Overexpression of MMP-1 and MMP-3 by Cultured Conjunctivochalasis Fibroblasts

Abstract

purpose. To determine whether conjunctivochalasis, denoting redundant, loose, nonedematous inferior bulbar conjunctiva, is associated with increased expression and activity of matrix metalloproteinases (MMPs) over their tissue inhibitors (TIMPs).

methods. Expression of transcripts and proteins of MMPs, TIMPs, and urokinase plasminogen activator (uPA) by cultured normal human conjunctival and conjunctivochalasis fibroblasts was determined by Northern hybridization, enzyme-linked immunosorbent assay (ELISA), and Western blot analysis, respectively. Gelatin and casein zymography and quantitative collagenase activity assay were performed in the serum-free conditioned media.

results. Compared with normal conjunctival fibroblasts from six subjects, conjunctivochalasis fibroblasts from eight patients showed markedly increased transcript expression of MMP-1 (5- to 32-fold) and MMP-3 (4- to 30-fold), whereas that of MMP-2, TIMP-1, TIMP-2, and uPA was similar between the two groups. Protein levels were increased in the serum-free conditioned media of conjunctivochalasis fibroblasts for MMP-1 (3.5- to 7.6-fold) and MMP-3 (2.3- to 13-fold), determined by ELISA and Western blot analysis. There was increased caseinolytic activity of MMP-3 and collagenolytic activity of MMP-1 (2.2-fold) by conjunctivochalasis fibroblasts, whereas no difference was noted between these two types of fibroblasts in the protein and gelatinolytic activity of MMP-2 or expression of TIMP-1 and TIMP-2 proteins, although that of TIMP-1 transcript was slightly higher in some conjunctivochalasis fibroblasts. No expression of MMP-9 was detected.

conclusions. Overexpression of MMP-1 and MMP-3 mRNA by conjunctivochalasis fibroblasts is correlated with their increased protein levels and proteolytic activities. Collectively, these data help explain how conjunctivochalasis manifests excessive degradation of the conjunctival matrix and Tenon’s capsule.

Conjunctivochalasis, defined as a redundant, loose, nonedematous inferior bulbar conjunctiva interposed between the globe and the lower eyelid, tends to be bilateral and is more prevalent in older populations. ^{1 2 3 4 5 6} Conjunctivochalasis in its mild form causes and aggravates an unstable tear film by depleting the tear meniscus and interfering with eyelid blinking, in its moderate form causes intermittent epiphora by interfering with tear clearance, and in its severe form causes exposure-related problems such as nocturnal lagophthalmos and dellen formation. ⁷ The underlying cause of conjunctivochalasis is unknown, but it has been suggested that it involves abnormalities in conjunctival extracellular components. Hughes ¹ considered it to be a senile change. Existing pathologic data are scanty and conflicting. Degeneration of elastic fibers has been noted, ⁸ but no fragmentation or other abnormality of the elastic fibers was observed using hematoxylin and eosin and Weigert’s elastic tissue stain. ¹ A similar elastotic degeneration is known as a feature of pingueculae, pterygium, and photoaged skin and has been regarded as a hallmark for actinic damage. ⁹ Pinguecular lesions are frequently associated with conjunctivochalasis. ¹⁰ The clinical sign of redundant tissue suggests that extracellular matrix–degrading enzymes may contribute to the pathogenesis of conjunctivochalasis. 

