A Novel Splicing Variant of Peroxisome Proliferator-Activated Receptor-γ (Pparγ1sv) Cooperatively Regulates Adipocyte Differentiation with Pparγ2

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors that regulate expression of a number of genes associated with the cellular differentiation and development. Here, we show the abundant and ubiquitous expression of a newly identified splicing variant of mouse Pparγ (Pparγ1sv) that encodes PPARγ1 protein, and its importance in adipogenesis. The novel splicing variant has a unique 5′-UTR sequence, relative to those of Pparγ1 and Pparγ2 mRNAs, indicating the presence of a novel transcriptional initiation site and promoter for Pparγ expression. Pparγ1sv was highly expressed in the white and brown adipose tissues at levels comparable to Pparγ2. Pparγ1sv was synergistically up-regulated with Pparγ2 during adipocyte differentiation of 3T3-L1 cells and mouse primary cultured preadipocytes. Inhibition of Pparγ1sv by specific siRNAs completely abolished the induced adipogenesis in 3T3-L1 cells. C/EBPβ and C/EBPδ activated both the Pparγ1sv and Pparγ2 promoters in 3T3-L1 preadipocytes. These findings suggest that Pparγ1sv and Pparγ2 synergistically regulate the early stage of the adipocyte differentiation.


Introduction
Obesity has become a growing worldwide health problem in recent years. An excessive accumulation of white adipose tissue caused by increases in the cell number and size of newly differentiated white adipocytes from preadipocytes is a major cause of obesity. Thus, the elucidation of mechanisms of adipocyte differentiation is essential for understanding the pathogenesis of obesity and obesity-associated diseases.
3T3-L1, a cell line derived from mouse 3T3 fibroblast, has been widely used as a model of adipocyte differentiation [1]. The addition of chemicals and hormones such as dexamethasone or insulin into culture media of 3T3-L1 cells induces the synthesis and accumulation of intracellular triglycerides and changes in their morphology from fibroblast-like to adipocyte-like [2]. During the progression, a number of adipocyte-related genes are up-regulated by a sequential induction of transcription factors such as peroxisome proliferator-activated receptor c (PPARc) and members of the CCAAT/enhancer-binding proteins (C/EBPa, C/ EBPb, and C/EBPd) [3]. PPARc is a member of the liganddependent nuclear receptor superfamily and plays a pivotal role in adipogenesis and intracellular lipid accumulation. C/EBPs belong to a family of the basic region-leucine zipper (bZIP) transcription factors. C/EBPb and C/EBPd are transiently expressed very early during adipocyte differentiation [4], which in turn transactivate gene expression of PPARc and C/EBPa [5]. Both proteins cooperatively promote downstream adipocyte-related genes such as the adipocyte-specific fatty acid-binding protein gene (FABP4) to develop functional adipocytes.
PPARc is expressed as at least two splicing variants, the ubiquitously expressed Pparc1 and adipocyte-specific Pparc2 [6,7]. PPARc2 protein that is translated from Pparc2 mRNA is longer than PPARc1 (from Pparc1) by 30 amino acid residues at the Nterminus in mice. PPARc2 protein has been considered to play a critical role in the adipogenesis, because Pparc2 mRNA, but not Pparc1, is abundantly expressed in the adipose tissues. However, PPARc1 expression also has been observed in adipocytes at similar level to PPARc2 in the previous reports [8][9][10], which complicated the role of PPARc1 in adipogenesis.
In addition to Pparc1 and Pparc2, several unique splicing variants of Pparc has been reported [11,12]. We have recently reported a novel PPARc splicing variant in humans that is regulated by circadian rhythmic D-site binding protein, DBP [13]. However, the involvement of this splicing variant in adipogenesis has not been uncovered.
