Regulatory elements of the leukaemia inhibitory factor (LIF) promoter in murine bone marrow stromal cells.
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-1beta, TNF-alpha, and the cAMP analogue 8-bromoadenosine 3':5'-monophosphate (8BrcAMP). LIF mRNA expression is controlled at the transcriptional level. Different fragments from -542 to -45 bp 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 249 bp (pLIF249) in size. When tested with the 314 bp LIF-promoter construct, incubation of stromal cells with IL-1beta (500 U/ml) resulted in a 1.57-fold stimulation, with TNF-alpha (500 U/ml) in 2.06-fold stimulation, and with 8BrcAMP (0.5 mM) in a 3. 42-fold stimulation of luciferase activity. By testing different deletion mutants we could narrow the IL-1 and TNF-alpha responsive promoter areas to the region -249 to -145 bp and the 8BrcAMP responsive area from -145 to -82 bp. Mobility shift experiments revealed that nuclear proteins from stromal cells form a DNA-protein complex by binding to the region from -249 to -145 bp of the LIF promoter.
Göllner
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III. Dept. of Medicine, Johannes Gutenberg University, Langenbeckstrasse 1, Mainz, D-55131, Germany.
Bug
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Rupilius
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Peschel
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Huber
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Derigs
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D015124:8-Bromo Cyclic Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D001854:Bone Marrow Cells / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D005786:Gene Expression Regulation / Q000187:drug effects; D017930:Genes, Reporter; D006131:Growth Inhibitors / Q000096:biosynthesis / Q000235:genetics*; D006801:Humans; D007375:Interleukin-1 / Q000494:pharmacology; D015850:Interleukin-6; D053605:Leukemia Inhibitory Factor; D008222:Lymphokines / Q000096:biosynthesis / Q000235:genetics*; D051379:Mice; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000096:biosynthesis; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000494:pharmacology; D012045:Regulatory Sequences, Nucleic Acid; D017154:Stromal Cells / Q000187:drug effects / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D006131:Growth Inhibitors; D007375:Interleukin-1; D015850:Interleukin-6; C507837:LIF protein, human; D053605:Leukemia Inhibitory Factor; C507838:Lif protein, mouse; D008222:Lymphokines; D012333:RNA, Messenger; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha; D015124:8-Bromo Cyclic Adenosine Monophosphate