LIF-mediated maintenance of PACAP-induced neurite outgrowths in human SH-SY5Y neuroblastoma cells require PI3K but not STAT or ERK signaling pathways

Previously, we have shown that pituitary adenylate cyclase-activating polypeptide (PACAP) induces neuritogenesis in human SH-SY5Y cells through a cAMP-dependent but protein kinase A-independent mechanism involving activation of the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein kinase (p38 MAPK) signaling pathways. However, the morphological changes induced by PACAP are transient, with the increase in number of neurite-bearing cells peaking at day 4 before returning to control cell levels over an 8-day time course. Control cells maintain an undifferentiated neuroblast morphology during the 8-day experiment. On the other hand, neurite extensions are maintained by cells co-treated with PACAP and the neuropoietic cytokine leukemia inhibitory factor (LIF), with similar levels in the number of neurite-bearing cells observed at day 8 to that at day 4. Interestingly, the induction of neurite outgrowths in SH-SY5Y cells requires a minimal 2-h treatment with PACAP, with 4–6 h of treatment inducing similar peak levels of neurite-bearing cells observed at day 4 to that of continual PACAP treatment. LIF is not required until day 4 after which continual treatment with LIF is necessary for neurite maintenance in PACAP-treated cells. The signaling pathways activated by LIF were investigated also. LIF elicits signal transducer and activator of transcription 3 (STAT3) but not STAT1 tyrosine phosphorylation along with a transient activation of the ERK and prolonged activation of phosphatidylinositol-3 kinase (PI3K)/protein kinase B (PKB/Akt) in PACAP-treated and control SH-SY5Y cells. Phosphorylation of serine 727 on STAT3 was detected in PACAP-treated but not control cells, and this was prevented by the MEK-1 inhibitor PD98059. Activation of p38 MAPK or c-jun N-terminal kinase (JNK) were not detected, however, an elevated basal level of phosphorylated p38 MAP kinase was observed in PACAP-treated cells. Neither PD98059 nor siRNA knockdown of STAT3 prevented LIF-mediated maintenance of PACAP-induced neurite extensions. However, the PI3K inhibitor wortmannin was found to effectively block LIF maintenance of neurite outgrowths in PACAP-treated cells. Thus, the ability of LIF to stabilize and maintain PACAP-induced neurite outgrowths requires PI3K but not the STAT3 or ERK signaling pathways in SH-SY5Y cells.