Development 110, 211-220 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

211

Expression of extracellular matrix-degrading metalloproteinases and

metalloproteinase inhibitors is developmental^ regulated during endoderm

differentiation of embryonal carcinoma cells

RICHARD R. ADLER,* CAROL A. BRENNERt and ZENA WERBt

Laboratory of Radiobiology and Environmental Health, and Department of Anatomy, University of California, San Francisco, California94143-0750, USA

•Present address: Department of Biology, Western Carolina University, Cullowhee, NC 28723, USAt Present address: University of Tennessee Medical Center, 1924 Alcoa Highway, Knoxville, TN 37920-6999, USA$ Author for correspondence

Summary

The differentiation of F9 and PSA-1 embryonal carci-noma cells to embryoid bodies composed of a mixture ofparietal and visceral endoderm was accompanied bychanges in their secretion of metalloproteinases. Differ-entiation was induced by retinoic acid and dibutyrylcyclic AMP (for F9 cells) or by removing cells from asubstrate of feeder cells to alter cell-cell interaction andadhesion (for. PSA-1 cells). The embryoid bodies at-tached to gelatin-coated dishes, and the parietal endo-derm cells spread out over the matrix. The differentiatedcells secreted specific gelatin- and casein-degrading pro-teinases, including enzymes that comigrated with proen-zyme forms of collagenase and stromelysin. Total pro-teinase activity as well as specific collagenase activityincreased with the time of differentiation. All of thegelatin- and casein-degrading proteinases detectable bysubstrate gel zymography were inhibited by inhibitors ofmetalloproteinases but not by inhibitors of serine orcysteine proteinases, indicating that they were metallo-

proteinases. Both cell lines showed increased collageno-lytic activity, which was activated by treatment withplasmin. In addition, both cell lines showed increasedsecretion of specific metalloproteinase inhibitors, includ-ing tissue inhibitor of metalloproteinases, with differen-tiation. Analysis of mRNA from undifferentiated anddifferentiated F9 cells by RNA blot analysis or reversetranscription coupled with the polymerase chain reac-tion showed that increased expression of genes forcollagenase, stromelysin and tissue inhibitor of metallo-proteinases is associated with differentiation of thesecells. These results suggest that the expression of extra-cellular matrix-degrading metalloproteinases and theirinhibitors is developmentally regulated during the dif-ferentiation and spreading of the parietal endoderm.

Key words: embryonal carcinoma; endoderm; proteinase;extracellular matrix.

Introduction

Development is characterized by the changing interac-tions of cells with their extracellular matrix (ECM) andby accompanying changes in cellular phenotype andgene expression. The preimplantation mouse embryobegins to synthesize and secrete components of theECM during the 4-cell stage. By the time the blastocystforms, there is ECM between all of the cells and newlydifferentiated tissue layers, as well as on the surface ofthe mural trophectoderm facing the blastocyst cavity(Adamson, 1982). This suggests that all growth and cellmovement after the 4-cell stage requires remodeling ofthe ECM. For example, at about 5.5 days of gestationthe cells on the blastocoelic surface of the inner cellmass (ICM) differentiate to form primitive endoderm;

this tissue subsequently gives rise to the visceral endo-derm, which continues to cover the ICM, and theparietal endoderm, which migrates out of the ICM andspreads over the blastocoelic surface of the muraltrophectoderm. Because the inner surface of the muraltrophectoderm is coated with ECM composed of col-lagen, laminin and fibronectin (Adamson, 1982), theoutgrowth of parietal endoderm cells must requireinteraction with and remodeling of that matrix.

It has been shown that ECM remodeling ofteninvolves the activity of specific proteolytic enzymes ofthe metalloproteinase family, such as collagenase andstromelysin (reviewed in Werb, 1989; Alexander andWerb, 1989). These enzymes are expressed during earlymouse development, with a large increase in expressionat the blastocyst stage (Brenner et al. 1989). However,

212 R. R. Adler, C. A. Brenner and Z. Werb

owing to the small size of the embryo, it is often difficultto use whole embryos to determine the specific cell ortissue that is secreting these enzymes or to demonstratethe specific role that the enzymes play in cell migrationor differentiation. Nor is it possible to obtain sufficientmaterial from embryos to fully characterize these activi-ties. This problem has been solved with the use ofembryonal carcinoma (EC) stem cells, which not onlyprovide an unlimited supply of embryonic material butcan also be induced to differentiate in culture. The celllines PSA-1 and F9 have been studied extensivelybecause of their biochemical and morphological resem-blance to the ICM and because of their ability todifferentiate and form parietal endoderm in vitro (Mar-tin and Evans, 1975; Strickland et al. 1980; Hogan et al.1981; Grover and Adamson, 1986; Casanova and Gra-bel, 1988; Edwards et al. 1988; Pecorino et al. 1988;Dahl and Grabel, 1989). In the present study, these celllines were used to investigate the expression of ECM-degrading proteinases and their inhibitors during thedifferentiation of parietal endoderm. The results indi-cate that these proteins are developmentally regulatedduring EC cell differentiation.

