Epidermal Growth Factor Stimulation of Stromelysin ... - NCBI - NIH

MOLECULAR AND CELLULAR BIOLOGY, Aug. 1990, p. 4284-4293

Vol. 10, No. 8

0270-7306/90/084284-10$02.00/0 Copyright C 1990, American Society for Microbiology

Epidermal Growth Factor Stimulation of Stromelysin mRNA in Rat Fibroblasts Requires Induction of Proto-Oncogenes c-fos and c-jun and Activation of Protein Kinase C SUSAN E. McDONNELL, LAWRENCE D. KERR,

AND

LYNN M. MATRISIAN*

Department of Cell Biology, Vanderbilt University, Nashville, Tennessee 37232 Received 20 December 1989/Accepted 14 May 1990

Stromelysin (transin) is a secreted metalloprotease that is transcriptionally induced by a variety of growth factors and oncogenes. We examined the necessity of specific secondary (protein kinase C) and tertiary (c-fos and c-jun protein products) messengers in the transactivation of stromelysin gene expression by epidermal growth factor (EGF). Rat-i fibroblasts exposed to antisense c-fos DNA or RNA demonstrated that c-fos expression was necessary for complete EGF induction of stromelysin expression. Similar results demonstrating the necessity of c-jun protein in the EGF induction of stromelysin were obtained. We also demonstrated that protein kinase C activation is required for the EGF induction of stromelysin, since phorbol ester desensitization of C kinase proteins abolished the ability of EGF to induce stromelysin mRNA, protein, and promoter activity. In reconstitution experiments, neither c-fos, c-jun, nor C kinase activation alone induced significant stromelysin expression. Overexpression of c-fos and c-jun was able to induce stromelysin to a level similar to that of the growth factor, and stimulation of protein kinase C activity augmented this induction. The data suggest that the EGF induction of stromelysin in rat fibroblasts procedes through a pathway involving c-fos, c-jun, and protein kinase C. The profound biological effects of growth factors and cellular phenotype is likely to be ultimately due to induced changes in the pattern of gene expression. It is envisioned that a cascade of secondary and tertiary messengers induced and/or modified by these agents specifies a pathway that results in a distinctive pattern of gene expression. We have attempted to elucidate the mechanism by which a specific growth factor, EGF, modulates the expression of a specific gene, the stromelysin (transin) gene. Rat stromelysin (transin) belongs to a multigene family of metal-dependent proteinases known as matrix metalloproteinases. This enzyme has been referred to as transin in previous publications from this laboratory but will subsequently be called rat stromelysin and has been designated MMP-3 (H. Birkedal-Hensen, ed., Proceedings of the Matrix Metalloproteinases Conference, in press). The MMPs can be roughly grouped into three subclasses: the interstitial collagenases, the type IV collagenases (gelatinases), and members of the stromelysin or transin subclass. They have been implicated as key rate-limiting enzymes in extracellular matrix degradation and are believed to be involved in the development and progression of human cancers. Type IV collagenase (gelatinase) is frequently elevated in human and mouse tumors, and the levels of the enzyme correlate with the metastatic potential of a series of mouse melanoma cells (15). We have previously shown that stromelysin expression also correlates with tumor progression. Stromelysin mRNA is frequently detected in mouse skin carcinomas but rarely in papillomas (28) and is particularly high in tumors with high metastasic potential (37). Matrix metalloproteinases also play potential roles in wound repair and such nonpathological events as embryogenesis, angiogenesis, and organ morphogenesis (for reviews, see references 6 and 27 and Birkedal-Hensen, in press). Rat stromelysin was originally cloned as a cellular gene

differentially expressed in polyomavirus-transformed cells but not in the parental FR 3T3 rat fibroblasts (29). Subsequent studies have shown that stromelysin is both positively and negatively regulated at the transcriptional level by a variety of biologically active molecules including growth factors, oncogenes, cytokines, and steroids (13, 24, 29). An understanding of the mechanisms by which these agents regulate stromelysin gene expression may aid in the understanding of processes such as metastasis and matrix remodeling. Initial studies showed that rat stromelysin mRNA is induced by the growth factor EGF and that this increase in cytoplasmic mRNA is due to an increase in the rate of transcriptional initiation (29). EGF induction of rat stromelysin could be blocked by the action of protein synthesis inhibitors, which suggests that de novo protein synthesis is required for the response. EGF is known to effect a rapid increase in the expression of a set of immediate-early response genes including c-myc, c-fos and c-jun (12, 33, 38). These inductions occur within 1 h of EGF treatment and are not dependent on de novo protein synthesis. Recent evidence has demonstrated that the c-fos and c-jun proteins (Fos and Jun) function as transcriptional activators for genes that contain a specific DNA sequence in their promoter referred to as a 12-O-tetradecanoylphorbol-13-acetate (TPA)-responsive element (TRE) or activator protein-1 (AP1)-binding site (for a review, see reference 10). Both Jun homodimers and the Jun-Fos heterodimer complex have been found to possess DNA-binding properties, though the Jun-Fos heterodimer has a much greater affinity for the AP-1-binding site and is a more potent transcriptional activator than the Jun homodimers (18, 34, 39). Interestingly, an AP-1-binding site has been identified in the rat stromelysin promoter at the -71-base-pair position (30). These observations suggest the possibility that c-fos and/or c-jun may be "third messengers" in the EGF induction of stromelysin gene transcription. Relatively little is known about the signal

