A transcription factor of lipid synthesis, sterol regulatory
element-binding protein (SREBP)-1a causes G1 cell-cycle
arrest after accumulation of cyclin-dependent kinase (cdk)
inhibitors
Masanori Nakakuki1, Hitoshi Shimano1,2, Noriyuki Inoue1, Mariko Tamura1, Takashi Matsuzaka1,
Yoshimi Nakagawa1,2, Naoya Yahagi2, Hideo Toyoshima1, Ryuichiro Sato3 and Nobuhiro Yamada1
1 Department of Internal Medicine (Endocrinology and Metabolism), Graduate School of Comprehensive Human Sciences,
University of Tsukuba, Japan
2 Center for Tsukuba Advanced Research Alliance, University of Tsukuba, Japan
3 Department of Applied Biological Chemistry, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan

Keywords
cell growth; cholesterol; fatty acids; p21;
p27
Correspondence
H. Shimano, 1-1-1Tennodai, Tsukuba,
Ibaraki 305-8575, Japan
Fax: +81 29 853 3174
Tel: +81 29 853 3053
E-mail: shimano-tky@umin.ac.jp(Received 9 November 2006, revised
25 June 2007, accepted 2 July 2007)
doi:10.1111/j.1742-4658.2007.05973.x

Sterol regulatory element-binding protein (SREBP)-1a is a unique membrane-bound transcription factor highly expressed in actively growing cells
and involved in the biosynthesis of cholesterol, fatty acids, and phospholipids. Because mammalian cells need to synthesize membrane lipids for cell
replication, the functional relevance of SREBP-1a in cell proliferation has
been considered a biological adaptation. However, the effect of this potent
lipid-synthesis activator on cell growth has never been explored. Here, weshow that induction of nuclear SREBP-1a, but not SREBP-2, completely
inhibited cell growth in inducible Chinese hamster ovary (CHO) cell lines.
Growth inhibition occurred through G1 cell-cycle arrest, which is observedin various cell types with transient expression of nuclear SREBP-1a.
SREBP-1a caused the accumulation of cyclin-dependent kinase (cdk) inhibitors such as p27, p21, and p16, leading to reduced cdk2 and cdk4 activities and hypophosphorylation of Rb protein. In contrast to transactivationof p21, SREBP-1a activated p27 by enhancing stabilization of the protein
through inhibition of SKP2 and KPC1. In vivo, SREBP-1a-expressing liversof transgenic mice exhibited impaired regeneration after partial hepatectomy. SREBP-1-null mouse embryonic ﬁbroblasts had a higher cell proliferation rate than wild-type cells. The unexpected cell growth-inhibitory roleof SREBP-1a provides a new paradigm to link lipid synthesis and cell
growth.

Sterol regulatory element-binding protein (SREBP)
family members have been established as transcription
factors regulating the transcription of genes involvedin cholesterol and fatty acid synthesis [1,2]. SREBP
proteins are initially bound to the rough endoplasmic
reticulum membrane and form a complex with SREBP
cleavage-activating protein (SCAP), a sterol-sensing

molecule, and insulin-induced gene 1 (Insig-1) [3]. On
sterol deprivation, SREBP is cleaved to liberate the
N-terminal portion containing a basic helix–loop–helix
leucine zipper domain, and enters the nucleus where it
can bind to speciﬁc sterol response elements (SRE) in
the promoters of target genes and activate their transcription [1]. Three isoforms of SREBP are known:

Abbreviations
BrdU, bromodeoxyuridine; cdk, cyclin-dependent kinase; CHO, Chinese hamster ovary; DLS, delipidated serum; DMEM, Dulbecco’s
modified Eagle’s medium; FPP, farnesyl pyrophosphate; GGPP, geranylgeranyl pyrophosphate; Insig-1, insulin-induced gene 1; IPTG,
isopropyl thio-b-D-galactoside; KPC, Kip1 ubiquitylation-promoting complex; MEF, mouse embryonic fibroblast; SCAP, SREBP cleavage
activating protein; SCF, Skp1–Cullin1–F-box; SRE, sterol response element; SREBP, sterol regulatory element-binding protein.

4440

FEBS Journal 274 (2007) 4440–4452 ª 2007 The Authors Journal compilation ª 2007 FEBSSREBP-1a causes G1 arrest

M. Nakakuki et al.

CHO-Lac

100

50

0

0

1

2

3

CHO-BP1a

IPTG(-)

100

50
IPTG(+)
00

BrdU uptake (O.D.)

BrdU uptake (O.D.)

2

3

0.2
0.1
1

Time (days)

CHO-BP2

IPTG(+)
50

0

0

2

2.0

0.5
0

2

3

CHO-BP2
IPTG (-)

1.0

0.0

1

Time (days)

IPTG (-)
IPTG +

1.5

IPTG(-)

100

CHO-BP1a

IPTG (-)
IPTG +

0

1

150

2.0

0.3

0.0

To assess the effects of SREBP-1a on cell growth, we
examined the growth rates of a stable Chinese hamster
ovary (CHO) cell line, in which the mature formof human SREBP-1a (CHO-BP1a) was inducibly
expressed by addition of isopropyl thio-b-d-galactoside(IPTG) to the medium, by way of a coexpressed Lac
repressor [8]. CHO cells expressing only the Lac repressor (CHO-Lac) were used as a negative control, while
another inducible cell line for nuclear SREBP-2(CHO-BP2) was established for comparison [9]. Overexpression of SREBP-1a completely suppressed cellproliferation 24 h after IPTG induction and the effectwas sustained for up to 72 h (Fig. 1A). This observa-

Time (days)

B CHO-Lac
0.4

SREBP-1a inhibits cell growth at G1
in cultured cells

150Time (days)
0.5

Results

BrdU uptake (O.D.)

150

Cell number (×104 cells/dish)Cell number (×104 cells/dish)A

In this study, we investigated the potential effects of
SREBP-1a on cell growth when its active form was
induced.

