gling Jin

The reduction of SIRT1 in livers of old mice leads to impaired body homeostasis and to inhibition of liver proliferation

Age declines liver functions, leading to the development of age‐associated diseases. A member of the sirtuins family, SIRT1, is involved in the control of glucose homeostasis and fat metabolism. Because aging livers have alterations in glucose and fat metabolism, we examined a possible role of SIRT1 in these alterations. We found that aged livers have a reduced expression of SIRT1 and have lost proper control of the regulation of SIRT1 after partial hepatectomy (PH). Down‐regulation of SIRT1 in the liver of old mice is mediated by CCAAT/Enhancer Binding Protein/histone deacetylase 1 (C/EBPβ‐HDAC1) complexes, which bind to and repress the SIRT1 promoter. In the livers of young mice, SIRT1 is activated after PH and supports high levels of glucose and triglycerides during liver regeneration. In old mice, however, C/EBPβ‐HDAC1–mediated repression of the SIRT1 promoter blocks activation of SIRT1, leading to low levels of glucose and triglycerides during liver regeneration. Down‐regulation of SIRT1 in the livers of young mice resulted in alterations similar to those observed in the livers of old mice, whereas the normalization of SIRT1 in the livers of old mice corrects the levels of glucose and triglycerides after PH. The normalization of SIRT1 in old mice also improves liver regeneration via the elimination of the C/EBPα‐Brm complex. These studies showed a critical role of the reduction of SIRT1 in age‐associated liver dysfunctions and provide a potential tool for the correction of liver functions in old patients after surgical resections. (HEPATOLOGY 2011;)

The Age-Associated Decline of Glycogen Synthase Kinase 3β Plays a Critical Role in the Inhibition of Liver Regeneration

ABSTRACT Aging reduces the regenerative capacities of many tissues. In this paper, we show a critical role of the glycogen synthase kinase 3β (GSK3β)-cyclin D3 pathway in the loss of the regenerative capacity of the liver. In young animals, high levels of growth hormone (GH) increase expression of GSK3β, which associates with cyclin D3 and triggers degradation of cyclin D3. In livers of old mice, the GSK3β promoter is repressed by C/EBPβ-histone deacetylase 1 (HDAC1) complexes, leading to the reduction of GSK3β. The treatment of old mice with GH increases expression of GSK3β via removal of the C/EBPβ-HDAC1 complexes from the GSK3β promoter. We found that the GSK3β-cyclin D3 pathway is also altered in young GH-deficient Little mice and that treatment of Little mice with GH corrects the GSK3β-cyclin D3 pathway. We present evidence that GSK3β regulates liver proliferation by controlling growth-inhibitory activity of C/EBPα. The downregulation of GSK3β in young mice inhibits liver proliferation after partial hepatectomy via the cyclin D3-C/EBPα pathway, while the elevation of GSK3β in old mice accelerates liver proliferation. Thus, this paper shows that GSK3β is a critical regulator of liver proliferation and that the reduction of GSK3β with age causes the loss of regenerative capacities of the liver.

Elimination of C/EBPα through the ubiquitin-proteasome system promotes the development of liver cancer in mice

Despite significant advancements in our understanding of cancer development, the molecular mechanisms that underlie the formation of liver cancer remain largely unknown. C/EBPalpha is a transcription factor that regulates liver quiescence. Phosphorylation of C/EBPalpha at serine 193 (S193-ph) is upregulated in older mice and is thought to contribute to age-associated liver dysfunction. Because development of liver tumors is associated with increasing age, we investigated the role of S193-ph in the development of liver cancer using knockin mice expressing a phospho-mimetic aspartic acid residue in place of serine at position 193 (S193D) of C/EBPalpha. The S193D isoform of C/EBPalpha was able to completely inhibit liver proliferation in vivo after partial hepatectomy. However, treatment of these mice with diethylnitrosamine/phenobarbital (DEN/PB), which induces formation of liver cancer, actually resulted in earlier development of liver tumors. DEN/PB treatment was associated with specific degradation of both the S193-ph and S193D isoforms of C/EBPalpha through activation of the ubiquitinproteasome system (UPS). The mechanism of UPS-mediated elimination of C/EBPalpha during carcinogenesis involved elevated levels of gankyrin, a protein that was found to interact with the S193-ph isoform of C/EBPalpha and target it for UPS-mediated degradation. This study identifies a molecular mechanism that supports the development of liver cancer in older mice and potential therapeutic targets for the prevention of liver cancer.

Farnesoid X receptor inhibits gankyrin in mouse livers and prevents development of liver cancer

One of the early events in the development of liver cancer is a neutralization of tumor suppressor proteins Rb, p53, hepatocyte nuclear factor 4α (HNF4α), and CCAAT/enhancer binding protein (C/EBP) α. The elimination of these proteins is mediated by a small subunit of proteasome, gankyrin, which is activated by cancer. The aim of this study was to determine the mechanisms that repress gankyrin in quiescent livers and mechanisms of activation of gankyrin in liver cancer. We found that farnesoid X receptor (FXR) inhibits expression of gankyrin in quiescent livers by silencing the gankyrin promoter through HDAC1‐C/EBPβ complexes. C/EBPβ is a key transcription factor that delivers HDAC1 to gankyrin promoter and causes epigenetic silencing of the promoter. We show that down‐regulation of C/EBPβ in mouse hepatoma cells and in mouse livers reduces C/EBPβ‐HDAC1 complexes and activates the gankyrin promoter. Deletion of FXR signaling in mice leads to de‐repression of the gankyrin promoter and to spontaneous development of liver cancer at 12 months of age. Diethylnitrosoamine (DEN)‐mediated liver cancer in wild‐type mice also involves the reduction of FXR and activation of gankyrin. Examination of liver cancer in old mice and liver cancer in human patients revealed that FXR is reduced, while gankyrin is elevated during spontaneous development of liver cancer. Searching for animal models with altered levels of FXR, we found that long‐lived Little mice have high levels of FXR and do not develop liver cancer with age and after DEN injections due to failure to activate gankyrin and eliminate Rb, p53, HNF4α and C/EBPα proteins. Conclusion: FXR prevents liver cancer by inhibiting the gankyrin promoter via C/EBPβ‐HDAC1 complexes, leading to subsequent protection of tumor suppressor proteins from degradation. (HEPATOLOGY 2013)

