Hepcidin, a master regulator of iron homeostasis, is produced in small amounts by inflammatory monocytes/macrophages. Chronic immune activation leads to iron retention within monocytes/macrophages and the development of anemia of chronic disease (ACD). We questioned whether monocyte-derived hepcidin exerts autocrine regulation toward cellular iron metabolism. Monocyte hepcidin mRNA expression was significantly induced within 3 hours after stimulation with LPS or IL-6, and hepcidin mRNA expression was significantly higher in monocytes of ACD patients than in controls. In ACD patients, monocyte hepcidin mRNA levels were significantly correlated to serum IL-6 concentrations, and increased monocyte hepcidin mRNA levels were associated with decreased expression of the iron exporter ferroportin and iron retention in these cells. Transient transfection experiments using a ferroportin/EmGFP fusion protein construct demonstrated that LPS inducible hepcidin expression in THP-1 monocytes resulted in internalization and degradation of ferroportin. Transfection of monocytes with siRNA directed against hepcidin almost fully reversed this lipopolysaccharide-mediated effect. Using ferroportin mutation constructs, we found that ferroportin is mainly targeted by hepcidin when expressed on the cell surface. Our results suggest that ferroportin expression in inflammatory monocytes is negatively affected by autocrine formation of hepcidin, thus contributing to iron sequestration within monocytes as found in ACD. IntroductionA dysregulated iron homeostasis is a cornerstone of acute and chronic inflammatory processes involving cell-mediated immunity and frequently leads to the development of anemia, termed as anemia of chronic disease (ACD), or anemia of inflammation. 1,2 ACD is a multifactorial disease, where immune mechanisms play key pathogenetic roles. These include cytokine-mediated suppression of erythropoiesis, 3,4 a blunted erythropoietin response, [5][6][7] and an increased iron accumulation in and a defective iron recycling from the reticuloendothelial system. [8][9][10][11][12][13] The liver-derived acute phase protein hepcidin, which is induced by cytokines and iron, plays a key role in this concert. 14,15 It causes anemia when overexpressed, 16,17 whereas hepcidin mutations lead to hepatic iron overload, 18,19 which can be referred to its regulatory effects on cellular iron efflux. This is exerted after binding of hepcidin to the only known cellular iron exporter ferroportin, [20][21][22] leading to ferroportin internalization and blockade of duodenal iron absorption and macrophage iron recycling. 23 Because hepcidin expression is induced by inflammatory stimuli, including interleukin-6 (IL-6) or lipopolysaccharide (LPS), [24][25][26][27][28][29] an increased expression of this acute phase protein has been found to be associated with macrophage iron retention in ACD patients. 30,31 In addition, hepcidinindependent inhibition of ferroportin mRNA expression by inflammatory cytokines also contributes to macrophage iron rete...

NOS2-derived nitric oxide drives ferroportin-1–mediated iron export in Salmonella-infected macrophages, thus limiting bacterial growth.

