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(Investigative Ophthalmology and Visual Science. 2005;46:1706-1713.)
© 2005 by The Association for Research in Vision and Ophthalmology, Inc.
DOI:  10.1167/iovs.04-0993
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Acute Effects of PGF2{alpha} on MMP-2 Secretion from Human Ciliary Muscle Cells: A PKC- and ERK-Dependent Process

Shahid Husain,1 Farahdiba Jafri,2 and Craig E. Crosson1

1From the Hewitt Laboratory of the Ola B. Williams Glaucoma Center, Department of Ophthalmology, and the 2Department of Endocrinology, Medical University of South Carolina, Charleston, South Carolina.


   Abstract
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 Abstract
Methods
 Results
 Discussion
 References
 
PURPOSE. Studies were designed to evaluate the cellular mechanisms associated with prostaglandin (PG)F2{alpha}-induced matrix metalloproteinase (MMP)-2 secretion from human ciliary muscle (HCM) cells.

METHODS. The secretion and activity of MMP-2 was determined by Western blot analysis and zymography, using conditioned medium and HCM cells. ERK1/2 activity was measured by in-gel kinase assay and Western blot analysis with anti-phospho-ERK1/2 antibodies.

RESULTS. PGF2{alpha} increased the secretion of MMP-2 in a dose-dependent manner with an EC50 of 2.7 x 10–8 M. The addition of 1 µM PGF2{alpha} also increased MMP-2 secretion in a time-dependent manner with maximum secretion occurring at 4 hours after administration. At 4 hours, the maximum increase in MMP-2 secretion and activity were 112% ± 32% and 88% ± 18%, respectively. The secretory action of PGF2{alpha} was inhibited by pretreatment with a protein kinase C (PKC) inhibitor, chelerythrine chloride; the FP receptor antagonist, AL-8810; and the MEK inhibitor, PD-98059. The addition of PGF2{alpha} and latanoprost acid increased ERK1/2 activity by 117% ± 12% and 75% ± 9%, respectively. The PGF2{alpha}- and latanoprost-acid–induced ERK1/2 activation was blocked by the presence of PKC inhibitors and downregulation of PKC by prolonged incubation with a phorbol ester.

CONCLUSIONS. These data provide evidence that FP receptor activation leads to an increase in the secretion and activation of MMP-2 through PKC- and ERK1/2-dependent pathways. FP-agonist–induced activation of ERK1/2 was blocked by PKC inhibitors, indicating that PKC activation is required for ERK1/2 activation and MMP-2 secretion from HCM cells. In the ciliary muscle, the functional responses to ERK1/2 activation include secretion of MMP-2, supporting the hypothesis that increases in uveoscleral outflow facility induced by PG administration involves the secretion and activation of MMP-2.

In the eye, prostanoid F (FP) receptors have generated considerable interest as a therapeutic target because prostaglandin (PG)F2{alpha} lowers intraocular pressure (IOP) in glaucomatous humans1 and primates.2 3 PGF2{alpha} analogues such as travoprost,4 latanoprost,5 6 and unoprostone isopropyl ester7 have been shown to lower IOP in many mammals, including humans. In animals and humans, this IOP reduction results from increased uveoscleral outflow without significant changes in conventional outflow or aqueous production, which implies that ciliary muscles play an important role.1 8 9 10 Investigators have attributed this PG-induced increase in uveoscleral outflow to relaxation of the ciliary muscle11 12 and remodeling of the extracellular matrix (ECM) between the muscle bundles13 14 by a group of enzymes called matrix metalloproteinases (MMPs).

