©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Retinoic Acid Induction of Human Tissue-type Plasminogen Activator Gene Expression via a Direct Repeat Element (DR5) Located at 7 Kilobases (*)

(Received for publication, November 11, 1994)

Frank Bulens Ines Ibañez-Tallon (§) Petra Van Acker Astrid De Vriese Luc Nelles (¶) Alexandra Belayew Désiré Collen (**)

From the Center for Molecular and Vascular Biology, University of Leuven, B-3000 Leuven, Belgium

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
APPENDIX
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 alpha and retinoid X receptor alpha 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.


INTRODUCTION

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)(^1), 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 alpha(2)-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 RARbulletRXR 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 (alpha, beta, 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.


MATERIALS AND METHODS

Reagents

Human HT1080 fibrosarcoma and SK-N-SH neuroblastoma cells were obtained from the American Type Culture Collection and EA.hy926 endothelial hybrid cells from Dr. C.-J. Edgell (Dept. of Pathology, University of North Carolina, Chapel Hill). 9cis-RA was a gift from Dr. F. Schneider (Hoffman-La Roche Ltd., Basel, Switzerland), expression vectors encoding h-RARalpha (RARalphaø), h-RARbeta (h-RARbeta), and h-RAR (RAR1.ø) from Dr. P. Chambon (Laboratoire de Génétique Moléculaire des Eukaryotes, Strasbourg, France), the expression vector encoding h-RXRalpha (pRSh-RXRalpha) from Dr. R. M. Evans (The Salk Institute for Biological Studies), the firefly luciferase expression vector (pRSVL) from Dr. D. R. Helinski (Dept. of Biology, University of California, San Diego), epitope-tagged human RARalpha and RXRalpha expression plasmids from Dr. T. Gulick and Dr. D. D. Moore (Massachusetts General Hospital, Boston), and epitope-tagged hRARalpha and hRXRalpha as partially purified Escherichia coli expression products from Dr. M. Baes (Laboratory for Clinical Chemistry, KUL, Leuven, Belgium). Dulbecco's modified Eagle's growth medium (DMEM), DMEM-F12 growth medium, and all medium supplements were purchased from Life Technologies, Inc./BRL (Ghent, Belgium), tissue culture recipients from Corning Inc. and Becton Dickinson, RA, staurosporine, cycloheximide, thymidine, dexamethasone, cholecalciferol, 3,3`,5-triiodo-L-thyronine, and chloramphenicol from Sigma, the human genomic EMBL3 library from Clontech, DNA purification columns used for plasmid preparations from Qiagen (Chatsworth, CA) and Mackerey-Nagel (Düren, Germany), acetyl-CoA and [^3H]acetyl-CoA from ICN Biomedicals, [alpha-P]UTP and [alpha-P]dCTP from Amersham Corp. (Ghent, Belgium), SP6 RNA polymerase, pSP64 plasmid, the Erase-a-base kit, reporter lysis buffer, and luciferin substrate from Promega, Econofluor-2 from DuPont NEN, and Lipoluma from Lumac-LSC (Olen, Belgium).

Cell Culture

Cells were grown in DMEM (HT1080 cells and EA.hy926 cells) or DMEM-F12 (SK-N-SK cells) supplemented with glutamine (1 mM), penicillin (100 IU/ml), streptomycin (100 µg/ml), and 10% heat-inactivated fetal calf serum. Cells were seeded at a density of 2-4 times 10^4 cells/cm^2 and grown overnight in DMEM with 5% heat-inactivated, charcoal-stripped fetal calf serum. RA, 9cis-RA and staurosporine were dissolved in dimethyl sulfoxide at a concentration of 10 to 100 mM and stored at -80 °C. Cycloheximide, dexamethasone, cholecalciferol (vitamin D(3)), and 3,3`,5-triiodo-L-thyronine (T(3)) were dissolved in ethanol at a concentration of 10 to 100 mM and were stored at -20 °C. The appropriate concentration of these agents was added to the medium in a volume corresponding to 0.1% of the culture medium. Control medium contained an equal amount of excipient.

