Hepatocyte-specific CCAAT/enhancer binding protein α restricts liver fibrosis progression.

Metabolic dysfunction-associated steatohepatitis (MASH) - previously described as nonalcoholic steatohepatitis (NASH) - is a major driver of liver fibrosis in humans, while liver fibrosis is a key determinant of all-cause mortality in liver disease independent of MASH occurrence. CCAAT/enhancer binding protein α (CEBPA), as a versatile ligand-independent transcriptional factor, has an important function in myeloid cells, and is under clinical evaluation for cancer therapy. CEBPA is also expressed in hepatocytes and regulates glucolipid homeostasis; however, the role of hepatocyte-specific CEBPA in modulating liver fibrosis progression is largely unknown. Here, hepatic CEBPA expression was found to be decreased during MASH progression both in humans and mice, and hepatic CEBPA mRNA was negatively correlated with MASH fibrosis in the human liver. CebpaΔHep mice had markedly enhanced liver fibrosis induced by a high-fat, high-cholesterol, high-fructose diet or carbon tetrachloride. Temporal and spatial hepatocyte-specific CEBPA loss at the progressive stage of MASH in CebpaΔHep,ERT2 mice functionally promoted liver fibrosis. Mechanistically, hepatocyte CEBPA directly repressed Spp1 transactivation to reduce the secretion of osteopontin, a fibrogenesis inducer of hepatic stellate cells. Forced hepatocyte-specific CEBPA expression reduced MASH-associated liver fibrosis. These results demonstrate an important role for hepatocyte-specific CEBPA in liver fibrosis progression, and may help guide the therapeutic discoveries targeting hepatocyte CEBPA for the treatment of liver fibrosis.

[Epigenetic Regulation of Gene Expression and Hepatocyte Proliferation by Nuclear Receptor PPARA].

Chronic activation of the nuclear receptor, peroxisome proliferator-activated receptor alpha (PPARA), causes hepatocellular proliferation and increases the incidence of hepatocellular carcinoma in rodents. However, the molecular mechanisms underlying hepatocyte proliferation by activated PPARA remain ambiguous. This review focuses on the genes repressed by PPARA and describes the mechanism by which it promotes hepatocyte proliferation in mice. PPARA undergoes autoinduction, leading to its overexpression by an agonist. PPARA subsequently activates the E2F transcription factor 8 (E2f8), which then activates the ubiquitin-like protein containing the PHD and RING finger domains 1 (Uhrf1). UHRF1, in complex with histone deacetylase 1 and DNA methyltransferase 1, stimulates DNA methylation and recruitment of histone H3 containing trimethylated lysine 9 to the promoters of specific target genes, including E-cadherin/cadherin 1 (Cdh1), resulting in their downregulation. Decreased expression of CDH1 stimulates Wnt signaling, upregulation of oncogenes, including Myc and the cell cycle control genes, cyclin D1 and Jun, and enhances hepatocyte hyperproliferation. Therefore, the PPARA-E2F8-UHRF1-CDH1-Wnt signaling axis is involved in the epigenetic regulation of hepatocyte proliferation. This review provides insights into the mechanisms underlying hepatocarcinogenesis induced by non-genotoxic substances.

Transcriptional regulation of adipogenin expression in liver steatosis by hepatic peroxisome proliferator-activated receptor gamma.

The nuclear receptors peroxisome proliferator-activated receptor gamma (PPARγ) and adipogenin (ADIG) play vital roles in lipid metabolism. However, the interaction between PPARγ and ADIG during liver steatosis remains unclear. In this study, we aimed to investigate the role of PPARγ in the transcriptional regulation of hepatic ADIG expression. Adig was found to be highly expressed in various fatty liver mouse models. Although hepatic Adig was expressed at high levels in the fatty liver of type 2 diabetic ob/ob mice and was upregulated by PPARγ agonist treatment, it was expressed at significantly low levels in liver-specific Pparg-knockout mice. Moreover, hepatic Adig expression was observed in other mouse models of liver steatosis, such as the leptin receptor mutant db/db and alcohol-fed mice. Adig was also highly expressed in the white and brown adipose tissues, skeletal muscles, and heart of ob/ob mice. Reporter and electromobility shift assays showed that PPARγ positively regulates Adig transcriptional activity by directly binding to a functional PPARγ-responsive element in the promoter region. Our results indicate that Adig is a novel target gene of hepatic PPARγ in liver steatosis.

Secretoglobin 3A2 protects lung from developing cigarette smoke-induced pulmonary emphysema.

