Krüppel-Like Factor KLF8 Plays a Critical Role in Adipocyte Differentiation
Citation: Lee H, Kim HJ, Lee YJ, Lee M-Y, Choi H, et al. (
Kru¨ ppel-Like Factor KLF8 Plays a Critical Role in Adipocyte Differentiation
Haemi Lee 0 1
Hyo Jung Kim 0 1
Yoo Jeong Lee 0 1
Min-Young Lee 0 1
Hyeonjin Choi 0 1
Hyemin Lee 0 1
Jae-woo Kim 0 1
Michael Mu¨ ller, Wageningen University, The Netherlands
0 Current address: Joslin Diabetes Center , Boston, Massachusetts , United States of America
1 Department of Biochemistry and Molecular Biology, Integrated Genomic Research Center for Metabolic Regulation, Institute of Genetic Science, Yonsei University College of Medicine , Seoul , Korea , 2 Brain Korea 21 Project for Medical Science, Yonsei University , Seoul , Korea , 3 Department of Integrated OMICS for Biomedical Sciences, WCU Program of Graduate School, Yonsei University , Seoul , Korea
KLF8 (Kru¨ ppel-like factor 8) is a zinc-finger transcription factor known to play an essential role in the regulation of the cell cycle, apoptosis, and differentiation. However, its physiological roles and functions in adipogenesis remain unclear. In the present study, we show that KLF8 acts as a key regulator controlling adipocyte differentiation. In 3T3-L1 preadipocytes, we found that KLF8 expression was induced during differentiation, which was followed by expression of peroxisome proliferator-activated receptor c (PPARc) and CCAAT/enhancer-binding protein a (C/EBPa). Adipocyte differentiation was significantly attenuated by the addition of siRNA against KLF8, whereas overexpression of KLF8 resulted in enhanced differentiation. Furthermore, luciferase reporter assays demonstrated that overexpression of KLF8 induced PPARc2 and C/ EBPa promoter activity, suggesting that KLF8 is an upstream regulator of PPARc and C/EBPa. The KLF8 binding sites were localized by site mutation analysis to 2191 region in C/EBPa promoter and 2303 region in PPARc promoter, respectively. Taken together, these data reveal that KLF8 is a key component of the transcription factor network that controls terminal differentiation during adipogenesis.
Funding: This work was supported by a grant from the Korea Health Technology R&D Project, Ministry of Health & Welfare, Republic of Korea (A100475). The
funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
. These authors contributed equally to this work.
Obesity is the main cause of metabolic syndrome and leads to
various complications, including an increased risk of diabetes and
cardiovascular diseases [
]. Obesity is characterized by increased
adipose tissue mass due to increased adipocyte number
(hyperplasia) and increased adipocyte size (hypertrophy) [
number of adipocytes is determined to a large degree by the
adipocyte differentiation process, which generates mature
adipocytes from fibroblast-like preadipocytes. Therefore, understanding
the regulatory processes involved in adipocyte differentiation may
help to limit obesity and its pathological consequences.
Adipocyte differentiation is influenced by endocrine and
autocrine factors that promote or constrain adipogenesis by
intracellular mechanisms that induce the synthesis and activation
of adipogenic transcription factors [
]. Murine 3T3-L1 cells
differentiate into mature adipocytes when treated with
serumcontaining medium supplemented with 1-methyl
3-isobutylxanthine, dexamethasone, and insulin. After hormonal induction,
growth-arrested 3T3-L1 preadipocytes reenter the cell cycle for
additional two rounds of division, known as mitotic clonal
expansion, and then express genes required for the adipocyte
]. Several transcription factors are involved in
adipocyte differentiation. These include
CCAAT/enhancer-binding proteins (C/EBPs) [
] and peroxisome proliferator-activated
receptor c (PPARc) [
]. Both C/EBPb and C/EBPd are
induced immediately [
] and stimulate cell proliferation and
expression of PPARc and C/EBPa [
]. PPARc and C/EBPa
serve as pleiotropic transcriptional activators that coordinately
induce the expression of adipocyte-specific genes that lead to
formation of a mature adipocyte.
