Androgens Stimulate Lipogenic Gene Expression in Prostate Cancer Cells by Activation of the Sterol Regulatory Element-Binding Protein Cleavage Activating Protein/Sterol Regulatory Element-Binding Protein Pathway
Hannelore Heemers,
Bart Maes,
Fabienne Foufelle,
Walter Heyns,
Guido Verhoeven and
Johannes V. Swinnen
Laboratory for Experimental Medicine and Endocrinology (H.H., B.M.,
W.H., G.V., J.V.S.), Faculty of Medicine, Onderwijs en Navorsing,
Gasthuisberg, K.U. Leuven, B-3000 Leuven, Belgium; and U465 Institut
National de la Santé et de la Recherche Médicale (F.F.),
Centre de Recherches Biomédicales des Cordeliers,
Université Paris 6, 75270 Paris, France
 |
ABSTRACT
|
---|
Using two independent prostate cancer cell lines (LNCaP and
MDA-PCa-2a), we demonstrate that coordinated stimulation of lipogenic
gene expression by androgens is a common phenomenon in
androgen-responsive prostate tumor lines and involves activation of the
sterol regulatory element-binding protein (SREBP) pathway. We show 1)
that in both cell lines, androgens stimulate the expression of fatty
acid synthase and hydroxymethylglutaryl-coenzyme A
synthase, two key lipogenic genes representative for the fatty acid and
the cholesterol synthesis pathway, respectively; 2) that treatment with
androgens results in increased nuclear levels of active SREBP; 3) that
the effects of androgens on promoter-reporter constructs derived
from both lipogenic genes (fatty acid synthase and
hydroxymethylglutaryl-coenzyme A synthase) depend on the presence of
intact SREBP-binding sites; and 4) that cotransfection with
dominant-negative forms of SREBPs abolishes the effects of
androgens. Related to the mechanism underlying androgen activation of
the SREBP pathway, we show that in addition to minor effects on
SREBP precursor levels, androgens induce a major increase in
the expression of sterol regulatory element-binding protein
cleavage-activating protein (SCAP), an escort protein that transports
SREBPs from their site of synthesis in the endoplasmic reticulum to
their site of proteolytical activation in the Golgi. Both time course
studies and overexpression experiments showing that increasing levels
of SCAP enhance the production of mature SREBP and stimulate lipogenic
gene expression support the contention that SCAP plays a pivotal role
in the lipogenic effects of androgens in tumor cells.
 |
INTRODUCTION
|
---|
FOR ALMOST 60 yr, androgens have been known
to play a key role in the biology of prostate cancer (1).
As illustrated by the successful results of androgen ablation therapy
(at least during the initial stages of tumor development) androgens are
absolutely required for survival and proliferation of prostate cancer
cells. Moreover, they affect many other cellular functions, including
differentiated secretory function and lipogenesis (2, 3, 4).
The latter process is highly active in many cancer cells and has been
associated with malignant progression and poor prognosis in several
cancers, including prostate cancer (5, 6, 7, 8, 9, 10).
Examination of the mechanism by which androgens affect lipogenesis in
LNCaP prostate cancer cells revealed that (at least in this cell line)
androgens coordinately stimulate the expression of several genes
involved in the synthesis, transport, and metabolism of both fatty
acids and cholesterol (11). This finding led us to suspect
that androgens do not affect the expression of all these enzymes
individually but rather induce or activate a transcription factor that
modulates and coordinates the expression of all these genes. In view of
their central role in the coordinate control of lipogenic gene
expression, sterol regulatory element-binding proteins (SREBPs) were
put forward as potential mediators of the lipogenic effects of
androgens in LNCaP cells (11).
SREBPs are a family of three basic helix-loop-helix leucine zipper
transcription factors (SREBP-1a, -1c, and -2) that have been identified
as transacting factors involved in the maintenance of intracellular
cholesterol homeostasis, the control of fatty acid metabolism, and the
differentiation of adipocytes (12, 13, 14). Synthesized as
125-kDa precursor proteins, SREBPs are anchored to intracellular
membranes where they form a complex with SREBP cleavage-activating
protein (SCAP). In sterol-deprived cells this SREBP/SCAP complex
travels to the Golgi apparatus where a 68-kDa amino-terminal SREBP
segment is released by a two-step mechanism of regulated intramembrane
proteolysis (Rip). This transcriptionally active SREBP fragment
migrates to the nucleus, where it activates the transcription of a
large set of sterol-regulatory element (SRE)-containing genes encoding
lipogenic enzymes belonging to the two major lipogenic pathways (fatty
acid synthesis and cholesterol synthesis) (15, 16, 17).
Experiments aimed at assessing the (putative) role of SREBPs in the
induction of lipogenesis by androgens revealed that (at least in LNCaP
cells) androgens enhanced the expression of SREBP precursor proteins
and led to increased nuclear levels of mature active SREBP. In support
of the involvement of SREBPs as mediators of the lipogenic effects of
androgens, the stimulatory effects of androgens on the expression of a
key lipogenic enzyme (FAS) were shown to be mediated by a complex
cis-acting element harboring SREBP binding sites
(11). Nevertheless, as these experiments have been
restricted to LNCaP cells and as this cis-acting element has
also been shown to bind other transcription factors such as upstream
stimulatory factor and other E box binding proteins
(18), final proof for the existence of an SREBP-mediated
mechanism of androgen action in prostate cancer cells demanded further
studies. Moreover, the exact mechanism by which androgens would
affect the SREBP pathway remained to be investigated.
In this report, using a second independent androgen-responsive
prostate cancer cell line (MDA-PCa-2a), in addition to LNCaP cells,
alternative promoter-reporter constructs derived from a second
androgen-regulated lipogenic gene [hydroxymethylglutaryl-coenzyme
A (HMG-CoA)-synthase, belonging to the pathway of cholesterol
synthesis] as well as dominant-negative forms of SREBPs, we
provide this final proof. Moreover, we present evidence that induction
of SCAP expression may play a pivotal role in the activation of the
SREBP pathway by androgens.
 |
RESULTS
|
---|
Androgens Stimulate Lipogenic Gene Expression in Both LNCaP and
MDA-PCa-2a Prostate Cancer Cells
In a previous study we demonstrated that, in the prostate cancer
cell line LNCaP, androgens coordinately regulate the mRNA expression of
several genes involved in the synthesis of fatty acids and cholesterol
(11). To show that this regulation is not a peculiarity of
LNCaP cells, we examined the effect of androgens on the expression of
two key lipogenic genes (belonging to either the fatty acid synthesis
pathway or the cholesterol synthesis pathway) in another independent
androgen-responsive prostate cancer cell line, MDA-PCa-2a
(19). To this end, cells were incubated for 2 d in
the presence or absence of 10-8 M of
the synthetic androgen R1881. Total RNA was prepared and subjected to
Northern blot analysis with probes for both FAS and HMG-CoA-synthase.
