P2Y2 purinergic receptor gene deletion protects mice from bacterial endotoxin and sepsis-associated liver injury and mortality.

The liver plays a significant role in regulating a wide range of metabolic, homeostatic, and host-defense functions. However, the impact of liver injury on the host's ability to control bacteremia and morbidity in sepsis is not well understood. Leukocyte recruitment and activation lead to cytokine and chemokine release, which, in turn, trigger hepatocellular injury and elevate nucleotide levels in the extracellular milieu. P2Y2 purinergic receptors, G protein-coupled and activated by extracellular ATP/UTP, are expressed at the cell surface of hepatocytes and nonparenchymal cells. We sought to determine whether P2Y2 purinergic receptor function is necessary for the maladaptive host response to bacterial infection and endotoxin-mediated inflammatory liver injury and mortality in mice. We report that P2Y2 purinergic receptor knockout mice (P2Y2-/-) had attenuated inflammation and liver injury, with improved survival in response to LPS/galactosamine (LPS/GalN; inflammatory liver injury) and cecal ligation and puncture (CLP; polymicrobial sepsis). P2Y2-/- livers had attenuated c-Jun NH2-terminal kinase activation, matrix metallopeptidase-9 expression, and hepatocyte apoptosis in response to LPS/GalN and attenuated inducible nitric oxide synthase and nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3 protein expression in response to CLP. Implicating liver injury in the disruption of amino acid homeostasis, CLP led to lower serum arginine and higher bacterial load and morbidity in the WT mice, whereas serum arginine levels were comparable to sham-operated controls in P2Y2-/- mice, which had attenuated bacteremia and improved survival. Collectively, our studies highlight the pathophysiological relevance of P2Y2 purinergic receptor function in inflammatory liver injury and dysregulation of systemic amino acid homeostasis with implications for sepsis-associated immune dysfunction and morbidity in mice.NEW & NOTEWORTHY Our studies provide experimental evidence for P2Y2 purinergic receptor-mediated potentiation of inflammatory liver injury, morbidity, and mortality, in two well-established animal models of inflammatory liver injury. Our findings highlight the potential to target P2Y2 purinergic signaling to attenuate the induction of "cytokine storm" and prevent its deleterious consequences on liver function, systemic amino acid homeostasis, host response to bacterial infection, and sepsis-associated morbidity and mortality.

Constitutive activation of mitogen-activated protein kinase kinase (MEK1) in ileal enterocytes leads to dysplasia and a predisposition to cancer.

Activation of mitogen-activated protein kinases (MAPKs) is a key factor in the pathogenesis of cancer, although the specific role of mitogen-activated protein kinase kinase (MEK1) is not well understood. Villin promoter-driven Cre expression was used to excise a floxed stop cassette from a phosphomimetically constitutively activated MEK1 (caMEK1) expression construct in the intestine of C57BL/6 mice. Zygosity status of caMEK1 afforded assessment of the dose dependence of the effect. The expected mendelian distribution of genotypes and sex was observed in 443 progenies. Between 21 and 63 days of life, caMEK1 had no effect on body weight in male mice, but reduced body weight in female mice homozygous for caMEK1. At 10 wk of age, the ileum of caMEK1-expressing mice was characterized by the finding of dysplasia and profound changes in overall architecture. Paneth cells were nearly absent in caMEK1 homozygotes. Targeted proteomic profiling via reverse phase protein array analyses with confirmatory Western blotting revealed significant changes in protein and phosphoprotein expression, including upregulation of proteins downstream of MEK1, associated with enhanced markers of proliferation, diminished apoptosis, alterations in cell-fate determination, cell-cell interactions, and tight junctions. Long-term viability of caMEK1 homozygous mice was reduced with no survival beyond 1 yr. Invasive adenocarcinoma developed in three of ten older mice [15 wk (homozygous), 26 wk (homozygous), and 35 wk (heterozygous) of age]. Expression of caMEK1 in enterocytes leads to marked derangements in the intestinal epithelium, which is associated with a predisposition to the development of invasive cancer.NEW & NOTEWORTHY The ileum of mice with constitutive expression of activated MEK1 (via phosphomimetic changes) in enterocytes is markedly abnormal with architectural distortion and cytologic atypia, which evolves into an adenoma invasive carcinoma sequence. Phosphoproteomic analysis reveals upregulation of proteins downstream of MEK1, associated with enhanced markers of proliferation, diminished apoptosis, alterations in cell-fate determination, cell-cell interactions, and tight junctions. This novel model provides new insights into intestinal homeostasis and carcinogenesis.