Members of the Kru¨ ppel-like factor (KLF) family of
transcription factors are important regulators of development, growth,
differentiation, and a number of other physiological cellular
]. The KLF family is composed of at least 17
transcription factors that share homology in their tandem three
C2H2 zinc-fingers near their C-terminus [
]. In adipogenesis,
KLF family transcription factors act as both activators and
repressors in the transcriptional cascade. KLF5, for example, is
induced at an early stage of differentiation and activates the
PPARc2 promoter in concert with C/EBPb [
KLF4 acts as a regulator at an earlier stage of differentiation by
binding to the C/EBPb promoter together with Krox20, thereby
inducing adipocyte differentiation [
]. In contrast, KLF3
represses adipocyte differentiation by recruiting C-terminal
binding protein (CtBP) corepressors [
]. Other KLF family
proteins have also been proven to promote or inhibit adipocyte
In the present study, we analyzed the expression patterns of
KLF family proteins during 3T3-L1 differentiation using
microarray and found that KLF8 was significantly induced after the
mitotic clonal expansion period. Adipocyte differentiation was
significantly attenuated by KLF8 knockdown, whereas
overexpression of KLF8 resulted in enhanced differentiation.
Furthermore, overexpression of KLF8 induced PPARc2 and C/EBPa
transcriptional activities, as shown by luciferase assay, suggesting
that KLF8 is an upstream regulator of PPARc and C/EBPa.
These results indicate that KLF8 plays an important role in early
Materials and Methods
Cell Isolation from Mice and Ethic Statement
The stromal vascular fraction (SVF) and fat fraction were
isolated from mouse epididymal fat pads of 12-week-old mice by
digestion with type I collagenase as described [
]. All procedures
were approved by the Committee on Animal Investigations of the
Cell Culture and in vitro Differentiation
Methods for maintenance and induction of differentiation of
3T3-L1 preadipocytes have been described previously [
Briefly, 3T3-L1 preadipocytes were maintained in Dulbecco’s
modified Eagle’s medium (DMEM) containing 100 U/ml
penicillin, 100 mg/ml streptomycin, and 8 mg/ml biotin, supplemented
with 10% heat-inactivated calf serum at 37uC, in an atmosphere of
90% air and 10% CO2. To induce differentiation, 2-day
postconfluent 3T3-L1 cells (designated day 0) were incubated in
DMEM containing 10% FBS, 0.5 mM
3-isobutyl-1-methylzanthine, 1 mM dexamethasone, and 1 mg/ml of insulin (MDI)
for 2 days. Cells were then cultured in DMEM containing 10%
FBS and insulin for another 2 days, after which they were grown
in DMEM containing 10% FBS. Cell numbers were determined
on day 2, and oil red-O staining was performed on day 8.
Western Blot Analysis
At each time indicated, cells were washed in ice-cold
phosphatebuffered saline (PBS) and lysed in a buffer containing 1% SDS and
60 mM Tris-Cl, pH 6.8. Lysates were briefly vortexed, boiled for
10 min, and cleared by centrifugation at 12,000 g for 10 min at
4uC. The supernatants were collected, and protein concentrations
were measured using a BCA assay kit (Pierce). Protein samples of
equal amount were separated by SDS-PAGE and transferred to
nitrocellulose membranes. Anti-KLF8 rabbit polyclonal antibody
was generated against the murine KLF8 peptide (residues 5–16,
IDNMDVRIKSES) by Atigen (Korea). Immunoblot analyses were
performed using the following antibodies: polyclonal antibody
against KLF8, C/EBPa [
], C/EBPb [
], mouse monoclonal
antibodies against PPARc (Santa Cruz Biotechnology), FLAG
antibody (Sigma), and b-actin antibody (Santa Cruz
Biotechnology). The immunoreactive bands were detected using an enhanced
chemiluminescence detection system (Amersham) following the
RNA Isolation and Real-time RT-PCR
Total RNA was isolated from cultured cells using TRIzol
(Invitrogen) according to the manufacturer’s instructions. For
quantitative RT-PCR, cDNA was synthesized from 5 mg of total
RNA using random hexamer primers and SuperScript reverse
transcriptase II (Invitrogen), following the manufacturer’s
instructions. An aliquot (1/40) of the reaction was used for quantitative
PCR using the SYBR Green PCR Master Mix (Applied
