of the pre-metastatic niche in the lungs.

Primary tumor-derived factors (TDFs) act upon normal cells to generate a pre-metastatic niche, which promotes colonization of target organs by disseminated malignant cells. Here we report that TDFs-induced activation of the p38α kinase in lung fibroblasts plays a critical role in the formation of a pre-metastatic niche in the lungs and subsequent pulmonary metastases. Activation of p38α led to inactivation of type I interferon signaling and stimulation of expression of fibroblast activation protein (FAP). FAP played a key role in remodeling of the extracellular matrix as well as inducing the expression of chemokines that enable lung infiltration by neutrophils. Increased activity of p38 in normal cells was associated with metastatic disease and poor prognosis in human melanoma patients whereas inactivation of p38 suppressed lung metastases. We discuss the p38α-driven mechanisms stimulating the metastatic processes and potential use of p38 inhibitors in adjuvant therapy of metastatic cancers.

Silencing of H4R inhibits the production of IL-1β through SAPK/JNK signaling in human mast cells

Context: Mast cell (MC) activation through H4R releases various inflammatory mediators which are associated with allergic asthma.Objectives: To investigate the siRNA-mediated gene silencing effect of H4R on human mast cells (HMCs) functions and the activation of stress-activated protein kinases (SAPK)/jun amino-terminal kinases (JNK) signaling pathways for the release of ineterleukin-1β (IL-1β) in HMCs.Materials and methods: H4R expression was analyzed by RT-PCR and western blotting in human mast cell line-1 (HMC-1) cells and H4RsiRNA transfected cells. The effect of H4RsiRNA and H4R-antagonist on H4R mediated MC functions such as intracellular Ca2+ release, degranulation, IL-6 and IL-1β release, and the activation SAPK/JNK signaling pathways were studied. HMC-1 cells were stimulated with 10 μM of histamine (His) and 4-methylhistamine (4-MH) and pretreated individually with H4R-antagonist JNJ7777120 (JNJ), histamine H1 receptor (H1R)-antagonist mepyramine, and signaling molecule inhibitors SP600125 (SP) and Bay117082.Results: We found that the HMC-1 cells expressed H4R and H4RsiRNA treatment down regulated the H4R expression in HMC-1 cells. Both His and 4-MH induced the intracellular Ca2+ release and degranulation whereas; H4R siRNA and JNJ inhibited the effect. Furthermore, the activation of H4R caused the phosphorylation of SAPK/JNK pathways. H4R gene silencing and pretreatment with SP and JNJ decreased His and 4-MH induced phosphorylation of SAPK/JNK. We found that the activation of H4R caused the release of IL-1β (124.22 pg/ml) and IL-6 (122.50 pg/ml) on HMC-1 cells. Whereas, SAPK/JNK inhibitor (68.36 pg/ml) inhibited the H4R mediated IL-1β release.Conclusions: Taken together, the silencing of H4R inhibited the H4R mediated MC functions and SAPK/JNK phosphorylation. Furthermore, the H4R activation utilized SAPK/JNK signaling pathway for IL-1β release in HMC-1 cells.

Reactive Oxygen Species Play a Biphasic Role in Brain Ischemia

ABSTRACTObjective: Reactive oxygen species (ROS) are the essential mechanism involving in the ischemic process. Due to their complex characteristics, the precise effects of ROS on post-ischemic neurons remain uncertain. This study aimed to investigate the potential role of ROS in brain ischemia. Methods: Dynamic ROS levels in the perifocal cortex were evaluated after right middle cerebral artery occlusion (MCAO) of SD rats. Furthermore the role of ROS was assessed following delayed treatment with the ROS scavenger dimethylthiourea (DMTU) after brain ischemia. Results: ROS levels markedly increased at 1 hr after reperfusion and then gradually decreased as the post-reperfusion time interval increased. ROS levels reached their lowest point at 3 days after reperfusion before increasing and showing a second peak at 7 days after reperfusion. ROS levels negatively correlated with neurological function scores. Delayed DMTU treatment after stroke worsened neurological outcomes, decreased microvessel density and inhibited stress-activated protein kinase activation. Conclusion: ROS may play a biphasic role in cerebral ischemia. Namely, ROS may induce damage during the injury phase of brain ischemia and participate in improving neurological function during the recovery phase.

Blocking two-component signalling enhances Candida albicans virulence and reveals adaptive mechanisms that counteract sustained SAPK activation.

