Functional Peroxisomes Are Essential for Efficient Cholesterol Sensing and Synthesis.

Khanichi N Charles,Steven J Fliesler,Herbert Stangl,Phyllis L Faust,Werner J Kovacs,Janis E Shackelford

doi:10.3389/fcell.2020.560266

Abstract

Cholesterol biosynthesis is a multi-step process involving several subcellular compartments, including peroxisomes. Cells adjust their sterol content by both transcriptional and post-transcriptional feedback regulation, for which sterol regulatory element-binding proteins (SREBPs) are essential; such homeostasis is dysregulated in peroxisome-deficient Pex2 knockout mice. Here, we compared the regulation of cholesterol biosynthesis in Chinese hamster ovary (CHO-K1) cells and in three isogenic peroxisome-deficient CHO cell lines harboring Pex2 gene mutations. Peroxisome deficiency activated expression of cholesterogenic genes, however, cholesterol levels were unchanged. 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR) protein levels were increased in mutant cells, whereas HMGCR activity was significantly decreased, resulting in reduced cholesterol synthesis. U18666A, an inhibitor of lysosomal cholesterol export, induced cholesterol biosynthetic enzymes; yet, cholesterol synthesis was still reduced. Interestingly, peroxisome deficiency promoted ER-to-Golgi SREBP cleavage-activating protein (SCAP) trafficking even when cells were cholesterol-loaded. Restoration of functional peroxisomes normalized regulation of cholesterol synthesis and SCAP trafficking. These results highlight the importance of functional peroxisomes for maintaining cholesterol homeostasis and efficient cholesterol synthesis.

Highlights

Cholesterol is an essential lipid constituent of cellular membranes, in particular the plasma membrane, and an obligatory precursor for synthesis of steroid hormones, bile acids, and regulatory oxysterols, as well as a requisite ligand for activation of Hedgehog family proteins
As total cholesterol levels were similar in P10 Swiss Webster-129 (SW/129) Pex2−/− and control mice, we examined the expression of Srebf2, Insig1, Insig2, and cholesterol biosynthetic genes (Hmgcr, Pmvk, Mvd, Idi1, farnesyl diphosphate synthase (Fdps), Fdft1, Sqle, Sc4mol, Lss) in these tissues as well as skeletal muscle
To exclude the possibility that the altered GFP-sterol regulatory element-binding protein (SREBP) cleavage-activating protein (SCAP) localization in ZR-82 cells cultured in medium containing 10% fetal calf serum (FCS) was caused by general changes in endoplasmic reticulum (ER) morphology, we examined the localization of activating transcription factor 6 (ATF6) in Chinese hamster ovary (CHO)-K1 and ZR-82 cells

Summary

Introduction

Cholesterol is an essential lipid constituent of cellular membranes, in particular the plasma membrane, and an obligatory precursor for synthesis of steroid hormones, bile acids, and regulatory oxysterols, as well as a requisite ligand for activation of Hedgehog family proteins. Peroxisomes and Cholesterol Homeostasis studies assign the pre-squalene steps of cholesterol biosynthesis to peroxisomes (Figure 1A) [reviewed in Kovacs et al (2002), Faust and Kovacs (2014)]. With the exception of HMGCR, the enzymes of the pre-squalene segment contain functional peroxisomal targeting signals (PTS) that mediate their import into the peroxisomal matrix (Kovacs et al, 2002; Faust and Kovacs, 2014). We showed that acetyl-CoA derived from peroxisomal β-oxidation of very long-chain fatty acids (i.e., [1,2,3,4-13C4]docosanoate) and dicarboxylic acids (i.e., [U13C12]dodecanedioate) is channeled to cholesterol synthesis inside peroxisomes (Kovacs et al, 2007). The enzymes of the pre-squalene segment of the isoprenoid biosynthetic pathway are localized to peroxisomes in plants [reviewed in Faust and Kovacs (2014), Henry et al (2018)]. Several enzymes of the isoprenoid biosynthetic pathway are localized within peroxisome-related microbodies called glycosomes in trypanosomatid parasites (Concepcion et al, 1998; Urbina et al, 2002; Colasante et al, 2006; Carrero-Lérida et al, 2009; Güther et al, 2014; Ferreira et al, 2016; Acosta et al, 2019)

Objectives

Methods

Results

Conclusion