Materials and methods

Cell lines, culture, and differentiationThe following cell lines were used: STO, an embryonicfibroblast line derived from a SIM mouse (Martin et al. 1977;obtained from Gail Martin, University of California, SanFrancisco); PSA-1, an ICM-like cell line (Martin et al. 1977;obtained from Gail Martin); and F9, another ICM-like cellline (Strickland and Mahdavi, 1978; obtained from EileenAdamson, La Jolla Cancer Institute, La Jolla, CA).

PSA-1 cells were maintained on X-ray-inactivated feederlayers of STO cells cultured in Dulbecco's modified Eagle'smedium (DME) containing 10% calf serum. Confluent cul-tures of PSA-1 cells growing on STO feeder layers weredissociated in trypsin, then suspended in 50 ml DME contain-ing calf serum. These suspensions were incubated 3 times for15min each time in T150 culture flasks (Falcon) at 37° C toremove the more rapidly adhering STO cells. The PSA-1 cellswere then plated in 100 mm tissue culture dishes (Costar) at adensity of 1 x 105 cells/dish and incubated for 3 days, by whichtime they had formed small clusters of attached cells calledembryoid bodies. The embryoid bodies were removed fromthe culture dishes by gentle pipetting. Embryoid bodies fromthree dishes were pooled, suspended in 50ml fresh DMEcontaining calf serum, transferred to 150 mm bacteriologicaldishes, and grown in suspension for up to an additional 21days. The culture days are expressed as the total number ofdays off feeder layers.

F9 cells were maintained in DME containing 10% fetalbovine serum. For differentiation, F9 cells were plated in100 mm culture dishes at a density of lxlO5 cells/dish in a 1:1mixture of Ham's F12 and DME containing 10 % fetal bovineserum and 1 nM retinoic acid (Sigma). The cells were fed every2 days, and after 7 days the cells were removed from thedishes by gentle pipetting, resuspended in 50 ml of the samemedium plus 0.5 HIM dibutyryl cyclic AMP (db-cAMP, Sigma)and 1.0mM isobutylmethylxanthine (Sigma), then transferredto 150 mm bacteriological dishes and grown in suspension forup to 4 days.

To isolate parietal endoderm cells for analysis of proteinase

expression, outgrowth of parietal endoderm-like cells wasstimulated by transferring the suspension cultures of eitherPSA-1 or F9 onto gelatin-coated tissue culture dishes. Disheswere prepared by briefly coating them with 0.3 % gelatin andwashing with sterile H2O. Cells or embryoid bodies from one150 mm dish were suspended in 20 ml of the appropriateculture medium and distributed equally into two gelatin-coated dishes. Cells and embryoid bodies were incubated for24 h in medium containing serum to allow them to attach. Foranalysis of secreted proteinases, cells were transferred toDME containing 0.2% lactalbumin hydrolysate (DME-LH)for 48 h before collection of cell-conditioned medium. Rabbitsynovial fibroblasts were cultured and treated with 12-O-tetradecanoylphorbol-13-acetate (TPA) in DME-LH as de-scribed previously (Unemori and Werb, 1988).

Detection of proteinases and proteinase inhibitorsZymography was used to detect the secretion of proteinasesand inhibitors by cultured cells as described previously(Herron et al. 1986a,b; Brenner et al. 1989). Protein concen-tration in all conditioned medium samples was determined bythe Bio-Rad protein assay (Bradford, 1976), and sampleswere diluted with DME-LH to the lowest concentrationfound. Briefly, conditioned medium samples (5—20/̂ 1) weremixed with Laemmli sample buffer (without /3-mercaptoetha-nol and modified to a final concentration of 2.5% sodiumdodecyl sulfate [SDS]). Samples were then separated withoutboiling under nonreducing conditions on 10 or 12 % polyacryl-amide gels containing lmgml"1 type I gelatin (Sigma) orcasein (Sigma). After removal of SDS by washing the gels for30 min in 2.5 % Triton X-100, the gels were incubated for 24 to48 h in 50 mM Tris-HCl, 10mM CaCl2, pH7.6, alone or with1,10-phenanthroline (4mM), EDTA(4mM), phenylmethylsul-fonylfluoride (PMSF) (5mM), leupeptin (10 jig ml"1), orrecombinant human tissue inhibitor of metalloproteinases(TTMP) (10/igmP1; gift of D. Carmichael, Synergen Corp.,Boulder, CO), then stained with Coomassie Blue R250.

