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Loss of protein function is a driving force of ageing. We have identified peptidyl-prolyl isomerase A (PPIA or cyclophilin A) as a dominant chaperone in haematopoietic stem and progenitor cells. Depletion of PPIA accelerates stem cell ageing. We found that proteins with intrinsically disordered regions (IDRs) are frequent PPIA substrates. IDRs facilitate interactions with other proteins or nucleic acids and can trigger liquid-liquid phase separation. Over 20% of PPIA substrates are involved in the formation of supramolecular membrane-less organelles. PPIA affects regulators of stress granules (PABPC1), P-bodies (DDX6) and nucleoli (NPM1) to promote phase separation and increase cellular stress resistance. Haematopoietic stem cell ageing is associated with a post-transcriptional decrease in PPIA expression and reduced translation of IDR-rich proteins. Here we link the chaperone PPIA to the synthesis of intrinsically disordered proteins, which indicates that impaired protein interaction networks and macromolecular condensation may be potential determinants of haematopoietic stem cell ageing., (© 2024. The Author(s).)

Proteasome inhibitors have become the standard of care for multiple myeloma (MM). Blocking protein degradation particularly perturbs the homeostasis of short-lived polypeptides such as transcription factors and epigenetic regulators. To determine how proteasome inhibitors directly impact gene regulation, we performed an integrative genomics study in MM cells. We discovered that proteasome inhibitors reduce the turnover of DNA-associated proteins and repress genes necessary for proliferation through epigenetic silencing. Specifically, proteasome inhibition results in the localized accumulation of histone deacetylase 3 (HDAC3) at defined genomic sites, which reduces H3K27 acetylation and increases chromatin condensation. The loss of active chromatin at super-enhancers critical for MM, including the super-enhancer controlling the proto-oncogene c-MYC, reduces metabolic activity and cancer cell growth. Epigenetic silencing is attenuated by HDAC3 depletion, suggesting a tumor-suppressive element of this deacetylase in the context of proteasome inhibition. In the absence of treatment, HDAC3 is continuously removed from DNA by the ubiquitin ligase SIAH2. Overexpression of SIAH2 increases H3K27 acetylation at c-MYC-controlled genes, increases metabolic output, and accelerates cancer cell proliferation. Our studies indicate a novel therapeutic function of proteasome inhibitors in MM by reshaping the epigenetic landscape in an HDAC3-dependent manner. As a result, blocking the proteasome effectively antagonizes c-MYC and the genes controlled by this proto-oncogene., Competing Interests: Authors’ Disclosures: P.L. is an advisory board member of Karyopharm Therapeutics. All other authors declare they have no competing interests.

The ubiquitin-proteasome pathway precisely controls the turnover of transcription factors in the nucleus, playing an important role in maintaining appropriate quantities of these regulatory proteins. The transcription factor c-MYC is essential for normal development and is a critical cancer driver. Despite being highly expressed in several tissues and malignancies, the c-MYC protein is also continuously targeted by the ubiquitin-proteasome pathway, which can either facilitate or inhibit c-MYC degradation. Deubiquitinating proteases can remove ubiquitin chains from target proteins and rescue them from proteasomal digestion. This study sought to determine novel elements of the ubiquitin-proteasome pathway that regulate c-MYC levels. We performed an overexpression screen with 41 human proteases to identify which deubiquitinases stabilize c-MYC. We discovered that the highly expressed Otubain-1 (OTUB1) protease increases c-MYC protein levels. Confirming its role in enhancing c-MYC activity, we found that elevated OTUB1 correlates with inferior clinical outcomes in the c-MYC-dependent cancer multiple myeloma, and overexpression of OTUB1 accelerates the growth of myeloma cells. In summary, our study identifies OTUB1 as a novel amplifier of the proto-oncogene c-MYC.