Cell number (×104 cells/dish)

SREBP-1a, -1c, and -2. Whereas SREBP-2 plays acrucial role in the regulation of cholesterol synthesis,
SREBP-1c controls the gene expression of enzymes
involved in the synthesis of fatty acids and triglycerides
in lipogenic organs [4,5]. Meanwhile, SREBP-1a ishighly expressed in cells that are actively growing [6],and has strong transcriptional activity in a wide range
of genes involved in the synthesis of cholesterol, fatty
acids, and phospholipids. All mammalian cells require
these lipids for the duplication of membranes in cell
division. Depending on the cellular nutritional state
and the availability of exogenous lipids, nuclear
SREBP-1a is induced in growing cells. Therefore, thefunctional relevance of this potent lipid-synthesis regulator in cell proliferation has been considered a biological adaptation to meet the demand for cellular lipids.
It has never been intensively explored whether thisregulatory system for the synthesis of cellular lipids
could inversely control cell growth. Recently, we
reported that p21, a cyclin-dependent kinase (cdk)
inhibitor, is a direct SREBP target gene, suggestingthat the SREBP family may regulate the cell cycle [7].

1Time (days)

2

IPTG +

1.5
1.0
0.5
0.0

0

1

2

Time (days)

Fig. 1. Inhibition of cell proliferation by nuclear SREBP-1a. (A) Time courses of cell proliferation in CHO stable cell lines inducibly expressing
nuclear SREBP-1a (CHO-BP1a) or SREBP-2 (CHO-BP2) under the control of an IPTG-regulated promoter, or only Lac repressor as a control(CHO-Lac). CHO stable cell lines were incubated in the absence (white circles) or the presence (black circles) of 0.1mM IPTG to induce
expression of nuclear SREBPs. At the indicated days, the number of viable cells was measured using a hemocytometer. (B) BrdU uptake as
index of DNA synthesis in CHO stable cell lines that inducibly express nuclear SREBPs. The cells with (black columns) or without (white columns) IPTG induction received a 2 h pulse of BrdU and the incorporation of BrdU into DNA was determined. Data represent mean ± SD in
triplicate.

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M. Nakakuki et al.

tion was speciﬁc to SREBP-1a and was not seen with
SREBP-2, as the growth rates of CHO-Lac cells andthe SREBP-2-expressing cell line (CHO-BP2) were
almost identical and not affected by IPTG treatment(Fig. 1A). During the growth arrest of CHO-BP1a, celldetachment indicative of cell death was minimal (datanot shown). However, DNA synthesis was essentially
blocked in these cells, as evidenced by the lack of
bromodeoxyuridine (BrdU) incorporation (Fig. 1B),
whereas control CHO-Lac and CHO-BP2 cells did notshow signiﬁcant changes. The level of induction of
nuclear SREBPs in these cell lines was reported to bephysiological, as the amounts of the transgene products
were comparable with the levels of endogenous
SREBPs in control cells cultured in lipoprotein-deﬁcient medium, which is a standard manipulation for the
induction of nuclear SREBPs [8,9]. As shown in
CHO-Lac
CHO-BP1a

BrdU uptakerul/s

20000
15000
10000

B

1.0

0.8
0.6
0.4

0

0 1 2 5 10 20 50 100
IPTG (μΜ)

HeLa cells

0.8
0.6
0.4
0.2

0.2

5000
0

CHO-Lac
CHO-BP1a

1.0

MTT assay (OD)

25000

MTT assay (OD)A

0 1 2 5 10 20 50 100
IPTG (µM)

0

FBS

CHO-BP1a

HeLa cells

1

2

5

10

20

50

100

0.6

CHO-BP1a

FBS DLS FBS DLS
3days
2days
Incubation time

IPTG (μΜ)

CHO-Lac

Control
Simvastatin 10µM

MTT assay (OD)

0.6

0.4

0.2

0.0

0.5

GGPP
3μg/mL

IPTG

0.3
0.2
0.1
0.3

1

Vehicle Simvastatin

4442

0.2

0.8

Control

0.4

0.0

0.4

FPP
3μg/mL

CHO-Lac

0.1

0.3

Cerulenin

3 (µM)

Control
IPTG

0.0
VehicleMTT assay (OD)C
MTT assay (OD)DLS

SREBP-1
nuclear form
0

MTT assay (OD)

Fig. 2A,B, the level of endogenous human SREBP-1
nuclear protein induced in HeLa cells by incubation
with delipidated serum (DLS) was comparable withthat induced in CHO-BP1a cells by IPTG at 5 lm,
which had already exhibited inhibition of growth.
Addition of geranylgeranyl pyrophosphate (GGPP) or
farnesyl pyrophosphate (FPP) restored the growth
inhibition caused by a high dose of simvastatin, an
HMG-CoA reductase inhibitor, but did not do so in
CHO-BP1a (Fig. 2C). Thus, it is unlikely that the cellgrowth inhibition observed in CHO-BP1a cells was
attributable to altered prenylation, as observed withstatins. Simvastatin and cerulenin were added to CHOBP1a as inhibitors of the biosynthesis of cholesteroland fatty acids, respectively. Neither attenuated theeffect of SREBP-1a (Fig. 2C), excluding the possibility
that the antiproliferation effect was attributable to

VehicleFPP

GGPP
3μg/mL

3μg/mLCHO-BP1a

Control
IPTG

0.6
0.4
0.2
0.0

0.3

1

0.1

Vehicle Simvastatin

0.3

Cerulenin

3 (µM)

Fig. 2. (A) Dose-dependent inhibition of cellproliferation by nuclear SREBP-1a protein in(B) CHO-BP1a with a comparison withendogenous SREBP-1a induced by lipiddeprived condition in HeLa cells. CHO-BP1acells and CHO-Lac were treated with theindicated dose of IPTG. After 2 days of incubation, MTT assay and BrdU uptake were
estimated as described in Fig. 1. In thesame procedure, nuclear SREBP-1a protein
level in CHO-BP1a induced by IPTG wasanalyzed by immunoblotting. After HeLa
cells had been grown in medium containing
delipidated serum for 2 and 3 days, MTT
assay for live cell number and estimation of
nuclear SREBP-1a by immunoblotting analysis were performed. (C) The antiproliferative
action of SREBP-1a was not due to sterol
and prenyl synthesis inhibition and lipid
accumulation. Stable cell line CHO cells
were cultured with the indicated concentration of liposome containing GGPP or FPP,
non-sterol metabolites of mevalonate, andwith simvastatin or cerulenin to inhibit
cholesterol and fatty acid synthesis, under
IPTG 0.1 mM for 2 days. Live cell number
was estimated by MTT assay. Values aremean ± SD in triplicate.

