The pattern of metalloproteinase expression by corneal fibroblasts is

altered by passage in cell culture

M. ELIZABETH FINP and MARIE T. GIRARD

Eye Research Institute and Departments of Ophthalmology, and Anatomy and Cell Biology, Harvard Medical School, Boston,Massachusetts 02114, USA

* Author for correspondence

Summary

We have examined the pattern of expression of fourdifferent matrix metalloproteinases (MMPs), col-lagenase, stromelysin, 92 kD gelatinase, and 72 kDgelatinase, by primary and passaged cultures ofrabbit corneal fibroblasts. Primary cultures of thiscell type have previously been shown to reproducethe normal tissue regulation of collagenase ex-pression. We demonstrate qualitative and quantitat-ive changes in the pattern of MMP expression as thecells are passaged in culture. Only a single MMP,72 kD gelatinase, is constitutively expressed by pri-mary fibroblast cultures. Phorbol myristate acetate(PMA) treatment upregulates expression of 72 kDgelatinase and turns on the expression of collagenaseand stromelysin, as well as 92 kD gelatinase. How-ever, the degree to which MMP expression is inducedis minimal. Cells subcultured but a single timeconstitutively produce not only 72 kD gelatinase, butalso collagenase and stromelysin. In addition, PMAtreatment upregulates expression of collagenase,stromelysin and 92 kD gelatinase to high levels. Incontrast, the expression of 72 kD gelatinase isrepressed by treatment of passaged cell cultures with

PMA. Our data indicate that the cell does not simplyturn the MMP genes on or off, as a group, in responseto various agents, but that it has the capacity for finecontrol over which MMPs are expressed and thedegree to which each is expressed. Changes in MMPprotein expression induced by PMA treatment arecorrelated with changes in specific mRNA levels inpassaged cultures. The kinetics of mRNA accumu-lation suggest that the MMP genes can respond tomultiple intracellular signals initiated in a temporalcascade by PMA. It is the combined effects of theindividual signals on the accumulation of specificmRNAs that must determine the ultimate pattern ofMMP protein expression. The distinct patterns ofMMP expression produced by primary and passagedcell cultures may be analogous to patterns ofexpression that might occur under particular in vivoconditions.

Key words: metalloproteinase, cornea, phorbol myristateacetate.

Introduction

Members of the family of enzymes known as the matrixmetalloproteinases (MMPs) have the capacity to degradecomponents of the extracellular matrix. These enzymes,along with their specific inhibitors, the tissue inhibitors ofmetalloproteinases (TIMPs), can be synthesized andsecreted locally by the resident cells of a tissue or byrecruited inflammatory cells. The MMPs are neutralproteinases showing greatest activity at the pH of theextracellular space, and they share a common require-ment for a zinc metal cofactor. A number of the MMPfamily members have now been cloned and sequenced(Matrisian et al. 1985; Goldberg et al. 1986; Whitham et al.1986; Fini et al. 1986, 1987a,6; Brinckerhoff et al. 1987;Wilhelm et al. 1987, 1989; Muller et al. 1988; Saus et al.1988; Collier et al. 19886). The sequence information hasdemonstrated their close relationship at the level ofprimary structure and suggests that the MMPs evolvedfrom a common ancestral gene. Further evidence substan-tiating this hypothesis is the identical intron/exonorganization of the collagenase (Fini et al. 19876; Collier et

Journal of Cell Science 97, 373-383 (1990)Printed in Great Britain © The Company of Biologists Limited 1990

al. 1988a) and stromelysin (Matrisian et al. 1986;Breathnach et al. 1987) genes., On the basis of preferential activity toward a specific

complement of extracellular matrix components, theMMPs might be classified into three subfamilies: strome-lysins, collagenases and gelatinases. The stromelysins cancleave proteoglycans, as well as native type IV collagen,fibronectin and laminin (Chin et al. 1985). The collagen-ases act specifically on native collagen types I, II and III,with little activity against any other substrate (Gross,1982). They attack the collagen triple helix at a singlepoint, two-thirds of the way from the N terminus. After theinitial cleavage, the collagen triple helix becomes de-natured, and the dissociated polypeptide chains, gelatinmolecules, can then be degraded by gelatinases. Enzymesof the gelatinase subfamily have activity against de-natured collagen molecules (gelatin) and can also cleavenative type IV, V and VII collagens (Murphy et al. 1989).

Collagenase was the first of the MMPs to be character-ized (Gross and Lapiere, 1962) and cloned (Gross et al.1984), and thus its role in health and disease is the bestunderstood of all the MMPs (Wooley and Evanson, 1980).

373

Increased collagenase levels are correlated with thehomeostatic and developmental events of connectivetissue remodeling as well as the debridement andremodeling associated with wound healing. Expression ofcollagenase at abnormally high levels is thought to play acentral role in the pathological destruction of connectivetissue in such disorders as corneal ulceration, rheumatoidarthritis and recessive epidermolysis bullosa; over-ex-pression of stromelysins and gelatinases have also, morerecently, been correlated with these disease processes (forexample, see Okada et al. 1989). In addition, inappropriateexpression of all three types of MMPs has been associatedwith the invasive and metastatic capacities of transformedcells (for example, Ura et al. 1989).

Clearly, an understanding of the mechanisms control-ling MMP gene expression is important for developingrational means of managing disorders involving theseenzymes. Early experiments suggested that all the MMPgenes might be under coordinate control. For example,stromelysin mRNA accumulates in rabbit synovial cellswith similar kinetics to collagenase mRNA after treat-ment with the inflammatory agent phorbol myristateacetate (PMA; Brinckerhoff and Fini, 1989). Recently,however, a few studies have shown that expression of theMMP genes may be independently regulated by cells.Thus, collagenase and stromelysin mRNAs are notcoordinately induced in a rabbit mammary epithelial cellline in response to treatment with PMA (Werb and Clark,1989). In addition, expression of collagenase and 72 kDgelatinase proteins is reciprocally regulated by transform-ing growth factor-/? (TGF-/3) in cultures of human gingivalfibroblasts (Overall et al. 1989).

