Inflammation is a hallmark of many important human diseases. Appropriate inflammation is critical for host defense. However, overactive response is detrimental to the host. Thus inflammation must be tightly regulated. The molecular mechanisms underlying the tight regulation of inflammation remain largely unknown. Ecotropic viral integration site 1 (EVI1), a proto-oncogene and zinc finger transcription factor, plays important roles in the normal development and leukemogenesis. However, its role in regulating NF-κB-dependent inflammation remains unknown. Here, we show that EVI1 negatively regulates nontypeable Haemophilus influenzae (NTHi)- and TNF-α-induced NF-κB-dependent inflammation in vitro and in vivo. EVI1 directly binds to the NF-κB p65 subunit and inhibits its acetylation at lysine 310, thereby inhibiting its DNA binding activity. Moreover, expression of EVI1 itself is induced by NTHi and TNF-α in a NF-κB-dependent manner, thereby unveiling a novel inducible negative feedback loop to tightly control NF-κB-dependent inflammation. Thus our study may not only provide important insights into the novel role of EVI1 in negatively regulating NF-κB-dependent inflammation, but may also shed light on the future development of novel anti-inflammatory strategies.

had only a modest contribution to S-glutathionylation, and Cys 120 was modulated by extracellular oxidants but not intracellular GSSG. Simulation modeling of Kir6.1 S-glutathionylation suggested that after incorporation to residue 176, the GSH moiety occupied a space between the slide helix and two transmembrane helices. This prevented the inner transmembrane helix from undergoing conformational changes necessary for channel gating, retaining the channel in its closed state. ATP-sensitive Kϩ (K ATP ) 7 channels are expressed in a variety of tissues, including smooth muscles, pancreatic ␤-cells, myocardium, and neurons, where they play an important role in cellular function (1, 2). Activity of the K ATP channels is tuned by physiological or pathophysiological stimuli, including hypoxia, hyperglycemia, ischemia, and oxidative stress, allowing a regulation of cellular excitability according to the metabolic state (3). The vascular smooth muscle (VSM) isoform of K ATP channels regulates vascular tones (4, 5). Activation of the channel by vasodilators produces hyperpolarization of VSM cells, reduces activity of the voltage-dependent Ca 2ϩ channels, and relaxes VSMs. Inhibition of the channel leads to constriction of VSMs. Disruption of the vascular K ATP channel in mice results in vasospasm in coronary arteries and sudden cardiac death (6, 7).Other studies have further shown that disruption of the vascular K ATP channel has drastic effects on the systemic response to septic stress. With a forward genetic approach by genomewide random chemical mutagenesis, Croker et al. (8) screened a large population of mice and found four strains that are highly susceptible to multiple septic pathogens, including lipopolysaccharides (LPSs). The LPS hypersensitivity phenotype of these mice is due to a null allele of Kcnj8, encoding the Kir6.1 subunit of the vascular K ATP channel (8). Similar septic susceptibility has been observed in Kcnj8-knock-out mice that also show coronary hypoperfusion and myocardial ischemia during LPS exposure (9). These studies thus indicate that the vascular K ATP channel not only contributes to the vascular tone regulation at physiological conditions but also affects critically systemic stress responses.Our recent studies have shown that the vascular K ATP channel is strongly inhibited in oxidative stress by S-glutathionylation (10). S-Glutathionylation is a post-translational modification mechanism occurring in a variety of physiological or pathophysiological conditions (11). This protein modulation mechanism is remarkable especially in vasculatures because oxidative stress is a major contributing factor to several cardiovascular diseases, in which S-glutathionylation plays an important role (12). Although S-glutathionylation is often associated with the adverse effects of oxidative stress, such a protein modulation is reversible under certain circumstances and can act as a functional modulation mechanism like protein phosphorylation (11). Thus, demonstration of how S-glutathionylation * This work was s...