Multiple myeloma and its precursor plasma cell dyscrasias affect 3% of the elderly population in the US. Proteasome inhibitors are an essential part of several standard drug combinations used to treat this incurable cancer. These drugs interfere with the main pathway of protein degradation and lead to the accumulation of damaged proteins inside cells. Despite promising initial responses, multiple myeloma cells eventually become drug resistant in most patients. The biology behind relapsed/refractory multiple myeloma is complex and poorly understood. Several studies provide evidence that in addition to the proteasome, mitochondrial proteases can also contribute to protein quality control outside of mitochondria. We therefore hypothesized that mitochondrial proteases might counterbalance protein degradation in cancer cells treated with proteasome inhibitors. Using clinical and experimental data, we found that overexpression of the mitochondrial matrix protease LonP1 (Lon Peptidase 1) reduces the efficacy of proteasome inhibitors. Some proteasome inhibitors partially crossinhibit LonP1. However, we show that the resistance effect of LonP1 also occurs when using drugs that do not block this protease, suggesting that LonP1 can compensate for loss of proteasome activity. These results indicate that targeting both the proteasome and mitochondrial proteases such as LonP1 could be beneficial for treatment of multiple myeloma.

Transcription is regulated through a dynamic interplay of DNA-associated proteins, and the composition of gene-regulatory complexes is subject to continuous adjustments. Protein alterations include post-translational modifications and elimination of individual polypeptides. Spatially and temporally controlled protein removal is, therefore, essential for gene regulation and accounts for the short half-life of many transcription factors. The ubiquitin-proteasome system is responsible for site- and target-specific ubiquitination and protein degradation. Specificity of ubiquitination is conferred by ubiquitin ligases. Cullin-RING complexes, the largest family of ligases, require multi-unit assembly around one of seven cullin proteins. To investigate the direct role of cullins in ubiquitination of DNA-bound proteins and in gene regulation, we analyzed their subcellular locations and DNA-affinities. We found CUL4A and CUL7 to be largely excluded from the nucleus, whereas CUL4B was primarily nuclear. CUL1,2,3, and 5 showed mixed cytosolic and nuclear expression. When analyzing chromatin affinity of individual cullins, we discovered that CUL1 preferentially associated with active promoter sequences and co-localized with 23% of all DNA-associated protein degradation sites. CUL1 co-distributed with c-MYC and specifically repressed nuclear-encoded mitochondrial and splicing-associated genes. These studies underscore the relevance of spatial control in chromatin-associated protein ubiquitination and define a novel role for CUL1 in gene repression.

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., (© 2017 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Despite intensive investigations, mechanisms of liver cancer are not known. Here, we identified an important step of liver cancer, which is the neutralization of tumor suppressor activities of an RNA binding protein, CUGBP1. The translational activity of CUGBP1 is activated by dephosphorylation at Ser302. We generated CUGBP1-S302A knock-in mice and found that the reduction of translational activity of CUGBP1 causes development of a fatty liver phenotype in young S302A mice. Examination of liver cancer in diethylnitrosamine (DEN)-treated CUGBP1-S302A mice showed these mice develop much more severe liver cancer that is associated with elimination of the mutant CUGBP1. Searching for mechanisms of this elimination, we found that the oncoprotein gankyrin (Gank) preferentially binds to and triggers degradation of dephosphorylated CUGBP1 (de-ph-S302-CUGBP1) or S302A mutant CUGBP1. To test the role of Gank in degradation of CUGBP1, we generated mice with liver-specific deletion of Gank. In these mice, the tumor suppressor isoform of CUGBP1 is protected from Gank-mediated degradation. Consistent with reduction of CUGBP1 in animal models, CUGBP1 is reduced in patients with pediatric liver cancer. Thus, this work presents evidence that de-ph-S302-CUGBP1 is a tumor suppressor protein and that the Gank-UPS-mediated reduction of CUGBP1 is a key event in the development of liver cancer., (Copyright © 2017 American Society for Microbiology.)

The histone acetyltransferase p300 has been implicated in the regulation of liver biology; however, molecular mechanisms of this regulation are not known. In this paper, we examined these mechanisms using transgenic mice expressing a dominant negative p300 molecule (dnp300). While dnp300 mice did not show abnormal growth within 1 year, these mice have many alterations in liver biology and liver functions. We found that the inhibition of p300 leads to the accumulation of heterochromatin foci in the liver of 2-month-old mice. Transcriptome sequencing (RNA-Seq) analysis showed that this inhibition of p300 also causes alterations of gene expression in many signaling pathways, including chromatin remodeling, apoptosis, DNA damage, translation, and activation of the cell cycle. Livers of dnp300 mice have a high rate of proliferation and a much higher rate of proliferation after partial hepatectomy. We found that livers of dnp300 mice are resistant to CCl4-mediated injury and have reduced apoptosis but have increased proliferation after injury. Underlying mechanisms of resistance to liver injury and increased proliferation in dnp300 mice include ubiquitin-proteasome-mediated degradation of C/EBPα and translational repression of the p53 protein by the CUGBP1-eukaryotic initiation factor 2 (eIF2) repressor complex. Our data demonstrate that p300 regulates a number of critical signaling pathways that control liver functions., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

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/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., (© 2014 by the American Association for the Study of Liver Diseases.)