Matrix metalloproteinases (MMPs) are a family of enzymes that act to modify or degrade the extracellular matrix. ^{11 12 13} These enzymes are synthesized and secreted by a variety of cell types including fibroblasts. At least 22 members of the MMP family have been identified and categorized into five groups: collagenases (MMP-1, -8, and -13), gelatinases (MMP-2 and -9), stromelysins (MMP-3, -10, -11, -21, and -22), membrane-type MMPs and others. MMPs are normally coexpressed with a family of tissue inhibitors of metalloproteinases (TIMPs), which inhibit active forms of MMPs. At least four inhibitors, 28-kDa TIMP-1, 21-kDa TIMP-2, 23-kDa TIMP-3, and 24-kDa TIMP-4, have been characterized and are also produced by many cell types including fibroblasts. ^{11 13 14} The balance between the activity of MMPs and that of TIMPs determines the extent of proteolysis linked with tissue remodeling or degradation of extracellular matrix components including collagen and elastin. ^{12 13} Besides MMPs and TIMPs, another proteolytic cascade leading to tissue degradation and remodeling involves urokinase plasminogen activator (uPA), a serine protease. ^{15 16 17} In this study, we provided experimental evidence showing that conjunctivochalasis fibroblasts in culture overexpressed MMP-1 and MMP-3 but maintained no change in MMP-2, TIMP-1, TIMP-2, and uPA, when compared with cultured normal human conjunctival fibroblasts. 

Materials and Methods

Materials

Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), amphotericin B, phenol, DNA, or RNA size marker, and random primers DNA labeling kit were purchased from Gibco (Grand Island, NY). Cell culture dishes, six-well plates and 15-ml centrifuge tubes were from Becton Dickinson (Lincoln Park, NJ). BCA protein assay kit was from Pierce (Rockford, IL). Zymogram-ready gels containing gelatin or casein, 4% to 15% Tris-HCl polyacrylamide gradient-ready gel, sodium dodecyl sulfate (SDS), and electrophoresis equipment were from Bio-Rad (Hercules, CA). Human MMP-1 and MMP-3 enzyme-linked immunosorbent assay (ELISA) kits and the monoclonal antibodies against human MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2 were from Oncogene Research Products division of Calbiochem (Cambridge, MA). The ABC peroxidase kit (Vectastain Elite) was from Vector Laboratories (Burlingame, CA). Nitrocellulose membranes were from Schleicher and Schuell (Keene, NH). The RNA-polymerase chain reaction (PCR) kit (GeneAmp) was from Perkin–Elmer Cetus (Norwalk, CT), and the DNA purification kit (Wizard PCR Preps) was from Promega (Madison, WI).[α -³²P]-dCTP was from Du Pont NEN (Boston, MA). Films (XAR-5 and BioMax MS-1) and intensifying screens were from Eastman Kodak (Rochester, NY). All other reagents and chemicals were purchased from Sigma (St. Louis, MO). 

Human Conjunctival and Conjunctivochalasis Fibroblast Cultures

Normal human conjunctiva and conjunctivochalasis specimens were obtained from patients who were undergoing cataract and conjunctivochalasis surgery, respectively, after obtaining the patients’ informed written consent and explaining the study in keeping with the tenants of the Declaration of Helsinki. The method described in the following section was used to obtain six samples of normal conjunctival fibroblasts and eight of conjunctivochalasis fibroblasts. For this study, fibroblasts at the third or fourth passage were used. Normal conjunctival fibroblasts and conjunctivochalasis fibroblasts were obtained from explant cultures using a technique identical with a previously described method. ¹⁸ In brief, each tissue specimen was cut into explants of approximately 2 × 2 mm² and placed onto 100-mm tissue culture dishes. Ten to 20 minutes later, each explant was covered with a drop of DMEM containing 10% FBS (DMEM-FBS), 50 μg/ml gentamicin, and 1.25 μg/ml amphotericin B and placed overnight in an incubator at 37°C with 95% humidity and 5% CO₂. On the next day 10 ml of the same media was added, and the media were changed three times weekly thereafter. These fibroblasts were subcultured with 0.1% trypsin and 0.02% EDTA in calcium-free minimum essential medium (MEM) at 80% to 90% confluence with 1:2 to 3 split for several passages. 

