Recurrent Cerebral Artery Dissection Associated with Seronegative Antiphospholipid Antibody Syndrome.

Stroke in young patients requires thorough evaluation as they often lack risk factors. Antiphospholipid syndrome can cause arterial thrombosis and pregnancy loss; hence, differential diagnoses should include seronegative antiphospholipid syndrome. We report a case of recurrent ischemic stroke caused by recurrent dissection in a patient with a history of pregnancy loss. A 33-year-old woman was admitted with global aphasia and right hemiparesis. During intra-arterial thrombectomy, a left middle cerebral artery dissection was detected. After 5.5 years, she was re-admitted for dysarthria, left facial palsy, subtle left hemiparesis, and right middle cerebral artery dissection. She tested negative for autoimmune diseases and vasculitis. However, underlying pathologic conditions could not be excluded because of the unique disease course. Finally, she was diagnosed with seronegative antiphospholipid syndrome. The concept of seronegative antiphospholipid syndrome has been proposed for patients with clinical features suggestive of antiphospholipid syndrome but with negative titers. However, this syndrome can only be diagnosed by exclusion. Furthermore, arterial dissection should be considered to be its main pathology. Antiphospholipid syndrome itself can be a risk factor for arterial dissection because it weakens the vessel walls. Therefore, diagnosis is important to prevent future complications in young patients with recurrent cerebral artery dissection, especially those associated with pregnancy-related morbidities.

Reactive oxygen species induce Cys106-mediated anti-parallel HMGB1 dimerization that protects against DNA damage.

Oxidative stress can induce covalent disulfide bond formation between protein-protein thiol groups and generate hydroxyl free radicals that damage DNA. HMGB1 is a DNA chaperone and damage-associated molecular pattern molecule. As a redox-sensitive protein, HMGB1 contains three cysteine residues: Cys23, Cys45, and Cys106. In this study, we focused on the relationship between HMGB1 dimerization and DNA stabilization under oxidative stress conditions. HMGB1 dimerization was positively modulated by CuCl2 and H2O2. Mutation of the Cys106 residue blocked dimer formation. Treatment of HEK293T cells with CuCl2 and H2O2 enhanced the oxidative self-dimerization of HMGB1, whereas this dimerization was inhibited in mutant HMGB1C106A cells. Furthermore, we performed a bimolecular fluorescence complementation assay to visualize Cys106 oxidation-induced HMGB1 dimerization in live cells exposed to oxidative stress and were able to reproduce the dimerization effect of HMGB1 in fluorescence resonance energy transfer analysis. Interestingly, dimerized HMGB1 bound to DNA with higher affinity than monomeric HMGB1. Dimerized HMGB1 protected DNA from damage due to hydroxyl free radicals and prevented cell death. In conclusion, dimerized HMGB1 may play a regulatory role in DNA stabilization under oxidative stress.

Sulfatide Inhibits HMGB1 Secretion by Hindering Toll-Like Receptor 4 Localization Within Lipid Rafts.

The high mobility group box 1 (HMGB1) is a well-known late mediator of sepsis, secreted by multiple stimuli, involving pathways, such as the mitogen-activated protein kinase (MAPK) and nuclear factor kappa B (NF-κB) pathways, and reactive oxygen species (ROS) under inflammation. Sulfatide, in contrast, is a sphingolipid commonly found in myelin sheets with a disputed immunological role. We sought to determine the immunological characteristics of sulfatide in the periphery by analyzing the secretion of HMGB1 triggered by lipopolysaccharide (LPS) stimulation in Raw 264.7 cells. Suppression of HMGB1 secretion by inhibiting its cytosolic translocation was observed after pre-treatment with sulfatide before LPS stimulation. Further analysis of the downstream molecules of toll-like receptor (TLR) signaling revealed suppression of c-Jun N-terminal kinase (JNK) phosphorylation and p65 translocation. LPS-mediated ROS production was also decreased when sulfatide pre-treatment was provided, caused by the down-regulation of the phosphorylation of activators, such as IRAK4 and TBK1. Investigation of the upstream mechanism that encompasses all the aforementioned inhibitory characteristics unveiled the involvement of lipid rafts. In addition to the co-localization of biotinylated sulfatide and monosialotetrahexosylganglioside, a decrease in LPS-induced co-localization of TLR4 and lipid raft markers was observed when sulfatide treatment was given before LPS stimulation. Overall, sulfatide was found to exert its anti-inflammatory properties by hindering the co-localization of TLR4 and lipid rafts, nullifying the effect of LPS on TLR4 signaling. Similar effects of sulfatide were also confirmed in the LPS-mediated murine experimental sepsis model, showing decreased levels of serum HMGB1, increased survivability, and reduced pathological severity.

