A role for NRAGE in NF-κB activation through the non-canonical BMP pathway

<p>Abstract</p> <p>Background</p> <p>Previous studies have linked neurotrophin receptor-interacting MAGE protein to the bone morphogenic protein signaling pathway and its effect on p38 mediated apoptosis of neural progenitor cells via the XIAP-Tak1-Tab1 complex. Its effect on NF-κB has yet to be explored.</p> <p>Results</p> <p>Herein we report that NRAGE, via the same XIAP-Tak1-Tab1 complex, is required for the phosphorylation of IKK -α/β and subsequent transcriptional activation of the p65 subunit of NF-κB. Ablation of endogenous NRAGE by siRNA inhibited NF-κB pathway activation, while ablation of Tak1 and Tab1 by morpholino inhibited overexpression of NRAGE from activating NF-κB. Finally, cytokine profiling of an NRAGE over-expressing stable line revealed the expression of macrophage migration inhibitory factor.</p> <p>Conclusion</p> <p>Modulation of NRAGE expression revealed novel roles in regulating NF-κB activity in the non-canonical bone morphogenic protein signaling pathway. The expression of macrophage migration inhibitory factor by bone morphogenic protein -4 reveals novel crosstalk between an immune cytokine and a developmental pathway.</p

The Expression and Localization of N-Myc Downstream-Regulated Gene 1 in Human Trophoblasts

The protein N-Myc downstream-regulated gene 1 (NDRG1) is implicated in the regulation of cell proliferation, differentiation, and cellular stress response. NDRG1 is expressed in primary human trophoblasts, where it promotes cell viability and resistance to hypoxic injury. The mechanism of action of NDRG1 remains unknown. To gain further insight into the intracellular action of NDRG1, we analyzed the expression pattern and cellular localization of endogenous NDRG1 and transfected Myc-tagged NDRG1 in human trophoblasts exposed to diverse injuries. In standard conditions, NDRG1 was diffusely expressed in the cytoplasm at a low level. Hypoxia or the hypoxia mimetic cobalt chloride, but not serum deprivation, ultraviolet (UV) light, or ionizing radiation, induced the expression of NDRG1 in human trophoblasts and the redistribution of NDRG1 into the nucleus and cytoplasmic membranes associated with the endoplasmic reticulum (ER) and microtubules. Mutation of the phosphopantetheine attachment site (PPAS) within NDRG1 abrogated this pattern of redistribution. Our results shed new light on the impact of cell injury on NDRG1 expression patterns, and suggest that the PPAS domain plays a key role in NDRG1's subcellular distribution. © 2013 Shi et al

Application of Ni(II)-Assisted Peptide Bond Hydrolysis to Non-Enzymatic Affinity Tag Removal

In this study, we demonstrate a non-enzymatic method for hydrolytic peptide bond cleavage, applied to the removal of an affinity tag from a recombinant fusion protein, SPI2-SRHWAP-His6. This method is based on a highly specific Ni(II) reaction with (S/T)XHZ peptide sequences. It can be applied for the protein attached to an affinity column or to the unbound protein in solution. We studied the effect of pH, temperature and Ni(II) concentration on the efficacy of cleavage and developed an analytical protocol, which provides active protein with a 90% yield and ∼100% purity. The method works well in the presence of non-ionic detergents, DTT and GuHCl, therefore providing a viable alternative for currently used techniques

Cellular differentiation determines the expression of the hypoxia-inducible protein NDRG1 in pancreatic cancer

N-myc downstream-regulated gene-1 (NDRG1) is a recently described hypoxia-inducible protein that is upregulated in various human cancers. Pancreatic ductal adenocarcinoma, called pancreatic cancer, is a highly aggressive cancer that is characterised by its avascular structure, which results in a severe hypoxic environment. In this study, we investigated whether NDRG1 is upregulated in these tumours, thus providing a novel marker for malignant cells in the pancreas. By immunohistochemistry, we observed that NDRG1 was highly expressed in well-differentiated cells of pancreatic cancer, whereas the poorly differentiated tumour cells were negative. In addition, hyperplastic islets and ducts of nonquiescent pancreatic tissue were positive. To further explore its selective expression in tumours, two well-established pancreatic cancer cell lines of unequal differentiation status were exposed to 2% oxygen. NDRG1 mRNA and protein were upregulated by hypoxia in the moderately differentiated Capan-1 cells; however, its levels remained unchanged in the poorly differentiated Panc-1 cell line. Taken together, our data suggest that NDRG1 will not serve as a reliable marker of tumour cells in the pancreas, but may serve as a marker of differentiation. Furthermore, we present the novel finding that cellular differentiation may be an important factor that determines the hypoxia-induced regulation of NDRG1

