Impaired embryonic haematopoiesis yet normal arterial development in the absence of the Notch ligand Jagged1.

Specific deletion of Notch1 and RBPjkappa in the mouse results in abrogation of definitive haematopoiesis concomitant with the loss of arterial identity at embryonic stage. As prior arterial determination is likely to be required for the generation of embryonic haematopoiesis, it is difficult to establish the specific haematopoietic role of Notch in these mutants. By analysing different Notch-ligand-null embryos, we now show that Jagged1 is not required for the establishment of the arterial fate but it is required for the correct execution of the definitive haematopoietic programme, including expression of GATA2 in the dorsal aorta. Moreover, successful haematopoietic rescue of the Jagged1-null AGM cells was obtained by culturing them with Jagged1-expressing stromal cells or by lentiviral-mediated transduction of the GATA2 gene. Taken together, our results indicate that Jagged1-mediated activation of Notch1 is responsible for regulating GATA2 expression in the AGM, which in turn is essential for definitive haematopoiesis in the mouse.

IkappaBalpha and p65 regulate the cytoplasmic shuttling of nuclear corepressors: cross-talk between Notch and NFkappaB pathways.

Notch and NFkappaB pathways are key regulators of numerous cellular events such as proliferation, differentiation, or apoptosis. In both pathways, association of effector proteins with nuclear corepressors is responsible for their negative regulation. We have previously described that expression of a p65-NFkappaB mutant that lacks the transactivation domain (p65DeltaTA) induces cytoplasmic translocation of N-CoR leading to a positive regulation of different promoters. Now, we show that cytoplasmic sequestration of p65 by IkappaBalpha is sufficient to both translocate nuclear corepressors SMRT/N-CoR to the cytoplasm and upregulate transcription of Notch-dependent genes. Moreover, p65 and IkappaBalpha are able to directly bind SMRT, and this interaction can be inhibited in a dose-dependent manner by the CREB binding protein (CBP) coactivator and after TNF-alpha treatment, suggesting that p65 acetylation is modulating this interaction. In agreement with this, TNF-alpha treatment results in downregulation of the Hes1 gene. Finally, we present evidence on how this mechanism may influence cell differentiation in the 32D myeloid progenitor system.

Hematopoietic stem cell development requires transient Wnt/ß-catenin activity.

Understanding how hematopoietic stem cells (HSCs) are generated and the signals that control this process is a crucial issue for regenerative medicine applications that require in vitro production of HSC. HSCs emerge during embryonic life from an endothelial-like cell population that resides in the aorta-gonad-mesonephros (AGM) region. We show here that ß-catenin is nuclear and active in few endothelial nonhematopoietic cells closely associated with the emerging hematopoietic clusters of the embryonic aorta during mouse development. Importantly, Wnt/ß-catenin activity is transiently required in the AGM to generate long-term HSCs and to produce hematopoietic cells in vitro from AGM endothelial precursors. Genetic deletion of ß-catenin from the embryonic endothelium stage (using VE-cadherin-Cre recombinase), but not from embryonic hematopoietic cells (using Vav1-Cre), precludes progression of mutant cells toward the hematopoietic lineage; however, these mutant cells still contribute to the adult endothelium. Together, those findings indicate that Wnt/ß-catenin activity is needed for the emergence but not the maintenance of HSCs in mouse embryos.

Inhibition of specific NF-κB activity contributes to the tumor suppressor function of 14-3-3σ in breast cancer.

14-3-3σ is frequently lost in human breast cancers by genetic deletion or promoter methylation. We have now investigated the involvement of 14-3-3σ in the termination of NF-κB signal in mammary cells and its putative role in cancer relapse and metastasis. Our results show that 14-3-3σ regulates nuclear export of p65-NF-κB following chronic TNFα stimulation. Restoration of 14-3-3σ in breast cancer cells reduces migration capacity and metastatic abilities in vivo. By microarray analysis, we have identified a genetic signature that responds to TNFα in a 14-3-3σ-dependent manner and significantly associates with different breast and other types of cancer. By interrogating public databases, we have found that over-expression of this signature correlates with poor relapse-free survival in breast cancer patients. Finally, screening of 96 human breast tumors showed that NF-κB activation strictly correlates with the absence of 14-3-3σ and it is significantly associated with worse prognosis in the multivariate analysis. Our findings identify a genetic signature that is important for breast cancer prognosis and for future personalized treatments based on NF-κB targeting.

The Notch/Hes1 pathway sustains NF-κB activation through CYLD repression in T cell leukemia.

