A Novel Gene, Fad49, Plays a Crucial Role in the Immediate Early Stage Of

A novel gene, fad49, plays a crucial role in the immediate early stage of adipocyte differentiation via involvement in mitotic clonal expansion Tomoaki Hishida, Tsuyoshi Eguchi, Shigehiro Osada, Makoto Nishizuka and Masayoshi Imagawa

Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Nagoya City University, Aichi, Japan

Keywords Adipogenesis is accomplished via a complex series of steps, and the events 3T3-L1 cell; adipocyte differentiation; at the earliest stage remain to be elucidated. To clarify the molecular mech- CCAAT ⁄ enhancer-binding protein; obesity; anisms of adipocyte differentiation, we previously isolated 102 genes peroxisome proliferator-activated receptor c expressed early in mouse 3T3-L1 preadipocyte cells using a PCR subtrac- Correspondence tion system. About half of the genes isolated appeared to be unknown. M. Imagawa, Department of Molecular After isolating full-length cDNAs of the unknown genes, one of them, Biology, Graduate School of Pharmaceutical named factor for adipocyte differentiation 49 (fad49), appeared to be a novel Sciences, Nagoya City University, 3-1 gene, as the sequence of this clone showed no identity to known genes. Tanabe-dori, Mizuho-ku, Nagoya, Aichi 467- FAD49 contains a phox homology (PX) domain and four Src homology 3 8603, Japan (SH3) domains, suggesting that it may be a novel scaffold protein. We Fax: +81 52 836 3455 found that the PX domain of FAD49 not only has afﬁnity for phosphoi- Tel: +81 52 836 3455 E-mail: [email protected] nositides, but also binds to its third SH3 domain. Expression of fad49 was transiently elevated 3 h after differentiation was induced, and diminished (Received 24 July 2008, revised 7 24 h after induction. Induction of the fad49 gene was observed in adipocyte September 2008, accepted 11 differentiable 3T3-L1 cells, but not in non-adipogenic NIH-3T3 cells. September 2008) RNAi-mediated knockdown of fad49 signiﬁcantly impaired adipocyte differentiation. Moreover, the knockdown of fad49 by RNAi inhibited mitotic doi:10.1111/j.1742-4658.2008.06682.x clonal expansion, and reduced the expression of CCAAT ⁄ enhancer-binding protein b (C ⁄ EBPb) and C ⁄ EBPd at the immediate early phase. Taken together, these results show that fad49, a novel gene, plays a crucial role in the immediate early stage of adipogenesis.

Obesity is a serious and growing health problem that dysregulated production of hormones and cytokines by is a key risk factor in several obesity-related diseases, adipose tissue, such as tumor necrosis factor a, adipo- such as type 2 diabetes, hypertension, hyperlipidemia nectin and resistin, which leads to various diseases, and cardiac infarction [1–3]. Obesity may occur such as type 2 diabetes, stroke and cardiac infarction through excessive accumulation of white adipose tissue [3–6]. (WAT), composed mainly of adipocytes, which play an Obesity, the pathological development of adipose tis- important role in the storage of energy and secretion sue, results from an increase in the cell size of individ- of a variety of hormones and cytokines that regulate ual adipocytes and an increase in total adipocyte cell metabolic activities in the body [1]. Such pathological numbers through differentiation of preadipocytes in accumulation of WAT in the body results in adipose tissue into mature adipocytes. Therefore, in

Abbreviations aP2, adipocyte lipid-binding protein; C ⁄ EBP, CCAAT ⁄ enhancer-binding protein; DAPI, 4¢,6-diamidino-2-phenylindole; DMEM, Dulbecco’s modiﬁed Eagle’s medium; fad, factor for adipocyte differentiation; FBS, fetal bovine serum; GST, glutathione S-transferase; IBMX, 3-isobutyl-1- methylxanthine; MCE, mitotic clonal expansion; PI(3)P, phosphatidylinositol 3-phosphate; PI(3,4)P2, phosphatidylinositol 3,4-bisphosphate;

PI(4)P, phosphatidylinositol 4-phosphate; PI(4,5)P2, phosphatidylinositol 4,5-bisphosphate; PI(5)P, phosphatidylinositol 5-phosphate; PPARc, peroxisome proliferator-activated receptor c; PX, phox homology; SH3, Src homology 3; shRNA, short hairpin RNA; SREB-1, sterol regulatory element-binding protein-1; WAT, white adipose tissue.

