MOLECULAR AND CELLULAR BIOLOGY, Oct. 2005, p. 8938–8947
0270-7306/05/$08.00⫹0 doi:10.1128/MCB.25.20.8938–8947.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Vol. 25, No. 20

Lipid Synthetic Transcription Factor SREBP-1a Activates
p21WAF1/CIP1, a Universal Cyclin-Dependent
Kinase Inhibitor
Noriyuki Inoue,1 Hitoshi Shimano,1* Masanori Nakakuki,1 Takashi Matsuzaka,1
Yoshimi Nakagawa,2 Takashi Yamamoto,1 Ryuichiro Sato,3 Akimitsu Takahashi,1Hirohito Sone,1 Naoya Yahagi,2 Hiroaki Suzuki,1Hideo Toyoshima,1 and Nobuhiro Yamada1
Department of Internal Medicine, Institute of Clinical Medicine,1 and Center for Tsukuba Advanced Research
Alliance,2 University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8575, Japan, and Department ofApplied Biological Chemistry, Graduate School of Agricultural and Life Sciences,
University of Tokyo, Tokyo, Japan3
Received 30 March 2005/Returned for modification 3 May 2005/Accepted 1 July 2005

Sterol regulatory element-binding proteins (SREBPs) are membrane-bound transcription factors that regulate lipid synthetic genes. In contrast to SREBP-2, which regulates cellular cholesterol level in normal cells,
SREBP-1a is highly expressed in actively growing cells and activates entire programs of genes involved in lipid
synthesis such as cholesterol, fatty acids, triglycerides, and phospholipids. Previously, the physiologicalrelevance of this potent activity of SREBP-1a has been thought to regulate the supply of membrane lipids in
response to cell growth. Here we show that nuclear SREBP-1a and SREBP-2 bind directly to a novel SREBP
binding site in the promoter of the p21WAF1/CIP1 gene, the major cyclin-dependent kinase inhibitor, andstrongly activate its promoter activity. Only the SREBP-1a isoform consistently causes induction of p21 at boththe mRNA and protein levels. Colony formation assays and polyploidy of livers from transgenic mice suggestthat activation of p21 by SREBP-1a could inhibit cell growth. Activation of endogenous SREBPs in lipid
deprivation conditions was associated with induction of p21 mRNA and protein. Expression of p21 was reducedin SREBP-1 null mice. These data suggest a physiological role of SREBP-1a in p21 regulation. Identificationof p21 as a new SREBP target might implicate a new paradigm in the link between lipid synthesis and cell
growth.

branes in cell division. Depending upon cellular nutritional
states and extracellular availability of lipids, nuclear SREBP-1a
is induced in growing cells. Therefore, the functional relevanceof this potent lipid synthetic regulator to cell proliferation has
been believed to be a biological adaptation to meet demands
for cellular lipids. However, it has never been intensively explored whether this regulatory system for synthesis of cellularlipids could inversely control cell growth.
p21WAF1/CIP1 is the major cyclin-dependent kinase inhibitor,
which halts the cell cycle at G1 (6, 8, 14, 27; reviewed inreference 10). In this study, we found that p21 is an SREBPtarget, and especially SREBP-1a activates and stabilizes p21
expression, leading to cell growth arrest.

Sterol regulatory element-binding protein (SREBP) family
members have been established as transcription factors regulating transcription of genes involved in cholesterol and fatty
acid synthesis (4, 5). SREBP proteins are initially bound to therough endoplasmic reticulum membrane and form a complex
with SREBP cleavage-activating protein (SCAP), a sterol-sensing molecule, and insulin-induced gene 1 (INSIG-1) (18).
Upon sterol deprivation, SREBP is cleaved to liberate theamino-terminal portion containing a basic helix-loop-helix
leucine zipper domain, and enters the nucleus where it canbind to specific sterol response elements (SREs) in the promoters of target genes and activate their transcription (4).
Three isoforms of SREBP, 1a, 1c, and 2, are known.
Whereas SREBP-2 plays a crucial role in regulation of cholesterol synthesis in any type of cell, SREBP-1c controls gene
expression of enzymes involved in synthesis of fatty acids andtriglycerides in lipogenic organs (9, 21). Meanwhile, SREBP-1a
is highly expressed in cells which are actively growing (24) andhas a strong transcriptional activity for a wide range of synthetic genes for cholesterol, fatty acids, and phospholipids. All
mammalian cells require these lipids for duplication of mem-

