Associate Professor of Biochemistry & Molecular Biology
Visit Dr. Chalfant's Website
PO Box 980614
Richmond, VA 23298-0614
Email: cechalfant@vcu.edu
Telephone: 804-828-9526
Education
- B.S., 1992, University of Tampa
- Ph.D., 1977, University of South Florida College of Medicine
Training
- National Research Service Award, Postdoctoral Fellow, Duke University
- Medical University of South Carolina, Advisor: Yusuf Hannun, M.D.
Research
Project 1: Ceramide Regulates The Alternative
Splicing Of Bcl-x
The long-term objectives of this
project focus on the elucidation of the pathways that
mediate programmed cell death (PCD) in response to
extracellular agents. Furthermore and importantly,
how dysregulation of apoptotic pathways confers resistance
to PCD and induction of a disease phenotype. In this
proposal, we will specifically define the mechanisms
involved in regulating the alternative splicing of
the apoptosis regulator, Bcl-x. Multiple lines of evidence
point to a role for the Bcl-2 family in regulating
PCD. Bcl-x(L), a member of the Bcl-2 family, has been
implicated as an inhibitor of PCD, and many studies
have shown that overexpression of Bcl-x(L) in cells
confers PCD resistance to many apoptotic stimuli including
chemotherapy, Fas activation, TNF?, and ?-irradiation.
Furthermore, many cell types spontaneously resistant
to chemotherapeutic agents demonstrate increased levels
of Bcl-x(L).
An essential component for understanding how Bcl-x(L)
levels are increased in chemotherapeutic-resistant
cancer cells is to identify and establish how Bcl-x(L)
expression is regulated. To date, the regulation of
Bcl-x(L) expression is a complex mechanism consisting
of both transcriptional and post-transcriptional processes.
The post-transcriptional processing of the Bcl-x gene
gives rise to at least 5 different Bcl-x isoforms via
alternative splicing (Bcl-x(L), Bcl-x(s), Bcl-x?, Bcl-x?TM,
and Bcl-x?) and studies have shown that these isoforms
have antagonistic functions in some cases. For example,
several studies have clearly demonstrated that the
Bcl-x splice variant, Bcl-x(s), in contrast to Bcl-x(L),
promotes apoptosis instead of inhibiting apoptosis.
Bcl-x(s) is produced by activation of an upstream 5’ splice
site within the Bcl-x exon 2. Recent studies have shown
that blockage of the downstream Bcl-x(L) specific 5’ splice
site in Bcl-x exon 2 using oligonucleotides induces
Bcl-x(s) expression while downregulating Bcl-x(L) levels
and sensitizing A549 lung adenocarcinoma cells to chemotherapy.
Thus, regulation of 5’ splice site selection
within the Bcl-x exon 2 can determine whether a cell
is susceptible or resistant to apoptosis.
Multiple lines of evidence point to roles for ceramide
in regulating apoptosis in response to extracellular
stimuli and published findings from our laboratory
have shown that ceramide regulates the 5’ splice
site selection within the Bcl-x exon 2. Treatment of
A549 lung adenocarcinoma cells with cell-permeable
ceramide downregulated Bcl-x(L) mRNA and immunoreactive
protein levels with a concomitant increase in mRNA
and immunoreactive protein levels of Bcl-x(s). This
effect was demonstrated to be through regulation of
Bcl-x pre-mRNA processing. Downregulation of Bcl-x(L)
by ceramide-induced Bcl-x(s) 5’ splice site activation
correlated with increased sensitivity of A549 cells
to daunorubicin. Furthermore, A549 cells resistant
to chemotherapeutic agents and cell-permeable ceramides
demonstrated increased Bcl-x(L) levels due to dysregulated
Bcl-x alternative pre-mRNA processing.
In further mechanistic studies by the PI, it was shown
that SR proteins, a family of RNA splicing factors
and substrates for protein phosphatases 1 (a ceramide-activated
protein phosphatases) are dephosphorylated in a time-
and dose-dependent manner by cell- permeable ceramide.
