 |
INTRODUCTION |
The conversion of cholesterol to pregnenolone, the first enzymatic
step in the biosynthesis of all steroid hormones, is catalyzed by the
mitochondrial cytochrome P450 side chain cleavage
(P450scc)1 enzyme (For review
see Ref. 1). Pregnenolone is the common precursor in the
steroidogenesis pathway, which leads to the synthesis of
mineralocorticoids, glucocorticoids, and sex hormones. The enzyme
P450scc is encoded by a single human gene, CYP11A1 (2), on
chromosome 15q23-24 (3), and its expression is regulated hormonally at
the transcriptional level (4). Although expression of the gene has been
detected in the central nervous system (5), P450scc transcript and
protein are expressed for the most part in steroidogenic tissues such
as adrenals, ovaries (6), testis, and the placenta (7-9). The hormonal
regulation and developmental pattern of expression of P450scc are
species- and tissue-specific (10). ACTH increases steroidogenesis and
accumulation of P450scc mRNA in the human adrenal zonae fasciculata
and reticularis. Luteinizing hormone (LH), follicle-stimulating hormone
(FSH), and human chorionic gonadotrophin (hCG) have the same effects in
Leydig cells and ovarian granulosa cells (11-13). It is apparent that
the cis-acting elements of the 5'-flanking region of the
P450scc gene involved in basal and cAMP-dependent
regulation are utilized in a developmentally programmed, tissue- and
cell type-dependent manner. For example, in human adrenal
NCI-H295 cells, the basal transcriptional activity lies within the
first 79 bp upstream from the transcriptional start site of the P450scc
gene (14), whereas the region between 
89 and 108 was found to
significantly increase promoter activity in human placenta JEG-3 cells
(15-17).
To identify tissue-specific transcription factors that regulate the
expression of cytochrome P450 hydroxylase genes, two separate groups
cloned the orphan nuclear receptor steroidogenic factor-1 (SF-1), also
known as Ad4BP (18, 19). SF-1 was identified based on its ability to
interact with a shared promoter element, PyCAAGGTCA, found in steroid
hydroxylase genes and was shown to activate their expression (20-22).
It has subsequently become apparent that SF-1 is a central regulator of
the endocrine and reproductive system (For review see Refs. 23 and 24).
It was shown in mouse and human that SF-1 is expressed at all levels of
the hypothalamic-pituitary-adrenal/gonadal axis, where it is required
for the expression of diverse genes that are essential for steroid
hormones biosynthesis. For instance, SF-1 is expressed in adrenal and
gonadal steroid-producing cells (5, 25), where it precedes P450scc gene
expression (26). In these cells, SF-1 was shown to activate the
expression of P450 hydroxylases (22, 27, 28), the ACTH receptor (29),
and the gene encoding steroidogenic acute regulatory protein (StAR) (30). In addition, it was demonstrated that SF-1 is expressed in
Sertoli cells, where it participates directly in the process of male
sex determination, and synergizes with SOX9 and WT1 in activation of
Müllerian inhibitory substance (MIS) gene expression (31-33). SF-1 is also expressed in pituitary gonadotropes, where it
activates the promoter for 
-gonadotropin, LH-
, and
gonadotropin-releasing hormone (GnRH) (24, 34). Also, the expression of
SF-1 in the ventromedial hypothalamic nucleus is correlated with the
synthesis of LH, FSH, and the GnRH receptor (34, 35). In agreement with the role of SF-1 in tissues involved in steroidogenesis and sexual differentiation, targeted SF-1 gene disruption produced knockout mice
which completely lacked the ventromedial hypothalamic nucleus, adrenal
glands, and gonads (reviewed in Ref. 23). Consequently, these
developmental defects were associated with phenotypic male-to-female sex reversal and adrenocortical insufficiency, resulting in neonatal death (36, 37). However, despite the many studies describing the
physiological functions and mechanisms of action of SF-1, it is
apparent that additional regulatory mechanisms involving this factor
remain to be elucidated.
As with other nuclear receptors, different mechanisms, including
phosphorylation and interaction with co-regulators, have been shown to
regulate basal and cAMP-responsive activity of SF-1. For example, a
consensus protein kinase A phosphorylation motif has been identified in
SF-1 (38) and has been found to mediate cAMP-responsive P450c17
hydroxylase gene expression (39). Furthermore, SF-1 has already been
shown to be a component of several multiprotein complexes involved in
the tissue- and promoter-specific hormonal responses of target genes.
For instance, SF-1 interacts with CBP/p300 (40) to activate the P450scc
gene promoter. Moreover, it was recently suggested that the c-Jun
protein acts synergistically with SF-1 to activate this promoter (41).
Identifying new factors that interact with SF-1 in protein complexes,
and understanding how they function with SF-1 to regulate the P450scc
gene will provide important insight into understanding how SF-1
modulates transcription.
The transcriptional regulating protein of 132 kDa (TReP-132) was
recently cloned and shown to activate the P450scc gene via the
5'-flanking DNA region from nucleotides 155 to 131 (42). Analysis
of the predicted primary structure of TReP-132 revealed motifs
characteristic of transcription factors and coactivators, which include
regions rich in glutamate, proline, and glutamine residues, as well as
two LXXLL motifs, suggesting the interaction of the protein
with nuclear receptors (43). SF-1 and TReP-132 transcripts colocalize
in many tissues, including the testis and adrenal cortex, which is
consistent with their functional interaction in steroidogenesis (5,
42).
This present study addresses the role of TReP-132 in the regulation of
the human P450scc gene mediated by SF-1 in human adrenal carcinoma
NCI-H295 cells. The overexpression of exogenous TReP-132 in these cells
led to increased pregnenolone production, which concurs with the
ability of this nuclear protein to increase P450scc expression.
