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-Demethylase (CYP51) in Spermatids
Institute of Biochemistry (D.R., M.F.) Medical Center for
Molecular Biology Medical Faculty University of Ljubljana 1000
Ljubljana, Slovenia
Institute de Génétique et de
Biologie Moléculaire et Cellulaire (G.M.F., P.S.-C.) Centre
Nationale de la Recherche Scientifique-INSERM-ULP 67404
Illkrich Strasbourg, France
Department of Biochemistry
(M.R.W.) Vanderbilt University School of Medicine Nashville,
Tennessee 37232-0146
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-demethylase (CYP51) produces MAS
sterols, intermediates in cholesterol biosynthesis that can reinitiate
meiosis in mouse oocytes. As a cholesterogenic gene, CYP51 is regulated
by a sterol/sterol-regulatory element binding protein (SREBP)-dependent
pathway in liver and other somatic tissue. In testis, however,
cAMP/cAMP-responsive element modulator CREM
-dependent regulation
of CYP51 predominates, leading to increased levels of shortened CYP51
mRNA transcripts. CREM-/- mice lack the abundant germ cell-specific
CYP51 mRNAs in testis while expression of somatic CYP51 transcripts is
unaffected. The mRNA levels of squalene synthase (an enzyme preceding
CYP51 in cholesterol biosynthesis in testis of CREM-/- mice are
unchanged as compared with wild-type animals, showing that
regulation by CREM is not characteristic for all
cholesterogenic genes expressed during spermatogenesis. The
-334/+314 bp CYP51 region can mediate both the sterol/SREBP-dependent
as well as the cAMP/CREM-dependent transcriptional activation.
SREBP-1a from somatic cell nuclear extracts binds to a conserved
CYP51-SRE1 element in the CYP51 proximal promoter. The
cAMP-dependent transcriptional activator CREM from germ cell
nuclear extracts binds to a conserved CYP51-CRE2 element while no
SREBP-1 binding is observed in germ cells. The two regulatory pathways
mediating expression of CYP51 describe this gene as a cholesterogenic
gene (SREBP-dependent expression in liver and other somatic cells) and
also as a haploid expressed gene (CREM-dependent expression in
haploid male germ cells). While in somatic cells all genes involved in
cholesterol biosynthesis are regulated coordinately by the
sterol/SREBP-signaling pathway, male germ cells contain alternate
routes to control expression of cholesterogenic genes. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-demethylase (CYP51, P45014DM) is a
cytochrome P450 enzyme involved in the postsqualene portion of
cholesterol biosynthesis. It is encoded by the CYP51 gene, the most
evolutionarily conserved gene in the large cytochrome P450 superfamily
(2). The postsqualene portion of cholesterol synthesis consists of at
least eight enzymatic steps and only certain of the corresponding genes
have been cloned (3, 4, 5, 6). CYP51 removes the 14-methyl group from
lanosterol producing the 14-demethylated sterol FF-MAS (follicular
fluid meiosis-activating sterol). FF-MAS is further metabolized by
sterol 14-reductase to produce T-MAS (testis meiosis activating sterol)
(Fig. 1
). Unlike the presqualene portion
of the pathway where intermediates have well known functions in
additional biological processes (i.e. farnesylation of
proteins, dolichol, heme A synthesis), the postsqualene portion of the
pathway has been considered as exclusively dedicated to cholesterol
biosynthesis. Recently, however, FF-MAS and T-MAS were found to
accumulate in ovary or testis and to have the capacity to reinitiate
meiosis in an in vitro mouse oocyte assay (7, 8). Being
intermediates of cholesterol biosynthesis, MAS sterols are not detected
in most tissues, but are easily observed in gonads (7, 9). These
findings suggest the existence of tissue-specific regulatory
pathways mediating expression of genes involved in MAS sterol
production.
