Liver-specific deletion of prohibitin 1 results in spontaneous liver injury, fibrosis, and hepatocellular carcinoma in mice

Abstract

Prohibitin 1 (PHB1) is a highly conserved, ubiquitously expressed protein that participates in diverse processes including mitochondrial chaperone, growth and apoptosis. The role of PHB1 in vivo is unclear and whether it is a tumor suppressor is controversial. Mice lacking methionine adenosyltransferase 1A (MAT1A) have reduced PHB1 expression, impaired mitochondrial function, and spontaneously develop hepatocellular carcinoma (HCC). To see if reduced PHB1 expression contributes to the Mat1a knockout (KO) phenotype, we generated liver-specific Phb1 KO mice. Expression was determined at the messenger RNA and protein levels. PHB1 expression in cells was varied by small interfering RNA or overexpression. At 3 weeks, KO mice exhibit biochemical and histologic liver injury. Immunohistochemistry revealed apoptosis, proliferation, oxidative stress, fibrosis, bile duct epithelial metaplasia, hepatocyte dysplasia, and increased staining for stem cell and preneoplastic markers. Mitochondria are swollen and many have no discernible cristae. Differential gene expression revealed that genes associated with proliferation, malignant transformation, and liver fibrosis are highly up-regulated. From 20 weeks on, KO mice have multiple liver nodules and from 35 to 46 weeks, 38% have multifocal HCC. PHB1 protein levels were higher in normal human hepatocytes compared to human HCC cell lines Huh-7 and HepG2. Knockdown of PHB1 in murine nontransformed AML12 cells (normal mouse hepatocyte cell line) raised cyclin D1 expression, increased E2F transcription factor binding to cyclin D1 promoter, and proliferation. The opposite occurred with PHB1 overexpression. Knockdown or overexpression of PHB1 in Huh-7 cells did not affect proliferation significantly or sensitize cells to sorafenib-induced apoptosis. Conclusion: Hepatocyte-specific PHB1 deficiency results in marked liver injury, oxidative stress, and fibrosis with development of HCC by 8 months. These results support PHB1 as a tumor suppressor in hepatocytes. (HEPATOLOGY 2010;52:2096-2108 P rohibitin (PHB) proteins are highly conserved and ubiquitously expressed proteins that have diverse cellular functions.1,2 Two PHB proteins, PHB1 and PHB2, encoded by genes located on different chromosomes, form a large multimeric complex (PHB complex) that is found largely in the inner mitochondrial membrane where it exerts a chaperone-like function to stabilize newly synthesized mitochondrial

Prohibitin 1 Regulates the H19-Igf2 Axis and Proliferation in Hepatocytes

“…Despite these findings, the mechanism of action of PHB1 as a cell proliferation control point in the liver is not clearly established. Although liver-specific Phb1 KO mice develop HCC spontaneously, this may have occurred due to the severe liver injury and regenerative response (12). Nevertheless, we observed an inverse relationship between PHB1 expression and growth in the nonmalignant AML12 hepatocyte cell line, suggesting that PHB1 plays a direct growth modulatory role (12).…”

“…PHB1 controls the H19-Igf2 axis by interacting and co-localizing with CTCF on the ICR regulatory region controlling both genes. Our previously published work showed that Phb1 KO mice develop HCC (12). To examine whether Phb1 modulation could lead to tumorigenesis in part via H19, we examined the effect of silencing or overexpressing Phb1 or H19 on proliferation of the liver cancer cell line, S-adenosylmethionine-deficient (SAMe-D), which is derived from the methionine adenosyltransferase 1a KO (Mat1a KO) mouse model of HCC (21).…”

“…To examine molecular mechanisms of action of Phb1 in the liver, we previously developed the liver-specific Phb1 knockout (Phb1 KO) mouse model (12). Phb1 KO mice livers develop severe oxidative stress, fibrosis, and liver cancer (12).…”

“…To examine molecular mechanisms of action of Phb1 in the liver, we previously developed the liver-specific Phb1 knockout (Phb1 KO) mouse model (12). Phb1 KO mice livers develop severe oxidative stress, fibrosis, and liver cancer (12). Microarray analysis of Phb1 KO mice livers revealed a significant upregulation of the long noncoding RNA, H19 and insulin-like growth factor 2 (Igf2) genes (12).…”

“…Phb1 KO mice livers develop severe oxidative stress, fibrosis, and liver cancer (12). Microarray analysis of Phb1 KO mice livers revealed a significant upregulation of the long noncoding RNA, H19 and insulin-like growth factor 2 (Igf2) genes (12). These genes are part of a well characterized cluster that contains the paternally expressed Igf2 and maternally expressed H19.…”