5576 FEBS Journal 275 (2008) 5576–5588 ª 2008 The Authors Journal compilation ª 2008 FEBS T. Hishida et al. fad49 plays a crucial role in adipogenesis the context of the prevention and treatment of obesity- differentiation. FAD49 contains a phox homology related diseases, it is important to elucidate the mecha- (PX) domain, which has affinity for phosphoinositides, nisms of adipocyte differentiation, as well as adipocyte and four Src homology 3 (SH3) domains, which bind enlargement. to polyproline PXXP ligands, suggesting that FAD49 Much knowledge of adipogenesis has derived from is a novel scaffold protein. RNAi experiments demon- studies using mouse 3T3-L1 cells as model cells of strated that fad49 is crucial in adipogenesis, and that it adipocyte differentiation. 3T3-L1 cells are grown to con- plays important roles in events early in the differentia- fluence and growth arrested. Growth-arrested 3T3-L1 tion process, including MCE and the induction of cells differentiate into mature adipocytes after the addi- C ⁄ EBPb and C ⁄ EBPd genes. Taken together, these tion of insulin, 3-isobutyl-1-methylxanthine (IBMX), results imply that fad49, encoding a novel scaffolding dexamethasone and fetal bovine serum (FBS) [7–9]. protein, plays an important role in the immediate early After treatment with the induction cocktail, they stage of adipocyte differentiation. undergo approximately two cycles of synchronized cell division, a process known as mitotic clonal expansion Results (MCE) [10,11]. MCE is a requisite step for adipocyte differentiation, followed by terminal differentiation, in Cloning of full-length mouse fad49 cDNA which peroxisome proliferator-activated receptor c (PPARc) and CCAAT ⁄ enhancer-binding protein a Using the PCR subtraction method, we originally (C ⁄ EBPa) play important roles as master regulators isolated fad49 as one of many unknown genes the [12]. In terminal differentiation, PPARc and C ⁄ EBPa expression of which was elevated at 3 h after induction transactivate each other and upregulate the expression of adipocyte differentiation. The PCR-subtraction of many adipogenic genes, causing the cells to acquire method used in the previous study gave cDNA frag- an adipogenic phenotype. ments only 300–900 bp long because the amplified frag- Expression of these transcriptional factors starts to ments were digested using RsaI for non-bias cloning increase in the middle stage of adipocyte differentia- [16]. The length of fad49 was 870 bp. Therefore, we tion, partly through transactivation of C ⁄ EBPb and attempted to isolate a full-length cDNA of fad49 using C ⁄ EBPd, the expression of which is immediately upreg- 5¢ and 3¢ RACE methods, and expressed sequence tag ulated after hormonal induction [13,14]. Several other (EST)-walk method, which is a combination method of factors that are involved in regulating the expression predicting exons of interest in genes, utilizing the mouse and transcriptional activity of PPARc and C ⁄ EBPa genome and EST followed by RT-PCR (Fig. 1A). 5¢ have been identified by other studies [15]. Therefore, and 3¢ RACE were performed using cDNA prepared events in the middle and late stage of adipocyte differ- from 3T3-L1 cells 3 h after induction. As a result, a entiation have been studied relatively thoroughly. In 1109 bp cDNA fragment containing an initiation contrast, the overall mechanisms of events in the early codon at 6 bp was isolated by 5¢ RACE. The sequence stage of the differentiation programme, including (GCCATGC) including initiation codon is close to MCE and induction of the C ⁄ EBPb and C ⁄ EBPd the consensus sequence for translation initiation genes, remain to be elucidated. (A/GCCATGA/G). A 1809 bp cDNA fragment con- In order to clarify the molecular mechanisms in the taining a stop codon was isolated by RT-PCR. A early phase of adipocyte differentiation, we previously 1007 bp cDNA containing a poly(A) tail was obtained isolated 102 genes for which expression early in the by 3¢ RACE. By combining these cDNA fragments, differentiation process was induced using a PCR sub- fad49 was found to consist of 7258 bp with an ORF of traction system [16,17]. These genes included transcrip- 910 amino acids (GenBank accession number tion factors and signaling molecules [17–19]. About AB430861). Because blast database searches identified half of them were unknown genes, whose functions no significant matches against proteins of known func- remain unclear. As the fragments obtained by PCR tion, fad49 appears to be a novel gene. subtraction are small, we needed to isolate the full- We next analyzed the genomic distribution of fad49 length cDNAs of the unknown genes. We have previ- using the mouse genome database (http://www. ously revealed that several of them are novel genes, ncbi.nlm.nih.gov/genome/seq/BlastGen/BlastGen.cgi? such as factor for adipocyte differentiation (fad) 24, taxid=10090), which was made public by the Mouse fad104 and fad158, which play crucial roles in adipo- Genome Sequencing Consortium. The result indicated genesis [20–23]. that fad49 exists at locus 11A4 of mouse chromo- Here, we report the isolation of another novel gene, some 11 and consists of 13 exons divided by 12 introns fad49, and the close involvement of fad49 in adipocyte (Fig. 1B). Sequencing of the exon ⁄ intron junctions in

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AB430862). A blast search of the human genome database revealed a human homolog of fad49 on chro- mosome 5 at locus 5q35. The protein encoded by human fad49 also contained a PX domain and four SH3 domains. A comparison of the human and mouse FAD49 showed 87.1% conservation at the full-length protein level, and more than 96% at the domain level (Fig. 2B).

