Rb and p53 Liver Functions Are Essential for Xenobiotic Metabolism and Tumor Suppression

The tumor suppressors Retinoblastoma (Rb) and p53 are frequently inactivated in liver diseases, such as hepatocellular carcinomas (HCC) or infections with Hepatitis B or C viruses. Here, we discovered a novel role for Rb and p53 in xenobiotic metabolism, which represent a key function of the liver for metabolizing therapeutic drugs or toxins. We demonstrate that Rb and p53 cooperate to metabolize the xenobiotic 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). DDC is metabolized mainly by cytochrome P450 (Cyp)3a enzymes resulting in inhibition of heme synthesis and accumulation of protoporphyrin, an intermediate of heme pathway. Protoporphyrin accumulation causes bile injury and ductular reaction. We show that loss of Rb and p53 resulted in reduced Cyp3a expression decreased accumulation of protoporphyrin and consequently less ductular reaction in livers of mice fed with DDC for 3 weeks. These findings provide strong evidence that synergistic functions of Rb and p53 are essential for metabolism of DDC. Because Rb and p53 functions are frequently disabled in liver diseases, our results suggest that liver patients might have altered ability to remove toxins or properly metabolize therapeutic drugs. Strikingly the reduced biliary injury towards the oxidative stress inducer DCC was accompanied by enhanced hepatocellular injury and formation of HCCs in Rb and p53 deficient livers. The increase in hepatocellular injury might be related to reduce protoporphyrin accumulation, because protoporphrin is well known for its anti-oxidative activity. Furthermore our results indicate that Rb and p53 not only function as tumor suppressors in response to carcinogenic injury, but also in response to non-carcinogenic injury such as DDC

Surgical resection and radiofrequency ablation initiate cancer in cytokeratin-19(+)- liver cells deficient for p53 and Rb

The long term prognosis of liver cancer patients remains unsatisfactory because of cancer recurrence after surgical interventions, particularly in patients with viral infections. Since hepatitis B and C viral proteins lead to inactivation of the tumor suppressors p53 and Retinoblastoma (Rb), we hypothesize that surgery in the context of p53/Rb inactivation initiate de novo tumorigenesis. We, therefore, generated transgenic mice with hepatocyte and cholangiocyte/liver progenitor cell (LPC)-specific deletion of p53 and Rb, by interbreeding conditional p53/Rb knockout mice with either Albumin-cre or Cytokeratin-19-cre transgenic mice. We show that liver cancer develops at the necrotic injury site after surgical resection or radiofrequency ablation in p53/Rb deficient livers. Cancer initiation occurs as a result of specific migration, expansion and transformation of cytokeratin-19+-liver (CK-19+) cells. At the injury site migrating CK-19+ cells formed small bile ducts and adjacent cells strongly expressed the transforming growth factor β (TGFβ). Isolated cytokeratin-19+ cells deficient for p53/Rb were resistant against hypoxia and TGFβ-mediated growth inhibition. CK-19+ specific deletion of p53/Rb verified that carcinomas at the injury site originates from cholangiocytes or liver progenitor cells. These findings suggest that human liver patients with hepatitis B and C viral infection or with mutations for p53 and Rb are at high risk to develop tumors at the surgical intervention site

Rb and p53 Liver Functions Are Essential for Xenobiotic Metabolism and Tumor Suppression