Immunological Significance of HMGB1 Post-Translational Modification and Redox Biology.

Most extracellular proteins are secreted via the classical endoplasmic reticulum (ER)/Golgi-dependent secretion pathway; however, some proteins, including a few danger-associated molecular patterns (DAMPs), are secreted via non-classical ER/Golgi-independent secretion pathways. The evolutionarily conserved high mobility group box1 (HMGB1) is a ubiquitous nuclear protein that can be released by almost all cell types. HMGB1 lacks signal peptide and utilizes diverse non-canonical secretion mechanisms for its extracellular export. Although the post-translational modifications of HMGB1 were demonstrated, the oxidation of HMGB1 and secretion mechanisms are not highlighted yet. We currently investigated that peroxiredoxins I and II (PrxI/II) induce the intramolecular disulfide bond formation of HMGB1 in the nucleus. Disulfide HMGB1 is preferentially transported out of the nucleus by binding to the nuclear exportin chromosome-region maintenance 1 (CRM1). We determined the kinetics of HMGB1 oxidation in bone marrow-derived macrophage as early as a few minutes after lipopolysaccharide treatment, peaking at 4 h while disulfide HMGB1 accumulation was observed within the cells, starting to secrete in the late time point. We have shown that HMGB1 oxidation status, which is known to determine the biological activity in extracellular HMGB1, is crucial for the secretion of HMGB1 from the nucleus. This review summarizes selected aspects of HMGB1 redox biology relevant to the induction and propagation of inflammatory diseases. We implicate the immunological significance and the need for novel HMGB1 inhibitors through mechanism-based studies.

HMGB1 modulates the balance between senescence and apoptosis in response to genotoxic stress.

High mobility group box-1 (HMGB1) is involved in various diseases and is associated with the resistance of many types of human cancers to chemotherapy; however, its role in cancer metastasis remains unexplored. This study examined the HMGB1 status of both highly and poorly metastatic cancer cells in response to genotoxic stress. The weakly and highly metastatic mouse melanoma cell lines (B16 vs. B16-F10), human melanoma cell lines (SK-MEL-28 vs. SK-MEL-24), colon cancer cell lines (DLD-1 vs. LS174T), and wild-type (WT) vs. HMGB1 knockout (KO) mouse embryonic fibroblasts (MEFs) were treated with doxorubicin (Dox) and camptothecin (CPT), and then cellular morphology, senescence-associated ß-galactosidase staining, lactate dehydrogenase release, and caspase-3 activation were used to assess cell fate. To investigate the role of HMGB1 in p21 expression, HMGB1 and p21 expressions were examined by Western blotting, and the HMGB1-mediated p21 promoter luciferase assay was performed after small interfering RNA or overexpression of HMGB1 prior to Dox treatment. Although highly metastatic mouse melanoma B16-F10 cells preferred senescence, with persistent HMGB1 expression, poorly metastatic B16 cells entered apoptosis, with decreasing HMGB1 levels via cleavage under Dox treatment. Similarly, more metastatic human melanoma SK-MEL-24 and human colon cancer LS174T cells underwent senescence, whereas fewer metastatic melanoma SK-MEL-28 and DLD-1 cells exhibited apoptosis under Dox stimulation. In senescent B16-F10, SK-MEL-24, and LS174T cells treated with Dox, p21 levels were increased by persistent HMGB1 expression. Furthermore, HMGB1 depletion caused a senescence-apoptosis shift with p21 down-regulation in B16-F10 cells, and HMGB1 overexpression switched from apoptosis to senescence concomitantly with increased p21 expression in B16 cells after Dox treatment. The same effects were observed in both cell pairs of mouse melanoma and human colon cancer cells treated with CPT, another genotoxic stressor. Indeed, although WT MEF entered senescence accompanied by p21 increase, HMGB1 KO underwent apoptosis with p21 decrease by Dox treatment. In our cell model system, we demonstrated that highly metastatic cancer cells preferentially enter senescence, whereas apoptosis predominates in weakly metastatic cancer cells under genotoxic stress, which depends on the presence or absence of HMGB1, suggesting that the HMGB1-p21 axis is required for genotoxic stress-induced senescence. These findings suggest that HMGB1 modulation of cancers with different metastatic status could be a strategy for selectively enforcing tumor suppression.-Lee, J.-J., Park, I. H., Rhee, W. J., Kim, H. S., Shin, J.-S. HMGB1 modulates the balance between senescence and apoptosis in response to genotoxic stress.

