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

• Published in: Cytokine (Philadelphia, Pa.) Pa.), 1999-09, Vol.11 (9), p.656-663
• Main Authors: Göllner, G, Bug, G, Rupilius, B, Peschel, C, Huber, C, Derigs, H.G
• Format: Article
• Language: English
• Subjects: 8-Bromo Cyclic Adenosine Monophosphate - pharmacology; Animals; Bone Marrow Cells - drug effects; Bone Marrow Cells - metabolism; bone marrow stromal cells/Leukaemia Inhibitory Factor (LIF)/promoter activity/transcriptional control; Cells, Cultured; Gene Expression Regulation - drug effects; Genes, Reporter; Growth Inhibitors - biosynthesis; Growth Inhibitors - genetics; Humans; Interleukin-1 - pharmacology; Interleukin-6; Leukemia Inhibitory Factor; Lymphokines - biosynthesis; Lymphokines - genetics; Mice; Promoter Regions, Genetic; Recombinant Fusion Proteins - biosynthesis; Recombinant Proteins - pharmacology; Regulatory Sequences, Nucleic Acid; RNA, Messenger - biosynthesis; Stromal Cells - drug effects; Stromal Cells - metabolism; Tumor Necrosis Factor-alpha - pharmacology
• ISSN: 1043-4666; 1096-0023
• DOI: 10.1006/cyto.1998.0475

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.