oncogenes on

*

Corresponding author. 4284

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transduction mechanisms by which EGF elicits its biological action. The interaction of EGF and its receptor at the cell surface activates the tyrosine kinase activity of the receptor. This results in a reported increase in inositol-1,4,5-trisphosphate (1P3) and intracellular Ca2" levels in several cell types (46 and references within), possibly mediated by tyrosine phosphorylation of phospholipase C (46). EGF has also been reported to induce protein kinase C activity in mouse keratinocytes (32) and in PC12 pheochromocytoma cells (20) and induce diacylglycerol in Rat-I cells (B. E. Magun, personal communication), although it is not clear that protein kinase C activation is a general mechanism for EGF signal transduction. The recent cloning and characterization of new family members of protein kinase C-related genes (35) has allowed speculation that individual isoforms may be capable of transducing growth-factor-specific signals. We investigated the involvement of the proto-oncogenes c-fos and c-jun and the activation of protein kinase C in the EGF induction of stromelysin in rat fibroblasts. By using antisense RNA and DNA technologies and pharmacological approaches, we demonstrated the necessity of c-fos, c-jun and the activation of protein kinase C in the EGF stimulation of stromelysin mRNA in rat fibroblasts. We were also able to mimic the EGF effect on stromelysin expression by using c-fos and c-jun expression vectors. A model for intracellular signals used by EGF in inducing stromelysin gene expression is presented.

MATERIALS AND METHODS Cell culture and materials. Rat-1, Rat-2, RatjunS.17, and RatjunFBR cells were grown in Dulbecco modified Eagle medium (DMEM) containing 10% calf serum (Colorado Serum Company) and gentamicin. RatASfos.16 cells were grown in DMEM without phenol red and supplemented with 10% steroid-stripped calf serum (treated with activated charcoal [Sigma Chemical Co.] at 55°C for 30 min). All cells were maintained at 37°C in a humidified 95% air-5% CO2 environment. EGF (receptor grade) was purchased from Amgen Biologicals; TPA and 1,2-dioctanoyl-ras-glycerol (DAG) were purchased from Sigma and dissolved in dimethyl sulfoxide (DMSO) (final concentration of DMSO in cultures was 0.1%). Northern blot analysis. For Northern (RNA) blot analysis, total cytoplasmic RNA was isolated and fractionated by electrophoresis on a 1.5% agarose gel containing 2.2 M formaldehyde, as previously described (29). Poly(A)+ RNA was isolated as described previously (43). The RNA was transferred to nitrocellulose membranes and dried under vacuum at 80°C. Prehybridization and hybridization were performed in the presence of either 50% formamide (in the case of stromelysin and cyclophilin) or 40% formamide (in the case of c-fos and c-jun). Probes were sucrose gradientpurified inserts of the following: stromelysin (1.7-kilobase EcoRI fragment from pTR1 [29]), c-fos (1.1-kilobase PstI fragment from pBK28, generously provided by I. Verma [26]), c-jun (1-kilobase EcoRI fragment from a c-jun cDNA, generously provided by J. Minna [42]), and cyclophilin (IB15) (11). The cDNAs were radiolabeled with [32P]dCTP by using random primer synthesis (Boehringer Mannheim Biochemicals) to a specific activity greater than 5 x 108 cpm/,lg. Plasmids. pJun(cDNA sense) was a gift from J. Schutte (42). The EcoRI fragment was subcloned in the sense orientation into the EcoRI site of pKCR3 (28), so that the full-length c-jun protein is expressed under control of the

