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 3T3-L1 cell; adipocyte differentiation; CCAAT ⁄ enhancer-binding protein; obesity; peroxisome proliferator-activated receptor c Correspondence M Imagawa, Department of Molecular Biology, Graduate School of Pharmaceutical Sciences, Nagoya City University, 3-1 Tanabe-dori, Mizuho-ku, Nagoya, Aichi 4678603, Japan Fax: +81 52 836 3455 Tel: +81 52 836 3455 E-mail: imagawa@phar.nagoya-cu.ac.jp (Received 24 July 2008, revised September 2008, accepted 11 September 2008) doi:10.1111/j.1742-4658.2008.06682.x Adipogenesis is accomplished via a complex series of steps, and the events at the earliest stage remain to be elucidated To clarify the molecular mechanisms of adipocyte differentiation, we previously isolated 102 genes expressed early in mouse 3T3-L1 preadipocyte cells using a PCR subtraction system About half of the genes isolated appeared to be unknown After isolating full-length cDNAs of the unknown genes, one of them, named factor for adipocyte differentiation 49 (fad49), appeared to be a novel gene, as the sequence of this clone showed no identity to known genes FAD49 contains a phox homology (PX) domain and four Src homology (SH3) domains, suggesting that it may be a novel scaffold protein We found that the PX domain of FAD49 not only has affinity for phosphoinositides, but also binds to its third SH3 domain Expression of fad49 was transiently elevated h after differentiation was induced, and diminished 24 h after induction Induction of the fad49 gene was observed in adipocyte differentiable 3T3-L1 cells, but not in non-adipogenic NIH-3T3 cells RNAi-mediated knockdown of fad49 significantly impaired adipocyte differentiation Moreover, the knockdown of fad49 by RNAi inhibited mitotic 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 is a key risk factor in several obesity-related diseases, such as type diabetes, hypertension, hyperlipidemia and cardiac infarction [1–3] Obesity may occur through excessive accumulation of white adipose tissue (WAT), composed mainly of adipocytes, which play an important role in the storage of energy and secretion of a variety of hormones and cytokines that regulate metabolic activities in the body [1] Such pathological accumulation of WAT in the body results in dysregulated production of hormones and cytokines by adipose tissue, such as tumor necrosis factor a, adiponectin and resistin, which leads to various diseases, such as type diabetes, stroke and cardiac infarction [3–6] Obesity, the pathological development of adipose tissue, results from an increase in the cell size of individual adipocytes and an increase in total adipocyte cell numbers through differentiation of preadipocytes 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-1methylxanthine; 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 the context of the prevention and treatment of obesityrelated diseases, it is important to elucidate the mechanisms of adipocyte differentiation, as well as adipocyte enlargement Much knowledge of adipogenesis has derived from studies using mouse 3T3-L1 cells as model cells of adipocyte differentiation 3T3-L1 cells are grown to confluence and growth arrested Growth-arrested 3T3-L1 cells differentiate into mature adipocytes after the addition of insulin, 3-isobutyl-1-methylxanthine (IBMX), dexamethasone and fetal bovine serum (FBS) [7–9] After treatment with the induction cocktail, they undergo approximately two cycles of synchronized cell division, a process known as mitotic clonal expansion (MCE) [10,11] MCE is a requisite step for adipocyte differentiation, followed by terminal differentiation, in which peroxisome proliferator-activated receptor c (PPARc) and CCAAT ⁄ enhancer-binding protein a (C ⁄ EBPa) play important roles as master regulators [12] In terminal differentiation, PPARc and C ⁄ EBPa transactivate each other and upregulate the expression of many adipogenic genes, causing the cells to acquire an adipogenic phenotype Expression of these transcriptional factors starts to increase in the middle stage of adipocyte differentiation, partly through transactivation of C ⁄ EBPb and C ⁄ EBPd, the expression of which is immediately upregulated after hormonal induction [13,14] Several other factors that are involved in regulating the expression and transcriptional activity of PPARc and C ⁄ EBPa have been identified by other studies [15] Therefore, events in the middle and late stage of adipocyte differentiation have been studied relatively thoroughly In contrast, the overall mechanisms of events in the early stage of the differentiation programme, including MCE and induction of the C ⁄ EBPb and C ⁄ EBPd