ICANCERRESEARCH54,3611-3616,July 1. 1994)

can; and gelatinases (MMP2 and MMP9) on type IV collagen (3).Increased expression of stromelysins, matrilysin, or gelatinases hasbeen reported in tumor cells with an invasive or metastatic phenotype(4—12).The observed association between secretion of MMPs andinvasive or metastatic properties of tumor cells implies that theirexpression could be crucial for progression of a tumor to malignancy.Elucidation of the mechanisms of how the secretion of these MMPsis regulated is therefore of prime importance to understanding ofmalignant progression.

We previously reported that cultured murine tumor cells secrete Mr60,000 and 95,000 gelatinases/type IV collagenases (MMP2 andMMP9, respectively) and that those with high spontaneous metastaticpotential to the lung produce remarkably higher amounts of MMP9than their counterparts with poorly metastatic potential (6). Thepresent study was undertaken to elucidate mechanisms underlying theaugmented secretion of MMP9 by murine metastatic colon carcinomacolon 26 cells. As a result, an autocrine factor enhancing secretion ofMMP9 was detected in medium conditioned with metastatic cells.

MATERIALS AND METHODS

Cells. Highly and poorly metastatic LuMI and NMI I, respectively. cells

were isolated by in vivo selection from mumine colon carcinoma colon 26 cellsaccording to the method described by Tsuruo ci a!. (13). Briefly, cultured colon26 colon cells (106cells) were implanted intrademmallyinto the abdominal skinof a 5-week-old BALB/Cfemale mouse. After 4 weeks, the lung of the mousebearing colon 26 tumors was dissected out and transplanted to another mouse.This was then repeated, and cloned metastatic cells in culture (LuM1) wereobtained from metastatic lung nodules after 7 cycles of lung transplantation.Poorly metastatic cells (NM) I) were cloned from cultured cells obtained from

theprimarytumor.Averagenumbersof metastaticnodulesin the lungsof mice5 weeks after intrademmal implantation of 5 X l0@ LuMI or NM1I cells to

6-week-old BALB/c female mice were 98 and 4, respectively. Cell lines weremaintained in RPM! 1640 medium with 10% FCS.

Detection and Quantitatlon of Gelatinase. Zymography with gelatin as asubstrate was used to detect gelatinases in serum-free culture medium (6).Quantitation was performed by densitometric tracing of zymograms using a

Shimadzu CS-930 densitometer. Gelatinase activity, detected as a band ofreduced Coomassie blue staining, showed a peak in the negative direction frombackground staining, and the peak area of the detected band was proportionalto the amount of gelatinase. Calculated values calibrated with the numbers ofcells in culture were used for relative amounts of gelatinase. In all experiments,two independent culture supernatants were obtained and used for measurementof gelatinase activity. All data are means ±range of points and represent theresults of at least three independent experiments. For immunochemical detection of Mr 95,000 gelatinase in the culture, 100 p.1 of supematant wereconcentrated using an ultrafree membrane (Millipore) and subjected to SDSPAGE.Fractionatedproteinswereblottedonto nitrocellulosepaperandprobedwith a monoclonal antibody raised in a rat to mouse M@95.000 gelatinase(MMP9)from LuM1cells.

Preparation ofSerum-free Medium COnditiOned with LuM1 Cells. Themedium of confluent culture LuM1 cells was changed to serum-free RPM!1640 medium, and cells were cultured overnight. Then, medium was againchanged, and after 48 h, culture supematant was obtained and centrifuged to

3611

Autocrine Factor Enhancing the Secretion of Mr 95,000 Gelatinase

(Matrix Metalloproteinase 9) in Serum.free Medium Conditioned

with Murine Metastatic Colon Carcinoma Cells'

Sumiko Hyuga, Yohko Nishikawa, Keita Sakata, Hidekazu Tanaka, Sadako Yamagata, Kenji Sugita, Shinsuke Saga,Mutsushi Matsuyama, and Satoru Shimizu2

Pathophysiology Unit, Aichi Cancer Center Research institute, 1—1Kanokoden. Chikusa-ku, Nagoya 464 fS. H., Y. N.. S. Y.. S. 5.1; Second Department of Pathology, NagoyaUniversity School ofMedicine, Showa-ku, Nagoya 466 [K. S., S. Sa., M. MI; and Shionogi Research Laboratory, Shionogi and Company, bd. Sagisu. Fukushima-ku, Osaka 533(H. T.. K. Su.J. Japan

ABSTRACT

We previously reported that murine tumor cells with a high spontaneoils metastatic potential to the lung secrete higher amounts of M@95,000gelatinase (matrix metalloprOtelnase 9, MMP9) than do poorly metastaticcells. The present study, conducted to clarify the mechanisms underlyingthe increase In MMP9, revealed an autocrine factor that enhances thesecretion of M@95,000 gelatinase (MMP9). The secretion of MMP9 byhighly metastatic colon cardnoma LuM1 cells, detected by zyinography,was augmented 10-fold when cultured In medium supplemented withserum-free medium conditioned with LuM1 cells. Because the secretion ofMr 60,000 gelatinase (MMP2), as well as total protein, by the same cells

was not affected under these conditions, the augmentation appears specificfor MMP9. The steady-state level of MMP9 mRNA was elevated In LuM1cells CUltUredIn the presence ofthe supernatant. The amount ofthe factorin the culture medium Increased with time In culture, indicating that itwas produced by the LuM1 cells. It was found to be heat stable butsensitive to try@n digestion. Conditioned medium from poorly metastaticNM11 cells did not stimulate the secretion of gelatinases by NM11 cells,suggesting that autocrine StImUlatiOnof MMP9 secretion Is a characterlade of metastatic cells. This factor could account for the augmentedsecretion of MMl@ by marine tumor cells with spontaneous metastaticpotential to the lung.

