Inflammation induced down-regulation of BMPER is mediated by NFκB and KLF2. (A) HUVECs were treated with TNFα (50 ng/mL) alone or in combination with NFκB inhibitor PS1145 (10μM). After 24 hours cells were lysed and RNA was reverse transcribed. BMPER levels were quantified by real-time PCR. (B) NFκB inhibition by overexpression of superrepressor IκBα-SR in endothelial cells abolished down-regulation of BMPER RNA by TNFα. (C) KLF2 knockdown by transfection of specific KLF2 siRNA reduced BMPER protein levels shown by Western blotting with anti–human BMPER antibody. β-tubulin was used as loading control. (D) KLF2 increased BMPER promoter activity in endothelial cells. HUVECs were transiently cotransfected by a KLF2 coding plasmid and indicated BMPER promoter constructs p(-1370)luc or p(-297)luc. After 24 hours cells were lysed and BMPER promoter activity was quantified by reporter assays. Values represent the mean ± SD of 3 independent experiments normalized to β-galactosidase. (E) Overexpression of KLF2 increases BMPER RNA level and attenuates down-regulation of BMPER RNA (E) and protein (F) by TNFα. At day 1 HUVECs were transfected with a KLF2 coding plasmid and after 24 hours stimulated with TNFα (50 ng/μL). Afterward, HUVECs were lysed and RNA was isolated and reverse transcribed. KLF2, BMPER, and hrpII were quantified by real-time PCR. Each experiment was performed at least 3 times with similar results. (G) LPS reduced KLF2 protein expression in vivo. KLF2 protein levels were analyzed in LPS treated lungs and compared with control lungs using Western blotting (*P < .05 versus control; #P < .05 versus pcDNA3 + TNFα).