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M. Aga, J. M. Bradley, M. J. Kelley, T. S. Acott; Src Signaling and Trabecular Meshwork Podosome- or Invadopodia-Like Structures (PILS). Invest. Ophthalmol. Vis. Sci. 2009;50(13):4869.
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© ARVO (1962-2015); The Authors (2016-present)
Extracellular matrix (ECM) turnover by trabecular meshwork (TM) cells is critical to intraocular pressure homeostasis. Focal ECM turnover, mediated by TM cells, appears to occur at podosome- or invadopodia-like structures (PILS), which are rich in matrix metalloproteinase (MMP)-2 and -14. We evaluated the possible involvement of Src and ERK signaling pathways in controlling these specialized adhesion and degradation structures in TM cells.
Cultured porcine and human TM cells were subjected to mechanical stretching, the Src inhibitor PP2, and the MEK inhibitor U0126. Confocal immunohistochemistry was used to evaluate treatment effects on localization of MMP-2, MMP-14, cortactin, actin isoforms, Src, ERK1/2 and phosphorylated forms of cortactin, Src and ERK.
Cortactin colocalized with MMP-2 and MMP-14, at PILS in TM cells. Of the actin isoforms, smooth muscle actin was more prevalent at PILS than either γ or β1. Stretching TM cells for 2, 24 and 48 hours increased phosphorylation of cortactin at Y466/470 and Src at Y416, particularly as they were associated with TM cell PILS. Much of the total Src also colocalized with cortactin at PILS, while phospho-ERK1/2 did not. PP2, U0126 and stretch increased colocalization of cortactin with MMP-2 and MMP-14 at PILS and appeared to increase the number and size of PILS. However, these inhibitors had a more profound effect on the shape and structural organization of PILS. PILS are seen as small discrete dots, 5 µm circular rosettes, larger irregular rings, lattice- like structures and irregular triangular and rhombus shaped structures. These inhibitors appear to change the distribution between these PILS forms, although they have not yet been clearly categorized in terms of functional differences or progression sequences. They may also actually modify the turnover or stability of PILS.
TM cell PILS are affected by Src and ERK signaling pathways, particularly in conjunction with mechanical stretch. Auto-phosphorylation of Src at Y416 and Src phosphorylation of cortactin at Y466/470 appear to be key steps in regulating TM cell PILS structures. ERKs can affect PILS structures, but ERK1/2 do not colocalize with them. These changes in PILS shape and structural organization may reflect a functional progression of these ECM turnover units, modulated by mechanical stretch, Src and ERK pathways.
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