The significant role of transforming growth factor (TGF)-β in lens pathology has been documented in association with anterior subcapsular and anterior polar cataracts.1–5 We have shown that lens epithelial cells (LECs) transdifferentiate into myofibroblast-like cells and form fibrotic plaques consisting of abnormal extracellular matrix (ECM) components such as type I collagen, fibronectin, and βig-h3.6,7 We also demonstrated that treatment of LECs with TGF-β increases the synthesis of pathologic ECM proteins characteristic of fibroblasts. Although the exact mechanism of the development of anterior polar cataract is not known, our studies support the possibility that it is associated with the abnormal deposition of extracellular proteins from LECs.6
A disintegrin and metalloproteinase (ADAM) is a large family of membrane-bound glycoproteins that contains a disintegrin and metalloprotease domain and possesses cell adhesion and protease activities. In addition to its well-defined role in sperm–egg interaction, myocyte binding, and fusion, it is associated with renal epithelial cellular interactions with the basal lamina and adjoining cells.8–11 Recent studies of the expression of known ADAM genes have shown that the genes possess specific tissue distribution and functions in particular biological systems. For instance, ADAM10 is involved in various aspects of development, such as neurogenesis, by releasing a soluble form of a Notch ligand that regulates cell fate determination.12 Among the known ADAMs, 17 have a metalloproteinase active site with a functional protease and 12 are thought to lack protease activity.10,11,13–18 Recently, ADAMTS (ADAM with thrombospondin motifs)-1 was shown to play a role in the cell–matrix interaction, which is crucial for the development of liver fibrosis.19
Little is known about the presence of ADAMs or their roles in LECs. Because ADAM is also known as a sheddase, which plays a role in the release and cleavage of a variety of membrane-anchored proteins, we hypothesized that this protein may be implicated in the abnormal accumulation of ECM seen in anterior polar cataracts. To test this, we looked at whether ADAM mRNA is aberrantly present in LECs from noncataractous and cataractous lenses. We also present evidence that the expression of ADAM is differentially regulated by TGF-β in cultured LECs.
Materials and Methods
Human Lens Capsules
Lens capsules with attached LECs were obtained during cataract surgery as previously described.6 Cataractous lenses were obtained from 21 patients aged 24 to 95 years with the clinical diagnosis of anterior polar and nuclear cataracts. Capsules of noncataractous lenses were obtained during clear lens extraction for correction of high myopia. Briefly, after viscoelastic material was injected into the anterior chamber, a continuous curvilinear capsulorhexis was performed and the anterior capsule was removed carefully with a forceps. Lens capsules were immediately placed in TriZol reagent (Gibco) for RNA preparation or frozen in liquid nitrogen and stored at –70°C for further analysis. Three independent experiments were performed. The data shown are 1 of the 3 independent experiments that produced similar results.
Postmortem eyes from BALB/c wild mice (Sam YuK Laboratory Animal, Inc.) were dissected to retrieve the lenses for storage in liquid nitrogen until RNA extraction. All animal procedures conformed to institutional guidelines and the ARVO Statement for Use of Animals in Ophthalmic and Vision Research.
Cell Culture and Growth Factor Treatments
The murine LEC line, αTN-4, was grown in Dulbecco's modified Eagle medium supplemented with 10% fetal bovine serum, 2 mM glutamine, 100 units/mL penicillin, and 100 μg/mL streptomycin. The HLE B-3 cell lines were propagated in minimum essential medium (MEM) supplemented with 20% fetal bovine serum and 50 μg/mL gentamicin. Eyes were removed from 8-week-old mice under sterile conditions, placed in the medium (ie, M199 containing bovine serum albumin and antibiotic agents), and preincubated at 37°C in 5% CO2/air. Lenses were removed and incubated in 2 mL for 1 hour. Epithelia were peeled away from fibers and pinned with the cellular surface uppermost in cultured dishes containing 1 mL medium. The entire epithelium was used unless otherwise specified, and each dish contained 9 or 10 explants. Approximately 3 hours after the explants were prepared, the medium was replaced and TGF-β1 was added to 5 final concentrations of 10 ng/mL.
