Insulin-like growth factor-1 stimulates rat prolactin gene expression by a Ras, ETS and phosphatidylinositol 3-kinase dependent mechanism.

We have examined the influence of insulin-like growth factor I (IGF-1) on prolactin gene expression in rat pituitary GH4C1 cells. Incubation with IGF-1 increases prolactin mRNA levels and activates the prolactin promoter in transient transfection assays. A similar degree of activation is observed with constructs extending to -3000 and -176 base pairs of the prolactin 5' flanking region, indicating that the IGF-1 response element is located in the proximal promoter sequences. A plasmid containing 101 base pairs shows a partial stimulation by IGF-1, and the response is lost in a deletion to -76 base pairs. The Ras oncoproteins have been implicated as a critical signaling component in mediating the effect of growth factor receptor tyrosine kinases. Expression of oncogenic RasVal12 mimics the effect of IGF-1 on the prolactin promoter, and a dominant negative Ras, RasAsn17, blocks IGF-1-mediated stimulation. Dominant negative mitogen-activated protein kinase (MAPK) also reduces the activation of the prolactin promoter by IGF-1. Ets transcription factors have been described to lie downstream of Ras and MAPK in the signaling pathway leading to prolactin gene activation. Mutation of two Ets binding sites in the promoter region between -101 and -76 abolishes the response to IGF-1. Furthermore, a dominant negative Ets vector strongly reduces the response of the prolactin promoter to IGF-1 and Ras. The endogenous concentration of Ets-related proteins is not limiting in GH4C1 cells for the IGF-1 effect. However, c-Ets-1 and GHF-1 act synergistically in HeLa cells with the IGF-1 receptor, reconstituting pituitary IGF-1 responsiveness. The response to IGF-1 in GH4C1 cells is still observed after transfection with RasVal12 suggesting that, although Ras is required, IGF-1 could stimulate other pathway/s in addition to Ras. Wortmanin, an inhibitor of phosphatidylinositol-3 kinase (PI-3 kinase), also prevents the response of the prolactin promoter to IGF-1. These results show that both the Ras/MAPK/Ets pathway, as well as the activation of PI-3 kinase are involved in the signaling mechanism leading to prolactin expression by IGF-1 in GH4C1 cells.