NITRIC OXIDE DECREASES SUBVENTRICULAR ZONE STEM CELL PROLIFERATION BY INHIBITION OF EPIDERMAL GROWTH FACTOR RECEPTOR AND PI3-K/AKT PATHWAY

¹ Área de Fisiología, Facultad de Medicina, Universidad de Cádiz, Cádiz, Spain
² Unidad de Investigación, Hospital Universitario Puerta del Mar, Cádiz, Spain

^* To whom correspondence should be addressed. E-mail: carmen.estrada{at}uca.es.

	Abstract

Nitric oxide (NO) inhibits proliferation of subventricular zone (SVZ) neural precursor cells in adult mice in vivo, under physiological conditions. The mechanisms underlying this NO effect have been now investigated using SVZ-derived neural stem cells, which generate neurospheres in vitro when stimulated by epidermal growth factor (EGF). In these cultures, NO donors decreased the number of newly-formed neurospheres as well as their size, which indicates that NO was acting on the neurosphere-forming neural stem cells and the daughter neural progenitors. The effect of NO was cytostatic, not pro-apoptotic, and did not involve cGMP synthesis. Neurosphere cells expressed the neuronal and endothelial isoforms of NO synthase (NOS), and produced NO in culture. Inhibition of NOS activity by N-nitro-L-arginine methyl ester (L-NAME) promoted neurosphere formation and growth, thus, revealing an autocrine/paracrine action of NO on the neural precursor cells. Both exogenous and endogenous NO impaired the EGF-induced activation of the EGF receptor (EGFR) tyrosine kinase and prevented the EGF-induced Akt phosphorylation in neurosphere cells. Inhibition of the PI3-K/Akt pathway by LY294002 significantly reduced the number of newly-formed neurospheres, which indicates that this is an essential pathway for neural stem cell self-renewal. Chronic administration of L-NAME to adult mice enhanced phospho-Akt staining in the SVZ and reduced nuclear p27^Kip1 in the SVZ and olfactory bulb. The inhibition of EGFR and PI3-K pathway by NO explains, at least in part, its antimitotic effect on neurosphere cells, and may be a mechanism involved in the physiological role of NO as a negative regulator of SVZ neurogenesis in adult mice.

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