Toxicology 319 (2014) 69–74
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Microcystin-LR stabilizes c-myc protein by inhibiting protein phosphatase 2A in HEK293 cells Huihui Fan a,b,1 , Yan Cai b,d,1 , Ping Xie b,∗ , Wuhan Xiao c,∗∗ , Jun Chen b , Wei Ji c , Sujuan Zhao b a
College of Fisheries, Huazhong Agricultural University, Wuhan, China Donghu Experimental Station of Lake Ecosystems, State Key Laboratory of Freshwater Ecology and Biotechnology of China, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China c Key Laboratory of Biodiversity and Conservation of Aquatic Organisms, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China d School of Petrochemical Engineer, Changzhou University, Changzhou, China b
a r t i c l e
i n f o
Article history: Received 15 January 2014 Received in revised form 24 February 2014 Accepted 26 February 2014 Available online 7 March 2014 Keywords: Microcystin-LR c-myc Protein phosphatase 2A Carcinogenicity
a b s t r a c t Microcystin-LR is the most toxic and the most frequently encountered toxin produced by the cyanobacteria in the contaminated aquatic environment. Previous studies have demonstrated that Microcystin-LR is a potential carcinogen for animals and humans, and the International Agency for Research on Cancer has classified Microcystin-LR as a possible human carcinogen. However, the precise molecular mechanisms of Microcystin-LR-induced carcinogenesis remain a mystery. C-myc is a proto-oncogene, abnormal expression of which contributes to the tumor development. Although several studies have demonstrated that Microcystin-LR could induce c-myc expression at the transcriptional level, the exact connection between Microcystin-LR toxicity and c-myc response remains unclear. In this study, we showed that the c-myc protein increased in HEK293 cells after exposure to Microcystin-LR. Coexpression of protein phosphatase 2A and two stable c-myc protein point mutants (either c-mycT58A or c-mycS62A ) showed that Microcystin-LR increased c-myc protein level mainly through inhibiting protein phosphatase 2A activity which altered the phosphorylation status of serine 62 on c-myc. In addition, we also showed that Microcystin-LR could increase c-myc promoter activity as revealed by luciferase reporter assay. And the TATA box for P1 promoter of c-myc might be involved. Our results suggested that Microcystin-LR can stimulate c-myc transcription and stabilize c-myc protein, which might contribute to hepatic tumorigenesis in animals and humans. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Toxic cyanobacterial blooms have become a worldwide environmental concern. Microcystins (MCs) are produced by cyanobacteria, which are harmful to aquatic organisms and mammals including humans (Azevedo et al., 2002; Carmichael et al., 2001). In 1996, 60 dialysis patients in Caruaru of Brazil were died due to the acute exposure to MCs in water used for dialysis (Pouria et al., 1998). Epidemiological studies have indicated that MCs might be responsible for the increased incidence of liver cancer in
∗ Corresponding author at: Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China. Tel.: +86 27 68780622; fax: +86 27 68780622. ∗∗ Corresponding author at: Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China. Tel.: +86 27 68780087; fax: +86 27 68780087. E-mail addresses: [email protected] (P. Xie), [email protected] (W. Xiao). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.tox.2014.02.015 0300-483X/© 2014 Elsevier Ireland Ltd. All rights reserved.
certain areas of China (Ueno et al., 1996; Yu, 1989). So far, more than 80 structural analogs of MCs have been identified, of which microcystin-LR (MC-LR) is the most studied and the most toxic (Hoeger et al., 2005). MC-LR is characterized with regard to hepatotoxicity, nephrotoxicity, neurotoxicity, reproductive toxicity and carcinogenicity (Chen et al., 2013; Feurstein et al., 2011; Li et al., 2012a; Suput, 2011; Wang et al., 2010a; Zhao et al., 2012). The International Agency for Research on Cancer (IARC) has classified MC-LR as a possible human carcinogen (Cogliano et al., 2008; Grosse et al., 2006). In recent years, MC-LR has attracted increasing attention because of its tumor-promoting capacity. The c-myc gene, a proto-oncogene, encodes a transcription factor involved in the control of cell proliferation, differentiation and apoptosis (Cole, 1986; Dang, 1999; Prendergast, 1999). Overexpression of c-myc plays a significant role in cancer development (Nesbit et al., 1999; Rapp et al., 2009). Because of its potent effects on cell fate, cells have evolved sophisticated methods to ensure proper expression of c-myc. The expression of c-myc is regulated at
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multiple levels: transcription, post-transcription and posttranslation (Jones and Cole, 1987; Kelly et al., 1983; Sears et al., 1999). The half-life of c-myc is very short in cells due to proteasomal degradation. Previous study has indicated that the phosphorylation of threonine 58 and serine 62, two phosphorylation sites in the N-terminus of c-myc, can regulate c-myc protein stability (Sears et al., 2000). Threonine 58 phosphorylation destabilizes c-myc, while serine 62 phosphorylation stabilizes c-myc. The well-known regulatory cycle of c-myc, from synthesis to degradation, includes: (1) new c-myc protein synthesis; (2) serine 62 of c-myc is phosphorylated by extracellular receptor kinase (ERK); (3) threonine 58 is phosphorylated by glycogen synthase kinase (GSK-3); (4) Pin1 prolyl isomerase recognizes the threonine 58 and serine 62 phosphorylated c-myc and catalyzes c-myc protein conformational change; (5) serine 62 is then dephosphorylated by protein phosphatase 2A (PP2A); (6) c-myc protein is degraded by the ubiquitin/proteasome system (Sears, 2004). The most studied and accepted mechanism of MC-LR toxicity is the inhibition of protein phosphatase 1 and 2A (PP1 and PP2A) by directly binding to the catalytic subunits of these enzymes (Mackintosh et al., 1995; Xing et al., 2006). PP2A is a major serine/threonine phosphatase in cells and is involved in regulating proliferation, growth, differentiation, and apoptosis (Janssens and Goris, 2001). Unlike c-myc, PP2A is generally regarded as a tumor suppressor (Janssens et al., 2005). Several studies have shown that PP2A might be able to affect the phosphorylation status of threonine 58 and serine 62 of c-myc in the direct or indirect pathways (Arnold and Sears, 2008; Dias et al., 2010; Henriksson et al., 1993; Lutterbach and Hann, 1994; Pulverer et al., 1994; Takumi et al., 2010). So, theoretically, given its potent effect on PP2A activity, MC-LR might be able to regulate c-myc protein stability. In addition, previous studies have shown that MC-LR could induce c-myc expression at the transcriptional level (Li et al., 2009; Takumi et al., 2010). Therefore, MC-LR might induce c-myc expression at both transcriptional and post-translational levels. However, the exact connection and molecular mechanisms between MC-LR toxicity and c-myc response remains unclear. In the present study, we used HEK293 cells because kidney is an important target organ of MC-LR and the endogenous c-myc protein level in HEK293 cells is low (Supplementary Fig. 1). To clarify the exact connection between MC-LR toxicity and c-myc response, two stable c-myc protein point mutants (c-mycT58A and c-mycS62A ) and three promoter mutants (P1 mut1, P1 mut2, P2 mut) were employed. The results suggested that MC-LR up-regulates the cmyc protein level mainly via the MC-LR/PP2A/serine 62 pathway.
