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Loss of sphingosine kinase-1 activates the intrinsic pathway of programmed cell death: modulation of sphingolipid levels and the induction of apoptosis

Tarek A. Taha^¶, Kazuyuki Kitatani, Mazen El-Alwani^¶, Jacek Bielawski, Yusuf A. Hannun and Lina M. Obeid^¶,,1

^¶ Division of General Internal Medicine, Ralph H. Johnson Veterans Administration Hospital, Charleston, South Carolina, USA; and ¶Department of Medicine and
Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, South Carolina, USA

¹Correspondence: Department of Medicine, Medical University of South Carolina, 114 Doughty St., P.O. Box 250779, Charleston, South Carolina 29425, USA. E-mail: obeidl{at}musc.edu

SPECIFIC AIMS

Studies investigating the regulation of the sphingosine kinase (SK)/sphingosine-1-phosphate (S1P) pathway have provided insight into the contribution of activated rather than basal SK activity to downstream biological effects. Recently, it was shown that sphingosine kinase-1 (SK1) is down-regulated by DNA damage and TNF, suggesting that endogenous SK1 is modulated under stress. Studies on down-regulation of endogenous SK1, however, have been limited by the lack of specific SK1 inhibitors. The aim of this study was to determine the role that basal endogenous SK1 contributes to cell regulation.

PRINCIPAL FINDINGS

1. Knockdown of SK1 induces changes in the cell cycle profile of MCF-7 cells
Our earlier studies showed that SK1 knockdown reduces the percentage of viable MCF-7 breast cancer cells. To explore the mechanisms possibly accounting for this, we determined the cell cycle profile of cells after the reduction of SK1 expression. Loss of SK1 produced a shift of 16% of the cell population from the S to the G1 phase. Moreover, at 72 h a sub-G1 peak also became evident in the SK1 siRNA-treated cells, signifying that 4% of the cells have undergone DNA fragmentation compared with < 0.5% in the scrambled (SCR) siRNA-treated cells. Together, these results suggest that SK1 function may be necessary to maintain basal MCF-7 progression through the cell cycle.

2. Loss of SK1 causes caspase activation, cytochrome c release, and Bax oligomerization
To confirm further whether SK1 loss induces the apoptotic pathway of cell death, activation of caspase proteases, another hallmark event of apoptosis, was tested. Figure 1 A shows that SK1 knockdown induced the activation of caspase 7, as indicated by the appearance of the cleaved active form of this protease. An In vitro assay using a caspase 3/7 DEVD substrate also showed a time dependent increase in cleavage activity in the lysates of cells treated with SK1 siRNA but not in cells treated with SCR siRNA (Fig. 1B ). Furthermore, PARP, a well-known substrate for caspase 7, was cleaved in the SK1 knockdown cells (Fig. 1A ). These data suggest that the executioner caspase 7 is activated after knockdown of SK1.

View larger version (29K): [in this window] [in a new window]   Figure 1. Effects of SK1 knockdown on parameters of apoptosis. A) SK1 was knocked down in MCF-7 cells, and Western blot was performed on the cell lysates for SK1, PARP, and caspases 7. B) In vitro DEVDase cleavage assay on lysates from MCF-7 cells after SK1 knockdown for 24 h, 48 h, and 72 h. C) MCF-7 cells were transfected with SCR or SK1 siRNA for 3 days, then cell lysates were fractionated into heavy membrane and cytosolic components. Fractions were then probed for cytochrome c. D) Confocal imaging of GFP-Bax in MCF-7 cells after transfection with SCR or SK1 siRNA showing an increase in Bax aggregation after SK1 knockdown. SCR, scrambled siRNA; SK1, SK1 siRNA.

Because the mitochondrion is a key integrator of apoptotic signals, the effects of SK1 loss on mitochondrial function were assessed. When MCF-7 cells were transfected with siRNA for 3 days, and their subcellular components were isolated by differential centrifugation, a striking increase of cytochrome c protein levels was observed in the cytosolic fraction of SK1 but not of SCR siRNA-treated cells (Fig. 1C ). This result strongly suggests that SK1 loss induces mitochondrial membrane permeabilization (MMP) and the release of proapoptotic cytochrome c from the intermembrane space into the cytosol.

Several studies have shown that MMP can be mediated by the proapoptotic members of the Bcl-2 family. In particular, Bax translocation from the cytosol to the mitochondrion and its oligomerization in the mitochondrial membrane is a frequent event in mitochondrial pathways of cell death. To determine the role of Bax in mediating MMP induction by SK1 loss, we used an MCF-7 cell line stably overexpressing GFP-tagged Bax. As shown in the left panel of Fig. 1D , cells transfected with SCR siRNA displayed a diffuse cytosolic distribution of Bax, along with a mitochondrial localization pattern. After treatment with SK1 siRNA for 3 days, a striking redistribution of GFP-Bax was observed in up to 10% of the cells, where Bax showed an aggregated mitochondrial signal characteristic of its oligomerization pattern observed under apoptotic conditions (Fig. 1D , right panel). These results suggest that MMP caused by SK1 loss may be driven by Bax oligomerization at the level of the mitochondria.

