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(Investigative Ophthalmology and Visual Science. 2007;48:2105-2114.)
© 2007 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.06-1089
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Influence of Actin Cytoskeletal Integrity on Matrix Metalloproteinase-2 Activation in Cultured Human Trabecular Meshwork Cells

Krishna Sanka,1 Rupalatha Maddala,1 David L. Epstein,1 and P. Vasantha Rao1,2

1From the Departments of Ophthalmology and 2Pharmacology and Cancer Biology, Duke University School of Medicine, Durham, North Carolina.


   Abstract
Top
 Abstract
Materials and Methods
 Results
 Discussion
 References
 
PURPOSE. The goal of this study was to investigate the possible link between actin cytoskeletal integrity and the activation of matrix metalloproteinases (MMPs) in trabecular meshwork (TM) cells.

METHODS. Primary human TM (HTM) cells treated with different actin cytoskeleton–interfering agents, including cytochalasin D, latrunculin A, ethacrynic acid (ECA), a Rho kinase inhibitor (Y-27632), and H-7 (serine/threonine kinase inhibitor), were examined for changes in actin cytoskeletal organization by phalloidin staining, MMP-2 activation by gelatin zymography, expression of MT1-MMP by quantitative real-time PCR analysis, levels of tissue inhibitor of metalloproteinases (TIMP-1 and TIMP-2), and activation of p38 mitogen-activated protein kinase (p38 MAPK) and extracellular signal-regulated protein kinase (ERK) by immunoblotting.

RESULTS. Treatment of HTM cells with cytochalasin D and latrunculin A led to significant activation of MMP-2, p38 MAPK, and ERK1/2, which appeared to correlate with changes in cell morphology and actin depolymerization. Additionally, treatment with these cytoskeleton-disrupting agents elicited increased expression of MT1-MMP in HTM cells, concomitant with a decrease in the levels of secreted TIMP-1 and TIMP-2. In contrast, treatment with ECA, Y-27632, or H-7 triggered changes in cell shape and reduced actin stress fibers in HTM cells but did not exert significant effects on MMP-2 activation or MT1-MMP expression.

CONCLUSIONS. These studies indicate that cytochalasin D– and latrunculin A–induced alteration of actin cytoskeletal integrity in HTM cells is associated with MMP-2 activation, most likely through the upregulation of its activator, MT1-MMP. These data provide a mechanistic connection between actin cytoskeletal organization and MMP-2 activation in TM cells and offer new insights into extracellular matrix remodeling in the aqueous outflow pathway.

Glaucoma is a leading cause of blindness and affects millions of people worldwide. It is a disease of both the optic nerve and the trabecular meshwork (TM) and is commonly associated with increased intraocular pressure (IOP).1 2 3 4 The TM displays smooth musclelike characteristics and participates in the regulation of aqueous humor outflow.3 5 6 7 In most forms of glaucoma, IOP rises because of an inability of the aqueous humor to drain through the TM and the Schlemm canal (SC). This increase in pressure can eventually lead to optic nerve degeneration, resulting in blindness.1 2 3 4

Elucidation of mechanisms involved in the regulation of aqueous humor outflow through the TM and SC could yield further insight into the biology of the TM and the treatment and pathophysiology of glaucoma.1 2 3 5 7 The TM and the SC cytoskeleton have been shown to play a potentially critical role in this process.3 8 9 10 11 12 Previous studies have shown that cellular contractility, which is mediated by the phosphorylation state of the myosin light chain,13 14 influences the status of actomyosin cytoskeletal organization, cell morphology, cell adhesions, and cell junctions in TM and SC cells.5 6 8 12 15 16 Additionally, other studies have shown that agents that alter actin cytoskeletal organization (microfilament perturbation) increase aqueous humor outflow, which would make them useful in exploring the modulation of aqueous outflow through TM, juxtacanalicular region (JCT), and SC.5 8 9 11 12 15 16 17 However, little is known about the mechanisms that result in altered aqueous outflow, even though a variety of hypotheses have been proposed to explain the effects of cytoskeletal changes on aqueous outflow facility.8 10 11 12 15

Patients with glaucoma have increased accumulations of extracellular matrix (ECM) in the aqueous outflow pathway,3 7 18 and an interaction exists between the cells of aqueous outflow system (TM, JCT, SC) and its ECM that plays an important role in regulating aqueous humor outflow.15 19 20 21 22 Some investigators have noted that abnormalities in TM–ECM interaction may lead to the increased amounts of ECM seen in the JCT of patients with glaucoma.7 15 19 23 24 25 26 27 Additionally, the interplay between the ECM and the actin cytoskeleton through integrin receptor modulation is well known22 28 29 and led us to speculate about whether cytoskeletal dynamics in the cells of outflow pathway are involved in ECM remodeling and turnover through their influence on the activation of matrix metalloproteinases (MMPs).30 31 32 33 34

MMPs are a family of zinc-dependent proteinases that take part in physiological and pathological processes by regulating ECM remodeling through its degradation. This family consists of more than 20 members that share domain structures and functional features but that have different substrate reaction profiles.35 36 37 38 39 MMPs are secreted from cells as zymogens and are activated by one of six membrane-type MMPs (MT-MMPs) that reside at the cell membrane because of the possession of a transmembrane domain at the C-terminal.37 40 41 Active forms of the MMP family members are inhibited by one of four tissue inhibitors of metalloproteinases (TIMPs).39 42 ECM turnover is thus determined, in large part, by the homeostasis between ECM synthesis and degradation. Expression and activity of the TIMPs and MMPs directly participate in and influence ECM degradation.37 38 39 42

Changes in the cell–ECM interaction occur during actin cytoskeletal reorganization.28 29 32 33 34 Additionally, the relationship between actin cytoskeletal reorganization and MMP activity has been studied extensively in different cell types.30 31 32 33 34 The regulation of MMPs has also been studied in the TM38 43 44 under several scenarios, such as mechanical stretching45 and manipulation of signaling pathways.46 Based on the evidence displaying a role for cytoskeletal changes in aqueous humor outflow modulation, support for the participation of MMPs in the TM with regard to outflow regulation and the data linking the cytoskeleton to MMP activation and ECM turnover in other cell types,30 31 32 33 34 we hypothesize that actin cytoskeletal integrity in the cells of outflow pathway influences MMP activation, which would subsequently modulate aqueous humor outflow facility through the remodeling of ECM.

