Latanoprost's effects on TIMP-1 and TIMP-2 expression in human ciliary muscle cells.
OBJECTIVE
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 micro M) 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 micro M 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.
Anthony
Todd L
TL
Hamilton Glaucoma Center, University of California San Diego, La Jolla 92093, USA.
Lindsey
James D
JD
Weinreb
Robert N
RN
D000959:Antihypertensive Agents / Q000494:pharmacology*; D015153:Blotting, Western; D002478:Cells, Cultured; D002924:Ciliary Body / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D000077338:Latanoprost; D020782:Matrix Metalloproteinases / Q000378:metabolism; D009130:Muscle, Smooth / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D011461:Prostaglandins F, Synthetic / Q000494:pharmacology*; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D012333:RNA, Messenger / Q000096:biosynthesis; D020133:Reverse Transcriptase Polymerase Chain Reaction; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000096:biosynthesis / Q000235:genetics*; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000096:biosynthesis / Q000235:genetics*
D000959:Antihypertensive Agents; D004791:Enzyme Inhibitors; D011461:Prostaglandins F, Synthetic; D012333:RNA, Messenger; D019715:Tissue Inhibitor of Metalloproteinase-1; D019716:Tissue Inhibitor of Metalloproteinase-2; D000077338:Latanoprost; D011493:Protein Kinase C; D020782:Matrix Metalloproteinases; D013755:Tetradecanoylphorbol Acetate