Cytoplasmic localization of amyotrophic lateral sclerosis-related TDP-43 proteins modulates stress granule formation.

TDP-43 is an RNA/DNA-binding protein associated with amyotrophic lateral sclerosis (ALS). Under pathological conditions, TDP-43 exported from the nucleus accumulates in the cytoplasm, forming inclusion bodies. However, the molecular mechanisms that contribute to such aggregation are unclear. The pathogenic processes that lead to aggregation in ALS were investigated by analysing the effects of wildtype human TDP-43 or with mutations in the nuclear localization sequence (NLS) or those associated with ALS in stress granule formation. TDP-43 (WT, ∆NLS or G348C), with or without a GFP-tag, was expressed in SH-SY5Y neuroblastoma or HeLa cells and stress granules induced by oxidative stress or heat shock. Stress granule formation was altered in cells strongly expressing GFP-TDP-∆NLS, or untagged TDP-43-∆NLS in the cytoplasm but not the negative controls, GFP or GFP-UtrCH. In contrast, there was no reduction in stress granule formation by cells that expressed untagged TDP-43 (WT or G348C) in the nucleus upon stress induction. GFP labelling of TDP-43 (WT or G348C) promotes high cytoplasmic expression and nuclear aggregation. Stress granule formation was impaired in cells expressing GFP-TDP-43 (WT or G348C) in the cytoplasm. Overall, these results suggest that stress granule formation may be inhibited by high levels of TDP-43 protein in the cytoplasm. As stress granules serve a protective function, their deregulation may promote neurodegeneration due to an aberrant stress response.

Interleukin-18 produced by bone marrow-derived stromal cells supports T-cell acute leukaemia progression.

Development of novel therapies is critical for T-cell acute leukaemia (T-ALL). Here, we investigated the effect of inhibiting the MAPK/MEK/ERK pathway on T-ALL cell growth. Unexpectedly, MEK inhibitors (MEKi) enhanced growth of 70% of human T-ALL cell samples cultured on stromal cells independently of NOTCH activation and maintained their ability to propagate in vivo. Similar results were obtained when T-ALL cells were cultured with ERK1/2-knockdown stromal cells or with conditioned medium from MEKi-treated stromal cells. Microarray analysis identified interleukin 18 (IL-18) as transcriptionally up-regulated in MEKi-treated MS5 cells. Recombinant IL-18 promoted T-ALL growth in vitro, whereas the loss of function of IL-18 receptor in T-ALL blast cells decreased blast proliferation in vitro and in NSG mice. The NFKB pathway that is downstream to IL-18R was activated by IL-18 in blast cells. IL-18 circulating levels were increased in T-ALL-xenografted mice and also in T-ALL patients in comparison with controls. This study uncovers a novel role of the pro-inflammatory cytokine IL-18 and outlines the microenvironment involvement in human T-ALL development.

Thrombopoietin promotes NHEJ DNA repair in hematopoietic stem cells through specific activation of Erk and NF-κB pathways and their target, IEX-1.

Loss of hematopoietic stem cell (HSC) function and increased risk of developing hematopoietic malignancies are severe and concerning complications of anticancer radiotherapy and chemotherapy. We have previously shown that thrombopoietin (TPO), a critical HSC regulator, ensures HSC chromosomal integrity and function in response to γ-irradiation by regulating their DNA-damage response. TPO directly affects the double-strand break (DSB) repair machinery through increased DNA-protein kinase (DNA-PK) phosphorylation and nonhomologous end-joining (NHEJ) repair efficiency and fidelity. This effect is not shared by other HSC growth factors, suggesting that TPO triggers a specific signal in HSCs facilitating DNA-PK activation upon DNA damage. The discovery of these unique signaling pathways will provide a means of enhancing TPO-desirable effects on HSCs and improving the safety of anticancer DNA agents. We show here that TPO specifically triggers Erk and nuclear factor κB (NF-κB) pathways in mouse hematopoietic stem and progenitor cells (HSPCs). Both of these pathways are required for a TPO-mediated increase in DSB repair. They cooperate to induce and activate the early stress-response gene, Iex-1 (ier3), upon DNA damage. Iex-1 forms a complex with pERK and the catalytic subunit of DNA-PK, which is necessary and sufficient to promote TPO-increased DNA-PK activation and NHEJ DSB repair in both mouse and human HSPCs.