As demonstrated in Fig. 1
, and rogens
stimulate the expression of these genes in both LNCaP and MDA-PCa-2a
cells. The absolute expression levels and the relative fold induction
of these genes were different, however, in both cell lines. Both basal
and androgen-induced FAS expression was higher in MDA-PCa-2a cells than
in LNCaP cells. In contrast, HMG-CoA-synthase expression and induction
were more pronounced in LNCaP cells.

View larger version (72K):
[in this window]
[in a new window]
|
Figure 1. Androgen Regulation of Lipogenic Gene Expression in
Two Independent Prostate Cancer Cell Lines
LNCaP cells and MDA-PCa-2a cells were cultured in the absence (-) or
in the presence (+) of 10-8 M of the synthetic
androgen R1881 for 2 d. Total RNA was prepared and 20 µg
aliquots were subjected to Northern blot analysis with
32P-labeled probes for fatty acid synthase (FAS) or
HMG-CoA-synthase (SYN) followed by autoradiography. Blots were stripped
and rehybridized with 18S to demonstrate that equal amounts of RNA were
present in all lanes. The data shown are representative of two
independent experiments.
|
|
Androgen Treatment Results in Increased Nuclear Levels of Mature
SREBP-1 in both LNCaP and MDA-PCa-2a Cell Lines
To test whether the observed increase in the expression of
lipogenic enzymes is accompanied by activation of the SREBP pathway,
nuclear extracts were prepared from cells cultured in the absence or
presence of 10-8 M R1881 for 2
d. Equal amounts of protein were analyzed by immunoblot analysis using
an antibody directed against the mature amino-terminal part of SREBP-1
(recognizes both SREBP-1a and SREBP-1c). As Fig. 2
shows, androgen treatment clearly
results in an increase in mature SREBP-1 levels in nuclei of both LNCaP
and MDA-PCa-2a cells, indicating androgen-mediated SREBP activation in
both prostate cancer cell lines. SREBP-2 levels could not be studied
because of the lack of suitable antibodies recognizing mature SREBP-2
in human prostate cancer cells.

View larger version (30K):
[in this window]
[in a new window]
|
Figure 2. Effects of Androgens on the Nuclear Content of
Mature SREBP-1
After incubation for 2 d in the absence (-) or presence (+) of
10-8 M R1881, LNCaP, and MDA-PCa-2 cells were
harvested and nuclear fractions were prepared. Equal amounts of protein
were subjected to SDS-PAGE and Western blot analysis with IgG-2A4, a
monoclonal antibody directed against the N-terminal domain of SREBP-1.
Immunoreactive bands were visualized using a peroxidase-coupled goat
antimouse secondary antibody and a chemiluminescence Western blot
analysis system. The data shown are representative of two independent
experiments.
|
|
Effects of Androgens on FAS and HMG-CoA-Synthase Promoter-Reporter
Constructs Depend on the Presence of Intact SREBP-Binding Sites
To investigate whether this androgen-induced elevation of nuclear
SREBP levels is responsible for the observed increase in lipogenic gene
expression, we performed transient transfection experiments using
promoter-reporter constructs derived from the two lipogenic genes
studied above. In a first series of experiments, LNCaP and MDA-PCA-2a
cells were transiently transfected with a 178-bp FAS promoter-reporter
construct (FASwt) harboring a complex SREBP-binding site
(20), or with a truncated construct that lacks this region
(FASdelSRE) (Fig. 3A
). Androgen treatment
of LNCaP cells resulted in a 3-fold increase in reporter activity for
the wild-type construct, and this stimulation was completely abolished
when the SREBP-binding site was deleted (Fig. 3B
). Very similar results
were obtained in MDA-PCa-2a cells (Fig. 3C
). A second set of
experiments using promoter-reporter constructs derived from the
HMG-CoA-synthase gene further demonstrated the importance of intact
SREBP-binding sites for the androgen induction of lipogenic gene
expression. As shown previously (21) and as confirmed in
our studies (data not shown), the HMG-CoA-synthase gene harbors two
well defined SREs (SRE-1 and SRE-2) (Fig. 4A
), the first of which is the most
critical one to confer responsiveness to the SREBP pathway. Figure 4B
shows that androgen exposure of LNCaP cells transfected with the
wild-type HMG-CoA-synthase promoter-reporter construct (SYNwt) gave
rise to a 5- to 6-fold induction of reporter activity. Disruption of
SRE-1 (SYNmutSRE-1) completely abolished the effects of androgens
whereas mutation of SRE-2 (SYNmutSRE-2) partially decreased the
response. Androgen treatment of MDA-PCa-2a cells transfected with these
promoter-reporter constructs led to similar conclusions (Fig. 4C
). The
lower reporter activities obtained by transfection of HMG-CoA-synthase
reporter constructs in MDA-PCa-2a cells both under basal and under
androgen-stimulated conditions are consistent with the lower basal
expression and lower androgen induction of the HMG-CoA-synthase gene in
this cell line (see Fig. 1
).

View larger version (20K):
[in this window]
[in a new window]
|
Figure 3. Involvement of SREBP-Binding Sites in the
Activation of FAS Promoter-Reporter Constructs by Androgens
LNCaP cells and MDA-PCa-2a cells were transiently transfected with a
plasmid containing a luciferase reporter gene driven by a 178-bp FAS
promoter fragment harboring a SREBP-binding site (SRE), flanked by
auxiliary NF-Y and Sp-1 sites (FASwt) or with a similar
construct in which the SRE was deleted (FASdelSRE) (A). The next day,
cells were treated with 10-8 M R1881. After
3 d of androgen exposure, cells were lysed and luciferase
acitivity was measured. Luciferase activity obtained from LNCaP cells
(B) and MDA-PCa-2a cells (C) was expressed as relative luciferase units
(RLU). Data shown are representative of three independent experiments.
Columns, Mean of three incubations performed in
triplicate; bars, SEM.
|
|

View larger version (19K):
[in this window]
[in a new window]
|
Figure 4. Involvement of SREBP-Binding Sites in the
Activation of HMG-CoA-Synthase Promoter-Reporter Constructs by
Androgens
LNCaP cells and MDA-PCa-2a cells were transiently transfected with a
plasmid containing a luciferase reporter gene driven by a
HMG-CoA-synthase promoter fragment harboring two well defined SREs
(SRE-1 and SRE-2) flanked by auxiliary binding sites for NF-Y
and Sp-1 (SYNwt), or with similar constructs in which either SRE-1 or
SRE-2 was mutated (SYNmutSRE-1 and SYNmutSRE-2) (A). The next day,
cells were treated with 10-8 M R1881. After
3 d, cells were lysed and luciferase activity was measured.
Luciferase activity obtained from LNCaP cells (B) and MDA-PCa-2a cells
(C) was expressed as relative luciferase units (RLU). Data shown are
representative of three independent experiments.
