Abstract
Purpose: :
. Stromal scarring due to corneal trauma, infection, or refractive surgery is the result of a complex cascade of multiple growth factors, cytokines, chemokines, and proteases. The transforming growth factor-β (TGF-β) system has been determined to play a key role in the formation of scar tissue. Furthermore, it is now known that connective tissue growth factor (CTGF) is a fibrogenic cytokine that is a down stream mediator of many of the fibrotic actions of TGF-β, including stimulation of synthesis of extracellular matrix and differentiation of fibroblasts into myofibroblasts. CTGF has the ability to stimulate two opposing and mutually exclusive biological functions, proliferation and differentiation. These diverse functions may be associated with proteolytic processing of CTGF.
Methods: :
Human corneal fibroblasts (HCF) that were serum starved for 48 hours were stimulated with 5ng/mL of TGF-β1. At 0, 6, 12, 24, 48, and 72 hours, cell extracts and conditioned media were removed and stored with the addition of a protease inhibitor cocktail. In a second experiment, serum starved HCF cultures had differing amounts of protease inhibitor cocktails with or without EDTA added. After 48 hours, the cells were stimulated with 5ng/mL of TGF-β1. At 24 hours, cell extracts and conditioned media were removed. All collected samples were separated by SDS-PAGE and analyzed using western blots.
Results: :
The addition of TGF-β1 to the HCF increased levels of full length (~38kDa) CTGF in both the cell extracts and conditioned media. In serum starved media, higher molecular weight bands (~75 &150kDa) were observed, but with the addition of TGF-β1 these bands decreased. Also, the addition of TGF-β1 caused the level of the ~18kDa CTGF fragment to decrease, whereas the ~20kDa CTGF fragment remained constant. Finally, the addition of the any protease inhibitor cocktail reduced levels of ~20kDa CTGF fragment in serum starved media.
Keywords: wound healing • growth factors/growth factor receptors • protein structure/function