ENHANCEMENT OF OVINE TROPHOBLAST INTERFERON BY GRANULOCYTE MACROPHAGE-COLONY-STIMULATING FACTOR - POSSIBLE INVOLVEMENT OF PROTEIN-KINASE-C

Interferon-tau (oIFN tau), the major secretory product of ovine concep tuses between days 13 and 21 (day 0=day of estrus) of pregnancy, is im plicated in the process of maternal recognition of pregnancy. Culturin g of day-14 and day-16 conceptus tissues in the presence of human gran ulocyte macrophage-colony stimulating factor (hGM-CSF) or interleukin- 3 (IL-3) produces a marked increase in oIFN tau mRNA and protein expre ssion. Since GM-CSF and IL-3 are localized at the luminal and glandula r epithelia of the ovine endometrium, maternally derived GM-CSF and IL -3 may affect conceptus production of oIFN tau in a paracrine manner. However, the molecular mechanisms by which endometrial GM-CSF and IL-3 up-regulate oIFN tau production have not been defined. As an initial investigation of the signaling pathway regulating the GM-CSF induction of the oIFN tau gene, day-16 conceptuses were treated with an inducer , phorbol 12-myristate 13-acetate (PMA) calphostin C of the protein pa thway. Treatment with either 150 units/ml hGM-CSF (P < 0.01) or 10 nM PMA (P < 0.05) resulted in a significant increase in oIFN tau mRNA exp ression. Pretreatment of conceptuses with 1 mu M PMA for 12 h to produ ce PKC-deficient tissues or treatment with 50 mM calphostin C abolishe d the hGM-CSF-induced increase in oIFN tau mRNA. An in vitro expressio n system was established for the analysis of oIFN tau gene regulatory sequences. The oIFN tau 010 gene has been isolated previously and foun d to be the principal oIFN tau gene up-regulated during the preimplant ation period. 5'-Flanking regions of the oIFN tau 010 gene, 2 kb and 0 .8 kb, were cloned into a basic chloramphenicol acetyltransferase repo rter plasmid. These oIFN tau 010 promoter constructs, along with expre ssion controls, were transfected into human choriocarcinoma cells (JAR and JEG3) and their responsiveness to hGM-CSF and second messenger sy stem activators including PMA, calcium ionophore (A23187) and 8-bromo- cAMP were characterized. The oIFN tau 010 promoter constructs were up- regulated by hGM-CSF and PMA treatments (P < 0.01). Combined treatment with PMA and A23187 prevented the promoter activation seen with PMA a lone. The conceptus culture data, along with the results from the tran sfection experiments, suggest that the stimulatory effect of GM-CSF on oIFN tau is mediated through the PKC second messenger system.