Biosystems) and gene-specific primers. RT-PCR products were
quantified using the ABI PRISM 7300 RT-PCR System (Applied
Biosystems). RT-PCR was performed using the following primers:
KLF8-sense, 59-CAAGC CATTA TGGTG CCTAC-39;
KLF8antisense, 59-ATAGA GCCCG GAGTG AGAAC-39. All
reactions were performed in triplicate. The relative amounts of the
mRNA were calculated using the comparative cycle-time method
(Applied Biosystems). GAPDH mRNA was also measured as an
Small Interfering RNA (siRNA)
Preadipocytes (3T3-L1) were plated into 60-mm-diameter
dishes 18–24 h prior to transfection. Cells were transfected with
control or gene-specific siRNA at 50 nM (Dharmacon) in
OPTIMEM medium using Lipofectamine RNAiMAX (Invitrogen),
according to the manufacturer’s protocol. The next day, the
medium was replaced with fresh DMEM containing 10% calf
serum and the cells were incubated for 24 h before the induction
of differentiation. Total RNA and protein extracts were prepared
from the cells at the indicated time points, and RT-PCR and
immunoblot analyses were performed. Oil red-O staining of KLF8
knockdown was performed at day 8. The siRNA sequences are as
follows: si-C/EBPb, 59-AGUAG AAGUU GGCCA CUUCC
AUGGG-39; si-KLF8a, 59-UGAAG UAGGC ACCAU AAUGG
CUUGA -39; and si-KLF8b, 59-UCAAG CCAUU AUGGU
GCCUA CUUCA-39. We used the Stealth RNA siRNA negative
control (Invitrogen) as si-RNA control.
Transient Transfection Assay
KLF8 overexpressing vector (pcDNA3.0-KLF8-FLAG) was
generated by inserting the whole open reading frame of mouse
KLF8 with a C-terminal FLAG tag into pcDNA3.0 (Invitrogen).
To maximize the transfection efficiency, microliter volume
electrophoration of 3T3-L1 preadipocytes was performed with
OneDrop MicroPorator MP-100 (Digital Bio). The cells were
trypsinized, washed with 16 PBS, and finally resuspended in 10 ml
of resuspension buffer R and 0.5 mg of plasmid at a concentration
of 200,000 cells per pipette. The cells were then microporated at
1,300 V, with a 20-ms pulse width, 2 pulses. Following
microporation, the cells were seeded in 35-mm cell culture dishes
and placed at 37uC in a 10% CO2-humidified atmosphere. For
luciferase assays of the promoter constructs, Lipopfectamin and
Plus Reagent (Invitrogen) was used. Briefly, NIH3T3 cells were
cultured at a density of 2.56105 cells/well in DMEM. The next
day, cells were transfected with the indicated luciferase reporter
plasmids using Lipofectamine and Plus Reagent following the
manufacturer’s instructions. After 3 h of incubation, the cell
medium was replaced with fresh complete medium. After 48 h of
incubation, the cells were washed with PBS and harvested in
200 ml of passive lysis buffer (Promega). The cells were mixed
vigorously for 15 s and centrifuged at 12,000 g for 10 min at 4uC.
The supernatants were transferred into a fresh tube, and 5-ml
aliquots of the cleared whole-cell lysate were assayed for luciferase
activity using a Dual-Luciferase Reporter Assay System (Promega).
Each transfection experiment was performed in triplicate.
PCR amplification of the wild-type luciferase reporter plasmid
was performed using site-directed mutation primers
(C/EBPamtC/EBP-sense 59-AGCGC AGGAG TCAGT GGGCG TTGat
aCACG ATCTC-39, C/EBPamt-C/EBP-antisense 59-GAGAT
CGTGt atCAA CGCCC ACTGA CTCCT GCGCT-39; C/
EBPamt-KLF-sense 59-AGCGC AGGAG TCAGT GGtgt
TTGCG CCACG ATCTC-39, C/EBPamt-KLF-antisense
59GAGAT CGTGG CGCAA acaCC ACTGA CTCCT
GCGCT39, PPARc2mtKLF-sense 59-AACTA CTGTA CAGTT acaGC
CCCTC ACAGA-39, PPARc2mt-KLF-antisense 59-TCTGT
GAGGG GCtgt AACTG TACAG TAGTT-39). The substituted
bases are indicated in small case letter. PCR amplification was
performed using 50 ng template DNA and 15 cycles of 95uC for
1 min, 55uC for 1 min and 72uC for 7 min. PCR products were
digested with DpnI for 2 h at 37uC, prior to transformation into
Escherichia coli DH5a competent cells. Colonies were screened by
Electrophoretic Mobility Shift Assays (EMSA)
Briefly, pcDNA3.0-C/EBPb-FLAG and KLF8-FLAG were
used for in vitro-translation reaction using TNT T7 quick master
mix (Promega). EMSAs were performed using in vitro-translated
protein as previously described [
]. Double-stranded probes were
labeled with [c-32P]ATP, using T4 polynucleotide kinase.
