Pro-survival Kinase Akt Research Articles

Dynamic Interplay of Smooth Muscle α-Actin Gene-Regulatory Proteins Reflects the Biological Complexity of Myofibroblast Differentiation.

Myofibroblasts (MFBs) are smooth muscle-like cells that provide contractile force required for tissue repair during wound healing. The leading agonist for MFB differentiation is transforming growth factor β1 (TGFβ1) that induces transcription of genes encoding smooth muscle α-actin (SMαA) and interstitial collagen that are markers for MFB differentiation. TGFβ1 augments activation of Smad transcription factors, pro-survival Akt kinase, and p38 MAP kinase as well as Wingless/int (Wnt) developmental signaling. These actions conspire to activate β-catenin needed for expression of cyclin D, laminin, fibronectin, and metalloproteinases that aid in repairing epithelial cells and their associated basement membranes. Importantly, β-catenin also provides a feed-forward stimulus that amplifies local TGFβ1 autocrine/paracrine signaling causing transition of mesenchymal stromal cells, pericytes, and epithelial cells into contractile MFBs. Complex, mutually interactive mechanisms have evolved that permit several mammalian cell types to activate the SMαA promoter and undergo MFB differentiation. These molecular controls will be reviewed with an emphasis on the dynamic interplay between serum response factor, TGFβ1-activated Smads, Wnt-activated β-catenin, p38/calcium-activated NFAT protein, and the RNA-binding proteins, Purα, Purβ, and YB-1, in governing transcriptional and translational control of the SMαA gene in injury-activated MFBs.

The MYC-Associated Protein CDCA7 Is Phosphorylated by AKT To Regulate MYC-Dependent Apoptosis and Transformation

Cell division control protein A7 (CDCA7) is a recently identified target of MYC-dependent transcriptional regulation. We have discovered that CDCA7 associates with MYC and that this association is modulated in a phosphorylation-dependent manner. The prosurvival kinase AKT phosphorylates CDCA7 at threonine 163, promoting binding to 14-3-3, dissociation from MYC, and sequestration to the cytoplasm. Upon serum withdrawal, induction of CDCA7 expression in the presence of MYC sensitized cells to apoptosis, whereas CDCA7 knockdown reduced MYC-dependent apoptosis. The transformation of fibroblasts by MYC was reduced by coexpression of CDCA7, while the non-MYC-interacting protein Δ(156-187)-CDCA7 largely inhibited MYC-induced transformation. These studies provide insight into a new mechanism by which AKT signaling to CDCA7 could alter MYC-dependent growth and transformation, contributing to tumorigenesis.

An insert-based enzymatic cell culture system to rapidly and reversibly induce hypoxia: investigations of hypoxia-induced cell damage, protein expression and phosphorylation in neuronal IMR-32 cells

SUMMARYIschemia-reperfusion injury and tissue hypoxia are of high clinical relevance because they are associated with various pathophysiological conditions such as myocardial infarction and stroke. Nevertheless, the underlying mechanisms causing cell damage are still not fully understood, which is at least partially due to the lack of cell culture systems for the induction of rapid and transient hypoxic conditions. The aim of the study was to establish a model that is suitable for the investigation of cellular and molecular effects associated with transient and long-term hypoxia and to gain insights into hypoxia-mediated mechanisms employing a neuronal culture system. A semipermeable membrane insert system in combination with the hypoxia-inducing enzymes glucose oxidase and catalase was employed to rapidly and reversibly generate hypoxic conditions in the culture medium. Hydrogen peroxide assays, glucose measurements and western blotting were performed to validate the system and to evaluate the effects of the generated hypoxia on neuronal IMR-32 cells. Using the insert-based two-enzyme model, hypoxic conditions were rapidly induced in the culture medium. Glucose concentrations gradually decreased, whereas levels of hydrogen peroxide were not altered. Moreover, a rapid and reversible (onoff) generation of hypoxia could be performed by the addition and subsequent removal of the enzyme-containing inserts. Employing neuronal IMR-32 cells, we showed that 3 hours of hypoxia led to morphological signs of cellular damage and significantly increased levels of lactate dehydrogenase (a biochemical marker of cell damage). Hypoxic conditions also increased the amounts of cellular procaspase-3 and catalase as well as phosphorylation of the pro-survival kinase Akt, but not Erk1/2 or STAT5. In summary, we present a novel framework for investigating hypoxia-mediated mechanisms at the cellular level. We claim that the model, the first of its kind, enables researchers to rapidly and reversibly induce hypoxic conditions in vitro without unwanted interference of the hypoxia-inducing agent on the cultured cells. The system could help to further unravel hypoxia-associated mechanisms that are clinically relevant in various tissues and organs.