genes, remain to be elucidated In order to clarify the molecular mechanisms in the early phase of adipocyte differentiation, we previously isolated 102 genes for which expression early in the differentiation process was induced using a PCR subtraction system [16,17] These genes included transcription factors and signaling molecules [17–19] About half of them were unknown genes, whose functions remain unclear As the fragments obtained by PCR subtraction are small, we needed to isolate the fulllength cDNAs of the unknown genes We have previously revealed that several of them are novel genes, such as factor for adipocyte differentiation (fad) 24, fad104 and fad158, which play crucial roles in adipogenesis [20–23] Here, we report the isolation of another novel gene, fad49, and the close involvement of fad49 in adipocyte fad49 plays a crucial role in adipogenesis differentiation FAD49 contains a phox homology (PX) domain, which has affinity for phosphoinositides, and four Src homology (SH3) domains, which bind to polyproline PXXP ligands, suggesting that FAD49 is a novel scaffold protein RNAi experiments demonstrated that fad49 is crucial in adipogenesis, and that it plays important roles in events early in the differentiation process, including MCE and the induction of C ⁄ EBPb and C ⁄ EBPd genes Taken together, these results imply that fad49, encoding a novel scaffolding protein, plays an important role in the immediate early stage of adipocyte differentiation Results Cloning of full-length mouse fad49 cDNA Using the PCR subtraction method, we originally isolated fad49 as one of many unknown genes the expression of which was elevated at h after induction of adipocyte differentiation The PCR-subtraction method used in the previous study gave cDNA fragments only 300–900 bp long because the amplified fragments were digested using RsaI for non-bias cloning [16] The length of fad49 was 870 bp Therefore, we attempted to isolate a full-length cDNA of fad49 using 5¢ and 3¢ RACE methods, and expressed sequence tag (EST)-walk method, which is a combination method of predicting exons of interest in genes, utilizing the mouse genome and EST followed by RT-PCR (Fig 1A) 5¢ and 3¢ RACE were performed using cDNA prepared from 3T3-L1 cells h after induction As a result, a 1109 bp cDNA fragment containing an initiation codon at bp was isolated by 5¢ RACE The sequence (GCCATGC) including initiation codon is close to the consensus sequence for translation initiation (A/GCCATGA/G) A 1809 bp cDNA fragment containing a stop codon was isolated by RT-PCR A 1007 bp cDNA containing a poly(A) tail was obtained by 3¢ RACE By combining these cDNA fragments, fad49 was found to consist of 7258 bp with an ORF of 910 amino acids (GenBank accession number AB430861) Because blast database searches identified no significant matches against proteins of known function, fad49 appears to be a novel gene We next analyzed the genomic distribution of fad49 using the mouse genome database (http://www ncbi.nlm.nih.gov/genome/seq/BlastGen/BlastGen.cgi? taxid=10090), which was made public by the Mouse Genome Sequencing Consortium The result indicated that fad49 exists at locus 11A4 of mouse chromosome 11 and consists of 13 exons divided by 12 introns (Fig 1B) Sequencing of the exon ⁄ intron junctions in FEBS Journal 275 (2008) 5576–5588 ª 2008 The Authors Journal compilation ª 2008 FEBS 5577 fad49 plays a crucial role in adipogenesis T Hishida et al AB430862) A blast search of the human genome database revealed a human homolog of fad49 on chromosome 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 Fig Cloning and genomic structures of mouse fad49 (A) The fulllength cDNA for mouse fad49 was isolated by RT-PCR, 5¢ and 3¢ RACE ‘S’ is the fragment obtained by the original PCR-subtraction method ‘RT’, ‘5¢-R’ and ‘3¢-R’ are fragments obtained by RT-PCR, 5¢ RACE and 3¢ RACE, respectively The combined sequence is shown as fad49 The initiation and stop codon are also indicated The deduced amino acid sequence revealed a 910 amino acid protein for mouse FAD49 (B) Schematic representation of the mouse fad49 gene structure The thirteen exons of the fad49 gene on chromosome 11 are represented by vertical bars in the top part of the figure The size of each exon is indicated in the bottom panel the database revealed that the GT ⁄ AG rule was maintained in all cases (data not shown) The deduced protein primary structure of mouse and human fad49 The ORF of fad49 encodes a putative protein of 910 amino acids that contains a PX domain (solid underlining) and four SH3 domains (dotted underlining) (Fig 2A) Moreover, the encoded protein also contained ten polyprolines (boxed), which could be possible ligands for the SH3 domain Thus, fad49 encodes a protein containing many protein-binding domains, suggesting that this protein may be a novel scaffold protein We next tried to isolate the full-length ORF of human fad49 We first used the human genome database to predict the ORF region of human fad49 by splicing out the introns and combining the exons of the ORF of human fad49 To isolate human fad49 including the entire ORF, we next constructed primer sets as described in Experimental procedures, and performed RT-PCR using template cDNA prepared from mRNA extracted from HeLa cells From sequence analyses of the resultant fragments, the full-length cDNA of human fad49 comprised a 2733 bp ORF encoding 911 amino acids (GenBank accession number 5578 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 lipidbinding specifities, although specificity for phosphatidylinositol 3-phosphate [PI(3)P] appears to be the most common [28,31–34] For example, the PX domain of p40phox interacts with PI(3)P, the PX domains of p47phox and Fish, which contains five SH3 domains and a PX domain, binds to PI(3,4)P2 [24,27], and the PX domain of C2 containing Ptdlns kinase (CPK) class of PI 3-kinase selectively binds to PI(4,5)P2 [32] Moreover, the conserved polyproline motif (PXXP) in many PX domains suggests that it may act as a target for SH3 domains In fact, it has been reported that the PX domain of p47phox binds intramolecularly to the SH3 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 p47phox releases the binding, resulting in the active state, i.e open conformation [25,35,36] As FAD49 contains a PX domain as described above, we determined whether the FAD49 PX domain could bind to phosphoinositides To test whether the FAD49 PX domain has affinity for phosphoinositides, we bacterially expressed the FAD49 PX domain fused to glutathione S-transferase (GST–PX) and used GST protein (GST) as a negative control Lipid binding was then measured using overlay blotting as described in Experimental procedures As shown in Fig 3A, GST–PX bound most strongly to PI(3,5)P2, and to a lesser extent to PI(3)P, PI(4)P and PI(5)P, but no binding to any of the lipid species was observed for GST as a control The PXXP motif was found in the FAD49 PX domain, as in many PX domains, suggesting that the PX domain could bind to an SH3 domain of FAD49 To test whether the PX domain could interact with an SH3 domain in FAD49, we performed in vitro binding assays using various bacterially expressed proteins: GST–PX and FLAG fusion proteins of individual SH3 domains of FAD49 We found that the PX 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 Fig 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 potential SH3 binding sites are boxed (B) Schematic 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 domain (Fig 3B) Subcellular localization of FAD49 To further characterize fad49, the subcellular localization of FAD49 was determined by transient transfection of an N-terminally Myc-tagged full-length FAD49 (Myc–FAD49) expression plasmid into 3T3L1 cells Cells were immunostained using monoclonal anti-Myc IgG1 As shown in Fig 4A, Myc–FAD49 was found predominantly in the cytoplasm The same result was obtained using a C-terminally Myc-tagged FAD49 (FAD49–Myc) expression plasmid To examine the role of the FAD49 PX domain or SH3 domains on the subcellular localization of FAD49, we next determined the subcellular localization of GFP proteins fused to full-length FAD49 (GFP–FL), the PX domain (GFP–PX) and FAD49 lacking its PX domain (GFP–SH3) (Fig 4B,C) GFP–FL was mostly detected in the cytoplasm, consistent with Fig 4A The truncated mutant GFP–PX, which only contains 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 not contain the PX domain, was localized in punctate structures that seem to differ from those in which GFP–PX was found These results suggest that the PX and SH3 domains of FAD49 play a role in the localization of FAD49 Expression profiles of fad49 in differentiating and non-differentiating cells and tissue distribution To investigate the role of fad49 during adipocyte differentiation, we first determined the mRNA expression levels of fad49 by Northern blotting To monitor changes in the levels of fad49 during adipocyte differentiation, 3T3-L1 cells were stimulated with an induction cocktail, and then total RNA was prepared at various time points The expression of fad49 increased quickly after differentiation was induced, reaching a FEBS Journal 275 (2008) 5576–5588 ª 2008 The Authors Journal compilation ª 2008 FEBS 5579 fad49 plays a crucial role in adipogenesis T Hishida et al Fig Characterization of