Transcriptional activation of fibroblast collagenase gene expression by

THEJOURNAL OF BIOLOGICAL CHEMISTRY 0 1992 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 267,No.8. Issue of March 15,pp. 5644-5648,1992 Printed in U.S.A.

Transcriptional Activationof Fibroblast Collagenase Gene Expression by a Novel Lymphokine, Leukoregulin* (Received for publication, July 26, 1991)

Alain MauvielS, Veli-Matti Kiihari, Charles H. Evans#, andJouni Uittoll From the Departments of Dermatology, and Biochemistry and Molecular Biology, Jefferson Medical College,Section of Molecular Dermatology, Jefferson Institute of Molecular Medicine, Thomas Jefferson University, Philadelphia, Pennsylvania 19107, and the $Tumor Biology Section, Laboratory of Biology, Division of Cancer Etiology, National Cancer Institute, Bethesda, Maryland 20892

Leukoregulin (LR) is a novel T-cell derived cytokine ing tumor metastasisand certain inflammatory diseases, such with unique anti-tumor properties. We have recently as rheumatoid arthritis (for review, see Refs. 1 and 2). The demonstrated that LR is also able to modulate the predominant component of the extracellular matrix in most biosynthetic repertoire of normal human skin fibro- connective tissues is collagen, a family of closely related, yet blasts in culture (Mauviel, A., Ridini, F., Hartmann, genetically distinct proteins (3). The predominant tissue colD. J., Pujol, J.-P., and Evans,C. H. (1991) J. Cell Biol. lagens, type I and type 111, form relatively broad fibers which 113, 1466-1462). In this study, we have examined in provide tensile properties to the tissues. Breakdown of these detail theeffects of LR on collagenase gene expression fibers is initiated by collagenase, a calcium-dependent endoin human skin fibroblastcultures. The results indicatedpeptidase, which is synthesized as a -60-kDa precursor protime-and dose-dependent induction of collagenase mRNA steady-state levels, the maximum elevation tein encoded by a -2.1 kb' mRNA (4, 5). The precursor form being -36-fold. In contrast, themRNA levels for tissue is proteolytically activated by removal of a peptide segment inhibitor of metalloproteases remained unchanged in of -10 kDa (6, 7). Collagenase activity in the tissues is also the same RNA preparations. The enhancement of col- modulated by the presence of inhibitor molecules, including lagenase mRNA levels was shown to be dependent on the tissue inhibitor of metalloproteases (TIMP), which forms protein synthesis, and itcould be counteracted by dex- stoichiometric 1:l complexes with the enzyme protein to amethasone or all-trum-retinoic acid. Transient trans- inactivate collagenase. In addition, serum factors, including a human collagen- az-macroglobulin, can serve as inhibitorsof collagenase activfections of cultured fibroblasts with ase promoter/reporter gene constructindicate4 up- ity (see Ref. 2). The activity of collagenase in inflammatory processes is regulation of the promoter activity, which could be blocked by dexamethasone and all-trans-retinoicacid. modulated by a variety of cytokines, including interleukin-1 The observation suggested regulation at the transcrip- (IL-1) and tumor necrosis factor-a (TNF-a)which are derived tional level of collagenase gene expression. LR was primarily from activated macrophages within the inflammaalso shown to induce the mRNA levels for junB, sug- tory cell infiltrates (1). BothIL-1and TNF-a have been gesting possible involvement of the AP-1 complex in shown to up-regulate collagenase gene expression in cell culthe regulation. The ability of LR to selectively induce tures, and this activation is mediated by the transcription collagenase gene expression in skin fibroblasts sug- factor complex AP-1 (8-10).On the other hand, IL-1 and gests that this cytokine may significantly contributeto TNF-a have been shown to enhance TIMP expression in skin the degradation of the extracellular matrix inphysio- fibroblasts, which may regulate extracellular collagenase aclogical situations, such as tissue development and re- tivity (11, 12). Thus, these cytokines are potentially able to pair, and in diseases characterized by excessive deg- modulate tissue destruction mediated by collagenase (1). radation and turnoverof collagen. Recently, a novel cytokine, leukoregulin (LR), has been purified from activated T-cells (13). This cytokine, a -50 kDa glycoprotein, exhibits unique antiproliferative effects on tuDegradation of extracellular matrix macromolecules by me- mor cells, and produces a rapid and reversible increase in talloproteases is an essential feature of a number of physio- plasma membrane permeability of the target cells (14-16). logical processes involving connective tissue remodeling, such We have recently demonstrated that, inaddition to itsgrowth as embryonic development, wound repair, and maintenance regulatory effects on neoplastic cells or on cells undergoing of tissue homeostasis. In addition, degradation of the extra- malignant transformation, LR canmodulate the biosynthetic cellular matrix plays a role in pathological conditions, includ- functions of normal human dermal fibroblasts in vitro (17). It was demonstrated that LR differentially regulates the syn* This work was supported by the United States Public Health thesis of major components of the extracellular matrix. SpeService, National Institutes of Health Grants R01-AB5297 and T32- cifically, LR reduced the synthesis andsecretion of type I and AR-0751, and theDermatology Foundation. The costs of publication type 111 collagens and fibronectin, while the production of of this article were defrayed in part by the payment of page charges. hyaluronic acid and several metalloproteases was increased This article must therefore be hereby marked "advertisement" in (17). Thus, LR appears to have the intrinsic capability of accordance with 18 U.S.C. Section 1734 solelyto indicate this fact. $VisitingScientist from the CentreNational de la Recherche regulating matrix turnover andtissue remodeling. Scientifique, Prance. (I To whom correspondence should be addressed Thomas Jefferson University, Dept. of Dermatology, Bluemle Life Sciences Bldg., 233 South 10th St., Suite 450, Philadelphia, PA 19107.Tel.:215-9555785. Fax: 215-955-5788.