MATERIALS AND METHODS
Construction of the p21 luciferase gene reporter plasmids. The p21 promoter
reporter plasmid WWP-Luc (6) was a kind gift from Bert Vogelstein. The
promoter cDNAs were subcloned into pGL3-Basic (Promega) and designatedp21–2338–Luc and p21–2258–Luc. Other luciferase constructs were generated by
PCR.
Transfections and luciferase assay. HepG2 cells and Saos-2 cells were cultured in Dulbecco’s modified Eagle’s medium containing 25 mM glucose and 50
mM glucose, respectively. All medium contained 100 units/ml penicillin and 100
␮g/ml streptomycin sulfate supplemented with 10% fetal calf serum. Cells wereplated on 12-well dishes at 5.0 ⫻ 104 cells/well. Each luciferase reporter plasmid(0.5 ␮g) and Renilla luciferase control plasmid pRL-SV40 (Promegi; 0.05 ␮g)were cotransfected into the cells using FuGENE6 transfection reagent (Roche)
according to the manufacturer’s protocol. The total amount of DNA in each

* Corresponding author. Mailing address: Center for Tsukuba Advanced Research Alliance, University of Tsukuba, 1-1-1 Tennodai,
Tsukuba, Ibaraki 305-8575, Japan. Phone: 81298533053. Fax:
81298533053. E-mail: shimano-tky@umin.ac.jp.
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transfection was adjusted to 1 ␮g/well with vector DNA. Luciferase activity intransfectants was normalized to the amount of Renilla luciferase activity as
measured by a kit (dual-luciferase reporter assay system; Promega).
In vitro translation of SREBPs. The pcDNA3.1(⫹)-SREBP-1a, -SREBP-1c,
and -SREBP-2 vectors were translated in the TNT T7 Quick Coupled transcription/translation reticulocyte lysate system (Promega). Each coupled transcription-translation reaction contained 1 ␮g of plasmid DNA in a final volume of 50
␮l and incubated at 30°C for 1.5 h. For radiolabeling, the vectors were translated
in a methionine-free amino acid mixture supplemented with [35S]methionine
according to the manufacturer’s protocol.
Gel mobility shift assay. The DNA probes were prepared by annealing bothstrands of the low-density lipoprotein receptor SRE site, GAAAATCAC
CCCACTGCAAA; LDLR-SRE, the SRE site in the p21 promoter, GCGGT
GGGCCGAGCG; p21-SRE and this mutation version, GCGGTACAAAAT
GCG; p21-SRE Mut, GCGGTACGCCGTGCG; p21-SRE Mut1, GCGGTACACCGAGCG; p21-SRE Mut2, GCGGTGGGCAATGCG; p21-SRE Mut3, GC
GTGGGCCAAACG; and p21-SRE Mut4, which were labeled with [␣-32P]dCTP(3,000 to 10,000 cpm) using the Megaprime DNA labeling system (Amersham
Pharmacia Biotech), followed by purification on mini Quick Spin oligo columns(Roche). The labeled DNA was incubated with 5 ␮l of in vitro transcriptiontranslation mixture with SREBPs in 40 mM HEPES-KOH (pH 7.8), 50 mM KCl,
5 mM MgCl2, 10% glycerol, 5 mM dithiothreitol, 25 ␮g/ml poly(dI-dC), 0.1%
Triton X-100, and 1 mg/ml skim milk for 1 h at room temperature. The DNAprotein complexes were resolved on a 3.75% polyacrylamide gel at 90 V for 1 h
at room temperature. Gels were dried and exposed to BAS 2000 with BAStation
software (Fuji Photo Film).
Animals. SREBP transgenic mice overexpressing human SREBP-1a, SREBP1c, and SREBP-2 under the control of the rat phosphoenolpyruvate carboxykinase promoter were established as described (22). p21 knockout mice wereobtained from Jackson Laboratory. SREBP-1a transgenic and p21 knockout
mice were generated by intercrossing these mice. The animals were fasted 12 hprior to sacrifice.
Preparation of recombinant adenovirus. cDNAs encoding the active aminoterminal fragment of human SREBP-1a (amino acids 1 to 460), SREBP-1c(amino acids 1 to 436), SREBP-2 (amino acids 1 to 460), and dominant-negative
SREBP-1, which lacked the amino-terminal transactivation domain (89 aminoacids), were integrated into the adenovirus vector (11). SREBP adenoviral vectors were propagated in 293 cells and purified by cesium chloride density centrifugation.
Northern blot analysis. Total RNA was prepared from mouse livers andculture cells using TRIzol reagent (Life Technologys, Inc.); 24 h after transfection, the mRNA of HEK293 cells was extracted by Oligotex-dT30 ⬍Super⬎ kit(Roche). For Northern gel analysis, equal aliquots of total RNA from three miceand culture cells were pooled (total 10 ␮g or 300 ng of mRNA), denatured withformaldehyde and formamide, subjected to electrophoresis in a 1% agarose gel,and transferred to Hybond N membranes (Amersham Pharmacia Biotech) for
hybridization. cDNA probes were labeled with [␣-32P]dCTP (3,000 to 10,000cpm) using Megaprime DNA labeling system (Amersham Pharmacia Biotech).
The filters were hybridized with the radiolabeled probe in Rapid-hyb buffer(Amersham Pharmacia Biotech) at 65°C and washed in 0.1⫻ (1⫻ SSC is 0.15 M
NaCl plus 0.015 M sodium citrate)–0.1% sodium dodecyl sulfate at 65°C. Blots
were exposed to Kodak BIOMAX MS1 (EASTMAN KODAK COMPANY) andBAS 2000 with BAStation software (Fuji Photo Film).
Western analysis. Cells were cultured for 24 or 48 h after transfection ortreated with 0.1 mM isopropylthiogalactopyranoside (IPTG) and harvested in
NP-40 lysis buffer [50 mM Tris (pH 7.5), 250 mM NaCl, 5 mM EDTA, 0.5%
Nonidet P-40, 1 mM DTT and protease inhibitor (Roche)]. Protein concentrations determined by the BCA protein assay kit (PIERCE). The samples werefractionated on 8, 12, or 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, followed by transfer to Immobilon Transfer membranes (MILLIPORE). Blots were subsequently incubated in TBS-T (10 mM Tris-HCl, pH 7.4,
150 mM NaCl, 0.05% Tween 20) overnight at 4°C with 1:2,500 anti-SREBP1
anti-p21WAF1 (Santa Cruz). Following incubation with horseradish peroxidaseconjugated secondary antibodies, blots were detected by the ECL or ECL Advance Western blotting detection kit (Amersham).
Colony formation assay. Saos-2 cells which achieved ⬃70% confluence weretransfected with each plasmids (pcDNA3.1[⫹]-empty, -SREBPs, and -p21waf1).
The cells growth media were supplemented with G418 (500 ␮g/ml) 48 h later.
Approximately 3 weeks later, when microscopic colonies become detectable, thecells were washed with phosphate-buffered saline, fixed in 10% acetic acid/10%
methanol for 15 min, and stained with 0.4% crystal violet, 20% ethanol for 15
min (17) (31).