Secretoglobin (SCGB) 3A2 is a bioactive molecule exhibiting various functions such as improving allergic airway inflammation and pulmonary fibrosis and promoting bronchial branching and proliferation during lung development. To determine if and how SCGB3A2 is involved in chronic obstructive pulmonary disease (COPD), a multifactorial disease with both airway and emphysematous lesions, a COPD mouse model was created by exposing Scgb3a2-deficient (KO), Scgb3a2-lung-specific overexpressing (TG), and wild type (WT) mice to cigarette smoke (CS) for 6 months. The KO mice showed loss of lung structure under control condition, and CS exposure resulted in more expansion of airspace and destruction of alveolar wall than WT mouse lungs. In contrast, TG mouse lungs showed no significant changes after CS exposure. SCGB3A2 increased the expression and phosphorylation of signal transducers and activators of transcription (STAT)1 and STAT3, and the expression of α1-antitrypsin (A1AT) in mouse lung fibroblast-derived MLg cells and mouse lung epithelial-derived MLE-15 cells. In MLg cells, A1AT expression was decreased in Stat3-knockdown cells, and increased upon Stat3 overexpression. STAT3 formed a homodimer when cells were stimulated with SCGB3A2. Chromatin immunoprecipitation and reporter assays demonstrated that STAT3 binds to specific binding sites on the Serpina1a gene encoding A1AT and upregulates its transcription in lung tissues of mice. Furthermore, nuclear localization of phosphorylated STAT3 upon SCGB3A2 stimulation was detected by immunocytochemistry. These findings demonstrate that SCGB3A2 protects the lungs from the development of CS-induced emphysema by regulating A1AT expression through STAT3 signaling.

Oxysterol-binding protein-like 3 is a novel target gene of peroxisome proliferator-activated receptor γ in fatty liver disease.

Oxysterol-binding protein-like 3 (OSBPL3) plays a key role in the development of fatty liver disease. Herein, we found that OSBPL3 is highly expressed in the fatty liver of humans and mice. Although high expression of Osbpl3 was observed in the fatty liver of type 2 diabetic ob/ob mice, liver-specific Pparg knockout ameliorated this increase in these mice. Moreover, high hepatic Osbpl3 expression was observed in other mice models of fatty liver disease, such as leptin receptor-mutant db/db and alcohol-fed mice. Analysis of the human liver transcriptome data revealed that hepatic OSBPL3 expression is higher in patients with advanced non-alcoholic fatty liver disease (NAFLD) when compared to those with mild NAFLD. Reporter and electrophoretic mobility shift assays showed that PPARγ positively regulates Osbpl3 transcription by binding to the two functional PPARγ-responsive elements present in the 5' upstream region. Overall, our results indicate that Osbpl3 is a novel PPARγ target in the fatty liver.

Gene repression through epigenetic modulation by PPARA enhances hepatocellular proliferation.

Peroxisome proliferator-activated receptor α (PPARA) is a key mediator of lipid metabolism and inflammation. Activation of PPARA in rodents causes hepatocyte proliferation, but the underlying mechanism is poorly understood. This study focused on genes repressed by PPARA and analyzed the mechanism by which PPARA promotes hepatocyte proliferation in mice. Activation of PPARA by agonist treatment was autoregulated, and induced expression of the epigenetic regulator UHRF1 via activation of the newly described PPARA target gene E2f8, which, in turn, regulates Uhrf1. UHRF1 strongly repressed the expression of CDH1 via methylation of the Cdh1 promoter marked with H3K9me3. Repression of CDH1 by PPARA activation was reversed by PPARA deficiency or knockdown of E2F8 or UHRF1. Furthermore, a forced expression of CDH1 inhibited expression of the Wnt signaling target genes such as Myc after PPARA activation, and suppressed hepatocyte hyperproliferation. These results demonstrate that the PPARA-E2F8-UHRF1-CDH1 axis causes epigenetic regulation of hepatocyte proliferation.

Lipase family member N is a novel target gene for CCAAT/enhancer-binding protein α in type 2 diabetic model mouse liver.