3. Knockdown of SK1 elevates ceramide levels
We next examined the effect of SK1 loss on the mass levels of ceramide, a well-established proapoptotic lipid. After 48 h knockdown of SK1, a significant 1.5-fold increase in total ceramide levels was observed. Total ceramide levels were also increased in the heavy membrane fraction of cells after SK1 loss. The dihydroC16-ceramide and the C16-ceramide species were consistently elevated. The changes in ceramide mass were accompanied by increased flux of palmitate into ceramide in cells were SK1 was knocked down. These results therefore indicate that basal SK1 may potentially regulate the levels of the growth-inhibiting proapoptotic lipid ceramide and that knocking down SK1 may shift the balance toward growth inhibition and cell death.

4. Myriocin reverses SK1 loss-induced Bax translocation and caspase activation
The enhanced ceramide accumulation after SK1 knockdown prompted us to examine the effect of myriocin, the serine palmitoyltransferase blocker, on the apoptotic pathway activated after loss of SK1. Figure 2 A shows that SK1 siRNA causes a significant increase in Bax translocation, an event inhibited by myriocin in a dose dependent manner. Reversal of Bax aggregation was accompanied by a concomitant inhibition of downstream caspase activation (Fig. 2B ). These results strongly suggest that Bax translocation and downstream caspase activation are dependent on sphingolipid alterations after knockdown of SK1, and that inhibition of these changes can rescue from apoptosis.

View larger version (17K): [in this window] [in a new window]   Figure 2. Effect of myriocin on Bax translocation and caspase activation. A) GFP-Bax expressing MCF-7 cells were transfected with SCR or SK1 siRNA in the presence or absence of 100 nM myriocin. Cells showing punctate Bax were then counted under a confocal microscope, and reported as a percentage of total GFP-Bax expressing cells. B) DEVD cleavage assay on SCR or SK1 siRNA-transfected MCF-7 cells in the presence or absence of 100 nM myriocin or 20 µM ZVAD. SCR, scrambled siRNA; SK1, SK1 siRNA.

CONCLUSIONS AND SIGNIFICANCE

In this study we examined the role of basal SK1 function in MCF-7 breast cancer cells. Our results show that SK1 is an important mediator of the progression through the cell cycle, and that loss of the enzyme induces cell cycle arrest and activates the intrinsic mitochondrial pathway of apoptosis. Knockdown of SK1 enhances the oligomerization of the proapoptotic protein Bax in the mitochondrial membrane, resulting in the release of cytochrome c and downstream caspase activation (Fig. 3 ). Associated with the biological effects of SK1 loss are significant increases in ceramide. The sphingolipid changes that occur after SK1 knockdown appear to be required for the downstream apoptotic response, since inhibition of the de novo pathway of ceramide generation significantly rescues from Bax translocation and caspase activation induced by down-regulation of SK1 (Fig. 3) .

View larger version (20K): [in this window] [in a new window]   Figure 3. Schematic diagram of the apoptotic pathway activated after endogenous SK1 knockdown. Cyt c, cytochrome c.

In most studies thus far, whether via overexpression of SK1 or agonist-mediated stimulation of the enzyme, the focus has been on the role of activated SK1 in biological effects, rather than of the endogenous enzyme. Recent studies have shown that during programmed cell death, SK1 may be down-regulated, suggesting that cells respond to stimuli not only by activating SK but also by inhibiting it. One advantage of siRNA is its ability to significantly and specifically reduce basal SK1 activity, therefore allowing dissection of responses that are maintained by basal enzyme activity. This study shows for the first time that knockdown of endogenous SK1 activity induces G1 arrest and also activates the mitochondrial pathway of apoptosis. Therefore, it appears that basal SK1 provides for important cellular functions.

The ability of SK1 to act as an antiapoptotic factor has been mostly attributed to its metabolic role. Ceramide and sphingosine mediate apoptosis, cell cycle arrest, and differentiation, whereas S1P promotes proliferation, survival, and inhibition of apoptosis. This study shows that knockdown of SK1 causes a significant increase in ceramide levels, suggesting that basal SK1 is an important regulator of cellular levels of this toxic lipid. The ceramide species consistently elevated by SK1 knockdown include the long chain dihydroC16-ceramide and C16-ceramide species. C16-ceramide, in particular, has been recently shown to mediate apoptotic responses, possibly by acting upstream or at the level of the mitochondrion. Indeed, our data show that in the heavy membrane fraction (which is mitochondria-enriched), C16 ceramide was elevated. In vitro studies have shown that ceramide can induce Bax translocation and cytochrome c release in isolated mitochondria. In vivo, the accumulation of ceramide in mitochondria has been associated with Bax oligomerization and the induction of cell death. Therefore, a proximal connection between ceramide and Bax may exist and ceramide may be a regulator of Bax-mediated responses in cell death pathways. In this respect, the results from this study suggest that SK1 knockdown induces its effect through metabolic perturbation of sphingolipids, and most probably of C16-ceramide. The ability of myriocin, the de novo ceramide synthesis blocker, to significantly attenuate Bax oligomerization and caspase activation further supports a sphingolipid-Bax connection.

In conclusion, this study has examined the role of endogenous SK1 in the regulation of cell function. The enzyme appears to be indispensable for the normal progression through the cell cycle and its loss activates the intrinsic pathway of programmed cell death. Furthermore, SK1 is an important regulator of ceramide levels in the cell, and its down-regulation results in enhanced ceramide synthesis and its accumulation in the mitochondrion, which may be key in initiating the mitochondrial events leading to apoptosis.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4412fje;

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