2. Materials and methods 2.1. Cell culture and reagents HEK293 cells were obtained from the American Type Culture Collection and were maintained in Dulbecco’s modified Eagle’s medium (Hyclone, UT) supplemented with 10% (v/v) fetal bovine serum (Hyclone, UT) at 37 ◦ C in a humidified atmosphere with 5% CO2 . Cells were counted using an automated cell counter (TC10, Bio-Rad, CA). MC-LR (purity ≥ 95%) was extracted and purified from the freeze-dried surface blooms collected from Lake Dianchi, Yunnan, China, following the method described previously (Wang et al., 2008). The MC-LR concentration (10 M) was chosen according to the previous study (Li et al., 2012b).
2.2. Plasmids The wild-type c-myc and two stable c-myc protein point mutants (c-mycT58A and c-mycS62A ) were cloned into the pCMV-HA expression vector (Clontech, CA). The PP2A catalytic ␣ subunit (PP2AC␣) was cloned into the pCMV-Myc plasmid at EcoR I and Bgl II sites. The wild-type c-myc promoter and its three mutated form (P1 mut1, P1 mut2, P2 mut) were cloned into the pGL3-Basic vector (Promega, WI).
2.3. Western blotting HEK293 cells were grown in 6-well plates and transfected using vigofect (Vigoruse, China) according to the manufacturer’s instruction. After incubation with MC-LR for 24 h, cells were lysed with ice-cold protein extraction buffer (Biyuntian, China) supplemented with 1 mM PMSF. Following homogenization, the lysates were centrifuged at 12,000 × g for 10 min and supernatants were collected. About 20 g of protein from each sample was denatured, electrophoresed, and transferred onto a PVDF membrane (Millipore, France). Western blotting analysis was performed with specific antibodies against c-myc (Santa Cruz, CA) and -actin (Proteintech Group, China). 2.4. Luciferase reporter assay HEK293 cells were grown in 24-well plates and transfected using vigofect. Renilla luciferase gene as an internal control was co-transfected into HEK293 cells with the target vectors. The luciferase activity was assayed after 24 h treatment with MC-LR. The luciferase activity in cell extracts was determined by Dual-luciferase reporter assay system (Promega, WI) according to the protocol supplied by the manufacturer. The relative light units were measured using a luminometer (Sirius, Zylux Corporation, Oak Ridge, TN). Data were normalized to Renilla luciferase activity. 2.5. cDNA preparation and real-time PCR Total RNA isolation, synthesis of first-strand cDNA, and real-time PCR were performed followed the method described previously (Li et al., 2011). Briefly, HEK293 cells were cultured with 0 or 10 M MC-LR, and the cells were harvested 10 h after treatment. Total cellular RNA was extracted from cells by using Trizol reagent (TaKaRa, Japan). cDNA was synthesized by reverse transcription of total RNA using first strand cDNA synthesis kit (Fermentas, EU). Real-time PCR was performed with a SYBR Green PCR kit (Toyobo, Japan) using a Chromo4 Real-Time Detection System (MJ Research, Cambridge, MA). GAPDH was chosen as an internal control to normalize the data. Relative quantification between different samples was then determined according to the 2−Ct method (Livak and Schmittgen, 2001). The primers are as follows: c-myc-F, 5 -ACA GCT ACG GAA CTC TTG TGC GTA-3 and c-myc-R, 5 -GCC CAA AGT CCA ATT TGA GGC AGT-3 ; GAPDH-F, 5 -TGG GTG TGA ACC ATG AGA AGT-3 and GAPDH-R, 5 -GGC ATG GAC TGT GGT CAT GA-3 . 2.6. Statistical analysis Statistical analysis was undertaken using Statistica 6.0 software. All values expressed as mean ± SD were subjected to one-way analysis of variance (ANOVA). Differences in mean values between groups were assessed and were considered statistically different at p < 0.05, p < 0.01.