CCAAT/enhancer-binding protein α (C/EBPα) is a transcription factor abundantly expressed in the liver and white adipose tissue (WAT). In this study, we investigated the mechanism by which C/EBPα regulates the lipase family member N (Lipn) gene in the mouse liver. Mouse Lipn consists of non-coding exon 1 and the translation start site located in exon 2. Lipn expression in the fatty liver of ob/ob mice was significantly higher than that in OB/OB mice and was significantly repressed by liver-specific C/EBPα deficiency. Lipn expression in ob/ob mice was detected in the liver, epididymal WAT (eWAT), subcutaneous WAT (sWAT), brown adipose tissue (BAT), and skeletal muscle, but not in the kidney, brain, and heart. Lipn expression in the liver, eWAT, and sWAT of wild-type mice was undetectable, although C/EBPα was highly expressed in these tissues. The database analysis revealed four putative C/EBP-responsive elements (CEBPREs), highly homologous with the typical CEBPRE consensus sequence at positions -2,686/-2,678, -1,364/-1,356, -106/-98, and -45/-37 from the transcription start site (+1) of Lipn. Reporter assays using reporter constructs with serial or internal deletions of the 5'-flanking regions of Lipn showed that two functional CEBPREs (-106/-98 and -45/-37) in the Lipn promoter region are essential for enhancing Lipn transcriptional activity by C/EBPα. Electrophoretic mobility shift assay showed that C/EBPα/ß binds to CEBPRE (-106/-98). These results suggest that C/EBPα and type 2 diabetic environment may be required for hepatic Lipn expression.

Feedback repression of PPARα signaling by Let-7 microRNA.

Peroxisome proliferator-activated receptor α (PPARα) controls hepatic lipid homeostasis and is the target of lipid-lowering fibrate drugs. PPARα activation represses expression of let-7 microRNA (miRNA), but the function of let-7 in PPARα signaling and lipid metabolism is unknown. In the current study, a hepatocyte-specific let-7b/c2 knockout (let7b/c2ΔHep) mouse line is generated, and these mice are found to exhibit pronounced resistance to diet-induced obesity and fatty liver. Let-7 inhibition by hepatocyte-specific let-7 sponge expression shows similar phenotypes as let7b/c2ΔHep mice. RNA sequencing (RNA-seq) analysis reveals that hepatic PPARα signaling is repressed in let7b/c2ΔHep mice. Protein expression of the obligate PPARα heterodimer partner retinoid X receptor α (RXRα) is reduced in the livers of let7b/c2ΔHep mice. Ring finger protein 8 (Rnf8), which is a direct target of let-7, is elevated in let7b/c2ΔHep mouse liver and identified as a E3 ubiquitin ligase for RXRα. This study highlights a let-7-RNF8-RXRα regulatory axis that modulates hepatic lipid catabolism.

Suppressing the intestinal farnesoid X receptor/sphingomyelin phosphodiesterase 3 axis decreases atherosclerosis.

Intestinal farnesoid X receptor (FXR) signaling is involved in the development of obesity, fatty liver disease, and type 2 diabetes. However, the role of intestinal FXR in atherosclerosis and its potential as a target for clinical treatment have not been explored. The serum levels of fibroblast growth factor 19 (FGF19), which is encoded by an FXR target gene, were much higher in patients with hypercholesterolemia than in control subjects and were positively related to circulating ceramide levels, indicating a link between intestinal FXR, ceramide metabolism, and atherosclerosis. Among ApoE-/- mice fed a high-cholesterol diet (HCD), intestinal FXR deficiency (in FxrΔIE ApoE-/- mice) or direct FXR inhibition (via treatment with the FXR antagonist glycoursodeoxycholic acid [GUDCA]) decreased atherosclerosis and reduced the levels of circulating ceramides and cholesterol. Sphingomyelin phosphodiesterase 3 (SMPD3), which is involved in ceramide synthesis in the intestine, was identified as an FXR target gene. SMPD3 overexpression or C16:0 ceramide supplementation eliminated the improvements in atherosclerosis in FxrΔIE ApoE-/- mice. Administration of GUDCA or GW4869, an SMPD3 inhibitor, elicited therapeutic effects on established atherosclerosis in ApoE-/- mice by decreasing circulating ceramide levels. This study identified an intestinal FXR/SMPD3 axis that is a potential target for atherosclerosis therapy.

Long non-coding RNA Gm15441 attenuates hepatic inflammasome activation in response to PPARA agonism and fasting.