In this paper, we report the identification of a novel Pparc splicing variant, Pparc1sv, in mice that is synergistically upregulated with Pparc2 during adipocyte differentiation of 3T3-L1 cells and mouse primary cultured preadipocytes. Knock-down experiments using siRNA specifically targeting to Pparc1sv revealed that PPARc1 protein expressed during adipogenesis is derived from Pparc1sv mRNA. Thus, this novel splicing variant could explain the induced PPARc1 protein during adipocyte differentiation. Furthermore, knock-down of Pparc1sv abolished the induced adipogenesis of 3T3-L1 cells, indicating that PPARc1 from Pparc1sv plays a crucial and synergistic role with PPARc2 in adipogenesis.

Materials and Methods
Cell Culture, Differentiation, and Staining 3T3-L1 and ST2 cells were obtained from the Japan Health Science Foundation, Health Science Research Resources Bank (Osaka, Japan) and RIKEN Cell Bank (Tsukuba, Japan), respectively. Mouse primary cultured preadipocytes isolated from white adipose tissues of newborn mice were purchased from Primary Cell Co., Ltd (Hokkaido, Japan). 3T3-L1 and ST2 cells were maintained in DMEM and RPMI1640 (Life Technologies), respectively, supplemented with 10% fetal bovine serum (Sigma-Aldrich) and penicillin-streptomycin at 37uC in a humidified atmosphere of 5% CO 2 . Cells were passaged every 3 days. For adipocyte differentiation, we plated cells in 3-cm or 6-cm dishes, allowed them to grow at 95-100% confluency, and then changed the culture medium to DMEM containing 0.25 mM dexamethasone, 500 mM isobutylmethylxanthine, and 1 mM insulin. Primary preadipocytes were cultured in DMEM containing 2.5 mM dexamethasone and 10 mg/ml insulin for two days to start differentiation into adipocytes according to the manufacturer's instructions. We estimated the adipocyte differentiation by staining intracellular lipid droplets with Oil Red O or quantifying cellular triglycerides content with AdipoRed assay reagent (Lonza).

Cloning of a Novel Splicing variant of Mouse Pparc
Total RNA was purified from adipocyte-differentiated 3T3-L1 cells (9 days after the chemical induction) using ISOGEN (Nippon Gene). 59-and 39-Ready SMART cDNA was synthesized from 1 mg of total RNA using the SMART RACE cDNA synthesis kit according to the manufacturer's instructions (Takara Bio). The 59end of mouse Pparc cDNA was amplified from 59-Ready SMART cDNA using mPPARg_5RACE_LP2, 59-TTGGGTCAGCTCTTGTGAATGGAATG-39 and Universal Primer Mix (UPM) (Takara Bio). After sequencing the 59-rapid amplification of cDNA end (RACE) product, full-length cDNA was amplified from 39-Ready SMART cDNA using mPPARg_-novel_59term, 59-GGGGCCTGGACCTCTGCTGGGGATCT-39 and UPM, cloned into pGEM-T Easy vector (Promega) and sequenced.

Western Blotting and Antibodies
Cells cultured in 6-cm dishes were trypsinized and harvested in 1 ml phosphate buffered saline. Nuclear extracts were prepared using NE-PER nuclear and cytoplasmic extraction reagents (Thermo scientific), mixed with 56 sodium dodecyl sulfate (SDS) sample buffer containing 2-mercaptoethanol, heated at 95uC for 3 min, and then loaded onto a 12.5% SDS-polyacrylamide gel. Proteins were transferred to a PVDF membrane and incubated in 1% Western blocking reagent (Roche Applied Science) at room temperature for 1 hr. The membrane was then incubated overnight with anti-PPARc (A3409A) (Perseus Proteomics), anti-C/EBPb (Santa Cruz Biotechnology), anti-C/EBPa (Santa Cruz Biotechnology), anti-Lamin B1 (abcam), anti-FABP4 (Cell Signaling Technology), anti-DLK (Santa Cruz Biotechnology), or anti-atubulin (Sigma-Aldrich) antibody diluted in 0.5% Western blocking reagent (1:1,000). HRP-conjugated goat anti-mouse IgG (Sigma-Aldrich) or anti-rabbit IgG (GE healthcare) antibody was used as the secondary antibody and detected with ECL prime reagent (GE Healthcare). The chemiluminescent signal was exposed to Hyperfilm ECL (GE Healthcare).