3. Results 3.1. Effect of MC-LR on c-myc protein stability To determine whether MC-LR could affect the stability of c-myc protein, HEK293 cells were transfected with exogenous wild-type c-myc expression vector and then treated with MC-LR. The expression of exogenous c-myc was verified by Western blot. As shown in Supplementary Fig. 1, the exogenous c-myc was clearly indicated by the c-myc antibody. While the endogenous c-myc protein level could hardly be detected, being much lower than the exogenous c-myc. Western blot assay was performed to assess the effect of MC-LR on c-myc protein stability. For the better comparability of the following study (c-mycT58A and c-mycS62A are both the exogenous c-myc), only exogenous c-myc was focused. As shown in Fig. 1A, c-myc protein level was increased after exposure to MCLR. To determine whether PP2A was involved in MC-LR-induced up-regulation of c-myc protein level, either myc empty vector or myc-tagged PP2AC␣ expression vector was co-transfected with c-myc expression vector into HEK293 cells. As shown in Fig. 1B, the myc-tagged PP2AC␣, marked by a black arrow, could decrease the MC-LR-induced up-regulation of c-myc protein level. The PP2A could also obviously down-regulate c-myc protein without MC-LR (Supplementary Fig. 2). To further study the underlying mechanism, two stable c-myc protein point mutants (c-mycT58A and c-mycS62A ) were used to identify whether MC-LR-induced upregulation of c-myc protein level was resulted from the alteration of the phosphorylation status of threonine 58 or serine 62 in c-myc.
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Fig. 2. Role of threonine 58 and serine 62 in the regulation of MC-LR-induced upregulation of c-myc. (A) MC-LR increased c-mycT58A protein level in the HEK293 cells (lane 1 and lane 2; lane 3 and lane 4, from left to right). PP2A could decrease the MC-LR-induced up-regulation of c-myc protein level (lanes 2 and 4). (B) After mutating serine 62 to alanine, MC-LR no longer increases c-myc protein level (lanes 1 and 2; lanes 3 and 4) and PP2A lost its ability to reduce the c-myc protein level (lanes 1 and 3; lanes 2 and 4). Fig. 1. Effect of MC-LR treatment and overexpression of PP2AC␣ on c-myc protein level. (A) C-myc protein level in HEK293 cells was significantly increased after treating with MC-LR. (B) Overexpression of PP2AC␣ (myc-tagged PP2AC␣, marked with a black arrow) significantly decreased the protein level of c-myc in HEK293 cells.
The results showed that MC-LR stimulate the c-mycT58A protein and PP2A down-regulate it (Fig. 2A). However, after mutation of serine 62, PP2A could not reduce the c-myc protein level and MC-LR no longer increases the protein level of c-myc (Fig. 2B). These data suggested that MC-LR could increase c-myc protein level by altering the phosphorylation status of serine 62.
3.2. Activation of the c-myc promoter reporter by MC-LR To determine whether MC-LR could induce c-myc transcription, we examined c-myc promoter activity after MC-LR treatment by a luciferase reporter assay. The activity of c-myc promoter was increased following exposure to MC-LR (Fig. 3C). Then, in order to identify the elements in c-myc promoter response to MC-LR toxicity, three mutants (P1 mut1, P1 mut2, P2 mut) of c-myc promoter were employed (Fig. 3A and B). The results showed that MC-LR could not affect the activity of P1 mut1 and P1 mut2, while still could activate the P2 mut significantly (Fig. 3C). These results
Fig. 3. MC-LR increases c-myc promoter activity. (A) The schematic depiction of the c-myc promoter with mutations in P1/P2 TATA box. (B) Detailed description of mutations. (C) Effect of MC-LR treatment on the different c-myc promoter mutants, *p < 0.05.
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Fig. 4. Effect of MC-LR on the expression of c-myc mRNA in HEK293 cells. **p < 0.01.
suggested that the TATA box for P1 might plays an important role in MC-LR-induced up-regulation of c-myc mRNA. 3.3. Analysis of c-myc mRNA expression To confirm that MC-LR could up-regulate c-myc mRNA level, we analyzed the endogenous mRNA level of c-myc in HEK293 cells after treatment with MC-LR by real-time PCR analysis. The mRNA level of c-myc was significantly increased after 10 h of MC-LR treatment (Fig. 4). 4. Discussion MC-LR has attracted increasing interest because of its tumorpromoting activity in hepatocytes of animals and humans. IARC has classified MC-LR as a possible human carcinogen (Cogliano et al., 2008). In this study, we showed that MC-LR increase c-myc protein stability by altering the phosphorylation status of serine 62. Furthermore, we identified that the TATA box of P1 promoter might play a critical role in MC-LR-induced up-regulation of c-myc promoter activity. These results suggested that MC-LR can up-regulate c-myc protein level at both transcriptional and post-translational levels, which might contribute to hepatic tumorigenesis in animals and humans (Fig. 5).
Fig. 5. Proposed model for the up-regulation of c-myc protein in HEK293 cells induced by MC-LR. MC-LR induces c-myc protein mainly by stimulating the phosphorylation of serine 62 of c-myc, which was mediated by the inhibition of the activity of PP2A. Moreover, the TATA box for P1 of c-myc may be involved in the activation of c-myc promoter which may contribute to the MC-LR-induced upregulation of c-myc protein. However, MC-LR may also have a relatively weak negative effect on c-myc protein through the proteasome pathway.
