(Received for publication, November 11, 1994)
From the
All-trans-retinoic acid (RA) and retinoids induce
synthesis of tissue-type plasminogen activator (t-PA) in endothelial
and neuroblastoma cells in vitro and in rats in vivo.
In HT1080 fibrosarcoma cells, induction of t-PA-related antigen
secretion and t-PA mRNA steady state levels by RA were found to depend
on de novo protein and mRNA synthesis. Fragments derived from
the 5`-flanking region of the t-PA gene (+197 to -9578 base
pairs (bp)) were linked to the chloramphenicol acetyltransferase gene.
Transfection studies demonstrated that the region spanning bp
-7145 to -9578 mediated induction by RA. A functional
retinoic acid response element (RARE), consisting of a direct repeat of
the GGGTCA motif spaced by 5 nucleotides (t-PA/DR5), was localized at
-7.3 kilobases. The t-PA/DR5 element interacted with the
heterodimer composed of retinoic acid receptor and retinoid X
receptor
in vitro, whereas its mutation abolished
induction by RA in transient expression. In human EA.hy926 hybrid
endothelial and in SK-N-SH neuroblastoma cells, the activity of
t-PA/DR5 was found to be independent of the intervening sequence
(-632 to -7144 bp) and of its distance from the
transcription initiation site. Staurosporine, an inhibitor of protein
kinase activity, inhibited induction by RA, suggesting that it required
protein phosphorylation.
The fibrinolytic system has been suggested to play a role in
several biological processes such as blood clot dissolution, smooth
muscle cell migration, angiogenesis, ovulation, embryogenesis, and
brain function and possibly in a number of other (patho)physiological
processes(1, 2, 3) . The system is composed
of the physiological plasminogen activators (PA)(),
tissue-type PA (t-PA) and urokinase-type PA (u-PA), which activate the
zymogen plasminogen to plasmin by cleavage of the
Arg
-Val
peptide bond. Inhibition occurs on
two levels: plasminogen activator inhibitor-1 (PAI-1) counteracts both
t-PA and u-PA while
-antiplasmin rapidly inactivates
plasmin(1) .
Endothelial cells constitute the major source of circulating t-PA(1) . Vitamin A, retinoic acid (RA), and some (synthetic) retinoids induce t-PA-related antigen secretion in association with increased t-PA mRNA levels in human umbilical vein endothelial cells (HUVEC) in vitro and in rats in vivo(4, 5, 6) . A potential role of t-PA in brain function is suggested by its early induction during seizure, kindling, and long-term potentiation(7) , and its association with neurite outgrowth and neuronal migration(8) . Furthermore, mice deficient for t-PA expression show impaired learning capabilities(9) . In vivo, RA is indispensable for the development of the central nervous system during embryogenesis(10) , while in vitro, RA induces differentiation of neuroblastoma cells resulting in axon formation and neurite growth which is often associated with increased levels of t-PA expression(11, 12, 13) .
RA response
elements (RARE) in the proximal promoter region of some genes have been
found to mediate RA-induced transcription (cf. for reviews, (14, 15, 16) ), but such cis-acting
elements have not been identified in the t-PA gene. RA and its
stereoisomer 9cis-RA induce gene transcription by activation of nuclear
receptors: 9cis-RA binds selectively to both retinoic acid nuclear
receptor (RAR) and retinoid X nuclear receptor (RXR), while RA binds to
RAR only. RXR homo- and RARRXR heterodimers bind variably to RARE
consisting of repeats of the (A/G)G(G/T)T(G/C)A motif, depending on the
spacing between these repeats (14, 15, 16) and their sequence
context(17) . Several subtypes of RAR and RXR, encoded by
distinct genes (
,
, and
), some of which show
differential splicing, have been identified. The tissue-specific
expression of these subtypes and their isoforms may explain the
pleiotropic effects of RA on gene regulation and embryonic
development(14, 15, 16) .
In the present study, a functional RARE is characterized which consists of a direct repeat of the GGGTCA motif spaced by 5 nucleotides (t-PA/DR5) and which is localized 7.3 kb upstream from the transcription start site of the human t-PA gene. This element mediates the direct regulation by RA in human fibrosarcoma, endothelial, and neuroblastoma cells.
