Yuzhi Jia

Protein kinase involved in lung injury susceptibility: Evidence from enzyme isoform genetic knockout and in vivo inhibitor treatment

Acute lung injury (ALI) associated with sepsis and iatrogenic ventilator-induced lung injury resulting from mechanical ventilation are major medical problems with an unmet need for small molecule therapeutics. Prevailing hypotheses identify endothelial cell (EC) layer dysfunction as a cardinal event in the pathophysiology, with intracellular protein kinases as critical mediators of normal physiology and possible targets for drug discovery. The 210,000 molecular weight myosin light chain kinase (MLCK210, also called EC MLCK because of its abundance in EC) is hypothesized to be important for EC barrier function and might be a potential therapeutic target. To test these hypotheses directly, we made a selective MLCK210 knockout mouse that retains production of MLCK108 (also called smooth-muscle MLCK) from the same gene. The MLCK210 knockout mice are less susceptible to ALI induced by i.p. injection of the endotoxin lipopolysaccharide and show enhanced survival during subsequent mechanical ventilation. Using a complementary chemical biology approach, we developed a new class of small-molecule MLCK inhibitor based on the pharmacologically privileged aminopyridazine and found that a single i.p. injection of the inhibitor protected WT mice against ALI and death from mechanical ventilation complications. These convergent results from two independent approaches demonstrate a pivotal in vivo role for MLCK in susceptibility to lung injury and validate MLCK as a potential drug discovery target for lung injury.

Coactivators in PPAR-Regulated Gene Expression.

Peroxisome proliferator-activated receptor (PPAR)α, β (also known as δ), and γ function as sensors for fatty acids and fatty acid derivatives and control important metabolic pathways involved in the maintenance of energy balance. PPARs also regulate other diverse biological processes such as development, differentiation, inflammation, and neoplasia. In the nucleus, PPARs exist as heterodimers with retinoid X receptor-α bound to DNA with corepressor molecules. Upon ligand activation, PPARs undergo conformational changes that facilitate the dissociation of corepressor molecules and invoke a spatiotemporally orchestrated recruitment of transcription cofactors including coactivators and coactivator-associated proteins. While a given nuclear receptor regulates the expression of a prescribed set of target genes, coactivators are likely to influence the functioning of many regulators and thus affect the transcription of many genes. Evidence suggests that some of the coactivators such as PPAR-binding protein (PBP/PPARBP), thyroid hormone receptor-associated protein 220 (TRAP220), and mediator complex subunit 1 (MED1) may exert a broader influence on the functions of several nuclear receptors and their target genes. Investigations into the role of coactivators in the function of PPARs should strengthen our understanding of the complexities of metabolic diseases associated with energy metabolism.

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.

Sustained activation of PPARα by endogenous ligands increases hepatic fatty acid oxidation and prevents obesity in ob/ob mice

Obesity, a major health concern, results from an imbalance between energy intake and expenditure. Leptin‐deficient ob/ob mice are paradigmatic of obesity, resulting from excess energy intake and storage. Mice lacking acyl‐CoA oxidase 1 (Acox1), the first enzyme of the peroxisomal fatty acid β‐oxidation system, are characterized by increased energy expenditure and a lean body phenotype caused by sustained activation of peroxisome proliferator‐activated receptor α (PPARα) by endogenous ligands in liver that remain unmetabolized in the absence of Acox1. We generated ob/ob mice deficient in Acox1 (Acox1‐/‐) to determine how the activation of PPARα by endogenous ligands might affect the obesity of ob/ob mice. In contrast to Acox1‐/‐ (14.3± 1.2 g at 6 mo) and the Acox1‐deficient (ob/ob) double‐mutant mice (23.8±4.6 g at 6 mo), the ob/ob mice are severely obese (54.3±3.2 g at 6 mo) and had significantly more (P<0.01) epididymal fat content. The resistance of Acox1‐/‐/ob/ob mice to obesity is due to increased PPARα‐mediated up‐regulation of genes involved in fatty acid oxidation in liver. Activation of PPARα in Acox1‐deficient ob/ob mice also reduces serum glucose and insulin (P<0.05) and improves glucose tolerance and insulin sensitivity. Further, PPARα activation reduces hepatic steatosis and increases hepatocellular regenerative response in Acox1‐/‐/ob/ob mice at a more accelerated pace than in mice lacking only Acox1. However, Acox1‐/‐/ob/ob mice manifest hepatic endoplasmic reticulum (ER) stress and also develop hepatocellular carcinomas (8 of 8 mice) similar to those observed in Acox1‐/‐ mice (10 of 10 mice), but unlike in ob/ob (0 of 14 mice) and OB/OB (0 of 6 mice) mice, suggesting that superimposed ER stress and PPARα activation contribute to carcinogenesis in a fatty liver. Finally, absence of Acox1 in ob/ob mice can impart resistance to high‐fat diet (60% fat)‐induced obesity, and their liver had significantly (P<0.01) more cell proliferation. These studies with Acox1‐/‐/ob/ob mice indicate that sustained activation of lipid‐sensing nuclear receptor PPARα attenuates obesity and restores glucose homeostasis by ameliorating insulin resistance but increases the risk for liver cancer development, in part related to excess energy combustion.—Huang, J., Jia, Y., Fu, T., Viswakarma, N., Bai, L., Sambasiva Rao, M., Zhu, Y., Borensztajn, J., Reddy, J. K. Sustained activation of PPARα by endogenous ligands increases hepatic fatty acid oxidation and prevents obesity in ob/ob mice. FASEB J. 26, 628–638 (2012). www.fasebj.org

