Latanoprost’s Effects on TIMP-1 and TIMP-2 Expression in Human Ciliary Muscle Cells

Abstract

purpose. To determine the effect of treatment with latanoprost on the tissue inhibitors of metalloproteinase (TIMP)-1 and -2 in cultured human ciliary muscle (HCM) cells.

methods. Confluent serum-starved HCM cells were exposed to increasing concentrations of latanoprost acid (LA, 1 nM to 10 μM) for 6, 18, and 24 hours. TIMP-1 and -2 mRNA transcripts were evaluated by RT-PCR. Gelatin zymography was used to measure changes in the amount of matrix metalloproteinase (MMP) in the culture medium. To evaluate the potential role of PKC, HCM cells were treated with phorbol 12-myrisate 13-acetate (PMA) in the absence or presence of the PKC inhibitor bisindolylmaleimide I (Bis I) or the PKA inhibitor KT5720. Data were quantitated by densitometry and statistically analyzed with the Student-Newman-Keuls test.

results. TIMP-1 and -2 mRNA transcripts and proteins were detected in primary cultures of HCM cells. TIMP-1 mRNA levels were unchanged at 6 hours, but increased 45% ± 17% and 54% ± 13% in cultures exposed for 18 hours to 1 and 10 μM LA, respectively (n = 3). In contrast, 6 hours of exposure to LA increased expression of TIMP-2 mRNA by up to 11.3% ± 0.2% (n = 3). However, no significant induction of TIMP-2 mRNA was observed at either 18 or 24 hours (n = 3). TIMP-1 protein was significantly increased in cultures exposed to LA for 18 and 24 hours. In contrast, TIMP-2 protein expression was insignificantly different from control cultures at 6, 18, and 24 hours of treatment. HCM cells exposed to PMA for 24 hours produced similar increases in TIMP-1 mRNA levels, as seen with latanoprost (n = 5). However, no significant induction of TIMP-2 mRNA was observed. Zymographic analysis of the media from these cultures confirmed dose-dependent increases of MMP-1 at 6, 18, and 24 hours, whereas dose-dependent increases in MMP-2 were seen only after 24 hours’ exposure to LA (n = 3). TIMP-1 protein levels were increased 27% ± 9.3% and 15% ± 11% in the media of cells exposed for 24 hours to 100 nM LA and 100 nM PMA, respectively (n = 5). The increases in TIMP-1 protein induced by LA were essentially eliminated by Bis I (n = 3) and unaffected by KT5720 (n = 3).

conclusions. For the most part, TIMP-1, and not TIMP-2, contributes to regulation of MMP within the uveoscleral outflow pathway after exposure to latanoprost. Moreover, this induction appears to be meditated by activation of PKC.

Topical application to the cynomolgus monkey eye of prostaglandin (PG) F_2α or latanoprost, a PGF_2α analogue, lowers intraocular pressure (IOP) and increases uveoscleral outflow. ^{1 2 3 4} This response appears to be mediated by activation of the FP receptor. ⁵ Topical treatment with PG also reduces collagens in the tissues of the uveoscleral outflow pathway, ^{6 7 8} and increases production of matrix metalloproteinases (MMPs), ^{9 10} a family of neutral proteases that can initiate degradation of the extracellular matrix (ECM). ^{11 12} These observations support the hypothesis that MMP-mediated reduction of collagen within the extracellular spaces of ciliary muscle and sclera reduces hydraulic resistance to uveoscleral outflow. ^{13 14}  

The activity of MMP can be regulated by a family of extracellular inhibitory proteins called tissue inhibitors of metalloproteinases (TIMPs). ^{15 16 17} Currently, four TIMPs (TIMP-1, -2, -3, and -4) have been identified in mammals, and each targets only certain MMPs. ^{18 19} The role of TIMPs in the response of ciliary muscle cells to PGs remains largely unknown. Examination of the promoter regions of the MMP and TIMP genes has identified activation protein (AP)-1 sites in both promoters, ²⁰ suggesting that they may be regulated by similar mechanisms. PGF_2α and 17-phenyl trinor-PGF_2α have been observed to induce the AP-1 activators c-Fos and c-Jun in cultured human ciliary muscle (HCM) cells. ^{21 22} Also, it has been demonstrated that stimulation of bovine luteal cells with PGF_2α induces expression of c-Fos through a protein kinase C (PKC)–dependent pathway. ²³ Thus, it is possible that latanoprost, a PGF_2α analogue, may induce expression of TIMP through the PKC-dependent pathway. 

