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INTRODUCTION |
The adipose tissue is an important organ for energy storage and
lipid homeostasis (1-3). In addition, it secretes many cytokines and
other proteins, such as leptin, adiponectin, adipsin, tumor necrosis
factor-
, and plasminogen activator inhibitor-1. These secreted
proteins have crucial roles in regulating food intake and the insulin
sensitivity/resistance in diabetes mellitus, as well as in obesity (1,
3, 4). Obesity is a ringleader for many diseases, such as diabetes,
hypertension, hyperlipoidemia, and also arteriosclerosis (5).
Therefore, further insight into the molecular basis of obesity is required.
Three families of transcription factor proteins have been identified
and characterized as the master regulators of adipocyte differentiation
(6, 7). Peroxisome proliferator-activated receptor
(PPAR
)1 is a key
transcription factor for the differentiation of preadipocytes into
mature adipocytes and activates the many adipocyte-specific genes
including the aP2, lipoprotein lipase, and resistin genes (8,
9). The CCAAT/enhancer-binding protein (C/EBP) family also has been
identified as master regulators. Notably, C/EBP
activates the
expression of PPAR
, as well as leptin and the insulin receptor (7,
10, 11). The expression of C/EBP
and C/EBP
is preceded by that of
C/EBP
and induces PPAR
expression (6, 12). Sterol regulatory
element-binding protein 1 (SREBP-1) functions as a regulator for lipid
homeostasis, and this protein is also known to be an activator for the
ligand production of PPAR
(7, 13, 14). Thus, the middle and late
stages of adipocyte differentiation are relatively well characterized.
However, the events at the early stage of adipogenesis are not fully understood.
In previous reports, we identified 102 genes as inducible at the
earliest stage of adipocyte differentiation by a polymerase chain
reaction (PCR) subtraction protocol (15, 16). These include genes for
transcription factors and signaling proteins. It is of interest that
almost half of them are unknown genes that are not found in the data
bases. The main reason for this is likely to be that the isolated
fragments are too short to identify the genes. Therefore, in a previous
study, we performed a preliminary analysis using the rapid
amplification of cDNA ends (RACE) technique and, by partial
sequencing, identified clone 26 as mouse TC10, similar in sequence to
human TC10, one of the Rho family (15). However, recent findings by two
groups have revealed the existence of one more Rho family member
(17-19). TC10-like (TCL) was identified from human and mouse EST
(expressed sequence tag) data bases sharing 95% sequence similarity
with human TCL, which has 10 more amino acids in the N-terminal portion
(17). TC10
was identified as a mouse ortholog of TC10 (18).
Interestingly, TC10
has an isoform, TC10
L, which has 10 more
amino acids at the N terminus. In the present study, we isolated the
full-length cDNA of clone 26 and by sequence analysis revealed that
our mouse clone 26 is identical to TC10
L (18) and also to the
longer form of TCL (17). Therefore, in the present paper we refer to
clone 26 as TCL/TC10
L.
The Rho family of small GTPases makes up a large gene family, which can
be divided into two groups; one group includes RhoA, -B, -C, -D, -E,
and -L and Rnd1-3, and the other includes Rac1-3, Rho G,
TTF/RhoH, Chp, Cdc42, TC10, and TCL/TC10
L (20). Rho proteins
act as molecular switches to control cellular processes by cycling
between the active, GTP-bound and inactive, GDP-bound states. Several
lines of evidence in the past few years have revealed that Rho proteins
have important roles in many cellular events, such as membrane
trafficking, transcriptional regulation, control of cell growth, and
development (20-23). Recently, Chiang et al. (24) suggested
that the activation of TC10 was essential for insulin-stimulated
glucose uptake and GLUT4 translocation in fully differentiated
adipocytes. However, the roles of Rho proteins have not been elucidated
in terms of adipocyte differentiation.
In our previous report (15), clone 26 (TCL/TC10
L in the present
paper) was strongly expressed at the early stage of adipocyte differentiation; this induction is specific to growth-arrested cells, a
state that is essential for adipocyte differentiation. These findings
prompted us to further characterize the functional roles of
TCL/TC10
L. In the present study, we blocked the expression of
TCL/TC10
L using the Lac Switch mammalian expression system, which
produces antisense TCL/TC10
L. The inhibition of TCL/TC10
L expression in 3T3-L1 cells prevented the cytoplasmic accumulation of
triglyceride and decreased the expression of adipogenic related genes.