Columns, Mean of three incubations performed in
triplicate; bars, SEM.
|
|
Dominant-Negative Forms of SREBPs Abolish the Effects of Androgens
on Lipogenic Gene Expression
To further corroborate the role of SREBPs in the transcriptional
activation of lipogenic genes by androgens, we introduced a
dominant-negative form of SREBP-1 (DN-SREBP) in LNCaP cells. This
dominant-negative form was generated by deleting the amino-terminal
transactivation domain of SREBP-1. The resulting protein is still able
to bind to SREs but is transcriptionally inactive and blocks the access
of wild-type endogenous SREBPs to SREs (22).
Cotransfection of LNCaP cells with increasing amounts of an expression
construct encoding DN-SREBP almost completely abolished the stimulatory
effects of androgen treatment on the activity of the HMG-CoA-synthase
promoter-reporter construct (Fig. 5A
). A similar decrease in androgen
responsiveness was also observed when the FAS promoter-reporter
construct was used, although in this case the effects were complicated
by a stimulatory effect of DN-SREBP on basal FAS expression.
Cotransfection with empty expression vector was without effect (data
not shown).

View larger version (24K):
[in this window]
[in a new window]
|
Figure 5. Effects of Dominant-Negative SREBP-1 on the
Induction of FAS and HMG-CoA-Synthase Promoter Activity By Androgens
A, LNCaP cells were transiently transfected with the same wild-type FAS
(FAS) and HMG-CoA-synthase (SYN) promoter-reporter constructs as in
Figs. 3 and 4 and increasing amounts of a construct encoding a
dominant-negative form of SREBP-1 (DN-SREBP). The next day, cells were
treated with 10-8 M R1881. After 3 d,
cells were lysed, and luciferase activity was measured and expressed as
relative luciferase units (RLU). Data shown are representative for two
independent experiments. Columns, Mean of incubations
performed in triplicate; bars, SEM. B,
MDA-PCa-2a cells were cultured either without adenovirus (No virus),
with adenovirus expressing a dominant-negative form of SREBP-1 (Ad-DN,
30 pfu/cell), or with adenovirus without insert (Ad-Null). The next
day, 10-8 M R1881 (+) or ethanol vehicle (-)
was added. Two days later, cells were harvested, followed by RNA
isolation and Northern blot analysis of FAS expression.
|
|
To further strengthen these findings we used an alternative
dominant-negative form of SREBP. This alternative DN-SREBP was
generated by introduction of a point mutation in the amino-terminal
fragment of SREBP-1c (amino acids 1403), replacing tyrosine at amino
acid 320 by alanine. The resulting protein no longer binds to SREs but
is still able to dimerize, leading to a decreased availability of
endogenous SREBP-1 (23). We used an adenoviral delivery
system allowing the study of the effect of DN-SREBP on endogenous
genes. As shown in Fig. 5B
, infection with adenovirus encoding this
DN-SREBP completely abolished the effects of androgens on endogenous
FAS expression. Also here, the dominant-negative SREBP caused a slight
elevation of basal FAS expression. Similar experiments were performed
with an adenoviral vector lacking the DN insert to exclude the
possibility that the observed reduction of FAS induction was due to
nonspecific adverse effects related to the viral infection. Infection
with the empty virus (Ad-Null) had little or no effect on the androgen
induction of FAS.
Androgens Markedly Stimulate the Expression of SCAP in Prostate
Cancer Cells
Having established that the involvement of SREBPs in the
coordinate regulation of lipogenic gene expression by androgens is not
an event limited to LNCaP cells, we attempted to unravel the mechanism
underlying the androgen activation of the SREBP pathway. We first
investigated whether androgens somehow affect the expression of
essential components of the SREBP pathway, such as the SREBP precursor
proteins (SREBP-1a, -1c, and -2), the enzymes involved in the
proteolytic cleavage of these precursor proteins [site 1 protease
(S1P) and site 2 protease (S2P)], or the transporter protein SCAP. To
this end, LNCaP cells were grown for 2 d in the presence or
absence of R1881 (10-8 M). Total RNA
was prepared and subjected to Northern blot analysis with probes for
the above mentioned components. As shown by Fig. 6A
, and rogen treatment caused a slight
increase in mRNA expression for SREBP-1c and SREBP-2. SREBP-1a and S1P
were not affected. Slight changes were also observed in the levels of
S2P mRNA expression. Major stimulatory effects were noticed only for
SCAP. This androgen-induction of SCAP expression was confirmed at the
protein level (Fig. 6B
).

View larger version (42K):
[in this window]
[in a new window]
|
Figure 6. Effect of Androgens on the Expression of Essential
Components of the SREBP Pathway
A, LNCaP cells were cultured in the presence (+) or absence (-) of
10-8 M R1881 for 2 d. Total RNA was
isolated and Northern blot analysis was performed with probes for the
indicated genes. Hybridization with 18S was performed to correct for
loading differences (not shown). Data shown are representative of two
independent experiments. B, After exposure for 2 d to
10-8 M R1881 (+) or ethanol vehicle (-),
LNCaP cells were harvested and total cell extracts were prepared. Equal
amounts of protein were subjected to Western blot analysis with a
polyclonal antibody directed against SCAP. Data shown are
representative of two independent experiments.
|
|
Enhanced Expression of SCAP Activates the SREBP Pathway
Given the presence of an SRE-like element in the 5'-flanking
region of the human SCAP gene (24), the key question was
addressed whether increased SCAP expression is actively involved in
androgen-induced SREBP activation or is merely a consequence of this
activation. To address this question, we cultured LNCaP cells under
conditions that activate the SREBP pathway independently of
androgens, i.e. sterol depletion. As shown in Fig. 7
, sterol depletion induced a marked
increase in the expression of HMG-CoA-synthase but had little
effect on SCAP expression.

View larger version (54K):
[in this window]
[in a new window]
|
Figure 7. Effect of Sterol Depletion on SCAP mRNA Expression
LNCaP cells were incubated under conditions of sterol depletion (-) or
sterol repletion (+) for 1 d. Total RNA was isolated and Northern
blot analysis was performed with a probe for SCAP. As a control for
sterol-regulated expression, blots were stripped and reprobed with a
probe directed against HMG-CoA-synthase (SYN).
|
|
To more directly demonstrate that SCAP has an active role in the
activation of the SREBP pathway, an expression vector encoding SCAP was
generated (pcDNA1.1-SCAP). COS-7 cells were transiently transfected
with increasing amounts of pcDNA1.1-SCAP and an expression vector
encoding a herpes simplex virus (HSV)-labeled SREBP precursor protein
(pTK-HSV-BP2). Two days later, nuclear extracts were prepared and
immunoblot analysis was performed using an antibody directed against
the HSV-epitope. Figure 8
shows that
gradual enhancement of cellular SCAP levels does indeed result in a
pronounced increase in mature nuclear SREBP.