Luciferase Reporter Assay
Preadipocyte 3T3-L1 cells in the 12-well plate were transiently transfected with 0.6 mg of promoter/luciferase reporter construct (pGL3-Basic), 0.5 mg of overexpression construct (pcDNA3.1), and 0.5 mg of the constitutive Renilla luciferase expression vector (pGL4.74) (Promega) for normalization in a well using Lipofectamine 2000 transfection reagent (Life Technologies). For the assessment of siRNA specificity, 3T3-L1 cells were simultaneously transfected with each of siRNAs, the reporter plasmid pGL3-Control containing the Pparc1sv or Pparc2 cDNA, and pGL4.74 for normalization. Cells were harvested 1 or 2 days after the transfection, and luciferase assays were performed using the Dual-luciferase reporter assay system (Promega). Luminescence was counted for 10 sec using a MiniLumat LB 9506 luminometer (Berthold).

A Novel Mouse Pparc Splicing variant and its Gene Structure
We amplified the 59-ends of mouse Pparc cDNAs using a reverse primer based on the sequence of exon 1 and a terminal adaptor primer from a cDNA library prepared from adipocyte differentiated 3T3-L1 cells (day 9). The amplified 59-end products (,350 bp) contained the coding sequence and 59-UTR of mouse Pparc cDNAs. We sequenced 22 clones, nine of which contained 59-end sequences of Pparc2, one contained the 59-end of Pparc1. Remaining 12 clones possessed a unique 59-UTR that was different from those of Pparc1 and Pparc2 (Fig. 1A). Full-length cDNAs of the novel splicing variant were then amplified using the 39-end adapter primer and 59-end gene-specific primer that was designed based on the sequences of 59RACE products. Sequencing of the full-length cDNA showed that the novel splicing variant encoded an identical amino acid sequence of mouse PPARc1. We have designated this novel splicing variant as Pparc1sv. The complete sequence of Pparc1sv cDNA was deposited in the DDBJ/ EMBL/GenBank database under the accession number AB644275. The transcription initiation site of the novel splicing variant was located on the novel exon C (68 bp) by aligning its sequence with the mouse Pparc genomic sequence on chromosome 6 ( Fig. 1B). Exon C is located far (,60 kbp) from exon B of Pparc2 whereas it is relatively close (,1 kbp) to exon A1 of Pparc1 (Fig. 1B). A homology search using the BLAST program revealed that exon C shared 81% sequence identity with porcine exon A' (GenBank no. AB121691) and 77% with exon C (or A') of human PPARc transcript variant 3 (NM_138711). Alignment of the nucleotide sequence of mouse exon C with those of corresponding exons of other mammals are shown in Fig 1C. Additional information about mouse Pparc1sv and its homologous transcription variants in other mammals is summarized in Table 1.

Tissue Distribution and Relative Abundance of Pparc1sv in Mice
We designed unique forward primers for Pparc1sv, Pparc1, and Pparc2, respectively and a common reverse primer for all Pparc transcripts on exon 1 to quantify their expression levels ( Fig. 2A). The relative expression levels of the three transcripts were analyzed by qPCR using normalized cDNAs prepared from 16 mouse tissues and embyos (Fig. 2B). Pparc1sv was expressed abundantly in the stomach, placenta, heart, spleen, lung, skeletal muscle, and 17-day mouse embryo. Pparc1sv was also abundantly expressed in the white and brown adipose tissues at higher levels than that of Pparc2 (Fig. 2C).

Kinetics of Pparc1sv Expression during Adipocyte Differentiation of 3T3-L1 and Primary Cells
To further clarify the involvement of Pparc1sv in adipogenesis, we examined its expression and kinetics in 3T3-L1 and primary cells from white adipose tissue of newborn mice during adipocyte differentiation. Both Pparc1sv and Pparc2 mRNAs were induced in the early phase (day 1) of adipocyte differentiation of 3T3-L1 cells, and continued to increase up to day 9 (Fig. 3A). Pparc1sv and Pparc2 mRNA levels were approximately 15-and 234-fold higher at day 9, respectively, than those of cells at day 0 (Fig. 3A). In primary cultured cells, the kinetics of Pparc1sv and Pparc2 induction were similar to those of 3T3-L1 cells. The expression of Pparc1sv and Pparc2 significantly increased upon differentiation up to day 6, and reached a plateau at day 9, respectively ( Fig. 3B). No appreciable induction of Pparc1 mRNA was observed in the course of adipocyte differentiation of both 3T3-L1 and primary cells.
