Down-regulation of GRP78 in human glaucomatous trabecular meshwork cells

PURPOSE: Since the 78 kDa glucose-regulated protein (GRP78) is a key marker of endoplasmic reticulum (ER) stress, we investigated and analyzed GRP78 expression levels in the trabecular meshwork (TM) by eyes with primary open angle glaucoma (POAG) and normal eyes to understand the role of GRP78 in human TM cells apoptosis. METHODS: Trabecular meshwork cells from POAG patients (GTM) and age-matched non-diseased individuals (NTM) were cultured and treated with or without the following chemical chaperones: tunicamycin (Tm), which induces ER stress, and staurosporine (STS), which induce apoptosis, and dimethylsulfoxide (DMSO) as a control, for 6–24 h. Expression of GRP78 mRNA and protein was measured using real-time PCR and western blot analysis. The intracellular distribution of GRP78 with myocilin was analyzed using confocal double immunofluorescence. RESULTS: Both real-time PCR and western blot analysis showed similar results, revealing that GRP78 mRNA expression and GRP78 protein levels were attenuated in GTM cells compared with NTM cells (65.97±3.8% and 80.49±4.2%, respectively, p<0.05). After exposure to Tm and following ER stress, increased GRP78 protein levels were detected in all cells. However, a low fold change of the protein (2.564 versus 2.710 for a 24 h exposure) and lower cell viability were found in GTM cells compared to NTM cells (p<0.05). Confocal microscopy showed that GRP78 was partly colocalized with myocilin in GTM cells, but less in NTM cells. After Tm and STS treatment, the colocalization of GRP78 with myocilin was found in both NTM and GTM cells. CONCLUSIONS: The authors propose that the down-regulation of GRP78 plays a role in the degeneration of TM cells in POAG patients, thus providing molecular insights into the pathogenesis of POAG and suggesting that GRP78 may have the potential to be a target for developing new modalities for ER stress-induced TM cell apoptosis.