Inhibitors were detected by reverse zymography on 15 %polyacrylamide gels containing gelatin by treatment of thegels for 30 min in conditioned medium from cultured rabbitskin that contained active metalloproteinases (Herron et al.1986a; Unemori and Werb, 1986,1988), followed by 24 to 48hof incubation as described above. In this assay the rabbitmetalloproteinases (largely the 72 x l O 3 ^ gelatinase) bind toand degrade the gelatin substrate of the gel. Where inhibitorsfrom the EC cells were present, the background gelatin wasnot degraded, and these bands are visualized after staining asdark blue bands against a pale blue background.

Specific collagenase activity was determined by the collagenfibril assay (Aggeler et al. 1984a). Activities are expressed inunits (U), where 1 U of collagenase activity degrades 1/ig ofcollagen fibrils min"1 at 37°C.

Activation of metalloproteinase proenzymesConditioned medium samples were activated by overnightincubation with the serine proteinase, plasmin (Sigma). Con-ditioned medium (100 yi) was incubated with plasmin(15/igmr')for 12hat37°C; 15 j*l of 150n%ml"' PMSF wasthen added to block further plasmin activity.

RNA preparationTotal RNA used for RNA blot analysis was prepared fromdifferentiated PSA-1 and F9 cells by the standard GuSCN/CsCl procedure (Chirgwin et al. 1979). The concentration ofRNA was determined by A260 and then normalized so thatafter the samples were run on agarose gels and stained withethidium bromide, the rRNA bands were of equal strength.

Differentiation induces proteinases 213

cDNA probesThe cDNA probes used in this study were obtained from thefollowing sources: rabbit collagenase, pCLl (Frisch et al.1987); rabbit stromelysin, pSL2 (Frisch et al. 1987); mousetissue-type plasminogen activator (tPA), gift of R. Rickles,SUNY-Stony Brook (Rickles et al. 1988); chicken actin, PA1,gift of M. Kirschner, UCSF (Cleveland et al. 1980); rattransin-1, pTRl, gift of R. Breathnach, University of Stras-bourg (Matrisian et al. 1985); human stromelysin, gift of G.Goldberg, Washington University, St Louis (Wilhelm et al.1987); human 68 x 1&3 MT gelatinase/type IV collagenase, giftof G. Goldberg, Washington University, St. Louis (Collier etal. 1988); mouse TIMP, gift of B. Williams, University ofToronto (Gewert et al. 1987), afl (IV) collagen, gift of M.Kurkinen, UMDNJ/Robert Wood Johnson Medical Center(Saus et al. 1989; Kurkinen et al. 1983). A mouse stromelysincDNA (Ostrowski et al. 1988) identical to embryonic mousestromelysin (EMS-2) (Brenner et al. 1989) was a gift of Dr L.Matrisian, Vanderbilt University, Nashville. For mouse col-lagenase cDNA, amplified fragments generated by reversetranscription followed by the polymerase chain reaction (RT-PCR) from F9 or PSA-1 RNA were purified by agarose gelelectrophoresis as described previously (Brenner et al. 1989).

RNA analysisRNA isolated from F9 and PSA-1 cells cultured as describedfor proteinase assays was separated and run on denaturingagarose gels, transferred to nylon membranes by capillaryaction (Thomas, 1980; Reed and Mann, 1985), and then UVcross-linked (Lehrach et al. 1977; Church and Gilbert, 1984).The RNA was then hybridized with inserts from cDNA clonesor PCR-amplified cDNA (Brenner et al. 1989) radiolabeledwith 32P-dCTP by nick translation (Maniatis et al. 1982). RT-PCR analysis of mRNA was performed as described pre-viously (Rappolee et al. 1988a,b, 1989), with stromelysinoligonucleotide primers (Brenner et al. 1989) and glyceralde-

A Gelatin

Days in Culture

STO 0 3 6 9 12 15 18 21 24

200-

•« —37

25- J

hyde-3-phosphate dehydrogenase primers (5' primer: 5'-TGATGACATCAAGAAGGTGGTGAAG-3'; 3' primer:5'TCCTTGGAGGCCATGTAGGCCAT-3') designed fromthe rat sequence (Fort et al. 1985) to give an amplified cDNAfragment of 240 bp.

