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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online August 2, 2005 as doi:10.1096/fj.04-3626fje. |
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* Department of Experimental and Clinical Pharmacology and Toxicology, Friedrich Alexander University Erlangen-Nürnberg, Erlangen, Germany;
Institute of Human Anatomy and Embryology, University of Regensburg, Regensburg, Germany
1 Correspondence: Department of Experimental and Clinical Pharmacology and Toxicology, Friedrich Alexander University Erlangen-Nürnberg, Fahrstrasse, Erlangen 17 D-91054, Germany. E-mail: hinz{at}pharmakologie.uni-erlangen.de
SPECIFIC AIMS
Prostaglandins (PGs) have been implicated in the reduction of intraocular pressure (IOP) by facilitating the outflow of aqueous humor. Evidence suggesting a role of cyclooxygenase-2 (COX-2)-derived PGs in this process comes from a recent study showing that COX-2 is constitutively expressed in the nonpigmented secretory epithelium (NPE) of the ciliary body, but is completely lost in the NPE of patients with end-stage primary open-angle glaucoma (POAG). These data raise the question of whether antiglaucomatous drugs may induce intraocular COX-2 expression thereby enhancing the production of outflow-facilitating PGs. The present study was conducted to investigate the effect of the antiglaucomatous PGF2
analog latanoprost on the production of COX-2-dependent PGs and its possible association with the induction of matrix metalloproteinases (MMPs) in human NPE cells, the primary intraocular site involved in the production of aqueous humor.
PRINCIPAL FINDINGS
1. Latanoprost induces COX-2 expression
Incubation of human NPE cells (ODM-2) for up to 24 h with latanoprost acid (10 µM) led to a continuous increase of PGE2 levels in cell culture supernatants during the first 12 h up to a 2-fold induction over vehicle. Significant induction of PGE2 synthesis by latanoprost became evident at 8 h after stimulation. At 24 h poststimulation an 
1.7-fold induction of PGE2 synthesis was observed. To examine the source of PGE2 release, cells were treated with latanoprost in the presence of the selective COX-2 inhibitor NS-398. NS-398 abolished latanoprost-induced PGE2 release. The F-prostaglandin (FP) receptor antagonist AL-8810 significantly reduced latanoprost-induced PGE2 levels.
To determine whether induction of PGE2 synthesis by latanoprost was reflected in the expression of COX-2, COX-2 mRNA levels were analyzed using real-time RT-PCR. Incubation of cells with latanoprost resulted in a transient stimulation with a COX-2 mRNA peak after 2 h stimulation (Fig. 1
A). Further analyses revealed a concentration-dependent increase of COX-2 mRNA levels by latanoprost (Fig. 1B
).
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Western blot analysis of cell lysates from latanoprost-treated cells revealed an increase of COX-2 protein levels after stimulation periods of 24 and 48 h, respectively. Under the same experimental conditions the expression of COX-1 remained unchanged.
2. MAP kinases are involved in latanoprost-induced COX-2 expression
Since mitogen-activated protein kinases (MAPKs) have been implicated in the expression of immediate-early genes, the involvement of these enzymes in latanoprost-induced COX-2 expression was investigated by Western blot analysis using antibodies against the phosphorylated forms of p38 and p42/44 MAPKs. An increase in the phosphorylated form of p38 MAPK was detected 5 min after latanoprost treatment. Phosphorylation of p38 MAPK continued to increase during the 4 h incubation. Phosphorylation of p42/44 MAPK began to increase within 15 min and declined after 2 h stimulation.
To confirm a causal link between activation of p38 and p42/44 MAPKs and induction of COX-2 expression by latanoprost, the impact of specific inhibitors of p38 MAPK (SB203580) and p42/44 MAPK activation (PD98059) on COX-2 expression was assessed in further experiments. Latanoprost-induced COX-2 mRNA expression was abolished in the presence of both inhibitors. Latanoprost-induced COX-2 expression was suppressed by the intracellular calcium chelator, BAPTA/AM, and the protein kinase C inhibitor bisindolylmaleimide II.
3. Latanoprost induces MMP-1 expression through a COX-2-dependent pathway
To study one potential functional consequence of latanoprost-induced COX-2 expression, we next analyzed MMP-1 mRNA levels in latanoprost-stimulated NPE cells. Latanoprost produced a significant increase of MMP-1 mRNA levels after an incubation of as long as 24 h.
To link products of COX-2 induction to the observed increase in MMP-1 expression, cells were treated with latanoprost in the presence of NS-398 at a concentration that fully inhibits latanoprost-induced PGE2 synthesis. As shown in Fig. 2
, NS-398 abolished the induction of MMP-1 expression by latanoprost at both the mRNA and protein level. The involvement of COX-2 in latanoprost-induced MMP-1 expression was confirmed by experiments showing that transfection of cells with COX-2 siRNA significantly inhibited both MMP-1 mRNA and protein expression. At the concentration tested COX-2 siRNA was shown to abolish latanoprost-induced COX-2 protein expression. Control experiments revealed no significant effect of nonsilencing siRNA on latanoprost-induced MMP-1 and COX-2 protein expression.
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To further demonstrate the role of COX-2 in MMP-1 induction, additional experiments were performed using the major COX-2 product PGE2. PGE2 induced the expression of MMP-1 mRNA and protein in a concentration-dependent manner.
