Multiple effects of curcumin on promoting expression of the exon 7-containing SMN2 transcript
© Springer-Verlag Berlin Heidelberg 2015
Received: 4 May 2015
Accepted: 14 August 2015
Published: 19 September 2015
Survival of motor neuron 2 (SMN2) is a modifier gene for spinal muscular atrophy (SMA), a neurodegenerative disease caused by insufficient SMN protein mostly due to SMN1 defect. SMN2 is nearly identical to SMN1 but unfortunately only able to produce a small amount of SMN protein due to exon 7 skipping. The exon 7-containing SMN2 transcript (SMN2_E7+) can be increased by a dietary compound, curcumin, but the involved molecular changes are not clear. Here we have found that in fibroblast cells of a SMA type II patient, curcumin enhanced the inclusion of SMN2 exon 7. Examination of the potential splicing factors showed that curcumin specifically increased the protein and transcript levels of SRSF1. The increased SRSF1 protein was mainly nuclear and hyperphosphorylated. Interestingly, the curcumin effects on the SMN2 and SRSF1 transcripts were inhibited by a protein deacetylase inhibitor, trichostatin A. Moreover, in support of its role in the SMN2 splicing, knocking down SRSF1 reduced the inclusion of SMN2 exon 7. Thus, curcumin appears to have multiple effects on the SMN2 transcript and its splicing regulators, including the change of alternative splicing and transcript/protein level as well as phosphorylation. Protein deacetylases and phosphatases are likely involved in these effects. Interestingly, the effects all seem to favor production of the SMN2_E7+ transcript in SMA patient cells.
Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder with an estimated incidence of 1 in 6000 live births, representing the primary genetic cause of infant mortality (Monani 2005; Burghes and Beattie 2009). The disease is characterized by degeneration of α-motor neurons in the anterior horn of the spinal cord and by consequent skeletal muscle atrophy (Monani 2005; Burghes and Beattie 2009). More than 96 % of SMA patients have insufficient amount of the survival of motor neuron (SMN) protein due to the homozygous deletion of the SMN1 gene (Coovert et al. 1997; Lefebvre et al. 1995). Interestingly, a paralogous human gene SMN2 encodes the same but only a small amount of the SMN protein (Lefebvre et al. 1995; Kashima et al. 2007), due to its various extents of exon 7 skipping in different cells/tissues (Burnett et al. 2009; Lorson et al. 1998). Of the many SMN-deficient tissues (Zhang et al. 2008), spinal cord is the most affected in terms of function and survival (Burghes and Beattie 2009; Chen et al. 2008).
A C6-to-T transition in exon 7 of the SMN2 gene is shown to contribute to the skipping, by disrupting the binding of a splicing activator SRSF1 (serine-/arginine-rich splicing factor 1) (Cartegni and Krainer 2002; Cartegni et al. 2006) and (or) promoting the binding of a splicing repressor hnRNP A1 (heterogeneous nuclear ribonucleoprotein A1) (Kashima et al. 2007; Kashima and Manley 2003), in splicing reporter assays. Another splicing repressor of the SMN2 exon 7 is SAM68 (Src-associated substrate in Mitosis of 68 kDa) (Pedrotti et al. 2010). There are likely more factors involved in the regulation, as suggested by the effects of anti-sense oligonucleotides targeting other regions of the SMN2 pre-mRNA (Singh et al. 2006; Hua et al. 2010).
Though the expression of SMN2 is not sufficient to compensate for the homozygous loss of SMN1 (Lefebvre et al. 1995), multiple copies of SMN2 increase SMN protein level and inversely correlate with disease severity in SMA patients and transgenic mice (Hsieh-Li et al. 2000; Monani et al. 2000; Wirth et al. 2006; Swoboda et al. 2005). Thus, as a modifier gene for SMA, the alternative splicing of SMN2 exon 7 provides a promising target for SMA therapy (Hua et al. 2010).
Curcumin is a dietary polyphenol compound enriched in the turmeric root. It has been used in clinical trials of numerous human diseases (Gupta et al. 2013; Darvesh et al. 2012), likely involving its regulation of multiple targets including histone acetyl-transferase (Shishodia 2013; Shishodia et al. 2007). It has also been reported to increase the SMN2_E7+ transcript and SMN protein in fibroblast cells from a patient with SMA type I (Sakla and Lorson 2008); however, the underlying molecular changes have been unclear. For the treatment of human diseases (Gupta et al. 2012), it is necessary to identify its potential targets and effects.
In this study, we report that curcumin increases expression of the SMN2_E7+ transcript and SMN protein in fibroblast cells from a SMA patient with multiple effects: enhancing SMN2 exon 7 inclusion, increasing transcript/protein level, and phosphorylation of the splicing activator SRSF1. These effects likely involve deacetylases and phosphatases.
