Biphasic increase in c-jun mRNA is required for induction of AP-1-mediated gene transcription: differential effects of muscarinic and thrombin receptor activation.
Activation of either muscarinic cholinergic or thrombin receptors increases phosphoinositide turnover, Ca2+ mobilization, and redistribution of protein kinase C and induces rapid transient increases in c-fos mRNA and c-jun mRNA in 1321N1 cells. To determine whether the increases in c-fos and c-jun mRNA induced by carbachol and thrombin are sufficient to stimulate AP-1-mediated transactivation, 1321N1 cells were transfected with a reporter carrying two copies of the tetradecanoyl phorbol acetate response element and the firefly luciferase gene. Thrombin was significantly more effective than carbachol at stimulating AP-1-mediated transactivation. To identify the factors underlying the difference in AP-1 activity induced by carbachol and thrombin, members of the fos and jun families which encode components of AP-1 were examined. Carbachol and thrombin have similar effects on expression of c-fos, fosB, fra-2, junB, and junD, both acutely and over a 24-h time course. However, whereas carbachol leads only to transient induction of c-jun (maximal at 0.5 h), thrombin induces a biphasic increase in c-jun mRNA--an initial peak at 0.5 h and a second, more-prolonged increase at 12 h. Thrombin but not carbachol also induces a late increase in fra-1 mRNA, which peaks at 12 h. The secondary increase in c-jun mRNA is associated with marked increases in c-Jun protein levels and AP-1 DNA-binding activity. The late induction of c-jun and fra-1 mRNA can be prevented by adding the antagonist hirudin 30 min after thrombin, which results in loss of thrombin-stimulated increases in c-Jun protein, AP-1 DNA-binding activity, and AP-1-mediated transactivation. These findings suggest that rapid and transient conduction of c-fos and c-jun mRNA is insufficient to induce prominent changes in gene transcription, while the sustained increase in c-jun mRNA and perhaps the late induction of fra-1 mRNA are required for generation of AP-1 DNA-binding activity and transactivation through AP-1.
Trejo
J
J
Department of Pharmacology, University of California, San Diego, La Jolla 92093.
Chambard
J C
JC
Karin
M
M
Brown
J H
JH
D015152:Blotting, Northern; D002217:Carbachol / Q000378:metabolism; D005786:Gene Expression Regulation; D006801:Humans; D007700:Kinetics; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism*; D011956:Receptors, Cell Surface / Q000378:metabolism*; D011976:Receptors, Muscarinic / Q000378:metabolism*; D018179:Receptors, Thrombin; D013917:Thrombin / Q000378:metabolism; D014158:Transcription, Genetic
D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012333:RNA, Messenger; D011956:Receptors, Cell Surface; D011976:Receptors, Muscarinic; D018179:Receptors, Thrombin; D002217:Carbachol; D013917:Thrombin