The MMP family and tissue inhibitor of matrix metalloproteinases (TIMPs) are integrally involved in regulating the turnover of ECM. These proteinases have been implicated in a variety of pathologic conditions, including arthritis, angiogenesis, and metastatic invasion.15 16 17 More recently, studies have provided evidence that these enzymes may take part in the regulation of aqueous humor outflow.18 19 In the anterior segment tissues, a number of different ligands, such as growth factors, cytokines, PGs, and phorbol esters have been shown to regulate MMP secretion.15 20 21 22 23 24 However, the cell signaling events that mediate PGF2{alpha}-induced secretion of MMP-2 from ciliary muscle have not been investigated. The purpose of the present study was to evaluate the signaling events associated with FP-receptor–mediated secretion of MMP-2 from human ciliary muscle (HCM) cells. We provide evidence that FP receptor agonists increase MMP-2 secretion from HCM cells via PKC- and ERK1/2-dependent pathways.


   Methods
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 Methods
Results
 Discussion
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Supplies
PGF2{alpha}, latanoprost acid, and 11ß-fluoro-15-epi-indanyl PGF2{alpha} (AL-8810) were purchased from Cayman (Ann Arbor, MI); polyclonal anti-ERK1/2 antibodies from Upstate Biotechnology (Lake Placid, NY); [{gamma}-32P] adenosine triphosphate (ATP; specific activity, 3000 Ci mmol–1) from Amersham Life Science (Arlington Heights, IL); chelerythrine chloride, calphostin C, and 2'-amino-3'methoxyflavone (PD-98059) from Calbiochem (La Jolla, CA); myelin basic protein and phorbol 12,13-dibutyrate (PDBu) from Sigma-Aldrich (St. Louis, MO); and fetal bovine serum (FBS) from Hyclone (Logan, UT). All cell culture supplies were obtained from Cell Gro (Herndon, VA). The PKC activity assay kit was obtained from StressGen Biotechnology (Victoria, British Columbia, Canada).

HCM Cells
HCM cells were prepared from normal human eyes with a procedure described earlier.25 The human eyes were obtained from the National Disease Research Interchange (Philadelphia, PA) and Life-Point Ocular Tissue Division (Storm Eye Institute; MUSC, Charleston, SC). Briefly, ciliary muscles were dissected with the aid of a dissecting microscope under sterile conditions, cleaned, and cut into 1- to 2-mm pieces. The explants were placed in DMEM containing 2 mg/mL collagenase type IA, 10% fetal bovine serum (FBS), and 50 µg/mL gentamicin and then incubated for 1 to 2 hours at 37°C, with occasional shaking. When a major part of the explant was dispersed into single cells or groups of cells, the cell suspension was centrifuged at 200g for 10 minutes and resuspended in DMEM 199 supplemented with 10% FBS, 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin B and maintained in a 5% CO2 humidified atmosphere. The confluent cells were subcultured at a split ratio of 1:4 in 0.05% trypsin and 0.02% EDTA.

In-Gel Kinase Assay for ERK1/2 Activity
The activity of ERK1/2 was measured by the in situ myelin basic protein (MBP) phosphorylation assay, as described elsewhere.26 Briefly, cells were serum starved for 16 hours before the addition of any agents. Cells were treated with FP agonists for 5 minutes. In experiments evaluating the FP receptor antagonists MEK inhibitor or PKC inhibitor, cells were pretreated for 30 minutes with the inhibitor before the addition of the agonist. At the end of the incubation period, cells were rinsed with ice-cold PBS and extracted in buffer (20 mM ß-glycerophosphate, 20 mM NaF, 2 mM EDTA, 0.2 mM Na3VO4, 1 mM phenylmethylsulfonyl fluoride [PMSF], 25 µg/mL leupeptin, 10 µg/mL aprotinin, and 0.3% [vol/vol] ß-mercaptoethanol [pH 7.5]). The cell extracts were centrifuged at 6000g for 10 minutes at 4°C, and the supernatant was resolved on 10% SDS-PAGE copolymerized with 0.5 mg/mL MBP. After electrophoresis, the gels were washed with 50 mM Tris-HCl buffer (pH 8.0) containing 20% (vol/vol) propanol to remove SDS, then washed with denaturing buffer (50 mM Tris-HCl [pH 8.0], containing 6 M guanidine hydrochloride and 5 mM ß-mercaptoethanol). The enzymes on the gels were then renatured by washing with 50 mM Tris-HCl buffer (pH 8.0) containing 0.04% Tween-40 (vol/vol) and 5 mM ß-mercaptoethanol at 4°C for 21 hours. The gels were then preincubated with assay buffer containing: 40 mM HEPES (pH 8.0), 10 mM MgCl2, 2 mM dithiothreitol, and 0.1 mM EGTA at 30°C for 30 minutes. The ERK1/2 activity was determined by incubating the gels with 20 mL of the assay buffer, which contained 20 µM ATP and 100 µCi [{gamma}-32-P] ATP at 30°C for 1 hour. After extensive washing in 5% (wt/vol) trichloroacetic acid containing 10 mM sodium pyrophosphate, the gels were dried and autoradiographed at –70°C.

