Charles E Chalfant, Ph.D.Associate Professor of Biochemistry & Molecular Biology
Visit Dr. Chalfant's Website
PO Box 980614
Richmond, VA 23298-0614
- B.S., 1992, University of Tampa
- Ph.D., 1977, University of South Florida College of Medicine
- National Research Service Award, Postdoctoral Fellow, Duke University
- Medical University of South Carolina, Advisor: Yusuf Hannun, M.D.
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
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?
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.