Results

EC cell differentiation is associated with secretion ofgelatin- and casein-degrading proteinasesPSA-1 cells remain undifferentiated as long as they arecultured on STO feeder cells. Once removed from thefeeder layers they form small aggregates of ICM-likecells with some primitive endoderm-like cells on theoutside (Martin et al. 1977). When these colonies arethen grown in suspension culture, they form hollowballs of cells, known as embryoid bodies, that arecomposed of a core of undifferentiated stem cellssurrounded by a mixture of differentiated visceral andparietal endoderm (Martin and Evans, 1975). Whengrown on gelatin-coated surfaces, the embryoid bodiesattach, and the parietal endoderm cells grow out ontothe matrix (Grabel and Watts, 1987).

PSA-1 cells were differentiated for up to 24 days andthe embryoid bodies were grown on gelatin, thencultured in DME-LH for 48 h. The resulting con-ditioned medium was then assayed for proteinase ac-tivity. ZymogTams from gelatin-SDS gels showed thatboth undifferentiated and differentiated cells secretedthree major gelatinases migrating at 68, 85, and96 xltfM; (Fig. 1A). The STO feeder cells also pro-duced copious amounts of the 68, 85, and 96 xlCr MT

gelatinases, and contamination from these cells may

B Casein

Days in Culture

STO 0 3 6 9 12 15 18 21 24

-200

-96

-68

-43

-25

Fig. 1. (A) Gelatin- and (B) casein-degrading proteinases secreted by PSA-1 cells during differentiation. UndifferentiatedPSA-1 cells and embryoid bodies from cells cultured for 0-24 days off feeder layers (days in culture) were incubated for 48 hon gelatin-coated dishes in DME-LH. Samples of conditioned medium containing 9.0 fig protein were then separated onsubstrate gels. Molecular weight markers (xlO"~3) are shown on the left in A and on'the right in B. Bands comigrating withprocoUagenase (A) and prostromelysin (B) are marked by arrows. The zymograms are shown as negative images.

214 R. R. Adler, C. A. Brenner and Z. Werb

A Gelatin B Casein

F9-RA + db-cAMP F9-RA + db-cAMP

F9 RA 1 2 3 4davs

-43

43-

-25

Fig. 2. (A) Gelatin- and (B) casein-degrading proteinases secreted by F9 cells during differentiation. Undifferentiated F9cells, embryoid bodies from F9 cells treated with retinoic acid (F9-RA), and embryoid bodies from F9 cells treated withretinoic acid followed by 1-4 days with db-cAMP were incubated for 48 h on gelatin-coated dishes in DME-LH. Samples ofconditioned medium containing 7.5 jig protein were then separated on substrate gels. Molecular weight markers (xlO~3) areshown on the left in A and on the right in B. Bands comigrating with procoUagenase (A) and prostromelysin (B) are markedby arrows. The zymograms are shown as negative images.

have accounted, in part, for the enzymes seen on day 0and day 3 of cells off feeders. However, in mixingexperiments that included contaminating STO cells,proteinases from these cells did not contribute substan-tially at days 0 and 3 and had no detectable contributionafter day 3. The undifferentiated cells also secreted a37 x 103 MT gelatinase that was initially lost on differen-tiation and then reexpressed by day 12. After 12 days ofdifferentiation, new gelatin-degrading proteinases ap-peared at 51, 53, and 100 xlO3 MT. The activity of mostof the gelatinases increased with differentiation through24 days (Fig. 1A). Casein gels also showed constitutiveand differentiation-induced proteinase activity(Fig. IB). The undifferentiated cells showed severalcaseinase bands above 75 x 103 MT. At 3 days of differ-entiation new bands appeared at 51, 53, 72 and100 x 103 Mr. With further differentiation all of the newbands showed increased activity. In general, the caseingels and gelatin gels showed distinct enzymes.

