Urokinase Receptor mRNA Level and Gene Transcription Are Strongly ...

Vol. 266, No.8, Issue of March 15, pp. 5177-5181,1391 Printed in U.S. A .

THEJOURNALOF BIOLOGICAL CHEMISTRY 0 1991 by The American Society for Biochemistry and Molecular Biology, Inc.

Urokinase Receptor mRNA Level and Gene Transcription Are Strongly and Rapidly Increased by Phorbol Myristate Acetate in Human Monocyte-like U937 Cells* (Received for publication, June 25, 1990)

Leif R. LundSQ, Ebbe R~nneS,Ann L. RoldanlT, Niels BehrendtS, John R~merS,Francesco BlasilT, and Keld Dan@$ From the $Finsen Laboratory, Rigshospitalet, Strandboukuarden 49, DK-2100 Copenhagen 0,Denmark and the Tlnstitute of Microbiology, University of Copenhagen, DK-1553 Copenhagen K, Denmark

al., 1985, 1988; Gelehrter and Sznycer-Laszuk, 1986; Sprengers and Kluft, 1987; Saksela and Rifkin, 1988; Schleef and Loskutoff, 1988; Laiho and Keski-Oja, 1989; Andreasen et al., 1990; Blasi et al., 1990). A new mode of regulation of the u-PA pathway of plasminogen activation has been identified. A specificcell surface receptor for u-PA (u-PAR) was first detected by a specific and saturable binding of u-PA to monocyte and monocytelike cells (Vassalli et aZ., 1985; Stoppelli et al., 1985) and has since been found on the surface of many types of cells, including many cell lines of neoplastic origin (Bajpai and Baker, 1985; Plow et al., 1986; Stoppelli et al., 1986; Boyd et al., 1988; Nielsen et al., 1988; for reviews, see Blasi et al., 1987, 1990). In some cell types u-PAR focalize u-PA to cell-cell and cell-substratumcontactsites(Pollanen et al., 1987,1988; Hebert and Baker, 1988). The receptor binds both u-PA and its proenzyme pro-u-PA (Cubellis et al., 1986). Concomitant binding of pro-u-PA to u-PAR and of plasminogen to yet unidentified binding sites at cell surfaces (Plow et al., 1986) strongly enhances plasmin formation (Ellis et al., 1989; Stephens et al., 1989). Receptor-bound u-PA can be inhibited by PAI-1and PAI-2 (Cubellis et al., 1989; Ellis et al., 1990; Estreicher et al., 1990),and thereceptor provides a mechanism Activation of the zymogen plasminogen to plasmin is a key for internalization of PAI-1-andPAI-2-inactivated u-PA step in the regulation of extracellular proteolysis. Plasmin, a (Cubellis et al., 1990; Estreicher et al., 1990). The u-PAR thus broad spectrum protease with trypsin-like specificity, is in- plays a crucial role both in localizing and modulating cell volved in the breakdown of proteins of the extracellular surface plasminogen activation. matrix and basement membrane during tissue degradation, Previous studies have shown that differentiation of the either directly or through activation of latent collagenases. monocyte-like U937 cells induced by the tumor promotor Activation of plasminogen can be catalyzed by at least two phorbol myristate acetate (PMA) leads to a strong increase different enzymes: urokinase-type (u-PA)’ and tissue-type of the number of u-PA receptors as well as a pronounced plasminogen activators, thelatter also being involved in decrease in their affinity for u-PA (Stoppelli et al., 1985; thrombolyses. Plasminogen activation is finely regulated by several factors, including two specificand fast acting plasmin- Nielsen et al., 1988; Picone et al., 1989). Similar data have ogen activator inhibitors, PAI-1 and PAI-2. Synthesis of the been obtained with HeLa cells (Estreicher et al., 1989). We plasminogen activators and their inhibitors is regulated by a recently purified human u-PAR and cloned its full-length variety of hormones, growth factors, and cytokines (Dan0 et cDNA (Behrendt et al., 1990; Roldan et al., 1990),and we now report that hybridization studies with this cDNA show that * This work wassupported financially by the Danish Biotechnology the PMA-induced increase in u-PAR proteinin the U937 cells program Center for Molecular Cell Biology, the Danish Cancer So- can be traced back to anincrease in the level of u-PAR mRNA ciety, and the Danish Medical Research Council. The costs of publi- which is due at least partly to an increased transcription of cation of this article were defrayed in part by the payment of page the u-PAR gene.

