A-kinase anchoring proteins: Molecular regulators of the cardiac stress response

In response to stress or injury the heart undergoes a pathological remodeling process, associated with hypertrophy, cardiomyocyte death and fibrosis, that ultimately causes cardiac dysfunction and heart failure. It has become increasingly clear that signaling events associated with these pathological cardiac remodeling events are regulated by scaffolding and anchoring proteins, which allow coordination of pathological signals in space and time. A-kinase anchoring proteins (AKAPs) constitute a family of functionally related proteins that organize multiprotein signaling complexes that tether the cAMP-dependent protein kinase (PKA) as well as other signaling enzymes to ensure integration and processing of multiple signaling pathways. This review will discuss the role of AKAPs in the cardiac response to stress. Particular emphasis will be given to the adaptative process associated with cardiac hypoxia as well as the remodeling events linked to cardiac hypertrophy and heart failure. This article is part of a Special Issue entitled: Cardiomyocyte Biology: Cardiac Pathways of Differentiation, Metabolism and Contraction.

An inhibitor of HIV-1 protease modulates constitutive eIF2α dephosphorylation to trigger a specific integrated stress response.

Inhibitors of the HIV aspartyl protease [HIV protease inhibitors (HIV-PIs)] are the cornerstone of treatment for HIV. Beyond their well-defined antiretroviral activity, these drugs have additional effects that modulate cell viability and homeostasis. However, little is known about the virus-independent pathways engaged by these molecules. Here we show that the HIV-PI Nelfinavir decreases translation rates and promotes a transcriptional program characteristic of the integrated stress response (ISR). Mice treated with Nelfinavir display hallmarks of this stress response in the liver, including α subunit of translation initiation factor 2 (eIF2α) phosphorylation, activating transcription factor-4 (ATF4) induction, and increased expression of known downstream targets. Mechanistically, Nelfinavir-mediated ISR bypassed direct activation of the eIF2α stress kinases and instead relied on the inhibition of the constitutive eIF2α dephosphorylation and down-regulation of the phophatase cofactor CReP (Constitutive Repressor of eIF2α Phosphorylation; also known as PPP1R15B). These findings demonstrate that the modulation of eIF2α-specific phosphatase cofactor activity can be a rheostat of cellular homeostasis that initiates a functional ISR and suggest that the HIV-PIs could be repositioned as therapeutics in human diseases to modulate translation rates and stress responses.

Mitochondrial reactive oxygen species generation triggers inflammatory response and tissue injury associated with hepatic ischemia-reperfusion: Therapeutic potential of mitochondrially targeted antioxidants.

Mitochondrial reactive oxygen species generation has been implicated in the pathophysiology of ischemia-reperfusion (I/R) injury; however, its exact role and its spatial-temporal relationship with inflammation are elusive. Herein we explore the spatial-temporal relationship of oxidative/nitrative stress and inflammatory response during the course of hepatic I/R and the possible therapeutic potential of mitochondrial-targeted antioxidants, using a mouse model of segmental hepatic ischemia-reperfusion injury. Hepatic I/R was characterized by early (at 2h of reperfusion) mitochondrial injury, decreased complex I activity, increased oxidant generation in the liver or liver mitochondria, and profound hepatocellular injury/dysfunction with acute proinflammatory response (TNF-α, MIP-1α/CCL3, MIP-2/CXCL2) without inflammatory cell infiltration, followed by marked neutrophil infiltration and a more pronounced secondary wave of oxidative/nitrative stress in the liver (starting from 6h of reperfusion and peaking at 24h). Mitochondrially targeted antioxidants, MitoQ or Mito-CP, dose-dependently attenuated I/R-induced liver dysfunction, the early and delayed oxidative and nitrative stress response (HNE/carbonyl adducts, malondialdehyde, 8-OHdG, and 3-nitrotyrosine formation), and mitochondrial and histopathological injury/dysfunction, as well as delayed inflammatory cell infiltration and cell death. Mitochondrially generated oxidants play a central role in triggering the deleterious cascade of events associated with hepatic I/R, which may be targeted by novel antioxidants for therapeutic advantage.