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simian virus 40 (SV40) promoter to create pSVjunS. The EcoRI fragment of c-jun was subcloned in the antisense orientation and in the same expression vector to create pSVhjunAS. pBK28 expresses c-fos under the control of a the viral long terminal repeat and was a gift from I. Verma. Transfections and CAT assays. Cells were plated at a density of 2.5 x 105 cells per 100-mm (diameter) dish 1 day prior to transfection. Calcium phosphate-DNA precipitates were prepared by the method of Graham and Van der Eb (17). For transient transfections, 10 ,ug of p750TRCAT (23), 2 ,ug of pCH110 (containing the SV40 promoter-linked Pgalactosidase sequences [40]), and 10 ,ug of either pBK28 or pSVjun were added, as indicated previously (17). Four hours after the addition of precipitates, cells were glycerol shocked for 3 min (10% glycerol in HEPES [N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid]-buffered saline) and refed with DMEM containing 10% calf serum. After 24 h, cells were serum starved for 16 h and stimulated with EGF (20 ng/ml) or TPA (100 ng/ml), as indicated previously (17). Cell lysates were prepared 8 h after the addition of growth factors and analyzed for chloramphenicol acetyltransferase (CAT) activity, as previously described (17). Cell lysates were also assayed for P-galactosidase activity, as described previously (19, 23), to control for slight variations in transfection efficiency. The percent ['4C]chloramphenicol converted to acetylated forms was determined by cutting out the appropriate areas of the thin-layer chromatography sheets and assaying for 14C radioactivity by scintillation counting. Results were normalized for P-galactosidase activity and expressed as percent CAT converted to the acetylated form. Stable transfections. Rat-1 fibroblasts were transfected with the plasmid pHfosAS (a gift from J. Holt [22]) or pSVjunS and pSVneo at a ratio of 1:5. Following calcium phosphate-mediated transfection, the cells were selected for neomycin resistance in medium containing G418 (400 p.g/ml). Individual clones were isolated, amplified, and characterized for the incorporation and expression of the construct by Southern and Northern blot analyses. One of these clones, RatjunS.17, was subsequently infected with a v-fos-containing virus, FBR (45). RatjunS.17 cells were plated at 1 x 105 cells per ml and allowed to attach overnight. The following day, the supernatant was removed and 0.5 ml of viral stock with 10 ,ug of Polybrene per ml was added. The plates were rocked every 15 min for 1 h, the supernatant was removed, and the cells were rinsed and refed with DMEM supplemented with 10% calf serum. After 10 days, plates were checked for focus formation and cells with transformed morphologies were selected and amplified. Oligonucleotide competition experiments. Oligonucleotides specific to the 5' end of c-fos and c-jun were synthesized in both the sense (FOS and JUN) and antisense (SOF and NUJ) orientation. The sequences of these oligonucleotides are as follows: FOS: 5'-ATGATGTTCTCGGGCTTG-3' SOF: 5'-GAAGCCCGAGAACATCAT-3' JUN: 5'-ATGACTGCAAAGATGGAA-3' NUJ: 5'-TTCCATCTTTGCAGTCAT-3' Following synthesis, the oligonucleotides were purified by urea-polyacrylamide gel electrophoresis (PAGE), eluted from the gel, and further purified by G-25 chromatography (Pharmacia, Inc.) to remove trace contaminants. p80 MARCKS phosphorylation assay. Protein kinase C enzyme activity was assayed by phosphorylation of the 80-kilodalton (kDa) myristylated alanine-rich C kinase sub-

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strate (MARCKS). First, Rat-1 fibroblasts were allowed to reach confluence in 60-mm (diameter) petri dishes before being serum deprived for 24 h in DMEM. Certain dishes were treated with 400 nM TPA during the 24 h of serum deprivation. All plates were changed to phosphate-free medium for 2 h, labeled with 100 ,uCi of [32P]orthophosphate per ml for 3 h, and stimulated with EGF (50 ng/ml) for 5 min. The cells were then placed on ice and rinsed three times with 0.15 M NaCl-20 mM Tris hydrochloride, pH 7.5. The cells were extracted with 5% trichloroacetic acid two times for 10 min each, rinsed three times with NaCl-Tris buffer as described above, and dissolved in 200 RI of 0.1% Nonidet P-40 in reticulocyte buffering saline (NaCl [10 mM], Tris hydrochloride [pH 7.5] [10 mM], MgCl2 [0.15 mM]). Cell lysates were centrifuged at 16,000 x g for 10 min, and the supernatant was transferred to a new tube. Equal trichloroacetic acid-precipitable counts (5.4 x 106) were separated by isoelectric focusing (pH 2.5 to 5) in the first dimension followed by sodium dodecyl sulfate (SDS)-PAGE (10% polyacrylamide) in the second dimension. Protein kinase C enzyme activity was also determined by using the RPN 77 protein kinase C enzyme assay system (Amersham Corp.). This system is based upon the proteinkinase-C-catalyzed transfer of the T-phosphate group of adenosine-5'-triphosphate to a peptide which is specific for protein kinase C. Briefly, cells were sonicated for 15 s in homogenization isotonic buffer [20 mM Tris hydrochloride (pH 7.4), 10 mM ethylene glycol-bis(,-aminoethyl ether)N,N,N',N'-tetraacetic acid (EGTA), 2 mM EDTA, 300 mM sucrose, 10 ,uM phenylmethylsulfonyl fluoride, 5 ,ug of leupeptin per ml, 1% Triton X-100]. The cell lysates were then centrifuged at 100,000 x g for 1 h at 4°C, and the supernatants analyzed for protein kinase C activity by using the Amersham kit at 25°C for 30 min. Phosphorylated peptide was separated on binding paper, and the extent of phosphorylation was detected by scintillation counting. Western blot analysis. For Western blot (immunoblot) analysis, nuclear proteins were isolated according to Bos et al. (4), except that the nuclei were not sheared after lysis. Total protein was determined by a protein assay method (Bio-Rad Laboratories), and the lanes were loaded for equal protein. Proteins were separated by SDS-PAGE on 10% polyacrylamide gels and transferred to nitrocellulose by using a Hoefer Transphor unit. Filters were placed in blocking buffer (5% nonfat dried milk [Carnation] plus 0.2% Nonidet P-40 in Tris-buffered saline solution) for 1 h at 37°C and then incubated in blocking buffer overnight at 4°C with affinity-purified polyclonal antibodies from Oncogene Science as follows: c-fos (Ab-2) and c-jun-AP-1 (Ab-2) at 10 jxg/ml. Filters were washed three times with blocking buffer and then incubated for 1 h with 125I-protein A at a concentration of 1 ,uCi/ml. Filters were then washed twice with blocking buffer and once with Tris-buffered saline, air dried, and exposed to X-Omat film (Eastman Kodak Co.) overnight at -70°C. Immunoprecipitation. Rat-2 cells were labeled with 150 ,uCi of [35S]methionine after preincubation in methioninefree growth medium at 37°C for 3 h. EGF (20 ng/ml) was then added, and the medium was collected 8 h later, clarified by centrifugation, and processed for immunoprecipitation, as previously described, by using 3 x 105 cpm of each sample and a rabbit polyclonal antibody to a synthetic peptide based on the deduced rat stromelysin sequence (28). Following clarification of antibody-bound protein A-Sepharose, samples were denatured in Laemmli sample buffer (25) and subjected to electrophoresis in an 8% polyacrylamide gel.

