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peg10, an imprinted gene, plays a crucial role in adipocyte differentiation
FEBS Letters 581 (2007) 4272–4278
peg10, an imprinted gene, plays a crucial role in adipocyte differentiation Tomoaki Hishida, Kumiko Naito, Shigehiro Osada, Makoto Nishizuka, Masayoshi Imagawa* Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan Received 28 July 2007; accepted 31 July 2007 Available online 10 August 2007 Edited by Robert Barouki
Abstract An imprinted gene, paternally expressed gene (peg) 10, was isolated as one of the genes expressed early in adipogenesis. The expression of peg10 was elevated after the addition of inducers, and was detected in adipocyte differentiable 3T3-L1 cells, but not observed in the non-adipogenic cell line NIH3T3. Moreover, the knockdown of peg10 by RNA interference (RNAi) inhibited the differentiation of 3T3-L1 cells into lipid-laden adipocytes. Interestingly, peg10 RNAi-treatment reduced the expressions of C/EBPb and C/EBPd, and inhibited mitotic clonal expansion. These findings strongly indicate that peg10 plays a crucial role at the immediate early stage of adipocyte differentiation. 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Adipocyte differentiation; Adipogenesis; Obesity; Peroxisome proliferator-activated receptor c; CCAAT/ enhancer-binding protein; 3T3-L1 cell
1. Introduction Obesity is related to many diseases, such as hypertension, heart disease, and diabetes. Obesity results from an imbalance between energy intake and energy expenditure, which leads to a pathological accumulation of adipose tissue [1]. This development of adipose tissue is caused by hypertrophy and hyperplasia of adipocytes. Therefore, an understanding of the molecular basis of hyperplasia as well as hypertrophy would contribute to the establishment of medical treatments to prevent health risks that can cause serious illness and death. One explanation for the increase in the number of adipocytes is that preadipocytes differentiate into lipid-laden adipocytes, and newly generated cells function as mature adipocytes. Recent studies have revealed that several genes, such as those for peroxisome proliferator-activated receptor c (PPARc) and CCAAT/enhancer-binding protein a (C/EBPa), play important roles in the mid and late stages of adipocyte differentiation [2–4]. The events occurring during the mid- to late* Corresponding author. Fax: +81 52 836 3455. E-mail address: [email protected] (M. Imagawa).
Abbreviations: C/EBP, CCAAT/enhancer-binding protein; DMEM, Dulbecco’s modified Eagle’s medium; fad, factor for adipocyte differentiation; FBS, fetal bovine serum; ORF, open reading frame; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PPARc, peroxisome proliferator-activated receptor c; RNAi, RNA interference; RT-PCR, reverse transcriptase coupled polymerase chain reaction; shRNA, short hairpin RNA; UTR, untranslated region
phase of differentiation are relatively well characterized, but the molecular mechanisms underlying the early stages of adipogenesis remain unknown. To elucidate the initial step of adipocyte differentiation, we have previously isolated genes whose expression is induced in the early stages of the differentiation process, using mouse 3T3-L1 fibroblastic cells. Using the PCR-subtraction system, we isolated 102 genes, including genes for transcription factors and signaling proteins [5–7]. Of these genes, 46 seem to be unknown genes, whose functions remain unclear. We previously isolated some full-length cDNAs of unknown genes and revealed that several of them are novel genes, such as fad24, fad104 and fad158, which positively regulate adipogenesis [8–10]. Here, we report the identification of one of the unknown genes as paternally expressed gene peg10 and the involvement of peg10 in adipocyte differentiation. Its expression was elevated during the differentiation of 3T3-L1 cells. Up-regulation of peg10 expression was detected in adipocyte differentiable 3T3L1, but not in the non-adipogenic cell line NIH-3T3. RNA interference (RNAi)-mediated knockdown of peg10 inhibited the differentiation of 3T3-L1 cells. Moreover, peg10 RNAitreatment impaired mitotic clonal expansion (MCE), which is necessary for adipocyte differentiation. Interestingly, the expression of C/EBPb and C/EBPd at the immediate early stage of the differentiation process was inhibited by the knockdown of peg10. Taken together, these results indicate that peg10 plays important roles in the early stages of adipocyte differentiation.