Exploring the molecular mechanisms that prevent inflammation during caloric restriction may yield promising therapeutic targets. During fasting, activation of the nuclear receptor peroxisome proliferator-activated receptor α (PPARα) promotes the utilization of lipids as an energy source. Herein, we show that ligand activation of PPARα directly upregulates the long non-coding RNA gene Gm15441 through PPARα binding sites within its promoter. Gm15441 expression suppresses its antisense transcript, encoding thioredoxin interacting protein (TXNIP). This, in turn, decreases TXNIP-stimulated NLR family pyrin domain containing 3 (NLRP3) inflammasome activation, caspase-1 (CASP1) cleavage, and proinflammatory interleukin 1ß (IL1B) maturation. Gm15441-null mice were developed and shown to be more susceptible to NLRP3 inflammasome activation and to exhibit elevated CASP1 and IL1B cleavage in response to PPARα agonism and fasting. These findings provide evidence for a mechanism by which PPARα attenuates hepatic inflammasome activation in response to metabolic stress through induction of lncRNA Gm15441.

Insulin induces expression of the hepatic vaspin gene.

Visceral adipose tissue-derived serine protease inhibitor (vaspin), initially identified in the visceral adipose tissue, is an adipokine that improves endoplasmic reticulum stress in obesity or insulin sensitivity and glucose tolerance. However, the transcriptional regulation of the hepatic vaspin gene remains elusive. We have previously shown that CCAAT-enhancer-binding protein α, a transcription factor of the basic leucine zipper class, positively regulates the vaspin gene. The present study aimed to investigate the nutritional or hormonal regulators of vaspin expression in the liver. For the fasting and refeeding study, mice in the fasting group were subjected to fasting for 24 h and then sacrificed. Mice in the refeeding group were subjected to fasting for 24 h and then refed with a 50% (w/w) sucrose/MF diet for further 24 h and then sacrificed. For the streptozotocin (STZ) study, STZ (50 mg/kg) was intraperitoneally injected into C57BL/6JJc1 mice for 5 d. Hepatic vaspin was repressed due to fasting for 24 h and was induced upon refeeding with a high-sucrose diet. In studies on liver-specific C/EBPα-deficient mice, C/EBPα was not involved in the induction of hepatic vaspin upon refeeding. In addition, the depletion of insulin by streptozotocin treatment markedly decreased hepatic vaspin expression. Finally, fasting-repressed vaspin expression in the liver was significantly increased by direct injection of insulin into fasting mice. In conclusion, our results suggest that insulin is a positive regulator of hepatic vaspin expression.

Fat-specific protein 27b is regulated by hepatic peroxisome proliferator-activated receptor γ in hepatic steatosis.

The fat-specific protein 27 gene (Fsp27) belongs to the cell death-inducing DNA fragmentation factor 45-like effector family. Fsp27 is highly expressed in adipose tissue and fatty liver. In adipocytes, FSP27 localizes to the membrane of lipid droplets and promotes lipid droplet hypertrophy. Recently, FSP27 was shown to consist of two isoforms, FSP27α and FSP27ß. Previously, we demonstrated that Fsp27a is directly regulated by peroxisome proliferator-activated receptor γ (PPARγ) in fatty livers of genetically obese leptin deficient ob/ob mice and that Fsp27b may potentially be regulated by different factors transcriptionally as they both have a different promoter region. Thus, the aim of the present study was to elucidate whether Fsp27b is regulated by PPARγ in fatty liver. Fsp27a and Fsp27b were markedly induced in fatty liver of ob/ob mice compared with those in the normal liver. However, both Fsp27a/b were expressed at markedly lower levels in liver-specific PPARγ knockout mice with an ob/ob background. Further, the PPAR response element (PPRE) for the PPARγ-dependent promotion of Fsp27b promotor activity was revealed at position -1,163/-1,151 from the transcriptional start site (+1). Interestingly, the cis-element responsible for the PPARγ-dependent induction of Fsp27b was the same as that responsible for PPARγ-dependent induction of Fsp27a. These results suggest that PPARγ regulates not only Fsp27a but also Fsp27b in fatty liver of ob/ob mice through a common PPRE.

Vaspin is a novel target gene of hepatic CCAAT-enhancer-binding protein.

Vaspin, initially identified in visceral adipose tissue, is an adipokine, and administration of recombinant vaspin leads to lowering of the endoplasmic reticulum stress which is elevated in obesity or enhancement of insulin sensitivity. CCAAT/enhancer binding protein (C/EBP), as a basic leucine zipper transcription factor, plays a critical role in adipocyte development and glucose and lipid metabolisms in liver. The present study aimed to investigate the effect of C/EBPα on vaspin gene expression. The expression of hepatic vaspin was markedly decreased in liver-specific C/EBPα knockout mice. A reporter assay indicated that two C/EBP-responsive elements (CEBPREs) are necessary for C/EBPα-dependent induction of vaspin promoter activities. Furthermore, electrophoretic mobility shift assay showed that C/EBPα in mouse liver is capable of directly binding the two CEBPREs. These results suggest that C/EBPα positively regulates hepatic vaspin expression through two functional CEBPREs. Thus, vaspin is a novel C/EBPα target gene in the liver.