Both SR protein dephosphorylation and Bcl-x alternative
splicing were blocked by inhibitors of serine-threonine
protein phosphatases and of the de novo ceramide pathway,
suggesting a role for protein phosphatases 1 (PP1)
and endogenous ceramide in regulating this mechanism.
Furthermore, dephosphorylation of SR proteins has been
shown to affect 5’ splice site selection strongly
implicating at least one SR protein family member in
regulating Bcl-x 5’ splice site selection.
Hypothesis: The above results lead us to hypothesize that RNA transactivating
factors, including at least one SR protein isoform,
interacting with specific RNA cis-elements in Bcl-x
pre-mRNA mediate the activation of the Bcl-x exon 2
upstream 5’ splice site (Bcl-x(s) specific 5’ splice
site), thereby, producing Bcl-x(s) mRNA following ceramide
treatment. We are currently testing this hypothesis.
Highlights of current findings: We have identified
the ceramide-responsive RNA cis-element (CRCE) and
have found that SR proteins, indeed, bind
specifically to this sequence.
Project 2: The Role of Ceramide-1-Phosphate In Prostanoid
Synthesis
The production of arachidonic acid by
phospholipases is the rate-limiting step in prostaglandin
biosynthesis, and the major phospholipase that regulates
prostaglandin synthesis in response to inflammatory cytokines
(e.g. IL-1? and TNF?) is type IVA cytosolic phospholipase
A2 (cPLA2) (1). Activation/translocation of cPLA2 in
cells requires the association of cPLA2 with membranes
in a Ca2+-dependent manner via a Ca2+-dependent lipid
binding domain (CaLB domain) located near the N-terminus
(2,3,4,5). However, the specific membrane lipids that
regulate this binding or whether activation of cPLA2
also requires the generation of activating lipids is
unknown.
An essential component for understanding cPLA2 activation
is to identify and establish the bioactive lipids responsible
for interacting with the CaLB domain and regulating
the membrane association of cPLA2. Ceramide-1-phosphate
(C-1-P) is a new addition to bioactive sphingolipids
generated by the phosphorylation of ceramide by ceramide
kinase. C-1-P is one such potential lipid regulator
of cPLA2. Indeed, the main component of the venom from
Loxosceles reclusa (brown recluse spider) is the enzyme
sphingomyelinase D (SMase D) which hydrolyzes sphingomyelin
to produce ceramide-1-phosphate (C-1-P) (6). The pathology
of a wound generated from the bite of this spider is
that of an intense inflammatory response mediated by
arachidonic acid (AA) and prostaglandins (7,8,9). The
production of endogenous C-1-P by the action of SMase
D raised the possibility of C-1-P acting as a patho-physiologic
link in the activation of cPLA2 and the inflammatory
response mediated by AA and prostaglandins.
Preliminary results from our laboratory concur with
this patho-physiologic link and demonstrate a specific
biology regulated by ceramide-1-phosphate. We found
that treatment of several cell types with C-1-P (nanomolar
concentrations) induced AA release and the synthesis
of prostanoids. Further exploration of this effect
demonstrated that C-1-P induced AA release in various
cell types, and this effect was also lipid-specific
as the closely related lipids, phosphatidic acid, ceramide,
diacylglycerol, and sphingosine phosphate had either
minimal or no effects on AA release and prostanoid
synthesis. Preliminary findings also show that C-1-P
induced activation/translocation of full-length cPLA2
as well as the truncated CaLB/C2 domain of cPLA2. siRNA
technology was employed to downregulate cPLA2 which
demonstrated that the induction of AA release by C-1-P
was strictly dependent on cPLA2 activation. These preliminary
findings also disclose that C-1-P directly binds to
cPLA2 in a Ca+2 enhanced manner via the CaLB/C2 domain,
and C-1-P also increased the enzymatic activity of
cPLA2 in vitro as well as increasing the affinity of
cPLA2 for Ca+2 by approximately 10-fold. Furthermore,
studies using pulse labeling demonstrate a marked increase
in C-1-P concurrent with the release of AA and PGE2
in response to inflammatory cytokines. Preliminary
results using an in vitro inhibitor of ceramide kinase
activity and siRNA technology to downregulate ceramide
kinase blocked cPLA2 activation, AA release and prostanoid
production in response to inflammatory cytokines. Lastly,
our preliminary results demonstrate that ceramide-1-phosphate
is downstream of calcium mobilization in the activation
of cPLA2.