F9 cells differentiate to form primitive endoderm onthe surface of embryoid bodies when they are treatedwith retinoic acid. When the embryoid bodies weretreated with 0.5 HIM db-cAMP, they further differen-tiated to form a mixture of parietal and visceral endo-derm (Strickland and Mahdavi, 1978; Strickland et al.1980; Hogan et al. 1981; Grover and Adamson, 1986).Culture of these embryoid bodies on gelatin allowed the

parietal endoderm cells to spread out onto the matrix.The embryoid bodies from differentiated and undiffer-entiated F9 cells were incubated for 48 h in DME-LH,and the conditioned medium was analyzed by zymogra-phy. With differentiation there was a large increase inthe secretion of specific gelatinases, particularly at 51,92, 96, and ISOxKPM,., and of a caseinase at52xlO%r(Fig. 2A,B).

ECM-degrading proteinases secreted by EC cells aremetalloproteinasesTo characterize these proteinases, the proteins secretedby the undifferentiated and differentiated cells wereseparated on polyacrylamide gels containing gelatin orcasein, and the gels were incubated for 24 or 48 h withvarious enzyme inhibitors. As positive controls, themetalloproteinases secreted by TPA-treated rabbitsynovial fibroblasts were also separated on these gels.PMSF and leupeptin, inhibitors of serine proteinases,had no effect on the gelatinase or caseinase activity inany of the conditioned medium analyzed; however,TIMP, 1,10-phenanthroline and EDTA, which arepotent inhibitors of metalloproteinases, blocked all ofthe proteolytic activity seen by zymography (Fig. 3,Table 1), as shown previously (Brenner et al. 1989). Atthe concentration used, all the enzymes were inhibitedin parallel by the metalloproteinase inhibitors. The

Differentiation induces proteinases 215

Fig. 3. Inhibition of gelatin-degrading enzymes secreted byF9 embryonal bodies treated with retinoic acid for 4 days.Lanes from the zymograms were incubated in substratebuffer alone (control) or in buffer containing 5 mM PMSF,4mM 1,10-phenanthroline, 4mM EDTA, or lO/zgml"1

TIMP. Molecular weight markers (xlO~3) are shown on theleft. The zymograms are shown as negative images.

ethanol and dimethyl sulfoxide solvents of the inhibi-tors had no appreciable effect on the proteolytic ac-tivity.

Latent and active collagenase activity increases withEC cell differentiationBecause most major metalloproteinases are secreted aszymogens (Werb, 1989), the effect of proteolytic acti-vation on the proteinases secreted by PSA-1 and F9cells was studied by zymography. There was a signifi-cant increase in both gelatin- and casein-degradingactivity in samples treated with plasmin. In addition,the intensity of the 51 and 53 xlfj3 Afr gelatinase bandsthat migrated with rabbit procollagenase decreasedafter plasmin treatment, and two bands of greatergelatinase activity appeared at 41 and 43xlO3Mr.Similarly, the 52 X l(r Mr proteinase comigrating withrabbit stromelysin seen on the casein gels disappearedwith plasmin treatment, and a new band appeared at42 xlO3 MT (data not shown).

The specific collagenase activity present in thesesamples was then analyzed by the collagen fibril assay.The secreted proteins from PSA-1 and F9 cells that haddifferentiated for various times were assayed directly tomeasure active collagenase, or after activation of the

proenzyme with plasmin to determine total active andlatent enzyme. A small amount of active collagenase(lOmUmg"1 protein) was secreted by undifferentiatedPSA-1 cells, and the collagenase activity increaseddramatically with differentiation after 9 days off feederlayers, reaching a maximum of 190mUmg~1 at 18 days(Fig. 4A). Activation with plasmin increased themeasurable collagenase activity in conditioned mediumfrom undifferentiated PSA-1 cells 6-fold to61mUmg~1, and from 18-day differentiated PSA-1cells 2-fold to SrjOmUmg^1 (Fig. 4A). The appearanceof collagenolytic activity paralleled the appearance ofthe 51 and 53 xl(PMT gelatinase bands on the zymo-grams.

The F9 cells had a similar pattern of collagenaseexpression. The unactivated secreted proteins fromundifferentiated F9 cells had 16 mU of collagenase permg, which increased to STmUmg"1 with activation.With differentiation the collagenolytic activityincreased rapidly, reaching lSOmUmg^1 without acti-vation and 270mUmg~1 with activation (Fig. 4B).These data suggest that collagenase secretion increaseswith differentiation of EC cells. Although the majorityof the collagenase activity was found in an activatableproenzyme form, a significant amount of active enzymewas present in the serum-free conditioned medium.

