(Received for publication, May 13, 1996, and in revised form, September 30, 1996)
From the Center for Molecular and Vascular Biology, University of Leuven, B-3000 Leuven, Belgium
A 2.4-kilobase (kb) DNA fragment, located 7.1 kb
upstream from the human tissue-type plasminogen activator (t-PA) gene
(t-PA2.4), acts as an enhancer which is activated by glucocorticoids,
progesterone, androgens, and mineralocorticoids. Transient expression
of t-PA-chloramphenicol acetyltransferase reporter constructs in HT1080
human fibrosarcoma cells identified a glucocorticoid responsive unit
with four functional binding sites for the glucocorticoid receptor,
located between bp 7,501 and
7,974. The region from bp
7,145 to
9,578 (t-PA2.4) was found to confer a cooperative induction by
dexamethasone and all-trans-retinoic acid (RA) to its
homologous and a heterologous promoter, irrespective of its
orientation. The minimal enhancer, defined by progressive deletion
analysis, comprised the region from
7.1 to
8.0 kb (t-PA0.9) and
encompassed the glucocorticoid responsive unit and the previously
identified RA-responsive element located at
7.3 kb (Bulens, F.,
Ibañez-Tallon, I., Van Acker, P., De Vriese, A., Nelles, L.,
Belayew, A., and Collen, D. (1995) J. Biol. Chem. 270, 7167-7175). The amplitude of the synergistic response to dexamethasone
and RA increased by reducing the distance between the enhancer and the
proximal t-PA promoter. The synergistic interaction was also observed
between the aldosterone and the RA receptors. It is postulated that the
t-PA0.9 enhancer might play a role in the hormonal regulation of the
expression of human t-PA in vivo.
Vascular patency is the result of a dynamic equilibrium between
blood coagulation and fibrinolysis. Injury of the vessel wall can
initiate the blood clotting cascade resulting in the formation of a
hemostatic clot. To counterbalance fibrin deposition, blood contains
the fibrinolytic system, one main function of which is to dissolve
blood clots in the circulation. It is composed of the inactive
precursor plasminogen, which can be converted into the proteolytic
enzyme plasmin by the plasminogen activators
(PAs)1 tissue-type and urokinase-type PA
(t-PA and u-PA, respectively) leading to the degradation of fibrin.
Fibrinolytic activity can be inhibited both at the level of the PAs and
plasmin by PA inhibitors (PAI-1 and -2) and
2-antiplasmin, respectively (for a review, see ref. 1).
Phenotypic analysis of mice deficient for either t-PA, u-PA, or both
suggested that t-PA and u-PA were complementary, not only in the
prevention of uncontrolled fibrin deposition in vivo but
also in distinct processes which require local extracellular proteolytic activity, such as wound healing and gonadotropin-induced ovulation, as reviewed elsewhere (2).
Dexamethasone, a synthetic glucocorticoid, and androgens increase t-PA
synthesis in vitro (3, 4, 5) and in vivo (6, 7). The
level of t-PA expression in breast carcinoma cells correlates with the
endogenous level of progesterone receptor (8), and progesterone induces
t-PA-related antigen secretion in primary human endometrial cells (9).
Vitamin A, retinoic acid (RA), and some of its (synthetic) analogues
induce t-PA-related antigen secretion by human umbilical vein
endothelial cells in vitro (10, 11, 12) and in the plasma as
well as in specific tissues of vitamin A-deficient rats in
vivo (11, 13, 14), suggesting that circulating RA might regulate
t-PA expression in the vessel wall. The induction of t-PA expression by
glucocorticoids and RA can be reproduced in HT1080 human fibrosarcoma
cells, and interestingly, both agents induce t-PA mRNA steady state
levels and t-PA-related antigen secretion in a cooperative
manner.2 Both steroid hormones and RA are
triggers of ligand-dependent activation of their respective
receptors, which are members of the nuclear receptor superfamily, a
class of transcription factors that specifically bind to
cis-elements in the regulatory regions of given genes (15).
Most hormone responsive genes have a functional RA or steroid
responsive element located in the close vicinity of the transcription
start site. In the human t-PA gene, however, an RA-responsive element
(RARE) consisting of a direct repeat of the GGGTCA motif was identified
at 7.3 kb (t-PA/DR5) and shown to mediate induction of t-PA
expression by RA (16). The present study provides evidence that the
t-PA/DR5 RARE is part of a multihormone responsive enhancer covering
the upstream fragment from
7.1 to
8.0 kb (t-PA0.9) which contains
an unusually complex GRU composed of four binding sites for the GR. It
is suggested that the enhancer mediates the synergistic response of
t-PA gene transcription to RA and steroids.
