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P53-dependent upregulation of neutral sphingomyelinase-2: role in doxorubicin-induced growth arrest.

Shamseddine AA, Clarke CJ, Carroll B, Airola MV, Mohammed S, Rella A, Obeid LM, Hannun YA - Cell Death Dis (2015)

Bottom Line: DNA-binding mutants as well as acetylation mutants of p53 were unable to induce nSMase2, suggesting a role of nSMase2 in growth arrest.Moreover, knockdown of nSMase2 prevented doxorubicin-induced growth arrest.Finally, p53-induced nSMase2 upregulation appears to occur via a novel transcription start site upstream of exon 3.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Stony Brook University, Health Science Center, Stony Brook, NY 11794-8430, USA.

ABSTRACT
Neutral sphingomyelinase-2 (nSMase2) is a ceramide-generating enzyme that has been implicated in growth arrest, apoptosis and exosome secretion. Although previous studies have reported transcriptional upregulation of nSMase2 in response to daunorubicin, through Sp1 and Sp3 transcription factors, the role of the DNA damage pathway in regulating nSMase2 remains unclear. In this study, we show that doxorubicin induces a dose-dependent induction of nSMase2 mRNA and protein with concomitant increases in nSMase activity and ceramide levels. Upregulation of nSMase2 was dependent on ATR, Chk1 and p53, thus placing it downstream of the DNA damage pathway. Moreover, overexpression of p53 was sufficient to transcriptionally induce nSMase2, without the need for DNA damage. DNA-binding mutants as well as acetylation mutants of p53 were unable to induce nSMase2, suggesting a role of nSMase2 in growth arrest. Moreover, knockdown of nSMase2 prevented doxorubicin-induced growth arrest. Finally, p53-induced nSMase2 upregulation appears to occur via a novel transcription start site upstream of exon 3. These results identify nSMase2 as a novel p53 target gene, regulated by the DNA damage pathway to induce cell growth arrest.

No MeSH data available.


Related in: MedlinePlus

nSMase2 upregulation is independent of known transcriptional regulators of nSMase2. (a) MCF7 cells were seeded in 60 mm dishes and transfected with siRNA to AllStars Negative Control (AS), Sp1, Sp3 or both together. After 24 h, cells were treated with vehicle, and 0.2 or 0.6 μM doxorubicin. After 24 h, cells were collected and immunoblotted for nSMase2, Sp1, Sp3 and actin. (b) MCF7 cells were seeded in 60 mm dishes. One hour before stimulation with doxorubicin or vehicle, they were pretreated with N-acetylcysteine (NAC). Cells were collected and immunoblotted for nSMase2 and actin. (c) MCF7 cells were seeded in 60 mm dishes and transfected with siRNA to AS or CREB3L1. After 24 h, they were treated with vehicle or 0.6 μM doxorubicin. Cells were collected and immunoblotted for nSMase2 and actin
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fig3: nSMase2 upregulation is independent of known transcriptional regulators of nSMase2. (a) MCF7 cells were seeded in 60 mm dishes and transfected with siRNA to AllStars Negative Control (AS), Sp1, Sp3 or both together. After 24 h, cells were treated with vehicle, and 0.2 or 0.6 μM doxorubicin. After 24 h, cells were collected and immunoblotted for nSMase2, Sp1, Sp3 and actin. (b) MCF7 cells were seeded in 60 mm dishes. One hour before stimulation with doxorubicin or vehicle, they were pretreated with N-acetylcysteine (NAC). Cells were collected and immunoblotted for nSMase2 and actin. (c) MCF7 cells were seeded in 60 mm dishes and transfected with siRNA to AS or CREB3L1. After 24 h, they were treated with vehicle or 0.6 μM doxorubicin. Cells were collected and immunoblotted for nSMase2 and actin

Mentions: Transcriptional regulation of nSMase2 has been described in response to different stimuli. Sp1 and Sp3 were shown to regulate nSMase2 transcriptionally in response to daunorubicin and ATRA.11, 12 Formation of reactive oxygen species (ROS) was shown to modulate nSMase2, and doxorubicin is a potent generator of ROS.32, 33, 34 In addition, Denard et al. described regulation of ceramide production by CREB3L1 following doxorubicin in MCF7 cells.35 As such, it was next essential to determine the effect of these transcriptional regulators on nSMase2 in response to doxorubicin. As can be seen, siRNA knockdown of Sp1 and Sp3 did not prevent nSMase2 upregulation after doxorubicin treatment (Figure 3a). Moreover, pre-treatment with N-acetylcysteine, a quencher of ROS, had no effect of nSMase2 induction (Figure 3b). Finally, knockdown of CREB3L1 by siRNA did not inhibit nSMase2 upregulation in response to doxorubicin (Figure 3c). CREB3L1 knockdown was verified by qRT-PCR (Supplementary Figure S3). Taken together, these results suggest that nSMase2 transcriptional activation is independent of known regulators, and is possibly due to a new previously undescribed mechanism.


