Unique Regulation of the Matrix Metalloproteinase, Gelatinase B

M. Elizabeth Fini, Marie T. Girard, Masao Matsubara, and John D. Bartlett

Purpose. The matrix metalloproteinase (MMP), gelatinase B, is expressed by both corneal cell types found at the epithelial-stromal tissue interface, the site of basement membrane repair in the healing cornea. This study investigates the relative regulation of gelatinase B compared to other MMPs in response to agents related to those found in the corneal repair environment or in corneal ulcers. Methods. A culture model of corneal cells isolated from rabbit was used. Results. Gelatinase B is the major MMP expressed by corneal epithelial cells, whereas stromal fibroblasts produce gelatinase B along with three other MMPs: collagenase, stromelysin, and gelatinase A. Phorbol-12-myristate 13-acetate (PMA) stimulates gelatinase B mRNA and protein synthesis by corneal cells, which is similar to its effect on the other MMPs. Stimulation occurs, at least partially, at the transcriptional level. PMA-stimulated MMP expression follows biphasic kinetics, with the major effect on collagenase, stromelysin, and gelatinase A occurring during the late component. In contrast, the major gelatinase B response occurs during the early component. Transforming growth factor-beta (TGF-/?) has no effect on constitutive expression of gelatinase B by fibroblasts; however, expression stimulated by PMA is enhanced. In contrast, constitutive expression of collagenase and stromelysin is inhibited by TGF-/3. However, in the presence of PMA, the initial inhibitory effect of TGF-/3 is reversed after treatment. Conclusion. Gelatinase B expression is regulated differently from other corneal MMPs. This provides a mechanism for control of basement membrane repair independent of repair processes in the stroma. Invest Ophthalmol Vis Sci. 1995;36:622-633.

•specialized mechanisms are required for the degra- structurally related, probably as a result of descent dation of extracellular matrix (ECM) ultrastructures from a common ancestral gene.6'7 The MMPs share because of their highly polymerized nature. Primary several properties. These include a requirement of mediators of this process are thought to be enzymes zinc for activity, secretion in a latent proenzyme form of the matrix metalloproteinase (MMP) family.1"4 Ten activated by proteolytic cleavage, and the capacity for different MMPs have been characterized and named inhibition by members of the tissue inhibitor of metal- according to a standard numbering system.5 For exam- loproteinases family. Each enzyme, however, has a ple, the enzyme, interstitial collagenase has been des- unique reactivity against ECM components. Together, ignated MMP-1, and stromelysin is MMP-3. Sequenc- the known MMPs have the capacity to degrade ECM ing studies have demonstrated that these enzymes are structures composed of different molecules. The 92-kd matrix metalloproteinase, gelatinase B (MMP-9), is one of the most recently characterized From the MCH/Narvard Cutaneous Biology Research Center, Massachusetts members of the MMP family.8'9 This enzyme specifi- General Hospital, Charlestown, and the Department of Dermatology, Harvard Medical School, Boston, Massachusetts. cally catalyzes cleavage of native basement membrane Supported by National Institutes of Health grants EY0840S, EY09S2S, and collagens, as well as denatured collagens of all types AR42981 (MJEF) and by an agreement between Massachusetts General Hospital and the Shiseido Company of Japan. MM was the 1989 Bausch and Lomb Japan (gelatins). Gelatinase B was originally identified as a Fellow. product of polymorphonuclear leukocytes and macro- Submitted for publication April 20, 1994; revised September 29, 1994; accepted 10 October 21, 1994. phages. However, it is now known that it can also be Proprietary interest category: N. synthesized by other cells, including corneal epithelial Reprint requests: M. Elizabeth Fini, Massachusetts General Hospital, Cutaneous 11 r* Biology Research Center, CNY3, 13th Street, Charlestown, MA 02129. cells and corneal stromal cells. This observation has