TGR5 activation induces cytoprotective changes in the heart and improves myocardial adaptability to physiologic, inotropic, and pressure-induced stress in mice.

INTRODUCTION: Administration of cholic acid, or its synthetic derivative, 6-alpha-ethyl-23(S)-methylcholic acid (INT-777), activates the membrane GPCR, TGR5, influences whole body metabolism, reduces atherosclerosis, and benefits the cardiovascular physiology in mice. Direct effects of TGR5 agonists, and the role for TGR5, on myocardial cell biology and stress response are unknown. METHODS: Mice were fed chow supplemented with 0.5% cholic acid (CA) or 0.025% INT-777, a specific TGR5 agonist, or regular chow for 3 weeks. Anthropometric, biochemical, physiologic (electrocardiography and echocardiography), and molecular analysis was performed at baseline. CA and INT-777 fed mice were challenged with acute exercise-induced stress, acute catecholamine-induced stress, and hemodynamic stress induced by transverse aortic constriction (TAC) for a period of 8 weeks. In separate experiments, mice born with constitutive deletion of TGR5 in cardiomyocytes (CM-TGR5del ) were exposed to exercise, inotropic, and TAC-induced stress. RESULTS: Administration of CA and INT-777 supplemented diets upregulated TGR5 expression and activated Akt, PKA, and ERK1/2 in the heart. CA and INT-777 fed mice showed improved exercise tolerance, improved sensitivity to catecholamine and attenuation in pathologic remodeling of the heart under hemodynamic stress. In contrast, CM-TGR5del showed poor response to exercise and catecholamine challenge as well as higher mortality and signs of accelerated cardiomyopathy under hemodynamic stress. CONCLUSIONS: Bile acids, specifically TGR5 agonists, induce cytoprotective changes in the heart and improve myocardial response to physiologic, inotropic, and hemodynamic stress in mice. TGR5 plays a critical role in myocardial adaptability, and TGR5 activation may represent a potentially attractive treatment option in heart failure.

P2X3 purinergic receptor overexpression is associated with poor recurrence-free survival in hepatocellular carcinoma patients.

UNLABELLED: P2 purinergic receptors are overexpressed in certain cancer tissues, but the pathophysiologic relevance of purinergic signaling in hepatocellular carcinoma (HCC) remains unknown. To examine the role of P2 purinergic signaling in the pathogenesis of HCC and characterize extracellular nucleotide effects on HCC cell proliferation, two independent HCC patient cohorts were analyzed for P2 purinergic receptor expression, and nucleotide treated HCC cell lines were evaluated for effects on proliferation and cell cycle progression. Our studies suggest that multiple P2 purinergic receptor isoforms are overexpressed in liver tumors, as compared to uninvolved liver, and dysregulation of P2 purinergic receptor expression is apparent in HCC cell lines, as compared to human primary hepatocytes. High P2X3 purinergic receptor expression is associated with poor recurrence-free survival (RFS), while high P2Y13 expression is associated with improved RFS. Extracellular nucleotide treatment alone is sufficient to induce cell cycle progression, via activation of JNK signaling, and extracellular ATP-mediated activation of P2X3 receptors promotes proliferation in HCC cells. CONCLUSION: Our analysis of HCC patient livers and HCC cells in vitro identifies a novel role for dysregulation of P2 purinergic signaling in the induction of hyper-proliferative HCC phenotype and identifies P2X3 purinergic receptors as potential new targets for therapy.

SRC-2 orchestrates polygenic inputs for fine-tuning glucose homeostasis.