Curcumin increases the SMN protein and the proportion of SMN2_E7+ transcript in fibroblast cells form a patient with SMA type II
To investigate potential curcumin effect on the expression of SMN protein and the usage of SMN2 exon 7, a SMA dermal fibroblast cell line (BJ301J) was established by using the skin biopsy of a patient (SMA type II, 6-month, male), who is deficient in both copies of the SMN1 gene but contains three copies of SMN2 (Figs. S1 and S2). In our initial tests, 15–25 μM of curcumin increased the SMN2_E7+ transcript, but only 25 μM was sufficient to upregulate the SMN protein level. We thus used 25 μM of curcumin on these cells in the following experiments.
Curcumin-induced increase in the SMN2_E7+ variant is inhibited by trichostatin A, a deacetylase inhibitor
SRSF1 is specifically increased at both protein and transcript levels by curcumin
Curcumin increases the hyperphosphorylated isoform of SRSF1 in the nucleus
The lower band of SRSF1 at about 32 kD is clearly visible in the presence of 4 mM of NaF in the protein lysates (Fig. 4a); however, it was not detectable when a higher concentration of NaF (10 mM) was used (Fig. 4b), suggesting that it is a less phosphorylated isoform. To explore the phosphorylation status of the two SRSF1 isoforms upon curcumin treatment, we immunoprecipitated (IP) the SRSF1 protein from the cytoplasmic fraction, followed by treatment with calf intestinal alkaline phosphatase (CIAP) (Fig. 4c). Upon CIAP treatment, the ~34-kD SRSF1 disappeared, compared to the untreated cell lysate or SRSF1 precipitate in western blot of the IP samples (lane 2 vs. lanes 1 and 3). Simultaneously, an extra much lower band appeared at about 30 kD in the same CIAP-treated sample (lane 2). Thus, it is likely that the ~34-kD SRSF1 increased by curcumin is a hyperphosphorylated isoform.
Curcumin-induced increase in SRSF1 is inhibited by trichostatin A
SRSF1 knockdown reduces the inclusion of SMN2 exon 7
Taken together, these data indicate that the dietary compound curcumin has multiple effects on SMN2 and its splicing regulators to promote the expression of the SMN2_E7+ transcripts in SMA fibroblast cells. These effects likely involve protein deacetylation and/or phosphorylation.
SMA patient fibroblast cells from a type I patient have been used to investigate the regulation of SMN2 pre-mRNA splicing by small compounds (Sakla and Lorson 2008; Dayangac-Erden et al. 2011). In this study, the BJ301J cells were derived from a patient (male, 9 months old) with SMA type II. They are homozygously deleted of the SMN1 but have three copies of the SMN2 gene (Fig. S1), with one more copy of SMN2 than the previously reported GM3813 cells (from a SMA type I, 3-year old male) (Sakla and Lorson 2008; Dayangac-Erden et al. 2011). In both types of cells, the fold increases in the percentages of SMN2_E7+ transcripts are similar (about 1.5-fold) upon 24-h treatment with 25 µM of curcumin. This similar change implies that the curcumin targets in promoting the production of full-length SMN2 transcript might be the same in different sources and types of SMA cells.
Curcumin has pleiotropic effects on a number of targets in cells (Shishodia 2013; Shishodia et al. 2007), particularly the histone acetyltransferase p300-/CREB-binding protein (p300/CBP) (Balasubramanyam et al. 2004). Of the different effects by curcumin, changes in splicing and transcript levels (Figs. 1, 2, 3) could be contributed by its inhibition of p300/CBP and protein/histone acetylation (Balasubramanyam et al. 2004). This is supported by the inhibition of these curcumin effects by the histone deacetylase (HDAC) inhibitor TSA (Figs. 2, 5). Particularly for SRSF1, we have shown that curcumin inhibits the histone acetylation/transcription of factors involved in the nonsense-mediated decay (NMD) pathway, consequently increasing the level of NMD-targeted SRSF1 variant transcripts in cells (Feng et al. 2015). The HDAC inhibitor may also increase the processivity of RNA Polymerase II and reduce co-transcriptional association of splicing regulators with certain alternative exons (Hnilicova et al. 2011), which may counteract the curcumin-enhanced exon 7 usage.