2. Materials and methods 2.1. RNA isolation and real-time quantitative RT-PCR (Q-PCR) Total RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The total RNA was converted to the single cDNA using a random primer and ReverTra Ace (Toyobo, Osaka, Japan). The cDNA was used as template for Q-PCR. An ABI PRISM 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA) was used to perform the QPCR. The pre-designed primers and probe sets for peg10, PPARc, C/EBPa, C/EBPb, C/EBPd, aP2, and 18S rRNA were obtained from Applied Biosystems. The reaction mixture was prepared using a TaqMan Universal PCR Master Mix (Applied Biosystems) according to the manufacturer’s instructions. The mixture was incubated at 50 C for 2 min and at 95 C for 10 min, and then the PCR was performed at 95 C for 15 s and at 60 C for 1 min for 40 cycles. Relative standard curves were generated in each experiment to calculate the input amounts of the unknown samples. 2.2. Cell culture and differentiation Mouse 3T3-L1 (ATCC CL173) preadipocyte cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% calf serum. For the differentiation experiment, the medium was replaced with DMEM containing 10% fetal bovine serum (FBS), 10 lg/ml
0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.07.074
T. Hishida et al. / FEBS Letters 581 (2007) 4272–4278 insulin, 0.5 mM 3-isobutyl-1-methylxantine, and 1 lM dexamethasone at 2 days post-confluence. After 2 days, the medium was changed to DMEM containing 5 lg/ml insulin and 10% FBS, then the cells were refed every 2 days. Mouse NIH-3T3 (clone 5611, JCRB 0615) fibroblastic cells were maintained in DMEM containing 10% calf serum. 2.3. RNAi experiment The three target regions in the open reading frame (ORF) of mouse peg10 (GenBanke Accession No. NM_130877); region 1 at 481– 501 bp (nucleotide A in the translation initiation codon is the first nucleotide), region 2 at 659–679 bp, and region 3 at 1201–1221 bp, were selected according to the Qiagen siRNA online design tool (http://sirna.qiagen.com/) for the RNAi of peg10. A 19-nucleotide short-hairpin RNA (shRNA)–coding fragment with a 5 0 -TTCAAGAGA-3 loop was subcloned into the ApaI/EcoRI site of pSilencer 1.0-U6 (Ambion, Inc. Austin, TX, USA). As a negative control, the scrambled fragment 5 0 -GTAAGATGAGGCAATGGAG-3 0 which does not have similarity with any mRNA listed in GenBank was generated. The transfection of shRNA expression vectors into 3T3-L1 cells was performed with Nucleofector (Amaxa, Cologne, Germany) using Cell Line Nucleofector Kit V (Amaxa). 3T3-L1 cells were harvested and resuspended in Nucleofector solution at 2.0 · 106 cells/100 ll. After the addition of shRNA expression vectors (9 lg), the cells were transfected using program ‘T-20’ of Nucleofector. Then, they were plated on 12- or 24-well plates. The 3T3-L1 cells were subjected to differentiation experiments 3 days after the transfection. Differentiation experiments were carried out using the same medium as described above. The cell counts were performed with 24-well plates. 2.4. Cell counts Cells were gently rinsed with PBS (phosphate-buffered saline) without Ca2+ and Mg2+ (PBS-), and trypsinized with the use of trypsin (0.25%)-EDTA (0.1%) solution in PBS- for 5 min at 37 C, 5% CO2. The trypsinized cells were subjected to cell counts using a hemocytometer.
3. Results and discussion 3.1. Expression of peg10 during the early stage of adipocyte differentiation With the PCR-subtraction method, we isolated 46 unknown genes, the expression of each of which was up-regulated 3 h after the differentiation was induced. Since the fragments obtained with this protocol were only 300–500 bp long, it was necessary to isolate the full-length cDNAs and identify the ORF of unknown genes. One of these unknown genes was identified as peg10, because the subtracted fragment showed identity to the 3 0 -UTR of mouse peg10, which was later listed in updated databases. Mouse peg10 is a single-copy gene located on proximal chromosome 6. peg10 is a retrotransposon-derived gene, which encodes pol-gag like peptides [11,12]. peg10 has two overlapping ORFs (RF1 and RF2), which results in two proteins being generated from a single mRNA: PEG10-RF1, which is a non-shifting product, and PEG10RF1/2, which is generated as a RF1-RF2 fusion protein by 1 ribosomal frameshifting [12,13]. Recently, it was reported that the knockout of peg10 in mice resulted in early embryonic death owing to defects in the placenta [14]. Moreover, studies showed that human peg10 was also involved in human hepatocellular carcinogenesis [15,16]. However, it is unclear whether peg10 is related to adipocyte differentiation or not. To investigate the role of peg10 in adipocyte differentiation, we first determined the peg10 expression profile during the differentiation process by Q-PCR (Fig. 1). The level of peg10 mRNA was immediately increased, reaching a peak at 3 h, after the differentiation was induced, and returned to basal lev-
