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.

Fig 1

Confirmation of deletion of Phb1 in DNA, mRNA and protein levels. Genotyping of Phb1 exon 2 region including loxP flanking Phb1 exon 2 sites shows the same size of bands (predicted 731 bp) in all of the organs tested (A, left) in WT control mice. However, deletion of loxP flanking Phb1 exon 2 sequence (predicted 334 bp) is detected only in liver and hepatocytes but not in other organs from liver-specific Phb1 KO mice (A, right), which indicated that Phb1 exon 2 is deleted only in the liver of liver-specific Phb1 KO mice. Northern blots of total RNA from the livers of WT control and liver-specific Phb1 KO mice show that Phb1 exon 2 is significantly removed in the livers of liver-specific Phb1 KO mice compared to those of WT control littermates (B, left). The graph on the right shows densitometric changes in % of WT control, which is expressed as mean±SEM from 2 in each group (B, right), *p<0.01. Protein expression of PHB1 and PHB2 by Western blots shows only PHB1 protein expression is dramatically decreased in whole livers and hepatocytes, but not in kidney (C, top). PHB2 expression is also decreased along with PHB1 protein in the whole livers and hepatocytes in liver-specific Phb1 KO mice (C, top). The graph shows densitometric changes in % of WT control, which is expressed as mean±SEM from 2 mice each for kidneys and hepatocytes and 8 mice each for livers (C, bottom), *p<0.01.

Fig 2

Histologic changes in liver-specific Phb1 KO livers. A to D (×400), E (×800) and F (×200) show H&E staining of livers from 3-week old WT control (A) and liver-specific Phb1 KO (B to E) mice (except for F, 14-week old KO). Characteristic findings in liver-specific Phb1 KO livers are (B) foci of necrosis, (C) increased inflammatory cell infiltrate, (D) bile duct metaplasia as indicated by arrow, and (E) anisocytosis (varying sizes) of hepatic nuclei as indicated by arrows, consistent with hepatocyte dysplasia. Consistent with this, 14-week old KO mouse exhibits dysplastic nodule (F, ×200).

Fig 3

Liver-specific Phb1 livers exhibit increased staining for oval cell and preneoplastic markers. OV6 staining is negative in 3-week old WT control (A, ×800) but positive in 3-week old KO mice (bright green staining, B, ×800). GSTP staining with DAPI co-staining for nuclei shows negative GSTP in 3-week old WT control (C, ×800) but positive cells in 3-week old KO (D, red staining, ×800). Note these GSTP positive cells are also OV6 positive (B). A and C show the same microscopic field as B and D, double OV6/GSTP immunofluorescent staining. AFP staining was negative in 38-week old WT control (E, ×400) but strongly positive in KO (F, ×400).

Fig 4

Changes in liver-specific Phb1 KO livers from 3-week to 14-week of age. Apoptosis is detected by immunohistochemical staining for active caspase 3 (top row, ×200). Slightly Increased number of active caspase 3 positive cells (brown staining) was found in the liver of 3-week old liver-specific Phb1 KO mouse, which progressed as the mice aged. Proliferation is detected by immunohistochmical staining for PCNA (middle row, ×400). Liver-specific Phb1 KO mice have more PCNA positive cells (brown staining) regardless of their age than those of their control littermates. Fibrosis is detected by reticulin staining (bottom row, ×200 except for 14-week WT, ×400). Significant amount of reticulin is accumulated in the liver of 3-week old liver-specific Phb1 KO mouse, which progressed in the 14-week old KO livers.

Fig 5

Development of HCC in the liver-specific Phb1 KO mice and PHB1 expression in human liver cancer cell lines. Histologic changes on H&E are shown below the gross picture of each liver. A shows no significant change in the liver of WT mouse on gross or histologic exam (×200). B shows multiple nodules (arrows) on the surface of the liver of a 35-week old KO mouse, one of which shows well differentiated HCC with fat deposit on H&E staining (×400). C shows different stages of HCC development in the liver of a 46-week KO mouse (arrows), where neoplastic cells (arrows) are clearly observed on H&E staining (×800), and D shows multi-focal HCC in a 38-week KO mouse by gross exam (arrows) and on H&E (×600). PHB1 protein level as determined by Western blot analysis (E) is much lower in Huh-7 and HepG2 cells as compared to normal primary human hepatocytes (PHH). Densitometric analyses from three independent experiments are shown below the blot and are expressed as mean±SEM % of PHH, p<0.05 between Huh-7 or HepG2 and PHH.

Fig 6

Effects of Phb1 siRNA treatment in AML12 cell line. AML12 cells were treated with siRNA of Phb1 for 18 (expression) or 24 (growth and ChIP) hours. PHB1 mRNA level was reduced by 90% as compared to control and scrambled siRNA (SC) (A, n=6) and PHB1 protein expression decreased by 30% (B, n=3). C (n=4 to 6) shows mRNA levels in Phb1 siRNA treated AML12 cells for the genes that are up- or down-regulated most in the livers of liver-specific Phb1 KO mice. All of mRNA expression changes in C are statistically significant against control and/or scrambled siRNA (p<0.05, Supplement Table 3). D shows Phb1 siRNA treated AML12 cells have increased proliferation rate measured by BrDU incorporation into DNA for 4 hours (n=4). *p<0.005 vs. control and scrambled siRNA. E shows the effect of Phb1 siRNA on E2F binding to different regions of the cyclin D1 promoter (numbers are in reference to the transcription start site). E2F binding is increased in all four regions but particularly to the region −513~−697. These changes were measured densitometrically and were significantly different from SC control with p<0.05.

Fig 7

Effects of PHB1 overexpression in AML12 and Huh-7 cell line. AML12 and Huh-7 cells were treated with the human PHB1-pcDNA3.1 PHB1 overexpression plasmid for 48 hours. A and B show the efficiency of PHB1 overexpression in protein level by Western blot. PHB1 protein level increased by 35 to 60% Huh-7 or AML12 cells treated with the overexpression vector (oePHB1) as compared to empty vector (EV) control, respectively. Densitometric analyses from three independent experiments are shown below the blots and are expressed as mean±SEM % of control, p<0.05 between oePHB1 and EV. Proliferation was measured by BrDU incorporation and are shown in C and D. AML12 cells overexpressing PHB1 had a significant decrease in proliferation (D) as compared to control and EV, while no significant change was found in Huh-7 cells (C). *p<0.05 vs. control and EV.

Fig 8

Effects of Phb1 siRNA and sorafenib (SF) treatments in Huh-7 cells. Huh-7 cells were treated with Phb1 siRNA or SC for 48 hours and sorafenib was added during the last 24 hours. PHB1 siRNA reduced PHB1 protein level by 50% as shown on Western blot (A, n=3, results are mean±SEM densitometric values expressed as % of control). B shows % over control apoptosis in Huh-7 cells treated with siPHB1 or SC control, with or without SF. Randomly selected 10× optic magnification fields were taken and apoptotic cells to normal cells ratio was calculated in 4 fields in each group. siPHB1 treatment did not affect apoptosis or sensitize cells to apoptosis induced by SF. C shows proliferation changes in similarly treated Huh-7 cells. SF inhibited proliferation and this was not affected by siPHB1. *p<0.05 vs. DMSO control.