Alternative splicing is an important mechanism to generate protein diversity, and regulates lots of cellular processes, such as apoptosis(Merdzhanova, Edmond et al. 2008). A large number of proteins that are involved in apoptosis are regulated by alternative splicing. Apoptosis protease activating factor 1(Apaf-1) and Caspase-9, which are factors involved in apoptosis, both have transcript variants generated by alternative splicing. Apaf-1 is an adaptor protein that can bind cytochrome C to activate caspase in mitochondria apoptosis pathway. One splice isoform, which includes exon 18, can activate procaspase-9. While the one that excludes exon 18, lose the ability to activate procaspase-9. Caspase-9, which is an initiator caspase, has two transcript variants by inclusion or exclusion of exon3 to exon6. Caspase-9a, which includes the four exons is pro-apoptotic. Caspase-9b, which excludes exon 3 to exon 6, is anti-apoptotic.
Digoxin is a plant derived compound that is used to treat congestive heart disease. Genestein is a plant-derived isoflavones. Studies have demonstrated that digoxin and genistein can modulate alternative splicing(Anderson, Liu et al. 2012). The goal of this study is to test whether digoxin and genistein treatment can modulate alternative splicing of Apaf-1 and Caspase-9. RT-PCR, qRT-PCR and western-blot shows that treatment of cells with digoxin can down-regulates the level of exon-18 containing transcript of Apaf-1 and exon 3 to exon 6-containing transcript of caspase-9.
Materials and Methods
Cell culture and treatment
HEK 293 cells, A549 cells and Hela cells were purchased from the ATCC. Cells were treated with 100ng/ml digoxin and 50ug/ml genistein for 48h.
RT-PCR analysis of alternative splice variants
Total cellular RNA is extracted using RNeasy Plus Mini Kits (Qiagen) according to the manufacturer's directions. 20ng of total RNA was used as template to in 20 μl RT-PCR reactions using OneStep RT-PCR Kits (Qiagen). Primers that can pick up both splice isoform were used (Benedict, Hu et al. 2000; Merdzhanova, Edmond et al. 2008). One-step RT-PCR was carried out as follows: one cycle of 50 °C x 30 min and 95 °C x 15 min, followed by 50 cycles of 94 °C x 30 s, 55°C or 61°C x 30 s, and 72 °C x 30 s, and then a final extension of 72 °C x 7min.
Real-time PCR analysis of alternative splice variants
Quantitect SYBR Green RT-PCR Kits (Qiagen) were used for real-time RT-PCR (qRT-PCR) analysis. ABI PRISM 7000 and 7500 Sequence Detection Systems (Applied Biosystems) was used for real-time RT-PCR. Results were then expressed as relative level of transcript in treated cells compared with untreated cells.
Western-blot analysis of alternative splice variants
Cells treated as described were washed twice with PBS and lysed in NuPAGE LDS sample buffer (Invitrogen). Equal amounts of proteins samples were analyzed on 10% NuPAGE Bis-Tris gel (Invitrogen). Proteins were then transferred onto nitrocellulose (Bio-Rad) at 100V for 1h. Blots were probed with anti-caspase-9 monoclonal primary antibody (Cell Signaling Technology, Inc).
Treatment with Digoxin modulates alternative splicing of Apaf-1
Inclusion and exclusion of exon18 in Apaf-1 can generate two transcript isoforms. RT-PCR result shows that digoxin decreases the exon 18-containing transcript in all the cell lines we tested. (Fig. 1). Real-time PCR was performed with primers that are specific for exon 18-containing transcript of Apaf-1. Real-time PCR shows that in digoxin treated HEK293 cells, relative level of exon 18-containing transcript mRNA decreases (Fig. 2).
Treatment with Digoxin modulates alternative splicing of Caspase-9
Alternative splicing can create two splice variants from caspase-9 gene by inclusion or exclusion of exon3 to exon 6 cassette. RT-PCR result shows that digoxin decreases the amount of Caspase-9a mRNA in all the cell lines tested (Fig.3). Real-time PCR with primers that are specific for Caspase-9a shows that caspase-9a transcript level decreases in digoxin treated cells (Fig. 4). Western-blot performed with anti-caspase-9 antibody shows that protein level of Caspase-9a decreases in cells treated with digoxin (Fig. 5).
By regulating alternative splicing of Apaf-1 and Caspase-9, digoxin increases the mRNA level of anti-apoptotic Apaf-1 and Caspase-9, which suggests a potential role of digoin to regulate apoptosis. However, some studies have shown that digoxin can trigger prostate cancer cell death by Cdk5/p25 dependent pathway. Ca2+ also plays important role in cytotoxic effect of digoxin (Lin, Juang et al. 2004). So, digoxin might have dual effects in regulating apoptosis through different pathways. Studies have shown that digoxin can regulate the expression level of one of the SR proteins, which plays important role in regulating alternative splicing (Data not published). This might be the mechanism that digoxin used to enhance the skipping of exons of Apaf-1 and Caspase-9.
Alternative splicing plays important role in regulating apoptosis, which is an important cellular process during embryotic development and homeostasis. We highlight the potential role of digoxin in regulation of apoptosis by modulating alternative splicing in this study.
Both RT-PCR and qRT-PCR show that digoxin decreases the inclusion of exon 18 of Apaf-1 in three cell lines tested. RT-PCR, qRT-PCR and Western-blot demonstrate that treatment of cells with digoxin decreases inclusion of exon 3 to exon 6 of Caspase-9.
Benedict, M. A., Y. Hu, et al. (2000). "Expression and functional analysis of Apaf-1 isoforms. Extra Wd-40 repeat is required for cytochrome c binding and regulated activation of procaspase-9." J Biol Chem 275(12): 8461-8468.
Merdzhanova, G., V. Edmond, et al. (2008). "E2F1 controls alternative splicing pattern of genes involved in apoptosis through upregulation of the splicing factor SC35." Cell Death Differ 15(12): 1815-1823.