FEBS Journal 274 (2007) 4440–4452 ª 2007 The Authors Journal compilation ª 2007 FEBSSREBP-1a causes G1 arrest

M. Nakakuki et al.

increased accumulation of cellular lipids. Flow cytometry revealed that the cessation of growth of CHO-BP1aoccurred through G1 cell-cycle arrest (Table 1).
SREBP-1a and not SREBP-2 evoked a marked
decrease in the number of cells in the S phase with aconcomitant increase in the G1 population. In transienttransfection studies with an SREBP-1a expression plasmid and SREBP-inducible enhanced green ﬂuorescentprotein (EGFP) reporter, similar changes in the cell
cycle were observed in various cell lines such as
HEK293 cells, mouse ﬁbroblast Swiss-3T3 cells, andhuman osteoblastoma Saos-2, a p53-deﬁcient cell line
(Table 2) [10]. These data show that the G1 arrest
induced by SREBP-1a is a universal phenomenon andis not mediated through p53, a well-known tumor suppressor that activates the transcription of p21, a cdk
Table 1. Cell-cycle profile of CHO-BP1a and CHO-BP2 cells inducibly expressing nuclear SREBP-1a and SREBP-2, respectively, with
CHO-Lac cells as control. The three types of CHO stable cell line,
after 24 h of culture with 0.1 mM IPTG, were trypsinized, collected,
and stained with propidium iodide and analyzed by flow cytometry.
Each value is mean ± SD. G2/M, total of G2 and mitotic S phase
populations.
Cell

IPTG

G0 ⁄ G1S

G2 ⁄ M

CHO-Lac

–
+
–
+
–
+

40.7 ± 1.3
40.4 ± 3.1
49.4 ± 1.5
73.7 ± 0.6**
34.7 ± 1.4
33.2 ± 1.5

37.5 ± 1.6
38.8 ± 1.9
22.7 ± 3.8
6.6 ± 1.0**
43.8 ± 4.0
41.5 ± 0.6

21.8 ± 2.0
20.8 ± 3.2
27.9 ± 5.3
19.6 ± 0.9
21.6 ± 2.5
25.3 ± 1.5

CHO-BP1a
CHO-BP2**P < 0.01 compared with IPTG non-treated group by Student’s
t-test.

Table 2. REBP-1a induces G1 arrest in the three types of celllines – HEK293, mouse fibroblast Swiss-3T3 cells, and humanosteoblastoma Saos-2 cells. Cells were transiently transfectedwith the indicated expression vectors and the SRE-EGFP vector.
Twenty-four hours later, cells were fixed in paraformaldehyde andpermeabilized with ethanol followed by staining with propidium
iodide. Cell-cycle profiles were estimated within the gate of
EGFP-positive cell population. Each value is mean ± SD.
Cell strain GroupG0 ⁄ G1S

G2 ⁄ M

HEK293

pcDNA3.1(+) 38.5 ± 2.9
21.2 ± 3.3
40.2 ± 2.4
SREBP-1a50.4 ± 2.0** 13.8 ± 0.1** 35.7 ± 2.1
p2155.6 ± 0.4** 22.3 ± 1.5
22.1 ± 1.2**
p27
81.9 ± 1.5** 5.8 ± 0.5** 12.2 ± 1.8
Swiss-3T3 pcDNA3.1(+) 49.7 ± 1.1
18.7 ± 0.6
32.0 ± 1.3
SREBP-1a59.5 ± 1.6** 2.0 ± 1.1** 28.5 ± 2.6
Saos-2
pcDNA3.1(+) 45.4 ± 2.0
15.1 ± 2.8
39.6 ± 3.3
SREBP-1a53.2 ± 5.0* 10.3 ± 2.2* 36.5 ± 4.7*P < 0.05, **P < 0.01 compared with pcDNA3.1(+) group by Dunnnett’s multiple comparison test.

Table 3. Mutated SREBP-1a does not induce G1 arrest in HEK293
cells. DTA–SREBP-1a lacks the N-terminal trans-activation domain.
YR–SREBP-1a loses the capability of binding to sterol response element of the target gene promoter. Each value is mean ± SD.
Cell strainGroup

G0 ⁄ G1S

G2 ⁄ M

HEK293

pcDNA3.1(+)
DTA–SREBP-1a
YR–SREBP-1a
SREBP-1a40.8 ± 2.9
37.2 ± 2.6
40.8 ± 6.2
51.7 ± 2.6**

20.4 ± 2.4
23.0 ± 2.6
19.6 ± 3.5
15.0 ± 3.7

38.8 ± 3.6
39.8 ± 1.2
39.7 ± 8.2
33.3 ± 2.7

inhibitor [11]. To elucidate the functional domains of
SREBP-1a involved in this growth-arrest effect, mutational analysis was performed (Table 3). When the
N-terminal transactivation domain was deleted (DTA–
SREBP-1a) [12], SREBP-1a-induced G1 arrest was
abolished. Its action was also cancelled by the introduction of a point mutation (YR–SREBP-1a) throughwhich SREBP-1 loses its ability to bind to an SRE,
which is generally found in promoters of known
SREBP target genes, but still binds to an E-box as aconsensus cis-element for bHLH proteins [13,14](Table 3). Therefore, the effect of SREBP-1a on the cell
cycle may be mediated through the transactivation ofsome SREBP target gene(s).
Involvement of cdk inhibitors in the
antiproliferaive action of SREBP-1a
It is highly plausible that cdk inhibitors and cell-cyclerelated genes could be involved in the G1 arrest caused
by SREBP-1a [15]. We have recently identiﬁed p21 asa direct target of SREBP-1 in the screening of upregulated genes in the liver of SREBP-1a transgenic miceusing a DNA microarray [7]. Northern blot analysis
showed that gene expression of p27 and p16 ⁄ p19, in
addition to p21, was highly elevated only in CHOBP1a cells, along with key enzymes in the biosynthetic
pathways for cholesterol, fatty acids, and phosphophatidylcholine (HMG-CoA synthase, FPP synthase,fatty acid synthase, and CTP : phosphocholine cytidylyltransferase a) (Fig. 3A), all of which are well-established SREBP-1a target genes. Luciferase reporter
assays in HEK293 cells revealed that SREBP-1a
activated mouse p16 and p21 promoters, though only
marginally compared with an authentic SRE reporter,
consistent with the increased mRNA levels in SREBPinducible cells; however, it did not activate the promoters of p19 and p27 (Fig. 3B). Although a precise
mechanism for the accumulation of p27 with SREBP1a has yet to be clariﬁed, p27 is known to be regulated
mainly at the post-transcriptional level. Recent reports
indicate that p27 protein is regulated through a