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Flow cytometry analysis. For flow cytometry, livers were suspended in phosphate-buffered saline and through a cell strainer. The hepatocytes were diluted
in 0.1% Triton X-100 and stained with 25 ␮g/ml propidium iodide and 1 mg/ml
RNase. Total cellular DNA content was determined on flow cytometry andanalyzed by FACScalibur (Becton Dickinson).

RESULTS
p21 is a novel target gene for SREBP-1a and SREBP-2.
DNA microarray analysis revealed that the bulk of lipid synthetic genes are highly upregulated in livers overexpressing
nuclear SREBP-1a from transgenic mice, confirming that
SREBP-1a is a potent transactivator of lipid synthetic genes.
Intriguingly, it was also found that expression of p21, a universal cyclin-dependent kinase inhibitor, was highly (7.5-fold) increased compared to wild-type mouse livers. To see whether
this induction was due to direct transactivation by SREBP,
promoter analysis on the human p21 gene was performed.
The human p21 promoter (2.4 kb) has been shown to fully
contain known elements for its gene regulation, including p53
binding sites (Fig. 1) (6, 7, 13). In Saos-2 cells, deficient in p53,
the basal p21 promoter activity, as estimated by luciferase
reporter assays, was very low and coexpression of p53 caused amarked induction of p21 promoter activity, confirming that p53
plays a dominant role in a basal p21 expression (Fig. 1a).
Expression of nuclear SREBP-1a induced p21 promoter activity even more strongly than p53. When the p53 binding sites
were deleted, the p53 activation of the p21 promoter was
eliminated, whereas the absence of p53 binding sites did not
influence activation of p21 by SREBP-1a. These data suggestthat SREBP activation of the p21 promoter is completely independent of p53. In HepG2 cells, which endogenously express
p53, the p21 promoter activity was robust, but still highly activated by SREBP-1a (Fig. 1b).
To identify the promoter region responsible for SREBP
activation, sequential deletion constructs were tested for
SREBP-1a activation. Through these assays in HepG2 cells(Fig. 1b) and Saos-2 cells (data not shown), an SREBP activation site was identified far downstream of p53 sites between⫺98 bp and ⫺90 bp. This region of the p21 promoter is not
related to any other known cis elements for reported p21
activators (7). As depicted in Fig. 1c, the DNA sequence of thisregion, TGGGCCGAG, was novel as an SREBP binding sitewith slight similarity to that of Diazepam binding protein/acyl
CoA binding protein, another SREBP target (25). The direct
and specific binding of SREBP to this region was also shown by
gel mobility retardation assays (Fig. 1d). Several mutations inthis region caused loss of SREBP binding and confirmed thespecificity of this sequence as p21-SRE (data not shown). The
three isoforms of the SREBP family: SREBP-1a, SREBP-1c,
and SREBP-2 have different target specificities and activities
depending upon target gene promoters (1). Upon comparisonof p21 activation among the isoforms in HepG2 cells (Fig. 2a)and Saos-2 cells (data not shown), SREBP-2 had similar oreven stronger activity than SREBP-1a whereas SREBP-1c was
the weakest. Consistently, in vitro-translated SREBP proteins
all bound to this p21-SRE (Fig. 2b).
Transcriptional regulation of p21 is mediated throughtransactivation domain of SREBPs. A mutation of tyrosine forarginine in the basic region of the basic helix-loop-helx portionof SREBP (YR mutation) is known to abolish the binding

