NF-κB-repressing factor phosphorylation regulates transcription elongation via its interactions with 5'→3' exoribonuclease 2 and negative elongation factor.

NF-κB-repressing factor (NKRF) inhibits transcription elongation by binding to specific sequences in target promoters. Stimuli such as IL-1 have been shown to overcome this inhibitory action and enable the resumption of transcription elongation machinery by an unknown mechanism. Using mass spectrometry and in vitro phosphorylation analyses, we demonstrate that NKRF is phosphorylated within 3 different domains in unstimulated HeLa cells. Phosphoamino acid mapping and mutation analysis of NKRF further suggest that only Ser phosphorylation within aa 421-429 is regulated by IL-1 stimulation. In copurification studies, aa 421-429 is required for interactions between NKRF, 5'→3' exoribonuclease 2 (XRN2) and the negative elongation factor (NELF)-E in HeLa cells. Chromatin immunoprecipitation experiments further show that IL-1 stimulation leads to decrease in NKRF aa 421-429 phosphorylation and dissociation of NELF-E and XRN2 by concomitant resumption of transcription elongation of a synthetic reporter or the endogenous NKRF target gene, IL-8. Together, NKRF phosphorylation modulates promoter-proximal transcription elongation of NF-κB/NKRF-regulated genes via direct interactions with elongation complex in response to specific stimuli.

Differential effects of multiplicity of infection on Helicobacter pylori-induced signaling pathways and interleukin-8 gene transcription.

Interleukin-8 (IL-8) plays a central role in the pathogenesis of Helicobacter pylori infection. We used four different H. pylori strains isolated from patients with gastritis or duodenal ulcer disease to examine their differential effects on signaling pathways and IL-8 gene response in gastric epithelial cells. IL-8 mRNA level is elevated in response to high (100) multiplicity of infection (MOI) independent of cagA, vacA, and dupA gene characteristics. By lower MOIs (1 or 10), only cagA ( + ) strains significantly induce IL-8 gene expression. This is based on differential regulation of IL-8 promoter activity. Analysis of intracellular signaling pathways indicates that H. pylori clinical isolates induce IL-8 gene transcription through NF-κB p65, but by a MOI-dependent differential activation of MAPK pathways. Thus, the major virulence factors of H. pylori CagA, VacA, and DupA might play a minor role in the level of IL-8 gene response to a high bacterial load.

Threonine 66 in the death domain of IRAK-1 is critical for interaction with signaling molecules but is not a target site for autophosphorylation.

Ligand binding in the TLR/IL-1R family results in the transient formation of an intracellular signaling complex, which contains, amongst others, the serine/threonine-specific kinase IL-1R-associated kinase 1 (IRAK-1). Concomitantly, the kinase function of IRAK-1 becomes activated, resulting in massive autophosphorylation and finally in the dissociation of the initially constituted signaling complex. The death domain (DD) of IRAK-1 mediates the interaction with other molecules of the signaling complex, e.g., the adaptor MyD88, the silencer Tollip, and the activator kinase IRAK-4. The conserved threonine at position 66 (T66), located within the DD, is a putative autophosphorylation target site. Here, we provide evidence that T66 critically impacts the secondary structure of the IRAK-1 DD. Thereby, it ensures the transient manner of interactions between IRAK-1 and the other signaling molecules. This essential role, however, is not regulated by phosphorylation of T66 itself.

NF-kappaB-repressing factor inhibits elongation of human immunodeficiency virus type 1 transcription by DRB sensitivity-inducing factor.

Human immunodeficiency virus type 1 (HIV-1) is able to establish a latent infection during which the integrated provirus remains transcriptionally silent. In response to specific stimuli, the HIV-1 long terminal repeat (LTR) is highly activated, enhancing both transcriptional initiation and elongation. Here, we have identified a specific binding sequence of the nuclear NF-kappaB-repressing factor (NRF) within the HIV-1 LTR. The aim of this work was to define the role of NRF in regulating the LTR. Our data show that the endogenous NRF is required for transcriptional activation of the HIV-1 LTR in stimulated cells. In unstimulated cells, however, NRF inhibits HIV-1 LTR activity at the level of transcription elongation. Binding of NRF to the LTR in unstimulated cells prevents recruitment of elongation factor DRB sensitivity-inducing factor and formation of processive elongation complexes by hyperphosphorylated RNA polymerase II. Our data suggest that NRF interrupts the regulatory coupling of LTR binding factors and transcription elongation events. This inhibitory mechanism might contribute to transcriptional quiescence of integrated HIV-1 provirus.