To evaluate this hypothesis, we chose a variety of actin-cytoskeleton–altering agents, including cytochalasin D, latrunculin A, ECA, H-7, and Rho kinase inhibitor (Y-27632), and determined their effects on MMP-2 activation in cultured primary human TM cells. We evaluated the effects of these compounds on TM cell morphology, the actin cytoskeleton, MMP-2 activation, and MT1-MMP expression. Further investigation to better characterize the nature of MMP-2 regulation and MT1-MMP expression was then conducted with cytochalasin D and latrunculin A by assessing the impact of cytoskeletal reorganization in the TM on TIMP-2 and TIMP-1 protein levels and the activation status of MAP kinases, including p38 and ERK.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results
 Discussion
 References
 
Materials
The following materials were used: ethacrynic acid (ECA), cytochalasin D, and tetramethyl rhodamine isothiocyanate (TRITC)–labeled phalloidin (Sigma-Aldrich, St. Louis, MO); H-7, latrunculin A, SB202190, PD98059, mouse monoclonal antibody for MMP-2, and rabbit polyclonal antibody for TIMP-1 (Calbiochem, San Diego, CA); Rho kinase-specific inhibitor Y-27632 (Welfide Corporation, Osaka, Japan); precast zymography gels with 0.1% gelatin (Invitrogen, Carlsbad, CA); rabbit polyclonal antibodies for TIMP-2, ERK total, and phospho-ERK (Santa Cruz Biotechnology, Santa Cruz, CA); polyclonal antibodies raised against phospho-p38 and total p38 MAP kinases (Cell Signaling Technology, Danvers, MA) and New England Biolabs (Ipswich, MA), respectively; enhanced chemiluminescence (ECL) detection reagents (Amersham Pharmacia Biotech, Piscataway, NJ); RNeasy Mini kit (Qiagen, Valencia, CA); Advantage RT-for-PCR kit and Advantage cDNA PCR kit (BD Biosciences Clontech, Palo Alto, CA); iQSYBR Green supermix kit (Bio-Rad Laboratories, Philadelphia, PA); ultracentrifugal filter devices (Amicon; Millipore, Bedford, MA). Specific oligonucleotide sequences were custom synthesized (Integrated DNA Technologies, Inc., Coralville, IA). All other chemicals were of analytical grade.

Cell Cultures
Porcine TM (PTM) and human TM (HTM) cells (from 13-, 25-, and 52-year-old donors) were isolated as described previously.11 HTM and PTM cells were cultured at 37°C under 5% CO2, in Dulbecco modified Eagle medium (DMEM) containing 10% fetal bovine serum (FBS) and penicillin (100 U/mL)–streptomycin (100 µg/mL)–glutamine (292 µg/mL). Cells were used at passages 4 to 6. All experiments were conducted using confluent cell cultures. Before treatment, cells were serum starved for 24 hours and treated with different agents for 24 hours. Cell culture medium was then removed, centrifuged (2800 rpm), and used for the quantification of active MMP-2 by gelatin zymography and of TIMP-1 and TIMP-2 by immunoblot analysis. Protein content of the cell culture medium was determined using the Bradford method.47

Drug concentrations chosen for this study were based on our initial dose–response experiments and also on published studies.11 12

Gelatin Zymography
Equal amounts of TM cells (5 x 106) were cultured in six-well plates to confluence and then were serum starved for 24 hours. This was followed by 24-hour treatment with test compounds. Treatment with ethanol (used to dissolve certain test compounds) served as the control. Cell culture medium was then removed and concentrated using ultracentrifugal filter devices (Amicon; Millipore) with a 10-kDa cutoff. Concentrated medium sample volumes were equalized with phosphate-buffered saline (PBS). Equal amounts (based on volume) of concentrated samples were mixed with Tris/glycine/sodium dodecyl sulfate (SDS) sample buffer (2x) and were allowed to stand at room temperature for 10 minutes, and samples were loaded onto precast zymography gels and electrophoresed at a constant voltage of 125 V in a running buffer (25 mM Tris, 192 mM glycine, and 0.1% SDS, pH 8.3). Gels were then incubated for 30 minutes at room temperature in a renaturation buffer (2.5% Triton X-100 [Invitrogen]), followed by overnight incubation in a development buffer (50 mM Tris, pH 7.6 containing 10 mM CaCl2, 50 mM NaCl, 0.05% Brij 35 [Invitrogen]) at 37°C. Developed gels were then stained with 0.25% Coomassie blue solution for 30 minutes, followed by destaining, until clear bands were visible against the blue background. Gels were scanned and subjected to densitometric analysis (perfection 2450 scanner; Epson, Nagano, Japan) and National Institutes of Health Image software (Image J software, ver. 1.30; available by ftp at http://rsb.info.nih.gov/nih-image; developed by Wayne Rasband, National Institutes of Health, Bethesda, MD).