P53-dependent upregulation of neutral sphingomyelinase-2: role in doxorubicin-induced growth arrest.

Shamseddine AA, Clarke CJ, Carroll B, Airola MV, Mohammed S, Rella A, Obeid LM, Hannun YA - Cell Death Dis (2015)

nSMase2 upregulation is independent of known transcriptional regulators of nSMase2. (a) MCF7 cells were seeded in 60 mm dishes and transfected with siRNA to AllStars Negative Control (AS), Sp1, Sp3 or both together. After 24 h, cells were treated with vehicle, and 0.2 or 0.6 μM doxorubicin. After 24 h, cells were collected and immunoblotted for nSMase2, Sp1, Sp3 and actin. (b) MCF7 cells were seeded in 60 mm dishes. One hour before stimulation with doxorubicin or vehicle, they were pretreated with N-acetylcysteine (NAC). Cells were collected and immunoblotted for nSMase2 and actin. (c) MCF7 cells were seeded in 60 mm dishes and transfected with siRNA to AS or CREB3L1. After 24 h, they were treated with vehicle or 0.6 μM doxorubicin. Cells were collected and immunoblotted for nSMase2 and actin
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4632297&req=5

fig3: nSMase2 upregulation is independent of known transcriptional regulators of nSMase2. (a) MCF7 cells were seeded in 60 mm dishes and transfected with siRNA to AllStars Negative Control (AS), Sp1, Sp3 or both together. After 24 h, cells were treated with vehicle, and 0.2 or 0.6 μM doxorubicin. After 24 h, cells were collected and immunoblotted for nSMase2, Sp1, Sp3 and actin. (b) MCF7 cells were seeded in 60 mm dishes. One hour before stimulation with doxorubicin or vehicle, they were pretreated with N-acetylcysteine (NAC). Cells were collected and immunoblotted for nSMase2 and actin. (c) MCF7 cells were seeded in 60 mm dishes and transfected with siRNA to AS or CREB3L1. After 24 h, they were treated with vehicle or 0.6 μM doxorubicin. Cells were collected and immunoblotted for nSMase2 and actin
Mentions: Transcriptional regulation of nSMase2 has been described in response to different stimuli. Sp1 and Sp3 were shown to regulate nSMase2 transcriptionally in response to daunorubicin and ATRA.11, 12 Formation of reactive oxygen species (ROS) was shown to modulate nSMase2, and doxorubicin is a potent generator of ROS.32, 33, 34 In addition, Denard et al. described regulation of ceramide production by CREB3L1 following doxorubicin in MCF7 cells.35 As such, it was next essential to determine the effect of these transcriptional regulators on nSMase2 in response to doxorubicin. As can be seen, siRNA knockdown of Sp1 and Sp3 did not prevent nSMase2 upregulation after doxorubicin treatment (Figure 3a). Moreover, pre-treatment with N-acetylcysteine, a quencher of ROS, had no effect of nSMase2 induction (Figure 3b). Finally, knockdown of CREB3L1 by siRNA did not inhibit nSMase2 upregulation in response to doxorubicin (Figure 3c). CREB3L1 knockdown was verified by qRT-PCR (Supplementary Figure S3). Taken together, these results suggest that nSMase2 transcriptional activation is independent of known regulators, and is possibly due to a new previously undescribed mechanism.

Bottom Line: DNA-binding mutants as well as acetylation mutants of p53 were unable to induce nSMase2, suggesting a role of nSMase2 in growth arrest.Moreover, knockdown of nSMase2 prevented doxorubicin-induced growth arrest.Finally, p53-induced nSMase2 upregulation appears to occur via a novel transcription start site upstream of exon 3.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Stony Brook University, Health Science Center, Stony Brook, NY 11794-8430, USA.

ABSTRACT
Neutral sphingomyelinase-2 (nSMase2) is a ceramide-generating enzyme that has been implicated in growth arrest, apoptosis and exosome secretion. Although previous studies have reported transcriptional upregulation of nSMase2 in response to daunorubicin, through Sp1 and Sp3 transcription factors, the role of the DNA damage pathway in regulating nSMase2 remains unclear. In this study, we show that doxorubicin induces a dose-dependent induction of nSMase2 mRNA and protein with concomitant increases in nSMase activity and ceramide levels. Upregulation of nSMase2 was dependent on ATR, Chk1 and p53, thus placing it downstream of the DNA damage pathway. Moreover, overexpression of p53 was sufficient to transcriptionally induce nSMase2, without the need for DNA damage. DNA-binding mutants as well as acetylation mutants of p53 were unable to induce nSMase2, suggesting a role of nSMase2 in growth arrest. Moreover, knockdown of nSMase2 prevented doxorubicin-induced growth arrest. Finally, p53-induced nSMase2 upregulation appears to occur via a novel transcription start site upstream of exon 3. These results identify nSMase2 as a novel p53 target gene, regulated by the DNA damage pathway to induce cell growth arrest.

No MeSH data available.


Related in: MedlinePlus