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INTRODUCTION |
Obesity is recognized as a worldwide public health problem that
contributes to a wide range of disease conditions (1, 2). Accumulating
evidence derived from both clinical and experimental observations
highlight the association of obesity with a number of chronic diseases
such as type II diabetes, atherosclerosis, restrictive lung disease,
cancer, and degenerative arthritis. Furthermore, there is growing
recognition that a greater understanding of mechanisms regulating fat
cell differentiation and the modulation of specific genes in the
adipocyte may allow for novel strategies to limit obesity and its
attendant consequences (1, 2).
Current models of the transcriptional basis for adipocyte
differentiation highlight an interplay among members of several major
families, the CCAAT/enhancer-binding protein
(C/EBP),1 the peroxisome
proliferator-activated receptor (PPAR) family, and the
basic-helix-loop-helix protein ADD1/SREBP1c (reviewed in Refs. 3 and
4). Studies to date suggest that C/EBP
and C/EBP
induce PPAR
,
which in turn initiates the adipogenic program (3, 4). Both gain and
loss of function experiments argue strongly that PPAR
is necessary
and sufficient to promote fat cell differentiation (5, 6). C/EBP
has
also been shown to play a critical role in certain aspects of
adipogenesis (7, 8). Recent studies using C/EBP
null fibroblasts
suggest that the critical role of this factor is to induce and maintain
PPAR
levels as well as to confer insulin sensitivity to adipocytes (7). Finally, ADD1/SREBP1c can also promote adipogenesis. This probably
occurs through several mechanisms including direct stimulation of
PPAR
expression as well as production of the endogenous ligand that
activates PPAR
(9-11).
Krüppel-like factors are zinc finger proteins that constitute an
important class of transcriptional regulators. Members of this family
are characterized by multiple zinc fingers containing regions with
conserved sequences
CX2CX3FX5LX2HX3H
(X is any amino acid; underlined cysteine and histidine
residues coordinate zinc) (12). The zinc fingers are usually found at
the C terminus of the protein and bind to the consensus sequence
5'-CNCCC'. The N terminus is involved in transcriptional activation
and/or repression as well as protein-protein interaction (12). Previous
studies show that Krüppel-like proteins typically regulate
critical aspects of cellular differentiation and function in diverse
cell types (13-16). A role for this gene family in adipocyte biology
was recently demonstrated by studies from our laboratory showing that
KLF15 is highly expressed in adipose tissue and can induce GLUT4
expression (17). Upon further investigation, we found that
KLF2/LKLF was also highly expressed in white and brown adipose
tissue in mice. However, in contrast to KLF15, KLF2/LKLF is
expressed in preadipocytes but not in mature adipocytes. In this report
we provide evidence that KLF2 can function as a negative regulator of
adipogenesis via inhibition of PPAR
.
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MATERIALS AND METHODS |
Cell Culture--
Adipocyte differentiation using 3T3L1 cells
was performed as described previously (18). Cells were cultured in 10%
FBS/DMEM until 2 days post-confluence (day 0). Medium was changed to
10% FBS/DMEM supplemented with 0.5 mM MIX
(3-isobutyl-1-methyl-xanthine, 1 µM dexamethasone, and
1.7 µM insulin). After 48 h (day 2), medium was
changed to 10% FBS/DMEM supplemented with 0.25 M
insulin used at day 0. The media of the cultured cells were
changed every 48 h during the experiment. Insulin was removed from
the maintenance medium on day 4.
For retroviral studies, KLF2 cDNA was cloned into the retroviral
vector of green fluorescent protein (a gift of K. Murphy), and
retrovirus was generated as described previously (19). For infection of
target cells, retroviral supernatant and 10% FBS/DMEM culture medium
were mixed at 1:1 ratio and added to cells along with 4 µg/ml
polybrene. Within 24-48 h, nearly 100% infectivity was noted by
assessment for green fluorescent protein. Differentiation of cells was
then performed as described above.
Oil Red-O Staining--
Oil Red-O staining of intracellular
lipid droplets was performed on plates overexpressing empty virus (EV),
KLF2, and KLF15 as described previously (8).