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suggested a possible role for gelatinase B in corneal Ophthalmic and Vision Research. Rabbit corneal cell basement membrane repair and remodeling and in isolation and primary cell culture were performed ac- the loss of the epithelial basement membrane, which cording to the method of Johnson-Wint and Gross19 precedes stromal ulceration in injuries due to thermal as described.1117 Briefly, corneas (from New Zealand or chemical burn.12 In support of this hypothesis, ex- White rabbits) were dissected along with a narrow rim pression of gelatinase B by resident cells is stimulated of scleral tissue. Central disks of tissue were cut with a after the initiation of healing processes in the injured 9-mm trephine, and the endothelial layer was removed cornea.13 In addition, increased gelatinase B expres- with forceps. Disks were incubated overnight in 0.25% sion is correlated with degradation of basement mem- trypsin at 4°C to digest the epithelial basement membrane and epithelial-stromal adhesion complexes in brane that lies between the epithelial and stromal lay- 14 the corneal ulcer, and specific inhibition of MMP ers. The epithelium was then scraped gently from the activity improves integrity of adhesion complexes in stromas with a scalpel; stromal cells were freed from these lesions (Fini et al, submitted for publication). the isolated stromal matrices by a 2- to 4-hour incuba- Expression of different MMPs often occurs coordi- tion in bacterial collagenase (Worthington, Freehold, nately in response to stimuli. Cell culture studies have NJ) dissolved at 4 mg/ml in complete medium con- made it increasingly clear, though, that each enzyme can sisting of minimal essential medium (Gibco, Grand respond to unique regulatory mechanisms.1"4'15 During Island, NY) containing 10% supplemented calf serum corneal repair, the expression of three MMPs—gela- (Hyclone, Logan, UT). Cells derived from epithelium tinase A (MMP-2), collagenase (MMP-1), and stromely- or stroma were separately pelleted by centrifugation; sin (MMP-3)—is coordinately stimulated1316 in the re- each pellet was resuspended in 1 ml of complete me- pair fibroblasts of the healing stroma. Expression dium. reaches a peak at about 2 weeks after injury, and this pattern of stimulated expression is maintained for many Stromal Fibroblast Culture months as the repair tissue is remodeled. In contrast, Passaging of stromal fibroblasts was performed as pre- gelatinase B expression reaches its peak early in the viously described.17 Briefly, cells derived from six cor- wound healing process, and enzyme expression is no neas were plated in a single 100-mm diameter dish longer detectable within a few weeks after healing be- with complete medium. Cells were cultured until they 13 gins. This enzyme also is expressed in both the epidie- had multiplied to form a confluent monolayer (about lium and the stroma of the healing cornea. These results 3 days). They were then treated with trypsin to remove indicate that gelatinase B is regulated by different mech- them from the plate, redistributed into three new anisms than are the other MMPs produced by corneal plates, and again left to multiply to confluence (3 days cells. An understanding of the nature of these mecha- to 1 week). Stromal fibroblasts between passages 1 to nisms would give important insight into regulation of 4 were used for experiments. the corneal repair process. Phorbol-12-myristate 13-acetate (PMA) has been Plating and Treatment of Cells for Secreted well documented as a stimulator of MMP expression in Protein Analysis cell culture.6"81117 This substance has the capacity to Epithelial cells, which do not separate completely activate intracellular signaling pathways by direct bind- from one another with trypsin treatment, were plated ing of protein kinase C located at the plasma membrane. as corneal equivalents (CE), with 1 CE equalling the In this respect, PMA may mimic the action of a number number of cells obtained from a single 9-mm corneal of specific cytokines found in healing wounds, many of disk. The number of cells from 1 CE is approximately which stimulate MMP expression.'"4 Interestingly, only a 2-4 X 106 cells, as we determined by DNA quantita- few substances have been demonstrated to inhibit MMP tion using a fluorometric method.20 One-fourth CE expression; outstanding among these is transforming was plated to each 16-mm diameter well of a 24-well growth factor-beta (TGF-/3). This cytokine, which cluster plate. Stromal fibroblasts were plated at equal strongly stimulates deposition of ECM when injected in density (2.0-2.5 X 105 cells/well) in 24-well cluster vivo,18 may be an important regulator of the synthetic dishes. Complete medium was used for plating of both phase of tissue remodeling. In this article, we report cell types because cell adherence and spreading on studies on regulation of gelatinase B by PMA and TGF- the culture plastic is facilitated by 10% calf serum. P in primary cultures of corneal epidielial cells and in However, 16 to 24 hours after plating, cells were early passage cultures of corneal stromal cells. washed and changed to serum-free medium for experiments; an equal volume of medium was added to each MATERIALS AND METHODS culture well. PMA at 10"6 M, TGF-/? at 1 ng/ml, or a Primary Corneal Cell Isolation combination of these agents was included in appropriate wells. When biosynthetic labeling of newly syn- All animal procedures were performed in accordance 35 with the ARVO Statement for the Use of Animals in thesized proteins was desired, S-methionine (New