The Ypd1 phosphorelay protein is a central constituent of fungal two-component signal transduction pathways. Inhibition of Ypd1 in Saccharomyces cerevisiae and Cryptococcus neoformans is lethal due to the sustained activation of the ‘p38-related’ Hog1 stress-activated protein kinase (SAPK). As two-component signalling proteins are not found in animals, Ypd1 is considered to be a prime antifungal target. However, a major fungal pathogen of humans, Candida albicans, can survive the concomitant sustained activation of Hog1 that occurs in cells lacking YPD1. Here we show that the sustained activation of Hog1 upon Ypd1 loss is mediated through the Ssk1 response regulator. Moreover, we present evidence that C. albicans survives SAPK activation in the short-term, following Ypd1 loss, by triggering the induction of protein tyrosine phosphatase-encoding genes which prevent the accumulation of lethal levels of phosphorylated Hog1. In addition, our studies reveal an unpredicted, reversible, mechanism that acts to substantially reduce the levels of phosphorylated Hog1 in ypd1Δ cells following long-term sustained SAPK activation. Indeed, over time, ypd1Δ cells become phenotypically indistinguishable from wild-type cells. Importantly, we also find that drug-induced down-regulation of YPD1 expression actually enhances the virulence of C. albicans in two distinct animal infection models. Investigating the underlying causes of this increased virulence, revealed that drug-mediated repression of YPD1 expression promotes hyphal growth both within murine kidneys, and following phagocytosis, thus increasing the efficacy by which C. albicans kills macrophages. Taken together, these findings challenge the targeting of Ypd1 proteins as a general antifungal strategy and reveal novel cellular adaptation mechanisms to sustained SAPK activation.

TNF-α and IGF-1 differentially modulate ionizing radiation responses of lung cancer cell lines

The mechanism by which tumor microenvironment derived cytokine network modulates therapy response is of great concern in lung cancer but is not completely understood. In this study, we evaluated the effects of tumor necrosis factor α (TNF-α) and insulin-like growth factor 1 (IGF-1) on response of lung cancer cell lines to ionizing radiation (IR). While TNF-α increased radio sensitivity and inhibited cell migration, treatment with IGF-1 promoted cell growth and increased migration. These effects of TNF- α were mediated by increased immediate activation of stress-activated protein kinases (SAPK)/jun amino-terminal kinases (JNK) and p38. IR induced DNA damage was increased by TNF- α and not altered by IGF-1. However, in IGF-1 treated cells, there was decreased γ- H2AX along with an increase in mitotic index, resulting in abnormal chromosomal segregation in the cells. Bio informatics analysis of 982 lung cancer patients revealed that higher expression of TNF- α was associated with low risk of cancer progression while overexpression of IGF-1 was correlated with high risk. Collectively, these results reveal that the cytokines in the tumor microenvironment differentially modulate radiation therapy through a variety of signaling mechanisms.

APOL1 kidney disease risk variants cause cytotoxicity by depleting cellular potassium and inducing stress-activated protein kinases

Two specific genetic variants of the apolipoprotein L1 (APOL1) gene are responsible for the high rate of kidney disease in people of recent African ancestry. Expression in cultured cells of these APOL1 risk variants, commonly referred to as G1 and G2, results in significant cytotoxicity. The underlying mechanism of this cytotoxicity is poorly understood. We hypothesized that this cytotoxicity is mediated by APOL1 risk variant-induced dysregulation of intracellular signaling relevant for cell survival. To test this hypothesis, we conditionally expressed WT human APOL1 (G0), the APOL1 G1 variant, or the APOL1 G2 variant in human embryonic kidney cells (T-REx-293) using a tetracycline-mediated (Tet-On) system. We found that expression of either G1 or G2 APOL1 variants increased apparent cell swelling and cell death compared with G0-expressing cells. These manifestations of cytotoxicity were preceded by G1 or G2 APOL1-induced net efflux of intracellular potassium as measured by X-ray fluorescence, resulting in the activation of stress-activated protein kinases (SAPKs), p38 MAPK, and JNK. Prevention of net K(+) efflux inhibited activation of these SAPKs by APOL1 G1 or G2. Furthermore, inhibition of SAPK signaling and inhibition of net K(+) efflux abrogated cytotoxicity associated with expression of APOL1 risk variants. These findings in cell culture raise the possibility that nephrotoxicity of APOL1 risk variants may be mediated by APOL1 risk variant-induced net loss of intracellular K(+) and subsequent induction of stress-activated protein kinase pathways.

