Songtao Yu

PPARalpha: energy combustion, hypolipidemia, inflammation and cancer.

The peroxisome proliferator-activated receptor α (PPARα, or NR1C1) is a nuclear hormone receptor activated by a structurally diverse array of synthetic chemicals known as peroxisome proliferators. Endogenous activation of PPARα in liver has also been observed in certain gene knockout mouse models of lipid metabolism, implying the existence of enzymes that either generate (synthesize) or degrade endogenous PPARα agonists. For example, substrates involved in fatty acid oxidation can function as PPARα ligands. PPARα serves as a xenobiotic and lipid sensor to regulate energy combustion, hepatic steatosis, lipoprotein synthesis, inflammation and liver cancer. Mainly, PPARα modulates the activities of all three fatty acid oxidation systems, namely mitochondrial and peroxisomal β-oxidation and microsomal co-oxidation, and thus plays a key role in energy expenditure. Sustained activation of PPARα by either exogenous or endogenous agonists leads to the development of hepatocellular carcinoma resulting from sustained oxidative and possibly endoplasmic reticulum stress and liver cell proliferation. PPARα requires transcription coactivator PPAR-binding protein (PBP)/mediator subunit 1(MED1) for its transcriptional activity.

Peroxisome proliferator-activated receptors, fatty acid oxidation, steatohepatitis and hepatocarcinogenesis

Fatty acids are metabolized in the liver by beta-oxidation in mitochondria and peroxisomes and by omega-oxidation in microsomes. Peroxisomal beta-oxidation is responsible for the metabolism of very long chain fatty acids and mitochondrial beta-oxidation is responsible for the oxidation of short, medium and long chain fatty acids. Very long chain fatty acids are also metabolized by the cytochrome P450 CYP4A omega-oxidation system to dicarboxylic acids. Both peroxisomal beta-oxidation and microsomal omega-oxidation lead to the generation of H2O2. The genes encoding peroxisomal, microsomal and some mitochondrial fatty acid metabolizing enzymes in the liver are transcriptionally regulated by peroxisome proliferator-activated receptor alpha (PPAR alpha). Sustained activation of PPAR alpha by peroxisome proliferators has been shown to induce hepatocellular carcinomas in rats and mice. The peroxisome proliferator-induced carcinogenic effect has been attributed to transcriptional activation of PPAR alpha regulated genes and the resulting excessive generation of H2O2. Evidence from mice lacking fatty acyl-CoA oxidase (AOX), PPAR alpha and PPAR alpha/AOX has confirmed the role of PPAR alpha in the development of hepatocellular carcinomas. In addition, mice lacking AOX developed steatohepatitis and provided clues regarding the molecular mechanism responsible for steatosis and steatohepatitis and the role of unmetabolized AOX substrates in the activation of PPAR alpha.

Identification of a transcriptionally active peroxisome proliferator-activated receptor α-interacting cofactor complex in rat liver and characterization of PRIC285 as a coactivator

Peroxisome proliferator-activated receptor α (PPARα) plays a central role in the cell-specific pleiotropic responses induced by structurally diverse synthetic chemicals designated as peroxisome proliferators. Transcriptional regulation by liganded nuclear receptors involves the participation of cofactors that form multiprotein complexes to achieve cell- and gene-specific transcription. Here we report the identification of such a transcriptionally active PPARα-interacting cofactor (PRIC) complex from rat liver nuclear extracts that interacts with full-length PPARα in the presence of ciprofibrate, a synthetic ligand, and leukotriene B4, a natural ligand. The liganded PPARα-PRIC complex enhanced transcription from a peroxisomal enoyl-CoA hydratase/l-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme gene promoter template that contains peroxisome proliferator response elements. Rat liver PRIC complex comprises some 25 polypeptides, and their identities were established by mass spectrometry and limited sequence analysis. Eighteen of these peptides contain one or more LXXLL motifs necessary for interacting with nuclear receptors. PRIC complex includes known coactivators or coactivator-binding proteins (CBP, SRC-1, PBP, PRIP, PIMT, TRAP100, SUR-2, and PGC-1), other proteins that have not previously been described in association with transcription complexes (CHD5, TOG, and MORF), and a few novel polypeptides designated PRIC300, -285, -215, -177, and -145. We describe the cDNA for PRIC285, which contains five LXXLL motifs. It interacts with PPARα and acts as a coactivator by moderately stimulating PPARα-mediated transcription in transfected cells. We conclude that liganded PPARα recruits a distinctive multiprotein complex from rat liver nuclear extracts. The composition of this complex may provide insight into the basis of tissue and species sensitivity to peroxisome proliferators.

