STAT 5B AND THE ORPHAN NUCLEAR RECEPTORS REGULATE EXPRESSION OF THE ALPHA-2-MACROGLOBULIN (ALPHA-2M) GENE IN RAT OVARIAN GRANULOSA-CELLS

alpha 2-Macroglobulin (alpha 2M) is a serine protease inhibitor and cy tokine inactivator associated with inflammation and tissue remodeling. The gene encoding this protein is selectively induced in the rat corp us luteum by the luteotropic hormone and cytokine, PRL. The promoter o f the alpha 2M gene contains two regulatory regions that bind a divers e set of transcription factors and confer functional activity in ovari an granulosa-luteal cells. The PRL response element (PRLRE) binds PRL- activated (tyrosine-phosphorylated) signal transducers and activators of transcription (Stat 5b and Stat 5a). 5'-Deletion of the Stat-bindin g sites or mutation of either one or both of these sites within the co ntext of the intact promoter abolished PRL inducibility of alpha 2M pr omoter-reporter constructs in granulosaluteal cells. Cotransfection wi th a vector expressing a dominant negative, truncated form of Stat 5b abolished PRL-induced activation of alpha 2M transgenes. 5'-Deletion o f the Stat-binding sites abolished all promoter-reporter activity in r esponse to PRL. Internal deletion of a second functional domain 3' of the PRLRE also abolished PRL inducibility and markedly reduced basal a ctivity, indicating that functional interactions between these two reg ions might occur. The 3'-region was shown to bind orphan members of th e nuclear receptor superfamily, steroidogenic factor 1 (SF-1) and chic ken ovalbumin upstream promoter-transcription factor (COUP-TF) and has been called the orphan receptor response element (ORRE). When site-sp ecific mutations were made in either the SF-1-binding site or the two COUP-TF direct repeat (DR1 and DR2) binding sites in the context of th e intact promoter, specific changes in the functional activity of this novel region of the alpha 2M promoter were observed. Mutation of the SF-1 site drastically reduced basal activity of the alpha 2M promoter. Mutation of the COUP-TF sites caused the basal activity of the alpha 2M promoter to increase markedly. Neither mutation altered the PRL ind ucibility of these constructs. Lastly, differentiation of cultured gra nulosa cells was required for functional activity of both the PRLRE an d the ORRE. Collectively, these results document for the first time th at Stat 5b, SF-1, and COUP-TF each exert specific effects on the funct ion of the alpha 2M promoter: basal activity is controlled by the bala nce of SF-1 (positive) and COUP-TF (negative) activities and PRL induc ibility is mediated by activation of Stat 5b. These results add alpha 2M to the list of nonsteroidal genes regulated by SF-1 in the gonads a nd provide the first evidence that COUP-TF has a specific role in regu lating ovarian gene activity. In addition, the ORRE and PRLRE act inde pendently of, rather than synergistically with, each other to regulate basal and PRL-induced expression of alpha 2M in ovarian luteal cells.