H1 histones, isolated from logarithmically growing and mitotically enriched human lymphoblastic T-cells (CCRF-CEM), were fractionated by reversed phase and hydrophilic interaction liquid chromatography, subjected to enzymatic digestion, and analyzed by amino acid sequencing and mass spectrometry. During interphase the four H1 subtypes present in these cells differ in their maximum phosphorylation levels: histone H1.5 is tri-, H1.4 di-, and H1.3 and H1.2, only monophosphorylated. The phosphorylation is site-specific and occurs exclusively on serine residues of SP(K/A)K motifs. The phosphorylation sites of histone H1.5 from mitotically enriched cells were also examined. In contrast to the situation in interphase, at mitosis there were additional phosphorylations, exclusively at threonine residues. Whereas the tetraphosphorylated H1.5 arises from the triphosphosphorylated form by phosphorylation of one of two TPKK motifs in the C-terminal domain, namely Thr 137 and Thr 154 , the pentaphosphorylated H1.5 was the result of phosphorylation of one of the tetraphosphorylated forms at a novel nonconsensus motif at Thr 10 in the N-terminal tail. Despite the fact that histone H1.5 has five (S/T)P(K/A)K motifs, all of these motifs were never found to be phosphorylated simultaneously. Our data suggest that phosphorylation of human H1 variants occurs nonrandomly during both interphase and mitosis and that distinct serineor threonine-specific kinases are involved in different cell cycle phases. The order of increased phosphorylation and the position of modification might be necessary for regulated chromatin decondensation, thus facilitating processes of replication and transcription as well as of mitotic chromosome condensation.The nucleosome core, which consists of 146 bp of DNA wrapped 1.75 times around an octamer of core histones, represents the fundamental subunit of chromatin (for review, see Ref. 1). The H1 or linker histones are associated with the core histone-DNA complex and with the linker DNA between adjacent nucleosomes. Histone H1 is phosphorylated in a cell cycle-dependent manner: levels of H1 phosphorylation are usually lowest in the G 1 phase and rise continuously during S and G 2 . The M phase, where chromatin is highly condensed, shows the maximum number of phosphorylated sites. The individual H1 subtypes, however, differ in their degree of phosphorylation during the cell cycle (2, 3). A number of studies indicate that H1 phosphorylation is more likely involved in chromatin decondensation than in condensation (4). H1 phosphorylation seems to destabilize chromatin structure, thus weakening its binding to DNA. This decondensation of chromatin may give the DNA access to factors involved in transcription and replication in G 1 and S as well as to condensing factors active during mitosis (5). Recent studies demonstrate that H1 phosphorylation regulates specific gene expression in vivo and that it acts by mimicking the partial removal of H1 (6).The H1 histones consist of a globular central region flanked by short N...

In stimulating effector functions of mononuclear phagocytes, IFN-c is of pivotal importance in host defense against intramacrophage pathogens including salmonellae. As the activity of IFN-c is modulated by iron and since a sufficient availability of iron is essential for the growth of pathogens, we investigated the regulatory effects of IFN-c on iron homeostasis and immune function in murine macrophages infected with Salmonella typhimurium. In Salmonella-infected phagocytes, IFN-c caused a significant reduction of iron uptake via transferrin receptor 1 and resulted in an increased iron efflux caused by an enhanced expression of the iron exporter ferroportin 1. Moreover, the expression of haem oxygenase 1 and of the siderophore-capturing antimicrobial peptide lipocalin 2 was markedly elevated following bacterial invasion, with IFN-c exerting a super-inducing effect. This observed regulatory impact of IFN-c reduced the intracellular iron pools within infected phagocytes, thus restricting the acquisition of iron by engulfed Salmonella typhimurium while concomitantly promoting NO and TNF-a production. Our data suggest that the modulation of crucial pathways of macrophage iron metabolism in response to IFN-c concordantly aims at withdrawing iron from intracellular Salmonella and at strengthening macrophage immune response functions. These regulations are thus consistent with the principles of nutritional immunity. IntroductionHost defense against intracellular microbes such as salmonellae or mycobacteria strongly depends on cell-mediated immunity, a major component of which is characterized by interactions between Th1 cells and macrophages [1]. By secreting Th1 cytokines, particularly IFN-c, antigen-specific Th1 cells activate a plethora of microbicidal mechanisms in infected macrophages. Specifically, in mononuclear phagocytes infected with Salmonella enterica serovar typhimurium (S. typhimurium), IFN-c promotes the internalization of bacteria and stimulates their elimination by various mechanisms including reactive oxygen and nitrogen species (ROS and RNS), generated via NADPH phagocyte oxidase and iNOS, respectively [2][3][4][5]. In Salmonella-infected mice, treatment with recombinant IFN-c increases host survival and decreases bacterial numbers in liver and spleen [6]. Conversely, neutralization of murine IFN-c functions with specific antibodies results in reduced host survival and increased bacterial counts [7]. Eur. J. Immunol. 2008. 38: 1923-1936 Immunity to infection In humans, the central importance of IFN-c for immune response against salmonellae is highlighted by the fact that patients with genetic defects in the IL-12-induced production of IFN-c or in the IFN-c receptor 1 selectively suffer from infections with salmonellae and otherwise weakly pathogenic mycobacteria since their phagocytes fail to eliminate these microbes [8,9]. Iron serves as an essential nutrient for nearly all pathogenic microorganisms, and the expression of iron acquisition systems by infectious agents is associated with their virule...

