Glucagon-like peptide-1 protects beta-cells against apoptosis by increasing the activity of an IGF-2/IGF-1 receptor autocrine loop.

OBJECTIVE: The gluco-incretin hormones glucagon-like peptide (GLP)-1 and gastric inhibitory peptide (GIP) protect beta-cells against cytokine-induced apoptosis. Their action is initiated by binding to specific receptors that activate the cAMP signaling pathway, but the downstream events are not fully elucidated. Here we searched for mechanisms that may underlie this protective effect. RESEARCH DESIGN AND METHODS: We performed comparative transcriptomic analysis of islets from control and GipR(-/-);Glp-1-R(-/-) mice, which have increased sensitivity to cytokine-induced apoptosis. We found that IGF-1 receptor expression was markedly reduced in the mutant islets. Because the IGF-1 receptor signaling pathway is known for its antiapoptotic effect, we explored the relationship between gluco-incretin action, IGF-1 receptor expression and signaling, and apoptosis. RESULTS: We found that GLP-1 robustly stimulated IGF-1 receptor expression and Akt phosphorylation and that increased Akt phosphorylation was dependent on IGF-1 but not insulin receptor expression. We demonstrated that GLP-1-induced Akt phosphorylation required active secretion, indicating the presence of an autocrine activation mechanism; we showed that activation of IGF-1 receptor signaling was dependent on the secretion of IGF-2. We demonstrated, both in MIN6 cell line and primary beta-cells, that reducing IGF-1 receptor or IGF-2 expression or neutralizing secreted IGF-2 suppressed GLP-1-induced protection against apoptosis. CONCLUSIONS: An IGF-2/IGF-1 receptor autocrine loop operates in beta-cells. GLP-1 increases its activity by augmenting IGF-1 receptor expression and by stimulating secretion; this mechanism is required for GLP-1-induced protection against apoptosis. These findings may lead to novel ways of preventing beta-cell loss in the pathogenesis of diabetes.

Downregulation of cannabinoid receptor 1 from neuropeptide Y interneurons in the basal ganglia of patients with Huntington's disease and mouse models.

Cannabinoid receptor 1 (CB(1) receptor) controls several neuronal functions, including neurotransmitter release, synaptic plasticity, gene expression and neuronal viability. Downregulation of CB(1) expression in the basal ganglia of patients with Huntington's disease (HD) and animal models represents one of the earliest molecular events induced by mutant huntingtin (mHtt). This early disruption of neuronal CB(1) signaling is thought to contribute to HD symptoms and neurodegeneration. Here we determined whether CB(1) downregulation measured in patients with HD and mouse models was ubiquitous or restricted to specific striatal neuronal subpopulations. Using unbiased semi-quantitative immunohistochemistry, we confirmed previous studies showing that CB(1) expression is downregulated in medium spiny neurons of the indirect pathway, and found that CB(1) is also downregulated in neuropeptide Y (NPY)/neuronal nitric oxide synthase (nNOS)-expressing interneurons while remaining unchanged in parvalbumin- and calretinin-expressing interneurons. CB(1) downregulation in striatal NPY/nNOS-expressing interneurons occurs in R6/2 mice, Hdh(Q150/Q150) mice and the caudate nucleus of patients with HD. In R6/2 mice, CB(1) downregulation in NPY/nNOS-expressing interneurons correlates with diffuse expression of mHtt in the soma. This downregulation also occludes the ability of cannabinoid agonists to activate the pro-survival signaling molecule cAMP response element-binding protein in NPY/nNOS-expressing interneurons. Loss of CB(1) signaling in NPY/nNOS-expressing interneurons could contribute to the impairment of basal ganglia functions linked to HD.

