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Prostaglandins increase matrix metalloproteinase release from human ciliary smooth muscle cells
R N Weinreb1, K Kashiwagi, F Kashiwagi
1Glaucoma Center, University of California San Diego, La Jolla 92093, USA.
Purpose:
To identify matrix metalloproteinases (MMPs) released by ciliary smooth muscle cells in vitro and to determine whether MMP release is altered by exposure to prostaglandins (PGs).
Methods:
Human ciliary smooth muscle cells were grown to confluence in monolayer cultures and treated with PGF2 alpha, 11-deoxy-PGE1, or PhXA85 (the nonesterified analogue of PhXA41) for 12 to 72 hours. The activity of MMP in the medium was assayed using gelatin and casein zymography. Identification of the specific MMP associated with each band was made by Western blot analysis. Band intensity, which reflects activity, was measured with a scanning laser densitometer.
Results:
Three major bands appeared in the gelatin zymographs at positions corresponding to molecular weights of 62 kDa, 68 kDa, and 97 kDa. A single band at 50 kDa predominated in the casein zymograms. Substitution of EDTA for calcium and zinc in the development solution eliminated the appearance of these bands, indicating that they reflect MMP activity. Immunoblotting, using MMP-specific antibodies, confirmed that the three bands in the gelatin zymographs were MMP-1, MMP-2, and MMP-9, respectively; the single band in the casein zymographs was MMP-3. Treatment with 200 nM PGF2 alpha, 11-deoxy-PGE1, or PhXA85 for 72 hours increased the combined density scores for MMP-1 and MMP-2 by 37%, 64%, and 27%; the density scores for MMP-9 by 268%, 253%, and 125%; and the density scores for MMP-3 by 35%, 71%, and 22%, respectively.
Conclusions:
These results indicate that ciliary smooth muscle cells can secrete MMP-1, MMP-2, MMP-3, and MMP-9. In addition, exposure to PGF2 alpha, 11-deoxy-PGE1, or PhXA85 increases production of all four MMPs. These observations support the hypothesis that increased MMP production by ciliary muscle cells has a role in increasing uveoscleral outflow facility after topical PG administration.
Insights
Ciliary smooth muscle cells release matrix metalloproteinases (MMPs). Prostaglandins (PGs) significantly increase the production of MMP-1, MMP-2, MMP-3, and MMP-9, suggesting a role in uveoscleral outflow.
Area of Science:
- Ophthalmology
- Cell Biology
- Biochemistry
Background:
- Ciliary smooth muscle cells play a role in ocular physiology.
- Matrix metalloproteinases (MMPs) are enzymes involved in tissue remodeling.
- Prostaglandins (PGs) are used therapeutically in ophthalmology.
Purpose of the Study:
- To identify specific matrix metalloproteinases (MMPs) secreted by human ciliary smooth muscle cells in vitro.
- To investigate the effect of prostaglandin (PG) exposure on MMP release from these cells.
Main Methods:
- Human ciliary smooth muscle cells were cultured and treated with different prostaglandins (PGF2 alpha, 11-deoxy-PGE1, PhXA85).
- Matrix metalloproteinase activity was assessed using gelatin and casein zymography.
- Specific MMPs were identified and quantified using Western blot analysis and scanning laser densitometry.
Main Results:
- Ciliary smooth muscle cells secreted MMP-1, MMP-2, MMP-3, and MMP-9.
- Treatment with PGF2 alpha, 11-deoxy-PGE1, or PhXA85 significantly increased the production of all identified MMPs.
- The most substantial increases were observed in MMP-9 production.
Conclusions:
- Human ciliary smooth muscle cells are a source of MMP-1, MMP-2, MMP-3, and MMP-9.
- Prostaglandin administration enhances the secretion of these MMPs.
- Increased MMP production may contribute to enhanced uveoscleral outflow following topical PG treatment.