Assays for t-PA, u-PA, and PAI-1 Antigen and mRNA Levels

t-PA, u-PA, and PAI-1-related antigens in the conditioned medium were determined by specific enzyme-linked immunosorbent assays (18, 19, 20) . Total cellular RNA was isolated from HT1080 cells by the guanidinium isothiocyanate method (21) and analyzed using P-labeled antisense RNA probes specific for t-PA or PAI-1 as described(6) . For the generation of a u-PA-specific RNA probe, the full-length cDNA coding for human u-PA (22) was subcloned as a HindIII fragment in HindIII linearized pSP64 plasmid (pSPscuPAnc). The RNA probe was generated from a PvuII linearized vector using SP6 polymerase. Quantitation was performed by scanning of the autoradiograms with the Ultrascan XLR laser scanner (Pharmacia Biotech, Roosendaal, The Netherlands) using the GELSCAN software package (Pharmacia Biotech) or by direct quantitation of radioactivity using the PhosphorImager (Molecular Dynamics). Results of t-PA, PAI-1, and u-PA mRNA were expressed as a fraction of the 18 S rRNA signal.

Reporter Constructs

A recombinant phage which contained 22 kb of human t-PA genomic sequence including 17 kb of the upstream region, was isolated from a human genomic EMBL3 library using a human t-PA cDNA probe(6) . All genomic sequences analyzed in this study are numbered relative to the transcription initiation site as determined by Henderson and Sleigh (23) and are schematically presented in Fig. 2A. A 7.3-kb (+197 to -7144 bp) and a 9.8-kb upstream fragment (+197 to -9578 bp) obtained by partial BamHI digestion were linked to the chloramphenicol acetyltransferase (CAT) gene in the reporter plasmid pBLCAT3 (24) to yield t-PA7144-CAT and t-PA9578-CAT, respectively. Deletion mutants t-PA632-CAT, t-PA1564-CAT, and t-PA3070-CAT with, respectively, 632, 1564, and 3070 bp of t-PA gene upstream sequence were obtained from the t-PA7144-CAT construct by progressive exonuclease III deletion mutagenesis (Erase-a-base kit). The 2.4-kb BamHI upstream genomic fragment (-7145 to -9578 bp; t-PA2.4) was cloned in front of these constructs yielding t-PA632down triangle2.4-CAT, t-PA1564down triangle2.4-CAT, and t-PA3070down triangle2.4-CAT, respectively (cf. Fig. 4E), and in either orientation in front of the thymidine kinase (TK) promoter linked to the CAT gene in pBLCAT2 (24) yielding t-PA2.4-TK-CAT (cf. Fig. 2A) and t-PA2.4INV-TK-CAT.


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 (box), and the first exon (&cjs2106;) of the human t-PA gene are indicated. B, induction of CAT activity by RA (10M, 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 (10M, 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 (10M) and/or 9cis-RA (10M) 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 (box) 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-PA2.4Delta0.5-TK-CAT and t-PA2.4Delta0.1-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-RARbeta and h-RXRalpha, 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 RARbeta and RXRalpha 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).

Transfection Analysis

To obtain stable expression of t-PA-CAT constructs in HT1080 cells, the calcium phosphate co-precipitation method (26) was applied using a DNA mixture which contained 20 to 60 µg of PvuI-linearized CAT reporter plasmid (quantity was according to the plasmid size) and 4 µg of PvuI-linearized pCMbetaNeo selection plasmid (27, 1:5 ratio). After 36-48 h of incubation, neomycin was added to the medium (500 µg/ml final concentration), and selection was performed for approximately 10 days. In order to avoid positional effects on the CAT reporter expression, experiments were performed using a large pool of neomycin-resistant colonies (routinely 10^2-10^3). After growing to confluency, cells were harvested and seeded in 6-well dishes in DMEM containing 0.25% (w/v) bovine serum albumin and 0.5% charcoal-stripped serum. After overnight incubation, the medium was replaced with medium containing RA or control solution. After 24 h of incubation, cells were analyzed as described below.

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) . 10^7 synchronized cells suspended in cytomix (29) were added to approximately 50 µg of PvuI-linearized reporter plasmid together with XmnI-linearized h-RARbeta and with NdeI-linearized h-RXRalpha 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^2 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 TrisbulletHCl, 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 [^3H]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 ^3H-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.

