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Remodeling of cellular cytoskeleton by the acid sphingomyelinase/ceramide pathway.

Zeidan YH, Jenkins RW, Hannun YA - J. Cell Biol. (2008)

Bottom Line: This translocation is blocked upon overexpression of a dominant-negative (DN) ASMase(S508A) mutant and by a DN PKCdelta.Importantly; knockdown of ASMase protects MCF-7 cells from cisplatin-induced cytoskeletal changes including ezrin dephosphorylation.Reciprocally, exogenous delivery of D-e-C16-Cer, but not dihydro-C16-Cer, recapitulates the morphotropic effects of cisplatin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA.

ABSTRACT
The chemotherapeutic agent cisplatin is widely used in treatment of solid tumors. In breast cancer cells, cisplatin produces early and marked changes in cell morphology and the actin cytoskeleton. These changes manifest as loss of lamellipodia/filopodia and appearance of membrane ruffles. Furthermore, cisplatin induces dephosphorylation of the actin-binding protein ezrin, and its relocation from membrane protrusions to the cytosol. Because cisplatin activates acid sphingomyelinase (ASMase), we investigate here the role of the ASMase/ceramide (Cer) pathway in mediating these morphological changes. We find that cisplatin induces a transient elevation in ASMase activity and its redistribution to the plasma membrane. This translocation is blocked upon overexpression of a dominant-negative (DN) ASMase(S508A) mutant and by a DN PKCdelta. Importantly; knockdown of ASMase protects MCF-7 cells from cisplatin-induced cytoskeletal changes including ezrin dephosphorylation. Reciprocally, exogenous delivery of D-e-C16-Cer, but not dihydro-C16-Cer, recapitulates the morphotropic effects of cisplatin. Collectively, these results highlight a novel tumor suppressor property for Cer and a function for ASMase in cisplatin-induced cytoskeletal remodeling.

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PP2A mediates dephosphorylation of ezrin by cisplatin. Cells plated on 10-cm dishes (5 × 105 cells/dish) were pretreated with 10 nM of either okadaic acid (A) or tautomycin (B). After 1 h, cells were incubated with DMSO or cisplatin (5 μg/ml) for 1 h. Phospho-ezrin levels were evaluated by Western blotting using a specific polyclonal antibody. (C) Cells were transfected with 20 nM of either SCR or PP2A (catalytic subunit) specific oligonucleotides. After 48 h, cells were subjected to DMSO or cisplatin treatments for 1 h. Lysates (30 μg) were analyzed by Western blotting for levels of p-ezrin and PP2A. Blots shown are representative of at least three independent experiments. Densitometric analysis was performed using NIH Image software. (D) MCF-7 cells expressing YFP-ezrin and CFP-PP2A (catalytic subunit) were subjected to sensitized emission FRET analysis. After 24 h of plasmid transfection, cells were treated with DMSO or cisplatin for 30 and 60 min. A representative FRET image of at least 30 cells imaged in three experiments is shown. FRET efficiencies are encoded by using the color bar scale shown on the left. Colors range between blue (lowest FRET) and red (highest FRET).
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fig7: PP2A mediates dephosphorylation of ezrin by cisplatin. Cells plated on 10-cm dishes (5 × 105 cells/dish) were pretreated with 10 nM of either okadaic acid (A) or tautomycin (B). After 1 h, cells were incubated with DMSO or cisplatin (5 μg/ml) for 1 h. Phospho-ezrin levels were evaluated by Western blotting using a specific polyclonal antibody. (C) Cells were transfected with 20 nM of either SCR or PP2A (catalytic subunit) specific oligonucleotides. After 48 h, cells were subjected to DMSO or cisplatin treatments for 1 h. Lysates (30 μg) were analyzed by Western blotting for levels of p-ezrin and PP2A. Blots shown are representative of at least three independent experiments. Densitometric analysis was performed using NIH Image software. (D) MCF-7 cells expressing YFP-ezrin and CFP-PP2A (catalytic subunit) were subjected to sensitized emission FRET analysis. After 24 h of plasmid transfection, cells were treated with DMSO or cisplatin for 30 and 60 min. A representative FRET image of at least 30 cells imaged in three experiments is shown. FRET efficiencies are encoded by using the color bar scale shown on the left. Colors range between blue (lowest FRET) and red (highest FRET).

Mentions: Ceramide-activated protein phosphatases (CAPPs) are well established effectors of ceramide generating pathways of which PP2A and PP1 remain the best studied thus far (Ruvolo, 2003). Therefore, it became important to determine whether the observed dephosphorylation of ezrin is mediated by CAPPs. In previous studies, it was demonstrated that okadaic acid potently inhibits PP2A when used at low doses (Chalfant et al., 1999, 2001). Cells were pretreated with either okadaic acid (10 nM) or vehicle (DMSO) 1 h before cisplatin addition. Western blot analysis revealed that inhibition of PP2A with okadaic acid restored levels of p-ezrin (Fig. 7 A). We also entertained the potential involvement of another well-established ceramide activated phosphatase, PP1. However, treatment with tautomycin (10 nM), a specific PP1 inhibitor, had little effect on cisplatin-induced ezrin dephosphorylation (Fig. 7 B). These results were further corroborated using RNAi technology. For these experiments, MCF-7 cells were transfected with 20 nM of either control (SCR) or PP2A-specific 21-bp oligonucleotides. After 48 h, marked knockdown (>70%) of PP2A protein was observed by Western blotting (Fig. 7 C). Importantly, loss of PP2A conferred protection from cisplatin-induced ezrin dephosphorylation (Fig. 7 C). These findings provide strong pharmacologic and genetic evidence for PP2A in ezrin dephosphorylation.