View larger version (33K):
[in this window]
[in a new window]
|
Figure 8. Effect of Increased Cellular SCAP Levels on Nuclear
Accumulation of Mature SREBP
COS-7 cells were transiently transfected with pTK-HSV-BP2, encoding a
HSV-labeled SREBP-2 precursor, and increasing amounts of an expression
construct encoding human SCAP (pcDNA1.1-SCAP). Two days later,
nuclear extracts were prepared. Nuclear SREBP (68 kDa) was visualized
by performing Western blot analysis with an antibody directed against
the HSV epitope.
|
|
In support of this finding, transfection of increasing amounts of
pcDNA1.1-SCAP in LNCaP cells gave rise to a dose-dependent increase in
FAS and HMG-CoA-synthase reporter activities only when intact SREs were
present (Fig. 9
). Moreover, addition of
DN-SREBP gradually decreased the stimulatory effects of SCAP expression
on reporter activities (Fig. 9C
). Cotransfection with empty expression
vectors (no SCAP or DN-SREBP insert) did not affect reporter activities
(data not shown).

View larger version (21K):
[in this window]
[in a new window]
|
Figure 9. Effects of Increased Cellular SCAP Levels on
Transcription of the FAS and HMG-CoA-Synthase Gene
FAS (A) and HMG-CoA-synthase (SYN) (B) promoter-reporter
constructs, described in the legends to Figs. 3 and 4 , were
cotransfected into LNCaP cells with increasing amounts of an expression
construct encoding human SCAP (pcDNA1.1-SCAP). To further examine the
involvement of nSREBP in the observed effects, increasing amounts of a
plasmid encoding a dominant-negative form of SREBP (DN-SREBP) were
added (C). Two days later, cells were lysed. Luciferase activity was
measured and was expressed in relative luciferase units (RLU). Results
shown are representative of at least three independent experiments.
Columns, Means of incubations performed in triplicate;
bars, SEM.
|
|
To further evaluate whether androgen-induced elevation in SCAP levels
causes the maturation of SREBP precursor proteins and leads to
coordinated activation of transcription of SREBP target genes, time
course studies were performed. To this end, LNCaP cells were incubated
in the presence or absence of R1881 (10-8
M). After the indicated periods of time, cells were
harvested and either RNA was isolated or nuclear extracts were
prepared. Dot blot analysis was performed with probes for SCAP, FAS,
and HMG-CoA-synthase. Expression of nuclear SREBP-1 (nSREBP-1) was
evaluated by immunoblot analysis. As Fig. 10
shows, SCAP mRNA levels were clearly
enhanced within 8 h of androgen treatment. An increase in the
nuclear levels of SREBP-1 was first observed after 8 h of androgen
exposure. In agreement with earlier reports (3), an
elevation of the mRNA levels of the SREBP-target genes FAS and
HMG-CoA-synthase was not noticed until the 16-h point. These time
courses support the hypothesis that androgen-induced stimulation of
SCAP expression may be an important element in the activation of
lipogenesis.

View larger version (38K):
[in this window]
[in a new window]
|
Figure 10. Time Course of the Response of SCAP, FAS, and
HMG-CoA-Synthase mRNA and Nuclear SREBP Protein to Androgens in LNCaP
Cells
LNCaP cells were grown in the presence (hatched bars) or
absence (black bars) of 10-8 M
R1881. After the indicated periods of time, cells were harvested and
either total RNA was isolated or nuclear extracts were prepared.
Northern blot analysis was performed with probes for SCAP, FAS, or
HMG-CoA-synthase (SYN). mRNA levels were quantitated using
PhosphorImager screens, and hybridization signals were expressed as
relative densitometric units taking the values obtained from cells at
the 0 h time point as 1. nSREBP was analyzed by Western blotting
with an antibody directed against SREBP-1. Immunoreactive signals were
quantitated using ImageMaster 1D Elite software (Amersham Pharmacia Biotech).
|
|
Finally, to investigate whether androgens might directly affect
transcription of the gene encoding SCAP, LNCaP cells were pretreated
with actinomycin D, an inhibitor of transcription, for 30 min, followed
by addition of androgens (R1881 10- 8
M) or ethanol vehicle. At the indicated time points, cells
were harvested and SCAP mRNA expression was evaluated. At each time
point studied, the stimulatory effects of androgens on SCAP mRNA levels
were abolished after actinomycin D treatment (Fig. 11
), indicating that SCAP induction
requires transcriptional activity.

View larger version (23K):
[in this window]
[in a new window]
|
Figure 11. Effects of Actinomycin D on the Induction of SCAP
mRNA by Androgens in LNCaP Cells
LNCaP cells were pretreated with actinomycin D (10 µg/ml) or ethanol
vehicle for 30 min, after which time 10-8 M
R1881 or ethanol was added. After the indicated periods of time, cells
were harvested. Total RNA was prepared and Northern blot analysis was
carried out with a probe for SCAP. Hybridization signals were
quantitated using PhosphorImager screens. mRNA values were expressed as
relative densitometric units, taking the values obtained from cells at
the 0 h time point as 1. Black bars, Ethanol;
white bars, R1881; dotted bars,
actinomycin D + ethanol; hatched bars, actinomycin D +
R1881)
|
|
 |
DISCUSSION
|
---|
In the present report we show that marked and coordinated
stimulation of lipogenic gene expression may be a common characteristic
of androgen action in androgen-responsive prostate tumor lines.
Moreover, we provide conclusive evidence that both the effects of
androgens on genes involved in fatty acid synthesis and their effects
on genes involved in cholesterol synthesis are mediated by activation
of the SREBP pathway. Finally, we demonstrate that SCAP is one of the
major sites at which androgens act to stimulate the SREBP pathway.
In a previous study we demonstrated that in the human prostate cancer
cell line LNCaP, androgens coordinately stimulate the expression of
several genes belonging to two major lipogenic pathways: fatty acid
synthesis and cholesterol synthesis (11). Here, using a
second independent prostate cancer cell line (MDA-PCa-2a), we
demonstrate that this regulation is not a peculiarity of LNCaP cells,
but occurs also in other androgen-responsive prostate cancer cell
lines. Recent findings (data not shown) confirm these observations in a
third androgen-responsive tumor line, PC346c (25),
suggesting that stimulation of lipogenic gene expression may be a
common characteristic of androgen action in androgen-responsive tumor
cell lines.
In support of the involvement of SREBPs in this regulation, we show
that androgen treatment leads to increased levels of mature nuclear
SREBP-1 in LNCaP and MDA-MCa-2a. That this increase in nuclear SREBP is
central to the regulation of lipogenic gene expression by androgens, is
unambiguously demonstrated using promoter-reporter constructs derived
from two key lipogenic genes, one belonging to the FAS pathway (FAS)
and one crucial for cholesterol synthesis (HMG-CoA synthase). These
genes harbor different, but well characterized SREBP-binding sites.
Deletion or mutation of the sites that are crucial for regulation of
these genes by the SREBP pathway completely abolishes the effects of
androgens. Moreover, ectopic expression of dominant negative forms of
SREBP-1 counteracts the stimulatory effects of androgens both on
endogenous lipogenic genes and on promoter-reporter constructs derived
from these genes, further supporting the concept that SREBPs function
as mediators of the lipogenic effects of androgens.
