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

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

Dependency of Neurotrophin Receptor-Interacting Melanoma Associated Antigen Protein in Bone Morphogenetic Protein-Mediated Apoptosis

We created a small peptide that can inhibit NF-KB activation and apoptosis in P19 neural progenitor cells. NF-KB is a ubiquitous protein complex found in almost all mammalian cell types that regulates transcription of genes in response to inflammation, immunological insults, cell proliferation, and apoptosis. Apoptosis is a form of programmed cell death, which is the death of a cell by any means that is mediated by an intracellular program. This process is used in normal growth and development of organisms by removing cells that are unhealthy or by reducing a surplus of an unnecessary population as in shaping organs. However, uncontrolled apoptosis is the basis of many diseases that can endanger survival of the organism. For example, it can be overcompensating causing too much cell death as in neurodegenerative disorders such as Parkinson\u27s and Alzheimer\u27s diseases or under compensating allowing too much cell proliferation as in some cancers. Here, we investigated the interaction between neurotrophin receptor-interacting melanoma-associated antigen (NRAGE) protein and X-linked inhibitor of apoptosis (XIAP) protein in a specific apoptotic cell signaling pathway that is mediated by bone morphogenetic protein (BMP). We used fluorescence resonance energy transfer (FRET) to conclude that a direct interaction is very likely occurring between XIAP and the NRAGE domain consisting of 25 hexameric amino acid repeats of tryptophan-glutamine-x-proline-x-x (x=any amino acid). FRET is a microscopy technique in which a fluorescent donor molecule transfers its energy to a fluorescent acceptor molecule if the molecules are within 10 nanometers of each other (along with other satisfied parameters) strongly suggesting a direct interaction. The repeat domain in NRAGE, which is not found in any other protein except for NRAGE homologs in human, mouse, and rat, was used to create a peptide mimetic that inhibits apoptosis from BMP signaling. Additionally, it was also found to inhibit NF-KB activation precluding transcription of NF-KB target proteins. NRAGE-XIAP interaction in BMP signaling for apoptosis may be a candidate pathway to inhibit in certain diseases in which there is too much cell death and initiates an immune response from NF-KB activation. One such disease is multiple sclerosis which is an autoimmune neurodegenerative disease where the myelin sheaths around the axons in the central nervous system are damaged due to the apoptosis of oligodendrocytes

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