Phosphorylation of Transcriptional Coactivator Peroxisome Proliferator-activated Receptor (PPAR)-binding Protein (PBP) STIMULATION OF TRANSCRIPTIONAL REGULATION BY MITOGEN-ACTIVATED PROTEIN KINASE

Peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP) is an important coactivator for PPARγ and other transcription factors. PBP is an integral component of a multiprotein thyroid hormone receptor-associated protein (TRAP)/vitamin D3 receptor-interacting protein (DRIP)/activator-recruited cofactor (ARC) complex required for transcriptional activity. To study the regulation of PBP by cellular signaling pathways, we identified the phosphorylation sites of PBP. Using a combination of in vitro and in vivo approaches and mutagenesis of PBP phosphorylation sites, we identified six phosphorylation sites on PBP: one exclusive protein kinase A (PKA) phosphorylation site at serine 656, two protein kinase C (PKC) sites at serine 796 and serine 1345, a common PKA/PKC site at serine 756, and two extracellular signal-regulated kinase 2 sites of the mitogen-activated protein kinase (MAPK) family at threonine 1017 and threonine 1444. Binding of PBP to PPARγ1 or retinoid-X-receptor for 9-cis-retinoic acid (RXR) is independent of their phosphorylation states, implying no changes in protein-protein interaction after modification by phosphorylation. Overexpression of RafBXB, an activated upstream kinase of the MAPK signal transduction pathway, exerts a significant additive inductive effect on PBP coactivator function. This effect is significantly diminished by overexpression of RafBXB301, a dominant negative mutant of RafBXB. These results identify phosphorylation as a regulatory modification event of PBP and demonstrate that PBP phosphorylation by Raf/MEK/MAPK cascade exerts a positive effect on PBP coactivator function. The functional role of PKA and PKC phosphorylation sites in PBP remains to be elucidated.

Transcriptional Regulation of Cidea, Mitochondrial Cell Death-inducing DNA Fragmentation Factor α-Like Effector A, in Mouse Liver by Peroxisome Proliferator-activated Receptor α and γ

Cidea (cell death-inducing DNA fragmentation factor α-like effector A), a member of a novel family of proapoptotic proteins, is expressed abundantly in the brown adipose tissue of the mouse. Although Cidea mRNA is not detectable in the mouse liver, we now show that peroxisome proliferator-activated receptor (PPAR) α ligands Wy-14,643 and ciprofibrate increase the Cidea mRNA level in a PPARα-dependent manner, whereas Cidea induction in liver by PPARγ overexpression is PPARα independent. Increase in Cidea mRNA content in liver did not alter the expression of uncoupling protein 1 (Ucp1) gene, which regulates thermogenesis, lipolysis, and conservation of energy. Although Cidea is considered to be a proapoptotic factor, Cidea induction in liver did not result in increased apoptosis. To elucidate the mechanism by which PPARα and PPARγ regulate Cidea gene expression in the liver, we analyzed the promoter region of the Cidea gene. Three putative peroxisome proliferator response elements (PPREs) are found in the Cidea gene promoter. Transactivation, gel-shift, and chromatin immunoprecipitation assays indicated that the proximal PPRE in Cidea gene (Cidea-PPRE1 at -680/-668) is functional for both PPARα and -γ. We conclude that Cidea is a novel target gene for both PPARα and -γ in the liver where these two transcription factors utilize the same PPRE region for dual regulation. The induction of Cidea in liver with these PPARα and -γ agonists suggests a possible role for Cidea in energy metabolism and a less likely role in hepatocyte apoptosis.

