Aleksander Koj

Interleukin-1-inducible MCPIP protein has structural and functional properties of RNase and participates in degradation of IL-1β mRNA

In human monocyte‐derived macrophages, the MCPIP gene (monocyte chemoattractant protein‐induced protein) is strongly activated by interleukin‐1β (IL‐1β). Using bioinformatics, a PIN domain was identified, spanning amino acids 130‐280; such domains are known to possess structural features of RNases. Recently, RNase properties of MCPIP were confirmed on transcripts coding for interleukins IL‐6 and IL‐12p40. Here we present evidence that siRNA‐mediated inhibition of the MCPIP gene expression increases the level of the IL‐1β transcript in cells stimulated with LPS, whereas overexpression of MCPIP exerts opposite effects. Cells with an increased level of wild‐type MCPIP showed lower levels of IL‐1β mRNA. However, this was not observed when mutant forms of MCPIP, either entirely lacking the PIN domain or with point mutations in this domain, were used. The results of experiments with actinomycin D indicate that lower levels of IL‐1β mRNA are due to shortening of the IL‐1β transcript half‐life, and are not related to the presence of AU‐rich elements in the 3′ UTR. The interaction of the MCPIP with transcripts of both IL‐1β and MCPIP observed in an RNA immunoprecipitation assay suggests that this novel RNase may be involved in the regulation of expression of several genes.

Regulatory feedback loop between NF‐κB and MCP‐1‐induced protein 1 RNase

A novel gene ZC3H12A, encoding MCP‐1‐induced protein 1 (MCPIP), was recently identified in human peripheral blood monocytes treated with monocyte chemotactic protein 1 (MCP‐1) and in human monocyte‐derived macrophages stimulated with interleukin (IL)‐1β. These experiments revealed that the gene undergoes rapid and potent transcription induction upon stimulation with proinflammatory molecules, such as MCP‐1, IL‐1β, tumour necrosis factor α and lipopolysaccharide. Here we show that the induction of ZC3H12A by IL‐1β is predominantly NF‐κB‐dependent because inhibition of this signalling pathway results in the impairment of ZC3H12A transcription activation. Our results indicate the presence of an IL‐1β‐responding region within the second intron of the ZC3H12A gene, which contains four functional NF‐κB‐binding sites. Therefore, we propose that this transcription enhancer transduces a ZC3H12A transcription‐inducing signal after IL‐1β stimulation. Recent reports suggest that MCPIP acts as a negative regulator of inflammatory processes because it is engaged in the degradation of transcripts coding for certain proinflammatory cytokines. Our observations provide evidence for a novel negative feedback loop in the activation of NF‐κB and point to potential significance of MCPIP in the treatment of various pathological states, such as diabetes or cancer that involve disturbances in the functioning of the NF‐κB system.

Termination of Acute-Phase Response: Role of Some Cytokines and Anti-Inflammatory Drugs

1. The acute-phase response is triggered by changes in intracellular mediators that activate stress-sensitive kinases and transcription factors controlling the synthesis of proinflammatory cytokines such as TNF-alpha, IL-1, IL-8 or IFN-gamma. 2. Natural extinguishing of acute-phase response occurs due to short half-lives of inflammatory mediators and production of anti-inflammatory cytokines such as IL-10, IL-4, IL-13, TGF-beta and some others. 3. Excess proinflammatory cytokines are removed by soluble cytokine receptors and receptor antagonists. 4. Synthesis of proinflammatory mediators and cytokines can be blocked by glucocorticoids, some nonsteroidal anti-inflammatory drugs suppressing cyclooxygenase and by specific inhibitors of cytokine induction. 5. The most promising approach in effective termination of acute-phase response appears to be a combined use of anti-inflammatory cytokines and specific drugs.

Subcellular distribution and intramitochondrial localization of three sulfurtransferases in rat liver.

