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Camptothecin and topotecan inhibit adipocyte differentiation by inducing degradation of PPARγ
Biochemical and Biophysical Research Communications 463 (2015) 1122e1128
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Camptothecin and topotecan inhibit adipocyte differentiation by inducing degradation of PPARg Jung-Hoon Kim a, Manhyung Jeong a, Sang-sik Lee b, Jaewhan Song a, * a b
Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea Department of Biomedical Engineering, Catholic Kwandong University, Gangneung, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 June 2015 Accepted 9 June 2015 Available online 12 June 2015
Camptothecin is an anti-cancer drug extracted from Camptotheca acuminata, a tree native to mainland China. Phase III clinical trials for camptothecin have been completed, and it is now used as a chemotherapeutic reagent. We identified a novel function of camptothecin that affects adipocyte differentiation. Following treatment with camptothecin, endogenous or overexpressed PPARg becomes destabilized; this was prevented in the presence of MG132, a proteasome inhibitor. Our findings suggest that camptothecin is able to induce proteasome-dependent degradation of PPARg. The ubiquitylation of PPARg increased in the presence of camptothecin. Adipogenic differentiation of 3T3-L1 cells was prevented by campothecin and topotecan, but not by irinotecan, confirming our initial findings. Our results suggest a possible role for camptothecin analogs in the regulation of PPARg. © 2015 Elsevier Inc. All rights reserved.
Keywords: PPARg Adipocyte differentiation Camptothecin Topotecan
1. Introduction The critical metabolic regulator PPARg is involved in obesity, cardiovascular diseases, inflammation, and diabetes mellitus [1e3]. As a transcription factor heterodimerizing with RXR (retinoic X receptor), PPARg translocates to the nucleus and induces a variety of target genes, including CD36, fatty acid binding protein 4, adiponectin, and CCAAT/enhancer binding protein a. PPARg also interacts with several other molecules, such as members of the C/EBP family (a,b, and d), SREBPs, PRDM16, PGC-1a, ZFP423, REV-ERBa, GATA3, and several microRNAs, thereby coordinating adipogenesis [4e9]. The induction of lipodystrophy in mice and humans when PPARg is defective highlights its significance and major roles in adipogenesis [1]. Moreover, forced expression of PPARg could stimulate adipocyte-like phenotypic changes in fibroblasts [10].
Abbreviations: PPARg, peroxisome proliferator-activated receptor g; TZDs, Thiazolidinediones; SREBPs, sterol regulatory element-binding protein; PRDM16, PR domain containing protein 16; PGC-1a, Peroxisome proliferator-activated receptor gamma coactivator 1- a; ZFP423, zinc-finger protein 423; GATA3, GATA binding protein 3; TGF-b, Transforming growth factor-b; BMP, Bone morphogenetic protein; MKRN1, makorin RING finger protein 1; PMSF, Phenylmethanesulfonylfluoride; NEM, N-ethylmaleimide; IBMX, 3-isobutyl-1methylxanthine. * Corresponding author. Department of Biochemistry, Yonsei University, Yonseiro 50, Seodaemun-gu, Seoul 120-749, Republic of Korea. E-mail address: [email protected] (J. Song). http://dx.doi.org/10.1016/j.bbrc.2015.06.069 0006-291X/© 2015 Elsevier Inc. All rights reserved.
The complex roles of PPARg in adipogenesis and regulation of metabolic pathways are demonstrated by its crosstalk with a variety of signal transduction factors, such as WNT, TGF-b, and BMP [11e13]. Therefore, PPARg is considered to have a major influence on lipid-related metabolic pathways at the cellular and systemic levels. Clinically, PPARg is important as a chemical agonist, affecting systemic glucose and lipid homeostasis [14]. Thiazolidinediones (TZDs) were first identified as a class of insulin-sensitizing drugs; they were later identified as artificial ligands of PPARg [15]. Genetic evidence from PPARg gain of function and loss of function experiments, further confirmed that PPARg in adipose tissue is the biological receptor of TZDs. The stimulation of PPARg by TZDs increases lipid influx into adipocytes and their synthesis, thereby lowering the levels of lipids and glucose in the blood of the diabetic patient [16]. Unfortunately, the use of TZDs results in a variety of side effects, such as weight gain, fluid retention, edema, and higher risk for congestive heart failure in patients. These side effects have raised questions regarding the efficacy and suitability of TZDs as therapeutic drugs for metabolic diseases [17]. This issue is now being circumvented through the development of highly potent and more selective PPARg modulators (SPPARMg), and activators of PPARg expression, such as harmine or phenamil [18,19]. In addition to PPARg regulation via artificial drugs, results from recent studies suggest that targeting post-translational modifications might help to control PPARg activities. As an example,
