PROLACTIN INDUCTION OF THE ALPHA(2)-MACROGLOBULIN GENE IN RAT OVARIANGRANULOSA-CELLS - STAT-5 ACTIVATION AND BINDING TO THE INTERLEUKIN-6 RESPONSE ELEMENT

alpha(2)-Macroglobulin (alpha 2M) is expressed at high levels in the c orpus luteum of pregnant rats in response to PRL and rat placental lac togens. These studies document that PRL induction of alpha 2M mRNA occ urs rapidly in granulosa cells differentiated to the preovulatory phen otype in the presence of FSH and steroid, is hormone specific [induced by PRL but not by LH or interleukin-6 (IL-6)], and involves tyrosine kinase activity. To analyze the cellular signaling events stimulated b y PRL, transient transfections of granulosa cells and electrophoretic mobility;shift assays were done using the IL-6 response element (IL-6R E) of the alpha 2M promoter. The IL-6RE consists of two gamma-activati ng like sequences (GAS) that bind the acute phase response factor (APR F/Stat 3) in rat liver and the mammary gland factor (MGF/Stat 5) from mammary tissue. By transfecting various alpha 2M promoter-luciferase r eporter transgenes into the granulosa cell cultures, we show that the GAS-like sites together with the minimal -48 base pairs of the alpha 2 M promoter can confer PRL inducibility to the luciferase reporter gene . These same GAS-like sequences of the alpha 2M promoter were used to analyze the DNA-binding activity of proteins in whole cell extracts pr epared from differentiated granulosa cells exposed to PRL for 0.25, 0. 5, 4, and 20 h. PRL rapidly stimulated the binding of a specific prote in to labeled alpha 2M GAS-like oligonucleotide, and this PRL-induced binding activity was shown to contain Stat 5 but not Stat 1 or Stat 3, using specific antibodies in the electrophoretic mobility shift assay s. Because both Stat 5 and Stat 3 proteins are present in the whole ce ll extracts of differentiated granulosa cells, PRL appears to activate detectable amounts of Stat 5 (and not Stat 3). Thus, the initial indu ction of the alpha 2M gene by PRL in differentiated rat granulosa cell s involves, at least in part, the activation (tyrosine phosphorylation ?) of Stat 5.