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England Nuclear, Boston, MA) was added to the cul- were either left untreated or were treated with PMA ture medium (at 80 mCi/ml) 4 hours before termina- for 5 or 24 hours. Total RNA was isolated from each tion of treatment. Cell-conditioned culture medium cell sample using a miniprep method.a2 Purified RNA was collected after the treatment period for analysis was dissolved in water, and the concentration of the of secreted proteinases. solution was determined by spectrophotometry.22 For Northern blot analysis,23 the total RNA sample Analysis of Secreted Proteinases isolated from six culture wells was denatured by glyoxy- Zymography was one method used to identify and esti- lation, electrophoresed on a 1% agarose gel, and blot- mate the levels of MMPs accumulated in samples of ted to Hybond membrane (Amersham, Arlington conditioned cell culture medium. The method of Heights, IL). The RNA ladder (BRL, Bethesda, MD) Heusen and Dowdle21 was performed as previously was coelectrophoresed as a size standard. The large described" 15 using gels copolymerized with either ribosomal subunit (appoximately 5 kb) and the small type I gelatin (Gibco) or /3-casein substrates (Sigma). ribosomal subunit (approximately 2 kb) served as in- Crude, cell-conditioned medium samples of equal size ternal size standards for each sample and as an indica- were electrophoresed in each gel lane. After develop- tion that the RNA was intact. These RNAs were visual- ment and staining, the migration positions of enzy- ized by ethidium bromide staining of the gel before matic species could be identified easily as clear bands blotting. A rabbit genomic clone, p25, containing the where the substrate background of the gel had been 5' end of the rabbit gelatinase B gene was labeled24'26 removed by enzymatic activity. Enzymatic species cor- with S2P and used as a probe for gelatinase B mRNA. responding to gelatinase B were identified by electro- Transcription analysis was performed using a re- phoretic mobility, greater activity against a gelatin verse transcription-polymerase chain reaction (RT- substrate than a casein substrate, and immunopre- PCR) assay reported to give information comparable cipitation with an antiserum raised against human gel- to the nuclear run-on assay.23 The method uses PCR atinase B.'° primers that hybridize to sequences located on either Gel electrophoresis and autoradiography was the side of an intron interrupting the sequence of a partic- second method used to identify and quantitate MMP ular gene. If the primary, unspliced transcript serves expression. Equal-sized aliquots of medium con- as the template for amplification with these primers, taining secreted proteins were added to sample buffer a product will result that is of a predictably longer containing 5% mercaptoethanol and electrophoresed length (by the length of the intron) than the product on SDS-polyacrylamide gels.'5 Gels were dried and resulting if amplification occurs from spliced mRNA. autoradiographed to display labeled proteins. Gel Increased detectability of the primary transcript serves bands that corresponded to collagenase (MMP-1), to indicate that transcription has been stimulated; if stromelysin (MMP-3), or gelatinase B (MMP-9) were the increase occurs only in the spliced mRNA form, identified by their electrophoretic mobility and by an increase in mRNA half-life is more likely to be the their immunoprecipitation with antisera raised against cause. One advantage of this method over nuclear rabbit collagenase or stromelysin7 or with the antise- run-on is that it requires only a small amount of cellu- rum to gelatinase B described above. lar material. This was a key issue for the study reported For qualitative analysis, treatments were per- here because the primary epithelial cells we use do formed in duplicate to ensure reproducibility. When not multiply in culture. quantitation of enzyme levels was desired, treatments Gelatinase B cDNA was reverse-transcribed (Su- were performed in quadruplicate. The amount of pro- perscript, BRL, Bethesda, MD) from 5 or 11 fig of tein in specific autoradiographic or zymographic gel total RNA in a 20 (j,\ reaction. The reaction was bands was quantitated by volume densitometry (Mo- primed with the antisense oligonucleotide, 5'-AAA lecular Dynamics, Sunnyvale, CA). When quantitation CTG GAT GAC GAT GTC TGC GTC CCG-3', which was performed from autoradiographs, film was ex- is complementary to sequences from the third exon atl posed over a range of times to obtain the best linearity of the rabbit gelatinase B gene. Five //I ('/4) of of film response. The mean of replicates was deter- the reverse-transcribed products were amplified by the mined, and the standard error of the mean (SEM) polymerase chain reaction with 1 U of Taq DNA poly- was calculated. Statistical analysis for significance (P< merase (Perkin-Elmer Cetus, Norwalk, CT) using the 0.05) of differences among treatments was performed reverse transcription primer and a second sense using a sample Student's i-test. primer, 5'-AC CTG TTC CGC TAT GGT TAC ACC CGC GTA-3', complementary to sequences from the Epithelial Cell RNA Analysis second exon of the rabbit gelatinase B gene. A 362-bp One CE of epithelial cells was plated in each 35-mm product would be expected if amplification originated diameter well of a six-well cluster plate as described from the spliced form of gelatinase B mRNA. A 623-bp above. After changing to serum-free medium, the cells product would be expected if amplification originated

Downloaded from iovs.arvojournals.org on 10/02/2021 Unique Regulation of Gelatinase B 625 from the primary nuclear transcript of the gelatinase A. 1 2 N B gene. Amplification was performed in a 50-/xl reaction in a Perkin-Elmer Cetus Thermocycler program- mable heat block under the following cycling conditions: 93°C for 1 minute, 58°C for 1 minute, and 70°C for 1 minute; 30 cycles of amplification were performed. kD As a control for equal mRNA loading in each of the reverse transcription reactions, 5 ^g of total 97- RNA was also used for parallel reverse-transcription and amplification of rabbit glyceraldehyde-3-phosphate dehydrogenase (GAP) message; the levels of this message do not change with PMA or TGF-/3 treatment.l7'27 The sequence of rabbit GAP was obtained from the GenBank database and used to con- -68 struct the following primers for RT-PCR: antisense primer, 5'-CGC GGG CTC CTG GAA GAT GGT RE° GAT-3'; sense primer, 5'-CGC GGG CCA TTC ATT FIGURE l. Gelatinase B expression in corneal epithelial cells. GAC CTC CAC TAC A-3'. A 147-bp PCR product Corneal epithelial cell cultures were left untreated or were would be expected to originate from the GAP treated with PMA for 24 hours. At 20 hours, :i5S-methionine mRNA. was added to cultures to pulse label newly synthesized pro- Five microliters of the total PCR reaction products teins. The migration position of size standards is indicated (Vio of the total) were electrophoresed on 2% Nu- in kd. (A) Analysis of total secreted proteins. Lanes 1 and seive agarose (FMC Bioproducts, Rockland, ME). The 2: untreated cells. Lanes 3 and 4: PMA-treated cells. (B) gel was stained with ethidium bromide, and the PCR Immunoprecipitation analysis with gelatinase B-specific anti- products were sized by comparison to a DNA size stan- serum. Arrows indicate the migration position of gelatinase dard (100-bp ladder, BRL). The results were then re- B. Lane S: total profile of conditioned medium from PMA- treated cells. Lane N: precipitated with normal serum. Lane corded by photography on a transilluminator using a A: precipitated with rabbit gelatinase B antiserum. (top) Zy- wavelength of 300 nm. To confirm the identity of the mographic analysis of proteinases accumulated in culture PCR product, the contents of the gel were then trans- 23 medium over 24 hours, (bottom) Autoradiographic analysis ferred to nylon membranes and hybridized with the of proteins pulse labeled during the last 4 hours of treat- rabbit gelatinase B probe (described earlier) or with ment. PMA = phorbol-12-myristate 13-acetate. a cDNA clone to human GAP.28

