The tumor necrosis factor-related apoptosis-inducing ligand receptors TRAIL-R1 and TRAIL-R2 have distinct cross-linking requirements for initiation of apoptosis and are non-redundant in JNK activation.

Overexpression of the tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) receptors, TRAIL-R1 and TRAIL-R2, induces apoptosis and activation of NF-kappaB in cultured cells. In this study, we have demonstrated differential signaling capacities by both receptors using either epitope-tagged soluble TRAIL (sTRAIL) or sTRAIL that was cross-linked with a monoclonal antibody. Interestingly, sTRAIL was sufficient for induction of apoptosis only in cell lines that were killed by agonistic TRAIL-R1- and TRAIL-R2-specific IgG preparations. Moreover, in these cell lines interleukin-6 secretion and NF-kappaB activation were induced by cross-linked or non-cross-linked anti-TRAIL, as well as by both receptor-specific IgGs. However, cross-linking of sTRAIL was required for induction of apoptosis in cell lines that only responded to the agonistic anti-TRAIL-R2-IgG. Interestingly, activation of c-Jun N-terminal kinase (JNK) was only observed in response to either cross-linked sTRAIL or anti-TRAIL-R2-IgG even in cell lines where both receptors were capable of signaling apoptosis and NF-kappaB activation. Taken together, our data suggest that TRAIL-R1 responds to either cross-linked or non-cross-linked sTRAIL which signals NF-kappaB activation and apoptosis, whereas TRAIL-R2 signals NF-kappaB activation, apoptosis, and JNK activation only in response to cross-linked TRAIL.

Activated protein C, severe sepsis and 28-day mortality.

Protein C (PC) de ciency is prevalent in severe sepsis, studies showing that more than 80% of patients with severe sepsis have a baseline PC level below the lower limit of normal [1,2]. The aim of the study was to relate the anticoagulation activity evaluated by PC, with clinical parameters and 28-day mortality.

Activated protein C consumption and coagulation parameters in severe sepsis and septic shock.

Introduction Activated protein C (APC) deC ciency is prevalent in severe sepsis and septic shock patients. The aim of the study was to relate the anticoagulation activity evaluated by APC with other coagulation parameters adjusted to 28-day mortality. Methods A cohort study of 150 critically ill adults. Age, sex, sources of infection and coagulation markers within 24< hours from severe sepsis or septic shock onset, deC ned according to Surviving Sepsis Campaign (SSC) criteria, were studied. We analyzed APC activity using a hemostasis laboratory analyzer (BCS® XP; Siemens). A descriptive and comparative statistical analysis was performed using SPSS version 15.0 (SPSS Inc., Chicago, IL, USA). Results We analyzed 150 consecutive episodes of severe sepsis (16%) or septic shock (84%) admitted to the UCI. The median age of the study sample was 64 (interquartile range (IQR): 22.30.001). See Figure 1. Conclusion Low levels of PC are associated with poor outcome and severity in severe sepsis, and it is well correlated with antithrombin III and INR.

Phosphorylation of the Na,K-ATPase alpha-subunit by protein kinase A and C in vitro and in intact cells. Identification of a novel motif for PKC-mediated phosphorylation.

Na,K-ATPase is a potential target for regulatory phosphorylation by protein kinase A and C (PKA and PKC). To identify the phosphorylation sites, we have mutated the alpha 1-subunit of Bufo marinus in a highly conservative PKA and in 20 different PKC consensus sequences. The mutants were expressed in Xenopus oocytes and their phosphorylation capacity tested in homogenates upon stimulation of PKA or PKC. While serine 943 (Ser-943) was identified as a unique target site for PKA, none of the PKC consensus serine or threonine residues are implicated in PKC phosphorylation. Controlled trypsinolysis of phosphorylated alpha-subunits of various purified enzyme preparations and of alpha/beta complexes from oocyte homogenates revealed that PKC phosphorylation was exclusively associated with the N terminus. A fusion protein containing the first 32 amino acids of the Bufo alpha-subunit was phosphorylated in vitro and serine and threonine residues (Thr-15 and Ser-16) in this region were identified by site-directed mutagenesis as the PKC phosphorylation sites. Finally, the Bufo alpha-subunit was phosphorylated by protein kinases in transfected COS-7 cells. In intact cells, PKA stimulation induced phosphorylation exclusively on Ser-943 and PKC stimulation mainly on Thr-15 and Ser-16, which are contained in a novel PKC phosphorylation motif.