AFT3 as a new therapeutic target for skin field cancerization in antagonism with compromised Notch/CSL signaling

Les interactions épithélio-mésenchymateuses jouent un rôle important dans le contrôle du développement normal de la peau, son homéostasie et sa tumorigenèse. Les fibroblastes dermiques (DFs) représentent la catégorie cellulaire la plus abondante dans le stroma et leur rôle est de plus en plus considéré. En ce qui concerne particulièrement la tumorigenèse, des facteurs diffusibles produits par les fibroblastes entourant les tumeurs épithéliales, appelés 'fibroblastes associés au cancer (CAF)', interagissent au niveau de l'inflammation impliquée directement ou indirectement dans la signalisation paracrine, entre le stroma et les cellules épiéliales cancéreuses. Le risque de cancer de la peau augmente de façon exponentielle avec l'âge. Comme un lien probable entre les deux, la sénescence des fibroblastes résulte de la production du sécrétome favorisant la sénescence (SMS), un groupe de facteurs diffusibles induisant une stimulation paracrine de la croissance, l'inflammation et le remodelage de la matrice. De façon fort intéressante, l'induction de ces gènes est aussi une caractéristique des CAFs. Cependant, le lien entre les deux événements cellulaires sénescence et activation des CAFs reste en grande partie inexploré. L'ATF3 (Activating Transcription Factor 3) est un facteur de transcription induit en réponse au stress, dont les fonctions sont hautement spécifiques du type cellulaire. Bien qu'il ait été découvert dans notre laboratoire en tant que promoteur de tumeurs dans les kératinocytes, ses fonctions biologique et biochimique dans le derme n'ont pas encore été étudiées. Récemment, nous avons constaté que, chez la souris, l'abrogation de la voie de signalisation de Notch/CSL dans les DFs, induisait la formation de tumeurs kératinocytaires multifocales. Ces dernières proviennent de la cancérisation en domaine, un phénomène associé à une atrophie du stroma, des altérations de la matrice et de l'inflammation. D'autres études ont montré que CSL agissait comme un régulateur négatif de gènes impliqués dans sénescence des DFs et dans l'activation des CAFs. Ici, nous montrons que la suppression ou l'atténuation de l'expression de ATF3 dans les DFs induit la sénescence et l'expression des gènes liés aux CAFs, de façon similaire à celle déclenchée par la perte de CSL, tandis que la surexpression de ATF3 supprime ces changements. Nous émettons l'hypothèse que ATF3 joue un rôle suppresseur dans l'activation des CAFs et dans la progression des tumeurs kératinocytaires, en surmontant les conséquences de l'abrogation de la voie de signalisation Notch/CSL. En concordance avec cette hypothèse, nous avons constaté que la perte de ATF3 dans les DFs favorisait la tumorigénicité des kératinocytes via le contrôle négatif de cytokines, des enzymes de la matrice de remodelage et de protéines associées au cancer, peut-être par liaison directe des effecteurs de la voie Notch/CSL : IL6 et les gènes Hes. Enfin, dans les échantillons cliniques humains, le stroma sous-jacent aux lésions précancéreuses de kératoses actiniques montre une diminution significative de l'expression de ATF3 par rapport au stroma jouxtant la peau normale. La restauration de l'expression de ATF3 pourrait être utilisée comme un outil thérapeutique en recherche translationnelle pour prévenir ou réprimer le processus de cancérisation en domaine. - Epithelial-mesenchymal interactions play an important role in control of normal skin development, homeostasis and tumorigenesis. The role of dermal fibroblasts (DFs) as the most abundant cell type in stroma is increasingly appreciated. Especially during tumorigenesis, fibroblasts surrounding epithelial tumors, called Cancer Associated Fibroblasts (CAFs), produce diffusible factors (growth factors, inflammatory cytokines, chemokines and enzymes, and matrix metalloproteinases) that mediate inflammation either directly or indirectly through paracrine signaling between stroma and epithelial cancer cells. The risk of skin cancer increases exponentially with age. As a likely link between the two, senescence of fibroblasts results in production of the senescence-messaging-secretome (SMS), a panel of diffusible factors inducing paracrine growth stimulation, inflammation, and matrix remodeling. Interestingly, induction of these genes is also a characteristic of Cancer Associated Fibroblasts (CAFs). However, the link between the two cellular events, senescence and CAF activation is largely unexplored. ATF3 is a key stress response transcription factor with highly cell type specific functions, which has been discovered as a tumor promoter in keratinocytes in our lab. However, the biological and biochemical function of ATF3 in the dermal compartment of the skin has not been studied yet. Recently, we found that compromised Notch/CSL signaling in dermal fibroblasts (DFs) in mice is a primary cause of multifocal keratinocyte tumors called field cancerization associated with stromal atrophy, matrix alterations and inflammation. Further studies showed that CSL functions as a negative regulator of genes involved in DFs senescence and CAF activation. Here, we show that deletion or silencing of the ATF3 gene in DFs activates senescence and CAF-related gene expression similar to that triggered by loss of CSL, while increased ATF3 suppresses these changes. We hypothesize that ATF3 plays a suppressing role in CAF activation and keratinocyte tumor progression, overcoming the consequences of compromised Notch/CSL signaling. In support of this hypothesis, we found that loss of ATF3 in DFs promotes tumorigenic behavior of keratinocytes via negative control of cytokines, matrix-remodeling enzymes and cancer-associated proteins, possibly through direct binding to Notch/CSL targets, IL6 and Hes genes. On the other hand, in human clinical samples, stromal fields underlying premalignant actinic keratosis lesions showed significantly decreased ATF3 expression relative to stroma of flanking normal skin. Restoration of ATF3, which is lost in cancer development, may be used as a therapeutic tool for translational research to prevent or suppress the field cancerization process.

