171 CELLULAR EXPRESSION, LOCALIZATION, AND SECRETION OF OVINE Mx2 (oMx2) IN OVINE GLANDULAR EPITHELIAL CELLS (oGE)

Abstract

Mx proteins are antiviral proteins that belong to the dynamin super-family of large GTPases. In a number of species Mx proteins were shown to be important components of the innate response to viral infection. Work in our laboratory showed that during early pregnancy Mx proteins are up-regulated in the ruminant uterus by conceptus-derived IFN tau. Transient oMx1 knockdown in ovine glandular epithelial cells (oGE) reduced oMx1 secretion and secretion of other unconventionally secreted proteins without affecting secretion via the classical secretory pathway. We further showed that oMx1 was present in uterine flushes from pregnant ewes. We recently characterized another Mx protein, oMx2, in sheep. The newly characterized oMx2 has higher homology with bovine Mx2 (93%) than with oMx1 (60%). The purpose of this study was to characterize cellular expression and localization of oMx2 in oGE cells and in cell lines derived from the luminal epithelium (oLE) and stroma (oSC). In addition we determined if oMx2 was secreted by oGE cells in vitro. Expressions of oMx1 and oMx2 were low to undetectable in all three lines in the absence of IFN tau and were increased by IFN tau (P < 0.01). Induction of oMx1 was greatest in oGE cells (8.4-fold increase; P < 0.01), whereas expression of oMx2 was greatest in oSC cells (19.9-fold increase; P < 0.01). Immunofluorescence analysis confirmed the high level of expression of oMx1 and oMx2 in response to IFN tau in oGE cells. Immunofluorescence analysis revealed that oMx2 co-localized with lamin A/C and nucleoporins, whereas oMx1 was distributed throughout the cytoplasm in oGE cells, suggesting that oMx2 is a nuclear membrane-associated protein. SignalP analysis indicated that oMx2 lacked a traditional leader sequence. Using an inhibitor of the classical secretory pathway, Monensin, we showed that oMx2 levels were not reduced in response to Monensin treatment. The oMx1, oMx2, and ISG15 levels in oGE-conditioned culture medium actually increased in secretions in response to Monensin (2.7-fold, 4.6-fold, and 2.9-fold increases, respectively; P < 0.1), whereas there was no effect of Monensin on �2MG levels (P > 0.1). Results show that oMx2 expression, like that of oMx1, is regulated by IFN tau. The functional significance of the different cellular localization between oMx1 and oMx2 is not clear, but may suggest that these related proteins possess distinct cellular functions. Finally, this is the first report that oMx2 is secreted, perhaps via poorly described unconventional secretory pathways. The potential intracellular and extracellular functions of oMx2 are currently being investigated. This work was supported by USDA grant 2002-02398 and NIH NCRR grant P20-RR15587-01 to T.L.O.