Based on these data, our central hypothesis is that
ceramide phosphate (C-1-P) produced from the phosphorylation
of ceramide by ceramide kinase is an important mediator
of prostaglandin synthesis through activation of cPLA2
in response to inflammatory cytokines. To validate
our hypothesis, we are currently answering the following
basic questions: 1) How is ceramide-1-phosphate generated
in response to inflammatory cytokines? 2) Is ceramide
kinase involved in the signal transduction of cPLA2
activation, AA release, and prostanoid production?
3) Does ceramide-1-phosphate act as a novel and direct
signaling molecule in the activation of cPLA2 with
subsequent induction of AA release and prostanoid synthesis
in response to inflammatory cytokines?
Project 3: The role of the alternative splicing of caspase 9 in oncogenesis.
The long-term objectives of this project focus on determining how dysregulation of apoptotic pathways confers
resistance to chemotherapy and increases the susceptibility of cells to oncogenic transformation. Caspase 9 (caspase
9a) has been shown to be an important factor in the apoptotic pathway and required for cell death induced by various
chemotherapies, stress agents, and radiation. Studies have shown that the expression of an RNA splice variant of
caspase 9, termed caspase 9b, confers the opposite effect by inducing resistance to many apoptotic stimuli. The post-
transcriptional processing of caspase 9 pre-mRNA is a complex mechanism involving the inclusion or exclusion of a
four exon cassette (exons 3, 4, 5, and 6). Inclusion of these four exons into the mature transcript produces the pro-
apoptotic caspase 9 while exclusion of this cassette produces the anti-apoptotic caspase 9b. The caspase 9b protein
lacks the catalytic domain, but retains all other amino acid sequence such as the APAF-1 association region. Caspase
9b competes with the full-length caspase 9 for binding to the apoptosome, and caspase 9b has also been shown to
heterodimerize with full-length caspase 9, thereby inhibiting the activation of this caspase. Thus, regulation of the
inclusion of this four exon cassette is a critical factor in determining whether a cell is susceptible or resistant
to apoptosis, and thus oncogenic transformation.
In corroboration with these reports and hypothesis, preliminary results from the PI’s laboratory demonstrate that the
direct modulation of the alternative splicing of caspase 9 using RNAi and anti-sense RNA oligonucleotides (ASROs)
significantly affected the susceptibility of A549 cells to daunorubicin (as measured by WST and clonogenic assays).
Induced expression of caspase 9b by a caspase 9a-specific ASRO in non-transformed cells also increased the oncogenic
ability of c-Myc/H-rasV12 as measured by colony formation in soft agar. In novel mechanistic studies by the PI, the
generation of the lipid second messenger, ceramide, and the activation of protein phosphatase-1 (PP1) were defined as
major components of the signal transduction pathway that induces the inclusion of the four exon cassette into the
mature caspase 9 transcript. Furthermore, we demonstrated that SR proteins, a family of RNA splicing factors, were
dephosphorylated in response to the generation of de novo ceramide in a PP1-dependent manner and within the same time
frame as the inclusion of the four exon cassette into the mature caspase 9 transcript. Preliminary results by the
PI’s laboratory also disclose that the alternative splicing of caspase 9 is intrinsically linked to the SR protein,
SRp30a (ASF/SF2). We found that downregulation of SRp30a using RNA interference technology (RNAi) dramatically
inhibited the inclusion of the 3, 4, 5, 6 exon cassette in the mature caspase 9 transcript. Furthermore, six possible
interaction sites for SRp30a were identified within and downstream of each exon in the exon 3, 4, 5, and 6 cassette
of the caspase 9 gene. Interestingly, lung adenocarcinoma tumors demonstrated a dysregulated ratio of caspase
9/caspase 9b that would produce an anti-apoptotic/chemotherapy resistance phenotype. The culmination of these data
suggest a role for SRp30a and the pre-mRNA processing of caspase 9 in the apoptotic mechanism of lung adenocarcinoma
tumors. In other mechanistic studies, the protein kinase, CLK/STY, was found to regulate the phospho-status of SR
proteins and the alternative splicing of caspase 9 in A549 cells. Furthermore, sphingosine-1-phosphate, a mitogenic
bioactive lipid, induced an increase in the phosphorylation of SR proteins.
