Role of CCAAT/Enhancer-Binding Protein Site in Transcription of Human Neutrophil Peptide-1 and -3 Defensin Genes (original) (raw)
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Human α-Defensin Expression Is Not Dependent on CCAAT/Enhancer Binding Protein-ε in a Murine Model
PLoS ONE, 2014
Specific granule deficiency (SGD) is a rare congenital disorder characterized by recurrent infections. The disease is caused by inactivating mutations of the CCAAT/enhancer binding protein-e (C/EBP-e) gene. As a consequence, specific and gelatinase granules lack most matrix proteins. Furthermore, azurophil granules contain diminished amounts of their most abundant proteins, a-defensins, also known as human neutrophil peptides (HNPs). In accordance with this, in vitro models have demonstrated induction of HNPs by C/EBP-e. Since mice do not express myeloid defensins, they cannot per se be used to characterize the role of C/EBP-e in controlling HNP expression in vivo. We therefore crossed a transgenic HNP-1-expressing mouse with the Cebpe -/mouse to study the in vivo significance of C/EBP-e for HNP-1 transcription and expression. Surprisingly, neither expression nor processing of HNP-1 was affected by lack of C/EBP-e in these mice. Transduction of C/ EBP-e into primary bone marrow cells from HNP-1 mice induced some HNP-1 expression, but not to levels comparable to expression human cells. Taken together, our data infer that the HNP-1 of the transgenic mouse does not show an expression pattern equivalent to endogenous secondary granule proteins. This limits the use of these transgenic mice as a model for human conditions.
Transcription Factors Human Neutrophils Involves C/EBP Inflammatory Cytokine Production by
2010
A growing number of neutrophil-derived cytokines have proven to be crucial to various inflammatory and immune processes in vivo. Whereas C/EBP (CCAAT/enhancer-binding protein) transcription factors are important for neutrophil differentiation from myeloid precursors, we report herein that they also regulate cytokine production in mature neutrophils. All known C/EBP proteins but C/EBP␥ are expressed in neutrophils; most isoforms localize to the nucleus, except for C/EBP␣, which is cytoplasmic. Neutrophil stimulation does not alter the overall levels, cellular distribution, or turnover of C/EBP proteins; it also does not further induce the constitutive DNA-binding activity detected in nuclear extracts, consisting of C/EBP and C/EBP. However, nuclear C/EBP is rapidly phosphorylated upon cell stimulation, suggesting that it can activate cytokine promoters. Indeed, the transactivation of an IL-8 promoter-luciferase construct in a human neutrophil-like cell line was impaired when its C/EBP or NF-B sites were mutated. Overexpression of a C/EBP repressor also impeded IL-8 promoter transactivation, as well as the generation of IL-8, Mip-1␣, and Mip-1 in this cellular model, whereas TNF-␣ generation was mostly unaffected. Finally, overexpression of a C/EBP mutant (T235A) as well as chromatin immunoprecipitation assays unveiled an important role for this residue in cytokine induction. This is the first demonstration that C/EBP factors are important regulators of cytokine expression in human neutrophils.
The structure of neutrophil defensin genes
FEBS Letters, 1993
Defensins are a family of microbicidal peptides abundant in the granules of mammalian neutrophils, in rabbit alveolar macrophages, and in human and murine intestinal Paneth cells. We cloned and sequenced the genes of three neutrophil-specific defensins. Human HNP-1 and HNP-3 are nearly identical and rabbit NP-3a is closely related. The four known neutrophil-specific defensin genes are strikingly similar in the structure and organization of their three exons and two introns, but the three defensin genes expressed in macrophages (MCP-1 and-2) or Paneth cells (HD-5) are organized differently: HD-5 has only two exons, and MCP-1 and-2 have a comparatively short first intron. The diverse genomic organization of defensin genes may contribute to their cell-specific expression.