Protein kinase A-dependent phosphorylation of GLUT2 in pancreatic beta cells.

In pancreatic beta cells, cyclic AMP-dependent protein kinase regulates many cellular processes including the potentiation of insulin secretion. The substrates for this kinase, however, have not been biochemically characterized. Here we demonstrate that the glucose transporter GLUT2 is rapidly phosphorylated by protein kinase A following activation of adenylyl cyclase by forskolin or the incretin hormone glucagon-like peptide-1. We show that serines 489 and 501/503 and threonine 510 in the carboxyl-terminal tail of the transporter are the in vitro and in vivo sites of phosphorylation. Stimulation of GLUT2 phosphorylation in beta cells reduces the initial rate of 3-O-methyl glucose uptake by approximately 48% but does not change the Michaelis constant. Similar differences in transport kinetics are observed when comparing the transport activity of GLUT2 mutants stably expressed in insulinoma cell lines and containing glutamates or alanines at the phosphorylation sites. These data indicate that phosphorylation of GLUT2 carboxyl-terminal tail modifies the rate of transport. This lends further support for an important role of the transporter cytoplasmic tail in the modulation of catalytic activity. Finally, because activation of protein kinase A stimulates glucose-induced insulin secretion, we discuss the possible involvement of GLUT2 phosphorylation in the amplification of the glucose signaling process.

Phosphatidylethanolamine critically supports internalization of cell-penetrating protein C inhibitor.

Although their contribution remains unclear, lipids may facilitate noncanonical routes of protein internalization into cells such as those used by cell-penetrating proteins. We show that protein C inhibitor (PCI), a serine protease inhibitor (serpin), rapidly transverses the plasma membrane, which persists at low temperatures and enables its nuclear targeting in vitro and in vivo. Cell membrane translocation of PCI necessarily requires phosphatidylethanolamine (PE). In parallel, PCI acts as a lipid transferase for PE. The internalized serpin promotes phagocytosis of bacteria, thus suggesting a function in host defense. Membrane insertion of PCI depends on the conical shape of PE and is associated with the formation of restricted aqueous compartments within the membrane. Gain- and loss-of-function mutations indicate that the transmembrane passage of PCI requires a branched cavity between its helices H and D, which, according to docking studies, precisely accommodates PE. Our findings show that its specific shape enables cell surface PE to drive plasma membrane translocation of cell-penetrating PCI.

Impaired activation of protein kinase C-zeta by insulin and phosphatidylinositol-3,4,5-(PO4)3 in cultured preadipocyte-derived adipocytes and myotubes of obese subjects.

Insulin resistance in obesity is partly due to diminished glucose transport in myocytes and adipocytes, but underlying mechanisms are uncertain. Insulin-stimulated glucose transport requires activation of phosphatidylinositol (PI) 3-kinase (3K), operating downstream of insulin receptor substrate-1. PI3K stimulates glucose transport through increases in PI-3,4,5-(PO(4))(3) (PIP(3)), which activates atypical protein kinase C (aPKC) and protein kinase B (PKB/Akt). However, previous studies suggest that activation of aPKC, but not PKB, is impaired in intact muscles and cultured myocytes of obese subjects. Presently, we examined insulin activation of glucose transport and signaling factors in cultured adipocytes derived from preadipocytes harvested during elective liposuction in lean and obese women. Relative to adipocytes of lean women, insulin-stimulated [(3)H]2-deoxyglucose uptake and activation of insulin receptor substrate-1/PI3K and aPKCs, but not PKB, were diminished in adipocytes of obese women. Additionally, the direct activation of aPKCs by PIP(3) in vitro was diminished in aPKCs isolated from adipocytes of obese women. Similar impairment in aPKC activation by PIP(3) was observed in cultured myocytes of obese glucose-intolerant subjects. These findings suggest the presence of defects in PI3K and aPKC activation that persist in cultured cells and limit insulin-stimulated glucose transport in adipocytes and myocytes of obese subjects.