TAK1 is required for survival of mouse fibroblasts treated with TRAIL, and does so by NF-kappaB dependent induction of cFLIPL.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is known as a "death ligand"-a member of the TNF superfamily that binds to receptors bearing death domains. As well as causing apoptosis of certain types of tumor cells, TRAIL can activate both NF-kappaB and JNK signalling pathways. To determine the role of TGF-beta-Activated Kinase-1 (TAK1) in TRAIL signalling, we analyzed the effects of adding TRAIL to mouse embryonic fibroblasts (MEFs) derived from TAK1 conditional knockout mice. TAK1-/- MEFs were significantly more sensitive to killing by TRAIL than wild-type MEFs, and failed to activate NF-kappaB or JNK. Overexpression of IKK2-EE, a constitutive activator of NF-kappaB, protected TAK1-/- MEFs against TRAIL killing, suggesting that TAK1 activation of NF-kappaB is critical for the viability of cells treated with TRAIL. Consistent with this model, TRAIL failed to induce the survival genes cIAP2 and cFlipL in the absence of TAK1, whereas activation of NF-kappaB by IKK2-EE restored the levels of both proteins. Moreover, ectopic expression of cFlipL, but not cIAP2, in TAK1-/- MEFs strongly inhibited TRAIL-induced cell death. These results indicate that cells that survive TRAIL treatment may do so by activation of a TAK1-NF-kappaB pathway that drives expression of cFlipL, and suggest that TAK1 may be a good target for overcoming TRAIL resistance.

MALT1 and Caspase-10 : two cysteine proteases controlling lymphocyte activation

Τ cell activation via the Τ cell receptor (TCR) through antigen recognition is one of the key steps to initiate the adaptive immune response. The mechanisms controlling TCR-induced signaling pathways are the subject of intense research, since deregulated signaling in lymphocytes can lead to immunodeficiency, autoimmunity or lymphomas. In Τ lymphocytes a complex composed of CARMA1, BCL10 and MALT1 has been identified to receive signals from TCR proximal events and to induce further signals crucial for Τ cell activation. MALT1 is scaffold protein and a cysteine protease and both functions have been shown, among other effects, to be crucial to initiate the activation of the transcription factors of the nuclear factor κΒ (NF-κΒ) family after TCR-stimulation. Several proteolytic targets have been described recently and all of them play roles in modulating NF-κΒ activation or other aspects of Τ cell activation. In this study, we describe a novel target of MALT1, Caspase-10. Two isoforms of Caspase-10 are cleaved by MALTI in Τ and Β cells after antigen receptor stimulation. Caspases are a family of cysteine proteases that are known for their roles in cell death and certain immune functions. Caspase-10 has so far only been reported to be involved in the induction of apoptosis. However it is very closely related to the well-characterized Caspase-8 that has been reported to be involved in Τ cell activation. In the present study, we describe a crucial role for Caspase-10, but not Caspase-8, in Τ cell activation after TCR stimulation. Jurkat Τ cells silenced for Caspase-10 expression exhibit a dramatic reduction in IL-2 production following stimulation. The data obtained revealed that this is due to severely reduced activation of activator protein-1 (AP-1), another transcription factor family with key functions in the process of Τ cell activation. We observed strongly reduced expression levels of the AP-1 family member c-Fos after Τ cell stimulation. This transcription factor is expressed upon TCR stimulation and is a crucial component of AP-1 transcription factor dimers required for Τ cell activation. In further analysis, it was shown that this defect is not based on reduced transcription, as the c-Fos mRNA levels are not altered, but rather seems to be caused by a defect in translation or protein stability in the absence of Caspase-10. Furthermore, we report a potential interaction of the c-Fos protein and Caspsae-10. This role of Caspase-10 in AP-1 activation however is independent of its cleavage by MALT1, leaving the role of Caspase-10 cleavage in activated lymphocytes unclear. Taken together, these results give new insights into the complex matter of lymphocyte activation whose understanding is crucial for the development of new drugs modulating the immune response or inhibiting lymphoma progression.

Mutated regions of nucleophosmin 1 elicit both CD4+ and CD8+ T-cell responses in patients with acute myeloid leukemia.

