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

Novel roles of miR-199b in regulating fat and bone metabolism

Public Health Problem: The incidence of obesity has reached epidemic proportions worldwide and has contributed to an increase in the risk of numerous chronic disorders-type 2 diabetes-liver pathologies, dyslipidemia, and cardiovascular diseases. Obesity can have negative effects on bone remodeling-reduced mineral density-osteoporosis Imbalance between food intake and energy expenditure-obesity-accumulation of fat mass and energy storage in white adipose tissue (WAT).https://knowledgeconnection.mainehealth.org/lambrew-retreat-2021/1036/thumbnail.jp

Two novel human NUMB isoforms provide a potential link between development and cancer

We previously identified four functionally distinct human NUMB isoforms. Here, we report the identification of two additional isoforms and propose a link between the expression of these isoforms and cancer. These novel isoforms, NUMB5 and NUMB6, lack exon 10 and are expressed in cells known for polarity and migratory behavior, such as human amniotic fluid cells, glioblastoma and metastatic tumor cells. RT-PCR and luciferase assays demonstrate that NUMB5 and NUMB6 are less antagonistic to NOTCH signaling than other NUMB isoforms. Immunocytochemistry analyses show that NUMB5 and NUMB6 interact and complex with CDC42, vimentin and the CDC42 regulator IQGAP1 (IQ (motif) GTPase activating protein 1). Furthermore, the ectopic expression of NUMB5 and NUMB6 induces the formation of lamellipodia (NUMB5) and filopodia (NUMB6) in a CDC42- and RAC1-dependent manner. These results are complemented by in vitro and in vivo studies, demonstrating that NUMB5 and NUMB6 alter the migratory behavior of cells. Together, these novel isoforms may play a role in further understanding the NUMB function in development and cancer

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

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

Spermatogonial Type 3 Deiodinase Regulates Thyroid Hormone Target Genes in Developing Testicular Somatic Cells.

Premature overexposure to thyroid hormone causes profound effects on testis growth, spermatogenesis, and male fertility. We used genetic mouse models of type 3 deiodinase (DIO3) deficiency to determine the genetic programs affected by premature thyroid hormone action and to define the role of DIO3 in regulating thyroid hormone economy in testicular cells. Gene expression profiling in the neonatal testis of DIO3-deficient mice identified 5699 differentially expressed genes. Upregulated and downregulated genes were, respectively, involved according to DAVID analysis with cell differentiation and proliferation. They included anti-Müllerian hormone and genes involved in the formation of the blood-testis barrier, which are specific to Sertoli cells (SCs). They also included steroidogenic genes, which are specific to Leydig cells. Comparison with published data sets of genes enriched in SCs and spermatogonia, and responsive to retinoic acid (RA), identified a subset of genes that were regulated similarly by RA and thyroid hormone. This subset of genes showed an expression bias, as they were downregulated when enriched in spermatogonia and upregulated when enriched in SCs. Furthermore, using a genetic approach, we found that DIO3 is not expressed in SCs, but spermatogonia-specific inactivation of DIO3 led to impaired testis growth, reduced SC number, decreased cell proliferation and, especially during neonatal development, altered gene expression specific to somatic cells. These findings indicate that spermatogonial DIO3 protects testicular cells from untimely thyroid hormone signaling and demonstrate a mechanism of cross-talk between somatic and germ cells in the neonatal testis that involves the regulation of thyroid hormone availability and action

Human NUMB6 induces epithelial-mesenchymal transition and enhances breast cancer cells migration and Invasion.

Mammalian NUMB is alternatively spliced generating four isoforms NUMB1-NUMB4 that can function as tumor suppressors. NUMB1-NUMB4 proteins, which normally determine how different cell types develop, are reduced in 21% of primary breast tumors. Our previous work has, however, indicated that two novel NUMB isoforms, NUMB5 and NUMB6 have the pro-oncogenic functions. Herein, we address a novel function of human NUMB isoform 6 (NUMB6) in promoting cancer cell migration and invasion. We found that NUMB6 induced expression of embryonic transcription factor Slug, which in turn actively repressed E-cadherin, prompting cells to undergo epithelial-mesenchymal transition (EMT). Low-metastatic breast cancer cells DB-7 stably expressing NUMB6, lost their epithelial phenotype, exhibited migratory and pro-invasive behavior, and ultimately elevated expression of mesenchymal markers. Among these markers, increased vimentin, β-catenin, and fibronectin expression elicited metalloproteinase 9 (MMP9) production. Our results revealed that NUMB6-DB-7 cells have significantly increased level of Akt1 and Akt2 phosphorylation. Therefore, antagonizing Akt signaling using a chemical inhibitor LY294002, we found that NUMB6-induced Slug expression was reduced, and ultimately accompanied with decreased cell migration and invasion. In summary, this study identified a novel molecular determinant of breast cancer progression, uncovering a potential oncogenic role for the NUMB6 protein in cancer cell migration and invasion, coupled to the maintenance of mesenchymal-like cells. J. Cell. Biochem. 118: 237-251, 2017. © 2016 Wiley Periodicals, Inc