Date of Award

12-2011

Level of Access

Campus-Only Thesis

Degree Name

Master of Science (MS)

Department

Biochemistry

Advisor

Joseph M. Verdi

Second Committee Member

Robert Friesel

Third Committee Member

Douglas Spicer

Abstract

Neural progenitor cell apoptosis is regulated in part by activation of the mitogen-activated protein kinase (MAPK) through the bone morphogenic pathway (BMP) pathway independent of classical SMAD signaling. Research has revealed that 'non-canonical' BMP signaling requires the formation and accumulation of a NRAGE/XIAP/Tab1/Tak1 complex at the BMP receptors present on the cell membrane, culminating in the phosphorylation of Tak1. Phosphorylated Tak1 is then able to activate the MAPK, JNK, and NF-kB pathways. The scaffolding protein NRAGE (neurotrophin receptor-interacting melanoma-associated antigen protein) contains a highly conserved MAGE Homology Domain (MHD), a less well-conserved MHD domain (MHD2), and a unique WQXPXX hexapeptide repeat region. Unlike the other MAGE family of genes whose protein expression is highly specific, NRAGE protein expression is found in most embryonic and adult tissues, suggesting a critical role in basic cellular function. While BMP-4 signaling has previously been linked to NF-KB activation, we wanted to determine if the scaffolding protein NRAGE was required. Using 293HEK cells as a model, the expression of NRAGE was systematically altered as well as other parts of the non-canonical BMP pathway. It was found that BMP-4 required the presence of NRAGE to activate the NF-kB pathway, and the overexpression of NRAGE constitutively activated NF-kB. The consequence of NF-kB activation was the dramatic increase in expression of the anti-apoptotic cytokine, macrophage migration inhibitory factor (MIF). Using P19 cells as a model for neural progenitor apoptosis, it was found that overexpression of a MIF:EGFP protein or stimulation of cells with rMIF decreased the amount of phospho-p53 and decreased the number of Annexin V and 7AAD double positive apoptotic cells. In contrast, the amount of apoptosis was increased when MIF expression was ablated. Overall, the studies herein provide novel insight into how neural progenitors escape BMP-4 induced apoptosis leaving behind a small subset of stem cells.

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