Expression of different Jun and Fos proteins during the G0-to-G1 transition in mouse fibroblasts: in vitro and in vivo associations.

We have characterized the expression of c-Jun, JunB, JunD, c-Fos, and FosB proteins following serum stimulation of quiescent Swiss 3T3 cells by immunoprecipitation analyses. The synthesis of the three Jun proteins rapidly increases following stimulation, remaining at a significant level for at least 8 h. JunB protein presents the highest expression of all. FosB, like c-Fos, is transiently induced. Pulse-chase experiments show that all of the proteins except JunD are short-lived. We have shown that c-Fos and FosB form complexes in vivo with the different Jun proteins and that JunB complexes are predominant. In vitro association and competition experiments show that the affinities between the different Fos and Jun proteins are similar. This finding, together with the in vivo observations described above, suggests that the proportion of the different Jun/Fos heterodimers is governed by the concentration of the different components. The Fos and Jun proteins are phosphoproteins, and some remain relatively highly phosphorylated in their heterodimeric form.     

Expression of c-fos and jun protooncogenes in ovine trophoblasts in relation to interferon-tau expression and early implantation process

Expression of c-fos and jun protooncogenes was analyzed in the ovine extraembryonic trophoblast from days 14–18 of gestation, using Northern and Western blotting and immunohistochemistry. This study was carried out in relation to the early implantation process and the expression of interferon-tau, which is secreted in large amounts for a few days before implantation. Our results demonstrated that c-fos, c-jun, and junB were differently expressed in the ovine trophoblast around the time of implantation. The c-fos mRNA and protein were detected at high levels prior to attachment and decreased thereafter, following the pattern of expression of interferon-tau, whereas c-jun expression was maintained at relatively high levels during the implantation process. By contrast, the levels of junB mRNA and protein decreased prior to attachment. Immunohistochemical studies indicated that JunB, like C-Fos and interferon tau, was no longer expressed in the trophoblastic cells which had established cellular contacts with the uterine epithelium. A striking finding in this study is the temporal correlation between the accumulation of c-Fos and c-Jun proteins and the expression of the interferon-tau (days 14 and 15 of gestation). We also showed by gel-retardation assays that an AP-1-like site present in the promoter of one interferon-tau gene was functional in vitro, as judged by its ability to bind day-15 trophoblast nuclear protein extracts. Nuclear proteins binding to this site had the characteristics of AP-1, as judged by the ability to be competed efficiently by a consensus TRE (12.0-tetradecanoyl phorbol 13-acetate-responsive element)-site oligonucleotide and by antibodies to c-Fos and Jun proteins. These results suggest that Fos and Jun could form regulatory complexes of interferon-tau expression and/or are regulated by common mechanisms which are still unknown. Mol Reprod Dev 46:127–137, 1997. © 1997 Wiley-Liss, Inc.     

JunB forms the majority of the AP-1 complex and is a target for redox regulation by receptor tyrosine kinase and G protein-coupled receptor agonists in smooth muscle cells

To understand the role of redox-sensitive mechanisms in vascular smooth muscle cell (VSMC) growth, we have studied the effect of N-acetylcysteine (NAC), a thiol antioxidant, and diphenyleneiodonium (DPI), a potent NADH/NADPH oxidase inhibitor, on serum-, platelet-derived growth factor BB-, and thrombin-induced ERK2, JNK1, and p38 mitogen-activated protein (MAP) kinase activation; c-Fos, c-Jun, and JunB expression; and DNA synthesis. Both NAC and DPI completely inhibited agonist-induced AP-1 activity and DNA synthesis in VSMC. On the contrary, these compounds had differential effects on agonist-induced ERK2, JNK1, and p38 MAP kinase activation and c-Fos, c-Jun, and JunB expression. NAC inhibited agonist-induced ERK2, JNK1, and p38 MAP kinase activation and c-Fos, c-Jun, and JunB expression except for platelet-derived growth factor BB-induced ERK2 activation. In contrast, DPI only inhibited agonist-induced p38 MAP kinase activation and c-Fos and JunB expression. Antibody supershift assays indicated the presence of c-Fos and JunB in the AP-1 complex formed in response to all three agonists. In addition, cotransfection of VSMC with expression plasmids for c-Fos and members of the Jun family along with the AP-1-dependent reporter gene revealed that AP-1 with c-Fos and JunB composition exhibited a higher transactivating activity than AP-1 with other compositions tested. All three agonists significantly stimulated reactive oxygen species production, and this effect was inhibited by both NAC and DPI. Together, these results strongly suggest a role for redox-sensitive mechanisms in agonist-induced ERK2, JNK1, and p38 MAP kinase activation; c-Fos, c-Jun, and JunB expression; AP-1 activity; and DNA synthesis in VSMC. These results also suggest a role for NADH/NADPH oxidase activity in some subset of early signaling events such as p38 MAP kinase activation and c-Fos and JunB induction, which appear to be important in agonist-induced AP-1 activity and DNA synthesis in VSMC.     

A novel AP-1 site is critical for maximal induction of the follicle-stimulating hormone beta gene by gonadotropin-releasing hormone

Regulation of follicle-stimulating hormone (FSH) synthesis is a central point of convergence for signals controlling reproduction. The FSHbeta subunit is primarily regulated by gonadotropin-releasing hormone (GnRH), gonadal steroids, and activin. Here, we identify elements in the mouse FSHbeta promoter responsible for GnRH-mediated induction utilizing the LbetaT2 cell line that endogenously expresses FSH. The proximal 398 bp of the mouse FSHbeta promoter is sufficient for response to GnRH. This response localizes primarily to an AP-1 half-site (-72/-69) juxtaposed to a CCAAT box, which binds nuclear factor-Y. Both elements are required for AP-1 binding, creating a novel AP-1 site. Multimers of this site confer GnRH induction, and mutation or internal deletion of this site reduces GnRH induction by 35%. The same reduction was achieved using a dominant negative Fos protein. This is the only functional AP-1 site identified in the proximal 398 bp, since its mutation eliminates FSHbeta induction by c-Fos and c-Jun. GnRH regulation of the FSHbeta gene occurs through induction of multiple Fos and Jun isoforms, forming at least four different AP-1 molecules, all of which bind to this site. Mitogen-activated protein kinase activity is required for induction of FSHbeta and JunB protein. Finally, AP-1 interacts with nuclear factor-Y, which occupies its overlapping site in vivo.