IDENTIFICATION OF THE FUNCTIONAL COMPONENTS OF THE RAS SIGNALING PATHWAY REGULATING PITUITARY CELL-SPECIFIC GENE-EXPRESSION

Ras, a small GTP-binding protein, is required for functional receptor tyrosine kinase signaling. Ultimately, Ras alters the activity of spec ific nuclear transcription factors and regulates novel patterns of gen e expression. Using a rat prolactin promoter construct in transient tr ansfection experiments, we show that both oncogenic Ras and activated forms of Raf-1 kinase selectively stimulated the cellular rat prolacti n promoter in GH(4) rat pituitary cells. We also show that the Ras sig nal is completely blocked by an expression vector encoding a dominant- negative Raf kinase. Additionally, using a molecular genetic approach, we determined that inhibitory forms of p42 mitogen-activated protein kinase and an Ets-2 transcription factor interfere with both the Ras a nd the Raf activation of the rat prolactin promoter. These findings de fine a functional requirement for these signaling constituents in the activation of the prolactin gene, a cell-specific gene which marks the lactotroph pituitary cell type. Further, this analysis allowed us to order the components in the Ras signaling pathway as it impinges on re gulation of prolactin gene transcription as Ras->Raf kinase->mitogen-a ctivated protein kinase->Ets. In contrast, we show that intact c-Jun e xpression inhibited the Ras-induced activation of the prolactin promot er, defining it as a negative regulator of this pathway, whereas c-Jun was able to enhance the Ras activation of an AP-1-driven promoter in GH(4) cells. These data show that c-Jun is not the nuclear mediator of the Ras signal for the highly specialized, pituitary cell-specific pr olactin cellular promoter. Thus, we have defined a model system which provides an ideal paradigm for studying Ras/Raf signaling pathways and their effects on neuroendocrine cell-specific gene regulation.