A Phosphoinositide 3-Kinase/Phospholipase Cgamma1 Pathway Regulates Fibroblast Growth Factor-Induced Capillary Tube Formation

Background: The fibroblast growth factors (FGFs) are key regulators of embryonic development, tissue homeostasis and tumour angiogenesis. Binding of FGFs to their receptor(s) results in activation of several intracellular signalling cascades including phosphoinositide 3-kinase (PI3K) and phospholipase C (PLC)gamma 1. Here we investigated the basic FGF (FGF-2)-mediated activation of these enzymes in human umbilical vein endothelial cells (HUVECs) and defined their role in FGF-2-dependent cellular functions.Methodology/Principal Findings: We show that FGF-2 activates PLC gamma 1 in HUVECs measured by analysis of total inositol phosphates production upon metabolic labelling of cells and intracellular calcium increase. We further demonstrate that FGF-2 activates PI3K, assessed by analysing accumulation of its lipid product phosphatidylinositol-3,4,5-P-3 using TLC and confocal microscopy analysis. PI3K activity is required for FGF-2-induced PLC gamma 1 activation and the PI3K/PLC gamma 1 pathway is involved in FGF-2-dependent cell migration, determined using Transwell assay, and in FGF-2-induced capillary tube formation (tubulogenesis assays in vitro). Finally we show that PI3K-dependent PLC gamma 1 activation regulates FGF-2-mediated phosphorylation of Akt at its residue Ser473, determined by Western blotting analysis. This occurs through protein kinase C (PKC)alpha activation since dowregulation of PKC alpha expression using specific siRNA or blockade of its activity using chemical inhibition affects the FGF-2-dependent Ser473 Akt phosphorylation. Furthermore inhibition of PKC alpha blocks FGF-2-dependent cell migration.Conclusion/Significance: These data elucidate the role of PLC gamma 1 in FGF-2 signalling in HUVECs demonstrating its key role in FGF-2-dependent tubulogenesis. Furthermore these data unveil a novel role for PLC gamma 1 as a mediator of PI3K-dependent Akt activation and as a novel key regulator of different Akt-dependent processes

Changes in LXR signaling influence early-pregnancy lipogenesis and protect against dysregulated fetoplacental lipid homeostasis

Human pregnancy is associated with enhanced de novo lipogenesis in the early stages followed by hyperlipidemia during advanced gestation. Liver X receptors (LXRs) are oxysterol-activated nuclear receptors that stimulate de novo lipogenesis and also promote the efflux of cholesterol from extrahepatic tissues followed by its transport back to the liver for biliary excretion. Although LXR is recognized as a master regulator of triglyceride and cholesterol homeostasis, it is unknown whether it facilitates the gestational adaptations in lipid metabolism. To address this question, biochemical profiling, protein quantification, and gene expression studies were used, and gestational metabolic changes in T0901317-treated wild-type mice and Lxrab-/- mutants were investigated. Here, we show that altered LXR signaling contributes to the enhanced lipogenesis in early pregnancy by increasing the expression of hepatic Fas and stearoyl-CoA desaturase 1 (Scd1). Both the pharmacological activation of LXR with T0901317 and the genetic ablation of its two isoforms disrupted the increase in hepatic fatty acid biosynthesis and the development of hypertriglyceridemia during early gestation. We also demonstrate that absence of LXR enhances maternal white adipose tissue lipolysis, causing abnormal accumulation of triglycerides, cholesterol, and free fatty acids in the fetal liver. Together, these data identify LXR as an important factor in early-pregnancy lipogenesis that is also necessary to protect against abnormalities in fetoplacental lipid homeostasis

LKB1 is required for hepatic bile acid transport and canalicular membrane integrity in mice