Human HT1080 fibrosarcoma cells were obtained from
the American Type Culture Collection (Rockville, MD). The expression
vector encoding the human androgen receptor (AR, pRSV-hAR) was a gift from Dr. A. O. Brinkmann (Erasmus University, Rotterdam, The
Netherlands), expression vectors encoding the human estrogen receptor
(pRSV-hER) and the human progesterone receptor (pRSV-hPR) from Dr. P. Chambon (Institut de Génétique et de Biologie
Moléculaire et Cellulaire, Illkirch, France), the expression
vector encoding the human GR (pRSV-hGR) and the human mineralocorticoid
receptor (pRSV-hMR) from Dr. R. M. Evans (The Salk Institute for
Biological Studies, La Jolla, CA), the firefly luciferase expression
vector (pRSVLuc) from Dr. D. R. Helinski (Department of Biology,
University of California, San Diego, CA). G418, Dulbecco's modified
Eagle's medium, and all medium supplements were purchased from Life
Technologies, Inc. (Ghent, Belgium), tissue culture recipients from
Corning Inc. (New York, NY) and Becton Dickinson (Franklin Lakes, NJ), D-aldosterone, RA, chloramphenicol, dexamethasone,
17-estradiol, and progesterone from Sigma. DNA
purification columns from Qiagen (Chatsworth, CA), the synthetic
androgen methyltrienolone (R1881), acetyl-CoA, and
[3H]acetyl-CoA from ICN Biomedicals (Costa Mesa, CA),
reporter lysis buffer and luciferin substrate from Promega (Madison,
WI), Galacto-Light kit from Tropix (Bedford, Mass), and Lipoluma from
Lumac-LSC (Olen, Belgium).
HT1080 cells were grown in supplemented
Dulbecco's modified Eagle's medium, containing glutamine (1 mM), penicillin (100 IU/ml), streptomycin (100 µg/ml),
and 10% heat-inactivated fetal calf serum. For experiments cells were
seeded at a density of 2-4 × 104
cells/cm2 and grown overnight at 37 °C in a humidified
95%, 5% air, CO2 atmosphere in medium with 5%
heat-inactivated, charcoal-stripped fetal calf serum. RA was dissolved
in dimethyl sulfoxide at a concentration of 10-100 mM and
stored at 80 °C. D-Aldosterone, dexamethasone,
17
-estradiol, progesterone, and the synthetic androgen R1881 were
dissolved in ethanol at a concentration of 10-100 mM and
stored at
80 °C. The appropriate concentration was added to the
medium in a volume corresponding to 0.1% of the culture medium.
Control medium contained an equal amount of excipient.
Isolation of t-PA upstream sequences
and their incorporation in chloramphenicol acetyltransferase (CAT)
reporter plasmids has been described previously (16). All genomic
sequences analyzed in this study are numbered relative to the
transcription initiation site as determined by Henderson and Sleigh
(17). The t-PA7144-CAT and t-PA9578-CAT constructs contain a 7.3 kb (bp
+197 to 7, 144) and a 9.8-kb upstream fragment (bp +197 to
9,578), respectively (see Figs. 4 and 5), linked to the CAT gene in
the reporter plasmid pBLCAT3 (18). Deletion mutants t-PA632-CAT,
t-PA1564-CAT, and t-PA3070-CAT contain 632, 1564, and 3070 bp of t-PA
gene upstream sequence, respectively. The 2.4-kb BamHI
upstream genomic fragment (bp
7,145 to
9,578: t-PA2.4) was inserted
in front of these constructs yielding t-PA632
2.4-CAT,
t-PA1564
2.4-CAT, and t-PA3070
2.4-CAT, respectively (see Fig. 5),
and in either orientation in front of the thymidine kinase
(TK)-promoter linked to the CAT gene in pBLCAT2 (18) yielding
t-PA2.4-TK-CAT and t-PA2.4INV-TK-CAT (see Figs. 4 and 6).
The t-PA2.0-TK-CAT construct was obtained by progressive exonuclease
III deletion mutagenesis from t-PA2.4-TK-CAT. Internal deletions of the
t-PA2.4 fragment (from bp 7,145 to
9,578) were created by
recombining deletion fragments from t-PA2.4-TK-CAT and from
t-PA2.4INV-TK-CAT, yielding t-PA2.4
GREahi-TK-CAT, t-PA2.1-TK-CAT, t-PA2.0
GREahi-TK-CAT, and t-PA2.0
GREfg-TK-CAT (coordinates of the
deleted regions are shown in Fig. 2). The region from bp
7,896 to
8,041 was removed from t-PA2.0
GREahi-TK-CAT by deletion of a
RsaI-PstI fragment (146 bp) yielding
t-PA2.0
GREafghi-TK-CAT.
Transfection Analysis
To obtain stable expression of
t-PA-CAT reporter fragments in HT1080 cells, the calcium phosphate
coprecipitation method (19) was applied to a 10-cm dish using a DNA
mixture which contained 20-60 µg of PvuI-linearized CAT
reporter plasmid (5 µg/kb of plasmid) and 4 µg of
PvuI-linearized pCMVNeo selection plasmid (20). After
48 h of incubation, G418 (500 µg/ml) was added to the medium and
selection was performed for 10 days. In order to avoid positional effects, expression experiments were performed using a large pool of
resistant colonies (102-103). After growing to
confluency, cells were harvested and seeded in 6-well dishes in
Dulbecco's modified Eagle's medium containing 0.25% (w/v) bovine
serum albumin and 0.5% charcoal-stripped serum. After overnight
incubation fresh medium was added containing dexamethasone and/or RA or
excipient. After 24 h of incubation, cells were analyzed as
described below.
To obtain transient expression of the t-PA promoter constructs in
HT1080 cells the calcium phosphate coprecipitation method (19) was
applied to a six well dish using a DNA mixture of 20-60 µg reporter
plasmid (5 µg/kb plasmid) with 0.1 µg of pRSVLuc or pCMVGAL
plasmid and 0.7 µg of the indicated nuclear receptor expression
plasmid. Cells were stimulated with the indicated hormone immediately
(treatment for 36 h) or 16 h after the glycerol shock (treatment for 24 h).
