High-mobility group box 1 promotes metalloproteinase-9 upregulation through Toll-like receptor 4 after cerebral ischemia.
OBJECTIVE
HMGB1 is a nuclear protein and an alarmin that signals cell damage in response to injury. It is believed that after release from injured cells, HMGB1 binds to its receptors to stimulate cross-talk among cells and to drive components of the inflammatory cascade. This study was intended to investigate the role of extracellular HMGB1 in ischemic stroke by examining the response of the zymogen matrix metalloproteinase-9 (MMP-9) to HMGB1 in vivo and in vitro.


METHODS
Toll-like receptor 2 (TLR2), TLR4, receptor for advanced glycation endproducts (RAGE), and MMP-9 expression was examined using quantitative RT-PCR in primary cultured neurons, astrocytes, and mouse brain after HMGB1 addition. MMP-9 expression/activity was examined using zymography. Middle cerebral artery occlusion was induced for 60 minutes using a filament model.


RESULTS
TLR4 is constitutively expressed in neurons, astrocytes, and mouse brain. HMGB1 addition to neuronal and glial cell cultures caused MMP-9 upregulation in a dose- and time-dependent manner. Lack of TLR4 function attenuated MMP-9 expression induced by HMGB1 in vitro. After striatal microinjection of HMGB1, MMP-9 was upregulated, and the response was independent of tumor necrosis factor-alpha. Interestingly, MMP-9 upregulation was reduced in TLR4 missense mutant mice after ischemia compared with wild-type controls, as was infarct volume.


CONCLUSIONS
Our results suggest that HMGB1 triggers MMP-9 upregulation in neurons and astrocytes predominantly via TLR4 after cerebral ischemia. Hence, targeting HMGB1/TLRs signaling pathway may reduce the acute inflammatory response and reduce tissue damage in cerebral ischemia.
Qiu
Jianhua
J
Department of Neurology and Radiology, Massachusetts General Hospital, Harvard Medical School, Boston, Mass 02129, USA. jqiu@partners.org
Xu
Jian
J
Zheng
Yi
Y
Wei
Ying
Y
Zhu
Xiaoxia
X
Lo
Eng H
EH
Moskowitz
Michael A
MA
Sims
John R
JR
D000704:Analysis of Variance; D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D002545:Brain Ischemia / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004305:Dose-Response Relationship, Drug; D024243:HMGB1 Protein / Q000378:metabolism* / Q000494:pharmacology; D007150:Immunohistochemistry; D008297:Male; D020780:Matrix Metalloproteinase 9 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D008822:Mice, Transgenic; D008845:Microinjections; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000187:drug effects; D051195:Toll-Like Receptor 2 / Q000235:genetics / Q000378:metabolism; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000378:metabolism*; D014409:Tumor Necrosis Factor-alpha / Q000235:genetics / Q000378:metabolism; D015854:Up-Regulation
D024243:HMGB1 Protein; D000067759:Receptor for Advanced Glycation End Products; D011971:Receptors, Immunologic; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D014409:Tumor Necrosis Factor-alpha; D020780:Matrix Metalloproteinase 9