The abbreviations used are: kb, kilobase; TIMP, tissue inhibitor of metalloproteases; IL-1, interleukin-1; TNF-a, tumor necrosis factor-a; LR, leukoregulin; CAT, chloramphenicol acetyltransferase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

5644

Expression Collagenase Gene In thisstudy, we have examined in further detail the effects of LR on the expression of collagenase and TIMPby cultured human skin fibroblasts. The results revealed induction of collagenase gene expression wile there was little, if any, effect on TIMP gene expression in the same cultures. Transient transfection experiments suggested that the up-regulation of collagenase gene expression occurred, a t least in part, at the transcriptional level. MATERIALS ANDMETHODS

Cell Culture-Adult human skin fibroblast cultures, established from tissue specimens obtained from cosmetic surgery procedures, and newborn foreskin fibroblast cultures were utilized in passages 38. The cell cultures were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, 2 mM glutamine, 100 units/ml of penicillin, and 50 pg/ml of streptomycin. Mouse NIH3T3 cells were purchased from the American Type CultureCollection, Rockville, MD, and maintained in Dulbecco's modified Eagle's medium supplemented with 10% calf serum, 2 mM glutamine, and the antibiotics indicated above. Northern Analyses-Confluent human skin fibroblast cultures incubated with or without growth factors to be tested were subjected to isolation of total cellular RNA, as described previously (18).RNA, 10-20 pg/lane, was fractionated on0.8% agarose gels (19), transferred t o nylon filters (Zeta probe, Bio-Rad), and immobilized by heating a t 80 "C for 30 min under vacuum. The filters were then prehybridized and hybridized with cDNA probes labeled radioactive by nick translation with both [m3*P]dGTP and [cY-~*P]~CTP (20). After hybridizations at 42 "C, the filters were washed in solutions with decreasing ionic strength andincreasing temperature, andin case of homologous cDNAs the final stringency of the washes was 0.1 X SSC at 65 "C and mouse junB and while in case of a heterologous probe (rat GAPDH cDNAs) the final stringency was 0.1 X SSC at 55 "C. The ["'PlcDNA-mRNA hybrids were visualized by autoradiography, and the corresponding steady-state levels of mRNAs were quantitated by scanning densitometry using a He-Ne laser scanner at 633 nm (LKB Produkter, Bromma, Sweden). The following cDNAs were used for Northern hybridizations: for collagenase mRNA,a 2.0-kb human cDNA (21); for TIMP-1 mRNA, a 0.7-kb human cDNA, pUC9-F5 (kindly provided by Dr. D. Carmichael, Synergen, Boulder, CO); for c-jun mRNA, a human cDNA (22); for junB mRNA, a full-length mouse cDNA in pRSV expression vector (23); for glyceraldehyde-3phosphate dehydrogenase (GAPDH) mRNA, a 1.3-kb rat cDNA (pRGAPDH13) (24). To performrehybridizationswith another cDNA probe, the Northern filters were boiled in 0.1 X SSC, 0.5% sodium dodecyl sulfate twice for 5 min. The filters were then prehybridized and hybridized as indicated above. Transient Transfections of Cultured Cells-Human foreskin fibroblastsandNIH-3T3 cells in late logarithmic growth phase were plasmid DNA pCLCAT3, which transfectedwith 10 or 20pgof contains -3.8 kb of 5"flanking DNA of human collagenase gene linked to the CAT reporter gene (25) (kindly provided by Dr. Steven Frisch, Washington University School of Medicine, St. Louis, MO). The transfections were performed with the calcium phosphate/DNA co-precipitation method, followed by 1 (human skin fibroblasts) or 2 min (NIH-3T3 cells) glycerol shock (15%) (26). After the glycerol shock, the cells were placed in medium supplemented either with 0.5% calf serum (NIH-3T3 cells) or 1% heat-inactivated fetal calf serum (human skinfibroblasts).After3hincubation, the growth factors to be tested were added, and the incubations were continued for 40 h. After incubation