Further studies addressing the molecular mechanism by which androgens
affect the SREBP pathway revealed that androgens induce a major
increase in the expression of SCAP. Since alternative routes of SREBP
activation, e.g. by cholesterol depletion, do not result in
a similar stimulation of SCAP expression, it is concluded that the
observed increase of SCAP expression is not an indirect effect of SREBP
activation, but indeed represents a more direct effect of androgens. In
support of this contention is the finding that SCAP is up-regulated
concordant with the accumulation of mature SREBP and well before the
activation of lipogenic gene expression. The hypothesis of a direct
link between the increase of SCAP expression and the stimulation of
lipogenic gene expression by androgens was further corroborated by the
finding that forced overexpression of SCAP led to enhanced nuclear
accumulation of mature SREBP and to activation of lipogenic gene
expression in an SRE- and SREBP-dependent manner. These data suggest
that androgens may activate the SREBP pathway by a mechanism that
differs from the one observed under conditions of sterol depletion. Low
intracellular concentrations of cholesterol are sensed by SCAP and
reduce its interaction with an as-yet-incompletely-characterized
retention protein (26) that holds the SREBP-SCAP complex
in the endoplasmic reticulum. As a result the SREBP-SCAP complex is
released from the endoplasmic reticulum and moves to the Golgi complex
where the SREBP is cleaved and activated. Androgens might reach the
same effect by promoting SCAP production and changing the balance
between SCAP and its retention protein. How androgens affect SCAP
expression remains to be clarified. Preliminary evidence using the
transcription inhibitor actinomycin D suggests that transcriptional
activity is required to activate this gene. Whether this involves a
direct or an indirect action of the AR is being investigated.
In addition to SCAP, other components of the SREBP pathway may also be
affected by androgens. In this respect it worth mentioning that
androgens slightly stimulate the expression of SREBP-1c and SREBP-2
precursors (Fig. 6
). As both these genes are subject to autoregulation
(27, 28), and as the increase in nuclear SREBP levels
precedes the observed effects on the SREBP precursors (data not shown),
it is unlikely that these genes are primary targets of androgens. The
changes in SREBP precursor expression evoked by androgen treatment may,
however, represent a secondary response and may provide a positive
feedback mechanism to further enhance SREBP activation. Moreover, as
only certain isoforms are affected, prolonged androgen exposure may
cause a shift in the predominant active SREBP isoforms involved and
result in a change in the set of genes that is ultimately affected.
Recent evidence from other research teams indicates that androgen
regulation of lipogenic gene expression is not restricted to prostate
cancer cell lines in culture, but is found also in prostate cancer
xenografts in vivo (29). Castration of male
mice carrying the CWR22 xenograft results in a significant decrease in
the expression of FAS. Readministration of androgens restores FAS
expression. The role of SREBPs has not been investigated in this system
but seems likely in view of our recent finding that in clinical
prostate cancer tissues lipogenic genes are also coordinately activated
(9). In this respect it is noteworthy that in the breast
cancer cell line MCF7, FAS expression has been shown to be affected by
progestagens and that this effect seems also to be mediated by the
SREBP pathway (30).
The fact that steroid hormones activate the SREBP pathway and stimulate
lipogenic gene expression in prostate and breast cancer cells suggests
that steroid receptor activation of the SREBP pathway may actively
contribute to the overexpression of lipogenic genes in the
corresponding cancers. In fact, overexpression of multiple lipogenic
genes has been found to correlate with SREBP expression and with the
expression of steroid receptors (31, 32, 33). Steroid receptor
activation may, however, not be sufficient to explain increased
lipogenic gene expression in these cancers. In this respect, recent
evidence from our laboratory indicates that growth factors, which may
enhance the function of steroid receptors and whose signaling is
frequently dysregulated and constitutively activated in cancer, may
also activate the SREBP pathway and lead to increased lipogenesis
(34). The extent to which these effects are mediated by
the AR remains to be examined. Another interesting question that is
under further investigation is whether activation of the SREBP pathway
by androgens (and other steroid hormones) is restricted to cancer cells
or represents a more general physiological regulation in noncancerous
tissues as well.
 |
MATERIALS AND METHODS
|
---|
Cell Culture
The human prostate carcinoma cell line LNCaP was obtained from
the American Type Culture Collection (ATCC,
Manassas, VA) and was maintained as described previously
(3).
The MDA-PCa-2a cell line was kindly provided by Dr. Navone (University
of Texas M.D. Anderson Cancer Center, Houston, TX) (19).
Cells were maintained in a humidified atmosphere of 5%
CO2 in air in BRFF-HPC1 medium (Biological
Research Faculty & Facility Inc., Ijamsville, MD) supplemented with
20% FCS (Life Technologies, Inc., Paisley, Scotland,
UK).
The COS-7 cell line was obtained from ATCC. Cells were
maintained in a humidified atmosphere of 5% CO2
in air in DMEM medium (Life Technologies, Inc.)
supplemented with 10% FCS (Life Technologies, Inc.), 3
mM L-glutamine, 100 µg/ml streptomycin, and
100 U/ml penicillin.
In experiments assessing the effects of androgens, FCS was pretreated
with dextran-coated charcoal (CT-FCS) (35) to reduce the
background levels of steroids. To study the effects of sterols, cells
were incubated in medium supplemented with 5% lipoprotein-deficient
serum (LPDS) (Intracel Corp., Rockville, MD).
The synthetic androgen R1881 (methyltrienolone) was purchased from
DuPont/NEN Life Science Products (Boston, MA), dissolved
in absolute ethanol, and added to the cultures from a 1,000-fold
concentrated stock. Cholesterol and 25-hydroxycholesterol were obtained
from Sigma (St. Louis, MO), dissolved in absolute ethanol,
and added to the cultures at a final concentration of 10 and 1 µg/ml,
respectively. Actinomycin D was purchased from Sigma,
dissolved in absolute ethanol, and added to the cultures at a final
concentration of 10 µg/ml. Control cultures received similar amounts
of ethanol only. Final ethanol concentrations did not exceed 0.2%.
cDNA Probes
cDNA probes for SREBP-1a, SREBP-1c, S1P, S2P, and SCAP were
prepared by PCR as follows. cDNA was generated by reverse transcription
of RNA from LNCaP cells by using Superscript II (Life Technologies, Inc.) and oligo(dT) as primer. This cDNA was used
as template for PCR with the primer pairs listed in Table 1
. PCR products were cloned into pGEM-T
(Promega Corp., Madison, WI) and verified by nucleotide
sequencing using an automated laser fluorescence automated sequencer
(Pharmacia Biotech, Piscataway, NJ). Single-stranded
radiolabeled PCR probes were produced as described (11).
Probes for SREBP-2, FAS, HMG-CoA-synthase, and 18S rRNA probe (18S)
were described previously (11).
