Yasuo Horie

Hepatocyte-specific Pten deficiency results in steatohepatitis and hepatocellular carcinomas

PTEN is a tumor suppressor gene mutated in many human cancers, and its expression is reduced or absent in almost half of hepatoma patients. We used the Cre-loxP system to generate a hepatocyte-specific null mutation of Pten in mice (AlbCrePten(flox/flox) mice). AlbCrePten(flox/flox) mice showed massive hepatomegaly and steatohepatitis with triglyceride accumulation, a phenotype similar to human nonalcoholic steatohepatitis. Adipocyte-specific genes were induced in mutant hepatocytes, implying adipogenic-like transformation of these cells. Genes involved in lipogenesis and beta-oxidation were also induced, possibly as a result of elevated levels of the transactivating factors PPARgamma and SREBP1c. Importantly, the loss of Pten function in the liver led to tumorigenesis, with 47% of AlbCrePten(flox/flox) livers developing liver cell adenomas by 44 weeks of age. By 74-78 weeks of age, 100% of AlbCrePten(flox/flox) livers showed adenomas and 66% had hepatocellular carcinomas. AlbCrePten(flox/flox) mice also showed insulin hypersensitivity. In vitro, AlbCrePten(flox/flox) hepatocytes were hyperproliferative and showed increased hyperoxidation with abnormal activation of protein kinase B and MAPK. Pten is thus an important regulator of lipogenesis, glucose metabolism, hepatocyte homeostasis, and tumorigenesis in the liver.

Control of cell polarity and motility by the PtdIns(3,4,5)P3 phosphatase SHIP1

Proper neutrophil migration into inflammatory sites ensures host defense without tissue damage. Phosphoinositide 3-kinase (PI(3)K) and its lipid product phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) regulate cell migration, but the role of PtdIns(3,4,5)P3-degrading enzymes in this process is poorly understood. Here, we show that Src homology 2 (SH2) domain-containing inositol-5-phosphatase 1 (SHIP1), a PtdIns(3,4,5)P3 phosphatase, is a key regulator of neutrophil migration. Genetic inactivation of SHIP1 led to severe defects in neutrophil polarization and motility. In contrast, loss of the PtdIns(3,4,5)P3 phosphatase PTEN had no impact on neutrophil chemotaxis. To study PtdIns(3,4,5)P3 metabolism in living primary cells, we generated a novel transgenic mouse (AktPH–GFP Tg) expressing a bioprobe for PtdIns(3,4,5)P3. Time-lapse footage showed rapid, localized binding of AktPH–GFP to the leading edge membrane of chemotaxing ship1+/+AktPH–GFP Tg neutrophils, but only diffuse localization in ship1−/−AktPH–GFP Tg neutrophils. By directing where PtdIns(3,4,5)P3 accumulates, SHIP1 governs the formation of the leading edge and polarization required for chemotaxis.

Regulation of the MDM2-P53 pathway and tumor growth by PICT1 via nucleolar RPL11

PICT1 (also known as GLTSCR2) is considered a tumor suppressor because it stabilizes phosphatase and tensin homolog (PTEN), but individuals with oligodendrogliomas lacking chromosome 19q13, where PICT1 is located, have better prognoses than other oligodendroglioma patients. To clarify the function of PICT1, we generated Pict1-deficient mice and embryonic stem (ES) cells. Pict1 is a nucleolar protein essential for embryogenesis and ES cell survival. Even without DNA damage, Pict1 loss led to p53-dependent arrest of cell cycle phase G1 and apoptosis. Pict1-deficient cells accumulated p53, owing to impaired Mdm2 function. Pict1 binds Rpl11, and Rpl11 is released from nucleoli in the absence of Pict1. In Pict1-deficient cells, increased binding of Rpl11 to Mdm2 blocks Mdm2-mediated ubiquitination of p53. In human cancer, individuals whose tumors express less PICT1 have better prognoses. When PICT1 is depleted in tumor cells with intact P53 signaling, the cells grow more slowly and accumulate P53. Thus, PICT1 is a potent regulator of the MDM2-P53 pathway and promotes tumor progression by retaining RPL11 in the nucleolus.

Colorectal carcinomas in mice lacking the catalytic subunit of PI(3)K|[gamma]|

Phosphoinositide-3-OH kinases (PI(3)Ks) constitute a family of evolutionarily conserved lipid kinases that regulate a vast array of fundamental cellular responses, including proliferation, transformation, differentiation and protection from apoptosis. PI(3)K-mediated activation of the cell survival kinase PKB/Akt, and negative regulation of PI(3)K signalling by the tumour suppressor PTEN (refs 3, 4) are key regulatory events in tumorigenesis. Thus, a model has arisen that PI(3)Ks promote development of cancers. Here we report that genetic inactivation of the p110γ catalytic subunit of PI(3)Kγ (ref. 8) leads to development of invasive colorectal adenocarcinomas in mice. In humans, p110γ protein expression is lost in primary colorectal adenocarcinomas from patients and in colon cancer cell lines. Overexpression of wild-type or kinase-dead p110γ in human colon cancer cells with mutations of the tumour suppressors APC and p53 , or the oncogenes β-catenin and Ki-ras, suppressed tumorigenesis. Thus, loss of p110γ in mice leads to spontaneous, malignant epithelial tumours in the colorectum and p110γ can block the growth of human colon cancer cells.

