May 2005
Volume 46, Issue 5
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Physiology and Pharmacology  |   May 2005
Acute Effects of PGF on MMP-2 Secretion from Human Ciliary Muscle Cells: A PKC- and ERK-Dependent Process
Author Affiliations
  • Shahid Husain
    From the Hewitt Laboratory of the Ola B. Williams Glaucoma Center, Department of Ophthalmology, and the
  • Farahdiba Jafri
    Department of Endocrinology, Medical University of South Carolina, Charleston, South Carolina.
  • Craig E. Crosson
    From the Hewitt Laboratory of the Ola B. Williams Glaucoma Center, Department of Ophthalmology, and the
Investigative Ophthalmology & Visual Science May 2005, Vol.46, 1706-1713. doi:10.1167/iovs.04-0993
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      Shahid Husain, Farahdiba Jafri, Craig E. Crosson; Acute Effects of PGF on MMP-2 Secretion from Human Ciliary Muscle Cells: A PKC- and ERK-Dependent Process. Invest. Ophthalmol. Vis. Sci. 2005;46(5):1706-1713. doi: 10.1167/iovs.04-0993.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

purpose. Studies were designed to evaluate the cellular mechanisms associated with prostaglandin (PG)F-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. PGF increased the secretion of MMP-2 in a dose-dependent manner with an EC50 of 2.7 × 10−8 M. The addition of 1 μM PGF 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 PGF 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 PGF and latanoprost acid increased ERK1/2 activity by 117% ± 12% and 75% ± 9%, respectively. The PGF- 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)F lowers intraocular pressure (IOP) in glaucomatous humans 1 and primates. 2 3 PGF analogues such as travoprost, 4 latanoprost, 5 6 and unoprostone isopropyl ester 7 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 muscle 11 12 and remodeling of the extracellular matrix (ECM) between the muscle bundles 13 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 PGF-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
Supplies
PGF, latanoprost acid, and 11β-fluoro-15-epi-indanyl PGF (AL-8810) were purchased from Cayman (Ann Arbor, MI); polyclonal anti-ERK1/2 antibodies from Upstate Biotechnology (Lake Placid, NY); [γ-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 β-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] β-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 β-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 β-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 [γ-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 β-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 PGF 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
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  
Effects of PGF on MMP-2 Secretion from HCM Cells
PGF produced a dose-dependent increase in MMP-2 (molecular mass, 72 kDa; pro-MMP-2) secretion with an EC50 of 2.7 × 10−8 M (Figs. 1A 1B) . The addition of 1 μM PGF 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 PGF-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 PGF-induced MMP-2 secretion by 85% (P < 0.05) from HCM cells (Fig. 2) . To determine whether PGF-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 PGF-induced secretion of MMP-2 (PGF 109% ± 17% vs. PGF+actinomycin D 118% ± 35% above control levels; n = 3). 
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 PGF 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 PGF (Fig. 2) . PGF-induced MMP-2 secretion was not inhibited by Go-6976, a classic PKC isoform inhibitor (PGF 99% ± 6% vs. PGF+Go-6976 105% ± 18% above control levels; n = 3). The PGF-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 PGF can influence PKC activity, HCM cells were treated with PGF for 5 minutes, followed by measurement of PKC activity in whole-cell lysates. PGF increases PKC activity by 74% ± 12% (n = 4; P < 0.05) above control levels in HCM cells. 
Zymographic analysis demonstrated that the addition of PGF 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 PGF and Latanoprost Acid on ERK1/2 Activation in HCM Cells
The addition of PGF (1 μM) or latanoprost acid (1 μM) for 5 minutes increased ERK1/2 activity by 117% ± 12% and 75% ± 9%, respectively. Moreover, the PGF 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 PGF-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 PGF and latanoprost acid treatment by Western blot techniques. The addition of PGF 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, PGF-induced activation of ERK1/2 is inhibited by 66%, 59%, and 63% in the presence of AL-8810, PGF-dimethyl amide, and PGF-dimethyl amine, respectively (Fig. 5)
To further confirm the involvement of PKC in PGF and latanoprost-acid-induced ERK1/2 activation, HCM cells were treated with phorbol esters (PDBu) for 16 hours. The PGF 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)
Discussion
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 PGF-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 PGF 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 PGF-induced MMP-2 secretion is mediated through the activation of FP receptors in HCM cells (Fig. 2) . Moreover, treatment of HCM cells with PGF 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 PGF, 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 PGF-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 cells 32 and human trabecular meshwork cells 33 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, PGF 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 pathways 41 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 PGF-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 PGF-induced secretion of MMP-2 was inhibited in the presence of PKC inhibitor chelerythrine chloride (Fig. 2) . We have not seen inhibition in PGF-induced MMP-2 secretion in the presence of Go-6976, a classic PKC isoform inhibitor, suggesting that PKC isoform(s) (other than α, β, and γ) are involved in PGF-induced MMP-2 secretion from HCM cells. Furthermore, activity and secretion of MMP-2 in response to PGF 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 PGF and latanoprost acid to HCM cells produced a rapid increase in the activation and phosphorylation of ERK1/2 (Fig. 4) . The inhibition of PGF-induced ERK1/2 activation by FP receptor antagonists AL-8810, PGF-dimethyl amide, and PGF-dimethyl amine also demonstrate that these stimulatory responses are mediated through the activation of FP receptors (Fig. 5) . PGF-dimethyl amide, and PGF-dimethyl amine have been shown to act as FP receptor antagonists. 53 54  
To investigate the role of PKC in PGF 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 PGF- 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 PGF and latanoprost-acid–induced activation of ERK1/2 requires activation of PKC (Fig. 6)
The cellular mechanism of action of the ocular hypotensive prostaglandins PGF 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. 
 