INTRODUCTION

The process of tumor metastasis consists of many complex steps,each involving specific interaction of tumor cells with host cells or theextracellular matrix. Migration of tumor cells across the basementmembrane is considered to be essential for the tumor cells to achievesuccessful metastasis (1). Thus, the secretion of enzymes that candegrade basement membrane components would clearly favor metastasis.Thebasementmembraneextracellularmatrixseparatingepithelium, endothelium, and methothelium from underlying stroma is pre

dominantly made up of type IV collagen, laminin, and heparan sulfateproteoglycans. These components of basement membranes can all bedegraded by MMPs,3 a family of proteinases acting specifically onextracellular matrix proteins and therefore playing important roles innormal and pathological processes involving their degradation. Nineseparate members of the MMP family have been identified thus far,each having a distinct substrate specificity (2). Some members of theMMP family act on specific components of basement membranes:

stromelysins (MMP3 and MMP1O) on type IV collagen, laminin, andproteoglycan; matrilysin (MMP7) on type IV collagen and proteogly

Received l2/2@W93;accepted 4129/94.Thecostsof publicationof thisarticleweredefrayedin partby thepaymentof page

charges. This article must therefore be hereby marked advertisement in accordance with18U.S.C.Section1734solelyto indicatethis fact.

I This study was supported in part by Special Coordination Funds of the Science and

Technology Agency of the Japanese Government.2 To whom requests for reprints should be addressed.

3 The abbreviations used are: MMP, matrix metalloproteinase; BSA, bovine serum

albumin;EGF,epidermalgrowthfactor,FCS,fetalcalfserum;IL-l, interleukin-l;PAGE,polyacrylamide gel electmophoresis; SDS. sodium dodecyl sulfate; TGF, transforminggrowth factor, liMP, tissue inhibitor of metallopmoteinases; cDNA, complementary DNA.

Research. on August 18, 2021. © 1994 American Association for Cancercancerres.aacrjournals.org Downloaded from

AUTOCRINE FACIOR ENHANCEMENT OF GELA11NASE SECREI1ON

with that OfMr 60'000 gelatinase, which increased linearly with time.Secretion of both Mr 60,000 and 95,000 gelatinases by NM11 cellsalso occurred linearly. These results suggested the presence of somefactor(s) that enhanced the secretion of Mr 95,000 gelatinase in theculture medium of LuM1 cells.

In order to detect the factor enhancing M@95,000 gelatinase secrelion, the effect of serum-free culture medium conditioned with LuM1cells, after included gelatinases were eliminated (fraction 0), wasexamined on gelatinase secretion by LuM1 cells. When cultured inmedium supplemented with fraction G, LuM1 cells showed enhancedsecretion of Mr 95,000 gelatinase (MMP9) as shown in Fig. 2, A andB, the amount being about 10-fold higher than in control culturewithout fraction 0. Enhancement of gelatinase secretion by fraction Gwas specific for Mr 95,000 gelatinase, and the secretion of Mr 60'000gelatinase was not affected by its addition to the culture medium.Immunochemical detection of Mr 95,000 gelatinase using a monoclo

nal antibody raised against mouse M1 95,000 gelatinase also showeda remarkable increase of the amount of secreted Mr 95,000 gelatinasein the culture supernatant with fraction 0 (Fig. 2C); total proteinsecreted by LuM1 cells, however, was not seriously affected by theaddition of fraction G (Table 1). The effect of fraction 0 on thesecretion of Mr 95,000 gelatinase was dose dependent up to a maximum of 20—50 @gprotein/mI (Fig. 3). The amount of the enhancingactivity of Mr 95,000 gelatinase secretion in the culture supernatantincreased with time in culture (Fig. 4), specific activity (enhancingactivity/mg protein) being almost the same during the same time,suggesting that LuM1 cells continuously secrete the autocrine factorinvolved. Results of Northern analysis are illustrated in Fig. 5. LuM1cells cultured in serum-free medium expressed mRNAs of bothMMP9 and MMP2. Elevated steady-state levels of both 3.5- and2.7-kilobase pair mRNAs of MMP9 were observed in LuM1 cellscultured in the presence of fraction 0, which is consistent with theenhanced secretion of Mr 95,000 gelatinase (MMP9). In contrast, thesteady-state level of MMP2 mRNA was not effected by the presenceof fraction G in the culture medium.

Effects of heating or trypsin digestion of fraction 0 on the enhancement of the gelatinase secretion are shown in Fig. 6. The activity infraction 0 was relatively heat stable, 80% being retained after heatingin boiling water for 10 min. After digestion with trypsin, however,fraction 0 lost a considerable proportion of its enhancing activity ongelatinase secretion. In the process ofpreparing fraction 0, serum-freeculture supernatant was concentrated using a membrane filter with amolecular sieve size of 30,000. These results suggest that the factorhaving an enhancing activity on gelatinase secretion is a heat-stableprotein with a molecular weight of >30,000.

