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Unique Regulation of the Matrix Metalloproteinase, Gelatinase B

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Unique Regulation of the Matrix Metalloproteinase, Gelatinase B 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 pro- tein 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 Investigative Ophthalmology & Visual Science, March 1995, Vol. 36, No. 3 622 Copyright © Association for Research in Vision and Ophthalmology Downloaded from iovs.arvojournals.org on 10/02/2021 Unique Regulation of Gelatinase B 623 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 mem- brane 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 exper- iments; 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 appro- priate 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 Downloaded from iovs.arvojournals.org on 10/02/2021 624 Investigative Ophthalmology & Visual Science, March 1995, Vol. 36, No. 3 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.
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