We have studied the effect of the tumor promotor phorbol myristate acetate (PMA) on the level of mRNA for the receptor for urokinase-type plasminogen activator (u-PAR) in the human monocyte-like cell line U937. PMA causes an early increase in the u-PAR mRNA level which reaches a maximal 50-foldenhancement after 24 h of treatment. Half-maximal stimulation occurs at = 5 nM PMA. The effect is observed only with phorbol esters that also act as tumor promotors. The protein synthesis inhibitor cycloheximide (10 pg/ml) also increases the level of u-PAR mRNA. Nuclear run-on experiments show a time-dependent increase in the u-PAR gene transcription rate after exposure of the cells to PMA. The PMA-induced increase in u-PAR mRNAis paralleled by a time-dependent increase in u-PAR protein as detected by crosslinking studies with radiolabeled ligand. We conclude that PMA stimulates transcription of the u-PAR gene in U937 cells, and this is responsible at least in part for the accumulation of the u-PAR mRNA and for the subsequent increase in urokinase-binding capacity.

charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. To whom all correspondence should be sent. Tel.: 45-35-453545 (ext. 5750); Fax: 45-31-385450. The abbreviations used are: u-PA, urokinase-type plasminogen activator; u-PAR, u-PA receptor; pro-u-PA, proenzyme form of uPA; PAI-1, plasminogen activator inhibitor type 1; PAI-2, plasminogen activator inhibitortype 2; PMA, phorbol 12-myristate 13-acetate; ATF, amino-terminal fragment of u-PA.

EXPERIMENTALPROCEDURES

Materials-Phorbol 12-myristate 13-acetate (PMA), phorbol 12, 13-didecanoate, phorbol 12-myristate 13-acetate 4-0-methyl ether, 4a-phorbol 12,13-didecanoate, Mezerein, and cycloheximidewere obtained from Sigma. Deoxycytidine 5’-[a-32P]triphosphate (specific activity, 3,000 Ci/mmol), uridine 5’-[a-32P]triphosphate(specific activity, 400 Ci/mmol), and Rainbow 14C-protein molecular weight markers were purchased from the Radiochemical Center, Amersham

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PMA Regulation of u-PAR

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Corp. RNase-free DNase, RNase A, RNase TI, anda kit for random primed labeling reactionwere purchased from Boehringer Mannheim. All other materials have been described previously (Andreasen et al., 1986; Lund et al., 1987; Mayer et al., 1988; Riccio et al., 1988) or were of the best grade commercially available. Cell Culture-The human monocyte-like cell line U937, originally isolated from the pleural fluid of a patient with histiocytic lymphoma (Sundstrom andNilson, 1976) (ATCC CRL1593), was obtained from Dr. A. Fattorossi(Research Laboratory of AeronauticaMilitare, Rome, Italy) andwas cultured in RPMI 1640 medium with 10%heatinactivated fetal calf serum and 2 mM L-glutamine a t a density of 0.5 X IO6 cells/ml at the onset of the experiment. The medium was supplemented with 200 units/ml penicillin and 25 pg/ml streptomycin. Thehuman larynxepidermoidcarcinoma cell line (HEp-2) (ATCC CCL 23) was obtained from Flow Laboratories, Irvine, United Kingdom, and kept in Dulbecco's modified Eagle's medium supplemented with 10% fetalcalf serum (Mayer etal., 1988). Adherent cells were released by a rubber policeman,and thecells were harvested for RNA analysis as described (Mayer et al., 1988). The cell lines were tested for and found free from mycoplasma infection. RNA Analysis-Total cellular RNA was isolated from cells by acid guanidinium thiocyanate-phenol-chloroformextraction as described by Chomczynsky and Sacchi (1987). Total RNA was analyzed by hybridizing Northern blots or quantitative dot blots as described (Lund et al., 1987) except that cDNA probes were radiolabeled with ['"PIdCTP by the random primer technique to a specific activity greater than 1 X lo9 dpm/pg (Feinberg and Vogelstein, 1983). The plasmid used as a probe for u-PAR mRNA (p-u-PAR-1) carriedcDNA covering the entirecoding region and the3'- and the 5'-untranslated regions (Roldan et al., 1990). Scanning of the areas and intensities of the autoradiographic films was performed with a Shimadzu dual wavelength TLC scanner CS930. The relative amounts of u-PAR mRNA were normalized against the corresponding relative amounts of @-actin mRNA, on which the PMA treatment was found to have no effect (see below). Among the dots representing a serial dilution of an RNA sample the areas and intensities were linearly related to the total amountsof RNA in the dots. The slopes of these lines and thecorresponding standard deviation were calculated by unweighted linear regression analysis (Lund et al., 1987). The relativeslopes of the linesfor different RNA preparations were takenas representing the relative amounts of specific mRNA in these preparations. Nuclear Transcription Assay-Preparation of nuclei in vitro, RNA elongation and isolation were performed as described by Greenberg and Ziff (1984) except that the nuclei were frozen and stored a t -80 "C between preparation and analysis and that the '*P-labeled RNA was treated with 10 pg/ml DNase (RNase free) for 30 min a t 37 "C. 2 X 10' nuclei were used for each assay. About 2.0 X lo6 cpm were used for hybridization. The hybridization conditions were 50% formamide, 5 X SSC, 5 X Denhardt's solution, 50 mM sodium phosphate buffer, pH 7.0, 0.1% sodium dodecyl sulfate, 100 pg/ml yeast RNA, and 10 pg/ml pUC19 DNA, a t 37 "C for 3 days. The following DNA probes were used p-u-PAR-1, a plasmid carrying the humanuPAR cDNA (Roldan et al., 1990); pPAI 57, a human PAI-2 cDNA probe (Schleuning et al., 1987); pHUK8, carrying a 1.6-kilobase PstI fragment of a human u-PA cDNA (Verde et al., 1984); pHF@A-3'UT, coding for the 3"untranslated region of human @-actin (Ponte et al., 1983); pGAPDH, carrying a full-length cDNA probe coding for rat glyceraldehyde phosphate dehydrogenase inserted into the PstI site of pUC19 (Fort et al., 1985) and pUC18 plasmid (Yanisch-Perron et al., 1985). Chemical Cross-linking Assay-Preparation of the detergentphase from U937 cells treated with PMA for different timeperiods and the chemical cross-linking of u-PAR to '251-labeledATF were performed as described (Behrendt et al., 1990). RESULTS

