IOVS
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

(Investigative Ophthalmology and Visual Science. 2007;48:4192-4199.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.06-1371
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hodgkinson, L. M.
Right arrow Articles by Wormstone, I. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hodgkinson, L. M.
Right arrow Articles by Wormstone, I. M.

MMP and TIMP Expression in Quiescent, Dividing, and Differentiating Human Lens Cells

Lisa M. Hodgkinson, George Duncan, Lixin Wang, Caroline J. Pennington, Dylan R. Edwards, and I. Michael Wormstone

From the School of Biological Sciences, University of East Anglia, Norwich, United Kingdom.


   Abstract
Top
 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. Matrix metalloproteinases (MMPs) and the tissue inhibitors of the MMPs (TIMPs) have been implicated in lens differentiation, growth, remodeling, and cataract. Hence, a gene expression analysis was undertaken in epithelial and fiber cells dissected from clear human donor lenses.

METHODS. The human lens was dissected into three regions: anterior epithelial, equatorial, and fiber cells. Primary lens cell cultures were also analyzed. cDNA was generated by reverse transcription of the mRNA portion of the total RNA isolated from each sample. Gene expression data were generated using quantitative real-time reverse transcription PCR. Data were analyzed in terms of cycle threshold number (CT) and were normalized to endogenous 18S expression. Western blot analyses were carried out to confirm the presence of two critical MMPs.

RESULTS. Anterior and equatorial samples were uncontaminated by fiber cells because they showed high expression of {alpha}-crystallin genes but low expression of ß- and {gamma}-crystallins. The fibers had high expression of these genes and of MIP. MMP genes were expressed at uniformly low levels in the native tissues except for MMP-14 and -15 (MT1- and MT2-MMP, respectively). In fact, MT1-MMP declined in expression from the anterior epithelium to fibers, whereas MT2-MMP increased. The presence of MT1 and MT2-MMP proforms and faster migrating bands, indicating processed or activated forms, was confirmed at the protein level. TIMP genes were uniformly highly expressed in native tissues, with TIMP-3 having the highest expression in the epithelial tissues and TIMP-2 in the fibers. MMP expression was generally elevated in both sets of cultured cells, including MMP-2 and -9. TIMP genes were also relatively highly expressed in the cultured cells.

CONCLUSIONS. MMP expression is generally well regulated in native tissues, with relatively low expression of MMPs and high expression of TIMPs. Membrane-type MMPs (MT1 and 2-MMPs) were the most highly expressed; this is important in a tissue with relatively high membrane content but low extracellular space. The striking reciprocal patterns of expression of MT1-MMP and MT2-MMP indicate that these enzymes are of particular significance in lens function.

The lens is an ideal organ in which to study gene expression because it can be removed from the eye intact and without the complication of attendant nerve and blood supplies. In addition, constituent epithelia and fiber cells are arranged in a regular and accessible manner. The anterior epithelium, which faces the aqueous humor, represents a quiescent, nondividing population in the adult lens, and the cell division necessary to give rise to new fibers occurs strictly in the equatorial region. Newly formed fibers undergo no further cell division; rather, they elongate to form some of the longest cells in the body (for a review, see Forrester et al.1 ). Ultimately, in the fully differentiated form, they lose their cell organelles. In this study, we chose to investigate the gene expression of the matrix metalloproteinases (MMP) and the tissue inhibitors of the matrix metalloproteinases (TIMP) because not only are they thought to play a role in differentiation,2 they have roles to play in the development of cataract2 3 and posterior capsule opacification (PCO).4

Collectively, the MMPs form a family of zinc-dependent endopeptidases involved in the degradation of extracellular matrix (ECM) to participate in the remodeling of their cellular environment. These enzymes are classified into groups associated with their substrate specificity and structural biology. The collagenases, gelatinases, stromelysins, matrilysins, membrane-type MMPs, and others5 generally consist of a prodomain, a catalytic domain, a hinge region, and a hemopexin domain and can be secreted from the cell or anchored at the cell surface. MMPs are specifically regulated at many levels (for reviews, see Woessner6 and Vosse and Nagase7 ).

Of particular interest in the lens are the gelatinases A and B (MMP-2 and -9), which are involved in corneal wound-healing processes8 and disorders.9 Specifically, they play different roles, MMP-9 in the early stages of wounding10 and MMP-2 in the ensuing ECM remodeling. MMP-2 and -9 are both present in aqueous and vitreous humors.11 12 In the normal lens, MMP-213 and -9 are expressed at low levels, and both are increased when the lens is stressed.3 4 Endogenously, MMPs are regulated by their inhibitors, the TIMPs, which control enzyme activity and proenzyme activation.14

It will be shown here that members of the membrane-type MMP (MT-MMP) subfamily became a particular focus for this study. Six have been identified to date.15 16 17 18 19 All are anchored to the cell surface, extending an extracellular catalytic domain through a transmembrane domain (MT1, MT2, MT3, and MT5-MMP, also referred to as MMP-14, -15, -16, and -24, respectively) or a glycophosphatidylinositol link (MT4 and MT6-MMP, also referred to as MMP-17 and -25, respectively). Additionally, MT5-MMP species are shed from the cell surface.18