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.

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., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

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.

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., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Unlabelled: 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., (Copyright © 2012 American Association for the Study of Liver Diseases.)

Myotonic dystrophy type 1 (DM1) is a complex neuromuscular disease characterized by skeletal muscle wasting, weakness, and myotonia. DM1 is caused by the accumulation of CUG repeats, which alter the biological activities of RNA-binding proteins, including CUG-binding protein 1 (CUGBP1). CUGBP1 is an important skeletal muscle translational regulator that is activated by cyclin D3-dependent kinase 4 (CDK4). Here we show that mutant CUG repeats suppress Cdk4 signaling by increasing the stability and activity of glycogen synthase kinase 3β (GSK3β). Using a mouse model of DM1 (HSA(LR)), we found that CUG repeats in the 3' untranslated region (UTR) of human skeletal actin increase active GSK3β in skeletal muscle of mice, prior to the development of skeletal muscle weakness. Inhibition of GSK3β in both DM1 cell culture and mouse models corrected cyclin D3 levels and reduced muscle weakness and myotonia in DM1 mice. Our data predict that compounds normalizing GSK3β activity might be beneficial for improvement of muscle function in patients with DM1.

Expansions of noncoding CUG and CCUG repeats in myotonic dystrophies type 1 (DM1) and DM2 cause complex molecular pathology, the features of which include accumulation of RNA aggregates and misregulation of the RNA-binding proteins muscleblind-like 1 (MBNL1) and CUG-binding protein 1 (CUGBP1). CCUG repeats also decrease amounts of the nucleic acid binding protein ZNF9. Using tetracycline (Tet)-regulated monoclonal cell models that express CUG and CCUG repeats, we found that low levels of long CUG and CCUG repeats result in nuclear and cytoplasmic RNA aggregation with a simultaneous increase of CUGBP1 and a reduction of ZNF9. Elevation of CUGBP1 and reduction of ZNF9 were also observed before strong aggregation of the mutant CUG/CCUG repeats. Degradation of CUG and CCUG repeats normalizes ZNF9 and CUGBP1 levels. Comparison of short and long CUG and CCUG RNAs showed that great expression of short repeats form foci and alter CUGBP1 and ZNF9; however, long CUG/CCUG repeats misregulate CUGBP1 and ZNF9 much faster than high levels of the short repeats. These data suggest that correction of DM1 and DM2 might be achieved by complete and efficient degradation of CUG and CCUG repeats or by a simultaneous disruption of CUG/CCUG foci and correction of CUGBP1 and ZNF9., (Copyright © 2011 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2011 American Association for the Study of Liver Diseases.)

Liver cancer is the fifth most common cancer and the third most common cause of cancer related death in the world. The recent development of new techniques for the investigations of global change in the gene expression, signaling pathways and wide genome binding has provided novel information for the mechanisms underlying liver cancer progression. Although these studies identified gene expression signatures in hepatocellular carcinoma, the early steps of the development of hepatocellular carcinomas (HCC) are not well understood. The development of HCC is a multistep process which includes the progressive alterations of gene expression leading to the increased proliferation and to liver cancer. This review summarizes recent progress in the identification of the key steps of the development of HCC with the focus on early events of carcinogenesis and on the role of translational and epigenetic alterations in the development of HCC. Quiescent stage of the liver is supported by several tumor suppressor proteins including p53, Rb and C/EBPα. Studies with chemical models of liver carcinogenesis and with human HCC have shown that the elevation of gankyrin is responsible for the elimination of these three proteins at early steps of carcinogenesis. Later stages of progression of the liver cancer are associated with alterations in many signaling pathways including translation which leads to epigenetic silencing/activation of many genes. Particularly, recent reports suggest a critical role of histone deacetylase 1, HDAC1, in the development of HCC through the interactions with transcription factors such as C/EBP family proteins., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

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., (© 2010 The Authors Aging Cell © 2010 Blackwell Publishing Ltd/Anatomical Society of Great Britain and Ireland.)