FEBS Journal 274 (2007) 4440–4452 ª 2007 The Authors Journal compilation ª 2007 FEBS

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SREBP-1a causes G1 arrest

M. Nakakuki et al.

A
CHO-Lac CHO-BP1a CHO-BP2
IPTG
SREBP-1
FAS
HMG-CoA synthase
FPP synthaseCT
p16/p19
p21
p27
36B4

B
p16 promoter

pcDNA3.1(+)
SREBP-1a
SREBP-2
0

10

20

30

40

50

60

0

10

20

30

40

50

60

0

100

200

500

600

700

0

10

20

40

50

60

pcDNA3.1(+)

p19 promoter

SREBP-1a
SREBP-2

pcDNA3.1(+)

p21 promoter

SREBP-1a
SREBP-2300

400

induction, at the mRNA level in both cases and at theprotein level in SKP2, potentially explaining the p27
protein elevation (Fig. 4A,D). The data show that
SREBP-1a regulates an assortment of genes involvedin the control of cell proliferation.
On induction of exogenous SREBP-1a protein in
CHO-BP1a cells, p21 and p27 proteins were markedly
induced, as shown by immunoblot analysis (Fig. 4B).
In accordance with the induction of these cdk inhibitors, SREBP-1a-expressing cells exhibited inhibition of
cdk2 and cdk4 activities without any change in total
protein level (Fig. 4C,D); in particular, the activity of
cdk2, which plays an essential role in DNA synthesis
and transition into the S phase [19], was almost abolished. Cyclins D and E were slightly decreased. Consequently, Rb protein, the major target of the cdk ⁄ cyclin
complex, was mainly in a phosphorylated form in thegrowing control CHO cells (Fig. 4D) [20]. SREBP-1a
expression caused a shift to the dephosphorylated formof Rb protein 24 h after induction by IPTG. Our data
show that SREBP-1a inhibits the ability of cdk ⁄ cyclin
complexes to phosphorylate Rb protein, resulting incell-cycle arrest at the G1 phase [16], and that thispartly occurs through the induction of p21 and p27.

pcDNA3.1(+)

p27 promoter

SREBP-1a
SREBP-2

Inhibition of cell growth by SREBP-1a in vivo
30

pcDNA3.1(+)

SRE-LUC

SREBP-1a
SREBP-2
0

20

40

60

80

100

120

Relative luciferase activity
Fig. 3. Induction of cdk inhibitors by nuclear SREBP-1a. (A) Expression of genes involved in lipid synthesis and cdk inhibitors in relation to cell-cycle progression. Total RNAs (10 lg) were preparedfrom each CHO stable cell line (CHO-BP1a, CHO-BP-2 and CHOLac as control) 24 h after IPTG addition and used for northern blot
analysis with the indicated cDNA probes. Fatty acid synthase(FAS), CTP : phosphocholine cytidylyltransferase a (CTa), 36B4 asloading control. (B) Transcriptional activation of SREBP-dependent
promoter-reporter of cdk inhibitors. HEK293 cells were transfectedwith cdk inhibitor promoter–luciferase constructs fused to the5¢-flanking region of p16, p19, p21, p27 genes and SRE–luciferase
reporter as positive control in the absence or presence of nuclear
form of SREBP expression plasmids. The cells were subjected to
firefly-luciferase reporter assays with Renilla luciferase as reference. Values are means ± SD.

ubiquitin-dependent proteasome system [16]. Twoubiquitin ligase complexes, Skp1–Cullin1–F-box (SCF)
and Kip1 ubiquitylation-promoting complex (KPC),
are involved in p27 degradation at the G2 and G1
phases, respectively [17,18]. In CHO-BP1a cells, SKP2
and KPC1, which are key components of SCF
and KPC, were markedly decreased by SREBP-1a
4444

The antiproliferative activity of SREBP-1a observed incultured cells was also tested in vivo. Partial hepatectomy is an established method for the synchronizedinduction of cell proliferation in a differentiated organ.
Partial hepatectomy was conducted in wild-type andtransgenic mice that overexpressed nuclear SREBP-1ain the liver [21] (Fig. 5). After 70% resection, wild-type
mouse livers recovered to their original size in 10 days.
SREBP-1a transgenic mice have huge, fatty livers containing large amounts of triglycerides and cholesterylesters due to the activation of lipid synthetic genes [21].
In contrast to wild-type mice, SREBP-1a transgenic
mice showed marked impairment in liver regeneration,with essentially no growth of the remaining liver, andabout half of the mice died 1–2 days after partial hepatectomy. DNA synthesis in the livers was estimated byincorporation of injected BrdU (Fig. 5A). Consistent
with the notion that most normal hepatocytes are in aquiescent stage, BrdU incorporation was very low inboth wild-type and SREBP-1a transgenic livers prior topartial hepatectomy. At 36 and 48 h after partial hepatectomy, the number of BrdU-positive cells was dramatically increased in wild-type livers, indicating
synchronized entry of the hepatocytes into the S phase.
In contrast, overexpression of nuclear SREBP-1a completely suppressed BrdU incorporation in hepatocytes

FEBS Journal 274 (2007) 4440–4452 ª 2007 The Authors Journal compilation ª 2007 FEBSSREBP-1a causes G1 arrest

