Ulrike Resch

Tristetraprolin Impairs NF-κB/p65 Nuclear Translocation

Tristetraprolin (TTP) is a prototypic family member of CCCH-type tandem zinc-finger domain proteins that regulate mRNA destabilization in eukaryotic cells. TTP binds to AU-rich elements (AREs) in the 3′-untranslated region of certain mRNAs, including tumor necrosis factor α, granulocyte macrophage colony-stimulating factor, and immediate early response 3, thereby facilitating their ARE-mediated decay. Expression of TTP is up-regulated by a variety of agents, including inflammatory mediators such as tumor necrosis factor α, a prominent activator of the nuclear factor κB (NF-κB) family of transcription factors. Accordingly, TTP is involved in the negative feedback regulation of NF-κB through promoting mRNA degradation. We describe here a novel, ARE-mediated decay-independent function of TTP on the termination of NF-κB response: TTP suppresses the transcriptional activity of NF-κB-dependent promoters independent of its mRNA-destabilizing property. In TTP knock-out mouse embryonic fibroblasts, lack of TTP leads to enhanced nuclear p65 levels, which is associated with the up-regulation of specific, ARE-less NF-κB target genes. We find that attenuation of NF-κB activity is at least in part due to an interference of TTP with the nuclear import of the p65 subunit of the transcription factor. This novel role of TTP may synergize with its mRNA-degrading function to contribute to the efficient regulation of proinflammatory gene expression.

Antioxidant status in thyroid dysfunction.

Abstract Our study was designed to test and compare the levels of enzymatic antioxidants (ARS), endogenous peroxides (POX), non-enzymatic antioxidants (Antiox-cap) and anti-oxidized low-density lipoprotein (LDL) antibody titers (oLAb) in hyperthyroid and hypothyroid patients. We measured POX, ARS, Antiox-cap and oLAb in the plasma from 68 patients (34 patients with hyperthyroidism, thyroid-stimulating hormone (TSH) <0.04; and 34 patients with hypothyroidism, TSH >4.0) and 34 healthy euthyroid controls. POX were highest in hyperthyroid patients, but differences between hyperthyroid and hypothyroid patients and controls were not significant. ARS were significantly higher in patients compared to controls. Antiox-cap were significantly lower in patients compared to controls. oLAb were significantly higher in hypothyroid patients compared to controls and hyperthyroid patients. Our study shows that both hyperthyroidism and hypothyroidism are associated with enhanced oxidative stress involving enzymatic and non-enzymatic antioxidants. Higher POX in hyperthyroid patients might reflect the hypermetabolic state of these patients. oLAb, indicating progression of atherosclerosis, were highest in hypothyroid patients.

XIAP regulates intracellular ROS by enhancing antioxidant gene expression

XIAP (X chromosome-linked inhibitor of apoptosis) is a member of the anti-apoptotic IAP gene family and an inhibitor of caspase-3. We show here that loss of XIAP renders cells highly sensitive to oxidative stress. Stimulation of XIAP(-/-) MEF with hydrogen peroxide, or other agents that generate reactive oxygen species (ROS) results in increased apoptosis assessed by caspase-3 activity and PARP cleavage. Furthermore, we observed increased levels of ROS and diminished expression of antioxidative genes, e.g., SOD1, -2, NQO1, HO-1, and Txn2 in XIAP(-/-) cells. In addition, stimulation of XIAP(-/-) MEF with hydrogen peroxide resulted in enhanced phosphorylation of JNK. Our findings reveal that XIAP, in addition to its well described caspase-inhibitory function, prevents prolonged JNK activation and is critically involved in modulating ROS levels through regulation of antioxidative genes, thereby inhibiting ROS-induced apoptosis.

XIAP regulates bi-phasic NF-κB induction involving physical interaction and ubiquitination of MEKK2

The transcription factor NF-kappaB is transiently activated by a wide variety of stress signals, including pro-inflammatory mediators, and regulates the expression of genes with e.g., immune, inflammatory, and anti-apoptotic functions. The strength and kinetics of its induction, as well as its ultimate down-regulation is subject to multiple levels of regulation. One such regulatory protein is X chromosome-linked inhibitor of apoptosis (XIAP) that, besides its anti-apoptotic properties, has been shown to enhance NF-kappaB activity, however, the underlying molecular mechanism has remained elusive. We show here that following TNFalpha stimulation XIAP regulates a second wave of NF-kappaB activation. XIAP interacts with and ubiquitinates MEKK2, a kinase that has previously been associated with bi-phasic NF-kappaB activation. We conclude that, through interaction with MEKK2, XIAP functions in an ubiquitin ligase dependent manner to evoke a second wave of NF-kappaB activation, resulting in the modulation of NF-kappaB target gene expression.

