Epidermal growth factor receptor signaling is partially responsible for the increased matrix metalloproteinase-1 expression in ocular epithelial cells after UVB radiation

Abstract

Pterygia are inflammatory, invasive, and proliferative lesions of the human ocular surface in which the matrix metalloproteinase (MMP) collagenase-1 (MMP-1) is highly expressed. Pterygia development may involve MMP-1 activity against interstitial fibrillar collagen, an abundant extracellular matrix component of the cornea, and its induction by ultraviolet light (UVB). We examined the pathways responsible for enhanced expression of MMP-1 in pterygium epithelial cells after UVB exposure and/or treatment with chemical inhibitors of mitogen-activated protein kinases or epidermal growth factor receptor. The induction of MMP-1 by UVB was comparable to that mediated by heparin-binding epidermal growth factor-like growth factor and epidermal growth factor. The epidermal growth factor receptor inhibitor PD153035 partially blocked the UVB-mediated induction of MMP-1 and totally abrogated its production after stimulation with either heparin-binding epidermal growth factor-like growth factor or epidermal growth factor. UVB exposure enhanced the phosphorylated form of ERK1/2 in a time-dependent manner whereas the ERK1/2 inhibitor PD98059 decreased this induction by at least fivefold. Transcripts for c-jun and c-fos were detected as early as 2 hours after UVB exposure and were suppressed by PD98059. The identification of a specific intracellular signaling pathway responsible for the enhanced production of a key enzyme that denatures intact fibrillar collagen has important implications for understanding the pathophysiology and future therapy for pterygia.

Figure 1

Inhibition of MMP-1 by PD98059. PECs were cultured under control conditions, irradiated with 20 mJ/cm² UVB, or treated with either PD98059 (25 μmol/L) or SB203580 (10 μmol/L). Cell supernatants and total RNA were harvested after 48 hours (A and B) or 24 hours (C) and analyzed by enzyme-linked immunosorbent assay (A), gelatin zymography (B), or RT-PCR (C). The arrowhead in B points to the region of gelatinolytic activity expected for latent MMP-9. The bars in A represent the mean ± STD of triplicate experiments. **Significance set at P < 0.001.

Figure 2

PD98059 inhibited the UVB-induced ERK1/2 activation. PECs were seeded at equal density and cultured until 95% confluence was established. Cells were serum starved overnight then either irradiated with 20 mJ/cm² UVB (A and G) or mock irradiated (B), and immediately treated with the ERK inhibitor PD98059 (25 μmol/L final) (C, D, and H). Total cell lysates were prepared at the indicated times ranging from 5 minutes to 7 hours. Western blot analysis was performed (A–D, G, and H) with phospho (P)-specific P-p44/p42 (A–C) and phospho-specific P-p38 (G and H) antibodies or antibodies directed against total (T)-p44/p42 (ERK) (D). Equal protein loading was confirmed by staining the SDS-PAGE gels with Coomassie Blue. The Western blot results were further semiquantified and comparisons made between UVB-irradiated and mock-irradiated PECs (E) and UVB-exposed and PD98059-treated PECs (F) with respect to phospho-ERK (pERK) expression. These results are representative of three different cell lines analyzed.

Figure 3

Localization of ERK1/2 in pterygia. Pterygia (A–I) and normal conjunctiva (J–O) were serially sectioned and stained for total ERK (A, D, G, J, and M) or pERK (B, E, H, K, and N). Control reactions included omitting the primary antibody (C and F) or applying a relevant isotype control IgG antibody (I, L, and O). Tissue sections in A to C and J to L were derived from one subject and tissue in G to I and M to O were derived from another patient. Pterygium tissue in D to F was obtained from a third donor. Red staining denoted positive immunoreactivity. Arrows in D and G indicate a positively stained blood vessel. Whereas the arrows in E and H point to the same unstained vessel. Arrowheads in J and M illustrate mild reactivity for total ERK in the basal conjunctival epithelium, whereas the arrowheads in K and N indicate the absence of any reactivity for phospho ERK (pERK) in the same region of the conjunctiva. This pattern of expression was representative of all diseased and normal tissue specimens analyzed. Original magnifications, ×500.