Low density lipoprotein signaling and pancreatic beta cells protection

SummaryLow-density lipoproteins (LDLs) have an important physiological role in organism transporting cholesterol and other fatty substances to target tissues. However, elevated LDL levels in the blood are associated with the formation of arterial plaques and consequently atherosclerosis. It is therefore important to characterize the intracellular pathways induced upon LDL stimulation as they might be involved in the pathological properties of these lipoproteins. It has been previously found that LDL stimulation of mouse embryonic fibroblasts activates p38 mitogen activated protein kinases (MAPKs). This leads to cell spreading and increase in the wound healing capabilities of the cells. These two responses might occur within atherosclerotic plaques.The aim of this project is to reveal the missing links between LDL particle and activation of p38 MAPK kinase. As previously shown in our lab activation of p38 MAPK kinase by the LDL particles occur independently of classical LDL receptor (LDLR). In this study we have shown that scavenger receptor type Β class I (SR-BI) is responsible for the signal transduction from the LDLs to the p38 MAPK. We have also shown that Mitogen activated kinase kinases (MKKs) that can directly activate ρ 38 MAPK in these conditions are MKK3 and MKK6 but not MKK4. We have also tested some of the intermediate components of the pathway like Ras and PI3 kinase but found that they do not play a role.The data obtained in this study showed a part of molecular mechanism responsible for p38 MAPK activation and subsequent wound healing and can contribute to our knowledge on function of the fibroblasts in the development of the atherosclerotic plaques.Diabetes Mellitus is a condition caused by disordered metabolism of blood glucose level. It is one of the most commonly spread disease in the western world, with the incidence reaching 8% of population in United States. Two most common types of diabetes are type 1 and 2 that differs slightly in the mechanism of the development. However in the basis of both types lies the cell death of pancreatic beta cells. The aim of this work is to improve beta cells survival in different pathophysiological settings. This could be extrapolated to the conditions in which Diabetes develops in humans. We decided to use RasGAP- derived fragment Ν with its strong antiapoptotic effect in beta cells. In our lab we have demonstrated that in the mild stress conditions RasGAP can be cleaved by caspases at the position 455 producing two fragments, fragment Ν and fragment C. Fragment Ν exerts

Adenosine A2B receptor-mediated leukemia inhibitory factor release from astrocytes protects cortical neurons against excitotoxicity.

ABSTRACT: BACKGROUND: Neuroprotective and neurotrophic properties of leukemia inhibitory factor (LIF) have been widely reported. In the central nervous system (CNS), astrocytes are the major source for LIF, expression of which is enhanced following disturbances leading to neuronal damage. How astrocytic LIF expression is regulated, however, has remained an unanswered question. Since neuronal stress is associated with production of extracellular adenosine, we investigated whether LIF expression in astrocytes was mediated through adenosine receptor signaling. METHODS: Mouse cortical neuronal and astrocyte cultures from wild-type and adenosine A2B receptor knock-out animals, as well as adenosine receptor agonists/antagonists and various enzymatic inhibitors, were used to study LIF expression and release in astrocytes. When needed, a one-way analysis of variance (ANOVA) followed by Bonferroni post-hoc test was used for statistical analysis. RESULTS: We show here that glutamate-stressed cortical neurons induce LIF expression through activation of adenosine A2B receptor subtype in cultured astrocytes and require signaling of protein kinase C (PKC), mitogen-activated protein kinases (MAPKs: p38 and ERK1/2), and the nuclear transcription factor (NF)-κB. Moreover, LIF concentration in the supernatant in response to 5'-N-ethylcarboxamide (NECA) stimulation was directly correlated to de novo protein synthesis, suggesting that LIF release did not occur through a regulated release pathway. Immunocytochemistry experiments show that LIF-containing vesicles co-localize with clathrin and Rab11, but not with pHogrin, Chromogranin (Cg)A and CgB, suggesting that LIF might be secreted through recycling endosomes. We further show that pre-treatment with supernatants from NECA-treated astrocytes increased survival of cultured cortical neurons against glutamate, which was absent when the supernatants were pre-treated with an anti-LIF neutralizing antibody. CONCLUSIONS: Adenosine from glutamate-stressed neurons induces rapid LIF release in astrocytes. This rapid release of LIF promotes the survival of cortical neurons against excitotoxicity.

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Neuroinflammation is observed in many brain pathologies: in neurodegenerative diseases and multiple sclerosis as well as in chemically induced lesions. It is characterized by the reactivity of microglial cells and astrocytes, activation of inducible NO-synthase (i-NOS), and increased expression and/or release of cytokines and chemokines. Clearly, cell-to-cell signaling between the different brain cell types plays an important role in the initiation and propagation of neuroinflammation, but despite the growing list of known molecular actors, the underlying pathways and the sequence of events remain to be fully elucidated. The present chapter presents an example of how to assess neuroinflammation in complex brain tissues, using aggregating brain cell cultures as an in vitro model. This three-dimensional cell culture system provides optimal cell-to-cell interactions crucial for histotypic cellular maturation and control of neuroinflammatory processes. The techniques described here comprise immunocytochemistry to assess the reactivity of microglia and astrocytes and the expression of cytokines; quantitative RT-PCR to measure the mRNA expression of cytokines (TNF-α, IL-1β, IL-6, IL-1ra, TGF-β, IL-15, IFN-γ), chemokines (ccl5, cxcl1, cxcl2), and i-NOS; and immunoblotting to assess MAP kinase pathway activation (phosphorylation of p38 and p44/42 MAP kinases).

