Matrix Metalloproteinase-2 Activated by Ultraviolet-B Degrades Human Ciliary Zonules In Vitro

Abstract

The ciliary zonules, also known as the zonules of Zinn, help to control the thickness of the lens during focusing. The ciliary zonules are composed of oxytalan fibers, which are synthesized by human nonpigmented ciliary epithelial cells (HNPCEC). The ciliary zonules are exposed to ultraviolet (UV), especially UV-A and UV-B, throughout life. We previously demonstrated that UV-B, but not UV-A, degrades fibrillin-1- and fibrillin-2-positive oxytalan fibers. However, the mechanism by which UV-B degrades oxytalan fibers remains unknown. In this study, we investigate the involvement of matrix metalloproteinase-2 (MMP-2) in the UV-B-induced degradation of fibrillin-1- and fibrillin-2-positive oxytalan fibers in cultured HNPCECs. Enzyme-linked immunosorbent assay revealed that UV-B irradiation at levels of 100 and 150 mJ/cm² significantly increased the level of active MMP-2. Notably, MMP-2 inhibitors completely suppressed the degradation of fibrillin-1- and fibrillin-2-positive oxytalan fibers. In addition, we show that UV-B activates MMP-2 via stress-responsive kinase p38. Taken together, the results suggest that UV-B activates a production of active type of MMP-2 via the p38 pathway, and subsequently, an active-type MMP-2 degrades the fibrillin-1- and fibrillin-2-positive oxytalan fibers in cultured HNPCECs.

Keywords: ciliary zonule; fibrillin; matrix metalloproteinase-2; p38; ultraviolet-B.

Fig. 1.

Degradation of fibrillin-1 and fibrillin-2 by UV-B irradiation. Double immunofluorescence for fibrillin-1 (upper first panels) and fibrillin-2 (second panels) in cultures of human nonpigmented ciliary epithelial cells. The cells were irradiated with the indicated dose of UV-B irradiation and then cultured for 24 hr, and doubly labeled for fibrillin-1 (green; first panels) and fibrillin-2 (red; second panels). Superimposition of both labels is shown in the third panels. DAPI for nuclear staining is shown in the fourth panels. Fibrillin-1-positive oxytalan fibers disappeared at a UV-B irradiation level of 150 mJ/cm² and fibrillin-2-positive oxytalan fibers disappeared at a UV-B irradiation level of 100 mJ/cm² and higher. Bar = 50 μm.

Fig. 2.

UV-B irradiation increased matrix metalloproteinase-2 (MMP-2) in cell lysates. Human nonpigmented ciliary epithelial cells were cultured for 4 weeks and then irradiated with UV-B at levels of 0, 50, 100, and 150 mJ/cm². Then, the cells were cultured for another 24 hr under the same culture conditions. Next, cell/matrix layers were collected at 12 (A) and 24 (B) hr after UV irradiation and analyzed by ELISA. The level of active MMP-2 increased depending on the irradiation level of UV-B. (A) The levels of total and active MMP-2 at 150 mJ/cm² were significantly increased compared to the control (0 mJ/cm²). (A) The levels of total and active MMP-2 at 100 and 150 mJ/cm² were significantly increased compared to the control (0 mJ/cm²). (B) The level of active MMP-2 at 100 mJ/cm² were significantly increased compared to the control (0 mJ/cm²). The amounts of total and active MMP-2 at 150 mJ/cm² were significantly increased compared to the control (0 mJ/cm²). The values for each control sample (0 mJ/cm²) were arbitrarily assigned a value of 1. The results represent the mean ± standard deviation of four independent determinations. **P < 0.05 versus control.

Fig. 3.

Inhibitory effect by addition of MMP-2 inhibitor. Human nonpigmented ciliary epithelial cells were cultured for 4 weeks and then irradiated with UV-B at levels of 0, 50, 100, and 150 mJ/cm². Then, 1.5 μM MMP-2 inhibitor was added to the medium for an additional 24 hr. After 24 hr, the cells were simultaneously labeled for fibrillin-1 (green; first panels) and fibrillin-2 (red; second panels). Superimposition of both labels is shown in the third panels. DAPI for nuclear staining is shown in the fourth panels. Bar = 50 μm.

Fig. 4.

Inhibitory effect by addition of MMP-2/MMP-9 inhibitor. Human nonpigmented ciliary epithelial cells were cultured for 4 weeks and then irradiated with UV-B at levels of 0, 50, 100, and 150 mJ/cm². Then, 50 nM MMP-2/MMP-9 inhibitor was added to the medium for an additional 24 hr. After 24 hr, the cells were doubly labeled for fibrillin-1 (green; first panels) and fibrillin-2 (red; second panels). Superimposition of both labels is shown in the third panels. DAPI for nuclear staining is shown in the fourth panels. Bar = 50 μm.

Fig. 5.

Negative control for the effect of MMP inhibitors. Human nonpigmented ciliary epithelial cells were cultured for 4 weeks and then irradiated with UV-B at levels of 0, 50, 100, and 150 mJ/cm². Then, DMSO at an equivalent concentration (0.01%) as in Figures 3 and 4 in DMEM with 10% serum was added to the medium for an additional 24 hr. After 24 hr, the cells were doubly labeled for fibrillin-1 (green; first panels) and fibrillin-2 (red; second panels). Superimposition of both labels is shown in the third panels. DAPI for nuclear staining is shown in the fourth panels. Bar = 50 μm.

Fig. 6.

UV-B irradiation at 150 mJ/cm² induced p38 activation. (A) Human nonpigmented ciliary epithelial cells were cultured for 24 hr and then irradiated with UV-B at a level of 150 mJ/cm². The cell/matrix layers were collected at 0, 5, 10, 15, and 30 min after UV irradiation and analyzed by Western blot against p38 and phosphor-p38. The protein levels of p38 did not change, whereas those of phosphor-p38 were maximally detected at 10 min. (B) 10 min after irradiation, the cells were labeled for phosphor-p38. Phospho-p38 is labeled in the nucleus at 150 mJ/cm², whereas it is not labeled at 0 mJ/cm². Bar = 50 μm.

Fig. 7.

UV-B irradiation increases the level of MMP-2 via p38 activation. Human nonpigmented ciliary epithelial cells were cultured for 24 hr and then the cells pretreated with SB203580 (p38 inhibitor) were irradiated with UV-B at levels of 0, 50, 100, and 150 mJ/cm². Then, the cells were cultured for another 12 and 24 hr under serum-free culture conditions. (A) Cell/matrix layers were collected 10 min after UV irradiation and analyzed by Western blot against phosphor-p38. The level of phosphor-p38 treated with SB203580 was inhibited at the same level as the control (0 mJ/cm²). (B) Immunofluorescence showed that phosphor-p38 was labeled in the cells at 150 mJ/cm², but not in SB203580-treated cells. Cell/matrix layers were collected at 12 (C) and 24 (D) hours after UV irradiation and analyzed by ELISA. The active MMP-2 in the SB203580-treated cells was suppressed compared with the SB203580-untreated vehicle cells. The values for each control sample (0 mJ/cm²) without SB203580 were arbitrarily assigned a value of 1. The results represent the mean ± standard deviation of four independent determinations. **P < 0.05 versus control.