title: Down-regulation of GRP78 in human glaucomatous trabecular meshwork cells.
abstract: OBJECTIVE
  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 immuno fluorescence.
  
  
  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.
authors.affiliation: State Key Laboratory of Ophthalmology, Zhongshan Ophthalmic Center, Sun Yat-sen University, Guangzhou, People's Republic of China.
authors.forename: Fang
authors.initials: F
authors.lastname: Chai
authors.forename: Rongjiang
authors.initials: R
authors.lastname: Luo
authors.forename: Yiqing
authors.initials: Y
authors.lastname: Li
authors.forename: Yujing
authors.initials: Y
authors.lastname: Bai
authors.forename: Yuan
authors.initials: Y
authors.lastname: He
authors.forename: Yantao
authors.initials: Y
authors.lastname: Wei
authors.forename: Zhichao
authors.initials: Z
authors.lastname: Yan
authors.forename: Jian
authors.initials: J
authors.lastname: Ge
authors.forename: Yehong
authors.initials: Y
authors.lastname: Zhuo
mesh_terms: D000293:Adolescent; D000328:Adult; D016923:Cell Death / Q000187:drug effects; D049109:Cell Proliferation / Q000187:drug effects; D048430:Cell Shape / Q000187:drug effects; D003598:Cytoskeletal Proteins / Q000378:metabolism; D015536:Down-Regulation / Q000187:drug effects / Q000235:genetics*; D000091342:Endoplasmic Reticulum Chaperone BiP; D005136:Eye Proteins / Q000378:metabolism; D005901:Glaucoma / Q000235:genetics* / Q000473:pathology*; D006023:Glycoproteins / Q000378:metabolism; D006360:Heat-Shock Proteins / Q000235:genetics* / Q000378:metabolism; D006801:Humans; D008875:Middle Aged; D021381:Protein Transport / Q000187:drug effects; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D019311:Staurosporine / Q000494:pharmacology; D013997:Time Factors; D014129:Trabecular Meshwork / Q000187:drug effects / Q000378:metabolism* / Q000473:pathology*; D014415:Tunicamycin / Q000494:pharmacology; D015854:Up-Regulation / Q000187:drug effects; D055815:Young Adult; D000099179:Myocilin
chemical_list: D003598:Cytoskeletal Proteins; D000091342:Endoplasmic Reticulum Chaperone BiP; D005136:Eye Proteins; D006023:Glycoproteins; D006360:Heat-Shock Proteins; D012333:RNA, Messenger; D019311:Staurosporine; D014415:Tunicamycin; D000099179:Myocilin; C000717247:HSPA5 protein, human