- Email: [email protected]
Spliceostatin A treatment inhibits mitotic clonal expansion and adipogenesis
Biochemical and Biophysical Research Communications 514 (2019) 848e852
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Spliceostatin A treatment inhibits mitotic clonal expansion and adipogenesis Daisuke Kaida Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 April 2019 Accepted 27 April 2019 Available online 10 May 2019
Adipogenesis is a differentiation process from mesenchymal stem cells to adipocytes. It has been reported that adipogenesis is regulated by a highly orchestrated transcriptional cascade. However, the effects of modulation of mRNA splicing on adipogenesis remain unknown. To investigate these effects, 3T3-L1 preadipocyte were treated with the potent splicing inhibitor spliceostatin A, which revealed that splicing inhibition suppressed adipogenesis. In addition, treatment of 3T3-L1 cells with spliceostatin A during the early phase of adipogenesis was sufficient to inhibit adipogenesis. In the early phase of adipogenesis, the cells re-entered the cell cycle, which is referred to as mitotic clonal expansion. As mitotic clonal expansion is required for adipogenesis, it was assumed that splicing inhibition would suppress mitotic clonal expansion, and consequently inhibit adipogenesis. As expected, spliceostatin A treatment caused G1 phase arrest and inhibited cell proliferation, i.e., inhibition of mitotic clonal expansion. These results suggest that splicing activity is required for mitotic clonal expansion and adipogenesis. © 2019 Elsevier Inc. All rights reserved.
Keywords: Splicing Spliceostatin A Adipogenesis Mitotic clonal expansion
1. Introduction Obesity is one of the most serious health problems in developed and developing countries because it causes obesity-related diseases including metabolic syndrome, high blood pressure, type 2 diabetes and cardiovascular diseases [1]. Obesity is characterized by an excessive accumulation of body fat, which is caused by an increase in the size or number of adipocytes, or both [2]. Therefore, the molecular mechanisms of adipogenesis, which are associated with the increase in the number of adipocytes, have been extensively studied to understand the nature of obesity and to identify novel therapeutic targets for obesity. In these studies, mostly 3T3-L1 preadipocytes have been used, as they are easy to culture and to differentiate into adipocytes [2,3]. Treatment of post-confluent, growth arrested 3T3-L1 cells with adipogenesis inducers initiates the differentiation process. One of the earliest steps in the differentiation process is resuming cell cycle progression, which is called mitotic clonal expansion (MCE) [4]. MCE is followed by the expression of adipogenesis-related genes, including peroxisome
Abbreviations: SSA, spliceostatin A; MCE, mitotic clonal expansion; CDK, cyclin dependent kinase. E-mail address: [email protected]. https://doi.org/10.1016/j.bbrc.2019.04.180 0006-291X/© 2019 Elsevier Inc. All rights reserved.
proliferator-activated receptor gamma and CCAAT enhancer binding protein family proteins. MCE is a prerequisite for adipogenesis, because cell cycle arrest by treatment with a cyclin-dependent kinase inhibitor suppressed the expression of adipogenesis-related genes and adipogenesis [5]. Adipogenesis is regulated by a highly orchestrated transcriptional cascade, hence proper transcriptional regulation is required [3]. However, the effects of modulation of mRNA splicing, which is necessary for proper gene expression, on adipogenesis remain unknown. To investigate the effects of splicing modulation, potent splicing modulators are useful. Spliceostatin A (SSA) is a potent splicing inhibitor [6]. In this study, I investigated the effects of splicing modulation on adipogenesis using SSA. 2. Materials and methods 2.1. Cell lines and reagents NIH 3T3-L1 cells were cultured in Dulbecco's modified Eagle's medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) containing 10% heat-inactivated fetal bovine serum (Life Technologies, Eugene, OR, USA) at 37 C with 5% CO2. Adipocyte differentiation was performed using the AdipoInducer Reagent (Takara bio, Shiga, Japan). SSA was a kind gift from Dr Minoru Yoshida (RIKEN,
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Fig. 1. Splicing inhibition suppresses adipogenesis. (A) Scheme of the experiment design. Three days after seeding, the post-confluent 3T3-L1 cells were cultured in adipogenesis induction medium for 2 days. The medium was then changed to the maintenance medium and cultured for 4 days. The maintenance medium was changed to fresh maintenance medium every 2 days. (B) 3T3-L1 cells were treated with the indicated concentrations of SSA for 6 days and stained with Oil Red O. (C) 3T3-L1 cells were treated with the indicated concentrations of SSA for the indicated time periods and stained with Oil Red O. (D) 3T3-L1 cells were treated with the indicated concentrations of SSA for the indicated time periods. The amounts of triglycerides in the cells were measured. Error bars indicate SD (n ¼ 3). Statistical significance was investigated by one-way ANOVA with Tukey's multiple comparisons test (*, P < 0.05; **, P < 0.01).
