CELLULAR FIBRONECTIN SUPRESSES NORMAL HUMAN TRABECULAR MESHWORK CELL METALLOPROTEINASE EXPRESSION AND ACTIVATES TRANSFORMING GROWTH FACTOR-BETA 2 SIGNALING PATHWAY

Glaucoma is one of the leading causes of blindness, set off generally by changes on eye pressure and tissue modifications. One of the common tissue modifications includes induction of extracellular proteins expression on the human eye trabecular meshwork. This study provides important information on one of the tissue changes caused by a factor involved in glaucoma, Transforming Growth Factor-beta 2, the induction of the expression of the extracellular protein fibronectin. Fibronectin is a protein that can be found in two types of forms, a plasma soluble form and a cellular insoluble form. This study showed how interaction with cellular fibronectin form can affect normal human trabecular meshwork cells. This type of tissue modification may be part of the trabecular meshwork changes that can lead to glaucoma. Understanding the tissue changes and factors affecting the eye conditions leading to glaucoma will help in the discovery of agents protecting the eye from glaucoma damage. Purpose (a): The expression of cellular fibronectin isoforms (cFN) are induced by transforming growth factor-beta 2 (TGF-β2) in cultured human trabecular meshwork (TM) cells, and TGF-β2 expression is elevated in glaucomatous TM tissues. Cellular interaction with cFN isoforms can affect extracellular matrix (ECM) homeostasis, as well as the cellular interaction and response to the surrounding microenvironment. Our purpose is to determine the impact of the interaction of normal HTM (NTM) cells with cFN isoforms on the metalloproteinase (MMP) expression and the TGF-β2 signaling pathway. Methods (b): NTM cell strains were cultured for up to 2 days on surfaces coated with cFN, and the responses were compared to control uncoated surfaces. In addition, to show that the EDA domain of cFN was involved, NTM cells were cultured in the presence of anti-EDA antibodies. Changes on gene and protein expression and cellular distribution of MMPs and TGF-β2 signaling pathway components were analyzed using qRT-PCR, Western immunoblots and immunocytochemistry. Results (c): NTM cell strains exposed to cFN isoforms significantly decreased MMP-1 and MMP-3 expression, and this effect was blocked by anti-EDA pre-incubation. cFN significantly altered the expression of TGF-β2 signaling pathway components, including regulatory and inhibitory SMADs. The phosphorylation and nuclear translocation of regulatory SMADs also was increased, indicating activation of the TGF-β signaling pathway. Conclusions (d): Our results demonstrate that NTM cell interactions with cFN isoforms decreases levels of critical components involved in ECM homeostasis. Furthermore, we show that interaction with cFN affects different TGF-β2 signaling components further activating this signaling pathway. In summary, our data suggest that interaction of NTM cells with a glaucoma-like ECM (i.e. cFN) further exacerbates TGF-β2 signaling leading to decreased ECM turnover and fibrosis.