Reporter gene assays showed that TReP-132 cooperates with SF-1 to
induce P450scc promoter activity; and the proximal SF-1 binding site at
position 46 to 38 was sufficient to confer responsiveness to both
proteins. The physical interaction between SF-1 and TReP-132 was
demonstrated and was shown to involve the LXXLL sequence
found at the amino-terminal region of TReP-132 as well as the proximal
activation domain and the AF-2 hexamer of SF-1. Considering that p300
was previously shown to interact with SF-1 and TReP-132 to regulate the
P450scc gene promoter (40, 42), coexpression of these three factors
showed cooperativity among them, leading to synergistic activation of
the promoter. The results of this study taken together demonstrate the
ability of TReP-132 to form a complex with SF-1 and CBP/p300 to
regulate gene transcription involved in steroidogenesis.
| |
EXPERIMENTAL PROCEDURES |
Cell Culture--
Human NCI-H295 adrenal tumor cells were
obtained from the American Type Culture Collection (Manassas,
VA). NCI-H295 were cultured in monolayers as described previously (14)
in Dulbecco's modified Eagle's medium/F12 medium supplemented with
penicillin (50 mg/liter), streptomycin (105 units/liter)
(Invitrogen), 5% fetal bovine serum (Hyclone, Logan, UT), 1% ITS (containing insulin, transferrin, selenium), Roche 1% red
phenol, 10
8 M, and 108
M OH-cortisone.
Plasmids--
The following constructs were made as described by
Monté et al. (40): P450scc luciferase reporter
constructs containing fragments of the human P450scc gene
spanning from nucleotides 1676, 155, or 110 at the 5'-end
to nucleotide +49 at the 3'-end subcloned in pGL3; the construct used
to express the GST-SF-1 fusion protein for electrophoretic mobility
shift assays; and the construct used to express an SF-1 fusion protein
containing a hemagglutinin (HA) tag at the carboxyl end (SF-1-HA). The
P450scc genomic clone was kindly provided by Dr. Bon-Chu Chung
(Academia Sinica, Nankang, Taipei). The SF-1 expression construct
containing the mouse SF-1 cDNA in the pCMV5 expression plasmid was
kindly supplied by Dr. Keith L. Parker (University of Southwestern Texas).
The TReP-132 mutants TReP-132m1, TReP-132m2, and TReP-132m1-2 were
mutated in NR-box 1, NR-box 2, and in both boxes, respectively. Leucines were changed to alanines (see Fig. 4A for
the sequence).
The human p300 cDNA in the pCMV-p300-HA and the mouse pCMV-CBP-HA
were kindly provided by Dr. Richard Goodman (Oregon Health Sciences
University) (44). The constructs expressing GST-CBP fusion proteins
were supplied by Dr. Ralph Janknecht (Hanover Medical School, Hanover,
Germany) (45).
The pFR-LUC plasmid, which comprises five GAL4 elements upstream of a
minimal E1b TATA-box followed by the luciferase gene, was purchased
from Stratagene. The pcDNA3-GAL4 vector was generated as
described previously (42) from the pSG424 plasmid (a gift from Dr.
Michael R. Green, Howard Hughes Medical Institute Research Laboratories, University of Massachusetts Medical Center, Worcester, MA). The GAL4-TReP-132 plasmid was created by subcloning a PCR product
corresponding to the entire coding region of TReP-132 into the
pcDNA3-GAL4 vector.
Transfections and Luciferase Assays--
NCI-H295 cells were
cultured in 12-well plates at a density of 3 × 105
cells/plate and grown for 24 h before transfection. The
medium was then changed, and transient transfections were carried out for 12 h. NCI-H295 cells were transfected with Effectene
transfection reagent (Qiagen, Mississauga, Ontario, Canada) at a ratio
of 1 µg of DNA to 25 µl of Effectene. Following transfection for
12 h, the cells were washed and incubated in fresh medium for
36 h in NCI-H295 cells. For luciferase assays, cells were
harvested, and 20 µl of the cell lysate were assayed for luciferase
activities with the Dual-LuciferaseTM reporter assay system
(Promega, Madison, WI). Firefly luciferase activities were normalized
to Renilla luciferase activity. The efficiency of
transfection was verified by green fluorescent protein expression
plasmid and efficiencies obtained were 60% or better.
Quantification by HPLC of Pregnenolone Levels Secreted by
NCI-H295 Cells--
NCI-H295 cells were cultured in 60 mm tissue
culture plates at a density of 1 × 106/plate, grown
for 24 h before transfection, and then transfected with 2 µg of pcDNA3 or TReP-132. At 24 or 48 h after transfection, two extractions of culture media (5 ml) were performed with 5 ml of
ethyl ether, and the organic solution was evaporated under nitrogen.
The dried extract was dissolved in 100 µl of isopropanol, and a
10-µl aliquot was diluted with 90 µl of HPLC elution solvent for
injection. The HPLC was carried out using the Waters Associates Millenium system (6000-A solvent pump, C-18 Novapack reverse phase column, and model 440 absorbance detector at 205 nm) and isocratic elution at 1 ml/min with acetonitrile-water (1:1). The retention time
of the standard pregnenolone was 13.8 min. Results are expressed as
nmol/mg protein.
Reverse Transcription and Quantitative PCR--
NCI-H295 cells
were first transfected with pcDNA3 or TReP-132 as described above.
Then, total RNA was isolated using Trizol reagent according to the
manufacturer's protocol, and the levels of P450scc mRNAs were
assessed by quantitative reverse transcription-PCR. Total RNA was
reverse transcribed using random hexamer primers and Superscript
reverse transcriptase (Invitrogen). Next, quantification by Real-Time
PCR was performed on a MX 4000 apparatus (Stratagene, La Jolla, CA)
using specific pairs of oligonucleotide primers, 5'-gaggccaccaagaactttttgcccc-3' and 5'-gatggcatcaatgaatcgctgggc-3', chosen by using the gene sequence XM_007646. Actin transcripts were
quantified simultaneously for normalization using the following oligonucleotide primers: 5'-gatgacccagatcatgtttga-3' and
5'-cggatgtcaacgtcacacttcatg-3'. PCR amplification was performed in a
final volume of 25 µl containing 100 nM each primer, 4 mM MgCl2, the Brilliant Quantitative PCR Core Reagent Kit mix as recommended by the manufacturer (Stratagene), and SYBR Green 0.33X (Sigma). PCR conditions were 95 °C for 10 min,
followed by 40 cycles of 30 s at 95 °C, 30 s at 55 °C,
and 30 s at 72 °C. P450scc mRNA levels were subsequently
normalized to actin mRNA levels.
In Vitro Protein Binding Assay--
SF-1 protein was expressed
as a GST fusion protein and was immobilized on glutathione-coupled
Sepharose as described (46) prior to incubation with radiolabeled
TReP-132 protein and washing, also as described previously (40).