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Herein we report that lanosterol 14-demethylase follows the pattern
of cholesterogenic gene expression in somatic cells and the pattern of
haploid gene expression in spermatids. Haploid male germ cells seem to
contain alternative routes to mediate expression of certain
cholesterogenic genes. While increased expression of CYP51 in
spermatids is mediated by binding of the cAMP-dependent transcriptional
activator CREM (cAMP response element modulator-) to CYP51-CRE2
and no SREBP (sterol response element binding protein) binding is
observed, the expression of squalene synthase, another enzyme of
cholesterol biosynthesis is independent of CREM in spermatids.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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). Two SRE-like
sequences similar to the SREBP recognition site ATCACCCCAC in the
low-density lipoprotein (LDL) receptor promoter are present in the
sense orientation in the 5'-part of the human CYP51 gene (12). One of
the SRE-like motifs (ATCACCTCAG; CYP51-SRE1; Fig. 3) is 80% identical to the functional
SRE-1 element in the LDL promoter gene (ATCACCCCAC) and is 100%
conserved between human (12) and rat (11). The other sense human
SRE-like element (GGCACCCCGC; CYP51-SRE2; Fig. 3) is not found in the
rat CYP51 gene (11). The purified transcription factor SREBP-1a binds
only to CYP51-SRE1 but not to CYP51-SRE2 (Fig. 4A). SREBP-1 from nuclear extracts of
cell lines derived from human liver (HepG2), human adrenal (H295R), and
human choriocarcinoma (JEG-3) cells binds specifically to the conserved
CYP51-SRE1 as determined by supershift and competition studies. For
example, CYP51-SRE1-bound SREBP proteins from H295R cells (Fig. 4B, lane 3) give a higher mol wt complex (supershift) in the presence of
the crude anti-SREBP-1 (Fig. 4B, lane 2), and the binding is competed
with excess of unlabeled CYP51-SRE1 probe (Fig. 4B, lane 5). Similarly,
a SREBP protein complex is formed with CYP51-SRE1 using JEG-3 nuclear
extracts (Fig. 4C). Anti-CREB (cAMP-response element binding protein)
antibody doesn’t shift this complex (Fig. 4C, lane 1), while purified
anti-SREBP-1 disrupts the complex (Fig. 4C, lane 5), and unlabeled
CYP51-SRE1 competes the binding (Fig. 4C, lane 4).
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in
Spermatids). Only background levels of CYP51 mRNA
are detected in steroidogenic Leydig cells while strong signals are
observed in these cells with a probe for mRNA encoding the
steroidogenic enyzme 17-hydroxylase/17,20-lyase cytochrome P450
(CYP17, Fig. 5E). The intensity of the CYP51 signal varies greatly
between different seminiferous tubule cross-sections (Fig. 5B),
suggesting stage-specific expression. Spermatogenesis in mouse
seminiferous tubules can be divided into 12 stages of cellular
associations, which are identified by their morphology and the
presence/location of distinct germ cells in each stage (13). Using the
mouse CYP51 antisense riboprobe, only background signal is detected in
stages I-III of the mouse seminiferous wave (Fig. 5D). In contrast, the
signal is very strong in stages VII–VIII and is restricted to late
round and early elongating spermatids immediately underneath the most
mature elongated spermatids, which face the lumen (Fig. 5, C and D). It
is clear that the CYP51 signal is largely absent from the cell layer
containing the most mature elongating spermatids that are ready to be
released to the lumen and whose condensed nuclei form a narrow band
lining the lumen (Fig. 5, C and D). The pattern of CYP51 expression in
mouse testis seems to be identical to that observed in rat (10).
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(L. Monaco, P. Sassone-Corsi,
unpublished data). This suggested a role for CREM in CYP51
transcription during spermatogenesis. To prove this, expression of
CYP51 has been monitored in mice with the disrupted CREM gene (15). The
expression of CYP51 mRNA in liver (Fig. 6A) is not affected by the absence of
CREM and is controlled by SREBP-1a as shown in Fig. 2
. The expression
of the two abundant CYP51 transcripts in testis depends on the presence
of at least one functional CREM gene. These transcripts in wild-type
and CREM+/- mouse testis are analogous to the shortest CYP51
transcript in rat (10) expressed only in postmeiotic germ cells. The
testis of CREM-/- mice lack the shortest CYP51 mRNAs and contain only
the somatic CYP51 transcripts expressed to similar levels as in liver
(Fig. 6A). This shows that haploid expression of CYP51 in mouse testis
is CREM-dependent in vivo.