Characterization of the PX domain of FAD49 The PX domain has been reported to be implicated in highly diverse functions, such as cell signaling, vesicular trafficking and protein sorting [24–30]. Recent studies have demonstrated that PX domains are important phosphoinositide-binding modules with varying lipid- binding specifities, although specificity for phosphati- Fig. 1. Cloning and genomic structures of mouse fad49. (A) The full- dylinositol 3-phosphate [PI(3)P] appears to be the most length cDNA for mouse fad49 was isolated by RT-PCR, 5¢ and 3¢ RACE. ‘S’ is the fragment obtained by the original PCR-subtraction common [28,31–34]. For example, the PX domain of phox method. ‘RT’, ‘5¢-R’ and ‘3¢-R’ are fragments obtained by RT-PCR, 5¢ p40 interacts with PI(3)P, the PX domains of phox RACE and 3¢ RACE, respectively. The combined sequence is shown p47 and Fish, which contains five SH3 domains and as fad49. The initiation and stop codon are also indicated. The a PX domain, binds to PI(3,4)P2 [24,27], and the PX deduced amino acid sequence revealed a 910 amino acid protein for domain of C2 containing Ptdlns kinase (CPK) class of mouse FAD49. (B) Schematic representation of the mouse fad49 PI 3-kinase selectively binds to PI(4,5)P2 [32]. Moreover, gene structure. The thirteen exons of the fad49 gene on chromo- the conserved polyproline motif (PXXP) in many PX some 11 are represented by vertical bars in the top part of the figure. The size of each exon is indicated in the bottom panel. domains suggests that it may act as a target for SH3 domains. In fact, it has been reported that the PX the database revealed that the GT ⁄ AG rule was main- domain of p47phox binds intramolecularly to the SH3 tained in all cases (data not shown). domain in the same protein, and that this intramolecular interaction suppresses the lipid-binding activity of the PX domain in the resting state; phosphorylation of The deduced protein primary structure of mouse p47phox releases the binding, resulting in the active state, and human fad49 i.e. open conformation [25,35,36]. The ORF of fad49 encodes a putative protein of 910 As FAD49 contains a PX domain as described above, amino acids that contains a PX domain (solid underlin- we determined whether the FAD49 PX domain could ing) and four SH3 domains (dotted underlining) bind to phosphoinositides. To test whether the FAD49 (Fig. 2A). Moreover, the encoded protein also con- PX domain has affinity for phosphoinositides, we bacte- tained ten polyprolines (boxed), which could be possible rially expressed the FAD49 PX domain fused to gluta- ligands for the SH3 domain. Thus, fad49 encodes a pro- thione S-transferase (GST–PX) and used GST protein tein containing many protein-binding domains, suggest- (GST) as a negative control. Lipid binding was then ing that this protein may be a novel scaffold protein. measured using overlay blotting as described in Experi- We next tried to isolate the full-length ORF of mental procedures. As shown in Fig. 3A, GST–PX human fad49. We first used the human genome data- bound most strongly to PI(3,5)P2, and to a lesser extent base to predict the ORF region of human fad49 by to PI(3)P, PI(4)P and PI(5)P, but no binding to any of splicing out the introns and combining the exons of the lipid species was observed for GST as a control. the ORF of human fad49. To isolate human fad49 The PXXP motif was found in the FAD49 PX including the entire ORF, we next constructed primer domain, as in many PX domains, suggesting that the sets as described in Experimental procedures, and per- PX domain could bind to an SH3 domain of FAD49. formed RT-PCR using template cDNA prepared from To test whether the PX domain could interact with an mRNA extracted from HeLa cells. From sequence SH3 domain in FAD49, we performed in vitro binding analyses of the resultant fragments, the full-length assays using various bacterially expressed proteins: cDNA of human fad49 comprised a 2733 bp ORF GST–PX and FLAG fusion proteins of individual encoding 911 amino acids (GenBank accession number SH3 domains of FAD49. We found that the PX

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Fig. 2. Amino acid sequence and domain structure of FAD49. (A) Amino acid sequence of mouse FAD49. The PX domain is underlined by a solid line and the four SH3 domains are underlined by dotted lines. Ten polyproline motifs that represent poten- tial SH3 binding sites are boxed. (B) Sche- matic structure of mouse and human FAD49. The PX domain and four SH3 domains are highly conserved between mice and humans. domain of FAD49 can interact with its third SH3 The truncated mutant GFP–PX, which only contains domain (Fig. 3B). the PX domain of FAD49, was found in punctate structures in the nuclei as well as in the cytoplasm. The other truncated mutant, GFP–SH3, which does Subcellular localization of FAD49 not contain the PX domain, was localized in punctate To further characterize fad49, the subcellular localiza- structures that seem to differ from those in which tion of FAD49 was determined by transient transfec- GFP–PX was found. These results suggest that the tion of an N-terminally Myc-tagged full-length PX and SH3 domains of FAD49 play a role in the FAD49 (Myc–FAD49) expression plasmid into 3T3- localization of FAD49. L1 cells. Cells were immunostained using monoclonal anti-Myc IgG . As shown in Fig. 4A, Myc–FAD49 1 Expression proﬁles of fad49 in differentiating and was found predominantly in the cytoplasm. The same non-differentiating cells and tissue distribution result was obtained using a C-terminally Myc-tagged FAD49 (FAD49–Myc) expression plasmid. To exam- To investigate the role of fad49 during adipocyte dif- ine the role of the FAD49 PX domain or SH3 ferentiation, we ﬁrst determined the mRNA expression domains on the subcellular localization of FAD49, we levels of fad49 by Northern blotting. To monitor next determined the subcellular localization of GFP changes in the levels of fad49 during adipocyte differ- proteins fused to full-length FAD49 (GFP–FL), the entiation, 3T3-L1 cells were stimulated with an induc- PX domain (GFP–PX) and FAD49 lacking its PX tion cocktail, and then total RNA was prepared at domain (GFP–SH3) (Fig. 4B,C). GFP–FL was mostly various time points. The expression of fad49 increased detected in the cytoplasm, consistent with Fig. 4A. quickly after differentiation was induced, reaching a