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/β 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/β-p300 complexes and do not develop age-dependent hepatic steatosis. Notably, the p300-C/EBPα/β 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.

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

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/EBPβ‐HDAC1 complexes. The C/EBPβ‐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. (Hepatology 2015;61:315–325)

Epigenetic changes play critical role in age-associated dysfunctions of the liver

CCAAT/Enhancer Binding Proteins family proteins are important regulators of liver functions. Here, we show the critical role of C/EBPα‐mediated chromatin remodeling in the age‐associated dysfunctions of the liver and in the maintenance of physiological homeostasis. Because ph‐S193 isoform of C/EBPα is increased in livers of old mice, we have generated C/EBPα‐S193D knockin mice, which mimic the ph‐S193 isoform of C/EBPα. Analyses of these mice showed that the S193D mutation causes chromatin remodeling leading to histological appearance of ‘foci‐like’ nodules, which are also observed in livers of old mice. These ‘foci‐like’ structures contain K9 trimethylated histone H3, a marker of heterochromatin. The increase of heterochromatin regions in S193D mice correlates with the elevation of S193D‐C/EBPα‐HDAC1 complexes and with dys‐regulation of gene expression including epigenetic silencing of cyclin D1 and D2 promoters and the inhibition of liver proliferation. The elimination of C/EBPα‐HDAC1 complexes in S193D mice by inhibition of HDAC1 corrects chromatin structure and normalizes expression of cyclin D1 and D2. We found that epigenetic dys‐regulation is also associated with the elevation of C/EBPβ and with the increase of C/EBPα/β heterodimers in S193D mice. The C/EBPα/β heterodimers activate transcription of Glut4 and increase the levels of Glut4. As the result, S193D livers have accelerated uptake of glucose and accumulation of glycogen in the liver. Thus, this study demonstrates that the phosphorylation of C/EBPα at S193 leads to the appearance of heterochromatin regions, which correlates with the development of age‐related dysfunctions of the liver.

Transcriptional and Translational Regulation of C/EBPβ-HDAC1 Protein Complexes Controls Different Levels of p53, SIRT1, and PGC1α Proteins at the Early and Late Stages of Liver Cancer

Background: Development of liver cancer involves alterations of multiple pathways of gene expression. Results: Translational activation of C/EBPβ-HDAC1 complexes represses p53, SIRT1, and PGC1α, leading to liver cancer. Conclusion: Modulation of levels of C/EBPβ-HDAC1 complexes at different stages of cancer is involved in liver cancer. Significance: Understanding the mechanisms of liver cancer is a critical step for the development of therapeutic approaches for 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 β 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 β-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/EBPβ and HDAC1, resulting in an increase of C/EBPβ-HDAC1 complexes at later stages of liver cancer. We found that C/EBPβ-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/EBPβ-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/EBPβ-HDAC1 complexes on the levels of transcription, translation, and posttranslational modifications.

GSK3β-cyclin D3-CUGBP1-eIF2 pathway in aging and in myotonic dystrophy

Epigenetic silencing of the cell cycle proteins is one of the major pathways of the inhibition of liver proliferation in old mice. We recently identified glycogen synthase 3β, GSK3β, as an enzyme which controls the epigenetic regulation of the liver proliferation via reduction of cyclin D3-cdk4. The elevation of cyclin D3 in livers of old mice leads to the accumulation of a transcriptional C/EBPα-HDAC1-Brm complex and translational CUGBP1-eIF2 complex. The accumulation of the CUGBP1-eIF2 increases translation of HDAC1 and C/EBPβ in livers of old mice leading to formation of C/EBPβ-HDAC1 complex which represses C/EBPβ-dependent genes by interacting with their promoters. The translational CUGBP1-eIF2 complex also plays critical role in the regulation of biological processes in young livers and needs to be tightly regulated. Our recent paper showed that this complex is un-appropriately up-regulated in skeletal muscle precursors of patients affected with multisystemic disease, Myotonic Dystrophy 2 (DM2) leading to translational elevation of several proteins. These data suggest that the age-specific translational dysfunctions might be involved in DM2 pathogenesis. Our data also suggest that the regulation of translation is conserved in liver and skeletal muscle. In this review, we discuss biological consequences of the age-associated alterations of translation in the liver and a possible role of the elevation of the CUGBP1-eIF2 complex in development of DM2 pathology.

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

Background: Older patients are more sensitive to drug-mediated development of liver disorders. Results: Age and CCl4 treatments change expression of C/EBP proteins leading to repression of key regulators of liver biology. Conclusion: The age-associated alterations of C/EBP proteins cause severe liver injury after CCl4 treatments. Significance: Understanding of mechanisms of age-associated severe liver injury is important for development of therapeutic approaches. 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/EBPβ-LIP, and subsequent repression of C/EBPα, FXR, and TERT promoters. C/EBPβ-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.