Mediators of interferon gamma-initiated signaling in bovine luteal cells

Interferon gamma (IFN gamma) has been implicated as a mediator of luteal st eroidogenesis and cell fate. IFN gamma -initiated signaling events, althoug h implied by studies in cell lines, have yet to be described in primary lut eal cells. The objective of these studies was to begin to characterize IFN gamma -initiated signaling within luteal cells. Dispersed bovine luteal cel l cultures were challenged with increasing levels of bovine recombinant IFN gamma (0-1000 U) or IFN gamma (200 U) in the presence or absence of tumor necrosis factor cu (TNF alpha, 10 ng/ml) over time (short term, 0-60 min; l ong term, 0, 24, 48 h). Fractionated or total cell lysates were evaluated b y the Western blotting technique to determine the changes in the levels of signal transducers and activators of transcription (STAT), interferon regul atory factor 1 (IRF-1), and I kappa B alpha (I kappaB-alpha). Utilizing ant ibodies that recognize the nonphosphorylated forms of STAT-1 and STAT-3, it was determined that levels of STAT-1 and STAT-3 in total cell lysates were constitutively expressed and did not change in response to treatment with IFN gamma or TNFa. In contrast, nuclear levels of STAT-1 and phosphorylated STAT-3 were elevated in a time-dependent manner in response to IFN gamma t reatment. Furthermore, IFN gamma and TNF alpha treatment elevated levels of IRF-1 within 2 h. TNF alpha -induced increases in the levels of IRF-1 were transient, whereas the levels of IRF-1 in response to IFN gamma treatment remained elevated at 48 h. These data suggest that IFN gamma treatment can activate members of the STAT pathway, resulting in increased levels of IRF- 1. TNF alpha treatment induced a rapid decrease in the levels of I kappaB-a lpha. IFN gamma treatment did not alter the levels of 1 kappaB-alpha and fa iled to inhibit the TNF alpha -initiated decrease in the levels of I kappaB -alpha, The present experiment demonstrates that the steroidogenic cells of the corpus luteum have the capacity to respond to IFN gamma via activation of STAT and IRF-1, providing further evidence that IFN gamma may be involv ed in the luteolytic process, These data also suggest that IFN gamma does n ot signal through the nuclear factor kappa B cell survival signaling pathwa y.