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els by 12 h. From 1 day after the induction, the peg10 mRNA expression was re-induced and continued to increase until day 8. It seems that there was an another peak at day 3. Similar expression profiles were obtained from three independent Northern blot analyses (data not shown). We also determined the gene expression of early and late markers of adipocyte differentiation during the differentiation process. The increases in the expression of late markers, such as aP2, PPARc, and C/ EBPa, were initially observed from 2 to 3 days after the induction and continued as the differentiation progressed. The expression of C/EBPb and C/EBPd, which are early markers and play early catalytic roles in the differentiation pathway, was immediately elevated, reached a peak at 1–3 h post-induction, and declined gradually at the later stages. In summary, peg10 mRNA expression was transiently induced at the early stages of adipocyte differentiation, having the same timing as the immediate early expression of C/EBPb and C/ EBPd. In the late stages of the differentiation process, the expression of peg10 mRNA was re-induced, ahead of the increases in the expression of aP2, PPARc, and C/EBPa, and continued to increase as the differentiation progressed. These results suggest that peg10 may have several different functions during adipocyte differentiation. 3.2. Expression profiles of peg10 in differentiating and nondifferentiating cells We next determined whether peg10 expression was restricted to cells in a state of differentiation or not. 3T3-L1 cells give rise to adipocytes when exposed to inducers 2 days after reaching a state of confluence, whereas proliferating 3T3-L1 cells do not differentiate even in the presence of inducers. Another mouse fibroblastic cell line, NIH-3T3, does not differentiate into adipocytes in either a postconfluent or proliferating state. These two cell lines were treated with inducers for 3 h while in a postconfluent (growth-arrested) or proliferating state. Q-PCR showed that remarkable induction only occurred in the growth-arrested 3T3-L1 cells, and that peg10 mRNA was not detected in either proliferating or growth-arrested NIH3T3 cells, suggesting that the elevation of peg10 expression was restricted to the adipocyte differentiable state (Fig. 2). The same results were obtained by Northern blot analyses (data not shown). This result implies that peg10 plays some functional role in adipocyte differentiation. 3.3. Effect of the knockdown of peg10 by RNAi on differentiation of 3T3-L1 cells into adipocytes To characterize the functional role of this gene in adipogenesis, we next performed RNAi-mediated knockdown of peg10 during adipocyte differentiation. For the RNAi experiments, three shRNA expression vectors named shpeg10-1, shpeg102, and shpeg10-3 were constructed for targeting regions 1, 2, and 3, respectively, in the ORF of the peg10 gene. These shRNA expression vectors were transfected into 3T3-L1 cells. Three hours after induction for differentiation, total RNA was isolated, and then, the expression level of peg10 was determined by Q-PCR. We found that all three shRNA expression vectors achieved an effective silencing of peg10 expression, with shpeg10-1 seeming to give the greatest reduction in activity (Fig. 3A). Therefore, we used shpeg10-1 to perform RNAi experiments for further analyses. Next, we determined the expression level of peg10 in the cells transfected with
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shpeg10-1 at each point after the induction by Q-PCR. We confirmed that the RNAi-treatment reduced the expression of peg10 mRNA at each time-point in the differentiation process (Fig. 3B). After 8 days, the cells were fixed and stained with Oil red O and the amounts of triacylglycerol were determined. The number of Oil red O-stained cells and the accumulation of triacylglycerol were reduced among the RNAi-treated cells (Fig. 3C). Next, we determined the expression levels of adipogenic marker genes by Q-PCR. The levels of aP2, PPARc, and C/ EBPa were remarkedly decreased at all the points examined in the peg10 RNAi-treated cells, indicating that the knockdown of peg10 inhibited the expression of adipogenic marker genes as well as the accumulation of triacylglycerol. Taken together, the knockdown of peg10 inhibited the adipocyte differentiation. At the same time points, we also determined the expression levels of C/EBPb and C/EBPd. Interestingly, the knockdown did not have a remarkable effect on the expression of C/EBPb and C/EBPd, although C/EBPd levels at 0 and 8 days were decreased (Fig. 3D). This is discussed below. Finally, to determine the effects of two other vectors for the knockdown of peg10 on adipogenesis, we performed RNAi experiments using shpeg10-2, shpeg10-3, as well as shpeg10-1, and determined the amounts of triacylglycerol in each of the transfected cells at 8 days after the induction. As shown in
T. Hishida et al. / FEBS Letters 581 (2007) 4272–4278
Fig. 2. Expression profile of peg10 in the adipocyte differentiating and non-differentiating cells. Total RNA isolated from growth-arrested and proliferating 3T3-L1 and NIH-3T3 cells before and 3 h after induction was subjected to Q-PCR. Each column represents the mean with standard deviation (n = 3). N.D., not detected.
Fig. 3E, we confirmed that peg10 RNAi-treatment with shpeg10-1 impaired adipocyte differentiation. In addition, the
Fig. 1. Time course of peg10 mRNA expression during adipocyte differentiation. Total RNA was prepared from 3T3-L1 cells at various time points after treatment with inducers, and was subjected to Q-PCR, and normalized with 18S rRNA expression determined by Q-PCR. Each column represents the mean with standard deviation (n = 3).
