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.

Imatinib mesilate-induced phosphatidylinositol 3-kinase signalling and improved survival in insulin-producing cells: role of Src homology 2-containing inositol 5'-phosphatase interaction with c-Abl.

AIMS/HYPOTHESIS: It is not clear how small tyrosine kinase inhibitors, such as imatinib mesilate, protect against diabetes and beta cell death. The aim of this study was to determine whether imatinib, as compared with the non-cAbl-inhibitor sunitinib, affects pro-survival signalling events in the phosphatidylinositol 3-kinase (PI3K) pathway. METHODS: Human EndoC-ßH1 cells, murine beta TC-6 cells and human pancreatic islets were used for immunoblot analysis of insulin receptor substrate (IRS)-1, Akt and extracellular signal-regulated kinase (ERK) phosphorylation. Phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] plasma membrane concentrations were assessed in EndoC-ßH1 and MIN6 cells using evanescent wave microscopy. Src homology 2-containing inositol 5'-phosphatase 2 (SHIP2) tyrosine phosphorylation and phosphatase and tensin homologue deleted on chromosome 10 (PTEN) serine phosphorylation, as well as c-Abl co-localisation with SHIP2, were studied in HEK293 and EndoC-ßH1 cells by immunoprecipitation and immunoblot analysis. Gene expression was assessed using RT-PCR. Cell viability was measured using vital staining. RESULTS: Imatinib stimulated ERK(thr202/tyr204) phosphorylation in a c-Abl-dependent manner. Imatinib, but not sunitinib, also stimulated IRS-1(tyr612), Akt(ser473) and Akt(thr308) phosphorylation. This effect was paralleled by oscillatory bursts in plasma membrane PI(3,4,5)P3 levels. Wortmannin induced a decrease in PI(3,4,5)P3 levels, which was slower in imatinib-treated cells than in control cells, indicating an effect on PI(3,4,5)P3-degrading enzymes. In line with this, imatinib decreased the phosphorylation of SHIP2 but not of PTEN. c-Abl co-immunoprecipitated with SHIP2 and its binding to SHIP2 was largely reduced by imatinib but not by sunitinib. Imatinib increased total ß-catenin levels and cell viability, whereas sunitinib exerted negative effects on cell viability. CONCLUSIONS/INTERPRETATION: Imatinib inhibition of c-Abl in beta cells decreases SHIP2 activity, which results in enhanced signalling downstream of PI3 kinase.

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.

2'-adenylated derivatives of Ap3A activate RNase L.

The exact physiological function of Ap3A (A5'ppp5"A, 5'5" diadenosine triphosphate) remains unclear. Previously we have demonstrated that the human p46 2-5A synthetase (OAS1) efficiently utilises Ap3A as an acceptor substrate for oligoadenylate synthesis. Here we show that Ap3A(2'p5'A)n oligonucleotides can activate the 2-5A-dependent RNase (RNase L), when the number of 2',5'-linked adenyl residues is two or more. Under the experimental conditions applied the half-maximal activation (AC50) of RNase L for 2'-adenylated Ap3A derivatives was determined to be in nanomolar range while the AC50 for 2-5A3 was 0.4 nM. The Ap3A(2'p5'A)n oligonucleotides are thus less effective in activating RNase L than 2-5A. We also investigated the occurrence of 2'-adenylated Ap3A in interferon and poly(I).poly(C)-treated HeLa cells. In purified trichloroacetic acid-soluble extracts about 40% of RNase L-activating material is resistant to phosphatase treatment, whereas the removal of 5'-terminal phosphates greatly reduces the activating properties of 2-5A. We assume that this activity at least partly may be associated with the presence of 2'-adenylated ApnA derivatives with blocked 5'-terminal phosphates.

Ap3A and Ap4A are primers for oligoadenylate synthesis catalyzed by interferon-inducible 2-5A synthetase.

The biological role of Ap3A synthesized in cells by tryptophanyl-tRNA synthetase (WRS) is unknown. Previously we have demonstrated that the cellular level of Ap3A significantly increases after interferon treatment. Here we show that the human 46 kDa 2-5A synthetase efficiently utilizes Ap3A as a primer for oligoadenylate synthesis. The Km for Ap3A is several-fold lower than for Ap4A and 100-fold lower than for ATP. This implies that Ap3A might be a natural primer for the 2'-adenylation reaction catalysed by 2-5A synthetase. Since WRS and 2-5A synthetase are both interferon-inducible proteins, a new link between two interferon-dependent enzymes is established.

Interferons induce accumulation of diadenosine triphosphate (Ap3A) in human cultured cells.