The MT-MMPs process proMMPs in the pericellular environment (proMMP-2) and degrade ECM components and cell surface molecules (for a review, see Noel et al.20 ). MT1-MMP is thought to be the main MT-MMP involved in the processing of proMMP-2 to activation when in complex with TIMP-2 and {alpha}Vß3 integrin,21 22 23 and indeed MT1-MMP has been detected in the normal human lens.13 MT2-MMP, however, activates proMMP-2 in a TIMP-2–independent manner.24

Lens fiber cells are continuously formed by differentiation of the equatorial epithelial cells. Differentiating cells withdraw from the cell cycle and specialize by synthesizing lens crystallins. The activity of the MMPs and TIMPs has been implicated in normal and pathologic lens growth, development, and remodeling. MMP substrates include molecules involved in lens fiber migration and differentiation, such as cytokines, cell adhesion molecules, and growth factors.25 26 27

Cataract and PCO are major causes of visual acuity loss and blindness worldwide.28 Gene expression of the MMP and TIMP families was, therefore, investigated in regions of the normal adult human lens using quantitative real-time PCR as a prelude to a cataract study. Given these observations, we chose to investigate further some important MMPs by analyzing protein expression with the use of Western blotting techniques. To provide adequate quantities of starting material, previous gene expression studies used sample pools obtained from multiple donors assessed by an experienced observer according to the Lens Opacification Classification System (LOCS).28 29 30 Because of the difficult nature of this assessment and the fact that many cataracts have, for example, nuclear and cortical involvement, it is important to study the properties of individual cataractous lenses.31 The purpose of this study was to analyze individual lenses. Hence, we developed the necessary techniques for gene expression analyses in small samples from normal, clear lenses to analyze expression patterns in different regions of the same lens.


   Methods
Top
 Abstract
 Methods
Results
 Discussion
 References
 
Donor Lenses and Culture-Derived Cells
Human donor eyes were obtained with full ethical permission (REC 04/Q0102/57) from the East Anglia Eye Bank or the Corneal Transplant Service (Eye Bank, Bristol, UK) after the cornea had been removed for transplantation purposes. Human tissue in the study was used in accordance with the tenets of the Declaration of Helsinki. All eyes were stored in individual sterile pots in an antibiotic wash medium before use. Dissection and primary culture methods have been described in detail elsewhere.32 33

For regional analyses, human donor eyes were obtained from the East Anglia Eye Bank (n = 6; age range, 55–77 years; mean, 67.5 years) less than 24 hours postmortem, and the lenses were dissected into three regions: anterior epithelium, equator, and fibers. The iris was removed, and a circular anterior epithelium was torn away. The lens nucleus was then released by hydroejection, and residual fibers were sampled after careful removal using forceps. Finally, the lens capsule was dissected from the zonules. Each sample was immediately snap-frozen in liquid nitrogen.

Primary cultures generated from combined anterior and equatorial epithelial cells were investigated as a growth and remodeling system. For primary cell culture, isolated human donor lenses were obtained from the Corneal Transplant Service (Eye Bank, Bristol, UK). The lenses were transported in individual tubes containing sterile Eagle minimal essential medium (EMEM; Sigma, Dorset, UK). The center of the cell-free posterior capsule was punctured, and an incision was made across the diameter of the posterior capsule. Entomology pins (D1; Watkins and Doncaster Ltd., Kent, UK) were inserted at the edges of the capsule to secure the capsule at either end of the incision. Small cuts were then made in the capsule, near the pins, so that most of the posterior capsule could be removed with two curvilinear tears. The remaining capsule (anterior and equatorial regions) was then further secured with six additional pins, and the major fiber mass and residual fibers were carefully removed with forceps. Explants were cultured in EMEM for 24 hours and split into two halves, again pinned to the culture dish and cultured in the presence of 10% fetal calf serum (FCS; Gibco, Paisley, UK) for 1 to 2 weeks. Explants were then removed from culture dishes, and epithelial cells were left to grow to confluence in the presence of serum.

Total RNA Extraction and cDNA Generation
Total RNA was extracted from tissue and primary cultured cells (RNeasy micro kits; Qiagen, West Sussex, UK) in accordance with the manufacturer's instructions. In the initial step, RLT buffer (containing ß-mercaptoethanol) was added to Eppendorf tubes containing snap-frozen tissues, or they were added directly to PBS-washed culture monolayers. Cultured cell lysates were removed with a cell scraper. Frozen tissue was homogenized with an Eppendorf homogenizer, and both sample sets were then passed through a needle and syringe. The remainder of the protocol was as described by the manufacturer and included a DNAse step. Quality control was maintained with an RNA analyzer (Bioanalyzer 2100; Agilent, West Lothain, UK) and an RNA lab chip (6000 Nano lab chip; Agilent) to ensure that 28S and 18S rRNA bands were clearly evident in total RNA samples. RNA was quantified with a spectrophotometer (ND-1000; NanoDrop, Wilmington, DE). For the 18 samples analyzed, the 260:280 ratio ranged from 1.8 to 2.2 (mean, 2.0). Where possible, total RNA was immediately used for cDNA generation or was briefly stored at –80°C. Generation of cDNA was performed with reverse transcriptase (Superscript II; Invitrogen, Paisley, UK) and random primers (Promega, Southampton, UK), according to standard protocols.