Cell extracts were prepared by three freeze-thaw cycles (Tris·HCl 100 mM, pH 7.8, EDTA 5 mM) or by using reporter
lysis buffer. Equal amounts of protein were used for the determination
of CAT activity by the liquid scintillation method (21), a mixture of
[3H]acetyl-CoA (0.1 µCi), acetyl-CoA, and
chloramphenicol (final concentration 0.1 and 0.9 mM,
respectively) was added to equal amounts of cell extracts, overlayered
with scintillation solution (Lipoluma) and the rate of
3H-labeled acetyl-chloramphenicol generation was measured
using a liquid scintillation counter. Luciferase or -galactosidase activity used as an indicator for the transfection efficiency was
measured in a luminometer after addition of luciferin or Galacton chemiluminescent substrate, respectively. Data obtained from stable and
transient expression experiments were corrected for endogenous CAT
activity or apparent luciferase or
-galactosidase activity.
All data shown represent values obtained from at least two independent experiments, each performed in triplicate (n = 6 or 9) and for which at least two different plasmid preparations were used.
Electrophoretic Mobility Shift Assay and DNase I Protection AnalysisDNA oligonucleotides or fragments were labeled by
filling in with the Klenow fragment of DNA polymerase I in the presence of [32P]dCTP. Electrophoretic mobility shift assays
(EMSAs) were performed according to Fried and Crothers (22) as modified
by Baes et al. (23). Purified fusion protein (at 50 ng/µl
as determined by polyacrylamide gel electrophoresis and Western blot
analysis) of protein A and the DNA-binding domain of the glucocorticoid or the androgen receptor (GRF and ARF, respectively) (24) were added to
the reaction mixture containing 10,000 cpm of labeled DNA fragment or
oligonucleotide followed by incubation at 4 °C for 15 min. EMSA
reactions were analyzed by a 4-5% polyacrylamide gel electrophoresis
at 4 °C in 0.5 × Tris borate buffer. Bands were visualized by
autoradiography. In vitro DNase I protection analysis was
performed according to Galas and Schmitz (25) as modified by Claessens
et al. (26). The DNA fragments were end-labeled as described
above and incubated with the ARF in a Tris·HCl buffer (20 mM, pH 7.9) containing 50 mM KCl, 6.25 mM MgCl2, 0.5 mM EDTA, 1 µg
poly(dI-dC), 2% polyvinyl alcohol, 1 mM dithiothreitol in a final volume of 50 µl. After incubation for 20 min on ice, 50 µl
of a 10 mM MgCl2, 5 mM
CaCl2 solution were added containing 0.1 unit of DNase I. The reaction was stopped after 1 min by the addition of 100 µl of
10% SDS, 200 mM NaCl, and 20 mM EDTA. Samples were extracted once with phenol-chloroform, and DNA was precipitated with ethanol and resuspended in gel loading dye (98% formamide, 10 mM EDTA, 0.2% bromophenol blue and 0.2% xylene cyanol).
The DNA samples were analyzed together with the A and the A/G Maxam and
Gilbert degradation reactions of the same radiolabeled DNA fragment as
described previously (26).
Co-expression of the t-PA9578-CAT construct with
different steroid hormone receptors in HT1080 cells revealed a similar
induction by 107 M progesterone,
10
7 M D-aldosterone,
10
8 M synthetic androgen R1881, and
10
7 M 17
-estradiol. Inductions varied from
2.5 ± 0.13-fold to 4.1 ± 0.73-fold compared to 3.1 ± 0.12-fold for 10
7 M dexamethasone, the latter
in the absence of co-expressed receptor (see Fig. 1). In
contrast, the t-PA7144-TK-CAT construct was not induced by
dexamethasone, progesterone, aldosterone, and R1881. A reproducible
induction of 1.8 ± 0.04-fold was observed with 10
7
M 17
-estradiol when the human estrogen receptor was
co-expressed. In order to assay putative enhancer activity of the
t-PA2.4 fragment deleted in the shorter construct, it was fused to the
TK promoter yielding t-PA2.4-TK-CAT. This construct was not responsive
to 17
-estradiol (1.1 ± 0.13-fold) but dexamethasone,
progesterone, aldosterone, and R1881 were equally potent, resulting in
inductions varying from 6.1 ± 0.64-fold to 10 ± 1.6-fold.
In conclusion, the t-PA2.4 enhancer was equally responsive to all steroid hormones except estrogens and able to confer this induction to both its natural or a heterologous promoter. The regulatory sequences involved in the response of the human t-PA gene to estrogens seemed to be located downstream from the t-PA2.4 enhancer.
