Project 1: Ceramide regulation of the alternative splicing of Bcl-x pre-mRNA (ongoing for  5 years)

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 research project, our goal is to 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. We have shown that treatment of A549 lung adenocarcinoma cells with cell-permeable ceramide and chemotherapies that induce the synthesis of de novo ceramide downregulated Bcl-x(L) mRNA and immunoreactive protein levels with a concomitant increase in mRNA and immunoreactive protein levels of Bcl-x(s). 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 led 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-elements (CRCEs), CRCE 1 and CRCE 2 within Exon 2 of the Bcl-x pre-mRNA. Further studies have identified CRCE 1 as the critical RNA cis-element for the induction of the Bcl-X(s) 5’ splice site by de novo ceramide. Analysis of RNA trans-factors that bind to CRCE 1 demonstrated that SAP155, a spliceosomal-associated protein, specifically bound to CRCE 1 and regulated the activation of the Bcl-X(s) 5’ splice site by ceramide. Formerly, SAP155 was thought to only regulate constitutive RNA processing, but these findings show a clear role for SAP155 in modulating alternative splicing! Ongoing collaborations with other researchers (e.g. Dr. Claudio Sette) have shown other binding partners for SAP155 that are well known regulators of alternative splicing. Current studies are focusing on these interactions, the role of the SAP155 phospho-state in this mechanism, and examining the heterozygous SAP155 knockout mouse for susceptibility to cancer.

Funding Source: The Veterans Administration (VA MERIT I)

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Project 2: The role of ceramide Kinase and ceramide-1-phosphate in eicsosanoid synthesis. (ongoing for  5 years)

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-1beta and TNFalpha) is type IVA cytosolic phospholipase A2 (cPLA2alpha) (1). Activation/translocation of cPLA2alpha in cells requires the association of cPLA2 alpha 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 cPLAalpha 2 also requires the generation of activating lipids is unknown.

An essential component for understanding cPLA2alpha activation is to identify and establish the bioactive lipids responsible for interacting with the CaLB domain and regulating the membrane association of cPLA2alpha. Ceramide-1-phosphate (C1P) is a new addition to bioactive sphingolipids generated by the phosphorylation of ceramide by ceramide kinase. C1P is one such potential lipid regulator of cPLA2alpha. 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 (C1P) (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 eicosanoids (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 cPLA2alpha and the inflammatory response mediated by AA and eicosanoids.

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 over 12 cell types with C1P (nanomolar concentrations) induced AA release and the synthesis of eicosanoids. Further exploration of this effect demonstrated that C1P 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 C1P induced activation/translocation of full-length cPLA2alpha as well as the truncated CaLB/C2 domain of cPLA2alpha. siRNA technology was employed to downregulate cPLA2alpha which demonstrated that the induction of AA release by C-1-P was strictly dependent on cPLA2alpha activation. These preliminary findings also disclose that C-1-P directly binds to cPLA2alpha in a Ca+2 enhanced manner via the CaLB/C2 domain, and C-1-P also increased the enzymatic activity of cPLA2alpha in vitro as well as increasing the affinity of cPLA2 for Ca+2 by approximately 10-fold. Furthermore, studies using pulse labeling and mass spectrometry demonstrate a marked increase in C1P concurrent with the release of AA and PGE2 in response to inflammatory cytokines. siRNA technology to downregulate ceramide kinase blocked cPLA2alpha activation, AA release and eicosanoid production in response to inflammatory cytokines, ATP, and A23187 calcium ionophore. Lastly, our results demonstrate that ceramide-1-phosphate is concurrent or downstream of calcium mobilization in the activation of cPLA2alpha.

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 eicosanoid synthesis through activation of cPLA2alpha in response to inflammatory agonists. To validate our hypothesis, we are currently answering the following basic questions: 1) How is ceramide-1-phosphate generated in response to inflammatory agonists? 2) How is ceramide kinase regulated by inflammatory agonists? 3) What is the interaction site for C1P in the C2 domain of cPLA2alpha? 4) Is the interaction of cPLA2alpha and C1P required for eicosanoid synthesis is response to agonists? 5) Are there any other enzymes regulated by C1P in the same manner as cPLA2alpha?

Highlights of current findings: This project has been steadily advancing over the past few years. Recently, we have determined the ceramide kinase utilizes ceramide provided by ceramide transport protein (CERT). We have localized the enzyme to the trans-Golgi Network (TGN) as well as early and late endosomes. Furthermore, activated cPLA2alpha co-localizes with ceramide kinase in cells. Our laboratory has also undertaken a comprehensive study of calcium-dependent and –independent mechanisms of CERK activation as they relate to inflammatory pathways.

Our understanding of the C1P/cPLA2alpha interaction has also progressed. We have now demonstrated that C1P enhances the calcium affinity for the enzyme as well as its membrane affinity. These findings strongly suggest that C1P is a “trigger” for cPLA2alpha translocation by lowering the dissociation of the enzyme from PC-rich membranes. Recently, we have also identified several critical amino acids for C1P interaction in the C2 domain of cPLA2alpha. We are currently defining whether this interaction site is required for cPLA2alpha translocation in response to various inflammatory agonists.

Funding Source: R01 award (1 R01 HL072925-01) from NIH specifically the National Heart, Lung, and Blood Institute.

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 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:  R01 award (1 R01 CA117950-02) from NIH specifically the National Cancer Institute.

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Thanks to our collaborators without whose help, these studies could not be done!

 1)      Dr. Alfred H. Merrill, Jr.  at Georgia Tech

Dr. Cameron Sullards

Samuel Kelly

Elaine Wang

Jeremy Allegood

2)      Dr. Sarah Spiegel at VCU

Dr. Shawn Payne

Dr. Mike Maceyka

3)      Dr. Yusuf Hannun at MUSC

Dr. Ben Pettus

Patrick Roddy

Dr. Alicja Bielawska

Dr. Zsdislaw Szulc

4)      Dr. Besim Ogretmen at MUSC

5)      Dr. Lina Obeid at MUSC

6)      Dr. Wonhwa Cho at Univ. of Illinois at Chicago

Dr. Rob Stahelin (currently moving into an independent position!)

7)      All current and past Chalfant Lab Members!!!