391 Increased hEKLF Levels in HIV-1 Infected Macrophages: A Differential Role for hEKLF in Regulating IL-12 p40 Promoter through Newly Identified CACCC Region and Modulation of the NFkB Pathway Q. Luo*1, X. Ma2, N. Ngoubilly1, S. Wahl3, J. Bieker4, M. Crossley5, L. J. Montaner1 1Wistar Inst, Philadelphia, PA; 2Weill Med Coll of Cornell Univ, New York, NY; 3Natl Inst of Hlth, Bethesda, MD; 4Mt Sinai Sch of Med, New York, NY; and 5Univ of Sydney, Australia
Background: The mechanism for suppression of IL-12 production in HIV infection remains undetermined. Here we describe a CACCC box (-224 to -220) as a newly identified transcriptional cis-element on the IL-12 p40 proximal promoter associated with constitutive repression, which is modulated in IFNg/LPS and IL-10 regulation of IL-12 p40 transcription.
Methods: Cell culture; Electropheratic mobility shift assay; site directed mutagenesis; transfection assay; multiplex RT-PCR; and western blot, flow cytometry, and multiple probe-ribonuclease protection assay.
Results: A CACCC binding protein is present in human primary macrophages which is modulated by IFNg/LPS/IL-10 in IL-12 p40 expression. The CACCC BP was identified as human Erythroid Kruppel Like Factor (EKLF). Studies of hEKLF over-expression with wild-type and CACCC-mutated IL-12 p40 promoters uncovered a differential role of hEKLF in regulating unstimulated (acting to induce) and IFNg/LPS stimulated (acting to repress) IL-12 p40 transcription, in association with hEKLF affecting NFkB activity. Consistently, over-expression of hEKLF enhanced endogenous NFkB target host genes, including IL-1a, IL-1b, IL-1Ra in the absence of stimulation, whereas a selective repression of IL-12 p40 and IL-6 by hEKLF over-expression was observed following IFNg/LPS stimulation in RAWs. Furthermore, HIV-1 infection in human MDMs enhanced hEKLF protein expression.
Conclusions: 1) There is a CACCC box binding protein present and binding with the CACCC (-224 to -220) cis-element on the IL-12 p40 promoter in human primary macrophages; 2) binding of this protein with the CACCC box represses transcription activity of IL-12 p40 promoter and is modulated by IFNg/LPS/IL-10; 3) the CACCC BP is hEKLF, expressed in primary macrophages; 4) studies of hEKLF overexpression show that exogenous hEKLF cross-talks with NFkB pathway and this interaction is associated with its role in modulating transcription activity of IL-12 p40 promoter; 5) over-expression of hEKLF acts as inhibitor of a subset of selected endogenous NFkB target genes in IFNg/LPS stimulated cells and as a widespread activator of NFkB target genes in unstimulated cells; and 6) HIV-1 infection increases levels of hEKLF protein in macrophages. Therefore, HIV regulation of hEKLF expression may represent a mechanism of suppressed IL-12 production by macrophages upon HIV-1 infection.
