Cysteine 106 Controls HMGB1 Nucleocytoplasmic Distribution and Function: Role of Sex and Implication to Pulmonary Hypertension

Abstract

IntroductionHigh mobility group box protein 1 (HMGB1) is a nuclear protein that gets released during cell damage and stimulates inflammatory and proliferative pathways in the survived neighboring cells. Our previous research discovered a significant sex difference in HMGB1 release and downstream signaling and confirmed its critical importance in the pathobiology of pulmonary hypertension (PH).HypothesisHMGB1 contains three cysteine residues (23/45/106) involved in the extracellular HMGB1 oligomerization and receptor binding, with Cys106 being especially critical for HMGB1 signaling. However, the role of cysteines in the intracellular HMGB1 activity is less established. We hypothesize that Cys106 regulates intracellular HMGB1 homeostasis and function and contributes to the pathogenic mechanisms relevant to PH.MethodsTo study the activities of HMGB1 cysteines, we designed peptides that bind to the specific regions of HMGB1 and, by shielding either Cys106 or Cys23/45, prevent their interaction with the downstream targets. The peptides with cell‐penetrating TAT sequences were used to study the intracellular role of HMGB1 cysteines, while peptides without TAT served as extracellular controls. Fractionating of pulmonary artery smooth muscle cells (PASMC) allowed the evaluation of HMGB1 cytosol/nuclear distribution. Calf thymus double‐stranded DNA cellulose was used to study the binding of recombinant fully reduced HMGB1 in the presence or absence of shielding peptide. The protective role of peptide in vivo was estimated using the rat Sugen/Hypoxia model of PH. Given the discovered sex difference in the HMGB1‐mediated signaling, the in vitro and in vivo studies were replicated in male and female animals and cells.ResultsThe cytosol and nuclear levels of HMGB1 were affected by cell‐penetrating peptide designed to shield Cys106 (TAT‐α‐HMGB1Cys106, or α‐Cys106) and not any other peptide. Further investigation revealed that α‐Cys106 peptide produced ~ 5.9 fold decrease in HMGB1 binding to DNA, enhanced the exit of nuclear HMGB1 to the cytosol, which was especially evident in male cells, and induced its proteasomal degradation (1±0.097 vs. 0.428±0.045 vs. 0.885±0.049 in untreated, α‐Cys106 and α‐Cys106/proteasome inhibitor (MG132, 10μM, 1hr) treated PASMC, p<0.001 and p<0.003, N=6). The peptide‐mediated decrease in HMGB1/DNA binding negatively affected the expression of TLR4 and p53 and reduced cell apoptosis. In females, treatment with α‐Cys106 (2.5 mg/kg/day, i.p during the wks 1‐2 or wks 3‐5) produced an anti‐apoptotic effect and reduced plasma HMGB1 levels, which corresponded to a decreased right ventricle (RV) systolic pressure at the early stage of PH and protected the RV at the later stage. In contrast, male PH rats showed no apoptosis activation in the lungs, and α‐Cys106 treatment produced no changes in plasma HMGB1 or hemodynamic parameters.ConclusionHMGB1Cys106 is the critical regulator of intracellular HMGB1 function, including HMGB1/DNA binding, regulation of p53 expression and apoptosis activation, the intracellular distribution of HMGB1, and its extracellular release. This study also confirms the significant sex difference in the levels of pulmonary apoptosis, HMGB1 release, and its contribution to the pathobiology of PH.