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A. M. Fitzgerald, C. Benz, A. F. Clark, R. J. Wordinger; Follistatin and Activin Expression in Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2010;51(13):3212.
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Glaucoma is a leading cause of blindness. Increased intraocular pressure is a major risk factor for glaucoma, resulting from increased outflow resistance of aqueous humor (AH) through the trabecular meshwork (TM). Transforming growth factor beta-II (TGF- β2) levels are increased in the AH and TM of glaucoma patients. This increased TGF- β2 up-regulates extracellular matrix proteins (ECM) and there deposition. Our lab has reported that TGF-β2 increases the expression of BMP antagonist gremlin in TM cells. Gremlin blocks the BMP inhibitory effects on TGF- ß2 induced ECM deposition. Follistatin (FST), a secreted BMP antagonist, is an important inhibitor of BMPs and activins (Act). Act are ligands in the TGF- β superfamily that play a role in cell growth and regulation. The purpose of the present study is to determine in cultured human TM cells (HTM): A)mRNA and protein expression of FST and Act, B) effects of TGF-β2 on FST expression and C) whether FST affects ECM proteins.
HTM cells were cultured and treated with or without recombinant human TGF-β2 protein for 12-72 hours, and with varying concentrations of recombinant human FST for 48 hrs. Total RNA was isolated and Reverse-Transcriptase Polymerase Chain Reaction (RT-PCR) was used to examine the expression of FST and Act. Western blotting and immunocytochemistry (IHC) was used to study the protein levels of FST, and ECM proteins in the presence and absence TGF-β2.
FST and Act A mRNA were expressed in HTM cells. TGF-β2 increased FST and Act A mRNA expression. TGF-β2 induced FST mRNA levels were associated with increased FST proteins determined by western blotting and IHC. Multiple forms of secreted FST were detected. The most active form of secreted FST (288) was increased in a time dependent manner upon TGF-β2 stimulation. FST up-regulated fibronectin and PAI-1 in a dose dependent manner.
This is the first report of FST and Act A expression in HTM cells. These data suggest that TGF-β2 increases the production and secretion of FST. This BMP/Act antagonist regulates the expression of ECM proteins.
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