Accumulation of mutant myocilins in ER leads to ER stress and potential cytotoxicity in human trabecular meshwork cells q Myung Kuk Joe, 1 Seongsoo Sohn, 1 Wonhee Hur, Younkyong Moon, Young Ran Choi, and Changwon Kee * Department of Ophthalmology, Samsung Medical Center, School of Medicine, Sungkyunkwan University, Seoul, Republic of Korea Received 26 September 2003 Abstract MYOC encoding a 55 kDa secretory glycoprotein named myocilin is closely linked to primary open-angle glaucoma (POAG). To understand a role played by MYOC in glaucoma, we examined the cellular fate of various mutant myocilins that were adenovirally expressed in human trabecular meshwork cells. Most myocilins with mutations such as G364V, Q368X, K423E, Y437H, and I477N were intrinsically stable, and appeared to have interactions with wild-type myocilin but not with stromelysin and thereby selectively inhibited the secretion of the former protein. The myocilins expressed were identified to be concentrated into fine punctate ag- gregates in endoplasmic reticulum, but never developed into the formation of aggresomes. In endoplasmic reticulum, the accu- mulation of the myocilins resulted in the upregulation of 78 kDa glucose-regulated protein and protein disulfide isomerase. In addition, the expression of the myocilins led to deformed cellular morphology and diminished cell proliferation, an effect postulated to result in the dysfunction of trabecular cells that could be a cause of glaucoma. Therefore, our results support the statement that gain of function rather than haploinsufficiency is a critical mechanism for POAG in individuals with mutations on MYOC. Ó 2003 Elsevier Inc. All rights reserved. Keywords: Myocilin; Glaucoma; Trabecular meshwork cell; Aggregation; Unfolded protein response; Cytotoxicity; Adenoviral vector Primary open-angle glaucoma (POAG), characterized by the progressive excavation of optic disc, typical visual- field defect, and optic-nerve damage, is the most common type of glaucoma [1]. The disease manifests hereditary predisposition in an autosomal dominant fashion and its juvenile-onset form is mapped by linkage analysis on chromosome 1q23–q25 designated as GLC1A [2–5]. Subsequent positional cloning on the locus identified MYOC, also known as TIGR (trabecular meshwork-in- ducible glucocorticoid response protein), as a candidate gene for POAG as well as juvenile-onset form of POAG [6]. It has been reported that more than 40 mutations in MYOC occur in about 4% of all cases of POAG [6–9]. MYOC comprises three exons and encodes a gly- coprotein consisting of 504 amino acids, named myo- cilin because of the homology of its N-terminal region to nonmuscle myosin [10–12]. The protein is present both intracellularly and extracellularly, and is normally expressed in a variety of ocular and nonocular tissues [10,11]. In ocular tissues, a high amount of myocilin is detectable in trabecular extracellular matrix (ECM), a likely area for outflow resistance [13,14]. Since the el- evated intraocular pressure (IOP) due to decreased outflow facility through trabecular meshwork (TM) is the major risk factor for POAG, it is suggested that one of the roles played by myocilin is the outflow regulation in outflow tissues [15,16]. There are, how- ever, no conclusive evidence for this, and the mecha- nism by which myocilin is involved in POAG remains unclear. Recently, a genetic study identified an individual who was homozygous for R46X mutation on MYOC but did not develop glaucoma [9]. Similarly, a study on MYOC q Supported by grant from the Korea Science and Engineering Foundation, Science Research Center (SRC) project endowed to the Molecular Therapeutic Research Center (MTRC). * Corresponding author. Fax: +82-2-3410-0074. E-mail address: cwkee@smc.samsung.co.kr (C. Kee). 1 These authors contributed equally to this work. 0006-291X/$ - see front matter Ó 2003 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2003.10.162 Biochemical and Biophysical Research Communications 312 (2003) 592–600 BBRC www.elsevier.com/locate/ybbrc