the PX domain of FAD49 (A) Phosphoinositide binding specificity of the PX domain of FAD49 Bacterially expressed GST or GST–PX were incubated with PIP arrays prespotted 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 antiGST serum (B) Interaction of the FAD49 PX domain with its third SH3 domain FLAG–SH3 proteins (10 pmol) were tested in co-precipitation 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 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 3T3L1 cells differentiate to mature adipocytes when stimulated with adipogenic inducers days after reaching a state of confluence, whereas proliferating 3T3-L1 cells not differentiate into adipocytes 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 in a postconfluent (growth-arrested) or proliferating state Total RNA was prepared from these cells before or h after differentiation was induced and subjected to quantitative PCR, which showed that marked induction only occurred in growth-arrested 3T3-L1 cells, suggesting that the elevation in expression of fad49 is restricted to the adipocyte differentiable state (Fig 5B) This result strongly suggests that fad49 plays a functional role in adipogenesis 5580 Fig Subcellular localization of FAD49 (A) 3T3-L1 cells transiently transfected with the Myc–FAD49 or FAD49–Myc expression plasmid were fixed and blocked for immunofluorescence staining with anti-Myc serum (B) Schematic representation of GFP fusion proteins for each FAD49 deletion mutant used in this study (C) Subcellular localization of EGFP–FAD49 fusion proteins 3T3-L1 cells were transiently transfected with an EGFP–FAD49-expressing plasmid or empty vector One day after transfection, the cells were fixed and stained with DAPI EGFP signals were detected using a fluorescence microscope To investigate the tissue distribution of fad49, we determined the expression levels of fad49 by quantitative PCR in various tissues isolated from adult male mice, including WAT and brown adipose tissue (BAT) (Fig 5C) WAT samples were separated into two fractions: the stromal–vascular fraction enriched with preadipocytes and the mature adipocyte fraction Tissue distribution studies revealed that high expression of 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 Fig 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 loading 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 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 WAT, and moderate expression was detected in heart, skeletal muscle and the mature adipocyte fraction of WAT Thus, the expression of fad49 in the stromal– vascular fraction was higher than that in mature adipocytes, suggesting that expression of fad49 is predominant in preadipocytes As expression of fad49 was observed in skeletal muscle, we analyzed the expression levels of fad49 during the myogenesis of mouse C2C12 cells Expression of fad49 was weak, and the level was unchanged during myogenesis of C2C12 cells (data not shown) Effect of fad49 knockdown on differentiation of 3T3-L1 cells into adipocytes As described above, the expression of mouse fad49 is rapidly upregulated early in the differentiation of 3T3L1 cells into adipocytes, and seems to play a role in adipogenesis To characterize the function of this gene during adipogenesis, we performed RNAi to silence the expression of fad49 during adipogenesis For the RNAi experiments, two short hairpin RNA (shRNA) expression plasmids named shfad49-1 and shfad49-2 were constructed to target regions and 2, respectively (as defined in Experimental procedures), in the ORF of the fad49 gene Each of the shRNA expression plasmids was transfected into 3T3-L1 cells Three hours after induction for adipocyte differentiation, total RNA was isolated, and the expression levels of fad49 were determined by quantitative PCR We found that shfad49-2 had a strong silencing effect on fad49 expression, while the effect of shfad49-1 was milder (Fig 6A) Therefore, we used shfad49-2 to perform the RNAi experiments Next, we determined the expression levels of fad49 by quantitative PCR in the cells transfected with shfad49-2 at each time point after induction of differentiation We confirmed that RNAi treatment reduced fad49 mRNA levels (Fig 6B) After days, the cells were fixed and stained with Oil red O and the amounts of triacylglycerol were determined The number of FEBS Journal 275 (2008) 5576–5588 ª 2008 The Authors Journal compilation ª 2008 FEBS 5581 fad49 plays a crucial role in adipogenesis T Hishida et al Fig 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 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 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) 