Increases in p53 expression induce CTGF synthesis by mouse and human hepatocytes and result in liver fibrosis in mice
Takahiro Kodama, Tetsuo Takehara, Hayato Hikita, Satoshi Shimizu, Minoru Shigekawa, Hinako Tsunematsu, Wei Li, Takuya Miyagi, Atsushi Hosui, Tomohide Tatsumi, Hisashi Ishida, Tatsuya Kanto, Naoki Hiramatsu, Satoshi Kubota, Masaharu Takigawa, Yoshito Tomimaru, Akira Tomokuni, Hiroaki Nagano, Yuichiro Doki, Masaki Mori, Norio Hayashi
Takahiro Kodama, Tetsuo Takehara, Hayato Hikita, Satoshi Shimizu, Minoru Shigekawa, Hinako Tsunematsu, Wei Li, Takuya Miyagi, Atsushi Hosui, Tomohide Tatsumi, Hisashi Ishida, Tatsuya Kanto, Naoki Hiramatsu, Satoshi Kubota, Masaharu Takigawa, Yoshito Tomimaru, Akira Tomokuni, Hiroaki Nagano, Yuichiro Doki, Masaki Mori, Norio Hayashi
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Research Article Hepatology

Increases in p53 expression induce CTGF synthesis by mouse and human hepatocytes and result in liver fibrosis in mice

Abstract

The tumor suppressor p53 has been implicated in the pathogenesis of non-cancer-related conditions such as insulin resistance, cardiac failure, and early aging. In addition, accumulation of p53 has been observed in the hepatocytes of individuals with fibrotic liver diseases, but the significance of this is not known. Herein, we have mechanistically linked p53 activation in hepatocytes to liver fibrosis. Hepatocyte-specific deletion in mice of the gene encoding Mdm2, a protein that promotes p53 degradation, led to hepatocyte synthesis of connective tissue growth factor (CTGF; the hepatic fibrogenic master switch), increased hepatocyte apoptosis, and spontaneous liver fibrosis; concurrent removal of p53 completely abolished this phenotype. Compared with wild-type controls, mice with hepatocyte-specific p53 deletion exhibited similar levels of hepatocyte apoptosis but decreased liver fibrosis and hepatic CTGF expression in two models of liver fibrosis. The clinical significance of these data was highlighted by two observations. First, p53 upregulated CTGF in a human hepatocellular carcinoma cell line by repressing miR-17-92. Second, human liver samples showed a correlation between CTGF and p53-regulated gene expression, which were both increased in fibrotic livers. This study reveals that p53 induces CTGF expression and promotes liver fibrosis, suggesting that the p53/CTGF pathway may be a therapeutic target in the treatment of liver fibrosis.

Authors

Takahiro Kodama, Tetsuo Takehara, Hayato Hikita, Satoshi Shimizu, Minoru Shigekawa, Hinako Tsunematsu, Wei Li, Takuya Miyagi, Atsushi Hosui, Tomohide Tatsumi, Hisashi Ishida, Tatsuya Kanto, Naoki Hiramatsu, Satoshi Kubota, Masaharu Takigawa, Yoshito Tomimaru, Akira Tomokuni, Hiroaki Nagano, Yuichiro Doki, Masaki Mori, Norio Hayashi

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Figure 7

p53 regulates CTGF synthesis in hepatocytes.

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p53 regulates CTGF synthesis in hepatocytes. (A) Hepatocytes and NPCs we...
(A) Hepatocytes and NPCs were isolated from Mdm2fl/fl [Cre(–)] mice and Mdm2fl/flalb-cre [Cre(+)] mice by collagenase-pronase perfusion of the liver. Ctgf mRNA levels in the isolated hepatocytes (left panel) and NPCs (right panel) were determined by real-time RT-PCR; 4–6 mice per group. (B) Expression of CTGF protein in liver sections was assessed by immunohistochemistry; black arrows indicate cholangiocytes. Original magnification, ×200. (C and D) HepG2 cells (1.0 × 105) were treated with nutlin-3a (20 μM) or vehicle for the indicated time courses. (C) Western blot analysis of p53, CTGF, and p21 proteins. (D) Real-time RT-PCR analysis of CTGF mRNA expression; n = 3/group; *P < 0.01 versus the other 3 groups. (E and F) HepG2 cells (1.0 × 105) were treated with Adriamycin (1 μM) or vehicle for indicated time courses. (E) Western blot analysis of p53, CTGF, and p21 proteins. (F) Real-time RT-PCR analysis of CTGF expression; n = 3/group; *P < 0.01 versus the other 3 groups, **P < 0.01 versus 0- and 6-hour groups. (G and H) HepG2 cells were transfected with p53 siRNA or control siRNA for 3 days and then cultured for 24 hours with nutlin-3a (20 μM), Adriamycin (1 μM), or vehicle. (G) Western blot analysis of p53, CTGF and p21 proteins. (H) Real-time RT-PCR analysis of CTGF mRNA expression; n = 3/group, statistical analyses were performed by the paired t test.