Retinoic acid-induced neuritogenesis of human neuroblastoma SH-SY5Y cells is ERK independent and PKC dependent.
Retinoic acid (RA), an active metabolite of vitamin A, is a natural morphogen involved in development and differentiation of the nervous system. To elucidate signaling mechanisms involved in RA-induced neuritogenesis, we used human neuroblastoma SH-SY5Y cells, an established in vitro model for studying RA action, to examine the role of extracellular signal-regulated kinase (ERK) 1 and 2 in RA-induced neuritogenesis and cell survival. From immunoblotting experiments, we observed that RA induced delayed but persistent ERK1 and ERK2 phosphorylation (until 96 hr) that was reduced significantly by the specific mitogen-activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor U0126. For the subsequent studies we chose 24 hr as the reference time. Inhibition of ERK activation did not affect RA-induced neuritogenesis (percentage of neurite-bearing cells and neurite length) but significantly reduced cell survival. In addition, we analyzed the signaling pathway that mediates ERK activation. Our results suggest that RA-induced ERK phosphorylation does not follow the classic Raf kinase-dependent pathway. Protein kinase C (PKC) and phosphatidylinositol 3-kinase (PI 3-K) are possible alternative kinases involved in the ERK signaling pathway. In fact, in the presence of the specific PKC inhibitor GF 109203X, or the specific PI 3-K inhibitor wortmannin, we observed a significant dose-dependent reduction in ERK phosphorylation. RA-induced neuritogenesis and cell survival were reduced by GF 109203X in a concentration-dependent manner. These results suggest that rather than ERK1 and ERK2, it is PKC that plays an important role during early phases of RA-induced neuritogenesis.
Miloso
Mariarosaria
M
Dipartimento di Neuroscienze e Tecnologie Biomediche, Università degli Studi di Milano-Bicocca, Monza, Italy.
Villa
Daniela
D
Dipartimento di Neuroscienze e Tecnologie Biomediche, Università degli Studi di Milano-Bicocca, Monza, Italy.
Crimi
Marco
M
Centro Dino Ferrari, Dipartimento di Scienze Neurologiche, Universita' di Milano, IRCCS Ospedale Maggiore Policlinico, Milano, Italy.
Galbiati
Stefania
S
Dipartimento di Neuroscienze e Tecnologie Biomediche, Università degli Studi di Milano-Bicocca, Monza, Italy.
Donzelli
Elisabetta
E
Dipartimento di Neuroscienze e Tecnologie Biomediche, Università degli Studi di Milano-Bicocca, Monza, Italy.
Nicolini
Gabriella
G
Dipartimento di Neuroscienze e Tecnologie Biomediche, Università degli Studi di Milano-Bicocca, Monza, Italy.
Tredici
Giovanni
G
Dipartimento di Neuroscienze e Tecnologie Biomediche, Università degli Studi di Milano-Bicocca, Monza, Italy.
D002454:Cell Differentiation / Q000187:drug effects* / Q000502:physiology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D019950:Mitogen-Activated Protein Kinase 1 / Q000187:drug effects / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000187:drug effects / Q000378:metabolism*; D009420:Nervous System / Q000166:cytology / Q000201:enzymology; D016501:Neurites / Q000187:drug effects / Q000201:enzymology* / Q000648:ultrastructure; D009447:Neuroblastoma / Q000201:enzymology*; D019869:Phosphatidylinositol 3-Kinases / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000187:drug effects / Q000378:metabolism*; D019908:Proto-Oncogene Proteins c-raf / Q000187:drug effects / Q000378:metabolism; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014212:Tretinoin / Q000378:metabolism* / Q000494:pharmacology
D004791:Enzyme Inhibitors; D014212:Tretinoin; D019869:Phosphatidylinositol 3-Kinases; D019908:Proto-Oncogene Proteins c-raf; D011493:Protein Kinase C; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases