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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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 3

HMGB1 protects beclin 1 and ATG5 from cleavage during murine colitis.

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HMGB1 protects beclin 1 and ATG5 from cleavage during murine colitis. (A...
(A) Immunoblot for beclin 1 using an antibody that recognizes aa 171–291 of the protein. Blots are representative of Hmgb1fl/fl (n = 4) and Vil-Cre Hmgb1fl/fl (n = 4) mice. (B) Immunoblot for beclin 1 in Hmgb1fl/fl Il10–/– (n = 4) and Vil-Cre Hmgb1fl/fl Il10–/– (n = 4) mice. (C) Beclin 1 immunoblot of the products from an in vitro cleavage assay of beclin 1 by calpain 1 with decreasing amounts of HMGB1. (D) MYC-DDK (FLAG) immunoblot of the products of the beclin 1 in vitro cleavage assay. Recombinant beclin 1 contained a C-terminal MYC-DDK epitope tag. (E) Immunoblot for ATG5 using an antibody that recognizes aa 2–15 of the protein. Blots are representative of Hmgb1fl/fl (n = 4) and Vil-Cre Hmgb1fl/fl (n = 4) mice. (F) Immunoblot for ATG5 in Hmgb1fl/fl Il10–/– (n = 4) and Vil-Cre Hmgb1fl/fl Il10–/– (n = 4) mice. (G) ATG5 immunoblot of the products from an in vitro cleavage assay of ATG5 by calpain 1 with decreasing amounts of HMGB1. (H) Co-IP of HMGB1 and beclin 1 or ATG5 in colonic mucosal lysates from Hmgb1fl/fl (n = 4) and Vil-Cre Hmgb1fl/fl (n = 4) mice treated with DSS for 3 days. (I) Calpain activity assay evaluating cleavage of a fluorogenic calpain 1/2 substrate (Suc-LLVY-AMC) in samples of colonic mucosa from Hmgb1fl/fl (n = 3) and Vil-Cre Hmgb1fl/fl mice (n = 3) on day 3 of DSS treatment (mean ± SEM). (J) Immunoblot for the active p20 fragment of caspase 1 in Hmgb1fl/fl (n = 4) and Vil-Cre Hmgb1fl/fl mice (n = 4) on day 3 of DSS treatment. (K) qRT-PCR for calpastatin in cDNA from Hmgb1fl/fl (n = 6) and Vil-Cre Hmgb1fl/fl mice (n = 6) on day 3 of DSS treatment (mean ± SEM). Data were analyzed by 2-tailed Student’s t tests. *P < 0.05; **P < 0.01; ***P < 0.005; ****P < 0.001. RFU, relative fluorescence units.