Critical Role for Transcription Coactivator Peroxisome Proliferator-activated Receptor (PPAR)-binding Protein/TRAP220 in Liver Regeneration and PPARα Ligand-induced Liver Tumor Development

Disruption of the gene encoding for the transcription coactivator peroxisome proliferator-activated receptor (PPAR)-binding protein (PBP/TRAP220/DRIP205/Med1) in the mouse results in embryonic lethality. Here, we have reported that targeted disruption of the Pbp/Pparbp gene in hepatocytes (PbpΔLiv) impairs liver regeneration with low survival after partial hepatectomy. Analysis of cell cycle progression suggests a defective exit from quiescence, reduced BrdUrd incorporation, and diminished entry into G2/M phase in PbpΔLiv hepatocytes after partial hepatectomy. PbpΔLiv hepatocytes failed to respond to hepatocyte growth factor/scatter factor, implying that hepatic PBP deficiency affects c-met signaling. Pbp gene disruption also abolishes primary mitogen-induced liver cell proliferative response. Striking abrogation of CCl4-induced hepatocellular proliferation and hepatotoxicity occurred in PbpΔLiv mice pretreated with phenobarbital due to lack of expression of xenobiotic metabolizing enzymes necessary for CCl4 activation. PbpΔLiv mice, chronically exposed to Wy-14,643, a PPARα ligand, revealed a striking proliferative response and clonal expansion of a few Pbpfl/fl hepatocytes that escaped Cre-mediated gene deletion in PbpΔLiv livers, but no proliferative expansion of PBP null hepatocytes was observed. In these PbpΔLiv mice, none of the Wy-14,643-induced hepatic adenomas and hepatocellular carcinomas was derived from PBPΔLiv hepatocytes; all liver tumors developing in PbpΔLiv mice maintained non-recombinant Pbp alleles and retained PBP expression. These studies provide direct evidence in support of a critical role of PBP/TRAP220 in liver regeneration, induction of hepatotoxicity, and hepatocarcinogenesis.

Upregulation of calpastatin in regenerating and developing rat liver: Role in resistance against hepatotoxicity

Acute liver failure induced by hepatotoxic drugs results from rapid progression of injury. Substantial research has shown that timely liver regeneration can prevent progression of injury leading to a favorable prognosis. However, the mechanism by which compensatory regeneration prevents progression of injury is not known. We have recently reported that calpain released from necrotic hepatocytes mediates progression of liver injury even after the hepatotoxic drug is cleared from the body. By examining expression of calpastatin (CAST), an endogenous inhibitor of calpain in three liver cell division models known to be resistant to hepatotoxicity, we tested the hypothesis that increased CAST in the dividing hepatocytes affords resistance against progression of injury. Liver regeneration that follows CCl4‐induced liver injury, 70% partial hepatectomy, and postnatal liver development were used. In all three models, CAST was upregulated in the dividing/newly divided hepatocytes and declined to normal levels with the cessation of cell proliferation. To test whether CAST overexpression confers resistance against hepatotoxicity, CAST was overexpressed in the livers of normal SW mice using adenovirus before challenging them with acetaminophen (APAP) overdose. These mice exhibited markedly attenuated progression of liver injury and 57% survival. Whereas APAP‐bioactivating enzymes and covalent binding of the APAP‐derived reactive metabolites remained unaffected, degradation of calpain specific target substrates such as fodrin was significantly reduced in these mice. In conclusion, CAST overexpression could be used as a therapeutic strategy to prevent progression of liver injury where liver regeneration is severely hampered. (HEPATOLOGY 2006;44:379–388.)