Despite extensive efforts to understand the monogenic contributions to perturbed glucose homeostasis, the complexity of genetic events that fractionally contribute to the spectrum of this pathology remain poorly understood. Proper maintenance of glucose homeostasis is the central feature of a constellation of comorbidities that define the metabolic syndrome. The ability of the liver to balance carbohydrate uptake and release during the feeding-to-fasting transition is essential to the regulation of peripheral glucose availability. The liver coordinates the expression of gene programs that control glucose absorption, storage, and secretion. Herein, we demonstrate that Steroid Receptor Coactivator 2 (SRC-2) orchestrates a hierarchy of nutritionally responsive transcriptional complexes to precisely modulate plasma glucose availability. Using DNA pull-down technology coupled with mass spectrometry, we have identified SRC-2 as an indispensable integrator of transcriptional complexes that control the rate-limiting steps of hepatic glucose release and accretion. Collectively, these findings position SRC-2 as a major regulator of polygenic inputs to metabolic gene regulation and perhaps identify a previously unappreciated model that helps to explain the clinical spectrum of glucose dysregulation.

Cardiomyopathy reverses with recovery of liver injury, cholestasis and cholanemia in mouse model of biliary fibrosis.

BACKGROUND: Triggers and exacerbants of cirrhotic cardiomyopathy (CC) are poorly understood, limiting treatment options in patients with chronic liver diseases. Liver transplantation alone reverses some features of CC, but the physiology behind this effect has never been studied. AIMS: We aimed to determine whether reversal of liver injury and fibrosis in mouse affects cardiac parameters. The second aim was to determine whether cardiomyopathy can be induced by specifically increasing systemic bile acid (BA) levels. METHODS: 6-8 week old male C57BL6J mice were fed either chow (n = 5) or 3,5-diethoxycarbonyl-1,4-dihydroxychollidine (DDC) (n = 10) for 3 weeks. At the end of 3 weeks, half the mice in the DDC fed group were randomized to chow (the reversed [REV] group). Serial ECHOs and electrocardiographic analysis was conducted weekly for 6 weeks followed by liver tissue and serum studies. Hearts were analysed for key components of function and cell signalling. Cardiac physiological and molecular parameters were similarly analysed in Abcb11(-/-) mice (n = 5/grp) fed 0.5% cholic acid supplemented diet for 1 week. RESULTS: Mice in the REV group showed normalization of biochemical markers of liver injury with resolution of electrocardiographic and ECHO aberrations. Catecholamine resistance seen in DDC group resolved in the REV group. Cardiac recovery was accompanied by normalization of cardiac troponin-T2 as well as resolution of cardiac stress response at RNA level. Cardiovascular physiological and molecular parameters correlated with degree of cholanemia. Cardiomyopathy was reproduced in cholanemic BA fed Abcb11(-/-) mice. CONCLUSIONS: Cardiomyopathy resolves with resolution of liver injury, is associated with cholanaemia, and can be induced by BA feeding.

P2Y2 purinergic receptor activation is essential for efficient hepatocyte proliferation in response to partial hepatectomy.

Extracellular nucleotides via activation of P2 purinergic receptors influence hepatocyte proliferation and liver regeneration in response to 70% partial hepatectomy (PH). Adult hepatocytes express multiple P2Y (G protein-coupled) and P2X (ligand-gated ion channels) purinergic receptor subtypes. However, the identity of key receptor subtype(s) important for efficient hepatocyte proliferation in regenerating livers remains unknown. To evaluate the impact of P2Y2 purinergic receptor-mediated signaling on hepatocyte proliferation in regenerating livers, wild-type (WT) and P2Y2 purinergic receptor knockout (P2Y2-/-) mice were subjected to 70% PH. Liver tissues were analyzed for activation of early events critical for hepatocyte priming and subsequent cell cycle progression. Our findings suggest that early activation of p42/44 ERK MAPK (5 min), early growth response-1 (Egr-1) and activator protein-1 (AP-1) DNA-binding activity (30 min), and subsequent hepatocyte proliferation (24-72 h) in response to 70% PH were impaired in P2Y2-/- mice. Interestingly, early induction of cytokines (TNF-α, IL-6) and cytokine-mediated signaling (NF-κB, STAT-3) were intact in P2Y2-/- remnant livers, uncovering the importance of cytokine-independent and nucleotide-dependent early priming events critical for subsequent hepatocyte proliferation in regenerating livers. Hepatocytes isolated from the WT and P2Y2-/- mice were treated with ATP or ATPγS for 5-120 min and 12-24 h. Extracellular ATP alone, via activation of P2Y2 purinergic receptors, was sufficient to induce ERK phosphorylation, Egr-1 protein expression, and key cyclins and cell cycle progression of hepatocytes in vitro. Collectively, these findings highlight the functional significance of P2Y2 purinergic receptor activation for efficient hepatocyte priming and proliferation in response to PH.