Determination of Phosphorylated ERK1/2
Cells were maintained in serum-free medium for 16 hours before the addition of any agent. Unless otherwise noted, cells were treated with FP agonists for 5 minutes. In experiments evaluating the FP receptor antagonist, MEK inhibitor or the PKC inhibitor, cells were pretreated for 30 minutes with the inhibitor before the addition of the agonist. At the end of the incubation periods, cells were rinsed with ice-cold PBS and lysed by the addition of lysis buffer (50 mM Tris-HCl buffer [pH 8.0], containing 100 mM NaCl, 1 mM EDTA, 1% Nonidet P-40, 0.1% SDS, 0.5% deoxycholate, 50 mM NaF, 1 mM Na3VO4, 5 mM PMSF, 10 µg/mL leupeptin, and 50 µg/mL aprotinin) for 20 minutes on ice. To determine the level of ERK1/2 activation (phosphorylation), equivalent amounts of protein (15 µg) were loaded onto 10% SDS-polyacrylamide gels, and the proteins separated according to molecular weight using standard SDS-PAGE protocols, and transferred to nitrocellulose membranes. The membranes were then probed with anti-phospho-ERK1/2 antibodies for 2 hours at room temperature. Bands were visualized by the addition of anti-rabbit horseradish peroxidase (HRP)-conjugated secondary antibodies (at 1:3000) and ECL reagents. Blots were then stripped by incubation in stripping buffer (62.5 mM Tris-HCl [pH 6.7], 100 mM ß-mercaptoethanol, and 2% SDS) for 30 minutes at 50°C, and total ERK levels (phosphorylated and nonphosphorylated forms) were determined by immunoblot techniques using polyclonal anti-ERK1/2 antibodies. Band densities were quantified with a densitometer (TM 2200 documentation and analysis system; Alpha Innotech Corp., San Leandro, CA). Specific immunoreactive bands were expressed as arbitrary units (AU), which were calculated from the selected band areas scanned by the densitometer. The level of phosphorylated ERK1/2 isoforms normalized for differences in loading, using the total ERK protein band intensities.

Western Blot Analysis and Zymography
HCM cells were starved in serum-free medium for 16 hours to minimize nonspecific induction of MMP. They were treated with vehicle or FP agonist for the indicated time, and the medium was collected and stored at –80°C until analyzed. In experiments evaluating the FP receptor antagonist (AL-8810), MEK inhibitor (PD-98059), or PKC inhibitor (chelerythrine chloride), cells were pretreated for 30 minutes with the inhibitor before the addition of the agonist. Medium was concentrated by using an ultrafiltration centrifugal concentrator (30-kDa cutoff; Centricon-0; Amicon Beverly, MA) and adjusted to a final concentration ratio of 10:1. Equivalent volumes (40 µL) of medium were loaded onto 10% SDS-polyacrylamide gels followed by transfer to a nitrocellulose membrane. The membranes were then probed with anti-MMP-2 antibodies overnight at 4°C. Bands were visualized by the addition of anti-mouse HRP-conjugated secondary antibodies (at 1:3000) and ECL reagents. The band intensities were quantified by densitometry and normalized with total cellular protein. This normalization to total cellular protein was used to correct for the differences in the number of cells within each experimental assay for MMP-2 secretion studies. Purified MMP-2 was run in parallel as a positive control to identify the MMP-2.