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is involved in mediating expression of
other cholesterogenic genes, expression of squalene synthase was
monitored in CREM-/- mice. We have shown previously that in addition
to CYP51, transcription of squalene synthase is also up-regulated in a
stage-specific manner during spermatogenesis (10). Increased levels of
squalene synthase mRNA were observed before meiosis in pachytene
spermatocytes (10) where the activator transcription factor CREM is
not yet present (16). Monitoring expression of squalene synthase in
CREM-/- mice showed that expression of this early cholesterogenic
gene is not controlled by the cAMP/CREM mechanism. Similar levels of
squalene synthase mRNA are present in testis of normal, CREM-/+, and
CREM-/- mice (Fig. 6B). Also, no CRE is found in the 5'-flank of the
human squalene synthase gene (17).
CREM Binds to the CYP51-CRE Elements Residing in the Promoter of
CYP51
The human CYP51 gene contains two potential cAMP-response elements
(CYP51-CRE1 and CYP51-CRE2) upstream of the major transcription start
site (Fig. 3). We wanted to evaluate whether purified CREM or
CREM present in nuclear extracts of testis and germ cells can bind
to these CYP51-CREs. Proteins in nuclear extracts from whole rat testis
(somatic cells and germ cells) bind strongly to human CYP51-CRE1 (Fig. 7A, lane 1). Both anti-CREB (Fig. 7A, lane 2) and anti-CREM (Fig. 7A, lane 3) antibodies disrupt this gel
shift pattern, giving different supershifts. Thus, it appears that
isoforms of both CREM and CREB proteins can bind to CYP51-CRE1. Binding
is competed with the consensus CRE from the somatostatin gene, showing
that most of the strong signal belongs to CRE-binding proteins (Fig. 7B).
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), albeit more weakly than to
CYP51-CRE1. The data show that CREM isoforms from the rat testis and
germ cell nuclear extracts efficiently bind to the CYP51-CRE2 sequence.
The anti-CREM antibody supershifts the complex in testis (Fig. 8A, lane
5) and germ cells (Fig. 8B, lane 5), while the anti-CREB antibody
doesn’t influence nuclear protein binding to CYP51-CRE2 (Fig. 8, A and
B, lanes 2 and 3). The anti-CREM antibody has a 100-fold higher
affinity for CREM than for CREB (16). The E. coli expressed
CREM-protein binds to CYP51-CRE1, CYP51-CRE2, and
CYP51-CRE2new elements, yet with a lower affinity than to
the consensus CRE TGACGTCA (Fig. 8C). CYP51-CRE2 and
CYP51-CRE2new differ in two bases flanking the CRE octamere
TGACGCGA.
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, lane 3), but
no CYP51-SRE1 binding proteins can be detected in such extracts (Fig. 9, lane 4). Using nuclear extracts from mature rat testis,
CYP51-SRE1-binding proteins are readily detected because these extracts
are derived from a mixture of Sertoli cells, Leydig cells, and germ
cells (Fig. 9, lane 2). This pattern is identical to that obtained with
nuclear extracts from human somatic cells (Fig. 4). Thus, based on gel
mobility shift binding assays, SREBP is not present in the majority of
male germ cell types isolated by the trypsin-DNAse method.
