Báo cáo khoa học: A novel gene, fad49, plays a crucial role in the immediate early stage of adipocyte differentiation via involvement in mitotic clonal expansion docx

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
Obesity is a serious and growing health problem that
is a key risk factor in several obesity-related diseases,
such as type 2 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, adipo-
nectin and resistin, which leads to various diseases,
such as type 2 diabetes, stroke and cardiac infarction
[3–6].
Obesity, the pathological development of adipose tis-
sue, results from an increase in the cell size of individ-
ual adipocytes and an increase in total adipocyte cell
numbers through differentiation of preadipocytes in
adipose tissue into mature adipocytes. Therefore, in
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 467-
8603, Japan
Fax: +81 52 836 3455
Tel: +81 52 836 3455
E-mail:
(Received 24 July 2008, revised 7
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 mech-
anisms of adipocyte differentiation, we previously isolated 102 genes
expressed early in mouse 3T3-L1 preadipocyte cells using a PCR subtrac-
tion 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 3
(SH3) domains, suggesting that it may be a novel scaffold protein. We
found that the PX domain of FAD49 not only has affinity for phosphoi-
nositides, but also binds to its third SH3 domain. Expression of fad49 was
transiently elevated 3 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 dif-
ferentiation. 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.
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)P
2
, phosphatidylinositol 3,4-bisphosphate;
PI(4)P, phosphatidylinositol 4-phosphate; PI(4,5)P
2
, 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
the context of the prevention and treatment of obesity-
related diseases, it is important to elucidate the mecha-
nisms 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 con-
fluence and growth arrested. Growth-arrested 3T3-L1
cells differentiate into mature adipocytes after the addi-
tion 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 differentia-
tion, partly through transactivation of C ⁄ EBPb and
C ⁄ EBPd, the expression of which is immediately upreg-
ulated 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 differ-
entiation 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 sub-
traction system [16,17]. These genes included transcrip-
tion 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 full-

length cDNAs of the unknown genes. We have previ-
ously revealed that several of them are novel genes,
such as factor for adipocyte differentiation (fad) 24,
fad104 and fad158, which play crucial roles in adipo-
genesis [20–23].
Here, we report the isolation of another novel gene,
fad49, and the close involvement of fad49 in adipocyte
differentiation. FAD49 contains a phox homology
(PX) domain, which has affinity for phosphoinositides,
and four Src homology 3 (SH3) domains, which bind
to polyproline PXXP ligands, suggesting that FAD49
is a novel scaffold protein. RNAi experiments demon-
strated that fad49 is crucial in adipogenesis, and that it
plays important roles in events early in the differentia-
tion 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 3 h after induction
of adipocyte differentiation. The PCR-subtraction
method used in the previous study gave cDNA frag-
ments only 300–900 bp long because the amplified frag-
ments 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 3 h after induction. As a result, a
1109 bp cDNA fragment containing an initiation
codon at 6 bp was isolated by 5¢ RACE. The sequence
(GCC
ATGC) including initiation codon is close to
the consensus sequence for translation initiation
(A/GCC
ATGA/G). A 1809 bp cDNA fragment con-
taining 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 func-
tion, 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 chromo-
some 11 and consists of 13 exons divided by 12 introns
(Fig. 1B). Sequencing of the exon ⁄ intron junctions in
T. Hishida et al. fad49 plays a crucial role in adipogenesis

FEBS Journal 275 (2008) 5576–5588 ª 2008 The Authors Journal compilation ª 2008 FEBS 5577
the database revealed that the GT ⁄ AG rule was main-
tained 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 underlin-
ing) and four SH3 domains (dotted underlining)
(Fig. 2A). Moreover, the encoded protein also con-
tained ten polyprolines (boxed), which could be possible
ligands for the SH3 domain. Thus, fad49 encodes a pro-
tein containing many protein-binding domains, suggest-
ing 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 data-
base 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 per-
formed 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
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-
dylinositol 3-phosphate [PI(3)P] appears to be the most
common [28,31–34]. For example, the PX domain of
p40
phox
interacts with PI(3)P, the PX domains of
p47
phox
and Fish, which contains five SH3 domains and
a PX domain, binds to PI(3,4)P
2
[24,27], and the PX
domain of C2 containing Ptdlns kinase (CPK) class of
PI 3-kinase selectively binds to PI(4,5)P
2
[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 p47
phox