For zymography, concentrated medium was separated onto 10% SDS-PAGE containing 1 mg/mL gelatin under nonreducing conditions. After electrophoresis, gels were washed twice in 50 mM Tris-HCl buffer (pH 7.5) containing 2.5% Triton X-100 for 30 minutes followed by incubation in activation buffer (50 mM Tris-HCl [pH 7.5], 150 nM NaCl, and 10 mM CaCl2) for 18 hours at 37°C, to allow enzymatic degradation of the substrate. Gels were stained with Coomassie blue R-250 and destained. Digestion of the substrate (gelatin) at the position of the enzyme was observed as a clear area in the otherwise uniformly dark-staining gel. The density of digested areas was measured by densitometry and normalized with total cellular protein.

Protein Kinase C Assay
HCM cells were starved in serum-free medium for 16 hours followed by PGF2{alpha} treatment for 5 minutes. At the end of the incubation period, cells were rinsed with ice-cold PBS and lysed by the addition of lysis buffer (20 mM Tris-HCl buffer [pH 7.5], 20 mM EGTA, 20 mM NaF, 1 mM sodium vanadate, 0.3% mercaptoethanol, and protease inhibitor cocktail). Protein kinase C activity in whole-cell lysate (10 µg) was measured with a PKC activity assay kit (non-radioactive), according to the directions of the manufacturer (StressGen Biotechnology).


   Results
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 Methods
 Results
Discussion
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Cell Culture
HCM cells were prepared from normal human donors without a history of ocular disease. The characteristics of the donors are given in Table 1 . Cultured cells from passages 2 to 5 were used in the present study. Ciliary muscle cells in culture were identified by their pattern of growth, morphology, and immunocytochemical staining, as described earlier.25 These cells formed a confluent monolayer of spindle-shaped cells organized into a "hill-and-valley" distribution, characteristically typical of ciliary smooth muscle cells.25 27


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  		TABLE 1. Summary of Normal Eyes from White Donors Used to Prepare Human Ciliary Muscle Cells

 
Effects of PGF2{alpha} on MMP-2 Secretion from HCM Cells
PGF2{alpha} produced a dose-dependent increase in MMP-2 (molecular mass, 72 kDa; pro-MMP-2) secretion with an EC50 of 2.7 x 10–8 M (Figs. 1A 1B) . The addition of 1 µM PGF2{alpha} produced a time-dependent increase in MMP-2 secretion, with the maximum secretion occurring at 4 hours (112% ± 32% above control levels; Figs. 1C 1D ). A gradual increase in control, nonstimulated levels of MMP-2 over time was also noted. Furthermore, latanoprost acid (a selective FP agonist) increased the secretion of MMP-2 by 73% ± 26% at 4 hours. To confirm that PGF2{alpha}-induced secretion of MMP-2 resulted from FP receptor stimulation, cells were pretreated with the FP antagonist AL-8810. Addition of the FP receptor antagonist, AL-8810 (1 µM for 30 minutes), inhibited the PGF2{alpha}-induced MMP-2 secretion by 85% (P < 0.05) from HCM cells (Fig. 2) . To determine whether PGF2{alpha}-induced secretion of MMP-2 is regulated at the transcriptional level, cells were treated with the transcriptional inhibitor actinomycin D (50 nM for 30 minutes). Pretreatment with actinomycin D did not significantly alter PGF2{alpha}-induced secretion of MMP-2 (PGF2{alpha} 109% ± 17% vs. PGF2{alpha}+actinomycin D 118% ± 35% above control levels; n = 3).