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). JEG-3 are human
choriocarcinoma cells that contain the sterol-dependent signal
transduction pathway and also a cAMP-dependent signal transduction
pathway (18). The functional importance of SREBP-1a and CREM in
CYP51 transcription has been measured by cotransfection experiments in
these cells. The human CYP51 -334/+314 promoter activity is
essentially unchanged (1.4-fold) in delipidated serum (Fig. 10, lane
2), stimulated 17-fold when activated by the mature form of the
transcription factor SREBP-1a (lane 3), and 18.5-fold when delipidated
serum and SREBP-1a activation are combined. These data show a marked
stimulation of CYP51 promoter activity by transcription factor
SREBP-1a. The same promoter shows stimulation of activity also by the
cAMP-dependent pathway. Activity is stimulated 4.3-fold by forskolin
(lane 6), indicating activation of CYP51 transcription by endogenous
cAMP-responsive element-binding proteins in these somatic cells. The
cAMP-dependent transcription activator CREM is highly expressed only
in spermatids, the haploid male germ cells (19). Since no spermatid
cell line is available, JEG-3 cells were cotransfected with CREM to
mimic the physiology of haploid male germ cells. The human CYP51
promoter activity is stimulated 2.6-fold by CREM (lane 5) and
6.1-fold by the combined action of forskolin and CREM (lane 7),
showing that CREM which is the most abundant cAMP-dependent
transcriptional activator in spermatids (19), and a master switch of
postmeiotic gene expression (15, 20) can mediate expression of
CYP51.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-demethylase, a ubiquitously
expressed gene of the postsqualene part of cholesterol biosynthesis.
Transcription of this gene in somatic cells (liver, adrenal, placenta)
follows the pattern of coordinate, sterol/SREBP-dependent regulation
characteristic of genes involved in cholesterol biosynthesis and
homeostasis (1, 21). Expression of somatic mouse CYP51 mRNAs is
4.6-fold higher in livers of SREBP-1a(460) transgenic mice, an increase
similar to that of mRNAs for two cholesterol biosynthetic genes before
squalene (hydroxymethylglutaryl-coenzyme A synthase 6.1-fold and
squalene synthase 5.4-fold) but is lower than
hydroxymethylglutaryl-coenzyme A reductase (37-fold), which is the
major regulatory point of cholesterol biosynthesis (22). The CYP51-SRE1
sequence found in the promoter of human and rat CYP51 genes and
recently also in the mouse CYP51 gene (Debeljak and D. Rozman,
unpublished), is not identical to any of the previously established
SREs (23). The sequence ATCACCTCAG (CYP51-SRE1) is 100% conserved in
human, rat, and mouse CYP51 promoters and is readily recognized by the
purified SREBP-1. SREBP-1 does not bind to the second CYP51-SRE-like
element GGCACCCCGC (CYP51-SRE2; Fig. 4A). We thus believe that
CYP51-SRE1 is a major point of SREBP interaction within the CYP51
promoter and is responsible for the sterol-dependent activation of
CYP51 transcription as observed in the CAT reporter assay. These data,
together with those in Fig. 10, strongly indicate that ATCACCTCAG is a
functional SRE sequence.
Surprisingly, the ubiquitously expressed mammalian CYP51 mRNA encoding
cholesterogenic lanosterol 14-demethylase is expressed to highest
levels in the testis (3, 10). The pattern of CYP51 mRNA expression in
rat (10) and mouse testis (this paper) is very similar, peaking in
round and early elongating spermatids that match the timing of
expression of transcription activator CREM (L. Monaco and P.
Sassone-Corsi, unpublished data). Germ cells of the testis contain
various activator as well as inhibitory CREB and CREM transcription
factors that arise by differential splicing of both genes (24, 25, 26).
CREM expression is developmentally switched during spermatogenesis from
antagonist forms (CREM, CREMß, CREM
) in premeiotic germ cells
to the activator forms (CREM, CREM1,
CREM2) in postmeiotic germ cells (19). CREM plays a
key role in transcriptional activation in haploid male germ cells and
acts as a master switch responsible for the cAMP-dependent
transcriptional activation of several genes expressed at this specific
time during spermatogenesis (27, 28). The homozygous CREM-/- males
are infertile despite normal mating behavior (15, 29) while the females
remain fertile. The list of CREM-regulated genes includes genes with
established roles in spermatogenesis, such as protamines 1 and 2,
transition protein 1, calspermine, ACE, etc., and a longer list of
genes having roles in germ cell maturation not yet fully established
(16, 30). Our results show that cholesterogenic lanosterol
14-demethylase belongs to this group of haploid-expressed
transcripts induced by the transcription activator CREM. High
expression of CYP51 in spermatids results from the appearance of
shorter, germ cell-specific CYP51 transcripts (Fig. 9) (10). The two
highly expressed mouse CYP51 mRNAs are absent in the testis of
CREM-/- mice, showing that a cAMP-dependent transcription
activator of the CREM family is needed to promote their synthesis.