Comparative analyses of lung transcriptomes in patients with alveolar capillary dysplasia with misalignment of pulmonary veins and in foxf1 heterozygous knockout mice.

Alveolar Capillary Dysplasia with Misalignment of Pulmonary Veins (ACDMPV) is a developmental disorder of the lungs, primarily affecting their vasculature. FOXF1 haploinsufficiency due to heterozygous genomic deletions and point mutations have been reported in most patients with ACDMPV. The majority of mice with heterozygous loss-of-function of Foxf1 exhibit neonatal lethality with evidence of pulmonary hemorrhage in some of them. By comparing transcriptomes of human ACDMPV lungs with control lungs using expression arrays, we found that several genes and pathways involved in lung development, angiogenesis, and in pulmonary hypertension development, were deregulated. Similar transcriptional changes were found in lungs of the postnatal day 0.5 Foxf1+/- mice when compared to their wildtype littermate controls; 14 genes, COL15A1, COL18A1, COL6A2, ESM1, FSCN1, GRINA, IGFBP3, IL1B, MALL, NOS3, RASL11B, MATN2, PRKCDBP, and SIRPA, were found common to both ACDMPV and Foxf1 heterozygous lungs. Our results advance knowledge toward understanding of the molecular mechanism of ACDMPV, lung development, and its vasculature pathology. These data may also be useful for understanding etiologies of other lung disorders, e.g. pulmonary hypertension, bronchopulmonary dysplasia, or cancer.

Delayed liver regeneration after partial hepatectomy in adipose differentiation related protein-null mice.

BACKGROUND & AIMS: Adult hepatocytes undergo cell cycle progression and proliferation in response to partial hepatectomy (PH). Transient lipid accumulation within hepatocytes preceding the peak proliferative phase is a characteristic feature of regenerating livers. However, the molecular mediators and mechanisms responsible for lipid accumulation in regenerating livers are not well understood. Adipose differentiation related protein (ADRP; Plin2) regulates hepatic triglyceride storage and Plin2-deficient (Plin2(-/-)) mice have significantly reduced triglyceride (TG) content in the liver. We sought to determine the functional significance of PLIN2 in liver regeneration in response to PH and toxic liver injury and examined whether absence of Plin2 expression modulates hepatocyte proliferation and liver regeneration. METHODS: We subjected wild-type (WT) and Plin2(-/-) mice to 70% PH or acute carbon tetrachloride (CCL4) treatment and examined the hepatic lipid content, the expression profile of lipid metabolism-related genes, the rate of cellular proliferation and the dynamics of liver regeneration in the treated animals. RESULTS: In response to PH, Plin2(-/-) mice showed decreased hepatic triglyceride accumulation and delayed cell cycle progression, which was associated with impaired liver regeneration. Fatty acid (FA) synthesis and lipid transfer gene expression profile were comparable between Plin2(-/-) and wild-type mice, while VLDL secretion rate was higher in the Plin2(-/-) mice. Downregulated ß-oxidation and reduced cytosolic FA level in Plin2(-/-) mice may have contributed to the attenuation of the liver regeneration capacity in these animals. In parallel experiments, we also observed attenuated hepatic lipid accumulation and proliferation in response to CCl4-mediated acute toxic liver injury in Plin2(-/-) mice. CONCLUSIONS: We conclude that PLIN2-mediated lipid accumulation and utilization by the liver is important for efficient liver regeneration in response to PH and toxic liver injury.

Evaluation of asymmetric immunoliposomal nanoparticles for cellular uptake.

