INSULIN-LIKE GROWTH-FACTOR-I STIMULATES RAT PROLACTIN GENE-EXPRESSIONBY 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 activa tion is observed with constructs extending to -3000 and -176 base pair s of the prolactin 5' flanking region, indicating that the IGF-I respo nse element is located in the proximal promoter sequences. A plasmid c ontaining 101 base pairs shows a partial stimulation by IGF-1, and the response is lost in a deletion to -76 base pairs. The Ras oncoprotein s have been implicated as a critical signaling component in mediating the effect of growth factor receptor tyrosine kinases. Expression of o ncogenic Ras(Val12) mimics the effect of IGF-1 on the prolactin promot er, and a dominant negative Ras, Ras(Asn17), blocks IGF-1-mediated sti mulation. Dominant negative mitogen-activated protein kinase (MAPK) al so reduces the activation of the prolactin promoter by IGF-1. Ets tran scription factors have been described to lie downstream of Ras and MAP K in the signaling pathway leading to prolactin gene activation. Mutat ion of two Ets binding sites in the promoter region between -101 and - 76 abolishes the response to IGF-1. Furthermore, a dominant negative E ts vector strongly reduces the response of the prolactin promoter to I GF-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, reco nstituting pituitary IGF-1 responsiveness. The response to IGF-1 in GH 4C1 cells is still observed after transfection with Ras(Val12) suggest ing that, although Ras is required, IGF-1 could stimulate other pathwa y/s in addition to Ras. Wortmanin, an inhibitor of phosphatidylinosito l-3 kinase (Pl-3 kinase), also prevents the response of the prolactin promoter to IGF-1. These results show that both the Ras/MAPK/Ets pathw ay, as well as the activation of Pl-3 kinase are involved in the signa ling mechanism leading to prolactin expression by IGF-1 in GH4C1 cells .