In contrast to CYP51, expression of another cholesterogenic gene,
squalene synthase, does not depend on the presence of CREM during
spermatogenesis. Increased levels of squalene synthase mRNA are
observed before meiosis in pachytene spermatocytes (10) where the
activator transcription factor CREM is not yet present (16).
Squalene synthase expression in the testis of CREM-/- mice is
identical to that in wild-type animals, showing that CREM-dependent
regulation is not characteristic for all cholesterogenic genes that are
highly expressed during spermatogenesis. In the absence of evidence for
the presence of sterol responsive element-binding proteins in germ
cells, the mechanism for squalene synthase expression in
spermatogenesis remains an enigma.
We wanted to evaluate which elements of the CYP51 promoter may be
involved in mediating the cAMP/CREM-dependent expression of CYP51 in
spermatids. CYP51-CRE2 (Fig. 3) is conserved across species, showing
high homology (7/8) to the aligned sequences of the rat (11) and mouse
(N. Debeljak and D. Rozman, unpublished). CYP51-CRE1 shows a
lower (5/8) evolutionary conservation in these species. Since the
evolutionary conserved CYP51-CRE2 binds CREM proteins present in rat
testis specifically or with a preference over CREB proteins, it is the
major candidate for binding of CREM in germ cells of different
species in vivo. In accordance to this, transfection assays
(Fig. 10) show that CYP51 promoter containing only CYP51-CRE2 can
efficiently mediate the CREM-dependent transcriptional
activation.
It is now established that cAMP regulates expression of a
cholesterogenic gene CYP51 in addition to the sterol type of gene
regulation. Both regulatory processes are mediated by the same
-334/+314 CYP51 promoter as determined by the CAT reporter analysis.
Thus, the availability of transcription factors in various cell types
may differentially control transcription of the CYP51 gene. While
expression of the germ cell-specific CYP51 mRNAs is under CREM
control, the expression of longer CYP51 somatic-type transcripts in
germ cells may be regulated in a housekeeping manner due to the
presence of a GC box that binds ubiquitous transcription factors of the
Sp family. It has recently been established that transcription factor
Sp1 plays a major role in determining expression of lactate
dehydrogenase gene during spermatogenesis (31).
In conclusion, CYP51 produces FF-MAS, an oocyte meiosis-activating
sterol (7), which is an intermediate in the coordinately regulated
housekeeping process of cholesterol biosynthesis. Interestingly, in
haploid male germ cells the cAMP/CREM-dependent regulation of CYP51
predominates over the sterol/SREBP regulation, which coordinates
cholesterol biosynthesis in somatic cells. The haploid expressed mRNAs
of the CYP51 gene belong to the group of CREM-regulated transcripts
whose detailed role in spermatogenesis (cholesterol production
vs. FF-MAS production) has yet to be determined. Since
another cholesterogenic gene (squalene synthase) is not regulated by
CREM and apparently no coordinate-type regulation of cholesterogenic
genes occurs in germ cells, it is likely that the
cAMP/CREM-dependent induction of CYP51 in spermatids does not serve
increased cholesterol synthesis and may thus be associated with
overproduction of MAS sterols, signaling molecules produced by the
gonads.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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In Situ Hybridization
Adult mouse testes were rinsed in PBS, pH 7.4, and immersion
fixed overnight in 4% paraformaldehyde in PBS, pH 7.4. The tissue was
dehydrated in ethanol and xylene solutions, embedded in paraffin, cut
into 6-µm thick sections and hybridized with the mouse CYP51 cDNA
probe. Sense and antisense mouse CYP51 riboprobes were prepared from
pM14DM by in vitro transcription with T3 and T7 RNA
polymerases in the presence of [35S]UTP. CYP17 riboprobe
was prepared by in vitro transcription from the cloned mouse
743 bp CYP17 fragment (PCR-Direct cloning system,
CLONTECH Laboratories, Inc., Palo Alto, CA)
(34) and used as a positive control.
