Down-regulation of Sphingosine Kinase-1 by DNA Damage: DEPENDENCE ON PROTEASES AND p53

Tarek A Taha,Walid Osta,Lina Kozhaya,Jacek Bielawski,Korey R Johnson,William E Gillanders,Ghassan S Dbaibo,Yusuf A Hannun,Lina M Obeid

doi:10.1074/jbc.m401259200

Abstract

Sphingosine kinase 1 (SK1), a key enzyme in sphingosine 1-phosphate (S1P) synthesis, regulates various aspects of cell behavior, including cell survival and proliferation. DNA damaging anti-neoplastic agents have been shown to induce p53, ceramide levels, and apoptosis; however, the effects of anti-neoplastic agents on SK have not been assessed. In this study, we investigated the effects of a DNA damaging agent, actinomycin D (Act D), on the function of sphingosine kinase (SK1). Act D caused a reduction in the protein levels of SK1, as indicated by Western blot analysis, with a concomitant decrease in SK activity. The down-regulation was post-transcriptional, because the mRNA levels of SK1 remained unchanged. Similar decreases in SK1 protein were observed with other DNA damaging agents such as doxorubicin, etoposide, and gamma-irradiation. ZVAD, the pancaspase inhibitor, and Bcl-2 annulled the effect of Act D on SK1, demonstrating a role for cysteine proteases downstream of Bcl-2 in the down-regulation of SK1. Inhibition of caspases 3, 6, 7, and 9 only partially reversed Act D-induced SK1 loss. Inhibition of cathepsin B, a lysosomal protease, produced a significant reversal of SK1 decline by Act D, suggesting that a multitude of ZVAD-sensitive cysteine proteases downstream of Bcl-2 mediated the SK1 decrease. When p53 up-regulation after Act D treatment was inhibited, SK1 down-regulation was rescued, demonstrating p53 dependence of SK1 modulation. Treatment of cells with S1P, the product of SK1, partially inhibited Act D-induced cell death, raising the possibility that a decrease in SK1 may be in part necessary for cell death to occur. Furthermore, the knockdown of SK1 by small interfering RNA in MCF-7 cells resulted in a significant reduction in cell viability. These studies demonstrate that SK1 is down-regulated by genotoxic stress, and that basal SK1 function may be necessary for the maintenance of tumor cell growth.

Highlights

Sphingosine kinase 1 (SK1), a key enzyme in sphingo- cell regulation
These cells exhibited a gross morphological change upon treatment with SK1 siRNA (Fig. 5B), accompanied by a 40% reduction in cell viability (Fig. 5C). These data strongly suggest that SK1 is required for cell survival and that attenuation of SK1 may be important for the execution of cell death. Both ceramide and sine 1-phosphate (S1P) have emerged as key and reciprocal regulators of anti-neoplastic action, studies examining the effects of DNA damaging chemotherapeutic agents on the key sphingolipid metabolizing enzyme sphingosine kinase are lacking
This work shows, for the first time, a regulated decline of SK1 protein and SK activity, events that are likely to be mediated in a p53-dependent manner by proteases downstream of Bcl-2, and that are accompanied by increases in sphingosine and ceramide

Summary

Introduction

Sphingosine kinase 1 (SK1), a key enzyme in sphingo- cell regulation. Key lipids in the sphingolipid pathway are sine 1-phosphate (S1P) synthesis, regulates various as- ceramide, sphingosine, and sphingosine 1-phosphate (S1P). pects of cell behavior, including cell survival and proliferation. The knockdown of SK1 by small interfering RNA in MCF-7 cells resulted in a significant reduction in cell viability These studies demonstrate that SK1 is down-regulated by genotoxic stress, and that basal SK1 function may be necessary for the maintenance of tumor cell growth. When cells are committed to die by the intrinsic pathway, mitochondrial function is disrupted, resulting in the release of cytochrome c from the intermembrane space and leading to the activation of downstream effector caspases 3, 6, and 7. The latter execute the program of cell death by cleaving key proteins that maintain cellular integrity and function. The common absence of p53 in tumors allows them to circumvent the apoptotic responses that normal cells launch when they are exposed to DNA damaging stimuli

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