Transendoscopic ultrasound in ventricular lesions.

BACKGROUND: After transendoscopic sonocatheters had been tested in the laboratory for imaging characteristics and practicability, clinical application was studied with special reference to imaging and navigation capabilities, practicability, safety, and preliminary indications. METHODS: Intraoperative ENS images prepared during surgery on 75 selected patients between 1996 and 2005 were examined. There were 35 female and 40 male patients, and their mean age was 42 years (range, 2-69 years). Within this series, there were 28 cases of ventricular lesions (ventricular hematomas, tumors, and colloid cyst included, 35 cases) with different diagnosis. In most cases, Aloka sono equipment (Aloka Deutschland, Düsseldorf, Germany) was used because equipment supplied by this company had yielded superior imaging results in the laboratory. This work with patients differed from the laboratory work in that 2 sizes (diameters) of catheters were used: 6-F catheters for block-shaft endoscopes and 8-F for hollow-shaft endoscopes. RESULTS: Imaging: In clinical use, the sonocatheter has superior imaging and navigation abilities to those seen in anatomical laboratory work. Real-time and online characteristics represent changes such as shifting, pulsation, CSF flow, blood flow, and changes in size and form of structures. When confronted with clinical problems, this technique still has some limitations such as short penetration depth of 3-cm radius and lack of scanning anterior to the endoscope. Navigation: The scan is radial 360 degrees and in an orthogonal plane to the axis of the endoscope. At the tip of the endoscope it delivers an image that looks geometrically like a "brain radar." Because of its real-time characteristic, ENS has a navigation capacity that markedly differs from usual neuronavigation, but is intuitively usable. Endoneurosonography was applied in 8 hydrocephali, 3 colloid cysts, 5 intraventricular hematomas, 1 septostomy, 11 ETVs, 2 cystostomies, 4 multiple cysts, and 1 tumor biopsy cases. Three illustrative cases are presented. CONCLUSION: Endoneurosonography is a tool for intraoperative real-time and online high-resolution imaging, and neuronavigation of endoscopes with a working channel at least 2 mm in diameter; it also has application in a wide variety of ventricular lesions. Endoneurosonography is limited by short penetration depth and not scanning ahead to the endoscope anteriorly.

Distinct domains of AU-rich elements exert different functions in mRNA destabilization and stabilization by p38 mitogen-activated protein kinase or HuR.

AU-rich elements (AREs) control the expression of numerous genes by accelerating the decay of their mRNAs. Rapid decay and deadenylation of beta-globin mRNA containing AU-rich 3' untranslated regions of the chemoattractant cytokine interleukin-8 (IL-8) are strongly attenuated by activating the p38 mitogen-activated protein (MAP) kinase/MAP kinase-activated protein kinase 2 (MK2) pathway. Further evidence for a crucial role of the poly(A) tail is provided by the loss of destabilization and kinase-induced stabilization in ARE RNAs expressed as nonadenylated forms by introducing a histone stem-loop sequence. The minimal regulatory element in the IL-8 mRNA is located in a 60-nucleotide evolutionarily conserved sequence with a structurally and functionally bipartite character: a core domain with four AUUUA motifs and limited destabilizing function on its own and an auxiliary domain that markedly enhances destabilization exerted by the core domain and thus is essential for the rapid removal of RNA targets. A similar bipartite structure and function are observed for the granulocyte-macrophage colony-stimulating factor (GM-CSF) ARE. Stabilization in response to p38/MK2 activation is seen with the core domain alone and also after mutation of the AUUUA motifs in the complete IL-8 ARE. Stabilization by ARE binding protein HuR requires different sequence elements. Binding but no stabilization is observed with the IL-8 ARE. Responsiveness to HuR is gained by exchanging the auxiliary domain of the IL-8 ARE with that of GM-CSF or with a domain of the c-fos ARE, which results in even stronger responsiveness. These results show that distinct ARE domains differ in function with regard to destabilization, stabilization by p38/MK2 activation, and stabilization by HuR.