Real-Time PCR
Quantitative real-time PCR was used to analyze mRNA expression for all genes in native tissue and culture-derived cells (for a comprehensive description of MMPs and TIMPs primer/probe sets used, see Nuttall et al.34 ). Predesigned gene expression assays were purchased (TaqMan; Applied Biosystems, Foster City, CA) for the following genes: MIP (NCBI reference sequence [RefSeq] at the time of publication; NM_012064.1), {gamma}A-crystallin (NM_014617.2), ßA1-crystallin (NM_005208.3), {alpha}A-crystallin (NM_000394.2), and {alpha}B-crystallin (NM_001885.1). Assuming 100% efficiency in the reverse transcription reaction, either 1 ng or 5 ng cDNA was used in real-time PCR reactions performed using a real-time PCR machine (ABI7700; Applied Biosystems). Reagent-based assays (TaqMan Universal PCR Master Mix, No AmpErase UNG; Applied Biosystems) with all PCR reagents were used according to the manufacturer's instructions. The amount of amplification associated with priming from genomic DNA contamination was evaluated with control reverse transcription reactions containing all reagents without reverse transcriptase. Conditions for the PCR reaction were 2 minutes at 50°C, 10 minutes at 95°C, and then 40 cycles, each consisting of 15 seconds at 95°C and 1 minute at 60°C. The cycle number at which amplification entered the exponential phase (cycle threshold [CT]) was determined, and this number was used as an indicator for the amount of target RNA in each tissue analyzed. To determine the relative RNA levels in the samples, standard curves for each primer/probe set were prepared by taking cDNA from one sample and making twofold serial dilutions covering the range equivalent to 20 ng to 0.625 ng RNA (for 18S analysis, the range was 1 ng to 0.03125 ng). Differences in the total amount of RNA present in each sample were normalized to endogenous 18S ribosomal RNA gene expression, as previously described.34

Protein Extraction, Quantification, and Western Blotting
Each experiment was performed at least three times. Frozen tissues were incubated at 4°C for 10 minutes in buffer: 50 mM HEPES, pH 7.5, 150 mM NaCl, 1% Triton X-100, 1 mM EDTA, 10% glycerol, 10 mM sodium pyrophosphate, 2 mM sodium orthovanadate, 10 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 10 µg/mL aprotinin.35 Samples were homogenized with an Eppendorf homogenizer, followed by passage through needle and syringe, and then were further incubated at 4°C for 10 minutes. Lysates were precleared by centrifugation at 13,000 rpm at 4°C for 10 minutes, and the protein content of the soluble fraction was assayed by a bicinchoninic acid protein assay (Pierce, Rockford, IL). Detergent-insoluble fractions were produced by solubilizing pelleted material in reducing sample loading buffer containing 10% (wt/vol) SDS, 20% glycerol, 0.1% (wt/vol) bromophenol blue, 125 mM Tris-HCl, pH 6.8, and 5% ß-mercaptoethanol. Equal amounts of reduced protein (100 µg) per sample were loaded onto 10% SDS-PAGE gels for electrophoresis and transfer to polyvinylidene difluoride (PVDF) membrane (NEN Life Science Products, Boston, MA) with a semidry transfer cell (Trans-Blot; Bio-Rad, Herts, UK). Proteins were detected with a chemiluminescent blot analysis system (ECL+; Amersham Biosciences, Amersham, UK) with primary antibodies against MT1-MMP (AB8102; Chemicon, Temecula, CA) and MT2-MMP (M4442 [Sigma] and MAB3320 [Chemicon]).

Statistical Analysis
When statistical analysis was performed, one-way ANOVA with Tukey post hoc analysis was used to confirm a significant difference in the data between lens regions.


   Results
Top
 Abstract
 Methods
 Results
Discussion
 References
 
Distribution of Lens-Specific Gene Expression in Lens Regions
It was important to demonstrate that the dissected regions of the lens contained relatively pure populations of cells and, in particular, that the anterior and equatorial cells were not contaminated with fiber remnants. The ß- and {gamma}-crystallins are the major soluble components of the fibers. Indeed, PCR analysis of the different regions corresponded with the expected distributions (Fig. 1) . In particular, {gamma}A-crystallin gene expression was absent from the epithelial cell populations, in concurrence with Wang et al.,36 because this protein is produced relatively late in fiber cell differentiation.37 Similarly, the expression of the major intrinsic protein (MIP), which was largely absent from the epithelial populations, was characteristically associated with fiber cells. Human lens epithelial cells contain little or no {alpha}A crystallin but a significant amount of {alpha}B-crystallin.36 Again, this is borne out by the present PCR data shown in Figure 1 . Next, MMP and TIMP expression were investigated in detail.


Figure 1
View larger version (17K):
[in this window]
[in a new window]

  		FIGURE 1. Crystallin and major intrinsic protein real-time PCR analysis in lens regions. The y-axis represents the gene of interest/18S expression expressed as mean ± SEM (n = 4). Data were analyzed by one-way ANOVA and Tukey post hoc analysis. Significance was determined at P ≤ 0.05 (*) in that region compared with the other two regions and with the region indicated.