Figure 4

The intermediate-early genes c-jun and c-fos are induced after UVB. Semiconfluent cultures of PECs were exposed to 20 mJ/cm² UVB. Total RNA was isolated at 0 to 24 hours after UVB and analyzed by RT-PCR to determine MMP-1 (A), c-fos (B), c-jun (C), and GAPDH (D) mRNA expression using gene-specific primers. Control reactions included RNA that was not reverse transcribed (−RT) or reactions in which the primer pairs were omitted (−1°). No products formed in reactions in which a DNA template was not included (not shown). Molecular weight of the products was estimated by comparison to a 100-bp ladder. These results were further semiquantified as fold-increase that was standardized to GAPDH levels (E). These results are representative of three different cell lines analyzed.

Figure 5

Localization of c-jun and c-fos in ocular tissue. Pterygia (A–H) and normal conjunctiva (I–P) were serially sectioned and stained for c-jun (A, E, I, and M), c-fos (B, F, J, and N), and Sp1 (C, G, K, and O). Control reactions included omitting the primary antibody (H and P) or applying a relevant isotype control IgG antibody (D and L). Tissue sections in A to D and I to L were derived from one donor and tissue in E to H and M to P were derived from a second patient. Red staining denoted positive immunoreactivity. To illustrate the extent and location of the immunoreactivity, sections in A to C, E to G, I to K, and M to O were not counterstained. Because no reactivity was observed in any of the control reactions (D, H, L, and P), these sections were counterstained with hematoxylin. This pattern of expression is representative of all diseased and normal tissue specimens analyzed. Original magnifications, ×500.

Figure 6

Differential expression of c-fos in epithelial cells. Semiconfluent cultures of PECs (A) and CECs (B, C) were exposed to 20 mJ/cm² UVB. Total RNA was isolated at 0 to 48 hours after UVB and analyzed by RT-PCR to determine c-fos (A, B) and GAPDH (C) mRNA levels using gene-specific primers. Control reactions included RNA that was not reverse transcribed (−RT) or reactions in which the primer pairs were omitted (−1°). No products formed in reactions that did not include a DNA template (not shown). Molecular weight of the products was estimated by comparison to a 100-bp ladder.

Figure 7

Inhibition of c-jun, c-fos, and MMP-1 mRNA by PD98059. Semiconfluent PECs were exposed to 20 mJ/cm² UVB. Total RNA was isolated at intermediate (2 to 6 hours) and at a late (24 hours) time point after UVB and analyzed by RT-PCR to determine c-fos (A), c-jun (B), MMP-1 (C), and GAPDH (D) mRNA expression using gene-specific primers. Control reactions included RNA that was not reverse transcribed (−RT) or reactions in which the primer pairs were omitted (−1°). Molecular weight of the products was estimated by comparison to a 100-bp ladder. These results were semiquantified and graphically represented as fold-induction for c-jun (E), c-fos (F), and MMP-1 (G) that was standardized to GAPDH levels (D).

Figure 8

Expression of the EGFR and its ligands in pterygia and cultured PECs. Serial sections of pterygium (A–D), cultured PECs (E, F, H, I), or CECs (G and J) were stained for the EGFR (B, E–G), the active phosphorylated EGFR (D, H–J), HB-EGF (C), and EGF (A). Control reactions included omitting the primary antibody (not shown) or applying a relevant isotype control IgG antibody (A, inset). Some cells were mock irradiated [−UVB, (F and I)] or exposed to 20 mJ/cm² UVB [+UVB (E, G, H, J)]. Red staining denoted positive immunoreactivity. All tissue sections and cultured cells were counterstained with hematoxylin. Arrowheads in H and J point to perinuclear immunoreactivity for the phospho-EGFR. This pattern of expression was representative of all diseased tissue specimens and cultured cells analyzed. Original magnifications: ×500 (A–D, E, G, I, J); ×640 (F and H).

Figure 9

Abrogation of MMP-1 production by PD153035. PECs were cultured under control conditions, irradiated with 20 mJ/cm² UVB (A, D, E), stimulated with either recombinant human HB-EGF (B) or EGF (C), and then treated with the EGFR inhibitor PD153035 (A–C), the PDGFR inhibitor AG1295 (D), or the G-protein desensitizer pertussis toxin (E). Supernatants were harvested after 48 hours and analyzed by enzyme-linked immunosorbent assay (A–E) or gelatin zymography (gel insets). Each bar represents the mean ± STD of triplicate experiments. *P < 0.01 or **P < 0.001. The arrowheads point to the region of gelatinolytic activity expected for MMP-9.