Electrophoretic Mobility Shift Assay

DNA oligonucleotides or DNA fragments were labeled by filling in with the Klenow fragment of DNA polymerase I using [alpha-P]dCTP. Electrophoretic mobility shift assays were performed according to Fried and Crothers (31) as modified by Baes et al.(32) : RARalpha (NH(2)-terminally tagged with the Hemophilus influenzae agglutinin nonapeptide: Flu-RARalpha) and/or RXRalpha (tagged with an epitope from c-Myc: Myc-RXRalpha) were incubated for 15 min at 25 °C, added to the reaction mixture containing 10,000 cpm of labeled DNA fragment or oligonucleotide which was then incubated at 25 °C for 30 min. Specific monoclonal antibodies directed against the Flu epitope (anti-Flu) or the c-Myc epitope (anti-Myc) were added to the reaction mixture as preformed complex with their respective epitope-tagged receptor (Flu-RARalpha or Myc-RXRalpha). As a negative control, the fibrin D-dimer directed 15C5 monoclonal antibody (33) was preincubated with either h-RARalpha or h-RXRalpha. Band shift reactions were analyzed on a 4-5% polyacrylamide gel electrophoresis at 4 °C in 0.5 times Tris borate buffer. Bands were visualized by autoradiography.


RESULTS

RA Induction of t-PA Gene Expression in HT1080 Cells

RA induced a dose-dependent secretion of t-PA-related antigen by HT1080 human fibrosarcoma cells (cf. Fig. 1A), reaching a 10 ± 0.4-fold (mean ± S.E.) induction at 10M within 48 h, in the absence of morphological changes of the cells. Higher RA concentrations (geq10M), however, affected cell viability. t-PA-related antigen secretion with 10M RA was progressive, reaching 7.2 ± 0.3-fold the control value within 24 h (Fig. 1B). Corresponding induction of u-PA-related antigen was 1.8 ± 0.2-fold and of PAI-1-related antigen 1.1 ± 0.1-fold (data not shown). Addition of 10M cycloheximide, an inhibitor of protein synthesis, abolished the induction of t-PA-related antigen by RA (1.1 ± 0.06-fold, cf. Fig. 1B). Neither cholecalciferol (vitamin D(3), 10M) nor 3,3`,5-triiodo-L-thyronine (T(3), 10M) had any effect on t-PA-related antigen secretion both in the absence and the presence of 10M RA (data not shown).


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, 10M 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 10M 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).

Identification of the RA-inducible Enhancer at -7 Kb

A phage containing about 17 kb of human t-PA gene upstream sequence was cloned of which 9.5 kb was sequenced and used for the construction of CAT reporter vectors as schematically represented in Fig. 2A (for details see ``Materials and Methods''). Results obtained by stable integration of these constructs in HT1080 cells are shown in Fig. 2B: t-PA9578-CAT reporter activity was 6.2 ± 0.2-fold induced by RA while the t-PA7144-CAT construct showed no response; t-PA2.4-TK-CAT and t-PA2.4INV-TK-CAT were 5.5 ± 0.5-fold and 9.5 ± 0.1-fold induced, respectively, while the TK promoter alone (pTK-CAT) showed no induction. Results of transient expression of the t-PA promoter constructs are presented in Fig. 2C: t-PA9578-CAT and t-PA2.4-TK-CAT were only 1.8 ± 0.4-fold and 2.2 ± 0.2-fold induced, respectively. However, as shown in Fig. 2D, transient co-expression of the t-PA2.4-TK-CAT construct with expression plasmids encoding RA nuclear receptors increased CAT expression by RA and 9cis-RA. Similar induction (12 ± 1.0-fold) was observed when t-PA2.4-TK-CAT was co-expressed with both human RARbeta and RXRalpha compared to RARbeta alone. Combination of RA and 9cis-RA (both 10M) did not cause additional induction, whereas 9cis-RA alone revealed a smaller increase. Similar induction was obtained with h-RARalpha instead of h-RARbeta (Fig. 2D) but not with h-RAR (data not shown). When h-RXRalpha alone was co-expressed, maximal CAT induction required both RA and 9cis-RA (cf. Fig. 2D). Surprisingly, similar experiments with the t-PA9578-CAT construct revealed no CAT induction at ratios of RARbeta and RXRalpha expression vector to CAT reporter plasmid of 2 to 20. Apparently, expression of the RA nuclear receptors repressed the basal promoter activity of the t-PA reporter constructs (data not shown).

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.