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  		FIGURE 1. Dose–response (A, B) and time-course study (C, D) of MMP-2 secretion after treatment with PGF2{alpha}. Serum-deprived HCM cells were treated with vehicle or different concentrations of PGF2{alpha} for 4 hours for the dose–response study. The HCM cells were treated with 1 µM PGF2{alpha} for 0, 2, 4, and 6 hours for the time-course study. The media were collected, concentrated, and analyzed for MMP-2 by Western blot analysis using anti-MMP-2 antibodies. (A, C) Representative immunoblots of MMP-2. (B, D) Data are the mean ± SE of normalized densitometry measurements from Western blots of concentrated media (n = 4; *P < 0.05).

 


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  		FIGURE 2. Effects of an FP antagonist and a PKC inhibitor on MMP-2 activity and secretion from HCM cells. Serum-deprived HCM cells were treated with vehicle, 1 µM AL-8810, or 1 µM chelerythrine chloride (Chel) for 30 minutes followed by 1 µM PGF2{alpha} treatment for 4 hours. The media were collected, concentrated, and analyzed for MMP-2 activity by zymography and for secretion by Western blot analysis. (A) Representative zymograms of concentrated media showing the activity of MMP-2 in gelatin-SDS-PAGE. (B) A representative immunoblot for MMP-2 secretion. (C) Data are the mean ± SE of normalized densitometry measurements from Western blots of concentrated media (n = 3). *Significant difference (P < 0.05) from the agonist alone.

 
To determine whether the secretion of MMP-2 was mediated through the activation of PKC and/or ERK1/2, HCM cells were pretreated with a PKC or ERK pathway inhibitor for 30 minutes before the addition of PGF2{alpha} for 4 hours. Pretreatment with the PKC inhibitor, chelerythrine chloride (1 µM), significantly (P < 0.05) inhibited the secretion of MMP-2 in response to PGF2{alpha} (Fig. 2) . PGF2{alpha}-induced MMP-2 secretion was not inhibited by Go-6976, a classic PKC isoform inhibitor (PGF2{alpha} 99% ± 6% vs. PGF2{alpha}+Go-6976 105% ± 18% above control levels; n = 3). The PGF2{alpha}-induced secretion of MMP-2 was also significantly inhibited in the presence of the MEK inhibitor, as determined by Western blot analysis using anti-MMP-2 antibodies (Fig. 3A) . The addition of chelerythrine chloride or PD-98059, alone, did not significantly alter the basal secretion of MMP-2 from HCM cells. To determine whether PGF2{alpha} can influence PKC activity, HCM cells were treated with PGF2{alpha} for 5 minutes, followed by measurement of PKC activity in whole-cell lysates. PGF2{alpha} increases PKC activity by 74% ± 12% (n = 4; P < 0.05) above control levels in HCM cells.



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  		FIGURE 3. Effect of MEK inhibitor on MMP-2 secretion (A) and activity (B) from HCM cells. Serum-deprived HCM cells were treated with vehicle or 1 µM PGF2{alpha} for 4 hours. To determine the effect of the inhibitor, cells were treated with 1 µM PD-98059 for 30 minutes before the addition of PGF2{alpha}. The media then collected, concentrated, and analyzed for MMP-2 activity by zymography and for secretion of MMP-2 by Western blot analysis. (A) Representative immunoblot of MMP-2. (B) Representative zymogram in concentrated media showing activity of MMP-2 in gelatin-SDS-PAGE. Histogram showing mean ± SE data of normalized densitometry measurements from zymogram and Western blots (n = 3, *P < 0.05).

 
Zymographic analysis demonstrated that the addition of PGF2{alpha} increased MMP-2 activity by 88% ± 18% (molecular mass, 66 kDa; active-MMP-2). This increase in activity was completely inhibited in the presence of 1 µM chelerythrine chloride or PD-98059 (Figs. 2 3B) .