Skin fibroblast model to study an impaired glutathione synthesis: consequences of a genetic polymorphism on the proteome.

An impaired glutathione (GSH) synthesis was observed in several multifactorial diseases, including schizophrenia and myocardial infarction. Genetic studies revealed an association between schizophrenia and a GAG trinucleotide repeat (TNR) polymorphism in the catalytic subunit (GCLC) of the glutamate cysteine ligase (GCL). Disease-associated genotypes of this polymorphism correlated with a decrease in GCLC protein expression, GCL activity and GSH content. To clarify consequences of a decreased GCL activity at the proteome level, three schizophrenia patients and three controls have been selected based on the GCLC GAG TNR polymorphism. Fibroblast cultures were obtained by skin biopsy and were challenged with tert-butylhydroquinone (t-BHQ), a substance known to induce oxidative stress. Proteome changes were analyzed by two dimensional gel electrophoresis (2-DE) and results revealed 10 spots that were upregulated in patients following t-BHQ treatment, but not in controls. Nine corresponding proteins could be identified by MALDI mass spectrometry and these proteins are involved in various cellular functions, including energy metabolism, oxidative stress response, and cytoskeletal reorganization. In conclusion, skin fibroblasts of subjects with an impaired GSH synthesis showed an altered proteome reaction in response to oxidative stress. Furthermore, the study corroborates the use of fibroblasts as an additional mean to study vulnerability factors of psychiatric diseases.

Physico-chemical characterization and oxidative reactivity evaluation of aged brake wear particles

Brake wear dust is a significant component of traffic emissions and has been linked to adverse health effects. Previous research found a strong oxidative stress response in cells exposed to freshly generated brake wear dust. We characterized aged dust collected from passenger vehicles, using microscopy and elemental analyses. Reactive oxygen species (ROS) generation was measured with acellular and cellular assays using 2′7-dichlorodihydrofluorescein dye. Microscopy analyses revealed samples to be heterogeneous particle mixtures with few nanoparticles detected. Several metals, primarily iron and copper, were identified. High oxygen concentrations suggested that the elements were oxidized. ROS were detected in the cell-free fluorescent test, while exposed cells were not dramatically activated by the concentrations used. The fact that aged brake wear samples have lower oxidative stress potential than fresh ones may relate to the highly oxidized or aged state of these particles, as well as their larger size and smaller reactive surface area.

