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J. Biol. Chem., Vol. 277, Issue 15, 12587-12595, April 12, 2002
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From the Previous studies have demonstrated that several
splice variants are derived from both the caspase 9 and
Bcl-x genes in which the Bcl-x splice variant, Bcl-x(L) and
the caspase 9 splice variant, caspase 9b, inhibit apoptosis in contrast
to the pro-apoptotic splice variants, Bcl-x(s) and caspase 9. In a
recent study, we showed that ceramide induces the dephosphorylation of
SR proteins, a family of protein factors that regulate alternative
splicing. In this study, the regulation of the alternative processing
of pre-mRNA of both caspase 9 and Bcl-x(L) was examined in response to ceramide. Treatment of A549 lung adenocarcinoma cells with cell-permeable ceramide, D-e-C6 ceramide, down-regulated
the levels of Bcl-x(L) and caspase 9b mRNA and immunoreactive
protein with a concomitant increase in the mRNA and immunoreactive
protein levels of Bcl-x(s) and caspase 9 in a dose- and
time-dependent manner. Pretreatment with calyculin A (5 nM), an inhibitor of protein phosphatase-1 (PP1) and
protein phosphatase 2A (PP2A) blocked ceramide-induced alternative
splicing in contrast to okadaic acid (10 nM), a specific
inhibitor of PP2A at this concentrations in cells, demonstrating a
PP1-mediated mechanism. A role for endogenous ceramide in regulating
the alternative splicing of caspase 9 and Bcl-x was demonstrated using
the chemotherapeutic agent, gemcitabine. Treatment of A549 cells with
gemcitabine (1 µM) increased ceramide levels 3-fold via
the de novo sphingolipid pathway as determined by pulse
labeling experiments and inhibition studies with myriocin (50 nM), a specific inhibitor of serine palmitoyltransferase
(the first step in de novo synthesis of ceramide).
Treatment of A549 cells with gemcitabine down-regulated the levels of
Bcl-x(L) and caspase 9b mRNA with a concomitant increase in the
mRNA levels of Bcl-x(s) and caspase 9. Again, inhibitors of
ceramide synthesis blocked this effect. We also demonstrate that the
change in the alternative splicing of caspase 9 and Bcl-x occurred
prior to apoptosis following treatment with gemcitabine. Furthermore,
doses of D-e-C6 ceramide that induce the alternative
splicing of both caspase 9 and Bcl-x-sensitized A549 cells to
daunorubicin. These data demonstrate a role for protein phosphatases 1 (PP1) and endogenous ceramide generated via the de novo
pathway in regulating this mechanism. This is the first report on the
dynamic regulation of RNA splicing of members of the Bcl-2 and caspase
families in response to regulators of apoptosis.
De Novo Ceramide Regulates the Alternative Splicing
of Caspase 9 and Bcl-x in A549 Lung Adenocarcinoma Cells
DEPENDENCE ON PROTEIN PHOSPHATASE-1*
§,,,,§¶,, and
Department of Biochemistry & Molecular
Biology, Medical University of South Carolina, Charleston, South
Carolina 29425, § Research and Development, Ralph H. Johnson
Veterans Affairs Medical Center, Charleston, South Carolina 29401, and
the ¶ Department of Medicine, Medical University of South
Carolina, Charleston, South Carolina 29425
*
This work was supported by National Institutes of Health
Grants CA87584 and GM43825 (to Y. A. H.), National Research
Service Award GM19953-02 (to C. E. C.) from the National
Institutes of Health, and a VA Merit Review (to C. E. C.)
from the Veterans Administration.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of
Biochemistry & Molecular Biology, Rm. 501, Basic Science Building,
Medical University of South Carolina, 173 Ashley Ave., P.O. Box 250509, Charleston, SC 29425. Tel.: 843-792-4321; Fax: 843-792-4322; E-mail: hannun@musc.edu.
Copyright © 2002 by The American Society for Biochemistry and Molecular Biology, Inc.
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