ProteinDNA complexes were resolved from the free probe by
electrophoresis on a 4%(wt/vol) polyacrylamide gel in 0.256TBE buffer.
The dried gels were exposed to X-ray film with an intensifying
screen. Probe sequences are as follows; b-globin, sense 59-TAGAG
CCACA CCCTG GTAAG-39, antisense 59-CTTAC CAGGG
TGTGG CTCTA-39, C/EBPa, sense 59-AGCGC AGGAG
TCAGT GGGCG TTGCG CCACG-39, antisense 59-CGTGG
CGCAA CGCCC ACTGA CTCCT GCGCT-39, PPARc2, sense
59-CTGTA CAGTT CACGC CCCTC ACAGA-39, antisense
59TCTGT GAGGG GCGTG AACTG TACAG-39. Also, mutated
probe sequences were used as described in site-directed
Chromatin Immunoprecipitation (ChIP) Assays
ChIP analysis was performed following the protocol of the ChIP
assay kit (Upstate). DNA-protein complexes were
immunoprecipitated with antibodies against C/EBPb and KLF8 for 4 h and then
collected with protein A-agarose for 3 h at 4uC with rotation. The
beads were washed, and chromatin complexes were eluted from
the beads. After reversal of the cross-links, the DNA was purified.
Input control and ChIP samples were used as PCR templates to
amplify the PPARc and C/EBPa promoters containing the C/
EBP and KLF8 binding sites using the following primers: C/
EBPa, the region from 2335 to 283 was amplified, sense
59TCCCT AGTGT TGGCT GGAAG-39, antisense 59-CAGTA
GGATG GTGCC TGCTG-39, PPARc2, the region from 2618
to 2119 was amplified, sense 59-ATTTA AATTT TACTA
GCCTT-39, antisense 59- GACAA AATGG TGTGT
All results are expressed as mean 6 SD. Statistical comparisons
of groups were made using an unpaired Student’s t test and
KLF8 is Induced After Mitotic Clonal Expansion in 3T3-L1
In order to analyze the roles of KLF family proteins in
adipogenesis, we conducted microarray during 3T3-L1 cell
differentiation induced by the standard hormone cocktail (MDI)
(Table 1). In this analysis, it is remarkable that KLF8, KLF9,
KLF12, and KLF17 were induced during adipocyte
differentiation. Interestingly, the expression pattern of KLF8, KLF12, and
KLF17 were similar, as they reached peak expression at day 2
after differentiation induction, suggesting that these KLFs may
play roles in terminal differentiation during adipogenesis.
To confirm the microarray data, the expression levels of these
KLFs were analyzed by RT-PCR at different time points. As
shown in Figure 1A, the amount of KLF8 mRNA was increased
during 3T3-L1 differentiation, reaching its maximum at 36–48 h
after induction. In contrast, the mRNA level of KLF5 was elevated
at an earlier point during differentiation. Similar to KLF8, KLF17
also was upregulated at the mRNA level during 3T3-L1
differentiation. Contrary to the microarray data, however,
KLF12 was not found to be induced during differentiation by
RT-PCR. Therefore, we focused on the potential roles of KLF8
and KLF17 in the differentiation program of adipogenesis. We
generated polyclonal anti-KLF8 antibody (Figure 1B) and verified
the increase of KLF8 protein during adipocyte differentiation
(Figure 1C), suggesting that KLF8 is involved in the terminal
differentiation of adipocytes. It is interesting to note that KLF8
was mainly expressed in the stromal vascular fraction (SVF)
compared to the fat fraction (Figure 1D). The SVF is known to
have many adipocyte progenitor cells [
], whereas the fat fraction
contains mature adipocytes. This suggests that KLF8, like KLF5,
which is also expressed in the SVF, is not required for
maintenance of the mature adipocyte phenotype, but instead
plays a role in the differentiation of adipocytes.
KLF8, but not KLF17, is Necessary for Adipocyte
Differentiation in 3T3-L1 Preadipocytes
To confirm the function of KLF8 directly, we observed the
effect of KLF8 knockdown in 3T3-L1 preadipocytes. Two
KLF8specific siRNAs (designated as si-KLF8a and b) were used in
3T3L1 cell transfection prior to hormonal induction of differentiation.