 
Distribution of MMP and TIMP Gene Expression in Lens Regions Determined by CT Analysis
Figure 2 demonstrates MMP and TIMP gene expression data prepared from six donors and primary epithelial cultures derived from four donors. The CT value (see Methods) was used to classify gene expression as very high (CT ≤ 20), high (CT = 21–25), moderate (CT = 26–30), low (CT = 31–38), or absent (CT = 39–40). Absent or low levels of MMP gene expression were determined in the three lens regions, except for the membrane-type MMPs—MT1-MMP (MMP-14) and MT2-MMP (MMP-15). There was low expression of gelatinase A (MMP-2) and negligible gelatinase B (MMP-9). Generally, regional MMP expression levels occurred in the order of anterior cells > equator cells > fiber cells. However, though MT1-MMP expression declined from anterior to fiber cells, MT2-MMP expression increased. Relatively high levels of the four inhibitory TIMPs were maintained throughout; the lowest in abundance was TIMP-4.


Figure 2
View larger version (45K):
[in this window]
[in a new window]

  		FIGURE 2. MMP and TIMP gene expression profiles were characterized in lens regions (donors 1–6) and primary cultured lens epithelium (four separate cultures from four donors). CT was used to classify gene expression as either very high (CT ≤ 20), high (CT = 21–25), moderate (CT = 26–30), low (CT = 31–38), or absent (CT = 39–40).

 
CT Analysis of MMP and TIMP Gene Expression in Cultured Primary Lens Cells
A different picture emerges after primary cell culture. Upregulation of several MMPs was apparent, including the gelatinases (MMP-2, -9) and the stromelysins (MMP-3, -10, -11). Of the collagenases, MMP-1 was high but -8 and -13 were low. MMP-19 and MT5-MMP (MMP-24) were also elevated. Upregulation of TIMP-1 gene expression occurred in primary culture.

Distribution of MMP and TIMP Gene Expression in Lens Regions and Cultured Cells Using the Relative Standard Curve Method
A more quantitative analysis of these data was obtained by expressing the data normalized with respect to the level of 18S message (Fig. 3) . Of the MMPs, MMP-14 and -15 showed the greatest levels of expression, with MMP-23 and -24 at a much lower level. In primary cultured cells, MMP-2, -9, -10, -23, and -24 were expressed at higher levels than those observed in native cells, whereas MMP-14 and -15 were expressed at levels similar to those observed in the native cells. Regarding inhibitors, TIMP-3 and -2 were the most highly expressed. In native anterior and equatorial cells, the relative expression of TIMPs followed the sequence 3 > 2 > 4 > 1; this was similar to the sequence determined in primary cultured cells. In fibers, the sequence was 2 > 3 = 4 = 1.


Figure 3
View larger version (28K):
[in this window]
[in a new window]

  		FIGURE 3. MMP and TIMP gene expression in each of the three regions of the lens. The x-axis represents the gene of interest/18S expression expressed as mean ± SEM. Data were analyzed by one-way ANOVA and Tukey post hoc analysis. Significance was determined at P ≤ 0.05 (*) in that region compared with the other two regions and with the region indicated.

 
Protein Expression of MT1-MMP in the Normal Human Lens
Two bands were detected on Western blot analysis of extracts from anterior epithelial (AE), equatorial (E), and fiber (F) protein fractions using an antibody that recognizes the catalytic domain of human MT1-MMP (Fig. 4) . These bands likely corresponded to the previously reported pro- and active forms at 63 kDa and 60 kDa,38 as judged from the migration of a recombinant MT1-MMP standard representing the human prodomain, catalytic domain, and hemopexin domain (CC1043; Chemicon).


Figure 4
View larger version (16K):
[in this window]
[in a new window]

  		FIGURE 4. Detection of MT1-MMP in anterior epithelial (AE), equatorial (E), and fiber protein fractions using anti-MMP14 catalytic domain. Fiber fractions were electrophoresed along side control human MMP-14 prodomain, catalytic domain, and hemopexin domain with a molecular weight of 58 kDa. Bands representing proprotein and activated enzyme were running at approximately 63 kDa and 60 kDa, respectively.

 
Protein Expression of MT2-MMP in the Normal Human Lens
Detection of MT2-MMP is shown in Figure 5 . In detergent-soluble protein fractions prepared from all lens regions, the proenzyme was shown at 72 kDa, activated enzyme at 55 kDa, and activation/breakdown products at 48 kDa. These were the most predominant, as described by the manufacturer. In detergent-soluble fiber fractions, however, little proenzyme or active enzyme banding was evident. Smaller bands were seen when MT2-MMP was detected in soluble fractions. These lower molecular weight bands were likely to have been MT2-MMP breakdown products, which could have included the hemopexin domain, the transmembrane region, the cytoplasmic domain, or various combinations of the three. Activated MT2-MMP at 55 kDa was determined in detergent-insoluble protein fractions.


Figure 5
View larger version (24K):
[in this window]
[in a new window]

  		FIGURE 5. Detection of MT2 MMP in (A) soluble and (B) detergent-insoluble protein fractions from anterior epithelium (AE), equator (E), and fiber (F), using anti–MMP-15 hinge region (SIGMA M4442) in all cases. A similar pattern was obtained with antibody (MAB3320). Arrows indicate bands identified by the antibody at 72 kDa and 55 kDa and prominent activation/breakdown products at 48 kDa seen when the enzyme was fully activated, as described by the antibody manufacturer. Marker lane (M) sizing is indicated (kDa).