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Fig. 3. Characterization of the PX domain of FAD49. (A) Phospho- inositide binding specificity of the PX domain of FAD49. Bacterially expressed GST or GST–PX were incubated with PIP arrays pre- spotted with the indicated phosphpoinositide (100, 50, 25, 12.5, 6.25, 3.13, 1.56 pmol). The membranes were washed and the GST fusion proteins bound to the membrane were detected using anti- GST serum. (B) Interaction of the FAD49 PX domain with its third SH3 domain. FLAG–SH3 proteins (10 pmol) were tested in co-pre- cipitation experiments with GST–PX or GST as a control (1 lg). The co-precipitating samples were subject to SDS–PAGE and detected by Western blotting. maximum at 3 h, and then decreased to 12 h (Fig. 5A). This result indicates that fad49 is transiently expressed in the early phase of adipocyte differentiation. We next determined whether or not fad49 expression was restricted to cells in a state of differentiation. 3T3- Fig. 4. Subcellular localization of FAD49. (A) 3T3-L1 cells transiently L1 cells differentiate to mature adipocytes when stimu- transfected with the Myc–FAD49 or FAD49–Myc expression plas- lated with adipogenic inducers 2 days after reaching a mid were fixed and blocked for immunofluorescence staining with state of confluence, whereas proliferating 3T3-L1 cells anti-Myc serum. (B) Schematic representation of GFP fusion proteins for each FAD49 deletion mutant used in this study. (C) Sub- do not differentiate into adipocytes even in the pres- cellular localization of EGFP–FAD49 fusion proteins. 3T3-L1 cells ence of inducers. Another mouse fibroblastic cell line, were transiently transfected with an EGFP–FAD49-expressing plas- NIH-3T3, does not differentiate into adipocytes in mid or empty vector. One day after transfection, the cells were either a postconfluent or proliferating state. These two fixed and stained with DAPI. EGFP signals were detected using a cell lines were treated with inducers in a postconfluent fluorescence microscope. (growth-arrested) or proliferating state. Total RNA was prepared from these cells before or 3 h after dif- To investigate the tissue distribution of fad49,we ferentiation was induced and subjected to quantitative determined the expression levels of fad49 by quantita- PCR, which showed that marked induction only tive PCR in various tissues isolated from adult male occurred in growth-arrested 3T3-L1 cells, suggesting mice, including WAT and brown adipose tissue (BAT) that the elevation in expression of fad49 is restricted to (Fig. 5C). WAT samples were separated into two frac- the adipocyte differentiable state (Fig. 5B). This result tions: the stromal–vascular fraction enriched with prea- strongly suggests that fad49 plays a functional role in dipocytes and the mature adipocyte fraction. Tissue adipogenesis. distribution studies revealed that high expression of

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Fig. 5. Expression profiles of fad49 in differentiating and non-differentiating cells and tissue distribution. (A) Time course of fad49 mRNA expression in the early stage of adipocyte differentiation. Total RNA prepared from 3T3-L1 cells at various time points after treatment with inducers was loaded (20 lg) in each column. Staining with ethidium bromide (EtBr) for ribosomal RNA is shown as a load- ing control. (B) Expression profile of fad49 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 quantitative PCR. Each column represents the mean ± SD (n = 3). (C) Distribution of fad49 mRNA in various tissues. The expression level of fad49 in various tissues isolated from C57B1 ⁄ 6J mice was determined by quantitative PCR, and normalized to 18S rRNA expression determined by quantitative PCR. Each column represents the mean ± SD (n = 3). fad49 was observed in the stromal–vascular fraction of the expression of fad49 during adipogenesis. For the WAT, and moderate expression was detected in heart, RNAi experiments, two short hairpin RNA (shRNA) skeletal muscle and the mature adipocyte fraction of expression plasmids named shfad49-1 and shfad49-2 WAT. Thus, the expression of fad49 in the stromal– were constructed to target regions 1 and 2, respectively vascular fraction was higher than that in mature (as defined in Experimental procedures), in the ORF adipocytes, suggesting that expression of fad49 is pre- of the fad49 gene. Each of the shRNA expression plas- dominant in preadipocytes. As expression of fad49 was mids was transfected into 3T3-L1 cells. Three hours observed in skeletal muscle, we analyzed the expression after induction for adipocyte differentiation, total levels of fad49 during the myogenesis of mouse C2C12 RNA was isolated, and the expression levels of fad49 cells. Expression of fad49 was weak, and the level was were determined by quantitative PCR. We found unchanged during myogenesis of C2C12 cells (data not that shfad49-2 had a strong silencing effect on fad49 shown). expression, while the effect of shfad49-1 was milder (Fig. 6A). Therefore, we used shfad49-2 to perform the RNAi experiments. Effect of fad49 knockdown on differentiation of Next, we determined the expression levels of fad49 3T3-L1 cells into adipocytes by quantitative PCR in the cells transfected with As described above, the expression of mouse fad49 is shfad49-2 at each time point after induction of differ- rapidly upregulated early in the differentiation of 3T3- entiation. We confirmed that RNAi treatment reduced L1 cells into adipocytes, and seems to play a role in fad49 mRNA levels (Fig. 6B). After 8 days, the cells adipogenesis. To characterize the function of this gene were fixed and stained with Oil red O and the amounts during adipogenesis, we performed RNAi to silence of triacylglycerol were determined. The number of