The pGEX2TK-containing GST-SF-1 fusion proteins were transformed in
Escherichia coli strain BL21(DE3) pLysS following induction
with 0.1 mM
isopropyl-1-thio--D-galactopyranoside at 28 °C
overnight. The [35S]methionine-labeled TReP-132 was
synthesized using the rabbit reticulocyte lysate and T7 RNA polymerase
system (Promega) according to the manufacturer's protocol. Bound
proteins, released from the Sepharose by boiling in SDS sample buffer,
were resolved by 8% SDS-PAGE (47). The gels were stained
with Coomassie Blue to ascertain that equal amounts of GST proteins
were loaded, after which the gels were incubated for 30 min with
Amplify (Amersham Biosciences) and visualized by autoradiography.
Co-immunoprecipitation--
For co-immunoprecipitation
experiments, 1 × 107 HeLa cells were plated in a
100-mm culture dish and incubated for 24 h. Cells were transfected
using Exgen 500 at 40 µl/5 µg of DNA of SF-1 tagged with a HA
epitope (SF-1-HA) and 5 µg of TReP-132 tagged with a FLAG epitope
(TReP-132-FLAG) or pcDNA3 for 24 h. Immunoprecipitation was
performed according to the protocol of Santa Cruz Biotechnology, Inc. Cells were collected in radioimmune precipitation assay buffer (1× PBS, 0.1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, and
protease inhibitors). Cell debris was removed by centrifugation, and
the resulting cell extract was preincubated for 30 min with a
rabbit non-immune antibody (sc-2027, Santa Cruz Biotechnology). The
quantity of protein in the supernatant was assessed by Bradford assay
as instructed by the manufacturer (BioRad, Hercules, CA). 1 mg of
protein was incubated overnight at 4 °C with 10 µg of the rabbit
anti-SF-1 (06-431, Upstate Biotechnology) or non-immune antibody.
Immunoprecipitates were subjected to 8-12% SDS-PAGE and analyzed by
Western blot using an anti-FLAG antiserum (O5-447, Upstate
Biotechnology) at a ratio of 1/10,000 as the first antibody. After
three washes with 0.2% Tween-20 in PBS, membranes were incubated with
a horseradish peroxidase-coupled anti-mouse antisera (1/10,000, Jackson
Immunoresearch Laboratories, West Grove, PA) at room temperature for 45 min. Membranes were washed three times with 0.2% Tween 20 in PBS then
with 2% Tween 20 in 10× TBS and revealed with the Renaissance ECL
Plus kit according to the manufacturer's protocol (PerkinElmer Life Sciences).
| |
RESULTS |
TReP-132 Increases Pregnenolone and P450scc Transcript Levels in
Human Adrenal Cells--
In a previous study it was demonstrated that
TReP-132 increases P450scc gene promoter activity (42), which would
indicate that this transcriptional regulating protein is potentially
able to increase steroid synthesis. To confirm that an increased level of TReP-132 can stimulate the conversion of cholesterol to pregnenolone catalyzed by P450scc, NCI-H295 cells were transfected with a pcDNA3 expression plasmid encoding TReP-132 or with the empty pcDNA3 vector alone. As determined by reverse HPLC, overexpression of exogenous TReP-132 led to an increased level of pregnenolone in the
medium of NCI-H295 cells (Fig.
1A). Thus it is apparent that TReP-132 expression increases pregnenolone production in intact human
adrenal cells, probably via its positive activity on P450scc gene
expression.
View larger version (10K):
[in this window]
[in a new window]
|
Fig. 1.
TReP-132 enhances pregnenolone synthesis and
P450scc gene expression in NCI-H295 cells. NCI-H295 cells were
transfected with a pcDNA3 expression plasmid encoding TReP-132 or
with the empty pcDNA3 vector alone. A, 24 and 48 h
after transfection, pregnenolone secreted in the cell medium was
assessed by reverse HPLC. The results are expressed as nmol of
pregnenolone normalized to total cellular protein. Values are the mean
of five independent experiments ± S.D. B, 24 h
after transfection, P450scc mRNA levels were measured by
quantitative reverse transcription-PCR and normalized to actin mRNA
levels as control. Values are expressed as fold induction of P450scc
mRNA levels in cells transfected with TReP-132 versus
cells transfected with pcDNA3. Results are the average of three
independent experiments ± S.D. P450scc transcript levels are
up-regulated by TReP-132 transfection.
|
|
To determine whether the overexpression of exogenous TReP-132 can
indeed increase the level of P450scc transcript, quantitative reverse
transcription-PCR analyses was performed on RNA isolated from NCI-H295
cells transfected with the pcDNA3 expression plasmid encoding
TReP-132. The overexpression of TReP-132 led to an increased level of
P450scc transcript 2.9-fold over that obtained when cells were
transfected with the empty pcDNA3 vector alone, thus suggesting that TReP-132 increases expression of the human P450scc gene.
TReP-132 Increases SF-1 Activation of the Human P450scc Gene
Promoter--
During characterization of TReP-132, it was demonstrated
that this protein can increase the expression of reporter plasmids via
the 5'-flanking region of the human P450scc gene. Considering that
TReP-132 was isolated based on its ability to interact with the
155/-131 element by screening a human placenta cDNA expression library, this protein was demonstrated to interact with the 155/131 element in electrophoretic mobility shift assays and to activate promoter activity via this element (42). TReP-132 was shown to activate
expression of the P450scc gene promoter via a fragment of the gene from
1676 to +49 (42). However, our subsequent studies in NCI-H295 cells
showed the ability of TReP-132 to activate the 110/+49 sccLuc
reporter plasmid which does not have the 155/131 element but
contains a single putative SF-1 binding site at position 46. The
primary structure of TReP-132 contains two copies of the
LXXLL motif, which is found in transcriptional
coregulators and is involved in the interaction with nuclear receptors.
Considering that the SF-1 nuclear receptor is an important regulator of
P450scc gene expression, the present study has ascertained
whether TReP-132 interaction with SF-1 is one mechanism by which
TReP-132 can influence promoter activity.