 

   Discussion
Top
 Abstract
 Methods
 Results
 Discussion
References
 
Several methods have been used for illustrating gene expression in a wide range of tissues. In the fluorometric method used here, the data could be expressed in terms of cycle threshold number, CT, and as relative RNA expression in terms of a standard housekeeping gene such as GAPDH or 18S.34 The CT values indicated low or very low expression for most of the MMPs in native cells, whereas primary cultures predicted upregulation of several of the MMPs. On the other hand, with the CT method, TIMP expression (of at least 1–3) was uniformly high throughout. When CT values were analyzed by the standard curve method and normalized to 18S, it was possible to conduct a more quantitative analysis of the relative expression across the different tissues. The resultant pattern was, however, similar to that obtained for CT analysis, except for TIMP-1 expression, in which curve analysis resulted in lowering of expression relative to the other TIMPS.

Regardless of which method is used, it is important to have well-characterized tissues from which to generate data. The present study demonstrated that it was possible to obtain reproducible data from single lenses when they are dissected into three regions (Figs. 1 2 3) . Furthermore, several studies from this laboratory and others (for a review, see Duncan et al.39 ) have emphasized the multifactorial nature of cataract and have demonstrated the importance of analyzing individual, well-characterized lens samples. As far as the native tissues were concerned, though it was easy to generate anterior samples simply by dissection, it was not obvious that the equatorial samples were similarly pure. It was, therefore, encouraging that the latter samples were uncontaminated by ßA1- or {gamma}A-crystallin and yet showed high expression of {alpha}B-crystallin (Fig. 1) . Consequently, we were confident in conducting further gene analysis in these regions.

In the present study, an investigation of the MMPs and TIMPs was performed because there was evidence that they have roles in the differentiating lens and in cataract.2 In immunohistochemical and ELISA analyses, Sachdev et al.2 reported low MMP-1, -2, -3, and -9 and TIMP-1, -2, and -3 reactivity in normal human donor lenses. ELISA analysis showed significant upregulation of MMP-1 and TIMP-1 in cortical cataract. In the present study, we did find significant gene upregulation of MMP-1 and TIMP-1 during culture of lens cells (Figs. 2 3) . The general low level of expression of MMP-2 and -9 in normal lens has previously been reported2 3 11 12 13 by immunocytochemistry, zymography, and ELISA techniques, and the present PCR data would support this. Interestingly, culture stimulates the expression of a number of MMPs, including MMP-2 and -9; hence, primary cell cultures should not be taken as adequate models for the anterior epithelium but rather for the events that follow cataract surgery and give rise to PCO. The elevation of MMPs in culture is supported by Wong et al.,40 who demonstrated an ability of MMP inhibitors to suppress cell migration in the capsular bag after culture. Dwivedi et al.41 also showed that the specific MMP-2 and -9 inhibitors significantly suppressed the formation of TGF-induced anterior subcapsular cataractous plaques in the rat lens. TGFß has been shown to greatly stimulate the expression of MMP-2 and -94 ; it would be interesting to know whether autocrine production of TGFß after culture was responsible for the upregulation seen.

One novel finding in the present study is the important role played by the MT-MMPs in the adult human lens. Interestingly, all the MMPs with a transmembrane domain and a cytoplasmic tail (MT1-, 2-, 3-, and 5-MMP) had their highest expression in one particular region of the lens, indicating that they may play a role in the maintenance, or indeed the production, of the three different lens compartments. MT4- and MT6-MMP have a GPI anchor rather than a transmembrane domain and cytoplasmic tail. Although the former is expressed solely in anterior cells, the latter is expressed at low levels in all three compartments. The relative importance of the MT-MMPs is perhaps not surprising in a tissue such as the lens, which has a very high density of membrane components. It should also be remembered that the MT-MMPs are generally important because they appear to have the ability to activate proMMPs (for a review, see Nuttall et al.34 ). Recent evidence indicates that activated MMP-9 has a particularly vital role to play in anterior subcapsular cataract (Dwivedi DJ, et al. IOVS 2006;47:ARVO E-Abstract 4740). Numerous studies34 42 43 44 of malignant cells have correlated the expression of the MT-MMPs with invasion and metastasis through proMMP-2 activation. Previous analysis of human lens epithelium suggested that the most widely studied MMPs, MMP-2 and -9, were undetectable using Western blot techniques but that the membrane-bound MT1-MMP (MMP-14) was present.13 This latter protease is associated with the activation of proMMP-2, but with the absence of MMP-2, its functional role in the lens is unknown. However, evidence in the cancer field suggests endogenous MT1-MMP can perform a variety of functions independently of proMMP-2 activation.45 46

With the exception of MT1-MMP, normal corneal cells express low levels of MMP. However, Dushku et al.47 show that when they are activated in the formation of pterygia, all MMPs, especially MT2-MMP, are upregulated. 47 The authors conclude that altered fibroblasts in areas of degeneration behind pterygia constitute a type of noninvasive tumor (stimulated cell growth).47

A unique aspect of this study has been the ability to assess the relative intensity of MMP/TIMP expression in different regions of the lens. The most significant finding has been the relative downregulation of expression in MT1-MMP and the concomitant upregulation of MT2-MMP in the transition from quiescent epithelia to differentiated fiber cells. Although the upregulation of MT2-MMP is reportedly associated with an increase in growth drive,47 no reports have been published of its association with differentiation. One report states that MT2-MMP acts as an antiapoptotic factor in cancer cells.48 It is interesting that fiber cells ultimately lose contact with the basement membrane capsule; normally, epithelia-derived cells undergo apoptosis when this occurs. Perhaps one role of MT2-MMP is to inhibit cell death in elongating nucleated fiber cells. One potential substrate for MT2-MMP in the migratory equatorial epithelia and fiber cells of the lens could be laminin49 because these cells express {alpha}6ß1 integrin, which is the putative laminin receptor for lens fiber cells.50 51 52 The Western blot analysis carried out to support the PCR data indicated that not only is the MT2-MMP protein expressed in fiber cells, it is also processed and activated.