We next used a retroviral system to over-express TCL/TC10
L stably in
NIH-3T3 cells. The constitutive over-expression of TCL/TC10
L is
sufficient to cause the adipocyte differentiation of these cells in the
presence of 3-isobutyl-1-methylxanthine (IBMX), dexamethasone (Dex),
insulin (Ins), 10% fetal bovine serum (FBS), and the PPAR
ligand
BRL49653. Furthermore, using the PCR-cDNA subtraction system, we
isolated several clones as candidates for the downstream target genes
of TCL/TC10
L. Some of the isolated genes were also induced during
the differentiation of 3T3-L1 cells. These results strongly suggest
that TCL/TC10
L has crucial roles in the program of adipocyte differentiation.
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EXPERIMENTAL PROCEDURES |
Cloning of Mouse TCL/TC10
L--
The full-length
cDNA for clone 26 was isolated by 5'-RACE analysis. The 5'-RACE
reaction was performed using the Marathon cDNA amplification kit
(Clontech, Palo Alto, CA) according to the
manufacturer's instructions. The sequences were determined with the
automated DNA sequencer DSQ 1000 (Shimadzu Corp., Kyoto, Japan) and ABI
PRISM 310 (PerkinElmer Life Sciences).
Cell Culture and Differentiation--
The mouse 3T3-L1 (ATCC
CL173) preadipocytes and mouse NIH-3T3 (clone 5611, JCRB 0615, Japanese
Cancer Research Resources Bank) fibroblast cells were maintained in
Dulbecco's modified Eagle's medium (DMEM) containing 10% calf serum.
For the differentiation experiment, the medium was changed to DMEM
supplemented with 0.5 mM IBMX, 10 µg/ml Ins, 1 µM Dex, and 10% FBS at 2 days postconfluence (15). After
2 days, the cells were transferred to DMEM containing 5 µg/ml Ins and
10% FBS, and then the cells were refed every 2 days. BRL49653, the
ligand for PPAR
(a gift from GlaxoSmithKline), was also added at a
final concentration of 0.5 µM. The packaging cell line
PT67 (Clontech) was maintained in DMEM containing
10% FBS.
Establishment of Stable Cell Lines Expressing Antisense
TCL/TC10
L--
Stable transformants expressing antisense
TCL/TC10
L under the regulation of
isopropyl-1-thio-
-D-galactopyranoside (IPTG) were
developed using a Lac Switch II inducible mammalian expression system
(Stratagene, La Jolla, CA) according to the manufacturer's instructions. The fragment containing the stretch between
11 and +241
(assigning A in start codon ATG for the first methionine from
the 5'-portion as +1) was amplified by PCR using the primers with the
restriction sites for SpeI and XhoI,
respectively. After enzyme digestion of the amplified fragment, the
resultant fragment was subcloned into a site in pOPRSVI/MCS in the
antisense orientation. The sequence of the inserted fragment was
confirmed as described above. 3T3-L1 cells were grown to 60-70%
confluency and co-transfected with pCMVLac1, which expresses the Lac
repressor protein, and pOPRSV1/MCS antisense TCL/TC10
L or pOPRSVI
without the insert using LipofectAMINE (Invitrogen). The stable
transformant was selected in the presence of both 400 µg/ml neomycin
(G418) and 150 µg/ml hygromycin. The drug-resistant clones were
removed individually and stored.
