Dae-Myung Jue

Thiol-Reactive Metal Compounds Inhibit NF-κB Activation by Blocking IκB Kinase

Gold compounds are used in the treatment of rheumatoid arthritis. NF-κB is a transcription factor implicated in the expression of many inflammatory genes. NF-κB is activated by signal-induced phosphorylation and subsequent degradation of inhibitory IκB (inhibitory protein that dissociates from NF-κB) proteins, and a multisubunit IκB kinase (IKK) has been identified previously. We tested the effect of various gold compounds on the activation of NF-κB and IKK in LPS-stimulated RAW 264.7 mouse macrophages. A lipophilic gold compound, auranofin, suppressed the LPS-induced increase of nuclear κB-binding activity, degradation of IκB proteins, and IKK activation. Auranofin also blocked IKK activation induced by TNF and PMA/ionomycin, suggesting that the target of auranofin action is common among these diverse signal pathways. In vitro IKK activity was suppressed by addition of hydrophilic gold compounds, such as aurothiomalate, aurothioglucose, and AuCl3. Other thiol-reactive metal ions such as zinc and copper also inhibited IKK activity in vitro, and induction of IKK in LPS-stimulated macrophages. In vitro IKK activity required the presence of reducing agent and was blocked by addition of thiol group-reactive agents. Two catalytic subunits of IKK complex, IKKα and IKKβ, were both inhibited by these thiol-modifying agents, suggesting the presence of a cysteine sulfhydryl group in these subunits, which is critical for enzyme activity. The antiinflammatory activity of gold compounds in the treatment of rheumatoid arthritis may depend on modification of this thiol group by gold.

Gold compound auranofin inhibits IκB kinase (IKK) by modifying Cys-179 of IKKβ subunit

Antirheumatic gold compounds have been shown to inhibit NF-κB activation by blocking IκB kinase (IKK) activity. To examine the possible inhibitory mechanism of gold compounds, we expressed wild type and mutant forms of IKKα and β subunits in COS-7 cells and determined the effect of gold on the activity of these enzymes both in vivo and in vitro. Substitution of Cys-179 of IKKβ with alanine (C179A) rendered the enzyme to become resistant to inhibition by a gold compound auranofin, however, similar protective effect was not observed with an equivalent level of IKKα (C178A) mutant expressed in the cells. Auranofin inhibited constitutively active IKKα and β and variants; IKKα (S176E, S180E) or IKKβ (S177E, S181E), suggesting that gold directly cause inhibition of activated enzyme. The different inhibitory effect of auranofin on IKKα (C178A) and IKKβ (C179A) mutants indicates that gold could inhibit the two subunits of IKK in a different mode, and the inhibition of NF-κB and IKK activation induced by inflammatory signals in gold-treated cells appears through its interaction with Cys-179 of IKKβ.

Dual effect of oxidative stress on NF-κB activation in HeLa cells

Reactive oxygen species (ROS) has been implicated as an inducer of NF-κB activity in numbers of cell types where exposure of cells to ROS such as H2O2 leads to NF-κB activation. In contrast, exposure to oxidative stress in certain cell types induced reduction of tumor necrosis factor (TNF)-induced NF-κB activation. And various thiol-modifying agents including gold compounds and cyclopentenone prostaglandins inhibit NF-κB activation by blocking IκB kinase (IKK). To understand such conflicting effect of oxidative stress on NF-κB activation, HeLa cells were incubated with H2O2 or diamide and TNF-induced expression of NF-κB reporter gene was measured. NF-κB activation was significantly blocked by these oxidizing agents, and the inhibition was accompanied with reduced nuclear NF-κB and inappropriate cytosolic IκB degradation. H2O2 and diamide also inhibited IKK activation in HeLa and RAW 264.7 cells stimulated with TNF and lipopolysaccharide, respectively, and directly blocked IKK activity in vitro. In cells treated with H2O2 alone, nuclear NF-κB was induced after 2 h without detectible degradation of cytosolic IkBa or activation of IKK. Our results suggest that ROS has a dual effect on NF-κB activation in the same HeLa cells: it inhibits acute IKK-mediated NF-κB activation induced by inflammatory signals, while longer-term exposure to ROS induces NF-κB activity through an IKK-independent pathway.