The mRNA levels of KLF8 decreased approximately to 75% and
40% by si-KLF8a and b, respectively, compared to control siRNA
(Figure 2A). Importantly, oil red-O staining on day 8 showed that
KLF8 siRNA significantly diminished the accumulation of lipid
droplets (Figure 2B). Moreover, the expression levels of PPARc
and C/EBPa were effectively suppressed by KLF8 siRNA during
differentiation compared to expression levels in the control siRNA
group, whereas C/EBPb expression was relatively unaffected
(Figure 2C). This suggests that KLF8 acts as an upstream regulator
of C/EBPa and PPARc, independent of C/EBPb. Meanwhile,
although the expression of KLF17 was induced during
differentiation, knockdown of KLF17 did not affect 3T3-L1 cell
differentiation (data not shown).
While expression of C/EBPb increases after induction of
differentiation, it has been reported that the upregulation of C/
EBPa is delayed [
], suggesting the presence of another regulator
between the two transcription factors. Because C/EBPb is also
required for mitotic clonal expansion [
], knockdown of C/EBPb
resulted in the inhibition of cell proliferation (Figure 2D).
Meanwhile, cell numbers at day 2 indicate that knockdown of
KLF8 did not inhibit mitotic clonal expansion to the level
observed in si-C/EBPb (Figure 2D), suggesting that the role of
KLF8 is downstream of C/EBPb and mitotic clonal expansion.
This is consistent with the result that expression of KLF8 increases
at 36–48 h after the first round of division during clonal expansion
(Figure 1A). Thus, KLF8 might not affect cell cycle or the
expression of C/EBPb but play a critical role in terminal
differentiation during adipogenesis.
We next examined whether overexpression of KLF8 might
induce adipogenesis in 3T3-L1 cells. KLF8 was overexpressed in
3T3-L1 preadipocytes by microporation prior to hormonal
induction of differentiation. As expected, KLF8 overexpression
increased the accumulation of lipid droplets (Figure 3A). In
addition, PPARc and C/EBPa were induced earlier in
KLF8overexpressing cells than in control cells, whereas no difference
was observed in the induction of C/EBPb (Figure 3B). Thus, it
appears that KLF8 is able to induce the expression of PPARc and
KLF8 Directly Controls and Binds to the PPARc2 and C/
Because we found that KLF8 plays an important role in PPARc
and C/EBPa expression, we next examined whether KLF8
directly controls transcription of these genes. To clarify which
region is responsible for KLF8 transactivation, serial deletion
constructs of a 2450 fragment of the C/EBPa promoter were
]. KLF8, similar to C/EBPb, significantly stimulated C/
EBPa promoter activity in reporter assays (Figure 4A). This
activation was almost completely abolished when the minimal
proximal promoter (C/EBPa-89) was used (Figure 4A), indicating
that KLF8 plays a critical role in C/EBPa promoter activity and
the direct binding site exists between 2205 and 289 bp in the C/
EBPa promoter. Also, we found that KLF8 stimulated PPARc
promoter activity directly (Figure 4B). Sequence analysis of the C/
EBPa promoter revealed two potential binding sites for KLF
family members, the consensus sequence for which has been
identified as CNCCC [
]. Of these, site-specific mutation of the
2191 KLF site abolished luciferase activity driven by KLF8
overexpression (Figure 4C). It should be noted that the KLF8
binding site is a GC-rich region right next to the C/EBP
regulatory element [
]. The overexpression of both C/EBPb and
KLF8 resulted in the activation of the C/EBPa-205-luciferase
construct in a partially additive manner, not synergistically.
Consistently, the mutation study revealed that C/EBPb and
KLF8 bind to the C/EBPa promoter separately, without affecting
the action of the other (Figure 4C). Similarly, KLF8 site was
localized to 2303 region of the PPARc2 promoter, which has
been identified as a KLF5 binding site previously [
] (Figure 4D).
Mutation of this sequence also abolished KLF8-driven
transactivation of the promoter, without affecting C/EBPb action.