RT-PCR in Human Preadipocytes and Adipocytes--
Human
preadipocyte and adipocyte mRNA were kindly provided by Dr. H. Hauner (Dusseldorf, Germany), and RT-PCR was performed as
described previously (20, 21). The primer sequences used for KLF2 were
sense 5'-CACCGACGACGACCTCAACA-3' and antisense
5'-CGCACAGATGGCACTGGAAT-3' (Invitrogen). The primer sequences for
PPAR
were sense 5'-CCAGCATTTCTACTCCACATTA-3' and antisense
5'-TCGTTCAAGTCAAGATTTACAA-3' (Invitrogen). The primer sequences for
GLUT4 were sense 5'-CGCAGAGAGCCACCCCAGGAAA-3' and antisense
5'-GGGTGACGGGGTGGACGGAGAG-3' (Invitrogen). For Sp1, the primer
sequences were sense 5'-GTTCAGAGCATCAGACCCCTC-3' and antisense
5'-GAGAGTGGCTCACAGCCTGTC-3' (Invitrogen) (21).
Generation of Promoter and Site-directed Mutagenesis
Constructs--
The PPAR
2 promoter was kindly provided by J. K. Reddy (Northwestern University, Chicago, IL). This plasmid was used
as a template for the generation of the
250-bp Luc construct using the following primers: sense 5'-CGGGGTACCTTGTAATGTACCAAGTCTTG-3' and
antisense 5'-GGAAGATCTCATAACAGCATAAAACAGAG-3'. The PCR products were
digested with KpnI and BglII and ligated into the
KpnI and BglII site of the luciferase expression
vector pGL2-basic (Promega). The KLF binding site of PPAR
2-250-bp
Luc was mutated using the QuikChange mutagenesis kit according to
manufacturer's recommendations (Stratagene). This mutation changed the
wild-type sequence 5'-CCCACCTCTCCCA-3' to
5'-GTGACCTCTGTGA-3'.
Transient Transfection and Luciferase Assay--
For the
transfection experiment, 3T3-L1 cells were plated in 6-well dishes at a
density of 1 × 106 cells/well, and transient
transfections were performed the following day using FuGENE 6 transfection reagent (Roche Molecular Biochemicals) according to
manufacturer's recommendations. The total amount of DNA used for the
transfection assay per well was always held constant to 1 µg. The
KLF2 expression plasmid was kindly provided by J. Leiden (Abbott
Laboratories, Chicago, IL). Luciferase and
-galactosidase assays
were carried out as described previously (16). Promoter activity of
each construct was expressed as the ratio of luciferase/
-galactoside
activity. All transfections were performed in triplicate from three
independent experiments.
Electrophoretic Mobility Shift Assays--
Gel mobility shift
assays using the GST-KLF2 fusion protein were performed as described
previously (22). Modifications of this procedure included a
reduction in the amount of PMSF to 200 µg/ml. The radioactive
wild-type probe was ~10,000 cpm, and each reaction tube contained 1 µl of rabbit preimmune serum containing dextran (for enhanced
binding). Competition studies were performed using 10×, 100×, and
1000× wild-type and mutant competitor. The sequence for the mutant
competitor is the same as that used for the mutated primers above. For
supershift studies, the anti-KLF2 antibody was kindly provided by J. Leiden (Abbott Laboratories).
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RESULTS |
Expression of KLF2 in Adipose Tissue--
We recently reported
that a member of the KLF family termed KLF15 is expressed in mature
adipocytes and regulates GLUT4 (17). In the course of these studies, we
assessed the expression of several other KLFs in adipose tissue. KLF1
(erythroid KLF) and KLF4 (Guit KLF) mRNA were barely
detected in mouse adipose tissue (Fig.
1A) (data not shown). However,
as shown in Fig. 1A, KLF2 mRNA was strongly expressed in
both white and brown adipose tissues. Given the availability of cell
lines that faithfully recapitulate adipogenesis in vitro, we
assessed the expression of KLF2 during 3T3-L1 differentiation (23).