LKB1 is a ‘master’ protein kinase implicated in the regulation of metabolism, cell proliferation, cell polarity and tumorigenesis. However, the long-term role of LKB1 in hepatic function is unknown. In the present study, it is shown that hepatic LKB1 plays a key role in liver cellular architecture and metabolism. We report that liver-specific deletion of LKB1 in mice leads to defective canaliculi and bile duct formation, causing impaired bile acid clearance and subsequent accumulation of bile acids in serum and liver. Concomitant with this, it was found that the majority of BSEP (bile salt export pump) was retained in intracellular pools rather than localized to the canalicular membrane in hepatocytes from LLKB1KO (liver-specific Lkb1-knockout) mice. Together, these changes resulted in toxic accumulation of bile salts, reduced liver function and failure to thrive. Additionally, circulating LDL (low-density lipoprotein)-cholesterol and non-esterified cholesterol levels were increased in LLKB1KO mice with an associated alteration in red blood cell morphology and development of hyperbilirubinaemia. These results indicate that LKB1 plays a critical role in bile acid homoeostasis and that lack of LKB1 in the liver results in cholestasis. These findings indicate a novel key role for LKB1 in the development of hepatic morphology and membrane targeting of canalicular proteins

Investigation of the pathological mechanism of cataract formation in Myotonic Dystrophy 1

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Progesterone Metabolites as Farnesoid X Receptor Inhibitors

Sulfated progesterone metabolites rise 100-fold in the third trimester of human pregnancy and have been shown to be elevated further in the gestational disorder intrahepatic cholestasis of pregnancy (ICP). Typical concentrations of progesterone sulfates range from 1 to 10 µmol/L in an uncomplicated pregnancy and rise to approximately 40 µmol/L in ICP. At this level they can influence bile acid and lipid metabolism. Studies using human and rodent specimens have shown that sulfated metabolites of progesterone competitively inhibit bile acid homeostasis pathways by functioning as partial agonists of farnesoid X receptor (FXR). This explains the loss of induction of FXR target genes in ICP, and may explain susceptibility to hypercholanaemia and dyslipidaemia in the second half of human pregnancy. Furthermore, progesterone sulfates are competitive inhibitors of biliary influx (NTCP) and efflux (BSEP) transport proteins, actions likely to further exacerbate hypercholanaemia and cholestasis.</jats:p

Nuclear receptor-driven alterations in bile acid and lipid metabolic pathways during gestation

AbstractNuclear receptor signalling is essential for physiological processes such as metabolism, development, and reproduction. Alterations in the endocrine state that naturally occur during pregnancy result in maternal adaptations to support the feto-placental unit. A series of studies have shown that nuclear receptor signalling is involved in maternal adaptations of bile acid, cholesterol, and lipid homeostasis pathways to ensure maintenance of the nutritional demands of the fetus. We discuss regulation of hepatic nuclear receptors and their target genes in pregnancy and their impact on the development of disorders such as intrahepatic cholestasis of pregnancy and oestrogen-induced hepatotoxicity. This article is part of a Special Issue entitled: Translating nuclear receptors from health to disease

Flow-dependent increase of ICAM-1 on saphenous vein endothelium is sensitive to apamin

The potassium channel blocker tetraethylammonium blocks the flow-induced increase in endothelial ICAM-1. We have investigated the subtype of potassium channel that modulates flow-induced increased expression of ICAM-1 on saphenous vein endothelium. Cultured human saphenous vein endothelial cells (HSVECs) or intact saphenous veins were perfused at fixed low nd high flows in a laminar shear chamber or flow rig, respectively, in the presence or absence of potassium channel blockers. Expression of K+ channels and endothelial ICAM-1 was measured by real-time polymerase chain reaction and/or immunoassays. In HSVECs, the application of 0.8 N/m2 (8 dyn/cm2) shear stress resulted in a two- to fourfold increase in cellular ICAM-1 within 6 h (P &lt; 0.001). In intact vein a similar shear stress, with pulsatile arterial pressure, resulted in a twofold increase in endothelial ICAM-1/CD31 staining area within 1.5 h (P &lt; 0.001). Both increases in ICAM-1 were blocked by inclusion of 100 nM apamin in the vein perfusate, whereas other K+ channel blockers were less effective. Two subtypes of small conductance Ca 2+-activated K+ channel (selectively blocked by apamin) were expressed in HSVECs and vein endothelium (SK3&gt;SK2). Apamin blocked the upregulation of ICAM-1 on saphenous vein endothelium in response to increased flow to implicate small conductance Ca2+-activated K+ channels in shear stress/flow-mediated signaling pathways