Abstract
Fibroblast growth factors (FGFs) have been implicated in pituitary lactotroph tumorigenesis; however, little is known about the molecular mechanisms of FGF signal transduction. We used a transient transfection approach, in GH4 cells, to identify components of the FGF signaling pathway leading to activation of the rat prolactin (rPRL) promoter. Using dominant-negative constructs of p21(Ras), Raf-1 kinase, and mitogen-activated protein (MAP) kinase, we show that FGF activation of the rPRL promoter is independent of Ras and Raf-1 but requires MAP kinase. Furthermore, MAP kinase but not Raf-1 kinase catalytic activity is stimulated by FGFs. The rPRL promoter FGF response maps to two Ets binding sites, centered at -212 (FRE1) and -96 (FRE2), and co-transfection of dominant-negative Ets inhibits FGF activation. FRE1 co-localizes with a composite, Ets/GHF-1, Ras response element. However, overexpression of Ets-1 and GHF-1, which potentiate the Ras response, inhibits FGF stimulation of the rPRL promoter, implying that Ras and FGF signaling pathways target distinct factors to elicit their effects. These data suggest that Ets factors serve to sort and integrate MAP kinase-dependent growth factor signals, allowing highly specific transcriptional responses to be mediated via the interaction of distinct Ets proteins and cofactors at common response elements.
Highlights
Fibroblast growth factors (FGFs) have been implicated in pituitary lactotroph tumorigenesis; little is known about the molecular mechanisms of FGF signal transduction
FGF Activation of the rPRL Promoter Is Independent of Ras—FGFs have been shown to selectively activate the rat (r) prolactin (PRL) promoter but not the ancestrally related rat growth hormone (GH) promoter, in GH4 pituitary cells [7]
To determine the functional role of Ras in FGF-2 and FGF-4 activation of the FGF Signaling in Neuroendocrine Cells rPRL promoter, a dominant-negative N17Ras construct [40] was transiently transfected into GH4 cells along with the rPRL promoter fused to the luciferase gene
Summary
(Received for publication, July 30, 1997, and in revised form, September 16, 1997). Rebecca E. GH4 cells are highly differentiated, retain cell-specific functions and hormonal responses, and express the phenotypic marker, PRL, under the control of the pituitary-specific POU homeodomain transcription factor, GHF-1/Pit-1 [28] This model system has been successfully employed to study the mechanisms of the epidermal growth factor and Ras signal transduction pathways as they impinge on the rPRL promoter (29 –32). We have identified critical functional components of the FGF signaling pathway in pituitary cells, and we present evidence that different growth factors, acting via MAP kinase, act to target distinct Ets members that bind common cis-acting response elements, serving to sort and integrate multiple signaling pathways resulting in specific changes in gene expression
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