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Saitama, Japan). 2.2. Oil Red O staining and triglyceride measurement Cells were washed twice with PBS, and then treated with 10% formaldehyde for 10 min at room temperature. Next, the cells were washed once with PBS and treated with 60% 2-propanol for 1 min at room temperature, and then stained with Oil Red O solution (60% 2propanol, 3 mg/mL Oil Red O) for 10 min at room temperature. The cells were then washed once with 60% 2-propanol and twice with PBS. Triglyceride measurement was performed using the LabAssay Triglyceride kit (FUJIFILM Wako Pure Chemical Corporation). 2.3. Cell viability assay After trypsinization, the number of viable cells and dead cells was counted using the trypan blue exclusion assay. Cells were counted manually with a hemocytometer. The cell number was determined by averaging the number of cells in eight squares, multiplying by the dilution factor and adjusting for cell volume to determine cells/well. 2.4. Cell cycle analysis Cells were fixed in 70% ethanol, rinsed with PBS and stained with a solution containing 20 mg/mL propidium iodide (Life Technologies), 0.05% Triton X-100 and 0.1 mg/mL RNase A (Life Technologies). Cell cycle progression was monitored by the image-based cytometer Tali (Life Technologies). 2.5. Western blot analysis
were stained with Oil Red O (Fig. 1B). After 6 days of culture, the cells showed Oil Red O staining, suggesting that they differentiated to adipocytes (Fig. 1B, Mock). After treatment with MeOH, the vehicle of SSA, or treatment with low concentrations of SSA, the cells were stained with Oil Red O, suggesting that MeOH or low SSA concentrations did not affect adipogenesis (Fig. 1B). However, after treatment with 1 or 1.5 ng/mL SSA, the cells showed significantly less staining than those treated with MeOH or low SSA concentrations, suggesting that these SSA concentrations inhibited adipogenesis (Fig. 1B). As described above, in the adipogenesis induction procedure, the 3T3-L1 cells were treated with induction medium in the early phase (Days 0e2), and then treated with maintenance medium in the late phase (Days 2e6). Next, I investigated in which phase SSA suppresses adipogenesis more efficiently. To this end, 3T3-L1 cells were treated with various SSA concentrations in the early (Fig. 1C, Days 0e2), late phase (Fig. 1C, Days 2e6) or both (Fig. 1C Days 0e6). Treatment with 1.5 ng/mL SSA in both phases inhibited adipogenesis, suggesting that splicing activity is required for both phases. Interestingly, 1 ng/mL SSA treatment in the early phase inhibited adipogenesis as effectively as the 6 days treatment, whereas 1 ng/ mL SSA treatment in the late phase was less effective (Fig. 1C). In addition to the Oil Red O staining, I measured the amounts of stored triglycerides in the differentiated adipocytes. Consistent with the Oil Red O staining, 1 ng/mL SSA treatment in the early phase (Days 0e2) inhibited triglyceride accumulation as effectively as the 6 days treatment (Days 0e6; Fig. 1D). In contrast, treatment in the late phase (Days 2e6) was less effective than treatment in the early phase (Fig. 1D). These results suggest that splicing activity is more important for adipogenesis in the early phase than in the late phase.
Cells were directly lysed on plates with 1 SDS-PAGE sample buffer. Proteins were separated by SDS-PAGE. After electrophoresis, proteins were transferred onto a PVDF membrane by electroblotting. Following the incubation of the membrane with primary and secondary antibodies using standard techniques, protein bands were detected using the NOVEX ECL Chemiluminescent Substrate Reagent Kit (Life Technologies) on an ImageQuant LAS 4000mini (GE Healthcare, Little Chalfont, UK). Anti-a-tubulin antibody (B-51-2) was purchased from Sigma-Aldrich (St. Louis, MO, USA). AntiCyclin B1 antibody was purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-p27 (C-19) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 2.6. Statistical analysis Statistical analysis was performed using R commander. Oneway ANOVA with Dunnett's multiple comparisons test or Tukey's multiple comparisons test was used to determine statistical significance. Data are presented as mean ± SD. The sample size (n) in each experiment is mentioned in the figure legends. P < 0.05 was considered statistically significant. 3. Results 3.1. Splicing inhibition suppresses adipogenesis To investigate the effects of splicing inhibition on adipogenesis, 3T3-L1 cells were cultured in an induction medium containing adipogenesis inducers (insulin, dexamethasone, 3-isobutyl-1methylxanthine) for 2 days (Days 0e2), and then cultured in maintenance medium containing insulin for 4 additional days (Day 2e6) with or without the potent splicing inhibitor, SSA (Fig. 1A). To determine whether the cells differentiated to adipocytes, the cells
Fig. 2. Splicing inhibition suppresses cell proliferation. (A, B) Post-confluent 3T3-L1 cells were cultured in the induction medium for 2 days with the indicated concentrations of SSA. Cell number and cell viability were determined. Error bars indicate SD (n ¼ 3). Statistical significance was investigated by one-way ANOVA with Dunnett's multiple comparisons test (*, P < 0.05; **, P < 0.01).