with or without the growth factors, the cells were harvested and lysed by three cycles of freeze-thawing in 100 pl of 0.25 M Tris-HC1, pH 7.8. The protein concentrationof each extract was determinedwitha protein assay kit (Bio-Rad) and identical amounts (5-10 pg/assay) of protein from each cell extract were used for parallel determinations of CAT activity using ["C] chloramphenicol as substrate(27). The acetylated and nonacetylated forms of radioactive chloramphenicol were separated by thin-layer chromatography and visualized by autoradiography. The enzyme activity was quantified by cutting outpieces of thin-layer chromatography plates containingdifferent forms of [14C]chloramphenicolconverted to its acetylated forms, after correction for @-galactosidase activity in each extract as a control of transfection efficiency (20). T o explore the specificity of LR effect, parallel transfections were performed with pSVSCAT, a construct containingSV40 early region promoter and SV40 enhancer linked to the CAT gene (27).

5645

Growth Factors and Cytokines-LR with a PIof 5.1 and a molecular mass of -50 kDa was purified from human peripheral blood leukocytes as previously described (28). In brief, normal human lymphocytes were stimulated withphytohaemagglutinin (leukoagglutinin isomer; Sigma), and LR was purified using sequential diafiltration, anion exchange, isoelectric focusing, and high performance molecular sieving liquid chromatography (28). One unit of LR was defined as the amountof activity causing 50% increase in the plasma membrane permeability of K562 erythroleukemia cells, 1 X 10' cells/ml, during a2-hincubation (15). Human recombinant interleukin-l@ (IL-l@) was purchased from Boehringer Mannheim; dexamethasone, cycloheximide, and all-trans-retinoicacid were purchased from Sigma. RESULTS

LR Enhances Collagenase but Not TIMP-1 mRNA Steadystate Levels in Skin Fibroblasts-Human skin fibroblasts in culture have been shown to actively express collagenase gene, as determined at the mRNA level. To explore the effects of LR on collagenase gene expression, human skin fibroblast cultures maintained in the presence of 10% fetal calf serum were incubated with varying concentrations of LR, and the collagenase mRNA levels were determined at 24 h of incubation by hybridizations with a human fibroblast collagenase cDNA. In control cultures incubated without LR, low levels of mRNA with the characteristic size of 2.1 kb were detected (Fig. 1).Incubation of parallel cultureswith LR in concentrations varying from 0.1 to 2 units/ml resulted in a marked enhancement of the collagenase mRNA abundance (Fig. 1). Quantitation of the mRNA steady-state levels by scanning densitometry of the Northern blots revealed that incubation of cells with 0.1 unit/ml of LR resulted in -12-fold enhancement, and themRNA levels were further increased up to -17fold in the presence of 2 units/ml of LR, after correction of collagenase mRNA steady-state levels for GAPDH mRNA abundance in the same preparations. Re-hybridization of the same filter with the TIMP cDNA revealed that the levels of TIMP mRNA remained essentially unchanged (Fig. 1). The selective enhancement of collagenase gene expression, as detected at the mRNA level, was also shown to be timedependent (Fig. 2). Specifically, incubation of skin fibroblast cultures with1unit/ml of LR resulted in an -7-fold enhancement at 6 h and -20-fold enhancement at 24 h of incubation, and the maximal induction of -35-fold was noted at 48 h (Fig. 2). Re-hybridization of the same filter demonstratedthat the levels of GAPDH mRNA were only slightly altered at different time points (Fig. 2). To determine whetherthe up-regulation of collagenase gene expression by LR is dependent on the on-going protein synthesis, cycloheximide (10 pg/ml) was added to fibroblast cultures either alone or l h prior to the addition of LR (1unit/ LR (U/mll