M. Nakakuki et al.

AC
CHO CHO CHO-Lac -BP1a -BP2

CHO-Lac CHO-BP1a CHO-BP2
IPTG

Cdk2

SKP2

Cdk4
KPC1

D

36B4

CHO-Lac CHO-BP1a CHO-BP2
IPTG

B
CHO-Lac

CHO-BP1a CHO-BP2

IPTG

SREBP-1

Cdk2
Cdk4
Cyclin D1

SREBP-2
p21

Cyclin E
SKP2

p27
α-Tubulin

Rb

Fig. 4. Effects of nuclear SREBP-1a on the cell-cycle regulators, p21(Cip1), p27(KIP1), S-phase kinase-associated protein 2 (SKP2), ubiquitin
ligase KPC1, cyclin D1, cyclin E expression, cdk2, cdk4 expression and related kinase activities and Rb protein phosphorylation. (A) Repression of SKP2 and KPC1 which regulate the ubiquitin-dependent degradation of p27 at G1 and G2 phase, respectively, in CHO cells inducibly
expressing nuclear SREBP-1a (CHO-BP1a) and -2 (CHO-BP2) and control cells (CHO-Lac) as estimated by northern blot analysis. (B) Nuclear
SREBPs, cdk inhibitor proteins cdk2, cdk4, cyclin D1, cyclin E, SKP2 protein levels, and phosphorylation of Rb protein in CHO-BP1a, CHOBP2, and CHO-Lac after induction by IPTG. Cells were treated with IPTG for 1 day, and nuclear extracts and cell lysates were subjected toimmunoblot analysis with antibodies against the indicated proteins. Alpha-tubulin was shown is the loading control. (C) Activities of cdk2 andcdk4 by SREBP-1a. cdk assay was carried out with cdk2 or cdk4 immunoprecipitates from 200 lg of protein of the cell lysates using Rb protein fragment and histone HI as substrate, respectively.

in transgenic mice, explaining the impaired liver regeneration. It has been established that partial hepatectomy leads to hepatic polyploidy, which reﬂects anincrease in nuclear DNA content [22]. Hepatocytes
from SREBP-1a transgenic mice had a higher proportion of 2N cells than did normal hepatocytes (Fig. 5B).
SREBP-1a inhibited a change in the polyploidy patternthat was observed in livers from wild-type mice by ﬂow
cytometry 10 days after partial hepatectomy. The data
provide supporting evidence that SREBP-1a overexpression inhibits cell proliferation in vivo as well as incultured cells, though it is possible that the accumulation of huge amounts of lipids in the transgenic hepatocytes may contribute to the inhibition of cell growth.
Effects of endogenous SREBP-1 on cell growth
To determine the physiological relevance of thegrowth-inhibitory action of SREBP-1a, the role ofendogenous SREBP-1a in cell proliferation was examined in SREBP-1-null mice. Both cell growth anduptake of BrdU in mouse embryonic ﬁbroblast (MEF)

cells prepared from SREBP-1-null mice were signiﬁcantly elevated compared with wild-type cells(Fig. 5C,D). Uptake of BrdU also tended to increasein hepatocytes from SREBP-1-null mice after partial
hepatectomy (Fig. 5E). The data suggest that endogenous SREBP-1a plays a substantial role in the regulation of cell proliferation, though it is possible that
SREBP-1c also makes a contribution.
The amounts of nuclear SREBPs, and thus theirendogenous activities, in cultured cells are known tobe highly induced under lipid-deprived conditions suchas culture in DLS or lipoprotein-deﬁcient serum, orwith HMG-CoA reductase inhibitors due to activationof the SCAP ⁄ Insig system [23]. These lipid-deprivation
manipulations induce endogenous nuclear SREBP-1a,
as shown by immunoblot analysis of nuclear extracts
from HeLa cells (Fig. 6A). The induction of nuclear
SREBP-1 accompanied a reduction in cell proliferation
and an increase in the population of cells at G1(Fig. 6A,C). The G1-arrest antiproliferative effect in
DLS was cancelled when an unsaturated fatty acid(oleate) was added to the medium in accordance with

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SREBP-1a causes G1 arrest

Tg SREBP-1a

50
40
30
DAPI

20
10
BeforePHx

Wild type MEF

80

*

60

**

40

20

0

*
0

1
2
Time (days)

8N

8N

8N
4N

80
4N

4N

4N

60
40
2N

20
0

48
24
36
Time (h) after PHx

D
N=5
SREBP-1 KO MEF N = 7

C

8N

2N

2N

2N

ND

ND

ND

B 100
Relative cell number (%)

Tg SREBP-1a hepatocyte N = 20

Cell number ( 104 cells/dish)Wild type
BrdU

Wild type hepatocyte N = 2

3

Wild type MEF

N=5

SREBP-1 KO MEF N = 7

60

*

40

20

0

1

2
Time (days)

3

E
10
BrdU positive nuclei ratio (%)

60

BrdU uptake(Chemiluminescence ( 104rlu/sec/well)

BrdU positive nuclei ratio (%)

A

M. Nakakuki et al.

Before After
Before After
PHx
PHx
Wild type
Tg SREBP-1a
Wild type hepatocyte N = 3
SREBP-1 KO hepatocyte N = 3

8
6
4
2
0

24
48
Time (h) after PHx

Fig. 5. Effects of SREBP-1a on cell growth in vivo. Impaired liver regeneration after partial hepatectomy (PHx) in SREBP-1a transgenic mice
(A, B) and enhanced cell growth in MEF cells (C, D) and livers from SREBP-1-null mice (E). SREBP-1a transgenic mice overexpressing
nuclear human SREBP-1a under the control of rat phosphoenolpyruvate carboxykinase promoter were established as described previously[21]. Non-transgenic littermates (wild-type) were used as controls. Each group of animals was fed a high protein ⁄ low carbohydrate diet for
5 days to induce transgene expression. Animals were deprived of food from 6 h before partial hepatectomy. (A) BrdU uptake of hepatocytes
at the indicated times (h) after partial hepatectomy from SREBP-1a transgenic mice and wild-types (left graph). BrdU immunostaining and
DAPI staining for nuclei were performed as described in Experimental procedures (right panels at 48 h). The incorporation rates of BrdU inlivers from SREBP-1a transgenic wild-type mice were represented with the ratio BrdU-positive nuclei to DAPI-stained nuclei. ND, no detection of BrdU-positive nuclei. (B) Analysis of ploidy in hepatocyte cell nuclei by flow cytometry. Nuclei were isolated from resected liver(pre PHx) at the time of partial hepatectomy and from remnant liver 9–10 days after partial hepatectomy (post PHx). Hepatocyte ploidy isshown as 2n, 4n, and 8n. (C) Cell proliferation and (D) BrdU uptake in MEF cells from SREBP-1-null mice and wild-type littermate mice.
Results are expressed as the means ± SD of five or seven independent experiments. **P < 0.01, *P < 0.05 compared with littermates byStudent’s t-test. (E) Uptake of BrdU in hepatocytes from SREBP-1-null mice and wild-type littermate mice after partial hepatectomy.

the suppression of nuclear SREBP-1 (Fig. 6A,C).
Meanwhile, cholesterol did not suppress nuclear
SREBP-1 or restore cell growth. Similar regulation byoleate was observed in Swiss-3T3 ﬁbroblasts (Fig. 6B).
Our data indicate that lipid regulation by endogenous
SREBP-1a contributes to the cell cycle and growth.