Mutations in the nucleophosmin gene (NPM1(mut)) are one of the most frequent molecular alterations in acute myeloid leukemia (AML), and immune responses may contribute to the favorable prognosis of AML patients with NPM1(mut). In the present study, we were able to demonstrate both CD4(+) and CD8(+) T-cell responses against NPM1(mut). Ten peptides derived from wild-type NPM1 and NPM1(mut) were subjected to ELISPOT analysis in 33 healthy volunteers and 27 AML patients. Tetramer assays against the most interesting epitopes were performed and Cr(51)-release assays were used to show the cytotoxicity of peptide-specific T cells. Moreover, HLA-DR-binding epitopes were used to test the role of CD4(+) T cells in NPM1 immunogenicity. Two epitopes (epitopes #1 and #3) derived from NPM1(mut) induced CD8(+) T-cell responses. A total of 33% of the NPM1(mut) AML patients showed immune responses against epitope #1 and 44% against epitope #3. Specific lysis of leukemic blasts was detected. To obtain robust immune responses against tumor cells, the activation of CD4(+) T cells is crucial. Therefore, overlapping (OL) peptides were analyzed in ELISPOT assays and OL8 was able to activate both CD8(+) and CD4(+) T cells. The results of the present study show that NPM1(mut) induces specific T-cell responses of CD4(+) and CD8(+) T cells and therefore is a promising target for specific immunotherapies in AML.

Activation mechanisms of the protease MALT1 and cleavage of one of its targets - the ribonuclease Regnase-1- in lymphocytes and lymphoma cell lines