Stimulation of these cells with an empirically derived
prodifferentiation mixture leads to a characteristic pattern of
induction for various transcription factors and target genes involved
in adipogenesis and lipogenesis. We observed that KLF2 mRNA was
expressed in 3T3-L1 preadipocytes and markedly diminished upon
adipocyte differentiation (Fig 1B). As expected, other
factors such as PPAR
, members of the C/EBP family, and adipocyte
fatty acid-binding protein were induced with 3T3-L1
differentiation. The expression pattern of KLF2 was most similar to two
other transcription factors that have been shown to inhibit
adipogenesis, termed GATA3 and 2 (Fig 1B) (data not shown)
(24). To verify this expression pattern in primary cells, we assessed
the expression of KLF2 in primary human preadipocytes and adipocytes by
RT-PCR (21). Similar to the KLF2 expression pattern in 3T3-L1 cells,
KLF2 is highly expressed in human preadipocytes and is markedly reduced
in mature adipocytes (Fig. 1C). Consistent with previous
studies, GLUT4 and PPAR
expression are induced with differentiation,
whereas Sp1 levels are minimally affected (Fig 1C).

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Fig. 1.
Expression of KLF2 in adipose tissue and
adipocytes. A, Northern analysis of KLF2, KLF4,
and KLF15 expression in selected mouse tissues. B, KLF2
expression during 3T3-L1 differentiation. 3T3-L1 cells were induced to
differentiate using hormonal agents as described under "Materials and
Methods." Cells were harvested at the indicated number of days of
post-induction, and total RNA was isolated and subjected to Northern
analysis using indicated probes. C, KLF2 expression in human
preadipocytes and adipocytes. Semi-quantitative RT-PCR was performed as
described under "Materials and Methods."
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Overexpression of KLF2 Inhibits Adipogenesis in Vitro--
The
reduced expression of KLF2 mRNA expression during 3T3-L1
differentiation suggested a potentially inhibitory role in
adipogenesis. To test this possibility directly, we retrovirally
overexpressed KLF2 and EV as a control in 3T3-L1 cells and induced
differentiation. In parallel, we also overexpressed KLF15, which has
been shown to promote 3T3-L1 differentiation. As shown in Fig.
2A, compared with EV-infected
cells, KLF2-overexpressing cells showed a decrease in intracellular
lipid accumulation as evidenced by reduced Oil-Red-O staining (Fig
2A). In contrast, KLF15-infected cells exhibited increased
lipid accumulation.

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Fig. 2.
Constitutive expression of KLF2 inhibits
adipocyte differentiation. A, effect of KLF2
overexpression on lipid accumulation in 3T3-L1 cells. 3T3-L1 cells were
retrovirally infected with EV, KLF2, and KLF15 and subjected to
Oil-Red-O staining to assess for lipid accumulation at day 4 differentiation. B, effect of KLF2 overexpression on
adipocyte gene expression. 3T3-L1 cells were retrovirally infected with
EV and KLF2 and differentiated as described under "Materials and
Methods." Total RNA was harvested at the indicated number of days
post-induction of differentiation and subjected to Northern analysis
using the indicated probes. C, effect of KLF2 and KLF15 on
PPAR expression. 3T3-L1 cells were retrovirally infected with EV,
KLF2, and KLF 15 and differentiated. Cells were harvested at the
indicated time points, and Northern analysis performed using the
indicated probes.
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To determine the mechanism by which KLF2 may inhibit 3T3-L1
differentiation, we considered several possibilities. In theory, inhibition of adipocyte differentiation may occur via inhibition of
factors that promote differentiation or induction of factors that
inhibit adipogenesis. With respect to the latter, we did not observe
any effect of KLF2 overexpression on known negative regulators of
adipogenesis such as GATA3 (24),
-catenin (25), Smad proteins (26),
c/EBP-homologous protein (27), or pref-1 (25) (data not shown).
Furthermore, overexpression of KLF2 did not significantly alter
mRNA levels of C/EBP
or C/EBP
, two upstream factors that
promote adipogenesis (Fig. 2B). In contrast, KLF2 overexpression potently inhibited PPAR
expression at all time points
tested (Fig. 2B). In addition, a strong inhibitory effect is
seen on the expression of ADD1/SREBP1c and C/EBP
, two positive regulators of PPAR
expression and adipogenesis. Finally, the expression of downstream gene characteristics of a mature adipocyte such as adipocyte fatty acid-binding protein, adipsin, and fatty acid synthase was also inhibited (Fig. 2B) (data not
shown). The inhibitory effect of KLF2 was specific because the other
family member expressed in adipose tissue, KLF15, did not inhibit
PPAR
expression (Fig. 2C).