For Northern blot analysis, fibroblasts were cultured for 7 to 9 days in 100-mm dishes containing DMEM-FBS until confluence, before extraction of total RNA. For ELISA, Western blot analysis, zymography, and quantitative collagenase assay, the same passages of these two types of fibroblasts were seeded in triplicate at the same density (2.5 × 10⁵ cells/well) in six-well plates and grown for 7 to 9 days until confluence. After they were washed four times with PBS, cultures were switched to the same volume (1 ml) of serum-free DMEM containing 5 μg/ml insulin, 5 μg/ml transferrin, and 5 ng/ml sodium selenite (DMEM-ITS) and incubated for an additional 48 hours. The conditioned media were then collected and stored at− 20°C before use, and the adherent cells were lysed in phosphate-buffered saline (PBS, pH 7.3), containing 1.5 M NaCl and 0.039% Triton X-100 for BCA protein assay. 

Probe Preparation

Five human DNA probes, including a 185-bp fragment of MMP-1, 480 bp of MMP-2, 155 bp of MMP-3, 551 bp of TIMP-1, and 590 bp of TIMP-2 were kindly provided by Velidi H. Rao (University of Nebraska Medical Center, Omaha). Three cDNA probes, 640 bp of MMP-9, 519 bp of uPA, and 498 bp of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), were purified from reverse transcription–polymerase chain reaction (RT-PCR) products by electrophoresis through a 1.2% low-melting-point agarose gel using the DNA purification kit (Promega) according to the manufacturer’s protocol. The primers used for PCR were 1502 to 1531 (sense) and 2111 to 2140 (antisense) for MMP-9 (accession number J05070), 487 to 506 (sense) and 982 to 1002 (antisense) for uPA (accession number A18397), 541 to 561 (sense) and 1018 to1038 (antisense) for GAPDH (accession number M33197). The ³²P-labeled cDNA probes (1–2 × 10⁹ cpm/μg DNA) were prepared with[α -³²P]-dCTP (3000 Ci/mmol) using a random primer DNA labeling system. 

RNA Isolation and Northern Hybridization

Total RNA isolation and Northern hybridization were performed using a previously described method. ¹⁸ Briefly, total RNA was isolated from fibroblast cultures by acid guanidium thiocyanate-phenol-chloroform extraction. Total RNA at 20 μg per lane was electrophoresed through 1.2% agarose containing formaldehyde, transferred to nitrocellulose membranes, and hybridized with ³²P-labeled cDNA probes at 2 to 4 × 10⁶ cpm per 3 to 8 ng/ml in the hybridization solution. After the hybridization product was visualized in the x-ray film, the ³²P-label on the membrane was stripped by washing the membranes at 65°C for 1 hour twice in 5 mM Tris-HCl (pH 8.0), 0.2 mM EDTA, 0.05% sodium pyrophosphate, and 0.1× Denhardt’s solution and rehybridized with other ³²P-labeled probes. The relative amount of each mRNA of interest was determined by scanning its autoradiofluorogram with a laser scanning densitometer (model FB910; Fisher Scientific, Pittsburgh, PA), and normalized as a ratio to that of the GAPDH mRNA band. 

MMP-1 and MMP-3 ELISA

Human MMP-1 or MMP-3 double-sandwiched ELISA was performed using commercial ELISA kits according to the manufacturer’s protocol (Oncogene). In brief, 100-μl standard dilutions of recombinant human MMP-1 or MMP-3 and experimental conditioned media were dispensed into a 96-well microtiter plate coated with mouse anti-MMP-1 or MMP-3 monoclonal antibody, respectively. The plate was sealed, incubated at room temperature (RT) for 2 hours or at 4°C for 1 hour, respectively, and washed four times with PBS containing 0.033% Tween 20. After addition of 100 μl diluted rabbit anti-MMP-1 serum to each well and incubation at RT for 2 hours followed by four washes, 100 μl diluted donkey anti-rabbit horseradish peroxidase conjugates was added and incubated for 1 hour at RT. For MMP-3, 100 μl diluted rabbit anti-MMP-3 horseradish peroxidase was added into each well and incubated at 4°C for 2 hours. Aliquots of 100 μl of the color reagent 3,3′,5,5′-tetramethylbenzidine were then applied for 20 to 30 minutes to develop a blue color, and the reaction was stopped by adding 100 μl 1 M H₂SO₄. Absorbance was read at 450 nm by an automatic plate reader with a reference wavelength of 570 nm. 