Abstract 18789: Pharmacologically Induced Hypothermia with Cannabinoid Receptor Agonist WIN55,212-2 vs Mild Therapeutic Hypothermia in a Rat Model of Cardiac Arrest with Extracorporeal Life Support

BACKGROUND: To compare the hemodynamic, cardioprotective and metabolic effects of cannabinoid receptor agonist WIN55,212-2 with mild therapeutic hypothermia and normothermic control on post-resuscitation myocardial function in a rat model of extracorporeal life support (ECLS). METHODS AND RESULTS: Ventricular fibrillation (VF) was induced in male Wistar rats. After 10 min of untreated VF, venoarterial ECLS was instituted for 60 min. At the beginning of ECLS animals were randomized to three groups of ten: normothermia, hypothermia (32°C) and WIN55,212 intravenous infusion (1 mg/kg/h). Cooling to 32°C or normothermia or drug infusion lasted for the entire ECLS. Plasma samples and myocardial biopsies were obtained and left-ventricular (LV) function was assessed by a conductance catheter at baseline and after weaning from ECLS. WIN55,212-2 administration produced pharmacologic hypothermia (32-34°C) and led to a significantly better recovery of the slope of the LV end-systolic pressure volume relationship (Ees) and Preload recruitable stroke work (PRSW) than hypothermia and normothermia: as percent of baseline 87+12 vs 54+9 vs 39+11 and 88+11 vs 59+10 vs 33+8 respectively (p<.01). LV stiffness expressed by end-diastolic pressure volume relationship (EDPVR) was significantly lower after WIN55,212-2 administration (p<.05). LV relaxation described by Tau was preserved after WIN55,212-2 treatment but not after hypothermia or normothermia (p<.01). WIN55,212-2 and not hypothermia significantly increased phosphorylation of the kinase ERK1 and 2 (2.8+0.4 and 2.5+0.3 vs 0.5+0.2 and 0.4+0.1 -fold of baseline levels) (p<.01). Both WIN55,212-2 and hypothermia but not normothermia increased phosphorylation of Akt. This resulted in a reduced cardiomyocyte apoptotic index (TUNEL, p<.01). WIN55,212-2 but not hypothermia nor normothermia preserved high energy phosphates content and energy charge and reduced lactate plasma concentration. CONCLUSIONS: Pharmacologically induced hypothermia with WIN55,212-2 was more effective than mild therapeutic hypothermia in recovering myocardial function after ECLS and in improving myocardial protection through activation of the pro-survival kinases Akt and ERK1/2.

Local anesthetic ‘in-situ’ toxicity during peripheral nerve blocks

Peripheral nerve blocks induce undesired side-effects linked to the toxicity of local anesthetics on neuron and myocytes via different cell targets. The effects of local anesthetics on these targets are now well known and summarized in this review. Local anesthetic-induced local cell toxicity involved different pathways leading to cell death, necrosis and different factors closely associated with the clinical practice modulated this toxicity. High concentration and prolonged duration of local anesthetic administration are closely associated with severe lesions. Phenotypic analyses revealed that local anesthetics could induce histological damage with lesions ranging from local to extreme in skeletal muscle. Metabolic alterations were also described involving sarcoplasmic reticulum and calcium dysregulation, alteration of mitochondrial physiology and of oxidative phosphorylation with associated overproduction of harmful reactive oxygen species, typically leading to apoptosis or necrosis. Biochemical and cell biology investigations now indicate that local anesthetics interact with different molecular targets in mammalian cells as respiratory chain complex I or the prosurvival kinase Akt. Functional dysfunction in both muscle and neuron remains to be investigated with caution in patients, as local anesthetic toxicity remains under-evaluated. Likewise, the use of adapted local anesthetics in patients with particular diseases and neuromuscular disorder could further reduce the risk of undesired effect.We need to improve our practice, and the optimization of our clinical protocol could prevent from these side-effects. Lastly, experimental studies highlight the preventive effects of antioxidant drugs or of recombinant human erythropoietin but the pharmacokinetic feature of such strategies remain to be evaluated.