T. Hishida et al. / FEBS Letters 581 (2007) 4272–4278
inhibitory effect of peg10’s knockdown on adipocyte differentiation was also observed in RNAi experiments performed with shpeg10-2, or shpeg10-3. Taken together, these results strongly suggest that peg10 plays a crucial role in adipocyte differentiation. 3.4. Effect of peg10 RNAi-treatment on MCE Peg10 was reported to promote cell growth in human hepatocellular carcinoma [15,16]. We focused on the ability of peg10 to promote cell growth, and next investigated the effect of peg10 on MCE. MCE is synchronous, transient cell growth that can be observed after post-confluent 3T3-L1 cells have been treated with an optimal mixture of adipogenic stimulants [17,18]. This phase is requisite for adipocyte differentiation, followed by the expression of adipogenic genes, such as
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PPARc and C/EBPa [19]. To elucidate the role of peg10 in MCE, we determined the effect of the knockdown of peg10 on MCE. This perceived effect on MCE was quantified by taking cell counts with a hemocytometer at 1-day intervals throughout the differentiation program. Cell numbers in control cultures increased 3.0-fold between days 0 and 4, and remained constant between days 4 and 5. In comparison, cell numbers in peg10 knockdown cultures only increased 2.4-fold by day 4 (Fig. 4A). Similar results were obtained from RNAi experiment performed with three independent shRNA expression vectors for peg10 (Fig. 4B), indicating that peg10 is critical for MCE in adipogenesis. Although little is known about the molecular mechanism of the regulation of MCE, C/EBPb seems to play an important role [20]. In this respect, we next investigated the expression
Fig. 3. Effect of the knockdown of peg10 by RNAi on adipocyte differentiation. (A) The effects of three different shRNAs on the expression of peg10. Total RNA obtained from 3T3-L1 cells transfected with shpeg10-1, shpeg10-2, shpeg10-3 or the scrambled shRNA expression plasmid as a control (control) at 3 h after differentiation was induced was subjected to Q-PCR. The expression level was normalized with 18S rRNA expression. Each column represents the mean with standard deviation (n = 3). (B) peg10 expression in peg10-knockdown 3T3-L1 cells was determined by Q-PCR. Total RNA obtained from 3T3-L1 cells transfected with shpeg10-1 (open bars) or with the scrambled shRNA expression plasmid as a control (solid bars) at each time point was subjected to Q-PCR. The expression level was normalized with 18S rRNA expression. Each column represents the mean with standard deviation (n = 3). (C) Adipocyte differentiation of peg10-knockdown 3T3-L1 cells. The cells transfected with shpeg10-1 (shpeg10-1) or the scramble shRNA expression vector as a control (control) were stimulated with inducers. After 8 days, the cells were stained with Oil red O to detect oil droplets. The amount of triglyceride measured in peg10 knockdown cells (open bar) or control cells (solid bar) 8 days after the induction is also shown. Each column represents the mean with standard deviation (n = 3). *P < 0.05 vs. control. (D) Effect of peg10 RNAi-treatment on the expression of various adipogenic genes. Total RNA obtained from peg10 knockdown cells (open bars) or control cells (solid bars) at each time point was subjected to Q-PCR, and normalized with 18S rRNA expression determined by Q-PCR. Each column represents the mean with standard deviation (n = 3). *P < 0.05; **P < 0.01 vs. control. (E) Effects of three independent shRNA expression vectors for peg10 knockdown on adipogenesis. The amounts of triglyceride were measured at 8 days after the induction in 3T3-L1 cells transfected with shpeg10-1, shpeg10-2, shpeg10-3 or the scrambled shRNA expression plasmid as a control (control). Each column represents the mean with standard deviation (n = 3). *P < 0.05; **P < 0.01 vs. control.
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Fig. 3 (continued)
of C/EBPb at the early stages of adipogenesis in peg10 RNAitreated cells. Additionally, we determined the levels of C/ EBPd, which is transiently expressed immediately after the differentiation is induced (Fig. 1) and known to facilitate the expression of PPARc and C/EBPa, in cooperation with C/ EBPb [2,21]. Notably, RNAi-mediated knockdown of peg10 resulted in a reduction in the expression of C/EBPb and C/ EBPd in the immediate early stage of adipocyte differentiation (Fig. 4C), although it did not have a clear effect on the expression of these two genes at the late stage (Fig. 3D). These results strongly indicate that peg10 lies upstream of C/EBPb and C/
EBPd in the signaling pathway for adipocyte differentiation, contributing to the expression of these two genes at the earliest stage of differentiation. On the basis of these findings, we now speculate that peg10 plays some role in MCE by regulating the expression of C/ EBPb and C/EBPd in the immediate early stage of the differentiation process. Although CREB (cAMP response element binding protein) is reported to regulate the expression of C/ EBPb and C/EBPd at the early stages of adipocyte differentiation [22], the overall mechanisms regulating C/EBPb and C/EBPd expression remain to be elucidated. In this regard,
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Fig. 4. Effect of peg10 RNAi-treatment on MCE and the expression of C/EBPb and C/EBPd at the immediate early stage of adipogenesis. (A) Effect of peg10 knockdown on MCE. The cells transfected with shpeg10-1 (peg10 knockdown) or the scramble shRNA expression vector as a control (control) were stimulated with inducers. Parallel cultures of peg10 knockdown cells or control cells were harvested at the indicated days after differentiation was induced. Cell numbers were determined by taking counts with a hemocytometer. Each column represents the mean with standard deviation (n = 4). *P < 0.05; **P < 0.01 vs. control. (B) Effects of three independent shRNA expression vectors for peg10 knockdown on MCE. The cells transfected with shpeg10-1, shpeg10-2, shpeg10-3 or the scrambled shRNA expression plasmid as a control (control) were stimulated with inducers. Parallel cultures of transfected cells were harvested at each time point after differentiation was induced. Cell numbers were determined by taking counts with a hemocytometer. Each column represents the mean with standard deviation (n = 4). *P < 0.05; **P < 0.01 vs. control. (C) The expression of C/EBPb and C/EBPd mRNA at the immediate early stage of adipogenesis was determined. Total RNA obtained from peg10 knockdown cells (open bars) or control cells (solid bars) at each time point was subjected to Q-PCR. Expression levels were normalized with 18S rRNA expression determined by Q-PCR. Each column represents the mean with standard deviation (n = 3). *P < 0.05; **P < 0.01 vs. control.
further studies of the molecular function of peg10 should provide new insights into the early stages of adipocyte differentiation. For the purpose of investigating the function of peg10 in adipocyte differentiation in detail, analyses of PEG10 proteins, namely PEG10-RF1 and PEG10-RF1/2, are ongoing. Acknowledgements: This study was supported in part by a Grant-inAid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (JSPS).
