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Metalloproteinase gene transcription in human ciliary muscle cells with latanoprost
Robert N Weinreb1, James D Lindsey
1Glaucoma Center, University of California San Diego, 9500 Gilman Drive, La Jolla, CA 92093-0946, USA. weinreb@eycenter.ucsd.edu
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.