There is a long history of drugs initially developed to treat one or more ocular conditions that are later found to carry an increased risk for cataract formation.53 It is, therefore, worth noting that latanoprost, which has proved useful in reducing intraocular pressure, also increases MMP-1 expression while it downregulates MT-MMPs in trabecular meshwork and in ciliary body cells.54 55 No study of the effect of latanoprost on MT-MMP expression in lens cells has yet been carried out.

In conclusion, this is the first study to report a global analysis of MMP and TIMP family members in the lens. Interestingly, relatively high expression of the membrane-type MMPs was observed throughout the human lens and showed regional differences. The striking reciprocal patterns of expression of MT1-MMP and MT2-MMP reported here indicate that these enzymes are of particular significance in lens function.


   Acknowledgements
 
The authors thank Gillian Murphy, Rob Nuttall, and James Hadfield for advice, technical assistance, and discussions. The authors also thank the staffs of the East Anglian and Bristol Eye Banks for providing donor material.


   Footnotes
 
Supported by Biotechnology and Biological Sciences Research Council, the Humane Research Trust, and Norfolk and Norwich Big C Appeal.

Submitted for publication November 14, 2006; revised April 16, 2007; accepted June 13, 2007.

Disclosure: L.M. Hodgkinson, None; G. Duncan, None; L. Wang, None; C.J. Pennington, None; D.R. Edwards, None; I.M. Wormstone, None

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Corresponding author: Lisa M. Hodgkinson, School of Biological Sciences, University of East Anglia, Norwich, United Kingdom; l.hodgkinson{at}uea.ac.uk.