Identification of Glucocorticoid Responsive ElementsTable I and Fig. 2, panels A and C, represent putative glucocorticoid responsive elements (GREs; t-PA/GREa, t-PA/GREd, t-PA/GREf, and t-PA/GREg) and GRE half-sites (t-PA/GREh and t-PA/GREi) identified in the t-PA2.4 fragment by the presence of the imperfect palindromic TGTTCT motif (30).
|
Wild type and mutant t-PA2.0-TK-CAT constructs were evaluated in HT1080
cells by co-expression with the glucocorticoid receptor (GR) in order
to obtain a maximal response (see Fig. 2, panel B). Whereas
the t-PA2.0-TK-CAT wild-type construct was induced 11 ± 2.4-fold,
elimination of the region from 7,318 to
7,535 (containing the
t-PA/GREa, GREh, and GREi elements, construct t-PA2.0
GREahi) or the
region from
7,896 to
8,041 bp (containing the t-PA/GREf and GREg,
construct t-PA2.0
GREfg) reduced the stimulatory effect of
dexamethasone to 5.3 ± 0.65-fold and 6.7 ± 1.6-fold, respectively. Further elimination of the t-PA/GREa, GREh, and GREi
elements in the latter construct by deletion of the region from
7,318
to
7,535 bp (t-PA2.0
GREafghi-TK-CAT) reduced the induction to
2.0 ± 0.12-fold compared to a 1.6 ± 0.15-fold induction for
the TK promoter alone.
Constructs containing site-specific mutations were evaluated in the
absence of co-expressed GR in order to increase the sensitivity of the
analysis (see Fig. 2, panel D). Site-specific mutation of
either the GREa or the GREd element alone had very little effect on the
induction by dexamethasone (6.7 ± 0.65 and 6.5 ± 0.25-fold, respectively, compared to 7.5 ± 0.85-fold induction for the wild type construct t-PA2.0-TK-CAT), but a construct with both mutated GREa
and GREd elements was only induced 2.6 ± 0.25-fold (Fig. 2,
panels C and D). Simultaneous mutation of the
GREh and GREi half sites in the wild-type construct and in the
t-PA2.0GREadMUT construct did not alter their response (6.9 ± 0.25-fold compared to 7.5 ± 0.85-fold, data not shown, and
2.5 ± 0.34-fold compared to 2.6 ± 0.25-fold, respectively).
In contrast to the results obtained in the presence of co-expressed GR
(see Fig. 2, panel B), elimination of the GREf and GREg
elements by deletion of the region from bp 7,896 to
8,041
(construct t-PA2.0
GREfg-TK-CAT) had no significant effect on the
response of the t-PA enhancer (6.0 ± 1.5-fold versus
7.5 ± 0.65-fold for the wild-type construct t-PA2.0-TK-CAT, data
not shown). Elimination of this region and simultaneous mutation of
GREa and GREd (t-PA2.0GREadMUT
fg-TK-CAT) abolished the response to
dexamethasone completely (0.9 ± 0.15-fold).
Putative GRE elements were linked in two copies to the TK-CAT fusion gene and assayed in transient transfection. Combination of the t-PA/GREf and the GREg elements (with a 3-bp intervening sequence as in their natural context) conferred a 22 ± 0.72-fold induction compared to a 2.5 ± 0.18- and a 1.2 ± 0.1-fold induction for the t-PA/GREf and the GREg elements alone (data not shown). A TK-CAT construct with two copies of the GREa element, the GREd element or two copies of both the GREa and GREd elements did not show any induction (1.1 ± 0.02, 1.0 ± 0.02-fold and 1.0 ± 0.05, respectively; data not show). A 86 ± 1.1-fold induction was observed for the positive control, the distal GRE located in the long terminal repeat of the mouse mammary tumor virus (MMTV-LTR).
In conclusion, the regulation of the t-PA2.4 enhancer is mediated by a
complex hormone responsive unit composed of several GREs, GREa (located
at bp 7,501), GREd (bp
7,703), GREg, and GREf (located at bp
7,942 and
7,960, respectively).
Binding of the GR to the t-PA/GREs was evaluated
in vitro by using recombinant proteins consisting of the DNA
binding domain of the GR or the AR linked to protein A (GRF and ARF,
respectively) since the GR and AR were equipotent
trans-activators of the t-PA2.4 enhancer (see above). The
similarity of the GREs to the consensus sequence is indicated in Table
I. In EMSA, GRF specifically bound to a radiolabeled oligonucleotide
harboring t-PA/GREa (compare lanes j-l with lane
i in Fig. 3A), t-PA/GREf (compare
lanes n-p with lane m) and t-PA/GREg (compare
lanes r-t with lane q). An oligonucleotide
combining both t-PA/GREf and t-PA/GREg with a spacing of 3 bp (as in
their natural environment), showed strong binding (compare lanes
v-x with lane u) with the appearance of a slower
migrating band probably representing tetrameric binding of GRF (see
lane x). The consensus GRE located in the MMTV-LTR was used
as a positive control (MMTV/GREa, lanes a-d). No binding was observed for a radiolabeled aspecific oligonucleotide (RAR/DR5, lanes e-h) and with the mutated GREa, GREd, and GREfg
elements (data not shown). Whereas the interaction of GRF with GREa,
GREf, and GREg was of the dimeric type, the interaction with GREd
in vitro was primarily monomeric; compared to the retarded
complex formed with the labeled MMTV/GREa oligonucleotide only a minor retarded complex migrated with similar mobility, whereas the major retarded complex of the labeled t-PA/GREd oligonucleotide migrated at a
lower apparent molecular weight (data not shown). The GREh and GREi
half-sites were not analyzed by EMSA.