A truncated isoform of C/EBPbeta, C/EBPbeta-LIP, is required for liver proliferation. This isoform is expressed at high levels in proliferating liver and in liver tumors. However, high levels of C/EBPbeta-LIP are also observed in non-proliferating livers during acute phase response (APR). In this paper we present mechanisms by which liver regulates activities of C/EBPbeta-LIP. We found that calmodulin (CaM) inhibits the ability of C/EBPbeta-LIP to promote liver proliferation during APR through direct interactions. This activity of CaM is under negative control of Ca(2+), which is reduced in nuclei of livers with APR, whereas it is increased in nuclei of proliferating livers. A mutant CaM, which does not interact with C/EBPbeta-LIP, also fails to inhibit the growth promotion activity of C/EBPbeta-LIP. Down-regulation of CaM in livers of LPS-treated mice causes liver proliferation via activation of C/EBPbeta-LIP. Overexpression of C/EBPbeta-LIP above levels of CaM also initiates liver proliferation in LPS-treated mice. In addition, CaM regulates transcriptional activity of another isoform of C/EBPbeta, C/EBPbeta-LAP, and might control liver biology through the regulation of both isoforms of C/EBPbeta. In searching for molecular mechanisms by which C/EBPbeta-LIP promotes cell proliferation, we found that C/EBPbeta-LIP releases E2F.Rb-dependent repression of cell cycle genes by a disruption of E2F1.Rb complexes and by a direct interaction with E2F-dependent promoters. CaM inhibits these growth promotion activities of C/EBPbeta-LIP and, therefore, supports liver quiescence. Thus, our findings discover a new pathway of the regulation of liver proliferation that involves calcium-CaM signaling.

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.

Liver tumor cells arise from normal hepatocytes that escape negative control of proliferation. The transcription factor C/EBPalpha maintains quiescence of hepatocytes through two pathways: inhibition of cdks and repression of E2F. Nevertheless, liver tumors and cultured hepatoma cell lines proliferate in the presence of C/EBPalpha. In this paper, we present evidence that the activation of the PI3K/Akt pathway in liver tumor cells blocks the growth inhibitory activity of C/EBPalpha through the PP2A-mediated dephosphorylation of C/EBPalpha on Ser 193, leading to a failure of C/EBPalpha to interact with and inhibit cdks and E2F. Mutation of Ser 193 to Ala also abolishes the ability of C/EBPalpha to cause growth arrest because of a lack of interactions with cdk2 and E2F-Rb complexes. These data provide a molecular basis for the development of liver tumors in which the activation of PI3K/Akt pathway neutralizes C/EBPalpha growth inhibitory activity.

Accumulation of RNA CUG repeats in myotonic dystrophy type 1 (DM1) patients leads to the induction of a CUG-binding protein, CUGBP1, which increases translation of several proteins that are required for myogenesis. In this paper, we examine the role of overexpression of CUGBP1 in DM1 muscle pathology using transgenic mice that overexpress CUGBP1 in skeletal muscle. Our data demonstrate that the elevation of CUGBP1 in skeletal muscle causes overexpression of MEF2A and p21 to levels that are significantly higher than those in skeletal muscle of wild type animals. A similar induction of these proteins is observed in skeletal muscle of DM1 patients with increased levels of CUGBP1. Immunohistological analysis showed that the skeletal muscle from mice overexpressing CUGBP1 is characterized by a developmental delay, muscular dystrophy, and myofiber-type switch: increase of slow/oxidative fibers and the reduction of fast fibers. Examination of molecular mechanisms by which CUGBP1 up-regulates MEF2A shows that CUGBP1 increases translation of MEF2A via direct interaction with GCN repeats located within MEF2A mRNA. Our data suggest that CUGBP1-mediated overexpression of MEF2A and p21 inhibits myogenesis and contributes to the development of muscle deficiency in DM1 patients.