Prior to determining the ability of TReP-132 to regulate P450scc gene
promoter activity via SF-1 and its binding site at position 46, the
relevance of this site was assessed. Previous experiments have
demonstrated the ability of SF-1 to confer significant promoter activity via the 5'-flanking region of the human P450scc gene in
NCI-H295 cells. It was shown that a fragment of the promoter region
from nucleotides 110 to +49, which contains a single SF-1 binding
site at position 46, is sufficient to confer response to SF-1 (14,
40). However, the 5'-flanking region of the gene from nucleotides
1676 to 110 contains four additional putative SF-1 binding sites;
thus the implication of the site at position 46 in the context of the
other upstream putative SF-1 cis-acting elements remained to
be determined. In agreement with previous results, SF-1 is able to
activate the 1676/+49sccLuc construct, which contains the five
putative SF-1 sites, as well as the 155/+49sccLuc and 110/+49sccLuc
constructs, which contain the single site (Fig. 2, A and B). In
addition, mutation of the SF-1 binding site at position 46 in the
1676/+49sccLuc, 155/+49sccLuc, and 110/+49sccLuc constructs
diminished the basal promoter activities and abolished the responses to
SF-1. Together these results indicate that SF-1 activates the human
P450scc gene promoter via the site at position 46, which was
previously shown to form a protein-DNA complex with the nuclear
receptor (40).
View larger version (24K):
[in this window]
[in a new window]
|
Fig. 2.
TReP-132 potentiates the transactivation of
P450scc mediated by SF-1. A, schematic representation of the
luciferase reporter constructs used in transcriptional activity assays
in NCI-H295 cells, containing progressive deletions of the human
P450scc gene 5'-flanking region from nucleotides 1676, 155, and
110 to +49. The constructs denoted as 1676/+49msccLuc,
155/+49msccLuc, and 110/+49msccLuc contain a mutated SF-1 binding
site found between nucleotides 38 and 46. B, the
promoter-reporter constructs (0.2 µg) were cotransfected with the
plasmid expressing SF-1 (0.1 µg). The results are expressed as
relative light units of luciferase activity. Transfections were
normalized to Renilla luciferase activity expressed from a
cotransfected plasmid. SF-1 increases expression of the reporter
constructs containing wild-type P450scc promoter regions, but the
transactivation is abolished by mutation of the SF-1 binding site.
C, the promoter-reporter constructs (0.1 µg) were
cotransfected with plasmids expressing SF-1 (0.1 µg) and TReP-132
(0.4 µg). The results are expressed as fold increase of luciferase
activity over the value obtained from the empty pGL3 reporter vector
alone. Coexpression of TReP-132 yielded significantly higher P450scc
promoter activities than obtained with expression of SF-1 alone.
TReP-132-mediated transactivation was abolished by mutation of the SF-1
binding site. Results represent the mean of four independent
experiments ± S.D.
|
|
To determine the ability of TReP-132 to influence SF-1 regulation of
the human P450scc gene, the 1676/+49sccLuc, 155/+49sccLuc, and
110/+49sccLuc reporter constructs were cotransfected with the
expression plasmids encoding SF-1 and TReP-132 in NCI-H295 cells (Fig.
2C). The expression of exogenous SF-1 or TReP-132 alone
increased the expression of all the wild-type reporter constructs tested, and the coexpression of both proteins led to the greatest increase in promoter activity. However, the constructs bearing a
mutated SF-1 binding site at position 46 exhibited no response to
SF-1 or TReP-132 when expressed either alone or in combination. These
results suggest a functional interaction between SF-1 and TReP-132,
which confers increased promoter activity via the SF-1 binding
site at position 46.
The TReP-132 and SF-1 Proteins Interact Directly--
To assess
whether TReP-132 and SF-1 interact directly, a pull-down assay was
performed. The immobilized glutathione S-transferase (GST)-SF-1 fusion protein was shown to bind specifically to
35S-labeled TReP-132 (Fig.
3A). To determine whether
TReP-132 interacts directly with SF-1 in intact cells,
immunoprecipitation analyses were performed on cell extracts in which
TReP-132 tagged with the FLAG epitope (TReP-Flag) was coexpressed with
SF-1 in HeLa cells, which do not express endogenous SF-1. Potential
protein complexes were immunoprecipitated using the anti-SF-1 antibody and subjected to Western blot analysis using a monoclonal anti-Flag antibody (Fig. 3B). The results show that the anti-SF-1
antibody precipitated a complex that contained the TReP-Flag protein.
The exogenous TReP-132 was precipitated neither in the absence of transfected SF-1 nor when the immunoprecipitation was performed with
non-immune serum. Thus, these results are indicative of a specific
interaction between SF-1 and TReP-132 in intact cells.
View larger version (20K):
[in this window]
[in a new window]
|
Fig. 3.
TReP-132 and SF-1 interact physically.
A, TReP-132 and SF-1 proteins interact in vitro
in a GST pull-down assay. SF-1 was expressed as a GST fusion protein
(GST-SF-1) and was immobilized on glutathione-coupled
Sepharose prior to incubation with
35[S]methionine-labeled TReP-132. The left
lane contains one-tenth the amount of TReP-132 protein used in all
incubation. Proteins were separated by 10% SDS-PAGE. B,
TReP-132 and SF-1 proteins interact in vivo in
coimmunoprecipitation assay. HeLa cells were transfected with
pcDNA3, TReP-132-Flag, or SF-1-HA or with both SF-1-HA and
TReP-132-Flag. The total extract of transfected cells were harvested,
immunoprecipitated with the polyclonal anti-SF-1 antibody, and
immunoblotted with an anti-Flag antibody. An immunoprecipitation was
performed with a rabbit non-immune serum as a negative control
(right lane). The coimmunoprecipitation of a
SF-1·TReP-132-Flag complex is obtained with the anti-SF-1 antibody
and not with the non-immune serum. C, TReP-132 and SF-1 proteins interact by
two-hybrid assay. The chimeric constructs composed of the GAL4
DNA-binding domain (GAL4) fused to the amino end of
full-length TReP-132 (GAL4-TReP) and the activation domain
of VP16 fused to full-length SF-1 (VP16-SF-1) were
transfected in HeLa cells with 1 µg of the pFR-Luc reporter plasmid
containing five GAL4 elements in front of the minimal E1b promoter. The
coexpression of SF-1 and TReP-132 fusion proteins increased the
luciferase activity above the control levels, thus indicating the
interaction between the proteins. The results are expressed as relative
light units of luciferase activity normalized to Renilla
luciferase activity. Values are the mean of three independent
experiments ± S.D.
|
|
To further confirm that SF-1 interacts with TReP-132 in intact cells,
two-hybrid assays were performed in HeLa cells. Two chimeric proteins
consisting of TReP-132 fused to the GAL4 DNA-binding domain (GAL4-TReP)
and SF-1 fused to the transactivation domain of VP16 (VP16-SF-1) were
coexpressed with the pFRLuc reporter plasmid containing five GAL4
binding sites (Fig. 3C). The coexpression of both fusion
proteins yielded an induction of promoter activity over the levels
obtained by coexpression of VP16-SF-1 with GAL4 or GAL4-TReP with VP16,
which is consistent with an interaction between TReP-132 and SF-1.