In view of these observations, in the present study we investigated the effect of treatment with latanoprost on the expression of TIMP-1 and -2 in HCM cells. In addition, we investigated whether regulation of the expression of TIMP by the activation of FP receptors is dependent on activation of PKC. 

Methods

Human Ciliary Smooth Muscle and Trabecular Meshwork Cultures

Eyes of three donors (obtained from the San Diego Eye Bank) were enucleated within 12 hours of death and stored at 4°C. Donors had no known history of glaucoma or other eye diseases. All procedures in this study adhered to the guidelines of the University of California, San Diego, and the provisions of the Declaration of Helsinki for the use of human tissue in research. 

Ciliary smooth muscle cell cultures were prepared as previously described. ²⁴ Briefly, strips of isolated HCM were explanted into 35-mm culture dishes (Falcon, Lincoln Park, NJ). The medium was DMEM/F-12 supplemented with 10% FCS (Gemini, Calabasas, CA), 100 U/mL penicillin, 100 μg/mL streptomycin, 2.5 μg/mL amphotericin B (all antibiotics from Gibco BRL, Grand Island, NY), and 1 ng/mL basic fibroblast growth factor (R&D Systems, Minneapolis, MN). The cultures were incubated in a humidified atmosphere of 95% air-5% CO₂. The medium was changed every 3 days. Confluent cultures were trypsinized and subcultured in the same medium at a ratio of 1:3. For this study, cells were used up to fifth passage. 

Experimental Design

Primary cultures of HCM cells were seeded in six-well plates and allowed to grow to confluence. The confluent cultures were then switched to serum-free medium and incubated for 2 days at 37°C. Serum-free medium was used to minimize the potential induction of MMPs or TIMPs from agents present in serum. ²⁵ On day 2, 10⁻⁹, 10⁻⁸, 10⁻⁷, 10⁻⁶, or 10⁻⁵ M latanoprost acid (Cayman Chemicals, Ann Arbor, MI) or 1, 10, or 100 nM phorbol 12-myrisate 13-acetate (PMA; Sigma Chemical Co., St. Louis, MO) was diluted in serum-free medium and incubated with the cells for 6, 18, or 24 hours. These time points were chosen based on previous experiments in which MMP activity was monitored in cultured HCM cells. ⁹ For zymogram analysis, media samples were collected, precipitated with 60% ethanol and stored at −80°C until analyzed. The RNA in the cells was then harvested. 

Isolation of RNA

The RNA was isolated from the cultures with 1 mL of extraction reagent (TRIzol; Gibco BRL), and the suspensions were transferred to 1.5-mL microcentrifuge tubes. Chloroform (200 μL) was added to each tube. The tubes were mixed by brief agitation and incubated for 3 minutes at 25°C. The samples were then centrifuged (12,000g) for 15 minutes at 4°C, and the aqueous phase was carefully transferred to fresh sterile 1.5-mL microcentrifuge tubes. Isopropanol (500 μL/tube) was added, and samples were incubated for 10 minutes at 25°C to initiate the precipitation of RNA. Samples were then centrifuged (12,000g) for 10 minutes at 4°C and the supernatant was removed. The RNA pellet was washed with 75% ethanol and diethyl pyrocarbonate (DEPC)–treated water and air dried. RNA was resuspended in a total volume of 20 μL DEPC-treated water. Gel electrophoresis and spectrophotometry were used to determine the quality and quantity of isolated RNA. 

