Transcription Factor KLF2 and Its Role in the Regulation of Inflammatory Processes.

KLF2 is a member of the Krüppel-like transcription factor family of proteins containing highly conserved DNA-binding zinc finger domains. KLF2 participates in the differentiation and regulation of the functional activity of monocytes, T lymphocytes, adipocytes, and vascular endothelial cells. The activity of KLF2 is controlled by several regulatory systems, including the MEKK2,3/MEK5/ERK5/MEF2 MAP kinase cascade, Rho family G-proteins, histone acetyltransferases CBP and p300, and histone deacetylases HDAC4 and HDAC5. Activation of KLF2 in endothelial cells induces eNOS expression and provides vasodilatory effect. Many KLF2-dependent genes participate in the suppression of blood coagulation and aggregation of T cells and macrophages with the vascular endothelium, thereby preventing atherosclerosis progression. KLF2 can have a dual effect on the gene transcription. Thus, it induces expression of multiple genes, but suppresses transcription of NF-κB-dependent genes. Transcription factors KLF2 and NF-κB are reciprocal antagonists. KLF2 inhibits induction of NF-κB-dependent genes, whereas NF-κB downregulates KLF2 expression. KLF2-mediated inhibition of NF-κB signaling leads to the suppression of cell response to the pro-inflammatory cytokines IL-1ß and TNFα and results in the attenuation of inflammatory processes.

Translation Factor eIF5A, Modification with Hypusine and Role in Regulation of Gene Expression. eIF5A as a Target for Pharmacological Interventions.

Translation factor eIF5A participates in protein synthesis at the stage of polypeptide chain elongation. Two eIF5A isoforms are known that are encoded by related genes whose expression varies significantly in different tissues. The eIF5A1 isoform is a constitutively and ubiquitously expressed gene, while the eIF5A2 isoform is expressed in few normal tissues and is an oncogene by a number of parameters. Unique feature of eIF5A isoforms is that they are the only two proteins that contain amino acid hypusine. Modification with hypusine is critical requirement for eIF5A activity. Another distinctive feature of eIF5A is its involvement in the translation of only a subset of the total population of cell mRNAs. The genes for which mRNAs translation requires eIF5A are the members of certain functional groups and are involved in cell proliferation, apoptosis, inflammatory processes, and regulation of transcription and RNA metabolism. The involvement of eIF5A is necessary for the translation of proteins containing oligoproline fragments and some other structures. Modification of eIF5A by hypusine is implemented by two highly specialized enzymes, deoxyhypusine synthase (DHS) and deoxyhypusine hydroxylase (DOHH), which are not involved in other biochemical reactions. Intracellular activity of these enzymes is closely associated with systems of protein acetylation, polyamine metabolism and other biochemical processes. Inhibition of DHS and DOHH activity provides the possibility of pharmacological control of eIF5A activity and expression of eIF5A-dependent genes.

Keap1-Nrf2 signaling pathway: mechanisms of regulation and role in protection of cells against toxicity caused by xenobiotics and electrophiles.

The transcription factor Nrf2 governs the expression of a considerable group of genes involved in cell protection against oxidants, electrophiles, and genotoxic compounds. The activity of Nrf2 is sensitive to xenobiotics and endogenous electrophiles. Nrf2 is negatively regulated by specific suppressor protein Keap1, which is also a receptor of electrophiles and adapter for Cul3 ubiquitin ligase. Electrophiles react with critical thiol groups of Keap1 leading to the loss of its ability to inhibit Nrf2. The Keap1-Nrf2 signaling pathway also down-regulates NF-κB transcriptional activity and attenuates cytokine-mediated induction of proinflammatory genes. Pharmacological activation of the Keap1-Nrf2 pathway can be used for treatment and prevention of many diseases. Widely known natural Keap1-Nrf2 activators include curcumin, quercetin, resveratrol, and sulforaphane. The most effective Keap1-Nrf2 activators are synthetic oleanane triterpenoids.

[The role of nitric oxide in the transcription of histone gene].

Nitric oxide (NO) is a transmitter for intracellular and extracellular signals. It is known that nitric oxide suppresses DNA replication and expression of genes responsible for cell growth and proliferation. In this study we investigated the effect of NO on histone H2B gene expression in human and murine cell lines. We have shown that treatment of cells with chemical NO donors leads to decreasing the histone H2B mRNA level. Using luciferase assay with reporter gene regulated by H2B gene promoter, we showed that NO reduced the reporter gene activity and mRNA level simultaneously. From these data we conclude that NO negatively regulates histone H2B transcription. We believe the affect of nitric acid on the transcription of histone gene plays important role for NO-induced cytostatic effects.

[Role of transcription factor AP-1 in integration of cellular signalling systems].

Transcription factor AP-1 is a dimer complex composed by DNA-binding proteins of Jun, Fos, and ATF families. AP-1 mediates cell response on growth factors, cytokines, neurotransmitters and other intercellular signaling molecules. AP-1 activity is mediated by G-proteins, adapter proteins, MAP kinases and other elements of cellular signaling systems. AP-1 dependent genes play a pivotal role in regulation of cell proliferation, morphogenesis, apoptosis, and differentiation.