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peg10, an imprinted gene, plays a crucial role in adipocyte differentiation Tomoaki Hishida, Kumiko Naito, Shigehiro Osada, Makoto Nishizuka, Masayoshi Imagawa* Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467-8603, Japan Received 28 July 2007; accepted 31 July 2007 Available online 10 August 2007 Edited by Robert Barouki
Abstract An imprinted gene, paternally expressed gene (peg) 10, was isolated as one of the genes expressed early in adipogenesis. The expression of peg10 was elevated after the addition of inducers, and was detected in adipocyte differentiable 3T3-L1 cells, but not observed in the non-adipogenic cell line NIH3T3. Moreover, the knockdown of peg10 by RNA interference (RNAi) inhibited the differentiation of 3T3-L1 cells into lipid-laden adipocytes. Interestingly, peg10 RNAi-treatment reduced the expressions of C/EBPb and C/EBPd, and inhibited mitotic clonal expansion. These findings strongly indicate that peg10 plays a crucial role at the immediate early stage of adipocyte differentiation. 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. Keywords: Adipocyte differentiation; Adipogenesis; Obesity; Peroxisome proliferator-activated receptor c; CCAAT/ enhancer-binding protein; 3T3-L1 cell
1. Introduction Obesity is related to many diseases, such as hypertension, heart disease, and diabetes. Obesity results from an imbalance between energy intake and energy expenditure, which leads to a pathological accumulation of adipose tissue [1]. This development of adipose tissue is caused by hypertrophy and hyperplasia of adipocytes. Therefore, an understanding of the molecular basis of hyperplasia as well as hypertrophy would contribute to the establishment of medical treatments to prevent health risks that can cause serious illness and death. One explanation for the increase in the number of adipocytes is that preadipocytes differentiate into lipid-laden adipocytes, and newly generated cells function as mature adipocytes. Recent studies have revealed that several genes, such as those for peroxisome proliferator-activated receptor c (PPARc) and CCAAT/enhancer-binding protein a (C/EBPa), play important roles in the mid and late stages of adipocyte differentiation [2–4]. The events occurring during the mid- to late* Corresponding author. Fax: +81 52 836 3455. E-mail address: [email protected] (M. Imagawa).
Abbreviations: C/EBP, CCAAT/enhancer-binding protein; DMEM, Dulbecco’s modified Eagle’s medium; fad, factor for adipocyte differentiation; FBS, fetal bovine serum; ORF, open reading frame; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; PPARc, peroxisome proliferator-activated receptor c; RNAi, RNA interference; RT-PCR, reverse transcriptase coupled polymerase chain reaction; shRNA, short hairpin RNA; UTR, untranslated region
phase of differentiation are relatively well characterized, but the molecular mechanisms underlying the early stages of adipogenesis remain unknown. To elucidate the initial step of adipocyte differentiation, we have previously isolated genes whose expression is induced in the early stages of the differentiation process, using mouse 3T3-L1 fibroblastic cells. Using the PCR-subtraction system, we isolated 102 genes, including genes for transcription factors and signaling proteins [5–7]. Of these genes, 46 seem to be unknown genes, whose functions remain unclear. We previously isolated some full-length cDNAs of unknown genes and revealed that several of them are novel genes, such as fad24, fad104 and fad158, which positively regulate adipogenesis [8–10]. Here, we report the identification of one of the unknown genes as paternally expressed gene peg10 and the involvement of peg10 in adipocyte differentiation. Its expression was elevated during the differentiation of 3T3-L1 cells. Up-regulation of peg10 expression was detected in adipocyte differentiable 3T3L1, but not in the non-adipogenic cell line NIH-3T3. RNA interference (RNAi)-mediated knockdown of peg10 inhibited the differentiation of 3T3-L1 cells. Moreover, peg10 RNAitreatment impaired mitotic clonal expansion (MCE), which is necessary for adipocyte differentiation. Interestingly, the expression of C/EBPb and C/EBPd at the immediate early stage of the differentiation process was inhibited by the knockdown of peg10. Taken together, these results indicate that peg10 plays important roles in the early stages of adipocyte differentiation.
