Ralf Marienfeld

Exogenous CNTF stimulates axon regeneration of retinal ganglion cells partially via endogenous CNTF

Intravitreal injections of exogenous CNTF stimulate axon regeneration of RGCs in vivo. Nevertheless, controversy exists over the ability of exogenous CNTF to directly stimulate axon regeneration of mature RGCs. Here we demonstrate that CNTF potently stimulated axon outgrowth of mature RGCs in culture in a JAK/STAT3- and PI3K/AKT-signaling pathway-dependent fashion and stronger than oncomodulin. Additional cAMP elevation or inhibition of MAPK activity increased these effects. In vivo intravitreal injections of exogenous CNTF induced endogenous CNTF expression in astrocytes in a manner that depended on the MAPK/ERK-signaling pathway activation. Reduction of endogenous CNTF expression by MAPK/ERK pathway inhibitors or its absence in CNTF deficient mice markedly reduced the neurite growth-promoting effects of exogenous CNTF. These data demonstrate that CNTF is a potent axon growth-promoting factor for mature RGCs. However, exogenously applied CNTF stimulates RGCs in vivo partially indirectly via a mechanism that depends on astrocyte-derived CNTF.

Signal-specific and phosphorylation-dependent RelB degradation: a potential mechanism of NF-κB control

RelB is an unusual member of the Rel/NF-κB family of transcription factors which are involved in oncogenic processes. Due to a relaxed control by the IκBs, the cytosolic NF-κB inhibitors, RelB is constitutively expressed in the nuclei of lymphoid cells. We show here that RelB is inducibly degraded upon activation of T cells in a fashion similar to the IκBs. However, RelB degradation differs from that of IκBs since it is not induced by TNFα but only by T cell receptor or TPA/ionomycin stimulation. Moreover, RelB degradation occurs in three steps: (i) after stimulation RelB is rapidly phosphorylated at amino acids Thr84 and Ser552 followed by (ii) an N-terminal cut and, finally, (iii) the complete degradation in the proteasomes. Since mutation of the two phosphoacceptor sites to non-acceptor sites abolished RelB phosphorylation in vivo and led to the stabilization of the mutated RelBDM, site-specific phosphorylation appears to be a necessary prerequisite for RelB degradation. RelB is a crucial regulator of NF-κB-dependent gene expression. Thus, the signal-induced degradation of RelB should be an important control mechanism of NF-κB activity.

The Ca2+-dependent Phosphatase Calcineurin Controls the Formation of the Carma1-Bcl10-Malt1 Complex during T Cell Receptor-induced NF-κB Activation

T cell receptor (TCR) ligation induces increased diacylglycerol and Ca2+ levels in T cells, and both secondary messengers are crucial for TCR-induced nuclear factor of activated T cells (NF-AT) and NF-κB signaling pathways. One prominent calcium-dependent enzyme involved in the regulation of NF-AT and NF-κB signaling pathways is the protein phosphatase calcineurin. However, in contrast to NF-AT, which is directly dephosphorylated by calcineurin, the molecular basis of the calcium-calcineurin dependence of the TCR-induced NF-κB activity remains largely unknown. Here, we demonstrate that calcineurin regulates TCR-induced NF-κB activity by controlling the formation of a protein complex composed of Carma1, Bcl10, and Malt1 (CBM complex). For instance, increased calcium levels induced by ionomycin or thapsigargin augmented the phorbol 12-myristate 13-acetate-induced formation of the CBM complex and activation of NF-κB, whereas removal of calcium by the calcium chelator EGTA-acetoxymethyl ester (AM) attenuated both processes. Furthermore, inhibition of the calcium-dependent phosphatase calcineurin with the immunosuppressive agent cyclosporin A (CsA) or FK506 as well as siRNA-mediated knockdown of calcineurin A strongly affected the PMA + ionomycin- or anti-CD3 + CD28-induced CBM complex assembly. Mechanistically, the positive effect of calcineurin on the CBM complex formation seems to be linked to a dephosphorylation of Bcl10. For instance, Bcl10 was found to be hyperphosphorylated in Jurkat T cells upon treatment with CsA or EGTA-AM, and calcineurin dephosphorylated Bcl10 in vivo and in vitro. Furthermore, we show here that calcineurin A interacts with the CBM complex. In summary, the evidence provided here argues for a previously unanticipated role of calcineurin in CBM complex formation as a molecular basis of the inhibitory function of CsA or FK506 on TCR-induced NF-κB activity.