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FIG. 1. The induction of c-fos, c-jun, and stromelysin mRNAs in Rat-1 fibroblasts after treatment with EGF. Density-arrested cultures of Rat-1 fibroblasts were serum deprived for 16 h prior to stimulation with 5 ng of EGF per ml. At the times indicated, poly(A)+ mRNA was collected, fractionated, and analyzed by Northern blot analysis, as described in Materials and Methods. Appropriately exposed autoradiograms were scanned with an Ultrascan XL densitometer (LKB Instruments, Inc.), and results are plotted as percent maximal induction.

Fractionated samples were prepared for fluorography and dried onto filter paper for autoradiography.

RESULTS Induction of c-fos, c-jun, and stromelysin RNA in Rat-i cells after stimulation with EGF. To examine the induction of c-fos, c-jun, and stromelysin mRNAs in rat fibroblasts, density-arrested cultures of Rat-1 cells were serum deprived for 24 h prior to stimulation with EGF. Poly(A)+ mRNA was collected at the indicated times and analyzed by Northern blot analysis (Fig. 1). EGF stimulated c-fos mRNA in these cells, with maximal induction observed at 30 min. c-fos mRNA levels rapidly decreased and were reduced to approximately 9% of maximal induction by 1 h and were undetectable at 2 h. c-jun mRNA was also maximal at 30 min, but its induction was more prolonged; at 2 h, 39% of the maximal signal remained. Stromelysin mRNA was induced over a much longer time period. It was first detected at 2 h and reached maximal induction at 8 h. These results demonstrated quite clearly that c-fos and c-jun mRNAs precede the induction of stromelysin mRNA after EGF stimulation. Necessity of Fos and Jun. To determine if the presence of Fos and Jun is a prerequisite for EGF induction of stromelysin, we used several different antisense methods to block EGF induction of Fos or Jun in order to determine their effect on subsequent stromelysin expression. By using an antisense DNA approach, oligonucleotides corresponding to small portions of the c-fos and c-jun sequences were synthesized in both the sense (FOS and JUN) and antisense (SOF and NUJ) orientations. The ability of these oligonucleotides to reduce EGF induction of Jun and Fos proteins was confirmed by Western blot analysis (Fig. 2). A comparison of EGF-induced c-jun protein in both the EGF control (untreated oligonucleotide) and control sense oligonucleotide cells revealed no significant effect from the sense oligonucleotide treatment (data not shown and Fig. 2A). However, in the

EGF STIMULATION OF STROMELYSIN mRNA

VOL. 10, 1990

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FIG. 2. Effect of antisense oligonucleotides on EGF-induced Fos and Jun proteins. Rat-2 fibroblasts were grown to confluency and serum deprived for 16 h. The appropriate oligonucleotide (10 ,ug) in 3 ml of DMEM was added to the cells, and the plates were rocked for 3 h at 37°C on a platform. EGF (50 ng/ml) was then added for 50 min. Nuclear proteins were isolated and separated on 8% polyacrylamide gels, transferred to nitrocellulose, and reacted with affinitypurified polyclonal antibodies (10 F.g/ml) to Jun (A) or Fos (B). Blots were reacted with 125I-protein A (10 ,uCi) and exposed to X-Omat film (Kodak) at -70°C. Con, Control. presence of the antisense oligonucleotide, EGF induction of Jun was reduced to control levels (Fig. 2A). Similar results were obtained with the c-fos antisense oligonucleotides. EGF stimulated Fos in the presence of the control sense oligonucleotide, but Fos levels were reduced to 36% of control levels in the presence of antisense c-fos oligonucleotides (Fig. 2B). In addition, a protein of approximately 41 kDa was also stimulated by EGF but was not significantly

reduced by the antisense oligonucleotide. It is possible that this protein represents a member of the class of c-fos-related antigens (FRAs). Stromelysin transcription was measured following treatment with antisense oligonucleotides by transient transfection of p750TRCAT, a plasmid containing 750 bp of the stromelysin promoter linked to bacterial CAT sequences, and CAT activity was assayed. Rat-2 fibroblasts were transfected with 10 ,ug of p750TRCAT, allowed to recover after glycerol shock, and then serum deprived for 24 h. Cells were then exposed to 0.5 ,uM of the appropriate oligonucleotide for 3 h prior to the addition of EGF. Cells were collected 8 h later, and cell lysates were processed for CAT activity. FOS oligomers in the sense orientation had no effect on EGFinduced, stromelysin-promoted CAT activity (Fig. 3). However, the antisense oligomer (SOF) inhibited EGF induction to 33% of control EGF levels. Similar results were obtained with c-jun oligomers. The sense oligomers (JUN) had no significant effect upon EGF-induced activity, whereas the antisense c-jun (NUJ) reduced CAT activity to 50o of control EGF levels (Fig. 3). None of the oligonucleotides had reproducible effects on transcription of the SV40-13galactosidase control construct. Both Jun and Fos proteins are therefore involved in the EGF induction of stromelysin gene expression. To further confirm the role of Fos in EGF induction of stromelysin gene expression, we also utilized a cell line stably transfected with an inducible expression vector producing antisense c-fos RNA in rat fibroblasts and measured stromelysin RNA levels by Northern blot analysis. The construct pHfosAS, containing a fragment of the human c-fos

