Abstract
Purpose: :
Cytochrome P450 1b1 (Cyp1b1) is a member of the cytochrome P450 super family of proteins with mono-oxygenase activity. Although mutations in Cyp1b1 gene have been reported in patients with congenital glaucoma, the role Cyp1b1 plays in the development and function of trabecular meshwork (TM) remains unknown. Here we have determined the impact of Cyp1b1 deficiency on TM cell function.
Methods: :
Primary cultures of TM cells were prepared from Cyp1b1+/+ and Cyp1b1-/- mice. The constitutive and inducible expression of Cyp1b1 protein was confirmed by Western blotting. The impact of Cyp1b1-deficiency on proliferation was evaluated using EdU DNA labeling as well as counting the number of cells. The rates of apoptosis, both basal and under oxidative challenge, was determined using TUNEL Click-iT and caspase 3/7 assays. The adhesion to various matrix proteins, cell contractility, cytoskeletal organization, and production of various extracellular matrix proteins were also evaluated.
Results: :
Cyp1b1-/- TM cells exhibited a significant increase in adhesion to various matrix proteins, especially collagens, and were less contractile. They also expressed an increased level of osteopontin but lower levels of tenascin C compared to Cyp1b1+/+ cells. The Cyp1b1-/- TM cells were also significantly more sensitive to oxidative stress compared to Cyp1b1+/+ cells.
Conclusions: :
Cyp1b1 deficiency has a significant impact on TM cell adhesive and contractile properties, and its metabolic activity may contribute to oxidative homeostasis, development and function of TM.
Keywords: trabecular meshwork • cytoskeleton • cell adhesions/cell junctions