Western Blot Analysis

To identify MMP and TIMP proteins present in each fibroblast-conditioned medium, Western blot analysis was performed using their specific antibodies. Appropriate volumes (25–30 μl) of conditioned media from different fibroblast cultures were adjusted to represent the same quantity of cellular protein (15 μg) and electrophoresed at 4°C under a reducing condition in a 4% to 15% gradient polyacrylamide gel. After electrophoretic transfer to a nitrocellulose membrane at 4°C, the membrane was immersed with 0.1% (vol/vol) Tween 20 in Tris-buffered saline (100 mM Tris, 0.9% NaCl[ pH 7.5]; TTBS) for 30 minutes with agitation. The primary antibody (i.e., 1 μg/ml mouse monoclonal antibody against human MMP-1, MMP-2, MMP-3, TIMP-1, or TIMP-2) in TTBS containing 1% horse serum was placed on each membrane and incubated at RT for 60 minutes with agitation. After a washing with three to four changes of TTBS over 15 minutes, each membrane was transferred to a 1:200 diluted solution of biotinylated second antibody (goat anti-mouse IgG from the ABC kit; Vector) in TTBS containing 1% horse serum and incubated for 30 minutes. After three to four washes with the same solution, they were incubated with a 1:50 diluted ABC reagent conjugated with peroxidase for 30 minutes and processed for color development in 0.5 μg/ml diaminobenzidine in 50 mM Tris-HCl [pH 7.2] containing 0.05% H₂O₂ for 10 to 20 minutes. 

Zymography of MMP Activity

To determine gelatinolytic and stromelysin activities of the fibroblast cultures, zymography was performed using a method similar to that previously described. ¹⁹ For gelatin zymography, 25 to 30 μl of each conditioned medium was used, which was adjusted to represent the same quantity of cellular protein (15 μg). For casein zymography, 75 to 90 μl of media, representing 45 μg cellular protein, was concentrated to 30 μl by a speed vacuum drier before use. The medium samples were treated with sample buffer without boiling or reduction. Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) was performed using a 10% polyacrylamide gel containing 0.1% gelatin or a 12% gel containing 0.1% casein at 100 V for 90 minutes at 4°C. The gels were soaked in 2.5% Triton X-100 for 30 minutes at RT to remove the SDS and incubated in a reaction buffer [50 mM Tris-HCl (pH 7.5), 200 mM NaCl, 5 mM CaCl₂ and 0.02% 23 lauryl ether (Brij-35)] containing 5 mM phenylmethylsulfonyl fluoride, a serine protease inhibitor, at 37°C overnight to allow proteinase digestion of its substrate. Gels were rinsed again in distilled water, stained with 0.5% Coomassie brilliant blue R-250 in 40% methanol and 10% acetic acid for 2 hours, and destained with 40% methanol and 10% acetic acid. Proteolytic activities appeared as clear bands of lysis against a dark background of stained gelatin or casein. To verify that the detected gelatinolytic and caseinolytic activities were specifically derived from MMPs, the gels were treated in parallel experiments with the Triton X-100 solution and the Tris-NaCl-CaCl₂ reaction buffer containing 10 mM EDTA. 