Peroxiredoxin-mediated disulfide bond formation is required for nucleocytoplasmic translocation and secretion of HMGB1 in response to inflammatory stimuli.

The nuclear protein HMGB1 (high mobility group box 1) is secreted by monocytes-macrophages in response to inflammatory stimuli and serves as a danger-associated molecular pattern. Acetylation and phosphorylation of HMGB1 are implicated in the regulation of its nucleocytoplasmic translocation for secretion, although inflammatory stimuli are known to induce H2O2 production. Here we show that H2O2-induced oxidation of HMGB1, which results in the formation of an intramolecular disulfide bond between Cys23 and Cys45, is necessary and sufficient for its nucleocytoplasmic translocation and secretion. The oxidation is catalyzed by peroxiredoxin I (PrxI) and PrxII, which are first oxidized by H2O2 and then transfer their disulfide oxidation state to HMGB1. The disulfide form of HMGB1 showed higher affinity for nuclear exportin CRM1 compared with the reduced form. Lipopolysaccharide (LPS)-induced HMGB1 secretion was greatly attenuated in macrophages derived from PrxI or PrxII knockout mice, as was the LPS-induced increase in serum HMGB1 levels.

Sodium butyrate sensitizes human glioma cells to TRAIL-mediated apoptosis through inhibition of Cdc2 and the subsequent downregulation of survivin and XIAP.

In TNF-related apoptosis-inducing ligand (TRAIL)-resistant glioma cells, co-treatment with nontoxic doses of sodium butyrate and TRAIL resulted in a marked increase of TRAIL-induced apoptosis. This combined treatment was also cytotoxic to glioma cells overexpressing Bcl-2 or Bcl-xL, but not to normal human astrocytes, thus offering an attractive strategy for safely treating resistant gliomas. Cotreatment with sodium butyrate facilitated completion of proteolytic processing of procaspase-3 that was partially blocked by treatment with TRAIL alone. We also found that treatment with sodium butyrate significantly decreased the protein levels of survivin and X-linked inhibitor of apoptosis protein (XIAP), two major caspase inhibitors. Overexpression of survivin and XIAP attenuated sodium butyrate-stimulated TRAIL-induced apoptosis, suggesting its involvement in conferring TRAIL resistance to glioma cells. Furthermore, the kinase activities of Cdc2 and Cdk2 were significantly decreased following sodium butyrate treatment, accompanying downregulation of cyclin A and cyclin B, as well as upregulation of p21. Forced expression of Cdc2 plus cyclin B, but not Cdk2 plus cyclin A, attenuated sodium butyrate/TRAIL-induced apoptosis, overriding sodium butyrate-mediated downregulation of survivin and XIAP. Therefore, Cdc2-mediated downregulation of survivin and XIAP by sodium butyrate may contribute to the recovery of TRAIL sensitivity in glioma cells.