TIMP

GAPOH

0 0.1 0.5

1

2

r I

FIG.1. Effect of different concentrations of LR oncollagenase andTIMP mRNA steady-state levels in human skin fibroblasts. Confluent cultures of human skin fibroblasts were incubated withvarying concentrations of LR (0.1-2 units/ml) for 24 h in medium containing 10% fetal calf serum. Total RNA (20 pg/lane) was analyzed by Northern blot hybridizations with cDNA probes for collagenase, TIMP, andGAPDH.

5646

Expression

Gene

time (h)

1

0

2

6

Collagenase 24 48

Collagenase

t

1

- + + + - -

LA OEX

A

Collagenase RR TIMP

- - + - + -

w . L ' I

4

FIG. 2. Time-dependent enhancement of collagenase mRNA levels by LR. Confluentfibroblast cultures were incubatedfor various times with LR (1 unit/ml) in medium containing 10% fetal calf serum. Total RNA (12 pgllane) extracteda t different time points was hybridized with cDNA probes for collagenase and GAPDH after electrophoretic size fractionation.

- + " + - - + +

LA CHX I

-1

Collagenase

-

I

'1I

FIG. 3. Effect of cycloheximide on LR-induced up-regulation of collagenase gene expression. Confluent fibroblastcultures were incubated in medium containing 1%fetal calf serum with (+) or without (-) LR (1 unit/ml) added 1 h afterthe addition of cycloheximide ( C H X ) (10 pglml). Total RNA (10 pg/lane) was extracted after a 24-h incubation and analyzed by Northern hybridizations with cDNA probes for collagenase and GAPDH.

ml) for a total of 24 h. As shown above, LR alone enhanced the collagenase mRNA levels by -19-fold (Fig. 3). Addition of cycloheximide alone had no effect on collagenase mRNA levels, but it completely abolished the up-regulatory effect elicited by LR (Fig. 3). Thus, the enhancementof collagenase mRNA levels in cultured skin fibroblasts by LR appears to require on-going protein synthesis. Demonstration That Dexamethasone and all-trans-Retirwic Acid Counteract LR-elicited Up-regulation of Collagenase Gene Expression-Previous studies have indicated that dexamethasone and all-trans-retinoic acid are able to inhibit collagenase gene expression in different cell types, including human skin fibroblasts and synovial cells in culture (29-31). In this study, we investigated the possibility that these two effector molecules would counteract the LR-elicited up-regulation of collagenase gene expression, as detected at themRNA level. Dexamethasone M) or all-trans-retinoic acid M) were added to skinfibroblast cultureseither alone or 1h prior to theaddition of LR (1unit/ml). Theaddition of dexamethasone or retinoic acid alone reduced the collagenase mRNA levels by -60-7095 when examined a t 24 h of incubation (Fig. 4). The addition of either one of these compounds partially counteracted the LR-elicited up-regulation of collagenase mRNA levels (Fig. 4A). Specifically, LR alone increased the mRNA steady-state abundance in this experiment by -20fold, while this effect in the presence of dexamethasone or retinoic acid was reduced to -3- and -7-fold, respectively, above the control cultures (Fig. 4B). We also examined the effects of dexamethasone and retinoic acid on TIMP mRNA levels in the same cells. Rehybridization of the filter with TIMP cDNA indicated that dexamethasone and all-trans-retinoic acid alone increased TIMP mRNA levels by 2.3- and 2.7-fold, respectively, after correction for GAPDH mRNA levels in the same samples (Fig. 4). However, this effect was not observed in cultures into which LR (1 unit/ml)was added 1h after theaddition of dexameth-

"