Discussion
SREBP-1a causes G1 arrest through cdk inhibitors
SREBP-1a is highly expressed in actively growing cells
and has been considered to be a master transcription
4446

factor in lipid synthesis. This study clearly demonstrates that nuclear SREBP-1a can also regulate thecell cycle and growth. Thus, lipid synthesis in proliferating cells is not simply a secondary event under the
regulation of cell growth [24], but rather, actively controls cell growth. This unexpected observation explains
the difﬁculty in obtaining cell lines that highly express
nuclear SREBP-1a, unlike those that express SREBP1c and SREBP-2.
Recently, we reported that both SREBP-1a and
SREBP-2 directly activate the promoter of the p21
gene, partially explaining this hypothesis [7]. However,current studies on various cell types show that an

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M. Nakakuki et al.

B Swiss3T3 fibroblast C

HeLa cellp<0.01

1.6

1.8

1.4

1.6

0.8
p<0.01
0.6

MTT assay (O.D.)

1.0

0.4

1.2
1.0
0.8
0.6
0.4

0.2

0.2

0.0

0.0

FBS DLS
VehicleFBS DLS
OleateFBS DLS
Cholesterol

100µM

5µM

nuclear SREBP-1 protein

100
G2/M

1.4

1.2

MTT assay (O.D.)

p<0.01

G2/M
S
G1

HeLa cell% of cells in the phase

A

G2/M

G2/M

G2/M

S

S

S

G1

G1

G1

FBS

DLS

80

60

S

40

G1
20

VehicleOleate
100µM

LPDS

0

FBS

DLS

VehicleOleate 100µM

nuclear SREBP-1 protein

Fig. 6. Effects of endogenous SREBP-1a on cell growth and cell cycle in lipid deprivation. (A) Effects of DLS, and rescue effect of oleate(unsaturated fatty acid) and cholesterol on cell proliferation in cultured cells were tested. HeLa cells were plated at 0.5 · 104 cells per wellin 24-well plates. After 1 day’s incubation, the medium was switched to DMEM containing 5% DLS, in the presence or absence of 100 lM
oleate or 5 lM cholesterol. DMEM containing 10% fetal bovine serum was control. For estimation of cell number, MTT assay (A) and cellcycle analysis by FACS (C) were performed after 2- and 1-day incubation, respectively. In another set of experiments, nuclear forms of
SREBP-1 protein were detected by immunoblot analysis on nuclear extracts from the cells (A, lower). (B) Swiss-3T3 fibroblasts subjected tolipid starvation by lipoprotein deficient medium were incubated with 100 lM oleate. MTT assay and immunoblotting for detection of nuclear
SREBP-1 protein (lower) after two and one days, respectively, were performed in the same procedure described previously. P < 0.01 compared with fetal bovine serum control group by Student’s t-test. Values are mean ± SD.

abundance of nuclear SREBP-1a induces various cdk
inhibitors, such as p27 and p16, in addition to p21,
leading to G1 arrest in cell growth. In the current
experimental setting, the antiproliferative action wasobserved only with SREBP-1a; however, SREBP-2
might have a similar, though less efﬁcient, action. The
mechanisms for the activation of individual cdk inhibitors are diverse and complex (scheme shown in Fig. 7).
Because the ability of SREBP-1a to cause G1 arrest
depends on its transcriptional activity (Table 3), some
unknown SREBP-1a-regulated genes may also be
involved in the mechanisms in addition to direct activation of p21 [7], and repression of SKP2 and KPC1.
The relative contributions of factors such as p27, p21,
p16, to this new action of SREBP-1a remain unknown,
but presumably depend on cell type. Further investigations are needed to clarify the more detailed mechanisms and identify the major upstream mediator(s).
It is well established that the amounts of nuclear
SREBPs are regulated by the sterol-regulated cleavage
system and primarily depend on cellular demand forsterols. In previous reports, enhanced proliferation on
activation of the phosphatidylinositol 3-kinase ⁄ Akt
pathway, has been linked to activation of SREBP-1a[25,26]. More recently, it has been reported that activation of SREBP-1a is crucial for cell growth [27,28]. In
contrast, our data imply that the presence of abundantnuclear SREBP-1a, indicating that cells are deﬁcient inlipid stores, not only activates transcription of its target genes involved in lipid synthesis, but also delays
cell growth, particularly in case of severe depletion
with very strong activation of SREBP-1a, until a time
when sufﬁcient lipids are available for membrane synthesis. In this respect, our data apparently contradict
previous reports indicating a link between SREBP-1aand cell growth. However, SREBP-1a may have biphasic effects depending on its nuclear amount. In theabsence of IPTG, incorporation of BrdU was greaterin CHO-BP1a and CHO-BP2 cells than in CHO-Lac
cells (Figs 1,2). Because expression of SREBP-1a in
CHO-BP1a cells may be leaky (Fig. 3A), one interpretation is that both transcription factors promote proliferation at low expression levels (i.e. in the absence of
IPTG), whereas overexpression of SREBP-1a blocks
proliferation. In knockout studies, trends of increasingcell growth and uptake of BrdU in SREBP-1-null
MEFs or hepatocytes were marginal and may berelated to compensated activation of SREBP-2.