The direct binding of KLF8 to the promoters was also
confirmed by EMSA. As shown in Figure 5A, in vitro translated
KLF8 bound to 2191 region of the C/EBPa promoter and 2303
region of the PPARc2 promoter, respectively. A known sequence
of the b-globin promoter was shown as a positive control. As
mentioned above, 2191 region of the C/EBPa promoter has an
interesting feature that KLF8 and C/EBPb binding sites exist in a
short sequence. Thus, we tested whether KLF8 and C/EBPb bind
separately using mutated probes. The Figure 5B and 5C show that
KLF8 or C/EBPb binding was not basically affected by each other
factor. This is consistent with the luciferase result: KLF8 and C/
EBPb transactivate the promoter in an additive manner
To further confirm the role of KLF8 on these promoters, we
next performed ChIP to analyze the binding of KLF8 to these
target genes. Chromatin samples were prepared from 3T3-L1 cells
before (day 0) and after the induction of adipocyte differentiation
(days 1 and 2) and were then immunoprecipitated with a
KLF8specific antibody. We found that KLF8 bound to these promoters
(day 1) and the strength of binding increased further after 2 days of
MDI treatment (Figure 5D). Anti-C/EBPb and anti-IgG were
used as positive and negative control, respectively. Taken together,
the results of this study implicate KLF8 as a key component of the
transcription factor network that controls the regulation of C/
EBPa and PPARc during adipogenesis.
In this study, we showed that KLF8 expression increased at
both the mRNA and protein level during adipocyte differentiation
in 3T3-L1 cells. In addition, KLF8 controlled the expression of
PPARc and C/EBPa by directly binding to the promoter regions
but had no effect on C/EBPb. These results suggest that KLF8 is a
key intermediate component of the transcription factor network
Previous studies identified that differentiation of the 3T3-L1
preadipocyte cell line into adipocytes requires sequential activation
of various transcription factors, including C/EBPb, C/EBPd,
PPARc, and C/EBPa [
]. C/EBPb and C/EBPd are expressed in
the early stage of adipogenesis (at ,4 h), whereas the activation of
the C/EBPa or PPARc gene is initiated ,36 h after
differentiation is induced. Therefore, it is suggested that additional events
are required for the terminal differentiation. The
phosphorylationdependent activation process of C/EBPb by MAPK and GSK3b
], as well as redox control of DNA-binding  were
suggested for those events. In addition, we also reported that
upstream stimulatory factors are required for the full activation of
C/EBPa promoter [
Meanwhile, the involvement of KLF family proteins provides
another example for the completion of transcriptional cascade in
adipogenesis. KLF family proteins are known to play diverse roles
in cell differentiation and development in mammals. Although
KLF proteins exhibit homology in their carboxyl-terminal zinc
finger domains, different amino-terminal sequences provide
unique regions for interaction with specific binding partners. It
is well established that KLF8 acts in critical cellular processes, such
as differentiation, cell cycle progression, transformation,
epithelialto-mesenchymal transition, migration, and invasion [
]. In the
present study, we first report the involvement of KLF8 in the
transcriptional cascade of adipocyte differentiation. Importantly,
KLF8 was expressed at ,36 h just prior to the activation of C/
EBPa and PPARc promoter. In addition, KLF8 overexpression in
3T3-L1 cells strongly upregulated PPARc and C/EBPa
expression by directly binding to these gene promoters. Furthermore, our
analysis showed that the role of KLF8 is initiated after mitotic
clonal expansion, suggesting that KLF8 is a critical regulator of
terminal differentiation in adipogenesis.
We found that KLF8 binding site on the C/EBPa promoter is a
GC-rich region right next to the C/EBP regulatory element. This
GC-rich region has been analyzed thoroughly, demonstrating that
Sp1 occupies the GC-box, which prevents access of the C/EBP
protein in preadipocytes [
]. Upon differentiation stimuli, Sp1
level is decreased and C/EBP then binds to the regulatory region,
thereby activating the C/EBPa promoter activity [
]. In our
data, KLF8 and C/EBPb bind to and activate the C/EBPa
promoter, in an additive manner. We also tested whether these
two transcription factors interact with each other by
proteinprotein interaction; however, we could not observe any detectible
interaction by immunoprecipitation (data not shown). Therefore,
we suggest a possible mechanism involved in an activation process
of the C/EBPa promoter as follows; in preadipocytes, Sp1
expression is high enough to repress the C/EBPa promoter, by
competing the regulatory element with C/EBPb. When the
differentiation is induced, the cells down-regulate Sp1 and begin to
express C/EBPb and KLF8. These two transcription factors
occupy the critical regulatory elements of the C/EBPa promoter,
thereby leading to a steady expression of C/EBPa protein. It is
also possible that this combination of KLF8 and C/EBPb recruits
specific co-activators to the promoter region. In previous studies,
KLF8 has been found to recruit p300/CBP in order to activate
expression of the cyclin D1 gene and promote acetylation of
nearby histones [
]. Post-translational modification of KLF8 via
SUMOlyation attenuates this ability [
]. Whether recruitment of
p300/CBP or post-translational modification of KLF8 are
involved in adipogenesis needs to be further investigated.