Identification of a DR5-RARE Element at -7 Kb

Fig. 3A provides a schematic overview of the t-PA genomic sequence spanning -7145 to -9578 bp. The (A/G)G(G/T)T(G/C)A repeats indicated are described in Table 1. Fig. 3B represents the results of transient co-expression of t-PA2.4-TK-CAT mutants with RARbeta and RXRalpha. Deletion of the DR0 motif (in t-PA2.0-TK-CAT and t-PA1.6-TK-CAT), the DR4 motif (in t-PA1.4TK-CAT), and the DR2 motif (in t-PA0.4-TK-CAT and t-PA2.4Delta0.5-TK-CAT) did not affect the induction by RA (9.3 ± 1.0-fold to 6.8 ± 0.03-fold) as compared to the full-length t-PA2.4-TK-CAT construct (8.0 ± 0.8-fold). The t-PA1.9-TK-CAT construct, lacking the ER8 and DR5 elements, showed no CAT induction (1.1 ± 0.1-fold), whereas reintroduction of these elements in t-PA2.4Delta0.1-TK-CAT restored the induction (7.4 ± 0.2-fold). The t-PA0.2-TK-CAT construct, containing the DR5 element but not the ER8 element, showed reduced RA induction (3.7 ± 0.1-fold). Elimination of the DR5 element by site-specific mutagenesis (t-PA2.0/DR5MUT-TK-CAT; for the exact sequence, cf. Table 1) abolished RA induction. Transient co-expression of CAT reporter constructs containing two copies of putative recognition motifs in front of the TK promoter ( Table 1and Fig. 3C) revealed a 27 ± 7-fold and a 5.1 ± 0.2-fold induction for the t-PA/DR5 (t-PA/DR5-TK-CAT) and the t-PA/DR2 element (t-PA/DR2-TK-CAT), respectively, compared to a 96 ± 22-fold induction observed for the DR5 element identified in the murine RARbeta2 promoter used as a positive control (RARbeta/DR5-TK-CAT, (34) ). The distal glucocorticoid response element of the MMTV-long terminal repeat (MMTV/GREa-TK-CAT, (35) ) and the t-PA/ER8 element (t-PA/ER8-TK-CAT) conferred no induction.

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.

Involvement of the t-PA/DR5 RARE in the Direct Regulation of the Human t-PA Promoter by RA in EA.hy926 Hybrid Endothelial and SK-N-SH Neuroblastoma Cells

RA (10M) also caused an increase in t-PA-related antigen secretion by EA.hy926 hybrid endothelial and SK-N-SH human neuroblastoma cells (3.1 ± 0.2-fold and 4.0 ± 0.2-fold after 30 and 24 h, respectively, data not shown).

Evaluation of t-PA promoter constructs in the EA.hy926 hybrid endothelial cell line required co-transfection of h-RARbeta and h-RXRalpha 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 RARbulletRXR 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.

Effect of the Distance from the Transcription Initiation Site and of the Intervening Sequence on the Activity of the t-PA/DR5 RARE

The t-PA2.4 upstream fragment was cloned at different distances (632, 1564, 3070, and 7144 bp) from the t-PA transcription start site with the use of homologous upstream genomic sequences (cf. Fig. 4E). In EA.hy926 endothelial cells, a 3.0 ± 0.03- to 3.3 ± 0.02-fold induction was observed with promoter deletion constructs containing the t-PA2.4 fragment, while no induction was observed with constructs lacking this fragment (Fig. 4C). Similar results were obtained in the SK-N-SH neuroblastoma cells (cf. Fig. 4D).

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.

Binding of RA Receptors to the t-PA/DR5 RARE Element

The electrophoretic mobility shift assay was performed using c-Myc epitope-tagged RXRalpha (Myc-RXRalpha) and Flu epitope-tagged RARalpha (Flu-RARalpha), the latter being equipotent to RARbeta in transient expression assays (cf. Fig. 2D). A radiolabeled DNA fragment spanning t-PA genomic sequences from -7145 to -7324 bp (t-PA0.2) and containing the t-PA/DR5 element, showed strong binding in the presence of both Flu-RARalpha and Myc-RXRalpha (Fig. 5A, lane g versus lane f) whereas with Flu-RARalpha or Myc-RXRalpha alone no binding was seen (data not shown). Displacement of the RARbulletRXR complex was observed upon competition with a 10- and 100-fold molar excess of t-PA/DR5 oligonucleotide (lanes l and m, respectively) as well as with the positive control, the RARE sequence of the RARbeta promoter (RARbeta/DR5, lanes h and i). Competition with an unrelated DNA oligonucleotide (MMTV/GREa) had no effect on complex formation (lanes j and k). When electrophoretic mobility shift assay was performed with a radiolabeled RARbeta/DR5 oligonucleotide (positive control), similar results were obtained (lanes a to e).