Mammalian tissues contain three enzymes involved in the transfer of bivalent sulphur to a variety .of nucleophilic acceptors: rhodanese (thiosulfate sulfurtransferase, EC 2.8.1. l), 3-mercaptopyruvate sulfurtransferase (EC 2.8.1.2) and thiosulfate reductase (no EC number, for definition see [l-4] ). These enzymes participate in the metabolism of some amino acids and low molecular weight sulphur compounds [5-71, show detoxifying activity against sulphide [8] and cyanide [9, IO], are probably involved in the formation of iron-sulfur chromophore of ferredoxin [ 11,121 and restoration of labile sulfur in succinate dehydrogenase (EC 1.3.99. I [ 131). Rhodanese is present solely in the mitochondria [ 14,151 and shows a characteristic latency [ 161, but the subcellular distribution of the two other sulfurtransferases is still disputed. Kun and Fanshier [ 171 and Van der Hamer et al. [ 181 observed that most of the mercaptopyruvate sulfurtransferase activity was in the soluble fraction of rat liver, while recently Taniguchi and Kimura [ 121 found the enzyme in both mitochondria and the cytosol of bovine adrenal cortex. Preliminary observations indicate that also thiosulfate reductase occurs in these two subcellular compartments [ 191. The experiments described here confirm the bimodal intracellular localization of mercaptopyruvate sulfurtransferase and thiosulfate reductase. They also indicate that, within the

Synthesis of tissue inhibitor of metalloproteinase-1 (TIMP-1) in human hepatoma cells (HepG2) Up-regulation by interleukin-6 and transforming growth factor β1

Metalloproteinases and their specific inhibitors, believed to play a role in extracellular matrix metabolism, are regulated by inflammatory cytokines. Here we have addressed the question of whether liver, the major site of Synthesis of plasma proteinase inhibitors, is also capable of synthesizing the tissue inhibitor of metalloproteinase‐1 (TIMP‐1). We show at mRNA and protein levels that TIMP‐1 is expressed in differentiated human hepatoma cells (HepG2) and that its synthesis is up‐regulated by interleukin‐6 (IL‐6), transforming growth factor β1 and phorbol 12‐myristate 13‐acetate. The physiological role of this phenomenon is underlined by the fact that lipopolysaccharide administration into rats in vivo, as well as IL‐6‐stimulation of rat hepatocytes in primary culture, also leads to an increase of TIMP‐1 mRNA in liver cells.

Monocyte chemotactic protein-1-induced protein-1 (MCPIP1) is a novel multifunctional modulator of inflammatory reactions

The generalized inflammatory response leads to activation of hundreds of genes transcribed in an established sequence in specialized cells. Transcriptome analysis of human monocyte-derived cells stimulated with IL-1beta or with monocyte chemotactic protein-1 (MCP-1) has led to the identification of a new inflammation-related gene ZC3H12A encoding a chain of 599 amino acids corresponding to a 66-kDa protein. The protein, given a provisional name of MCPIP1 (monocyte chemotactic protein-induced protein-1), is expressed in several human and murine tissues such as bone marrow, spleen, heart and placenta. In in vivo studies, mice with inactivated MCPIP1-encoding gene showed growth retardation, lymphadenopathy, splenomegaly and enhanced inflammatory symptoms. Principal molecular features of MCPIP1 include a single zinc finger motif, an RNase-like PIN domain and ubiquitin-binding domain. Reports from independent laboratories suggest that MCPIP1 may function also as a deubiquitinase. Although MCPIP1 is regarded by some authors as a new transcription factor or cell differentiation factor modulating angiogenesis or adipogenesis, its principal function appears to be downregulation of inflammatory responses through at least two independent mechanisms: increased degradation of cytokine mRNAs and inhibition of LPS- and IL-1-induced NF-kappaB signaling pathway. The interference with NF-kappaB activation is highly complex and includes TRAF6 and TANK interaction with the ubiquitin-associated (UBA) domain of MCPIP1. Purified MCPIP1 protein was reported to degrade specific mRNA and cleave K48- and K63-linked polyubiquitin chains. Although some structural features and the mechanism of action of MCPIP1 are not fully explained yet, its importance in the regulation of inflammatory reactions has been firmly established.