Effect of PGF2{alpha} and Latanoprost Acid on ERK1/2 Activation in HCM Cells
The addition of PGF2{alpha} (1 µM) or latanoprost acid (1 µM) for 5 minutes increased ERK1/2 activity by 117% ± 12% and 75% ± 9%, respectively. Moreover, the PGF2{alpha} and latanoprost-acid–induced ERK1/2 activity was completely inhibited in the presence of the MEK inhibitor PD-98059 (1 µM). Pretreatment with the PKC inhibitors chelerythrine chloride (1 µM) or calphostin C (1 µM) inhibited the PGF2{alpha}-induced activity of ERK1/2 by 64% ± 5% and 75% ± 4%, respectively. Latanoprost-acid–induced ERK1/2 activity was also completely blocked in the presence of these inhibitors. The addition of PKC activator, phorbol esters (PDBu) increases ERK1/2 activity by 260% ± 24% (Fig. 4) . As ERK1/2 activation requires dual phosphorylation at both the tyrosine and serine/threonine residues, we measured the appearance of the phosphorylated form of ERK1/2 after PGF2{alpha} and latanoprost acid treatment by Western blot techniques. The addition of PGF2{alpha} or latanoprost acid produced a significant increase in phosphorylation of ERK1/2. Again, this response was inhibited by pretreatment with PKC and MEK inhibitors (Fig. 4) . Furthermore, PGF2{alpha}-induced activation of ERK1/2 is inhibited by 66%, 59%, and 63% in the presence of AL-8810, PGF2{alpha}-dimethyl amide, and PGF2{alpha}-dimethyl amine, respectively (Fig. 5) .



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  		FIGURE 4. Inhibition of ERK1/2 phosphorylation and ERK1/2 activity in the presence of PKC and MEK inhibitors. (A) Representative immunoblot of phosphorylated ERK1/2 from HCM cell lysates. Serum-deprived HCM cells were treated with vehicle or inhibitor (1 µM) for 30 minutes followed by incubation with the FP agonist (1 µM) for 5 minutes. Cell lysates (15 µg protein/lane) were analyzed for phospho-ERK1/2 by Western blot analysis using anti-phospho-ERK1/2 antibodies. (B) Representative immunoblot of total ERK1/2 from HCM cell lysates. (C) Data from a representative experiment of ERK1/2 activity. Cell lysates (15 µg protein/lane) were analyzed for ERK1/2 activity using an in-gel kinase assay. (D) Data are mean ± SE of densitometry measurements from in-gel kinase assays (n = 4). *Significant difference (P < 0.05) from agonist alone. Data were normalized using total ERK1/2 protein band intensities. Chel, chelerythrine chloride.

 


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  		FIGURE 5. Inhibition of PGF2{alpha}-induced ERK1/2 activity in the presence of FP antagonists. Serum-deprived HCM cells were treated with vehicle or FP antagonist (1 µM) for 30 minutes followed by 1 µM PGF2{alpha} treatment for 5 minutes. After incubation, cells were analyzed for ERK1/2 activity using an in-gel kinase assay. (A) Data are from a representative experiment for ERK1/2 activity. (B) Data are the mean ± SE of densitometry measurements from in-gel kinase assays (n = 3). *Significant difference (P < 0.05) from agonist alone.

 
To further confirm the involvement of PKC in PGF2{alpha} and latanoprost-acid-induced ERK1/2 activation, HCM cells were treated with phorbol esters (PDBu) for 16 hours. The PGF2{alpha} and latanoprost-acid-induced ERK1/2 activation was completely abolished when HCM cells were treated with 1 µM PDBu for 16 hours. However, total ERK1/2 protein levels were not affected by this treatment (Fig. 6) .



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  		FIGURE 6. PKC downregulation by prolonged PDBu treatment. (A) Data are from a representative experiment for ERK1/2 activity. Serum-deprived HCM cells were incubated with 1 µM PDBu or without for 16 hours followed by vehicle or FP agonist (1 µM) treatment for 5 minutes. Cell lysates (15 µg protein/lane) were analyzed for ERK1/2 activity using an in-gel kinase assay. (B) Representative immunoblot of total ERK1/2 from HCM cell lysates.