D. Kaida / Biochemical and Biophysical Research Communications 514 (2019) 848e852
3.2. Splicing inhibition suppresses mitotic clonal expansion SSA treatment in the early phase of differentiation inhibited adipogenesis as effectively as treatment during the entire period. In the early phase of adipogenesis, MCE occurs as one of the earliest events of adipogenesis upon treatment with adipogenesis inducers [4]. Because MCE is a prerequisite for adipogenesis, I assumed that SSA treatment inhibits MCE, resulting in inhibition of adipogenesis. To investigate this hypothesis, 3T3-L1 cells were treated with various concentrations of SSA during the early phase of differentiation, and the cell proliferation rate was determined, because if MCE occurs the cell number should increase. As expected, the cell number did not increase upon 1 or 1.5 ng/mL SSA treatment (Fig. 2A), supporting the idea that SSA treatment inhibits MCE. However, it is also possible that SSA causes cell death, consequently the cell number would not increase. To rule out this possibility, the cell viability after SSA treatment was examined. After 48 h of treatment with 1.5 ng/mL SSA, more than 90% of the cells were still viable (Fig. 2B), suggesting that SSA treatment causes cell cycle arrest, but not cell death. It has previously been reported that SSA treatment causes cell cycle arrest at the G1 and G2/M phases [6,7]. To investigate at which phase the 3T3 cells arrest upon SSA treatment, the DNA contents of the SSA-treated cells was measured using a cytometer. More than 40% of the MeOH-treated cells were in the G2/M phase at 24 h after induction (Fig. 3A), suggesting that the cells resumed cell cycle progression. In contrast, the population of SSA-treated cells in the G2/M phase did not change after SSA treatment (Fig. 3B). These results suggest that SSA treatment causes cell cycle arrest at the G1
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phase. In addition to the cell cycle analysis using a cytometer, the expression level of cell cycle-related factors was examined. Cyclin B1 protein, a G2/M phase marker, was observed at 24 h in the MeOH-treated cells, suggesting that at least a portion of the cells was in the G2/M phase (Fig. 3C). This result is consistent with the result above (Fig. 3A). However, cyclin B1 was not observed in SSAtreated cells (Fig. 3C). This result also supports the idea that SSAtreated cells were arrested at the G1 phase. Furthermore, the protein levels of p21 and p27, which are CDK inhibitors, were examined as these proteins inhibit cell cycle progression at the G1 phase and they have been reported to be upregulated in SSA-treated HeLa cells [6,8,9]. In the control cells, the p27 protein level decreased at 12 and 24 h, suggesting that the cells exited the G1 phase at these time points (Fig. 3C). However, in the SSA-treated cells, there was no accumulation of p27, in fact a decrease in p27 expression was observed compared with the control (Fig. 3C). The protein level of p21 decreased at 24 h in the control cells, suggesting that a portion of these cells were in S phase, because the p21 protein level decreases during S phase [10]. In contrast, SSA treatment upregulated the p21 protein level, which is consistent with a previous report [6]. Taken together, these results suggest that the control cells exited the G1 phase and re-entered cell cycle, whereas SSA-treated cells were arrested at the G1 phase. 4. Discussion Splicing is one of the most vital molecular mechanisms for proper gene expression in eukaryotes. As more than 97% and 80% of
Fig. 3. Splicing inhibition suppresses mitotic clonal expansion. (A, B, C) Post-confluent 3T3-L1 cells were cultured in the induction medium for 2 days with the indicated concentrations of SSA. Cells were harvested at the indicated time points and DNA contents were measured (A, B). The protein levels of Cyclin B1, p21, p27 and a-tubulin were analyzed (C). a-Tubulin was used as an internal control.
