Stat 5b and the Orphan Nuclear Receptors Regulate Expression of the α2-Macroglobulin (α2M) Gene in Rat Ovarian Granulosa Cells

Abstract

Alpha2-macroglobulin (alpha2M) is a serine protease inhibitor and cytokine inactivator associated with inflammation and tissue remodeling. The gene encoding this protein is selectively induced in the rat corpus luteum by the luteotropic hormone and cytokine, PRL. The promoter of the alpha2M gene contains two regulatory regions that bind a diverse set of transcription factors and confer functional activity in ovarian 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-binding sites or mutation of either one or both of these sites within the context of the intact promoter abolished PRL inducibility of alpha2M promoter-reporter constructs in granulosa-luteal cells. Cotransfection with a vector expressing a dominant negative, truncated form of Stat 5b abolished PRL-induced activation of a2M transgenes. 5'-Deletion of the Stat-binding sites abolished all promoter-reporter activity in response to PRL. Internal deletion of a second functional domain 3' of the PRLRE also abolished PRL inducibility and markedly reduced basal activity, indicating that functional interactions between these two regions might occur. The 3'-region was shown to bind orphan members of the nuclear receptor superfamily, steroidogenic factor 1 (SF-1) and chicken ovalbumin upstream promoter-transcription factor (COUP-TF) and has been called the orphan receptor response element (ORRE). When site-specific 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 the intact promoter, specific changes in the functional activity of this novel region of the alpha2M promoter were observed. Mutation of the SF-1 site drastically reduced basal activity of the alpha2M promoter. Mutation of the COUP-TF sites caused the basal activity of the alpha2M promoter to increase markedly. Neither mutation altered the PRL inducibility of these constructs. Lastly, differentiation of cultured granulosa cells was required for functional activity of both the PRLRE and the ORRE. Collectively, these results document for the first time that Stat 5b, SF-1, and COUP-TF each exert specific effects on the function of the alpha2M promoter: basal activity is controlled by the balance of SF-1 (positive) and COUP-TF (negative) activities and PRL inducibility is mediated by activation of Stat 5b. These results add alpha2M to the list of nonsteroidal genes regulated by SF-1 in the gonads and provide the first evidence that COUP-TF has a specific role in regulating ovarian gene activity. In addition, the ORRE and PRLRE act independently of, rather than synergistically with, each other to regulate basal and PRL-induced expression of alpha2M in ovarian luteal cells.