binds intramolecularly to the SH3
domain in the same protein, and that this intramolecu-
lar interaction suppresses the lipid-binding activity of
the PX domain in the resting state; phosphorylation of
p47
phox
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 bacte-
rially expressed the FAD49 PX domain fused to gluta-
thione S-transferase (GST–PX) and used GST protein
(GST) as a negative control. Lipid binding was then
measured using overlay blotting as described in Experi-
mental procedures. As shown in Fig. 3A, GST–PX
bound most strongly to PI(3,5)P
2
, 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

Fig. 1. Cloning and genomic structures of mouse fad49. (A) The full-
length 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 chromo-
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.
fad49 plays a crucial role in adipogenesis T. Hishida et al.
5578 FEBS Journal 275 (2008) 5576–5588 ª 2008 The Authors Journal compilation ª 2008 FEBS
domain of FAD49 can interact with its third SH3
domain (Fig. 3B).
Subcellular localization of FAD49
To further characterize fad49, the subcellular localiza-
tion of FAD49 was determined by transient transfec-
tion of an N-terminally Myc-tagged full-length
FAD49 (Myc–FAD49) expression plasmid into 3T3-
L1 cells. Cells were immunostained using monoclonal
anti-Myc IgG
1
. 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 exam-
ine 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 dif-
ferentiation, we first determined the mRNA expression
levels of fad49 by Northern blotting. To monitor
changes in the levels of fad49 during adipocyte differ-
entiation, 3T3-L1 cells were stimulated with an induc-
tion cocktail, and then total RNA was prepared at
various time points. The expression of fad49 increased
quickly after differentiation was induced, reaching a
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.
T. Hishida et al. fad49 plays a crucial role in adipogenesis
FEBS Journal 275 (2008) 5576–5588 ª 2008 The Authors Journal compilation ª 2008 FEBS 5579
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-
L1 cells differentiate to mature adipocytes when stimu-
lated with adipogenic inducers 2 days after reaching a
state of confluence, whereas proliferating 3T3-L1 cells
do not differentiate into adipocytes even in the pres-
ence 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 3 h after dif-
ferentiation 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.
To investigate the tissue distribution of fad49,we
determined the expression levels of fad49 by quantita-

tive PCR in various tissues isolated from adult male
mice, including WAT and brown adipose tissue (BAT)
(Fig. 5C). WAT samples were separated into two frac-
tions: the stromal–vascular fraction enriched with prea-
dipocytes and the mature adipocyte fraction. Tissue
distribution studies revealed that high expression of
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.
Fig. 4. Subcellular localization of FAD49. (A) 3T3-L1 cells transiently
transfected with the Myc–FAD49 or FAD49–Myc expression plas-
mid were fixed and blocked for immunofluorescence staining with
anti-Myc serum. (B) Schematic representation of GFP fusion pro-
teins for each FAD49 deletion mutant used in this study. (C) Sub-
cellular localization of EGFP–FAD49 fusion proteins. 3T3-L1 cells
were transiently transfected with an EGFP–FAD49-expressing plas-
mid or empty vector. One day after transfection, the cells were
fixed and stained with DAPI. EGFP signals were detected using a
fluorescence microscope.
fad49 plays a crucial role in adipogenesis T. Hishida et al.
5580 FEBS Journal 275 (2008) 5576–5588 ª 2008 The Authors Journal compilation ª 2008 FEBS
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 pre-
dominant 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 3T3-
L1 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 1 and 2, respectively
(as defined in Experimental procedures), in the ORF
of the fad49 gene. Each of the shRNA expression plas-
mids 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 differ-
entiation. We confirmed that RNAi treatment reduced
fad49 mRNA levels (Fig. 6B). After 8 days, the cells
were fixed and stained with Oil red O and the amounts
of triacylglycerol were determined. The number of
Fig. 5. Expression profiles of fad49 in differ-
entiating and non-differentiating cells and
tissue distribution. (A) Time course of fad49
mRNA expression in the early stage of adipo-
cyte differentiation. Total RNA prepared from
3T3-L1 cells at various time points after treat-
ment 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-differ-
entiating 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 deter-