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genes contain introns in humans and mice, respectively [11,12], treatment with splicing inhibitors causes numerous effects on cellular functions [13]. One of the most apparent effects is cell cycle arrest [6,14]. Upon treatment with adipogenesis inducers, postconfluent 3T3-L1 cells resume cell cycle progression, i.e., MCE. MCE is one of the earliest events in adipogenesis and a prerequisite for adipogenesis [5]. In this report, I found that SSA treatment inhibited MCE, therefore splicing inhibition may suppress adipogenesis through MCE inhibition. Although splicing inhibition can cause cell cycle arrest at both the G1 and G2/M phases [6,14], 3T3-L1 cells seemed to be arrested only at the G1 phase after SSA treatment. One of the reasons for the G1 phase arrest in SSA-treated HeLa cells is overexpression of p27 [6,15]. However, SSA treatment of 3T3-L1 cells did not cause p27 overexpression, suggesting it was not the reason for the G1 phase arrest and the suppressed adipogenesis. In addition, it has recently been reported that SIRT6-deficient 3T3-L1 cells arrested at the S and M phases through stabilization of p27, suggesting that p27 overexpression causes S or M phase arrest rather than G1 phase arrest in 3T3-L1 cells [16]. These findings support the idea that p27 overexpression was not the reason for the G1 phase arrest of 3T3-L1 cells. In SSA-treated 3T3-L1 cells, another CDK inhibitor, p21, was overexpressed [6]. Because p21 inhibits cell cycle progression at the G1 and S phases [8], p21 overexpression may be the reason for the G1 phase arrest. However, it is also possible that downregulation of G1 cyclins, such as cyclin D and E, was the reason for the G1 phase arrest caused by SSA treatment. The detailed molecular mechanism of the G1 phase arrest of 3T3-L1 cells will be explored in a future study. A variety of small compounds have been reported as antiobesity drugs. Some of them have been approved by the Food and Drug Administration and/or the European Medicines Agency. However, most of them have been withdrawn from the market because of serious side effects, and very few are still used as antiobesity drugs [1]. As obesity is a serious health risk, more effective anti-obesity drugs with fewer side effects are highly soughtafter. The mRNA splicing mechanism has not been reported as a target of anti-obesity drugs, therefore this report will pave the way for the development of an anti-obesity drug with a novel therapeutic target, and will improve our understanding of the nature of adipogenesis. Conflicts of interest The author declares no competing interests. Acknowledgements Spliceostatin A was a kind gift from Dr M Yoshida (RIKEN, Japan).
This work was supported in part by the JSPS KAKENHI (24659129 and 26670134).
Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.04.180.
References [1] R.J. Rodgers, M.H. Tschop, J.P. Wilding, Anti-obesity drugs: past, present and future, Dis. Model Mech. 5 (2012) 621e626. [2] F.J. Ruiz-Ojeda, A.I. Ruperez, C. Gomez-Llorente, A. Gil, C.M. Aguilera, Cell models and their application for studying adipogenic differentiation in relation to obesity: a review, Int. J. Mol. Sci. 17 (2016). [3] W. Kuri-Harcuch, C. Velez-delValle, A. Vazquez-Sandoval, C. HernandezMosqueira, V. Fernandez-Sanchez, A cellular perspective of adipogenesis transcriptional regulation, J. Cell. Physiol. 234 (2019) 1111e1129. [4] Q.Q. Tang, M.D. Lane, Adipogenesis: from stem cell to adipocyte, Annu. Rev. Biochem. 81 (2012) 715e736. [5] Q.Q. Tang, T.C. Otto, M.D. Lane, Mitotic clonal expansion: a synchronous process required for adipogenesis, Proc. Natl. Acad. Sci. U. S. A. 100 (2003) 44e49. [6] D. Kaida, H. Motoyoshi, E. Tashiro, T. Nojima, M. Hagiwara, K. Ishigami, H. Watanabe, T. Kitahara, T. Yoshida, H. Nakajima, T. Tani, S. Horinouchi, M. Yoshida, Spliceostatin A targets SF3b and inhibits both splicing and nuclear retention of pre-mRNA, Nat. Chem. Biol. 3 (2007) 576e583. [7] H. Nakajima, Y. Hori, H. Terano, M. Okuhara, T. Manda, S. Matsumoto, K. Shimomura, New antitumor substances, FR901463, FR901464 and FR901465. II. Activities against experimental tumors in mice and mechanism of action, J. Antibiot. (Tokyo) 49 (1996) 1204e1211. [8] W.S. el-Deiry, J.W. Harper, P.M. O'Connor, V.E. Velculescu, C.E. Canman, J. Jackman, J.A. Pietenpol, M. Burrell, D.E. Hill, Y. Wang, et al., WAF1/CIP1 is induced in p53-mediated G1 arrest and apoptosis, Cancer Res. 54 (1994) 1169e1174. [9] C.J. Sherr, J.M. Roberts, CDK inhibitors: positive and negative regulators of G1phase progression, Genes Dev. 13 (1999) 1501e1512. [10] A.R. Barr, S. Cooper, F.S. Heldt, F. Butera, H. Stoy, J. Mansfeld, B. Novak, C. Bakal, DNA damage during S-phase mediates the proliferation-quiescence decision in the subsequent G1 via p21 expression, Nat. Commun. 