 

   Discussion
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 Abstract
 Methods
 Results
 Discussion
References
 
In the present study, we investigated signal transduction pathways involved in FP agonist–induced secretion of MMP-2 and ERK1/2 activation in HCM cells. Our results demonstrate that PGF2{alpha}-induced increased secretion of MMP-2 from HCM cells in a dose- and time-dependent fashion. Initial increases were measured as early as 2 hours; and this secretory response peaked 4 hours after PGF2{alpha} administration (Fig. 1) . The secretion of MMP-2 was significantly inhibited in the presence of the selective FP receptor antagonist AL-8810,28 suggesting that PGF2{alpha}-induced MMP-2 secretion is mediated through the activation of FP receptors in HCM cells (Fig. 2) . Moreover, treatment of HCM cells with PGF2{alpha} also increases the active form of MMP-2 as determined by zymography (Fig. 3) . A previous study using HCM cells was unable to identify a significant change in mRNA expression of MMP-2 after latanoprost acid treatment after 24 hours.29 Our study used 4 hours of treatment with PGF2{alpha}, and we noted substantial differences in the secretion of MMP-2. This early secretory event was not inhibited in the presence of 50 nM actinomycin D, indicating that PGF2{alpha}-induced MMP-2 secretion is not regulated at the transcriptional level. Unlike other MMPs, a study has shown that MMP-2 is often constitutively expressed and regulated at the level of secretion.30 Our data provide evidence that MMP-2 secretion is involved in the acute response of ciliary muscle to FP agonists. Several studies have reported that in trabecular meshwork cells the expression and secretion of MMPs in response to several different stimuli, including phorbol esters, growth factor, cytokines, and mechanical stress.15 18 20 31 Treatment periods during which these changes have been observed were generally 24 hours or more, indicating that MMPs may not be involved in the acute response to the agent that enhances conventional outflow facility. However, recent results in our laboratory have shown that cyclohexyladenosine (an adenosine A1 receptor agonist) induces secretion of MMP-2 from trabecular meshwork cells within 2 hours and is regulated primarily at the level of secretion.21 Hence, we hypothesize that activation of FP receptors would lead to a similar rapid secretion of MMP-2 from HCM cells.

PGs exert a broad range of physiological and pharmacological effects in a variety of tissues through interaction with specific cell surface G-protein-coupled receptors. FP receptor activation in HCM cells32 and human trabecular meshwork cells33 has been shown to result in the generation of second-messengers such as inositol-1,4,5-trisphosphates (IP3) and diacylglycerol (DAG). These second messengers eventually activate several protein kinases, including protein kinase C and mitogen-activated protein (MAP) kinase. The protein kinase C family, a serine-threonine kinase, has been shown to be involved in diverse cellular functions including differentiation, growth control, migration, paracellular permeability, smooth muscle contraction, cytoskeleton organization, and modulation of aqueous humor outflow.34 35 36 37 Mitogen-activated protein (MAP) kinases convey signals that regulate cell growth and differentiation, gene expression, protein synthesis, and secretion, activating several substrates located in the nucleus, the cytoplasm, and the membrane.38 39 The mammalian MAP kinase family is subdivided into three groups: the extracellular responsive kinases (extracellular signal-regulated kinase 1 and 2 or ERK1/2); the c-Jun N-terminal kinase (JNK/SAPK); and the p38 MAP kinase.