Western Blot Analysis
HTM cells were cultured in Petri dishes and treated as described. Cell medium samples were concentrated, and volumes were equalized. Protein concentration was estimated by the Bradford method.47 Samples with equal amounts of protein (5 or 10 µg protein/lane) were mixed with Laemmli buffer and separated by SDS-polyacrylamide gel electrophoresis (10% or 12.5% acrylamide), followed by transfer of resolved proteins to nitrocellulose membranes. Membranes were then blocked for 2 hours at room temperature in Tris-buffered saline containing 0.1% Tween-20 and 3% (wt/vol) nonfat dry milk. Membranes were then probed using antibodies specifically directed against MMP-2 (monoclonal), TIMP-1 (polyclonal), and TIMP-2 (polyclonal) overnight at 4°C. Membranes were washed and incubated for 2 hours at room temperature with peroxidase-labeled secondary antibodies, and detection of immunoreactivity was carried out by ECL according to manufacturer’s recommendations. Membranes or ECL films were then scanned for densitometric analysis using NIH Image software.

Levels of total and phosphorylated forms of p38 and ERK1/2 kinases were determined in HTM cell lysates derived from cytochalasin D and latrunculin A treatments by immunoblot analysis using the respective polyclonal antibodies. For these analyses, cell lysates were prepared in lysis buffer containing 20 mM Tris (pH 7.4), 0.5 mM sodium orthovanadate, 0.2 mM EDTA, 10 mM PMSF, 0.1 M NaCl, 50 mM NaF, 25 µg/mL each of aprotinin and leupeptin, and 1 µM okadaic acid, and the cell lysates prepared were briefly spun at 800g. Supernatants were used for determining the total protein content by the Bradford method.47

Reverse Transcription—Polymerase Chain Reaction
HTM cells were cultured and treated in Petri dishes as described. Total RNA from HTM cells was extracted (RNeasy Mini kit; Qiagen) according to the manufacturer’s instructions. During the extraction, HTM cell homogenates were passed through a 20-gauge needle several times to shear DNA, and total RNA was treated with DNAse I to eliminate contamination from genomic DNA. Purified RNA was quantitated spectrophotometrically at 260/280 nm. Equal amounts of RNA were then reverse transcribed (Advantage RT-for-PCR kit; BD Biosciences Clontech) according to the manufacturer’s instructions. Controls lacking reverse transcriptase (RT) were also set up to confirm the absence of genomic DNA-generated signals. PCR amplification was then performed on the resultant HTM RT-derived complementary DNA libraries (Advantage RT-for-PCR kit) according to the manufacturer’s instructions with sequence-specific forward and reverse oligonucleotide primers for MT1-MMP (5'-TCC ATC AAC ACT GCC TAC GAG AG; 3'-TGA GCT CTT CGT TGA AAC GGT AGT; 228-bp fragment). PCR cycle numbers were chosen such that the amplified DNA product was within the linear range. PCR products were then subjected to agarose gel electrophoresis with ethidium bromide for UV visualization. Using sequence-specific forward (5'-CCG AGC TGA GCA TAG ACA TT) and reverse (3'-TCC ACC ACC CTG TTG CTG TA) oligonucleotide primers, the housekeeping gene G3PDH (464 bp) was amplified as an internal control for normalizing the cDNA content of control and test compound–treated samples in PCR reactions. In addition to this semiquantitative analysis, we performed real-time quantitative PCR analysis to further substantiate the semiquantitative observations.

Real-Time Quantitative PCR
Real-time quantification of MT1-MMP expression in cytochalasin D– and latrunculin A–treated HTM cells was performed (iCycler iQ Detection System; Bio-Rad, Philadelphia, PA). cDNA from different treated samples were normalized with an endogenous housekeeping gene, G3PDH (glyceraldehyde 3-phosphaste dehydrogenase). The same MT1-MMP oligonucleotide primers described for the RT-PCR method were used for real-time PCR analysis. For the G3PDH, the oligonucleotide primers 5'-CTG GCA TTG CCC TCA ACG ACC and 3'-CTT GCT GGG GCT GGT GGT CC (140 bp) were used. Briefly, the PCR master mix (iQ supermix; Bio-Rad) consisted of 1 µL template cDNA in 20-µL reaction, 2x PCR master mix, 10 nM fluorescein calibration dye (Bio-Rad), 1 µL of a 1:1500 dilution of 10,000x nucleic acid dye (iQSYBR Green 1; Molecular Probes), and 500 nM each of a gene-specific oligonucleotide pair. Duplicate PCR reactions were carried out using the following amplification protocol: 95°C for 2 minutes followed by 50 cycles of 95°C for 15 seconds, 60°C for 15 seconds, and 72°C for 15 seconds. The increase in fluorescence was measured in real time during the extension step, and melt curves were obtained immediately after amplification by increasing temperature in 0.4°C increments from 65°C for 85 cycles of 10 seconds each and analyzed (iCycler software; Bio-Rad). The fold difference in MT1-MMP expression between control, cytochalasin D–, and latrunculin A–treated samples was normalized to the housekeeping gene (G3PDH) and was calculated by the comparative threshold (CT) method, as described by the manufacturer (Prism 7700 Sequence Detection System; Applied Biosystem, Inc., Foster City, CA).

Cytoskeletal Staining and Cell Morphology
HTM cells were cultured on gelatin (2%)–coated glass coverslips to confluence and were treated as described. Cells were fixed with 3.7% formaldehyde in cytoskeletal buffer (10 mM MES [2-N-morpholino-(ethanesulfonic ether) N,N,N,N,-tetra acetic acid], 150 mM NaCl, 5 mM EGTA, 5 mM MgCl2, 5 mM glucose, pH 6.1) and permeabilized with 0.1% Triton X-100 in PBS at room temperature. Actin was stained with rhodamine-phalloidin, as described elsewhere.11 Micrographs were recorded using a fluorescence microscope (Axioplan-II; Carl Zeiss, Oberkochen, Germany). Changes in cell shape were recorded with a phase-contrast microscope (IM 35; Carl Zeiss).