Reverse Transcription–Polymerase Chain Reaction

RT-PCR was performed as described, ²⁶ with the total RNA isolated from HCM cells. Primers were chosen to amplify unique regions within the individual human TIMP-1 and -2 coding sequences. All PCR primer pairs were 100% homologous with the reported human TIMP-1, TIMP-2, and GAPDH cloned sequences. The PCR reaction mix (final volume, 50 μL) contained 5 μL of the RT reaction, 5 μL of 10× PCR buffer (Gibco BRL), 1 μL of 10 mM dNTP mixture, 1.5 μL of 50 mM MgCl₂, 2.5 μL of the sense and antisense primers (20 μM), and 0.5 μL of 5 U/μLTaq polymerase (Gibco BRL). The PCR program consisted of an initial step at 95°C for 5 minutes; followed by 30 cycles at 95°C for 1 minute, 55°C for 1 minute, and 72°C for 1 minute; and a final step at 72°C for 7 minutes. Products were analyzed by electrophoresis in 1% agarose gels. The PCR product sizes were 527 bp for TIMP-1, 583 bp for TIMP-2, and 280 bp for GAPDH. After each PCR reaction, the gels were scanned and densitometry was performed to measure the mRNA transcripts. The relative increase in band intensity was expressed as a percentage of GAPDH band intensities. 

Detection of TIMP Proteins by Western Blot

Samples of conditioned media were collected from HCM cultures after treatment with latanoprost. Samples were precipitated at −80°C after addition of ice-cold ethanol (final concentration 60%) for 36 to 48 hours. Protein pellets (12,000g for 20 minutes) were washed once with 0.5 mL of ethanol and subsequently lyophilized by a heated rotary vacuum evaporator (Speed Vac; Savant Instrument Inc., Farmingdale, NY). The lyophilized proteins were resuspended in PAN buffer (10 mM piperazine-N-N′-bis(2-ethanesulfonic acid) [PIPES; pH 7.0], 1% aprotinin, and 100 mM NaCl). SDS-PAGE sample buffer (250 mM Tris-HCl [pH 6.8], 8% SDS, 0.4% bromophenol blue, 40% glycerol, 1% 2-mercaptoethanol) was added to samples and to prestained molecular weight standards containing myosin H-chain, β-galactosidase, bovine serum albumin, carbonic anhydrase, soybean trypsin inhibitor, lysozyme, and aprotinin (BioRad Laboratories, Hercules, CA). The samples were boiled for 3 minutes at 100°C and then separated on a 12% polyacrylamide gel in SDS running buffer (25 mM Tris-HCl [pH 8.3], 192 mM glycine, 0.1% SDS). After electrophoresis, proteins were transferred to 0.45-μm nitrocellulose in a buffer containing 25 mM Tris-HCl [pH 8.0], 192 mM glycine, and 20% methanol at 4°C. Nitrocellulose membranes were washed once in TBST (100 mM Tris-HCl [pH 8.0] 150 mM NaCl, and 0.2% Tween-20) and blocked for 30 minutes with TBST containing 3% nonfat dry milk. Primary antibody to TIMP-1 (1:500, IM32L; Calbiochem, San Diego, CA) or TIMP-2 (1:500, IM11L; Calbiochem) were added to the blocking solution and allowed to incubate overnight at 4°C. After four washes, 15 minutes each in TBST, and reblocking with 3% nonfat dry milk for 30 minutes, secondary antibody rabbit anti-mouse HRP conjugated antibody (1:1000, A-9044; Sigma) was added and allowed to incubate for 1 hour at room temperature. Blots were developed with the enhanced chemiluminescence detection system (Pierce, Rockford, IL). 

Gelatin Zymography

Media samples were prepared as described earlier, except the samples were reconstituted in a nondenaturing sample buffer (250 mM Tris-HCl [pH 6.8], 8% SDS, 0.4% bromophenol blue, 40% glycerol). Samples were loaded on 10% polyacrylamide gels containing 0.1% gelatin. After electrophoresis, the gels were incubated in 2.5% Triton X-100 in water at room temperature for 30 minutes. Gels were washed twice in developing buffer (50 mM Tris [pH 7.6], 200 mM NaCl, 5 mM CaCl₂, and 5 μM ZnCl₂), once for 30 minutes at room temperature and again overnight at 37°C. Gels were stained with Coomassie blue for 1 hour at room temperature. After the gels were destained in water containing 40% MeOH and 10% glacial acetic acid, the positions of the MMPs were observed as clear areas within the dark-stained background. 

Densitometry Measurements

The band densities were measured from scanned images of each gel photograph or Western blot film on computer (imaging software; Scion Corp., Frederick, MD). The densities were determined by calculating the area under the curve for each band histogram and expressed as a percentage of control band densities. 

