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The PI3 kinase (PI3K) family plays a complex role in cell biology and metabolism. Signaling through the PI3Ks is frequently activated in many human cancers, including glioblastoma, because of gain-of-function mutations in PIK3CA or loss of PTEN. Experiments involving genetic mouse models and small molecule inhibitors have helped to elucidate the roles of the regulatory and catalytic subunits of PI3K in metabolism and cancer. Downstream of PI3K is Akt, a critical effector of growth, proliferation and survival. The suggested dependence of glioblastoma tumors on PI3K signaling implies that PI3K inhibitors should lead to effective killing of these cancer cells, but that has been shown not to be the case. The engagement of other survival pathways in response to PI3K inhibition prompts the need to develop combination therapies that promote cytotoxicity in cancer cells. [ABSTRACT FROM AUTHOR]

In multicellular epithelial tissues, the orientation of polarity of each cell must be coordinated. Previously, we reported that for Madin–Darby canine kidney cells in three-dimensional collagen gel culture, blockade of β1-integrin by the AIIB2 antibody or expression of dominant-negative Rac1N17 led to an inversion of polarity, such that the apical surfaces of the cells were misorientated towards the extracellular matrix. Here, we show that this process results from the activation of RhoA. Knockdown of RhoA by short hairpin RNA reverses the inverted orientation of polarity, resulting in normal cysts. Inhibition of RhoA downstream effectors, Rho kinase (ROCK I) and myosin II, has similar effects. We conclude that the RhoA–ROCK I–myosin II pathway controls the inversion of orientation of epithelial polarity caused by AIIB2 or Rac1N17. These results might be relevant to the hyperactivation of RhoA and disruption of normal polarity frequently observed in human epithelial cancers. [ABSTRACT FROM AUTHOR]

Midkine and pleiotrophin comprise a family of heparin-binding growth factors, and are expressed in overlapping tissues during the mid- to late-gestation periods of mouse development. Their distinct expression during early mouse development, as revealed by in situ hybridization, was reported. Midkine was expressed in the embryonic ectoderm from as early as embryonic day (E5.5). In the neural tube midkine was expressed specifically in the neuroepithelium, that is, in the whole area of the neural tube at E9.5, and in the ventricular zone from E10.5–13.5. At E15.5, when the neuroepithelium disappeared, midkine concomitantly became undetectable. In contrast, pleiotrophin expression started exclusively in the neural plate at E8.5, and in the lateral plate of the neural tube at E9.5. It then became restricted to a dorsal ventricular zone from E11.5–13.5, and finally to the central gray neurons at E15.5. Moreover, pleiotrophin was expressed in the ventral horns. Among placental tissues, midkine was detected in the chorion, the fetal component of the placenta, whereas pleiotrophin was found in the decidua basalis, the maternal component of the placenta. The distinct expression of midkine and pleiotrophin suggests their differential role in early development. [ABSTRACT FROM AUTHOR]

Many studies have shown that apolipoprotein E (apoE) plays important roles in maintaining intracellular lipid homeostasis in nonneuronal cells. However, little is known about the extracellular transport of lipids in the CNS. In this study, we determined whether and to what degree lipid efflux from astrocytes and neurons depended on apoE. Our results showed that exogenously added apoE promoted the efflux of cholesterol and phosphatidylcholine from both astrocytes and neurons in culture, resulting in the generation of high-density lipoprotein-like particles. The order of potency of the apoE isoforms as lipid acceptors was apoE2 > apoE3 = apoE4 in astrocytes and apoE2 > apoE3 > apoE4 in neurons. Treatment with brefeldin A, monensin, and a protein kinase C inhibitor, H7, abolished the ability of apoE to promote cholesterol efflux from cultured astrocytes, without altering apoE-mediated phosphatidylcholine efflux. In contrast, the efflux of both cholesterol and phosphatidylcholine promoted by apoE was abolished following treatment with heparinase or lactoferrin, which block the interaction of apoE with heparan sulfate proteoglycans (HSPGs) or low-density lipoprotein receptor-related protein (LRP), respectively. This study suggests that apoE promotes lipid efflux from astrocytes and neurons in an isoform-specific manner and that cell surface HSPGs and/or HSPG-LRP pathway may mediate this apoE-promoted lipid efflux. [ABSTRACT FROM AUTHOR]