Cell Viability and Cytotoxicity
TM cells treated with different cytoskeletal agents were evaluated for cytotoxic response using fluorescein diacetate and propidium iodide staining, as described earlier.11 Additionally, after washing of the drugs the reversibility of drug-induced changes in cell morphology and actin cytoskeletal changes was evaluated. Cells that were detached because of treatment with certain cytoskeletal drugs were collected from the medium by centrifugation at 2800 rpm and recultured in regular medium in the absence of cytoskeletal drugs. These cells recovered normal morphology in a time-dependent manner.

Statistical Analysis
Results are presented as the mean ± SE, and statistical significance was evaluated by paired Student’s t-test. P < 0.05 was considered significant. All experiments were conducted with n = 4 to 6 using cells derived from at least two different donors.


   Results
Top
 Abstract
 Materials and Methods
 Results
Discussion
 References
 
Effects of Actin Cytoskeleton Interfering Drugs on HTM Cell Morphology and Actin Cytoskeletal Organization
Treatment of serum-starved HTM cells that were grown to confluence with ECA (100 µM), H-7 (50 µM), cytochalasin D (25 µM), latrunculin A (0.25 µM), and Y-27632 (25 µM) for 24 hours induced changes in cell morphology (Fig. 1A) . Treatment with H-7, Y-27632, and ECA resulted in cell–cell separation and a stellate appearance in cells compared with control cells. These changes were more pronounced with ECA treatment, which also resulted in cell detachment. Cells treated with cytochalasin D and latrunculin A showed the greatest degree of change in cell morphology, with rounding up of cells, cell–cell separation, and cell detachment. These treatments also induced alterations in staining patterns for F-actin (Fig. 1B) . Actin stress fibers were observed throughout the control cells, whereas a significant decrease was noted in the treated cells. ECA, H-7, and Y-27632 displayed some stress fibers at the peripheral region of the cell body but weak to undetectable fibers in the center of the cell body, whereas cells treated with cytochalasin D and latrunculin A showed complete loss of stress fibers. Similar results were recorded from four independent experiments. Under these conditions, though the morphology of TM cells changed and they detached from their surfaces, they were found to be viable based on fluorescein diacetate and propidium iodide in vivo labeling (data not shown). Further, reculturing of the detached cells in the medium lacking cytoskeletal drugs led to recovery of normal cell shape over a period of 24 to 36 hours (data not shown).


Figure 1
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  		FIGURE 1. ECA, H-7, cytochalasin D, latrunculin A, and Y-27632 induced changes in human TM cell morphology and actin cytoskeletal organization. Treatment of serum-starved HTM cells with ECA, H-7, cytochalasin D, latrunculin A, and Y-27632 for 24 hours induced changes in cell morphology and actin cytoskeletal organization compared with control cells. (A) ECA, H-7, and Y-27632 treatment induced cell–cell separation along with a stellate appearance. Cytochalasin D and latrunculin A treatment produced a strong effect on cell morphology, as observed with a complete rounding of cells along with cell-cell separation and cell detachment. (B) Actin stress fibers (phalloidin staining) are significantly decreased in TM cells treated with different test compounds compared with control cells. Although cells treated with ECA, H-7, and Y-27632 exhibited some actin stress fibers at the peripheral region of the cell body, treatment with cytochalasin D and latrunculin A resulted in the near complete disappearance of stress fibers.

 
Activation of MMP-2 in TM Cells Treated with Actin Cytoskeleton Interfering Agents
To assess whether actin cytoskeleton interfering agents can influence MMP-2 activation, TM cells were serum-starved for 24 hours and treated with ECA (100 µM), H-7 (50 µM), cytochalasin D (25 µM), latrunculin A (0.25 µM), and Y-27632 (25 µM) for 24 hours. Cell medium was then collected, concentrated, and analyzed using gelatin zymography. HTM cells treated with cytochalasin D and latrunculin A exhibited a dramatic and significant increase (P < 0.05) in the active form of MMP-2 by approximately 23-fold and 25-fold, respectively. ECA, H-7, and Y-27632 displayed marginal (statistically insignificant) effect on the activation of MMP-2 (Fig. 2A) . Zymography data were further corroborated by Western blot analysis, which confirmed the results observed with cytochalasin D and latrunculin A, revealing the presence of a band corresponding to the active form of MMP-2 (66 kDa) in conditioned media. Additionally, the band corresponding to the proform of MMP-2 (72 kDa) was clearly reduced in samples derived from cytochalasin D– and latrunculin A–treated cells compared with control cells, indicating that the reduction in the proform was associated with the increase in the activated form of MMP-2 (Fig. 2B) . Porcine primary TM cells were treated in a fashion identical to that for HTM cells and then underwent gelatin zymography analysis. In contrast to HTM cells, treatment of PTM cells with ECA and H-7 was also observed to result in MMP-2 activation, in addition to activation of MMP-2 by cytochalasin D and latrunculin A. Y-27632 did not appear to exert much effect on the activity of MMP-2 (Fig. 2C) . Furthermore, in contrast to HTM cells, PTM cells treated with the various cytoskeletal-interacting agents and kinase inhibitors were noted to produce a prominent 58-kDa MMP-2 fragment in addition to the 62-kDa fragment of MMP-2 (Fig. 2C) and also revealed a prominent and additional gelanolytic protein band migrating more slowly than the pro-MMP-2 protein band (Fig. 2C) .