Reverse Transcribed-Polymerase Chain Reaction
Total cellular RNA was isolated by using TriZol reagent. One microgram of RNA was reverse transcribed (RT) using a cDNA synthesis kit (Boehringer Mannheim). The cDNA was then amplified in a 20 μL reaction mixture by polymerase chain reaction (PCR) with the following conditions: 0.4 μM each primer, 0.2 mM deoxynucleoside triphosphate mixture (Perkin Elmer Corp.), 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 1.5 mM MgCl2, and 1 unit Taq polymerase (Perkin Elmer Corp.). The reaction mixtures were incubated in a thermal controller (model PTC-100, MJ Research) for 35 cycles of denaturation for 45 seconds at 95°C, annealing for 1 minute at 58°C, and extended for 45 seconds at 72°C. The amount of amplified products was analyzed using an image-documentation system (ImageMaster VDS, Pharmacia Biotech, Inc.). The primer sequences specific for the genes examined and the predicted sizes for human ADAM9 integrin isoform cDNA were 5′-AATGCGAGTTATGGGATTGA-3′ and 5′-ATTACCACAGGAGGGAGCA-3′, yielding an amplication product of 844 bp; for mouse ADAM1 integrin isoform cDNA, they were 5′-GACCTTCCCGAGTATTGTGA-3′ and 5′-GCTTACCAACAGGACCACTG-3′, yielding an amplification product of 750 bp. Beta-actin was amplified as an internal control; primers for human and mouse cDNA were 5′-ATCATGTTTGAGACCTTCAACACC-3′ and 5′-CATGGTGGTGCCGCCAGACAG-3′.
As shown in Figure 1, top, the expression of ADAM9 mRNA was readily detectable in LECs from noncataractous lenses and nuclear cataracts but was markedly reduced in anterior polar cataracts. The amount of β-actin product as an internal control for PCR amplification was similar among the samples.
The results in Figure 1, bottom, correlated with those in Figure 1, top, demonstrating a reduced expression of ADAM9 in TGF-β1-treated LECs. In comparison, the levels of ADAM9 mRNA for epidermal growth factor (EGF), fibroblast growth factor (FGF)-2, and insulin growth factor (IGF)-1 were similar in LECs from control samples. The EGF, FGF-2, and IGF-1 were added because these cytokines are known to be aberrantly secreted in LECs at the capsulotomy site as an expression of wound healing after cataract surgery. The reduction in ADAM9 mRNA levels was similar to that in anterior polar cataractous LECs.
Only ADAM1 mRNA was inherently expressed in the mouse lens tested (Figure 2, top). As indicated in Figure 2, middle, ADAM1 mRNA was detected in the αTN-4 cell line and mouse explants. Figure 2, middle, indicates that the expression of ADAM1 mRNA was reduced in αTN-4 from TGF-β1. In comparison, levels of ADAM1 mRNA were reduced in NIH-3T3 for FGF-2, indicating a specific regulation of ADAM1 mRNA in the lens (data not shown). Because the mouse LEC line (αTN-4) has a limited number of LECs, mouse explants were used to examine the regulation of ADAM1 mRNA in response to TGF-β1 and FGF-2. Figure 2, bottom, shows that TGF-β1 mediated the reduction of mRNA for ADAM1 in mouse lens explants within 24 hours, while there was no effect on the regulation of ADAM1 mRNA by FGF-2.
