Matrix Metalloproteinase 19 Regulates Insulin-like Growth Factor-mediated Proliferation, Migration, and Adhesion in Human Keratinocytes through Proteolysis of Insulin-like Growth Factor Binding Protein-3

Abstract

Unlike most other matrix metalloproteinases (MMPs) MMP-19 is expressed in undifferentiated basal keratinocytes of healthy human skin. The human keratinocyte cell line HaCaT, which like basal keratinocytes constitutively expresses MMP-19, down-regulated the expression of MMP-19 at high calcium concentrations. Calcium-regulation occurred through E-cadherin mediated cell-cell contacts because neutralizing anti-E-cadherin antibodies restored MMP-19 expression in high calcium. Overexpression of MMP-19 in HaCaT cells (HaCaT-WT) increased cellular proliferation, as well as migration and adhesion on type I collagen. This was due to proteolysis of the insulin-like growth factor (IGF) binding protein-3 by MMP-19, which augmented signaling through the IGF-I receptor, as evidenced by its increased autophosphorylation. Conversely, these effects were not observed in cells transfected with MMP-2 or a catalytically inactive MMP-19 mutant. As further proof that increased IGF-signaling promoted adhesion and migration in HaCaT-WT cells, we reproduced these effects by treating parental HaCaT with IGF-I. We observed dephosphorylation of the focal adhesion kinase in HaCaT-WT as well as IGF-I-treated HaCaT cells, suggesting that inactivating focal adhesion kinase is a mechanism by which IGF-I enhances adhesion. Furthermore, IGF-I-triggered motility on type I collagen was mediated by MMP activity, which, however, was distinct from MMP-19. Considering the coexpression of IGFBP-3 and MMP-19 in the skin, we conclude that MMP-19 is a likely candidate to be the major IGFBP-3 degrading MMP in the quiescent epidermis. This activity might have widespread consequences for the behavior of epidermal keratinocytes.

Catalytic activity of matrix metalloproteinase‐19 is essential for tumor suppressor and anti‐angiogenic activities in nasopharyngeal carcinoma

“…8,17 Our current study provides the first evidence to demonstrate the anti-angiogenesis effect of MMP19 is associated with its catalytic activity through the inhibition of VEGF production. When the catalytic activity of MMP19 is disrupted by a single point mutation in the zinc-binding motif, 12,32 then suppression of tube-formation in HUVEC and HMEC-1 cells, and VEGF production in tumor cell is abolished. MMP19 was reported to efficiently cleave VEGF into smaller fragments.…”

“…20 In brief, slides were incubated with human MMP-19 CK8/4 antibody at 1:3000 dilution. 12 The positive control for MMP19 staining was lung adenocarcinoma. Replacing the primary antibody with blocking serum served as the negative control (Supporting Information Figure 1).…”

“…NPC cell lines were treated with 5 lM 5-Aza-dC (Sigma) every day for 5 days and harvested for RNA or conditioned media. 26 Transfection and colony formation assay (CFA) The 1.6 kb human WT full-length MMP19 cDNA and mutant MMP19 (MMP19 EA) 12 were cloned into pCR3.1 (Invitrogen). The inactive mutant (EA) was previously constructed by site-directed mutagenesis to exchange glutamic acid (E) for alanine (A) at position 213 of MMP19 to disrupt the zinc-binding motif and inactivate the catalytic function of MMP19.…”

“…11 A mutation in the zinc-binding motif from glutamate (E) to alanine (A) causes a loss of MMP19 function in cell proliferation, migration, and protein degradation in a human keratinocyte cell model. 12 MMP19 was first isolated as an autoantigen from the synovium of a rheumatoid arthritis patient. 13 In contrast to most MMPs, MMP19 is expressed in many types of normal tissues such as lung, pancreas, placenta, ovary, spleen, intestine, breast, and skin, but is down-regulated or lost during neoplastic progression in breast, skin, and colon carcinomas.…”

“…Functional assays were performed using NPC cell lines transfected with wild-type (WT) and mutant MMP19, which contains a single point mutation in the zinc-binding motif to inactive the catalytic function. 12 In vivo nude mouse tumorigenicity, in vitro colony formation, tube formation assays together with ELISA quantitation of VEGF and blood vessel staining in tumor sections were performed to demonstrate the importance of the catalytic activity of MMP19 in anti-tumor formation and anti-angiogenesis in NPC.…”