PLoS ONE, 2012
The azurophilic granules of human neutrophils contain four a-defensins called human neutrophil peptides (HNPs 1-4). HNPs are tridisulfide-linked antimicrobial peptides involved in the intracellular killing of organisms phagocytosed by neutrophils. The peptides are produced as inactive precursors (proHNPs) which are processed to active microbicides by as yet unidentified convertases. ProHNP1 was expressed in E. coli and the affinity-purified propeptide isolated as two species, one containing mature HNP1 sequence with native disulfide linkages (''folded proHNP1'') and the other containing non-native disulfide linked proHNP1 conformers (misfolded proHNP1). Native HNP1, liberated by CNBr treatment of folded proHNP1, was microbicidal against Staphylococcus aureus, but the peptide derived from misfolded proHNP1 was inactive. We hypothesized that neutrophil elastase (NE), proteinase 3 (PR3) or cathepsin G (CG), serine proteases that co-localize with HNPs in azurophil granules, are proHNP1 activating convertases. Folded proHNP1 was converted to mature HNP1 by both NE and PR3, but CG generated an HNP1 variant with an N-terminal dipeptide extension. NE and PR3 cleaved folded proHNP1 to produce a peptide indistinguishable from native HNP1 purified from neutrophils, and the microbicidal activities of in vitro derived and natural HNP1 peptides were equivalent. In contrast, misfolded proHNP1 conformers were degraded extensively under the same conditions. Thus, NE and PR3 possess proHNP1 convertase activity that requires the presence of the native HNP1 disulfide motif for high fidelity activation of the precursor in vitro.
Blood, 2003
In the bone marrow of C/EBP⑀ ؊/؊ mice, expression of neutrophil secondary and tertiary granule mRNAs is absent for lactoferrin (LF), neutrophil gelatinase (NG), murine cathelinlike protein (MCLP), and the cathelin B9; it is severely reduced for neutrophil collagenase (NC) and neutrophil gelatinase-associated lipocalin (NGAL). In addition, the expression of eosinophil granule genes, major basic protein (MBP), and eosinophil peroxidase (EPX) is absent. These mice express C/EBP␣, C/EBP, and C/EBP␦ in the bone marrow at levels similar to those of their wild-type counterparts, suggesting a lack of functional redundancy among the family in vivo. Stable inducible expression of C/EBP⑀ and C/EBP␣ in the murine fibroblast cell line NIH 3T3 activated expression of mRNAs for B9, MCLP, NC, and NGAL but not for LF. In transient transfections of C/EBP⑀ and C/EBP␣, B9 was strongly induced with weaker induction of the other genes. C/EBP and C/EBP␦ proteins weakly induced B9 expression, but C/EBP␦ induced NC expression more efficiently than the other C/EBPs. The expression of MBP was inefficiently induced by C/EBP⑀ alone and weakly induced with C/EBP⑀ and GATA-1, but the addition of PU.1 resulted in a striking cooperative induction of MBP in NIH 3T3 cells. Mutation of a predicted PU.1 site in the human MBP promoter-luciferase reporter construct abrogated the response to PU.1. Gel-shift analysis demonstrated binding of PU.1 to this site. MBP and EPX mRNAs were absent in a PU.1-null myeloid cell line established from the embryonic liver of PU.1 ؊/؊ mice. Restitution of PU.1 protein expression restored MBP and EPX protein expression. This study demonstrates that C/EBP⑀ is essential and sufficient for the expression of a particular subset of neutrophil secondary granule genes. Furthermore, it indicates the importance of PU.1 in the cooperative activation of eosinophil granule genes.