To compare autocrine enhancement of Mr 95,000 gelatinase secretion between LuM1 and NM11 cells, 0 fractions were prepared fromboth, and enhancing activity was assessed in the two cell types(Fig. 7). The Mr 95,000 gelatinase secretion of LuM1 cells wasenhanced by fraction 0 from either LuM1 or NM11 cells, the extentbeing lower in the latter case. With NM11 cells, only the LuM1 cellfraction 0 enhanced gelatinase secretion, and no stimulation by fraction G from NM11 cells was evident, indicating that the autocrineenhancing mechanism of MMP9 secretion observed in LuM1 cellsdoes not operate in the NM11 case.

DISCUSSION

In the present study, aimed at clarifying the mechanism underlyinghigher amounts of secretion of MMP9 (Mr 95,000 gelatinase) bymetastatic LuM1 cells than poorly metastatic NM1 1 cells, both derived from murine colon carcinoma colon 26 cells (6), there was clearevidence that an autocrine factor might be responsible.

remove cell debris. To eliminate gelatinases, the centrifuged supernatant (1.5liters) was subjected to column chromatography using gelatin-cellulofine (bedvolume of 90 ml; Seikagaku Kogyo, Tokyo), and the fraction passing throughthe column was obtained. This procedure was repeated three times to removeany trace amounts of remaining gelatinase, and the resulting passing-throughfraction was concentrated to 50 ml. The concentrated fraction (fraction 0),after testing for the absence of gelatinase activity, was used for detection of

factor(s) enhancing gelatinase secretion. Amounts of protein were determinedwith a protein assay reagent from Bio-Rad Laboratories using BSA as a

standard.

Detection of a Factor Enhancing the Secretion OfMr 95,000 GCintiIthSC(MMP9). Trypsinized cells were washed once with 10% FCS-RPMI 1640medium and twice with serum-free RMPI 1640 medium. They were thensuspended in serum-free RPM! 1640 medium at the concentration of 10@cells/mI, and 1 ml of cell suspension was seeded in a culture dish of 30 mmdiameter. After culture overnight, medium was changed to RPM! 1640 medium containing 100 @g/mlof BSA with or without fraction G, and culturesupernatant was collected after 24 h. The gelatinase activities in the supematant with fraction 0 were measured and compared with those in the culturesupernatant without fraction 0. To examine the effect of fraction 0 on thesecretion of total protein, 10 @Ciof [35S]methionine(Trans 35S-label; ICNBiochemicals, Inc.) were added to the culture, and after 24 h, protein in theculture supernatant was precipitated with 5% trichloroacetic acid. The amountof secreted protein was expressed as total radioactivity in the precipitate, andthe radioactivity from the culture supernatant with fraction G was comparedwith that without fraction 0. The heat stability of the factor enhancinggelatinase secretion was determined by measuring the activity of the fraction0 after heating for 10 mm in boiling water. To test the sensitivity of the factorto trypsin digestion, fraction 0 was incubated with one-twentieth of its amountby weight of trypsin for 20 h at 37°C.After soybean trypsin inhibitor wasadded, digested fraction 0 was examined for enhancing activity.

Northern Analysl& To analyze the expression of gelatinases, total cellular

RNA was prepared by acid guanidine thiocyanate-phenol-chloroform extraction (14). RNA (20 @g)was fractionated on a 1% agarose gel containing 2.2M formaldehyde, transferred to Hybond N (Amersham), and hybridized with

DNA probe randomly labeled with [a-32PJdCT'P. cDNA for murine MMP9

(15) and human MMP2 (16) were used as DNA probes.

RESULTS

Time courses of secretion of Mr 60,000 and 95,000 gelatinases(MMP2 and MMP9, respectively) by LuM1 and NM11 cells intoserum-free RPMI 1640 medium are shown in Fig. 1. The amount ofM1 95,000 gelatinase in the serum-free culture medium of LuM1 cellsincreased exponentially, not linearly, with time in culture, contrasting

@003

I:

Fig. 1. Gelatinase secretion into serum-free culture medium by LuM1 and NM11 Cells.Samples of 2.5 x 1O@cells in culture dishes of 60 mm diameter were cultured overnightin 4 ml of RPMI 1640 medium with 10% FCS, the medium was changed to serum-freeRPM) 1640, and the culture supernatants obtained at 24-h intervals were analyzed forgelatinases by zymography. M, 95,000 and 60,000 gelatinase activities detected werequantitated by densitometrictracing ofthe zymograms. Points(bars), means(±ranges ofpoints) from duplicate determinations. Insets, zymograms of 48-h culture supematants.

0 20 40 60 80 0 20 40 60 80Time (h) Time (h)

3612

Research. on August 18, 2021. © 1994 American Association for Cancercancerres.aacrjournals.org Downloaded from

Table1 EffectoffractionG onproteinsecretionbyLuMJcellsLuM1cells (5 x 10') were cultured overnight in 1 ml of serum-free RPMI 1640

medium containing 100 pg/mi of BSA and 2 pCi/mi of [35S]methionine in thepresenceandabsenceof Fraction0. Proteinin theculturemediumwascollectedbyprecipitationwith5% trichloroacetic acid. Amount of prutcin secreted into the medium wasexpressedas

radiOactiVityin thisprecipitatedfraction.Dataaremeans±therangeofvaluesoftwoindependentdeterminations.Protein

secretedCulturesupernatant (10@ X cpm/10'cells)Without

fraction0 3.91±0.32Withfraction G 4.53 ±0.05

5

00.01 0.1 1 10

Fraction G (@sg/mIprotein)

Fig. 3. Effect of the amount of fraction 0 on gelatinase secretion of LuM1 cells. Cellswere cultured as described for Fig. 2 with varying amounts of added fraction 0, and theculture supernatant was analyzed for gelatinase secretion. Points (bars), means (±rangesof points) from duplicate determinations.