Table 1.
 
Summary of Normal Eyes from White Donors Used to Prepare Human Ciliary Muscle Cells
Table 1.
 
Summary of Normal Eyes from White Donors Used to Prepare Human Ciliary Muscle Cells
Donor Code Donor Age (y) Sex of Donor Time of Death to Isolation of Cells (h)
1 64 Male 28
2 70 Female 36
3 60 Male 39
4 72 Female 27
5 80 Female 24
Figure 1.
 
Dose–response (A, B) and time-course study (C, D) of MMP-2 secretion after treatment with PGF. Serum-deprived HCM cells were treated with vehicle or different concentrations of PGF for 4 hours for the dose–response study. The HCM cells were treated with 1 μM PGF 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).
Figure 1.
 
Dose–response (A, B) and time-course study (C, D) of MMP-2 secretion after treatment with PGF. Serum-deprived HCM cells were treated with vehicle or different concentrations of PGF for 4 hours for the dose–response study. The HCM cells were treated with 1 μM PGF 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).
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 PGF 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.
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 PGF 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.
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 PGF 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 PGF. 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).
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 PGF 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 PGF. 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).
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.
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.
Figure 5.
 
Inhibition of PGF-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 PGF 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.
Figure 5.
 
Inhibition of PGF-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 PGF 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.
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.
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.
The authors thank Luanna Bartholomew, PhD (Dept. of Ophthalmology, Medical University of South Carolina, Charleston, SC) for critical review of the manuscript. 
BitoLZ, StjernschantzJ, ResulB, MirandaOC, BasuS. 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. [PubMed]
BitoLZ. Prostaglandins: a new approach to glaucoma management with a new, intriguing side effect. Surv Ophthalmol. 1997;41(suppl 2)S1–S14. [CrossRef]
WangRF, CamrasCB, LeePY, PodosSM, BitoLZ. Effects of prostaglandins F2 alpha, A2, and their esters in glaucomatous monkey eyes. Invest Ophthalmol Vis Sci. 1990;31:2466–2470. [PubMed]
HellbergMR, SalleeVL, McLaughlinMA, et al. Preclinical efficacy of travoprost, a potent and selective FP prostaglandin receptor agonist. J Ocul Pharmacol Ther. 2001;17:421–432. [CrossRef] [PubMed]
AlmA, StjernschantzJ. Effects on intraocular pressure and side effects of 0.005% latanoprost applied once daily, evening or morning: a comparison with timolol. Scandinavian Latanoprost Study Group. Ophthalmology. 1995;102:1743–1752. [CrossRef] [PubMed]
StjernschantzJ, SelenG, SjoquistB, ResulB. Preclinical pharmacology of latanoprost, a phenyl-substituted PGF2 alpha analogue. Adv Prostaglandin Thromboxane Leukot Res. 1995;23:513–518. [PubMed]
TaniguchiT, HaqueMS, SugiyamaK, HoriN, KitazawaY. Ocular hypotensive mechanism of topical isopropyl unoprostone, a novel prostaglandin metabolite-related drug, in rabbits. J Ocul Pharmacol Ther. 1996;12:489–498. [CrossRef] [PubMed]
GabeltBT, KaufmanPL. Prostaglandin F2 alpha increases uveoscleral outflow in the cynomolgus monkey. Exp Eye Res. 1989;49:389–402. [CrossRef] [PubMed]
CrawfordK, KaufmanPL. Pilocarpine antagonizes prostaglandin F2 alpha-induced ocular hypotension in monkeys: evidence for enhancement of uveoscleral outflow by prostaglandin F2 alpha. Arch Ophthalmol. 1987;105:1112–1116. [CrossRef] [PubMed]
NilssonSF, SamuelssonM, BillA, StjernschantzJ. Increased uveoscleral outflow as a possible mechanism of ocular hypotension caused by prostaglandin F2 alpha-1-isopropylester in the cynomolgus monkey. Exp Eye Res. 1989;48:707–716. [CrossRef] [PubMed]
PoyerJF, MillarC, KaufmanPL. Prostaglandin F2 alpha effects on isolated rhesus monkey ciliary muscle. Invest Ophthalmol Vis Sci. 1995;36:2461–2465. [PubMed]