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FIG. 3. Effect of antisense c-fos and c-jun oligonucleotides on EGF-induced, stromelysin-promoted CAT activity. Rat-2 fibroblasts were transfected with 10 ,ug of p750TRCAT, allowed 24 h to recover after glycerol shock, and then serum deprived for 16 h. The appropriate oligonucleotide (10 pg) in 3 ml of DMEM was added to the cells, and the plates were rocked gently at 37°C for 3 h prior to the addition of 20 ng of EGF per ml. Cells were collected 8 h later, and cell lysates were processed for CAT activity. Results were normalized for slight differences in transfection efficiency by assaying for 3-galactosidase activity. Data represent fold induction over that of the EGF control of five separate transfection experiments.

gene in the antisense orientation under the control of the steroid-inducible mouse mammary tumor virus promoter, was transfected into Rat-1 cells. After transfection and selection in medium containing G418, individual clones were isolated, amplified, and characterized for the incorporation and expression of the construct (data not shown). One of these clones (RatASfos.16) was grown to confluency, serum deprived for 24 h, and treated with dexamethasone for 30 min prior to the addition of EGF. Total cytoplasmic RNA was isolated 8 h later and analyzed for stromelysin RNA by Northern analysis. These cells were stimulated by EGF to produce stromelysin mRNA (Fig. 4). In the presence of dexamethasone, which induces antisense c-fos RNA from this construct and inhibits c-fos protein production (22, 23), EGF induction of stromelysin was inhibited by at least 50%. Similar levels of dexamethasone had no inhibitory effect on EGF induction of stromelysin RNA in parental cells (Fig. 4), although higher levels of dexamethasone did inhibit stromelysin RNA expression in these cells, with a half-maximal effect at 10 nM (data not shown). We were able to confirm the effects of antisense c-jun on stromelysin gene expression by using a transient transfection assay with an expression vector containing human c-jun in the antisense orientation (pSVhjunAS). Although not all cells receive the antisense construct by using this approach, both the antisense-producing construct and the reporter gene are transfected into the same cell population and the effect of the antisense RNA on stromelysin expression can be measured. Rat-2 fibroblasts were cotransfected with 10 ,ug of p750TRCAT and 10 ,ug of pSVhjunAS. EGF-induced CAT activity was reduced to 50o of control values in the presence of the antisense c-jun (data not shown). These studies suggest that the c-jun product is involved in EGF induction of CAT activity and are in agreement with the results obtained in the oligonucleotide competition experiments described above. Both Fos and Jun induction were therefore necessary for EGF stimulation of stromelysin gene expression. Activation of protein kinase C by EGF. We have examined the ability of EGF to activate protein kinase C in our system

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- Sg 0 FIG. 4. Effect of antisense c-fos on EGF-induced stromelysin mRNA in Rat-1 fibroblasts. Rat-1 and RatASfos.16 cells were grown to confluency, serum deprived for 16 h, and treated with dexamethasone (1 nM) for 30 min prior to the addition of EGF (20 ng/ml). Total cytoplasmic RNA was isolated and subjected to Northern blot analysis by using a random primed rat stromelysin cDNA insert. Appropriately exposed autoradiograms were scanned with an Ultrascan XL densitometer (LKB), and results were expressed as a percentage of the EGF value. Inset, autoradiogram for RatASfos.16

cells.

by assaying the phosphorylation of the 80-kDa MARCKS (44) by two-dimensional gel electrophoresis. EGF stimulated an approximate fivefold increase in the amount of 32P associated with the MARCKS protein in Rat-2 fibroblasts (Fig. 5). Stimulation of protein kinase C activity by EGF has also been observed in other cell types (20, 32). Phosphorylation of the MARCKS protein by EGF is inhibited by prolonged prior treatment with phorbol ester (Fig. 5), thereby supporting the involvement of protein kinase C in the EGF response. In addition, EGF stimulation of Rat-2 cells results in an increase in protein kinase C activity, as measured by phosphorylation of a specific substrate peptide (Amersham). The degree of EGF stimulation varied from experiment to experiment but was generally two- to eightfold over control values (data not shown). Role of protein kinase C activation in EGF stimulation of stromelysin, c-fos, and c-jun mRNAs. In order to examine the role of protein kinase C activity in the EGF induction of stromelysin mRNA, Rat-2 cells were first pretreated with 400 nM TPA for 24 h. This treatment down regulates phorbol-ester-sensitive protein kinase C activity so that subsequent treatment with phorbol esters is no longer effective in stimulating protein kinase C activity (9). Down regulation of protein kinase C activity was confirmed in these cells by examining the phosphorylation of the 80-kDa MARCKS protein (44). TPA was no longer capable of stimulating MARCKS phosphorylation following prolonged TPA pretreatment (Fig. 6A). Prolonged TPA pretreatment had no significant effect on EGF receptor number or affinity or on autophosphorylation activity of the EGF receptor (data not shown). The prolonged treatment also did not reduce EGF-induced [3H]thymidine incorporation or growth in these cells, demonstrating no cytotoxic effect of the treatment (data not shown). Rat fibroblasts were treated with EGF with or without prior down regulation of protein kinase C activity and were assayed for stromelysin mRNA, protein, and transcriptional activity. Under the conditions of protein kinase C down regulation, EGF is no longer capable of inducing stromelysin RNA, as measured by Northern blot