Differentiating EC cells secrete inhibitors ofmetalloproteinasesMany cells and tissues that secrete metalloproteinasesalso secrete inhibitors of these enzymes, such as TIMPor other inhibitors of metalloproteinases (such asTIMP-2), which are members of the same gene family(Herron et al. 1986a; Werb, 1989; Stetler-Stevenson etal. 1989; Apodaca et al. 1990). To determine if EC cellsalso secrete these inhibitors, secreted proteins fromundifferentiated and differentiated PSA-1 and F9 cellswere analyzed for metalloproteinase inhibitor activityby an adaptation of the substrate gel procedure (Herronet al. 1986a). The PSA-1 cells showed a band at29xlO3Mr, comigrating with human TIMP, that

Table 1. Effect of inhibitors on gelatin- and casein-degrading proteinases

Inhibitor

NoneDMSOEthanol1,10-phenanthrolineEDTAPMSFLeupeptinTIMP

Concentration

1%1%

4 n w4mM5mM

10/^ ml"1

lOueml"1

RSF(relative

4+3+4+-—

4+4+—

PSA-1 F9expression)*

3+2+2+-—

2+3+—

3+2+2+-—

2+3+—

* Expression refers to appearance of gelatin- and casein-degrading proteinases on SDS-substrate gels. All of the bands seenin Figs 1 and 3 were inhibited by 1,10-phenanthroline, EDTA andTIMP. —, no activity; 2+, moderate activity; 3+, high activity; 4+,very high activity. RSF, rabbit synovial fibroblasts; DMSO,dimethyl sulfoxide.

216 R. R. Adler, C. A. Brenner and Z. Werb400

300

200-

1 0 20

Day* In Culture

Fig. 4. Time course of appearance of specific collagenase inthe culture medium of (A) PSA-1 and (B) F9 cells withdifferentiation. PSA-1 and F9 cells were differentiated andthen incubated for 48 h in DME-LH as described in thelegends to Figs 1 and 2. Samples of conditioned mediumwere then assayed for collagenase activity in the collagenfibril assay either directly or after activation of proenzymeforms by plasmin. The data are expressed as mU ofcollagenase mg""1 cell protein.

polyacrylamide gels without a protein substrate, indi-cating that they were major secreted proteins but notinhibitors.

Metalloproteinase and TIMP gene expression isregulated during EC cell differentiationRNA from undifferentiated and differentiated F9 cellswas then analyzed by RNA blot analysis (Fig. 6A). The2 kb collagenase mRNA band seen by hybridizationwith mouse collagenase cDNA at high stringency wasdevelopmentally regulated. In several experiments thistranscript appeared highest at 2 days of endodermdifferentiation, decreasing thereafter. This mRNA hada different time course of induction than the 51 andSSXVPMT gelatinases. Thus, it is possible that theenzymes seen by zymography are additional enzymes.A 1 kb TIMP mRNA band also increased with differen-tiation. Differentiation was shown by the induction ofmRNA for tPA and type IV collagen, as describedpreviously (Kurkinen etal. 1983; Rickles etal. 1988). Incontrast, there was no increase with differentiation inactin mRNA (Fig. 6A) or in the mRNA for the68xlOiMT gelatinase/type IV collagenase, which ap-peared to be secreted constitutively (data not shown).The expression of mRNA for major excreted protein(cathepsin L), a cysteine proteinase, increased onlyafter treatment of cells with retinoic acid, suggestingthat its increased expression may be selective forvisceral endoderm. Similar evidence for regulation ofthese mRNAs with differentiation was obtained withPSA-1 cells (data not shown).

Although an mRNA band hybridizing with a humanstromelysin cDNA probe at low stringency recognized a2 kb mRNA in F9 and PSA-1 cells (Brenner et al. 1989),this washed off at high stringency. cDNA probes for ratand mouse stromelysin hybridized poorly to 10 u% oftotal RNA from PSA-1 or F9 cells, indicating thatstromelysin mRNA was not abundant. Therefore, RT-PCR was used to analyze differentiating F9 cells forstromelysin mRNA. As shown in Fig. 6B, stromelysinmRNA increased with differentiation of F9 cells. Theappearance of this mRNA paralleled the appearance ofthe 52 xlf^Af,. caseinolytic proteinase by zymography.