Northern Analysis
Five SREBP-1a transgenic mice (22) and five littermate wild-type
controls were placed on a high protein/low carbohydrate diet (No.
5789C-3) from Purina Mills Inc. (St. Louis, MO) containing 71% (wt/wt)
casein and 4.25% (wt/wt) sucrose for 2 weeks before study. All mice
were killed after a 12-h fast in the early phase of the light cycle.
Isolation of total RNA from liver and Northern blot analysis was
performed exactly as described (22) using the mouse CYP51 647-bp RT-PCR
fragment as probe. Liver RNA was pooled from five transgenic animals
and from five wild-type animals. Equal amounts (3 µg) of RNA isolated
from individual animals were mixed in the five-animal pool (total 15
µg of RNA). CYP51 analysis was performed in the laboratory of Drs.
J. L. Goldstein and M. S. Brown on RNAs from the same group
of transgenic animals previously used to study expression of other
cholesterogenic genes (22). The bands detected by Northern analysis
were quantified by exposing the filter to a BAS1000 Phosphor-Imager
Fuji Photo Film Co., Ltd., Tokyo, Japan), and results were
normalized to the signal generated by glyceraldehyde-3-phosphate
dehydrogenase mRNA. The analysis was performed twice with identical
outcome.
Total RNA from the liver and testis of CREM-/-, CREM+/- and the wild-type mice was prepared (15) and 20 µg of each were used for Northern analysis. Equal loading was verified by staining the gel with ethidium bromide. Partial mouse CYP51 cDNA and a partial rat squalene synthase cDNA were used as probes (10). Northern analysis was performed in the laboratory of Dr. Paolo Sassone-Corsi on RNA samples isolated from the same group of mice previously used to study expression of other genes, with equivalent results obtained from five different sets of mutant and normal mice (15). CYP51 analysis was performed three times with identical outcome.
Preparation of Nuclear Extracts from Rat Testis and Germ
Cells
Nuclear extracts were prepared at the same time as cytosolic
protein extracts from sexually mature rats (10). Testes of one adult or
five prepubertal Harlan Sprague Dawley (Indianapolis, IN)
rats were decapsulated and homogenized on ice by 50–100 strokes with a
glass-Teflon hand homogenizer in assay buffer (100 mM
K3PO4, 0.1 M dithiothreitol (DTT),
0.1 mM EDTA, 20% glycerol). Homogenates were filtered
through a 50-µm filter and spun for 15 min at 1500 x
g and +4 C. The pellet was resuspended by hand
homogenization in 10 ml of ice-cold buffer A (0.25 M
sucrose, 10 mM potassium phosphate buffer, pH 6.8, 5
mM MgCl2) containing the proteinase inhibitor
mixture of 1 µg/ml aprotinin, 1 µg/ml pepstatin, 1 µg/ml
leupeptin, 0.5 mM phenylmethylsulfonylfluoride, and 0.5
mM DTT. After centrifugation at 1500 x g
for 10 min at +4 C, the resulting pellet was suspended in 1 ml of
ice-cold buffer B (0.25 M sucrose, 20 mM HEPES,
pH 7.9, 0.3 M NaCl, 5 mM MgCl2)
containing the same proteinase inhibitor mixture. It was transferred to
Eppendorf tubes, kept 20 min on ice with occasional
vortexing and spun 10 min in a microcentrifuge at +4 C. The supernatant
was recentrifuged at 100,000 x g at +4 C for 30 min,
aliquoted, and frozen in liquid nitrogen.