Interaction of TReP-132 with SF-1 Involves the Putative NR-box
LRQLL of TReP-132--
To further characterize the interaction between
TReP-132 and SF-1, which leads to increased promoter activity of the
P450scc gene, the interacting domains involved in each of the proteins were further defined. To identify the domain(s) implicated in TReP-132,
the full-length GAL4-TReP protein as well as fusion proteins containing
5' or 3' deletions of TReP-132 were coexpressed with VP16-SF-1 in HeLa
cells. The deletions were designed to remove specific regions
corresponding to domains that are potentially important in
protein-protein interaction and transcriptional activity, such as the
glutamine-, proline-, and glutamic acid-rich regions and the
zinc-finger domains (Fig. 4A).
In the constructs containing progressive deletion from the 3'-end of
TReP-132, removal of putative functional domains in GAL4-TRePdel2 to
del3 led to slight variations on basal promoter activity and SF-1
interaction (Fig. 4B). However, it is clear that the
shortest fusion protein (GAL4-TRePdel4) containing a single
LXXLL motif (LRQLL) at residue 181 retained the ability to
interact with SF-1. All of the GAL4-TReP constructs that were deleted
from the amino-terminal end, which do not contain the LRQLL motif, also
did not interact with SF-1. To further determine the functional
relevance of the LRQLL sequence, the leucine residues were changed to
alanines, thereby abolishing the LXXLL motif in the
full-length GAL4-TRePm protein, which resulted in abolition of the
ability of the fusion protein to interact with SF-1. Similar results
were obtained when the same mutations were introduced in the
GAL4-TRePdel4 construct (Fig. 4B).
View larger version (37K):
[in this window]
[in a new window]
|
Fig. 4.
SF-1 and TReP-132 interaction is dependent on
the putative NR-box LRQLL of TReP-132. A, schematic diagram
depicting GAL4-TReP-132 constructs containing progressive deletions of
the carboxyl-terminal end (GAL4-TRePdel1 to
-del4) and the amino-terminal end (GAL4-TRePdel5
to -del7) of TReP-132 fused to the DNA-binding domain of
GAL4. GAL4-TReP constructs mutated in the putative NR-box, LRQLL, are
labeled GAL4-TRePm and GAL4-TRePdel4m.
B, the GAL4 reporter construct, pFR-Luc (0.55 µg), was
cotransfected in SF-1-deficient HeLa cells with the various GAL4-TReP
fusion constructs (190 ng) in the presence or absence of VP16-SF-1 (340 ng). Transfections were normalized to Renilla luciferase
activity. The values are expressed as relative luciferase activity
(mean ± S.D.) and represent three independent experiments, each
performed in triplicate. The cotransfection of VP16-SF-1 with each
GAL4-TReP construct yielded significant activation of promoter
activity, which was abolished by deletion or mutation of the putative
LRQLL NR-box. C, transfection studies were performed in
human NCI-H295 cells with the 110/+49sccLuc reporter construct (0.2 µg) to determine the function of the two putative NR-boxes (LRQLL and
LEMLL) in full-length TReP-132 in the presence or absence of exogenous
SF-1. Cells were transfected with wild-type TReP-132, mutated at either
the LRQLL (TReP-132m1) or LEMLL (TReP-132m2)
site, or mutated at both sites (TReP-132m1-2). The results
are expressed as fold increase of luciferase activity over the value
obtained from the empty pGL3 vector. Transfections were normalized to
Renilla luciferase activity. The proteins SF-1 and TReP-132
interact to activate the P450scc gene promoter, but the cotransfection
of SF-1 with TReP-132 mutated in the LRQLL NR-box did not confer
luciferase activity above the values obtained with transfection of the
SF-1 vector alone. Results represent the mean of three independent
experiments ± S.D.
|
|
To further determine the functional importance of the two
LXXLL motifs in TReP-132, the sequences were mutated
individually and in combination in the full-length protein, and the
effects on the ability of TReP-132 to activate the P450scc gene
promoter was assessed with the 110/+49sccLuc reporter construct
transfected in the presence and absence of exogenous SF-1 in NCI-H295
cells. Mutation of the LRQLL sequence either alone or in combination with the mutation of the LEMLL sequence abolished the ability of
TReP-132 to increase promoter activity (Fig. 4C). The
increased promoter activity obtained when wild-type TReP-132 is
coexpressed with SF-1 was also abolished with these mutations. Although
the individual mutations of the LEMLL sequence also led to decreased transcriptional activation, the effect was consistently less than that
seen when TReP-132 is mutated at the LRQLL motif.
TReP-132 Interacts with SF-1 via the AF-2 Domain and the Proximal
Activation Domain--
As reported by Crawford et al. (48),
the carboxyl-terminal end of SF-1 contains an AF-2 hexamer, shown to be
included in the essential conserved motif for the transcriptional
activation function 2 (AF-2) domain of many nuclear receptors (49-56).
The AF-2 hexamer was shown to be required for transcriptional
activation, for functional interaction with SRC-1 (57, 58), and for
the ability of SF-1 to confer the steroidogenic phenotype to
embryonic stem cells by stable transfection (57). Another
functional domain found in SF-1, described by Crawford et
al. (48), is the proximal activation domain, located between
residues 187 and 245 and involved in interaction with SRC-1.
Additionally, the FTZ-F1-box and the proline cluster of SF-1 were
previously proposed to interact with TFIIB and c-Jun (59). To test
which domain of SF-1 is required for interaction with TReP-132, a
mammalian two-hybrid analysis was performed in HeLa cells by expressing
a series of VP16-SF-1 chimeric proteins coexpressed with GAL4-TReP or
with GAL4-TRePdel4 (Fig. 5). The
progressive deletion of the amino-terminal end of SF-1 showed
that removal of the DNA-binding domain did not diminish interaction
with TReP-132. However, removal of the proximal activation domain and
the upstream amino-end region between amino acids 119 and 187 led to
diminished interaction. Deletions of the carboxyl-terminal region of
SF-1 showed that removal of the regions from nucleotides 119-462
(VP16-SF-1del5), 245-462 (VP16-SF-1del4), or of just the AF-2
hexamer motif (VP16-SF-1-
AF-2) abolished the interaction between
SF-1 and TReP-132. Thus, it is apparent that the proximal activation
domain and the AF-2 hexamer both have essential roles in the
interaction with TReP-132.