Quantification of the carbapenem antibiotic ertapenem in human plasma by a validated liquid chromatography-mass spectrometry method.

BACKGROUND: Ertapenem (Invanz) is a newly developed carbapenem beta-lactam antibiotic. LC-MS is the method of choice for therapeutic drug monitoring (TDM) of a variety of drugs including antibiotics. No validated LC-MS method for ertapenem quantification is described in the literature so far. METHODS: A rapid and robust LC-MS quantification method for ertapenem was developed and validated for clinical routine application in plasma samples. After immediate stabilisation with MES buffer (pH 6.5), samples were prepared for LC-MS analysis using simple protein precipitation. LC-MS coupling was realised by the use of a Phenomenex Synergi 4micro Polar-RP A80 Mercury LC column (10 x 2.0 mm) in combination with a Single-MS (Agilent 1100 LC-MSD SL) operating in negative selected ion monitoring (SIM) detection mode with ceftazidime as internal standard for adequate selective and sensitive analysis. RESULTS: LC-MS method validation by means of determination of limit of detection (LOD 0.1 microg mL-1), lower limit of quantification (LLOQ 1 microg mL-1), linearity (0.1-50 microg mL-1), recovery (> 90%), intra- and inter-day precision (RSD < 10%), accuracy (> 90%), inter-subject variability (< 10% at LLOQ), drug stability in plasma (> 3 months) and in post-extracted samples (> 99% for 24 h), and matrix effects (process efficiency > 90%) showed excellent performance parameters considering Guidance for Industry - Bioanalytical Method Validation. CONCLUSION: This method is perfectly appropriate for routine quantification of ertapenem and possibly other polar carbapenem beta-lactam antibiotics.

Sirolimus/cyclosporine/tacrolimus interactions on bile flow and biliary excretion of immunosuppressants in a subchronic bile fistula rat model.

The new immunosuppressive agent sirolimus generally is combined in transplant patients with cyclosporine and tacrolimus which both exhibit cholestatic effects. Nothing is known about possible cholestatic effects of these combinations which might be important for biliary excretion of endogenous compounds as well as of immunosuppressants. Rats were daily treated with sirolimus (1 mg kg(-1) p.o.), cyclosporine (10 mg kg(-1) i.p.), tacrolimus (1 mg kg(-1) i.p.), or a combination of sirolimus with cyclosporine or tacrolimus. After 14 days a bile fistula was installed to investigate the effects of the immunosuppressants and their combinations on bile flow and on biliary excretion of bile salts, cholesterol, and immunosuppressants. Cyclosporine as well as tacrolimus reduced bile flow (-22%; -18%), biliary excretion of bile salts (-15%;-36%) and cholesterol (-15%; -47%). Sirolimus decreased bile flow by 10%, but had no effect on cholesterol or bile salt excretion. Combination of sirolimus/cyclosporine decreased bile flow and biliary bile salt excretion to the same extent as cyclosporine alone, but led to a 2 fold increase of biliary cholesterol excretion. Combination of sirolimus/tacrolimus reduced bile flow only by 7.5% and did not change biliary bile salt and cholesterol excretion. Sirolimus enhanced blood concentrations of cyclosporine (+40%) and tacrolimus (+57%). Sirolimus blood concentration was increased by cyclosporine (+400%), but was not affected by tacrolimus. We conclude that a combination of sirolimus/tacrolimus could be the better alternative to the cotreatment of sirolimus/cyclosporine in cholestatic patients and in those facing difficulties in reaching therapeutic ranges of sirolimus blood concentration.

[Endosonographic anatomy of the ventricular system]. / A kamrarendszer endoszonográfiás anatómiája.

A preclinical cadaver study was performed to test a transendoscopic sonographic probe for neurosurgery. In 25 fresh post-mortem adult human cadaver with a total of 39 endo-sonographic dissections in the ventricular system were carried out. A sonograph with an outer diameter of 6 F was used and radial sonograms were made by a real-time image technique. First results showed precise imaging, comparable to a CT in a neighbouring area of 3 cm. In this publication, the authors describe the endo-neurosonographic anatomy of the ventricular system. The sonographic probe was advanced through the working canal of a ventriculoscope, then the endoscopic and sonographic imaging were compared. Results were documented by parallel sonographic and endoscopic photo and video recordings. Based on the authors experience, it is revealed that the additional sonographic view could also be used as a navigation tool.