Effective and targeted in vivo delivery of polynucleotide therapeutics is the key for the treatment of many diseases. Asymmetric immunoliposomes can be used as vehicles to deliver polynucleotides effectively because the two leaflets of the bilayer can have different compositions, which enhance the delivery capacity. The formation and in vitro cellular uptake of asymmetric immunoliposomes containing polynucleotide cargoes were studied here. Maleimide-functionalised DSPE-PEG (2000) were incorporated into the outer leaflet to produce asymmetric liposomes capable of covalently attaching antibodies. Thiolated antibodies from both human and rabbit origin were conjugated to produce asymmetric pendant-type immunoliposomes that retain their specificity towards detection antibodies through the formation process. Human IgG-conjugated asymmetric immunoliposomes were readily internalised (>20 per cell) by macrophage, HEPG2, and CV-1 monkey kidney cells. The cells internalised the liposomal nanoparticles by the endocytic pathway. The immunoliposome-encapsulated endosomes were intact for at least 5 days and sequestered the plasmid from expression by the cell.

Endothelial nitric oxide synthase is a key mediator of hepatocyte proliferation in response to partial hepatectomy in mice.

UNLABELLED: Endothelial nitric oxide synthase (eNOS) is a critical modulator of vascular tone and blood flow and plays major roles in liver physiology and pathophysiology. Nitric oxide (NO) is widely recognized as one of the key humoral factors important for the initiation of liver regeneration in response to partial hepatectomy. Liver regeneration in response to partial hepatectomy is dependent on the efficiency of growth factor-mediated cell-cycle progression. Epidermal growth factor receptor (EGFR) is a critical mediator of multiple hepatic mitogens, such as epidermal growth factor (EGF), transforming growth factor alpha, amphiregulin, and heparin-binding EGF in regenerating livers. However, the functional significance of endothelial nitric oxide synthase (eNOS) expressed in hepatocytes, and its potential role in EGFR-mediated hepatocyte proliferation, remains unexplored. We sought to determine whether eNOS is essential for hepatocyte proliferation in response to partial hepatectomy (PH). Our studies with eNOS knockout (eNOS(-/-) ) mice suggest that eNOS activation is essential for the efficient induction of early events and elicitation of a robust hepatocyte proliferative response to PH. Moreover, eNOS expression is essential for the efficient early induction of matrix metalloprotease-9, a known mediator of extracellular matrix remodeling and growth factor activation in regenerating livers. Our in vitro studies suggest that eNOS is a critical mediator of EGF-induced hepatocyte proliferation, potentially via its influence on the induction of early growth response-1 (Egr-1) and phosphorylation of c-Jun--known mediators of cell-cycle progression. EGF-induced eNOS phosphorylation at Ser 1177 is dependent on the phosphorylation and activation of EGFR/PI3 kinase/AKT signaling in hepatocytes. CONCLUSION: Collectively, these results highlight a hitherto unrecognized role for eNOS activation in hepatocyte proliferation with implications for targeted therapies to enhance liver regenerative response in chronic disorders.

Hypertrophic cardiomyopathy and dysregulation of cardiac energetics in a mouse model of biliary fibrosis.

UNLABELLED: Cardiac dysfunction is a major cause of morbidity and mortality in patients with end-stage liver disease; yet the mechanisms remain largely unknown. We hypothesized that the complex interrelated impairments in cardiac structure and function secondary to progression of liver diseases involve alterations in signaling pathways engaged in cardiac energy metabolism and hypertrophy, augmented by direct effects of high circulating levels of bile acids. Biliary fibrosis was induced in male C57BL/6J mice by feeding a 0.1% 3,5-diethoxycarbonyl-1,4-dihydroxychollidine (DDC) supplemented diet. After 3 weeks, mice underwent live imaging (dual energy x-ray absorptiometry [DEXA] scanning, two-dimensional echocardiography [2DE], electrocardiography, cardiac magnetic resonance imaging), exercise treadmill testing, and histological and biochemical analyses of livers and hearts. Compared with chow-fed mice, DDC-fed mice fatigued earlier on the treadmill, with reduced VO(2). Marked changes were identified electrophysiologically (bradycardia and prolonged QT interval) and functionally (hyperdynamic left ventricular [LV] contractility along with increased LV thickness). Hearts of DDC-fed mice showed hypertrophic signaling (activation of v-akt murine thymoma viral oncogene/protein kinase B [AKT], inhibition of glycogen synthase kinase-3beta [GSK3beta], a 20-fold up-regulation of beta myosin heavy chain RNA and elevated G(s)alpha/G(i)alpha ratio. Genes regulating cardiac fatty acid oxidation pathways were suppressed, along with a threefold increase in myocardial glycogen content. Treatment of mouse cardiomyocytes (which express the membrane bile acid receptor TGR5) with potent natural TGR5 agonists, taurochenodeoxycholic acid and lithocholic acid, activated AKT and inhibited GSK3beta, similar to the changes seen in DDC-fed mouse hearts. This provides support for a novel mechanism whereby circulating natural bile acids can induce signaling pathways in heart associated with hypertrophy. CONCLUSION: Three weeks of DDC feeding-induced biliary fibrosis leads to multiple functional, metabolic, electrophysiological, and hypertrophic adaptations in the mouse heart, recapitulating some of the features of human cirrhotic cardiomyopathy.