Background: Midkine (MK) is a growth factor implicated in the development and repair of various tissues, especially neural tissues. However, its in vivo function has not been clarified. Results: Knockout mice lacking the MK gene (Mdk) showed no gross abnormalities. We closely analysed postnatal brain development in Mdk(–/–) mice using calcium binding proteins as markers to distinguish neuronal subpopulations. Intense and prolonged calretinin expression was found in the dentate gyrus granule cell layer of the hippocampus of infant Mdk(–/–) mice. In infant Mdk(+/+) mice, calretinin expression in the granule cell layer was weaker, and had disappeared by 4 weeks after birth, when calretinin expression still persisted in Mdk(–/–) mice. Furthermore, 4 weeks after birth, Mdk(–/–) mice showed a deficit in their working memory, as revealed by a Y-maze test, and had an increased anxiety, as demonstrated by the elevated plus-maze test. Conclusion: Midkine plays an important role in the regulation of postnatal development of the hippocampus. [ABSTRACT FROM AUTHOR]

The phagocyte NADPH oxidase is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The formation of the active oxidase complex at the membrane requires translocation of the Rac GTPase and two specialized cytosolic proteins that harbor SH3 domains, p67phox and p47phox. Another SH3-domain-containing protein p40 phox, which is constitutively associated with p67phox in phagocytes, also enters the complex upon cell stimulation. Here we describe how we cloned mouse cDNAs encoding p40 phox and its partner in phagocytes, p67phox. Both p40 phox and p67phox comprise several protein-binding modules that are structurally and functionally well conserved between mouse and human, indicating their nature as adaptor proteins. We have also systematically investigated expression of the gene for p40 phox in comparison with those for p67phox and p47phox. Distributions of the mRNAs for the three proteins among tissues are similar, with the most abundant expression in the spleen. The messages are abundant not only in phagocytic cells, but also in B cell lineage. The p40 phox gene, but not the other two, is expressed in some types of cells such as plasma cells and T lymphocytes. Furthermore, in situ hybridization analysis shows that the p40 phox mRNA is distributed in neuronal cells of mouse brain, providing evidence that one of the genes for the specialized oxidase factors is expressed in neurons. These observations raise the possibility that the adaptor protein p40 phox plays a heretofore unsuspected role via interacting with other proteins in the cells that do not express p67phox or p47phox. [ABSTRACT FROM AUTHOR]

AbstractMicrotubule-associated protein 2 (MAP2) is a neuron-specific cytoskeletal protein enriched in dendrites and cell bodies. MAP2 regulates microtubule stability in a phosphorylation-dependent manner, which has been implicated in dendrite outgrowth and branching. We have previously reported that cholesterol deficiency causes tau phosphorylation and microtubule depolymerization in axons (Fan et al . 2001). To investigate whether cholesterol also modulates microtubule stability in dendrites by modulating MAP2 phosphorylation, we examined the effect of compactin, a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, and TU-2078 (TU), a squalene epoxidase inhibitor, on these parameters using cultured neurons. We have found that cholesterol deficiency induced by compactin and TU, inhibited dendrite outgrowth, but not of axons, and attenuated axonal branching. Dephosphorylation of MAP2 and microtubule depolymerization accompanied these alterations. The amount of protein phosphatase 2 A (PP2A) and its activity in association with microtubules were decreased, while those unbound to microtubules were increased. The synthesized ceramide levels and the total ceramide content were increased in these cholesterol-deficient neurons. These alterations caused by compactin were prevented by concurrent treatment of cultured neurons with β-migrating very-low-density lipoproteins (β-VLDL) or cholesterol. Taken together, we propose that cholesterol-deficiency causes a selective inhibition of dendrite outgrowth due to the decreased stability of microtubules as a result of inhibition of MAP2 phosphorylation. [ABSTRACT FROM AUTHOR]