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 p
Trang 1early 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: imagawa@phar.nagoya-cu.ac.jp
(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)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.
Trang 2the 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 inducdifferentia-tion 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 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 (GCCATGC) including initiation codon is close to the consensus sequence for translation initiation (A/GCCATGA/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, fad49was 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
Trang 3the 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 underlinunderlin-ing)
(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 propro-tein-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 p40phox interacts with PI(3)P, the PX domains of p47phoxand 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 intramolecu-lar interaction suppresses the lipid-binding activity of the PX domain in the resting state; phosphorylation of p47phoxreleases 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)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
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.
Trang 4domain 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 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
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.
Trang 5maximum 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.
Trang 6fad49was 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-differdiffer-entiating 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).
Trang 7Fig 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.
Trang 8Oil 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
fad49on 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 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 contain-ing this domain [40] In particular, p47phox, which con-tains 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 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)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
intra-or intermolecular remains to be established
To further characterize fad49, we examined the sub-cellular localization of FAD49 Subsub-cellular 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
Trang 9exposure 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
fad49is involved in MCE, partly through its
contribu-tion to the induccontribu-tion 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
arrays 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)
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 50C for 2 min and at 95 C for
10 min, and then PCR was performed at 95C for 15 s and
at 60C 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
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
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
Trang 10KS+ (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
(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 30C 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 arrays (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
4C 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)1insulin, 0.5 mm IBMX and 1 lm 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