Establishment of TCL/TC10
L-over-expressing Cell
Lines--
The TCL/TC10
L expression vector (pDON-AI-TCL/TC10
L)
was constructed by ligating the full-length cDNA encoding mouse
TCL/TC10
L. The full-length mouse TCL/TC10
L cDNA was amplified
from 3T3-L1 cells from total RNA by RT-PCR. The PCR products were
ligated into the SalI site of pDON-AI (Takara Biomedicals,
Kusatsu, Japan). PT67 cells were cultured in 100-mm dishes and
transfected by calcium phosphate coprecipitation with 14 µg of
pDON-AI-TCL/TC10
L plasmid. After a 48-h transfection, the viral
supernatants were collected and used to infect NIH-3T3 fibroblasts at
50% confluence in 100-mm dishes. Polybrene (4 µg/ml) was added to
the viral supernatants before treatment. After a 24-h incubation with
viral supernatant, cells were split and replaced in DMEM containing
10% calf serum and 400 µg/ml of neomycin (G418) to select infected
cells for 2 weeks. The drug-resistant clones were removed individually
and stored.
RNA Isolation, RT-PCR, and Northern Blot Analyses--
The cells
were harvested at a specific time. Total RNA was extracted using TRIzol
(Invitrogen). The mRNA was isolated from total RNA using
Oligotex-dT30 (Daiichi Pure Chemicals, Tokyo, Japan) for 5'-RACE. For
RT-PCR, cDNA was prepared using AMV Reverse Transcriptase XL
(Takara Biomedicals) following the manufacturer's recommended
procedures. The PCR was done under the appropriate conditions. The PCR
fragments were stained with ethidium bromide, and the intensities were
determined with a fluoroimager (FluorImager 595, Amersham Biosciences).
For Northern blot analyses, 25 µg of total RNA was electrophoresed on
a 1.0% agarose gel containing 2% formaldehyde and then transferred to
a Hybond-N+ nylon membrane (Amersham Biosciences). The filter was
hybridized with each probe, which was labeled with
[
-32P]dCTP using a random labeling kit (Takara
Biomedicals). The radioactivity corresponding to each band was measured
with a bioimage analyzer (BAS2000, Fuji Film, Tokyo, Japan).
PCR Subtraction Method--
The subtraction cloning with PCR was
performed using a PCR-select cDNA subtraction kit
(Clontech) as described previously (15). For this
purpose, two stable cell lines, NIH-TCL/TC10
L (a stable cell line
integrated with pDON-AI-TCL/TC10
L) and NIH-vector (a stable cell
line integrated with the pDON-AI control vector) were used. These cells
were harvested before and at 2 days after induction, and Poly
(A)+ RNA was prepared from each cell.
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RESULTS |
Cloning of Mouse TCL/TC10
L--
We previously
isolated genes expressed at the beginning of the differentiation of
mouse 3T3-L1 cells (15, 16). Of these, 46 were unknown genes not found
in any data bases. Clone 26 was expressed very strongly and
specifically during adipocyte differentiation. This induction is not
caused by just a change in medium without the differentiation mixture,
because the expression of clone 26 was not observed in fresh medium
without inducers (15). Moreover, when IBMX was omitted from the
differentiation mixture, the induction did not occur, indicating that
IBMX is a principal inducer (15). Because clone 26 is obtained
as a small DNA fragment, we next attempted to isolate the full-length
cDNA of this gene. Using 5'-RACE analysis, PCR products ~800 bp
long were obtained, and sequence analysis showed a predicted protein
that encodes 214 amino acids (data not shown).
In amino acid sequence, clone 26 showed 79.1% similarity to human
TC10, which is one of the Rho family (19). As a result of a data base
search, we first regarded this gene as a mouse homolog of human TC10.
However, Vignal et al. (17) reported human and mouse TCL
(TC10-like), a new GTPase of the Rho family related to TC10. The
sequence analysis revealed 95% similarity between human and mouse,
with the only major difference being found in the N-terminal portion,
where human TCL is 10 amino acids longer than mouse TCL. Interestingly,
the amino acid sequence of our clone 26 is the same as that of mouse
TCL, except it has 10 more amino acids at the N terminus, which
is exactly the same length as the human TCL sequence.
While we were preparing this manuscript, Chiang et al. (18)
reported a related TC10 isoform involved in insulin-stimulated glucose
transport and referred to TC10 and the new isoform as TC10
and
TC10
, respectively (18). TC10
has two isoforms, and they named
the longer variant TC10
L (TC10
Long). It was found that mouse
TC10
L is identical to our clone 26 and of the same length as human
TCL and also that TC10
is equal to mouse TCL. Therefore, we refer to
our clone 26 as TCL/TC10
L, as a longer form of TCL, throughout this
study. The expression of TCL/TC10
L was markedly elevated, whereas
TC10
was involved in glucose transport in a late stage of adipocyte
differentiation. These results indicated functional differences between
TC10
and -
. Therefore, we next determined the expression profiles
of Rho family genes including TCL/TC10
L and TC10
.