increased with differentiation, particularly after day 15,as well as a 20 xlO3 MT band (Fig. 5A). A weak inhibi-tor at 18 x 103 MT was also seen, suggesting that EC cellsproduce additional metalloproteinase inhibitors, as hasbeen found for other cells (Apodaca et al. 1990). The F9cells also secreted metalloproteinase inhibitors thatincreased with differentiation. The largest increaseoccurred after day 1 in db-cAMP, with smaller incre-ments to day 4. However, the major inhibitor in F9 cellswas a band at 21xlO3Mr, comigrating with humanTIMP-2 (Fig. 5B). In some experiments a weak29 x 103 MT band was also seen. These data suggest thatTIMP is the major inhibitor produced by PSA-1 cells,whereas TIMP-2 is the major inhibitor of F9 cells. Thedark bands migrating above the inhibitors on both ofthese gels were also visible on conventional SDS-

Discussion

In previous studies, it was shown that metalloprotei-nases and their inhibitors are expressed during mouseembryogenesis and by certain EC cell types (Brenner etal. 1989). In the present study, PSA-1 and F9 cells wereused to determine if the expression of ECM-degradingmetalloproteinases and their inhibitors during parietalendoderm differentiation and spreading on gelatin-coated surfaces is developmentally regulated. The re-sults showed that differentiation of parietal endoderm isassociated with an increase in the secretion of specificgelatin- and casein-degrading proteins, which can bedetected by zymography. Two of these proteinasescomigrated with the metalloproteinases collagenaseand stromelysin. Collagenase activity also increased

Differentiation induces proteinases 217

Days in Culture B•o •o

0QT - CM

>

o o

.a•o>.oa

43* ***"*—***"""""

25'TIMP

TIMP II*

PSA-1 F9Fig. 5. Substrate gel analysis of metalloproteinase inhibitors secreted by (A) PSA-1 and (B) F9 cells during differentiation.Samples of conditioned medium prepared as described in the legends to Figs 1 and 2 were separated on SDS-gelatin gelsand analyzed for inhibitors as described in Materials and Methods. The inhibitors TIMP and IMP appear as dark bandsmigrating at 29 and 20xl03Mr, respectively. STO, feeder layer cells for PSA-1; H, human TIMP and IMP standards; F9,undifferentiated F9 cells; F9-RA, F9 cells treated with retinoic acid; 1-4 days db-cAMP, F9 cells treated with retinoic acidfollowed by 1-4 days with db-cAMP. Molecular weight markers (xlO~3) are shown on the left. The dark bands seen above45xlO3Afr are major secreted proteins from F9 and PSA-1 cells that were also visible on conventional SDS-polyacrylamidegels. Zymograms are shown as positive images.

significantly with differentiation. Expression of theseproteins was mirrored by expression of mRNA: BothRNA blot analysis and RT-PCR showed the presence ofcollagenase and stromelysin mRNA in F9 and PSA-1cells (Brenner et al. 1989), with an increase in theexpression of these mRNA species during differen-tiation. Thus, the expression of these metalloprotei-nases is developmentally regulated during parietal en-doderm differentiation. Inhibitor studies showed thatall of the proteinases detected by zymography weremetalloproteinases. Together, these metalloproteinasescan degrade the basement membrane collagens, glyco-proteins and proteoglycans, and the interstitial ECM,producing the remodeling necessary for changes inECM-cell adhesion, migration and embryonic expan-sion as the fetal membranes containing parietal endo-derm differentiate and grow. The correlation betweencollagenase activity, bands comigrating with authenticcollagenase on zymograms, and collagenase mRNAlevels was not strong. Because TIMP and other metallo-proteinase inhibitors were also expressed by the, ECcells, this would have effects on the measurable activityin biochemical assays (Herron et al. 1986a,b). Addition-ally, collagenase activity is also increased about 10-foldby treatment with stromelysin (Alexander and Werb,1989). Thus, the relative expression of these two

proteinases could modify the catalytic activity of col-lagenase. Similarly, the other gelatinases may facilitatecollagen peptide solubilization in the quantitative assay.A third possibility includes post-transcriptional regu-lation of metalloproteinase expression. This mechanismhas been observed for collagenase (Werb, unpublishedresults). Finally, the comigrating bands seen on zymo-grams may be due to uncharacterized metalloprotei-nases. The appearance of a major caseinolytic band at52 x 103 MT did not correlate strongly with very lowamounts of stromelysin mRNA. The strong hybridiz-ation of F9 RNA with a rabbit stromelysin probe at lowstringency further points to this possibility.