Effect of PMA on the u-PAR mRNA Level-Total RNA was extracted from control U937 cells and from U937 cells treated with PMA at different time periods. The size and relative concentration of mRNA specific for u-PAR from a typical experiment were analyzed by Northern blots, hybridized with a plasmid containing a full-length cDNA coding for u-PAR (Roldan et al., 1990). A signal for u-PAR mRNA is not visible in the control cells after standardexposure (Fig. IA)but was observed after prolonged exposure (result not shown). After

28s 18S-

"b-acti"

mRNA

loL"-~ 00

10

20

30

40

50

Time (hours)

FIG. 1. Northern blot analysis of u-PAR mRNA in U937

cells treated with PMA. The cells were seeded a t 0.5

X 106/ml in RPMI 1640 medium containing 10% fetal calf serum and incubated with 150 nM PMA for the indicated number of h. After the incubation both adherent and nonadherentcells were collected and pooled, and total RNA was isolated as described under "ExperimentalProcedures." For the Northern blotanalyses, 30 pg of total RNA was electrophoresed in 1.5% agarose gels under denaturingconditions and blotted onto a nitrocellulose filter. The filterswere hybridized with a randomlyprimed"P-labeled u-PAR cDNAprobe (A),andafter stripping, they were rehybridized to a @-actin cDNA probe ( B ) .The positions of the ribosomal RNAs are indicated to the left and the positions of the mRNA bands to the right. The relative amount of uPAR mRNA a t each time point in the experiment shown in A was estimated by spectrophotometric scanning of autoradiograms of the Northern filtershybridized with the u-PARprobe after normalization against thecorresponding relative amounts of @-actinmRNA. The uPAR level a t time 0 has been set equal to 1 and is at subsequent time points expressed as fold induction ( C ) .

1.5 h of PMA treatment, a signal for u-PAR mRNA is also seen after the standard exposure and obviously increases with the time(Fig. 1A). As a control for equal loading of RNA, the Northern filter was stripped and rehybridized with a human @-actincDNA probe (Ponte etal., 1983) (Fig. 1B). A moderate variation(less than 2.1-fold) in p-actinmRNA signal was seen. This variation was due to loading artifacts because serial dilution of the same RNA preparations showed no effect of the PMA treatment on the @-actin mRNAlevel in dot blots (result not shown); the @-actin mRNA level was therefore used to normalize the level of hybridization with the u-PAR probe as measured by scanning of the autoradiographic films (Fig. IC). By this method the effect of PMA on the u-PAR mRNA level in U937 cells is detectable already after 0.5 h of PMA treatment;a maximal40-50-fold increase inthe u-PAR mRNA level is reached after 24 h of treatment, and thislevel remains stable until a t least 48 h after the startof the PMA treatment. Similar results were obtained in threeindependent experiments. Fig. 2 shows the dose dependence of the induction of uPAR mRNA inU937 cells with a48-h PMA treatment. There is ahalf-maximalresponse at about 5 nM PMA, and the maximal effect is obtained with about 15 nM PMA. A small but reproducible decrease in the u-PAR mRNA level is seen a t very high concentrations of PMA (100-150 nM), in agreement with previous data on the effect of PMA on the binding of u-PA to these cells and the induction of PAI-1 protein in RD cells (Mayer et al., 1988; Picone et al., 1989). Similar data have been obtained in two independent experiments. The specificity of the response of U973 cells with respect to the structure of the inducing agent was studied in the experiment shown in Fig. 3. PMA, phorbol12,13-didecanoate, and Mezerein are biologically active tumor promotors and activators of protein kinase C, whereas phorbol 12-myristate 13-acetate 4-O-methyl etherand 4a-phorbol 12,13-didecanoate are inactive. Treatment of the cells for 48 h with each