Matrix metalloproteinases are secreted as zymogens that have noactivity without activation in vivo by proteolytic cleavage of the

@2t@mn'nalpeptide or in vitro by treatment with various reagentssuch as amino phenyl mercuric acetate and SDS. Activated metalloproteinases degrade the components of extracellular matrix accordingto their substrate specificities: interstitial collagens by interstitialcollagenase (MMP1); type N collagen by gelatinases (MMP2 andMMP9), stromelysins (MMP3 and MMP1O), and matrilysin (MMP7);and proteoglycans by MMP3, MMP1O, and MMP7 (2). However, theactivity of these metalloproteinases is inhibited by inhibitors such asa@-macroglobulin in serum or liMP in the body fluid (2). The liMPsare secreted by the same cells that secrete the metalloproteinases. Theactivity of metalloproteinases is thus strictly controlled throughout the

AUTOCRINE FACFOR ENHANCEMENT OF GELATINASE SECREI1ON

A BkDa

95@

60-i

C40 kDa

. -95

@@!;@

Fig. 2. Effect of fraction G on gelatinasesecretion by LuM1 cells. Samples of 1 X 10'trypsinized LuM1 cells were cultured overnight inculture dishes of 30 mm diameter with 1 ml ofserum-free RPM! 1640 medium, the medium waschanged to the RPMI 1640 medium containingBSA (100 g.@g/ml)with or without fraction 0(Fr.G), and after 24 h, culture supernatants wereanalyzed for gelatinase secretion. A, zymogramsof culturesupernatantswith (+) andwithout(—)fraction G. B, relative gelatinase activities quantitated by densitometric tracing of the zymogram.Columns (bars), means (±ranges of points) fromduplicate determinations. C, immunostaining ofM, 95,000 gelatinase in culture supernatant with(+) or without (—)fraction0, after concentration, fractionationby SDS-PAGE,and blottingonto nitrocellulose,using monoclonalantibodyraisedin a rat againstmouseM@95,000gelatinase(MMP9).

95kDa 60 kDa

processes of secretion, activation, and inhibition. This means thatthere are various difficulties in estimating the activity of metalloproteinases in crude samples, because they must be activated at the sametime as inhibitors are eliminated. Zymography overcomes these diificulties and, therefore, is considered to be the best method fordetection of gelatinases, with proteins in the samples being firstseparated by SDS-PAGE so that the liMPs and the molecular speciesof gelatinases are located according to their molecular size. Theinactive form of gelatinases, zymogens, are activated by SDS in theprocess. After samples are renatured with Triton X-100, all of themolecular species of gelatinases can be easily detected without anyspecial treatment to activate gelatinases or to eliminate the inhibitorsin the crude samples.

Quantitative measurements reflecting secretion of Mr 60,000 and95,000 gelatinases (MMP2 and MMP9, respectively) by metastaticLuM1 cells showed that the accumulation of the latter in serum-freemedium was not linearly proportional to time in culture but ratherexponential, in contrast to the secretion of MMP2 by LuM1 cells andboth MMP2 and MMP9 by poorly metastatic NM11 cells. Because theamount of MMP9 secreted in 24 h by LuM1 cells, tested after dailyexchange of the culture medium, did not change for several days (data

60C0

Fig. 4. Amount of factor enhancing the secretion OfM, 95,000 gelatinase in serum-freeculture supernatant of LuM1 cells. LuMI cells, after reaching approximate confluence,were cultured in serum-free RPM! 1640, and culture supernatants were obtained every24 h after changing to further serum-free medium. Enhancing activity of Mr 95,000gelatinase in culture supernatants was estimated and expressed as units/ml (1 unit ofenhancing activity equals that giving half-maximum enhancement of gelatinase secretionin the standard assay). Specific activity (units/mg protein) was calculated from units ofenhancing activity, and the amount of protein in the supernatant was determined with aprotein assay reagent from Bio-Rad Laboratories. Columns and points (bars), means(±ranges of points) from duplicate determinations.

3613

20

:@

w 10

C

C

3100

24 48 72Time (h)

Research. on August 18, 2021. © 1994 American Association for Cancercancerres.aacrjournals.org Downloaded from

Control FractionG Heated TIYPSinIZOdFraCtionG FractionG

AUTOCRINEFACFORENHANCEMENTOF GELAT1NASESECREI1ON

collagen, and Matrigel has been reported (21—23),and perturbation ofcell-extracellular matrix contacts with antibodies to cellular receptorsis known to influence the secretion of type IV collagenase (24, 25).Soluble factors such as growth factors and cytokines have been shownto stimulate the secretion of matrix metalloproteinases: collagenase(MMP1) and stromelysin (MMP3) by IL-1f3, tumor necrosis factor-a,EGF, basic fibroblasts growth factor, and platelet-derived growthfactor (26—30),and Mr 92,000 gelatinase (MMP9) by EGF, IL-1@3,and tumor necrosis factor-a (31—33).TGF-f3 suppresses the secretionof MMP1 and MMP3 (30, 34) but augments that of MMP2 andMMP9 (35—37).Thus, the expression of MMPs by tumor cells appears to depend on what kinds of receptors for growth factors orcytokines they possess or whether the soluble factors enhancing orinhibiting secretion of MMPs are supplied from outside. For example,compounds from fibroblasts in the stroma surrounding tumor tissuesaugment their secretion of MMP2 and MMP9, influencing the metastatic potential of tumor cells (38, 39).

