Cooperation of C/EBP family proteins and chromatin remodeling proteins is essential for termination of liver regeneration.

UNLABELLED: Liver cancer is the fifth most common cancer. A highly invasive surgical resection of the liver tumor is the main approach used to eliminate the tumor. Mechanisms that terminate liver regeneration when the liver reaches the original size are not known. The aims of this work were to generate an animal model that fails to stop liver regeneration after surgical resections and elucidate mechanisms that are involved in termination of liver regeneration. Because epigenetic control of liver function has been previously implicated in the regulation of liver proliferation, we generated C/EBPα-S193A knockin mice, which have alterations in formation of complexes of C/EBP family proteins with chromatin remodeling proteins. The C/EBPα-S193A mice have altered liver morphology and altered liver function leading to changes of glucose metabolism and blood parameters. Examination of the proliferative capacity of C/EBPα-S193A livers showed that livers of S193A mice have a higher rate of proliferation after birth, but stop proliferation at the age of 2 months. These animals have increased liver proliferation in response to liver surgery as well as carbon tetrachloride (CCl4 )-mediated injury. Importantly, livers of C/EBPα-S193A mice fail to stop liver regeneration after surgery when livers reach the original, preresection, size. The failure of S193A livers to stop regeneration correlates with the epigenetic repression of key regulators of liver proliferation C/EBPα, p53, FXR, SIRT1, PGC1α, and TERT by C/EBPß-HDAC1 complexes. The C/EBPß-HDAC1 complexes also repress promoters of enzymes of glucose synthesis PEPCK and G6Pase. CONCLUSION: Proper cooperation of C/EBP and chromatin remodeling proteins is essential for the termination of liver regeneration after surgery and for maintenance of liver functions.

Age-associated change of C/EBP family proteins causes severe liver injury and acceleration of liver proliferation after CCl4 treatments.

The aged liver is more sensitive to the drug treatments and has a high probability of developing liver disorders such as fibrosis, cirrhosis, and cancer. Here we present mechanisms underlying age-associated severe liver injury and acceleration of liver proliferation after CCl4 treatments. We have examined liver response to CCl4 treatments using old WT mice and young C/EBPα-S193D knockin mice, which express an aged-like isoform of C/EBPα. Both animal models have altered chromatin structure as well as increased liver injury and proliferation after acute CCl4 treatments. We found that these age-related changes are associated with the repression of key regulators of liver biology: C/EBPα, Farnesoid X Receptor (FXR) and telomere reverse transcriptase (TERT). In quiescent livers of old WT and young S193D mice, the inhibition of TERT is mediated by HDAC1-C/EBPα complexes. After CCl4 treatments, TERT, C/EBPα and FXR are repressed by different mechanisms. These mechanisms include the increase of a dominant negative isoform, C/EBPß-LIP, and subsequent repression of C/EBPα, FXR, and TERT promoters. C/EBPß-LIP also disrupts Rb-E2F1 complexes in C/EBPα-S193D mice after CCl4 treatments. To examine if these alterations are involved in drug-mediated liver diseases, we performed chronic treatments of mice with CCl4. We found that C/EBPα-S193D mice developed fibrosis much more rapidly than WT mice. Thus, our data show that the age-associated alterations of C/EBP proteins create favorable conditions for the increased liver proliferation after CCl4 treatments and for development of drug-mediated liver diseases.

Transcriptional and translational regulation of C/EBPß-HDAC1 protein complexes controls different levels of p53, SIRT1, and PGC1α proteins at the early and late stages of liver cancer.

Cancer changes biological processes in the liver by altering gene expression at the levels of transcription, translation, and protein modification. The RNA binding protein CUGBP1 is a key regulator of translation of CCAAT enhancer binding protein ß and histone deacetylase 1 (HDAC1). These proteins form complexes that are involved in the regulation of liver biology. Here we show a critical role of the translational activation of CCAAT/enhancer binding protein ß-HDAC1 complexes in the development of liver cancer mediated by diethylnitrosamine. We found that diethylnitrosamine increases the levels of CUGBP1 and activates CUGBP1 by phosphorylation, leading to the formation of the CUGBP1-eIF2 complex, which is an activator of translation of CUGBP1-dependent mRNAs. The elevation of the CUGBP1-eIF2 complex increases translation of C/EBPß and HDAC1, resulting in an increase of C/EBPß-HDAC1 complexes at later stages of liver cancer. We found that C/EBPß-HDAC1 complexes repress promoters of three key regulators of liver functions: p53, SIRT1, and PGC1α. As the result of this suppression, the p53-, SIRT1-, and PGC1α-dependent downstream pathways are reduced, leading to increased liver proliferation. We also found that the proper regulation of C/EBPß-HDAC1 complexes is required for the maintenance of biological levels of p53, SIRT1, and PGC1α in quiescent livers and at early stages of liver cancer. Taken together, these studies showed that the development of liver cancer includes a tight regulation of levels of C/EBPß-HDAC1 complexes on the levels of transcription, translation, and posttranslational modifications.