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Fig. 6. Effect of fad49 knockdown by RNAi on adipocyte differentiation. (A) The effects of two different shRNAs on the expression of fad49. Total RNA, obtained from 3T3-L1 cells transfected with shfad49-1, shfad49-2 or the scrambled shRNA expression plasmid as a control at 3 h after differentiation induction, was subjected to quantitative PCR. The expression level of fad49 was normalized to 18S rRNA expression. Each column represents the mean ± SD (n = 3). (B) fad49 expression in fad49 knockdown 3T3-L1 cells was determined by quantitative PCR. Total RNA obtained from 3T3-L1 cells transfected with shfad49-2 (open bars) or with the scrambled shRNA expression plasmid as a control (solid bars) at each time point was subjected to quantitative PCR. The expression level was normalized to 18S rRNA expression. Each column represents the mean ± SD (n = 3). (C) Adipocyte differentiation of fad49 knockdown 3T3-L1 cells. The cells transfected with shfad49-2 or the scrambled shRNA expression plasmid as a 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 fad49 knockdown cells (open bars) or control cells (solid bars) 8 days after the induction is also shown. Each column represents the mean ± SD (n = 3). **P < 0.01 versus control. (D) Effect of fad49 RNAi treatment on the expression of various adipogenic genes. Total RNA obtained from fad49 knockdown cells (open bars) or control cells (solid bars) at each time point was subjected to quantitative PCR, and normalized to 18S rRNA expression determined by quantitative PCR. Each column represents the mean ± SD (n = 3). *P < 0.05 and **P < 0.01 versus control. (E) Effect of fad49 RNAi treatment on the expression of C ⁄ EBPb and C ⁄ EBPd in the immediate early stage of adipogenesis. Total RNA obtained from fad49 knockdown cells (open bars) or control cells (solid bars) at each time point was subjected to quantitative PCR. Expression levels were normalized to 18S rRNA expression determined by quantitative PCR. Each column represents the mean ± SD (n = 3). *P < 0.05 and **P < 0.01 versus control. (F) Effect of fad49 RNAi treatment on MCE. The cells transfected with shfad49-2(fad49 KD) or the scrambled shRNA expression plasmid as a control were stimulated with inducers. Parallel cultures of fad49 knockdown cells or control cells were harvested on the indicated days after differentiation had been induced. Cell numbers were determined by taking counts with a hemocytometer. Each column represents the mean ± SD (n = 4). **P < 0.01 versus control.

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Oil red O-stained cells and the accumulation of triacyl- the full-length cDNA of a novel gene, named fad49, glycerol were significantly reduced in the RNAi-treated which showed no identity to known genes. Isolation of cells (Fig. 6C). An inhibitory effect of knockdown of the full-length ORFs of mouse and human fad49 fad49 on adipocyte differentiation was also observed in revealed that the protein encoded by fad49 has a PX RNAi experiments performed with shfad49-1 (data not domain, four SH3 domains and several PXXP motifs, shown). Next, we determined the expression levels of suggesting that FAD49 may be a novel scaffold adipogenic marker genes by quantitative PCR protein. (Fig. 6D). The levels of PPARc,C⁄ EBPa, adipocyte The PX domain, described as a phosphpoinositide- lipid-binding protein (aP2) and sterol regulatory ele- binding module, was first identified in p40phox and ment-binding protein-1 (SREBP-1) decreased in fad49 p47phox, two cytosolic subunits of NADPH oxidase, and knockdown cells, indicating that RNAi-mediated has been found since in a variety of proteins involved in knockdown of fad49 inhibits adipocyte differentiation cell signaling and membrane traffic [27,28,30,39]. of 3T3-L1 cells. The levels of C ⁄ EBPb and C ⁄ EBPd Several studies have demonstrated that PX domains were altered in fad49 knockdown cells compared to play important roles in the function of proteins contain- control cells transfected with scrambled shRNA. As ing this domain [40]. In particular, p47phox, which con- C ⁄ EBPb and C ⁄ EBPd were dramatically expressed tains the PX domain and two SH3 domains, has been early in adipogenesis, we determined the expression intensely studied. In unstimulated neutrophils, p47phox levels of C ⁄ EBPb and C ⁄ EBPd in fad49 knockdown shows an intramolecular interaction between its PX cells at 0–6 h after the differentiation was induced. domain and the second SH3 domain, preventing Interestingly, fad49 RNAi treatment significantly membrane association. Stimulation of neutrophils reduced the levels of C ⁄ EBPd and partially reduced results in release of the inhibitory intramolecular inter- those of C ⁄ EBPb (Fig. 6E). These results imply that action, allowing its PX domain to associate with the fad49 is crucial in the immediate early stage of membrane by binding to lipids [25,35,36]. adipocyte differentiation. In order to examine the functional role of the FAD49 As fad49 appears to play an important role in the PX domain, we characterized its PX domain. Lipid early stages of adipocyte differentiation, we focused on binding studies and in vitro binding studies showed that MCE, which is synchronous transient cell growth that the PX domain of FAD49 has affinity for PI(3)P, can be observed after postconfluent 3T3-L1 cells have PI(4)P, PI(5)P and PI(3,5)P2, and interacts with the been treated with an optimal mixture of adipogenic third SH3 domain. Thus, binding of the FAD49 PX stimulants [10,37]. It has been reported that this phase domain to phosphoinositides might be regulated by the is required for adipocyte differentiation [38]. To eluci- interaction between the PX domain and the third SH3 date the role of fad49 in MCE, we determined the domain, as in p47phox, although whether the interaction effect of fad49 RNAi treatment on MCE (Fig. 6F). of the PX domain with the third SH3 domain is intra- The effect on MCE was quantified by determining the or intermolecular remains to be established. cell count using a hemocytometer at 1-day intervals To further characterize fad49, we examined the sub- throughout the differentiation program. Cell numbers cellular localization of FAD49. Subcellular localization in control cultures increased 3.1-fold between days 0 studies showed that it is found in the cytoplasm, and and 4, and remained constant between days 4 and 5. that the PX domain and SH3 domains are important In comparison, cell numbers in fad49 knockdown in FAD49 localization. Two deletion mutants, GFP– cultures only increased 2.2-fold by day 4. This result PX and GFP–SH3, localized to punctate structures, indicates that fad49 plays an important role in MCE while GFP–FL was diffusely cytoplasmic, suggesting during adipogenesis. that, in the context of the full-length protein, the PX domain and SH3 domains of FAD49 might not be able to contact lipids and or protein targets due to Discussion ⁄ interaction between them. We are now investigating To elucidate the molecular mechanisms of adipocyte the role of this interaction on FAD49 localization. differentiation, we previously used PCR subtraction to Although we tested whether the inducers for adipocyte isolate 102 genes that were induced 3 h after differenti- differentiation induce a change in the distribution of ation was induced [16,17]. About half of them were FAD49, we did not observe any changes in FAD49 unknown genes. As a result of attempts to isolate full- localization after induction. length cDNAs for the unknown genes, we previously In this study, we also demonstrated an important isolated several novel genes that are crucial for adipo- role of fad49 in adipocyte differentiation. The expres- cyte differentiation [20–22]. In this study, we cloned sion of fad49 was transiently upregulated 3 h after