Induction of p21 in senescent human fibroblasts plays a key role in the inactivation of cyclin-dependent kinases and the resulting irreversible growth arrest in the early stages of cell senescence. We found that RNA-binding proteins are critical regulators of p21 during senescence. Two RNA-binding proteins, CUGBP1 and calreticulin (CRT), interact with the same nucleotide sequences within the 5' region of p21 mRNA, but have opposite effects on the translation of p21 mRNA. CUGBP1 increases translation of p21 mRNA, whereas CRT blocks translation of p21 via stabilization of a stem-loop structure within the 5' region of the p21 mRNA. CUGBP1 and CRT compete for binding to p21 mRNA and thereby the regulation of p21 translation. In senescent fibroblasts, CUGBP1 displaces CRT from the p21 mRNA and releases CRT-dependent repression of p21 translation leading to growth arrest and development of a senescent phenotype. These data present evidence that competition between RNA-binding proteins for the regulation of p21 translation determines cell fate.

The liver is capable of completely regenerating itself in response to injury and after partial hepatectomy. In liver of old animals, the proliferative response is dramatically reduced, the mechanism for which is unknown. The liver specific protein, C/EBPalpha, normally arrests proliferation of hepatocytes through inhibiting cyclin dependent kinases (cdks). We present evidence that aging switches the liver-specific pathway of C/EBPalpha growth arrest to repression of E2F transcription. We identified an age-specific C/EBPalpha-Rb-E2F4 complex that binds to E2F-dependent promoters and represses these genes. The C/EBPalpha-Rb-E2F4 complex occupies the c-myc promoter and blocks induction of c-myc in livers of old animals after partial hepatectomy. Our results show that the age-dependent switch from cdk inhibition to repression of E2F transcription causes a loss of proliferative response in the liver because of an inability to induce E2F target genes after partial hepatectomy providing a possible mechanism for the age-dependent loss of liver regenerative capacity.

We previously identified an RNA binding protein, CUGBP1, which binds to GCN repeats located within the 5' region of C/EBPbeta mRNAs and regulates translation of C/EBPbeta isoforms. To further investigate the role of RNA binding proteins in the posttranscriptional control of C/EBP proteins, we purified additional RNA binding proteins that interact with GC-rich RNAs and that may regulate RNA processing. In HeLa cells, the majority of GC-rich RNA binding proteins are associated with endogenous RNA transcripts. The separation of these proteins from endogenous RNA identified several proteins in addition to CUGBP1 that specifically interact with the GC-rich 5' region of C/EBPbeta mRNA. One of these proteins was purified to homogeneity and was identified as calreticulin (CRT). CRT is a multifunctional protein involved in several biological processes, including interaction with and regulation of rubella virus RNA processing. Our data demonstrate that both CUGBP1 and CRT interact with GCU repeats within myotonin protein kinase and with GCN repeats within C/EBPalpha and C/EBPbeta mRNAs. GCN repeats within these mRNAs form stable SL structures. The interaction of CRT with SL structures of C/EBPbeta and C/EBPalpha mRNAs leads to inhibition of translation of C/EBP proteins in vitro and in vivo. Deletions or mutations abolishing the formation of SL structures within C/EBPalpha and C/EBPbeta mRNAs lead to a failure of CRT to inhibit translation of C/EBP proteins. CRT-dependent inhibition of C/EBPalpha is sufficient to block the growth-inhibitory activity of C/EBPalpha. This finding further defines the molecular mechanism for posttranscriptional regulation of the C/EBPalpha and C/EBPbeta proteins.

CCAAT/enhancer binding protein alpha (C/EBPalpha) causes growth arrest via direct interaction with the cyclin-dependent kinases cdk2 and cdk4. In this paper, we present evidence showing that C/EBPalpha enhances a proteasome-dependent degradation of cdk4 during growth arrest in liver of newborn mice and in cultured cells. Overexpression of C/EBPalpha in several biological systems leads to a reduction of cdk4 protein levels, but not mRNA levels. Experiments with several tissue culture models reveal that C/EBPalpha enhances the formation of cdk4-ubiquitin conjugates and induces degradation of cdk4 through a proteasome-dependent pathway. As a result, the half-life of cdk4 is shorter and protein levels of cdk4 are reduced in cells expressing C/EBPalpha. Gel filtration analysis of cdk4 complexes shows that a chaperone complex cdk4-cdc37-Hsp90, which protects cdk4 from degradation, is abundant in proliferating livers that lack C/EBPalpha, but this complex is weak or undetectable in livers expressing C/EBPalpha. Our studies show that C/EBPalpha disrupts the cdk4-cdc37-Hsp90 complex via direct interaction with cdk4 and reduces protein levels of cdk4 by increasing proteasome-dependent degradation of cdk4.

