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Phospholipase D1 is an effector of Rheb in the mTOR pathway.

ABSTRACT: The mammalian target of rapamycin (mTOR) assembles a signaling network essential for the regulation of cell growth, which has emerged as a major target of anticancer therapies. The tuberous sclerosis complex 1 and 2 (TSC1/2) proteins and their target, the small GTPase Rheb, constitute a key regulatory pathway upstream of mTOR. Phospholipase D (PLD) and its product phosphatidic acid are also upstream regulators of the mitogenic mTOR signaling. However, how the TSC/Rheb and PLD pathways interact or integrate in the rapamycin-sensitive signaling network has not been examined before. Here, we find that PLD1, but not PLD2, is required for Rheb activation of the mTOR pathway, as demonstrated by the effects of RNAi. The overexpression of Rheb activates PLD1 in cells in the absence of mitogenic stimulation, and the knockdown of Rheb impairs serum stimulation of PLD activation. Furthermore, the overexpression of TSC2 suppresses PLD1 activation, whereas the knockdown or deletion of TSC2 leads to elevated basal activity of PLD. Consistent with a TSC-Rheb-PLD signaling cascade, AMPK and PI3K, both established regulators of TSC2, appear to lie upstream of PLD as revealed by the effects of pharmacological inhibitors, and serum activation of PLD is also dependent on amino acid sufficiency. Finally, Rheb binds and activates PLD1 in vitro in a GTP-dependent manner, strongly suggesting that PLD1 is a bona fide effector for Rheb. Hence, our findings reveal an unexpected interaction between two cascades in the mTOR signaling pathways and open up additional possibilities for targeting this important growth-regulating network for the development of anticancer drugs.

SUBMITTER: Sun Y

PROVIDER: S-EPMC2448829 | biostudies-other | 2008 Jun

REPOSITORIES: biostudies-other

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Phospholipase D1 is an effector of Rheb in the mTOR pathway.

Sun Y Y Fang Y Y Yoon M-S MS Zhang C C Roccio M M Zwartkruis F J FJ Armstrong M M Brown H A HA Chen J J

Proceedings of the National Academy of Sciences of the United States of America 20080611 24

The mammalian target of rapamycin (mTOR) assembles a signaling network essential for the regulation of cell growth, which has emerged as a major target of anticancer therapies. The tuberous sclerosis complex 1 and 2 (TSC1/2) proteins and their target, the small GTPase Rheb, constitute a key regulatory pathway upstream of mTOR. Phospholipase D (PLD) and its product phosphatidic acid are also upstream regulators of the mitogenic mTOR signaling. However, how the TSC/Rheb and PLD pathways interact o ...[more]

PMID: 18550814

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Project description:Objective：Phospholipase D1 (PLD1), a phosphatidylcholine-hydrolyzing enzyme, has been found to be involved in cellular lipid metabolism. However, how PLD1 involves in hepatocyte lipid metabolism and thus affects non-alcoholic fatty liver diseases (NAFLD) has not been explicitly explored. Method: NAFLD is induced in hepatocyte-specific Pld1 knockout (Pld1(H)-KO) mice or control (Pld1-Flox) mice with a high fat diet (HFD) for 20 weeks. Plasma glucose and lipid levels, liver lipid and function parameters and inflammation- and fibrogenesis-related gene expression levels were studied. Changes in lipid composition of liver were detected by lipidomic analyses. Changes in gene expression profile were detected by mRNA sequencing. In vitro, alpha mouse liver 12 (AML12) cells were incubated with sodium palmitate (SP) to explore the mechanisms of PLD1 on development of NAFLD. The protein levels of PLD1 and CD36 were detected in the liver tissues of NAFLD patients. Finally, the therapeutic effect of NAFLD by inhibiting PLD with 5-Fluoro-2-indolyl deschlorohalopemide (FIPI) was examined. Results: PLD1 expression levels were increased in the liver tissues of NAFLD patients and the hepatocytes of HFD-induced mice. Compared with Pld1-Flox mice, Pld1(H)-KO mice exhibited lower plasma glucose and lipids levels, and decreased lipid accumulation, inflammation and fibrosis related gene expression levels in the liver tissues after HFD feeding. Inhibition of hepatocyte PLD1 significantly altered lipid composition, especially phosphatidic acids (PA) and lyso-PAs (LPA) levels in the liver tissues after NAFLD. Transcriptomic analysis showed that hepatocyte specific deficiency of PLD1 mainly decreased CD36 expression levels in the NAFLD liver tissues, which was confirmed at the protein and gene expression levels. In vitro, inhibition of PLD1 markedly decreased CD36 expression and lipid accumulation in SP treated AML12 cells. Furthermore, PA, the downstream product catalyzed by PLD1, increased the expression levels of CD36 and lipid accumulation in vitro, which was reversed by PPARγ antagonist. These suggested that PPARγ played an important role in transcriptional regulation of CD36 expression after PA stimulation. Finally, PLD inhibitor FIPI had significant therapeutic effect on HFD-induced NAFLD. Conclusion: Hepatocyte-specific deficiency of PLD1 ameliorates lipid accumulation and NAFLD development by inhibiting PPARγ/CD36 pathway conducted by PA.

Dataset Information

Phospholipase D1 is an effector of Rheb in the mTOR pathway.

Publications

Phospholipase D1 is an effector of Rheb in the mTOR pathway.

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