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 5582 FEBS Journal 275 (2008) 5576–5588 ª 2008 The Authors Journal compilation ª 2008 FEBS T Hishida et al Oil red O-stained cells and the accumulation of triacylglycerol were significantly reduced in the RNAi-treated cells (Fig 6C) An inhibitory effect of knockdown of fad49 on adipocyte differentiation was also observed in RNAi experiments performed with shfad49-1 (data not shown) Next, we determined the expression levels of adipogenic marker genes by quantitative PCR (Fig 6D) The levels of PPARc, C ⁄ EBPa, adipocyte lipid-binding protein (aP2) and sterol regulatory element-binding protein-1 (SREBP-1) decreased in fad49 knockdown cells, indicating that RNAi-mediated knockdown of fad49 inhibits adipocyte differentiation of 3T3-L1 cells The levels of C ⁄ EBPb and C ⁄ EBPd were altered in fad49 knockdown cells compared to control cells transfected with scrambled shRNA As C ⁄ EBPb and C ⁄ EBPd were dramatically expressed early in adipogenesis, we determined the expression levels of C ⁄ EBPb and C ⁄ EBPd in fad49 knockdown cells at 0–6 h after the differentiation was induced Interestingly, fad49 RNAi treatment significantly reduced the levels of C ⁄ EBPd and partially reduced those of C ⁄ EBPb (Fig 6E) These results imply that fad49 is crucial in the immediate early stage of adipocyte differentiation As fad49 appears to play an important role in the early stages of adipocyte differentiation, we focused on MCE, which is synchronous transient cell growth that can be observed after postconfluent 3T3-L1 cells have been treated with an optimal mixture of adipogenic stimulants [10,37] It has been reported that this phase is required for adipocyte differentiation [38] To elucidate the role of fad49 in MCE, we determined the effect of fad49 RNAi treatment on MCE (Fig 6F) The effect on MCE was quantified by determining the cell count using a hemocytometer at 1-day intervals throughout the differentiation program Cell numbers in control cultures increased 3.1-fold between days and 4, and remained constant between days and In comparison, cell numbers in fad49 knockdown cultures only increased 2.2-fold by day This result indicates that fad49 plays an important role in MCE during adipogenesis Discussion To elucidate the molecular mechanisms of adipocyte differentiation, we previously used PCR subtraction to isolate 102 genes that were induced h after differentiation was induced [16,17] About half of them were unknown genes As a result of attempts to isolate fulllength cDNAs for the unknown genes, we previously isolated several novel genes that are crucial for adipocyte differentiation [20–22] In this study, we cloned fad49 plays a crucial role in adipogenesis the full-length cDNA of a novel gene, named fad49, which showed no identity to known genes Isolation of the full-length ORFs of mouse and human fad49 revealed that the protein encoded by fad49 has a PX domain, four SH3 domains and several PXXP motifs, suggesting that FAD49 may be a novel scaffold protein The PX domain, described as a phosphpoinositidebinding module, was first identified in p40phox and p47phox, two cytosolic subunits of NADPH oxidase, and has been found since in a variety of proteins involved in cell signaling and membrane traffic [27,28,30,39] Several studies have demonstrated that PX domains play important roles in the function of proteins containing this domain [40] In particular, p47phox, which contains the PX domain and two SH3 domains, has been intensely studied In unstimulated neutrophils, p47phox shows an intramolecular interaction between its PX domain and the second SH3 domain, preventing membrane association Stimulation of neutrophils results in release of the inhibitory intramolecular interaction, allowing its PX domain to associate with the membrane by binding to lipids [25,35,36] In order to examine the functional role of the FAD49 PX domain, we characterized its PX domain Lipid binding studies and in vitro binding studies showed that the PX domain of FAD49 has affinity for PI(3)P, PI(4)P, PI(5)P and PI(3,5)P2, and interacts with the third SH3 domain Thus, binding of the FAD49 PX domain to phosphoinositides might be regulated by the interaction between the PX domain and the third SH3 domain, as in p47phox, although whether the interaction of the PX domain with the third SH3 domain is intraor intermolecular remains to be established To further characterize fad49, we examined the subcellular localization of FAD49 Subcellular localization studies showed that it is found in the cytoplasm, and that the PX domain and SH3 domains are important in FAD49 localization Two deletion mutants, GFP– PX and GFP–SH3, localized