Fat-specific protein 27 is a novel target gene of liver X receptor α.

Fat-specific protein 27 (FSP27) is highly expressed in the fatty liver of genetically obese ob/ob mice and promotes hepatic triglyceride (TG) accumulation. The nuclear hormone receptor liver X receptor α (LXRα) also plays a critical role in the control of TG levels in the liver. The present study demonstrated transcriptional regulation of Fsp27a and Fsp27b genes by LXRα. Treatment with the LXR ligand T0901317 markedly increased Fsp27a and Fsp27b mRNAs in wild-type C57BL/6J and ob/ob mouse livers. A reporter assay indicated that two LXR-responsive elements (LXREs) are necessary for LXRα-dependent induction of Fsp27a and Fsp27b promoter activities. Furthermore, the LXRα/retinoid X receptor α complex is capable of directly binding to the two LXREs both in vitro and in vivo. These results suggest that LXRα positively regulates Fsp27a and Fsp27b expression through two functional LXREs. Fsp27a/b are novel LXR target genes in the ob/ob fatty liver.

Insulin Represses Fasting-Induced Expression of Hepatic Fat-Specific Protein 27.

The fat-specific protein 27 (Fsp27) gene belongs to the cell death-inducing DNA fragmentation factor 45-like effector family. Fsp27 is highly expressed in adipose tissue as well as the fatty liver of ob/ob mice. Fsp27 is directly regulated by the peroxisome proliferator-activated receptor γ (PPARγ) in livers of genetically obese leptin deficient ob/ob mice. In the present study, Fsp27 was markedly induced by 24 h fasting in genetically normal mouse livers and repressed by refeeding a high sucrose diet. In contrast with the liver, Fsp27 expression was decreased in adipose tissue by fasting and increased by refeeding. Interestingly, fasting-induced Fsp27 liver expression was independent of PPARγ. Moreover, Fsp27 expression was induced in the insulin-depleted livers of streptozotocin-treated mice. Finally, Fsp27 expression was repressed by direct injection of glucose or insulin in fasting mice. These results suggest that insulin represses Fsp27 expression in the fasting liver.

Hepatic PPARγ and LXRα independently regulate lipid accumulation in the livers of genetically obese mice.

The nuclear hormone receptors liver X receptor α (LXRα) and peroxisome proliferator-activated receptor γ (PPARγ) play key roles in the development of fatty liver. To determine the link between hepatic PPARγ and LXRα signaling and the development of fatty liver, a LXRα-specific ligand, T0901317, was administered to normal OB/OB and genetically obese (ob/ob) mice lacking hepatic PPARγ (Pparγ(ΔH)). In ob/ob-Pparγ(ΔH) and OB/OB-Pparγ(ΔH) mice, as well as ob/ob-Pparγ(WT) and OB/OB-Pparγ(WT) mice, the liver weights and hepatic triglyceride levels were markedly increased in response to T0901317 treatment. These results suggest that hepatic PPARγ and LXRα signals independently contribute to the development of fatty liver.

Expression of hepatic fat-specific protein 27 depends on the specific etiology of fatty liver.

Fat-specific protein 27 gene (FSP27), isolated by screening for genes specifically expressed in fully differentiated mouse adipocytes, belongs to the cell death-inducing DNA fragmentation factor, alpha subunit-like effector family. FSP27 is induced in not only adipose tissue but also the liver of ob/ob mice, and it promotes the development of fatty liver. The FSP27 gene is expressed in a fatty liver-specific manner and is not detected in the normal mouse liver. FSP27 expression is directly regulated by the induction of the hepatic peroxisome proliferator-activated receptor γ (PPARγ) in ob/ob fatty liver. In the present study, expression of hepatic FSP27 mRNA was determined in non-genetic fatty liver models. The FSP27 gene was markedly induced in the high-fat- or methionine- and choline-deficient (MCD) diet-induced fatty liver, but it was not elevated in alcohol-induced fatty liver. Interestingly, the induction of FSP27 mRNA due to the MCD diet was independent of PPARγ levels and completely absent in the liver from PPARγ-null mice. These results suggest that FSP27 mRNA expression in the liver depends on the etiology of fatty liver.