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 2

The autophagic response to DSS in the intestinal epithelium is blunted in the absence of HMGB1.

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The autophagic response to DSS in the intestinal epithelium is blunted i...
(A) Confocal microscopic images of colons from untreated (n = 3) or DSS-treated (day 3) (n = 3) Hmgbfl/fl mice stained for the DNA marker Hoechst (blue) and HMGB1 (red) (original magnification, ×400). Colocalization was evaluated using Pearson’s correlation coefficient with the Costes correction (mean ± SEM) (B) qRT-PCR (mean ± SEM) and immunoblot for HMGB1 expression in colonic mucosal scrapings from untreated (n = 4) and DSS-treated (n = 4) Hmgb1fl/fl mice. (C) Immunoblot for HMGB1 in intestinal mucosal scrapings from Hmgb1fl/fl Il10–/– mice with and without signs of colitis (n = 4). (D) Immunoblot for LC3 and p62 in Hmgb1fl/fl (n = 4) and Vil-Cre Hmgb1fl/fl (n = 4) mice on day 3 of DSS treatment. LC3II represents the lipidated form of LC3 and is increased during autophagy. LC3II/LC3I (mean ± SEM) represents the ratio between the lipidated and unlipidated forms of the protein. (E) Confocal microscopic images of endogenous LC3 staining in frozen colonic sections from Hmgb1fl/fl (n = 3) and Vil-Cre Hmgb1fl/fl (n = 3) mice on day 3 of DSS treatment (original magnification, ×630). Autophagosomes have the appearance of LC3-positive punctate structures. (F) TUNEL staining on frozen colonic sections from Hmgb1fl/fl (n = 3) and Vil-Cre Hmgb1fl/fl (n = 3) mice on day 5 of DSS treatment (original magnification, ×400). Data were analyzed using 2-tailed Student’s t tests, except for the data in F, which were analyzed by 2-way Anova with Bonferroni’s multiple comparisons test as well as the 2-tailed Student’s t test for between-genotype comparisons within a treatment group. *P < 0.05; ****P < 0.001.