8 (2017) 14728. [11] E.A. Grzybowska, Human intronless genes: functional groups, associated diseases, evolution, and mRNA processing in absence of splicing, Biochem. Biophys. Res. Commun. 424 (2012) 1e6. [12] K.R. Sakharkar, M.K. Sakharkar, C.T. Culiat, V.T. Chow, S. Pervaiz, Functional and evolutionary analyses on expressed intronless genes in the mouse genome, FEBS Lett. 580 (2006) 1472e1478. [13] K.A. Effenberger, V.K. Urabe, M.S. Jurica, Modulating splicing with small molecular inhibitors of the spliceosome, Wiley Interdiscip. Rev. RNA 8 (2017). [14] Y. Mizui, T. Sakai, M. Iwata, T. Uenaka, K. Okamoto, H. Shimizu, T. Yamori, K. Yoshimatsu, M. Asada, Pladienolides, new substances from culture of Streptomyces platensis Mer-11107. III. In vitro and in vivo antitumor activities, J. Antibiot. (Tokyo) 57 (2004) 188e196. [15] T. Satoh, D. Kaida, Upregulation of p27 cyclin-dependent kinase inhibitor and a C-terminus truncated form of p27 contributes to G1 phase arrest, Sci. Rep. 6 (2016) 27829. [16] Q. Chen, W. Hao, C. Xiao, R. Wang, X. Xu, H. Lu, W. Chen, C.X. Deng, SIRT6 is essential for adipocyte differentiation by regulating mitotic clonal expansion, Cell Rep. 18 (2017) 3155e3166.
Contents lists available at ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Spliceostatin A treatment inhibits mitotic clonal expansion and adipogenesis Daisuke Kaida Graduate School of Medicine and Pharmaceutical Sciences, University of Toyama, 2630 Sugitani, Toyama, 930-0194, Japan
a r t i c l e i n f o
a b s t r a c t
Article history: Received 11 April 2019 Accepted 27 April 2019 Available online 10 May 2019
Adipogenesis is a differentiation process from mesenchymal stem cells to adipocytes. It has been reported that adipogenesis is regulated by a highly orchestrated transcriptional cascade. However, the effects of modulation of mRNA splicing on adipogenesis remain unknown. To investigate these effects, 3T3-L1 preadipocyte were treated with the potent splicing inhibitor spliceostatin A, which revealed that splicing inhibition suppressed adipogenesis. In addition, treatment of 3T3-L1 cells with spliceostatin A during the early phase of adipogenesis was sufficient to inhibit adipogenesis. In the early phase of adipogenesis, the cells re-entered the cell cycle, which is referred to as mitotic clonal expansion. As mitotic clonal expansion is required for adipogenesis, it was assumed that splicing inhibition would suppress mitotic clonal expansion, and consequently inhibit adipogenesis. As expected, spliceostatin A treatment caused G1 phase arrest and inhibited cell proliferation, i.e., inhibition of mitotic clonal expansion. These results suggest that splicing activity is required for mitotic clonal expansion and adipogenesis. © 2019 Elsevier Inc. All rights reserved.
Keywords: Splicing Spliceostatin A Adipogenesis Mitotic clonal expansion
1. Introduction Obesity is one of the most serious health problems in developed and developing countries because it causes obesity-related diseases including metabolic syndrome, high blood pressure, type 2 diabetes and cardiovascular diseases [1]. Obesity is characterized by an excessive accumulation of body fat, which is caused by an increase in the size or number of adipocytes, or both [2]. Therefore, the molecular mechanisms of adipogenesis, which are associated with the increase in the number of adipocytes, have been extensively studied to understand the nature of obesity and to identify novel therapeutic targets for obesity. In these studies, mostly 3T3-L1 preadipocytes have been used, as they are easy to culture and to differentiate into adipocytes [2,3]. Treatment of post-confluent, growth arrested 3T3-L1 cells with adipogenesis inducers initiates the differentiation process. One of the earliest steps in the differentiation process is resuming cell cycle progression, which is called mitotic clonal expansion (MCE) [4]. MCE is followed by the expression of adipogenesis-related genes, including peroxisome
Abbreviations: SSA, spliceostatin A; MCE, mitotic clonal expansion; CDK, cyclin dependent kinase. E-mail address: [email protected]. https://doi.org/10.1016/j.bbrc.2019.04.180 0006-291X/© 2019 Elsevier Inc. All rights reserved.