Figure 2:
RA-mediated transactivation of t-PA-CAT
constructs following stable or transient expression in HT1080 human
fibrosarcoma cells. The data represent mean ± S.E. A,
schematic representation of the genomic sequences upstream from the
transcription initiation site of the human t-PA gene and the
t-PA-CAT-reporter constructs obtained thereof. Numbering is according
to Henderson and Sleigh(23) . CRE/AP1 and AP2 binding sites
involved in basal promoter activity (42) are indicated by an arrow, and BamHI restriction sites are indicated by a filled triangle. The CAT reporter gene (), the TK
promoter (
), and the first exon (&cjs2106;) of the human t-PA
gene are indicated. B, induction of CAT activity by RA
(10
M, hatched bars) versus control (open bars) by stably expressed human t-PA
promoter constructs (a, p-CAT; b, t-PA9578-CAT; c, t-PA7144-CAT; d, pTK-CAT; e,
t-PA2.4-TK-CAT; f, t-PA2.4INV-TK-CAT). Cells were harvested 24
h after RA treatment. C, induction of CAT activity by RA
(10
M, hatched bars) versus control (open bars) by transiently expressed t-PA-CAT
constructs (a to e). D, effect of transient
co-expression of plasmids encoding different RA nuclear receptors with
the t-PA2.4-TK-CAT construct. Stimulation was performed for 36 h with
RA (10
M) and/or 9cis-RA (10
M) and compared with control (Co) as
indicated.
Figure 4:
Induction of t-PA promoter activity by RA
in EA.hy926 hybrid endothelial and SK-N-SH neuroblastoma cells. A and B, induction of CAT activity by RA (10M, hatched bars) versus control (open bars) in EA.hy926 hybrid endothelial cells (A)
and SK-N-SH neuroblastoma cells (B), transiently expressing
the t-PA9578-CAT (a), t-PA7144-CAT (b), wild-type
t-PA2.0-TK-CAT (c), and mutant t-PA2.0/DR5MUT-TK-CAT construct (d). C and D, RA response of t-PA-CAT
constructs with increasing deletions of intervening sequence between
the enhancer and the promoter in EA.hy926 hybrid endothelial cells (C) and in SK-N-SH neuroblastoma cells (D). t-PA2.4
enhancer was fused (
) or not (
) to the t-PA promoter
length indicated. Experiments were performed as described in A and B. E, schematic representation of reporter
constructs containing the t-PA2.4 fragment linked to deletion mutants
of the t-PA7144-CAT construct. The CAT reporter gene (
) and the
first exon (&cjs2106;) of the human t-PA gene are
indicated.
Constructs obtained by progressive exonuclease III deletion
mutagenesis from the t-PA2.4-TK-CAT construct (t-PA2.0-TK-CAT,
t-PA1.6-TK-CAT, t-PA1.4-TK-CAT, t-PA0.4-TK-CAT, and t-PA0.2-TK-CAT) and
from t-PA2.4INV-TK-CAT (t-PA1.9TK-CAT) are shown in the left panel of Fig. 3B. Internal deletions were created by
recombining deletion fragments from t-PA2.4-TK-CAT and
t-PA2.4INV-TK-CAT, yielding t-PA2.40.5-TK-CAT and
t-PA2.4
0.1-TK-CAT with an internal deletion from -8042 bp to
-8574 bp and from -7535 to -7650 bp, respectively.
The presumed DR5 RARE identified in the upstream 2.4-kb t-PA gene
fragment was mutated (t-PA/DR5MUT; cf. Table 1) in the
t-PA2.0-TK-CAT construct by site-specific mutagenesis using polymerase
chain reaction(25) , yielding t-PA2.0/DR5MUT-TK-CAT.
Figure 3:
Localization of the RA response element in
the human t-PA promoter by mutagenesis of the t-PA2.4 genomic fragment.
The data represent mean ± S.E. of cells treated with RA
(10M, hatched bars) versus control (open bars). A, schematic representation
of the genomic sequence from -7145 to -9578 bp upstream
from the human t-PA gene. Motifs resembling repeats of the
(A/G)G(G/T)T(G/C)A half-site are represented: direct (DR) and
everted repeats (ER) are indicated with the number of the
intervening nucleotides. B, induction of CAT activity by RA of
the indicated mutant constructs derived from t-PA2.4-TK-CAT. HT1080
cells, transiently co-expressing reporter constructs with h-RAR
and h-RXR
, were treated with RA for 36 h. The mutations in the DR5
element of t-PA2.0/DR5MUT-TK-CAT are marked with vertical bars (for the corresponding sequence, cf. Table 1). C, induction of CAT activity by RA of different DNA
oligonucleotide constructs fused to TK-CAT and transiently co-expressed
with RAR
and RXR
in HT1080 cells. Oligonucleotide sequences
are shown in Table 1. Experiments were performed as outlined in
the legend of Fig. 3B.