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FIG. 1. Activation of p21 gene promoter by SREBP-1a. a. Activation of p21 promoter-luciferase by SREBP-1a and p53. Saos-2 cells,
p53-deficient osteosarcoma cell line, were transfected with the human p21 promoter firefly luciferase plasmid (p21–2338–Luc or p21–1363–Luc),
expression vector for nuclear SREBP-1a or p53, and a Renilla luciferase reference plasmid, pRL-SV40. The luciferase activity was measured andnormalized to the Renilla luciferase activity after 24 h incubation. p21–2338–Luc contains and p21–1363–Luc lacks two p53-binding sites. b.
Identification of an SREBP binding site (SRE) in p21 gene promoter. A series of deletion luciferase constructs for the human p21-promoter are
indicated in the left panel. HepG2 cells, human hepatocellular carcinoma cell line, were transfected with the indicated reporter plasmid, acytomegalovirus expression plasmid for SREBP-1a or empty plasmid, and a reference plasmid, pRL-SV40. Relative luciferase activity wasmeasured after 24 h incubation. Fold activation represents ratio of values for SREBP-1a versus a cytomegalovirus empty vector. c. The SREBP
binding site (p21-SRE) and its neighboring region in human p21 gene promoter. d. SREBP-1a directly binds to p21-SRE. Gel mobility shift assays
was performed to show the direct binding of SREBP-1a to the p21 promoter. Double-stranded DNA fragments corresponding to the LDLR-SRE(1 to 4), the original binding site for SREBP, p21-SRE (5 to 8), and p21-SRE-Mut (9 to 12) where the GTGGGCCGAG was replaced by
GTACAAAATG, labeled with [␣-32P]dCTP and incubated in the reaction mixture with (lanes 2 to 4, 6 to 8, and 10 to 12) or without (lane 1, 5,
and 9) in vitro synthesized nuclear SREBP-1a protein. Specificity of SREBP-1a binding to the p21 SRE probe was confirmed by a supershift after
the addition of SREBP-1 antibody (lane 8). In competition assays (Competitor), a 50-fold molar excess of an unlabeled SRE complex DNA (lane7) was added to the incubation. RL, reticulocyte lysate; 1a, SREBP-1a.