Because it is well known that tumor cells can also, themselves,secrete growth factors or cytokines, the possibility of autocrine stirsulation must be taken into account as demonstrated by the presentstudy. Paracrine stimulation by factors derived from tumor cells hasbeen reported for production of MMPs by fibroblasts. The paracrinefactor, tumor cell-derived collagenase stimulatory factor, was originally found in conditioned medium of human tumor cells and, whenpurified from the cell surface, strongly stimulated the secretion ofMMP1, MMP3, and MMP2 (40). While tumor cell-derived collagenase stimulatory factor does not act on the tumor cells themselves,autocrine factors stimulating MMP secretion have been reported.Rabbit synovial fibroblasts, when stimulated by 12-O-tetradecanoylphorbol-13-acetate or IL-1@3,synthesize and secrete autocrinefactors, which in turn induce secretion of MMP1. Purification of thefactors revealed them to be serum amyloid A and @32-microglobulin(41, 42). In addition, autocrine intermediate stimulating factors havebeen reported in SPARC (osteonectin)-treated fibroblasts (43). Auto

not shown), the presence of a factor enhancing its production wassuggested. A similar pattern was recently reported for 12-O-tetradecanoylphorbol-13-acetate-induced secretion of MMP1 and liMP byU937 cells (17) and for interleukin-1@3- and TGF-a/EGF-inducedexpression of MMP9 and MMP1 by rat mucosal keratinocytes (18). Afactor(s) enhancing MMP9 secretion could actually be demonstratedin culture medium conditioned with LuM1 cells for 48 h, without anyeffects on secretion of MMP2. The fact that the amount of the factorin the culture medium increased with time in culture directly implicated the LuM1 cells in its production, thereby indicating autocrineenhancement.

Microenvironmental conditions have been shown to regulate theexpression of MMPs: cell-cell or cell-extracellular matrix interactions Fig.7. Comparisonof autocrineenhancementof Mr95,000gelatinasesecretionbeand transport of soluble factors such as growth factors and cytokines tweenLuM1andNM11cells.G fractions(Fr.G)werepreparedfromLuM1andNM!!

cells, and enhancmg activity of M 95,000 gelatmase secretion of each was examined.are involved. Signal transduction through the fibronectin receptor has SccretionofM,95,000gelatinaseofLuMicellswasstimulatedbyfraction0 preparationsbeen reported to induce collagenase and stromelysin expression frombothLuM1andNM!! cells,butthatof NM!! cellswasenhancedonlyby the

. . fraction G from LuMI cells. Fraction G from NM! 1 cells did not stimulate M 95,000

(19, 20). Augmented secretlon of type IV collagenase due to interac- gelatinasesecretionbyNM!1cells,incontrasttotheclearstimulationintheLuM1case.tion of tumor cells with extracellular matrix proteins such as laminin, Points(bars),means(±rangesof points)fromduplicatedeterminations.

3614

MMP9 MMP2

28S—@ •0 *@•

18S—

—+ —+

Fig. 5. Northern analysis of mRNA levels of MMP9 and MMP2. Total cellular RNAwas preparedby acid guanidinethincyanate-phenol-chlomformextractionfromLuM!cells cultured in the presence (+) or absence (—)of fraction 0. RNA (20 @g)wasfractionated on a 1% agarose gel containing 2.2 Mformaldehyde, transferred to HybondN (Amersham), and hybridized with DNA probes randomly labeled with [a-32P]dCTP.cDNAsusedas probeswerefor murineMMP9andhumanMMP2.

80

Fig. 6. Stability of enhancing activity of gelatinase secretion after heating or trypsindigestion. Fraction G (20 @gprotein in 200 @d)was incubated with ! @.tgtrypsin for24 h at 37°Cand then tested for activity after 5 @gsoybean trypsin inhibitor was added.Fortheheatstabilitytest,fractionGwaskeptinboilingwaterfor10mm.Columns(bars),means (±ranges of points) from duplicate determinations.

I0.E

0C

0 20 40 60 80 100Fracdon G Added (pg/mi protein)

Research. on August 18, 2021. © 1994 American Association for Cancercancerres.aacrjournals.org Downloaded from

AtTFOCRINE FACrOR ENHANtEMENT OF GELATINASE SECRETION

crine stimulation of MMP3 has been reported with culture mediumconditioned with metastatic murine mammary carcinoma cells, although the nature of the factor has not yet been characterized (44). Aunique autocrmnefactor with an Mr of 70,000, the invasion-stimulatingfactor that stimulates secretion of MMP2 and invasion of tumor cells,has also been purifiedfrom culturemediumconditionedwith metastatic human prostatic cancer cells (45). Invasion-stimulating factordid not directly stimulate an increase in mRNA levels of MMP2 but

was suggested to regulate the receptor-mediated activation of processes controlling protease release (46). The description of an autocrine stimulation of MMP9 secretion by TGF-f3 released from fibrosarcoma cells transfected with a plasmid containing a porcine TGF-@1regulated by a metallothionein promoter has been reported (47), andthe present results further suggest that autocrine control of MMPs isa widespread phenomenon, occurring in a variety of cells.