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phosphorylation of PPARg by CDK5 in adipose tissue and pancreatic b-cells induces glucose intolerance; however, the non-TZD drug, SR1664, blocks the effects of CDK5 on PPARg [20]. Sumoylation by PIAS1 or ubiquitylation by MKRN1 and Siah affects PPARg stability and activities, highlighting the possibility that novel regulators can be developed to target these systems [21e23]. Camptothecin, which is isolated from the tree Camptotheca acuminata, is used as a chemotherapeutic agent for various cancers [24]. It is a negative regulator of class 1 topisomerases, and induces the apoptosis of cancer cells. Based on the structure of camptothecin, topotecan and irinotecan were developed for use as anticancer drugs [25]. Screening of registered US Food and Drug Administration (FDA) drugs was used to identify chemicals affecting adipogenesis, with camptothecin identified as a negative regulator of PPARg. Topotecan, but not irinotecan, exhibited effects similar to those of camptothecin on the function of PPARg. Camptothecin and topotecan induced destabilization of PPARg via ubiquitylation and proteasome-dependent degradation. Our findings of an additional target of camptothecin and its analog might require further studies on metabolic complications induced by these drugs. 2. Materials and methods 2.1. Plasmids We used pcDNA3.1-PPARg2 [22] and site-directed mutagenesis to generate pcDNA3.1-PPARg2 C139A mutants (TAKARA Bio Company, Otsu, Japan), with specific primers employed (50 -AAC TCC CTC ATG GCC ATT GAG GCA CGA GTC TGT GGG GAT-30 and 50 -ATC CCC ACA GAC TCG TGC CTC AAT GGC CAT GAG GGA GTT-30 ). The pTKPPREx3-luc plasmid was kindly provided by H. W. Lee (Yonsei University, Seoul, Korea). 2.2. Antibodies and chemicals We used antibodies against PPARg (mouse sc-7273X, rabbit sc7196X; Santa Cruz Biotechnology, CA, USA), aP2 (goat sc-18661; Santa Cruz Biotechnology, CA, USA), C/EBPa (rabbit sc-61X; Santa Cruz Biotechnology, CA, USA), hemagglutinin (HA; mouse sc-7392; Santa Cruz Biotechnology, CA, USA), myc (sc-40; Santa Cruz Biotechnology, CA, USA), MKRN1 (A300-990A; Bethyl Laboratories, Montgomery, USA), and b-actin (A5316; SigmaeAldrich, St. Louis, MO, USA) for western blotting analyses. Camptothecin (C9911), topotecan (T2705), irinotecan (I1406), Aprotinin (A1153), Pepstatin A (P5318), Leupeptin (L2884), Phenylmethanesulfonylfluoride (PMSF; P7626), N-ethylmaleimide (NEM; E3876), dimethyl sulfoxide (DMSO; D8418), dexamethasone (D1756), 3-isobutyl-1methylxanthine (IBMX; I5879), and Oil Red O (O0625) were purchased from SigmaeAldrich (St. Louis, MO, USA), while insulin (11 376 497 001) was from Roche (Mannheim, Germany). 2.3. Transfection Human lung carcinoma cells H1299 were transfected using Lipofectamine® 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. PC-3 cells were transfected with the short interfering RNAs MKRN1 #5 (50 -GGC GAA GCT GAG TCA AGA A-30 ) and MKRN1 #6 (50 -GGA TCC TCT CCA ACT GCA A-30 ) using Lipofectamine® RNAiMAX™ (Invitrogen, Carlsbad, CA, USA). 2.4. Cell culture and adipocyte differentiation H1299 cells were maintained in DMEM (Gibco, NY 14072, USA) containing 10% FBS. Adipocyte differentiation was performed with
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3T3-L1 pre-adipocyte cells. The 3T3-L1 cells were cultured in DMEM with 10% FBS for 3e5 days. Once cultures were 90e100% confluent, cells were differentiated with DMEM containing 10% FBS, 1 mM dexamethasone, 520 mM IBMX, and 1 mg/mL insulin for 2 days. Cells were then cultured in DMEM containing 10% FBS and 5 mg/mL insulin for an additional 2 days. Differentiated cells were observed by staining with Oil Red O, as previously described [22]. The Oil Red O stained cells were visualized with a CKX41SF microscope (Olympus Inc., Tokyo 163-0914, Japan). The Oil Red O dye was extracted from stained cells using isopropanol, and density measurements were conducted at 500 nm using a Benchmark Plus microplate reader (Bio-Rad Laboratories Inc., Hercules, CA 94547, USA).
2.5. Screening chemicals FDA approved drug library (L1300, Selleckchem, Houston, TX 77054 USA) was employed to find chemicals regulating adipocyte differentiation. The 3T3-L1 cells differentiated for 2 days were treated with each chemical (10 mM) for additional 2 days followed by the Oil Red O staining and extraction analyses as mentioned above.
2.6. Quantitative RT-PCR analysis Extraction of total RNA was performed using Trizol reagent, according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). The isolated total RNA was used to synthesize cDNA with the aid of M-MLV reverse transcriptase (TAKARA Bio Company, Otsu, Japan). Samples were then analyzed by quantitative PCR using a QuantiTect SYBR Green PCR Kit (Qiagen, CA 91355, USA) and primers specific for 36B4 (50 -AGA TGC AGC AGA TCC GCA T-30 and 50 -GTT CTT GCC CAT CAG CAC C-30 ), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 50 -GGC TGC TTT TAA CTC TGG TA-30 and 50 -ACT TGA TTT TGG AGG GAT CT-30 ), mouse PPARg (50 -CCA TTC TGG CCC ACC AAC-30 and 50 -AAT GCG AGT GGT CTT CCA TCA-30 ), human PPARg (50 -TTC AGA AAT GCC TTG CAG TG-30 and 50 -CCA ACA GCT TCT CCT TCT CG-30 ), and aP2 (50 -CAC CGC AGA CGA CAG GAA G30 and 50 -GCA CCT GCA CCA GGG C-30 ). The expression level of each gene was determined from 3T3-L1 cell samples, and was normalized to the expression of the 36B4 gene in the same sample. The expression levels of genes in PC-3 and H1299 cell lines were normalized to GAPDH gene expression levels.
2.7. Ubiquitylation assays We transfected H1299 cells with ubiquitin conjugated to HA (PRK5-HA-ub), pcDNA3.1 and pcDNA3.1-PPARg plasmids. Cell were harvested with PBS containing 10 nM NEM, and then lysed by boiling in 1% SDS for 10 min. Samples were then diluted using lysis buffer containing protease inhibitor cocktail (2 mM Aprotinin, 1 mM Pepstatin A, 2 mM Leupeptin, 200 mM PMSF), and NEM to achieve a final SDS concentration of 0.1%. Lysates were subjected to immunoprecipitation using an antibody against mouse PPARg, followed by western blotting with appropriate antibodies.
2.8. Statistical analyses Statistical analyses were conducted using GraphPad Prism (GraphPad Software Inc., USA) and results presented as the mean ± SD. A P-value less than 0.05 was considered statistically significant.