The capacity to express each of the MMP genesindependently would give a tissue additional capacity forfine control over the composition of its extracellularmatrix. Thus, it is important to document the patterns ofMMP expression that occur in tissues under differentconditions. All studies to date on coordination of MMPgene expression have made use of culture-passaged cells orcell lines. This limits the conclusions that can be drawnfrom the data, since it is well known that gene expressionin passaged cells may not accurately reflect what occurswhen the cells are situated in the tissue. Thus, we havechosen to study coordination of MMP expression usingprimary cultures of corneal fibroblasts, a cell typepreviously shown to reproduce faithfully the normal tissuepattern of expression for the MMP, collagenase (Johnson-Muller and Gross, 1979; Wagoner and Johnson-Wint,1987). However, we have taken advantage of the fact thatthese cells change with passage in cell culture to explorefurther the potential of corneal fibroblasts to modulatetheir MMP expression from the normal tissue pattern. Wedemonstrate qualitative and quantitative changes in thepattern of constitutive, and PMA-induced MMP ex-pression as the cells are passaged in culture. The capacityof primary and passaged cells to respond to the samestimulus in different ways appears to be due to the abilityof the MMP genes to respond to multiple, intracellularsignals. The distinct patterns of MMP expression producedby primary and passaged cell cultures may be analogous topatterns of expression that might occur under particularin vivo conditions.

Materials and methods

Corneal cell cultureRabbit corneas were obtained from New Zealand White rabbits

(2.5 kg) killed by intravenous injection of sodium pentobarbitaljust prior to harvesting the tissues. The endothelial layer at theback of the cornea was removed manually. Using a trephine,9 mm corneal discs were cut from the remaining corneal tissue,placed in a solution of 0.25% trypsin dissolved in Hanks'Balanced Salt Solution (GIBCO, Grand Island, NY), and left at4°C overnight. The next day the epithelial layer of the cornealdisc was gently scraped from the stromal layer with a scalpel.Stromas were incubated in a solution of 4mgml~1 of bacterialcollagenase (Worthington, Freehold, NJ) for several hours, torelease fibroblasts. The collagenase was dissolved in completemedium consisting of Minimum Essential Medium (GIBCO)containing 10 % supplemented bovine serum (Hyclone, Utah, NY)and antibiotic/antimycotic (GIBCO).

To prepare passaged cell cultures, the fibroblasts obtained fromsix corneal discs were plated in a single, 100 mm culture dish withcomplete medium. These were considered to be primary cultures(rabbit fibroblast primaries or RF° cultures). Once cells reachedconfluency (about 3 days to a week), they were trypsinized briefly(less than lOmin) in calcium/magnesium-free Hanks' BalancedSalt Solution, then split into three new culture dishes (rabbitfibroblasts, first passage, or RF1 cultures). Once these subcul-tured cells reached confluency, they were again split three waysinto new dishes (RF2 cultures). Subculturing was continued inthis manner; cells were routinely used for an experiment by thefourth passage.

For experiments, fibroblasts were plated at the desired density(1 x 106 to 5 x 105 cells per well) in the 16 mm diameter wells of 24-well cluster dishes. Cells were left overnight in complete mediumto attach to the culture well. Before an experiment, serum-containing medium was removed, and the cells were washed threetimes with Hanks' Balanced Salt Solution to remove traces ofserum albumin. Then 350^1 of fresh, serum-free medium wasplaced on cells.

The rabbit corneal epithelial cell line, SIRC (Grabner et al.1983), was obtained from American Type Culture Collection(Rockville, MD). Cells were grown and passaged by the sameprocedures used for fibroblasts.

PMA (Sigma, St Louis, MO) was dissolved in dimethylsulfoxide(DMSO) at 5 X 1 0 " 4 M . For use in an experiment, this stock wasdiluted into culture medium. Fourth-passage fibroblasts wereused to perform a dosimetric analysis of the effects of PMA oncollagenase expression. Collagenase expression was induced in adose-dependent manner at PMA concentrations ranging from10~8M to 10~6M, but began to drop by a concentration of 10~6M.Thus PMA was used to treat cells at 10~6 M for all experiments.

Cytochalasin B (CB; Aldrich, Milwaukee, WI) was dissolved atlmgml"1 in DMSO. Cells were treated with this agent at7 fig ml'1 as recommended (Johnson-Muller and Gross, 1978).

Since DMSO was used as the vehicle for PMA and cytochalasinB treatment of cells, the effect of this agent, alone, on MMPexpression was tested. Even when DMSO was included in the cellculture medium at 3.5 %, which is higher than any concentrationused in PMA or CB experiments reported here, no change inexpression of any MMP was observed.

Labelling of cell culture proteins, gel electrophoresis andimmunoprecipitation[35S]methionine was added to serum-free culture medium at80/iCiml"1 for biosynthetic labelling of proteins. Passagedcultures were routinely labelled for 4h. Primary cultures wereless active in protein synthesis and incorporated much less labelinto secreted proteins than did passaged cell cultures. Thus, tomaximize detection of secreted proteins, these cultures werelabelled for 16-48 h. After labelling, medium containing labelled,secreted cell proteins was collected. Samples of 10—15;

and autoradiographed to display labelled proteins. The amount ofsynthesized and secreted collagenase and stromelysin wasquantitated by densitometry. In selected cases, the identities ofgel bands that corresponded to procollagenase or prostromelysinwere verified by immunoprecipitation using sheep anti-rabbitantisera (Fini et al. 1987a), gifts from C. Brinckerhoff (DartmouthMedical School).

ZymographyZymography was performed by the method of Birkedahl-Hansenand Taylor (1982). With this technique, proteolytic species areseparated on the basis of molecular size by electrophoresisthrough an SDS-polyacrylamide gel within which a substrate forthe enzyme of interest is co-polymerized. Subsequently, theposition of each enzyme in the gel is visualized by its ability todegrade the substrate. Proenzyme species as well as proteolyti-cally activated species can often be visualized; many proenzymescan be activated without a change in molecular size as a result ofthe change in their protein structure produced by the SDS in thegel. SDS-gels (11%) were prepared using a 37.5:1 stock ofacrylamide to bis-acrylamide, and gelatin (from bovine skin,Sigma, St Louis, MO) was included in the gel at a concentration of0.1 %. Samples of crude cell-conditioned medium were diluted 2:5in gel sample buffer before loading on a gel. After electrophoresisof the samples, the gel was shaken in a 2.5 % solution of TritonX-100 for lh , to remove SDS and then developed in reactionbuffer (10mM Tris-HCl, pH 7.6, 50mi CaCl2) overnight at 37°C.After the gel was stained with Coomassie Brilliant Blue R, thepositions of enzymatic species could be easily identified as clearbands in the stained gelatin-substrate background.