Northern Blot Analyses of TCL/TC10
L and Other Rho
Family Genes during Adipocyte Differentiation--
We investigated the
expression of TCL/TC10
L and other family members during the
differentiation of 3T3-L1 preadipocytes into adipocytes. The expression
levels of TCL/TC10
L quickly elevated after the induction, reaching a
peak at 3 h. The expression of TC10
was only slightly
detectable before the induction, and it decreased after the induction.
However, it increased clearly in the late stage (Fig.
1). The levels of other Rho family
members, such as RhoA, Rac1, and Cdc42, were unchanged throughout the
incubation, indicating the specific expression of TCL/TC10
L and
TC10
in adipocyte differentiation.

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Fig. 1.
Northern blot analyses of Rho family
mRNAs during adipocyte differentiation. Total RNA was prepared
from mouse 3T3-L1 cells at various time points after treatment with
inducers. The total RNA (20 µg) was loaded, and the filter was
hybridized with each probe. Staining with ethidium bromide
(EtBr) for ribosomal RNA is shown as a control.
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As described above, TC10
has two isoforms that differ in the
N-terminal portion. Therefore, we next performed RT-PCR analyses to
determine the expression levels of these two isoforms during adipogenesis. Because the first three amino acids were the same between
the short and long forms of TC10
, we speculated that the shorter
form of TC10
is a splicing isoform and designed PCR primers for the
detection of both isoforms. The PCR analyses detected that only
TC10
L, suggesting that the long form of TC10
(TCL/TC10
L) was
specifically expressed in adipocytes (data not shown); however, we
cannot rule out the possibility of sequence differences in the
5'-untranslated region due to the usage of different promoters.
Antisense TCL/TC10
L Prevents the Differentiation of
Fibroblasts into Adipocytes--
To gain insight into the biological
functions of TCL/TC10
L, we first attempted to block the expression
of TCL/TC10
L during preadipocyte differentiation using the Lac
Switch mammalian expression system. We co-transfected
pOPRSV1-TCL/TC10
L antisense and pCMVLac1 into mouse 3T3-L1 cells. As
a control, a cell line transfected with pCMVLac1 and pOPRSVI without
the insert was also developed. By selection, using G418 and hygromycin,
we isolated stable transformants, antisense
TCL/TC10
L-expressing cells and control cells. 5 mM IPTG
was added to the medium 12 h before treatment with inducers for
the differentiation. The level of expression of the TCL/TC10
L mRNA was determined by RT-PCR. In TCL/TC10
L antisense cells, the
expression level of glyceraldehyde-3-phosphate dehydrogenase, used as a
control, did not change with or without IPTG. In contrast, the
expression of TCL/TC10
L decreased at 3 h in the presence of
IPTG (Fig. 2A). In control
cells, the expression levels of TCL/TC10
L and
glyceraldehyde-3-phosphate dehydrogenase were not affected by the
addition of IPTG.

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Fig. 2.
Antisense TCL/TC10 L
inhibits adipocyte differentiation. A, the
expression of TCL/TC10 L mRNA was inhibited by the addition of
IPTG in stable transformants expressing antisense TCL/TC10 L. The
mouse 3T3-L1 cells, stably expressing antisense TCL/TC10 L under the
regulation of IPTG, and integrated empty vector (control)
were treated with inducers. IPTG was added to the medium 12 h
before the inducers at a final concentration of 5 mM. The
expression levels of TCL/TC10 L and glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) were determined by RT-PCR, and the
relative intensities of the products are also shown. B,
adipocyte conversion was inhibited by expressing antisense
TCL/TC10 L. The stable transformants expressing antisense
TCL/TC10 L and control cells were treated with inducers. IPTG was
added to the medium 12 h before the inducers at a final
concentration of 5 mM. After 8 days of treatment the cells
were stained with Oil Red O, and the oil droplets were stained
red. C, Northern blot analyses of various
adipogenic marker genes. The stable transformants expressing antisense
TCL/TC10 L were treated with inducers with or without IPTG. Total RNA
was isolated from cells at various time points of incubation and used
for Northern blot analyses.