ATP release after partial hepatectomy regulates liver regeneration in the rat.

BACKGROUND & AIMS: Paracrine interactions are critical to liver physiology, particularly during regeneration, although physiological involvement of extracellular ATP, a crucial intercellular messenger, remains unclear. The physiological release of ATP into extracellular milieu and its impact on regeneration after partial hepatectomy were investigated in this study. METHODS: Hepatic ATP release after hepatectomy was examined in the rat and in human living donors for liver transplantation. Quinacrine was used for in vivo staining of ATP-enriched compartments in rat liver sections and isolated hepatocytes. Rats were treated with an antagonist for purinergic receptors (Phosphate-6-azo(benzene-2,4-disulfonic acid), PPADS), and liver regeneration after hepatectomy was analyzed. RESULTS: A robust and transient ATP release due to acute portal hyperpressure was observed immediately after hepatectomy in rats and humans. Clodronate liposomal pre-treatment partly inhibited ATP release in rats. Quinacrine-stained vesicles, co-labeled with a lysosomal marker in liver sections and isolated hepatocytes, were predominantly detected in periportal areas. These vesicles significantly disappeared after hepatectomy, in parallel with a decrease in liver ATP content. PPADS treatment inhibited hepatocyte cell cycle progression after hepatectomy, as revealed by a reduction in bromodeoxyuridine incorporation, phosphorylated histone 3 immunostaining, cyclin D1 and A expression and immediate early gene induction. CONCLUSION: Extracellular ATP is released immediately after hepatectomy from hepatocytes and Kupffer cells under mechanical stress and promotes liver regeneration in the rat. We suggest that in hepatocytes, ATP is released from a lysosomal compartment. Finally, observations made in living donors suggest that purinergic signalling could be critical for human liver regeneration.

Redundant roles for cJun-N-terminal kinase 1 and 2 in interleukin-1beta-mediated reduction and modification of murine hepatic nuclear retinoid X receptor alpha.

BACKGROUND/AIMS: Retinoid X receptor alpha (RXRalpha), the heterodimeric partner for multiple nuclear receptors (NRs), was shown to be an essential target for inflammation-induced cJun-N-terminal kinase (JNK) signaling in vitro. This study aimed to explore the role of hepatic JNK signaling and its effects on nuclear RXRalpha levels downstream of interleukin-1beta (IL-1beta) in vivo. METHODS: Effects of IL-1beta on hepatic NR-dependent gene expression, nuclear RXRalpha levels, and roles for individual JNK isoforms were studied in wild-type, Jnk1(-/-), and Jnk2(-/-) mice and in primary hepatocytes of each genotype. RESULTS: IL-1beta administration showed a time-dependent reduction in expression of the hepatic NR-dependent genes Ntcp, Cyp7a1, Cyp8b1, Abcg5, Mrp2, and Mrp3. IL-1beta treatment for 1h activated JNK and resulted in both post-translational modification and reduction of nuclear RXRalpha. In wild-type primary hepatocytes, IL-1beta modified and reduced nuclear RXRalpha levels time dependently, which was prevented by chemical inhibition of JNK as well as by inhibition of proteasomal degradation. Individual absence of either JNK1 or JNK2 did not significantly influence the reduction or modification of hepatic nuclear RXRalpha by IL-1beta both in vivo and in primary hepatocytes. CONCLUSIONS: Functional redundancy exists for JNK1 and JNK2 in IL-1beta-mediated alterations of hepatic nuclear RXRalpha levels, stressing the importance of this pathway in mediating the hepatic response to inflammation.