Persistent infection induces an adaptive immune response that is mediated by T and B lymphocytes. Upon triggering with an antigen, these cells become activated and turn into fast expanding cells able to efficiently defend the host. Lymphocyte activation is controlled by a complex composed of CARMA1, BCL10 and MALT1 which regulates the NF-KB signaling pathway upon antigen triggering. Abnormally high expression or activity of either one of these three proteins can favor the development of lymphomas, while genetic defects in the pathway are associated with immunodeficiency. MALT1 was identified as a paracaspase sharing homology with other cysteine proteases, namely caspases and metacaspases. In order to be active, caspases need to dimerize. Based on their sequence similarity with MALT1, we hypothesized that dimerization might also be a mechanism of activation employed by MALT1. To address this assumption, we performed a bioinformatics modelling based on the crystal structures of several caspases. Our model suggested that the MALT1 caspase-like domain can indeed form dimers. This finding was later confirmed by several published crystal structures of MALT1. In the dimer interface of our model, we noticed the presence of charged amino acids that could potentially form salt bridges and thereby hold both monomers together. Mutation of one of these residues, E549, into alanine completely blocked the catalytic activity of MALT1. Additionally, we provided evidence for a role of E549 in promoting the MALTl-dependent growth of cells derived from diffuse large B cell lymphoma (DLBCL) of the aggressive B cell-like type (ABC). To our initial surprise, the E549A mutation showed only a partial defect in dimerization, indicating that additional residues are essential to form a stable dimer. The MALT1 crystal structures revealed a key function for E549 in stabilizing the catalytic site of the protease via its interaction with an arginine which is located next to the catalytic active cysteine. In an additional study, we discovered that MALT1 monoubiquitination is required for the catalytic activity of the protease. Interestingly, we found that the MALT1 dimer interface mutant E549A could not be monoubiquitinated. Based on these findings, we suggest that correct formation of the dimer interface is a prerequisite for monoubiquitination. In a second project, we discovered a novel target of the protease MALT1, the ribonuclease Regnase¬la It was described that the RNase activity of Regnase-1 negatively regulates immune responses. We could show that in ABC DLBCL cell lines, Regnase-1 is not only cleaved by MALT1 but also phosphorylated, at least in part, by the inhibitor of KB kinase (IKK). Both regulations appear to restrain the RNase function of Regnase-1 and thereby allow the production of pro-survival proteins. In conclusion, our studies further highlight and explain the importance of the catalytic activity of MALT1 for the activation of lymphocytes and provide additional knowledge for the development of specific drugs targeting the catalytic activity of MALT1 for immunomodulation and treatment of lymphomas.  SUMMARY IN FRENCH PhD Thesis Katrin Cabalzar 2 SUMMARY IN FRENCH Une infection persistante induit une réponse immunitaire adaptative par l'intermédiaire des lymphocytes T et B. Quand elles reconnaissent l'antigène, ces cellules sont activées et se multiplient très rapidement pour défendre efficacement l'hôte. L'activation des lymphocytes est transmise par un complexe composé de trois protéines, CARMA1, BCL10 et MALT1, qui régule la voie de signalisation NF-KB lorsque l'antigène est reconnu. L'expression ou l'activité anormalement élevée de l'une de ces trois protéines peut favoriser le développement de lymphomes, tandis que des défauts génétiques de cette voie de signalisation sont associés à l'immunodéficience. MALT1 a été identifiée comme étant une paracaspase qui partage des séquences homologues avec d'autres protéases à cystéine, comme les caspases et les métacaspases. Pour être actives, les caspases ont besoin de dimériser. Etant donné leur similarité de séquence avec MALT1, nous avons supposé que la dimérisation pouvait aussi être un mécanisme d'activation utilisé par MALT1. Pour vérifier cette hypothèse, nous avons conçu un modèle bioinformatique à partir des structures cristallographiques de plusieurs caspases. Et notre modèle a suggéré que le domaine catalytique de MALT1 était effectivement capable de former des dimères. Cette découverte a été confirmée plus tard par des publications qui montrent des structures cristallographiques dimériques de MALT1. Dans l'interface du dimère de notre modèle, nous avons remarqué la présence d'acides aminés chargés qui pouvaient former des liaisons ioniques et ainsi réunir les deux monomères. La mutation de l'un de ces résidus, E549, pour une alanine, a complètement inhibé l'activité catalytique de MALT1. De plus, nous avons mis en évidence un rôle d'E549 dans la croissance dépendante de MALT1, des cellules dérivées de lymphomes B diffus à grandes cellules (DLBCL) de sous-type cellules B actives (ABC). Dans un premier temps nous avons été surpris de constater que cette mutation révélait seulement un défaut partiel de dimérisation, ce qui indique que des acides aminés supplémentaires sont indispensables pour former un dimère stable. Les structures cristallographiques de MALT1 ont révélé un rôle primordial d'E549 dans la stabilisation du site catalytique de la protéase via son interaction avec une arginine qui se trouve à côté de la cystéine du site actif. Dans une autre étude, nous avons découvert que la monoubiquitination de MALT1 est requise pour l'activité catalytique de la protéase. A remarquer que nous avons trouvé que le mutant E549A de l'interface dimère de MALT1 n'a pas pu être monoubiquitiné. Sur la base de ces résultats, nous suggérons que la formation correcte de l'interface du dimère est une condition préalable pour la monoubiquitination. Dans un second projet, nous avons découvert une nouvelle cible de la protéase MALT1, la ribonucléase Regnase-1. Il a été décrit que l'activité RNase de Regnase-1 régulait négativement les réponses immunitaires. Nous avons pu montrer que dans les lignées cellulaires ABC DLBCL, la Regnase-1 n'était pas seulement clivée par MALT1 mais également phosphorylée, au moins en partie, par la kinase de l'inhibiteur de KB (IKK). Les deux régulations semblent supprimer la fonction RNase de Regnase-1 et permettre ainsi la stabilisation de certains ARN messagers et la production de protéines favorisant la survie. En conclusion, nos études mettent en évidence le rôle-clé de la dimérisation de MALT1 et expliquent l'importance de l'activité catalytique de MALT1 pour l'activation des lymphocytes. Ainsi, nos résultats apportent des connaissances supplémentaires pour le développement de médicaments spécifiques ciblant l'activité catalytique de MALT1, qui pourraient être utiles pour modifier les réponses immunitaires et traiter des lymphomes.

Regulation of lymphocyte proliferation and death by FLIP.

Lymphocyte homeostasis is a balance between lymphocyte proliferation and lymphocyte death. Tight control of apoptosis is essential for immune function, because its altered regulation can result in cancer and autoimmunity. Signals from members of the tumour-necrosis-factor receptor (TNF-R) family, such as Fas and TNF-R1, activate the caspase cascade and result in lymphocyte death by apoptosis. Anti-apoptotic proteins, such as FLIP (also known as FLICE/caspase-8 inhibitory protein) have recently been identified. FLIP expression is tightly regulated in T cells and might be involved in the control of both T-cell activation and death. Abnormal expression of FLIP might have a role not only in autoimmune diseases, but also in tumour development and cardiovascular disorders.