Based on the above findings, we hypothesize that the alternative splicing of caspase 9 is a critical factor in
determining the susceptibility of cells to chemotherapy and transformation by oncogenes. Furthermore, we hypothesize
that SRp30a is an important regulator of caspase 9 pre-mRNA processing in response to ceramide via interaction with
specific RNA cis-elements, and that SRp30a regulates the inclusion of the exon 3, 4, 5, and 6 cassette of caspase 9
via its phospho-status (Scheme 1). Lastly, we hypothesize that prosurvival agonists (e.g. S-1-P) induce the
phosphorylation of SRp30a via activation of CLK/STY, which in turn increases the expression of caspase 9b (Scheme 1).
Highlights of current findings: We have essentially demonstrated that SRp30a is a required factor for both basal and
ceramide-induced expression of caspase 9a via regulation of exon inclusion. We have also determined two cis-elements
that regulate ceramide effects on the inclusion of the exon 3,4,5,6 cassette of caspase 9 pre-mRNA as well as shown
that SRp30a interacts specifically with these RNA cis-elements. We have also determined a repressor element in exon 3
of the caspase 9 pre-mRNA, but the function and RNA trans-factors associated with this element are currently unknown.
We have also determined the protein kinase that regulates the phospho-state of SRp30a. Studies are ongoing to
determine whether the phospho-state of SRp30a has a role in regulating the alternative splicing of caspase 9. Lastly,
we have developed all of the technologies required to manipulate the alternative splicing of caspase 9 and are
examining the role of this mechanism in oncogenesis and sensitivity of cells to various chemotherapies.
We believe these studies will demonstrate that the alternative splicing of caspase 9 is a key mechanism for
regulating the susceptibility of cells to chemotherapy-induced cell death and oncogenic transformation. These studies
will also largely define the signal transduction pathway leading to the inclusion of the exon 3, 4, 5, and 6 cassette
of caspase 9 in response to apoptotic agonists. Furthermore, these studies will begin to define factors involved in
the signal transduction pathway that regulates the pro-survival activation of the exclusion the exon 3, 4, 5, and 6
cassette of caspase 9. This cannot be understated because the definition of these signal transduction pathways
creates, not one, but many new targets, for anti-cancer therapies. These are exciting studies, and our laboratory
group looks forward to pursuing the identification of both the apoptotic and pro-survival pathways of signal
transduction that regulate the fate of a cell, and thus, a whole organism.
Funding Source: Currently none. Hopefully an R01 award (1 R01 CA117950-02) from NIH specifically the National Cancer
Institute (priority score = 150, percentile = 13.1%) starting in July 2006. We have our Ju Ju and rosary beads out
and are saying prayers!
New Projects On-Going:
- Role of ceramide kinase and C1P in exocytosis and phagocytosis.
- Identification of other C1P-interacting proteins.
- Identification of CERK interacting proteins if any.
- Conditional Knock-out mice for all projects.
- Crystallization of CERK and a cPLA2alpha/C1P complex.
- Role of CERK in asthma animal models.
Publications
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