mined by quantitative PCR. Each column
represents the mean ± SD (n = 3).
T. Hishida et al. fad49 plays a crucial role in adipogenesis
FEBS Journal 275 (2008) 5576–5588 ª 2008 The Authors Journal compilation ª 2008 FEBS 5581
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 expres-
sion. 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 treat-
ment 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 deter-
mined 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 differentia-
tion 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.
fad49 plays a crucial role in adipogenesis T. Hishida et al.
5582 FEBS Journal 275 (2008) 5576–5588 ª 2008 The Authors Journal compilation ª 2008 FEBS
Oil red O-stained cells and the accumulation of triacyl-
glycerol 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 ele-
ment-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 eluci-
date 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 0
and 4, and remained constant between days 4 and 5.
In comparison, cell numbers in fad49 knockdown
cultures only increased 2.2-fold by day 4. 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 3 h after differenti-
ation was induced [16,17]. About half of them were
unknown genes. As a result of attempts to isolate full-
length cDNAs for the unknown genes, we previously
isolated several novel genes that are crucial for adipo-
cyte differentiation [20–22]. In this study, we cloned
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 phosphpoinositide-
binding module, was first identified in p40
phox
and
p47
phox

, 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 contain-
ing this domain [40]. In particular, p47
phox
, which con-
tains the PX domain and two SH3 domains, has been
intensely studied. In unstimulated neutrophils, p47
phox
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 inter-
action, 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)P
2
, 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 p47
phox
, although whether the interaction

of the PX domain with the third SH3 domain is intra-
or intermolecular remains to be established.
To further characterize fad49, we examined the sub-
cellular 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 expres-
sion of fad49 was transiently upregulated 3 h after
T. Hishida et al. fad49 plays a crucial role in adipogenesis
FEBS Journal 275 (2008) 5576–5588 ª 2008 The Authors Journal compilation ª 2008 FEBS 5583
exposure to an induction cocktail, and this upregula-
tion was restricted to the adipocyte differentiable state.
Moreover, RNAi experiments demonstrated that fad49
is closely involved in adipocyte differentiation. Inter-
estingly, 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 expres-
sion remain to be elucidated. In this regard, further
studies of the molecular function of fad49 should pro-
vide 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 mecha-
nism for regulation of MCE, C ⁄ EBPb and mitogen-
activated 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 contribu-
tion 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 pur-
chased 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 IgG
1
(Sigma) and anti-c-Myc
IgG
1
(BD Biosciences Clontech, Palo Alto, CA, USA),
and polyclonal anti-GST IgG (Amersham Biosciences,
Piscataway, NJ, USA).
RNA isolation, real-time quantitative RT-PCR and
northern blotting
Total RNA was extracted using TRIzol (Invitrogen, Carls-
bad, 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 Bio-
systems) according to the manufacturer’s instructions. The

mixture was incubated at 50 °C for 2 min and at 95 °C for
10 min, and then PCR was performed at 95 °C for 15 s and
at 60 °C for 1 min 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
3 h after induction. mRNA was isolated from total RNA
using Oligotex-dT30 (Daiichi Pure Chemicals, Tokyo,
Japan) according to the manufacturer’s instructions. First-
strand 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 1 and E. coli DNA ligase. PCR for 5¢ RACE
was performed using the AP1 primer (5¢-CCATCCTAAT
ACGACTCACTATAGGGC-3¢) and one of three fad49-
specific 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 pre-
pared from 3T3-L1 cells 3 h after induction. PCRs were per-

formed 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
fad49 plays a crucial role in adipogenesis T. Hishida et al.
5584 FEBS Journal 275 (2008) 5576–5588 ª 2008 The Authors Journal compilation ª 2008 FEBS
KS+ (Stratagene, Agilent Technologies, Santa Clara, CA,
USA) and analyzed by DNA sequencing as described below.
Cloning of the full-length cDNA of human fad49
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 ampli-
fied 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.
Plasmid construction