Northern Blot and Dot Blot Analysis
LNCaP cells were plated in 150-mm dishes at a density of 3
x 106 cells per dish in RPMI 1640 medium
containing 5% CT-FCS. Two days later, medium was replaced, and cells
were treated with 10-8 M R1881 or
ethanol vehicle. For the actinomycin D experiments, LNCaP cells were
pretreated with actinomycin D (10 µg/ml) for 30 min. MDA-PCa-2a cells
were plated in 6-mm dishes at a density of 4 x
106 cells per dish in BRFF-HPC1 medium
supplemented with 20% FCS. After 2 or 3 d, medium was changed to
F12 medium supplemented with 10% CT-FCS, and cells were treated with
androgens (10-8 M R1881) or ethanol
vehicle. After the indicated period of androgen exposure, cells were
washed with PBS, snap-frozen in liquid nitrogen, and stored at -80 C.
Total RNA was prepared using a modified guanidinium/CsCl
ultracentrifugation method as described (3). Northern blot
and dot blot analyses were carried out as previously described
(11). Blots were autoradiographed by exposure to
Amersham Pharmacia Biotech hyperfilm-MP or to
Kodak Biomax film (Amersham Pharmacia Biotech, Arlington Heights, IL). Hybridization signals were
quantitated with PhosphorImager screens (Molecular Dynamics, Inc., Sunnyvale, CA) and corrected for differences in RNA
loading as revealed by hybridization with an 18S ribosomal RNA
probe.
Western Blot Analysis
To study the impact of androgen treatment on SREBP maturation,
LNCaP cells were seeded in 150-mm plates at a density of 3 x
106 cells per plate in RPMI 1640 medium
containing 5% CT-FCS. Two days later, medium was replaced and cells
were treated with 10-8 M R1881 or
ethanol for 2 d. MDA-PCa-2a cells were seeded in 60- mm plates at
a density of 4 x 106 cells per plate in
BRFF-HPC-1 medium supplemented with 20% FCS. Three days later, medium
was replaced by F12 medium containing 10% CT-FCS, and cells were
treated with 10-8 M R1881 or
vehicle. After 2d of treatment, LNCaP cells and MDA-PCa-2a cells were
harvested and nuclear fractions were prepared as described by Wang
et al. (36) and Hua et al.
(37) with modifications. Cells were scraped in 500 µl of
ice-cold PBS supplemented with 2.2 µg/ml aprotinin, 10 µg/ml
leupeptin, 0.5 mM phenylmethylsulfonyl fluoride
(PMSF), 0.5 mM dithiothreitol (DTT), and 50
µg/ml N-acetyl-leucyl-leucyl-norleucinal (ALLN). After
centrifugation at 1,000 x g at 4 C for 5 min, cells
were resuspended in 10 mM HEPES-KOH (pH 7.4), 10
mM KCl, 1.5 mM
MgCl2, 0.5 mM EDTA, 0.5
mM EGTA, 1 mM PMSF, 10
µg/ml aprotinin, 10 µg/ml leupeptin, 1 mM
DTT, and 25 µg/ml ALLN. After incubation at 4 C for 10 min, the cell
suspensions were passed through a 22-gauge needle 25 times (LNCaP
cells) or through a 24.5-gauge needle 20 times (MDA-PCa-2a cells) and
centrifuged at 1,000 x g at 4 C for 5 min. The pellets
were resuspended in 10 mM HEPES-KOH (pH 7.4),
0.42 M NaCl, 2.5% (vol/vol) glycerol, 1.5
mM MgCl2, 0.5
mM EDTA, 0.5 mM EGTA, 1
mM DTT, 1 mM PMSF, 10
µg/ml aprotinin, 10 µg/ml leupeptin, and 25 µg/ml ALLN. After
incubation at 4 C for 30 min with gentle agitation, the suspension was
centrifuged at top speed in a microcentrifuge at 4 C. The supernatant
was collected, the protein concentration was determined, and the
proteins were denatured by boiling for 3 min in SDS-gel loading buffer.
Equal amounts of protein were subjected to 7.5% SDS-PAGE. Proteins
were blotted onto PVDF Western blotting membranes (Roche Diagnostics, Mannheim, Germany) and incubated with an
antibody against the N-terminal part of SREBP-1 (IgG2A4). The
hybridoma cell line producing this monoclonal antibody against the
basic helix-loop-helix leucine zipper domain of human SREBP-1 (IgG-2A4)
was obtained from the American Type Culture Collection. Immunoreactive proteins were visualized with
horseradish peroxidase-conjugated goat antimouse IgG (Amersham Pharmacia Biotech) by using an enhanced chemilumi-nescence
detection kit (Renaissance, NEN Life Science Products,
Dreiech, Germany).
To detect SCAP protein in total cell extracts, LNCaP cells were seeded
in RPMI 1640 medium supplemented with 5% CT-FCS in 60-mm dishes at a
density of 5 x 105 cells per dish. Two days
later medium was replaced and cells were treated with
10-8 M R1881 or ethanol vehicle.
After the indicated period of treatment, cells were collected in SDS
lysis buffer and boiled for 3 min. Equal amounts of protein were
analyzed by 7.5% SDS-PAGE as described above, using a polyclonal
antibody against SCAP (K-19, Santa Cruz Biotechnology, Inc., Santa Cruz, CA).
Transient Transfection
The promoter-reporter constructs, FASwt and FASdelSRE, and the
HMG-CoA-synthase promoter-reporter constructs, SYNwt, SYNmutSRE-1, and
SYNmutSRE-2, were described previously (11, 34). The
construct DN-SREBP, encoding amino acids 90460 of SREBP-1, was
generated by cloning the corresponding PCR fragmentusing pSREBP-1a
(ATCC79810) as a templateinto the pIRES vector
(CLONTECH Laboratories, Inc., Palo Alto, CA). The
construct pTK-HSV-BP2 was obtained from ATCC (no. 99530).
pcDNA1.1-SCAP was constructed by cloning the entire coding sequence for
SCAP into the pcDNA1.1 vector (Invitrogen, San Diego,
CA).
In experiments assessing the effects of androgens or sterols, LNCaP
cells were seeded in 60-mm dishes at a density of 1 x
106 cells per dish in RPMI 1640 medium containing
5% CT-FCS. The next day, transfection mixtures were prepared. For each
dish, 2 ml of serum-free DMEM medium (Life Technologies, Inc.) were supplemented with 5 µg of promoter-reporter
construct. In the case of cotransfection experiments, the indicated
amounts of the DN-SREBP construct were added. After addition of 15 µl
Transfast (Promega Corp.) the transfection mixture was
incubated for 15 min at room temperature. Cells were washed with
serum-free medium, the transfection mixture was added, and cell
cultures were incubated for 30 min at 37 C. Four milliliters of RPMI
medium with 5% CT-FCS (androgen effects) or 5% LPDS (sterol effects)
were added. The next day, medium was replaced (RPMI with either 5%
CT-FCS or 5% LPDS), and cells were treated with either
10-8 M R1881 for 3 d (androgen
effects) or cholesterol (10 µg/ml) + 25-hydroxycholesterol (1
µg/ml) or mevastatin (10 µM) (Sigma) for
1 d. After the indicated time of treatment, cells were washed with
PBS and lysed in 500 µl passive lysis buffer (Promega Corp.). Aliquots of 10 µl of cleared lysate were assayed for
luciferase activity by using a luciferase reporter assay kit from
Promega Corp. and a Berthold Microlumat LB96P
luminometer.