2. Materials and methods 2.1. RNA isolation and real-time quantitative RT-PCR (Q-PCR) Total RNA was extracted with TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions. The total RNA was converted to the single cDNA using a random primer and ReverTra Ace (Toyobo, Osaka, Japan). The cDNA was used as template for Q-PCR. An ABI PRISM 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA) was used to perform the QPCR. The pre-designed primers and probe sets for peg10, PPARc, C/EBPa, C/EBPb, C/EBPd, aP2, and 18S rRNA were obtained from Applied Biosystems. The reaction mixture was prepared using a TaqMan Universal PCR Master Mix (Applied Biosystems) according to the manufacturer’s instructions. The mixture was incubated at 50 C for 2 min and at 95 C for 10 min, and then the PCR was performed at 95 C for 15 s and at 60 C for 1 min for 40 cycles. Relative standard curves were generated in each experiment to calculate the input amounts of the unknown samples. 2.2. Cell culture and differentiation Mouse 3T3-L1 (ATCC CL173) preadipocyte cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% calf serum. For the differentiation experiment, the medium was replaced with DMEM containing 10% fetal bovine serum (FBS), 10 lg/ml
0014-5793/$32.00 2007 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2007.07.074
T. Hishida et al. / FEBS Letters 581 (2007) 4272–4278 insulin, 0.5 mM 3-isobutyl-1-methylxantine, and 1 lM dexamethasone at 2 days post-confluence. After 2 days, the medium was changed to DMEM containing 5 lg/ml insulin and 10% FBS, then the cells were refed every 2 days. Mouse NIH-3T3 (clone 5611, JCRB 0615) fibroblastic cells were maintained in DMEM containing 10% calf serum. 2.3. RNAi experiment The three target regions in the open reading frame (ORF) of mouse peg10 (GenBanke Accession No. NM_130877); region 1 at 481– 501 bp (nucleotide A in the translation initiation codon is the first nucleotide), region 2 at 659–679 bp, and region 3 at 1201–1221 bp, were selected according to the Qiagen siRNA online design tool (http://sirna.qiagen.com/) for the RNAi of peg10. A 19-nucleotide short-hairpin RNA (shRNA)–coding fragment with a 5 0 -TTCAAGAGA-3 loop was subcloned into the ApaI/EcoRI site of pSilencer 1.0-U6 (Ambion, Inc. Austin, TX, USA). As a negative control, the scrambled fragment 5 0 -GTAAGATGAGGCAATGGAG-3 0 which does not have similarity with any mRNA listed in GenBank was generated. The transfection of shRNA expression vectors into 3T3-L1 cells was performed with Nucleofector (Amaxa, Cologne, Germany) using Cell Line Nucleofector Kit V (Amaxa). 3T3-L1 cells were harvested and resuspended in Nucleofector solution at 2.0 · 106 cells/100 ll. After the addition of shRNA expression vectors (9 lg), the cells were transfected using program ‘T-20’ of Nucleofector. Then, they were plated on 12- or 24-well plates. The 3T3-L1 cells were subjected to differentiation experiments 3 days after the transfection. Differentiation experiments were carried out using the same medium as described above. The cell counts were performed with 24-well plates. 2.4. Cell counts Cells were gently rinsed with PBS (phosphate-buffered saline) without Ca2+ and Mg2+ (PBS-), and trypsinized with the use of trypsin (0.25%)-EDTA (0.1%) solution in PBS- for 5 min at 37 C, 5% CO2. The trypsinized cells were subjected to cell counts using a hemocytometer.
3. Results and discussion 3.1. Expression of peg10 during the early stage of adipocyte differentiation With the PCR-subtraction method, we isolated 46 unknown genes, the expression of each of which was up-regulated 3 h after the differentiation was induced. Since the fragments obtained with this protocol were only 300–500 bp long, it was necessary to isolate the full-length cDNAs and identify the ORF of unknown genes. One of these unknown genes was identified as peg10, because the subtracted fragment showed identity to the 3 0 -UTR of mouse peg10, which was later listed in updated databases. Mouse peg10 is a single-copy gene located on proximal chromosome 6. peg10 is a retrotransposon-derived gene, which encodes pol-gag like peptides [11,12]. peg10 has two overlapping ORFs (RF1 and RF2), which results in two proteins being generated from a single mRNA: PEG10-RF1, which is a non-shifting product, and PEG10RF1/2, which is generated as a RF1-RF2 fusion protein by 1 ribosomal frameshifting [12,13]. Recently, it was reported that the knockout of peg10 in mice resulted in early embryonic death owing to defects in the placenta [14]. Moreover, studies showed that human peg10 was also involved in human hepatocellular carcinogenesis [15,16]. However, it is unclear whether peg10 is related to adipocyte differentiation or not. To investigate the role of peg10 in adipocyte differentiation, we first determined the peg10 expression profile during the differentiation process by Q-PCR (Fig. 1). The level of peg10 mRNA was immediately increased, reaching a peak at 3 h, after the differentiation was induced, and returned to basal lev-
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els by 12 h. From 1 day after the induction, the peg10 mRNA expression was re-induced and continued to increase until day 8. It seems that there was an another peak at day 3. Similar expression profiles were obtained from three independent Northern blot analyses (data not shown). We also determined the gene expression of early and late markers of adipocyte differentiation during the differentiation process. The increases in the expression of late markers, such as aP2, PPARc, and C/ EBPa, were initially observed from 2 to 3 days after the induction and continued as the differentiation progressed. The expression of C/EBPb and C/EBPd, which are early markers and play early catalytic roles in the differentiation pathway, was immediately elevated, reached a peak at 1–3 h post-induction, and declined gradually at the later stages. In summary, peg10 mRNA expression was transiently induced at the early stages of adipocyte differentiation, having the same timing as the immediate early expression of C/EBPb and C/ EBPd. In the late stages of the differentiation process, the expression of peg10 mRNA was re-induced, ahead of the increases in the expression of aP2, PPARc, and C/EBPa, and continued to increase as the differentiation progressed. These results suggest that peg10 may have several different functions during adipocyte differentiation. 