Nitric oxide (NO) inhibits vascular inflammation, but the molecular basis for its anti-inflammatory properties is unknown. We show that NO inhibits exocytosis of Weibel-Palade bodies, endothelial granules that mediate vascular inflammation and thrombosis, by regulating the activity of N-ethylmaleimide-sensitive factor (NSF). NO inhibits NSF disassembly of soluble NSF attachment protein receptor (SNARE) complexes by nitrosylating critical cysteine residues of NSF. NO may regulate exocytosis in a variety of physiological processes, including vascular inflammation, neurotransmission, thrombosis, and cytotoxic T lymphocyte cell killing.

The mitogen-activated protein kinase (MAPK) pathway plays a critical role in Toll-like receptor (TLR) signaling. MAPK phosphatase-1 (MKP-1) inhibits the MAPK pathway and decreases TLR signaling, but the regulation of MKP-1 is not completely understood. We now show that MKP-1 is acetylated, and that acetylation regulates its ability to interact with its substrates and deactivate inflammatory signaling. We found that LPS activates acetylation of MKP-1. MKP-1 is acetylated by p300 on lysine residue K57 within its substrate-binding domain. Acetylation of MKP-1 enhances its interaction with p38, thereby increasing its phosphatase activity and interrupting MAPK signaling. Inhibition of deacetylases increases MKP-1 acetylation and blocks MAPK signaling in wild-type (WT) cells; however, deacetylase inhibitors have no effect in cells lacking MKP-1. Furthermore, histone deacetylase inhibitors reduce inflammation and mortality in WT mice treated with LPS, but fail to protect MKP-1 knockout mice. Our data suggest that acetylation of MKP-1 inhibits innate immune signaling. This pathway may be an important therapeutic target in the treatment of inflammatory diseases.

Background and Purpose Carbon monoxide (CO) is a gaseous second messenger produced when heme oxygenase (HO) enzymes catabolize heme. We have demonstrated that CO can be therapeutic in ischemia-reperfusion brain injury; however, it is unclear whether CO can also offer protection in permanent ischemic stroke or what mechanism(s) underlies the effect. HO1 neuroprotection was shown to be regulated by Nrf2; therefore, we investigated whether CO might partially exert neuroprotection by modulating the Nrf2 pathway. Methods To evaluate potential protective effects of CO, we exposed male wildtype and Nrf2 knockout mice to 250 ppm CO or control air for 18 hours immediately after permanent middle cerebral artery occlusion. Infarct volume and neurological deficits were assessed on day 7. Molecular mechanisms of Nrf2 pathway activation by CO were also investigated. Results Mice exposed to CO after permanent ischemia had 29.6±12.6% less brain damage than did controls at 7 days, though amelioration in neurological deficits did not reach significance. Additionally, 18-hour CO treatment led to Nrf2 dissociation from Keap1, nuclear translocation, increased binding activity of Nrf2 to HO1 antioxidant response elements, and elevated HO1 expression 6–48 hours after CO exposure. The CO neuroprotection was completely abolished in Nrf2 knockout mice. Conclusions Low-concentration CO represent a neuroprotective agent for combination treatment of ischemic stroke, and its beneficial effect would be at least partially mediated by activation of the Nrf2 pathway.

Interest in histone deacetylase (HDAC)-based therapeutics as a potential treatment for stroke has grown dramatically. The neuroprotection of HDAC inhibition may involve multiple mechanisms, including modulation of transcription factor acetylation independent of histones. The transcription factor Nrf2 has been shown to be protective in stroke as a key regulator of antioxidant-responsive genes. Here, we hypothesized that HDAC inhibition might provide neuroprotection against mouse cerebral ischemia by activating the Nrf2 pathway. We determined that the classic HDAC inhibitor trichostatin A increased neuronal cell viability after oxygen-glucose deprivation (from an OD value of 0.10±0.01 to 0.25±0.08) and reduced infarct volume in wild-type mice with stroke (from 49.1±3.8 to 21.3±4.6%). In vitro studies showed that HDAC inhibition reduced Nrf2 suppressor Keap1 expression, induced Keap1/Nrf2 dissociation, Nrf2 nuclear translocation, and Nrf2 binding to antioxidant response elements in heme oxygenase 1 (HO1), and caused HO1 transcription. Furthermore, we demonstrated that HDAC inhibition upregulated proteins downstream of Nrf2, including HO1, NAD(P)H:quinone oxidoreductase 1, and glutamate-cysteine ligase catalytic subunit in neuron cultures and brain tissue. Finally, unlike wild-type mice, Nrf2-deficient mice were not protected by pharmacologic inhibition of HDAC after cerebral ischemia. Our studies suggest that activation of Nrf2 might be an important mechanism by which HDAC inhibition provides neuroprotection.