Immunoblotting of 3T3-L1 (Fig. 3C) and primary cultured cells (Fig. 3D) with anti-PPARc antibody revealed that the PPARc1 protein was abundant at day 3, expressed at day 6 with similar amount to the PPARc2 protein, but reduced at day 9. We confirmed adipocyte differentiation of both 3T3-L1 (Fig. 3E) and primary cultured cells (Fig. 3F) by staining intracellular lipid accumulation with Oil Red O.
In ST2 cells, the kinetics of Pparc1sv and Pparc2 induction were different from 3T3-L1 cells. The expression of Pparc1sv and Pparc2 slightly increased upon differentiation but was down-regulated at days 6 and 9, respectively (Fig. S1A). This is probably due to the lower extent of adipocyte differentiation of ST2 cells. Upon stimulation with bone morphogenetic proteins, ST2 cells alternatively differentiate into osteoblasts. To assess if the up-regulation of Pparc1sv is specific to adipogenesis in ST2 cells, we examined the expression level of Pparc1sv in the course of osteoblast differentiation of ST2 cells. Expression levels of both Pparc1sv and Pparc2 mRNAs were low and not markedly changed during differentiation (Fig. S1B). Alkaline phosphatase staining showed an increase in alkaline phosphatase activity, a hallmark of osteoblastic differentiation, in ST2 cells at 9 days after induction (Fig. S1B, inset photos).

Pparc1sv is Indispensable for Adipogenesis in 3T3-L1
To evaluate whether the expression of Pparc1sv is essential for the adipogenesis, we specifically knocked down Pparc1sv mRNA in the early phase of the differentiation. We designed three specific siRNAs for respective targets, Pparc1sv (sic1sv22, sic1sv30, and sic1sv38) and Pparc2 (sic2_8, sic2_48, and sic2_88) mRNAs. Positions of target sequences for designed siRNAs were indicated in Fig. 4A. 3T3-L1 cells were transfected with either of the siRNAs, and subjected to adipogenic induction in the following day. PPARc1 and PPARc2 protein levels were examined 2 days after induction by Western blotting using anti-PPARc antibody (Fig. 4B). Introduction of all siRNAs for Pparc1sv greatly reduced PPARc1 protein levels relative to differentiated 3T3-L1 cells transfected with negative control siRNA (siControl in Fig. 4B). This indicates that most PPARc1 protein was originated from Pparc1sv mRNA in 3T3-L1 cells during adipocyte differentiation. In contrast, introduction of siRNAs for Pparc2 significantly suppressed PPARc2 proteins at day 2. We also confirmed effective knock-down of both PPARc1 and PPARc2 proteins by introducing siRNA for the common region of the Pparc coding sequence (siccommon in Figs. 4A and 4B). In Pparc1sv knock-down cells, PPARc2 protein levels were notably reduced along with PPARc1 proteins compared with siControl cells (Fig. 4B). Similarly, PPARc1 proteins were partially reduced in Pparc2 knock-down cells (Fig. 4B). These results prompted us to evaluate specificity of these siRNAs in quantitative method. For this purpose, we used the luciferase-based reporter system, in which the full-length Pparc1sv or Pparc2 cDNA was linked to luciferase gene (luc+) in sense or antisense direction (Fig. 4A). We excluded sic1sv38 and sic2_88 siRNAs from this validation assay because they showed less specificity in the knock-down of PPARc1 and PPARc2 proteins. In Fig. 4C, sic1sv22 and sic1sv30 siRNAs for Pparc1sv achieved more than 95% knock-down of the reporter gene with    Neither of siRNAs affected the activity of the reporter with Pparc1sv or Pparc2 in antisense direction (Fig. 4C, right upper and right lower panel). These results confirmed that each siRNA could suppress its target mRNA with high specificity. We thus concluded that depletion