Both FGF2- and protein kinase C-mediated signaling determine differentiation of SH-SY5Y human neuroblastoma cells
Dokas LA, Watson SJ, Akil H
Society for Neuroscience. 2016.
The SH-SY5Y cell line is an N-type neuroblastoma with catecholaminergic properties. These cells can be further differentiated by several mechanisms including the combination of a growth factor and a phorbol ester that activates protein kinase C (PKC). One of the most effective growth factors in this regard is FGF2. Given the phenotype of SH-SY5Y cells, differentiation with FGF2 and phorbol ester may model development from a neuroblastic stage to more mature neuronal morphology. We have previously reported that FGF2 and the phorbol ester, phorbol 12, 13-dibutyrate (PDB) each affect the morphology of differentiating cells: PDB produces lamellipodial profiles while FGF2 elongates cells and processes. In combination, highly networked cells result. In order to understand the signaling that underlies these changes, interactions between FGF2 and PKC are being characterized in undifferentiated and differentiated cells. Both FGF2 and PDB activate the extracellular stimulus-regulated kinase1/2 (ERK1/2) pathway, measured as phosphorylation of the activation domain of ERK1/2 at Thr202/Tyr204, with a considerable degree of crosstalk in undifferentiated cells. Although only the effect of FGF2 is blocked by the FGF receptor (FGFR)-selective inhibitor, PD 173074, the PKC inhibitor, GF 109203X, blocks both PDB- and FGF2-mediated ERK1/2 phosphorylation. One likely site of this crosstalk is at the level of raf since inhibition of MEK1/2, the protein kinase target of raf, affects ERK1/2 phosphorylation in response to either stimulus. Furthermore, ERK1/2 phosphorylation in response to FGF2 is lost in differentiated cells indicating that a change in FGFR sensitivity or coupling occurs during the process of differentiation. As a second means of characterizing involvement of PKC in SH-SY5Y cell differentiation, phosphorylation of two major substrate proteins, myristoylated alanine-rich C kinase substrate (MARCKS) and growth-associated protein-43 (GAP-43) have been compared. Both expression and phosphorylation of GAP-43 at Ser41 are increased in differentiated cells, principally in response to PDB. MARCKS phosphorylation at Ser167/170 is also PDB-sensitive; however, in this case, co-exposure to FGF2 reduces levels of phospho-MARCKS. Since both MARCKS and GAP-43 are involved in actin-based remodeling of the neuronal cytoskeleton, differential regulation of their phosphorylated states by PKC and FGF2 could underlie the changes in cell morphology seen during differentiation of SH-SY5Y cells.