Results

Expression of TIMP Protein in Human Ciliary Muscle Cells

TIMP-1 protein from HCM cells appeared in Western blot analysis as a single band at 28 kDa (Fig. 1) . TIMP-2 also appeared as a single band but migrated to a position corresponding to 20 kDa. Both proteins were detected in the medium of vehicle-treated cells (lane C). After a 24-hour exposure to latanoprost acid (1 nM to 10 μM), the expression of TIMP-1 protein increased in a dose-dependent manner from 5% to 42%. In contrast, exposure to latanoprost had little effect on the expression level of TIMP-2. Similar results were obtained from HCM cells treated with PGF_2α. These results indicate that expression of TIMP-1 protein was induced during exposure to latanoprost in HCM cells, whereas expression TIMP-2 was unaffected. 

Expression of TIMP-1 and -2 mRNAs in HCM Cells

To determine whether changes in induction of TIMP-1 and -2 by latanoprost reflects pre- or posttranslational regulation, we used RT-PCR to examine the TIMP-1 and -2 mRNA levels at various time points. Serum-starved cultures of HCM cells were exposed to latanoprost acid (1 nM to 10 μM) for 6, 18, and 24 hours. For statistical comparison, each experiment was performed in triplicate. After 6 hours of treatment, there was minimal change in TIMP-1 in any of the latanoprost-treated cultures compared with the control cultures (Fig. 2) . In contrast, 18 and 24 hours of treatment induced similar dose-dependent increases of TIMP-1 protein. These increases were statistically significant at 1 and 10 μM in the cultures treated for 18 hours and at all tested latanoprost concentrations in the cultures treated for 24 hours. The increases in TIMP-1 mRNA levels ranged from 20% to 53% after 18 hours of exposure and 21% to 37% after 24 hours of exposure. 

The same samples were used to assess the effect of latanoprost on TIMP-2 mRNA levels. In contrast to the expression of TIMP-1 mRNA , we observed a modest but significant increase in TIMP-2 mRNA relative to untreated control cultures at 6 hours at each concentration of latanoprost acid tested (Fig. 2B) . The increases in TIMP-2 mRNA ranged from 6% to 11%. However, we did not observe any significant difference in levels of TIMP-2 mRNA at the 18- or 24-hour time point. Taken together, these data indicate that exposure of HCM cells to latanoprost acid produces differential effects on the transcriptional regulation of the TIMP-1 and -2 genes. 

Zymographic Analysis

Gelatin zymograms were used to assess the activities of the various MMPs induced after exposure to latanoprost. Media samples from HCM cells exposed to vehicle (Fig. 3 , lane C) or to increasing concentrations of latanoprost acid were separated on 12% polyacrylamide gels. Three major bands were detected in the gelatin zymogram in both vehicle- and latanoprost-treated samples that had apparent molecular masses of 62, 68, and 95 kDa (Fig. 3) . Western immunoblot analysis identified these bands as MMP-1, -2, and -9, respectively. The Western blot experiments were optimized to show the position of each band to facilitate identification of the zymography bands. The 62-kDa MMP-1 immunoreactivity and 95-kDa MMP-9 immunoreactivity corresponds most closely to the pro forms of these MMPs. In contrast, 68-kDa MMP-2 immunoreactivity corresponds most closely to the activated form of MMP-2. This is consistent with the small bands for MMP-1 and -9, and the large band for MMP-2 in the zymogram. The presence of these MMPs is consistent with our previous investigations of the expression of MMP by cultured HCM cells. ¹⁰  

The time course of the induction of MMP by latanoprost was analyzed to determine whether it correlates with the induction of TIMP-1 mRNA. Parallel serum-starved HCM cultures were exposed to various concentrations of latanoprost acid for 6, 18, and 24 hours, and the conditioned media were analyzed in gelatin zymograms. For statistical comparison, each experiment was performed in triplicate. We examined the inductions of MMP-1 (62 kDa) and -2 (68 kDa), because they are known to be inhibited by TIMP-1. ¹⁶ Exposure to increasing concentrations of latanoprost acid induced dose-dependent increases in the secretion of both MMP-1 and -2 (Fig. 4) . In the case of MMP-1, these increases were significant after exposure to 1 nM to 10 μM latanoprost for 18 and for 24 hours. In the case of MMP-2, these increases were significant after exposure to 1 nM to 10 μM latanoprost for 24 hours, only. This difference probably reflects the larger magnitude of the increases in MMP-1 than of MMP-2. 