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FIG. 2. Binding characteristics of SREBP isoforms to the p21 gene promoter. a. Effects of SREBP isoforms and YR mutants on the p21
promoter. Luciferase assays for the p21–2338–Luc were compared among nuclear SREBP isoforms, and their respective YR mutants (SREBP1aM ⫺1 cM and ⫺2 M), in which the tyrosine residue in the basic region of each SREBP isoform was replaced by arginine. These mutants werecapable to bind to E-boxes as an innate feature of basic helix-loop-helx protein, but not to the SRE sites, SREBP-specific binding sites (12). cont,cytomegalovirus empty vector; BP1a, SREBP-1a; BP1c, SREBP-1c; BP2, SREBP-2. b. Gel mobility shift assay of nuclear SREBP-1 and -2. A
double-stranded DNA fragment corresponding to p21-SRE was labeled with [␣-32P]dCTP and incubated in the reaction mixture with (lanes 2, 3,
5, and 6) or without (lanes 1 and 4) in vitro synthesized nuclear SREBP-1a (1 to 3) or -2 (4 to 6) protein. Since SREBP-1a and -1c share thecommon basic helix-loop-helix domain, these data indicate that all the SREBP isoforms bind to the p21-SRE with similar affinities. c. The p21-SRE
has an enhancer activity in the activation of p21 gene promoter by SREBPs. An SV40 promoter luciferase reporter for estimation of a region
containing the upstream Sp1 site and p21-SRE as an enhancer was constructed as indicated. Deletion and mutation analysis were performed with
reporter genes in the indicated positions of the promoter. HepG2 cells were transfected with each reporter plasmid, an expression plasmid fornuclear SREBP-1a or SREBP-2, reference plasmid pRL-SV40. Luciferase activity was measured and normalized to pRL-SV40 activity after 24 hincubation. Fold induction of luciferase activity by SREBPs is shown. d. Deletion of transactivation domain of SREBP-1 suppresses activation ofp21 promoter by SREBP-1a and SREBP-2 in dominant negative fashion. SREBP-1 dominant negative form (DN-SREBP-1) lacks the aminoterminal transactivation domain (89 amino acids) and has the ability to bind to SRE site but inhibits transcriptional activities by native SREBPs
for known SREBP target promoters (23). HepG2 cells were transfected with the p21–2338–Luc, SREBP expression vector (SREBP-1a or -2), andthe indicated DNA amount of the dominant-negative SREBP-1 expression vector. Luciferase activity was measured after 24 h incubation.

activity of SREBP to SRE, but retains binding to the E-box asa general property of basic helix-loop-helix proteins (12). The
YR mutation of SREBPs markedly reduced activation of thep21 promoter activity by SREBPs, suggesting that SREBPs
bind to the p21-SRE in an SREBP-specific manner. This p21-

SRE possessed an enhancer activity when it was put next to thesimian virus promoter for the test of SREBP activation (Fig.
2c).
Previous reports demonstrated that coactivators such as Sp1and NF-Y are essential for SREBP activation in the promoters

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FIG. 3. SREBP-1a induces p21 at mRNA and protein levels. a. p21 mRNA induction by overexpression of SREBPs in HEK293 cells. HEK293
cells, human kidney cell line were transfected with the indicated cytomegalovirus promoter expression plasmids. Total RNA was isolated from cells
24 h after transfection for Northern blot analysis (10 ␮g per lane). cont, cytomegalovirus empty vector; BP1a, SREBP-1a; BP1cm, SREBP-1c; BP2,
SREBP-2. b. p21 protein induction by overexpression of SREBPs in HEK293 and Saos-2 cells. The cells were transfected with indicatedcytomegalovirus promoter expression plasmid. Total cellular proteins from cells, 24 h after transfection of indicated expression plasmids. The
protein was subjected to immunoblot analysis for SREBP-1 and SREBP-2 and p21 (42.5 ␮g per lane). c. p21 mRNA induction by overexpressionof SREBPs in hepatocytes. Rat primary hepatocytes were infected with control adenovirus expressing GFP or an adenoviral vector expressing
SREBPs at an MOI of 100. Total RNA was isolated from cells 48 h after infection for Northern blot analysis (10 ␮g per lane). GFP, green
fluorescent protein for controls; DNBP, dominant-negative form of SREBP-1 described in the legend to Fig. 2d. d. p21 gene expression in livers
from SREBP transgenic mice. Mice overexpressing a nuclear active form of human SREBP-1a, SREBP-1c, and SREBP-2 in the liver were fed ahigh-protein/low-carbohydrate diet for a week and fasted overnight to induce the transgene under the phosphoenolpyruvate carboxykinase(PEPCK) promoter. Total liver RNAs were subjected to Northern blot analysis to estimate gene expression with the indicated cDNA probes. WT,
wild-type; BP1a, SREBP-1a transgenic mice; BP1c, SREBP-1c transgenic mice; BP2, SREBP-2 transgenic mice. e. Cellular p21 protein level in
SREBP-1a overexpression. In the left panel, nuclear extract proteins from livers of wild-type or SREBP-1a transgenic mice were subjected toimmunoblot analysis with antibody against SREBP-1 and p21. In the center panel, total cellular proteins from HepG2, 24 or 48 h after transfectionof SREBP-1a expression plasmid, were subjected to immunoblot analysis with antibody against p21 (40 ␮g per lane). In the right panel, CHO stable
cell lines that inducibly express nuclear SREBPs under IPTG-regulated promoter (18) were incubated in 0.1 mM IPTG-containing medium. Eachcell was harvested in NP-40 buffer at the indicated time. Proteins were subjected to immunoblot analysis for SREBP-1 and p21 (100 ␮g per lane).