The curcumin effect on SMN2 splicing and SRSF1 phosphorylation (Figs. 1, 2, 4) may also involve the inhibition of phosphatases such as PP1 and PP2A. PP1 binds to the beta-4 sheet of the SRSF1 RNA recognition motif (RRM1) domain, thus dephosphorylating the splicing factor (Novoyatleva et al. 2008). PP1 inhibition promotes SMN2 exon 7 inclusion in fibroblast cells of SMA patients and spinal cord of SMA mice (Novoyatleva et al. 2008). Both PP1 and PP2A can be inhibited by okadaic acid (OA). PP2A activity can also be inhibited by curcumin (Han et al. 2012). However, in our experiments, OA pretreatment did not change the curcumin effect on exon 7 inclusion in cells (Fig. 2). A reasonable explanation is that curcumin and OA have overlapping effects on the phosphatases and thus the phosphorylation of the splicing factors for SMN2 exon 7 usage.
In the immunoprecipitation experiment (Fig. 4c), we were not able to efficiently immunoprecipitate the nuclear SRSF1 protein, likely due to occupation of its RRM1 domain by phosphatases (Novoyatleva et al. 2008), which is part of the N-terminal antigen targeted by the SRSF1 antibody.
In summary, we have observed multiple effects of curcumin on the expression of genes involved in the production of the full-length SMN2 protein. These effects on gene transcription/splicing and protein phosphorylation could be contributed by the inhibitory effect of curcumin on the histone acetyl-transferase p300/CBP and protein phosphatases. These two targets are likely important for the overall beneficial effects for producing the full-length SMN2 protein.
Materials and methods
Cell culture and treatment
Human fibroblast cells (BJ301J) were derived from a type II SMA patient (male, 9 months old) with three copies of the SMN2 gene and maintained in Dulbecco’s modified Eagle’s medium supplemented with 10 % fetal bovine serum, 2 mM glutamine, and 1 % penicillin–streptomycin solution (Invitrogen) at 37 °C in a humidified atmosphere containing 5 % CO2. Cells were treated with DMSO or 25 μM of curcumin (Sigma-Aldrich) for 0, 24, or 48 h. In the assay of inhibitors on transcript of SMN2 or SRSF1, cells were pretreated with each inhibitor for 2 h followed by 24-h addition of DMSO or 25 μM of curcumin.
Knockdown of splicing factors using lentiviral vector-mediated transduction
Lentiviral particles were prepared using the shRNA plasmids pGIPZ-shSRSF1 (Open Biosystems, RMM4431-99938975, 5′-TCG AGA TCG AGA TCT TCC A-3′), pGIPZ-shhnRNPA1 (5′-GTG TAA AGC ATT CCA ACG A-3′), and pGIPZ-scrambled (5′-TAG TGA AGC CAC AGA ATA T-3′) according to our previous procedures (Liu et al. 2012; Yu et al. 2009). Cells were treated with DMSO or curcumin 7 days after transduction. The silencing effects were confirmed by RT-PCR and immunoblotting.
Reverse transcription polymerase chain reaction
Cytoplasmic RNA was fractionated according to our previous procedure (Feng et al. 2015; Ma et al. 2007) and extracted using the GenElute Mammalian Total RNA Miniprep Kit (Sigma-Aldrich). One microgram of cytoplasmic RNA was included in a 10 µl reverse transcription reaction. PCR reactions were carried out for 26–30 cycles. The sequences (5′–3′) of the primer pairs were as follows, with the forward primer listed first followed by the reverse primer for each gene. SMN2: AAG ACT GGG ACC AGG AAA GC, TAT CTT CTA TAA CGC TTC ACA TTC CAG; SRSF1: CCT CCA GAC ATC CGA ACC AAG, TGC TAC GGC TTC TGC TAC GAC; hnRNP A1: GTC TAA GTC AGA GTC TCC TAA AGA GCC, TCT CAT TAC CAC ACA GTC CGT G; SAM68: GCT GAC GGC AGA AAT TGA GAA G, TTG ACA GGT ATC AGC ACT CGC TC; GAPDH: GTC AAC GGA TTT GGT CGT ATT G, AAC CAT GTA GTT GAG GTC AAT GAA G.
PCR products were resolved in 2–3 % agarose gels containing 0.5 μg/ml ethidium bromide. The gels presented in figures are inverted digital images. The abundance of the SMN2_E7+ splice variants is expressed as molar percentages relative to the total of the SMN2 variants (E7+ and E7−).
