Ceramide Induces the Dephosphorylation and Inhibition of Constitutively Activated Akt in PTEN Negative U87MG Cells

Zinda, Michael; Vlahos, Chris J.; Lai, Mei‐Huei T.

doi:10.1006/bbrc.2000.4248

Cited by 74 publications

(59 citation statements)

References 33 publications

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“…Against the first hypothesis, we have shown that C2-ceramide inhibitory action on Akt kinase activity is not bypassed by the expression of a constitutively active form of Akt (transiently transfected in primary cells). Findings from another recent study, in which the ability of C2-ceramide to inhibit the constitutive phosphorylation and activity of Akt in phosphatase and tensin homolog delete from chromosome 10 -negative U87MG cells was demonstrated (50), support these data. Indeed, C2-ceramideϪinduced dephosphorylation of Akt was prevented by pretreatment with the phosphatase inhibitor okadaic acid, thereby indicating that ceramide inhibits insulin signaling by maintaining Akt in an inactive dephosphorylated state through a mechanism involving an okadaic acidϪsensitive phosphatase, such as PP2A or PP1, in brown adipocytes.…”

Section: Discussionsupporting

confidence: 54%

Ceramide Mediates Insulin Resistance by Tumor Necrosis Factor-α in Brown Adipocytes by Maintaining Akt in an Inactive Dephosphorylated State

2001

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Tumor necrosis factor (TNF)-␣ causes insulin resistance on glucose uptake in fetal brown adipocytes. We explored the hypothesis that some effects of TNF-␣ could be mediated by the generation of ceramide, given that TNF-␣ treatment induced the production of ceramide in these primary cells. A short-chain ceramide analog, C2-ceramide, completely precluded insulin-stimulated glucose uptake and insulin-induced GLUT4 translocation to plasma membrane, as determined by Western blot or immunofluorescent localization of GLUT4. These effects were not produced in the presence of a biologically inactive ceramide analog, C2-dihydroceramide. Analysis of the phosphatidylinositol (PI) 3-kinase signaling pathway indicated that C2-ceramide precluded insulin stimulation of Akt kinase activity, but not of PI-3 kinase or protein kinase C-activity. C2-ceramide completely abolished insulin-stimulated Akt/protein kinase B phosphorylation on regulatory residues Thr 308 and Ser 473, as did TNF-␣, and inhibited insulin-induced mobility shift in Akt1 and Akt2 separated in PAGE. Moreover, C2-ceramide seemed to activate a protein phosphatase (PP) involved in dephosphorylating Akt because 1) PP2A activity was increased in C2-ceramide؊ and TNF-␣؊ treated cells, 2) treatment with okadaic acid concomitantly with C2-ceramide completely restored Akt phosphorylation by insulin, and 3) transient transfection of a constitutively active form of Akt did not restore Akt activity. Our results indicate that ceramide produced by TNF-␣ induces insulin resistance in brown adipocytes by maintaining Akt in an inactive dephosphorylated state.

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Section: Discussionsupporting

confidence: 54%

Ceramide Mediates Insulin Resistance by Tumor Necrosis Factor-α in Brown Adipocytes by Maintaining Akt in an Inactive Dephosphorylated State

2001

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“…Furthermore, now included in the therapeutic strategy design against such tumors is the inhibition of the PI3K/Akt pathway (12,22,24) and the up-regulation of Cer (26 -30). Indeed, a reciprocal control between PI3K/Akt and Cer signaling in glioma cell survival/death is suggested by data indicating that Cer may induce apoptosis also by inactivation of the prosurvival kinase Akt (38,54), possibly through the activation of a Cer-dependent protein phosphatase, and by data demonstrating a protective role of PI3K/Akt on Cer-induced cell death in glial cells (31,34,55). This led us to wonder whether the PI3K-Akt-PTEN pathway is able to regulate Cer metabolism; indeed, if such regulation does occur, some insight could be gained into crucial aspects that could, after further investigations, strongly influence our knowledge of the sensitivity of glioma cells to Cer-based cytotoxic treatments.…”

Section: Discussionmentioning

confidence: 99%

Phosphatidylinositol 3-Kinase/AKT Pathway Regulates the Endoplasmic Reticulum to Golgi Traffic of Ceramide in Glioma Cells