Siva1 is a XIAP-interacting protein that balances NFκB and JNK signalling to promote apoptosis

XIAP is known as a potent inhibitor of apoptosis, but in addition is involved in cellular signalling, including the NFκB, JNK and TGFβ pathways. Our search for XIAP-interacting partners led us to Siva1, a proapoptotic protein that is known to play a role in T-cell apoptosis through a caspase-dependent mitochondrial pathway. The interaction sites between XIAP and Siva1 were mapped to the RING domain of XIAP and the N-terminal, SAH-containing and death-homology-region-containing domains of Siva1. Co-immunoprecipitation experiments showed that XIAP, Siva1 and TAK1 form a ternary complex in Jurkat T cells. Reporter-gene analysis revealed that Siva1 inhibits XIAP- and TAK1-TAB1-mediated NFκB activation. By contrast, Siva1 increased XIAP- and TNFα-mediated AP1 activity and prolonged TNFα-induced JNK activation, whereas knock down of Siva1 resulted in reduced JNK activation. This suggests that Siva1 differentially modulates signalling by JNK and NFκB and shifts the balance between these pathways towards enhanced JNK activation, a situation that promotes apoptosis. Ectopically expressed Siva1 increased caspase-3 activity, which was inhibited by XIAP in a ubiquitin-ligase-dependent manner. In line with this, Siva1 was lysine-48-linked polyubiquitylated by XIAP. Our findings suggest that, via physical interaction with XIAP and TAK1, Siva1 diminishes NFκB and enhances JNK activity to favour apoptosis.

|[alpha]|-Catulin, a Rho signalling component, can regulate NF-|[kappa]|B through binding to IKK-|[beta]|, and confers resistance to apoptosis

Rho GTPases regulate diverse cellular functions including adhesion, cytokinesis and motility, as well as the activity of the transcription factors NF-κB, serum response factor and C/EBP. α-Catulin, an α-catenin-related protein that shares structural similarities with cytoskeletal linker proteins, facilitates Rho signalling by serving as a scaffold for the Rho-specific guanine nucleotide exchange factor Lbc. We report here that α-catulin also interacts with a key component of the NF-κB signalling pathway, namely the IκB kinase (IKK)-β. In co-immunoprecipitations, α-catulin can bind IKK-β and Lbc. Ectopic expression of α-catulin augmented NF-κB activity, promoted cell migration and increased resistance to apoptosis, whereas knockdown experiments showed the opposite effects. Together, these features suggest that α-catulin has tumorigenic potential.

Novel phosphorylation-dependent ubiquitination of tristetraprolin by mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase 1 (MEKK1) and tumor necrosis factor receptor-associated factor 2 (TRAF2).

Background: During inflammation, tristetraprolin displays a dual function as mRNA destabilizer and regulator of NF-κB signaling. Results: Tristetraprolin is inducibly hypermodified by phosphorylation and ubiquitination, affecting cell viability. Conclusion: Tristetraprolin hypermodification provokes its functional shift toward the activation of JNK signaling. Significance: The NF-κB/JNK cross-talk is central for our understanding of chronic inflammatory diseases and cancer development. Acute versus chronic inflammation is controlled by the accurate activation and regulation of interdependent signaling cascades. TNF-receptor 1 engagement concomitantly activates NF-κB and JNK signaling. The correctly timed activation of these pathways is the key to account for the balance between NF-κB-mediated cell survival and cell death, the latter fostered by prolonged JNK activation. Tristetraprolin (TTP), initially described as an mRNA destabilizing protein, acts as negative feedback regulator of the inflammatory response: it destabilizes cytokine-mRNAs but also acts as an NF-κB inhibitor by interfering with the p65/RelA nuclear import pathway. Our biochemical studies provide evidence that TTP contributes to the NF-κB/JNK balance. We find that the MAP 3-kinase MEKK1 acts as a novel TTP kinase that, together with the TNF receptor-associated factor 2 (TRAF2), constitutes not only a main determinate of the NF-κB-JNK cross-talk but also facilitates “TTP hypermodification”: MEKK1 triggers TTP phosphorylation as prerequisite for its Lys-63-linked, TRAF2-mediated ubiquitination. Consequently, TTP no longer affects NF-κB activity but promotes the activation of JNK. Based on our data, we suggest a model where upon TNFα induction, TTP transits a hypo- to hypermodified state, thereby contributing to the molecular regulation of NF-κB versus JNK signaling cascades.