The tumor suppressors Retinoblastoma (Rb) and p53 are frequently inactivated in liver diseases, such as hepatocellular carcinomas (HCC) or infections with Hepatitis B or C viruses. Here, we discovered a novel role for Rb and p53 in xenobiotic metabolism, which represent a key function of the liver for metabolizing therapeutic drugs or toxins. We demonstrate that Rb and p53 cooperate to metabolize the xenobiotic 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC). DDC is metabolized mainly by cytochrome P450 (Cyp)3a enzymes resulting in inhibition of heme synthesis and accumulation of protoporphyrin, an intermediate of heme pathway. Protoporphyrin accumulation causes bile injury and ductular reaction. We show that loss of Rb and p53 resulted in reduced Cyp3a expression decreased accumulation of protoporphyrin and consequently less ductular reaction in livers of mice fed with DDC for 3 weeks. These findings provide strong evidence that synergistic functions of Rb and p53 are essential for metabolism of DDC. Because Rb and p53 functions are frequently disabled in liver diseases, our results suggest that liver patients might have altered ability to remove toxins or properly metabolize therapeutic drugs. Strikingly the reduced biliary injury towards the oxidative stress inducer DCC was accompanied by enhanced hepatocellular injury and formation of HCCs in Rb and p53 deficient livers. The increase in hepatocellular injury might be related to reduce protoporphyrin accumulation, because protoporphrin is well known for its anti-oxidative activity. Furthermore our results indicate that Rb and p53 not only function as tumor suppressors in response to carcinogenic injury, but also in response to non-carcinogenic injury such as DDC

Differential regulation of porcine beta-defensins 1 and 2 upon Salmonella infection in the intestinal epithelial cell line IPI-2I

Intestinal epithelial cells represent the first line of defence against pathogenic bacteria in the lumen of the gut. Besides acting as a physical barrier, epithelial cells orchestrate the immune response through the production of several innate immune mediator molecules including beta-defensins. Here, we establish the porcine intestinal cell line IPI-2I as a new model system to test the regulation of porcine beta-defensins 1 and 2. Gene expression of both defensins was highly upregulated by foetal calf serum components in normal growth medium. In serum-free medium, baseline expression remained low, but pBD-2 gene expression was increased 10-fold upon infection with Salmonella Typhimurium. Arcobacter cryaerophilus and Salmonella Enteritidis, pathogenic bacteria with comparable adhesion and invasion characteristics, failed to increase pBD-2 mRNA levels. Heat killed or colistin-treated Salmonella Typhimurium had no effect, showing that the upregulation of pBD-2 was dependent on the viability of the Salmonella Typhimurium. Gene expression of pBD-1 was regulated differently since an increase in pBD-1 mRNA was observed by Salmonella Enteritidis infection. We conclude that the IPI-2I cells can serve as a new model to study porcine beta-defensin regulation and that pBD-1 and pBD-2 are differentially regulated in this cell line

Loss of Rb and p53 results in increased hepatocellular injury, hepatocyte proliferation and polyploidization after DDC.

<p>(<b>A</b>) ALT and AST serum levels (<b>B</b>) Representative pictures of TUNEL, Ki67 and PH3 staining on liver sections (<b>C</b>) Percentage of TUNEL positive hepatocyte nuclei (<b>D</b>) Percentage of PH3 positive hepatocyte nuclei (<b>E</b>) Mitotic index of hepatocytes (<b>F</b>) Multinucleated hepatocytes (G) Anisokaryosis score of hepatocytes from mice of indicated genotypes fed with normal chow or 3-weeks DDC diet. Data presented as average ± SEM. * P < 0.05 compared to +DDC, <i>Rb</i>^{<i>f/ f</i>}<i>; p53</i>^<i>f/f</i>, # P < 0.05 compared to +DDC, <i>Rb</i>^Δ/Δ, $ P < 0.05 compared to +DDC, <i>p53</i>^Δ/Δ.</p

Combined loss of Rb and p53 suppresses protoporphyrin accumulation, ductular reaction and Cyp3a expression in livers of DDC-fed mice.

<p>(<b>A</b>) Representative pictures of H&E and CK19 staining on liver sections from mice of indicated genotypes after 3 weeks of DDC feeding. Arrows indicate protoporphyrin accumulation. (<b>B</b>) Semi-quantitative analysis of percentage area of protoporphyrin accumulation from mice fed with normal chow or 3-weeks DDC diet (n = 5 per group). (<b>C</b>) (<b>D</b>) (<b>E</b>) Expression of <i>CK19</i>, <i>Cyp3a11</i> and <i>FECH</i> was measured by qPCR in livers of indicated genotypes. -DDC, <i>Rb</i>^{<i>f/ f</i>}<i>; p53</i>^<i>f/f</i>, were normalized to 1. Data presented as average ± SEM. * P < 0.05 compared to +DDC, <i>Rb</i>^{<i>f/ f</i>}<i>; p53</i>^<i>f/f</i>, # P < 0.05 compared to +DDC, <i>Rb</i>^Δ/Δ.</p

Inactivation of Rb and p53 shortens life span and accelerates hepatocellular carcinoma formation after DDC exposure.