PMA Regulation of u-PAR ___

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U-PAR

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FIG. 2. Dose dependence of PMAinduction of u-PAR mRNA in U937 cells. The cells were cultured and harvested as described in the legend to Fig. 1except that they all were incubated for 48 h in the presence of the indicated concentrations of PMA. Total RNA was purified, and dotblots (insets) were prepared by applying 2-fold serial dilutions of total RNA starting from 2 pg onto a nitrocellulose filter. The filter was hybridized with a randomly primed "P-labeled u-PAR cDNA probe (inset, left) and was subsequently stripped and rehybridized with a @-actinprobe (inset, right).The relative amount of u-PAR mRNA was determined by scanning the autoradiographic film with a laser densitometer and was normalized against the corresponding relative amounts of @-actin. The numbers in the insets indicates nontreated cells (lane I), 0.0015 nM PMA (lane 2), 0.015 nM PMA (lane 3),0.15 nM PMA (lane 4 ) , 1.5 nM PMA (lane 5), 15 nM (lane 6), 75 nM PMA (lane 7),and 150 nM PMA (lane 8).The bars indicate standard deviations. 1 2 3 4 5 6

FIG.4. Time courseof PMA induction of u-PAR protein as detected by chemical cross-linking to "'1-ATF. Nontreated cells (lane 1 ) and cells treated with 150 nM PMA for 3 h (lane 2), 9 h (lane 3), 24 h (lane 4 ) , and 48 h (Lane 5 ) were acid treated to remove endogenous u-PA, and detergent extracts were prepared by temperature-inducedphaseseparation as described under "Experimental Procedures." Nondiluted extracts, a buffer control sample (lane 6) and a 1%Triton X-114 total lysate (diluted 1/25) from HEp-2 cells (lane 7)were incubated with I2'II-ATF, cross-linked with disuccinimidyl suberate and run in a 6-16% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gradient gel followed by autoradiography. Electrophoretic mobility of molecular weight standard proteins are indicated to theleft. 1 2 3 4 5 6 7

"u-PAR mRNA

A 28s- I

18s- '

h!

y - U-PAR mRNA FIG.5. Northern blot analysis of the effect of cycloheximide, alone and in combination with PMA, on u-PAR and 8actin mRNA in U937 cells. Total RNA was isolated from cells

FIG. 3. Effect of different phorbol esters and Mezerein on the u-PAR mRNA level in U937 cells. U937 cells cultured as described in the legend to Fig. 1 were incubated in the presence of the indicated compounds (150 nM) for 48 h. After incubation the RNA was isolated and analyzed by Northern blotting as described in Fig. 1. Lane 1, no treatment; lane 2, phorbol 12-myristate 13-acetate; lane 3, phorbol 12,13-didecanoate; lane 4, phorbol 12-myristate 13acetate 4-0-methyl ether;lane 5,4a-phorbol 12,13-didecanoate; lane 6, Mezerein.

of the threeformer compounds (lanes 2,3, and 6, respectively) increases the cellular level of u-PAR mRNA stronglywhereas the two latter phorbol esters (lanes 4 and 5 ) have no effect on the u-PAR mRNA level in theU937 cells. Virtually identical results were obtained in two independent experiments. Time Course of PMA Induction of u-PAR Protein-The time course of the induction of u-PAR protein was studied by a chemical cross-linkingexperiment. u-PAR binds to the amino-terminal fragment (ATF) of the u-PA molecule. After treatment with 150 nM PMA for different time periods Triton X-114 extracts were prepared from U937 cells by temperatureinduced phase separationand cross-linked to '2sI-labeled ATF (see Behrendt et al., 1990). As shown in Fig. 4 there was a weak signal of"'1-ATF cross-linked to u-PAR in control U937 cells ( l a n e 1). After 9 h of PMA treatment ( l a n e 3 ) there was a clear increase in the strength of the signal, and this increase continuedthroughout the 48-h incubation period. Similar data have been obtained in two independent

