Distribution of myocilin, a glaucoma gene product, in human corneal fibroblasts

Purpose: Myocilin is a gene linked to open-angle glaucomas. In this study, the expression and distribution of myocilin in corneal fibroblasts with or without dexamethasone (DEX) treatment were investigated. Methods: Human corneal fibroblasts were treated with 100 nM DEX for 10-14 days. Immunofluorescence staining for myocilin was performed. Cell lysates and ultracentrifugation fractions were assessed by western blotting for distribution of myocilin and its possible association with various organelles. Staurosporine was used to induce apoptosis and apoptotic cells were detected using a monoclonal single stranded DNA antibody. Results: By immunofluorescence, myocilin protein was found to distribute throughout the cytoplasm of corneal fibroblasts including perinuclear regions. Myocilin distribution overlapped to varying degrees with that of the Golgi complex, endoplasmic reticulum, and mitochondria. Subsequent examination by subcellular fractionation however revealed that myocilin, while co-sedimenting with the Golgi complex, lysosomes, and endoplasmic reticulum, did not fractionate or associate with mitochondria. On western blots, protein bands at approximately 66, 57, and 55 kDa were detected and the intensity of the bands was not affected by DEX treatment in corneal fibroblasts. Apoptosis was induced by staurosporine to a similar extent in both DEX-treated and untreated corneal cultures. Conclusions: In corneal fibroblasts, myocilin expression is not enhanced by DEX treatment and the protein was not associated with mitochondria, in contrast to what were found in human trabecular meshwork (TM) cells. Such differences suggest that the expression and distribution of myocilin may be distinctive for TM cells and may explain why pathology with myocilin mutations is only evident in glaucoma even though myocilin is expressed ubiquitously in ocular and nonocular tissues.