Fibroblast RNA Analysis RESULTS Fibroblasts plated in their fourth passage were al- Gelatinase B Is the Major MMP Synthesized lowed to multiply in culture until they formed nearly and Secreted by Corneal Epithelial Cells in confluent monolayers. Medium was then changed Culture to serum free, and cells were treated with PMA, TGF- In previous work", we determined that the 92-kd P, or a combination of these agents, as described, MMP, gelatinase B, is the principal gelatinase pro- for up to 72 hours. At scheduled times after treat- duced by corneal epithelial cells in primary culture. ment, a sample of cells was collected, lysed in a solu- In this study, we performed experiments to learn tion of 8 M guanidine HC1. Total RNA was then whether these epithelial cell cultures synthesize gela- 21 purified by centrifugation through a CsCl pad. tinase B exclusively, reproducing the selectivity of Equal samples of total RNA (20 /ig) were analyzed MMP expression in the healing corneal epithelium, by Northern blot, as described. Blots were probed or whether they also express MMPs, collagenase, and sequentially with a full-length rabbit cDNA for stro- stromelysin as do stromal fibroblasts in culture. 29 melysin, the rabbit genomic clone for gelatinase The zymographic profile of gelatinolytic enzymes B described earlier, and a human cDNA for GAP, secreted by corneal epithelial cells revealed a single also described earlier. After hybridization, the blots major band at approximately 92 kd (Fig. 1 A, top) with were exposed to film over a range of times to ensure multiple minor bands at higher molecular weight. linearity of film response. The amount of specific PMA treatment of cells resulted in increased accumu- message in each sample was quantitated by densi- lation of these enzymes in the culture medium. The tometry, and loading errors in each gel lane were molecular sizes and gelatin substrate reactivity of the corrected by normalizing to the GAP message levels. zymogen visualized on the substrate gel is appropriate Downloaded from iovs.arvojournals.org on 10/02/2021 626 Investigative Ophthalmology & Visual Science, March 1995, Vol. 36, No. 3

for the proenzyme form of the matrix metallopro- in untreated and treated lanes is probably much smaller teinase, gelatinase B. Minor gelatinase forms at sizes than die ratio between die actual amounts of enzyme between 92 kd and the top of the gel seemed likely diat produced the clearing. The fact that enzyme can to be forms of the same enzyme. Gelatinase B can be visualized by zymography is testimony to die extreme form multimers via intramolecular disulfide bridges sensitivity of this technique for visualizadon of gela- via free cysteine residues; these multimers are not dis- tinases. rupted under the nonreducing conditions of zymogra- We conclude that gelatinase B is the major MMP phy.30 In addition, there is some evidence that gela- synthesized by rabbit corneal epithelial cells in pri- tinase B can also associate by disulfide bridges with mary culture, demonstrating that this culture system other members of the MMP family or with tissue inhib- reproduces the in vivo expression pattern of repairing itor of metalloproteinases. The proposed identity of corneal epithelium."1617 the major zymogen was confirmed by immunoprecipi- Stimulation of Gelatinase B Gene Transcription tation analysis shown in Figure IB. An antiserum by PMA Treatment of Corneal Epithelial Cell raised against gelatinase B precipitated the 92-kd zy- Cultures mogen, which coelectrophoresed with a serum pro- To characterize the size of the gelatinase B mRNA tein. The antiserum also precipitated higher molecu- produced by corneal epithelial cells, Northern blot lar weight zymogen forms, providing additional evi- analysis was performed on the total RNA from cells dence for our proposal that these represent additional that had been PMA-treated for 24 hours (Fig. 2). This gelatinase B forms. Only the darkly staining serum pro- revealed a gelatinase B mRNA of 2.3 kb, similar to the tein was precipitated by a nonimmune serum. These reported size of the message isolated from human results reproduce those previously reported," confirm- fibroblasts.9 ing that corneal epithelial cells in culture produce pri- To learn whether PMA stimulates gelatinase B syn- marily gelatinase B and negligible gelatinase A. thesis by stimulating transcription of the gelatinase B Zymography of epithelial conditioned media was gene, we used an RT-PCR assay that is reported to give >r further performed against casein, a substrate for colla- comparable information to the nuclear run-on assay.29 genase and stromelysin. However, this revealed no evi- Results from two different experiments are shown in dence of either enzyme (data not shown). In the event Figure 3. The 147-bp GAP PCR product, which served that collagenase and stromelysin were syndiesized by epi- as a control for RNA loading into each reaction, was thelial cells, but at low levels, we further analyzed pro- equally detectable in all RNA samples from each ex- teins synthesized and secreted into epithelial cell-condi- periment. However, in neither experiment could a tioned media by pulse labeling and autoradiography; we gelatinase B PCR product be detected in total RNA have found this method to be a more sensitive assay for isolated from untreated cell cultures. After 5 hours collagenase and stromelysin than zymography. Shown of PMA treatment, a gelatinase B product was still in the bottom panel of Figtire 1A is such an analysis of undetected in the first experiment, but two gelatinase the same medium assayed by zymography in the top B products were easily detected in the second experi- panel. No proteins of the correct size to be collagenase ment. The 623-bp product was the appropriate size to (53 kd) or stromelysin (51 kd) were visible in either be derived from the primary nuclear transcript, a untreated or PMA-treated cultures. Furthermore, itnmu- short-lived RNA form whose presence in detectable noprecipitation analysis did not reveal even low levels of amounts is indicative of active transcription. The 362- these proteins (data not shown). However, PMA treat- bp was the appropriate size for the more stable spliced ment stimulated synthesis and secretion of a closely mRNA form. Both of these gelatinase B PCR products spaced protein doublet of 92 kd as visualized in the were detectable in both experiments by 24 hours. electrophoretic profile of 35S-pulse-labeled secreted pro- However, in the second experiment, the gelatinase B teins (Fig. 1A, bottom). This protein was identified by message level appeared by 24 hours to have dropped immunoprecipitation analysis (Fig. IB, bottom) to be from the level expressed at 5 hours because a gela- gelatinase B. The closely spaced doublet most likely tinase B product could be detected easily from 11 ng indicates partial glycosylation31 because all enzyme of total mRNA but not so easily from 5 \ig. These multimers would be disrupted by the reducing condi- results indicate that PMA stimulates expression of gel- tions of die standard gel electrophoresis conditions used atinase B at least in part by stimulating transcription in diis experiment. Visualization of the 92-kd zymogen of the gene. in untreated and PMA-treated lanes of the zymogram, Differential Effects of TGF-/J on Constitutive but only the 92-kd protein doublet in PMA-treated lanes and PMA-Stimulated Expression of Gelatinase of the autoradiograph, is not inconsistent with their B Compared to Three Other MMPs in Stromal coidentity. The area of the cleared band on a substrate Cell Cultures gel does not increase linearly as the amount of enzyme The experiment shown in Figure 4 is representative increases.15 Thus, die ratio between the 92-kd clearing of the results obtained in a number of similar experi-