<p>(<b>A</b>) Survival and time to tumor curves of mice of indicated genotypes after feeding with DDC diet for 3 weeks at young ages and aged with normal chow. (<b>B</b>) Representative macroscopic pictures of H&E staining of liver tumors of mice feeding with DDC diet for 3 weeks at young ages and aged with normal chow. White and black dotted lines indicate the borders between normal liver tissue (L) and tumor (T).</p

Scheme for the role of RB and p53 in DDC metabolism.

<p>DDC interacts with Cyp in particular Cyp3a to form <i>N</i>-methyl protoporphyrin IX which inhibits ferrochelatase causing an accumulation of protoporphyrin in the bile ducts. Bile duct injury induces ductular reaction and inflammation. In addition, DDC induces reactive oxygen species (ROS) and oxidative stress causing hepatocellular injury. Protoporphyrin, on the other hand, acts as an antioxidant and protects hepatocytes. Rb and p53 are important for Cyp expression. Oxidative stress activates Rb and p53. Loss of Rb and p53 results in reduced expression of Cyp3a, therefore less production of the ferrochelatase inhibitor and thereby prevents the accumulation of its substrate protophorphyrin. Consequently, less bile injury and ductular reaction was observed. The reduction of protoporphyrin, an antioxidant, increases oxidative induced hepatocellular injury and later liver tumors develop.</p

E2F8 is essential for polyploidization in mammalian cells

Polyploidization is observed in all mammalian species and is a characteristic feature of hepatocytes, but its molecular mechanism and biological significance are unknown. Hepatocyte polyploidization in rodents occurs through incomplete cytokinesis, starts after weaning and increases with age. Here, we show in mice that atypical E2F8 is induced after weaning and required for hepatocyte binucleation and polyploidization. A deficiency in E2f8 led to an increase in the expression level of E2F target genes promoting cytokinesis and thereby preventing polyploidization. In contrast, loss of E2f1 enhanced polyploidization and suppressed the polyploidization defect of hepatocytes deficient for atypical E2Fs. In addition, E2F8 and E2F1 were found on the same subset of target promoters. Contrary to the long-standing hypothesis that polyploidization indicates terminal differentiation and senescence, we show that prevention of polyploidization through inactivation of atypical E2Fs has, surprisingly, no impact on liver differentiation, zonation, metabolism and regeneration. Together, these results identify E2F8 as a repressor and E2F1 as an activator of a transcriptional network controlling polyploidization in mammalian cells

Atypical E2f functions are critical for pancreas polyploidization

The presence of polyploid cells in the endocrine and exocrine pancreas has been reported for four decades. In rodents, pancreatic polyploidization is initiated after weaning and the number of polyploid cells increases with age. Surprisingly the molecular regulators and biological functions of polyploidization in the pancreas are still unknown. We discovered that atypical E2f activity is essential for polyploidization in the pancreas, using an inducible Cre/LoxP approach in new-born mice to delete ubiquitously the atypical E2f transcription factors, E2f7 and E2f8. In contrast to its critical role in embryonic survival, conditional deletion of both of both atypical E2fs in newborn mice had no impact on postnatal survival and mice lived until old age. However, deficiency of E2f7 or E2f8 alone was sufficient to suppress polyploidization in the pancreas and associated with only a minor decrease in blood serum levels of glucose, insulin, amylase and lipase under 4 hours starvation condition compared to wildtype littermates. In mice with fewer pancreatic polyploid cells that were fed ad libitum, no major impact on hormones or enzymes levels was observed. In summary, we identified atypical E2fs to be essential for polyploidization in the pancreas and discovered that postnatal induced loss of both atypical E2fs in many organs is compatible with life until old age