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

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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 dif- ferentiation. 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 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 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].
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