Calcineurin inhibitors activate the proto-oncogene Ras and promote protumorigenic signals in renal cancer cells.

The development of cancer is a major problem in immunosuppressed patients, particularly after solid organ transplantation. We have recently shown that calcineurin inhibitors (CNI) used to treat transplant patients may play a critical role in the rapid progression of renal cancer. To examine the intracellular signaling events for CNI-mediated direct tumorigenic pathway(s), we studied the effect of CNI on the activation of proto-oncogenic Ras in human normal renal epithelial cells (REC) and renal cancer cells (786-0 and Caki-1). We found that CNI treatment significantly increased the level of activated GTP-bound form of Ras in these cells. In addition, CNI induced the association of Ras with one of its effector molecules, Raf, but not with Rho and phosphatidylinositol 3-kinase; CNI treatment also promoted the phosphorylation of the Raf kinase inhibitory protein and the downregulation of carabin, all of which may lead to the activation of the Ras-Raf pathway. Blockade of this pathway through either pharmacologic inhibitors or gene-specific small interfering RNA significantly inhibited CNI-mediated augmented proliferation of renal cancer cells. Finally, it was observed that CNI treatment increased the growth of human renal tumors in vivo, and the Ras-Raf pathway is significantly activated in the tumor tissues of CNI-treated mice. Together, targeting the Ras-Raf pathway may prevent the development/progression of renal cancer in CNI-treated patients.

Fibronectin-binding proteins and clumping factor A in Staphylococcus aureus experimental endocarditis: FnBPA is sufficient to activate human endothelial cells.

Surface molecules of Staphylococcus aureus are involved in the colonization of vascular endothelium which is a crucial primary event in the pathogenesis of infective endocarditis (IE). The ability of these molecules to also launch endothelial procoagulant and proinflammatory responses, which characterize IE, is not known. In the present study we investigated the individual capacities of three prominent S. aureus surface molecules; fibronectin-binding protein A (FnBPA) and B (FnBPB) and clumping factor A (ClfA), to promote bacterial adherence to cultured human endothelial cells (ECs) and to activate phenotypic and functional changes in these ECs. Non-invasive surrogate bacterium Lactococcus lactis, which, by gene transfer, expressed staphylococcal FnBPA, FnBPB or ClfA molecules were used. Infection of ECs increased 50- to 100-fold with FnBPA- or FnBPB-positive recombinant lactococci. This coincided with EC activation, interleukin-8 secretion and surface expression of ICAM-1 and VCAM-1 and concomitant monocyte adhesion. Infection with ClfA-positive lactococci did not activate EC. FnBPA-positive L. lactis also induced a prominent tissue factor-dependent endothelial coagulation response that was intensified by cell-bound monocytes. Thus S. aureus FnBPs, but not ClfA, confer invasiveness and pathogenicity to non-pathogenic L. lactis organisms indicating that bacterium-EC interactions mediated by these adhesins are sufficient to evoke inflammation as well as procoagulant activity at infected endovascular sites.

DAI/ZBP1 recruits RIP1 and RIP3 through RIP homotypic interaction motifs to activate NF-kappaB.

Detection of viral nucleic acids is central to antiviral immunity. Recently, DAI/ZBP1 (DNA-dependent activator of IRFs/Z-DNA binding protein 1) was identified as a cytoplasmic DNA sensor and shown to activate the interferon regulatory factor (IRF) and nuclear factor-kappa B (NF-kappaB) transcription factors, leading to type-I interferon production. DAI-induced IRF activation depends on TANK-binding kinase 1 (TBK1), whereas signalling pathways and molecular components involved in NF-kappaB activation remain elusive. Here, we report the identification of two receptor-interacting protein (RIP) homotypic interaction motifs (RHIMs) in the DAI protein sequence, and show that these domains relay DAI-induced NF-kappaB signals through the recruitment of the RHIM-containing kinases RIP1 and RIP3. We show that knockdown of not only RIP1, but also RIP3 affects DAI-induced NF-kappaB activation. Importantly, RIP recruitment to DAI is inhibited by the RHIM-containing murine cytomegalovirus (MCMV) protein M45. These findings delineate the DAI signalling pathway to NF-kappaB and suggest a possible new immune modulation strategy of the MCMV.