Although the technique of zymography has been used by anumber of investigators, we know of no report of a method forquantitating the enzyme activity in a sample that is visualized bythis method. In an attempt to devise such a technique, wemeasured the optical density volume of the cleared region of thesubstrate gels produced by gelatinase activity with a laserdensitometer (Molecular Dynamics, Sunnyvale, CA). Quanti-tation of serial dilutions of a sample of culture medium by thistechnique revealed that a twofold change in the amount ofenzyme loaded in the gel lane was not always reflected in acorresponding twofold change in the optical density volume ofsubstrate clearing. However, the values obtained from thedilution curve could be used to construct a standard curve. Thesubstrate clearing produced by the undiluted sample wasconsidered to represent 100 % activity of the sample, the twofolddilution was 50%, the fourfold dilution, 25%, etc. A relativeactivity value for all experimental samples could then be obtainedby comparison with the standard curve. A 1:2 stepwise dilution ofa standard sample was routinely run each time zymogramanalysis was performed; the standard curve constructed fromthese data was used only for zymograms performed togetherunder the same conditions as the dilution series. Activity valuesobtained for different cell culture treatments were compared onlyif all samples were electrophoresed on the same gel with the samestandard curve. If necessary, the standard curve was extrapolatedto cover enzyme activity in samples higher than the higheststandard value measured (for example, 150 % activity).

When quantitation of gelatinase activity was desired, exper-imental treatments to cell cultures were always performed inquadruplicate. The mean of all four samples was determined, andthe standard error of the mean calculated. The significance ofdifferences between two experimental treatments was deter-mined by use of a paired sample t-test.

RNA preparation and Northern blot analysisTotal RNA was isolated from cells as described (Fini et al. 1987a),and poly(A)+ RNA was prepared in some cases (Maniatis et al.1982). Northern blotting was performed by the glyoxal method(Maniatis et al. 1982). The probe for stromelysin mRNA was afull-length rabbit cDNA (Fini et al. 1987a); the human cDNA fortype IV collagenase (gelatinase; Collier et al. 1988a), a gift fromDr G. Goldberg, was used as a probe for the 72 kD gelatinasemRNA. A full-length, human cDNA clone for tissue inhibitor ofmetalloproteinases (TIMP-1), kindly contributed by Dr D.

Carmichael of Synergen Inc. (Boulder, CO), was also used as aprobe (Carmichael et al. 1986). Probes were labelled with 32Pusing the oligo-labelling method (Feinberg and Vogelstein, 1985).

Results

Differential inducibility of collagenase/stromelysin byPMA in primary or passaged corneal fibroblast culturesExpression of the MMPs, collagenase and stromelysin, bycorneal fibroblasts was assayed at different stages of cellculture passage. Constitutive expression was examined aswell as expression after treatment with the inflammatoryagent, PMA, which is known to stimulate collagenaseexpression in many systems (for example, Fini et al.1987a). In a number of different experiments, we consist-ently observed certain quantitative and qualitative differ-ences in the pattern of collagenase and stromelysinexpression by primary cell cultures as compared withculture-passaged cells. These differences were apparentboth before and after treatment of the cells with PMA. Arepresentative example of these results is shown in Fig. 1.This figure depicts the results from two different exper-iments in which secreted protein expression was examinedin fibroblasts that were freshly isolated from cornea (RF°),or fibroblasts that had been passaged but a single time inculture (RF1). Equal numbers of cells were plated inindividual wells of a 24-well cluster plate. Cultures werechanged the next day to serum-free medium, with orwithout the addition of PMA or CB. Proteins synthesizedand secreted by the cells were labelled by addition of[35S]methionine to the culture medium. First-passagecultures constitutively synthesized and secreted a varietyof different proteins (Fig. 1C, RF1); included among thesewas a protein migrating at 53 kD and one at 51 kD, whichwere prominently induced by PMA treatment of culturedcells. In addition, a minor protein of 57 kD was induced byPMA treatment. We have previously used immunoprecipi-tation analysis to identify these proteins as collagenaseand stromelysin (Fini and Girard, 1990). The immuno-precipitation experiment was repeated here (Fig. ID, IP).Both the 57 and the 53 kD proteins precipitated withcollagenase antibody; the sizes are appropriate for theglycosylated (57 kD) and unglycosylated (53 kD) forms ofprocollagenase. The 51 kD protein precipitated withstromelysin antibody, and its size also indicates theproenzyme form. In untreated cultures, collagenase andstromelysin represented 1.7% and 1.3% of the secretedproteins, respectively, as determined by densitometry.PMA treatment of cells for 24 h induced the combined rateof synthesis of the two collagenase proteins by 22.3-fold.Stromelysin was also induced, but to a lesser extent (11.4-fold). The greater inducibility of collagenase as opposed tostromelysin is a typical result seen in passaged cellcultures. In the experiment shown, collagenase rep-resented 18.7 % and stromelysin 8.8 % of the total secretedprotein produced by PMA-stimulated cells. In contrast,total synthesized and secreted proteins increased only 1.7-fold after PMA treatment.

Autoradiographic analysis demonstrated that primarycorneal fibroblast cultures, unlike passaged cultures,secreted very little protein in the right size range to beprocollagenase or prostromelysin (Fig. 1A, RF°). Primarycultures synthesized and secreted less protein thanpassaged cultures in general. However, even when thisproblem was alleviated by longer labelling periods forproteins (as shown in the figure), and longer exposure ofautoradiographs to film (not shown), no procollagenase or

Metalloproteinase expression by corneal fibroblasts 375

M,X1CT3

2 0 0 -

97-

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Mr

RF°

1 2 3 4 5 6

Fig. 1. MMPs secreted byuntreated, PMA-treated or CB-

1 3 A 5 A 7 ft O in n n treated cultures of primary and;i ., . passaged corneal fibroblasts. Size