To investigate the impact of latanoprost on other MMPs and TIMPs (tissue inhibitors of MMPs), further experiments focused on the regulation of MMP-2 and MMP-9 as well as on TIMP-1 (endogenous inhibitor of MMP-1 and MMP-9) and TIMP-2 (endogenous inhibitor of MMP-2). According to RT-PCR analyses, the expression of the investigated MMPs and TIMPs was not significantly increased over 24 h incubation with latanoprost. Likewise, no induction was observed at the protein level.
CONCLUSIONS AND SIGNIFICANCE
The present study demonstrates a stimulatory effect of latanoprost on the expression of the PG-synthesizing enzyme COX-2 in human NPE cells. Enhanced expression of COX-2 was accompanied by increased synthesis of PGE2. The involvement of COX-2 in the PG-elevating effect of latanoprost was corroborated by experiments demonstrating that the selective inhibitor of COX-2 activity, NS-398, abolished this response. Moreover, PGE2 formation by latanoprost was blocked by the FP receptor antagonist AL-8810, confirming the involvement of FP receptor signaling in this process. In support of a causal link between Gq-protein-coupled FP receptor activation and COX-2 expression by latanoprost, further experiments revealed a significant contribution of calcium and protein kinase C to this pathway.
To provide evidence for further targets within the latanoprost-mediated pathway of COX-2 induction, our interest has focused on the MAPK signaling. Western blot analysis using antibodies specific for phospho-p38 MAPK and phospho-p42/44 MAPK revealed an activation of both kinases by latanoprost. Consistent with these findings, latanoprost-induced COX-2 expression was markedly suppressed by SB203580, a selective p38 MAPK inhibitor, and PD98059, a specific inhibitor of p42/44 MAPK activation, confirming that both p38 and p42/44 MAPKs play a crucial role in mediating up-regulation of COX-2 by latanoprost.
Though the precise mechanism by which latanoprost lowers IOP is unknown, increases in the biosynthesis of certain MMPs and subsequent remodeling of the extracellular matrix appear to be critical for latanoprost’s facilitating action on water flow through the ciliary muscle. In an attempt to link the expression of COX-2 with that of MMPs and to provide evidence for a potential target of PGs synthesized upon treatment of NPE cells with latanoprost, the expression of MMPs was focused on in further experiments. RT-PCR and Western blot analyses revealed an induction of MMP-1 by latanoprost. The stimulatory effect of latanoprost was specific in that latanoprost did not induce the expression of other members of the MMP and TIMP family. The expression of TIMP-1 remained unaffected, suggesting that the availability and subsequent action of newly synthesized MMP-1 is not impaired in the investigated cells. There are several lines of evidence supporting a function of COX-2 in MMP-1 expression by latanoprost: 1) inhibition of COX-2 activity by NS-398 was associated with a significant reduction of latanoprost-induced MMP-1; 2) transfection of cells with COX-2 siRNA mimicked the inhibitory effect of NS-398 on latanoprost-induced MMP-1 expression; 3) induction of MMP-1 mRNA expression had a delayed onset and was not evident at early times of COX-2 mRNA formation when induction of PGE2 production had not yet occurred; and 4) a role for COX-2 as effector of MMP-1 expression was supported by our observation that incubation of NPE cells with PGE2, a major product of the COX-2 pathway, mimicked the stimulatory effect of latanoprost on MMP-1 expression.
Other investigations showing induction of MMP and TIMP synthesis by latanoprost have been performed before in several tissues involved in uveoscleral outflow such as ciliary muscle, iris root, anterior choroids, and sclera. However, this study provides the first evidence for a significant contribution of de novo synthesized PGs to the MMP-1-inducing action of latanoprost. Since COX-2 expression and formation of outflow-facilitating PGE2 is significantly impaired in patients with POAG, stimulation of COX-2 expression appears to be a conceivable and meaningful mechanism underlying the pharmacological action of an antiglaucomatous drug. As the NPE is the primary source of aqueous humor, MMPs synthesized by these cells and secreted into the aqueous humor subsequently, could theoretically be involved in the degradation of extracellular matrix of both uveoscleral and trabecular tissues (Fig. 3
). MMP-1, also referred to as interstitial collagenase, efficiently targets a specific site found in the fibrillar collagen types I and III. In the case of the uveoscleral pathway, collagen types I and III have been described as the major components of the fibrillar collagen in the extracellular matrix of the interstitial spaces among ciliary muscle fiber bundles. Effects of PGs on trabecular outflow facility have also been reported, although this topic is presently controversially discussed and requires further investigation. Our data raise the question of whether the use of COX inhibitors prior to or concurrent with latanoprost may attenuate the therapeutic efficacy of this compound. This issue merits further investigation given that COX inhibitors are often co-administered in elderly patients suffering from POAG. An involvement of PG formation in the antiglaucomatous action of latanoprost was recently suggested by a clinical study showing that topical application of the COX inhibitor bromfenac sodium diminished the IOP-lowering action of latanoprost in healthy volunteers.
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In summary, we have demonstrated for the first time that latanoprost is capable of activating the COX-2 pathway in NPE cells. Increases in COX-2 activity result in an enhanced expression of MMP-1 that has been related to IOP decrease by facilitating the outflow of aqueous humor. Therefore, this novel pathway seems to be of functional relevance and may contribute to the ocular hypotensive effect of latanoprost. In light of our investigations, activation of COX-2 expression and subsequent formation of PGs could be an important mechanism by which latanoprost exerts its pharmacological action in patients with POAG.
FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-3626fje;
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