Recently, it was reported that the closely related family member
KLF3 is highly expressed in undifferentiated preadipocytes and
reduced upon differentiation into adipocytes [
]. Other study
demonstrated that KLF3 repressed expression of KLF8 in other
cell types [
], raising a possibility of network between KLF family
of proteins during adipocyte differentiation. In this regard, it is
possible that the activities of KLF3 and KLF8 are reciprocally
regulated during adipogenesis. Thus, it would be interesting to
investigate the KLF family protein network in adipogenesis,
including regulation of the KLF3-KLF8 axis.
In summary, the present study provides interesting evidence for
the pivotal role played by KLF8 in adipocyte differentiation.
Further studies of the mechanisms by which KLF8 expression and
function are regulated should provide additional insight into
Conceived and designed the experiments: Haemi Lee HJK MYL YJL
JwK. Performed the experiments: Haemi Lee HJK YJL MYL HC Hyemin
Lee. Analyzed the data: Haemi Lee HJK MYL YJL JwK. Wrote the paper:
Haemi Lee HJK MYL JwK.
1. Kopelman PG ( 2000 ) Obesity as a medical problem . Nature 404 : 635 - 643 .
2. Otto TC , Lane MD ( 2005 ) Adipose development: from stem cell to adipocyte . Crit Rev Biochem Mol Biol 40 : 229 - 242 .
3. Rosen ED , Spiegelman BM ( 2000 ) Molecular regulation of adipogenesis . Annu Rev Cell Dev Biol 16 : 145 - 171 .
4. MacDougald OA , Lane MD ( 1995 ) Transcriptional regulation of gene expression during adipocyte differentiation . Annu Rev Biochem 64 : 345 - 373 .
5. Tang QQ , Otto TC , Lane MD ( 2003 ) Mitotic clonal expansion: a synchronous process required for adipogenesis . Proc Natl Acad Sci U S A 100 : 44 - 49 .
6. Rosen ED , MacDougald OA ( 2006 ) Adipocyte differentiation from the inside out . Nat Rev Mol Cell Biol 7 : 885 - 896 .
7. Christy RJ , Kaestner KH , Geiman DE , Lane MD ( 1991 ) CCAAT/enhancer binding protein gene promoter: binding of nuclear factors during differentiation of 3T3-L1 preadipocytes . Proc Natl Acad Sci U S A 88 : 2593 - 2597 .
8. Freytag SO , Paielli DL , Gilbert JD ( 1994 ) Ectopic expression of the CCAAT/ enhancer-binding protein alpha promotes the adipogenic program in a variety of mouse fibroblastic cells . Genes Dev 8 : 1654 - 1663 .
9. Tontonoz P , Hu E , Spiegelman BM ( 1994 ) Stimulation of adipogenesis in fibroblasts by PPAR gamma 2, a lipid-activated transcription factor . Cell 79 : 1147 - 1156 .
10. Morrison RF , Farmer SR ( 2000 ) Hormonal signaling and transcriptional control of adipocyte differentiation . J Nutr 130 : 3116S - 3121S .
11. Cao Z , Umek RM , McKnight SL ( 1991 ) Regulated expression of three C/EBP isoforms during adipose conversion of 3T3-L1 cells . Genes Dev 5 : 1538 - 1552 .
12. Rosen ED , Hsu CH , Wang X , Sakai S , Freeman MW , et al. ( 2002 ) C/EBPalpha induces adipogenesis through PPARgamma: a unified pathway . Genes Dev 16 : 22 - 26 .
13. Bieker JJ ( 2001 ) Kruppel-like factors: three fingers in many pies . J Biol Chem 276 : 34355 - 34358 .
14. McConnell BB , Yang VW ( 2010 ) Mammalian Kruppel-like factors in health and diseases . Physiol Rev 90 : 1337 - 1381 .