incubated for 3 h (lanes 1 - 4 ) or 6 h (lanes 5-7) with no additions (lane I), with 150 nM PMA (lunes 2 and 5),with 10 pg/ml cycloheximide (lanes 3 and 6), and their combination (lanes 4 and 7). For Northern blot analysis, 30-pg portions of total RNA were electrophoresed in agarose gels under denaturing conditions and blotted onto a nitrocellulose filter. The filters were hybridized to a random primed "P-labeled u-PAR probe ( A ) or @-actinprobe (€3). The positions of the u-PAR and /3-actin mRNAs are indicated to the right.

experiments. These results are in good agreement with the time course of the increase in u-PAR mRNA level (see Fig. 1C). Concomitantly with the increase in u-PARprotein level, PMA induced a change to a lower electrophoretic mobility of the u-PAR. ATF complex in agreement withprevious findings (Nielsen et al., 1988). This change has been shown to be due to a difference in glycosylation of the u-PAR molecule (Behrendt etal., 1990). Effect of Cycloheximide on the u-PAR mRNALevel-To test whether protein synthesis is required for the effect of PMA on the u-PAR mRNA level, U937 cells were incubated for 3 or 6 h with PMA (Fig. 5, lanes 2 and 5 ) , cycloheximide (lanes 3 and 6)) and a combination of the two compounds (lanes 4 and 7). Total RNA was isolated from the cells and analyzed by Northern blotting. Scanning of the autoradiographs showed that cycloheximide itself produced a maximal 5-8-fold increase in the u-PAR mRNA level after 3 and 6 h of cycloheximide treatment. A virtually identical increase in the u-PAR mRNA level was observed in four independent experiments. Incubation of the U937 cells with cycloheximide in addition to PMA for 3 and 6 h reduced the effect of PMA on the mRNA level for u-PAR mRNA a t both time points.

PMA Regulation of u-PAR

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This effect was also observed in four independent experiments. Effect of PMA Treatment onthe Transcriptional Rateof the u-PAR Gene-In order to determine whether the PMA-induced increase in the u-PAR mRNA levels was due to an increased rate of transcription, we have measured the transcriptional activity of the u-PAR gene using a nuclear transcription assay (Greenberg and Ziff, 1984). Nuclei were isolated from U937 cells treated with PMA for different time periods. The nuclear RNA was elongated in the presence of ["PIUTP for 30 min, extracted, and hybridized to u-PAR cDNA and other cDNA probes immobilized on nitrocellulose filter. Autoradiographs of the filters are showed in Fig.6. Similar results have been obtained in four independent experiments. In nuclei from untreated cells u-PAR has a low rate of transcription which is reproducibly increased after 1h of treatment with PMA. This fast induction of u-PAR gene transcription is followed bya furtherincrease after continued exposure to PMA, similar to a continued increase observed for transcription of the PAI-2 gene, which was used as a positive control in this experiment (see Schleuning et al., 1987).