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SREBP-1a causes G1 arrest

M. Nakakuki et al.

SREBP-1a expression
membrane SREBP-1a
Cleavage system
SCAP/Insig

Lipid
depletion

Growthstimulation

nuclear SREBP-1a

Lipid synthesis
Expression level

Cell growth

nuclear
SREBP-1a
SREBP-1a target genes
SKP2,KPC1

p27 stability
p21

p27

p16/p19

CDK inhibitors

CDK4

P

CDK2
pRb

P

pRb
E2F

E2F

progression of cell proliferation
Fig. 7. Schematic diagram illustrating the mechanisms by which
SREBP-1a causes cell-cycle G1 arrest.

Considering these biphasic actions, the physiologicalroles of SREBP-1a in the regulation of cell growth
may be complex, and should be investigated carefully.
Unsaturated fatty acids suppressed the cleavage of
SREBP-1, consistent with previous studies [29,30], andcancelled the cell-growth inhibition (Fig. 6). These datasuggest that regulation of SREBP-1a may be related tocellular fatty acid metabolism linked to cell growth,
although a lack of oleate could affect cell growth independent of SREBP-1.
Physiological relevance of SREBP-1a activation
Recently, an intriguing study was reported suggestingthat SREBP-1a is involved in regulation of the cell
cycle. Nuclear SREBP-1a is hyperphosphorylated at

4448

G2 ⁄ M, which is associated with increased transcriptional activity, explaining the activation of lipid synthetic genes at mitosis [31]. In addition to G1 arrest,
our data suggest that nuclear SREBP-1a could potentially modify the cell cycle at the G2 ⁄ M phase. No
marked reduction in the number of cells in the G2 ⁄ Mphase was observed despite a marked decrease in
S-phase cells in SREBP-1a overexpression, indicating aconcomitant G2 ⁄ M arrest by SREBP-1a.
The nuclear forms of SREBPs have been speculated
to be degraded by the ubiquitin–proteasome pathway,
because N-acetyl-leucyl-leucyl-norleucinal, a calpain
inhibitor, stabilizes them experimentally [32]. Recently,
it was reported that Fbw7, an F-box and a componentof an SCF-type ubiquitin ligase complex, is responsible
for the degradation of SREBP-1a after phosphorylation by GSK-3 [33]. Fbw7 in SCF also regulates thestability of c-Myc, cyclin E, and c-Jun and the JNK
signal, supporting its involvement in cell growth. It
can be speculated that the cellular lipid balance
regulates SREBP-1a activity through cleavage by the
SCAP ⁄ Insig system, whereas cell-cycle-associated regulation involves the stability of nuclear SREBP-1athrough Fbw7 activity. Thus, both SREBP-1a and p27
are regulated by SCF ubiquitin pathways in a cellcycle-dependent manner and could thereby regulate thecell cycle and growth. It is important to investigate
endogenous Fbw7 activity in relation to the cell cycle
and lipid availability.
Our data also suggest a new mechanism for the antiproliferative activity of statins, which are HMG-CoA
reductase inhibitors [34], through the activation of
nuclear SREBP-1a, though the main mechanism has
been considered to be inhibition of protein prenylation[35]. Further studies of this strong lipid synthetic factor will reveal new aspects of a link between the regulation of lipid synthesis and the cell cycle and growth.

Experimental procedures
Cell proliferation and cell-cycle analysis of CHO
stable cell lines
CHO cell lines, CHO-BP1a and CHO-BP2, expressing amature form of human SREBP-1a (amino acids 1–487) andhuman SREBP-2 (amino acids 1–481), respectively, with a
Lacswitch inducible mammalian expression system, andCHO cells constitutively expressing the Lac repressor(CHO-Lac) were constructed as described previously [8,9].
Cells were grown in Dulbecco’s modiﬁed Eagle’s medium(DMEM) containing 10% fetal bovine serum, 100 UÆmL)1
penicillin, and 100 lgÆmL)1 streptomycin and incubated at
37C in a humidiﬁed 5% CO2 atmosphere. For induction

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M. Nakakuki et al.

of SREBP, IPTG was added to the medium at 0.1 mm. For
cell-proliferation analysis, cells were seeded at 1 · 105 per
10 cm dish. At the indicated time after treatment with
0.1 mm IPTG, the cells were trypsinized, collected, andcounted using a hemocytometer. To determine of the BrdU
uptake, 1.5 · 103 cells per well were harvested in a 96-wellplate. After 24 h of treatment with 0.1 mm IPTG, the cells
were incubated with 10 lm BrdU for 4 h in a CO2 incubator at 37C, and BrdU uptake was measured with a BrdU
Labeling and Detection kit (Roche Diagnostics, Basel,
Switzerland) or cell proliferation ELISA, BrdU (chemiluminesence) (Roche Applied Science Inc., Basel, Switzerland). For determination of the cell-cycle proﬁle, the cells
were harvested and resuspended with 0.1% Triton X-100 in
NaCl ⁄ Pi solution containing 0.1 mgÆmL)1 of RNAse and25 lgÆmL)1 of propidium iodide (Sigma Chemical Co., St
Louis, MO). The stained cells were examined by ﬂow
cytometry (FACScaliber; Becton Dickinson, Franklin
Lakes, NJ). For all experiments, the cells were harvested at
pre-conﬂuency, the stage of exponential proliferation.

Expression plasmids and cell-cycle analysis oftransiently transfected cell lines
cDNAs encoding a mature form of human SREBP-1a(amino acids 1–487) and human SREBP-2 (amino acids
1–481), a transactivation domain-deleted form of SREBP1a, and a YR-mutant of SREBP-1a (substitution of tyrosine at amino acid 335 for arginine) were inserted into a
pcDNA3.1(+) expression plasmid [13,14] (Invitrogen,
Carlsbad, CA). An SRE–EGFP vector encoding anenhanced green ﬂuorescent protein under control of the
SRE was prepared by subcloning a region containing the
SRE and Sp1 site derived from the human LDL receptor[36] into pEGFP-1 (Clontech Laboratories Inc., Palo Alto,CA). Transfection studies were conducted with cells plated
on 10 cm dishes using Transfection Reagent Fugene 6(Roche Diagnostics). For suppression of intrinsic SREBP,
25-hydroxycholesterol was added to the medium 4 h after
transfection. Twenty-four hours after transfection, the cells
were harvested, ﬁxed, permeabilized, and resuspended in
NaCl ⁄ Pi containing propidium iodide and RNAse. EGFPpositive cell populations expressing transfected nuclear
srebps were analyzed by ﬂow cytometry [37].