Phosphorylation of Serine 68 in the IκB Kinase (IKK)-binding Domain of NEMO Interferes with the Structure of the IKK Complex and Tumor Necrosis Factor-α-induced NF-κB Activity

The IκB-Kinase (IKK) complex is a multisubunit protein complex crucial for signal-induced phosphorylation of the IκB proteins and thus controls the activity of the transcription factor NF-κB. Besides the two kinases IKKα and IKKβ, the IKK complex contains NEMO/IKKγ, an additional subunit with regulatory and adaptor functions. NEMO not only confers structural stability to the IKK complex but also participates in the activation process of the IKK complex by linking the IKK subunits to upstream activators. In this study we analyze the IKKβ-mediated phosphorylation of the IKK-binding domain of NEMO. In vitro, IKKβ phosphorylates three serine residues in the domain of NEMO at positions 43, 68, and 85. However, mutational analysis revealed that only the phosphorylation of serine 68 in the center of the IKK-binding domain plays an essential role for the formation and the function of the IKK complex. Thus, Ser68 phosphorylation attenuates the amino-terminal dimerization of NEMO as well as the IKKβ-NEMO interaction. In contrast, the NEMO-IKKα interaction was only mildly affected by the phosphorylation of Ser68. However, functional analysis revealed that Ser68 phosphorylation primarily affects the activity of IKKα. Furthermore, in complementation experiments of NEMO-deficient murine embryonic fibroblasts, a S68A-NEMO mutant enhanced, whereas a S68E mutant decreased, TNF-α-induced NF-κB activity, thus emphasizing the inhibitory role of the Ser68 phosphorylation on the signal-induced NF-κB activity. Finally, we provide evidence that the protein phosphatase PP2A is involved in the regulation of the Ser68-based mechanism. In summary, we provide evidence for a signal-induced phosphorylation-dependent alteration of the IKK complex emphasizing the dynamic nature of this multisubunit kinase complex.

Critical Role of RelB Serine 368 for Dimerization and p100 Stabilization

In mature B cells RelB-containing complexes are constitutively present in the nucleus, and they are less susceptible to inhibitory κB proteins. In most other cell types inhibitory κB proteins prevent nuclear translocation and activation of NFκB. We reasoned that this characteristic might be because of post-translational modifications of RelB. In Drosophila, signal-dependent phosphorylation of the Rel homologue Dorsal at serine 317 has been shown to be critical for nuclear import. The evolutionary conservation of this serine prompted us to analyze the function of the corresponding site in RelB. As a model system we used the murine S107 plasmacytoma cell line, which lacks endogenous RelB expression. Analysis of S107 cells expressing wild type RelB and serine 368 mutants reveals that serine 368 is not required for nuclear import but that it is critical for RelB dimerization with other members of the NFκB family. Similar effects were obtained when the conserved serine in RelA was mutated. We further demonstrate that expression of functional RelB, but not of serine 368 mutants, severely reduces p52 generation and strongly increases expression of the p52 precursor, p100. Wild type RelB, but not mutant RelB, prolonged p100 half-life. We therefore suggest an inhibitory effect of RelB on p100 processing, which is possibly regulated in a signal-dependent manner.

NF-κB signaling in prostate cancer: A promising therapeutic target?

Prostate carcinoma (PCa) displays a wide variety of genetic alterations, versatile expression profiles as well as cell surface markers. Despite this heterogeneity, a common treatment for advanced PCa is androgen deprivation therapy (ADT). ADT targets the androgen receptor—a member of the nuclear receptor superfamily—which is required for development and function of the prostate and critical for PCa growth and survival. After an initial regression of the tumor during ADT, a large fraction of tumors progress to so-called castration-resistant prostate carcinoma (CRPca) which is highly resistant toward chemotherapy. The ensuing high mortality rates illustrate the importance of novel therapeutic targets for CRPCa. The transcription factor NF-κB was recently proposed as such a potential target for therapeutic intervention in CRPCa. Although NF-κB is essential for the regulation of innate and adaptive immunity recent data suggest a role of NF-κB in cancer initiation and progression. However, the exact function of NF-κB signaling in PCa is still a matter of debate. Here, we review known roles of NF-κB signaling in PCa and emphasize the crosstalk of NF-κB and androgen receptor signaling. Finally, we discuss potential therapeutic relevance of blocking NF-κB in PCa.

