Abstract
Retinoic acid (RA) induces neural differentiation of SH-SY5Y neuroblastoma cells. We show that the mRNA levels of the differentiation-inhibiting basic helix-loop-helix transcription factors ID1, ID2, and ID3 are down-regulated during RA-induced differentiation of SH-SY5Y cells. The levels of ID proteins decreased in parallel to the observed transcriptional repression. The expression of other basic helix-loop-helix genes changed during RA-induced differentiation: expression of neuroblast-specific ASCL1 (HASH-1) gene was promptly reduced after RA treatment, whereas expression of differentiation-promoting genes NEUROD6 (NEX-1, HATH-2) and NEUROD1 was increased. Treatments with 12-O-tetradecanoylphorbol-13-acetate, another inducer of neuroblastoma cell differentiation, also resulted in coordinated down-regulation of ID gene expression, underscoring the role of ID genes in differentiation. Down-regulation of ID gene expression by RA involves a complex mechanism because full transcriptional repression required newly synthesized proteins and signaling by phosphatidylinositol 3-kinase (PI3K). RA treatment activates the PI3K/Akt signaling pathway, resulting in increased PI3K activity in extracts from RA-treated cells and a rapid increase in phosphorylation of Akt in Ser-473. Inhibition of PI3K by LY294002 impaired RA-induced differentiation, as assessed by morphological and biochemical criteria. We propose that RA, by activating the PI3K/Akt signaling pathway, plays an important role in the regulation of neuronal cell survival.
Highlights
Cell differentiation is a complex process regulated by an interplay among intrinsic cellular programs, cell-cell and cellsubstrate interactions, and a plethora of soluble extracellular signaling molecules, including hormones, growth factors, cyto
Because different members of the ID gene subfamily of bHLH factors have been involved in the regulation of differentiation in many cell types [33, 34], we investigated whether other ID genes are expressed in SH-SY5Y neuroblastoma cells and how Retinoic acid (RA) treatment could modify their expression levels
We show that ID gene expression is coordinately down-regulated during RA-induced differentiation of SH-SY5Y neuroblastoma cells and that ID protein levels follow a parallel decrease
Summary
SH-SY5Y human neuroblastoma cells were cultured in RPMI 1640 medium with 10% fetal calf serum and 0.15 mg/ml gentamycin sulfate in a humidified incubator at 37 °C with 5% CO2. The medium was replaced every 3 days, and the cells were split before they reached confluence. RA (all-trans retinoic acid), TPA (12-O-tetradecanoylphorbol-13-acetate), cycloheximide, and LY294002 (all from Sigma) were dissolved in ethanol or dimethyl sulfoxide and added to the culture medium at the indicated concentrations
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