Therapeutic effect of TMZ-POH on human nasopharyngeal carcinoma depends on reactive oxygen species accumulation.

Nasopharyngeal carcinoma (NPC) is a common head and neck malignancy without efficient chemotherapeutic agents for it. In our current study, we demonstrated the cytotoxicity effects of a newly patented compound temozolomide-perillyl alcohol (TMZ-POH) on NPC in vitro and in vivo, and the possible mechanisms involved. Human NPC cell lines CNE1, CNE2, HNE2, and SUME-α were treated with control (DMSO), TMZ, POH, TMZ plus POH, and TMZ-POH. Our data indicated that TMZ-POH could inhibit NPC cell proliferation, cause G2/M arrest and DNA damage. TMZ-POH triggered apoptosis in NPC cells via significant activation of caspase-3 and poly(ADP-ribose) polymerase (PARP). Importantly, TMZ-POH-induced cell death was found to be associated with (i) the loss of inner mitochondrial membrane potential (ΔΨm) and release of mitochondrial Cytochrome c, (ii) the increase in ROS generation, and (iii) the activation of stress-activated protein kinases (SAPK)/c-Jun N-terminal kinases (JNK) signaling pathway. The generation of ROS in response to TMZ-POH seems to play a crucial role in the cell death process since the blockage of ROS production using the antioxidant N-acetyl-L-cysteine or catalase reversed the TMZ-POH-induced JNK activation, DNA damage, and cancer cell apoptosis. These results provide the rationale for further research and preclinical investigation of the antitumor effect of TMZ-POH against human NPC.

Disulfiram Suppresses Growth of the Malignant Pleural Mesothelioma Cells in Part by Inducing Apoptosis

Dithiocarbamate compound Disulfiram (DSF) that binds with copper and functions as an inhibitor of aldehyde dehydrogenase is a Food and Drug Administration approved agent for treatment of alcoholism. Copper complexed DSF (DSF-Cu) also possesses anti-tumor and chemosensitizing properties; however, its molecular mechanisms of action remain unclear. Here we investigated malignant pleural mesothelioma (MPM) suppressive effects of DSF-Cu and the molecular mechanisms involved. DSF-Cu inhibited growth of the murine as well as human MPM cells in part by increasing levels of ubiquitinated proteins. DSF-Cu exposure stimulated apoptosis in MPM cells that involved activation of stress-activated protein kinases (SAPKs) p38 and JNK1/2, caspase-3, and cleavage of poly-(ADP-ribose)-polymerase, as well as increased expression of sulfatase 1 and apoptosis transducing CARP-1/CCAR1 protein. Gene-array based analyses revealed that DSF-Cu suppressed cell growth and metastasis-promoting genes including matrix metallopeptidase 3 and 10. DSF inhibited MPM cell growth and survival by upregulating cell cycle inhibitor p27Kip1, IGFBP7, and inhibitors of NF-κB such as ABIN 1 and 2 and Inhibitory κB (IκB)α and β proteins. DSF-Cu promoted cleavage of vimentin, as well as serine-phosphorylation and lysine-63 linked ubiquitination of podoplanin. Administration of 50 mg/kg DSF-Cu by daily i.p injections inhibited growth of murine MPM cell-derived tumors in vivo. Although podoplanin expression often correlates with metastatic disease and poor prognosis, phosphorylation of serines in cytoplasmic domain of podoplanin has recently been shown to interfere with cellular motility and migration signaling. Post-translational modification of podoplanin and cleavage of vimentin by DSF-Cu underscore a metastasis inhibitory property of this agent and together with our in vivo studies underscore its potential as an anti-MPM agent.

CARP-1 Functional Mimetics Are a Novel Class of Small Molecule Inhibitors of Malignant Pleural Mesothelioma Cells