In vitro binding of glucocorticoid and
androgen receptors to the t-PA/GRE elements. A, EMSA was
performed using 32P end-labeled oligonucleotides
representing MMTV/GREa (lanes a-d), RAR/DR5 (lanes
e-h) and the t-PA/GRE elements identified at
7,501 (lanes
i-l),
7,960 (lanes m-p), and
7,942 (lanes
q-t) upstream from the human t-PA gene (respectively, t-PA/GREa,
t-PA/GREf, and t-PA/GREg) or the combined t-PA/GREfg element
(lanes u-x). Oligonucleotides were incubated in the absence
or the presence of increasing amounts of a fusion protein of protein A
and the DNA binding domain of the GRF (50 ng/µl) as indicated.
Differences in relative intensity and migration distance of the
retarded complex between the oligonucleotides are due to different
exposure times and because data were obtained from separate experiments. B, a competition
EMSA was performed using a 32P end-labeled oligonucleotide
representing MMTV/GREa and different amounts (10-250 ng) of competing
unlabeled oligonucleotides representing t-PA/GREa, t-PA/GREd,
t-PA/GREf, t-PA/GREa, and t-PA/GREfg were added. The respective mutant
oligonucleotides were evaluated at a dose of 250 ng: GREaMUT, GREdMUT,
and GREfgMUT. An unlabeled oligonucleotide representing MMTV/GREa and
an aspecific oligonucleotide (NF1) were used as a positive and negative
control, respectively. Experiments were performed as described above.
C, a 32P end-labeled DNA probe covering genomic
sequences from bp
7,535 to
7,850 upstream from the t-PA gene was
incubated in the absence or the presence of a fusion protein of protein
A and the DNA binding domain of the ARF (50 ng/µl, lanes a
and b, respectively). Lane c represents the A/G
reaction of the Maxam and Gilbert sequencing method of the same
fragment. Reaction mixtures were subjected to denaturing polyacrylamide
gel electrophoresis and analyzed by autoradiography. The sequence of
the protected region is shown.
Unlabeled oligonucleotides harboring wild-type t-PA/GREa, GREd, GREf, GREg and GREfg competed for binding of GRF to a labeled MMTV/GREa oligonucleotide (see Fig. 3B). Such competition was also observed for an unlabeled oligonucleotide harboring the MMTV/GREa (used as a positive control) but not for the respective mutant GRE oligonucleotides (GREaMUT, GREdMUT, and GREfgMUT) and not for an aspecific oligonucleotide (NF1).
Interaction of the ARF with the fragment from bp 7,145 to
7,896 was
evaluated in the DNase I protection analysis. Only one region showed an
altered DNase I sensitivity (both protection and appearance of
hypersensitive sites), which mapped to the GREd element (compare
lane a to b in Fig. 3C). This element
is composed of a 3
half-site similar to the consensus and a more
degenerated 5
half-site. It is not clear from this experiment whether
the interaction involves monomeric rather than dimeric ARF binding. The
GREa element, which formed a complex with GRF in vitro in EMSA (see above), was not protected (data not shown). The GREf and GREg
were not evaluated by DNase I protection analysis.
In conclusion, the data suggest that all four GREs, which are involved in the regulation of the t-PA2.4 enhancer by dexamethasone (GREa, GREd, GREg, and GREf), have specific affinity for the GR or the AR in vitro.
Identification of an Enhancer (t-PA2.4) Responsive to Dexamethasone and RA, 7 kb Upstream from the Human t-PA GeneAddition of
dexamethasone (107 M) or RA
(10
6 M) to cells stably expressing
t-PA9578-CAT led to a 3.7 ± 0.11-fold or 6.2 ± 0.21-fold induction, respectively, whereas the hormone combination had a 21 ± 0.23-fold effect (Fig. 4, panels A and
B). The t-PA7144-CAT construct showed no
response to dexamethasone nor RA. The t-PA2.4-TK-CAT construct was
5.1 ± 0.14-fold and 5.5 ± 0.50-fold induced by
dexamethasone and RA alone, respectively, whereas the combination had a
18 ± 1.5-fold effect. Similar effects were observed for the
t-PA2.4INV-TK-CAT construct which contained the enhancer in the reverse
orientation. The TK promoter alone (construct TK-CAT) showed no or only
a minor induction.
Both dexamethasone and RA induced t-PA2.4-TK-CAT expression in a
dose-dependent fashion (see Fig. 4, panels C and
D, respectively). Values ranged from 11 ± 0.75-fold
(104 M dexamethasone) to 1.8 ± 0.08-fold (10
8 M dexamethasone) and from
8.9 ± 0.29-fold (10
5 M RA) to 1.9 ± 0.05-fold (10
9 M RA). RA showed a maximal
level of induction at 10
6 M which was not
further increased at higher concentrations. However, for dexamethasone
a 2-fold higher induction was seen at 10
4 M
compared to the level observed for 10
6 to
10
7 M probably representing aspecific effects
at this high concentration.
Synergistic response of t-PA2.4-TK-CAT to RA and dexamethasone was
confirmed in the following way; low doses were identified from the
dexamethasone and RA dose-response curves for stably integrated
t-PA2.4-CAT that were equipotent for transcriptional induction
(1.9 ± 0.08- and 2.0 ± 0.11-fold with 108
M dexamethasone and 10
9 M RA,
respectively). When a combined treatment with half of these doses was
used, a significantly higher 3.7 ± 0.1-fold induction was
observed (see Fig. 4, panel E).