   References
Top
 Abstract
 Methods
 Results
 Discussion
 References
 

  1. Forrester JV, Dick AD, McMenamin PG, Lee WR. Embryology and early development of the eye and adnexa. The Eye: Basic Sciences in Practice. 2002;99–129. WB Saunders Philadelphia.
  2. Sachdev NH, Di Girolamo N, Nolan TM, et al. Matrix metalloproteinases and tissue inhibitors of matrix metalloproteinases in the human lens: implications for cortical cataract formation. Invest Ophthalmol Vis Sci. 2004;45:4075–4082.[Abstract/Free Full Text]
  3. Tamiya S, Wormstone I, Marcantonio J, Gavrilovic J, Duncan G. Induction of matrix metalloproteinases 2 and 9 following stress to the lens. Exp Eye Res. 2000;71:591–597.[CrossRef][ISI][Medline][Order article via Infotrieve]
  4. Wormstone IM, Tamiya S, Anderson I, Duncan G. TGF-ß2-induced matrix modification and cell transdifferentiation in the human lens capsular bag. Invest Ophthalmol Vis Sci. 2002;43:2301–2308.[Abstract/Free Full Text]
  5. Vu TH, Werb Z. Matrix metalloproteinases: effectors of development and normal physiology. Genes Dev. 2000;14:2123–2133.[Free Full Text]
  6. Woessner J., Jr. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J. 1991;5:2145–2154.[Abstract]
  7. Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res. 2003;92:827–839.[Abstract/Free Full Text]
  8. Ye HQ, Azar DT. Expression of gelatinases A and B, and TIMPs 1 and 2 during corneal wound healing. Invest Ophthalmol Vis Sci. 1998;39:913–921.[Abstract/Free Full Text]
  9. Sobrin L, Liu ZG, Monroy DC, et al. Regulation of MMP-9 activity in human tear fluid and corneal epithelial culture supernatant. Invest Ophthalmol Vis Sci. 2000;41:1703–1709.[Abstract/Free Full Text]
  10. Mohan R, Chintala SK, Jung JC, et al. Matrix metalloproteinase gelatinase B (MMP-9) coordinates and affects epithelial regeneration. J Biol Chem. 2002;277:2065–2072.[Abstract/Free Full Text]
  11. Schlotzer-Schrehardt U, Lommatzsch J, Kuchle M, Konstas AGP, Naumann GOH. Matrix metalloproteinases and their inhibitors in aqueous humor of patients with pseudoexfoliation syndrome/glaucoma and primary open-angle glaucoma. Invest Ophthalmol Vis Sci. 2003;44:1117–1125.[Abstract/Free Full Text]
  12. Plantner JJ, Smine A, Quinn TA. Matrix metalloproteinases and metalloproteinase inhibitors in human interphotoreceptor matrix and vitreous. Curr Eye Res. 1998;17:132–140.[CrossRef][ISI][Medline][Order article via Infotrieve]
  13. Smine A, Plantner JJ. Membrane type-1 matrix metalloproteinase in human ocular tissues. Curr Eye Res. 1997;16:925–929.[CrossRef][ISI][Medline][Order article via Infotrieve]
  14. Kleiner DE, Stetler-Stevenson WG. Structural biochemistry and activation of matrix metalloproteases. Curr Opin Cell Biol. 1993;5:891–897.[CrossRef][Medline][Order article via Infotrieve]
  15. Will H, Hinzmann B. cDNA sequence and mRNA tissue distribution of a novel human matrix metalloproteinase with a potential transmembrane segment. Eur J Biochem. 1995;231:602–608.[ISI][Medline][Order article via Infotrieve]
  16. Takino T, Sato H, Shinagawa A, Seiki M. Identification of the second membrane-type matrix metalloproteinase (MT-MMP-2) gene from a human placenta cDNA library. J Biol Chem. 1995;270:23013–23020.[Abstract/Free Full Text]
  17. Puente XS, Pendas AM, Llano E, Velasco G, Lopez-Otin C. Molecular cloning of a novel membrane-type matrix metalloproteinase from a human breast carcinoma. Cancer Res. 1996;56:944–949.[Abstract/Free Full Text]
  18. Pei D. Identification and characterization of the fifth membrane-type matrix metalloproteinase MT5-MMP. J Biol Chem. 1999;274:8925–8932.[Abstract/Free Full Text]
  19. Pei D. Leukolysin/MMP25/MT6-MMP: a novel matrix metalloproteinase specifically expressed in the leukocyte lineage. Cell Res. 1999;9:291–303.[CrossRef][ISI][Medline][Order article via Infotrieve]
  20. Noel A, Maillard C, Rocks N, et al. Membrane associated proteases and their inhibitors in tumour angiogenesis. J Clin Pathol. 2004;57:577–584.[Abstract/Free Full Text]
  21. Strongin AY, Collier I, Bannikov G, et al. Mechanism of cell surface activation of 72-kDa type IV collagenase. J Biol Chem. 1995;270:5331–5338.[Abstract/Free Full Text]
  22. Zucker S, Drews M, Conner C, et al. Tissue inhibitor of metalloproteinase-2 (TIMP-2) binds to the catalytic domain of the cell surface receptor, membrane type 1-matrix metalloproteinase 1 (MT1-MMP). J Biol Chem. 1998;273:1216–1222.[Abstract/Free Full Text]
  23. Deryugina EI, Ratnikov B, Monosov E, et al. MT1-MMP initiates activation of pro-MMP-2 and integrin {alpha}vß3 promotes maturation of MMP-2 in breast carcinoma cells. Exp Cell Res. 2001;263:209–223.[CrossRef][ISI][Medline][Order article via Infotrieve]
  24. Morrison CJ, Butler GS, Bigg HF, et al. Cellular activation of MMP-2 (gelatinase A) by MT2-MMP occurs via a TIMP-2-independent pathway. J Biol Chem. 2001;276:47402–47410.[Abstract/Free Full Text]
  25. Richiert DM, Ireland ME. Matrix metalloproteinase secretion is stimulated by TGF-ß in cultured lens epithelial cells. Curr Eye Res. 1999;19:269–275.[CrossRef][ISI][Medline][Order article via Infotrieve]
  26. Ueda Y, Chamberlain CG, Satoh K, McAvoy JW. Inhibition of FGF-induced {alpha}A-crystallin promoter activity in lens epithelial explants by TGFß. Invest Ophthalmol Vis Sci. 2000;41:1833–1839.[Abstract/Free Full Text]
  27. Hales AM, Chamberlain CG, Dreher B, McAvoy JW. Intravitreal injection of TGFß induces cataract in rats. Invest Ophthalmol Vis Sci. 1999;40:3231–3236.[Abstract/Free Full Text]
  28. Ruotolo R, Grassi F, Percudani R, et al. Gene expression profiling in human age-related nuclear cataract. Mol Vis. 2003;9:538–548.[ISI][Medline][Order article via Infotrieve]
  29. Segev F, Mor O, Segev A, Belkin M, Assia EI. Downregulation of gene expression in the ageing lens: a possible contributory factor in senile cataract. Eye. 2005;19:80–85.[CrossRef][ISI][Medline][Order article via Infotrieve]