Malignant pleural mesothelioma (MPM) is an asbestos-related thoracic malignancy that is characterized by late metastases, and resistance to therapeutic modalities. The toxic side-effects of MPM therapies often limit their clinical effectiveness, thus necessitating development of new agents to effectively treat and manage this disease in clinic. CARP-1 functional mimetics (CFMs) are a novel class of compounds that inhibit growth of diverse cancer cell types. Here we investigated MPM cell growth suppression by the CFMs and the molecular mechanisms involved. CFM-1, -4, and -5 inhibited MPM cell growth, in vitro, in part by stimulating apoptosis. Apoptosis by CFM-4 involved activation of pro-apoptotic stress-activated protein kinases (SAPKs) p38 and JNK, elevated CARP-1 expression, cleavage of PARP1, and loss of the oncogene c-myc as well as mitotic cyclin B1. Treatments of MPM cells with CFM-4 resulted in depletion of NF-κB signaling inhibitor ABIN1 and Inhibitory κB (IκB)α and β, while increasing expression of pro-apoptotic death receptor (DR) 4 protein. CFM-4 enhanced expression of serine-phosphorylated podoplanin and cleavage of vimetin. CFMs also attenuated biological properties of the MPM cells by blocking their abilities to migrate, form colonies in suspension, and invade through the matrix-coated membranes. Both podoplanin and vimentin regulate processes of cell motility and invasion, and their expression often correlates with metastatic disease, and poor prognosis. The fact that phosphorylation of serines in the cytoplasmic domain of podoplanin interferes with processes of cellular motility, CFM-4-dependent elevated phosphorylated podoplanin and cleavage of vimentin underscore a metastasis inhibitory property of these compounds, and suggest that CFMs and/or their future analogs have potential as anti-MPM agents.

ATF3 Expression in the Corpus Luteum: Possible Role in Luteal Regression†

The present study investigated the induction and possible role of activating transcription factor 3 (ATF3) in the corpus luteum. Postpubertal cattle were treated at midcycle with prostaglandin F2α(PGF) for 0-4 hours. Luteal tissue was processed for immunohistochemistry, in situ hybridization, and isolation of protein and RNA. Ovaries were also collected from midluteal phase and first-trimester pregnant cows. Luteal cells were prepared and sorted by centrifugal elutriation to obtain purified small (SLCs) and large luteal cells (LLCs). Real-time PCR and in situ hybridization showed that ATF3 mRNA increased within 1 hour of PGF treatment in vivo. Western blot and immunohistochemistry demonstrated that ATF3 protein was expressed in the nuclei of LLC within 1 hour and was maintained for at least 4 hours. PGF treatment in vitro increased ATF3 expression only in LLC, whereas TNF induced ATF3 in both SLCs and LLCs. PGF stimulated concentration- and time-dependent increases in ATF3 and phosphorylation of MAPKs in LLCs. Combinations of MAPK inhibitors suppressed ATF3 expression in LLCs. Adenoviral-mediated expression of ATF3 inhibited LH-stimulated cAMP response element reporter luciferase activity and progesterone production in LLCs and SLCs but did not alter cell viability or change the expression or activity of key regulators of progesterone synthesis. In conclusion, the action of PGF in LLCs is associated with the rapid activation of stress-activated protein kinases and the induction of ATF3, which may contribute to the reduction in steroid synthesis during luteal regression. ATF3 appears to affect gonadotropin-stimulated progesterone secretion at a step or steps downstream of PKA signaling and before cholesterol conversion to progesterone.

Editorial:Molecular EndocrinologyArticles in the Spotlight for December 2013

Welcome to the December issue of Molecular Endocrinology. Among this month’s papers you’ll find the following research to be of great interest, particularly to those interested in reproductive biology. “ATF3 Expression in the Corpus Luteum: Possible Role in Luteal Regression” by Mao et al shows that the luteolytic hormone prostaglandin F2 (PGF2 ) selectively stimulated the expression of activating transcription factor 3 (ATF3) mRNA and ATF3 protein in large steroidogenic cells of the bovine corpus luteum in vivo and in vitro. The action of PGF2 was associated with the rapid activation of stress-activated protein kinases and the induction of ATF3 in large luteal cells. Furthermore, expression of ATF3 in large or small steroidogenic cells reduced LH-stimulated cAMP response element (CRE)mediated transcription downstream of protein kinase A (PKA) signaling and inhibited progesterone synthesis at a step before conversion of cholesterol to progesterone. Therefore, ATF3 may be primarily responsible for the PGF2 -dependent reduction in steroid synthesis that occurs during luteal regression. In “COUP-TFII Regulates Human Endometrial Stromal Genes Involved in Inflammation,” Li et al demonstrate that chicken ovalbumin upstream promoter-transcription factor II (COUP-TFII) was spatially and temporally expressed in endometrial stroma cells during the menstrual cycle in women. Furthermore, COUP-TFII was found to be reduced in stroma cells of ectopic lesions in endometriosis patients. Cistromic and transcriptomic analyses demonstrated that COUP-TF regulated processes critical for human endometrial stroma cell decidualization in vitro. Specifically, COUP-TFII was responsible for the down-regulation of inflammatory genes during decidualization. This may contribute to the function of decidual stroma cells in pregnancy and the pathology observed in endometriosis. In “BMP15 Suppresses Progesterone Production by Down-regulating StAR via ALK3 in Human Granulosa Cells” (freely available at http://mend.endojournals.org/), Chang et al found that bone morphogenetic protein 15 (BMP15), but not growth differentiation factor 9 (GDF9), reduced basal progesterone production by suppressing the expression of the rate-limiting steroidogenic regulatory (StAR) protein. Neither affected the expression of cytochrome P450 side chain cleavage (P450scc) enzyme or 3 -hydroxysteroid dehydrogenase (3 -HSD). The authors also showed that knockdown of ALK2 dramatically decreased basal StAR levels, indicating a positive role for ALK2 in maintaining StAR expression. Therefore, these findings suggest that oocyte-derived BMP15 plays a role in preventing premature luteinization by modulating progesterone production during the late stage of follicle development. Congratulations to all the authors in this issue for their fine work and research. Donald B. DeFranco, PhD Editor-in-Chief, Molecular Endocrinology