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4, top left). The sizes of these proteins suggested that they were the proenzyme forms of gelatinase A and gelatinase B, respectively. Analysis by substrate gel electrophoresis performed on samples of the same cell-conditioned medium used for the analysis described above (Fig. 4, bottom left) displayed the expected gelatinolytic proteinases of 65 kd and 92 kd. The 65-kd proteinase is the appropriate size for the proenzyme form of gelatinase A, which migrates more rapidly than would be predicted from its molecular weight under the nonreducing conditions of zymography." Immunoprecipitation analysis of radiolabeled

FIGURE 2. Northern blot analysis of gelatinase B mRNA in corneal epithelial cells after treatment with PMA. mRNA FIGURE 3. RT-PCR transcription analysis of gelatinase B ex- extracted from cells treated with PMA for 24 hours was dis- pression in epithelial cells after treatment with PMA. Ethid- played by Northern blotting and probed with a radiolabeled ium bromide-stained gels of PCR amplified products of re- gelatinase B gene fragment. The result of blot autoradiogra- verse-transcribed epithelial cell RNA. Lanes 1 to 3 (EXP-1) phy is shown. The arrow points to the gelatinase B message. and lanes 4 to 9 (EXP-2) represent results from two separate The migration position of the large and small ribosomal sets of cell cultures. Lane L is a size standard (the 100-bp subunits is indicated. Some nonspecific sticking of the probe ladder); sizes of specific bands are indicated in bp. Arrows to the large ribosomal subunit occurred. PMA = phorbol- indicate the positions of the PCR products. (A) Primers 12-myristate 13-acetate. used were specific for gelatinase B. The 363-bp PCR product (bottom arrow) is a size consistent with its derivation from the gelatinase B mRNA; the 623-bp band could have originated ments designed to assess the response of corneal fi- either from the unspliced primary transcript of gelatinase broblast-synthesized MMPs to PMA, as well as to the B or from contaminating genomic DNA. EXP 1: lane 1 = cytokine TGF-/3. Pulse-labeling of cultures for the last untreated; lane 2 = PMA for 5 hours; lane 3 = PMA for 24 4 hours of the 24-hour treatment period revealed that hours. Eight micrograms of total RNA was used as the start- the synthesis and secretion rate of two constitutively ing material. EXP 2: lanes 4 and 5 = untreated; lanes 6 and expressed proteins of 53 and 51 kd were selectively 7 = PMA for 5 hours; lanes 8 and 9 = PMA for 24 hours. altered after treatment with PMA and/or TGF-/3 (Fig. In lanes 4, 6, and 8, 5 /ig of total RNA was used as starting material. In lanes 5, 7, and 9, 11 fig of total RNA was used 4, top left). These proteins are the appropriate sizes as starting material. (B) Primers used were specific for the to be the proenzyme forms of collagenase (53 kd) and GAP message. An equal amount of RNA was used as starting stromelysin (51 kd). Immunoprecipitation analysis material for each sample. The 147-bp product is indicated confirmed these identifications (Fig. 4, top right). The with an arrow. Lanes 1 and 4 = untreated; lanes 2 and 5 = synthesis and secretion rate of two less prominent pro- PMA for 5 hours; lanes 3 and 6 = PMA for 24 hours. RT-PCR teins, one of 72 kd and one a closely spaced protein = reverse transcription-polymerase chain reaction; PMA = doublet of 92 kd, were also affected by treatment (Fig. phorbol-12-myristate 13-acetate. Downloaded from iovs.arvojournals.org on 10/02/2021 628 Investigative Ophthalmology & Visual Science, March 1995, Vol. 36, No. 3

CONTROL TGF-p TGF-p+PMA PMA 92 kD 197.8 ±2 2.8 29.5 ± 21.1 53 kD 408.2 ±71.0 206.8 ±34.0 529.8 ±51.0 636.0 ±136.9 51 kD 244.8 ±45.7 113.2 ± 5.5 334.8 ±4 0.4 380.0 ± 42.3 fc©