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of most of SREBP target genes (3) (26) (2). Although there areseveral Sp1 sites in the p21 promoter which were reported to
be involved in p21 gene regulation (Fig. 1c), Sp1 was dispensable in SREBP activation of p21 promoter, as shown in deletion and mutation analyses (Fig. 2c). A dominant negative
mutant of SREBP-1 in which the amino-terminal transactivation domain was deleted had no activity for the p21 promoter,and further inhibited activation of the p21-SRE construct by
cotransfected native SREBP-1 or SREBP-2 in a dose-dependent manner (Fig. 2d). These data imply that the transactivation domain of SREBP-1 is essential for its activation of thep21 promoter, as reported in other regular SREBP target
genes, presumably through recruitment of coactivators such as
CBP/p330 (3).
Only SREBP-1a strongly and consistently induces p21 protein. Expression of p21 following SREBP activation of the p21
promoter was investigated. In 293 cells, both endogenous p21
mRNA and protein were detected by Northern and immunoblot analyses, respectively (Fig. 3a and b). After the transient
expression, SREBP-1a and SREBP-2 but not SREBP-1c elevated p21 mRNA levels with resultant increases in cellular p21
levels. Induction of p21 protein by SREBP-1a and SREBP-2
expression was more prominent in p53-deficient Saos-2 cells(Fig. 3b). Conversely, overexpression of dominant-negative
SREBP reduced endogenous p21 protein in these cell lines,
confirming the specific SREBP activation of p21. These datasuggest that overexpression of SREBP-1a and SREBP-2 canactivate p21 expression in a manner consistent with the resultsfrom the luciferase assays. In contrast, when rat primary hepatocytes were infected with adenoviral SREBP expression vectors, only SREBP-1a caused appreciable activation of p21 expression (Fig. 3c).
As a model for sustained SREBP activation, hepatic expression of p21 was compared in livers overexpressing nuclear
SREBPs from transgenic mice (22). SREBP-1a markedly increased hepatic p21 mRNA levels, consistent with DNA microarray results (Fig. 3d). The p21 protein was also markedly
induced in SREBP-1a-expressing livers (Fig. 3e). Elevation ofp21 protein by SREBP-1a was also observed in HepG2 cells
after the transient transfection and stably inducible CHO cells(19). In contrast, constitutive activation of SREBP-2 only modestly elevated p21 expression in transgenic livers, althoughmRNA for LDLR, an authentic SREBP target gene, was fully
activated in this transgenic line, whereas SREBP-1c was essentially inactive for p21 (Fig. 3d). These data suggest that although both SREBP-1a and SREBP-2 have similar transcription activities for the p21 promoter, there is differential
posttranscriptional regulation of p21 depending upon SREBP
isoform and cell type.
Nuclear SREBP-1a inhibits cell growth. To see the consequences of SREBP activation of p21 expression in cell growth,colony formation assays were performed in stably transfected
Saos-2 cells. As shown in Fig. 4a, permanent transfection of acontrol vector containing the neomycin resistance gene generated a substantial number of surviving colonies in the presenceof G418. Cotransfection of the p21 expression plasmid significantly decreased the number of resistant colonies, showing its
inhibitory effect on cell growth. SREBP-1a and SREBP-2 exhibited an inhibitory effect slightly less than that of p21,
whereas SREBP-1c and dominant-negative SREBP-1 had es-

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FIG. 4. Induction of p21 by SREBP-1a leads to cell growth arrestas evidenced by colony formation assays with Saos-2 cells (a) andhepatocyte polyploidy of SREBP-1a transgenic and p21 null mice (b).
a. Saos-2 cells were transfected with 10 ␮g of the indicated expression
plasmids containing neomycin resistance gene; 24 h after transfection,
the cells were cultured in medium containing 500 ␮g/ml of G418 for 3
weeks. The resultant survival cell colonies were fixed and stained withviolet blue as representatively indicated in the upper panel. The values
represent relative colony numbers normalized to an empty vector control. b. The hepatocytes were prepared for livers from the indicatedmouse lines. The DNA content was determined by flow cytometry after
DNA staining with propidium iodide. Hepatocyte ploidy is shown as
2n, 4n, and 8n. WT, C57BL/6J; Tg1a, SREBP-1a transgenic mice;
p21KO, p21 knockout mice; Tg1a⫻p21KO, SREBP-1a transgenic andp21 knockout double mutant mice. **, P ⬍ 0.01, and *, P ⬍ 0.05 versus
wild-type; #, P ⬍ 0.05 versus SREBP-1a transgenic mice (Student’s ttest).