Involvement of the betaTrCP in the ubiquitination and stability of the HIV-1 Vpu protein.

The human immunodeficiency virus type 1 (HIV-1) Vpu protein binds to the CD4 receptor and targets it to the proteasome for degradation. This process requires the recruitment of human betaTrCP, a component of the Skp1-Cullin-F box (SCF) ubiquitin ligase complex, that interacts with phosphorylated Vpu molecules. Vpu, unlike other ligands of betaTrCP, has never been reported to be degraded. We provide evidence that Vpu, itself, is ubiquitinated and targeted for degradation by the proteasome. We demonstrate that the mutant Vpu2.6, which cannot interact with betaTrCP, is stable and, unlike wild-type Vpu, is not polyubiquitinated. These results suggest that betaTrCP is involved in Vpu polyubiquitination.

Overexpression of human beta TrCP1 deleted of its F box induces tumorigenesis in transgenic mice.

Genetic alterations affecting beta-catenin, adenomatous polyposis coli or axin proteins are associated with the pathogenesis of numerous human tumors. All these mutations result in the synthesis of unphosphorylated beta-catenin that escapes recognition by the beta transducin repeat protein (beta TrCP1), the receptor of an ubiquitin. The stabilized beta-catenin translocates to the nucleus and activates the transcription of genes crucial for tumorigenesis. Recent evidence implicates mutations and overexpresssion of beta TrCP1 in human prostate and colon tumors, respectively, suggesting that deregulated beta TrCP1 may be involved in tumorigenesis. To explore this possibility further, we generated transgenic mice that specifically express a dominant-negative mutant of beta TrCP1 (Delta F beta TrCP1) or full-length beta TrCP1 in intestine, liver and kidney. We found that 46% (16/35) of the transgenic mice that overexpressed the transgenes developed either intestinal adenomas (10/35) or hepatic (4/35) or urothelial (2/35) tumors. Immunohistological analysis of the tumors revealed that upregulation of cyclin D1, glutamine synthetase and chemotaxin 2 was associated with nuclear accumulation of beta-catenin. These results show that the overexpression of Delta F beta TrCP1 or beta TrCP1 in vivo induce tumors through beta-catenin activation.

HIV-1 Vpu sequesters beta-transducin repeat-containing protein (betaTrCP) in the cytoplasm and provokes the accumulation of beta-catenin and other SCFbetaTrCP substrates.

The human immunodeficiency virus type 1 Vpu protein acts as an adaptor for the proteasomal degradation of CD4 by recruiting CD4 and beta-transducin repeat-containing protein (betaTrCP), the receptor component of the multisubunit SCF-betaTrCP E3 ubiquitin ligase complex. We showed that the expression of a Vpu-green fluorescent fusion protein prevented the proteosomal degradation of betaTrCP substrates such as beta-catenin, IkappaBalpha, and ATF4, which are normally directly targeted to the proteasome for degradation. Beta-catenin was translocated into the nucleus, whereas the tumor necrosis factor-induced nuclear translocation of NFkappaB was impaired. Beta-catenin was also up-regulated in cells producing Vpu+ human immunodeficiency virus type 1 but not in cells producing Vpu-deficient viruses. The overexpression of ATF4 also provoked accumulation of beta-catenin, but to a lower level than that resulting from the expression of Vpu. Finally, the expression of Vpu induces the exclusion of betaTrCP from the nucleus. These data suggest that Vpu is a strong competitive inhibitor of betaTrCP that impairs the degradation of SCFbetaTrCP substrates as long as Vpu has an intact phosphorylation motif and can bind to betaTrCP.