The Transcription Factor SOX18 Regulates the Expression of Matrix Metalloproteinase 7 and Guidance Molecules in Human Endothelial Cells

Background Mutations in the transcription factor SOX18 are responsible for specific cardiovascular defects in humans and mice. In order to gain insight into the molecular basis of its action, we identified target genes of SOX18 and analyzed one, MMP7, in detail. Methodology/Principal Findings SOX18 was expressed in HUVEC using a recombinant adenoviral vector and the altered gene expression profile was analyzed using microarrays. Expression of several regulated candidate SOX18 target genes was verified by real-time PCR. Knock-down of SOX18 using RNA interference was then used to confirm the effect of the transcription factor on selected genes that included the guidance molecules ephrin B2 and semaphorin 3G. One gene, MMP7, was chosen for further analysis, including detailed promoter studies using reporter gene assays, electrophoretic mobility shift analysis and chromatin-immunoprecipitation, revealing that it responds directly to SOX18. Immunohistochemical analysis demonstrated the co-expression of SOX18 and MMP7 in blood vessels of human skin. Conclusions/Significance The identification of MMP7 as a direct SOX18 target gene as well as other potential candidates including guidance molecules provides a molecular basis for the proposed function of this transcription factor in the regulation of vessel formation.

Inhibition of low-density lipoprotein and high-density lipoprotein oxidation by raloxifene.

BACKGROUND Raloxifene (RX), a selective estrogen receptor modulator (SERM), has demonstrated hypolipidemic and in vitro antioxidant properties. In the pathogenesis of atherosclerosis, the oxidative modification of low-density lipoprotein (LDL) is implicated to play a crucial role. METHODS In this study, we investigated the antioxidant properties of RX during in vitro LDL and high-density lipoprotein (HDL) oxidation. The concentrations of RX, chosen ranged from 5.1 to 5100 nM. Isolated lipoproteins were supplemented with RX, and copper-mediated oxidation was monitored via the formation of conjugated dienes (CD), and oxidation resistance (lag time) was calculated. The formation of thiobarbituric acid-reactive substances (TBARS) was measured after 90, 120, 210, and 300 minutes of oxidation. RESULTS We found a dose-dependent inhibition of LDL and HDL oxidation in terms of both increased lag time and decreased formation of TBARS. Differences in lag time became significant at 51 nM RX added to LDL and HDL, respectively. TBARS formation in LDL was significantly reduced after 120 minutes incubation with 510 and 5100 nM RX and also after 300 minutes with 51 nM RX added. In HDL, significant reduction in TBARS formation was observed after 90 minutes by the addition of 51, 510, and 5100 nM RX. CONCLUSIONS These results confirm data from previous studies concerning inhibition of in vitro oxidation of LDL by RX and additionally show that RX efficiently inhibits in vitro oxidation of HDL in a broad concentration range.

The Transcription Factor ZNF683/HOBIT Regulates Human NK-Cell Development

We identified ZNF683/HOBIT as the most highly upregulated transcription factor gene during ex vivo differentiation of human CD34+ cord blood progenitor cells to CD56+ natural killer (NK) cells. ZNF683/HOBIT mRNA was preferentially expressed in NK cells compared to other human peripheral blood lymphocytes and monocytes. During ex vivo differentiation, ZNF683/HOBIT mRNA started to increase shortly after addition of IL-15 and further accumulated in parallel to the generation of CD56+ NK cells. shRNA-mediated knockdown of ZNF683/HOBIT resulted in a substantial reduction of CD56−CD14− NK-cell progenitors and the following generation of CD56+ NK cells was largely abrogated. The few CD56+ NK cells, which escaped the developmental inhibition in the ZNF683/HOBIT knockdown cultures, displayed normal levels of NKG2A and KIR receptors. Functional analyses of these cells showed no differences in degranulation capacity from control cultures. However, the proportion of IFN-γ-producing cells appeared to be increased upon ZNF683/HOBIT knockdown. These results indicate a key role of ZNF683/HOBIT for the differentiation of the human NK-cell lineage and further suggest a potential negative control on IFN-γ production in more mature human NK cells.