MOL. CELL. BIOL.

analysis (Fig. 6B), or stromelysin protein, as measured by immunoprecipitation with an anti-stromelysin antibody (Fig. 6C). In addition, transcription from the rat stromelysin promoter was inhibited by down regulation of protein kinase C, as determined by transfection of p750TRCAT into rat fibroblasts and assaying for CAT activity (Fig. 6D). We also utilized several well-characterized pharmacological inhibitors to desensitize C kinase activity to further stimulation by growth factors. Palmitoylcarnitine and sphingosine inhibit protein kinase C activation by interaction with the lipid-binding domain (7, 14), quercitin recognizes the phorbol-ester-binding domain of the C kinase (5), and H-7 is thought to interfere with ATP binding within the catalytic subunit (21). All four inhibitors, at the optimal concentration to inhibit C kinase, are capable of repressing the EGF induction of stromelysin mRNA (data not shown). We also examined the role of protein kinase C activation on the EGF induction of c-fos and c-jun mRNA. Protein kinase C activity was down regulated, as described above, and cells were stimulated with EGF for 50 min, poly(A)+ RNA isolated, and analyzed by Northern blotting. EGF stimulation of rat fibroblasts induced both c-fos and c-jun mRNAs (Fig. 1 and 7). Down regulation of protein kinase C activity did not decrease the EGF induction of either c-fos or c-jun mRNA (Fig. 7) and in fact may have a slight stimulatory effect on the induction of both c-fos and c-jun mRNAs. Sufficiency of Fos and Jun. In order to determine if induction of Fos and Jun is sufficient to induce stromelysin RNA, Rat-2 fibroblasts were transfected with p750TRCAT and expression vectors in which the human c-fos (pBK28) and c-jun (pSVjunS) cDNAs are driven by constitutive viral promoters. Following transfection and serum deprivation, cultures were treated with EGF or with the protein kinase C activator DAG for 8 h and then collected and assayed for CAT activity. DAG was utilized in place of TPA in these experiments because it is believed to activate a broader range of C kinase isoforms than TPA, although identical results were obtained when TPA was used at the same concentration. Stimulation with EGF causes a threefold induction of stromelysin-promoted CAT activity (Fig. 8). Neither c-jun expression nor activation of protein kinase C by treatment with DAG was sufficient for stromelysin induction (Fig. 8). The addition of c-fos by itself, however, gives a twofold induction of CAT activity, possibly due to a relatively high level of endogenous c-jun. When the two oncogenes were added together, they were able to cooperate to induce stromelysin-promoted CAT activity 4.2-fold. The addition of DAG to either Jun- or Fos-transfected cells resulted in a synergistic increase in stromelysin-promoted CAT activity. When the overexpression of Fos and Jun was supplemented by the activation of protein kinase C, we observed a sixfold increase in stromelysin levels, exceeding the level produced by stimulation with EGF or any other growth factor (23). To confirm the synergistic effect of the oncogenes and C kinase activity observed with transient transfection assays, Rat-1 fibroblasts were stably transfected with a construct bearing the human c-jun cDNA driven by the SV40 promoter (pSVjunS) and a construct expressing the gene for neomycin resistance (SVneo). Cells were selected in media containing G418, and individual clones were isolated and amplified. DNA and RNA were extracted and analyzed for c-jun integration and expression by Southern and Northern analyses, respectively (data not shown). One of these clones, RatjunS.17, expressed high levels of c-jun mRNA and was selected for further studies. These cells were then infected

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with the FBR virus (45) at a high multiplicity of infection, and populations of cells demonstrating transformed morphologies were further analyzed for stromelysin gene expression. RatjunS. 17 cells with and without FBR infection were grown to confluency, serum starved, and treated with EGF and TPA for 8 h. Total cytoplasmic mRNA was isolated and analyzed by Northern blot analysis for induction of stromelysin mRNA. Stromelysin transcripts were detectable in the RatjunS.17 cells following stimulation with both EGF and TPA (Fig. 9). This is in contrast to the parental Rat-1 cells, in which EGF, but not TPA, is capable of inducing stromelysin RNA (29). Infection of these cells with FBR results in an increase in the basal level of expression of stromelysin to levels observed following EGF or TPA stimulation of Jun-expressing cells. Further stimulation of these cells with EGF or TPA results in a marked stimulation in stromelysin levels. These results are in agreement with those observed with transient transfections (Fig. 8) and suggest that Fos and Jun cooperate to stimulate stromelysin

RNA to a level equivalent to that seen with EGF and that activation of protein kinase C further enhances this stimulation.