After incubation of human monocytes J96 and human myeloid leukemia HL60 cells with interferons (IFN) alpha or gamma, the Ap3A concentration considerably increases in parallel with accumulation of tryptophanyl-tRNA synthetase (TrpRS, EC 6.1.1.2). The Ap3A formation in response to IFNs is catalysed by an excessive amount of TrpRS. Although the Ap3A function still remains unknown, its accumulation may imply the Ap3A involvement in the IFN-signalling pathway.

The mechanisms of the cyclic AMP-dependent regulation of the enzymes of the 2',5'-oligoadenylate system.

The cAMP-dependent induction of 2,5-oligoadenylate (2-5A) synthetase and cAMP-dependent inhibition of 2-5A phosphodiesterase are shown. Variations in activities of cAMP-dependent protein kinase and the enzymes of 2-5A metabolism in the cells deepening into the resting state were found to be compatible with the above finding. A scheme of coordinated action of cAMP and 2-5A is proposed.

Regulation of 2',5'-oligo(A) synthetase activity in theophylline-treated NIH 3T3 cells.

Sequential treatment of NIH 3T3 cells with theophylline and actinomycin D results in an enhancement of 2',5'-oligo(A) synthetase activity 2-3-fold exceeding that induced by treatment with theophylline only. This 'superinduction' phenomenon suggests the existence of a negative control of the enzyme that involves a labile, transcription-dependent factor.

Induction of vascular endothelial growth factor by nitric oxide in cultured human articular chondrocytes.

We investigated the role of nitric oxide (NO) in the control of vascular endothelial growth factor A (VEGF) gene expression in cultured human articular chondrocytes. Cell treatment with the NO-generating compound nitrosoglutathione (GSNO) caused a significant accumulation of 4.4 kb VEGF mRNA, a major VEGF mRNA isoform expressing in chondrocytes. This is the first demonstration that NO can induce VEGF mRNA expression in chondrocytes. VEGF mRNA level was not affected in cells exposed to dibutyryl cGMP, a non-hydrolyzable analog of cGMP, suggesting that the cGMP system is not involved in NO-dependent transcriptional activation of VEGF gene. The GSNO-stimulated induction of VEGF mRNA was slightly attenuated by MAP protein kinase inhibitors PD98058 and SB203580, but was completely blocked in cells incubated with GSNO in the presence of catalase and superoxide dismutase, enzymes scavenging reactive oxygen species (ROS), or in the presence of thiol-containing antioxidants, N-acetyl cysteine and reduced glutathione. These results suggest that in articular chondrocytes the GSNO-induced VEGF gene transcriptional activation is dependent on endogenous ROS production and oxidative thiol modifications.

Regulation of 2-5 A phosphodiesterase activity by cAMP-dependent phosphorylation: mechanism and biological role.

The results of the present study permit the explanation of one of the mechanisms of the interconnection between the regulatory systems of cAMP and 2-5A. cAMP-dependent regulation of 2'-PDE was found to involve phosphorylation of the specific protein inhibitor. Originally, a similar way of regulation of the enzyme activity was discovered for protein phosphatase I. This enzyme has a specific protein inhibitor type 1, which is phosphorylated by cAMP-dependent protein kinase and is activated by phosphorylation (18). It is interesting that the molecular weights of 2'-PDE protein inhibitor and of the inhibitor type 1 of protein phosphatase I are essentially the same. There is also a certain similarity between the above described mechanism and phosphorylation of the regulatory subunit of cAMP-dependent protein kinase type 2. The regulatory subunit can also act as a protein inhibitor of the enzyme and change its properties as a result of phosphorylation (19). The results obtained permit as well a more detailed explanation for cAMP-dependent inhibition of cell proliferation. Evidently, cAMP elevation causes activation of cAMP-dependent phosphorylation which, in turn, leads to the induction of 2-5A synthetase and inhibition of 2'-PDE. As a result of variations in the activities of these enzymes, the level of 2-5A rises. The latter brings about the changes characteristic of the resting state. They involve activation of RNase L and the succeeding acceleration of RNA hydrolysis, inhibition of protein synthesis and cell proliferation. The resting state is characterized by a rapid turnover of macromolecules due to their intensive degradation (20). The above described scheme suggested that the rapid turnover of RNA during inhibition of cell proliferation can be partially accounted for by activation of 2-5A-dependent RNase L. Thus, it can be thought that at least one of the mechanisms of the antiproliferative effect of cAMP-dependent phosphorylation of proteins involves cAMP-dependent elevation of intracellular 2-5A. Evidently, a number of properties of the resting cells are determined by the elevated content of 2-5A. Finally, it should be noted that the interconnection between the systems of cAMP and 2-5A is a multiple process. We have earlier demonstrated (12) that 2-5A activates cAMP phosphodiesterase in NIH 3T3 cell homogenates. These data suggest that the mutual regulation of cAMP and 2-5A levels involves the negative feedback mechanism (Fig. 8).

