The mechanism by which activation of common signal transduction pathways can elicit cell-specific responses remains an important question in biology. To elucidate the molecular mechanism by which the Ras signaling pathway activates a cell-type-specific gene, we have used the pituitary-specific rat prolactin (rPRL) promoter as a target of oncogenic Ras and Raf in GH4 rat pituitary cells. Here we show that expression of either c-Ets-1 or the POU homeo-domain transcription factor GHF-1/Pit-1 enhance the Ras/Raf activation of the rPRL promoter and that coexpression of the two transcription factors results in an even greater synergistic Ras response. By contrast, the related GHF-1-dependent rat growth hormone promoter fails to respond to Ras or Raf, indicating that GHF-1 alone is insufficient to mediate the Ras/Raf effect. Using amino-terminal truncations of c-Ets-1, we have mapped the c-Ets-1 region required to mediate the optimal Ras response to a 40-amino-acid segment which contains a putative mitogen-activated protein kinase site. Finally, dominant-negative Ets and GHF constructs block Ras activation of the rPRL promoter, and each blocks the synergistic activation mediated by the other partner protein, further corroborating that a functional interaction between c-Ets-1 and GHF-1 is required for an optimal Ras response. Thus, the functional interaction of a pituitary-specific transcription factor, GHF-1, with a widely expressed nuclear proto-oncogene product, c-Ets-1, provides one important molecular mechanism by which the general Ras signaling cascade can be interpreted in a cell-type-specific manner.

Activation of the rat prolactin (rPRL) promoter by Ras is a prototypical example of tissue-specific transcriptional regulation in a highly differentiated cell type. Using a series of site-specific mutations and deletions of the proximal rPRL promoter we have mapped the major Ras/Raf response element (RRE) to a composite Ets-1/ GHF-1 binding site located between positions ؊217 and ؊190. Mutation of either the Ets-1 or GHF-1 binding sites inhibits Ras and Raf activation of the rPRL promoter, and insertion of this RRE into the rat growth hormone promoter confers Ras responsiveness. We show that Ets-1 is expressed in GH 4 cells and, consistent with their functional synergistic interaction, both Ets-1 and GHF-1 are able to bind specifically to this bipartite RRE. We confirm that Ets-1 or a related Ets factor is the nuclear target of the Ras pathway leading to activation of the rPRL promoter and demonstrate that Elk-1 and Net do not mediate the Ras response. Thus, the pituitary-specific POU homeodomain transcription factor, GHF-1, serves as a cell-specific signal integrator by functionally interacting with an Ets-1-like factor, at uniquely juxtaposed binding sites, thereby targeting an otherwise ubiquitous Ras signaling pathway to a select subset of cell-specific GHF-1-dependent genes.The p21 Ras proto-oncogene is a critical component of a network of signaling pathways that mediate the control of cell growth, metabolism, and differentiation (1) Signals initiated at transmembrane receptors are transduced via Ras and propagated, by a phosphorylation cascade, to the nucleus, resulting in changes in the activity of specific transcription factors (2, 3). Distinct signaling components of the Ras pathway may be present in different cell types, allowing the signal to be interpreted in a cell-specific manner (4, 5) Indeed, cell-specific, phenotypic sequelae of Ras activation are exemplified by the differential effects of oncogenic Ras in PC12 pheochromocytoma, TT medullary carcinoma, and FRTL5 thyroid cells (6 -8), whereby V-12 Ras induces terminal differentiation of the first two cell lines but causes transformation of the last. Thus, the characterization of cell-specific endogenous nuclear factors that may act as effectors of the Ras signaling pathway and the identification of specific Ras-responsive cis-acting DNA elements are important unanswered questions. Several Ras/Raf response elements (RREs) 1 or oncogene response units have been identified to date, implicating members of the Ets, AP-1, and ATF/CREB families of transcription factors as nuclear components of the Ras signaling pathway (2, 3).Tandem c-Ets-2 binding sites have been shown to confer Ras responsiveness in NIH-3T3 cells (9), and dominant-negative Ets constructs inhibit both Ras-induced mitogenesis (10) and transformation (11). The serum response is governed by MAP kinase phosphorylation of Elk-1, a member of the Ets family of transcription factors (12, 13), thus facilitating its interaction with serum response factor (SRF). Similarly, the Drosophila Ets fact...

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 elfects on neuroendocrine cell-specific gene regulation.The precise control of cell growth, metabolism, and differentiation is mediated by the interaction of extracellular stimuli such as hormones and growth factors with discrete surface membrane receptors. The mechanisms by which cells respond to extracellular signals are not yet understood and present a key problem in biology. It is known that many peptide growth factors initiate signals by activating transmembrane receptors which contain a cytoplasmic tyrosine kinase domain. Transduction of the tyrosine kinase signal initiated at the membrane is thought to be mediated by a phosphorylation cascade, in which the serine and threonine phosphorylations of cytoplasmic and nuclear regulatory proteins result in the amplification and transduction of the initial extracellular signal to the various compartments of the cell, culminating in the activation of specific transcription factors (8, 27, 32). The nuclear machinery responds to a variety of signal pathways through the activation of nuclear transcription factors, which leads to DNA replication and/or cellular proliferation or to novel patterns of gene expression. Although the precise mechanisms which switch the membrane tyrosine kinase signal to an intracellular Ser/Thr kinase component remain elu...