Quantitative Collagenase Activity Assay

Collagenase activity was verified and quantified by incubation with soluble, telopeptide-free collagen extracted from rat skin and labeled with [³H]-acetic anhydride. ²⁰ For assay of collagenase with labeled substrate, it was necessary to inactivate the TIMPs in media (500 μl) from fibroblast cultures by reduction in 2 mM dithiothreitol at 37°C for 30 minutes, followed by alkylation in 5 mM iodoacetamide at 37°C for 30 minutes. This step also inactivates any α-2-macroglobulin. The samples were chilled on ice and dialyzed against the assay buffer (50 mM Tris-HCl, 200 mM NaCl, 10 mM CaCl₂, and 0.005% Brij-35 [pH 7.5]) for 4 hours before use. Each sample was then tested in triplicate at different volumes (30, 60, and 90 μl), and the assay was performed twice for accuracy. One of each triplicate sample was added with aminophenylmercuric acetate (APMA) to a final concentration of 0.5 mM to activate latent procollagenase. Another was added with 1,10-phenanthroline to a final concentration of 2.0 mM in the presence of 0.5 mM APMA to chelate the zinc and inactivate the collagenase. The blanks were prepared by replacing the conditioned medium with an equivalent volume of the assay buffer.[ ³H]-acetic collagen (115,400 cpm per 15.82μ g/5 μl) was added to each sample, and the final volume was adjusted to 110 μl with the assay buffer. The reaction was incubated at 30°C for 18 hours and then stopped by placing the tubes in an ice bath. After adding 120 μl of the assay buffer containing 200 μg acid-soluble intact collagen as a cold carrier, 20 μg trypsin, 20μ g chymotrypsin, and 30 mM EDTA, the second digestion was performed at 31.5°C for 90 minutes. The soluble digested products were separated from the undigested collagen by precipitating with an equal volume of ice-cold 20% trichloroacetic acid (TCA). After centrifugation at 13,000 rpm for 5 minutes, triplicate aliquots (100μ l each) of the supernatant (representing TCA-soluble peptides) were counted by liquid scintillation for 3 minutes. The collagenase activity was reported as units per milliliter (1 unit of enzyme digests 1 μg of collagen per minute at 30°C). 

Statistical Analysis

Student’s t-test was used for statistical comparison for the data of Northern hybridization, ELISA, and collagenase activity assay. 

Results

Transcript Expression of uPA, MMPs, and TIMPs

The transcripts of 2.3 kb uPA, three MMPs (2.2 kb MMP-1, 3.1 kb MMP-2, and 1.9 kb MMP-3) and two TIMPs, (0.9 kb TIMP-1 and 3.5 kb TIMP-2), were expressed by cultured normal conjunctival (HJF) and conjunctivochalasis (Chalasis) fibroblasts (Figs. 1 and 2) . The sizes of these six transcripts were consistent with those previously reported. ^{15 16 21 22} No MMP-9 transcript was detected in these two types of fibroblasts. Among the three MMPs, the MMP-2 transcript did not show any difference between normal conjunctival and conjunctivochalasis fibroblasts when normalized to the GAPDH transcript used as a loading control (Fig. 1) . In contrast, there was a marked increase in the expression of MMP-1 and MMP-3 transcripts by conjunctivochalasis fibroblasts (Fig. 1) . The amount of the MMP-1 transcript expressed by six different strains of normal conjunctival fibroblasts was low to undetectable, whereas that of the MMP-1 transcript was markedly increased from 5- to 32-fold (mean ± SD: 13.4 ± 8.6-fold) in all eight strains of conjunctivochalasis fibroblasts (P < 0.005). The expression of the MMP-3 transcript was low to undetectable in five of six strains of normal conjunctival fibroblasts (except strain 6) but was markedly increased from 4- to-30 fold (14.1 ± 9.1-fold) in seven of eight strains of conjunctivochalasis fibroblasts (except strain 3; P < 0.05). There was no significant difference in the transcript expression of uPA, TIMP-1, and TIMP-2 between these two types of fibroblasts, although the expression of TIMP-1 transcript was slightly higher in four of eight conjunctivochalasis fibroblasts (Fig. 2) . 