Mild hypothermia does not affect liver regeneration after partial hepatectomy in mice.

BACKGROUND: The use of mild hypothermia has been suggested to be therapeutically useful in treating acute liver failure. It is not known if hypothermia influences liver regeneration. AIM: To assess the effect of hypothermia on liver regeneration in mice. METHODS: After partial (70%) hepatectomy (PHx), C57BL6/J mice were randomly assigned to either a hypothermic group or a normothermic group. Controlled mild hypothermia was maintained for up to 3 h after surgery. In addition, assessment of liver mass restitution was examined by studying the induction of key cell cycle proteins (cyclin A, D1 and E) and hepatocyte proliferation [assessment of proliferating cell nuclear antigen (PCNA) protein expression] by Western blotting and DNA synthesis by measuring 5-bromo-2-deoxyuridine (BrdU) incorporation by immunohistochemical techniques 45 h after PHx. RESULTS: Partial hepatectomy induced a vigorous proliferative response in the remnant livers of both groups of mice (normothermic and hypothermic groups), as evidenced by the induction of key cyclins, PCNA and incorporation of BrdU after PHx. The liver/body weight ratio and both cyclin and PCNA protein expression as well as BrdU incorporation did not differ between the regenerating livers of hypothermic and normothermic groups. CONCLUSION: Mild hypothermia does not influence liver regeneration in mice.

Evaluation of asymmetric liposomal nanoparticles for encapsulation of polynucleotides.

Conventional lipid bilayer liposomes have similar inner and outer leaflet compositions; asymmetric liposomes have different lipid leaflet compositions. The goal of this work is to place cationic lipids in the inner leaflet to encapsulate negatively charged polynucleotides and to place neutral/anionic lipids on the outer leaflet to decrease nonspecific cellular uptake/toxicity. Inverse emulsion particles have been developed with a single lipid leaflet of cationic and neutral lipids surrounding an aqueous core containing a negatively charged 21-mer DNA oligo. The particles are accelerated through an oil-water interface, entrapping a second neutral lipid to form oligo encapsulated unilamellar liposome nanoparticles. Inverse emulsion particles can be consistently produced to encapsulate an aqueous environment containing negatively charged oligo. The efficiency of encapsulated liposome formation is low and depends on the hydrocarbon used as the oil phase. Dodecane, mineral oil, and squalene were tested, and squalene, a branched hydrocarbon, yielded the highest efficiency.

Rosiglitazone attenuates suppression of RXRalpha-dependent gene expression in inflamed liver.

BACKGROUND/AIMS: A recently determined target of lipopolysaccharide (LPS) and cytokine signaling in liver is the central Type II nuclear receptor (NR) heterodimer partner, retinoid X receptor alpha (RXRalpha). We sought to determine if Rosiglitazone (Rosi), a peroxisome proliferator activated receptor gamma (PPARgamma) agonist with anti-inflammatory properties, can attenuate LPS and cytokine-induced molecular suppression of RXRalpha-regulated genes. METHODS: In vivo, mice were gavage-fed Rosi for 3 days, prior to intraperitoneal injection of LPS, followed by harvest of liver and serum. In vitro, HepG2 cells were treated with IL-1beta, +/- short-term Rosi pretreatment. RNA was analyzed by quantitative RT-PCR, while nuclear and cytoplasmic proteins were analyzed by immunoblotting and gel shifts. RESULTS: Rosi attenuated LPS-mediated suppression of RNA levels of several Type II NR-regulated genes, including bile acid transporters and the major drug metabolizing enzyme, Cyp3a11, without affecting cytokine expression, suggesting a novel, direct anti-inflammatory effect in hepatocytes. Rosi suppressed the inflammation-induced nuclear export of RXRalpha, in both LPS-injected mice and IL-1beta-treated HepG2 cells, leading to maintenance of nuclear RXRalpha levels and heterodimer binding activity. CONCLUSIONS: Rosi directly attenuates the suppressive effects of inflammation-induced cell signaling on nuclear RXRalpha levels in liver.