DISCUSSION We examined the molecular mechanisms by which a specific growth factor, EGF, induces the expression of a specific gene, the stromelysin (transin) gene. Altered expression of matrix-degrading metalloproteinases such as stromelysin may be involved in mediating the effects of growth factors and oncogenes, e.g., in promoting tumor invasion during carcinogenic progression or in affecting matrix remodeling during embryonic development. On the basis of results presented in this paper, we propose that there are at least three distinct components involved in EGF induction of stromelysin RNA in rat fibroblasts. EGF stimulation of rat fibroblasts results in the expression of the proto-oncogenes c-fos and c-jun (Fig. 1) and activation of

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FIG. 7. Effect of down regulation of protein kinase C activity on the EGF induction of c-jun (A) and c-fos (B) mRNA in rat fibroblasts. Rat fibroblasts were grown to confluency and serum starved for 24 h in the presence of 400 nM TPA or the carrier alone (dimethyl sulfoxide). The following day, cells were stimulated with EGF (20 ng/ml) and TPA (100 ng/ml) for 45 min. Cells were collected, and poly(A)+ mRNA was isolated. A total of 2 ,ug of poly(A)+ mRNA from control (CON), EGF-stimulated, and TPA-stimulated cells were fractionated as described in Materials and Methods. Following Northern blot analyses for c-fos and c-jun mRNAs, blots were stripped by immersion in water at 90°C for 1 min and reprobed with the probe IB15 encoding cyclophilin. Bar graphs (with bars corresponding to the lanes directly above) represent the relative c-fos and c-jun mRNA levels normalized to the IB15 levels and show the means of two or three separate experiments.

protein kinase C activity (Fig. 5). By using antisense DNA and RNA, we demonstrated that induction of both Fos and Jun was required for subsequent stromelysin expression (Fig. 3 and 4). This finding is consistent with our previous observations that de novo protein synthesis is required for EGF induction of stromelysin RNA (30). The antisense oligonucleotides significantly reduce but do not abolish the stromelysin response to EGF (Fig. 3). This may be explained by the low levels of Fos and Jun protein that are still detectable in the presence of the antisense oligonucleotide following 50 min of EGF stimulation (Fig. 2). It is also not clear that the oligonucleotides remain intact during the 8-h incubation required for CAT activity analysis, perhaps allowing a later increase in proto-oncogene protein levels. This p750TRCAT, serum deprived, TPA pretreated, and stimulated with EGF for 8 h, as described above. Lanes are as marked for panel B. Cell lysates were processed for CAT activity. Numerical data represent the average fold induction over the control value from three separate transfection experiments.

EGF STIMULATION OF STROMELYSIN mRNA

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could be particularly significant in the case of Jun, since the time course of c-jun mRNA induction is prolonged compared with that of c-fos (Fig. 1). The expression of either Fos or Jun alone is not sufficient to significantly induce stromelysin expression, though c-fos expression is capable of slightly inducing stromelysin-promoted CAT activity (Fig. 8). This may be due to cooperation with endogenous c-jun, since the constitutive level of c-jun mRNA is higher than that of c-fos in rat fibroblasts (Fig. 7 and data not shown). The combination of c-fos and c-jun expression closely mimics the effect of EGF (Fig. 8 and 9). The ability of antisense c-fos and c-jun to reduce EGFstimulated stromelysin expression and the ability to reconstitute the stimulatory effect of EGF by constitutive expression of Fos and Jun suggest that these proto-oncogenes are necessary and, together, are sufficient for stromelysin gene expression.

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further infected with the FBR virus (FBR/JUN). Cells were grown to confluency, serum starved overnight, and treated with EGF (20 ng/ml) and TPA (100 ng/ml) for 8 h. Total cytoplasmic RNA was isolated and analyzed by Northern blot analysis for induction of stromelysin mRNA. Following Northern blot analysis for stromelysin, the blot was stripped by immersion in water at 90°C for 1 min and reprobed with a cyclophilin probe. CON, Control.

