A20 ameliorates inflammatory bowel disease in mice via inhibiting NF-κB and STAT3 activation.

A20 is a zinc finger protein that effectively inhibits the activation of nuclear factor (NF)-κB to downregulate the expression of tumor necrosis factor-α, interleukin (IL)-1ß, and IL-17. A20 also plays a crucial role as a feedback inhibitor of the inflammatory response. Due to its inhibitory role, A20 may be useful in regulating diseases resulting from chronic inflammation and excessive pro-inflammatory cytokine production, such as colitis. Patients with colitis produce high levels of pro-inflammatory cytokines in the intestine. Therefore, this study aimed to investigate whether A20 improves experimental colitis by reducing high levels of inflammation in the intestine. An A20 overexpression vector was administered to mice by intrarectal injection after colitis induction. Histological analysis by immunohistochemistry was used to score sections of the intestine. Confocal laser scanning microscopy was used to identify the expression of IL-17 and forkhead box p (FOXP) 3 protein in spleen tissues. Protein expression induced by STAT3 and NF-κB signaling was analyzed by western blot. We found that A20 reduced the colitis activity index score and the histological score of the intestine. A20 also decreased inflammatory cytokine levels in the intestine and increased colon length. Additionally, A20 overexpression downregulated the activation of NF-kB and STAT3. A20 also reduced IL-17 expression in CD4+ T cells from spleen sections. In contrast, A20 overexpression enhanced the expression of FOXP3 in CD4+ T cells. These results suggest that A20 may inhibit the progression of colitis by decreasing inflammation via inhibition of NF-κB, phosphorylated STAT3, and IL-17.

Intracelluar delivery of A20 protein inhibits TNFα-induced NF-κB activation.

A20 (also known as TNFAIP3) is a potent anti-inflammatory protein that suppresses many intracellular signaling pathways induced by inflammatory cytokines and bacterial and viral pathogens. The anti-inflammatory function of A20 depends on its modulation of or binding to polyubiquitin chains on key signaling proteins in the nuclear factor-κB (NF-κB) pathway. To test whether A20 can be used as therapeutic agent in these inflammatory diseases, we prepared a recombinant cell-penetrating form of A20 (TAT-A20) for intracellular delivery and examined its effect on tumor necrosis factor-α (TNFα)-induced NF-κB activation. We observed that TAT-A20 was effectively transduced into cells within 30 min, whereas A20 protein without TAT motive was not. TAT-A20 also inhibited NF-κB induction in fibroblasts stimulated with TNFα. These results suggest that increasing intracellular level of A20 can be an effective means to suppress NF-κB activation and treat inflammatory diseases.

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.

The Fos-Related Antigen 1-JUNB/Activator Protein 1 Transcription Complex, a Downstream Target of Signal Transducer and Activator of Transcription 3, Induces T Helper 17 Differentiation and Promotes Experimental Autoimmune Arthritis.

Dysfunction of T helper 17 (Th17) cells leads to chronic inflammatory disorders. Signal transducer and activator of transcription 3 (STAT3) orchestrates the expression of proinflammatory cytokines and pathogenic cell differentiation from interleukin (IL)-17-producing Th17 cells. However, the pathways mediated by STAT3 signaling are not fully understood. Here, we observed that Fos-related antigen 1 (FRA1) and JUNB are directly involved in STAT3 binding to sites in the promoters of Fosl1 and Junb. Promoter binding increased expression of IL-17 and the development of Th17 cells. Overexpression of Fra1 and Junb in mice resulted in susceptibility to collagen-induced arthritis and an increase in Th17 cell numbers and inflammatory cytokine production. In patients with rheumatoid arthritis, FRA1 and JUNB were colocalized with STAT3 in the inflamed synovium. These observations suggest that FRA1 and JUNB are associated closely with STAT3 activation, and that this activation leads to Th17 cell differentiation in autoimmune diseases and inflammation.

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 H(2)O(2) 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 H(2)O(2) significantly delayed the TNFα-induced expression of an NF-κB reporter gene and inflammatory proteins (intercellular adhesion molecule-1 and IL-1ß). The degradation of inhibitor of NF-κB α (IκBα) and the nuclear translocation of NF-κB were also delayed by H(2)O(2) treatment, whereas IκBα phosphorylation and IκB kinase activity were not changed. When we examined the ubiquitin/proteosome pathway in H(2)O(2)-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.

Aurora-a contributes to radioresistance by increasing NF-kappaB DNA binding.