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Using these transformants, we performed a differentiation experiment.
The isolated clones were brought to confluence in 10% calf serum
containing G418 and hygromycin. After 2 days incubation, the medium was
changed to the differentiated medium with or without IPTG. After 8 days, the cells were stained with Oil Red O to detect oil droplets. The
control cells stored the oil droplets well regardless of the presence
of IPTG. On the other hand, the accumulation of oil droplets in the
cells expressing antisense TCL/TC10
L was clearly inhibited by the
addition of IPTG (Fig. 2B).
To test whether the stage of adipogenesis was really blocked by the
expression of antisense TCL/TC10
L, we next determined the expression
levels of the fat differentiation-linked genes. The expression of
PPAR
, SREBP-1, and C/EBP
, which are all known to be master
regulators of adipogenesis, was inhibited to a certain extent (Fig.
2C). Lipoprotein lipase, an adipogenic marker, was also
inhibited during the differentiation, whereas the expression of
C/EBP
was decreased only slightly, and that of C/EBP
was unchanged (Fig. 2C).
Over-expression of TCL/TC10
L Promotes Adipose
Differentiation of NIH-3T3 Cells--
We next generated stable
transformants constitutively expressing sense TCL/TC10
L using a
retrovirus system. The full-length sense TCL/TC10
L was cloned into a
retroviral vector, pDON-AI. NIH-3T3 cells were infected with viruses
containing pDONAI-TCL/TC10
L (TCL/TC10
L) or empty vector alone
(control), which were produced in PT67 cells. After selection using
G418, the drug-resistant clones were isolated. The Northern blot
analysis showed that the endogenous TCL/TC10
L (~3.5 kb) was rarely
detected in TCL/TC10
L and control cells, whereas the exogenous
TCL/TC10
L derived from retrovirus was found as a ~4.6-kb long
mRNA (Fig. 3A).

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Fig. 3.
TCL/TC10 L promotes
adipocyte differentiation in the presence of PPAR ligand.
A, the ectopic expression of TCL/TC10 L in NIH-3T3 stable
transformants. Northern blot analyses were performed for RNAs prepared
from two stable transformants: control integrated with empty vector
only and TCL/TC10 L integrated with sense TCL/TC10 L in
NIH-3T3 cells. The blots were hybridized with TCL/TC10 L. The
arrowhead and arrow indicate the endogenous
expression and the exogenous expression of TCL/TC10 L derived from
integrated retrovirus, respectively. B, differentiation of
TCL/TC10 L-expressing stable transformants into adipocytes in the
presence of PPAR ligand, BRL49653. The TCL/TC10 L-expressing
stable transformant and control (control: integrated
with empty vector) stable transformant were treated with the
differentiation medium containing IBMX, Dex, Ins, and FBS in the
presence of BRL49653. After 10 days of treatment, the cells were
stained with Oil Red O. C, Northern blot analyses of various
adipogenic marker genes. The stable transformant expressing sense
TCL/TC10 L and control transformant were treated with the
differentiation medium containing IBMX, Dex, Ins, and FBS in the
presence of BRL49653. Total RNA isolated from cells at various time
points of incubation was used for Northern blot analyses.
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Then, using these established cell lines, a differentiation experiment
was performed. The TCL/TC10
L and control cells were cultured to
confluence and treated with IBMX, Dex, Ins, and 10% FBS. Under the
conditions, no morphological change or accumulation of oil droplets was
observed after 10 days in either of the cells (data not shown).
However, it is of interest that TCL/TC10
L cells started to
differentiate into adipocytes when 0.5 mM BRL49653, a
PPAR
ligand, was added to the differentiation medium. As shown in
Fig. 3B, the oil droplets were
detected in TCL/TC10
L cells, whereas neither morphological change
nor the accumulation of oil droplets was observed in control cells
(Fig. 3B).

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Fig. 4.