Van AlphenGW, KernR, RobinetteSL. Adrenergic receptors of the intraocular muscles: comparison to cat, rabbit and monkey. Arch Ophthalmol. 1965;74:253–259. [CrossRef] [PubMed]
LindseyJD, KashiwagiK, KashiwagiF, WeinrebRN. Prostaglandin action on ciliary smooth muscle extracellular matrix metabolism: implications for uveoscleral outflow. Surv Ophthalmol. 1997;41:S53–S59. [CrossRef] [PubMed]
Lütjen-DrecollE, TammE. Morphological study of the anterior segment of cynomolgus monkey eyes following treatment with prostaglandin F2 alpha. Exp Eye Res. 1988;47:761–769. [CrossRef] [PubMed]
AlexanderJP, SamplesJR, Van BuskirkEM, AcottTS. Expression of matrix metalloproteinases and inhibitor by human trabecular meshwork. Invest Ophthalmol Vis Sci. 1991;32:172–180. [PubMed]
Birkedal-HansenH, MooreWG, BoddenMK, et al. Matrix metalloproteinases: a review. Crit Rev Oral Biol Med. 1993;4:197–250. [PubMed]
WoessnerJF., Jr. Matrix metalloproteinases and their inhibitors in connective tissue remodeling. FASEB J. 1991;5:2145–2154. [PubMed]
BradleyJM, KelleyMJ, ZhuX, AnderssohnAM, AlexanderJP, AcottTS. Effects of mechanical stretching on trabecular matrix metalloproteinases. Invest Ophthalmol Vis Sci. 2001;42:1505–1513. [PubMed]
BradleyJM, VrankaJ, ColvisCM, et al. Effect of matrix metalloproteinases activity on outflow in perfused human organ culture. Invest Ophthalmol Vis Sci. 1998;39:2649–2658. [PubMed]
AlexanderJP, SamplesJR, AcottTS. Growth factor and cytokine modulation of trabecular meshwork matrix metalloproteinase and TIMP expression. Curr Eye Res. 1998;17:276–285. [CrossRef] [PubMed]
ShearerTW, CrossonCE. Adenosine A1 receptor modulation of MMP-2 secretion by trabecular meshwork cells. Invest Ophthalmol Vis Sci. 2002;43:3016–3020. [PubMed]
SagaraT, GatonDD, LindseyJD, GabeltBT, KaufmanPL, WeinrebRN. Reduction of collagen type I in the ciliary muscle of inflamed monkey eyes. Invest Ophthalmol Vis Sci. 1999;40:2568–2576. [PubMed]
LindseyJD, KashiwagiK, BoyleD, KashiwagiF, FiresteinGS, WeinrebRN. Prostaglandins increase proMMP-1 and proMMP-3 secretion by human ciliary smooth muscle cells. Curr Eye Res. 1996;15:869–875. [CrossRef] [PubMed]
LindseyJD, KashiwagiK, KashiwagiF, WeinrebRN. Prostaglandins alter extracellular matrix adjacent to human ciliary muscle cells in vitro. Invest Ophthalmol Vis Sci. 1997;38:2214–2223. [PubMed]
HusainS, Kaddour-DjebbarI, Abdel-LatifAA. Alterations in arachidonic acid release and phospholipase C-beta(1) expression in glaucomatous human ciliary muscle cells. Invest Ophthalmol Vis Sci. 2002;43:1127–1134. [PubMed]
HusainS, Abdel-LatifAA. Effects of PGF2alpha and carbachol on MAP kinase, cytosolic phospholipase A2 and arachidonic acid release in cat iris sphincter smooth muscle cells. Exp Eye Res. 2001;72:581–590. [CrossRef] [PubMed]
TammE, FlugelC, BaurA, Lütjen-DrecollE. Cell cultures of human ciliary muscle: growth, ultrastructural and immunocytochemical characteristics. Exp Eye Res. 1991;53:375–387. [CrossRef] [PubMed]
GriffinBW, KlimkoP, CriderJY, SharifNA. AL-8810: a novel prostaglandin F2 alpha analog with selective antagonist effects at the prostaglandin F2 alpha (FP) receptor. J Pharmacol Exp Ther. 1999;290:1278–1284. [PubMed]
WeinrebRN, LindseyJD. Metalloproteinase gene transcription in human ciliary muscle cells with latanoprost. Invest Ophthalmol Vis Sci. 2002;43:716–722. [PubMed]
SternlichtMD, WerbZ. How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol. 2001;17:463–516. [CrossRef] [PubMed]
SamplesJR, AlexanderJP, AcottTS. Regulation of the levels of human trabecular matrix metalloproteinases and inhibitor by interleukin-1 and dexamethasone. Invest Ophthalmol Vis Sci. 1993;34:3386–3395. [PubMed]
SharifNA, CriderJY, HusainS, Kaddour-DjebbarI, AnsariHR, Abdel-LatifAA. Human ciliary muscle cell responses to FP-class prostaglandin analogs: phosphoinositide hydrolysis, intracellular Ca(2+) mobilization and MAP kinase activation. J Ocul Pharmacol Ther. 2003;19:437–455. [CrossRef] [PubMed]
SharifNA, KellyCR, CriderJY. Human trabecular meshwork cell responses induced by bimatoprost, travoprost, unoprostone, and other FP prostaglandin receptor agonist analogues. Invest Ophthalmol Vis Sci. 2003;44:715–721. [CrossRef] [PubMed]
