Anthony Todd L, Lindsey James D, Weinreb Robert N
Hamilton Glaucoma Center, University of California San Diego, La Jolla 92093, USA.
Invest Ophthalmol Vis Sci. 2002 Dec;43(12):3705-11.
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.
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.
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).
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.
确定用拉坦前列素治疗对培养的人睫状肌（HCM）细胞中金属蛋白酶组织抑制剂（TIMP）-1和-2的影响。
将汇合的血清饥饿的HCM细胞暴露于浓度递增的拉坦前列素酸（LA，1 nM至10 μM）中6、18和24小时。通过逆转录聚合酶链反应（RT-PCR）评估TIMP-1和-2信使核糖核酸（mRNA）转录本。用明胶酶谱法测量培养基中基质金属蛋白酶（MMP）量的变化。为了评估蛋白激酶C（PKC）的潜在作用，在不存在或存在PKC抑制剂双吲哚基马来酰亚胺I（Bis I）或蛋白激酶A（PKA）抑制剂KT5720的情况下，用佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）处理HCM细胞。数据通过光密度测定法定量，并采用学生-纽曼-库尔斯检验进行统计分析。
在HCM细胞的原代培养物中检测到TIMP-1和-2 mRNA转录本及蛋白质。TIMP-1 mRNA水平在6小时时未改变，但在暴露于1 μM和10 μM LA 18小时的培养物中分别增加了45%±17%和54%±13%（n = 3）。相比之下，暴露于LA 6小时使TIMP-2 mRNA表达最多增加11.3%±0.2%（n = 3）。然而，在18或24小时时未观察到TIMP-2 mRNA的显著诱导（n = 3）。暴露于LA 18和24小时的培养物中TIMP-1蛋白显著增加。相比之下，在处理6、18和24小时时，TIMP-2蛋白表达与对照培养物无显著差异。暴露于PMA 24小时的HCM细胞产生的TIMP-1 mRNA水平增加与拉坦前列素相似（n = 5）。然而，未观察到TIMP-2 mRNA的显著诱导。对这些培养物的培养基进行酶谱分析证实，在6、18和24小时时MMP-1呈剂量依赖性增加，而仅在暴露于LA 24小时后才观察到MMP-2呈剂量依赖性增加（n = 3）。暴露于100 nM LA和100 nM PMA 24小时的细胞培养基中TIMP-1蛋白水平分别增加了27%±9.3%和15%±11%（n = 5）。LA诱导的TIMP-1蛋白增加基本上被Bis I消除（n = 3），且不受KT5720影响（n = 3）。
在很大程度上，TIMP-1而非TIMP-2在暴露于拉坦前列素后对葡萄膜巩膜流出途径中的MMP起调节作用。此外，这种诱导似乎是由PKC的激活介导的。
