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HR3/RORα-mediated cholesterol sensing regulates TOR signaling

ABSTRACT: Cells and organisms adjust their growth based on the availability of cholesterol, which is essential for cellular functions. However, the mechanisms by which cells sense cholesterol levels and translate these into growth signals are not fully understood. We report that cholesterol rapidly activates the master growth-regulatory TOR pathway in Drosophila tissues. We identify the nuclear receptor HR3, an ortholog of mammalian RORα, as an essential factor in cholesterol-induced TOR activation. We demonstrate that HR3 binds cholesterol and promotes TOR pathway activation through a non-genomic mechanism acting upstream of the Rag GTPases. Similarly, we find that RORα is necessary for cholesterol-mediated TOR activation in human cells, suggesting that HR3/RORα represents a conserved mechanism for cholesterol sensing that couples cholesterol availability to TOR-pathway activity. These findings advance our understanding of how cholesterol influences cell growth, with implications for cholesterol-related diseases and cancer. Here, we demonstrate that dietary cholesterol intake leads to rapid and dynamic activation of the TOR pathway in tissues of Drosophila. This response is modulated by the Drosophila RORα ortholog, HR3, which – like RORα – binds cholesterol and is activated by this ligand. Although HR3 is known to be transcriptionally upregulated by the ecdysone steroid hormone EcR, our results reveal that HR3 regulates growth through the TOR pathway in response to cholesterol independently of ecdysone-mediated effects. This regulation involves rapid cholesterol-induced TOR activation that in part is independent of the transcriptional functions of HR3, through an isoform of HR3 that lacks a DNA-binding domain (DBD). Reducing HR3 levels in cells mitigate the overactivation of TOR caused by the intralysosomal accumulation of cholesterol resulting from depletion of Npc1a. This indicates that HR3 is necessary for TOR activation by lysosomal cholesterol. Our findings suggest that HR3 activates the TOR pathway upstream of Rag proteins. Furthermore, our findings indicate that RORα is involved in cholesterol-induced TOR activation in human cells, suggesting that a conserved function of HR3/RORα is to couple cholesterol levels to TOR-pathway activation

INSTRUMENT(S):

ORGANISM(S): Homo Sapiens (human) Drosophila Melanogaster (fruit Fly)

SUBMITTER: Ivan Bradić

LAB HEAD: Kim Rewitz

PROVIDER: PXD074014 | Pride | 2026-02-10

REPOSITORIES: Pride

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			Action	DRS
	Cholesterolrefeedingphosphorylation.msf	Msf
	CholesterolrefeedingphosphorylationF10.raw	Raw
	CholesterolrefeedingphosphorylationF11.raw	Raw
	CholesterolrefeedingphosphorylationF12.raw	Raw
	CholesterolrefeedingphosphorylationF13.raw	Raw

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Project description:Cholesterol has attracted significant attention as a possible lifespan regulator. It has been reported that serum cholesterol levels have an impact on mortality due to age-related disorders such as cardiovascular disease. Diet is also known to be an important lifespan regulator. Dietary restriction retards the onset of age-related diseases and extends lifespan in various organisms. Although cholesterol and dietary restriction are known to be lifespan regulators, it remains to be established whether cholesterol is involved in dietary restriction-induced longevity. Here, we show that cholesterol deprivation suppresses longevity induced by intermittent fasting, which is one of the dietary restriction regimens that effectively extend lifespan. We also found that cholesterol is required for the fasting-induced upregulation of transcriptional target genes such as the insulin/IGF-1 pathway effector DAF-16 and that cholesterol deprivation suppresses the long lifespan of the insulin/IGF-1 receptor daf-2 mutant. Remarkably, we found that cholesterol plays an important role in the fasting-induced nuclear accumulation of DAF-16. Moreover, knockdown of the cholesterol-binding protein NSBP-1, which has been shown to bind to DAF-16 in a cholesterol-dependent manner and to regulate DAF-16 activity, suppresses both fasting-induced longevity and DAF-16 nuclear accumulation. Furthermore, this suppression was not additive to the cholesterol deprivation-induced suppression, which suggests that NSBP-1 mediates, at least in part, the action of cholesterol to promote fasting-induced longevity and DAF-16 nuclear accumulation. These findings identify a novel role for cholesterol in the regulation of lifespan. Two independent replicates were performed. Total RNA was extracted with TRIzol (Invitrogen). The extracted RNA was purified with PureLink RNA Micro Kit (Invitrogen) and analyzed with Agilent 2100 Bioanalyzer to assess the RNA integrity. The microarray procedures were performed according to Affymetrix protocols. Hybridized arrays were scanned using an Affymetrix GeneChip Scanner.

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HR3/RORα-mediated cholesterol sensing regulates TOR signaling

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