Remodeling of cellular cytoskeleton by the acid sphingomyelinase/ceramide pathway.

Zeidan YH, Jenkins RW, Hannun YA - J. Cell Biol. (2008)

PP2A mediates dephosphorylation of ezrin by cisplatin. Cells plated on 10-cm dishes (5 × 105 cells/dish) were pretreated with 10 nM of either okadaic acid (A) or tautomycin (B). After 1 h, cells were incubated with DMSO or cisplatin (5 μg/ml) for 1 h. Phospho-ezrin levels were evaluated by Western blotting using a specific polyclonal antibody. (C) Cells were transfected with 20 nM of either SCR or PP2A (catalytic subunit) specific oligonucleotides. After 48 h, cells were subjected to DMSO or cisplatin treatments for 1 h. Lysates (30 μg) were analyzed by Western blotting for levels of p-ezrin and PP2A. Blots shown are representative of at least three independent experiments. Densitometric analysis was performed using NIH Image software. (D) MCF-7 cells expressing YFP-ezrin and CFP-PP2A (catalytic subunit) were subjected to sensitized emission FRET analysis. After 24 h of plasmid transfection, cells were treated with DMSO or cisplatin for 30 and 60 min. A representative FRET image of at least 30 cells imaged in three experiments is shown. FRET efficiencies are encoded by using the color bar scale shown on the left. Colors range between blue (lowest FRET) and red (highest FRET).
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Related In: Results  -  Collection

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fig7: PP2A mediates dephosphorylation of ezrin by cisplatin. Cells plated on 10-cm dishes (5 × 105 cells/dish) were pretreated with 10 nM of either okadaic acid (A) or tautomycin (B). After 1 h, cells were incubated with DMSO or cisplatin (5 μg/ml) for 1 h. Phospho-ezrin levels were evaluated by Western blotting using a specific polyclonal antibody. (C) Cells were transfected with 20 nM of either SCR or PP2A (catalytic subunit) specific oligonucleotides. After 48 h, cells were subjected to DMSO or cisplatin treatments for 1 h. Lysates (30 μg) were analyzed by Western blotting for levels of p-ezrin and PP2A. Blots shown are representative of at least three independent experiments. Densitometric analysis was performed using NIH Image software. (D) MCF-7 cells expressing YFP-ezrin and CFP-PP2A (catalytic subunit) were subjected to sensitized emission FRET analysis. After 24 h of plasmid transfection, cells were treated with DMSO or cisplatin for 30 and 60 min. A representative FRET image of at least 30 cells imaged in three experiments is shown. FRET efficiencies are encoded by using the color bar scale shown on the left. Colors range between blue (lowest FRET) and red (highest FRET).
Mentions: Ceramide-activated protein phosphatases (CAPPs) are well established effectors of ceramide generating pathways of which PP2A and PP1 remain the best studied thus far (Ruvolo, 2003). Therefore, it became important to determine whether the observed dephosphorylation of ezrin is mediated by CAPPs. In previous studies, it was demonstrated that okadaic acid potently inhibits PP2A when used at low doses (Chalfant et al., 1999, 2001). Cells were pretreated with either okadaic acid (10 nM) or vehicle (DMSO) 1 h before cisplatin addition. Western blot analysis revealed that inhibition of PP2A with okadaic acid restored levels of p-ezrin (Fig. 7 A). We also entertained the potential involvement of another well-established ceramide activated phosphatase, PP1. However, treatment with tautomycin (10 nM), a specific PP1 inhibitor, had little effect on cisplatin-induced ezrin dephosphorylation (Fig. 7 B). These results were further corroborated using RNAi technology. For these experiments, MCF-7 cells were transfected with 20 nM of either control (SCR) or PP2A-specific 21-bp oligonucleotides. After 48 h, marked knockdown (>70%) of PP2A protein was observed by Western blotting (Fig. 7 C). Importantly, loss of PP2A conferred protection from cisplatin-induced ezrin dephosphorylation (Fig. 7 C). These findings provide strong pharmacologic and genetic evidence for PP2A in ezrin dephosphorylation.

Bottom Line: This translocation is blocked upon overexpression of a dominant-negative (DN) ASMase(S508A) mutant and by a DN PKCdelta.Importantly; knockdown of ASMase protects MCF-7 cells from cisplatin-induced cytoskeletal changes including ezrin dephosphorylation.Reciprocally, exogenous delivery of D-e-C16-Cer, but not dihydro-C16-Cer, recapitulates the morphotropic effects of cisplatin.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC 29425, USA.

ABSTRACT
The chemotherapeutic agent cisplatin is widely used in treatment of solid tumors. In breast cancer cells, cisplatin produces early and marked changes in cell morphology and the actin cytoskeleton. These changes manifest as loss of lamellipodia/filopodia and appearance of membrane ruffles. Furthermore, cisplatin induces dephosphorylation of the actin-binding protein ezrin, and its relocation from membrane protrusions to the cytosol. Because cisplatin activates acid sphingomyelinase (ASMase), we investigate here the role of the ASMase/ceramide (Cer) pathway in mediating these morphological changes. We find that cisplatin induces a transient elevation in ASMase activity and its redistribution to the plasma membrane. This translocation is blocked upon overexpression of a dominant-negative (DN) ASMase(S508A) mutant and by a DN PKCdelta. Importantly; knockdown of ASMase protects MCF-7 cells from cisplatin-induced cytoskeletal changes including ezrin dephosphorylation. Reciprocally, exogenous delivery of D-e-C16-Cer, but not dihydro-C16-Cer, recapitulates the morphotropic effects of cisplatin. Collectively, these results highlight a novel tumor suppressor property for Cer and a function for ASMase in cisplatin-induced cytoskeletal remodeling.

Show MeSH
Related in: MedlinePlus