In addition to proteolytic activity, PSA-1 and F9 cellsalso showed increased secretion of specific metallopro-teinase inhibitors with differentiation. However, PSA-1cells secreted inhibitor activity comigrating with humanTIMP, whereas the major inhibitor activity of F9 cellscomigrated with human TIMP-2 (Stetler-Stevenson etal. 1989). In both F9 and PSA-1 cells, mRNA for TIMPincreased with differentiation, although the amountswere low. In recent studies using nuclease protectionassays, Nomura et al. (1989) found TIMP mRNA inmouse fetal membranes, including parietal endoderm.These inhibitors may regulate the amount of degra-dation of Reichert's membrane so that net accumu-

218 R. R. Adler, C. A. Brenner and Z. Werb

A

CLi

« * =* =*re to to (0

O) <LL CC l- CN CO "tf

TIMP

tPA

MEP

type IV

Act in

I

SL

GAPDH

Fig. 6. Analysis of specific mRNA transcripts expressed byF9 cells during differentiation. Cells were cultured asdescribed in the legend to Fig. 2 and RNA was isolated and(A) 10 ̂ tg analyzed by RNA blot analysis or (B) 0.1 nganalyzed by RT-PCR analysis. GL, 68 xl(PMT gelatinase;CL, collagenase; MEP, major excreted protein (cathepsinL); type IV, type IV collagen; SL, stromelysin; GAPDH,glyceraldehyde-3-phosphate dehydrogenase. The RT-PCRamplification products in B are shown as negative images.

lation takes place while the ECM is remodeled to allowgrowth.

Previous studies have shown that the differentiationof parietal endoderm from either F9 or PSA-1 cellsrequires specific cell-cell and cell-ECM interactionsand may be mediated by cell shape (Hogan et al. 1981;Grabel and Watts, 1987; Casanova and Grabel, 1988;Dahl and Grabel, 1989). F9 differentiation is alsoaccompanied by the development of a fibrillar fibronec-tin matrix (Dahl and Grabel, 1989). All of these types ofinteractions have been implicated in the stimulation ofmetalloproteinase gene expression by fibroblasts andendothelial cells (Aggeler et al. 1984b; Unemori andWerb, 1986; Werb, 1989; Werb et al. 1989). Further-more, F9 cell differentiation is associated with theexpression of tPA (Pecorino et al. 1988; Sabbag et al.1989) and c-fos (Edwards et al. 1988), both of whichhave been shown to be associated with metalloprotei-nase expression in other systems (Mignatti et al. 1986;Kerref al. 1988).

The present study has demonstrated that mouse ECcell metalloproteinases are developmentally regulatedand produced, in part, in enzymatically active form,and that the latent zymogens can be activated byplasmin. It is possible that tPA expression by these cellsmay be directly related to the plasminogen-dependentactivation of the observed metalloproteinases. Theabundant expression of cathepsin L by these cells alsoprovides a potential mechanism for plasminogen-inde-pendent activation of the metalloproteinases (Werb,1989).

Finally, it is encouraging that similar results wereobserved for parietal endoderm differentiation in bothF9 and PSA-1 cells. Because PSA-1 cell differentiationis stimulated by a change in cell-cell interactions andcell shape, whereas F9 differentiation requires stimu-lation by retinoic acid and db-cAMP, the results indi-cate that the metalloproteinase and inhibitor geneexpression and secretion observed were characteristicof the parietal endoderm phenotype and not the type ofstimulation. These data suggest that the signalsrequired for differentiation regulate expression of themetalloproteinase genes in development. These signals,including retinoid-receptor binding and modulation ofcAMP-mediated second messenger pathways, as well aspathways induced by changes in cell-cell and cell-ECMinteraction, have all been elucidated in fibroblast cellsin vitro (Unemori and Werb, 1986, 1988; Kerr et al.1988; Werb et al. 1989; Werb, 1989). Our results may beapplicable to an understanding of normal developmentin vivo.

Differentiation induces proteinases 219

This work was supported by a grant (HD 23539) from theNational Institutes of Health, a National Research ServiceAward (5 T32 ES07106) from the National Institute ofEnvironmental Health Sciences, and contract DE-AC03-76SF01012 from the Office of Health and EnvironmentalResearch, U.S. Department of Energy. We thank Joey Clarkand Leslie Rail for performing the RT-PCR analyses, RandiNeff for assistance with the zymograms, Ole Behrendtsen forassistance with the photography, Rick Lyman for typing themanuscript, and Mary McKenney for editing it.

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(Accepted 31 May 1990)