Peroxisomal L-bifunctional enzyme (Ehhadh) is essential for the production of medium-chain dicarboxylic acids

L-bifunctional enzyme (Ehhadh) is part of the classical peroxisomal fatty acid β-oxidation pathway. This pathway is highly inducible via peroxisome proliferator-activated receptor α (PPARα) activation. However, no specific substrates or functions for Ehhadh are known, and Ehhadh knockout (KO) mice display no appreciable changes in lipid metabolism. To investigate Ehhadh functions, we used a bioinformatics approach and found that Ehhadh expression covaries with genes involved in the tricarboxylic acid cycle and in mitochondrial and peroxisomal fatty acid oxidation. Based on these findings and the regulation of Ehhadhs expression by PPARα, we hypothesized that the phenotype of Ehhadh KO mice would become apparent after fasting. Ehhadh mice tolerated fasting well but displayed a marked deficiency in the fasting-induced production of the medium-chain dicarboxylic acids adipic and suberic acid and of the carnitine esters thereof. The decreased levels of adipic and suberic acid were not due to a deficient induction of ω-oxidation upon fasting, as Cyp4a10 protein levels increased in wild-type and Ehhadh KO mice.We conclude that Ehhadh is indispensable for the production of medium-chain dicarboxylic acids, providing an explanation for the coordinated induction of mitochondrial and peroxisomal oxidative pathways during fasting.

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.

Transcription Coactivator Mediator Subunit Med1 is Required for the Development of Fatty Liver in the Mouse

Peroxisome proliferator‐activated receptor‐γ (PPARγ), a nuclear receptor, when overexpressed in liver stimulates the induction of adipocyte‐specific and lipogenesis‐related genes and causes hepatic steatosis. We report here that Mediator 1 (MED1; also known as PBP or TRAP220), a key subunit of the Mediator complex, is required for high‐fat diet–induced hepatic steatosis as well as PPARγ‐stimulated adipogenic hepatic steatosis. Mediator forms the bridge between transcriptional activators and RNA polymerase II. MED1 interacts with nuclear receptors such as PPARγ and other transcriptional activators. Liver‐specific MED1 knockout (MED1ΔLiv) mice, when fed a high‐fat (60% kcal fat) diet for up to 4 months failed to develop fatty liver. Similarly, MED1ΔLiv mice injected with adenovirus‐PPARγ (Ad/PPARγ) by tail vein also did not develop fatty liver, whereas mice with MED1 (MED1fl/fl) fed a high‐fat diet or injected with Ad/PPARγ developed severe hepatic steatosis. Gene expression profiling and northern blot analyses of Ad/PPARγ–injected mouse livers showed impaired induction in MED1ΔLiv mouse liver of adipogenic markers, such as aP2, adipsin, adiponectin, and lipid droplet‐associated genes, including caveolin‐1, CideA, S3‐12, and others. These adipocyte‐specific and lipogenesis‐related genes are strongly induced in MED1fl/fl mouse liver in response to Ad/PPARγ. Re‐expression of MED1 using adenovirally‐driven MED1 (Ad/MED1) in MED1ΔLiv mouse liver restored PPARγ‐stimulated hepatic adipogenic response. These studies also demonstrate that disruption of genes encoding other coactivators such as SRC‐1, PRIC285, PRIP, and PIMT had no effect on hepatic adipogenesis induced by PPARγ overexpression. Conclusion: We conclude that transcription coactivator MED1 is required for high‐fat diet–induced and PPARγ‐stimulated fatty liver development, which suggests that MED1 may be considered a potential therapeutic target for hepatic steatosis. (HEPATOLOGY 2011;)

Mistargeting of Peroxisomal EHHADH and Inherited Renal Fanconi's Syndrome

BACKGROUND In renal Fanconis syndrome, dysfunction in proximal tubular cells leads to renal losses of water, electrolytes, and low-molecular-weight nutrients. For most types of isolated Fanconis syndrome, the genetic cause and underlying defect remain unknown. METHODS We clinically and genetically characterized members of a five-generation black family with isolated autosomal dominant Fanconis syndrome. We performed genomewide linkage analysis, gene sequencing, biochemical and cell-biologic investigations of renal proximal tubular cells, studies in knockout mice, and functional evaluations of mitochondria. Urine was studied with the use of proton nuclear magnetic resonance ((1)H-NMR) spectroscopy. RESULTS We linked the phenotype of this familys Fanconis syndrome to a single locus on chromosome 3q27, where a heterozygous missense mutation in EHHADH segregated with the disease. The p.E3K mutation created a new mitochondrial targeting motif in the N-terminal portion of EHHADH, an enzyme that is involved in peroxisomal oxidation of fatty acids and is expressed in the proximal tubule. Immunocytofluorescence studies showed mistargeting of the mutant EHHADH to mitochondria. Studies of proximal tubular cells revealed impaired mitochondrial oxidative phosphorylation and defects in the transport of fluids and a glucose analogue across the epithelium. (1)H-NMR spectroscopy showed elevated levels of mitochondrial metabolites in urine from affected family members. Ehhadh knockout mice showed no abnormalities in renal tubular cells, a finding that indicates a dominant negative nature of the mutation rather than haploinsufficiency. CONCLUSIONS Mistargeting of peroxisomal EHHADH disrupts mitochondrial metabolism and leads to renal Fanconis syndrome; this indicates a central role of mitochondria in proximal tubular function. The dominant negative effect of the mistargeted protein adds to the spectrum of monogenic mechanisms of Fanconis syndrome. (Funded by the European Commission Seventh Framework Programme and others.).

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.