and function, we have analyzed the effect of phorbol ester on u-PAR gene expression in the U937 cell line. The increase in specific binding capacity can be traced back to a strong and rapid increase in the cellular level of u-PAR mRNA caused at least in part by an increased transcription of the u-PAR gene. The u-PA pathway of pericellular plasminogen activation requires the presence of u-PA and u-PAR and theabsence of the inhibitors PAI-1 and PAI-2. Synthesis of u-PA, PAI-1, and PAI-2 is regulated at the transcriptional level by hormones, cytokines, and growth factors (for reviews, see Dan0 et al., 1985, 1988; Schleef and Loskutoff, 1988; Laiho and Keski-Oja, 1989; and Andreasen et al., 1990). The present study demonstrates also that u-PAR synthesis is transcriptionally regulated. The pattern of regulation of these components of the plasminogen activation system appears to be different in different cell types. Furthermore, the regulation is in some cases concerted, i.e. the same compound, which in a certain cell type increases both u-PA and u-PAR synthesis, decreases that of inhibitors. In other cases the regulation is not concerted (for reviews, see Dan0 et al., 1988; Saksela and Rifkin, 1988; Laiho and Keski-Oja, 1989; Andreasen et al., 1990). This pattern provides a versatile regulatory system in DISCUSSION which certain conditions inthe microenvironment will induce The receptor for urokinase is a key protein in the surface pericellular proteolysis in one cell type but not in others. A pathway of localized extracellular plasminogen activation clarification of the detailed molecular mechanisms involved since it focuses proteolytic activity of the cell surface and in this regulation is crucial for understanding why the reguregulates its extent. It has been observed previously that lation fails in some pathological conditions, as for example specific binding capacity for u-PA was increased in human cancer, leading to an increase of pericellular proteolysis and U937 cells by PMA treatment (Stoppelli et al., 1986; Nielsen a change in the invasive attitude of cells. The effect of PMA observed in this study is compatible et al., 1988; Picone et al., 1989) and in HeLa cells by PMA with an action via protein kinase C since the active concenand epidermal growth factor (Estreicher et al., 1989). In order to gain an insight in theregulation of u-PAR level tration range as well as the specificity of the response with respect to the structureof phorbol esters paralleled the affinA 0 1 3 6 2 4 ity andspecificity for activation and binding to protein kinase "-PAR-!.' %@ C (for reviews, see Ashendel, 1985; Nishizuka, 1986,1988; u-pA-1 . . .Castagna, 1987). However, the sustained effect of PMA on PAI-Z-: .. ..;vi both the protein (Nielsen et al., 1988; Picone et al., 1989) and P-actin-iIr; the mRNA level is in contrast to a desensitization to the pUC18PMA regulation of other molecules, observed in some cell GApDH- ...-. types (see for example Degen et al., 1985). Possible explanations for this difference are that in the U937 cells an early desensitization of protein kinase C does not occur, that the specific message sent by protein kinase C has along half-life, that PMA prolongs the half-life of u-PAR mRNA, or that PMA also induces synthesis of growth factors or cytokines which in turn may induce a lateincrease in the u-PAR mRNA level. With respect to thelast possibility it is noteworthy that treatment of U937 cells with PMA has been reported to increase the platelet-derived growth factor A chain mRNA level and that this level remained elevated for at least 48 h (Makela et al., 1987). These possible mechanisms for the prolonged effect of PMA on the u-PAR mRNA level are now under investigation. TIME (hours) Cycloheximide treatment was found to increase the u-PAR FIG.6. Transcriptional activity of the human u-PAR gene mRNA level in the U937 cells. A similar increase (5-10-fold) following stimulation by PMA. A, nitrocellulose filter with imin the u-PAR mRNA level is induced by cycloheximide in the mobilized DNA were hybridized to R'P-labeled RNA prepared from nuclei isolated from control orcells treated with 150 nM PMA for the human A549 carcinoma cell line.*Despite this effect of cyclotop. The filters were exposed for 10 heximide alone, it reduced the PMA-induced increase in unumber of hours indicated on the PAR mRNA in the U937cell. A similar effect has been days. @.-actin,human @-actin; pGAPDH, rat glyceraldehyde-3-phosphate dehydrogenase. B, quantification of changes ingene transcrip- described for cycloheximide alone and for cycloheximide in tion. The relative gene transcription of u-PAR (O),PAI-2 (A), and combination with dexamethasone on the PAI-2 mRNA level u-PA (0)a t each time point in the experiment shown in A was in HT-1080 cells (Medcalf et al., 1988). The explanation for determined by spectrophotometric scanning of the autoradiograms presented in A after normalization against the corresponding relative this phenomenon remains unknown, but it suggests a complex amount of @-actin signal. The level for u-PAR, PAI-2, and u-PA at regulation of u-PAR synthesis which may involvethe inacti,

:

time 0 has been set equal to 1 and is at subsequent time points expressed as fold induction. The bars indicate standard deviation.

* L. R. Lund, unpublished result.