Methylation and acetylation of position-specific lysine residues in the N-terminal tail of histones H3 and H4 play an important role in regulating chromatin structure and function. In the case of H3-Lys 4 , H3-Lys 9 , H3-Lys 27 , and H4-Lys 20 , the degree of methylation was variable from the mono-to the di-or trimethylated state, each of which was presumed to be involved in the organization of chromatin and the activation or repression of genes. Here we investigated the interplay between histone H4-Lys 20 mono-and trimethylation and H4 acetylation at induced (␤-major/␤-minor globin), repressed (c-myc), and silent (embryonic ␤-globin) genes during in vitro differentiation of mouse erythroleukemia cells. By using chromatin immunoprecipitation, we found that the ␤-major and ␤-minor promoter and the ␤-globin coding regions as well as the promoter and the transcribed exon 2 regions of the highly It has been proposed that distinct post-translational histone modifications act sequentially or in combination to form a "histone code" within chromatin (1). Acetylation and methylation of specific histone lysine residues can serve as a mark of either euchromatin or silent heterochromatin. Although methylation of H3-Lys 4 , H3-Lys 36 , and H3-Lys 79 has been linked to transcriptional activation and protection of euchromatin, H3-Lys 9 , H3-Lys 27 , and H4-Lys 20 methylation is generally thought to be associated with gene repression and heterochromatin formation (2-4). In this regard it must be noted that histone lysine residues can be mono-, di-, or trimethylated (5), thus extending the coding potential of a methylatable lysine position. Previous studies, however, focused only on detection of H3 (for review see Refs. 3 and 4) or H4 (6 -8) lysine methylation regardless of the methylation status. Recently, it was shown that a distinction between di-and trimethylation of various lysines of histone H3 is important for processes of transcriptional regulation or gene silencing (9 -11). Moreover, studies that focused on the in vivo distribution of mono-, di-, and trimethylated H3-Lys 9 and H3-Lys 27 demonstrate that mono-and dimethylated H3-Lys 9 and H3-Lys 27 are specifically localized to silent domains within euchromatin, whereas trimethylated H3-Lys 9 and monomethylated H3-Lys 27 were enriched at pericentric heterochromatin (12, 13). In contrast to findings suggesting a role for H4-Lys 20 methylation in regulating gene expression, a recently published study demonstrates that H4-Lys et al. (19), who found that the Drosophila epigenetic activator ASH-1, a histone methyltransferase, activates transcription by dimethylation of H3-Lys 4 , H3-Lys 9 , and H4-Lys 20 at the promoter of target genes. Significant differences in subnuclear localization of the mono-and trimethyl versions of histone H4-Lys

In vivo phosphorylation of the five histone H1 variants H1a-H1e including H1(0) in NIH 3T3 mouse fibroblasts was examined during the cell cycle by using a combination of HPLC techniques and conventional AU gel electrophoresis. Phosphorylation starts during the late G1 phase and increases throughout the S phase. In the late S phase, the H1 variants exist as a combination of molecules containing 0 or 1 (H1a, H1c), 0-2 (H1d), or 0-3 (H1b, H1e) phosphate groups with a share of unphosphorylated protein ranging between 35% and 75%, according to the particular subtype. Pulse-chase experiments show that phosphorylation during the S phase is a dynamic phosphorylation process with a limited phosphorylation maximum. In most H1 subtypes, phosphorylation occurs very rapidly at the G2/M transition with only small amounts of intermediate phosphorylated molecules. Phosphorylation of mouse H1c, however, occurs stepwise during this transition. Phosphorylated mouse histone subtypes from cells in mitosis contain four phosphate groups in the case of H1a, H1c, and H1e and five in the case of H1b and H1d. Comparison of the mouse phosphorylation pattern to that in rat C-6 glioma cells showed differences for H1e and H1d. By comparing the different phosphorylation patterns of the individual H1 variants during the cell cycle, we were able to classify the H1 histones into subtypes with low (H1a, H1c, H1(0)) and high (H1b, H1d, H1e) phosphorylation levels.