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 RARbeta promoter (RARbeta/DR5). The distal glucocorticoid response element from the MMTV long terminal repeat promoter (MMTV/GREa), the RARbeta/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 RARbeta/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 RARalpha (Flu-RARalpha) and RXRalpha (Myc-RXRalpha) 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-RARalpha alone, weakly with Myc-RXRalpha 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 RARalpha (anti-Flu) or the Myc epitope of RXRalpha (anti-Myc) caused a partial disruption of the retarded complex (lane l) and a clear supershift (lane m), respectively, suggesting that both RARalpha and RXRalpha were present in the retarded complex. Similar results were obtained with the radiolabeled t-PA/DR2 (cf. Table 1) and RARbeta/DR5 oligonucleotides, except that no interaction was observed with RXRalpha 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 RARalphabulletRXRalpha (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 RARbulletRXR heterodimers in vitro, although only the t-PA/DR5 element is functionally active in the t-PA gene.

Role of Phosphorylation in the Induction of t-PA Gene Expression by RA

Staurosporine (25 nM), a potent inhibitor of the protein kinase C pathway(36) , reduced the induction of t-PA-related antigen by 10M RA from 5.8 ± 0.2-fold to 2.1 ± 0.05-fold and from 3.2 ± 0.2-fold to 1.1 ± 0.07-fold in HT1080 fibrosarcoma and EA.hy926 hybrid endothelial cells, respectively (cf. Table 2).



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.


DISCUSSION

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 RARbulletRXR 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. (^2)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-kappaB-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 RARbulletRXR 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.


APPENDIX

DNA Sequence Analysis

The DNA sequence of the human t-PA gene spanning bp +197 to -9578 relative to the transcription start site (23; nucleotide 9579 of the enclosed sequence) was determined in both directions by using a set of 5` progressive deletion constructs derived from the t-PA9578-CAT construct (cf. Fig. 2A) in combination with ``primer walking.''

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 times 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.


FOOTNOTES

*
This work was supported by the Geconcerteerde Onderzoeksacties (90-95) from the Belgian Government. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank(TM)/EMBL Data Bank with accession number(s) Z48484[GenBank].

§
Present address: Laboratorio di Genetica Molecolare, Department of Biological and Technological Research, DIBIT-HSR, Milano, Italy.

Present address: Celgen, University of Leuven, Leuven, Belgium.

**
To whom correspondence and reprint requests should be addressed: Center for Molecular and Vascular Biology, University of Leuven, Campus Gasthuisberg, O & N Herestraat 49, B-3000 Leuven, Belgium. Tel.: 32-16-345-772; Fax: 32-16-345-990.

(^1)
The abbreviations used are: PA, plasminogen activator; t-PA, tissue-type PA; u-PA, urokinase-type PA; PAI-1, plasminogen activator inhibitor-1; RA, retinoic acid; RARE, RA response elements; RAR, retinoic acid nuclear receptor; RXR, retinoid X nuclear receptor; bp, base pair(s); kb, kilobase(s); DMEM, Dulbecco's modified Eagle's medium; CAT, chloramphenicol acetyltransferase; TK, thymidine kinase; MMTV, murine mammary tumor virus.

(^2)
During the preparation of this manuscript, Åström et al.(48) described the identification of a DR5 RARE 5.6 kb upstream of the human cellular retinoic acid-binding protein-II gene.


ACKNOWLEDGEMENTS

We are grateful to Dr. M. Baes for helpful discussions and for providing the purified h-RARalpha and h-RXRalpha preparations, Dr. P. Chambon for the h-RARalpha, h-RARbeta, and h-RAR expression plasmids, Dr. R. Evans for the h-RXRalpha expression plasmid, Dr. C.-J. Edgell for the EA.hy926 hybrid endothelial cell line, Dr. T. Gulick and Dr. D. D. Moore for the h-RARalpha and h-RXRalpha bacterial expression vectors, Dr. F. Schneider for the 9cis-RA, Dr. D. Helinski for the RSVluc expression vector, Dr. L. Hendrickx for the 18 S ribosomal RNA probe, and Brigitte Verheyden for secretarial assistance.


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