15. Oishi Y , Manabe I , Tobe K , Tsushima K , Shindo T , et al. ( 2005 ) Kruppel-like transcription factor KLF5 is a key regulator of adipocyte differentiation . Cell Metab 1 : 27 - 39 .
16. Birsoy K , Chen Z , Friedman J ( 2008 ) Transcriptional regulation of adipogenesis by KLF4 . Cell Metab 7 : 339 - 347 .
17. Sue N , Jack BH , Eaton SA , Pearson RC , Funnell AP , et al. ( 2008 ) Targeted disruption of the basic Kruppel-like factor gene (Klf3) reveals a role in adipogenesis . Mol Cell Biol 28 : 3967 - 3978 .
18. Siersbaek R , Nielsen R , Mandrup S ( 2012 ) Transcriptional networks and chromatin remodeling controlling adipogenesis . Trends Endocrinol Metab: TEM 23 : 56 - 64 .
19. Sugii S , Kida Y , Kawamura T , Suzuki J , Vassena R , et al. ( 2010 ) Human and mouse adipose-derived cells support feeder-independent induction of pluripotent stem cells . Proc Natl Acad Sci U S A 107 : 3558 - 3563 .
20. Lee H , Lee YJ , Choi H , Ko EH , Kim JW ( 2009 ) Reactive oxygen species facilitate adipocyte differentiation by accelerating mitotic clonal expansion . J Biol Chem 284 : 10601 - 10609 .
21. Kim JW , Tang QQ , Li X , Lane MD ( 2007 ) Effect of phosphorylation and S-S bond-induced dimerization on DNA binding and transcriptional activation by C/EBPbeta . Proc Natl Acad Sci U S A 104 : 1800 - 1804 .
22. Tang W , Zeve D , Suh JM , Bosnakovski D , Kyba M , et al. ( 2008 ) White fat progenitor cells reside in the adipose vasculature . Science 322 : 583 - 586 .
23. Tang QQ , Lane MD ( 1999 ) Activation and centromeric localization of CCAAT/enhancer-binding proteins during the mitotic clonal expansion of adipocyte differentiation . Genes Dev 13 : 2231 - 2241 .
24. Tang QQ , Otto TC , Lane MD ( 2003 ) CCAAT/enhancer-binding protein beta is required for mitotic clonal expansion during adipogenesis . Proc Natl Acad Sci U S A 100 : 850 - 855 .
25. Kim JW , Monila H , Pandey A , Lane MD ( 2007 ) Upstream stimulatory factors regulate the C/EBP alpha gene during differentiation of 3T3-L1 preadipocytes . Biochem Biophys Res Commun 354 : 517 - 521 .
26. Crossley M , Whitelaw E , Perkins A , Williams G , Fujiwara Y , et al. ( 1996 ) Isolation and characterization of the cDNA encoding BKLF/TEF-2, a major CACCC-box-binding protein in erythroid cells and selected other cells . Mol Cell Biol 16 : 1695 - 1705 .
27. Tang QQ , Gronborg M , Huang H , Kim JW , Otto TC , et al. ( 2005 ) Sequential phosphorylation of CCAAT enhancer-binding protein beta by MAPK and glycogen synthase kinase 3beta is required for adipogenesis . Proc Natl Acad Sci U S A 102 : 9766 - 9771 .
28. Tang QQ , Jiang MS , Lane MD ( 1999 ) Repressive effect of Sp1 on the C/ EBPalpha gene promoter: Role in adipocyte differentiation . Mol Cell Biol 19 : 4855 - 4865 .
29. Urvalek AM , Wang X , Lu H , Zhao J ( 2010 ) KLF8 recruits the p300 and PCAF co-activators to its amino terminal activation domain to activate transcription . Cell Cycle 9 : 601 - 611 .
30. Wei H , Wang X , Gan B , Urvalek AM , Melkoumian ZK , et al. ( 2006 ) Sumoylation delimits KLF8 transcriptional activity associated with the cell cycle regulation . J Biol Chem 281 : 16664 - 16671 .
31. Eaton SA , Funnell AP , Sue N , Nicholas H , Pearson RC , et al. ( 2008 ) A network of Kruppel-like Factors (Klfs). Klf8 is repressed by Klf3 and activated by Klf1 in vivo . J Biol Chem 283 : 26937 - 26947 .