TGFβ1 induces apoptosis in invasive prostate cancer and bladder cancer cells via Akt-independent, p38 MAPK and JNK/SAPK-mediated activation of caspases

Recent findings indicate that advanced stage cancers shun the tumor suppressive actions of TGFβ and inexplicably utilize the cytokine as a tumor promoter. We investigated the effect of TGFβ1 on the survival and proliferation of invasive prostate (PC3) and bladder (T24) cancer cells. Our study indicated that TGFβ1 decreased cell viability and induced apoptosis in invasive human PC3 and T24 cells via activation of p38 MAPK-JNK-Caspase9/8/3 pathway. Surprisingly, no change in the phosphorylation of pro-survival Akt kinase was observed. We postulate that TGFβ1 pathway may be utilized for specifically targeting urological cancers without inflicting side effects on normal tissues.

Cardioprotective effect of δ-opioid receptor agonist vs mild therapeutic hypothermia in a rat model of cardiac arrest with extracorporeal life support

BackgroundTo compare the effect of δ-opioid receptor agonist, d-Ala2-d-Leu5 enkephalin (DADLE) with normothermic control and therapeutic hypothermia on post resuscitation myocardial function in a model of extracorporeal life support (ECLS). MethodsVentricular fibrillation (VF) was induced in male Wistar rats. After 10min of untreated VF, venoarterial ECLS was instituted for 60min. At the beginning of ECLS animals were randomized to three groups of ten: normothermia, hypothermia (32°C) and DADLE intravenous infusion (1mg/kg/h). Cooling to 32°C or normothermia or drug infusion lasted for the entire ECLS. Plasma samples and myocardial biopsies were obtained and left-ventricular (LV) function was assessed by a conductance catheter at baseline and after weaning from ECLS. ResultsDADLE administration resulted in a significantly enhanced recovery of LV systolic function expressed by slope of the LV end-systolic pressure volume relationship (Ees) and preload recruitable stroke work (PRSW) than hypothermia and normothermia. LV stiffness indicated by end-diastolic pressure volume relationship (EDPVR) was significantly lower after DADLE administration (P<0.01). LV relaxation described by Tau was preserved after DADLE treatment but not after normothermia or mild hypothermia (P<0.01). Plasma lactate concentrations were lower in DADLE group (P<0.05). DADLE and not conventional hypothermia significantly increased phosphorylation of the kinases ERK1 and 2 (3.9±0.3 and 3.1±0.5 vs 0.4±0.1 and 0.3±0.1-fold of baseline levels) (P<0.001). Both DADLE and hypothermia but not normothermia increase phosphorylation of Akt. ConclusionsDADLE was more effective than mild therapeutic hypothermia in recovering myocardial function and activation of the pro-survival kinases Akt and ERK after ECLS.

Salicylic acid induces apoptosis in colon carcinoma cells grown in-vitro: Influence of oxygen and salicylic acid concentration

In solid tumors the hypoxic environment can promote tumor progression and resistance to therapy. Recently, acetylsalicylic acid a major component of analgesic drugs and its metabolite salicylic acid (SA) have been shown to reduce the risk of colon cancer, but the mechanisms of action remain still unclear. Here we elucidate the effects of physiologically relevant concentrations of SA on colon carcinoma cells (CaCo-2) grown under normoxic and hypoxic conditions. Western blotting, caspase-3/7 apoptosis assays, MTS cell-proliferation assays, LDH cytotoxicity assays and hydrogen peroxide measurements were performed to investigate the effects of 1 and 10μM SA on CaCo-2 cells grown under normoxic conditions and cells exposed to hypoxia. Under normoxic conditions, SA did not influence cell proliferation or LDH release of CaCo-2 cells. However, caspase-3/7 activity was significantly increased. Under hypoxia, cell proliferation was reduced and LDH release and caspase-3/7 activities were increased. None of these parameters was altered by the addition of SA under hypoxic conditions. Hypoxia increased hydrogen peroxide concentrations 300-fold and SA significantly augmented the release of hydrogen peroxide under normoxic, but not under hypoxic conditions. Phosphorylation of the pro-survival kinases akt and erk1/2 was not changed by SA under hypoxic conditions, whereas under normoxia SA reduced phosphorylation of erk1/2 after 2 hours. We conclude that in colon carcinoma cells effects of SA on apoptosis and cellular signaling are dependent on the availability of oxygen.