The cdc7 protein kinase is a dosage dependent regulator of septum formation in fission yeast.

Mutation of the Schizosaccharomyces pombe cdc7 gene prevents formation of the division septum and cytokinesis. We have cloned the cdc7 gene and show that it encodes a protein kinase which is essential for cell division. In the absence of cdc7 function, spore germination, DNA synthesis and mitosis are unaffected, but cells are unable to initiate formation of the division septum. Overexpression of p120cdc7 causes cell cycle arrest; cells complete mitosis and then undergo multiple rounds of septum formation without cell cleavage. This phenotype, which is similar to that resulting from inactivation of cdc16 protein, requires the kinase activity of p120cdc7. Mutations inactivating the early septation gene, cdc11, suppress the formation of multiple septa and allow cells to proliferate normally. If formation of the division septum is prevented by inactivation of either cdc14 or cdc15, p120cdc7 overproduction does not interfere with other events in the mitotic cell cycle. Septation is not induced by overexpression of p120cdc7 in G2 arrested cells, indicating that it does not bypass the normal dependency of septation upon initiation of mitosis. These findings indicate that the p120cdc7 protein kinase plays a key role in initiation of septum formation and cytokinesis in fission yeast and suggest that p120cdc7 interacts with the cdc11 protein in the control of septation.

Regulation of endothelial cell integrin function and angiogenesis by COX-2, cAMP and Protein Kinase A.

Angiogenesis, the development of new blood vessels from preexisting vessels, is a key step in tumor growth, invasion and metastasis formation. Inhibition of tumor angiogenesis is considered as an attractive approach to suppress cancer progression and spreading. Adhesion receptors of the integrin family promote tumor angiogenesis by mediating cell migration, proliferation and survival of angiogenic endothelial cells. Integrins up regulated and highly expressed on neovascular endothelial cells, such as alphaVbeta3 and alpha5beta1, have been considered as relevant targets for anti-angiogenic therapies. Small molecular integrin antagonists or blocking antibodies suppress angiogenesis and tumor progression in many animal models, and some of them are currently being tested in cancer clinical trials as anti-angiogenic agents. COX-2 inhibitors exert anti-cancer effects, at least in part, by inhibiting tumor angiogenesis. We have recently shown that COX-2 inhibitors suppress endothelial cell migration and angiogenesis by preventing alphaVbeta3-mediated and cAMP/PKA-dependent activation of the small GTPases Rac and Cdc42. Here we will review the evidence for the involvement of vascular integrins in mediating angiogenesis and the role of COX-2 metabolites in modulating the cAMP/Protein Kinase A pathway and alphaVbeta3-dependent Rac activation in endothelial cells.

The gene MAPK8IP1, encoding islet-brain-1, is a candidate for type 2 diabetes.

Type 2 diabetes is a polygenic and genetically heterogeneous disease . The age of onset of the disease is usually late and environmental factors may be required to induce the complete diabetic phenotype. Susceptibility genes for diabetes have not yet been identified. Islet-brain-1 (IB1, encoded by MAPK8IP1), a novel DNA-binding transactivator of the glucose transporter GLUT2 (encoded by SLC2A2), is the homologue of the c-Jun amino-terminal kinase-interacting protein-1 (JIP-1; refs 2-5). We evaluated the role of IBi in beta-cells by expression of a MAPK8IP1 antisense RNA in a stable insulinoma beta-cell line. A 38% decrease in IB1 protein content resulted in a 49% and a 41% reduction in SLC2A2 and INS (encoding insulin) mRNA expression, respectively. In addition, we detected MAPK8IP1 transcripts and IBi protein in human pancreatic islets. These data establish MAPK8IP1 as a candidate gene for human diabetes. Sibpair analyses performed on i49 multiplex French families with type 2 diabetes excluded MAPK8IP1 as a major diabetogenic locus. We did, however, identify in one family a missense mutation located in the coding region of MAPK8IP1 (559N) that segregated with diabetes. In vitro, this mutation was associated with an inability of IB1 to prevent apoptosis induced by MAPK/ERK kinase kinase 1 (MEKK1) and a reduced ability to counteract the inhibitory action of the activated c-JUN amino-terminal kinase (JNK) pathway on INS transcriptional activity. Identification of this novel non-maturity onset diabetes of the young (MODY) form of diabetes demonstrates that IB1 is a key regulator of 3-cell function.