ELISA for MMP-1 and MMP-3 Proteins Produced in Media

The protein levels of MMP-1 and MMP-3 were determined by their respective ELISAs in the serum-free conditioned media from three samples of normal conjunctival fibroblasts and three samples of conjunctivochalasis fibroblasts. As shown in Figure 3 , the amount of MMP-1 protein in the media from three normal conjunctival fibroblasts was 8.3 ± 1.0 (J1), 4.8 ± 2.4 (J2), and 7.7 ± 2.4 (J3) ng/ml, and was 24.21 ± 8.29 (C1), 52.88 ± 7.81 (C2), and 27.43 ± 4.31 (C3) ng/ml for three conjunctivochalasis fibroblasts. The latter were 3.5 to 7.6 times higher than the former (P < 0.05; n = 3). The amount of MMP-3 protein in the media from three normal conjunctival fibroblasts was 23.65 ± 1.62 (J1), 17.20 ± 2.37 (J2), and 13.23 ± 3.91 (J3) ng/ml, and was 48.31 ± 4.56 (C1), 82.85 ± 13.47 (C2), and 269.50 ± 16.40 (C3) ng/ml for three conjunctivochalasis fibroblasts. The latter were 2.3 to 13 times higher than the former (P < 0.05; n = 3). 

Protein Expression of MMPs and TIMPs by Western Blot Analysis

The protein expression of MMPs and TIMPs was studied in the serum-free conditioned media derived from three samples of normal conjunctival and three samples of conjunctivochalasis fibroblasts using their specific monoclonal antibodies, which recognize both latent and active forms. As shown in Figure 4 , the amounts of a 54-kDa MMP-1 band and a 57-kDa MMP-3 band secreted by conjunctivochalasis fibroblasts (C1, C2, and C3) were markedly increased when compared with those from normal conjunctival fibroblasts (J1, J2, and J3), which revealed nondetectable bands in the Western blot condition. In contrast, protein levels of 72-kDa MMP-2, 28-kDa TIMP-1, and 21-kDa TIMP-2 were similar between both fibroblasts. 

Zymography for Gelatinolytic and Caseinolytic Activities of MMP-2 and MMP-3

The gelatin zymogram showed a strong but comparable intensity of a 72-kDa band corresponding to MMP-2 in both types of fibroblasts (Fig. 5 , top). Both latent (predominantly) and active forms of MMP-2 existed. This gelatinolytic activity of 72-kDa MMP-2 was completely inhibited by incubating the gel with solutions containing 10 mM EDTA, one of the MMP inhibitors (not shown). The gelatinolytic activity of 92-kDa MMP-9 was not detected in any of these fibroblast-conditioned media. The casein zymogram disclosed caseinolytic activities of a 57-kDa MMP-3 band and a weak 72-kDa MMP-2 band in these conditioned media (Fig. 5 , bottom). MMP-3 activity produced by conjunctivochalasis fibroblasts (C1, C2, and C3) was significantly higher than that by normal conjunctival fibroblasts (J1, J2, and J3), which produced very little activity. The two bands of MMP-3 with molecular weights close to each other may be of the latent and active (predominant) forms. In contrast, weak caseinolytic activity of a 72-kDa MMP-2 band was produced in a similar amount by these two types of fibroblasts. The caseinolytic activity of 57-kDa MMP-3 and 72-kDa MMP-2 disappeared after the gel was incubated with a solution containing 10 mM EDTA (not shown), confirming that the increased caseinolytic activity of 57-kDa MMP-3 by conjunctivochalasis fibroblasts was indeed derived from an MMP. 