Dimerization of the IκB Kinase-Binding Domain of NEMO Is Required for Tumor Necrosis Factor Alpha-Induced NF-κB Activity

ABSTRACT Previous studies have demonstrated that peptides corresponding to a six-amino-acid NEMO-binding domain from the C terminus of IκB kinase alpha (IKKα) and IKKβ can disrupt the IKK complex and block NF-κB activation. We have now mapped and characterized the corresponding amino-terminal IKK-binding domain (IBD) of NEMO. Peptides corresponding to the IBD were efficiently recruited to the IKK complex but displayed only a weak inhibitory potential on cytokine-induced NF-κB activity. This is most likely due to the formation of sodium dodecyl sulfate- and urea-resistant NEMO dimers through a dimerization domain at the amino terminus of NEMO that overlaps with the region responsible for binding to IKKs. Mutational analysis revealed different α-helical subdomains within an amino-terminal coiled-coil region are important for NEMO dimerization and IKKβ binding. Furthermore, NEMO dimerization is required for the tumor necrosis factor alpha-induced NF-κB activation, even when interaction with the IKKs is unaffected. Hence, our data provide novel insights into the role of the amino terminus of NEMO for the architecture of the IKK complex and its activation.

Sustained JNK Activation in Response to Tumor Necrosis Factor Is Mediated by Caspases in a Cell Type-specific Manner

In most cell types, tumor necrosis factor (TNF) induces a transient activation of the JNK pathway. However, in NFκB-inhibited cells, TNF stimulates also a second sustained phase of JNK activation, which has been implicated in cell death induction. In the present study, we have analyzed the relationship of cell death induction, caspase activity, JNK, and NFκB stimulation in the context of TNF signaling in four different cellular systems. In all cases, NFκB inhibition enhanced TNF-induced cell death and primed most, but not all, cells for sustained JNK activation. The caspase inhibitor Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethyl ketone (Z-VAD-fmk) and overexpression of the antiapoptotic proteins FLIP-L and Bcl2 differentially blocked transient and sustained JNK activation in NFκB-inhibited KB and HaCaT cells, indicating that the two phases of TNF-induced JNK activation occur at least in these cellular models by different pathways. Although the broad range caspase inhibitor Z-VAD-fmk and the antioxidant butylated hydroxyanisole interfered with TNF-induced cell death to a varying extent in a cell type-specific manner, inhibition of JNK signaling had no or only a very moderate effect. Notably, the JNK inhibitory effect of neither Z-VAD-fmk nor butylated hydroxyanisole was strictly correlated with the capability of these compounds to rescue cells from TNF-induced cell death. Thus, sustained JNK activation by TNF has no obligate role in TNF-induced cell death and is mediated by caspases and reactive oxygen species in a cell type-specific manner.

Pyrosequencing of BRAF V600E in routine samples of hairy cell leukaemia identifies CD5+ variant hairy cell leukaemia that lacks V600E.

Mikkael A. Sekeres Heesun J. Rogers Ramon V. Tiu Department of Hematologic Oncology and Blood Disorders, Taussig Cancer Institute, Cleveland Clinic, Clinical Pathology, Cleveland Clinic, and Translational Hematology and Oncology Research, Taussig Cancer Institute, Cleveland Clinic, Cleveland, OH, USA. E-mail: tiur@ccf.org

FTO Deficiency Induces UCP-1 Expression and Mitochondrial Uncoupling in Adipocytes

Variants in the fat mass- and obesity-associated (FTO) gene are associated with obesity and body fat mass in genome-wide association studies. However, the mechanism by which FTO predisposes individuals to obesity is not clear so far. First mechanistic evidence was shown in Fto-negative mice. These mice are resistant to obesity due to enhanced energy expenditure, whereas the mass of brown adipose tissue remains unchanged. We hypothesize that FTO is involved in the induction of white adipose tissue browning, which leads to mitochondrial uncoupling and increases energy expenditure. Uncoupling protein 1 (Ucp-1) was significantly higher expressed in both gonadal and inguinal adipose depots of Fto(-/-) compared with Fto(+/+) littermates accompanied by the appearance of multivacuolar, Ucp-1-positive adipocytes in these tissues. By using lentiviral short hairpin RNA constructs, we established FTO-deficient human preadipocytes and adipocytes and analyzed key metabolic processes. FTO-deficient adipocytes showed an adipogenic differentiation rate comparable with control cells but exhibited a reduced de novo lipogenesis despite unchanged glucose uptake. In agreement with the mouse data, FTO-deficient adipocytes exhibited 4-fold higher expression of UCP-1 in mitochondria compared with control cells. The up-regulation of UCP-1 in FTO-deficient adipocytes resulted in enhanced mitochondrial uncoupling. We conclude that FTO deficiency leads to the induction of a brown adipocyte phenotype, thereby enhancing energy expenditure. Further understanding of the signaling pathway connecting FTO with UCP-1 expression might lead to new options for obesity and overweight treatment.