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Bruce A Pfeffer, Sriganesh Ramachandra Rao, Libin Xu, Ned A. Porter, Steven J Fliesler; Up-regulation of ER and oxidative stress markers induced by oxysterols in a photoreceptor-derived cell line, with relevance to Smith-Lemli-Opitz syndrome. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2394.
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© ARVO (1962-2015); The Authors (2016-present)
Smith-Lemli-Opitz syndrome (SLOS) involves defective biosynthesis of cholesterol (CHOL) and accumulation of 7-dehydrocholesterol (7DHC) in bodily tissues and fluids. We previously reported toxicity of 7-ketocholesterol (7kCHOL) and a 7DHC-derived oxysterol, EPCD, using 661W cells, a photoreceptor surrogate. Previous gene array analysis identified up-regulated pathways for endoplasmic reticulum (ER) stress, DNA damage and repair, autophagy, and oxidative stress following exposure to these two oxysterols, relative to vehicle control and CHOL. Here we employed immunohistochemical (IHC) and biochemical methods to confirm protein changes for associated genes that were up-regulated by ≥ 2.5-fold (vs. controls) in oxysterol-treated 661W cells.
Cells were incubated with 7kCHOL, EPCD, or with their respective vehicles (VC) alone (hydroxypropyl-β-cyclodextrin or DMSO). At appropriate time points preceding global cell detachment and death, cell lysates were prepared for PAGE-Western blot (WB) analysis; parallel cultures were fixed and processed for IHC/confocal microscopy. The following proteins were probed: heme oxygenase-1 (hox1), CHOP, tribbles homologue-3 (trb3), cysteine dioxygenase-1 (cdo1), and homocysteine ER-stress inducible-1 (herpud1).
Increased cytoplasmic immunoreactivity (IR) for hox1 was observed in cells treated with 7kCHOL or EPCD, compared to VC; quantitative WB revealed a 4-fold increase for hox1 in EPCD-treated cells. 7kCHOL-treated cells showed higher cdo1 IR, correlating well with 3-fold transcriptional up-regulation by microarray; this was not observed for EPCD treatment, which caused a 1.5-fold down-regulation of cdo1 by microarray. CHOP was increased (vs. VC) and localized primarily to nuclei in EPCD-treated cells, while 7kCHOL increased cytoplasmic CHOP IR. Subcellular localization of trb3 IR was restricted to mitotic spindles of unaffected cells, and its cytoplasmic IR was greatly increased in rounded-up cells following oxysterol incubation. For herpud1 (linked to ER stress), punctate IR was substantially elevated in oxysterol-treated cells (vs. VC).
Gene array data were predictive of oxysterol-induced expression of proteins involved in cellular stress responses. These results are consistent with the involvement of ER and oxidative stress in the mechanism of photoreceptor cell death in SLOS.
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