P.1.g.062 Reduction in serotonin 5-HT2C receptor antagonist-induced effects on theta power after chronic escitalopram treatment in rats

Background: The mammalian response to stress results in the activation of stress-activated protein kinases (also known as cJun N-terminal kinases; SAPKs or JNKs), which are a sub-group of the mitogen-activated protein (MAP) kinase family. The SAPKs are involved in the upregulation of activity of the transcription factor AP-1 by post-translational modification of two of its components, cJun and ATF2. AP-1 activity can also be elevated by increased expression of the Fos protein, a further AP-1 component. Elk-1 (also called p62TCF), a transcription factor involved in the induction of the expression from the c-fos promoter through the promoter's serum response element, is known to be activated as a result of phosphorylation by the MAP kinases ERK1 and ERK2. However, induction of c-fos expression in response to noxious agents takes place in the absence of ERK activation. We therefore investigated whether SAPKs similarly upregulate c-fos expression by phosphorylating Elk-1.Results Elk-1 is activated in response to stimuli other than mitogenic signals. Both p46SAPK and p54SAPK interact physically with, and phosphorylate, Elk-1. The capacity of Elk-1 to form a ternary complex with serum response factor in vitro is thereby elevated. In vivo, selective activation of SAPKs stimulates formation of the ternary complex containing Elk-1, serum response factor and the serum response element, and enhances Elk-1-dependent transcription. Expression of the SAPK upstream-activator kinase, MEKK1, induces SAPK activation and c-fos transcription in the absence of ERK activity. Phosphopeptide mapping of Elk-1 phosphorylated with p46SAPK or p54SAPK reveals Ser383, a residue critical for ternary complex formation and transcriptional activation, to be the major phosphorylation site.Conclusion Elk-1 responds to stress-induced, as well as mitogenic, signals by stimulating c-fos transcription through the serum response element. Phosphorylation of Elk-1 by SAPKs and the ensuing expression of Fos protein thus constitutes an additional mechanism by which cells can upregulate AP-1 activity in response to stress.

SAPK activity is a better marker than AMPK activity for measuring stressful O2 levels in stem cells

To test whether stress-activated protein kinase (SAPK) or AMP-activated protein kinase (AMPK) correlates with stress as induced by non-physiologic O2 levels during culture of mouse trophoblast stem cells (TSC).

The Small GTPase RALA Controls c-Jun N-terminal Kinase-mediated FOXO Activation by Regulation of a JIP1 Scaffold Complex

FOXO (forkhead box O) transcription factors are tumor suppressors and increase the life spans of model organisms. Cellular stress, in particular oxidative stress caused by an increase in levels of reactive oxygen species (ROS), activates FOXOs through JNK-mediated phosphorylation. Importantly, JNK regulation of FOXO is evolutionarily conserved. Here we identified the pathway that mediates ROS-induced JNK-dependent FOXO regulation. Following increased ROS, RALA is activated by the exchange factor RLF (RalGDS-like factor), which is in complex with JIP1 (C-Jun-amino-terminal-interacting protein 1) and JNK. Active RALA consequently regulates assembly and activation of MLK3, MKK4, and JNK onto the JIP1 scaffold. Furthermore, regulation of FOXO by RALA and JIP1 is conserved in C. elegans, where both ral-1 and jip-1 depletion impairs heat shock-induced nuclear translocation of the FOXO orthologue DAF16.