Disruption of the Interface between the Pleckstrin Homology (PH) and Kinase Domains of Akt Protein Is Sufficient for Hydrophobic Motif Site Phosphorylation in the Absence of mTORC2

The pro-survival kinase Akt requires phosphorylation at two conserved residues, the activation loop site (Thr-308) and the hydrophobic motif site (Ser-473), for maximal activation. Previous reports indicate that mTORC2 is necessary for phosphorylation of the hydrophobic motif and that this site is not phosphorylated in cells lacking components of the mTORC2 complex, such as Sin1. Here we show that Akt can be phosphorylated at the hydrophobic motif site (Ser-473) in the absence of mTORC2. First, increasing the levels of PIP(3) in Sin1(-/-) MEFs by (i) expression of a constitutively active PI3K or (ii) relief of a negative feedback loop on PI3K by prolonged inhibition of mTORC1 or S6K is sufficient to rescue hydrophobic motif phosphorylation of Akt. The resulting accumulation of PIP(3) at the plasma membrane results in Ser-473 phosphorylation. Second, constructs of Akt in which the PH domain is constitutively disengaged from the kinase domain are phosphorylated at the hydrophobic motif site in Sin1(-/-) MEFs; both myristoylated-Akt and Akt lacking the PH domain are phosphorylated at Ser-473. Thus, disruption of the interface between the PH and kinase domains of Akt bypasses the requirement for mTORC2. In summary, these data support a model in which Akt can be phosphorylated at Ser-473 and activated in the absence of mTORC2 by mechanisms that depend on removal of the PH domain from the kinase domain.

Activation of Akt by the bacterial inositol phosphatase, SopB, is wortmannin insensitive.

Salmonella enterica uses effector proteins translocated by a Type III Secretion System to invade epithelial cells. One of the invasion-associated effectors, SopB, is an inositol phosphatase that mediates sustained activation of the pro-survival kinase Akt in infected cells. Canonical activation of Akt involves membrane translocation and phosphorylation and is dependent on phosphatidyl inositide 3 kinase (PI3K). Here we have investigated these two distinct processes in Salmonella infected HeLa cells. Firstly, we found that SopB-dependent membrane translocation and phosphorylation of Akt are insensitive to the PI3K inhibitor wortmannin. Similarly, depletion of the PI3K regulatory subunits p85α and p85ß by RNAi had no inhibitory effect on SopB-dependent Akt phosphorylation. Nevertheless, SopB-dependent phosphorylation does depend on the Akt kinases, PDK1 and rictor-mTOR. Membrane translocation assays revealed a dependence on SopB for Akt recruitment to Salmonella ruffles and suggest that this is mediated by phosphoinositide (3,4) P2 rather than phosphoinositide (3,4,5) P3. Altogether these data demonstrate that Salmonella activates Akt via a wortmannin insensitive mechanism that is likely a class I PI3K-independent process that incorporates some essential elements of the canonical pathway.

β2-adrenergic receptors mediate cardioprotection through crosstalk with mitochondrial cell death pathways