Cells were rinsed three times with ice-cold PBS, harvested using cell scrapers, pelleted by centrifugation at 14,000 rpm for 30 s at 4 °C, and lysed in RIPA buffer (containing 2 mM PMSF, 2 mM Na3VO4 and 10 mM NaF) (Feng et al. 2015). Protein was quantified using the Bradford method, and samples were run on 10 or 12 % Tris–glycine acrylamide gels and then transferred to polyvinylidene fluoride membranes. The membranes were blocked in 5 % dry milk and probed with the following mouse antibodies, which were all purchased from Santa-Cruz Biotechnology unless otherwise indicated: anti-SMN (H-7, 1:400), anti-SRSF1 (clone 96, 1:500), anti-hnRNP A1 (9H10, 1:1000), anti-SAM68 (7-1, 1:250), anti-nucleolin (H-6, 1:1000), anti-β-actin (C4, 1:1000), and anti-GAPDH (Sigma, 1G5, 1:2000). After incubation with peroxidase-conjugated goat anti-mouse immunoglobulin M or G secondary antibodies (Sigma-Aldrich, 1:2000), proteins were visualized using enhanced chemiluminescence (GE Healthcare). Densitometry of the resulting bands was analyzed by Image J (developed by the U.S. National Institutes of Health and available at http://rsb.info.nih.gov/ij/).
BJ301J fibroblast cells were plated over the slides in six-well plates, treated with DMSO or curcumin for 24 h, rinsed twice in ice-cold phosphate-buffered saline (PBS) with 1 % BSA, fixed with 4 % paraformaldehyde (PFA) for 15 min and then permeabilized with 0.2 % Triton X-100 for 10 min at room temperature. The fixed cells were incubated overnight at 4 °C with mouse monoclonal antibodies anti-SRSF1, anti-hnRNP A1, and anti-Sam68, respectively. The primary antibodies were diluted at 1:100 in TBS (20 mM TrisCl, 500 mM NaCl) containing 1 % BSA. Cells were rinsed twice with TBS and incubated with goat anti-mouse fluorescent secondary antibody (conjugated with FITC, 1:1000) in the dark for 1 h at room temperature. Cell nuclei were counterstained with DAPI (1:6000). The stained cells were mounted with mounting media (Sigma-Aldrich). Images were taken at 100× magnification with an Olympus microscope.
Fractionation of nuclear and cytoplasmic proteins
BJ301J fibroblast cells were rinsed with ice-cold PBS three times in the dishes then harvested in 1 ml of ice-cold PBS using rubber scrapers. Cell pellets were collected into 1.5-ml tubes by centrifuging at 14,000 rpm for 30 s and then resuspended in ice-cold NP-40 buffer supplemented with 2 mM PMSF, 2 mM Na3VO4 and 10 mM NaF. After centrifugation at 14,000 rpm for 2 min, the supernatant was used as cytoplasmic fraction by additional centrifugation at 14,000 rpm through 24 % (w/v) sucrose cushion, the nuclear pellets were washed twice using 1 ml of ice-cold NP-40 buffer followed by resuspension in RIPA buffer supplemented with 2 mM PMSF, 2 mM Na3VO4 and 10 mM NaF, then used as nuclear fraction after sonication.
Immunoprecipitaion and phosphatase assay
BJ301J fibroblast cells were rinsed with cold PBS for three times. The cytoplasmic and nuclear lysates were prepared according to the protocol described in the section of “Cytoplasm and nuclei fractionation.” Protein G beads were washed with cold PBS for five times, then packed with 2 μg of anti-SRSF1 antibody and incubated at 4 °C under rotary agitation for 4 h. After washing with cold PBS, the packed beads were incubated overnight with lysates at 4 °C. When the incubation time was over, the supernatant was removed by centrifugation, and the beads were washed in lysis buffer three times for further analysis.
The precipitations were suspended in 1× reaction buffer with 10 units of CIAP and incubated at 37 °C for 60 min. After that, the suspensions were mixed with 6× SDS loading buffer, heated at 95 °C for 5 min to denature the proteins, and separated them from the protein G beads, and the supernatants were used for western blotting analysis.
Data were analyzed by two-tailed Student’s t test. A p value <0.05 was considered significant.
We sincerely thank the SMA patient and his family for donating the skin biopsy sample, Ms. Weimin Zhang for help establishing the primary culture of BJ301J, Dr. Guodong Liu for providing the shhnRNP A1 plasmid, Drs. Aleh Razanau and Wenguang Cao for helpful discussions, and Lei Lei and Nan Liang for proofreading the manuscript. This work was supported by a Manitoba Research Chair Fund and in part by a Canadian Institutes of Health Research (CIHR) Operating Grant FRN_106608 to JX, by FRN_2006BIA05A07 and 2006BIA05A08 from the National Key Technology R&D Program of China to SH.
Compliance with ethical standards
Conflict of interest
All of the authors declare no conflict of interests.
All procedures performed in this study involving the SMA patient were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 Declaration of Helsinki and its later amendments or comparable ethical standards. Informed consent was obtained from the SMA patient included in the study.
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