Identification of interleukin-1 and interleukin-6-responsive genes in human monocyte-derived macrophages using microarrays

The transcriptome profile of human monocyte-derived macrophages stimulated in vitro by low doses of IL-1 or IL-6 was analyzed by microarrays (Affymetrix, HG-U133A) in 5 independent experiments. Out of 4886 probe sets consistently detected in all 5 array replicates we found approximately 300 genes (FDR<5%) modulated by IL-1 and/or IL-6, among which 34 may be regarded as novel cytokine-responsive macrophage genes of various function. Detailed analysis indicates that cytokine-responsive genes include 125 transcripts significantly up-regulated by IL-1 and only 39 transcripts up-regulated by IL-6, whereas the number of down-regulated transcripts is lower and almost equal for both cytokines. These data indicate that, in comparison to liver cells, IL-1 is more potent than IL-6 in modulating gene expression of human macrophages. Hierarchical clustering analysis of these transcripts yielded 7 separate gene clusters. The most abundant group contains genes strongly activated by IL-1 alone and coding for chemokines, cytokines and their receptors, the components of intracellular signaling as well as transcription factors from NF-kB family. In order to validate the results obtained by microarray analysis the expression of 5 genes from various clusters was determined by quantitative RT-PCR. Moreover, the putative promoter regions of all cytokine-responsive genes were subjected to the in silico identification of transcription factor binding sites (TFBS). We found that TFBS corresponding to RelA/NF-kB is the most strongly over-represented group and we demonstrated involvement of NF-kB in the expression of selected genes.

Subcellular compartmentation of rhodanese and 3-mercaptopyruvate sulphurtransferase in the liver of some vertebrate species

Abstract 1. 1. Activities of two sulphurtransferases were determined in lysed red cells and in mitochondrial and cytosolic fractions from the liver of eight vertebrate species, with glutamate dehydrogenase as the marker enzyme for mitochondria. 2. 2. Specific activities of 3-mercaptopyruvate sulphurtransferase were found low in fish, amphiba and reptilia in comparison with birds and mammals. On the other hand, the highest activities of rhodanese were observed in rat, hamster and frog. 3. 3. In fish, amphibia and reptilia majority of liver sulphurtransferases was found in the cytosol, while in birds and mammals the enzymes were localized predominantly in the mitochondrial fraction.

Mechanism of plasminogen activator inhibitor-1 regulation by oncostatin M and interleukin-1 in human astrocytes

Glial cells that produce and respond to various cytokines mediate inflammatory processes in the brain. Here, we show that oncostatin M (OSM) and interleukin‐1 (IL‐1) regulate the expression of plasminogen activator inhibitor‐1 (PAI‐1) and urokinase‐type plasminogen activator (uPA) in human astrocytes. Using the PAI‐1 reporter constructs we show that the −58 to −51 proximal element mediates activation by both cytokines. This element is already bound by c‐fos/c‐jun heterodimers in unstimulated astrocytes, and treatment with cytokine strongly stimulates both expression of c‐fos and binding of c‐fos/c‐jun heterodimers. In addition, IL‐1 activates an inhibitory mechanism that down‐regulates PAI‐1 expression after longer exposure to this cytokine. Overexpression of dominant‐negative signal transducer and activator of transcription‐1 (STAT1), STAT3, STAT5 and inhibitor of nuclear factor‐κB (IκB) suppressed OSM/IL‐1‐induced expression of the PAI‐1 reporter construct. We conclude that OSM and IL‐1 regulate the PAI‐1 gene expression via up‐regulating c‐fos levels and subsequent binding of c‐fos/c‐jun heterodimers to the proximal element of the PAI‐1 gene.

Different preparations of natural and recombinant human interleukin-6 (ifn-β2, bsf-2) similarly stimulate acute phase protein synthesis and uptake of α-aminoisobutyric acid by cultured rat hepatocytes

1. Rat hepatocytes were cultured for 2 days in Williams E medium containing 1 microM insulin and dexamethasone. 2. Production of five plasma proteins was determined by electroimmunoassay in the media, and amino acid uptake was measured by [alpha-14C]aminoisobutyric acid accumulation in hepatocytes. 3. Supernatants from rat peritoneal macrophages and IL-6/IFN-beta 2/BSF-2 obtained from four different laboratories similarly stimulated synthesis of fibrinogen, alpha 1-cysteine proteinase inhibitor and alpha 2-macroglobulin, as well as [14C]-accumulation in cultured hepatocytes. 4. It is concluded that IL-6 is the principal hepatocyte stimulating factor responsible for typical features of the acute phase response of liver cells.