Aurora-A, a serine/threonine kinase that is overexpressed in certain human cancer cell lines, plays an important role in mitotic progression. Aurora-A has also been reported to be involved in the activation of nuclear factor kappa B (NF-kappaB). The purpose of the present study was to identify the role of Aurora-A in the radiation-induced activation pathway of NF-kappaB. Wild-type and Aurora-A knockdown (Aurora-A(KD)) HeLa cells were irradiated with 4 Gy of gamma rays and the EMSA, luciferase reporter gene assay and immunoblot analysis were performed. The siRNA-based gene knockdown and overexpression system was adopted to elucidate the role of Aurora-A in radiation-induced NF-kappaB pathway activation. The clonogenic survival study indicated that Aurora-A(KD) cells and the wild-type cells transfected with Aurora-A siRNA or RelA/p65 siRNA were more radiosensitive than the wild-type cells. In both the wild-type and Aurora-A(KD) cells, radiation caused IkappaB kinase-mediated phosphorylation, degradation of IkappaBalpha and phosphorylation of RelA/p65. The nuclear translocation of RelA/p65 was also similar in the wild-type and Aurora-A(KD) cells. However, RelA/p65-DNA binding was markedly suppressed in Aurora-A(KD) cells compared to that in wild-type cells. It was concluded that Aurora-A enhances the binding of NF-kappaB to DNA, thereby increasing the gene transcription by NF-kappaB and decreasing the radiosensitivity of the cells.

Piceatannol, a catechol-type polyphenol, inhibits phorbol ester-induced NF-{kappa}B activation and cyclooxygenase-2 expression in human breast epithelial cells: cysteine 179 of IKK{beta} as a potential target.

There are multiple lines of evidence supporting that chronic inflammation is linked to carcinogenesis. Nuclear factor-kappaB (NF-kappaB), a major redox-sensitive transcription factor responsible for the induction of a wide array of pro-inflammatory genes, is frequently overactivated in many tumors. Moreover, constitutive activation of IkappaB kinase (IKK), a key regulator of NF-kappaB signaling, has been implicated in inflammation-associated tumorigenesis. Piceatannol (trans-3,4,3',5'-tetrahydroxystilbene; PIC) derived from grapes, rhubarb and sugarcane exhibits immunosuppressive and antitumorigenic activities in several cell lines, but the underlying mechanisms are poorly understood. In the present study, we found that PIC inhibited migration and anchorage-independent growth of human mammary epithelial cells (MCF-10A) treated with the prototypic tumor promoter, 12-O-tetradecanoylphorbol-13-aceate (TPA). PIC treatment suppressed the TPA-induced activation of NF-kappaB and expression of cyclooxygenase-2 (COX-2) in MCF-10A cells. We speculate that an electrophilic quinone formed as a consequence of oxidation of PIC bearing the catechol moiety may directly interact with critical cysteine thiols of IKKbeta, thereby inhibiting its catalytic activity. In support of this speculation, the reducing agent dithiothreitol abrogated the inhibitory effects of PIC on TPA-induced activation of NF-kappaB signaling and expression of COX-2. In addition, the inhibitory effects of PIC on NF-kappaB activation and COX-2 induction were blunted in cells expressing mutant IKKbeta (C179A) in which cysteine 179 was replaced by alanine. In conclusion, our results show that direct modification of IKKbeta by PIC, presumably at the cysteine 179 residue, blocks NF-kappaB activation signaling and COX-2 induction in TPA-treated MCF-10A cells and also migration and transformation of these cells.

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.

Treatment with N-tosyl-l-phenylalanine chloromethyl ketone after the onset of collagen-induced arthritis reduces joint erosion and NF-kappaB activation.

N-tosyl-l-phenylalanine chloromethyl ketone (TPCK) is known to inhibit NF-kappaB activation and the expression of inflammation mediators in cultured cells. We measured the potential of TPCK to inhibit the pathogenesis of collagen-induced arthritis by blocking NF-kappaB activation. Arthritis was induced in DBA/1J mice by the injection of bovine type II collagen in adjuvant on days 0 and 14. Mice received either TPCK (3 or 10 mg/kg, i.p.) or vehicle three times a week for 3 weeks starting on day 21. TPCK moderately reduced clinical disease activity scores, whereas it markedly suppressed histological indications of joint destruction. In vitro production of tumor necrosis factor-alpha, interleukin-6, and monocyte chemotactic protein-1 from lipopolysaccharide-stimulated spleen cells was also reduced by in vivo treatment with TPCK. Proliferation of cells isolated from spleen or draining lymph nodes and production of interferon-gamma and interleukin-17 in response to stimulation with type II collagen was decreased by TPCK. Moreover, nuclear NF-kappaB activity induced by collagen immunization was significantly reduced in mice treated with TPCK. Finally, osteoclast differentiation of bone marrow cells induced by macrophage colony-stimulating factor and receptor activator of NF-kappaB ligand was completely inhibited by TPCK. These results indicate that TPCK attenuates collagen-induced arthritis and bone erosion by suppressing NF-kappaB activation and thus expression of inflammatory and osteoclastogenic genes.

