Pleiotropic effects of FGF2 on the SH-SY5Y human neuroblastoma cell line

Dokas LA, Beals JL, Watson SJ, Akil H
Society for Neuroscience. 2011.


Developing and mature midbrain dopaminergic neurons and their targets are responsive to fibroblast growth factor (FGF) with potential implications for the pathologies of Parkinson's disease, schizophrenia and addictive disorders. The SH-SY5Y cell line is a human N-type neuroblastoma that possesses catecholaminergic properties, can be differentiated to a more mature neuronal phenotype, expresses endogenous FGF receptors and has been used as a model of dopaminergic neurons. In SH-SY5Y cells, FGF2 rapidly increases phosphorylation at the activation domain (Thr202/Tyr204) of extracellular stimulus-regulated kinase1/2 (ERK1/2) that is blocked by preincubation with PD 173074, a selective inhibitor of the FGF receptor (FGFR). Maximal phosphorylation is seen at 5 min of incubation with FGF2. Thereafter, FGF-stimulated phosphorylation of ERK1/2 declines but remains elevated above basal levels for as long as 90 min. Although ERK1 and ERK2 are present at near-equal amounts in undifferentiated SH-SY5Y cells, normalized phosphorylation of ERK2 is higher than that of ERK1 following incubation with FGF2. Immunoblotting demonstrates that SH-SY5Y cells express the FGFR1 subtype. FGF9, which has little affinity for FGFR1, neither increases ERK1/2 phosphorylation substantially nor does it interfere with the stimulatory effect of FGF2. More extended culture of SH-SY5Y cells with FGF2 in serum-free medium modestly increases cell growth. Phase contrast microscopy and confocal imaging of the actin cytoskeleton with rhodamine phalloidin were used to examine cell morphology following differentiation in response to phorbol 12, 13-dibutyrate (PDB) and/or FGF2. Incubation for 5 days in serum-free medium with PDB ± FGF2 increases the number of filopodia on SH-SY5Y cells, an effect not seen with FGF2 alone. In response to PDB, cells express high levels of the neuronal marker, GAP-43. Although FGF2 only slightly induces GAP-43 in comparison to PDB, it potentiates PDB-dependent differentiation causing elaboration of long, highly networked processes with prominent terminations. In addition, the combination of PDB with FGF2 significantly increases phosphorylation of ERK1/2 above control levels in differentiated cells. Thus, the SH-SY5Y neuroblastoma is an appropriate model system for examination of signaling pathways mediating FGF-dependent responses in cells that mimic human dopaminergic neurons.