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3. Results 3.1. Adipocyte differentiation was repressed by camptothecin To identify small molecules that regulate the function of PPARg, we screened FDA-approved chemicals for their abilities to affect adipocyte differentiation of 3T3-L1 cells (Fig. 1A). Of the 460 chemicals we screened, a large number induced cell death because of their toxicity. Treatment of cells with ivermetin and rosiglitazone, known activators of adipocyte differentiation, also resulted in the differentiation of cells in our hand confirming the validity of our assays [26e28]. Among chemicals tested, adefovir dipivoxil, Oxandrolone, Pemetrexed disodium, Camptothecin and topotecan were identified as inhibitors of adipocyte differentiation (Fig. 1A and B, data not shown). The decreased levels of adipocyte differentiation were not due to toxicity, as cell numbers were similar to those in control cultures, regardless if those chemicals were present (Fig. 1C). Among these chemicals, we decided to work on camptothecin and topotecan for further analyses as they are well known chemotherapeutic agents. Interestingly, irinotecan, a chemical analog of camptothecin, did not have any effect on the differentiation of adipocyte (data not shown). For more convincing results, we retested the abilities of camptothecin, topotecan and irinotecan to affect adipocyte differentiation (Fig. 2A). Topotecan, but not irinotecan, suppressed adipocyte differentiation (Fig. 2B and C). There was little difference in cell numbers among cultures; therefore, suppression of differentiation did not affect the proliferation or survival of cells (Fig. 2B). Confirming these results, we observed that PPARg, C/EBP-a and aP2 levels decreased following treatment with camptothecin or topotecan, but not with irinotecan (Fig. 2D and E). The efficacy of camptothecin and topotecan appeared to be similar, with both chemicals suppressing adipocyte differentiation at similar
concentrations (Fig. 3A and B). Camptothecin and topotecan negatively regulated adipocyte differentiation by suppressing the expression of PPARg. 3.2. Camptothecin and topotecan induce destabilization of PPARg via proteasome-dependent pathways We treated the PC3 cell line, which constitutively expresses PPARg [29], with camptothecin, topotecan, or irinotecan. Following treatment, PPARg levels were determined using western blotting. Treatment with camptothecin or topotecan resulted in a decrease of endogenous PPARg levels, while irinotecan did not affect PPARg expression (Fig. 4A). There was no change in the levels of PPARg mRNAs following treatment with camptothecin or topotecan, suggesting that PPARg levels are regulated post-translationally (Fig. 4A). PPARg levels were decreased when we increased the concentration of camptothecin or topotecan, or subjected cells to longer treatments with these chemicals, suggesting that PPARg instability was a direct result of camptothecin or topotecan treatments (Fig. 4B and C). The degradation of PPARg by these two chemicals was recovered using MG132, indicating that PPARg might be destabilized by proteasome-dependent pathways (Fig. 4D). We showed that the negative regulation of PPARg by camptothecin and topotecan possibly occurs via post-translational modifications. Proteasome-dependent pathways appear to play an important role in the destabilization of PPARg during camptothecin and topotecan treatments. 3.3. Camptothecin and topotecan induce increased ubiquitylation of PPARg Camptothecin and topotecan induce increased destabilization of PPARg through proteasome-dependent pathways. Ubiquitylation
Fig. 1. Camptothecin strongly suppresses adipocyte differentiation. (a) The suppressive effects of US FDA-approved chemicals upon adipocyte differentiation were investigated. Murine 3T3-L1 cells were differentiated into adipocytes and stained with Oil Red O. The Oil Red O dye was extracted with isopropanol and measured at 500 nm using a microplate reader. (b) 3T3-L1 cells were differentiated into adipocytes in the presence of camptothecin and stained with Oil Red O. (c) Oil Red O-stained cells were examined by microscopy.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. Camptothecin and topotecan suppress adipocyte differentiation. (a) The chemical structure of camptothecin, topotecan, and irinotecan. (b, c) Murine 3T3-L1 cells were differentiated with the indicated chemical for 6 days. Cells were stained with Oil Red O and examined by microscopy. (d) Expression levels of PPARg and other target proteins in differentiated cell lysates were determined by western blotting. (e) The mRNA expression of PPARg and aP2 in cell lysates was analyzed by quantitative RT-PCR. Data are presented as mean ± SD; n ¼ 3 with ***P < 0.001 compared to each lane. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Camptothecin and topotecan inhibit adipocyte differentiation. (a, b) Differentiated 3T3-L1 cells were treated with the indicated concentration of camptothecin or topotecan. Cells were stained with Oil Red O, which was then extracted and quantified. Data are presented as mean ± SD; n ¼ 3 with ***P < 0.001 compared to each lane. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Camptothecin and topotecan induce the degradation via ubiquitylation of PPARg and reduce its stability. (a) PC-3 Cells were treated with or without 10 mM Camptothecin, Topotecan, or Irinotecan for 6 h. Cell lysates were then analyzed by western blotting and quantitative RT-PCR. (b) Expression of the PPARg2 protein was suppressed following treatment with camptothecin or topotecan, however mRNA expression levels were unaffected. H1299 cells were transfected with pcDNA3.1-PPARg2 or 6XMYC and treated with increasing concentrations of camptothecin or topotecan for 6 h. Cell lysates were analyzed by western blotting and quantitative RT-PCR. (c) PC-3 cells were treated at the indicated times with 10 mM camptothecin or topotecan. Cell lysates were analyzed by western blotting. The stability of the PPARg protein was reduced following treatment. (d) H1299 cells were transfected with pcDNA3.1-PPARg or 6XMYC, and treated with camptothecin or topotecan at the indicated concentration, in the presence or absence of MG132. Cell lysates were analyzed by western blotting using the indicated antibodies. (e) H1299 cells were transfected with PRK5-HA-ub or pcDNA3.1-PPARg, and treated with MG132 for 6 h, in the presence or absence of 10 mM camptothecin or topotecan for 6 h. Cell lysates were immunoprecipitated using monoclonal PPARg antibody, were analyzed by western blotting.