Nuclear export of retinoid X receptor alpha in response to interleukin-1beta-mediated cell signaling: roles for JNK and SER260.

As the obligate heterodimer partner to class II nuclear receptors, the retinoid X receptor alpha (RXRalpha) plays a vital physiological role in the regulation of multiple hepatic functions, including bile formation, intermediary metabolism, and endobiotic/xenobiotic detoxification. Many RXRalpha-regulated genes are themselves suppressed in inflamed liver via unknown mechanisms, which constitute a substantial component of the negative hepatic acute phase response. In this study we show that RXRalpha, generally considered a stable nuclear resident protein, undergoes rapid nuclear export in response to signals initiated by the pro-inflammatory cytokine interleukin-1beta (IL-1beta), a central activator of the acute phase response. Within 30 min of exposure to IL-1beta, nuclear levels of RXRalpha are markedly suppressed in human liver-derived HepG2 cells, temporally coinciding with its appearance in the cytoplasm. The nuclear residence of RXRalpha is maintained by inhibiting c-jun N-terminal kinase (JNK, curcumin or SP600125) or CRM-1-mediated nuclear export (Leptomycin B). Pretreatment with the proteasome inhibitor MG132 blocks IL-1beta-mediated reductions in nuclear RXRalpha levels while increasing accumulation in the cytoplasm. Mutational studies identify one residue, serine 260, a JNK phosphoacceptor site whose phosphorylation status had an unknown role in RXRalpha function, as critical for IL-1beta-mediated nuclear export of transfected human RXRalpha-green fluorescent fusion constructs. These findings indicate that inflammation-mediated cell signaling leads to rapid and profound reductions in nuclear RXRalpha levels, via a multistep, JNK-dependent mechanism involving Ser260, nuclear export, and proteasomal degradation. Thus, inflammation-meditated cell signaling targets RXRalpha for nuclear export and degradation; a potential mechanism that explains the broad suppression of RXRalpha-dependent gene expression in the inflamed liver.

Endotoxin leads to rapid subcellular re-localization of hepatic RXRalpha: A novel mechanism for reduced hepatic gene expression in inflammation.

BACKGROUND: Lipopolysaccharide (LPS) treatment of animals down-regulates the expression of hepatic genes involved in a broad variety of physiological processes, collectively known as the negative hepatic acute phase response (APR). Retinoid X receptor alpha (RXRalpha), the most highly expressed RXR isoform in liver, plays a central role in regulating bile acid, cholesterol, fatty acid, steroid and xenobiotic metabolism and homeostasis. Many of the genes regulated by RXRalpha are repressed during the negative hepatic APR, although the underlying mechanism is not known. We hypothesized that inflammation-induced alteration of the subcellular location of RXRalpha was a common mechanism underlying the negative hepatic APR. RESULTS: Nuclear RXRalpha protein levels were significantly reduced (~50%) within 1-2 hours after low-dose LPS treatment and remained so for at least 16 hours. RXRalpha was never detected in cytosolic extracts from saline-treated mice, yet was rapidly and profoundly detectable in the cytosol from 1 hour, to at least 4 hours, after LPS administration. These effects were specific, since the subcellular localization of the RXRalpha partner, the retinoic acid receptor (RARalpha), was unaffected by LPS. A potential cell-signaling modulator of RXRalpha activity, c-Jun-N-terminal kinase (JNK) was maximally activated at 1-2 hours, coincident with maximal levels of cytoplasmic RXRalpha. RNA levels of RXRalpha were unchanged, while expression of 6 sentinel hepatic genes regulated by RXRalpha were all markedly repressed after LPS treatment. This is likely due to reduced nuclear binding activities of regulatory RXRalpha-containing heterodimer pairs. CONCLUSION: The subcellular localization of native RXRalpha rapidly changes in response to LPS administration, correlating with induction of cell signaling pathways. This provides a novel and broad-ranging molecular mechanism for the suppression of RXRalpha-regulated genes in inflammation.