The role of calcium influx in cellular proliferation induced by interaction of endogenous ganglioside GM1 with the B subunit of cholera toxin.
The B subunit of cholera toxin, which binds specifically to ganglioside GM1, is mitogenic for quiescent Swiss 3T3 fibroblasts. It was previously shown that the B subunit had no effect on cAMP, protein kinase C or phosphoinositide turnover, but did cause an increase in the influx of calcium from extracellular sources (Spiegel, S. and Panagiotopoulos, C. (1988) Exp. Cell Res. 177, 414-427). In contrast to the action of known growth factors, the B subunit induced significant DNA synthesis after only a 1-3 h treatment. We utilized this unique property to determine whether the increase in calcium influx plays a role in B subunit-induced mitogenicity. Cells were briefly treated with the B subunit in the presence of calcium channel blockers, followed by removal of the blockers and further incubation in B subunit-free medium for the remaining time required to measure DNA synthesis. When 1 mM cobalt was only present during the first 3 h incubation. DNA synthesis induced by either the B subunit or fetal bovine serum was completely abolished. However, both nickel (1 mM) adn the L-type voltage-gated calcium channel inhibitor nicardipin (10 microM) inhibited B subunit-induced cell proliferation without abrogating the response to fetal bovine serum. Using a gel retardation assay, we found that the B subunit markedly stimulated specific DNA-binding activity of the transcription factor, activator protein-1 (AP-1), which functions as a major convergence point coupling early events induced by a variety of mitogens to long term growth responses. Presence of c-Fos protein in the AP-1 complex was demonstrated as a supershift band in the gel mobility assay using c-Fos polyclonal antibody. Cobalt, which markedly inhibited B subunit-induced DNA synthesis, also completely abolished AP-1 DNA-binding activity stimulated by the B subunit. In sharp contrast, cobalt had no effect on DNA-binding activity of AP-1 induced by the tumor promoter, 12-O-tetradecanoylphorbol 13-acetate. Our results suggest that calcium influx is a key element for both DNA-binding activity of AP-1 and cell proliferation induced by binding of the B subunit of cholera toxin to cell surface ganglioside GM1.
Buckley
N E
NE
Department of Biochemistry and Molecular Biology, Georgetown University, Medical Center, Washington, DC 20007, USA.
Su
Y
Y
Milstien
S
S
Spiegel
S
S
D016475:3T3 Cells / Q000187:drug effects; D000818:Animals; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002121:Calcium Channel Blockers / Q000494:pharmacology; D002455:Cell Division / Q000187:drug effects; D002772:Cholera Toxin / Q000737:chemistry / Q000378:metabolism* / Q000494:pharmacology; D003035:Cobalt / Q000494:pharmacology; D004247:DNA / Q000096:biosynthesis; D004268:DNA-Binding Proteins / Q000378:metabolism; D005677:G(M1) Ganglioside / Q000378:metabolism*; D051379:Mice; D008969:Molecular Sequence Data; D009532:Nickel / Q000494:pharmacology; D009687:Nuclear Proteins / Q000378:metabolism; D000071701:Regulatory Factor X Transcription Factors; D018808:Transcription Factor AP-1 / Q000494:pharmacology; D014157:Transcription Factors
D002121:Calcium Channel Blockers; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D000071701:Regulatory Factor X Transcription Factors; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D005677:G(M1) Ganglioside; D003035:Cobalt; D009532:Nickel; D004247:DNA; D002772:Cholera Toxin; D002118:Calcium