Northern blot analysis and immunoblot analysis
Total RNA was isolated from the cells using Trizol
reagents (Life Technologies, Rockville, MD) and subjected
to northern blot analysis as described previously [38] using
the indicated 32P-labeled cDNA probe. Total cell lysatesand nuclear extracts from CHO cells were prepared as
described previously [39,40] and subjected to immunoblot
analysis using the indicated monoclonal or polycolonal
antibodies (IgG). Horseradish peroxidase-linked mouse or

SREBP-1a causes G1 arrest

rabbit IgG was used as a secondary antibody and the target
protein was visualized using an ECL kit (Amersham Pharmacia Biotech, Piscataway, NJ).

Cloning of promoter of cdk inhibitor andtransfection and luciferase assay
A SacI–XhoI fragment of human p16 INK4A, an NheI–HindIII fragment of human p19INK4D, and a BglII–HindIII
fragment of mouse p27(KIP1) extending from the 5¢-UTR
to each promoter region were subcloned into a pGL3 basic
vector (Promega, Madison, WI). The primers used for PCR
were as follows: P16: 3¢ primer, 5¢-TGCCTGCTCTCCCC
CTCTCC-3¢, 5¢ primer, 5¢-GCCACCGCGTCCTGCTCCA
AAG-3¢; p19: 3¢ primer, 5¢-ACACTGGCGGCCTGACAA
AG-3¢, 5¢ primer, 5¢-AGCTCGTAGTAAGGGCCAATGA
ATGTTCT-3¢; p27: 3¢ primer, 5¢-CAAAACCGAACAAA
AGCGAAACGCCA-3¢, 5¢ primer, 5¢-CAACCCATCCAA
ATCCAGACAAAAT-3¢. All constructs were conﬁrmed bysequencing. The p21 (Waf1 ⁄ Cip1) promoter luciferase construct has been described previously [7]. For transfection
and luciferase assay, HEK293 cells were cultured in DMEM
containing 25 mM glucose, 100 unitÆmL)1 penicillin, and100 lgÆmL)1 streptomycin sulfate supplemented with 10%
fetal bovine serum. On day 0, cells were plated on a 24-wellplate at 2.5 · 104 per well. On day 1, each luciferase reporter plasmid (0.25 lg) and pRL-SV40 reference plasmid(0.02 lg) (Promega) were transfected into cells using thetransfection reagent Fugene 6 (Roche Diagnostics) according to the manufacturer’s protocol. Expression plasmid(pcDNA3.1(+)–SREBP-1a, -1c, or -2) (0.25 lg) or basic
plasmid pcDNA3.1(+) as a negative control were also
cotransfected. Four hours after transfection, cells were
exchanged into fresh medium, followed by culture for 1 day
before harvesting. The luciferase activity was measured
and normalized to the activity of co-transfected pRL-SV40
Renila luciferase reporter.

Immunoprecipitation kinase assay
of cdk2 and cdk4
Cdk2 and cdk4 were immunoprecipitated with mousemonoclonal anti-cdk2 and anti-cdk4 sera (Santa Cruz Biotechnology, Santa Cruz, CA), respectively. The immunocomplexes were then subjected to an in vitro kinase assay
with cdk2 substrate histone HI protein (Santa Cruz Biochemistry) and the cdk4 substrate, Rb protein fragment(Santa Cruz Biochemistry), as described previously [41].

Partial hepatectomy of SREBP-1a transgenic miceand SREBP-1 knockout mice
All animal studies were approved by the Animal Care
Committee of the University of Tsukuba. The mice were

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SREBP-1a causes G1 arrest

M. Nakakuki et al.

housed in colony cages, maintained on a 12 h light ⁄ 12 hdark cycle, and given free access to water and a standard
chow diet (MF, Oriental yeast).
Transgenic mice expressing a mature form of human
SREBP-1a [21], SREBP-1-null mice, and littermates (wildtype) were subjected to partial hepatectomy as described
previously [42]. Approximately 70% of each liver was
resected. For in vivo BrdU incorporation experiments, mice
were given an intravenous injection of BrdU (60 mgÆkg) 2 h
before sacriﬁce. Liver tissue was immediately ﬁxed in 10%
formalin, dehydrated, embedded in parafﬁn, and sectioned.
Brdu immunohisochemistry was performed using the Amersham cell proliferation kit. The number of BrdU-positive
hepatocytes was counted. For cell-cycle proﬁle analysis ofhepatocytes, resected and remnant livers were minced andﬁltered through a ﬁlter mesh (BD Falcon cell strainer), andexamined by ﬂow cytometry.

Growth rate of embryonic fibroblasts fromSREBP-1 knockout mice

Acknowledgements
We are grateful to Alyssa H. Hasty for critical readingof this manuscript. We also thank Drs Tomotaka
Yokoo, Takashi Yamamoto, Akimitsu Takahashi,
Hirohito Sone, and Hiroaki Suzuki for helpful discussion. This work was supported by grants-in-aid from
the Ministry of Science, Education, Culture, and Technology of Japan.

References

Primary MEFs were prepared from post-implantationembryos (day 13–15) derived from the mating of hemizygote SREBP-1-null mice. The genotype for SREBP1+ ⁄ + and – ⁄ – was determined by Southern blot analysis[43]. MEFs of both genotypes were grown in DMEM supplemented with 10% fetal bovine serum. For cell-growth
assays, MEFs were seeded at a density of 0.5 · 105 cells per
10 cm dish on day 0. Cell numbers were counted dailyusing the hemocytometer. Data were collected from triplicate cell counts from ﬁve to seven independent experiments.
For DNA synthesis measurements, MEFs were plated at adensity of 0.5 · 104 cells per well in a 24-well plate. On theindicated day, a BrdU uptake assay was performed according to the manufacturer’s protocol.

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4450

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followed by the MTT assay and cell-cycle analysis. For estimation of the nuclear form of SREBP-1 protein, nuclear
extracts from HeLa cells and Swiss-3T3 ﬁbroblasts wereprepared and subjected to immunoblot analysis as described
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