Expression of Ras-GRF in the SK-N-BE neuroblastoma accelerates retinoic-acid-induced neuronal differentiation and increases the functional expression of the IRK1 potassium channel.
Ras-GRF, a neuron-specific Ras exchange factor of the central nervous system, was transfected in the SK-N-BE neuroblastoma cell line and stable clones were obtained. When exposed to retinoic acid, these clones showed a remarkable enhancement of Ras-GRF expression with a concomitant high increase in the level of active (GTP-bound) Ras already after 24 h of treatment. In the presence of retinoic acid, the transfected cells stopped growing and acquired a differentiated neuronal-like phenotype more rapidly than the parental ones. Cells expressing Ras-GRF also exhibited a more hyperpolarized membrane potential. Moreover, treatment with retinoic acid led to the appearance of an inward rectifying potassium channel with electrophysiological properties similar to IRK1. This current was present in a large number of cells expressing Ras-GRF, while only a small percentage of parental cells exhibited this current. However, Northern analysis with a murine cDNA probe indicated that IRK1 mRNA was induced by retinoic acid at a similar level in both kinds of cells. Brief treatment with a specific inhibitor of the mitogen-activated protein kinase (MAPK) pathway reduced the number of transfected cells showing IRK1 activity. These findings suggest that activation of the Ras pathway accelerates neuronal differentiation of this cell line. In addition, our results suggest that Ras-GRF and/or Ras-pathway may have a modulatory effect on IRK1 channel activity.
Tonini
R
R
Dipartimento di Fishologia e Biochimica Generali, Univeritá degli Studi di Milano, Via Celoria 26, 20133 Milano, Italy.
Mancinelli
E
E
Balestrini
M
M
Mazzanti
M
M
Martegani
E
E
Ferroni
A
A
Sturani
E
E
Zippel
R
R
D000970:Antineoplastic Agents / Q000494:pharmacology*; D015152:Blotting, Northern; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D018797:Cell Cycle Proteins / Q000235:genetics; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology; D002455:Cell Division / Q000187:drug effects / Q000502:physiology; D029721:Drosophila Proteins; D004594:Electrophysiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005419:Flavonoids / Q000494:pharmacology; D005434:Flow Cytometry; D015972:Gene Expression Regulation, Neoplastic; D020662:Guanine Nucleotide Exchange Factors; D006801:Humans; D015640:Ion Channel Gating / Q000502:physiology; D009447:Neuroblastoma; D009474:Neurons / Q000737:chemistry / Q000166:cytology* / Q000201:enzymology; D010749:Phosphoprotein Phosphatases / Q000235:genetics; D015221:Potassium Channels / Q000235:genetics*; D024661:Potassium Channels, Inwardly Rectifying; D011506:Proteins / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D016271:Proto-Oncogene Proteins c-myc / Q000235:genetics; D051096:Proto-Oncogene Proteins c-ret; D012333:RNA, Messenger / Q000032:analysis; D020794:Receptor Protein-Tyrosine Kinases / Q000235:genetics; D014162:Transfection; D014212:Tretinoin / Q000494:pharmacology*; D014407:Tumor Cells, Cultured; D020663:ras Guanine Nucleotide Exchange Factors; D020666:ras-GRF1
D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D029721:Drosophila Proteins; D004791:Enzyme Inhibitors; D005419:Flavonoids; D020662:Guanine Nucleotide Exchange Factors; D015221:Potassium Channels; D024661:Potassium Channels, Inwardly Rectifying; D011506:Proteins; D011518:Proto-Oncogene Proteins; D016271:Proto-Oncogene Proteins c-myc; D012333:RNA, Messenger; D020663:ras Guanine Nucleotide Exchange Factors; D020666:ras-GRF1; D014212:Tretinoin; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C416649:Ret protein, Drosophila; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; C093973:2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one