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. 2013 Feb 15;288(7):4522-37.
doi: 10.1074/jbc.M112.440792. Epub 2012 Dec 28.

Fam57b (Family With Sequence Similarity 57, Member B), a Novel Peroxisome Proliferator-Activated Receptor γ Target Gene That Regulates Adipogenesis Through Ceramide Synthesis

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Fam57b (Family With Sequence Similarity 57, Member B), a Novel Peroxisome Proliferator-Activated Receptor γ Target Gene That Regulates Adipogenesis Through Ceramide Synthesis

Yzumi Yamashita-Sugahara et al. J Biol Chem. .
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Abstract

This report identifies a novel gene encoding Fam57b (family with sequence similarity 57, member B) as a novel peroxisome proliferator-activated receptor γ (PPARγ)-responsive transmembrane gene that is related to obesity. The gene was identified based on an integrated bioinformatics analysis of the following three expression profiling data sets: adipocyte differentiation of mouse stromal cells (ST2 cells), adipose tissues from obesity mice, and siRNA-mediated knockdown of Pparγ using ST2 cells. Fam57b consists of three variants expressed from different promoters and contains a Tram-Lag1-CLN8 domain that is related to ceramide synthase. Reporter and ChIP assays showed that Fam57b variant 2 is a bona fide PPARγ target gene in ST2 cells. Fam57b was up-regulated during adipocyte differentiation, suggesting that FAM57B is involved in this process. Surprisingly, FAM57B overexpression inhibited adipogenesis, and siRNA-mediated knockdown promoted adipocyte differentiation. Analysis of the ceramide content by lipid assay found that ceramides were in fact augmented in FAM57B-overexpressing ST2 cells. We also confirmed that ceramide inhibits adipogenesis. Therefore, the aforementioned results of FAM57B overexpression and siRNA experiments are reconciled by ceramide synthesis. In summary, we present in vitro evidence showing that PPARγ regulates Fam57b transcription during the adipogenesis of ST2 cells. In addition, our results suggest that PPARγ activation contributes to the regulation of ceramide metabolism during adipogenesis via FAM57B.