In experiments determining the effect of SCAP (over)expression on
lipogenic gene transcription, LNCaP cells were seeded in 60 mm dishes
at a density of 1 x 106 cells in RPMI 1640
medium supplemented with 10% FCS. The next day, transfection was
performed as described using 3 µg promoter-reporter construct, the
indicated amounts of pcDNA1.1 and/or DN-SREBP, and 9 µl Transfast per
dish. The following day, medium was replaced. One day later, cells were
lysed and luciferase activity was assayed as described.
In experiments assessing the impact of SCAP (over)expression on SREBP
maturation, COS-7 cells were seeded in 60-mm dishes at a density of
4 x 105 cells per dish in DMEM medium
containing 10% FCS. The next day, cells were transfected2.5 µg
pTK-HSV-BP2 and the indicated amounts of pcDNA1.1-SCAP per dishusing
FuGene 6 Transfection Reagent (Roche Molecular Biochemicals) in accordance with the manufacturers
instructions. The following day, medium was replaced. Nuclear extracts
were prepared 24 h later as described above. Equal amounts of
protein were subjected to 7.5% SDS-PAGE and analyzed for recombinant
SREBP expression, using an antibody directed against the HSV epitope
(Novagen, Madison, WI).
Treatment with Recombinant Adenovirus
The construction and propagation of recombinant adenoviruses
expressing a dominant-negative form of SREBP-1 (Ad-DN) and of
adenoviruses without insert (Ad-Null) were described previously
(38, 39). MDA-PCa-2a cells were seeded in 60-mm dishes at
a density of 4 x 106 cells per dish in
BRFF-HPC1 medium supplemented with 20% FCS. Three days later, medium
was removed and replaced by 2 ml F12 medium supplemented with 10%
CT-FCS. Virus (30 pfu/cell) was added as indicated. Cultures were
incubated for 4 h with frequent agitation. Fresh medium was added
to a final volume of 5 ml. The next day, cells were treated with
10-8 M R1881 or ethanol vehicle.
After 2 d, cells were washed and snap-frozen for RNA isolation as
described previously (11).
 |
ACKNOWLEDGMENTS
|
---|
We acknowledge the excellent technical assistance of Frank
Vanderhoydonc.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Dr. J.V. Swinnen, LEGENDO, Gasthuisberg, K. U. Leuven, Onderwijs en Navorsing 9, Herestraat 49, B-3000 Leuven, Belgium. E-mail:
Johan.Swinnen{at}med.kuleuven.ac.be
This work was supported by a grant "Geconcerteerde Onderzoeksactie
van de Vlaamse Gemeenschap," by research grants from the Fund for
Scientific Research-Flanders (Belgium) (F.W.O.), by a Cancer Research
Grant from FB Insurance and VIVA, by a grant from the Belgische
Federatie tegen Kanker, by a specialization grant from the Flemish
Institute for the Enhancement of Scientific-Technological Research in
the Industry (IWT) (to H.H.), and by a grant Interuniversity Poles of
Attraction Programme-Belgian State, Prime Ministers Office, Federal
Office for Scientific, Technical and Cultural Affairs.
Abbreviations: ALLN,
N-acetyl-leucyl-leucyl-norleucinal; CT-FCS,
Charcoal-treated FCS; DTT, dithiothreitol; FAS, fatty acid synthase;
HMG-CoA, hydroxymethylglutaryl-coenzyme A; HSV, herpes simplex virus;
LPDS, lipoprotein-deficient serum; PMSF, phenylmethylsulfonyl fluoride;
18S, 18S rRNA probe; SCAP, SREBP cleavage activating protein; SRE,
sterol-regulatory element; SREBP, SRE-binding protein; S1P, site 1
protease; S2P, site 2 protease.
Received for publication November 15, 2000.
Accepted for publication June 11, 2001.
 |
REFERENCES
|
---|
-
Huggins C, Hodges CV 1941 Studies on prostatic cancer:
effect of castration, of estrogen, and of androgen injection on serum
phosphatases in metastatic carcinoma of the prostate. Cancer Res 1:293297
-
Swinnen JV, Van Veldhoven PP, Esquenet M, Heyns W, Verhoeven
G 1996 Androgens markedly stimulate the accumulation of neutral lipids
in the human prostatic adenocarcinoma cell line LNCaP. Endocrinology 137:44684474[Abstract]
-
Swinnen JV, Esquenet M, Goossens K, Heyns W, Verhoeven G 1997 Androgens stimulate fatty acid synthase in the human prostate cancer
cell line LNCaP. Cancer Res 57:10861090[Abstract]
-
Swinnen JV, Verhoeven G 1998 Androgens and the control of
lipid metabolism in human prostate cancer cells. J Steroid Biochem Mol
Biol 65:191198[CrossRef][Medline]
-
Kuhajda FP 2000 Fatty-acid synthase and human cancer: new
perspectives on its role in tumor biology. Nutrition 16:202208[CrossRef][Medline]
-
Shurbaji MS, Kuhajda FP, Pasternack GR, Thurmond TS 1992 Expression of oncogenic antigen 519 (OA-519) in prostate cancer is a
potential prognostic indicator. Am J Clin Pathol 97:686691[Medline]
-
Shurbaji MS, Kalbfleish JH, Thurmond TS 1996 Immunohistochemical detection of a fatty acid synthase (OA-519) as
predictor of progression of prostate cancer. Hum Pathol 27:917921[Medline]
-
Epstein JI, Carmichael M, Partin AW 1995 OA-519 (fatty acid
synthase) as an independent predictor of pathologic stage in
adenocarcinoma of the prostate. Urology 45:8186[CrossRef][Medline]
-
Swinnen JV, Vanderhoydonc F, Elgamal AA, et al. 2000 Selective activation of the fatty acid synthesis pathway in human
prostate cancer. Int J Cancer 88:176179[CrossRef][Medline]
-
Pizer ES, Pflug BRL, Bova S, Han WF, Udan MS, Nelson JB 2001 Increased fatty acid synthase as a therapeutic target in
androgen-independent prostate cancer progression. Prostate 47:102110[CrossRef][Medline]
-
Swinnen JV, Ulrix W, Heyns W, Verhoeven G 1997 Coordinate
regulation of lipogenic gene expression by androgens. Evidence for a
cascade mechanism involving sterol regulatory element binding proteins.