Figure 2
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  		FIGURE 2. Activation of MMP-2 induced by actin-depolymerizing agents in TM cells. Human and porcine TM cells were serum starved for 24 hours and were treated with ECA, H-7, cytochalasin D, latrunculin A, and Y-27632 for 24 hours. An equal volume of cell medium was then collected and analyzed. (A) Gelatin zymography shows a marked increase in active MMP-2 in HTM cells treated with cytochalasin D and latrunculin A compared with control cells. Densitometric analysis based on four independent observations revealed that cytochalasin D and latrunculin A produced a statistically significant increase in the active form of MMP-2. *Significant at P < 0.05 versus control. (B) Western blot analysis confirms the results observed in zymography (A) and exhibits a positive immunoreactive band corresponding to the activated form of MMP-2 with cytochalasin D and latrunculin A treatment in HTM cells. (C) Gelatin zymography of porcine TM cells treated with cytochalasin D, latrunculin A, ECA and H-7 shows an increase in active MMP-2 with the exception of Y-27632.

 
Expression of MT1-MMP in HTM Cells Treated with Cytochalasin D and Latrunculin A
After establishing that cytochalasin D and latrunculin A significantly increased the amount of active MMP-2 in HTM cells, we wanted to assess the impact of treatment of these agents on the expression of MT1-MMP, an activator that facilitates the conversion of the proform of MMP-2 to the active form.35 37 48 To accomplish this, HTM cells were serum starved and treated with cytochalasin D (25 µM), latrunculin A (0.25 µM), ECA (100 µM), H-7 (50 µM), and Y-27632 (25 µM) for 24 hours. Total RNA was then isolated and reverse transcribed using RT. PCR was conducted on the resultant cDNA to amplify a specific fragment of the MT1-MMP coding region. After normalization of the RT-derived cDNA content in all samples through a PCR amplification of the housekeeping gene G3PDH, it was observed that the expression of MT1-MMP was markedly increased in cells treated with cytochalasin D and latrunculin A (Fig. 3A) . ECA, H-7, and Y-27632, produced no observable increase in MT1-MMP expression compared with control cells (data not shown).


Figure 3
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  		FIGURE 3. Cytochalasin D and latrunculin A induce an increased expression of MT1-MMP in HTM cells. HTM cells were serum starved for 24 hours and treated with cytochalasin D and latrunculin A for 24 hours. Expression of MT1-MMP was then determined by RT-PCR analysis of total RNA extracted from cell cultures. The content of reverse-transcribed RNA derived from control and drug-treated samples was normalized with the expression of the G3PDH gene (A). Semiquantitative RT-PCR showed that cytochalasin D and latrunculin A induced an increase in MT1-MMP expression compared with control cells (A). To determine the relative difference in MT1-MMP expression induced by cytochalasin D and latrunculin A in HTM cells, total RNA extracted from the 4 sets of control and drug-treated samples was subjected to quantitative real-time PCR analysis (B). Based on the mean values of 4 individual samples, the increase in MT1-MMP expression with cytochalasin D and latrunculin A was found to be greater than 10-fold and significant (*P < 0.01; **P < 0.003). HTM cells derived from two human subjects were used in these analyses.

 
The increase in MT1-MMP expression in HTM cells treated with cytochalasin D and latrunculin A (Fig. 3A) was confirmed further by quantitative real-time PCR analysis. Serum-starved HTM cells treated with cytochalasin D and latrunculin A for 24 hours demonstrated a significant (P < 0.01; n = 4) and greater than 10-fold increase in MT1-MMP expression compared with untreated HTM cells (Fig. 3B) .

TIMP-1 and TIMP-2 Protein Levels in HTM Cells Treated with Cytochalasin D and Latrunculin A
To gain a comprehensive perspective on the nature of MMP-2 regulation in HTM cells after treatment with cytochalasin D and latrunculin A, we decided to evaluate the effects of treatment on the levels of TIMP-2 and TIMP-1 proteins. HTM cells were serum starved and treated with cytochalasin D (25 µM) and latrunculin A (0.25 µM) for 24 hours. Cell medium was then collected, and samples of equal amounts of protein underwent Western blot analysis. A significant decrease in the levels of TIMP-1 (Fig. 4A) and TIMP-2 (Fig. 4B) was observed when HTM cells were treated with cytochalasin D (1.6-fold decrease for TIMP-1 [P < 0.01] and 1.75-fold decrease for TIMP-2 [P < 0.01]) and latrunculin A (1.4-fold decrease for TIMP-1 [P < 0.05] and 3-fold decrease for TIMP-2 [P < 0.01]).


Figure 4
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  		FIGURE 4. Cytochalasin D and latrunculin A decrease TIMP-1 and TIMP-2 secretion in HTM cells. HTM cells were serum starved for 24 hours and treated with cytochalasin D and latrunculin A for 24 hours. Equal amounts of a protein from cell medium were analyzed by Western blot analysis. Western blot shows that cytochalasin D and latrunculin A reduced the levels of TIMP-1 (A) and TIMP-2 (B) compared with control cells. Densitometric analysis based on four independent observations revealed that the decreased expression produced by both compounds was statistically significant. Significant at *P < 0.05 or **P < 0.01 versus control.

