Blockage of TRPM7 channel induces hepatic stellate cell death through endoplasmic reticulum stress-mediated apoptosis.
OBJECTIVE
Proliferation is a 'multiplier' for extracellular matrix production and contraction of activated hepatic stellate cells (HSC) in fibrotic liver. Transient receptor potential melastatin-like 7 channels (TRPM7) are implicated in the survival and proliferation of several kinds of cells. This study was aimed to investigate the effect of TRPM7 blocker 2-APB on survival and proliferation of HSC and the underlying mechanisms.


METHODS
Rat HSC were stimulated by 2-APB for 24 h and then collected for further use. Cell viability was detected by MTT, and apoptosis was determined by AnnexinV/PI staining and TUNEL assay. Gene expressions of TRPM7, α-SMA, bcl-2, bax, and endoplasmic reticulum (ER) stress key members CHOP, caspase-12, ATF4, ATF6, Xbp1, GRP78 and calnexin were evaluated with quantitative RT-PCR. Quantifications of α-SMA, TRPM7, CHOP and GRP78 proteins were carried out by Western blot. Transmission electron microscopy and Xbp1 mRNA splicing analysis were also used for detection of ER stress.


RESULTS
2-APB decreased TRPM7 and α-SMA expressions in primary HSC, and inhibited proliferation of activated HSC in a dose-dependent manner. 2-APB also decreased total count of activated HSC and increased the number of apoptotic cells. 2-APB increased expressions of bax and ER stress key factors CHOP, caspase-12, ATF4, ATF6, Xbp1, GRP78 and calnexin. Meanwhile, ultra-structural ER changes and spliced Xbp1 mRNA were also observed in 2-APB treated HSC.


CONCLUSIONS
Blockage of TRPM7 could inhibit activation and proliferation of primary HSC and induce apoptotic death of activated cells, in which ER stress was identified as one of possible underlying molecular bases.
Division of Digestive Diseases, West China Hospital, Sichuan University, Chengdu 610041, China.
Yongjun
Y
Zhu
Division of Digestive Diseases, West China Hospital, Sichuan University, Chengdu 610041, China.
Ruoting
R
Men
Division of Digestive Diseases, West China Hospital, Sichuan University, Chengdu 610041, China.
Maoyao
M
Wen
Division of Digestive Diseases, West China Hospital, Sichuan University, Chengdu 610041, China; Department of Internal Medicine, Hospital of Southwest University, Chongqing 400715, China.
Xiaolin
X
Hu
Laboratory of Cardiovascular Diseases, West China Hospital, Sichuan University, Chengdu 610041, China.
Xiaojing
X
Liu
Division of Digestive Diseases, West China Hospital, Sichuan University, Chengdu 610041, China. Electronic address: linayang63@gmail.com.
Li
L
Yang
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D015153:Blotting, Western; D001896:Boron Compounds / Q000008:administration & dosage / Q000494:pharmacology*; D049109:Cell Proliferation / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D059865:Endoplasmic Reticulum Stress / Q000187:drug effects*; D005786:Gene Expression Regulation / Q000187:drug effects; D055166:Hepatic Stellate Cells / Q000187:drug effects* / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D008297:Male; D046529:Microscopy, Electron, Transmission; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D020133:Reverse Transcriptase Polymerase Chain Reaction; D050053:TRPM Cation Channels / Q000037:antagonists & inhibitors* / Q000378:metabolism
D001896:Boron Compounds; D012333:RNA, Messenger; D050053:TRPM Cation Channels; C109986:2-aminoethoxydiphenyl borate; C486584:Trpm7 protein, rat
Apoptosis; Endoplasmic reticulum stress; Hepatic stellate cells; Transient receptor potential melastatin-like 7