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 specific nuclear transcription factors and regulates novel patterns of gene expression. Using a rat prolactin promoter construct in transient transfection experiments, we show that both oncogenic Ras and activated forms of Raf-1 kinase selectively stimulated the cellular rat prolactin promoter in GH4 rat pituitary cells. We also show that the Ras signal 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 and the Raf activation of the rat prolactin promoter. These findings define 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 regulation of prolactin gene transcription as Ras-->Raf kinase-->mitogen-activated protein kinase-->Ets. In contrast, we show that intact c-Jun expression inhibited the Ras-induced activation of the prolactin promoter, 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 GH4 cells. These data show that c-Jun is not the nuclear mediator of the Ras signal for the highly specialized, pituitary cell-specific prolactin 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.
Conrad
K E
KE
Department of Medicine, University of Colorado Health Services Center, Denver 80262.
Oberwetter
J M
JM
Vaillancourt
R
R
Johnson
G L
GL
Gutierrez-Hartmann
A
A
D000595:Amino Acid Sequence; D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000502:physiology*; D002460:Cell Line; D005786:Gene Expression Regulation; D016758:Genes, jun; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010903:Pituitary Gland, Anterior / Q000502:physiology*; D011388:Prolactin / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000502:physiology; D050783:Proto-Oncogene Proteins c-ets; D016755:Proto-Oncogene Proteins c-jun / Q000502:physiology; D019908:Proto-Oncogene Proteins c-raf; D016283:Proto-Oncogene Proteins p21(ras) / Q000502:physiology*; D051381:Rats; D015398:Signal Transduction; D013329:Structure-Activity Relationship; D014157:Transcription Factors
D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D016755:Proto-Oncogene Proteins c-jun; D014157:Transcription Factors; D011388:Prolactin; D019908:Proto-Oncogene Proteins c-raf; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D016283:Proto-Oncogene Proteins p21(ras)