DNA oligonucleotides representing recognition motifs for distinct nuclear receptors are shown in Table 1, some of which were cloned as two copies in front of the TK promoter linked to the CAT gene (cf. Fig. 3C, left panel).
To obtain transient expression of the t-PA promoter constructs in HT1080 and SK-N-SH cells, the calcium phosphate co-precipitation method (26) was applied to a 6-well dish using a DNA mixture of 12 to 46 µg of CAT reporter plasmid (according to the size of the reporter construct) with 0.1 µg of pRSVL plasmid and 2.4 µg of the indicated nuclear receptor expression plasmid. HT1080 and SK-N-SH cells were stimulated with RA immediately and 16 h after the glycerol shock, respectively.
EA.hy926 cells were transiently transfected by electroporation
according to a (modified) procedure described by Schwachtgen et
al.(28) . 10 synchronized cells suspended in
cytomix (29) were added to approximately 50 µg of PvuI-linearized reporter plasmid together with XmnI-linearized h-RAR
and with NdeI-linearized
h-RXR
expression vectors (ratio 20:1) followed by electroporation
at a voltage of 274 V and a capacitance of 1,800 microfarads (time
constant
= 26 ± 0.3 ms, mean ± S.E., n = 10). Cells were immediately added to 12 ml of
supplemented DMEM containing 5% charcoal-stripped serum and divided in
six 10-cm
wells. After overnight incubation, cells were
washed with phosphate-buffered saline, and growth medium containing RA
or excipient was added, followed by a 30-h incubation.
All cell
extracts were prepared by three freeze-thaw cycles (100 mM TrisHCl, pH 7.8, 5 mM EDTA) or by using reporter
lysis buffer. CAT activity was quantified using the liquid
scintillation method(30) : a mixture of
[
H]acetyl-CoA (0.1 µCi), acetyl-CoA (final
concentration 0.1 mM), and chloramphenicol (final
concentration 0.9 mM) was added to equal amounts of cell
extracts, overlayered with scintillation solution (either Econofluor-2
or Lipoluma), and the rate of
H-labeled
acetylchloramphenicol generation was measured using a liquid
scintillation analyzer. Luciferase activity, used as an indicator for
the transfection efficiency, was measured in a luminometer after
addition of luciferin substrate. Data obtained from stable and
transient expression experiments were corrected for endogenous CAT
activity and apparent luciferase activity.
Figure 1:
Regulation of human t-PA gene
expression by RA in HT1080 fibrosarcoma cells. The data represent mean
± S.E. A, dose-response effect of RA on t-PA-related
antigen (AG) secreted in the conditioned medium within 48 h. B, time course of RA (10M, hatched bars) induced secretion of t-PA-related antigen (AG)
relative to controls (open bars); cyclo,
10
M cycloheximide added. Samples of the
conditioned medium were taken at the indicated time points. C,
Northern blot analysis of t-PA, u-PA, and PAI-1 mRNA in the absence (open bars) or the presence (hatched bars) of
10
M RA. Total RNA was extracted 12 h after
addition of RA. mRNA levels were expressed relative to the 18 S rRNA
level and to control.
Northern blot
analysis of total RNA extracted from HT1080 cells showed that RA caused
a significant increase of the t-PA steady state mRNA level (8.0
± 2.0-fold, cf. Fig. 1C) which was
totally inhibited by simultaneous treatment with cycloheximide
(10M, data not shown). The t-PA mRNA
stability was not affected by RA treatment (data not shown). The 3.4-
and the 2.8-kb PAI-1 mRNAs and the u-PA mRNA were only marginally
affected by RA (1.3 ± 0.03-fold, 1.2 ± 0.03-fold, and 1.5
± 0.02-fold the control value, respectively; cf. Fig. 1C).
In aggregate, these results are indicative of a RA response sequence localized in the 2.4-kb genomic fragment spanning the region from -7145 to -9578 bp upstream from the human t-PA gene.
In conclusion, the t-PA2.4 genomic fragment contains two elements, t-PA/DR2 and t-PA/DR5, that confer RA inducibility to the TK promoter, but only t-PA/DR5 appears to be functionally active in the t-PA gene.
Evaluation of t-PA
promoter constructs in the EA.hy926 hybrid endothelial cell line
required co-transfection of h-RAR and h-RXR
expression
plasmids and linearization of the plasmids to obtain maximal induction.