Diabetes mellitus is characterized by hyperglycemia and excessive production of intermediary metabolites including methylglyoxal (MGO), a reactive carbonyl species that can lead to cell injuries. Interacting with proteins, lipids, and DNA, excessive MGO can cause dysfunction of various tissues, especially the vascular walls where diabetic complications often take place. However, the potential vascular targets of excessive MGO remain to be fully understood. Here we show that the vascular Kir6.1/SUR2B isoform of ATP-sensitive K(+) (K(ATP)) channels is likely to be disrupted with an exposure to submillimolar MGO. Up to 90% of the Kir6.1/SUR2B currents were suppressed by 1 mM MGO with a time constant of ∼2 h. Consistently, MGO treatment caused a vast reduction of both Kir6.1 and SUR2B mRNAs endogenously expressed in the A10 vascular smooth muscle cells. In the presence of the transcriptional inhibitor actinomycin-D, MGO remained to lower the Kir6.1 and SUR2B mRNAs to the same degree as MGO alone, suggesting that the MGO effect is likely to compromise the mRNA stability. Luciferase reporter assays indicated that the 3'-untranslated regions (UTRs) of the Kir6.1 but not SUR2 mRNA were targeted by MGO. In contrast, the SUR2B mRNAs obtained with in vitro transcription were disrupted by MGO directly, while the Kir6.1 transcripts were unaffected. Consistent with these results, the constriction of mesenteric arterial rings was markedly augmented with an exposure to 1 mM MGO for 2 h, and such an MGO effect was totally eliminated in the presence of glibenclamide. These results therefore suggest that acting on the 3'-UTR of Kir6.1 and the coding region of SUR2B, MGO causes instability of Kir6.1 and SUR2B mRNAs, disruption of vascular K(ATP) channels, and impairment of arterial function.

Otitis media (OM) is the most common childhood bacterial infection, and leading cause of conductive hearing loss. Nontypeable Haemophilus influenzae (NTHi) is a major bacterial pathogen for OM. OM characterized by the presence of overactive inflammatory responses is due to the aberrant production of inflammatory mediators including C-X-C motif chemokine ligand 5 (CXCL5). The molecular mechanism underlying induction of CXCL5 by NTHi is unknown. Here we show that NTHi up-regulates CXCL5 expression by activating IKKβ-IκBα and p38 MAPK pathways via NF-κB nuclear translocation-dependent and -independent mechanism in middle ear epithelial cells. Current therapies for OM are ineffective due to the emergence of antibiotic-resistant NTHi strains and risk of side effects with prolonged use of immunosuppressant drugs. In this study, we show that curcumin, derived from Curcuma longa plant, long known for its medicinal properties, inhibited NTHi-induced CXCL5 expression in vitro and in vivo. Curcumin suppressed CXCL5 expression by direct inhibition of IKKβ phosphorylation, and inhibition of p38 MAPK via induction of negative regulator MKP-1. Thus, identification of curcumin as a potential therapeutic for treating OM is of particular translational significance due to the attractiveness of targeting overactive inflammation without significant adverse effects.

Diabetes is characterized by hyperglycemia and excessive production of metabolites. One of these metabolites is the highly reactive carbonyl, methylglyoxal (MGO). MGO can readily react with biomolecules leading to cellular dysfunction. Here we showed that acute MGO application led to a dose-dependent activation of KATP channels, a major vascular tone regulator and a critical pharmacological target for treating diabetes. Both Kir6.1 and Kir6.2 containing KATP channels were targeted by MGO in a SUR subunit independent manner. Single channel analysis showed that MGO mediated KATP channel activation via enhancement of channel open probability, leaving the channel conductance unaltered. This modulation appeared to be due to the direct interaction of MGO with the KATP channel, without the need for additional cell signaling pathways. Moreover, MGO mediated KATP channel activity was completely reversed with bath solution washout. Taken together, these data suggest that acute exposure to MGO activates KATP channels through direct, non-covalent and reversible interactions with the Kir6.x subunit, suggest a potential target for pharmacological intervention towards vascular complications of diabetes.