p38 MAPK-Mediated Bmi-1 Down-Regulation and Defective Proliferation in ATM-Deficient Neural Stem Cells Can Be Restored by Akt Activation Jeesun Kim1, Jeon Hwangbo2, Paul K. Y. Wong 1 * 1 Department of Molecular Carcinogenesis, MD Anderson Cancer Center, The University of Texas, Smithville, Texas, United States of America, 2 Department of Genetic Engineering and Graduate School of Biotechnology, Kyung Hee University, Suwon, Korea Abstract A-T (ataxia telangiectasia) is a genetic disease caused by a mutation in the Atm (A-T mutated) gene that leads to neurodegeneration. Despite an increase in the numbers of studies in this area in recent years, the mechanisms underlying neurodegeneration in human A-T are still poorly understood. Previous studies demonstrated that neural stem cells (NSCs) isolated from the subventricular zone (SVZ) of Atm-/- mouse brains show defective self-renewal and proliferation, which is accompanied by activation of chronic p38 mitogen-activated protein kinase (MAPK) and a lower level of the polycomb protein Bmi-1. However, the mechanism underlying Bmi-1 down-regulation and its relevance to defective proliferation in Atm-/- NSCs remained unclear. Here, we show that over-expression of Bmi-1 increases self-renewal and proliferation of Atm-/- NSCs to normal, indicating that defective proliferation in Atm-/- NSCs is a consequence of down-regulation of Bmi-1. We also demonstrate that epidermal growth factor (EGF)-induced Akt phosphorylation renders Bmi-1 resistant to the proteasomal degradation, leading to its stabilization and accumulation in the nucleus. However, inhibition of the Akt-dependent Bmi-1 stabilizing process by p38 MAPK signaling reduces the levels of Bmi-1. Treatment of the Atm-/- NSCs with a specific p38 MAPK inhibitor SB203580 extended Bmi-1 posttranscriptional turnover and H2A ubiquitination in Atm-/- NSCs. Our observations demonstrate the molecular basis underlying the impairment of self-renewal and proliferation in Atm-/- NSCs through the p38 MAPK-Akt-Bmi-1-p21 signaling pathway. Citation: Kim J, Hwangbo J, Wong PKY (2011) p38 MAPK-Mediated Bmi-1 Down-Regulation and Defective Proliferation in ATM-Deficient Neural Stem Cells Can Be Restored by Akt Activation. PLoS ONE 6(1): e16615. doi:10.1371/journal.pone.0016615 Editor: Hitoshi Okazawa, Tokyo Medical and Dental University, Japan Received September 20, 2010; Accepted January 6, 2011; Published January 28, 2011 Copyright: 2011 Kim et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by fellowship from the Odyssey Program of MD Anderson Cancer Center, and by funds from the Longevity Foundation (formally, A-T Foundation) in Austin, Texas and A-T Children’s Project in Deerfield Beach, Florida. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: pkwong@mdanderson.org Introduction ATM (A-T mutated) kinase plays a critical role in the regulation of cell cycling, DNA repair, and cellular redox status. ATM expression is abundant in neural stem cells (NSCs), but is gradually reduced as the cell differentiates [1], suggesting that ATM may play an essential role in NSC survival and function. In normal brain tissue, the number of NSCs is the result of a tightly controlled balance between self-renewal, proliferation, differenti- ation and death. This means that proper control of these events is critical for maintaining a normal numbers of neurons, astrocytes, and oligodendrocytes in the brain [2]. The hypothesis that NSCs are functionally defective in the absence of ATM has not been conclusively determined. Abnormal neuronal and astrocytic development was reported in ATM knockout mice [1], [3–5], which could be the result of abnormal differentiation of NSCs. We previously reported that ATM is required to maintain normal self- renewal and proliferation of NSCs, due to its role in controlling the redox status. Loss of ATM renders NSCs defective in proliferation through oxidative stress-dependent p38 MAPK (hereafter called p38) signaling [6]. However, despite the mounting interest, the mechanisms regulating NSC survival and proliferation in response to oxidative stress remain elusive in this mouse model. In the present study we found that Bmi-1 is highly downreg- ulated in Atm-/- NSCs, or when normal NSCs are treated with H2O2. Bmi-1 functions as a component of the polycomb repressive protein complex [7–8]. It interacts with RING1B, another component of polycomb repressive protein complex 1, causing H2A ubiquitination and modification of chromatin structure leading to epigenetically suppress gene expression [9]. In NSCs, particularly those derived from the SVZ and hippocampus, Bmi-1 is necessary for normal NSC self-renewal and survival, because it silences genes that encode the cell cycle inhibitors p16, p19, and p21 [8], [10–11]. Accordingly, shRNA knockdown of bmi-1 results in up-regulation of p21, which in turn causes suppression of NSC self-renewal and proliferation [11]. Interestingly, Bmi-1 can also separately regulate mitochondrial function [12] and redox homeostasis by reducing the intracellular levels of ROS [13]. Thus, down-regulation of Bmi-1 in cells results in oxidative stress, just as ATM deficiency does. Furthermore, Bmi-1 deficient mice resemble Atm-/- mice, as they exhibit oxidative stress and a progressive postnatal depletion of NSCs that leads to neurological abnormalities and ataxia [11–12]. Therefore, further investigation into the mechanisms underlying Bmi-1 down-regulation in Atm-/- NSCs is necessary to better understand the role of ATM in regulating self-renewal and proliferation of NSCs. PLoS ONE | www.plosone.org 1 January 2011 | Volume 6 | Issue 1 | e16615