Quantitative Collagenase Activity Assay for MMP-1

The collagenolytic activity of MMP-1 was determined with a soluble, telopeptide-free collagen extracted from rat skin and labeled with [³H]-acetic anhydride. This collagenolytic activity was mainly present in a latent form in the serum-free fibroblast-conditioned media. After activation by APMA, the collagenolytic activity produced by three normal conjunctival fibroblasts was 0.073 ± 0.010 (J1), 0.078 ± 0.004 (J2), and 0.035 ± 0.019 (J3) U/ml, whereas the activity produced by three conjunctivochalasis fibroblasts was 0.148 ± 0.025 (C1), 0.126 ± 0.015 (C2), and 0.124 ± 0.02 (C3) U/ml. The latter was an average of 2.2 times higher than that from normal conjunctival fibroblasts (P < 0.05; n = 3; Fig. 6 ). 

Discussion

MMPs and their inhibitors (TIMPs) play a vital role in connective tissue degradation and remodeling (for review see References 12 and 13). In the human eye, studies of MMPs and TIMPs have been performed in the aqueous humor, ^{23 24} vitreous, ^{25 26} retina, ²⁷ trabecular meshwork, ²⁸ keratoconus corneas, ^{29 30} and corneas during wound healing. ^{31 32} These studies have focused primarily on gelatinase A (MMP-2), gelatinase B (MMP-9), TIMP-1, and TIMP-2. Our present study concludes that under the same culture condition (cell passage, seeding density, presence or absence of FBS, and so forth) the expression and activity of MMP-1 and MMP-3, but not those of MMP-2 or MMP-9, are significantly increased in conjunctivochalasis fibroblasts, whereas the expression of TIMP-1 and TIMP-2 is not changed, compared with that in normal conjunctival fibroblasts. These results suggest that increased gene expression levels of MMP-1 and MMP-3 in conjunctivochalasis fibroblasts may contribute to the histopathologic abnormalities seen in conjunctivochalasis. Although uPA, a serine protease, was found to be responsible for tissue degradation and tumor cell invasion, ^{15 17 33} its expression is not changed in normal conjunctival and conjunctivochalasis fibroblasts. We suggest that the looseness of conjunctival tissue observed in conjunctivochalasis may be correlated to fibroblast overexpression of MMP-1 and MMP-3. 

Overexpression of MMP-1 and MMP-3 transcript and protein by conjunctivochalasis fibroblasts was demonstrated by Northern hybridization (Figs. 1 and 2) , ELISA (Fig. 3) , and Western blot analysis (Fig. 4) , respectively. For comparison, the protein levels of TIMP-1 and TIMP-2 were unchanged (Fig. 4) , although TIMP-1 transcript expression was slightly higher in some conjunctivochalasis fibroblasts (Fig. 2) . These data suggest that the ratio between MMPs and TIMPs produced by conjunctivochalasis fibroblasts is higher than those produced by normal conjunctival fibroblasts. This notion is further supported by a higher caseinolytic activity of MMP-3 (Fig. 5) and a significantly higher collagenolytic activity of MMP-1 produced by conjunctivochalasis fibroblasts (Fig. 6) . It is known that MMP-1 and MMP-3 can degrade collagen and elastin fibers. MMP-1, an interstitial collagenase, can degrade native fibrillar collagen types I, II, III, IX, and XI. ^{12 13 34} MMP-3, or stromelysin-1, has a broad substrate specificity that includes casein, proteoglycans, fibronectin, elastin, and laminin, as well as collagen types III, IV, V, IX, and IX. ^{11 12 13 35} Cooperative actions of MMP-1 and MMP-3 further augment the final proteolytic action. Therefore, it is conceivable that overproduction of MMP-1 and MMP-3 relative to their TIMPs by conjunctivochalasis fibroblasts may facilitate the degradation of the extracellular matrix. 