To our knowledge, this study possibly provides the first direct evidence of promoting effects of MC-LR on c-myc protein stability. C-myc, as an important tumor-promoting factor, has been well studied. In this study, we showed that c-myc protein level was increased in HEK293 cells after exposure to MC-LR. Previous studies have established that MC-LR is a potent inhibitor of PP2A by directly binding to the PP2A catalytic subunit (Mackintosh et al., 1990; Toivola et al., 1994; Xing et al., 2006). On the other hand, studies have suggested that PP2A negatively regulates c-myc protein level by directly dephosphorylating the serine 62 of c-myc, which is a key event in c-myc degradation pathway (Arnold and Sears, 2006; Sears, 2004). Moreover, serine 62 is phosphorylated by ERK and threonine 58 is phosphorylated by GSK-3 (Sears et al., 2000). Previous studies have shown that PP2A could inactivate ERKs and GSK-3 (Kins et al., 2003; Seeling et al., 1999). So, in order to evaluate the effect of PP2A on MC-LR-induced up-regulation of c-myc protein level, we overexpressed the PP2AC␣, which is the direct target of MC-LR (Xing et al., 2006). The result showed that PP2A could decrease the MC-LR-induced up-regulation of c-myc protein level. These results are consistent with what we expected. To further investigate the exact mechanism of MC-LR-induced up-regulation of c-myc protein, the c-mycS62A and c-mycT58A mutants were used. The results showed that, after mutation of serine 62, PP2A lost its ability to reduce the c-myc protein level and MC-LR no longer increases the protein level of c-myc. Based on these results, we proposed that MC-LR could increase c-myc protein stability by altering the phosphorylation status of serine 62. Moreover, interestingly, we found that the protein level of c-mycS62A was decreased after exposure to MC-LR, which suggest that MC-LR also could induce c-myc protein degradation by a PP2A/serine 62-independent pathway. Previous study has shown that the half-life of c-mycS62A is shorter than the wild-type, and the level of c-mycS62A is enhanced in the presence of the proteasome inhibitor, which suggested that there is another, more effective serine 62-independent proteasome pathway for c-myc degradation (Sears et al., 2000). Several proteomics studies showed that MC-LR could affect ubiquitin/proteasome system (Chen et al., 2005; Wang et al., 2010b), which might be responsible for the MC-LR induced down-regulation of c-mycS62A . The connection between MC-LR and ubiquitin/proteasome system is worth to be further investigated. Hence, these results suggested that the MC-LR has dual effects on c-myc protein stability, both stabilizing and destabilizing c-myc protein. And the former is stronger than the latter on the wild-type c-myc. Moreover, after mutation of threonine 58, our results showed that there was no significant association between MC-LR and threonine 58 of c-myc. However, we found that PP2A still could facilitate c-mycT58A degradation. Previous study has shown that the c-MycT58A mutant could not be dephosphorylated by PP2A (Yeh et al., 2004). So, the protein level of c-MycT58A mutant cannot be affected by MC-LR or PP2A in the PP2A/serine 62 pathway. However, it also has been shown that the mutant c-mycT58A protein is increased by stimulation of Ras/Raf/MEK/ERK pathway (Sears et al., 2000). And it has been shown that PP2A could inactivate ERKs (Kins et al., 2003). Therefore, the PP2A-induced down-regulation of c-mycT58A protein is probably caused by the inhibition of ERK. These results confirmed that MC-LR can stabilize c-myc protein by altering the phosphorylation status of serine 62. It has been well established that MC-LR could induce c-myc expression at the transcriptional level (Li et al., 2009; Takumi et al., 2010). However, the underlying mechanism is still unclear. In this study, we showed that MC-LR up-regulated the c-myc promoter activity in HEK293 cells. Next, to define the possible response elements in c-myc promoter for MC-LR-induced up-regulation of c-myc promoter activity, three mutants (P1 mut1, P1 mut2, P2 mut) of c-myc promoter were employed. The results showed that the P1
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mut1 and the P1 mut2 could reverse the up-regulation of c-myc promoter activity induced by MC-LR, while the P2 mut couldn’t. These results suggested that the TATA box for P1 might play an important role in MC-LR-induced up-regulation of c-myc mRNA. Previous studies have suggested that ERK could also induce c-myc expression at the transcriptional level (Kerkhoff et al., 1998; Verykokakis et al., 2007). So ERK might be an effective approach to further study the mechanism of MC-LR-induced up-regulation of c-myc promoter activity. In addition, the effect of MC-LR on c-myc promoter activity was further confirmed the result of real-time PCR. However, although the c-myc mRNA was significantly increased after 10 h of MC-LR treatment, it is only about 1.3-fold higher than the control. This result in our study was probably caused by the relatively low sensitivity of HEK293 cells to MC-LR (Fischer et al., 2010). On the other hand, we found that the c-myc mRNA in HEK293 cells was not changed significantly after 24 h of MC-LR treatment (data not shown), which might be caused by an autoregulatory mechanism. However, the result showed that the c-myc protein level was significantly increased after 24 h of MC-LR treatment. So we suggested that the post-translational level pathway is stronger than the transcriptional level pathway of c-myc response to MC-LR toxicity. So, in this study, we proposed that MC-LR up-regulated the c-myc protein level mainly via the PP2A/serine 62 pathway. 5. Conclusions In the present study, we showed that MC-LR increased c-myc protein level mainly by altering the phosphorylation status of serine 62, which was mediated by the inhibition of the activity of PP2A. Moreover, we demonstrated that the TATA box for P1 of cmyc might play an important role in MC-LR-induced up-regulation of c-myc mRNA, which may also contribute to MC-LR-induced up-regulation of c-myc protein level. And we also found that MCLR may have a relatively weak negative effect on c-myc protein through the proteasome pathway. The connection between MC-LR and ubiquitin/proteasome system is interesting and worth to be further investigated. The results of this study may contribute to the knowledge of the potential carcinogenicity of MC-LR. Conflict of interest statement The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgements The authors thank Dr. Xiaoyang Wan for helping to revise the manuscript. This work was supported by the National Natural Science Foundation of China (31322013) and the State Key Laboratory of Freshwater Ecology and Biotechnology (2011FBZ07). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tox.2014.02.015. References Arnold, H.K., Sears, R.C., 2006. Protein phosphatase 2A regulatory subunit b56 alpha associates with c-Myc and negatively regulates c-Myc accumulation. Mol. Cell. Biol. 26, 2832–2844.
