2-O, 3-O Desulfated Heparin Blocks High Mobility Group Box 1 Release by Inhibition of p300 Acetyltransferase Activity

Abstract

High mobility group box 1 (HMGB1) is an alarmin released from macrophages after infection or inflammation and is a biomarker of lung disease progression in patients with cystic fibrosis. We reported that 2-O, 3-O desulfated heparin (ODSH) inhibits the release of HMGB1 from murine macrophages triggered by neutrophil elastase both in vivo and in vitro. HMGB1 shuttles between the nucleus and the cytoplasm. When acetylated at lysine residues in the nuclear localization signal domains, HMGB1 is sequestered in the cytoplasm and is fated for secretion. In this study, we investigated the mechanism by which ODSH blocks HMGB1 secretion. We tested whether ODSH inhibits the activity of p300, a histone acetyltransferase that has been linked to HMGB1 acetylation and release. ODSH inhibited both neutrophil elastase and LPStriggered HMGB1 release from the murine macrophage cell line RAW264.7 in a concentration-dependent manner. Fluoresceinlabeled ODSH was taken up by RAW264.7 cells into the cytoplasm as well as the nucleus, suggesting an intracellular site of action of ODSH for blocking HMGB1 release. ODSH inhibited RAW264.7 cell nuclear extract, human macrophage nuclear extract, and recombinant p300 HAT activity in vitro, resulting in the failure to acetylate HMGB1. In silico molecular modeling predicted that of the numerous possible ODSH sequences, a small number preferentially recognizes a specific binding site on p300. Fluorescence binding studies showed that ODSH bound p300 tightly (dissociation constant z1 nM) in a highly cooperative manner. These results suggest that ODSH inhibited HMGB1 release, at least in part, by direct molecular inhibition of p300 HAT activity.Keywords: p300; heparin; high mobility group box 1 Clinical RelevanceWe report that a modified heparin, 2-O, 3-O desulfated heparin (ODSH), inhibits high mobility group box 1 release from macrophages via inhibition of a histone acetyltransferase, p300, required for acetylation. This is a novel mechanism by which ODSH blocks inflammation, and this finding supports the development of ODSH as an antiinflammatory therapy for cystic fibrosis.High mobility group box 1 (HMGB1) is found at high concentrations in the airways of patients with chronic inflammatory lung diseases. Increased airway HMGB1 is associated with bronchopulmonary dysplasia in preterm infants (1), severe asthma in children (2), and chronic obstructive pulmonary disease in adults (3, 4). HMGB1 is a biomarker for lung disease progression in patients with cystic fibrosis (CF) (4, 5). Furthermore, a polymorphism in the gene Ager, which encodes the HMGB1 receptor, receptor for advanced glycation end products (RAGE), results in increased RAGE expression

Non-Anticoagulant Heparins as Heparanase Inhibitors

“…Along with a high potency in inhibiting heparanase, 2OdesH showed in vivo antitumor activity in CaPan-2 pancreatic adenocarcinoma xenografts and antimetastatic activity in the B16-F10 mouse melanoma experimental model [88,89]. Recently, 2OdesH has been described to block the release of the inflammation mediator, high mobility group box 1, by inhibition of p300 acetyltransferase activity [90]. A 2OdesH is currently under clinical investigation as CX01 in combination treatment of acute myeloid leukemia [Sect.…”

Molecular principles for heparin oligosaccharide–based inhibition of neutrophil elastase in cystic fibrosis

“…To assess whether DNA and heparins compete for an overlapping site of binding on NE, we used computational molecular modeling. We used combinatorial virtual library screening (CVLS) to identify sites of heparin binding on proteins (21)(22)(23). The algorithm predicts the binding site residues as well as the most preferred heparin sequence for the target site.…”

“…Molecules built were capped on the termini, and AMBER charges were added and energy-minimized for 10,000 iterations with a termination gradient of 0.05 kcal/ (mol⅐Å). A library of 1,728 ODSH hexasaccharide sequences was constructed based on earlier reports (21,22,37,38) and energy-minimized using Tripos force field with Gasteiger-Hückel charges, a fixed dielectric constant of 80, and a nonbonded cutoff radius of 8 Å.…”

Emerging Role of High Mobility Group Box-1 in Thrombosis-Related Diseases

“…Besides, 2-O,3-O-desulfated heparin (ODSH) inhibited HMGB-1/RAGEmediated airway inflammation [173]. In macrophages, ODSH blocked HMGB-1 secretion via the inhibition of the activity of acetyltransferase p300 [174]. Thus, HMGB-1 might be a new target for heparin during inflammation and thrombosis.…”