It was previously shown that the NF-κB pathway is downstream of oncogenic Notch1 in T cell acute lymphoblastic leukemia (T-ALL). Here, we visualize Notch-induced NF-κB activation using both human T-ALL cell lines and animal models. We demonstrate that Hes1, a canonical Notch target and transcriptional repressor, is responsible for sustaining IKK activation in T-ALL. Hes1 exerts its effects by repressing the deubiquitinase CYLD, a negative IKK complex regulator. CYLD expression was found to be significantly suppressed in primary T-ALL. Finally, we demonstrate that IKK inhibition is a promising option for the targeted therapy of T-ALL as specific suppression of IKK expression and function affected both the survival of human T-ALL cells and the maintenance of the disease in vivo.

Efficient nuclear export of p65-IkappaBalpha complexes requires 14-3-3 proteins.

IkappaB are responsible for maintaining p65 in the cytoplasm under non-stimulating conditions and promoting the active export of p65 from the nucleus following NFkappaB activation to terminate the signal. We now show that 14-3-3 proteins regulate the NFkappaB signaling pathway by physically interacting with p65 and IkappaBalpha proteins. We identify two functional 14-3-3 binding domains in the p65 protein involving residues 38-44 and 278-283, and map the interaction region of IkappaBalpha in residues 60-65. Mutation of these 14-3-3 binding domains in p65 or IkappaBalpha results in a predominantly nuclear distribution of both proteins. TNFalpha treatment promotes recruitment of 14-3-3 and IkappaBalpha to NFkappaB-dependent promoters and enhances the binding of 14-3-3 to p65. Disrupting 14-3-3 activity by transfection with a dominant-negative 14-3-3 leads to the accumulation of nuclear p65-IkappaBalpha complexes and the constitutive association of p65 with the chromatin. In this situation, NFkappaB-dependent genes become unresponsive to TNFalpha stimulation. Together our results indicate that 14-3-3 proteins facilitate the nuclear export of IkappaBalpha-p65 complexes and are required for the appropriate regulation of NFkappaB signaling.

Phosphorylation by glycogen synthase kinase-3 beta down-regulates Notch activity, a link for Notch and Wnt pathways.

Phosphorylation of Notch proteins has been indirectly correlated with Notch activation and nuclear translocation as well as cellular transformation. There is evidence that the Wnt signaling pathway, which results in glycogen synthase kinase-3 beta (GSK-3 beta) inhibition, cross-talks with the Notch pathway. In this study, we show that GSK-3 beta is able to bind and phosphorylate Notch2 in vitro and in vivo. We identify three specific phosphorylation sites in the Notch2 serine/threonine-rich domain that are dependent on GSK-3 beta activity. Phosphorylation of the serine/threonine-rich domain has been shown previously to be crucial in regulating cytokine-specific cell differentiation. Coimmunoprecipitation experiments show that full-length Notch2 binds more efficiently than intracellular Notch2 to GSK-3 beta. Nevertheless, only the processed Notch2 is a substrate for the kinase, thus suggesting that GSK-3 beta-dependent phosphorylation may be specifically regulating the activated Notch molecule. Consistent with this, GSK-3 beta inhibits the transcriptional activation of Notch target genes both in vitro and in vivo, whereas lithium chloride treatment or Wnt-1 overexpression that results in GSK-3 beta inhibition leads to the up-regulation of the Hes-1 promoter. Together, our results suggest that cross-talk between Notch and Wnt pathways may be partially mediated by specific regulation of GSK-3 beta-dependent Notch phosphorylation.

p65-NFkappaB synergizes with Notch to activate transcription by triggering cytoplasmic translocation of the nuclear receptor corepressor N-CoR.

Notch/RBP-Jkappa and nuclear factor-kappaB (NFkappaB) complexes are key mediators of the progression of many cellular events through the activation of specific target gene transcription. Independent observations have shown that activation of Notch-dependent transcription generally correlates with inhibition of differentiation. In contrast, activated NFkappaB complexes are required for progression of differentiation in several systems. Although some interactions between both pathways have been observed, the physiological significance of their connection is unclear. We have now demonstrated that the increase in p65-NFkappaB protein levels enhances Notch-mediated activation of the Hes1 promoter up to three-fold. This effect does not require NFkappaB transcriptional activity, and it is independent of the previously described interaction between Notch and p50-NFkappaB. Furthermore, we show that p65-NFkappaB can modulate subcellular localization of the transcriptional corepressor N-CoR, abrogating N-CoR mediated repression of the Hes1 promoter. In addition, p65-NFkappaB is able to upregulate not only the Hes1 but also other promoters containing SRE and AP-1 sites, which are repressed by N-CoR. Thus, we conclude that p65-NFkappaB can regulate gene expression by a general mechanism that involves cytoplasmic translocation of the transcriptional corepressor protein N-CoR.