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Microcystin-LR stabilizes c-myc protein by inhibiting protein phosphatase 2A in HEK293 cells Huihui Fan a,b,1 , Yan Cai b,d,1 , Ping Xie b,∗ , Wuhan Xiao c,∗∗ , Jun Chen b , Wei Ji c , Sujuan Zhao b a
College of Fisheries, Huazhong Agricultural University, Wuhan, China Donghu Experimental Station of Lake Ecosystems, State Key Laboratory of Freshwater Ecology and Biotechnology of China, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China c Key Laboratory of Biodiversity and Conservation of Aquatic Organisms, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, China d School of Petrochemical Engineer, Changzhou University, Changzhou, China b
a r t i c l e
i n f o
Article history: Received 15 January 2014 Received in revised form 24 February 2014 Accepted 26 February 2014 Available online 7 March 2014 Keywords: Microcystin-LR c-myc Protein phosphatase 2A Carcinogenicity
a b s t r a c t Microcystin-LR is the most toxic and the most frequently encountered toxin produced by the cyanobacteria in the contaminated aquatic environment. Previous studies have demonstrated that Microcystin-LR is a potential carcinogen for animals and humans, and the International Agency for Research on Cancer has classified Microcystin-LR as a possible human carcinogen. However, the precise molecular mechanisms of Microcystin-LR-induced carcinogenesis remain a mystery. C-myc is a proto-oncogene, abnormal expression of which contributes to the tumor development. Although several studies have demonstrated that Microcystin-LR could induce c-myc expression at the transcriptional level, the exact connection between Microcystin-LR toxicity and c-myc response remains unclear. In this study, we showed that the c-myc protein increased in HEK293 cells after exposure to Microcystin-LR. Coexpression of protein phosphatase 2A and two stable c-myc protein point mutants (either c-mycT58A or c-mycS62A ) showed that Microcystin-LR increased c-myc protein level mainly through inhibiting protein phosphatase 2A activity which altered the phosphorylation status of serine 62 on c-myc. In addition, we also showed that Microcystin-LR could increase c-myc promoter activity as revealed by luciferase reporter assay. And the TATA box for P1 promoter of c-myc might be involved. Our results suggested that Microcystin-LR can stimulate c-myc transcription and stabilize c-myc protein, which might contribute to hepatic tumorigenesis in animals and humans. © 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Toxic cyanobacterial blooms have become a worldwide environmental concern. Microcystins (MCs) are produced by cyanobacteria, which are harmful to aquatic organisms and mammals including humans (Azevedo et al., 2002; Carmichael et al., 2001). In 1996, 60 dialysis patients in Caruaru of Brazil were died due to the acute exposure to MCs in water used for dialysis (Pouria et al., 1998). Epidemiological studies have indicated that MCs might be responsible for the increased incidence of liver cancer in
∗ Corresponding author at: Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China. Tel.: +86 27 68780622; fax: +86 27 68780622. ∗∗ Corresponding author at: Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan 430072, China. Tel.: +86 27 68780087; fax: +86 27 68780087. E-mail addresses: [email protected] (P. Xie), [email protected] (W. Xiao). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.tox.2014.02.015 0300-483X/© 2014 Elsevier Ireland Ltd. All rights reserved.
certain areas of China (Ueno et al., 1996; Yu, 1989). So far, more than 80 structural analogs of MCs have been identified, of which microcystin-LR (MC-LR) is the most studied and the most toxic (Hoeger et al., 2005). MC-LR is characterized with regard to hepatotoxicity, nephrotoxicity, neurotoxicity, reproductive toxicity and carcinogenicity (Chen et al., 2013; Feurstein et al., 2011; Li et al., 2012a; Suput, 2011; Wang et al., 2010a; Zhao et al., 2012). The International Agency for Research on Cancer (IARC) has classified MC-LR as a possible human carcinogen (Cogliano et al., 2008; Grosse et al., 2006). In recent years, MC-LR has attracted increasing attention because of its tumor-promoting capacity. The c-myc gene, a proto-oncogene, encodes a transcription factor involved in the control of cell proliferation, differentiation and apoptosis (Cole, 1986; Dang, 1999; Prendergast, 1999). Overexpression of c-myc plays a significant role in cancer development (Nesbit et al., 1999; Rapp et al., 2009). Because of its potent effects on cell fate, cells have evolved sophisticated methods to ensure proper expression of c-myc. The expression of c-myc is regulated at
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multiple levels: transcription, post-transcription and posttranslation (Jones and Cole, 1987; Kelly et al., 1983; Sears et al., 1999). The half-life of c-myc is very short in cells due to proteasomal degradation. Previous study has indicated that the phosphorylation of threonine 58 and serine 62, two phosphorylation sites in the N-terminus of c-myc, can regulate c-myc protein stability (Sears et al., 2000). Threonine 58 phosphorylation destabilizes c-myc, while serine 62 phosphorylation stabilizes c-myc. The well-known regulatory cycle of c-myc, from synthesis to degradation, includes: (1) new c-myc protein synthesis; (2) serine 62 of c-myc is phosphorylated by extracellular receptor kinase (ERK); (3) threonine 58 is phosphorylated by glycogen synthase kinase (GSK-3); (4) Pin1 prolyl isomerase recognizes the threonine 58 and serine 62 phosphorylated c-myc and catalyzes c-myc protein conformational change; (5) serine 62 is then dephosphorylated by protein phosphatase 2A (PP2A); (6) c-myc protein is degraded by the ubiquitin/proteasome system (Sears, 2004). The most studied and accepted mechanism of MC-LR toxicity is the inhibition of protein phosphatase 1 and 2A (PP1 and PP2A) by directly binding to the catalytic subunits of these enzymes (Mackintosh et al., 1995; Xing et al., 2006). PP2A is a major serine/threonine phosphatase in cells and is involved in regulating proliferation, growth, differentiation, and apoptosis (Janssens and Goris, 2001). Unlike c-myc, PP2A is generally regarded as a tumor suppressor (Janssens et al., 2005). Several studies have shown that PP2A might be able to affect the phosphorylation status of threonine 58 and serine 62 of c-myc in the direct or indirect pathways (Arnold and Sears, 2008; Dias et al., 2010; Henriksson et al., 1993; Lutterbach and Hann, 1994; Pulverer et al., 1994; Takumi et al., 2010). So, theoretically, given its potent effect on PP2A activity, MC-LR might be able to regulate c-myc protein stability. In addition, previous studies have shown that MC-LR could induce c-myc expression at the transcriptional level (Li et al., 2009; Takumi et al., 2010). Therefore, MC-LR might induce c-myc expression at both transcriptional and post-translational levels. However, the exact connection and molecular mechanisms between MC-LR toxicity and c-myc response remains unclear. In the present study, we used HEK293 cells because kidney is an important target organ of MC-LR and the endogenous c-myc protein level in HEK293 cells is low (Supplementary Fig. 1). To clarify the exact connection between MC-LR toxicity and c-myc response, two stable c-myc protein point mutants (c-mycT58A and c-mycS62A ) and three promoter mutants (P1 mut1, P1 mut2, P2 mut) were employed. The results suggested that MC-LR up-regulates the cmyc protein level mainly via the MC-LR/PP2A/serine 62 pathway.