Differentiation of skeletal muscle is affected in myotonic dystrophy (DM) patients. Analysis of cultured myoblasts from DM patients shows that DM myoblasts lose the capability to withdraw from the cell cycle during differentiation. Our data demonstrate that the expression and activity of the proteins responsible for cell cycle withdrawal are altered in DM muscle cells. Skeletal muscle cells from DM patients fail to induce cytoplasmic levels of a CUG RNA binding protein, CUGBP1, while normal differentiated cells accumulate CUGBP1 in the cytoplasm. In cells from normal patients, CUGBP1 up-regulates p21 protein during differentiation. Several lines of evidence show that CUGBP1 induces the translation of p21 via binding to a GC-rich sequence located within the 5' region of p21 mRNA. Failure of DM cells to accumulate CUGBP1 in the cytoplasm leads to a significant reduction of p21 and to alterations of other proteins responsible for the cell cycle withdrawal. The activity of cdk4 declines during differentiation of cells from control patients, while in DM cells cdk4 is highly active during all stages of differentiation. In addition, DM cells do not form Rb/E2F repressor complexes that are abundant in differentiated cells from normal patients. Our data provide evidence for an impaired cell cycle withdrawal in DM muscle cells and suggest that alterations in the activity of CUGBP1 causes disruption of p21-dependent control of cell cycle arrest.

The transcription factor CCAAT/enhancer binding protein alpha (C/EBPalpha) is a strong inhibitor of cell proliferation. We found that C/EBPalpha directly interacts with cdk2 and cdk4 and arrests cell proliferation by inhibiting these kinases. We mapped a short growth inhibitory region of C/EBPalpha between amino acids 175 and 187. This portion of C/EBPalpha is responsible for direct inhibition of cyclin-dependent kinases and causes growth arrest in cultured cells. C/EBPalpha inhibits cdk2 activity by blocking the association of cdk2 with cyclins. Importantly, the activities of cdk4 and cdk2 are increased in C/EBPalpha knockout livers, leading to increased proliferation. Our data demonstrate that the liver-specific transcription factor C/EBPalpha brings about growth arrest through direct inhibition of cdk2 and cdk4.

We have previously found that loss of C/EBPalpha in hepatocytes of newborn livers leads to increased proliferation, to a reduction in p21 protein levels and to an induction of S phase-specific E2F/p107 complexes. In this paper, we investigated C/EBPalpha-dependent regulation of E2F complexes in a well-characterized cell line, 3T3-L1, and in stable transformants that conditionally express C/EBPalpha. C/EBPalpha and C/EBPbeta proteins are induced in 3T3-L1 preadipocytes during differentiation with different kinetics and potentially may regulate E2F/Rb family complexes. In pre-differentiated cells, three E2F complexes are observed: cdk2/E2F/p107, E2F/p130 and E2F4. cdk2/E2F/p107 complexes are induced in nuclear extracts of 3T3-L1 cells during mitotic expansion, but are not detectable in nuclear extracts at later stages of 3T3-L1 differentiation. The reduction in E2F/p107 complexes is associated with elevation of C/EBPalpha, but is independent of C/EBPbeta expression. Bacterially expressed, purified His-C/EBPalpha is able to disrupt E2F/p107 complexes that are observed at earlier stages of 3T3-L1 differentiation. C/EBPbeta, however, does not disrupt E2F/p107 complexes. A short C/EBPalpha peptide with homology to E2F is sufficient to bring about the disruption of E2F/p107 complexes from 3T3-L1 cells in vitro. Induction of C/EBPalpha in stable 3T3-L1 clones revealed that C/EBPalpha causes disruption of p107/E2F complexes in these cells. In contrast, E2F/p130 complexes are induced in cells expressing C/EBPalpha. Our data suggest that induction of p130/E2F complexes by C/EBPalpha occurs via up-regulation of p21, which, in turn, leads to association with and inhibition of, cdk2 kinase activity. The reduction in cdk2 kinase activity correlates with alterations of p130 phosphorylation and with induction of p130/E2F complexes in 3T3-L1 stable clones. Our data suggest two pathways of C/EBPalpha-dependent regulation of E2F/Rb family complexes: disruption of S phase-specific E2F/p107 complexes and induction of E2F/p130 complexes.