Cytosolic HMGB1 controls the cellular autophagy/apoptosis checkpoint during inflammation
Xiaorong Zhu, … , David L. Boone, Eugene B. Chang
Xiaorong Zhu, … , David L. Boone, Eugene B. Chang
Published February 2, 2015
Citation Information: J Clin Invest. 2015;125(3):1098-1110. https://doi.org/10.1172/JCI76344.
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Research Article Gastroenterology

Cytosolic HMGB1 controls the cellular autophagy/apoptosis checkpoint during inflammation

Abstract

The intracellular protein HMGB1 is released from cells and acts as a damage-associated molecular pattern molecule during many diseases, including inflammatory bowel disease (IBD); however, the intracellular function of HMGB1 during inflammation is poorly understood. Here, we demonstrated that cytosolic HMGB1 regulates apoptosis by protecting the autophagy proteins beclin 1 and ATG5 from calpain-mediated cleavage during inflammation. Colitis in mice with an intestinal epithelial cell–specific Hmgb1 deletion and patients with IBD were both characterized by increased calpain activation, beclin 1 and ATG5 cleavage, and intestinal epithelial cell (IEC) death compared with controls. In vitro cleavage assays and studies of enteroids verified that HMGB1 protects beclin 1 and ATG5 from calpain-mediated cleavage events that generate proapoptotic protein fragments. Together, our results indicate that HMGB1 is essential for mitigating the extent and severity of inflammation-associated cellular injury by controlling the switch between the proautophagic and proapoptotic functions of beclin 1 and ATG5 during inflammation. Moreover, these studies demonstrate that HMGB1 is pivotal for reducing tissue injury in IBD and other complex inflammatory disorders.

Authors

Xiaorong Zhu, Jeannette S. Messer, Yunwei Wang, Fanfei Lin, Candace M. Cham, Jonathan Chang, Timothy R. Billiar, Michael T. Lotze, David L. Boone, Eugene B. Chang

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Figure 6

IBD in humans is associated with decreased HMGB1, beclin 1, and ATG5 cleavage and increased cell death.

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IBD in humans is associated with decreased HMGB1, beclin 1, and ATG5 cle...
(A) HMGB1 expression by qRT-PCR in controls (n = 6) and in patients with active UC (n = 5) or indeterminate colitis (IC) (n = 1) (mean ± SEM). Immunoblot for HMGB1 protein in lysates of snap-frozen endoscopic biopsies. Lanes 1–4, controlled CD patients; lanes 5–8, active CD patients (mean ± SEM). (B) Confocal microscopic images of endoscopic biopsies stained for Hoechst (blue) and HMGB1 (red) in controls (n = 3) and in patients with active UC (n = 4) (original magnification, ×400). Colocalization evaluated using Pearson’s correlation coefficient with the Costes correction (mean ± SEM). (C) Beclin 1 immunoblot. Lane 1, control/normal; lane 2, control/normal; lane 3, control/quiescent CD; lane 4, moderate UC; lane 5, moderate UC; and lane 6, severe CD (mean ± SEM). (D) Immunoblot for ATG5 in lysates of snap-frozen endoscopic biopsies (mean ± SEM). Samples were loaded as in A. (E) Immunoblot with antibody recognizing the active p19/p17 fragments of cleaved caspase 3. Samples were loaded as in Figure 4D. (F) Immunoblot for the active p20 fragment of cleaved caspase 1. Samples were loaded as in Figure 4D. (G) Calpain activity as evaluated by cleavage of the fluorescent substrate in samples from controls (n = 3) and from patients with active UC (n = 3) (mean ± SEM). (H) Calpastatin levels analyzed by qRT-PCR in controls (n = 6) and in patients with active UC (n = 5) or IC (n = 1) (mean ± SEM). Data were analyzed by 2-tailed Student’s t tests, except for data in G, which were determined by the 1-tailed Student’s t test. *P < 0.05; **P < 0.01; ****P < 0.001.