Mitogenesis of 3T3 fibroblasts induced by endogenous ganglioside is not mediated by cAMP, protein kinase C, or phosphoinositides turnover.
The B subunit of cholera toxin, which binds specifically to ganglioside GM1, stimulates DNA synthesis in quiescent Swiss 3T3 fibroblasts grown in chemically defined medium. The mitogenic response to the B subunit was potentiated by insulin and other growth factors. To elucidate the mechanism by which the B subunit stimulates cell growth , its effects on several transmembrane signaling systems which have been suggested to play a vital role in cell growth regulation were examined. The B subunit did not increase cAMP levels nor activate adenylate cyclase. The B subunit induced a rapid and profound increase in intracellular free Ca2+ as measured with the fluorescent Ca2+-sensitive dye quin 2/AM. Removal of external Ca2+ completely inhibited the signal, thus suggesting that the B subunit elevates intracellular Ca2+ through a net influx of extracellular Ca2+ rather than by causing the release of Ca2+ from intracellular stores. These findings are consistent with the observations that the B subunit induced reinitiation of DNA synthesis without activation of phospholipase C. There was no increase in the formation of inositol trisphosphate, the second messenger that mediates release of Ca2+ from intracellular stores. In addition, the B subunit still stimulated DNA synthesis in Swiss 3T3 cells pretreated with phorbol ester to down-regulate protein kinase C. These results suggest that the mitogenic effects of the B subunit are mediated mainly by facilitation of Ca2+ influx and that activations of adenylate cyclase, phospholipase C, or protein kinase C are not obligatory steps in the initiation of cell growth by the B subunit. Furthermore, the observation that Ca2+ ionophores, such as ionomycin and A23187, are not mitogenic implies that additional undefined growth signaling pathways may exist in this system.
Spiegel
S
S
Membrane Biochemistry Section, National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, Maryland 20892.
Panagiotopoulos
C
C
D000262:Adenylyl Cyclases / Q000378:metabolism; D000818:Animals; D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line / Q000187:drug effects; D002772:Cholera Toxin / Q000494:pharmacology; D000242:Cyclic AMP / Q000494:pharmacology*; D004261:DNA Replication / Q000187:drug effects; D004987:Ethers / Q000494:pharmacology; D015759:Ionomycin; D051379:Mice; D010716:Phosphatidylinositols / Q000378:metabolism*; D011493:Protein Kinase C / Q000378:metabolism*; D010738:Type C Phospholipases / Q000378:metabolism
D004987:Ethers; D010716:Phosphatidylinositols; D000001:Calcimycin; D015759:Ionomycin; D002772:Cholera Toxin; D000242:Cyclic AMP; D011493:Protein Kinase C; D010738:Type C Phospholipases; D000262:Adenylyl Cyclases