analyses were conducted using HA-ub and overexpressed PPARg in H1299 cells, in the presence or absence of camptothecin or topotecan, and in the presence of MG132. We precipitated PPARg, and then conducted western blotting analyses under denaturing conditions as previously reported [22]. Levels of ubiquitinated PPARg were increased following treatment with camptothecin or topotecan (Fig. 4E). The overall levels of ubiquitinated proteins in whole cell lysates were unchanged, indicating that increased PPARg ubiquitylation was due to the effects of camptothecin and topotecan (Fig. 4E). MKRN1 is a known E3 ligase of PPARg; therefore, the possibility of MKRN1 acting as an E3 ligase of PPARg was tested in the presence of camptothecin [22]. When MKRN1 expression was knocked down, there was an increase in PPARg protein levels (Sup.Fig. 1A). However, accumulation of PPARg was not observed when either camptothecin or topotecan was added, suggesting that MKRN1 might not be involved in the camptothecin- or topotecan-induced degradation of PPARg (Sup.Fig. 1A). PPARg is also known to possess E3 ligase activities; therefore, we investigated the possibility of self-ubiquitylation and degradation induced by camptothecin or topotecan. We used the mutant PPARg C139A, whose E3 ligase activities were defective, in conjunction with WT PPARg [30]. Camptothecin and topotecan facilitated degradation of the WT and mutant PPARg, indicating that self-ubiquitylation was not involved (Sup.Fig. 1B). In summary, treatment with camptothecin or
topotecan resulted in increased PPARg ubiquitylation, thus accelerating its degradation. During these processes, the known E3 ligase of PPARg did not appear to be involved, implying the existence of other PPARg regulatory pathways affected by camptothecin or topotecan. 4. Discussion PPARg is a key transcription factor that induces adipogenesis [31]. The formation of a PPARg-RXR complex in the presence of ligands, such as fatty acids, leads to the activation of PPARg, a necessary step for adipocyte differentiation [32]. This process increases the volume of systemic body lipid deposits; however, levels of blood triacylglycerol, free fatty acids, and glucose are reduced. This results in a decrease in insulin resistance, making PPARg a suitable target for the treatment of patients with diabetes. Agonists of PPARg, such as TZDs, are currently used for the treatment of type 2 diabetes [33]. Agonists of PPARg have long been regarded as cures for metabolic disorders, while antagonists such as GW9662 and T0070907, are known to have anti-tumor effects [34]. We observed that two topoisomerase inhibitors, camptothecin and topotecan, which are also well-known anti-tumor agents that are widely used, could function as PPARg antagonists [35]. Camptothecin and topotecan caused the degradation of PPARg via ubiquitylation and proteasome-dependent pathways. Verifying our initial findings,
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these two chemicals suppressed the adipocyte differentiation of 3T3L1 cells. While their ability to induce the degradation of PPARg is clear, the mechanisms leading to this outcome are yet to be identified. Ablation of MKRN1 expression, the E3 ligase of PPARg, failed to suppress PPARg degradation, suggesting that MKRN1 is not involved in camptothecin-mediated PPARg degradation. PPARg is also known to possess E3 ligase activity that can results in selfubiquitylation and possible degradation. When PPARg C139A, an E3 ligase-defective mutant of PPARg, was treated with camptothecin, it was also degraded. Further research is required to uncover the cellular factors involved in PPARg regulatory pathways. Camptothecin and topotecan have pentacyclic planar ring structures, which are important with respect to their ability in inhibiting topoisomerases [36]. Another camptothecin derivative, irinotecan, was unable to induce the degradation of PPARg. This suggests that the attachment of a pyridine to C10 of camptothecin by an ester bond, to form irinotecan, somehow interferes with its ability to induce PPARg degradation. Camptothecin and its analogs have been previously shown to induce the degradation of certain target proteins. As an example, FL118, an analog of irinotecan and topotecan, induced the proteasomal degradation of HDMX, resulting in the activation of p53 [37]. In contrast, camptothecin was unable to affect HDMX. Camptothecin and topotecan were also able to activate NF-kB, by inducing the degradation of IkBa through a ubiquitylation-dependent proteasome pathway [38]. Topotecan and camptothecin are also able to induce degradation of topoisomerase I, suppressing the release of DNA supercoils, and leading to the death of cancer cells [39]. To date, there is no sufficient mechanistic explanation of how treatment with camptothecin, or its analogs, results in the degradation of target molecules. Camptothecin and its analogs could directly regulate ubiquitylation and degradation; however, we cannot exclude the possibility that these chemicals might indirectly affect degradation by associating with other post-translational modifications, such as sumoylation and phosphorylation. Post-translational modifications have been intricately linked with ubiquitylation-mediated proteasomal degradation. Acknowledgments This study was supported by the National Cancer Center, Korea (NCC-1420300), and by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), which was funded by the Ministry of Health & Welfare of the Republic of Korea (HI12C1280). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2015.06.069. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.06.069. References [1] Y. Barak, M.C. Nelson, E.S. Ong, Y.Z. Jones, P. Ruiz-Lozano, K.R. Chien, A. Koder, R.M. Evans, PPAR gamma is required for placental, cardiac, and adipose tissue development, Mol. Cell 4 (1999) 585e595. [2] P. Tontonoz, B.M. Spiegelman, Fat and beyond: the diverse biology of PPARgamma, Annu Rev. Biochem. 77 (2008) 289e312. [3] K.W. Park, D.S. Halperin, P. Tontonoz, Before they were fat: adipocyte progenitors, Cell Metab. 8 (2008) 454e457. [4] A.G. Cristancho, M.A. Lazar, Forming functional fat: a growing understanding of adipocyte differentiation, Nat. Rev. Mol. Cell Biol. 12 (2011) 722e734.