Inhibition of prostaglandin synthase activity of sheep seminal vesicular gland by human serum haptoglobin

SummarySerum haptoglobin was added to the reaction mixture of prostaglandin synthase (EC 1.14.99.1) and its inhibitory effect was studied. [1-14C]Arachidonic acid was used as substrate and the enzyme activity was estimated by monitoring the radioactivity of the products after thin layer chromatography. With or without addition of hemoglobin to the reaction mixture, both the purified haptoglobin 1-1 and 2-2 showed inhibitory activity. In the presence of 5 μM hematin, however, inhibitory activity of haptoglobin was not observed. Inhibition of prostaglandin synthesis in the system depended on the molar ratio of haptoglobin to hemoglobin in the reaction mixture. These results demonstrate that haptoglobin inhibits prostaglandin synthase by restricting available heme group for the enzyme activity through complexing with hemoglobin. However, haptoglobin did not inhibit completely the stimulatory effect of free hemoglobin. Relevant significance of this effect was discussed.

N-tosyl-L-phenylalanine chloromethyl ketone inhibits NF-kappaB activation by blocking specific cysteine residues of IkappaB kinase beta and p65/RelA.

N-Tosyl-L-phenylalanine chloromethyl ketone (TPCK), a serine/cysteine protease inhibitor, has been reported to inhibit expression of inflammatory mediators by blocking nuclear factor-kappaB (NF-kappaB) activation. We examined the effect of TPCK on the NF-kappaB activation pathway in HeLa cells by measuring the activity of IkappaB kinase (IKK) and p65/RelA-DNA binding. TPCK inhibited tumor necrosis factor-alpha-induced IKK activation and directly blocked IKK activity in vitro. TPCK-induced inhibition of NF-kappaB and IKK activation was abrogated by addition of the thiol-reducing agent dithiothreitol, suggesting that the effect of TPCK occurred through modification of a thiol group in IKK. Consistent with this, an IKKbeta mutant in which Cys-179 was substituted with alanine was not more susceptible to TPCK. Our result also showed that TPCK inhibits the DNA binding of transiently expressed p65/RelA in HeLa cells. Inhibition of p65/RelA-DNA binding was recovered in the presence of dithiothreitol, and substitution of Cys-38 with Ser in p65/RelA rendered the protein resistant to inhibition by TPCK. Mass spectrometry analysis of IKKbeta and p65/RelA isolated from cells treated with TPCK by UPLC-ESI-Q-TOF tandem MS revealed the labeling of Cys-179 of IKKbeta and Cys-38 of p65/RelA with a tosylphenylalanylmethyl group. These results suggest that TPCK inhibits NF-kappaB activation by directly modifying thiol groups on two different targets: Cys-179 of IKKbeta and Cys-38 of p65/RelA.

Cysteine-179 of IκB kinase β plays a critical role in enzyme activation by promoting phosphorylation of activation loop serines

IκB kinase β (IKKβ) subunit of IKK complex is essential for the activation of NF-κB in response to various proinflammatory signals. Cys-179 in the activation loop of IKKβ is known to be the target site for IKK inhibitors such as cyclopentenone prostaglandins, arsenite, and antirheumatic gold compounds. Here we show that a mutant IKKβ in which Cys-179 is substituted with alanine had decreased activity when it was expressed in HEK-293 cells, and TNF stimulation did not restore the activity. Phosphorylation of activation loop serines (Ser-177 and Ser-181) which is required for IKKβ activation was reduced in the IKKβ (C179A) mutant. The activity of IKKβ (C179A) was partially recovered when its phosphorylation was enforced by coexpression with mitogen-activated protein kinase kinase kinases (MAPKKK) such as NF-κB inducing kinase (NIK) and MAPK/extracellular signal-regulated kinase kinase kinase 1(MEKK1) or when the serine residues were replaced with phospho-mimetic glutamate. The IKKβ (C179A) mutant was normal in dimer formation, while its activity abnormally responded to the change in the concentration of substrate ATP in reaction mixture. Our results suggest that Cys-179 of IKKβ plays a critical role in enzyme activation by promoting phosphorylation of activation-loop serines and interaction with ATP.

Chloroquine decreases cell-surface expression of tumour necrosis factor receptors in human histiocytic U-937 cells