FEBS Journal 275 (2008) 5576–5588 ª 2008 The Authors Journal compilation ª 2008 FEBS 5583 fad49 plays a crucial role in adipogenesis T. Hishida et al. exposure to an induction cocktail, and this upregula- RNA isolation, real-time quantitative RT-PCR and tion was restricted to the adipocyte differentiable state. northern blotting Moreover, RNAi experiments demonstrated that fad49 Total RNA was extracted using TRIzol (Invitrogen, Carls- is closely involved in adipocyte differentiation. Inter- bad, CA, USA) according to the manufacturer’s instructions. estingly, fad49 is involved in the induction of C EBPb ⁄ The total RNA was converted to single-stranded cDNA and C ⁄ EBPd genes. Although the cAMP response using a random primer and ReverTra Ace (Toyobo, Osaka, element-binding protein has been reported to regulate Japan). The cDNA was used as a template for quantitative the expression of C ⁄ EBPb and C ⁄ EBPd in the early PCR. An ABI PRISM 7000 sequence detection system stages of adipocyte differentiation [41], the overall (Applied Biosystems, Foster City, CA, USA) was used to mechanisms regulating C ⁄ EBPb and C ⁄ EBPd expres- perform the quantitative PCR. The pre-designed primers and sion remain to be elucidated. In this regard, further probe sets for fad49, aP2, PPARc,C⁄ EBPa,C⁄ EBPb, studies of the molecular function of fad49 should pro- C ⁄ EBPd, SREBP-1 and 18S rRNA were obtained from vide new insights into the regulation of C ⁄ EBPb and Applied Biosystems. The reaction mixture was prepared C ⁄ EBPd expression early in adipocyte differentiation. using a TaqMan Universal PCR Master Mix (Applied Bio- In addition, RNAi-mediated knockdown of fad49 systems) according to the manufacturer’s instructions. The resulted in significant inhibition of cell growth after mixture was incubated at 50 C for 2 min and at 95 C for differentiation was induced, suggesting that fad49 is 10 min, and then PCR was performed at 95 C for 15 s and crucial in MCE. As MCE is synchronous transient cell at 60 C for 1 min for 40 cycles. Relative standard curves growth and is required for adipocyte differentiation, were generated in each experiment to calculate the input the effect of FAD49 on MCE may be critical for amounts for the unknown samples. Northern blotting was adipogenesis. performed as described previously [22]. Although little is known about the molecular mecha- nism for regulation of MCE, C ⁄ EBPb and mitogen- Cloning of the full-length cDNA of mouse fad49 activated protein kinase are likely to play important roles in MCE [12,38]. In addition, we found that To isolate the full-length cDNA of mouse fad49,5¢ and 3¢ C ⁄ EBPd is also required for MCE (unpublished data). RACE and RT-PCR were performed. 5¢ and 3¢ RACE were As fad49 RNAi treatment results in reduction of performed using a Marathon cDNA amplification kit (BD Biosciences Clontech) according to the manufacturer’s C ⁄ EBPb and C ⁄ EBPd expression, we speculate that instructions. Total RNA was prepared from 3T3-L1 cells fad49 is involved in MCE, partly through its contribu- 3 h after induction. mRNA was isolated from total RNA tion to the induction of C ⁄ EBPb and C ⁄ EBPd genes. using Oligotex-dT30 (Daiichi Pure Chemicals, Tokyo, Further analysis of fad49 will give new insight into the Japan) according to the manufacturer’s instructions. First- signaling pathway in the immediate early stage of strand cDNA was amplified using the oligo(dT) primer and adipocyte differentiation. avian myeloblastosis virus reverse transcriptase (Clontech). The second strand was synthesized using a second-strand Experimental procedures enzyme mixture containing RNase H, Escherichia coli DNA polymerase 1 and E. coli DNA ligase. PCR for 5¢ RACE Materials was performed using the AP1 primer (5¢-CCATCCTAAT ACGACTCACTATAGGGC-3¢) and one of three fad49- Dexamethasone, insulin and 4¢,6-diamidino-2-phenylindole specific primers: 5¢-GCCTGTGAGCGCCTAGCATGG (DAPI) were purchased from Sigma (St Louis, MO, USA). TTC-3¢,5¢-GGATTCCTGCAGAGCGTGGGTGTGG-3¢ IBMX was purchased from Nacalai Tesque (Kyoto, Japan). and 5¢-CCTGGGGTGGGATGGGGGGCTTCGGCAG-3¢ Dulbecco’s modified Eagle’s medium (DMEM) was pur- for 5¢-R-1, 5¢-R-2 and 5¢-R-3, respectively. PCR for 3¢ chased from NISSUI Pharmaceutical (Tokyo, Japan). PIP RACE was performed using the AP1 primer and an arrays were purchased from Echelon Biosciences (Salt fad49-specific primer (5¢-GGCCATCTCGGCCCCTTCGC Lake City, UT, USA). GTGGC-3¢). RT-PCR was performed using total RNA prepared from 3T3-L1 cells 3 h after induction. PCRs were performed using KOD plus (Toyobo) with fad49-specific Antibodies forward primer 5¢-CATGAGATGACCCAGCT-3¢ and The following antibodies were obtained commercially: reverse primer 5¢-GCTTCTGGTAACATGG-3¢ for P-1, and monoclonal anti-FLAG M2 IgG1 (Sigma) and anti-c-Myc fad49-specific forward primer 5¢-TGTTGGACAAGTTCCC IgG1 (BD Biosciences Clontech, Palo Alto, CA, USA), CAT-3¢ and reverse primer 5¢-GCGGCTCCATCTTCTGTC and polyclonal anti-GST IgG (Amersham Biosciences, TTTCCC-3¢ for P-2. The fragments obtained from RT-PCR, Piscataway, NJ, USA). 5¢ RACE and 3¢ RACE were subcloned into pBluescript