of one PPARc isoform affect the other's protein level in 3T3-L1 cells during adipocyte differentiation. We next examined the effect of knock-down of Pparc1sv mRNA on the adipogenesis by Oil Red O staining at day 9 (Fig. 4D) and quantitation of intracellular triglycerides at day 6 ( Fig. 4E). Both results showed that knock-down of Pparc1sv completely (by sic1sv22) or substantially (by sic1sv30) inhibited the lipid accumulation as observed in Pparc2 knock-down cells, implying the importance of Pparc1sv in adipogenesis. We further characterized sic1sv-and sic2-transfected cells by analyzing the expression of the adipocyte-related proteins C/EBPa, C/EBPb, FABP4 (aP2), and DLK (pref-1) by Western blotting (Fig. 4F). Transfection of sic1sv22 or sic1sv30 resulted in no apparent change in protein levels of C/EBPb at days 2 and 6 compared with those of the control cells whereas a slight inhibition in protein levels of C/ EBPa at day 6. Induction of FABP4, an adipogenic marker protein, was markedly inhibited in sic1sv22 and sic2_8 cells at day 6, but neither in siControl nor in sic1sv30 cells. The expression of DLK, a preadipocyte marker, was drastically down-regulated upon differentiation in siControl cells. It was also decreased but could be detected at a very low level in all sic1sv, sic2, and siccommon knock-down cells at day 2 (Fig. 4F).

Pparc1sv Expression is Dependent on C/EBPb and C/EBPd in 3T3-L1 Cells
The significant induction of mouse Pparc1sv mRNA during adipocyte differentiation raised a question of how transcription of Pparc1sv is regulated. C/EBPb and C/EBPd are induced within a day during adipogenesis of 3T3-L1 cells [4]. This in turn activates expression of Pparc and C/EBPa. The two reciprocally stimulate each other by forming a positive feedback loop, and synergistically promote the downstream gene expression required to accomplish adipogenesis. The promoter of Pparc2 contains two C/EBP recognition elements, and Pparc2 is directly up-regulated by C/ EBPa and C/EBPd [14]. To clarify whether C/EBPs up-regulate Pparc1sv as well, we performed a luciferase reporter assay using the Pparc1sv promoter (2969 to +50) that had been subcloned into the luciferase reporter vector pGL3-Basic. The reporter construct was co-transfected with the expression vector harboring either of the coding sequence of C/EBPa, C/EBPb, or C/EBPd. C/EBPa and C/EBPb have several isoforms, which include full-length and Nterminally truncated proteins [15]. In differentiating 3T3-L1 cells, we detected three C/EBPb isoforms, full-length (p34) and two Nterminally truncated C/EBPb proteins (p30 and p20). Of the three, C/EBPb (p30) was the dominant isoform (Fig. 5A). As shown in Fig. 5B, C/EBPb (p30) and C/EBPd markedly increased the promoter activities of Pparc1sv and Pparc2, while full-length C/ EBPb (p34) did not. Overexpression of C/EBPa gave a slight but significant increment (P,0.05) in both Pparc1sv and Pparc2 promoter activities compared to control cells, respectively. Pparc1 promoter activity was not altered by co-transfection with each of the overexpression vectors (Fig. 5B). We next examined the effect of C/EBPb knock-down on the expression levels of Pparc1sv and Pparc2 mRNAs after the induction of adipocyte differentiation. Each of two discrete siRNAs targeting C/EBPb were transfected into 3T3-L1 cells. As shown in Fig. 5C, expression of both Pparc1sv and Pparc2 mRNAs were markedly inhibited in C/EBPb knockdown cells at day 3 of induction. Protein levels of PPARc1 and PPARc2 were also significantly suppressed at days 2 and 6 of induction by transfecting C/EBPb siRNA (#1) relative to those of control siRNA (Fig. 5D).

