Signaling from G protein coupled receptors to the c-jun promoter involves the MEF2 transcription factor - Evidence for a novel c-Jun amino-terminal kinase-independent pathway

The c-Jun amino-terminal kinases (JNKs) are a subfamily of mitogen-activated protein kinases that phosphorylate c-Jun and ATF2, and it has been postulated that phosphorylated c-Jun enhances its own expression through AP-1 sites on the c-jun promoter. In this study, we asked whether signals activating JNK regulate the c-jun promoter, Using NIH 3T3 cells expressing G protein-coupled mi acetylcholine receptors as an experimental model, we have recently shown that the cholinergic agonist carbachol, but not platelet derived growth factor, potently elevates JNK activity, Consistent with these findings, carbachol, but not platelet-derived growth factor, increased the activity of a c-jun promoter-driven reporter gene (for chloramphenicol acetyltransferase), However, coexpression of JNK kinase kinase (MEKK) effectively increased JNK activity, but resulted in surprisingly limited induction of the c- jun promoter, This raised the possibility that pathway(s) distinct from JNK control the c-jun promoter, and prompted us to explore which of its regulatory elements participate in transcriptional control. We observed that deletion of the 3' AP-1 site diminished chloramphenicol acetyltransferase activity in response to carbachol, but only to a limited extent, In contrast, deletion of a MEF2 site dramatically reduced expression, and deletion of both the MEF2 and 3' AP-1 sites abolished induction, Furthermore, cotransfection with MEF2C and MEF2D cDNAs potently enhanced the activity of the c-jun promoter in response to carbachol, and stimulation of mi receptors, but not direct JNK activation, induced expression of a MEF2-responsive plasmid. Taken together, these data strongly suggest that MEF2 mediates c-jun promoter expression by G protein-coupled receptors through a yet to be identified pathway, distinct from that of JNK.