As illustrated in Fig. 4A, transient expression of the
t-PA9578-CAT construct (a) resulted in a 2.8 ± 0.1-fold
induction of CAT activity by RA while the t-PA7144-CAT construct (b) was not induced. The t-PA2.0TK-CAT construct (c)
showed a 6.0 ± 0.5-fold induction by RA, while no induction was
observed for the t-PA2.0/DR5MUT-TK-CAT construct (d) in which
the t-PA/DR5 RARE is eliminated by site-specific mutagenesis. Similar
results were obtained in the SK-N-SH neuroblastoma cell line (Fig. 4B). Co-transfection of RAR
RXR expression
plasmids was not required for full induction of the RA response,
possibly as a result of higher levels of endogenous RA nuclear
receptors in this cell line. Thus, the t-PA/DR5 motif appears to be
involved in the regulation of t-PA gene expression by RA in both
endothelial and neuroblastoma cells.
In conclusion, RA-mediated activation of the t-PA promoter through the t-PA/DR5 motif is independent on the distance between both and does not require the intervening sequence.
Figure 5:
Characterization of the t-PA RARE by the
electrophoretic mobility shift assay. A, electrophoretic
mobility shift assay of P-end-labeled fragment t-PA0.2
(-7145 to -7324 bp) and the control DR5/RARE from the mouse
RAR
promoter (RAR
/DR5). The distal glucocorticoid response
element from the MMTV long terminal repeat promoter (MMTV/GREa), the
RAR
/DR5, and the DR5 element identified at -7319 bp upstream
of the human t-PA gene (t-PA/DR5) were added as cold competitor (10-
and 100-fold molar excess) where indicated. The band marked with an open triangle represents an aspecific band; the free probe and
the specific retarded complexes are indicated by a filled triangle and by a filled triangle marked with a filled circle,
respectively. B, electrophoretic mobility shift assay of
P-end-labeled oligonucleotides representing the
RAR
/DR5, t-PA/DR5, and the t-PA/DR2 element identified -8396
bp upstream from the human t-PA gene (t-PA/DR2). Reactions were
performed with epitope-tagged human RAR
(Flu-RAR
) and
RXR
(Myc-RXR
) in the presence or the absence of an aspecific
antibody (15C5) or specific antibodies (anti-Flu and anti-Myc). The
difference in migration and relative intensity of the bands obtained
for t-PA/DR2 were due to slightly different conditions applied during
the electrophoresis of these reaction
mixtures.
In Fig. 5B, a radiolabeled t-PA/DR5
oligonucleotide did not interact with Flu-RAR alone, weakly with
Myc-RXR
alone (lanes i and j, respectively) and
strongly with the mixture of both (lane k). Addition of
specific monoclonal antibodies directed against the Flu epitope of
RAR
(anti-Flu) or the Myc epitope of RXR
(anti-Myc) caused a
partial disruption of the retarded complex (lane l) and a
clear supershift (lane m), respectively, suggesting that both
RAR
and RXR
were present in the retarded complex. Similar
results were obtained with the radiolabeled t-PA/DR2 (cf. Table 1) and RAR
/DR5 oligonucleotides, except that no
interaction was observed with RXR
alone (respectively, lanes o to u and lanes a to g). A radiolabeled
t-PA/ER8 oligonucleotide showed virtually no binding (data not shown),
and experiments performed with a modified DR5 oligonucleotide, in which
the first repeat of the DR5 motif was mutated but the ER2 repeats were
left intact or a modified DR2 oligonucleotide in which only the ER1 was
conserved (cf. Table 1), revealed only weak binding of
RAR
RXR
(data not shown).
In conclusion, two RARE
elements were identified in the t-PA2.4 upstream genomic fragment
(t-PA/DR5 and t-PA/DR2) with affinity for RARRXR heterodimers in vitro, although only the t-PA/DR5 element is functionally
active in the t-PA gene.
In HT1080 and EA.hy926 cells transiently expressing the t-PA2.4-TK-CAT construct, staurosporine (25 nM) reduced the induction of CAT activity by RA from 6.8 ± 0.2-fold to 1.5 ± 0.01-fold and from 6.5 ± 0.2-fold to 1.2 ± 0.1-fold, respectively (cf. Table 2). These data suggest that protein phosphorylation prior to RA-mediated transactivation of t-PA gene expression is required.