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Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
Camptothecin and topotecan inhibit adipocyte differentiation by inducing degradation of PPARg Jung-Hoon Kim a, Manhyung Jeong a, Sang-sik Lee b, Jaewhan Song a, * a b
Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, Republic of Korea Department of Biomedical Engineering, Catholic Kwandong University, Gangneung, Republic of Korea
a r t i c l e i n f o
a b s t r a c t
Article history: Received 2 June 2015 Accepted 9 June 2015 Available online 12 June 2015
Camptothecin is an anti-cancer drug extracted from Camptotheca acuminata, a tree native to mainland China. Phase III clinical trials for camptothecin have been completed, and it is now used as a chemotherapeutic reagent. We identified a novel function of camptothecin that affects adipocyte differentiation. Following treatment with camptothecin, endogenous or overexpressed PPARg becomes destabilized; this was prevented in the presence of MG132, a proteasome inhibitor. Our findings suggest that camptothecin is able to induce proteasome-dependent degradation of PPARg. The ubiquitylation of PPARg increased in the presence of camptothecin. Adipogenic differentiation of 3T3-L1 cells was prevented by campothecin and topotecan, but not by irinotecan, confirming our initial findings. Our results suggest a possible role for camptothecin analogs in the regulation of PPARg. © 2015 Elsevier Inc. All rights reserved.
Keywords: PPARg Adipocyte differentiation Camptothecin Topotecan
1. Introduction The critical metabolic regulator PPARg is involved in obesity, cardiovascular diseases, inflammation, and diabetes mellitus [1e3]. As a transcription factor heterodimerizing with RXR (retinoic X receptor), PPARg translocates to the nucleus and induces a variety of target genes, including CD36, fatty acid binding protein 4, adiponectin, and CCAAT/enhancer binding protein a. PPARg also interacts with several other molecules, such as members of the C/EBP family (a,b, and d), SREBPs, PRDM16, PGC-1a, ZFP423, REV-ERBa, GATA3, and several microRNAs, thereby coordinating adipogenesis [4e9]. The induction of lipodystrophy in mice and humans when PPARg is defective highlights its significance and major roles in adipogenesis [1]. Moreover, forced expression of PPARg could stimulate adipocyte-like phenotypic changes in fibroblasts [10].
Abbreviations: PPARg, peroxisome proliferator-activated receptor g; TZDs, Thiazolidinediones; SREBPs, sterol regulatory element-binding protein; PRDM16, PR domain containing protein 16; PGC-1a, Peroxisome proliferator-activated receptor gamma coactivator 1- a; ZFP423, zinc-finger protein 423; GATA3, GATA binding protein 3; TGF-b, Transforming growth factor-b; BMP, Bone morphogenetic protein; MKRN1, makorin RING finger protein 1; PMSF, Phenylmethanesulfonylfluoride; NEM, N-ethylmaleimide; IBMX, 3-isobutyl-1methylxanthine. * Corresponding author. Department of Biochemistry, Yonsei University, Yonseiro 50, Seodaemun-gu, Seoul 120-749, Republic of Korea. E-mail address: [email protected] (J. Song). http://dx.doi.org/10.1016/j.bbrc.2015.06.069 0006-291X/© 2015 Elsevier Inc. All rights reserved.
The complex roles of PPARg in adipogenesis and regulation of metabolic pathways are demonstrated by its crosstalk with a variety of signal transduction factors, such as WNT, TGF-b, and BMP [11e13]. Therefore, PPARg is considered to have a major influence on lipid-related metabolic pathways at the cellular and systemic levels. Clinically, PPARg is important as a chemical agonist, affecting systemic glucose and lipid homeostasis [14]. Thiazolidinediones (TZDs) were first identified as a class of insulin-sensitizing drugs; they were later identified as artificial ligands of PPARg [15]. Genetic evidence from PPARg gain of function and loss of function experiments, further confirmed that PPARg in adipose tissue is the biological receptor of TZDs. The stimulation of PPARg by TZDs increases lipid influx into adipocytes and their synthesis, thereby lowering the levels of lipids and glucose in the blood of the diabetic patient [16]. Unfortunately, the use of TZDs results in a variety of side effects, such as weight gain, fluid retention, edema, and higher risk for congestive heart failure in patients. These side effects have raised questions regarding the efficacy and suitability of TZDs as therapeutic drugs for metabolic diseases [17]. This issue is now being circumvented through the development of highly potent and more selective PPARg modulators (SPPARMg), and activators of PPARg expression, such as harmine or phenamil [18,19]. In addition to PPARg regulation via artificial drugs, results from recent studies suggest that targeting post-translational modifications might help to control PPARg activities. As an example,