JKTBP1 is involved in stabilization and IRES-dependent translation of NRF mRNAs by binding to 5' and 3' untranslated regions.

Heterogeneous nuclear ribonucleoprotein D-like protein (JKTBP) 1 was implicated in cap-independent translation by binding to the internal ribosome entry site in the 5' untranslated region (UTR) of NF-κB-repressing factor (NRF). Two different NRF mRNAs have been identified so far, both sharing the common 5' internal ribosome entry site but having different length of 3' UTRs. Here, we used a series of DNA and RNA luciferase reporter constructs comprising 5', 3' or both NRF UTRs to study the effect of JKTBP1 on translation of NRF mRNA variants. The results indicate that JKTBP1 regulates the level of NRF protein expression by binding to both NRF 5' and 3' UTRs. Using successive deletion and point mutations as well as RNA binding studies, we define two distinct JKTBP1 binding elements in NRF 5' and 3' UTRs. Furthermore, JKTBP1 requires two distinct RNA binding domains to interact with NRF UTRs and a short C-terminal region for its effect on NRF expression. Together, our study shows that JKTBP1 contributes to NRF protein expression via two disparate mechanisms: mRNA stabilization and cap-independent translation. By binding to 5' UTR, JKTBP1 increases the internal translation initiation in both NRF mRNA variants, whereas its binding to 3' UTR elevated primarily the stability of the major NRF mRNA. Thus, JKTBP1 is a key regulatory factor linking two pivotal control mechanisms of NRF gene expression: the cap-independent translation initiation and mRNA stabilization.

Different curcuminoids inhibit T-lymphocyte proliferation independently of their radical scavenging activities.

PURPOSE: We investigated the inhibitory effects of curcumin, curcumin derivatives and degradation products on OKT3-induced human peripheral blood mononuclear cell (PBMC) proliferation and the role of their radical scavenging activity. METHODS: OKT3-induced human PBMC proliferation was determined by measuring 3H-thymidine incorporation. Radical scavenging activity was evaluated by using an in vitro DPPH assay. RESULTS: OKT3-induced PBMC proliferation was inhibited by curcumin, isocurcumin, bisdesmethoxy-, diacetyl-, tetrahydro-, hexahydro-, and octahydrocurcumin as well as by vanillin, ferulic acid, and dihydroferulic acid with IC50-values of 2.8, 2.8, 6.4, 1.0, 25, 38, 82, 729, 457, and >1,000 microM, respectively. The investigated substances with the strongest effect on radical scavenging were tetrahydro-, hexahydro-, and octahydrocurcumin with IC50 values of 10.0, 11.7, and 12.3 microM, respectively. IC50-values of dihydroferulic acid, ferulic acid, and curcumin were 19.5, 37, and 40 microM. The substances with the lowest radical scavenging activities were vanillin, isocurcumin, diacetylcurcumin, and bisdesmethoxycurcumin with IC50 values higher than 100 microM each. CONCLUSIONS: Curcuminoid-induced inhibition of OKT3-induced PBMC proliferation depends on the number of carbon atoms and double bonds of the 1,6-heptadiene-3,5-dione structure as well as on the phenolic ring substitutes of the curcuminoids but is not correlated to their respective radical scavenging activity.

c-Jun controls histone modifications, NF-kappaB recruitment, and RNA polymerase II function to activate the ccl2 gene.