View larger version (28K):
[in this window]
[in a new window]
|
Fig. 5.
The interaction between SF-1 and TReP-132
involves the proximal activation domain and the AF-2 domain of SF-1.
A, the structure of truncated forms of SF-1 fused to the
VP16 transactivation domain are shown. The functional domains are
denoted: DBD, DNA-binding domain; P, proline
cluster; AF-2, activation function-2. Regions denoted as
RII and RIII are involved in ligand binding with
AF-2 (49). B, HeLa cells were transiently cotransfected with
the luciferase reporter plasmid pFRLuc (550 ng) and the VP-16-SF-1
deletion constructs (340 ng) in combination with 190 ng of the
GAL4-TReP or GAL4-TRePdel4 expression constructs (depicted in Fig. 4).
The results are expressed relative to the luciferase activity obtained
with the wild-type VP16-SF-1 plasmid cotransfected with the GAL4-TReP
(left side) or GAL4-TRePdel4 plasmid (right
side). The GAL4-TReP and VP16-SF-1 proteins interact to activate
the pFRLuc reporter activity. Interaction between SF-1 and TReP-132 is
abolished by deletion of the AF-2 hexamer motif and the proximal
activation domain present in the SF-1 protein. The results are
normalized to Renilla luciferase activity and represent the
mean of four independent experiments ± S.D.
|
|
CBP/p300 Potentiates Activation of the P450scc Promoter by SF-1 and
TReP-132--
CBP/p300 was previously shown to interact with SF-1 (40)
to increase the activity of the P450scc gene promoter, and it was subsequently demonstrated in a separate study that CBP/p300 can also
interact with TReP-132 to increase expression of the same gene promoter
(42). Pull-down analyses performed in the present study demonstrate
that full-length TReP-132 can interact with CBP at three independent
regions (Fig. 6A). Also having
shown in this study that TReP-132 can interact with SF-1, it was next ascertained if these three factors can interact synergistically to regulate P450scc gene promoter activity. The greatest
increase of reporter gene expression (1676/+49sccLuc and
110/+49sccLuc) was obtained when expression plasmids encoding SF-1,
TReP-132, and p300 were cotransfected into NCI-H295 cells (Fig.
6B). The synergistic effect was observed only during
coexpression of the three factors and was not seen when they were
expressed alone or in any combination of only two of the proteins.
Therefore these results demonstrate the cooperativity between SF-1,
TReP-132, and p300, which interact synergistically to activate P450scc
gene expression.
View larger version (23K):
[in this window]
[in a new window]
|
Fig. 6.
TReP-132, SF-1, and CBP/p300 interaction
confers synergistic activation of the P450scc promoter. A,
TReP-132 interacts with three distinct regions of CBP by pull-down
assay. Different regions of CBP between the amino acid residues as
indicated at the top of the figure were expressed as GST
fusion proteins, and approximately equal amounts were immobilized on
glutathione-coupled Sepharose prior to incubation with TReP-132 labeled
with [35S]methionine. The regions of CBP between residues
1-451, 1099-1460, and 1892-2441 interacted with TReP-132, whereas
the other regions did not show increased interaction above the
background levels seen with incubation of TReP-132 with GST alone
(GST control). The leftmost lane
(TReP-132 input) contains one-tenth the amount of TReP-132
protein used in all incubations. Proteins were separated by 10%
SDS-PAGE. B, the human P450scc gene promoter-reporter
constructs 1676/+49sccLuc, 1676/+49msccLuc, and 110/+49sccLuc
(0.1 µg) were each transiently cotransfected with plasmids expressing
SF-1 (0.1 µg), TReP-132 (0.2 µg), and p300 (0.4 µg) or a
combination of these proteins, as indicated at the bottom of
the graph. The results are expressed as fold increase of luciferase
activity over the value obtained from pGL3 alone (± S.D.) and were
normalized to Renilla luciferase activities. The
coexpression of p300, SF-1, and TReP-132 leads to a synergistic
increase of promoter activity. This synergistic effect is abolished by
mutation of the SF-1 binding site found between nucleotides 38 and
46.
|
|
| |
DISCUSSION |
Cytochrome P450scc, which catalyzes the first step in the
synthesis of steroid hormones from cholesterol, is a key determinant of
steroid synthesis in steroidogenic tissues (for review see Ref. 60). It
is clear that the temporal and spatial specific expression of this gene
is required for steroid synthesis, which is necessary for many
physiological processes (6, 10, 61-63). However, relatively little is
known about the mechanism(s) of its regulation and the factors involved
therein. Recently, a novel zinc-finger protein, TReP-132, was isolated
and shown to increase P450scc gene promoter activity in human placental
JEG-3 and adrenal NCI-H295 cells (42). Although the TReP-132 transcript
is expressed in many of the tissues examined, the highest levels were
found in the adrenal cortex and the testis, which is consistent with a
role for TReP-132 in the regulation of P450scc gene expression in
steroidogenic tissues. In the present study, the overexpression of
exogenous TReP-132 in adrenal NCI-H295 cells led to increased pregnenolone production, which concurs with the ability of this nuclear
protein to increase P450scc expression.
To determine the mechanism(s) by which TReP-132 increases P450scc
expression in adrenal cells, it was found that exogenous TReP-132 can
increase the expression of a reporter plasmid under the control of the
5'-flanking region of the P450scc gene from nucleotides 110 to +49.
Although it has been shown that TReP-132 can increase P450scc gene
promoter activity via a cis-acting element between
nucleotides 155 and 131, the present study addressed the ability of
TReP-132 to regulate promoter activity via interaction with SF-1.
In previous studies, the region of the P450scc gene from 79 to +49,
which contains an inverted SF-1 binding site TCAAGGCCA between
nucleotides 38 and 46, was demonstrated to confer basal and
cAMP-responsive promoter activity in NCI-H295 cells (14). Because the
primary structure of TReP-132 contains putative NR-box sequences, which
suggests that it may interact with nuclear receptors to confer
increased promoter activity, the function of the SF-1 binding site at
position 46 was ascertained in the 1676/+49sccLuc reporter
construct that contains four additional upstream putative SF-1 binding
sites. In agreement with previous results (40), the presence of the
additional upstream sites did not confer significant additional basal
activity nor did it increase responsiveness to SF-1 in NCI-H295 cells.