Germ cells from groups of two sexually mature male rats were prepared by mechanical treatment and trypsinization (10). Briefly, decapsulated testes were thoroughly minced with an array of sealed razor blades and treated with DNAse and trypsin. This procedure destroys most of the Sertoli and Leydig cells while germ cells remain intact (35). Germ cells were pelleted, washed and suspended in 5 ml of assay buffer, and hand homogenized on ice by 100 strokes. After centrifugation at 1500 x g for 15 min at +4 C, the pellet was suspended in 10 volumes of ice-cold buffer A and nuclear extracts were prepared.
Preparation of Nuclear Extracts from Cell Lines
Human adrenocortical cells, NCI-H295R (36, 37), were grown in a
mixture of DMEM/Ham’s F12 medium/Nuserum IV (45:45:10). Cells were
split 1:2 twice a week. Human hepatoma HepG2 cells and human
choriocarcinoma JEG-3 cells were grown in DMEM with 10% bovine serum.
Cells were split twice a week: HepG2, 1:7 and JEG-3, 1:5. All cultures
were grown at 37 C and 5% CO2. Nuclear extracts were
isolated from two 150-mm culture dishes containing two thirds confluent
cells as described (38). Protein concentrations were determined by the
Bio-Rad dye binding assay according to the manufacturer’s instructions
(Bio-Rad Laboratories, Inc., Richmond, CA).
Expression and Purification of Recombinant CREM and SREBP-1a
Proteins
Purification of bacterially expressed CREM protein was
previously described (16). Recombinant SREBP-1a protein (amino acids
1–490) was expressed in E. coli as a 6xHis-tagged fusion
protein at the amino terminus (39) and purified by Ni2+
affinity chromatography by the QIAexpressionist system
(Qiagen, Chatsworth, CA) as recommended by the
manufacturer. The expression plasmid was a gift of T. F. Osborne
(University of California, Irvine, CA). Protein concentrations for both
recombinant proteins were determined by the Bio-Rad Laboratories, Inc. dye binding assay according to the manufacturer’s
instructions. The homogenity of samples was checked by SDS/PAGE
followed by Comassie blue staining.
Production and Purification of the anti-SREBP-1
The SREBP-1 monoclonal antibody was produced from the IGG-2A4
(CRL 2121) hybridoma cell line (ATCC, Manassas, VA) and
purified by protein A-Sepharose affinity chromatography (Sigma Chemical Co., St. Louis, MO) as described (40). Antibody is
directed against amino acids 301–407 of SREBP-1 and therefore
recognizes both SREBP-1a and SREBP-1c isoforms, which differ only in
the sequence of the first exon (41). Isoform 1c is a weaker
transcription activator than isoform 1a in most cell types (42).
Gel Shift Analysis
Oligonucleotides used to generate double-stranded fragments
containing CRE and SRE elements were: CYP51-SRE1
(5'-GGCCGAGATCACCTCAGGCGCT-3' and 5'-GCGAGCGCC TGAGGTGATCTCG-3');
CYP51-SRE2 (5'-CGTGTCCCGGCACCCCGCACCCGG-3' and
5'-TGCCCGGGTGCGGGGTGCCGGGAC-3'); LDL-SRE
(5'-TTTGAAAATCACCC CACTGCA-3' and
5'-GTTTGCAGTGGGGTGATTTTC-3'); CYP51-CRE1
(5'-GGGACGGGGCTGACCTCACC GTCCT-3' and 5'-AGGACGGTGAGGTCAGCCCCGT-3');
CYP51-CRE2 (5'-GCCCCGTTGA CGCGATGTAGGCCGA-3' and
5'-GATCTCGGCCTACATCGCGTCAACGG-3'); CYP51-CRE2new
(5'-GCCCCGCTGACGCGATGTAGGCCGA-3' and 5'-GATCTCGGCCTACATCGCGTCAGCGG).