KLF2 Inhibits the PPAR
Promoter--
Recent studies support a
central role for PPAR
in adipogenesis. Given the potent inhibitory
effect of KLF2 overexpression on PPAR
expression, we sought to
determine the mechanistic basis for this observation. We considered
several possibilities such as (a) direct inhibition of the
PPAR
promoter or (b) inhibition of regulatory factor(s)
that can induce PPAR
expression. With respect to the second
possibility, we did observe a marked inhibition of two factors that
have been demonstrated to positively regulate PPAR
function,
C/EBP
and ADD1/SREBP1c (Fig. 2B). To assess the former
possibility, we tested whether KLF2 may directly inhibit the promoter
of PPAR
2, the major isoform in adipose tissue. Cotransfection of
KLF2, the
250-bp Luc-PPAR
2 promoter, resulted in an ~70% inhibition of promoter activity (Fig.
3A, left panel).
This inhibition was not seen with a mutant KLF2 construct consisting of
only the zinc finger DNA-binding domain. Consistent with previous
reports, members of the C/EBP family are able to transactivate the
PPAR
2 promoter (Fig. 3A, right panel) (28).
These data suggest that KLF2 inhibition of PPAR
2 may be mediated at
the level of the promoter within the
250-bp proximal promoter region.
An examination of this 250-bp region revealed the presence of a
two-tandem Krüppel binding site,
5'-CCCACCTCTCCCA-3' (base pairs
82 to >93). To
determine whether KLF2 was able to bind this sequence, electrophoretic
mobility gel shift studies were performed. As shown in Fig.
3B, KLF2 is able to bind to this site (Fig. 3B, arrow), and the specificity of this interaction was
confirmed by competition with identical but not a mutated oligomer
(5'-GTGACCTCTGTGA-3'). Furthermore, this
DNA-protein complex was supershifted in the presence of an anti-KLF2
antibody (Fig. 3B, arrowhead) but not IgG. To
verify that this site was important in KLF2-mediated repression, we
mutated the KLF binding site within the context of the
250-bp Luc-PPAR
2 promoter. As shown in Fig. 3C, mutation of this site significantly attenuated the inhibitory effect of KLF2, suggesting that
this site is important for KLF2-mediated inhibition of the PPAR
2
promoter.

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Fig. 3.
KLF2 represses PPAR 2
promoter activity. A, effect of KLF2 on PPAR 2
promoter activity. 3T3-L1 cells were transfected with PPAR 2 promoter
( 250-bp Luc) and either KLF2 or ZnF expression construct (left
panel). 3T3-L1 cells were transfected with PPAR 2 promoter and
C/EBP and C/EBP expression constructs (right panel).
Luciferase and -galactosidase assays were performed as described
under "Materials and Methods." Results are expressed as fold
repression compared with vector (pCDNA3.1) alone (n = 6-10/group). *, p < 0.001 compared with the empty
vector. B, KLF2 binds to a CACCC element. Electrophoretic
mobility shift assays were performed as described under "Materials
and Methods." The GST-KLF2 fusion protein was incubated with a
32P-labeled wild-type oligomer extending from base pairs
82->93 of the PPAR 2 promoter. A single dominant DNA-protein
complex was seen (arrow). Competition and supershift studies
(arrowhead) were performed to verify specificity.
C, effect of mutation of the CACCC element on KLF2-mediated
inhibition of the PPAR 2 promoter. 3T3-L1 cells were transfected with
KLF2 and the PPAR 2 promoter ( 250-bp Luc and mutated 250-bp Luc).
n = 9/group; *, p < 0.001;
#, p < 0.001 statistically significant by
comparison to KLF2 on 250-bp Luc promoter).
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DISCUSSION |
Members of the Krüppel-like family of transcription factors
have been shown to play important roles in cellular differentiation and
growth (12). KLF2 was initially identified through a homology screening
strategy using the zinc finger domain of KLF1 as a probe (29). Studies
targeting KLF2 in mice have shown an essential role in embryonic
development as these animals die during mid-gestation because of
abnormal blood vessel development (30). In addition, T-cell maturation
(15) as well as lung development (31) is impaired. However, despite the
importance of KLF2, very few direct targets have been identified (22).