A Small Peptide Modeled after the NRAGE Repeat Domain Inhibits XIAP-TAB1-TAK1 Signaling for NF-κB Activation and Apoptosis in P19 Cells

In normal growth and development, apoptosis is necessary to shape the central nervous system and to eliminate excess neurons which are not required for innervation. In some diseases, however, apoptosis can be either overactive as in some neurodegenerative disorders or severely attenuated as in the spread of certain cancers. Bone morphogenetic proteins (BMPs) transmit signals for regulating cell growth, differentiation, and apoptosis. Responding to BMP receptors stimulated from BMP ligands, neurotrophin receptor-mediated MAGE homolog (NRAGE) binds and functions with the XIAP-TAK1-TAB1 complex to activate p38MAPK and induces apoptosis in cortical neural progenitors. NRAGE contains a unique repeat domain that is only found in human, mouse, and rat homologs that we theorize is pivotal in its BMP MAPK role. Previously, we showed that deletion of the repeat domain inhibits apoptosis, p38MAPK phosphorylation, and caspase-3 cleavage in P19 neural progenitor cells. We also showed that the XIAP-TAB1-TAK1 complex is dependent on NRAGE for IKK-α/β phosphorylation and NF-κB activation. XIAP is a major inhibitor of caspases, the main executioners of apoptosis. Although it has been shown previously that NRAGE binds to the RING domain of XIAP, it has not been determined which NRAGE domain binds to XIAP. Here, we used fluorescence resonance energy transfer (FRET) to determine that there is a strong likelihood of a direct interaction between NRAGE and XIAP occurring at NRAGE's unique repeat domain which we also attribute to be the domain responsible for downstream signaling of NF-κB and activating IKK subunits. From these results, we designed a small peptide modeled after the NRAGE repeat domain which we have determined inhibits NF-κB activation and apoptosis in P19 cells. These intriguing results illustrate that the paradigm of the NRAGE repeat domain may hold promising therapeutic strategies in developing pharmaceutical solutions for combating harmful diseases involving excessive downstream BMP signaling, including apoptosis

Genome-Wide Association Analysis of Oxidative Stress Resistance in Drosophila melanogaster

Background: Aerobic organisms are susceptible to damage by reactive oxygen species. Oxidative stress resistance is a quantitative trait with population variation attributable to the interplay between genetic and environmental factors. Drosophila melanogaster provides an ideal system to study the genetics of variation for resistance to oxidative stress. Methods and Findings: We used 167 wild-derived inbred lines of the Drosophila Genetic Reference Panel for a genomewide association study of acute oxidative stress resistance to two oxidizing agents, paraquat and menadione sodium bisulfite. We found significant genetic variation for both stressors. Single nucleotide polymorphisms (SNPs) associated with variation in oxidative stress resistance were often sex-specific and agent-dependent, with a small subset common for both sexes or treatments. Associated SNPs had moderately large effects, with an inverse relationship between effect size and allele frequency. Linear models with up to 12 SNPs explained 67–79 % and 56–66 % of the phenotypic variance for resistance to paraquat and menadione sodium bisulfite, respectively. Many genes implicated were novel with no known role in oxidative stress resistance. Bioinformatics analyses revealed a cellular network comprising DNA metabolism and neuronal development, consistent with targets of oxidative stress-inducing agents. We confirmed associations of seven candidate genes associated with natural variation in oxidative stress resistance through mutational analysis. Conclusions: We identified novel candidate genes associated with variation in resistance to oxidative stress that hav

Microarray analysis of altered gene expression in murine fibroblasts transformed by nickel(II) to nickel(II)-resistant malignant phenotype.

