Combinatorial roles of protein kinase A, Ets2, and 3′,5′-cyclic-adenosine monophosphate response element-binding protein-binding protein/p300 in the transcriptional control of interferon-γ expression in a trophoblast cell line

In ruminants, conceptus interferon-T (IFNT) production is necessary for maintenance of pregnancy. We examined the role of protein kinase A (PKA) in regulating IFNT expression through the activation of Ets2 in JAr choriocarcinoma cells. Although overexpression of the catalytic subunit of PKA or the addition of 8-bromo-cAMP had little ability to up-regulate boIFNT1 reporter constructs on their own, coexpression with Ets2 led to a large increase in gene expression. Progressive truncation of reporter constructs indicated that the site of PKA/Ets2 responsiveness lay in a region of the promoter between -126 and -67, which lacks a cAMP response element but contains the functional Ets2-binding site and an activator protein 1 (AP1) site. Specific mutation of the former reduced the PKA/Ets2 effects by more than 98%, whereas mutation of an AP1-binding site adjacent to the Ets2 site or pharmacological inhibition of MAPK kinase 2 led to a doubling of the combined Ets2/PKA effects, suggesting there is antagonism between the Ras/MAPK pathway and the PKA signal transduction pathway. Although Ets2 is not a substrate for PKA, lowering the effective concentrations of the coactivators, cAMP response element-binding protein-binding protein (CBP)/p300, known PKA targets, reduced the ability of PKA to synergize with Ets2, suggesting that PKA effects on IFNT regulation might be mediated through CBP/p300 coactivation, particularly as CBP and Ets2 occupy the proximal promoter region of IFNT in bovine trophoblast CT-1 cells. The up-regulation of IFNT in the elongating bovine conceptus is likely due to the combinatorial effects of PKA, Ets2, and CBP/p300 and triggered via growth factors released from maternal endometrium.