Oligonucleotides were purified on 20% acrylamide gels. Pairs were
annealed in equimolar concentrations and end labeled with
32P-ATP by T4 polynucleotide kinase. In some experiments
the commercial CREB consensus oligonuleotide (Promega Corp., Madison, WI) was used. Gel retardation assay was
performed as follows: 10 µg of nuclear proteins or 4–7 µg of the
overexpressed transcription factor were incubated in a 20 µl reaction
volume containing 20 mM HEPES, pH 7.6, 32 mM
KCl, 80 nM EDTA, 8% glycerol, 0.8 mM DTT, 1
µg poly dI-dC, 1 µg yeast tRNA at room temperature for 5 min.
Labeled probe (3 x 104 cpm) was added and incubated
on ice for an additional 10 min. DNA-protein complexes were resolved on
a 5% native acrylamide gel in 0.5 x TBE buffer at room
temperature and detected by autoradiography. For gel supershift
analyses, different volumes of rabbit sera containing the anti-CREM
antibody (16) or anti-CREB antibody were added. Anti-rat CREB antibody
was prepared by injecting rabbits with the E. coli expressed
rat CREB protein by standard procedures (43). Anti-SREBP1 antibody was
used before (crude antibody) or after purification on the protein A
Sepharose.
Preparation of CAT-Reporter Constructs
The 5'-untranslated region (-334/+314) of the human CYP51 gene
was amplified by the cloned Pfu polymerase
(Stratagene) using sense (5'-GGAGGAGGGTGAGGTGCC
ACAGTTCGAGGT-3') and antisense (5'-ACCTCGAACTGTGGCACCTCACCCTTCTCC-3')
primers from the cosmid 121G12 containing the whole human CYP51 gene
(12). The fragment was cloned into the SmaI-digested pCAT
basic plasmid (Promega Corp.). Two independent clones were
completely sequenced.
Cell Culture, Transfections, and CAT Assay
JEG-3 (human choriocarcinoma cells) were cultured in DMEM
supplemented with 10% bovine calf serum and 1%
L-glutamine. Cells were maintained in a humidified
incubator with 5% CO2 at 37 C. They were split 1:3 twice a
week and plated into culture dishes (90 mm diameter) 1 day before
transfection. Cells were transfected at approximately 50% confluency
by the calcium precipitate method (33) with 20 µg of plasmid DNA: 10
µg of the CYP51-CAT construct, 5 µg of the RSVß-gal plasmid (for
normalization), and 3 µg of pSV CREM or pCMVh SREBP 1a plasmids.
The pCAT basic plasmid was used as the carrier DNA to 20 µg. The CAT
activity of this plasmid is negligible. Twenty four hours after
transfection cells were treated either with forskolin, the artificial
inducer of the cAMP response (25 µM final concentration),
or the medium was removed and changed to DMEM, 10% delipidated serum,
and 1% L-glutamine, which is the media for induction of
the SREBP-dependent response. Cells were harvested 48 h after
transfection (24 h after addition of forskolin or delipidated serum).
CAT assays were performed as described (44). Protein concentrations
were determined by the Bio-Rad dye binding assay according to the
manufacturer’s instructions (Bio-Rad Laboratories, Inc.).
The ß-galactosidase activity assay was performed as described (33).
CAT activity was normalized by the formula: [CAT activity
(cpm)/ß-gal activity (A420)]/[protein concentration]
(mg/ml). Every transfection was performed at least three times with two
parallel samples in each experiment. The average value and
SEM were calculated with the Excel program (Microsoft Corp.).
Experimental Animals
Animal studies were conducted in accord with the principles and
procedures outlined in Guidelines for Care and Use of Experimental
Animals.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by USPHS Grants DK-28350, ES 00267, by Grant 9650310N from the American Heart Association and by Grants Z1–7225, J1–1380, and SLO-USA 0002 from the Ministry of Science of Slovenia.
Received for publication July 1, 1999. Revision received July 23, 1999. Accepted for publication August 6, 1999.
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REFERENCES |
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