Ionizing radiation induces vascular smooth muscle cell senescence through activating NF-κB/CTCF/p16 pathway.

Radiation injury of blood vessels (RIBV) is a serious long-term complication of radiotherapy, characterized by the development of atherosclerosis. The involvement of vascular smooth muscle cells (VSMCs) senescence in the pathogenesis of radiation-induced atherosclerosis has been implicated, yet the precise mechanisms governing VSMCs senescence remain inadequately comprehended. In this study, the senescence of VSMCs was examined by employing SA-ß-gal staining and assessing the expression of p16 and p21, both in vivo and in vitro. Our findings revealed that ionizing radiation (IR) has the potential to augment cellular senescence. In addition, IR significantly activated the NF-κB pathway, as evidenced by increased p65 nuclear translocation, phospho-p65 expression, and enhanced binding ability of p65 (EMSA). Furthermore, a decrease in HMGB2 expression following exposure to IR was observed via Western blot analysis, while CTCF expression remained unchanged. Interestingly, the formation of CTCF spatial clustering was detected under super-resolution fluorescence microscopy. Concurrently, the ChIP technique identified the facilitation of the interaction between CTCF and p16 gene through IR. The inhibition of CTCF or the overexpression of HMGB2 through lentiviruses effectively eliminates the formation of CTCF clusters and the upregulation of p16 and p21 after IR. Inhibition of NF-κB activation induced by IR by PDTC (100 µM) led to a decrease in the staining of SA-ß-gal, a reduction in p16 expression, an increase in HMGB2 protein expression and a decrease in CTCF clusters formation. This study provided significant insights into the role and mechanism of IR in VSMCs senescence by regulating NF-κB/CTCF/p16 pathway.

Targeting HMG-CoA synthase 2 suppresses tamoxifen-resistant breast cancer growth by augmenting mitochondrial oxidative stress-mediated cell death.

AIMS: In this study, we aimed to investigate previously unrecognized lipid metabolic perturbations in tamoxifen-resistant breast cancer (BC) by conducting comprehensive metabolomics and transcriptomics analysis. We identified the role of 3-hydroxy-3-methylglutary-coenzyme-A-synthase 2 (HMGCS2), a key enzyme responsible for ketogenesis, in tamoxifen-resistant BC growth. MAIN METHODS: Comprehensive metabolomics (CE-TOFMS, LC-TOFMS) and transcriptiomics analysis were performed to characterize metabolic pathways in tamoxifen-resistant BC cells. The upregulation of HMGCS2 were verified thorugh immunohistochemistry (IHC) in clinical samples obtained from patients with recurrent BC. HMGCS2 inhibitor was discovered through surface plasmon resonance analysis, enzyme assay, and additional molecular docking studies. The effect of HMGCS2 suppression on tumor growth was studied thorugh BC xenograft model, and intratumoral lipid metabolites were analyzed via MALDI-TOFMS imaging. KEY FINDINGS: We revealed that the level of HMGCS2 was highly elevated in both tamoxifen-resistant T47D sublines (T47D/TR) and clinical refractory tumor specimens from patients with ER+ breast cancer, who had been treated with adjuvant tamoxifen. Suppression of HMGCS2 in T47D/TR resulted in the accumulation of mitochondrial reactive oxygen species (mtROS) and apoptotic cell death. Further, we identified alphitolic acid, a triterpenoid natural product, as a novel HMGCS2-specific inhibitor that elevated mtROS levels and drastically retarded the growth of T47D/TR in in vitro and in vivo experiments. SIGNIFICANCE: Enhanced ketogenesis with upregulation of HMGCS2 is a potential metabolic vulnerability of tamoxifen-resistant BC that offers a new therapeutic opportunity for treating patients with ER+ BC that are refractory to tamoxifen treatment.

Puerarin from Pueraria lobate attenuates ischemia-induced cardiac injuries and inflammation in vitro and in vivo: The key role of miR-130a-5p/HMGB2 pathway.

Acute myocardial infarction (AMI) is a common cardiovascular disease and puerarin (Pue) is an active compound from Pueraria lobate with cardio-protective potential. In the current study, the mechanism underlying the cardio-protective effects of Pue was explored by focusing miR-130a-5p/HMGB2 pathway. MiR expression profile was determined and myocardial infarction was induced in cardiomyocytes and rats, which was treated with Pue. The role of miR-130a-5p and downstream HMGB2/NF-κB axis in the cardio-protective effects of Pue was also explored. Pue increased viability and suppressed inflammation in OGD cardiomyocytes, which was associated with the deactivation of HMGB2/NF-κB pathway. After the suppression of miR-130a-5p, the cardio-protective effects of Pue were compromised. In rat models, Pue attenuated structure deterioration and inflammatory response in heart. At the molecular level, miR-130a-5p was up-regulated, and HMGB2 were down-regulated. It was demonstrated that Pue induced the expression of miR-130a-5p, which suppressed the activity of HMGB2/NF-κB, contributing to the attenuation of infarct heart tissues.