However, mutation of the site at 46 decreased basal promoter activity
and abolished the response to SF-1, which concurs with the recent
in vivo results of Hu et al. (64) demonstrating in transgenic mice that mutation of the same site greatly reduces human
CYP11A1 gene promoter activity in adrenals. Despite the loss
of responsiveness to SF-1 in all the reporter constructs that contain
the mutated SF-1 site at position 46, the 5'-flanking region retained
the ability to express a minimal amount of promoter activity. This is
consistent with the results of Guo and Chung (65) showing that the
first 34 bp upstream from the transcriptional start site, containing a
TATA-box as the only known functional transcriptional element, is still
an active promoter and is able to direct cAMP-dependent
transcription in Y1 and NCI-H295 cells. With all of the reporter
constructs used in the present study, mutation of the SF-1 binding site
at position 46 also abolished the response to TReP-132, which
suggests an interaction between the two proteins where the ability of
TReP-132 to increase P450scc promoter activity is dependent on SF-1
binding to its cis-acting element.
As an initial approach to determining whether TReP-132 is able to
interact directly with SF-1, pull-down analysis demonstrated that the
GST-SF-1 fusion protein is capable of binding full-length TReP-132. To
confirm this interaction in vivo it was demonstrated in
cotransfection experiments in HeLa cells that SF-1 protein coprecipitates with TReP-132, which is indicative of the two proteins interacting in intact cells. A third approach, consisting of two-hybrid analyses, showed an interaction between the GAL4-TReP-132 and VP16-SF-1
fusion proteins. The results obtained using these three approaches are
thus consistent with the possibility that these two proteins interact
without the necessity of TReP-132 to bind DNA.
TReP-132 contains two nuclear receptor box (NR-box) LXXLL
motifs at amino acids 181 and 863, which are frequently found in the
nuclear receptor interaction domains of transcriptional coregulatory proteins such as p/CIP, NcoA-1, NcoA-2, RAC3, TIF-2/GRIP-1, SRC-1/p160, CBP/p300, RIP-140, TIF-1, and TRIP-1/SUG-1 (43, 66-70). To determine whether these two putative NR-boxes are involved in the interaction with SF-1, two-hybrid experiments were performed with TReP-132 amino-
and carboxyl-terminal deletion mutants fused to the DNA-binding domain
of GAL4. The GAL4-TRePdel4 protein, which comprises the amino-terminal
region of TReP-132 from residues 1 to 248 and contains the LRQLL motif
at residue 181, is sufficient to retain interaction with SF-1. In
contrast, the GAL4-TRePde15 to del7 proteins, which contain the LEMLL
motif at position 862 but not the LRQLL sequence at residue 181, were
not able to interact with SF-1. Thus, only the TReP-132 constructs
containing the LRQLL motif are able to interact with VP16-SF-1.
Moreover, the interactions of GAL4-TReP and GAL4-TRePdel4 with
VP16-SF-1 were abolished when the leucines of the LRQLL sequence were
changed to alanines, thus further implicating the LRQLL motif in the
interaction with SF-1. To confirm the importance of the LRQLL motif in
the function of TReP-132 as a coactivator of SF-1, mutation of this
sequence in full-length TReP-132 abolished the stimulatory effect on
P450scc gene promoter activity in NCI-H295 cells cotransfected with
SF-1.
The two-hybrid experiments also implicated the two
carboxyl-terminal zinc-fingers of TReP-132 in transcriptional
regulation. Deletion of the region containing the zinc-finger motifs in
the GAL4-TRePdel1 construct decreased the ability of TReP-132 to
activate basal promoter activity and decreased the functional
interaction between TReP-132 and SF-1. The C2H2
zinc-finger domains of transcription factors have previously been
implicated in protein-protein interactions. For example, both the aryl
hydrocarbon receptor (AhR) and the AhR nuclear translocator (Arnt),
which form a trans-acting heterodimer, interact with the
zinc-finger domain of Sp1 to confer drug inducible expression of
CYP1A1 (71). Similarly, the carboxyl-terminal zinc-fingers
of GATA1, which interact and synergize with Sp1 and EKLF (72), are
sufficient to induce megakaryocytic differentiation (73). As other
examples, p53 and par-4 were shown to interact with the
C2H2-type zinc-fingers located at the carboxyl
terminus of WT1 (74, 75) and to repress the activity of WT1. Because the transcriptional activation and repression domains of WT1 have been
mapped to regions outside of the zinc-finger domains, it was suggested
that par-4 does not play a role as a classical cofactor for
WT1-mediated transcription but is nonetheless a critical modulator that
contributes to the determination of WT1 function as either a
transcriptional activator or repressor. Although it is apparent that
the zinc-fingers at the carboxyl region of TReP-132 are involved in its
transcriptional activity, the mechanism(s) of action and factors
implicated remain to be elucidated.
Unlike the classical nuclear receptors, which have known high affinity
ligands that are essential for AF-2-mediated activation (50, 52, 53,
76, 77), a high affinity ligand for SF-1 remains to be identified (78,
79). Mutations in the AF-2 domain of SF-1 suppresses protein kinase
A-dependent transactivation of the bovine CYP17
gene (80). Moreover, the AF-2 domain was shown to interact with SRC-1
and to be required for GAL4-SF-1 transactivation (58). In the present
study, two-hybrid experiments show that the AF-2 domain of SF-1 is
required for interaction with TReP-132, because deletions of the AF-2
hexamer motif in the VP16-SF-1 chimeric proteins alleviated interaction
with GAL4-TReP and GAL4-TRePdel4.
In addition to the AF-2 domain, other regions of SF-1 have been shown
to be involved in protein-protein interactions. Although SF-1 does not
harbor a transcriptionally active AF-1 domain, this nuclear receptor
has been shown to contain other active domains in the amino-terminal
region. Crawford et al. (48) have shown that full
interaction of the SRC-1 coregulator with SF-1 requires an intact AF-2
hexamer motif and the proximal activation domain located between amino
acids 187 and 245. The present study shows that the same domains are
involved in the interaction between SF-1 and TReP-132, because the
deletion of either the proximal activation domain or the AF-2 hexamer
motif in SF-1 alleviated interaction with TReP-132. It is also shown
here that the addition of an amino region to the proximal activation
domain of SF-1 between amino acids 119 and 187 increased interaction
with TReP-132, which concurs with the results of Crawford et
al. (48) showing that this region also increases the interaction
between SF-1 and SRC-1. It can be speculated that SF-1 interacts
differentially with coregulatory proteins to regulate gene
transcription in a promoter- and cell type-specific manner. However,
with the recent reports demonstrating the implication of other factors
such as Dax-1 (32, 81-84) and N-CoR (85) in SF-1 function, it is clear
that other studies will be required to fully understand the mechanisms
by which SF-1 regulates the expression of its many target genes.