3.2. Expression profiles of peg10 in differentiating and nondifferentiating cells We next determined whether peg10 expression was restricted to cells in a state of differentiation or not. 3T3-L1 cells give rise to adipocytes when exposed to inducers 2 days after reaching a state of confluence, whereas proliferating 3T3-L1 cells do not differentiate even in the presence of inducers. Another mouse fibroblastic cell line, NIH-3T3, does not differentiate into adipocytes in either a postconfluent or proliferating state. These two cell lines were treated with inducers for 3 h while in a postconfluent (growth-arrested) or proliferating state. Q-PCR showed that remarkable induction only occurred in the growth-arrested 3T3-L1 cells, and that peg10 mRNA was not detected in either proliferating or growth-arrested NIH3T3 cells, suggesting that the elevation of peg10 expression was restricted to the adipocyte differentiable state (Fig. 2). The same results were obtained by Northern blot analyses (data not shown). This result implies that peg10 plays some functional role in adipocyte differentiation. 3.3. Effect of the knockdown of peg10 by RNAi on differentiation of 3T3-L1 cells into adipocytes To characterize the functional role of this gene in adipogenesis, we next performed RNAi-mediated knockdown of peg10 during adipocyte differentiation. For the RNAi experiments, three shRNA expression vectors named shpeg10-1, shpeg102, and shpeg10-3 were constructed for targeting regions 1, 2, and 3, respectively, in the ORF of the peg10 gene. These shRNA expression vectors were transfected into 3T3-L1 cells. Three hours after induction for differentiation, total RNA was isolated, and then, the expression level of peg10 was determined by Q-PCR. We found that all three shRNA expression vectors achieved an effective silencing of peg10 expression, with shpeg10-1 seeming to give the greatest reduction in activity (Fig. 3A). Therefore, we used shpeg10-1 to perform RNAi experiments for further analyses. Next, we determined the expression level of peg10 in the cells transfected with
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shpeg10-1 at each point after the induction by Q-PCR. We confirmed that the RNAi-treatment reduced the expression of peg10 mRNA at each time-point in the differentiation process (Fig. 3B). After 8 days, the cells were fixed and stained with Oil red O and the amounts of triacylglycerol were determined. The number of Oil red O-stained cells and the accumulation of triacylglycerol were reduced among the RNAi-treated cells (Fig. 3C). Next, we determined the expression levels of adipogenic marker genes by Q-PCR. The levels of aP2, PPARc, and C/ EBPa were remarkedly decreased at all the points examined in the peg10 RNAi-treated cells, indicating that the knockdown of peg10 inhibited the expression of adipogenic marker genes as well as the accumulation of triacylglycerol. Taken together, the knockdown of peg10 inhibited the adipocyte differentiation. At the same time points, we also determined the expression levels of C/EBPb and C/EBPd. Interestingly, the knockdown did not have a remarkable effect on the expression of C/EBPb and C/EBPd, although C/EBPd levels at 0 and 8 days were decreased (Fig. 3D). This is discussed below. Finally, to determine the effects of two other vectors for the knockdown of peg10 on adipogenesis, we performed RNAi experiments using shpeg10-2, shpeg10-3, as well as shpeg10-1, and determined the amounts of triacylglycerol in each of the transfected cells at 8 days after the induction. As shown in
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Fig. 2. Expression profile of peg10 in the adipocyte differentiating and non-differentiating cells. Total RNA isolated from growth-arrested and proliferating 3T3-L1 and NIH-3T3 cells before and 3 h after induction was subjected to Q-PCR. Each column represents the mean with standard deviation (n = 3). N.D., not detected.
Fig. 3E, we confirmed that peg10 RNAi-treatment with shpeg10-1 impaired adipocyte differentiation. In addition, the
Fig. 1. Time course of peg10 mRNA expression during adipocyte differentiation. Total RNA was prepared from 3T3-L1 cells at various time points after treatment with inducers, and was subjected to Q-PCR, and normalized with 18S rRNA expression determined by Q-PCR. Each column represents the mean with standard deviation (n = 3).
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inhibitory effect of peg10’s knockdown on adipocyte differentiation was also observed in RNAi experiments performed with shpeg10-2, or shpeg10-3. Taken together, these results strongly suggest that peg10 plays a crucial role in adipocyte differentiation. 3.4. Effect of peg10 RNAi-treatment on MCE Peg10 was reported to promote cell growth in human hepatocellular carcinoma [15,16]. We focused on the ability of peg10 to promote cell growth, and next investigated the effect of peg10 on MCE. MCE is synchronous, transient cell growth that can be observed after post-confluent 3T3-L1 cells have been treated with an optimal mixture of adipogenic stimulants [17,18]. This phase is requisite for adipocyte differentiation, followed by the expression of adipogenic genes, such as
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PPARc and C/EBPa [19]. To elucidate the role of peg10 in MCE, we determined the effect of the knockdown of peg10 on MCE. This perceived effect on MCE was quantified by taking cell counts with a hemocytometer at 1-day intervals throughout the differentiation program. Cell numbers in control cultures increased 3.0-fold between days 0 and 4, and remained constant between days 4 and 5. In comparison, cell numbers in peg10 knockdown cultures only increased 2.4-fold by day 4 (Fig. 4A). Similar results were obtained from RNAi experiment performed with three independent shRNA expression vectors for peg10 (Fig. 4B), indicating that peg10 is critical for MCE in adipogenesis. Although little is known about the molecular mechanism of the regulation of MCE, C/EBPb seems to play an important role [20]. In this respect, we next investigated the expression