Characterization of the promoters of the guinea pig neutrophil cationic peptide-1 and -2 genes
FEBS Letters, 1994
Guinea pig neutrophils contain the antimicrobial cationic peptides GNCP-1 and GNCP-2 in the granules. To understand the regulation of the gene expression, the promoters for the GNCP-1 and GNCP-2 genes were characterized. Sequencing analysis of the genomic clones showed that the nucleotide sequences of the 5'-upstream regions (1.7 kb) from exon 1 were homologous @O-93%) between the GNCP-1 and GNCP-2 genes. However, transient transfection assays using luciferase reporter gene constructs revealed that the promoter activity of GNCP-1 was 2-fold greater than that of GNCP-2. Furthermore, DNase I footprint analysis demonstrated that three regions (I, II and III) were protected on the GNCP-1 promoter, whereas only two protected regions (II and III) were identified on the GNCP-2 promoter. Together these observations indicate that GNCP-1 and GNCP-2 are encoded by homologous genes, but the expression of the GNCP-1 and GNCP-2 genes is likely to be different at the level of transcription.
C/EBP, c-Myb, and PU.1 cooperate to regulate the neutrophil elastase promoter
Molecular and Cellular Biology, 1996
The murine neutrophil elastase (NE) gene is expressed specifically in immature myeloid cells. A 91-bp NE promoter region contains three cis elements which are conserved evolutionarily and are essential for activation of the promoter in differentiating 32D cl3 myeloid cells. These elements bound c-Myb (at -49), C/EBPalpha (at -57), and PU.1 (at -82). In NIH 3T3 cells, the NE promoter was activated by c-Myb, C/EBPalpha, and PU.1, via their respective binding sites. Cooperative activation was seen by any combination of c-Myb, C/EBPalpha, and PU.1, including all three together, again via their DNA-binding sites. In CV-1 cells, but not in NIH 3T3 cells, cooperation between Myb and C/EBPalpha depended on the integrity of the PU.1-binding site. In addition to C/EBPalpha, C/EBPdelta strongly activated the NE promoter, alone or with c-Myb, but C/EBPbeta was less active. Either of C/EBPalpha's two transactivation domains cooperatively activated the promoter with c-Myb, in both NIH 3T3 and...
Genomic modulators of gene expression in human neutrophils
Nature Communications, 2015
Neutrophils form the most abundant leukocyte subset and are central to many disease processes. Technical challenges in transcriptomic profiling have prohibited genomic approaches to date. Here we map expression quantitative trait loci (eQTL) in peripheral blood CD16 þ neutrophils from 101 healthy European adults. We identify cis-eQTL for 3281 neutrophil-expressed genes including many implicated in neutrophil function, with 450 of these not previously observed in myeloid or lymphoid cells. Paired comparison with monocyte eQTL demonstrates nuanced conditioning of genetic regulation of gene expression by cellular context, which relates to cell-type-specific DNA methylation and histone modifications. Neutrophil eQTL are markedly enriched for trait-associated variants particularly autoimmune, allergy and infectious disease. We further demonstrate how eQTL in PADI4 and NOD2 delineate risk variant function in rheumatoid arthritis, leprosy and Crohn's disease. Taken together, these data help advance understanding of the genetics of gene expression, neutrophil biology and immune-related diseases.
Blood, 2003
In the bone marrow of C/EBPε−/− mice, expression of neutrophil secondary and tertiary granule mRNAs is absent for lactoferrin (LF), neutrophil gelatinase (NG), murine cathelinlike protein (MCLP), and the cathelin B9; it is severely reduced for neutrophil collagenase (NC) and neutrophil gelatinase-associated lipocalin (NGAL). In addition, the expression of eosinophil granule genes, major basic protein (MBP), and eosinophil peroxidase (EPX) is absent. These mice express C/EBPα, C/EBPβ, and C/EBPδ in the bone marrow at levels similar to those of their wild-type counterparts, suggesting a lack of functional redundancy among the family in vivo. Stable inducible expression of C/EBPε and C/EBPα in the murine fibroblast cell line NIH 3T3 activated expression of mRNAs for B9, MCLP, NC, and NGAL but not for LF. In transient transfections of C/EBPε and C/EBPα, B9 was strongly induced with weaker induction of the other genes. C/EBPβ and C/EBPδ proteins weakly induced B9 expression, but C/EBPδ i...