to punctate structures, while GFP–FL was diffusely cytoplasmic, suggesting 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 interaction between them We are now investigating the role of this interaction on FAD49 localization Although we tested whether the inducers for adipocyte differentiation induce a change in the distribution of FAD49, we did not observe any changes in FAD49 localization after induction In this study, we also demonstrated an important role of fad49 in adipocyte differentiation The expression of fad49 was transiently upregulated 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 upregulation was restricted to the adipocyte differentiable state Moreover, RNAi experiments demonstrated that fad49 is closely involved in adipocyte differentiation Interestingly, fad49 is involved in the induction of C ⁄ EBPb and C ⁄ EBPd genes Although the cAMP response element-binding protein has been reported to regulate the expression of C ⁄ EBPb and C ⁄ EBPd in the early stages of adipocyte differentiation [41], the overall mechanisms regulating C ⁄ EBPb and C ⁄ EBPd expression remain to be elucidated In this regard, further studies of the molecular function of fad49 should provide new insights into the regulation of C ⁄ EBPb and C ⁄ EBPd expression early in adipocyte differentiation In addition, RNAi-mediated knockdown of fad49 resulted in significant inhibition of cell growth after differentiation was induced, suggesting that fad49 is crucial in MCE As MCE is synchronous transient cell growth and is required for adipocyte differentiation, the effect of FAD49 on MCE may be critical for adipogenesis Although little is known about the molecular mechanism for regulation of MCE, C ⁄ EBPb and mitogenactivated protein kinase are likely to play important roles in MCE [12,38] In addition, we found that C ⁄ EBPd is also required for MCE (unpublished data) As fad49 RNAi treatment results in reduction of C ⁄ EBPb and C ⁄ EBPd expression, we speculate that fad49 is involved in MCE, partly through its contribution to the induction of C ⁄ EBPb and C ⁄ EBPd genes Further analysis of fad49 will give new insight into the signaling pathway in the immediate early stage of adipocyte differentiation Experimental procedures Materials Dexamethasone, insulin and 4¢,6-diamidino-2-phenylindole (DAPI) were purchased from Sigma (St Louis, MO, USA) IBMX was purchased from Nacalai Tesque (Kyoto, Japan) Dulbecco’s modified Eagle’s medium (DMEM) was purchased from NISSUI Pharmaceutical (Tokyo, Japan) PIP arraysÔ were purchased from Echelon Biosciences (Salt Lake City, UT, USA) Antibodies The following antibodies were obtained commercially: monoclonal anti-FLAG M2 IgG1 (Sigma) and anti-c-Myc IgG1 (BD Biosciences Clontech, Palo Alto, CA, USA), and polyclonal anti-GST IgG (Amersham Biosciences, Piscataway, NJ, USA) 5584 RNA isolation, real-time quantitative RT-PCR and northern blotting Total RNA was extracted using TRIzol (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions The total RNA was converted to single-stranded cDNA using a random primer and ReverTra Ace (Toyobo, Osaka, Japan) The cDNA was used as a template for quantitative PCR An ABI PRISM 7000 sequence detection system (Applied Biosystems, Foster City, CA, USA) was used to perform the quantitative PCR The pre-designed primers and probe sets for fad49, aP2, PPARc, C ⁄ EBPa, C ⁄ EBPb, C ⁄ EBPd, SREBP-1 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 and at 95 °C for 10 min, and then PCR was performed at 95 °C for 15 s and at 60 °C for for 40 cycles Relative standard curves were generated in each experiment to calculate the input amounts for the unknown samples Northern blotting was performed as described previously [22] Cloning of the full-length cDNA of mouse fad49 To isolate the full-length cDNA of mouse fad49, 5¢ and 3¢ RACE and RT-PCR were performed 5¢ and 3¢ RACE were performed using a Marathon cDNA amplification kit (BD Biosciences Clontech) according to the manufacturer’s instructions Total RNA was prepared from 3T3-L1 cells h after induction mRNA was isolated from total RNA using Oligotex-dT30 (Daiichi Pure Chemicals, Tokyo, Japan) according to the manufacturer’s instructions Firststrand cDNA was amplified using the oligo(dT) primer and avian myeloblastosis virus reverse transcriptase (Clontech) The second strand was synthesized using a second-strand enzyme mixture containing RNase H, Escherichia coli DNA polymerase and E coli DNA ligase PCR for 5¢ RACE was performed using the AP1 primer (5¢-CCATCCTAAT ACGACTCACTATAGGGC-3¢) and one of three fad49specific primers: 5¢-GCCTGTGAGCGCCTAGCATGG TTC-3¢, 5¢-GGATTCCTGCAGAGCGTGGGTGTGG-3¢ and 