Transient expression of these reporter constructs in HT1080 cells
revealed a similar effect of dexamethasone on t-PA9578-CAT and
t-PA2.4-TK-CAT as compared to the results obtained by stable integration of the same constructs (respectively 3.1 ± 0.12-fold and 8.8 ± 0.65-fold as compared to 3.7 ± 0.11-fold and
5.1 ± 0.14-fold). However, a substantially lower induction of
t-PA9578-CAT and t-PA2.4-TK-CAT reporter activity by RA is seen in
transient expression experiments (respectively 1.8 ± 0.25-fold
and 2.2 ± 0.15-fold as compared to 6.2 ± 0.21-fold and
5.5 ± 0.50-fold). In HT1080 cells the synergistic effect of
dexamethasone and RA on both t-PA9578-CAT and t-PA2.4-TK-CAT was only
observed for stably integrated constructs. An increased response to RA
of the t-PA2.4-TK-CAT but not of the t-PA9578-CAT construct is obtained
by co-expression of the RA nuclear receptors RAR and RXR
as shown
previously (16). However, even under these conditions the synergistic
activation of t-PA2.4-TK-CAT and t-PA9578-CAT by dexamethasone and RA
could not be reproduced in HT1080 cells (data not shown).
To investigate whether either dexamethasone had an influence on the RA
signal transduction pathway or vice versa, control reporter constructs
containing two copies of a consensus glucocorticoid responsive element
(distal glucocorticoid responsive element of the MMTV-LTR) (27) or two
copies of a RA consensus responsive element (DR5 element identified in
the murine RAR2 promoter) (28) linked to the TK promoter (MMTV/GREa-
and RAR
/DR5-TK-CAT, respectively) were evaluated in transient
expression (data not shown). RA alone had no effect on the induction of
MMTV/GREa-TK-CAT promoter activity (1.0 ± 0.07-fold compared to
the basal level) and did not alter the induction by dexamethasone
(37 ± 5.2-fold in the presence of both dexamethasone and RA
compared to 35 ± 3.3-fold in the presence of dexamethasone
alone). Similarly, dexamethasone alone did not have any effect on
RAR
/DR5-TK-CAT promoter activity (1.1 ± 0.04-fold compared to
the basal level) and did not alter the induction by RA (42 ± 6.1-fold in the presence of both dexamethasone and RA compared to
45 ± 4.0-fold in the presence of RA alone).
In summary, stable expression of various t-PA reporter constructs
identified the region from 7.1 to
9.6 kb (t-PA2.4) as a
dexamethasone- and RA-inducible enhancer since it functioned irrespective of orientation and distance from the promoter and activated a heterologous promoter. Control experiments did not reveal
an effect of dexamethasone or RA on the trans-activation potential of RA receptors or the GR, respectively.
The
t-PA2.4 upstream fragment was cloned at various distances (632, 1564, and 3070 bp) from the transcription start site of the human t-PA gene
using its homologous upstream genomic sequence as "spacer" (16).
Transient expression of the enhancer-less constructs t-PA632-CAT,
t-PA1564-CAT, t-PA3070-CAT, and t-PA7144-CAT in HT1080 cells
revealed no effect of dexamethasone (107 M,
36 h; values ranged from 0.81 ± 0.14- to 0.98 ± 0.05-fold control, data not shown) but the presence of the t-PA2.4
enhancer 5
from these promoter fragments rendered them inducible by
dexamethasone (from 3.3 ± 0.16- to 4.8 ± 0.15-fold, data
not shown). Similar data have been reported previously for the response
of these constructs to RA revealing no or a minor increase in the
response with decreasing distance between the enhancer and the promoter
(16).
Stable expression in HT1080 cells of promoter deletion constructs
containing the t-PA2.4 fragment showed that the induction by
dexamethasone (108 M) and RA
(10
7 M) alone is only influenced to a minor
extent by the distance or the intervening sequence; whereas the
synergistic effect of dexamethasone (5 × 10
9
M) together with RA (5 × 10
8
M) increased with decreasing distance between the enhancer
and the t-PA proximal promoter elements (from 3.3 ± 0.06-fold for t-PA9578-CAT to 7.3 ± 0.09-fold for t-PA632
2.4-CAT, see Fig. 5).
In aggregate, these data indicate that the t-PA2.4 enhancer did not require the presence of the intervening sequence to confer dexamethasone and RA responsiveness to its natural promoter. The response to dexamethasone and RA alone was not or weakly affected by decreasing the distance between the enhancer and the proximal promoter elements. However, the synergistic effect of both hormones together increased considerably with decreasing distance.
Synergistic Regulation of the t-PA2.4 Enhancer by D-Aldosterone and RAIn order to determine whether
the synergy would also occur with other members of the steroid hormone
family, the mineralocorticoid receptor was transiently expressed in
HT1080 cells which contained the t-PA2.4-TK-CAT construct stably
integrated in the genome. Whereas treatment with
D-aldosterone and RA (both 107 M)
induced CAT-activity 3.0 ± 0.18- and 4.9 ± 0.11-fold,
respectively, combined treatment with D-aldosterone and RA
at half these concentration (both 5 × 10
8
M) led to a 8.4 ± 0.3-fold induction suggesting a
synergistic effect.