The DNA fragments encoding fad49 were amplified by
RT-PCR from total RNA extracted from 3T3-L1 cells 3 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-termi-
nally Myc-tagged FAD49 (Myc–FAD49), the DNA frag-
ment 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
(Amersham Biosciences) and FLAG M2 beads (Sigma),
according to the manufacturer’s instructions.
Phospholipid binding

Lipid binding studies were performed using PIP arraysÔ
(Echelon Biosciences) according to the manufacturer’s
instructions.
In vitro binding assay
For in vitro binding experiments, glutathione–Sepharose-
bound 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 buf-
fer [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
4 °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. Fol-
lowing 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 lgÆmL
)1
insulin,
0.5 mm IBMX and 1 l m dexamethasone 2 days postconflu-
ence. After 2 days, the medium was changed to DMEM
containing 5 lgÆmL
)1
insulin and 10% FBS, and then the
cells were re-fed every 2 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
T. Hishida et al. fad49 plays a crucial role in adipogenesis
FEBS Journal 275 (2008) 5576–5588 ª 2008 The Authors Journal compilation ª 2008 FEBS 5585
serum. For determination of cell counts, cells were gently
rinsed with NaCl ⁄ P
i
, and trypsinized with trypsin
(0.25%) ⁄ EDTA (0.1%) solution in NaCl ⁄ P
i
for 5 min at
37 °C, 5% CO
2
. Cell counts were determined for the trypsi-
nized 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 6 weeks, washed with sterile NaCl ⁄ P
i
, minced,
and washed using Krebs–Ringer bicarbonate buffer,
pH 7.4. Then, the minced tissue was digested with
1.5 mgÆmL
)1
of collagenase type II (Sigma-Aldrich) in
Krebs–Ringer bicarbonate buffer, containing 4% w ⁄ v BSA,
at 37 °C for 1 h on a shaking platform. The undigested tis-
sue was removed with 250 lm nylon mesh and the digested
fraction was centrifuged at 500 g for 5 min. The adipocytes
were obtained from the uppermost layer, washed with buf-
fer, and centrifuged at 500 g for 5 min at 4 °C to remove
other cells. Stromal–vascular cells were resuspended in an
erythrocyte lysis buffer (150 mm NH
4
Cl, 25 mm NH
4
HCO
3
and 1 mm EDTA pH 7.7), filtered through 28 mm nylon
mesh and then precipitated at 500 g for 5 min.
Subcellular localization of FAD49
For immunostaining, 3T3-L1 cells transfected with pCMV-
Myc-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 ⁄ P
i
and fixed in 4% w ⁄ v paraformaldehyde for 15 min
at room temperature. After washing the cells three times
with NaCl ⁄ P
i
, they were permeabilized with 0.2% w ⁄ v gela-
tin in NaCl ⁄ P
i
with 0.2% Triton X-100 for 30 min, washed
three times with NaCl ⁄ P
i
, and then blocked using blocking
solution (1% BSA in NaCl ⁄ P
i
with 0.1% Tween-20) for 1 h
at room temperature. After blocking, each disk was
incubated with the mouse monoclonal c-Myc antibody
overnight at 4 °C. After washing three times with 0.1%
Tween-20 in NaCl ⁄ P
i
, each disk was incubated with anti-
mouse fluorescein isothiocyanate (FITC) (Sigma) for 1 h at
room temperature. After three more washes with NaCl ⁄ P
i
,
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 6 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 min at room temperature. GFP signals were detected
using a fluorescence microscope.
RNAi experiment
The two target regions in the ORF of mouse fad49
(GenBank accession number AB430861); region 1 at 2515–
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
( for RNAi of fad49. A 19-nucleo-
tide 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 con-
trol, 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 · 10
6
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 3 days after the
transfection. Differentiation experiments were performed
using the same medium as described above. Cell counts were
performed using 24-well plates.
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 Sci-
entific Research on Priority Areas from the Ministry
of Education, Culture, Sports, Science and Technology
(MEXT), Japan, and a Grant-in-Aid for Scientific
Research (B) from the Japan Society for the Promo-
tion of Science (JSPS).
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