Expression patterns of genes isolated from
the TCL/TC10 L-expressing cell line. Total
RNA was prepared from mouse 3T3-L1 cells at various times after
treatment with inducers. The total RNA (20 µg) was loaded, and the
filter was hybridized with each probe.
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Northern blot analyses were performed to characterize further the
phenotype of the TCL/TC10
L cells during the incubation with
inducers. Total RNAs were prepared from TCL/TC10
L and control cells
at various times after the treatment with differentiation medium
containing 0.5 mM BRL49653. The expression of PPAR
and SREBP-1, which are master transcription factors for adipogenesis, was
elevated in TCL/TC10
L cells during incubation (Fig. 3C). The expression of aP2 and lipoprotein lipase was also increased, although these increases were slight in control cells. However, the
expression levels of C/EBP
and C/EBP
were unchanged in both TCL/TC10
L and control cells. The expression of C/EBP
was almost undetectable throughout the incubation in both cells. These results strongly suggest that TCL/TC10
L has important functions in the promotion of adipogenesis.
Isolation of cDNA Clones Up-Regulated in
TCL/TC10
L-over-expressing Cells--
The sense and
antisense experiments indicated that TCL/TC10
L has critical roles in
the early stage of adipocyte differentiation. Because the TCL/TC10
L
gene has been reported only recently (17, 18), the functions of its
product are not well understood yet, and the signaling network through
TCL/TC10
L in adipogenesis has not been characterized at all. To
solve this issue, we next isolated the genes that seem to be downstream
targets of TCL/TC10
L, by subtraction between TCL/TC10
L and
control cells.
We designed two different conditions for the characterization of
TCL/TC10
L in the differentiation of adipocytes. First, we performed
PCR subtraction and isolated the genes in which levels are increased by
the expression of ectopic TCL/TC10
L, without the inducers described
under "Experimental Procedures" (condition 1). Second, the induced
genes were cloned from cells incubated with PPAR
ligand in addition
to the inducers for 2 days (condition 2).
After performing the PCR subtraction protocol, we chose 300 colonies in
each condition and sequenced them. As a result, 231 and 234 independent
clones were isolated in conditions 1 and 2, respectively. It seems that
56 clones in conditions 1 and 58 clones in condition 2 are unknown
genes as determined from a data base search. Next, we chose 60 and 43 clones, respectively, from known genes in each condition and
performed Northern blot analyses by using total RNA prepared from
TCL/TC10
L and control cells at 0 day (condition 1) and 2 days after
treatment with inducers (condition 2). Finally, we identified 21 and 13 genes, the expression of which was enhanced by TCL/TC10
L in
conditions 1 and 2, respectively. These genes are listed in Table
I.
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Table I
Up-regulated genes in the TCL/TC10 L-expressing stable
transformant
The PCR subtraction was performed under two different conditions as
described under "Experimental Procedures." The nucleotide sequences
of isolated clones were searched in the DNA data bases, and the names
were shown when identical to mouse clones. When the clones showed high
similarity to other species, the name of the source is given in
parentheses.
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As expected, the isolated genes include the ones contributing to the
signal transduction and cytoskeletal and extracellular structures.
Northern blot analyses were performed for some genes during the
adipogenesis of 3T3-L1 cells. As shown in Fig. 4, the expression
patterns of several genes were found to be similar, or slightly
delayed, compared with the expression of TCL/TC10
L.
 |
DISCUSSION |
The differentiation of adipocytes is a very complex process. The
expression patterns and the functions of several transcription factors
have been well characterized. Experiments using knockout mice for
PPAR
showed the requirement of this gene for the differentiation into adipocytes (25, 26). The double knockout of C/EBP
and C/EBP
impaired the synthesis of fat in mice (27). Furthermore, it is reported
that the ectopic expression of C/EBP
promotes adipogenesis as well
as PPAR
(28). The dominant negative form of SREBP-1 prevented
adipocyte differentiation (13). These findings strongly suggested that
these three transcription factor families have indispensable roles in
adipocyte differentiation. Thus, even though the middle and late
stages of adipocyte differentiation have been relatively well
characterized, the events in the early stage of adipogenesis have not
been fully elucidated.