5584 FEBS Journal 275 (2008) 5576–5588 ª 2008 The Authors Journal compilation ª 2008 FEBS T. Hishida et al. fad49 plays a crucial role in adipogenesis

KS+ (Stratagene, Agilent Technologies, Santa Clara, CA, (Amersham Biosciences) and FLAG M2 beads (Sigma), USA) and analyzed by DNA sequencing as described below. according to the manufacturer’s instructions.

Cloning of the full-length cDNA of human fad49 Phospholipid binding

First, we predicted the full-length ORF of human fad49 Lipid binding studies were performed using PIP arrays using the human genome sequence and the full-length ORF (Echelon Biosciences) according to the manufacturer’s of mouse fad49. Next, based on the predicted sequence for instructions. human fad49, RT-PCR was performed as described above using total RNA prepared from HeLa cells. The 5¢ region In vitro binding assay of the human cDNA of fad49 was amplified using KOD - plus (Toyobo) with a human fad49-specific forward primer For in vitro binding experiments, glutathione–Sepharose- (5¢-GCGGCCATGCCGCCGCGGCGCAGCATCG-3¢) and bound proteins, GST or GST–PX, were prepared, and then reverse primer (5¢-TTTCTCGATCACCTCGAC-3¢). In the incubated with each of the purified FLAG fusion proteins same way, the 3¢ region of the human cDNA of fad49 was for individual SH3 domains of FAD49 in GST-binding buf- amplified with a human fad49-specific forward primer fer [20 mm Tris ⁄ HCl pH 8.0, 180 mm KCl, 0.2 mm EDTA, (5¢-ACATGACCATTCCTCGAG-3¢) and reverse primer 0.5% w ⁄ v Nonidet P-40 (Nacalai Tesque)] overnight at (5¢-TCTAGGCAGAAAGGGAGT-3¢). As both the ampli- 4 C. Samples were washed three times in GST wash buffer fied 5¢ and 3¢ regions harbor the EcoRI site, the fragments (20 mm Tris ⁄ HCl pH 8.0, 180 mm KCl, 0.2 mm EDTA, obtained from RT-PCR were digested with EcoRI, purified, 1% Nonidet P-40), eluted from the resin by boiling in an subcloned into the HincII ⁄ EcoRI site of pBluescript KS+ SDS sample buffer (62.5 mm Tris ⁄ HCl pH 6.8, 10% v ⁄ v and analyzed by DNA sequencing as described below. glycerol, 2% w ⁄ v SDS, 5% v ⁄ v b-mercaptoethanol, 0.01% w ⁄ v bromophenol blue), subjected to SDS–PAGE and transferred to poly(vinylidene difluoride) membranes. Fol- Plasmid construction lowing transfer, membranes were blocked with 4% w ⁄ v The DNA fragments encoding fad49 were amplified by skim milk in Tris-buffered saline with 0.1% Tween-20 RT-PCR from total RNA extracted from 3T3-L1 cells 3 h (TBS-T), and probed using primary antibodies, secondary after differentiation induction. The resulting fragments were antibodies, conjugated horseradish peroxidase and an cloned, using appropriate restriction sites, in-frame into enhanced chemiluminescence detection kit (GE Healthcare, several expression vectors as described below. Fragments Chalfont St Giles, UK) to detect specific proteins. encoding the PX domain and individual SH3 domains of FAD49 were subcloned into the pGEX4T-1 vector DNA sequencing and database analyses (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and pFLAG-MAC vectors (Sigma), respectively. For N-termi- The sequence was determined using a DSQ 1000 automated nally Myc-tagged FAD49 (Myc–FAD49), the DNA frag- sequencer (Shimadzu Corp., Kyoto, Japan) and an ABI ment encoding full-length FAD49 was subcloned into PRISM 3100 genetic analyzer (Applied Biosystems). The the pCMV-Myc vector (BD Biosciences Clontech). For search for a human ortholog in human genome databases was C-terminally Myc-tagged FAD49 (FAD49–Myc), the DNA performed using blast programs accessed via the National fragment that encodes full-length FAD49 followed by the Center for Biotechnology Information (NCBI) homepage. Myc tag sequence and stop codon was subcloned into pEGFP-N3 (BD Biosciences Clontech). For three constructs, full-length FAD49 (GFP–FL), the FAD49 PX domain Cell culture, differentiation and cell counts (GFP–PX) and FAD49 lacking its PX domain (GFP–SH3), Mouse 3T3-L1 (ATCC CL173) preadipocyte cells were individual DNA fragments encoding full-length FAD49 maintained in DMEM containing 10% v ⁄ v calf serum. For (amino acids 1–910), the FAD49 PX domain (amino acids the differentiation experiment, the medium was replaced ) 1–130) and FAD49 lacking the PX domain (amino acids with DMEM containing 10% v ⁄ v FBS, 10 lgÆmL 1 insulin, 126–910) were subcloned into pEGFP-C1 vectors (BD 0.5 mm IBMX and 1 lm dexamethasone 2 days postconflu- Biosciences Clontech). All constructs described above were ence. After 2 days, the medium was changed to DMEM analyzed by DNA sequencing as described below. containing 5 lgÆmL)1 insulin and 10% FBS, and then the cells were re-fed every 2 days. Adipogenesis was determined Preparation of fusion proteins by staining the cells with Oil Red O (Amresco, Salon, OH, USA). Mouse NIH-3T3 fibroblastic cells (clone 5611, JCRB GST and FLAG fusion proteins were expressed in E. coli 0615) were maintained in DMEM containing 10% calf BL21 at 30 C and purified using glutathione–Sepharose 4B