The factor detected in the culture medium of metastatic murinecolon carcinoma LuM1 cells in the present study demonstrated aspecificity for MMP9 with no stimulation of MMP2. The steady-statelevel of MMP9 mRNA was elevated, indicating that transcription orstabilization of mRNA is switched on in contrast to the prostaticcancer cell case. The comparison of autocrine enhancement of MMP9

secretion between LuM1 and NM11 cells revealed a complex naturefor the factor stimulation. The fact that the MMP9 secretion of LuM1cells was stimulated by fraction 0 from both sources, but in the NM11case, only that from LuM1 cells demonstrated activity, precludes asingle factor and receptor explanation and suggests that multiplefactors or steps such as secretion and activation are involved in theenhanced secretion of MMP9 by LuM1 cells. However, the finding

that culture supernatant conditioned with poorly metastatic NM11cells did not give any enhancement of MMP9 secretion by the samecells suggests that the autocrine function is directly related to themetastatic nature of LuM1 cells. Thus, expression of the factor(s) bycolon tumor cells enhancing MMP9 secretion might be intimatelylinked with tumor progression.

ACKNOWLEDGMENTS

We gratefully acknowledge the kind gift of human MMP2 cDNA fromDr. G. I. Goldberg, Washington University School of Medicine.

REFERENCES

1. Liotta, L A., Rao, C. N., and Wewer, U. M. Biochemical interactions of tumor cellswith the basement membrane. Annu. Rev. Biochem., 55: 1037—!057,1986.

2. Matrisian, L M. The matrix-degrading metalloproteinases. Bioessays, 14: 455-463,1992

3. Woessner, J. F., Jr. Matrix metalloproteinases and their inhibitors in connective tissueremodeling. FASEB J., 5: 2145—2154,1991.

4. Liotta, L A., Tryggvason, K., GarbiSa, S., Hart, I., Foltz, C. M., and Shafle, S.Metastatic potential correlates with enzymatic degradation of basement membranecollagen. Nature (Lund.), 284: 67—68,1980.

5. Garbisa,S., Poziatti,R@,Muschel,R. J., Saffiotti,U., Ballin,M.,Goldfarb,R. H.,Khoury, 0., and Uotta, L A. Secretion of type IV collagenolytic protease andmetastaticphenotype:inductionby transfectionwithc-Ha-msbutnotc-Ha-rasplusAd2-Ela. Cancer Res., 47: 1523—1528,1987.

6. Yamagata, S., Ito, Y., Tanaka, R., and Shimizu, S. Gelatinases of metastatic cell linesof murinecoloniccarcinomaas detectedby substrate-gelelectrophoresis.Biochem.Biophys.Res.Commun.,151: 158—162.,1988.

7. Yamagata, S., Tanaka, R., Ito, Y., and Shimizu, S. Gelatinases of murine metastatictumor cells. Biochem. Biophys. Res. Commun., 158: 228—234, 1989.

8. Bernhard, E. J., Muschel, R. J., and Hughes, E. N. M, 92,000 gclatinasc releasecorrelates with the metastatic phenotype in transformed rat embryo cells. Cancer Rca.,50: 3872—3877,1990.

9. Margulies, I. M. K.. Hoyhtya, M., Evans, C., Stracke, M. L, Uotta, L A., andStctler-Stevenson, W. 0. Urinary type N collagcnasc: elevated levels are associatedwith bladder transitional cell carcinoma. Cancer EpidemioL Biomarkers Prey., 1:467—474,1992.

10. Srcenath, T., Matrisian, L M., Stetler-Stevenson, W., Gattom-Celli, S., and Pozzatti,R. 0. Expressionof matrixmetallopmteinasegenesin transformedrat cell linesofhigh and low metastatic potential. Cancer Res., 52: 4942—4947,1992.

11. McDonnell, S., and Matrisian, L M. Stromelysin in tumor progression and metasta

sis Cancer Metastasis Rev., 9: 305-319, !990.12. McDonnell, S., Navre, M., Coffey, R. J., and Matrisian, L M. Expression and

localization of the matrix metalloprotcinase pump-! (MMP-7) in human gastric andcolon carcinomas. Mol. Carcinog., 4: 527—533,1991.

13. Tsuruo, T., Yamori, T., Naganuma, K., Tsukagoshi, S., and Sakurai, Y. Characterizationof metastaticclones derivedfrom a metastaticvariant of mouse colonadenocarcinoma 26. Cancer Rca., 43: 5437-5442, 1983.

!4. Chomczynski, P., and Sacehi, N. Single-step method of RNA isolation by acidguanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162:156—159,1987.

15. Tanaka, H., Hojo, K., Yoshida, H., Yoshioka, T., and Sugita, K. Molecular cloningand expression of the mouse 105-kDa gelatinase cDNA. Biochem. Biophys. Res.Commun., 190: 732—740,1993.

