Jana V. Maier

Aplidin induces JNK-dependent apoptosis in human breast cancer cells via alteration of glutathione homeostasis, Rac1 GTPase activation, and MKP-1 phosphatase downregulation.

Aplidin® is an antitumor agent in phase II clinical trials that induces apoptosis through the sustained activation of Jun N-terminal kinase (JNK). We report that Aplidin® alters glutathione homeostasis increasing the ratio of oxidized to reduced forms (GSSG/GSH). Aplidin® generates reactive oxygen species and disrupts the mitochondrial membrane potential. Exogenous GSH inhibits these effects and also JNK activation and cell death. We found two mechanisms by which Aplidin® activates JNK: rapid activation of Rac1 small GTPase and downregulation of MKP-1 phosphatase. Rac1 activation was diminished by GSH and enhanced by L-buthionine (SR)-sulfoximine, which inhibits GSH synthesis. Downregulation of Rac1 by transfection of small interfering RNA (siRNA) duplexes or the use of a specific Rac1 inhibitor decreased Aplidin®-induced JNK activation and cytotoxicity. Our results show that Aplidin® induces apoptosis by increasing the GSSG/GSH ratio, a necessary step for induction of oxidative stress and sustained JNK activation through Rac1 activation and MKP-1 downregulation.

Cross-talk between the p38α and JNK MAPK Pathways Mediated by MAP Kinase Phosphatase-1 Determines Cellular Sensitivity to UV Radiation

MAPK phosphatase-1 (DUSP1/MKP-1) is a mitogen and stress-inducible dual specificity protein phosphatase, which can inactivate all three major classes of MAPK in mammalian cells. DUSP1/MKP-1 is implicated in cellular protection against a variety of genotoxic insults including hydrogen peroxide, ionizing radiation, and cisplatin, but its role in the interplay between different MAPK pathways in determining cell death and survival is not fully understood. We have used pharmacological and genetic tools to demonstrate that DUSP1/MKP-1 is an essential non-redundant regulator of UV-induced cell death in mouse embryo fibroblasts (MEFs). The induction of DUSP1/MKP-1 mRNA and protein in response to UV radiation is mediated by activation of the p38α but not the JNK1 or JNK2 MAPK pathways. Furthermore, we identify MSK1 and -2 and their downstream effectors cAMP-response element-binding protein/ATF1 as mediators of UV-induced p38α-dependent DUSP1/MKP-1 transcription. Dusp1/Mkp-1 null MEFs display increased signaling through both the p38α and JNK MAPK pathways and are acutely sensitive to UV-induced apoptosis. This lethality is rescued by the reintroduction of wild-type DUSP1/MKP-1 and by a mutant of DUSP1/MKP-1, which is unable to bind to either p38α or ERK1/2, but retains full activity toward JNK. Importantly, whereas small interfering RNA-mediated knockdown of DUSP1/MKP-1 sensitizes wild-type MEFs to UV radiation, DUSP1/MKP-1 knockdown in MEFS lacking JNK1 and -2 does not result in increased cell death. Our results demonstrate that cross-talk between the p38α and JNK pathways mediated by induction of DUSP1/MKP-1 regulates the cellular response to UV radiation.

Restriction to Fos Family Members of Trip6-Dependent Coactivation and Glucocorticoid Receptor-Dependent Trans-Repression of Activator Protein-1

The term activator protein (AP)-1 describes homodimeric and heterodimeric transcription factors composed of members of the Jun, Fos, and cAMP response element-binding protein (CREB)/activating transcription factor (ATF) families of proteins. Distinct AP-1 dimers, for instance the prototypical c-Jun:c-Fos and c-Jun:ATF2 dimers, are differentially regulated by signaling pathways and bind related yet distinct response elements in the regulatory regions of AP-1 target genes. Little is known about the dimer-specific regulation of AP-1 activity at the promoter of its target genes. We have previously shown that nTrip6, the nuclear isoform of the LIM domain protein Trip6, acts as an AP-1 coactivator. Moreover, nTrip6 is an essential component of glucocorticoid receptor (GR)-mediated trans-repression of AP-1, in that it mediates the tethering of GR to the promoter-bound AP-1. We have now discovered a striking specificity of nTrip6 actions determined by the binding preference of its LIM domains. We show that nTrip6 interacts only with Fos family members. Consequently, nTrip6 is a selective coactivator for AP-1 dimers containing Fos. nTrip6 also assembles activated GR to c-Jun:c-Fos-driven promoters. Neither nTrip6 nor GR are recruited to a promoter occupied by c-Jun:ATF2. Thus, only Fos-containing dimers are trans-repressed by GR. Thus, the dimer composition of AP-1 determines the mechanism of both the positive and negative regulation of AP-1 transcriptional activity. Interestingly, on a second level of action, GR represses the increase in transcriptional activity of c-Jun:ATF2 induced by c-Jun N-terminal kinase (JNK)-dependent phosphorylation. This repression depends on GR-mediated induction of MAPK phosphatase 1 (MKP-1) expression, which results in c-Jun N-terminal kinase inactivation.

Allergen Arrays for Antibody Screening and Immune Cell Activation Profiling Generated by Parallel Lipid Dip‐Pen Nanolithography

Multiple-allergen testing for high throughput and high sensitivity requires the development of miniaturized immunoassays that allow for a large test area and require only a small volume of the test analyte, which is often available only in limited amounts. Developing such miniaturized biochips containing arrays of test allergens needs application of a technique able to deposit molecules at high resolution and speed while preserving its functionality. Lipid dip-pen nanolithography (L-DPN) is an ideal technique to create such biologically active surfaces, and it has already been successfully applied for the direct, nanoscale deposition of functional proteins, as well as for the fabrication of biochemical templates for selective adsorption. The work presented here shows the application of L-DPN for the generation of arrays of the ligand 2,4-dinitrophenyl[1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine-N-[6-[(2,4-dinitrophenyl)amino]hexanoyl] (DNP)] onto glass surfaces as a model system for detection of allergen-specific Immunoglobin E (IgE) antibodies and for mast cell activation profiling.

PEST-domain-enriched tyrosine phosphatase and glucocorticoids as regulators of anaphylaxis in mice

To cite this article: Obiri DD, Flink N, Maier JV, Neeb A, Maddalo D, Thiele W, Menon A, Stassen M, Kulkarni RA, Garabedian MJ, Barrios AM, Cato ACB. PEST‐domain‐enriched tyrosine phosphatase and glucocorticoids as regulators of anaphylaxis in mice. Allergy 2012; 67: 175–182.

Depletion of the cellular levels of Bag-1 proteins attenuates phorbol ester-induced downregulation of IκBα and nuclear accumulation of NF-κB

Bag-1 consists in humans of four isoforms generated from the same RNA by alternative translation. Overexpression of single Bag-1 isoforms has identified Bag-1 as a negative regulator of action of many proteins including the glucocorticoid receptor (GR). Here we have analysed the ability of Bag-1 to regulate the transrepression function of the GR. Silencing Bag-1 expression only marginally affects the transrepression action of the GR but decreased the action of the transcription factor NF-κB. Furthermore phosphorylation and degradation of the inhibitor protein IκBα and nuclear accumulation of p65 and p50 NF-κB proteins in response to phorbol ester was attenuated following Bag-1 depletion in HeLa cells. Reconstitution of Bag-1 in depleted cells partially restored IκBα and NF-κB activation. Knock-down of Bag-1 expression also did not significantly alter GR-mediated transactivation but affected the basal transcription of some of the target genes. Thus Bag-1 proteins function as regulators of the action of selective transcription factors.