A study on the relationship between the interferon enzyme system and the system of cyclic nucleotide metabolism.

In order to study interrelationships between the components of the interferon enzyme system and the cyclic AMP system, NIH 3T3 cells were incubated in the presence of theophylline or adrenaline that cause a rise of intracellular cAMP, respectively, through inhibition of phosphodiesterase of cAMP and activation of adenylate cyclase. In doses that caused a transient, 2-to 3-fold elevation of the cAMP level, theophylline and adrenaline elicited about 2.5-fold elevation of 2',5'-oligoadenylate synthetase (2-5A synthetase) activity. This increase could be prevented by actinomycin D. This suggests that the elevation of the enzyme activity in the cells was due to a transcription-dependent induction process. Theophylline and adrenaline treatment of the cell cultures also led to a 2-to 3-fold fall of the activity of the phosphodiesterase of 2',5'-oligoadenylate (2'-phosphodiesterase). This effect of adrenaline was prevented by propanolol but not by actinomycin D. In the case of adrenaline, the fall of 2'-phosphodiesterase activity was accompanied by at least 5-fold increase in the enzyme activity which did not occur if actinomycin D was present in the culture. Similarities and differences between these effects and those induced by interferon are discussed. It is concluded that cAMP is an important regulator of the enzyme system of the 2',5'-oligoadenylate metabolism. 2',5'-Oligoadenylate, in turn, was found to act on the activity of phosphodiesterase of cyclic AMP. The cAMP phosphodiesterase activity in the NIH 3T3 cell lysates was activated 2- to 2.5-fold at physiological concentrations (10(-9) to 10(-7) M) of both the phosphorylated form of oligoisoadenylate, ppp(5'A2'p)n5'A2'OH, and the dephosphorylated form, HO(5'A2'p)25'A2'OH. The phosphorylated form of oligoisoadenylate also activated partially purified preparations of cAMP phosphodiesterase. The data obtained in this study allow us to consider cAMP and 2',5'-oligoadenylate as the key metabolites that may be used in the cells to form a complex, interconnected, multifunctional circuit that involves the interferon enzyme system and the system of cyclic AMP metabolism and governs essential cell functions, as regulation of RNA metabolism and protein synthesis, cell growth and differentiation.

[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.

[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.

[Study of 2'-phosphodiesterase activity in cultured NIH 3T3 cells during activation of cAMP-dependent phosphorylation]. / Issledovanie aktivnosti 2'-fosfodiésterazy v kul'ture kletok NIH 3T3 pri aktivatsii cAMP-zavisimogo fosforilirovaniia.

A rapid and transient decrease in 2'-phosphodiesterase activity in NIH 3T3 mouse cells was observed after adrenaline addition. The decrease of activity was accompanied by an elevation of intracellular cAMP level. The 2'-phosphodiesterase activity changed similarly when cells sink deeper into the resting state. In the latter case, the fall of the enzyme activity was correlated with elevation of the activity of cAMP-dependent proteinkinase and, moreover, a considerable increase of the intracellular level of 2',5'-oligoadenylate was observed. Phosphorylation of proteins by cAMP-dependent proteinkinase in the cell lysate also produced a pronounced drop of 2'-phosphodiesterase activity. Exogenous 2',5'-oligo (A) treatment of the cells resulted in the rise of 2'-phosphodiesterase activity; actinomycin D prevented this effect. The data presented suggest the involvement of two different mechanisms in regulation of 2'-phosphodiesterase activity: cAMP-dependent phosphorylation and induction of 2'-phosphodiesterase by 2',5'-oligoadenylate.

[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.

[Effect of modulation of the production of interferon by L-929 cells treated with theophylline]. / Effekt moduliatsii produktsii interferona kletkami L-929 pri obrabotke teofillinom.

The specific inhibitor of cAMP phosphodiesterase theophylline has been shown to evoke in L929 cells 2.3-fold induction of 2-5A-synthetase activity and 3.5-fold superinduction of the same enzyme activity while acting in combination with actinomycin D. It has been shown also that temporal coincidence of 2-5A-synthetase induction with the active period of interferon production resulted in 8-16 times decrease in the level of interferon production. The result was supported by the experiments of superinduced cells (containing the high stable level of 2-5A-synthetase) fusion with monolayer of poly(I).poly(C)-induced L929 cells (taken at the start of interferon production). In this case the production of interferon was dramatically decreased in comparison with the control. Possible role of 2-5A-synthetase in regulation of interferon production is discussed.