Hyperplasia and Carcinomas in Pten-Deficient Mice and Reduced PTEN Protein in Human Bladder Cancer Patients

PTEN is a tumor suppressor gene mutated in many human cancers. We used the Cre-loxP system to generate an urothelium-specific null mutation of Pten in mice [FabpCrePten(flox/flox) (FPten(flox/flox)) mice]. Histologic examination revealed that all FPten(flox/flox) mice exhibited urothelial hyperplasia in which component cells showed enlarged nuclei and increased cell size. With time, 10% of FPten(flox/flox) mice spontaneously developed pedicellate papillary transitional cell carcinomas (TCC). This type of tumor also arose in FPten(flox/flox) mice treated with the chemical carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine. FPten(flox/flox) urothelial cells were hyperproliferative and showed increased activation of the survival signaling molecules Akt and extracellular signal-regulated kinase. In humans, 53% of primary bladder cancer patients exhibited decreased or absent expression of PTEN protein in either the cytoplasm or nucleus of tumor cells. In early bladder cancers, PTEN expression was repressed in 42% of superficial papillary TCC but in only 8% of cases of carcinoma in situ (CIS). In advanced bladder cancers, PTEN protein was significantly reduced (particularly in the nucleus) in 94% of cases, and this decrease in PTEN correlated with disease stage and grade. Thus, PTEN deficiency may contribute to bladder cancer both by initiating superficial papillary TCC and by promoting the progression of CIS to advanced invasive and metastatic forms.

Eicosapentaenoic acid ameliorates steatohepatitis and hepatocellular carcinoma in hepatocyte-specific Pten-deficient mice☆

BACKGROUND/AIMS Eicosapentaenoic acid (EPA) has been known as a reagent for improving lipid metabolism and inflammation. Hepatocyte-specific Pten-deficient mice exhibit hepatic lesions analogous to non-alcoholic steatohepatitis (NASH). Therefore, we administered EPA to Pten-deficient mice to investigate the mechanisms of NASH. METHODS Pten-deficient mice were assigned to a control group fed with a standard chow or an EPA group fed with a 5% EPA-supplemented standard chow. At 40 weeks, livers from each group were processed to measure triglyceride content, gene expression analysis, Western blotting analysis, and histological examination. Level of serum reactive oxygen species (ROS) was also determined. Forty- and 76-week-old mice were used in tumor burden experiments. RESULTS EPA-ameliorated hepatic steatosis in Pten-deficient mice was based on decreased expression of AMPKalpha1-mediated SREBP-1c and increased PPARalpha expression. The EPA group exhibited less severe chronic hepatic inflammation compared to the control group, resulting from decreased ROS formation and a dramatically low ratio of arachidonic acid to EPA. Moreover, EPA inhibited development of hepatocellular carcinoma (HCC) in Pten-deficient mice based on an inhibition of MAPK activity and a low ratio of oleic to stealic acid, and a reduction in ROS formation. CONCLUSIONS EPA ameliorated steatohepatitis and development of HCC in Pten-deficient mice.

The PtdIns(3,4)P2 phosphatase INPP4A is a suppressor of excitotoxic neuronal death

Phosphorylated derivatives of phosphatidylinositol, collectively referred to as phosphoinositides, occur in the cytoplasmic leaflet of cellular membranes and regulate activities such as vesicle transport, cytoskeletal reorganization and signal transduction. Recent studies have indicated an important role for phosphoinositide metabolism in the aetiology of diseases such as cancer, diabetes, myopathy and inflammation. Although the biological functions of the phosphatases that regulate phosphatidylinositol-3,4,5-trisphosphate (PtdIns(3,4,5)P3) have been well characterized, little is known about the functions of the phosphatases regulating the closely related molecule phosphatidylinositol-3,4-bisphosphate (PtdIns(3,4)P2). Here we show that inositol polyphosphate phosphatase 4A (INPP4A), a PtdIns(3,4)P2 phosphatase, is a suppressor of glutamate excitotoxicity in the central nervous system. Targeted disruption of the Inpp4a gene in mice leads to neurodegeneration in the striatum, the input nucleus of the basal ganglia that has a central role in motor and cognitive behaviours. Notably, Inpp4a-/- mice show severe involuntary movement disorders. In vitro, Inpp4a gene silencing via short hairpin RNA renders cultured primary striatal neurons vulnerable to cell death mediated by N-methyl-d-aspartate-type glutamate receptors (NMDARs). Mechanistically, INPP4A is found at the postsynaptic density and regulates synaptic NMDAR localization and NMDAR-mediated excitatory postsynaptic current. Thus, INPP4A protects neurons from excitotoxic cell death and thereby maintains the functional integrity of the brain. Our study demonstrates that PtdIns(3,4)P2, PtdIns(3,4,5)P3 and the phosphatases acting on them can have distinct regulatory roles, and provides insight into the unique aspects and physiological significance of PtdIns(3,4)P2 metabolism. INPP4A represents, to our knowledge, the first signalling protein with a function in neurons to suppress excitotoxic cell death. The discovery of a direct link between PtdIns(3,4)P2 metabolism and the regulation of neurodegeneration and involuntary movements may aid the development of new approaches for the treatment of neurodegenerative disorders.