In ciliary muscle cells, PGF2{alpha} and other FP agonists have been shown to modulate cell function and stimulate MMP secretion.11 23 40 Recently, it has been shown that FP receptors in HCM cells are coupled to the activation of ERK1/2.32 In other systems, PGs have been shown to activate MAP kinase signaling pathways41 42 43 44 45 46 47 and the activation of these pathways modulates the secretion of MMP.48 49 50 51 Furthermore, ERK1/2 pathways have been shown to play a role in the regulation of MMP secretion from trabecular meshwork cells.52 However, the cellular event controlling MMP secretion remains poorly understood. In our initial studies to delineate the signaling events that are associated with PGF2{alpha}-induced MMP-2 secretion, HCM cells were treated with an FP agonist in the absence or presence of protein kinase C and MAP kinase pathway inhibitors. Our results demonstrate that PGF2{alpha}-induced secretion of MMP-2 was inhibited in the presence of PKC inhibitor chelerythrine chloride (Fig. 2) . We have not seen inhibition in PGF2{alpha}-induced MMP-2 secretion in the presence of Go-6976, a classic PKC isoform inhibitor, suggesting that PKC isoform(s) (other than {alpha}, ß, and {gamma}) are involved in PGF2{alpha}-induced MMP-2 secretion from HCM cells. Furthermore, activity and secretion of MMP-2 in response to PGF2{alpha} was completely inhibited by the pretreatment of cells with the MEK inhibitor, PD-98059 (Fig. 3) , demonstrating that PKC and ERK1/2 activation are involved in the secretion of MMP-2. In addition, administration of PGF2{alpha} and latanoprost acid to HCM cells produced a rapid increase in the activation and phosphorylation of ERK1/2 (Fig. 4) . The inhibition of PGF2{alpha}-induced ERK1/2 activation by FP receptor antagonists AL-8810, PGF2{alpha}-dimethyl amide, and PGF2{alpha}-dimethyl amine also demonstrate that these stimulatory responses are mediated through the activation of FP receptors (Fig. 5) . PGF2{alpha}-dimethyl amide, and PGF2{alpha}-dimethyl amine have been shown to act as FP receptor antagonists.53 54

To investigate the role of PKC in PGF2{alpha} and latanoprost-acid–induced ERK1/2 activation, experiments were performed using agents that either stimulate (e.g., PDBu) or inhibit PKC activity (e.g., chelerythrine chloride and calphostin C). The cellular response to phorbol esters is biphasic: the initial response involves translocation and activation of PKC; however, prolonged activation of the enzyme results in the downregulation of PKC. The PGF2{alpha}- and latanoprost-induced activation of ERK1/2 was inhibited in the presence of PKC inhibitors, whereas PDBu resulted in a robust increase in ERK1/2 activation and phosphorylation (Fig. 4) . These data suggest that PKC plays a central role in FP-agonist–induced activation of ERK1/2. Furthermore, our experiments evaluating PKC downregulation via long-term PDBu exposure confirmed that PGF2{alpha} and latanoprost-acid–induced activation of ERK1/2 requires activation of PKC (Fig. 6) .

The cellular mechanism of action of the ocular hypotensive prostaglandins PGF2{alpha} and latanoprost is believed to involve MMP secretion from ciliary muscle to promote uveoscleral outflow.40 Recent investigations have established that MMPs can decrease outflow resistance in uveoscleral outflow pathways. Our results demonstrate that FP receptor activation leads to the acute secretion of MMP-2, and this functional response is mediated by a PKC and ERK1/2 signaling pathway.


   Acknowledgements
 
The authors thank Luanna Bartholomew, PhD (Dept. of Ophthalmology, Medical University of South Carolina, Charleston, SC) for critical review of the manuscript.


   Footnotes
 
Supported in part by the American Health Assistance Foundation (SH), National Eye Institute Grants EY09741 (CEC) and EY14793, and an unrestricted grant to the Storm Eye Institute from Research to Prevent Blindness.

Submitted for publication August 17, 2004; revised November 10, 2004; accepted December 20, 2004.

Disclosure: S. Husain, None; F. Jafri, None; C.E. Crosson, 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: Shahid Husain, Storm Eye Institute, 167 Ashley Avenue, Charleston, SC 29425; husain{at}musc.edu.


   References
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 Abstract
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 Discussion
 References
 

  1. Bito LZ, Stjernschantz J, Resul B, Miranda OC, Basu S. The ocular effects of prostaglandins and the therapeutic potential of a new PGF2 alpha analog, PhXA41 (latanoprost), for glaucoma management. J Lipid Mediat. 1993;6:535–543.[Web of Science][Medline][Order article via Infotrieve]
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