Purchase this article with an account.
Bruce A Pfeffer, Paul Wong, Libin Xu, Zihua Hu, Alison C Ziesel, Sriganesh Rao, Ned A Porter, Steven J Fliesler; Transcriptomic analysis of oxysterol effects on a photoreceptor-derived cell line, with relevance to Smith-Lemli-Opitz syndrome. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1708.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
Smith-Lemli-Opitz syndrome (SLOS) is a recessive disease involving defective biosynthesis of cholesterol (CHOL) and aberrant accumulation of its immediate precursor, 7-dehydrocholesterol (7DHC), in body tissues and fluids. 7DHC gives rise to several oxysterol by-products, some of which are cytotoxic, including 7-ketocholesterol (7kCHOL), as well as 7DHC-specific oxysterols, notably 5,9-endoperoxy-cholest-7-en-3β,6α-diol (EPCD). Selective degeneration and loss of photoreceptors (PRs) is a hallmark of the AY9944-induced rat SLOS model, which we hypothesize is due to the action of such oxysterols on PR cells. We previously showed that such oxysterols accumulate in the retina in this model, but not in untreated control rat retinas. We also have shown that EPCD is far more toxic than 7kCHOL to 661W cells (a mouse PR-derived line), and that RPE and rMC-1 cells are comparatively more resistant to such cytotoxicity. To elucidate molecular mechanisms of oxysterol-induced PR cell death, we carried out microarray analysis on 661W cells following incubations with oxysterols.
Cultured 661W cells were treated (N=3 replicates per condition) with 7kCHOL, EPCD, or CHOL at defined concentrations, vs. vehicle control (VC; hydroxypropyl-β-cyclodextrin). At selected time points (5 and 24 h), prior to overt morphological signs of cell death, RNA was harvested and subjected to gene microarray analysis. Gene annotation analysis was performed using gene sets with ≥ 2.0-fold change (FC) and false discovery rate (FDR) ≤ 0.05, with particular emphasis on canonical pathways associated with stress responses and cell death/ survival.
Both 7kCHOL and EPCD up-regulated transcription of nuclear receptors LXR and AHR, modulating their canonical downstream effector genes (P≤0.05), including virtually complete down-regulation of cholesterol pathway genes, as also seen in microarray analysis of AY9944 rat retinas. Notably, key genes associated with ER stress, mitochondrial dysfunction, autophagy, and cell death/ survival were significantly dysregulated. Some oxysterol-specific (7kCHOL vs. EPCD) effects also were observed. While CHOL (negative control) did not elicit such effects, it did exert some differential gene expression changes compared to VC treatment.
Our results suggest that EPCD and 7kCHOL may mediate some of their cytotoxic effects via divergent mechanisms.
This PDF is available to Subscribers Only