Abstract
Purpose::
Myocilin (MYOC) is the protein product of a glaucoma-associated gene. MYOC appears extracellularly in vitro and in vivo in association with extracellular vesicles called exosomes, which function in the transport of lipid-associated ligands between cell neighbors. The goal of the present study was to determine ways to regulate the release of MYOC associated exosomes from trabecular meshwork (TM) cells.
Methods::
Conditioned media from TM cells were analyzed for MYOC associated exosomes after treatment with dexamethasone (1µM) or γ-interferon (500 units/mL) for 5 days. Exosomes from conditioned medium were purified by differential centrifugation as described previously. Proteins in exosome and soluble fractions were separated by SDS-PAGE and analyzed for MYOC content by western blotting.
Results::
The appearance of extracellular MYOC in association with exosomes isolated from dexamethasone treated TM cell conditioned media increased by 261% and the total extracellular fraction increased 37.9%. Treatment with γ- interferon resulted in the appearance of the exosome marker HLA-DR and increased MYOC association with exosomes by 40.5% over untreated cells in culture. By comparison, total extracellular MYOC decreased by 29.5% upon γ- interferon treatment.
Conclusions::
Both dexamethasone and γ-interferon significantly upregulate the expression of MYOC and its association with exosomes in conditioned media from TM cells.
Keywords: trabecular meshwork • proteins encoded by disease genes • protein purification and characterization