2. Materials and methods 2.1. Cell culture and reagents HEK293 cells were obtained from the American Type Culture Collection and were maintained in Dulbecco’s modified Eagle’s medium (Hyclone, UT) supplemented with 10% (v/v) fetal bovine serum (Hyclone, UT) at 37 ◦ C in a humidified atmosphere with 5% CO2 . Cells were counted using an automated cell counter (TC10, Bio-Rad, CA). MC-LR (purity ≥ 95%) was extracted and purified from the freeze-dried surface blooms collected from Lake Dianchi, Yunnan, China, following the method described previously (Wang et al., 2008). The MC-LR concentration (10 M) was chosen according to the previous study (Li et al., 2012b).
2.2. Plasmids The wild-type c-myc and two stable c-myc protein point mutants (c-mycT58A and c-mycS62A ) were cloned into the pCMV-HA expression vector (Clontech, CA). The PP2A catalytic ␣ subunit (PP2AC␣) was cloned into the pCMV-Myc plasmid at EcoR I and Bgl II sites. The wild-type c-myc promoter and its three mutated form (P1 mut1, P1 mut2, P2 mut) were cloned into the pGL3-Basic vector (Promega, WI).
2.3. Western blotting HEK293 cells were grown in 6-well plates and transfected using vigofect (Vigoruse, China) according to the manufacturer’s instruction. After incubation with MC-LR for 24 h, cells were lysed with ice-cold protein extraction buffer (Biyuntian, China) supplemented with 1 mM PMSF. Following homogenization, the lysates were centrifuged at 12,000 × g for 10 min and supernatants were collected. About 20 g of protein from each sample was denatured, electrophoresed, and transferred onto a PVDF membrane (Millipore, France). Western blotting analysis was performed with specific antibodies against c-myc (Santa Cruz, CA) and -actin (Proteintech Group, China). 2.4. Luciferase reporter assay HEK293 cells were grown in 24-well plates and transfected using vigofect. Renilla luciferase gene as an internal control was co-transfected into HEK293 cells with the target vectors. The luciferase activity was assayed after 24 h treatment with MC-LR. The luciferase activity in cell extracts was determined by Dual-luciferase reporter assay system (Promega, WI) according to the protocol supplied by the manufacturer. The relative light units were measured using a luminometer (Sirius, Zylux Corporation, Oak Ridge, TN). Data were normalized to Renilla luciferase activity. 2.5. cDNA preparation and real-time PCR Total RNA isolation, synthesis of first-strand cDNA, and real-time PCR were performed followed the method described previously (Li et al., 2011). Briefly, HEK293 cells were cultured with 0 or 10 M MC-LR, and the cells were harvested 10 h after treatment. Total cellular RNA was extracted from cells by using Trizol reagent (TaKaRa, Japan). cDNA was synthesized by reverse transcription of total RNA using first strand cDNA synthesis kit (Fermentas, EU). Real-time PCR was performed with a SYBR Green PCR kit (Toyobo, Japan) using a Chromo4 Real-Time Detection System (MJ Research, Cambridge, MA). GAPDH was chosen as an internal control to normalize the data. Relative quantification between different samples was then determined according to the 2−Ct method (Livak and Schmittgen, 2001). The primers are as follows: c-myc-F, 5 -ACA GCT ACG GAA CTC TTG TGC GTA-3 and c-myc-R, 5 -GCC CAA AGT CCA ATT TGA GGC AGT-3 ; GAPDH-F, 5 -TGG GTG TGA ACC ATG AGA AGT-3 and GAPDH-R, 5 -GGC ATG GAC TGT GGT CAT GA-3 . 2.6. Statistical analysis Statistical analysis was undertaken using Statistica 6.0 software. All values expressed as mean ± SD were subjected to one-way analysis of variance (ANOVA). Differences in mean values between groups were assessed and were considered statistically different at p < 0.05, p < 0.01.