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phosphorylation of PPARg by CDK5 in adipose tissue and pancreatic b-cells induces glucose intolerance; however, the non-TZD drug, SR1664, blocks the effects of CDK5 on PPARg [20]. Sumoylation by PIAS1 or ubiquitylation by MKRN1 and Siah affects PPARg stability and activities, highlighting the possibility that novel regulators can be developed to target these systems [21e23]. Camptothecin, which is isolated from the tree Camptotheca acuminata, is used as a chemotherapeutic agent for various cancers [24]. It is a negative regulator of class 1 topisomerases, and induces the apoptosis of cancer cells. Based on the structure of camptothecin, topotecan and irinotecan were developed for use as anticancer drugs [25]. Screening of registered US Food and Drug Administration (FDA) drugs was used to identify chemicals affecting adipogenesis, with camptothecin identified as a negative regulator of PPARg. Topotecan, but not irinotecan, exhibited effects similar to those of camptothecin on the function of PPARg. Camptothecin and topotecan induced destabilization of PPARg via ubiquitylation and proteasome-dependent degradation. Our findings of an additional target of camptothecin and its analog might require further studies on metabolic complications induced by these drugs. 2. Materials and methods 2.1. Plasmids We used pcDNA3.1-PPARg2 [22] and site-directed mutagenesis to generate pcDNA3.1-PPARg2 C139A mutants (TAKARA Bio Company, Otsu, Japan), with specific primers employed (50 -AAC TCC CTC ATG GCC ATT GAG GCA CGA GTC TGT GGG GAT-30 and 50 -ATC CCC ACA GAC TCG TGC CTC AAT GGC CAT GAG GGA GTT-30 ). The pTKPPREx3-luc plasmid was kindly provided by H. W. Lee (Yonsei University, Seoul, Korea). 2.2. Antibodies and chemicals We used antibodies against PPARg (mouse sc-7273X, rabbit sc7196X; Santa Cruz Biotechnology, CA, USA), aP2 (goat sc-18661; Santa Cruz Biotechnology, CA, USA), C/EBPa (rabbit sc-61X; Santa Cruz Biotechnology, CA, USA), hemagglutinin (HA; mouse sc-7392; Santa Cruz Biotechnology, CA, USA), myc (sc-40; Santa Cruz Biotechnology, CA, USA), MKRN1 (A300-990A; Bethyl Laboratories, Montgomery, USA), and b-actin (A5316; SigmaeAldrich, St. Louis, MO, USA) for western blotting analyses. Camptothecin (C9911), topotecan (T2705), irinotecan (I1406), Aprotinin (A1153), Pepstatin A (P5318), Leupeptin (L2884), Phenylmethanesulfonylfluoride (PMSF; P7626), N-ethylmaleimide (NEM; E3876), dimethyl sulfoxide (DMSO; D8418), dexamethasone (D1756), 3-isobutyl-1methylxanthine (IBMX; I5879), and Oil Red O (O0625) were purchased from SigmaeAldrich (St. Louis, MO, USA), while insulin (11 376 497 001) was from Roche (Mannheim, Germany). 2.3. Transfection Human lung carcinoma cells H1299 were transfected using Lipofectamine® 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. PC-3 cells were transfected with the short interfering RNAs MKRN1 #5 (50 -GGC GAA GCT GAG TCA AGA A-30 ) and MKRN1 #6 (50 -GGA TCC TCT CCA ACT GCA A-30 ) using Lipofectamine® RNAiMAX™ (Invitrogen, Carlsbad, CA, USA). 2.4. Cell culture and adipocyte differentiation H1299 cells were maintained in DMEM (Gibco, NY 14072, USA) containing 10% FBS. Adipocyte differentiation was performed with
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3T3-L1 pre-adipocyte cells. The 3T3-L1 cells were cultured in DMEM with 10% FBS for 3e5 days. Once cultures were 90e100% confluent, cells were differentiated with DMEM containing 10% FBS, 1 mM dexamethasone, 520 mM IBMX, and 1 mg/mL insulin for 2 days. Cells were then cultured in DMEM containing 10% FBS and 5 mg/mL insulin for an additional 2 days. Differentiated cells were observed by staining with Oil Red O, as previously described [22]. The Oil Red O stained cells were visualized with a CKX41SF microscope (Olympus Inc., Tokyo 163-0914, Japan). The Oil Red O dye was extracted from stained cells using isopropanol, and density measurements were conducted at 500 nm using a Benchmark Plus microplate reader (Bio-Rad Laboratories Inc., Hercules, CA 94547, USA).
2.5. Screening chemicals FDA approved drug library (L1300, Selleckchem, Houston, TX 77054 USA) was employed to find chemicals regulating adipocyte differentiation. The 3T3-L1 cells differentiated for 2 days were treated with each chemical (10 mM) for additional 2 days followed by the Oil Red O staining and extraction analyses as mentioned above.
2.6. Quantitative RT-PCR analysis Extraction of total RNA was performed using Trizol reagent, according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). The isolated total RNA was used to synthesize cDNA with the aid of M-MLV reverse transcriptase (TAKARA Bio Company, Otsu, Japan). Samples were then analyzed by quantitative PCR using a QuantiTect SYBR Green PCR Kit (Qiagen, CA 91355, USA) and primers specific for 36B4 (50 -AGA TGC AGC AGA TCC GCA T-30 and 50 -GTT CTT GCC CAT CAG CAC C-30 ), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 50 -GGC TGC TTT TAA CTC TGG TA-30 and 50 -ACT TGA TTT TGG AGG GAT CT-30 ), mouse PPARg (50 -CCA TTC TGG CCC ACC AAC-30 and 50 -AAT GCG AGT GGT CTT CCA TCA-30 ), human PPARg (50 -TTC AGA AAT GCC TTG CAG TG-30 and 50 -CCA ACA GCT TCT CCT TCT CG-30 ), and aP2 (50 -CAC CGC AGA CGA CAG GAA G30 and 50 -GCA CCT GCA CCA GGG C-30 ). The expression level of each gene was determined from 3T3-L1 cell samples, and was normalized to the expression of the 36B4 gene in the same sample. The expression levels of genes in PC-3 and H1299 cell lines were normalized to GAPDH gene expression levels.
2.7. Ubiquitylation assays We transfected H1299 cells with ubiquitin conjugated to HA (PRK5-HA-ub), pcDNA3.1 and pcDNA3.1-PPARg plasmids. Cell were harvested with PBS containing 10 nM NEM, and then lysed by boiling in 1% SDS for 10 min. Samples were then diluted using lysis buffer containing protease inhibitor cocktail (2 mM Aprotinin, 1 mM Pepstatin A, 2 mM Leupeptin, 200 mM PMSF), and NEM to achieve a final SDS concentration of 0.1%. Lysates were subjected to immunoprecipitation using an antibody against mouse PPARg, followed by western blotting with appropriate antibodies.
2.8. Statistical analyses Statistical analyses were conducted using GraphPad Prism (GraphPad Software Inc., USA) and results presented as the mean ± SD. A P-value less than 0.05 was considered statistically significant.
