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Mini Aga, John M Bradley, Ted S Acott; Matrix metalloproteinase-14 dimerization orchestrates matrix degradation at podosome- or invadopodia-like structures (PILS) in trabecular meshwork.. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3283.
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© ARVO (1962-2015); The Authors (2016-present)
PILS on TM cells are dynamic and distinctive structures where MMP14, MMP2, cortactin, β1 integrin, caveolin 1 and 2, N-WASP and numerous other cellular components colocalize. PILS carry out ECM degradation by proteolysis. Although TM cells usually exhibit 3-10 PILS, but only a subset are actively degrading gelatin at any one time. We previously showed that MMP14 is involved in the extracellular matrix (ECM) remodeling process that mediates the intraocular pressure (IOP) homeostasis effect. Here, we conducted studies on MMP14 regulation and involvement with PILS, attempting to differentiate active PILS from quiescent PILS.
Cultured TM cells were plated either on uncoated Bioptech dishes or on fluorescent-labeled gelatin to assess degradation. A small peptide, NSC405020, was used to block dimerization of MMP14 at its hemopexin domains. Immunohistochemistry was used to identify phosphorylated and specific protein localizations. The effect of NSC405020 was assessed on association of cortactin and MMP14 with PY-99, PY-100, Arg and integrin β1.
PILS markers MMP14 and the other components of colocalization were used as indicators of PILS locations. At 24-48 hrs only a subset of total PILS were observed actively degrading labeled gelatin. Previously we reported that cortactin as well as MMP14 is present at sites of ECM degradation. Blocking MMP14 dimerization using NSC405020 diminishes discernable gelatin degradation as early as 2 hrs and at 24 hrs. Src mediated MMP14 phosphorylation on Y573, in the small cytoplasmic tail, was detected using pY99 or pY100 antibody binding and showed intense localization in the cellular tips of TM PILS-containing structures at the cellular tips. NSC405020 also affects phosphorylation levels of PY 99 and PY100 while it reduces and alters MMP14 and β1 integrin cellular distributions. In this study we observed pCort 421 is partially coincident with sites of degradation as well. Arg levels increased in response to the MMP14 inhibitor treatment.
Although TM cells normally contain 3-10 PILS/cell, only a small portion of PILS are actively degrading gelatin. MMP14 dimerization by their hemopexin domain is required for gelatin degradation. Arg and Integrin β1 might interact to stabilize the PILS (Macpherson et al., 2012 ) and phosphorylation of MMP14 and cortactin may have a role in PILS maturation.
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