Interleukin-1 (IL-1)-induced mRNA expression of ccl2 (also called MCP-1), a prototypic highly regulated inflammatory gene, is severely suppressed in cells lacking c-Jun or Jun N-terminal protein kinase 1 (JNK1)/JNK2 genes and is only partially restored in cells expressing a c-Jun(SS63/73AA) mutant protein. We used chromatin immunoprecipitation to identify three c-Jun-binding sites located in the far 5' region close to the transcriptional start site and in the far 3' region of murine and human ccl2 genes. Mutational analysis revealed that the latter two sites contribute to ccl2 transcription in response to the presence of IL-1 or of ectopically expressed c-Jun-ATF-2 dimers. Further experiments comparing wild-type and c-Jun-deficient cells revealed that c-Jun regulates Ser10 phosphorylation of histone H3, acetylation of histones H3 and H4, and recruitment of histone deacetylase 3 (HDAC3), NF-kappaB subunits, and RNA polymerase II across the ccl2 locus. c-Jun also coimmunoprecipitated with p65 NF-kappaB and HDAC3. Based on DNA microarray analysis, c-Jun was required for full expression of 133 out of 162 IL-1-induced genes. For inflammatory genes, these data support the idea of an activator function of c-Jun that is executed by multiple mechanisms, including phosphorylation-dependent interaction with p65 NF-kappaB and HDAC3 at the level of chromatin.

The death domain of IRAK-1: an oligomerization domain mediating interactions with MyD88, Tollip, IRAK-1, and IRAK-4.

Ligand binding in the Toll-like/interleukin-1 receptor family results in the recruitment of an intracellular signaling complex. IRAK-1, which is centrally involved in this complex, is able to homo-oligomerize and to bind to Tollip and the adapters MyD88 and IRAK-4. The interactions of IRAK-1 with MyD88 or Tollip are mediated by the N-terminal part of IRAK-1, containing the death domain with the highly conserved threonine at position 66 (T66). Mutation of this amino acid into alanine or aspartic acid stabilized binding to MyD88, Tollip, and IRAK-4, allowing the definitive experimental proof, that all these interactions are mediated by the death domain of IRAK-1. Homo-oligomerization of IRAK-1, which is mediated by the death domain too, is not affected by mutation of T66. Finally, mutation of IRAK-1 at T66 not only allowed stable binding to the signaling adapters, but also enhanced its signaling capacity.

Endoneurosonography: technique and equipment, anatomy and imaging, and clinical application.

OBJECTIVE: To evaluate the usefulness of transendoscopic ultrasound in neurosurgery, we studied two new sonoprobes measuring 6 and 8 French in diameter in 20 fresh specimens. The application and indication are discussed in the first clinical series of 75 patients. METHODS: Sonocatheters (ALOKA, Meerbusch, Germany) 1.9 mm (6 French) and 2.4 mm (8 French) in diameter were introduced into the working channel of an endoscope. The preparations were done in nonfixed skulls in a surgical simulation-setting laboratory. Based on these experiences with imaging possibilities, intraoperative transendoscopic ultrasound was applied in 75 patients and a variety of lesions. It was used for imaging (41 patients), targeting (18 patients), and neuronavigation (16 patients) in neuroendoscopy. RESULTS: The sonoprobe adds a transverse scan at the tip of the probe to the anterior endoscopic view. This axial scan to the longitudinal axis of the endoscope is geometrically comparable with radar scanning. Three probes working with 10, 15, and 20 MHz were used, resulting in a short penetration with a radius of 3 cm. The orthogonal scanning plane had limitations, which were documented. We observed precise imaging of well known anatomic structures and, moreover, achieved an additional dimension in endoscopy. The axial scan presents the anatomic landmarks like a map at the tip of the endoscope where the endoscope is represented as a spot. The real-time imaging and representation of the tip of the endoscope showed a capacity for navigation. This preclinical study rectified clinical application. The real-time imaging of this technique showed the ability of the navigation of endoscopes to detect more overall movements, such as blood flow or change of ventricle size during endoscopy. The primary benefit in this first clinical series was witnessed in difficult endoscopy cases and complex lesions, but benefit was also observed in cases in which vision through the endoscope alone was obscured. The main limitation was the result of little penetration depth and lack of anterior scanning. CONCLUSION: Application of transendoscopic ultrasound is appropriate in neurosurgery. Training is necessary to understand the imaging and the geometry of scans because this technique does not scan along the axis of the endoscope. Further development to overcome the current limits of this technique and more clinical experience are needed.

Peptide-mediated disruption of NFkappaB/NRF interaction inhibits IL-8 gene activation by IL-1 or Helicobacter pylori.