Members of the ADAM family differ in the range of cell types that are normally expressed and in the biological functions they affect. With the recent demonstration of the role of ADAM9 in the kidney and ADAM1 in fertilization,10 it is likely that more ADAMs will be identified and that previously known members will be found to possess new functions. The ADAMs identified in this study include ADAM1 and ADAM9, containing active metalloprotease functions, and other members whose functions require further investigation. The results of this study provide the first evidence that ADAM1 and ADAM9 are inherently and differentially regulated in LECs. The expression of ADAM1 and ADAM9 was unequivocally demonstrated at the RNA levels, suggesting that these genes may play a significant role in the normal and pathological processes of the lens.
Previous studies demonstrate that LECs of anterior subcapsular cataracts and anterior polar cataracts are transdifferentiated into fibroblast-like cells and produce a large amount of ECM not normally expressed in lens capsules.6,20 In the present study, we demonstrated that the LECs of anterior polar cataracts had reduced expression levels of ADAM9 mRNA compared with specimens taken from age-related nuclear cataracts and high myopia. The reduced levels of mRNAs in ADAM9 suggest the existence of another mode of action by which TGF-β may modulate transdifferentiation and ECM production of LECs. Growing evidence suggests the physiological roles of ADAMs in regulating epithelial interaction with the basal ECM and adjoining cells.10 Recently, ADAM9, a widely expressed non-RGD-containing member of the ADAM family, was shown to be localized in the basolateral portion of the renal cortical tubule and glomerular visceral epithelial cells, colocalizing with the β1 integrin chain.9,10 Also, ADAM9 has been shown to mediate cell−cell adhesion via an interaction with the αvβ5 integrin.21 However, correlation of the regulation of ADAM and TGF-β accessible to LECs in vivo is still difficult.
Inherent expression of ADAM1 mRNA is thought to be limited to the mouse and has not been observed in human LECs. The data presented suggest that the expression of the ADAM1 mRNA in the lens may be regulated by endogenous lens growth factors that regulate development in the mouse lens. The data further reveal that the transcriptional regulation of ADAM1 is mediated by various growth factors in LECs. Considering the downregulated expression of ADAMTS-1 in endothelial cells in the development of liver fibrotic rat models, downregulated expression of ADAM1 might play a role during the process of fibrogenesis acting in a paracrine or autocrine way.19 The transcription of ADAM1 has been shown to be involved in sperm–egg fertilization.8,22–24 What then is the functional relevance in the lens of ADAM, whose functions are thought to be restricted to sperm–egg adhesion? Wong et al.25 recently reported that the disintegrin-like domain of mouse ADAM1 participates in sperm–egg adhesion during fertilization and identified a region of this domain that partially mediates adhesive activity.
Evidence has also shown that the ability of an integrin to organize a specific ECM ligand binding site can be developmentally regulated.16,17,26 Hence, it appears that there may be developmental situations, in addition to fertilization, in which cells use differential regulation of an integrin for binding ECM ligands and cell surface receptors, such as ADAMs. For example, alpha 6 integrin is expressed in developing LECs at sites of cell–cell contact and their coreceptors at these contacts sites are not known.27 If their coreceptors are ADAMs such as ADAM1 or ADAM2, a switch in the affinity of the alpha 6 integrin from a laminin to an ADAM binding mode may be a key step in the switch from an LEC–ECM mode, for binding to the basement membrane, to a cell–cell binding mode in the ocular lens. Further studies are needed for the distribution and localization of ADAM1 during the developmental stage of the lens.
In summary, this report presents the first study analyzing mRNA for ADAMs associated with pathology in LECs obtained from patients with anterior polar cataracts. Our results demonstrate the reduced expression of ADAM1 and ADAM9 in the lens epithelium in vitro. Recent studies point to ADAM as a new target for a diverse set of biological processes, including fertilization, neurogenesis, myogenesis, and the inflammatory responses. Recent studies have also focused on the functional role of ADAMs in proteolysis, processing of cytokines, the shedding of cytokine receptors, remodeling of ECM components, and cell adhesive activity.25,28 In this regard, the present work may provide a new insight into the cellular and molecular mechanisms involved in the pathogenesis of cataract and the differentiation of the lens.
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