5¢-CCTGGGGTGGGATGGGGGGCTTCGGCAG-3¢ for 5¢-R-1, 5¢-R-2 and 5¢-R-3, respectively PCR for 3¢ RACE was performed using the AP1 primer and an fad49-specific primer (5¢-GGCCATCTCGGCCCCTTCGC GTGGC-3¢) RT-PCR was performed using total RNA prepared from 3T3-L1 cells h after induction PCRs were performed using KOD plus (Toyobo) with fad49-specific forward primer 5¢-CATGAGATGACCCAGCT-3¢ and reverse primer 5¢-GCTTCTGGTAACATGG-3¢ for P-1, and fad49-specific forward primer 5¢-TGTTGGACAAGTTCCC CAT-3¢ and reverse primer 5¢-GCGGCTCCATCTTCTGTC TTTCCC-3¢ for P-2 The fragments obtained from RT-PCR, 5¢ RACE and 3¢ RACE were subcloned into pBluescript 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, USA) and analyzed by DNA sequencing as described below (Amersham Biosciences) and FLAG M2 beads (Sigma), 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 using the human genome sequence and the full-length ORF of mouse fad49 Next, based on the predicted sequence for human fad49, RT-PCR was performed as described above using total RNA prepared from HeLa cells The 5¢ region of the human cDNA of fad49 was amplified using KOD plus (Toyobo) with a human fad49-specific forward primer (5¢-GCGGCCATGCCGCCGCGGCGCAGCATCG-3¢) and reverse primer (5¢-TTTCTCGATCACCTCGAC-3¢) In the same way, the 3¢ region of the human cDNA of fad49 was amplified with a human fad49-specific forward primer (5¢-ACATGACCATTCCTCGAG-3¢) and reverse primer (5¢-TCTAGGCAGAAAGGGAGT-3¢) As both the amplified 5¢ and 3¢ regions harbor the EcoRI site, the fragments obtained from RT-PCR were digested with EcoRI, purified, subcloned into the HincII ⁄ EcoRI site of pBluescript KS+ and analyzed by DNA sequencing as described below Lipid binding studies were performed using PIP arraysÔ (Echelon Biosciences) according to the manufacturer’s instructions Plasmid construction The DNA fragments encoding fad49 were amplified by RT-PCR from total RNA extracted from 3T3-L1 cells h after differentiation induction The resulting fragments were cloned, using appropriate restriction sites, in-frame into several expression vectors as described below Fragments encoding the PX domain and individual SH3 domains of FAD49 were subcloned into the pGEX4T-1 vector (Amersham Pharmacia Biotech, Piscataway, NJ, USA) and pFLAG-MAC vectors (Sigma), respectively For N-terminally Myc-tagged FAD49 (Myc–FAD49), the DNA fragment encoding full-length FAD49 was subcloned into the pCMV-Myc vector (BD Biosciences Clontech) For C-terminally Myc-tagged FAD49 (FAD49–Myc), the DNA fragment that encodes full-length FAD49 followed by the 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 (GFP–PX) and FAD49 lacking its PX domain (GFP–SH3), individual DNA fragments encoding full-length FAD49 (amino acids 1–910), the FAD49 PX domain (amino acids 1–130) and FAD49 lacking the PX domain (amino acids 126–910) were subcloned into pEGFP-C1 vectors (BD Biosciences Clontech) All constructs described above were analyzed by DNA sequencing as described below Preparation of fusion proteins GST and FLAG fusion proteins were expressed in E coli BL21 at 30 °C and purified using glutathione–Sepharose 4B In vitro binding assay For in vitro binding experiments, glutathione–Sepharosebound proteins, GST or GST–PX, were prepared, and then incubated with each of the purified FLAG fusion proteins for individual SH3 domains of FAD49 in GST-binding buffer [20 mm Tris ⁄ HCl pH 8.0, 180 mm KCl, 0.2 mm EDTA, 0.5% w ⁄ v Nonidet P-40 (Nacalai Tesque)] overnight at °C Samples were washed three times in GST wash buffer (20 mm Tris ⁄ HCl pH 8.0, 180 mm KCl, 0.2 mm EDTA, 1% Nonidet P-40), eluted from the resin by boiling in an SDS sample buffer (62.5 mm Tris ⁄ HCl pH 6.8, 10% v ⁄ v 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 Following transfer, membranes were blocked with 4% w ⁄ v skim milk in Tris-buffered saline with 0.1% Tween-20 (TBS-T), and probed using primary antibodies, secondary antibodies, conjugated horseradish peroxidase and an enhanced chemiluminescence detection kit (GE Healthcare, Chalfont St Giles, UK) to detect specific proteins DNA sequencing and database analyses The sequence was determined using a DSQ 1000 automated sequencer (Shimadzu Corp., Kyoto, Japan) and an ABI PRISM 3100 genetic analyzer (Applied Biosystems) The search for a human ortholog in human genome databases was performed using blast programs accessed via the National Center for Biotechnology Information (NCBI) homepage Cell culture, differentiation and cell counts Mouse 3T3-L1 (ATCC CL173) preadipocyte cells were maintained in DMEM containing 10% v ⁄ v calf serum For the differentiation experiment, the medium was replaced with DMEM containing 10% v ⁄ v FBS, 