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Mini Aga, Samuel J Berk, John M Bradley, Ted S Acott; Novel extracellular protein kinases Fam20A and Fam20C differentially distribute with MMP14 at PILS in TM cells. Invest. Ophthalmol. Vis. Sci. 2016;57(12):4701. doi: https://doi.org/.
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© ARVO (1962-2015); The Authors (2016-present)
To examine the relationship of Fam20A, B & C, a family of kinases involved in phosphorylation of extracellular matrix (ECM) proteins, with MMP14 and other podosome- or invadopodia-like structure (PILS) components, which are involved in mediating focal ECM turnover.
Porcine trabecular meshwork (PTM) cells were subjected to mechanical stretch on collagen-coated flex cell plates and/or treated with the matrix metalloproteinase 14 (MMP14) dimerization inhibitor (NSC405020). For substrate degradation assays, cells were cultured on FITC-labeled collagen-coated flex cell plates. Confocal microscopy, immunohistochemistry, and western immunoblots were used to evaluate changes in levels or distribution. Human anterior segments were also used for microarray analyses.
Mechanical stretch for 2 hours increased collagen degradation at PILS and at perinuclear regions beneath the Golgi apparatus. Partial colocalization of Fam20A and Fam20C with MMP14 was observed at these regions. NSC405020 modestly reduced collagen degradation at PILS. Confocal imaging showed strong colocalization of Fam20A with cortactin, particularly at smoke ring-shaped PILS, following mechanical stretch or NSC405020 treatment. Fam20C was found primarily in the perinuclear region or at cortical regions of the cell and showed only minor colocalization with Fam20A or MMP14. Mechanical stretch augmented association of Fam20A/20C with each other and with tenascin-c, versican, fibronectin and the v7 isoform of CD44. Microarray analyses showed high expression levels of all three Fam20 transcripts. Western immunoblots showed Fam20C in TM cell culture media, while Fam20B was not detected, and Fam20A determinations were inconclusive.
Microarray studies showed high transcript levels for all three Fam20 kinases. Mechanical stretch dramatically increased Fam20A colocalization with MMP14 and cortactin at PILS and this correlated with substratum degradation regions. Interestingly, Fam20A and Fam20C colocalization was also observed with, tenascin-c, versican and v7-CD44 isoform and this was increased with stretching. Reportedly, Fam20C has protein kinase activity and Fam20A does not, but increases Fam20C activity. Our studies suggest that Fam20A may recruit Fam20C to specific substrates at PILS or that Fam20A has an independent function at PILS.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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