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Gunther R. Schlunck, Hong Han, Daniel Kampik, Franz J. Grehn; TGF-β2-induced Invadosomes In Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2011;52(14):5326.
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Invadopodia and podosomes (invadosomes) are distinct sites of cell-matrix interaction and localized matrix-metalloprotease (MMP) activity. Their presence in human trabecular meshwork (HTM) cells has been suggested earlier. We studied the effects of TGF-β2 on invadosomes in this cell type to gain further insight into their possible role in trabecular meshwork physiology and glaucoma.
HTM cells were derived from corneal donor buttons. Confluent cultures were stimulated with vehicle or TGF-β2 for 2d. Zymography and Western Blot analysis were performed in parallel to assess MMP activity and protein expression. To study localized gelatinolysis, cells were plated on Oregon-green-tagged gelatin in the presence of MMP-inhibitors and allowed to spread for 24h. Synchronized MMP activation was achieved by MMP inhibitor washout, the cells were fixed 16h later and stained for actin, cortactin, Grb2 or Nck1. Image-J software was used for quantitative assessment of confocal micrographs. ECM transcription was studied by qPCR.
HTM form podosomes and invadopodia as characterized by Grb2 and Nck1 localization to sites of gelatinolysis. TGF-β2 enhances HTM cell gelatinolytic activity and invadosomal matrix digestion with a concomitant increase in expression of PAI-1 and increased transcription of fibronectin and collagens 1, 4 and 6. ROCK inhibiton blocks the TGF-β-induced increase in gelatinolysis and PAI-1 expression.
TGF-β-induced ECM deposition and remodelling are accompanied by an increase rather than inhibition of MMP activity. This underlines the physiological role of MMPs in tissue turnover and sheds a new light on possible mechanisms of ECM deposition in glaucoma. The distinct subcellular localization of MMP activity to invadosomes suggests important contributions of MMP-related signaling to HTM function.
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