CTL

LR

LR+DEH L R + R R

DEH

RA

FIG. 4. Effect of dexamethasone and all-trans-retinoic acid on LR-induced up-regulation of collagenase gene expression. Dexamethasone (DEX)and retinoic acid ( R A ) , both M, were added to confluent fibroblastcultures maintained in medium containing 1% fetal calf serum. One hour later LR (1unit/ml) was added to the same cultures (+). Total RNA (15 pgllane) was extracted after a 24-h incubation and analyzed by Northern hybridizations with cDNA probes for collagenase, TIMP, and GAPDH. A, autoradiograms; B, densitometric analysis after correction for GAPDH mRNA levels. CTL,control cultures.

asone or retinoic acid (Fig. 4B). Evidence That Leukoregulin Modulates CollagenaseGene Expression at the Transcriptional Level-Transient cell transfections with acollagenase promoter/CAT reportedgene construct were performed to examine whether LR enhances collagenase mRNA steady-state levels through activation of the transcription at thepromoter level. NIH-3T3 cells were transfected with the promoter-CAT construct, and the cultures were then treatedwith LR (1unit/ml). Assay of CAT activity after 40 h of incubation indicated that LR enhanced the promoter activity by -5-fold on the average (Fig. 5). Similar enhancement of the promoter activity was also noted in transient transfections of human foreskin fibroblasts (data not shown). Thus,the enhancement of collagenase gene expression, as detected at the mRNA level, appears to be mediated, a t least in part, by enhancement of the transcriptional activity of the corresponding gene. Parallel transfections with pSV2CAT showed no effect by LR (not shown), suggesting that the up-regulation of collagenase promoter activity by LR is selective. The level of inhibition of collagenase gene expression as elicited by dexamethasone and retinoic acid was also examined in transienttransfections withthe collagenase promoterCAT construct. Dexamethasone alone inhibited the collagenase promoter activity by -70%, while retinoic acid had only a slight effect on the basal promoter activity (Fig. 5B).The enhancement of promoter activity by LR was abrogated by M dexamethasone. Specifically, the CAT activity in cultures incubated with dexamethasone and LR was essentially the same as incontrolcultures (Fig. 523). By contrast, M retinoic acid demonstrated only partial inhibition of LR-

Expression Collagenase Gene

5647

time (h) c- j u n

0

6

1

1m

24

48

0

1

2

6

24

48

1

I

jun-B

"

FIG. 6. Effects of LR and IL-1/3 on c-jun and junB mRNA levels. Confluent fibroblast cultures were incubated for various time periods with LR or IL-10 (1unit/ml each) in medium containing 10% fetal calf serum. Total RNA (12 pg/lane) extracted at thetime points indicated was hybridized with cDNA probes for c-jun andjunB protooncogenes.

CTL

LR

LR+DEH LR+RR

DEX

RR

FIG. 5. Enhancement of collagenase promoter activity by LR in transient cell transfections. Confluent cultures of NIH3T3 fibroblasts were transfected with the human collagenase promoter/CAT gene construct pCLCAT3, as indicated under "Materials and Methods." Three hours after the glycerol shock, the cells were exposed to dexamethasone (DEX) or retinoic acid ( R A ) , both M, in medium containing 0.5% calf serum. LR (1unit/ml) was added t o some cultures 1 h later. After 40 h of additional incubation, the cells were harvested and CAT activity was determined as described under "Materials and Methods." A, autoradiogram of the CAT assay depicting the separation of acetylated (AC) and unacetylated (C) forms of [14C]chloramphenicol by thin-layer chromatography; B, quantitation of CAT activity, expressed as fold stimulation, ascomThe values are the mean of five pared to control cultures (CTL). independent experiments, each performed in duplicate.

was noted at 6, 24, and 48 h of incubation (Fig. 6). Even though the reasons for the apparent decline of junB mRNA below the control levels are not clear, these data indicate a rapid and transientinduction ofjunB expression by LR. Thus, these differential effects of the two cytokines on c-jun and junB expression further attest to the fact that the cellular responses to LR are distinct from those elicited by IL-1p. DISCUSSION

