Mitochondrial cholesterol trafficking: impact on inflammatory mediators

Abstract

Macrophage ‘foam cells’ are the hallmark of early, and developing, atherosclerotic lesions. Generation of 27-oxygenated derivatives of cholesterol, one of the most abundant oxysterols in human atheroma, via mitochondrial sterol 27-hydroxylase (CYP27A1), achieves ligand-activation of liver X nuclear receptors (LXR), which marshal cholesterol homeostatic mechanisms leading to cholesterol efflux and nascent high-density lipoprotein generation. The rate-limiting step controlling activity of CYP27A1 is supply of cholesterol from the outer to the inner (cholesterol-poor) mitochondrial membrane, and can be facilitated by steroidogenic acute regulatory protein (StAR). However, LXR activation also exerts indirect control (transrepression) over gene expression of a range of inflammatory mediators, via interference with nuclear factor-kappa B transcription factors, integrating metabolic and inflammatory signalling. Here, we considered the impact of increased cholesterol delivery to CYP27A1 on the expression of inflammatory mediators: Toll-like receptor 3 (Tlr3), Toll-like receptor 6 (Tlr6) and lymphotoxin alpha (Lta). Murine RAW 264.7 macrophages stably transfected with pCMV.5 (empty vector control) and pCMV.5_Stard1 (StAR over-expressing) were challenged for 24 h, in the presence or absence of dibutyryl cAMP (0.3 mM), lipopolysaccharide (LPS; 0.1 mg/ml), LXR agonist (T0901317; 10 mM) and combinations thereof. Following isolation of RNA and cDNA synthesis, qualitative polymerase chain reaction (PCR) was used to determine the presence and expression of StAR (355 bp) and housekeeping gene, glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 410 bp), in each cell line. Levels of Stard1, Tlr3, Tlr6 and Lta mRNA were determined by quantitative PCR and expressed as a ratio to Gapdh. Over-expression of StAR significantly altered expression of genes implicated in the innate immune response, increasing Tlr3, Tlr6 and Lta expression under basal conditions, or following the addition of cAMP to increase StAR activity. Addition of LPS decreased intracellular levels of Stard1 mRNA; preliminary evidence of Tlr6 transrepression was also noted in StAR over-expressing cells following this inflammatory challenge. In contrast, induction of Tlr3 was noted in control following addition of LXR agonist, T0901317, suggesting Tlr3 may be a direct LXR target; Lta expression was also enhanced in StAR over-expressing cells in the presence of this agonist. These results should be considered carefully when developing StAR as a possible therapeutic strategy for human metabolic disease.