16. Collier, I. E., Wilhelm, S. M., Eiscn, A. Z., Manner, B. L, Grant, G. A., Seltzer,J. L, Kronberger, A.. He, C., Bauer, E. A., and Goldberg, G. I. H-ms oncogenetransformed human bronchial epithelial cells (TBE-!)secrete a single metalloproteasecapable ofdegrading basement membrane collagen. J. Biol. Chem., 263: 6579—6587,1988.

17. ShapirO,S. D., Doyle, 0. A. R., Ley, T. J., Parks, W. C., and Webs, H. 0. Molecularmechanisms regulating the production of collagenase and TIMP in U937 cells:evidence for involvement of delayed transcriptional activation and enhanced mRNAstability. Biochemistry, 32: 4286—4292, 1993.

18. Lyons, J. G., Birkedal-Hansen, B., Pierson, M. C., \Vhitelock, 1. M., andBirkedal-Hansen, H. Interleukin-1@ and transforming growth factor-a/epidermalgrowth factor induce expression of M, 95,000 type IV collagenase/gelatinase andinterstitial fibroblast-type collagenasc by rat mucosal keratinocytes. 1. Biol. Chem.,268: 19143—19151,1993.

19. Werb, Z., Tremble, P. M., Behrendtsen, 0., @rowley,E., and Damsky, C. H. Signaltransduction through the fibronectin receptor induces collagenase and stromelysingeneexpression.J. CellBiol.,109:877—889,1989.

20. Werb, Z., Tremble, P., and Damsky, C. H. Regulation of extracellular matrix degradationbycdll-extracellularmatrixinteractions.CellDiffer.Dcv.,32: 299-306,1990.

21. Turpeenniemi-Hujanen, T., Thorgeirsson, U. P., Rao, C. N., and Uotta, L A. Lamininincreases the release oftype N collagenase from malignantcells. 1. Biol. Chem., 261:1883—1889,1986.

22. Kanemoto, T., Reich, R., Royce, L, Greatorex, D., Adler, S. H., Shiraishi, N., Martin,G. R., Yamada, Y., and Kleinman, H. K. Identification of an amino acid sequencefrom the laminin A chain that stimulates metastasis and collagenasc IV production.Proc. Nat!. Acad. Sci. USA, 87: 2279-2283, 1990.

23. Emonard, H., Christiane, Y., Smet, M., Grimaud, J. A., and Foidart, 1. M. Type IVand interstitial collagenolytic activities in normal and malignant trophoblast cells arespecifically regulated by the extracellular matrix. Invasion Metastasis, 10: !70—l77,1990.

24. Seftor, R. E. B., Seftor, E. A., Stetler-Stevenson, W. G., and Hendrix, M. J. C. The72 kDa type IV collagenase is modulated via differential expression aj3, and a5@1integrins during human melanoma cell invasion. Cancer Rca., 53: 3411—3415,1993.

25. Latjava, H., Lyons, J. 0., Salo, T., Macla, M., Koivisto, L, Birkedal-Hansen, H.,Akiyama, S. K., Yamada, K. M., and Heino, J. Anti-integrin antibodies induce typeIV collagenase expression in keratinocytes. J. Cell. Physiol., 157: 190—200,1993.

26. Chua, C. C., Geiman, D. E., Keller, 0. H., and Ladda, R. L Induction of collagenasesecretion in human fibroblast cultures by growth promoting factors. J. Biol. (1cm.,260: 5213—5216,1985.

27. Dayer, J-M., Beutler, B., and Cerami, A. Cachectin/tumor necrosis factor stimulatescollagenase and prostaglandin E@production by human synovial cells and dermalfibroblasts. J. Exp. Med., 162: 2163—2168, 1985.

28. Matrisian, L M., Leroy, P., Ruhlmann, C., Gesnel, M-C., and Breathnach, R.Isolation of the oncogene and epidermal growth factor-induced transin gene: complexcontrol in rat fibroblasts. Mol. Cell. Biol., 6: 1679—1686,1986.

29. Saus, J., Quinones, S., Otani, Y., Nagase, H., Harris, E. D., Jr., and Kurkinen, M. Thecomplete primary structure of human matrix metalloproteinase-3. Identity withstromelysin.J. Biol.(1cm., 263:6742—6745,1988.

30. Edwards, D. R., Murphy, 0., Reynolds, J. J., Vlhitham, S. E., Docherty, A. J. P.,Angel, P., and Heath, J. K. Transforming growth factor beta modulates the expressionof collagenase and metalloproteinase inhibitor. EMBO J., 6: 1899—1904,1987.

31. Wilhelm, S. M., Collier, I. E., Manner, B. L, Eisen, A. Z., Grant, 0. A., andGoldberg,0. I. SV4O-transformedhumanlungfibroblastssecretea 92-Wa typeIVcollagenase which is identical to that secreted by normal human macrophages. J. Biol.(1cm.,264:17213—17221,1989.

32. Okada, Y., Tsuchiya, H., Shimizu, H., Tomita, K., Nakanishi, I., Sato, H., Sciki, M.,Yamashita, K., and Hayakawa, T. Induction and stimulation of 92-kDa gelatinasc/type IV collagenase production in osteosarcoma and fibrosarcoma cell lines by tumornecrosis factor a. Biochem. Biophys. Rca. Commun., 171: 610—617, 1990.