Progressive Endoplasmic Reticulum Stress Contributes to Hepatocarcinogenesis in Fatty Acyl-CoA Oxidase 1–Deficient Mice

Fatty acyl-coenzyme A oxidase 1 (ACOX1) knockout (ACOX1(-/-)) mice manifest hepatic metabolic derangements that lead to the development of steatohepatitis, hepatocellular regeneration, spontaneous peroxisome proliferation, and hepatocellular carcinomas. Deficiency of ACOX1 results in unmetabolized substrates of this enzyme that function as biological ligands for peroxisome proliferator-activated receptor-α (PPARα) in liver. Here we demonstrate that sustained activation of PPARα in ACOX1(-/-) mouse liver by these ACOX1 substrates results in endoplasmic reticulum (ER) stress. Overexpression of transcriptional regulator p8 and its ER stress-related effectors such as the pseudokinase tribbles homolog 3, activating transcription factor 4, and transcription factor CCAAT/-enhancer-binding protein homologous protein as well as phosphorylation of eukaryotic translation initiation factor 2α, indicate the induction of unfolded protein response signaling in the ACOX1(-/-) mouse liver. We also show here that, in the liver, p8 is a target for all three PPAR isoforms (-α, -β, and -γ), which interact with peroxisome proliferator response elements in p8 promoter. Sustained activation of p8 and unfolded protein response-associated ER stress in ACOX1(-/-) mouse liver contributes to hepatocyte apoptosis and liver cell proliferation culminating in the development of hepatocarcinogenesis. We also demonstrate that human ACOX1 transgene is functional in ACOX1(-/-) mice and effectively prevents metabolic dysfunctions that lead to ER stress and carcinogenic effects. Taken together, our data indicate that progressive PPARα- and p8-mediated ER stress contribute to the hepatocarcinogenesis in ACOX1(-/-) mice.

Conditional Ablation of Mediator Subunit MED1 (MED1/PPARBP) Gene in Mouse Liver Attenuates Glucocorticoid Receptor Agonist Dexamethasone-Induced Hepatic Steatosis

Glucocorticoid receptor (GR) agonist dexamethasone (Dex) induces hepatic steatosis and enhances constitutive androstane receptor (CAR) expression in the liver. CAR is known to worsen hepatic injury in nonalcoholic hepatic steatosis. Because transcription coactivator MED1/PPARBP gene is required for GR- and CAR-mediated transcriptional activation, we hypothesized that disruption of MED1/PPARBP gene in liver cells would result in the attenuation of Dex-induced hepatic steatosis. Here we show that liver-specific disruption of MED1 gene (MED1(delta Liv)) improves Dex-induced steatotic phenotype in the liver. In wild-type mice Dex induced severe hepatic steatosis and caused reduction in medium- and short-chain acyl-CoA dehydrogenases that are responsible for mitochondrial beta-oxidation. In contrast, Dex did not induce hepatic steatosis in mice conditionally null for hepatic MED1, as it failed to inhibit fatty acid oxidation enzymes in the liver. MED1(delta Liv) livers had lower levels of GR-regulated CAR mRNA compared to wild-type mouse livers. Microarray gene expression profiling showed that absence of MED1 affects the expression of the GR-regulated genes responsible for energy metabolism in the liver. These results establish that absence of MED1 in the liver diminishes Dex-induced hepatic steatosis by altering the GR- and CAR-dependent gene functions.

Induction of nuclear translocation of constitutive androstane receptor by peroxisome proliferator-activated receptor α synthetic ligands in mouse liver

Peroxisome proliferators activate nuclear receptor peroxisome proliferator-activated receptor α (PPARα) and enhance the transcription of several genes in liver. We report here that synthetic PPARα ligands Wy-14,643, ciprofibrate, clofibrate, and others induce the nuclear translocation of constitutive androstane receptor (CAR) in mouse liver cells in vivo. Adenoviral-enhanced green fluorescent protein-CAR expression demonstrated that PPARα synthetic ligands drive CAR into the hepatocyte nucleus in a PPARα- and PPARβ-independent manner. This translocation is dependent on the transcription coactivator PPAR-binding protein but independent of coactivators PRIP and SRC-1. PPARα ligand-induced nuclear translocation of CAR is not associated with induction of Cyp2b10 mRNA in mouse liver. PPARα ligands interfered with coactivator recruitment to the CAR ligand binding domain and reduced the constitutive transactivation of CAR. Both Wy-14,643 and ciprofibrate occupied the ligand binding pocket of CAR and adapted a binding mode similar to that of the CAR inverse agonist androstenol. These observations, therefore, provide information for the first time to indicate that PPARα ligands not only serve as PPARα agonists but possibly act as CAR antagonists.