KhuranaN, DengPF, EpsteinDL, VasanthaRP. The role of protein kinase C in modulation of aqueous humor outflow facility. Exp Eye Res. 2003;76:39–47. [CrossRef] [PubMed]
KhalilRA, MorganKG. PKC-mediated redistribution of mitogen-activated protein kinase during smooth muscle cell activation. Am J Physiol. 1993;265:C406–C411. [PubMed]
QuestAF. Regulation of protein kinase C: a tale of lipids and proteins. Enzyme Protein. 1996;49:231–261. [PubMed]
ThiemeH, NassJU, NuskovskiM, et al. The effects of protein kinase C on trabecular meshwork and ciliary muscle contractility. Invest Ophthalmol Vis Sci. 1999;40:3254–3261. [PubMed]
MarshallCJ. MAP kinase kinase kinase, MAP kinase kinase and MAP kinase. Curr Opin Genet Dev. 1994;4:82–89. [CrossRef] [PubMed]
MarshallCJ. Specificity of receptor tyrosine kinase signaling: transient versus sustained extracellular signal-regulated kinase activation. Cell. 1995;80:179–185. [CrossRef] [PubMed]
WeinrebRN, KashiwagiK, KashiwagiF, TsukaharaS, LindseyJD. Prostaglandins increase matrix metalloproteinase release from human ciliary smooth muscle cells. Invest Ophthalmol Vis Sci. 1997;38:2772–2780. [PubMed]
MelienO, ThoresenGH, SandnesD, OstbyE, ChristoffersenT. Activation of p42/p44 mitogen-activated protein kinase by angiotensin II, vasopressin, norepinephrine, and prostaglandin F2alpha in hepatocytes is sustained, and like the effect of epidermal growth factor, mediated through pertussis toxin-sensitive mechanisms. J Cell Physiol. 1998;175:348–358. [CrossRef] [PubMed]
MigginSM, KinsellaBT. Thromboxane A(2) receptor mediated activation of the mitogen activated protein kinase cascades in human uterine smooth muscle cells. Biochim Biophys Acta. 2001;1539:147–162. [CrossRef] [PubMed]
MilneSA, JabbourHN. Prostaglandin (PG) F(2alpha) receptor expression and signaling in human endometrium: role of PGF(2alpha) in epithelial cell proliferation. J Clin Endocrinol Metab. 2003;88:1825–1832. [CrossRef] [PubMed]
MinuzP, GainoS, ZulianiV, et al. Functional role of p38 mitogen activated protein kinase in platelet activation induced by a thromboxane A2 analogue and by 8-iso-prostaglandin F2alpha. Thromb Haemost. 2002;87:888–898. [PubMed]
NilssenLS, HegeTG, ChristoffersenT, SandnesD. Differential role of MAP kinases in stimulation of hepatocyte growth by EGF and G-protein-coupled receptor agonists. Biochem Biophys Res Commun. 2002;291:588–592. [CrossRef] [PubMed]
OhmichiM, KoikeK, KimuraA, et al. Role of mitogen-activated protein kinase pathway in prostaglandin F2alpha-induced rat puerperal uterine contraction. Endocrinology. 1997;138:3103–3111. [PubMed]
StoccoCO, LauLF, GiboriG. A calcium/calmodulin-dependent activation of ERK1/2 mediates JunD phosphorylation and induction of nur77 and 20alpha-hsd genes by prostaglandin F2alpha in ovarian cells. J Biol Chem. 2002;277:3293–3302. [CrossRef] [PubMed]
GumR, WangH, LengyelE, JuarezJ, BoydD. Regulation of 92 kDa type IV collagenase expression by the jun amino terminal kinase- and the extracellular signal-regulated kinase-dependent signaling cascades. Oncogene. 1997;14:481–493. [CrossRef] [PubMed]
KurataH, ThantAA, MatsuoS, et al. Constitutive activation of MAP kinase kinase (MEK1) is critical and sufficient for the activation of MMP-2. Exp Cell Res. 2000;254:180–188. [CrossRef] [PubMed]
SudbeckBD, BaumannP, RyanGJ, et al. Selective loss of PMA-stimulated expression of matrix metalloproteinase 1 in HaCaT keratinocytes is correlated with the inability to induce mitogen-activated protein family kinases. Biochem J. 1999;339:167–175. [CrossRef] [PubMed]
ThantAA, SeinTT, LiuE, et al. Ras pathway is required for the activation of MMP-2 secretion and for the invasion of src-transformed 3Y1. Oncogene. 1999;18:6555–6563. [CrossRef] [PubMed]
ShearerT, CrossonCE. Activation of extracellular signal-regulated kinase in trabecular meshwork cells. Exp Eye Res. 2001;73:25–35. [CrossRef] [PubMed]
MaddoxYT, RamwellPW, ShinerCS, CoreyEJ. Amide and 1-amino derivatives of F prostaglandins as prostaglandin antagonists. Nature. 1978;273:549–552. [CrossRef] [PubMed]
StingerRB, FitzpatrickTM, CoreyEJ, RamwellPW, RoseJC, KotPA. Selective antagonism of prostaglandin F2 alpha-mediated vascular responses by N-dimethylamino substitution of prostaglandin F2 alpha. J Pharmacol Exp Ther. 1982;220:521–525. [PubMed]
Figure 1.
 
Dose–response (A, B) and time-course study (C, D) of MMP-2 secretion after treatment with PGF. Serum-deprived HCM cells were treated with vehicle or different concentrations of PGF for 4 hours for the dose–response study. The HCM cells were treated with 1 μM PGF 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).
Figure 1.
 
Dose–response (A, B) and time-course study (C, D) of MMP-2 secretion after treatment with PGF. Serum-deprived HCM cells were treated with vehicle or different concentrations of PGF for 4 hours for the dose–response study. The HCM cells were treated with 1 μM PGF 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).
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 PGF 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.
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 PGF 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.
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 PGF 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 PGF. 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).
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 PGF 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 PGF. 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).
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.
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.
Figure 5.
 
Inhibition of PGF-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 PGF 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.
Figure 5.
 
Inhibition of PGF-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 PGF 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.
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.
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.
Table 1.
 
Summary of Normal Eyes from White Donors Used to Prepare Human Ciliary Muscle Cells
Table 1.
 
Summary of Normal Eyes from White Donors Used to Prepare Human Ciliary Muscle Cells
Donor Code Donor Age (y) Sex of Donor Time of Death to Isolation of Cells (h)
1 64 Male 28
2 70 Female 36
3 60 Male 39
4 72 Female 27
5 80 Female 24
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