In this regard, the observations from this study may bear important
implications not only for adipocyte biology but also for the role of
KLF2 in other tissues.
In this report, we provide evidence that the Krüppel-like factor
KLF2 is a negative regulator of adipogenesis. Consistent with an
inhibitory role, KLF2 is expressed in preadipocytes and expression is
markedly reduced in mature adipocytes (Fig. 1). In addition,
overexpression of KLF2 markedly inhibits lipid accumulation in 3T3-L1
cells (Fig. 2). This ability is specific to KLF2, whereas another
member of this family, KLF15, augments lipid accumulation. To
understand the basis for the inhibitory effect of KLF2, we considered
several possibilities such as (a) inhibition of factors that
have been shown to positively regulate adipocyte differentiation or
(b) induction of factors identified as negative regulators of adipocyte differentiation. With respect to the latter possibility, we did not observe any effect on the expression of several previously identified negative regulators such as GATA3 (24),
-catenin (25),
Smad proteins (26), CHOP (27), or pref-1 (32). With respect to factors
that promote adipogenesis, KLF2 did not significantly affect the
expression of C/EBP
and C/EBP
, two upstream regulators of
adipogenesis. In contrast, KLF2 potently inhibits the expression of
PPAR
, the central regulator of adipogenesis. Given that PPAR
expression is essential for adipocyte development, it is probable that
this inhibition is critical for KLF2-mediated inhibition of adipocyte
differentiation. In addition to PPAR
, KLF2 also inhibits expression
of both C/EBP
and ADD1/SREBP1c, two factors that play important
roles in adipocyte differentiation. A recent study (8) suggests
that the principal role of C/EBP
is to induce and maintain PPAR
expression. As such, the inhibition of C/EBP
expression may
contribute to the reduced levels of PPAR
seen in KLF2-overexpressing
cells, thereby leading to the inhibition of adipocyte differentiation.
Furthermore, ADDI/SREBP1c has been shown to augment adipogenesis via
direct induction of PPAR
expression as well as through production of
an endogenous PPAR
ligand (9-11). The inhibition of ADD1/SREBP1c
expression by KLF2 may therefore lead to both a reduction in PPAR
expression and activation. Thus, KLF2 may arrest the adipogenic program
through inhibition of PPAR
expression and inhibition of factors that
positively regulate PPAR
expression and function.
The mechanism by which KLF2 is able to inhibit PPAR
expression has
yet to be fully elucidated. As suggested above, part of the mechanism
may be via the inhibition of factors such as C/EBP
and ADD1/SREBP
1c, which positively regulate PPAR
expression and function. However,
our data also suggest that KLF2 can inhibit PPAR
2 promoter activity,
thus supporting a direct mechanism. As shown in Fig. 3C,
KLF2 inhibited PPAR
2 promoter activity by ~70%. An examination of
the PPAR
2 promoter revealed the presence of a single consensus
Krüppel binding site. Gel shift studies demonstrate that KLF2 can
bind to this site (Fig. 3B), and promoter mutation analyses
support a role for this site in mediating the inhibitory effect of KLF2
(Fig. 3C). Importantly, however, the mutation of this site
alone was not sufficient to completely abrogate KLF2-mediated
inhibition of promoter activity, suggesting that non-DNA
binding-dependent mechanisms are probably involved for the
full inhibitory effect. It is also noteworthy that the KLF binding site
lies in between the two binding sites identified by Tong et
al. (24) as essential for GATA-mediated inhibition of the PPAR
2
promoter activity. The proximity of these two sites raises the
possibility of a cooperative interaction between these two factors and
is the subject of future investigations.
Our current understanding of the transcriptional basis for adipogenesis
highlights the function of several major positive and negative
regulatory families such as the C/EBPs, PPARs, and GATA factors. We
recently reported (17) a role for the Krüppel-like factor
KLF15 in adipocyte biology by virtue of its ability to induce the
insulin-sensitive glucose transporter GLUT4. In this report, we provide
a novel function for another KLF family member, KLF2, as a negative
regulator of adipogenesis. Taken together, these studies support a role
for the Krüppel-like factors as potential regulators of
adipogenesis. Future studies evaluating these factors in
vivo will help elucidate more precisely the role of
Krüppel-like factors in adipocyte biology.