PMA Regulation of u-PAR vation of negative and activation or induction of positive regulatory factors acting not only at the transcriptional but maybe also at the level of mRNA stability and at the translational level. The u-PAR mRNA contains the sequence AUUUA (Roldan et al., 1990) common to those mRNAs that have been shown to be subject to a selective degradation reported for granulocyte macrophage colony-stimulating factor mRNA (Shaw and Kamen, 1986). This sequence is also present in PAI-1 mRNA (the largest transcript) and in the PAI-2 transcript(Ny et al., 1986; Pannekoek et al., 1986; Schleuning et al., 1987); levels of both these mRNAs were increased by PMA (Schleuning et al., 1987;Mayer et al., 1988). Although the molecular events between activation of protein kinase C and increased expression of responsive genes remain largely unknown, recent studies have identified several PMA-responsive enhancer elements in the5'-flanking region of the responsive genes (for a recent review, see Mitchell and Tjian, 1989). Future studies require the isolation and characterization of the u-PAR gene to establish the molecular mechanism behind the PMA-induced increase inthe u-PAR mRNA level. Acknowledgments-We thank Dr. Larry Kedes for the 0-actin probe, Dr. W.-D. Schleuning for the PAI-2 probe, and Dr. P. Fort for the glyceraldehyde-3-phosphate dehydrogenase probe. We also acknowledge gratefully the excellent technical assistance of Lene Ahrenst, Kirsten Lund Jakobsen, John Post, Anna Margrethe Poulsen, and Nine Scherling. REFERENCES Andreasen, P. A., Riccio, A., Welinder, K. G., Douglas, R., Sartorio, R., Nielsen, L. S., Oppenheimer, C., Blasi, F., and Dan@,K. (1986) FEBS Lett. 209,213-218 Andreasen, P. A., Georg, B., Lund, L. R., Riccio, A., and Stacey, S. N. (1990) Mol. Cell. Endocrinol. 68, 1-19 Ashendel, C. L. (1985) Biochim. Biophys. Acta822,219-242 Bajpai, A., and Baker, J. B. (1985) Biochem. Biophys. Res. Commun. 133,475-482 Behrendt, N., Rbnne, E., Ploug, M., Petri, T., Lbber, D., Nielsen, L. S., Schleuning, W.-D., Blasi, F., Appella, E., and Dan@,K. (1990) J. Biol. Chem. 265,6453-6460 Blasi, F., Vassalli, J.-D., and Dan@,K. (1987) J. Cell Biol. 104, 801804 Blasi, F., Cubellis, M. V., Roldan, A.L., Masucci, M. T., Behrendt, N., Ellis, V., Appella, E., and Dan@,K. (1990) In Serine Proteases and Their Serpins in the Nervous System: Regulation in Deuelopment and in Degenerative and Malignant Diseases (Festoff, B. W., Mantain, D., and Barlovats-Meimon, G., eds.) pp. 21-30, Plenum Publishing Corp., New York Boyd, D., Florent, G., Kim, P., and Brattain, M. (1988) Cancer Res. 48,3112-3116 Castagna, M. (1987) Biol. Cell. 5 9 , 3-14 Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 1 6 2 , 156159 Cubellis, M. V., Nolli, M. L., Cassani, G., and Blasi, F. (1986) J. Biol. Chem. 261,15819-15822 Cubellis, M. V., Andreasen, P. A., Ragno, P., Mayer, M., Danb, K., and Blasi, F. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 4828-4832 Cubellis, M. V., Wun, T.-C., and Blasi, F. (1990) EMBO J. 9, 10791085 Dan@,K., Andreasen, P. A., Gr@ndahl-Hansen,J., Kristensen, P., Nielsen, L. S., and Skriver, L. (1985) Adv. Cancer Res. 44, 139266 Dan@,K., Andreasen, P. A., Behrendt, N., Grondahl-Hansen, J., Kristensen, P., and Lund, L. R. (1988) in Development and Function of the Reproductive Organs (Parvinen, M., Huhtaniem, I., and Pelliniemi, L. J., eds) vol. 2, pp. 259-278, Ares-Serona Symposia, Rome, Italy Degen, J. L., Estensen, R. D., Nagamine, Y., and Reich, E. (1985) J. Biol. Chem. 260, 12426-12433