The coregulators CBP/p300 have been demonstrated to interact with SF-1
and to increase the promoter activity of the human P450scc gene via the
SF-1 binding site located between nucleotides 38 and 46 (40).
Subsequently, it has been shown that CBP/p300 also interact with
TReP-132, leading to increased promoter activity of the human P450scc
gene (42). With the finding of the present study that TReP-132
interacts with SF-1, another question we addressed was whether the
three factors CBP/p300, TReP-132, and SF-1 can function in association
to increase transcriptional activity. The transfection experiments
clearly demonstrate that combined coexpression of all three proteins
leads to a synergistic activation of the P450scc gene promoter that is
much higher than when coexpressing either of the two proteins alone.
This cooperativity is similar to the situation described for the
carboxyl-terminal transactivation domains of HIF-1, which was shown
to function in synergy with SRC-1 and CBP to increase transcription
(86).
In the study of Monté et al. (40) SF-1 was shown to
interact with both the amino- and carboxyl-terminal regions of CBP between amino acids 1-451 and 1460-1891. In a subsequent study, a
partial TReP-132 protein that was truncated at the amino-terminal end
and thus contained residues 439 to 1200 was shown to interact with CBP
at the region from amino acids 1460 to 1891 (42). In the present study,
the full-length TReP-132 protein was shown to interact with the regions
of CBP between residues 1-451, 1460-1891, and 1892-2441. These three
regions of CBP involved in interaction with SF-1 and TReP-132 are also
involved in the interaction with other distinct nuclear receptors and
transcriptional coregulators (for review see Refs. 87 and 88). The
region 1-460 was previously shown to interact with RAR,
RXR, TR (89, 90), ER (91), CREB, c-Jun (44, 92), and
PPAR
2 (93). The region between residues 1460 and 1891 contains the
histone acetyltransferase domain and is involved in interaction
with transcriptional activators such as MyoD (94), TFIIB (95), and
E2F-1 (96). The most carboxyl region of CBP from residues 1892 to 2441, which interacts with TReP-132, contains the domain shown to interact
with the coregulator SRC-1 (97, 98). The interaction of TReP-132 and
SF-1, with more than one domain of CBP, is not too surprising
considering that this has been observed with other transcription
factors such as p/CAF (99), STAT1 (100), p65 (101), and AP-1 (102).
TReP-132 was isolated based on its ability to bind to the region
between nucleotides 155 and 131 of the P450scc gene promoter. However, the interaction of TReP-132 with SF-1 and the requirement of
an intact SF-1 binding site for TReP-132 activity in human adrenal
NCI-H295 cells indicate that this protein also functions as a
coactivator of the nuclear receptor. Moreover, TReP-132 was shown to
activate the reporter construct containing three copies of the SF-1
binding site upstream of the minimal thymidine kinase promoter in
NCI-H295 cells; and TReP-132 does not interact with the SF-1 binding
site, as determined by electrophoretic mobility shift analyses (data
not shown). The function of TReP-132 as a coregulator of SF-1 resembles
other factors that have been demonstrated to interact with SF-1 and to
influence its activity on gene promoter regulation. The Ptx1 homeobox
transcription factor was initially demonstrated to bind to and activate
expression of the pituitary pro-opiomelanocortin (POMC) gene
(103, 104). Subsequently, Ptx1 was also shown to interact and synergize
with SF-1 to activate the pituitary LH gene promoter, where Ptx1
apparently does not bind DNA (105). It has also been reported that the
orphan nuclear receptor Dax-1 (dosage-sensitive sex reversal-adrenal
hypoplasia congenita critical region on the X chromosome, gene 1)
suppresses SF-1 activity through direct physical interaction (81, 85) and inhibits the expression of several SF-1 target genes including those encoding MIS (32), the high density
lipoprotein receptor (82), and hP450c17 (84). In the study of Crawford
et al. (85), Dax-1 was shown to recruit the corepressor
N-CoR to SF-1 to repress promoter activity. In the study of Nachtigal
et al. (32), Dax-1 was demonstrated to interact with SF-1
and to antagonize the synergy between SF-1 and WT1 involved in
regulation of MIS gene expression. It is interesting that
Dax-1 and WT1 are two transcription factors that, under certain
promoter contexts, interact directly with cis-acting
elements (106, 107), but in regulation of the MIS gene their
mode of action apparently does not involve direct DNA binding when
interacting with SF-1 (82, 83).
Results taken from the present and previous studies (40, 42) indicate
that TReP-132, SF-1, and CBP/p300 interact together to regulate gene
transcription. As described for other transcription factors and
coregulators, it is tempting to speculate on a mechanism by which SF-1
and TReP-132 are bridged by CBP/p300 and influence the interaction of
p300 with other factors to influence RNA polymerase II transcription.
An example of this is the role of CBP/p300 as a "bridging molecule"
in the positive cross-talk between STAT3 and Smad1 (108) and the role
of CBP/p300 between Myb and C/EBP to activate Mim-2 in
myelomonocytic differentiation (109). Considering the different
coregulators that interact with SF-1 (such as Dax-1, N-CoR, and WT-1),
it is possible that TReP-132 also interacts with these proteins to
recruit or displace factors in a SF-1-dependent complex
required to regulate gene expression. The coexpression of TReP-132 with
these SF-1-interacting proteins in the same tissues, which include the
adrenal and testis, is consistent with their interaction. It is clear
that additional studies will be required to further decipher and
understand the mechanism(s) by which TReP-132 and SF-1 interact to
regulate gene expression, which may play a major role in
steroidogenesis, organogenesis, and reproductive function.
,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Genfit, Parc
Eurasanté, 885, Ave. Eugène Avinée, 59120 Loos,
France. Tel.: 33-3-20-16-40-00; Fax: 33-3-20-16-40-01; E-mail:
Dean.Hum@genfit.com.
