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Tara Tovar-Vidales, Ashley Fitzgerald, Abbot Clark, Robert Wordinger; Transforming Growth Factor-β2 (TGF-β2) Induces Expression of Biologically Active Bone Morphogenetic Protein-1 (BMP1) in Human Trabecular Meshwork Cells. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3531.
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© ARVO (1962-2015); The Authors (2016-present)
Previous studies have shown that TGF-β2 increases the deposition of extracellular matrix (ECM) in the trabecular meshwork (TM), which may account for increased aqueous humor outflow resistance in glaucoma. However, there are limited studies on the factors that regulate the processing of TGF-β2 and ECM proteins into their mature form. Bone morphogenetic protein 1 (BMP1) is an enzyme responsible for the cleavage and maturation of growth factors and ECM proteins. The purpose of this study was to determine whether: (a) cultured human TM cells express BMP1, (b) BMP1 expression is regulated by TGF-β2, (c) BMP1 is biologically active, and (d) BMP1 regulates LOX activity.
Primary human TM cell strains were grown until confluent, and total RNA and conditioned medium (CM) were isolated and subjected to qPCR and Western immunoblotting for BMP1. BMP1 immunolocalization was performed in 3 pairs of normal (NTM) and 3 pairs of glaucomatous (GTM) human donor eyes. Human TM cells were treated with or without TGF-β2 (5ng/ml) for 6, 12, 24 and 48 hours in serum free medium. qPCR (NTM=3) was used to determine BMP1 mRNA expression and Western immunoblotting (NTM=3; GTM=2) was used to determine BMP1 protein expression. BMP1 protein secretion was measured in CM of TM cell cultures (NTM=5; GTM=5). BMP1 activity was measured (NTM=3) in TM cells treated with TGF-β2 or with a combination of TGF-β2 /UK383367 at 1, 3, and 5 μM for 24 hours. Lysyl oxidase (LOX) enzyme activity (NTM=3) was evaluated by WB in TM cells treated with BMP1 or with a combination of BMP1/LOX activity inhibitor β-aminoprorionitrile (BAPN) for 48 hours.
Human TM cells expressed mRNA and protein for BMP1. Exogenous TGF-β2 increased mRNA expression at all time points for 6, 12, and 24 hours compared with their controls (p<0.05). GTM BMP1 secretion in the CM were increased compared to NTM (p<0.05). An ELISA showed TGF-β2 induced BMP1 secretion compared to their controls in all cell strains (p<0.05). BMP1 stimulated LOX enzymatic activity in TM cells.
BMP1 is expressed in the human TM. TGF-β2 induction of BMP1 may be responsible for increased processing of growth factors and ECM proteins into their mature forms resulting in TM stiffness and resistance to ECM degradation. BMP1 may be involved in normal TM function as well as the pathogenesis of glaucoma.
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