FEBS Journal 275 (2008) 5576–5588 ª 2008 The Authors Journal compilation ª 2008 FEBS 5585 fad49 plays a crucial role in adipogenesis T. Hishida et al. serum. For determination of cell counts, cells were gently RNAi experiment rinsed with NaCl ⁄ Pi, and trypsinized with trypsin (0.25%) ⁄ EDTA (0.1%) solution in NaCl ⁄ Pi for 5 min at The two target regions in the ORF of mouse fad49 37 C, 5% CO2. Cell counts were determined for the trypsi- (GenBank accession number AB430861); region 1 at 2515– nized cells using a hemocytometer. 2535 bp (for which nucleotide A in the translation initiation codon is the first nucleotide) and region 2 at 778–798 bp were selected using the Qiagen siRNA online design tool Fractionation of fat cells (http://sirna.qiagen.com/) for RNAi of fad49. A 19-nucleo- The fat cells were prepared as described previously [42]. In tide shRNA coding fragment with a 5¢-TTCAAGAGA-3¢ brief, epidermal fat pads were isolated from male C57Bl ⁄ 6J loop was subcloned into the ApaI ⁄ EcoRI site of pSilencer mice aged 6 weeks, washed with sterile NaCl ⁄ Pi, minced, 1.0-U6 (Ambion, Inc., Austin, TX, USA). As a negative con- and washed using Krebs–Ringer bicarbonate buffer, trol, the scrambled fragment 5¢-GTAAGATGAGGC pH 7.4. Then, the minced tissue was digested with AATGGAG-3¢, which does not have similarity with any 1.5 mgÆmL)1 of collagenase type II (Sigma-Aldrich) in mRNA listed in GenBank, was generated. Transfection of Krebs–Ringer bicarbonate buffer, containing 4% w ⁄ v BSA, shRNA expression plasmids into 3T3-L1 cells was performed at 37 C for 1 h on a shaking platform. The undigested tis- using Nucleofector (Amaxa) with cell line Nucleofector kit V sue was removed with 250 lm nylon mesh and the digested (Amaxa). 3T3-L1 cells were harvested and resuspended in fraction was centrifuged at 500 g for 5 min. The adipocytes Nucleofector solution at 1.5 · 106 cells per 100 lL. After were obtained from the uppermost layer, washed with buf- addition of shRNA expression plasmids (9 lg), the cells were fer, and centrifuged at 500 g for 5 min at 4 C to remove transfected using the T-20 Nucleofector program. Then they other cells. Stromal–vascular cells were resuspended in an were plated on 12- or 24-well plates. The 3T3-L1 cells were erythrocyte lysis buffer (150 mm NH4Cl, 25 mm NH4HCO3 subjected to differentiation experiments 3 days after the and 1 mm EDTA pH 7.7), filtered through 28 mm nylon transfection. Differentiation experiments were performed mesh and then precipitated at 500 g for 5 min. using the same medium as described above. Cell counts were performed using 24-well plates. Subcellular localization of FAD49 Accession numbers For immunostaining, 3T3-L1 cells transfected with pCMV- Myc-FAD49 using the Nucleofector system (Amaxa, The sequences of the cloned mouse and human fad49 Cologne, Germany) were seeded onto cell disks (Sumitomo cDNAs have been deposited in the GenBank database with Bakelite Co. Ltd, Tokyo, Japan) with a collagen coating. accession numbers AB430861 and AB430862, respectively. One day after transfection, the cells were washed with NaCl ⁄ P and fixed in 4% w ⁄ v paraformaldehyde for 15 min i Acknowledgements at room temperature. After washing the cells three times with NaCl ⁄ Pi, they were permeabilized with 0.2% w ⁄ v gela- We thank Asako Shimada and Aya Fujii for technical tin in NaCl ⁄ Pi with 0.2% Triton X-100 for 30 min, washed assistance (Nagoya City University, Aichi, Japan). This three times with NaCl ⁄ Pi, and then blocked using blocking study was supported in part by a Grant-in-Aid for Sci- solution (1% BSA in NaCl ⁄ Pi with 0.1% Tween-20) for 1 h entific Research on Priority Areas from the Ministry at room temperature. After blocking, each disk was of Education, Culture, Sports, Science and Technology incubated with the mouse monoclonal c-Myc antibody (MEXT), Japan, and a Grant-in-Aid for Scientific overnight at 4 C. After washing three times with 0.1% Research (B) from the Japan Society for the Promo- Tween-20 in NaCl P , each disk was incubated with anti- ⁄ i tion of Science (JSPS). mouse fluorescein isothiocyanate (FITC) (Sigma) for 1 h at room temperature. After three more washes with NaCl ⁄ Pi, FITC signals were detected by fluorescence microscopy. References For subcellular localization studies using the three 1 Kopelman PG (2000) Obesity as a medical problem. 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