Cysteine-179 of IkappaB kinase beta plays a critical role in enzyme activation by promoting phosphorylation of activation loop serines.

IkappaB kinase beta (IKKbeta) subunit of IKK complex is essential for the activation of NF-kappaB in response to various proinflammatory signals. Cys-179 in the activation loop of IKKbeta 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 IKKbeta 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 IKKbeta activation was reduced in the IKKbeta (C179A) mutant. The activity of IKKbeta (C179A) was partially recovered when its phosphorylation was enforced by coexpression with mitogen-activated protein kinase kinase kinases (MAPKKK) such as NF-kappaB 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 IKKbeta (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 IKKbeta plays a critical role in enzyme activation by promoting phosphorylation of activation-loop serines and interaction with ATP.

Interleukin-18 induces the production of vascular endothelial growth factor (VEGF) in rheumatoid arthritis synovial fibroblasts via AP-1-dependent pathways.

Interleukin-18 (IL-18) is a novel pro-inflammatory cytokine which has been implicated to play a pathogenic role in rheumatoid arthritis (RA). Vascular endothelial growth factor (VEGF) plays an important role in angiogenesis in rheumatoid synoviocytes. In present study, we examined the effect of IL-18 on VEGF production in fibroblast-like synoviocytes (FLS) isolated from the patients with RA. FLS were prepared from the synovial tissues of patients with RA and osteoarthritis (OA) and cultured in the presence of IL-18. The production of VEGF from FLS was measured in culture supernatants by enzyme-linked immunosorbent assay (ELISA). The VEGF messenger RNA (mRNA) expression and AP-1 binding activity of VEGF transcript were determined by reverse transcription-polymerase chain reaction (RT-PCR) and electrophoretic mobility shift assay (EMSA). IL-18 and VEGF levels of sera and synovial fluids (SF) of RA patients (n=30) were significantly higher than those of OA patients (n=20). IL-18 dose-dependently increased the production of VEGF. The effect of IL-18 on VEGF production appeared to be as potent as IL-1beta, whereas tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma showed little effects on VEGF production. AP-1-specific inhibitor Curcumin dose-dependently abrogated the effect of IL-18 on VEGF production. The VEGF enhancement of IL-18 was associated with increased AP-1 binding activity to the VEGF promoter site. These findings suggest IL-18 as an angiogenic factor in RA and down-regulation of IL-18 activity or AP-1 signal pathway can be potential therapeutic targets for RA.

Nox 2 stimulates muscle differentiation via NF-kappaB/iNOS pathway.

The NF-kappaB/iNOS pathway stimulates muscle differentiation downstream of the PI 3-kinase/p38 MAPK pathway and diverse antioxidants block muscle differentiation. Therefore, we here investigated whether Nox 2 links those two myogenic pathways in H9c2 and C2C12 myoblasts. Compared with the proliferation stage, ROS generation was enhanced from the early stage of differentiation and gradually increased as differentiation progressed. Antioxidants suppressed the activated NF-kappaB/iNOS pathway during muscle differentiation. Nox 2 activity was also increased during muscle differentiation. Treatment with DPI and apocynin, two inhibitors of NADPH oxidase, and suppression of Nox 2 expression using siRNA, but not Nox 1, inhibited NADPH oxidase activity, muscle differentiation, and the NF-kappaB/iNOS pathway. Inhibition of PI 3-kinase and p38 MAPK suppressed the Nox 2/NF-kappaB/iNOS pathway. Nitric oxide restored muscle differentiation blocked by treatment with antioxidants or suppression of the Nox 2/NF-kappaB/iNOS pathway. In conclusion, Nox 2 stimulates muscle differentiation downstream of the PI 3-kinase/p38 MAPK pathway by activating the NF-kappaB/iNOS pathway via ROS generation.

Gold compound auranofin inhibits IkappaB kinase (IKK) by modifying Cys-179 of IKKbeta subunit.

Antirheumatic gold compounds have been shown to inhibit NF-kappaB activation by blocking IkappaB kinase (IKK) activity. To examine the possible inhibitory mechanism of gold compounds, we expressed wild type and mutant forms of IKKalpha and beta 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 IKKbeta 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 IKKalpha (C178A) mutant expressed in the cells. Auranofin inhibited constitutively active IKKalpha and beta and variants; IKKalpha (S176E, S180E) or IKKbeta (S177E, S181E), suggesting that gold directly cause inhibition of activated enzyme. The different inhibitory effect of auranofin on IKKalpha (C178A) and IKKbeta (C179A) mutants indicates that gold could inhibit the two subunits of IKK in a different mode, and the inhibition of NF-kappaB and IKK activation induced by inflammatory signals in gold-treated cells appears through its interaction with Cys-179 of IKKbeta.

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.

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

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