β-adrenergic receptors (β-ARs) modulate cardiotoxicity/cardioprotection through crosstalk with multiple signaling pathways. We have previously shown that β2-ARs are cardioprotective during exposure to oxidative stress induced by doxorubicin (DOX). DOX cardiotoxicity is mediated in part through a Ca 2+-dependent opening of the mitochondrial permeability transition (MPT), however the signals linking a cell surface receptor like the β2-AR to regulators of mitochondrial function are not clear. The objective of this study was to assess mechanisms of crosstalk between β2-ARs and mitochondrial cell death pathways. DOX administered to WT mice resulted in no acute mortality, however 85% of β2-/- mice died within 30 min. Several pro- and anti-survival pathways were altered. The pro-survival kinase, εPKC, was decreased by 64% in β2-/- after DOX vs WT (p < 0.01); the εPKC activator ψεRACK partially rescued these mice (47% reduction in mortality). Activity of the pro-survival kinase Akt decreased by 76% in β2-/- after DOX vs WT (p < 0.01). The α1-antagonist prazosin restored Akt activity to normal and also partially reversed the mortality (45%). Deletion of the β2-AR increased rate of Ca 2+ release by 75% and peak [Ca 2+] i by 20% respectively in isolated cardiomyocytes; the Ca 2+ channel blocker verapamil also partially rescued the β2-/- (26%). Mitochondrial architecture was disrupted and complex I and II activities decreased by 40.9% and 34.6% respectively after DOX only in β2-/-. The MPT blocker cyclosporine reduced DOX mortality by 41% and prazosin plus cyclosporine acted synergistically to decrease mortality by 85%. β2-ARs activate pro-survival kinases and attenuate mitochondrial dysfunction during oxidative stress; absence of β2-ARs enhances cardiotoxicity via negative regulation of survival kinases and enhancement of intracellular Ca 2+, thus predisposing the mitochondria to opening of the MPT.

Development of severe skeletal defects in induced SHP-2-deficient adult mice: a model of skeletal malformation in humans with SHP-2 mutations

SUMMARYSHP-2 (encoded by PTPN11) is a ubiquitously expressed protein tyrosine phosphatase required for signal transduction by multiple different cell surface receptors. Humans with germline SHP-2 mutations develop Noonan syndrome or LEOPARD syndrome, which are characterized by cardiovascular, neurological and skeletal abnormalities. To study how SHP-2 regulates tissue homeostasis in normal adults, we used a conditional SHP-2 mouse mutant in which loss of expression of SHP-2 was induced in multiple tissues in response to drug administration. Induced deletion of SHP-2 resulted in impaired hematopoiesis, weight loss and lethality. Most strikingly, induced SHP-2-deficient mice developed severe skeletal abnormalities, including kyphoses and scolioses of the spine. Skeletal malformations were associated with alterations in cartilage and a marked increase in trabecular bone mass. Osteoclasts were essentially absent from the bones of SHP-2-deficient mice, thus accounting for the osteopetrotic phenotype. Studies in vitro revealed that osteoclastogenesis that was stimulated by macrophage colony-stimulating factor (M-CSF) and receptor activator of nuclear factor kappa B ligand (RANKL) was defective in SHP-2-deficient mice. At least in part, this was explained by a requirement for SHP-2 in M-CSF-induced activation of the pro-survival protein kinase AKT in hematopoietic precursor cells. These findings illustrate an essential role for SHP-2 in skeletal growth and remodeling in adults, and reveal some of the cellular and molecular mechanisms involved. The model is predicted to be of further use in understanding how SHP-2 regulates skeletal morphogenesis, which could lead to the development of novel therapies for the treatment of skeletal malformations in human patients with SHP-2 mutations.

Initial testing (stage 1) of the Akt inhibitor GSK690693 by the pediatric preclinical testing program

GSK690693 is a small molecule ATP-competitive inhibitor of the pro-survival kinase Akt. Since Akt regulates multiple downstream targets including transcription factors, glycogen synthase 3, the pro-apoptotic protein Bad, as well as MDM2 and mTORC1, it was tested against the in vitro and in vivo panels of the Pediatric Preclinical Testing Program (PPTP). GSK690693 was tested in vitro at concentrations from 1 nM to 10 µM, and against the in vivo panel of xenografts at a dose of 30 mg/kg daily × 5 for 6 consecutive weeks. Three measures of in vivo antitumor activity were used: (1) an objective response measure modeled after the clinical setting; (2) a treated to control (T/C) tumor volume measure; and (3) a time to event measure based on the median event-free survival (EFS) of treated and control animals for each xenograft. GSK690693 inhibited cell growth in vitro with IC(50) values between 6.5 nM and >10 µM. In vivo, GSK690693 significantly increased EFS in 11 of 34 (32%) solid tumor xenografts, most notably in all 6 osteosarcoma models, but not in any of the 8 ALL xenografts tested. No objective responses were observed and only one solid tumor met EFS T/C criteria for intermediate activity. GSK690693 demonstrated broad activity in vitro, however our results against both the solid tumor and ALL PPTP in vivo panels demonstrate that, as single agent at the dose and schedule used, GSK690693 has only modest antitumor activity.

