MMP-7 knock-in corneal fibroblast cell lines secrete MMP-7 with proteolytic activity towards collagen XVIII

Abstract

Purpose: To determine whether matrix metalloproteinase-7 (MMP-7) that is stably overexpressed by mouse corneal fibroblast cell lines exhibits proteolytic activity against the NC1 fragment of collagen XVIII.

Methods: Corneal fibroblasts isolated from MMP-7 knockout (7ko) mice were subjected to SV40 T-antigen immortalization and stably transfected with a bicistronic retroviral vector encoding green fluorescence protein and active MMP-7. The resulting MMP-7 knock-in fibroblasts (7ko-MMP-7 cells) were isolated and enriched by fluorescence activated cell sorting (FACS). Culture media samples from 7ko and 7ko-MMP-7 cells were then incubated with the recombinant NC1 fragment of collagen XVIII, and NC1 degradation was monitored by immunoblotting.

Results: Immunoblot analysis revealed that MMP-7 was present in lysates and culture media from 7ko-MMP-7 fibroblasts, but not media from immortalized 7ko fibroblasts. Importantly, lower amounts of the NC1 fragment were present in in vitro enzymatic reaction mixtures containing concentrated 7ko-MMP-7 media than in those containing concentrated 7ko media.

Conclusion: Immortalized fibroblasts stably transfected with MMP-7 secrete active MMP-7 with proteolytic activity towards the NC1 fragment of collagen XVIII.

FIGURE 1

PCR analysis of the MMP-7 allele in 7ko and WT cell lines. Bands of 535 bp and 685 bp were expected for 7ko and WT cell lines, respectively (A). No immunostaining for MMP-7 was observed in WT or 7ko immortalized fibroblasts [(B) and (C); PI nuclei, red], and MMP-7 was localized to the cytoplasm of the immortalized WT corneal epithelial cells (D).

FIGURE 2

Characterization of immortalized WT and 7ko fibroblasts. WT and 7ko fibroblasts were visualized by phase contrast [WT in (A), 7ko in (B)] and immunostained for vimentin [WT in (C), 7ko in (D)], integrin α5β1 [WT in (E), 7ko in (F)], and epithelial keratin [using AE1/AE3 antibody; WT in (I), 7ko in (J)]. A subset of cells was stimulated with TGF-β and stained for α-SMA (myofibroblast marker) [WT in (G), 7ko in (H)]. Corneal epithelial cells immunostained for epithelial keratin served as a positive control (K), and TGF-β-stimulated corneal epithelial cells immunostained for α-SMA served as a negative control (L). Scale bar, 50 μm.

FIGURE 3

Enrichment of GFP-expressing cells with FACS. Initial sorting yielded only 1.65% GFP-expressing 7ko-MMP-7 fibroblasts (A) and 3.11% 7ko-vector fibroblasts (B). After three sequential sortings, the population of GFP-expressing cells exceeded 90% purity for both the 7ko-MMP-7 (C) and 7ko-vector group (D).

FIGURE 4

GFP and MMP-7 expression in cultured fibroblasts. Phase contrast images of 7ko (A), 7ko-vector (B), and 7ko-MMP-7 knock-in fibroblasts (C). No GFP expression was observed in 7ko fibroblasts (E). Confocal images of 7ko-vector (F) and 7ko-MMP-7 (knock-in) fibroblasts [(G) and (H)]. There was detectable MMP-7 immunostaining in 7ko-MMP-7 knock-in fibroblasts (D).

FIGURE 5

Immunoblot analysis of MMP-7 expression in media (lanes 1–3) and lysates (lanes 4–6) from WT, 7ko, and 7ko-MMP-7 fibroblasts (A). The 7ko and WT groups served as negative controls. Immunoblot analysis of collagen XVIII NC1 degradation induced by media collected from 7ko, 7ko-vector, and 7ko-MMP-7 fibroblast cultures (B). Three independent experiments were performed to quantify the intensity of remaining, unprocessed NC1 fragments. The NC1 fragment density in the 7ko-MMP-7 group is 0.72 ± 0.15% of that of the control 7ko group (C).