10 lgỈmL)1 insulin, 0.5 mm IBMX and lm dexamethasone days postconfluence After days, the medium was changed to DMEM containing lgỈmL)1 insulin and 10% FBS, and then the cells were re-fed every days Adipogenesis was determined by staining the cells with Oil Red O (Amresco, Salon, OH, USA) Mouse NIH-3T3 fibroblastic cells (clone 5611, JCRB 0615) were maintained in DMEM containing 10% calf 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 rinsed with NaCl ⁄ Pi, and trypsinized with trypsin (0.25%) ⁄ EDTA (0.1%) solution in NaCl ⁄ Pi for at 37 °C, 5% CO2 Cell counts were determined for the trypsinized cells using a hemocytometer Fractionation of fat cells The fat cells were prepared as described previously [42] In brief, epidermal fat pads were isolated from male C57Bl ⁄ 6J mice aged weeks, washed with sterile NaCl ⁄ Pi, minced, and washed using Krebs–Ringer bicarbonate buffer, pH 7.4 Then, the minced tissue was digested with 1.5 mgỈmL)1 of collagenase type II (Sigma-Aldrich) in Krebs–Ringer bicarbonate buffer, containing 4% w ⁄ v BSA, at 37 °C for h on a shaking platform The undigested tissue was removed with 250 lm nylon mesh and the digested fraction was centrifuged at 500 g for The adipocytes were obtained from the uppermost layer, washed with buffer, and centrifuged at 500 g for at °C to remove other cells Stromal–vascular cells were resuspended in an erythrocyte lysis buffer (150 mm NH4Cl, 25 mm NH4HCO3 and mm EDTA pH 7.7), filtered through 28 mm nylon mesh and then precipitated at 500 g for RNAi experiment The two target regions in the ORF of mouse fad49 (GenBank accession number AB430861); region at 2515– 2535 bp (for which nucleotide A in the translation initiation codon is the first nucleotide) and region at 778–798 bp were selected using the Qiagen siRNA online design tool (http://sirna.qiagen.com/) for RNAi of fad49 A 19-nucleotide shRNA coding fragment with a 5¢-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¢-GTAAGATGAGGC AATGGAG-3¢, which does not have similarity with any mRNA listed in GenBank, was generated Transfection of shRNA expression plasmids into 3T3-L1 cells was performed using Nucleofector (Amaxa) with cell line Nucleofector kit V (Amaxa) 3T3-L1 cells were harvested and resuspended in Nucleofector solution at 1.5 · 106 cells per 100 lL After addition of shRNA expression plasmids (9 lg), the cells were transfected using the T-20 Nucleofector program Then they were plated on 12- or 24-well plates The 3T3-L1 cells were subjected to differentiation experiments days after the transfection Differentiation experiments were performed using the same medium as described above Cell counts were performed using 24-well plates Subcellular localization of FAD49 For immunostaining, 3T3-L1 cells transfected with pCMVMyc-FAD49 using the NucleofectorÔ system (Amaxa, Cologne, Germany) were seeded onto cell disks (Sumitomo Bakelite Co Ltd, Tokyo, Japan) with a collagen coating One day after transfection, the cells were washed with NaCl ⁄ Pi and fixed in 4% w ⁄ v paraformaldehyde for 15 at room temperature After washing the cells three times with NaCl ⁄ Pi, they were permeabilized with 0.2% w ⁄ v gelatin in NaCl ⁄ Pi with 0.2% Triton X-100 for 30 min, washed three times with NaCl ⁄ Pi, and then blocked using blocking solution (1% BSA in NaCl ⁄ Pi with 0.1% Tween-20) for h at room temperature After blocking, each disk was incubated with the mouse monoclonal c-Myc antibody overnight at °C After washing three times with 0.1% Tween-20 in NaCl ⁄ Pi, each disk was incubated with antimouse fluorescein isothiocyanate (FITC) (Sigma) for h at room temperature After three more washes with NaCl ⁄ Pi, FITC signals were detected by fluorescence microscopy For subcellular localization studies using the three GFP-fad49 chimeric protein expression plasmids described above, 3T3-L1 cells were transfected with one of the three expression plasmids or an empty vector as a control using FuGENE transfection reagent (Roche Applied Science, Indianapolis, IN, USA) according to the manufacturer’s instructions One day after transfection, the cells were washed three times and fixed with 4% paraformaldehyde for 15 at room temperature GFP signals were detected using a fluorescence microscope 5586 Accession numbers The sequences of the cloned mouse and human fad49 cDNAs have been deposited in the GenBank database with accession numbers AB430861 and AB430862, respectively Acknowledgements We thank Asako Shimada and Aya Fujii for technical assistance (Nagoya City University, Aichi, Japan) This study was supported in part by a Grant-in-Aid for Scientific Research on Priority 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