Asiatic acid preserves beta cell mass and mitigates hyperglycemia in streptozocin‐induced diabetic rats

Type 1 diabetes is a chronic condition in which the pancreas produces little or no insulin due to the loss or dysfunction of pancreatic beta cells. This study investigated the beneficial effects of asiatic acid-a triterpenoid compound-preserved beta mass and mitigated hyperglycemia in streptozocin-induced diabetic rats. Diabetes mellitus was induced in adult male Wistar rats by a single intraperitoneal injection of streptozocin (60 mg/kg body weight). The diabetic rats were divided into untreated and asiatic acid (25 mg/kg) groups. Controls were intraperitoneal injection with citrate buffer. Blood glucose level, plasma insulin, and pancreas immunohistochemistry analysis were examined after a 2-week experimental period. AKT and Bcl-xL expression in the pancreatic islets of rats were evaluated by Western blot methods. Blood glucose levels were significantly reduced in rats receiving asiatic acid after streptozocin administration. Asiatic acid concomitantly increased serum insulin levels in diabetic rats. Immunohistochemical staining revealed a marked preservation by asiatic acid of insulin-producing beta cells in the pancreatic islets of the diabetic rats. Furthermore, asiatic acid in vivo induced pro-survival Akt kinase activation and Bcl-xL expression in the pancreatic islets of diabetic rats. These results suggest that asiatic acid exerts its glucose-lowering effects, in part through influences on beta-cell mass. Asiatic acid administration resulted in preservation and restoration of beta-cell mass and function in diabetic rodent models.

Mixed Lineage Kinase-3 Stabilizes and Functionally Cooperates with TRIBBLES-3 to Compromise Mitochondrial Integrity in Cytokine-induced Death of Pancreatic Beta Cells

Mixed lineage kinases (MLKs) have been implicated in cytokine signaling as well as in cell death pathways. Our studies show that MLK3 is activated in leukocyte-infiltrated islets of non-obese diabetic mice and that MLK3 activation compromises mitochondrial integrity and induces apoptosis of beta cells. Using an ex vivo model of islet-splenocyte co-culture, we show that MLK3 mediates its effects via the pseudokinase TRB3, a mammalian homolog of Drosophila Tribbles. TRB3 expression strongly coincided with conformational change and mitochondrial translocation of BAX. Mechanistically, MLK3 directly interacted with and stabilized TRB3, resulting in inhibition of Akt, a strong suppressor of BAX translocation and mitochondrial membrane permeabilization. Accordingly, attenuation of MLK3 or TRB3 expression each prevented cytokine-induced BAX conformational change and attenuated the progression to apoptosis. We conclude that MLKs compromise mitochondrial integrity and suppress cellular survival mechanisms via TRB3-dependent inhibition of Akt.

Targeting PI3K/Akt/HSP90 Signaling Sensitizes Gastric Cancer Cells to Deoxycholate-Induced Apoptosis