Ghrelin deletion protects against age-associated hepatic steatosis by downregulating the C/EBPα-p300/DGAT1 pathway.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. NAFLD usually begins as low-grade hepatic steatosis which further progresses in an age-dependent manner to nonalcoholic steatohepatitis (NASH), fibrosis, cirrhosis, and hepatocellular carcinoma in some patients. Ghrelin is a hormone known to promote adiposity in rodents and humans, but its potential role in hepatic steatosis is unknown. We hypothesized that genetic ghrelin deletion will protect against the development of age-related hepatic steatosis. To examine this hypothesis, we utilized ghrelin knockout (KO) mice. Although no different in young animals (3 months old), we found that at 20 months of age, ghrelin KO mice have significantly reduced hepatic steatosis compared to aged-matched wild-type (WT) mice. Examination of molecular pathways by which deletion of ghrelin reduces steatosis showed that the increase in expression of diacylglycerol O-acyltransferase-1 (DGAT1), one of the key enzymes of triglyceride (TG) synthesis, seen with age in WT mice, is not present in KO mice. This was due to the lack of activation of CCAAT/enhancer binding protein-alpha (C/EBPα) protein and subsequent reduction of C/EBPα-p300 complexes. These complexes were abundant in livers of old WT mice and were bound to and activated the DGAT1 promoter. However, the C/EBPα-p300 complexes were not detected on the DGAT1 promoter in livers of old KO mice resulting in lower levels of the enzyme. In conclusion, these studies demonstrate the mechanism by which ghrelin deletion prevents age-associated hepatic steatosis and suggest that targeting this pathway may offer therapeutic benefit for NAFLD.

Increased expression of enzymes of triglyceride synthesis is essential for the development of hepatic steatosis.

Molecular mechanisms underpinning nonalcoholic fatty liver disease (NAFLD) are not well understood. The earliest step of NAFLD is hepatic steatosis, which is one of the main characteristics of aging liver. Here, we present a molecular scenario of age-related liver steatosis. We show that C/EBPα-S193D knockin mice have age-associated epigenetic changes and develop hepatic steatosis at 2 months of age. The underlying mechanism of the hepatic steatosis in old wild-type (WT) mice and in young S193D mice includes increased amounts of tripartite p300-C/EBPα/ß complexes that activate promoters of five genes that drive triglyceride synthesis. Knockdown of p300 in old WT mice inhibits hepatic steatosis. Indeed, transgenic mice expressing dominant-negative p300 have fewer C/EBPα/ß-p300 complexes and do not develop age-dependent hepatic steatosis. Notably, the p300-C/EBPα/ß pathway is activated in the livers of patients with NAFLD. Thus, our results show that p300 and C/EBP proteins are essential participants in hepatic steatosis.

Farnesoid X receptor directly regulates xenobiotic detoxification genes in the long-lived Little mice.

Activation of xenobiotic metabolism pathways has been linked to lifespan extension in different models of aging. However, the mechanisms underlying activation of xenobiotic genes remain largely unknown. Here we showed that although farnesoid X receptor (FXR, Nr1h4) mRNA levels do not change significantly, FXR protein levels are elevated in the livers of the long-lived Little mice, leading to increased DNA binding activity of FXR. Hepatic FXR expression is sex-dependent in wild-type mice but not in Little mice, implying that up-regulation of FXR might be dependent on the reduction of growth hormone in Little mice. Growth hormone treatment decreased hepatic expression of FXR and xenobiotic genes Abcb1a, Fmo3 and Gsta2 in both wild-type and Little mice, suggesting an association between FXR and xenobiotic gene expression. We found that Abcb1a is transactivated by FXR via direct binding of FXR/retinoid X receptor α (RXRα) heterodimer to a response element at the proximal promoter. FXR also positively controls Fmo3 and Gsta2 expression through direct interaction with the response elements in these genes. Our study demonstrates that xenobiotic genes are direct transcriptional targets of FXR and suggests that FXR signaling may play a critical role in the lifespan extension observed in Little mice.