Neurotrophic actions of PACAP-38 and LIF on human neuroblastoma SH-SY5Y cells.
The neurotrophic actions of pituitary adenylate cyclase-activating polypeptide (PACAP)-38 and leukemia inhibitory factor (LIF) were investigated in human neuroblastoma SH-SY5Y cells. Effects on differentiation were assessed through monitoring morphological changes and Western blot analysis of the expression of neuronal marker proteins. In contrast to PACAP-38, which induced a 5.5-fold increase in the number of neurite-bearing cells, LIF had no significant effect on cell morphology compared to control cells over the 4-day time course. Cells co-treated with PACAP-38+LIF showed a similar increase in neurite-bearing cells compared to those treated with PACAP-38 alone. Cell morphology was similar for PACAP-38-treated and PACAP-38+LIF-co-treated cells, with the formation of bipolar neuron-like cells with long thin neurites, topped by growth cone-like structures and varicosities. SH-SY5Y cells express tyrosine hydroxylase (TH) but only low levels of the neuronal marker proteins: Bcl-2, GAP-43 and choline acetyltransferase (ChAT). Treatment of cells with PACAP-38 induced the expression of Bcl-2, GAP-43, and ChAT but did not appear to alter the expression of TH. LIF failed to induce the expression of GAP-43 and had little effect on the expression of TH, but did induce the expression of Bcl-2 and upregulated the expression of ChAT. Co-treatment with LIF had no effect on PACAP-38-induced expression of Bcl-2, GAP-43, and ChAT. Cells differentiated for 4 days with PACAP-38 or treated with LIF also displayed increased resistance to hypoxic conditions and to treatment with H2O2 and TNFalpha. The increased resistance to hypoxic conditions for PACAP-differentiated cells was blocked by the p38 MAP kinase inhibitor, SB203580, but not by the MEK1 inhibitor, PD98059. Additionally, cell proliferation assays show that LIF, but not PACAP-38, stimulates proliferation of SH-SY5Y cells, and this observed increase by LIF is not attenuated by co-treatment with PACAP. Further investigation of the intracellular signaling pathways mediating the neurotrophic effects of PACAP on SH-SY5Y cells indicate that neither phospholipase C activation nor Ca2+/calmodulin-dependent kinase II (CAMKII) are involved.
Strathclyde Institute of Pharmacy and Biomedical Sciences, Royal College, 204 George Street, Glasgow, G1 1XW, UK.
T K
TK
Monaghan
C
C
Pou
C J
CJ
MacKenzie
R
R
Plevin
E M
EM
Lutz
D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2 / Q000378:metabolism; D002454:Cell Differentiation / Q000187:drug effects; D045744:Cell Line, Tumor; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape; D006801:Humans; D053605:Leukemia Inhibitory Factor / Q000494:pharmacology*; D009414:Nerve Growth Factors / Q000494:pharmacology*; D009447:Neuroblastoma; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide / Q000494:pharmacology*; D053606:Receptors, OSM-LIF / Q000235:genetics / Q000378:metabolism; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I / Q000235:genetics / Q000378:metabolism; D010738:Type C Phospholipases / Q000378:metabolism
D015415:Biomarkers; C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; D009414:Nerve Growth Factors; D051219:Pituitary Adenylate Cyclase-Activating Polypeptide; D053606:Receptors, OSM-LIF; D051237:Receptors, Pituitary Adenylate Cyclase-Activating Polypeptide, Type I; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D010738:Type C Phospholipases