Loss of Endogenous HMGB2 Promotes Cardiac Dysfunction and Pressure Overload-Induced Heart Failure in Mice.

BACKGROUND: The rapid increase in the number of heart failure (HF) patients in parallel with the increase in the number of older people is receiving attention worldwide. HF not only increases mortality but decreases quality of life, creating medical and social problems. Thus, it is necessary to define molecular mechanisms underlying HF development and progression. HMGB2 is a member of the high-mobility group superfamily characterized as nuclear proteins that bind DNA to stabilize nucleosomes and promote transcription. A recent in vitro study revealed that HMGB2 loss in cardiomyocytes causes hypertrophy and increases HF-associated gene expression. However, it's in vivo function in the heart has not been assessed. Methods and Results: Western blotting analysis revealed increased HMGB2 expression in heart tissues undergoing pressure overload by transverse aorta constriction (TAC) in mice. Hmgb2 homozygous knockout (Hmgb2-/-) mice showed cardiac dysfunction due to AKT inactivation and decreased sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a activity. Compared to wild-type mice, Hmgb2-/- mice had worsened cardiac dysfunction after TAC surgery, predisposing mice to HF development and progression. CONCLUSIONS: This study demonstrates that upregulation of cardiac HMGB2 is an adaptive response to cardiac stress, and that loss of this response could accelerate cardiac dysfunction, suggesting that HMGB2 plays a cardioprotective role.

HMGB2 stabilizes p53 by interfering with E6/E6AP-mediated p53 degradation in human papillomavirus-positive HeLa cells.

We investigated the effect of HMGB2 on the stability of p53 protein in HeLa cells. Overexpression of HMGB2 led to accumulation of the p53 protein, whereas HMGB2 knockdown with siRNA resulted in a substantial decrease in the p53 protein level. The HMGB2-dependent increase of p53 stability was specific for HPV-positive HeLa cells as HCT116 and MCF7 cell lines did not demonstrate this response. Co-expression of HMGB2 and HPV E6 prevented HPV E6 protein-mediated ubiquitination and degradation of p53. FACS analysis exhibited that HeLa cells transfected with HMGB2 displayed decreased cell proliferation, with a concomitant increase of the p53 protein and arrest of the cell cycle, predominantly in G1 phase. Our findings collectively suggest that HMGB2 could stabilize p53 by interfering with E6/E6AP-mediated p53 degradation in HPV-positive HeLa cells.

The interaction of the estrogen receptor with mononucleosomes.

To directly activate specific gene expression, the estrogen receptor (ER) must bind to estrogen receptor response elements (EREs) in the context of nucleosomes. In order to investigate the interaction of the ER with mononucleosomes, we developed a mononucleosome gel shift assay. A 164 bp high specific activity [(32)P]probe DNA (32 bp consensus ERE with flanking regions separated by 23 nucleotides from an artificial nucleosome positioning sequence) was prepared. Nuclear extracts from MCF-7 cells or recombinant human ERalpha were incubated with the labeled ERE +/- excess ERE. A retarded band was seen which was completely obliterated with excess ERE, confirming the specificity of binding. This probe was then used to make reconstituted mononucleosomes by sequential dilution of a high salt histone preparation. The nucleosomes were purified by sucrose density gradients and footprinting analysis was performed to demonstrate that the mononucleosomes were rotationally phased as seen by a periodic digestion pattern (10 bp) of the nucleosomes versus ERE. Nucleosomes were incubated with nuclear extracts containing ER or recombinant ERalpha. Dose dependence in the shift of the mononucleosomes with increasing concentrations of ER was observed. Specificity was demonstrated in experiments with excess ERE and anti-ER antibody. Footprinting analysis was also performed. We also determined that addition of high mobility group protein-2 (HMGB-2, a protein closely related to HMGB-1) with the ER increased the interaction of ER with mononucleosomes. These studies will allow us to address the interactions of ER with core histones containing a multiplicity of variants and modifications in nucleosomal structure.