3. Results 3.1. Effect of MC-LR on c-myc protein stability To determine whether MC-LR could affect the stability of c-myc protein, HEK293 cells were transfected with exogenous wild-type c-myc expression vector and then treated with MC-LR. The expression of exogenous c-myc was verified by Western blot. As shown in Supplementary Fig. 1, the exogenous c-myc was clearly indicated by the c-myc antibody. While the endogenous c-myc protein level could hardly be detected, being much lower than the exogenous c-myc. Western blot assay was performed to assess the effect of MC-LR on c-myc protein stability. For the better comparability of the following study (c-mycT58A and c-mycS62A are both the exogenous c-myc), only exogenous c-myc was focused. As shown in Fig. 1A, c-myc protein level was increased after exposure to MCLR. To determine whether PP2A was involved in MC-LR-induced up-regulation of c-myc protein level, either myc empty vector or myc-tagged PP2AC␣ expression vector was co-transfected with c-myc expression vector into HEK293 cells. As shown in Fig. 1B, the myc-tagged PP2AC␣, marked by a black arrow, could decrease the MC-LR-induced up-regulation of c-myc protein level. The PP2A could also obviously down-regulate c-myc protein without MC-LR (Supplementary Fig. 2). To further study the underlying mechanism, two stable c-myc protein point mutants (c-mycT58A and c-mycS62A ) were used to identify whether MC-LR-induced upregulation of c-myc protein level was resulted from the alteration of the phosphorylation status of threonine 58 or serine 62 in c-myc.
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Fig. 2. Role of threonine 58 and serine 62 in the regulation of MC-LR-induced upregulation of c-myc. (A) MC-LR increased c-mycT58A protein level in the HEK293 cells (lane 1 and lane 2; lane 3 and lane 4, from left to right). PP2A could decrease the MC-LR-induced up-regulation of c-myc protein level (lanes 2 and 4). (B) After mutating serine 62 to alanine, MC-LR no longer increases c-myc protein level (lanes 1 and 2; lanes 3 and 4) and PP2A lost its ability to reduce the c-myc protein level (lanes 1 and 3; lanes 2 and 4). Fig. 1. Effect of MC-LR treatment and overexpression of PP2AC␣ on c-myc protein level. (A) C-myc protein level in HEK293 cells was significantly increased after treating with MC-LR. (B) Overexpression of PP2AC␣ (myc-tagged PP2AC␣, marked with a black arrow) significantly decreased the protein level of c-myc in HEK293 cells.
The results showed that MC-LR stimulate the c-mycT58A protein and PP2A down-regulate it (Fig. 2A). However, after mutation of serine 62, PP2A could not reduce the c-myc protein level and MC-LR no longer increases the protein level of c-myc (Fig. 2B). These data suggested that MC-LR could increase c-myc protein level by altering the phosphorylation status of serine 62.
3.2. Activation of the c-myc promoter reporter by MC-LR To determine whether MC-LR could induce c-myc transcription, we examined c-myc promoter activity after MC-LR treatment by a luciferase reporter assay. The activity of c-myc promoter was increased following exposure to MC-LR (Fig. 3C). Then, in order to identify the elements in c-myc promoter response to MC-LR toxicity, three mutants (P1 mut1, P1 mut2, P2 mut) of c-myc promoter were employed (Fig. 3A and B). The results showed that MC-LR could not affect the activity of P1 mut1 and P1 mut2, while still could activate the P2 mut significantly (Fig. 3C). These results
Fig. 3. MC-LR increases c-myc promoter activity. (A) The schematic depiction of the c-myc promoter with mutations in P1/P2 TATA box. (B) Detailed description of mutations. (C) Effect of MC-LR treatment on the different c-myc promoter mutants, *p < 0.05.
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Fig. 4. Effect of MC-LR on the expression of c-myc mRNA in HEK293 cells. **p < 0.01.
suggested that the TATA box for P1 might plays an important role in MC-LR-induced up-regulation of c-myc mRNA. 3.3. Analysis of c-myc mRNA expression To confirm that MC-LR could up-regulate c-myc mRNA level, we analyzed the endogenous mRNA level of c-myc in HEK293 cells after treatment with MC-LR by real-time PCR analysis. The mRNA level of c-myc was significantly increased after 10 h of MC-LR treatment (Fig. 4). 4. Discussion MC-LR has attracted increasing interest because of its tumorpromoting activity in hepatocytes of animals and humans. IARC has classified MC-LR as a possible human carcinogen (Cogliano et al., 2008). In this study, we showed that MC-LR increase c-myc protein stability by altering the phosphorylation status of serine 62. Furthermore, we identified that the TATA box of P1 promoter might play a critical role in MC-LR-induced up-regulation of c-myc promoter activity. These results suggested that MC-LR can up-regulate c-myc protein level at both transcriptional and post-translational levels, which might contribute to hepatic tumorigenesis in animals and humans (Fig. 5).
Fig. 5. Proposed model for the up-regulation of c-myc protein in HEK293 cells induced by MC-LR. MC-LR induces c-myc protein mainly by stimulating the phosphorylation of serine 62 of c-myc, which was mediated by the inhibition of the activity of PP2A. Moreover, the TATA box for P1 of c-myc may be involved in the activation of c-myc promoter which may contribute to the MC-LR-induced upregulation of c-myc protein. However, MC-LR may also have a relatively weak negative effect on c-myc protein through the proteasome pathway.