IntroductionOsteoarthritis (OA) is a common joint disease that can cause gradual disability among the aging population. Nuclear factor (erythroid-derived 2)-like 2 (Nrf2) is a key transcription factor that regulates the expression of phase II antioxidant enzymes that provide protection against oxidative stress and tissue damage. The use of histone deacetylase inhibitors (HDACi) has emerged as a potential therapeutic strategy for various diseases. They have displayed chondroprotective effects in various animal models of arthritis. Previous studies have established that Nrf2 acetylation enhances Nrf2 functions. Here we explore the role of Nrf2 in the development of OA and the involvement of Nrf2 acetylation in HDACi protection of OA.MethodsTwo OA models—monosodium iodoacetate (MIA) articular injection and destabilization of the medial meniscus (DMM)—were used with wild-type (WT) and Nrf2-knockout (Nrf2-KO) mice to demonstrate the role of Nrf2 in OA progression. A pan-HDACi, trichostatin A (TSA), was administered to examine the effectiveness of HDACi on protection from cartilage damage. The histological sections were scored. The expression of OA-associated matrix metalloproteinases (MMPs) 1, 3, and 13 and proinflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6 were assayed. The effectiveness of HDACi on OA protection was compared between WT and Nrf2-KO mice.ResultsNrf2-KO mice displayed more severe cartilage damage in both the MIA and DMM models. TSA promoted the induction of Nrf2 downstream proteins in SW1353 chondrosarcoma cells and in mouse joint tissues. TSA also reduced the expression of OA-associated proteins MMP1, MMP3, and MMP13 and proinflammatory cytokines TNF-α, IL-1β, and IL-6. TSA markedly reduced the cartilage damage in both OA models but offered no significant protection in Nrf2-KO mice.ConclusionsNrf2 has a major chondroprotective role in progression of OA and is a critical molecule in HDACi-mediated OA protection.

CBFb-SMMHC is expressed from the inv(16) chromosome in M4Eo AML. Mice lacking CBF subunits or expressing the CBFb-SMMHC or AML1-ETO oncoproteins failed to develop de®nitive hematopoiesis. To investigate these e ects on hematopoiesis, we expressed CBFb-SMMHC from the metallothionein promoter, in both 32D cl3 myeloid cells and Ba/F3 B-lymphoid cells. Addition of zinc increased CBFb-SMMHC levels more than tenfold, with higher levels evident in Ba/F3 lines. Levels obtained in 32D cl3 cells were similar to those of endogenous CBFb. Indirect immuno¯uorescence revealed zinc-inducible speckled, nuclear staining in Ba/F3 cells and di use nuclear staining in 32D cl3 cells. CBFb-SMMHC reduced endogenous CBF DNA-binding ®vefold in both cell types, increased cell generation time 1.9-fold, on average, in 32D cl3 cells and 1.5-fold in Ba/ F3 cells and decreased tritiated thymidine incorporation into DNA correspondingly. CBFb-SMMHC increased the proportion of cells in G1 1.7-fold, on average, in 32D cl3 and Ba/F3 cells, and decreased the proportion of cells in S phase by a similar degree. CBFb-SMMHC induced a marked increase in hypophosphorylated Rb, but did not alter IL-3 Receptor a or b subunit levels. Neither apoptosis nor 32D di erentiation was induced by zinc in IL-3 in these lines. Induction of CBFb-SMMHC in 32D cl3 cells did not inhibit their di erentiation to neutrophils or their expression of myeloperoxidase mRNA in G-CSF, and did not produce an eosinophilic phenotype. Additional, proliferative genetic changes in M4eo AMLs might potentiate inhibition of di erentiation by CBFb-SMMHC by allowing its increased expression.

Hemoproteins undergo degradation during hypoxic/ischemic conditions, but the pro-oxidant free heme that is released cannot be recycled and must be degraded. The extracellular heme associates with its high-affinity binding protein, hemopexin (HPX). Hemopexin is shown here to be expressed by cortical neurons and it is present in mouse cerebellum, cortex, hippocampus, and striatum. Using the transient ischemia model (90-min middle cerebral artery occlusion followed by 96-h survival), we provide evidence that HPX is protective in the brain, as neurologic deficits and infarct volumes were significantly greater in HPX−/− than in wild-type mice. Addressing the potential protective HPX cellular pathway, we observed that exogenous free heme decreased cell survival in primary mouse cortical neuron cultures, whereas the heme bound to HPX was not toxic. Heme–HPX complexes induce HO1 and, consequently, protect primary neurons against the toxicity of both heme and prooxidant tert-butyl hydroperoxide; such protection was decreased in HO1−/− neuronal cultures. Taken together, these data show that HPX protects against heme-induced toxicity and oxidative stress and that HO1 is required. We propose that the heme–HPX system protects against stroke-related damage by maintaining a tight balance between free and bound heme. Thus, regulating extracellular free heme levels, such as with HPX, could be neuroprotective.

The MAPK pathway mediates TLR signaling during innate immune responses. We discovered previously that MKP-1 is acetylated, enhancing its interaction with its MAPK substrates and deactivating TLR signaling. As HDACs modulate inflammation by deacetylating histone and nonhistone proteins, we hypothesized that HDACs may regulate LPS-induced inflammation by deacetylating MKP-1. We found that mouse macrophages expressed a subset of HDAC isoforms (HDAC1, HDAC2, and HDAC3), which all interacted with MKP-1. Genetic silencing or pharmacologic inhibition of HDAC1, -2, and -3 increased MKP-1 acetylation in cells. Furthermore, knockdown or pharmacologic inhibition of HDAC1, -2, and -3 decreased LPS-induced phosphorylation of the MAPK member p38. Also, pharmacologic inhibition of HDAC did not decrease MAPK signaling in MKP-1 null cells. Finally, inhibition of HDAC1, -2, and -3 decreased LPS-induced expression of TNF-α, IL-1β, iNOS (NOS2), and nitrite synthesis. Taken together, our results show that HDAC1, -2, and -3 deacetylate MKP-1 and that this post-translational modification increases MAPK signaling and innate immune signaling. Thus, HDAC1, -2, and -3 isoforms are potential therapeutic targets in inflammatory diseases.