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Ellis, V., Scully, M. F., and Kakkar, V. V. (1989) J. Biol. Chem. 264, 2185-2188 Ellis, V., Wun, T.-C., Behrendt, N., Rbnne, E., and Dan@,K. (1990) J. Biol. Chem. 265,9904-9908 Estreicher, A., Wohlwend, A., Belin, D., Schleuning, W.-D., and Vassalli, J.-D. (1989) J. Biol. Chem. 264, 1180-1189 Estreicher, A., Muhlhauser, J., Carpentier, J.-L., Orci, L., and Vassalli, J.-D. (1990) J. Cell Biol. 111,783-792 Feinberg, A. P., and Vogelstein, B. (1983)A d . Biochem. 132, 6-13 Fort, P., Marty, L., Piechaczyk, M., El Sabrauty, S., Dani, C., Jeanteur, P., and Blanchard, J. M. (1985) Nucleic Acids Res.13, 14311442 Gelehrter, T. D., and Sznycer-Laszuk, R. (1986) J. Clin. Invest. 77, 165-169 Greenberg, M. E., and Ziff, E. B. (1984) Nature 311, 432-438 Hebert, C. A., and Baker, J. B. (1988) J. Cell Biol. 1 0 6 , 1241-1247 Laiho, M., and Keski-Oja, J. (1989) Cancer Res. 49, 2533-2553 Lund, L. R., Riccio, A., Andreasen, P. A., Nielsen, L. S., Kristensen, P., Laiho, M., Saksela, O., Blasi, F., and Danb, K. (1987) EMBO J. 6, 1281-1286 Makela, T. P., Alitalo, R., Paulson, Y., Westermark, B., Heldin, C.H., and Alitalo, K. (1987) Mol. Cell. Biol. 7,3656-3662 Mayer, M., Lund, L. R., Riccio, A., Skouv, J., Nielsen, L. S., Stacey, S. N., Dan@,K., and Andreasen, P. A. (1988) J. Biol. Chem. 263, 15688-15693 Medcalf, R. L., Van den Berg, E., and Schleuning, W.-D. (1988) J. Cell Biol. 106, 971-978 Mitchell, P. J., and Tjian, R. (1989) Science 245, 371-378 Nielsen, L. S., Kellerman, G. M., Behrendt, N., Picone, R., Dan@,K., and Blasi, F. (1988) J. Biol. Chem. 263,2358-2363 Nishizuka, Y. (1986) Science 233, 305-312 Nishizuka, Y. (1988) Nature 3 3 4 , 661-665 Ny, T., Sawdey, M., Lawrence, D., Millan, J. L., and Loskutoff, D. J. (1986) Proc. Natl. Acad. Sci. U. S. A. 83,6776-6780 Pannekoek, H., Voerman, H., Lambers, H., Diergaarde, P., Verweig, C. L., van Zonnenveld, A.-J., and van Mourik, J.-A. (1986) EMBO J. 5,2539-2544 Picone, R., Kajtaniak, E., Nielsen, L. S., Behrendt, N., Mastronicola, M. R., Cubellis, M.V., Stoppelli, M. P., Pedersen, S., Dan@,K., and Blasi, F. (1989) J. Cell Bwl. 108,693-702 Plow, E. F., Freaney, D. E., Plescia, J., andMiles, L. A. (1986) J.Cell Biol. 1 0 3 , 2411-2420 Andreasen, P. A., Nielsen, Pollanen, J., Saksela, O., Salonen, E."., L. S., Dan@,K., and Vaheri, A. (1987) J. Cell Biol. 1 0 4 , 1085-1096 Pollanen, J., Hedman, K., Nielsen, L. S., Dan@,K., and Vaheri, A. (1988) J. Cell Biol. 106, 87-95 Ponte, P., Gunning, P., Blau, H., and Kedes, L. (1983) Mol. Cell. Biol. 3,1783-1791 Riccio, A., Lund, L.R., Sartorio, R., Lania, A., Andreasen, P. A., Danb, K., and Blasi, F. (1988) Nucleic Acids Res. 16, 2805-2824 Roldan, A. L., Cubellis, M. V., Masucci, M. T., Behrendt, N., Lund, L. R., Dan@,K., Appella, E., and Blasi, F. (1990) EMBO J. 9,467474 Saksela, O., and Rifkin, D. B. (1988) Annu. Rev. Cell Biol. 4, 93-126 Schleef, R. R., and Loskutoff, D. J. (1988) Haemostasis 18,328-341 Schleuning, W.-D., Medcalf, R. L., Hession, C., Rothenbuhler, R., Shaw, A., and Kruithof, E. K. 0. (1987) Mol. Cell. Biol. 7,45644567 Shaw, G., and Kamen, R. (1986) Cell 46,659-667 Sprengers, E. D., and Kluft, C. (1987) Blood 69,381-387 Stephens, R. W., Pollanen, J., Tapiovaara, H., Leung, K.-C., Sim, P.S., Salonen, E."., Rbnne, E., Behrendt, N., Dan@,K., and Vaheri, A. (1989) J. Cell Biol. 108, 1987-1995 Stoppelli, M. P., Corti, A., Soffentini, A., Cassani, G., Blasi, F., and Assoian, R. K. (1985) Proc. Natl. Acad. Sci. U. S. A. 82,4939-4943 Stoppelli, M. P., Tacchetti, C., Cubellis, M. V., Corti, A., Hearing, V. J., Cassani, G., Appella, E., and Blasi, F. (1986) Cell 45, 675-684 Sundstrom, C., and Nilsson, K. (1976) Znt. J. Cancer 17,565-577 Vassalli, J.-D., Baccino, D., and Belin, D. (1985) J. CellBiol. 100, 86-92 Verde, P., Stoppelli, M. P., Galeffi, P., Di Nocera, P., and Blasi, F. (1984) Proc. Natl. Acad. Sci. U. S. A. 81,4727-4731 Yanisch-Perron, C., Vieira, J., and Messing, J. (1985) Gene (Amst.) 3 3 , 103-119