Neuroendocrine, metabolic, and immune functions during the acute phase response of inflammatory stress in monosodium L-glutamate-damaged, hyperadipose male rat.

In rats, neonatal treatment with monosodium L-glutamate (MSG) induces several metabolic and neuroendocrine abnormalities, which result in hyperadiposity. No data exist, however, regarding neuroendocrine, immune and metabolic responses to acute endotoxemia in the MSG-damaged rat. We studied the consequences of MSG treatment during the acute phase response of inflammatory stress. Neonatal male rats were treated with MSG or vehicle (controls, CTR) and studied at age 90 days. Pituitary, adrenal, adipo-insular axis, immune, metabolic and gonadal functions were explored before and up to 5 h after single sub-lethal i.p. injection of bacterial lipopolysaccharide (LPS; 150 microg/kg). Our results showed that, during the acute phase response of inflammatory stress in MSG rats: (1) the corticotrope-adrenal, leptin, insulin and triglyceride responses were higher than in CTR rats, (2) pro-inflammatory (TNFalpha) cytokine response was impaired and anti-inflammatory (IL-10) cytokine response was normal, and (3) changes in peripheral estradiol and testosterone levels after LPS varied as in CTR rats. These data indicate that metabolic and neroendocrine-immune functions are altered in MSG-damaged rats. Our study also suggests that the enhanced corticotrope-corticoadrenal activity in MSG animals could be responsible, at least in part, for the immune and metabolic derangements characterizing hypothalamic obesity.

Endocrine response and perceived stress test during an experimental challenge task in adult survivors of a childhood cancer.

BACKGROUND: Although long-term implications of cancer in childhood or adolescence with regard to medical conditions are well documented, the impact on mental health and on response to stress, which may be an indicator of psychological vulnerability, is not yet well understood. In this study, psychological and physiological responses to stress were examined.¦PROCEDURE: Fifty-three participants aged 18-39 years (n = 25 survivors of childhood or adolescence cancer, n = 28 controls) underwent an experimental stress test, the Trier Social Stress Test (TSST). Participants were asked to provide repeated evaluations of perceived stress on visual-analogical scales and blood samples were collected before and after the TSST to measure plasma cortisol.¦RESULTS: The psychological perception of stress was not different between the two groups. However, the cancer survivors group showed a higher global plasma cortisol level as well as higher amplitude in the response to the TSST. The global cortisol level in cancer survivors was increased when depression symptoms were present. The subjective perception of stress and the plasma cortisol levels were only marginally correlated in both groups.¦CONCLUSIONS: It is suggested that the exposure to a life-threatening experience in childhood/adolescence increases the endocrine response to stress, and that the presence of depressive symptoms is associated with an elevation of plasma cortisol levels. A better knowledge of these mechanisms is important given that the dysregulations of the stress responses may cause psychological vulnerability. Pediatr Blood Cancer 2012; 59: 138-143. © 2011 Wiley Periodicals, Inc.

The caspase-3-p120-RasGAP module generates a NF-κB repressor in response to cellular stress.

The nuclear factor κB (NF-κB) transcription factor is a master regulator of inflammation. Short-term NF-κB activation is generally beneficial. However, sustained NF-κB might be detrimental, directly causing apoptosis of cells or leading to a persistent damaging inflammatory response. NF-κB activity in stressed cells needs therefore to be controlled for homeostasis maintenance. In mildly stressed cells, caspase-3 cleaves p120 RasGAP, also known as RASA1, into an N-terminal fragment, which we call fragment N. We show here that this fragment is a potent NF-κB inhibitor. Fragment N decreases the transcriptional activity of NF-κB by promoting its export from the nucleus. Cells unable to generate fragment N displayed increased NF-κB activation upon stress. Knock-in mice expressing an uncleavable p120 RasGAP mutant showed exaggerated NF-κB activation when their epidermis was treated with anthralin, a drug used for the treatment of psoriasis. Our study provides biochemical and genetic evidence of the importance of the caspase-3-p120-RasGAP stress-sensing module in the control of stress-induced NF-κB activation.