Collectively, these data show that the time course of TIMP-1 induction by latanoprost acid was similar to the induction of MMP-1 and -2. The earliest significant increase in expression of MMP-1 was observed after 6 hours of treatment. In the case of expression of TIMP-1, the earliest significant increase was observed after 18 hours of treatment. Hence, the onset of MMP induction may precede the onset of TIMP-1 induction. 

Effect of PKC on TIMP-1 mRNA Levels

mRNA transcription and protein synthesis of MMP-1, MMP-3, and TIMP-1 increase in human synovial fibroblasts after stimulation of PKC. ²⁷ Because activation of the FP receptor in these cells leads to the activation of PKC, the following experiments were performed to examine whether direct activation of PKC can mimic latanoprost acid–mediated induction of TIMP mRNA transcription in HCM cells. PMA is a well-characterized direct activator of PKC. ²⁸ TIMP-1 mRNA levels were induced in serum-starved HCM cells after 24-hour exposure to increasing concentrations of PMA (Fig. 5) . The increase in TIMP-1 transcription was significant at concentrations as low as 10 nM and appeared to increase only slightly at 50 and 100 nM. These treatments with PMA had no significant effect on the transcription of TIMP-2 mRNA (n = 5). These results suggest that the induction of TIMP-1 mRNA transcription by latanoprost acid can be mimicked by direct activation of the PKC-dependent pathway. 

PKC Dependence of Expression of TIMP-1 Protein

The selective PKC inhibitor bisindolylmaleimide I (Bis I) ²⁹ was used to assess further the role of PKC in the latanoprost-induced expression of TIMP-1 protein. HCM cultures were pretreated with 200 nM Bis I for 15 minutes before the addition of 100 nM latanoprost acid. The presence of Bis I alone had minimal effect on the expression of TIMP-1 protein after 24 hours (data not shown). Twenty-four-hour treatment with latanoprost acid produced a significant increase in TIMP-1 protein levels. In the presence of Bis I, the latanoprost acid–mediated increase in TIMP-1 was significantly inhibited (P < 0.05, Fig. 6A ). Although the Bis K _i for protein kinase A (PKA) is 2 μM, it is important to exclude the possibility that this response might in part reflect activation of PKA. Hence, parallel studies were conducted in which HCM cells were exposed to the specific PKA inhibitor KT5720 (168 nM, K _i = 56 nM) ³⁰ for 15 minutes before the addition of 100 nM latanoprost acid. In these studies,, the increase in TIMP-1 induced by latanoprost acid was not inhibited by KT5720 (Fig. 6B) . These results indicate that the increase in TIMP-1 by latanoprost acid is mediated by PKC and not by PKA. 

Discussion

The present results show that exposure of HCM cells to latanoprost acid induced a time- and dose-dependent increase in TIMP-1 mRNA and protein. In contrast, there was only a brief and minor increase in TIMP-2 mRNA, and insignificant increases of TIMP-2 protein in the medium. These results suggest that TIMP-1 is important for regulating the activities of MMPs in HCM cells. In addition, the time course of induction of TIMP-1 was similar to the that of induction of MMP-1 and -2, as shown by comparison of the results of the TIMP Western blot and the gelatin zymogram analyses. Thus, latanoprost appears to induce TIMP-1, MMP-1, and MMP-2 in a coordinated manner. 

Several indirect lines of evidence suggest that promoter similarities among the genes for MMP-1, MMP-2, and TIMP-1 may be the basis for the coordinated induction of these proteins in latanoprost-treated HCM cells. First, the promoter sequences of the MMP and TIMP genes contain several essential elements for basal and induced transcriptional activation in common, including numerous individual AP-1 sites (which bind c-Fos and c-Jun) and PEA-3 sites (which bind Ets transcription factors). ²⁰ Second, treatment of human ciliary smooth muscle cells with PGF_2α, the naturally occurring parent prostaglandin for latanoprost, induced transient expression of the nuclear regulatory protein c-Fos. ²¹ Induction of c-Fos has been linked with induction of both MMPs and TIMP-1 in other cell systems. ³¹ Third, several in vitro studies of reporter gene constructs demonstrated that the transcription of the MMP and TIMP genes are similarly induced by extracellular signals, such as growth factors, phorbol esters, and inflammatory cytokines. ^{32 33 34} Thus, the present observation that latanoprost coinduces TIMP-1, MMP-1, and MMP-2 in HCM cells probably reflects the activation of nuclear regulatory signals that are recognized by transcription elements common in the promoters of each of these genes. 