In order to delineate the
minimal t-PA enhancer, progressive 5 deletions of the t-PA2.4 fragment
linked to TK-CAT were evaluated by stable expression in HT1080 cells
(see Fig. 6). Stimulation with equipotent concentrations
of dexamethasone and RA (10
7 M and
10
8 M RA, respectively) led to a 4.5 ± 0.35-fold and a 4.8 ± 0.2-fold induction of the t-PA2.4-TK-CAT
construct, whereas the combination of 5 × 10
8
M dexamethasone and 5 × 10
9
M RA had a 12 ± 2-fold effect. The TK promoter alone
showed no response. Deletion of the region spanning bp
9,213 to
9,578 (t-PA2.0-TK-CAT), bp
8,559 to
9,578 (t-PA1.4-TK-CAT) or bp
8,008 to
9,578 (t-PA0.9-TK-CAT) did only minimally influence the
activation by dexamethasone and RA and did not eliminate the
cooperative effect of dexamethasone and RA (see Fig. 6). Deletion of
the region from bp
7,897 to
9,578 (t-PA0.7-TK-CAT) reduced the
response to dexamethasone or RA as well as the cooperative effect of
both agents as compared to the t-PA0.9-TK-CAT construct (from 3.0 ± 0.18- to 2.0 ± 0.69-fold for dexamethasone, from 3.6 ± 0.08- to 2.6 ± 0.58-fold for RA and from 10 ± 2.2- to
5.0 ± 1.0-fold for the combined treatment). Deletion of the
regions spanning bp
7,535 to
9,578 or bp
7,324 to
9,578 in
(t-PA0.4-TK-CAT and t-PA0.2-TK-CAT, respectively) eliminated or
strongly reduced the induction by dexamethasone (0.86 ± 0.1- and
1.4 ± 0.04-fold, respectively); likewise, the induction by RA was
strongly reduced (1.9 ± 0.38- and 2.1 ± 0.56-fold,
respectively) and was not further increased by the combination of
dexamethasone and RA (2.0 ± 0.2- and 2.4 ± 0.23-fold).
Similar data were obtained by transient co-expression of the t-PA
reporter constructs with the GR, RAR, and RXR
nuclear receptors
in the SK-N-SH neuroblastoma cell line (data not shown). In conclusion,
these results show that the enhancer can be reduced to a minimal size
of 863 bp spanning the region from bp
7,145 to
8,008.
The present data show that the region from bp 7,145 to
9,578
upstream from the human t-PA gene (t-PA2.4) acts as an enhancer which
mediates the effect of glucocorticoids, aldosterone, mineralocorticoids and androgens but not estrogens. Complete elimination of
dexamethasone-mediated induction of the t-PA2.4 enhancer activity
required the site-specific mutation of GREa (bp
7,501) and GREd (bp
7,703) in combination with deletion of the GREg (bp
7,942) and GREf
(bp
7,960) elements. Therefore, the human t-PA gene is a direct
target for glucocorticoid action through a unusually complex
glucocorticoid responsive unit (GRU) composed of multiple binding sites
for the GR, which is located between bp
7,501 and
7,974. All four
sites contained the crucial nucleotides of the 3
half site of the
consensus GRE motif shown to direct initial binding of one GR of the
dimeric complex (29, 30), and they interacted specifically with a truncated recombinant form of the GR or AR in vitro. In
contrast to the other t-PA/GREs, in EMSA the interaction of GR with
t-PA/GREd was primarily of the monomeric type but dimeric binding might occur in vivo due to the binding of GR to the GREa and GREfg
elements and of potential co-activator factors in the GRU. The
observation that t-PA/GREa interacted with GR or AR in the
electrophoretic mobility shift assay but not in the DNase I protection
analysis might be due to the different amounts of purified receptor
used in these in vitro assays. Deletion of a fragment
containing only GREa but not GREd, GREf, and GREg did also lead to a
reduced level of induction. This result is not necessarily conflicting
the site-specific mutagenesis data since deletion of a DNA fragment
rather than single mutations might eliminate binding sites for
transcriptional co-activators which are required for the dexamethasone
response of the enhancer.
The GREf and GREg elements activate transcription in response to dexamethasone in a cooperative manner when linked in two copies to the heterologous TK promoter. In contrast, a similar construct with the GREa, GREd, or the combination of GREa and GREd was not responsive to dexamethasone. The reason why GREa and GREd are active within the t-PA enhancer might be the regular spacing of about 200 bp (202 and 239 bp, respectively) observed between the GREa, GREd, and the combination of GREf and GREg. Nucleosome phasing (32) might therefore bring the GREs close enough to each other to allow biological activity for the GREa and GREd by providing multiple contacts with the RNA polymerase II initiation complex. Similar protein-protein interactions mediated by the progesterone receptor bound to distal hormone responsive elements have been reported for the uteroglobin gene (33). Interaction between distal regulatory loci and the basic transcription complex requires looping which depends on protein/protein interactions between transcription factors bound, respectively, to the enhancer and to the proximal promoter (34). The fact that the intervening sequence between the t-PA enhancer and promoter can be deleted without reducing their response to dexamethasone and RA is suggestive for such a mechanism. The Sp1 transcription factor has been shown to mediate looping of DNA (35, 36). Putative Sp1 binding sites are present in the t-PA2.4 enhancer as well as in the promoter. Whether interactions between distally and proximally bound Sp1 are involved in the regulation of the t-PA gene expression by hormones is under investigation.
