Expression of mitochondrial membrane–linked SAB determines severity of sex-dependent acute liver injury
Sanda Win, Robert W.M. Min, Christopher Q. Chen, Jun Zhang, Yibu Chen, Meng Li, Ayako Suzuki, Manal F. Abdelmalek, Ying Wang, Mariam Aghajan, Filbert W.M. Aung, Anna Mae Diehl, Roger J. Davis, Tin A. Than, Neil Kaplowitz
Sanda Win, Robert W.M. Min, Christopher Q. Chen, Jun Zhang, Yibu Chen, Meng Li, Ayako Suzuki, Manal F. Abdelmalek, Ying Wang, Mariam Aghajan, Filbert W.M. Aung, Anna Mae Diehl, Roger J. Davis, Tin A. Than, Neil Kaplowitz
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Research Article Hepatology

Expression of mitochondrial membrane–linked SAB determines severity of sex-dependent acute liver injury

Abstract

SH3 domain–binding protein that preferentially associates with Btk (SAB) is an outer-membrane docking protein for JNK-mediated impairment of mitochondrial function. Deletion of Sab in hepatocytes inhibits sustained JNK activation and cell death. The current study demonstrates that an increase in SAB expression enhanced the severity of acetaminophen-induced (APAP-induced) liver injury. Female mice were resistant to liver injury and exhibited markedly decreased hepatic SAB protein expression compared with male mice. The mechanism of SAB repression involved a pathway from ERα to p53 expression that induced miR34a-5p. miR34a-5p targeted the Sab mRNA coding region, thereby repressing SAB expression. Fulvestrant or p53 knockdown decreased miR34a-5p and increased SAB expression in female mice, leading to increased injury from APAP and TNF/galactosamine. In contrast, an ERα agonist increased p53 and miR34a-5p, which decreased SAB expression and hepatotoxicity in male mice. Hepatocyte-specific deletion of miR34a also increased the severity of liver injury in female mice, which was prevented by GalNAc-ASO knockdown of Sab. Similar to mice, premenopausal women expressed elevated levels of hepatic p53 and low levels of SAB, whereas age-matched men expressed low levels of p53 and high levels of SAB, but there was no difference in SAB expression between the sexes in the postmenopausal stage. In conclusion, SAB expression levels determined the severity of JNK-dependent liver injury. Female mice expressed low levels of hepatic SAB protein because of the ERα/p53/miR34a pathway, which repressed SAB expression and accounted for the resistance to liver injury seen in these females.

Authors

Sanda Win, Robert W.M. Min, Christopher Q. Chen, Jun Zhang, Yibu Chen, Meng Li, Ayako Suzuki, Manal F. Abdelmalek, Ying Wang, Mariam Aghajan, Filbert W.M. Aung, Anna Mae Diehl, Roger J. Davis, Tin A. Than, Neil Kaplowitz

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Figure 5

ERα and p53 regulation of miR34a-5p expression.

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ERα and p53 regulation of miR34a-5p expression. (A) Liver tissue from ma...
(A) Liver tissue from male and female littermates was snap-frozen in RNAlater RNA stabilizing solution, and miRNA-Seq analysis was performed. n = 3 mice/group. Volcano plot shows the fold-change (FC) difference in miRNA expression in liver tissue from female and male mice. Sab-targeting miR34a-5p was expressed at a 3.2-fold significantly higher level in female mice than in their male littermates. The log2 fold change (0.59) or (–0.59) is equivalent to a 1.5-fold change in expression in either direction. The arrow points to the dot representing miR34a-5p. (B) miR34a-5p and Sab mRNA alignment. (C) qPCR analysis of miR34a-5p expression. RNA-Seq analysis showed that miR191-5p expression did not differ between male and female mice and was used as a loading control. n = 5 mice/group, *P < 0.05 versus male mice, by unpaired, 2-tailed Student’s t test. (D) The miR-34a-5p mimic was transfected into HEK293 cells and cultured for 3 days. Cell survival was the same as that of nontransfected cells (data not shown). SAB levels were determined by immunoblotting with rabbit anti-SAB. For the control (C), cells were transfected with a scrambled oligonucleotide. “<” indicates the nonspecific band. (E and F) Basal levels of p53 and ERα protein expression were determined in male and female littermate mice (n = 10 mice/group; *P < 0.05 versus male mice, by unpaired, 2-tailed Student’s t test) and in human liver tissue from men and premenopausal women (n = 5 pairs; *P < 0.05 versus men, by paired, 2-tailed Student’s t test). (G) Male mice were treated with PPT to activate ERα, and female mice were treated with fulvestrant to deplete ERα. Liver protein extract was examined for p53 expression. Immunoblot is representative of 3 separate experiments. n = 3 per group. *P < 0.05 versus vehicle control males; #P < 0.05 versus vehicle control females, by unpaired, 2-tailed Student’s t test. All data are presented as the mean ± SEM.