REGULATORY ELEMENTS OF THE LEUKAEMIA INHIBITORY FACTOR (LIF) PROMOTER IN MURINE BONE MARROW STROMAL CELLS

Abstract

Leukaemia inhibitory factor (LIF) plays an important role as a haematopoietically active cytokine. As described earlier in a murine model, interleukin 1 (IL-1) induced LIF mRNA and protein expression. We utilized the murine cell line +/+-1.LDA11 to further define regulatory mechanisms of LIF expression in bone marrow stromal cells. The production of LIF mRNA is stimulated by IL-1β, TNF-α, and the cAMP analogue 8-bromoadenosine 3′:5′-monophosphate (8BrcAMP). LIF mRNA expression is controlled at the transcriptional level. Different fragments from −542 to −45bp 5′ upstream of the transcriptional start site of the murine LIF gene were fused to the luciferase gene. All LIF-promoter luciferase constructs exhibited constitutive luciferase activity under serum free conditions. The level of luciferase activity decreased with LIF-promoter constructs of less than 249bp (pLIF249) in size. When tested with the 314bp LIF-promoter construct, incubation of stromal cells with IL-1β (500U/ml) resulted in a 1.57-fold stimulation, with TNF-α (500U/ml) in 2.06-fold stimulation, and with 8BrcAMP (0.5mM) in a 3.42-fold stimulation of luciferase activity. By testing different deletion mutants we could narrow the IL-1 and TNF-α responsive promoter areas to the region −249 to −145bp and the 8BrcAMP responsive area from −145 to −82bp. Mobility shift experiments revealed that nuclear proteins from stromal cells form a DNA-protein complex by binding to the region from −249 to −145bp of the LIF promoter.