Figure 3
NFκB and C/EBP binding sites are required for MALP-2-induced Lcn2 expression from both the mouse and human promoters. (A) An alignment of human and mouse proximal promoters with the C/EBP and NFkB site identified. Human C/EBP-1 was mutated in this study. Sources of the sequences were GenBank: x81627 (mouse) and x99133 (human). (B) HC11 cells infected with mycoplasma (myco+) or uninfected (myco-) were co-transfected with a Renilla luciferase expression control plasmid and firefly luciferase reporter plasmids each containing one of a series of truncated Lcn2 promoters (starting at the listed gene position and ending at +53 bp). The cells were harvested and measured for luciferase activity 48 h after transfection. The relative luciferase activities are shown normalized to the value obtained from cells transfected by a plasmid from which firefly luciferase expression was driven by the SV40 promoter. Lcn2-1477, Lcn2-1003, Lcn2-711 and Lcn2-438 had similar activity levels as Lcn2-2108 in M. arginini infected HC11 cells (data not shown). (C,D) HC11 cells were co-transfected with a Renilla luciferase expression control plasmid and a firefly reporter plasmid with one of the indicated Lcn2 or NGAL promoter segments. Twenty-four hours after transfection the cells were treated with or without 10 ng/ml MALP-2, harvested 12 h later and measured for luciferase activity. Data was normalized to the Renilla transfection control. (B) Expression from the mouse Lcn2 promoter and mutant versions. (C) Expression from the NGAL promoter and mutant versions. (A–C) hatched bars, controls; red bars, M. arginini infected cells, green bars, MALP-2 treated uninfected cells.