Selective inhibition of proinflammatory chemokines such as IL-8 is an important approach to combat inflammatory and infection diseases. Previous studies suggested that interaction of transcription factors NFkappaB repressing factor (NRF) and NFkappaB play a crucial role in activation of IL-8 gene expression. In a search for a specific inhibitor of IL-8 expression, we applied tandem affinity purification to investigate interaction of NRF and NFkappaB p65 in cells. We identified a synthetic peptide corresponding to aa 223-238 of NRF interfering with binding of endogenous p65 to NRF. Furthermore, nucleofection experiments were established to introduce this inhibitory peptide into the nucleus of IL-1 stimulated human cervical and Helicobacter pylori infected gastric epithelial cells. Our data clearly show that the specific peptide disturbing NRF/NFkappaB interaction is able to significantly decrease endogenous IL-8 gene transcription in response to IL-1 or Helicobacter pylori infection. Thus, our study provides novel insights into NRF and NFkappaB interaction in vivo and may facilitate the design of new anti-IL-8 drugs based on novel strategies.

NRF IRES activity is mediated by RNA binding protein JKTBP1 and a 14-nt RNA element.

The mRNA of human NF-kappaB repressing factor (NRF) contains a long 5'-untranslated region (UTR) that directs ribosomes to the downstream start codon by a cap-independent mechanism. Comparison of the nucleotide (nt) sequences of human and mouse NRF mRNAs reveals a high degree of identity throughout a fragment of 150 nt proximal to the start codon. Here, we show that this region constitutes a minimal internal ribosome entry segment (IRES) module. Enzymatic RNA structure analysis reveals a secondary structure model of the NRF IRES module. Point mutation analysis of the module determines a short, 14-nt RNA element (nt 640-653) as a mediator of IRES function. Purification of IRES binding cellular proteins and subsequent ESI/MS/MS sequence analysis led to identification of the RNA-binding protein, JKTBP1. EMSA experiments show that JKTBP1 binds upstream to the 14-nt RNA element in the NRF IRES module (nt 579-639). Over-expression of JKTBP1 significantly enhances activity of the NRF IRES module in dicistronic constructs. Moreover, siRNA experiments demonstrate that down-regulation of endogenous JKTBP1 decreases NRF IRES activity and the level of endogenous NRF protein. The data of this study show that JKTBP1 and the 14-nt element act independently to mediate NRF IRES activity.

Direct and fast determination of antiretroviral drugs by automated online solid-phase extraction-liquid chromatography-tandem mass spectrometry in human plasma.

BACKGROUND: In this study antiretroviral drugs of the classes protease inhibitors (PI) and non-nucleoside reverse transcriptase inhibitors (NNRTI) were quantified for the first time directly in patient plasma samples by means of an automated and validated online solid-phase extraction-liquid chromatography-tandem mass spectrometry (XLC-MS/MS) method using the Symbiosis Pharma system (Spark Holland) for XLC coupled to an API 2000 for MS/MS analysis. METHODS: The PI drugs amprenavir, nelfinavir, indinavir, lopinavir, saquinavir, ritonavir, and atazanavir, and the NNRTI drugs nevirapine and efavirenz in real patient samples were analysed in a 25-microL sample volume, which was only diluted with 200 microL of H2O (containing 500 ng/mL of the internal standard reserpine) to minimise the matrix concentration and to add the internal standard. No additional tedious and time-consuming sample preparation steps such as protein precipitation, centrifugation, and pipetting were per-formed for sample clean-up. RESULTS: The high-throughput method developed allowed the simultaneous analysis of two samples (first analysis 6.6 min, subsequent analyses 3.3 min between injections) and has been validated in terms of the limit of detection (LOD, 2-70 ng/mL), lower limit of quantification (LLOQ, 78-156 ng/mL), linearity (R2, 0.9971-0.9989), linear concentration range (from LLOQ to 10,000 ng/mL), intra- and inter-day precision (< 13.5% at LLOQ, < 7.5% at high concentrations), proficiency testing accuracy (78-127%), laboratory internal accuracy (86-113%), recovery (60-110%), and drug stability (freeze-thaw, short-term temperature, long-term and post-preparative) and inter-subject variability. CONCLUSION: Although direct analysis of diluted plasma was performed, post-column experiments showed efficient matrix minimisation by the XLC-MS/MS technique, which is perfectly appropriate for routine therapeutic drug monitoring of HIV/AIDS patient samples.