Subconjunctival Injection of XG-102, a c-Jun N-Terminal Kinase Inhibitor Peptide, in the Treatment of Endotoxin-Induced Uveitis in Rats.

Abstract Purpose: XG-102, a TAT-coupled dextrogyre peptide inhibiting the c-Jun N-terminal kinase, was shown efficient in the treatment of experimental uveitis. Preclinical studies are now performed to determine optimal XG-102 dose and route of administration in endotoxin-induced uveitis (EIU) in rats with the purpose of clinical study design. METHODS: EIU was induced in Lewis rats by lipopolysaccharides (LPS) injection. XG-102 was administered at the time of LPS challenge by intravenous (IV; 3.2, 35 or 355 μg/injection), intravitreal (IVT; 0.08, 0.2 or 2.2 μg/eye), or subconjunctival (SCJ; 0.2, 1.8 or 22 μg/eye) routes. Controls received either the vehicle (saline) or dexamethasone phosphate injections. Efficacy was assessed by clinical scoring, infiltrating cells count, and expression of inflammatory mediators [inducible nitric oxide synthase (iNOS), cytokine-induced neutrophil chemoattractant-1 (CINC-1)]. The effect of XG-102 on phosphorylation of c-Jun was evaluated by Western blot. RESULTS: XG-102 demonstrated a dose-dependent anti-inflammatory effect in EIU after IV and SCJ administrations. Respective doses of 35 and 1.8 μg were efficient as compared with the vehicle-injected controls, but only the highest doses, respectively 355 and 22 μg, were as efficient as dexamethasone phosphate. After IVT injections, the anti-inflammatory effect of XG-102 was clinically evaluated similar to the corticoid's effect with all the tested doses. Regardless of the administration route, the lowest efficient doses of XG-102 significantly decreased the ration of phospho c-Jun/total c-Jun, reduced cells infiltration in the treated eyes, and significantly downregulated iNOS and CINC-1 expression in the retina. CONCLUSION: These results confirm that XG-102 peptide has potential for treating intraocular inflammation. SCJ injection appears as a good compromise to provide a therapeutic effect while limiting side effects.

Zoledronate sensitizes endothelial cells to tumor necrosis factor-induced programmed cell death: evidence for the suppression of sustained activation of focal adhesion kinase and protein kinase B/Akt.

Bisphosphonates are potent inhibitors of osteoclast function widely used to treat conditions of excessive bone resorption, including tumor bone metastases. Recent evidence indicates that bisphosphonates have direct cytotoxic activity on tumor cells and suppress angiogenesis, but the associated molecular events have not been fully characterized. In this study we investigated the effects of zoledronate, a nitrogen-containing bisphosphonate, and clodronate, a non-nitrogen-containing bisphosphonate, on human umbilical vein endothelial cell (HUVEC) adhesion, migration, and survival, three events essential for angiogenesis. Zoledronate inhibited HUVEC adhesion mediated by integrin alphaVbeta3, but not alpha5beta1, blocked migration and disrupted established focal adhesions and actin stress fibers without modifying cell surface integrin expression level or affinity. Zoledronate treatment slightly decreased HUVEC viability and strongly enhanced tumor necrosis factor (TNF)-induced cell death. HUVEC treated with zoledronate and TNF died without evidence of enhanced annexin-V binding, chromatin condensation, or nuclear fragmentation and caspase dependence. Zoledronate inhibited sustained phosphorylation of focal adhesion kinase (FAK) and in combination with TNF, with and without interferon (IFN) gamma, of protein kinase B (PKB/Akt). Constitutive active PKB/Akt protected HUVEC from death induced by zoledronate and TNF/IFNgamma. Phosphorylation of c-Src and activation of NF-kappaB were not affected by zoledronate. Clodronate had no effect on HUVEC adhesion, migration, and survival nor did it enhanced TNF cytotoxicity. Taken together these data demonstrate that zoledronate sensitizes endothelial cells to TNF-induced, caspase-independent programmed cell death and point to the FAK-PKB/Akt pathway as a novel zoledronate target. These results have potential implications to the clinical use of zoledronate as an anti-angiogenic or anti-cancer agent.