Proc Natl Acad Sci USA 94:1297512980[Abstract/Free Full Text]
-
Briggs MR, Yokoyama C, Wang X, Brown MS, Goldstein JL 1993 Nuclear protein that binds sterol regulatory element of low density
lipoprotein receptor promotor. I. Identification of the protein and
delineation of its target nucleotide sequence. J Biol Chem 268:1449014496[Abstract/Free Full Text]
-
Wang X, Briggs MR, Hua X, Yokoyama C, Goldstein JL, Brown MS 1993 Nuclear protein that binds sterol regulatory element of the low
density lipoprotein receptor promotor. II. Purification and
characterisation. J Biol Chem 268:1449714504[Abstract/Free Full Text]
-
Tontonoz P, Kim JB, Graves RA, Spiegelman BM 1993 ADD1: a
novel helix-loop helix-transcription factor associated with adipocyte
determination and differentiation. Mol Cell Biol 13:47534759[Abstract]
-
Brown MS, Ye J, Rawson RB, Goldstein JL 2000 Regulated
intramembrane proteolysis: a control mechanism conserved from bacteria
to humans. Cell 100:391398[Medline]
-
Brown MS, Goldstein JL 1999 A proteolytic pathway that
controls the cholesterol content of membranes, cells and blood. Proc
Natl Acad Sci USA 96:1104111048[Abstract/Free Full Text]
-
Nohturfft A, Yabe D, Goldstein JL, Brown MS, Espenshade PJ 2000 Regulated step in cholesterol feedback localized to budding of
SCAP from ER membranes. Cell 102:315323[Medline]
-
Roder K, Wolf SS, Sickinger S, Schweizer M 1999 FIRE3 in the
promoter of the rat fatty acid synthase (FAS) gene binds the ubiquitous
transcription factors CBF and USF but does not mediate an insulin
response in a rat hepatoma cell line. Eur J Biochem 260:743751[Abstract/Free Full Text]
-
Navone NM, Olive M, Ozen M, et al. 1997 Establishment of two
human prostate cancer cell lines derived from a single bone metastasis.
Clin Cancer Res 3:24932500[Abstract]
-
Magana MM, Osborne TF 1996 Two tandem binding sites for sterol
regulatory element binding proteins are required for sterol regulation
of fatty-acid synthase promoter. J Biol Chem 271:3268932694[Abstract/Free Full Text]
-
Inoue J, Sato R, Maeda M 1998 Multiple DNA elements for sterol
regulatory element binding protein and NF-Y are responsible for
sterol-regulated transcription of the genes for human
3-hydroxy-3-methylglutaryl coenzyme 198 A synthase and squalene
synthase. J Biochem 123:11911198[Abstract]
-
Sato R, Yang J, Wang X, et al. 1994 Assignment of the membrane
attachment, DNA binding, and transcriptional activation domains of
sterol regulatory element binding protein-1 (SREBP-1). J Biol Chem 269:1725717273[Abstract/Free Full Text]
-
Kim JB, Spiegelman BM 1996 ADD1/SREBP1 promotes adipocyte
differentiation and gene expression linked to fatty acid metabolism.
Genes Dev 10:10961107[Abstract]
-
Nakajima T, Hamakubo T, Kodama T, Inazama J, Emi M 1999 Genomic structure and chromosomal mapping of the human sterol
regulatory element binding protein (SREBP) cleavage-activating protein
(SCAP) gene. J Hum Genet 44:402407[CrossRef][Medline]
-
van Weerden WM, de Ridder CM, Verdaasdonk CL, et al. 1996 Development of seven new human prostate tumor xenograft models and
their histopathological characterization. Am J Pathol 149:10551062[Abstract]
-
Yang T, Goldstein JL, Brown MS 2000 Overexpression of membrane
domain of SCAP prevents sterols from inhibiting SCAP-SREBP exit from
endoplasmic reticulum. J Biol Chem 275:2988129886[Abstract/Free Full Text]
-
Sato R, Inoue J, Kawabe Y, Kodama T, Takano T, Maeda M 1996 Sterol-dependent transcriptional regulation of sterol regulatory
element-binding protein-2. J Biol Chem 271:2646126464[Abstract/Free Full Text]
-
Amemiya-Kudo M, Shimano H, Yoshikawa T, et al. 2000 Promoter
analysis of the mouse sterol regulatory element-binding protein-1c
gene. J Biol Chem 275:3107831085[Abstract/Free Full Text]
-
Myers RB, Oelschlager DK, Weiss HL, Frost AR, Grizzle WE 2001 Fatty acid synthase: an early molecular marker of progression of
prostatic adenocarcinoma to androgen independence. J Urol 165:10271032[Medline]
-
Lacasa D, Le Liepvre X, Ferre P, Dugail I 2001 Progesterone
stimulates adipocyte determination and differentiation 1/sterol
regulatory element-binding protein 1c gene expression. Potential
mechanism for the lipogenic effect of progesterone in adipose tissue.
J Biol Chem 276:1151211516[Abstract/Free Full Text]
-
Li JN, Mahmoud MA, Han WF, Ripple M, Pizer ES 2000 Sterol
regulatory element-binding protein-1 participates in the regulation of
fatty acid synthase expression in colorectal neoplasia. Exp Cell Res 261:159165[CrossRef][Medline]
-
Pizer ES, Lax SF, Kuhajda FP, Pasternack GR, Kurman RJ 1998 Fatty acid synthase expression in endometrial carcinoma: correlation
with cell proliferation and hormone receptors. Cancer 83:528537[CrossRef][Medline]
-
Alo PL, Visca P, Trombetta G, et al. 1999 Fatty acid synthase
(FAS) predictive strength in poorly differentiated early breast
carcinomas. Tumori 85:3540[CrossRef][Medline]
-
Swinnen JV, Heemers H, Deboel L, Foufelle F, Heyns W,
Verhoeven G 2000 Stimulation of tumor-associated fatty acid synthase
expression by growth factor activation of the sterol regulatory
element-binding protein pathway. Oncogene:51735181
-
Leake RE, Freshney RI, Munir I 1987 Steroid response in
vivo and in vitro. In: Green B, Leake RE, eds. Steroid
hormones: a practical approach. Washington DC: IRL Press; 213214
-
Wang X, Sato R, Brown M, Hua X, Goldstein JL 1994 SREBP-1, a
membrane-bound transcription factor released by sterol-regulated
proteolysis. Cell 77:5362[Medline]
-
Hua X, Sakai J, Brown MS, Goldstein JL 1996 Regulated cleavage
of sterol regulatory element binding proteins requires sequences on
both sides of the endoplasmic reticulum membrane. J Biol Chem 271:1037910384[Abstract/Free Full Text]
-
Foretz M, Guichard C, Ferré P, Foufelle F 1999 Sterol
regulatory element binding protein-1c is a major mediator of insulin
action on the hepatic expression of glucokinase and lipogenesis-related
genes. Proc Natl Acad Sci USA 96:1273712742[Abstract/Free Full Text]
-
Foretz M, Pacot C, Dugail I, et al. 1999 ADD1/SREBP-1c is
required in the activation of hepatic lipogenic gene expression by
glucose. Mol Cell Biol 19:37603768[Abstract/Free Full Text]