The role of inflammatory cells in the pathogenesis of a variety of connective tissue diseases involving degradation of the extracellular matrixhas been well established (1, 2). Previous studies have concentrated on the role of IL-1 and TNF-a, cytokines produced primarily by activated macrophages. Both IL-1 and TNF-a have been shown to enhance collagenase gene expression in a number of different cell types (9-11,32). These affector molecules also enhance the expression of stromelysin, a metalloprotease capable of degrading a variety of extracellular matrix components and also necessary for maximal activation of collagenase in tissues (33). At the same time, these two cytokines have differential effects on the expression of TIMP, depending on the cell type (11, 12, elicited up-regulation of the collagenase promoteractivity (Fig. 5 B ) . It should be noted that these inhibitory effects of 34). Consequently, these macrophage-derived cytokines are dexamethasone and retinoic acid in general paralleled those capable of initiating degradation of the extracellular matrix through induction of collagenase activity. noted atthe mRNA level (Fig. 4), even though theLR In this study, we have examined a novel cytokine, leukoreenhancement at the mRNA level was quantitatively higher than noted at thepromoter level (Fig. 4B). It appears, there- gulin, which is derived from activated T-cells. Although the fore, that the down-regulatory effects of dexamethasone and initial studies on LR have concentrated on its anti-tumor properties, our recent study has indicated that LR is also retinoic acid also occur at the transcriptionallevel. capable of modulating the biosynthetic repertoire of normal Specific Induction of JunB Expression by kukoregulinPreviously, IL-1 has been shown to enhance collagenase gene skin fibroblasts in culture (17). In this study, we have examexpression ina variety of experimentalsystems (1). Our ined in detailthe effects of LR on the expression of collagenprevious studies suggested that theeffects of LR are notdue ase gene both in human and rodent fibroblasts. The results to the presence of contaminating IL-1 (17). This conclusion indicated that LR is a potent up-regulator of collagenase gene was based on the inability of neutralizing polyclonal antibod- expression, as determined at the mRNA level. In fact, incuies to IL-la and -p to inhibit the LR effects on collagen bation of fibroblasts for 24 h with 1 unit/ml of LR resulted biosynthesis (17). In this study, we examined the effects of in -20-fold elevation of the collagenase mRNA steady-state 48-h point of incubation as high as -35-fold LR furtherin relation to those elicited by IL-1. In particular, levels, and at the the effects of LR and IL-lpon the expression of members of induction was seen. The steady-state levels of TIMP mRNA the jun family of oncogens, c-jun and jun B, components of in the same cell cultures remained essentially unaltered. Thus, the AP-1 trans-actingregulatory protein complex, were deter- LR appears to selectively enhance the collagenase gene mined at themRNA level. Time course experiments indicated expression, and this cytokine is therefore capable of potenthat IL-1p (1 U/ml) markedly enhanced both c-jun andjunB tiating matrixdegradation mediated by collagenase activity. Transient transfections of fibroblasts with human collagenexpression (Fig. 6 ) . In contrast, LR (1 unit/ml) when incubated in parallel hadonly minimal effects on c-junexpression ase promoter-CAT reporter gene construct indicated that LR (Fig. 6). However, junB mRNA levels were markedly in- was capable of enhancing the collagenase promoter activity. creased at 1 h of incubation, but the mRNA levels returned It should be noted, however, that thehighest enhancement of was up to-7-fold. to thecontrol level a t 2 h, and even lower mRNA abundance the promoter activity in these experiments