[Effect of the antioxidants on NO-dependent induction of heme oxigenase 1 gene in U937 monocytes].

Previously it was shown that thiol antioxidants are potent inhibitors of the NO-dependent induction of heme oxygenase 1 (HOX-1) gene. However, the mechanism of HOX-1 gene down-regulation by thiol antioxidants and underlying signaling pathway remain unclear. In this study we have examined, whether the scavenging of reactive oxygen and reactive nitrogen species (ROS and RNS) is the major cause for thiol-mediated suppression of the HOX-1 induction by NO. Further, to identify the ROS family members implicated in the HOX-1 induction, we also exposed cells to various non-thiol antioxidants: dimethyl sulfoxide, dimetylthiourea, sodium salicylate, sodium formate, uric acid, catalase, and superoxide dismutase. A partial inhibition of HOX-1 induction occurred in the presence of non-polar hydroxyl radical scavengers, dimethyl sulfoxide and dimetylthiourea. The other non-thiol antioxidants were ineffective towards HOX-1 expression. Then, in order to determine, whether RNS scavenging is implicated in the HOX-1 down-regulation by thiol antioxidants, we took advantage of the capacity of suboptimal concentrations of the NO scavenger PTIO (2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl-3-oxide) to oxidize NO to nitrosating species. We showed that simultaneous cell treatment with NO donor and PTIO significantly enhanced the rate of the HOX-1 gene NO-dependent induction indicating that RNS are mediators of HOX-1 gene transcriptional activation. Thiol antioxidants completely suppressed PTIO stimulatory action. These findings imply that inhibitory action of thiol antioxidants is mediated by RNS scavenging. The study provides an approach for pharmacologycal modulation of cell response to NO and its derivatives through the use of antioxidants.

[Redox-dependent regulation of gene expression induced by nitric oxide]. / Redoks-zavisimaia reguliatsiia ékspressii genov, indutsiruemykh okis'iu azota.

Nitric oxide (NO) is a mobile, highly reactive signal molecule, and changes the expression of specific genes in effector cells. Under physiological conditions, NO reacts with molecular oxygen and with reactive oxygen species (ROS) to produce intermediates known as reactive nitrogen species (RNS). The production of NO and RNS in the cell is controlled by hormones, neurotransmitters, cytokines, and growth factors. Hence NO and its derivatives act as secondary paracrinous factors and transmit the signal from NO-producing to neighboring cells. Intracellular reception of NO and RNS is due to Src-related tyrosine protein kinases, G-protein Ras, cytochrome oxidase, and guanylate cyclase. Receptor proteins mostly contain heme, active thiol, or iron-sulfur groups, and are both on the plasma membrane and in internal cell compartments. Many of the NO receptors are the key components of cell regulatory systems controlling the transcription factors AP-1, HIF-1, NF-kappa B, and p53 and the expression of their target genes. A distinguishing feature of NO signaling is that changes in redox potential of the cell switch the NO receptor and, consequently, modify the NO effect. Depending on the ROS level, NO activates different signal transduction pathways to induce (or suppress) different gene sets. The data considered indicate that antioxidants may be used to directionally change the transcriptional response of the cell to NO.

[Identification of genes induced to chondrocytes by nitric oxide]. / Poisk i identifikatsiia genov, indutsiruemykh v khondrotsitakh pod deistviem okisi azota.

Nitric oxide (NO) acts as a short-lived paracrine factor and selectively activates transcription of certain genes. The spectrum of inducible genes was studied in primary chondrocytes. A cDNA library was obtained by subtraction hybridization with RNAs isolated from rabbit chondrocytes before and after treatment with nitrosoglutathione, an NO-generating agent. Some of the cloned cDNAs were homologous to known mammalian genes and human EST. NO-dependent transcriptional activation was demonstrated for the stromelysin 1 and cyclooxygenase 2 genes and, for the first time, for mcl1 coding for an apoptosis suppressor.

Reactive oxygen species and regulation of gene expression.

In eucaryotic cells reactive oxygen species (ROS) are produced in the reactions catalyzed by NAD(P)H oxidase and by some other specialized oxidases and also as an inevitable by-product of many redox reactions. Intracellular ROS synthesis is regulated by various hormones, cytokines, and growth factors. An increase in the ROS levels above a certain threshold (so-called oxidative stress) is accompanied by processes that are harmful for cell survival, such as lipid peroxidation and oxidative modification of proteins and nucleic acids. However, at low concentrations ROS act as secondary messengers responsible for a signal transduction from extracellular signaling molecules and their membrane receptors to the intracellular regulatory systems which control gene expression. Cellular transcriptional response to ROS is mediated mainly by activation of MAP protein kinases and submitted transcription factors AP-1, ATF, and NF-kappaB. A number of specific genes is also induced under hypoxia, i.e., under conditions opposite to oxidative stress. Cellular transcriptional response on hypoxia is mediated by activation of transcription factors HIF-1 and AP-1. Together with ROS, nitric oxide fulfills the role of a mobile and highly reactive redox-sensitive signaling molecule. Chemical reactions of NO with the superoxide anion and with other free radicals leads to production of highly reactive intermediates. Depending on the ratio of their intracellular concentrations, NO and ROS can either enhance or attenuate their reciprocal effects on cells.

