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Mini Aga, John M. Bradley, Mary J. Kelley, Ted S. Acott; Gleevec Alters Endosomal Arrangement at PILS and Inhibits Extracellular Matrix (ECM) Degradation in Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3251.
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© ARVO (1962-2015); The Authors (2016-present)
Podosome- or invadopodia-like structures (PILS), specialized complexes at the basal surface of trabecular meshwork (TM) cells, are sites of adhesion and extracellular matrix turnover. Very little is known about PILS mediated regulation of ECM and aqueous humor outflow facility. Studies were conducted to understand the role of endosomes and ECM regulation at PILS using Gleevec.
Effects of mechanical stretch and Gleevec on PILS component localization and matrix degradation were determined by confocal microscopy and live cell imaging of cultured porcine TM cells. Cellular extracts were analyzed by western immunoblotting.
Mechanical stretch increases cortactin and Rab4 levels within 5 minutes. Exposure to Gleevec attenuated this increase. PILS were more diffuseand lattice-like after Gleevec treatment. Actin fibers became more cortical in distribution; microtubules thickened and shortened to encase vesicular structures, which stained positive for various endosome markers. After 15 minutes of stretch or Gleevec treatment EEA1, the very early endocytic marker, appears to be unchanged. However, a more diffused and perinuclear distribution of Rab4 and Rab5 was observed in TM cells. Interestingly, Rab7 (a late endosome marker involved in sorting) levels increased with stretch. This was inhibited by Gleevec at 15 minutes. Both Rab9 and Rab21 had a perinuclear distribution in untreated cells. Their levels were reduced after mechanical stretch as well as Gleevec treatment. Mechanical stretch increased cortactin and Rab4 levels within 10 minutes but Gleevec inhibited the levels of both proteins. Furthermore, stretch as well as Gleevec enhanced ERK1/2 activity within 15 minutes. Gleevec also decreased cell motility and ECM degradation within one hour of exposure. Stretching PTM cells for 5 days increased tenacin-C and thrombospondin matrix protein expression. Gleevec treated TM cells had shorter and disrupted tenacin-C fibrils, however an increase in thrombospondin expression was observed in response to Gleevec.
Mechanical stretch and Gleevec change the organization of TM cell PILS. Gleevec appears to regulate the change in ECM expression and degradation by altering the vesicular trafficking of endosomes and modulation of the ERK pathway.
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