5648

Collagenase Ge! n e Expression

Although this enhancement is clearly less than the -20-fold expression.* Thus, the differential effects of these cytokines elevation of the collagenase mRNA levels, these results sug- on the proto-oncogene and TIMP expression further attests gest that LR can increase the collagenase mRNA levels, at to thefact the LR is distinct from IL-1 and TNF-a. least in part, by activation of the transcription of the gene. In summary, LR is a novel T-cell-derived inflammatory The difference inthe enhancement of collagenase gene mediator, and one of its functions, clearly established in this expression, as determined at themRNA levelv e r s u transient study, is to enhance collagenase gene expression. It is, therecell transfections, could be explained by somewhat different fore, conceivable then thatLR may play an important role in cell culture conditions required for these two assay techniques. the cascade leading to tissuedestructionin inflammatory diseases. Nevertheless, it is possible that additional, perhapsposttranscriptional regulatory mechanisms may contribute to furAcknowledgments-We thank Debra Pawlicki for expert secretarial ther enhancement of the collagenase mRNA levels. In support help. Drs. D. Carmichael, E. Bauer, S. Frisch, M. Karin, and P. Fort of the latter possibility is the demonstration that phorbol kindly provided cDNAs for this study. esters, such as 12-O-tetradecanoylphorbol-13-acetate, can inREFERENCES crease the half-life of collagenase mRNA in rabbit synovial 1. Krane, S. M., Conca, W., Stephenson, M. L., Amento, E. P., and Goldring, cells (15). M. B. (1990)Ann. N. Y. Acad. Sci. 680,340-354 Previously, the modulation of collagenase gene expression 2. Woessner, J. F., Jr. (1991)FASEB J. 6,2145-2154 E., and de Crombrugghe, B. (1990)Annu. Reu. Bioehem. 69,837by IL-1, TNF-a, and 12-0-tetradecanoylphorbol-13-acetate 3. Vuorio, R73 has been shown to involve induction of the transcription 4. StA&n, G. P., Bauer, E.A., Jeffrey, J. J., and Eisen, A. Z. (1977) Biochemistry 16,1607-1615 factor AP-1 (8-10). Specifically,the AP-1 complex consisting 5. Brinckerhoff, C. E., Pluicinska, I. M., Sheldon, L. A., and O’Connor, G. T. (1986)Bioehemistrv 26.6378-6384 of subunit polypeptides of the junand fos gene families, has . ase,’H., Brinckerhoff,’C. E., Vater, C. A., and Harris, E. D., Jr. (1983) been shown to bind to the 12-0-tetradecanoylphorbol-13- 6. Nywchem. J. 214,281-288 acetate-responsive cis-element in thecollagenase promoter (8, 7. Harris, E. D., Jr., Welgus, H. G., and Krane, S. M. (1989)Collagen Relut. Res. 6. 493-610 -- - - 35;for review, see Ref. 1).Similarly, the effect of dexameth- 8. An-el; P-,,-Imagawa, M., Chiu, R., Stein, B., Imbra, R. J., Rahmsdorf, H. J., fonat;C., Herrlich, P., and Karin, M. (1987)Cell 49,729-739 asone on the collagenase gene expression has been shown to 9. Brenner. D. A.. O’Hara. M.. Aneel. P.. Choikier. M.. and Karin. M. (1989) . involve binding of the steroid receptor-ligand complex to the Nature 337;661-663 “ ’ W., Kaplan, P. B., and Krane, S. M. (1989)J. Clin. Inuest. 83, AP-1 protein complex, thus preventing its attachment to the 10. Conca, 1752-1 - .-- -757 .- . responsive cis-element (29-31). Thus, AP-1 appears toplay a 11. Murphy, G., Reynolds, J. J., and Werb, Z.(1985) Biol. J. Chem. 260,3079gnu2 role in the modulation of collagenase gene expression by a 12. Ma%~ul, K. L., Chartrain, N., Lark, M., Tocci, M. J., and Hutchinson,N. variety of effector molecules. In this study, we demonstrated I. (1990) BJ. ~ chem. L 266,17238-17245 13. Ransom, J. H., Evans, C. H., McCabe, R. P., Pomato, N., Heinbaugh, J. that LR transiently induces expression of junB, a subunit A., Chin, M., and Hanna, M. G., Jr. (1985)Cancer Res. 46,851-862 component of the AP-1 complex. Also, our results demon- 14. Barnett, S. C., and Evans, C. H. (1986)Cancer Res. 46,2686-2692 C. H., Heinbaugh, J. A,, and Ransom, J. H. (1987)Lymphokine Res. strated thatdexamethasone was capable of counteracting the 15. Evans, R~ ,-377-997 . . -- . LR-induced activation of collagenase gene expression, and 16. Evans, C. H.,(1987)in Leukolysins and Cancer (Ransom,J. H., and Ortaldo, J. R., eds) pp. 3-23,Humana Press, NJ this effect was noted both at themRNA and at thepromoter 17. Mauviel, A., W n i , F., Hartmann, D. J., Pujol, J.