NRC publication: Ye

MicroRNA199b regulates mouse hematopoietic stem cells maintenance

The preservation of hematopoieticstem cell pool in bone marrow (BM) is crucial for sustained hematopoiesisin adults. Studies assessing adult hematopoieticstem cells functionality had been shown that for example loss of quiescence impairs hematopoieticstem cells maintenance. Although, miR‐199b is frequently down‐regulated in acute myeloid leukemia, its role in hematopoieticstem cells quiescence, self‐renewal and differentiation is poorly understood. Our laboratory investigated the role of miR‐199b in hematopoieticstem and progenitor cells (HSPCs) fate using miR‐199b‐5p global deletion mouse model. Characterization of miR‐199b expression pattern among normal HSPC populations revealed that miR‐199b is enriched in LT‐HSCsand reduced upon myeloablativestress, suggesting its role in HSCsmaintenance. Indeed, our results reveal that loss of miR‐199b5p results in imbalance between long‐term hematopoieticstem cells (LT‐HSCs), short‐term hematopoieticstem cells (ST‐HSCs) and multipotentprogenitors (MMPs) pool. We found that during homeostasis, miR‐199b‐null HSCshave reduced capacity to maintain quiescent state and exhibit cell‐cycle deregulation. Cell cycle analyses showed that attenuation of miR‐199b controls HSCspool, causing defects in G1‐S transition of cell cycle, without significant changes in apoptosis. This might be due to increased differentiation of LT‐HSCs into MPPs. Indeed, cell differentiation assay in vitro showed that FACS‐sorted LT‐HSCs(LineagenegSca1posc‐Kitpos CD48neg CD150pos) lacking miR‐199b have increased differentiation potential into MPP in the presence of early cytokines. In addition, differentiation assays in vitro in FACS‐sorted LSK population of 52 weeks old miR‐199b KO mice revealed that loss of miR‐199b promotes accumulation of GMP‐like progenitors but decreases lymphoid differentiation, suggesting that miR199b may regulate age‐related pathway. We used noncompetitive repopulation studies to show that overall BM donor cellularitywas markedly elevated in the absence of miR‐199b among HSPCs, committed progenitors and mature myeloid but not lymphoid cell compartments. This may suggest that miR‐199b‐null LT‐HSC render enhanced self‐renewal capacity upon regeneration demand yet promoting myeloid reconstitution. Moreover, when we challenged the self‐renewal potential of miR‐199b‐null LT‐HSC by a secondary BM transplantation of unfractionatedBM cells from primary recipients into secondary hosts, changes in PB reconstitution were dramatic. Gating for HSPCspopulations in the BM of secondary recipients in 24 weeks after BMT revealed that levels of LT‐HSC were similar between recipients reconstituted with wild‐type and miR‐199b‐KO chimeras, whereas miR‐199b‐null HSCscontributed relatively more into MPPs. Our data identify that attenuation of miR‐199b leads to loss of quiescence and premature differentiation of HSCs. These findings indicate that loss of miR‐199b promotes signals that govern differentiation of LT‐HSC to MPP leading to accumulation of highly proliferativeprogenitors during long‐term reconstitution. Hematopoieticregeneration via repopulation studies also revealed that miR‐199b‐deficient HSPCshave a lineage skewing potential toward myeloid lineage or clonalmyeloid bias, a hallmark of aging HSCs, implicating a regulatory role for miR‐199b in hematopoietic aging

A role for NRAGE in NF-kappaB activation through the non-canonical BMP pathway.

BACKGROUND: Previous studies have linked neurotrophin receptor-interacting MAGE protein to the bone morphogenic protein signaling pathway and its effect on p38 mediated apoptosis of neural progenitor cells via the XIAP-Tak1-Tab1 complex. Its effect on NF-kappaB has yet to be explored. RESULTS: Herein we report that NRAGE, via the same XIAP-Tak1-Tab1 complex, is required for the phosphorylation of IKK -alpha/beta and subsequent transcriptional activation of the p65 subunit of NF-kappaB. Ablation of endogenous NRAGE by siRNA inhibited NF-kappaB pathway activation, while ablation of Tak1 and Tab1 by morpholino inhibited overexpression of NRAGE from activating NF-kappaB. Finally, cytokine profiling of an NRAGE over-expressing stable line revealed the expression of macrophage migration inhibitory factor. CONCLUSION: Modulation of NRAGE expression revealed novel roles in regulating NF-kappaB activity in the non-canonical bone morphogenic protein signaling pathway. The expression of macrophage migration inhibitory factor by bone morphogenic protein -4 reveals novel crosstalk between an immune cytokine and a developmental pathway