Previously we isolated many genes induced early in adipocyte
differentiation (15, 16). The present paper describes the functional
role of TCL/TC10
L. The pattern of TCL/TC10
L expression during
adipocyte differentiation was specific and different from that of other
Rho family members such as RhoA, Rac1, and Cdc42. Interestingly,
although TCL/TC10
L shares high similarity to TC10
, the expression
pattern of the TC10
gene was also different. TCL/TC10
L was
expressed at the beginning of adipogenesis, whereas the expression of
TC10
peaked late in the differentiation. It is also reported that
TC10
and TCL/TC10
L differ in their impact on insulin-stimulated GLUT4 translocation (18). These results indicated that TCL/TC10
L and
TC10
have quite different functions in adipocyte differentiation and
adipose tissue. Saltiel's group (18) also reported that there were two
isoforms, TC10
and TC10
L. In our experiments, only the longer
form, TC10
L, was detected at the early stage of differentiation,
which suggests different roles for the isoforms, although further
characterization is definitely needed.
When NIH-3T3 cells expressing TCL/TC10
L differentiated into
adipocytes, the PPAR
ligand was required. In our previous paper, we
demonstrated that the regulator of G-protein signaling 2 (RGS2) promoted the differentiation in the presence of ligand for PPAR
(29). In this case, the expression patterns of adipogenic genes were
quite similar to those of TCL/TC10
L; that is, the expression of
PPAR
, but not C/EBPs, was observed. Rosen et al. (30)
recently showed that C/EBP
induces adipogenesis through PPAR
and
has no ability to promote adipocyte differentiation in the absence of
PPAR
, strongly indicating that the adipocytes developed through PPAR
as a unified pathway and that PPAR
seems to be the proximal effector of adipogenesis. It is likely that both TCL/TC10
L and RGS2
are involved in this PPAR
-linked signaling pathway, and this is one
reason that neither gene has an effect on the expression of C/EBPs.
Sakaue et al. (31) reported the requirement of fibroblast
growth factor 10 (FGF10) in the development of white adipose tissue. By
experiments using anti-FGF10, and also FGF10 knockout mice and
embryonic fibroblasts, they showed that FGF10 contributes to the
expression of C/EBP
through an autocrine/paracrine mechanism. Therefore, it seems that this signaling pathway may be different from
the TCL/TC10
L and/or RGS2 pathway, although it is finally linked to
a unified pathway through PPAR
.
The functional role of PPAR
ligand in the adipogenesis of
TCL/TC10
L-expressing NIH-3T3 cells remains to be investigated. There
are at least two possibilities. One is that the endogenous PPAR
, activated by the added ligand, enhances the functions of TCL/TC10
L and RGS2 and also activates the PPAR
pathway. The other
possibility is that the ligand BRL49653 binds and activates different
receptor proteins.
There is no direct evidence of a relationship between the signaling
pathways through TCL/TC10
L and RGS2 at this moment. Therefore, we
newly isolated the genes induced to expression by TCL/TC10
L, and found that the expression patterns of several were similar to that
of TCL/TC10
L. It is not clear that these genes are the real targets
of TCL/TC10
L signaling, because we do not have any experimental
evidences as to their potential function in relation to TCL/TC10
L.
Especially, we have no information on the relationship between the Rho
GTPase activity of TCL/TC10
L and these isolated genes. However, it
is of interest that Ras p21 protein activator 3 was identified as a
GTPase-activating protein modulating Ras activity during normal
brain development (32). A cellular nucleic acid-binding protein (Cnbp)
is a zinc finger DNA-binding protein (33), and a cellular thyroid
hormone-binding protein (CTHBP) is known to be involved in insulin
action (34). The further characterization of these genes would help us
to understand the signaling pathway at the early stage of adipocyte
differentiation. The relationship between TCL/TC10
L activity (Rho
GTPase activity) and the early stage of adipocyte differentiation is
also still unclear. Further studies, including experiments using
constitutively active and/or dominant negative mutants of TCL, and also
transient inhibition at the various time points using the RNA
interference method, are definitely needed to determine whether
TCL/TC10
L has a crucial role during the early stage of adipogenesis.