Figures

FIGURE 1.
FIGURE 1.
Screening of PPARγ target genes that regulate metabolic disease. A, we performed an expression analysis of genes under the following three conditions: 1) genes that were up-regulated more than 2-fold in adipose tissue from mice that were fed a high fat diet (HFD) compared with a normal diet as a control (ND), 2) genes up-regulated in ST2 cells that had undergone adipogenesis, and 3) genes down-regulated in ST2 cells that had been treated with siRNA targeting Pparγ and undergone adipogenesis. The number in the Venn diagram represents the number of probes on the microarray. There were 14 common genes that contained a signal sequence and/or transmembrane region (which was screened with the signal peptide (SP) and transmembrane hidden Marcov method (Tmhmm) programs) as shown in Table 5. B, the structures of three variants of the Fam57b gene. The last four exons are common exons in these three variants. +1, transcription start site. C, the common amino acid sequence of the three FAM57B variants is indicated with a star below each amino acid, in which the lined amino acid sequence indicates the TLC domain. The sequences were obtained from the NCBI Web site.
FIGURE 2.
FIGURE 2.
Expression profiling of Fam57b in ST2 adipogenesis and mouse different tissues. A, samples were harvested on the indicated days after adipogenic induction, and total RNA was extracted and analyzed by real-time RT-PCR with primers targeting a common sequence (indicated by Fam57b_c2) and variant-specific sequences (indicated by Fam57b_var1, -2, and -3). As a control, samples left uninduced were also analyzed by qRT-PCR using the common sequence primers (Fam57b_c2(C)). B, 3T3L1 cells were differentiated into adipocytes, and samples were harvested on the indicated days. Total RNA was extracted and analyzed for Fam57b expression using common primers (Fam57b_c2) as well as specific primers for variants 1 and 2 (Fam57b_var1 and Fam57b_var2). C, ST2 cells were induced with rosiglitazone instead of a total adipogenic mixture and analyzed on the indicated days of differentiation. The mRNA expression of three Fam57b variants was analyzed by real-time RT-PCR as indicated above. D, the expression of the Fam57b variants in normal tissues from C57bBL/6J mice was analyzed by real-time RT-PCR with variant-specific primers. BAT, brown adipose tissue; WAT, white adipose tissue; AdreG, adrenal gland; CortB, cortical bone; Calv, calvaria; BM, bone marrow. E, the expression of the Fam57b variants was examined in adipose tissue derived from obese mice induced by a high fat diet (HFD), and from mice fed a normal diet (ND). Two different primers were used for variants 1 and 2 (a and b). Results are the means ± S.D. (error bars) (n = 3). *, p < 0.05; **, p < 0.01; ***, p < 0.001.
FIGURE 3.
FIGURE 3.
PPARγ targets a promoter-proximal site of the Fam57b variant 2. A, the predicted PPRE sequence sites are indicated with arrows on each Fam57b promoter, and heavy lines indicate the PCR amplicons used for ChIP-qPCR, whose numbers correspond to those indicated in Table 2. B, ST2 cells that were induced into adipocytes for 2 and 6 days (A2d and A6d, respectively) as well as uninduced cells were analyzed by ChIP-qPCR as indicated. Fabp4 was used as a positive control, and the promoter site of each Fam57b variant was examined. Results are means ± S.D. (error bars) (n = 3); **, p < 0.01. +1, transcription start site.
FIGURE 4.
FIGURE 4.
PPARγ binds and activates the gene promoter of Fam57b variant 2. A, schematic illustration of Fam57b variant 2 promoter and luciferase reporter construct. The five candidates for the PPRE are numbered 1–5 in the promoter region of Fam57b variant 2. These promoter sequences were inserted into the pGL4.10 promoterless reporter vector. The sequences of WT and mutant types (mut1 and mut2) for the second PPRE are indicated. B, NIH3T3 cells were transfected with the reporter vectors together with PPARγ or the control expression vector and treated with rosiglitazone (Rosi) at 24 h post-transfection for another 24 h. The cells were harvested, and luciferase activity was measured with ARVO according to the manufacturer's instructions. The luciferase activity was shown as FL/RL. FL, firefly luciferase activity was normalized by RL, renilla luciferase. C, the pro1–5 wild type (pro_wt), pro1–5 mutants 1 and 2 (pro_mut1 and pro_mut2), and pro1–5 PPRE2 deleted (pro_del) were analyzed as described above. D, EMSA was performed using a 32P-labeled oligonucleotide containing the above second PPRE WT and mut1 of Fam57b var2 promoter and ap2 PPRE as a positive control. The labeled probes were incubated with nuclear extracts of adipocytes (Ad NE) and preadipocytes (Pread NE) of ST2 cells as a negative control. For the supershift assay, the adipocyte nuclear extract was preincubated with labeled WT or ap2 PPRE and then incubated with an anti-PPARγ antibody or mouse IgG for 20 min. The DNA-protein complexes were resolved by PAGE. Lane 1, free probe; lanes 2–4, WT, mut1, and ap2 PPRE with preadipocyte nuclear extract, respectively; lanes 5–7, WT, mut1, and ap2 PPRE with adipocyte nuclear extract, respectively; lanes 8 and 10, WT and ap2 with mouse IgG and Ad NE, respectively; lanes 9 and 11, WT and ap2 PPRE with PPARγ-specific antibody and adipocyte nuclear extract, respectively. Results are means ± S.D. (error bars) (n = 3).
FIGURE 5.
FIGURE 5.
Adipocyte differentiation of ST2 cells is inhibited by overexpression of FAM57B. A, ST2 cells overexpressing FAM57B-FLAG were analyzed on day 6 of adipocyte differentiation by Western blotting using an anti-FLAG antibody. B, ST2 cells were infected with retroviruses expressing three different FAM57B variants. Three days after infection, adipogenesis was induced by replacing the medium with fresh medium containing an adipogenic mixture as indicated under “Experimental Procedures.” On day 6 of differentiation, the cells were fixed and stained with Oil Red O in which lipids are stained red, at a magnification of ×10. Scale bar, 100 μm. C, triglyceride (TG) content of ST2 cells expressing the FAM57B variants was analyzed on day 6 of adipogenesis. D, mRNA level of adipocyte markers was analyzed by qRT-PCR. E, we measured the Fam57b mRNA levels in ST2 cells overexpressing each FAM57B variant. F, ST2 cells overexpressing FAM57B var3 were treated with MG132 at the indicated concentrations for 6–12 h. The cells were harvested and subjected to Western blot analysis using anti-FLAG and anti-β-actin antibodies. Results are means ± S.D. (error bars) (n = 3); *, p < 0.05; **, p < 0.01; ***, p < 0.001.