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3. Results 3.1. Adipocyte differentiation was repressed by camptothecin To identify small molecules that regulate the function of PPARg, we screened FDA-approved chemicals for their abilities to affect adipocyte differentiation of 3T3-L1 cells (Fig. 1A). Of the 460 chemicals we screened, a large number induced cell death because of their toxicity. Treatment of cells with ivermetin and rosiglitazone, known activators of adipocyte differentiation, also resulted in the differentiation of cells in our hand confirming the validity of our assays [26e28]. Among chemicals tested, adefovir dipivoxil, Oxandrolone, Pemetrexed disodium, Camptothecin and topotecan were identified as inhibitors of adipocyte differentiation (Fig. 1A and B, data not shown). The decreased levels of adipocyte differentiation were not due to toxicity, as cell numbers were similar to those in control cultures, regardless if those chemicals were present (Fig. 1C). Among these chemicals, we decided to work on camptothecin and topotecan for further analyses as they are well known chemotherapeutic agents. Interestingly, irinotecan, a chemical analog of camptothecin, did not have any effect on the differentiation of adipocyte (data not shown). For more convincing results, we retested the abilities of camptothecin, topotecan and irinotecan to affect adipocyte differentiation (Fig. 2A). Topotecan, but not irinotecan, suppressed adipocyte differentiation (Fig. 2B and C). There was little difference in cell numbers among cultures; therefore, suppression of differentiation did not affect the proliferation or survival of cells (Fig. 2B). Confirming these results, we observed that PPARg, C/EBP-a and aP2 levels decreased following treatment with camptothecin or topotecan, but not with irinotecan (Fig. 2D and E). The efficacy of camptothecin and topotecan appeared to be similar, with both chemicals suppressing adipocyte differentiation at similar
concentrations (Fig. 3A and B). Camptothecin and topotecan negatively regulated adipocyte differentiation by suppressing the expression of PPARg. 3.2. Camptothecin and topotecan induce destabilization of PPARg via proteasome-dependent pathways We treated the PC3 cell line, which constitutively expresses PPARg [29], with camptothecin, topotecan, or irinotecan. Following treatment, PPARg levels were determined using western blotting. Treatment with camptothecin or topotecan resulted in a decrease of endogenous PPARg levels, while irinotecan did not affect PPARg expression (Fig. 4A). There was no change in the levels of PPARg mRNAs following treatment with camptothecin or topotecan, suggesting that PPARg levels are regulated post-translationally (Fig. 4A). PPARg levels were decreased when we increased the concentration of camptothecin or topotecan, or subjected cells to longer treatments with these chemicals, suggesting that PPARg instability was a direct result of camptothecin or topotecan treatments (Fig. 4B and C). The degradation of PPARg by these two chemicals was recovered using MG132, indicating that PPARg might be destabilized by proteasome-dependent pathways (Fig. 4D). We showed that the negative regulation of PPARg by camptothecin and topotecan possibly occurs via post-translational modifications. Proteasome-dependent pathways appear to play an important role in the destabilization of PPARg during camptothecin and topotecan treatments. 3.3. Camptothecin and topotecan induce increased ubiquitylation of PPARg Camptothecin and topotecan induce increased destabilization of PPARg through proteasome-dependent pathways. Ubiquitylation
Fig. 1. Camptothecin strongly suppresses adipocyte differentiation. (a) The suppressive effects of US FDA-approved chemicals upon adipocyte differentiation were investigated. Murine 3T3-L1 cells were differentiated into adipocytes and stained with Oil Red O. The Oil Red O dye was extracted with isopropanol and measured at 500 nm using a microplate reader. (b) 3T3-L1 cells were differentiated into adipocytes in the presence of camptothecin and stained with Oil Red O. (c) Oil Red O-stained cells were examined by microscopy.(For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 2. Camptothecin and topotecan suppress adipocyte differentiation. (a) The chemical structure of camptothecin, topotecan, and irinotecan. (b, c) Murine 3T3-L1 cells were differentiated with the indicated chemical for 6 days. Cells were stained with Oil Red O and examined by microscopy. (d) Expression levels of PPARg and other target proteins in differentiated cell lysates were determined by western blotting. (e) The mRNA expression of PPARg and aP2 in cell lysates was analyzed by quantitative RT-PCR. Data are presented as mean ± SD; n ¼ 3 with ***P < 0.001 compared to each lane. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3. Camptothecin and topotecan inhibit adipocyte differentiation. (a, b) Differentiated 3T3-L1 cells were treated with the indicated concentration of camptothecin or topotecan. Cells were stained with Oil Red O, which was then extracted and quantified. Data are presented as mean ± SD; n ¼ 3 with ***P < 0.001 compared to each lane. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
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Fig. 4. Camptothecin and topotecan induce the degradation via ubiquitylation of PPARg and reduce its stability. (a) PC-3 Cells were treated with or without 10 mM Camptothecin, Topotecan, or Irinotecan for 6 h. Cell lysates were then analyzed by western blotting and quantitative RT-PCR. (b) Expression of the PPARg2 protein was suppressed following treatment with camptothecin or topotecan, however mRNA expression levels were unaffected. H1299 cells were transfected with pcDNA3.1-PPARg2 or 6XMYC and treated with increasing concentrations of camptothecin or topotecan for 6 h. Cell lysates were analyzed by western blotting and quantitative RT-PCR. (c) PC-3 cells were treated at the indicated times with 10 mM camptothecin or topotecan. Cell lysates were analyzed by western blotting. The stability of the PPARg protein was reduced following treatment. (d) H1299 cells were transfected with pcDNA3.1-PPARg or 6XMYC, and treated with camptothecin or topotecan at the indicated concentration, in the presence or absence of MG132. Cell lysates were analyzed by western blotting using the indicated antibodies. (e) H1299 cells were transfected with PRK5-HA-ub or pcDNA3.1-PPARg, and treated with MG132 for 6 h, in the presence or absence of 10 mM camptothecin or topotecan for 6 h. Cell lysates were immunoprecipitated using monoclonal PPARg antibody, were analyzed by western blotting.
