Abstract
Cholesterol homeostasis is regulated not only by cholesterol, but also by oxygenated cholesterol species, referred to as oxysterols. Side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), regulate cholesterol homeostasis through feedback inhibition and feed-forward activation of transcriptional pathways that govern cholesterol synthesis, uptake, and elimination, as well as through direct nongenomic actions that modulate cholesterol accessibility in membranes. Elucidating the cellular distribution of 25-HC is required to understand its biological activity at the molecular level. However, studying oxysterol distribution and behavior within cells has proven difficult due to the lack of fluorescent analogs of 25-HC that retain its chemical and physical properties. To address this, we synthesized a novel intrinsically fluorescent 25-HC mimetic, 25-hydroxycholestatrienol (25-HCTL). We show that 25-HCTL modulates sterol homeostatic responses in a similar manner as 25-HC. 25-HCTL associates with lipoproteins in media and is taken up by cells through LDL-mediated endocytosis. In cultured cells, 25-HCTL redistributes among cellular membranes and, at steady state, has a similar distribution as cholesterol, being enriched in both the endocytic recycling compartment as well as the plasma membrane. Our findings indicate that 25-HCTL is a faithful fluorescent 25-HC mimetic that can be used to investigate the mechanisms through which 25-HC regulates sterol homeostatic pathways.
Highlights
Cholesterol homeostasis is regulated by cholesterol, and by oxygenated cholesterol species, referred to as oxysterols
25-HC can bind the liver X receptors (LXRs) to active LXR-mediated transcription that results in increased cholesterol efflux and elimination [10, 11]. 25-HC inhibits sterol regulatory element-binding protein (SREBP) maturation and subsequent transcription of genes involved in cholesterol biosynthesis and uptake [1]
In contrast to cholesterol, which directly interacts with SREBP cleavage-activating protein (SCAP), 25-HC enhances the interaction between SCAP and Insig proteins, resulting in retention of the SREBP-2-SCAP complex in the endoplasmic reticulum (ER) [12, 13]
Summary
Cholesterol homeostasis is regulated by cholesterol, and by oxygenated cholesterol species, referred to as oxysterols. Side-chain oxysterols, such as 25-hydroxycholesterol (25-HC), regulate cholesterol homeostasis through feedback inhibition and feed-forward activation of transcriptional pathways that govern cholesterol synthesis, uptake, and elimination, as well as through direct nongenomic actions that modulate cholesterol accessibility in membranes. 25-HCTL associates with lipoproteins in media and is taken up by cells through LDL-mediated endocytosis. 25-HCTL redistributes among cellular membranes and, at steady state, has a similar distribution as cholesterol, being enriched in both the endocytic recycling compartment as well as the plasma membrane. Our findings indicate that 25-HCTL is a faithful fluorescent 25-HC mimetic that can be used to investigate the mechanisms through which 25-HC regulates sterol homeostatic pathways.— Iaea, D.
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