Posttranslational modification of histones is a common means of regulating chromatin structure and thus diverse nuclear processes. Using a hydrophilic interaction liquid chromatographic separation method in combination with mass spectrometric analysis, the present study investigated the alterations in histone H4 methylation/acetylation status and the interplay between H4 methylation and acetylation during in vitro differentiation of mouse erythroleukemia cells and how these modifications affect the chromatin structure. Independently of the type of inducer used (dimethyl sulfoxide, hexamethylenebisacetamide, butyrate, and trichostatin A), we observed a strong increase in non-and monoacety- In eukaryotes, histone proteins associate with DNA to form nucleosomes that are folded into higher order chromatin structures. Differences in higher order chromatin structures, which are important prerequisites for numerous biological processes including cellular proliferation, differentiation, development, gene expression, genome stability, and cancer, are thought to be realized by a variety of posttranslational modifications of histone N termini, particularly of histones H3 and H4. Besides acetylation, histones are subjected to phosphorylation, methylation, ubiquitination, ADP-ribosylation, and deamidation (1, 2). Distinct combinations of covalent histone modifications including lysine acetylation, lysine and arginine methylation, and serine phosphorylation form the basis of the histone code hypothesis (3-5). This hypothesis proposes that a pre-existing modification affects subsequent modifications on histone tails and that these modifications generate unique surfaces for the binding of various proteins or protein complexes responsible for higher order chromatin organization and gene activation and inactivation. Some of the histone-modifying enzymes (e.g. lysine methyltransferases) are, when deregulated, considered to be involved in carcinogenesis (6).Histone H4 is typically acetylated at lysines 5, 8, 12, and 16, methylated at arginine 3 and lysine 20, and phosphorylated at serine 1 (3,7,8). Unlike the dynamic process of histone acetylation and phosphorylation, histone methylation is regarded as a relatively static long-term signal with a low turnover of the methyl group. Whereas arginine can be either mono-or dimethylated (the latter in symmetric or asymmetric form), lysine methylation can occur as a mono-, di-, or trimethylated derivative. In contrast to histone H3 methylation, H4-Lys 20 was long considered to be maximally dimethylated in mammals (9). Most recently, however, Sarg et al. (10) conducted a mass spectrometric analysis with a newly developed hydrophilic interaction chromatographic method enabling the simultaneous separation of methylated and acetylated forms and, for the first time, found in vivo evidence that H4-Lys 20 is also trimethylated in mammalian tissue. Moreover, in rat liver and kidney the proportion of trimethylated histone H4 increases during aging. In Raji and K562 cells the trimethylated form was ...

The cell cycle-associated phosphorylation of histone H1.5 is manifested as three discrete phosphorylated forms, occurring exclusively on Ser(17), Ser(172), and Ser(188) during interphase. During late G2 and mitosis the up-phosphorylation occurs exclusively on threonine at either Thr(137) or Thr(154) to build the tetraphosphorylated forms of H1.5, whereas the pentaphosphorylated forms result from phosphorylation at Thr(10). To determine the kinetic and spatial distribution of histone H1 phosphorylation within the nucleus of synchronized Hela cells we localized three distinct phosphorylation sites of histone subtype H1.5 using affinity-purified polyclonal antibodies generated against phosphorylated Ser(17), Ser(172), and Thr(10). Immunofluorescence labeling of synchronized HeLa cells using the specific antibodies revealed that phosphorylation of H1.5 Ser(17) appeared early in G1 at discrete speckles followed by phosphorylation of Ser(172). Thr(10) phosphorylation started during prophase, showed highest phosphorylation levels during metaphase, and disappeared clearly before chromatin decondensation occurred. Experiments using the kinase inhibitor staurosporine indicate the involvement of different kinases at the various phospho-sites. Colocalization studies revealed that Ser(172) phosphorylation of H1.5 and H1.2 does colocalize to DNA replication and transcription sites. These results favor the idea that the various site-specifically phosphorylated forms of H1.5 and H1.2 localized at distinct regions of the nucleus are related to different functions during the cell cycle.