proliferator-activated receptor gamma and CCAAT enhancer binding protein family proteins. MCE is a prerequisite for adipogenesis, because cell cycle arrest by treatment with a cyclin-dependent kinase inhibitor suppressed the expression of adipogenesis-related genes and adipogenesis [5]. Adipogenesis is regulated by a highly orchestrated transcriptional cascade, hence proper transcriptional regulation is required [3]. However, the effects of modulation of mRNA splicing, which is necessary for proper gene expression, on adipogenesis remain unknown. To investigate the effects of splicing modulation, potent splicing modulators are useful. Spliceostatin A (SSA) is a potent splicing inhibitor [6]. In this study, I investigated the effects of splicing modulation on adipogenesis using SSA. 2. Materials and methods 2.1. Cell lines and reagents NIH 3T3-L1 cells were cultured in Dulbecco's modified Eagle's medium (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan) containing 10% heat-inactivated fetal bovine serum (Life Technologies, Eugene, OR, USA) at 37 C with 5% CO2. Adipocyte differentiation was performed using the AdipoInducer Reagent (Takara bio, Shiga, Japan). SSA was a kind gift from Dr Minoru Yoshida (RIKEN,
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Fig. 1. Splicing inhibition suppresses adipogenesis. (A) Scheme of the experiment design. Three days after seeding, the post-confluent 3T3-L1 cells were cultured in adipogenesis induction medium for 2 days. The medium was then changed to the maintenance medium and cultured for 4 days. The maintenance medium was changed to fresh maintenance medium every 2 days. (B) 3T3-L1 cells were treated with the indicated concentrations of SSA for 6 days and stained with Oil Red O. (C) 3T3-L1 cells were treated with the indicated concentrations of SSA for the indicated time periods and stained with Oil Red O. (D) 3T3-L1 cells were treated with the indicated concentrations of SSA for the indicated time periods. The amounts of triglycerides in the cells were measured. Error bars indicate SD (n ¼ 3). Statistical significance was investigated by one-way ANOVA with Tukey's multiple comparisons test (*, P < 0.05; **, P < 0.01).
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Saitama, Japan). 2.2. Oil Red O staining and triglyceride measurement Cells were washed twice with PBS, and then treated with 10% formaldehyde for 10 min at room temperature. Next, the cells were washed once with PBS and treated with 60% 2-propanol for 1 min at room temperature, and then stained with Oil Red O solution (60% 2propanol, 3 mg/mL Oil Red O) for 10 min at room temperature. The cells were then washed once with 60% 2-propanol and twice with PBS. Triglyceride measurement was performed using the LabAssay Triglyceride kit (FUJIFILM Wako Pure Chemical Corporation). 2.3. Cell viability assay After trypsinization, the number of viable cells and dead cells was counted using the trypan blue exclusion assay. Cells were counted manually with a hemocytometer. The cell number was determined by averaging the number of cells in eight squares, multiplying by the dilution factor and adjusting for cell volume to determine cells/well. 2.4. Cell cycle analysis Cells were fixed in 70% ethanol, rinsed with PBS and stained with a solution containing 20 mg/mL propidium iodide (Life Technologies), 0.05% Triton X-100 and 0.1 mg/mL RNase A (Life Technologies). Cell cycle progression was monitored by the image-based cytometer Tali (Life Technologies). 2.5. Western blot analysis
were stained with Oil Red O (Fig. 1B). After 6 days of culture, the cells showed Oil Red O staining, suggesting that they differentiated to adipocytes (Fig. 1B, Mock). After treatment with MeOH, the vehicle of SSA, or treatment with low concentrations of SSA, the cells were stained with Oil Red O, suggesting that MeOH or low SSA concentrations did not affect adipogenesis (Fig. 1B). However, after treatment with 1 or 1.5 ng/mL SSA, the cells showed significantly less staining than those treated with MeOH or low SSA concentrations, suggesting that these SSA concentrations inhibited adipogenesis (Fig. 1B). As described above, in the adipogenesis induction procedure, the 3T3-L1 cells were treated with induction medium in the early phase (Days 0e2), and then treated with maintenance medium in the late phase (Days 2e6). Next, I investigated in which phase SSA suppresses adipogenesis more efficiently. To this end, 3T3-L1 cells were treated with various SSA concentrations in the early (Fig. 1C, Days 0e2), late phase (Fig. 1C, Days 2e6) or both (Fig. 1C Days 0e6). Treatment with 1.5 ng/mL SSA in both phases inhibited adipogenesis, suggesting that splicing activity is required for both phases. Interestingly, 1 ng/mL SSA treatment in the early phase inhibited adipogenesis as effectively as the 6 days treatment, whereas 1 ng/ mL SSA treatment in the late phase was less effective (Fig. 1C). In addition to the Oil Red O staining, I measured the amounts of stored triglycerides in the differentiated adipocytes. Consistent with the Oil Red O staining, 1 ng/mL SSA treatment in the early phase (Days 0e2) inhibited triglyceride accumulation as effectively as the 6 days treatment (Days 0e6; Fig. 1D). In contrast, treatment in the late phase (Days 2e6) was less effective than treatment in the early phase (Fig. 1D). These results suggest that splicing activity is more important for adipogenesis in the early phase than in the late phase.