Proinflammatory cytokine tumour necrosis factor (TNF) mediates its diverse effects through cell surface receptors. A variety of inflammatory signals are known to modulate TNF activities by changing expression and shedding of cell‐surface TNF receptors. We have examined the effects of anti‐rheumatic drug chloroquine on the expression of cell surface and soluble TNF receptors in human histiocytic U‐937 cells. Chloroquine partially reduced production of soluble p55 and p75 TNF receptors in cells stimulated with phorbol 12‐myristate 13‐acetate (PMA). In these cells, induction of both TNF receptor mRNA was not changed and the levels of cell‐associated TNF receptors were rather increased by chloroquine. Flow cytometric analysis revealed that chloroquine does not inhibit the PMA‐triggered shedding of TNF receptors from cell surface, while it was suppressed by a metalloproteinase inhibitor BB‐3103. Treatment of U‐937 cells with chloroquine significantly reduced the level of cell surface TNF receptors and a similar effect was observed with human peripheral blood monocytes. Other weak‐base amines, including hydroxychloroquine, ammonium chloride and methylamine, also induced reduction of cell surface TNF receptors, whereas lysosomal proteinase inhibitor, leupeptin, and BB‐3013 were without effect. Our results suggest that chloroquine down‐regulates cell surface TNF receptors by retarding their transport to the cell surface, while cleavage of cell surface receptors is not inhibited by chloroquine.

Simple spectrophotometric determination of haptoglobin-haemoglobin complex in haemolysed samples

The concentration of haptoglobin-haemoglobin complex in haemolysed serum was measured by reducing the sample with sodium hydrosulphite. The absorption spectra of serum before and after sodium hydrosulphite treatment were compared, and the absorbance change was used to determine the amount of haemoglobin in the sample. By analysing the difference absorption spectra of various types of haemoglobin-containing samples, it was also possible to determine the haptoglobin level in samples haemolysed severely beyond their haemoglobin-binding capacities. When the present methods are used, along with the spectrophotometric method for free haptoglobin, one can measure separately the amounts of free haptoglobin, haptoglobin-haemoglobin complex, and free haemoglobin in samples with varying degrees of haemolysis.

Transient exposure to hydrogen peroxide inhibits the ubiquitination of phosphorylated IκBα in TNFα-stimulated HEK293 cells

During ischemia-reperfusion injury, brief pre-exposure to oxidative stress renders organs resistant to subsequent severe damage. NF-κB is a transcription factor that is involved in reperfusion-induced inflammatory and immune responses. The activity of NF-κB has been shown to be modulated by oxidative stress in various cell types through different pathways. We studied the effect of pre-exposure to oxidative stress on subsequent NF-κB activation in TNFα-stimulated HEK293 cells. The cells were transiently exposed to 0.5 mM H2O2 for 20 min, prior to stimulation with TNFα, and the subsequent expression of NF-κB-dependent genes and the levels of NF-κB signaling molecules were measured. Pre-exposure to H2O2 significantly delayed the TNFα-induced expression of an NF-κB reporter gene and inflammatory proteins (intercellular adhesion molecule-1 and IL-1β). The degradation of inhibitor of NF-κB α (IκBα) and the nuclear translocation of NF-κB were also delayed by H2O2 treatment, whereas IκBα phosphorylation and IκB kinase activity were not changed. When we examined the ubiquitin/proteosome pathway in H2O2-treated cells, we could not detect significant changes in proteosomal peptidase activities, but we were able to detect a delay of IκBα poly-ubiquitination. Our results suggest that transient exposure to oxidative stress temporally inhibits NF-κB-dependent gene expression by suppressing the poly-ubiquitination of phosphorylated IκBα in HEK293 cells.

Suppression of NFAT5-mediated Inflammation and Chronic Arthritis by Novel κB-binding Inhibitors

Nuclear factor of activated T cells 5 (NFAT5) has been implicated in the pathogenesis of various human diseases, including cancer and arthritis. However, therapeutic agents inhibiting NFAT5 activity are currently unavailable. To discover NFAT5 inhibitors, a library of > 40,000 chemicals was screened for the suppression of nitric oxide, a direct target regulated by NFAT5 activity, through high-throughput screening. We validated the anti-NFAT5 activity of 198 primary hit compounds using an NFAT5-dependent reporter assay and identified the novel NFAT5 suppressor KRN2, 13-(2-fluoro)-benzylberberine, and its derivative KRN5. KRN2 inhibited NFAT5 upregulation in macrophages stimulated with lipopolysaccharide and repressed the formation of NF-κB p65-DNA complexes in the NFAT5 promoter region. Interestingly, KRN2 selectively suppressed the expression of pro-inflammatory genes, including Nos2 and Il6, without hampering high-salt-induced NFAT5 and its target gene expressions. Moreover, KRN2 and KRN5, the latter of which exhibits high oral bioavailability and metabolic stability, ameliorated experimentally induced arthritis in mice without serious adverse effects, decreasing pro-inflammatory cytokine production. Particularly, orally administered KRN5 was stronger in suppressing arthritis than methotrexate, a commonly used anti-rheumatic drug, displaying better potency and safety than its original compound, berberine. Therefore, KRN2 and KRN5 can be potential therapeutic agents in the treatment of chronic arthritis.