sentially no effect. These data suggest that p21 activation by
SREBP-1a and SREBP-2 could cause cell growth inhibition.
The effects of p21 on cell cycle were also estimated in thelivers of SREBP-1a transgenic mice. As shown in Fig. 4b, flow
cytometry demonstrated that a considerable proportion of normal hepatocytes exhibit polyploidy (4n and 8n). The absence of
p21 did not changes these patterns of polyploidy in normal
hepatocytes. Compared to control livers, SREBP-1a expression caused a significant increase in 2n cells, indicating theG0/G1 stage, with concomitant decreases in 4n and 8n cells,
supporting increased activity of p21 for G1 arrest induced by
SREBP-1a. p21 contributed roughly 50% to these changes ofpolyploidy by SREBP-1a, as estimated in transgenic SREBP-

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FIG. 5. Activation of p21 promoter and induction of p21 protein by endogenous SREBPs in Saos-2 cells. Saos-2 cells were transfected with eachreporter plasmid, SRE-Luc (a and c) or p21–2338–Luc (b and d), a reference plasmid pRL-CMV, and incubated at the indicated conditions toactivate endogenous SREBPs by delipidated serum (a to d) and further pravastatin, an HMGCoA reductase inhibitor (e to h). Luciferase activity
was measured and normalized to pRL-CMV activity after 24 h incubation (a, b, e, and f). Total cellular (40 ␮g) (c and d) and nuclear (10 ␮g) (gand h) proteins were subjected to immunoblot analysis for SREBP-1 and SREBP-2 (c and g), and p21 (d and h). FCS, 5% fetal calf serum; DLS,
5% delipidated serum; statin, 50 ␮M pravastatin; sterols, 10 ␮g/ml cholesterol and 1 ␮g/ml 25-hydroxychoresterol.

1a/p21-knockout double mutant mice. These data provide supporting evidence that SREBP-1a overexpression could activate
p21 and could affect cell proliferation in vivo as well as incultured cells.
Endogenous SREBP-1a regulates p21 expression. To test
the physiological relevance to SREBP regulation of p21, theeffects of endogenous SREBPs on p21 expression were esti-

mated. Saos-2 cells were cultured in delipidated serum andfurther treated with pravastatin, a 3-hydroxy-3-methylglutaryl
coenzyme A reductase inhibitor. These treatments are known
to increase endogenous nuclear SREBP-1a and SREBP-2
through activation of the SCAP/INSIG system. As shown by
luciferase assays in Fig. 5a, the SRE-Luc reporter as an indicator of endogenous SREBPs was strongly activated by delipi-

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FIG. 6. Disruption of endogenous SREBPs suppressed p21 expression. a. Saos-2 cells infected with control adenovirus expressing GFP or an
adenoviral vector expressing dominant-negative SREBP-1 at a multiplicity of infection of 30. Total cellular proteins were isolated from cells
incubated at the indicated conditions 24 h after infection. Total proteins (35 ␮g) were subjected to immunoblot analysis for SREBP-1, SREBP-2,
and p21. b. Northern blot analysis was performed to measure p21 mRNA in livers and white adipose tissue (WAT) from wild-type (WT) and
SREBP1 knockout (BPKO) mice. The animals were refed a high-sucrose/fat-free diet for 12 h after 24 h of starvation.

dated serum. Addition of sterols (25OH cholesterol and cholesterol) completely cancelled this activation. With a similar
pattern, the p21 promoter was also activated and repressed bysterol deprivation and supplementation, respectively (Fig. 5b).
In the same set of experiments using immunoblot analysis, it
was shown that p21 protein was consistently regulated in accordance with changes in endogenous nuclear SREBP-1a and
SREBP-2 proteins depending upon cellular sterol states (Fig.
5c and d). Treatment with pravastatin, another manipulation
for sterol deprivation, and sterols provided similar results (Fig.
5e to h). The regulation of p21 promoter activity by endogenous SREBPs by delipidated serum, statin, and sterols was also
observed in 293 cells (data not shown). Endogenous SREBPs
were responsible for the induction of p21 by incubation indelipidated serum because adenoviral overexpression of thedominant-negative form of SREBP-1 cancelled the inductionof p21 observed in adenovirus-green fluorescent protein-infccted Saos-2 cells incubated in delipidated serum (Fig. 6a).
These data demonstrate that endogenous SREBPs can regulate p21 promoter in a physiological range of sterol regulation.
Finally, p21 expression was significantly decreased in livers andadipose tissue of SREBP-1 null mice compared to wild-type

mice, providing more support that SREBP-1 can participate inendogenous p21 expression (Fig. 6b and c).