If concomitant gene transcription is responsible for the induction of TIMP-1 and MMP in ciliary muscle cells, it probably reflects specific activation of certain intracellular signaling cascades. Molecular and pharmacologic studies have demonstrated that latanoprost is a potent activator of the prostaglandin FP receptor. ⁵ Activation of that receptor can stimulate multiple intracellular signaling pathways, including formation of inositol phosphate, release of intracellular calcium, and activation of PKC. ^{35 36} PKC’s involvement has been demonstrated in transcriptional regulation of the MMPs and TIMPs in several tissues. ^{23 31 37} In this study, direct activation of PKC produced a 52% increase in TIMP-1 mRNA after 24-hour treatment with PMA. This effect was similar to the 37% increase in mRNA transcription after exposure to latanoprost. Similar effects on TIMP-1 mRNA levels have been reported in human synovial fibroblasts after treatment with PMA. ³⁸ Thus, activation of PKC appears to play a role in the induction of TIMP-1 in HCM cells. 

It was important to address the possibility that increased activation of PKC may be unrelated to the increase in TIMP-1 mRNA. Thus, the induction of TIMP-1 by latanoprost was examined while activation of PKC was blocked. We observed that latanoprost acid’s induction of TIMP-1 protein could be blocked with pretreatment with the PKC inhibitor Bis I. This block was complete in the presence of latanoprost acid at concentrations that occur in aqueous humor after topical treatment with latanoprost (0.1 μM). ³⁹ Hence, activation of PKC appears to be essential for expression of TIMP-1. This result is similar to a recent study that showed induction of TIMP-1 by 12-tetradecanoylphorbol-13-acetate (TPA) in porcine trabecular meshwork cells was inhibited by the PKC inhibitor Bis I. ⁴⁰ Further studies are needed to confirm whether induction of MMP in HCM cells also depends on the activation of PKC. 

Substantial evidence supports the view that the increased uveoscleral outflow that occurs after topical application of latanoprost is accompanied by reductions of the ECM and increased MMPs in the ciliary muscle cells. ^{7 8 10 13 41 42} In other tissues, the release of MMP accompanied by release of TIMP occurs during the normal turnover of the ECM, which occurs during the development of connective tissue, morphogenesis, and wound healing. ¹² In contrast, release of MMP in the absence of release of TIMP has been observed in disease states such as arthritis and cancer and is associated with pathologically excessive ECM degradation. ¹¹ Histologic analysis showed that the overall structural organization of monkey ciliary muscle remains intact after 10 days of topical treatment with latanoprost, even though there were reductions of various ECM components including collagen types I, III, and IV; fibronectin; and laminin. ⁸ This study also demonstrated increased activated MMP-2 in the treated eyes, similar to the present investigation in cultured HCM cells. This MMP may be primarily responsible for these changes in the ECM, because MMP-2 can degrade collagen types I, III, and IV; fibronectin; and laminin. ⁴³ Also, although latanoprost increased MMP-1 and -9, their migration in the zymography gels at 62 and 95 kDa, respectively, indicates that they were present primarily as pro-MMPs. Thus, the induction of the potent MMP-2 inhibitor TIMP-1 along with MMP-2 observed in the present study suggests that the changes in the ECM induced by latanoprost are more akin to the regulated remodeling observed during development, morphogenesis, and wound healing. This likely limitation of the activity of MMP-2 by TIMP-1 after exposure to latanoprost is consistent with the observation that long-term clinical use of latanoprost is well tolerated by most patients. ⁴⁴  

The current results show that the induction of MMPs by latanoprost is accompanied by the simultaneous induction of TIMP-1. In addition, we demonstrated that this response reflects increased transcription of TIMP-1 mRNA and is critically dependent on latanoprost-mediated activation of PKC. Activation of PKC may also be involved in the induction of MMPs in ciliary muscle cells, based on the similarities in their promoter regions. Therefore, the MMP-dependent hypotensive effects of latanoprost appear to reflect a modulated upregulation of ECM turnover due to increases in both MMPs and TIMP-1 in HCM cells that occur through activation of a common intracellular signaling pathway. 