12 3 4 kD 97 -MMP 9 -MMP 2 6S

UUD 1 ^ffffUTIr I -MMP 3 —

Immunoprecipitation

92 kD 68 8 ±7 .5 67.e ±6 . 1 609 .2 ±7 . 6 518.2 ±6 . 6 65 kD 57 .0 ±2 . 3 60.3 ±5 . 4 34 .0 ±4 . 7 27 .2 ±3 . 7 FIGURE 4. Independent regulation of gelatinase B protein synthesis and secretion by PMA and TGF-/3 in comparison to three other MMPs in corneal stromal fibroblasts.Quadruplicat e cultures of corneal fibroblasts were left untreated (control) or were treated with TGF-/3, PMA, or a combination of TGF-p + PMA for 24 hours. Newly synthesized proteins were pulse labeled during the last 4 hours of treatment. Samples of culture medium were then analyzed by gel electrophoresis. The electrophoretic migration of size standards are indicated in kilodaltons (kd). The gel bands corresponding to specific MMP proenzyme forms are also indicated, (lap, left) The profile of radiolabeled proteins secreted into the medium of each culture as visualized by autoradiography. Mean band densities ± SEM for gelatinase B proenzyme (92 kd), unglycosylated MMP-1 proenzyme (53 kd), and MMP-3 proenzyme (51 kd) are indicated over the autoradiograph for each treatment group. (top, right)Immuno - precipitations: the identity of autoradiography bands determined by immunoprecipitation with specific antisera. Lane 1 = nonimmune serum; lane 2 = human gelatinase B antiserum; lane 3 = MMP-1 antiserum; lane 4 = MMP-3 antiserum. (bottom, left) Analysis of the same culture medium samples by gelatin zymography to determine the accumulation of specific gelatinases over the complete 24-hour period. Mean band densities ± SEM for gelatinase B proenzyme (92 kd), unglycosylated MMP-1 proenzyme (53 kd), and MMP-3 proenzyme (51 kd) were corrected to a standard curve, and relative values are indicated under the zymogram for each treatment group. PMA = phorbol-12-myristate 13-acetate; TGF/3 = transforming growth factor-/?; MMP = matrix metalloproteinase.

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culture medium provided further evidence for the The rate of synthesis and secretion was 6.7-fold higher identity of the 92-kd proteins as gelatinase B (Fig. 4, than the level stimulated by PMA treatment alone top right). (Fig. 4, top left). This difference was statistically sig- Quantitation of the changes in MMP expression nificant. These results demonstrate that gelatinase B was performed by densitometric measurements of spe- is regulated differently from the other corneal synthe- cific protein bands from the autoradiographs. We sized MMPs in response to PMA or to TGF-/3. have demonstrated the validity of this technique for quantitating the rate of collagenase and stromelysin Early Stimulation of Gelatinase B mRNA synthesis.15 Normally, this method is not useful for Accumulation by PMA and Enhancement by quantitating gelatinase B expression because this en- TGF-/J in Corneal Stromal Cell Cultures zyme is expressed by fibroblasts at very low levels. As The change in the gelatinase B mRNA steady state a result, the biosynthetically labeled gelatinase B pro- level in response to PMA or PMA plus TGF-/3 in cor- tein is often obscured on autoradiographs by the pres- neal fibroblasts was examined by Northern blot analy- ence of a stronger band at 95 kd, which probably sis (Fig. 5). Gelatinase B mRNA was not detectable represents a collagen chain. However, we have found before treatment and was not induced to detectable that different cultures (derived from different rabbits) levels by treatment with TGF-/3. However, the gela- show some variability; in the present study, gelatinase tinase B message was detectable within 2 hours after B expression was high enough and collagen expres- treatment with PMA and increased steadily up to 8 sion low enough for densitometric quantitation of gel- hours after treatment. After this time, the level rapidly atinase B to be possible. Using this method, we deter- declined to baseline. TGF-/3 enhanced the PMA-stimu- mined that TGF-/3 treatment coordinately decreased lated accumulation of gelatinase B mRNA; the induc- the rate of constitutive collagenase and stromelysin tion curve paralleled the PMA-stimulated curve, but synthesis and secretion by approximately twofold com- at a higher level, over the first 8 hours. However, after pared to controls (Fig. 4, top left) but did not alter this time, message levels did not drop to baseline but the rate of gelatinase A synthesis and secretion in this instead reached a plateau at approximately one third 24-hour experiment. The rate of constitutive gela- of peak stimulation. Similar results were obtained in tinase B synthesis and secretion was too low in this a second repetition of this experiment (data not experiment to be visualized by autoradiography. How- shown). ever, quantitation of substrate clearing on the zymo- The time course of stromelysin expression fol- gram (Fig. 4, bottom left) demonstrated that TGF-/? lowed a much different pattern. In the experiment did not alter the amount of gelatinase B that accumu- shown here (Fig. 5), very low levels of message were lated in the culture medium in the 24 hours of treat- detected between 0 and 10 hours after PMA treatment. ment. These levels were not large enough to show up on the PMA treatment resulted in a coordinately stimu- graph, but we have published results demonstrating lated rate of collagenase and stromelysin synthesis and that a slight increase in the stromelysin mRNA level secretion by approximately 1.5-fold over controls dur- occurs during this time.17 At 24 hours, stromelysin ing the 20- to 24-hour pulse-labeling period (Fig. 4, message reached a level that was easily detectable, top left). In addition, the rate of gelatinase B synthesis which also was in agreement with our previous work. and secretion was stimulated to levels detectable by This level was further increased at 48 hours. TGF-/3 pulse labeling. As assayed by zymography, the 92-kd decreased the PMA-stimulated message levels slightly gelatinase B accumulated in the culture medium of at 24 hours, but this pattern had switched by 48 hours PMA-treated cultures to a level about 7.5-fold higher so that stromelysin mRNA levels were higher than they than control cultures in the 24 hours of treatment were with PMA alone. In the second time course (data (Fig. 4, bottom left). In contrast, PMA treatment in- not shown), a similar result was obtained. hibited gelatinase A accumulation by about twofold. These results indicate that stimulation of gela- TGF-/3 treatment decreased PMA-stimulated syn- tinase B expression occurs primarily as an early re- thesis and secretion of collagenase and stromelysin sponse to treatment of cells with PMA or PMA plus only slightly in this experiment (Fig. 4, top left). How- TGF-/3, in contrast to the late response of collagenase ever, in other experiments, greater inhibition oc- and stromelysin. curred (see below). In contrast, TGF-/9 treatment appeared to enhance the PMA-stimulated accumulation of gelatinase B during the 24 hours of the experiment, DISCUSSION as assayed by zymography (Fig. 4, bottom left). Autora- Four MMPs—gelatinase A, collagenase, stromelysin, diographic analysis of 20- to 24-hour pulse-labeled pro- and gelatinase B—are expressed by resident corneal teins more clearly derrionstrated the enhancement of cells in the repair tissue that forms after corneal in- PMA-stimulated gelatinase B expression by TGF-/3. jury.1316 The first three of these four MMPs are ex-