Discussion
In this study, we analyzed 59-ends of mouse Pparc cDNAs and isolated full-length cDNA of the novel splicing variant, Pparc1sv, that encodes PPARc1 protein. Pparc1sv was remarkably upregulated with an induced adipogenesis. Knock-down of Pparc1sv in adipocytes resulted in the substantial reduction of the PPARc1 protein and intracellular lipid accumulation, indicating an indispensable role of Pparc1sv in adipocyte differentiation.
Several Pparc splicing variants except for Pparc1 and Pparc2 have been identified in humans [12,13,[16][17][18], monkeys [19], and pigs [11]. Mouse Pparc1sv and corresponding splicing variants in the above 3 mammals and rats share a unique exon (named C in mouse) (Table 1), which implies that the expression of this splicing variant is ubiquitous in mammals.
The expression profiling of three Pparc transcripts showed that their different abundance in mouse tissues (Figs. 2B and 2C). In several tissues, Pparc1sv is expressed at higher levels than the others. For example, expression level of Pparc1sv in spleen was 3.3 and 5.3 times higher than those of Pparc1 and Pparc2, respectively. Thus, Pparc1sv could be a major transcript and contribute to the PPARc protein expression the most in those tissues. While localization of Pparc1sv in mouse embryo is undetermined, Pparc1sv is dramatically up-regulated during the late stages of fatal development (15-and 17-day, Fig. 2B), implying that Pparc1sv is deeply involved in cell differentiation in embryo. The pathological analyses of PPARc-deficient mice revealed PPARc functions in multiple tissues such as the adipose tissue, the placenta, and the developing heart during pre-and postnatal development [20]. Recently, overlapping and distinct functions of PPARc1 and PPARc2 in prostate epithelial cells have been reported [21]. We are presently generating Pparc1svand/or Pparc1-deficient mouse to assess the specific roles of each isoform in development, which will provide some answers to the meaning and importance of the production of multiple transcripts in Pparc.
To date, PPARc2 protein but not PPARc1 is thought to play an essential role in adipogenesis, because Pparc2 mRNA is upregulated during the initiation of adipocyte differentiation whereas Pparc1 is not. In this study, we showed that Pparc1sv is highly expressed in the white and brown adipose tissues (Fig. 2C), which indicates considerable expression of not only PPARc2 but also PPARc1 protein in the adipose tissues. In fact, both PPARc1 and PPARc2 proteins drastically increased during adipogenesis of 3T3-L1 and primary cultured cells (Figs. 3C and 3D). We showed that Pparc1sv is markedly up-regulated during adipocyte differentiation (Figs. 3A and 3B). The knock-down of Pparc1sv using siRNAs resulted in significant suppression of PPARc1 protein during adipocyte differentiation of 3T3-L1 cells (Fig. 4B). These results strongly support that PPARc1 protein expressed during adipogenesis is derived from Pparc1sv mRNA. Knock-down of Pparc1sv also greatly inhibited the accumulation of intracellular triglyceride (Fig. 4E) and the induction of an adipocyte marker FABP4 (sic1sv22 in Fig. 4F) in 3T3-L1 cells. Incomplete adipocyte differentiation of sic1sv-and sic2-transfected cells was also confirmed by the partial expression of a preadipocyte marker, DLK at day 2 (Fig. 4F). It was likely that inhibition of adipocyte differentiation evaluated by lipid accumulation and marker proteins was dependent on the abundance of PPARc1 and PPARc2 proteins in sic1sv-and sic2-treated cells. We thus concluded that the up-regulation of PPARc1 proteins originated The luciferase reporter construct, the pGL3-Basic, containing the Pparc1sv, Pparc1, or Pparc2 promoter was co-transfected with the C/EBP overexpression construct to 3T3-L1 cells. Cells were harvested 2 days after transfection and assayed using Dual-luciferase reporter assay reagents. The values represent the mean of triplicate measurements. The activity obtained from cells transfected with empty vector is defined as 1. (C) Effect of C/EBPb depletion by siRNA on Pparc expression. Each of two discrete C/EBPb siRNAs (siC/ EBPb #1 and #2) was transfected to 3T3-L1 cells. Cells were harvested at days 0 and 3 of differentiation, and expression of Pparc1sv (black bar) and Pparc2 (gray bar) mRNAs was evaluated by qPCR. Values were normalized to those of 18S rRNA. The RNA expression of siControl cells at day 3 is defined as 100%. (D) Immunoblotting of the nuclear extracts of siC/EBPb #1-treated cells detected by antibodies specific to each of C/EBPb, PPARc, or Lamin B1 (control). doi:10.1371/journal.pone.006558h.g005 from Pparc1sv during adipogenesis is indispensable to accomplish the differentiation process.
