Potential sites for regulation of FGF2-mediated ERK1/2 pathway activation in human SH-SY5Y neuroblastoma cells

Dokas LA, Watson SJ, Akil H
Society for Neuroscience. 2017.


The complexity of the fibroblast growth factor (FGF) system is determined by multiple ligands grouped into several functional classes, four major subtypes of receptor (FGFR) with splice variants, and co-regulatory factors that modulate ligand-receptor interactions. FGF ligands that bind to membrane-bound receptors commonly activate the extracellular signal-regulated kinase 1/2 (ERK1/2) pathway. If FGF responses are to be selectively targeted in the brain, one approach may be to determine whether any aspects of ERK1/2 pathway regulation or its downstream effectors are specific to neurons and glia. Use of the SH-SY5Y neuroblastoma cell line is an appropriate starting point for such comparisons. It is a homogeneous system in which FGF2, but not FGF9, activates the ERK1/2 pathway consistent with predominant expression of FGFR1 in these cells. Being an N-type neuroblastoma, SH-SY5Y cells can also be differentiated to a mature neuronal phenotype in multiple ways including with a combination of FGF2 and a phorbol ester to activate protein kinase C (PKC). Immunoblotting for phosphorylated ERK1/2 at Thr202/Tyr204 as a measure of FGFR activation in SH-SY5Y cells demonstrates several potential sites that could be targeted for selective regulation. Firstly, ERK isoforms differ in their response to FGF2: ERK2 phosphorylation is greater than that of ERK1, while ERK1 phosphorylation is more sensitive to inhibition by the PKC inhibitor, GF 109203X. Secondly, heparin modulates FGF2-mediated ERK1/2 phosphorylation in a biphasic manner: At low concentrations of FGF2, heparin is inhibitory but this effect reverses to a stimulatory one when FGF2 is used at higher concentrations. Finally, FGF2 and insulin-like growth factor-1 (IGF-1), which have both been shown by this laboratory and others to differentiate SH-SY5Y cells, produce different patterns of cell signaling. Both growth factors activate ERK1/2 phosphorylation although with varying sensitivities. Moreover, IGF-1 has a large stimulatory effect on Akt phosphorylation at Ser473, a marker of mTOR pathway activation, in contrast to only a minimal increase with FGF2. Thus, more detailed characterization of the downstream effector systems of FGF2 and IGF-1 in SH-SY5Y cells will likely identify concentration-dependent or temporally-sensitive responses that are specific to each growth factor. Overall, these experiments suggest that comparison of the responses of SH-SY5Y cells to FGF2 with those of other cultured cell types to alternative FGF ligands may identify potential targets to selectively intervene in FGFR subtype-mediated signaling pathways.