  30. Hawse JR, Hejtmancik JF, Huang Q, et al. Identification and functional clustering of global gene expression differences between human age-related cataract and clear lenses. Mol Vis. 2003;9:515–537.[ISI][Medline][Order article via Infotrieve]
  31. Marcantonio JM, Duncan G, Davies PD, Bushell AR. Classification of human senile cataracts by nuclear colour and sodium content. Exp Eye Res. 1980;31:227–237.[CrossRef][ISI][Medline][Order article via Infotrieve]
  32. Liu CS, Wormstone IM, Duncan G, et al. A study of human lens cell growth in vitro: a model for posterior capsule opacification. Invest Ophthalmol Vis Sci. 1996;37:906–914.[Abstract/Free Full Text]
  33. Wang L, Prescott AR, Spruce BA, Sanderson J, Duncan G. Sigma receptor antagonists inhibit human lens cell growth and induce pigmentation. Invest Ophthalmol Vis Sci. 2005;46:1403–1408.[Abstract/Free Full Text]
  34. Nuttall RK, Pennington CJ, Taplin J, et al. Elevated membrane-type matrix metalloproteinases in gliomas revealed by profiling proteases and inhibitors in human cancer cells. Mol Cancer Res. 2003;1:333–345.[Abstract/Free Full Text]
  35. Daub H, Wallasch C, Lankenau A, Herrlich A, Ullrich A. Signal characteristics of G protein-transactivated EGF receptor. EMBO J. 1997;16:7032–7044.[CrossRef][ISI][Medline][Order article via Infotrieve]
  36. Wang X, Garcia CM, Shui Y-B, Beebe DC. Expression and regulation of {alpha}-, ß-, and {gamma}-crystallins in mammalian lens epithelial cells. Invest Ophthalmol Vis Sci. 2004;45:3608–3619.[Abstract/Free Full Text]
  37. McAvoy JW. Developmental biology of the lens. Duncan G eds. Mechanisms of Cataract Formation in the Human Lens. 1981; Academic Press Inc. London.
  38. Rathke-Hartlieb S, Budde P, Ewert S, et al. Elevated expression of membrane type 1 metalloproteinase (MT1-MMP) in reactive astrocytes following neurodegeneration in mouse central nervous system. FEBS Lett. 2000;481:227–234.[CrossRef][ISI][Medline][Order article via Infotrieve]
  39. Duncan G, Williams MR, Riach RA. Calcium, cell signaling and cataract. Prog Retinal Eye Res. 1994;13:623–652.[CrossRef][ISI]
  40. Wong TTL, Daniels JT, Crowston JG, Khaw PT. MMP inhibition prevents human lens epithelial cell migration and contraction of the lens capsule. Br J Ophthalmol. 2004;88:868–872.[Abstract/Free Full Text]
  41. Dwivedi DJ, Pino G, Banh A, et al. Matrix metalloproteinase inhibitors suppress transforming growth factor-ß-induced subcapsular cataract formation. Am J Pathol. 2006;168:69–79.[Abstract/Free Full Text]
  42. Ueno H, Nakamura H, Inoue M, et al. Expression and tissue localization of membrane-types 1, 2, and 3 matrix metalloproteinases in human invasive breast carcinomas. Cancer Res. 1997;57:2055–2060.[Abstract/Free Full Text]
  43. Nakada M, Nakamura H, Ikeda E, et al. Expression and tissue localization of membrane-type 1, 2, and 3 matrix metalloproteinases in human astrocytic tumors. Am J Pathol. 1999;154:417–428.[Abstract/Free Full Text]
  44. Nakamura H, Ueno H, Yamashita K, et al. Enhanced production and activation of progelatinase a mediated by membrane-type 1 matrix metalloproteinase in human papillary thyroid carcinomas. Cancer Res. 1999;59:467–473.[Abstract/Free Full Text]
  45. Egeblad M, Werb Z. New functions for the matrix metalloproteinases in cancer progression. Nat Rev Cancer. 2002;2:161–174.[ISI][Medline][Order article via Infotrieve]
  46. Lafleur MA, Handsley MM, Knauper V, Murphy G, Edwards DR. Endothelial tubulogenesis within fibrin gels specifically requires the activity of membrane-type-matrix metalloproteinases (MT-MMPs). J Cell Sci. 2002;115:3427–3438.[Abstract/Free Full Text]
  47. Dushku N, John MK, Schultz GS, Reid TW. Pterygia pathogenesis: corneal invasion by matrix metalloproteinase expressing altered limbal epithelial basal cells. Arch Ophthalmol. 2001;119:695–706.[Abstract/Free Full Text]
  48. Abraham R, Schafer J, Rothe M, et al. Identification of MMP-15 as an anti-apoptotic factor in cancer cells. J Biol Chem. 2005;280:34123–34132.[Abstract/Free Full Text]
  49. d'Ortho MP, Will H, Atkinson S, et al. Membrane-type matrix metalloproteinases 1 and 2 exhibit broad-spectrum proteolytic capacities comparable to many matrix metalloproteinases. Eur J Biochem. 1997;250:751–757.[ISI][Medline][Order article via Infotrieve]
  50. de Iongh RU, Lovicu FJ, Overbeek PA, et al. Requirement for TGFß receptor signaling during terminal lens fiber differentiation. Development. 2001;128:3995–4010.[ISI][Medline][Order article via Infotrieve]
  51. Menko AS, Philip NJ. Beta 1 integrins in epithelial tissues: a unique distribution in the lens. Exp Cell Res. 1995;218:516–521.[CrossRef][ISI][Medline][Order article via Infotrieve]
  52. Walker JL, Menko AS. {alpha}6 Integrin is regulated with lens cell differentiation by linkage to the cytoskeleton and isoform switching. Dev Biol. 1999;210:497–511.[CrossRef][ISI][Medline][Order article via Infotrieve]
  53. Shaffer RN, Hetherington J. Anticholinesterase drugs and cataracts. Am J Ophthalmol. 1966;62:613–618.[ISI][Medline][Order article via Infotrieve]
  54. Oh D-J, Martin JL, Williams AJ, et al. Effect of latanoprost on the expression of matrix metalloproteinases and their tissue inhibitors in human trabecular meshwork cells. Invest Ophthalmol Vis Sci. 2006;47:3887–3895.[Abstract/Free Full Text]
  55. Oh D-J, Martin JL, Williams AJ, et al. Analysis of expression of matrix metalloproteinases and tissue inhibitors of metalloproteinases in human ciliary body after latanoprost. Invest Ophthalmol Vis Sci. 2006;47:953–963.[Abstract/Free Full Text]




This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a response
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via ISI Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Hodgkinson, L. M.
Right arrow Articles by Wormstone, I. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Hodgkinson, L. M.
Right arrow Articles by Wormstone, I. M.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS