March 2002
Volume 43, Issue 3
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Glaucoma  |   March 2002
Metalloproteinase Gene Transcription in Human Ciliary Muscle Cells with Latanoprost
Author Affiliations
  • Robert N. Weinreb
    From the Glaucoma Center, University of California San Diego, La Jolla, California.
  • James D. Lindsey
    From the Glaucoma Center, University of California San Diego, La Jolla, California.
Investigative Ophthalmology & Visual Science March 2002, Vol.43, 716-722. doi:https://doi.org/
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      Robert N. Weinreb, James D. Lindsey; Metalloproteinase Gene Transcription in Human Ciliary Muscle Cells with Latanoprost. Invest. Ophthalmol. Vis. Sci. 2002;43(3):716-722. doi: https://doi.org/.

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

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Abstract

purpose. The present study was undertaken to determine whether treatment of ciliary muscle cells with the prostaglandin (PG) analogue latanoprost acid alters transcription of mRNA for matrix metalloproteinase (MMP)-1, -2, -3, and -9.

methods. Human ciliary smooth muscle cell cultures were grown to confluence and treated for 24 hours with medium supplemented with latanoprost acid or vehicle. Total RNA was then isolated, and the expression of mRNAs for MMP-1, -2, -3, and -9 were determined using Taqman and energy-transfer real-time PCR analyses. All results were normalized according to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA in each sample.

results. Specificity and linearity of each real-time PCR assay were confirmed by electrophoresis and serial dilution analysis of oligonucleotides containing the amplicon sequence. Addition of latanoprost acid for 24 hours increased expression of MMP-1 by 3- to 13-fold in three of five primary ciliary muscle lines. Addition of 8, 40, and 200 nM latanoprost acid for 24 hours increased MMP-1 mRNA in a dose-dependent manner. Analysis of cultures exposed to 200 nM latanoprost acid for 4, 6, 12, or 24 hours revealed an increase and then a decline of MMP-1 mRNA, with peak expression at 6 to 12 hours after initiation of treatment. Parallel assessments of RNA from ciliary muscle cultures exposed to latanoprost acid for 24 hours revealed increased MMP-1, -3, and -9 mRNAs and reduced MMP-2 mRNA, when compared with RNA from vehicle-treated cultures.

conclusions. Latanoprost acid induced a dose-dependent increase of MMP-1, -3, and -9 gene transcription in cultured human ciliary smooth muscle cells. These results are consistent with increased MMPs contributing to the increased uveoscleral outflow facility observed after topical latanoprost.

Topical treatment of cynomolgus monkeys with prostaglandin (PG)-F and of humans with latanoprost, a PG analogue, lowers intraocular pressure and increases uveoscleral outflow facility. 1 2 Extracellular spaces among ciliary muscle fiber bundles that also contain extracellular matrix molecules, such as collagens, are a major component of the uveoscleral outflow pathway. 3 4 5 6 The PGF-induced increase in uveoscleral outflow facility has been associated with several changes in ciliary muscle, including expansion of the extracellular spaces, 7 reduction of collagens, 8 9 and increased matrix metalloproteinases (MMPs). 10 11 12 Reduced collagens and increased MMPs in ciliary muscle also have been noted after treatment with latanoprost. 11 13 These studies support an association between PG-mediated lowering of IOP and increased MMP expression. 
MMPs are neutral proteinases that specifically degrade extracellular matrix molecules. 14 15 They can initiate degradation of collagen and other extracellular matrix components within ciliary muscle extracellular matrix. 16 After such cleavage, the resultant collagen fragments unwind at body temperature 17 and are then further degraded by MMPs and nonspecific extracellular proteases or by lysosomal enzymes after phagocytosis of the fragments. 18 We have hypothesized previously that increased MMPs plays a pivotal role in the reduction of extracellular matrix in the ciliary muscle after PG treatment and the concomitant increase in uveoscleral outflow. 19 Support for another possible role of MMPs, in normal regulation of uveoscleral outflow, comes from the observation of measurable MMP-1, -2, and -3 immunoreactivity in the iris, the ciliary muscle, choroid, and sclera of untreated normal human and primate eyes. 20 21 22 However, little is known about how PGs, or PG analogues such as latanoprost influence MMP gene transcription. 
Previous studies in various cell types have found that increases in MMP secretion in tissues typically are preceded by changes in corresponding MMP gene transcription. 23 24 However, this is not always the case. 25 26 The present study was therefore undertaken to characterize the induction of MMP-1, -2, -3, and -9 mRNAs in cultures of human ciliary smooth muscle cells after exposure to latanoprost acid, the biologically active form of latanoprost. 
Methods
Cell Cultures
Eyes from eight individuals ranging from 56 to 89 years of age were enucleated within 6 hours after death and stored at 4°C for less than 24 hours. Ciliary muscle cells were cultured from these eyes, as previously described. 27 28 Briefly, the globe was bisected between the ora serrata and equator. The anterior segment was placed in a dish with the corneal epithelium down. Under a dissecting microscope, the lens was removed and the iris disinserted. The ciliary body was gently removed from the sclera and placed in a sterile dish. Against the dark background of the pigmented epithelium, the muscle was easily identified as a broad, pale, circular band. The outermost portion of the muscle was dissected. Strips of the muscle were explanted into 35-mm culture dishes (Falcon, Lincoln Park, NJ) filled with Dulbecco’s modified Eagle’s medium and Ham’s F12 nutrient mixture (DMEM-F12; Gibco BRL, Grand Island, NY) supplemented with 10% fetal bovine serum (J. R. Scientific, Woodland, CA) and 1 ng/mL recombinant human basic fibroblast growth factor (R&D Systems, Inc., Minneapolis, MN). The cultures were incubated in a humidified atmosphere of 95% air, 5% CO2. The medium was changed every 3 to 4 days. Primary explant cultures reached confluence within 30 days. These and subsequent confluent cultures were trypsinized and subcultured at a ratio of 1:3. The subsequent passage cultures reached confluence within 7 to 10 days. Cells in these cultures express smooth muscle actin and a moderate amount of desmin, similar to ciliary smooth muscle cells in vivo. 27 In addition, electron microscopic evaluation of the cultures in the same study revealed parallel microfilament bundles in the cultured cells similar to those found in ciliary muscle cells in vivo. The ciliary muscle cells to be experimentally analyzed were plated into T-75 culture flasks and allowed to grow to confluence. Once confluent, the cultures were maintained for an additional week to maximize their differentiation. 
To minimize dedifferentiation that can occur in high-passage primary cell lines, cultures were used in the present experiments that had been passaged five to seven times. Because the number of low-passage cells that can be generated from the ciliary muscle of a single pair of human donor eyes is limited, each experiment with a particular cell line was performed once. 
Experimental Treatments of the Cultures
Stock solutions containing 10 mM latanoprost acid (Cayman Chemical Co., Ann Arbor, MI) were prepared in ethanol and diluted to appropriate test concentrations with DMEM/F12 nutrient mixture. The vehicle control was DMEM/F12 nutrient mixture containing 0.1% ethanol (the same concentration of ethanol as was present in the highest agonist concentration experimental test media). 
To directly assess the role of MMP transcription changes in the ocular response to latanoprost, the ciliary smooth muscle cells were exposed to latanoprost acid. Test concentrations were 8, 40, 200, and 1000 nM. Previous investigation had shown that the average peak concentration of latanoprost in human anterior chamber aqueous after a standard clinical dose is approximately 100 nM, although concentrations as high as 200 nM have been observed (Sjostrand B, personal communication, May 2000). Therefore, the test concentrations encompassed the pharmacological range observed in aqueous humor with clinical doses. Treatments were initiated by exposing cultures to the test media and terminated by addition of the lysis buffer for RNA harvesting. 
Reverse Transcriptase Reaction Conditions
Total RNA was harvested by using acid guanidine phenol chloroform extraction, as described previously. 29 30 To assess the accuracy of the procedures, triplicate aliquots of total RNA were used to produce cDNA in parallel reactions. First-strand cDNA was synthesized using RNase H reverse transcriptase purified form Escherichia coli containing the pol gene of Moloney murine leukemia virus (Superscript II; Gibco BRL). The 20 μL reaction volume contained 1 to 5 μg total RNA, 0.5 μg oligo (dT), 50 mM Tris (pH 8.3 at room temperature), 75 mM KCl, 3 mM MgCl2, 0.01 M dithiothreitol, 0.5 mM dNTPs, and 200 U reverse transcriptase. The reaction mixture was incubated at 42°C for 50 minutes and terminated by incubation at 70°C for 15 minutes. 
Taqman Real-Time PCR
MMP-1 gene transcription in the treated ciliary muscle cells was measured using a real-time polymerase chain reaction (PCR) system (Taqman Real-Time PCR; PE-Applied Biosystems, Inc., Foster City, CA). 31 The protocol was exactly as described previously, 30 except that the primers and probe were specific for MMP-1. Briefly, the probe, which contains both a fluorophore and a quencher on opposite ends, was designed to bind specifically to a region of the MMP-1 sequence between the primers. During each PCR cycle, the bound probe was digested by the 5′-nuclease activity of Taqman polymerase as the primers were extended. The amount of fluorophore that remained unquenched during each cycle was assessed by illuminating the reaction mixture with a laser beam and measuring the resultant fluorescence. Expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the same specimens also was measured and used to normalize the MMP-1 results. Primers and probe for MMP-1 and GAPDH are shown in Table 1
Energy-Transfer Real-Time PCR
MMP-1, -2, -3, and -9 mRNAs were measured using a modification of the Taqman real-time PCR method referred to as molecular energy-transfer real-time PCR. With this method (Amplifluor Universal Detection System; Intergen Co., Purchase, NY), a hairpin configuration oligonucleotide containing a fluorophore and quencher becomes incorporated into the specific PCR product produced with each cycle. 32 Similar to the Taqman real-time PCR method, the amount of fluorescence present in each reaction was monitored during each PCR cycle. Although in both cases, the signal arose from separation of a quencher and a fluorophore, separation in the present method occurred by opening up the hairpin rather than from digestion of the probe. With each cycle, the signal increases and then plateaus the same as in Taqman real-time PCR. Therefore, analysis of the results determined the threshold cycle (CT), in the same way as described earlier. 
Reaction differences include the addition of a short oligonucleotide sequence, 5′-ACTGAACCTGACCGTACA-3′, to the 5′ end of the forward primer, inclusion of a hairpin configuration oligonucleotide primer containing the quenched fluorophore (Amplifluor Uniprimer; Intergen Co.), and elimination of the specific probe used in Taqman real-time PCR. The primers used for measuring the various MMP mRNAs are shown in Table 2 . Each determination of specific MMP mRNA amount was normalized according to GAPDH mRNA also present in each sample by using Taqman real-time PCR primers and probe. 
Specificity
The specificity of each assay was confirmed with PCR reaction products generated using the cDNA from latanoprost acid–treated human ciliary muscle cells. For each assay, 15 μL of the reaction mixture containing PCR products from amplified reaction plates was separated by electrophoresis in an 8% polyacrylamide gel (Novex/Invitrogen, Carlsbad, CA) for the Taqman real-time PCR products and in a 4% agarose gel (NuSeive; Cambrex Corp., East Rutherford, NJ) for the energy-transfer real-time PCR products. The running buffer contained 89 mM Tris base, 89 mM boric acid, and 2 mM EDTA (pH 8.3; TBE buffer). Each gel included 100- and 25-bp ladder standards (Promega, Madison, WI). No-template control gels contained probes and primer (in the case of the Taqman real-time PCR samples), and PCR reagents were present but cDNA template was not added. Experimental assays contained probes, primer, PCR reagents, and the MMP-1 amplicon oligonucleotide or OCM1 cDNA. The no-template control gels were used to evaluate the possibility of nonspecific amplification of primer or probe sequences. The gels were developed using ethidium bromide and photographed on a light box with 360-nm excitation (Transilluminator 4000; Stratagene, La Jolla, CA). 
Standard Curves
To evaluate linearity of the Taqman assay, a standard curve was produced using a synthetic oligonucleotide generated according to the sequence of the MMP-1 amplicon defined by the corresponding primers. The concentration of RNA was determined by measuring optical density (OD) at 260 nm and comparing this against an OD curve generated by a serial dilution of purified calf liver RNA (Sigma, St. Louis, MO). A serial dilution of the oligonucleotide was generated extending from 50 to 0.78 ng, and each dilution was assayed by Taqman real-time PCR to determine CT. For the energy-transfer real-time PCR assays, standard curves for MMP-1, -2, -3, and -9 and GAPDH were generated using RNA from an MMP-secreting human melanoma cell line (OCM1). A serial dilution of this RNA was made, and the CT for MMP-1, -2, -3, and -9 in each dilution was determined using the corresponding primers (Amplifluor Uniprimers; Intergen Co.). The CT for GAPDH in each of the OCM1 RNA dilutions also was assessed using Taqman real-time PCR. 
Analysis
The mean ± SD of the measurements for each RNA sample was determined to evaluate the reproducibility of the measurements. 
Results
Specificity of the Taqman and Energy-Transfer PCR Assays
To assess that the origin of the signal-reflected amplification of the target cDNA, reaction mixtures from a completed real-time PCR run were separated by electrophoresis and then stained using ethidium bromide. The product of no-template controls amplified using the Taqman MMP-1 assay contained a single band with mobility corresponding to approximately 35 bp (Fig. 1A) . This corresponded to the Taqman real-time PCR probe. The product of the complete Taqman real-time PCR reaction contained a single band with mobility corresponding to approximately 71 bp. This is consistent with specific amplification of MMP-1 cDNA only. In the case of the energy-transfer assays, no band was observed in the no-template control lanes and a single band of appropriate size was observed in the complete reactions for each of the four MMP mRNAs analyzed (Fig. 1B)
Standard Curves
To calibrate experimental measurements, standard curves for MMP were generated, using serial dilution of the amplicon oligonucleotides. As shown in Figure 2A , the relationship between CT and the log of concentration in the Taqman real-time PCR reaction was linear over the range of 0.1 to 30 pg of oligonucleotide (R 2 = 0.99). GAPDH amplification was shown to be linear in a previous study. 30 Linear relationships also were observed in the energy-transfer real-time PCR reactions (Fig. 2B) . Formulas for these linear relationships were determined and used to calculate the magnitude of the specific mRNA induction in the treated cultures from the obtained CT values. 
Induction of MMP-1 mRNA Expression by Latanoprost Acid
Confluent ciliary muscle cultures were exposed to 200 nM latanoprost acid for 24 hours. RNA was isolated and the amount of MMP-1 mRNA measured by Taqman real-time PCR. All results were normalized according to expression of GAPDH in the same sample and compared with expression of MMP-1 mRNA in parallel cultures exposed to vehicle control. As shown in Figure 3 , latanoprost acid increased MMP-1 mRNA greater than twofold in three of the five culture lines established from different donors. 
Dose-Response Analysis
Exposure of parallel cultures to increasing concentrations of latanoprost acid induced increased MMP-1 mRNA in a dose-dependent manner (Fig. 4A) . Treatment with concentrations of latanoprost acid of 1000 nM or more usually did not result in any further increase in MMP-1 mRNA than was observed at 200 nM. Repeated experiments with various cell lines also resulted in dose-dependent increases in MMP-1 mRNA (Fig. 4B)
Time Course of MMP-1 mRNA Induction by Latanoprost Acid
Ciliary muscle cultures were exposed to 200 nM latanoprost acid for 4, 6, 12, or 24 hours, and RNA was isolated for analysis by Taqman real-time PCR. Increased MMP-1 mRNA typically was transient, with peak expression observed at either 6 or at 12 hours after initiation of treatment (Fig. 5) . By 24 hours, MMP-1 mRNA expression usually was less than peak but remained higher than expression in parallel vehicle-treated cultures. 
Energy-Transfer Real-Time PCR Analysis of MMP-1, -2, -3, and -9 Induction by Latanoprost Acid
Ciliary muscle cultures generated from five eyes of different donors were exposed to 50, 200, 500, and 1000 nM latanoprost acid or to vehicle control for 24 hours and the RNA harvested. Parallel aliquots of this RNA were analyzed to determine MMP-1, -2, -3, and -9 mRNA content, using energy-transfer real-time PCR. As shown in Figure 6A , exposure of ciliary muscle cell cultures to latanoprost acid increased expression of MMP-1 mRNA in all five primary cell lines examined. In some cases, this increase was biphasic, first increasing and then diminishing with increased concentration. In other cases it was linear, continuously increasing with increased concentration. 
MMP-3 mRNA expression was increased in the ciliary muscle cultures from three of the five donors and unchanged in the other two after exposure to latanoprost acid (Fig. 6B) . This increase was biphasic in two of the responding culture lines and increased linearly in the other responding culture line. 
MMP-9 mRNA expression was increased in the ciliary muscle cultures from four of the five donors and unchanged in the remaining one after exposure to latanoprost acid (Fig. 6C) . This increase was biphasic in two of the responding cultures and increased linearly in the other two responding cultures. 
In contrast, to MMP-1, -3, and -9, MMP-2 mRNA expression was reduced in the ciliary muscle cultures from three of the five donors and unchanged in the other two after exposure to latanoprost acid (Fig. 6D) . In each responding culture line, the overall trend was greater reduction with increasing latanoprost acid concentration. 
Discussion
Exposure of human ciliary muscle cells to latanoprost acid increased transcription of MMP-1, -3, and -9 mRNA. This response was variable among ciliary muscle cell lines generated from different donor eyes. Increased MMP-1 expression typically was dose dependent within the range of concentrations previously found in aqueous humor after topical latanoprost treatment. Significant MMP-1 mRNA induction was observed in many of the cultures by 6 hours after treatment was initiated. In contrast to MMP-1, -3, and -9 mRNAs, the expression of MMP-2 mRNA was either reduced or unchanged in ciliary muscle cultures. In responding cultures, this reduction was generally related to the concentration of latanoprost acid. 
The time course of MMP-1 mRNA induction was consistent with its accounting for the increased MMP-1 secretion observed after exposure of ciliary muscle cells to latanoprost acid. Zymographic analysis found no MMP-1 protein difference in the medium from ciliary muscle cells treated with latanoprost acid for 12 hours or the medium from vehicle-treated cultures. 11 However, marked increases were observed after treatment for 24 or 72 hours. In the present study, increased MMP-1 mRNA was observed at 6 or 12 hours after exposure to latanoprost acid. Thus, the induction of MMP-1 mRNA preceded the increase in MMP-1 protein secretion. 
Although increases in MMP-1, -3, and -9 mRNAs were seen in most of the cultures treated overnight with 200 nM latanoprost acid, there was a portion of cell lines in which there was no detectable response. This was probably not the result of choosing GAPDH to serve as a reference for mRNA loading in the real-time PCR measurements. In previous experiments that directly compared the amount of cardiac myocyte mRNA loaded in real-time PCR measurements, the investigators observed that the ratio of GAPDH mRNA to total mRNA was the same in cells exposed to control medium or to 1 μM PGF, a treatment known to induce myocyte hypertrophy. 33 At the same time, transcription of genes for c-Fos and atrial natriuretic factor in these cells were increased 35-fold and 800-fold, respectively. However, a doubling of total mRNA per cell in the treated cultures was noted. If an increase in total mRNA per cell occurred in the present cultures, it would have resulted in an underestimation of transcription induction by latanoprost acid. Unlike cardiac myocytes, however, no change was noted in the appearance or survival of cultured human ciliary smooth muscle cells exposed to 10 μM PGF, a concentration 10 times higher than that used in the present study. 34 Therefore, the present increases in mRNA measurements relative to GAPDH mRNA in the treated cultures is likely either to directly reflect or to underestimate the increase in MMP mRNA copies per cell. 
The observation that MMP-1, -3, and -9 mRNAs increased, whereas MMP-2 mRNA decreased in latanoprost acid-treated ciliary muscle cells suggests there is a different mechanism of gene regulation for MMP-2 than for MMP-1, -3, and -9 in these cells. The basis of this difference may be due in part to the presence of several regulatory element types in the MMP-1, -3, and -9 promoters that are absent from the MMP-2 promoter. 24 35 The physiological significance of this promoter difference is supported by the increased MMP-1, -3, and -9 secretion and decreased MMP-2 secretion by corneal fibroblasts exposed to the AP-1 regulatory element activator phorbol myristate acetate. Similar results were observed in human cervical smooth muscle cells exposed to TNFα, 36 a cytokine that also activates AP-1. 37 Therefore, it is plausible that these promoter differences are important for the differences in MMP mRNA induction. 
The significance of the differential MMP-2 gene regulation to MMP-2 protein secretion may be different in ciliary muscle cells than in corneal fibroblasts. Zymographic analysis of medium from human ciliary muscle cell cultures exposed to various PGs, including PG F and latanoprost acid, have found increased secretion of MMP-1, -3, and -9, as well as MMP-2. 11 13 Stimulated increases in MMP-2 secretion that are not accompanied by increases in MMP-2 mRNA have been demonstrated in macrophages and vascular tumor cells. 25 26 In the latter case, it was also shown that this secreted MMP-2 originated from intracellular stores. Further experiments are needed to determine whether this applies to ciliary muscle cells or whether MMP-2 mRNA induction by PGs is merely delayed, compared with the induction of MMP-1, -3, and -9 mRNAs. This latter possibility is supported by the increased MMP-2 immunoreactivity observed in paraffin sections of monkey ciliary muscle after 5 days of daily treatment with PGF-isopropyl ester. 12  
In conclusion, this study has provided evidence that the increased MMPs seen in ciliary muscle cells after latanoprost acid treatment reflects increased transcription of MMP-1, -3, and -9 mRNAs. These observations are consistent with a role for increased MMPs in the regulation of uveoscleral outflow. 
 
Table 1.
 
Sequences of the Primers and Probes for Taqman Real-Time PCR
Table 1.
 
Sequences of the Primers and Probes for Taqman Real-Time PCR
MMP-1
Forward primer GGC TGT TTT GTA CTG CCT GCT
Reverse primer AGG AGA CAC AGG CTC TAG GGA A
Probe FAM-AGT TTC CAG ACC TCC GCT GGC CA-TAMRA
GAPDH
Forward primer TGC ACC ACC AAC TGC TTA
Reverse primer GGA TGC AGG GAT GAT GTT C
Probe FAM-CAG AAG ACT GTG GAT GGC CCC TC-TAMRA
Table 2.
 
Sequences of the Primers for Energy-Transfer Real-Time PCR
Table 2.
 
Sequences of the Primers for Energy-Transfer Real-Time PCR
MMP-1
Forward primer GGC TGA AAG TGA CTG GGA AA
Reverse primer CAC ATC AGG CAC TCC ACA TC
MMP-2
Forward primer AGG ACT ACG ACC GCG ACA AG
Reverse primer GTT CCC ACC AAC AGT GGA CAT
MMP-3
Forward primer AAC CTG TCC CTC CAG AAC CT
Reverse primer CAG CAT CAA AGG ACA AAG CA
MMP-9
Forward primer GGC GCT CAT GTA CCC TAT GT
Reverse primer GCC ATT CAC GTC GTC CTT AT
Figure 1.
 
Electrophoretic analysis of PCR reaction products. (A) Reaction products from Taqman real-time PCR reactions separated in an 8% polyacrylamide gel using TBE buffer. Samples included 25-bp ladder standards (lane S); a sample in which probes, primer, and PCR reagents were present but to which cDNA template was not added (lane 1−); and a sample containing probes, primer, PCR reagents, and the MMP-1 amplicon oligonucleotide (lane 1+). Both samples were processed through thermal cycling. The no-template experiment is a control for nonspecific amplification of primer or probe sequences. Note that the probe is visible in lane 1−. In lane 1+, the probe is depleted and the 71-bp amplified product appears. (B) Reaction products from energy-transfer real-time PCR reactions separated in a 4% agarose gel. Samples included 25-bp ladder standards (lane S); reaction products of MMP-1, -2, -3, and -9 energy-transfer PCR reactions run without cDNA template (lanes 1−, 2−, 3−, and 9−, respectively); and corresponding energy-transfer PCR reactions run with cDNA from ciliary muscle cells treated for 24 hours with 200 nM latanoprost acid (lanes 1+, 2+, 3+, and 9+, respectively).
Figure 1.
 
Electrophoretic analysis of PCR reaction products. (A) Reaction products from Taqman real-time PCR reactions separated in an 8% polyacrylamide gel using TBE buffer. Samples included 25-bp ladder standards (lane S); a sample in which probes, primer, and PCR reagents were present but to which cDNA template was not added (lane 1−); and a sample containing probes, primer, PCR reagents, and the MMP-1 amplicon oligonucleotide (lane 1+). Both samples were processed through thermal cycling. The no-template experiment is a control for nonspecific amplification of primer or probe sequences. Note that the probe is visible in lane 1−. In lane 1+, the probe is depleted and the 71-bp amplified product appears. (B) Reaction products from energy-transfer real-time PCR reactions separated in a 4% agarose gel. Samples included 25-bp ladder standards (lane S); reaction products of MMP-1, -2, -3, and -9 energy-transfer PCR reactions run without cDNA template (lanes 1−, 2−, 3−, and 9−, respectively); and corresponding energy-transfer PCR reactions run with cDNA from ciliary muscle cells treated for 24 hours with 200 nM latanoprost acid (lanes 1+, 2+, 3+, and 9+, respectively).
Figure 2.
 
Standard curves plotting CT as a function of a serial dilution of an oligonucleotide corresponding to the amplicon for MMP-1 defined by the Taqman real-time PCR primers (A) and a serial dilution of RNA from MMP-secreting OCM1 cells analyzed with the specific energy-transfer primers (B). In the latter case, specific energy-transfer primers for MMP-1, -2, -3, and -9 were analyzed. Also, the CT of GAPDH in each of these RNA dilutions, as determined by Taq real-time PCR, is plotted. Error bars, SD of triplicate determinations. Note that the increase of the signal in each case was linear.
Figure 2.
 
Standard curves plotting CT as a function of a serial dilution of an oligonucleotide corresponding to the amplicon for MMP-1 defined by the Taqman real-time PCR primers (A) and a serial dilution of RNA from MMP-secreting OCM1 cells analyzed with the specific energy-transfer primers (B). In the latter case, specific energy-transfer primers for MMP-1, -2, -3, and -9 were analyzed. Also, the CT of GAPDH in each of these RNA dilutions, as determined by Taq real-time PCR, is plotted. Error bars, SD of triplicate determinations. Note that the increase of the signal in each case was linear.
Figure 3.
 
Taqman real-time PCR analysis of MMP-1 mRNA in human ciliary muscle cells exposed to 200 nM latanoprost acid for 24 hours. Primary cell lines from five different donors ranging from 54 to 87 years of age were analyzed.
Figure 3.
 
Taqman real-time PCR analysis of MMP-1 mRNA in human ciliary muscle cells exposed to 200 nM latanoprost acid for 24 hours. Primary cell lines from five different donors ranging from 54 to 87 years of age were analyzed.
Figure 4.
 
Taqman real-time PCR analysis of MMP-1 in ciliary muscle cells exposed to increasing concentrations of latanoprost acid for 24 hours. In both (A) a low-responder culture (passage 7 cells from an 87-year-old donor) and (B) a high-responder culture (passage 5 cells from a 74-year-old donor), increases in latanoprost acid concentration were associated with increases in MMP-1 mRNA expression.
Figure 4.
 
Taqman real-time PCR analysis of MMP-1 in ciliary muscle cells exposed to increasing concentrations of latanoprost acid for 24 hours. In both (A) a low-responder culture (passage 7 cells from an 87-year-old donor) and (B) a high-responder culture (passage 5 cells from a 74-year-old donor), increases in latanoprost acid concentration were associated with increases in MMP-1 mRNA expression.
Figure 5.
 
Time course of changes in MMP-1 mRNA in ciliary muscle cells treated with 200 nM latanoprost acid and analyzed using Taqman real-time PCR. Cultures were passage-7 cells from a 56-year-old donor (A), passage-6 cells from a 79-year-old donor (B), passage-5 cells from a 56-year-old donor (C), and passage-6 cells from an 87-year-old donor (D). Induction of MMP-1 mRNA reached a maximum by 6 or 12 hours and was reduced at later time points.
Figure 5.
 
Time course of changes in MMP-1 mRNA in ciliary muscle cells treated with 200 nM latanoprost acid and analyzed using Taqman real-time PCR. Cultures were passage-7 cells from a 56-year-old donor (A), passage-6 cells from a 79-year-old donor (B), passage-5 cells from a 56-year-old donor (C), and passage-6 cells from an 87-year-old donor (D). Induction of MMP-1 mRNA reached a maximum by 6 or 12 hours and was reduced at later time points.
Figure 6.
 
Comparison of MMP mRNA expression in ciliary muscle cells treated for 24 hours with vehicle or increasing amounts of latanoprost acid and analyzed using energy-transfer real-time PCR. After treatment, RNA from each culture was isolated, and the expression of MMP-1 (A), -2 (B), -3 (C), and -9 (D) mRNAs was determined.
Figure 6.
 
Comparison of MMP mRNA expression in ciliary muscle cells treated for 24 hours with vehicle or increasing amounts of latanoprost acid and analyzed using energy-transfer real-time PCR. After treatment, RNA from each culture was isolated, and the expression of MMP-1 (A), -2 (B), -3 (C), and -9 (D) mRNAs was determined.
The authors thank Mila Angert for technical assistance and Jacques Corbeil, PhD, University of California San Diego (UCSD) Department of Medicine, for assistance with the design of the primers and probes and helpful discussions regarding the real-time PCR method; the San Diego Eye Bank for providing donor eyes; and the Genomics Core of the UCSD Center for AIDS Research for analysis of the PCR plates. 
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Figure 1.
 
Electrophoretic analysis of PCR reaction products. (A) Reaction products from Taqman real-time PCR reactions separated in an 8% polyacrylamide gel using TBE buffer. Samples included 25-bp ladder standards (lane S); a sample in which probes, primer, and PCR reagents were present but to which cDNA template was not added (lane 1−); and a sample containing probes, primer, PCR reagents, and the MMP-1 amplicon oligonucleotide (lane 1+). Both samples were processed through thermal cycling. The no-template experiment is a control for nonspecific amplification of primer or probe sequences. Note that the probe is visible in lane 1−. In lane 1+, the probe is depleted and the 71-bp amplified product appears. (B) Reaction products from energy-transfer real-time PCR reactions separated in a 4% agarose gel. Samples included 25-bp ladder standards (lane S); reaction products of MMP-1, -2, -3, and -9 energy-transfer PCR reactions run without cDNA template (lanes 1−, 2−, 3−, and 9−, respectively); and corresponding energy-transfer PCR reactions run with cDNA from ciliary muscle cells treated for 24 hours with 200 nM latanoprost acid (lanes 1+, 2+, 3+, and 9+, respectively).
Figure 1.
 
Electrophoretic analysis of PCR reaction products. (A) Reaction products from Taqman real-time PCR reactions separated in an 8% polyacrylamide gel using TBE buffer. Samples included 25-bp ladder standards (lane S); a sample in which probes, primer, and PCR reagents were present but to which cDNA template was not added (lane 1−); and a sample containing probes, primer, PCR reagents, and the MMP-1 amplicon oligonucleotide (lane 1+). Both samples were processed through thermal cycling. The no-template experiment is a control for nonspecific amplification of primer or probe sequences. Note that the probe is visible in lane 1−. In lane 1+, the probe is depleted and the 71-bp amplified product appears. (B) Reaction products from energy-transfer real-time PCR reactions separated in a 4% agarose gel. Samples included 25-bp ladder standards (lane S); reaction products of MMP-1, -2, -3, and -9 energy-transfer PCR reactions run without cDNA template (lanes 1−, 2−, 3−, and 9−, respectively); and corresponding energy-transfer PCR reactions run with cDNA from ciliary muscle cells treated for 24 hours with 200 nM latanoprost acid (lanes 1+, 2+, 3+, and 9+, respectively).
Figure 2.
 
Standard curves plotting CT as a function of a serial dilution of an oligonucleotide corresponding to the amplicon for MMP-1 defined by the Taqman real-time PCR primers (A) and a serial dilution of RNA from MMP-secreting OCM1 cells analyzed with the specific energy-transfer primers (B). In the latter case, specific energy-transfer primers for MMP-1, -2, -3, and -9 were analyzed. Also, the CT of GAPDH in each of these RNA dilutions, as determined by Taq real-time PCR, is plotted. Error bars, SD of triplicate determinations. Note that the increase of the signal in each case was linear.
Figure 2.
 
Standard curves plotting CT as a function of a serial dilution of an oligonucleotide corresponding to the amplicon for MMP-1 defined by the Taqman real-time PCR primers (A) and a serial dilution of RNA from MMP-secreting OCM1 cells analyzed with the specific energy-transfer primers (B). In the latter case, specific energy-transfer primers for MMP-1, -2, -3, and -9 were analyzed. Also, the CT of GAPDH in each of these RNA dilutions, as determined by Taq real-time PCR, is plotted. Error bars, SD of triplicate determinations. Note that the increase of the signal in each case was linear.
Figure 3.
 
Taqman real-time PCR analysis of MMP-1 mRNA in human ciliary muscle cells exposed to 200 nM latanoprost acid for 24 hours. Primary cell lines from five different donors ranging from 54 to 87 years of age were analyzed.
Figure 3.
 
Taqman real-time PCR analysis of MMP-1 mRNA in human ciliary muscle cells exposed to 200 nM latanoprost acid for 24 hours. Primary cell lines from five different donors ranging from 54 to 87 years of age were analyzed.
Figure 4.
 
Taqman real-time PCR analysis of MMP-1 in ciliary muscle cells exposed to increasing concentrations of latanoprost acid for 24 hours. In both (A) a low-responder culture (passage 7 cells from an 87-year-old donor) and (B) a high-responder culture (passage 5 cells from a 74-year-old donor), increases in latanoprost acid concentration were associated with increases in MMP-1 mRNA expression.
Figure 4.
 
Taqman real-time PCR analysis of MMP-1 in ciliary muscle cells exposed to increasing concentrations of latanoprost acid for 24 hours. In both (A) a low-responder culture (passage 7 cells from an 87-year-old donor) and (B) a high-responder culture (passage 5 cells from a 74-year-old donor), increases in latanoprost acid concentration were associated with increases in MMP-1 mRNA expression.
Figure 5.
 
Time course of changes in MMP-1 mRNA in ciliary muscle cells treated with 200 nM latanoprost acid and analyzed using Taqman real-time PCR. Cultures were passage-7 cells from a 56-year-old donor (A), passage-6 cells from a 79-year-old donor (B), passage-5 cells from a 56-year-old donor (C), and passage-6 cells from an 87-year-old donor (D). Induction of MMP-1 mRNA reached a maximum by 6 or 12 hours and was reduced at later time points.
Figure 5.
 
Time course of changes in MMP-1 mRNA in ciliary muscle cells treated with 200 nM latanoprost acid and analyzed using Taqman real-time PCR. Cultures were passage-7 cells from a 56-year-old donor (A), passage-6 cells from a 79-year-old donor (B), passage-5 cells from a 56-year-old donor (C), and passage-6 cells from an 87-year-old donor (D). Induction of MMP-1 mRNA reached a maximum by 6 or 12 hours and was reduced at later time points.
Figure 6.
 
Comparison of MMP mRNA expression in ciliary muscle cells treated for 24 hours with vehicle or increasing amounts of latanoprost acid and analyzed using energy-transfer real-time PCR. After treatment, RNA from each culture was isolated, and the expression of MMP-1 (A), -2 (B), -3 (C), and -9 (D) mRNAs was determined.
Figure 6.
 
Comparison of MMP mRNA expression in ciliary muscle cells treated for 24 hours with vehicle or increasing amounts of latanoprost acid and analyzed using energy-transfer real-time PCR. After treatment, RNA from each culture was isolated, and the expression of MMP-1 (A), -2 (B), -3 (C), and -9 (D) mRNAs was determined.
Table 1.
 
Sequences of the Primers and Probes for Taqman Real-Time PCR
Table 1.
 
Sequences of the Primers and Probes for Taqman Real-Time PCR
MMP-1
Forward primer GGC TGT TTT GTA CTG CCT GCT
Reverse primer AGG AGA CAC AGG CTC TAG GGA A
Probe FAM-AGT TTC CAG ACC TCC GCT GGC CA-TAMRA
GAPDH
Forward primer TGC ACC ACC AAC TGC TTA
Reverse primer GGA TGC AGG GAT GAT GTT C
Probe FAM-CAG AAG ACT GTG GAT GGC CCC TC-TAMRA
Table 2.
 
Sequences of the Primers for Energy-Transfer Real-Time PCR
Table 2.
 
Sequences of the Primers for Energy-Transfer Real-Time PCR
MMP-1
Forward primer GGC TGA AAG TGA CTG GGA AA
Reverse primer CAC ATC AGG CAC TCC ACA TC
MMP-2
Forward primer AGG ACT ACG ACC GCG ACA AG
Reverse primer GTT CCC ACC AAC AGT GGA CAT
MMP-3
Forward primer AAC CTG TCC CTC CAG AAC CT
Reverse primer CAG CAT CAA AGG ACA AAG CA
MMP-9
Forward primer GGC GCT CAT GTA CCC TAT GT
Reverse primer GCC ATT CAC GTC GTC CTT AT
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