-P., and Evans, C. H. (1991) Cell J. Biol. 113,1455-1462 levels. In addition, retinoic acid was able to partially block 18. Chirwin, J. M., Pnybyla, A. E., MacDonald, R. J., and Rutter, W. J. the LR-induced enhancement of collagenase gene expression. (1979)Biochemistry 18,5294-5299 19. Thomas, P. S. (1980)Proc. Natl. Acad. Sci. U.S. A. 77,5201-5205 Previously, the inhibition of stromelysin-1 gene expression 20. Sambrook, J., Fritsch, E. F., and Maniatis,T. (1989)in Moleculur Cloning; by retinoic acid has also been shown to involve the AP-1 A Laboratory Manual, Second Ed, Cold Spring HarborLaboratory, Cold S ring Harbor, NY element (36). Collectively, these observations suggest that 21. Godberg, G. I., Wilhelm, S. M., Kronberger, A., Bauer, E. A., Grant, G. A., J. Chem. 261,6600-6605 and Eisen, A. Z.(1986) Biol. activation of collagenase gene expression by LR may involve 22. AnFl, P., Allegretto, E. A., Okino, S. T., Hattori, K., Boyle, W. J., Hunter, the AP-1 regulatory element. ., and Karin, M. (1988)Nature 332,166-171 Our previous studies have suggested that LR is clearly 23. Chiu, R., Angel, P., and Karin, M. (1989)Cell 69,979-986 P., Marty, L., Piechaczyk, M., E! Sabrouty, S., Danz, C., Jeanteur, P., distinct from IL-1 and TNF-a (17). This conclusion was based 24. Fort, and Blanchard,J. M. (1985)N~lclelcAcrds Res. 13,1431-1442 S. M., Reich, R., Collier, I. E., Genrich, L. T., Martin, G., and on the inability of neutralizing polyclonal antibodies against 25. Frisch, Goldber G I (1990)Oncogene 6,75-83 IL-1 or TNF-a to block the LR effects on the synthesis of 26. Graham, lf and’Van der Eb, A. (1973)Virobgy 62,456-457 C. M., Moffat, L. F., and Howard, B. H. (1982)Mol. Cell. Bwl. 2, collagen by skin fibroblasts. In contrast to IL-1 and TNF-a, 27. Gorman, 1044-1051 which up-regulate TIMP expression in human skin fibroblasts 28. Evans, C. H., Wilson, A. C., and Gelleri, B. A. (1989)Anal. Bioehem. 177, 74117~1 (11, 12), we found that LR has no effect on TIMP mRNA 29. Jonat C. Rahmsdorf H. J. Park, K.-K., Cato, A. C. B., Gebel, S., Ponta, H. ’and Herrlich P. (1996)Cell 62, 1189-1204 levels in this cell type. In this study, we also compared the H. F. dhambard J.-C., Sun, Y.L., Smeal, T., Schmidt, T. J., effects of LR and IL-1/3 on the expression profile of c-jun and 30. Yang”Yen Drouin,’J., and Karin, M. i1990)Cell 62, 1205-1215 R., Ran arajan P , Kliewer, S., Ransone, L. J., Bolado, J., Yang, junB. As indicated under “Results,” the effects of LR and IL- 31. Schule, N., Verma, I. h.,and Eians,R. M. (1990)Cell 62, 1217-1226 18were clearly distinct. Specifically,LR transiently enhanced 32. Mitchell, T.I., Coon, C. I., and Brinckerhoff, C. E. (1991) Clin. J. Inuest. 87,1177-1185 the junBexpression but had only a relatively small effect on 33. Brinckerhoff C.E., Susuki, K., Mitchell, T. I., Oram, F., Coon, C. I., c-jun expression. In contrast, IL-lB strongly enhanced the Palmiter, R. D. and Na me, H. (1990) Bioj. J. Gem. 266,22262-22269 34. Chua, C. C., and khua, B.%. L. (1990)Connectwe %sue Res. 26,161-170 expression of both c-jun and junB. Both cytokines appeared 35. An el, P., Baumann, I., Stein, B., Delius, H., Rahmsdorf, H. J.,and herrlich, P. (1987)Mol. Cell. Biol. 7 , 2256-2266 to have no effect on the c-fos expression in these cells (results R. C., Mader, S., Na al, S., Leid, M., Rochette-Egly, C., and not shown). We have also previously demonstrated that TNF- 36. Nicholson, Chambon, P. (1990)EMBO J.%,4443-4454 a transiently enhances the expression of c-jun in skin fibroV.-M. Kahiiri, unpublished observations. blasts while there is minimal, if any, effect on the junB I

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