A steroid hormone responsive unit of similar complexity, as described here, has only been identified in the long terminal repeat of the mouse mammary tumor virus (27). The presence of multiple steroid receptor binding sites increases the amplitude and reduces the dose dependence of the response through cooperative binding (37, 38) and a reduced dissociation rate of the DNA-receptor complex (38).
Treatment of HT1080 fibrosarcoma cells with both dexamethasone and RA led to a cooperative induction of t-PA-related antigen secretion by increasing the level of mRNA steady state levels through a mechanism which requires protein synthesis.2 This effect is mediated by the t-PA2.4 enhancer as shown by stable expression experiments performed with t-PA promoter constructs. The response of the enhancer to dexamethasone and RA alone increased to some extent when the distance between the enhancer and the proximal promoter elements was decreased, whereas the synergistic effect of both hormones together increased more strongly. Combination of half of multiple equipotent doses of dexamethasone and RA induced significantly higher t-PA2.4 activity than was observed for the corresponding doses alone. According to the definition of Berenbaum (39) and as more recently described by Collen (40), this is indicative for a synergistic rather than an additive interaction between dexamethasone and RA.
The effect of RA in HT1080 cells was found to be more pronounced in stably than in transiently transfected HT1080 cells (16). Moreover, the synergistic interaction on the t-PA2.4 enhancer between dexamethasone and RA in HT1080 cells was only observed for stably integrated CAT-reporter constructs. These phenomena could be due to the more physiological environment of the stably integrated reporter constructs (41). Alternatively, the lower copy number of the reporter construct in stably transfected cell lines may prevent depletion of trans-activating factors. Indeed, stronger induction by RA and/or the synergistic activation by dexamethasone and RA of transiently expressed t-PA-TK-CAT constructs were restored by co-expression of the RA receptors and/or the GR.
RA is able to enhance the translocation of the GR to the nucleus (42), whereas dexamethasone increases the expression of RXR genes in certain cell lines in vitro (43). Neither of these phenomena seem to play a role in the synergistic induction of t-PA expression by dexamethasone and RA; RA was not able to enhance the induction of a consensus glucocorticoid responsive element by dexamethasone when linked to TK-CAT and, similarly, dexamethasone did not influence the RA-mediated induction of a consensus RARE. Therefore, it is concluded that the synergistic response is due to an interaction between the steroid- and RA-signal transduction pathways at the level of the enhancer.
The size of the enhancer could be restricted to the region spanning bp
7,145 to
8,007 (863 bp, t-PA0.9) without loss of response to both
dexamethasone and RA. This enhancer contains both the complex GRU and
the previously identified RARE located at bp
7,319 and which are
responsible for the effect of dexamethasone and RA, respectively (see
above) (16). Binding of the GR homodimer as well as the RAR/RXR
heterodimer is the initial event in the formation of a functional
trans-activation complex in vivo (44, 45).
Binding of the GR homodimer has been shown to disrupt the nucleosome
structure of the MMTV-LTR (46). Therefore, it is conceivable that
the binding of the GR to the t-PA/GREs might facilitate binding of the
RAR/RXR receptors to the t-PA/DR5 element and/or vice versa.
Alternatively, simultaneous binding of GR and RAR/RXR receptors might
facilitate the binding of co-regulators involved in the hormonal
response of the enhancer by opening the chromatin structure more
efficiently than either the GR or the RAR/RXR would do
separately. More experiments are required to confirm such a hypothesis,
which would explain the synergistic interaction between both pathways
on the t-PA0.9 enhancer.
The present data indicate that in the appropriate cellular context the glucocorticoid and the RA signal transduction pathways can up-regulate t-PA gene expression in a cooperative manner resulting in either an enhanced response or a higher sensitivity of the t-PA gene toward these hormones in vivo. The amplitude of the synergistic response increased considerably with decreasing distance between the enhancer and the promoter. Therefore, it is suggested that the remote localization of the enhancer permits the dexamethasone and RA pathways to interact synergistically on the level of t-PA expression without exceeding physiological limits of trans-activation. An increased effect was also observed with the aldosterone and the RA receptors suggesting that the synergism could also occur with other steroid receptors (except the estrogen receptor).
Evaluation of 3 kb of upstream human t-PA gene sequence led to an expression pattern in transgenic mice of a linked reporter gene which did not fully resemble the pattern of endogenous t-PA-related antigen and mRNA expression (47). The presence of a far upstream enhancer in the human t-PA gene which is inducible by steroid hormones (except estrogen) and by RA is therefore proposed to be important for the expression of the t-PA gene in vivo.
We thank Dr. P. Chambon (Institut de Génétique et de Biologie Moléculaire et Cellulaire, Illkirch, France) for the hER and hPR expression plasmids, Dr. R. Evans (The Salk Institute for Biological Studies, La Jolla, CA) for the hGR and hMR expression plasmids, Dr. A. O. Brinkmann (Erasmus University, Rotterdam, The Netherlands) for the hAR expression vector and Dr. D. R. Helinksi (Department of Biology, University of California, San Diego, CA) for the RSVluc expression vector. We thank Brigitte Verheyden from the Center for Molecular and Vascular Biology for secretarial assistance.