 
Cytochalasin D– and Latrunculin A–Induced Effects on the Activation Status of p38 MAPK and ERK Kinase in HTM Cells
To understand mechanistically the influence of actin depolymerizing agents on expression and activation of MT1-MMP and MMP-2, respectively, in TM cells, we determined the effects of cytochalasin D and latrunculin A on the activation status of p38 and ERK1/2 kinase activities. Both these kinase activities are known to be influenced by actin cytoskeletal organization and, in turn, influence the expression of MT1-MMP and the secretion and activation of MMP-2 in different cell types.28 45 48 49 50 51 52 53 54 55 Therefore, total cell lysates derived from HTM cells treated with cytochalasin D and latrunculin A for 24 hours (under serum-starved conditions, as described earlier) were examined for changes in the protein levels of total p38 and ERK1/2 kinases (both p42/44) and their phosphorylated forms by immunoblot analysis. Analysis of equal amounts of protein from control, cytochalasin D–, and latrunculin A–treated HTM cells revealed a significant increase in the levels of phospho-p38 MAPK (Fig. 5) . This effect was relatively stronger with latrunculin A than with cytochalasin D. Under similar conditions, levels of p38 MAPK total protein were increased marginally with cytochalasin D and latrunculin A compared with untreated control cells, and the difference was found to be significant between latrunculin A–treated and control samples (Fig. 5) . Levels of total ERK1/2 were not altered with cytochalasin D and latrunculin A treatments in HTM cells (Fig. 6) ; however, the levels of phospho-ERK were increased significantly (P < 0.05) with latrunculin A treatment compared with control or cytochalasin D treatment (Fig. 6) . Total protein of p38 and ERK kinases in HTM cells was considered loading controls for the analysis of phosphorylated p38 and ERK by immunoblotting.


Figure 5
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  		FIGURE 5. Activation of p38 MAP kinase in cytochalasin D– and latrunculin A–treated HTM cells. Confluent cultured HTM cells were serum starved and treated with cytochalasin D (25 µM) or with latrunculin A (0.25 µM) for 24 hours. Equal amounts of protein from the total cell lysates derived from the different treatments were analyzed for changes in total and phosphorylated p38 MAP kinase by immunoblot analysis. (A, B) Representative immunoblots reflecting the changes in HTM cell phospho-p38 MAP kinase and total p38 protein, respectively, with cytochalasin D and latrunculin treatment. Densitometric analysis based on six (for total p38) to eight (for phospho-p38) independent observations revealed a significant (P < 0.001) increase in the phosphorylated form of p38 MAP kinase with cytochalasin D and latrunculin A treatment. Latrunculin A also showed a significant (P < 0.004) but marginal increase in total protein of p38 MAP kinase. HTM cells derived from two human donors were used in these analyses.

 

Figure 6
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  		FIGURE 6. Activation of ERK1/2 in latrunculin A–treated HTM cells. Equal amounts of protein from total cell lysates derived from cytochalasin D and latrunculin A treatments along with control were analyzed for changes in the total ERK1/2 (p42/44) and phosphorylated ERK kinase by immunoblot analysis. (A, B) Representative immunoblots of the HTM cell phospho-ERK1/2 and total ERK1/2 protein levels, respectively, with cytochalasin D and latrunculin A treatment. Densitometric analysis based on five independent observations showed that latrunculin A produced a statistically significant (P < 0.008) increase in the phosphorylated form of ERK.

 
After finding that treatment of HTM cells with cytochalasin D and latrunculin A activates p38 MPAK and ERK kinase (Figs. 5 6) , we set out to determine whether this response was mechanistically linked to the activation of MMP-2. Toward this end, we pretreated (30 minutes) serum-starved HTM cells with specific inhibitors of p38 MAPK (5 or 20 µM SB202190) or ERK kinase (25 µM PD98059) before adding cytochalasin D and latrunculin A. After 24 hours of treatment, the conditioned medium was collected, concentrated, and examined for stimulation of MMP-2 activity by gelatin zymography. Neither inhibitor exhibited significant effects on cytochalasin D– or latrunculin A–induced activation of MMP-2 (data not shown).


   Discussion
Top
 Abstract
 Materials and Methods
 Results
 Discussion
References
 
It is well established that an increase in aqueous humor outflow facility occurs after actin cytoskeletal disruption in the outflow pathway.8 9 10 11 12 16 17 56 Other data indicate that ECM remodeling through MMP activity participates in the modulation of aqueous humor outflow.3 15 18 38 44 However, the possibility of actin cytoskeleton organization influencing MMP activity has not been explored in the cells of aqueous outflow pathway, and we hypothesized that cross talk may occur between cytoskeleton integrity and MMP activation. Our goal, therefore, was to provide information that would corroborate the connection between actin cytoskeleton organization in TM cells and MMP activation, which may eventually influence ECM remodeling and aqueous humor outflow. The data presented herein demonstrate that the disruption of actin cytoskeletal organization induced by direct inhibitors of actin polymerization increases the activation of MMP-2 in TM cells and that this increase in active MMP-2 is correlated with increases in the expression of MT1-MMP, the activator of MMP-2, and with decreases in protein levels of the MMP inhibitors TIMP-1 and TIMP-2.

The interaction between the actin cytoskeleton and ECM through inside-out and outside-in cellular responses at the cell surface is known to influence ECM organization, ECM synthesis, and cell adhesion formation through the integrin receptors.15 22 28 29 57 58 Importantly, changes in cell morphology and actin cytoskeletal organization influence MMP activity in different cell types.30 31 32 33 34 Therefore, to obtain insight into the possible link between actin cytoskeletal organization and ECM turnover through MMP activity in the aqueous humor outflow pathway, we chose to use an array of actin cytoskeleton–altering pharmacologic compounds. When selecting our compounds, we took note of the fact that these compounds have been documented to increase aqueous humor outflow facility.8 9 10 11 12 59 In addition, we chose direct (cytochalasin D and latrunculin A) and indirect (ECA, H-7, and Y-27632) modulators of actin polymerization to assess whether there would be any difference between them regarding MMP-2 activity.