In the present study, the direct regulation of human t-PA
gene transcription by RA was investigated in three different cell types
of human origin: fibrosarcoma HT1080 cells, EA.hy926 endothelial, and
SK-N-SH neuroblastoma cells. A functional DR5 RARE binding RARRXR
heterodimers in vitro was identified 7.3 kb upstream from the
human t-PA gene. This element, which probably constitutes the general
physiological target for direct RA-mediated transactivation, has
enhancer-like properties since its activity did not depend on the
orientation and distance from the basic promoter elements of either the
t-PA or a heterologous TK gene. Finally, the induction of t-PA gene
expression required intermediate protein synthesis and phosphorylation.
RA induction was found to be more pronounced in stably than in transiently transfected HT1080 cells. It is unclear whether this is due to the more physiological chromatin environment (37) or the lower copy number of the reporter construct, preventing depletion of trans-activating factors, in the stable transfectants. Indeed, strong RA induction from transiently expressed t-PA-TK-CAT constructs could be restored by co-expression of RA receptors.
The presence of
a RARE at such a long distance from a promoter is unusual. ()The fact that the intervening sequence can be deleted
without affecting the RA response is suggestive of a mechanism
involving DNA looping to bring this enhancer in contact with the
promoter. As demonstrated in other systems(38) , stabilization
of DNA loops requires a number of protein/protein interactions between
transcription factors bound on the enhancer and those bound on the
proximal promoter. Cooperation between nuclear receptors forming
heterodimers with RXR has been shown for Sp1 and factors binding to a
CRE and an NF-
B-like
element(39, 40, 41) . Several Sp1 and AP-2
consensus binding sites are indeed present in the vicinity of the
t-PA/DR5 element as well as a functional CRE-like and AP-2 element
close to the transcription start site of the human t-PA
gene(42) .
In contrast to the t-PA/DR5 element, the t-PA/DR2
element was not indispensable for the RA response of the t-PA2.4
enhancer. However, both elements bind RARRXR heterodimers with
comparable affinity in vitro and two copies of either element
conferred RA responsiveness to a linked TK-CAT construct. This
discrepancy may be due to the presence of several binding sites for
transcription factors (Sp1, NF1) in the TK promoter which have been
shown to cooperate with other nuclear
receptors(43, 44) . Such binding sites might be
lacking in the t-PA/DR2 natural environment.
In addition to the RA response element described here, a perfect DR4 element (t-PA/DR4) was localized 8.7 kb upstream from the human t-PA gene. Repeats of the (A/G)G(G/T)T(G/C)A sequence spaced by 4 nucleotides have been shown to bind a heterodimer consisting of thyroid hormone nuclear receptor with RXR, conferring transactivation by thyroid hormones to the nearby gene(14) . Induction of t-PA-related antigen secretion by thyrotropin in ovine thyroid cells has been reported(45) , but a potential role of the t-PA/DR4 element in T3-mediated gene regulation of t-PA remains to be investigated.
Our finding of a functional DR5 RARE, 7.3 kb upstream from the human t-PA gene, is not necessarily in contradiction with the results obtained by Darrow et al.(46) who previously demonstrated that, in murine F9 teratocarcinoma cells, the induction of t-PA gene transcription during differentiation by cAMP and RA is mediated by two proximal GC-rich boxes. Direct involvement of RA nuclear receptors in this process, which required treatment with RA for 24 h to several days, was not substantiated.
In the present experiments, staurosporine, which is a potent but rather aspecific inhibitor of protein kinases(36) , inhibited RA-induced t-PA-related antigen secretion and t-PA2.4-TK-CAT activity, suggesting a role for protein phosphorylation, as previously demonstrated for RAR (47) .
In summary, RA induction of human t-PA gene expression appears to be mediated by a DR5 RARE located 7.3 kb upstream from the transcription initiation site. This element mediates RA response in different cell types in vitro (fibroblast, endothelial, and neuronal cells) and thus may be involved in the modulation of t-PA expression in the vessel wall and in the brain.
For the DNA dideoxy
sequencing reactions, the AutoRead Sequencing kit of Pharmacia Biotech
(Roosendaal, the Netherlands) was used with either fluorescent-labeled
or unlabeled primers (Pharmacia Biotech) combined with
fluorescent-labeled dATP (Pharmacia Biotech). Samples were analyzed by
electrophoresis on a 6% polyacrylamide-8 M urea gel (0.6
Tris borate buffer) using the Automated Laser Fluorescent DNA
sequencer system from Pharmacia Biotech. Generated sequences were
analyzed using the PC Gene software from Intelligenetics.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z48484[GenBank].