Two pathways of the nitric oxide-induced cytotoxycal action.

Nitric oxide is a diffusible messenger with multiple biological functions. We show here that NO-generating compound, S-nitrosoglutathione (GSNO) induces apoptosis in human chondrocytes and causes necrosis-like cell death in human epithelial CaOv cell line. Pretreatment of chondrocytes with low-dose GSNO or with gamma-interferon enhances their tolerance to the second high-concentration GSNO exposure. On the contrary, in CaOv cells low-dose GSNO pretreatment diminishes the resistance and increases cytolysis at the second GSNO exposure. We conclude that human chondrocytes possess specific and inducible mechanism preventing cell killing by nitric oxide.

Alternative processing of the tryptophanyl-tRNA synthetase mRNA from interferon-treated human cells.

We have analysed the structure of mRNA isoforms of the human gene encoding tryptophanyl-tRNA synthetase (Trp-tRNA synthetase) expressed in the epithelial CaOv cells and MT-4 lymphocytes. The Trp-tRNA synthetase gene is induced by interferon-gamma in both lines and, in MT-4 lymphocytes, also by interferon-alpha. Four Trp-tRNA synthetase mRNA isoforms have different combinations of the first exons IA, IB and II. Two transcription initiation sites (P1 and P2) were detected 90 bp from each other. Processing of the primary transcript initiated from the P1 start site generates the mRNA isoform where exon IA joins to exon II. The other three isoforms are produced by alternative splicing of the primary transcript produced from the P2 start site. Isoform 2 has a 3'-end fragment of exon IA joined to exon II. Isoform 3 contains exons IA and IB. Isoform 4 contains exon IA and exon III and lacks exon II encoding the N-terminus of the Trp-tRNA synthetase. Therefore, the two primary transcripts of the Trp-tRNA synthetase gene differ only in the 5' flank sequence between P1 and P2, and this fragment regulates their processing. Both interferon-alpha and interferon-gamma induce exon IA-containing and exon IB-containing isoforms of the Trp-tRNA synthetase mRNA.

[Statistical analysis of enhancers of a series of eukaryotic genes]. / Statisticheskii analiz énkhanserov riada éukarioticheskikh genov.

Consensus sequences of transcription factors IRF, E12/E47, MBF1 and MyoD1 were represented as combination of the short oligonucleotide patterns ("enhansones"). The ad hoc computer program was employed for analysis of distribution and localization of these patterns on the sequences of interferons, immunoglobulins, metallothioneins and muscle-specific genes. The set of patterns was strictly specific for each gene family. In enhancer regions of almost all genes from mentioned families patterns were gathered in compact groups surrounding the operator sites. This empirical rule is true for genes where two conditions were fulfilled. (1) Operator site consists of tandem or inverted repeat of the short oligonucleotide motif, for instance GAAA-GAA in interferons or CCA-TGG in immunoglobulins. (2) Multiplication of the operator site leads to increase of enhancer activity because of cooperative interactions between transcriptions factors.

Binding of proteins of HeLa S3 cell extract to oligonucleotides containing the consensus interferon-response sequence (IRS) and to the IRS-containing fragment of the human c-myc gene.

The human c-myc proto-oncogene was recently found to contain a regulatory sequence similar to the consensus interferon-response sequence (IRS) of interferon-activating genes. Binding of regulatory protein(s) to this sequence of cloned fragment of c-myc, lacking the main part of 5'-nontranscribing region, regulates in vitro transcription from I1/I2 initiation sites located in the first intron of the gene. Here, we have shown that HeLa S3 nuclear extract contains different protein factors, at least two, that bind preferentially to the IRS sequence of either the c-myc gene or the interferon-dependent 6-16 gene. Moreover, each of these factors 'cross-binds' to the region of the other gene, although affinity of this interaction is lower. Binding constants of these proteins to oligonucleotide fragments of c-myc and 6-16 genes were determined. In vitro transcription of the human full-length c-myc gene (i.e. the gene containing the complete 5'-noncoding region) initiated from I1/I2 sites, that is controlled by the IRS region, was demonstrated to be blocked. A possible physiological role for the mechanisms described is discussed.

[Nuclear protein factors binding with specific DNA sequences]. / Iadernye belkovye faktory, vzaimodeistvuiushchie so spetsificheskimi posledovatel'nostiami DNK.

Primary structure of thousands of genes is being determined in many laboratories worldwide. While it is relatively easy to analyse the coding region(s) of genes, it is usually hard to understand what is located in non-coding regions. A non-coding region may contain very valuable information about the mode of functioning of a given gene, e. g. promoters, enhancers, silencers etc. The regulatory function of these sequences is determined by their interaction with certain sequence-specific proteins, i. e. the presence of a certain DNA sequence in a non-coding region of a gene may suggest that the gene is regulated by a specific protein factor. This minireview summarizes recent data on most known eukaryotic sequence-specific DNA-binding protein factors, including their origin, DNA consensus, and their role in expression of corresponding genes.