We began our study by confirming that treating human TM cells with the cytoskeleton-altering agents caused changes in cell morphology (Fig. 1A) and reduced actin stress fiber staining (Fig. 1B) . Treatment with cytochalasin D and latrunculin A, the direct inhibitors of actin polymerization, produced a complete rounding of cells along with cell–cell separation and cell detachment. Additionally, almost no actin stress fiber staining was observed, and only a few actin filaments were observed at the periphery of rounded cells (Fig. 1B) . Under these conditions TM cells were confirmed as viable. Treatment with ECA, H-7, and Y-27632, the indirect modulators of actin polymerization, resulted in changes that were less severe than cell–cell separation, and a stellate cellular appearance was observed without marked cell detachment (Fig. 1A) . Although actin stress fibers were not reduced to the degree they were with cytochalasin D and latrunculin A, one could clearly see cells retaining some actin stress fibers at the peripheral region of the cell body and a small number of fibers in the central region (Fig. 1B) .

The direct inhibitors of actin polymerization, cytochalasin D and latrunculin A, yielded a dramatic activation of MMP-2 in HTM cells, represented by the bands at 66 kDa, compared with control cells (Fig. 2A) . On the other hand, treatment with the indirect inhibitors of actin polymerization—ECA, H-7, and Y-27632—resulted in MMP-2 activation that showed a rising trend compared with control cells, but it was without statistical significance. Although we noted activation after 24 hours, we did not detect MMP-2 activation after 6 or 12 hours with any of the agents used (data not shown). In contrast to HTM cells, however, direct (cytochalasin D and latrunculin A) and indirect (H-7 and ECA) modulators of the actin cytoskeleton influenced MMP-2 activity in porcine TM cells, indicative of species-specific differences in terms of the roles played by actin cytoskeletal integrity and organization in the regulation of MMP-2 activation (Fig. 2C) .

These results demonstrate a proportional correlation between the degree of MMP-2 activation and the degree of change seen in HTM cell morphology and actin cytoskeletal organization, with the direct inhibitors of actin polymerization, cytochalasin D, and latrunculin A eliciting marked variation from control cells. Further study could determine whether change in one of these characteristics is sufficient for MMP-2 activation in the TM or whether a combination of the two is necessary. However, a study in fibroblasts suggests that organization of the actin cytoskeleton, and not cell shape change, influences MMP-2 activation.33 Along with our data, this could indicate that modifying the time of treatment and concentrations of our indirect inhibitors to promote increased cytoskeletal reorganization may increase the activation of MMP-2 in HTM cells. Although our data did not reveal increased MMP-2 activation with shorter treatment times (data not shown), other studies have shown an increased amount of MMP-2 activation when different cells were treated with cytoskeletal disrupting agents for periods longer than 24 hours.32 60 Therefore, it is possible that increasing the length of treatment (more than 24 hours) with ECA, H-7 and Y-27632 may result in an increase of activated MMP-2.

We supported our zymography data by confirming that cytochalasin D– and latrunculin A–induced MMP-2 activation was observed through an independent technique, Western blot analysis (Fig. 2B) . The blot displayed a clear activation of MMP-2 (bands at 66 kDa) compared with control cells. Additionally, it revealed a reduction in the proform of MMP-2 (bands at 72 kDa) when cells were treated with cytochalasin D and latrunculin A, suggesting that the transcription of MMP-2 may not be altered because of actin depolymerization and that the presence of the active form of MMP-2 is a result of proteolytic cleavage of the latent MMP-2 present at the time of treatment.

To gain more insight into how MMP-2 activation was regulated in human TM cells, we evaluated the effects of cytochalasin D and latrunculin A on the expression of MT1-MMP, the activator for MMP-2 (Fig. 3) . We observed that treatment with both compounds resulted in a robust increase of MT1-MMP expression compared with control cells. We also explored MT1-MMP expression in human TM cells treated with ECA, H-7, and Y-27632, the indirect inhibitors of actin polymerization. We did not observe an increase in MT1-MMP expression compared with control cells, which is consistent with the previously obtained data showing no significant increase in MMP-2 activation (data not shown).

Results from our experiments on MT1-MMP expression lend support to MMP-2 activation in HTM cells through the commonly accepted mechanism described earlier.39 40 41 We investigated this further by examining the influence of treatment of HTM cells with cytochalasin D and latrunculin A on the protein levels of TIMP-2 (Fig. 4) . Cytochalasin D and latrunculin A both considerably diminished the protein levels of TIMP-2 compared with control cells, with latrunculin A providing greater reduction than cytochalasin D.

Although the data we obtained on TIMP-2 experiments provided important insight into the possible mechanism involved in the activation of MMP-2, further study is needed to determine whether the reduction in TIMP-2 resulted from increased degradation, reduced transcription, or another process. One study showing cytochalasin D–induced MMP-2 activation with no change in TIMP-2 gene expression postulated that TIMP-2 was sequestered at the cell membrane by activated MT1-MMP, which had a higher affinity for TIMP-2.61 This would have resulted in the formation of the MMP-2/TIMP-2/MT1-MMP complex and would have led to MMP-2 activation. When checking the levels of extracellular TIMP-2, one would see a reduction in levels caused by sequestration rather than by decreased transcription or protein degradation. This could explain the reduction of TIMP-2 in our experiments, but we cannot make that conclusion at this time, especially given the fact that studies in other cell types have shown variability in TIMP-2 gene expression and protein levels during MMP-2 activation.32 62

As described earlier, a key difference between the compounds was that only the direct inhibitors of actin polymerization increased MT1-MMP expression in HTM cells. Actin cytoskeletal change has been previously shown in different cells to increase MMP-2 activation through the upregulation of MT1-MMP expression, though the precise mechanism leading to this upregulation is not yet known.32 33 34 61 This leads us to speculate that direct inhibition of actin polymerization through the administration of cytochalasin D or latrunculin A may trigger a greater number and variety of signaling pathways compared with cells that undergo a smaller degree of cytoskeletal change from treatment with the indirect inhibitors, which could account for why the induction of MT1-MMP expression is only seen in the former case.