The antiviral adaptor proteins Cardif and Trif are processed and inactivated by caspases

The outcome of a viral infection depends on the interplay between the host's capacity to trigger potent antiviral responses and viral mechanisms that counteract them. Although Toll-like receptor (TLR)-3, which recognizes virally derived double-stranded (ds) RNA, transmits downstream antiviral signaling through the TIR adaptor Trif (TICAM-1), viral RNA-sensing RIG-like helicases (RLHs) use the mitochondrial-bound CARD protein Cardif (IPS-1/MAVS/VISA). The importance of these two antiviral signaling pathways is reflected by the fact that both adaptors are inhibited through specific cleavage triggered by the hepatitis C virus serine protease NS3-4A. Here, we show that inactivation can also occur through cellular caspases activated by various pro-apoptotic signals. Upon caspase-dependent cleavage both adaptors loose their capacity to activate the transcription factors interferon regulatory factors (IRF) and NF-kappaB. Importantly, poliovirus infection triggers a caspase-dependent cleavage of Cardif, suggesting that some viruses may activate caspases not only as a mean to facilitate shedding and replication, but also to impair antiviral responses

Retinoid X receptor and peroxisome proliferator-activated receptor activate an estrogen responsive gene independent of the estrogen receptor.

Estrogen receptors regulate transcription of genes essential for sexual development and reproductive function. Since the retinoid X receptor (RXR) is able to modulate estrogen responsive genes and both 9-cis RA and fatty acids influenced development of estrogen responsive tumors, we hypothesized that estrogen responsive genes might be modulated by RXR and the fatty acid receptor (peroxisome proliferator-activated receptor, PPAR). To test this hypothesis, transfection assays in CV-1 cells were performed with an estrogen response element (ERE) coupled to a luciferase reporter construct. Addition of expression vectors for RXR and PPAR resulted in an 11-fold increase in luciferase activity in the presence of 9-cis RA. Furthermore, mobility shift assays demonstrated binding of RXR and PPAR to the vitellogenin A2-ERE and an ERE in the oxytocin promoter. Methylation interference assays demonstrated that specific guanine residues required for RXR/PPAR binding to the ERE were similar to residues required for ER binding. Moreover, RXR domain-deleted constructs in transfection assays showed that activation required RXR since an RXR delta AF-2 mutant completely abrogated reporter activity. Oligoprecipitation binding studies with biotinylated ERE and (35)S-labeled in vitro translated RXR constructs confirmed binding of delta AF-2 RXR mutant to the ERE in the presence of baculovirus-expressed PPAR. Finally, in situ hybridization confirmed RXR and PPAR mRNA expression in estrogen responsive tissues. Collectively, these data suggest that RXR and PPAR are present in reproductive tissues, are capable of activating estrogen responsive genes and suggest that the mechanism of activation may involve direct binding of the receptors to estrogen response elements.

Localization of HLA-A2.1-restricted T cell epitopes in the circumsporozoite protein of Plasmodium falciparum.

Localization of human MHC class I-restricted T cell epitopes in the circumsporozoite (CS) protein of the human parasite Plasmodium falciparum is an important objective in the development of antimalarial vaccines. To this purpose, we synthesized a series of overlapping synthetic 20-mer peptides, spanning the entire sequence of the 7G8 CS molecule except for the central repeat B cell domain. The P.f.CS peptides were first tested for their ability to bind to the human MHC class I HLA-A2.1 molecule on T2, a human cell line. Subsequently, the use of a series of shorter peptide analogues allowed us to determine the optimal A2.1 binding sequence present in several of the 20-mers. Binding P.f.CS peptides were further tested for their capacity to activate PBL from HLA-A2.1+ immune donors living in a malaria-endemic area. Specific IFN-gamma production was detected in the supernatant of cultures of PBL from exposed individuals. Cytotoxic T cell lines and clones were derived from the PBL of one responder, and their activity was shown to be HLA-A2.1-restricted and specific for the peptide 334-342 of the CS protein. In addition, double transgenic HLA-A2.1 x human beta 2-microglobulin mice were immunized with peptide 1-10 of the CS protein. T cells derived from immune lymph nodes displayed a peptide-specific HLA-A2.1-restricted cytolytic activity after one in vitro stimulation.

