In preliminary studies done during his fellowship, the Dr. Bradford demonstrated a role for FGF in regulation of the prolactin (PRL) promoter in GH4 pituitary cells. These preliminary studies indicate that FGF-2 and FFG-4 activate PRL promoter activity in a Ras and Raf independent manner, but involve MAP kinase and members of the Ets family of transcription factors. This regulation is specific and not observed for the growth hormone promoter in the same cells. Preliminary studies are also presented to suggest that this pathway is protein kinase C (PKC) dependent through the use of inhibitors and down-regulation of the enzyme. Based on these preliminary data, four specific aims are described: To map precisely the FGF response elements in the PRL promoter, the investigator will use a combination of conventional techniques. These will include mutagenesis with functional analysis of the PRL promoter using a luciferase assay, production of heterologous FRE promoter constructs which can be inserted upstream of a minimal viral promoter, insertion of PRL promoter element in the GH promoter which is normally not FGF responsive, and analysis of the nuclear factors binding to the FRE by both electrophoretic mobility shift assay and southwestern analysis. Analysis of FGF receptors and their responses in GH4 pituitary cells. FGF receptor expression will be determined by RTPCR. Receptor activation in tyrosine phosphorylation will be characterized, as will the mitogenic response to FGF 2 and FGF 4. The investigator will also determine whether there are changes in synthesis and secretion of prolactin in response to FGF treatment. Attempts will be made to identify and characterize the cytoplasmic components of the FGF signaling pathway. Here the investigator will focus on novel, non-Ras components which might account for the preliminary data. He will specifically investigate the possibility that B-Raf and Rap I are involve as well as phospholipase C and protein kinase C. Several other pathways will also be tested for their participation. Finally, the investigator will attempt to identify and characterize nuclear components of the FGF signal transduction pathway with a focus on the Ets transcription factors. These will be done through a series of transfections with dominant negative Ets and other potential interacting factors, through studies of the roles of GHF-1 and GHF-2 in the process and functional studies of the potential role of Jun kinase, nuclear MAP kinase and FGF induced phosphorylation of Ets factors.
| Jackson, Twila A; Koterwas, David M; Bradford, Andrew P (2006) Differential regulation of cell growth and gene expression by FGF-2 and FGF-4 in pituitary lactotroph GH4 cells. Mol Cell Endocrinol 247:183-91 |
| Tentler, John J; Bradford, Andrew P; Schweppe, Rebecca E et al. (2003) Selective repression of rat prolactin gene by stable expression of dominant-negative Ets in GH4 pituitary cells. Endocrine 20:3-12 |
| Jackson, T A; Schweppe, R E; Koterwas, D M et al. (2001) Fibroblast growth factor activation of the rat PRL promoter is mediated by PKCdelta. Mol Endocrinol 15:1517-28 |
| Bradford, A P; Brodsky, K S; Diamond, S E et al. (2000) The Pit-1 homeodomain and beta-domain interact with Ets-1 and modulate synergistic activation of the rat prolactin promoter. J Biol Chem 275:3100-6 |
