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HMGB1 is ubiquitously expressed in most cells to maintain a large "pool" of pre-formed protein in the nucleus[1-2] owing to the existence of two nuclear-localization sequences(NLS) that facilitate its nuclear transportation[3]. Within the nucleus, HMGB1 binds chromosomal DNA, and fulfills its nuclear functions in maintaining nucleosomal structures and regulating gene expression[4]. The complete depletion of HMGB1 expression renders animals more susceptible to infectious[5] or injurious insults[6-7], suggesting an overall protective role of intracellular HMGB1 against stresses[8].
In response to microbial toxins(such as CpG-DNA and endotoxin)[9-10], cytokines[e.g., interferon (IFN)-γ and Cold-inducible RNA-binding protein (CIRP)][11-13] or oxidative free radicals(e.g., hydrogen peroxide)[14], macrophages/monocytes acetylate and/or phosphorylate the NLS of HMGB1[8, 15-17], enabling its sequestration into cytoplasmic vesicles destined for subsequent secretion[2, 11, 18]. Cytoplasmic HMGB1 can be secreted through several pathways, including the double-stranded RNA-activated protein kinase R (PKR)-and Caspase-1/Caspase-11-mediated inflammasome activation and pyroptosis. For instance, genetic disruption PKR expression or pharmacological inhibition of PKR phosphorylation similarly reduces NLRP3 or NLRP1 agonists-induced inflammasome activation[19-20], pyroptosis[19-20] and HMGB1 release[19].
In addition to active secretion, HMGB1 can be passively released from damaged cells[21] following ischemia/reperfusion[22-23], trauma[24-25], or toxemia[26-28], thereby serving as damage-associated molecular pattern(DAMP) molecule. Although radiation emits high energy photons that can ionize atoms and disrupt molecular bonds, it was previously unknown whether it similarly induces HMGB1 cytoplasmic translocation and release. Here we provided evidence that X-ray irradiation induces a time- and dose-dependent HMGB1 cytoplasmic translocation and release by tumor cells in vitro, and stimulates systemic HMGB1 accumulation in vivo.
Ionizing Radiation Induces HMGB1 Cyto-plasmic Translocation and Extracellular Release
Ionizing Radiation Induces HMGB1 Cyto-plasmic Translocation and Extracellular Release
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Abstract:
Objective A nucleosomal protein, HMGB1, can be secreted by activated immune cells or passively released by dying cells, thereby amplifying rigorous inflammatory responses. In this study we aimed to test the possibility that radiation similarly induces cytoplasmic HMGB1 translocation and release. Methods Human skin fibroblast (GM0639) and bronchial epithelial (16HBE) cells and rats were exposed to X-ray radiation, and HMGB1 translocation and release were then assessed by immunocytochemistry and immunoassay, respectively. Results At a wide dose range(4.0-12.0 Gy), X-ray radiation induced a dramatic cytoplasmic HMGB1 translocation, and triggered a time- and dose-dependent HMGB1 release both in vitro and in vivo. The radiation-mediated HMGB1 release was also associated with noticeable chromosomal DNA damage and loss of cell viability. Conclusions Radiation induces HMGB1 cytoplasmic translocation and extracellular release through active secretion and passive leakage processes. -
Key words:
- X-ray /
- HMGB1 /
- Tumor cells /
- Inflammatory response /
- Damage-associated molecule pattern molecules
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Figure 1. Radiation induced cytoplasmic HMGB1 translocation in tumor cells. Human skin fibroblast(GM0639) and bronchial epithelial(16HBE) cells were subjected to 6 MeV X-ray radiation at a dose of 8 Gy for 24 h, and assayed for HMGB1 cytoplasmic translocation by immunohistochemistry using HMGB1-specific antibodies. Note that HMGB1 was predominantly localized in the nuclear region of un-treated cells("control"), but found in both cytoplasmic and nuclear regions of X-ray radiated cells ("6 MeV-X").
Figure 2. Radiation inversely altered nuclear and cytoplasmic HMGB1 levels. Following X-ray radiation, cytoplasmic and nuclear fractions were isolated, and assayed for levels of HMGB1 along with a nuclear(Lamin B1) or cytoplasmic(β-actin) marker by Western blotting analysis. Equal loading of samples was confirmed by Western blotting analysis of respective fractions with cytoplasmic (β-actin) or nuclear (Lamin B1) protein markers.
Figure 3. Radiation induced a dose- and time-dependent HMGB1 release. Human skin fibroblast(GM0639) and/or bronchial epithelial (16HBE) were exposed to X-ray radiation at various doses for different time periods, and extracellular levels of HMGB1 were determined by Western blotting analysis. Note that proteins were recovered from equal volume of cell-conditioned medium, and sample loading was normalized by equal volume of cell-conditioned medium.
Figure 4. Radiation caused DNA damage and loss of cell viability. Human skin fibroblast (GM0639) and bronchial epithelial (16HBE) cells exposed to X-ray at a dose of 4 Gy, and cells were stained with γ-H2AX-specific antibodies to detect DNA damage. In parallel, the cell viability was determined by MTT assay, and expressed as a % of controls in the absence of X-ray radiation. *, P < 0.05 versus untreated control at respective time points.
Figure 5. Radiation elevated circulating HMGB1 levels in vivo. Male Sprague-Dawley rats were exposed to X-ray radiation at various doses and for different time periods, and blood samples were collected to measure serum HMGB1 levels by ELISA. *, P < 0.05 versus untreated controls(no radiation, Panel A; or immediately prior to X-ray radiation, Panel B).
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