Role for Mammalian Neutral Sphingomyelinase 2 in Confluence-induced Growth Arrest of MCF7 Cells

Marchesini, Norma; Osta, Walid; Bielawski, Jacek; Luberto, Chiara; Obeid, Lina M.; Hannun, Yusuf A.

doi:10.1074/jbc.m313662200

Cited by 144 publications

(146 citation statements)

References 67 publications

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“…1A). As reported previously [8], nSMase2 was localized at the plasma membrane when overexpressed in the confluent culture of MCF-7 cells (Fig. 1B).…”

Section: Plasmid Constructionsupporting

confidence: 87%

Analysis of membrane topology of neutral sphingomyelinase 2

Tani

Hannun

2007

FEBS Letters

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Neutral sphingomyelinase 2 (nSMase2), which has two hydrophobic segments at its NH 2 -terminus, plays an important role in ceramide-mediated cell regulation. Here, we investigated the membrane topology of nSMase2. When a double-tagged nSMase2 at both the NH 2 and COOH termini, was overexpressed in MCF-7 cells, the signals from both tags were detected in the inner leaflet of the plasma membrane. Furthermore, insertion of a tag into the internal sequence and green fluorescent protein-fused deletion mutants revealed that the entire catalytic region of the protein was located on the cytosolic face of the membranes and each hydrophobic segment is integrated into the membranes, but unlikely to span the entire membrane. These results indicate the presence of the enzyme in the inner leaflet of plasma membrane.

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“…1A). As reported previously [8], nSMase2 was localized at the plasma membrane when overexpressed in the confluent culture of MCF-7 cells (Fig. 1B).…”

Section: Plasmid Constructionsupporting

confidence: 87%

Analysis of membrane topology of neutral sphingomyelinase 2

Tani

Hannun

2007

FEBS Letters

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“…1 A very recent report demonstrated that endogenous nSMase2 levels are induced upon confluence and are involved in induction of cell cycle arrest in MCF7 cells. 40,41 Furthermore, nSMase overexpression and treatment with cell-permeable ceramide analogues can induce growth arrest in a number of cells types including SK-Hep1, 6 U937 7 and HT-29. 8 Over 1000 research papers have described the induction of programmed cell death (apoptosis) by interventions that elevate the cell content of ceramide.…”

Section: Discussionmentioning

confidence: 99%

Ceramide triggers an NF-κB-dependent survival pathway through calpain

et al. 2005

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We have shown that C2 ceramide, a cell-permeable analog of this lipid second messenger, triggers an NF-jB dependent survival pathway that counteracts cell death. Activation of NF-jB and subsequent induction of prosurvival genes relies on calpain activity and is prevented on silencing of the calpain small subunit (Capn4) that is required for the function of ubiquitous calpains. We have demonstrated that p105 (NFjB1) and its proteolytic product p50 can be targets of microand milli-calpain in vitro and that a p50 deletion mutant, lacking both the N-and the C-terminal ends, is resistant to calpain-mediated degradation. Capn4 silencing results in stabilization of endogenous p105 and p50 in diverse human cell lines. Furthermore, p105 processing and activation of NFjB survival genes in response to C2 ceramide is impaired in Capn4À/À mouse embryonic fibroblasts defective in calpain activity. Altogether, these data argue for the existence of a ceramide-calpain-NF-jB axis with prosurvival functions.

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“…Scyphostatin was provided by Sankyo (Tokyo, Japan). NSMase2 (smpd3)-specific short interfering ribonucleic acid (siRNA) (AAtgttactggctggtggacc 32 ) and scrambled control (scr) siRNA (Cat. # 1022076) were from Qiagen (Valencia, CA).…”

Section: Methodsmentioning

confidence: 99%

“…Hepatocytes were isolated from ether-anesthe-tized young (3-4 months) and aged (20-22 months) male Fisher 344 rats by in situ collagenase perfusion and cultured in culture dishes coated with 6.3 mg/ml Matrigel in Waymouth's medium supplemented with insulin (0.15 M) and penicillin/streptomycin (100 U/ml) 21 for 5 days at 37°C in 5% CO 2 . Murine nSMase2 (smpd3)-specific siRNA 32 was used to silence the endogenous NSMase-2. Transfections (100 pmol siRNA per dish) using Lipofectamine were performed on day 3 of cell culture.…”

Section: Methodsmentioning

confidence: 99%

Aging in rat causes hepatic hyperresposiveness to interleukin-1β which is mediated by neutral sphingomyelinase-2

et al. 2007

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The process of aging has recently been shown to substantially affect the ability of cells to respond to inflammatory challenges. We demonstrate that aging leads to hepatic hyperresponsiveness to interleukin 1␤ (IL-1␤), and we examine the factors that could be responsible for this phenomenon. IL-1␤-induced phosphorylation of c-jun N-terminal kinase (JNK) in hepatocytes isolated from aged rats was 3 times more potent than that in hepatocytes from young rats. Moreover, JNK was activated by substantially lower doses of IL-1␤. These age-related changes in JNK phosphorylation correlated with diminished IL-1␤-induced degradation of interleukin-1 receptor-associated kinase-1 (IRAK-1). Expression levels of IL1␤ receptor I, total JNK, IRAK-1, and transforming growth factor-␤-activated kinase-1 (TAK-1) were not affected by aging. However, increased neutral sphingomyelinase activity was observed in hepatocytes from old animals, which we show is caused by induction of the plasma membrane localized neutral sphingomyelinase-2 (NSMase-2). We provide evidence that NSMase-2 is both required and sufficient for the onset of IL-1␤ hyperresponsiveness during aging. Overexpression of NSMase-2 in hepatocytes from young rats leads both to a reduction in IRAK-1 degradation and potentiation of JNK phosphorylation, mimicking that seen in hepatocytes from old animals. More importantly, inhibition of NSMase activity in hepatocytes from aged rats using either scyphostatin or short interfering ribonucleic acid (siRNA) leads to reversion to the "young" phenotype of IL-1␤ response. Conclusion: These results show that the process of aging causes increased basal NSMase-2 activity in hepatocytes, which in turn leads to IRAK-1 stabilization, JNK potentiation, and ultimately IL-1␤ hyperresponsiveness. (HEPATOLOGY 2007;46:1166-1176

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Role for Mammalian Neutral Sphingomyelinase 2 in Confluence-induced Growth Arrest of MCF7 Cells

Abstract: Recently, we reported that neutral sphingomyelinase 2 (nSMase2) functions as a bona fide neutral sphingomyelinase and that overexpression of nSMase2 in MCF7 breast cancer cells caused a decrease in cell growth

Cited by 144 publications

References 67 publications

Analysis of membrane topology of neutral sphingomyelinase 2

Analysis of membrane topology of neutral sphingomyelinase 2

Ceramide triggers an NF-κB-dependent survival pathway through calpain

Aging in rat causes hepatic hyperresposiveness to interleukin-1β which is mediated by neutral sphingomyelinase-2

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