2 0 0 " ' ' standards on all autoradiographsshown are indicated in Mr(xl0~

3).97_ ' (A,B) Equal numbers of primary

. _ L fibroblasts, plated in wells of a 24-well cluster plate were left

atj, untreated or treated with PMASm " "' (10"6M)orCB(7,ugmr1)for48h

,-• \ . _- in serum-free medium containing[36S]methionine. Expression of

4 3 " secreted proteins were assayed bygel electrophoresis andautoradiography, or byimmunoprecipitation analysis, of a

2 9 - sample from each culture well.(A) Total labelled proteins from the

g 48-h media samples. Lanes 1 and 2,__O untreated cells; lanes 3 and 4," PMA-treated cells; lanes 5 and 6,

CB-treated cells. The locations ofthe 53 and 51 kD proteins, inducedby PMA are indicated by dots onthe figure. (B) Proteins

^ 2 3 immunoprecipitated by anti-collagenase or anti-stromelysinantibody. Lanes 1-4, untreatedcells; lanes 5-8, PMA-treated cells;lanes 9-12, CB-treated cells. Lanes1, 3, 5, 7, 9, and 11, collagenaseantiserum; lanes 2, 4, 6, 8, 10, and12, stromelysin antiserum.(C) Equal numbers of first passage

43— if 4 | •• M f | fibroblasts were plated as above andtreated with PMA (10~6M) or CB

m 9 •• •• • • D (7/gmr1) for 20 h. At 20 h,^ _ — ^ IP [

35S]methionine was added to29— culture wells and incubation was

continued for four more hours.** • ~m~"^*^ Expression of secreted proteins were** "^ assayed by gel electrophoresis and

RF1 autoradiography. Lanes 1 and 2,untreated cells; lanes 3 and 4,

PMA-treated cells; lanes 5 and 6, CB-treated cells. The position on the gels of the two forms of procollagenase, glycosylated andunglycosylated (col), and stromelysin (str) are indicated as determined from immunoprecipitation analysis similar to that shown inD. (D) Autoradiograph showing immunoprecipitation analysis of a sample of culture medium containing secreted proteins from anuntreated culture of passaged fibroblasts. Lane 1, proteins immunoprecipitated with non-immune serum. Lane 2, proteinsimmunoprecipitated with anti-collagenase antiserum. Lane 3, proteins immunoprecipitated with anti-stromelysin antiserum. Inthis case, a single protein was precipitated by stromelysin antibody, but at other times a closely spaced doublet isimmunoprecipitated (data not shown). The precipitation of the doublet corresponds to the appearance of a doublet at 51 kD in thetotal protein profile (as in untreated lanes 1 and 2 of RF1 cultures shown in C). It seems likely that the occasional appearance ofthe doublet is the result of allelic variation in the stromelysin gene in our rabbit population.

97-«»

6 8 -

Mr

xicr3

97-68-

43-

prostromelysin could be visualized. Moreover, neitheranti-collagenase nor anti-stromelysin antibodies specifi-cally precipitated any labelled proteins secreted byuntreated cultures (Fig. IB, RF°). However, PMA treat-ment of cultures induced the appearance in the totalsecreted protein profile of a 53 kD and a 51 kD protein of asize appropriate to be procollagenase and prostromelysin(Fig. 1A, RF°). The 53 kD protein could be precipitatedwith anti-collagenase antibody and the 51 kD protein byanti-stromelysin antibody (Fig. IB, RF°). The degree ofinduction by PMA in primary cell cultures, however, wasnot as great as that in passaged cultures. In theexperiment shown, collagenase and stromelysin rep-resented only 2.5% and 3.1%, respectively, of the totalproteins synthesized and secreted by PMA-treated cul-tures.

The results with another reported inducer of collagenaseexpression, cytochalasin B (CB), on expression of secretedproteins by primary or passaged fibroblast cultures werequite similar to the results with PMA. CB inducedcollagenase and stromelysin expression in passaged cells,much as did PMA (Fig. 1C, RF1). However, primary cellcultures were refractory in their response to CB. In fact,primary fibroblast cultures treated with CB were notstimulated to produce collagenase and stromelysin at eventhe low rate induced by PMA treatment (Fig. 1A,B, RF°).

Differences in the pattern of 92 kD and 72 kD gelatinaseexpression in response to PMA in primary or passagedcorneal fibroblast culturesAnother protein of interest in the total profile of proteinssecreted by corneal fibroblasts was one that electrophor-

376 M. E. Fini and M. T. Girard

eses at 72 kD as shown on the autoradiograph in Fig. 1.Like collagenase and stromelysin, this protein showeddifferences in expression with cell passage. The 72 kDprotein was constitutively expressed in both primary andfirst-passage fibroblasts cultures. However, after PMAtreatment, expression of the 72 kD protein was induced inprimary fibroblast cultures, but repressed in the passagedcell cultures. The size of this protein suggested that itmight be the proenzyme form of 72 kD gelatinase, alsocalled type IV collagenase (Collier et al. 19886), which wepreviously demonstrated was synthesized by cornealfibroblasts (Fini and Girard, 1990). We utilized zymogramanalysis to quantitate the changes in 72 kD gelatinaseexpression in response to PMA in primary and passagedcorneal fibroblast cultures; a representative example of

this type of experiment is shown in Fig. 2. Both primaryand passaged cell cultures produced a gelatinolytic speciesthat migrated at 65 kD with respect to reduced proteinstandards. This electrophoretic behavior is appropriate for72 kD gelatinase, which migrates at 65 kD under non-reducing conditions. In primary fibroblast cultures treatedwith PMA, 72 kD gelatinase accumulation in the culturemedium was 3.3-fold greater than in untreated culturesafter 1 day and 2.4-fold greater after 2 days. Both of thesedifferences were statistically significant (P=0.003;P=0.006). In contrast, accumulation of 72 kD gelatinasewas repressed by PMA treatment of fourth-passagefibroblast cultures. After 1 day of treatment, accumulationof 72 kD gelatinase was 3.2-fold greater in untreated thanin PMA-treated cultures and after 2 days the difference