Fig. 3. Effect of the knockdown of peg10 by RNAi on adipocyte differentiation. (A) The effects of three different shRNAs on the expression of peg10. Total RNA obtained from 3T3-L1 cells transfected with shpeg10-1, shpeg10-2, shpeg10-3 or the scrambled shRNA expression plasmid as a control (control) at 3 h after differentiation was induced was subjected to Q-PCR. The expression level was normalized with 18S rRNA expression. Each column represents the mean with standard deviation (n = 3). (B) peg10 expression in peg10-knockdown 3T3-L1 cells was determined by Q-PCR. Total RNA obtained from 3T3-L1 cells transfected with shpeg10-1 (open bars) or with the scrambled shRNA expression plasmid as a control (solid bars) at each time point was subjected to Q-PCR. The expression level was normalized with 18S rRNA expression. Each column represents the mean with standard deviation (n = 3). (C) Adipocyte differentiation of peg10-knockdown 3T3-L1 cells. The cells transfected with shpeg10-1 (shpeg10-1) or the scramble shRNA expression vector as a control (control) were stimulated with inducers. After 8 days, the cells were stained with Oil red O to detect oil droplets. The amount of triglyceride measured in peg10 knockdown cells (open bar) or control cells (solid bar) 8 days after the induction is also shown. Each column represents the mean with standard deviation (n = 3). *P < 0.05 vs. control. (D) Effect of peg10 RNAi-treatment on the expression of various adipogenic genes. Total RNA obtained from peg10 knockdown cells (open bars) or control cells (solid bars) at each time point was subjected to Q-PCR, and normalized with 18S rRNA expression determined by Q-PCR. Each column represents the mean with standard deviation (n = 3). *P < 0.05; **P < 0.01 vs. control. (E) Effects of three independent shRNA expression vectors for peg10 knockdown on adipogenesis. The amounts of triglyceride were measured at 8 days after the induction in 3T3-L1 cells transfected with shpeg10-1, shpeg10-2, shpeg10-3 or the scrambled shRNA expression plasmid as a control (control). Each column represents the mean with standard deviation (n = 3). *P < 0.05; **P < 0.01 vs. control.
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Fig. 3 (continued)
of C/EBPb at the early stages of adipogenesis in peg10 RNAitreated cells. Additionally, we determined the levels of C/ EBPd, which is transiently expressed immediately after the differentiation is induced (Fig. 1) and known to facilitate the expression of PPARc and C/EBPa, in cooperation with C/ EBPb [2,21]. Notably, RNAi-mediated knockdown of peg10 resulted in a reduction in the expression of C/EBPb and C/ EBPd in the immediate early stage of adipocyte differentiation (Fig. 4C), although it did not have a clear effect on the expression of these two genes at the late stage (Fig. 3D). These results strongly indicate that peg10 lies upstream of C/EBPb and C/
EBPd in the signaling pathway for adipocyte differentiation, contributing to the expression of these two genes at the earliest stage of differentiation. On the basis of these findings, we now speculate that peg10 plays some role in MCE by regulating the expression of C/ EBPb and C/EBPd in the immediate early stage of the differentiation process. Although CREB (cAMP response element binding protein) is reported to regulate the expression of C/ EBPb and C/EBPd at the early stages of adipocyte differentiation [22], the overall mechanisms regulating C/EBPb and C/EBPd expression remain to be elucidated. In this regard,
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Fig. 4. Effect of peg10 RNAi-treatment on MCE and the expression of C/EBPb and C/EBPd at the immediate early stage of adipogenesis. (A) Effect of peg10 knockdown on MCE. The cells transfected with shpeg10-1 (peg10 knockdown) or the scramble shRNA expression vector as a control (control) were stimulated with inducers. Parallel cultures of peg10 knockdown cells or control cells were harvested at the indicated days after differentiation was induced. Cell numbers were determined by taking counts with a hemocytometer. Each column represents the mean with standard deviation (n = 4). *P < 0.05; **P < 0.01 vs. control. (B) Effects of three independent shRNA expression vectors for peg10 knockdown on MCE. The cells transfected with shpeg10-1, shpeg10-2, shpeg10-3 or the scrambled shRNA expression plasmid as a control (control) were stimulated with inducers. Parallel cultures of transfected cells were harvested at each time point after differentiation was induced. Cell numbers were determined by taking counts with a hemocytometer. Each column represents the mean with standard deviation (n = 4). *P < 0.05; **P < 0.01 vs. control. (C) The expression of C/EBPb and C/EBPd mRNA at the immediate early stage of adipogenesis was determined. Total RNA obtained from peg10 knockdown cells (open bars) or control cells (solid bars) at each time point was subjected to Q-PCR. Expression levels were normalized with 18S rRNA expression determined by Q-PCR. Each column represents the mean with standard deviation (n = 3). *P < 0.05; **P < 0.01 vs. control.
further studies of the molecular function of peg10 should provide new insights into the early stages of adipocyte differentiation. For the purpose of investigating the function of peg10 in adipocyte differentiation in detail, analyses of PEG10 proteins, namely PEG10-RF1 and PEG10-RF1/2, are ongoing. Acknowledgements: This study was supported in part by a Grant-inAid for Scientific Research on Priority Areas from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan, and a Grant-in-Aid for Scientific Research (B) from the Japan Society for the Promotion of Science (JSPS).
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