 Supported in part by National Eye Institute Grant EY05990 (RNW) and NRSA EY07047 (TLA).

 Submitted for publication December 13, 2001; revised June 17, 2002; accepted June 26, 2002.

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Figure 1.

 

Immunoblot analysis of HCM cell conditioned media stained with anti TIMP-1 and -2 antibodies. Extracellular proteins were precipitated from conditioned media, normalized, and loaded on a 10% SDS-polyacrylamide gel. Western blot analysis showed distinct bands at approximately 28 kDa (A) and at 21 kDa (B) which correspond to TIMP-1 and TIMP-2, respectively. One representative experiment of three is shown. Lane C: vehicle treatment. Lane −9: 10⁻⁹ M latanoprost, etc.

Figure 2.

 

Expression of TIMP-1 and -2 mRNA determined by PCR in HCM cells after exposure to increasing concentrations of latanoprost or vehicle control (con). Confluent cultures were exposed to increasing concentrations of latanoprost acid (1 nM to 1 μM) for 6 hours (n = 3), 18 hours (n = 3), or 24 hours (n = 4). Relative band intensities for each concentration are expressed as a percentage of GAPDH levels for each condition. Data are expressed as the mean ± SE. Significance of P < 0.05 was determined by the Student-Newman-Keuls test. Con, control; −9, 10⁻⁹ M latanoprost, etc.

Figure 3.

 

Analysis of MMP induction by latanoprost. (A) Gelatin zymogram of cultured media from confluent HCM cells exposed to increasing amounts of latanoprost (−9, 10⁻⁹ M latanoprost, etc.). (B) Western blot probed with antibodies recognizing MMP-1 (lane 1), -2 (lane 2), and -9 (lane 3). Bands were observed in the gelatin zymogram at 62, 68, and 95 kDa. Media samples from HCM cells exposed to vehicle (lane C) or increasing concentrations of latanoprost acid (1 nM to 1 μM). Western blot analysis confirmed that these bands were MMP-1 (62 kDa), -2 (68 kDa), and -9 (95 kDa).

Figure 4.

 

Densitometric analysis of matrix metalloproteinase bands at 62 kDa (A, n = 3) and 68 kDa (B, n = 3) in zymograms of ciliary smooth muscle cultures exposed to latanoprost acid or vehicle control for 6, 18, or 24 hours. Cultures were treated with increasing concentrations (1 nM to 1 μM) of latanoprost. Protein samples were normalized to total protein concentration and ran on 10% SDS-PAGE gels containing 0.1% gelatin.

Figure 5.

 

RT-PCR analysis of TIMP-1 and TIMP-2 mRNA induction in HCM cells exposed to PMA. Parallel cultures were exposed to increasing concentrations of PMA (1–100 nM) for 24 hours (n = 5). Representative gel analysis of each PCR product is shown (A). Relative band intensities for each concentration are expressed as a percentage of control for each condition (B). Data are expressed as the mean ± SE. *Significant difference (P < 0.05), as determined by the Student-Newman-Keuls test.

Figure 6.

 

Western blot analysis of TIMP-1 protein in media of HCM cells exposed to latanoprost in the absence or presence of the PKC inhibitor Bis I (A) or the PKA inhibitor KT5720 (B). Results are expressed as multiples of change compared with expression in inhibitor alone. Extracellular proteins were precipitated from conditioned media, normalized, and loaded on a 12% SDS-polyacrylamide gel. Western blot analysis was performed with anti TIMP-1 antibody. *Significant difference of P < 0.05 was determined by Student’s t-test (n = 3 for each inhibitor).

The authors thank Mila Angert and Eleanore Hewitt (Glaucoma Center, University of California, San Diego, La Jolla, CA) for assistance with Western blot analysis and cell culture.