Small interfering RNAs generated by recombinant dicer induce inflammatory gene expression independent from the TAK1-NFkappaB-MAPK signaling pathways.

Generation of mixtures of small interfering (si) RNAs by recombinant dicer avoids selection of efficient target sites within mRNAs but little is known about off-target effects of this approach. Using recombinant human dicer we generated siRNA mixtures (dsiRNA) directed against the protein kinase TAK1 and its subunit TAB1, important upstream molecules in the pathways activated by IL-1, TNF, and toll-like receptors (TLR). dsiRNA against TAK1 or TAB1 significantly suppressed their target proteins as well as TAK1-mediated activation of NFkappaB, p38 MAPK, and JNK, and of IL-8 transcription. However, microarray analysis of 136 endogenous inflammatory genes revealed that dsiRNA against TAB1 or TAK1 did not suppress IL-1 or TNF-induced genes but rather induced a broader range of 15 inflammatory genes as well as seven known interferon-response genes. The same genes were induced by dsiRNA directed against luciferase but not by a synthetic control siRNA molecule. Hence, our results show that complex mixtures of siRNA induce an inflammatory gene response that is independent from TAK1-mediated signal transduction. In the light of the increasing usage of enzymatically prepared libraries of siRNA these results provide important insight into potential off-target effects of this approach.

Injection of IL-12- and IL-18-encoding plasmids ameliorates the autoimmune pathology of MRL/Mp-Tnfrsf6lpr mice: synergistic effect on autoimmune symptoms.

IL-12 and IL-18 are mediators involved in the onset and progression of the autoimmune disease developing in MRL/Mp-Tnfrsf6(lpr) (lpr) mice, which display symptoms similar to the human systemic lupus erythematosus (SLE). The pathology is characterized by progressive lymphadenopathy and auto-antibody-mediated multiple organ failure, e.g. glomerulonephritis, or pneumonitis and a concomitant increase in serum levels for IFNgamma and tumor necrosis factor-alpha (TNFalpha). In this study, we intramuscularly injected lpr mice with plasmids encoding IL-12 and IL-18, either alone or in combination, in order to affect the development of the autoimmune disease. Five biweekly injections of the combined plasmids starting at 4-5 weeks of age diminished serum levels of TNFalpha and reduced the ability of lymphocytes from treated mice to produce IFNgamma in vitro. Injection of both plasmids synergistically attenuated the development of autoimmune syndromes, lymphoproliferation in secondary lymphoid organs, proteinuria and kidney damage, and pneumonitis. We conclude that IL-12 and IL-18 synergistically affect the pathogenesis of the T(h)1-dependent autoimmune syndrome of lpr mice and that approaches that target both IL-12 and IL-18 may be a therapeutic option in the treatment of autoimmune SLE.

The Yersinia enterocolitica effector YopP inhibits host cell signalling by inactivating the protein kinase TAK1 in the IL-1 signalling pathway.

The mechanism by which YopP simultaneously inhibits mitogen-activated protein kinase (MAPK) and nuclear factor-kappaB pathways has been elusive. Ectopic expression of YopP inhibits the activity and ubiquitination of a complex consisting of overexpressed TGF-beta-activated kinase 1 (TAK1) and its subunit TAK1-binding protein (TAB)1, but not of MEK kinase 1. YopP, but not the catalytically inactive mutant YopP(C172A), also suppresses basal and interleukin-1-inducible activation of endogenous TAK1, TAB1 and TAB2. YopP does not affect the interaction of TAK1, TAB1 and TAB2 but inhibits autophosphorylation of TAK1 at Thr 187 and phosphorylation of TAB1 at Ser 438. Glutathione S-transferase-tagged YopP (GST-YopP) binds to MAPK kinase (MAPKK)4 and TAB1 but not to TAK1 or TAB2 in vitro. Furthermore, YopP in synergy with a previously described negative regulatory feedback loop inhibits TAK1 by MAPKK6-p38-mediated TAB1 phosphorylation. Taken together, these data strongly suggest that YopP binds to TAB1 and directly inhibits TAK1 activity by affecting constitutive TAK1 and TAB1 ubiquitination that is required for autoactivation of TAK1.