Activation and Repression of Interleukin-12 p40 Transcription by Erythroid Kruppel-like Factor in Macrophages

Abstract

Transcription of interleukin (IL)-12 p40 in myeloid cells is attributed to the recruitment of multiple activated transcription factors such as nuclear factor B (NFB), CCAAT enhancer-binding protein ␤, ets-2, PU.1, and so forth. We now provide the first description of the human erythroid Kruppel-like factor (EKLF) in human primary macrophages and identify the role of EKLF in IL-12 p40 expression. EKLF-specific binding to the CACCC element (؊224 to ؊220) on the human IL-12 p40 promoter was observed in resting human primary macrophages. Functional analysis of the CACCC element revealed a dependent role for EKLF binding in activating IL-12 p40 transcription in resting RAW264.7 cells, whereas EKLF overexpression in the presence or absence of this element repressed IL-12 p40 transcription in interferon ␥/lipopolysaccharide-stimulated RAW264.7 cells. Murine endogenous IL-12 p40 mRNA was consistently induced by overexpressed EKLF in resting RAW264.7 cells, whereas EKLF suppressed IL-12 p40 expression in activated RAW264.7 cells. Modulation of nuclear binding activities at the IL-12 p40 NFB halfsite was induced by EKLF for down-regulation of IL-12 p40 transcription in activated RAW264.7 cells, but no effect of EKLF on NFB activity was observed in resting RAW264.7 cells. Taken together, we identify EKLF as a transcription factor in macrophages able to regulate IL-12 p40 transcription depending on the cellular activation status. The bifunctional control of IL-12 p40 by EKLF and its modulation of NFB support a potential function for this factor in orchestrating IL-12 p40 production in macrophages.

Role of Phosphatidylinositol-3 Kinase in Transcriptional Regulation of TLR-Induced IL-12 and IL-10 by Fcγ Receptor Ligation in Murine Macrophages

“…The regulation of IL-12 mRNA expression has been described extensively in a recent review (69) and is dependent upon the coordinated expression of IL-12 IL-12b and IL-12a genes, which are encoded on different chromosomes. Many studies have confirmed that the primary regulatory step for the expression of both IL-12 IL-12b and IL-12a genes is at the level of transcription: multiple transcription factors are known to be involved in regulation of these genes, e.g., NF-B (47), C/EBP␤ (41), PU.1 (70), IRF-1 (37, 71), IRF-2 (37), IRF-8/ICSBP (50,51), NFAT (72), AP-1 (73), and activator and repressor factors such as Kruppellike factor (74). TLR-mediated NF-B binding and translocation is dependent upon the degradation of an inhibitory protein, IB␣.…”

Extrinsic and intrinsic control by EKLF (KLF1) within a specialized erythroid niche

“…There have been hints of EKLF expression in macrophage (Luo et al, 2004), although our analysis of adult bone marrow hematopoietic material had shown no evidence (Frontelo et al, 2007). To address this we used the mouse strain derived from ESCs that contain a single copy of the EKLF promoter directly upstream of a GFP reporter, integrated into the HPRT locus ( Fig.…”

PKCδ-Induced PU.1 Phosphorylation Promotes Hematopoietic Stem Cell Differentiation to Dendritic Cells

“…The cDNAs were amplified with 35 cycles of PCR using the Phusion polymerase (NEB, Frankfurt am Main, Germany, http://www.neb-online.de/de/) under the following conditions: denaturation for 10 seconds at 98 C, primer annealing for 30 seconds at the temperature of 50 C, and primer extension for 1 minute at 72 C. SiRNA-mediated knockdown of human ERK, PKCd, and PU.1 in HeLa cells was performed by transfection of RNA duplexes: MAPK/ERK: Hs_MAPK3_5/Hs_MAPK1_10 siRNA (SI00300776, SI00300755; Qiagen) PKCd: Hs_PRKCD_7 siRNA (SI00301329; Qiagen). For PU.1 silencing, we used a PU.1-specific siRNA with the target sequence 5 0 -AAGCTCACCT ACCAGTTC-3 0 (Eurofins MWG Biotech, Ebersberg, Germany, http://www.eurofinsdna.com/de/home.html?gclid¼CI_ZkIXg4aUCFQGHDgodx Rmc0Q) as previously described [22]. For control, a nonspecific siRNA with the target sequence 5 0 -AAUUCUCCGAACGUGU-CACGT-3 0 was used (1022076; Qiagen).…”