Earlier, we noted that the expression of MMP-2 transcript and protein was unchanged in normal conjunctival and conjunctivochalasis fibroblasts (Figs. 1 4 and 5) . The finding that MMP-2 expression was unaltered is consistent with the view that MMP-2 expression tends to be constitutive and is thought to perform a surveillance function. ³¹ This unique feature is due to the unusual promoter structure of MMP-2, which does not have a TATA box or AP-1 elements commonly found and critical for gene activation in the promoters of MMP-1, MMP-3, and other inducible MMPs. ¹¹  

Interestingly, upregulation of proteins and activities of MMP1, MMP3, and MMP-9 but not MMP-2 has been reported in cultured skin fibroblasts from patients with cutis laxa, ³⁶ a disorder exhibiting loose and sagging skin with reduced elasticity. It has been suggested that overexpression of MMPs in these skin fibroblasts may contribute to the finding of abnormal collagen and elastin fibers in this disease. ³⁶ Hours after exposure to UVB radiation, the skin also shows increased mRNAs, proteins and activities of MMP-1, MMP-3, and MMP 9 but not MMP-2. ³⁷ Recently, we also noted overexpression of MMP-1 and MMP-3 by cultured pterygial head fibroblasts. ³⁸ Because photoaged skin shows a similar pathologic change of elastotic degeneration, which is also found in pingueculae and pterygium (diseases associated with UV exposure ^{9 10} ), and because pingueculae are frequently associated with conjunctivochalasis, ⁸ future studies are needed to investigate whether conjunctivochalasis may be causatively linked with UV exposure leading to overexpression of MMP-1 and MMP-3. 

 Presented in part as an abstract at the annual meeting of the Association for Research in Vision and Ophthalmology, May 9–14, 1999, Fort Lauderdale, Florida.

 Supported in part by Public Health Service Research Grant EY-06819 (SCGT) from the National Eye Institute, by an unrestricted grant from Research to Prevent Blindness, and by Research Fellowship Grant Me 1623/1-1 from the Deutsche Forschungsgemeinschaft (DM).

 Submitted for publication June 10, 1999; revised August 27, 1999; accepted September 23, 1999.

 Commercial relationships policy: N.

 

Figure 1.

 

Northern hybridization. The patterns of transcript expression of MMP-1, MMP-2, and MMP-3 were compared among six samples of normal conjunctival fibroblasts (HJF) and eight samples of conjunctivochalasis fibroblasts (Chalasis). All 14 samples of the two groups of fibroblasts were grown to confluence in DMEM-FBS and subjected to total RNA extraction and Northern blot analysis. The RNA blots were then individually hybridized with ³²P-labeled specific cDNA probes with GAPDH as a loading control.

Figure 2.

 

Northern hybridization. The patterns of transcript expression of uPA, TIMP-1, and TIMP-2 were compared among six samples of normal conjunctival fibroblasts (HJF) and eight samples of conjunctivochalasis fibroblasts (Chalasis). See legend to Figure 1 for method used.

Figure 3.

 

ELISA. Protein levels of MMP-1 and MMP-3 secreted in serum-free conditioned media from three samples each of normal conjunctival fibroblasts (J1, J2, and J3) and conjunctivochalasis fibroblasts (C1, C2, and C3).

Figure 4.

 

Western blot analysis. Proteins of MMP-1, MMP-2, MMP-3, TIMP-1, and TIMP-2 expressed in the serum-free conditioned media from three samples each of normal conjunctival fibroblast (J1, J2, and J3) and conjunctivochalasis fibroblasts (C1, C2, and C3).

Figure 5.

 

Zymograms. The gelatinolytic activity of MMP-2 (top) and the caseinolytic activity of MMP-3 (bottom) were demonstrated in the serum-free conditioned medium from three samples each of normal conjunctival fibroblasts (J1, J2, and J3) and conjunctivochalasis fibroblasts (C1, C2, and C3).

Figure 6.

 

Collagenase activity. The collagenase activity of MMP-1 was measured by a quantitative collagenase assay in serum-free conditioned media from three samples each of normal conjunctival fibroblasts (J1, J2, and J3) and conjunctivochalasis fibroblasts (C1, C2, and C3). One unit of enzyme digests 1 μg collagen per minute at 30°C.