33. Shima, I., Sasagun, Y., Kusukawa, J., Nakano, R, Yamana, H., Fujita, H., Kakegawa,T., andMorimatsu,M.Productionof matrixmetalloproteinase9 (92-kDagelatinase)by humanoesophagealsquamouscell carcinomain responseto epidermalgrowthfactor. Br. J. Cancer, 67: 721—727,1993.

34. Machida, C. M., Muldoon, L L, Rodland, K. D., and Magun, B. E. Transcriptionalmodulation of transin gene expression by epidermal growth factor and transforminggrowth factor beta. Mol. Cell. Biol., 8: 2479—2483,1988.

35. Overall, C. M., Wrana, J. L., and Sodek, J. Independent regulation of collagenase,72-kDa progelatinase, and metalloendoproteinase inhibitor expression in humanfibroblasts by transforming growth factor-a. J. Biol. Chem., 264: 1860—!869,1989.

36. Welch, D. R., Fabra, A., and Nakajima, M. Transforming growth factor@ stimulatesmammary adenocarcinoma cell invasion and metastatic potential. Proc. Nat!. Acad.Sd. USA, 87: 7678—7682,1990.

37. Salo, T., Lyons, J. G., Rahemtulla, F., Birkedal-Hansen, H., and Larjava, H. Trans

3615

Research. on August 18, 2021. © 1994 American Association for Cancercancerres.aacrjournals.org Downloaded from

AUTOCRINE FACI'OR ENHANCEMENT OF GELATINASE SECRETION

forminggrowthfactor-a! up-regulatestype IV collagenaseexpressionin culturedhumankeratinocytes.J. Biol.(1cm., 266: 11436—1144!,199!.

38. Nakajima, M., Morikawa, K., Fabra, A., Bucana, C. D., and Fidler, I. J. Influence oforgan environment on extracellular matrix degradative activity and metastasis ofhuman colon carcinoma cells. J. Nati. Cancer Inst., 82: 1890—1898, 1990.

39. Fabra,A.,Nakajima,M.,Bucana,C. D.,andFidler,I. J. Modulationof the invasivephenotypeofhumancoloncarcinomacellsbyorganspecificfibroblastsofnudemice.Differentiation, 52: 101—110,1992.

40. Kataoka, H., DeCastro, R., Zucker, S., and Biswas, C. Tumor cell-derived collagenase-stimulatory factor increases expression of interstitial collagenase, stromelysin,and 72-kDa gelatinase. Cancer Rca., 53: 3154—3!58,1993.

41. Brinckerhoff,C. E.,andMitchell,T. I.Autocrinecontrolofcollagenasesynthesisbysynovial fibroblasts. J. Cell. Physiol., 136: 72—80,1988.

42. Brinckerhoff, C. E., Mitchell, 1. 1., Karmilowicz, M. J., Kluve-Beckerman, B., andBenson, M. D. Autocrine induction of collagenase by serum amyloid A-like and

@2-microglobulin-like proteins. Science (Washington DC), 243: 655—657, 1989.43. Tremble, P. M., Lane, T. F., Sage, E. H., and Werb, Z. SPARC, a secreted protein

associated with morphogenesis and tissue remodeling, induces expression of metalloproteinasesin fibroblaststhrougha novelextracellularmatrix-dependentpathway.J. Cell Biol., 121: 1433—1444,1993.

44. Korczak, B., Kerbel, R. S., and Dennis, J. W. Autocrine and paracrine regulation oftissue inhibitor of metalloproteinases, transin, and urokinasc gene expression inmetastatic and nonmetastatic mammary carcinoma cells. Cell Growth Differ.,2: 335—341,1991.

45. Stearns, M. E., and Wang, M. Regulation of kinesin expression and type IV collagenase secretion in invasive human prostate PC-3 tumor sublines. Cancer Rca., 51:5866—5875,199!.

46. Stearns, M. E., and Stearns, M. Autocrine factors, type IV collagenase secretion andprostaticcancercell invasion.CancerMetastasisRev.,12: 39—52,1993.

47. Samuel, S. K., Hurts, R. A. R., Kondaiah, P., Khalil, N., Turley, E. A., Wright, J. A..andGreenberg,A. H.Autocrineinductionof tumorproteaseproductionandinvasionby a metallothionein-regulatedTGF-@1(Ser223,225).EMBOJ., 11: 1599—1605,1992.

3616

Research. on August 18, 2021. © 1994 American Association for Cancercancerres.aacrjournals.org Downloaded from

1994;54:3611-3616. Cancer Res   Sumiko Hyuga, Yohko Nishikawa, Keita Sakata, et al.   Conditioned with Murine Metastatic Colon Carcinoma CellsGelatinase (Matrix Metalloproteinase 9) in Serum-free Medium

95,000rMAutocrine Factor Enhancing the Secretion of

  Updated version

  http://cancerres.aacrjournals.org/content/54/13/3611

Access the most recent version of this article at:

   

   

   

  E-mail alerts related to this article or journal.Sign up to receive free email-alerts

  Subscriptions

Reprints and

  .pubs@aacr.orgDepartment at

To order reprints of this article or to subscribe to the journal, contact the AACR Publications

  Permissions

  Rightslink site. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC)

.http://cancerres.aacrjournals.org/content/54/13/3611To request permission to re-use all or part of this article, use this link

Research. on August 18, 2021. © 1994 American Association for Cancercancerres.aacrjournals.org Downloaded from