Involvement of NOS3 in RA‐Induced neural differentiation of human NT2/D1 cells

Nitric oxide (NO) plays a key role in neurogenesis as a regulator of cell proliferation and differentiation. NO is synthesized from the amino acid L-arginine by nitric oxide synthases (NOS1, NOS2, and NOS3), which are encoded by separate genes and display different tissue distributions. We used an in vitro model of RA-induced neural differentiation of NT2 cells to examine which of the three NO-synthesizing enzymes is involved in this process. The results revealed a transient induction of NOS3 (known as the constitutively expressed endothelial nitric oxide synthase; eNOS) during the time course of the RA treatment. The peak of gene expression and the nuclear presence of NOS3 protein coincided with cell cycle exit of NT2-derived neuronal precursors. The subsequent analysis of cytosine methylation and histone H3 acetylation of the human NOS3 5' regulatory sequences indicated that epigenetic modifications, especially upstream of the proximal promoter (-734 to -989, relative to exon 2 TSS at +1), were also taking place. NOS1 was expressed only in the differentiated neurons (NT2-N), whereas NOS2 was not expressed at all in this cellular model. Thus, a burst of NO production, possibly required to inhibit neural cell proliferation, was generated by the transient expression of NOS3. This pattern of gene expression, in turn, required epigenetic remodeling of its regulatory region.