To our knowledge, this study possibly provides the first direct evidence of promoting effects of MC-LR on c-myc protein stability. C-myc, as an important tumor-promoting factor, has been well studied. In this study, we showed that c-myc protein level was increased in HEK293 cells after exposure to MC-LR. Previous studies have established that MC-LR is a potent inhibitor of PP2A by directly binding to the PP2A catalytic subunit (Mackintosh et al., 1990; Toivola et al., 1994; Xing et al., 2006). On the other hand, studies have suggested that PP2A negatively regulates c-myc protein level by directly dephosphorylating the serine 62 of c-myc, which is a key event in c-myc degradation pathway (Arnold and Sears, 2006; Sears, 2004). Moreover, serine 62 is phosphorylated by ERK and threonine 58 is phosphorylated by GSK-3 (Sears et al., 2000). Previous studies have shown that PP2A could inactivate ERKs and GSK-3 (Kins et al., 2003; Seeling et al., 1999). So, in order to evaluate the effect of PP2A on MC-LR-induced up-regulation of c-myc protein level, we overexpressed the PP2AC␣, which is the direct target of MC-LR (Xing et al., 2006). The result showed that PP2A could decrease the MC-LR-induced up-regulation of c-myc protein level. These results are consistent with what we expected. To further investigate the exact mechanism of MC-LR-induced up-regulation of c-myc protein, the c-mycS62A and c-mycT58A mutants were used. The results showed that, after mutation of serine 62, PP2A lost its ability to reduce the c-myc protein level and MC-LR no longer increases the protein level of c-myc. Based on these results, we proposed that MC-LR could increase c-myc protein stability by altering the phosphorylation status of serine 62. Moreover, interestingly, we found that the protein level of c-mycS62A was decreased after exposure to MC-LR, which suggest that MC-LR also could induce c-myc protein degradation by a PP2A/serine 62-independent pathway. Previous study has shown that the half-life of c-mycS62A is shorter than the wild-type, and the level of c-mycS62A is enhanced in the presence of the proteasome inhibitor, which suggested that there is another, more effective serine 62-independent proteasome pathway for c-myc degradation (Sears et al., 2000). Several proteomics studies showed that MC-LR could affect ubiquitin/proteasome system (Chen et al., 2005; Wang et al., 2010b), which might be responsible for the MC-LR induced down-regulation of c-mycS62A . The connection between MC-LR and ubiquitin/proteasome system is worth to be further investigated. Hence, these results suggested that the MC-LR has dual effects on c-myc protein stability, both stabilizing and destabilizing c-myc protein. And the former is stronger than the latter on the wild-type c-myc. Moreover, after mutation of threonine 58, our results showed that there was no significant association between MC-LR and threonine 58 of c-myc. However, we found that PP2A still could facilitate c-mycT58A degradation. Previous study has shown that the c-MycT58A mutant could not be dephosphorylated by PP2A (Yeh et al., 2004). So, the protein level of c-MycT58A mutant cannot be affected by MC-LR or PP2A in the PP2A/serine 62 pathway. However, it also has been shown that the mutant c-mycT58A protein is increased by stimulation of Ras/Raf/MEK/ERK pathway (Sears et al., 2000). And it has been shown that PP2A could inactivate ERKs (Kins et al., 2003). Therefore, the PP2A-induced down-regulation of c-mycT58A protein is probably caused by the inhibition of ERK. These results confirmed that MC-LR can stabilize c-myc protein by altering the phosphorylation status of serine 62. It has been well established that MC-LR could induce c-myc expression at the transcriptional level (Li et al., 2009; Takumi et al., 2010). However, the underlying mechanism is still unclear. In this study, we showed that MC-LR up-regulated the c-myc promoter activity in HEK293 cells. Next, to define the possible response elements in c-myc promoter for MC-LR-induced up-regulation of c-myc promoter activity, three mutants (P1 mut1, P1 mut2, P2 mut) of c-myc promoter were employed. The results showed that the P1
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mut1 and the P1 mut2 could reverse the up-regulation of c-myc promoter activity induced by MC-LR, while the P2 mut couldn’t. These results suggested that the TATA box for P1 might play an important role in MC-LR-induced up-regulation of c-myc mRNA. Previous studies have suggested that ERK could also induce c-myc expression at the transcriptional level (Kerkhoff et al., 1998; Verykokakis et al., 2007). So ERK might be an effective approach to further study the mechanism of MC-LR-induced up-regulation of c-myc promoter activity. In addition, the effect of MC-LR on c-myc promoter activity was further confirmed the result of real-time PCR. However, although the c-myc mRNA was significantly increased after 10 h of MC-LR treatment, it is only about 1.3-fold higher than the control. This result in our study was probably caused by the relatively low sensitivity of HEK293 cells to MC-LR (Fischer et al., 2010). On the other hand, we found that the c-myc mRNA in HEK293 cells was not changed significantly after 24 h of MC-LR treatment (data not shown), which might be caused by an autoregulatory mechanism. However, the result showed that the c-myc protein level was significantly increased after 24 h of MC-LR treatment. So we suggested that the post-translational level pathway is stronger than the transcriptional level pathway of c-myc response to MC-LR toxicity. So, in this study, we proposed that MC-LR up-regulated the c-myc protein level mainly via the PP2A/serine 62 pathway. 5. Conclusions In the present study, we showed that MC-LR increased c-myc protein level mainly by altering the phosphorylation status of serine 62, which was mediated by the inhibition of the activity of PP2A. Moreover, we demonstrated that the TATA box for P1 of cmyc might play an important role in MC-LR-induced up-regulation of c-myc mRNA, which may also contribute to MC-LR-induced up-regulation of c-myc protein level. And we also found that MCLR may have a relatively weak negative effect on c-myc protein through the proteasome pathway. The connection between MC-LR and ubiquitin/proteasome system is interesting and worth to be further investigated. The results of this study may contribute to the knowledge of the potential carcinogenicity of MC-LR. Conflict of interest statement The authors declare that there are no conflicts of interest. Transparency document The Transparency document associated with this article can be found in the online version. Acknowledgements The authors thank Dr. Xiaoyang Wan for helping to revise the manuscript. This work was supported by the National Natural Science Foundation of China (31322013) and the State Key Laboratory of Freshwater Ecology and Biotechnology (2011FBZ07). Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.tox.2014.02.015. References Arnold, H.K., Sears, R.C., 2006. Protein phosphatase 2A regulatory subunit b56 alpha associates with c-Myc and negatively regulates c-Myc accumulation. Mol. Cell. Biol. 26, 2832–2844.
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