Giussani¹,

Brioschi²,

Bassi

et al. 2009

Journal of Biological Chemistry

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World Health Organization classifies gliomas according to their cellular features and their grade of malignancy (from I to IV) (4). Glioblastoma multiforme (GBM), 3 also referred to as grade IV astrocytoma, is the most frequent class of malignant primary brain tumor and one of the most aggressive forms of cancer. There is a poor prognosis for patients with GBM, the median survival being 9 -12 months, and this does not significantly improve even after aggressive treatment with multiple approaches, including surgery, chemotherapy, and radiotherapy (5). This poor survival rate seems to be mainly due to the marked invasiveness and proliferation of GBM, as well as to their apoptotic resistance and aberrant angiogenesis. These malignancy features could be related to the varying mutations frequently found in GBM tumors that impact different key pathways involved in the control of cell proliferation, survival, differentiation, migration, and DNA repair (6 -9).Among the disregulated signaling pathways in GBMs, PI3K-Akt-PTEN has been identified as being an important oncogenic pathway (10 -12). PI3Ks are members of a unique and conserved family of lipid kinases that phosphorylate the 3Ј-hydroxyl group of phosphatidylinositol and phosphoinositides such as phosphatidylinositol(4,5)P 2 (PIP 2 ), which is converted to phosphatidylinositol(3,4,5)P 3 (PIP 3 ). The activation of PI3Ks can occur through growth factor receptors, G-protein-coupled receptors, cell adhesion molecules, and oncogenes such as Ras. The generated polyphosphoinositides bind to the pleckstrin homology (PH) domain of different proteins, including PDK1 * This work was supported by grants from the University of Milan (to P. G., L. R., and P. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. N-[hexanoyl-6-3 H]D-erythro-hexanoyl-sphingosine; HPTLC, high performance thin layer chromatography; NBD-C 6 -Cer, 6-((N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)hexanoyl) sphingosine; FAPP, four phosphate adaptor protein; EGF-FAPP1-PH, PH domain of FAPP1 protein fused with enhanced green fluorescent protein; DN-AKT, dominant negative mutant of Akt; PBS, phosphate-buffered

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“…Evidence suggests that these lipids modify a number of target proteins, including protein kinases and phosphatases, to induce a cascade of enzymatic and transcriptional activities; however, the exact mechanisms by which they trigger apoptotic signals are unclear. Exogenous ceramide induces ecient antiproliferative signals (Yoshimura et al, 1997;Zinda et al, 2001), and our initial experiments showed that ceramide at 50 mM was required for eective elimination of U87MG cells. Therefore, we examined the survival of U87MG/hDkk cells (clone 2, which expressed the least amount of Dkk-1 mRNA) in the presence of 50 mM ceramide over a 5-day period.…”

Section: Stable Transfection Of Hdkk-1 In U87mg Cells Promotes Ceramimentioning

confidence: 99%

Human Dkk-1, a gene encoding a Wnt antagonist, responds to DNA damage and its overexpression sensitizes brain tumor cells to apoptosis following alkylation damage of DNA

et al. 2002

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The human Dkk-1 (hDkk-1) gene, a transcriptional target of the p53 tumor suppressor, encodes a powerful inhibitor of the Wnt signaling pathway and regulates the spatial patterning/morphogenesis of the mammalian central nervous system. We investigated the p53-related functions of the hDkk-1 gene by studying its response to DNA damage and its modulation of apoptosis in human glioma cells. Various chemotherapeutic and other agents that induce DNA adducts and compromise its integrity (1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU), cisplatin, H 2 O 2 and UV rays) enhanced the expression of hDkk-1 signi®cantly. The damage-induced increase in hDkk-1 mRNA levels occurred in many human tumor cell lines, irrespective of their p53 gene status. The human glioblastoma cell line, U87MG, which had undetectable hDkk-1 expression, was engineered to express moderate levels of the hDkk protein by stable transfection. The engineered cells did not show any morphological changes, but underwent marked apoptosis after ceramide treatment. Further, the DNA cross-linking drugs BCNU and cisplatin, but not the microtubule poison vincristine, induced signi®cant cell death in U87MG/hDkk cells, and this was accompanied by altered Bcl-2/Bax expression and a reduction in the amount of telomere DNA as visualized by¯uorescence in situ hybridization. These results show that hDkk-1 is a pro-apoptotic gene and suggest that it may play important roles in linking the oncogenic Wnt and p53 tumor suppressor pathways.

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Ceramide Induces the Dephosphorylation and Inhibition of Constitutively Activated Akt in PTEN Negative U87MG Cells

Cited by 74 publications

References 33 publications

Ceramide Mediates Insulin Resistance by Tumor Necrosis Factor-α in Brown Adipocytes by Maintaining Akt in an Inactive Dephosphorylated State

Ceramide Mediates Insulin Resistance by Tumor Necrosis Factor-α in Brown Adipocytes by Maintaining Akt in an Inactive Dephosphorylated State

Phosphatidylinositol 3-Kinase/AKT Pathway Regulates the Endoplasmic Reticulum to Golgi Traffic of Ceramide in Glioma Cells

Human Dkk-1, a gene encoding a Wnt antagonist, responds to DNA damage and its overexpression sensitizes brain tumor cells to apoptosis following alkylation damage of DNA

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