DISCUSSION
The current studies first demonstrate that the cyclin-dependent kinase inhibitor p21 is an SREBP target gene.
SREBPs activate the p21 promoter as strongly as p53. The
p21 mRNA promoter contains a new SREBP binding site
(p21-SRE). Promoter analysis with mutated SREBPs andDNA probes supports that p21 activation is mediatedthrough transcriptional activity of SREBPs by binding tothis site. Although both SREBP-1a and SREBP-2 highly
activate the p21 promoter, there is a discrepancy in the
resultant elevation of p21 mRNA and protein levels depending upon the experimental conditions and cell types. Only
SREBP-1a strongly and consistently activated p21 protein inliver. To sustain high p21 expression, it is likely to require
some posttranscriptional regulation where SREBP-1a but
not other isoforms could stabilize p21 mRNA and protein
levels.
The importance of posttranscriptional regulation of p21 has

8946

INOUE ET AL.

been recognized, especially with regard to the involvement of
mitogen-activated protein kinases in stabilization of p21 (15).
Supportively, we observed that induction of SREBP-1a and not
SREBP-2 activates ERK1/2 (p42/44) in the inducibly expressing CHO cell line, which might contribute to high p21 protein
level in the CHO-SREBP-1a line (data not shown). Neither
p38 nor JNK was changed in these cells. Further studies areneeded to clarify the precise mechanism by which nuclear
SREBPs determine the p21 protein level.
The physiological relevance to p21 activation of SREBP-1a
is yet to be fully clarified. Reduced p21 expression in SREBP1-deficient mice suggested that SREBP-1 could physiologicallycontribute to p21 regulation. The data from cultured cells in
conditions of lipid deprivation demonstrated that SREBP activation of p21 can occur in a physiological range of sterol
regulation. For cellular cholesterol homeostasis, nuclear
SREBP-2 is a key player that is strictly regulated by the sterolregulated cleavage system composed of SCAP/INSIG (30) depending upon the cellular cholesterol balance. However, actively growing cells could still be in danger of a deficiency oflipids, where SREBP-1a should be activated to support
SREBP-2 and in some severe conditions could transiently sustain cell growth at G1 for lipid synthesis through p21 activation.
This hypothesis is applicable to cells and tissues highly expressing SREBP-1a: cultured cells, embryonic tissues, adult intestine, and lymphoid tissues.
SREBP-1a is partly under control of the sterol-sensing
SCAP/INSIG system, but seems to be activated independently,
which suggests the presence of an unidentified regulatory function(s) of SREBP-1a other than sterol regulation. In contrast,
SREBP-1c is the major SREBP-1 isoform in differentiatedtissues and not in cultured cells, is nutritionally regulated, andcontrols synthesis of fatty acids and triglycerides (9, 20). p21
was not induced by this nutritional regulator.
p21 gene is also known as a p53 target, induced by DNA
damage such as radiation and UV (6). One potential mechanism to be considered is that accumulation of lipids in cells
caused by SREBP-1a could be a cellular stress to induce p53
and thus p21. We recently reported that p53 and p21 areactivated in nutritionally dysregulated tissues such as in enlarged adipose tissues and fatty livers of leptin-deficient ob/ob
mice (28, 29). In fatty livers from mutant mice with different
genotypes of leptin and SREBP-1 genes, there was a strong
correlation between hepatic triglyceride and p21 mRNA levels.
This hepatosteatosis-associated mechanism, presumably related to some stress signal, could contribute to the marked induction of p21 in SREBP-1a transgenic fatty livers in addition to thedirect and p53-independent induction by SREBP-1a.
p21 is also induced by contact inhibition and differentiation,and numerous factors such as STAT family, C/EBP, retinoic
acid receptor, vitamin D receptor, MyoD, Sp1, Sp3, AP2, andcalcitonin have been identified by extensive promoter analysis(reviewed in reference 7). In this aspect, activation of p21 by
SREBP-1a is unique because SREBP-1a is linked to growth,which is opposed to the generally accepted functions of p21. In
our preliminary data (M. Nakakuki and H. Shimano, unpublished observation), nuclear SREBP-1a causes G1 cell cycle
arrest and inhibits cell proliferation through diverse mechanisms which are currently under investigation. Activation of
p21 as a new SREBP target at least partially explains thisMOL. CELL. BIOL.

unexpected antiproliferative activity of SREBP-1a. Our current data imply that lipid synthesis is not simply under regulation of growth, but is actively involved in the regulation of cell
growth. Further investigations on the link between lipid synthesis and cell growth, especially in the light of the cell cycle,
should be pursued.
ACKNOWLEDGMENTS
We thank Bert Vogelstein for p21 promoter plasmid WWP-Luc andA. H. Hasty for critical reading of the manuscript.
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