The heat shock protein 90 (HSP90) plays a crucial role in the stability of several proteins that are essential for cell survival and for malignant transformation. The binding of HSP90 with pro-survival kinase Akt prevents proteosomal degradation of Akt and contributes to the functional stabilization of PI3K/Akt signaling and cell survival. Akt kinase and HSP90 are therefore highly over-expressed in a large panel of cancer cell lines and are present in multi-chaperoning complexes. In this paper, we investigated whether targeting both Akt and HSP90 would inhibit the survival pathway in AGS cells (human gastric mucosal cells), and how Akt/HSP90 inhibition modulates the deoxycholate (DC)-induced apoptosis. AGS cells in the presence of Akt inhibitors (LY294002 and wortmannin), or HSP90 inhibitor (geldanamycin, GA) for 30 min or 18 h, respectively, were treated with DC (50 µM). Activation of PI3K/Akt signaling was evaluated by measuring the Akt and PTEN phosphorylation. HSP90, caspase-3 and caspase-9 were detected in whole lysates by Western blot analysis. AGS cells, transiently transfected with Akt siRNA, were treated with DC, and apoptosis was measured by caspase-3 activation. Apoptotic-positive cells were counted according to changes of cell morphology by Hoechst staining and fluorescence microscopy. The intrinsic level of phospho-Akt (pAkt; active form), phospho-PTEN (pPTEN; inactive enzyme) and HSP90 were highly expressed in AGS cells indicating the active PI3K/Akt/HSP90 signaling. Although, deoxycholate at low concentration (50 µM) slightly inhibited the expression of pAkt and cleaved HSP90 to 55 KDa fragment, no significant effect on apoptosis induction, up to 4 h (as assessed by caspase-3 activation) was observed. The higher concentrations of DC (100 µM-300 µM) resulted in progressive inhibition of pAkt, activation of PTEN, and specific cleavage of HSP90 to approximately 45 KDa fragments with significant induction of apoptosis. Although DC (50 µM) had no profound effect on Akt/HSP90 and did not induce apoptosis, it became an inducer of apoptosis when cells were pretreated with LY294002, wortmannin, or geldanamycin. Consistent with these findings, significant activation of apoptosis in response to DC (50 µM) was observed in cells with depleted Akt protein. These results demonstrate that down-regulation of PI3K/Akt pathway with specific cleavage of HSP90 to 45 KDa modulates the pro-apoptotic effects of DC in gastric cells. They further indicate the importance of stable Akt/HSP90 complex in regulation of survival/death responses.

UPR-mediated TRIB3 expression correlates with reduced AKT phosphorylation and inability of interleukin 6 to overcome palmitate-induced apoptosis in RINm5F cells

Unfolded protein response (UPR)-mediated pancreatic beta-cell death has been described as a common mechanism by which palmitate (PA) and pro-inflammatory cytokines contribute to the development of diabetes. There are evidences that interleukin 6 (IL6) has a protective action against beta-cell death induced by pro-inflammatory cytokines; the effects of IL6 on PA-induced apoptosis have not been investigated yet. In the present study, we have demonstrated that PA selectively disrupts IL6-induced RAC-alpha serine/threonine-protein kinase (AKT) activation without interfering with signal transducer and activator of transcription 3 phosphorylation in RINm5F cells. The inability of IL6 to activate AKT in the presence of PA correlated with an inefficient protection against PA-induced apoptosis. In contrast to PA, IL6 efficiently reduced apoptosis induced by pro-inflammatory cytokines. In addition, we have demonstrated that IL6 is unable to overcome PA-stimulated UPR, as assessed by activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP) expression, X-box binding protein-1 gene mRNA splicing, and pancreatic eukaryotic initiation factor-2 alpha kinase phosphorylation, whereas no significant induction of UPR by pro-inflammatory cytokines was detected. This unconditional stimulation of UPR and apoptosis by PA was accompanied by the stimulation of CHOP and tribble3 (TRIB3) expression, irrespective of the presence of IL6. These findings suggest that IL6 is unable to protect pancreatic beta-cells from PA-induced apoptosis because it does not repress UPR activation. In this way, CHOP and ATF4 might mediate PA-induced TRIB3 expression and, by extension, the suppression of IL6 activation of pro-survival kinase AKT.

Beneficial Dysregulation of the Time Course of Inflammatory Mediators in Lipopolysaccharide-Induced Tumor Necrosis Factor Alpha Factor-Deficient Mice

To begin to understand the surprising survival of macrophage-specific lipopolysaccharide-induced tumor necrosis factor alpha factor-deficient (macLITAF(-/-)) animals after a lethal dose of lipopolysaccharide (LPS), as reported earlier, the present follow-up study focuses on the role of LITAF in the regulation of inflammatory cytokines secreted in response to lethal or sublethal doses of LPS administered to wild-type (WT) and macLITAF(-/-) mice. A time course study of kinase expression in peritoneal macrophages revealed increased phosphorylation of prosurvival kinases Akt, Erk1/2, and ribosomal S6 kinase (RSK) in macLITAF(-/-) mice compared to that in WT mice (n = 8), confirming their role in LPS-mediated diseases. macLITAF(-/-) mice (n = 8) survived a lethal dose of LPS plus d-galactosamine (d-GalN), expressing lower serum levels of pro- and anti-inflammatory cytokines than the WT levels. To extend our knowledge on LPS-induced inflammatory events, an effective sublethal dose of LPS was administered to the animals (n = 14). WT animals exhibited an acute inflammatory response that decreased after 4 h. Interestingly, macLITAF(-/-) mice exhibited an initial delay in the secretion of proinflammatory cytokines that peaked after 8 h and reached WT levels after 18 h. Anti-inflammatory cytokine secretions were initially delayed but increased after 4 h and remained elevated compared to WT levels, even after 18 h. Our results demonstrate that LITAF deficiency in vivo affects cytokines other than TNF-alpha and influences the balance between the pro- and anti-inflammatory cytokines, which protects the animals from the deleterious effects of an LPS-induced inflammatory response, resulting in a beneficial host regulation of inflammatory cytokines and in enhanced survival. Therapeutic intervention aimed at reducing LITAF via kinase modulators may prove useful in preventing LPS-induced mortality.