FIGURE 6.
FIGURE 6.
Adipogenic differentiation is promoted by Fam57b down-regulation in ST2 cells. A, ST2 cells were transfected with three classes of Fam57b siRNA (siFam57b-A, -B, and -C) targeting a common site in all of the Fam57b variants. Non-targeting control siRNA (siNC) and Pparγ siRNA (siPparγ) were used as a negative and positive control, respectively. At 6 h post-transfection, adipogenesis was induced, and total RNA was extracted from the cells on day 2. The knockdown efficiency on day 2 of differentiation was analyzed by real-time RT-PCR. B, ST2 cells were treated with siRNA and adipogenic induction and then fixed and stained with Oil Red O on day 6. Magnification was ×10. Scale bar, indicates 100 μm. C, the triglyceride (TG) content was analyzed on day 6 of differentiation. D, markers of adipocyte differentiation were analyzed on day 6 of differentiation by qRT-PCR. Pparγ, AdipoQ and Glut4 were used as mature adipocyte markers, and perilipin 1 (Plin1) was used as a lipid droplet marker. Results are means ± S.D. (error bars) (n = 3); *, p < 0.05; **, p < 0.01; ***, p < 0.001.
FIGURE 7.
FIGURE 7.
Total ceramide content is augmented in ST2 cells overexpressing FAM57B. A, ST2 cells were treated with the indicated concentration of C6-ceramide during adipogenesis and then stained with Oil Red O on day 6 of differentiation. The Oil Red O that accumulated in lipid droplets was extracted with isopropyl alcohol and measured at OD 535 nm. B, the triglyceride content was measured on day 6 of adipogenesis and is presented as the triglyceride levels normalized to the protein levels and the control. C, ST2 cells overexpressing each FAM57B variant with (var1F–var3F) and without FLAG (var1–var3) were differentiated into adipocytes for 6 days. Total lipids were extracted from cell lysates containing 200 μg of protein and subjected to the diacylglycerol kinase assay that enzymatically phosphorylates and labels lipids with 32P (Ceramide-1-[32P]), as described under “Experimental Procedures.” The samples were spotted onto thin layer chromatography plates and developed in organic solvents (bottom panel). The ceramide levels were digitized by measuring the band intensity with an Image analyzer (Image Gauge version 3.4, FUJIFILM), normalized to the protein levels, and then expressed relative to the control (EGFP-expressing cells). γ2 represents the ceramide levels that accumulated upon PPARγ overexpression as a positive control. The experiment was performed at least three times. D, the ceramide content of siFam57b-A treated ST2 cells was examined in a similar manner with or without 20 μm FB1, a ceramide synthase inhibitor, which was added from day 5 to 6 of differentiation. The ceramide levels were measured 6 days after differentiation using 100 μg of protein. The relative ceramide levels between siNC and siFam57b are shown for both conditions with and without FB1. E, ST2 cells were treated with siRNA (siNC and siFam57b-A, -B, and -C) and induced to undergo adipogenesis. On day 6, total RNA was extracted and analyzed by qRT-PCR. The mRNA expression of Cers2, -4, -5, and -6 was measured using the primer sets listed in Table 2. Results are means ± S.D. (error bars) for three individual experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
FIGURE 8.
FIGURE 8.
FAM57B overexpression does not affect the mRNA expression levels of ceramide synthase or sphingomyelinase activity. A, the mRNA expression levels of four ceramide synthases (Cers2, -4, -5, and -6) in differentiated FAM57B-overexpressing ST2 cells on day 6 were analyzed by qRT-PCR. B, the total sphingomyelinase activity was analyzed in differentiated FAM57B-overexpressing ST2 cells on day 6 using a sphingomyelinase assay kit. Results are means ± S.D. (error bars) for three individual experiments. *, p < 0.05; **, p < 0.01; ***, p < 0.001.
FIGURE 9.
FIGURE 9.
Ceramide synthase activity of the FAM57B protein. A, in vitro ceramide synthase activity was measured using differentiated ST2 cell extracts (50 μg of protein), which overexpressed each FAM57B variant (var1–var3) with different substrates, such as palmitoyl-CoA (C16:0), stearoyl-CoA (C18:0), arachidoyl-CoA (C20:0), and behenoyl-CoA (C22:0). B, an in vitro ceramide synthase assay was performed using undifferentiated FAM57B-FLAG or ceramide synthase 2-FLAG (CERS2F)-overexpressed ST2 cell extracts (50 μg of protein) that were preincubated with or without 20 μm FB1, followed by adding C18-acyl-CoA as substrate. C, in vitro ceramide synthase activity with or without fumonisin B1 was assayed using 293FT cell extracts (50 μg of protein), which transiently overexpressed FAM57B var2F or CERS2F. D, ST2 cells overexpressing CERS2F, EGFP, or FAM57B-F (each variant) were immunoprecipitated (IP) with an anti-FLAG antibody, which was released with 3× FLAG peptide, and subjected to Western blot analysis (WB) using an anti-FLAG antibody. E, in vitro ceramide synthase activity was measured using purified FAM57B-FLAG protein (var1F–var3F) and CERS2F, with different substrates, such as C16-, C18- and C20-acyl-CoA. Results are representative of three independent experiments.
FIGURE 10.
FIGURE 10.
Effect of C18- and C20-ceramide on ST2 adipogenesis. A, ST2 cells were treated with 20 μm C18-ceramide (C18-cer), or 15 μm C20-ceramide (C20-cer) during adipogenesis. The cells were stained with Oil Red O on day 6 of differentiation. Scale bar, 100 μm. B, the Oil Red O, which accumulated in lipid droplets, was extracted with isopropyl alcohol and measured at OD 535 nm. C, ST2 cells were treated with 20 or 25 μm C18-ceramide and 7 μm or 15 μm C20-ceramide during adipogenesis. The relative triglyceride levels were measured on day 6 of differentiation and normalized to the total protein levels. *, p < 0.05; **, p < 0.01; ***, p < 0.001. Error bars, S.D.
FIGURE 11.
FIGURE 11.
The proposed role of FAM57B in adipogenesis. PPARγ directly controls FAM57B var2 expression, and consequently FAM57B synthesizes ceramides during adipogenesis. PPARγ is the master regulator of adipogenesis. FAM57B also regulates adipogenesis through ceramide metabolism. High ceramide levels could inhibit adipogenesis, whereas low levels might promote adipogenesis by being converted to sphingosin 1-phosphate (S1P), which is another sphingolipid that promotes adipogenesis and generally works contrary to ceramides.

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