No Mycobacterium paratuberculosis detected in intestinal tissue, including Peyer's patches and lymph follicles, of Crohn's disease.

Abstract: To clarify the etiologic significance of Mycobacterium paratuberculosis in Crohns disease, we investigated whether M. paratuberculosis was detected in intestinal tissues, including Peyers patches, where M. paratuberculosis invades, and colonic lymph follicles, where early lesions appear. Fifty-one samples of intestinal tissues, either therapeutically resected or biopsied, including 34 specimens from 30 patients with Crohns disease, were studied. Four Peyers patches and eight lymph follicles were included in the intestinal tissue samples of Crohns disease. They were visualized by acetic acid fixation. DNA extracted from intestinal tissues by proteinase K treatment was used for nested polymerase chain reaction (PCR) for detection of IS900, which is specific for M. paratuberculosis. PCR products were analyzed by agarose gel electrophoresis and subsequent Southern blot analysis. Our amplification system could detect 7.5 fg of M. paratuberculosis DNA. None of the tissue samples showed positive IS900 amplification, whereas they all showed amplification of the positive control human leukocyte antigen (HLA)-DQA DNA. Spiked experiments of tissue samples with M. paratuberculosis demonstrated that inhibitors of IS900 amplification were not present in the samples. Our study does not support the etiologic significance of M. paratuberculosis in Crohns disease.

PPARγ contributes to PKM2 and HK2 expression in fatty liver

Rapidly proliferating cells promote glycolysis in aerobic conditions, to increase growth rate. Expression of specific glycolytic enzymes, namely pyruvate kinase M2 and hexokinase 2, concurs to this metabolic adaptation, as their kinetics and intracellular localization favour biosynthetic processes required for cell proliferation. Intracellular factors regulating their selective expression remain largely unknown. Here we show that the peroxisome proliferator-activated receptor gamma transcription factor and nuclear hormone receptor contributes to selective pyruvate kinase M2 and hexokinase 2 gene expression in PTEN-null fatty liver. Peroxisome proliferator-activated receptor gamma expression, liver steatosis, shift to aerobic glycolysis and tumorigenesis are under the control of the Akt2 kinase in PTEN-null mouse livers. Peroxisome proliferator-activated receptor gamma binds to hexokinase 2 and pyruvate kinase M promoters to activate transcription. In vivo rescue of peroxisome proliferator-activated receptor gamma activity causes liver steatosis, hypertrophy and hyperplasia. Our data suggest that therapies with the insulin-sensitizing agents and peroxisome proliferator-activated receptor gamma agonists, thiazolidinediones, may have opposite outcomes depending on the nutritional or genetic origins of liver steatosis.

Loss of Pten, a tumor suppressor, causes the strong inhibition of autophagy without affecting LC3 lipidation

1Pten (phosphatase and tensin homolog deleted on chromosome ten), a tumor suppressor, is a phosphatase with a variety of substrate specificities. Its function as a negative regulator of the class I phosphatidyl-inositol 3-kinase/Akt pathway antagonizes insulin-dependent cell signaling. The targeted deletion of Pten in mouse liver leads to insulin hypersensitivity and the upregulation of the phosphatidyl-inositol 3-kinase/Akt signaling pathway. In this study, we investigated the effects of Pten deficiency on autophagy, a major cellular degradative system responsible for the turnover of cell constituents. The autophagic degradation of [14C]-leucine-labeled proteins of hepatocytes isolated from Pten-deficient livers was strongly inhibited, compared with that of control hepatocytes. However, no significant difference was found in the levels of the Atg12-Atg5 conjugate and LC3-II, the lipidated form of LC3, an intrinsic autophagosomal membrane marker, between control and Pten-deficient livers. Electron microsopic analyses showed that numerous autophagic vacuoles (autophagosomes plus autolysosomes) were present in the livers of control mice that had been starved for 48 hours, whereas they were markedly reduced in Pten-deficient livers under the same conditions. In vivo administration of leupeptin to control livers caused the inhibition of autophagic proteolysis, resulting in the accumulation of autolysosomes. These autolysosomes could be separated as a denser autolysosomal fraction from other cell membranes by Percoll density gradient centrifugation. In leupeptin-administered mutant livers, however, the accumulation of denser autolysosomes was reduced substantially. Collectively, we conclude that enhanced insulin signaling in Pten deficiency suppresses autophagy at the formation and maturation steps of autophagosomes, without inhibiting ATG conjugation reactions.