Effect of Thymoquinone on Mouse Dendritic Cells

Thymoquinone, a component of Nigella sativa is known to confer protection against tumour growth due to stimulation of tumour cell apoptosis. Moreover, thymoquinone has remarkable anti-inflammatory potency. Surprisingly, despite its powerful influence on inflammation and its immunomodulatory effects, little is known about its effect on dendritic cells (DCs), key players in the regulation of innate and adaptive immunity. DC maturation and cytokine release is triggered by bacterial components such as lipopolysaccharides (LPS). The present study explored whether thymoquinone modifies LPS-induced DC maturation, survival and cytokine release. To this end, mouse bone marrow derived DCs were treated with LPS and different concentrations of thymoquinone and the surface expression of CD11c, CD86, MHCII, CD54 and CD40 was determined by FACS analysis, the formation of the interleukins 10 (IL-10) and 12 (IL-12p70) as well as TNF-α by ELISA, caspase activation by FITC-labelled antibodies (FACS), cell membrane scrambling by annexin V binding (FACS) and Akt and ERK1/2 phosphorylation by Western blotting. LPS increased the percentage of CD11c⁺CD86⁺, CD11c⁺MHCII⁺, CD11c⁺CD40⁺ and CD11c⁺CD54⁺ cells and stimulated the release of IL-10, IL-12p70 and TNF-α. These effects were blunted by thymoquinone in a concentration dependent manner (1-20 µM). Moreover, LPS decreased and thymoquinone increased caspase 3 and caspase 8 activation and annexin V binding. Moreover, LPS-induced phosphorylation of prosurvival kinases Akt and ERK1/2 was abrogated by thymoquinone. In conclusion, thymoquinone compromises the maturation, cytokine release and survival of DCs.

Cardioprotective effects of mineralocorticoid receptor antagonists at reperfusion

Pre-treatment with mineralocorticoid receptor (MR) antagonists is reported to reduce myocardial infarct size from ischaemia/reperfusion. Here, we tested whether the MR antagonists potassium canrenoate and eplerenone could protect in the more clinically relevant schedule of administration at the end of ischaemia. In all models, hearts were subjected to 30 min regional ischaemia followed by 120 min (rabbits 4 h) reperfusion. A bolus of canrenoate 5 min prior to reperfusion in open-chest mice decreased infarct size in a dose-dependent manner. Maximum protection was seen at 1 mg/kg where infarction was 18% of that in the control (P < 0.001). Ecto-5'-nucleotidase (CD73) as well as adenosine A(2b) receptor knock-out mice could no longer be protected, suggesting a role for adenosine and the A(2b) receptor in the mechanism. A 1 mg/kg bolus of canrenoate prior reperfusion also reduced infarct size in open-chest rabbits. To explore the underlying mechanisms, we studied isolated rat hearts. Eplerenone (10 microM) at the end of ischaemia was similarly protective in the rat heart and the protection was abolished by co-treatment with inhibitors of the adenosine receptor, protein kinase C, PI3-kinase, and ERK. In addition, eplerenone or canrenoate treatment increased phosphorylation of the pro-survival kinases Akt and ERK1/2 at reperfusion in the rat hearts. Taken together, MR antagonists when given at the end of ischaemia are highly effective and potent cardioprotective drugs with a signalling similar to that of ischaemic pre-conditioning and, hence, could be a very promising candidate for the treatment of acute myocardial infarction in man.