Cells were directly lysed on plates with 1 SDS-PAGE sample buffer. Proteins were separated by SDS-PAGE. After electrophoresis, proteins were transferred onto a PVDF membrane by electroblotting. Following the incubation of the membrane with primary and secondary antibodies using standard techniques, protein bands were detected using the NOVEX ECL Chemiluminescent Substrate Reagent Kit (Life Technologies) on an ImageQuant LAS 4000mini (GE Healthcare, Little Chalfont, UK). Anti-a-tubulin antibody (B-51-2) was purchased from Sigma-Aldrich (St. Louis, MO, USA). AntiCyclin B1 antibody was purchased from Cell Signaling Technologies (Danvers, MA, USA). Anti-p27 (C-19) antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). 2.6. Statistical analysis Statistical analysis was performed using R commander. Oneway ANOVA with Dunnett's multiple comparisons test or Tukey's multiple comparisons test was used to determine statistical significance. Data are presented as mean ± SD. The sample size (n) in each experiment is mentioned in the figure legends. P < 0.05 was considered statistically significant. 3. Results 3.1. Splicing inhibition suppresses adipogenesis To investigate the effects of splicing inhibition on adipogenesis, 3T3-L1 cells were cultured in an induction medium containing adipogenesis inducers (insulin, dexamethasone, 3-isobutyl-1methylxanthine) for 2 days (Days 0e2), and then cultured in maintenance medium containing insulin for 4 additional days (Day 2e6) with or without the potent splicing inhibitor, SSA (Fig. 1A). To determine whether the cells differentiated to adipocytes, the cells
Fig. 2. Splicing inhibition suppresses cell proliferation. (A, B) Post-confluent 3T3-L1 cells were cultured in the induction medium for 2 days with the indicated concentrations of SSA. Cell number and cell viability were determined. Error bars indicate SD (n ¼ 3). Statistical significance was investigated by one-way ANOVA with Dunnett's multiple comparisons test (*, P < 0.05; **, P < 0.01).
D. Kaida / Biochemical and Biophysical Research Communications 514 (2019) 848e852
3.2. Splicing inhibition suppresses mitotic clonal expansion SSA treatment in the early phase of differentiation inhibited adipogenesis as effectively as treatment during the entire period. In the early phase of adipogenesis, MCE occurs as one of the earliest events of adipogenesis upon treatment with adipogenesis inducers [4]. Because MCE is a prerequisite for adipogenesis, I assumed that SSA treatment inhibits MCE, resulting in inhibition of adipogenesis. To investigate this hypothesis, 3T3-L1 cells were treated with various concentrations of SSA during the early phase of differentiation, and the cell proliferation rate was determined, because if MCE occurs the cell number should increase. As expected, the cell number did not increase upon 1 or 1.5 ng/mL SSA treatment (Fig. 2A), supporting the idea that SSA treatment inhibits MCE. However, it is also possible that SSA causes cell death, consequently the cell number would not increase. To rule out this possibility, the cell viability after SSA treatment was examined. After 48 h of treatment with 1.5 ng/mL SSA, more than 90% of the cells were still viable (Fig. 2B), suggesting that SSA treatment causes cell cycle arrest, but not cell death. It has previously been reported that SSA treatment causes cell cycle arrest at the G1 and G2/M phases [6,7]. To investigate at which phase the 3T3 cells arrest upon SSA treatment, the DNA contents of the SSA-treated cells was measured using a cytometer. More than 40% of the MeOH-treated cells were in the G2/M phase at 24 h after induction (Fig. 3A), suggesting that the cells resumed cell cycle progression. In contrast, the population of SSA-treated cells in the G2/M phase did not change after SSA treatment (Fig. 3B). These results suggest that SSA treatment causes cell cycle arrest at the G1