C/EBPb and C/EBPd play key roles in the early phase of the adipogenic molecular cascade. Expression of both proteins is enhanced during the initial few hours of differentiation in 3T3-L1, which in turn activate expression of Pparc2 and C/EBPa. We have found that C/EBPb and/or C/EBPd also activated the Pparc1sv promoter (Fig. 5B). Unexpectedly, N-terminally truncated C/ EBPb (p30) significantly activated the both Pparc1sv and Pparc2 promoters (Fig. 5B), but full-length C/EBPb isoform (p34) did not. We demonstrated that the major product in adipogenesis of 3T3-L1 cells was p30 (Fig. 5A). Therefore, it is possible that p30 and C/ EBPd directly initiate the synergistic expression of Pparc1sv and Pparc2 mRNA in the early period of adipogenesis.
Intriguingly, inhibition of either of Pparc transcript by specific siRNA resulted in suppression of both PPARc proteins (Fig. 4B). Validation of designed siRNAs using the luciferase reporter system showed their highly effective and specific knock-down properties (Fig. 4C). These results imply that expression level of PPARc1 protein could affect that of PPARc2 and vice versa during adipogenesis of 3T3-L1 cells. One possible explanation is the direct up-regulation of the Pparc transcription by PPARc proteins. It has been demonstrated that Pparc2 gene expression is regulated by binding of the PPARc/RXRa heterodimer to the Pparc2 promoter during adipocyte differentiation of 3T3-L1 [8]. Although direct interaction of the PPARc1 protein to the Pparc2 promoter has not been clarified, it is probable that depletion of the PPARc1 protein by siRNA targeting to Pparc1sv caused reduction in the amount of the PPARc/RXRa heterodimer, which resulted in less activation of the Pparc2 promoter and down-regulation of the PPARc2 protein (Fig. 6, arrow with an asterisk). On the other hand, binding of the PPARc/RXRa heterodimer to the regions of the Pparc1sv promoter (Fig. 6, arrow with a sharp) was not observed [8]. We could not identify the consensus sequence for PPARc and RXRa binding in the Pparc1sv promoter (,1 kb). Therefore, down-regulation of Pparc1sv by the introduction of Pparc2-specific siRNA might be involved in downstream factors that are regulated by PPARc2 protein and activate the Pparc1 promoter.
The present study suggests the importance of Pparc1sv in the adipocyte differentiation and a real need to elucidate a detailed mechanism of the Pparc1sv regulation and the precise function of PPARc1 protein in cell differentiation. Figure S1 Relative expression of three PPARc transcripts during adipocytic and osteoblastic differentiation of ST2 cells. (A) Confluent ST2 cells were cultured in RPMI1640 medium supplemented with 10% FBS, 0.25 mM dexamethasone, 500 mM isobutylmethylxanthine, 1 mM insulin, and 1 mM rosiglitazone to induce adipocytic differentiation. Cells were harvested at the indicated time and analyzed by real-time RT-PCR. (B) ST2 cells were cultured in RPMI1640 medium supplemented with 10% FBS and 100 ng/ml BMP-4 (Wako, Japan) to induce osteoblastic differentiation. Cells were analyzed by real-time RT-PCR or fixed with 10% formalin for 20 min and stained using an alkaline phosphatase staining kit (Primary Cell Co., Ltd). (TIF)