Interestingly, in this study, cytochalasin D– and latrunculin A–induced activation of MMP-2 and increased MT1-MMP expression in HTM cells were associated with concomitant activation of p38 MAPK (Fig. 5) . Latrunculin A was noted to be consistently more effective than cytochalasin D at stimulating MMP2 and p38 MAPK in HTM cells. Further, the levels of total p38 were also found to be marginally but significantly higher in latrunculin A–treated HTM cells (Fig. 5) . Moreover, in contrast to cytochalasin D, latrunculin A also stimulated significant increases in ERK1/2 activity in HTM cells. Although we have not carried out quantitative analysis of G actin/F actin ratios as a function of dose of cytoskeletal agents, latrunculin A is known to be a more potent inhibitor of actin polymerization than cytochalasin D.63 Thus, it may be that the degree of actin depolymerization is related to the degree of MMP-2 activation and stress kinase activities in TM cells. p38 and ERK kinase activities have been shown to regulate the secretion and activation of MMPs, including MMP-2, in various cell types.45 46 48 49 53 54 64 Further, in cancer cells, the inhibition of p38 and ERK kinase activities has been found to suppress MMP-2 activation and MT-MMP expression.53 64 65 Intriguingly, the inhibition of p38 and ERK kinase activities in HTM cells did not affect the activation of MMP-2 activity induced by cytochalasin D and latrunculin A. However, further studies are required for an understanding of the specific role of p38 MAPK and ERK activities in the regulation of expression of MT1-MMP and TIMPs in HTM cells.

In addition to p38 and ERK, src-kinases may have a role to play in cytochalasin D–induced MMP-2 activation.60 Clostridium difficile toxin B, which inhibits the Rho-family GTPases (Rho, Rac, and Cdc42), has been shown to increase the activation of MMP-2 and the expression of MT1-MMP in bovine smooth muscle cells. Y-27632, which works in a more specific fashion further downstream by inhibiting Rho-kinase, was unable to do the same.32 Importantly, different Rho GTPases, including Rho, Rac, and Cdc42, which regulate various aspects of actin cytoskeletal organization through distinct cellular mechanisms, have been shown to influence the activities of MMPs and TIMPs in different cell types.32 50 52 62 66 67 This contention is further supported by a group who found that Rac1 increased MMP-2 activation and MT1-MMP expression and postulated that the modulation of interleukin-1{alpha} (IL-1{alpha}) was responsible for the induction of MT1–MMP. Therefore, studies targeting these individual Rho GTPases, in addition to their downstream specific kinases in TM cells, might provide important insight into the regulation of ECM turnover in the aqueous outflow pathway given that these GTPases are thought to exert master control in the regulation of actin cytoskeletal organization and cell–ECM interactions.68

In addition to trying to determine why direct and indirect modulators of actin polymerization act differently with regard to MMP-2 activation, we found it interesting that cytoskeletal change in human TM cells occurred soon after the initiation of treatment without a change in MMP-2 activation at the same time, as has been observed in human capillary endothelial cells.34 Previous studies that have examined the effects of cytoskeleton-altering compounds on aqueous humor outflow facility have observed changes in aqueous outflow earlier than 24 hours.3 8 9 12 17 Therefore, cytochalasin D– and latrunculin A–induced changes in aqueous outflow facility in a short-term perfusion model might not be directly related to their effects on MMP-2 activation. Thus, mechanisms involved in aqueous humor outflow regulation, such as the modulation of cell–cell junctions, cell adhesions, and paracellular fluid flow, could still play a predominant role in the rapid effects of cytoskeletal reorganization on aqueous outflow.3 8 11 12 56 Increased MMP-2 activation, on the other hand, may possibly account for the delayed response of aqueous outflow to cytoskeletal drug–induced changes in cytoskeletal integrity.69

In summary, the data presented here indicate that actin cytoskeletal reorganization, induced by direct inhibitors of actin polymerization in the TM can lead to the activation of MMP-2, which may influence ECM remodeling in the aqueous outflow pathway and eventually in aqueous outflow. Mechanistically, this response of actin-depolymerizing agents appears to link to the upregulation and decrease of the physiological activator, MT1-MMP, and inhibitors, TIMP-1/ TIMP-2, respectively, thus providing a possible association among actin cytoskeletal changes, MMP-2 activation, ECM remodeling, and modulation of aqueous humor outflow. Not only does this work provide additional insight into the mechanism of hypotensive effects of actin-depolymerizing agents on ocular pressure, it may assist in the development of innovative therapeutic approaches to lower increased intraocular pressure in glaucoma patients.


   Acknowledgements
 
The authors thank Peifeng Deng, Jianming Qiu, and Min Zhang for their assistance with various experiments.


   Footnotes
 
Supported by National Institutes of Health/National Eye Institute Grants EY12201 and EY013573 (PVR) and by the Research to Prevent Blindness Wasserman Merit Award (PVR) and Medical Student Fellowship (RKS).

Submitted for publication September 12, 2006; revised December 10 and December 31, 2006; accepted March 6, 2007.

Disclosure: K. Sanka, None; R. Maddala, None; D.L. Epstein, None; P. Vasantha Rao, 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: P. Vasantha Rao, Department of Ophthalmology, Duke University School of Medicine, Box 3802, Durham, NC 27710; rao00011{at}mc.duke.edu.


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