8 24 h

RF42 3 4 5 6 7 8

- 9 2

- 7 2

48 h

0-24h 0-48hTime in culture

0-24h 0-48hTime in culture

Fig. 2. (A) Zymogram analysis of gelatinases accumulated in the culture medium from primary (RF°) or fourth-passage (RF4)corneal fibroblasts, with or without PMA treatment. Equal numbers of cells were plated in the wells of a 24-well cluster plate.Cultures were changed to serum-free medium, and quadruplicate culture wells were left untreated or treated with PMA. After theindicated time, culture medium was collected and assayed for secreted gelatinases. Lanes 1-4, medium from untreated cellsassayed after 24 or 48 h of culture. Lanes 5-8, medium from cultures treated with PMA at 10~ 6 M PMA and assayed after 24 or48 h after treatment. The positions on the gels of 92 kD gelatinase (92) and 72 kD gelatinase (72) are indicated in the figure. Notethat additional zymogram bands, besides those representing 92 and 72 kD gelatinases, are produced by the samples from theprimary cultures. These bands are often seen in primary cultures but their identity is unknown. (B) Quantitation of theaccumulation of 72 kD gelatinase activity in the culture medium samples shown above. The optical density volume of the substrateclearing in the zymogram produced by the 72 kD gelatinase in each sample was determined as described in Materials and methods.Each raw value was converted to % activity of a standard sample by comparison to a standard curve produced by serial dilution ofthe standard sample. The mean was then calculated for the quadruplicate determinations of the same experimental treatment andplotted on the graph; the standard error of the mean is shown.

Metalloproteina.se expression by corneal fibroblasts 377

was 2.4-fold. The difference at day 2 was statisticallysignificant (P=0.002).

A higher molecular weight gelatinase, which wepreviously identified as 92 kD gelatinase (Fini and Girard,1990), also called the 92 kD type IV collagenase (Wilhelmet al. 1989) or type V collagenase (Hibbs et al. 1987) is alsoproduced by corneal fibroblast cultures. This enzyme is theproduct of a different gene from the one that encodes 72 kDgelatinase. The zymogram analysis in Fig. 2 shows thatthis enzyme is expressed in a pattern quite unlike thepattern of 72 kD gelatinase expression. 92 kD gelatinasewas detectable in conditioned medium from untreatedprimary fibroblasts cultured 1 or 2 days. Visual inspectiondemonstrated that PMA induced the accumulation of thisprotein in the culture medium slightly; however, the levelof expression in either untreated or PMA-treated cultureswas too small to measure by densitometry. No 92 kDgelatinase could be detected in the 1- or 2-day conditionedmedium from untreated cultures of fourth-passage fibro-blasts. PMA treatment of these cell cultures, however,induced expression of 92 kD gelatinase to a level that wasquite easily detectable by zymography. This expressionpattern seemed much like that of collagenase andstromelysin.

The accumulation of gelatinases in the culture mediumover 48 h would be determined by the rate of synthesis andsecretion of the enzymes as well as the rate of degradationof the enzyme in the cell culture medium. To determinewhich factor was more important in causing the differ-ences in enzyme accumulation seen with cell culturepassage, the accumulation of gelatinases in the culturemedium was measured over a much shorter time period, tominimize the contribution of degradation to the totalaccumulation. In addition, synthesized and secretedproteins were biosynthetically labelled during this time,so that expression of gelatinases could be directlycompared with expression of collagenase and stromelysin.In the experiment shown in Fig. 3, fourth-passage fibro-blasts were left untreated or treated with PMA for 24 h.After this time, cell culture medium was removed andfresh medium containing [35S]methionine was added; thismedium was left on cells for 4h. In untreated cultures,expression of collagenase and stromelysin was undetect-able by autoradiography, but, as expected from the resultsof the previous experiments, synthesis and secretion ofthese proteins was induced by PMA treatment. However,synthesis and secretion of the 72 kD protein, which wesuspect is 72 kD gelatinase, was repressed by the PMAtreatment. Zymography of the 4-h culture medium showedthe expected appearance of 92 kD gelatinase in themedium from PMA-treated cultures. However, in corre-lation with the results of autoradiography, there was 1.7-fold less of the 72 kD gelatinase in the 4-h medium fromPMA-treated cultures than from untreated cultures.These data suggest that alterations in gelatinase accumu-lation in the culture medium of cell cultures as they arepassaged are determined by changes in the synthesis andsecretion of the proteins by the cells.

Changes in metalloproteinase expression after PMAtreatment of passaged cell cultures is correlated withchanges in specific mRNA levelsNorthern blot analysis was performed to learn if changesin the expression of the MMPs in response to PMA could becorrelated with the changes in the levels of specificmRNAs. Total RNA was prepared from cells that had beenchanged to serum-free medium 50 h before RNA isolation

r

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97—

72—

43—

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— »tr

Fig. 3. Rate of MMP synthesis and secretion by passagedfibroblasts. Equal numbers of fourth-passage fibroblasts wereplated in the wells of a 24-well cluster plate. The cultures werechanged to serum-free medium, and quadruplicate cultureswere either left untreated (lanes 1—4) or treated with PMA at10~ 6 M (lanes 5-8). After 24 h, the culture medium wasremoved and fresh medium containing [36S]methionine at80/iCiml"1 was added. Cultures were pulse-labelled for 4h,then the labelled medium was harvested. Analysis of newlysynthesized proteins was analyzed by gel electrophoresis (top),and autoradiography or zymography (bottom). Size standards(Mrxl0~

3) are indicated to the left of the autoradiograph. Thepositions of the collagenase (col) and stromelysin (str) proteinsare indicated to the right of the autoradiograph. Note thatcollagenase and stromelysin are not as well resolved as in theautoradiograph in Fig. 1. This is because an 11 % gel instead ofan 8 % gel was used in this experiment. On the zymogram, thepositions of 92 (92 gel) and 72 (72 gel) kD gelatinases arenoted. The relative amount of enzyme in zymographic bandswas quantitated, with reference to a standard curve, asdescribed in Materials and methods; the quantitative resultsare described in the text.

and from cells that had been treated with PMA for 50 h inserum-free medium. Also, to determine whether thechange to serum-free medium alone could affect mRNAlevels for MMPs, RNA was prepared from cells grown inmedium containing 10% serum. In addition, RNA wasprepared from cells of the SIRC cell line, a rabbit cornealepithelial cell line (Grabner et al. 1983). Since these cellsdo not secrete any neutral gelatinase activity nor do theysynthesize and secrete collagenase or stromelysin (M. E.Fini, unpublished observations), RNA from these cellscould serve as a negative control.

When the Northern blot was probed with a cDNA clonefor the 72 kD human gelatinase, two distinct mRNA bandswere visualized in RNA from corneal fibroblasts (Fig. 4).One of these RNAs migrated on the gel at the sameposition (3.1kilobases, kb) as the single mRNA speciesdetected by Northern blotting of mRNA from human

378 M. E. Fini and M. T. Girard

3 4

kb

4 4 -

2 / 1 -

1.4-

f *

1 IFig. 4. Northern blot of RNA prepared from fourth-passagefibroblasts and SIRC cells. All experimental treatments werebegun simultaneously on cells that were plated in culture atthe same time and the same cell density. Left: blot probed withcDNA for 72 kD gelatinase. Right: the same blot as shown atleft was stripped and reprobed with a cDNA for stromelysin.Lane 1, RNA from SIRC cells grown to confluence in mediumwith 10 % serum. Lane 2, RNA from fibroblasts grown toconfluence in medium with 10 % serum, then changed toserum-free medium for 50 h. Lane 3, RNA from fibroblastsgrown to confluence in medium containing 10 % serum, thenchanged to serum-free medium and treated with PMA at10~6 M for 50 h. Lane 4, RNA from fibroblasts grown toconfluence in medium containing 10 % serum. Size standardsare indicated to the left of the blots in kilobases (kb).

fibroblasts (Collier et al. 19886). In addition, a mRNAmigrating more slowly on the gel (4.1kb) was visualized.These bands gave approximately equal autoradiographicsignals when the Northern blot was washed underconditions of moderate stringency (0.3 M NaCl at 65°C)and did not change in relative intensity under high-stringency conditions (0 .15 M NaCl at 65°C), demonstrat-ing the close relationship in sequence between the twoRNAs. Neither message was expressed by SIRC cells. Wedo not currently understand the meaning of two mRNAsfor 72 kD gelatinase, but are investigating the possibilitythat they are the result of allelic variation in the 72 kDgelatinase gene in our rabbit population (Fini and Girard,1990). Probing of the Northern blot with a cDNA for rabbitstromelysin revealed a 2.1kb message in corneal fibro-blasts of the same size as found in rabbit synovialfibroblasts (Fini et al. 1987a). SIRC cells did not producethis message.

A reciprocal relationship was observed in messagelevels for 72 kD gelatinase and stromelysin in untreatedcells and cells treated with PMA. Approximately equalamounts of gelatinase message were detected in the RNAfrom untreated cells grown in serum-free or serum-containing medium. PMA treatment drastically reducedthe levels of message (by 17.6-fold). In contrast, nostromelysin message could be visualized in untreated cellsgrown under either serum-free or serum-containingconditions. However, easily detectable message was foundin RNA from PMA-treated cells. The changes in mRNAlevels thus reflect the pattern of MMP accumulation in theconditioned culture medium, strongly suggesting thataccumulation of enzyme, or the lack of it, is due to changesin MMP mRNA levels.

Time course of the changes in specific MMP mRNAlevels in passaged fibroblasts after PMA treatmentsuggests a multistep mechanism coordinating MMPmRNA levelsTo determine the timing of the changes in MMP mRNAlevels after PMA treatment of corneal fibroblast cultures,RNA was harvested at various intervals up to 50 h afterPMA treatment. A Northern blot of this RNA wasprepared and hybridized with three different probes inturn: the 72 kD gelatinase cDNA, stromelysin cDNA, anda cDNA against human TIMP-1 (Carmichael et al. 1986).Finally, the blot was stripped one last time and hybridizedto a human cDNA clone for glyceraldehyde-3-phosphatasedehydrogenase (GAP; Allen et al. 1987). Hybridizationwith this clone enabled us to normalize the data fordifferences in total RNA loading per gel lane, since thelevel of GAP mRNA is known to remain constant in cellsafter PMA treatment (Edwards et al. 1987). The results inFig. 5 show that mRNA levels for stromelysin were quitelow over the first 6h after PMA treatment. In longerautoradiographic exposures than the one shown, a verysmall amount of message could be visualized at time zero;this was increased slightly by 6 h. However, by 20 h afterPMA treatment, a large amount of stromelysin mRNAwas present in cells. Stromelysin mRNA had increasedeven further by 50 h after PMA treatment so that the finalinduction of message was approximately 65-fold. Theseresults are quite similar to those obtained using rabbitsynovial fibroblasts (Fini et al. 1987a).

In contrast to stromelysin, hybridization of the North-ern blot with the 72 kD gelatinase probe yielded unexpec-ted results. Messenger RNA for 72 kD gelatinase was quiteeasily detectable in untreated cells. From our analysis ofprotein expression, we expected the specific mRNA level todrop after PMA treatment of cells. This was not the case;in fact, 72 kD gelatinase mRNA increased in amount overthe first 6 h of PMA treatment to approximately five timeshigher than the constitutive level. However, by 20 h afterPMA treatment, the increase in 72 kD gelatinase mRNAhad been reversed. The level of 72 kD gelatinase mRNAwas less than half the constitutive value by 20 h, and by50 h the level of mRNA was undetectable. It is notable thatthe timing of the reversal of 72 kD mRNA accumulationwas coincident with the dramatic increase in mRNA forstromelysin.

The kinetics of changes in mRNA levels after PMAtreatment showed a third pattern for TIMP-1 mRNA.Expression of this mRNA, like that of 72 kD gelatinase,could be detected before PMA treatment. TIMP mRNAincreased steadily over the first 6 h of PMA treatment to alevel approximately 6-fold higher than at time zero. By20 h after PMA treatment, TIMP mRNA had increasedeven further, to a level 14.5-fold higher than at time zero.However, by 50 h after PMA treatment, the amount ofTIMP mRNA had dropped and was only 4.4-fold higherthan the constitutive level.

Taken together, these data suggest that multipleintracellular signals must be initiated by treatment ofpassaged corneal fibroblasts with PMA, each of which hasthe potential to affect MMP gene expression. It is thecombined effects of the individual signals on the accumu-lation of specific mRNAs that must determine the ultimatechanges in the expression of MMP or TIMP proteins.

Discussion

Regulation of the expression of the genes for matrix

Metalloproteinase expression by corneal fibroblasts 379

Hours2 4 6 20 50 0

GAP r

72 kDGelatinase

I10 20 30 40 50 60

80 Stromelysin

0 10 20 30 40 50 60

TIMP

10 20 30 40 50 60Hours after PMA

Fig. 5. Northern blot of RNAprepared from fourth-passagefibroblasts over a time courseafter treatment with PMA.Right: fibroblasts were grown toconfluence in medium containing10 % serum, then changed toserum-free medium and treatedwith PMA at 10~6 M. Samplecultures were harvested at 0, 2,4, 6, 20 and 50 h after PMA wasadded to the cultures. Poly(A)+

RNA was prepared from the cellsand equal amounts (the poly A+

RNA from 200 ^g of total RNA)were used for Northern blotting.The blot was then probedserially with 72 kD gelatinasecDNA, stromelysin cDNA andTIMP-1 cDNA. The amount ofspecific RNA in each lane wasquantitated by densitometry.Finally, the blot was probed withGAP cDNA. Densitometricquantitation of the amount ofGAP mRNA in each lane servedas a control for variation in totalmRNA run in each gel lane.Left: densitometric valuesobtained with the other probeswere normalized to the GAPvalues. The normalized resultsare depicted graphically.

380 M. E. Fini and M. T. Girard

metalloproteinases, in coordination with expression ofgenes for matrix components, may be an importantmechanism for the dynamic maintenance of a tissue'sextracellular matrix. Here we have examined the patternof expression of four different MMPs, collagenase, strome-lysin, 92 kD gelatinase and 72 kD gelatinase, in untreatedcultures of corneal fibroblasts and in cultures treated withPMA. We show qualitative and quantitative changes inthe pattern of MMP expression as the cells are passaged inculture. Our data indicate that the cell does not simplyturn the MMP genes on or off, as a group, in response tovarious agents, but that it has the capacity for fine controlover which MMPs are expressed and over the degree towhich each is expressed.

Kuter et al. (1989) observed that primary cultures ofcorneal fibroblasts secrete no detectable collagenaseactivity and can not be induced to secrete this activityeven after treatment with CB or PMA. By directmeasurement of the amount of biosynthetically labelledcollagenase protein, we confirmed these results for CBtreatment. However, in our experiments, PMA treatmentwas effective in inducing primary cell cultures tosynthesize and secrete collagenase, although at low levels(2.5% of the secreted proteins). Perhaps this difference inresults is due to the different methods used to measurecollagenase expression; the activity assay may be lesssensitive than the biosynthetic-labelling assay. In ad-dition, we used a considerably higher concentration ofPMA for treating cells than did Kuter et al. (10~ 6 M versus3 .33X10~ 9 M) . In any event, the conclusion from bothlaboratories is still essentially the same: that is, primarycultures of corneal fibroblasts are quite limited in theircapacity to produce collagenase, even after treatment withPMA or CB. In addition, we extend these results to includethe MMPs, stromelysin and 92 kD gelatinase.

We also observed that, following subculture, cornealfibroblasts develop the capacity to synthesize collagenaseas a major gene product in response to PMA or CB. Again,this agrees with the findings of Kuter et al. (1989), whoshowed that collagenase activity could be highly inducedby PMA after cell culture passage. In our experiments, asingle passage was enough for this change in the cellresponse to occur. After treatment with PMA, 18.5 % of thetotal protein secreted by first-passage cultures wascollagenase. Stromelysin was induced similarly, althoughnot to as great an extent. Finally, 92 kD gelatinase couldalso be highly induced in passaged cells.

We found that a fourth MMP, 72 kD gelatinase, isexpressed constitutively by primary or passaged cellcultures at a level that can be easily visualized byzymography or autoradiography, unlike the other threeMMPs studied. Expression in primary culture is appar-ently a simple continuation of expression in vivo, since72 kD gelatinase can be extracted from the normal cornealstroma (Fini and Girard, 1990). PMA treatment ofprimary cell cultures resulted in a minimal increase insecretion of 72 kD gelatinase (2.4-fold) similar to the effecton secretion of collagenase, stromelysin, and 92 kDgelatinase. However, in passaged cell cultures, PMAtreatment repressed secretion of 72 kD gelatinase, at thesame time that it increased secretion of the other threeMMPs.

It is of interest that expression of 72 kD gelatinase and92 kD gelatinase is under such different control. Thereports in the literature, to date, indicate that these twodifferent enzymes, the products of different genes, havesimilar if not identical substrate specificities (Murphy et

al. 1989). Yet, the capacity of the fibroblasts for differentialexpression of these two enzymes suggests they play quitedifferent roles in biological processes. Expression of 92 kDgelatinase, in coordination with collagenase, might func-tion in proteolytic debridement of a wound, or in woundremodeling. On the other hand, 72 kD gelatinase may playa surveillance role in maintaining the integrity of thematrix structure, participating in the removal of theoccasional collagen molecule that becomes damaged anddenatured, or aiding in the proper assembly of newcollagen fibrils.

In passaged cell cultures, the reciprocal change in thelevel of 72 kD gelatinase and stromelysin secretionobserved after PMA treatment was correlated with asimilar effect on the levels of specific mRNAs whenassayed 50 h after drug treatment. These data indicatethat the effect of PMA on 72 kD gelatinase and stromely-sin expression is mediated by changes in mRNA accumu-lation; PMA represses 72 kD gelatinase mRNA accumu-lation and induces accumulation of stromelysin mRNA.However, this effect did not occur until later times afterPMA treatment. During the first 6 h after addition of PMAto the cells, there was only a minimal increase instromelysin mRNA and 72 kD gelatinase levels actuallyincreased by 5-fold. A third mRNA, TIMP-1, shows adifferent pattern of accumulation in response to PMA.Like 72 kD gelatinase, TIMP-1 mRNA increased over thefirst 6 h after PMA treatment. However, this accumulationof message was not reversed and continued to increase upto 20 h after PMA treatment.

Our results suggest that a single substance that acts atthe cell surface can initiate a multistep signallingpathway that serves to alter expression of MMP genes andtheir inhibitor, TIMPs, in non-coordinate ways. Thekinetics of specific mRNA accumulation suggest that stepsin the pathway progress in cascade fashion, each beingactivated by a previous signal. Which signalling pathwaysare functioning in a given cell, in combination with thecapacity of individual MMP/TIMP genes to respond toeach signal step, must determine the final MMP/TIMPexpression pattern produced by the surface-active agent.