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phase. In addition to the cell cycle analysis using a cytometer, the expression level of cell cycle-related factors was examined. Cyclin B1 protein, a G2/M phase marker, was observed at 24 h in the MeOH-treated cells, suggesting that at least a portion of the cells was in the G2/M phase (Fig. 3C). This result is consistent with the result above (Fig. 3A). However, cyclin B1 was not observed in SSAtreated cells (Fig. 3C). This result also supports the idea that SSAtreated cells were arrested at the G1 phase. Furthermore, the protein levels of p21 and p27, which are CDK inhibitors, were examined as these proteins inhibit cell cycle progression at the G1 phase and they have been reported to be upregulated in SSA-treated HeLa cells [6,8,9]. In the control cells, the p27 protein level decreased at 12 and 24 h, suggesting that the cells exited the G1 phase at these time points (Fig. 3C). However, in the SSA-treated cells, there was no accumulation of p27, in fact a decrease in p27 expression was observed compared with the control (Fig. 3C). The protein level of p21 decreased at 24 h in the control cells, suggesting that a portion of these cells were in S phase, because the p21 protein level decreases during S phase [10]. In contrast, SSA treatment upregulated the p21 protein level, which is consistent with a previous report [6]. Taken together, these results suggest that the control cells exited the G1 phase and re-entered cell cycle, whereas SSA-treated cells were arrested at the G1 phase. 4. Discussion Splicing is one of the most vital molecular mechanisms for proper gene expression in eukaryotes. As more than 97% and 80% of
Fig. 3. Splicing inhibition suppresses mitotic clonal expansion. (A, B, C) Post-confluent 3T3-L1 cells were cultured in the induction medium for 2 days with the indicated concentrations of SSA. Cells were harvested at the indicated time points and DNA contents were measured (A, B). The protein levels of Cyclin B1, p21, p27 and a-tubulin were analyzed (C). a-Tubulin was used as an internal control.
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genes contain introns in humans and mice, respectively [11,12], treatment with splicing inhibitors causes numerous effects on cellular functions [13]. One of the most apparent effects is cell cycle arrest [6,14]. Upon treatment with adipogenesis inducers, postconfluent 3T3-L1 cells resume cell cycle progression, i.e., MCE. MCE is one of the earliest events in adipogenesis and a prerequisite for adipogenesis [5]. In this report, I found that SSA treatment inhibited MCE, therefore splicing inhibition may suppress adipogenesis through MCE inhibition. Although splicing inhibition can cause cell cycle arrest at both the G1 and G2/M phases [6,14], 3T3-L1 cells seemed to be arrested only at the G1 phase after SSA treatment. One of the reasons for the G1 phase arrest in SSA-treated HeLa cells is overexpression of p27 [6,15]. However, SSA treatment of 3T3-L1 cells did not cause p27 overexpression, suggesting it was not the reason for the G1 phase arrest and the suppressed adipogenesis. In addition, it has recently been reported that SIRT6-deficient 3T3-L1 cells arrested at the S and M phases through stabilization of p27, suggesting that p27 overexpression causes S or M phase arrest rather than G1 phase arrest in 3T3-L1 cells [16]. These findings support the idea that p27 overexpression was not the reason for the G1 phase arrest of 3T3-L1 cells. In SSA-treated 3T3-L1 cells, another CDK inhibitor, p21, was overexpressed [6]. Because p21 inhibits cell cycle progression at the G1 and S phases [8], p21 overexpression may be the reason for the G1 phase arrest. However, it is also possible that downregulation of G1 cyclins, such as cyclin D and E, was the reason for the G1 phase arrest caused by SSA treatment. The detailed molecular mechanism of the G1 phase arrest of 3T3-L1 cells will be explored in a future study. A variety of small compounds have been reported as antiobesity drugs. Some of them have been approved by the Food and Drug Administration and/or the European Medicines Agency. However, most of them have been withdrawn from the market because of serious side effects, and very few are still used as antiobesity drugs [1]. As obesity is a serious health risk, more effective anti-obesity drugs with fewer side effects are highly soughtafter. The mRNA splicing mechanism has not been reported as a target of anti-obesity drugs, therefore this report will pave the way for the development of an anti-obesity drug with a novel therapeutic target, and will improve our understanding of the nature of adipogenesis. Conflicts of interest The author declares no competing interests. Acknowledgements Spliceostatin A was a kind gift from Dr M Yoshida (RIKEN, Japan).
This work was supported in part by the JSPS KAKENHI (24659129 and 26670134).
Transparency document Transparency document related to this article can be found online at https://doi.org/10.1016/j.bbrc.2019.04.180.
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