analyses were conducted using HA-ub and overexpressed PPARg in H1299 cells, in the presence or absence of camptothecin or topotecan, and in the presence of MG132. We precipitated PPARg, and then conducted western blotting analyses under denaturing conditions as previously reported [22]. Levels of ubiquitinated PPARg were increased following treatment with camptothecin or topotecan (Fig. 4E). The overall levels of ubiquitinated proteins in whole cell lysates were unchanged, indicating that increased PPARg ubiquitylation was due to the effects of camptothecin and topotecan (Fig. 4E). MKRN1 is a known E3 ligase of PPARg; therefore, the possibility of MKRN1 acting as an E3 ligase of PPARg was tested in the presence of camptothecin [22]. When MKRN1 expression was knocked down, there was an increase in PPARg protein levels (Sup.Fig. 1A). However, accumulation of PPARg was not observed when either camptothecin or topotecan was added, suggesting that MKRN1 might not be involved in the camptothecin- or topotecan-induced degradation of PPARg (Sup.Fig. 1A). PPARg is also known to possess E3 ligase activities; therefore, we investigated the possibility of self-ubiquitylation and degradation induced by camptothecin or topotecan. We used the mutant PPARg C139A, whose E3 ligase activities were defective, in conjunction with WT PPARg [30]. Camptothecin and topotecan facilitated degradation of the WT and mutant PPARg, indicating that self-ubiquitylation was not involved (Sup.Fig. 1B). In summary, treatment with camptothecin or
topotecan resulted in increased PPARg ubiquitylation, thus accelerating its degradation. During these processes, the known E3 ligase of PPARg did not appear to be involved, implying the existence of other PPARg regulatory pathways affected by camptothecin or topotecan. 4. Discussion PPARg is a key transcription factor that induces adipogenesis [31]. The formation of a PPARg-RXR complex in the presence of ligands, such as fatty acids, leads to the activation of PPARg, a necessary step for adipocyte differentiation [32]. This process increases the volume of systemic body lipid deposits; however, levels of blood triacylglycerol, free fatty acids, and glucose are reduced. This results in a decrease in insulin resistance, making PPARg a suitable target for the treatment of patients with diabetes. Agonists of PPARg, such as TZDs, are currently used for the treatment of type 2 diabetes [33]. Agonists of PPARg have long been regarded as cures for metabolic disorders, while antagonists such as GW9662 and T0070907, are known to have anti-tumor effects [34]. We observed that two topoisomerase inhibitors, camptothecin and topotecan, which are also well-known anti-tumor agents that are widely used, could function as PPARg antagonists [35]. Camptothecin and topotecan caused the degradation of PPARg via ubiquitylation and proteasome-dependent pathways. Verifying our initial findings,
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these two chemicals suppressed the adipocyte differentiation of 3T3L1 cells. While their ability to induce the degradation of PPARg is clear, the mechanisms leading to this outcome are yet to be identified. Ablation of MKRN1 expression, the E3 ligase of PPARg, failed to suppress PPARg degradation, suggesting that MKRN1 is not involved in camptothecin-mediated PPARg degradation. PPARg is also known to possess E3 ligase activity that can results in selfubiquitylation and possible degradation. When PPARg C139A, an E3 ligase-defective mutant of PPARg, was treated with camptothecin, it was also degraded. Further research is required to uncover the cellular factors involved in PPARg regulatory pathways. Camptothecin and topotecan have pentacyclic planar ring structures, which are important with respect to their ability in inhibiting topoisomerases [36]. Another camptothecin derivative, irinotecan, was unable to induce the degradation of PPARg. This suggests that the attachment of a pyridine to C10 of camptothecin by an ester bond, to form irinotecan, somehow interferes with its ability to induce PPARg degradation. Camptothecin and its analogs have been previously shown to induce the degradation of certain target proteins. As an example, FL118, an analog of irinotecan and topotecan, induced the proteasomal degradation of HDMX, resulting in the activation of p53 [37]. In contrast, camptothecin was unable to affect HDMX. Camptothecin and topotecan were also able to activate NF-kB, by inducing the degradation of IkBa through a ubiquitylation-dependent proteasome pathway [38]. Topotecan and camptothecin are also able to induce degradation of topoisomerase I, suppressing the release of DNA supercoils, and leading to the death of cancer cells [39]. To date, there is no sufficient mechanistic explanation of how treatment with camptothecin, or its analogs, results in the degradation of target molecules. Camptothecin and its analogs could directly regulate ubiquitylation and degradation; however, we cannot exclude the possibility that these chemicals might indirectly affect degradation by associating with other post-translational modifications, such as sumoylation and phosphorylation. Post-translational modifications have been intricately linked with ubiquitylation-mediated proteasomal degradation. Acknowledgments This study was supported by the National Cancer Center, Korea (NCC-1420300), and by the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), which was funded by the Ministry of Health & Welfare of the Republic of Korea (HI12C1280). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2015.06.069. Transparency document Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2015.06.069. References [1] Y. Barak, M.C. Nelson, E.S. Ong, Y.Z. Jones, P. Ruiz-Lozano, K.R. Chien, A. Koder, R.M. Evans, PPAR gamma is required for placental, cardiac, and adipose tissue development, Mol. Cell 4 (1999) 585e595. [2] P. Tontonoz, B.M. Spiegelman, Fat and beyond: the diverse biology of PPARgamma, Annu Rev. Biochem. 77 (2008) 289e312. [3] K.W. Park, D.S. Halperin, P. Tontonoz, Before they were fat: adipocyte progenitors, Cell Metab. 8 (2008) 454e457. [4] A.G. Cristancho, M.A. Lazar, Forming functional fat: a growing understanding of adipocyte differentiation, Nat. Rev. Mol. Cell Biol. 12 (2011) 722e734.
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