Alteration of gene expression by PMA is thought to bethe result of direct binding and activation of proteinkinase C (Castagna et al. 1982). This protein, in turn, isthought to transmit the initial signal of PMA binding byphosphorylation of specific proteins on a signal transduc-tion pathway that ends at the gene (Nishizuka, 1984).Thus, changes in PMA inducibility with passage of cells inculture might be mediated by differential expression ofproteins in the signal transduction pathway. One possiblecandidate for differential expression is protein kinase C.This enzyme has recently been shown to exist as severalisoforms that are differentially expressed in developmentand that might have different signalling properties(Hanks et al. 1987). Other possible candidates are thecellular proto-oncogene products, several of which appearto influence collagenase gene expression (Schonthal et al.1988).

Of particular relevance when considering the PMA-triggered signal transduction pathway is a nuclear proteincomplex, called AP-1. Composed, at least in part, of aheterodimer of the proto-oncogene products, c-jun andc-fos (Halazonetis et al. 1988), AP-1 is a component of thesignal transduction pathway that activates transcriptionof a collagenase gene transfected into certain cell linesafter treatment of the cells with PMA (Angel et al. 1987).The stromelysin gene is apparently under similar control

Metalloproteinase expression by corneal fibroblasts 381

(Sirum and Brinckerhoff, 1989). The complex activates thetranscriptional machinery by binding to a specific DNAsequence, the TRE, which lies just upstream from thetranscriptional start site of the collagenase or stromelysingene (Chiu et al. 1988). It seems likely that AP-1 is alsoinvolved in activation of the endogenous collagenase genein passaged fibroblasts. However, this mechanism alonecannot fully explain the high levels to which collagenaseand stromelysin mRNAs accumulate (40- to 65-foldinduction) in these cells after PMA treatment (Gross et al.1984; Fini et al. 1987a; this paper), since AP-1 does notactivate transcription of the collagenase gene so dramati-cally (11- to 12-fold, at most). An additional problem isthat induction of gene transcription by the AP-1 factorrequires no new protein synthesis and cannot be inhibitedby treating cells with cycloheximide. However, inductionof collagenase and stromelysin mRNA by PMA in manypassaged fibroblast types can be blocked by treating cellswith cycloheximide (for example, Frisch et al. 1987). Thisindicates that additional factors must amplify the initialAP-1 signal and production or activation of these newfactors requires protein synthesis. In contrast, to theresults that we and others have obtained with passagedcell cultures, the small degree of induction of collagenaseand stromelysin expression by PMA in primary cornealfibroblasts could be explained solely by AP-1-mediatedactivation of gene expression. Perhaps in primary cul-tures, PMA activates the same set of intracellular signalsthat are activated in passaged cultures, including AP-1activation, however, progression to the secondary part ofthe signalling pathway cannot occur. It will be interestingto learn whether the gelatinase genes might also becontrolled by AP-1, or if they are regulated by a divergentsignalling pathway.

The cell culture studies reported here have revealedseveral different patterns of MMP gene expressionproduced by corneal fibroblasts. Could the conditions ofcell culture that are permissive for each of these patternshave in vivo correlates? One might argue that the behaviorof corneal fibroblasts in primary culture is an artifact ofcell preparation. The extensive protease treatmentsrequired to isolate corneal fibroblasts for primary culturemight strip off cell surface proteins, such as matrixattachment factors or cytokine receptors, that are essen-tial for constitutive and inducible expression of MMPs.The more gentle protease treatment required for cellculture passage might not damage these essential surfaceproteins, resulting in a greater capacity of passaged cellsto produce MMPs. However, we think that this expla-nation of the differences in MMP expression betweenprimary and passaged cultures is not very likely. We allowprimary cell cultures to recover from their proteasetreatment for 16 h before using them for an experiment -ample time for resynthesis of surface proteins. Further-more, cells in primary culture demonstrate similar typesof behavior to those of cells in situ. Corneal stromalfibroblasts from unwounded tissue show no evidence ofconstitutive production of collagenase in vivo and the cellsin corneal stromas that are cultured intact, withoutprotease treatment to release cells from the matrix, are asrefractory as primary cell cultures in their response toPMA (Wagoner et al. 1987; Fini and Girard, unpublisheddata). These observations suggest that primary culturesaccurately reflect the pattern of gene regulation thatoccurs in vivo.

It is tempting to suggest that the changes that cornealfibroblasts undergo when passaged in cell culture are

analogous to changes that occur in corneal stromalfibroblasts after wounding of the corneal stroma. Cornealfibroblasts from unwounded adult tissues are quiescent,undergoing few, if any, cell divisions, while woundfibroblasts are highly mitotic and metabolically active, asare culture-passaged cells. In addition, culturing ofcorneal stroma results in little incorporation of methion-ine. Corneal fibroblasts in the vicinity of a stromallaceration undergo morphological differentiation to amore fibroblastic phenotype, with a steady transition ofcell nuclei from an oval to a long narrow shape, anincrease in number and size of nucleoli per nucleus, and anincrease in the amount of cytoplasm surrounding eachnucleus (Weimar, 1960). Kuter et al. (1989) have describedsimilar cell shape changes during the transition of cornealfibroblast cultures from primary to passaged. We haveshown here that the differentiated stromal fibroblastsproduced by passage in cell culture are particularlyreceptive to inflammatory mediators such as PMA and cansecrete quite high levels of collagenase, stromelysin and92 kD gelatinase in response to this agent. If analogouschanges occur in a wound, then a wounded cornea mightbecome particularly vulnerable to self-destruction by theproducts of its own stromal cells if infiltrated byinflammatory cells, which have the capacity to secretemembrane-active cytokines with effects similar to PMA(Dinarello, 1988). This could explain the propensity of thecorneal stroma to ulcerate after severe injury caused byalkali or thermal burn (Kenyon, 1985). Because it may beimportant to the understanding of corneal health anddisease processes, the molecular mechanisms causingtransition in the pattern of MMP expression as primarycorneal fibroblasts are passaged in culture should continueto be investigated.

The generous gifts of collagenase and stromelysin antisera fromDr Constance Brinckerhoff were greatly appreciated. We thankDr Gregory Goldberg for the human cDNA clone for 72 kDgelatinase, Dr Robert Allen for the human GAP cDNA clone, andDr David Carmichael for the cDNA clone to human TIMP.

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(Received 18 April 1990 - Accepted 9 July 1990)

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