Role of salt-inducible kinase 1 in the activation of MEF2-dependent transcription by BDNF.

Substantial evidence supports a role for myocyte enhancer factor 2 (MEF2)-mediated transcription in neuronal survival, differentiation and synaptic function. In developing neurons, it has been shown that MEF2-dependent transcription is regulated by neurotrophins. Despite these observations, little is known about the cellular mechanisms by which neurotrophins activate MEF2 transcriptional activity. In this study, we examined the role of salt-inducible kinase 1 (SIK1), a member of the AMP-activated protein kinase (AMPK) family, in the regulation of MEF2-mediated transcription by the neurotrophin brain-derived neurotrophic factor (BDNF). We show that BDNF increases the expression of SIK1 in primary cultures of rat cortical neurons through the extracellular signal-regulated kinase 1/2 (ERK1/2)-signaling pathway. In addition to inducing SIK1 expression, BDNF triggers the phosphorylation of SIK1 at Thr182 and its translocation from the cytoplasm to the nucleus of cortical neurons. The effects of BDNF on the expression, phosphorylation and, translocation of SIK1 are followed by the phosphorylation and nuclear export of histone deacetylase 5 (HDAC5). Blockade of SIK activity with a low concentration of staurosporine abolished BDNF-induced phosphorylation and nuclear export of HDAC5 in cortical neurons. Importantly, stimulation of HDAC5 phosphorylation and nuclear export by BDNF is accompanied by the activation of MEF2-mediated transcription, an effect that is suppressed by staurosporine. Consistent with these data, BDNF induces the expression of the MEF2 target genes Arc and Nur77, in a staurosporine-sensitive manner. In further support of the role of SIK1 in the regulation of MEF2-dependent transcription by BDNF, we found that expression of wild-type SIK1 or S577A SIK1, a mutated form of SIK1 which is retained in the nucleus of transfected cells, is sufficient to enhance MEF2 transcriptional activity in cortical neurons. Together, these data identify a previously unrecognized mechanism by which SIK1 mediates the activation of MEF2-dependent transcription by BDNF.

Differential involvement of MEK kinase 1 (MEKK1) in the induction of apoptosis in response to microtubule-targeted drugs versus DNA damaging agents.

MEK kinase 1 (MEKK1) is a 196-kDa enzyme that is involved in the regulation of the c-Jun N-terminal kinase (JNK) pathway and apoptosis. In cells exposed to genotoxic agents including etoposide and cytosine arabinoside, MEKK1 is cleaved at Asp874 by caspases. The cleaved kinase domain of MEKK1, itself, stimulates caspase activity leading to apoptosis. Kinase-inactive MEKK1 expressed in HEK293 cells effectively blocks genotoxin-induced apoptosis. Treatment of cells with taxol, a microtubule stabilizing agent, did not induce MEKK1 cleavage in cells, and kinase-inactive MEKK1 expression failed to block taxol-induced apoptosis. MEKK1 became activated in HEK293 cells exposed to taxol, but in contrast to etoposide-treatment, taxol failed to increase JNK activity. Taxol treatment of cells, therefore, dissociates MEKK1 activation from the regulation of the JNK pathway. Overexpression of anti-apoptotic Bcl2 blocked MEKK1 and taxol-induced apoptosis but did not block the caspase-dependent cleavage of MEKK1 in response to etoposide. This indicates Bcl2 inhibition of apoptosis is, therefore, downstream of caspase-dependent MEKK1 cleavage. The results define the involvement of MEKK1 in the induction of apoptosis by genotoxins but not microtubule altering drugs.