1$alpha;-Hydroxylase transactivation by $gamma;-interferon in murine macrophages requires enhanced C/EBP$beta; expression and activation*1

Abstract

γ-Interferon [γ-IFN] induction of macrophage 1α-hydroxylase mRNA and activity causes severe hypercalcemia in granulomatous disorders. These studies demonstrate transcriptional regulation. γ-IFN induces the activity of the murine 1α-hydroxylase [−1651; +22] promoter in the murine macrophage cell line Raw 264.7 only after a 24 h exposure. This slow kinetics is incompatible with classical γ-IFN-mediated transactivation. In fact, γ-IFN response mapped to the minimal [−85; +11] promoter, which lacks GAS or ISRE sites but contains a putative C/EBPβ site. C/EBPβ is a γ-IFN inducible gene and a novel mediator of γ-IFN-regulated transcription. As expected for a C/EBPβ-driven transcription, ectopic C/EBPβ expression was sufficient to increase 1α-hydroxylase activity, enhance minimal promoter activity and potentiate the induction of this promoter by γ-IFN. Importantly, the dominant negative C/EBPβ isoform antagonized C/EBPβ-transcriptional activity. γ-IFN induction of C/EBPβ expression is not sufficient for γ-IFN induction of minimal promoter activity. There is also a cell-specific induction of C/EBPβ-transcriptional activity by γ-IFN. In Raw cells, specific inhibition of γ-IFN induction of endogenous-C/EBPβ phosphorylation by MEKK1 markedly reduced basal promoter activity and the response to γ-IFN. We conclude that γ-IFN-induction of C/EBPβ expression and activation by phosphorylation contributes to γ-IFN-transcriptional control of 1α-hydroxylase expression in murine macrophages.