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Inhibition of protein kinase C activity by a variety of methods blocked EGF stimulation of stromelysin expression, strongly suggesting that activation of C kinases was required for EGF stimulation of stromelysin RNA (Fig. 6). We noticed, however, that occasionally prolonged culturing of rat fibroblasts can result in a cell population that no longer requires protein kinase C activation for stromelysin induction. These cells appear to have a slightly higher basal level of protein kinase C activity. Although we have not fully characterized the increased protein kinase C activity in these cells, we speculate that it may be the result of the activation of a phorbol-ester-insensitive isotype of protein kinase C (36) or possibly that our usual dose of TPA is not sufficient to completely desensitize C kinase in these cells. This result would explain the ability of EGF to induce stromelysin expression in these cells following prolonged TPA pretreatment. The activation of protein kinase C is not required for the EGF induction of c-fos and c-jun mRNAs (Fig. 7). Assuming that Fos and Jun protein levels correspond to the mRNA levels, this suggests that C kinase is not required in the pathway regulating c-fos or c-jun protein levels in these cells. This was further supported by the observation that the addition of TPA to cells expressing high levels of c-fos and c-jun results in an additional increase in stromelysin RNA expression (Fig. 8 and 9). TPA stimulation of Rat-2 cells weakly induces mRNA levels for c-fos and c-jun (Fig. 7), and experiments in which levels of c-fos or c-jun are maximized by transfection with increasing amounts of plasmid DNA still show TPA-induced increases in stromelysin expression, even with the highest levels of c-fos and c-jun (data not shown). We therefore speculate that C kinase activation is not acting to increase stromelysin transcription by elevating levels of c-fos or c-jun mRNA. C kinase activation may alter the phosphorylation state of proteins involved in the transcription complex. It is possible that the substrate for C kinase activity may be the Fos or Jun protein itself. Fos is known to be phosphorylated by protein kinase C (3), although no effect of this phosphorylation on transcriptional activity has been described. Jun does not appear to be directly phosphorylated by protein kinase C (W. W. Boyle, personal communication), although there may be an alteration in the phosphorylation state of Jun through a proteinkinase-C-initiated cascade of phosphorylation or dephosphorylation. One model that would fit the data we have described is that a specific C-kinase-dependent phosphorylation state of either Jun or Fos is required for transcriptional activation of stromelysin RNA. One would therefore predict that a reduction in the levels of Jun or of Fos or an alteration in protein kinase C activity would negatively effect stromelysin transcription. We have depicted the pathways used by EGF to stimulate stromelysin gene expression to involve three distinct, essential components: c-jun protein, c-fos protein, and a protein-kinase-C-dependent step, though we cannot at this time define the relationship between the protein-kinase-C-dependent step and the proto-oncogenes. It is envisioned that the three necessary components of the signaling pathway converge at the AP-1 site in the rat stromelysin promoter. This sequence is required for EGF stimulation of stromelysin-promoted CAT activity in NIH 3T3 cells and Rat-2 fibroblasts, as determined by in vivo oligonucleotide competition experiments (23, and data not shown). The Fos-Jun protein complex has been shown by several laboratories to bind this sequence (for a review, see reference 10) and to activate transcription through this element in the human collagenase promoter (8). This se-

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quence has also been described as a TPA-responsive element and has been shown to be responsible for TPA induction of gene expression from several genes and from synthetic promoter constructs (1, 2). It is therefore very likely that the AP-1 site in the stromelysin promoter is the cis-acting DNA element responsible for EGF stimulation of stromelysin gene expression. The role of Fos as a third messenger has been previously described. Schonthal et al. (41) have demonstrated Fos involvement in ras and mos induction of collagenase gene expression. We have demonstrated that Fos is required for platelet-derived growth factor induction of stromelysin RNA in murine NIH 3T3 cells (23). However, in these NIH 3T3 cells, EGF induction of stromelysin RNA occurs through a c-fos-independent pathway (23). The reason for the cell type differences in the requirement for c-fos expression in EGF stimulation of stromelysin gene expression is unclear. However, preliminary experiments suggest that C kinase isoforms are different in the two cell types. We have been unable to detect protein kinase Cp1 transcripts in Rat-2 fibroblasts, though they are detectable in NIH 3T3 cells. It is possible that the presence of protein kinase Cp1 in NIH 3T3 cells abrogates the absolute need for increased levels of Fos protein. It is interesting that TPA does not induce stromelysin RNA in Rat-2 fibroblasts, though it does in the NIH 3T3 cells (Fig. 8) (24). This may also be related to the lack of protein kinase C,1l in rat fibroblasts. This raises the possibility that a specific protein-kinase-Cpl-mediated phosphorylation cascade greatly enhances the ability of Fos to act as a transactivator for stromelysin and other AP-1binding-site-containing genes. ACKNOWLEDGMENTS We thank Jeffrey Holt for helpful advice and assistance with the antisense experiments, Nancy Olashaw for assistance with the phosphorylation experiments, and L. Bernstein and N. Colburn for helpful advice with the Western blots. We thank Beth Fields and Farideh Bagheri for technical assistance and Jane Wright and Nancee Thomas for help with the photography. This work was supported by a Public Health Service grant from the National Institutes of Health (CA 46843 to L.M.M.) and grants from the American Cancer Society (JFRA-192 to L.M.M. and IN-25-29 to L.D.K.). LITERATURE CITED 1. Angel, P., I. Baumann, B. Stein, H. Delius, H. J. Rahmsdorf, and P. Herrlich. 1987. 12-0-Tetradecanoyl-phorbol-13-acetate induction of the human collagenase gene is mediated by an inducible enhancer element located in the 5'-flanking region. Mol. Cell. Biol. 7:2256-2266. 2. Angel, P., M. Imagawa, R. Chiu, B. Stein, R. J. Imbra, H. J. Rahmsdorf, C. Jonat, P. Herrlich, and M. Karin. 1987. Phorbol ester-inducible genes contain a common cis element recognized by a TPA-modulated trans-acting factor. Cell 49:729-739. 3. Barber, J. R., and I. M. Verma. 1987. Modification of fos proteins: phosphorylation of c-fos but not v-fos, is stimulated by 12-tetradecanoyl-phorbol-13-acetate and serum. Mol. Cell. Biol. 7:2201-2211. 4. Bos, T. J., D. Bohmann, H. Tsuchie, R. Tjian, and P. K. Vogt.

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