Nathan Eklund

Several different oncogenes and growth factors promote G1 phase progression. Cyclin D1, the regulatory subunit of several cyclin-dependent kinases, is required for, and capable of shortening, the G1 phase of the cell cycle. The present study demonstrates that transforming mutants of p21ras (Ras Val-12, Ras Leu-61) induce the cyclin D1 promoter in human trophoblasts (JEG-3), mink lung epithelial (Mv1.Lu), and in Chinese hamster ovary fibroblast cell lines. Site-directed mutagenesis of AP-1-like sequences at -954 abolished p21ras-dependent activation of cyclin D1 expression. The AP-1-like sequences were also required for activation of the cyclin D1 promoter by c-Jun. In electrophoretic mobility shift assays using nuclear extracts from cultured cells and primary tissues, several AP-1 proteins (c-Jun, JunB, JunD, and c-Fos) bound the cyclin D1 -954 region. Cyclin D1 promoter activity was stimulated by overexpression of mitogen-activated protein kinase (p41MAPK) or c-Ets-2 through the proximal 22 base pairs. Expression of plasmids encoding either dominant negative MAPK (p41MAPKi) or dominant negatives of ETS activation (Ets-LacZ), antagonized MAPK-dependent induction of cyclin D1 promoter activity. Epidermal growth factor induction of cyclin D1 transcription, through the proximal promoter region, was antagonized by either p41MAPKi or Ets-LacZ, suggesting that ETS functions downstream of epidermal growth factor and MAPK in the context of the cyclin D1 promoter. The activation of cyclin D1 transcription by p21ras provides evidence for cross-talk between the p21ras and cell cycle regulatory pathways.

The P-450 side chain cleavage (CYP11A1) gene encodes the enzyme that catalyzes the initial step in steroid biosynthesis, resulting in the conversion of cholesterol to pregnenolone. Expression of the CYP11A1 gene is increased by hormones, such as adrenocorticotropin and luteinizing hormone, as well as by a number of growth factors, suggesting that its promoter may contain regulatory elements that respond to multiple signal transduction pathways. Using transient expression assays of the ovine CYP11A1 promoter in JEG-3 placental cells, distinct regulatory elements were found to mediate transcriptional stimulation by cAMP and epidermal growth factor (EGF). The cAMP response was mediated through a GC-rich sequence localized between -117 and -92. In contrast, EGF induced CYP11A1 transcription through an adjacent but distinct sequence (-92 to -77 base pairs) that was shown previously to bind nuclear proteins in DNase I footprinting reactions. This EGF-responsive element (EGF-RE) resembles an activator protein-1 (AP-1) site and was also required for transactivation by co-transfected c-Jun. A point mutation within the EGF-RE impaired stimulation by both EGF and c-Jun, suggesting that these pathways converge on a common regulatory element. Transfer of single or multiple copies of the EGF-RE upstream of an heterologous promotor conferrd EGF and c-Jun responses, providing further evidence that this element is sufficient for both responses. Transfection studies employing mutant c-Jun proteins confirmed a requirement for its DNA binding, leucine zipper and amino-terminal domains, each of which are required for activation of a classical AP-1 reporter. Gel shift studies demonstrated that protein binding to the CYP11A1 EGF-RE was competed specifically by a canonical AP-1 site, and the addition of an anti-JUN antibody confirmed the presence of AP-1 proteins. Consistent with the possibility that EGF may act in part via c-Jun, EGF stimulated the activity of a chimeric GAL4 c-Jun protein, indicating that JUN can serve as a potential target of EGF in JEG-3 cells. EGF also induced mitogen-activated protein kinase activity, and a dominant negative mutant of mitogen-activated protein kinase partially blocked EGF stimulation of GAL4 c-Jun activity. We conclude that EGF stimulates the CYP11A1 promoter through an AP-1 like element and that c-Jun is one of the targets of EGF action.