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1500 regulate MMP expression. In fact, we previously reported" results using a primary-passaged fibroblast culture system that faithfully reproduces the change in the metalloproteinase expression profile between 1000- the uninjured corneal stroma and the stromal repair tissue deposited after injury. We used this system to investigate regulatory mechanisms that might contrib- ute to corneal repair.15 Corneal epithelial cell cultures also seemed likely to be useful for similar experiments, 500- based on earlier work.'' In that earlier study, we determined that the 92-kd gelatinase B is the principle gelatinase produced by epithelial cells. This enzyme was found to be synthesized constitutively by corneal epithelial cells, much as occurs in the repair epithelium 10 20 30 40 50 that forms after injury. Very small amounts of a 65- Time (hours) kd gelatinase, which might have been gelatinase A, accumulated in epithelial cell culture medium when left on cells for prolonged periods (72 hours). How- ever, the 65-kd enzyme obviously represents minor component of MMP expression in this cell type; we 1500 could not visualize an enzyme of this size in the current study that used only 24-hour accumulation times. Furthermore, the enzyme could not be visualized by biosynthetic pulse labeling and autoradiography. 1000- Thus, at least for gelatinases, this model also seemed to reproduce accurately the wound situation. In this study, we performed the necessary additional experiments to learn whether primary cultures

500- of corneal epithelial cells maintain the tissue selectivity of metalloproteinase expression seen in the healing epithelium. We found that, faithful to the healing expression pattern,131416 no collagenase or stromelysin is produced by these cultures. Thus, gelatinase B is the major MMP expressed by corneal epithelial cells under constitutive conditions or after treatment with Time (hours) PMA. This is in contrast to stromal cells, which express collagenase, gelatinase A, stromelysin, and gelatinase FIGURE 5. Kinetic analysis of MMP mRNA levels in fibroblasts 3 after treatment with PMA or PMA plus TGF-/?. Northern B.' Corneal epithelial cells were found to produce a blot analysis was used to visuallize specific mRNAs. Plotted gelatinase B mRNA of the same size (2.3 kb) reported are autoradiographic band densities for specific messages for other cell types.9 PCR analysis revealed that PMA normalized to the GAP message, which served as an internal stimulates mRNA levels in this cell type, at least par- standard. MMP = matrix metalloproteinase; TGF/? = trans- tially by stimulating gelatinase B gene transcription. forming growth factor-/?; PMA = phorbol-12-myristate 13- The selectivity of MMP expression in corneal epi- acetate. thelial cells revealed by the current study is different from the reports on the expression pattern in cultured pressed coordinately over the prolonged period of keratinocytes from the skin. These investigations have healing and repair tissue remodeling. Gelatinase B, demonstrated significant expression of MMPs in addi- however, is expressed in repairing cornea according tion to gelatinase B, including collagenase.32 At this to a different temporal and spatial pattern than the time, it is unclear whether the difference in our find- 13 16 other corneal-synthesized MMPs. ' Here we have ings represents a true difference in MMP selectivity in used a model of primary and early passage cell cultures the two types of epithelial cells or whether differences from corneal epithelium and stroma to study the fac- in cell culture methods are responsible. We isolate tors regulating gelatinase B expression. corneal epithelial cells and culture them directly; es- Cell culture bears much in common with the envi- tablishment of skin epithelial cells in culture often ronment of the healing wound, and it seemed likely makes use of a fibroblast feeder layer. Nevertheless, to us to offer a useful system for studying factors that expression of large amounts of collagenase mRNA in

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the leading epidermal edge of healing skin ulcers was normal diploid cells, TGF-/3 can, in some circum- recently documented.33 Although there is a report that stances, act as a growth stimulator.18 Thus, TGF-/? en- collagenase mRNA expression can be stimulated in hances the growth stimulatory effects of platelet-de- the epithelium of organ-cultured corneas by platelet- rived growth factor on Fischer rat 3T3 fibroblasts activating factor,34 in vivo situations in which corneal transformed with a myc oncogene, but it inhibits epithelial cells express significant amounts of colla- growth stimulated by epidermal growth factor.37 A genase remain to be demonstrated. connection between the TGF-/3 effects on cell growth Having established the relevance of our corneal cell and MMP expression is suggested by the recent report culture model to the wound situation, we examined the that TGF-(5 can stimulate expression of the collagen- effects on gelatinase B expression produced by treat- ase gene in a human lung adenocarcinoma cell line ment with TGF-/3 or with TGF-/? plus PMA. TGF-/? is that responds positively to TGF-/3 in terms of cell 38 thought to be an important regulatory cytokine control- growth. ling the wound healing response. It strongly stimulates Another important new finding of this study re- deposition of ECM when injected in vivo.18 This action lates to the kinetics with which PMA affects the levels has been attributed to the simultaneous stimulation of of gelatinase B mRNA. We found that changes in ECM synthesis and inhibition of matrix degradation. mRNA levels for gelatinase B after PMA treatment The latter effect is thought to be partially mediated by follow a biphasic pattern of early and late expression, a general inhibition of proteinase synthesis and activity. similar to the pattern that we observed for collagen- However, recent data have challenged this concept. Con- ase,39 stromelysin, and gelatinase A,17 and that we re- stitutive expression of the gelatinolytic metalloprotei- produced here for stromelysin. However, unlike these nase, gelatinase A, is stimulated by TGF-/? in early pas- other MMPs, the major change in gelatinase B mRNA sage cultures of human gingival35 or rabbit corneal fi- levels occurred during the early portion of the re- broblasts,15 whereas expression of collagenase and sponse. Our results are also consistent with a similar stromelysin is inhibited. In another report, TGF-/3 was early gelatinase B response by epithelial cells. This shown to stimulate expression of gelatinase B in early early stimulation of gelatinase B transcription indi- passage cultures of human mucosal and skin epithelial cates that the gene is activated primarily by direct sig- cells.36 On the other hand, human gingival fibroblasts nals initiated by PMA treatment of cells, unlike the failed to respond to TGF-/3 by stimulation of gelatinase genes for collagenase, stromelysin, and gelatinase A B expression. that require synthesis of intermediate proteins before 17 40 Consistent with these previous reports, we found the major alteration in expression. ' Although it is well known that PMA stimulates gelatinase B expres- in this study that constitutive expression of gelatinase 9 B in corneal stromal fibroblasts is not affected by TGF- sion in a variety of cell types, this finding of early P treatment. However, we extend these findings by response to PMA has not been previously reported. showing that TGF-/3 has a clear enhancing effect on Such induction kinetics are consistent with the early appearance of gelatinase B, compared to the other gelatinase B expression stimulated by PMA. Enhance- 316 ment was apparent at the earliest time point examined MMPs, in the healing corneal wound.' at the mRNA level (2 hours) and was correlated with Deposition and remodeling of the epithelial base- an enhancement in the levels of protein synthesis and ment membrane during corneal wound healing in- secretion. In contrast, TGF-/0 inhibited constitutive ex- volves progressive synthesis, degradation, and resyn- pression of collagenase/stromelysin in stromal fibro- thesis of ECM,41 and this must occur in coordination blast cultures. A dual effect of TGF-/3 on PMA-stimu- with the remodeling of the stromal ECM. Epithelial lated expression of collagenase/stromelysin by stro- cells and fibroblasts come into direct contact at the mal fibroblasts was observed. This was most clearly epithelial-stromal interface of the healing corneal revealed by kinetic analysis of changes in mRNA levels wound. The capacity of gelatinase B to be expressed in response to TGF-/3 in PMA-treated fibroblasts. Ex- by each of these cell types, and for regulation indepen- pression of stromelysin was clearly inhibited at the dent of the other MMPs synthesized by corneal cells, mRNA level by TGF-/9 at early times after treatment, provides a mechanism for fine control over basement but this inhibition was reversed at later time points membrane repair in the healing corneal wound. The until a clear enhancement of message accumulation studies reported here, describing the unique tran- was seen. We think this is the first clearly documented scriptional regulation of the gelatinase B gene com- study demonstrating a positive effect of TGF-/3 on col- pared to other MMPs, provide the essential rationale lagenase/stromelysin gene expression in normal cells. for exploring regulatory factors involved using the gel- The potential for a dual MMP response to TGF- atinase B gene's transcriptional promoter as a tool. /3 is consistent with the bifunctional nature of TGF-/3 Key Words action on other cell functions. For example, though matrix metalloproteinase, collagenase, tumor promotor, generally recognized as a potent growth inhibitor of transforming growth factor, cornea

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Acknowledgments ment membrane dissolution preceding corneal ulceration. Invest Ophthalmol Vis Sci. 1991;32:3221-3237. The authors thank Dr. Constance Brinckerhoff (Dartmouth 15. Girard MT, Matsubara M, Fini ME. Transforming Medical School, Hanover, NH) for rabbit collagenase and growth factor-beta and interleukin-1 modulate expres- stromelysin antisera and Dr. Karen Hasty (University of Ten- sion of metalloproteinases by corneal stromal cells. nessee, Memphis, TN) for human gelatinase B antiserum. Invest Ophthalmol Vis Sci. 1991; 32:2441-2454. 16. Girard MT, Matsubara M, Kublin C, Tessier M, Cin- References tron C, Fini ME. Stromal fibroblasts synthesize colla- 1. Alexander CM, Werb Z. Proteinases and extracellular genase and stromelysin during long-term remodeling matrix remodeling. Curr Opin Cell Biol. 1989; 1:974- of repair tissue. J Cell Sci. 1993; 104:1001-1011. 982. 17. Fini ME, Girard MT. The pattern of metalloproteinase 2. Woessner JF. 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